A cdk4 and cdk9 dual-target inhibitor and application thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a CDK4 and CDK9 double-target inhibitor and application thereof. The present application provides, for the first time, a kind of compound with general structure shown in formula (I), (II) or (III) based on brand-new structural design. The compound can simultaneously and efficiently inhibit CDK4 and CDK9, two different targets belonging to key nodes of cell cycle regulation and transcription regulation. Through the dual inhibition of CDK4 and CDK9, the compound can synergistically act on the proliferation and survival mechanism of tumor cells, thereby realizing the double attack on tumor cells. This property is expected to overcome the problems of insufficient efficacy and easy drug resistance of single-target inhibitors, and provides a brand-new compound entity and solution for the research and development of anti-tumor drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a CDK4 and CDK9 dual-target inhibitor and its application. Background Technology

[0002] Cyclin-dependent kinases (CDKs) are a class of serine / threonine protein kinases that play important roles in key biological processes such as cell cycle regulation and transcriptional regulation. Among them, cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 9 (CDK9) have become important targets for anti-tumor drug development. CDK4 / 6 inhibitors can inhibit tumor proliferation by blocking the G1 / S phase transition of the cell cycle, while CDK9 inhibitors promote tumor cell apoptosis by inhibiting transcriptional elongation.

[0003] Currently, although some selective CDK4 or CDK9 inhibitors have entered clinical use or research stages, existing CDK inhibitors still have significant limitations. First, tumor development and progression often involve the synergistic effects of multiple pathways; inhibitors targeting only a single CDK subtype often have limited efficacy and are prone to drug resistance. Second, due to the highly conserved kinase domains of the CDK family, many inhibitors are not highly selective for CDK4 or CDK9 and may also inhibit other members such as CDK1 and CDK2, causing adverse reactions such as myelosuppression and limiting their therapeutic window.

[0004] Therefore, developing a dual-target inhibitor that can simultaneously and efficiently inhibit both CDK4 and CDK9 with good selectivity is of great significance for improving anti-tumor efficacy, overcoming drug resistance, and reducing side effects. Summary of the Invention

[0005] The purpose of this invention is to address the problems of insufficient efficacy, easy development of drug resistance, and poor selectivity of existing CDK inhibitors, and to provide a dual-target inhibitor of CDK4 and CDK9 and its application.

[0006] To achieve the above objectives, the present invention provides a dual-target inhibitor of CDK4 and CDK9, wherein the inhibitor is selected from compounds having the structure shown in formula (I), formula (II) or formula (III), or pharmaceutically acceptable salts, solvates, hydrates or prodrugs of said compounds; (I) (II) (III); R1, R4, and R7 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, cyano, trifluoromethyl, difluoromethyl, fluoromethyl, vinyl, ethynyl, methoxy, hydroxy, formyl, tert-butyl, isopropyl, nitro, or amino. R2, R3, R5, R6, R8, and R9 are each independently selected from hydrogen, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, propynyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, 3-methylpentyl, 1,1,1-trifluoroethyl, 1,1,1-trifluoropropyl, 1,1,1-trifluorobutyl, 1,1,1-trifluoropentyl, 1,1,1-trifluorohexyl, methylcyclopentadiene, 2-furanmethyl, 2-pyrrolemethyl, or 2-thiophenmethyl; X and Y are each independently selected from nitrogen atoms, carbon atoms, or carbon atoms with fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, methyl, methoxy, trifluoromethyl, difluoromethyl, fluoromethyl, formyl, nitro, ethyl, isopropyl, tert-butyl, or amino atoms attached to them. L1 is selected from , , , , , or ; L2 is selected from , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0007] The present invention also provides a pharmaceutical composition comprising the aforementioned dual-target inhibitors of CDK4 and CDK9, and one or more pharmaceutically acceptable carriers or excipients.

[0008] The present invention also provides the use of the CDK4 and CDK9 dual-target inhibitor or the pharmaceutical composition thereof in the preparation of a medicament for inhibiting CDK4 and / or CDK9 activity.

[0009] The present invention also provides the use of the CDK4 and CDK9 dual-target inhibitor or the pharmaceutical composition of claim 2 in the preparation of a medicament for treating or preventing diseases associated with abnormal CDK4 and / or CDK9 activity.

[0010] In one alternative implementation, the disease includes breast cancer, lung cancer, colorectal cancer, leukemia, or lymphoma.

[0011] Unless otherwise expressly defined, the terms and phrases used in this invention shall be understood in their usual meaning in the art.

[0012] In equation (II), L1 is connected to the lower half by a straight line and to the upper half by a wavy line. When L1 is... hour, The wavy line on the left corresponds to the upper half of the connecting formula (II), and the straight line on the right corresponds to the lower half of the connecting formula (II).

[0013] The beneficial effects of this invention are as follows: This invention, based on a novel structural design, provides for the first time a class of compounds with the general formulas shown in (I), (II), or (III). These compounds can simultaneously and efficiently inhibit CDK4 and CDK9, two different targets belonging to key nodes in cell cycle and transcriptional regulation, respectively. Through dual inhibition of CDK4 and CDK9, the compounds of this invention can synergistically act on the proliferation and survival mechanisms of tumor cells, thereby achieving a dual attack on tumor cells. This characteristic is expected to overcome the potential problems of insufficient efficacy and easy development of drug resistance in single-target inhibitors, providing a novel compound entity and solution for anti-tumor drug development. Detailed Implementation

[0014] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0015] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0016] Example 1 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, with the following structural formula: R1, R2, and R3 are all hydrogen atoms, and X is a carbon atom; it is labeled A-01.

[0017] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: Compound 1 (1 mmol) and compound 2 1 mmol was dissolved in 20 mL of 1,4-dioxane, and potassium carbonate (0.5 mmol) and catalyst diphenylphosphine ferrocene palladium dichloride (CAS: 72287-26-4; 0.05 mmol) were added. Under nitrogen protection, the reaction mixture was refluxed at 110 °C overnight. The reaction progress was monitored by TLC (thin-layer chromatography). After the reaction was complete, the reaction solution was poured into a saturated sodium chloride solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the intermediate. .

[0018] The intermediate (1 mmol) and compound 3 (1 mmol) was dissolved in 20 mL of ethanol, and 0.5 mL of 37.5% hydrochloric acid solution was added. The reaction mixture was heated at 100 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound A-01.

