An EZH2 degrading agent, its preparation method and uses

Abstract

This invention provides an EZH2 degrading agent, its preparation method, and its uses, belonging to the field of chemical pharmaceuticals. Specifically, this invention provides a compound of Formula I, which is composed of an EZH2 targeting ligand and a degradation determinant. This compound can effectively inhibit the proliferation of various tumor cells and effectively degrade EZH2 protein in tumor cells. It can serve as an EZH2 degrading agent and has broad application prospects in the preparation of drugs for the prevention and / or treatment of cancer.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceuticals, specifically relating to an EZH2 degrading agent, its preparation method, and its uses. Background Technology

[0002] The human homolog of the Drosophila zeste gene enhancer 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2). It inhibits target gene transcription by trimethylating lysine 27 of histone H3 and participates in regulating physiological and pathological processes such as cell cycle, cellular senescence, and cell differentiation. Studies have found that EZH2 overexpression and abnormal regulation can be detected in various cancer cells, including prostate cancer, breast cancer, myeloma, and lymphoma. During tumorigenesis, EZH2 expression levels remain consistently elevated and are typically directly associated with high cancer invasiveness and poor prognosis. Therefore, inhibiting EZH2 protein activity could be used to treat related tumors.

[0003] Traditional small-molecule EZH2 inhibitors exhibit significant anti-proliferative capabilities in tumors by inhibiting protein methyl transfer activity. However, long-term use of small-molecule drugs inevitably leads to drug resistance, and to achieve the desired effect, small-molecule compounds need to be maintained at a certain concentration within cells. Higher concentrations of small molecules can cause adverse reactions due to off-target effects. Therefore, finding compounds that can overcome these shortcomings is of great significance in new drug development.

[0004] Protein degradation-targeting chimeric compounds (PROTACs) have attracted widespread attention as compounds capable of inducing the degradation of target proteins. As small molecule probes, PROTACs can simultaneously bind to both the target protein and E3 ubiquitinase, thereby promoting the ubiquitination of the target protein, which is then recognized and degraded by the proteasome. However, currently reported PROTACs with EZH2 degradation activity are generally composed of three parts: a small molecule compound that binds to the target protein (POI), a linker introduced at an appropriate position, and a small molecule compound that binds to the E3 ubiquitinase. This traditional PROTAC structure is complex, and the synthesis process is cumbersome. Furthermore, PROTACs possess bifunctional chemical characteristics. To simultaneously bind to two proteins, the microstructure of these traditional PROTACs is inevitably large and complex. Combined with the spacer of the linker, the increased molecular size leads to unfavorable pharmacokinetic and biopharmaceutics characteristics, resulting in lower bioavailability, poor pharmacokinetic properties, and increased metabolic sites. Summary of the Invention

[0005] In order to overcome the above-mentioned problems in the prior art, the purpose of this invention is to provide a new EZH2 degrading agent, its preparation method and uses.

[0006] This invention provides compounds of Formula I, their stereoisomers, their pharmaceutically acceptable salts, and their solvates:

[0007]

[0008] The structure of the EZH2 targeting ligand is shown in Formula II:

[0009]

[0010] R1 is selected from C 1-6 Alkyl or C 1-6 Alkoxy;

[0011] R2 is C 1-6 Alkyl group, R3 is NR 3a R 3b R 3a Selected from C 1-6 Alkyl, R 3b Selected from 5- to 6-membered saturated heterocyclic groups; or, R2 and R3 are linked to form unsubstituted or substituted groups with one or more R groups. 3c Substituted 5-6 member unsaturated heterocycles, R 3c Each is independently selected from C 1-6 alkyl;

[0012] E is selected from acetylene or

[0013] X is CH or N;

[0014] Z represents none, CH2, or CO;

[0015] Cy is selected from the following groups that are unsubstituted or substituted with one or more substituents: 3-14 membered heterocyclic groups, 4-14 membered heterobridged cyclic groups,

[0016] Among them, ring A is selected from 3-14 membered heterocyclic groups, and ring B is selected from 3-14 membered heterocyclic groups;

[0017] Each of the substituents is independently selected from C. 1-6 Alkyl, C 1-6 Alkoxy, hydroxyl, halogen.

[0018] Furthermore, the structure of the EZH2 targeting ligand is shown as EZH2-TL1, EZH2-TL2, EZH2-TL3, or EZH2-TL4:

[0019]

[0020]

[0021] Among them, R1 is selected from C 1-4 Alkyl or C 1-4 Alkoxy;

[0022] X is CH or N;

[0023] Z represents none, CH2, or CO;

[0024] Cy is selected from the following groups that are unsubstituted or substituted with one or more substituents: 3-14 membered heterocyclic groups, 4-14 membered heterobridged cyclic groups,

[0025] Among them, ring A is selected from 3-14 membered heterocyclic groups, and ring B is selected from 3-14 membered heterocyclic groups;

[0026] Each of the substituents is independently selected from C. 1-4 Alkyl, C 1-4 Alkoxy, hydroxyl, halogen.

[0027] Furthermore, R1 is selected from methyl, ethyl, propyl, or methoxy;

[0028] X is CH or N;

[0029] Z represents none, CH2, or CO;

[0030] Cy is selected from the following groups that are either unsubstituted or substituted by one or more substituents:

[0031]

[0032] Among them, m1, m2, m3, and m4 are each independently selected from 1 or 2;

[0033] m5, m6, m7, and m8 are each independently selected from 1 or 2;

[0034] Each of the substituents is independently selected from methyl, hydroxyl, and halogen.

[0035] Furthermore, the structure of the degradation determinant is shown in formula DG-1, DG-2, or DG-3:

[0036]

[0037] Among them, W is selected from CH and N;

[0038] Y is selected from none or CH2;

[0039] U is selected from CH and N;

[0040] V is selected from CO and CH2;

[0041] R2 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Alkoxy, hydroxyl, halogen;

[0042] R3 is selected from hydrogen, C 1-4 Alkyl, C 1-4 Alkoxy, hydroxyl, halogen.

[0043] Furthermore, the structure of DG-1 is as shown in DG-1a: W is selected from CH and N;

[0044] The structure of DG-2 is shown in either DG-2a or DG-2b:

[0045] Furthermore, the structure of the compound is shown below:

[0046]

[0047]

[0048]

[0049] The present invention also provides a pharmaceutical composition, which is a formulation prepared by using the above-mentioned compound, its stereoisomer, its pharmaceutically acceptable salt, its solvate as active ingredients, plus pharmaceutically acceptable excipients.

[0050] The present invention also provides the use of the above-mentioned compounds, their stereoisomers, their pharmaceutically acceptable salts, and their solvates in the preparation of EZH2 degrading agents.

[0051] Furthermore, the EZH2 degrading agent is a drug for the prevention and / or treatment of cancer.

[0052] Furthermore, the cancers mentioned are prostate cancer, breast cancer, bone marrow cancer, lymphoma, ovarian cancer, colorectal cancer, neuroblastoma, lung cancer, liver cancer, malignant rhabdomyosarcoma, glioma, esophageal cancer, stomach cancer, pancreatic cancer, or leukemia.

[0053] In this invention, the minimum and maximum carbon atom content in the hydrocarbon groups are indicated by a prefix, for example, C. 1-6 Alkyl groups refer to any straight-chain or branched alkyl group containing 1-6 carbon atoms; C 1-6 Alkoxy refers to any straight-chain or branched alkoxy group containing 1 to 6 carbon atoms.

[0054] In this invention, "heterocyclic cyclic group" refers to a multi-ring heterocyclic group in which two rings share two non-adjacent carbon atoms or heteroatoms.

[0055] Halogens are fluorine, chlorine, bromine or iodine.

[0056] Compared with the prior art, the compound provided by the present invention has the following beneficial effects:

[0057] 1. The compounds of this invention consist of an EZH2 targeting ligand and a degradation determinant linked together, without any linker. Compared to conventional PROTACs in the prior art, this invention, through analysis of EZH2 and its targeting ligand, directly links the EZH2 targeting ligand and the degradation determinant without a linker. The resulting compounds have a more compact stereoconformity, forming a tight degradation ternary complex that is more effective in inducing EZH2 protein degradation. Furthermore, the linker-free compounds of this invention have advantages in bioavailability and metabolic stability.

[0058] 2. The compounds of the present invention can effectively inhibit the proliferation of various tumor cells, and most of the compounds have better inhibitory effects than known small molecule EZH2 inhibitors: EPZ6438 and GSK126.

[0059] 3. The compound of the present invention can effectively degrade EZH2 protein in tumor cells and can be used as an EZH2 degrading agent.

[0060] As is well known to those skilled in the art, EZH2 degrading agents can be used as drugs for the prevention and / or treatment of cancers (including prostate cancer, breast cancer, bone marrow cancer, lymphoma, ovarian cancer, colorectal cancer, neuroblastoma, lung cancer, liver cancer, malignant rhabdomyosarcoma, glioma, esophageal cancer, gastric cancer, pancreatic cancer, or leukemia, etc.). Therefore, the compounds provided by this invention have broad application prospects as EZH2 degrading agents in the preparation of drugs for the prevention and / or treatment of cancer.

[0061] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0062] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0063] Figure 1 2 μM ED4 can degrade and stabilize EZH2 protein in KELLY cells in a time-dependent manner.

[0064] Figure 2 2 μM ED12 can degrade and stabilize EZH2 protein in KELLY cells in a time-dependent manner.

[0065] Figure 3 1 μM ED16 can degrade and stabilize EZH2 protein in MV4-11 cells in a time-dependent manner.

