Keap1 degradation agent as well as preparation method and application thereof
By designing Keap1 degrading agents and using PROTAC technology to induce KEAP1 protein degradation and activate the NRF2 signaling pathway, the efficacy and off-target effects of existing KEAP1-targeting drugs have been addressed, enabling effective treatment of a variety of chronic diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-11
- Publication Date
- 2026-03-10
AI Technical Summary
Existing KEAP1-targeting drugs, such as NRF2 agonists, suffer from insufficient efficacy, off-target effects, and drug resistance, making it difficult to effectively regulate cellular oxidative stress.
A class of Keap1 degraders was developed. Highly specific molecules were designed using PROTAC technology to induce KEAP1 protein degradation, activate the NRF2 signaling pathway, and regulate cellular oxidative homeostasis.
It achieves efficient degradation of KEAP1 and upregulation of NRF2, with a clear mechanism of action and high selectivity, and is used to treat a variety of chronic diseases caused by oxidative stress, such as neurodegenerative diseases, metabolic diseases and cardiovascular diseases.
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Figure CN121627675A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of medicine, and particularly relates to a Keap1 degrader, a preparation method and application thereof, and use of the compound for preparing a drug for treating or preventing a Keap1-mediated disease. BACKGROUND
[0002] The Kelch-like ECH-associated protein 1 (KEAP1)-NRF2 signaling pathway can sense active oxygen and regulate cellular oxidative stress, and is a main regulator of the protective response of cells to electrophilic chemicals or active oxygen. Under normal physiological conditions, KEAP1 recognizes NRF2 and ubiquitinates NRF2, and degrades NRF2 through a proteasome pathway, and the expression amount of NRF2 in cells is very low; under the condition of the presence of exogenous oxidants, the active cysteine of KEAP1 is modified, causing a conformational change, weakening the degradation regulation of NRF2, triggering the nuclear accumulation of NRF2, and activating downstream proteins to play an antioxidant role. When the body is in a stressed state due to excessively high active oxygen levels, oxidative damage to biological macromolecules such as DNA is caused, further inducing cell senescence, apoptosis or necrosis. Inhibition of KEAP1 to activate NRF2 antioxidant response and regulation of the oxidative environment disorder caused by oxidative stress are expected to treat various chronic diseases including neurodegenerative diseases, respiratory diseases, metabolic diseases, cardiovascular diseases, rheumatoid arthritis, fatty liver, and are the most popular treatment strategy in the field of chronic disease drug development.
[0003] Development of a proteolysis targeting chimera (PROTAC) degrader targeting KEAP1 can achieve the purpose of regulating NRF2 function through KEAP1 and further regulating cellular oxidative homeostasis. Traditional NRF2 agonists (such as dimethyl fumarate, used for treating multiple sclerosis) have defects such as insufficient efficacy, off-target effects and drug resistance. The KEAP1 degrader can up-regulate the expression amount of NRF2 from the mechanism, producing stronger and more persistent NRF2 activation effect; at the same time, using the PROTAC degradation technology, a highly specific molecule can be designed to degrade only KEAP1, reducing the toxic side effects caused by off-target. Therefore, KEAP1 degradation induced by PROTAC is a potential treatment for improving the oxidative stress state of cells. SUMMARY
[0004] The present application aims to provide a Keap1 degrader, a preparation method and application thereof. The present application provides a compound or a pharmaceutically acceptable salt, optical isomer, stereoisomer or metabolite thereof, and a corresponding solvate, metabolite, co-crystal or prodrug and pharmaceutical composition for preventing and / or treating a Keap1-mediated related disease.
[0005] To achieve the above object, the technical scheme provided by the present application is as follows: In a first aspect, a compound as shown in formula I or a pharmaceutically acceptable salt, optical isomer, stereoisomer or metabolite thereof is provided: , wherein X is selected from when Y is selected from , and m is 7 or 10; X is selected from when Y is selected from , and n is 7.
[0006] Further, it is selected from any one of the following compounds: , , .
