PROTAC molecule and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a PROTAC molecule and application thereof in preparation of a targeted HPK1 inhibitor, a targeted HPK1 degradation agent and a medicine for treating and / or preventing cancers, a target protein ligand in the PROTAC molecule is an indolone derivative, and the PROTAC small molecule provided by the invention can selectively degrade HPK1 protein.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a class of PROTAC molecules and their use in preparing targeted HPK1 inhibitors, targeted HPK1 degraders, and drugs for treating and or preventing cancer. Background Art

[0002] Hematopoietic progenitor kinase 1 (HPK1), also known as MAP4K1, is a member of the STE20-like serine / threonine kinase family. Other members of the MAP4K1 family include GCK / MAP4K2, GLK / MAP4K3, HGK / MAP4K4, KHS / MAP4K5, and MINK / MAP4K6. HPK1 has been shown to have an inhibitory effect on tumor immunity. In T cells, activation of the T cell receptor (TCR) signaling pathway leads to the recruitment of HPK1 in the cytoplasm to the cell membrane, binding to and phosphorylating the adaptor protein SLP76, promoting the binding of SLP76 to the E3 ligase 14-3-3, leading to the degradation of the SLP76 / LAT signalosome, and then negatively regulating the TCR pathway, inhibiting T cell activation and effector T cell function. In addition, HPK1 is also a negative regulator of B cell and dendritic cell activation, and plays an important role in maintaining Treg cell function. Early knockout studies showed that HPK1-deficient mice had enhanced T cell proliferation, increased production of helper T type 1 (Th1) cytokines (such as IL-2, interferon-γ), and improved dendritic cell-mediated antigen presentation, indicating that HPK1 kinase activity plays an important role in regulating T cell function. Genetic studies using HPK1 knockout and kinase-dead mice have shown that inhibiting HPK1 alone or in combination with immune checkpoint blockade may be a promising cancer immunotherapy. Studies have found that HPK1 is associated with the occurrence and development of a variety of malignant tumors, such as pancreatic cancer, breast cancer, colorectal cancer, etc. Therefore, HPK1 is a potential target for tumor immunotherapy and autoimmune diseases.

[0003] Proteolysis Targeting Chimera (PROTAC) is a bifunctional molecule consisting of a ligand that binds to an E3 ubiquitin ligase and a ligand that binds to a target protein connected by a linker. The PROTAC molecular structure allows the E3 ubiquitin ligase to approach the protein, allowing the target protein to be tagged with ubiquitin and then degraded through the ubiquitin-proteasome pathway. Traditional inhibitors mainly block the catalytic activity of druggable proteins through occupancy-driven pharmacological effects. In contrast, PROTACs degrade target proteins through an event-driven mode of action, thereby removing functions associated with the target protein. This technology has received widespread attention in recent years.

[0004] At present, major pharmaceutical companies, research institutes and universities are competing to develop drugs related to the HPK1 target, most of which are HPK1 inhibitors. At present, at least 10 HPK1 inhibitors have entered clinical research, and most of them are in Phase I clinical research. However, the development of HPK1 degraders is relatively small, and the relevant patents that have been disclosed include: CN115873018A, WO2020227325Al, WO2023006063A1, WO2023023941A1, CN112552293A, etc.

[0005] The HPK1 PROTAC disclosed herein provides a new mechanism for treating HPK1-mediated diseases. In addition, the ability of degraders to target HPK1 for degradation, rather than simply inhibiting the catalytic activity of HPK1, is expected to overcome drug resistance, whether due to drugs used in previous treatments or acquired resistance caused by genetic mutations, amplifications or other reasons. Summary of the invention

[0006] The present invention discloses a class of indolone degraders with novel structure and high selectivity targeting HPK1 protein, which have high HPK1 protein inhibitory activity, degradation activity and good pharmacokinetic properties and can be used to treat HPK1-related diseases such as cancer.

[0007] To achieve the above object, the present invention provides a PROTAC molecule or a stereoisomer, tautomer, deuterated product, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal thereof, wherein the target protein ligand in the PROTAC molecule is an indolinone derivative, and the PROTAC molecule is selected from the compounds represented by the general formula (I), and the general formula (I) is as follows:

[0008]

[0009] In the formula: HI is a ligand that binds to the target protein HPK1, EL is an E3 ligase ligand, L is a connecting chain connecting HI and EL, and HI is the following structure, where "--" indicates the position of connection with L:

[0010]

[0011] Among them, R 1 and R 2 Both are independently selected from any one of an aromatic ring, an aromatic heterocyclic ring containing a N atom, and an aromatic cyclic ring;

[0012] R 3 and R 4Both are independently selected from any one of -H, -F, -Cl, -CH3, -CH2CH3, -OCH3; M and K are both independently selected from C, N, O, S or CR 5 Any one of the following, where R 5 is any one of H, -CH3, -CH2CH3, or -CH(CH3)2; n is 0, 1 or 2; X is C or N.

[0013] As a preferred embodiment of the PROTAC molecule of the present invention, the compound represented by Formula I or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof, wherein EL is the following structure:

[0014]

[0015] Where Y is a hydrogen atom, a deuterium atom, a fluorine atom, a chlorine atom or an alkyl substituent; Z is a carbon atom or a carbonyl group; G is a hydrogen atom or a methyl group; Ring B is C 6-12 aryl, 5-12 membered heteroaryl or 4-12 membered heterocyclic group, each of which is optionally substituted by 1 to 3 R a Replace; where each R a are independently selected from hydrogen, oxo, imino, sulfoxime, halo, nitro, -CN, C 1～6 Alkyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 3～15 Cycloalkyl, C 1～8 Haloalkyl, C 6～12 Aryl, 5- to 12-membered heteroaryl, 4- to 12-membered heterocyclic group, -OR b 、-C(O)-R b 、-C(O)OR b 、-C(O)-N(R b )(R b )、-N(R b )(R b )、-N(R b )C(O)-R b 、-N(R b )C(O)OR b 、-N(R b )C(O)N(R b )(R b )、-N(R b )S(O)2(R b ),-NR b S(O)2N(R b )(R b )、-N(R b )S(O)2O(R b)、-OC(O)R b 、-OC(O)-N(R b )(R b )、-Si(R b )3. -SR b ,

[0016] -S(O)R b 、-S(O)(NH)R b 、-S(O)2R b or -S(O)2N(R b )(R b ), where C 1～6 Alkyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 3～15 Cycloalkyl, C 1～8 Haloalkyl, C 6～12 Each of the aryl group, the 5- to 12-membered heteroaryl group, and the 4- to 12-membered heterocyclic group can be optionally substituted by 1 to 3 R c Replacement; where R b is hydrogen or C 1～6 Alkyl; each R c are independently halo, oxo, -CN, -OH, C optionally substituted with 1 to 3 fluorine groups 1～6 Alkyl, or C 3～8 Cycloalkyl, or -OC optionally substituted by 1 to 3 fluorine groups 1～6 Alkyl; W is a carbon atom or a nitrogen atom.

[0017] In even more specific embodiments, the compounds shown in formula IV, V, and VI are characterized in that the compound structure is preferably the following structure:

[0018]

[0019]

[0020] In an embodiment of the present invention, the compounds represented by formula II and III are preferably the following structures, where “--” represents the position where L is connected.

[0021]

[0022] In an embodiment of the present invention, L is the following structure:

[0023]

[0024]

[0025] Wherein, Q and G are independently selected from carbon atoms, oxygen atoms, nitrogen atoms, -(CO)-, -NH(CO)-, -N(CH3)-,

[0026]

[0027] n1, m1, m2 are integers between 0 and 12; Q 1 and G 2 are independently selected from a carbon atom or a nitrogen atom; 2 , Q 3 , G 2 and G 3 are independently selected from a covalent bond, a carbon atom, an oxygen atom, a nitrogen atom, and -(CO)-, -NH(CO)-, a nitrogen-containing five-membered ring, and a nitrogen-containing six-membered ring;

[0028] In the embodiments of the present invention, the compound represented by formula I can be selected from compounds of any of the following structures, but this does not mean that the compound represented by formula I of the present invention is limited to the following compounds.

[0029]

[0030]

[0031]

[0032] In an embodiment of the present invention, there is provided a use of a compound as shown in Formula I, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in the preparation of a drug for treating and or preventing cancer.

[0033] The pharmaceutically acceptable salts refer to salts of the compounds of the present invention, prepared from compounds with specific substituents found in the present invention and relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid (forming carbonate or bicarbonate), phosphoric acid (forming phosphate, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid (forming sulfate or bisulfate), hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid. Acids such as tartaric acid and methanesulfonic acid; organic acid salts also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups, and can be converted into any base or acid addition salt. Preferably, the salt is contacted with a base or acid in a conventional manner, and the parent compound is separated, thereby regenerating the free form of the compound. The free form of the compound differs from its various salt forms in certain physical properties, such as different solubility in polar solvents.

[0034] It should be noted that the isomers of the present invention refer to compounds having the same chemical formula but different atomic arrangements. The metabolites of the present invention refer to pharmaceutically active products produced by the metabolism of the compound shown in Formula I or its salt in vivo. Such products can be produced, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, glucuronidation, enzymatic cleavage, etc. of the administered compound. Therefore, the present invention includes metabolites of the compounds of the present invention, including compounds produced by a method of contacting the compounds of the present invention with mammals for a period of time sufficient to obtain their metabolites. In addition to the form of salts, the compounds provided by the present invention also exist in the form of prodrugs. The prodrugs of the compounds described herein are easily chemically changed under physiological conditions to be converted into the compounds of the present invention. Any compound that can be converted in vivo to provide a biologically active substance (i.e., a compound shown in Formula I) is a prodrug within the scope and spirit of the present invention. For example, a compound containing a carboxyl group can form a physiologically hydrolyzable ester, which acts as a prodrug by hydrolyzing in vivo to obtain the compound shown in Formula I itself. The prodrug is preferably administered orally, because hydrolysis occurs mainly under the influence of digestive enzymes in many cases. Parenteral administration may be used when the ester itself is active or when hydrolysis occurs in the blood.

[0035] The cancer refers to one or more of gastric cancer, lung cancer, breast cancer, prostate cancer, leukemia, ovarian cancer, esophageal cancer, liver cancer, colon cancer, and brain cancer.

[0036] Beneficial effects:

[0037] The PROTAC molecules provided by the present invention all have significant enzyme activity that inhibits HPK1 kinase and have good protein degradation activity on HPK1, and can therefore be used to prepare targeted HPK1 inhibitors or targeted HPK1 degraders. The PROTAC molecules provided by the present invention also have good selective degradation effects on HPK1, wherein the protein degradation activity of some compounds on HPK1 is more than 10 times that of GLK, and compound C48 in particular exhibits a high selectivity for HPK1 / GLK protein degradation activity greater than 1000 times. Therefore, the PROTAC molecules provided by the present invention have good application potential in the treatment of diseases mediated by HPK1.

[0038] The present application verified through experiments that the PROTAC molecules provided can significantly inhibit the phosphorylation of SLP76 downstream of HPK1, and are superior to the positive control compounds. This indicates that the compounds of the present invention can inhibit the signal transduction of HPK1 kinase or enhance the anti-tumor immune response by degrading HPK1 protein.

[0039] This application also verifies through experiments that the PROTAC molecule provided has high exposure in rats, a long half-life, and good oral absorption. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a gel electrophoresis photograph of the degradation of HPK1 protein by compound C48;

[0041] Figure 2 This is the gel electrophoresis photograph of the degradation of GLK protein by compound C48. DETAILED DESCRIPTION

[0042] The present invention will be further described below in conjunction with specific examples, but these examples do not limit the scope of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial sources.

[0043] Example 1: Synthesis of Intermediate A1

[0044]

[0045] Step 1: Dissolve 2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxylic acid (10 g, 1.0 eq), HATU (34.1 g, 1.5 eq), and DIPEA (15.5 g, 2.0 eq) in DMF (80 ml), stir and react at 10 ° C for 30 min, then add tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (16.5 g, 1.2 eq), stir and react at 25 ° C for 1 h. Add 100 ml of water to the reaction solution, extract with dichloromethane, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography (PE: EA = 15: 1) to obtain 16.3 g of light white solid, namely intermediate 2, with a yield of 72%. MS m / z (ESI): 379.23 [M+H + ].

[0046] Step 2: Dissolve the intermediate 2 (10 g) in dichloromethane (50 ml), maintain 0°C, slowly add trifluoroacetic acid (9 g, 3 eq), and stir at room temperature for 1 h. The reaction solution was adjusted to a weakly alkaline pH with a 2 mol / L sodium hydroxide solution, washed with water, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to obtain 6.9 g of a brown oil (intermediate A1), with a yield of 93%. MS m / z (ESI): 279.18 [M+H + ].

[0047] Example 2: Synthesis of Intermediate B1

[0048]

[0049] Step 1: Dissolve 5-bromo-2-chloro-4-methyl-3-nitropyridine (8.8 g, 1 eq) in 1,4-dioxane (70 ml), cool to below 10°C, add NaH (2.8 g, 2 eq) and ethyl 2-hydroxyacetate (7.3 g, 2 eq), and maintain the reaction at room temperature for 3 h. Slowly add the reaction solution to 1N hydrochloric acid solution at low temperature, extract with ethyl acetate three times, combine the organic phases, wash with water, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography (PE: EA = 10: 1) to obtain 7 g of yellow solid (B-02), with a yield of 63%. MS m / z (ESI): 318.99 [M+H + ].

[0050] Step 2: Add intermediate B-02 (4g, 1eq) into a sealed tube, add acetic acid (20ml), anhydrous ethanol (8ml) and zinc powder (4.48g, 5eq), and maintain the sealed reaction at 85°C for 3h. Cool the reaction to room temperature, filter, concentrate the filtrate, dilute with ethyl acetate, adjust the base with saturated sodium carbonate solution, extract with ethyl acetate three times, combine the organic phases, wash with water, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, and concentrate to obtain 3g of off-white solid (B-03), with a yield of 98.5%. MS m / z (ESI): 242.98 [M+H + ].

[0051] Step 3: Add intermediate B-03 (3 g, 1 eq) into a sealed tube, add tetrahydrofuran (15 ml) and borane-tetrahydrofuran complex (36.5 ml, 3 eq), and maintain the reaction at room temperature for 2 h. Add saturated sodium bicarbonate solution to the reaction solution to adjust the base, extract with ethyl acetate three times, combine the organic phases, wash with water and saturated sodium chloride solution, dry with anhydrous sodium sulfate, and concentrate to obtain 1.5 g of white solid (B-04), with a yield of 53%. MS m / z (ESI): 229.00 [M+H + ].

[0052] Step 4: Dissolve B-04 (10.5 g, 1 eq) in tetrahydrofuran (68 ml), cool to below 0°C, add LiHMDS (91 ml, 2 eq) and di-tert-butyl dicarbonate (30 g, 3 eq), and maintain the reaction at room temperature for 2 h. Add saturated sodium bicarbonate solution to quench, extract with EA, combine the organic phases, wash with water, dry with anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography to obtain 7 g of yellow solid (B-05), with a yield of 46%. MS m / z (ESI): 329.05 [M+H + ].

[0053] Step 5: Dissolve the intermediate B-05 (1.5 g, 1 eq) in 1,4-dioxane (15 ml), add diboric acid pinacol ester (2.39 g, 2 eq), potassium acetate (1.92 g, 3.5 eq) and Pd(pddf)Cl2 (0.84 g, 0.2 eq), replace with N2, and maintain the reaction at 90°C for 3 h. The reaction solution was concentrated and purified by silica gel column chromatography (PE: EA = 10: 1) to obtain 1.5 g of oil (B-06), with a yield of 93%. MS m / z (ESI): 377.22 [M+H + ].

[0054] Step 6: Dissolve 5-bromoindole (0.34 g, 1 eq) in 1,4-dioxane (4 ml), add intermediate B-06 (0.59 g, 1 eq), 2N K2CO3 aqueous solution (1.6 ml) and Pd(pddf)Cl2 (115 mg, 0.1 eq), replace with N2, and maintain the reaction at 90°C for 3 h. The reaction solution was filtered, extracted with ethyl acetate three times, and the organic phases were combined, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (PE: EA = 6: 1) to obtain yellow solid B-07 with a yield of 75%. MS m / z (ESI): 382.18 [M+H + ].

[0055] Step 7: Dissolve the intermediate B-07 (0.8 g, 1 eq) in anhydrous ethanol (8 ml), add 2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxylic acid (0.38 g, 1.05 eq) and 1 drop of piperidine, and maintain the reaction at 70°C for 4 h. The reaction solution was cooled to room temperature and filtered to obtain 0.5 g of a brick-red solid (B1), which was used directly in the next step without purification, with a yield of 45%. MS m / z (ESI): 531.22 [M+H + ].

[0056] Example 3: Synthesis of Intermediate B2

[0057]

[0058] The synthesis method is the same as the seventh step of intermediate B1, except that only 2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxylic acid needs to be replaced with A1, and the equivalent weight is the same. Finally, intermediate B2 is prepared with a yield of 69%. MS m / z (ESI): 642.34 [M+H + ].

[0059] Example 4: Synthesis of Intermediate B3

[0060]

[0061] Step 1: The synthesis method is the same as the sixth step of intermediate B1, except that 5-bromoindole oxide is replaced with 5-bromo-6-fluoroindolin-2-one with the same equivalent weight to obtain B-08. MS m / z (ESI): 400.17 [M+H + ].

[0062] Step 2: The synthesis method is the same as the seventh step of intermediate B1, except that B-07 is replaced by B-08, 2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxylic acid is replaced by A1, and the equivalent is the same, and finally intermediate B3 is prepared. MS m / z (ESI): 660.33 [M+H + ].