[0066] Figure 4 ED25 can degrade PRC2 complex protein subunits in a concentration-dependent manner after 48 hours of treatment in MV4-11 cells. Detailed Implementation

[0067] Unless otherwise specified, the raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0068] The following is a method for preparing the intermediate.

[0069] Preparation of intermediate A6:

[0070]

[0071] Step 1: Methyl 2-methyl-3-bromo-5-nitrobenzene A1 (20 mmol) and ammonium chloride (100 mmol) were dissolved in an aqueous ethanol solution (60 mL, H2O:EtOH = 1:3). The solution was heated to 80 °C, and then iron powder (200 mmol) was added in three portions. The reaction was completed after 1 hour. The mixture was filtered with diatomaceous earth as an aid. The solvent was removed by vacuum distillation of the filtrate. The residue was extracted with DCM, dried over Na2SO4, and concentrated to obtain product A2. No further purification was required. MS (m / z) [M+H] + :244.0.

[0072] Step 2: A2 (4 g, 17.4 mmol) and tetrahydropyranone (4.4 g, 52.2 mmol) were dissolved in 50 mL of chloroform. Acetic acid (2.1 g, 34.8 mmol) was added, and the mixture was stirred at room temperature for 3 h. Then, sodium triacetylborohydride (2.7 g, 43.6 mmol) was added, and the mixture was stirred overnight. After the reaction was complete, the solvent was removed by vacuum distillation, and the residue was purified by silica gel column chromatography to obtain product A3 (3.3 g, 57.8%). MS (m / z) [M+H] + 328.1.

[0073] Step 3: Under nitrogen protection, 3g of A3 was added to 1,2-dichloroethane, and anhydrous acetaldehyde (2.3g) was slowly added with stirring. Acetic acid (1.1g, 18.2mmol, 2.0eq) was then added over 30 minutes. The reaction mixture was allowed to naturally reach room temperature and stirred for 1 hour. Sodium triacetoxyborohydride (1.48g) was slowly added in portions, controlling the addition rate to maintain the reaction system temperature below 5°C. After 2 hours, the mixture was inverted to room temperature and stirred overnight. After the TLC reaction was complete, the reaction system was cooled to 0°C, and 100mL of ice water was added. An excess of saturated sodium bicarbonate solution was slowly added with stirring. After the addition was complete, the mixture was stirred for 30 minutes and allowed to stand. The aqueous phase was separated, and the aqueous phase was extracted with dichloromethane. The organic layers were combined, washed twice with water, and separated again. The organic phase was separated and concentrated under reduced pressure to constant weight, yielding a yellow oily liquid. MS (m / z) [M+H] + 356.1.

[0074] Step 4: Under nitrogen protection, 2d (2g, 5.6mmol) was added to 25mL of methanol in one go. The temperature was raised to 60℃, and then 20mL of sodium hydroxide aqueous solution (2M) was slowly added dropwise while maintaining the temperature. The reaction solution gradually changed from a light green clear liquid to an emulsion, and finally to a light green clear liquid. After maintaining the temperature for 1 hour, the reaction was monitored by TLC to indicate completion. The reaction solution was transferred to a rotary evaporator, and most of the methanol was removed under reduced pressure. 100mL of water was added to the residue, and the mixture was stirred for 10 minutes until the solid was completely dissolved. The temperature was raised to 65℃, and hydrochloric acid (2M) was added to adjust the pH to 2-3, causing precipitation. The heating bath was stopped, and the mixture was cooled to room temperature and stirred for 0.5 hours. The mixture was filtered, and the filter cake was thoroughly washed with ice water. After drying the filter cake, it was vacuum dried to obtain A5 (1.75g, 91.2%) white solid. MS (m / z) [M+H] + :342.1.

[0075] Step 5: A5 (1.5 g, 4.4 mmol) and 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (0.61 g, 4 mmol) were dissolved in DMSO (20 mL), and HOAT (0.55 g, 1.5 mmol) and EDCI (0.84 g, 2.2 mmol) were added. The reaction mixture was stirred at 45 °C for 20 h. After the reaction was completed by TLC monitoring, the reaction mixture was poured into ice water (100 mL), stirred for 30 min, and a precipitate was formed. The precipitate was filtered, washed with water, dried, dissolved in a methanol-chloroform mixture (10:1), mixed, and purified by silica gel column chromatography to obtain a yellow solid A6 (1.42 g, 68%). MS (m / z) [M+H] + :476.2.

[0076] Preparation of intermediates B4, B5, and B6:

[0077]

[0078] Step 1: Methyl 6-bromo-1H-indazole-4-carboxylate B1 (20 mmol), isopropane iodophosphate (24 mmol), and cesium carbonate (30 mmol) were dissolved in acetonitrile (60 mL). The mixture was heated to 60 °C and stirred for 8 hours until the reaction was complete. The mixture was filtered with diatomaceous earth as an aid. The solvent was removed by vacuum distillation of the filtrate. The residue was purified by silica gel column chromatography to obtain product B2. MS (m / z) [M+H] + :297.0.

[0079] Step 2: Add 10 mmol of B2 to 25 mL of methanol all at once. Heat to 60°C, then slowly add 20 mL of 2 M sodium hydroxide aqueous solution while maintaining the temperature. The reaction solution gradually changes from a light green clear liquid to an emulsion, and finally to a light green clear liquid. After maintaining the temperature for 1 hour, monitor the reaction for completion using TLC. Transfer the reaction solution to a rotary evaporator, remove most of the methanol under reduced pressure, add 100 mL of water to the residue, and stir for 10 minutes until the solid is completely dissolved. Heat to 65°C, add hydrochloric acid (2 M) to adjust the pH to 2-3, causing precipitation. Stop the heating process, cool to room temperature, and stir for 0.5 hours. Filter, wash the filter cake thoroughly with ice water, and dry the filter cake under vacuum to obtain a white solid, B3. MS (m / z) [M+H] + :282.9.

[0080] Step 3: B3 (4 mmol) and 3-(aminomethyl)-4,6-dimethylpyridin-2(1H)-one (4.8 mmol) were dissolved in DMSO (20 mL), and HOAT (6 mmol) and EDCI (6 mmol) were added. The reaction mixture was stirred at 45 °C for 20 h. After the reaction was completed by TLC monitoring, the reaction mixture was poured into ice water (100 mL), stirred for 30 min, and a precipitate was formed. The precipitate was filtered, washed with water, dried, dissolved in a methanol-chloroform mixture (10:1), mixed, and purified by silica gel column chromatography to obtain a yellow solid B4. MS (m / z) [M+H] + :417.1.

[0081] The synthesis methods of intermediates B5 and B6 are similar to those of B4. They are prepared by amide condensation reaction of 3-(aminomethyl)-6-methyl-4-propyl-6-methylpyridin-2(1H)-one and 3-aminomethyl-4-methoxy-6-methylpyridin-2(1H)-one with B3, respectively.

[0082] The following are examples of the preparation of the target compound of the present invention.

[0083] Example 1: ED-1

[0084]

[0085] Preparation of intermediate (1): A6 (1 mmol) and 4-(4-BOC-piperazinylmethyl)phenylboronic acid pinacol ester (1.2 mmol) were dissolved in a mixed solution of 1,4-dioxane and water (4:1, 30 mL), K2CO3 (2 mmol) and Pd(dppf)Cl2 (0.05 mmol) were added. After nitrogen protection, the mixture was reacted at 100 °C for 8 h, and then cooled to room temperature. The solvent was removed by vacuum distillation, and the mixture was dissolved in ethyl acetate and filtered with diatomaceous earth as an aid. The filtrate was extracted, dried with anhydrous sodium sulfate, concentrated, mixed, and purified by silica gel column chromatography to obtain the product. No further purification was required. The product was directly added to 25 mL of trifluoroacetic acid / dichloromethane solution (10%) and reacted at room temperature for 1 h. The solvent was then removed by vacuum distillation. The residue was neutralized to pH 8 with saturated sodium carbonate solution (2 M), precipitating out a precipitate. The precipitate was filtered, washed with water, and dried to obtain intermediate (I). MS (m / z) [M+H] + :573.4.

[0086] Preparation of ED-1: Intermediate (I) (0.25 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq), and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.3 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3–8 h. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then obtained by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H),11.07(s,1H),8.19(t,J=4.9Hz,1H),7.68(d,J=8.5Hz,1H),7.60(d,J=7.8Hz,2H),7.47–7.37(m,3 H),7.34(d,J=2.3Hz,1H),7.25(dd,J=9.5,2.0Hz,2H),5.86(s,1H),5.07(dd,J=12.9,5.4Hz,1H),4.30(d,J=5.0Hz,2H),3.83(d,J=11.3 Hz,2H),3.56(s,2H),3.46(t,J=4.9Hz,4H),3.25(t,J=11.5Hz,2H),3.10(t,J=6.9Hz,2H),3.06–2.98(m,1H),2.88(m,1H),2.60(m,2H), 2.53(s,4H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.06–1.97(m,1H),1.67(d,J=12.4Hz,2H),1.60–1.46(m,2H),0.84(t,J=6.9Hz,3H).

[0087] Example 2: ED-2

[0088]

[0089] Preparation of intermediate (2): The preparation method was the same as that of intermediate (I). A6 (0.5 mmol) was coupled with 4-(4-BOC-piperazine-1-carbonyl)phenylboronic acid pinacol ester (0.6 mmol) via a SUZUKI coupling reaction, followed by deprotection of the Boc layer to obtain intermediate (2). MS (m / z) [M+H] + :586.1.

