An EZH2 ligand PROTAC derivative and its preparation method and use

Abstract

An EZH2 ligand PROTAC derivative, its preparation method, and use, belonging to the field of pharmaceutical technology, relates to an EZH2 ligand derivative represented by structural formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof. Also provided are methods for preparing such EZH2 ligand derivatives, and their use in the preparation of therapeutic and / or preventive agents for tumors. In structural formulas (I), (II), and (III), E3 and L are as described in the specification and claims. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and specifically relates to an EZH2 ligand derivative with anti-tumor activity, or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition containing the compound. It also relates to a method for preparing the compound and its use in preparing a drug for treating and / or preventing tumors. Background Art

[0002] Recent advances in cancer immunotherapy have emphasized the immune system's ability to control tumors, yet only a small fraction of patients respond to current immunotherapies. Emerging evidence suggests that targeting epigenetic factors that promote tumor progression and suppress immune cell activity can enhance anti-tumor immunity by reshaping the tumor microenvironment (TME). Recent studies have uncovered pleiotropic functions of the enhancer of zeste homolog 2 (EZH2) in both tumor and immune cells, suggesting that EZH2 inhibition offers a promising approach to enhance existing immunotherapies and improve outcomes for patients with certain cancers.

[0003] EZH2 is a histone methyltransferase and catalytic subunit of the polycomb repressive complex 2 (PRC2). EZH2 catalyzes the mono-, di-, and tri-methylation of lysine 27 of histone H3 (H3K27me3), a histone mark associated with compact chromatin and transcriptional repression. EZH2 plays a role in the normal biology of multiple cell types, including immune cells. Dysfunctional EZH2 has been implicated in the development and progression of multiple cancer types in mice and humans and can promote immune evasion by inhibiting intratumoral antigen presentation, immune cell migration, and enhancing the suppressive activity of CD4+ T regulatory cells (Tregs). These functions make EZH2 an attractive therapeutic target that could complement existing immunotherapy approaches.

[0004] Proteolysis targeting chimeras (PROTACs) are bifunctional small molecules in which the target protein ligand and the E3 ubiquitin ligase ligand are linked together by a linker to form a triplet compound. As a potential therapeutic approach, proteolysis targeting chimeras (PROTACs) can target specific proteins for degradation. Proteolysis targeting chimeras are specific biological functional molecules, usually consisting of a compound molecule that binds to the protein target, a ligand that recruits the E3 ubiquitin ligase, and a linker. PROTAC-induced selective polyubiquitination of the target protein and subsequent degradation on the proteasome. Compared with traditional small molecule inhibitors, PROTACs have multiple advantages, including the ability to exert their effects without binding to the active site of the target protein, the ability to degrade difficult-to-drug targets, and their event-driven and catalytic nature. They can act at lower doses, and therefore have great potential, particularly in the development of anti-cancer drugs. Summary of the Invention

[0005] The primary object of the present invention is to provide an EZH2 ligand PROTAC derivative, or a pharmaceutically acceptable salt thereof.

[0006] A secondary object of the present invention is to provide a method for preparing the EZH2 ligand PROTAC derivative and its use in preparing drugs for treating and / or preventing tumors.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] An EZH2 ligand PROTAC derivative of the present invention has a structural formula as shown in (I), (II) or (III):

[0009]

[0010] E3 is one of the E3 ligase ligands CRBN, VHL, MDM2, CIAP, UBR7, RNF114, CBLB, and KEAP1. When E3 is a ligand for CRBN ligase, it is selected from thalidomide and its derivatives, lenalidomide and its derivatives, and pomalidomide and its derivatives;

[0011] Preferably, the E3 structure is any one of the following structural formulas:

[0012]

[0013] in:

[0014] W is selected from CH2, C=O, SO2, NH, N-alkyl;

[0015] X is selected from O, S;

[0016] Z is selected from alkyl, cycloalkyl, -Cl, -F, -H;

[0017] G, G' are each independently selected from -H, alkyl, -OH, -CH2-heterocycle;

[0018] R 1 Selected from -H, -D, -F, -Cl, -Br, -I, -NO2, -CN, -NH2, -OH, -CH3, -CH2F, -CHF2, -CF3, -CH2D, -CHD2, -CD3, -CH2CH3;

[0019] Q is selected from -CH2, C=O, -NH-C=O, -NH2, -NHBoc;

[0020] M is selected from amide, ester, carboxyl, and acyl chloride;

[0021] A is selected from piperazinyl derivatives, piperidinyl derivatives, heterocyclic groups and connecting groups represented by the following structural formula (IV); in the structural formula (IV), n is an integer from 0 to 5; and the heterocyclic group is one of piperazinone, pyrrolyl, pyrazolyl, furanyl, thienyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrimidinyl, pyrazinyl and pyridazinyl.

[0022]

[0023] L is a connecting arm selected from aliphatic chains, aromatic chains, ether chains and amide chains; the intermediate compound and E3 are connected by a covalent bond to form a multifunctional molecular compound; preferably, L is any one of the structural formulas (V):

[0024]

[0025] Among them, 1≤m≤10, preferably 1≤m≤5.

[0026] The EZH2 ligand PROTAC derivative of the present invention is preferably a compound represented by the following structural formula or any one of its pharmaceutically acceptable salts and stereoisomers:

[0027]

[0028] Wherein: a represents the connection mode between the intermediate compound and the connecting arm L,

[0029] aSelected from:

[0030] R 1 Selected from -H, -D, -F, -Cl, -Br, -I, -NO2, -CN, -NH2, -OH, -CH3, -CH2F, -CHF2, -CF3, -CH2D, -CHD2, -CD3, -CH2CH3, preferably H;

[0031] R is selected from H, CH3;

[0032] A is selected from piperazinyl derivatives, piperidinyl derivatives, heterocyclic groups and connecting groups represented by the structural formula (IV), preferably piperazinyl derivatives;

[0033] Q is selected from -CH2, C=O, -NH-C=O-, -NH2, -NHBoc, preferably -NH2 or -NHBoc;

[0034] M is selected from amide, ester, carboxyl, acyl chloride, preferably ester or amide, more specifically -COOCH3;

[0035] L is any one of the structural formulas (V);

[0036] The EZH2 ligand derivative of the present invention is further preferably any one of the following compounds or pharmaceutically acceptable salts thereof:

[0037]

[0038] The EZH2 ligand derivatives may contain asymmetric or chiral centers and thus may exist in different stereoisomeric forms. The present invention includes all stereoisomeric forms, including but not limited to diastereomers, enantiomers and atropisomers, and mixtures thereof, such as racemates, which are included within the scope of the present invention.

[0039] The pharmaceutically acceptable salts of the EZH2 ligand derivative include addition salts formed between the EZH2 ligand derivative and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalene disulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, pyruvic acid, and succinic acid.

[0040] A pharmaceutical composition comprising the EZH2 ligand derivative, a stereoisomer of the ligand derivative, a pharmaceutically acceptable salt of the ligand derivative, a hydrate of the ligand derivative, or a prodrug of the ligand derivative, and may further comprise a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle, or a combination thereof.

[0041] Wherein, the dosage form of the pharmaceutical composition is injection, tablet or capsule.

[0042] A method for preparing an EZH2 ligand derivative comprises the following steps:

[0043] Step 1: Using methyl 5-bromo-2-methyl-3-nitrobenzoate 2a as the starting material, the nitro group was first reduced to an amino group, and then EDCI, HOBt, propiolic acid, and TEA were added and the reaction was moved to room temperature to prepare compound 1;

[0044]

[0045] Step 2: Dissolve 2 g of the intermediate compound in a solvent, add EDCI, HOBt, 4-pentynoic acid, and TEA under ice-cooling and stirring, and move the mixture to room temperature to react to obtain compound 2;

[0046]

[0047] Step 3: Dissolve 2 g of the intermediate compound in a solvent, add EDCI, HOBt, a tyrosine alkynyl derivative, and TEA under ice-cooling and stirring, and move the mixture to room temperature to react to obtain compound 3;

[0048]

[0049] Step 4: The prepared compound 1 and the azide-linked E3 ligand are dissolved in a solvent and linked by a Click reaction to obtain compounds of series 1-5; namely: compound E-nP-Tha (n = 2-5) (series 5), compound E-nCH2-VHL1 / 2 (n = 1, 3, 5, 7) (series 1), compound E-nCH2-B4 (n = 1) (series 2), compound E-nP-B5 (n = 2-4) (series 3), compound E-nP-MDM2 (n = 1-4) (series 4);

[0050]

[0051] The prepared compound 2 and the azide-linked E3 ligand were dissolved in a solvent and linked via a click reaction to obtain compounds of series 6-8; namely, compound E-4W-nCH2V2 (n=7) (series 6), compound E-4W-nCH2B4 (n=1,3) (series 7), and compound E-4W-nPB5 (n=2-4) (series 8);

[0052]

[0053] Step 5: The obtained series 8 compound was deprotected in HCl in ethyl acetate to obtain series 9 compound E-4W-nPB5T (n=2-4);

[0054]

[0055] Step 6: The prepared compound 3 and the azide-linked E3 ligand were dissolved in a solvent and linked via a click reaction to obtain series 10 compounds MP-EC-n (n=2-4);

[0056]

[0057] The solvent in the above preparation method is one or a combination of two of acetone, dichloromethane, THF, ethyl acetate and water.

[0058] The present invention studies the molecular mechanism of EZH2 protein and a series of synthetic compounds in inhibiting EZH2 protein degradation; studies the anti-tumor activity and mechanism of action of the derivatives at the cellular level and the animal level, as well as the effects on tumor animals.

[0059] Use of the EZH2 ligand PROTAC derivative of the present invention or its pharmaceutically acceptable salt, stereoisomer, or pharmaceutical composition comprising the same in the preparation of a medicament for treating and / or preventing tumors.

[0060] Use of the EZH2 ligand PROTAC derivative of the present invention or its pharmaceutically acceptable salt, stereoisomer, or pharmaceutical composition comprising the above substance in the preparation of a histone methyltransferase degrader in SU-DHL-6 lymphoma cells.

[0061] The EZH2 ligand PROTAC derivative of the present invention, its preparation method and use, have the following beneficial effects:

[0062] The EZH2 ligand PROTAC derivatives of the present invention can inhibit EZH2 by targeting it. Using the intermediate compound 2g as the core, they are linked to the E3 ligase ligand via the linker arm L to form EZH2 derivatives that can target EZH2. Research has shown that these derivatives have broad application prospects in the preparation of drugs for treating and / or preventing tumors. The present methods provide methods for preparing the following compounds. Furthermore, studies have found that the EZH2 derivatives E-2P-MDM2, E-3P-MDM2, and E-4P-MDM2 have excellent EZH2 degradation activity, thus providing useful value in treating and / or preventing tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Compounds' ability to degrade EZH2 in SU-DHL-6 cells; (A) Compounds MP-EC-2 / 3 / 4 showed no effective degradation of EZH2 at concentrations between 0.3 and 30 μM. (B) Effect of E-4W-2 / 3 / 4PB5 on EZH2 expression in SU-DHL-6 cells after 48 hours of treatment. E-4W-2PB5 and E-4W-4PB5 had no effect on EZH2 expression, while E-4W-3PB5 showed some effect at 10 and 30 μM. (C) Compounds E-2 / 3 / 4 / 5P-Tha showed some degradation activity in SU-DHL-6 cells. All compounds in this series showed some degradation activity, with E-4P-Tha being the most effective, showing an effect on EZH2 expression at concentrations between 3 and 30 μM. (D) EZH2 degradation activity of E-CH2-B4 and E-4W-1 / 3CH2-B4 in SU-DHL-6 cells. E-CH2-B4 had the ability to degrade EZH2 at 10 μM, but the degradation ability of E-4W-1 / 3CH2-B4 was weak.

