A chimeric body based on molecular chaperone HSP90 mediated targeted degradation of GPX4 and preparation method and application thereof

Abstract

The application discloses a chimeric body based on targeted degradation of GPX4 mediated by a molecular chaperone HSP90 and a preparation method and application thereof, and belongs to the technical field of biological medicines. Five chimeric bodies for targeted degradation of GPX4 are prepared by using the technology. The molecular chaperone HSP90 mediated protein targeted degradation chimeric body disclosed by the application can effectively induce degradation of GPX4, thereby triggering ferroptosis. Meanwhile, the chimeric body has obvious anti-proliferation activity on cell strains with high expression of GPX4, and has important significance for tumor targeted therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a chimera based on the molecular chaperone HSP90-mediated targeted degradation of GPX4, its preparation method, and its application. Background Technology

[0002] Unlike apoptosis, necrosis, and pyroptosis, ferroptosis is a programmed cell death process characterized by the accumulation of iron-dependent lipid reactive oxygen species (ROS). Numerous studies have found that glutathione peroxidase 4 (GPX4) can serve as an indicator of ferroptosis. GPX4's catalytic active site is selenocysteine, with GSH as a cofactor. GPX4 can reduce intracellular lipid hydroperoxides to non-toxic lipid alcohols, and it can also catalyze the reduction of other organic peroxides such as hydrogen peroxide, thus protecting cells from oxidative stress and inhibiting ferroptosis. Therefore, inhibiting GPX4 activity affects its ability to scavenge lipid peroxides, ultimately leading to ferroptosis. Furthermore, inhibiting GPX4 function can trigger persistent ferroptosis and prevent tumor recurrence, making it a strategy for overcoming drug resistance.

[0003] Currently, small molecule inhibitors targeting GPX4 remain challenging, and no GPX4 inhibitors have been reported to have entered clinical trials. The main reasons are: 1) GPX4 lacks a drug-like binding pocket on its molecular surface; 2) Currently reported inhibitors are all covalent inhibitors, exerting their effect by binding to selenocysteine, the active site of GPX4, but they suffer from low selectivity.

[0004] Inducing cancer protein degradation using protein degradation techniques is a widely studied topic recently. For example, the inventors have previously filed a patent for the targeted degradation of GPX4 using protein-targeting chimeras (PROTAC). Unlike the previous patent, this patent discloses for the first time the preparation and application of GPX4-targeted degradation chimeras mediated by molecular chaperones. Molecular chaperone-mediated targeted degradation technology is a novel protein degradation technique with several advantages, including overcoming drug resistance. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a chimera based on molecular chaperone-mediated targeted degradation of GPX4, its preparation method, and its application. The chimera of this invention can effectively degrade GPX4 protein, thereby inducing ferroptosis in tumor cells.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] A chimera based on the molecular chaperone HSP90-mediated targeted degradation of GPX4, wherein the chimera of the molecular chaperone HSP90-mediated targeted degradation of GPX4 is a compound represented by general formula (I) or a pharmacologically or physiologically acceptable salt thereof.

[0008]

[0009] In general formula (I), Linker is a linking group, representing -alkylene or -alkoxy or -piperazinyl or -1,2,3-triazolyl, wherein the -alkylene or -alkoxy or -piperazinyl or -1,2,3-triazolyl is selected from any one of the following groups or any combination thereof, wherein m and n represent natural numbers from 1 to 20;

[0010] -(CH2) n -C(O)NH(CH2CH2O) m -or-(CH2CH2O) n -C(O)NH(CH2CH2O) m -or

[0011]

[0012] Furthermore, the molecular chaperone-mediated targeted degradation chimera of GPX4 provided by the present invention is a compound as shown below or its stereoisomers, geometric isomers, tautomers, nitrogen oxides, hydrates, solvates, metabolites, pharmaceutically or physiologically acceptable salts or prodrugs.

[0013]

[0014] The pharmacologically or physiologically acceptable salt mentioned in this invention refers to the salt generated by the chimera of molecular chaperone-mediated targeted degradation of GPX4 and a pharmacologically or physiologically acceptable acid or base.

[0015] The present invention also proposes a pharmaceutical composition comprising the aforementioned chaperone-mediated targeted degradation of GPX4 chimera or its stereoisomers, geometric isomers, tautomers, nitrides, hydrates, solvates, metabolites, pharmaceutically acceptable salts, or prodrugs.