[0063] Example 5: Synthesis of Intermediate B4

[0064]

[0065] Step 1: Add tert-butyl acrylate (3.10 g, 1 eq) and ethylene glycol (5 g, 3.3 mmol) to the reaction mixture, dropwise add 5 ml of saturated potassium hydroxide aqueous solution, stir at room temperature, react overnight, TLC monitoring shows that the raw materials react completely, add 20 ml of water and 20 ml of dichloromethane, stir for 0.5 h, separate, wash the organic phase with brine, dry the organic phase, and remove the solvent under reduced pressure to obtain a milky white oil B4-1 (2.76 g, 60%). MS m / z (ESI): 191.13 [M+H + ].

[0066] Step 2: Dissolve the intermediate B4-1 (2 g, 1 eq) in DMSO (20 ml), add Desmartin (8.9 g, 2 eq) in batches at 0°C, stir at room temperature until the reaction is complete as detected by LC-MS, add an equal amount of ice water to the reaction mixture, adjust the pH to 10 with a saturated Na2CO3 aqueous solution, and extract the aqueous layer with dichloromethane, combine the organic phases, wash once with a saturated Na2S2O3 solution, wash twice with brine, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain 1.77 g of a yellow oil (B4-2), which can be directly used for the next step without purification. Yield 89%. MS m / z (ESI): 189.11 [M+H + ].

[0067] Step 3: Dissolve intermediate B2 (1.5 g, 1 eq) and B4-2 (660 mg, 1.5 eq) in tetrahydrofuran (20 ml), add acetic acid (2 eq), stir at room temperature for 30 minutes, add sodium triacetoxyborohydride (990 mg, 2 eq) in batches, and maintain the reaction at room temperature for 2 hours. Add saturated sodium carbonate solution to the reaction solution to adjust the base, extract with dichloromethane three times, combine the organic phases, wash with water and saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography to obtain a yellow oil B4-3 (1.2 g, yield 65%). MS m / z (ESI): 814.45 [M+H + ].

[0068] Step 4: Dissolve the intermediate B4-3 (1 g, 1 eq) in dichloromethane (10 ml), add TFA dropwise at 0°C, and return to room temperature for about 2 hours. After the reaction is complete, concentrate and purify by silica gel column chromatography to obtain B4 (754 mg) with a yield of 79%. MS m / z (ESI): 758.39 [M+H + ].

[0069] Example 6: Synthesis of Intermediate H1

[0070]

[0071] The first step: 4-bromo-2-hydroxybenzyl cyanide (5.00 g, 27.47 mmol), 4-methylthiazole (4.99 ml, 54.94 mmol, 2.0 eq), anhydrous potassium acetate (5.39 g, 54.94 mmol), palladium acetate (61.50 mg, 2.74 mmol) in DMF was bubbled with nitrogen for 10 min. The resulting mixture was stirred at 110 ° C for 5 hours under nitrogen. The reaction was complete after TLC monitoring. The reaction mixture was cooled to room temperature, diluted with water, and extracted with dichloromethane (2x50 ml). The combined organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (PE: EA = 10: 1) to give a yellow solid H1-1 (3.3 g) with a yield of 60%. MS m / z (ESI): 201.05 [M+H + ].

[0072] Step 2: H1-1 (5 g, 24.96 mmol) was dissolved in THF (40 ml), and 1 ml of LAH in THF (37.45 ml, 37.45 mmol) was slowly added at -10 °C under nitrogen. The resulting reaction mixture was stirred at room temperature for 5 h. After the reaction was complete as monitored by TLC, the reaction was quenched by adding Na2SO4·10H2O, extracted with ethyl acetate (2 x 100 ml), the organic layers were combined, washed with saturated sodium chloride (2 x 100 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The resulting crude product was dissolved in a methanol solution of hydrochloric acid (100 ml), stirred for 10 hours, and filtered. The filter cake was washed with a mixture of ethyl acetate and methanol (5:1) to obtain H1-2 (1.8 g), with a yield of 30%. MS m / z (ESI): 205.08 [M+H + ].

[0073] Step 3: Under nitrogen atmosphere, DIPEA (7.6 g, 3 eq) and HATU (11.2 g, 2 eq) were added to a DMF solution of trans-N-(tert-butyloxycarbonyl)-4-hydroxy-L-proline (5 g, 1.1 eq) cooled in an ice bath, and the resulting mixture was stirred at room temperature for 0.5 h. H1-2 (4.7 g, 1 eq) was slowly added at -20°C, and the resulting mixture was slowly returned to room temperature and stirred for 6 hours. The reaction was monitored by TLC, and an appropriate amount of water was added for dilution, and ethyl acetate was extracted (2 x 100 ml). The organic layers were combined, washed with saturated brine (2 x 100 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (DCM / MeOH) to obtain H1-3 as a pale yellowish white solid (4.9 g) with a yield of 60%. MS m / z (ESI): 418.18 [M+H + ].

[0074] Step 4: Add CF3COOH (15 ml) to a DCM solution of H1-3 (4.9 g 11.73 mmol) cooled in an ice-water bath, and stir the reaction mixture at room temperature for 3 h. TLC monitors the reaction to be complete. The mixture is concentrated to obtain H1-4 as a yellow oil (4.4 g) with a yield of 90%. MS m / z (ESI): 318.13 [M+H + ].

[0075] Step 5: (S)-2-[(tert-Butyloxycarbonyl)amino]-3,3-dimethylbutanoic acid (2.42 g, 10.46 mmol), HATU (5.4 g, 14.31 mmol), and DIPEA (3.70 g, 28.62 mmol) were dissolved in DMF (35 ml) and cooled in an ice bath. H1-4 (4.00 g, 9.54 mmol) was slowly added to the mixture. Stirring was performed at room temperature for 6 h. The reaction was complete after TLC monitoring. The reaction solution was diluted with water, extracted with ethyl acetate (2 x 50 ml), the organic phases were combined, washed with saturated brine (2 x 50 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (DCM: MeOH = 20: 1) to obtain H1-5 (2.28 g) with a yield of 45%. MS m / z (ESI): 531.26 [M+H + ].

[0076] Step 6: Slowly add 2M HCl in ethyl acetate (7.54 ml, 15.08 mmol) to an ethyl acetate solution of H1-5 (2.00 g, 3.77 mmol) cooled in an ice-water bath, stir the reaction mixture at room temperature for 3 h, filter the reaction mixture, wash the filter cake with ethyl acetate, and obtain the hydrochloride of H1 (1.36 g), with a yield of 77%. MS m / z (ESI): 431.21 [M+H + ].

[0077] Example 7: Synthesis of Intermediate H2

[0078]

[0079] The first step: a DCM solution of (R)-1-(4-bromophenyl)ethylamine (3 g, 1 eq) was cooled in an ice bath and treated with TEA (303 mg, 0.2 eq) and tert-butyl dicarbonate (910 mg, 1.5 eq), and the reaction mixture was returned to room temperature and stirred for 3 hours. The crude reaction product was diluted with DCM and washed with dilute aqueous hydrochloric acid, saturated aqueous NaHCO3 and brine in sequence, dried over anhydrous sodium sulfate, and desolventized to obtain H2-1 (4.18 g), with a yield of 93%. MS m / z (ESI): 300.06 [M+H + ].

[0080] Step 2: The synthesis method is the same as the synthesis step of intermediate H1-1 in Example 6, except that the raw material 4-bromo-2-hydroxybenzyl cyanide is replaced with H2-1 to obtain H2-2 (2.53 g) with a yield of 57%. m / z (ESI): 319.15 [M+H +The subsequent synthesis steps were the same as the second to sixth steps of the synthesis of intermediate H1, except that H1-1 was replaced by H2-2, and finally H2 (703 mg) was obtained with a yield of 70%. MS m / z (ESI): 445.23 [M+H+].

[0081] Example 8: Synthesis of intermediates L1b and L1c

[0082]

[0083] Step 1: Dissolve 2-(2,6-dioxapiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (10 g, 1 eq) in DMSO (70 ml), add tert-butyl (6-aminohexyl)carbamate (9.4 g, 1.2 eq) and DIPEA (11.7 g, 2.5 eq), and maintain the reaction at 90 ° C for 2 h. The reaction solution was diluted with water, extracted with ethyl acetate three times, and the organic phases were combined, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (PE: EA = 6: 1) to obtain 12.6 g of yellow solid (L1-1b, yield 74%). MS m / z (ESI): 473.24 [M+H + ].

[0084] Step 2: Dissolve L1-1b (5 g, 1 eq) in dichloromethane (25 ml), add trifluoroacetic acid (20 ml), and maintain the reaction at room temperature for 1 h. The reaction solution was concentrated to obtain a yellow-green solid L1b, which was directly used in the next step without purification. MS m / z (ESI): 373.19 [M+H + ].

[0085] The synthesis of intermediate L1c was based on the synthesis of intermediate L1b. Only (6-aminohexyl)carbamic acid tert-butyl ester was replaced with (7-aminoheptyl)carbamic acid tert-butyl ester with the same equivalent weight to obtain L1c. MS m / z (ESI): 387.20 [M+H + ].

[0086] Example 9: Synthesis of Intermediates L2a and L2b

[0087]

[0088] The first and second steps: The first and second steps of the synthesis steps of intermediates L2a and L2b are different from the synthesis steps of intermediate L1b in Example 8 only in that (6-aminohexyl)carbamic acid tert-butyl ester is replaced by mono-Boc-ethylenediamine (1.5eq) to obtain intermediate L2-2.

[0089] Step 3: Dissolve the intermediate L2-2 (2 g, 1 eq) in DMF (20 ml), add DIPEA (2.2 ml, 2 eq) and HATU (2.9 g, 1.2 eq), stir at room temperature for 30 minutes, add suberic acid (1.32 g, 1.2 eq), and react at room temperature for 2 h. The reaction solution was diluted with water, extracted with dichloromethane, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (DCM: MeOH = 60: 1) to obtain a green oil L2a (2.2 g), with a yield of 84%. MS m / z (ESI): 417.14 [M+H + The synthesis method of intermediate L2b refers to the synthesis of L2a. Only suberic acid is replaced with adipic acid, and the equivalent weight is the same to obtain L2b. MS m / z(ESI):445.17[M+H + ].

[0090] Example 10: Synthesis of Intermediate L3

[0091]

[0092] Step 1: Dissolve 2-(2,6-dioxapiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (3 g, 1 eq), 4-piperidinol (1.6 g, 1.3 eq), and DIPEA (2.8 g, 2 eq) in DMSO (30 ml), and stir at 110°C for 3 h. Dilute with water, extract with dichloromethane, wash with saturated sodium chloride solution, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain the crude product L3-1, which is used directly in the next step without further purification, with a yield of 85%. MS m / z (ESI): 372.16 [M+H + ].

[0093] Step 2: Add compound L3-1 (3.4 g, 1 eq) and DMSO (17 ml) into a 250 ml round-bottom flask, and add Dess-Martin reagent (7.7 g, 2 eq) in batches at 0°C. Stir and react for 2 h at room temperature. Dilute with water, adjust pH to 10 with saturated sodium carbonate aqueous solution, extract with dichloromethane, combine the organic phases, wash with saturated Na2S2O3 solution, dry over anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography to obtain the target product L3, yield: 67%. MS m / z (ESI): 370.14 [M+H + ].

[0094] Example 11: Synthesis of Intermediate L4

[0095]

[0096] The synthesis method of intermediate L4 is the same as that of intermediate L3, except that 4-piperidinemethanol is replaced with 4-hydroxypiperidine with the same equivalent weight to obtain intermediate L4. MS m / z (ESI): 356.26 [M+H + ].

[0097] Example 12: Synthesis of Intermediates L5, L6 and L7

[0098]

[0099] The synthesis method of intermediate L5 is different from that of intermediate L3 only in that the starting material 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione is replaced with 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione, and the equivalent weight is the same, and intermediate L5 can be obtained. MS m / z(ESI):370.14[M+H + ].

[0100] The synthesis methods of L6 and L7 differ from those of L5 only in that 4-piperidinemethanol is replaced by 4-hydroxypiperidine and azetidine-3-ol, respectively, with the same equivalents, to obtain intermediate L6 (MS m / z (ESI): 356.12 [M+H + ]), intermediate L7 (MS m / z (ESI): 328.09 [M+H + ]).

[0101] Example 13: Synthesis of Intermediate L8

[0102]

[0103] The first step of the synthesis of intermediate L8 is the same as the synthesis step of intermediate L3-1, and only 4-piperidinol is replaced with piperidine-4-carboxylic acid tert-butyl ester (1.5 eq) to obtain L8-1. MS m / z (ESI): 442.20 [M+H + ].

[0104] The second step of the synthesis of intermediate L8 is the same as the fourth step of the synthesis of intermediate B4, except that B4-3 is replaced by L8-1 with the same equivalent weight to obtain intermediate L8. MS m / z (ESI): 386.13 [M+H + ].

[0105] Example 14: Synthesis of intermediates and L10

[0106]

[0107] Step 1: Dissolve 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (1 g, 1 eq) in DMF (10 ml), add potassium iodide (61 mg, 0.1 eq), sodium bicarbonate (613 mg, 2 eq) and 2-(2-(2-bromoethoxy)ethoxy)ethanol (1.4 g, 1.8 eq), and maintain the reaction at 80°C for 3 h; dilute the reaction solution with ethyl acetate, wash with water and saturated sodium chloride solution, dry over anhydrous sodium sulfate, concentrate, and dry to obtain a yellow solid L10-1 (1.1 g), with a yield of 74%. MS m / z (ESI): 407.14 [M+H + ];

[0108] Step 2: The same as the second step of the synthesis method of intermediate L3, only L3-1 needs to be replaced by L10-1, and the equivalent weight is the same to obtain intermediate L10. MS m / z (ESI): 405.13 [M+H + ].

[0109] Example 15: Synthesis of Intermediate L11

[0110]

[0111] The synthesis method is the same as that of L10, except that the starting material 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione is replaced by 2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindoline-1,3-dione; 2-(2-(2-bromoethoxy)ethoxy)ethanol is replaced by 2-(2-bromoethoxy)ethanol, and the equivalent weight is the same, and the intermediate L11 can be obtained. MS m / z(ESI):361.10[M+H + ].

[0112] Example 16: Synthesis of intermediates L12 and L13

[0113]

[0114] Dissolve 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (1.00 g, 1 eq) and 2-(2-azidoethoxy)ethylamine (1.43 g, 1.1 eq) in 20 ml of dry DMF, cool down, add N,N-diisopropylethylamine DIPEA (0.93 g, 2 eq) dropwise, and stir at 80°C for about 2 h. Dilute with water, extract with ethyl acetate, wash with brine, dry with anhydrous sodium sulfate, concentrate, and purify the residue with silica gel column chromatography (DCM: MeOH = 30: 1) to obtain yellow solid L12 (0.84 g) with a yield of 60%. MSm / z (ESI): 387.14 [M+H + ].

[0115] The synthesis of L13 refers to L12. It is only necessary to replace 2-(2-azidoethoxy)ethylamine with 2-(2-(2-azidoethoxy)ethoxy)ethylamine with the same equivalent to obtain intermediate L13. MS m / z (ESI): 431.17 [M+H + ].

[0116] Example 17: Synthesis of Intermediate L14

[0117]

[0118] Step 1: 2-tert-Butyloxycarbonyl-2,7-diazaspiro[3.5]nonane (2.00 g, 1 eq) and benzyl bromoacetate (2.02 g, 1 eq) were dissolved in DMF (20 ml), cesium carbonate (0.14 g, 0.05 eq) was added, and the mixture was stirred at room temperature for 5 h. The mixture was diluted with water, extracted with dichloromethane, washed with brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. L14-1 (3.3 g) was obtained with a yield of 73%. MS m / z (ESI): 375.23 [M+H + ].

[0119] Step 2: Add trifluoroacetic acid (15 ml) to a solution of L14-1 (3.3 g, 6.44 mmol) in DCM (33 ml) and stir at room temperature for 3 h. The mixture was concentrated to obtain L14-2 (1.6 g) with a yield of 90%. MS m / z (ESI): 275.18 [M+H + ].

[0120] Step 3: 2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindoline-1,3-dione (1.60 g, 1 eq), L14-2 (1.59 g, 1 eq), N,N-diisopropylethylamine (1.49 g, 2 eq) were dissolved in NMP (32 ml) and stirred at 85 ° C for about 10 h. After the reaction was complete, the mixture was purified by column chromatography (PE: EA = 1: 1) to obtain L14-3 (1.38 g) with a yield of 45%. MS m / z (ESI): 531.22 [M+H + ].

[0121] Step 4: Add 10% Pb / C (0.68 g) to a solution of L14-3 (1.38 g) in EtOH (100 ml) and DCM (45 ml). Pass hydrogen into the reaction solution and keep it under one atmosphere. Stir the reaction at room temperature for about 3 hours. Filter the reaction mixture through diatomaceous earth and concentrate to obtain L14 (0.96 g) with a yield of 84%. MS m / z (ESI): 441.18 [M+H + ].

[0122] Example 18: Synthesis of Intermediate L15

[0123]

[0124] Step 1: Dissolve 3-(4-amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione (2.13 g, 1 eq) and tert-butyl 6-bromohexanoate (2.07 g, 1 eq) in NMP (35 ml) and stir at 110°C for 16 hours. Dilute with water, extract with ethyl acetate, wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate. Purify by silica gel column chromatography to obtain L15-1 (2.3 g) with a yield of 65%. MS m / z (ESI): 430.23 [M+H + ].

[0125] Step 2: Referring to the first step of the synthesis of H2-1, only (R)-1-(4-bromophenyl)ethylamine needs to be replaced with L15-1 to obtain L15-2. MS m / z (ESI): 530.29 [M+H + ].

