A gpx4 protein degradation agent based on a hydrophobic tag, and a preparation method and application thereof

Abstract

The application discloses a GPX4 protein degrading agent based on a hydrophobic label, a preparation method and application thereof, and belongs to the technical field of medicines, wherein a structural general formula of the GPX4 protein degrading agent based on the hydrophobic label and pharmaceutically acceptable salts thereof is shown as (I). Pharmacological experiments show that the compound has strong anti-proliferation activity on ferroptosis-sensitive cells HT1080, and can effectively down-regulate the GPX4 protein level in the HT1080 cells. The compound has application value of the GPX4 degrading agent, can be applied to preparation of an anti-tumor drug, and has a good market application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a GPX4 protein degrading agent based on a hydrophobic label, a preparation method and application thereof. BACKGROUND

[0002] In 2012, the Stockwell group proposed the concept of "ferroptosis". As a new form of cell death, ferroptosis is an iron-dependent, lipid peroxidation accumulation-induced programmed cell death. Ferroptosis is closely related to cancer, neurodegenerative diseases and many other diseases in the human body, and the regulation of ferroptosis may be a potential breakthrough in the treatment of these diseases. In the study of the mechanism of ferroptosis, it was found that the cystine / glutamate antiporter system (Xc-)-glutathione peroxidase 4 (GPX4) axis is an important part of the ferroptosis defense. GPX4 can use glutathione to reduce toxic lipid peroxide to nontoxic alcohol, thereby avoiding ferroptosis. Studies have shown that inhibition of the function of GPX4 can induce ferroptosis.

[0003] GPX4 is a selenium-containing protein, and the catalytic center is selenocysteine (Sec). The drug binding pocket found so far is a shallow pocket on the surface of the protein, so the inhibitors targeting GPX4 usually have a covalent binding warhead, which plays an inhibitory role by irreversible covalent binding with the active center selenocysteine of GPX4. However, these inhibitors generally have the disadvantages of high toxicity, poor pharmacokinetics, and easy off-target.

[0004] In recent years, targeted protein degradation (TPD) technology has developed rapidly. Compared with traditional small molecule inhibitors, TPD drugs have the advantages of rapidly degrading target proteins, allowing protein site mutations, overcoming non-druggable targets, and reducing drug resistance. Therefore, converting small molecule drugs into corresponding degrading agents can overcome the shortcomings of drugs or targets themselves, thereby achieving more ideal results. A research group designed a PROTAC degrading agent based on GPX4 inhibitor, and experiments proved that it can target the down-regulation of GPX4. Hydrophobic tag is a new targeted degradation technology. By linking a large hydrophobic fragment to the target protein ligand with a suitable linker, a hydrophobic fragment is introduced on the surface of the target protein ligand when it recognizes the target protein, which simulates the misfolding of the protein, thereby causing the target protein to be degraded by the protein quality control system. Compared with PROTAC, it maintains the advantages of targeted protein degradation technology and has a smaller molecular weight without the risk of potential teratogenicity. However, the specific action principle of the hydrophobic tag degradation technology is still unknown, so the expansion and application of this technology are in urgent need, which is conducive to the development and improvement of the hydrophobic tag degradation technology. SUMMARY

[0005] The purpose of the present application is to provide a GPX4 protein degrading agent based on hydrophobic tag and its preparation method and application. The present application provides the activity screening results of GPX4 degrading agent based on hydrophobic tag on iron death sensitive cell HT1080 and its application as GPX4 degrading agent, and experiments prove that the compound of the present application can be used as GPX4 degrading agent for preparing antitumor drugs.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The first aspect of the present application provides a GPX4 protein degrading agent based on hydrophobic tag and its pharmaceutically acceptable salt, which has the structure shown in the following formula (1):

[0008]

[0009] Among them, R1 is selected from the following groups:

[0010]

[0011] X is selected from the following linking groups:

[0012]

[0013] n is 1-20, and m is 1-20.

[0014] Further preferred, the above-mentioned compounds of general formula I are the following compounds:

[0015] Compound R-A1 : (1 S,3R)-1 -(4-((2-(2-((3R,5R,7R)-adamantan-1 - yl)acetamido)ethoxy)ethyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro- 1 H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0016] Compound R-A2: (1 S,3R)-1 -(4-((2-(2-(2-(2-((3R,5R,7R)-adamantan-1 - yl)acetamido)ethoxy)ethoxy)ethyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9- tetrahydro-1 H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0017] Compound R-A3: (1 S,3R)-1 -(4-((1 -((3R,5R,7R)-adamantan-1 -yl)-2-oxo-6,9,12- trioxa-3-azatetradecan-14-yl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9- tetrahydro-1 H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0018] Compound R-B1 : (1 S,3R)-1 -(4-((2-(2-((3R,5R,7R)-adamantan-1 - yl)acetamido)ethyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H- pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0019] Compound R-B2: (1 S,3R)-1 -(4-((4-(2-((3R,5R,7R)-adamantan-1 - yl)acetamido)butyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H- pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0020] Compound R-B3: (1 S,3R)-1 -(4-((5-(2-((3R,5R,7R)-adamantan-1 - yl)acetamido)pentyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H- pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0021] Compound R-B4: (1S,3R)-1-(4-((6-(2-((3R,5R,7R)-adamantan-1-yl)acetylamino)hexyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0022] Compound R-B5: (1S,3R)-1-(4-((7-(2-((3R,5R,7R)-adamantan-1-yl)acetylamino)heptyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0023] Compound R-B6: (1S,3R)-1-(4-((8-(2-((3R,5R,7R)-adamantan-1-yl)acetylamino)octyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0024] Compound R-B7: (1S,3R)-1-(4-((10-(2-((3R,5R,7R)-adamantan-1-yl)acetylamino)decyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0025] Compound R-C1: (1S,3R)-1-(4-(4-(2-((3R,5R,7R)-adamantan-1-yl)acetyl)piperazine-1-carbonyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0026] Compound R-C2: (1S,3R)-1-(4-(4-(2-(2-((3R,5R,7R)-adamantan-1-yl)acetylamino)ethyl)piperazine-1-carbonyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0027] Compound R-C3: (1S,3R)-1-(4-(4-(4-(2-((3R,5R,7R)-adamantan-1-yl)acetylamino)butyl)piperazine-1-carbonyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester;

[0028] Compound R-C4: (1S, 3R)-1-(4-(4-(6-(2-((3R, 5R, 7R)-adamantan-1-yl)acetylamino)hexyl)piperazine-1-carbonyl)phenyl)-2-(2-chloroacetyl)-2, 3, 4, 9-tetrahydro-1H-pyrido[3, 4-b]indole-3-carboxylic acid methyl ester;