[0007] In another aspect, the present application provides a preparation method of a Keap1 degrading agent, and the synthesis route is as follows: , wherein when m is 7, it is KPD-3; and when m is 10, it is KPD-4; wherein in the preparation of compound V from compound IV, the acid is trifluoroacetic acid; in the preparation of compound VI from compound V, the organic base is triethylamine; and in the preparation of KPD-3 or KPD-4 from compound VII, the organic base is N,N-diisopropyl ethylamine.
[0008] In another aspect, the synthesis route is as follows: , wherein in the preparation of compound X from compound IX, the inorganic base is potassium carbonate; in the preparation of compound XI from compound X, the acid is trifluoroacetic acid; and in the preparation of KPD-5 from compound XII, the organic base is N,N-diisopropyl ethylamine.
[0009] In a third aspect, the present application also relates to a pharmaceutical composition comprising the compound of the first aspect or a pharmaceutically acceptable salt, optical isomer, stereoisomer or metabolite thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.
[0010] In a fourth aspect, the present application also relates to the use of the compound of the first aspect or a pharmaceutically acceptable salt thereof, a solvate, a metabolite, a co-crystal or a prodrug thereof, or the aforementioned composition in the preparation of a medicament for treating / preventing a Keap1-mediated disease.
[0011] The Keap1 degrading agents mentioned above are used to prevent and treat metabolic diseases, cardiovascular diseases, rheumatoid arthritis, or fatty liver disease.
[0012] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Nevertheless, this invention still intends to provide a more detailed description and explanation of these terms and phrases. In the event of any inconsistency between the terms and phrases mentioned and their known meanings, the meanings expressed in this invention shall prevail.
[0013] Unless otherwise stated, the terms used in the specification and claims shall have the following meanings.
[0014] In this invention, the definition and conventional use of stereochemistry are generally referred to in the following literature: SP Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984); McGraw-Hill Book Company, New York; and Elielj E. and Wilenj S., Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York, 1994. "Stereoisomers" are compounds that have the same chemical structure but whose atoms or groups are arranged differently in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotational isomers), geometric isomers (cis / trans) isomers, and hindered isomers, etc.
[0015] In some embodiments, the compositions of this disclosure may comprise two or more enantiomers or diastereomers of the compound, wherein a single enantiomer or diastereomer accounts for at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 98% by weight, or at least about 99% by weight or more of the total weight of all stereoisomers. Methods for producing substantially pure enantiomers are well known to those skilled in the art.
[0016] In addition, heavier isotopes, especially deuterium (i.e., 2 Substitution with H or D can provide certain therapeutic advantages. These advantages result from increased metabolic stability, such as increased in vivo half-life, reduced dose requirement, or improved therapeutic index. It should be understood that deuterium in this invention is considered a reasonable substituent for the compound.
[0017] Unless otherwise stated, the compounds described herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds where hydrogen is replaced by deuterium or tritium, or where carbon is enriched. 13C or 14 Compounds having the structure of this invention, other than carbon substitution of C, are within the scope of this disclosure. The compounds of this disclosure optionally contain atomic isotopes in non-natural proportions at one or more atoms constituting such compounds. For example, the compounds may use isotopes such as, for example, deuterium (₂H), tritium (₃H), etc. 3 H), iodine 125 ( 125 I) or carbon 14 ( 14 C) Mark. Use 2 H, 11 C 13 C 14 C 15 C 12 N、 13 N、 15 N、 16 N、 16 O、 17 O、 14 F, 15 F, 16 F, 17 F, 18 F, 33 S, 34 S, 35 S, 36 S, 35 Cl、 37 Cl、 79 Br、 81 Br and 125 Isotopic substitutions of I are all considered. All isotopic variants of the compounds of this invention, whether or not they are radioactive, are covered within the scope of this invention.
[0018] "Pharmaceutically acceptable salt" means a salt of the compound of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. Such salts include: salts formed by addition to inorganic acids or acids formed with organic acids; salts formed when an acidic proton present on the parent compound is replaced by a metal ion; or coordination compounds formed with organic bases.
[0019] "Pharmaceutical composition" refers to a mixture of one or more of the compounds of the present invention or their pharmaceutically acceptable salts, solvates, hydrates or prodrugs with other chemical components, such as pharmaceutically acceptable carriers.
