Pyridone CDK2 degradation agent as well as preparation method and application thereof

Abstract

The invention discloses a pyridone CDK2 degradation agent and a preparation method and application thereof, the compound has a structure as shown in a formula (P), and a series of compounds designed and synthesized by the invention have wide pharmacological activity, have a function of degrading CDK2 protein, and can be used for preventing or / and treating various cancers.

Description

Technical Field

[0001] This invention relates to compounds, their preparation methods, and applications; specifically, it relates to a compound that targets the degradation of CDK2 protein, its preparation method, and its applications. Background Technology

[0002] Cyclin-dependent kinases (CDKs) play a crucial role in regulating cell cycle progression and have significant regulatory effects on cell proliferation. Abnormal upregulation of CDK activity accelerates cell cycle progression, thereby promoting abnormal cell proliferation and driving cancer development and progression. CDK2, in particular, is primarily responsible for mediating the cell cycle transition from G1 to S phase and participates in regulating DNA synthesis during the S phase. Therefore, inhibiting CDK2 can significantly arrest cell cycle progression, effectively suppress cancer cell proliferation, and demonstrate promising anti-tumor potential.

[0003] However, most current CDK2 inhibitors suffer from low selectivity, significant toxic side effects, and potential off-target risks. In contrast, technologies such as protein degradation-targeting chimeras (PROTACs) specifically degrade CDK2, enabling more efficient and selective inhibition of its function. CDK2 degraders hold promise for overcoming the limitations of traditional inhibitors, significantly reducing toxic side effects, and improving treatment safety. Summary of the Invention

[0004] Objective of the invention: This invention provides a highly selective and safe compound for targeting the degradation of CDK2 protein, or a pharmaceutically acceptable salt thereof. This invention also provides a method for preparing this compound and its applications.

[0005] Technical solution: This invention provides a compound or a pharmaceutically acceptable salt thereof that targets the degradation of CDK2 protein, the structure of which is shown in general formula (P):

[0006]

[0007] (P)

[0008] In the formula, the linker is selected from: , , , Wherein, L1 is absent or selected from C1-C3 alkylene groups, L2 is selected from -NH- and C1-C3 alkylene groups, and k, n, m, p, and q are each independently selected from 0, 1, and 2.

[0009] Preferably, the linker is selected from: , , , Wherein, L1 is absent or selected from C1-C3 alkylene groups, and n is selected from: 0, 1, 2.

[0010] Preferably, the linker is selected from the following structures:

[0011]

[0012] Most preferably, the compound represented by formula (P) or a pharmaceutically acceptable salt thereof is selected from any of the following structures:

[0013]

[0014] The pharmaceutically acceptable salts mentioned above are acid addition salts of compounds of general formula (P), wherein the acid used for salt formation is selected from inorganic acids and organic acids, wherein the inorganic acid is selected from hydrochloric acid, sulfuric acid, and phosphoric acid, and the organic acid is selected from acetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, methanesulfonic acid, malonic acid, trichloroacetic acid, trifluoroacetic acid, fumaric acid, citric acid, digluconic acid, camphoric acid, cinnamic acid, aspartic acid, and tartaric acid.

[0015] The present invention discloses a method for preparing a compound targeting CDK2 protein degradation or a pharmaceutically acceptable salt thereof, comprising the following steps:

[0016] Compound A and compound B were dissolved in N,N-dimethylformamide, and N,N-diisopropylethylamine was added. The mixture was heated and stirred until the reaction was complete, yielding compound P.

[0017]

[0018] R1 is selected from , , , Wherein, L1 is absent or selected from C1-C3 alkylene groups, and n is selected from: 0, 1, 2.

[0019] Specifically, compound A and compound B were dissolved in N,N-dimethylformamide, N,N-diisopropylethylamine was added, the mixture was heated and stirred, and after the reaction was completed, compound P, which degrades CDK2 protein, was obtained by purification.

[0020] The present invention provides a pharmaceutical composition comprising a compound of general formula (P) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0021] Furthermore, the dosage form of the pharmaceutical composition is selected from at least one of tablets, capsules, granules, injections, powder for injection, eye drops, powders, pills, transdermal patches, ointments, aerosols, emulsions, liniments, suppositories, films, controlled-release formulations, and nanoformulations.

