GSPT1 degradation agent and application

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a compound capable of being used as a GSPT1 degradation agent, pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer of the compound, a pharmaceutical preparation containing the compound, the pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer of the compound, and a composition containing the compound, the pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer of the compound. And uses of the compounds, pharmaceutically acceptable salts, solvates, crystals, prodrugs or stereoisomers thereof. The compound is shown as a formula (1). # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a compound that can be used as a GSPT1 degrading agent, a pharmaceutically acceptable salt, solvate, crystal, prodrug or a stereoisomer thereof, a pharmaceutical preparation and composition containing the compound, a pharmaceutically acceptable salt, solvate, crystal, prodrug or a stereoisomer thereof, and uses of the compound, a pharmaceutically acceptable salt, solvate, crystal, prodrug or a stereoisomer thereof. Background Art

[0002] MYC is an oncogene, whose expression is often dysregulated and causes many diseases, including tumors. Currently, patients with MYC-high-expressing tumors are mainly treated with chemotherapy and some immune checkpoint inhibitors, but the clinical benefits are limited. Studies have found that MYC protein lacks typical kinase activity pockets and is difficult to be targeted by small molecules. At the same time, directly acting on MYC protein to inhibit killing can easily bring safety risks.

[0003] GSPT1 (G1 To S Phase Transition 1) is a GTP hydrolase that forms a complex with the translation termination factor eRF1 to mediate translation termination.

[0004] Studies have found that MYC-driven tumors are addicted to protein translation. MYC amplification is accompanied by high expression of related eIF4E and 4EBP1 genes, and the termination of this protein translation is extremely dependent on the translation factor GSPT1. Therefore, GSPT1-related protein degraders are developed to ultimately kill and control MYC-driven tumors by degrading GSPT1.

[0005] Molecular glues (MGs) are small molecules that can bind cooperatively at the interface of protein-protein interactions (PPIs). Typically, MGs have low affinity for protein ligands and can enhance or strengthen the interactions at the interface after binding. MGs can bind to both native PPIs and non-native or novel PPIs.

[0006] Molecular glue degraders (MGDs) are a type of MGs that bind to E3 ubiquitin ligases (E3 ligases). E3 ligases transfer ubiquitin to substrate proteins, typically marking the protein for degradation via the proteasome. MGDs cause the native or non-native protein of interest to be ubiquitinated by E3, which in turn leads to protein degradation via the proteasome.

[0007] CRBN (cereblon) is the substrate recognition subunit of the CRL4CRBN E3 ligase complex, which can ubiquitinate many other proteins, thereby initiating protein degradation. Studies have found that GSPT1 is a new substrate of Cereblon. When it is effectively degraded by GSPT1 degraders, the proliferation of related cells is effectively controlled, thereby achieving the purpose of treating MYC-high tumor-related diseases. Therefore, the development of degraders that can degrade GSPT1 to achieve the killing and control of MYC-driven tumors is of great significance for the treatment of MYC-high tumor-related diseases.

[0008] In order to achieve the above purpose, the invention patent with publication number WO2023 / 069708A1 discloses a compound of the general formula shown below, which can degrade GSPT1 and is used to treat tumor-related diseases;

[0009]

[0010] However, our company has found the following problems through research: 1) Under the above general formula, when the substitutable groups are different, there are great differences in the degradation activity of GSPT1, and the substitutable groups disclosed in the patent have general GSPT1 degradation activity; 2) When the substitutable groups of the compounds represented by the above general formula are different, their sensitivity to different tumor cells varies greatly, resulting in large differences in their efficacy in treating different types of tumor diseases. Therefore, screening for substitutable groups with good degradation activity of GSPT1 on ABC-1 cells (human lung adenocarcinoma cell line) and CAL-51 cells (human triple-negative breast cancer cell line) is of great significance for improving the degradation activity of compounds on GSPT1, improving the therapeutic effect of MYC high-expression tumor-related diseases, especially improving the therapeutic effect of lung cancer or breast cancer. Summary of the invention

[0011] The purpose of the present invention is to overcome the defects in the prior art and provide a GSPT1 degradation agent and application thereof, which greatly improves the degradation activity of GSPT1.

[0012] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0013] The compound represented by general formula (1), its pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer, is characterized in that:

[0014]

[0015] wherein L is independently selected from -O- or -NH-;

[0016] The R1 is selected from a heterocyclic group, a phenyl heterocyclic group, a 6-11 membered aryl group or a 6-13 membered heteroaryl group.

[0017] R2 is selected from the group consisting of hydrogen, hydroxy, alkyl, chloroalkyl, alkoxy, haloalkoxy, alkylacyl, cycloalkyl, heterocycloalkyl and heteroaryl.

[0018] As a further technical solution, the R1 is independently selected from

[0019] Q1, Q2, Q3, Q4, Q5 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, amino, hydroxyl, carboxyl, cyano, C1-6 alkyl, C1-6 alkoxy, halo C1-6 alkyl, C1-6 alkynyl, C1-6 cycloalkyl alkynyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxyalkoxy, amino, alkylacylamino, alkylacyl, alkylaminoacyl, 3-6 membered heterocyclyl, 3-6 membered cycloalkyl;

[0020] Ring A is selected from 5-10 membered cycloalkyl, 5-10 membered heterocyclic group, 5-10 membered heteroaromatic ring group, benzofuranyl;

[0021] Ring B is selected from 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, and 6 membered aryl;

[0022] Ring C, Ring D, and Ring E are each independently selected from a 5-10 membered heterocyclic group;

[0023] R 3 is selected from the group consisting of hydrogen, hydroxy, alkyl, chloroalkyl, alkoxy, haloalkoxy, alkylacyl, cycloalkyl, heterocycloalkyl, and heteroaryl;

[0024] wherein m is selected from an integer of 1-3, n is selected from 1 or 2, p is selected from an integer of 1-3; s is selected from an integer of 1-3; t is selected from 1 or 2, and i is selected from 1 or 2.

[0025] As a further technical solution, the R1 is independently selected from

[0026] Among them, R 3 is selected from the group consisting of hydrogen, hydroxy, alkyl, chloroalkyl, alkoxy, haloalkoxy, alkylacyl, cycloalkyl, heterocycloalkyl and heteroaryl.

[0027] A compound, a pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer thereof, having the structure shown in the following table:

[0028]

[0029]

[0030] A pharmaceutical preparation contains the compound, its pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer.

[0031] As a further technical solution, it also contains one or more pharmaceutical carriers and / or diluents; the pharmaceutical preparation is any dosage form that is clinically or pharmaceutically acceptable.