[0126] Step 3: Dissolve L15-2 (2.47 g, 1 eq) in dichloromethane (50 ml), add paraformaldehyde (1.39 g, 10 eq) and 5 ml glacial acetic acid, stir at room temperature for 1 h, add sodium triacetoxyborohydride (1.97 g, 2 eq) in batches, and stir at room temperature overnight. After the reaction mixture is concentrated, it is purified by silica gel column chromatography to obtain L15-3 (2.10 g), with a yield of 83%. MS m / z (ESI): 544.30 [M+H + ].

[0127] Step 4: Synthesis method: Refer to the fourth step of B4 synthesis, only replace B4-3 with L15-3, the equivalent is the same, and L15 can be obtained. MS m / z (ESI): 388.19 [M+H + ].

[0128] Example 19: Synthesis of Intermediate L16

[0129]

[0130] Adipic acid (1.00 g, 1.1 eq), HATU (3.90 g, 1.5 eq), and N,N-diisopropylethylamine (1.76 g, 3 eq) were dissolved in tetrahydrofuran (10 ml), stirred at 0 °C for 30 min, and H2 (2.6 g, 1 eq) was added. The mixture was stirred at room temperature for 5 h. The mixture was diluted with water, extracted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography to obtain L16 (1.82 g) with a yield of 56%. MS m / z (ESI): 587.29 [M+H + ].

[0131] Example 20: Synthesis of Intermediate L17

[0132]

[0133] The synthesis method of intermediate L17 is different from that of L16 only in that the starting material H2 is replaced by H1, and adipic acid is replaced by 3,3'-oxydipropionic acid, and the equivalent weight is the same, and L17 can be obtained. MS m / z (ESI): 575.25 [M+H + ].

[0134] Example 21: Synthesis of Intermediate L18

[0135]

[0136] Step 1: Dissolve 3-(4-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1g, 1eq), 8-nonyn-1-ol (1.1g, 2.5eq), Pd(dppf)C12 (904mg, 0.4eq), CuI (118mg, 0.2eq) and triethylamine (938mg, 3eq) in DMF (20ml) and maintain the reaction at 80°C for 4h. Cool to room temperature, dilute with water, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain L18-1 (700mg), with a yield of 59%. MS m / z (ESI): 383.20 [M+H + ].

[0137] Step 2: Synthesis method: Refer to the second step of synthesizing intermediate L3, and only replace L3-1 with L18-1, with the same equivalent, to obtain intermediate L18. MS m / z (ESI): 381.18 [M+H + ].

[0138] Example 22: Synthesis of Intermediate L19

[0139]

[0140] Step 1: Dissolve 4-bromo-2,5-difluorobenzonitrile (3.0 g, 1 eq) and methylhydrazine (1.9 g, 3 eq) in ethanol (50 ml) and stir in a microwave reactor at 120°C for 1 h. Quench with ice water, extract with ethyl acetate, wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate. The residue is purified by silica gel column chromatography (PE:EA=2:1) ​​to obtain yellow solid L19-1 (2.0 g) with a yield of 64%. MS m / z (ESI): 228.98 [M+H + ].

[0141] Step 2: Dissolve L19-1 (2.0 g, 1 eq), ethyl acrylate (8.7 g, 10 eq) and DBU (13.3 g, 10 eq) in tetrahydrofuran (20 ml) and stir at 90°C for 3 h. The reaction mixture was quenched with ice water, extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (PE: EA = 1: 1) to obtain a yellow solid L19-2 (1.1 g) with a yield of 35%. MS m / z (ESI): 344.04 [M+H + ].

[0142] Step 3: Dissolve L19-2 (1.1 g, 1 eq) in ethanol (20 ml), add cyanogen bromide (507 mg, 1.5 eq) and sodium acetate (523 mg, 2 eq) in sequence. Stir the reaction at 90 ° C for 5 h. Quench with ice water, extract with ethyl acetate, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The residue is purified by silica gel column chromatography (PE: EA = 1: 1) to obtain the product L19-3 (482 mg) with a yield of 41%. MSm / z (ESI): 368.04 [M+H + ].

[0143] Step 4: Dissolve L19-3 (500 mg, 1 eq), acetaldehyde oxime (241 mg, 3 eq) and indium chloride (25 mg, 0.1 eq) in toluene (10 ml) and stir at 110 ° C for 1 h. Cool to room temperature, dilute with water, extract with ethyl acetate, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The residue was purified by silica gel column chromatography (PE: EA = 10: 1) to obtain a yellow solid L19-4 (316 mg) with a yield of 60%. MSm / z (ESI): 387.05 [M+H + ].

[0144] Step 5: Dissolve L19-4 (300 mg, 1 eq) in acetonitrile (5 ml) and add benzyltrimethylammonium hydroxide (194 mg, 1.5 eq). Stir the reaction at room temperature for 1 h. Dilute with water, extract with ethyl acetate, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The residue is purified by silica gel column chromatography (PE: EA = 1: 1) to obtain the product L19-5 (114 mg) with a yield of 43%. MS m / z (ESI): 341.00 [M+H + ].

[0145] Step 6: Dissolve L19-5 (500 mg, 1 eq) in 1,4-dioxane (10 ml), add cesium carbonate (1.2 g, 2.5 eq), tert-butyl 2-(piperidin-4-yl) acetate (582 mg, 2 eq), Pd-PEPPSI-IHept-Cl catalyst (142 mg, 0.1 eq) in turn, replace with nitrogen for 10 min, stir at 105 ° C for 3 h. Cool to room temperature, dilute with water, extract with ethyl acetate, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The residue was purified by silica gel column chromatography to obtain the product L19-6 (349 mg) with a yield of 52%. MS m / z (ESI): 460.24 [M+H + ].

[0146] Step 7: Dissolve L19-6 (346 mg, 0.75 mmol) in dichloromethane (2 ml), cool to below 0°C in an ice bath, add 1 ml of trifluoroacetic acid, and stir at room temperature for 1 h. Dilute with water, extract with dichloromethane, dry with anhydrous sodium sulfate, and concentrate under reduced pressure to obtain intermediate L19 (221 mg) with a yield of 73%. MS m / z (ESI): 404.17 [M+H + ].

[0147] Example 23: Synthesis of Intermediate L20

[0148]

[0149] Step 1: Dissolve 7-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (500 mg, 1 eq) in tetrahydrofuran (10 ml), add potassium tert-butoxide (1M in THF, 1.2 eq) dropwise at 10°C, keep warm for 30 minutes, slowly add a solution of [1-[(4-methoxyphenyl)methyl]-2,6-dioxopiperidin-3-yl]trifluoromethylsulfonate (923 mg, 1.1 eq) in tetrahydrofuran (5 ml), and stir at low temperature for 2 hours. Add saturated aqueous ammonium chloride solution to quench, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate. The residue was purified by silica gel column chromatography (PE:EA=1:1) to obtain white solid L20-1 (736 mg) with a yield of 73%. MS m / z(ESI):458.07[M+H + ].

[0150] Step 2: Dissolve L20-1 (736 mg, 1 eq) in toluene (6 ml) and stir at 100°C for 3 hours. Cool to room temperature, add the reaction solution to 6 ml of water, solid precipitates, and collect the solid by filtration. Obtain L20-2 (467 mg), yield 86%. MS m / z (ESI): 338.01 [M+H + ].

[0151] Step 3: Dissolve L20-2 (1.1 g, 1 eq), 8-nonyn-1-ol (1.08 g, 2.5 eq), [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (900 mg, 0.4 eq), CuI (118 mg, 0.2 eq) and triethylamine (1.3 ml, 3 eq) in DMF (10 ml) and stir at 80 ° C for 4 h. Cool to room temperature, dilute with water, extract with ethyl acetate, wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate to obtain a white solid L20-3 (836 mg) with a yield of 67%. MS m / z (ESI): 384.19 [M+H + ].

[0152] Step 4: The synthesis method is the same as the second step of intermediate L3, only L3-1 needs to be replaced with L20-3, and the equivalent weight is the same to obtain intermediate L20. MS m / z (ESI): 382.18 [M+H + ].

[0153] Example 24: Synthesis of Intermediate L21

[0154]

[0155] Step 1: Dissolve 6-bromo-1-nitronaphthalene (1.0 g, 1 eq) in ethanol / water (3 v: 1 v, 10 ml), add ammonium chloride (1.6 g, 7 eq) and zinc powder (1.9 g, 7 eq), and stir at 60 ° C for 2 h. Cool to room temperature and filter. The filtrate is concentrated. The residue is diluted with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. Purification by silica gel column chromatography (PE: EA = 3: 2) gave L21-1 (723 mg) with a yield of 82%. MS m / z (ESI): 221.99 [M+H + ].

[0156] Step 2: Dissolve L21-1 (723 mg, 1 eq) in toluene (10 ml), add acrylic acid (1.4 g, 6 eq), and stir the reaction at 110°C for 6 h under nitrogen atmosphere. Concentrate the reaction mixture to obtain L21-2 (908 mg), with a yield of 95%. MS m / z (ESI): 294.01 [M+H + ].

[0157] Step 3: Dissolve L21-2 (908 mg, 1 eq) and urea (462 mg, 2.5 eq) in acetic acid (20 ml), and stir at 120°C for 20 hours under nitrogen atmosphere. Pour the reaction mixture slowly into ice water, filter, and concentrate under reduced pressure to obtain L21-3 (698 mg), with a yield of 71%. MS m / z (ESI): 319.01 [M+H + ].

[0158] Step 4: Dissolve L21-3 (698 mg, 1 eq), potassium phosphate (1.4 g, 3 eq), 4-piperidinemethanol (502 mg, 2 eq) and RuPhos Pd G3 (365 mg, 0.2 eq) in 1,4-dioxane (15 ml), stir at 90 ° C for 6 h under nitrogen atmosphere. Cool to room temperature, filter, and concentrate the filtrate under reduced pressure. The crude product was purified by silica gel column chromatography (PE: EA = 10: 1) to obtain L21-4 (539 mg), with a yield of 70%. MS m / z (ESI): 354.18 [M+H + ].

[0159] Step 5: The synthesis method is the same as the second step of intermediate L3, only L3-1 needs to be replaced with L21-4, and the equivalent weight is the same to obtain intermediate L21. MS m / z (ESI): 352.17 [M+H + ].

[0160] Example 25: Synthesis of Intermediate L22

[0161]

[0162] Step 1: Dissolve 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (2 g, 1 eq) in acetonitrile (20 ml), add N-bromosuccinimide (1.4 g, 1.1 eq) in batches, and stir at room temperature for 6 h. Add water to precipitate, filter, wash the filter cake with petroleum ether and dry to obtain L22-1 (2.2 g), with a yield of 81%. MS m / z (ESI): 354.97 [M+H + ].

[0163] Step 2: Dissolve L22-1 (2 g, 1 eq), potassium iodide (93 mg, 0.1 eq), sodium bicarbonate (946 mg, 2 eq) and tert-butyl 6-hydroxyhexanoate (1.6 g, 1.5 eq) in DMF (20 ml) and stir at 80 ° C for 4 h. Cool to room temperature, dilute with water, extract with ethyl acetate, wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate to obtain L22-2 (1.6 g), with a yield of 62%. MS m / z (ESI): 463.19 [M+H + ].

[0164] Step 3: The synthesis method is the same as step 7 of intermediate L19, except that L19-6 is replaced with L22-2 with the same equivalent weight to obtain intermediate L22. MS m / z (ESI): 463.19 [M+H + ].

[0165] Example 26: Synthesis of Intermediate L23

[0166]

[0167] The synthesis steps of intermediate L23 are different from those of intermediate L22 only in that L22-1 is replaced by L21-3, and 6-hydroxyhexanoic acid tert-butyl ester is replaced by 5-hydroxyvaleric acid tert-butyl ester, and the equivalent weight is the same to obtain intermediate L23. MS m / z (ESI): 371.16 [M+H + ].

[0168] Example 27: Synthesis of Intermediate L24

[0169]

[0170] Step 1: Dissolve 2-fluoro-4-cyanobenzoic acid (2.0 g, 1.2 eq), 3-amino-2,6-piperidindione (1.3 g, 1 eq), N,N-diisopropylethylamine (3.9 g, 3 eq), and PPACA (9.6 g, 3 eq) in DMF (20 ml). Stir and react at room temperature for 2 h. Dilute with water, extract with ethyl acetate, wash with brine, dry over anhydrous sodium sulfate, and concentrate to obtain L24-1 (2.3 g), with a yield of 83%. MS m / z (ESI): 276.08 [M+H + ].

[0171] Step 2: Dissolve L24-1 (2.3 g, 1 eq) in tetrahydrofuran (46 ml), cool to below -10°C in an ice bath, add 25 ml of borane tetrahydrofuran complex (1 M in THF, 3 eq), and react at 60°C for 2 h. Quench with saturated sodium bicarbonate solution, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate and concentrate under reduced pressure. Obtain L24-2 (1.3 g), yield 57%. MSm / z (ESI): 280.11 [M+H + ].

[0172] Step 3: The synthesis method is the same as the first step of intermediate H2, except that (R)-1-(4-bromophenyl)ethylamine is replaced with L24-2, and the equivalent weight is the same to obtain L24-3. MS m / z (ESI): 380.16 [M+H + ].

[0173] Step 4: The synthesis method is the same as the third step of intermediate H1, only H1-2 needs to be replaced with L24-3, and the equivalent weight is the same to obtain L24-4. MS m / z (ESI): 280.11 [M+H + ].

[0174] Step 5: Dissolve 6-hydroxyhexanoic acid (252 mg, 1.2 eq) in DMF (5 ml), add N,N-diisopropylethylamine (618 mg, 3 eq) and HATU (904 mg, 1.5 eq), stir at room temperature for 30 minutes, add intermediate L24-4 (500 mg, 1 eq), stir at room temperature for 2 hours. Dilute with water, extract with dichloromethane, wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate under reduced pressure. The residue is purified by silica gel column chromatography (DCM: MeOH = 40: 1) to obtain L24-5 (492 mg), with a yield of 79%. MS m / z (ESI): 397.18 [M+H + ].

[0175] Step 6: The synthesis method is the same as the second step of intermediate L3, only L3-1 needs to be replaced with L24-5, and the equivalent weight is the same to obtain intermediate L24. MS m / z (ESI): 392.16 [M+H + ].

[0176] Example 28: Synthesis of Intermediate L25

[0177]

[0178] Step 1: tert-Butyl 4-(4-(4,4,5-trimethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidine-1-carboxylate (33.4 g, 8.8 mmol), Pd(dppf)C12 (0.64 g, 0.88 mmol), 2,6-bis(benzyloxy)-3-bromopyridine (33.2 g, 8.8 mmol), cesium carbonate (5.7 g, 17.6 mmol) were placed in 1,4-dioxane / water (60 ml, 10:1), and the mixture was stirred at 100°C overnight. Cool to room temperature, filter and concentrate the filtrate, and the crude product was purified by silica gel column chromatography (PE:EA=1:1) to obtain L25-1 (1 g), with a yield of 21%. MS m / z (ESI): 551.29 [M+H + ].

[0179] Step 2: L25-1 (1 g, 1.82 mmol) was dissolved in methanol (10 ml), Pd / C (10%, w / w, 100 mg) was added to the solution at once, stirred overnight under hydrogen atmosphere, filtered and the filtrate was concentrated, and the crude product was purified by silica gel column chromatography to obtain L25-2 (380 mg), with a yield of 56%. MS m / z (ESI): 372.21 [M+H + ].

[0180] Step 3: The synthesis method is the same as the third step of intermediate H1, only H1-2 needs to be replaced with L25-2, and the equivalent weight is the same to obtain L25-3. MS m / z (ESI): 273.16 [M+H + ].

[0181] Step 4: Dissolve L25-3 (228 mg, 1 eq), 4-penten-1-ol (127 mg, 1.2 eq), and N,N-diisopropylethylamine (189 mg, 2 eq) in acetonitrile (8 ml), and stir the resulting mixture at 80°C overnight. Cool to room temperature, and concentrate under reduced pressure to obtain a crude product, which is purified by silica gel column chromatography (DCM: MeOH = 10: 1) to obtain the product L25-4 (159 mg), with a yield of 60%. MS m / z (ESI): 359.23 [M+H + ].

[0182] Step 5: The synthesis method is the same as the second step of L3, except that L3-1 is replaced with L25-4 with the same equivalent weight to obtain the intermediate L25. MS m / z (ESI): 357.22 [M+H + ].

[0183] Example 29: Synthesis of Intermediate L26

[0184]

[0185] The synthesis method is the same as that of intermediate L25, except that tert-butyl 4-(4-(4,4,5-trimethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidin-1-carboxylate is replaced with tert-butyl {4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy]piperidin-1-yl}carboxylate, and the equivalent weight is the same to obtain L26. MSm / z(ESI):373.21[M+H + ].

[0186] Example 30: Synthesis of Intermediate L27

[0187]

[0188] Step 1 to Step 4: The first to fourth steps of the synthesis of L27-4 are the same as the second to fifth steps of the synthesis of L19-5, except that L19-1 is replaced with 6-bromo-1H-indazole with the same equivalent weight to obtain the intermediate L27-4. MS m / z (ESI): 309.00 [M+H + ].

