CN116813620A - Chimeric body for degrading GPX4 based on HSP90 protein targeting, preparation method and application - Google Patents

Abstract

The application discloses a chimeric body based on HSP90 protein targeted degradation GPX4, a preparation method and application thereof, and belongs to the technical field of biological medicine. The application prepares 13 chimeras for targeted degradation of GPX4 by using the technology. The molecular chaperone HSP90 mediated protein targeted degradation chimeric body can effectively induce the degradation of GPX4. Meanwhile, the chimeras have obvious antiproliferative activity on GPX4 high-expression cell lines and have important significance on tumor targeted therapy.

Description

Chimeric body for degrading GPX4 based on HSP90 protein targeting, preparation method and application

Technical Field

The application belongs to the technical field of biological medicine, and particularly relates to a diaminopurine chimeric body with GPX4 degradation activity based on molecular chaperone HSP90 mediation, a preparation method thereof and application thereof in anti-tumor aspect.

Background

Unlike apoptosis, necrosis, and pyrodeath, iron death is characterized by a programmed pattern of cell death characterized by the accumulation of iron-dependent lipid reactive oxygen species (Reactive Oxygen Species, ROS) free radicals. Numerous studies have found that glutathione peroxidase 4 (Glutathione Peroxidase, GPX 4) can be used as one of the indicators for determining iron death in cells. GPX4 has selenocysteine as the catalytic active center, GSH as cofactor, GPX4 can reduce lipid hydroperoxide in cells into nontoxic fatty alcohol compounds, and can catalyze the reduction of other organic peroxides such as hydrogen peroxide, thus having the effects of protecting cells from oxidative stress and inhibiting iron death. Thus, inhibition of GPX4 activity affects GPX 4's ability to scavenge lipid peroxides, ultimately leading to the occurrence of cellular iron death. Furthermore, inhibition of GPX4 function triggers cell-sustained iron death and prevents tumor recurrence, and is therefore one of the strategies to address drug resistance.

There is still a challenge to small molecule inhibitors targeted to GPX4 at present, and there is no report of GPX4 inhibitors entering the clinical research stage. The main reason is that: 1) The molecular surface of GPX4 lacks a drug-like binding pocket; 2) The currently reported inhibitors are covalent inhibitors, and can be combined with selenocysteine of the active site of GPX4 to play a role, but have the problem of low selectivity and the like.

The induction of oncoprotein degradation using protein degradation techniques is one of the more widely studied topics recently. The molecular chaperone mediated targeting degradation technology is a novel protein degradation technology and has a plurality of advantages of overcoming drug resistance and the like. The application discloses preparation and application of diaminopurine compounds as GPX4 degradation agents by utilizing molecular chaperone mediated targeted protein degradation technology.

Disclosure of Invention

Based on the problems in the background art, the application aims to provide a diaminopurine chimeric body for degrading GPX4 based on HSP90 protein targeting, a preparation method and application thereof, and the chimeric body can effectively degrade GPX4 protein so as to induce iron death of tumor cells.

In order to achieve the above object, the present application adopts the following technical scheme:

the application provides a diaminopurine chimera of a molecular chaperone mediated target degradation GPX4 protein shown in a formula (I) or a pharmacologically or physiologically acceptable salt thereof:

wherein:

linker is a linking group representing an-alkylene or-alkoxy or-piperazinyl or-1, 2, 3-triazolyl group selected from any one of or any combination of the following groups, wherein p, m and n represent natural numbers from 1 to 20:

-(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

Further, the present application provides a chimeric compound as shown below or a stereoisomer, geometric isomer, tautomer, nitroxide, hydrate, solvate, metabolite, pharmaceutically or physiologically acceptable salt or prodrug thereof;

the pharmacologically or physiologically acceptable salt refers to a salt formed by the diaminopurine chimera of the HSP90 protein targeted degradation GPX4 and pharmacologically or physiologically acceptable acid or alkali.

The application also provides a pharmaceutical composition, which comprises the chimeric body of the molecular chaperone-mediated targeted degradation GPX4 or stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs thereof.

The pharmaceutical compositions also include a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, or combination thereof.

The pharmaceutical composition is injection, oral administration and mucosa administration.

The pharmaceutical composition further comprises other medicaments with the effect of treating or preventing tumors.

The application also provides an application of the chimeric body based on HSP90 protein targeted degradation GPX4 or a pharmaceutical composition containing the chimeric body. The method comprises the following steps:

the HSP90 protein-targeted GPX4 degradation-based chimeric or the application of a pharmaceutical composition containing the chimeric in preparation of a GPX4 degradation drug.

