PROTAC degrader targeting DNA-PK and preparation method and use thereof

Abstract

The application discloses a kind of PROTAC degradation agent targeting DNA-PK and preparation method and purposes thereof.The PROTAC degradation agent targeting DNA-PK provided in the application comprises DNA-PK ligand fragment, linker and E3 ligase ligand fragment, and its structure is shown as formula 1.The preparation route of the compound is mature and controllable, the reaction condition is mild, the compound can specifically bind to DNA-PK target protein, and shows good DNA-PK enzyme activity inhibition effect and target protein degradation potential in in-vitro experiment, and can inhibit tumor cell growth.Therefore, the compound can be used to prepare related drugs for treating and / or preventing tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical chemistry and drug development, and specifically relates to a class of PROTAC degrading agents targeting DNA-PK, their preparation methods and uses. Background Technology

[0002] Cancer is one of the major diseases threatening human health. Although molecular targeted therapy has made progress in recent years, tumors often exhibit multi-gene drive and pathway compensation characteristics, and single-target intervention still has limitations in terms of the breadth and durability of efficacy. In clinical practice, combination therapy is often used to improve efficacy, but the combination of different drugs often leads to problems such as the superposition of toxic side effects and difficulties in pharmacokinetic / pharmacodynamic (PK / PD) matching.

[0003] DNA-dependent protein kinases (DNA-PKs) are key kinases in DNA double-strand break repair (especially the NHEJ pathway); PARP1 (poly(ADP-ribose) polymerase-1) participates in DNA damage response and regulates the recruitment of related repair proteins. Tumor cells, due to genomic instability, are highly dependent on DNA repair networks for survival, especially in the context of BRCA and other homologous recombination defects, making them more sensitive to DDR (DNA damage response) targets.

[0004] PROTAC (proteolysis targeting chimera) technology induces ubiquitination of target proteins and their degradation via the proteasome through the formation of a ternary complex of target protein ligand-linker arm-E3 ligand, achieving a sustained inhibitory effect on the target within the cell. Based on this mechanism, the development of DNA-PK targeted degraders holds promise for reducing drug dosage, overcoming drug resistance, and enhancing efficacy; however, candidate structures with both fabricability and good therapeutic potential are currently lacking. Summary of the Invention

[0005] The first objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a compound.

[0006] A second object of the present invention is to provide a method for preparing the compound.

[0007] A third object of the present invention is to provide a pharmaceutical composition.

[0008] The fourth aspect of the present invention is to provide the use of the said compound or pharmaceutical composition.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A compound, said compound being a DNA-PK PROTAC degrader, having the structure shown in Formula 1;

[0011] Formula 1;

[0012] In Formula 1, R is independently selected from alkyl, aryl, or heterocyclic groups; and the compound comprises a DNA-PK ligand fragment, a linker, and an E3 ligase ligand fragment, wherein the E3 ligase ligand is preferably (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide ligand.

[0013] Preferably, in Formula 1, R is selected from any one of alkyl, aryl, or heterocyclic compounds.

[0014] Preferably, the compound includes any one of the following compounds (1) to (14):

[0015] .

[0016] The method for preparing the compound includes the following steps:

[0017] 1) The compound shown in Formula I is subjected to a substitution reaction with a methyl bromocarboxylic acid ester compound, followed by a reduction conversion to obtain the intermediate shown in Formula II;

[0018] Formula I; Formula II;

[0019] In Formula II, R is selected from any one of alkyl, aryl, and heterocyclic groups;

[0020] 2) The compound shown in Formula III was subjected to nucleophilic substitution and amination with 4-aminotetrahydropyran hydrochloride, followed by hydrolysis and transformation to obtain the intermediate shown in Formula IV;

[0021] Formula III; Formula IV;

[0022] 3) The intermediate shown in Formula IV is reacted with diphenyl azidophosphate to construct a key heterocyclic skeleton (or an activated intermediate), and then methylated with iodomethane to obtain the intermediate shown in Formula V;

[0023] Formula V;

[0024] 4) The intermediate shown in Formula V is coupled with the intermediate shown in Formula II, and then hydrolyzed to obtain the intermediate shown in Formula VI:

[0025] Formula VI;

[0026] 5) The intermediate shown in Formula VI is further condensed with (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide to obtain the target product DNA-PKPROTAC degrading agent, i.e., the compound.

[0027] Preferably, in step 1), the bromocarboxylic acid methyl ester compound includes at least one of methyl 4-bromobutyrate, methyl 5-bromopentanoate, methyl 6-bromohexanoate, methyl 7-bromoheptanoate, ethyl 6-bromo-2,2-dimethylhexanoate, ethyl 7-bromo-2,2-dimethylheptanoate, methyl 3-(bromomethyl)benzoate, methyl 2-(bromomethyl)benzoate, methyl 5-bromofuran-2-carboxylic acid, methyl 2-(4-(bromomethyl)phenyl)acetate, methyl 4-(bromomethyl)benzoate, methyl 11-bromoundecanoate, methyl 6-(bromomethyl)nicotinic acid, and methyl 3-amino-4-methylbenzoate.

[0028] Preferably, in step 1), the molar ratio of the compound represented by Formula I to the methyl bromocarboxylic acid ester compound is 1:(1.39-1.40); more preferably, it is 1:1.39.

[0029] Preferably, in step 1), the reaction conditions are: reacting at 80°C for 25 to 30 minutes.

[0030] Preferably, in step 2), the molar ratio of the compound represented by Formula III to 4-aminotetrahydropyran hydrochloride is 1:(1.0 to 1.1); more preferably, it is 1:1.

[0031] Preferably, in step 2), the reaction conditions are: stirring at room temperature for more than 10 hours.

[0032] Preferably, in step 3), the molar ratio of the compound represented by formula IV to diphenyl azidophosphate is 1:(1.0 to 1.1); more preferably, it is 1:1.

[0033] Preferably, in step 3), the reaction conditions are: react at room temperature for 1 hour, then react in an oil bath at 120°C for 10-12 hours.

[0034] Preferably, in step 4), the molar ratio of the compound represented by formula V to the compound represented by formula II is 1:(1.1 to 1.2); more preferably, it is 1:1.2.

[0035] Preferably, in step 4), the reaction conditions are: reacting at 105°C for 12 to 18 hours under nitrogen atmosphere.

[0036] Preferably, in step 5), the molar ratio of the compound represented by formula VI to (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide is 1:(1.1 to 1.2); more preferably, it is 1:1.2.

[0037] Preferably, in step 5), the reaction conditions are: reacting at room temperature for 1 to 2 hours.

[0038] A pharmaceutical composition comprising the above-mentioned compound (DNA-PK PROTAC degrader) or its stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or cocrystal.

[0039] The use of the compound or its stereoisomers, solvates, prodrugs, metabolites, pharmaceutically acceptable salts, or cocrystals in the preparation of drugs for the prevention, treatment, delay, and / or adjuvant therapy of tumors. This compound can specifically bind to DNA-PK target proteins, exhibiting good DNA-PK enzyme activity inhibition and target protein degradation potential, and can be used in the preparation of antitumor-related drugs.

[0040] Preferably, the tumor includes at least one of breast cancer, melanoma, and leukemia.

[0041] Preferably, the tumor drug includes a DNA-PK pathway intervention drug and / or a protein degradation drug based on (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazol-5-yl)benzyl]pyrrolidine-2-carboxamide mediated by the ubiquitin-proteasome system; wherein the protein degradation drug is preferably a PROTAC degrader mediated by (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazol-5-yl)benzyl]pyrrolidine-2-carboxamide.

[0042] The present invention has the following advantages and effects compared with the prior art:

[0043] 1. The compound provided by this invention has a novel structure, can selectively bind to DNA-PK, and promotes the degradation of DNA-PK target proteins through the PROTAC mechanism mediated by (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide. At the same time, it shows good DNA-PK-related functional inhibition and anti-tumor potential in in vitro experiments.

[0044] 2. The preparation route of this invention is mature and controllable, the reaction conditions are mild, the safety and scalability are good, and the environmental friendliness is good, giving it the advantage of industrial scale-up application; the compound can be used to prepare drug compositions related to PROTAC degrading agents targeting DNA-PK, and can also be used to prepare drugs for the treatment and / or prevention of tumors. Attached Figure Description

[0045] Figure 1 This is the chemical structure of the DNA-PK PROTAC degrading agent of this invention.

[0046] Figure 2 This is a synthetic reaction route diagram of the compounds of this invention.

[0047] Figure 3 This is a graph showing the experimental results of the DNA-PK degradation activity of the synthesized compound. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0049] The chemical structure of the compound of this invention is as follows: Figure 1 As shown, the synthetic reaction route is as follows: Figure 2 As shown. According to the reaction route, the preparation of the compound includes the following steps:

[0050] Route A:

[0051] .