[0020] "Solvate" refers to an association formed by one or more solvent molecules with the compounds of this invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. "Hydrate" refers to an association formed by solvent molecules that are water.
[0021] "Pharmaceutical carrier" refers to the inactive component in a pharmaceutical composition that does not cause significant irritation to the organism and does not interfere with the biological activity and properties of the given compound.
[0022] The beneficial effects of this invention are: This invention provides a class of Keap1 degrading agents, their preparation methods, and applications. By testing the ability of these compounds to induce KEAP1 protein degradation in HCT116 and HEK293-KEAP1 overexpressing cells, and further evaluating their effect on activating the NRF2 signaling pathway, the efficient degradation of KEAP1 and the upregulation of NRF2 by the compounds of this invention were verified. Furthermore, proteasome inhibitor experiments confirmed that these compounds specifically degrade KEAP1 via the proteasome pathway, exhibiting a clear mechanism of action and high selectivity. Therefore, the compounds of this invention can serve as potential KEAP1 degrading agents for the treatment of various chronic diseases caused by oxidative stress, such as neurodegenerative diseases, metabolic diseases, and cardiovascular diseases, demonstrating significant clinical application potential.
[0023] The abbreviations for the reaction reagents mentioned in this invention specification are as follows: DIEA: N,N-diisopropylethylamine; NMP: N-methylpyrrolidone; PyBOP: Benzotriazol-1-yl-oxytripyrrolidinyl hexafluorophosphate; DMF: N,N-dimethylformamide; DCM: Dichloromethane; THF: Tetrahydrofuran; TFA: Trifluoroacetic acid; K2CO3: Potassium carbonate; TEA: Triethylamine. Attached Figure Description
[0024] Figure 1 Western blot results for KPD-1~KPD-7 induced KEAP1 degradation; Figure 2 Western blot results and DC of KPD-3-induced KEAP1 degradation 50 Fitted curve; Figure 3 Western Blot results and DC of KPD-4-induced KEAP1 degradation 50 Fitted curve; Figure 4 Western Blot results and DC of KPD-5-induced KEAP1 degradation 50 Fitted curve; Figure 5 Western blot results for NRF2 upregulation induced by KPD-3, KPD-4, and KPD-5. Detailed Implementation
[0025] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented under the premise of the technical solution of the present invention. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.
[0026] Example 1: KPD-3 Synthesis
[0027] To a solution of N-Boc-heptandiamine (417.0 mg, 1.81 mmol) in NMP (5 mL), DIEA (630.6 μL, 3.62 mmol) and (2,6-dioxadiidine-3-yl)-4-fluoroisoindoline-1,3-dione (500 mg, 1.81 mmol) were added, and the mixture was heated to 110°C and stirred for 12 hours. The reaction was quenched with water (10 mL), and then diluted with ethyl acetate (10 mL). The aqueous phase was diluted with ethyl acetate (5 mL). 3) Extraction. Combine the organic phases, wash with saturated sodium chloride aqueous solution (10 mL), dry with sodium sulfate, filter, concentrate the filtrate under reduced pressure, and pass the residue through a rapid silica gel column (ISCO). ® Sepa Flash ® Purified by silica gel column chromatography (20 g; ethyl acetate-petroleum ether, 0–37%; 80 mL / min) to give a yellow oily substance KPD-3-1 (610 mg, yield 69.3%). MS (ESI) was then used for purification. + ): m / z 387.2 [M+H-C5H8O2] + .
[0028] TFA (1.0 mL, 13.46 mmol) was added to a DCM (5 mL) solution of KPD-3-1 (610 mg, 1.25 mmol), and the mixture was stirred at 20 °C for 12 hours. The reaction mixture was concentrated under reduced pressure to give crude KPD-3-2 trifluoroacetate (1.1 g) as a yellow oil. MS (ESI) + ): m / z 387.2 [M+H] + .
