Chimera for targeted degradation of GPX4 based on HSP70 protein as well as preparation method and application of chimera

Abstract

The invention discloses a chimera for targeted degradation of GPX4 based on HSP70 protein as well as a preparation method and application of the chimera, and belongs to the technical field of biological medicines. According to the invention, 12 chimeras for targeted degradation of GPX4 are prepared by using the technology. The HSP70 molecular chaperone complex mediated protein targeted degradation chimera provided by the invention can rapidly and durably induce the degradation of GPX4. Meanwhile, the chimeras have obvious anti-proliferative activity on GPX4 high-expression cell strains and high-selectivity induction of ferroptosis of cells, and have important significance on tumor targeted therapy and drug resistance overcoming.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a class of chimeras with GPX4 degradation activity mediated by molecular chaperone HSP70, a preparation method thereof, and an application thereof in anti-tumor aspects. Background Art

[0002] Different from apoptosis, necrosis, and pyroptosis, ferroptosis is a programmed cell death mode characterized by the accumulation of iron-dependent lipid reactive oxygen species (LPO) radicals. A large number of studies have found that glutathione peroxidase 4 (GPX4) can be used as one of the indicators for judging cell ferroptosis. The catalytic active center of GPX4 is selenocysteine, and with GSH as a cofactor, GPX4 can reduce lipid hydroperoxides in cells into non-toxic lipid alcohols, and can also catalyze the reduction of other organic peroxides such as hydrogen peroxide. Therefore, it has the function of protecting cells from oxidative stress and inhibiting the occurrence of ferroptosis. Therefore, inhibiting the activity of GPX4 will affect the ability of GPX4 to scavenge lipid peroxides, and ultimately lead to the occurrence of cell ferroptosis. In addition, inhibiting the function of GPX4 will trigger persistent ferroptosis of cells and prevent tumor recurrence, so it is one of the strategies to solve drug resistance.

[0003] At present, there are still certain challenges for GPX4-targeted small molecule inhibitors, and there is no report on GPX4 inhibitors entering the clinical research stage. The main reasons are as follows: 1) There is no suitable binding pocket on the molecular surface of GPX4; 2) The currently reported inhibitors are all covalent inhibitors, which play a role by binding to the selenocysteine in the active site of GPX4, but there are problems such as low selectivity and poor in vivo activity; 3) Some degraders that can induce the down-regulation of GPX4 have been developed based on CRBN and VHL. However, in cells with low expression or natural defects of these E3 enzymes, the degradation effect of GPX4 will be severely weakened. In addition, degraders developed based on a single E3 enzyme are prone to drug resistance. Therefore, there is an urgent need to develop new GPX4-targeted small molecules.

[0004] Inducing the degradation of oncoproteins using protein degradation technology is one of the hot research areas in recent years. The molecular chaperone-mediated targeted degradation technology is a novel protein degradation technology with multiple advantages such as overcoming drug resistance. Using the molecular chaperone-mediated targeted protein degradation technology, we previously developed a GPX4 degrader based on HSP90-mediated degradation, but there was a problem of slow degradation rate. This application discloses the preparation and application of a novel chimeric body based on HSP70 as a GPX4 degrader, which has the advantage of rapidly degrading GPX4. In addition, HSP70 is highly expressed in tumor tissues, which helps to improve its targeting. Multiple E3 ubiquitin ligases recruited by the HSP70 molecular chaperone complex can also avoid the drug resistance caused by the current PROTAC recruiting a single E3 ubiquitin ligase CRBN or VHL, or expand the application of the degrader in cells with low expression or mutation of CRBN or VHL. Summary of the Invention

[0005] Based on the problems existing in the above background technology, the object of the present invention is to provide a chimeric body for targeting the degradation of GPX4 based on HSP70 protein, its preparation method and application. The chimeric body of the present invention can efficiently and rapidly degrade GPX4 protein, thereby inducing ferroptosis of tumor cells and avoiding potential drug resistance and other problems, which are specifically realized through the following technical solutions:

[0006] A chimeric body for targeting the degradation of GPX4 based on HSP70 protein, the chimeric body has the structure shown in general formula (I) or its pharmaceutically or physiologically acceptable salt,

[0007]

[0008] In general formula (I), Linker is a linking group, representing -alkylene or -alkoxy or -piperazinyl or -1,2,3-triazolyl, and the -alkylene or -alkoxy or -piperazinyl or -1,2,3-triazolyl is selected from any one of the following groups or any combination thereof, where p, m, and n represent natural numbers from 1 to 20:

[0009] -(CH 2 ) n -C(O)NH(CH 2 CH 2 O) m - or -(CH 2 CH 2 O) n -C(O)NH(CH 2 CH 2 O) m - or

[0010]

[0011] Furthermore, the chimeric body is the following compound or its stereoisomer, geometric isomer, tautomer, N-oxide, hydrate, solvate, metabolite, pharmaceutically or physiologically acceptable salt or prodrug:

[0012]

[0013] The pharmaceutically or physiologically acceptable salt referred to in the present invention means the salt formed by the chimeric body targeting the degradation of GPX4 by the HSP70 protein described in the present invention and a pharmaceutically or physiologically acceptable acid or base.

[0014] The present invention also provides a pharmaceutical composition, which comprises the above-mentioned chimeric body targeting the degradation of GPX4 by the HSP70 protein or its pharmaceutically or physiologically acceptable salt, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof. The pharmaceutical composition is an injection, an oral preparation or a mucosal administration preparation.

[0015] The present invention also provides the use of the above-mentioned chimeric body targeting the degradation of GPX4 by the HSP70 protein or the pharmaceutical composition containing the chimeric body. Specifically as follows:

[0016] Use of the chimeric body targeting the degradation of GPX4 by the HSP70 protein or the pharmaceutical composition containing the chimeric body in the preparation of a drug for degrading GPX4.

[0017] Use of the chimeric body targeting the degradation of GPX4 by the HSP70 protein or the pharmaceutical composition containing the chimeric body in the preparation of a drug for treating GPX4-related diseases. The GPX4-related diseases are tumors, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease.