[0052] According to the above reaction route, the preparation of the compound specifically includes the following steps:

[0053] (1) 4-Methyl-3-nitrophenol (A) was dissolved in N,N-dimethylformamide (DMF), and 2.5 equivalents of potassium carbonate were added based on A. The mixture was stirred at 80°C for 30 minutes. Subsequently, 1.39 equivalents of compound B were added based on A, and the reaction was continued at 80°C for 30 minutes. After the reaction was completed by TLC monitoring, the reaction system was extracted with ethyl acetate, the organic phase was collected and washed with saturated brine, dried with anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure to obtain intermediate C. The specific reaction formula is as follows:

[0054] .

[0055] (2) The intermediate C obtained in step (1) was dissolved in N-N-dimethylformamide, and 3 equivalents of tetrahydroxydiboron were added under ice-water bath conditions. 0.00467 equivalents of 2,2'-bipyridine were added based on intermediate C. After the reaction was completed (approximately 5 minutes), the reaction system was extracted with ethyl acetate by TLC. The organic phase was collected, washed with saturated brine, dried with anhydrous sodium sulfate, filtered, and then desolvated under reduced pressure (to remove the solvent). The residue was purified by column chromatography to obtain intermediate D. The specific reaction formula is as follows:

[0056] .

[0057] Route B:

[0058] .

[0059] According to the above reaction route, the preparation of the compound specifically includes the following steps;

[0060] (a) Ethyl 2,4-dichloro-5-pyrimidinecarboxylate (E) was dissolved in acetonitrile. One equivalent of 4-aminotetrahydropyran hydrochloride (F) and 2.5 equivalents of potassium carbonate were added based on E. The mixture was stirred at room temperature for 10 hours, and the reaction was monitored by TLC until completion. Thin-layer chromatography was used to monitor the completeness of the reaction. After the reaction, the solvent was removed under reduced pressure, and the residue was purified by column chromatography to obtain intermediate G. The specific reaction formula is as follows:

[0061] .

[0062] (b) Dissolve intermediate G obtained in step (a) in tetrahydrofuran (THF). Weigh 2.5 equivalents of sodium hydroxide (using G as the stoichiometric standard), dissolve it in water, and add it to the reaction system. React in an oil bath at 60°C for 1 hour. Terminate the reaction after TLC shows that it is complete. Add water to the crude product and adjust the pH to soft acidity (pH≈5-6) with dilute hydrochloric acid under ice bath conditions. A precipitate forms, and a white solid, intermediate H, is obtained by filtration. The specific reaction formula is as follows:

[0063] .

[0064] (c) Dissolve intermediate H obtained in step (b) in dimethylacetamide (DMA), and add 1 equivalent of diphenyl azidophosphate and 1 equivalent of triethylamine based on H. After reacting at room temperature for 1 hour, transfer to an oil bath at 120°C and react for 12 hours. Terminate the reaction after confirming its completion by TLC, add ice water and stir rapidly to precipitate the solid. Filter to obtain a pale yellow solid intermediate I. The specific reaction formula is as follows:

[0065] .

[0066] (d) Dissolve intermediate I obtained in step (c) in anhydrous THF. Add 2.5 equivalents of sodium hydride as a stoichiometric basis under ice bath conditions. After removing the ice bath, react at room temperature for 30 minutes. Then add 2.5 equivalents of iodomethane and continue reacting at room temperature for 8 hours. Terminate the reaction when TLC shows completion. Extract the reaction system with ethyl acetate, collect the organic phase, wash with saturated brine, dry with anhydrous sodium sulfate, filter, and remove solvent under reduced pressure. Purify the residue by column chromatography to obtain intermediate J. The specific reaction formula is as follows:

[0067] .

[0068] III. The intermediate D synthesized in route A and the intermediate J synthesized in route B are further reacted, as shown in the following route:

[0069] .

[0070] According to the above reaction route, the preparation of the compound specifically includes the following steps;

[0071] (i) Intermediate J was dissolved in a 1,4-dioxane solution. 1.2 moles of intermediate D, 0.1 moles of tris(dibenzylacetone)palladium, 0.3 moles of 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene), and 2.5 moles of cesium carbonate were added according to the molar amount of intermediate J. The reaction was carried out at 105°C for 12 hours under nitrogen atmosphere. After the reaction was monitored by thin-layer chromatography, the reaction solution was extracted with dichloromethane. The organic phase was dried over saturated brine and anhydrous sodium sulfate, dissolved, and purified by column chromatography to obtain intermediate K. The specific reaction formula is as follows:

[0072] .

[0073] (ii) Dissolve intermediate K obtained in step (i) in methanol solution. Weigh 2.5 molar amounts of sodium hydroxide (calculated as the molar amount of intermediate K) and dissolve it in water. Add the sodium hydroxide to the reaction system and react at room temperature for 30 minutes. After the reaction is complete as monitored by a thin-layer chromatography plate, stop the reaction. Remove the solvent from the reaction solution by vacuum distillation to obtain the crude product. Add water to the crude product and adjust the pH to a soft acidic state (pH≈5-6) with dilute hydrochloric acid under ice bath conditions. A precipitate forms, and a white solid, i.e., intermediate L, is obtained by filtration. The specific reaction formula is as follows:

[0074] .

[0075] (iii) Take intermediate L obtained in step (ii) and add an appropriate amount of DMF as a solvent. Then, add 1.2 moles of (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide (CAS: 1448189-80-7) according to the molar amount of intermediate L. Add 1.2 moles of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), 1.5 moles of HOBT (1-hydroxybenzotriazole), and 2.5 moles of triethylamine according to the molar amount of intermediate L. Under room temperature, react for 2 hours. After the reaction is complete as monitored by thin-layer chromatography, add ice water, purge and filter to obtain the final product N. The specific reaction formula is as follows:

[0076] .

[0077] Example 1

[0078] The specific preparation steps of the compound in this embodiment are as follows:

[0079] 1) Synthesis of intermediate methyl 5-(4-methyl-3-nitrophenoxy)butyrate (C): 1.0 g of 4-methyl-3-nitrophenol (A) (6.53 mmol) was dissolved in 10 mL of N,N-dimethylformamide solution, and 2.26 g of potassium carbonate (16.33 mmol) was added. After reacting at 80 °C for 30 min, 1.64 g of methyl 4-bromobutyrate (B) (9.08 mmol) was added, and the reaction was continued at 80 °C for another 30 min. After monitoring the reaction to completion by thin-layer chromatography, the reaction solution was extracted with ethyl acetate (30 mL × 3), allowed to stand, and separated. The organic phase was washed successively with water (5 mL × 1) and saturated brine (5 mL × 3), then dried over anhydrous sodium sulfate, filtered, and the ethyl acetate was removed under reduced pressure to obtain 1.55 g of solid, with a yield of 93.9%. The obtained solid was identified by nuclear magnetic resonance (NMR), and the results showed that the solid was methyl 5-(4-methyl-3-nitrophenoxy)butyrate (C). Its structural formula is:

[0080] .

[0081] 2) Synthesis of intermediate methyl 5-(4-methyl-3-aminophenoxy)butyrate (D): 1.55 g of methyl 5-(4-methyl-3-nitrophenoxy)butyrate (C) (6.12 mmol) was added to a reaction flask and dissolved in 5 mL of DMF (NN-dimethylformamide). Under ice-water bath conditions, 1.65 g of tetrahydroxydiboron (18.36 mmol) and 4.46 mg of 2,2'-bipyridine (28.58 μmol) were added sequentially. The reaction was carried out for 5 minutes under ice-water bath conditions. The reaction solution was extracted with ethyl acetate, and the organic phase was dried over saturated brine and anhydrous sodium sulfate. After solvent removal, the solid was subjected to column chromatography with dichloromethane to obtain 1.2 g of white solid, with a yield of 87.6%. The white solid obtained by column chromatography was identified by nuclear magnetic resonance (NMR), and the results showed that the white solid was methyl 5-(4-methyl-3-aminophenoxy)butyrate (D). Its structural formula is:

[0082] .

[0083] 3) Synthesis of ethyl 2-chloro-4-((tetrahydro-2h-piperan-4-yl)amino)pyrimidine-5-carboxylate (G): 5 g (22.62 mmol) of ethyl 2,4-dichloro-5-pyrimidinecarboxylate (E) was added to a reaction flask, along with 3.11 g (22.62 mmol) of 4-aminotetrahydropyran hydrochloride (F) and 7.82 g (56.55 mmol) of potassium carbonate. The mixture was dissolved in 30 mL of acetonitrile. The reaction was allowed to proceed at room temperature for 10 hours. After the reaction was complete as monitored by a thin-layer chromatography plate, the insoluble solid was washed with 25 mL each of dichloromethane and ethanol. The liquid was removed, yielding a solid. The solid was subjected to column chromatography using 1 L of dichloromethane and 3 L of petroleum ether (volume ratio 1:3) to obtain 6 g of a white solid, with a yield of 87.6%. The white solid obtained by column chromatography was identified by nuclear magnetic resonance (NMR) as ethyl 2-chloro-4-((tetrahydro-2h-piperan-4-yl)amino)pyrimidine-5-carboxylate (G). Its structural formula is:

[0084] .