[0090] Preparation of ED-2: Intermediate (2) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.44(s,1H),11.03(s,1H),8.21(t,1H),7.68(d,J=8.5Hz,1H),7.58(d,J= 7.8Hz,2H),7.43(m,3H),7.36(d,1H),7.25(m,2H),5.86(s,1H),5.08(m,1H),4.29(d,2H),3.83(d, 2H),3.46(t,J=5.1Hz,4H),3.23(t,2H),3.14(t,2H),3.04(m,1H),2.88(m,1H),2.65(m,2H),2.58( s,4H),2.26(s,3H),2.20(s,3H),2.13(s,3H),2.01(m,1H),1.68(d,2H),1.56(m,2H),0.85(t,3H).

[0091] Example 3: ED-3

[0092]

[0093] Preparation of intermediate (3): The preparation method was the same as that of intermediate (I). A6 (0.5 mmol) was coupled with 4-[4-(N-BOC)piperazin-1-yl]phenylboronic acid pinacol ester (0.6 mmol) via a SUZUKI coupling reaction, followed by deprotection of the Boc layer to obtain intermediate (3). MS (m / z) [M+H] +:558.2.

[0094] Preparation of ED-3: Intermediate (3) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H),11.07(s,1H),8.18(t,1H),7.68(d,J=8.6Hz,1H),7.60(d,2H),7.47–7. 37(m,3H),7.34(d,1H),7.25(m,2H),5.86(s,1H),5.07(dd,J=12.9,5.4Hz,1H),4.29(d,J=4.9Hz,2H),3.83(d, 2H),3.73(t,4H),3.68–3.57(m,4H),3.25(t,2H),3.08(d,2H),3.02(s,1H),2.92(m,1H),2.59(d,J=13.9Hz,2 H),2.25(d,J=2.1Hz,3H),2.21(s,3H),2.11(s,3H),2.06–1.99(m,1H),1.67(d,2H),1.52(d,2H),0.87(t,3H).

[0095] Example 4: ED-4

[0096]

[0097] Preparation of intermediate (4): The preparation method was the same as that of intermediate (I). A6 (0.5 mmol) was coupled with 4-[4-(N-BOC)piperazin-1-yl]pyridineboronic acid pinacol ester (0.6 mmol) via a SUZUKI coupling reaction, followed by deprotection of the Boc layer to obtain intermediate (4). MS (m / z) [M+H] + :559.2.

[0098] Preparation of ED-4: Intermediate (4) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.46 (s, 1H), 11.09 (s, 1H), 8.44 (d, J = 2.5Hz, 1H), 8. 16(d,J=5.2Hz,1H),7.86(dd,J=9.0,2.5Hz,1H),7.72(d,J=8.6Hz,1H),7.39( dd,J=9.5,2.2Hz,2H),7.30(dd,J=8.8,2.3Hz,1H),7.19(d,J=2.0Hz,1H),6.9 6(d,J=8.9Hz,1H),5.86(s,1H),5.08(dd,J=12.9,5.4Hz,1H),4.29(d,J=4.9H z,2H),3.83(d,J=11.5Hz,2H),3.73(t,J=5.0Hz,4H),3.68–3.57(m,4H),3.25 (t,J=11.3Hz,2H),3.08(d,J=7.1Hz,2H),3.02(s,1H),2.94–2.84(m,1H),2.5 9(d,J=14.1Hz,2H),2.23(d,J=2.0Hz,3H),2.21(s,3H),2.11(s,3H),2.06–1. 99(m,1H),1.67(d,J=11.9Hz,2H),1.52(d,J=11.7Hz,2H),0.87–0.79(m,3H).

[0099] Example 5: ED-5

[0100]

[0101] Preparation of intermediate (5):

[0102] The preparation method was the same as for intermediate (I), using A6 (0.3 mmol) and tert-butyl6-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (0.36 mmol) via SUZUKI coupling reaction, followed by deprotection of Boc to obtain intermediate (5). MS (m / z) [M+H] + :584.4.

[0103] Preparation of ED-5: Intermediate (5) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.46 (s, 1H), 11.08 (s, 1H), 8.20 (t, J = 5.0Hz, 1H), 7. 64(d,J=8.2Hz,1H),7.57(d,J=7.8Hz,2H),7.40(d,J=1.9Hz,1H),7.35(d,J=7. 9Hz,2H),7.22(d,J=1.8Hz,1H),6.79(d,J=2.1Hz,1H),6.64(dd,J=8.4,2.2Hz ,1H),5.86(s,1H),5.06(dd,J=12.9,5.4Hz,1H),4.30(d,J=4.9Hz,2H),4.12(s ,4H),3.89–3.77(m,2H),3.60(s,2H),3.38(s,4H),3.25(t,J=11.5Hz,2H),3. 08(p,J=6.0,5.3Hz,2H),3.01(q,J=7.5,5.7Hz,1H),2.94–2.80(m,1H),2.58(d d,J=19.8,6.5Hz,2H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.04–1.95(m,1H ), 1.66 (d, J = 12.2Hz, 2H), 1.53 (tt, J = 12.1, 6.1Hz, 2H), 0.83 (t, J = 6.9Hz, 3H).

[0104] Example 6: ED-6

[0105]

[0106] Preparation of intermediate (5):

[0107] The preparation method was the same as for intermediate (I), using A6 (0.3 mmol) and tert-butyl6-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2,6-diazaspiro[3.4]octane-2-carboxylate (0.36 mmol) via SUZUKI coupling reaction, followed by deprotection of Boc to obtain intermediate (6). MS (m / z) [M+H] + :598.4.

[0108] Preparation of ED-6: Intermediate (6) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1H NMR (400MHz, DMSO-d6) δ11.47 (s, 1H), 11.08 (s, 1H), 8.20 (d, J = 4.9Hz, 1H), 7.61(dd,J=24.6,8.0Hz,3H),7.39(d,J=6.4Hz,3H),7.22(s,1H),6.90(s,1 H),6.80(d,J=8.6Hz,1H),5.86(s,1H),5.06(dd,J=12.9,5.4Hz,1H),4.30( d,J=4.8Hz,2H),3.88–3.79(m,2H),3.71(s,2H),3.57(s,2H),3.43(t,J=6.8 Hz,4H),3.26(d,J=12.0Hz,3H),3.20(d,J=18.3Hz,2H),3.08(p,J=6.6,6.1 Hz,2H),3.00(d,J=10.6Hz,1H),2.89(td,J=15.9,14.7,5.3Hz,1H),2.62–2 .54(m,1H),2.25(s,3H),2.21(s,5H),2.11(s,3H),2.01(d,J=11.6Hz,1H), 1.66(d,J=12.5Hz,2H), 1.53(tt,J=14.5,7.3Hz,2H), 0.83(t,J=7.0Hz,3H).

[0109] Example 7: ED-7

[0110]

[0111] Preparation of intermediate (7):

[0112] The preparation method was the same as for intermediate (I), using A6 (0.3 mmol) and tert-butyl7-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2,7-diazaspiro[3.5]nonane-2-carboxylate (0.36 mmol) via SUZUKI coupling reaction, followed by deprotection of Boc to obtain intermediate (7). MS (m / z) [M+H] + :612.4.

[0113] Preparation of ED-7: Intermediate (7) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.47(s,1H),11.07(s,1H),8.19(t,J=5.0Hz,1H),7.6 3(d,J=8.3Hz,1H),7.58(d,J=7.9Hz,2H),7.45–7.29(m,3H),7.24(d,J=1.8Hz,1 H),6.77(d,J=2.1Hz,1H),6.64(dd,J=8.4,2.1Hz,1H),5.86(s,1H),5.06(dd,J= 12.8,5.4Hz,1H),4.30(d,J=4.9Hz,2H),3.88–3.79(m,2H),3.74(s,4H),3.48(s ,2H),3.30–3.20(m,2H),3.09(q,J=7.2Hz,2H),3.01(dt,J=10.8,3.8Hz,1H),2. 88(ddd,J=17.3,13.9,5.5Hz,1H),2.64–2.53(m,2H),2.35(s,4H),2.25(s,3H), 2.21(s,3H),2.11(s,3H),2.01(dd,J=9.2,4.3Hz,1H),1.77(t,J=5.3Hz,4H),1. 67(dd,J=12.9,3.5Hz,2H), 1.53(qd,J=11.7,4.3Hz,2H), 0.84(t,J=7.0Hz,3H).

[0114] Example 8: ED-8

[0115]

[0116] Preparation of intermediate (8):

[0117] The preparation method was the same as for intermediate (I), using A6 (0.3 mmol) and tert-butyl2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2,6-diazaspiro[3.4]octane-6-carboxylate (0.36 mmol) via SUZUKI coupling reaction, followed by deprotection of Boc to obtain intermediate (8). MS (m / z) [M+H] + :598.2.

[0118] Preparation of ED-8: Intermediate (8) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.47(s,1H),11.08(s,1H),8.20(d,J=4.9Hz,1H),7.64(d,J=8.4Hz,1H),7.58(d,J=7.7Hz,2H),7.39(d,J=6.4Hz,3H), 7.22(s,1H),6.90(s,1H),6.80(d,J=8.5Hz,1H),5.86(s,1H),5.06(dd, J=13.0,5.3Hz,1H),4.30(d,J=4.8Hz,2H),3.90–3.78(m,2H),3.71(s,2H ),3.57(s,2H),3.41(s,4H),3.23(m,6H),3.08(p,J=6.2,5.4Hz,2H),3. 00(d,J=10.6Hz,1H),2.89(td,J=16.0,15.0,5.3Hz,1H),2.68–2.53(m, 2H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.01(d,J=11.5Hz,1H),1.6 6(d,J=12.5Hz,2H), 1.53(tt,J=14.6,7.4Hz,2H), 0.83(t,J=7.0Hz,3H).

