Compound for targeted ubiquitination degradation of G9a protein and pharmaceutical composition and application thereof

Abstract

The invention discloses a compound for targeted ubiquitination degradation of G9a protein as shown in a formula I and a medicinal composition and application thereof. The compound has the activity of inhibiting G9a protein and degrading G9a protein, shows good anti-tumor activity and can effectively inhibit malignant proliferation of pancreatic cancer PANC-1 cells. Therefore, the compound provided by the invention can be used for diseases related to abnormal expression of G9a protein, such as various cancers. (I).

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a class of compounds that target ubiquitination and degradation of G9a protein, their pharmaceutical compositions, and applications. Background Technology

[0002] Pancreatic cancer is one of the most common malignant tumors of the digestive tract, often referred to as the "king of cancers" in the field of oncology. Because the pancreas is a gland located deep in the abdomen, between the stomach and spine, early symptoms are often atypical and the onset is insidious. Most patients are diagnosed at an advanced stage, losing the opportunity for surgery, resulting in a poor prognosis; the survival rate for most patients after diagnosis is only about one year. The difficulty in diagnosis and poor treatment outcomes are the main reasons why pancreatic cancer is called the "king of cancers."

[0003] G9a is a widely studied methyltransferase that, along with its highly homologous analog GLP, catalyzes monomethylation and dimethylation of histone 3-lysine 9 (H3K9). Recent studies have reported that overexpression of G9a can induce various diseases, including cancer, Alzheimer's disease, sickle cell disease, and Prader-Willi syndrome. Therefore, G9a is a highly promising anti-tumor target.

[0004] Ubiquitin-mediated protein degradation is the most important negative regulatory mechanism for intracellular proteins. The ubiquitin-proteasome system (UPS) is responsible for clearing useless or harmful proteins from the cell, acting as the cell's "cleaner" and maintaining intracellular protein homeostasis. Proteolytic-targeting chimeras (PROTACs) are a class of heterobifunctional molecules that can simultaneously bind to a protein of interest and an E3 ubiquitin ligase, ubiquitinizing and degrading the protein of interest. They exhibit catalytic degradation characteristics and require low dosage.

[0005] Therefore, designing and synthesizing inhibitors with stronger activity against G9a protein and applying the protein hydrolysis-targeting chimera (PROTAC) technology to G9a protein can provide important references for G9a as a drug development target, the treatment of pancreatic cancer, and the development and application of PROTAC technology. Summary of the Invention

[0006] In one aspect, this invention provides a compound having the structure shown in Formula I, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, enantiomer, diastereomer, transisomer, racemate, or isotopically labeled compound thereof: (I); in: R 1 Selected from , , , , , , , , , , , , , and ; X 1 It can be -N- or -CH-; Y is either -CH2- or -C(=O)-; L is selected from -R 2 -C(=O)NH-R 3 C 4-18 Alkylene and C 4-18 alkeneoxy group; when L is selected from C 4-18 When alkylene, it includes, but is not limited to, C4 alkylene, C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene, C9 alkylene, C... 10 Alkylene, C 11 Alkylene, C 12 Alkylene, C 13 Alkylene, C 14 Alkylene, C 15 Alkylene, C 16 Alkylene, C 17 alkylene, or C 18 Alkylene; R 2 Selected from C 1-6 Alkylene, specifically it can be C1 alkylene, C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, or C6 alkylene; R 3 Selected from C 3-14 Alkylene and -CH2-(-CH2-O-CH2-) m -CH2-, m is an integer selected from 1 to 4; R 3 Specifically, it can be C3 alkylene, C4 alkylene, C5 alkylene, C6 alkylene, C7 alkylene, C8 alkylene, C9 alkylene, C 10 Alkylene, C 11 Alkylene, C 12 Alkylene, C 13 Alkylene, C 14Alkylene, -CH2-CH2-O-CH2-CH2-, -CH2(-CH2-O-CH2)2-CH2-, -CH2(-CH2-O-CH2)3-CH2-, or -CH2(-CH2-O-CH2)4-CH2-; R 3 Preferred C 6-11 Alkylene or -CH2(-CH2-O-CH2)3-CH2-; X 2 It can be -NH-, -O-, or -CH2-; A is selected from the following A-1, A-2, and A-3: A-1 is particularly preferred.

[0007] Preferably, the compound has the structure shown in Formula II:

[0008] (II); Among them, Y, L, X 2 The definition of A is the same as above. Preferably, Y is -CH2-, and X 2 It can be -NH- or -O-.

[0009] Preferably, the compound has the structure shown in Formula III: III; Where L is -R 2 -C(=O)NH-R 3 -; R 2 Selected from C 1-6 Alkylene; R 3 Selected from C 3-14 Alkyl groups and -CH2(-CH2-O-CH2) m -CH2-, m is an integer selected from 1 to 4; The definition of A is the same as above.

[0010] Preferably, the compound has the structure shown in Formula IV: (IV); Where L is -R 2 -C(=O)NH-R 3 -; R 2 Selected from C 1-6 Alkylene; R 3 Selected from C 3-14Alkylene and -CH2-(-CH2-O-CH2-) m -CH2-, m is an integer selected from 1 to 4; Further optimization yields L as -CH2CH2CONH(-CH2-). 7-10 -

[0011] Preferably, the compound is selected from:

[0012]

[0013]

[0014]

[0015]

[0016]

[0017]

[0018]

[0019] .

[0020] The compounds according to the present invention can be synthesized using the following synthetic scheme 1: Option 1:

[0021] III-b and or The reaction yields compound III; Where L2 is ; The definitions of R2, R3, and A are as described above.

[0022] The reaction in Scheme 1 described above can be carried out in the presence of a base, which includes inorganic bases (sodium carbonate, potassium carbonate, cesium carbonate, lithium hydroxide, sodium hydroxide, or potassium hydroxide, etc.) and organic bases (triethylamine, etc.). N , N (e.g., diisopropylamine or pyridine), with potassium carbonate being the preferred base. The reaction can be carried out in a polar solvent, including acetonitrile, etc. N , N -Dimethylformamide, N , N -Dimethylacetamide or dimethyl sulfoxide, etc., polar solvents are preferred. N,N -Dimethylformamide.

[0023] The compound III-b may be a commercially available product, or may be synthesized in a manner similar to that exemplified in the examples, or may be synthesized using the method described in Scheme 2 below.

[0024] Option 2:

[0025] M5 and The reaction yields intermediate III-a, which is then hydrolyzed to yield intermediate III-b. Where L1 represents ; L2 indicates ; The definition of R2 is as described above.

[0026] The reaction in Scheme 2 described above can be carried out in the presence of a base, which includes inorganic bases (sodium carbonate, potassium carbonate, cesium carbonate, lithium hydroxide, sodium hydroxide, or potassium hydroxide, etc.) and organic bases (triethylamine, etc.). N , N (e.g., diisopropylamine or pyridine), with potassium carbonate being the preferred base. The reaction can be carried out in a polar solvent, including acetonitrile, etc. N , N -Dimethylformamide, N , N -Dimethylacetamide or dimethyl sulfoxide, etc.), polar solvents are preferred. N,N -Dimethylformamide.