[0085] 4) Synthesis of 2-chloro-4-((tetrahydro-2h-piperan-4-yl)amino)pyrimidine-5-carboxylic acid (H): Dissolve 6 g (21 mmol) of ethyl 2-chloro-4-((tetrahydro-2h-piperan-4-yl)amino)pyrimidine-5-carboxylic acid (G) in tetrahydrofuran. Weigh 2.1 g (52.50 mmol) of sodium hydroxide and dissolve it in 5 ml of water. Add the sodium hydroxide solution to the reaction solution. Place the reaction flask in a 60°C oil bath and react for 1 hour. After the reaction is complete as monitored by a thin-layer chromatography plate, stop the reaction. Remove the solvent from the reaction solution by vacuum distillation to obtain the crude product. Add water to the crude product and adjust the pH to soft acidity (pH≈5-6) with dilute hydrochloric acid under ice bath conditions. A precipitate formed, and 4.4 g of a white solid was obtained by filtration, yielding a yield of 81%. The solid was identified by nuclear magnetic resonance (NMR) as 2-chloro-4-((tetrahydro-2h-piperan-4-yl)amino)pyrimidine-5-carboxylic acid (H). Its structural formula is:

[0086] .

[0087] 5) Synthesis of 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purine-8-one (I): Dissolve 2 g (7.76 mmol) of 2-chloro-4-((tetrahydro-2h-piperan-4-yl)amino)pyrimidine-5-carboxylic acid (H) in 2 ml of DMA, add 1.67 ml (7.76 mmol) of diphenyl azidophosphate and 1.08 ml (7.76 mmol) of triethylamine, react at room temperature for 1 hour, then react in an oil bath at 120 °C for 12 hours. After the reaction is complete as monitored by thin-layer chromatography, stop the reaction, add ice water and stir rapidly to precipitate, filter out 0.92 g of pale yellow solid, yield 46.54%. The obtained solid was identified by nuclear magnetic resonance (NMR), and the results showed that it was 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (I). Its structural formula is:

[0088] .

[0089] 6) Synthesis of 2-chloro-7-methyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (J): 920 mg (3.61 mmol) of 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (I) was dissolved in anhydrous tetrahydrofuran. Under ice bath conditions, 362 mg (9.03 mmol) of sodium hydride was added. After removing the ice, the mixture was allowed to react at room temperature for 30 min. Then, 563 μl (9.03 mmol) of iodomethane was added, and the reaction was continued at room temperature for 8 hours. The reaction was stopped after monitoring the reaction on a thin-layer chromatography plate to ensure its completeness. The reaction solution was extracted with ethyl acetate. The organic phase was dried over saturated brine and anhydrous sodium sulfate, dissolved, and purified by column chromatography to obtain 673 mg of a white solid, with a yield of 69.33%. The solid was identified by nuclear magnetic resonance (NMR) as 2-chloro-7-methyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (J). Its structural formula is:

[0090] .

[0091] 7) Synthesis of intermediate methyl 4,4-(4-methyl-3-((7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)butyrate (K): 0.4 g of 2-chloro-7-methyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one (J) (1.49 mmol) was added to a reaction flask, followed by the addition of 0.399 g of methyl 5-(4-methyl-3-aminophenoxy)butyrate (D) (1.79 mmol), 0.258 g of 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene) (0.447 mmol), and 0.73 g of cesium carbonate (3.725 mmol). The reaction mixture was prepared with 0.136 g of tris(dibenzylacetone)dipalladium (0.149 mmol) and 15 mL of 1,4-dioxane solution at 105 °C for 12 hours under nitrogen atmosphere. After the reaction was completed by monitoring with a thin-layer chromatography plate, the reaction solution was extracted with ethyl acetate (30 mL × 3), allowed to stand and separated, and the organic phase was washed successively with water (5 mL × 2) and saturated brine (5 mL × 5), then dried with anhydrous sodium sulfate, filtered, and the ethyl acetate was removed under reduced pressure to obtain a solid. The solid was subjected to column chromatography with 2 L of dichloromethane and 20 mL of methanol (v / v ratio 100:1) to obtain 0.3 g of white solid, with a yield of 44.2%. The colorless liquid obtained by column chromatography was identified by nuclear magnetic resonance (NMR). The results showed that the white solid was methyl 4,4-(4-methyl-3-((7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)butyrate (K). Its structural formula is:

[0092] .

[0093] 8) Synthesis of intermediate 4,4-(4-methyl-3-((7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)butyric acid (L): 0.3 g of methyl 4,4-(4-methyl-3-((7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)butyric acid (K) was dissolved in 5 mL of methanol in a reaction flask (0.658 mmol). 0.065 mg of NaOH (1.65 mmol) was dissolved in 2 mL of water. The NaOH aqueous solution was then slowly added dropwise to the reaction flask. The reaction was carried out at room temperature for 30 minutes. After the reaction was monitored by thin-layer chromatography, the solvent was removed by vacuum distillation to obtain the crude product. After adding water to the crude product, the pH was adjusted to a slightly acidic level (pH≈5-6) with dilute hydrochloric acid under rapid stirring. Filtering yielded 260 mg of a white solid, with a yield of 89.7%. The white solid was identified by nuclear magnetic resonance (NMR) as 4-(4-methyl-3-((7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)butyric acid (L). Its structural formula is:

[0094] .

[0095] 9) Synthetic product (2S,4R)-1-[(S)-3,3-dimethyl-2-({4-[3-methyl-4-({7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl}amino)phenoxy]butyryl}amino)butyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide: Take 0.04 g of the intermediate 4-(4-methyl-3-((7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purinyl) (L)-(S)-2-yl)amino)phenoxy)butyric acid (90.60 µmol) was added to a reaction flask, followed by the addition of 0.047 g of (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide (108.72 µmol), 0.015 g of EDCI (108.72 µmol), 0.026 g of HOBT (135.90 µmol), 0.023 g of triethylamine (226.51 µmol), and 1 mL of DMF. The reaction was allowed to proceed at room temperature for 2 hours. After the reaction was confirmed to be complete by thin-layer chromatography, ice water was added to the reaction flask, and the mixture was stirred rapidly. The mixture was then filtered to obtain 0.015 g of a white solid, with a yield of 19.4%. The white solid obtained by filtration was identified by nuclear magnetic resonance (NMR). The results showed that the white solid was (2S,4R)-1-[(S)-3,3-dimethyl-2-({4-[3-methyl-4-({7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl}amino)phenoxy]butyryl}amino)butyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide, denoted as compound (1). Its structural formula is:

[0096] .

[0097] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (1) are as follows: 11H NMR (600 MHz, DMSO) δ 8.98 (s, 1H), 8.56 (s, 1H), 8.23 (s, 1H), 8.07 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.40 (dd, J = 22.5, 7.1 Hz, 4H), 7.33 (s, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.54 (d, J = 7.1 Hz, 1H), 5.13 (s, 1H), 4.55 (d, J = 8.9 Hz, 1H), 4.47 - 4.30 (m, 4H), 4.23 (s, 1H), 4.03 - 3.85 (m, 4H), 3.65 (s, 2H), 3.45 - 3.37 (m, 2H), 3.29 (s, 3H), 2.42 (d, J = 26.3 Hz, 4H), 2.32 (d, J = 6.9 Hz, 1H), 2.16 (s, 3H), 2.03 (s, 1H), 1.91 (d, J = 6.4 Hz, 3H), 1.65 (d, J = 10.8 Hz, 2H), 0.92 (s, 9H). 13 13C NMR (151 MHz, DMSO) δ 172.03, 171.69, 169.73, 156.93, 155.67, 152.11, 151.53, 149.72, 147.80, 139.59, 139.43, 133.63, 131.24, 130.59, 129.72, 128.71, 127.50, 121.74, 116.54, 108.81, 68.96, 66.98, 66.63, 58.78, 56.48, 49.22, 41.72, 40.14, 38.01, 35.30, 31.46, 29.55, 27.15, 26.43, 25.27, 17.35, 16.02. HRMS (ESI-Q-TOF) m / z: [M+H] + calculated for C 44 H 56 N9O7S+ :854.4018, found: 854.4067. HPLC, t 1 / 2 = 12.624, purity = 96.898%.

[0098] Example 2

[0099] In the preparation of the compound in this embodiment, methyl 5-bromopentanoate was used in step 1) of compound (B), and the remaining steps were the same as in Example 1, yielding compound (2S,4R)-1-((S)-3,3-dimethyl-2-(5-(3-methyl-4-(7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)pentanoyl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxamide), 0.027 g of white solid product, with a yield of 34.56%, designated as compound (2). The structure is:

[0100] .