[0029] To a DMF (5 mL) solution of crude KPD-3-2 trifluoroacetate (480.0 mg, 959.1 μmol), PYBOP (1.50 g, 2.88 mmol), triethylamine (467.2 μL, 3.36 mmol), and 2-[(2-amino-1,3-benzothiazol-6-yl)oxy]acetic acid (215.1 mg, 959.1 μmol) were added, and the mixture was stirred at 20°C for 12 hours. The reaction was quenched with water (2 mL) and ethyl acetate (2 mL) was added. 3) Extraction. The organic phases were combined, washed with saturated sodium chloride aqueous solution (5 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative thin-layer chromatography (SiO2, ethyl acetate:methanol = 5:1) to give a yellow solid KPD-3-3 (327 mg, yield 57.5%). MS (ESI) + ): m / z 593.3 [M+H] + .
[0030] To a solution of KPD-3-3 (70 mg, 118.1 μmol) in THF (1 mL), DIEA (61.7 μL, 354.3 μmol) and chloroacetyl chloride (9.4 μL, 118.1 μmol) were added, and the mixture was stirred at 20°C for 0.5 hours. The reaction mixture was concentrated under reduced pressure, and the residue was subjected to preparative HPLC (Waters Xbridge BEH C18 100). Purified by [H2O(10 mM NH4HCO3)-ACN], 35%-65%, to obtain green solid KPD-3 (5.2 mg, yield 6.3%). MS (ESI) + ): m / z 669.2[M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 12.61 (br s, 1H), 11.08 (s, 1H), 8.07 (brt, J = 5.6 Hz, 1H), 7.71 - 7.63 (m, 1H), 7.60 - 7.51 (m, 2H), 7.44 - 7.31 (m,1H), 7.13 - 7.00 (m, 2H), 6.59 - 6.43 (m, 1H), 5.04 (dd, J = 5.2, 12.8 Hz,1H), 4.51 (s, 2H), 4.43 (s, 2H), 3.27 (br d, J = 5.6 Hz, 2H), 3.12 (q, J =6.8 Hz, 2H), 2.93 - 2.81 (m, 1H), 2.55 (br d, J = 10.0 Hz, 2H), 2.08 - 1.97(m, 1H), 1.53 (br d, J = 6.8 Hz, 2H), 1.48 - 1.37 (m, 2H), 1.34 - 1.17 (m,6H).
[0031] KPD-4 Synthesis
[0032] Following the synthesis method of KPD-3, N-Boc-decanediamine was used as a starting material to obtain green solid KPD-4. MS (ESI) + ):m / z 711.3 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 12.65 - 12.56 (m, 1H), 11.09 (s,1H), 8.06 (t, J = 5.6 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.61 - 7.53 (m, 2H),7.14 - 7.05 (m, 2H), 7.01 (d, J = 7.2 Hz, 1H), 6.51 (br t, J = 6.0 Hz, 1H),5.10 - 5.00 (m, 1H), 4.51 (s, 2H), 4.43 (s, 2H), 3.29 - 3.24 (m, 2H), 3.16 -3.06 (m, 2H), 2.93 - 2.81 (m, 1H), 2.62 - 2.55 (m, 2H), 2.06 - 1.98 (m, 1H), 1.60 - 1.51 (m, 2H), 1.46 - 1.37 (m, 2H), 1.35 - 1.25 (m, 4H), 1.24 - 1.13(m, 8H).
[0033] KPD-5 Synthesis
[0034] Potassium carbonate (1.66 g, 12.03 mmol) and N-Boc-7-bromoheptylamine (1.77 g, 6.02 mmol) were added to a DMF (12 mL) solution of 2-amino-6-hydroxy-1,3-benzothiazole (1 g, 6.02 mmol), and the mixture was heated to 80 °C and stirred for 12 hours. After diluting the reaction mixture with water (80 mL), ethyl acetate (50 mL) was added. 3) Extraction. Combine the organic phases, dry with sodium sulfate, filter, concentrate the filtrate under reduced pressure, and purify the residue by silica gel column chromatography (petroleum ether / ethyl acetate = 5 / 1-1 / 3) to give a yellow solid compound KPD-5-1 (790 mg, yield 34.60%).
[0035] MS (ESI + ): m / z 380.2 [M+H] + .
[0036] TFA (1 mL, 13.46 mmol) was added to an 8 mL solution of KPD-5-1 (790 mg, 2.08 mmol) in DCM, and the mixture was stirred at 20 °C for 1 hour. The reaction mixture was concentrated under reduced pressure to give crude KPD-5-2 trifluoroacetate (800 mg) as a yellow oil. MS (ESI) + ): m / z 280.2 [M+H] + .