[0022] Pharmaceutically acceptable carriers refer to excipients or diluents that do not cause significant irritation to the organism and do not interfere with the biological activity and properties of the compound being administered. Excipients include flavoring agents, antioxidants, aromatizers, solubilizers, emulsifiers, preservatives, binders, osmotic pressure regulators, disintegrants, colorants, fillers, lubricants, etc., while diluents include dextrin, starch, sucrose, physiological saline, lactose, etc.

[0023] The compounds described in this invention or their pharmaceutically acceptable salts can be used in the preparation of CDK2 degrading agents.

[0024] The compounds described in this invention, or pharmaceutically acceptable salts thereof, may be used in the preparation of medicaments for the prevention and / or treatment of tumor-related diseases.

[0025] Preferably, the tumor-related diseases are selected from: leukemia, ovarian cancer, lung cancer, gastric cancer, prostate cancer, testicular cancer, colon cancer, breast cancer, multiple myeloma, liver cancer, pancreatic cancer, melanoma, glioma, brain glioma, pituitary adenoma, and various solid tumors and hematologic malignancies.

[0026] The compound of general formula (P) or its pharmaceutically acceptable salt described in this invention has degradative activity against CDK2 protein and has significant therapeutic effects on related malignant tumors.

[0027] Beneficial effects: Compared with the prior art, the compound represented by the general formula (P) of this invention has degradation activity on CDK2 protein and can be used to prepare drugs for treating cancer or tumor-related diseases. Detailed Implementation

[0028] The present invention will be further described below. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were all purchased from conventional biochemical reagent stores. The present application will now be described in detail with reference to specific embodiments.

[0029] I. Synthesis of intermediate reactants

[0030] The specific preparation method of the independently developed intermediate reactant (M) is as follows:

[0031] (1) Synthesis of 5-bromo-1-(sec-butyl)-3-fluoropyridine-2(1H)-one (M1):

[0032]

[0033] 2-Hydroxy-3-fluoro-5-bromopyridine (3.84 g, 20 mmol) and sodium carbonate (6.36 g, 60 mmol) were dissolved in N,N-dimethylformamide (150 mL), and then 11.04 g, 60 mmol of iodosec-butane (11.04 g, 60 mmol) were added. The mixture was heated to 70 °C and reacted for 12 h. The mixture was then purified by rapid silica gel column chromatography to give compound M1 (2.58 g, yield 52%). 1 H NMR (400 MHz, Chloroform-d)δ 7.20 (dd, J = 2.5, 1.6 Hz, 1H), 7.16 (dd, J = 8.3, 2.5 Hz, 1H), 5.13 – 5.04(m, 1H), 1.76 – 1.66 (m, 2H), 1.36 (d, J = 6.8 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H).

[0034] (2) Synthesis of 1-(sec-butyl)-5-(2-chloro-5-fluoropyrimidin-4-yl)-3-fluoropyridin-2(1H)-one (M2):

[0035]

[0036] Compound M1 (2.48 g, 10.0 mmol) and pinacol diborate (2.79 g, 11 mmol) were dissolved in 1,4-dioxane (80 mL), followed by the addition of Pd(dppf)Cl2 (365.5 mg, 0.5 mmol) and potassium acetate (2.94 g, 30 mmol). The mixture was purged with argon three times and heated to 100 °C for 12 h. After the reaction was complete, the mixture was quenched with water, concentrated under reduced pressure using ethyl acetate, and used directly in subsequent reactions without further purification. 2,4-Dichloro-5-fluoropyrimidine (2.00 g, 12 mmol), Pd(PPh3)2Cl2 (351 mg, 0.5 mmol), sodium carbonate (2.65 g, 25 mmol), water (15 mL), and ethylene glycol dimethyl ether (20 mL) were added to a three-necked flask and purged with argon three times. Compound M1-2 dissolved in ethylene glycol dimethyl ether (80 mL) was slowly added to a three-necked flask, heated to 80 °C, and reacted for 12 h. The mixture was then rapidly purified by silica gel column chromatography to obtain compound M2 (1.62 g, yield 54%). 1H NMR (400 MHz, CDCl3) δ 8.49 (d, J = 3.5 Hz, 1H), 8.24 – 8.23 ​​(m,1H), 7.98 (dd, J = 10.4, 2.4 Hz, 1H), 5.19 – 5.10 (m, 1H), 1.84 – 1.79 (m,2H), 1.46 (d, J = 6.8 Hz, 3H), 0.93 (t, J = 7.4 Hz, 3H).