[0016] Furthermore, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, medium, or combination thereof.

[0017] Furthermore, the pharmaceutical composition is an injection, an oral preparation, or a mucosal delivery preparation.

[0018] Furthermore, the pharmaceutical composition further includes other drugs that have therapeutic or preventative effects on tumors.

[0019] Furthermore, the present invention also provides the use of the aforementioned molecular chaperone-mediated chimera targeting the degradation of GPX4, or a pharmaceutical composition comprising the chimera. Specifically:

[0020] The use of the molecular chaperone-mediated targeted degradation chimera of GPX4 or a pharmaceutical composition containing the chimera in the preparation of GPX4-degrading drugs.

[0021] The use of the aforementioned molecular chaperone-mediated targeted degradation of GPX4 chimeras or pharmaceutical compositions containing such chimeras in the preparation of medicaments for treating GPX4-related diseases. The GPX4-related diseases include tumors and neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.

[0022] The use of the aforementioned molecular chaperone-mediated targeted degradation of GPX4 chimeras or pharmaceutical compositions containing such chimeras in antitumor drugs. The tumor is defined as gastric cancer, breast cancer, lung cancer, ovarian cancer, colonic adenocarcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, lung adenocarcinoma, prostate cancer, rectal adenocarcinoma, thyroid cancer, and endometrial cancer. Further, the tumor is a tumor with high GPX4 expression.

[0023] This invention also proposes a synthetic route for the targeted degradation of GPX4 chimeras mediated by molecular chaperones, as shown in the general formula, which specifically includes the following steps:

[0024] The compounds shown in the general formula link the ligands of the HSP90 protein and the ligands of the GPX4 protein through reaction types such as amide condensation, deprotection, and nucleophilic substitution.

[0025] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0026] Unlike the PROTAC technology, which directly pulls on E3 ubiquitin ligases, this invention is based on the molecular chaperone HSP90-mediated ubiquitination of target proteins, thereby inducing the degradation of target proteins by the proteasome. The inventors demonstrated through Western blot experiments that the molecular chaperone-mediated protein-targeting degradation chimera of this invention can effectively degrade GPX4 and effectively kill GPX4-overexpressing cell lines. Attached Figure Description

[0027] Figure 1 Synthetic route diagram of chimeras GDPU-1 to 5;

[0028] Figure 2 Western blotting was used to detect the degradation activity of the chimera on GPX4. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. The technical and scientific terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. The basic raw materials and reagents are obtained commercially and have a purity of 97% or higher. The room temperature described in this invention is 25-30°C. The present invention provides a general and specific description of the materials and experimental methods used in the experiments. Although many materials and methods of operation used to achieve the objectives of this invention are well known in the art, they are still described herein as comprehensively as possible.

[0030] Example 1: Synthesis and structural confirmation of chimeras targeting GPX4 degradation

[0031] Synthetic routes for the final product GDPU 1-5, such as Figure 1 As shown:

[0032] Synthesis of Compound 2: Pyrimidine-4,5,6-triamine (1,42.75 mmol, 5.35 g), carbon disulfide (256.52 mmol, 19.59 g), and NaHCO3 (128.26 mmol, 10.77 g) were mixed using ethanol and water as solvents (H2O:EtOH = 2:1). The reaction system was heated to reflux at 90 °C for 3 days. Excess carbon disulfide was evaporated, and the pH was adjusted with acetic acid until a large amount of solid precipitated. The solid was filtered and dried to give approximately 6 g of a light yellow solid, 6-amino-9H-purine-8-thiol, with a yield of 83.94%. The product did not require further purification and could be used in the next reaction.

[0033] Synthesis of Compound 3: 6-amino-9H-purine-8-thiol (1 equivalent, 1 g), 5-iodobenzo[d][1,3]diazole (3 equivalent, 4.45 g), sodium tert-butoxide (2 equivalent, 1.15 g), cuprous iodide (0.1 equivalent, 110 mg), and neocuproine (0.1 equivalent, 140 mg) were dissolved in DMF and reacted at 110 °C for 12 hours. DMF was removed under reduced pressure, and the product was purified by column chromatography with dichloromethane:methanol = 15:1 as the developing solvent. The target product, 8-(benzo[d][1,3]dioxane-5-thio)-9H-purine-6-amine, was obtained as a yellowish-brown solid with a yield of 75%.