[0019] The proton NMR spectrum data of A-01 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.47 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.22 (d, J = 8.4 Hz, 2H), 8.12 (s, 1H), 8.04 (d, J = 8.4Hz, 2H), 7.66 (d, J = 9.0 Hz, 2H), 7.51 (s, 1H), 7.39 (d, J = 5.2 Hz, 1H), 6.92 (d, J= 9.0 Hz, 2H), 3.13 – 3.03 (m, 4H), 2.50 – 2.45 (m, 4H), 2.24 (s, 3H). 13 CNMR (75 MHz, DMSO- d 6) δ 167.81, 163.12, 160.83, 159.66, 146.66, 139.77,136.55, 133.05, 128.46 (2C), 127.16 (2C), 120.79 (2C), 116.34 (2C), 107.99,55.13, 49.27, 46.17. Example 2 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 1 in that R1 is modified to fluorine and labeled as A-02.

[0020] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0021] The proton NMR spectrum data of A-02 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.55 (s, 1H), 8.60 (d, J = 3.4 Hz, 1H), 8.15 (d, J = 10.3 Hz, 1H), 8.06 (t, J = 5.2 Hz, 4H), 7.59 (d, J = 8.9 Hz, 2H), 7.54 (s, 1H), 6.91 (d, J = 8.9 Hz, 2H), 3.07 (s, 4H), 2.48 (s, 4H), 2.24 (s, 3H). 13 C NMR (75 MHz, DMSO- d 6) δ 167.74, 157.31, 151.97,150.50, 148.19, 146.71, 136.57, 136.29, 133.02 (2C), 128.99, 128.29 (2C), 120.51 (2C), 116.35 (2C), 55.12, 49.24, 46.16. Example 3 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 1 in that R2 and R3 are modified to be pentyl, and X is a carbon atom with a methoxy group attached; it is labeled as A-03.

[0022] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0023] The proton NMR spectrum data of A-03 are as follows: 1 H NMR (300 MHz, CDCl3) δ 8.50 – 8.44 (m,2H), 8.40 (d, J = 8.6 Hz, 1H), 8.18 (d, J = 7.5 Hz, 1H), 7.89 (d, J = 7.8 Hz, 1H),7.61 (s, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.15 (d, J = 5.2 Hz, 1H), 6.59 (s, 1H), 6.15 (d, J = 6.6 Hz, 1H), 4.51 – 4.41 (m, 1H), 3.91 (s, 3H), 3.25 (s, 4H), 2.72(s, 4H), 2.45 (s, 3H), 2.15 (d, J = 6.6 Hz, 2H), 1.78 – 1.65 (m, 4H), 1.58 –1.50 (m, 2H). 13 C NMR (75 MHz, CDCl3) δ 166.77, 163.81, 160.40, 158.82, 149.18,147.06, 137.68, 135.55, 129.12, 129.08, 125.33, 122.58, 119.82, 108.31,107.81, 100.75, 55.71, 55.11, 51.85, 50.02, 45.94, 33.29, 23.88. Example 4 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 1 in that R3 is modified to a propynyl group and labeled as A-04.

[0024] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0025] The proton NMR spectrum data of A-04 are as follows: 1 H NMR (300 MHz, CDCl3) δ 8.26 (s, 1H),8.07 (d, J = 9.0 Hz, 2H), 7.93 (s, 1H), 7.69 (s, 1H), 7.18 (s, 1H), 7.00 (s,1H), 6.45 – 6.42 (m, 2H), 6.33 (s, 1H), 4.88 (s, 1H), 4.21 (s, 2H), 3.86 (s,3H), 3.44 (s, 4H), 3.08 (s, 1H), 2.35 (s, 4H), 2.21 (s, 3H). 13 C NMR (75 MHz, DMSO) δ 166.07, 163.48, 161.08, 159.67, 151.56, 147.05, 137.37, 134.91,130.15, 130.04, 129.50, 126.26, 123.04, 122.02, 107.85, 101.33, 81.70, 73.48,56.27, 52.93, 46.94, 44.18, 29.07. Example 5 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 1 in that X is replaced with a nitrogen atom and labeled as A-05.

[0026] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0027] The proton NMR spectrum data of A-05 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.62 (s, 1H), 8.60 (d, J = 5.2 Hz, 1H), 8.25 (d, J = 8.4 Hz, 2H), 8.16 (d, J = 9.0 Hz, 2H), 8.04(d, J= 8.4 Hz, 2H), 7.53 – 7.49 (m, 2H), 7.49 – 7.45 (m, 1H), 3.16 – 3.11 (m,4H), 2.47 (d, J = 4.9 Hz, 4H), 2.24 (s, 3H). 13 C NMR (75 MHz, DMSO- d 6) δ 167.37,162.82, 159.62, 159.38, 145.59, 142.73, 139.10, 136.29, 135.48, 128.12 (2C),126.87 (2C), 125.25, 113.63, 108.64, 62.73, 54.56, 52.12, 48.54, 45.85,40.43, 40.16, 39.88, 39.60, 39.32, 39.04, 38.77, 7.31. Example 6 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 1 in that R1 is replaced with fluorine and X is a carbon atom with a methoxy group attached; it is labeled as A-06.

[0028] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0029] The proton NMR spectrum data of A-06 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 8.54 (d, J = 3.5Hz, 1H), 8.24 (s, 1H), 8.13 (d, J = 5.2 Hz, 1H), 8.04 (s, 2H), 7.67 (d, J = 8.7Hz, 1H), 7.53 (s, 1H), 6.65 (d, J = 2.4 Hz, 1H), 6.51 (dd, J = 8.8, 2.4 Hz, 1H), 3.81 (s, 3H), 3.20 – 3.13 (m, 4H), 2.54 (dd, J = 8.4, 3.5 Hz, 4H), 2.29 (s,3H). 13C NMR (75 MHz, DMSO- d 6) δ 167.73, 157.83, 157.80, 152.10, 150.58,150.49, 148.79, 148.29, 148.10, 147.95, 136.57, 136.23, 136.20, 136.16,128.97, 128.89, 128.26 (2C), 123.53, 121.00, 107.27, 100.69, 56.08, 54.97,48.93, 45.92, 40.83, 40.56, 40.28, 40.00, 39.72, 39.44, 39.17. Example 7 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Example 1 in that R3 is modified to be methyl and labeled as A-07.