[0037] (3) Synthesis of 1-(sec-butyl)-3-fluoro-5-(5-fluoro-2-(piperidin-4-ylamino)pyrimidin-4-yl)pyridin-2-(1H)-one (M3):

[0038]

[0039] Compound M2 (299 mg, 1 mmol) and 1-Boc-4-aminopiperidine (250 mg, 1.25 mmol) were dissolved in 1,4-dioxane (6 mL), and then Pd2(dba)3 (46 mg, 0.05 mmol), Xantphos (58 mg, 0.1 mmol), and cesium carbonate (650 mg, 2 mmol) were added. Argon gas was purged three times, and the mixture was heated to 105 °C and reacted for 12 h. After cooling, the mixture was filtered and concentrated, and column chromatography was used to obtain compound M3-1 (185 mg, yield 40%) as a white solid. 1 H NMR (300 MHz, DMSO-d6) δ 8.36 (d,J = 3.8 Hz, 1H), 8.13 (t, J = 1.5 Hz, 1H), 7.88 – 7.84 (m, 1H), 7.26 (d, J =7.7 Hz, 1H), 4.97 – 4.88 (m, 1H), 3.95 – 3.88 (m, 3H), 2.86 (s, 2H), 1.88 –1.82 (m, 2H), 1.77 – 1.68 (m, 2H), 1.39 (s, 9H), 1.36 – 1.33 (m, 5H), 0.78(t, J = 7.4 Hz, 3H).

[0040] Compound M3-1 (185 mg, 0.4 mmol) was dissolved in dichloromethane (5 mL), and 2N hydrogen chloride-ethyl acetate solution (5 mL) was added. The reaction was carried out at room temperature for 4 h. The pH of the reaction solution was adjusted to neutral, and column chromatography was used to obtain compound M3 (130 mg, yield 90%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 3.8 Hz, 1H), 8.15 (d,J = 2.3 Hz, 1H), 7.93 (d, J = 11.2 Hz, 1H), 7.45 (d, J = 7.4 Hz, 1H), 4.98 –4.92 (m, 1H), 3.96 – 3.85 (m, 1H), 3.23 – 3.18 (m, 2H), 2.97 – 2.86 (m, 2H), 2.02 – 1.98 (m, 2H), 1.82 – 1.71 (m, 2H), 1.69 – 1.58 (m, 2H), 1.36 (d, J =6.8 Hz, 3H), 0.80 (t, J = 7.3 Hz, 3H).

[0041] (4) Synthesis of 5-(2-((1-((4-(bromomethyl)phenyl)sulfonyl)piperidin-4-yl)amino)-5-fluoropyrimidin-4-yl)-1-(sec-butyl)-3-fluoropyridine-2(1H)-one (M4):

[0042]

[0043] Compound M3 (290 mg, 0.8 mmol) and triethylamine (243 mg, 2.4 mmol) were dissolved in dichloromethane (5 mL), and the mixture was kept in an ice bath. 4-bromomethylbenzenesulfonyl chloride (312 mg, 1.2 mmol) was added dropwise, and the mixture was brought to room temperature. After stirring for 2 h, the mixture was concentrated and column chromatography was used to obtain compound M4 (276 mg, yield 58%), a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 8.14 (d,J = 4.0 Hz, 1H), 8.08 (s, 2H), 7.89 – 7.85 (m, 1H), 7.79 (t, J = 8.6 Hz, 2H),7.61 – 7.59(m, 2H), 5.22 – 5.11 (m, 2H), 4.67 (s, 1H), 3.81 – 3.72 (m, 3H), 2.65 – 2.59 (m, 2H), 2.18 – 2.12 (m, 2H), 1.84 – 1.74 (m, 2H), 1.70 – 1.63(m, 2H), 1.41 (d, J = 7.0 Hz, 3H), 0.91 (t, J = 7.3 Hz, 3H).