[0034] Synthesis of Compound 4: At 0 °C, 1.3 g of the reactant 8-(benzo[d][1,3]dioxacyclopenten-5-thio)-9H-purine-6-amine was dissolved in acetonitrile, and trifluoroacetic acid (5 equivalents, 2.6 g) was added. 6.1 g of N-iodosuccinimide (6 equivalents) was dissolved in acetonitrile and slowly added dropwise to the reaction system. The reaction was allowed to proceed overnight. After the reaction was complete, acetonitrile was removed under reduced pressure, and the product was purified by column chromatography using dichloromethane:methanol = 15:1. The target product 8-((6-iodobenzo[d][1,3]dioxacyclopenten-5-yl)thio)-9H-purine-6-amine was obtained as a brownish-brown solid with a yield of 74.88%.

[0035] Synthesis of Compound 7: One equivalent of 8-((6-iodobenzo[d][1,3]dioxacyclopenten-5-yl)thio)-9H-purine-6-amine and 2.5 equivalents of different Boc methylbenzenesulfonate were dissolved in DMF solution, and two equivalents of cesium carbonate were added. The reaction was carried out at 80°C for 12 hours. After the reaction was completed as detected by TLC, water was added to the reaction system, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography with dichloromethane:methanol = 15:1 as the developing solvent to obtain the corresponding product 6. The above product was dissolved in dichloromethane, and an appropriate amount of trifluoroacetic acid was added. After reacting at room temperature for 2 hours, the solvent was removed under reduced pressure. The obtained product could be used for the next reaction without further purification.

[0036] Synthesis of Compound 10: Product 7 was dissolved in DMF in a 1:1 ratio with carboxylic acid amino Boc. 1.5 equivalents of HATU and 3 equivalents of DIPEA were added, and the mixture was reacted overnight at room temperature. After the reaction was complete as monitored by TLC, the mixture was extracted three times with water and ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain the target product 9, which was dissolved in dichloromethane. An appropriate amount of trifluoroacetic acid was added, and the reaction was continued at room temperature for two hours. The mixture was then concentrated under reduced pressure. The product could be used in the next reaction without further purification.

[0037] Synthesis of Compound 11: The above product was dissolved in DMF in a 1:1 ratio with azidoacetic acid, and 1.5 equivalents of HATU and 3 equivalents of DIPEA were added. The mixture was reacted overnight at room temperature. After the reaction was completed by TLC monitoring, the mixture was extracted three times with water and ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by column chromatography using dichloromethane:methanol = 15:1 as the developing solvent. The combined and concentrated products yielded the target product, the azide-modified compound.

[0038] Synthesis of Compound 14: Propylene-substituted aniline (13g, 4.96g, 27.44mmol) and 2-thiophenecarboxaldehyde (3.08g, 27.44mmol) were dissolved in methanol (25mL), activated at 25°C for 1h, and then (2-isocyanoethyl)benzene (3g, 22.87mmol) and chloroacetic acid (2.16g, 22.87mmol) were added. The mixture was stirred overnight at room temperature. The reaction solution was concentrated to dryness under reduced pressure, and the concentrate was purified by silica gel column chromatography (ethyl acetate:petroleum ether, v / v ratio 1:1). The concentrate was dried at 25°C to give intermediate 4 (white solid, 2g, yield 20%).

[0039] Synthesis of Compound 15: Under ice bath conditions, the azide-modified compound and the alkyne compound were dissolved in DMF in a 1:1.1 ratio. Sodium ascorbate and copper sulfate were dissolved in water at 2.5 and 0.5 equivalents, respectively. The two systems were mixed and reacted overnight under nitrogen protection. After the reaction was complete, water was added, resulting in the precipitation of a large amount of solid. The solid was filtered and purified by column chromatography using dichloromethane:methanol in a 15:1 ratio as the developing solvent. After concentration, the final product was obtained.