The HSP90 protein-targeted GPX4 degradation-based chimera or the application of a pharmaceutical composition containing the chimera in preparing a medicament for treating GPX 4-related diseases. The GPX4 related diseases are tumor, neurodegenerative diseases such as Alzheimer's disease, parkinson's disease and Huntington's disease.

The chimeric body based on HSP90 protein targeted degradation GPX4 or the application of the pharmaceutical composition containing the chimeric body in antitumor drugs. The tumor is 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 tumor is a tumor with high expression of GPX4.

The application also provides a synthesis route of the chimeric body based on HSP90 protein targeted degradation GPX4 shown in the general formula, which specifically comprises the following steps:

the preparation of GPX4 ligand ML162-1 shown in the general formula and the preparation of ligand BIIB021 of HSP90 protein are connected through amide condensation, deprotection, nucleophilic substitution, click chemistry and other reaction types.

Compared with the prior art, the application has the beneficial effects that:

unlike the protoc technique, which directly pulls a certain E3 ubiquitin ligase, the present application is based on chaperone HSP90 mediated ubiquitination of target proteins by various types of E3 ubiquitin ligases, thereby inducing degradation of the target proteins by proteasomes. The inventor confirms through Western blot experiments that the diaminopurine chimera related to the application can effectively degrade GPX4 and can effectively kill GPX4 high expression cell lines.

Drawings

FIG. 1 is a synthetic scheme for chimeras GDCN-1-13;

FIG. 2Western Blot detection of the degradation activity of chimeras on GPX4.

Detailed Description

The present application will be described in detail below with reference to the drawings and specific examples, wherein technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The basic raw material reagent is obtained from commercial paths, and the purity is 97% or more. The room temperature of the application is 25-30 ℃. The materials used in the test and the experimental methods are described generally and specifically. Although many materials and methods of operation are known in the art for accomplishing the objectives of the present application, the present application is nevertheless described herein as much as possible.

Example 1: synthesis and structure confirmation of targeted degradation GPX4 chimera

The synthetic routes for the end products GDCN 1-23 are shown in FIG. 1:

synthesis of Compound 1:

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 2 times. The combined organic layers were treated with anhydrous Na ₂ SO ₄ Drying, filtering, and concentrating under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: ethyl acetate: petroleum ether in a volume ratio of 1:5) to give compound 1 (yellow liquid, 714mg, yield 84%).

Synthesis of Compound 2:

compound 1 (1000 mg,4.1 mmol) was dissolved in N, N-dimethylformamide (20 mL), potassium carbonate (851 mg,6.2 mmol) was added, and 3-bromopropane-1-yne (634 mg,5.3 mmol) was stirred overnight at room temperature. The reaction mixture was extracted 3 times with ethyl acetate, the combined organic layers were washed 1 time with water and saturated aqueous sodium chloride, respectively, and with anhydrous Na ₂ SO ₄ Drying, filtering, and concentrating under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: ethyl acetate: petroleum ether in a volume ratio of 1:5) to give compound 2 (yellow liquid, 850mg, yield 73%).

Synthesis of Compound 3:

compound 2 (1000 mg,3.55 mmol) was dissolved in dichloromethane (40 mL), trifluoroacetic acid (10 mL) was added and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and NaHCO was added ₃ Aqueous solution, and ethyl acetate extraction 3 times. Anhydrous Na for separated organic layer ₂ SO ₄ Drying, filtration and concentration under reduced pressure gave compound 3 (white solid, 480mg, 74% yield). Synthesis of Compound ML 162-1:

compound 3 (1.82 g,10 mmol) and 2-thiophenecarboxaldehyde (1.12 g,10 mmol) were dissolved in (25 mL) of methanol, activated at 25℃for 1 hour, and then (2-isocyanatoethyl) benzene (1.09 g,8.33 mmol), chloroacetic acid (787.45 mg,8.33 mmol) were added thereto 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 in a volume ratio of 1:1) to give compound ML162-1 (white solid, 850mg, yield 20%).

Synthesis of Compound 4:

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 cooling the reaction mixture, 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 give compound 4 (white solid, 10.5g, 70% yield).

Synthesis of Compound 5:

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, extraction was performed 3 times with ethyl acetate, the combined organic layers were washed 1 time with water and saturated aqueous sodium chloride solution, respectively, and with anhydrous Na ₂ SO ₄ Drying, filtration and concentration under reduced pressure gave compound 5 (white solid, 1.1g, 75% yield).

Synthesis of Compound 6:

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 give compound 6 (white solid, 650mg, yield 75%).

