Artesunate PROTAC derivative with GPX4 degradation activity, composition and application

Abstract

The present invention belongs to the technical field of anticancer drugs, and specifically relates to artesunate PROTAC derivatives, compositions and applications with GPX4 degradation activity, with the structural formula being as follows: #imgabs0#; wherein R is a Cereblon protein ligand or a VHL protein ligand, and the linker is a connecting group between artesunate and R; the present invention improves the anti-tumor effect thereof.

Description

Technical Field

[0001] The present 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 urinary tract malignancy in the world. Due to its high recurrence, bladder cancer usually requires long-term monitoring and multiple treatments for patients. 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 intracellular reactive oxygen species (ROS) levels. Multiple studies have demonstrated that ferroptosis plays a key role in tumor suppression and could be used for cancer therapy. Glutathione peroxidase 4 (GPX4) is a central regulator of ferroptosis; inactivation of GPX4 leads to the accumulation of phospholipid hydroperoxides (PLOOH), inducing cell membrane damage and ferroptosis. Numerous GPX4 inhibitors have been discovered, including RSL3, ML162, DPI compounds, FIN56, and FINO2. Currently, most reported small-molecule GPX4 inhibitors are covalent inhibitors containing a chloroacetamide group. Due to their strong electrophilicity, these compounds can bind to a wide range of proteins, resulting in low specificity for GPX4 binding. Therefore, the development of highly active and selective targeted GPX4 degraders has become a research hotspot in this field.

[0004] Proteolysis targeting chimeric molecules (PROTACs) are heterotypic bifunctional 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, PROTACs only require a brief interaction with their target protein to cause the loss of target protein function. PROTACs can significantly reduce the IC value of drug molecules due to their 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 semisynthetic, water-soluble derivative of artemisinin. As a first-line treatment for severe malaria, other pharmacological effects of artesunate are constantly being discovered. Currently, multiple studies have demonstrated that artesunate can exert certain anti-tumor effects by inducing ferroptosis, often accompanied by downregulation of GPX4 protein. However, the anti-cancer activity exhibited by artesunate is still significantly inferior to its anti-malarial activity. Furthermore, artesunate has drawbacks such as poor solubility, low oral bioavailability, and poor stability. Therefore, based on the advantages of PROTACs, 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. However, no similar reports have been reported to date. 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:

[0008] ;

[0009] The R is a Cereblon protein ligand or a VHL protein ligand, and the Linker is a connecting group between artesunate and R.

[0010] Optionally, the Cereblon protein ligand is thalidomide or a derivative thereof; the structural formula of the VHL protein ligand is:

[0011] .

[0012] The structural formula of the thalidomide is:

[0013] .

[0014] Optionally, the linker is X-R1-X, wherein X is N or O, and R1 is a saturated fatty chain; R2 is CH2-R2-CH2, and R2 is (CH2CH2O) n1 -(CH2) n2 ; Alternatively, X is N and R1 is (CH2)n-CO.

[0015] Optionally, the saturated fatty chain is a C3-11 saturated fatty chain, n1 is 3, n2 is 2, and n is 3-7.

[0016] Optionally, the artesunate derivative is the following compound,

[0017] .

[0018] Optionally, the artesunate derivative is the following compound,

[0019] 、 or ,

[0020] More preferably .

[0021] The present invention provides a composition comprising the artesunate derivative or a pharmacologically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, diluent, vehicle or a combination thereof.

[0022] 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.

[0023] Optionally, the drug is an anti-tumor drug.

[0024] The tumor is bladder cancer.

[0025] 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:

[0026] Step 1: Artesunate reacts with HO-R1-Br in the presence of an esterification catalyst to produce compounds 3a-3e;

[0027] Step 2: 2-(2,6-dioxo-piperidin-3-yl)-4-hydroxyisoindoline-1,3-dione reacts with intermediates 3a-3e to obtain artesunate PROTAC compounds A1-A5. The reaction scheme is as follows:

[0028] .

[0029] The linker is X-R1-X, wherein X is O, R1 is a saturated fatty chain, and R is thalidomide. The steps of the preparation method of artesunate derivatives are:

[0030] Step 1: 2-(2,6-dioxo-piperidin-3-yl)-4-hydroxyisoindoline-1,3-dione reacts with HO-R1-Br to give compounds 5f-5g;

[0031] Step 2: Compounds 5f-5g undergo esterification reaction with artesunate in the presence of an esterification catalyst to obtain artesunate PROTAC compounds A6-A7. The reaction scheme is as follows:

[0032] .

[0033] 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:

[0034] 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;

[0035] Step 2: Compounds 8a-8f are subjected to acid hydrolysis in the presence of trifluoroacetic acid to remove the Boc protecting group to obtain compounds 9a-9f;

[0036] 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:

[0037] .

