Artesunate PROTAC derivative with GPX4 degradation activity, composition and application

Abstract

The invention belongs to the technical field of anti-cancer drugs, and particularly relates to an artesunate PROTAC derivative with GPX4 degradation activity, a composition and application of the artesunate PROTAC derivative with GPX4 degradation activity. R is a Cerebron protein ligand or a VHL protein ligand, and Linker is a linking group of artesunate and R; according to the invention, the anti-tumor effect is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of anticancer drugs, and in particular relates to artesunate derivatives with GPX4 degradation activity, compositions and applications. Background Art

[0002] Bladder cancer is the second most common malignant tumor of the urinary system in the world. Due to the high recurrence of bladder cancer, patients usually need long-term monitoring and multiple treatments. It is considered to be the malignant tumor with the highest treatment cost.

[0003] Ferroptosis, defined as iron-dependent regulated necrosis caused by extensive lipid peroxidation-mediated membrane damage, is characterized by redox imbalance and elevated levels of intracellular reactive oxygen species (ROS). Several studies have shown that ferroptosis plays a key role in tumor suppression and can be used for cancer treatment. Glutathione peroxidase 4 (GPX4) is a central regulator of ferroptosis; inactivation of GPX4 leads to the accumulation of phospholipid hydroperoxides (PLOOH), thereby inducing cell membrane damage and ferroptosis. Researchers have discovered a number of GPX4 inhibitors, such as RSL3, ML162, DPI compounds, FIN56, and FINO2. At present, most of the reported GPX4 small molecule inhibitors are covalent inhibitors containing chloroacetamide groups. Due to their strong electrophilicity, these compounds can bind to a variety of proteins, resulting in low specificity for their binding to GPX4. Therefore, the development of highly active and highly selective targeted GPX4 degraders has become a current research hotspot in this field.

[0004] Proteolysis targeting chimeric molecules (PROTAC) are heterobifunctional molecules that degrade specific endogenous proteins through the E3 ubiquitin ligase pathway. The molecule consists of a ligand for the target protein (POI) and a ligand for the E3 ubiquitin ligase, which are covalently linked by a linker. Unlike traditional small molecule drugs, PROTAC only requires a brief interaction with its target protein to cause the loss of function of the target protein. PROTAC can significantly reduce the IC of drug molecules with its unique mode of action. 50 It also has the advantages of good cell permeability, good tissue specificity, and high oral bioavailability, and PROTAC also has better advantages in improving selectivity.

[0005] Artesunate is a semi-synthetic water-soluble derivative of artemisinin. As a first-line treatment for severe malaria, other pharmacological effects of artesunate are constantly being discovered. At present, many studies have shown that artesunate can exert a certain anti-tumor effect by inducing ferroptosis and the process is often accompanied by downregulation of GPX4 protein. However, the anti-cancer activity of artesunate is still far behind its anti-malarial activity. In addition, artesunate has defects such as poor solubility, low oral bioavailability and poor stability. Therefore, based on the advantages of PROTAC, screening artesunate derivatives that can specifically degrade GPX4 protein and applying them to the targeted degradation of GPX4 is of great significance for the treatment of tumors. No similar reports have been seen so far. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide an artesunate derivative with GPX4 degradation activity, a composition and an application thereof, so as to improve the anti-tumor effect thereof.

[0007] The present invention provides an artesunate derivative having GPX4 degradation activity, the structural formula of which is as follows: ; The R is a Cereblon protein ligand or a VHL protein ligand, and the Linker is a connecting group between artesunate and R.

[0008] Optionally, the Cereblon protein ligand is thalidomide or its derivatives; the structural formula of the VHL protein ligand is: .

[0009] The structural formula of the thalidomide is: .

[0010] Optionally, the Linker is X-R1-X, wherein X is N or O, wherein R1 is a saturated fatty chain, R2, CH2-R2-CH2, wherein R2 is (CH2CH2O) n1 -(CH2) n2 ; Or, X is N and R1 is (CH2)n-CO.

[0011] Optionally, the saturated fatty chain is a C3-11 saturated fatty chain, n1 is 3, n2 is 2, and n is 3-7.

[0012] Optionally, the artesunate derivative is the following compound, .

[0013] Optionally, the artesunate derivative is the following compound, More preferably

[0014] The present invention provides a composition, comprising the artesunate derivative or a pharmacologically acceptable salt thereof, and also comprising a pharmaceutically acceptable carrier, excipient, diluent, vehicle or a combination thereof.

[0015] The present invention provides an artesunate derivative or an application of the composition. The artesunate derivative or the composition is used as a drug for degrading GPX4 or inhibiting GPX4.

[0016] Optionally, the drug is an anti-tumor drug.

[0017] The tumor is bladder cancer.

[0018] The embodiment of the present invention provides a method for preparing the artesunate derivative, wherein the linker is X-R1-X, X is O, R1 is a saturated fatty chain, and R is thalidomide. The steps of the method for preparing the artesunate derivative are: Step 1: In the presence of an esterification catalyst, artesunate reacts with HO-R1-Br to obtain compounds 3a-3e; Step 2: 2-(2,6-dioxo-piperidin-3-yl)-4-hydroxyisoindoline-1,3-dione undergoes substitution reaction with intermediates 3a-3e to obtain artesunate PROTAC compounds A1-A5. The reaction scheme is as follows: .

[0019] The linker is X-R1-X, X is O, R1 is a saturated fatty chain, and R is thalidomide. The steps of the preparation method of artesunate derivatives are: Step 1: 2-(2,6-dioxo-piperidin-3-yl)-4-hydroxyisoindoline-1,3-dione reacts with HO-R1-Br to obtain compound 5f-5g; Step 2: Compounds 5f-5g are subjected to an esterification reaction with artesunate in the presence of an esterification catalyst to obtain artesunate PROTAC compounds A6-A7; the reaction scheme is as follows: .

[0020] The linker is X-R1-X, wherein X is N, R1 is a saturated fatty chain, R2, CH2-R2-CH2, wherein R2 is (CH2CH2O) n1 -(CH2) n2, wherein R is thalidomide, and the steps of preparing the artesunate derivative are: Step 1: In the presence of an acid-binding agent, 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione reacts with a compound represented by the formula H2N-R1-NHBoc to obtain compounds 8a-8f; Step 2: Compound 8a-8f is subjected to acid hydrolysis in the presence of trifluoroacetic acid to remove the Boc protecting group to obtain compound 9a-9f; Step 3: Compounds 9a-9f undergo amide condensation reaction with artesunate in the presence of an acylation activator and an amide condensation agent to obtain artesunate PROTAC compounds A8-A13; the reaction scheme is as follows: .

