Protein degradation agent targeting KDM5 and application thereof

Abstract

The invention discloses a KDM5-targeted protein degradation agent and application thereof, and belongs to the technical field of medicinal chemistry. The chemical structural formula of the protein degradation agent is shown in the specification; in the formula, R is an E3 ligase ligand. The invention provides a KDM5-targeted protein degradation agent with a novel structure, the protein degradation agent can effectively target KDM5, has excellent degradation activity on KDM5A and KDM5B, is a high-efficiency and potential dual-targeted degradation agent, solves the problems of low degradation efficiency and limited clinical application of the existing KDM5-targeted degradation agent, and provides a novel KDM5-targeted protein degradation agent. The compound has an excellent treatment effect on triple-negative breast cancer and acute liver injury, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to a protein degrader targeting KDM5 and its application. Background Technology

[0002] Histone lysine-specific demethylase 5 (KDM5) utilizes the cofactors α-ketoglutarate and Fe... 2+ KDM5 exhibits a mechanism-dependent ability to demethylate and trimethylate methyl groups at H3K4 sites, thereby regulating gene expression and transcriptional activity. In addition to its classic histone demethylase activity, KDM5 also possesses non-catalytic functions independent of enzyme activity, which play crucial roles in the occurrence and development of various diseases. Therefore, simultaneously targeting both the enzymatic and non-enzymatic functions of KDM5 may be a superior strategy for its effective development as a drug target. Targeted protein degraders are heterobifunctional molecules that recruit disease-related proteins to E3 ubiquitin ligases, utilizing the ubiquitin-proteasome mechanism to degrade target proteins, achieving dual regulation of protease and non-enzymatic functions. While KDM5-targeted degraders have been reported, their degradation efficiency is low due to the use of different KDM5 ligands or E3 ligase ligands, limiting their clinical application. Given the importance of KDM5 in disease treatment, there is an urgent need to develop safe, efficient, and structurally novel KDM5 degraders to provide a material basis for innovative drug research targeting KDM5. Summary of the Invention

[0003] To address the aforementioned technical problems, the present invention aims to provide a protein degrader targeting KDM5 and its application, thereby solving the problems of low degradation efficiency and limited clinical application of existing KDM5-targeting degraders.

[0004] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides a protein degrader targeting KDM5, having a chemical structure as shown in Formula I or Formula II: Formula I: Formula II: ; In Formula I, L is selected from , , , , , , , , , , , , and Any one of them; In Formula I, R is the E3 ligase ligand.

[0005] Furthermore, the E3 ligase ligand is selected from... , , , , , , , and Any one of them.

[0006] Furthermore, the protein degraders targeting KDM5 are selected from the following compounds: , , , , , , , , , , , , , , , , , , , , and Any one of them.

[0007] In a second aspect, the invention provides the use of the described KDM5-targeting protein degrader in the preparation of a medicament for treating triple-negative breast cancer and / or acute liver injury.

[0008] A third aspect of the present invention provides a pharmaceutical composition comprising at least one of the above-described protein degrader targeting KDM5 or a pharmaceutically acceptable salt, isomer, solvate, crystal, and prodrug thereof.

[0009] Furthermore, it also includes pharmaceutically acceptable excipients and / or pharmaceutically acceptable carriers.

[0010] A fourth aspect of the invention provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating triple-negative breast cancer and / or acute liver injury.

[0011] The present invention has the following beneficial effects: 1. This invention provides a novel type of protein degrader targeting KDM5. This protein degrader can effectively target KDM5 and has excellent degradation activity against both KDM5A and KDM5B. It is a highly efficient and promising dual-targeting degrader.

[0012] 2. The protein degrader targeting KDM5 provided by this invention has good biocompatibility and is non-toxic in vivo. It exhibits good anti-triple-negative breast cancer activity in vivo, inhibits lung metastasis of cancer cells and Ki67 expression in tumor tissue, alleviates tumor-induced splenomegaly, and inhibits the migration and drug resistance of paclitaxel-resistant triple-negative breast cancer.

[0013] 3. The protein degrader targeting KDM5 provided by this invention can effectively reduce the expression levels of alanine aminotransferase and aspartate aminotransferase in cells during acute liver injury, alleviate liver tissue necrosis and reduce the damaged area in liver tissue, improve the survival rate of mice with LPS / D-Gal-induced hepatocellular injury and improve histopathology. The effects are superior to dexamethasone, a positive drug for the treatment of acute liver injury, and the therapeutic effect on acute liver injury is excellent. Attached Figure Description

[0014] Figure 1 The results of screening for KDM5A and KDM5B targeted degradative agents are shown. A and B are the intracellular degradation activity analysis results of the protein degradative agents targeting KDM5 prepared in Examples 1-22 of this invention, and C and D are the concentration gradient degradation activity analysis results of KD-14, KD-19 and KD-22, respectively. Figure 2 The optimal degradation time and half-maximal concentration (MCC) of KD-22 are shown in Figure 1. Here, A represents the optimal degradation time of KD-22 on KDM5A / KDM5B, B represents the concentration gradient degradation result of KD-22, and C represents the half-maximal concentration (MCC). 50 value; Figure 3 The effects of KD-22 on tumors and tissues in a PTX-resistant 4T1 mouse model are shown in the graph. In the graph, A represents tumor volume, B represents mouse body weight, and CI represents changes in the weight of each tissue. Figure 4 The effect of KD-22 on cancer cells and cancer tissues in a PTX-resistant 4T1 mouse model is shown in Figure A, where A represents the histopathological results of cancer cell tissues and B represents the results of Ki67 expression detection in tumor tissues. Figure 5The effects of KD-22 on LPS / D-GalN-induced stem cell damage are shown in the following figures: A represents the effect of KD-22 on the degree effect of AML12 cells; B represents the effect of KD-22 on the cytotoxic effect of LX-2 cells; CE represents the effect of KD-22 on AML12 cytokine secretion; FH represents the effect of KD-22 on LX-2 cytokine secretion; I and J represent the effects of KD-22 on AST and ALT activities in AML12 cells; and K and L represent the effects of KD-22 on AST and ALT activities in LX-2 cells. Figure 6 The effects of KD-22 on LPS / D-GalN-induced acute liver injury in mice are shown in Figure 1. A represents the effect of KD-22 on the survival rate of mice with LPS / D-GalN-induced acute liver injury, B represents the effect of KD-22 on the morphology of the mouse liver, C represents the results of eosin methylene blue staining, and D represents the statistical results of the liver injury area. Detailed Implementation

[0015] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0016] Example 1: A protein degrader compound KD-1 targeting KDM5 has the following structural formula: ; The synthesis route is shown below:

