GPX4 protein degradation agent and application

Abstract

According to the GPX4 protein degradation agent and the application, the degradation agent serves as molecular glue to induce tumor cell ferroptosis and is different from an existing PROTAC technology, and the molecular glue can induce interaction between E3 ubiquitin ligase and target protein GPX4 and promote ubiquitination of the E3 ubiquitin ligase and the target protein GPX4. Compared with the existing GPX4 degradation agent, the molecular glue has the advantages of small molecular weight, high cell permeability and better druggability. The GPX4 molecular glue disclosed by the invention can be used for effectively degrading GPX4 and has a killing effect on various tumor cell lines. The preparation method is simple in synthesis route and mild in reaction condition, can be used for being developed into a new generation of GPX4 targeting drugs, and has great clinical application value and considerable market potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to a GPX4 protein degrading agent and application. BACKGROUND

[0002] The main treatment methods for cancer are surgical therapy, radiotherapy and chemotherapy, and there are still some problems in the actual application of various treatment methods. Therefore, finding efficient and novel anticancer drugs is one of the main tasks of pharmaceutical workers.

[0003] Inducing tumor cell ferroptosis is a new way of treating tumors in recent years. Unlike apoptosis, necrosis and pyroptosis, ferroptosis is a programmed cell death mode characterized by the accumulation of iron-dependent lipid reactive oxygen species (ROS) 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 GSH is used as a cofactor. GPX4 can reduce intracellular lipid hydroperoxide into nontoxic lipid alcohol compounds, and also catalyze the reduction of hydrogen peroxide and other organic peroxides, thereby protecting cells from oxidative stress and inhibiting the occurrence of ferroptosis. Therefore, inhibiting the activity of GPX4 will affect the ability of GPX4 to remove lipid peroxides, ultimately leading to the occurrence of cell ferroptosis. In addition, inhibiting the function of GPX4 will trigger persistent ferroptosis of cells and prevent tumor recurrence, and thus is one of the strategies to solve drug resistance.

[0004] At present, there are still some challenges for GPX4-targeted small molecules, and no GPX4 small molecule has entered the clinical research stage. For inhibitors, the main problems include: 1) the molecular surface of GPX4 lacks a drug-like binding pocket; 2) the currently reported inhibitors are covalent inhibitors, which bind to the active site selenocysteine of GPX4 to play a role, but have the problem of low selectivity. For GPX4-targeted degrading agents, although there are many reports of PROTAC molecules, which can also improve their selectivity, but due to their large molecular weight, poor drugability also limits their further development.

[0005] At present, there is no report on GPX4-targeted degrading molecules. Unlike PROTAC, molecular glue has the characteristics of dual-ligand structure for E3 ubiquitin ligase and target protein, and has the function of binding to two proteins, promoting ubiquitination of two proteins, and degrading non-drug targets and protein-protein interactions. Molecular glue has small molecular weight, high cell permeability, good oral absorption, meets the principles of drug-like substances, and has good drugability. The molecular glue strategy provides a new research idea for drug development in the field of targeted protein degradation. SUMMARY

[0006] In view of the problems in the prior art, the GPX4 protein degrading agent and application are designed to trigger tumor cell ferroptosis by targeting degradation of GPX4 as a molecular glue, and the technical solutions are as follows:

[0007] A GPX4 protein degrading agent has a structure shown in formula (I):

[0008]

[0009] The GPX4 protein degrading agent is prepared by the following steps:

[0010] 1) obtaining the ML162-yne intermediate 5 by Ugi four-component reaction; the reaction flow is as follows:

[0011] 2) reacting the acryloyl chloride derivative with the Boc monosubstituted piperazine derivative to obtain the intermediate 7;

[0012] 3) removing the Boc substituent of the intermediate 7 in the presence of trifluoroacetic acid to obtain the intermediate 8;

[0013] 4) the intermediate 8 is subjected to amide condensation reaction with azidoacetic acid under the action of a condensing agent to obtain the azide intermediate 9;

[0014] 5) the intermediate 9 is subjected to click reaction with the prepared ML162-yne to obtain the final products LDCWL-1 and LDCWL-2;

[0015] The reaction flow of step 2) step-5) is as follows:

[0016]

[0017] The application of the above GPX4 protein degrading agent in preparing an antitumor drug.

[0018] Further, the tumor includes a solid tumor and a blood tumor, the blood tumor includes lymphoma Krapas-299, human acute lymphoblastic leukemia CCRF-CEM, the solid tumor includes non-small cell lung cancer NCI-H3122, fibrosarcoma cell HT108, and melanoma B16-F10.