[0027] The compound M5 may be a commercially available product, or may be synthesized in a manner similar to that exemplified in the examples, or may be synthesized using the method described in Scheme 3 below.

[0028] Option 3:

[0029] S31: M1 reacts with 1-chloro-3-iodopropane in the presence of a base to give intermediate M2.

[0030] The alkali includes inorganic alkalis (sodium carbonate, potassium carbonate, cesium carbonate, lithium hydroxide, sodium hydroxide, or potassium hydroxide, etc.) and organic alkalis (triethylamine, etc.). N , N (e.g., diisopropylamine or pyridine), with potassium carbonate being the preferred base. This reaction can be carried out in polar solvents, including acetonitrile, etc. N , N -Dimethylformamide, N , N (e.g., dimethylacetamide or dimethyl sulfoxide), with acetonitrile being the preferred polar solvent.

[0031] S32: M2 reacts with pyrrole in the presence of a base to give intermediate M3.

[0032] The alkali includes inorganic alkalis (sodium carbonate, potassium carbonate, cesium carbonate, lithium hydroxide, sodium hydroxide, or potassium hydroxide, etc.) and organic alkalis (triethylamine, etc.). N , N -Diisopropylamine or pyridine, etc.), with potassium carbonate being the preferred base. Under these conditions, polar solvents include (acetonitrile, ... N , N -Dimethylformamide, N , N (e.g., dimethylacetamide or dimethyl sulfoxide), with acetonitrile being the preferred polar solvent.

[0033] S33: M3 reacts with a reducing agent to give intermediate M4.

[0034] The reducing agent includes iron powder, sulfide, tin chloride or zinc powder, etc., with iron powder being the preferred reducing agent.

[0035] S34: M4 reacts with cyclohexylformonitrile in the presence of acid, and then reacts with phosphorus oxychloride to give intermediate M5.

[0036] The acids include hydrochloric acid, sulfuric acid, etc. The reaction can be carried out in a solvent, including dioxane, acetonitrile, etc. N , N -Dimethylformamide, N , N (e.g., dimethylacetamide or dimethyl sulfoxide). After reacting with phosphorus oxychloride, it can be neutralized with an alkali, including sodium bicarbonate, sodium carbonate, sodium hydroxide, lithium hydroxide, potassium hydroxide, etc.

[0037] In another aspect, the present invention provides the use of the above-mentioned compound or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, enantiomer, diastereomer, transisomer, racemic mixture, or isotopically labeled compound in the preparation of a medicament for the prevention or treatment of diseases associated with abnormal expression of G9a protein activity.

[0038] Preferably, the diseases associated with abnormal expression of G9a protein activity include: tumors, hyperglycemia, diabetes, obesity, hyperlipidemia, hypercholesterolemia, hyperlipoproteinemia, hypertriglyceridemia, hypertension, hyperinsulinemia, hyperuricemia, Parkinson's disease, and Alzheimer's disease.

[0039] In another aspect, the present invention provides the use of the above-mentioned compound or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, enantiomer, diastereomer, transisomer, racemic mixture, or isotopically labeled compound in the preparation of a medicament for the prevention or treatment of tumors or for the prevention of postoperative recurrence of tumors.

[0040] Preferably, the tumor is selected from one or more of the following: non-small cell lung cancer, malignant melanoma, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, colon cancer, rectal cancer, breast cancer, cervical cancer, lung cancer, laryngeal cancer, nasopharyngeal cancer, pancreatic cancer, multiple myeloma, B lymphoma, and leukemia.

[0041] In another aspect, the present invention provides a pharmaceutical composition comprising one or more selected from the compounds described in any one of the preceding claims, pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, enantiomers, diastereomers, transisomers, racemates, isotopically labeled compounds, and pharmaceutically acceptable excipients.

[0042] Based on the above solution, the present invention has the following beneficial effects: The compounds provided by this invention can inhibit G9a protein activity and degrade G9a protein activity, and can effectively inhibit the malignant proliferation of pancreatic cancer PANC-1 cells. Therefore, this invention can be used for diseases related to abnormal G9a protein expression, such as various cancers. Moreover, the compounds of this invention can provide important references for the research and development of G9a as a drug development target, the treatment of leukemia, and the development of PROTAC technology. Attached Figure Description

[0043] Figure 1 The IC50 of compound 2 of this invention shows its inhibitory activity against various pancreatic cancer cells. 50 The bar chart.

[0044] Figure 2 This is an immunoblotting analysis of G9a protein in PANC-1 cells after treatment with blank control (DMSO), compounds 1-8 of this application, and negative control compound (UNC0638) at different concentrations for 24 hours.

[0045] Figure 3 This is an immunoblotting analysis of G9a and GLP proteins in PANC-1 cells after treatment with blank control (DMSO), negative control compound (UNC0638), and compound 9 of this application at different concentrations for 24 hours. Detailed Implementation

[0046] The following examples will illustrate the implementation of this application in detail, so that the process of how this application uses technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0047] Unless otherwise specified, all raw materials and equipment used in this application are commonly used in the field and are derived from commercially available products. Unless otherwise specified, all methods used in this application are conventional methods in the field.

[0048] There are many other feasible technical solutions in this application, which will not be listed here. All technical solutions claimed in the claims of this application are feasible.

[0049] The terms "comprising" or "including" are intended to indicate that a composition (e.g., a medium) and a method include the listed elements, but do not exclude other elements. When used to define compositions and methods, "consisting substantially of" means excluding other elements that are of any significance to the combination for the stated purpose. Therefore, a composition consisting substantially of the elements defined herein does not exclude other materials or steps that do not materially affect the essential and novel features of the claimed application. "Constitutes" means excluding trace elements and substantial method steps that are other components. Embodiments defined by each of these transitional terms are within the scope of this application.

[0050] The invention will now be further described with reference to the accompanying drawings.

[0051] The structures of compounds S1 to S8 used in the synthesis are as follows.