[0021] The linker is X-R1-X, X is N, R1 is (CH2)n-CO, and R is a VHL protein ligand. The steps of the preparation method of artesunate derivatives are: Step 1: (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide is esterified with H2N-CH2-COOH to give compounds 12a-12b; Step 2: Compounds 12a-12b undergo amide condensation reaction with artesunate in the presence of an acylation activator and an amide condensation agent to obtain artesunate PROTAC compounds B1-B2.

[0022] .

[0023] The beneficial effect of the present invention is that the artesunate PROTAC compound of the present invention can effectively degrade GPX4 protein, thereby inducing ferroptosis of tumor cells, can be used as a GPX4 degrader, can effectively inhibit the proliferation of various bladder cancer cells, and has good safety in normal bladder cells, and is an excellent anti-tumor candidate compound.

[0024] The present invention develops a novel and diverse artesunate PROTAC compound with GPX4 degradation activity based on artesunate and E3 ubiquitin ligase CRBN and VHL recruitment ligands. Compared with artesunate, the compound of the present invention has a significant cell proliferation inhibitory effect on tumor cells. The inventors confirmed through Western blot experiments that the protein targeted degradation chimera of the present invention can bind to GPX4 protein and induce effective degradation, thereby causing cell ferroptosis, and has the potential to be used as an anti-tumor drug to treat tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result of studying the concentration gradient degradation effect of compound A7 on the target protein GPX4 in RT4 cells.

[0026] Figure 2 This is the result of compound A7's degradation mechanism on the target protein GPX4 through ubiquitin-proteasome in RT4 cells.

[0027] Figure 3 This is the result of studying the concentration gradient degradation effect of compound A7 on the target protein GPX4 in T24 cells.

[0028] Figure 4 This is the result of compound A7's degradation mechanism on the target protein GPX4 through ubiquitin-proteasome in T24 cells. DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the technical solution of the present invention, the technical solution of the present invention is clearly and completely described by examples below. The present invention is further described below in conjunction with specific examples and drawings, but the examples cited are not intended to limit the present invention. The purpose of providing these embodiments is to make the understanding of the technical solution of the present invention more thorough and comprehensive. The ingredients or materials involved in the following methods, unless otherwise specified, are all commercially available. The relevant experimental methods are all conventional methods in the art unless otherwise specified.

[0030] Example 1: Preparation of Compound A1 .

[0031] 1 g of artesunate (0.0026 mol), 0.159 g of DMAP (0.0013 mol) and 0.249 g of EDCI (0.0013 mol) were completely dissolved in 15 mL of dichloromethane to prepare a uniform solution, and 3-bromo-1-propanol (0.3948 g, 0.0028 mol) was added dropwise under stirring; the reaction was carried out at room temperature for 3.5 h, and the reaction progress was monitored by TLC. After the reaction, the intermediate product 3a (0.4531 g, yield 34.6%) was obtained by separation and purification using a silica gel column.

[0032] The intermediate product 5a (0.4 g, 0.00079 mol) was dissolved in 10 mL DMF, and potassium iodide (0.0131 g, 0.000079 mol) and sodium bicarbonate (0.1333 g, 0.001587 mol) and 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (0.2609 g, 0.00095 mol) were added under stirring, and the reaction was carried out at 80°C in the dark for 12 h, and the reaction progress was monitored by TLC. After the reaction, the reaction solution was added to 100 mL of water, and then extracted three times with 80 mL of ethyl acetate. The organic phases were combined and concentrated by rotary evaporation, and then separated and purified by silica gel column to obtain the target compound A1 (0.022 g white solid, yield: 4%).

[0033] Data of compound A1: 3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)propyl ((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10-yl) succinate (A1). 1 H NMR (600MHz, Methanol- d 4) δ 11.10 (s, 1H), 7.82 (dd, J = 8.5, 7.3 Hz, 1H), 7.52 (d, J =8.5 Hz, 1H), 7.46 (d, J = 7.2 Hz, 1H), 5.63 (d, J = 9.8 Hz, 1H), 5.52 (d, J = 1.8Hz, 1H), 5.08 (dd, J = 12.9, 5.5 Hz, 1H), 4.25 (dt, J = 26.7, 5.8 Hz, 4H), 2.67 –2.64 (m, 1H), 2.62 – 2.59 (m, 2H), 2.27 (ddd, J = 9.8, 7.2, 4.3 Hz, 1H), 2.17(td, J = 14.0, 3.9 Hz, 1H), 2.08 (td, J= 6.1, 3.2 Hz, 2H), 2.05 – 1.96 (m, 3H), 1.83 – 1.77 (m, 1H), 1.61 – 1.55 (m, 1H), 1.55 – 1.49 (m, 1H), 1.39 (d, J =11.9 Hz, 2H), 1.33 (s, 2H), 1.29 (d, J = 3.8 Hz, 1H), 1.28 (d, J = 2.8 Hz, 3H),1.22 (s, 3H), 1.18 – 1.15 (m, 1H), 0.88 (d, J = 6.4 Hz, 3H), 0.73 (d, J = 7.0 Hz,3H). 13 C NMR (151 MHz, Methanol-d4) δ 172.79, 171.81, 170.90, 169.94, 166.83,165.30, 155.74, 137.10, 133.24, 119.78, 116.37, 115.37, 103.58, 91.81, 90.59,79.84, 65.53, 60.88, 51.11, 48.75, 44.54, 35.97, 35.88, 33.69, 31.54, 29.43,28.66, 28.46, 27.88, 25.50, 22.10, 22.00, 20.99, 20.06, 11.69. HRMS (ESI) (m / z) [M+NH4] + : 716.3041.

[0034] Example 2: Preparation of Compound A2: 1.0 g (0.0060 mol) of 5-bromo-1-pentanol was used to replace the 3-bromo-1-propanol in Example 1, and other conditions remained unchanged to obtain compound A2 (0.2076 g, yield: 5.2%).

[0035] .

