A protac compound based on flexible linker chain and preparation method and application thereof

Abstract

This invention relates to the field of targeted protein degradation technology, providing a PROTAC compound based on a flexible linker chain, its preparation method, and its applications. The general formula of the PROTAC compound is asteroidin group-Linker-CRBN ligand; the CRBN ligand is a phthalimide-based core structure; the asteroidin group is an indolecarbazole-based core structure; the Linker is a flexible linker chain, with at least one triazole ring group at one end and an amino group at the other end; the indolecarbazole-based core is linked to a carbon atom on the triazole ring group at one end of the flexible linker chain; the phthalimide-based core is linked to a nitrogen atom on the amino group at the other end of the flexible linker chain. The application of the PROTAC compound of this invention, or pharmaceutical compositions containing it, in the preparation of medicaments for the treatment and / or prevention of tumors, and the compounds of this invention exhibit excellent degradation effects on Fyn and other kinases.

Description

Technical Field

[0001] This invention relates to the field of targeted protein degradation technology, and in particular to a PROTAC compound based on a flexible linker chain, its preparation method, and its applications. Background Technology

[0002] Melanoma is a classic model for drug development. Melanoma, usually referring to malignant melanoma, is a highly malignant tumor originating from melanocytes. It commonly occurs in the skin, but can also appear in mucous membranes and internal organs, accounting for approximately 3% of all tumors. Cutaneous malignant melanoma ranks third among malignant skin tumors (approximately 6.8%–20%). In recent years, the incidence and mortality rates of malignant melanoma have been increasing annually, and compared to other solid tumors, its age of onset is lower. Besides early surgical resection, there is a lack of specific treatments for malignant melanoma, resulting in a poor prognosis. Therefore, early diagnosis and treatment of malignant melanoma are extremely important.

[0003] Targeted protein degradation (TPD) is a promising therapeutic and research strategy that relies on protein degradation-targeting chimeras (PROTAC) technology. PROTACs are heterobifunctional molecules consisting of two ends: a ligand for the target protein (POI) linked via a suitable linker and a specific ligand for an E3 ubiquitin ligase. They utilize the intracellular ubiquitin-proteasome system (UPS) to degrade the target protein. Compared to traditional small molecules, they are less likely to increase target overexpression or mutation, and can overcome acquired resistance caused by traditional inhibitors.

[0004] Astrocytocin and its derivatives (such as midostaurin) have been shown to possess broad-spectrum kinase inhibitory activity, but their potential as target protein ligands for PROTACs has not been fully explored. Furthermore, studies have shown that the structure of the linker significantly influences the activity, conformation, and ternary complex formation of PROTAC molecules. Developing a library of PROTAC molecules with different linker structures is of great significance for screening highly efficient degradative agents and for studying structure-activity relationships. Summary of the Invention

[0005] This invention provides a PROTAC compound based on a flexible linker chain, its preparation method, and its application. The compound connects an astrocytocin derivative to an E3 ubiquitin ligase ligand (CRBN ligand) via a specific flexible linker chain, aiming to efficiently degrade the kinase FYN, thereby providing a more effective drug option for tumor treatment.

[0006] To achieve the above objectives, embodiments of the present invention provide a PROTAC compound based on a flexible linker chain, wherein the general formula of the PROTAC compound is astrosporin group-Linker-CRBN ligand; The CRBN ligand has a phthalimide-based core structure. The astrosporin group has an indolecarbazole core structure. The Linker is a flexible connecting chain, with at least one triazole ring group at one end and an amino group at the other end; The indolecarbazole core is connected to the carbon atom on the triazole ring group at one end of the flexible linker chain; the phthalimide core is connected to the nitrogen atom on the amine group at the other end of the flexible linker chain.

[0007] Preferably, the flexible linker further comprises one or more ethoxy units and / or alkylene units; the CRBN ligand is linked to the Linker at the 4- or 5-position of the phthalimide core; and the astrocytocin group is linked to the Linker via an amide bond or a carbamate bond.