[0052]

[0053] Example 1: Synthesis of intermediate M5

[0054] Step 1: Synthesize M2 Compound M1 (13.6 g, 60 mmol) and potassium carbonate (20.7 g, 150 mmol) were dissolved in acetonitrile (120 mL), and 1-chloro-3-iodopropane (25.0 g, 122 mmol) was slowly added. The mixture was heated to 82 °C and reacted for 2 h. After the reaction was complete, the solid was removed by filtration and washed with ethyl acetate (100 mL). The filtrate was concentrated and purified by column chromatography to give a white solid M2 (13.3 g, 73% yield). 1 H NMR (400 MHz, methanol-) d 4) δ 7.58 (s, 1H), 7.24 (s, 1H), 4.25 (t, J = 5.9 Hz, 2H), 3.96 (s, 3H), 3.87 (s, 3H), 3.78 (t, J = 6.4 Hz,2H), 2.32 – 2.24 (m, 2H). MS (ESI): 304.20 [M + H] + . Step 2: Synthesize M3 Intermediate M2 (3.5 g, 13.5 mmol), NaI (3.4 g, 23 mmol), and tetrabutylammonium iodide (0.19 g, 0.5 mmol) were dissolved in acetonitrile (40 mL), and the mixture was stirred at 82 °C for 15 min. Then, a solution of tetrahydropyrrole (2.9 mL, 35 mmol) and potassium carbonate (4.1 g, 30 mmol) in acetonitrile (20 mL) was added, and the reaction mixture was stirred at 82 °C for 3 h. After the reaction was complete, the solid was removed by filtration and the mixture was washed with ethyl acetate (40 mL). The filtrate was concentrated and purified by column chromatography to give a yellow oily substance M3 (3.03 g, 78% yield). 1 H NMR (500 MHz, methanol-) d 4) δ 7.63 (s, 1H), 7.31 (s, 1H), 4.30 (t, J = 5.6 Hz, 2H), 4.00 (s, 3H), 3.90 (s, 3H), 3.55 – 3.43 (m, 6H), 2.37 – 2.29 (m, 2H), 2.18 – 2.12 (m, 4H).MS (ESI): 339.32 [M + H] + . Step 3: Synthesize M4 Intermediate M3 (2.7 g, 9.3 mmol), iron powder (2.1 g, 38 mmol), and ammonium acetate (4.4 g, 57 mmol) were dissolved in a mixed solvent of ethyl acetate and water (40 mL: 25 mL). The mixture was heated to 100 °C and stirred overnight. After the reaction was complete, solid impurities were removed by filtration, and the mixture was washed with a 100 mL solution of dichloromethane containing 5% methanol. The organic phases were combined, concentrated, and purified by column chromatography to give a brown oily substance M4 (1.66 g, 58% yield). 1 H NMR (600 MHz, DMSO- d 6) δ 9.40 – 9.25 (m, 1H), 7.15 (s, 1H), 6.54 – 6.41 (m, 1H), 6.37 (s,1H), 4.02 (t, J= 5.8 Hz, 2H), 3.75 (s, 3H), 3.66 (s, 3H), 3.65 – 3.59 (m, 2H), 3.32 – 3.27 (m, 2H), 3.09 – 3.00 (m, 2H), 2.20 – 2.11 (m, 2H), 2.08 – 1.99(m, 2H), 1.90 – 1.82 (m, 2H). MS (ESI): 309.27 [M + H] + . Step 4: Synthesize M5 Intermediate M4 (0.75 g, 2.43 mmol), cyclohexanenitrile (3.0 mL, 24.3 mmol), and a 1,4-dioxane solution of hydrogen chloride (4N, 10 mL, 40 mmol) were added to a sealed tube. After the starting materials were added, the mixture was heated at 100 °C for 12 h. After the reaction was complete, the reaction solution was neutralized with sodium bicarbonate solution, cooled, filtered, and the filter cake was washed with water. The filter cake was dried and then... N,N -Diisopropylethylamine (0.27 mL, 1.68 mmol) was dissolved in phosphorus oxychloride (15 mL) and heated to 108 °C for 6 h. After the reaction was complete, the solvent was removed by vacuum distillation and the solution was slowly poured into a cold saturated sodium bicarbonate solution. The resulting solution was extracted with dichloromethane (20 mL × 3), and the organic phases were combined, dried, concentrated, and purified by column chromatography to give a yellow solid M5 (0.57 g, 59% two-step yield). 1 H NMR (500 MHz, methanol-) d 4) δ 7.39 (s, 1H), 7.30 (s, 1H), 4.27 (t, J = 6.1 Hz, 2H), 4.00 (s, 3H), 2.87 (tt, J = 11.8, 3.5 Hz, 1H), 2.80 (t, 2H), 2.72 – 2.68 (m, 4H), 2.19 – 2.13 (m, 2H), 2.03 – 1.98 (m, 2H), 1.91 – 1.85(m, 6H), 1.75 – 1.66 (m, 2H), 1.51 – 1.42 (m, 2H), 1.39 – 1.30 (m, 2H). MS(ESI): 404.41 [M + H] + . Example 2: Synthesis of Compound 1