[0017] The preparation method specifically includes the following steps: Synthesis of S1 and Compound 3 Compound 1 (500 mg, 1.0 eq) and 7-aminoheptanoate (265 mg, 1.0 eq) were added to a reaction flask, followed by the addition of acetonitrile and triethylamine (810 μL, 4.0 eq). The reaction was carried out at 60 °C for 6 h. After the reaction was complete, the mixture was filtered and washed with acetonitrile to give 3419 mg of the compound as a white solid, with a yield of 73%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.57 (s, 1H), 6.39 (d, J = 10.7 Hz, 1H), 6.12 (d, J = 6.7 Hz, 1H), 5.73 (q, J= 5.5 Hz, 1H), 4.16 – 4.05(m, 1H), 3.97 – 3.84 (m, 1H), 3.66 – 3.46 (m, 3H), 3.02 – 2.87 (m, 3H), 2.27– 2.13 (m, 2H), 2.11 – 1.96 (m, 1H), 1.84 – 1.66 (m, 1H), 1.54 – 1.41 (m, 2H), 1.39 – 1.30 (m, 2H), 1.22 (d, J = 7.0 Hz, 10H). Synthesis of S2 and KD-1 Compound 3 (400 mg, 1.0 eq), HATU (428 mg, 1.1 eq), DIPEA (710 μl, 4.0 eq), and DMF (6 mL) were added to a reaction flask and stirred at room temperature for 30 min. Then, 3-amino-2,6-piperidinidone hydrochloride 4 (166 mg, 1.0 eq) was added, and the reaction was allowed to proceed overnight. The mixture was extracted with water / ethyl acetate, and the organic layers were combined, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by column chromatography to give 153 mg of compound KD-1 as a white solid, with a yield of approximately 30%. 1 H NMR (400 MHz, DMSO- d 6)δ 12.91 (s, 1H), 10.78 (s, 1H), 8.15 (d, J = 8.3 Hz, 1H), 6.38 (s, 1H), 6.13(d, J = 6.3 Hz, 1H), 5.77 – 5.70 (m, 2H), 4.59 – 4.47 (m, 1H), 4.09 (s, 1H), 3.96 – 3.87 (m, 1H), 3.66 – 3.47 (m, 3H), 3.01 – 2.90 (m, 4H), 2.75 – 2.66(m, 1H), 2.14 – 2.08 (m, 2H), 2.05 – 1.97 (m, 1H), 1.93 – 1.86 (m, 2H), 1.80 – 1.68 (m, 1H), 1.52 – 1.47 (m, 2H), 1.40 – 1.28 (m, 4H), 1.22 (d, J = 7.1 Hz, 8H). Example 2: A protein degrader compound, KD-2, targeting KDM5, has the following structural formula: ; The synthesis method of compound KD-2 in this embodiment is the same as in Example 1, except that the E3 enzyme ligand in this embodiment is... The compound KD-2 obtained in this example is a white solid with a yield of 15%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.90 (s, 1H), 11.19 (s, 1H), 8.42 (d, J = 7.5 Hz, 1H), 6.38 (s, 1H), 6.12 (d, J = 6.8 Hz, 1H), 5.76 – 5.69 (m, 1H), 4.42 – 4.31 (m, 1H), 4.13 –4.06 (m, 1H), 3.98 – 3.87 (m, 1H), 3.73 – 3.41 (m, 3H), 3.00 – 2.92 (m, 3H),2.90 – 2.81 (m, 1H), 2.49 – 2.42 (m, 1H), 2.10 – 2.05 (m, 2H), 2.05 – 1.94(m, 1H), 1.82 – 1.67 (m, 1H), 1.50 – 1.43 (m, 2H), 1.36 – 1.30 (m, 2H), 1.22(d, J = 6.9 Hz, 11H). Example 3: A protein degrader compound KD-3 targeting KDM5 has the following structural formula: ; The synthesis method of compound KD-3 in this embodiment is the same as in Example 1, except that the E3 enzyme ligand in this embodiment is... The compound KD-3 obtained in this example is a white solid with a yield of 12%. 1 H NMR (400 MHz, DMSO- d 6) δ 13.01 (s, 1H), 8.66 (t, J= 7.4 Hz, 1H), 8.15 – 8.05 (m, 1H), 7.26 – 7.21 (m, 4H), 7.20 – 7.15 (m, 1H), 6.42 – 6.34 (m, 2H), 5.96 – 5.88 (m,1H), 4.53 – 4.40 (m, 2H), 4.12 – 4.05 (m, 1H), 3.97 – 3.87 (m, 1H), 3.65 –3.47 (m, 3H), 3.01 – 2.88 (m,4H), 2.83 – 2.71 (m, 2H), 2.43 – 2.28 (m, 1H),2.07 – 1.98 (m, 3H), 1.83 – 1.68 (m, 1H), 1.35 – 1.26 (m, 4H), 1.22 (d, J =6.9 Hz, 6H), 1.17 – 1.11 (m, 2H), 1.09 – 1.01 (m, 2H). Example 4: A protein degrader compound, KD-4, targeting KDM5, has the following structural formula: ; The synthesis method of compound KD-4 in this embodiment is the same as in Example 1, the difference being that the E3 enzyme ligand in this embodiment is... The compound KD-4 obtained in this example is a white solid with a yield of 14%. 1 H NMR (400 MHz, DMSO- d6) δ 12.91 (s, 1H), 10.83 (s, 1H), 8.43 – 8.31 (m, 1H), 8.10 – 7.98 (m, 1H), 7.29 – 7.22 (m, 4H), 7.19 – 7.14 (m, 1H), 6.44 – 6.35 (m, 1H), 6.13(d, J = 6.7 Hz, 1H), 5.77 – 5.66 (m, 1H), 4.62 – 4.50 (m, 2H), 4.13 – 4.06(m, 1H), 3.97 – 3.86 (m, 1H), 3.72 – 3.44 (m, 3H), 3.00 – 2.87 (m, 4H), 2.81– 2.69 (m, 2H), 2.14 – 1.96 (m, 4H), 1.96 – 1.88 (m, 1H), 1.84 – 1.68 (m,2H), 1.36 – 1.27 (m, 4H), 1.22 (d, J = 7.0 Hz, 6H), 1.16 – 1.03 (m, 4H). Example 5: A protein degrader compound, KD-5, targeting KDM5, has the following structural formula: ; The synthesis method of compound KD-5 in this embodiment is the same as in Example 1, the difference being that the E3 enzyme ligand in this embodiment is... The compound KD-5 obtained in this example is a gray solid with a yield of 32%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.90 (s, 1H), 10.86 (s, 1H), 10.43 (s, 1H), 8.94 (d, J = 8.4 Hz,1H), 8.85 – 8.83 (m, 1H), 8.23 ​​(dd, J = 8.7, 2.4 Hz, 1H), 8.02 (d, J = 8.5 Hz,1H), 6.38 (s, 1H), 6.14 (d, J= 6.7 Hz, 1H), 5.80 – 5.70 (m, 1H), 4.84 – 4.72(m, 1H), 4.14 – 4.06 (m, 1H), 3.98 – 3.89 (m, 1H), 3.69 – 3.45 (m, 3H), 3.02– 2.91 (m, 3H), 2.86 – 2.73 (m, 1H), 2.59 – 2.53 (m, 1H), 2.41 – 2.34 (m, 2H)2.27 – 2.15 (m, 1H), 2.07 – 1.96 (m, 2H), 1.83 – 1.68 (m, 1H), 1.61 (d, J =7.4 Hz, 2H), 1.39 – 1.27 (m, 6H), 1.22 (d, J = 6.9 Hz, 6H). Example 6: A protein degrader compound KD-6 targeting KDM5 has the following structural formula: ; The synthesis route is shown below:

[0018] The preparation method specifically includes the following steps: Compound 1 (500 mg, 1.0 eq) and compound 5 (402 mg, 1.0 eq) were added to a reaction flask, followed by the addition of acetonitrile and triethylamine (812 μL, 4.0 eq). The reaction was carried out at 60 °C for 6 h. After the reaction was completed, compound KD-6 was purified by column chromatography to obtain 148 mg of compound KD-6 as a pale yellow solid, with a yield of 19%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.91 (s, 1H), 8.19 (d, J =6.6 Hz, 1H), 6.90 – 6.74 (m, 1H), 6.39 (s, 1H), 6.24 – 6.13 (m, 2H), 6.08(dd, J = 14.7, 3.2 Hz, 1H), 5.76 (d, J= 10.3 Hz, 1H), 4.43 – 4.24 (m, 2H), 3.99 – 3.84 (m, 3H), 3.81 – 3.71 (m, 2H), 3.66 – 3.48 (m, 2H), 3.01 – 2.91(m, 1H), 2.79 (q, J = 8.1 Hz, 2H), 2.39 (q, J = 7.2 Hz, 2H), 2.12 – 1.96 (m,1H), 1.86 – 1.71 (m, 1H), 1.30 – 1.26 (m, 2H), 1.22 (d, J = 7.0 Hz, 6H). 13 CNMR (151 MHz, DMSO- d 6) δ 174.04, 173.33, 172.39, 167.87, 157.39, 136.47,130.54, 128.55, 128.40, 103.79, 55.95, 50.41, 49.47, 48.01, 46.23, 44.93,38.95, 31.36, 30.34, 29.88, 28.30, 24.70, 22.76. Example 7: A protein degrader compound KD-7 that targets KDM5 has the following structural formula: ; The synthesis route is shown below:

[0019] The preparation method specifically includes the following steps: Synthesis of S1 and intermediate 7 Compound 1 (1 g, 2.92 mmol), N -Boc-1,6-hexanediamine 6 (631 mg, 2.92 mmol), acetonitrile (10 mL), and triethylamine (1.22 mL, 8.76 mmol) were added to a reaction flask and reacted at 60 °C for 7 h. After the reaction was complete, the mixture was filtered, and the upper solid was washed with acetonitrile. The upper solid was intermediate 7, a white solid. 1 H NMR (400 MHz, DMSO- d 6) δ 12.90 (d, J= 9.3 Hz, 1H), 6.81 – 6.68 (m, 1H), 6.37 (s, 1H), 6.18 – 6.00 (m, 1H), 5.71(d, J = 6.3 Hz, 1H), 4.09 (s, 1H), 3.99 – 3.83 (m, 1H), 3.69 – 3.45 (m, 2H), 2.99 – 2.92 (m, 3H), 2.91 – 2.84 (m, 2H), 2.09 – 1.93 (m, 1H), 1.84 – 1.65 (m, 1H), 1.37 (s, 9H), 1.35 – 1.29 (m, 4H), 1.22 (d, J = 7.0 Hz, 11H). Synthesis of S2 and intermediate 8 Intermediate 7 (300 mg, 0.65 mmol), dichloromethane (4 mL), and trifluoroacetic acid (1 mL) were added to a reaction flask and reacted at room temperature for 2 h. After the reaction was completed, the solvent was evaporated to obtain the trifluoroacetate of intermediate 8.

[0020] Synthesis of S3 and KD-7 Trifluoroacetate of intermediate 8, HATU (272 mg, 0.71 mmol), DIPEA (0.34 mL, 3.05 mmol), compound 9 (173 mg, 0.65 mmol), and DMF (5 mL) were added to a reaction flask. The mixture was reacted overnight at room temperature, extracted with ethyl acetate, and the organic layers were combined, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain compound KD-7. Compound KD-7 was a light brown solid with a yield of 11%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.90 (s, 1H), 10.53 (s, 1H), 8.57 (s, 1H), 7.94 (s, 1H), 7.84 (d, J= 8.4 Hz, 1H), 7.67 (d, J = 8.4Hz, 1H), 6.38 (s, 1H), 6.12 (d, J = 6.7 Hz, 1H), 5.79 – 5.69 (m, 1H), 4.14 –4.04 (m, 2H), 3.97 – 3.86 (m, 1H), 3.80 – 3.71 (m, 1H), 3.68 – 3.46 (m, 4H), 3.28 – 3.21 (m, 2H), 3.02 – 2.90 (m, 3H), 2.80 – 2.71 (m, 2H), 2.09 – 1.96(m, 1H), 1.82 – 1.66 (m, 1H), 1.55 – 1.47 (m, 2H), 1.40 – 1.32 (m, 3H), 1.29 – 1.24 (m, 5H), 1.22 (d, J = 7.0 Hz, 6H). Example 8: A protein degrader compound KD-8 that targets KDM5 has the following structural formula: ; The synthesis method of compound KD-8 in this embodiment is the same as that in Example 7, the difference being that the E3 enzyme ligand in this embodiment is... The compound KD-8 obtained in this example is a light brown solid with a yield of 32%. 1 H NMR (400 MHz, DMSO- d 6) δ 13.09 – 12.83 (m, 1H), 11.24 (s, 1H), 10.56 (s, 1H), 8.92 – 8.51 (m,1H), 6.38 (s, 1H), 6.12 (d, J = 6.7 Hz, 1H), 6.05 – 5.95 (m, 1H), 5.81 – 5.67(m, 1H), 4.17 – 4.02 (m, 1H), 3.98 – 3.87 (m, 2H), 3.68 – 3.48 (m, 2H), 3.22– 3.15 (m, 2H), 3.03 – 2.91 (m, 3H), 2.12 – 1.94 (m, 1H), 1.86 – 1.64 (m,1H), 1.52 – 1.42 (m, 2H), 1.39 – 1.32 (m, 2H), 1.31 – 1.25 (m, 4H), 1.22 (d,J = 6.9 Hz, 7H). Example 9: A protein degrader compound KD-9 that targets KDM5 has the following structural formula: ; The synthesis route is shown below:

[0021] The preparation method specifically includes the following steps: Synthesis of S1 and Compound 10 Compound 5 (1 g, 1.0 eq), N Boc-piperazine (543 mg, 1.0 eq), triethylamine (1.22 ml, 3.0 eq), and an appropriate amount of acetonitrile solvent were added to a reaction flask, and the reaction was carried out at 60 °C for 6 h. After the reaction was completed, 10,820 mg of the compound was obtained by filtration as a white solid, with a yield of 84%. 1 H NMR (400 MHz, DMSO- d 6) δ 13.07 – 12.79 (m, 1H), 6.57 (d, J = 6.0 Hz, 1H), 6.37 (s, 1H), 4.15 (d, J = 7.1 Hz, 1H), 4.07 – 3.86 (m, 0H), 3.69 – 3.38 (m, 2H), 3.27 (d, J = 5.8 Hz, 7H), 2.97 (d, J = 8.5 Hz, 1H), 2.09 –1.99 (m, 1H), 1.90 – 1.77 (m, 1H), 1.40 (d, J = 1.5 Hz, 8H), 1.22 (d, J = 7.0Hz, 6H). Synthesis of S2 and Compound 11 Add 4 mL of 2 mol / L ethyl hydrochloride solution to compound 11, stir overnight, evaporate the solvent to obtain the hydrochloride salt of compound 11, which can be used directly in the next step without purification.

[0022] Synthesis of S3 and Compound 12 Compound 11 hydrochloride (600 mg, 1.0 eq) NBoc-4-piperidinone (643 mg, 2.0 eq), acetic acid (95 μL, 1.0 eq), DMF (2 mL), and DCE (4 mL) were added to a reaction flask. After reacting at room temperature for 2 h, sodium triacetoxyborohydride (1.38 g, 4.0 eq) was added in portions, and the reaction was allowed to proceed overnight. After the reaction was complete, 412 mg of white compound 12 was obtained by column chromatography, with a yield of approximately 49%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.87 (s, 1H), 6.49 (s, 1H), 6.39 (d, J = 8.2 Hz, 66H), 4.14 (s, 1H), 4.06 – 3.80 (m, 4H), 3.69 – 3.56 (m,2H), 3.26 (s, 4H), 3.01 – 2.90 (m, 1H), 2.68 (s, 2H), 2.44 – 2.37 (m, 4H), 2.37 – 2.31 (m, 1H), 2.10 – 1.97 (m, 1H), 1.87 – 1.77 (m, 1H), 1.70 (d, J =11.3 Hz, 2H), 1.38 (s, 9H), 1.22 (d, J = 6.8 Hz, 8H). Synthesis of S4 and Compound 13 Compound 12 was added to a mixed solvent of DCM (2 mL) and TFA (593 μL, 10 eq), and the reaction was carried out for 2 h. After the reaction was completed, the solution was concentrated under reduced pressure to obtain the trifluoroacetate of compound 13.

[0023] Synthesis of S5 and KD-9 The trifluoroacetate of compound 13, 3-(6-fluoro-1-oxo-1-) H (220 mg, 1 eq) of isoindol-2-yl)piperidin-2,6-dione, DIPEA (554 μL, 4.0 eq), and DMSO (4 mL) were added to a reaction flask and reacted in an oil bath at 90 °C for 2 h. After the reaction was completed, the mixture was extracted with ethyl acetate / water, the organic phases were combined, concentrated under reduced pressure, and the crude product was subjected to column chromatography to give 80 mg of the yellow compound KD-9, with a yield of approximately 15%. 1H NMR (400 MHz, DMSO-d6) δ 12.83 (s, 1H), 11.08 (s, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H), 7.24 (d, J = 8.7 Hz, 1H), 6.51 (d, J= 5.1 Hz, 1H), 6.39 (d, J = 8.3 Hz, 1H), 5.06 (dd, J = 12.9, 5.4 Hz, 1H), 4.17 – 4.12 (m, 1H), 4.10 – 3.99 (m, 2H), 3.96 – 3.77 (m, 2H), 3.69 – 3.56(m, 2H), 3.27 (s, 4H), 3.01 – 2.91 (m, 3H), 2.90 – 2.80 (m, 1H), 2.64 – 2.53(m, 3H), 2.44 (s, 4H), 2.10 – 1.96 (m, 2H), 1.88 – 1.77 (m, 3H), 1.51 – 1.39(m, 2H), 1.22 (d, J = 7.0 Hz, 6H). Example 10: A protein degrader compound KD-10 targeting KDM5 has the following structural formula: ; The synthesis method of compound KD-10 in this embodiment is the same as that in Example 9, except that the Linker (L) in this embodiment is: The compound KD-10 obtained in this example is a yellow solid with a yield of 14%. 1H NMR (400 MHz, DMSO-d6) δ 12.88 (s, 1H), 11.08 (s, 1H), 7.65 (d, J = 8.2 Hz, 1H), 6.78 (s,1H), 6.65 (d, J = 8.4 Hz, 1H), 6.56 (s, 1H), 6.38 (s, 1H), 5.06 (dd, J =12.9, 5.4 Hz, 1H), 4.21 – 4.14 (m, 1H), 4.12 – 4.06 (m, 2H), 4.05 – 3.98 (m,1H), 3.87 (s, 2H), 3.69 – 3.56 (m, 2H), 3.51 – 3.42 (m, 1H), 2.98 – 2.83 (m, 2H), 2.62 – 2.59 (m, 1H), 2.54 (s, 4H), 2.32 (s, 4H), 2.05 – 1.97 (m, 2H), 1.92 – 1.78 (m, 1H), 1.22 (d, J = 7.1 Hz, 8H). Example 11: A protein degrader compound KD-11 that targets KDM5 has the following structural formula: ; The synthesis method of compound KD-11 in this embodiment is the same as that in Example 9, except that the Linker (L) in this embodiment is: The compound KD-11 obtained in this example is a yellow solid with a yield of 11%. 1H NMR (400 MHz, DMSO-d6) δ 12.92 (s, 1H), 11.08 (s, 1H), 7.63 (d, J = 8.3 Hz, 1H), 6.77 (s,1H), 6.64 (d, J = 8.1 Hz, 1H), 6.55 (s, 1H), 6.38 (s, 1H), 5.05 (dd, J =13.0, 5.4 Hz, 1H), 4.13 (t, J = 8.3 Hz, 3H), 4.06 – 3.84 (m, 2H), 3.72 – 3.57(m, 4H), 3.51 – 3.44 (m, 1H), 3.30 (s, 4H), 3.04 – 2.91 (m, 2H), 2.90 – 2.80(m, 1H), 2.61 (s, 2H), 2.56 (s, 1H), 2.55 (s, 1H), 2.35 (s, 3H), 2.05 – 1.98(m, 2H), 1.90 – 1.79 (m, 1H), 1.22 (d, J = 6.9 Hz, 6H). Example 12: A protein degrader compound, KD-12, targeting KDM5, has the following structural formula: ; The synthesis method of compound KD-12 in this embodiment is the same as that in Example 9, except that the Linker (L) in this embodiment is: The compound KD-12 obtained in this example is a yellow solid with a yield of 10%. 1H NMR (600MHz, DMSO-d6) δ 12.90 (s, 1H), 11.07 (s, 1H), 7.63 (d, J = 8.2 Hz, 1H), 6.75(s, 1H), 6.62 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 6.38 (s, 1H), 5.05 (dd, J =13.2, 5.5 Hz, 1H), 4.14 (s, 1H), 4.06 (s, 2H), 4.05 – 4.00 (m, 1H), 3.94 (s, 2H), 3.67 – 3.58 (m, 2H), 3.49 – 3.43 (m, 1H), 3.28 (s, 4H), 2.96 (s, 1H), 2.91 – 2.84 (m, 1H), 2.63 – 2.58 (m, 2H), 2.56 (s, 1H), 2.34 (s, 2H), 2.19 (s, 4H), 2.06 – 2.02 (m, 2H), 2.01 – 1.98 (m, 2H), 1.90 – 1.80 (m, 1H), 1.22(d, J = 7.1 Hz, 6H). Example 13: A protein degrader compound, KD-13, targeting KDM5, has the following structural formula: ; The synthesis route is shown below:

[0024] The preparation method specifically includes the following steps: Synthesis of S1 and intermediate 15 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione 14 (800 mg, 1.0 eq), N Boc-piperazine (593 mg, 1.1 eq), DIPEA (1 mL, 2.0 eq), and DMF (8 mL) were added to a reaction flask and reacted at 80 °C for 4 h. After the reaction was complete, the mixture was extracted with ethyl acetate, the organic layers were combined, dried over anhydrous magnesium sulfate, filtered, evaporated under reduced pressure, and purified by column chromatography to obtain 589 mg of yellow intermediate 15, with a yield of 46%. 1 H NMR (400 MHz, Chloroform- d ) δ 8.40 (s, 1H), 7.50 (t, J= 7.8 Hz, 1H), 7.11 (d, J = 7.1 Hz, 1H), 6.91 (d, J = 8.6 Hz,1H), 6.52 (s, 1H), 5.05 (s, 1H), 4.93 (s, 1H), 3.73 (t, J = 5.3 Hz, 2H), 3.65(s, 4H), 3.57 (t, J = 5.3 Hz, 2H), 3.52 – 3.45 (m, 2H), 3.32 (d, J = 5.8 Hz, 2H), 2.91 – 2.68 (m, 3H), 2.17 – 2.09 (m, 1H), 1.65 (s, 2H), 1.43 (s, 9H). Synthesis of S2 and intermediate 16 Intermediate 15, dichloromethane (4 mL), and trifluoroacetic acid (1 mL, 10 eq) were added to a reaction flask and reacted at room temperature for 3 h. After the reaction was completed, the solvent was evaporated to obtain the trifluoroacetate of intermediate 16, which was used directly in the next reaction without purification.

[0025] Synthesis of S3 and intermediate 17 Trifluoroacetate of intermediate 16, 1-tert-butoxycarbonylpiperidine-4-carboxaldehyde (425 mg, 1.5 eq), triethylamine (554 μL, 3.0 eq), and dichloromethane were added to a reaction flask and reacted at room temperature for 6 h. After the intermediate was formed, NaBH(OAc)3 (423 mg, 1.5 eq) was slowly added in portions, followed by a catalytic amount of glacial acetic acid, and the reaction was carried out at room temperature for 6 h. After the reaction was completed, the mixture was extracted with dichloromethane, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain 330 mg of pale yellow intermediate 17, with a yield of approximately 46%.

[0026] Synthesis of S4 and intermediate 18 Intermediate 17, dichloromethane (2 mL), and trifluoroacetic acid (455 μL, 10 eq) were added to a reaction flask and reacted at room temperature for 3 h. After the reaction was completed, the solvent was evaporated to obtain the trifluoroacetate of intermediate 18.

[0027] Synthesis of S5 and KD-13 Acetonitrile was added to dissolve the trifluoroacetate of intermediate 18, and triethylamine (339 μL, 4.0 eq) was added dropwise until no smoke was produced. Compound 5 (230 mg, 1.1 eq) was then added, and the reaction was carried out at 60 °C for 6 h. After the reaction was completed, the mixture was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain 201 mg of yellow solid compound KD-13, with a yield of approximately 48%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.89 (s, 1H), 11.06 (s, 1H), 7.70(t, J = 7.8 Hz, 1H), 7.40 – 7.28 (m, 2H), 6.48 – 6.34 (m, 2H), 5.13 – 5.02 (m,1H), 4.15 (s, 1H), 3.95 (d, J = 10.5 Hz, 3H), 3.71 – 3.56 (m, 2H), 3.51 – 3.40(m, 1H), 3.37 (d, J = 5.2 Hz, 1H), 3.29 (s, 4H), 3.00 – 2.91 (m, 1H), 2.90 –2.81 (m, 1H), 2.72 – 2.61 (m, 3H), 2.60 – 2.52 (m, 4H), 2.18 (t, J = 5.9 Hz,2H), 2.05 – 1.96 (m, 2H), 1.92 – 1.77 (m, 1H), 1.67 (d, J = 13.0 Hz, 3H), 1.22(d, J = 6.9 Hz, 6H), 0.95 (d, J = 9.1 Hz, 2H). Example 14: A protein degrader compound, KD-14, targeting KDM5, has the following structural formula: ; The synthesis route is shown below:

[0028] The preparation method specifically includes the following steps: Synthesis of S1 and intermediate 19 Commercially available 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione 14 (600 mg, 2.17 mmol), N-Boc-hexanediamine 22 (517 mg, 2.39 mmol), DIPEA (0.76 mL, 4.34 mmol), and DMF (6 mL) were added to a 25 mL reaction flask, and the mixture was reacted at 80 °C for 4 h. After the reaction was completed, the mixture was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and the crude product was separated by column chromatography to obtain intermediate 19.

[0029] Synthesis of S2 and intermediate 20 Intermediate 19 (300 mg, 0.64 mmol), trifluoroacetic acid (1 mL), and dichloromethane (5 mL) were added to a reaction flask and reacted at room temperature for 3 h. After the reaction was completed, the solvent was evaporated to obtain the trifluoroacetate of intermediate 20.