[0036] Data for compound A2: δ 7.99 (s, 1H), 7.77 (dd, J = 8.5, 7.3 Hz, 1H), 7.44 (dd, J = 7.9, 2.2 Hz, 2H), 5.73 (d, J = 9.8 Hz, 1H), 5.50 (d, J = 1.7 Hz,1H), 5.11 (dt, J = 11.4, 5.7 Hz, 1H), 4.20 (dt, J = 57.1, 6.2 Hz, 4H), 2.80 –2.65 (m, 4H), 2.47 – 2.40 (m, 1H), 2.35 – 2.28 (m, 1H), 2.16 – 2.11 (m, 1H),2.03 (ddd, J = 14.6, 5.0, 3.3 Hz, 1H), 1.92 – 1.86 (m, 3H), 1.79 – 1.61 (m ,4H), 1.57 (dt, J = 13.9, 4.7 Hz, 1H), 1.46 (d, J = 6.8 Hz, 1H), 1.44 – 1.40(m, 1H), 1.39 (s, 3H), 1.38 – 1.34 (m, 3H), 1.34 – 1.32 (m, 1H), 1.28 (s,3H), 1.22 (ddd, J = 11.3, 7.6, 3.9 Hz, 1H), 0.96 (d, J = 6.4 Hz, 3H), 0.85(d, J = 7.2 Hz, 3H). 13 C NMR (150 MHz, MeOD) δ 174.70, 174.04, 171.47, 168.70,167.30, 164.87, 158.04, 137.94, 135.15, 124.31, 120.55, 116.30, 105.65,93.73, 92.85, 81.35, 70.27, 65.64, 52.94, 50.42, 46.55, 38.25, 37.26, 35.29,33.04, 32.15, 30.08, 29.82, 29.54, 29.31, 25.88, 25.76, 23.68, 23.49, 22.81,20.56, 12.39.HRMS (ESI) (m / z) [M+NH4] + : 744.3361.

[0037] Example 3: Preparation of Compound A3: 1.1 g (0.0061 mol) of 6-bromo-1-hexanol was used to replace the 3-bromo-1-propanol in Example 1, and other conditions remained unchanged to obtain compound A3 (0.4558 g, yield: 11.2%).

[0038] .

[0039] Data of compound A3: 1 H NMR (600 MHz, Methanol- d 4) 1 H NMR (600 MHz, Methanol- d 4) δ 8.09 (s, 1H), 7.77 (ddd, J = 8.4, 7.3, 2.5 Hz, 1H), 7.44 (dt, J = 5.8, 3.5Hz, 2H), 5.75 (dd, J = 9.8, 2.9 Hz, 1H), 5.50 (s, 1H), 5.11 (dd, J = 12.8, 5.5Hz, 1H), 4.27 – 4.19 (m, 2H), 4.12 (t, J = 6.5 Hz, 2H), 2.87 (ddt, J = 9.6, 6.5,3.2 Hz, 1H), 2.79 – 2.60 (m, 5H), 2.49 – 2.42 (m, 1H), 2.31 (dd, J = 14.0, 4.0Hz, 1H), 2.14 (dtd, J = 10.1, 5.5, 2.8 Hz, 1H), 2.06 – 2.00 (m, 1H), 1.89 (dtd,J = 12.2, 6.7, 3.2 Hz, 3H), 1.77 – 1.65 (m, 4H), 1.62 – 1.55 (m, 4H), 1.53 –1.42 (m, 4H), 1.36 (s, 3H), 1.33 – 1.19 (m, 2H), 1.00 (ddd, J = 26.7, 13.6,4.3 Hz, 1H), 0.95 (d, J = 6.4 Hz, 3H), 0.86 (d, J = 7.1 Hz, 3H). 13C NMR (150MHz, MeOD) δ 174.66, 173.99, 172.85, 172.84, 171.46, 168.69, 158.07, 137.96,135.16, 120.58, 118.17, 116.31, 105.64, 93.72, 92.87, 81.36, 70.46, 65.80,52.95, 50.42, 46.55, 38.25, 37.26, 35.28, 33.06, 32.21, 30.10, 29.83, 29.81,29.58, 26.65, 26.60, 25.88, 25.77, 23.67, 22.82, 20.56, 12.41. HRMS (ESI) (m / z) [M+NH4] + : 758.3523.

[0040] Example 4: Preparation of Compound A4: 1.2 g (0.0061 mol) of 7-bromo-1-heptanol was used to replace the 3-bromo-1-propanol in Example 1, and other conditions remained unchanged to obtain compound A4 (0.3525 g, yield: 8.5%).

[0041] .

[0042] Data of compound A4: 1H NMR (600 MHz, DMSO-d6) δ 11.10 (s, 1H), 7.81 (dd, J= 8.5, 7.3 Hz, 1H), 7.52 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.66(d, J = 9.7 Hz, 1H), 5.55 (s, 1H), 5.08 (dd, J = 12.9, 5.5 Hz, 1H), 4.20 (t,J = 6.4 Hz, 2H), 4.01 (dd, J = 6.6, 1.5 Hz, 2H), 2.67 – 2.52 (m, 6H), 2.28(ddd, J = 9.8, 7.2, 4.4 Hz, 1H), 2.20 – 2.14 (m, 1H), 2.01 (dddd, J = 27.2,14.5, 5.3, 2.8 Hz, 2H), 1.80 – 1.72 (m, 3H), 1.60 – 1.54 (m, 4H), 1.48 – 1.43(m, 3H), 1.41 – 1.38 (m, 1H), 1.36 – 1.32 (m, 5H), 1.28 (s, 3H), 1.23 (d, J =5.6 Hz, 3H), 1.15 (td, J = 11.4, 6.7 Hz, 1H), 0.87 (d, J = 6.4 Hz, 3H), 0.76(d, J = 7.1 Hz, 3H). 13 C NMR (150 MHz, DMSO) δ 172.79, 171.83, 170.84, 169.95,166.86, 165.31, 162.31, 137.04, 133.25, 119.78, 116.21, 115.13, 103.57,91.79, 90.59, 79.84, 68.78, 64.08, 51.09, 48.73, 44.54, 35.95, 35.87, 35.78,33.68, 31.61, 31.54, 30.96, 30.77, 29.42, 28.67, 28.28, 28.03, 25.50, 25.30,25.20, 20.98, 20.04, 11.70. HRMS (ESI) (m / z) [M+NH4] + : 772.3676。

[0043] Example 5: Preparation of Compound A5 1.3 g (0.0061 mol) of 8-bromo-1-octanol was used to replace the 3-bromo-1-propanol in Example 1, and other conditions remained unchanged to obtain compound A5 (0.4017 g, yield: 9.5%).

[0044] .