[0064] Figure 2Compounds' ability to degrade EZH2 in SU-DHL-6 cells; (A) EZH2 degradation activity of compounds E-1 / 3 / 5 / 7CH2-V2 and E-4W-7CH2-V2 in SU-DHL-6 cells. Compounds E-1 / 3 / 5 / 7CH2-V2 all had some EZH2 degradation activity, with E-3 / 5CH2-V2 showing stronger degradation activity. E-4W-7CH2-V2 had no significant EZH2 degradation activity. (B) EZH2 degradation activity of E-1 / 5CH2-V1 at 48 and 72 hours. The compounds had little effect on EZH2 in cells at 48 hours, but showed some degradation activity at 72 hours. (C) EZH2 degradation activity of intermediate compound 2g and EP-MDM2. (D) EZH2 degradation activity of E-2 / 3 / 4P-MDM2 in cells at 48 hours. All three compounds have a degradation effect on EZH2, among which E-3 / 4P-MDM2 has the strongest degradation ability.

[0065] Figure 3 Effects of the compounds on mitochondrial membrane potential and apoptosis in SU-DHL-6 cells after 48 hours of treatment. (A) Effect of E-3P-MDM2 on mitochondrial membrane potential. E-3P-MDM2 significantly increased mitochondrial membrane potential in SU-DHL-6 cells, with the Q3 region increasing from 7.23% to 62.8%. (B) With increasing E-3P-MDM2 concentration, early apoptosis increased from 8.53% to 39.8%, and late apoptosis increased from 4.08% to 34.5%. DETAILED DESCRIPTION

[0066] The following is a detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0067] Example 1 Preparation of Intermediate Compound 2b

[0068]

[0069] The starting material, compound 2a (548.2 mg, 2 mmol), was dissolved in a 3 / 1 mixture of MeOH / H₂O. Ammonium chloride (534.9 mg, 10 mmol) was added with stirring, and the temperature was raised to 80°C. Reduced iron powder (1.12 g, 20 mmol) was added and allowed to react for 1 hour. Celite was used to remove impurities, and the filtrate was collected. The filter cake was rinsed three times with methanol. The methanol was evaporated, and the mixture was extracted with dichloromethane / water. The organic layer was collected, dried over anhydrous sodium sulfate, and evaporated to dryness to yield a colorless oil (445.6 mg, 99.4%). 1H NMR (600MHz, CDCl3) δ7.33 (t, J = 1.6Hz, 1H), 6.94 (s, 1H), 3.88 (s, 3H), 3.80 (s, 2H), 2.28 (s, 3H).

[0070] Example 2 Preparation of Intermediate Compound 2c

[0071]

[0072] Intermediate compound 2b (440 mg, 1.8 mmol) and tetrahydropyrone (540.6 mg, 5.4 mmol) were dissolved in 5 mL of DCM and stirred at 30°C. Acetic acid (206 μL, 3.6 mmol) was added dropwise and allowed to react for 3 hours. NaBH(OAc)₃ (953.7 mg, 4.5 mmol) was then slowly added and allowed to react overnight at room temperature. The DCM was evaporated, and the mixture was extracted with ethyl acetate / water. The organic layer was collected and dried. Column chromatography with petroleum ether:ethyl acetate = 5:1-3:1 (v / v) was performed to yield 344 mg of a white solid, yielding 58.2%. 1 H NMR (600MHz, CDCl3) δ7.23(d,J=1.9Hz,1H),6.84(s,1H),4.02(dt,J=12.0,3.7Hz,2H),3.88(s,3H),3.65(d,J=6.9Hz,1 H),3.55(td,J=12.0,11.6,2.2Hz,2H),3.52–3.47(m,1H),2.24(s,3H),2.08–2.02(m,2H),1.52(qd,J=10.8,4.2Hz,2H).

[0073] Example 3 Preparation of Intermediate Compound 2d

[0074]

[0075] Intermediate compound 2c (300 mg, 0.91 mmol) was dissolved in 5 mL of 1,2-dichloroethane. Acetaldehyde (120 mg, 2.73 mmol) and acetic acid (104 μL, 1.82 mmol) were added sequentially with stirring. The reaction system was heated to 30°C for 1 hour. NaBH(OAc)₃ (483 mg, 2.28 mmol) was slowly added, and the reaction was continued at room temperature for 1 hour. The 1,2-dichloroethane was evaporated, and the mixture was extracted with EA / H₂O. The organic phase was collected, dried over anhydrous sodium sulfate, and purified by column chromatography with petroleum ether:ethyl acetate = 8:1 (v / v) to yield 158 mg of a white solid, yielding 48.7%. 1H NMR (600MHz, DMSO-d6) δ7.60(d,J=2.1Hz,1H),7.53(d,J=2.2Hz,1H),3.83(s,5H),3.25(td,J=11.7,2.0Hz,2H),3.04(q,J=7.0 Hz,2H),2.97(tt,J=11.0,3.8Hz,1H),2.36(s,3H),1.61(d,J=12.7Hz,2H),1.49(qd,J=11.8,4.4Hz,2H),0.79(t,J=7.0Hz,3H).

[0076] Example 4 Preparation of Intermediate Compound 2e

[0077]

[0078] Intermediate compound 2d (150 mg, 0.42 mmol) was dissolved in 2 mL of methanol. A 2 mL aqueous solution of NaOH (58.9 mg, 1.47 mmol) was added with stirring and stirred at room temperature until the reaction was complete. The methanol was evaporated to dryness, and the aqueous layer was extracted with EA and adjusted to pH 5-6 with 2M HCl. EA extraction was repeated three times, and the organic phases were combined, dried over anhydrous sodium sulfate, and evaporated to dryness to yield 132.6 mg of a white solid (92.2%). 1 H NMR (600MHz, DMSO-d6) δ13.16(s,1H),7.57(d,J=2.1Hz,1H),7.47(d,J=2.2Hz,1H),3.85–3.79(m,2H),3.25(td,J=11.6,2.1Hz,2H),3. 03(q,J=7.1Hz,2H),2.96(tt,J=11.0,3.7Hz,1H),2.37(s,3H),1.63–1.58(m,2H),1.49(qd,J=11.8,4.4Hz,2H),0.79(t,J=7.0Hz,3H).

[0079] Example 5 Preparation of Intermediate Compound 2f

[0080]

[0081] Intermediate compound 2e (132.6 mg, 0.39 mmol) was dissolved in 5 mL of DMSO, and EDCI (51 mg, 0.27 mmol), HOBt (36 mg, 0.27 mmol), and 3-(aminomethyl)-4,6-dimethyl-1H-pyridin-2-one (54 mg, 0.355 mmol) were added sequentially with stirring. The mixture was reacted at 45°C for 20 hours and monitored by TLC. After the reaction of the starting material was complete, the mixture was extracted with ethyl acetate / water. The organic phase was collected, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography with dichloromethane:methanol = 50:1 (v / v) to obtain 135.3 mg of a white solid (80%). 1 H NMR (600MHz, DMSO-d6) δ11.47(s,1H),8.23(t,J=5.0Hz,1H),7.31(d,J=2.1Hz,1H),7.0 9(d,J=2.0Hz,1H),5.86(s,1H),4.25(d,J=5.0Hz,2H),3.82(d,J=11.6Hz,2H),3.27–3. 20(m,2H),3.01(q,J=7.0Hz,2H),2.94(tt,J=11.0,3.9Hz,1H),2.54(s,1H),2.19(s,3H ),2.15(s,3H),2.12–2.09(m,3H),1.60(d,J=12.6Hz,2H),1.50(qd,J=11.9,4.4Hz,2H).

[0082] Example 6 Preparation of Intermediate Compound 2g

[0083]

[0084] Intermediate compound 2f (100 mg, 0.21 mmol) was dissolved in a mixture of 1,4-dioxane and water (8 / 2). 4-(4-Boc-1-piperazinemethyl)phenylboronic acid pinacol ester (100.6 mg, 0.25 mmol), potassium carbonate (44.2 mg, 0.32 mmol), and Pd(dppf)Cl2 (14.6 mg, 0.02 mmol) were added sequentially with stirring. After degassing for 5 minutes, the mixture was reacted at 100°C for 8 hours. TLC monitored the complete reaction of the starting materials. The reaction system was diluted with ethyl acetate and filtered through Celite to remove impurities. The filtrate was extracted with ethyl acetate / water, and the organic layer was purified by column chromatography using dichloromethane:methanol = 30:1 (v / v) to obtain 2 g of a khaki solid (Boc). 1H NMR (600MHz, DMSO-d6) δ11.47(s,1H),8.20(t,J=5.0Hz,1H),7.58(d,J=8.0Hz,2H),7.40(d,J=1.9Hz,1H),7.37(d,J=7.8Hz,2 H),7.22(d,J=1.9Hz,1H),5.86(d,J=1.1Hz,1H),4.29(d,J=5.0Hz,2H),3.83(dt,J=9.6,2.9Hz,2H),3.50(s,2H),3.31(s,4H), 3.25(td,J=11.7,2.0Hz,2H),3.09(q,J=7.0Hz,2H),3.02(tt,J=11.0,3.8Hz,1H),2.33(d,J=5.3Hz,4H),2.25(s,3H),2.21(s ,3H),2.11–2.10(m,3H),1.66(dt,J=12.1,3.2Hz,2H),1.53(tdd,J=12.3,8.0,3.2Hz,2H),1.39(s,9H),0.83(t,J=7.0Hz,3H).

[0085] Compound 2g (Boc) was added with excess hydrogen chloride ethyl acetate solution and stirred. After the reaction was complete, the mixture was evaporated to dryness to obtain intermediate compound 2g.