[0018] Use of the chimeric body targeting the degradation of GPX4 by the HSP70 protein or the pharmaceutical composition containing the chimeric body in the preparation of an anti-tumor drug. The tumors are gastric cancer, breast cancer, lung cancer, ovarian cancer, colorectal adenocarcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, lung adenocarcinoma, prostate cancer, rectal adenocarcinoma, thyroid cancer and endometrial cancer.

[0019] Furthermore, the tumor is a tumor with high expression of GPX4.

[0020] The present invention also proposes a synthetic route of the chimeric body targeting the degradation of GPX4 by the HSP70 protein shown by the general formula, which specifically comprises the following steps:

[0021] Preparation of the GPX4 ligand ML162-yne shown by the general formula, preparation of the ligand of the HSP70 protein, and connecting the two through reaction types such as amide condensation, deprotection, nucleophilic substitution and Click chemistry.

[0022] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0023] Different from the PROTAC technology that directly pulls a certain E3 ubiquitin ligase, the protein degrader mediated by the molecular chaperone HSP70 according to the present invention can induce the ubiquitination of the target protein by various different types of E3 ubiquitin ligases, thereby triggering the degradation of the target protein by the proteasome. The present invention confirmed through Western blot experiments that the chimeras involved in the present invention can effectively degrade GPX4, thereby effectively killing cell lines with high expression or abnormal activation of GPX4. Description of the Drawings

[0024] Figure 1 is the synthetic route diagram of chimeras GDAz-1 to 12;

[0025] Figure 2 is the detection of the degradation activity of GPX4 by different concentrations of chimeras through Western Blot;

[0026] Figure 3 is the effect diagram of GDAz-3 on intracellular lipid peroxides;

[0027] Figure 4 is the comparison of the degradation effects of the GPX4 degrader GDAz-3 mediated by the HSP70 molecular chaperone complex and the GPX4 degrader GDCNF-2 / -11 mediated by the HSP90 molecular chaperone complex;

[0028] Figure 5 is the potential application effect diagram of the GPX4 degrader GDAz-3 mediated by the HSP70 molecular chaperone complex. Detailed Embodiments

[0029] The following further describes the present invention in combination with specific embodiments. The technical and scientific terms used in the following embodiments have the same meanings commonly understood by those skilled in the art to which the present invention belongs. The basic raw material reagents are obtained from commercial channels, and the purity is above 97%. The room temperature referred to in the present invention is 25-30°C. The present invention describes the materials and experimental methods used in the experiments in a general and specific manner. Although many materials and operation methods used to achieve the purpose of the present invention are well known in the art, the present invention still describes them as much as possible here.

[0030] Example 1: Synthesis and Structure Confirmation of Chimeras Targeting the Degradation of GPX4

[0031] The synthetic routes of the final products GDAz-1 to 12 are as Figure 1 shown. The synthetic methods will be specifically described below.

[0032] Synthesis of Compound 2:

[0033] A suspension of 4-amino-2-chlorophenol (500 mg, 3.48 mmol) shown in Compound 1 and di-tert-butyl dicarbonate (836 mg, 3.83 mmol) dissolved in tetrahydrofuran (20 mL) was stirred at room temperature for 24 hours. The reaction solution was concentrated under reduced pressure and extracted twice with ethyl acetate. The combined organic layers were dried over anhydrous Na 2 SO 4 2, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: ethyl acetate: petroleum ether with a volume ratio of 1:5) to obtain Compound 2 (yellow liquid, 714 mg, yield 84%).

[0034] Synthesis of Compound 3:

[0035] Compound 2 (1000 mg, 4.1 mmol) was dissolved in N,N-dimethylformamide (20 mL), potassium carbonate (851 mg, 6.2 mmol), 3-bromoprop-1-yne (634 mg, 5.3 mmol) were added, and the mixture was stirred at room temperature overnight. Water was added to the reaction solution, and it was extracted three times with ethyl acetate. The combined organic layers were washed once with water and once with saturated sodium chloride aqueous solution in sequence, and dried over anhydrous Na 2 SO 4 2, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: ethyl acetate: petroleum ether with a volume ratio of 1:5) to obtain Compound 3 (yellow liquid, 850 mg, yield 73%).

[0036] Synthesis of Compound 4:

[0037] Compound 3 (1000 mg, 3.55 mmol) was dissolved in dichloromethane (40 mL), and then trifluoroacetic acid (10 mL) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, an aqueous NaHCO 3 3 solution was added, and it was extracted three times with ethyl acetate. The separated organic layer was dried over anhydrous Na 2 SO 4 2, filtered, and concentrated under reduced pressure to obtain Compound 4 (white solid, 480 mg, yield 74%).

[0038] Synthesis of Compound 5:

[0039] Compound 4 (1.82 g, 10 mmol) and 2-thiophenecarboxaldehyde (1.12 g, 10 mmol) were dissolved in (25 mL) methanol, activated at 25°C for 1 h, and then (2-isocyanoethyl)benzene (1.09 g, 8.33 mmol) and chloroacetic acid (787.45 mg, 8.33 mmol) were added and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, and the residue was separated by silica gel column chromatography (the mobile phase was ethyl acetate: petroleum ether in a volume ratio of 1:1) to obtain compound 5, recorded as: ML162-yne (white solid, 850 mg, yield 20%).

[0040] Synthesis of compound 7

[0041] 1,2-bis(4-methoxyphenyl)ethane-1,2-dione (7) shown in compound 7: Oxalyl chloride (3 ml, 35.2 mmol) was slowly added to a mixture of anisole (11.4 g, 106 mmol) and anhydrous aluminum chloride (21.3 g, 250 mmol) shown in compound 6 at 0°C. After the addition was complete, the mixture was stirred at room temperature for 6 hours. After cooling, it was poured into ice water and extracted with dichloromethane. The collected organic phase was washed with 2N HCl and then with anhydrous Na 2 SO 4 Dry, filter and concentrate under reduced pressure. The residue is recrystallized from ethanol. The resulting precipitate is heated, washed several times in ethanol and dried to give 5.97 g (62%) of pure product as a yellow solid.