[0037] Add TEA (149.5 μL, 1.07 mmol) and (2,6-dioxadiazine-3-yl)-5-fluoroisoindoline-1,3-dione (148.3 mg, 536.9 μmol) to a DMF (2 mL) solution of crude KPD-5-2 trifluoroacetate (100 mg, 357.9 μmol), heat to 110°C and stir for 12 hours. Perform two replicates. Combine the two reaction mixtures, quench the reaction with water (6 mL), and ethyl acetate (5 mL) 3) Extraction, combining the organic phases, washing with saturated sodium chloride aqueous solution (5 mL), drying with sodium sulfate, filtering, and concentrating the filtrate under reduced pressure to obtain crude yellow oil KPD-5-3 (80 mg). MS (ESI) + ): m / z 536.1 [M+H] + .
[0038] To a THF (1 mL) solution of crude KPD-5-3 (40 mg, 74.7 μmol), DIEA (26.0 μL, 149.4 μmol) and chloroacetyl chloride (6.0 μL, 74.7 μmol) were added, and the mixture was stirred at 20°C for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was subjected to preparative HPLC (Waters Xbridge BEH C18 100). Purified at 30 mm and 10 μm (using [H₂O(10 mMNH₄HCO₃)-ACN], 50%-70%), yielding a green solid KPD-5 (9.4 mg, yield 20.35%). MS (ESI+): m / z 612.2 [M+H) + . 1H NMR (400 MHz, DMSO-d6) δ 12.66 - 12.47 (m, 1H), 11.05 (s,1H), 7.64 (d, J = 8.8 Hz, 1H), 7.60 - 7.52 (m, 2H), 7.10 (br t, J = 5.2 Hz,1H), 7.03 (dd, J = 2.4, 8.8 Hz, 1H), 6.94 (d, J = 1.2 Hz, 1H), 6.84 (dd, J =1.6, 8.4 Hz, 1H), 5.02 (dd, J = 5.2, 12.8 Hz, 1H), 4.43 (s, 2H), 4.01 (t, J =6.4 Hz, 2H), 3.21 - 3.12 (m, 2H), 2.93 - 2.81 (m, 1H), 2.62 - 2.51 (m, 2H), 2.04 - 1.94 (m, 1H), 1.81 - 1.69 (m, 2H), 1.58 (br d, J = 6.4 Hz, 2H), 1.50 -1.34 (m, 6H).
[0039] Comparative example: KPD-1 Synthesis
[0040] Following the KPD-5 synthesis method, using KPD-5-2 and (2,6-dioxadiazine-3-yl)-4-fluoroisoindoline-1,3-dione as starting materials, a green solid KPD-1 was obtained. MS (ESI) + ): m / z 612.2 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 12.64 - 12.48 (m, 1H), 11.09 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.60 -7.53 (m, 2H), 7.09 (d, J = 8.8 Hz, 1H), 7.05 - 6.99 (m, 2H), 6.54 (t, J = 5.6Hz, 1H), 5.05 (dd, J = 5.6, 12.8 Hz, 1H), 4.43 (s, 2H), 4.05 - 3.96 (m, 2H), 3.31 - 3.27 (m, 2H), 2.94 - 2.81 (m, 1H), 2.63 - 2.52 (m, 2H), 2.07 - 1.97 (m, 1H), 1.74 (quin, J = 6.8 Hz, 2H), 1.65 - 1.53 (m, 2H), 1.48 - 1.34 (m,6H).