[0032] As a further technical solution, the pharmaceutical preparation can be made into an oral preparation, for example, it can be made into a conventional oral solid preparation, such as tablets, capsules, pills, granules, etc. When it is made into tablets, it can be added with suitable fillers, binders, disintegrants, lubricants, etc. The oral preparation can also be an oral liquid, and the oral liquid can also be added with additives, etc.

[0033] As a further technical solution, it contains the compound according to claim 1 or 2, its pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer, and one or more second therapeutically active agents.

[0034] As a further technical solution, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers and / or diluents.

[0035] Use of the compound, its pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer thereof, or the pharmaceutical preparation, or the pharmaceutical composition in the preparation of a drug for treating MYC high-expression tumor diseases.

[0036] As a further technical solution, the MYC high-expression tumor diseases include breast cancer, liver cancer, gastric cancer, blood tumors, lung cancer, prostate cancer, pancreatic cancer, etc.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) The compounds of the present invention, their pharmaceutically acceptable salts, solvates, crystals, prodrugs or stereoisomers thereof have excellent degradation activity for GSPT1 and can treat MYC high-expression tumor-related diseases, such as breast cancer, liver cancer, gastric cancer, blood tumors, lung cancer, prostate cancer, pancreatic cancer, etc.

[0039] (2) The compounds of the present invention, their pharmaceutically acceptable salts, solvates, crystals, prodrugs or stereoisomers thereof have high sensitivity to ABC-1 cells (human lung adenocarcinoma cell line) and CAL-51 cells (human triple-negative breast cancer cell line). Therefore, compared with other replaceable groups of L and R, the replaceable groups defined in the present invention have better therapeutic effects on lung cancer and triple-negative breast cancer cells.

[0040] (3) The compounds of the present invention, their pharmaceutically acceptable salts, solvates, crystals, prodrugs or stereoisomers thereof have good safety.

[0041] (4) The compound of the present invention has a simple preparation process, high drug purity, stable quality, and is easy to carry out large-scale industrial production. DETAILED DESCRIPTION

[0042] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0044] In the present invention, the raw materials used are all commercially available unless otherwise specified.

[0045] Preparation Example 1: Preparation of 3-(4-(3-aminoazetidin-1-yl)-2,6-difluorophenyl)piperidine-2,6-dione (Formula D), comprising:

[0046]

[0047] 1. Preparation of tert-butyl (1-(4-bromo-3,5-difluorophenyl)azetidin-3-yl)carbamate (Formula A)

[0048] 3,5-difluoro-4-bromoiodobenzene (3.0 g, 9.4 mmol), 3-N-tert-butyloxycarbonylaminocyclobutylamine hydrochloride (3.91 g, 18.8 mmol), tris(dibenzylideneacetone)dipalladium (0.86 g, 0.94 mmol), 4,5-bis(diphenylphosphino-9,9-dimethylxanthene) (0.54 g, 0.94 mmol), cesium carbonate (9.18 g, 28.2 mmol) and toluene (60 mL) were added to a 100 mL single-necked flask. The reaction solution was stirred at 110 ° C for 16 h under nitrogen protection. TLC was used to detect the reaction until it was complete. The mixture was returned to room temperature and water (100 mL) was added. The mixture was extracted with ethyl acetate (100 mL) for 3 times. The organic phases were combined, washed with water (100 mL) twice, washed with saturated brine (100 mL) twice, dried and concentrated under reduced pressure to obtain the target product.

[0049] ESI-MS m / z of the target product: 362.8[M+H] + .

[0050] 2. Preparation of tert-butyl (1-(4-(2,6-bis(benzyloxy)pyridin-3-yl)-3,5-difluorophenyl)azetidin-3-yl)carbamate (Formula B)

[0051] To a 50 mL single-necked flask, tert-butyl (1-(4-bromo-3,5-difluorophenyl)azetidin-3-yl)carbamate (930 mg, 2.56 mmol), 2,6-di(benzyloxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine (1.3 g, 3.07 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (187 mg, 0.26 mmol), potassium carbonate (1.1 g, 7.68 mmol) and 1,4-dioxane / water (9 mL / 3 mL) were added. The reaction solution was stirred at 100°C for 8 h under nitrogen protection. TLC detected that the reaction of the raw material was complete. The mixture was cooled to room temperature and water (50 mL) was added. The mixture was extracted with ethyl acetate (50 mL) three times. The organic phases were combined, washed twice with water (50 mL) and twice with saturated brine (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to purify the target product.

[0052] ESI-MS m / z of the target product: 573.7[M+H] + .

[0053] 3. Preparation of tert-butyl (1-(4-(2,6-dioxopiperidin-3-yl)-3,5-difluorophenyl)azetidin-3-ylcarbamate (Formula C)

[0054] To a 50mL single-necked flask, add tert-butyl (1-(4-(2,6-bis(benzyloxy)pyridin-3-yl)-3,5-difluorophenyl)azetidin-3-yl)carbamate (870mg, 1.52mmol), palladium carbon (435mg, 50% water content), palladium hydroxide carbon (435mg), N,N-dimethylformamide (5mL) and isopropanol (5mL), replace with hydrogen 3 times, and stir the reaction solution at 25°C for 16h. The reaction is complete. The reaction solution is filtered, concentrated, and purified to obtain the target product.

[0055] ESI-MS m / z of the target product: 396.2[M+H] + .

[0056] 4. Preparation of 3-(4-(3-aminoazetidine-1-yl)-2,6-difluorophenyl)piperidine-2,6-dione (Formula D)

[0057] To a 25 mL single-necked bottle, add tert-butyl (1-(4-(2,6-dioxopiperidin-3-yl)-3,5-difluorophenyl)azetidin-3-ylcarbamate (118 mg, 0.3 mmol), add dichloromethane (2 mL), and dropwise add trifluoroacetic acid (681 mg, 6.0 mmol) under stirring. The reaction solution is stirred at 25 ° C for 1 h until the reaction is complete. The reaction solution is concentrated to obtain the target product.

[0058] ESI-MS m / z of the target product: 296.3[M+H] + .

[0059] Preparation Example 2: Preparation of 1-(5-(cyclopropylethynyl)-6-(1,1-difluoroethyl)pyridin-3-yl)-3-(1-(4-(2,6-dioxopiperidin-3-yl)-3,5-difluorophenyl)azetidin-3-yl)urea (Compound 1), comprising:

[0060]

[0061] 1. Preparation of 3-bromo-2-(1-ethoxyvinyl)-5-nitropyridine (Formula 1-1)

[0062] Add 2,3-dibromo-5-nitropyridine (5.0 g, 17.7 mmol) to a 500 mL single-necked flask, add dioxane (100 mL) to dissolve, add cesium fluoride (5.4 g, 35.5 mmol), tetrakistriphenylphosphine palladium (1.02 g, 8.9 mmol), and stir the reaction solution at 110 ° C for 2.5 h under argon protection. TLC detection shows that the reaction is complete. Add water (100 mL), extract with ethyl acetate (100 mL) 3 times, combine the organic phases, wash with water (100 mL), wash with saturated brine (100 mL), dry the organic phase, concentrate under reduced pressure, and purify to obtain the target product.

