Chimera based on hsp90 protein targeting degradation of gpx4 and preparation method and application

Abstract

The application discloses a chimeric body based on HSP90 protein targeted degradation of GPX4, a preparation method and application, and belongs to the technical field of biological medicines. The application utilizes the technology to prepare 13 chimeric bodies targeted degradation of GPX4. The chimeric body mediated by the molecular chaperone HSP90 involved in the application can effectively induce the degradation of GPX4. Meanwhile, the chimeric bodies have obvious anti-proliferation activity on cell strains with high expression of GPX4, and have important significance for tumor targeted therapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a class of chimeras based on molecular chaperone HSP90-mediated GPX4 degradation activity, a preparation method thereof, and application thereof in the aspect of anti-tumor. BACKGROUND

[0002] Different from apoptosis, necrosis and pyroptosis, the particularity of ferroptosis lies in that it is a programmed cell death mode characterized by accumulation of iron-dependent lipid reactive oxygen species (ROS) radicals. A large number of studies have found that glutathione peroxidase 4 (GPX4) can be 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 can also catalyze the reduction of hydrogen peroxide and other organic peroxides, thus having the effect 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 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.

[0003] At present, there are still certain challenges for small molecule inhibitors targeting GPX4, and no GPX4 inhibitor has been reported in the clinical research stage. The main reasons are as follows: 1) the molecular surface of GPX4 lacks a drug-like binding pocket; 2) the currently reported inhibitors are all covalent inhibitors, which act by binding to the active site selenocysteine of GPX4, but have the problem of low selectivity.

[0004] Using protein degradation technology to induce degradation of cancer proteins is one of the recent research topics. Molecular chaperone-mediated targeted degradation technology is a new protein degradation technology, which has the advantages of overcoming drug resistance and the like. Using molecular chaperone-mediated targeted protein degradation technology, the present application discloses preparation and application of a diamino purine compound as a GPX4 degradation agent. SUMMARY

[0005] Based on the problems in the above background technology, the purpose of the present application is to provide a diamino purine chimeric based on HSP90 protein targeted degradation of GPX4, a preparation method and application. The chimeric of the present application can effectively degrade GPX4 protein, thereby inducing ferroptosis of tumor cells.

[0006] To achieve the above application purposes, the present application adopts the following technical solutions:

[0007] The present application provides a chimeric molecule of a diamino purine class for targeted degradation of GPX4 protein as shown in formula (I) or a pharmacologically or physiologically acceptable salt thereof:

[0008]

[0009] wherein:

[0010] Linker is a linking group, representing -alkylene or -alkoxy or -piperazinyl or -1,2,3-triazolyl, said -alkylene or -alkoxy or -piperazinyl or -1,2,3-triazolyl is selected from any one of the following groups or any combination thereof, wherein p, m and n represent a natural number from 1 to 20:

[0011] -(CH2) n -C(O)NH(CH2CH2O) m - or -(CH2CH2O) n -C(O)NH(CH2CH2O) m - or

[0012]

[0013] Further, the present application provides a chimeric molecule as shown in the following compound or a stereoisomer, geometric isomer, tautomer, nitroxide, hydrate, solvate, metabolite, pharmaceutically or physiologically acceptable salt or prodrug thereof:

[0014]

[0015]

[0016] The pharmacologically or physiologically acceptable salt of the present application refers to the salt formed by the HSP90 protein targeted degradation of GPX4 of the diamino purine class chimeric molecule of the present application and a pharmacologically or physiologically acceptable acid or base.

[0017] The present application also provides a pharmaceutical composition comprising the chimeric molecule of the present application for targeted degradation of GPX4 or a stereoisomer, geometric isomer, tautomer, nitroxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug thereof.

[0018] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or a combination thereof.

[0019] The pharmaceutical composition is an injection, oral agent, mucosal administration agent.

[0020] The pharmaceutical composition further comprises other drugs with therapeutic or prophylactic effects on tumors.

[0021] The application also provides the use of the chimeric body targeting degradation of GPX4 based on HSP90 protein or the pharmaceutical composition comprising the chimeric body. The specific embodiments are as follows.

[0022] The use of the chimeric body targeting degradation of GPX4 based on HSP90 protein or the pharmaceutical composition comprising the chimeric body in the preparation of a drug for degrading GPX4.

[0023] The use of the chimeric body targeting degradation of GPX4 based on HSP90 protein or the pharmaceutical composition comprising 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, and Huntington's disease.