[0189] Step 5: Synthesis of intermediate L27-5. The synthesis steps of L27-5 are different from the sixth step of intermediate B1 in that B-06 is replaced by L27b and 5-bromoindole is replaced by L27-4 to obtain intermediate L27-5. MS m / z(ESI):583.31[M+H + ]. Among them, the synthesis steps of L27b are as follows: Step a: Add imidazole (1.1g, 1.2eq) to a solution of 7-bromo-1-heptanol (2.5g, 1eq) in dichloromethane (110ml). After stirring the reaction at room temperature for 35min, add tert-butyl (chloro) diphenylsilane (3.6g, 1eq) and stir the reaction at room temperature for 1h. Dilute with water, wash with saturated sodium chloride, dry with anhydrous sodium sulfate, and concentrate under reduced pressure. The crude product was purified by silica gel column chromatography (PE:EA=3:1) to obtain L27a (2.4g) with a yield of 42%. MS m / z(ESI):433.16[M+H +]. Step b: Dissolve L27a (2.0 g, 1 eq), bis(pinacolato)diboron (2.3 g, 2 eq), K2CO3 aqueous solution (2.5 eq) and Pd(pddf)Cl2 (336 mg, 0.1 eq) in 1,4-dioxane (30 ml) and react at 90 °C for 3 h under nitrogen atmosphere. The reaction solution was cooled to room temperature, filtered and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (PE: EA = 6: 1) to obtain a yellow solid L27b (1.8 g) with a yield of 85%. m / z (ESI): 481.33 [M+H + ].

[0190] Step 6: Add tetrabutylammonium fluoride (1M in THF, 1.38ml, 2eq) to a solution of L27-5 (402mg, 1eq) in THF (10ml) and stir at room temperature for 16h. Quench with saturated sodium bicarbonate aqueous solution, extract with ethyl acetate, wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate under reduced pressure. The crude product is purified by silica gel column chromatography (PE:EA=1:1) to obtain L27-6 (197mg), with a yield of 83%. MS m / z(ESI):345.19[M+H + ].

[0191] Step 7: The synthesis method is the same as the second step of L3, except that L3-1 is replaced with L27-6 with the same equivalent weight to obtain the intermediate L27. MS m / z (ESI): 343.18 [M+H + ].

[0192] Example 31: Synthesis of Intermediate L28

[0193]

[0194] Step 1: Dissolve 5-bromo-3-methyl-2-pyridinecarboxylic acid methyl ester (1.2g, 1eq) in tetrahydrofuran (24ml), add N-bromosuccinimide (1.4g, 1.5eq) and azobisisobutyronitrile (171mg, 0.2eq) in turn, and stir at 50°C for 4h. Cool to room temperature, quench with ice water, extract with ethyl acetate, wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate. Concentrate, and purify the crude product by silica gel column chromatography (PE:EA=1:1) to obtain L28-1 (1.2g), with a yield of 75%. MS m / z (ESI): 307.89 [M+H + ].

[0195] Step 2: Under nitrogen atmosphere, L28-1 (1.2 g, 1 eq) was dissolved in DMF (24 ml), and 3-amino-2,6-piperidindione hydrochloride (637 mg, 1 eq) and DIPEA (1.0 g, 2 eq) were added in sequence. The reaction temperature was maintained at 90°C and stirred for about 1.5 h. The reaction mixture was cooled to room temperature, poured into 300 ml of ice water, stirred for crystallization, filtered, and the filter cake was washed with water and dried to obtain an off-white solid L28-2 (1.0 g) with a yield of 82%. MS m / z (ESI): 324.00 [M+H + ].

[0196] Step 3: Under nitrogen atmosphere, L28-2 (1.0 g, 1 eq) was dissolved in DMF (20 ml), and Pd2(dba)3 (282 mg, 0.1 eq), 1,1'-bis(diphenylphosphino)ferrocene (342 mg, 0.2 eq), zinc cyanide (200 mg, 0.5 eq), and zinc acetate (284 mg, 0.5 eq) were added in sequence, and the reaction temperature was maintained at 120°C and stirred for 3 h. The mixture was cooled to room temperature, poured into 250 ml of ice water, stirred for crystallization, and filtered. The filter cake was washed with ethyl acetate and dried to obtain L28-3 (800 mg), with a yield of 96%. MS m / z (ESI): 271.08 [M+H + ].

[0197] Step 4: Dissolve L28-3 (800 mg, 1 eq) in DMF (10 ml), add di-tert-butyl dicarbonate (1.1 g, 2 eq) and Raney nickel, and stir and react at 50 ° C for 5 h under a hydrogen atmosphere. Cool to room temperature, filter, dilute the filtrate with water, extract with ethyl acetate, wash with water, dry over anhydrous sodium sulfate, and concentrate under reduced pressure to obtain L28-4 (707 mg), with a yield of 64%. MS m / z (ESI): 375.17 [M+H + ].

[0198] Step 5: The method is the same as the third step of the synthesis of intermediate L26, except that L26-2 is replaced by L28-4, and the equivalent weight is the same to obtain L28-5. MS m / z (ESI): 275.11 [M+H + ].

[0199] Step 6: The method is the same as the fifth step of the synthesis of intermediate L24, except that L24-4 is replaced by L28-5 and 6-hydroxyhexanoic acid is replaced by 5-hydroxypentanoic acid to obtain intermediate L28. MS m / z (ESI): 403.16 [M+H + ].

[0200] Example 32: Synthesis of Intermediate L29

[0201]

[0202] Step 1: Dissolve 4-aminophenol (1 g, 1 eq) and acrylic acid (3.8 g, 5.8 eq) in water (5 ml), maintain the reaction temperature at 80°C and stir for 3 h. Cool the reaction solution to room temperature, filter, wash the filter cake with an appropriate amount of water, and dry to obtain a white solid L29-1 (1.9 g), with a yield of 82%. MS m / z (ESI): 254.10 [M+H + ].

[0203] Step 2: Dissolve L29-1 (1.9 g, 1 eq) in acetic acid (10 ml), add urea (680 mg, 1.5 eq), and stir at 120 ° C for 5 h. Continue to add 10% hydrochloric acid (5 ml) and stir for 30 min. The reaction solution was cooled to room temperature, solid precipitated, filtered, and the filter cake was washed with petroleum ether and dried to obtain off-white solid L29-2 (830 mg), with a yield of 54%. MS m / z (ESI): 207.08 [M+H + ].

[0204] Step 3: Dissolve L29-2 (830 mg, 1 eq) in DMF (10 ml), add 8-bromo-n-octanol (606 mg, 2 eq) and potassium carbonate (268 mg, 2 eq), and stir at 80 ° C for 6 h. The reaction solution was cooled to room temperature, diluted with water, extracted with ethyl acetate, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a pink solid L29-3 (875 mg), with a yield of 65%. MS m / z (ESI): 335.20 [M+H + ].

[0205] Step 4: The synthesis method is the same as the second step reaction of L3, only L3-1 needs to be replaced with L29-3, and the equivalent weight is the same to obtain the intermediate L29. MS m / z (ESI): 333.18 [M+H + ].

[0206] Example 33: Synthesis of Intermediate L30

[0207]

[0208] Step 1: Dissolve 3-(5-bromo-1-oxoisoindol-2-yl)piperidine-2,6-dione (1g, 1eq) in DMF (10ml), add 8-nonyn-1-ol (1.08g, 2.5eq), [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (0.9g, 0.4eq), CuI (118mg, 0.2eq) and triethylamine (1.3ml, 3eq), and maintain the reaction at 80°C for 4h. Cool to room temperature, add water to the reaction solution and extract with ethyl acetate, combine the organic phases, wash with water, dry over anhydrous sodium sulfate, and concentrate. The crude product was purified by silica gel column chromatography to obtain L30-1 (890mg), with a yield of 74%. MS m / z (ESI): 383.20 [M+H + ].

[0209] Step 2: The synthesis method is the same as the second step reaction of L3, only L3-1 needs to be replaced with L30-1, and the equivalent weight is the same to obtain the intermediate L30. MS m / z (ESI): 381.18 [M+H + ].

[0210] Example 34: Synthesis of Intermediate L31

[0211]

[0212] Step 1: Dissolve 3-hydroxy-1-(4-methoxybenzyl)piperidine-2,6-dione (2g, 1eq) in dichloromethane (30ml), add pyridine (1.3g, 2eq) dropwise at 0°C, stir for 15 minutes, then add trifluoromethanesulfonic anhydride (2.7g, 1.2eq), and keep warm for 3 hours. The reaction solution was directly concentrated, the residue was dissolved in dichloromethane, washed with water, dried over anhydrous sodium sulfate, and desolventized to obtain L31-1 (1.3g), with a yield of 85%. MS m / z (ESI): 382.06 [M+H + ].

[0213] Step 2: Dissolve 7-bromo-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (2g, 1eq) in tetrahydrofuran (20ml), slowly add potassium tert-butoxide (980mg, 1eq) and stir at 0℃ for 30min, then add L31-1 (2.6g, 1eq). Maintain the reaction temperature at 40℃ and stir for 2h. Dilute with water, extract with ethyl acetate, wash with water, dry with anhydrous sodium sulfate, concentrate, and purify the residue by silica gel column chromatography (PE:EA=2:1) to obtain yellow oil L31-2 (323mg), with a yield of 16%. MS m / z(ESI):458.07[M+H + ].

[0214] Step 3: Dissolve L31-2 (323 mg, 1 eq) in 1,4-dioxane (10 ml), add tert-butyl 2-(piperidin-4-yl)acetate (211 mg, 1.5 eq), cesium carbonate (459 mg, 2 eq), Ruphos-Pd-G3 (125 mg, 0.2 eq) in sequence, purge with nitrogen and maintain at 100 ° C for 15 h. The reaction solution was cooled to room temperature and concentrated. The residue was purified by silica gel column chromatography (PE: EA = 2: 1) to obtain a yellow oil L31-3 (167 mg) with a yield of 41%. MS m / z (ESI): 577.30 [M+H + ].

[0215] Step 4: Dissolve L31-3 (167 mg, 1 eq) in toluene (1 ml) and trisulfonic acid methane (550 ul), and stir at 100 ° C for 2 h. Cool to room temperature, dilute with ethyl acetate, filter, and wash the filter cake with ethyl acetate. Dry in vacuum. Obtain off-white solid L31 (10 mg), yield 95%. MS m / z (ESI): 401.18 [M+H + ].

[0216] Example 35: Synthesis of Intermediate L32

[0217]

[0218] The synthesis method is the same as L3, except that the starting material 2-(2,6-dioxacyclic piperidin-3-yl)-4-fluoroisoindoline-1,3-dione is replaced with 3-(5,6-difluoro-1-oxoisoindoline-2-yl)piperidine-2,6-dione, and the equivalent weight is the same to obtain L32. MS m / z(ESI):374.15[M+H + ].

[0219] Example 36: Synthesis of Intermediate L33

[0220]

[0221] Step 1: Dissolve 5-fluoro-2-nitropyridine (1.0 g, 1 eq) in DMF (10 ml), add 4-(1,3-dioxolan-2-yl)piperidine (1.2 g, 1.2 eq) and cesium carbonate (6.7 g, 3 eq), and maintain the reaction at 80 ° C for 2 h. Quench with water, extract with dichloromethane, wash with saturated brine, dry with anhydrous sodium sulfate, concentrate, and purify the crude product by silica gel column chromatography to obtain L33-1 (1.5 g), with a yield of 76%. MS m / z (ESI): 280.13 [M+H + ].

[0222] Step 2: Dissolve the intermediate L33-1 (1.5 g, 7.31 mmol) in tetrahydrofuran (30 ml), add ammonium chloride (2.9 g, 10 eq), pure water (44 ml) and iron powder (3.1 g, 10 eq) in sequence, and maintain the reaction at 70 ° C for 12 h. Filter, dilute with water, extract with dichloromethane, wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate. The crude product is purified by silica gel column chromatography to obtain L33-2 (1.1 g), with a yield of 85%. MS m / z (ESI): 250.16 [M+H + ].

[0223] Step 3: Dissolve L33-2 (1.1 g, 1 eq) in DMF (20 ml), add 3-bromopiperidine-2,6-dione (1.3 g, 1.5 eq), Pd(dppf)Cl2 (324 mg, 0.1 eq) and sodium carbonate (1.4 g, 3 eq) in sequence, and maintain the reaction temperature at 100 ° C for 5 h. Add water to quench, extract with dichloromethane, wash with water, dry with anhydrous sodium sulfate, concentrate, and purify the crude product by silica gel column chromatography to obtain L33-3 (1.3 g), with a yield of 80%. MS m / z (ESI): 361.19 [M+H + ].

[0224] Step 4: Dissolve L33-3 (1.3 g, 1 eq) in trifluoroacetic acid (7 ml), maintain the reaction temperature at 80 ° C, react for 1 h, add 1.4 ml of concentrated hydrochloric acid to the system, maintain the reaction temperature at 80 ° C for 3 h. After the reaction is completed, add saturated sodium carbonate aqueous solution to adjust to weak alkalinity, extract with dichloromethane, wash with water, dry with anhydrous sodium sulfate, and concentrate to obtain L33 (878 mg), with a yield of 77%. MS m / z (ESI): 317.16 [M+H + ].

[0225] Example 37: Synthesis of Intermediate L34

[0226]

[0227] Step 1: At 0°C, under a nitrogen atmosphere, piperidin-4-ylmethanol (5.7g, 1.5eq), K3PO4 (14.1g, 2eq), CuI (12.6g, 2eq) and L-proline (7.6g, 0.2eq) were added to a solution of compound 1-bromo-4-iodobenzene (10.0g, 1eq) in DMSO (50mL). Stir overnight at 80°C under a nitrogen atmosphere. Cool to room temperature, dilute with water, and extract with ethyl acetate. Wash the organic layer with brine, dry over anhydrous sodium sulfate, and concentrate. The residue was purified by silica gel column chromatography. L34-1 (5.5g) was obtained with a yield of 56%. MS m / z (ESI): 271.18 [M+H + ].

[0228] Step 2: K2CO3 (7.1 g, 3 eq) and Pd(dppf)Cl2·CH2Cl2 (1.4 g, 0.1 eq) were added to a solution of L34-1 (5 g, 1 eq) and 2,6-bis(benzyloxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (9.4 g, 22.465 mmol, 1.3 eq) in 1,4-dioxane (75 mL) and pure water (10 mL). Stir overnight at 110 ° C under a nitrogen atmosphere. Cool to room temperature and filter, concentrate the filtrate under reduced pressure, and purify by silica gel column chromatography. L34-2 (5.1 g) was obtained with a yield of 57%. MS m / z (ESI): 481.25 [M+H + ].

[0229] Step 3: Dissolve L34-2 (2.9 g, 1 eq) in a solution of THF (29 mL) and EtOH (29 mL), add glacial acetic acid (1.1 g, 3 eq), 10% Pd / C (750 mg) and 20% Pd(OH)2 / C (876 mg). The suspension was degassed in vacuo and purged with H2 several times. The mixture was stirred at room temperature for 10 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain a crude product, which was recrystallized from isopropyl ether. An off-white solid L34-3 (1.2 g) was obtained with a yield of 67%. MS m / z (ESI): 303.17 [M+H + ].

[0230] Step 4: The synthesis method is the same as the second step reaction of L3, only L3-1 needs to be replaced with L34-3, and the equivalent weight is the same to obtain the intermediate L34. MS m / z (ESI): 301.15 [M+H + ].

[0231] Example 38: Synthesis of Intermediate L35

[0232]

[0233] The synthesis method is the same as that of L34, except that the starting material 1-bromo-4-iodobenzene is replaced with 2-fluoro-4-bromo-iodobenzene with the same equivalent weight to obtain L35. MS m / z (ESI): 319.15 [M+H + ].

[0234] Example 39: Synthesis of Intermediate L36

[0235]

[0236] The synthesis method is the same as that of L34, except that the starting material 1-bromo-4-iodobenzene is replaced with 1-bromo-3-fluoro-4-iodobenzene to obtain L36. MS m / z (ESI): 319.15 [M+H + ].

[0237] Example 40: Synthesis of Intermediate L37

[0238]

[0239] Step 1: Dissolve tert-butyl di(prop-2-yn-1-yl)carbamate (10 g, 1 eq) in DMF (100 ml), maintain 0 ° C, add 60% sodium hydride (3.1 g, 1.2 eq) in batches, return to room temperature and stir for 30 minutes, add 3-bromopropyne (7.6 g, 1 eq) dropwise. The reaction solution was stirred at 25 ° C for 5 hours. Quench with ice water, extract with ethyl acetate, wash the organic phase with water, wash with saturated brine, dry over anhydrous sodium sulfate, and concentrate. The residue was purified by silica gel column chromatography to obtain L37-1 (10.3 g) with a yield of 83%. MS m / z (ESI): 194.12 [M+H + ].

[0240] Step 2: Dissolve L37-1 (10.3 g, 1 eq) and dimethyl butynedioate (30.3 g, 4 eq) in ethanol (100 ml), add Wilkinson's catalyst (tris(triphenylphosphine)rhodium chloride) (990 mg, 0.02 eq), maintain 80°C under nitrogen atmosphere, stir and react for 12 h. Cool to room temperature, concentrate, dilute the residue with ethyl acetate, wash with water, dry over anhydrous sodium sulfate, concentrate and purify by silica gel column chromatography to obtain L37-2 (4.1 g), yield 23%. MS m / z (ESI): 336.14 [M+H + ].

[0241] Step 3: Add 3N sodium hydroxide solution (49ml) to a solution of L37-2 (4.1g, 1eq) in ethanol (41ml), maintain stirring at 80℃ for 3h. Concentrate under reduced pressure, dilute with ethyl acetate, adjust pH to 4 with 1N HCl at 0℃, wash the organic phase with water, dry with anhydrous sodium sulfate, and concentrate to obtain L37-3, which is used directly in the next step without further purification, with a yield of 85%. MS m / z(ESI):308.11[M+H + ].

[0242] Step 4: Dissolve L37-3 (3.2 g, 1 eq) in acetic anhydride (32 mL) and stir at 100 °C for 3 h. Cool to room temperature, dilute with ethyl acetate, wash with water, dry over anhydrous sodium sulfate, concentrate and purify by silica gel column chromatography to obtain L37-4 (1.8 g), with a yield of 62%. MS m / z (ESI): 290.10 [M+H + ].