[0063] ESI-MS m / z of the target product: 273.1[M+H] + .

[0064] 2. Preparation of 1-(3-bromo-5-nitropyridin-2-yl)ethane-1-one (Formula 1-2)

[0065] Add 3-bromo-2-(1-ethoxyvinyl)-5-nitropyridine (1.30 g, 4.76 mmol) to a 100 mL single-necked flask, add dioxane (20 mL) and 2M hydrochloric acid (20 mL), and stir at room temperature for 16 h. TLC detection shows that the reaction is complete. Add saturated sodium bicarbonate solution to adjust to alkalinity, extract with ethyl acetate (50 mL) 3 times, combine the organic phases, wash twice with water (50 mL), wash with saturated brine (100 mL), dry the organic phase, and concentrate under reduced pressure to obtain the target product.

[0066] ESI-MS m / z of the target product: 245.0 [M+H] + .

[0067] 3. Preparation of 3-bromo-2-(1,1-difluoroethyl)-5-nitropyridine (Formula 1-3)

[0068] Add 1-(3-bromo-5-nitropyridin-2-yl)ethane-1-one (1.0 g, 4.08 mmol), bis(2-methoxyethyl)aminosulfur trifluoride (4.5 g, 20.4 mmol) and 2 drops of methanol to a 25 mL fluorination bottle, and stir the reaction solution at 80 ° C for 16 h. TLC detection shows that the reaction is complete. Add water (50 mL) to the reaction solution, extract 3 times with ethyl acetate (50 mL), combine the organic phases, wash twice with water (50 mL), wash twice with saturated brine (100 mL), dry the organic phase, concentrate under reduced pressure, and purify to obtain the target product.

[0069] ESI-MS m / z of the target product: 267.0 [M+H] + .

[0070] 4. Preparation of 5-bromo-6-(1,1-difluoroethyl)pyridin-3-amine (Formula 1-4)

[0071] Add 3-bromo-2-(1,1-difluoroethyl)-5-nitropyridine (0.7 g, 2.62 mmol) to a 50 mL single-mouth bottle, dissolve in methanol (12 mL), add Ni (350 mg, 5.96 mmol), replace with hydrogen 3 times, and stir at room temperature for 16 h. TLC detection shows that the reaction is complete. Filter to remove nickel, and concentrate under reduced pressure to obtain the target product.

[0072] ESI-MS m / z of the target product: 237.2[M+H] + .

[0073] 5. Preparation of 5-(cyclopropylethynyl)-6-(1,1-difluoroethyl)pyridin-3-amine (Formula 1-5)

[0074] Add 5-bromo-6-(1,1-difluoroethyl)pyridin-3-amine (283 mg, 1.19 mmol), ethynylcyclopropane (237 mg, 3.6 mmol), cuprous iodide (46 mg, 0.24 mmol), bistriphenylphosphine palladium dichloride (84 mg, 0.12 mmol) and N,N-diisopropylethylamine (3 mL) to the sealed tube, replace the argon three times, and react at 90 ° C for 16 hours. TLC detection shows that the raw material has reacted completely. The reaction solution is added to water (20 mL), extracted with ethyl acetate (20 mL) three times, the organic phases are combined, washed twice with water (20 mL), washed twice with saturated brine (50 mL), and the organic phase is dried and concentrated under reduced pressure to purify the target product.

[0075] ESI-MS m / z of the target product: 223.2[M+H] + .

[0076] 6. Preparation of phenyl (5- (cyclopropylethynyl) -6- (1,1-difluoroethyl) pyridin-3-yl) carbamate (Formula 1-6)

[0077] Add 5-(cyclopropylethynyl)-6-(1,1-difluoroethyl)pyridin-3-amine (161 mg, 0.72 mmol) and pyridine (3 mL) to a 25 mL three-necked flask, protect with nitrogen, cool to 0 ° C, add 4-dimethylaminopyridine (88.5 mg, 0.72 mmol) and phenyl chloroformate (136 mg, 0.87 mmol). React at room temperature for 2.5 h. TLC detection shows that the reaction is complete. The reaction solution is added to water (10 mL), extracted with ethyl acetate (10 mL) 3 times, the organic phases are combined, washed twice with water (10 mL), washed twice with saturated brine (20 mL), and the organic phase is dried and concentrated under reduced pressure to purify the target product.

[0078] ESI-MS m / z of the target product: 343.2[M+H] + .

[0079] 7. Preparation of 1-(5-(cyclopropylethynyl)-6-(1,1-difluoroethyl)pyridin-3-yl)-3-(1-(4-(2,6-dioxopiperidin-3-yl)-3,5-difluorophenyl)azetidin-3-yl)urea (Compound 1)

[0080] Phenyl (5-(cyclopropylethynyl)-6-(1,1-difluoroethyl)pyridin-3-yl)carbamate (85 mg, 0.25 mmol) was added to a 50 mL single-necked flask, and dioxane (2 mL) was dissolved, and 3-(4-(3-aminoazetidin-1-yl)-2,6-difluorophenyl)piperidine-2,6-dione (Formula D) (80.6 mg, 0.27 mmol) and N,N-diisopropylethylamine (96 mg, 0.75 mmol) prepared in Preparation Example 1 were added. The mixture was stirred at 100°C for 16 h under nitrogen protection until the reaction was complete. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the target product directly.

[0081] ESI-MS m / z of the target product: 543.7[M+H] + ;

[0082] 1 H NMR (400MHz, DMSO-d6) δ10.88(s,1H),9.10(s,1H),8.44(d,J=2.4Hz,1H),8.07(d,J=2 .4Hz,1H),7.23(d,J=7.2Hz,1H),6.19(dd,J=12.0,4.0Hz,2H),4.63-4.50(m,1H),4.14 (t,J=8.0Hz,2H),4.10-4.00(m,1H),3.69(t,J=8.0Hz,2H),2.80-2.68(m,1H),2.48-2. 40(m,1H),2.20-2.18(m,5H),1.60-1.50(m,1H),0.90-0.80(m,2H),0.75-0.65(m,2H).