[0045] Data of compound A5: 1 H NMR (600 MHz, DMSO-d6) δ 11.10 (s, 1H), 7.83 – 7.78(m, 1H), 7.47 (dd, J = 43.9, 7.9 Hz, 2H), 5.65 (d, J = 9.8 Hz, 1H), 5.54 (s,1H), 5.10 – 5.05 (m, 1H), 4.10 (dt, J = 114.3, 6.6 Hz, 4H), 2.67 – 2.55 (m,5H), 2.53 – 2.51 (m, 1H), 2.32 – 2.25 (m, 1H), 2.20 – 2.13 (m, 1H), 2.06 –1.95 (m, 2H), 1.83 – 1.72 (m, 3H), 1.63 – 1.51 (m, 5H), 1.47 – 1.36 (m, 5H), 1.36 – 1.33 (m, 1H), 1.33 – 1.29 (m, 5H), 1.28 (s, 3H), 1.26 – 1.22 (m, 2H), 1.15 (td, J = 11.4, 6.6 Hz, 1H), 0.86 (d, J = 6.3 Hz, 3H), 0.76 (d, J = 7.1Hz, 3H). 13C NMR (150 MHz, DMSO) δ 172.94, 171.98, 171.00, 170.10, 167.01,165.45, 156.18, 137.19, 133.40, 119.92, 116.35, 115.27, 103.72, 91.94, 90.74,79.99, 68.93, 64.24, 51.25, 48.88, 44.70, 35.93, 33.83, 32.68, 31.76, 30.91,29.17, 28.81, 28.71, 28.57, 28.54, 28.20, 25.65, 25.41, 25.36, 24.33, 22.15,21.14, 20.19, 11.85. HRMS (ESI) (m / z) [M+NH4] + : 786.3827.

[0046] Example 6: Preparation of Compound A6 1.5 g of 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (0.005474 mol, 1 equivalent) was dissolved in 15 mL of DMF, and 0.091 g of potassium iodide (0.000547 mol, 0.1 equivalent) and 0.919 g of sodium bicarbonate (0.01095 mol, 2 equivalents) were added dropwise under stirring, and 10-bromo-1-decanol (1.421 g, 1.1 equivalent) was added dropwise, and the mixture was reacted at 80°C in the dark for 12 h. After the reaction, the reaction solution was added to 150 mL of water, and then extracted three times with 100 mL of ethyl acetate. The organic phases were combined, concentrated by rotary evaporation, and purified by silica gel column to obtain the intermediate product 5f (0.9811 g, yield: 41.7%).

[0047] The intermediate product 5f (0.9 g, 0.0021 mol 1 equivalent) was dissolved in 15 mL of dichloromethane, and artesunate (0.9630 g, 0.002512 mol, 1.2 equivalent), DMAP (0.1539 g, 0.00126 mol, 0.6 equivalent) and EDCI (0.1956 g, 0.00126 mol, 0.6 equivalent) were added under stirring, and the reaction was carried out at room temperature for 3.5 h. The reaction progress was monitored by TLC. After the reaction, the target compound A6 (0.2592 g, light yellow oily, yield: 15.5%) was obtained by separation and purification using a silica gel column.

[0048] .

[0049] Data of compound A6: 1 H NMR (600 MHz, Methanol-d4) δ 7.77 (dd, J = 8.4, 7.4Hz, 1H), 7.44 (s, 1H), 7.43 (d, J = 2.3 Hz, 1H), 5.76 (dd, J = 9.8, 2.0 Hz,1H), 5.52 (d, J = 3.4 Hz, 1H), 5.11 (ddd, J = 12.5, 5.6, 3.4 Hz, 1H), 4.13 –4.07 (m, 4H), 2.92 – 2.84 (m, 1H), 2.79 – 2.63 (m, 6H), 2.46 (ddd, J = 9.8,7.2, 4.5 Hz, 1H), 2.32 (td, J = 14.0, 4.0 Hz, 1H), 2.14 (ddt, J = 10.4, 4.2,2.2 Hz, 1H), 1.89 – 1.83 (m, 2H), 1.77 (dd, J = 13.5, 3.9 Hz, 1H), 1.71 (dt,J = 13.3, 3.6 Hz, 1H), 1.65 – 1.52 (m, 5H), 1.47 – 1.40 (m, 4H), 1.35 (s,9H), 1.30 (s, 2H), 1.29 – 1.20 (m, 5H), 0.95 (d, J = 6.4 Hz, 3H), 0.87 (d, J= 7.1 Hz, 3H). 13 C NMR (150 MHz, MeOD) δ 174.66, 173.99, 173.00, 172.83,171.49, 168.70, 158.08, 137.94, 135.16, 120.53, 118.15, 116.27, 105.64,93.72, 92.87, 81.36, 65.94, 61.54, 52.95, 50.41, 46.57, 38.25, 37.26, 35.29,33.07, 32.21, 30.49, 30.41, 30.31, 30.25, 30.10, 30.01, 29.81, 29.68, 26.98,25.88, 23.67, 22.86, 20.86, 20.57, 14.46, 12.42. HRMS (ESI) (m / z) [M+NH4]+ :814.4109.

[0050] Example 7: Preparation of Compound A7 1.4 g (0.0061 mol) of 11-bromo-1-undecanol was used to replace the 10-bromo-1-decanol in Example 6, and other conditions remained unchanged to obtain compound A7 (1.0474 g, yield: 23.5%).

[0051] .

[0052] Data of compound A7: 1 H NMR (600 MHz, DMSO-d6) δ 11.11 (s, 1H), 7.81 (dd, J= 8.5, 7.3 Hz, 1H), 7.52 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 5.67(d, J = 9.7 Hz, 1H), 5.55 (s, 1H), 5.09 (dd, J = 12.9, 5.4 Hz, 1H), 4.20 (t,J = 6.4 Hz, 2H), 4.03 – 3.99 (m, 2H), 2.69 – 2.60 (m, 2H), 2.63 – 2.58 (m,1H), 2.58 (d, J = 6.5 Hz, 2H), 2.58 – 2.47 (m, 1H), 2.34 – 2.25 (m, 1H), 2.18(td, J = 14.0, 4.0 Hz, 1H), 2.06 – 1.97 (m, 3H), 1.83 – 1.73 (m, 3H), 1.66 – 1.60 (m, 1H), 1.63 – 1.51 (m, 4H), 1.50 – 1.44 (m, 2H), 1.47 – 1.37 (m, 2H), 1.37 – 1.32 (m, 2H), 1.29 (s, 3H), 1.25 (d, J = 21.8 Hz, 10H), 1.18 (t, J =7.1 Hz, 2H), 0.95 (td, J = 12.9, 12.5, 3.7 Hz, 1H), 0.88 (d, J = 6.4 Hz, 3H), 0.77 (d, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 173.23, 172.26, 171.30,170.39, 167.32, 165.76, 156.50, 137.48, 133.72, 120.23, 116.69, 115.58,104.04, 92.25, 91.06, 80.29, 69.26, 64.55, 51.57, 49.20, 45.02, 36.43, 36.34,36.24, 34.16, 32.07, 31.43, 31.23, 29.44, 29.39, 29.15, 29.13, 28.90, 28.54,25.96, 25.80, 25.75, 24.65, 21.47, 21.22, 20.49, 14.55. HRMS (ESI) (m / z) [M+NH4] + : 828.4275.