[0029] Compound R-C5: (1S, 3R)-1-(4-(4-(8-(2-((3R, 5R, 7R)-adamantan-1-yl)acetylamino)octyl)piperazine-1-carbonyl)phenyl)-2-(2-chloroacetyl)-2, 3, 4, 9-tetrahydro-1H-pyrido[3, 4-b]indole-3-carboxylic acid methyl ester;

[0030] Compound R-C6: (1S, 3R)-2-propenoyl-1-(4-(4-(6-(2-((3R, 5R, 7R)-adamantan-1-yl)acetylamino)hexyl)piperazine-1-carbonyl)phenyl)-2, 3, 4, 9-tetrahydro-1H-pyrido[3, 4-b]indole-3-carboxylic acid methyl ester;

[0031] or Compound R-C7: (1S, 3R)-1-(4-(4-(6-(2-((3R, 5R, 7R)-adamantan-1-yl)acetylamino)hexyl)piperazine-1-carbonyl)phenyl)-2-propynoyl-2, 3, 4, 9-tetrahydro-1H-pyrido[3, 4-b]indole-3-carboxylic acid methyl ester.

[0032] Further preferably, the pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt; the inorganic acid is at least one selected from hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid; and the organic acid is at least one selected from methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, and mandelic acid.

[0033] The second aspect of the present application provides a preparation method of the GPX4 protein degradation agent based on the hydrophobic tag and pharmaceutically acceptable salts thereof, which comprises the following steps:

[0034] (1) Synthesis of intermediates

[0035] Formaldehyde benzene carboxylic acid 1 reacts with Boc 2O to generate intermediate 2; intermediate 2 is cyclized with D-tryptophan methyl carbonate hydrochloride 3 to obtain intermediate 4;

[0036]

[0037] (2) Synthesis of compounds

[0038] The intermediate 4 is subjected to condensation reaction with the oxygen chain 5a-5c to obtain the intermediate 6a-6c; the intermediate 6a-6c is subjected to Boc protection group removal with TFA and then subjected to condensation reaction with adamantane acetic acid 7 to obtain the intermediate 8a-8c; finally, the intermediate 8a-8c is subjected to reaction with chloroacetyl chloride 9 to obtain the target compound R-A1-R-A3;

[0039]

[0040] Alternatively, the intermediate 4 is subjected to condensation reaction with the alkane chain 10a-10g to obtain the intermediate 11a-11g; the intermediate 11a-11g is subjected to Boc protection group removal with TFA and then subjected to condensation reaction with adamantane acetic acid 7 to obtain the intermediate 12a-12g; finally, the intermediate 12a-12g is subjected to reaction with chloroacetyl chloride 9 to obtain the target compound R-B1-R-B7;

[0041]

[0042] Alternatively, the intermediate 4 is subjected to condensation reaction with N-Boc-piperazine to obtain the intermediate 14; the intermediate 14 is subjected to Boc protection group removal with TFA and then subjected to condensation reaction with adamantane acetic acid 7 to obtain the intermediate 15; finally, the intermediate 15 is subjected to reaction with chloroacetyl chloride 9 to obtain the target compound R-C1;

[0043]

[0044] Further, the preparation method further comprises synthesis of the compounds R-C2-R-C5:

[0045] The dibromoalkane with different lengths is subjected to substitution reaction with isoindoline-1,3-dione 16 in the presence of K2CO3 to obtain the intermediate 17a-17d; the intermediate 17a-17d is subjected to substitution reaction with N-Boc-piperazine 13 in the presence of K2CO3 to obtain the intermediate 18a-18d; the intermediate 18a-18d is subjected to reduction in the action of 85% hydrazine hydrate and then subjected to reaction with adamantane acetic acid 7 to obtain the intermediate 19a-19d; the intermediate 19a-19d and the intermediate 4 are subjected to Boc protection group removal in TFA respectively, and the obtained products are subjected to condensation reaction with each other to obtain the intermediate 20a-20d; finally, the intermediate 20a-20d is subjected to reaction with chloroacetyl chloride 9 to obtain the target compound R-C2-R-C5, the intermediate 20c is subjected to reaction with acryloyl chloride 21 to obtain the target compound R-C6, and the intermediate 20c is subjected to reaction with propiolic acid 22 to obtain the target compound R-C7;

[0046]

[0047] The application provides application of the GPX4 protein inhibitor based on the hydrophobic tag and pharmaceutically acceptable salts thereof in preparation of a GPX4 protein inhibitor.

[0048] The third aspect of the present application provides the use of the GPX4 protein degradation agent based on the hydrophobic tag and pharmaceutically acceptable salts thereof in the preparation of an antitumor drug.

[0049] Further, the tumor is an iron death sensitive tumor. The iron death sensitive tumor includes human breast cancer, human fibrosarcoma and renal cancer.

[0050] Compared with the prior art, the advantages and technical effects of the present application are that the newly synthesized GPX4 protein degradation agent based on the hydrophobic tag of the present application is tested for in vitro antitumor activity on HT1080 cells, and the relevant data are shown in Table 2, with the GPX4 inhibitor RSL3 reported in the literature as a positive drug. Experiments prove that the newly synthesized GPX4 protein degradation agent based on the hydrophobic tag of the present application exhibits significant antiproliferative activity on HT1080 cells. Among them, the activity of compounds such as R-B4, R-B5 and R-C1 is better than that of the positive drug RSL3, which has further research value and can be further developed.

[0051] The Western blot experiment of the newly synthesized GPX4 degradation agent based on the hydrophobic tag technology of the present application on HT1080 cells, and the relevant results are shown in Table 3 and Figure 1 Compounds with similar activity to RSL3 are further selected at two concentrations of 0.05 μM and 0.02 μM. At the same concentration, compounds R-B4, R-B5 and other compounds exhibit better GPX4 degradation activity than RSL3. R-C1 with better activity has already exhibited better GPX4 degradation activity than RSL3 at 0.2 μM at 0.02 μM, which also corresponds to their activity and can prove that the increase in the activity of the compound is due to the enhancement of GPX4 degradation.

[0052] Experiments show that the GPX4 protein degradation agent based on the hydrophobic tag provided by the present application has significant antiproliferative activity and GPX4 degradation activity on HT1080 cells. The compounds of the present application have application value as GPX4 degradation agents and can be applied to the preparation of antitumor drugs, which have good market application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 GPX4 degradation activity results of representative compound R-C1 on HT1080 cells. DETAILED DESCRIPTION

[0054] In order to more clearly illustrate the present application, the present application will be further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the specific description below is illustrative rather than limiting and should not limit the protection scope of the present application.