[0083] Preparation Example 3: Preparation of 1-(1-(4-(2,6-dioxopiperidin-3-yl)-3,5-difluorophenyl)azetidin-3-yl)-3-(4-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-6-yl)urea (Compound 2), comprising:

[0084]

[0085] 1. Preparation of 4-bromo-2-fluoro-6-methoxybenzaldehyde (Formula 2-1)

[0086] 4-Bromo-2-fluoro-6-hydroxybenzaldehyde (25g, 114.2mmol), iodomethane (17.8g, 125.6mmol), potassium carbonate (18.9g, 137.0mmol), N,N-dimethylformamide (200mL) were added to a 500mL single-necked flask. The reaction solution was stirred at 20°C for 16h under nitrogen protection. TLC detected that the reaction was complete. Water (200mL) was added, and ethyl acetate (200mL) was extracted three times. The organic phases were combined, washed twice with water (150mL), washed with saturated brine (100mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography to obtain the target product.

[0087] ESI-MS m / z of target product: 500.5[M+H] + .

[0088] 2. Preparation of (4-bromo-2-fluoro-6-methoxyphenyl)methanol (Formula 2-2)

[0089] 4-Bromo-2-fluoro-6-methoxybenzaldehyde (25.4 g, 109.0 mmol), 200 mL of ethanol and 100 mL of tetrahydrofuran were added to a 500 mL single-necked flask, and sodium borohydride (6.2 g, 163.5 mol) was added in batches. The mixture was stirred at room temperature under nitrogen protection and reacted overnight. After the reaction was complete, water was added to quench the mixture, and the mixture was extracted with ethyl acetate three times. The organic phases were combined, washed with saturated sodium chloride water, dried over anhydrous sodium sulfate, and purified by a concentration chromatography column to obtain the target product.

[0090] ESI-MS m / z of the target product: 235.1[M+H] + .

[0091] 3. Preparation of 4-bromo-2-fluoro-6-methoxybenzyl methanesulfonate (Formula 2-3)

[0092] Add (4-bromo-2-fluoro-6-methoxyphenyl)methanol (24.6 g, 104.7 mmol), triethylamine (31.8 g, 314.3 mol) and 250 mL of anhydrous dichloromethane to a 500 mL single-necked flask, add methylsulfonyl chloride (28.8 g, 251.4 mmol) dropwise at 0°C, and react at room temperature for 12 hours. After the reaction is complete, add 200 mL of water to the reaction solution, extract three times with ethyl acetate, combine the organic phases, wash once with saturated sodium chloride, and dry with anhydrous sodium sulfate to obtain the target product.

[0093] ESI-MS m / z of the target product: 313.1[M+H] + .

[0094] 4. Preparation of 2-(4-bromo-2-fluoro-6-methoxyphenyl)acetonitrile (Formula 2-4)

[0095] 4-Bromo-2-fluoro-6-methoxybenzyl methanesulfonate (28.8 g, 91.97 mmol), trimethylsilyl cyanide (10.9 g, 110.4 mmol), and 300 mL of acetonitrile were added to a 1000 mL single-necked flask, and tetrabutylammonium fluoride (28.8 g, 110.4 mmol) was added dropwise at 0°C, and stirred at room temperature for 16 hours. The reaction was complete, and the reaction solution was concentrated and purified by chromatography to obtain the target product.

[0096] ESI-MS m / z of the target product: 244.1[M+H] + .

[0097] 5. Preparation of 2-(4-bromo-2-fluoro-6-methoxyphenyl)acetic acid (Formula 2-5)

[0098] Add 2-(4-bromo-2-fluoro-6-methoxyphenyl)acetonitrile (20.6 g, 84.4 mmol), 100 mL of ethanol, and 100 mL of 10% sodium hydroxide solution to a 500 mL single-necked flask, and stir the reaction solution at 78°C for 1 h until the raw material disappears. Adjust the pH of the reaction solution to 2, and filter the filter cake to obtain the target product.

[0099] ESI-MS m / z of the target product: 263.1[M+H] + .

[0100] 6. Preparation of 2-(4-bromo-2-fluoro-6-hydroxyphenyl)acetic acid (Formula 2-6)

[0101] Add 100 mL of dichloromethane and 240 mL of boron tribromide to a 500 mL single-necked flask, add 2-(4-bromo-2-fluoro-6-methoxyphenyl)acetic acid (19 g, 72.55 mmol) to the reaction solution at -70 °C, and stir for 2 h. The reaction is complete. Add water to quench, extract three times with ethyl acetate, combine the organic phases, wash once with saturated sodium chloride, dry with anhydrous sodium sulfate, and purify with a concentration column to obtain the target product.

[0102] ESI-MS m / z of the target product: 249.1[M+H] + .

[0103] 7. Preparation of 6-bromo-4-fluorobenzofuran-2(3H)-one (Formula 2-7)

[0104] To a 500mL single-necked flask, add 2-(4-bromo-2-fluoro-6-hydroxyphenyl)acetic acid (9.5g, 38.15mmol), p-toluenesulfonic acid (13.14g, 76.30mmol) and 200mL of toluene. Stir at 110°C for 12h. When the reaction is complete, concentrate and purify by chromatography to obtain the target product.

[0105] ESI-MS m / z of the target product: 231.0 [M+H] + .

[0106] 8. Preparation of 6-bromo-4-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-2-one (Formula 2-8)

[0107] To a 250mL single-necked flask, add 6-bromo-4-fluorobenzofuran-2(3H)-one (2g, 8.66mmol), (2-bromoethyl)diphenylsulfonium trifluoromethanesulfonate (5.8g, 12.98mmol), N,N-dimethylformamide (100ml), and triethylamine (2.6g, 25.97mmol). React at room temperature for 30min. When the reaction is complete, extract three times with ethyl acetate, combine the organic phases, wash once with saturated sodium chloride, dry with anhydrous sodium sulfate, and concentrate to obtain the target product.

[0108] ESI-MS m / z of the target product: 257.1[M+H] + .

[0109] 9. Preparation of 5-bromo-3-fluoro-2-(1-(hydroxymethyl)cyclopropyl)phenol (Formula 2-9)

[0110] Add 6-bromo-4-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-2-one (1.5 g, 5.84 mmol) and 100 mL of tetrahydrofuran into a 250 mL single-necked flask, add lithium aluminum hydride (0.44 g, 11.67 mol) at 0°C, react at room temperature for 14 h, and after the reaction is complete, filter and concentrate to obtain the target product.

[0111] ESI-MS m / z of the target product: 261.1[M+H] + .

[0112] 10. Preparation of 6-bromo-4-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane] (Formula 2-10)

[0113] To a 50 mL single-necked flask, add 5-bromo-3-fluoro-2-(1-(hydroxymethyl)cyclopropyl)phenol (1.2 g, 4.62 mmol), 8 ml of dimethyl carbonate, and potassium tert-butoxide (1.0 g, 9.92 mmol). Under nitrogen protection, react at 85 °C for 1 h until the raw material disappears. Concentrate and purify directly by chromatography to obtain the target product.

[0114] ESI-MS m / z of the target product: 243.1[M+H] + .