[0119] Example 9: ED-9

[0120]

[0121] Preparation of intermediate (9):

[0122] The preparation method was the same as for intermediate (I), using A6 (0.3 mmol) and tert-butyl7-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate (0.36 mmol) via SUZUKI coupling reaction, followed by deprotection of Boc to obtain intermediate (9). MS (m / z) [M+H] + :612.2.

[0123] Preparation of ED-9: Intermediate (9) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.47(s,1H),11.07(s,1H),8.19(t,J=5.0Hz,1H),7.62(d,J=8.3Hz,1H),7.57(d,J=7.8Hz,2H),7.47–7.32(m,3H),7.23(s, 1H),6.88(s,1H),6.83–6.74(m,1H),5.86(s,1H),5.05(dd,J=12.8,5.4Hz ,1H),4.30(d,J=4.9Hz,2H),3.88–3.77(m,2H),3.64(s,2H),3.42(dd,J=1 6.8,8.6Hz,4H),3.24(t,J=11.4Hz,2H),3.08(q,J=7.7,7.3Hz,2H),3.03– 2.95(m,1H),2.89(m,1H),2.73–2.65(m,1H),2.65–2.58(m,2H),2.56(q,J =4.9Hz,2H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.00(m,4H),1.81(t, J=7.5Hz,2H),1.65(d,J=12.3Hz,2H),1.52(m,2H),0.83(t,J=6.9Hz,3H).

[0124] Example 10: ED-10

[0125]

[0126] Preparation of intermediate (10):

[0127] The preparation method was the same as for intermediate (I), using A6 (0.3 mmol) and tert-butyl8-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2,8-diazaspiro[4.5]decane-2-carboxylate (0.36 mmol) via SUZUKI coupling reaction, followed by deprotection of Boc to obtain intermediate (10). MS (m / z) [M+H] + :626.4.

[0128] Preparation of ED-10: Intermediate (10) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1H NMR (400MHz, DMSO-d6) δ11.48 (s, 1H), 11.08 (s, 1H), 8.20 (t, J = 5.0Hz, 1H), 7.63 (d, J=8.4Hz,1H),7.58(d,J=7.8Hz,2H),7.47–7.33(m,3H),7.24(d,J=1.8Hz,1H),6.92 (d,J=2.2Hz,1H),6.80(dd,J=8.6,2.2Hz,1H),5.86(s,1H),5.06(dd,J=12.8,5.4Hz ,1H),4.31(d,J=4.9Hz,2H),3.83(dd,J=11.4,3.3Hz,2H),3.51(s,2H),3.45(t,J=6 .9Hz,2H),3.31–3.21(m,4H),3.09(q,J=7.5,7.0Hz,2H),3.01(td,J=10.7,10.0,4. 5Hz,1H),2.95–2.83(m,1H),2.67–2.53(m,2H),2.45(s,2H),2.39(d,J=7.9Hz,2H), 2.26(s,3H),2.21(s,3H),2.11(s,3H),2.02(td,J=8.4,7.8,4.6Hz,1H),1.87(t,J= 7.0Hz,2H),1.70–1.63(m,2H),1.54(dt,J=17.7,5.3Hz,6H),0.84(t,J=6.9Hz,3H).

[0129] Example 11: ED-11

[0130]

[0131] Preparation of intermediate (11):

[0132] The preparation method was the same as for intermediate (I), using A6 (0.3 mmol) and tert-butyl2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (0.36 mmol) via SUZUKI coupling reaction, followed by deprotection of Boc to obtain intermediate (11). MS (m / z) [M+H] + :612.4.

[0133] Preparation of ED-11: Intermediate (11) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H),11.07(s,1H),8.19(t,J=5.0Hz,1H),7.64(d,J=8.5Hz,1H),7.55(d,J=7.9Hz,2H),7.43–7.27(m, 4H),7.27–7.14(m,2H),5.86(s,1H),5.06(dd,J=12.9,5.4Hz,1H),4.29(d,J=4.9Hz,2H),3.83(d,J=11.5Hz,2H),3.62(s,2H),3.44(t, J=5.3Hz,4H),3.23(d,J=11.4Hz,2H),3.09(q,J=7.1Hz,2H),3.00(s,4H),2.93–2.83(m,1H),2.63–2.53(m,2H),2.24(s,3H),2.21(s,3 H),2.10(s,3H),2.05–1.95(m,2H),1.76(t,J=5.2Hz,4H),1.66(d,J=12.3Hz,2H),1.52(qd,J=12.0,4.0Hz,2H),0.83(t,J=7.0Hz,3H).

[0134] Example 12: ED-12

[0135]

[0136] Preparation of intermediate (12):

[0137] The preparation method was the same as for intermediate (I), using A6 (0.3 mmol) and tert-butyl2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2,8-diazaspiro[4.5]decane-8-carboxylate (0.36 mmol) via SUZUKI coupling reaction, followed by deprotection of Boc to obtain intermediate (12). MS (m / z) [M+H] + :626.5.

[0138] Preparation of ED-12: Intermediate (12) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H),11.08(s,1H),8.19(t,J=5.0Hz,1H),7.64(d,J=8.4Hz,1H),7.58(d,J=7.7Hz,2H),7.43–7. 35(m,3H),7.31(d,J=2.2Hz,1H),7.27–7.18(m,2H),5.86(s,1H),5.06(dd,J=12.9,5.4Hz,1H),4.30(d,J=5.0Hz,2H),3.83(dd,J =11.5,3.6Hz,2H),3.59(s,2H),3.54–3.40(m,4H),3.25(t,J=11.4Hz,2H),3.09(q,J=7.1Hz,2H),3.03–2.96(m,1H),2.89(m,1H) ,2.57(m,4H),2.40(s,2H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.05–1.96(m,1H),1.72–1.47(m,10H),0.83(t,J=6.9Hz,3H).

[0139] Example 13: ED-13

[0140]

[0141] Preparation of intermediate (13):

[0142] The preparation method was the same as for intermediate (I), using A6 (0.3 mmol) and tert-butyl9-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (0.36 mmol) via SUZUKI coupling reaction, followed by deprotection of Boc to obtain intermediate (13). MS (m / z) [M+H] + :640.3.

[0143] Preparation of ED-13: Intermediate (13) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H),11.07(s,1H),8.19(t,J=5.0Hz,1H),7.65(d,J=8.4Hz,1H),7.58(d,J=7.8Hz,2H),7.45–7.33 (m,3H),7.29(s,1H),7.21(d,J=13.3Hz,2H),5.86(s,1H),5.06(dd,J=12.9,5.4Hz,1H),4.30(d,J=4.7Hz,2H),3.89–3.77(m,2H),3. 51(s,2H),3.44(d,J=5.7Hz,4H),3.25(t,J=11.4Hz,2H),3.08(m,2H),3.04–2.96(m,1H),2.88(m,1H),2.64–2.53(m,2H),2.39(s,4 H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.05–1.97(m,1H),1.66(d,J=12.2Hz,2H),1.52(h,J=6.7Hz,10H),0.83(t,J=6.9Hz,3H).

[0144] Example 14: ED-14

[0145]

[0146] Preparation of intermediate (14):

[0147] The preparation method was the same as for intermediate (I), using A6 (0.3 mmol) and tert-butyl5-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)hexahydropyrrolo[3,4-c]pyrrole-2(1H)-carboxylate (0.36 mmol) via SUZUKI coupling reaction, followed by deprotection of Boc to obtain intermediate (14). MS (m / z) [M+H] + :598.5.

[0148] Preparation of ED-14: Intermediate (14) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.58–11.32(m,1H),11.08(s,1H),8.18(t,J=5.0Hz,1H),7.66(d,J=8.3Hz,1H),7.57(d,J=7.8Hz,2H),7.43–7. 31(m,3H),7.22(s,1H),6.96(s,1H),6.87(d,J=8.6Hz,1H),5.85(s,1H),5.07(dd,J=12.9,5.4Hz,1H),4.29(d,J=5.0Hz,2H),3.82(d,J= 11.6Hz,2H),3.63(d,J=7.6Hz,4H),3.25(q,J=11.7Hz,4H),3.08(q,J=7.9,7.2Hz,2H),3.03–2.95(m,2H),2.89(m,1H),2.59(m,3H),2.2 4(s,3H),2.20(s,3H),2.10(s,3H),2.06–1.98(m,1H),1.65(d,J=12.2Hz,2H),1.52(qt,J=10.4,7.6,5.9Hz,2H),0.82(t,J=6.9Hz,3H).

[0149] Example 15: ED-15

[0150]

[0151] Preparation of intermediate (15): Dissolve A6 (0.5 mmol) and tert-butyl(1S,4S)-5-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate (0.6 mmol) in a mixed solution of 1,4-dioxane and water (4:1, 30 mL), add K2CO3 (1 mmol) and Pd(dppf)Cl2 (0.05 mmol), and react at 100 °C for 8 h under nitrogen protection, then cool to room temperature. Remove the solvent from the reaction solution by vacuum distillation, dissolve in ethyl acetate, and filter with diatomaceous earth as an aid. Extract the filtrate, dry with anhydrous sodium sulfate, concentrate, mix, and purify by silica gel column chromatography to obtain the product. No further purification was required. The sample was directly added to 25 mL of a 10% trifluoroacetic acid / dichloromethane solution and reacted at room temperature for 1 h. The solvent was then removed by vacuum distillation. The residue was neutralized to pH 8 with a saturated sodium carbonate solution (2 M), precipitating out a precipitate. The precipitate was filtered, washed with water, and dried to obtain intermediate (15). MS (m / z) [M+H] + :582.5.