[0055] The experimental methods in the following examples, unless otherwise specified, were generally carried out according to the conventional conditions or the conditions recommended by the manufacturers. The percentages described in the examples are percentages by weight, unless otherwise specified.

[0056] The specific compound structure and NMR data of the GPX4 protein degrading agent provided by the application are shown in Table 1 below:

[0057] Table 1. Structure and NMR data of the compound provided by the application

[0058]

[0059]

[0060]

[0061]

[0062]

[0063] Example 1: Preparation of (1S,3R)-methyl 1-(4-(tert-butoxycarbonyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate 4

[0064] (1) Preparation of intermediate 2: tert-butyl 4-formylbenzoate

[0065] The raw material p-formylbenzoic acid 1 (1 g, 6.66 mmol) was dissolved in 10 mL of THF, Boc2O (2.91 g, 3.06 mL, 13.32 mmol,) was added, DMAP (162.8 mg, 1.33 mmol), and the reaction was carried out at 80°C for 2h. TLC detection showed that the reaction was complete, the reaction liquid was quenched with 10 mL of saturated NaHCO3, and the water layer was extracted with dichloromethane (50 mL x 3). The combined organic layer was dried with anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA = 20:1) to obtain 800 mg of colorless oily liquid, which was intermediate 2, with a yield of 60%. 1 H NMR (400 MHz, Chloroform-d) δ: 10.08 (s, 1H), 8.16-8.12 (m, 2H), 8.07 (d, J = 8.4 Hz, 1H), 7.93-7.91 (m, 1H), 1.61 (s, 9H).

[0066] (2) Preparation of intermediate 4: (1S,3R)-methyl 1-(4-(tert-butoxycarbonyl)phenyl)-2,3,4,9-tetrahydropyrido[3,4-b]indole-3-carboxylate

[0067] D-tryptophan methyl ester hydrochloride (1.16 g, 4.54 mmol) was added to 20 mL of DCM, TEA (497.8 mg, 684 μL, 4.92 mmol) was added, and the mixture was stirred at room temperature for 1 h to dissolve completely, and then the solvent was evaporated under reduced pressure and dried. The dried product was dissolved in 30 mL of DCM. Intermediate 2 (780 mg, 3.78 mmol) and TFA (43.1 mg, 28 μL, 0.38 mmol) were added, and the mixture was reacted at 45 °C for 1 h, and then TFA (1.29 g, 843 μL, 11.4 mmol) was added, and the mixture was reacted at 45 °C for 8 h. The reaction was completed as detected by TLC, and the reaction solution was quenched with 20 mL of saturated NaHCO3, and the aqueous layer was extracted with dichloromethane (100 mL x 3). The combined organic layer was dried with anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA = 4:1) to obtain 351 mg of white solid, which was Intermediate 4, with a yield of 23%. 1 H NMR (400 MHz, Chloroform-d) δ: 7.94 (d, J = 8.2 Hz, 2H), 7.58 - 7.53 (m, 2H), 7.35 (d, J = 8.2 Hz, 2H), 7.26 - 7.22 (m, 1H), 7.15 (pd, J = 7.0, 1.1 Hz, 2H), 5.47 (s, 1H), 3.96 (t, J = 6.0 Hz, 1H), 3.72 (s, 3H), 3.21 (ddd, J = 50.8, 15.4, 6.4 Hz, 2H), 1.58 (s, 9H).

[0068]

[0069] Reagents and conditions: a: DMAP, Boc2O, THF, 80 °C, 2 h, yield 60%. b: TEA, TFA, DCM, 45 °C, 10 h, yield 23%.

[0070] Example 2: Preparation of (1S,3R)-1-(4-((2-(2-((3R,5R,7R)-adamantan-1-yl)acetamido)ethoxy)ethyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester R-A1

[0071] (1) Preparation of Intermediate 6a: (1S,3R)-1-(4-((2-((tert-butoxycarbonyl)amino)ethoxy)ethyl)carbamoyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester

[0072] Intermediate 4 (100 mg, 0.25 mmol) was dissolved in 4 mL of DCM, 2 mL of TFA was added, and the reaction was allowed to proceed at room temperature for 2 h. TLC detection showed that the reaction was complete, the solvent was evaporated under reduced pressure, and the product was used directly in the next step without purification. The resulting crude product was dissolved in 4 mL of DMF, HBTU (139.9 mg, 0.37 mmol) and DIPEA (47.7 mg, 64 μL, 0.37 mmol) were added, and the mixture was stirred for 5 min. Compound 5a (55.3 mg, 0.27 mmol) was then added, and the reaction was allowed to proceed at room temperature for 8 h. TLC detection showed that the reaction was complete, 100 mL of saturated NaCl solution was added, and the aqueous layer was extracted with ethyl acetate (50 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 50:1) to give 55 mg of yellow solid, which was intermediate 6a, in a yield of 42%. 1 H NMR (400 MHz, Chloroform-d) δ: 7.84 - 7.70 (m, 2H), 7.54 (d, J = 7.4 Hz, 1H), 7.33 (d, J = 7.1 Hz, 2H), 7.17 - 7.09 (m, 2H), 6.60 (s, 1H), 5.44 (s, 1H), 4.84 (s, 1H), 3.93 (t, J = 5.5 Hz, 1H), 3.71 (s, 3H), 3.63 (d, J = 8.2 Hz, 4H), 3.53 (t, J = 5.3 Hz, 2H), 3.30 (d, J = 7.5 Hz, 2H), 3.24 (d, J = 5.0 Hz, 1H), 3.18 - 3.08 (m, 1H), 1.39 (s, 9H).