[0008] Preferably, the general chemical formula of the PROTAC compound is selected from formula I, II, III or IV: ; Where n is 1-10; more preferably n is 1-5.

[0009] Preferably, the PROTAC compound is selected from any one of the following compounds 1-20: .

[0010] More preferably, the PROTAC compound is selected from any one of the following compounds 1-5 and 11-15: .

[0011] The embodiments of the present invention provide a method for preparing the above-mentioned PROTAC compound, comprising performing an azide-alkyne click cycloaddition reaction between an alkyne-containing asteroidin derivative and a CRBN ligand intermediate with a flexible linker containing an azide group.

[0012] Preferably, the azide-acetylene click cycloaddition reaction is carried out in a mixed solvent of tetrahydrofuran and water in the presence of copper sulfate and sodium ascorbate.

[0013] Embodiments of the present invention also provide a pharmaceutical composition comprising the above-described PROTAC compound or a pharmaceutically acceptable salt, solvate or prodrug thereof, and one or more pharmaceutically acceptable carriers.

[0014] The embodiments of the present invention provide the use of the above-described PROTAC compound or the above-described pharmaceutical composition in the preparation of a medicament for treating and / or preventing tumors.

[0015] Preferably, the tumor is a kinase-mediated tumor.

[0016] The above-described solution of the present invention has the following beneficial effects: (1) This invention successfully constructed a novel class of PROTAC molecules by linking asteroidin derivatives with CRBN ligands via a flexible chain containing a triazole ring. Bioactivity tests showed that these compounds (especially compounds 2, 3, 4, and 8) significantly degraded FYN protein after treatment of melanoma SK-MEL-5 cells at concentrations of 1 μM and 10 μM for 12 hours. Compound 2 showed a degradation rate as high as 72% at 10 μM concentration, and compound 3 showed a degradation rate as high as 80% at 1 μM concentration. This indicates that the flexible linker design of this invention facilitates the formation of a stable PROTAC-target protein-E3 ligase ternary complex, thereby achieving efficient targeted protein degradation.

[0017] (2) This invention systematically investigated the effects of flexible linker chain length (different chain lengths) and CRBN ligand linking position (4- or 5-position on the benzene ring) on ​​activity. Experimental results showed that compounds constructed with 4-position (ortho-) substituted CRBN ligands (1-5, 11-15) generally exhibited better degradation activity than compounds with 5-position (meta-) substituted ligands (6-10, 16-20). Meanwhile, the length of the linker chain significantly affected the activity, providing important data support for subsequent compound structure optimization.