[0152] Preparation of ED-15: Intermediate (15) (0.25 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.3 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1H NMR (400MHz, DMSO-d6) δ11.46(s,1H),11.08(s,1H),8.19(t,1H),7.69(d,J=8.4Hz,1H),7.60(d,J=7.8Hz,2H),7.39(m,3H ),7.35(d,1H),7.24(dd,2H),5.88(s,1H),5.07(m,1H),4.31(d,2H),3.83(d,J=11.3Hz,2H),3.56(s,2H),3.43(m,1H),3. 37(d,2H),3.25(t,J=11.3Hz,2H),3.10(t,J=6.9Hz,2H),3.06–2.98(m,1H),2.88(m,1H),2.60(m,2H),2.53(m,2H),2.38( m,1H),2.26(s,3H),2.21(s,3H),2.10(s,3H),2.03(m,1H),1.77(t,2H),1.68(d,2H),1.58(m,2H),0.84(t,J=6.9Hz,3H).

[0153] Example 16: ED-16

[0154]

[0155] Preparation of intermediate (16): 3-(1-oxo-5-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione hydrochloride (0.25 mmol), 4-bromomethylphenylboronic acid pinacol ester (0.24 mmol) and DIPEA (0.5 mmol) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3 h. After the reaction was completed, the product was extracted with ethyl acetate, dried with Na2SO4, and the solvent was removed by vacuum distillation before being used directly in the next step of the reaction.

[0156] Preparation of ED-16: A6 (0.15 mmol) and intermediate (16) (0.2 mmol) were dissolved in a mixed solution of 1,4-dioxane and water (4:1, 15 mL). K2CO3 (0.3 mmol) and Pd(dppf)Cl2 (0.015 mmol) were added. After nitrogen protection, the mixture was reacted at 100 °C for 4 h, then cooled to room temperature. The solvent was removed by vacuum distillation, and the mixture was dissolved in ethyl acetate and filtered with diatomaceous earth as an aid. The filtrate was extracted, dried over anhydrous sodium sulfate, concentrated, mixed, and purified by silica gel column chromatography to obtain the product. HRMS m / z calculated for C 47 H 55 N7O6[M+H] + :814.4287,found:814.4296.

[0157] Example 17: ED-17

[0158]

[0159] Preparation of intermediate (17): 3-(1-oxo-5-(piperazin-1-yl)isoindolin-2-yl)piperidine-2,6-dione hydrochloride (0.25 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)benzoyl chloride (0.24 mmol) and DIPEA (0.5 mmol) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation before being used directly in the next step of the reaction.

[0160] Preparation of ED-17: A6 (0.15 mmol) and intermediate (17) (0.2 mmol) were dissolved in a mixed solution of 1,4-dioxane and water (4:1, 15 mL). K2CO3 (0.3 mmol) and Pd(dppf)Cl2 (0.015 mmol) were added. After nitrogen protection, the mixture was heated to 100 °C for 4 h and then cooled to room temperature. The solvent was removed by vacuum distillation, and the mixture was dissolved in ethyl acetate and filtered with diatomaceous earth as an aid. The filtrate was extracted, dried over anhydrous sodium sulfate, concentrated, mixed, and purified by silica gel column chromatography to obtain the product. HRMS m / z calculated for C 47 H 55 N7O6[M+H] + :828.4079,found:828.4083.

[0161] Example 18: ED-18

[0162]

[0163] Preparation of intermediate (18): 3-(4-(piperazin-1-yl)phenyl)piperidin-2,6-dione (0.3 mmol, 1 eq), pinacol ester of 4-bromomethylphenylboronic acid (0.36 mmol, 1.2 eq), and DIPEA (0.6 mmol, 2 eq) were dissolved in DMF (5 mL) and reacted at 65 °C for 4 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then obtained by silica gel column chromatography. MS (m / z) [M+H] + :490.3.

[0164] Preparation of ED-18: Dissolve A6 (0.15 mmol) and intermediate (18) (0.2 mmol) in a mixed solution of 1,4-dioxane and water (4:1, 15 mL), add K2CO3 (0.3 mmol) and Pd(dppf)Cl2 (0.015 mmol), and react at 100 °C for 4 h under nitrogen protection, then cool to room temperature. Remove the solvent by vacuum distillation, dissolve in ethyl acetate, and filter with diatomaceous earth as an aid. Extract the filtrate, dry with anhydrous sodium sulfate, concentrate, mix, and purify by silica gel column chromatography to obtain the product. 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H),10.60(s,1H),8.19(t,J=5.0Hz,1H),7.57(d,J=7.7Hz,2H),7.49–7.28(m,3H),7.26(d,J=7.8Hz,2H),7.2 2(s,1H),6.68(d,J=7.8Hz,2H),5.86(s,1H),4.29(d,J=4.7Hz,2H),4.10(d,J=5.3Hz,1H),3.83(d,J=11.1Hz,2H),3.49(d,J=11.8Hz,2H),3.31 (s,2H),3.25(t,J=11.4Hz,2H),3.17(d,J=6.0Hz,2H),3.09(q,J=7.6,7 .2Hz,2H),3.02(s,1H),2.71–2.56(m,2H),2.38(s,4H),2.25(s,3H),2. 21(s,3H),2.11(s,3H),2.01(d,J=12.8Hz,1H),1.90–1.82(m,1H),1.67 (d,J=12.5Hz,2H),1.53(tt,J=12.5,6.3Hz,2H),0.83(t,J=7.1Hz,3H).

[0165] Example 19: ED-19

[0166]

[0167] Preparation of intermediate (19): 3-(4-(piperazin-1-yl)phenyl)aminopiperidine-2,6-dione (0.3 mmol, 1 eq), pinacol ester of 4-bromomethylphenylboronic acid (0.36 mmol, 1.2 eq), and DIPEA (0.6 mmol, 2 eq) were dissolved in DMF (5 mL) and reacted at 65 °C for 4 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. MS (m / z) [M+H] + :505.2.

[0168] Preparation of ED-19: A6 (0.15 mmol) and intermediate (19) (0.2 mmol) were dissolved in a mixed solution of 1,4-dioxane and water (4:1, 15 mL). K2CO3 (0.3 mmol) and Pd(dppf)Cl2 (0.015 mmol) were added. After nitrogen protection, the mixture was heated to 100 °C for 4 h and then cooled to room temperature. The solvent was removed by vacuum distillation, and the mixture was dissolved in ethyl acetate and filtered with diatomaceous earth as an aid. The filtrate was extracted, dried over anhydrous sodium sulfate, concentrated, mixed, and purified by silica gel column chromatography to obtain the product. HRMS m / z calculated for C 47 H 55 N7O6[M+H] + :774.4337,found:774.4331.

[0169] Example 20: ED-20

[0170]

[0171] Preparation of intermediate (20): 3-(4-(piperazin-1-yl)phenyl)aminopiperidine-2,6-dione (0.3 mmol, 1 eq), pinacol ester of 4-bromomethylphenylboronic acid (0.36 mmol, 1.2 eq), and DIPEA (0.6 mmol, 2 eq) were dissolved in DMF (5 mL) and reacted at 65 °C for 4 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. MS (m / z) [M+H] + :505.2.

[0172] Preparation of ED-20: A6 (0.15 mmol) and intermediate (19) (0.2 mmol) were dissolved in a mixed solution of 1,4-dioxane and water (4:1, 15 mL). K2CO3 (0.3 mmol) and Pd(dppf)Cl2 (0.015 mmol) were added. After nitrogen protection, the mixture was reacted at 100 °C for 4 h, and then cooled to room temperature. The solvent was removed by vacuum distillation, and the mixture was dissolved in ethyl acetate and filtered with diatomaceous earth as an aid. The filtrate was extracted, dried over anhydrous sodium sulfate, concentrated, mixed, and purified by silica gel column chromatography to obtain the product. HRMS m / z calculated for C 47 H 55 N7O6[M+H] + :774.4337,found:774.4331.

[0173] Example 21: ED-21

[0174]

[0175] Preparation of ED-21: Intermediate (I) (0.25 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (0.3 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3–8 h. After the reaction was complete, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.48(s,1H),11.10(s,1H),8.21(s,1H),7.75–7.66(m,1H),7.60(d,J=7.8Hz,2H),7.41(d,J=7.6Hz,3H),7. 34(dd,J=13.3,7.8Hz,2H),7.25(d,J=1.8Hz,1H),5.86(s,1H),5.10(dd,J=12.8,5.4Hz,1H),4.31(d,J=5.0Hz,2H),3.88–3.78(m,2H) ,3.58(s,2H),3.33–3.27(m,4H),3.26–3.17(m,2H),3.08(m,2H),3.02(q,J=7.1,5.4Hz,1H),2.93–2.82(m,1H),2.59(t,J=5.0Hz,4H ),2.51(d,2H),2.26(s,3H),2.22(s,3H),2.11(s,3H),2.07–1.99(m,1H),1.67(d,J=12.1Hz,2H),1.54(m,2H),0.84(t,J=6.9Hz,3H).