[0024] The use of the chimeric body targeting degradation of GPX4 based on HSP90 protein or the pharmaceutical composition comprising the chimeric body in the preparation of an anti-tumor drug. The tumors are gastric cancer, breast cancer, lung cancer, ovarian cancer, colon adenocarcinoma, renal chromophobe, renal clear cell carcinoma, lung adenocarcinoma, prostate cancer, rectal adenocarcinoma, thyroid cancer, and endometrial cancer. Further, the tumors are tumors with high expression of GPX4.

[0025] The application also provides a synthetic route of the chimeric body targeting degradation of GPX4 based on HSP90 protein, which specifically comprises the following steps:

[0026] The preparation of the GPX4 ligand ML162-1 and the preparation of the HSP90 protein ligand BIIB021 are connected by amide condensation, deprotection, nucleophilic substitution, and Click click chemistry reaction types.

[0027] Compared with the prior art, the application has the following beneficial effects:

[0028] Unlike the PROTAC technology which directly pulls a certain E3 ubiquitin ligase, the application is based on the molecular chaperone HSP90 to mediate the ubiquitination of the target protein by various types of E3 ubiquitin ligases, thereby inducing the degradation of the target protein by the proteasome. The inventors have confirmed through Western blot experiments that the diamino purine chimeric body involved in the application can effectively degrade GPX4 and can effectively kill GPX4 high expression cell lines. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Synthetic route map of chimeric body GDCN-1-13;

[0030] Figure 2 Western Blot detection of the degradation activity of the chimeric body on GPX4. DETAILED DESCRIPTION

[0031] The present application will be described in detail below with reference to the accompanying drawings and specific examples. The technical and scientific terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present application belongs. The base raw reagents are obtained from commercial channels, and the purity is 97% or above. The room temperature described in the present application is 25-30°C. The materials used in the test and the experimental methods are described generally and specifically. Although many materials and operating methods used to achieve the purpose of the present application are well known in the art, the present application is described as much as possible.

[0032] Example 1: Synthesis and structural confirmation of chimeras targeting degradation of GPX4

[0033] The synthesis route of the final product GDCN 1-23 is as shown in Figure 1

[0034] Synthesis of compound 1:

[0035] A suspension of 4-amino-2-chlorophenol (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 hours. The reaction solution was concentrated under reduced pressure, and extracted with ethyl acetate twice. The combined organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (the mobile phase was ethyl acetate: petroleum ether at a volume ratio of 1:5) to obtain compound 1 (yellow liquid, 714 mg, yield 84%).

[0036] Synthesis of compound 2:

[0037] Compound 1 (1000 mg, 4.1 mmol) was dissolved in N,N-dimethylformamide (20 mL), potassium carbonate (851 mg, 6.2 mmol) was added, 3-bromopropan-1- yne (634 mg, 5.3 mmol) was added, and stirred at room temperature overnight. The reaction solution was added with water, extracted with ethyl acetate three times, and the combined organic layer was washed with water and saturated aqueous sodium chloride solution respectively, and dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (the mobile phase was ethyl acetate: petroleum ether at a volume ratio of 1:5) to obtain compound 2 (yellow liquid, 850 mg, yield 73%).

[0038] Synthesis of compound 3:

[0039] ​Compound 2 (1000 mg, 3.55 mmol) was dissolved in dichloromethane (40 mL), trifluoroacetic acid (10 mL) was added, and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, an aqueous NaHCO3 solution was added, and extracted with ethyl acetate 3 times. The separated organic layer was dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 3 (white solid, 480 mg, yield 74 %). Synthesis of compound ML162-1:

[0040] Compound 3 (1.82 g, 10 mmol) and 2-thiophene carboxaldehyde (1.12 g, 10 mmol) were dissolved in (25 mL) methanol, and after activation at 25℃ 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: ethyl acetate: petroleum ether = 1:1 by volume) to obtain compound ML162-1 (white solid, 850 mg, yield 20 %).

[0041] Synthesis of compound 4:

[0042] 2-amino-6-chloropurine (8.0 g, 47.18 mmol) was dissolved in N,N-dimethylformamide (50 mL), and 2-(chloromethyl)-4-methoxy-3,5-dimethylpyridine hydrochloride (10.42 g, 47.18 mmol), sodium iodide (707 mg, 4.72 mmol), and potassium carbonate (19.56 g, 141.53 mmol) were added, and reacted at 40℃ for 6 hours. After the reaction mixture was cooled, it was filtered, and washed with DMF. The organic layer was diluted with water, filtered, and the precipitate was dried in an oven at 40℃ to obtain compound 4 (white solid, 10.5 g, yield 70 %).