[0101] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (2) are as follows: 1 H NMR (600 MHz, DMSO)δ 8.97 (s, 1H), 8.56 (s, 1H), 8.23 ​​(s, 1H), 8.07 (s, 1H), 7.89 (d, J = 8.0Hz, 1H), 7.40 (d, J = 17.3 Hz, 4H), 7.33 (s, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.54 (d, J = 6.2 Hz, 1H), 5.13 (s, 1H), 4.55 (d, J = 8.2 Hz, 1H), 4.50 -4.31(m, 4H), 4.22 (d, J = 12.5 Hz, 1H), 3.92 (d, J = 15.1 Hz, 4H), 3.66 (s, 2H), 3.29 (s, 3H), 2.44 (s, 3H), 2.33 (s, 1H), 2.16 (s, 4H), 2.03 (s, 1H), 1.91 (s, 1H), 1.66 (s, 6H), 0.93 (s, 9H). 13C NMR (151 MHz, DMSO) δ 172.04, 169.81,157.05, 155.71, 152.13, 151.53, 149.75, 147.81, 139.60, 139.44, 133.63,131.27, 130.59, 129.73, 128.73, 127.52, 121.72, 116.55, 109.25, 108.78,68.98, 67.15, 66.64, 58.80, 56.43, 49.24, 41.76, 40.14, 38.04, 35.32, 34.65,29.56, 28.55, 27.15, 26.47, 22.27, 17.37, 16.03. HRMS (ESI-Q-TOF) m / z: [M+H] + Calculated for C 45 H 58 N9O7S + : 868.4174, found: 868.4149. HPLC, t 1 / 2 = 12.767, purity = 97.568%.

[0102] Example 3

[0103] In the preparation of the compound in this embodiment, methyl 6-bromohexanoate was used in step 1) of compound (B), and the remaining steps were the same as in Example 1, yielding compound (2S,4R)-1-((S)-3,3-dimethyl-2-(6-(3-methyl-4-(7-methyl-8-oxa-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)hexamido)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxamide), 0.030 g of white solid product, yield 12.5%, designated as compound (3). The structure is:

[0104] .

[0105] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (3) are as follows: 1H NMR (600 MHz, DMSO)δ 8.98 (s, 1H), 8.55 (s, 1H), 8.23 (s, 1H), 8.07 (s, 1H), 7.86 (d, J = 9.1Hz, 1H), 7.40 (dd, J = 22.4, 7.5 Hz, 4H), 7.31 (s, 1H), 7.03 (d, J = 8.1 Hz,1H), 6.53 (d, J = 7.1 Hz, 1H), 5.12 (s, 1H), 4.54 (d, J = 9.1 Hz, 1H), 4.48 -4.31 (m, 4H), 4.25 - 4.16 (m, 1H), 3.95 (d, J = 8.3 Hz, 2H), 3.89 (s, 2H),3.65 (s, 2H), 3.40 (t, J = 11.6 Hz, 2H), 3.29 (s, 3H), 2.44 (s, 3H), 2.28(dd, J = 13.7, 7.1 Hz, 1H), 2.16 (s, 4H), 2.02 (d, J = 7.6 Hz, 1H), 1.90 (s,1H), 1.75 - 1.60 (m, 4H), 1.60 - 1.49 (m, 2H), 1.44 - 1.33 (m, 2H), 0.92 (s,9H). 13 C NMR (151 MHz, DMSO) δ 172.01, 171.94, 169.70, 156.95, 155.61, 152.02,151.44, 149.65, 147.71, 139.50, 139.34, 133.53, 131.16, 130.48, 129.63,128.63, 127.41, 121.61, 116.45, 109.14, 108.69, 68.86, 67.23, 66.53, 58.68,56.28, 49.12, 41.64, 40.05, 37.94, 35.19, 34.83, 29.46, 28.59, 27.05, 26.37,25.30, 17.27, 15.93. HRMS (ESI-Q-TOF) m / z: [M+H] + calculated for C 46 H 60 N9O7S +:882.4331, found: 882.4337. HPLC, t 1 / 2 = 12.640, purity = 98.691%.

[0106] Example 4

[0107] In the preparation of the compound in this embodiment, methyl 7-bromoheptanoate was used in step 1) of compound (B), and the remaining steps were the same as in Example 1, yielding compound (2S,4R)-1-((S)-3,3-dimethyl-2-(7-(3-methyl-4-(7-methyl-8-oxa-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)heptamido)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxamide), a white solid product of 0.019 g, with a yield of 10.1%, designated as compound (4). The structure is:

[0108] .

[0109] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (4) are as follows: 1 H NMR (600 MHz, DMSO)δ 8.98 (s, 1H), 8.56 (s, 1H), 8.22 (s, 1H), 8.07 (s, 1H), 7.85 (s, 1H), 7.40(d, J = 15.7 Hz, 4H), 7.32 (s, 1H), 7.03 (s, 1H), 6.53 (s, 1H), 5.13 (s, 1H), 4.54 (d, J = 6.6 Hz, 1H), 4.39 (d, J = 44.1 Hz, 3H), 4.22 (s, 1H), 3.94 (s, 2H), 3.89 (s, 2H), 3.65 (s, 2H), 3.39 (d, J = 11.3 Hz, 2H), 3.29 (s, 3H), 2.44 (s, 3H), 2.26 (s, 1H), 2.16 (s, 3H), 2.03 (s, 1H), 1.90 (s, 1H), 1.66(s, 4H), 1.50 (d, J = 26.8 Hz, 2H), 1.39 (s, 2H), 1.29 (s, 2H), 0.92 (s, 9H). 13C NMR (151 MHz, DMSO) δ 171.91, 169.56, 156.83, 155.45, 151.88, 151.28,149.49, 147.57, 139.36, 133.39, 131.01, 130.33, 129.49, 128.48, 127.27,121.42, 116.31, 108.96, 108.52, 68.72, 67.13, 66.39, 58.54, 56.13, 48.99,41.50, 37.80, 35.05, 34.68, 29.32, 28.60, 28.32, 26.91, 26.22, 25.21, 17.12,15.79. HRMS (ESI-Q-TOF) m / z: [M+H] + Calculated for C 47 H 62 N9O7S + : 896.4487, found:896.4483. HPLC, t 1 / 2 = 13.585, purity = 96.327%.

[0110] Example 5

[0111] In the preparation of the compound in this embodiment, ethyl 6-bromo-2,2-dimethylhexanoate was used in step 1) of compound (B). The remaining steps were the same as in Example 1, yielding compound (2S,4R)-1-((S)-2-(2,2-dimethyl-6-(3-methyl-4-(7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)hexamido-3,3-dimethylbutyramide)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxamide), a white solid product of 0.021 g, with a yield of 10.7%, designated as compound (5). The structure is:

[0112] .

[0113] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (5) are as follows: 1H NMR (600 MHz, DMSO)δ 8.98 (s, 1H), 8.56 (s, 1H), 8.23 (s, 1H), 8.07 (s, 1H), 7.93 (d, J = 8.8Hz, 1H), 7.40 (dd, J = 22.5, 7.1 Hz, 4H), 7.33 (s, 1H), 7.04 (d, J = 8.0 Hz,1H), 6.54 (d, J = 7.1 Hz, 1H), 5.13 (s, 1H), 4.55 (d, J = 8.9 Hz, 1H), 4.47 -4.30 (m, 4H), 4.23 (s, 1H), 4.03 - 3.85 (m, 4H), 3.65 (s, 2H), 3.45 - 3.37(m, 2H), 3.29 (s, 3H), 2.42 (d, J = 26.3 Hz, 4H), 2.32 (d, J = 6.9 Hz, 1H),2.16 (s, 3H), 2.03 (s, 1H), 1.91 (d, J = 6.4 Hz, 3H), 1.65 (d, J = 10.8 Hz,2H), 0.92 (s, 9H). 13 C NMR (151 MHz, DMSO) δ 176.02, 171.82, 169.68, 156.93,155.62, 152.02, 151.44, 149.66, 147.74, 139.43, 139.33, 133.50, 131.13,130.47, 129.70, 128.70, 127.49, 121.67, 116.45, 109.17, 108.77, 68.83, 67.21,66.53, 58.76, 56.40, 56.26, 49.11, 41.69, 41.61, 40.17, 40.06, 37.85, 35.65,29.45, 29.41, 27.05, 26.35, 25.15, 25.09, 21.06, 17.27, 15.92. HRMS (ESI-Q-TOF) m / z: [M+H] + calculated for C 48 H 64 N9O7S + : 910.4644, found: 910.4623. HPLC,t 1 / 2= 14.095, purity = 97.131%.

[0114] Example 6

[0115] In the preparation of the compound in this embodiment, ethyl 7-bromo-2,2-dimethylheptanoate was used in step 1) of compound (B). The remaining steps were the same as in Example 1, yielding compound (2S,4R)-1-((S)-2-(2,2-dimethyl-7-(3-methyl-4-(7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)heptamido-3,3-dimethylbutyric acid amide)-4-hydroxy-N-(4-(4-methylthiazolyl)phenyl)pyrrolidine-2-carboxamide), a white solid product of 0.027 g, with a yield of 11.6%, designated as compound (6). The structure is:

[0116] .