[0115] 11. Preparation of 4-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-6-amine (Formula 2-11)

[0116] 6-Bromo-4-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane] (600 mg, 2.45 mmol), benzophenone imine (449.4 mg, 2.45 mmol), 2-(dicyclohexylphosphine)-3,6-dimethoxy-2'-4'-6'-tri-1-propyl-11'-biphenyl (532.6 mg, 1 mmol), tris(dibenzylideneacetone)dipalladium 3 (453 mg, 2.45 mmol) were added to a 100 mL single-necked flask. .7mg, 0.5mmol), sodium tert-butoxide (285.6mg, 3mol), anhydrous tetrahydrofuran (20mL), react at 70℃ under nitrogen protection for 12h, the reaction is complete, filtered, the filtrate is concentrated, 2mol / L dilute hydrochloric acid (5mL) and tetrahydrofuran (5mL) are added and mixed and stirred for 5min, the reaction is complete, the pH is adjusted to 7-9 at 0℃, extracted with ethyl acetate three times, the organic phases are combined, and the target product is purified by concentration column chromatography.

[0117] ESI-MS m / z of the target product: 179.2[M+H] + .

[0118] 12. Preparation of phenyl (4-fluoro-2H-spiro [benzofuran-3,1'-cyclopropane] -6-yl) carbamate (Formula 2-12)

[0119] To a 100 mL single-necked flask, add 4-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-6-amine (310 mg, 1.73 mmol), phenyl chloroformate (406.3 mg, 2.60 mmol), pyridine (410.5 mg, 5.19 mmol), and dissolve in anhydrous N,N-dimethylformamide (10 mL). React at 0°C under nitrogen protection for 12 h. After the reaction is complete, add water (30 mL), extract three times with ethyl acetate, combine the organic phases, wash once with saturated sodium chloride, dry over anhydrous sodium sulfate, and purify by column chromatography to obtain the target product.

[0120] ESI-MS m / z of the target product: 300.1[M+H] + .

[0121] 13. Preparation of 1-(1-(4-(2,6-dioxopiperidin-3-yl)-3,5-difluorophenyl)azetidin-3-yl)-3-(4-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-6-yl)urea (Compound 2)

[0122] To a 250 mL single-necked flask was added phenyl(4-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-6-yl)carbamate (200 mg, 0.67 mmol), the target product of Preparation Example 1 3-(4-(3-aminoazetidine-1-yl)-2,6-difluorophenyl)piperidine-2,6-dione (Formula D) (310 mg, 1.05 mmol), dissolved in 10 mL of 1,4-dioxane, N,N-diisopropylethylamine (519 mg, 4.02 mmol), and reacted at 100 ° C for 3 h. The reaction was complete, and the temperature was restored to room temperature. The reaction solution was concentrated and purified by column chromatography to obtain the target product.

[0123] ESI-MS m / z of the target product: 501.2[M+H] + ;

[0124] 1 H NMR (400MHz, DMSO-d6) δ10.86 (s, 1H), 8.42 (s, 1H), 7.11 (dd, J = 13.1, 2.0Hz, 1H), 6.80 (d, J=7.3Hz,1H),6.60(d,J=2.0Hz,1H),6.24–6.14(m,2H),4.58–4.53(m,1H),4.52(s,2H),4 .12(t,J=7.6Hz,2H),4.04(dd,J=12.6,5.2Hz,1H),3.64(ddd,J=7.7,5.6,1.8Hz,2H),2.7 9(ddd,J=17.1,13.4,5.6Hz,1H),2.15–1.87(m,3H),1.14–1.08(m,2H),1.04–0.97(m,2H).

[0125] Preparation Example 4: Preparation of 1-(1-(4-(2,6-dioxopiperidin-3-yl)-3,5-difluorophenyl)azetidin-3-yl)-3-(5-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-6-yl)urea (Compound 3)

[0126]

[0127] 1. Preparation of (4-bromo-5-fluoro-2-methoxyphenyl)methanol (Formula 3-1)

[0128] Add methyl 4-bromo-5-fluoro-2-methoxybenzoate (20.0 g, 76.04 mmol) and a mixed solution (110 ml) of methanol and anhydrous tetrahydrofuran in a ratio of 1:10 to a 1000 mL single-necked flask, cool the system to 0°C, slowly add sodium borohydride (14.7 g, 387.8 mmol) in batches, replace with nitrogen three times after addition, and stir at room temperature for 16 hours. TLC detection shows that the reaction is complete, slowly add saturated ammonium chloride aqueous solution at 0°C to quench, concentrate under reduced pressure, extract the residual liquid three times with ethyl acetate, combine the organic phases, wash with saturated sodium chloride water, dry with anhydrous sodium sulfate, and concentrate to obtain the target product.

[0129] ESI-MS m / z of the target product: 235.1[M+H] + .

[0130] 2. Preparation of 1-bromo-4-(bromomethyl)-2-fluoro-5-methoxybenzene (Formula 3-2)

[0131] Add (4-bromo-5-fluoro-2-methoxyphenyl)methanol (16.8 g, 71.5 mmol) and dichloromethane (200 mL) to a 1000 mL single-necked flask, cool the system to 0°C, slowly add phosphorus tribromide (23.2 g, 103.1 mmol), and stir at room temperature for 2 h. TLC detection shows that the reaction is complete. The reaction solution is slowly quenched with water at 0°C, extracted three times with dichloromethane (200 mL), the organic phases are combined, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the target product.

[0132] ESI-MS m / z of the target product: 297.2[M+H] + .

[0133] 3. Preparation of 2-(4-bromo-5-fluoro-2-methoxyphenyl)acetonitrile (Formula 3-3)

[0134] 1-Bromo-4-(bromomethyl)-2-fluoro-5-methoxybenzene (16.9 g, 56.9 mmol), trimethylsilyl cyanide (6.77 g, 68.2 mmol), tetrabutylammonium fluoride (17.85 g, 68.3 mmol) and 200 ml of acetonitrile were added to a 500 mL single-necked flask, and the reaction solution was stirred at room temperature for 4 h. TLC detected that the reaction was complete. The reaction solution was slowly quenched with water at 0 ° C, then concentrated to remove acetonitrile, extracted with ethyl acetate (200 mL), combined organic phases, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the target product.

[0135] ESI-MS m / z of the target product: 244.1[M+H] + .