[0042] Synthesis of compound 8:

[0043] 4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)benzoic acid shown in compound 8: 4-(aminomethyl)benzoic acid (2 g, 13.2 mmol), 3,5-bis(trifluoromethyl)benzaldehyde (3.27 mL, 19.8 mmol), 1,2-bis(4-methoxyphenyl)ethane-1,2-dione (5.36 g, 19.8 mmol) and ammonium acetate (6.17 g, 79.4 mmol) were dissolved in acetic acid and stirred at 100 °C for 12 h. The mixture was cooled to room temperature, diluted with ethyl acetate, and washed with water, saturated NaHCO 3 The organic layer was washed with anhydrous Na 2 SO 4 Dry, filter, and concentrate under reduced pressure. The residue was purified by flash column chromatography (DCM: MeOH = 15: 1) and dried to give 1.6 g (52%) of pure product in the form of a white solid.

[0044] Synthesis of compounds 9 and 10:

[0045] A stirred solution of compound 8 (1 eq) in DMF was added to N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC-HCl) (1.2 eq) and 1-hydroxybenzotriazole (HOBt) (1.3 eq). The mixture was stirred at room temperature for 1 h, then different amino-substituted derivatives (1.5 eq) were added and stirred for 2 h. The reaction mixture was diluted with EtOAc, washed with brine, and dried over anhydrous Na 2 SO 4 dried, concentrated under reduced pressure, and the residue was purified by flash column chromatography (DCM:MeOH = 15:1) to obtain product 9 or 10.

[0046] Synthesis of compound 11:

[0047] Compound 10 (1.1 g, 3.14 mmol) was dissolved in dichloromethane (40 mL), and trifluoroacetic acid (10 mL) was added. The mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure to obtain a concentrate (white solid, 650 mg). The above-prepared concentrate (650 mg, 1.75 mmol) and azidoacetic acid (212.79 mg, 2.11 mmol) were dissolved in acetonitrile (20 mL), and then tetramethylchlorouronium hexafluorophosphate (589.2 mg, 2.1 mmol) and N-methylimidazole (573 mg, 7 mmol) were added. The mixture was stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure, water was added, and the mixture was extracted with ethyl acetate three times. The combined organic layers were washed successively with water and saturated sodium chloride aqueous solution once each, and dried over anhydrous Na 2 SO 4 dried, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: dichloromethane:methanol with a volume ratio of 25:1) to obtain compound 11 (white solid, 600 mg).

[0048] Synthesis of the final products GDAz-1 to 12:

[0049] To an aqueous solution of sodium ascorbate (2.5 eq) and copper sulfate pentahydrate (0.5 eq) at 0 °C, a DMF solution of ML162-yne (1.1 eq) and azido-substituted derivative 9 or 11 (1 eq) was added. The mixture was stirred overnight under nitrogen protection. The reaction mixture was diluted with water, extracted with DCM, washed with brine, and dried over Na 2 SO 4 dried, filtered, concentrated, and the residue was purified by column chromatography (DCM:MeOH = 15:1) to obtain the target compounds GDAz-1 to 12.

[0050] GDAz-1: 4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-(2-(2-(4-((2-chloro-4-(2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)benzamide.

[0051] 1 H NMR(400MHz,CDCl 3 )δ7.98(d,J = 1.7Hz,2H),7.74(s,1H),7.71–7.64(m,3H),7.46–7.40(m,2H),7.18–7.05(m,6H),7.04–7.00(m,2H),6.89(d,J = 8.0Hz,2H),6.85–6.77(m,3H),6.77–6.68(m,5H),6.18(t,J = 5.9Hz,1H),6.01(s,1H),5.06(s,3H),4.46(t,J = 4.9Hz,2H),3.80(t,J = 5.0Hz,2H),3.72(d,J = 5.5Hz,5H),3.68(s,3H),3.53(t,J = 4.2Hz,3H),3.46–3.41(m,1H),2.81(d,J = 31.6Hz,3H),2.72–2.67(m,1H). 13 C NMR(101MHz,CDCl 3 )δ166.99,165.71,165.59,161.56,159.16,157.61,153.18,143.11,142.15,139.43,137.81,137.60,133.75,132.76,131.22,131.05,130.77,130.71,130.57,129.45,129.09,128.39,127.79,127.59,127.55,127.22,126.96,126.86,125.54,125.46,125.43,124.81,123.33,122.95,121.18,120.91,120.62,113.65,112.71,112.51,68.74,67.92,62.12,54.27,54.17,49.23,47.13,41.38,40.04,38.61,35.48,34.41.HRMS(ESI)calcd for[C 62 H 54 Cl2 F 6 N 8 O 7 S+H] + 1239.3196, found 1239.3181。

[0052] GDAz-2: 4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-(2-(2-(4-((2-chloro-4-(2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethyl)benzamide。

[0053] 1 H NMR(400MHz, CDCl 3 ) δ 7.99 (d, J = 1.7 Hz, 2H), 7.74 (d, J = 5.3 Hz, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.9 Hz, 2H), 7.17 (d, J = 7.5 Hz, 3H), 7.11 (dd, J = 7.8, 4.0 Hz, 3H), 7.05 (d, J = 7.0 Hz, 2H), 6.89 (d, J = 8.0 Hz, 3H), 6.84–6.74 (m, 4H), 6.72 (d, J = 8.9 Hz, 2H), 6.65 (d, J = 5.4 Hz, 1H), 6.01 (d, J = 12.3 Hz, 2H), 5.14 (s, 1H), 5.06 (s, 2H), 4.44 (t, J = 5.0 Hz, 2H), 3.80 (t, J = 5.1 Hz, 2H), 3.73 (d, J = 6.4 Hz, 5H), 3.70 (s, 3H), 3.52 (d, J = 6.0 Hz, 8H), 3.46 (dd, J = 14.6, 6.9 Hz, 2H), 2.77–2.65 (m, 2H). 13 C NMR(101MHz, CDCl 3)δ166.92,165.65,165.61,159.15,157.62,153.27,143.09,142.04,139.35,137.79,137.57,133.70,133.00,131.20,131.07,130.81,130.73,130.53,129.41,129.10,128.35,127.80,127.57,127.29,126.95,126.77,125.54,125.49,124.81,123.32,123.14,122.14,121.21,120.88,120.61,113.65,112.72,112.56,69.47,69.15,68.78,68.28,62.22,59.74,54.28,54.18,49.31,47.12,41.33,40.04,38.75,34.43.HRMS(ESI)calcd for[C 64 H 58 Cl 2 F 6 N 8 O 8 S+H] + 1283.3458,found 1283.3431。