[0055] Step 1: Synthesize M8 Compounds M6 (2.0 g, 10 mmol), M7 (1.32 g, 10 mmol), and 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethylurea hexafluorophosphate (HATU, 3.8 g, 10 mmol) soluble in N,N Add 10 ml of dimethylformamide (DMF) to the solution, then add... N,N -Diisopropylethylamine (2.6 ml, 15 mmol) was added and the mixture was stirred at room temperature for 2 h. After the reaction was complete, the mixture was dissolved in water (40 ml) and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, dried, concentrated, and purified by column chromatography to give a white solid M8 (2.57 g, 82% yield). 1 H NMR (600 MHz, methanol-) d 4) δ4.40 – 4.30 (m, 1H), 4.01 – 3.90 (m, 1H), 3.68 (s, 3H), 3.66 – 3.56 (m, 1H), 3.24 – 3.15 (m, 1H), 2.88 – 2.80 (m, 1H), 2.74 – 2.67 (m, 2H), 2.64 – 2.58(m, 2H), 1.99 – 1.92 (m, 1H), 1.89 – 1.82 (m, 1H), 1.51 – 1.39 (m, 10H), 1.37– 1.29 (m, 1H). MS (ESI): 315.09 [M + H] + . Step 2: Synthesize M10 M8 (2.2 g, 7 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (5 mL, 70 mmol) was added. The mixture was stirred at room temperature for 4 h. After the reaction was complete, the solvent was removed by evaporation under reduced pressure, and the remaining liquid was transferred to a saturated sodium bicarbonate solution (20 mL). The solution was extracted with dichloromethane (20 mL × 3), and the organic phases were combined, dried, and concentrated to obtain intermediate M9. Intermediate M5 (95 mg, 0.235 mmol), M9 (0.24 g, 0.72 mmol), and potassium carbonate (330 mg, 2.4 mmol) from Example 1 were dissolved in... N,N In dimethylformamide, the reaction solution was stirred overnight at 60 °C. After the reaction was complete, water (10 mL) was added and the mixture was extracted with ethyl acetate (6 mL × 3). The organic phases were combined, dried, concentrated, and purified by column chromatography to give a white solid M10 (83 mg, yield 61%).1 H NMR (600 MHz, methanol-) d 4) δ 7.85 (s, 1H), 7.20 (s, 1H), 4.77 – 4.68 (m, 1H), 4.69 – 4.61 (m, 1H), 4.36 (t, J = 5.5 Hz, 2H), 4.21 – 4.13(m, 1H), 4.03 (s, 3H), 3.88 – 3.80 (m, 2H), 3.70 (s, 3H), 3.65 (s, 1H), 3.49(t, J = 7.2 Hz, 2H), 3.21 – 3.12 (m, 2H), 2.93 – 2.77 (m, 3H), 2.77 – 2.61 (m,3H), 2.42 – 2.35 (m, 2H), 2.25 – 2.19 (m, 3H), 2.18 – 2.12 (m, 1H), 2.10 –2.01 (m, 4H), 1.99 – 1.92 (m, 2H), 1.87 – 1.75 (m, 4H), 1.74 – 1.66 (m, 1H),1.56 – 1.46 (m, 2H), 1.43 – 1.36 (m, 1H). MS (ESI): 582.67 [M + H] + . Step 3: Synthesize M11 Intermediate M10 (83 mg, 0.143 mmol) was dissolved in a mixture of tetrahydrofuran (2.7 mL) and methanol (1.8 mL), followed by the addition of lithium hydroxide solution (2.5 M, 0.9 mL). The mixture was stirred at room temperature for 24 h. After the reaction was complete, the solution was acidified to pH 2 with dilute hydrochloric acid (1 M), extracted with ethyl acetate (10 mL × 3), and the organic phases were combined, dried, and concentrated to obtain intermediate M11, which could be used directly in the next reaction without further purification. MS (ESI): 566.40 [M - H] - . Step 4: Synthesize compound 1 Intermediate M11 (0.1 mmol), 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethylurea hexafluorophosphate (HATU, 0.12 mmol) N,N -Diisopropylethylamine (0.15 mmol) and compound S1 (0.1 mmol) dissolved in N,N- Dimethylformamide (2 ml) was mixed and stirred at room temperature for 2 h. After the reaction was complete, 10 ml of water was added, followed by extraction with ethyl acetate (6 mL × 3). The organic phases were combined, dried, concentrated, and purified by preparative liquid chromatography (mobile phase: acetonitrile containing 0.1% trifluoroacetic acid and water containing 0.1% trifluoroacetic acid) to give compound 1 (66 mg, yield 69%) as a yellow solid. 1 H NMR (500MHz, methanol-) d 4) δ 7.82 (s, 1H), 7.53 (dd, J = 8.6, 7.1 Hz, 1H), 7.19 (s, 1H), 7.02 (d, J = 8.6 Hz, 1H), 6.99 (d, J = 7.1 Hz, 1H), 5.05 (dd, J = 12.6, 5.5 Hz,1H), 4.75 – 4.61 (m, 2H), 4.35 (t, J = 5.5 Hz, 2H), 4.16 (d, J = 13.8 Hz, 1H), 4.02 (s, 3H), 3.88 – 3.77 (m, 2H), 3.49 (t, J = 7.2 Hz, 2H), 3.33 – 3.28 (m,2H), 3.23 – 3.12 (m, 4H), 2.93 – 2.82 (m, 3H), 2.80 – 2.64 (m, 4H), 2.62 –2.48 (m, 2H), 2.42 – 2.35 (m, 2H), 2.28 – 2.18 (m, 3H), 2.16 – 2.01 (m, 6H), 1.99 – 1.91 (m, 2H), 1.87 – 1.63 (m, 7H), 1.60 – 1.27 (m, 14H). 13 C NMR (126MHz, methanol-) d4) δ 173.30, 173.19, 171.19, 170.31, 169.36, 167.86, 165.47,159.21, 155.39, 150.26, 146.84, 135.80, 135.09, 132.44, 116.55, 110.27,109.50, 105.71, 102.71, 99.63, 66.81, 55.80, 54.03, 52.74, 49.92, 48.77,44.28, 44.15, 42.00, 40.77, 39.04, 31.05, 30.81, 30.56, 30.49, 30.40, 28.96,28.92, 28.88, 28.85, 27.83, 26.45, 26.43, 25.36, 25.25, 25.18, 22.60, 22.40.HPLC>95%; HRMS (ESI-TOF) m / z: [M + H] + Calculated value C 52 H 72 N9O8 + , 950.5498; Measured value, 950.5497. Example 3: Synthesis of compounds 2, 6, 7, and 8