[0030] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0031] The proton NMR spectrum data of A-07 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.48 (s, 1H), 8.59 (d, J = 4.5 Hz, 1H), 8.53 (d, J = 5.2 Hz, 1H), 8.23 ​​(d, J = 8.3 Hz, 2H), 7.99(d, J = 8.3 Hz, 2H), 7.66 (d, J = 8.9 Hz, 2H), 7.39 (d, J = 5.2 Hz, 1H), 6.93 (d, J =9.0 Hz, 2H), 3.12 (s, 4H), 2.82 (d, J = 4.4 Hz, 3H), 2.58 (s, 4H), 2.31 (s, 3H). 13 C NMR (75 MHz, DMSO- d6) δ 166.51, 163.11, 160.83, 159.66, 146.46,139.58, 136.76, 133.19, 128.05 (2C), 127.22 (2C), 120.80 (2C), 116.44 (2C),107.99, 54.86, 48.99, 45.74, 26.78. Example 8 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 1 in that R2 and R3 are modified to be methyl and labeled as A-08.

[0032] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0033] The proton NMR spectrum data of A-08 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.47 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H), 8.20 (d, J = 8.4 Hz, 2H), 7.66 (d, J = 9.0 Hz, 2H), 7.56(d, J = 8.4 Hz, 2H), 7.36 (d, J = 5.2 Hz, 1H), 6.93 (d, J = 9.1 Hz, 2H), 3.14 –3.08 (m, 4H), 2.98 (d, J = 22.0 Hz, 6H), 2.60 – 2.54 (m, 4H), 2.30 (s, 3H). 13 CNMR (75 MHz, DMSO- d 6) δ 170.03, 163.22, 160.83, 159.63, 146.48, 139.03,138.03, 133.19, 127.94 (2C), 127.24 (2C), 120.77 (2C), 116.44 (2C), 107.82,54.91, 49.05, 45.82, 35.21. Example 9 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 1 in that R3 is modified to be a methyl group and X is a nitrogen atom; and it is labeled as A-09.

[0034] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0035] The proton NMR spectrum data of A-09 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.75 (s, 1H), 8.65 – 8.57 (m, 2H), 8.26 (d, J = 8.4 Hz, 2H), 8.19 (d, J = 9.1 Hz, 1H), 8.07 (d, J = 2.9 Hz, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.54 – 7.44 (m, 2H), 3.20 (s, 4H), 2.83 (d, J = 4.5 Hz, 3H), 2.67 (s, 4H), 2.36 (s, 3H). 13 C NMR (75 MHz, DMSO- d 6) δ166.48, 163.23, 159.99, 159.76, 146.36, 142.53, 139.31, 136.90, 136.20,128.09 (2C), 127.34 (2C), 125.96, 114.00, 109.09, 54.04, 48.02, 44.85, 26.78. Example 10 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 1 in that R2 and R3 are modified to be methyl groups and X is a nitrogen atom; and it is labeled as A-10.

[0036] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0037] The proton NMR spectrum data of A-10 are as follows: 1 H NMR (300 MHz, DMSO- d6) δ 9.72 (s, 1H), 8.60 (d, J = 5.2 Hz, 1H), 8.21 (t, J = 8.1 Hz, 3H), 8.07 (d, J = 2.8 Hz, 1H), 7.57(d, J = 8.2 Hz, 2H), 7.49 (dd, J = 5.6, 3.5 Hz, 2H), 3.22 (s, 4H), 3.02 (s, 3H), 2.94 (s, 3H), 2.71 (s, 4H), 2.39 (s, 3H). 13 C NMR (75 MHz, DMSO- d 6) δ 170.00,163.34, 159.98, 159.75, 146.42, 142.44, 139.20, 137.76, 136.23, 127.97 (2C),127.37 (2C), 126.04, 113.94, 108.95, 53.93, 47.90, 44.68, 26.81. Example 11 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 1 in that R1 is modified to chlorine and labeled as A-11.

[0038] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0039] The proton NMR spectrum data of A-11 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.73 (s, 1H), 8.57 (s, 1H), 8.15 (s, 1H), 8.03 (d, J = 8.1 Hz, 2H), 7.86 (d, J = 8.1 Hz, 2H),7.62 – 7.49 (m, 3H), 6.89 (d, J = 8.8 Hz, 2H), 3.07 (s, 4H), 2.47 (s, 4H), 2.23(s, 3H). 13 C NMR (75 MHz, DMSO- d6) δ 167.83, 158.91, 158.78, 146.97, 139.07,135.92, 132.40, 129.32 (2C), 127.82 (2C), 120.98 (2C), 116.69, 116.26 (2C),55.06, 49.10, 46.11. Example 12 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Example 1 in that R1 is modified to ethyl and labeled as A-12.

[0040] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0041] The proton NMR spectrum data of A-12 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 8.39 (s, 1H),8.02 – 7.98 (m, 4H), 7.00 (s, 2H), 6.76 (s, 2H), 6.21 (s, 2H), 4.79 (s, 1H),3.44 (s, 4H), 2.71 (s, 2H), 2.35 (s, 4H), 2.21 (s, 3H), 1.18 (s, 3H). 13 C NMR (75 MHz, DMSO-) d 6) δ 168.02, 155.62, 152.74, 146.03, 145.30, 145.19, 138.63,135.53, 134.04, 129.32 (2C), 127.66 (2C), 119.75 (2C), 116.53 (2C), 57.69,55.14, 49.50, 46.11. Example 13 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Example 1 in that R1 is modified to be methyl and labeled as A-13.

[0042] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0043] The proton NMR spectrum data of A-13 are as follows: 1H NMR (300 MHz, DMSO- d 6) δ 9.29 (s, 1H), 8.34 (s, 1H), 8.08 (s, 1H), 8.01 (d, J = 8.3 Hz, 2H), 7.71 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 9.0 Hz, 2H), 7.45 (s, 1H), 6.85 (d, J = 8.7 Hz, 2H), 3.11 – 3.02 (m,4H), 2.51 (s, 4H), 2.26 (s, 3H), 2.20 (s, 3H). 13 C NMR (75 MHz, DMSO- d 6) δ167.99, 164.31, 160.25, 159.34, 146.19, 141.49, 135.03, 133.65, 128.91 (2C), 127.84 (2C), 120.21 (2C), 117.48, 116.46 (2C), 55.05, 49.33, 46.01, 16.22. Example 14 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 1 in that R1 is modified to be methoxy and labeled as A-14.