[0054] GDAz-3: 4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-(2-(2-(2-(2-(4-((2-chloro-4-(2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)benzamide。

[0055] 1 H NMR(400MHz,CDCl 3) δ 8.01 (s, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 7.9 Hz, 2H), 7.45 (d, J = 8.8 Hz, 2H), 7.19–7.15 (m, 3H), 7.11 (t, J = 7.5 Hz, 4H), 7.07–7.03 (m, 2H), 6.88 (d, J = 8.0 Hz, 2H), 6.86–6.75 (m, 6H), 6.73 (d, J = 8.9 Hz, 2H), 5.99 (s, 1H), 5.13 (s, 2H), 5.07 (s, 2H), 4.41 (t, J = 5.0 Hz, 2H), 3.80–3.67 (m, 11H), 3.60–3.41 (m, 15H), 2.88 (s, 1H), 2.81 (s, 1H). 13 C NMR (101 MHz, CDCl 3 ) δ 166.91, 165.62, 165.56, 161.55, 159.26, 157.80, 153.29, 142.88, 141.94, 137.56, 133.69, 133.14, 131.22, 131.15, 130.81, 130.51, 129.37, 129.10, 128.33, 127.80, 127.73, 127.58, 127.31, 127.07, 126.88, 125.54, 125.50, 124.73, 123.27, 123.24, 122.13, 121.51, 120.56, 113.70, 112.78, 112.55, 69.47, 69.34, 69.13, 68.76, 68.28, 62.20, 54.30, 54.20, 49.29, 47.20, 41.31, 40.04, 38.78, 34.44. HRMS (ESI) calcd for [C 66 H 62 Cl 2 F 6 N 8 O 9 S + H] + 1327.3720, found 1327.3690。

[0056] GDAz-4: 4-((2-(3,5-Bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-(14-(4-((2-chloro-4-(2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxatetradecyl)benzamide.

[0057] 1 H NMR(400MHz,CDCl 3 )δ8.00(d,J = 1.6Hz,2H),7.77(d,J = 11.4Hz,2H),7.70(d,J = 8.1Hz,2H),7.43(d,J = 8.8Hz,2H),7.16(d,J = 7.5Hz,3H),7.13–7.07(m,4H),7.04(d,J = 6.9Hz,3H),6.88(d,J = 8.0Hz,3H),6.83–6.74(m,4H),6.72(d,J = 8.9Hz,2H),6.08(t,J = 5.8Hz,1H),6.00(s,1H),5.13(s,1H),5.06(s,2H),4.41(t,J = 5.0Hz,2H),3.73(d,J = 3.7Hz,7H),3.69(s,3H),3.54(t,J = 8.6Hz,10H),3.48(d,J = 18.0Hz,8H),2.83(d,J = 30.4Hz,1H),2.75–2.69(m,1H). 13 C NMR(101MHz,CDCl 3 )δ166.95,165.62,165.58,159.15,157.61,153.32,143.07,141.87,139.21,137.79,137.59,133.73,133.06,131.19,131.09,130.75,130.73,130.53,129.44,129.10,128.35,127.80,127.56,127.50,127.26,126.93,125.54,125.48,124.65,123.39,123.32,121.18,120.89,120.61,113.65,112.71,112.53,69.45,69.43,69.41,69.39,69.29,69.11,68.90,68.29,62.15,54.28,54.18,49.26,47.14,41.35,40.04,38.81,34.43.HRMS(ESI)calcd for[C 68 H 66 Cl 2 F 6 N 8 O 10 S+H] + 1371.3982,found 1371.3958。

[0058] GDAz-5: 4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-(17-(4-((2-chloro-4-((2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15-pentaoxoheptadecyl)benzamide.

[0059] 1 H NMR(400MHz,CDCl 3 ) δ 8.00 (d, J = 1.7 Hz, 2H), 7.81 (s, 1H), 7.77–7.69 (m, 3H), 7.45–7.40 (m, 2H), 7.26 (s, 1H), 7.19–7.06 (m, 7H), 7.05–7.01 (m, 2H), 6.88 (d, J = 8.1 Hz, 2H), 6.86–6.73 (m, 5H), 6.73–6.69 (m, 2H), 6.14 (t, J = 5.9 Hz, 1H), 6.01 (s, 1H), 5.14 (s, 1H), 5.06 (s, 2H), 4.43 (t, J = 5.0 Hz, 2H), 3.73 (s, 7H), 3.69 (s, 3H), 3.60–3.49 (m, 13H), 3.46 (d, J = 8.1 Hz, 9H), 2.87–2.68 (m, 2H). 13 C NMR(101MHz,CDCl 3)δ166.98, 165.61, 165.57, 159.13, 157.58, 153.34, 143.09, 141.83, 139.18, 137.85, 137.61, 133.76, 133.04, 131.82, 131.18, 131.07, 130.74, 130.68, 130.54, 129.46, 129.10, 128.37, 127.80, 127.55, 127.50, 127.46, 127.24, 127.00, 126.92, 125.55, 125.51, 125.46, 124.62, 123.48, 123.33, 122.08, 121.13, 120.94, 120.62, 113.63, 112.70, 112.53, 69.43, 69.40, 69.37, 69.31, 69.07, 68.90, 68.27, 62.13, 59.67, 54.27, 54.18, 49.28, 47.15, 41.38, 40.05, 38.83, 34.43. HRMS(ESI) calcd for [C 70 H 70 Cl 2 F 6 N 8 O 11 S + H] + 1415.4244, found 1415.4225。