[0053] Example 8: Preparation of Compound A8 1.0 g of 2-(2,6-dioxapiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (0.0036 mol) was dissolved in 8 mL of DMF, and 818 μL of DIPEA (0.0047 mol) and 0.75 g of tert-butyl (4-aminobutyl) carbamate (0.0040 mol) were added dropwise under stirring. The mixture was reacted at 90 °C for 12 h, and the reaction progress was monitored by TLC. After the reaction, the reaction solution was poured into 80 mL of water and extracted three times with 60 mL of ethyl acetate. The organic phases were combined, washed twice with saturated sodium carbonate solution, dried over anhydrous magnesium sulfate, and then separated and purified by silica gel column to obtain intermediate 8a (0.7364 g, yield: 44.9%).

[0054] The intermediate product 1 (0.7 g, 0.0016 mol) was added to a mixed solution of TFA / DCM = 1 / 1 (6 mL) and stirred at room temperature for 1 h. After the reaction was completed, the product was spin-dried and purified by silica gel column to obtain the intermediate product 9a (0.49 g, yield: 89%).

[0055] Take 0.49 g (0.0014 mol) of intermediate product 2 and dissolve it in 8 mL DMF, add 0.8 g (0.0020 mol) of HATU and 0.6 g (0.0016 mol) of artesunate, add 1 mL (0.0057 mol) of DIPEA dropwise under stirring, react at room temperature for 3.5 h, and monitor the reaction progress by TLC. After the reaction, add the reaction solution into 80 mL of water, extract with 60 mL of ethyl acetate three times, combine the organic phases, concentrate by rotary evaporation, and separate and purify by silica gel column to obtain the target compound A8 (0.2889 g, total yield: 11.3%).

[0056] .

[0057] Data of compound A8: 1H NMR (600 MHz, DMSO-d6) δ 11.08 (s, 1H), 7.84 (t, J= 5.7 Hz, 1H), 7.57 (dd, J = 8.6, 7.1 Hz, 1H), 7.09 (d, J = 8.6 Hz, 1H), 7.01(d, J = 7.0 Hz, 1H), 6.53 (t, J = 6.0 Hz, 1H), 5.39 (d, J = 30.5 Hz, 1H),5.04 (dd, J = 12.9, 5.5 Hz, 1H), 4.58 (dd, J = 9.3, 4.5 Hz, 1H), 3.29 (q, J =6.8 Hz, 2H), 3.06 (q, J = 6.5 Hz, 2H), 2.87 (ddd, J = 17.1, 13.8, 5.4 Hz,1H), 2.61 – 2.47 (m, 5H), 2.40 (t, J = 7.0 Hz, 2H), 2.28 (t, J = 7.0 Hz, 2H),2.20 – 2.12 (m, 1H), 2.04 – 1.93 (m, 2H), 1.79 (ddd, J = 13.8, 6.9, 3.5 Hz,1H), 1.65 – 1.49 (m, 4H), 1.45 (p, J = 7.0 Hz, 2H), 1.40 – 1.28 (m, 2H), 1.26(d, J = 7.0 Hz, 3H), 1.22 (d, J = 5.1 Hz, 1H), 1.13 (td, J = 11.3, 6.6 Hz,1H), 0.87 (d, J = 6.3 Hz, 3H), 0.77 (d, J = 7.2 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 173.88, 172.84, 170.82, 170.13, 168.96, 167.33, 146.42, 136.30,132.21, 117.26, 110.41, 109.02, 103.18, 93.24, 90.04, 80.19, 52.16, 51.32,48.55, 45.09, 41.55, 38.14, 36.32, 36.08, 33.86, 30.99, 30.03, 29.20, 26.54,26.16, 25.68, 24.35, 22.17, 21.29, 20.14, 12.85. HRMS (ESI) (m / z) [M+Na] + :733.3045.

[0058] Example 9: Preparation of Compound A9 1.0 g (0.0049 mol) of tert-butyl (5-aminopentyl) carbamate was used to replace the tert-butyl (4-aminobutyl) carbamate in Example 8, and other conditions remained unchanged to obtain compound A9 (0.1075 g, yield: 10.6%).

[0059] .

[0060] Data of compound A9: 1H NMR (600 MHz, Methanol-d4) δ 8.73 (dd, J = 4.4, 1.4Hz, 1H), 8.43 (dd, J = 8.4, 1.4 Hz, 1H), 7.56 (dd, J = 8.6, 7.1 Hz, 1H), 7.05(dd, J = 9.9, 7.8 Hz, 2H), 6.63 (d, J = 8.3 Hz, 1H) 5.74 (d, J = 8.7 Hz, 1H),5.47 (d, J = 2.4 Hz, 1H), 5.07 (ddd, J = 12.6, 5.5, 2.2 Hz, 1H), 3.35 (d, J =7.0 Hz, 2H), 3.33 – 3.31 (m, 4H), 3.21 (td, J = 6.7, 4.3 Hz, 2H), 2.91 – 2.83(m, 1H), 2.79 – 2.70 (m, 4H), 2.45 (ddd, J = 9.8, 7.2, 4.5 Hz, 1H), 2.31(ddd, J = 14.6, 13.4, 3.9 Hz, 1H), 2.15 – 2.09 (m, 1H), 1.92 – 1.86 (m, 1H),1.77 – 1.65 (m, 4H), 1.57 (ddd, J = 8.8, 6.7, 4.1 Hz, 3H), 1.51 – 1.45 (m,2H), 1.44 – 1.39 (m, 2H), 1.35 (s, 3H), 1.33 – 1.28 (m, 2H), 0.94 (d, J = 6.3Hz, 3H), 0.86 (d, J = 7.1 Hz, 3H). 13C NMR (150 MHz, MeOD) δ 174.73, 174.10,173.02, 171.61, 170.80, 169.34, 148.83, 139.35, 134.73, 118.05, 111.75,109.52, 106.12, 95.55, 92.87, 82.07, 52.92, 50.21, 46.55, 43.30, 40.18,38.67, 37.26, 35.28, 33.06, 32.25, 31.26, 30.57, 30.01, 29.86, 25.89, 25.77,25.09, 23.83, 22.85, 20.58, 11.95. HRMS (ESI) (m / z) [M+Na] + : 747.3225.

[0061] Example 10: Preparation of Compound A10 1.0 g (0.0046 mol) of N-Boc-1,6-hexanediamine was used to replace the tert-butyl (4-aminobutyl) carbamate in Example 8, while other conditions remained unchanged, to obtain compound A10 (0.1302 g, yield: 12.3%).

[0062] .