[0019] The application provides a new acrylamide compound ML162, in addition to targeting solid tumors like PROTAC, the GPX4-targeting molecular glue degrader based on ML162 provided by the application has strong activity and wider applicability, and it has higher activity in both solid tumors and hematological tumors. From the cell level, it has significant inhibitory activity on hematological diseases including Krapas-299 lymphoma, human acute lymphoblastic leukemia CCRF-CEM, solid tumors such as non-small cell lung cancer NCI-H3122, fibrosarcoma cell HT108, and melanoma B16-F10 cell strain. The results show that the molecular glue of the application has more than 3 times the tumor inhibitory activity of the PROTAC degrader. From the molecular level detection, the degradation efficiency of GPX4 is more than 10 times that of the previous PROTAC degrader, indicating that the application has good potential clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Synthetic route of LDCWL-1 and LDCWL-2;

[0021] Figure 2 NMR hydrogen spectrum of LDCWL-1;

[0022] Figure 3 NMR carbon spectrum of LDCWL-1;

[0023] Figure 4 NMR hydrogen spectrum of LDCWL-2;

[0024] Figure 5 NMR carbon spectrum of LDCWL-2;

[0025] Figure 6 Growth inhibition of compounds LDCWL-1 and LDCWL-2 on different tumor cell strains;

[0026] Figure 7 Degradation of compounds LDCWL-1 and LDCWL-2 on GPX4 protein of HT1080 cell strain. DETAILED DESCRIPTION

[0027] The application will be further described below in conjunction with the accompanying drawings of the specification, so as to better understand the technical solution.

[0028] The technical and scientific terms used in the following examples have the same meaning as generally understood by those skilled in the art to which the application belongs. The basic raw reagents are obtained from commercial channels, and the purity is 97% or higher. The room temperature described in the application is 25-30 DEG C. The application generally and specifically describes the materials used in the test and the experimental method.

[0029] Example 1: Synthesis and structural characterization of LDCWL-1 and LDCWL-2

[0030] Synthesis of compound 2:

[0031] A suspension of compound 14-amino-2-chlorophenol (1,500 mg, 3.48 mmol) and di-tert-butyl dicarbonate (836 mg, 3.83 mmol) in tetrahydrofuran (20 mL) was stirred at room temperature for 24 h. The reaction was concentrated under reduced pressure and extracted with ethyl acetate twice. The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: ethyl acetate: petroleum ether at a volume ratio of 1:5) to give compound 2 (yellow liquid, 714 mg, yield 84%).

[0032] Synthesis of compound 3:

[0033] Compound 2 (1000 mg, 4.1 mmol) was dissolved in N,N-dimethylformamide (20 mL), potassium carbonate (851 mg, 6.2 mmol) and 3-bromoprop-1-yn (634 mg, 5.3 mmol) were added and stirred at room temperature overnight. The reaction was diluted with water and extracted with ethyl acetate three times. The combined organic layers were washed with water and saturated aqueous sodium chloride solution in turn, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: ethyl acetate: petroleum ether at a volume ratio of 1:5) to give compound 3 (yellow liquid, 850 mg, yield 73%).

[0034] Synthesis of compound 4:

[0035] Compound 3 (1000 mg, 3.55 mmol) was dissolved in dichloromethane (40 mL), trifluoroacetic acid (10 mL) was added and stirred at room temperature for 2 h. The reaction was concentrated under reduced pressure and diluted with aqueous NaHCO3 solution and extracted with ethyl acetate three times. The separated organic layers were dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give compound 4 (white solid, 480 mg, yield 74%).

[0036] Synthesis of compound 5 (ML162-yne):

[0037] Compound 4 (1.82 g, 10 mmol) and 2-thiophene carboxaldehyde (1.12 g, 10 mmol) were dissolved in (25 mL) methanol, after activation at 25 °C for 1 h, (2-isocyanomethyl) benzene (1.09 g, 8.33 mmol), 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 (mobile phase was ethyl acetate: petroleum ether at a volume ratio of 1:1) to obtain compound 5ML162-yne (white solid, 850 mg, yield 20%).

[0038] Synthesis of compound 7:

[0039] Compound 6 acyl chloride (1.0 eq, 20 mmol) and N-Boc-piperazine (1.1 eq, 22 mmol) were dissolved in 20 ml of anhydrous dichloromethane, and triethylamine (3 eq, 60 mmol) was 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 (mobile phase was ethyl acetate: petroleum ether at a volume ratio of 1:1) to obtain compound 7.

[0040] Synthesis of compound 8:

[0041] Compound 7 (1.0 eq, 20 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (5 mL) was added, and the solution was rotary evaporated to dryness after 2 hours of reaction, and dried in a 40 °C oven to obtain compound 8.