[0136] 4. Preparation of 2-(4-bromo-5-fluoro-2-methoxyphenyl)acetic acid (Formula 3-4)

[0137] Add 2-(4-bromo-5-fluoro-2-methoxyphenyl)acetonitrile (13.6 g, 45.8 mmol), 10% sodium hydroxide solution (50 mL), and 50 mL ethanol to a 500 mL single-necked flask. Stir and react at 80 ° C for 2 h under nitrogen protection. TLC detection shows that the reaction is complete. Concentrate the reaction solution, add 100 mL of dichloromethane for extraction, and discard. Adjust the acidity of the aqueous phase to PH 3-4, add 200 mL of ethyl acetate for extraction three times, combine the organic phases, dry over anhydrous sodium sulfate, and concentrate to obtain the target product.

[0138] ESI-MS m / z of the target product: 263.1[M+H] + .

[0139] 5. Preparation of 2-(4-bromo-5-fluoro-2-hydroxyphenyl)acetic acid (Formula 3-5)

[0140] Add dichloromethane (100 mL) to a 500 mL single-necked flask, slowly add boron tribromide (100 mL) at 0°C, cool the system to -78°C after addition, then add 2-(4-bromo-5-fluoro-2-methoxyphenyl)acetic acid (10.4 g, 39.7 mmol), stir the reaction solution at -78°C for 24 h, and the reaction is complete when detected by TLC. Slowly add 50 mL of water at -78°C to quench, extract three times with 200 mL of ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and purify by column chromatography to obtain the target product.

[0141] ESI-MS m / z of the target product: 249.1[M+H] + .

[0142] 6. Preparation of 6-bromo-5-fluorobenzofuran-2(3H)-one (Formula 3-6)

[0143] 2-(4-bromo-5-fluoro-2-hydroxyphenyl)acetic acid (3.0 g, 12 mmol), 4-toluenesulfonic acid (6.2 g, 36 mmol) and anhydrous toluene (30 ml) were added to a 500 mL single-necked flask. The reaction solution was stirred at 110 °C for 2 h. The reaction was complete after TLC detection. The product was concentrated and purified by chromatography column to obtain the target product.

[0144] ESI-MS m / z of the target product: 231.0 [M+H] + 。

[0145] 7. Preparation of 6-bromo-5-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-2-one (Formula 3-7)

[0146] To a 250 mL single-necked flask, 6-bromo-5-fluorobenzofuran-2(3H)-one (1.8 g, 7.8 mmol), (2-bromoethyl)diphenylsulfonium trifluoromethanesulfonate (5.18 g, 11.7 mmol), and anhydrous N,N-dimethylformamide (20 mL) were added. Triethylamine (2.36 g, 23.4 mmol) was added under stirring at room temperature. The mixture was reacted for 1 h at room temperature. TLC showed that the reaction was complete. Water (30 mL) was added, and the mixture was extracted three times with 150 mL of ethyl acetate. The organic phases were combined, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the target product.

[0147] ESI-MS m / z of the target product: 257.1[M+H] + .

[0148] 8. Preparation of 5-bromo-4-fluoro-2-(1-(hydroxymethyl)cyclopropyl)phenol (Formula 3-8)

[0149] To a 100 mL single-necked flask, add 6-bromo-5-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-2-one (1.3 g, 5 mmol), add lithium aluminum tetrahydride (286 mg, 10 mmol) and anhydrous tetrahydrofuran (20 mL) at 0°C, react at room temperature for 24 h, and filter after completion. Wash the filter cake with anhydrous tetrahydrofuran, concentrate the organic phase, and purify by column chromatography to obtain the target product.

[0150] ESI-MS m / z of the target product: 261.1[M+H] + .

[0151] 9. Preparation of 6-bromo-5-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane] (Formula 3-9)

[0152] To a 100 mL single-necked flask, add 5-bromo-4-fluoro-2-(1-(hydroxymethyl)cyclopropyl)phenol (850 mg, 3.3 mmol), potassium tert-butoxide (733.6 mg, 6.54 mmol), and dimethyl carbonate (10 ml). React at 85 °C under nitrogen protection for 24 h. After the reaction is complete, concentrate the reaction solution under reduced pressure and purify by column chromatography to obtain the target product.

[0153] ESI-MS m / z of the target product: 243.1[M+H] + .

[0154] 10. Preparation of 5-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-6-amine (Formula 3-10)

[0155] To a 100 mL single-necked flask were added 6-bromo-5-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane (600 mg, 2.45 mmol), benzophenone imine (449.4 mg, 2.45 mmol), 2-(dicyclohexylphosphine)-3,6-dimethoxy-2'-4'-6'-tri-1-propyl-11'-biphenyl (532.6 mg, 1 mmol), tris(dibenzylideneacetone)dipalladium (453.7 mg, 0 .5mmol), sodium tert-butoxide (285.6mg, 3mmol), anhydrous tetrahydrofuran (20mL) was dissolved, and the reaction was carried out at 70°C for 24h under argon protection. After the reaction was complete, the filtrate was filtered and concentrated. 2mol / L dilute hydrochloric acid (5mL) and tetrahydrofuran (5mL) were added and mixed and stirred for 5min. After the reaction was complete, the base was adjusted to pH 7-9 at 0°C, and extracted three times with 200mL of ethyl acetate. The organic phases were combined and concentrated and purified by column chromatography to obtain the target product.

[0156] ESI-MS m / z of the target product: 180.2[M+H] + .

[0157] 11. Preparation of phenyl (5-fluoro-2H-spiro [benzofuran-3,1'-cyclopropane] -6-yl) carbamate (Formula 3-11)

[0158] To a 100 mL single-necked flask, 5-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-6-amine (345 mg, 1.75 mmol), phenyl chloroformate (411.3 mg, 2.63 mmol), pyridine (457.4 mg, 5.78 mmol), and anhydrous N,N-dimethylformamide (10 mL) were added. The mixture was protected by nitrogen and stirred at 0°C for 24 h. After the reaction was complete, water (30 mL) was added, and the mixture was extracted with 100 mL of ethyl acetate, washed once with saturated sodium chloride, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain the target product.

[0159] ESI-MS m / z of the target product: 300.1[M+H] + .

[0160] 12. Preparation of 1-(1-(4-(2,6-dioxopiperidin-3-yl)-3,5-difluorophenyl)azetidin-3-yl)-3-(5-fluoro-2H-spiro[benzofuran-3,1'-cyclopropane]-6-yl)urea (Compound 3)

[0161] Add phenyl (5-fluoro-2H-spiro [benzofuran-3,1'-cyclopropane] -6-yl) carbamate (150 mg, 0.5 mmol) to a 250 mL single-necked flask, the product of Preparation Example 1 3- (4- (3-aminoazetidin-1-yl) -2,6-difluorophenyl) piperidine-2,6-dione (Formula D) (204.4 mg, 0.5 mmol), 1,4-dioxane 10 mL dissolved, N, N-diisopropylethylamine (193.9 mg, 1.5 mmol), react at 100 ° C for 3 hours, the reaction is complete, return to room temperature, concentrate the reaction solution, and purify by column chromatography to obtain the target product. ESI-MS m / z of the target product: 501.2 [M + H] + .