[0030] Synthesis of S3 and KD-14 To dissolve the trifluoroacetate of intermediate 20, 5 mL of acetonitrile was added. Triethylamine (0.27 mL, 1.90 mmol) was added dropwise until no smoke was produced. Then, compound 1 (217 mg, 0.64 mmol) was added, and the reaction was carried out at 60 °C for 6 hours. After the reaction was completed, the mixture was extracted three times with ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain a yellow solid compound KD-14, with a yield of 31%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.90 (d, J = 9.8 Hz, 1H), 11.09 (s, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.13 – 7.06 (m, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.52 (d, J = 7.5 Hz, 1H), 6.41 – 6.35 (m, 1H), 6.10(d, J = 6.6 Hz, 1H), 5.72 (d, J = 7.5 Hz, 1H), 5.11 – 5.00 (m, 1H), 4.08 (s,1H), 4.00 – 3.87 (m, 1H), 3.68 – 3.46 (m, 2H), 3.28 (d, J= 6.3 Hz, 3H), 3.01– 2.92 (m, 3H), 2.90 – 2.83 (m, 1H), 2.63 – 2.57 (m, 2H), 2.11 – 1.96 (m,2H), 1.85 – 1.68 (m, 1H), 1.62 – 1.52 (m, 2H), 1.41 – 1.27 (m, 6H), 1.22 (d, J = 6.9 Hz, 6H). Example 15: A protein degrader compound KD-15 that targets KDM5 has the following structural formula: ; The synthesis method of compound KD-15 in this embodiment is the same as that in Example 12, except that the Linker (L) in this embodiment is: The compound KD-15 obtained in this example is a yellow solid with a yield of 38%. 1 H NMR (400MHz, DMSO- d 6) δ 12.90 (s, 1H), 11.04 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.20– 7.04 (m, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.87 – 6.79 (m, 1H), 6.38 (s, 1H), 6.14 (d, J = 6.7 Hz, 1H), 5.77 (q, J = 6.1 Hz, 1H), 5.08 – 4.88 (m, 1H), 4.15 –4.04 (m, 1H), 3.91 (d, J = 28.2 Hz, 1H), 3.66 – 3.47 (m, 2H), 3.30 (s, 1H), 3.14 (d, J = 5.3 Hz, 2H), 3.12 – 3.05 (m, 3H), 3.02 – 2.92 (m, 3H), 2.90 –2.83 (m, 1H), 2.61 – 2.54 (m, 1H), 2.13 – 1.94 (m, 2H), 1.82 – 1.67 (m, 1H),1.61 – 1.43 (m, 1H), 1.36 (t, J= 7.1 Hz, 3H), 1.32 – 1.25 (m, 2H), 1.22 (d, J = 7.0 Hz, 6H). Example 16: A protein degrader compound, KD-16, targeting KDM5, has the following structural formula: ; The synthesis method of compound KD-16 in this embodiment is the same as that in Example 12, except that the Linker (L) in this embodiment is: The compound KD-16 obtained in this example is a yellow solid with a yield of 36%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.84 (s, 1H), 11.02 (s, 1H), 7.58 – 7.42 (m, 1H), 7.11 (t, J =8.2 Hz, 1H), 7.00 – 6.88 (m, 1H), 6.63 (d, J = 5.6 Hz, 1H), 6.38 – 6.23 (m,2H), 6.04 – 5.82 (m, 1H), 5.07 – 4.86 (m, 1H), 4.16 – 3.99 (m, 2H), 3.86 (d, J = 41.7 Hz, 1H), 3.59 – 3.53 (m, 1H), 3.50 – 3.40 (m, 1H), 3.18 – 3.09 (m,3H), 2.94 – 2.76 (m, 3H), 2.52 (d, J = 17.5 Hz, 2H), 2.05 – 1.89 (m, 2H), 1.70(d, J = 32.8 Hz, 1H), 1.15 (d, J = 6.9 Hz, 6H). Example 17: A protein degrader compound, KD-17, targeting KDM5, has the following structural formula: ; The synthesis method of compound KD-17 in this embodiment is the same as that in Example 12, except that the Linker (L) in this embodiment is: The compound KD-17 obtained in this example is a yellow solid with a yield of 36%. 1H NMR (400MHz, DMSO- d 6) δ 12.96 (s, 1H), 11.07 (s, 1H), 7.64 – 7.54 (m, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 7.0 Hz, 1H), 6.51 (t, J = 5.7 Hz, 1H), 6.37 (s,1H), 6.31 (s, 1H), 5.88 (d, J = 6.1 Hz, 1H), 5.11 – 4.99 (m, 1H), 4.15 – 4.04(m, 1H), 3.92 (d, J = 24.5 Hz, 1H), 3.65 – 3.44 (m, 2H), 3.29 (d, J = 6.9 Hz,2H), 3.01 – 2.91 (m, 3H), 2.91 – 2.83 (m, 1H), 2.63 – 2.56 (m, 1H), 2.08 –1.95 (m, 2H), 1.84 – 1.66 (m, 1H), 1.57 (s, 2H), 1.39 – 1.31 (m, 4H), 1.28(d, J = 15.9 Hz, 6H), 1.24 (s, 2H), 1.22 (d, J = 6.9 Hz, 6H). Example 18: A protein degrader compound KD-18 that targets KDM5 has the following structural formula: ; The synthesis method of compound KD-18 in this embodiment is the same as that in Example 12, except that the Linker (L) in this embodiment is: The compound KD-18 obtained in this example is a yellow solid with a yield of 32%. 1 H NMR (400MHz, DMSO- d 6) δ 12.89 (s, 1H), 11.09 (s, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.14(d, J = 8.6 Hz, 1H), 7.04 (d, J= 7.0 Hz, 1H), 6.60 (d, J = 6.5 Hz, 1H), 6.37(s, 1H), 6.29 (d, J = 6.6 Hz, 1H), 5.83 – 5.67 (m, 1H), 5.14 – 4.95 (m, 1H), 4.09 (s, 1H), 3.91 (d, J = 23.7 Hz, 1H), 3.69 – 3.58 (m, 3H), 3.56 (t, J = 5.0Hz, 2H), 3.54 – 3.48 (m, 3H), 3.48 – 3.42 (m, 2H), 3.41 – 3.35 (m, 3H), 3.18– 3.09 (m, 2H), 3.01 – 2.82 (m, 2H), 2.66 – 2.51 (m, 2H), 2.04 (s, 2H), 1.84– 1.65 (m, 1H), 1.22 (d, J = 7.0 Hz, 6H). Example 19: A protein degrader compound, KD-19, targeting KDM5, has the following structural formula: ; The synthesis method of compound KD-19 in this embodiment is the same as that in Example 12, except that the Linker (L) in this embodiment is: The compound KD-19 obtained in this example is a yellow solid with a yield of 32%. 1 H NMR (400 MHz, DMSO- d 6) δ 12.89 (d, J = 11.5 Hz, 1H), 11.09 (s, 1H), 7.71 (t, J = 7.8 Hz, 1H),7.41 – 7.32 (m, 2H), 6.63 (d, J = 5.9 Hz, 1H), 6.38 (s, 1H), 5.21 – 4.94 (m,1H), 4.18 (s, 1H), 4.09 – 3.87 (m, 2H), 3.73 – 3.57 (m, 2H), 3.56 – 3.46 (m,4H), 3.25 (d, J= 5.3 Hz, 4H), 3.02 – 2.81 (m, 2H), 2.64 – 2.51 (m, 2H), 2.04(d, J = 5.4 Hz, 2H), 1.97 – 1.78 (m, 1H), 1.22 (d, J = 6.9 Hz, 6H). Example 20: A protein degrader compound KD-20 targeting KDM5 has the following structural formula: ; The synthesis method of compound KD-20 in this embodiment is the same as that in Example 12, except that the Linker (L) in this embodiment is: The compound KD-20 obtained in this example is a yellow solid with a yield of 19%. 1 H NMR (600 MHz, DMSO-d6) δ 12.91 (d, J = 12.7 Hz, 1H), 11.05 (s, 1H), 7.69 (q, J = 5.8 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.28 (dd, J = 11.5, 7.9 Hz, 2H), 7.21 (dd, J =14.6, 7.9 Hz, 2H), 6.94 (s, 1H), 6.90 – 6.85 (m, 1H), 6.37 (s, 1H), 6.29 (s,1H), 6.25 – 6.21 (m, 1H), 5.01 (dd, J = 12.9, 5.4 Hz, 1H), 4.41 (t, J = 5.6Hz, 2H), 4.20 – 4.15 (m, 2H), 4.14 – 4.09 (m, 1H), 3.99 – 3.89 (m, 1H), 3.71 – 3.59 (m, 1H), 3.56 – 3.47 (m, 1H), 3.33 – 3.26 (m, 1H), 2.99 – 2.92 (m,1H), 2.90 – 2.83 (m, 1H), 2.59 – 2.54 (m, 1H), 2.53 – 2.45 (m, 1H), 2.09 –1.96 (m, 2H), 1.83 – 1.71 (m, 1H), 1.22 (d, J = 6.9 Hz, 6H). Example 21: A protein degrader compound, KD-21, targeting KDM5, has the following structural formula: ; The synthesis route is shown below:

[0031] The preparation method specifically includes the following steps: Synthesis of S1 and intermediate 23 Compound 21 (800 mg, 1.0 eq), tert-butyl hex-5-yn-1-ylcarbamate 22 (936 mg, 2.0 eq), TEA (2.5 mL), DMF (5 mL), cuprous iodide (90 mg, 0.2 eq), and palladium dichloride bis(triphenylphosphine) (167 mg, 0.1 eq) were added to a reaction flask and reacted at 70 °C for 3 h under nitrogen protection. After the reaction was complete, the mixture was extracted with ethyl acetate, the organic phases were combined, concentrated under reduced pressure, and the crude product was purified by column chromatography to give 345 mg of brown compound 23. 1 H NMR (600 MHz, Chloroform- d ) δ 8.22 (s, 1H), 7.85 (s, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.73(d, J = 7.8 Hz, 1H), 4.98 (dd, J = 12.6, 5.4 Hz, 1H), 4.59 (s, 1H), 3.19 (s,2H), 2.94 – 2.88 (m, 1H), 2.87 – 2.80 (m, 1H), 2.78 – 2.71 (m, 1H), 2.49 (s,2H), 2.18 – 2.12 (m, 1H), 1.68 – 1.64 (m, 4H), 1.45 (s, 9H). Synthesis of S2 and KD-21 Intermediate 23 (300 mg, 0.66 mmol), trifluoroacetic acid (1 mL), and dichloromethane (4 mL) were added to a reaction flask and reacted at room temperature for 2 h. After the reaction was complete, the solvent was evaporated under reduced pressure, and acetonitrile (5 mL), intermediate 1 (226 mg, 0.66 mmol), and triethylamine (0.28 mL, 1.98 mmol) were added. The reaction was carried out at 60 °C for 6 h. After the reaction was complete, compound KD-21 was obtained by column chromatography as a white solid with a yield of approximately 24%. 1H NMR (600 MHz, DMSO-d6) δ 13.07 – 12.71 (m,1H), 11.14 (s, 1H), 7.94 – 7.79 (m, 3H), 6.37 (d, J = 7.6 Hz, 1H), 6.14 (t,1H), 5.81 (dd, J = 11.3, 5.7 Hz, 1H), 5.16 (dd, J = 12.9, 5.4 Hz, 1H), 4.14 –4.07 (m, 1H), 3.99 – 3.88 (m, 1H), 3.68 – 3.59 (m, 1H), 3.57 – 3.52 (m, 1H),3.51 – 3.44 (m, 1H), 3.33 – 3.26 (m, 1H), 3.08 – 3.01 (m, 2H), 2.99 – 2.93(m, 1H), 2.92 – 2.86 (m, 1H), 2.63 – 2.58 (m, 1H), 2.56 – 2.52 (m, 1H), 2.50– 2.48 (m, 1H), 2.09 – 1.96 (m, 2H), 1.83 – 1.67 (m, 1H), 1.59 – 1.49 (m,4H), 1.22 (d, J = 5.9 Hz, 6H). Example 22: A protein degrader compound, KD-22, targeting KDM5, has the following structural formula: ; The synthesis route is shown below:

[0032] The preparation method specifically includes the following steps: Compound KD-22 (200 mg, 0.33 mmol) obtained in Example 21 was added to methanol (8 mL) and 10% palladium on carbon (55% water, 30 mg), and reacted overnight under hydrogen atmosphere. After the reaction was completed, compound KD-22 was obtained by column chromatography purification. It was a white solid with a yield of 76%. 1 H NMR (600 MHz, DMSO- d 6) δ 12.90 (d, J = 15.4 Hz, 1H), 11.12 (s, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.76 (d, J= 4.3 Hz, 1H), 7.72 – 7.68(m, 1H), 6.37 (d, J = 7.2 Hz, 1H), 6.10 (d, J = 6.0 Hz, 2H), 5.75 – 5.68 (m,1H), 5.14 (dd, J = 12.9, 5.4 Hz, 1H), 4.12 – 4.06 (m, 1H), 3.99 – 3.87 (m,1H), 3.67 – 3.58 (m, 1H), 3.56 – 3.47 (m, 1H), 3.32 – 3.27 (m, 1H), 3.13 –3.06 (m, 1H), 3.00 – 2.86 (m, 4H), 2.77 (q, J = 6.9 Hz, 2H), 2.63 – 2.58 (m,2H), 2.58 – 2.52 (m, 1H), 2.10 – 1.96 (m, 2H), 1.80 – 1.67 (m, 1H), 1.64 –1.59 (m, 2H), 1.37 – 1.31 (m, 2H), 1.30 – 1.24 (m, 4H), 1.22 (d, J = 6.6 Hz, 6H). Experimental example: (1) Evaluation of the degradation activity of PROTACs targeting KDM5A and KDM5B degradation in paclitaxel-resistant MDA-MB-231 cells (MDA-MB-231 / PTX) To evaluate the degradation ability of KDM5-targeting protein degraders on KDM5A and KDM5B, this invention selected paclitaxel (PTX)-resistant MDA-MB-231 / PTX cells with high KDM5A and KDM5B expression for screening. The compound was set at a concentration of 3.0 μmol / L, and after treating MDA-MB-231 / PTX cells for 24 h, cellular protein samples were collected for Western blotting experiments.