[0073] (2) Preparation of intermediate 8a: (1S,3R)-1-(4-((2-(2-((3R,5R,7R)-adamantan-1-yl)acetamido)ethoxy)ethyl)carbamoyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester

[0074] Compound 6a (55 mg, 0.1 mmol) was dissolved in 4 mL DCM, 2 mL TFA was added, and the reaction was carried out at room temperature for 2 h. TLC detection showed that the reaction was complete, the solvent was evaporated under reduced pressure, and the product was directly used in the next step without purification. The obtained crude product was dissolved in 4 mL DMF, HBTU (56.2 mg, 0.15 mmol) and DIPEA (19.1 mg, 26 μL, 0.15 mmol) were added, stirred for 5 min, then compound 7 (21.1 mg, 0.11 mmol) was added, and the reaction was carried out at room temperature for 8 h. TLC detection showed that the reaction was complete, 100 mL saturated NaCl solution was added, and the aqueous layer was extracted with ethyl acetate (50 mL x 3). The combined organic layers were dried with anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 100:3) to obtain 20 mg of yellow solid, which was intermediate 8a, with a yield of 33%. 1 H NMR (400 MHz, Chloroform-d) δ: 7.82 (s, 1H), 7.74 (d, J = 8.2 Hz, 2H), 7.54 (d, J = 7.3 Hz, 1H), 7.33 (d, J = 8.2 Hz, 2H), 7.23 (s, 1H), 7.14 (dtd, J = 14.4, 7.1, 1.2 Hz, 2H), 6.68 (s, 1H), 5.79 (t, J = 5.2 Hz, 1H), 5.45 (s, 1H), 3.94 (t, J = 6.0 Hz, 1H), 3.71 (s, 3H), 3.62 (d, J = 2.1 Hz, 4H), 3.54 (t, J = 5.1 Hz, 2H), 3.41 (q, J = 5.3 Hz, 2H), 3.30 - 3.10 (m, 2H), 1.88 (d, J = 8.8 Hz, 5H), 1.64 (d, J = 12.1 Hz, 3H), 1.55 (d, J = 2.4 Hz, 9H).

[0075] (3) Preparation of target compound R-A1:

[0076] Compound 8a (20 mg, 0.03 mmol) was dissolved in 5 mL DCM, TEA (5 mg, 7 μL, 0.05 mmol) was added, and compound 9 (5.5 mg, 4 μL, 0.05 mmol) was diluted in 1 mL DCM and slowly dropped into the above reaction system. The reaction was carried out at room temperature for 10 h. TLC detection showed that the reaction was complete, 50 mL saturated NaCl solution was added, and the aqueous layer was extracted with dichloromethane (20 mL x 3). The combined organic layers were dried with anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 100:3) to obtain 17 mg of white solid, which was compound R-A1, with a yield of 76%. 1H NMR(400MHz,Chloroform-d)δ:8.75(d,J=69.1Hz,1H),7.67(d,J=68.0Hz,2H),7.50(d, J=8.3Hz,1H),7.30(s,1H),7.19(s,1H),7.09(p,J=6.9Hz,2H),6.80(d,J=116.8Hz,1H) ,6.14(d,J=43.3Hz,1H),5.90(s,1H),5.20(s,1H),4.08(d,J=31.2Hz,2H),3.57(d,J=4 5.6Hz,14H),1.87(d,J=11.6Hz,5H),1.66(d,J=13.0Hz,3H),1.62(s,2H),1.53(s,7H). 13 C NMR (400MHz, DMSO) δ172.20,170.50,168.26,166.63,147.37,136.90,134.31,133.37,128.35,127.72,127.09,126.20,126.12,12 1.81,119.36,118.52,111.80,104.03,69.48,69.21,57.05,56.52,53.27,50.44,43.80,42.52,38.79,36.89,32.59,28.48,26.82.

[0077]

[0078] Reagents and conditions: a: (1) TFA, DCM, rt, 4h, yield 100%. (2) HBTU, DIPEA, rt, 8h, yield 20-95%. b: (1) TFA, DCM, rt, 4h, yield 100%. (2) HBTU, DIPEA, rt, 8h, yield 20-95%.c:TEA,DCM,rt,8h,yield40-85%.

[0079] Example 3

[0080] Preparation of compound R-A2: In Example 2, compound 5a was replaced with compound 5b in the first step, and the rest was the same as in Example 2. Compound R-A2 was prepared with a yield of 30%.

[0081] Example 4

[0082] Preparation of compound R-A3: In Example 2, compound 5a was replaced with compound 5c in the first step, and the rest was the same as in Example 2. Compound R-A3 was prepared with a yield of 33%.

[0083] Example 5

[0084] Preparation of compound R-B1 : replace compound 5a in the first step of example 2 with compound 10a, and other same as example 2, to prepare compound R-B1 with a yield of 37%.

[0085] Example 6

[0086] Preparation of compound R-B2: replace compound 5a in the first step of example 2 with compound 10b, and other same as example 2, to prepare compound R-B2 with a yield of 31%.

[0087] Example 7

[0088] Preparation of compound R-B3: replace compound 5a in the first step of example 2 with compound 10c, and other same as example 2, to prepare compound R-B3 with a yield of 39%.

[0089] Example 8

[0090] Preparation of compound R-B4: replace compound 5a in the first step of example 2 with compound 10d, and other same as example 2, to prepare compound R-B4 with a yield of 37%.

[0091] Example 9

[0092] Preparation of compound R-B5: replace compound 5a in the first step of example 2 with compound 10e, and other same as example 2, to prepare compound R-B5 with a yield of 34%.

[0093] Example 10

[0094] Preparation of compound R-B6: replace compound 5a in the first step of example 2 with compound 10f, and other same as example 2, to prepare compound R-B6 with a yield of 38%.

[0095] Example 11

[0096] Preparation of compound R-B7: replace compound 5a in the first step of example 2 with compound 10g, and other same as example 2, to prepare compound R-B7 with a yield of 35%.

[0097]

[0098] Reagents and conditions: a: (1) TFA, DCM, rt, 4 h, yield 100%. (2) HBTU, DIPEA, rt, 8 h, yield 20-95%. b: (1) TFA, DCM, rt, 4 h, yield 100%. (2) HBTU, DIPEA, rt, 8 h, yield 20-95%. c: TEA, DCM, rt, 8 h, yield 40-85%.

[0099] Example 12

[0100] Preparation of compound R-C1: replace compound 5a in the first step in example 2 with compound 13, and other as example 2, to give compound R-C1 in 26% yield.

[0101]

[0102] Reagents and conditions: a: (1) TFA, DCM, rt, 4 h, yield 100%. (2) HBTU, DIPEA, rt, 8 h, yield 89%. b: (1) TFA, DCM, rt, 4 h, yield 100%. (2), HBTU, DIPEA, rt, 8 h, yield 84%. c: TEA, DCM, rt, 8 h, yield 62%.