[0117] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (6) are as follows: 1H NMR (600 MHz, DMSO)δ 8.96 (d, J = 9.7 Hz, 1H), 8.55 (t, J = 6.0 Hz, 1H), 8.23 (s, 1H), 8.06 (d,J = 8.4 Hz, 1H), 7.46 - 7.34 (m, 4H), 7.31 (d, J = 2.5 Hz, 1H), 7.03 (d, J =8.5 Hz, 1H), 6.68 (d, J = 9.1 Hz, 1H), 6.52 (dd, J = 8.3, 2.5 Hz, 1H), 5.12(d, J = 3.3 Hz, 1H), 4.53 (d, J = 9.1 Hz, 1H), 4.47 - 4.40 (m, 1H), 4.39 (dd,J = 9.7, 5.3 Hz, 1H), 4.35 (d, J = 6.3 Hz, 1H), 4.26 (dd, J = 15.7, 5.7 Hz,1H), 3.94 (dd, J = 11.1, 3.9 Hz, 2H), 3.88 (t, J = 6.4 Hz, 2H), 3.64 (dd, J =10.5, 3.9 Hz, 1H), 3.59 (d, J = 10.7 Hz, 1H), 3.45 - 3.35 (m, 2H), 3.29 (d, J= 3.7 Hz, 3H), 2.52 (d, J = 4.2 Hz, 2H), 2.43 (d, J = 11.3 Hz, 3H), 2.16 (s,3H), 2.07 - 1.98 (m, 1H), 1.90 (ddd, J = 12.9, 8.7, 4.5 Hz, 1H), 1.66 (dd, J= 22.6, 7.9 Hz, 4H), 1.49 (dd, J = 10.7, 5.8 Hz, 2H), 1.35 (dt, J = 15.0, 7.5Hz, 2H), 1.21 (dd, J = 9.5, 5.6 Hz, 3H), 1.12 - 1.05 (m, 6H), 0.92 (s, 9H). 13CNMR (151 MHz, DMSO) δ 176.03, 171.81, 169.67, 156.95, 155.60, 152.01, 151.44,149.63, 147.74, 139.43, 139.32, 133.53, 131.12, 130.48, 129.69, 128.69,127.49, 121.58, 116.44, 109.10, 108.65, 68.82, 67.24, 66.53, 58.75, 56.39,56.22, 49.12, 41.68, 41.59, HRMS (ESI-Q-TOF) m / z:[M+H] + Calculated for C 49 H 66 N9O7S + : 924.4800, found: 924.4791. HPLC, t 1 / 2 = 14.619, purity = 98.144%.

[0118] Example 7

[0119] In the preparation of the compound in this embodiment, methyl 3-(bromomethyl)benzoate was used in step 1) of compound (B). The remaining steps were the same as in Example 1, yielding compound (2S,4R)-1-((S)-3,3-dimethyl-2-(3-((3-methyl-4-(7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)methyl)aniline)butyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxamide), a white solid product of 0.018 g, with a yield of 23.04%, designated as compound (7). The structure is:

[0120] .

[0121] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (7) are as follows: 1H NMR (600 MHz, DMSO)δ 8.97 (s, 1H), 8.59 (s, 1H), 8.26 (s, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.6Hz, 1H), 7.96 (s, 1H), 7.83 (d, J = 7.0 Hz, 1H), 7.61 (d, J = 6.6 Hz, 1H),7.48 (s, 2H), 7.40 (d, J = 8.3 Hz, 4H), 7.07 (d, J = 8.0 Hz, 1H), 6.64 (d, J= 6.9 Hz, 1H), 5.14 (d, J = 31.8 Hz, 3H), 4.79 (d, J = 8.7 Hz, 1H), 4.44(ddd, J = 32.1, 18.9, 11.7 Hz, 4H), 4.30 - 4.16 (m, 1H), 3.92 (d, J = 9.1 Hz,2H), 3.74 (s, 2H), 3.29 (s, 3H), 2.44 (s, 3H), 2.18 (s, 3H), 2.05 (d, J = 7.5Hz, 1H), 1.93 (s, 1H), 1.65 (d, J = 10.3 Hz, 2H), 1.03 (s, 9H). 13 C NMR (151MHz, DMSO) δ 171.92, 169.45, 166.32, 156.59, 155.55, 152.04, 151.45, 149.66,147.74, 139.48, 139.44, 137.50, 134.20, 133.54, 131.17, 130.57, 130.49,129.68, 128.70, 128.37, 127.47, 127.07, 126.88, 122.07, 116.51, 109.30,109.06, 68.94, 68.89, 66.53, 58.84, 57.32, 56.48, 49.16, 41.71, 40.06, 37.96,35.62, 29.49, 27.08, 26.52, 17.32, 15.95. HRMS (ESI-Q-TOF) m / z: [M+H] + calculated for C 48 H 56 N9O7S +: 902.4018, found: 902.3997. HPLC, t 1 / 2 = 13.613, purity = 96.429%.

[0122] Example 8

[0123] In the preparation of the compound in this embodiment, methyl 2-(bromomethyl)benzoate was used as compound (M) in step 7). The remaining steps were the same as in Example 1, yielding compound (2S,4R)-1-((S)-3,3-dimethyl-2-(2-((3-methyl-4-((7-methyl-8-oxa-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)methyl)aniline)butyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxamide), a white solid product of 0.021 g, with a yield of 26.88%, designated as compound (8). The structure is:

[0124] .

[0125] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (8) are as follows: 1 H NMR (600 MHz, DMSO)δ 8.97 (s, 1H), 8.56 (s, 1H), 8.25 (s, 2H), 8.05 (s, 1H), 7.60 - 7.52 (m,1H), 7.46 (dd, J = 14.6, 7.2 Hz, 2H), 7.43 - 7.33 (m, 6H), 7.03 (d, J = 8.2Hz, 1H), 6.59 (d, J = 7.6 Hz, 1H), 5.21 (d, J = 12.8 Hz, 1H), 5.15 (d, J =12.5 Hz, 2H), 4.69 (d, J = 8.8 Hz, 1H), 4.48 (t, J = 7.7 Hz, 1H), 4.45 - 4.31(m, 3H), 4.24 (dd, J = 15.7, 5.0 Hz, 1H), 3.91 (t, J = 11.0 Hz, 2H), 3.73 (s,2H), 3.29 (s, 3H), 2.42 (s, 3H), 2.15 (s, 3H), 2.08 - 1.99 (m, 1H), 1.93 (d,J = 8.0 Hz, 1H), 1.65 (d,J = 10.9 Hz, 2H), 0.99 (s, 9H).13 C NMR (151 MHz, DMSO) δ 171.92, 169.46, 168.16, 156.49, 155.59, 152.04, 151.44, 149.67,147.73, 139.47, 139.40, 135.25, 133.47, 131.16, 130.56, 129.77, 129.66,128.68, 127.89, 127.79, 127.46, 127.37, 122.27, 116.50, 109.44, 109.11,68.91, 66.96, 66.51, 58.82, 57.54, 56.28, 49.14, 41.68, 40.06, 37.88, 35.20,29.47, 27.07, 26.51, 17.31, 15.92. HRMS (ESI-Q-TOF) m / z: [M+H] + Calculated for C 48 H 56 N9O7S + : 902.4018, found: 902.3997. HPLC, t 1 / 2 = 13.377, purity = 97.629%.

[0126] Example 9

[0127] In the preparation of the compound in this embodiment, methyl 5-bromofuran-2-carboxylate (CAS: 2527-99-3) was used in step 1) of compound (B). The remaining steps were the same as in Example 1, yielding compound (2S,4R)-1-((S)-3,3-dimethyl-2-(5-(3-methyl-4-(7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)furan-2-carboxamide)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxylate, a white solid product of 0.024 g, with a yield of 30.72%, designated as compound (9). The structure is:

[0128] .