[0056] Step 1: Synthesize compound M13 Compound M12 (5 g, 27.62 mmol) was dissolved in potassium iodide (4.585 g, 27.62 mmol) in... N,N In dimethylformamide (50 mL), stir at 60 °C for 15 min. Then, slowly add M6 (5.0 g, 25 mmol) and triethylamine (10.5 mL, 75 mmol), and stir the mixture overnight at 60 °C. After the reaction is complete, filter to remove the solid insoluble matter, dissolve the filtrate in 150 mL of water, extract with ethyl acetate (100 mL × 3), combine the organic phases, dry, concentrate, and purify by column chromatography to give white solid M13 (7.416 g, yield 98.4%). 1 H NMR (500 MHz, chloroform-) d) δ 4.43(s, 1H), 3.67 (s, 3H), 3.45 (s, 1H), 2.88 – 2.74 (m, 2H), 2.37 – 2.28 (m,4H), 2.09 – 2.00 (m, 2H), 1.96 – 1.87 (m, 2H), 1.85 – 1.76 (m, 2H), 1.44 (s,9H), 1.41 – 1.34 (m, 2H). MS (ESI): 301.32 [M + H] + . Step 2: Synthesize compound M15 M13 (2.1 g, 7 mmol) was dissolved in dichloromethane (10 mL), and trifluoroacetic acid (5 mL, 70 mmol) was added. The mixture was stirred at room temperature for 4 h. After the reaction was complete, the solvent was removed by evaporation under reduced pressure, and the remaining liquid was transferred to a saturated sodium bicarbonate solution (20 mL). The solution was extracted with dichloromethane (20 mL × 3), and the organic phases were combined, dried, and concentrated to obtain intermediate M14. Intermediate M5 (403 mg, 1 mmol), M14 (600 mg, 3 mmol), and potassium carbonate (1375 mg, 10 mmol) prepared in Example 1 were dissolved in... N,N The reaction mixture was stirred overnight at 60 °C in 10 mL of dimethylformamide. After the reaction was complete, 40 mL of water was added, and the mixture was extracted with ethyl acetate (20 mL × 3). The organic phases were combined, dried, concentrated, and purified by column chromatography to give a white solid M15 (476 mg, 84% yield). 1 H NMR (400 MHz, methanol-) d 4) δ 7.88 (s, 1H), 7.22 (s,1H), 4.82 – 4.72 (m, 1H), 4.36 (t, J = 5.5 Hz, 2H), 4.03 (s, 3H), 3.91 – 3.76(m, 4H), 3.73 (s, 3H), 3.49 (t, J = 7.2 Hz, 2H), 3.32 – 3.21 (m, 4H), 3.20 –3.11 (m, 2H), 2.95 – 2.85 (m, 1H), 2.53 (t, J= 6.9 Hz, 2H), 2.47 – 2.34 (m,4H), 2.28 – 2.00 (m, 10H), 1.98 – 1.91 (m, 2H), 1.89 – 1.71 (m, 3H), 1.58 –1.45 (m, 2H), 1.45 – 1.35 (m, 1H). MS (ESI): 568.52 [M + H] + . Step 3: Synthesize M16 Intermediate M15 (264 mg, 0.5 mmol) was dissolved in a mixture of tetrahydrofuran (2.7 mL) and methanol (1.8 mL), followed by the addition of lithium hydroxide solution (2.5 M, 3 mL). The mixture was stirred at room temperature for 24 h. After the reaction was complete, the solution was acidified to pH 2 with dilute hydrochloric acid (1 M), extracted with ethyl acetate (15 mL × 3), and the organic phases were combined, dried, and concentrated to obtain intermediate M16, which could be used directly in the next reaction without purification. MS (ESI): 552.31 [M - H] - . Step 4: Synthesize compound 2 Intermediate M16 (0.1 mmol), 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethylurea hexafluorophosphate (HATU, 0.12 mmol) N,N -Diisopropylethylamine (0.15 mmol) and compound S1 (0.1 mmol) dissolved in N,N - Dimethylformamide (2 ml) was mixed and stirred at room temperature for 2 h. After the reaction was complete, 10 ml of water was added, followed by extraction with ethyl acetate (6 mL × 3). The organic phases were combined, dried, concentrated, and purified by preparative liquid chromatography (mobile phase: acetonitrile containing 0.1% trifluoroacetic acid and water containing 0.1% trifluoroacetic acid) to give compound 2 (61 mg, yield 65%) as a yellow solid. 1 H NMR (500MHz, methanol-) d 4) δ 7.82 (s, 1H), 7.56 – 7.48 (m, 1H), 7.18 (s, 1H), 7.00 (dd, J =7.8, 3.7 Hz, 2H), 5.03 (dd, J = 12.6, 5.5 Hz, 1H), 4.79 – 4.69 (m, 1H), 4.33(t, J= 5.4 Hz, 2H), 4.00 (s, 3H), 3.86 – 3.69 (m, 4H), 3.47 (t, J = 7.2 Hz, 2H),3.30 – 3.26 (m, 2H), 3.27 – 3.09 (m, 8H), 2.92 – 2.79 (m, 2H), 2.77 – 2.62(m, 2H), 2.49 – 2.32 (m, 6H), 2.24 – 2.16 (m, 2H), 2.15 – 1.99 (m, 9H), 1.97 – 1.89 (m, 2H), 1.86 – 1.71 (m, 3H), 1.69 – 1.59 (m, 2H), 1.53 – 1.28 (m,14H). 13 C NMR (126 MHz, methanol-) d 4) δ 174.88, 174.58, 171.72, 170.78, 169.25,166.97, 160.93, 156.97, 151.77, 148.26, 137.22, 136.62, 133.86, 117.95,111.70, 110.92, 107.16, 104.08, 100.99, 68.23, 58.35, 57.21, 55.43, 54.12,52.81, 50.16, 45.58, 43.37, 40.61, 34.22, 32.19, 31.93, 30.30, 30.22, 29.91,27.85, 26.76, 26.59, 26.56, 23.98, 23.79, 21.16. HPLC>95%; HRMS (ESI-TOF) m / z: [M + H] + Calculated value C 52 H 74 N9O7 + , 936.5706; Measured value, 936.5709. Step 5: Synthesize compound 6 Intermediate M16 (0.1 mmol), 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethylurea hexafluorophosphate (HATU, 0.12 mmol) N,N -Diisopropylethylamine (0.15 mmol) and compound S2 (0.1 mmol) dissolved in N,N- Dimethylformamide (2 ml) was mixed and stirred at room temperature for 2 h. After the reaction was complete, 10 ml of water was added, followed by extraction with ethyl acetate (6 mL × 3). The organic phases were combined, dried, concentrated, and purified by preparative liquid chromatography (mobile phase: acetonitrile containing 0.1% trifluoroacetic acid and water containing 0.1% trifluoroacetic acid) to give a yellow solid compound 6 (56 mg, yield 61%). 