[0103] Example 13

[0104] Preparation of (1S,3R)-1-(4-(4-(2-(2-((3R,5R,7R)-adamantan-1-yl)acetylamino)ethyl)piperazine-1- carbonyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester R-C2

[0105] (1) Preparation of intermediate 17a: 2-(2-bromoethyl)isoindoline-1,3-dione

[0106] Compound 16 (500 mg, 3.4 mmol) was dissolved in 15 mL of DMF, 1,2-dibromoethane (957.6 mg, 440 μL, 5.1 mmol), K2CO3(1.41 g, 10.2 mmol) were added, and stirred at room temperature overnight. TLC detected that the reaction was complete, 200 mL of saturated NaCl solution was added, and the aqueous layer was extracted with ethyl acetate (50 mL x 3). The combined organic layer was dried with anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (PE:EA = 10:1) to give 828 mg of white solid as intermediate 17a in 96% yield. 1H NMR (400 MHz, Chloroform-d) δ 7.86 (dt, J = 7.3, 3.6 Hz, 2H), 7.76 - 7.71 (m, 2H), 4.10 (t, J = 6.7 Hz, 2H), 3.61 (t, J = 6.7 Hz, 2H).

[0107] (2) Preparation of intermediate 18a: tert-butyl 4-(2-(l,3-dioxoisoindolin-2- yl)ethyl)piperazine-l-carboxylate

[0108] Intermediate 17a (828 mg, 3.26 mmol) was dissolved in 20 mL of acetonitrile, N-Boc-piperazine 13 (728.6 mg, 3.91 mmol), K2CO3(1.13 g, 8.15 mmol) and 3 drops of TEA were added and heated to reflux at 85 °C for 8 h. The reaction was monitored by TLC, it was first concentrated under reduced pressure, then extracted with water and dichloromethane, the obtained organic layer was dried using anhydrous Na2SO4, concentrated under reduced pressure and purified by column chromatography on silica gel (DCM:MeOH = 30: 1) to obtain 837 mg of intermediate 18a as a yellow oil with a yield of 72%. 1 H NMR (400 MHz, Chloroform-d) δ 7.82 (td, J = 5.2, 2.0 Hz, 2H), 7.71 (td, J = 5.5, 5.1, 1.9 Hz, 2H), 3.81 (t, J = 6.4 Hz, 2H), 3.39 - 3.30 (m, 4H), 2.63 (t, J = 6.4 Hz, 2H), 2.49 - 2.38 (m, 4H), 1.43 (s, 9H).

[0109] (3) Preparation of intermediate 19a: tert-butyl 4-(2-(2-((3R,5R,7R)-adamantan-l- yl)acetamido)ethyl)piperazine-l-carboxylate

[0110] Intermediate 18a (400 mg, 1.11 mmol) was dissolved in 25 mL of absolute ethanol, 85% hydrazine hydrate (841 mg, 964 μL, 16.7 mmol) was added, and the reaction was heated at 80 °C for 5 h. The reaction was cooled to room temperature, filtered, and the filtrate was concentrated under reduced pressure. Then 100 mL of dichloromethane was added, and the mixture was washed with water (30 mL x 3). The resulting organic layer was dried over anhydrous Na2SO4, and concentrated under reduced pressure to give the crude product, which was used directly in the next step without further purification. The crude product (225 mg, 0.98 mmol) was dissolved in 8 mL of DMF, and adamantane acetic acid 7 (229.1 mg, 1.18 mmol), HBTU (558.9 mg, 1.47 mmol), and DIPEA (190.5 mg, 257 μL, 1.47 mmol) were added. The reaction was stirred at room temperature overnight. The reaction was monitored by TLC, and 200 mL of saturated NaCl solution was added. The aqueous layer was extracted with ethyl acetate (50 mL x 3). The combined organic layer was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 100:3) to give 220 mg of yellow solid as intermediate 19a in 57% yield. 1 HNMR (400 MHz, Chloroform-d) δ 3.54 - 3.34 (m, 6H), 2.72 - 2.35 (m, 6H), 1.96 (d, J = 4.4 Hz, 5H), 1.70 (d, J = 12.2 Hz, 3H), 1.61 (d, J = 2.2 Hz, 9H), 1.46 (s, 9H).

[0111] (4) Preparation of intermediate 20a: (1S,3R)-1-(4-(4-(2-(2-((3r,5r,7r)-adamantan-1-yl)acetamido)ethyl)piperazine-1-carbonyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester

[0112] Compound 4 (50 mg, 0.12 mmol) was dissolved in 4 mL DCM, 2 mL TFA was added, and the reaction was allowed to proceed at room temperature for 2 h. After the reaction was completed as determined by TLC, the solvent was evaporated under reduced pressure, and the crude product was used directly in the next reaction without purification. Intermediate 19a (59.9 mg, 0.15 mmol) was dissolved in 4 mL DCM, 2 mL TFA was added, and the reaction was allowed to proceed at room temperature for 2 h. After the reaction was completed as determined by TLC, the solvent was evaporated under reduced pressure, and the crude product was used directly in the next reaction without purification. The two crude products obtained above were mixed and dissolved in 5 mL DMF, HBTU (70 mg, 0.18 mmol) and DIPEA (23.8 mg, 33 μL, 0.18 mmol) were added, and the reaction was allowed to proceed at room temperature overnight. After the reaction was completed as determined by TLC, 100 mL of saturated NaCl solution was added, and the aqueous layer was extracted with ethyl acetate (30 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 25:2) to obtain 67 mg of yellow solid as intermediate 20a with a yield of 85%. 1 H NMR (400 MHz, Chloroform-d) δ 7.65 (s, 1H), 7.56 (d, J = 7.2 Hz, 1H), 7.35 (d, J = 2.2 Hz, 4H), 7.25 (s, 1H), 7.20 - 7.10 (m, 2H), 5.43 (s, 1H), 3.97 (t, J = 6.0 Hz, 1H), 3.73 (s, 5H), 3.45 (s, 2H), 3.36 (q, J = 5.5 Hz, 2H), 3.31 - 3.24 (m, 1H), 3.19 - 3.11 (m, 1H), 2.59 - 2.39 (m, 6H), 1.95 (d, J = 10.0 Hz, 5H), 1.70 (d, J = 12.3 Hz, 3H), 1.64 - 1.60 (m, 9H).