[0043] Synthesis of compound 5:

[0044] Compound 4 (1 g, 3.14 mmol) was dissolved in n-butanol (20 mL), and tert-butyl (4-aminobutyl)carbamate (649.69 mg, 3.45 mmol) and N-ethyldiisopropylamine (3 mL) were added, and reacted at 120℃ for 8 hours. The reaction solution was concentrated under reduced pressure, water was added, extracted with ethyl acetate 3 times, and the combined organic layer was washed with water and saturated aqueous sodium chloride solution each 1 time, dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 5 (white solid, 1.1 g, yield 75 %).

[0045] Synthesis of compound 6:

[0046] Compound 5 (1.1 g, 3.14 mmol) was dissolved in dichloromethane (40 mL), and trifluoroacetic acid (10 mL) was added, and stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to obtain compound 6 (white solid, 650 mg, yield 75%).

[0047] Synthesis of compound 7:

[0048] Compound 6 (650 mg, 1.75 mmol) and azidoacetic acid (212.79 mg, 2.11 mmol) were dissolved in acetonitrile (20 mL), and tetramethylchloroformamidinium hexafluorophosphate (589.2 mg, 2.1 mmol), N-methylimidazole (573 mg, 7 mmol) were added, and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, water was added, extracted with ethyl acetate 3 times, and the combined organic layer was washed with water and saturated aqueous sodium chloride solution each once, and dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: dichloromethane:methanol at a volume ratio of 25:1) to obtain compound 7 (white solid, 600 mg, yield 75%).

[0049] Synthesis of final product GDCN-1:

[0050] Compound 7 (271 mg, 0.60 mmol) and compound ML162-1 (299.63 mg, 0.60 mmol) were dissolved in N,N-dimethylformamide (5 mL), and copper sulfate (47.88 mg, 0.3 mmol) and sodium ascorbate (297 mg, 1.5 mmol) were dissolved in water (3 mL), and mixed with each other under nitrogen protection, and reacted at 0°C for 8 hours. The reaction solution was added with water, extracted with ethyl acetate 3 times, and the combined organic layer was washed with water and saturated aqueous sodium chloride solution each once, and dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: dichloromethane:methanol at a volume ratio of 15:1) to obtain final product GDCN-1 (white solid, 80 mg, yield 14%). 1H NMR (400 MHz, CDC13) δ 8.04 (s, 1H), 7.84 (s, 1H), 7.45 (s, 2H), 7.19 - 7.05 (m, 4H), 7.00 (d, J = 7.2 Hz, 2H), 6.91 (s, 1H), 6.75 (d, J = 3.9 Hz, 3H), 6.41 (t, J = 5.7 Hz, 2H), 6.05 (s, 1H), 5.35 - 4.74 (m, 8H), 3.75 (s, 2H), 3.65 (s, 3H), 3.44 (dtd, J = 27.0, 13.3, 12.8, 5.9 Hz, 4H), 3.26 - 3.05 (m, 2H), 2.68 (p, J = 6.7 Hz, 2H), 2.18 (s, 3H), 2.13 (s, 3H).

[0051] Similarly, GDCN-3, GDCN-5~12 refer to the synthesis scheme of GDCN-1.

[0052] Synthesis of compound 8:

[0053] Compound 4 (1 g, 3.14 mmol) was dissolved in n-butanol (20 mL), and then 2-(2-(2-azidoethoxy)ethoxy)ethan-1-amine (753.17 mg, 3.45 mmol) and N-ethyldiisopropylamine (5 mL) were added, and the reaction was carried out at 120°C for 8 hours. The reaction solution was concentrated under reduced pressure, water was added, and extraction was carried out with ethyl acetate 3 times. The combined organic layer was washed with water and saturated sodium chloride aqueous solution each 1 time in order, and dried with anhydrous Na2S04, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: dichloromethane:methanol = 25:1 by volume) to obtain compound 8 (white solid, 1.1 g, yield 70%).