[0044] (5) Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(piperidin-4-ylamino)isoindoline-1,3-dione (M5):

[0045]

[0046] 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (1.38 g, 5 mmol) and 1-tert-butoxycarbonyl-4-aminopiperidine (1.5 g, 7.5 mmol) were dissolved in N,N-dimethylformamide (25 mL), and then N,N-diisopropylethylamine (1.03 g, 8 mmol) was added. The reaction was carried out at 80 °C for 12 h. After the reaction was completed, the solution was concentrated and purified by column chromatography to obtain M5-1 (1048 mg, yield 46%), a pale yellow solid. 1 H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.61 (d, J =8.3 Hz, 1H), 6.97 (d, J = 2.1 Hz, 1H), 6.75 (dd, J = 8.4, 2.2 Hz, 1H), 4.59 –4.51 (m, 1H), 3.57 – 3.52 (m, 1H), 2.84 – 2.76 (m, 6H), 2.19 – 2.10 (m, 4H), 2.07 – 2.03 (m, 2H), 1.47 (s, 9H).

[0047] Compound M5-1 (912 mg, 2 mmol) was dissolved in dichloromethane (20 mL), and 2N hydrogen chloride-ethyl acetate solution (20 mL) was added. The reaction was carried out at room temperature for 4 h. After the reaction was completed, the pH of the reaction solution was adjusted to neutral, and the solution was concentrated and column chromatography was used to obtain compound M5 (640 mg, yield 90%) as a yellow solid.

[0048] (6) Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-((piperidin-4-ylmethyl)amino)isoindoline-1,3-dione (M6):

[0049]

[0050] Following the synthesis method of M5, the product is a yellowish-green solid with a yield of 41%. 1H NMR (400 MHz, CDCl3) δ 8.85 (s,1H), 7.53 (d, J = 8.2 Hz, 1H), 6.89 (s, 1H), 6.70 (d, J = 7.4 Hz, 1H), 5.13(t, J = 5.8 Hz, 1H), 4.96 – 4.92 (m, 1H), 4.14 – 4.10 (m, 2H), 3.11 – 3.07(m, 2H), 2.95 (s, 1H), 2.88 – 2.68 (m, 6H), 1.75 – 1.72 (m, 4H), 1.45 (s,9H).

[0051] (7) Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-((2-(piperidin-4-yl)ethyl)amino)isoindoline-1,3-dione (M7):

[0052]

[0053] Following the synthesis method of M5, the product is a yellowish-green solid with a yield of 42%. 1 H NMR (400 MHz, Chloroform-d) δ8.14 (s, 1H), 7.64 (d, J = 8.2 Hz, 1H), 6.97 (d, J = 2.2 Hz, 1H), 6.76 (dd, J= 8.3, 2.1 Hz, 1H), 4.96 (dd, J = 12.0, 5.3 Hz, 1H), 4.55 (s, 1H), 4.14 (s,2H), 3.29 (t, J = 7.2 Hz, 2H), 2.97 – 2.84 (m, 1H), 2.86 – 2.73 (m, 2H), 2.73(s, 2H), 2.20 – 2.11 (m, 1H), 1.76 – 1.57 (m, 4H), 1.49 (s, 9H).

[0054] (8) Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(piperazin-1-yl)piperidin-1-yl)isoindoline-1,3-dione (M8):

[0055]

[0056] Following the synthesis method of M5, a pale yellow solid was obtained with a yield of 53%. 1H NMR (400 MHz, DMSO-d6) δ 11.10(s, 1H), 7.66 (d, J = 8.5 Hz, 1H), 7.33 (d, J = 2.3 Hz, 1H), 7.25 (dd, J =8.7, 2.3 Hz, 1H), 5.10 – 5.05 (m, 1H), 4.09 – 4.01 (m, 3H), 3.34 (s, 1H), 3.29 (d, J = 5.0 Hz, 4H), 2.98 – 2.92 (m, 2H), 2.89 (s, 2H), 2.43 (t, J = 5.1Hz, 4H), 1.99 (s, 1H), 1.84 – 1.81 (m, 2H), 1.49 – 1.43 (m, 2H), 1.39 (s,9H).