[0176] Example 22: ED-22

[0177]

[0178] Preparation of ED-22: Intermediate (3) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1H NMR (400MHz, DMSO-d6) δ11.49(s,1H),11.11(s,1H),8.18(t,J=5.0Hz,1H),7.74(t,J=7.8Hz,1H),7.53(d,J=8.3Hz,2H),7.43–7.33(m,3 H),7.21(s,1H),7.09(d,J=8.5Hz,2H),5.86(s,1H),5.13(dd,J=12.8,5.4Hz,1H),4.31(d,J=4.9Hz,2H),3.90–3.77(m,2H),3.51–3.42(m ,4H),3.39(d,J=4.9Hz,4H),3.30–3.21(m,2H),3.08(d,J=9.0Hz,2H),3.04–2.96(m,1H),2.90(m,1H),2.61(dt,J=18.8,4.1Hz,1H),2.5 4(d,1H),2.24(s,3H),2.22(s,3H),2.11(s,3H),2.05(dd,J=9.8,4.7Hz,1H),1.67(d,J=12.3Hz,2H),1.52(m,2H),0.84(t,J=6.9Hz,3H).

[0179] Example 23: ED-23

[0180]

[0181] Preparation of ED-23: Intermediate (1) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq), and 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3–8 h. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then obtained by silica gel column chromatography. HRMS m / z calculated for C 47 H 55 N7O6[M+H] + :683.3915,found:683.3918.

[0182] Example 24: ED-24

[0183]

[0184] Preparation of ED-24: GSK126 (0.15 mmol), 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (0.2 mmol), and DIPEA (0.6 mmol, 2 eq) were dissolved in DMSO (5 mL) and reacted at 65 °C for 4 hours. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.48(s,1H),11.11(s,1H),8.56(d,J=2.5Hz,1H),8.16(t,J=5.1Hz,1H),7.97(dd,J=8.9,2.5Hz,1 H),7.80–7.69(m,2H),7.41(dd,J=7.8,3.4Hz,2H),7.26(s,1H),7.23–7.16(m,1H),7.01(d,J=8.9Hz,1H),5.87(s,1H),5.1 4(dd,J=12.9,5.5Hz,1H),4.61(q,J=6.9Hz,1H),4.36(d,J=5.0Hz,2H),3.75(t,J=4.7Hz,4H),3.44(t,J=4.8Hz,4H),2.90( m,1H),2.69–2.54(m,2H),2.25(s,3H),2.17(s,3H),2.11(s,3H),2.09–2.00(m,1H),1.80(m,2H),1.41(d,3H),0.73(t,3H).

[0185] Example 25: ED-25

[0186]

[0187] Preparation of ED-25: GSK126 (0.15 mmol), 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.2 mmol), and DIPEA (0.6 mmol, 2 eq) were dissolved in DMSO (5 mL) and reacted at 65 °C for 4 hours. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1H NMR (400MHz, DMSO-d6) δ11.48(s,1H),11.10(s,1H),8.55(d,J=2.5Hz,1H),8.16(t,J=5.1Hz,1H),7.97(dd,J=8.8,2.6Hz,1H),7.80–7.67 (m,2H),7.41(d,J=2.2Hz,1H),7.31(dd,J=8.7,2.3Hz,1H),7.26(s,1H),7.22–7.16(m,1H),6.98(d,J=8.9Hz,1H),5.88(s,1H),5.09(dd,J =12.9,5.4Hz,1H),4.61(q,J=6.9Hz,1H),4.36(d,J=5.0Hz,2H),3.73(dd,J=7.2,3.6Hz,4H),3.63(dd,J=6.8,3.9Hz,4H),2.90(m,1H),2.6 8–2.56(m,1H),2.25(s,3H),2.17(s,3H),2.12(s,3H),2.08–1.99(m,1H),1.86–1.75(m,2H),1.41(d,J=6.6Hz,3H),0.73(t,J=7.3Hz,3H).

[0188] Example 26: ED-26

[0189]

[0190] Preparation of ED-26: GSK126 (0.15 mmol), 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.2 mmol), and DIPEA (0.6 mmol, 2 eq) were dissolved in DMSO (5 mL) and reacted at 65 °C for 4 hours. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1H NMR (400MHz, DMSO-d6) δ11.47(s,1H),10.66(s,1H),8.50(d,J=2.5Hz,1H),8.14(t,J=5.1Hz,1H),7.91(dd,J=8.8, 2.6Hz,1H),7.73(s,1H),7.25(s,1H),7.18(s,1H),6.91(d,J=8.9Hz,1H),5.86(s,1H),4.60(q,J=7.0Hz,1H),4.35( d,J=4.5Hz,2H),3.52(s,5H),2.83(d,J=10.7Hz,2H),2.74(s,2H),2.56(dt,J=8.8,4.8Hz,2H),2.24(s,3H),2.16( s,3H),2.11(s,3H),2.08(s,1H),1.91(d,J=12.9Hz,1H),1.80(m,2H),1.40(d,J=6.6Hz,3H),0.73(t,J=7.3Hz,3H).

[0191] Example 27: ED-27

[0192]

[0193] Preparation of intermediate (27): B4 (0.3 mmol, 1 eq), 6-(4-tert-butyloxycarbonylpiperazin-1-yl)pyridine-3-boronic acid pinacol ester (0.36 mmol, 1.2 eq), and DIPEA (0.6 mmol, 2 eq) were dissolved in a mixed solution of 1,4-dioxane and water (4:1, 15 mL). K2CO3 (0.3 mmol) and Pd(dppf)Cl2 (0.015 mmol) were added. After nitrogen protection, the mixture was heated to 100 °C for 4 h and then cooled to room temperature. The solvent was removed by vacuum distillation, and the mixture was dissolved in ethyl acetate and filtered with diatomaceous earth as an aid. The filtrate was extracted, dried over anhydrous sodium sulfate, concentrated, mixed, and purified by silica gel column chromatography to obtain the product. No further purification was required. The sample was directly added to 25 mL of a 10% trifluoroacetic acid / dichloromethane solution and reacted at room temperature for 1 h. The solvent was then removed by vacuum distillation. The residue was neutralized to pH 8 with a saturated sodium carbonate solution (2 M), precipitating out a precipitate. The precipitate was filtered, washed with water, and dried to obtain the intermediate (27). MS (m / z) [M+H] + :500.3.

[0194] Preparation of ED-27: Intermediate (27) (0.15 mmol), 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.2 mmol), and DIPEA (0.6 mmol, 2 eq) were dissolved in DMSO (5 mL) and reacted at 65 °C for 4 hours. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.54(s,1H),11.09(s,1H),8.69(d,J=2.5Hz,1H),8.60(t,J=5.0Hz,1H),8.36(s,1H),8 .14–8.03(m,2H),7.87–7.80(m,1H),7.72(d,J=8.5Hz,1H),7.41(d,J=2.2Hz,1H),7.31(dd,J=8.7,2.3Hz,1H),7. 02(d,J=8.9Hz,1H),5.91(s,1H),5.11(m,1H),4.42(d,J=4.9Hz,2H),3.87–3.71(m,4H),3.65(d,J=5.4Hz,4H),2. 95–2.84(m,1H),2.63(s,3H),2.18(s,3H)2.14(m,1H),2.03(m,2H),1.54(d,J=7.6Hz,1H),1.50(d,J=6.6Hz,6H).

[0195] Example 28: ED-28

[0196]

[0197] Preparation of intermediate (28): B5 (0.3 mmol, 1 eq), 6-(4-tert-butyloxycarbonylpiperazin-1-yl)pyridine-3-boronic acid pinacol ester (0.36 mmol, 1.2 eq), and DIPEA (0.6 mmol, 2 eq) were dissolved in a mixed solution of 1,4-dioxane and water (4:1, 15 mL). K2CO3 (0.3 mmol) and Pd(dppf)Cl2 (0.015 mmol) were added. After nitrogen protection, the mixture was heated to 100 °C for 4 h and then cooled to room temperature. The solvent was removed by vacuum distillation, and the mixture was dissolved in ethyl acetate and filtered with diatomaceous earth as an aid. The filtrate was extracted, dried over anhydrous sodium sulfate, concentrated, mixed, and purified by silica gel column chromatography to obtain the product. No further purification was required. The sample was directly added to 25 mL of a 10% trifluoroacetic acid / dichloromethane solution and reacted at room temperature for 1 h. The solvent was then removed by vacuum distillation. The residue was neutralized to pH 8 with a saturated sodium carbonate solution (2 M), precipitating out a precipitate. The precipitate was filtered, washed with water, and dried to obtain the intermediate (28). MS (m / z) [M+H] + :528.4.

[0198] Preparation of ED-28: Intermediate (28) (0.15 mmol), 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.2 mmol), and DIPEA (0.6 mmol, 2 eq) were dissolved in DMSO (5 mL) and reacted at 65 °C for 4 hours. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.54(s,1H),11.09(s,1H),8.69(d,J=2.5Hz,1H),8.60(t,J=5.0Hz,1H),8.36(s,1H),8.14–8.03(m ,2H),7.87–7.80(m,1H),7.72(d,J=8.5Hz,1H),7.41(d,J=2.2Hz,1H),7.31(dd,J=8.7,2.3Hz,1H),7.02(d,J=8.9Hz,1H),5.9 1(s,1H),5.11(ddd,J=18.1,13.1,6.1Hz,2H),4.42(d,J=4.9Hz,2H),3.87–3.71(m,4H),3.65(d,J=5.4Hz,4H),2.95–2.84(m ,1H),2.67–2.53(m,3H),2.14(s,3H),2.09–1.99(m,2H),1.54(d,J=7.6Hz,2H),1.50(d,J=6.6Hz,6H),0.88(t,J=7.3Hz,3H).