[0113] (5) Preparation of target compound R-C2

[0114] Intermediate 20a (67 mg, 0.11 mmol) was dissolved in 5 mL DCM, TEA (17.8 mg, 13 μL, 0.16 mmol) was added, and the reaction was allowed to proceed at 0 °C in an ice bath. Compound 9 (15.9 mg, 23 μL, 0.16 mmol) was diluted in 1 mL DCM and slowly added dropwise to the above reaction system, which was then slowly warmed to room temperature and allowed to react for 10 h. After the reaction was completed as determined by TLC, 50 mL of saturated NaCl solution was added, and the aqueous layer was extracted with dichloromethane (20 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 25:2) to obtain 60 mg of white solid as compound R-C2 with a yield of 80%. 1H NMR (400 MHz, Chloroform-d) δ 8.28 (s, 1H), 7.56 - 7.28 (m, 5H), 7.11 (q, J = 7.7, 6.8 Hz, 3H), 6.30 - 5.96 (m, 1H), 5.77 (s, 1H), 5.19 (s, 1H), 4.22 - 3.98 (m, 2H), 3.65 (s, 7H), 3.49 - 3.30 (m, 5H), 2.56 - 2.36 (m, 6H), 1.95 (d, J = 10.2 Hz, 5H), 1.70 (d, J = 12.2 Hz, 3H), 1.64 - 1.61 (m, 9H). 13 C NMR (400 MHz, DMSO) δ 172.18, 170.31, 169.15, 168.27, 145.61, 136.86, 134.49, 127.54, 127.33, 126.33, 126.24, 121.81, 119.40, 118.53, 111.89, 104.06, 57.30, 57.09, 56.49, 53.28, 50.60, 43.84, 42.57, 36.94, 36.21, 32.62, 31.45, 28.51, 26.82, 22.56, 14.45, 11.74.

[0115] Example 14

[0116] Preparation of compound R-C3: replace compound 1,2-dibromoethane in the first step of Example 13 with compound 1,4-dibromobutane, and other same as Example 13 to prepare compound R-C3 with a yield of 41%.

[0117] Example 15

[0118] Preparation of compound R-C4: replace compound 1,2-dibromoethane in the first step of Example 13 with compound 1,6-dibromohexane, and other same as Example 13 to prepare compound R-C4 with a yield of 40%.

[0119] Example 16

[0120] Preparation of compound R-C5: replace compound 1,2-dibromoethane in the first step of Example 13 with compound 1,8-dibromooctane, and other same as Example 13 to prepare compound R-C5 with a yield of 45%.

[0121] Example 17

[0122] Preparation of (1S,3R)-2-acryloyl-1-(4-(4-(6-(2-((3r,5r,7r)-adamantan-1-yl)acetylamino)hexyl)piperazine-1-carbonyl)phenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester R-C6

[0123] Intermediate 20a (100 mg, 0.14 mmol) was dissolved in 5 mL of DCM, DIPEA (22.4 mg, 30 μL, 0.17 mmol) and DMAP (9 mg, 0.07 mmol) were added, acryloyl chloride 21 (15.6 mg, 14 μL, 0.17 mmol) was diluted in 1 mL of DCM and slowly added to the above reaction, which was slowly warmed to room temperature and reacted for 8 h. TLC detected that the reaction was complete, 50 mL of saturated NaCl solution was added, and the aqueous layer was extracted with dichloromethane (20 mL x 3). The combined organic layers were dried with anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 25:2) to obtain 77 mg of white solid as compound R-C6 with a yield of 71%. 1 H NMR (400 MHz, Chloroform-d) δ: 7.52 - 7.28 (m, 5H), 7.10 (q, J = 7.3 Hz, 3H), 6.39 (dd, J = 16.8, 10.1 Hz, 1H), 6.14 (d, J = 65.1 Hz, 1H), 5.88 (dd, J = 16.8, 2.3 Hz, 1H), 5.78 (dd, J = 10.1, 2.4 Hz, 1H), 5.30 (s, 1H), 5.18 (s, 1H), 4.3 - 3.4 (m, 10H), 3.21 (m, 2H), 2.52 (d, J = 69.2 Hz, 6H), 1.98 (s, 3H), 1.89 (s, 2H), 1.63 (d, J = 10.1 Hz, 3H), 1.61 (m, 9H), 1.50 - 1.47 (m, 4H), 1.29 (s, 4H). 13 C NMR (101 MHz, DMSO) δ 173.11, 172.52, 170.35, 168.88, 137.52, 136.81, 134.53, 132.90, 127.96, 127.92, 127.83, 127.73, 127.20, 122.28, 119.91, 118.62, 111.32, 106.63, 56.54, 54.53, 53.03, 52.95, 52.73, 46.00, 45.79, 40.20, 38.61, 36.28, 35.24, 30.11, 28.62, 27.69, 27.37, 25.88, 22.35.

[0124] Example 18

[0125] Preparation of (1S,3R)-1-(4-(4-(6-(2-((3r,5r,7r)-adamantan-1-yl)acetylamino)hexyl)piperazine-1- carbonyl)phenyl)-2-propynoyl-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester R-C7

[0126] Intermediate 20a (100 mg, 0.14 mmol) was dissolved in 10 mL of DCM, and propynoic acid 22 (20.2 mg, 18 μL, 0.29 mmol), CMPI (88.4 mg, 0.35 mmol) and TEA (70 mg, 99 μL, 0.69 mmol) were added, and the reaction was allowed to proceed at room temperature for 8 h. TLC detection showed that the reaction was complete, 50 mL of saturated NaCl solution was added, and the aqueous layer was extracted with dichloromethane (20 mL x 3). The combined organic layers were dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM:MeOH = 25:2) to obtain 26 mg of compound R-C7 as a white solid, with a yield of 24%. 1 H NMR (400 MHz, Chloroform-d) δ 8.22 (s, 1H), 7.57 - 7.30 (m, 4H), 7.23 - 7.04 (m, 4H), 6.13 (d, J = 75.9 Hz, 1H), 5.37 (s, 1H), 5.19 (s, 1H), 4.20 - 3.78 (m, 4H), 3.68 - 3.32 (m, 7H), 3.24 - 3.18 (m, 2H), 2.52 (d, J = 47.8 Hz, 6H), 1.95 (s, 3H), 1.89 (s, 2H), 1.69 (d, J = 12.0 Hz, 3H), 1.61 (d, J = 10.5 Hz, 9H), 1.54 - 1.46 (m, 4H), 1.32 (s, 4H). 13 C NMR (101 MHz, DMSO) δ 173.11, 172.52, 170.35, 161.27, 137.52, 136.81, 134.53, 132.87, 127.91, 127.83, 127.20, 122.28, 119.91, 118.62, 111.32, 106.63, 77.52, 76.79, 56.54, 54.82, 52.95, 52.73, 52.25, 46.00, 45.79, 40.20, 38.61, 36.28, 35.24, 30.11, 28.62, 27.69, 27.37, 25.88, 22.35.