[0086] Example 7 Preparation of EZH2 Compound 1

[0087]

[0088] 2g (80mg, 0.14mmol) of the intermediate compound was dissolved in 3mL of dichloromethane. EDCI (54mg, 0.28mmol), HOBt (38mg, 0.28mmol), propiolic acid (12mg, 0.17mmol), and TEA (77μL, 0.55mmol) were added sequentially under ice-cooling and stirring. The ice-cooling bath was removed and the mixture was allowed to react at room temperature for 3-4 hours. The mixture was extracted three times with dichloromethane and water. The organic layer was purified by open silica gel column chromatography (dichloromethane:methanol = 30:1) to obtain EZH2 compound 1 in a 40% yield. 1H NMR(600MHz, DMSO-d6)δ11.46(s,1H),8.19(t,J=5.0Hz,1H),7.60–7.56(m,2H),7.41–7.36(m,3H),7.22( d,J=1.9Hz,1H),5.86(s,1H),4.29(d,J=5.0Hz,2H),3.83(dd,J=9.0,6.0Hz,2H),3.54(s,2H),3.50(s,3H) ,3.25(td,J=10.6,9.8,3.6Hz,4H),3.09(q,J=6.9Hz,2H),3.02(tt,J=11.1,3.8Hz,1H),2.45–2.33(m,4H) ,2.25(s,3H),2.21(s,3H),2.11(s,3H),1.66(d,J=12.5Hz,2H),1.57–1.48(m,2H),0.83(t,J=7.0Hz,3H).

[0089] Example 8 Preparation of EZH2 Compound 2

[0090] Using the preparation method of Example 7, with the same feed ratio and operation, the intermediate compound 2g is reacted with 4-pentynoic acid to obtain EZH2 compound 2. 1 H NMR (600MHz, DMSO-d6) δ11.48(s,1H),8.20(t,J=5.0Hz,1H),7.60–7.56(m,2H),7.42–7.35(m,3H),7.22(d,J=1.8Hz,1H),5. 87–5.84(m,1H),4.29(d,J=5.0Hz,2H),3.83(ddd,J=11.5,4.2,2.0Hz,2H),3.52(s,2H),3.44(dt,J=10.6,5.3Hz,3H),3.25( td,J=11.6,2.0Hz,2H),3.09(q,J=7.0Hz,2H),3.01(ddt,J=11.0,7.7,3.7Hz,1H),2.75(t,J=2.6Hz,1H),2.55–2.49(m,3H), 2.41–2.30(m,6H),2.25(s,3H),2.21(s,3H),2.12–2.09(m,3H),1.68–1.63(m,2H),1.57–1.48(m,2H),0.83(t,J=7.0Hz,3H).

[0091] Using the preparation method of Example 7, with the same feed ratio and operation, the intermediate compound 2g is reacted with a tyrosine alkynyl derivative to obtain compound 3.

[0092] Example 9 Preparation of Azide-Linked E3 Ligand N3-nCH2-VHL2 (n=1)

[0093]

[0094] To a flask containing 50.3 mg of azido-CH2-carboxylic acid was added 5 mL of DCM. Under ice-cooling and stirring, 159 mg of EDCI, 112 mg of HOBt, 289 μL of DIPEA, and 200 mg of compound VHL2 were added sequentially. The mixture was then extracted with 30 mL of water and 30 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated to obtain the crude product. The organic layer was then purified by silica gel column chromatography (elution with dichloromethane:methanol = 25:1) to give a yellow solid in a 60% yield. 1 H NMR (400MHz, CDCl3) δ8.70(s,1H),7.38(q,J=8.5Hz,4H),7.02(d,J=8.6Hz,1H),5.14–5.03(m,1H),4.73(t,J=7.8Hz,1H),4.57(d ,J=8.7Hz,2H),3.63(dd,J=11.3,3.7Hz,1H),2.60–2.50(m,4H),2.08(dd,J=19.9,6.3Hz,1H),1.48(d,J=6.9Hz,3H),1.06(s,8H).

[0095] Depending on the value of n, the azide-linked E3 ligands in series 1 and 6 were prepared using the preparation method of Example 9.

[0096] Example 10 Preparation of EZH2 Series 1 Compound E-CH2-VHL2 (Series 1, n=1)

[0097]

[0098] Compound 1 (30 mg, 0.050 mmol) was dissolved in 2 mL of tetrahydrofuran solution, and 2 mL of water was added. N3-nCH2-VHL2 (n=1) (29 mg, 0.055 mmol), VcNa (30 mg, 0.15 mmol), and CuSO4 (10 mg, 0.0625 mmol) were added in sequence. After TLC monitoring showed that there was no starting material, post-treatment was carried out, extraction was performed with ethyl acetate / saturated aqueous NaCl solution, and the organic layer was purified by open silica gel column chromatography (elution with dichloromethane:methanol = 15:1-5:1) to give a slightly yellow solid in a yield of 40%. 1H NMR (600MHz, DMSO) δ11.46(s,1H),8.19(t,J=5.0Hz,1H),7.60–7.55(m,2H),7.52(d,J=8.3 Hz,2H),7.43–7.39(m,3H),7.37–7.35(m,1H),7.22(t,J=2.4Hz,1H),7.03–6.96(m,3H),5. 86(s,1H),4.92(s,1H),4.42–4.38(m,1H),4.29(d,J=5.0Hz,2H),4.24(d,J=7.4Hz,1H),4. 19(dd,J=8.1,5.7Hz,1H),4.11(s,1H),3.85–3.81(m,2H),3.66(d,J=4.1Hz,1H),3.64(s,1 H),3.63(s,1H),3.51(s,2H),3.49(s,2H),3.46(s,2H),3.26–3.22(m,5H),3.17(s,6H),3. 09(d,J=7.1Hz,2H),3.01(d,J=11.3Hz,1H),2.73(s,1H),2.61(s,1H),2.39(p,J=1.9Hz,1H ),2.33(s,2H),2.25(d,J=2.2Hz,3H),2.21(s,3H),2.11(s,3H),2.01(d,J=24.2Hz,1H),1. 89(s,1H),1.66(d,J=12.6Hz,2H),1.53(dt,J=12.0,5.9Hz,2H),1.24(s,9H),0.83(s,3H).

[0099] The synthesis of other compounds in EZH2 series 1 was carried out according to the method of Example 10.

[0100] Example 11 Preparation of EZH2 Series 1 Compound E-3CH2-VHL2 (Series 1, n=3)

[0101] 1H NMR(600MHz,DMSO-d6)δ11.48(s,1H),8.99(s,1H),8.54(s,1H),8.40(d,J=7.8Hz,1H),8.21(t,J=5.0Hz,1H),7.96(d,J=9.2Hz,1H),7.61–7.57(m,2H),7.43(d,J=8.3Hz,4H),7.40–7.37(m,3H),7.23(d,J=1.8Hz,1H),5.86(s,1H),5.14(t,J=3.9Hz,1H),4.95–4.88(m,1H),4.61(s,1H),4.51(d,J=9.3Hz,1H),4.43–4.37(m,4H),4.29(d,J=5.1Hz,4H),3.83(d,J=9.9Hz,2H),3.61(d,J=5.3Hz,2H),3.55(s,2H),3.51(d,J=1.2Hz,4H),3.48(d,J=5.3Hz,2H),3.42(d,J=5.4Hz,2H),3.25(s,2H),3.09(q,J=7.0Hz,2H),3.04–2.99(m,1H),2.45(s,4H),2.25(s,3H),2.21(s,3H),2.12–2.09(m,4H),1.78(ddd,J=12.9,8.5,4.7Hz,1H),1.66(d,J=12.4Hz,2H),1.53(dd,J=11.9,3.9Hz,2H),1.36(t,J=6.6Hz,3H),1.25(s,3H),0.93(s,9H). 13 C NMR(101MHz,DMSO-d6)δ171.43,171.08,169.96,169.53,163.47,159.96,151.95,150.00,149.35,148.21,145.13,143.36,140.07,137.44,133.11,131.58,130.13,129.28,128.93,126.92,126.85,123.35,122.07,121.30,107.84,72.79,70.26,69.22,66.79,60.66,59.02,58.32,57.04,49.71,48.16,41.61,38.18,35.63,35.34,32.00,31.74,30.77,26.91,22.55,19.42,18.66,16.44,15.03,14.41.

[0102] Example 12 Preparation of EZH2 Series 1 Compound E-5CH2-VHL2 (Series 1, n=5)

[0103] 1 H NMR (600MHz, DMSO) δ11.46(s,1H),8.98(s,1H),8.53(s,1H),8.36(d,J=7.8Hz,1H),8.19(t, J=5.0Hz,1H),7.81(d,J=9.3Hz,1H),7.61–7.56(m,2H),7.45–7.36(m,7H),7.22(d,J=1.8Hz ,1H),5.86(s,1H),5.11(d,J=3.6Hz,1H),4.94–4.89(m,1H),4.50(d,J=9.3Hz,1H),4.42(t, J=8.1Hz,1H),4.37(t,J=7.1Hz,2H),4.29(d,J=5.0Hz,2H),4.07–4.04(m,1H),3.85–3.80(m, 2H),3.61(dt,J=14.1,3.6Hz,3H),3.55(s,2H),3.25(td,J=11.7,2.0Hz,3H),3.09(d,J=7.0 Hz,1H),3.02(tt,J=11.0,3.8Hz,1H),2.45(s,6H),2.25(s,3H),2.21(s,3H),2.15–2.11(m, 1H),2.11(s,3H),2.04–1.99(m,1H),1.91(s,1H),1.86–1.77(m,3H),1.69–1.63(m,2H),1.5 6–1.44(m,5H),1.37(d,J=7.0Hz,3H),1.25–1.22(m,5H),0.92(s,9H),0.84(d,J=7.0Hz,3H).

[0104] Example 13 Preparation of EZH2 Series 1 Compound E-7CH2-VHL2 (Series 1, n=7)

[0105] 1H NMR(600MHz,DMSO-d6)δ11.46(s,1H),8.98(s,1H),8.54(s,1H),8.37(d,J=7.8Hz,1H),8.19(t,J=5.1Hz,1H),7.78(d,J=9.3Hz,1H),7.59(d,J=7.6Hz,2H),7.40(s,4H),7.38(d,J=8.4Hz,3H),7.23(s,1H),5.86(s,1H),5.10(d,J=3.5Hz,1H),4.91(p,J=7.2Hz,1H),4.58(t,J=5.5Hz,1H),4.51(d,J=9.3Hz,1H),4.40(dt,J=21.6,7.6Hz,4H),4.33–4.24(m,4H),3.83(d,J=11.1Hz,2H),3.64–3.58(m,2H),3.56(d,J=17.8Hz,2H),3.51(d,J=1.1Hz,8H),3.48(q,J=5.4Hz,2H),3.41(t,J=5.3Hz,2H),3.25(t,J=11.5Hz,2H),3.09(q,J=7.0Hz,2H),3.06–2.99(m,1H),2.45(s,4H),2.25(s,3H),2.21(s,3H),2.11(s,4H),2.02(d,J=7.7Hz,1H),1.91(s,1H),1.85–1.75(m,2H),1.66(d,J=12.5Hz,2H),1.50(ddq,J=33.2,12.1,6.8,5.5Hz,6H),1.34(s,3H),0.93(s,9H). 13 C NMR(101MHz,DMSO-d6)δ172.48,171.08,170.81,170.07,163.47,159.97,151.95,149.99,149.36,148.22,145.12,131.58,130.14,129.28,126.90,122.08,121.32,107.82,72.80,70.26,69.21,66.80,60.67,60.22,59.00,58.33,50.02,48.16,38.19,35.65,31.75,31.61,30.77,30.29,29.95,29.51,26.90,22.88,22.55,21.23,19.42,18.65,16.44,14.55,14.41,13.19.