[0199] Example 29: ED-29

[0200]

[0201] Preparation of ED-29: Dissolve (27)(0.15mmol), 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (0.2mmol), and DIPEA (0.6mmol, 2eq) in DMSO (5mL), and react at 65℃ for 4 hours. After the reaction is complete, extract with ethyl acetate, dry with Na2SO4, remove the solvent by vacuum distillation, and then separate by silica gel column chromatography to obtain the corresponding product. 1 HNMR (400MHz, DMSO-d6) δ11.54(s,1H),11.10(s,1H),8.68(d,J=2.5Hz,1H),8.60(t,J=5.0Hz,1H),8.35(s,1H),8.15–8 .05(m,2H),7.89–7.79(m,1H),7.72(d,J=8.5Hz,1H),7.41(d,J=2.2Hz,1H),7.31(dd,J=8.5,2.2Hz,1H),7.02(d,J=8.9 Hz,1H),5.91(s,1H),5.12(ddt,J=18.1,13.0,6.0Hz,2H),4.42(d,J=4.9Hz,2H),3.86–3.72(m,4H),3.64(t,J=5.4Hz,4 H),2.97–2.83(m,1H),2.68–2.54(s,3H),2.14(s,3H),2.10–1.98(m,2H),1.54(d,J=7.6Hz,1H),1.50(d,J=6.6Hz,6H).

[0202] Example 30: ED-30

[0203]

[0204] Preparation of ED-30: Dissolve (27)(0.15mmol), 3-bromopiperidine-2,6-dione (0.2mmol), and DIPEA (0.6mmol, 2eq) in DMSO (5mL) and react at 65℃ for 4 hours. After the reaction is complete, extract with ethyl acetate, dry with Na2SO4, remove the solvent by vacuum distillation, and then separate by silica gel column chromatography to obtain the corresponding product. 1H NMR (400MHz, DMSO-d6) δ11.54(s,1H),11.10(s,1H),8.68(d,J=2.5Hz,1H),8.60(t,J=5.0Hz, 1H),8.35(s,1H),7.89–7.79(m,2H),7.72(d,J=8.5Hz,1H),7.02(d,J=8.9Hz,1H),5.91(s,1H) ,5.12(m,1H),4.42(d,J=4.9Hz,2H),3.81(m,4H),3.61(t,J=5.4Hz,4H),2.96–2.82(m,1H),2. 68–2.54(s,3H),2.14(s,3H),2.10–1.98(m,2H),1.54(d,J=7.6Hz,2H),1.51(d,J=6.6Hz,6H).

[0205] Example 31: ED-31

[0206]

[0207] Preparation of intermediate (31): 3-(4-(piperazin-1-yl)phenyl)piperidine-2,6-dione (0.3 mmol, 1 eq), bromopropyne (0.36 mmol, 1.2 eq), and DIPEA (0.6 mmol, 2 eq) were dissolved in DMF (5 mL) and reacted at 65 °C for 4 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then obtained by silica gel column chromatography. MS (m / z) [M+H] + :505.4.

[0208] Preparation of ED-31: Intermediate (31) (0.2 mmol, 1 eq) and A6 (0.24 mmol) were dissolved in DMF (5 mL), and CuI (0.02 mmol) and tetrakis(triphenylphosphine)palladium (0.02 mmol) were added. Under nitrogen protection, the mixture was reacted at 90 °C for 5 h, then cooled to room temperature. The solvent was removed by vacuum distillation, and the solution was dissolved in ethyl acetate, with diatomaceous earth as a filter aid. The filtrate was extracted, dried over anhydrous sodium sulfate, concentrated, mixed, and purified by silica gel column chromatography to obtain the product. HRMS m / z calculated for C 47 H 55 N7O6[M+H] + :707.1935.3915,found:707.3921.

[0209] Example 32: ED-32

[0210]

[0211] Preparation of intermediate (32): A6 (0.5 mmol) and 4-(prop-2-yn-1-yl)piperazine-1-carboxylic acid tert-butyl ester (0.6 mmol) were dissolved in DMF (5 mL), and CuI (0.02 mmol) and tetrakis(triphenylphosphine)palladium (0.02 mmol) were added. After nitrogen protection, the mixture was reacted at 90 °C for 5 h, and then cooled to room temperature. The solvent was removed by vacuum distillation, and the mixture was dissolved in ethyl acetate and filtered with diatomaceous earth as an aid. The filtrate was extracted, dried with anhydrous sodium sulfate, concentrated, mixed, and purified by silica gel column chromatography to obtain the product. The product was then directly added to 25 mL of trifluoroacetic acid / dichloromethane solution (10%) and reacted at room temperature for 1 h. The solvent was then removed by vacuum distillation, and the residue was neutralized to pH 8 with saturated sodium carbonate solution (2 M), precipitating out a precipitate. The precipitate was filtered, washed with water, and dried to obtain intermediate (32). MS (m / z) [M+H] + :520.4.

[0212] Preparation of ED-32: Intermediate (32) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H),11.08(s,1H),8.18(t,J=5.1Hz,1H),7.69(d,J=8.4Hz,1H),7.36(s,1H),7.27(d,J=9.3Hz,1H ),7.18(s,1H),6.99(s,1H),5.85(s,1H),5.07(dd,J=13.0,5.4Hz,1H),4.25(d,J=4.9Hz,2H),3.87–3.76(m,2H),3.59(s,2H),3.49( t,J=5.1Hz,4H),3.22(t,J=11.4Hz,2H),2.99(q,J=7.5,7.1Hz,2H),2.95–2.83(m,2H),2.67(t,J=4.8Hz,4H),2.59(dd,J=19.3,6.0H z,2H),2.19(d,J=6.6Hz,6H),2.10(s,3H),2.01(dd,J=12.3,6.4Hz,1H),1.59(d,J=11.0Hz,2H),1.47(m,2H),0.76(t,J=6.9Hz,3H).

[0213] Example 33: ED-33

[0214]

[0215] Preparation of intermediate (33): The preparation method was the same as that of intermediate (5). A6 (0.5 mmol) and 1-Boc-4-ethynylpiperidine (0.6 mmol) were dissolved in DMF (5 mL), and CuI (0.02 mmol) and tetrakis(triphenylphosphine)palladium (0.02 mmol) were added. Intermediate (33) was obtained by Sonogashira coupling reaction. MS (m / z) [M+H] + :505.4.

[0216] Preparation of ED-33: Intermediate (33) (0.2 mmol, 1 eq), DIPEA (0.5 mmol, 2.0 eq) and 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (0.24 mmol, 1.2 eq) were dissolved in DMSO (5 mL) and reacted at 85 °C for 3-8 h. After the reaction was completed, the product was extracted with ethyl acetate, dried over Na2SO4, and the solvent was removed by vacuum distillation. The product was then separated by silica gel column chromatography. 1 H NMR (400MHz, DMSO-d6) δ11.46(s,1H),11.08(s,1H),8.15(t,J=5.1Hz,1H),7.67(d,J=8.4Hz,1H),7.35(d,J=2.3Hz,1H),7 .26(dd,J=8.6,2.1Hz,1H),7.15(s,1H),6.97(s,1H),5.85(d,J=3.3Hz,1H),5.07(dd,J=12.9,5.4Hz,1H),4.25(d,J=4.6Hz ,2H),3.83(dd,J=11.7,7.4Hz,4H),3.22(t,J=11.4Hz,2H),3.07–2.83(m,6H),2.66–2.53(m,2H),2.19(d,J=5.0Hz,6H),2 .11(s,3H),2.06–1.98(m,2H),1.98–1.89(m,2H),1.67(m,2H),1.59(d,J=12.7Hz,2H),1.47(m,2H),0.76(t,J=7.0Hz,3H).

[0217] To illustrate the beneficial effects of the present invention, the following experimental examples are provided.

[0218] Experimental Example 1: Detection of the inhibitory activity of compounds on tumor cell proliferation

[0219] 1. Experimental Methods

[0220] The in vitro cell viability assay of the compounds of this invention against tumor cells (human neuroblastoma cells Kelly, human myeloid monocytic leukemia cells MV4-11, and human neuroblastoma cells BE2C) was performed using the MTT assay, with known small molecule EZH2 inhibitors EPZ6438 and GSK126 as positive controls.

[0221]

[0222] The method is as follows: Collect cells in the logarithmic growth phase, centrifuge, and resuspend in the appropriate culture medium to a concentration of 1.5–8 × 10⁻⁶. 4 Cell suspensions of varying concentrations (cells / mL) were seeded at 100 μL into 96-well cell culture plates and incubated overnight at 37°C in a 5% CO2 incubator. The next day, culture medium containing different concentrations of the drug was added to each well, with three replicates for each concentration. Medium containing 0.1% DMSO was used as a negative control. After incubating the 96-well plates at 37°C in a 5% CO2 incubator for different times, 20 μL of 5 mg / mL MTT solution was added to each well, and the plates were incubated at 37°C for 2.5 h. For adherent cells, the supernatant was discarded, and 150 μL of DMSO was added to each well to dissolve the purple formazan crystals. For suspension cells, 50 μL of 20% SDS solution was added to each well and incubated overnight to dissolve the purple formazan crystals. Finally, the absorbance value at 570 nm (A570) was measured using a microplate reader (A570 is proportional to the number of viable cells). The cell growth inhibition curve was fitted using Graph Prism 5.0 software, and the half-maximal inhibitory concentration (IC50) of the drug was calculated. 50 A represents IC. 50 <2.5μM; B indicates 2.5μM <IC 50 <5μM; C indicates 5μM <IC 50 <10μM; D indicates IC 50 >10μM.