1 H NMR (500MHz, methanol-) d 4) δ 7.85 (s, 1H), 7.57 – 7.48 (m, 1H), 7.22 (s, 1H), 7.06 – 6.98 (m, 2H), 5.06 (dd, J = 12.7, 5.4 Hz, 1H), 4.80 – 4.72 (m, 1H), 4.40 – 4.32 (m,2H), 4.02 (s, 3H), 3.89 – 3.72 (m, 4H), 3.49 (t, J = 7.2 Hz, 2H), 3.33 – 3.29(m, 2H), 3.30 – 3.11 (m, 8H), 2.97 – 2.81 (m, 2H), 2.80 – 2.65 (m, 2H), 2.55– 2.34 (m, 6H), 2.26 – 2.01 (m, 11H), 1.99 – 1.91 (m, 2H), 1.88 – 1.74 (m,3H), 1.72 – 1.64 (m, 2H), 1.61 – 1.23 (m, 12H). 13 C NMR (126 MHz, methanol-) d4) δ173.46, 173.18, 170.32, 169.37, 167.84, 165.55, 159.52, 155.56, 150.36,146.84, 135.81, 135.23, 132.45, 116.56, 110.29, 109.51, 105.76, 102.70,99.63, 66.83, 56.91, 55.82, 54.02, 52.71, 51.42, 48.77, 44.16, 41.97, 39.13,32.81, 30.80, 30.53, 28.83, 28.79, 28.63, 28.49, 26.48, 26.45, 25.36, 25.20,25.16, 22.59, 22.39, 19.79. HPLC>95%; HRMS (ESI-TOF) m / z: [M + H] + Calculated value C 51 H 72 N9O7 + , 922.5549; Measured value, 922.5548. Step 6: Synthesize compound 7 Intermediate M16 (0.1 mmol), 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethylurea hexafluorophosphate (HATU, 0.12 mmol) N,N -Diisopropylethylamine (0.15 mmol) and compound S3 (0.1 mmol) dissolved in N,N Dimethylformamide (2 ml) was mixed and stirred at room temperature for 2 h. After the reaction was complete, 10 ml of water was added, followed by extraction with ethyl acetate (6 mL × 3). The organic phases were combined, dried, concentrated, and purified by preparative liquid chromatography (mobile phase: acetonitrile containing 0.1% trifluoroacetic acid and water containing 0.1% trifluoroacetic acid) to give a yellow solid compound 7 (65 mg, yield 68%). 1 H NMR (500MHz, methanol-) d 4) δ 7.85 (s, 1H), 7.54 (dd, J = 8.6, 7.0 Hz, 1H), 7.22 (s, 1H), 7.06 – 6.98 (m, 2H), 5.06 (dd, J = 12.6, 5.5 Hz, 1H), 4.82 – 4.72 (m, 1H), 4.36(t, J= 5.5 Hz, 2H), 4.02 (s, 3H), 3.90 – 3.73 (m, 4H), 3.49 (t, J = 7.2 Hz, 2H),3.33 – 3.10 (m, 10H), 2.95 – 2.82 (m, 2H), 2.80 – 2.66 (m, 2H), 2.52 – 2.34(m, 6H), 2.28 – 2.01 (m, 11H), 1.99 – 1.92 (m, 2H), 1.87 – 1.73 (m, 3H), 1.71 – 1.62 (m, 2H), 1.57 – 1.30 (m, 16H). 13 C NMR (126 MHz, methanol-) d 4) δ 173.43,173.19, 170.33, 169.37, 167.86, 165.56, 159.53, 155.57, 150.37, 146.85,135.81, 135.23, 132.46, 116.55, 110.28, 109.51, 105.77, 102.69, 99.62, 66.83,56.92, 55.80, 54.02, 52.72, 51.42, 48.76, 44.17, 41.97, 39.22, 32.79, 30.80,30.53, 29.13, 28.92, 28.86, 28.83, 28.51, 26.53, 26.49, 25.36, 25.20, 25.16,22.59, 22.40, 19.77. HPLC>95%; HRMS (ESI-TOF) m / z: [M + H] + Calculated value C 53 H 76 N9O7 + ,950.5862; Measured value, 950.5866. Step 7: Synthesize compound 8 Intermediate M16 (0.1 mmol), 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethylurea hexafluorophosphate (HATU, 0.12 mmol) N,N -Diisopropylethylamine (0.15 mmol) and compound S4 (0.1 mmol) dissolved in N,N- Dimethylformamide (2 ml) was mixed and stirred at room temperature for 2 h. After the reaction was complete, 10 ml of water was added, followed by extraction with ethyl acetate (6 mL × 3). The organic phases were combined, dried, concentrated, and purified by preparative liquid chromatography (mobile phase: acetonitrile containing 0.1% trifluoroacetic acid and water containing 0.1% trifluoroacetic acid) to give a yellow solid compound 8 (66 mg, yield 68%). 1 H NMR (500MHz, methanol-) d 4) δ 7.85 (s, 1H), 7.53 (dd, J = 8.5, 7.1 Hz, 1H), 7.22 (s, 1H), 7.01 (dd, J = 8.6, 7.1 Hz, 2H), 5.06 (dd, J = 12.6, 5.5 Hz, 1H), 4.83 – 4.72 (m,1H), 4.35 (t, J = 5.5 Hz, 2H), 4.03 (s, 3H), 3.89 – 3.73 (m, 4H), 3.49 (t, J =7.2 Hz, 2H), 3.33 – 3.27 (m, 2H), 3.28 – 3.10 (m, 8H), 2.95 – 2.81 (m, 2H), 2.80 – 2.67 (m, 2H), 2.51 – 2.34 (m, 6H), 2.26 – 2.18 (m, 2H), 2.17 – 2.01(m, 9H), 1.98 – 1.90 (m, 2H), 1.86 – 1.72 (m, 3H), 1.71 – 1.62 (m, 2H), 1.57 – 1.48 (m, 4H), 1.48 – 1.31 (m, 14H). 13 C NMR (126 MHz, methanol-) d4) δ 173.38,173.19, 170.33, 169.36, 167.87, 165.56, 159.52, 155.56, 150.37, 146.85,135.81, 135.23, 132.45, 116.55, 110.27, 109.50, 105.77, 102.70, 99.63, 66.83,56.88, 55.81, 54.01, 52.71, 51.42, 48.77, 44.16, 42.00, 39.25, 32.75, 30.81,30.53, 29.15, 29.13, 28.98, 28.94, 28.88, 28.86, 28.49, 26.59, 26.52, 25.37,25.20, 25.16, 22.59, 22.40, 19.79. HPLC>95%; HRMS (ESI-TOF) m / z: [M + H] + Calculated value C 54 H 78 N9O7 + , 964.6019; Measured value, 964.6017. Example 4: Synthesis of compounds 3, 4, 5, and 9