[0129] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (9) are as follows: 11H NMR (600 MHz, DMSO) δ 8.97 (s, 1H), 8.59 (s, 1H), 8.45 (s, 1H), 8.08 (s, 1H), 7.67 (s, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.40 (s, 5H), 7.33 (s, 1H), 7.21 (d, J = 7.8 Hz, 1H), 6.78 (d, J = 7.2 Hz, 1H), 5.76 (s, 1H), 5.15 (s, 1H), 4.70 (d, J = 8.9 Hz, 1H), 4.49 - 4.41 (m, 2H), 4.37 (d, J = 21.1 Hz, 2H), 4.24 (d, J = 11.4 Hz, 1H), 3.93 (s, 2H), 3.67 (d, J = 10.7 Hz, 2H), 3.29 (s, 3H), 2.44 (s, 3H), 2.25 (s, 3H), 2.05 (s, 1H), 1.91 (s, 1H), 1.63 (d, J = 10.8 Hz, 2H), 0.98 (s, 9H). 13 13C NMR (151 MHz, DMSO) δ 171.80, 169.24, 158.03, 156.90, 155.16, 152.99, 152.05, 151.46, 149.68, 147.75, 140.01, 139.45, 138.67, 133.29, 131.16, 129.70, 128.71, 127.46, 126.36, 116.88, 116.63, 111.62, 89.70, 68.88, 66.51, 58.78, 56.20, 49.16, 41.69, 40.06, 37.93, 35.76, 29.42, 27.10, 26.33, 17.53, 15.94. HRMS (ESI-Q-TOF) m / z: [M+H] + calculated for C 45 H 52 N9O7S + : 878.3654, found: 878.3666. HPLC, t 1 / 2 = 13.085, purity = 97.576%.

[0130] Example 10

[0131] In the preparation of the compound in this embodiment, in step 1), compound (B) was prepared using methyl 2-(4-(bromomethyl)phenyl)acetate (CAS: 7398-42-7; methyl 4-bromomethylphenylacetate). The remaining steps were the same as in Example 1, yielding compound (2S,4R)-1-((S)-3,3-dimethyl-2-(2-(4-((3-methyl-4-(7-methyl-8-oxa-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)methyl)phenyl)propamidyl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxamide), a white solid product of 0.029 g, with a yield of 37.12%, designated as compound (10). The structure is as follows:

[0132] .

[0133] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (10) are as follows: 1 H NMR (600 MHz, DMSO)δ 8.98 (s, 1H), 8.58 (s, 1H), 8.25 (s, 1H), 8.13 (d, J = 7.9 Hz, 1H), 8.07(s, 1H), 7.40 (d, J = 14.8 Hz, 5H), 7.35 (d, J = 6.5 Hz, 2H), 7.29 (s, 2H), 7.05 (d, J = 6.6 Hz, 1H), 6.61 (s, 1H), 5.13 (s, 1H), 5.00 (s, 2H), 4.52 (d,J = 8.2 Hz, 1H), 4.44 (d, J = 8.4 Hz, 3H), 4.34 (s, 1H), 4.22 (d, J = 12.4Hz, 1H), 3.92 (s, 2H), 3.73 - 3.54 (m, 3H), 3.45 (d, J = 13.8 Hz, 2H), 3.30(s, 3H), 2.45 (s, 3H), 2.17 (s, 3H), 2.03 (s, 1H), 1.90 (s, 1H), 1.65 (d, J =9.3 Hz, 2H), 0.91 (s, 9H). 13C NMR (151 MHz, DMSO) δ 171.95, 169.93, 169.55,156.69, 155.56, 152.04, 151.46, 149.65, 147.73, 139.51, 136.19, 135.26,133.56, 131.18, 130.51, 129.66, 129.09, 128.65, 127.54, 127.44, 121.93,116.49, 109.26, 109.06, 69.08, 68.88, 66.53, 58.72, 56.50, 49.14, 41.67,41.52, 40.05, 37.94, 35.45, 29.48, 27.08, 26.33, 17.31, 15.96. HRMS (ESI-Q-TOF) m / z: [M+H] + Calculated for C 49 H 58 N9O7S + : 916.4174, found: 916.4166. HPLC,t 1 / 2 = 13.379, purity = 96.027%.

[0134] Example 11

[0135] In the preparation of the compound in this embodiment, methyl 4-(bromomethyl)benzoate was used in step 1) of compound (B). The remaining steps were the same as in Example 1, yielding the compound (2S,4R)-1-((S)-3,3-dimethyl-2-(4-((3-methyl-4-((7-methyl-8-oxa-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)methyl)aniline)butyryl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxamide), a white solid product of 0.013 g, with a yield of 16.64%, designated as compound (11). The structure is:

[0136] .

[0137] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (11) are as follows: 1H NMR (600 MHz, DMSO)δ 8.98 (s, 1H), 8.58 (s, 1H), 8.25 (s, 1H), 8.13 (d, J = 7.9 Hz, 1H), 8.07(s, 1H), 7.40 (d, J = 14.8 Hz, 5H), 7.35 (d, J = 6.5 Hz, 2H), 7.29 (s, 2H),7.05 (d, J = 6.6 Hz, 1H), 6.61 (s, 1H), 5.13 (s, 1H), 5.00 (s, 2H), 4.52 (d,J = 8.2 Hz, 1H), 4.44 (d, J = 8.4 Hz, 3H), 4.34 (s, 1H), 4.22 (d, J = 12.4Hz, 1H), 3.92 (s, 2H), 3.73 - 3.54 (m, 3H), 3.45 (d, J = 13.8 Hz, 2H), 3.30(s, 3H), 2.45 (s, 3H), 2.17 (s, 3H), 2.03 (s, 1H), 1.90 (s, 1H), 1.65 (d, J =9.3 Hz, 2H), 0.91 (s, 9H). 13 C NMR (151 MHz, DMSO) δ 171.95, 169.93, 169.55,156.69, 155.56, 152.04, 151.46, 149.65, 147.73, 139.51, 136.19, 135.26,133.56, 131.18, 130.51, 129.66, 129.09, 128.65, 127.54, 127.44, 121.93,116.49, 109.26, 109.06, 69.08, 68.88, 66.53, 58.72, 56.50, 49.14, 41.67,41.52, 40.05, 37.94, 35.45, 29.48, 27.08, 26.33, 17.31, 15.96. HRMS (ESI-Q-TOF) m / z: [M+H] + calculated for C 48 H 56 N9O7S + : 902.4018, found: 902.4059. HPLC,t 1 / 2= 12.560, purity = 97.976%.

[0138] Example 12

[0139] In the preparation of the compound in this embodiment, methyl 11-bromoundecanoate was used as compound (B) in step 1). The remaining steps were the same as in Example 1, yielding the compound (2S,4R)-1-((S)-3,3-dimethyl-2-(11-(3-methyl-4-(7-methyl-8-oxa-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)undecanoyl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxamide), a white solid product of 0.016 g, with a yield of 20.48%, designated as compound (12). The structure is:

[0140] .

[0141] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (12) are as follows: 1H NMR (600 MHz, DMSO)δ 8.97 (d, J = 6.7 Hz, 1H), 8.56 (t, J = 5.8 Hz, 1H), 8.22 (s, 1H), 8.07 (s,1H), 7.83 (d, J = 9.3 Hz, 1H), 7.40 (dd, J = 23.2, 8.0 Hz, 4H), 7.33 (d, J =1.7 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.53 (dd, J = 8.2, 2.1 Hz, 1H), 5.13(s, 1H), 4.54 (d, J = 9.3 Hz, 1H), 4.40 (ddd, J = 28.5, 16.4, 10.2 Hz, 4H),4.21 (dd, J = 15.7, 5.1 Hz, 1H), 4.02 - 3.84 (m, 4H), 3.80 (t, J = 6.5 Hz,1H), 3.70 - 3.60 (m, 2H), 3.40 (s, 3H), 3.29 (s, 3H), 2.53 (d, J = 14.0 Hz,2H), 2.44 (s, 3H), 2.30 - 2.20 (m, 1H), 2.14 (d, J = 19.8 Hz, 3H), 2.12 -2.05 (m, 1H), 2.05 - 1.99 (m, 1H), 1.96 - 1.86 (m, 1H), 1.66 (dd, J = 16.7,9.9 Hz, 4H), 1.55 - 1.41 (m, 2H), 1.36 (dd, J = 18.8, 11.3 Hz, 2H), 1.34 -1.17 (m, 11H), 0.93 (s, 9H). 13C NMR (151 MHz, DMSO) δ 172.12, 171.96, 169.73,156.99, 155.61, 152.03, 151.44, 149.63, 147.72, 139.52, 139.33, 133.56,131.17, 130.49, 129.64, 128.64, 127.43, 121.53, 116.46, 109.07, 108.64,68.88, 67.31, 66.55, 58.70, 56.35, 56.28, 49.14, 41.66, 40.06, HRMS (ESI-Q-TOF) m / z: [M+H] + Calculated for C 51 H 70 N9O7S + : 952.5113,found: 952.5120. HPLC, t 1 / 2 = 12.549, purity = 97.707%.

[0142] Example 13

[0143] In the preparation of the compound in this embodiment, methyl 6-(bromomethyl)nicotinic acid was used in step 1) of compound (B). The remaining steps were the same as in Example 1, yielding compound N-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazolyl-5-yl)phenyl)carbamoyl)pyrrolidine-1-yl)-3,3-dimethyl-1-oxobut-2-yl)-6-((3-methyl-4-((7-methyl-8-oxo-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purine-2-yl)amino)phenoxy)methyl)nicotinamide, a white solid product of 0.019 g, with a yield of 24.32%, designated as compound (13). The structure is:

[0144] .