[0060] GDAz - 6:4 - ((2 - (3,5 - bis(trifluoromethyl)phenyl) - 4,5 - bis(4 - methoxyphenyl) - 1H - imidazol - 1 - yl)methyl) - N - (2 - (2 - (4 - ((2 - chloro - 4 - (2 - chloro - N - (2 - oxo - 2 - (phenylethylamino) - 1 - (thiophen - 2 - yl)ethyl)acetamido)phenoxy)methyl) - 1H - 1,2,3 - triazol - 1 - yl)acetamido)ethyl)benzamide。

[0061] 1 H NMR(400MHz, CDCl 3)δ 7.96 (d, J = 1.7 Hz, 2H), 7.73 (d, J = 14.8 Hz, 3H), 7.63 (d, J = 8.1 Hz, 2H), 7.51–7.45 (m, 1H), 7.43–7.37 (m, 2H), 7.12–7.07 (m, 5H), 7.06–7.01 (m, 1H), 6.99–6.94 (m, 2H), 6.86 (d, J = 8.0 Hz, 2H), 6.80–6.75 (m, 2H), 6.72–6.68 (m, 3H), 6.67 (d, J = 2.1 Hz, 1H), 6.40 (t, J = 5.9 Hz, 1H), 6.02 (s, 1H), 5.03 (d, J = 5.7 Hz, 4H), 4.95 (s, 2H), 3.71 (s, 2H), 3.68 (s, 3H), 3.64 (s, 3H), 3.42 (d, J = 6.5 Hz, 2H), 3.37–3.31 (m, 2H), 2.65 (dq, J = 14.1, 6.9 Hz, 4H). 13 C NMR (101 MHz, CDCl 3 )δ 167.17, 166.57, 165.76, 165.37, 159.13, 157.59, 153.16, 143.11, 142.21, 139.64, 137.84, 137.56, 133.70, 132.33, 131.76, 131.19, 131.00, 130.70, 130.66, 130.57, 129.52, 129.20, 128.43, 127.74, 127.52, 127.24, 126.98, 126.91, 125.61, 125.47, 125.43, 124.83, 124.08, 123.32, 121.16, 120.86, 120.61, 113.65, 112.73, 112.54, 61.87, 59.75, 54.24, 54.15, 51.52, 47.11, 41.64, 40.06, 39.23, 38.96, 34.31. HRMS (ESI) calcd for [C 62 H 53 Cl 2 F 6 N 9 O 7 S + H] + 1252.3148, found 1252.3135。

[0062] GDAz-7: 4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-(3-(2-(4-((2-chloro-4-(2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamido)propyl)benzamide.

[0063] 1 H NMR(400MHz,CDCl 3 )δ7.96(d,J=1.7Hz,2H),7.81(d,J=6.9Hz,1H),7.74–7.63(m,4H),7.47–7.37(m,3H),7.11–7.05(m,5H),7.05–7.01(m,1H),6.97–6.93(m,2H),6.85(d,J=8.0Hz,2H),6.79–6.74(m,2H),6.71–6.65(m,4H),6.47(d,J=5.9Hz,1H),6.03(s,1H),5.12–4.94(m,6H),3.73–3.60(m,8H),3.24(dq,J=33.5,6.0Hz,4H),2.74(d,J=12.1Hz,2H),2.64(dd,J=15.4,8.2Hz,2H),1.91(s,1H),1.60(s,1H). 13 C NMR(101MHz,CDCl 3 )δ168.24,167.04,166.76,165.88,160.17,158.62,154.24,144.15,143.16,140.47,138.86,138.60,134.77,133.68,132.83,132.21,132.04,131.70,130.59,130.23,128.75,128.57,128.53,128.23,128.02,127.86,126.64,126.52,126.45,125.83,125.13,124.35,121.87,121.64,114.67,113.74,62.94,60.43,55.25,55.16,48.15,42.68,41.11,36.54,35.34,29.03.HRMS(ESI)calcd for

[0064] [C 63 H 55 Cl 2 F6 N 9 O 7 S+H] + 1266.3305, found 1266.3284。

[0065] GDAz-8: 4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-(6-(2-(4-((2-chloro-4-(2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamido)hexyl)benzamide。

[0066] 1 H NMR(400MHz, CDCl 3 ) δ 7.97(s, 2H), 7.81(s, 1H), 7.74(s, 1H), 7.64(d, J = 8.4Hz, 2H), 7.42(d, J = 8.9Hz, 2H), 7.17–7.12(m, 3H), 7.10(dd, J = 9.0, 2.5Hz, 3H), 7.04–7.00(m, 2H), 6.97(t, J = 5.8Hz, 1H), 6.88(d, J = 8.1Hz, 3H), 6.80(d, J = 8.8Hz, 2H), 6.75(d, J = 5.2Hz, 2H), 6.71(d, J = 8.9Hz, 2H), 6.55(t, J = 5.9Hz, 1H), 6.18(t, J = 5.9Hz, 1H), 6.01(s, 1H), 5.12(s, 1H), 5.05(s, 2H), 4.98(s, 2H), 3.75–3.67(m, 8H), 3.51–3.36(m, 2H), 3.30(q, J = 6.7Hz, 2H), 3.15(q, J = 6.5Hz, 2H), 2.74–2.65(m, 2H), 1.49–1.38(m, 3H), 1.24(s, 4H), 1.18(d, J = 7.1Hz, 1H). 13 C NMR(101MHz, CDCl 3)δ167.02,165.88,165.67,163.99,159.15,157.62,153.21,143.11,142.31,139.32,137.79,137.56,133.68,133.11,131.72,131.19,131.05,130.80,130.72,130.57,129.47,129.16,128.37,127.77,127.59,127.56,127.29,126.97,126.69,125.59,125.48,125.38,124.83,123.96,123.31,121.20,120.85,120.60,113.65,112.72,112.54,61.98,59.76,54.27,54.18,51.80,47.11,41.47,40.06,38.49,38.33,34.38,28.30,27.82,24.80,24.73.HRMS(ESI)calcd for[C 66 H 61 Cl 2 F 6 N 9 O 7 S+H] + 1308.3774,found 1308.3755。