[0018] (3) The preparation method of the present invention uses a highly efficient click chemistry reaction to construct a flexible linker chain. The reaction conditions are mild and the yield is moderate (up to 78%). It also provides detailed synthesis steps and purification methods for intermediates and target compounds, and has good reproducibility and industrial application prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 It is compound 1 of the present invention. 1 H NMR spectrum; Figure 2 It is compound 1 of the present invention. 13 C10 NMR spectrum; Figure 3 It is compound 2 of the present invention 1 H NMR spectrum; Figure 4 It is compound 2 of the present invention 13 C10 NMR spectrum; Figure 5 It is compound 3 of the present invention. 1 H NMR spectrum; Figure 6 It is compound 3 of the present invention. 13 C10 NMR spectrum; Figure 7 It is compound 4 of the present invention. 1 H NMR spectrum; Figure 8 It is compound 4 of the present invention. 13 C10 NMR spectrum; Figure 9 It is compound 5 of the present invention. 1 H NMR spectrum; Figure 10 It is compound 5 of the present invention. 13 C10 NMR spectrum; Figure 11 It is compound 6 of the present invention. 1 H NMR spectrum; Figure 12 It is compound 6 of the present invention. 13 C10 NMR spectrum; Figure 13 It is compound 7 of the present invention. 1 H NMR spectrum; Figure 14 It is compound 7 of the present invention. 13 C10 NMR spectrum; Figure 15 It is compound 8 of the present invention. 1 H NMR spectrum; Figure 16 It is compound 8 of the present invention. 13 C10 NMR spectrum; Figure 17 It is compound 9 of the present invention. 1 H NMR spectrum; Figure 18 It is compound 9 of the present invention. 13 C10 NMR spectrum; Figure 19 It is compound 10 of the present invention. 1 H NMR spectrum; Figure 20It is compound 10 of the present invention. 13 C10 NMR spectrum; Figure 21 It is compound 11 of the present invention. 1 H NMR spectrum; Figure 22 It is compound 11 of the present invention. 13 C10 NMR spectrum; Figure 23 It is compound 12 of the present invention. 1 H NMR spectrum; Figure 24 It is compound 12 of the present invention. 13 C10 NMR spectrum; Figure 25 It is compound 13 of the present invention. 1 H NMR spectrum; Figure 26 It is compound 13 of the present invention. 13 C10 NMR spectrum; Figure 27 It is compound 14 of the present invention. 1 H NMR spectrum; Figure 28 It is compound 14 of the present invention. 13 C10 NMR spectrum; Figure 29 It is compound 15 of the present invention. 1 H NMR spectrum; Figure 30 It is compound 15 of the present invention. 13 C10 NMR spectrum; Figure 31 It is compound 16 of the present invention. 1 H NMR spectrum; Figure 32 It is compound 16 of the present invention. 13 C10 NMR spectrum; Figure 33 It is compound 17 of the present invention. 1 H NMR spectrum; Figure 34 It is compound 17 of the present invention. 13 C10 NMR spectrum; Figure 35 This is a band diagram showing the effect of the compounds of this invention on the degradation of FYN protein in melanoma SK-MEL-5 cells; Figure 36 This is a graph showing the inhibitory activity of the compounds of this invention on the proliferation of melanoma A375 cells. Detailed Implementation

[0021] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0022] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0023] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0024] Example explanation: To avoid the chemical structural formula being too long and affecting the text display, the phrase "" in this article is shortened. The subscript number specifically refers to the " The number of structural repetitions is listed here, and other similar structures are listed in the same way.

[0025] Example 1 This embodiment provides a method for preparing an astrocytocin derivative, the synthetic route of which is shown below, and specifically includes the following steps:

[0026] Take a 25 mL round-bottom flask, add asteroidin (200 mg, 0.43 mmol), propargyl chloroformate (101.9 mg, 0.86 mmol), triethylamine (87.0 mg, 0.86 mmol), and dichloromethane (DCM, 2.1 mL), and react at 55 °C. After 2 days, TLC monitoring showed that the reaction was complete. The reaction solution was extracted three times with ethyl acetate (EA) and water. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation. The sample was loaded dry and purified by silica gel column chromatography. DCM and CH3OH at a mass ratio of 100:1 were used as the eluent to obtain asteroidin derivative 1 (210 mg, yield 89%).

[0027] Its structure is: prop-2-yn-1-yl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate.

[0028] Example 2 This embodiment provides a method for preparing an astrocytocin derivative, the synthetic route of which is shown below, and specifically includes the following steps:

[0029] Take a 10 mL reaction tube, add 4-acetylenic benzoic acid (99.2 mg, 0.68 mmol), add dichloromethane (DCM, 2.72 mL), add oxaloyl chloride (129.4 mg, 1.02 mmol) under ice bath, and add the catalytic amount of N,N-dimethylformamide (DMF). The system bubbled vigorously, and then reacted at room temperature. After the system gradually changed from turbid to clear, a small sample was taken and quenched with methanol. TLC was used to monitor the reaction of the starting materials until complete. The solvent was removed by rotary evaporation and the mixture was dried by pumping dry. Astrococcus (158.4 mg, 0.34 mmol), NaOH (54.4 mg, 1.36 mmol), and DMF (3.4 mL) were added, and the reaction was carried out at 55 °C for 2 days. After the reaction was complete, the mixture was extracted three times with 1N NaOH solution and DCM. The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and then evaporated to dryness. Dry loading and silica gel column chromatography (DCM:CH3OH=100:1) were used to purify the astrosporin derivative 2 (100 mg, yield 25%).