[0040] NMR characterization of the final product:

[0041] N-(4-((1-(2-(2-(6-amino-8-((6-iodobenzo[d][1,3]dioxolane-5-yl)thio)-9H-purine-9-yl)ethyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-chlorophenyl)-2-chloro-N-(2-oxo-2(phenylethylamino)-1-(thiophenyl-2)ethyl)acetamide (GDPU-1): pale yellow solid; 1H NMR (400MHz, DMSO) δ8.52(q,J=6.0Hz,1H),8.38(t,J=5.7Hz,1H),8.23(d,J=39.1Hz,1H),8.15(s,1H),7.48(s,1H),7.4 0(h,J=8.6,5.4Hz,2H),7.25(dd,J=7.8,6.4Hz,3H),7.20–7.15(m,3H),6.94(s,1H),6.85(dd,J=5.1,3.5Hz,1H),6.82( dd,J=3.6,1.3Hz,1H),6.24(s,1H),6.07(s,1H),5.76(s,1H),5.19(s,2H),5.05(d,J=15.0Hz,2H),4.32(dt,J=29.6,5. 5Hz,2H),4.10–3.92(m,2H),3.56(dq,J=53.5,5.9,5.1Hz,2H),3.46–3.37(m,1H),3.31–3.21(m,1H),2.81–2.63(m,2H).

[0042] N-(2-(6-amino-8-((6-iodobenzo[d][1,3]dioxolane-5-yl)thio)-9H-purine-9-yl)ethyl)-3-(2-(4-((2-chloro-4-(2-oxo-2-(phenethylamino)-1-(thiophene-2-yl)ethyl)acetamide)phenoxy)-1H-1,2,3-triazol-1-yl)acetamide)propionamide (GDPU-2): light brown solid; 1 H NMR (400MHz, DMSO) δ8.37(t,J=5.7Hz,2H),8.18(d,J=5.0Hz,1H),8.08(q,J=8.3,7.2Hz,1H),7.45(s,1H),7.40(d,J= 5.0Hz,2H),7.24(t,J=7.2Hz,3H),7.20–7.12(m,3H),6.90(s,1H),6.85(dd,J=5.1,3.5Hz,1H),6.81(d,J=3.5Hz,1H), 6.23(s,1H),6.05(s,1H),5.18(s,2H),5.10(d,J=13.9Hz,2H),4.39–4.17(m,2H),4.09–3.91(m,2H),3.45(d,J=6.3H z, 2H), 3.36 (s, 1H), 3.26 (q, J = 6.8Hz, 3H), 3.17 (d, J = 5.0Hz, 1H), 2.70 (dt, J = 12.3, 5.8Hz, 2H), 2.20 (t, J = 7.1Hz, 2H).

[0043] N-(2-(6-amino-8-((6-iodobenzo[d][1,3]dioxolane-5-yl)thio)-9H-purine-9-yl)ethyl)-6-(2-(4-((2-chloro-4-(2-oxo-2-(phenethylamino)-1-(thiophene-2-yl)ethyl)acetamide)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)acetamide)hexaamide (GDPU-3): pale yellow solid; 1 H NMR (400MHz, DMSO) δ8.36(dt,J=19.1,5.5Hz,2H),8.18(d,J=15.6Hz,2H),7.94(q,J=7.0,6.4Hz,1H),7.41 (dd,J=21.9,16.3Hz,3H),7.25(t,J=7.3Hz,3H),7.21–7.05(m,4H),6.94–6.77(m,3H),6.25(s,1H),6.05( d,J=6.6Hz,2H),5.19(s,2H),5.12(s,2H),4.28(dt,J=21.9,5.5Hz,2H),4.13–3.90(m,3H),3.60–3.33(m, 6H), 3.07 (q, J=6.7Hz, 2H), 2.73 (dq, J=13.3, 7.2Hz, 2H), 2.05–1.92 (m, 3H), 1.40 (dt, J=15.5, 7.4Hz, 4H).