[0106] Example 14 Preparation of Azide-Linked E3 Ligand N3-nCH2-B4 (n=1)

[0107]

[0108] To a flask containing 56.3 mg of azido-CH2-carboxylic acid was added 5 mL of DCM. The mixture was then added sequentially with stirring and ice-cooling, followed by the addition of 214 mg of EDCI, 150.7 mg of HOBt, 309 μL of TEA, and 200 mg of compound B4 (Bestadine-methyl ester). The mixture was then extracted with 30 mL of water and 30 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated to obtain a crude product, which was then purified by silica gel column chromatography. The organic layer was then eluted with open silica gel column chromatography (dichloromethane:methanol = 50:1) to give an oil in a 70% yield.

[0109] The preparation process of series 2 and 7 intermediates can be referred to Example 14.

[0110] Example 15 Preparation of EZH2 Series 2 Compound E-CH2-B4 (Series 2)

[0111]

[0112] Compound 1 (30 mg, 0.050 mmol) was dissolved in 2 mL of tetrahydrofuran solution, and 2 mL of water was added. N3-CH2-B4 (23 mg, 0.055 mmol), VcNa (30 mg, 0.15 mmol), and CuSO4 (10 mg, 0.0625 mmol) were added respectively. After TLC monitoring, no starting material was found, and post-treatment was performed. The mixture was extracted with ethyl acetate / water solution, and the organic layer was purified by silica gel column chromatography (elution with dichloromethane:methanol = 15:1-10:1) to give a slightly yellow solid in a yield of 50%. 1H NMR(600MHz,DMSO-d6)δ11.40(s,1H),8.22(d,J=1.1Hz,1H),8.13(t,J=5.0Hz,1H),7.92–7.88(m,1H),7.80(d,J=8.4Hz,1H),7.55–7.50(m,2H),7.36–7.32(m,2H),7.21(t,J=7.5Hz,2H),7.15(d,J=7.1Hz,3H),7.12(td,J=7.1,1.4Hz,1H),6.27(d,J=5.9Hz,1H),5.79(s,1H),5.69(s,1H),5.06(d,J=16.2Hz,1H),4.95(d,J=16.2Hz,1H),4.29(ddd,J=10.3,8.4,4.9Hz,1H),4.22(d,J=5.0Hz,1H),4.14(td,J=9.4,8.9,6.6Hz,1H),4.02–3.93(m,2H),3.86–3.83(m,1H),3.81–3.74(m,2H),3.57(s,2H),3.54(s,3H),3.51(s,2H),3.44(d,J=1.4Hz,2H),3.18(td,J=11.8,2.0Hz,4H),3.02(q,J=7.0Hz,2H),2.95(td,J=11.0,5.5Hz,1H),2.79(dd,J=13.4,7.2Hz,1H),2.60(dd,J=13.3,7.5Hz,1H),2.18(s,3H),2.14(s,3H),2.04(s,3H),1.62–1.56(m,3H),1.55–1.39(m,5H),0.81(d,J=6.5Hz,3H),0.77–0.73(m,6H). 13C NMR(101MHz,DMSO-d6)δ173.15,172.15,169.52,164.86,163.48,159.82,150.00,149.36,14 3.14,138.86,137.43,133.14,130.32,130.15,129.64,128.75,127.72,126.96,126.70,124. 94,122.08,119.59,110.10,107.83,71.03,70.24,66.80,58.34,55.38,54.25,52.40,52.01,50.14,41.61,37.29,35.35,30.77,29.46,24.63,23.22,21.65,19.41,18.65,15.03,13.20.

[0113] Example 16 Preparation of Azide-Linked E3 Ligand N3-nP-B5 (n=2)

[0114]

[0115] Compound B5 (Tert-butyl bestadin-methyl ester) was placed in an eggplant-shaped flask and added with 5 mL of DCM. 188 mg of EDCI, 132 mg of HOBt, 272 μL of TEA, and 94 μL of azido-2PEG-NH2 were added sequentially with stirring on an ice bath. The mixture was then extracted with 30 mL of water and 30 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated to obtain the crude product. The organic layer was then purified by open silica gel column chromatography (elution with dichloromethane:methanol = 50:1) to obtain an oil in a 70% yield. 1 H NMR (400MHz, CDCl3) δ7.33–7.27(m,2H),7.23(d,J=7.5Hz,3H),6.57(s,1H),5.01(d,J=8.0Hz,1H),4.45(d,J=4.7Hz,1H),4.13(s,1H),3.98(s,1H ),3.67–3.60(m,2H),3.59–3.52(m,2H),3.37(dd,J=21.2,16.4Hz,3H),3 .18(d,J=13.9Hz,1H),3.03(s,1H),1.43–1.33(m,8H),0.95–0.86(m,6H).

[0116] Depending on the value of n, the azide-linked E3 ligands in series 3 and 8 were prepared using the preparation method of Example 16.

[0117] Example 17 Preparation of EZH2 Series 3 Compound E-2P-B5 (Series 3, n=2)

[0118]

[0119] Compound 1 (30 mg, 0.050 mmol) was dissolved in 2 mL of tetrahydrofuran solution, and 2 mL of water was added. N3-nP-B5 (n = 2) (31 mg, 0.055 mmol), VcNa (30 mg, 0.15 mmol), and CuSO4 (10 mg, 0.0625 mmol) were added in sequence. The reaction was heated at 40°C. The reaction system was turbid, but soon became clear. TLC monitoring showed that there was no starting material. Post-treatment was performed, extraction was performed with ethyl acetate / saturated aqueous NaCl solution, and the organic layer was purified by silica gel column chromatography (elution with dichloromethane: methanol = 15:1-10:1) to obtain a slightly yellow solid in a yield of 50%. 1H NMR(600MHz,DMSO-d6)δ11.47(s,1H),8.49(s,1H),8.20(td,J=5.0,1.9Hz,1H),8.06(t,J=5.6Hz,1H),7.60(d,J=8.2Hz,3H),7.42–7.39(m,3H),7.26(dd,J=8.4,6.4Hz,2H),7.24–7.19(m,3H),7.19–7.16(m,1H),6.17(d,J=9.4Hz,1H),5.99(d,J=6.2Hz,1H),5.86(s,1H),4.56(t,J=5.2Hz,1H),4.34(td,J=9.3,5.2Hz,1H),4.29(d,J=5.0Hz,1H),3.95(ddt,J=11.6,8.8,4.3Hz,1H),3.85–3.79(m,5H),3.67(d,J=32.2Hz,2H),3.59(q,J=4.8Hz,2H),3.51(d,J=1.3Hz,8H),3.48(d,J=5.2Hz,2H),3.46–3.44(m,2H),3.41(t,J=5.3Hz,2H),3.29–3.22(m,2H),3.17(d,J=4.5Hz,2H),3.09(q,J=7.0Hz,2H),3.02(tt,J=11.1,3.3Hz,1H),2.78(dd,J=13.3,7.3Hz,1H),2.68–2.61(m,1H),2.25(s,3H),2.21(s,3H),2.11(s,3H),1.69–1.64(m,2H),1.53(dd,J=12.0,3.7Hz,2H),1.46(dt,J=10.4,4.9Hz,1H),1.41(s,1H),1.29(d,J=12.2Hz,10H),1.23(s,3H),0.86–0.83(m,6H). 13C NMR(101MHz,DMSO-d6)δ172.55,170.35,163.68,159.90,154.97,149.93,146.05,143 .81,139.17,134.49,129.69,128.60,127.87,127.06,124.90,119.66,115.12,110.09 ,107.83,78.02,12.80,10.25,69.86,66.91,66.74,60.67,54.92,50.67,49.97,42.78,42.17,39.00,37.92,35.35,31.75,28.58,23.60,22.55,22.18,19.40,18.65,14.42.

[0120] Using the preparation method of Example 14, with the same feed ratio and operation, compound 1 is reacted with N3-nP-B5 to obtain EZH2 series 3 compounds.

[0121] Example 18 Preparation of EZH2 Series 3 Compound E-3P-B5 (Series 3, n=3)

[0122] 1H NMR(600MHz,DMSO-d6)δ11.47(s,1H),8.48(s,1H),8.20(t,J=5.0Hz,1H),8.06(t,J=5.7Hz,1H),7.59(dd,J=8.5,2.3Hz,3H),7.40(dt,J=6.3,3.5Hz,3H),7.26(t,J=7.5Hz,2H),7.24–7.20(m,3H),7.19–7.16(m,1H),6.17(d,J=9.4Hz,1H),5.98(d,J=6.2Hz,1H),5.86(s,1H),4.57(q,J=5.1Hz,1H),4.34(td,J=9.3,5.2Hz,1H),4.29(d,J=5.0Hz,1H),3.94(dtd,J=10.0,7.5,2.6Hz,1H),3.85–3.78(m,5H),3.63(s,2H),3.58(d,J=10.3Hz,2H),3.53–3.43(m,12H),3.43–3.35(m,6H),3.25(td,J=11.6,2.0Hz,2H),3.20–3.15(m,2H),3.09(q,J=7.0Hz,2H),3.02(tt,J=11.0,3.7Hz,1H),2.78(dd,J=13.3,7.3Hz,1H),2.66(dd,J=13.3,7.7Hz,1H),2.25(s,3H),2.21(s,3H),2.11(s,3H),1.69–1.61(m,2H),1.55–1.51(m,2H),1.46(td,J=9.0,4.6Hz,1H),1.40(tt,J=8.2,2.7Hz,1H),1.30–1.25(m,10H),1.23(s,3H),0.85–0.83(m,6H). 13C NMR (101MHz, DMSO-d6) δ172.28,171.95,169.52,163.47,159.90,155.33,149.99,149.35,143.31,140. 08,139.19,137.43,133.13,130.11,129.68,129.38,128.59,126.95,126.48,123.34,122.08,121.30, 107.82,78.04,72.80,71.87,70.25,70.14,70.05,69.98,69.30,68.82,66.80,60.67,58.34,54.94,50.75,50.01,39.03,35.35,31.74,30.76,29.46,28.58,23.60,22.20,19.42,18.65,15.03,14.41,13.19.