[0057] (9) Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(4-(piperazin-1-ylmethyl)piperidin-1-yl)isoindoline-1,3-dione (M9):

[0058]

[0059] Following the synthesis method of M5, a pale yellow solid was obtained with a yield of 51%. 1 H NMR (400 MHz, DMSO-d6) δ 11.09(s, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 2.2 Hz, 1H), 7.23 (dd, J =8.8, 2.3 Hz, 1H), 5.09 – 5.05 (m, 1H), 4.06 – 4.01 (m, 2H), 3.34 – 3.29 (m,5H), 2.99 – 2.84 (m, 4H), 2.61 – 2.54 (m, 2H), 2.29 (t, J = 5.0 Hz, 4H), 2.14(d, J = 6.9 Hz, 2H), 2.05 – 1.98 (m, 1H), 1.79 (t, J = 8.1 Hz, 3H), 1.40 (s,9H).

[0060] (10) Synthesis of 2-(2,6-dioxadiazin-3-yl)-5-(2,6-diazaspiro[3.3]heptane-2-yl)isoindoline-1,3-dione (M10):

[0061]

[0062] Following the synthesis method of M5, a pale yellow solid was obtained, with a yield of 49%. 1 H NMR (300 MHz, DMSO-d6) δ 11.16(s, 1H), 8.02 (dd, J = 8.2, 4.5 Hz, 1H), 7.86 (dd, J = 7.5, 2.2 Hz, 1H), 7.79– 7.67 (m, 1H), 5.21 – 5.15 (m, 1H), 2.90 (s, 3H), 2.74 (s, 3H), 2.67 – 2.53(m, 1H), 2.13 – 2.03 (m, 1H), 1.92 (s, 3H), 1.45 – 1.11 (m, 1H).

[0063] (11) Synthesis of 2-(2,6-dioxadiazin-3-yl)-5-(3,9-diazaspiro[5.5]undecane-3-yl)isoindoline-1,3-dione (M11):

[0064]

[0065] Following the synthesis method of M5, the product is a white solid with a yield of 56%. 1 H NMR (300 MHz, DMSO-d6) δ 11.09(s, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.34 (d, J = 2.2 Hz, 1H), 7.27 (dd, J =8.7, 2.3 Hz, 1H), 5.10 – 5.04 (m, 1H), 3.49 – 3.45 (m, 4H), 3.03 (s, 4H), 2.90 (t, J = 4.3 Hz, 1H), 2.61 – 2.54 (m, 3H), 2.06 – 1.97 (m, 1H), 1.70 –1.56 (m, 8H).

[0066] II. Synthesis of compounds P-1–P-7

[0067] Example 1: Synthesis of 5-((1-(4-((4-((4-(1-(sec-butyl)-5-fluoro-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoropyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperidin-4-yl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-1):

[0068]

[0069] M4 (595 mg, 1 mmol) and M5 (427 mmol, 1.2 mmol) were dissolved in 10 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (387 mg, 3 mmol) was added. The mixture was reacted at 80 °C for 12 h. After the reaction was completed, the mixture was concentrated and precipitated by column chromatography to obtain P-1, a pale yellow solid with a yield of 63%. 1 H NMR (400 MHz, DMSO-d6) δ 11.08 (s, 1H), 8.36 (d,J = 3.8 Hz, 1H), 8.11 (d, J = 2.2 Hz, 1H), 7.88 – 7.85 (m, 1H), 7.73 (d, J =7.7 Hz, 2H), 7.61 – 7.55 (m, 3H), 7.31 (d, J = 7.4 Hz, 1H), 7.08 – 7.06 (m,1H), 7.00 (s, 1H), 6.90 (d, J = 8.5 Hz, 1H), 5.77 (s, 1H), 5.06 – 5.02 (m,1H), 4.97 – 4.88 (m, 1H), 3.67 – 3.56 (m, 4H), 3.19 – 3.12 (m, 1H), 2.93 –2.88 (m, 1H), 2.86 – 2.80 (m, 2H), 2.02 – 1.95 (m, 4H), 1.78 – 1.65 (m, HRMS (ESI) for C 43 H 47 F2N9O7S (M + H) + : calcd 872.3360; found872.3362.