[0223] 2. Experimental Results

[0224] Table 1. Inhibitory activity of each compound against the proliferation of different tumor cell lines

[0225]

[0226]

[0227] It can be seen that the compounds of the present invention can effectively inhibit the proliferation of various tumor cells, and most of the compounds have better inhibitory effects than the positive controls EPZ6438 and GSK126.

[0228] Experimental Example 2: Degradation efficiency of the compound on EZH2 protein in tumor cells

[0229] 1. Experimental Methods

[0230] Extraction of total cellular protein: For suspension cells, collect cells by centrifugation into 1.5 mL centrifuge tubes, wash the culture medium with pre-chilled PBS, disperse the cells at the bottom of the tube, add an appropriate volume of RIPA lysis buffer containing a 1:1000 cocktail protease inhibitor, and lyse the cells at 4°C. For adherent cells, aspirate the culture medium from the dish and wash away any residual culture medium with pre-chilled PBS, add trypsin to digest the cells for 3–5 min, collect the cells into 1.5 mL centrifuge tubes, wash away the trypsin and culture medium with pre-chilled PBS, disperse the cells at the bottom of the tube, add an appropriate volume of RIPA lysis buffer containing a 1:1000 cocktail protease inhibitor, and lyse the cells at 4°C. After lysing the cells for 30 min, sonicate the cells in an ice bath (sonicate for 5 s, 5 s intervals, for a total of 3 times) to ensure complete cell lysis. Then centrifuge at 13000 rpm / min for 15 min at 4°C, and gently aspirate the supernatant into a new 1.5 mL centrifuge tube. The concentration of the protein solution was determined using the BCA protein concentration assay kit. 1 / 4 volume of SDS-PAGE protein loading buffer (5×) was added to the remaining protein solution and mixed well. The mixture was boiled in a water bath for 8 minutes. After the sample was allowed to cool naturally to room temperature, it was aliquoted and stored in a -20°C freezer.

[0231] SDS-PAGE gel electrophoresis procedure and immunoblotting analysis: Select an appropriate concentration of SDS-PAGE gel based on the molecular weight of the protein to be detected, and prepare the gel according to the SDS-PAGE gel preparation kit instructions. Place the solidified gel into the electrophoresis tank and add an appropriate volume of electrophoresis buffer. Add equal volumes of the pre-stained protein marker and the prepared protein samples to the corresponding gel wells according to the preset loading order. Cover the electrophoresis tank, adjust the voltage to 80V, and begin electrophoresis. After the samples enter the separating gel, adjust the voltage to 120V and continue electrophoresis to separate the samples. Stop electrophoresis when the SDS-PAGE protein loading buffer blot reaches the bottom of the gel. Prepare 5 sheets of filter paper of the same size as the gel and moisten them with transfer buffer. Remove the gel from the transfer buffer. In a transfer sandwich holder, prepare the sandwich in the following order: black side - sponge - 3 layers of filter paper - gel - NC membrane - 2 layers of filter paper - sponge - white side. Remove any air bubbles between the layers and quickly assemble the transfer sandwich holder. Place an ice pack in the electrophoresis tank and replenish the transfer buffer. Adjust the voltage (80–100V) and transfer time (1–2 hours) according to the molecular weight of the protein to be detected, and begin the transfer. After transfer, wash the NC membrane in TBS / T buffer to remove any methanol adhering to the membrane. Then, place the NC membrane in blocking buffer and block it at 37°C on a shaker for 2 hours. Prepare the primary antibody using the dilution buffer according to the antibody's instructions. After blocking, cut the NC membrane into appropriate bands according to the molecular weight of the protein to be detected and place them in the corresponding antibody incubation solution. Incubate overnight at 4°C. After primary antibody incubation, place the NC membrane in TBS / T buffer and wash it three times on a horizontal shaker for 5 minutes each time. Following the dilution ratio in the instructions, prepare HRP-labeled secondary antibody at an appropriate concentration using blocking dilution buffer and incubate at 37°C in a shaker for 1 hour. After incubation, wash the NC membrane three times with TBS / T buffer, 15 minutes each time. After washing, immerse the NC membrane in UltraSignal high-sensitivity ECL chemiluminescent substrate for a few seconds and then expose it to light to obtain Western blot data. Quantitative analysis of protein expression levels was performed using ImageJ software. A indicates degradation rate >70%; B indicates 40% < degradation rate <70%; C indicates degradation rate <40%.

[0232] 2. Experimental Results

[0233] Table 2. Degradation rate of each compound on EZH2 tumor cell line protein 72 hours after administration at 2.5 μM

[0234] Kelly MV4-11 ED-1 C B ED-2 C C ED-3 B B ED-4 B A ED-5 C C ED-6 A B ED-7 A B ED-8 A A ED-9 B B ED-10 A A ED-12 A A ED-13 A B ED-14 C C ED-15 B B ED-16 - A ED-17 C C ED-18 B C ED-19 B A ED-20 B C ED-21 C B ED-22 B C ED-23 C C ED-24 C C ED-25 - A ED-26 C C ED-27 B B ED-29 B C ED-30 C C ED-31 B B ED-32 C B ED-33 - B

[0235] The experimental results are shown in Table 2 and Figures 1-4 As shown, the compound of the present invention can effectively degrade EZH2 protein in tumor cells and can be used as an EZH2 degrading agent for the treatment of tumors.

[0236] Experimental Example 3: Metabolic Stability Test of Compounds

[0237] 1. Experimental Methods

[0238] The liver microsome in vitro incubation method was used. The incubation system included phosphate buffer (0.1 M, pH 7.4), an NADPH generating system (1 mM NADP, 5 mM glucose-6-phosphate, 1 U / mL glucose-6-phosphate dehydrogenase, 3.3 mM magnesium chloride), 0.5 mg / mL liver microsome protein, and the analyte. Incubation was performed at 37°C in a water bath, with each sample in triplicate. A sample without the NADPH generating system served as a negative control. Incubation was performed at time gradients of 0, 5, 10, 15, 30, and 60 min, followed by termination of the reaction with an equal volume of pre-cooled acetic acid. The parent drug was detected using HPLC-MS. The half-life (t) of the analyte in liver microsomes was calculated. 1 / 2 ) and clearance rate (Cl int ).

[0239] 2. Experimental Results

[0240] Table 3. Half-life and scavenging rate of each compound

[0241] Compound <![CDATA[t 1 / 2 ]]> <![CDATA[Cl int ]]> ED-11 3.11 558.84 ED-25 11.38 152.76 E7 (Patent ZL202010451831.6) 2.07 838.83

[0242] The experimental results are shown in Table 3. It can be seen that the compounds of the present invention have excellent metabolic stability.

Claims

1. The compound represented by Formula I or a pharmaceutically acceptable salt thereof: Formula I in, The structure of the degradation determinant is shown in formula DG-1: ； Y is selected from none or NH2; The structures of the EZH2 targeting ligands are shown as EZH2-TL1, EZH2-TL2, EZH2-TL3, or EZH2-TL4: Among them, R1 is selected from C 1-4 alkyl; X is CH or N; Z represents none, CH2, or CO; Cy is selected from the following groups: .

2. The compound represented by Formula III or a pharmaceutically acceptable salt thereof: Formula III in, The structure of the degradation determinant is shown in formula DG-2: ； V is selected from CO and CH2; The structures of the EZH2 targeting ligands are shown as EZH2-TL1, EZH2-TL2, EZH2-TL3, or EZH2-TL4: Among them, R1 is selected from C 1-4 alkyl; Z represents none, CH2, or CO; Cy is selected from the following groups: , , ; Among them, m5, m6, m7, and m8 are each independently selected from 1 or 2.

3. The compound shown in Formula V or a pharmaceutically acceptable salt thereof: Formula V in, The structure of the degradation determinant is shown in formula DG-2: ； V is selected from CO and CH2; The structure of the EZH2 targeting ligand is shown as EZH2-TL1, EZH2-TL2 or EZH2-TL3: Among them, R1 is selected from C 1-4 alkyl; X is CH or N; Z represents none, CH2, or CO; Cy is selected from the following groups: , , ; Among them, m5, m6, m7, and m8 are each independently selected from 1 or 2.

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, characterized in that: R1 is selected from methyl, ethyl, or propyl.

5. A compound or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the compound is shown below:

6. A pharmaceutical composition, characterized in that: It is a formulation prepared by using the compound of any one of claims 1-5 or its pharmaceutically acceptable salt as the active ingredient, plus pharmaceutically acceptable excipients.

7. Use of the compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof in the preparation of an EZH2 degrading agent.

8. The use according to claim 7, characterized in that: The EZH2 degrading agent is a drug for the prevention and / or treatment of cancer.

9. The use according to claim 8, characterized in that: The cancers mentioned are prostate cancer, breast cancer, bone marrow cancer, lymphoma, ovarian cancer, colorectal cancer, neuroblastoma, lung cancer, liver cancer, malignant rhabdomyosarcoma, glioma, esophageal cancer, stomach cancer, pancreatic cancer, or leukemia.

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