[0067] GDAz-9:4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-(1-(2-(4-((2-chloro-4-(2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetyl)piperidin-4-yl)benzamide。

[0068] 1 H NMR(400MHz,CDCl 3) δ 7.97 (s, 2H), 7.74 (d, J = 6.5 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.15–7.04 (m, 6H), 6.98 (d, J = 7.3 Hz, 2H), 6.85 (t, J = 9.7 Hz, 4H), 6.78 (d, J = 8.3 Hz, 2H), 6.75–6.66 (m, 4H), 6.31 (t, J = 6.0 Hz, 1H), 6.01 (s, 1H), 5.32 (d, J = 16.3 Hz, 1H), 5.08 (dd, J = 16.3, 9.1 Hz, 5H), 4.41 (d, J = 12.9 Hz, 1H), 4.18–4.03 (m, 1H), 3.78 (d, J = 13.5 Hz, 1H), 3.70 (d, J = 3.1 Hz, 4H), 3.66 (s, 3H), 3.39 (dp, J = 30.2, 6.6 Hz, 2H), 3.15 (t, J = 12.5 Hz, 1H), 2.82 (s, 1H), 2.74 (s, 1H), 2.66 (q, J = 7.7 Hz, 2H), 2.39 (s, 2H), 1.95 (dd, J = 43.0, 11.7 Hz, 2H). 13 C NMR (101 MHz, CDCl 3 ) δ 167.10, 165.60, 165.08, 162.27, 161.57, 159.12, 157.58, 153.18, 143.10, 142.07, 139.49, 137.84, 137.60, 133.77, 132.72, 131.82, 131.19, 131.01, 130.73, 130.67, 129.49, 129.16, 127.76, 127.52, 127.23, 126.95, 126.90, 125.59, 125.50, 125.43, 124.74, 124.36, 123.33, 120.89, 120.62, 113.63, 112.71, 112.59, 62.01, 54.25, 54.16, 49.94, 47.10, 45.86, 43.32, 41.56, 40.05, 35.49, 34.35, 31.09, 30.40, 30.29. HRMS (ESI) calcd for [C 65 H 57 Cl 2 F 6 N 9 O 7 S + H] + 1292.3461, found 1292.3446。

[0069] GDAz-10: 4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-((1-(2-(4-((2-chloro-4-(2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetyl)piperidin-4-yl)methyl)benzamide.

[0070] 1 H NMR (400 MHz, CDCl 3 ) δ 7.97 (s, 2H), 7.87 (s, 1H), 7.74 (s, 2H), 7.65 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.17–7.05 (m, 6H), 7.01 (d, J = 7.3 Hz, 2H), 6.88 (d, J = 7.7 Hz, 4H), 6.80 (d, J = 8.3 Hz, 3H), 6.75–6.62 (m, 4H), 6.25 (t, J = 6.0 Hz, 1H), 6.01 (s, 1H), 5.15 (d, J = 16.1 Hz, 3H), 5.05 (s, 2H), 4.42 (d, J = 13.1 Hz, 1H), 3.87–3.69 (m, 6H), 3.67 (s, 2H), 3.40 (dd, J = 15.6, 8.3 Hz, 1H), 3.21 (dt, J = 22.6, 6.7 Hz, 2H), 3.02 (t, J = 12.9 Hz, 1H), 2.85 (s, 2H), 2.76 (s, 2H), 2.68 (d, J = 8.2 Hz, 1H), 2.54 (t, J = 12.9 Hz, 1H), 2.26 (s, 2H), 1.89–1.63 (m, 3H). 13 C NMR (101 MHz, CDCl 3)δ167.05,165.95,165.61,162.02,161.58,159.15,157.60,153.23,143.10,142.10,139.47,137.84,137.61,133.75,132.85,131.81,131.19,131.03,130.70,129.48,129.15,128.38,127.78,127.54,127.24,126.95,126.78,125.59,125.45,124.81,124.18,123.33,122.15,121.15,120.88,120.62,113.65,112.72,112.63,62.09,59.67,54.27,54.18,50.01,47.10,44.13,44.01,41.54,41.34,40.05,35.50,35.05,34.38,30.41,29.16,28.29.HRMS(ESI)calcd for[C 66 H 59 Cl 2 F 6 N 9 O 7 S+H] + 1306.3618,found 1306.3601。

[0071] GDAz-11:4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)-N-(2-(1-(2-(4-((2-chloro-4-(2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamido)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetyl)piperidin-4-yl)ethyl)benzamide。