[0030] Its structure is: 4-ethynyl-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-ethynyl-N-((5R,7R,8R,9S)-16 -hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide).

[0031] Example 3 This embodiment provides a method for preparing CRBN ligand intermediate 1-a. The synthetic route is shown below, and specifically includes the following steps:

[0032] Take 10 mL of the sealed tube, add 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (55.2 mg, 0.2 mmol), 2-(2-azidoethoxy)ethylamine (28.6 mg, 0.22 mmol), N,N-diisopropylethylamine (DIPEA, 51.7 mg, 0.4 mmol), and finally add N-methylpyrrolidone (NMP, 0.2 mL) to dissolve. React at 85 °C for 5 hours, and monitor the reaction completion by TLC. Extract the reaction solution three times with EA and H2O, dry the organic phase with anhydrous sodium sulfate, filter, and then evaporate to dryness. Dry-load the sample and purify by silica gel column chromatography (DCM:CH3OH = 100:1) to obtain CRBN ligand intermediate 1-a (33.6 mg, yield 44%). Its structure is: 4-((2-(2-azidoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione.

[0033] Example 4 This embodiment provides a method for preparing CRBN ligand intermediate 1-b. The difference from Example 3 is that the ethoxy group in 2-(2-azidoethoxy)ethylamine is doubled. All other steps and parameters are the same as in Example 1, and CRBN ligand intermediate 1-b (98.2 mg, yield 23%) is obtained. Its structure is: 4-((2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione.

[0034] Example 5 This embodiment provides a method for preparing CRBN ligand intermediate 1-c. The difference from Example 3 is that the number of ethoxy groups in 2-(2-azidoethoxy)ethylamine is 3. All other steps and parameters are the same as in Example 1, and CRBN ligand intermediate 1-c (110.4 mg, yield 23%) is obtained. Its structure is: 4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione).

[0035] Example 6 This embodiment provides a method for preparing CRBN ligand intermediate 1-d. The difference from Example 3 is that the number of ethoxy groups in 2-(2-azidoethoxy)ethylamine is 4. All other steps and parameters are the same as in Example 1, and CRBN ligand intermediate 1-d (92.2 mg, yield 17%) is obtained. Its structure is: 4-((14-azido-3,6,9,12-tetraoxatetradecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione.

[0036] Example 7 This embodiment provides a method for preparing CRBN ligand intermediate 1-e. The difference from Example 3 is that the number of ethoxy groups in 2-(2-azidoethoxy)ethylamine is 5. All other steps and parameters are the same as in Example 1, and CRBN ligand intermediate 1-e (85.1 mg, yield 15%) is obtained. Its structure is: 4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione).

[0037] Example 8 This embodiment provides a method for preparing CRBN ligand intermediate 2-a, which differs from Example 3 in that 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione is replaced with 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione, yielding CRBN ligand intermediate 2-a (65.1 mg, yield 34%). Its structure is: 5-((2-(2-azidoethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione.

[0038] Example 9 This embodiment provides a method for preparing CRBN ligand intermediate 2-b, which differs from Example 8 in that the ethoxy group in 2-(2-azidoethoxy)ethylamine is doubled, yielding CRBN ligand intermediate 2-b (64.2 mg, yield 30%). Its structure is: 5-((2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (5-((2-(2-(2-azidoethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione).

[0039] Example 10 This embodiment provides a method for preparing CRBN ligand intermediate 2-c, which differs from Example 8 in that the number of ethoxy groups in 2-(2-azidoethoxy)ethylamine is 3, yielding CRBN ligand intermediate 2-c (24.6 mg, yield 10%). Its structure is: 5-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (5-((2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione).

[0040] Example 11 This embodiment provides a method for preparing CRBN ligand intermediate 2-d, which differs from Example 8 in that the number of ethoxy groups in 2-(2-azidoethoxy)ethylamine is 4, yielding CRBN ligand intermediate 2-d (43.6 mg, yield 16%). Its structure is: 5-((14-azido-3,6,9,12-tetraoxatetradecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (5-((14-azido-3,6,9,12-tetraoxatetradecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione).