[0063] Data of compound A10: 1H NMR (600 MHz, Methanol-d4) δ 11.10 (s, 1H), 7.95(s, 1H), 7.79 (dd, J = 8.5, 7.3 Hz, 1H), 7.50 (d, J = 8.5 Hz, 1H), 7.43 (d, J= 7.2 Hz, 1H), 5.74 (s, 1H), 5.65 (d, J = 9.7 Hz, 1H), 5.53 (s, 1H), 5.07(dd, J = 12.8, 5.5 Hz, 1H), 4.18 (t, J = 6.4 Hz, 2H), 4.01 – 3.96 (m, 2H),2.67 – 2.62 (m, 2H), 2.59 – 2.55 (m, 2H), 2.52 – 2.47 (m, 2H), 1.81 – 1.70(m, 3H), 1.60 (ddd, J = 19.2, 14.6, 3.5 Hz, 2H), 1.53 (td, J = 7.8, 4.1 Hz, 3H), 1.47 – 1.36 (m, 5H), 1.33 (s, 4H), 1.28 (s, 2H), 1.27 (s, 5H), 1.16 (s,1H), 0.86 (d, J = 6.4 Hz, 3H), 0.75 (d, J = 7.0 Hz, 3H). 13 C NMR (150 MHz, MeOD) δ 172.77, 171.80, 170.84, 169.92, 166.86, 162.30, 156.04, 137.01,133.26, 119.74, 116.22, 115.11, 103.58, 91.79, 90.60, 79.82, 64.09, 59.76,51.11, 48.75, 44.57, 35.99, 33.70, 31.60, 30.97, 30.76, 28.94, 28.68, 28.44,28.09, 25.49, 25.35, 24.19, 22.03, 21.02, 20.75, 20.02, 14.08. HRMS (ESI) (m / z) [M+Na] + : 761.3378.

[0064] Example 11: Preparation of Compound A11 1.0 g (0.0041 mol) of N-Boc-1,8-octanediamine was used to replace the tert-butyl (4-aminobutyl) carbamate in Example 8, while other conditions remained unchanged, to obtain compound A11 (0.0902 g, yield: 8.4%).

[0065] .

[0066] Data of compound A11: 1 H NMR (600 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.03 (s,1H), 7.82 (s, 1H), 7.57 (dd, J = 8.5, 7.1 Hz, 1H), 7.08 (d, J = 8.6 Hz, 1H), 7.01 (d, J = 7.0 Hz, 1H), 5.64 (d, J = 9.7 Hz, 1H), 5.53 (s, 1H), 5.04 (dd, J= 12.9, 5.4 Hz, 1H), 4.02 (q, J = 7.1 Hz, 2H), 3.28 (q, J = 6.8 Hz, 2H), 3.00(q, J = 6.5 Hz, 2H), 2.87 (ddd, J = 17.1, 13.8, 5.5 Hz, 1H), 2.63 – 2.53 (m,3H), 2.42 – 2.33 (m, 2H), 2.27 (q, J = 6.8 Hz, 1H), 2.17 (td, J = 14.0, 3.9Hz, 1H), 2.05 – 1.95 (m, 4H), 1.63 – 1.49 (m, 5H), 1.38 – 1.30 (m, 8H), 1.27(s, 3H), 1.22 – 1.21 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H), 0.75 (d, J = 7.1 Hz, 3H). 13C NMR (150 MHz, DMSO) δ 173.28, 171.69,170.66, 170.56, 169.42, 167.77, 146.90, 136.76, 132.66, 117.64, 110.84,109.47, 104.03, 92.05, 91.04, 80.32, 51.58, 49.00, 45.04, 42.31, 38.97,36.43, 36.36, 34.16, 32.11, 32.00, 31.45, 30.00, 29.89, 29.55, 29.49, 29.19,26.81, 26.76, 25.98, 24.65, 22.62, 21.23, 20.51, 14.55. HRMS (ESI) (m / z) [M+Na] + : 789.3696.

[0067] Example 12: Preparation of Compound A12 1.0 g (0.0031 mol) of tert-butyl 12-amino-4,7,10-trioxadodecanoate was used to replace the tert-butyl (4-aminobutyl) carbamate in Example 8, while other conditions remained unchanged, to obtain compound A12 (0.2109 g, yield: 18.5%).

[0068] .

[0069] Data of compound A12: 1H NMR (600 MHz, DMSO-d6) δ 11.09 (s, 1H), 7.94 (t, J= 5.7 Hz, 1H), 7.58 (dd, J = 8.6, 7.1 Hz, 1H), 7.14 (d, J = 8.6 Hz, 1H), 7.04(d, J = 7.0 Hz, 1H), 6.60 (t, J = 5.9 Hz, 1H), 5.64 (d, J = 9.8 Hz, 1H), 5.53(s, 1H), 5.05 (dd, J = 12.9, 5.4 Hz, 1H), 3.62 (t, J = 5.4 Hz, 2H), 3.56 (dd,J = 5.8, 3.3 Hz, 2H), 3.54 – 3.50 (m, 4H), 3.50 – 3.44 (m, 4H), 3.37 (t, J =5.9 Hz, 2H), 3.17 (q, J = 5.8 Hz, 2H), 2.61 – 2.49 (m, 6H), 2.38 (td, J =6.9, 2.3 Hz, 2H), 2.27 (ddd, J = 9.8, 7.0, 4.3 Hz, 1H), 2.17 (td, J = 14.0,3.9 Hz, 1H), 2.08 – 1.95 (m, 3H), 1.64 – 1.50 (m, 3H), 1.45 – 1.30 (m, 3H),1.28 (s, 3H), 1.19 – 1.12 (m, 1H), 0.87 (d, J = 6.4 Hz, 3H), 0.75 (d, J = 7.1Hz, 3H). 13C NMR (150 MHz, DMSO) δ 172.82, 171.23, 170.57, 170.08, 168.95, 167.30, 146.42, 136.25, 132.10, 117.46, 110.69, 109.25, 103.58, 91.61, 90.58,79.87, 69.80, 69.78, 69.78, 69.60, 69.10, 68.89, 53.60, 51.12, 48.56, 44.58,41.70, 38.61, 35.98, 33.71, 31.64, 30.99, 29.46, 28.95, 25.52, 24.19, 22.15,21.02, 20.06, 11.72. HRMS (ESI) (m / z) [M+H] + : 815.3719.

[0070] Example 13: Preparation of Compound A13 2.6 g (0.0045 mol) of N-Boc-4,7,10-trioxa-1,13-tridecanediamine was used to replace the tert-butyl (4-aminobutyl) carbamate in Example 8, while other conditions remained unchanged, to obtain compound A13 (0.1946 g, yield: 16.5%).