[0044] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitors, which differs from Example 1 in that the raw materials are adaptively adjusted according to the substituents of the target compounds.

[0045] The proton NMR spectrum data of A-14 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.16 (s, 1H),8.40 (s, 1H), 8.10 (d, J = 8.4 Hz, 2H), 8.04 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.9 Hz, 2H), 7.42 (s, 1H), 6.86 (d, J= 8.9 Hz, 2H), 3.87 (s, 3H), 3.06 (s, 4H), 2.51 (s, 4H), 2.26 (s, 3H). 13 C NMR (75 MHz, DMSO- d 6) δ 168.02,155.62, 152.74, 146.03, 145.30, 145.19, 138.63, 135.53, 134.04, 129.32 (2C), 127.66 (2C), 119.75 (2C), 116.53 (2C), 57.69, 55.14, 49.50, 46.11. Example 15 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, with the following structural formula: Where R4 is fluorine, R5 and R6 are hydrogen, Y is carbon, and L1 is... Mark it as B-01.

[0046] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: Compound a 1 mmol of 2,4-dichloro-5-fluoropyrimidine and 1 mmol of ethanol were dissolved in 20 mL of ethanol, and 0.5 mmol of triethylamine was added. The reaction mixture was heated under reflux at 100 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium chloride solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the intermediate. .

[0047] The intermediate (1 mmol) and 4-(4-methylpiperazine)aniline (1 mmol) were dissolved in 20 mL of ethanol, and 0.5 mL of 37.5% hydrochloric acid solution was added. The reaction mixture was heated at 100 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound B-01. The proton NMR spectrum data of B-01 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.33 (s, 1H), 8.43 (d, J= 3.0 Hz, 1H), 8.07 (s, 1H), 8.01 (d, J = 8.7 Hz, 2H), 7.47 (s, 1H), 7.39 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 8.5 Hz, 2H), 6.63 (d, J = 8.9 Hz, 2H), 3.17(s, 3H), 3.00 – 2.95 (m, 4H), 2.44 – 2.39 (m, 4H), 2.20 (s, 3H). 13 C NMR (75MHz, DMSO- d 6) δ 167.58, 157.30, 155.72, 154.78, 146.53, 146.27, 140.03,132.54, 132.31, 129.74 (2C), 122.34 (2C), 120.21 (2C), 115.94 (2C), 55.13, 49.19, 46.25. Example 16 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 15 in that L1 is modified to... It was marked as B-02.

[0048] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: Compound b 1 mmol of N,N-dimethylformamide and 1 mmol of 2,4-dichloro-5-fluoropyrimidine were dissolved in 20 mL of N,N-dimethylformamide, and 0.5 mmol of sodium hydride was added. The reaction mixture was heated under reflux at 110 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium chloride solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the intermediate. .

[0049] The intermediate (1 mmol) and 4-(4-methylpiperazine)aniline (CAS: 170911-92-9; 1 mmol) were dissolved in 20 mL of ethanol, and 0.5 mL of 37.5% hydrochloric acid solution was added. The reaction mixture was heated under reflux at 100 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound B-02. The proton NMR spectrum data of B-02 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.49 (s, 1H),9.04 (s, 1H), 8.11 (d, J = 3.7 Hz, 1H), 7.93 (d, J = 8.8 Hz, 2H), 7.89 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 9.0 Hz, 2H), 7.25 (s, 1H), 6.87 (d, J = 9.1Hz, 2H), 3.10 – 3.03 (m, 4H), 2.49 – 2.44 (m, 4H), 2.23 (s, 3H). 13 C NMR (75MHz, DMSO- d 6) δ 167.94, 156.32, 149.66, 146.60, 142.48, 142.44, 140.60,133.36, 128.54 (2C), 128.48, 120.98 (2C), 120.03 (2C), 116.31 (2C), 55.16, 49.40, 46.21. Example 17 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 15 in that L1 is modified to... It is marked as B-03.

[0050] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: Compound C 1 mmol of EDCl and 1 mmol of 2,4-dichloro-5-fluoropyrimidine were dissolved in 20 mL of dichloromethane, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl; 1 mmol) and 0.05 mmol of 4-dimethylaminopyridine were added. The reaction mixture was reacted overnight at 37 °C, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated ammonium chloride solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the intermediate. .

[0051] The intermediate (1 mmol) and 4-(4-methylpiperazine)aniline (1 mmol) were dissolved in 20 mL of ethanol, and 0.5 mL of 37.5% hydrochloric acid solution was added. The reaction mixture was heated at 100 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound B-03. The proton NMR spectrum data of B-03 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.11 (s, 1H),8.18 (s, 4H), 8.05 (d, J = 9.0 Hz, 1H), 7.02 – 6.98 (m, 2H), 6.78 – 6.43 (m,2H), 6.19 (s, 2H), 4.79 (s, 1H), 3.46 – 3.42 (s, 4H), 2.37 – 2.33 (m, 4H),2.21 (s, 3H). 13 C NMR (75 MHz, DMSO- d 6) δ 168.55, 167.08, 151.96, 148.75,147.96, 146.22, 137.88, 136.94, 134.99, 133.30, 130.11 (2C), 128.31 (2C), 125.74 (2C), 121.10 (2C), 52.65, 50.29, 46.06. Example 18 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, with the following structural formula: R7 is fluorine, R8 and R9 are hydrogen, and L2 is... Mark it as C-01.

[0052] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: Compound m 1 mmol of 1,4-dichloro-5-fluoropyrimidine and 1 mmol of 20 mL of 1,4-dioxane were dissolved in 20 mL of potassium carbonate (0.5 mmol) and the catalyst diphenylphosphine ferrocene palladium dichloride (CAS: 72287-26-4; 0.05 mmol). Under nitrogen protection, the reaction mixture was refluxed at 110 °C overnight. The reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium chloride solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the intermediate. .