[0072] 1 H NMR(400MHz,CDCl 3)δ 7.97 (d, J = 1.6 Hz, 2H), 7.76 (d, J = 6.6 Hz, 2H), 7.62 (d, J = 8.2 Hz, 2H), 7.47–7.39 (m, 2H), 7.16 (t, J = 7.2 Hz, 3H), 7.11 (d, J = 8.7 Hz, 4H), 7.05–7.00 (m, 2H), 6.88 (d, J = 8.1 Hz, 3H), 6.80 (s, 3H), 6.78–6.74 (m, 2H), 6.72 (d, J = 8.9 Hz, 2H), 6.34 (t, J = 5.7 Hz, 1H), 6.11 (t, J = 5.9 Hz, 1H), 6.00 (s, 1H), 5.17 (d, J = 5.0 Hz, 2H), 5.05 (s, 2H), 4.44 (d, J = 13.6 Hz, 1H), 3.73 (d, J = 2.4 Hz, 5H), 3.69 (s, 3H), 3.49–3.36 (m, 4H), 3.08–2.96 (m, 1H), 2.71 (dt, J = 10.3, 6.9 Hz, 2H), 2.57 (d, J = 10.0 Hz, 1H), 1.96 (s, 1H), 1.80 (d, J = 13.8 Hz, 1H), 1.72 (d, J = 13.1 Hz, 1H), 1.58–1.43 (m, 3H), 1.21–1.03 (m, 3H). 13 C NMR (101 MHz, CDCl 3 )δ 166.98, 165.75, 165.63, 161.87, 159.15, 157.61, 153.26, 143.11, 142.19, 139.41, 137.82, 137.58, 133.69, 133.03, 131.76, 131.19, 131.05, 130.72, 129.43, 129.14, 128.31, 127.79, 127.56, 127.29, 126.95, 126.63, 125.57, 125.48, 125.40, 124.86, 124.05, 123.32, 121.19, 120.88, 120.61, 113.65, 112.72, 112.65, 62.16, 54.28, 54.18, 50.02, 47.09, 44.46, 41.67, 41.47, 40.04, 36.42, 35.07, 34.40, 32.53, 31.28, 30.47. HRMS (ESI) calcd for [C 67 H 61 Cl 2 F 6 N 9 O 7 S + H]+ 1320.3774, found 1320.3751。

[0073] GDAz-12: N-(4-((1-(2-(4-(4-((2-(3,5-bis(trifluoromethyl)phenyl)-4,5-bis(4-methoxyphenyl)-1H-imidazol-1-yl)methyl)benzoyl)piperazin-1-yl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-chlorophenyl)-2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophen-2-yl)ethyl)acetamide。

[0074] 1 H NMR(400MHz, CDCl 3 ) δ 7.96 (d, J = 1.7Hz, 2H), 7.77 (s, 1H), 7.74 (s, 1H), 7.46–7.41 (m, 2H), 7.27 (d, J = 8.0Hz, 2H), 7.20 (s, 1H), 7.16–7.11 (m, 5H), 7.10–7.07 (m, 1H), 7.04–6.99 (m, 2H), 6.94 (d, J = 7.9Hz, 3H), 6.85–6.80 (m, 2H), 6.77–6.68 (m, 4H), 6.24 (t, J = 5.9Hz, 1H), 6.03 (s, 1H), 5.27–4.99 (m, 6H), 3.73 (d, J = 3.9Hz, 6H), 3.68 (s, 4H), 3.60–3.24 (m, 8H), 2.80 (d, J = 31.7Hz, 1H), 2.71–2.65 (m, 1H). 13 C NMR(101MHz, CDCl 3 ) δ 168.87, 167.03, 165.59, 162.71, 159.19, 157.62, 153.20, 143.04, 142.33, 138.41, 137.85, 137.64, 133.76, 133.39, 131.82, 131.15, 130.94, 130.73, 130.60, 129.56, 129.13, 128.46, 127.79, 127.53, 127.23, 126.99, 126.89, 125.58, 125.44, 125.00, 124.11, 123.37, 120.87, 120.66, 113.72, 112.73, 62.10, 59.56, 54.29, 54.18, 49.91, 47.07, 41.55, 40.03, 34.39. HRMS(ESI) calcd for [C 64 H55 Cl 2 F 6 N 9 O 7 S+H] + 1278.3305, found 1278.3286。

[0075] Experimental Example 1: Verification of the degradation effect of the target compound on GPX4 in HT1080 cells

[0076] Western blot: HT1080 cells (3×10 5 cells) were seeded into a 6-well culture plate containing 2 mL of DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and cultured at 37 °C for 24 h. After the cells grew to 70% confluence, the original medium was discarded, and each well was replaced with 2 mL of DMEM medium containing a series of concentrations (0.1 μM, 0.3 μM, 1 μM, and 3 μM) of the test compound molecules supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. After incubation at 37 °C for 24 h, the culture medium was discarded, and the cells were washed twice with PBS. After discarding the washing solution, 100 μL of RIPA containing 1% phenylmethylsulfonyl fluoride (PMSF) and 10% phosphatase inhibitor was added to the culture wells, and the cells were lysed on ice for 10 min. Then, the cells were scraped off with a scraper and placed in a 1.5 mL EP tube. After collecting and quantifying the protein, 10 μL (5×) SDS loading buffer was added to the EP tube, and the mixture was heated at 100 °C for 10 min. The samples were separated by 15% SDS-PAGE and transferred to a PVDF membrane. After the membrane was blocked with 5% skim milk (in TBST buffer) at room temperature for 1.5 h, the membrane was cut at around 30 kD. The part of the membrane less than 30 kD was incubated with rabbit anti-anti-GPX4 (1:1000 dilution) at 4 °C overnight, and then HRP-conjugated goat anti-rabbit IgG (1:2000 dilution) was added and incubated at room temperature for 2 h; the PVDF membrane larger than 30 kD was incubated with HRP-conjugated mouse anti-GAPDH (1:100000 dilution) at 4 °C overnight, and then HRP-conjugated mouse anti-IgG (1:1000 dilution) was added and incubated at room temperature for 2 h. The blot was recorded using Invitrogen iBright 1500.

[0077] The experimental results are as Figure 2 shown. The WB results showed that multiple compounds such as GDAz-2 to GDAz-7 could significantly degrade GPX4.

[0078] Experimental Example 2: Verification of the killing effect of the synthesized chimera on GPX4 overexpressing tumor cell lines

[0079] Anti-cell proliferation activity assay: The CCK-8 method was used to evaluate the cytotoxicity and IC50 of all target compounds in HT1080 cells (DMEM medium). Cells were seeded in 96-well plates at a cell density of 5×10 3 cells / well for 24 hours. Then, the cells were treated with different concentrations of the compounds for 48 h. After discarding the original medium, 100 μL of the medium solution containing CCK-8 (1:10) was added to each well. After culturing for 1.5 h, the absorbance at 450 nm was measured using a microplate reader (TECAN). After converting the absorbance values into inhibition rates, the IC50 values were calculated using Graphpad Prism 5. The results are shown in Table 1.