[0042] Synthesis of compound 9:

[0043] Compound 8 (1.1 eq, 22 mmol) was dissolved in acetonitrile (20 mL), and 2-azidoacetic acid (1.0 eq, 20 mmol), N,N,N',N'-tetramethylchloroformamidinium hexafluorophosphate (TCFH, 1.2 eq, 24 mmol), N-methylimidazole (2.0 eq, 40 mmol) were added, and reacted at room temperature for 5 hours, then water was added and extracted with ethyl acetate, and the organic layer was dried with anhydrous sodium sulfate, and the solution was rotary evaporated to dryness, and the residue was separated by silica gel column chromatography (mobile phase was ethyl acetate: petroleum ether at a volume ratio of 1:1) to obtain compound 9.

[0044] Synthesis of compounds LDCWL-1 and LDCWL-2

[0045] Compound 9 (1.0 eq, 0.5 mmol) and compound ML162-yne (1.0 eq, 0.5 mmol) were dissolved in tetrahydrofuran (5 mL), copper sulfate (0.05 eq, 0.025 mmol) and sodium ascorbate (2.5 eq, 1.25 mmol) were dissolved in water (1 mL), and the reaction was carried out at room temperature under nitrogen protection overnight. The reaction solution was diluted with ethyl acetate (200 mL), washed with water and saturated sodium chloride aqueous solution in turn, and the organic phase was dried over sodium sulfate and then filtered under suction. The filtrate was rotary evaporated to dryness, and the concentrate was separated by silica gel column chromatography (eluent: ethyl acetate) to obtain target compounds LDCWL-1 and LDCWL-2.

[0046] The synthesis route of compounds LDCWL-1 and LDCWL-2 is shown in Figure 1 The nuclear magnetic hydrogen spectrum of LDCWL-1 is shown in Figure 2 The nuclear magnetic carbon spectrum is shown in Figure 3 The nuclear magnetic hydrogen spectrum of LDCWL-2 is shown in Figure 4 The nuclear magnetic carbon spectrum is shown in Figure 5 .

[0047] N-(4-((1-(2-(4-Propenoylpiperazin-1-yl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3- chlorophenyl)-2-chloro-N-(2-oxo-2-(phenethylamino)-1-(thiophen-2-yl)ethyl)acetamide (LDCWL-1) 1 H NMR (400 MHz, CDC13) δ 7.79 (s, 1H), 7.20 - 7.15 (m, 3H), 7.15 - 6.95 (m, 4H), 6.95 - 6.56 (m, 4H), 6.48 (dd, J = 16.8, 10.5 Hz, 1H), 6.27 (dd, J = 16.8, 1.8 Hz, 1H), 6.02 (d, J = 4.0 Hz, 2H), 5.70 (dd, J = 10.5, 1.8 Hz, 1H), 5.23 (s, 2H), 5.20 (s, 2H), 3.74 (d, J = 1.4 Hz, 2H), 3.70 - 3.49 (m, 8H), 3.49 - 3.34 (m, 2H), 2.84 - 2.62 (m, 2H). 13C NMR (101 MHz, CDC13) δ 166.92, 165.62, 164.60, 162.54, 153.22, 142.52, 137.59, 133.71, 130.87, 130.58, 129.12, 128.36, 128.19, 127.81, 127.59, 127.31, 125.72, 125.57, 125.51, 123.92, 122.31, 112.69, 64.83, 62.20, 59.71, 49.98, 45.71, 41.42, 41.23, 40.50, 40.04, 34.46.

[0048] (E)-2-chloro-N-(3-chloro-4-((l-(2-(4-(3-fluoroallyl)piperazin-l-yl)-2- oxoethyl)-lH-l,2,3-triazol-4-yl)methoxy)phenyl)-N-(2-oxo-2-(phenethylamino)- 1-(thiophen-2-yl)ethyl)acetamide (LDCWL-2) 1 H NMR (400 MHz, CDC13) δ 7.79 (s, 1H), 7.18 (dt, J = 5.9, 1.9 Hz, 3H), 7.15 - 7.03 (m, 4H), 6.97 - 6.72 (m, 4H), 6.03 (d, J = 6.9 Hz, 2H), 5.32 - 5.17 (m, 5H), 5.13 (dd, J = 16.8, 3.6 Hz, 1H), 3.74 (d, J = 1.3 Hz, 2H), 3.62 (dd, J = 6.9, 3.4 Hz, 4H), 3.57 (d, J = 6.1 Hz, 4H), 3.50 - 3.39 (m, 2H), 2.80 - 2.64 (m, 2H). 13 C NMR (101 MHz, CDC13) δ 166.93, 165.62, 162.57, 157.23, 154.53, 153.22, 142.51, 137.59, 133.72, 130.87, 130.59, 129.13, 128.37, 127.81, 127.58, 127.30, 125.57, 125.50, 123.92, 112.67, 100.03, 99.88, 76.24, 62.19, 59.70, 49.96, 41.42, 40.04, 35.50, 34.45, 30.44, 28.67.