[0145] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (13) are as follows: 1H NMR (600 MHz, DMSO)δ 8.97 (d, J = 6.5 Hz, 1H), 8.62 - 8.42 (m, 1H), 8.26 (dd, J = 25.6, 14.6 Hz,1H), 8.06 (d, J = 4.4 Hz, 1H), 7.64 - 7.46 (m, 2H), 7.41 (d, J = 9.1 Hz, 3H),7.12 - 7.03 (m, 1H), 6.83 - 6.59 (m, 1H), 6.38 - 6.14 (m, 1H), 5.63 (s, 1H),5.18 (d, J = 26.0 Hz, 2H), 4.92 - 4.72 (m, 1H), 4.44 (dt, J = 30.0, 12.3 Hz,3H), 4.24 (d, J = 10.4 Hz, 1H), 3.95 (s, 2H), 3.74 (s, 1H), 3.39 (d, J = 9.8Hz, 3H), 3.30 (s, 3H), 2.44 (s, 3H), 2.18 (s, 3H), 2.05 (dd, J = 29.1, 17.5Hz, 1H), 1.94 (d, J = 22.2 Hz, 2H), 1.66 (s, 2H), 1.14 - 0.74 (m, 9H). 13 C NMR(151 MHz, DMSO) δ 169.23, 156.18, 155.70, 155.54, 155.47, 152.03, 151.45,149.66, 148.40, 147.73, 139.46, 138.36, 136.40, 133.53, 130.61, 130.32,129.63, 128.83, 128.68, 127.46, 123.48, 122.17, 120.50, 116.55, 110.86,110.64, 108.94, 103.57, 101.96, 69.95, 68.92, 66.52, 58.85, 57.50, 49.15,49.06, 41.68, 40.06, 37.96, 37.91, 35.50, 29.47, 29.02, 27.07, 26.54, 17.36,17.28, 16.66, 16.13, 15.94. HRMS (ESI-Q-TOF) m / z: [M+H] +Calculated for C 47 H 55 N 10 O7S + : 903.5113, found: 903.4007. HPLC, t 1 / 2 = 11.993, purity = 98.777%.

[0146] Example 14

[0147] In the preparation of the compound in this embodiment, methyl 3-amino-4-methylbenzoate was used as compound (D) in step 1). The remaining steps were the same as in Example 1, yielding the compound (2S,4R)-1-((S)-3,3-dimethyl-2-(2-(3-methyl-4-(7-methyl-8-oxa-9-(tetrahydro-2H-pyran-4-yl)-8,9-dihydro-7H-purin-2-yl)amino)phenoxy)propionyl)-4-hydroxy-N-(4-(4-methylthiazolyl-5-yl)phenyl)pyrrolidine-2-carboxamide), denoted as compound (14). The structure is:

[0148] .

[0149] The proton NMR spectrum, carbon NMR spectrum, and mass spectrum of compound (14) are as follows: 1H NMR (600 MHz, DMSO)δ 8.97 (s, 1H), 8.60 (s, 1H), 8.29 (s, 1H), 8.06 (s, 1H), 7.72 (d, J = 9.1Hz, 1H), 7.39 (dd, J = 14.0, 8.0 Hz, 5H), 7.07 (d, J = 8.2 Hz, 1H), 6.59 (d,J = 7.5 Hz, 1H), 5.17 (s, 1H), 4.58 (d, J = 9.4 Hz, 1H), 4.55 (s, 2H), 4.41(ddd, J = 29.1, 18.3, 11.4 Hz, 4H), 4.25 (dd, J = 15.6, 4.7 Hz, 1H), 3.96 (d,J = 8.9 Hz, 2H), 3.64 (dd, J = 33.1, 8.8 Hz, 2H), 3.41 (d, J = 11.8 Hz, 2H),3.28 (s, 3H), 2.52 (s, 2H), 2.43 (s, 3H), 2.18 (s, 3H), 2.10 - 2.01 (m, 1H),1.90 (s, 1H), 1.65 (s, 2H), 0.91 (s, 9H). 13 C NMR (151 MHz, DMSO) δ 171.78,169.11, 167.20, 155.88, 155.55, 152.04, 151.44, 149.69, 147.74, 139.53,139.45, 133.45, 131.15, 130.58, 129.70, 128.68, 127.48, 122.76, 116.56,109.22, 109.08, 68.89, 66.80, 66.50, 58.78, 56.57, 55.99, 49.11, 41.69,40.06, 37.91, 35.82, 29.41, 27.05, 26.18, 17.32, 15.92. HRMS (ESI-Q-TOF) m / z:[M+H] + calculated for C 42 H 52 N9O7S + : 826.3705, found: 826.3701. HPLC, t 1 / 2= 11.991, purity = 98.778%.

[0150] Example 1

[0151] The effect of compounds on intracellular DNA-PK protein levels was measured using Western blotting.

[0152] Western blotting was used to detect changes in intracellular DNA-PK protein levels after compound treatment. Cells (4T1, B16-F10) were purchased from Fuheng Biotechnology Co., Ltd.; RPMI-1640 / DMEM basal medium, fetal bovine serum (FBS), and penicillin-streptomycin solution were all purchased from Fuheng Biotechnology Co., Ltd.; RIPA lysis buffer was purchased from Yaxin Biotechnology Co., Ltd.; protease inhibitors and phosphatase inhibitors were purchased from Kaiji Biotechnology Co., Ltd.; BCA protein quantification kit was purchased from Fode Biotechnology Co., Ltd.; SDS-PAGE gel reagent was prepared by Fode Biotechnology Co., Ltd.; PVDF membrane was purchased from Yaxin Biotechnology Co., Ltd.; ECL chemiluminescence reagent was purchased from Baisha Biotechnology Co., Ltd.; antibodies DNA-PKcs (PRKDC), Lamin B1, and secondary antibody HRP-anti-rabbit IgG were purchased from Huaan Biotechnology Co., Ltd.

[0153] The experimental procedure is as follows:

[0154] 1. Cell seeding and culture: After digestion and counting of logarithmic growth phase cells (4T1, B16-F10), the cells were seeded into 6-well plates, with approximately 2 × 10⁶ cells added to each well. 5 -5×10 5 Add 2 mL of complete culture medium (1640 basal medium + 10% (v / v) FBS) to each well for each cell, and set up 3 replicates for each experiment; incubate overnight at 37°C in a 5% CO2 incubator.

[0155] 2. Drug treatment: The next day, remove the old culture medium and add complete culture medium containing different concentrations of the test compounds (compounds (1)-(14) prepared in Examples 1-14); set up a blank control group (no compound added) and a positive control group (AZD-7648); continue to incubate in a 37°C incubator for a specified time.

[0156] 3. Cell Collection and Centrifugation: After incubation, use a cell scraper to completely scrape the cells from the bottom of the well, and transfer the culture medium along with the scraped cells to a centrifuge tube. Centrifuge at 4°C (2000 rpm, 5 minutes) to precipitate the cells, and discard the supernatant.

[0157] 4. PBS washing: Add pre-cooled PBS to the cell pellet to resuspend, gently pipette to mix, centrifuge again, and discard the supernatant to remove residual culture medium components.

[0158] 5. Lysis: Add RIPA lysis buffer to each sample, with an amount of lysis buffer of approximately 60 µL per well; mix thoroughly by pipetting and place on ice for lysis for 30 minutes.

[0159] 6. Ultrasonic Disruption: After lysis, the sample is ultrasonically treated to shear nucleic acids and reduce viscosity. The ultrasonic power is set to 10W, and the process is carried out in cycles of "5 seconds of sonication followed by 5 seconds of intermittent sonication". The total processing time for each sample is about 2 minutes. It is recommended to keep the sample on ice or in an ice bath throughout the ultrasonic process to avoid the temperature rise affecting protein stability.

[0160] 7. Centrifugation and supernatant collection and BCA quantification: After sonication, centrifuge at 4℃ (12000 rpm, 10 minutes) and collect the supernatant as the total protein sample; then determine the protein concentration according to the BCA protein quantification kit instructions and adjust the protein sample concentration of each group to be consistent.

[0161] 8. SDS-PAGE electrophoresis: Add the sample to the SDS-PAGE gel, set the stacking gel / separating gel concentration to 6%, and perform electrophoresis under constant voltage of 80V until the bromophenol blue front reaches the bottom of the gel.

[0162] 9. Transfer: Transfer the protein from the gel to the PVDF membrane; transfer at 100V for 150 minutes at 4℃.

[0163] 10. Blocking: After the transfer is completed, block with 5% (v / v) bovine serum albumin (BSA) at room temperature for 1 hour.

[0164] 11. Primary antibody incubation: Place the PVDF membrane in DNA-PK dilution buffer and incubate overnight at 4°C on a shaker.

[0165] 12. Washing the membrane: Wash the membrane with TBST 3 times the next day, 10 minutes each time, at room temperature on a shaker.