[0162] 1 H NMR (400MHz, DMSO-d6) δ10.86 (s, 1H), 8.19 (d, J = 2.7Hz, 1H), 7.49 (d, J = 6.4Hz, 1H), 7.24(d,J=7.2Hz,1H),6.72(d,J=10.6Hz,1H),6.22–6.16(m,2H),4.58–4.50(m,1H), 4.44(s,2H),4.14(t,J=7.7Hz,2H),4.05(dd,J=12.6,5.2Hz,1H),3.62(ddd,J=7.5,5 .4,1.5Hz,2H),2.79(ddd,J=17.8,13.3,5.5Hz,1H),2.16–1.90(m,3H),1.01(s,4H).

[0163] Application Example 1: In vitro test of cell activity and GSPT1 degradation activity of the compound of the present invention

[0164] 1. Experimental Materials

[0165] Test compounds: Compound 1 prepared in Preparation Example 2 of the present invention, Compound 2 prepared in Preparation Example 3, Compound 3 prepared in Preparation Example 4, and Compound 4 as shown in formula (2) (the preparation method of Compound 4 is shown in Patent WO2023 / 069708A1), Compound 5 as shown in formula (3) (the preparation method of Compound 5 is shown in Patent WO2022152822A1);

[0166]

[0167] Each compound was prepared into a 10mM stock solution with DMSO. The compounds in the ABC-1 cell experiment were finally diluted to 9 concentrations for detection: 2000nM, 400nM, 80nM, 16nM, 3.2nM, 1.6nM, 0.8nM, 0.4nM, 0.2nM / 30000nM, 10000nM, 2000nM, 400nM, 80nM, 16nM, 3.2nM, 0.64nM, 0.128nM; the compounds in the CAL-51 cell experiment were finally diluted to 6 concentrations for detection: 10000nM, 2000nM, 400nM, 80nM, 16nM, 3.2nM.

[0168] ABC-1 cells were provided by Nanjing Kebai Biotechnology Co., Ltd., and CAL-51 cells were provided by Jiangsu KeyGen Biotech Co., Ltd.

[0169] Reagents: MEM, catalog number Gibco 11095-080; 1% Non Essential Amino Acids (NEAA), catalog number Gibco11140-050; 1mM Sodium Pyruvate (NaP), catalog number Gibco 11360-070; DMEM, catalog number GibcoC11965500BT, Fetal Bovine Serum (FBS), catalog number Gibco A5669701; 0.25% trypsin solution, catalog number Keygen KGL2101-100; PBS, catalog number Solarbio P1020; Celltiter Glo assay kit (CTG), catalog number Promega G9683; 4% paraformaldehyde, catalog number biosharp BL539A; Triton X, catalog number Biosharp P0096; bovine serum albumin BSA, catalog number biosharp BS114; GSPT1 Polyclonal antibody, the catalog number is Proteintech 10763-1-AP; Alexa FluorTM 488goat anti-rabbit lgG(H+L), the catalog number is Invitrogen A11008; DAPI, the catalog number is Biotech P0131.

[0170] 2. Experimental Methods

[0171] 2.1 Cell culture and passaging

[0172] (1) All cells were cultured according to the method recommended by ATCC, and the experiments were performed in the exponential growth phase.

[0173] (2) Cell culture medium:

[0174] ABC-1 cells: MEM+10% FBS+1% Non Essential Amino Acids (NEAA)+1mM Sodium Pyruvate (NaP).

[0175] CAL-51 cells: DMEM + 10% FBS.

[0176] (3) Cell culture conditions: 37°C, 5% CO 2 , 95% air

[0177] (4) Depending on the growth of the cells, the culture medium should be changed every 2 to 3 days or the cells should be subcultured.

[0178] 2.2 Cell plating

[0179] ABC-1 and CAL-51 cells: Collect cells from cell culture flasks by centrifugation and resuspend in fresh medium. The cell density of ABC-1 cells is adjusted to 7.5 × 10 4 The cell density of CAL-51 cells was adjusted to 5×10 4 The cell resuspension was inoculated into a 96-well culture plate at 100 μL / well.

[0180] 2.3 Administration

[0181] 2.3.1 Cell viability assay

[0182] ABC-1 cells: 24 hours after cell inoculation, the original culture medium was discarded, 100 μL of different concentrations of drugs were added to the drug group, and fresh culture medium without drugs was added to the control group. Three replicate wells were set for each concentration group, and 5% CO was continued to be placed in the well. 2 Incubate for 72 hours. The compound was prepared as follows: 5 mg of the compound was weighed in advance and prepared into a 10 mM stock solution using DMSO. The drug was diluted with culture medium, with the drug concentration starting at 2 μM as the highest concentration, and then diluted to 9 concentration gradients in sequence. The final concentrations of the drug were: 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 1.6 nM, 0.8 nM, 0.4 nM, and 0.2 nM.

[0183] CAL-51 cells: 24 hours after cell inoculation, the original culture medium was discarded, 100 μL of drugs of different concentrations were added to the drug group, and fresh culture medium without drugs was added to the control group. Three replicate wells were set for each concentration group, and 5% CO was continued. 2The cells were incubated in an incubator for 72 hours. The compound was prepared as follows: 5 mg of the compound was weighed in advance and prepared into a 10 mM stock solution using DMSO. The drug was diluted using culture medium, with the drug concentration starting at 10 μM and then diluted to 6 concentration gradients, with the final concentrations being 10000 nM, 2000 nM, 400 nM, 80 nM, 16 nM, and 3.2 nM.

[0184] 2.3.2 GSPT1 degradation experiment

[0185] ABC-1 cells: 24 hours after cell inoculation, the original culture medium was discarded, 100 μL of different concentrations of drugs were added to the drug group, and fresh culture medium without drugs was added to the control group. Three replicate wells were set for each concentration group, and 5% CO was continued to be placed in the well. 2 Incubate for 6 hours. The compound was prepared as follows: 5 mg of the compound was weighed in advance and prepared into a 10 mM stock solution using DMSO. The drug was diluted with culture medium, with a drug concentration of 30 μM as the starting maximum concentration, and then diluted to 9 concentration gradients in sequence. The final concentrations of the drug were: 30000 nM, 10000 nM, 2000 nM, 400 nM, 80 nM, 16 nM, 3.2 nM, 0.64 nM, and 0.128 nM.