[0243] Step 5: Add 3-amino-2,6-piperidindione hydrochloride (1.1 g, 1.1 eq) and triethylamine (629 mg, 3 eq) to a toluene (18 ml) solution of L37-4 (1.8 g, 1 eq), and stir at 80°C for 3 h. Cool to room temperature, concentrate under reduced pressure, dissolve in ethyl acetate, wash with water, dry over anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography to obtain L37-5 (1 g), with a yield of 42%. MS m / z (ESI): 400.15 [M+H + ].

[0244] Step 6: The synthesis method is the same as the third step reaction of the synthetic intermediate L26, only L26-2 needs to be replaced with L37-5, and the equivalent weight is the same to obtain L37-6. MS m / z (ESI): 300.10 [M+H + ].

[0245] Step 7: Add DIPEA (3 g, 10 eq) and tert-butyl bromoacetate (489 mg, 1.1 eq) to an acetonitrile solution (8 ml) of L37-6 (782 mg, 1 eq), stir at room temperature for 6 hours, concentrate under reduced pressure, and purify the residue by silica gel column chromatography to obtain L37-7 (413 mg), with a yield of 43%. MS m / z (ESI): 414.17 [M+H + ].

[0246] Step 8: The synthesis method is the same as the seventh step of intermediate L19, only L19-6 needs to be replaced with L37-7, and the equivalent weight is the same to obtain intermediate L37. MS m / z (ESI): 358.10 [M+H + ].

[0247] Example 41: Synthesis of Intermediate L38

[0248]

[0249] Step 1: At room temperature, add NBS (15.9 g, 1.5 eq) to a solution of methyl 4-fluoro-2-methylbenzoate (10 g, 1 eq) in 1,2-dichloroethane (200 ml), followed by benzoyl peroxybenzoate (720 mg, 0.05 eq). Maintain stirring at 80 °C for 4 h. The reaction mixture was cooled to room temperature, washed with water, dried over anhydrous sodium sulfate, and concentrated to obtain L38-1, which was used directly in the next step without further purification. MS m / z (ESI): 246.98 [M+H + ].

[0250] Step 2: Add NMO (10.5 g, 2.2 eq) to a solution of L38-1 (10.1 g, 1 eq) in dichloromethane (200 ml) at room temperature and stir the reaction at room temperature for 5 hours. Filter, wash the filtrate with water, dry over anhydrous sodium sulfate and concentrate. The residue is purified by silica gel column chromatography to obtain L38-2 (5.4 g) with a yield of 73%. MS m / z (ESI): 183.05 [M+H + ].

[0251] Step 3: To a solution of L38-2 (5 g, 1 eq) in THF (125 ml) at room temperature, add a solution of lithium hydroxide monohydrate (5.8 g, 5 eq) in pure water (82 ml). After the reaction is complete as monitored by LC-MS, concentrate under reduced pressure. Acidify the reaction with dilute HCl to adjust the pH to 4 and extract with ethyl acetate. Dry over anhydrous sodium sulfate and concentrate to obtain L38-3 (4.4 g) with a yield of 96%. MS m / z (ESI): 169.03 [M+H + ].

[0252] Step 4: At room temperature, add hydrazine monohydrate (1.3 mg, 1 eq) to a solution of L38-3 (4.4 g, 1 eq) in MeOH (44 ml), heat to 80 ° C, and stir for 3 h. Cool to room temperature and concentrate under reduced pressure, dilute with water, and extract with ethyl acetate. The combined organic layer was dried over anhydrous sodium sulfate and concentrated to give L38-4 (3.3 g) with a yield of 77%. MS m / z (ESI): 165.05 [M+H + ].

[0253] Step 5: At 0°C, lithium diisopropylamide (2M in THE, 13ml, 1.3eq) was slowly added to a solution of L38-4 (3.3g, 1eq) in anhydrous THF (66ml), and a solution of 3-bromopiperidine-2,6-dione hydrochloride (4.8g, 1.5eq) in anhydrous tetrahydrofuran (50ml) was added dropwise, and the resulting mixture was stirred at room temperature for 5 hours. Cooled to room temperature, saturated ammonium chloride solution was added, extracted with ethyl acetate, the organic phase was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. L38-5 was obtained and used directly in the next step without further purification. MS m / z (ESI): 276.08 [M+H + ].

[0254] Step 6: The synthesis method is the same as the first step of L3, except that 2-(2,6-dioxacyclic piperidin-3-yl)-4-fluoroisoindoline-1,3-dione is replaced with L38-5, and the equivalent weight is the same to obtain L38-6. MS m / z (ESI): 371.17 [M+H + ].

[0255] Step 7: The synthesis method is the same as the second step reaction of L3, and the equivalent is the same. Only L3-1 needs to be replaced with L38-6 to obtain L38. MS m / z (ESI): 369.16 [M+H + ].

[0256] Example 42: Synthesis of Intermediate L39

[0257]

[0258] Step 1: At 0°C, add concentrated H2SO4 (500uL) to a solution of 4-bromo-3-fluoroaniline (10g, 1eq) in glacial acetic acid (40ml), and add acrylic acid (11.4g, 3eq) to the reaction mixture. Stir the reaction mixture at 100°C. After the reaction is complete, concentrate the resulting mixture under reduced pressure. After adding ice water, extract with ethyl acetate, combine the organic phases, wash with water, dry over anhydrous sodium sulfate, filter and concentrate. The resulting crude product is purified by silica gel chromatography to obtain L39-1, yield: 40%. MS m / z (ESI): 261.99 [M+H + ].

[0259] Step 2: To a solution of L39-1 (5.5 g, 1 eq) in glacial acetic acid (55 ml), urea (3.7 g, 3 eq) was added. The reaction mixture was stirred at 100 ° C overnight. The resulting solution was extracted with ethyl acetate and the organic layers were combined. Purification by silica gel column chromatography gave L39-2 in a yield of 42%. MS m / z (ESI): 286.95 [M+H + ].

[0260] The subsequent steps, namely the third and fourth steps, are the same as the fourth and fifth steps of the L21 synthesis process, except that L21-3 is replaced with L39-2, and the equivalent weight is the same to obtain L39. MS m / z (ESI): 320.14 [M+H + ].

[0261] Example 43: Synthesis of Intermediate L41

[0262]

[0263] Step 1: The synthesis method is the same as the fourth step reaction of intermediate B1, only B-04 needs to be replaced with 4-bromo-3,5-difluoroaniline to obtain L41-1. MS m / z(ESI):308.01[M+H + ].

[0264] Step 2: The synthesis method is the same as the fourth step reaction of L21, only L21-3 needs to be replaced with L41-1, and the equivalent weight is the same to obtain L41-2. MS m / z (ESI): 343.18 [M+H + ].

[0265] Step 3: The synthesis method is the same as the second step reaction of L14, only L14-1 needs to be replaced with L41-2 to obtain L41-3. MS m / z (ESI): 243.13 [M+H + ].

[0266] Step 4: Under nitrogen atmosphere, at 0°C, tert-butyl nitrite (1.6 g, 1.5 eq) was added to an acetonitrile solution of L41-3 (2.4 g, 1 eq), and the mixture was kept warm for 30 minutes, and then cuprous bromide (19 g, 0.86 eq) was added at 0°C. The resulting mixture was stirred at room temperature for 3 hours, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with ethyl acetate (100 ml), and ice water (80 ml) was added. At low temperature, NaHCO3 aqueous solution was used to adjust the pH to 9. The combined aqueous layer was extracted with ethyl acetate for three layers, and the combined organic layer was washed with water, washed with salt, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain L41-4 with a yield of 30%. MS m / z (ESI): 306.03 [M+H + ].

[0267] The subsequent steps are the same as L35, only L35-1 needs to be replaced by L41-4, and the equivalents are the same to obtain L41. MS m / z (ESI): 337.14 [M+H + ].

[0268] Example 44: Synthesis of Intermediate L42

[0269]

[0270] Step 1: The synthesis method is the same as the first step of L3, except that 2-(2,6-dioxacyclopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione is replaced with p-fluoronitrobenzene with the same equivalent weight to obtain L42-1. MS m / z(ESI):207.15[M+H + ].

[0271] Step 2: The synthesis method is the same as the first step of L21, except that 6-bromo-1-nitronaphthalene is replaced with L42-1, and the equivalent weight is the same to obtain L42-2. MS m / z (ESI): 207.15 [M+H+].

[0272] Step 3: Acrylic acid (3.8 g, 1.5 eq) was added to a toluene (73 ml) solution of L42-2 (7.3 g, 1 eq). The mixture was reacted at 90°C. After the reaction was complete as monitored by HPLC, it was cooled to room temperature, followed by the addition of AcOH (55 mL) and urea (10.6 g, 5 eq). The resulting mixture was reacted at 110°C for 15 hours, cooled to room temperature, and concentrated under reduced pressure. The residue was diluted with ethyl acetate, and then adjusted to pH = 7 with a saturated aqueous Na2CO3 solution. The organic phases were extracted with ethyl acetate, washed with a saturated aqueous NaCl solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel chromatography to obtain L42-3 in a yield of 43%. MS m / z (ESI): 346.18 [M+H + ].

[0273] Step 4: A solution of L42-3 (5.3 g, 1 eq) in HCl (2 M, 46 ml, 6 eq) was reacted at 100°C for 15 hours, then cooled to 0°C, adjusted to pH 7 with saturated NaHCO3 aqueous solution, and filtered. The filter cake was washed with water and dried in vacuo to obtain L42-4, which was used directly in the next step without further purification. Yield 70%. MS m / z (ESI): 304.17 [M+H + ].

[0274] Step 5: The synthesis method is the same as the second step of L3, only L3-1 needs to be replaced with L42-4, and the equivalent weight is the same, to obtain L42. MS m / z (ESI): 302.15 [M+H + ].

[0275] Example 45: Synthesis of Compound C02

[0276]

[0277] Step 1: Dissolve B1 (800 mg, 1 eq) in DMF (8 ml), maintain 0 ° C, and add N, N-diisopropylethylamine (390 mg, 2 eq) and 2-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (688 mg, 1.2 eq) in sequence. The reaction mixture was stirred at room temperature for 30 minutes and L1b (620 mg, 1.1 eq) was added. The progress of the reaction was monitored by TLC. After completion, the reaction mixture was diluted with water and extracted with 10% methanol / dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. Purification by silica gel column chromatography gave Bo-2 (932 mg) as a yellow solid with a yield of 70%. MSm / z(ESI):885.39[M+H + ].

[0278] Step 2: Add hydrogen chloride solution (4M in 1,4-dioxane, 10eq) to Bo-2 (600mg, 1eq) dissolved in 1,4-dioxane (10V) at 0°C, and then stir the reaction mixture at room temperature. After the reaction is complete by HPLC monitoring, filter the filter cake, adjust the pH to 8 with saturated sodium carbonate solution, extract with dichloromethane, combine the organic layers, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and separate and purify by silica gel column chromatography (DCM: MeOH = 20: 1) to obtain yellow solid C02 (495mg), with a yield of 93%. 1 H-NMR (300MHz, DMSO-d6): δ=13.61(s,1H),11.31(s,1H),10.84(s,1H),7.81(s,1H),7.75( s,1H),7.60-7.55(m,2H),7.22(s,1H),7.10(d,J=8.4Hz,1H),7.05(s,2H),6.97(d,J=7.4Hz ,1H),6.55(s,1H),5.60(s,1H),5.11-5.02(m,1H),4.28(s,2H),3.31(s,2H),3.26-3.15(m ,4H),2.60(s,3H),2.48-2.39(m,6H),2.12(s,4H),1.61-1.53(m,4H),1.41-1.30(m,4H).MS m / z(ESI):785.34[M+H + ].

[0279] Example 46: Synthesis of Compound C03

[0280]

[0281] The synthesis method is the same as that of compound C02, the only difference being that compound C03 can be obtained by simply replacing L1b with L1c with the same equivalent weight. 1 H-NMR (300MHz, DMSO-d6): δ=13.62(s,1H),11.30(s,1H),10.83(s,1H),7.82(s,1H),7.77( s,1H),7.61-7.55(m,2H),7.25(s,1H),7.12(d,J=8.8Hz,1H),7.04(s,2H),6.95(d,J=7.9Hz ,1H),6.54(s,1H),5.63(s,1H),5.12-5.03(m,1H),4.24(s,2H),3.32(s,2H),3.29-3.13(m ,4H),2.62(s,3H),2.47-2.36(m,6H),2.13(s,4H),1.61-1.50(m,4H),1.38-1.27(m,6H).MS m / z(ESI):799.35[M+H + ].

[0282] Example 47: Synthesis of Compound C05

[0283]

[0284] Step 1: Dissolve intermediate B2 (1.2 g, 1 eq) and intermediate L2a (1.1 g, 1.5 eq) in tetrahydrofuran (20 ml), add acetic acid (2 eq), stir at room temperature for 30 minutes, add sodium triacetoxyborohydride (793 mg, 2 eq) in batches, and maintain the reaction at room temperature for 2 h. Add saturated sodium carbonate solution to the reaction solution to adjust the base, extract with dichloromethane three times, combine the organic phases, wash with water and saturated sodium chloride solution, dry with anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography (DCM: MeOH = 50: 1) to obtain yellow solid Bo-5 (1.4 g), with a yield of 71%. MS m / z (ESI): 1040.46 [M+H + ].

[0285] Step 2: The synthesis method is the same as the second step reaction of compound C02, only replacing Bo-2 with Bo-5 to obtain compound C05. 1H-NMR (300MHz, DMSO-d6): δ = 13.66 (s, 1H), 11.13 (s, 1H), 10.85 (s, 1H), 8.72 (s, 1H), 8.13-7.99 (m, 1H), 7 .82(s,1H),7.75(s,1H),7.63-7.57(m,2H),7.12(d,J=8.4Hz,1H),7.04(s,2H),6.91(s,1H),6.86(d,J=7 .7Hz,1H),6.52(s,1H),5.07-5.00(m,1H),4.24(s,2H),3.64-3.53(m,2H),3.48-3.41(m,6H),3.37(dt,J =6.5Hz,2H),3.33(s,2H),2.57(s,3H),2.56-2.50(m,8H),2.42(s,2H),2.39-2.31(m,6H),2.08(s,4H).MS m / z(ESI):940.41[M+H + ].

[0286] Example 48: Synthesis of Compound C06

[0287]

[0288] The synthesis method is the same as that of compound C05, and compound C06 can be obtained by simply replacing L2a with L2b with the same equivalent weight. 1 H-NMR (300MHz, DMSO-d6): δ = 13.55 (s, 1H), 11.28 (s, 1H), 10.08 (s, 1H), 8.75-8.70 (m, 1H), 7.97 (s, 1H), 7.84 (s, 1H) ,7.64-7.56(m,2H),7.17(dd,J=7.8,5.1Hz,1H),7.08(d,J=8.4Hz,1H),6.95(s,2H),6.87(d,J=8.0Hz,1H),6.45(s,1 H),5.48(t,J=4.3Hz,1H),5.12-5.04(m,1H),4.27(s,2H),3.52-3.40(m,4H),3.35(dt,J=4.1Hz,2H),3.29(s,2H),3 .17-3.04(m,4H),2.61(s,3H),2.57-2.50(m,8H),2.46(s,2H),2.40-2.31(m,6H),2.13(s,4H),1.41-1.30(m,4H).MS m / z(ESI):968.44[M+H + ].

[0289] Example 49: Synthesis of Compound C07

[0290]

[0291] Step 1: Dissolve intermediate B2 (800 mg, 1 eq) and intermediate L3 (691 mg, 1.5 eq) in tetrahydrofuran (8 ml), add acetic acid (2 eq), stir at room temperature for 30 minutes, add sodium triacetoxyborohydride (528 mg, 2 eq) in batches, and maintain the reaction at room temperature for 2 hours. After the reaction is complete, add saturated sodium carbonate solution to adjust the pH to 8, extract three times with dichloromethane, combine the organic phases, wash with water and saturated sodium chloride solution, dry over anhydrous sodium sulfate, concentrate, and purify by silica gel column chromatography (DCM: MeOH = 50: 1) to obtain yellow solid Bo-7 (870 mg), with a yield of 69%. MS m / z (ESI): 1040.46 [M+H + ].

[0292] Step 2: The synthesis method is the same as the second step of compound C02, only Bo-2 needs to be replaced with Bo-07 to obtain compound C07. 1 H-NMR (300MHz, DMSO-d6): δ = 13.68 (s, 1H), 11.29 (s, 1H), 10.75 (s, 1H), 7.97 (s, 1H), 7.68 (s, 1H), 7.61-7.50 (m,2H),7.20(s,1H),7.11(d,J=8.2Hz,1H),7.03(s,2H),6.87(d,J=5.3Hz,1H),6.51(s,1H),5.24-5.13(m,1H ),4.21(s,2H),3.38(s,2H),3.38-3.29(m,2H),3.18-3.07(m,4H),2.87-2.76(m,1H),2.64(s,3H),2.54(d,J =7.31Hz,2H),2.49-2.40(m,6H),2.30-2.21(m,6H),2.11(s,4H),2.05(d,J=7.2Hz,3H),1.45-1.38(m,5H).MS m / z(ESI):895.42[M+H + ].