[0054] Synthesis of final product GDCN-2:

[0055] Compound 8 (946.26 mg, 1.99 mmol) and compound ML162-1 (1000 mg, 1.99 mmol) were dissolved in N,N-dimethylformamide (8 mL), and copper sulfate (158.81 mg, 0.995 mmol) and sodium ascorbate (985.54 mg, 4.98 mmol) were dissolved in water (4 mL), and mixed with each other under nitrogen protection, and the reaction was carried out at 0°C overnight. Water was added to the reaction solution, and extraction was carried out with ethyl acetate 3 times. The combined organic layer was washed with water and saturated sodium chloride aqueous solution each 1 time in order, and dried with anhydrous Na2S04, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: dichloromethane:methanol = 15:1 by volume) to obtain final product GDCN-2 (white solid, 300 mg, yield 15%). 1H NMR (400 MHz, CDC13) δ 8.10 (s, 1H), 7.82 (s, 1H), 7.19 (d, J = 4.8 Hz, 1H), 7.18 - 7.08 (m, 4H), 7.08 - 7.01 (m, 2H), 7.01 - 6.71 (m, 4H), 6.14 (t, J = 5.9 Hz, 1H), 6.02 (s, 2H), 5.16 (d, J = 12.1 Hz, 4H), 4.74 (s, 2H), 4.48 (t, J = 5.0 Hz, 2H), 3.82 - 3.77 (m, 2H), 3.74 (d, J = 1.6 Hz, 2H), 3.66 (s, 3H), 3.58 (t, J = 5.1 Hz, 2H), 3.54 (d, J = 1.3 Hz, 8H), 3.45 (dt, J = 13.8, 6.7 Hz, 2H), 2.77 - 2.68 (m, 2H), 2.18 (s, 3H), 2.16 (s, 3H).

[0056] Similarly, GDCN-4, and GDCN-13 were synthesized following the synthetic scheme of GDCN-2.

[0057] Example 2: Validation of the degradation effect of the synthesized chimera on GPX4 in cells

[0058] Immunoblotting: HT1080 cells (3 x 107) were treated with 10 mM of GDCN-2, GDCN-4, GDCN-13, or DMSO for 24 h. 5The cells were inoculated into a 6-well plate (Titan) added with 2 mL of DMEM medium containing 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 culture medium was discarded, 2 mL of DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin and a series of concentrations (0.1 μM, 0.3 μM, 1 μM and 3 μM) of the compound molecules to be tested were added to each well, and incubated at 37°C for 24 h. After the culture solution was discarded, the cells were washed twice with PBS, the washing solution was discarded, 100 μL of RIPA containing 1% phenylmethylsulfonyl fluoride (PMSF) and 10% phosphatase inhibitor was added to the culture well, and the cells were lysed on ice for 10 min. The cells were 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 heated at 99°C for 10 min. The sample was 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 from about 30 kD, the <30 kD membrane part was incubated with rabbit anti-anti-GPX4 (1:1000 dilution) overnight at 4°C, and then HRP-conjugated goat anti-rabbit IgG (1:2000 dilution) was added and incubated at room temperature for 2 h; the >30 kD PVDF membrane was incubated with HRP-conjugated mouse anti-GAPDH (1:100000 dilution) overnight at 4°C, 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.

[0059] The experimental results are shown in Table 1. Figure 2 As shown in Table 1, the WB results show that the diamino purine chimeras GDCN-2, GDCN-10 and GDCN-12, etc. can significantly degrade GPX4.

[0060] Example 3: Verification of the killing effect of chimeras on GPX4 high expression tumor cell lines

[0061] Anti-cell proliferation activity determination: the CCK8 method was used to evaluate the cytotoxicity and IC 50 of all target compounds in HT1080 cells (DMEM medium) and MGC803 cells (1640 medium). The cells were inoculated in a 96-well plate at a cell density of 5×10 3 cells / well for 24 hours. Then, the cells were treated with different concentrations of compounds for 48 h. Subsequently, 10 μL of CCK8 solution was added to each well, and after incubation for 1.5 h, the absorbance at 450 nm was detected using an enzyme marker (TECAN). After the absorbance value was converted into inhibition rate, the IC 50 value was calculated using Graphpad Prism5. The results are shown in Table 1.

[0062] Table 1. Evaluation of the anti-cell proliferation activity of chimeras

[0063]

[0064] As can be seen from the table, the diamino purine chimeras GDCN-1, GDCN-2, GDCN-11 and GDCN-12 and other compounds have obvious anti-cell proliferation activity.

Claims

1. A chimera targeting degradation of GPX4 based on HSP90 protein, characterized in that, The chimera is specifically any one of the following compounds GDPU-1 to GDPU-13:

2. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the chimera targeting degradation of GPX4 based on HSP90 protein or a pharmacologically or physiologically acceptable salt thereof according to any one of claim 1.

3. Use of the chimera targeting degradation of GPX4 based on HSP90 protein or a pharmacologically or physiologically acceptable salt thereof according to claim 1 in the preparation of an antitumor drug, the tumor being gastric cancer and human fibrosarcoma.

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