[0044] N-(4-((1-(2-((5-(6-amino-8-((6-iodobenzo[d][1,3]dioxolane-5-yl)thio)-9H-purine-9-yl)pentyl)amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-chlorophenyl)-2-chloro-N-(2-oxo-2(phenylethylamino)-1-(thiophenol-2)ethyl)acetamide (GDPU-4): Yellowish-white solid; 1H NMR (400MHz, DMSO) δ8.37(dt,J=15.3,5.6Hz,2H),8.18(d,J=10.5Hz,2H),7.50(s,1H),7.41(d,J=4.7Hz,3H ),7.25(t,J=7.3Hz,3H),7.17(t,J=8.5Hz,3H),6.89–6.80(m,3H),6.24(s,1H),6.06(s,2H),5.19(s,2H),5 .11(s,2H),4.23–3.91(m,4H),3.45–3.38(m,1H),3.27(dq,J=13.2,6.6Hz,1H),3.07(dq,J=12.8,6.2Hz,2H ),2.71(dt,J=12.4,5.9Hz,2H),1.68(p,J=7.4Hz,2H),1.44(t,J=7.4Hz,2H),1.26(dt,J=11.4,5.8Hz,2H).

[0045] N-(4-(1-(2-(2-(2-(2-(6-amino-8-((6-iodobenzo[d][1,3]dioxo-5-yl)thio)-9H-purine-9-yl)ethoxy)ethoxy)ethyl)ethyl)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-3-chlorophenyl)-2-chloro-N-(2-oxo-2-(phenylethylamino)-1-(thiophene-2-yl)ethyl)acetamide (GDPU-5): yellowish-white solid; 1 H NMR (400MHz, DMSO) δ8.41(dt,J=17.5,5.6Hz,2H),8.19(s,1H),7.47(s,1H),7.41(d,J=5.1Hz, 2H),7.21(dt,J=30.8,7.5Hz,7H),6.88–6.83(m,2H),6.82(d,J=3.6Hz,1H),6.24(s,1H),6.05( d,J=5.7Hz,2H),5.19(s,2H),5.14(d,J=5.6Hz,2H),4.38(t,J=5.5Hz,2H),4.08–3.95(m,2H), 3.86–3.72(m,2H),3.55–3.41(m,6H),3.24(tq,J=13.7,8.3,7.6Hz,4H),2.71(q,J=6.6Hz,2H).

[0046] Example 2: Verification of the degradation effect of the synthesized chimera on intracellular GPX4

[0047] Immunoblot: HT1080 cells (3×10⁻⁶) were used to bleach the cells. 5Cells were seeded into 6-well Titan plates containing 2 mL of 1640 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 a series of concentrations (0.1 μM, 0.3 μM, 1 μM, and 3 μM) of the target compound, supplemented with 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% 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.

[0048] Experimental results are as follows Figure 2 Therefore, the WB results showed that the chimeric GDPU-4 could significantly degrade GPX4.

[0049] Example 3: Verification of the killing effect of chimera on GPX4-overexpressing tumor cell lines

[0050] Anti-cell proliferation activity assay: The cytotoxicity and IC50 of all target compounds in HT1080 cells (DMEM medium) and MGC803 cells (1640 medium) were evaluated using the CCK8 assay. 50 Cells were loaded at 5 × 10⁻⁶ 3 Cells were seeded at a density of 10 cells / well in 96-well plates for 24 hours. Then, the cells were treated with different concentrations of the compound for 48 hours. Subsequently, 10 μL of CCK8 solution was added to each well, and after 1.5 hours of incubation, the absorbance at 450 nm was measured using a TECAN microplate reader. The absorbance values ​​were converted to inhibition rates, and the IC50 was calculated using Graphpad Prism 5. 50 Values. The results are shown in Table 1:

[0051] Table 1. Evaluation of the anti-cell proliferation activity of chimeras

[0052]

[0053] As can be seen from the table, chimeras GDPU-1, GDPU-4, and GDPU-5 have significant anti-cell proliferation activity.

Claims

1. A chimer based on the targeted degradation of GPX4 mediated by the chaperone HSP90, characterized in that, The chimeric body of the molecular chaperone HSP90-mediated targeted degradation of GPX4 is any one of the following compounds GDPU-1, GDPU-4, GDPU-5: 、 、 。 2. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the chimeric body of the molecular chaperone-mediated targeted degradation of GPX4 or a pharmacologically or physiologically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle or combination thereof.

3. Use of the chimeric body of the molecular chaperone-mediated targeted degradation of GPX4 or a pharmacologically or physiologically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a GPX4 degrading agent or a GPX4 inhibiting drug.

4. Use of the chimeric body of the molecular chaperone-mediated targeted degradation of GPX4 or a pharmacologically or physiologically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 2 in the preparation of a drug for treating a GPX4-related disease.

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