[0041] Example 12 This embodiment provides a method for preparing CRBN ligand intermediate 2-e, which differs from Example 8 in that the number of ethoxy groups in 2-(2-azidoethoxy)ethylamine is 5, yielding CRBN ligand intermediate 2-e (51.3 mg, yield 18%). Its structure is: 5-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (5-((17-azido-3,6,9,12,15-pentaoxaheptadecyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione).

[0042] Example 13 This embodiment provides a method for preparing a PROTAC compound based on a flexible connecting chain. The synthetic route is shown below, and specifically includes the following steps:

[0043] Take a 10 mL round-bottom flask, add asteroidin derivative 1 (40 mg, 0.072 mmol), anhydrous copper sulfate (4.6 mg, 0.029 mmol), and sodium ascorbate (28.9 mg, 0.146 mmol). Dissolve CRBN ligand intermediate 1-a (30.8 mg, 0.08 mmol) in tetrahydrofuran (THF, 0.72 mL) and H2O (0.36 mL) and add it to the flask. React at room temperature. After 4 hours, monitor the reaction of the starting material by TLC until complete. Remove the solvent by rotary evaporation and dry the mixture with a pump. Dry sample loading and purification by silica gel column chromatography (dichloromethane (DCM):CH3OH = 60:1) to give compound 1 (26.2 mg, yield 38%). Its structure is: (1-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene) Dibenzo[b,h]cyclonona[jkl]cyclopenta[e]-asymmetric indacen-7-yl)(methyl)carbamate ((1-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 1 , Figure 2 As shown.

[0044] Example 14 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 13 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 1-b to obtain compound 2 (25.3 mg, yield 35%). Its structure is: (1-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dimethyl) Benzo[b,h]cyclonona[jkl]cyclopenta[e]-asymmetric indacen-7-yl)(methyl)carbamate ((1-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 3 , Figure 4 As shown.

[0045] Example 15 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 13 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 1-c, yielding compound 3 (23.1 mg, yield 31%). Its structure is: (1-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo-p-2-yl) [b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate ((1-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 5 , Figure 6 As shown.

[0046] Example 16 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 13 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 1-d to obtain compound 4 (56.8 mg, yield 72%). Its structure is: (1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylenediphenyl) [b,h]cyclonona[jkl]cyclopenta[e]-asymmetric indacen-7-yl)(methyl)carbamate ((1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 7 , Figure 8 As shown.

[0047] Example 17 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 13 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 1-e, yielding compound 5 (40.1 mg, yield 50%). Its structure is: 1-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-3,6,9,12,15-pentoxaheptadecyl)-1H-1,2,3-triazol-4-yl)methyl((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo-p-2-yl) [b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate ((1-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaheptadecyl)-1H-1,2,3-triazol-4-yl)methyl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 9 , Figure 10 As shown.

[0048] Example 18 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 13 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 2-a, yielding compound 6 (32.4 mg, yield 68%). Its structure is: (1-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene) Dibenzo[b,h]cyclonona[jkl]cyclopenta[e]-asymmetric indacen-7-yl)(methyl)carbamate ((1-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 11 , Figure 12 As shown.

[0049] Example 19 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 13 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 2-b, yielding compound 7 (26.7 mg, yield 53%). Its structure is: (1-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dimethyl) Benzo[b,h]cyclonona[jkl]cyclopenta[e]-asymmetric indacen-7-yl)(methyl)carbamate ((1-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 13 , Figure 14 As shown.

[0050] Example 20 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 13 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 2-c, yielding compound 8 (40.4 mg, yield 78%). Its structure is: (1-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo-p-2-yl) [b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate ((1-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 15 , Figure 16 As shown.

[0051] Example 21 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 13 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 2-d to obtain compound 9 (39.4 mg, yield 73%). Its structure is: (1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylenediphenyl) [b,h]cyclonona[jkl]cyclopenta[e]-asymmetric indacen-7-yl)(methyl)carbamate ((1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)methyl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 17 , Figure 18 As shown.