[0041] KPD-2 Synthesis
[0042] Following the synthesis methods of KPD-5 and KPD-1, using N-Boc-10-bromodecylamine and (2,6-dioxadiazine-3-yl)-4-fluoroisoindoline-1,3-dione as starting materials, green solid KPD-2 was obtained. MS (ESI) + ): m / z 654.2 [M+H] + . 1H NMR(400 MHz, DMSO-d6) δ 12.74 - 12.41 (m, 1H), 11.09 (s, 1H), 7.69 - 7.52 (m,3H), 7.14 - 6.98 (m, 3H), 6.52 (br t, J = 6.0 Hz, 1H), 5.04 (dd, J = 5.6, 12.8 Hz, 1H), 4.43 (s, 2H), 3.99 (t, J = 6.4 Hz, 2H), 3.27 (br s, 1H), 2.87 (ddd, J = 5.6, 14.0, 17.6 Hz, 1H), 2.58 (br d, J = 16.4 Hz, 1H), 2.08 - 1.98 (m, 1H), 1.76 - 1.67 (m, 2H), 1.62 - 1.51 (m, 2H), 1.46 - 1.22 (m, 13H).
[0043] KPD-6 Synthesis
[0044] Following the synthesis method of KPD-2, using KPD-2-2 and (2,6-dioxadiazine-3-yl)-5-fluoroisoindoline-1,3-dione as starting materials, green solid KPD-6 was obtained. MS (ESI) + ): m / z 654.2 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 12.79 - 12.36 (m, 1H), 11.17 - 10.98 (m, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.60 - 7.52 (m, 2H), 7.09 (br t, J = 5.2 Hz, 1H), 7.02 (dd, J = 2.4, 8.8 Hz, 1H), 6.93 (d, J = 1.6 Hz, 1H), 6.83 (dd, J = 1.6, 8.4 Hz, 1H), 5.02 (dd, J =5.2, 12.8 Hz, 1H), 4.43 (s, 2H), 3.99 (t, J = 6.4 Hz, 2H), 3.19 - 3.09 (m,2H), 2.87 (ddd, J = 5.6, 14.4, 17.2 Hz, 1H), 2.62 - 2.52 (m, 2H), 2.04 - 1.94(m, 1H), 1.79 - 1.67 (m, 2H), 1.62 - 1.51 (m, 2H), 1.50 - 1.13 (m, 12H).
[0045] KPD-7 Synthesis
[0046] Following the synthesis method of KPD-3, using N-Boc-decanediamine and (2,6-dioxadiidine-3-yl)-5-fluoroisoindoline-1,3-dione as starting materials, green solid KPD-7 was obtained. MS (ESI) + ): m / z 711.2 [M+H] + . 1H NMR (400 MHz, DMSO-d6) δ 12.87 - 12.33 (m, 1H), 11.05 (s, 1H), 8.07 (t, J = 6.0 Hz, 1H), 7.68 (d, J = 8.8 Hz, 1H), 7.60 - 7.52 (m, 2H), 7.15 - 7.04 (m, 2H), 6.93 (s,1H), 6.83 (dd, J = 1.6, 8.4 Hz, 1H), 5.02 (dd, J = 5.6, 12.8 Hz, 1H), 4.51(s, 2H), 4.43 (s, 2H), 3.12 (quin, J = 6.4 Hz, 4H), 2.93 - 2.81 (m, 1H), 2.65- 2.51 (m, 2H), 2.04 - 1.94 (m, 1H), 1.55 (quin, J = 6.8 Hz, 2H), 1.45 - 1.30(m, 4H), 1.21 (br s, 10H).
[0047] Example 2: Evaluation of the effect of compound-induced degradation of KEAP1 at the cellular level 1) Preparation of cell lines, culture media and test compounds Table 1. Composition of culture media used for each cell line
[0048] As shown in Table 1, the cell lines to be tested were placed in a culture medium containing 10% heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, 100 U / mL penicillin, and 100 g / mL streptomycin, and incubated at 37°C. o C. Culture in a 5% CO2 cell culture incubator. Change the medium every two days. After the cells reach 80% confluence, digest with trypsin, passage, and maintain the cells in a good logarithmic growth phase.
[0049] 2) Methods for detecting the degradation effect of KEAP1 HCT116 cells in logarithmic growth phase at 2 10⁶ cells / plate were seeded in 6 cm cell culture dishes and cultured for 24 hours. Cells were washed with PBS and serum-free medium, respectively. Serum-free medium containing 2 μM of compounds KPD-1, KPD-2, KPD-3, KPD-4, KPD-5, KPD-6, and KPD-7 was prepared and added to the cell culture dishes. The cells were then incubated with the drugs in an incubator for 24 hours. The cell culture dishes were then placed on ice, and the cells were washed twice with pre-chilled PBS at 4°C. PBS was then added again, and the cells were scraped off and collected.