[0038] The linker is X-R1-X, wherein X is N, R1 is (CH2)n-CO, and R is a VHL protein ligand. The steps of the preparation method of the artesunate derivative are as follows:

[0039] Step 1: (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-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;

[0040] 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.

[0041] .

[0042] 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. It is an excellent anti-tumor candidate compound.

[0043] The present invention develops novel and diverse artesunate PROTAC compounds with GPX4 degradation activity based on artesunate and its E3 ubiquitin ligases CRBN and VHL recruitment ligands. Compared to artesunate, the compounds exhibit significant anti-proliferation effects on tumor cells. Western blot experiments confirm that the protein-targeted degradation chimeras of the present invention bind to GPX4 and trigger its effective degradation, thereby inducing ferroptosis in cells, demonstrating their potential as anti-tumor agents for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the result of studying the concentration gradient degradation effect of compound A7 on the target protein GPX4 in RT4 cells.

[0045] Figure 2 This is the result of compound A7's degradation mechanism on the target protein GPX4 through ubiquitin-proteasome in RT4 cells.

[0046] Figure 3 This is the result of studying the concentration gradient degradation effect of compound A7 on the target protein GPX4 in T24 cells.

[0047] 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

[0048] 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 below through examples. 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 examples 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. Unless otherwise specified in the relevant experimental methods, they are all conventional methods in the art.

[0049] Example 1: Preparation of Compound A1

[0050]

[0051] 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 form a homogeneous solution. 3-Bromo-1-propanol (0.3948 g, 0.0028 mol) was added dropwise with stirring. The reaction was allowed to proceed at room temperature for 3.5 h, and the reaction progress was monitored by TLC. After completion of the reaction, intermediate 3a was isolated and purified using a silica gel column to obtain 0.4531 g, 34.6% yield.

[0052] Intermediate product 5a (0.4 g, 0.00079 mol) was dissolved in 10 mL of DMF. Potassium iodide (0.0131 g, 0.000079 mol), 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 with stirring. The mixture was incubated at 80°C in the dark for 12 h, and the reaction progress was monitored by TLC. After completion of the reaction, the reaction mixture was added to 100 mL of water and extracted three times with 80 mL of ethyl acetate. The combined organic phases were concentrated by rotary evaporation and purified by silica gel column chromatography to obtain the target compound A1 (0.022 g, 4% yield, as a white solid).

[0053] Data for 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). 1H NMR (600MHz, Methanol-d4) 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。

[0054] Example 2: Preparation of Compound A2:

[0055] By substituting 1.0 g (0.0060 mol) of 5-bromo-1-pentanol for the 3-bromo-1-propanol in Example 1 and keeping other conditions unchanged, compound A2 (0.2076 g, yield: 5.2%) was obtained.

[0056]

[0057] 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). 13C 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.

[0058] Example 3: Preparation of Compound A3:

[0059] By substituting 1.1 g (0.0061 mol) of 6-bromo-1-hexanol for 3-bromo-1-propanol in Example 1 and keeping other conditions unchanged, compound A3 (0.4558 g, yield: 11.2%) was obtained.

[0060]

[0061] Data of compound A3: 1 H NMR (600 MHz, Methanol-d4) 1H NMR (600 MHz, Methanol-d4) 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). 13 C 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。

[0062] Example 4: Preparation of Compound A4:

[0063] 1.2 g (0.0061 mol) of 7-bromo-1-heptanol was used to replace the 3-bromo-1-propanol in Example 1, while other conditions remained unchanged, to obtain compound A4 (0.3525 g, yield: 8.5%).

[0064]

[0065] Data of compound A4: 1 H 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). 13C 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.

[0066] Example 5: Preparation of Compound A5

[0067] By substituting 1.3 g (0.0061 mol) of 8-bromo-1-octanol for the 3-bromo-1-propanol in Example 1 and keeping other conditions unchanged, compound A5 (0.4017 g, yield: 9.5%) was obtained.

[0068]

[0069] Data of compound A5: 1H 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). 13 C 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.

[0070] Example 6: Preparation of Compound A6

[0071] 1.5 g of 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (0.005474 mol, 1 eq) was dissolved in 15 mL of DMF. 0.091 g of potassium iodide (0.000547 mol, 0.1 eq) and 0.919 g of sodium bicarbonate (0.01095 mol, 2 eq) were added dropwise under stirring. 10-bromo-1-decanol (1.421 g, 1.1 eq) was then added dropwise. The mixture was reacted at 80°C in the dark for 12 h. After completion of the reaction, the reaction mixture was added to 150 mL of water and extracted three times with 100 mL of ethyl acetate. The combined organic phases were concentrated by rotary evaporation and purified on a silica gel column to afford intermediate 5f (0.9811 g, 41.7% yield).