[0123] Example 19 Preparation of EZH2 Series 3 Compound E-4P-B5 (Series 3, n=4)

[0124] 1H NMR(600MHz,DMSO-d6)δ11.47(s,1H),8.48(s,1H),8.20(t,J=5.0Hz,1H),8.09–8.04(m,1H),7.59(dd,J=8.6,3.2Hz,3H),7.42–7.38(m,3H),7.26(t,J=7.5Hz,2H),7.24–7.20(m,3H),7.17(t,J=7.5Hz,1H),6.17(d,J=9.4Hz,1H),5.98(d,J=6.2Hz,1H),5.86(s,1H),4.57(t,J=5.2Hz,1H),4.34(td,J=9.3,5.2Hz,1H),4.29(d,J=5.0Hz,1H),3.98–3.91(m,1H),3.82(dt,J=17.7,6.9Hz,5H),3.63(s,2H),3.56(s,2H),3.54–3.49(m,8H),3.48–3.44(m,10H),3.39(dt,J=22.1,5.7Hz,4H),3.28–3.22(m,2H),3.21–3.15(m,2H),3.09(q,J=7.0Hz,2H),3.02(td,J=10.9,5.4Hz,1H),2.78(dd,J=13.3,7.3Hz,1H),2.66(dd,J=13.3,7.7Hz,1H),2.25(s,3H),2.21(s,3H),2.11(s,3H),1.66(d,J=11.4Hz,2H),1.53(dt,J=11.8,6.1Hz,2H),1.46(dq,J=9.6,5.2,4.7Hz,1H),1.39(td,J=9.1,8.5,4.2Hz,1H),1.26(d,J=19.0Hz,10H),1.23(s,3H),0.85–0.81(m,6H). 13C NMR(101MHz,DMSO-d6)δ172.28,171.95,169.52,163.47,159.91,155.33,150.00,149.36,143.30,140.0 8,139.19,137.43,133.13,130.09,129.68,129.37,128.60,126.94,126.47,123.35,122.08,121.30,107 .83,78.04,72.80,71.87,70.22,70.04,69.96,69.30,68.83,66.80,60.68,58.34,54.93,50.76,50.02,42.19,41.62,39.03,38.02,35.35,30.76,29.48,28.58,24.44,23.60,22.20,19.41,18.65,15.03,13.20.

[0125] Example 20 Preparation of Azide-Linked E3 Ligand N3-nP-MDM2 (n=2)

[0126]

[0127] To a flask containing 200 mg of compound MDM2 (Nutlin carboxylic acid), 5 mL of DCM was added. 118.9 mg of EDCI, 83.8 mg of HoBt, 216 μL of DIPEA, and 59.2 mg of azido-2PEG-NH2 were added sequentially with stirring and ice-cooling. The mixture was then extracted with 30 mL of water and 30 mL of dichloromethane. The organic layer was dried over anhydrous sodium sulfate and concentrated to obtain the crude product. The organic layer was then purified by silica gel column chromatography (elution with dichloromethane:methanol = 25:1) to give a white solid in a 70% yield. 1H NMR (400MHz, CDCl3) δ7.54(d,J=8.5Hz,1H),7.03(d,J=7.6Hz,4H),6.86(dd,J=11.8,8.4Hz,4H),6.51( dd,J=8.5,2.2Hz,1H),6.45(d,J=2.1Hz,1H),6.27(t,J=5.4Hz,1H),5.59(d,J=9.9Hz,1H),5.45(d,J=9. 9Hz,1H),4.59(dt,J=12.0,6.0Hz,1H),3.94–3.78(m,5H),3.75–3.68(m,2H),3.68–3.59(m,2H),3.57– 3.39(m,6H),3.38–3.32(m,2H),3.31–3.22(m,1H),3.10(t,J=5.2Hz,2H),1.37(dd,J=16.5,6.0Hz,6H).

[0128] Depending on the value of n, different azido-nPEG-NH2 groups were used to prepare other azide-linked E3 ligands N3-nP-MDM2 according to the preparation method of Example 20.

[0129] Example 21 Preparation of EZH2 Series 4 Compound EP-MDM2 (Series 4, n=1)

[0130]

[0131] The preparation process and feed ratio of Example 17 were adopted to prepare the product using compound 1 and N3-P-MDM2 (n=1). 1HNMR(600MHz,DMSO-d6)δ11.46(s,1H),8.47(s,1H),8.19(t,J=5.0Hz,1H),7.98(d,J=8.5Hz,1H),7.92(t,J=5.7Hz,1H),7.74–7.70(m,1H),7.59(d,J=7.8Hz,2H),7.53(d,J=8.1Hz,1H),7.40(dd,J=7.0,4.0Hz,3H),7.23(d,J=1.8Hz,1H),7.14(d,J=8.3Hz,2H),7.11(d,J=8.2Hz,2H),7.04(d,J=8.2Hz,2H),6.97(d,J=8.1Hz,2H),6.61(s,1H),5.86(s,1H),5.65(d,J=9.7Hz,1H),5.58(d,J=9.7Hz,1H),4.71(p,J=6.0Hz,1H),4.56(dt,J=14.3,5.4Hz,3H),4.29(d,J=5.0Hz,2H),4.06(s,1H),3.82(q,J=5.3,4.8Hz,6H),3.75–3.67(m,2H),3.62(d,J=13.5Hz,2H),3.56(d,J=14.9Hz,2H),3.51(d,J=1.3Hz,6H),3.48(t,J=5.3Hz,2H),3.42(d,J=5.3Hz,2H),3.25(t,J=11.7Hz,2H),3.15(d,J=5.9Hz,2H),3.09(q,J=7.0Hz,2H),2.99(s,2H),2.25(s,3H),2.21(s,3H),2.10(s,3H),1.91(s,1H),1.66(d,J=12.4Hz,2H),1.53(dd,J=12.5,8.6Hz,2H),1.34(s,3H),1.26(s,6H). 13C NMR(151MHz,DMSO-d6)δ174.76,170.82,169.52,167.83,164.85,163.47,159.92,156 .98,150.00,139.31,130.15,130.12,129.20,127.93,127.90,126.98,115.10,99.79 ,72.80,70.28,70.25,69.09,68.56,66.80,60.67,60.23,55.90,50.01,49.06,41.59,40.52,38.75,31.75,31.62,30.30,27.02,25.58,24.96,22.56,21.23,14.56,14.42.

[0132] Example 22 Preparation of EZH2 Series 4 Compound E-2P-MDM2 (Series 4, n=2)

[0133]

[0134] The preparation process and feed ratio of Example 17 were adopted to prepare compound 1 and N 3 -2P-MDM 2 (n=2) was prepared. 1HNMR(600MHz,DMSO-d6)δ11.46(s,1H),8.49(s,1H),8.19(t,J=5.0Hz,1H),7.97(d,J=8.4Hz,1H),7.93(t,J=5.7Hz,1H),7.71(d,J=8.3Hz,1H),7.59(d,J=7.8Hz,2H),7.52(d,J=8.3Hz,1H),7.40(d,J=7.4Hz,3H),7.23(d,J=1.8Hz,1H),7.15(d,J=8.1Hz,2H),7.11(d,J=8.1Hz,2H),7.04(d,J=8.1Hz,2H),6.97(d,J=8.0Hz,2H),6.61(s,1H),5.86(s,1H),5.65(d,J=9.7Hz,1H),5.58(d,J=9.7Hz,1H),4.71(p,J=6.0Hz,1H),4.57(q,J=5.6,5.0Hz,3H),4.29(d,J=4.9Hz,2H),4.07(s,1H),3.83(d,J=5.3Hz,6H),3.75–3.67(m,2H),3.62(d,J=19.8Hz,2H),3.57(d,J=7.4Hz,2H),3.51(s,8H),3.47(dt,J=16.7,4.9Hz,4H),3.41(t,J=5.3Hz,2H),3.25(t,J=11.7Hz,2H),3.17(q,J=6.0Hz,2H),3.09(q,J=7.0Hz,2H),3.00(q,J=8.1,5.3Hz,2H),2.25(s,3H),2.21(s,3H),2.11(s,3H),1.91(s,1H),1.66(d,J=12.5Hz,2H),1.53(qd,J=12.2,4.5Hz,2H),1.34(s,3H),1.25(s,6H). 13 C NMR(101MHz,DMSO-d6)δ167.79,164.82,163.47,159.92,156.99,131.67,130.14,129.20,127.86,127.0 3 ,107.83,99.79,72.80,70.26,69.86,69.38,68.80,66.79,66.62,60.67,60.22,55.91,50.0 3,49.46,38.96,35.36,33.22,31.75,31.61,22.55,22.18,22.11,18.65,14.55,14.41.

[0135] Example 23 Preparation of EZH2 Series 4 Compound E-3P-MDM2 (Series 4, n=3)

[0136]

[0137] The preparation process and feed ratio of Example 17 were used to prepare the compound 1 and N3-3P-MDM 2 (n=3) were prepared. 1 HNMR(600MHz,DMSO-d6)δ11.40(s,1H),8.42(s,1H),8.13(t,J=5.0Hz,1H),7.88(t,J=5.7Hz,1H), 7.53(s,2H),7.46(d,J=8.4Hz,1H),7.37–7.30(m,3H),7.16(d,J=1.8Hz,1H),7.09–7.06(m,2H),7. 06–7.03(m,2H),6.97(d,J=8.4Hz,2H),6.91(d,J=8.1Hz,2H),6.54(s,1H),5.79(s,1H),5.59(d,J= 9.8Hz,1H),5.51(d,J=9.8Hz,1H),4.67–4.63(m,1H),4.51(dt,J=10.5,5.3Hz,3H),4.23(d,J=5.0H z,2H),3.76(d,J=5.9Hz,6H),3.68–3.60(m,2H),3.55(d,J=11.9Hz,2H),3.46(d,J=4.1Hz,2H),3.4 4(d,J=1.3Hz,10H),3.43–3.40(m,4H),3.39(d,J=4.5Hz,4H),3.36–3.31(m,4H),3.18(d,J=2.0Hz, 2H),3.10(dd,J=5.3,3.2Hz,2H),3.02(q,J=6.9Hz,2H),2.94(d,J=14.7Hz,2H),2.18(s,3H),2.14( s,3H),2.04(s,3H),1.59(d,J=12.2Hz,2H),1.49–1.44(m,2H),1.27(s,3H),1.19(t,J=3.0Hz,6H). 13C NMR(101MHz,DMSO-d6)δ172.89,169.28,167.86,164.82,163.29,160.03,156.70,154.53,153.69 ,149.90,149.31,147.62,143.12,139.32,132.36,131.75,130.15,129.20,127.92,126.95,126. 84,123.63,122.15,121.82,115.31,107.96,105.44,99.94,72.80,70.25,70.13,69.98,69.37,68.83,66.80,60.67,55.90,50.02,41.60,31.75,30.75,30.29,22.55,22.15,19.41,14.41,13.19.