[0293] Example 50: Synthesis of Compound C08

[0294]

[0295] The synthesis method is the same as that of compound C07, and compound C08 can be obtained by simply replacing L3 with L4 with the same equivalent weight. 1H-NMR (300MHz, DMSO-d6): δ=13.74(s,1H),11.38(s,1H),10.87(s,1H),7.79(s,1H),7.73(s,1H),7.64- 7.51(m,2H),7.45(s,1H),7.13(d,J=8.49Hz,1H),7.02(s,2H),6.91(d,J=5.6Hz,1H),6.58(s,1H),5.21 -5.12(m,1H),4.58(s,2H),3.42(s,2H),3.30-3.24(m,2H),3.14-3.08(m,4H),2.81-2.75(m,1H),2.64( s,3H),2.55(d,J=7.31Hz,2H),2.46-2.35(m,6H),2.31-2.19(m,8H),2.10(s,4H),1.45-1.38(m,4H).MS m / z(ESI):881.41[M+H + ].

[0296] Example 51: Synthesis of Compound C09

[0297]

[0298] The synthesis method is the same as that of compound C07, and compound C09 can be obtained by simply replacing L3 with L5 with the same equivalent weight. 1 H-NMR (300MHz, DMSO-d6): δ = 13.63 (s, 1H), 11.21 (s, 1H), 10.91 (s, 1H), 7.99 (s, 1H), 7.85 (s, 1H), 7.7 0-7.61(m,2H),7.19(s,1H),7.12(d,J=8.5Hz,1H),7.07(s,2H),6.79(d,J=7.32Hz,1H),6.59(s,1H),5 .28-5.19(m,1H),4.33(s,2H),3.48(s,2H),3.39-3.31(m,2H),3.17-3.07(m,4H),2.79-2.71(m,1H),2 .60(s,3H),2.55(d,J=5.9Hz,2H),2.48-2.37(m,6H),2.34-2.26(m,6H),2.17(s,4H),2.01(d,J=7.3Hz 3H),1.48-1.39(m,5H).MS m / z(ESI):895.42[M+H + ].

[0299] Example 52: Synthesis of Compound C10

[0300]

[0301] The synthesis method is the same as that of compound C07, and compound C10 can be obtained by simply replacing L3 with L6 with the same equivalent weight. 1 H-NMR (300MHz, DMSO-d6): δ = 13.69 (s, 1H), 11.41 (s, 1H), 10.74 (s, 1H), 8.14 (s, 1H), 7.87 (s, 1H), 7.74 -7.65(m,2H),7.51(s,1H),7.21(d,J=8.9Hz,1H),7.11(s,2H),6.89(d,J=7.3Hz,1H),6.54(s,1H),5.28 -5.19(m,1H),4.61(s,2H),3.48(s,2H),3.37-3.26(m,2H),3.19-3.10(m,4H),2.88-2.75(m,1H),2.63 (s,3H),2.57(d,J=7.8Hz,2H),2.51-2.45(m,6H),2.37-2.21(m,8H),2.15(s,4H),1.47-1.35(m,4H).MS m / z(ESI):881.41[M+H + ].

[0302] Example 53: Synthesis of Compound C11

[0303]

[0304] The synthesis method is the same as that of compound C07, and compound C11 can be obtained by simply replacing L3 with L7 with the same equivalent weight. 1 H-NMR (300MHz, DMSO-d6): δ = 13.67 (s, 1H), 11.32 (s, 1H), 10.79 (s, 1H), 7.71 (s ,1H),7.69(s,1H),7.61-7.50(m,2H),7.35(s,1H),7.22(d,J=9.30Hz,1H),7.08 (s,2H),6.89(d,J=7.31Hz,1H),6.47(s,1H),5.24-5.18(m,1H),4.54(s,2H),3. 91-3.85(m,4H),3.39(s,2H),3.36-3.30(m,3H),2.59(s,3H),2.46(d,J=8.12Hz 2H),2.47-2.39(m,6H),2.33-2.25(m,8H),2.17(s,4H).MS m / z(ESI):853.38[M+H + ].

[0305] Example 54: Synthesis of Compound C12

[0306]

[0307] The synthesis method is the same as that of compound C05, except that L2a is replaced by L8 with the same equivalent weight to obtain compound C12. 1 H-NMR (300MHz, DMSO-d6): δ = 13.69 (s, 1H), 11.41 (s, 1H), 10.74 (s, 1H), 8.14 (s, 1H) ,7.87(s,1H),7.74-7.65(m,2H),7.51(s,1H),7.21(d,J=8.97Hz,1H),7.11(s,2H),6 .89(d,J=7.21Hz,1H),6.54(s,1H),5.28-5.19(m,1H),4.61(s,2H),3.48(s,2H),3.3 7-3.26(m,2H),3.19-3.10(m,4H),2.88-2.75(m,1H),2.63(s,3H),2.57(d,J=7.81Hz 2H),2.51-2.45(m,6H),2.37-2.21(m,8H),2.15(s,4H),1.47-1.35(m,4H).MS m / z(ESI):909.40[M+H + ].

[0308] Example 55: Synthesis of Compound C14

[0309]

[0310] The synthesis method is the same as that of compound C07, and compound C14 can be obtained by simply replacing L3 with L10 with the same equivalent weight. 1H-NMR (300MHz, DMSO-d6): δ = 13.68 (s, 1H), 11.27 (s, 1H), 10.75 (s, 1H), 8.61 (s, 1H), 7.97 (s, 1H), 7.6 4(s,1H),7.61-7.50(m,2H),7.12(d,J=8.7Hz,1H),7.03(s,2H),6.87(d,J=7.4Hz,1H),6.51(s,1H),5. 25-5.14(m,1H),4.65(s,2H),4.35-4.23(m,2H),3.79-3.68(m,2H),3.54-3.47(m,6H),3.40(s,2H),3. 36-3.27(m,2H),2.67(s,3H),2.55-2.49(m,4H),2.41-2.39(m,6H),2.31-2.22(m,8H),2.12(s,4H).MS m / z(ESI):930.41[M+H + ].

[0311] Example 56: Synthesis of Compound C15

[0312]

[0313] The synthesis method is the same as that of compound C07, except that L3 is replaced by L11 with the same equivalent weight to obtain compound C15. 1 H-NMR (300MHz, DMSO-d6): δ = 13.69 (s, 1H), 11.26 (s, 1H), 10.79 (s, 1H), 8.65 (s, 1H), 8.05 (s, 2H), 7.9 6(s,1H),7.65(s,1H),7.62-7.53(m,2H),7.12(d,J=8.8Hz,1H),6.84(d,J=7.2Hz,1H),6.52(s,1H),5. 27-5.12(m,1H),4.59(s,2H),4.34-4.26(m,2H),3.79-3.68(m,2H),3.57-3.44(m,2H),3.39(s,2H),3. 32-3.28(m,2H),2.67(s,3H),2.54-2.49(m,4H),2.42-2.36(m,6H),2.29-2.21(m,8H),2.14(s,4H).MS m / z(ESI):886.39[M+H + ].

[0314] Example 57: Synthesis of Compound C16

[0315]

[0316] Step 1: EDCI (226 mg, 1.2 eq), HOBt (160 mg, 1.2 eq), and DIPEA (250 mg, 2 eq) were added to a dry dichloromethane solution of B2 (630 mg, 0.98 mmol, 1 eq) and 4-pentynoic acid (106 mg, 1.1 eq) in sequence. The reaction mixture was returned to room temperature and stirred, and the reaction was monitored by TLC. After the reaction was completed, dichloromethane and saturated aqueous sodium bicarbonate solution were added. The organic phase was separated, washed with brine, and then dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and purified by silica gel column chromatography (DCM: MeOH = 50: 1) to obtain 650 mg of white solid Bo-16-1 with a yield of 85%. MS m / z (ESI): 722.37 [M+H + ].

[0317] Step 2: Bo-16-1 (600 mg, 1 eq) and L36 (0.32 g, 1 eq) were added to 5 ml of THF, and the reaction mixture was stirred at room temperature. CuSO4 (0.13 g, 1 eq) and sodium ascorbate (0.33 g, 2 eq) were dissolved in 1 ml of water, and the solution was added to the reaction system. The resulting reaction mixture was stirred at room temperature for 3 h. After TLC monitoring, the reaction was complete, and 15 ml of water was added for dilution. The mixture was extracted with ethyl acetate (3 X 20ml), the organic layers were combined, washed with brine, dried over anhydrous sodium sulfate, filtered to collect the filtrate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to obtain Bo-16 (0.37g), with a yield of 41%. MS m / z (ESI): 1108.50 [M+H + ].

[0318] Step 3: The synthesis method refers to the second step reaction of C02. Just replace Bo-2 with Bo-16 with the same equivalent to obtain C16. 1H-NMR (300MHz, DMSO-d6): δ = 12.00 (s, 1H), 11.06 (s, 1H), 10.40 (s, 1H), 8.94 (d, J = 11.2Hz 2H), 8.74 (s, 1H), 8.05 (s, 1H), 7.79 (d, J = 9.7Hz 1H),7.68(s,1H),7.59(s,2H),7.57(s,1H),7.28(s,2H),6.98(d,J=8.2Hz,1H) ,4.44(t,J=4.1Hz,1H),4.17-4.10(m,2H),3.90-3.85(m,4H),3.64(d,J=5.3Hz ,2H),3.46-3.37(m,10H),2.83(dd,J=7.9,1.1Hz,2H),2.67-2.54(m,8H),2.50 -2.41(m,7H),2.21-2.15(m,1H),2.11-2.02(m,5H).MSm / z(ESI):1008.45[M+H + ].

[0319] Example 58: Synthesis of Compound C17

[0320]

[0321] The synthesis method is the same as the second and third steps of the synthesis of compound C16 in Example 62, using Bo-16-1 synthesized in the first step of Example 62 as the raw material, and compound C17 can be obtained by simply replacing the corresponding intermediates. 1 H-NMR (300MHz, DMSO-d6): δ = 11.98 (s, 1H), 11.01 (s, 1H), 10.38 (s, 1H), 8.95 (d, J = 10.4Hz 2H), 8.71 (s, 1H), 8.07 (s, 1H), 7.72 (d, J = 9.7Hz 1H),7.64(s,1H),7.58(s,2H),7.55(s,1H),7.30(s,2H),6.99(d,J=7.7Hz,1H),4.4 1(t,J=4.1Hz,1H),4.21-4.15(m,2H),3.91-3.84(m,4H),3.67(d,J=5.3Hz,2H),3.6 1-3.52(m,4H),3.43-3.38(m,10H),2.86(dd,J=7.9,1.1Hz,2H),2.61-2.53(m,8H), 2.50-2.42(m,7H),2.20-2.14(m,1H),2.11-2.05(m,5H).MSm / z(ESI):1052.47[M+H +].

[0322] Example 59: Synthesis of Compound C18

[0323]

[0324] Step 1: Dissolve intermediate L14 (247 mg, 1.2 eq) in dry DMF (5 ml), cool, add HATU (262 mg, 1.5 eq), DIPEA (178 mg, 3 eq), stir at room temperature for 30 minutes, add intermediate B2 (295 mg, 1 eq), maintain the reaction at room temperature for 3 hours, monitor the reaction completion by TLC, dilute the reaction mixture with water, extract with ethyl acetate (3X20 ml), wash with saturated brine, dry with anhydrous sodium sulfate, remove the solvent under reduced pressure, and purify the residue by silica gel column chromatography (DCM: MeOH = 30: 1). Bo-18 (362 mg) was obtained with a yield of 74%. MS m / z (ESI): 1064.50 [M+H + ].

[0325] Step 2: The synthesis method was the same as C01 to obtain C18 (184 mg) with a yield of 56%. 1 H-NMR (300MHz, DMSO-d6): δ=12.03(s,1H),10.98(s,1H),10.39(s,1H),8.96(s,1H),8.73(s,1H),8.05(s,1H),7.73(d,J=10.8Hz 1H),7.61(s,1H),7.56(s,1H),7.51(s,1H),7.32(s,2H),6.97(d,J=8.1Hz,1H),4.43(t,J=6.7Hz,1H),4.22-4.11(m,2H),3.78(s,2H), 3.58-3.49(m,10H),3.29(s,2H),2.63-2.55(m,6H),2.51-2.43(m,11H),2.28-2.19(m,1H),2.13-2.04(m,5H),1.35(t,J=6.8Hz,4H).MS m / z(ESI):964.45[M+H + ].

[0326] Example 60: Synthesis of Compound C19

[0327]

[0328] The synthesis method is the same as that of compound C05, except that L2a is replaced by L15 with the same equivalent weight to obtain compound C19. 1H-NMR (300MHz, DMSO-d6): δ = 12.06 (s, 1H), 10.91 (s, 1H), 10.40 (s, 1H), 8.96 (s, 1H), 8.75 (s, 1H), 8.05 (s, 1H), 7.74 (d, J = 12.3Hz 1H),7.62(s,1H),7.55(s,1H),7.48(s,1H),7.35(s,2H),6.94(d,J=8.1H z,1H),4.40(t,J=6.7Hz,1H),4.22-4.17(m,4H),3.54-3.48(m,8H),3.17- 3.10(m,2H),2.76(s,3H),2.67-2.60(m,6H),2.51-2.45(m,7H),2.27-2. 19(m,3H),2.17-2.09(m,5H),1.55-1.51(m,4H),1.33(t,J=5.2Hz,2H).MS m / z(ESI):911.46[M+H + ].

[0329] Example 61: Synthesis of Compound C20

[0330]

[0331] The synthesis method is the same as C05, only L2a needs to be replaced by L16 with the same equivalent weight to obtain C20. 1H-NMR (300MHz, DMSO-d6): δ=11.98(s,1H),10.54(s,1H),9.13(s,1H),8.96(s,1H),8.83(s,1H),8.74(s,1H),8.31(s,1H),8.08(s,1 H),7.87(d,J=7.4Hz,2H),7.80(s,1H),7.57-7.44(m,4H),7.31(s,1H),5.86(t,J=8.8Hz,1H),4.89(d,J=8.7Hz,1H),4.47(m,1H),4.2 5(s,1H),4.19(t,J=5.3Hz,1H),4.10-4.04(m,1H),3.87-3.75(m,1H),3.55-3.48(m,1H),3.41-3.32(m,8H),2.65-2.52(m,6H),2.49 -2.42(m,7H),2.28(d,J=9.8Hz,2H),2.20-2.13(m,8H),2.10(dd,J=11.1,8.2Hz,2H),1.58-1.52(m,4H),1.45(m,3H),0.97(s,9H).MS m / z(ESI):1096.54[M+H + ].

[0332] Example 62: Synthesis of Compound C21

[0333]

[0334] The synthesis method is the same as that of compound C18, and compound C21 can be obtained by simply replacing B2 with B4 and L14 with H2 with the same equivalents. 1H-NMR (300MHz, DMSO-d6): δ = 12.05 (s, 1H), 10.44 (s, 1H), 9.15 (s, 1H), 8.95 (s, 1H), 8.80 (s, 1H), 8.73 (s, 1H), 8.35 (s, 1H), 8.07 (s,1H),7.81(d,J=8.3Hz,2H),7.75(s,1H),7.54-7.46(m,4H),7.32(s,1H),5.87(t,J=9.6Hz,1H),4.86(d,J=9.3Hz,1H),4.49(m ,1H),4.24(s,1H),4.16(t,J=6.5Hz,1H),4.09-4.04(m,1H),3.84-3.76(m,1H),3.52-3.47(m,5H),3.40-3.34(m,8H),2.66-2.5 4(m,8H),2.48-2.43(m,7H),2.35(d,J=8.3Hz,2H),2.23-2.16(m,6H),2.11(dd,J=10.2,7.3Hz,2H),1.45(m,3H),0.97(s,9H).MS m / z(ESI):1084.54[M+H + ].

[0335] Example 63: Synthesis of Compound C22

[0336]

[0337] The synthesis method is the same as that of compound C05, except that L2a is replaced by L17 with the same equivalent weight to obtain compound C22. 1H-NMR (300MHz, DMSO-d6): δ = 12.01 (s, 1H), 10.45 (s, 1H), 9.07 (s, 1H), 8.94 (s, 1H), 8.87 (s, 1H), 8.74 (s, 1H), 8.32(s,1H),8.05(s,1H),7.79(d,J=2.0Hz,2H),7.68(s,1H),7.58-7.47(m,4H),7.37(s,1H),5.81(t,J=4.4Hz ,1H),4.50(d,J=9.9Hz,2H),4.46-4.32(m,4H),3.74-3.69(m,5H),3.56-3.46(m,3H),3.39(s,4H),2.67-2.52 (m,6H),2.50-2.45(m,7H),2.38-2.30(m,7H),2.21-2.15(m,5H),2.12(dd,J=12.1,7.1Hz,2H),0.95(s,9H).MS m / z(ESI):1098.52[M+H + ].

[0338] Example 64: Synthesis of Compound C23

[0339]

[0340] Step 1: Dissolve B2 (300 mg, 1 eq) and intermediate L18 (267 mg, 1.5 eq) in tetrahydrofuran (5 ml), cool to below 0°C in an ice bath, add acetic acid (56 uL, 2 eq), stir at room temperature for 30 minutes, add sodium triacetoxyborohydride (197 mg, 2 eq) in batches, and maintain the reaction at room temperature for 5 h. Monitor the reaction by TLC. After the reaction, pour the reaction mixture into ice water, adjust the base with saturated sodium carbonate solution, and extract with dichloromethane (3x100 ml); combine the organic phases, wash with water, wash with saturated sodium chloride solution, dry with anhydrous Na2SO4, concentrate, and purify by silica gel column chromatography (DCM: MeOH = 30: 1) to obtain yellow solid Bo-23 (197 mg), with a yield of 42%. MS m / z (ESI): 1006.52 [M+H + ].