[0166] 13. Secondary antibody incubation: Add HRP-labeled secondary antibody (HRP-anti-rabbit IgG) and incubate at room temperature for 1 hour.

[0167] 14. Wash the membrane again: Wash the membrane 3 times with TBST, 10 minutes each time, at room temperature on a shaker to reduce background.

[0168] 15. Color development and imaging: Add ECL chemiluminescent substrate, incubate for 1-2 minutes according to the reagent instructions, and then place it in a chemiluminescence imaging system for exposure and imaging to obtain the signal of each band.

[0169] 16. Gray-scale analysis: Use ImageJ or the software built into the imaging system to perform gray-scale analysis on the bands; normalize using β-actin or Lamin B1 as internal references, calculate the relative expression levels of DNA-PKcs protein in different treatment groups, and compare them with the control group.

[0170] Experimental results:

[0171] The test results of the compound's ability to degrade DNA-PK protein in tumor cells are as follows: Figure 3 As shown, compared with the untreated control group, compounds 2, 3, 4, 5, 8, 10, 12, and 14 exhibited stronger DNA-PK protein degradation ability, while compounds 1, 6, 7, 9, 11, and 13 showed weaker degradation ability.

[0172] Example 2

[0173] Tests on the inhibition of tumor cell growth by compounds.

[0174] (1) Tests on the cytotoxicity of the compound to 4T1 cells

[0175] The cytotoxicity of the compounds was determined using the MTT assay. 4T1 cells were purchased from Fuheng Biotechnology Co., Ltd., and the MTT solution was purchased from Bidex Pharmaceutical Co., Ltd. 1640 culture medium, bovine serum albumin solution, and penicillin-streptomycin antibacterial solution were all purchased from Fuheng Biotechnology Co., Ltd. The specific testing methods are as follows:

[0176] The experimental procedure is as follows:

[0177] 1) Prepare 4T1 cell suspension: cell count; 2) Seed approximately 3000 cells per well and 100 μL of cell suspension per well in a 96-well plate; 3) Incubate overnight at 37°C; 4) Aspirate the culture medium and add the drug diluted to 25 μmol (compound 1-14) and the positive control AZD-7648 sequentially to a 98-well plate, setting up three replicates; 5) Incubate at 37°C for 48 h; 6) Add 20 μL of MTT in the dark, immersing the pipette tip in the culture medium and gently tapping the plate after adding the reagent to aid mixing; 7) Incubate at 37°C for 4 h; 8) Measure the absorbance at 490 nm and 570 nm; 9) Calculate the inhibition rate.

[0178] The positive control AZD-7648 was compared with compounds 1-14, and the results are shown in Table 1. The experimental results showed that compounds 2, 3, 4, 5, 8, 10, 12, and 14 had strong cytotoxicity against 4T1 cells. Compounds 1, 6, 7, 9, 11, and 13, however, showed poor inhibitory effects.

[0179] Table 1 Results of the compound's cytotoxicity test on 4T1 cells

[0180]

[0181] (2) Tests on the cytotoxicity of the compound to B16-F10 cells

[0182] The cytotoxicity of the compounds was determined using the MTT assay. B16-F10 cells were purchased from Fuheng Biotechnology Co., Ltd., MTT was purchased from Biological Products Co., Ltd., and B16-F10-specific culture medium was purchased from Prologis Co., Ltd. The specific testing methods are as follows:

[0183] The experimental procedure is as follows:

[0184] 1) Prepare B16-F10 cell suspension: cell counting; 2) Seed approximately 3000 cells per well and 100 μL of cell suspension per well in a 96-well plate; 3) Incubate overnight at 37°C; 4) Aspirate the culture medium and add the drug diluted to 25 μmol (compound 1-14) and the positive control AZD-7648 sequentially to a 98-well plate, setting up three replicates; 5) Incubate at 37°C for 48 h; 6) Add 20 μL of MTT in the dark, immersing the pipette tip in the culture medium and gently tapping the plate after adding the reagent to aid mixing; 7) Incubate at 37°C for 4 h; 8) Measure the absorbance at 490 nm and 570 nm; 9) Calculate the inhibition rate.

[0185] The positive control AZD-7648 was compared with the sample compounds 1-14. The results are shown in Table 1: The experimental results showed that compounds 2, 3, 4, 5, 8, 10, 12, and 14 had strong toxicity to B16-F10 cells, and their activity was comparable to that of AZD-7648. However, compounds 1, 6, 7, 9, 11, and 13 showed weak inhibitory effects.

[0186] Table 2 Results of the compound's cytotoxicity test against B16-F10 cells

[0187]

[0188] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention. For example, the weight of the relevant components mentioned in the specification of the embodiments of the present invention can not only refer to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally increased or decreased according to the specification of the embodiments of the present invention, it is within the scope disclosed in the specification of the embodiments of the present invention.

Claims

1. A compound, characterized in that: The structure is shown in Equation 1; Formula 1; In Formula 1, R is independently selected from alkyl, aryl, or heterocyclic groups.

2. The compound according to claim 1, characterized in that: The compound includes any one of the following compounds (1) to (14): 。 3. A method for preparing the compound according to claim 1 or 2, characterized in that, Includes the following steps: 1) The compound shown in Formula I is subjected to a substitution reaction with a methyl bromocarboxylic acid ester compound, followed by a reduction conversion to obtain the intermediate shown in Formula II; Formula I; Formula II; In Formula II, R is selected from any one of alkyl, aryl, and heterocyclic groups; 2) The compound shown in Formula III was subjected to nucleophilic substitution and amination with 4-aminotetrahydropyran hydrochloride, followed by hydrolysis and transformation to obtain the intermediate shown in Formula IV; Formula III; Formula IV; 3) The intermediate shown in Formula IV was reacted with diphenyl azidophosphate to construct a key heterocyclic skeleton, and then methylated with iodomethane to obtain the intermediate shown in Formula V; Formula V; 4) The intermediate shown in Formula V is coupled with the intermediate shown in Formula II, and then hydrolyzed to obtain the intermediate shown in Formula VI: Formula VI; 5) The intermediate shown in Formula VI is further condensed with (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide to obtain the target product DNA-PK PROTAC degrader, i.e., the compound.

4. The method for preparing the compound according to claim 3, characterized in that: In step 1), the bromocarboxylic acid methyl ester compounds include at least one of methyl 4-bromobutyrate, methyl 5-bromopentanoate, methyl 6-bromohexanoate, methyl 7-bromoheptanoate, ethyl 6-bromo-2,2-dimethylhexanoate, ethyl 7-bromo-2,2-dimethylheptanoate, methyl 3-(bromomethyl)benzoate, methyl 2-(bromomethyl)benzoate, methyl 5-bromofuran-2-carboxylic acid, methyl 2-(4-(bromomethyl)phenyl)acetate, methyl 4-(bromomethyl)benzoate, methyl 11-bromoundecanoate, methyl 6-(bromomethyl)nicotinic acid, and methyl 3-amino-4-methylbenzoate.

5. The method for preparing the compound according to claim 3, characterized in that: In step 1), the molar ratio of the compound represented by Formula I to the methyl bromocarboxylic acid ester compound is 1:1.39 to 1.40; In step 2), the molar ratio of the compound represented by formula III to 4-aminotetrahydropyran hydrochloride is 1:1.0 to 1.1; In step 3), the molar ratio of the compound represented by formula IV to diphenyl azidophosphate is 1:1.0 to 1.1; In step 4), the molar ratio of the compound represented by formula V to the compound represented by formula II is 1:1.1 to 1.2; In step 5), the molar ratio of the compound represented by formula VI to (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide is 1:1.1 to 1.

2.

6. The method for preparing the compound according to claim 3, characterized in that: In step 1), the reaction conditions are: reacting at 80°C for 25-30 minutes; In step 2), the reaction conditions are: stirring at room temperature for more than 10 hours; In step 3), the reaction conditions are: react at room temperature for 1 hour, then transfer to an oil bath at 120°C for 10-12 hours. In step 4), the reaction conditions are: reacting at 105°C for 12–18 hours under nitrogen atmosphere; In step 5), the reaction conditions are: reacting at room temperature for 1 to 2 hours.

7. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the compound of claim 1 or 2 or its stereoisomers, solvates, prodrugs, metabolites, pharmaceutically acceptable salts, or cocrystals.

8. Use of the compound of claim 1 or 2 or its stereoisomers, solvates, prodrugs, metabolites, pharmaceutically acceptable salts or eutectics in the preparation of medicaments for the prevention, treatment, delay and / or adjuvant treatment of tumors.

9. The application according to claim 8, characterized in that: The tumors mentioned include at least one of breast cancer, melanoma, and leukemia.

10. The application according to claim 8, characterized in that: The tumor drugs include DNA-PK pathway intervention drugs and / or protein degradation drugs mediated by (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[4-(4-methylthiazolyl-5-yl)benzyl]pyrrolidine-2-carboxamide.

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