[0033] Experimental results are as follows Figure 1 As shown in the figure. The results showed that KD-14, KD-19, and KD-22 exhibited degradation activity against KDM5A and KDM5B. Figure 1(See Figures A and B). Further, three concentration gradients of 1.0 μmol / L, 3.0 μmol / L, and 10.0 μmol / L were set up for the three compounds to verify their degradation activities for KDM5A and KDM5B. The results showed that compound KD-22 exhibited the strongest degradation activity.

[0034] To further refine the time-gradient administration of the most active compound, KD-22, MDA-MB-231 / PTX cells was developed. Dosing times were set at seven time points: 0 h, 4 h, 8 h, 12 h, 16 h, 20 h, and 24 h. MDA-MB-231 / PTX cells were co-incubated with 3 μmol / L KD-22 for different durations, and cell samples were collected. The levels of KDM5A and KDM5B were then detected using immunoblotting. Similarly, based on the optimal degradation times of the two proteins, a gradient administration was implemented at seven concentrations: 10 nmol / L, 30 nmol / L, 100 nmol / L, 300 nmol / L, 1000 nmol / L, 3000 nmol / L, and 10000 nmol / L. The corresponding concentrations of KD-22 were co-incubated with cells for the optimal degradation time, and cell samples were collected. The levels of KDM5A and KDM5B were then detected using immunoblotting.

[0035] Experimental results are as follows Figure 2 As shown in the figure. The results show that the optimal degradation times for KD-22 of KDM5A and KDM5B are 12 h and 6 h, respectively. Figure 2 (Figure A). Meanwhile, KD-22 can effectively degrade KDM5A and KDM5B in a dose-dependent manner, with a half-maximal degradation concentration (DC). 50 The values ​​were 31.6 nmol / L (12 h) and 22.4 nmol / L (6 h), respectively. Figure 2 (B and 2C). The above data indicate that KD-22 is a highly efficient and promising dual-targeting degrader, laying an important foundation for further research on its mechanism of action and anti-TNBC activity.

[0036] (2) Evaluation of the therapeutic effect of KD-22 on paclitaxel-resistant triple-negative breast cancer KDM5A and KDM5B are key genes mediating TNBC resistance to PTX. Studies have found that KDM5A and KDM5B can activate Tenascin C in a non-methyltransferase-dependent manner, promoting tumor invasion and metastasis. Therefore, inhibitors targeting only KDM5A and KDM5B enzyme activity may not completely inhibit TNBC migration and drug resistance, while simultaneously targeting the degradation of KDM5A and KDM5B holds promise for reversing TNBC resistance to PTX and inhibiting TNBC migration. To this end, this invention established the PTX-resistant 4T1 cell line 4T1 / PTX, and then used this cell line to construct a 4T1 mouse orthotopic TNBC model. When the tumor volume reached 60 mm... 3 Subsequently, mice were administered different concentrations of KD-22 (25 mg / kg and 50 mg / kg) via gavage, and tumor size was measured daily. When the tumor volume in the untreated model group reached 1000 mm², the tumor growth was considered complete. 3 Afterwards, the experiment was terminated, and tumor, liver, spleen, kidney, heart, and lung tissues were removed.

[0037] Experimental results are as follows Figure 3 As shown. The results showed that KD-22 exhibited good activity against PTX-resistant TNBC in vivo ( Figure 3 (Figures A-D), but it showed no significant toxicity to vital organs in mice. Figure 3 Figure E-I). Histopathological examination results showed that KD-22 can effectively inhibit lung metastasis of cells (Figure E-I). Figure 4 (Figure A) and Ki67 expression in tumor tissue ( Figure 4 Figure B), to alleviate tumor-induced splenomegaly ( Figure 3 (See Figure F in the middle). The above results indicate that KD-22 can inhibit the migration and resistance of PTX-resistant TNBC.

[0038] (3) Evaluation of the therapeutic effect of KD-22 on acute liver injury KDM5A and KDM5B are upregulated in acute liver injury in humans and mice, promoting the progression of acute liver injury. This invention first used the CCK8 assay to detect the 48-h cytotoxicity of KD-22 on normal mouse hepatocytes AML12 and human hepatocytes LX-2, and then examined the effects of safe concentrations of KD-22 on LPS / D-GalN-induced expression of inflammatory factors and liver injury markers alanine aminotransferase (AST) and aspartate aminotransferase (ALT).

[0039] Experimental results are as follows Figure 4 As shown in the figure. The results showed that KD-22 did not exhibit significant toxicity to either mouse or human hepatocytes at concentrations below 50 μmol / L. Figure 5(See Figures A and B). Furthermore, KD-22 can reduce the expression of LPS / D-GalN-induced pro-inflammatory cytokines TNFα, IL-β, and IL-6 (see Figures A and B). Figure 5 (Figures C-H) KD-22 simultaneously reduced AST and ALT levels in cells, and at the same concentration, it was more effective than the positive control drug TK-129. Figure 5 (Figure I-L)

[0040] This invention further established an LPS / D-GalN-induced liver injury model in 6-8 week old male C57 mice and investigated the effect of KD-22 on its survival rate within 24 hours. Liver samples from these mice treated with different drugs for 6 hours were harvested for morphological observation and eosin methylene blue staining.

[0041] Experimental results are as follows Figure 5 As shown. The results showed that KD-22 could increase the survival rate of mice with LPS / D-GalN-induced liver injury in a dose-dependent manner ( Figure 6 (Figure A) KD-22 can alleviate LPS / D-GalN-induced liver necrosis ( Figure 6 (Figure B) and reducing the area of ​​damage in liver tissue ( Figure 6 (Figures C and D). KD-22 is superior to dexamethasone (DEX), a positive drug in the clinical treatment of acute liver injury, in improving mouse survival rate and histopathology.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A protein degrader targeting KDM5, characterized in that, The chemical structural formula is shown in Formula I or Formula II: Formula I: Formula II: ; In Formula I, L is selected from , , , , , , , , , , , , and Any one of them; In Formula I, R is the E3 ligase ligand.

2. The protein degrader targeting KDM5 according to claim 1, characterized in that, The E3 ligase ligand is selected from... , , , , , , , and Any one of them.

3. The protein degrader targeting KDM5 according to claim 1, characterized in that, Protein degraders targeting KDM5 are selected from the following compounds: , , , , , , , , , , , , , , , , , , , , and Any one of them.

4. The use of the KDM5-targeting protein degrader according to any one of claims 1-3 in the preparation of a medicament for treating triple-negative breast cancer and / or acute liver injury.

5. A pharmaceutical composition, characterized in that, Includes at least one of the protein degraders targeting KDM5 as described in any one of claims 1-3, or a pharmaceutically acceptable salt, isomer, solvate, crystal, and prodrug thereof.

6. The pharmaceutical composition according to claim 5, characterized in that, It also includes pharmaceutically acceptable excipients and / or pharmaceutically acceptable carriers.

7. Use of the pharmaceutical composition of claim 5 or 6 in the preparation of a medicament for treating triple-negative breast cancer and / or acute liver injury.

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