[0127]

[0128] Reagents and conditions: a: K2CO3, DMF, rt, 8 h, yield 71%. b: K2CO3, TEA, CH3CN, 85 °C, 8 h, yield 89%. c: (1): hydrazine hydrate, CH3CH2OH, 80 °C, 4 h, yield 100%. (2): HBTU, DIPEA, DMF, rt, 8 h, yield 70-95%. d: (1) TFA, DCM, rt, 4 h, yield 100%. (2) HBTU, DIPEA, rt, 8 h, 40-60%. e: TEA, DCM, 0 °C, 8 h, yield 40-85%. f: acryloyl chloride, DIPEA, DMAP, 0 °C, 8 h, yield 71%. g: propiolic acid, CPMI, TEA, rt, 8 h, yield 24%.

[0129] Example 19: In vitro anti-tumor activity test (IC50) of the target compounds 50 )

[0130] The tumor cell anti-proliferation ability of the compounds of the present application was tested on HT1080 cells (human fibrosarcoma cells) using the SRB method. The HT1080 cells in the logarithmic growth phase were trypsinized and then diluted to a single cell suspension with culture medium (DMEM + 10% FBS + 1% double antibody) to adjust the cell density to 3-4 x 10 4 The cell suspension was added to each well of a 96-well plate at 90 μL / well, and the plate was placed in a 37 °C, 5% CO2 incubator for 24 hours. The cells were observed under a microscope to confirm that they were well adhered. 10 μL of culture medium containing different concentrations of the sample was added to each well, and three replicate wells were prepared for each concentration. A corresponding DMSO control group was also prepared, and the plate was placed in a 37 °C, 5% CO2 incubator for 72 hours. The culture medium was removed, and the cells were fixed with 100 μL of TCA. The plate was placed in a 4 °C refrigerator for at least 1 hour, and the fixing solution was removed. The plate was washed with tap water along the edge for 5-6 times, and then dried at 50 °C for 2 hours. 100 μL of SRB was added to each well, and the plate was placed in the dark for 10 minutes. The unbound SRB was removed, and the plate was washed with 1% glacial acetic acid three times and then dried at 50 °C for 2 hours. Non-buffered 100-150 μL of tris base solution was added to dissolve the bound SRB, and the plate was shaken gently for 2 minutes. The plate was placed in an enzyme-linked immunoassay detector, and the optical density value (OD value) of each well was detected at a wavelength of 515 nm. The experiment was repeated three times. The survival rate was calculated according to the following formula: survival rate % = experimental group OD value / DMSO control group OD value x 100%.

[0131] Experimental results: the half-inhibitory concentration IC of the compounds of the present application on HT1080 cells is shown in Table 1 50 As shown in Table 2, the anti-tumor activity of most of the compounds in series B with methylene as the connecting group and series C with piperazine rigid ring as the connecting group is better than that of the positive control RSL3. Among different covalent binding warhead compounds, the propynylamide warhead R-C7 is more active than the chloroacetamide warhead R-C4 and the propenamide warhead R-C6. The anti-tumor activity of compound R-C1 (0.005±0.001 μM) is the best, which is about 18 times better than that of RSL3 (0.090±0.010 μM).

[0132] Table 2: Anti-tumor activity in vitro of target compounds

[0133]

[0134] Example 20: Test of GPX4 degradation activity of target compounds

[0135] The GPX4 degradation activity of the compounds of the present application on HT1080 cells was tested by Western blotting. The HT1080 cells in the logarithmic growth phase were inoculated in a 6-well plate at a density of 300,000 cells per well and 1800 μL per well, and incubated in a 37°C, 5% CO2 incubator. The control group was DMSO, and after co-culturing the compounds of the present application at different concentrations for 24 h, the cells were washed twice with PBS, and appropriate Loading Buffer was added to each well, and the cells were lysed at 4°C for 45 min. After lysis, the cells were collected in a centrifuge tube with a clean cell scraper and boiled in boiling water for 15 min. The proteins were separated by 10% SDS-PAGE and transferred to an NC membrane. The membrane was washed with TBST, incubated with blocking solution for 30 min, and then incubated with the first antibody at 4°C with gentle shaking overnight. The membrane was washed with TBST and incubated with the second antibody at room temperature for 2 h. The developed image was obtained by washing and imaging, and the obtained Western blotting image was processed and analyzed by Image J software.

[0136] Experimental results: the degradation effect of some compounds of the present application on GPX4 in HT1080 cells is shown in Table 3, wherein the degradation activity of R-B1, R-B4-B5, R-B7, R-C4-C7 is better than that of the positive drug RSL3. The activity of these compounds is better than that of RSL3, which can prove that the increase in the activity of these compounds is due to the enhanced degradation of GPX4. Since the activity of R-C1 is significantly better than that of other compounds, it is further selected to test its degradation effect on GPX4 at lower concentrations, i.e. four concentration gradients of 0.001, 0.005, 0.01 and 0.02 μM, so as to more accurately judge its degradation effect on GPX4. The results are shown in Table 4. Figure 1The results show that the compound R-C1 can degrade GPX4 in a concentration-dependent manner, and the degradation rate reaches 83±2% at a concentration of 0.02 μM.

[0137] Table 3: GPX4 degradation activity of target compounds

[0138]

[0139] The above description is only the preferred embodiment of the present application, and does not limit the present application in any form. Although the present application has been disclosed as above with the preferred embodiment, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above-mentioned technical content with equivalent embodiments without departing from the technical solution of the present application. Any simple modification, equivalent change and modification of the above embodiments made according to the technical essence of the present application, which does not depart from the technical solution of the present application, still belongs to the scope of the present application.

Claims

1. A GPX4 protein degradation agent based on a hydrophobic tag or a pharmaceutically acceptable salt thereof, characterized in that: It has the structure shown in the following formula (I): ； R1 is selected from the following groups: ； X is selected from the following linking groups: ； n is 1, and m is 1-20.