[0052] Example 22 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 13 is that the CRBN ligand intermediate 1-a is replaced with the CRBN ligand intermediate 2-e, yielding compound 10 (30.1 mg, yield 54%). Its structure is: (1-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)-3,6,9,12,15-pentoxaheptadecyl)-1H-1,2,3-triazol-4-yl)methyl((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylenediphenyl) [b,h]cyclonona[jkl]cyclopenta[e]-asymmetric indacen-7-yl)(methyl)carbamate ((1-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)-3,6,9,12,15-pentaoxaheptadecyl)-1H-1,2,3-triazol-4-yl)methyl ((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)(methyl)carbamate); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 19 , Figure 20 As shown.

[0053] Example 23 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 13 is that asteroidin derivative 1 (40 mg, 0.072 mmol) is replaced with asteroidin derivative 2 (20 mg, 0.033 mmol) to obtain compound 11 (14.2 mg, yield 43%). Its structure is: 4-(1-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-(1-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3- dioxoisoindolin-4-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetra hydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 21 , Figure 22 As shown.

[0054] Example 24 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 23 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 1-b to obtain compound 12 (15.9 mg, yield 47%). Its structure is: 4-(1-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-(1-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1, 3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9- tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 23 , Figure 24 As shown.

[0055] Example 25 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 23 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 1-c, yielding compound 13 (17.5 mg, yield 49%). Its structure is: 4-(1-(2-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxopiperidin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-(1-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)-1) ,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7, 8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 25 , Figure 26 As shown.

[0056] Example 26 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 23 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 1-d to obtain compound 14 (19.1 mg, yield 52%). Its structure is: 4-(1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-(1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxa-7-yl)-N-methylbenzamide) oisoindolin-4-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9 -tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 27 , Figure 28 As shown.

[0057] Example 27 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 23 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 1-e, yielding compound 15 (20.8 mg, yield 54%). Its structure is: 4-(1-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)amino)-3,6,9,12,15-pentoxaheptadecyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-(1-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxa-7-yl)-N-methylbenzamide) oisoindolin-4-yl)amino)-3,6,9,12,15-pentaoxaheptadecyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8 ,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 29 , Figure 30 As shown.

[0058] Example 28 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 23 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 2-a, yielding compound 16 (17.8 mg, yield 53%). Its structure is: 4-(1-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-(1-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3- dioxoisoindolin-5-yl)amino)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetra hydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 31 , Figure 32 As shown.

[0059] Example 29 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 23 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 2-b to obtain compound 17 (17.6 mg, yield 52%). Its structure is: 4-(1-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-(1-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1, 3-dioxoisoindolin-5-yl)amino)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9- tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide); its 1 H NMR spectra and 13 The C NMR spectra are as follows: Figure 33 , Figure 34 As shown.

[0060] Example 30 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 23 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 2-c, yielding compound 18 (21.8 mg, yield 61%). Its structure is: 4-(1-(2-(2-(2-(2-((2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-(1-(2-(2-(2-(2-(2,6-dioxopiperidin-3-yl)- 1,3-dioxoisoindolin-5-yl)amino)ethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7 ,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide).

[0061] Example 31 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 23 is that CRBN ligand intermediate 1-a is replaced with CRBN ligand intermediate 2-e, yielding compound 19 (19.4 mg, yield 53%). Its structure is: 4-(1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-(1-(14-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxa-7-yl)-N-methylbenzamide) oisoindolin-5-yl)amino)-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8, 9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide).

[0062] Example 32 This embodiment provides a method for preparing a PROTAC compound based on a flexible linker chain. The difference from Example 23 is that the CRBN ligand intermediate 1-a is replaced with the CRBN ligand intermediate 2-f, yielding compound 20 (23.7 mg, yield 62%). Its structure is: 4-(1-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)amino)-3,6,9,12,15-pentoxaheptadecyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-bridged methylene dibenzo[b,h]cyclononano[jkl]cyclopentano[e]-asymmetric indarsen-7-yl)-N-methylbenzamide (4-(1-(17-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxa-7-yl)-N-methylbenzamide) oisoindolin-5-yl)amino)-3,6,9,12,15-pentaoxaheptadecyl)-1H-1,2,3-triazol-4-yl)-N-((5R,7R,8R,9S)-16-hydroxy-8-methoxy-9-methyl-6,7,8 ,9-tetrahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-yl)-N-methylbenzamide).