[0138] Example 24 Preparation of EZH2 Series 4 Compound E-4P-MDM2 (Series 4, n=4)

[0139]

[0140] The preparation process and feed ratio of Example 17 were adopted to prepare the product using compound 1 and N3-4P-MDM2 (n=4). 1HNMR(600MHz,DMSO-d6)δ11.46(s,1H),8.48(s,1H),8.19(t,J=5.0Hz,1H),7.98(d,J=8.5Hz,1H),7.95(t,J=5.6Hz,1H),7.74–7.70(m,1H),7.59(d,J=7.6Hz,2H),7.52(d,J=8.3Hz,1H),7.40(q,J=5.3,4.6Hz,3H),7.23(d,J=1.8Hz,1H),7.15(d,J=8.4Hz,2H),7.11(d,J=8.3Hz,2H),7.04(d,J=8.3Hz,2H),6.97(d,J=8.1Hz,2H),6.61(s,1H),5.86(s,1H),5.65(d,J=9.8Hz,1H),5.58(d,J=9.7Hz,1H),4.72(p,J=6.0Hz,1H),4.57(q,J=5.6Hz,3H),4.29(d,J=5.0Hz,2H),4.05(s,1H),3.83(d,J=5.5Hz,6H),3.73(d,J=16.5Hz,2H),3.59(d,J=17.7Hz,2H),3.53(d,J=4.0Hz,2H),3.52(s,10H),3.48–3.46(m,8H),3.42(d,J=5.3Hz,2H),3.37(d,J=6.0Hz,2H),3.26(dd,J=11.6,2.2Hz,2H),3.19(d,J=6.0Hz,2H),3.09(d,J=7.1Hz,2H),3.00(d,J=6.1Hz,2H),2.25(s,3H),2.21(s,3H),2.10(s,3H),1.91(s,1H),1.66(d,J=12.5Hz,2H),1.55–1.51(m,2H),1.34(s,3H),1.27–1.25(m,6H). 13C NMR (151MHz, DMSO-d6) δ170.82,169.52,167.79,164.82,163.47,159.91,156.98,150.00,149.36,143.19,140. 09,132.43,131.72,131.58,130.14,129.39,129.19,127.92,126.96,123.35,121.30,107.81,105.48,99.78,72 .80,70.28,70.25,70.17,70.04,69.96,69.38,68.82,68.31,66.80,60.67,60.23,58.33,55.90,50.02,49.06,46.90,41.59,40.52,39.00,35.35,31.73,30.76,22.56,22.19,22.12,21.24,19.42,18.65,15.04,14.56,13.20.

[0141] Example 25 Synthesis of Azide-Linked E3 Ligand N3-nPEG-Tha (n=4)

[0142]

[0143] 58 mg of a thalidomide derivative was placed in an eggplant-shaped flask and 3 mL of DMF was added. 50 mg of azido-PEG4-amine and 47 μL of DIPEA were added sequentially with stirring. The mixture was reacted at 90°C for 3-4 hours. 30 mL of water and 30 mL of ethyl acetate were added for extraction. The organic layer was dried over anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography with a gradient elution ratio of petroleum ether:ethyl acetate = 1:2 to 1:4 to obtain a yellow oil in a 60% yield. 1 H NMR (400MHz, CDCl3) δ8.81 (br s,1H),7.48(dd,J=8.0,7.2Hz,1H),7.09(d,J=7.2Hz,1H),6.92(d,J=8.8Hz,1H),6.49(t,J=5.6Hz,1H),4.95–4.90(m,1H),3 .72(t,J=5.2Hz,2H),3.68–3.66(m,14H),3.48(q,J=5.6Hz,2H),3.38(t,J=5.0Hz,2H),2.88–2.72(m,3H),2.13–2.09(m,1H); 13C NMR (100MHz, CDCl3) δ171.6,169.4,168.8,167.8,146.9,136.1,132.6,116.9,111.7,1 10.3,70.8,70.74,70.71,70.7,70.65,70.62,70.1,69.6,50.8,48.9,42.5,31.5,22.9.

[0144] The preparation process of other azide-linked E3 ligands N3-nPEG-Tha in series 5 is the same as Example 25.

[0145] Example 26 Preparation of EZH2 Series 5 Compound E-2P-Tha (Series 5, n=2)

[0146]

[0147] Compound 1 and N3-2PEG-Tha were used as raw materials, and the preparation process and feeding ratio were referred to Example 10. 1HNMR(600MHz,DMSO-d6)δ11.46(s,1H),11.09(s,1H),8.43(s,1H),8.19(t,J=5.0Hz,1H),7.61– 7.57(m,2H),7.56–7.52(m,1H),7.42–7.37(m,3H),7.23(d,J=1.8Hz,1H),7.08(d,J=8.6Hz,1H), 7.01(d,J=7.0Hz,1H),6.59(t,J=6.0Hz,1H),5.86(s,1H),5.32(tt,J=4.5,1.3Hz,1H),5.04(td ,J=13.2,6.8Hz,1H),4.58(t,J=5.2Hz,2H),4.29(d,J=5.0Hz,2H),3.99(dd,J=14.4,7.8Hz,2H), 3.88(t,J=5.2Hz,2H),3.83(t,J=5.8Hz,2H),3.61(t,J=5.4Hz,3H),3.53(s,2H),3.44–3.41(m, 2H),3.25(td,J=11.7,2.0Hz,2H),3.09(q,J=7.0Hz,2H),3.04–3.01(m,1H),2.89–2.86(m,1H),2 .61–2.57(m,1H),2.55–2.51(m,1H),2.43(s,4H),2.25(s,3H),2.21(s,3H),2.11(d,J=0.8Hz,3H ),2.06–2.00(m,2H),1.66(d,J=12.5Hz,2H),1.55–1.51(m,2H),1.24(s,3H),0.85–0.83(m,3H).

[0148] Example 27 Preparation of EZH2 Series 5 Compound E-3P-Tha (Series 5, n=3)

[0149]

[0150] Compound 1 and N3-3PEG-Tha were used as raw materials, and the preparation process and feeding ratio were referred to Example 10. 1HNMR(600MHz,DMSO-d6)δ11.47(s,1H),11.09(s,1H),8.49(s,1H),8.20(t,J=5.0Hz, 1H),7.61–7.54(m,3H),7.43–7.36(m,3H),7.23(d,J=1.8Hz,1H),7.12(d,J=8.6Hz,1 H),7.04(d,J=7.0Hz,1H),6.59(t,J=5.8Hz,1H),5.86(s,1H),5.05(dd,J=12.8,5.4H z,1H),4.57(t,J=5.2Hz,2H),4.30(d,J=5.0Hz,2H),4.08–4.00(m,2H),3.87–3.81(m ,4H),3.55(ddt,J=18.4,9.6,3.8Hz,10H),3.44(q,J=5.5Hz,2H),3.25(td,J=11.6,2 .1Hz,2H),3.09(q,J=7.0Hz,2H),3.02(t,J=3.8Hz,1H),2.91–2.84(m,1H),2.62–2.5 2(m,2H),2.47–2.39(m,4H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.02(d,J=5.4Hz ,1H),1.71–1.63(m,2H),1.57–1.48(m,3H),1.29–1.21(m,3H),0.84(t,J=7.0Hz,3H).

[0151] Example 28 Preparation of EZH2 Series 5 Compound E-4P-Tha (Series 5, n=4)

[0152]

[0153] Compound 1 and N3-4PEG-Tha were used as raw materials, and the preparation process and feed ratio were referred to Example 10. 1HNMR (600MHz, DMSO-d6) δ11.47(s,1H),11.10(s,1H),8.47(s,1H),8.20(t,J=5. 0Hz,1H),7.62–7.54(m,3H),7.43–7.35(m,3H),7.23(d,J=1.9Hz,1H),7.13(d,J =8.6Hz,1H),7.04(d,J=7.1Hz,1H),6.59(t,J=5.8Hz,1H),5.86(s,1H),5.05(dd ,J=12.9,5.4Hz,1H),4.56(t,J=5.2Hz,2H),4.30(d,J=5.0Hz,2H),4.09–4.00(m, 2H),3.83(q,J=4.9,4.5Hz,4H),3.69–3.41(m,17H),3.25(td,J=11.6,2.0Hz,2H ),3.09(q,J=7.0Hz,2H),3.02(t,J=3.8Hz,1H),2.92–2.84(m,1H),2.62–2.52(m ,2H),2.48–2.37(m,4H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.02(s,2H),1. 69–1.63(m,2H),1.57–1.49(m,2H),1.24(d,J=4.8Hz,2H),0.84(t,J=7.0Hz,3H).

[0154] Example 29 Preparation of EZH2 Series 5 Compound E-5P-Tha (Series 5, n=5)

[0155]

[0156] Compound 1 and N3-5PEG-Tha were used as raw materials, and the preparation process and feeding ratio were referred to Example 10. 1HNMR(600MHz,DMSO-d6)δ11.46(s,1H),11.09(s,1H),8.47(s,1H),8.19(t,J=5.0Hz,1H) ,7.61–7.55(m,3H),7.41–7.36(m,3H),7.23(t,J=1.2Hz,1H),7.13(d,J=8.6Hz,1H),7.0 3(d,J=7.0Hz,1H),6.60(t,J=5.8Hz,1H),5.86(s,1H),5.05(dd,J=12.8,5.5Hz,1H),4.5 6(t,J=5.2Hz,2H),4.29(d,J=5.0Hz,2H),4.04(t,J=8.0Hz,2H),3.85–3.80(m,4H),3.61( t,J=5.5Hz,2H),3.55–3.51(m,7H),3.48–3.44(m,10H),3.25(td,J=11.6,2.0Hz,2H),3. 09(q,J=7.1,6.5Hz,2H),3.02(t,J=3.8Hz,1H),2.90–2.85(m,1H),2.61–2.52(m,2H),2. 44(d,J=14.7Hz,4H),2.25(s,3H),2.21(s,3H),2.11(d,J=0.8Hz,3H),2.05–1.96(m,2H) ,1.66(d,J=12.1Hz,2H),1.58–1.49(m,2H),1.24(d,J=4.0Hz,3H),0.83(t,J=7.0Hz,3H).

[0157] Example 30 Preparation of EZH2 Series 6 Compound E-4W-7CH2V2 (Series 6, n=7)

[0158] Compound 2 and N3-nCH2-VHL2 (n=7) were used as raw materials, and the preparation process and feed ratio were the same as those in Example 10. 1H NMR(600MHz,DMSO-d6)δ11.49(s,1H),8.98(s,1H),8.39(d,J=7.8Hz,1H),8.20(s,1H),7.81(d,J=13.6Hz,2H),7.58(d,J=7.9Hz,2H),7.45–7.41(m,2H),7.41–7.36(m,5H),7.23(d,J=1.9Hz,1H),5.86(s,1H),5.12(d,J=3.5Hz,1H),4.92(s,1H),4.52(d,J=9.3Hz,1H),4.42(s,1H),4.31–4.25(m,4H),3.85–3.80(m,2H),3.63–3.57(m,2H),3.50(s,2H),3.46–3.43(m,3H),3.36(s,8H),3.24(td,J=11.7,2.1Hz,2H),3.08(q,J=7.0Hz,2H),3.01(tt,J=11.0,3.8Hz,1H),2.82(t,J=7.5Hz,2H),2.65(t,J=7.6Hz,2H),2.45(s,3H),2.36–2.30(m,3H),2.25(s,3H),2.21(s,3H),2.11(s,3H),2.02(dd,J=12.8,7.9Hz,1H),1.78–1.74(m,2H),1.66(d,J=12.6Hz,2H),1.56–1.40(m,5H),1.37(d,J=7.1Hz,3H),1.22(d,J=4.0Hz,6H),0.93(s,9H). 13 C NMR(101MHz,DMSO-d6)δ172.50,171.09,170.09,169.53,163.49,151.93,150.00,149.35,148.21,146.47,145.13,143.22,140.08,137.45,133.12,131.58,129.94,129.28,126.91,126.85,123.33,122.37,122.08,121.30,107.85,69.22,66.80,59.01,58.33,56.80,49.58,48.17,41.62,38.19,35.65,35.32,32.31,30.77,30.21,29.48,28.91,28.56,26.90,26.24,25.76,22.88,21.42,19.42,18.65,15.03,13.19.