[0080] Table 1. Evaluation of the anti-cell proliferation activity of the synthesized chimeras

[0081]

[0082] As can be seen from Table 1: The vast majority of the compounds have strong anti-cell proliferation activity, among which the two compounds GDAz-3 and GDAz-5 are particularly significant.

[0083] Experimental Example 3: Lipid peroxide determination

[0084] The accumulation of lipid peroxides is a significant feature of ferroptosis in cells. Cultured HT1080 cells were seeded in 6-well plates at 6×10 5 cells per well. After treatment with the drug (5 μM), the cells were collected, suspended in 500 μL of PBS containing 2 μM C11-BODIPY (581 / 591), and incubated at 37 °C for 10 min, followed by flow cytometry analysis. In this experiment, the compound GDAz-3 was randomly selected as a representative to test its effect on intracellular lipid peroxides. The results are as Figure 3 shown.

[0085] From Figure 3 it can be seen that: GDAz-3 can increase the level of intracellular lipid peroxides in a concentration-dependent manner, and at the same concentration, GDAz-3 has a stronger ability to induce lipid peroxides than the positive control ML162. The accumulation of peroxides triggers ferroptosis, but can be reversed by the ferroptosis inhibitor Fer-1, indicating that this compound has a high selectivity for ferroptosis.

[0086] Experimental Example 4: Comparison of the degradation effects of GDAz-3, a GPX4 degrader mediated by the HSP70 molecular chaperone complex, and GDCNF-2 / -11, a GPX4 degrader mediated by the HSP90 molecular chaperone complex

[0087] To further verify that the compounds involved in the present invention have unique advantages compared with the GPX4 degrader mediated by the HSP90 chaperone complex previously mentioned by the inventors (selecting the active compounds GDCNF-2 / -11 as controls), we used Western blot to detect its degradation time-dependence and its ability to continuously degrade GPX4 after drug withdrawal. The results are as Figure 4 shown: Compared with the two GPX4 degraders GDCNF-2 / -11 mediated by the HSP90 chaperone complex reported previously by us, the novel GPX4 degrader GDAz-3 mediated by the HSP70 chaperone complex involved in the present invention has a faster degradation rate and a more persistent degradation effect.

[0088] Experimental Example 5: Potential application of the GPX4 degrader GDAz-3 mediated by the HSP70 chaperone complex

[0089] To continue to verify the potential application of the GPX4 degrader GDAz-3 mediated by the HSP70 chaperone complex, after knocking out the E3 ubiquitin ligases CRBN and VHL of HT-1080 cells, GDAz-3 still showed effective GPX4 degradation activity; in the renal cancer 786-O cell line that is naturally lacking in VHL, the previously reported active compounds dGPX4 and 8e (GPX4-targeted PROTACs based on CRBN and VHL respectively) and the previously reported active GPX4 degrader GDCNF-11 mediated by the HSP90 chaperone complex could not effectively degrade GPX4 in the 786-O cell line, while the GPX4 degrader GDAz-3 mediated by the HSP70 chaperone complex involved in the present invention could effectively degrade GPX4 in the 786-O cell line. The results are as Figure 5 shown, which indicates that the GPX4 degrader GDAz-3 mediated by the HSP70 chaperone complex has great application potential in CRBN / VHL low-expression or mutant, drug-resistant cell lines.

Claims

1. A chimera based on HSP70 protein targeted degradation of GPX4, characterized in that: The chimera is a structure represented by the general formula (I) or a pharmacologically or physiologically acceptable salt thereof. In the general formula (I), Linker is a linking group, which represents an -alkylene group or an -alkoxy group or a -piperazinyl group or a -1,2,3-triazolyl group, wherein the -alkylene group or the -alkoxy group or the -piperazinyl group or the -1,2,3-triazolyl group is selected from any one of the following groups or any combination thereof, wherein p, m and n represent natural numbers from 1 to 20: -(CH2) n -C(O)NH(CH2CH2O) m -or-(CH2CH2O) n -C(O)NH(CH2CH2O) m -or 2. A chimera based on HSP70 protein targeted degradation of GPX4 as claimed in claim 1, characterized in that: The chimera is any one of the following compounds GDAz-1 to GDAz-12:

3. A chimera based on HSP70 protein targeted degradation of GPX4 as claimed in claim 2, characterized in that: The synthetic route of the chimera is: 。 4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the chimera based on HSP70 protein targeted degradation of GPX4 as described in any one of claims 1-2 or a pharmacologically or physiologically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof.

5. Use of a chimera for targeted degradation of GPX4 based on HSP70 protein according to any one of claims 1 to 2 or a pharmacologically or physiologically acceptable salt thereof or a pharmaceutical composition according to claim 4 in the preparation of a GPX4 degrading agent or a drug for inhibiting GPX4.

6. Use of a chimera for targeting GPX4 degradation based on HSP70 protein as described in any one of claims 1 to 2 or a pharmacologically or physiologically acceptable salt thereof or a pharmaceutical composition as described in claim 4 in the preparation of a drug for treating GPX4-related diseases.

7. The use according to claim 6, characterized in that The GPX4-related diseases are tumors and neurodegenerative diseases.

8. Use of a chimera based on HSP70 protein targeted degradation of GPX4 according to any one of claims 1-2 or a pharmacologically or physiologically acceptable salt thereof or the pharmaceutical composition according to claim 4 in the preparation of an anti-tumor drug, wherein the tumor is gastric cancer, breast cancer, lung cancer, ovarian cancer, colon adenocarcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, lung adenocarcinoma, prostate cancer, rectal adenocarcinoma, thyroid cancer and endometrial cancer.

9. The use according to claim 8, characterized in that The tumor is a tumor with high expression of GPX4.

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