Synthesis of Compound 7:

compound 6 (650 mg,1.75 mmol) and azidoacetic acid (212.79 mg,2.11 mmol) were dissolved in acetonitrile (20 mL), and tetramethyl chlorourea hexafluorophosphate (589.2 mg,2.1 mmol), N-methylimidazole (573 mg,7 mmol) was added and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, water was added, extraction was performed 3 times with ethyl acetate, the combined organic layers were washed 1 time with water and saturated aqueous sodium chloride solution, respectively, and with anhydrous Na ₂ SO ₄ Drying, filtering, and concentrating under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: 25:1 by volume of dichloromethane: methanol) to give compound 7 (white solid, 600mg, 75% yield).

Synthesis of the final product GDCN-1:

compound 7 (271mg, 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 (293 mg,1.5 mmol) were dissolved in water (3 mL), mixed with each other and reacted at 0℃for 8 hours under nitrogen protection. The reaction solution was added with water, extracted 3 times with ethyl acetate, and the combined organic layers were washed 1 time with water and saturated aqueous sodium chloride solution, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was separated by silica gel column chromatography (15:1 by volume of mobile phase: methylene chloride: methanol) to give the final product GDCN-1 (white solid, 80mg, 14% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.04(s,1H),7.84(s,1H),7.45(s,2H),7.19–7.05(m,4H),7.00(d,J＝7.2Hz,2H),6.91(s,1H),6.75(d,J＝3.9Hz,3H),6.41(t,J＝5.7Hz,2H),6.05(s,1H),5.35–4.74(m,8H),3.75(s,2H),3.65(s,3H),3.44(dtd,J＝27.0,13.3,12.8,5.9Hz,4H),3.26–3.05(m,2H),2.68(p,J＝6.7Hz,2H),2.18(s,3H),2.13(s,3H)。

Similarly, GDCN-3, GDCN-5-12 refers to the synthetic scheme for GDCN-1.

Synthesis of Compound 8:

compound 4 (1 g,3.14 mmol) was dissolved in N-butanol (20 mL) and 2- (2- (2-azidoethoxy) ethoxy) ethan-1-amine (753.17 mg,3.45 mmol) and N-ethyldiisopropylamine (5 mL) were added and reacted at 120℃for 8 hours. The reaction solution was concentrated under reduced pressure, water was added, extraction was performed 3 times with ethyl acetate, the combined organic layers were washed 1 time with water and saturated aqueous sodium chloride solution, respectively, and with anhydrous Na ₂ SO ₄ Drying, filtering, and concentrating under reduced pressure. The residue was separated by silica gel column chromatography (mobile phase: 25:1 by volume of dichloromethane: methanol) to give compound 8 (white solid, 1.1g, yield 70%).

Synthesis of the final product GDCN-2:

compound 8 (946.26 mg,1.99 mmol) was reacted with compound ML162-1 (100)0mg,1.99 mmol) in N, N-dimethylformamide (8 mL), copper sulfate (158.81 mg,0.995 mmol) and sodium ascorbate (985.54 mg,4.98 mmol) in water (4 mL), and mixed with each other under nitrogen protection, and reacted overnight at 0 ℃. The reaction mixture was extracted 3 times with ethyl acetate, the combined organic layers were washed 1 time with water and saturated aqueous sodium chloride, respectively, and with anhydrous Na ₂ SO ₄ Drying, filtering, and concentrating under reduced pressure. The residue was chromatographed on a silica gel column (15:1 by volume of mobile phase dichloromethane: methanol) to give the final product GDCN-2 (white solid, 300mg, 15% yield). ¹ H NMR(400MHz,CDCl ₃ )δ8.10(s,1H),7.82(s,1H),7.19(d,J＝4.8Hz,1H),7.18–7.08(m,4H),7.08–7.01(m,2H),7.01–6.71(m,4H),6.14(t,J＝5.9Hz,1H),6.02(s,2H),5.16(d,J＝12.1Hz,4H),4.74(s,2H),4.48(t,J＝5.0Hz,2H),3.82–3.77(m,2H),3.74(d,J＝1.6Hz,2H),3.66(s,3H),3.58(t,J＝5.1Hz,2H),3.54(d,J＝1.3Hz,8H),3.45(dt,J＝13.8,6.7Hz,2H),2.77–2.68(m,2H),2.18(s,3H),2.16(s,3H)。

Similarly, GDCN-4, and GDCN-13 refer to the synthetic schemes for GDCN-2.