[0053] The intermediate (1 mmol) and 4-(4-Bocpiperazine)aniline (CAS: 57260-72-7; 1 mmol) were dissolved in 20 mL of ethanol, and 0.5 mL of 37.5% hydrochloric acid solution was added. The reaction mixture was heated at 100 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound C-01.

[0054] The proton NMR spectrum data of C-01 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 8.51 (d, J = 12.0Hz, 1H), 7.93 (m, 4H), 7.02 – 6.99 (m, 2H), 6.78 – 6.73 (m, 2H), 6.22 (s,2H), 4.78 (s, 1H), 3.35 – 3.26 (m, 8H), 1.42 (s, 9H). 13 C NMR (75 MHz, DMSO- d 6)δ 168.55, 156.56, 152.61, 151.96, 149.33, 145.69, 145.01, 136.11, 135.77,133.83 (2C), 133.30, 129.79 (2C), 125.74 (2C), 121.10 (2C), 81.20, 49.72,43.50, 28.33. Example 19 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 18 in that L2 is modified to... It is marked as C-02.

[0055] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: The C-01 (1 mmol) prepared in Example 18 was dissolved in 50 mL of dichloromethane, and 0.5 mL of 37.5% hydrochloric acid solution was added. The reaction mixture was reacted overnight at 37°C, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound C-02.

[0056] The proton NMR spectrum data for C-02 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 8.51 (d, J = 12.0Hz, 1H), 7.97 – 7.88 (m, 4H), 7.02 – 6.98 (m, 2H), 6.78 – 6.73 (m, 2H), 6.21(s, 2H), 4.75 (s, 1H), 3.48 – 3.43 (s, 4H), 2.83 – 2.73 (m, 4H). 13 C NMR (75MHz, DMSO- d 6) δ 168.55, 152.61, 151.93, 149.31, 145.69, 145.11, 136.08,135.71, 133.83 (2C), 133.30, 129.79 (2C), 125.74 (2C), 121.10 (2C), 50.10,45.05. Example 20 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 18 in that L2 is modified to... It is marked as C-03.

[0057] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: The C-02 (1 mmol) and acetic anhydride (1 mmol) prepared in Example 19 were dissolved in 20 mL of dichloromethane. The reaction mixture was reacted overnight at 37 °C. The reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound C-03.

[0058] The proton NMR spectrum data of C-03 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 8.49 (d, J = 12.0Hz, 1H), 7.95 – 7.90 (m, 4H), 7.02 – 6.95 (m, 2H), 6.75 – 6.70 (m, 2H), 6.20(s, 2H), 4.75 (s, 1H), 3.48 – 3.43 (s, 4H), 2.83 – 2.73 (m, 4H), 2.10 (s, 3H). 13 C NMR (75 MHz, DMSO- d 6) δ 168.51, 152.61, 151.89, 149.29, 145.63,145.09, 136.08, 135.71, 133.79 (2C), 133.30, 129.72 (2C), 125.69 (2C), 121.03(2C), 50.10, 45.05. Example 21 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 18 in that L2 is modified to... It is marked as C-04.

[0059] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: Compound n 1 mmol of 4-(4-ethylpiperazine)aniline and 1 mmol of 4-(4-ethylpiperazine)aniline were dissolved in 50 mL of ethanol, and 0.5 mL of 37.5% hydrochloric acid solution was added. The reaction mixture was heated at 100 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound C-04.

[0060] The proton NMR spectrum data for C-04 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 9.57 (s, 1H), 8.59 (d, J = 3.5 Hz, 1H), 8.16 (s, 1H), 8.06 (d, J = 8.1 Hz, 4H), 7.60 (d, J = 9.0Hz, 2H), 7.55 (s, 1H), 6.91 (d, J = 9.0 Hz, 2H), 3.12 (s, 4H), 2.62 (s, 4H), 2.50 – 2.40 (m, 2H), 1.06 (t, J = 7.1 Hz, 3H). 13 C NMR (75 MHz, DMSO- d 6) δ167.75, 157.31, 151.98, 150.57, 148.13, 146.52, 136.58, 136.22, 133.16,128.98 (2C), 128.31 (2C), 120.52 (2C), 116.41 (2C), 52.52, 51.94, 45.86,11.95. Example 22 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 18 in that L2 is modified to... It is marked as C-05.

[0061] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: Compound x 1 mmol of EDCl and 1 mmol of mono-Boc piperazine (CAS: 57260-72-7; 1 mmol) were dissolved in 20 mL of dichloromethane, and 1 mmol of EDCl and 0.05 mmol of 4-dimethylaminopyridine were added. The reaction mixture was reacted overnight at 37 °C, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated ammonium chloride solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the intermediate. .

[0062] The intermediate (1 mmol) and compound n (1 mmol) was dissolved in 20 mL of ethanol, and 0.5 mL of 37.5% hydrochloric acid solution was added. The reaction mixture was heated at 100 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound C-05. The proton NMR spectrum data for C-05 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 8.51 (d, J = 9.0Hz, 1H), 8.025 – 7.97 (m, 4H), 5 7.72 – 7.63 (m, 4H), 6.22 (s, 2H), 4.95 (s,1H), 3.615 – 3.57 (m, 4H), 3.36 – 3.32 (m, 4H), 1.42 (s, 9H). 13 C NMR (75 MHz, DMSO) δ 167.75, 157.31, 151.98, 150.57, 148.13, 146.52, 136.58, 136.22,133.16, 128.98 (2C), 128.31 (2C), 120.52 (2C), 116.41 (2C), 52.52, 51.94,45.86, 11.95. Example 23 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, which differs from Embodiment 18 in that L2 is modified to... It is marked as C-06.

[0063] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: Compound x 1 mmol of EDCl and 1-Boc-4-aminopiperidine (CAS: 87120-72-7; 1 mmol) were dissolved in 50 mL of dichloromethane, and 1 mmol of EDCl and 0.05 mmol of 4-dimethylaminopyridine were added. The reaction mixture was reacted overnight at 37 °C, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated ammonium chloride solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the intermediate. .