[0063] Comparative Example 1 This comparative example provides a method for preparing a PROTAC compound based on a flexible connecting chain. The difference from Example 13 is that water was not added as a solvent, and the reaction could not proceed.

[0064] Bioactivity test The degradation effects of compounds 1 to 20 on the kinase FYN were tested in the melanoma cell line SK-MEL-5, and the results are shown in Table 1. The specific procedures of the Western Blot experiment are as follows: After 12 hours of adherent cell culture, cells were treated with 1 μM and 10 μM of the target compound for 12 hours, respectively. Cell lysates were collected, separated by SDS-PAGE electrophoresis, and then detected by Western blotting using FYN-specific antibody, with GAPDH as an internal control. The results are as follows: Figure 35 As shown. Experimental results indicate that the flexible chain PROTAC compounds provided by this invention, wherein the ortho-substituted CRBN ligands are more effective than the meta-substituted ones, especially compounds 2, 3, 4, and 8, which can significantly degrade FYN protein at different concentrations, showing potential as a treatment for melanoma.

[0065] Table 1

[0066] CCK8 assay was used to detect the inhibitory activity of the compound on the proliferation of melanoma A375 cells. To test the inhibitory effects of compounds 1 to 20 of this invention on tumor cell proliferation, the CCK8 assay was used to detect the inhibitory effects of the compounds at concentrations of 1 μM and 10 μM on the proliferation of melanoma A375 cell lines. The results are as follows: Figure 36 As shown, compounds 1-20 of the present invention exhibit significant anti-melanoma proliferative activity at the cellular level.

[0067] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A PROTAC compound based on a flexible connecting chain, characterized in that, The general formula of the PROTAC compound is astrosporin group-Linker-CRBN ligand; The CRBN ligand has a phthalimide-based core structure. The astrosporin group has an indolecarbazole core structure. The Linker is a flexible connecting chain, with at least one triazole ring group at one end and an amino group at the other end; The indolecarbazole core is connected to the carbon atom on the triazole ring group at one end of the flexible linker chain; the phthalimide core is connected to the nitrogen atom on the amine group at the other end of the flexible linker chain.

2. The PROTAC compound according to claim 1, characterized in that, The flexible connecting chain further comprises one or more ethoxy units and / or alkylene units; The CRBN ligand is linked to the Linker at the 4- or 5-position of the phthalimide core. The connection between the astrosporin group and the linker is an amide bond or a carbamate bond.

3. The PROTAC compound according to claim 1, characterized in that, The general chemical formula is selected from formula I, II, III or IV: ； Where n is 1-10.

4. The PROTAC compound according to claim 1, characterized in that, The PROTAC compound is selected from any one of the following compounds 1-20: 。 5. The PROTAC compound according to claim 1, characterized in that, The PROTAC compound is selected from any one of the following compounds 1-5 and 11-15: 。 6. The method for preparing the PROTAC compound according to any one of claims 1 to 5, characterized in that, This includes the azido-acetylation click cycloaddition reaction of an alkyne-containing astrin derivative with a CRBN ligand intermediate containing a flexible linker chain with an azido group.

7. The preparation method according to claim 6, characterized in that, The azide-acetylene click cycloaddition reaction is carried out in a mixed solvent of tetrahydrofuran and water in the presence of copper sulfate and sodium ascorbate.

8. A pharmaceutical composition, characterized in that, It comprises the PROTAC compound of any one of claims 1-5 or a pharmaceutically acceptable salt, solvate or prodrug thereof, and one or more pharmaceutically acceptable carriers.

9. The use of the PROTAC compound according to any one of claims 1-5 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for the treatment and / or prevention of melanoma.

10. The application according to claim 8, characterized in that, The tumor is a kinase-mediated tumor.

Images