[0057] Step 1: Synthesize compound 3 Intermediate M16 (0.1 mmol), 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethylurea hexafluorophosphate (HATU, 0.12 mmol) N,N -Diisopropylethylamine (0.15 mmol) and compound S5 (0.1 mmol) dissolved in N,N Dimethylformamide (2 ml) was mixed and stirred at room temperature for 2 h. After the reaction was complete, 10 ml of water was added, followed by extraction with ethyl acetate (6 mL × 3). The organic phases were combined, dried, concentrated, and purified by preparative liquid chromatography (mobile phase: acetonitrile containing 0.1% trifluoroacetic acid and water containing 0.1% trifluoroacetic acid) to give a yellow solid compound 3 (61 mg, yield 65%). 1 H NMR (500MHz, methanol-) d 4) δ 7.83 (s, 1H), 7.76 – 7.70 (m, 1H), 7.43 – 7.36 (m, 2H), 7.20 (s, 1H), 5.08 (dd, J= 12.6, 5.5 Hz, 1H), 4.78 – 4.68 (m, 1H), 4.34 (t, J = 5.5Hz, 2H), 4.19 (t, J = 6.3 Hz, 2H), 4.00 (s, 3H), 3.87 – 3.79 (m, 2H), 3.79 –3.70 (m, 2H), 3.47 (t, J = 7.2 Hz, 2H), 3.29 – 3.10 (m, 8H), 2.93 – 2.81 (m,2H), 2.78 – 2.66 (m, 2H), 2.48 – 2.33 (m, 6H), 2.25 – 2.00 (m, 11H), 1.96 –1.88 (m, 2H), 1.88 – 1.70 (m, 5H), 1.59 – 1.30 (m, 14H). 13 C NMR (126 MHz, methanol-) d 4) δ 172.91, 172.66, 169.67, 166.70, 165.41, 165.06, 159.02, 156.10,155.06, 149.87, 136.04, 134.73, 133.16, 118.63, 116.17, 114.38, 105.26,102.19, 99.13, 68.63, 66.33, 56.39, 55.31, 53.51, 52.22, 50.92, 48.49, 43.66,38.74, 32.28, 30.28, 30.02, 28.33, 28.05, 27.99, 25.93, 24.97, 24.86, 24.70,24.65, 22.09, 21.77, 19.29. HPLC>95%; HRMS (ESI-TOF) m / z: [M + H] + Calculated value C 52 H 73 N8O8 + , 937.5546; Measured value, 937.5548. Step 2: Synthesize compound 4 Intermediate M16 (0.1 mmol), 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethylurea hexafluorophosphate (HATU, 0.12 mmol) N,N-Diisopropylethylamine (0.15 mmol) and compound S6 (0.1 mmol) dissolved in N,N - Dimethylformamide (2 ml) was mixed and stirred at room temperature for 2 h. After the reaction was complete, 10 ml of water was added, followed by extraction with ethyl acetate (6 mL × 3). The organic phases were combined, dried, concentrated, and purified by preparative liquid chromatography (mobile phase: acetonitrile containing 0.1% trifluoroacetic acid and water containing 0.1% trifluoroacetic acid) to give a yellow solid compound 4 (67 mg, yield 71%). 1 H NMR (500MHz, methanol-) d 4) δ 7.84 (s, 1H), 7.55 (dd, J = 8.6, 7.1 Hz, 1H), 7.22 (s, 1H), 7.09 (d, J = 8.6 Hz, 1H), 7.04 (d, J = 7.1 Hz, 1H), 5.07 (dd, J = 12.7, 5.5 Hz,1H), 4.82 – 4.71 (m, 1H), 4.36 (t, J = 5.5 Hz, 2H), 4.02 (s, 3H), 3.88 – 3.80(m, 2H), 3.76 (t, J = 5.2 Hz, 4H), 3.73 – 3.64 (m, 4H), 3.65 – 3.57 (m, 2H), 3.56 (s, 1H), 3.55 – 3.46 (m, 4H), 3.46 – 3.39 (m, 2H), 3.30 – 3.12 (m, 6H),2.95 – 2.82 (m, 2H), 2.81 – 2.63 (m, 2H), 2.54 – 2.42 (m, 3H), 2.43 – 2.35(m, 3H), 2.28 – 2.17 (m, 2H), 2.18 – 2.00 (m, 9H), 1.99 – 1.90 (m, 2H), 1.87– 1.72 (m, 3H), 1.56 – 1.45 (m, 2H), 1.45 – 1.28 (m, 2H). 13 C NMR (126 MHz, methanol-) d4) δ 173.62, 173.19, 170.35, 169.33, 167.77, 165.55, 159.51, 155.57,150.37, 146.75, 135.86, 135.23, 132.43, 116.85, 110.66, 109.88, 105.76,102.69, 99.61, 70.21, 69.86, 69.11, 69.06, 66.84, 56.82, 55.80, 54.02, 52.72,51.43, 48.80, 44.17, 41.80, 39.23, 32.67, 30.80, 30.53, 28.47, 25.36, 25.21,25.16, 22.59, 22.41, 19.70. HPLC>95%; HRMS (ESI-TOF) m / z: [M + H] + Calculated value C 50 H 70 N9O9 + , 940.5291; Measured value, 940.5295. Step 3: Synthesize compound 5 Intermediate M16 (0.1 mmol), 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethylurea hexafluorophosphate (HATU, 0.12 mmol) N,N -Diisopropylethylamine (0.15 mmol) and compound S7 (0.1 mmol) dissolved in N,N - Dimethylformamide (2 ml) was mixed and stirred at room temperature for 2 h. After the reaction was complete, 10 ml of water was added, followed by extraction with ethyl acetate (6 mL × 3). The organic phases were combined, dried, concentrated, and purified by preparative liquid chromatography (mobile phase: acetonitrile containing 0.1% trifluoroacetic acid and water containing 0.1% trifluoroacetic acid) to give a yellow solid compound 5 (63 mg, 67% yield). 1 H NMR (500MHz, methanol-) d 4) δ 7.83 (s, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.20 (s, 1H), 6.92 (d, J = 2.1 Hz, 1H), 6.79 (dd, J = 8.4, 2.2 Hz, 1H), 5.02 (dd, J= 12.8, 5.5 Hz, 1H),4.80 – 4.69 (m, 1H), 4.33 (t, J = 5.5 Hz, 2H), 4.00 (s, 3H), 3.86 – 3.71 (m,4H), 3.47 (t, J = 7.2 Hz, 2H), 3.29 – 3.10 (m, 10H), 2.94 – 2.78 (m, 2H), 2.77– 2.63 (m, 2H), 2.52 – 2.32 (m, 6H), 2.26 – 1.99 (m, 11H), 1.98 – 1.88 (m,2H), 1.84 – 1.70 (m, 3H), 1.69 – 1.59 (m, 2H), 1.56 – 1.30 (m, 14H). 13 C NMR (126 MHz, methanol-) d 4) δ 172.93, 172.71, 169.94, 167.72, 167.33, 165.06, 159.03,155.05, 154.28, 149.86, 134.72, 134.01, 124.26, 115.92, 114.61, 105.26,104.66, 102.20, 99.13, 66.34, 56.40, 55.31, 53.52, 52.23, 50.91, 48.38,43.66, 42.23, 38.70, 32.29, 30.30, 30.03, 28.44, 28.35, 28.00, 27.90, 26.13,25.97, 24.86, 24.70, 24.66, 22.09, 21.95, 19.29. HPLC>95%; HRMS (ESI-TOF) m / z: [M + H] + Calculated value C 52 H 74 N9O7 + , 936.5706; Measured value, 936.5711. Step 4: Synthesize compound 9 Intermediate M16 (0.1 mmol), 2-(7-azobenzotriazole)- N,N,N',N' -Tetramethylurea hexafluorophosphate (HATU, 0.12 mmol) N,N -Diisopropylethylamine (0.15 mmol) and compound S8 (0.1 mmol) dissolved inN,N - Dimethylformamide (2 ml) was mixed and stirred at room temperature for 2 h. After the reaction was complete, 10 ml of water was added, followed by extraction with ethyl acetate (6 mL × 3). The organic phases were combined, dried, concentrated, and purified by preparative liquid chromatography (mobile phase: acetonitrile containing 0.1% trifluoroacetic acid and water containing 0.1% trifluoroacetic acid) to give compound 9 as a yellow solid (60 mg, 65% yield). 1 H NMR (600MHz, methanol-) d 4) δ 7.83 (s, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.19 (s, 1H), 7.02 (d, J = 7.4 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 5.13 (dd, J = 13.3, 5.2 Hz, 1H), 4.76 –4.70 (m, 1H), 4.33 (t, J = 5.5 Hz, 2H), 4.31 – 4.21 (m, 2H), 4.00 (s, 3H), 3.84 – 3.79 (m, 2H), 3.76 – 3.71 (m, 2H), 3.47 (t, J = 7.2 Hz, 2H), 3.25 – 3.12 (m,9H), 2.94 – 2.85 (m, 2H), 2.81 – 2.76 (m, 1H), 2.47 – 2.43 (m, 2H), 2.43 –2.35 (m, 4H), 2.22 – 2.17 (m, 2H), 2.13 – 2.01 (m, 8H), 1.95 – 1.90 (m, 2H), 1.83 – 1.73 (m, 3H), 1.68 – 1.63 (m, 2H), 1.53 – 1.41 (m, 6H), 1.39 – 1.28(m, 10H). 13 C NMR (126 MHz, methanol-) d4) δ 174.64, 172.37, 166.97, 160.95, 156.98,151.78, 145.15, 136.65, 130.56, 128.00, 113.78, 111.79, 107.19, 104.10,101.01, 68.23, 58.32, 57.20, 55.43, 54.13, 53.58, 52.81, 47.37, 45.58, 44.41,40.61, 34.19, 32.38, 31.93, 30.42, 30.28, 30.24, 30.12, 29.90, 28.10, 27.88,26.76, 26.60, 26.56, 24.24, 23.98, 21.18. HPLC>95%; HRMS (ESI-TOF) m / z: [M +H] + Calculated value C 52 H 76 N9O6 + , 922.5913; Measured value, 922.5911. Test Example 1: IC 50 Test methods Based on different cell growth rates, cells in the logarithmic growth phase were seeded into 96-well plates at specific quantities, with a cell suspension volume of 100 μL. After overnight culture, different concentrations of the drug were added for 72 h of treatment, with each concentration in triplicate. Corresponding solvent controls and cell-free zeroing wells were also included. After treatment, adherent cells were decanted from the culture medium, and 10% (w / v) trichloroacetic acid (100 μL / well) was added for fixation at 4°C for 1 h. The cells were then washed five times with distilled water and dried at room temperature. 100 μL of sulfonylrhodamine B solution (4 mg / mL, dissolved in 1% glacial acetic acid) was added to each well, and the cells were incubated and stained at room temperature for 15 min. Afterward, the cells were washed five times with 1% glacial acetic acid to remove unbound sulfonylrhodamine B. After drying at room temperature, 150 μL of 10 mM Tris solution was added to each well, and the optical density (OD value) at 560 nm was measured using a SpectraMax 190 microplate reader. The inhibition rate of the compound on cell proliferation was calculated using the following formula: Inhibition rate (%) = [1 - (OD administration wells - OD negative control wells) / (OD positive control wells - OD negative control wells)] × 100%. IC50 50 The value was estimated using the four-parameter method.