[0064] The intermediate (1 mmol) and compound n (1 mmol) was dissolved in 20 mL of ethanol, and 0.5 mL of 37.5% hydrochloric acid solution was added. The reaction mixture was heated at 100 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound C-06. The proton NMR spectrum data for C-06 are as follows: 1 H NMR (300 MHz, DMSO- d 6) δ 8.51 (d, J = 9.0Hz, 1H), 8.02 – 7.92 (m, 4H), 7.72 – 7.62 (m, 4H), 6.37 (s, 1H), 6.22 (s,2H), 4.97 (s, 1H), 3.80 – 3.68 (m, 1H), 3.64 – 3.43 (m, 4H), 2.27 – 2.16 (s, 2H), 1.92 – 1.79 (m, 2H), 1.42 (s, 9H). 13 C NMR (125 MHz, DMSO- d 6) δ 168.55,167.65, 156.56, 152.61, 149.33, 145.69, 145.01, 140.22, 136.11, 135.77,133.83 (2C), 130.50, 129.79 (2C), 128.58 (2C), 116.90 (2C), 81.20, 47.90,45.41, 30.36, 28.33. Example 24 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, whose structure differs from that of Embodiment 18 in that L2 is modified to... Mark it as D-01.

[0065] This embodiment also provides a method for preparing the above-mentioned dual-target inhibitor of CDK4 and CDK9, including the following steps: Compound n (1 mmol) and compound d1 (1 mmol) was dissolved in 50 mL of ethanol, and 0.5 mL of 37.5% hydrochloric acid solution was added. The reaction mixture was heated at 100 °C overnight, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was poured into a saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent. The crude product was purified by column chromatography to obtain the target compound D-01. The proton NMR spectrum data of D-01 are as follows: 1 H NMR (300 MHz, DMSO) δ 9.60 (s, 1H), 8.96 (dd, J = 8.0, 3.1 Hz, 2H), 8.62 (t, J = 3.9 Hz, 1H), 8.21 (d, J = 6.7 Hz, 2H), 8.07 (d, J = 8.2 Hz, 2H), 7.61 (d, J = 8.9 Hz, 2H), 7.57 (s, 1H), 6.94 (d, J = 8.8Hz, 2H), 3.69 (d, J = 12.3 Hz, 2H), 3.12 (t, J = 11.3 Hz, 1H), 2.96 (q, J = 7.2 Hz, 2H), 2.66 (t, J = 11.9 Hz, 2H), 2.07 (d, J = 10.6 Hz, 2H), 1.68 (dd, J = 20.5, 12.0Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H). 13 C NMR (75 MHz, DMSO) δ 167.67, 165.60,157.30, 154.51, 148.47, 146.18, 137.02, 136.42, 135.39, 133.14, 129.23,129.08, 128.36, 120.62, 117.06, 54.20, 48.31, 39.29, 28.28, 11.83. Example 25 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, whose structure differs from that of Embodiment 18 in that L2 is modified to... Mark it as D-02.

[0066] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitor, the difference from Example 24 being that compound d1 is replaced with compound d2. .

[0067] The proton NMR spectrum data of D-02 are as follows: 1 H NMR (300 MHz, DMSO) δ 9.57 (s, 1H),8.59 (d, J = 3.5 Hz, 1H), 8.12 (d, J = 8.6 Hz, 1H), 8.06 (t, J = 6.0 Hz, 4H), 7.62(d, J = 9.0 Hz, 2H), 7.55 (s, 1H), 6.91 (d, J = 9.1 Hz, 2H), 3.77 – 3.70 (m, 4H), 3.07 – 3.01 (m, 4H). 13 C NMR (75 MHz, DMSO) δ 167.78, 157.30, 151.99, 150.49,148.30, 146.74, 136.57, 136.23, 133.32, 128.92, 128.29(2C), 120.53 (2C),116.10 (2C), 66.64 (2C), 49.71 (2C). Example 26 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, whose structure differs from that of Embodiment 18 in that L2 is modified to... Mark it as D-03.

[0068] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitor, the difference from Example 24 being that compound d1 is replaced with compound d3. .

[0069] The proton NMR spectrum data of D-03 are as follows: 1 H NMR (300 MHz, DMSO) δ 10.11 (s, 1H),8.72 (d, J = 3.3 Hz, 1H), 8.17 (s, 1H), 8.10 (q, J = 8.6 Hz, 4H), 7.87 (d,J = 8.7Hz, 2H), 7.56 (s, 1H), 7.42 (d, J = 8.6 Hz, 2H), 3.49 (s, 8H), 2.03 (s, 3H). 13 CNMR (75 MHz, DMSO) δ 169.82, 168.93, 167.72, 156.70, 152.51, 150.85, 148.09,142.45, 136.76, 135.90, 129.02, 128.71, 128.44, 128.38 (2C), 118.08 (2C), 45.84 (2C), 21.73 (2C). Example 27 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, whose structure differs from that of Embodiment 18 in that L2 is modified to... Mark it as D-04.

[0070] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitor, the difference from Example 24 being that compound d1 is replaced with compound d4. .

[0071] The proton NMR spectrum data of D-04 are as follows: 1 H NMR (300 MHz, DMSO) δ 9.55 (s, 1H),8.59 (d, J = 3.3 Hz, 1H), 8.16 (s, 1H), 8.09 – 8.04 (m, 4H), 7.62 – 7.57 (m,2H), 7.55 (s, 1H), 6.91 (d, J = 9.1 Hz, 2H), 3.59 (d, J = 12.5 Hz, 2H), 3.01 –2.89 (m, 1H), 2.67 (t, J = 11.3 Hz, 2H), 1.89 (d, J = 10.3 Hz, 2H), 1.52 (dt, J =11.4, 8.7 Hz, 2H). 13C NMR (75 MHz, DMSO) δ 167.78, 157.32, 150.59, 148.63,148.11, 146.54, 136.56, 136.23, 132.88, 128.91, 128.30 (2C), 120.61 (2C),116.91 (2C), 48.45, 48.41, 32.15. Example 28 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, whose structure differs from that of Embodiment 18 in that L2 is modified to... Mark it as D-05.

[0072] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitor, the difference from Example 24 being that compound d1 is replaced with compound d5. .