[0341] Step 2: The synthesis method is the same as that of compound C02, except that Bo-2 is replaced by Bo-23 with the same equivalent weight to obtain compound C23. 1H-NMR (300MHz, DMSO-d6): δ = 11.06 (s, 1H), 10.88 (s, 1H), 9.42 (s, 1H), 8.81 (s, 1H), 8.74 (d, J = 6.7Hz, 1H), 8.18 (s, 1H) ),7.85(s,1H),7.80(s,1H),7.65-7.61(m,2H),7.62(s,1H),7.51(s,1H),4.44-4.40(m,1H),4.19-4.15(m,4H),3.50 -3.46(m,5H),3.19-3.15(m,2H),3.10(s,1H),2.86-2.83(m,4H),2.55-2.51(m,2H),2.34-2.30(m,8H),2.25-2.21(s ,2H),2.17-2.14(m,6H),2.09(s,2H),2.05-2.00(m,2H),1.48-1.45(m,2H),1.35-1.30(m,4H),1.29-1.26(m,2H).MS m / z(ESI):906.47[M+H + ].

[0342] Example 65: Synthesis of Compound C27

[0343]

[0344] The synthesis method is the same as that of compound C05, except that L2a is replaced by L22 with the same equivalent weight to obtain compound C27. 1 H-NMR (300MHz, DMSO-d6): δ = 10.84 (s, 1H), 10.21 (s, 1H), 9.41 (s, 1H), 8.79 (s, 1H),8.70(d,J=6.4Hz,1H),8.28(s,1H),7.91(s,1H),7.63-7.59(m,2H),7.55( s,1H),7.50(s,1H),4.41-4.37(m,1H),4.17-4.13(m,2H),3.51-3.47(m,5H),3 .22-3.18(m,2H),3.15(s,1H),2.56-2.52(m,2H),2.35-2.31(m,8H),2.27-2.21

[0345] (s,4H),2.16-2.11(m,6H),2.08(s,2H),2.03-1.98(m,2H),1.74-1.69(m,2H),

[0346] 1.54-1.50(m,2H),1.37-1.34(m,2H).MS m / z(ESI):930.39[M+H + ].

[0347] Example 66: Synthesis of Compound C28

[0348]

[0349] The synthesis method is the same as that of compound C05, except that L2a is replaced with L23 with the same equivalent weight to obtain compound C28. 1 H-NMR (300MHz, DMSO-d6): δ = 11.15 (s, 1H), 10.60 (s, 1H), 9.37 (s, 1H), 8.95 (s, 1H), 8.73 (d, J = 5.1Hz, 1H), 8.15 (s, 1H), 8.05-8.01 (m, 1H) ,7.84(s,1H),7.91(d,J=7.3Hz,1H),7.68-7.63(m,2H),7.48(t,J=6.5Hz,1H),7.34(s,1H),7.06(d,J=6.4Hz,1H),6.82(s,1H),4.30-4.25 (m,4H),3.69-3.65(m,1H),3.45-3.41(m,5H),3.24-3.19(m,2H),3.02(s,1H),2.83-2.78(m,2H),2.55-2.50(m,2H),2.34-2.30(m,8H),2 .29-2.21(m,2H),2.19-2.16(m,6H),2.08-2.04(m,2H),1.78-1.74(m,2H),1.46-1.42(m,2H),1.31-1.28(m,2H).MSm / z(ESI):894.07[M+H + ].

[0350] Example 67: Synthesis of Compound C29

[0351]

[0352] The synthesis method is the same as that of compound C07, except that L3 is replaced by L24 with the same equivalent weight to obtain compound C29. 1H-NMR (300MHz, DMSO-d6): δ = 11.08 (s, 1H), 10.53 (s, 1H), 10.13-10.08 (m, 1H), 9.40 (s, 1H), 8.98 (s, 1H), 8.72 (d, J = 5.3Hz ,1H),8.21-8.18(m,1H),8.11(s,1H),7.75(s,1H),7.64-7.60(m,2H),7.13-7.09(m,1H),6.80(s,1H),6.32-6.29(m,1H), 4.30-4.26(m,4H),3.81-3.78(s,1H),3.43-3.39(m,5H),3.26-3.21(m,2H),3.18(s,1H),3.02-2.97(m,2H),2.52-2.48(m ,2H),2.34-2.30(m,8H),2.23-2.19(m,2H),2.16-2.11(m,8H),2.07-2.03(m,2H),1.43-1.40(m,2H),1.36-1.30(m,4H).MS m / z(ESI):889.45[M+H + ].

[0353] Example 68: Synthesis of Compound C30

[0354]

[0355] The synthesis method is the same as that of compound C07, except that L3 is replaced by L25 with the same equivalent weight to obtain compound C29. 1 H-NMR (300MHz, DMSO-d6): δ = 11.91 (s, 1H), 11.10 (s, 1H), 9.58 (s, 1H), 8.87 (s, 1H), 8.72 (d, J = 7.3Hz, 1H), 8. 17(s,1H),7.78(s,1H),7.64-7.60(m,2H),7.19-7.14(m,4H),4.32-4.25(m,2H),3.78-3.73(m,1H),3.47-3. 44(m,5H),3.28-3.23(m,2H),3.15(s,1H),3.02-2.97(m,4H),2.69-2.66(m,1H),2.57-2.54(m,6H),2.42-2. 38(m,8H),2.22-2.18(m,4H),2.13-2.09(m,6H),1.74-1.70(m,2H),1.52-1.48(m,4H),1.45-1.41(m,4H).MS m / z(ESI):882.50[M+H +].

[0356] Example 69: Synthesis of Compound C32

[0357]

[0358] The synthesis method is the same as that of compound C07, except that L3 is replaced by L27 with the same equivalent weight to obtain compound C32. 1 H-NMR (300MHz, DMSO-d6): δ = 11.93 (s, 1H), 10.26 (s, 1H), 9.55 (s, 1H), 8.81 (s, 1H), 8.78 (d, J = 8.7Hz, 1H), 8.58-8.53 (m ,1H),8.20(s,1H),7.81(s,1H),7.65-7.62(m,2H),7.54(s,1H),7.31(s,1H),6.58-6.55(m,1H),4.33-4.29(m,2H),3.76 -3.72(m,2H),3.47-3.44(m,5H),3.40-3.36(m,2H),3.25-3.21(m,2H),3.15(s,1H),3.05-3.01(m,2H),2.74-2.71(m,2 H),2.56-2.52(m,2H),2.45-2.41(m,8H),2.16-2.12(m,6H),1.68-1.64(m,2H),1.42-1.38(m,2H),1.29-1.25(m,6H).MS m / z(ESI):868.46[M+H + ].

[0359] Example 70: Synthesis of Compound C33

[0360]

[0361] The synthesis method is the same as that of compound C07, except that L3 is replaced by L28 with the same equivalent weight to obtain compound C33. 1H-NMR (300MHz, DMSO-d6): δ=11.81(s,1H),11.16(s,1H),9.51(s,1H),8.92(s,1H),8.87(s,1H),8.71(d ,J=6.5Hz,1H),8.28(s,1H),8.21(s,1H),8.15-8.11(m,1H),7.85(s,1H),7.68-7.65(m,2H),4.45-4.49 (m,2H),4.36-4.32(m,3H),4.21(s,2H),3.45-3.40(m,5H),3.27-3.24(m,2H),3.17(s,1H),2.58-2.55( m,2H),2.47-2.41(m,8H),2.24-2.19(m,4H),2.15-2.10(m,6H),2.21-2.18(m,4H),1.57-1.53(m,4H).MS m / z(ESI):926.43[M+H + ].

[0362] Example 71: Synthesis of Compound C34

[0363]

[0364] The synthesis method is the same as that of compound C07, except that L3 is replaced by L29 with the same equivalent weight to obtain compound C34. 1 H-NMR (300MHz, DMSO-d6): δ = 10.84 (s, 1H), 10.31 (s, 1H), 9.37 (s, 1H), 8.91 (s, 1H), 8.85 (d, J = 5.7Hz, 1H), 8.1 5(s,1H),7.95-7.93(m,2H),7.74(s,1H),7.67-7.63(m,2H),7.05-6.99(m,2H),4.25-4.19(m,2H),4.08-4.05 (m,2H),3.74-3.70(m,2H),3.55-3.49(m,5H),3.21-3.18(m,4H),3.11-3.15(m,1H),2.55-2.47(m,2H),2.36- 2.31(m,8H),2.27-2.23(s,2H),2.19-2.15(m,6H),1.75-1.74(m,2H),1.45-1.40(m,6H),1.29-1.26(m,4H).MS m / z(ESI):858.47[M+H + ].

[0365] Example 72: Synthesis of Compound C35

[0366]

[0367] The synthesis method is the same as that of compound C07, except that L3 is replaced by L30 with the same equivalent weight to obtain compound C35. 1 H-NMR (300MHz, DMSO-d6): δ = 12.12 (s, 1H), 11.07 (s, 1H), 10.35 (s, 1H), 8.92 (s, 1H), 8.66 (d, J = 8.6Hz, 1H), 8. 12(s,1H),8.03(m,1H),7.97(s,2H),7.89(s,1H),7.81(m,1H),7.69(s,1H),7.59-7.53(m,2H),4.65-4.42(s,2 H)4.31(s,2H),4.25-4.13(s,2H),3.58(s,2H),3.02-2.97(m,2H),2.85-2.74(m,4H),2.65-2.59(m,2H),2.42- 2.37(m,6H)2.23-2.20(m,8H),,2.15-2.10(m,3H),2.08-2.04(m,4H),1.55-1.46(m,6H),1.40-1.28(m,2H).MS m / z(ESI):906.47[M+H + ].

[0368] Example 73: Synthesis of Compound C37

[0369]

[0370] The synthesis method is the same as that of compound C07, except that L3 is replaced by L32 with the same equivalent weight to obtain compound C37. 1H-NMR (300MHz, DMSO-d6): δ=11.08(s,1H),10.87(s,1H),9.41(s,1H),8.89(s,1H),8.71(d,J=6.3Hz,1H),8.17(s,1 H),7.88(s,1H),7.67-7.62(m,2H),7.55(d,J=5.1Hz,1H),7.39(t,J=7.1Hz,1H),5.03(dd,J=12.1,7.5Hz,1H),4.16- 4.11(m,4H),3.49-3.45(m,5H),3.23-3.17(m,2H),3.09(s,1H),2.87-2.84(m,4H),2.54-2.50(m,2H),2.37-2.31(m ,8H),2.27-2.25(s,2H),2.16-2.11(m,6H),2.08(s,2H),2.04-1.99(m,2H),1.47-1.42(m,1H),1.31-1.25(m,4H).MS m / z(ESI):899.44[M+H + ].

[0371] Example 74: Synthesis of Compound C39

[0372]

[0373] The synthesis method is the same as that of compound C07, and compound C39 can be obtained by simply replacing B2 with B3 with the same equivalent weight.

[0374] 1 H-NMR (300MHz, DMSO-d6) δ=11.8(s,1H),11.35(s,1H),10.22(s,1H),9.10(s,1H),8.45(s,1H),8.24(d,J=7 .4Hz,1H),8.20(s,1H),7.91(s,1H),7.86-7.62(m,2H),7.43(d,J=7.1Hz,1H),7.09(d,J=7.0,Hz,1H),5.45( m,1H),5.11(m,2H),4.32-4.15(m,2H),3.90-3.67(m,2H),3.52-3.31(m,4H),3.28-3.21(m,2H),2.98-2.74( m,10H),2.58(s,3H),2.40(s,3H),2.25(s,3H),2.07(m,4H),1.76(s,1H),1.54-1.46(m,2H),1.38(m,2H).MS m / z(ESI):913.42[M+H+ ].

[0375] Example 75: Synthesis of Compound C40

[0376]

[0377] The synthesis method is the same as that of compound C07, except that L3 is replaced by L34 with the same equivalent weight to obtain compound C40.

[0378] 1 H-NMR (300MHz, DMSO-d6)δ=12.31(s,1H),11.95(s,1H),10.65(s,1H),8.87(s,1H),8.32(s,1H),7.85(d,J=4.5H z,1H),7.72-7.60(m,1H),7.43(s,1H),7.15-6.92(m,1H),6.64(s,1H),6.53-6.29(m,2H),5.67(s,2H),5.01(s,1 H),4.65(m,2H),4.52(m,4H),4.42(s,2H),3.92(s,3H),3.91(d,J=5.9Hz,2H),3.63-3.47(m,4H),3.25-3.11(s, 3H)2.94-2.71(s,3H),2.75-2.56(m,8H),2.53-2.42(m,1H),2.31(s,1H),2.07(m,1H),1.82-1.63(m,4H),1.32(m 1H).MS m / z(ESI):826.44[M+H + ].

[0379] Example 76: Synthesis of Compound C41

[0380]

[0381] The synthesis method is the same as that of compound C07, except that L3 is replaced by L35 with the same equivalent weight to obtain compound C41.

[0382] 1H-NMR (300MHz, DMSO-d6) δ=11.56(s,1H),10.97(s,1H),10.85(s,1H),9.23(s,1H),8.63(s,1H),8.21(d,J=7 .9Hz,1H),7.64-7.42(m,1H),6.97-6.69(m,1H),6.32(s,1H),6.12-5.97(m,2H),5.77(s,2H),4.78(s,1H),4. 70(m,2H),4.61(m,4H),4.23(s,2H),4.15(s,3H),3.89(d,J=8.6Hz,2H),3.57-3.49(m,4H),3.17-3.05(s,3H) 2.85-2.64(s,3H),2.51-2.33(m,8H),2.25-2.12(m,1H),1.96(s,1H),1.83(m,1H),1.54-1.43(m,4H),1.04(m 1H).MS m / z(ESI):844.43[M+H + ].

[0383] Example 77: Synthesis of Compound C42

[0384]

[0385] The synthesis method is the same as that of compound C07, except that L3 is replaced by L36 with the same equivalent weight to obtain compound C42.

[0386] 1 H-NMR (300MHz, DMSO-d6)δ=12.03(s,1H),11.46(s,1H),10.73(s,1H),9.57(s,1H),9.02(s,1H),8.96(s,1H),8 .23-8.06(m,1H),7.75(d,J=8.3Hz,1H),7.36(s,1H),7.01-6.87(m,2H),6.54(s,2H),5.39(s,1H),5.06(m,2H), 4.95(m,4H),4.74(s,2H),4.62(s,3H),4.29(d,J=5.3Hz,2H),4.06-3.97(m,4H),3.68-3.44(s,3H),3.39-3.16 (s,3H),2.90-2.75(m,6H),2.48(s,2H),2.33-2.02(m,1H),1.96(s,1H),1.78(m,1H),1.61-1.57(m,4H),1.40(m 1H).MS m / z(ESI):844.43[M+H+ ].

[0387] Example 78: Synthesis of Compound C43

[0388]

[0389] The synthesis method is the same as that of compound C05, except that L2a is replaced by L37 with the same equivalent weight to obtain compound C43.

[0390] 1 H-NMR (300MHz, DMSO-d6): δ = 11.96 (s, 1H), 11.35 (s, 1H), 10.66 (s, 1H), 9.38 (s, 1H), 8.96 (s, 1H) ),8.12(m,2H),7.65(s,1H),7.34(d,J=7.4Hz,1H),7.23(s,2H),6.97(d,J=5.5Hz,1H),5.96(m, 1H),5.34-5.11(m,2H),4.31(s,4H),3.85(s,4H),3.67-3.41(m,2H),3.33-3.06(m,4H),2.95-2 .73(m,2H),2.55(s,3H),2.46(d,J=5.7Hz,2H),2.16(m,4H),1.57(s,3H),1.40-1.32(m,5H).MS m / z(ESI):881.37[M+H + ].

[0391] Example 79: Synthesis of Compound C44

[0392]

[0393] The synthesis method is the same as that of compound C07, except that L3 is replaced by L38 with the same equivalent weight to obtain compound C44.

[0394] 1H-NMR (300MHz, DMSO-d6): δ = 11.36 (s, 1H), 11.02 (s, 1H), 10.96 (s, 1H), 8.57 (s, 1H), 8.23 ​​(s, 1H), 7.93 (s, 1H) ,7.82-7.63(m,2H),7.45(s,1H),7.36(d,J=8.0Hz,1H),6.95(s,1H),6.85(d,J=6.4Hz,1H),6.74(s,1H),6.69( m,1H),4.56(s,1H),4.23(m,2H),3.65-3.46(m,2H),3.39-3.20(m,4H),2.97-2.85(m,2H),2.73(s,3H),2.61(d ,J=6.9Hz,2H),2.52-2.41(m,8H),2.31-2.19(m,6H),2.15(s,3H),1.89(d,J=5.9Hz,3H),1.69-1.32(m,5H).MS m / z(ESI):894.44[M+H + ].

[0395] Example 80: Synthesis of Compound C45

[0396]

[0397] The synthesis method is the same as that of compound C07, except that L3 is replaced by L39 with the same equivalent weight to obtain compound C45.

[0398] 1 H-NMR (300MHz, DMSO-d6): δ=12.03(s,1H),11.35(s,1H),10.64(s,1H),8.52(s,1H),8.31(s,1H),8.32( s,1H),8.02(s,1H),7.85(m,1H),7.43(s,1H),6.95(d,J=6.4Hz,1H),6.76(s,1H),6.45-6.27(m,1H),6. 04(s,1H),3.99(s,2H),3.85-3.64(m,2H),3.45-3.30(m,2H),3.13-2.95(m,4H),2.78(s,2H),2.70(d,J =7.5Hz,2H),2.51-2.43(m,6H),2.35-2.18(m,2H),2.11-1.97(m,8H),1.88(m,5H),1.63-1.45(m,5H).MS m / z(ESI):845.43[M+H + ].

[0399] Example 81: Synthesis of Compound C47

[0400]

[0401] The synthesis method is the same as that of compound C07, except that L3 is replaced by L41 with the same equivalent weight to obtain compound C47.