[0070] Example 2: Synthesis of 5-(((1-(4-((4-((4-(1-(sec-butyl)-5-fluoro-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoropyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperidin-4-yl)methyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-2):

[0071]

[0072] Following the synthesis method of P-1, a pale yellow solid was obtained in 60% yield. 1 H NMR (600 MHz, DMSO-d6) δ 8.35(d, J = 3.8 Hz, 1H), 8.10 (s, 1H), 7.98 – 7.89 (m, 2H), 7.86 – 7.84 (m, 2H),7.81 (s, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 7.4 Hz, 1H), 6.99 – 6.96(m, 1H), 6.85 – 6.84 (m, 1H), 4.94 – 4.90 (m, 1H), 4.36 (s, 1H), 3.70 – 3.58(m, 6H), 3.13 (s, 1H), 3.08 – 3.04 (m, 6H), 2.92 (s, 2H), 1.99 – 1.91 (m,6H), 1.74 – 1.68 (m, 4H), 1.63 – 1.55 (m, 4H), 1.33 (d, J = 6.8 Hz, 3H), 0.76(t, J = 7.5 Hz, 3H). HRMS (ESI) for C 44 H 49 F2N9O7S (M + H) + :calcd 886.3516;found 886.3516.

[0073] Example 3: Synthesis of 5-((2-(1-(4-((4-((4-((1-(sec-butyl)-5-fluoro-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoropyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperidin-4-yl)ethyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-3):

[0074]

[0075] Following the synthesis method of P-1, a pale yellow solid was obtained, with a yield of 61%. 1H NMR (400 MHz, DMSO-d6) δ 8.35– 8.33 (m, 1H), 8.09 (d, J = 2.7 Hz, 1H), 7.96 – 7.94 (m, 1H), 7.87 – 7.82(m, 1H), 7.71 – 7.68 (m, 2H), 7.60 – 7.57 (m, 2H), 7.50 (t, J = 7.3 Hz, 1H), 7.29 (d, J = 7.3 Hz, 1H), 7.09 – 7.03 (m, 1H), 6.91 (s, 1H), 6.79 (dd, J =8.3, 2.5 Hz, 1H), 4.95 – 4.89 (m, 1H), 4.17 – 4.10 (m, 1H), 3.67 – 3.56 (m,6H), 3.18 – 3.13 (m, 2H), 3.03 – 2.93 (m, 1H), 2.83 – 2.75 (m, 2H), 2.60 –2.55 (m, 1H), 2.48 – 2.43 (m, 2H), 2.03 – 1.91 (m, 4H), 1.78 – 1.67 (m, 6H), 1.57 – 1.46 (m, 6H), 1.31 (d, J = 6.8 Hz, 3H), 0.75 (t, J = 7.2 Hz, 3H). HRMS(ESI) for C 45 H 51 F2N9O7S (M + H) + :calcd 900.3673; found 900.3859.

[0076] Example 4: Synthesis of 5-(4-(4-((4-((4-(1-(sec-butyl)-5-fluoro-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoropyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperazin-1-yl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-4):

[0077]

[0078] Following the synthesis method of P-1, a pale yellow solid was obtained in 68% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.11(s, 1H), 8.36 (d, J = 3.7 Hz, 1H), 8.11 (d, J = 2.2 Hz, 1H), 7.88 – 7.85 (m,1H), 7.71 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.3 Hz, 2H), 7.25 (dd, J = 8.7, 2.3 Hz, 1H), 5.10 – 5.06(m, 1H), 4.97 – 4.88 (m, 1H), 4.10 – 4.07 (m, 2H), 3.68 (s, 1H), 3.58 – 3.55 (m, 4H), 2.99 – 2.85 (m, 4H), 2.61 – 2.55 (m, 4H), 2.49 – 2.44 (m, 4H), 2.04– 1.85 (m, HRMS (ESI) for C 47 H 54 F2N 10 O7S (M + H) + : calcd941.3938; found 941.3937.