Example 2: verification of the degradation Effect of the synthesized chimera on intracellular GPX4

Immunoblotting: HT1080 cells (3X 10) ⁵ Individual cells) were inoculated into 2mL of 6-well culture plate (Titan) supplemented with 1640 medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin, and cultured at 37℃for 24 hours. After cells were grown to 70% confluence, the original medium was discarded, 2mL of DMEM medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin streptomycin at a range of concentrations (0.1 μm, 0.3 μm, 1 μm and 3 μm) containing the test compound molecules was changed per well, after incubation at 37 ℃ for 24 hours, the medium was discarded, the cells were washed twice with PBS, the washes were discarded, 100 μl of RIPA containing 1% phenylmethylsulfonyl fluoride (PMSF) and 10% phosphatase inhibitor was added to the culture wells, and after lysis on ice for 10min, the cells were scraped off with a spatula and placed in 1.5mL EP tubes. 20. Mu.L of 5 XSDS loading buffer was added to the EP tube and heated at 99℃for 10min. Samples were separated by 15% SDS-PAGE and transferred onto PVDF membrane. After blocking the membrane with 5% skim milk (in TBST buffer) for 1.5h at room temperature, the membrane was cut from around 30kD，<The 30kD membrane fraction was incubated with rabbit anti-GPX4 (1:1000 dilution) overnight at 4deg.C, then HRP conjugated goat anti-rabbit IgG (1:2000 dilution) was added and incubated for 2 hours at room temperature;>the 30kD PVDF membrane was incubated with HRP conjugated mouse anti-GAPDH (1:100000 dilution) overnight at 4deg.C, then HRP conjugated mouse anti-IgG (1:1000 dilution) was added and incubated for 2 hours at room temperature. And the print was recorded using Invitrogen iBright 1500.

The experimental results are shown in fig. 2, WB results show: the diaminopurine chimeras GDCN-2, GDCN-10, GDCN-12 and the like can obviously degrade GPX4.

Example 3: verification of the killing effect of the chimera on GPX4 high expression tumor cell lines

Determination of anti-cell proliferation Activity: cytotoxicity and IC of all target compounds in HT1080 cells (DMEM medium) and MGC803 cells (1640 medium) were evaluated using CCK8 method ₅₀ . The cells were grown at 5X 10 ³ Cell density of individual cells/well was seeded in 96-well plates for 24 hours. Cells were then treated with different concentrations of the compound for 48h. Subsequently, 10. Mu.L of CCK8 solution was added to each well, and after 1.5 hours of incubation, absorbance at 450nm was measured using a microplate reader (TECAN). After conversion of absorbance values to inhibition, IC was calculated using Graphpad Prism5 ₅₀ Values. The results are shown in Table 1:

TABLE 1 evaluation of chimeric anti-cell proliferation Activity

From the table it can be seen that: the diaminopurine chimeras GDCN-1, GDCN-2, GDCN-11, GDCN-12 and other compounds have obvious anti-cell proliferation activity.

Claims (9)

1. A chimeric body for degrading GPX4 based on HSP90 protein targeting is characterized in that the chimeric body is a diaminopurine chimeric body shown in a general formula (I) or a pharmacology or physiology acceptable salt thereof,

in the general formula (I), linker is a linking group, and represents-alkylene or-alkoxy or-piperazinyl or-1, 2, 3-triazole, wherein the-alkylene or-alkoxy or-piperazinyl or-1, 2, 3-triazole is selected from any one or any combination of the following groups, and m and n represent natural numbers from 1 to 20:

-(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

2. The chimeric body based on HSP90 protein targeted degradation of GPX4 according to claim 1, wherein the chimeric body is any one of the following compounds GDPU-1 to GDPU-13:

3. the method for preparing the chimera based on the HSP90 protein targeted degradation GPX4 as claimed in claim 2, which is characterized in that the synthesis route of the chimera is as follows:

(1)

4. a pharmaceutical composition comprising a HSP90 protein-targeted degradation GPX 4-based chimera according to any one of claims 1-2, or a pharmacologically or physiologically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or combination thereof.

5. Use of a HSP90 protein-targeted degradation GPX 4-based chimera according to any one of claims 1-2 or a pharmacologically or physiologically acceptable salt thereof or a pharmaceutical composition according to claim 4 for the preparation of a GPX4 degradation agent or a GPX4 inhibition drug.

6. Use of a HSP90 protein-targeted degradation GPX 4-based chimera according to any one of claims 1-2 or a pharmacologically or physiologically acceptable salt thereof or a pharmaceutical composition according to claim 4 for the preparation of a medicament for the treatment of a GPX 4-related disorder.

7. The use according to claim 6, wherein the GPX 4-related disease is a tumor, a neurodegenerative disease.

8. Use of a HSP90 protein-targeted degradation GPX 4-based chimera according to any one of claims 1-2 or a pharmacologically or physiologically acceptable salt thereof or a pharmaceutical composition according to claim 4 in an anti-tumor drug, said tumor being gastric cancer, breast cancer, lung cancer, ovarian cancer, colon adenocarcinoma, renal chromophobe cells, renal clear cell carcinoma, lung adenocarcinoma, prostate cancer, rectal adenocarcinoma, thyroid cancer and endometrial cancer.

9. The use according to claim 8, wherein the tumour is a tumour with high expression of GPX4.