[0050] The resulting cells were added to cell lysis buffer [PBS (ThermoFisher Scientific), 1% IGEPAL-CA-630 (Sigma-Aldrich), 0.2% SDS (Sigma-Aldrich), 1% EDTA-free protease inhibitor mixture (Sigma-Aldrich), 0.1% Benzonase (Beyotime)], and after sonication, were centrifuged at 4°C using a benchtop centrifuge (20000 g, 30 min). The supernatant was collected and the concentration was adjusted to 2 mg / mL with cell lysis buffer. The resulting proteins were separated by SDS-PAGE and then transferred to an NC membrane. After transfer, the NC membrane was immersed in Ponceau S staining solution, and the Ponceau S signal was read. The NC membrane was then incubated in 10 mL of 5% BSA / TBST at room temperature for 1 hour. The sample was incubated overnight at 4°C with the target protein antibody KEAP1 monoclonal antibody (Proteintech), followed by incubation for 1 hour with the secondary antibody (Alexa Fluor 488 labeled Goat Anti-Mouse IgG, Beyotime). The fluorescence signal of the secondary antibody was scanned using ChemiDoc imaging. Subsequently, the sample was incubated at room temperature for 1 hour with β-actin fluorescent antibody (Proteintech) to assess loading consistency.
[0051] 3) DC 50 Detection methods HEK293 cells in logarithmic growth phase at 1 10⁶ cells / plate were seeded in 6 cm cell culture dishes and cultured overnight. Two hours before transfection, the original culture medium was removed from the cells and replaced with fresh complete culture medium. 1 µg of KEAP1 plasmid DNA was diluted with 100 μL of serum-free diluent to prepare a DNA dilution buffer. 2 μL of Neofect™ was added directly to the DNA dilution buffer, gently mixed, and incubated at room temperature for 15–30 minutes. The transfection complex was then added to the cell culture medium and gently mixed. After culturing for 24 hours, the cells were washed with PBS and serum-free medium, respectively. Different concentrations of the test compound were added for 24 hours (proteasome inhibitors require 1 hour of pretreatment before adding the test compound), and the cells were harvested. Subsequent immunoblotting procedures were performed as described above.
[0052] Relative degradation rate (%) = 100 - (fluorescence signal of target band in sample group / fluorescence signal of target band in blank group) 100. The degradation curves were fitted using Graphpad with a four-parameter fitting.
[0053] The effects of 2 μM compounds KPD-1, KPD-2, KPD-3, KPD-4, KPD-5, KPD-6, and KPD-7 on the degradation of KEAP1 in HCT116 cells were characterized by Western blotting results. Figure 1 The results showed that KPD-3, KPD-4, and KPD-5 could induce the degradation of KEAP1, while the comparative compounds KPD-1, KPD-2, KPD-6, and KPD-7 had no effect on the KEAP1 protein level.
[0054] Using Western blotting, the degradation effects of KPD-3, KPD-4, and KPD-5 were further tested in HEK293-KEAP1 permeabilized cells by varying drug treatment concentrations. The half-maximal concentration (DC) was then calculated. 50 Compounds KPD-3, KPD-4, and KPD-5 exhibited excellent KEAP1 degradation activity, with KPD-4 showing the strongest activity. The results are shown in Table 2.
[0055] Simultaneously, when the proteasome inhibitors Bortezomib and MG-132 were introduced into the three compound treatment groups and co-incubated, the KEAP1 degradation effect induced by compounds KPD-3, KPD-4, and KPD-5 disappeared. This indicates that compounds KPD-3, KPD-4, and KPD-5 achieve KEAP1 degradation through the ubiquitin-proteasome pathway. (See...) Figure 2 , Figure 3 , Figure 4 .
[0056] Table 2. KPD-3, KPD-4, and KPD-5 induce KEAP1 degradation of DC. 50 (μM)
[0057] Example 3: Evaluation of the effect of compounds on NRF2 upregulation at the cellular level 1) Preparation of cell lines, culture media and test compounds For cell line types, culture medium components used for each cell line, and cell culture methods, please refer to step 1 of Example 2.