[0159] Example 31 Preparation of EZH2 Series 7 Compound E-4W-CH2B4 (Series 7, n=1)

[0160]

[0161] Compound 2 and N3-nCH2-B4 (n=1) are used as raw materials, and the preparation process and feeding ratio are referred to Example 10. 1 H NMR (600MHz, DMSO-d6) δ11.49(s,1H),8.21(t,J=5.0Hz,1H),8.17(d,J=9.0Hz,1H),7.87(d,J=8.5Hz ,1H),7.62(s,1H),7.58(d,J=8.0Hz,2H),7.40(d,J=1.9Hz,1H),7.38(d,J=7.9Hz,2H),7.28(t,J=7. 5Hz,2H),7.24–7.21(m,3H),7.21(d,J=1.4Hz,1H),6.36(dd,J=11.3,5.8Hz,1H),5.86(s,1H),5.02( d,J=16.2Hz,1H),4.89(d,J=16.0Hz,1H),4.37(ddd,J=10.3,8.5,4.9Hz,1H),4.30(d,J=5.0Hz,2H), 4.25–4.18(m,1H),3.91(dd,J=5.9,2.5Hz,1H),3.83(dt,J=9.3,2.7Hz,2H),3.61(s,3H),3.51(s,2H ),3.48–3.42(m,4H),3.25(td,J=11.6,2.0Hz,2H),3.08(q,J=7.0Hz,2H),3.01(dt,J=10.9,3.8Hz,1 H),2.89–2.80(m,3H),2.66(dt,J=21.3,7.4Hz,3H),2.34(dt,J=18.1,4.8Hz,4H),2.25(s,3H),2.21 (s,3H),2.10(s,3H),1.71–1.63(m,3H),1.62–1.46(m,4H),0.88(d,J=6.5Hz,3H),0.86–0.80(m,6H). 13C NMR(101MHz,DMSO-d6)δ173.15,172.16,170.05,169.53,165.32,163.49,150.02,149.35,146.34,1 43.22,140.06,139.11,138.89,137.46,133.10,129.95,129.65,128.75,126.91,126.69,123.76,12 3.33,122.08,121.29,107.85,71.01,66.80,61.94,58.33,54.13,53.21,52.76,52.40,51.86,50.18,45.23,41.59,37.33,35.35,32.30,30.75,24.62,23.23,21.67,21.28,19.41,18.65,15.03,13.19.

[0162] Example 32 Preparation of EZH2 Series 7 Compound E-4W-3CH2B4 (Series 7, n=3)

[0163]

[0164] Compound 2 and N3-3CH2-B4 are used as raw materials, and the preparation process and feeding ratio are the same as those in Example 10. 1H NMR(600MHz,DMSO-d6)δ11.49(s,1H),8.21(q,J=5.4,3.8Hz,1H),7.86(d,J=8.5Hz,1H),7.76(d,J=2.3Hz,1H),7.63(d,J=8.8Hz,1H),7.60–7.56(m,2H),7.40(s,1H),7.37(d,J=7.8Hz,2H),7.28–7.24(m,2H),7.22(d,J=7.6Hz,3H),7.19–7.14(m,1H),6.21(d,J=6.1Hz,1H),5.86(d,J=2.4Hz,1H),4.37–4.31(m,1H),4.30(d,J=4.9Hz,2H),4.22(q,J=8.4Hz,1H),4.16(q,J=4.8,2.4Hz,2H),3.88(dd,J=6.1,2.9Hz,1H),3.85–3.80(m,2H),3.60(d,J=2.3Hz,3H),3.51(s,2H),3.45(d,J=16.4Hz,4H),3.24(t,J=11.6Hz,2H),3.08(q,J=7.3Hz,2H),3.02(dt,J=6.7,3.4Hz,1H),2.82(q,J=6.7,6.1Hz,3H),2.65(t,J=7.6Hz,3H),2.33(d,J=19.0Hz,4H),2.25(d,J=2.2Hz,3H),2.21(d,J=2.2Hz,3H),2.11(d,J=2.3Hz,3H),2.06(dt,J=15.0,7.6Hz,1H),1.99(s,1H),1.88(ddp,J=20.9,14.0,7.1Hz,2H),1.69–1.59(m,3H),1.57–1.42(m,4H),0.82(ddd,J=14.1,6.7,2.2Hz,6H),0.76(dd,J=6.5,2.2Hz,3H). 13C NMR (101MHz, DMSO-d6) δ173.18,172.44,171.14,170.08,169.54,163.50,150.03,149.36,146.49,143. 23,140.07,139.23,139.12,137.46,133.12,129.96,129.66,128.61,126.91,126.55,123.35,122.48,1 22.08,121.29,107.87,71.65,66.80,61.94,58.33,53.63,53.24,52.76,52.36,50.06,49.11,45.24,41.60,37.39,35.36,32.39,32.33,30.62,26.52,24.49,23.23,21.60,21.40,19.41,18.65,15.02,13.18.

[0165] Example 33 Preparation of EZH2 Series 8 Compound E-4W-2PB5 (Series 8, n=2)

[0166]

[0167] Compound 2 and N3-2P-B5 were used as raw materials, and the preparation process and feeding ratio were the same as those in Example 10. 1 H NMR (600MHz, DMSO-d6) δ11.49(s,1H),8.21(t,J=5.0Hz,1H),8.07(t,J=5.7Hz,1H),7.80(s,1H),7.61(d,J=9.0Hz,1H),7.58(d,J=8.0Hz,2 H),7.40(d,J=1.9Hz,1H),7.37(d,J=8.0Hz,2H),7.26(t,J=7.5Hz,2H),7.24–7.19(m,3H),7.17(t,J=7.3Hz,1H),6.17(d,J=9.4Hz,1H),6.0 3(d,J=6.2Hz,1H),5.86(s,1H),4.44(t,J=5.2Hz,2H),4.35(dd,J=9.3,5.2Hz,1H),4.30(d,J=5.0Hz,2H),3.99–3.92(m,1H),3.85–3.80(m,3H ),3.76(t,J=5.2Hz,2H),3.53–3.48(m,4H),3.45(dd,J=6.3,3.9Hz,6H),3.35(d,J=6.0Hz,3H),3.28–3.21(m,2H),3.17(q,J=6.0Hz,2H),3.08 (q,J=7.0Hz,2H),3.01(tt,J=11.0,3.8Hz,1H),2.86–2.75(m,3H),2.65(q,J=7.6,6.7Hz,3H),2.33(dt,J=22.1,4.9Hz,4H),2.25(s,3H),2.21 (s,3H),2.11(s,3H),1.65(td,J=12.1,11.3,5.8Hz,2H),1.57–1.50(m,3H),1.28(s,10H),0.85(d,J=6.9Hz,3H),0.81(dd,J=9.0,6.7Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ172.31,172.01,170.11,169.55,163.50,155.34,150.04,149.35,146.45,143.23, 140.06,139.15,137.44,133.12,129.96,129.68,128.59,126.90,126.47,123.35,122.92,122.07,121.29, 107.89,78.05,71.87,69.92,69.27,66.79,61.94,58.33,54.94,53.23,52.74,50.79,49.67,45.23,42.12,41.60,39.02,38.04,35.36,32.32,30.62,28.57,24.44,23.61,22.16,21.36,19.41,18.65,15.02,13.18.

[0168] The preparation method of Example 10 was used, and the same feed ratio and operation were used to prepare compound 2 and N 3 -3P-B5、N 3 -4P-B5 reaction can produce series 8 compounds E-4W-3PB5 and E-4W-4PB5.

[0169] Example 34 Preparation of EZH2 Series 8 Compound E-4W-3PB5 (Series 8, n=3)

[0170] 1 H NMR (600MHz, DMSO-d6) δ11.49(s,1H),8.21(t,J=5.0Hz,1H),8.08(t,J=5.7Hz,1H),7.80(s,1H),7.61(d,J=9.0Hz,1H),7.58(d,J=8.1Hz,2H), 7.40(d,J=1.9Hz,1H),7.37(d,J=8.0Hz,2H),7.26(t,J=7.5Hz,2H),7.23–7.19(m,3H),7.17(td,J=7.1,1.5Hz,1H),6.17(d,J=9.4Hz,1H),6.0 3 (d,J=6.2Hz,1H),5.86(s,1H),4.45(t,J=5.2Hz,2H),4.35(qd,J=9.5,5. 2Hz,1H),4.30(d,J=5.0Hz,2H),3.95(dtd,J=10.0,7.5,2.6Hz,1H),3.87 –3.80(m,3H),3.77(t,J=5.2Hz,2H),3.52–3.48(m,4H),3.46(d,J=3.2Hz ,10H),3.37(d,J=6.1Hz,3H),3.24(td,J=11.6,2.0Hz,2H),3.18(q,J=5. 9Hz,2H),3.08(q,J=7.0Hz,2H),3.01(tt,J=11.0,3.8Hz,1H),2.86–2.75 (m,3H),2.66(dt,J=15.3,7.7Hz,3H),2.33(dt,J=21.4,4.7Hz,4H),2.25 (s,3H),2.21(s,3H),2.11(s,3H),1.68–1.63(m,2H),1.53(ddd,J=17.1, 10.0, 4.8Hz, 3H), 1.28 (s, 10H), 0.85 (d, J = 6.7Hz, 3H), 0.84–0.80 (m, 6H). 13C NMR(101MHz,DMSO-d6)δ172.31,172.00,170.10,169.55,163.51,155.34,150.05,1 49.35,146.43,143.23,140.06,139.14,137.47,133.12,129.96,129.69,128.59,12 6.90,126.48,123.35,122.92,122.07,121.29,107.90,78.05,71.86,70.29,69.28,66.80,61.95,58.33,54.93,53.23,52.75,50.78,49.67,45.24,42.15,41.60,39.0 3 ,38.04,35.36,32.32,30.77,28.57,24.43,23.61,22.17,21.37,19.41,18.65,15.02,13.18.