[0186] CAL-51 cells: 24 hours after cell inoculation, the original culture medium was discarded, 100 μL of drugs of different concentrations were added to the drug group, and fresh culture medium without drugs was added to the control group. Three replicate wells were set for each concentration group, and 5% CO was continued. 2 Incubate for 6 hours. The compound was prepared as follows: 5 mg of the compound was weighed in advance and prepared into a 10 mM stock solution using DMSO. The drug was diluted with culture medium, with a starting concentration of 30 μM, and then diluted to 6 concentration gradients, with the final concentrations being: 30000 nM, 10000 nM, 2000 nM, 400 nM, 80 nM, and 16 nM.

[0187] 2.4 Detection

[0188] 2.4.1 Cell viability assay

[0189] ABC-1 and CAL-51 cells: 72 hours after drug treatment, remove the Celltiter Glo assay kit and required culture medium 30 minutes in advance, equilibrate to room temperature, and dilute with culture medium 1:1 to obtain working solution. Add 100 μL of Celltiter-Glo reagent working solution to Blank wells, drug wells, and Control wells, incubate at room temperature in the dark for 10 minutes, and detect luminescent signals. According to the luminescent intensity value, calculate the inhibition rate of each well relative to the Control group:

[0190] Inhibition% = (Ave_H-Sample) / (Ave_H-Ave_L) × 100, Note: H = Ave (Control), L = Ave (Blank). According to different drug concentrations and their corresponding inhibition rates, GraphicPad 9.5 software was used to draw IC50 curves, analyze data, and obtain the final IC50 value. The experimental results are shown in Table 1.

[0191] 2.4.2 GSPT1 degradation experiment

[0192] ABC-1 and CAL-51 cells: After 6 hours of drug treatment, cells were taken out of the incubator, the culture medium was discarded, each well was washed with PBS, and then 40 μL of 4% paraformaldehyde was added to each well and fixed at room temperature for 15 min; after fixation, the fixative was discarded, and each well was washed with PBS, repeated 3 times, 5 min each time; after washing, 40 μL of 0.2% Trixon permeabilization solution was added to each well and incubated at room temperature for 10 min; after permeabilization, the permeabilization solution was discarded, and each well was washed with PBS, repeated 3 times, 5 min each time; after washing, 40 μL of 3% BSA blocking solution was added to each well and blocked at room temperature for 30 min; after blocking, 35 μL of freshly diluted primary antibody (GSPT1, Proteintech 10763-1-AP, 5% BSA diluted 1:400), incubated at 4℃ overnight; the next day (after 16 hours of incubation), the culture plate was taken out after the incubation, the primary antibody in each well was recovered, and then each well was washed with PBS, repeated 3 times, 5 minutes each time; after washing, 40μL of freshly diluted secondary antibody (Alexa FluorTM 488goat anti-rabbit lgG, diluted 1:100 with PBS) was added to each well, and incubated at room temperature in the dark for 1 hour; after incubation, each well was washed with PBS in the dark, repeated 3 times, 5 minutes each time; after washing, 40μL of DAPI was added to each well, and then placed under a fluorescence microscope, and the uniform shooting parameters were determined according to the fluorescence intensity of the Control group, and each well was photographed continuously. After shooting, the fluorescence intensity was identified using Image J software, and the degradation rate of each well relative to the Control group was calculated: Degradation%=(Ave_H-Sample) / (Ave_H)×100, Note: H=Ave(Control). According to different drug concentrations and their corresponding degradation rates, the DC50 curve was drawn using GraphicPad 9.5 software, and the data was analyzed to obtain the final DC50 value. The experimental results are shown in Table 1.

[0193] 3. Experimental results

[0194] Table 1

[0195]

[0196] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A compound represented by general formula (1), a pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer thereof, characterized in that: wherein L is independently selected from -O- or -NH-; The R1 is selected from a heterocyclic group, a phenyl heterocyclic group, a 6-11 membered aryl group or a 6-13 membered heteroaryl group. R2 is selected from the group consisting of hydrogen, hydroxy, alkyl, chloroalkyl, alkoxy, haloalkoxy, alkylacyl, cycloalkyl, heterocycloalkyl and heteroaryl.

2. The compound of general formula (1) according to claim 1, its pharmaceutically acceptable salt or its stereoisomer, characterized in that: The R1 is independently selected from Q1, Q2, Q3, Q4, Q5 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, amino, hydroxyl, carboxyl, cyano, C1-6 alkyl, C1-6 alkoxy, halo C1-6 alkyl, C1-6 alkynyl, C1-6 cycloalkyl alkynyl, alkyl, haloalkyl, hydroxyalkyl, alkoxy, hydroxyalkoxy, amino, alkylacylamino, alkylacyl, alkylaminoacyl, 3-6 membered heterocyclyl, 3-6 membered cycloalkyl; Ring A is selected from 5-10 membered cycloalkyl, 5-10 membered heterocyclic group, 5-10 membered heteroaromatic ring group, benzofuranyl; Ring B is selected from 3-10 membered cycloalkyl, 3-10 membered heterocyclyl, and 6 membered aryl; Ring C, Ring D, and Ring E are each independently selected from a 5-10 membered heterocyclic group; R3 is selected from hydrogen, hydroxy, alkyl, chloroalkyl, alkoxy, haloalkoxy, alkylacyl, cycloalkyl, heterocycloalkyl and heteroaryl; wherein m is selected from an integer of 1-3, n is selected from 1 or 2, p is selected from an integer of 1-3; s is selected from an integer of 1-3; t is selected from 1 or 2, and i is selected from 1 or 2.

3. The compound of general formula (1) according to claim 1, its pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer thereof, characterized in that: The R1 is independently selected from wherein R3 is selected from the group consisting of hydrogen, hydroxy, alkyl, chloroalkyl, alkoxy, haloalkoxy, alkylacyl, cycloalkyl, heterocycloalkyl and heteroaryl.

4. A compound, a pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer thereof, characterized in that: It has the structure shown in the following table:

5. A pharmaceutical preparation, characterized in that Containing the compound according to any one of claims 1 to 4, its pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer thereof.

6. The pharmaceutical preparation according to claim 5, characterized in that It also contains one or more pharmaceutical carriers and / or diluents; the pharmaceutical preparation is in any dosage form that is clinically or pharmaceutically acceptable.

7. A pharmaceutical composition, characterized in that It contains the compound according to any one of claims 1 to 4, its pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer and one or more second therapeutically active agents.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers and / or diluents.

9. Use of the compound according to any one of claims 1 to 4, its pharmaceutically acceptable salt, solvate, crystal, prodrug or stereoisomer thereof, or the pharmaceutical preparation according to claim 3 or 4, or the pharmaceutical composition according to claim 5 or 6 in the preparation of a medicament for treating MYC-high-expression tumor diseases.

10. The use according to claim 9, characterized in that The MYC high-expression tumor diseases include breast cancer, liver cancer, gastric cancer, blood tumors, lung cancer, prostate cancer, and pancreatic cancer.

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