[0058] 2) Detection methods HCT116 cells in logarithmic growth phase at 2 10⁶ cells / plate were seeded in 6 cm cell culture dishes and cultured for 24 hours. Cells were washed with PBS and serum-free medium, respectively. Serum-free medium containing 0.5 μM of compounds KPD-3, KPD-4, and KPD-5 was prepared and added to the cell culture dishes. The cells were then incubated with the drugs in an incubator for 24 hours. The cell culture dishes were then placed on ice, and the cells were washed twice with pre-chilled PBS at 4°C. PBS was then added again, and the cells were scraped off and collected.
[0059] The resulting cells were added to cell lysis buffer [PBS (ThermoFisher Scientific), 1% IGEPAL-CA-630 (Sigma-Aldrich), 0.2% SDS (Sigma-Aldrich), 1% EDTA-free protease inhibitor mixture (Sigma-Aldrich), 0.1% Benzonase (Beyotime)], and after sonication, were centrifuged at 4°C using a benchtop centrifuge (20000 g, 30 min). The supernatant was collected and the concentration was adjusted to 2 mg / mL with cell lysis buffer. The resulting proteins were separated by SDS-PAGE and then transferred to an NC membrane. After transfer, the NC membrane was immersed in Ponceau S staining solution, and the Ponceau S signal was read. The NC membrane was then incubated in 10 mL of 5% BSA / TBST at room temperature for 1 hour. The sample was incubated overnight at 4°C with the target protein antibody NRF2 Recombinant monoclonal antibody (Proteintech), followed by incubation for 1 hour with the secondary antibody (Alexa Fluor 488-labeled Goat Anti-Rabbit IgG, Beyotime). The fluorescence signal of the secondary antibody was scanned using ChemiDoc imaging. Subsequently, the sample was incubated at room temperature for 1 hour with β-actin fluorescent antibody (Proteintech) to assess loading consistency.
[0060] The effects of 0.5 μM compounds KPD-3, KPD-4, and KPD-5 on NRF2 upregulation in HCT116 cells were characterized using Western blotting results. Figure 5 The results showed that KPD-3, KPD-4, and KPD-5 induce NRF2 upregulation by causing KEAP1 degradation, thereby affecting changes in cellular oxidative stress levels.
[0061] The above description is merely an optional embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A compound as shown in Formula I or a pharmaceutically acceptable salt, optical isomer, stereoisomer or metabolite thereof: Formula I wherein the compound is selected from any one of the following compounds: ， wherein X is selected from Y is selected from m is 7 or 10; X is selected from Y is selected from n is 7.
2. The compound of claim 1, or a pharmaceutically acceptable salt, optical isomer, stereoisomer, or metabolite thereof, wherein, Synthetic route is as follows: 、 、 。 3. A process for the preparation of a compound according to claim 2, characterized in that, wherein when m is 7, it is KPD-3; when m is 10, it is KPD-4; ， wherein in the preparation of compound V from compound IV, the acid is trifluoroacetic acid; in the preparation of compound VI from compound V, the organic base is triethylamine; in the preparation of KPD-3 or KPD-4 from compound VII, the organic base is N,N-diisopropylethylamine. Synthetic route is as follows:
4. A process for the preparation of a compound according to claim 2, characterized in that, wherein in the preparation of compound X from compound IX, the inorganic base is potassium carbonate; in the preparation of compound XI from compound X, the acid is trifluoroacetic acid; in the preparation of KPD-5 from compound XII, the organic base is N,N-diisopropylethylamine. ， A compound as claimed in any one of claims 1-2 or a pharmaceutically acceptable salt, optical isomer, stereoisomer or metabolite thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.
5. A pharmaceutical composition, wherein, 6. Use of a compound as claimed in any one of claims 1-2 or a solvate, metabolite, co-crystal or prodrug thereof, or a composition as described above, in the manufacture of a medicament for the treatment / prevention of a Keap1-mediated disease.
7. Use according to claim 6, wherein the disease is a metabolic disease, a cardiovascular disease, rheumatoid arthritis or fatty liver disease.