[0079] Example 5: Synthesis of 5-(4-((4-((4-((4-((1-(sec-butyl)-5-fluoro-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoropyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperazin-1-yl)methyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-5):

[0080]

[0081] Following the synthesis method of P-1, a pale yellow solid was obtained, with a yield of 66%. 1H NMR (400 MHz, DMSO-d6) δ 11.09(s, 1H), 8.36 (d, J = 3.8 Hz, 1H), 8.10 (s, 1H), 7.86 (d, J = 11.1 Hz, 1H), 7.71 (d, J = 7.8 Hz, 2H), 7.65 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 7.9 Hz, 2H), 7.30 (d, J = 5.8 Hz, 2H), 7.22 (d, J = 8.5 Hz, 1H), 5.76 (s, 1H), 5.09 – 5.04(m, 1H), 4.95 – 4.90 (m, 1H), 4.05 – 4.02 (m, 2H), 3.68 (s, 2H), 3.58 – 3.55(m, 4H), 2.98 – 2.84 (m, 4H), 2.61 – 2.57 (m, 2H), 2.45 – 2.35 (m, 6H), 2.14(s, 2H), 2.03 – 1.93 (m, 5H), 1.79 – 1.69 (m, 6H), 1.58 – 1.55 (m, 2H), 1.32(d, J = 6.8 Hz, 3H), 0.76 (t, J = 7.4 Hz, 3H). HRMS (ESI) for C 48 H 56 F2N 10 O7S (M+ H) + : calcd 955.4095; found 955.4086.

[0082] Example 6: Synthesis of 5-(6-(4-((4-((4-(1-(sec-butyl)-5-fluoro-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoropyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)-2,6-diazaspiro[3.3]heptane-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-6):

[0083]

[0084] Following the synthesis method of P-1, a pale yellow solid was obtained, with a yield of 64%. 1H NMR (400 MHz, DMSO-d6) δ 11.08(s, 1H), 8.35 (d, J = 3.8 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.87 – 7.84 (m,1H), 7.69 (d, J = 8.0 Hz, 2H), 7.60 – 7.54 (m, 3H), 7.30 (t, J = 5.5 Hz, 2H), 7.11 (d, J = 2.1 Hz, 1H), 6.98 (dd, J = 8.5, 2.1 Hz, 1H), 5.07 – 5.02 (m,1H), 4.97 – 4.88 (m, 1H), 3.99 (s, 2H), 3.75 (s, 2H), 3.54 (t, J = 8.7 Hz,4H), 3.17 – 3.08 (m, 4H), 2.93 – 2.84 (m, 1H), 2.60 – 2.54 (m, 1H), 2.02 –1.91 (m, 3H), 1.78 – 1.65 (m, 2H), 1.59 – 1.51 (m, 2H), 1.32 (d, J = 6.8 Hz, 3H), 0.76 (t, J = 7.3 Hz, 3H). HRMS (ESI) for C 43 H 45 F2N9O7S (M + H) + : calcd870.3203; found 870.3193.

[0085] Example 7: Synthesis of 5-(9-(4-((4-((4-(1-(sec-butyl)-5-fluoro-6-oxo-1,6-dihydropyridin-3-yl)-5-fluoropyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)-3,9-diazaspiro[5.5]undecane-3-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (P-7):

[0086]

[0087] Following the synthesis method of P-1, a pale yellow solid was obtained in 60% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.10(s, 1H), 8.35 (d, J = 3.8 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.87 – 7.84 (m,1H), 7.72 (d, J = 7.7 Hz, 2H), 7.63 (t, J = 12.7 Hz, 3H), 7.31 (d, J = 6.8Hz, 2H), 7.21 (dd, J = 8.8, 2.3 Hz, 1H), 5.09 – 5.05 (m, 1H), 4.96 – 4.88 (m,1H), 4.16 – 4.12 (m, 1H), 3.59 – 3.56 (m, 4H), 3.47 – 3.44 (m, 4H), 3.17 (d,J = 5.0 Hz, 2H), 2.94 – 2.84 (m, 1H), 2.61 – 2.55 (m, 2H), 2.48 – 2.45 (m,2H), 2.41 – 2.39 HRMS (ESI) for C 47 H 53 F2N9O7S (M + H) + : calcd 926.3829; found 926.3833.

[0088] III. Biological Evaluation Experiment:

[0089] (1) Assay of CDK2 in vitro degradation activity:

[0090] The ability of the compounds in the examples to degrade CDK2 protein was evaluated using Western blotting, and Western blotting analysis was performed in leukemia HL60 cells.