[0058] The inhibitory activity of some compounds against pancreatic cancer PANC-1 cells was measured using the above method, and the results are shown in Table 1. These results indicate that the compounds of this application possess good inhibitory activity against PANC-1 cell proliferation.

[0059] Table 1

[0060] Note: +++<1 μM; 1 μM ≤ ++ <30 μM; 30 μM ≤ + <500 μM.

[0061] The inhibitory activity of compound 2 (G9D-4) against various pancreatic cancer cells was also compared. The cell lines used included KP-4, PANC-1, ASPC-1, HPAF-II, Panc10.05, SW1990, Panc08.13, Panc04.03, Panc05.04, and Panc02.03. The results are shown below. Figure 1 The above results indicate that the compounds of this application possess broad-spectrum inhibitory activity against pancreatic tumor cell proliferation.

[0062] Test Example 2: Degradation Ratio Test Method PANC-1 cells were seeded at 500,000 cells / well in six-well plates and cultured overnight. Then, 0.1 μM or 1 μM of the compounds described in this application (compounds 1–9) were added for 24 h. DMSO wells and 1 μM of the G9a inhibitor UNC0638 were included as controls. The plate volume was 2 mL. After cell collection, the expression level of the target protein was detected by Western blotting. Results are shown below. Figure 2 and Figure 3 .

[0063] The above results indicate that after 24 h of treatment with the compound of this application, PANC-1 cells showed a significant downregulation effect on G9a protein expression compared with the DMSO control group, indicating that the compound of this application can degrade G9a protein in PANC-1 cells.

[0064] The contents not described in detail in this application specification are common knowledge to those skilled in the art.

[0065] As used throughout the specification and claims, the term "comprising" is an open-ended term and should therefore be interpreted as "comprising but not limited to". "Approximately" means that within an acceptable margin of error, those skilled in the art can solve the technical problem and substantially achieve the technical effect within a certain margin of error.

[0066] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a product or system comprising a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a product or system. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the product or system that includes said element.

[0067] The foregoing description illustrates and describes several preferred embodiments of this application. However, as previously stated, it should be understood that this application is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the inventive concept described herein through the foregoing teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of this application should be protected within the scope of the appended claims.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, enantiomer, diastereomer, transisomer, racemate, or isotopically labeled compound thereof: (I)； in: R 1 Selected from , , , , , , , , , , , , , or ; X 1 It can be -N- or -CH-; Y is either -CH2- or -C(=O)-; L is selected from -R 2 -C(=O)NH-R 3 -、C 4-18 Alkylene and C 4-18 Alkyloxy; R 2 Selected from C 1-6 Alkylene; R 3 Selected from C 3-14 Alkylene and -CH2-(-CH2-O-CH2-) m -CH2-, m is an integer selected from 1 to 4; X 2 It can be -NH-, -O-, or -CH2-; A is selected from the following A-1, A-2, and A-3: 。 2. The compound according to claim 1, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, enantiomer, diastereomer, transisomer, racemic mixture, or isotopically labeled compound thereof, wherein, The compound is the compound shown in formula (II): (II); Among them, Y, L, X 2 The definition of A is the same as in claim 1.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, enantiomer, diastereomer, transisomer, racemic mixture, or isotopically labeled compound thereof, wherein, The compound is the compound shown in formula (III): (III); L is -R 2 -C(=O)NH-R 3 -; Among them, R 2 Selected from C 1-6 Alkylene; R 3 Selected from C 3-14 Alkylene and -CH2-(-CH2-O-CH2-) m -CH2-, m is an integer selected from 1 to 4; The definition of A is the same as in claim 1.

4. The compound according to claim 3, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, enantiomer, diastereomer, transisomer, racemic mixture, or isotopically labeled compound thereof, wherein, The compound is the compound shown in formula (IV): (IV); The L is the same as that defined in claim 3.

5. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, enantiomer, diastereomer, transisomer, racemic mixture, or isotopically labeled compound thereof, wherein, The compound is selected from: 。 6. The use of the compound of any one of claims 1-5 or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, enantiomer, diastereomer, transisomer, racemic mixture, or isotopically labeled compound in the preparation of a medicament for the prevention or treatment of diseases associated with abnormal expression of G9a protein activity.

7. The application according to claim 6, characterized in that, The diseases associated with abnormal expression of G9a protein activity include: tumors, hyperglycemia, diabetes, obesity, hyperlipidemia, hypercholesterolemia, hyperlipoproteinemia, hypertriglyceridemia, hypertension, hyperinsulinemia, hyperuricemia, Parkinson's disease, and Alzheimer's disease.

8. The application according to claim 7, characterized in that, The tumor is selected from one or more of the following: non-small cell lung cancer, malignant melanoma, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, colon cancer, rectal cancer, breast cancer, cervical cancer, lung cancer, laryngeal cancer, nasopharyngeal cancer, pancreatic cancer, multiple myeloma, B lymphoma, and leukemia.

9. A pharmaceutical composition comprising one or more of the following: a compound selected from any one of claims 1-5, a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, enantiomer, diastereomer, transisomer, racemic mixture, isotopically labeled compound, and a pharmaceutically acceptable excipient.

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