[0072] Intermediate product 5f (0.9 g, 0.0021 mol, 1 equivalent) was dissolved in 15 mL of dichloromethane. 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. The reaction was allowed to proceed at room temperature for 3.5 hours, with TLC monitoring of the reaction progress. After completion of the reaction, the target compound A6 was isolated and purified using a silica gel column to obtain the target compound A6 (0.2592 g, light yellow oil, yield: 15.5%).

[0073]

[0074] Data of compound A6: 1H 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。

[0075] Example 7: Preparation of Compound A7

[0076] By substituting 1.4 g (0.0061 mol) of 11-bromo-1-undecanol for the 10-bromo-1-decanol in Example 6 and keeping other conditions unchanged, compound A7 (1.0474 g, yield: 23.5%) was obtained.

[0077]

[0078] 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.

[0079] Example 8: Preparation of Compound A8

[0080] 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. 818 μL of DIPEA (0.0047 mol) and 0.75 g of tert-butyl (4-aminobutyl) carbamate (0.0040 mol) were added dropwise with stirring. The reaction was allowed to proceed at 90°C for 12 h, and the reaction progress was monitored by TLC. After completion of 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 purified using a silica gel column to obtain intermediate 8a (0.7364 g, 44.9% yield).

[0081] Intermediate product 1 (0.7 g, 0.0016 mol) was added to a mixture of TFA / DCM = 1 / 1 (6 mL) and stirred at room temperature for 1 h. After the reaction, the product was spin-dried and purified by silica gel column to obtain intermediate product 9a (0.49 g, yield: 89%).

[0082] 0.49 g (0.0014 mol) of intermediate 2 was dissolved in 8 mL of DMF. 0.8 g (0.0020 mol) of HATU and 0.6 g (0.0016 mol) of artesunate were added. 1 mL (0.0057 mol) of DIPEA was added dropwise with stirring. The reaction was allowed to react at room temperature for 3.5 hours, and the reaction progress was monitored by TLC. After completion of the reaction, the reaction solution was added to 80 mL of water and extracted three times with 60 mL of ethyl acetate. The combined organic phases were concentrated by rotary evaporation and purified on a silica gel column to obtain the target compound A8 (0.2889 g, total yield: 11.3%).

[0083]

[0084] 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.

[0085] Example 9: Preparation of Compound A9

[0086] By replacing the tert-butyl (4-aminobutyl) carbamate in Example 8 with 1.0 g (0.0049 mol) of tert-butyl (5-aminopentyl) carbamate and other conditions remaining unchanged, compound A9 (0.1075 g, yield: 10.6%) was obtained.

[0087]

[0088] 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.

[0089] Example 10: Preparation of Compound A10

[0090] By substituting 1.0 g (0.0046 mol) of N-Boc-1,6-hexanediamine for the tert-butyl (4-aminobutyl) carbamate in Example 8 and keeping other conditions unchanged, compound A10 (0.1302 g, yield: 12.3%) was obtained.

[0091]

[0092] 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.

[0093] Example 11: Preparation of Compound A11

[0094] By substituting 1.0 g (0.0041 mol) of N-Boc-1,8-octanediamine for the tert-butyl (4-aminobutyl) carbamate in Example 8 and keeping other conditions unchanged, compound A11 (0.0902 g, yield: 8.4%) was obtained.

[0095]

[0096] 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.

[0097] Example 12: Preparation of Compound A12

[0098] By replacing the tert-butyl (4-aminobutyl)carbamate in Example 8 with 1.0 g (0.0031 mol) of tert-butyl 12-amino-4,7,10-trioxadodecanoate and keeping other conditions unchanged, compound A12 (0.2109 g, yield: 18.5%) was obtained.

[0099]

[0100] 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.

[0101] Example 13: Preparation of Compound A13

[0102] By replacing the tert-butyl (4-aminobutyl) carbamate in Example 8 with 2.6 g (0.0045 mol) of N-Boc-4,7,10-trioxa-1,13-tridecanediamine and maintaining other conditions unchanged, compound A13 (0.1946 g, yield: 16.5%) was obtained.

[0103]

[0104] 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.

[0105] Example 14: Preparation of Compound B1

[0106] 1 g of (2S,4R)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.0023 mol) was dissolved in 20 mL of dichloromethane. 0.947 g of HATU (0.0025 mol) and 0.28 g of 4-aminobutyric acid (0.0027 mol) were added dropwise. 2.9 mL of TEA was added dropwise and the reaction was allowed to react at room temperature for 5 h. The reaction progress was monitored by TLC. After completion of the reaction, the reaction solution was diluted with 40 mL of dichloromethane and washed twice with 40 mL of saturated NH4Cl and then twice with 40 mL of distilled water. Finally, the product was dried over anhydrous magnesium sulfate and purified by silica gel column chromatography to obtain intermediate 12a (0.8359 g, 68.7% yield).