[0091] Experimental Procedure: HL60 cells were seeded in 6-well plates and incubated at 37°C with 5% CO2 for 30 min. The test compound was then added, and the cells were cultured for another 24 h. Cells were collected, centrifuged, and the supernatant was discarded. The cells were washed once with PBS. Lysis buffer containing protease and phosphatase inhibitors was added to the cell pellet. Protein samples were obtained after lysis and quantified. Protein samples were mixed with LDS loading buffer and boiled. After separation by SDS-PAGE, the samples were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 h, and then incubated overnight at 4°C with primary antibodies (β-Actin, 1:5000; CDK2, 1:1000). After washing with TBST, the membrane was incubated with HRP-labeled secondary antibody (1:5000) at room temperature for 1 h. After washing, chemiluminescence imaging was performed, and protein expression levels were analyzed using ImageJ.

[0092] Experimental results show that compounds P-1 to P-7 prepared in the embodiments of the present invention have significant degradation activity against CDK2 protein. The results are shown in Table 1, where the degradation rates of compounds against CDK2 protein at a concentration of 50 nM are classified according to the description:

[0093] Table 1. Degradation activity of the compounds of the present invention on CDK2 protein

[0094]

[0095] "A" indicates a CDK2 protein degradation rate of ≥70%; "B" indicates a CDK2 protein degradation rate of ≥50% but <70%; "C" indicates a CDK2 protein degradation rate of <50%.

[0096] (2) Assay for cell proliferation inhibition activity:

[0097] The inhibitory activity of compound P-4 in inhibiting the proliferation of leukemia cells HL-60, K562, and Molm13 was evaluated using the CCK-8 assay.

[0098] Experimental procedure: Leukemia cells were seeded at 8000 cells / well in 96-well plates and cultured for 30 min. Then, medium containing different concentrations of the test compound or DMSO was added, and the cells were cultured for another 72 h. 10 μL of CCK-8 reagent was added to each well, and after incubation for 4 h, the absorbance was measured at 450 nm. Data analysis was performed using GraphPad software.

[0099] Experimental results show that compound P-4 prepared in the embodiments of the present invention has significant inhibitory activity against leukemia cells HL-60, K562, and MOLM13, as shown in Table 2:

[0100] Table 2. Inhibitory activity of compound P-4 of the present invention against leukemia cell proliferation

[0101]

[0102] "+++" indicates the IC50 value for inhibiting cell proliferation. 50 Less than or equal to 0.5 μM; "++" indicates IC50 of inhibitory activity against cell proliferation. 50 Greater than 0.5 μM and less than or equal to 1 μM; "+" indicates IC50 of inhibitory activity against cell proliferation. 50 Greater than 1 μM and less than or equal to 5 μM.

Claims

1. A compound of formula (P) or a pharmaceutically acceptable salt thereof, In the formula, the linker is selected from: , , , ;in, L1 is absent or selected from C1-C3 alkylene groups, L2 is selected from -NH- and C1-C3 alkylene groups, and k, n, m, p, and q are each independently selected from 0, 1, and 2.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The linker is selected from: , , , Wherein, L1 is absent or selected from C1-C3 alkylene groups, and n is selected from: 0, 1, 2.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The linker can be selected from the following structures: 。 4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Selected from any of the following compounds: 。 5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The pharmaceutically acceptable salt is a salt formed by a compound of formula (P) with hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, acetic acid, trichloroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid, or tartaric acid.

6. A method for preparing the compound of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, Compound A and compound B were dissolved in N,N-dimethylformamide, and N,N-diisopropylethylamine was added. The mixture was heated and stirred until the reaction was complete, yielding compound P. R1 is selected from , , , Wherein, L1 is absent or selected from C1-C3 alkylene groups, and n is selected from: 0, 1, 2.

7. A pharmaceutical composition, characterized in that, It comprises the compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is selected from at least one of tablets, capsules, granules, injections, powder for injection, eye drops, powders, pills, transdermal patches, ointments, aerosols, emulsions, liniments, suppositories, films, controlled-release formulations, and nanoformulations.

9. The use of the compound according to any one of claims 1-5 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention and / or treatment of tumor diseases.

10. The application according to claim 9, characterized in that, Tumor diseases are selected from leukemia, ovarian cancer, lung cancer, stomach cancer, prostate cancer, testicular cancer, colon cancer, breast cancer, multiple myeloma, liver cancer, pancreatic cancer, melanoma, glioma, brain glioma, pituitary adenoma, and hematologic malignancies.

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