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WO2024255845A1 - Agent de dégradation de protéine, composition pharmaceutique et utilisation associées - Google Patents

Agent de dégradation de protéine, composition pharmaceutique et utilisation associées Download PDF

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Publication number
WO2024255845A1
WO2024255845A1 PCT/CN2024/099218 CN2024099218W WO2024255845A1 WO 2024255845 A1 WO2024255845 A1 WO 2024255845A1 CN 2024099218 W CN2024099218 W CN 2024099218W WO 2024255845 A1 WO2024255845 A1 WO 2024255845A1
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alkyl
substituted
unsubstituted
protein
replaced
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胡有洪
陈奕
曾艳萍
丁健
沈倩倩
方艳芬
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Shanghai Institute of Materia Medica of CAS
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Shanghai Institute of Materia Medica of CAS
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Definitions

  • the present invention relates to the field of medical technology, and relates to a class of protein degraders and pharmaceutical compositions thereof, and uses thereof in preparing drugs for treating, preventing and/or improving diseases associated with CDK, EZH2 or RAS.
  • Targeted protein degradation is a very promising tool for biological mechanism research and treatment.
  • the drug resistance caused by the target protein mutation can be overcome, and the non-enzymatic function of pathogenic proteins that the inhibitor does not have can be played.
  • PROTAC technology has made great progress, and several PROTAC drug candidates have entered clinical research.
  • PROTAC technology still faces problems such as the emergence of E3 ubiquitin ligase resistance and the fact that it relies on the ubiquitin proteasome system but there are still many substances in the cell that are not substrates for the proteasome to clear.
  • the inventors used CDK9 distributed in cells as a preliminary screening of degradation fragments, and found that by introducing a connecting chain at the 1-position of the structure shown in formula (A) and connecting it to the ligand of CDK9, a series of compounds with degradation effects on the CDK9-cyclin T1 complex were obtained, and it was clarified that it can achieve autophagic degradation of CDK9 by recruiting LC3B.
  • the degradation fragments of the structure shown in the relevant representative formula (A) were connected with binding fragments of other targets (such as EZH2, RAS or other CDK inhibitors, etc.), and the related proteins were degraded, indicating that the structure has a broad spectrum of protein degradation.
  • the present invention proposes for the first time that the structure shown in formula (A) can be used as an LC3B recruitment compound, and by connecting the structure shown in formula (A), especially the 2,4-quinazolinedione structure, to the ligand of the target protein to obtain a protein degrader, the autophagic degradation of the target protein can be achieved, biological effects can be produced, and related diseases can be treated and prevented.
  • one of the objectives of the present invention is to provide a class of protein degradation agents.
  • the second object of the present invention is to provide a method for preparing the above-mentioned protein degradation agent.
  • a third object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned protein degrading agent.
  • a fourth object of the present invention is to provide the use of the above-mentioned protein degrading agent or pharmaceutical composition in the preparation of a drug for treating, preventing and/or ameliorating diseases related to CDK, EZH2, or RAS.
  • the first aspect of the present invention provides a protein degradation agent or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof, wherein the protein degradation agent comprises:
  • LCBM part At least one part that can bind to LC3 (LC3Binding Moiety, hereinafter sometimes referred to as LCBM part);
  • At least one protein of interest ligand that can bind to CDK or EZH2 or RAS POIL
  • a linker is used to covalently link the LCBM part to the POIL part independently.
  • LC3 refers to microtubule-associated protein 1A/1B-light chain 3, which is a soluble protein with a molecular weight of about 17 kDa.
  • LC3 is commonly found in mammalian tissues and cultured cells and is a key component of autophagy (the recycling system of eukaryotic cells). It is incorporated into the inner and outer membranes of autophagosomes during autophagosome biosynthesis. Therefore, LC3 is a specific marker for autophagy (especially autophagosome formation).
  • CDK refers to the cyclin-dependent kinases of the serine/threonine kinase family.
  • EZH2 stands for Enhancer of zeste homologue 2.
  • RAS refers to RAS protein.
  • the present invention provides a protein degradation agent represented by formula (1) or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof,
  • LCBM indicates the moiety that can bind to LC3
  • Linker represents a covalently linked moiety
  • POIL represents the target protein ligand portion that can bind to CDK or EZH2 or RAS.
  • the LCBM part and the POIL part can be independently selected from small molecule compounds.
  • the molecular weight of the LCBM portion and the POIL portion is each independently about 100-about 2000 Da, preferably about 100-about 1000 Da, for example, about 100-about 900 Da, about 100-about 800 Da, about 100-about 700 Da, about 100-about 600 Da, about 100-about 500 Da.
  • the LCBM part can be linked to one or more POIL parts, and vice versa.