[0049] Example 2: Validation of the growth inhibitory effect of the synthetic molecular glue on tumor cells

[0050] The anti-proliferative activity of the drug against selected tumor cell lines was detected using CCK-8 assay: 3000 cells were seeded into each well of a 96-well plate (100 μL of serum-containing medium), with blank wells (medium only) and control wells (untreated cells). Cells were incubated at 37°C for 24 h. Complete medium with drug concentration gradients of 0.06, 0.1, 0.3, 0.6, and 1 μmol / ml was added according to the experimental design, and the cells were incubated at 24°C for 48 h. 10 μL of CCK-8 reagent was added directly to each well (avoiding air bubbles), and the cells were incubated at 37°C in the dark for 2 h. The OD value at 450 nm was measured using a microplate reader.

[0051] Experimental results are as follows Figure 6 As shown, from Figure 6 It can be seen that the synthesized molecular gels LDCWL-1 and LDCWL-2 have significant anti-tumor effects on the selected tumor cell lines, especially on the Karpas-299, CCRF-CEM and HT1080 cell lines, with IC50 values ​​as low as nanomolar.

[0052] Example 3: Verification of the degradation effect of the synthesized molecular glue on intracellular GPX4

[0053] Immunoblot: HT1080 cells (3×10⁻⁶) were used to bleach the cells. 5Cells were seeded into 6-well plates 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 reached 70% confluence, the original medium was discarded, and each well was replaced with 2 mL of DMEM medium containing different concentrations (0, 0.06, 0.1, 0.3, 0.6, 1 μM) of the target compound containing 10% 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. The wash buffer was discarded, and 100 μL of RIPA containing 1% phenylmethylsulfonyl fluoride (PMSF) and 10% phosphatase inhibitor was added to each well. The cells were lysed on ice for 10 min, and then scraped off with a spatula and placed in a 1.5 mL EP tube. 20 μL of 5×SDS loading buffer was added to the EP tube, and the tube was heated at 99°C for 10 min. Samples were separated by 15% SDS-PAGE and transferred to PVDF membranes. After blocking the membranes with 5% skim milk (in TBST buffer) for 1.5 h at room temperature, the membranes were cut at approximately 30 kDa. The portion <30 kDa was incubated overnight at 4°C with rabbit anti-GPX4 (1:1000 dilution), followed by the addition of HRP-conjugated goat anti-rabbit IgG (1:2000 dilution) and incubation at room temperature for 2 h. The PVDF membrane >30 kDa was incubated overnight at 4°C with HRP-conjugated mouse anti-GAPDH (1:100000 dilution), followed by the addition of HRP-conjugated mouse anti-IgG (1:1000 dilution) and incubation at room temperature for 2 h. Blots were recorded using an Invitrogen iBright 1500.

[0054] Experimental results are as follows Figure 7 As shown, from Figure 7 It can be seen that the synthesized molecular gels LDCWL-1 and LDCWL-2 both have significant degradation effects on GPX4. Degradation of GPX4 can effectively trigger ferroptosis in tumor cells. Compared with PROTAC degrading agents, molecular gel degrading agents have smaller molecular weights, higher drug-likeness, and broad application prospects.

Claims

1. A GPX4 protein degrading agent, characterized in that, It has the structure shown in equation (I):

2. The GPX4 protein degrading agent according to claim 1, characterized in that, The preparation is carried out using the following steps: 1) ML162-yne intermediate 5 was obtained via a four-component reaction using Ugi; 2) Acryloyl chloride derivatives react with Boc monosubstituted piperazine derivatives to give intermediate 7; 3) In the presence of trifluoroacetic acid, intermediate 7 is desubstituented to give intermediate 8; 4) Intermediate 8 undergoes an amide condensation reaction with azidoacetic acid under the action of a condensing agent to obtain azide intermediate 9; 5) Intermediate 9 reacts with the prepared ML162-yne via a click reaction to yield the final products LDCWL-1 and LDCWL-2.

3. The GPX4 protein degrading agent as described in claim 2, characterized in that, The reaction process for step 1) is as follows:

4. The GPX4 protein degrading agent as described in claim 2, characterized in that, The reaction flow for steps 2) and 5) is as follows:

5. The use of the GPX4 protein degrader according to any one of claims 1-4 in the preparation of antitumor drugs.

6. The application as described in claim 5, characterized in that... The tumors include solid tumors and hematologic tumors, including lymphoma Krapas-299, human acute lymphoblastic leukemia CCRF-CEM, solid tumors non-small cell lung cancer NCI-H3122, fibrosarcoma cells HT108, and melanoma B16-F10.

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