[0073] The proton NMR spectrum data of D-05 are as follows: 1 H NMR (300 MHz, DMSO) δ 9.54 (s, 1H), 8.60 (d, J = 3.5 Hz, 1H), 8.15 (d, J = 11.3 Hz, 1H), 8.12 – 8.05 (m, 4H), 8.03(s, 1H), 7.58 (d, J = 9.0 Hz, 2H), 7.54 (s, 1H), 6.92 (d, J = 9.1 Hz, 2H), 3.69(dd, J = 10.9, 7.1 Hz, 1H), 3.56 (d, J = 12.4 Hz, 2H), 2.72 (t, J = 11.0 Hz, 2H), 1.82 (d, J = 10.1 Hz, 2H), 1.53 (dd, J = 14.0, 6.1 Hz, 2H), 0.71 – 0.58 (m, 4H). 13C NMR (75 MHz, DMSO) δ 172.17, 167.75, 157.30, 150.61, 148.32, 147.98,146.74, 136.58, 136.23, 132.81, 129.00, 128.91 (2C), 128.29 (2C), 120.58(2C), 116.89, 48.94 (2C), 46.37, 40.82, 40.54, 40.27, 39.99, 39.71, 39.43,39.15, 31.82 (2C), 14.09, 6.63 (2C). Example 29 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, whose structure differs from that of Embodiment 18 in that L2 is modified to... Mark it as D-06.

[0074] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitor, the difference from Example 24 being that compound d1 is modified to compound d6. .

[0075] The proton NMR spectrum data of D-06 are as follows: 1 H NMR (300 MHz, DMSO) δ 8.82 (s, 1H),8.49 (d, J = 2.6 Hz, 1H), 8.11 (s, 1H), 8.01 (s, 4H), 7.53 (s, 1H), 7.27 (d, J =8.4 Hz, 1H), 6.84 (dd, J = 23.8, 10.6 Hz, 3H), 6.16 (d, J = 16.6 Hz, 1H), 5.72(d, J = 10.2 Hz, 1H), 3.70 (s, 4H), 3.37 (d, J = 2.5 Hz, 1H), 3.12 (s, 4H). 13C NMR(75 MHz, DMSO) δ 167.75, 164.70, 158.73, 150.50, 148.58, 148.09, 136.48,136.31, 134.25, 130.77, 128.92, 128.63, 128.20, 127.95, 126.94, 118.29 (2C), 114.22 (2C), 49.94, 49.27, 45.41, 41.83, 18.94. Example 30 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, whose structure differs from that of Embodiment 18 in that L2 is modified to... Mark it as D-07.

[0076] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitor, the difference from Example 24 being that compound d1 is replaced with compound d7. .

[0077] The proton NMR spectrum data of D-07 are as follows: 1 H NMR (300 MHz, DMSO) δ 9.81 (s, 1H),8.65 (d, J = 3.3 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.09 (q, J = 8.6 Hz, 4H), 7.72(d, J = 8.3 Hz, 2H), 7.56 (s, 1H), 7.21 (d, J = 8.3 Hz, 2H), 3.39 (s, 2H), 2.29(d, J = 45.1 Hz, 8H), 2.18 (s, 3H). 13 C NMR (75 MHz, DMSO) δ 167.76, 157.02,152.23, 150.59, 148.13, 139.76, 136.66, 136.16, 131.53 (2C), 129.61 (2C),128.96, 128.33 (2C), 118.85 (2C), 62.13, 55.02 (2C), 52.69 (2C), 45.93. Example 31 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, whose structure differs from that of Embodiment 18 in that L2 is modified to... Mark it as D-08.

[0078] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitor, the difference from Example 24 being that compound d1 is modified to compound d8. .

[0079] The proton NMR spectrum data of D-08 are as follows: 1 H NMR (300 MHz, DMSO) δ 9.81 (s, 1H),8.66 (d, J = 3.4 Hz, 1H), 8.13 (d, J = 3.1 Hz, 1H), 8.07 (t, J = 7.3 Hz, 4H), 7.72(d, J = 8.5 Hz, 2H), 7.55 (s, 1H), 7.21 (d, J = 8.5 Hz, 2H), 3.44 (s, 2H), 2.61 (s, 8H). 13 C NMR (75 MHz, DMSO) δ 167.74, 157.01, 152.23, 150.73, 148.89,148.39, 139.79, 136.66 (2C), 136.08, 131.30 (2C), 129.62, 129.05 (2C), 118.85(2C), 110.48, 62.76, 54.81 (2C), 27.66 (2C). Example 32 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, whose structure differs from that of Embodiment 18 in that L2 is modified to... Mark it as D-09.

[0080] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitor, the difference from Example 24 being that compound d1 is replaced with compound d9. .

[0081] The proton NMR spectrum data of D-09 are as follows: 1 H NMR (300 MHz, DMSO) δ 10.11 (s, 1H),8.71 (d, J= 3.4 Hz, 1H), 8.17 (d, J = 11.3 Hz, 2H), 8.10 (q, J = 8.6 Hz, 4H), 7.87(d, J = 8.7 Hz, 2H), 7.56 (s, 1H), 7.43 (d, J = 8.6 Hz, 2H), 3.64 (d, J = 64.2 Hz, 8H), 1.97 (d, J = 4.6 Hz, 1H), 0.77 (dd, J = 7.3, 5.8 Hz, 2H), 0.74 – 0.68 (m,2H). 13 C NMR (75 MHz, DMSO) δ 171.76, 169.81, 167.74, 156.70, 152.51, 150.95,149.15, 148.06, 142.46, 136.75 (2C), 135.90, 129.13, 128.73 (2C), 128.37(2C), 118.08 (2C), 45.85 (2C), 10.83, 8.91 (2C), 7.61 (2C). Example 33 This embodiment provides a dual-target inhibitor of CDK4 and CDK9, whose structure differs from that of Embodiment 18 in that L2 is modified to... It is marked as D-10.

[0082] This embodiment also provides a method for preparing the above-mentioned CDK4 and CDK9 dual-target inhibitor, the difference from Example 24 being that compound d1 is modified to compound d10. .