[0107] 0.8 g of 20-1 (0.0015 mol) was dissolved in 10 mL of DMF, and 0.9 g of artesunate (0.0024 mol) and 0.8 g of HATU (0.0021 mol) were added. 1 mL of DIPEA was added dropwise. The mixture was reacted 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.

[0108]

[0109] Data of compound B11 H 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.

[0110] Example 15: Preparation of Compound B2

[0111] 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%).

[0112]

[0113] 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.

[0114] This embodiment also provides artesunate-based PROTAC compounds prepared by the aforementioned method and their applications.

[0115] Test Example 1

[0116] The antitumor activity and cytotoxicity of the compounds were assessed using the MTT assay, with artesunate and CRBN used as positive controls. Human bladder cancer cells (J82, T24, and RT4) in the logarithmic phase and human normal bladder SV-HUC-1 cells were seeded at 7,000 cells per well. The seeded 96-well plates were incubated overnight in an incubator and treated with various concentrations of the compounds (0, 0.01, 0.1, 1, 10, and 100 μM). After treatment, the plates were returned to the incubator and incubated for 72 hours. The absorbance (OD) at 490 nm was read. The cell inhibition rate (%) was calculated as [(blank control OD value - drug OD value) / blank control OD value] * 100%. The compounds of the present invention demonstrated significant antiproliferative effects against human bladder cancer cell lines, as shown in Table 1.

[0117] Table 1

[0118]

[0119] By adopting the above technical scheme, the artesunate PROTAC derivatives synthesized by the present invention have an inhibitory effect on various bladder cancer cells. Among them, compound A7 has the strongest activity in RT4 cells, and its anti-proliferation activity is enhanced by more than 10 times compared with artesunate.

[0120] Test Example 2

[0121] Western Blot was used to detect the degradation activity of compound A7 on GPX4 protein.

[0122] Experimental steps: RT4 or T24 cells were cultured at 2.5 × 10 5 Cells were seeded at a specific density in 6-well plates and then treated with A7 at the specified concentrations, with PBS used as a control. Protein extracts were loaded and electrophoresed on SDS-PAGE before being transferred to a polyvinylidene fluoride (PVDF) membrane. The blots were blocked with 5% milk and incubated with GPX4 (CST) and GAPDH (CST) overnight at 4°C. After washing with TBST for 30 minutes, the blots were incubated with secondary antibodies for 1 hour. After another wash with PBS, the blots were visualized 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.

[0123] The test results are as shown in the instructions. 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.

[0124] Test Example 3

[0125] Western Blot was used to detect the degradation mechanism of GPX4 protein by compound A7.

[0126] Experimental steps: blank group, MG132-treated group, A7-treated group, and A7-treated group + MG132-treated group were set up. RT4 or T24 cells were cultured at 2.5 × 10 5 Cells were seeded at a density of 100 μg / ml in 6-well plates and grouped and treated as described above. Protein extracts were loaded and electrophoresed on SDS-PAGE before being transferred to polyvinylidene difluoride (PVDF) membranes. The blots were blocked with 5% milk and incubated with GPX4 (CST) and GAPDH (CST) overnight at 4°C. After washing with TBST for 30 minutes, the blots were incubated with secondary antibodies for 1 hour. After washing again with PBS, the blots were visualized 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.

[0127] The test results are as shown in the instructions. Figure 2 and attached Figure 4As shown, it can be observed that in different cell lines with high GPX4 expression, treatment of cells with the proteasome inhibitor MG132 or pretreatment with the proteasome inhibitor MG132 followed by the addition of compound A7 showed no degradation activity, while direct addition of compound A7 exhibited significant GPX4 degradation. This indicates that pretreatment with the 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.

[0128] Those 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 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.

[0129] The one or more embodiments of this application are intended to encompass all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of this application should be included in the scope of protection of this application.

Claims

1. An artesunate derivative having anti-bladder cancer activity, characterized in that: The artesunate derivative is the following compound, 。 2. The artesunate derivative according to claim 1, wherein The artesunate derivative is the following compound, 。 3. A composition characterized in that: The invention comprises the artesunate derivative or the pharmacologically acceptable salt thereof according to any one of claims 1 to 2, and further comprises a pharmaceutically acceptable carrier, excipient, diluent, vehicle or a combination thereof.

4. A use of the artesunate derivative according to any one of claims 1 to 2, or the composition according to claim 3, characterized in that: Application of the artesunate derivative or composition in the preparation of anti-bladder cancer drugs.

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