[0171] Example 35 Preparation of EZH2 Series 8 Compound E-4W-4PB5 (Series 8, n=4)

[0172] 1H NMR(600MHz,DMSO-d6)δ11.49(s,1H),8.21(t,J=5.0Hz,1H),8.09(t,J=5.7Hz,1H),7.81(s,1H),7.61(d,J=9.0Hz,1H),7.58(d,J=8.1Hz,2H),7.40(d,J=1.9Hz,1H),7.37(d,J=7.9Hz,2H),7.26(t,J=7.5Hz,2H),7.24–7.20(m,3H),7.17(td,J=7.1,1.5Hz,1H),6.17(d,J=9.4Hz,1H),6.04(d,J=6.2Hz,1H),5.86(s,1H),4.45(t,J=5.3Hz,2H),4.34(td,J=9.3,5.2Hz,1H),4.30(d,J=5.0Hz,2H),3.95(dtd,J=10.0,7.5,2.6Hz,1H),3.82(ddd,J=8.8,5.8,2.8Hz,3H),3.78(t,J=5.3Hz,2H),3.53–3.49(m,4H),3.47(dd,J=5.5,2.8Hz,14H),3.37(d,J=7.2Hz,3H),3.24(td,J=11.6,2.0Hz,2H),3.20–3.16(m,2H),3.08(q,J=7.0Hz,2H),3.04–2.98(m,1H),2.86–2.76(m,3H),2.66(dt,J=15.3,7.6Hz,3H),2.39–2.30(m,4H),2.25(s,3H),2.21(s,3H),2.11(s,3H),1.68–1.63(m,2H),1.53(ddt,J=12.6,9.3,3.1Hz,3H),1.28(s,10H),0.86–0.84(m,3H),0.84–0.80(m,6H). 13CNMR(101MHz,DMSO-d6)δ172.31,172.00,170.10,169.55,163.50,155.34,150.04,149.35,146.42,143.23,1 40.06,139.14,137.46,133.12,129.96,129.69,128.59,126.90,126.47,123.35,122.92,122.07,121.29,10 7.89,78.05,71.85,70.19,70.06,69.28,66.79,61.94,58.33,54.93,53.23,52.74,50.78,49.67,45.24,42.15,41.64,39.02,38.05,35.35,32.33,30.61,28.57,24.44,23.61,22.18,21.37,19.41,18.65,15.02,13.18.

[0173] Example 36 Preparation of EZH2 Series 9 Compound E-4W-2PB5(T) (Series 9, n=2)

[0174]

[0175] Compound E-4W-2PB5 (series 8, n=2) was dissolved in ethyl acetate and an excess of 5M / L HCl in ethyl acetate was added with stirring. TLC confirmed the complete reaction. The reaction system was then distilled under reduced pressure and the pH was adjusted to 9-10 with saturated sodium bicarbonate. The reaction mixture was extracted with ethyl acetate, and the organic layer was dried to yield E-4W-2PB5T.

[0176] By adopting the preparation method of Example 29, with the same feed ratio and operation, using compounds E-4W-3PB5 (series 8, n=3) and E-4W-4PB5 (series 8, n=4) as raw materials, compounds E-4W-3PB5T and E-4W-4PB5T of series 9 can be obtained.

[0177] The synthesis of series 10 compounds was carried out by referring to the synthesis method of series 5 compounds to obtain compounds MP-EC-2, MP-EC-3, and MP-EC-4, respectively.

[0178] Example 37

[0179] SU-DHL-6 cells in logarithmic phase growth were digested with trypsin and plated in 96-well plates at a density of 5000 cells / well. 24 hours later, they were treated with a gradient of compound concentrations (0–30 μM). The number of viable cells was determined 48 hours after administration using CCK-8 at a wavelength of 450 nm. IC was calculated using GraphPad Prism 8.0.1. 50 The results are shown in Table 1.

[0180] Experimental results show that compounds based on thalidomide (Tha) are less effective. The toxicity of this series of compounds increases with increasing chain length, but the activity is low when the chain length is 5 PEG. In addition, the cytotoxicity of PROTACs based on Bestatin derivatives (including B4 and B5) is generally better than that of PROTACs based on thalidomide. The cytotoxicity of PROTACs based on VHL is relatively low, but the activity of E-5CH2-V1 is much higher than that of other compounds. Among the molecules based on MDM2 ligands, the cytotoxicity of the compound is closely related to its chain length, and E-3P / 4P-MDM2 has stronger cytotoxicity.

[0181] Example 38 Western Blot Detection of Changes in Related Proteins

[0182] (1) Digest and collect cells that have grown logarithmically and have reached a confluence of 90%, add fresh culture medium to adjust the cell concentration, and then count them using a cell counting plate. Evenly inoculate the cells in a large dish and culture them in an incubator for 24 hours. Replace the culture medium containing drugs and continue to culture them in an incubator for 48 hours. After waiting for the end of culture, collect the cells and wash them twice with PBS. After washing, add an appropriate amount of RIPA lysis buffer according to the number of cells. Add phosphatase inhibitors and whole enzyme inhibitors to the lysis buffer in advance, and add PMSF in time. PMSF is an irreversible inhibitor of serine proteases that can inhibit serine proteases (trypsin and chymosin), as well as cysteine ​​proteases and acetylcholinesterase, thereby preventing protein degradation. After adding the lysis buffer, use a vortex oscillator to mix the cells and lyse the proteins on ice to prevent protein degradation. Lyse on ice for 20 minutes. After lysis, use a 4℃ centrifuge to centrifuge at 11500 rpm for 10 minutes. Wait for the centrifugation to end and aspirate the supernatant, which is protein; (2) This experiment uses the BCA method to detect protein content. Under alkaline conditions, the protein converts Cu 2+ Reduction to Cu + , Cu +It forms a purple-blue complex with the BCA reagent, measures its absorbance at 562 nm, and compares it with the standard curve to calculate the concentration of the protein to be tested; (3) Add the corresponding volume of 6× Loading buffer to the quantified protein sample according to its volume. Before use, add DTT to the 6× Loading buffer. DTT can open the disulfide bond on the cys, making it a linear peptide, which is convenient for the antibody to recognize and bind to specific sites. Use a vortex oscillator to mix the sample and place it in a 100℃ water bath for 5-10 minutes to fully denature the protein. If the amino acid composition of a protein contains a large number of hydrophobic amino acids (tryptophan W, alanine A, valine V, leucine L, isoleucine I, proline P, phenylalanine F, etc.), the protein is very hydrophobic and will easily form aggregates after being boiled. For such a protein rich in hydrophobic amino acids, it can be incubated at 37°C for 1 hour, or 60°C for 30 minutes instead of boiling in a 100°C water bath for 5-10 minutes; (4) Prepare SDS-PAGE gel and load the prepared sample; (5) Place the PVDF membrane in anhydrous methanol for 30 seconds to activate the PVDF membrane. Then soak the membrane in wet transfer solution. Place the sponge, filter paper and PVDF membrane in the white side of the electrotransfer clamp in sequence, and then place the gel, filter paper and sponge in sequence to make a sandwich structure. Avoid bubbles between the gel and the membrane. Insert the sandwich structure into the electrotransfer tank and place the electrotransfer device in an ice box mixed with ice and water to avoid excessive heat generated during the transfer process, which may cause transfer failure. Use 300mA to transfer the protein for 2 hours; (6) After the protein transfer is completed, open the sandwich structure, stain the membrane with Lichunhong to check the transfer effect and use pure water to clean the membrane. Cut the membrane according to the molecular weight of the protein, then dry the PVDF membrane with filter paper and use methanol for secondary activation again. After activation, clean the surface methanol with pure water, then put it into 5% skim milk and block it on a shaker for 2 hours; (7) Use TBST solution to clean the milk on the blocked membrane. Use 5% BSA liquid to dilute the primary antibody according to the dilution ratio specified in the antibody manual to prepare the required antibody. Then place the PVDF membrane in TBST on a wet box for primary antibody incubation, and place the wet box in a 4℃ refrigerator overnight to incubate the primary antibody; (8) Remove the wet box and wash the membrane three times with TBST on a shaker, each time for 5 minutes. Select different secondary antibodies according to the primary antibody manual. Place the washed membrane on the wet box again and add secondary antibody liquid and block it at room temperature for two hours; (9) Expose the target band using a gel imager.

[0183] The experimental results are as follows Figure 1-2The CRBN series of compounds showed poor degradation of EZH2, with only E-4P-Tha and E-4W-3P-B5 showing specific degradation. Within the VHL2 series, with the exception of E-5CH2-V2, the degradation of the other compounds was poor. Due to the high toxicity of VHL1, the administered concentration was reduced after 48 hours of exposure. Furthermore, the degradation activity of the compounds was examined at 72 hours. Results showed slight degradation of E-5CH2-V1 after 72 hours of administration. Within the VHL series, E-5CH2-V2 and E-5CH2-V1 affected EZH2 expression, but their degradation capacity was limited. Within the MDM2 series of compounds, with the exception of EP-MDM2, all other compounds showed varying degrees of EZH2 degradation.

[0184] Example 39 Detection of cell apoptosis and MMP by flow cytometry

[0185] (1) Mitochondrial membrane potential: Take 100,000-600,000 cells and resuspend them in 0.5 mL of cell culture medium, which may contain serum and phenol red. Add 0.5 mL of JC-1 staining working solution and mix by inverting several times. Incubate in a cell culture incubator at 37°C for 20 minutes. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1X) by adding 4 mL of distilled water to every 1 mL of JC-1 staining buffer (5X) and place it in an ice bath. After the incubation at 37°C, centrifuge at 600g for 3-4 minutes at 4°C to pellet the cells. Discard the supernatant, taking care not to remove the cells as much as possible. Wash twice with JC-1 staining buffer (1X): Add 1 mL of JC-1 staining buffer (1X) to resuspend the cells, centrifuge at 600g for 3-4 minutes at 4°C to pellet the cells, and discard the supernatant. Resuspend the cells in 1 mL of JC-1 Staining Buffer (1X). Centrifuge at 600g for 3-4 minutes at 4°C to pellet the cells. Discard the supernatant. Resuspend the cells in an appropriate amount of JC-1 Staining Buffer (1X) and analyze using a flow cytometer.

[0186] (2) Apoptosis: After stimulation of cell apoptosis, centrifuge at 1000 g for 5 min, discard the supernatant, collect the cells, gently resuspend the cells in PBS and count them. Take 50,000-100,000 resuspended cells, centrifuge at 1000 g for 5 min, discard the supernatant. Add 5 μL of Annexin V-FITC and mix gently. Add 10 μL of propidium iodide staining solution and mix gently. Incubate at room temperature (20-25°C) in the dark for 10-20 min, then place in an ice bath.

[0187] The experimental results are as follows Figure 3 As shown, E-3P-MDM2 can significantly induce mitochondrial depolarization at high concentrations, and as time goes by, the compound significantly induces cell apoptosis. This also indicates that the compound induces SU-DHL-6 cell apoptosis through the mitochondrial pathway.

[0188] Table 1 Cytotoxicity of compounds on SU-DHL-6 cells after 48 hours

[0189]

Claims

1. An EZH2 ligand derivative or a pharmaceutically acceptable salt thereof, characterized in that: Any one of the following structural formulas: Series 2: The compound was named E-CH2-B4; Series 4: n = 2, 3 or 4, when n = 2, the compound is named E-2P-MDM2, or when n = 3, the compound is named E-3P-MDM2, or when n = 4, the compound is named E-4P-MDM2; Series 5: n = 3 or 4, when n = 3, the compound is named E-3P-Tha, or when n = 4, the compound is named E-4P-Tha; Series 8: n = 3, the compound was named E-4W-3PB5; Series 9: n=3, the compound was named E-4W-3PB5T.

2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the EZH2 ligand derivative according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle or a combination thereof; the dosage form of the pharmaceutical composition is an injection, tablet or capsule.

3. A method for preparing the EZH2 ligand derivative according to claim 1, characterized in that: The following steps are involved:

4. Use of the EZH2 ligand derivative according to claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 2 in the preparation of a medicament for treating lymphoma.

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