[0083] The proton NMR spectrum data of D-10 are as follows: 1 H NMR (300 MHz, DMSO) δ 10.13 (s, 1H),8.73 (d, J = 3.4 Hz, 1H), 8.18 (d, J = 8.1 Hz, 2H), 8.16 – 8.13 (m, 2H), 8.11 (s,1H), 8.08 (d, J = 8.7 Hz, 2H), 7.85 (d, J= 8.0 Hz, 4H), 7.56 (s, 1H), 4.31 (dd, J = 25.6, 11.6 Hz, 2H), 4.15 – 4.01 (m, 1H), 3.21 (t, J = 11.7 Hz, 1H), 3.06 (dd, J = 7.1, 3.2 Hz, 2H), 2.70 (d, J = 11.2 Hz, 1H), 2.04 – 1.96 (m, 1H), 1.93 –1.77 (m, 2H), 1.48 (dd, J = 20.9, 11.4 Hz, 2H), 0.71 (t, J = 6.3 Hz, 4H). 13 C NMR(75 MHz, DMSO) δ 171.35, 167.72, 165.66, 156.62, 152.55, 150.67, 149.19,148.22, 143.60, 136.77 (2C), 129.08, 128.57 (2C), 128.37 (2C), 127.61, 117.71 (2C), 47.04, 45.89 (2C), 26.81, 10.74, 8.93 (2C), 7.30. Experimental Example 1 This experiment used the ADP-Glo ​​kinase assay to determine the in vitro inhibitory effect of the target compound on the activity of human CDK4 and CDK9 kinases. The specific experimental steps are as follows: (1) Preparation of working solution Using kinase reaction buffer (composed of 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol (DTT), 0.01% bovine serum albumin (BSA), and 2% dimethyl sulfoxide (DMSO)) as solvent, the following solutions were prepared: 2×ATP / substrate working solution: Contains a specific substrate with a final concentration of 2 times ATP and its corresponding kinase; 2× kinase working solution: Contains human CDK4 / cyclin D1 fusion protein or CDK9 / cyclin T1 fusion protein at a final concentration of 2 times.

[0084] (2) Compound sample addition Using an Echo 655 ultrasonic pipetting system, 100 nL of DMSO solutions with a series of concentration gradients were precisely transferred into the corresponding wells of a 384-well white opaque detection plate. After sample addition, the detection plate was centrifuged at 1000 rpm for 1 min to allow the liquid to collect at the bottom of the wells.

[0085] (3) Kinase pre-incubation Add 5 μL of 2× kinase working solution to each well, centrifuge to mix (1000 rpm, 1 min), and then incubate the detection plate at 25°C for 10 min to allow the kinase to fully bind with the compound.

[0086] (4) Kinase response initiation and incubation Add 5 μL of 2×ATP / substrate working solution to each well to initiate the kinase reaction. After centrifugation and remixing, incubate at 25°C for 60 min. The final concentrations of kinase, ATP, and substrate in the reaction system are all 1×, and the final concentrations of compounds are determined according to a pre-set gradient.

[0087] (5) Termination of reaction and consumption of remaining ATP After the kinase reaction was completed, 5 μL of ADP-Glo ​​reagent was added to each well, centrifuged and mixed, and then incubated at 25°C for 40 min to terminate the kinase reaction and specifically consume the unused ATP in the reaction system.

[0088] (6) Generation and detection of light emission signals Add 10 μL of kinase assay reagent to each well, centrifuge to mix, and incubate at 25°C for 40 min. This reagent can convert ADP generated by the kinase reaction back into ATP, and generate a chemiluminescent signal proportional to the ATP concentration through the luciferase-luciferin system. The relative luminescence intensity (RLI) of each well was detected using a BMG multi-microplate reader.

[0089] (7) Calculation of inhibition rate The following control group was set up for the experiment: Blank control group: Contains only reaction buffer, without kinase, used to measure background signal; Negative control group: Contains kinase, ATP and substrate, but no inhibitors, representing the maximum activity of the kinase; Positive control group: Contains kinase, ATP, substrate and a known CDK4 / CDK9 inhibitor, used to verify the effectiveness and sensitivity of the experimental system.

[0090] The inhibition rate of the compound on CDK4 or CDK9 kinase activity at each test concentration was calculated using the following formula: Inhibition rate (%) = [1 - (sample well RLI - blank control RLI) / (negative control RLI - blank control RLI)] × 100%.

[0091] (8) Record the test results in Table 1. In Table 1, ND is an abbreviation for Not Detected, which means not detected.

[0092] Table 1 Test Results

[0093] Therefore, by employing the aforementioned dual-target inhibitor, this invention can simultaneously and efficiently inhibit CDK4 and CDK9, two different targets belonging to key nodes of cell cycle regulation and transcriptional regulation, respectively.

[0094] Finally, it should be noted that the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A dual-target inhibitor of CDK4 and CDK9, characterized in that, The inhibitor is selected from compounds having the structure shown in formula (I), formula (II) or formula (III), or pharmaceutically acceptable salts, solvates, hydrates or prodrugs of said compounds; (I), (II) (III); R1, R4, and R7 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, ethyl, cyano, trifluoromethyl, difluoromethyl, fluoromethyl, vinyl, ethynyl, methoxy, hydroxy, formyl, tert-butyl, isopropyl, nitro, or amino. R2, R3, R5, R6, R8, and R9 are each independently selected from hydrogen, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, propynyl, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, 3-methylpentyl, 1,1,1-trifluoroethyl, 1,1,1-trifluoropropyl, 1,1,1-trifluorobutyl, 1,1,1-trifluoropentyl, 1,1,1-trifluorohexyl, methylcyclopentadiene, 2-furanmethyl, 2-pyrrolemethyl, or 2-thiophenmethyl; X and Y are each independently selected from nitrogen atoms, carbon atoms, or carbon atoms with fluorine atoms, chlorine atoms, bromine atoms, iodine atoms, methyl, methoxy, trifluoromethyl, difluoromethyl, fluoromethyl, formyl, nitro, ethyl, isopropyl, tert-butyl, or amino atoms attached to them. L1 is selected from , , , , , or ; L2 is selected from , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

2. A pharmaceutical composition, characterized in that, It comprises the CDK4 and CDK9 dual-target inhibitor as described in claim 1, and one or more pharmaceutically acceptable carriers or excipients.

3. The use of the CDK4 and CDK9 dual-target inhibitor of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for inhibiting CDK4 and / or CDK9 activity.

4. The use of the CDK4 and CDK9 dual-target inhibitor of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for treating or preventing diseases associated with abnormal CDK4 and / or CDK9 activity.

5. The application according to claim 4, characterized in that, Diseases include breast cancer, lung cancer, colorectal cancer, leukemia, or lymphoma.

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