[0071] .

[0072] Data of compound A13: 1H NMR (600 MHz, DMSO-d6) δ 11.08 (s, 1H), 7.84 (t, J= 5.6 Hz, 1H), 7.58 (dd, J = 8.6, 7.1 Hz, 1H), 7.10 (d, J = 8.6 Hz, 1H), 7.01(d, J = 7.0 Hz, 1H), 6.66 (t, J = 6.0 Hz, 1H), 5.64 (d, J = 9.7 Hz, 1H), 5.53(s, 1H), 5.04 (dd, J = 12.9, 5.4 Hz, 1H), 4.02 (q, J = 7.1 Hz, 1H), 3.57 –3.43 (m, 12H), 3.06 (dt, J = 9.0, 6.1 Hz, 2H), 2.92 – 2.83 (m, 1H), 2.58 (q,J = 6.9 Hz, 3H), 2.36 (t, J = 6.9 Hz, 2H), 2.27 (ddd, J = 9.8, 7.1, 4.4 Hz,1H), 2.17 (td, J = 14.0, 4.0 Hz, 1H), 2.05 – 1.95 (m, 4H), 1.83 – 1.76 (m,3H), 1.65 – 1.55 (m, 4H), 1.55 – 1.49 (m, 1H), 1.46 – 1.37 (m, 2H), 1.28 (s,3H), 1.17 (t, J = 7.0 Hz, 3H), 0.87 (d, J = 6.4 Hz, 3H), 0.75 (d, J = 7.1 Hz,3H). 13C NMR (150 MHz, DMSO) δ 172.83, 171.24, 170.33, 170.10, 168.85, 167.34,146.46, 136.30, 132.21, 117.10, 110.38, 109.06, 103.57, 91.61, 90.58, 79.87,69.80, 69.74, 69.71, 69.54, 68.22, 68.04, 59.77, 51.12, 48.53, 44.57, 40.05,35.97, 35.85, 33.71, 31.64, 30.98, 29.54, 29.34, 29.00, 28.89, 25.52, 24.19,22.17, 20.77, 20.06, 14.10. HRMS (ESI) (m / z) [M+Na] + : 865.3861.

[0073] Example 14: Preparation of Compound B1 1 g (2S,4R)-1-((S)-2-amino-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.0023 mol) was dissolved in 20 mL dichloromethane, 0.947 g HATU (0.0025 mol) and 0.28 g 4-aminobutyric acid (0.0027 mol) were added, and 2.9 mL TEA was added dropwise. The mixture was reacted at room temperature for 5 h, and the reaction progress was monitored by TLC. After the reaction was completed, 40 mL dichloromethane was added to dilute the reaction solution, and the mixture was washed twice with 40 mL saturated NH4Cl, and then washed twice with 40 mL distilled water. Finally, the mixture was dried over anhydrous magnesium sulfate and separated and purified by silica gel column to obtain intermediate 12a (0.8359 g, yield: 68.7%).

[0074] 0.8 g 20-1 (0.0015 mol) was dissolved in 10 mL DMF, 0.9 g artesunate (0.0024 mol) and 0.8 g HATU (0.0021 mol) were added, and 1 mL DIPEA was added dropwise. The reaction was carried out at room temperature for 5 h. The reaction progress was monitored by TLC. After the reaction, the target compound B1 (0.1956 g, total yield: 9.5%) was obtained by separation and purification on a silica gel column.

[0075] .

[0076] Data of compound B1 1H NMR (600 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.38 (d, J =7.8 Hz, 1H), 7.88 (s, 1H), 7.84 (d, J = 9.3 Hz, 1H), 7.45 – 7.42 (m, 2H),7.40 – 7.37 (m, 2H), 5.76 (s, 1H), 5.66 (d, J = 9.7 Hz, 1H), 5.56 (s, 1H),4.92 (t, J = 7.2 Hz, 1H), 4.53 – 4.50 (m, 1H), 4.43 (t, J = 8.1 Hz, 1H), 3.64– 3.57 (m, 3H), 3.06 – 2.99 (m, 2H), 2.64 – 2.57 (m, 2H), 2.46 (m, 3H), 2.39(d, J = 7.0 Hz, 2H), 2.31 – 2.10 (m, 5H), 2.01 (dtd, J = 14.7, 8.0, 4.0 Hz,2H), 1.84 – 1.76 (m, 2H), 1.67 – 1.55 (m, 4H), 1.50 – 1.42 (m, 2H), 1.38 (d,J = 7.0 Hz, 3H), 1.35 – 1.30 (m, 1H), 1.29 (s, 3H), 1.21 – 1.14 (m, 2H), 0.94(d, J = 2.3 Hz, 9H), 0.89 (d, J = 6.3 Hz, 3H), 0.77 (d, J = 7.1 Hz, 3H). 13CNMR (151 MHz, DMSO) δ 171.69, 171.25, 170.61, 170.32, 169.55, 151.49, 147.76,144.66, 131.11, 129.69, 128.83, 126.38, 103.57, 91.61, 90.57, 79.87, 68.76,58.55, 56.43, 54.92, 51.13, 47.69, 44.58, 40.05, 38.26, 37.71, 35.96, 35.22,33.71, 32.54, 31.64, 29.56, 28.99, 26.45, 26.44, 25.71, 25.52, 24.19, 22.45,21.01, 20.07, 15.99, 11.73. HRMS (ESI) (m / z) [M+Na] + : 918.4298.

[0077] Example 15: Preparation of Compound B2 0.5 g of 8-aminooctanoic acid (0.0031 mol) was used to replace the 4-aminobutyric acid in Example 14, and other conditions remained unchanged to obtain compound B2 (0.3655 g, yield: 16.7%).

[0078] .