2. A GPX4 protein degradation agent based on a hydrophobic tag or a pharmaceutically acceptable salt thereof, characterized in that: It is the following compound: Compound R-A1 : (1 S ,3 R )-1-((2-((2-((3 R ,5 R ,7 R )-adamantan-1-yl)acetylamino)ethoxy)ethyl)aminocarbonyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H -methyl 1,4-dioxa-9-azaspiro[5.5]undecane-8,10-dione; Compound R-A3: (1 S ,3 R )-1-((4-((1-((3 R ,5 R ,7 R )-adamantan-1-yl)-2-oxo-6,9,12-trioxa-3-azatetradecyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H -methyl pyrido[3,4-b]indole-3-carboxylate; Compound R-B3: (1 S ,3 R )-1-((4-((5-(2-((3 R ,5 R ,7 R )-adamantan-1-yl)acetamido)pentyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H - pyrido[3,4- b ]indole-3-carboxylic acid methyl ester; Compound R-B4: (1 S ,3 R )-1-((4-((6-(2-((3 R ,5 R ,7 R )-adamantan-1-yl)acetylamino)hexyl)carbamoyl)(phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H - pyrido[3,4- b ]indole-3-carboxylic acid methyl ester; Compound R-B5: (1 S ,3 R )-1-((4-((7-(2-((3 R ,5 R ,7 R )-adamantan-1-yl)acetamido)heptyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H - pyrido[3,4- b ]indole-3-carboxylic acid methyl ester; Compound R-B6: (1 S ,3 R )-1-((4-((8-(2-((3 R ,5 R ,7 R )-adamantan-1-yl)acetamido)octyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H - pyrido[3,4- b ]indole-3-carboxylic acid methyl ester; Compound R-B7: (1 S ,3 R )-1-((4-((10-((2-((3 R ,5 R ,7 R )-adamantan-1-yl)acetylamino)decyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H - pyrido[3,4- b ]indole-3-carboxylic acid methyl ester; Compound R-C1 : (1 S ,3 R )-1- (4- (4- (2- ( (3 R ,5 R ,7 R )-adamantan-1-yl)acetyl)piperazin-1-yl)carbonyl)phenyl)-2- (2-chloroacetyl)-2,3,4,9-tetrahydro-1 H pyrido [3,4- b ]indole-3-carboxylic acid methyl ester; Compound R-C2: (1 S ,3 R )-1-((1 R ,5 R ,7 R )-adamantan-1-yl)ethyl)piperazine-1-carbonyl)- phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H H-pyrido[3,4- b ]indole-3-carboxylic acid methyl ester; Compound R-C3: (1 S ,3 R )-1- (4- (4- (4- (2- ( (3 R ,5 R ,7 R )-adamantan-1-yl)acetamido)butyl) piperazine-1-carbonyl)phenyl)-2- (2-chloroacetyl)-2,3,4,9-tetrahydro-1 H pyrido [3,4- b ]indole-3-carboxylic acid methyl ester; Compound R-C4: (1 S ,3 R )-1-((4-((4-(6-(2-((3 R ,5 R ,7 R )-adamantan-1-yl)acetylamino)hexyl)carbamoyl)phenyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H - pyrido[3,4- b ]indole-3-carboxylic acid methyl ester; Compound R-C5: (1 S ,3 R )-1-((4-((4-(8-(2-((3 R ,5 R ,7 R )-adamantan-1-yl)acetamido)octyl)piperazin-1-yl)carbonyl)-2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1 H - pyrido[3,4- b ]indole-3-carboxylic acid methyl ester; Compound R-C6: (1 S ,3 R )-2-acryloyl-1-(4-(4-(6-(2-((3 r ,5 r ,7 r )-adamantan-1-yl)acetamido)hexyl)piperazin-1-ylcarbonyl)phenyl)-2,3,4,9-tetrahydro-1 H - pyrido[3,4- b ]indole-3-carboxylic acid methyl ester; or compound R-C7: (1 S ,3 R )-1- (4- (4- (6- (2- ( (3 r ,5 r ,7 r )-adamantan-1-yl) acetamido) hexyl) piperazine-1-carbonyl) phenyl) -2-propynoyl-2,3,4,9-tetrahydro-1 H pyrido [3,4- b ] indole-3-carboxylic acid methyl ester.

3. The GPX4 protein degradation agent or a pharmaceutically acceptable salt thereof according to claim 1, characterized by: The pharmaceutically acceptable salt is an inorganic acid salt or an organic acid salt; the inorganic acid is selected from at least one of hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid; and the organic acid is selected from at least one of methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, and mandelic acid.

4. A method of producing the GPX4 protein degradation agent or a pharmaceutically acceptable salt thereof according to claim 1, characterized by, The preparation method comprises the following steps: (1) Synthesis of intermediates p-Formaldehyde benzonic acid 1 reacts with Boc 2O to generate intermediate 2; intermediate 2 is cyclized with D-tryptophan methyl carbonate hydrochloride 3 to obtain intermediate 4; ； (2) Synthesis of target compounds Intermediate 4 is condensed with oxygen chain 5a / 5c to generate intermediate 6a / 6c; intermediate 6a / 6c is deprotected with TFA to remove the Boc protecting group, and then condensed with adamantane acetic acid 7 to obtain intermediate 8a / 8c; finally, intermediate 8a / 8c reacts with chloroacetyl chloride 9 to obtain the target compound R-A1 / R-A3; ； Alternatively, intermediate 4 is condensed with alkane chain 10a-e to generate intermediate 11a-e; intermediate 11a-e is deprotected with TFA to remove the Boc protecting group, and then condensed with adamantane acetic acid 7 to obtain intermediate 12a-e; finally, intermediate 12a-e reacts with chloroacetyl chloride 9 to obtain the target compound R-B3~R-B7; ； Alternatively, intermediate 4 is condensed with N-Boc-piperazine to generate intermediate 14; intermediate 14 is deprotected with TFA to remove the Boc protecting group, and then condensed with adamantane acetic acid 7 to obtain intermediate 15; finally, intermediate 15 reacts with chloroacetyl chloride 9 to obtain the target compound R-C1; 。 5. The method of claim 4, wherein: The step (2) further comprises: Different lengths of dibromoalkane are substituted with isoindoline-1,3-dione 16 in the presence of K2CO3 to obtain intermediate 17a-d; intermediate 17a-d is substituted with N-Boc-piperazine 13 in the presence of K2CO3 to obtain intermediate 18a-d; intermediate 18a-d is first reduced and then reacted with adamantane acetic acid 7 to obtain intermediate 19a-d; intermediate 19a-d and intermediate 4 are respectively deprotected with TFA to remove the Boc protecting group, and the obtained products are condensed with each other to obtain intermediate 20a-d; finally, intermediate 20a-d reacts with chloroacetyl chloride 9 to obtain the target compound R-C2~R-C5, intermediate 20c reacts with acryloyl chloride 21 to obtain the target compound R-C6, and intermediate 20c reacts with propargyl acid 22 to obtain the target compound R-C7; 。 6. Use of the GPX4 protein degrading agent or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the preparation of a GPX4 protein inhibitor.

7. Use of the GPX4 protein degrading agent or a pharmaceutically acceptable salt thereof according to any one of claims 1-3 in the preparation of an antitumor drug.

8. Use according to claim 7, characterized in that: The tumor is a ferroptosis-sensitive tumor.

9. Use according to claim 8, characterized in that: The ferroptosis-sensitive tumor is selected from human breast cancer, human fibrosarcoma, and renal cancer.

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