[0402] 1 H-NMR (300MHz, DMSO-d6): δ = 11.78 (s, 1H), 11.02 (s, 1H), 10.35 (s, 1H), 8.96 (s, 1H), 8.55 (s, 1H), 8. 01(s,1H),7.89(s,1H),7.64(m,1H),7.25(s,1H),6.66(d,J=5.3Hz,1H),6.53(s,1H),6.41-6.30(m,1 H),4.32(s,2H),4.01-3.89(m,2H),3.67-3.44(m,2H),3.35-3.02(m,6H),2.87(d,J=7.3Hz,2H),2.6 4-2.42(m,4H),2.35(s,2H),2.25-2.03(m,2H),1.87-1.64(m,8H),1.44(m,5H),1.35-1.26(m,5H).MS m / z(ESI):862.42[M+H + ].

[0403] Example 82: Synthesis of Compound C48

[0404]

[0405] The synthesis method is the same as that of compound C07, except that L3 is replaced by L42 with the same equivalent weight to obtain compound C48. 1H-NMR (400MHz, DMSO-d6): δ = 12.15 (s, 1H), 11.32 (s, 1H), 10.43 (s, 1H), 9.12 (s, 1H), 8.66 (s, 1H), 8.01 (d, J = 4. 7Hz,1H),7.82-7.71(m,1H),7.63(s,1H),7.42-7.21(m,1H),6.98-6.84(m,2H),6.70(s,1H),6.62-6.38(m,2H), 5.35(s,2H),4.98(s,2H),4.77(m,2H),4.36(m,4H),4.15(s,2H),3.12(s,2H),2.97(s,3H),2.86(d,J=6.2Hz,2H ),2.75-3.53(m,3H),2.51-2.40(s,3H),2.37-2.28(s,2H),2.22-2.09(m,8H),1.75-1.48(m,4H),1.14(m1H).MS m / z(ESI):828.01[M+H + ].

[0406] Test Example 1: Compound kinase activity assay

[0407] 1. Test method:

[0408] The inhibitory activity of the compounds of the present invention on HPK1 kinase was determined by Beijing Aisiyipu Company using the ADP-Glo ​​kinase assay. The specific steps are as follows:

[0409] 1) Prepare 2× ATP & Substrate Solution and 2× Kinase Solution in Assay Buffer.

[0410] 2) Transfer 20 nL of compound to 384 assay plate via Echo 655. Add 2 μL of 2× kinase solution, mix and incubate in 384 assay plate at 25° C. for 10 minutes.

[0411] 3) Add 2 μL of 2× substrate and ATP solution to the wells and incubate at 25°C for 60 minutes.

[0412] 4) Add 4 μL of ADP-Glo ​​reagent to the wells and incubate at 25°C for 40 minutes.

[0413] 5) Add 8 μL of kinase assay reagent to the wells and incubate at 25°C for 40 minutes.

[0414] 6) The luminescent signal was recorded on a microtiter plate reader. GraphPad software was used for data fitting to calculate the IC value of the inhibitory activity of the compound on HPK1 kinase. 50 value.

[0415] 2. Test results:

[0416] The reference compound HC58-76 is the compound reported in publication number WO2020227325. The chemical structure of HC58-76 is shown below. The compound HC58-76 used in this experiment was prepared according to the method disclosed in patent WO2020227325.

[0417]

[0418] The experimental results are shown in Table 1 below:

[0419] Table 1. Inhibitory activity of the compounds of the present invention on HPK1 kinase (IC 50 / nM)

[0420]

[0421]

[0422] Note: A in Table 1 represents IC 50 ≤10nM, B means 10nM<IC 50 Value ≤ 50nM, C means 50nM<IC 50 Value ≤ 100nM, D means 100nM<IC 50 ≤500nM value.

[0423] It can be seen from Table 1 that the compounds disclosed in the present invention have significant inhibitory activity on the HPK1 kinase enzyme. The IC values ​​of all compounds are 50 The value is below 50 nM, and some compounds can even reach below 10 nM. Compared with the compound HC58-76 disclosed in the prior art, the above compounds disclosed in the present invention have significantly better binding and inhibitory activity with HPK1 protein.

[0424] Test Example 2: Degradation activity of compounds on HPK1 and GLK proteins

[0425] 1. Test method:

[0426] Cell culture: Jurkat cells were incubated with compounds (0, 0.13 nM, 0.4 nM, 1.2 nM, 3.7 nM, 11 nM, 33 nM, 100 nM) for 12 hours at 37°C, 5% CO2, and RPMI-1640 (containing 10% serum).

[0427] Cell collection: After incubation of Jurkat cells for 12 hours, centrifuge at 4°C, 1000 rpm, 5 minutes to collect cells.

[0428] Protein extraction: discard the original culture medium, wash the cells with PBS and discard the PBS. Add an appropriate amount of strong RIPA lysis buffer (containing 1% protease inhibitor, 1% phosphatase inhibitor, 1‰ super nuclease) to each well and let stand at 4℃ for 5min. Centrifuge at 12500rpm for 15min to collect the supernatant.

[0429] Protein quantification: Use the BCA kit to quantify protein. Mix solution A and solution B in a ratio of 50:1 to obtain a working solution. Add 200 μL of the solution, 18 μL of PBS and 2 μL of protein sample into a 96-well plate. Mix well and incubate at 37°C for 30 min. Measure the absorbance at 562 nm with a full-wavelength microplate reader and calculate based on the standard curve.

[0430] Sample preparation: The quantified protein was mixed with 5x loading buffer solution at a volume ratio of 4:1, denatured in a metal bath for 10 min, and then placed on ice for 5 min. The prepared samples were stored at -20°C.

[0431] Prepare SDS-PAGE gel electrophoresis: the voltage of the concentrated gel is 80V, the voltage of the separation gel is 180V, and the membrane is wet-transferred at 220mA constant current for 45min at 4°C. After the membrane transfer is completed, the protein membrane is placed in a PBST solution containing 5% skimmed milk powder at room temperature for 2h.

[0432] Refer to the instructions of HPK1 (E6D2F) Rabbit mAb (53453S, CST), MAP4K3 (D1L4G) Rabbit mAb (92427S, CST), and β-actin (FD0060, Fdbio), dilute the primary antibody at 1:1000, incubate at 4°C overnight, and then wash three times with TBST, 10 min each time.

[0433] Select Peroxidase Conjugated Gaot anti-Mouse IgG(hl)(FDM007, Fdbio) and Peroxidase Conjugated Gaot anti-Rabbit IgG(h+l)(FDR007, Fdbio) according to the primary antibody, refer to the antibody instructions, dilute the secondary antibody at 1:5000, incubate at room temperature with slow shaking for 1 hour, and wash 3 times with TBST, 10 min / time.

[0434] ECL luminescent solution was added, the results were observed by the developer, and the Western results were quantified using Image J software. The degradation rate (HPK1 and GLK) and the half-degradation concentration DC were calculated by comparing with the control group without adding the compound. 50 .

[0435] 2. Experimental results:

[0436] Table 2 Degradation activity of the compounds of the present invention on HPK1 protein

[0437]

[0438]

[0439] Note: A in Table 2 represents DC 50 ≤10nM, B means 10nM<DC 50 Value ≤ 50nM, C means 50nM<DC 50 Value ≤ 100nM, D means 100nM<DC 50 ≤500nM value.

[0440] It can be seen from the data in Table 2 that the compounds of the present invention have good protein degradation activity on HPK1 and have good application potential in treating diseases mediated by HPK1.

[0441] Table 3 Selectivity of the compounds of the present invention for HPK1 / GLK protein degradation

[0442] Compound <![CDATA[GLKDC 50 (nM)]]> HPK1 / GLK selectivity (M) HC58-76 C - C09 B ++ C18 C +++ C23 C +++ C35 B ++ C39 B ++ C40 D +++ C43 C +++ C48 E ++++

[0443] Note: A in Table 3 represents DC 50 ≤10nM, B means 10nM<DC 50 Value ≤ 50nM, C means 50nM<DC 50 Value ≤ 100nM, D means 100nM<DC 50 ≤500nM value, E means 500nM<DC 50 In Table 3, “-” means M≤1 times, “+” means 1<M≤10 times, “++” means 10<M≤100 times, “+++” means 100<M≤1000 times, and “++++” means 1000 times<M.

[0444] From the data in Table 3, it can be seen that the compounds of the present invention have good selectivity for HPK1. The protein degradation activity of some compounds of the present invention for HPK1 is several times that of GLK and is more than 10 times, which indicates that the compounds of the present invention have good safety and good application potential in treating diseases mediated by HPK1. Among them, compound C48 showed a high selectivity of more than 1000 times for HPK1 / GLK protein degradation activity. Figure 1 As shown in Figure 2, it can be seen that compound C48 exhibits efficient and selective degradation of HPK1 protein, and has no degradation effect on GLK at a concentration of 10 μM. Figure 2 shown.

[0445] Test Example 3: SLP76 phosphorylation cytology test

[0446] Inhibition of HPK1 can inhibit the phosphorylation of its downstream SLP76. The phosphorylation of SLP76 protein was detected using Jurkat (ATCC.Clone E6-1TIB-152 TM On the first day of the experiment, the cells were diluted to 10 6 / ml, 100 μL per well, 10 5 The cells were plated in a 96-well cell culture plate and starved for 4 hours. The compounds in Table 3 were dissolved in 100% DMSO, and the stock concentration was 4 mM. The DMSO solution of the compound was diluted four times from 10 mM, with a total of 9 concentrations. 4 μL of the diluted compound was diluted to 196 μL, and preheated RPMI1640 at 37 degrees and mixed. Take 50 μL of the final diluted compound and add it to the cells, incubate at 37 degrees for 20 minutes, add 50 μL of the diluted human CD3 / CD28 T cell activator, so that the final concentration volume of the activator is 1 / 40 of the total system, and incubate at 37 degrees for 30 minutes. After the reaction, the cells were centrifuged at 1200 rpm at 4 degrees for 5 minutes, the culture medium was aspirated, 150 μL of cell lysis solution (provided in the ELISA kit, Cell Signaling) was added and placed on ice for 30 minutes to allow the cells to be fully lysed. After the lysed cells were blown evenly, they were centrifuged at 4000 rpm at 4 degrees for 5 minutes, and then 50 μL of supernatant was taken and added to the FastScanTm Phospho-SLP-76 (Ser376) ELISA Kit (Cell Signaling) to test the phosphorylation level of cell SLP76.

[0447] The average values ​​of the positive wells and negative wells were calculated as the positive control value (Signal pos) and the negative control value (Signal neg). The inhibition rate was calculated by the formula Inhibition rate = (Signal pos-Signal test) / (Signal pos-Signal neg) x 100% using the signal value of the working well (Signal test). The obtained inhibition rate was used to draw a concentration-inhibition rate curve in GraphPadPrism software by nonlinear fitting to calculate IC 50 .

[0448] Table 3. Inhibitory activity of the compounds of the present invention on SLP76 phosphorylation (IC 50 / nM)

[0449] Compound <![CDATA[HPK1p-SLP76IC 50 (nM)]]> Compound <![CDATA[HPK1p-SLP76IC 50 (nM)]]> B1 D C39 A C09 B C40 A C18 C C43 B C23 A C46 B C35 B C48 A

[0450] Note: A in Table 1 represents DC 50 ≤10nM, B means 10nM<DC 50 Value ≤ 50nM, C means 50nM<DC 50 Value ≤ 100nM, D means 100nM<DC 50 ≤500nM value.

[0451] The compounds of the present invention can significantly inhibit the phosphorylation of SLP76 downstream of HPK1, and are superior to the positive control compound B1. This indicates that the compounds of the present invention can inhibit the signal transduction of HPK1 kinase or enhance the anti-tumor immune response by degrading HPK1 protein.

[0452] Test Example 4: Pharmacokinetics test in rats

[0453] Experimental animals: 3 male SD rats and 3 female SD rats, provided by Qinglongshan Animal Breeding Farm, Jiangning District, Nanjing, with a body weight range of 180 g to 220 g.

[0454] Experimental procedures: The mice were fed for three days before use. The mice were fasted for 12 hours before administration and had free access to water during the experiment. The compound was mixed with the solvent (10% DMSO / 30% PEG400 / 60% Saline), vortexed and sonicated for 15 minutes. Clear injection solutions and oral solutions were prepared. 50 mg / kg was administered by gavage and 5 mg / kg was administered by intravenous injection into the tail vein.

[0455] About 200 μl of blood was collected by oral administration at 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, and 24 h before and after administration, respectively, and placed in an EP tube containing 1% sodium heparin. The blood was centrifuged at 8000 rpm for 5 min to separate the plasma, determine the plasma drug concentration, and calculate the pharmacokinetic parameters.

[0456] For intravenous administration, about 100 μL of blood was collected before and 2 minutes, 15 minutes, 45 minutes, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after administration, and placed in an EP tube containing 1% sodium heparin. The blood was centrifuged at 8000 rpm for 5 minutes to separate the plasma, determine the plasma drug concentration, and calculate the pharmacokinetic parameters (PK parameters were calculated using WinNonlin version 6.4). The results are shown in Table 4 below:

[0457] Table 4. Pharmacokinetic parameters of the compounds in rats

[0458]

[0459] The compound of the invention has high exposure in rats, long half-life and good oral absorption.

[0460] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A class of PROTAC molecules, characterized in that Compounds shown in formula I: In the formula: HI is a ligand that binds to the target protein HPK1, EL is an E3 ligase ligand, L is a connecting chain connecting HI and EL, and HI is the following structure, where "--" indicates the position of connection with L: Among them, R 1 and R 2 Both are independently selected from any one of an aromatic ring, an aromatic heterocyclic ring containing a nitrogen atom, and an aromatic cyclic ring; R 3 and R 4 Both are independently selected from any one of -H, -F, -Cl, -CH3, -CH2CH3, -OCH3; M and K are both independently selected from C, N, O, S or CR 5 Any one of the following, where R 5 is any one of H, -CH3, -CH2CH3, or -CH(CH3)2; n is 0, 1 or 2; X is C or N.

2. A pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate of the PROTAC molecule of claim 1.

3. A PROTAC molecule according to claim 1, characterized in that Formula II and III are the following structures, "--" indicates the position connected to L:

4. A PROTAC molecule according to claim 1, characterized in that EL has the following structure: Where Y is a hydrogen atom, a deuterium atom, a fluorine atom, a chlorine atom or an alkyl substituent; Z is a carbon atom or a carbonyl group; G is a hydrogen atom or a methyl group; Ring B is C 6-12 aryl, 5-12 membered heteroaryl or 4-12 membered heterocyclic group, each of which is optionally substituted by 1 to 3 R a Replace; where each R a are independently selected from hydrogen, oxo, imino, sulfoxime, halo, nitro, -CN, C 1～6 Alkyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 3～15 Cycloalkyl, C 1～8 Haloalkyl, C 6～12 Aryl, 5- to 12-membered heteroaryl, 4- to 12-membered heterocyclic group, -OR b 、-C(O)-R b 、-C(O)OR b 、-C(O)-N(R b )(R b )、-N(R b )(R b )、-N(R b )C(O)-R b 、-N(R b )C(O)OR b 、-N(R b )C(O)N(R b )(R b )、-N(R b )S(O)2(R b ),-NR b S(O)2N(R b )(R b ), -N(R b )S(O)2O(R b )、-OC(O)R b 、-OC(O)-N(R b )(R b )、-Si(R b )3. -SR b 、-S(O)R b 、-S(O)(NH)R b 、-S(O)2R b or -S(O)2N(R b )(R b ), where C 1～6 Alkyl, C 2～6 Alkenyl, C 2～6 Alkynyl, C 3～15 Cycloalkyl, C 1～8 Haloalkyl, C 6～12 Each of the aryl group, the 5- to 12-membered heteroaryl group, and the 4- to 12-membered heterocyclic group can be optionally substituted by 1 to 3 R c Replacement; where R b is hydrogen or C 1～6 Alkyl; each R c are independently halo, oxo, -CN, -OH, C optionally substituted with 1 to 3 fluorine groups 1～6 Alkyl, or C 3～8 Cycloalkyl, or -OC optionally substituted by 1 to 3 fluorine groups 1～6 Alkyl; W is a carbon atom or a nitrogen atom.

5. A PROTAC molecule according to claim 4, characterized in that The structures of formula IV, V and VI are as follows:

6. A PROTAC molecule according to claim 1, characterized in that Indicates the location connected to HI; Indicates the position connected to EL; L is the following structure: Wherein, Q and G are independently selected from carbon atoms, oxygen atoms, nitrogen atoms, -(CO)-, -NH(CO)-, -N(CH3)-, n1, m1, m2 are integers between 0 and 12; Q 1 and G 2 are independently selected from a carbon atom or a nitrogen atom; 2 , Q 3 , G 2 and G 3 They are independently selected from a covalent bond, a carbon atom, an oxygen atom, a nitrogen atom, -(CO)-, -NH(CO)-, a nitrogen-containing five-membered ring, and a nitrogen-containing six-membered ring.

7. A class of PROTAC molecules according to claim 1, wherein the compound represented by formula I can be selected from compounds of any of the following structures:

8. Use of a PROTAC molecule according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in the preparation of a targeted HPK1 inhibitor or a targeted HPK1 degrader.

9. Use of a PROTAC molecule according to any one of claims 1 to 7, or a pharmaceutically acceptable salt, isomer, metabolite, prodrug, solvate or hydrate thereof in the preparation of a medicament for treating and or preventing cancer.

10. The use according to claim 9, characterized in that: The cancer is one or more of gastric cancer, lung cancer, breast cancer, prostate cancer, leukemia, ovarian cancer, esophageal cancer, liver cancer, colon cancer, and brain cancer.

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