[0079] Data of compound B2: 1H NMR (600 MHz, DMSO-d6) δ 8.99 (s, 1H), 8.38 (d, J =7.8 Hz, 1H), 7.83 (s, 1H), 7.79 (d, J = 9.3 Hz, 1H), 7.46 – 7.42 (m, 2H),7.40 – 7.36 (m, 2H), 6.07 (s, 1H), 5.70 (d, J = 9.6 Hz, 1H), 5.10 (d, J = 3.6Hz, 1H), 4.92 (t, J = 7.2 Hz, 1H), 4.52 (d, J = 9.4 Hz, 1H), 4.42 (t, J = 8.0Hz, 1H), 4.28 (s, 1H), 3.77 (q, J = 7.9 Hz, 1H), 3.64 – 3.58 (m, 2H), 3.01(t, J = 6.3 Hz, 2H), 2.63 – 2.52 (m, 3H), 2.46 (s, 3H), 2.39 – 2.32 (m, 2H),2.24 (dt, J = 14.7, 7.6 Hz, 1H), 2.12 (dd, J = 8.1, 5.9 Hz, 2H), 2.01 (td, J= 9.3, 4.6 Hz, 2H), 1.90 – 1.76 (m, 4H), 1.71 (dt, J = 12.1, 4.1 Hz, 2H),1.54 – 1.40 (m, 5H), 1.40 – 1.30 (m, 7H), 1.24 (d, J = 4.7 Hz, 7H), 0.94 (s,9H), 0.89 (dd, J = 6.3, 2.9 Hz, 3H), 0.77 (dd, J = 7.1, 5.5 Hz, 3H). 13C NMR(150 MHz, DMSO) δ 172.03, 170.62, 170.17, 169.60, 168.59, 151.49, 147.76,144.67, 131.11, 129.69, 128.82, 126.38, 103.57, 92.71, 90.40, 79.20, 68.75,67.69, 58.53, 56.31, 54.21, 47.68, 46.34, 40.06, 38.54, 37.72, 35.19, 34.87,33.47, 29.94, 29.45, HRMS (ESI) (m / z) [M+Na] + :974.4937.

[0080] This embodiment also provides artesunate-based PROTAC compounds prepared by the aforementioned method and their applications.

[0081] Test Example 1 The MTT method was used to detect the cell anti-tumor activity and toxicity of the compound, and artesunate and CRBN were used as positive controls. Human bladder cancer cells (J82, T24, RT4) in the logarithmic phase and human normal bladder cells SV-HUC-1 were plated at 7000 cells per well, and the inoculated 96-well culture plates were placed in an incubator overnight. The 96-well plates were treated with different concentrations of compounds (0, 0.01, 0.1, 1, 10, 100 μM). After the treatment, the 96-well culture plates were returned to the incubator for 72 hours, and the absorbance value (OD) at a wavelength of 490 nm was read. Cell inhibition rate (%) = [(blank control OD value-drug OD value) / blank control group OD value] * 100%. After the test, the compounds of the present invention have a significant inhibitory effect on the proliferation of human bladder cancer cell lines, as shown in Table 1 below.

[0082] Table 1

[0083] By adopting the above technical scheme, the synthesized artesunate PROTAC derivatives of the present invention have an inhibitory effect on a variety of bladder cancer cells, among which compound A7 has the strongest activity in RT4 cells, and its inhibitory proliferation activity is enhanced by more than 10 times compared with artesunate.

[0084] Test Example 2 Western Blot was used to detect the degradation activity of compound A7 on GPX4 protein.

[0085] Experimental steps: RT4 or T24 cells were cultured at 2.5 × 10 5 The cells were seeded in 6-well plates at a density of 100 μg / ml and then treated with A7 at a specific concentration, while PBS was used as a control. The protein extracts were loaded and electrophoresed on SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. The blots were blocked with 5% milk and then incubated with GPX4 (CST) and GAPDH (CST) at 4 °C overnight. After washing with TBST for 30 minutes, the blots were incubated with secondary antibodies for 1 hour. After washing with PBS again, the blots were observed using the ChemiDoc system (Tanon 4600, Shanghai, China). Finally, the grayscale values ​​of the bands in the resulting images were measured using ImageJ software for protein quantification.

[0086] The test results are as attached in the instruction manual. Figure 1 And attached Figure 3 As shown, it can be seen that compound A7 can degrade GPX4 protein in human bladder cancer cells in a concentration-dependent manner.

[0087] Test Example 3 Western Blot was used to detect the degradation mechanism of GPX4 protein by compound A7.

[0088] Experimental steps: set up blank group, MG132 treatment group, A7 treatment group, A7 treatment group + MG132 treatment group. RT4 or T24 cells were cultured at 2.5 × 10 5 The cells were seeded in 6-well plates at a density of 100 μg / ml and treated as described above. The protein extracts were loaded and electrophoresed on SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked with 5% milk and then incubated with GPX4 (CST) and GAPDH (CST) at 4 °C overnight. After washing with TBST for 30 min, the blots were incubated with secondary antibodies for 1 h. After washing with PBS again, the blots were observed using the ChemiDoc system (Tanon 4600, Shanghai, China). Finally, the grayscale values ​​of the bands in the resulting images were measured using ImageJ software for protein quantification.

[0089] The test results are as attached in the instruction manual. Figure 2 And attached Figure 4As shown, it can be observed that in different GPX4 highly expressed cell lines, treatment of cells with proteasome inhibitor MG132 and pretreatment with proteasome inhibitor MG132 followed by addition of compound A7 showed no degradation activity, while direct addition of compound A7 showed significant GPX4 degradation. This indicates that pretreatment with proteasome inhibitor MG132 can competitively bind to or inhibit the degradation of compound A7, indicating that compound A7 degrades GPX4 through the ubiquitin-proteasome pathway.

[0090] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0091] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.

Claims

1. An artesunate derivative having GPX4 degradation activity, characterized in that the structural formula as follows: ； The R is a Cereblon protein ligand or a VHL protein ligand, and the Linker is a connecting group between artesunate and R.

2. The artesunate derivative according to claim 1, characterized in that: The Cereblon protein ligand is thalidomide or its derivatives; the structural formula of the VHL protein ligand is: 。 3. The structural formula of the thalidomide is: 。 4. The artesunate derivative according to claim 1 or 2, characterized in that: The linker is X-R1-X, wherein X is N or O, R1 is a saturated fatty chain, R2, CH2-R2-CH2, wherein R2 is (CH2CH2O) n1 -(CH2) n2 ; Or, X is N and R1 is (CH2)n-CO.

5. The artesunate derivative according to claim 3, characterized in that: The saturated fatty chain is a C3-11 saturated fatty chain, n1 is 3, n2 is 2; and n is 3-7.

6. The artesunate derivative according to claim 4, characterized in that: The artesunate derivative is the following compound, 。 7. The artesunate derivative according to claim 5, characterized in that: The artesunate derivative is the following compound, 8. A composition, characterized in that: The invention comprises the artesunate derivative or the pharmacologically acceptable salt thereof as claimed in any one of claims 1 to 6, and further comprises a pharmaceutically acceptable carrier, excipient, diluent, vehicle or a combination thereof.

9. Use of the artesunate derivative according to any one of claims 1 to 6, or the composition according to claim 7, wherein the artesunate derivative or the composition is used as a drug for degrading GPX4 or inhibiting GPX4.

10. The use according to claim 8, characterized in that: The drug is an anti-tumor drug.

11. The use according to claim 9, characterized in that: The tumor is bladder cancer.

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