  • the POIL parts can be selected independently, and the POIL parts can be the same or different.
  • the linkers used can also be selected independently.
  • This portion means a portion that can bind to LC3 and refers to a portion that has affinity for the LC3 protein.
  • the LCBM moiety is of formula (A),
  • Y1 and Y2 are each independently selected from O or S;
  • the Ar ring is selected from a C6-C10 aryl group or a 5-10 membered heteroaryl group;
  • R 1 is n substituents on the Ar ring, n is selected from an integer of 0-4, such as 0, 1, 2, 3, 4;
  • Each R 1 is independently selected from halogen, cyano (-CN), hydroxyl (-OH), amino (-NH 2 ), nitro (-NO 2 ), carboxyl (-COOH), unsubstituted or substituted C1-C20 alkyl, unsubstituted or substituted C1-C20 alkoxy, C1-C20 alkyl-NH-, (C1-C20 alkyl)(C1-C20 alkyl)N-, C1-C20 alkoxycarbonyl-NH-, unsubstituted or substituted C3-C16 cycloalkyl, unsubstituted or substituted 3-16 membered heterocyclyl, unsubstituted or substituted C6-C14 aryl, unsubstituted or substituted 5-15 membered heteroaryl, wherein the substitution means that one or more hydrogen in the defined group is replaced by one or more substituents selected from halogen, amino (-NH 2 ), hydroxyl (-OH);
  • R2 is selected from hydrogen, unsubstituted or substituted C1-C20 alkyl, amino C1-C20 alkyl, (C1-C6 alkyl)(C1-C6 alkyl)N-C1-C16 alkyl, C1-C6 alkyl-NH-C1-C16 alkyl, unsubstituted or substituted C3-C16 cycloalkyl, unsubstituted or substituted C3-C16 cycloalkylC1-C6 alkyl, unsubstituted or substituted 3-16 membered heterocycloalkyl, unsubstituted or substituted 3-10 membered heterocycloalkylC1-C6 Alkyl, unsubstituted or substituted C6-C14 aryl, unsubstituted or substituted 5-15 membered heteroaryl, unsubstituted or substituted C6-C14 aryl C1-C6 alkyl, unsubstituted
  • each R 1 is independently selected from halogen, cyano (-CN), hydroxyl (-OH), amino (-NH 2 ), nitro (-NO 2 ), carboxyl (-COOH), unsubstituted or substituted C1-C10 alkyl, unsubstituted or substituted C1-C10 alkoxy, C1-C10 alkyl-NH-, (C1-C10 alkyl)(C1-C10 alkyl)N-, C1-C10 alkoxycarbonyl-NH-, unsubstituted or substituted C3-C10 cycloalkyl, unsubstituted or substituted 3-10 membered heterocyclyl, unsubstituted or substituted C6-C10 aryl, unsubstituted or substituted 5-10 membered heteroaryl, and the substitution means that one or more hydrogens in the defined group are replaced by one or more substituents selected from halogen, amino (-NH 2 ), nitro (-NO 2 ),
  • R2 is selected from hydrogen, unsubstituted or substituted C1-C10 alkyl, aminoC1-C10 alkyl, (C1-C6 alkyl)(C1-C6 alkyl)N-C1-C10 alkyl, C1-C6 alkyl-NH-C1-C10 alkyl, unsubstituted or substituted C3-C10 cycloalkyl, unsubstituted or substituted C3-C10 cycloalkylC1-C6 alkyl, unsubstituted or substituted 3-10 membered heterocycloalkyl, unsubstituted or substituted 3-10 membered heterocycloalkylC1-C6 Alkyl, unsubstituted or substituted C6-C10 aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C6-C10 arylC1-C6 alkyl, unsubstituted
  • heteroaryl group and the heterocyclic group may contain 1 to 4, for example 1 to 2, heteroatoms selected from N, O and S.
  • the structure represented by formula (A) is selected from the structure represented by the following formula (A-1):
  • the Ar ring is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl;
  • R 1 is n substituents on the Ar ring, n is selected from an integer of 0-2, such as 0, 1, 2;
  • Each R 1 is independently selected from halogen, nitro, carboxyl, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted substituted C1-C6 alkoxy, wherein the substitution means that one or more hydrogens in the defined group are replaced by one or more substituents selected from halogen, amino, hydroxyl; preferably, n is 0 or 1; R1 is selected from halogen, nitro, methyl, methoxy;
  • R 2 is selected from hydrogen, unsubstituted or substituted C1-C10 alkyl, unsubstituted or substituted C3-C10 cycloalkyl C1-C6 alkyl, unsubstituted or substituted 3-10 membered heterocyclyl C1-C6 alkyl, unsubstituted or substituted C6-C10 aryl, unsubstituted or substituted 5-10 membered heteroaryl, unsubstituted or substituted C6-C10 aryl C1-C6 alkyl, unsubstituted or substituted 5-10 membered heteroaryl C1-C6 alkyl, wherein the substitution means that one or more hydrogen in the defined group is replaced by one or more substituents selected from halogen, amino, hydroxyl, C1-C3 alkyl (e.g.
  • R 2 is selected from hydrogen, ethyl, tert-pentyl, cyclohexylmethyl, N-methylpiperidinylmethyl, N,N-dimethylaminoethyl, phenyl, benzyl, benzyl substituted by methoxy or trifluoromethyl, phenethyl, pyridyl, N-methylpyrazolyl.
  • the structure represented by formula (A) is selected from the following structures:
  • This part refers to the part that can bind to CDK or EZH2 or RAS, and refers to the part that can interact with CDK or EZH2 or RAS, that is, the target of the POIL part is CDK or EZH2 or RAS.
  • the CDKs include CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, CDK14, CDK15, CDK16, CDK17, CDK18, CDK19, CDK20, CDK21 and all isoforms of the CDKs described herein.
  • the POIL moiety is capable of binding to CDK9.
  • the POIL moiety is capable of binding to CDK7.
  • the POIL moiety is capable of binding to CDK2.
  • the POIL moiety is capable of binding to CDK2, CDK4, and CDK6 simultaneously.
  • the POIL moiety is capable of binding to CDK5.
  • the POIL moiety is capable of binding to EZH2.
  • the POIL moiety is capable of binding to EZH2, EED, SUZ12, and EZH1 simultaneously.
  • the POIL moiety is capable of binding to RAS.
  • the POIL portion can be a CDK probe, CDK inhibitor, EZH2 probe, EZH2 inhibitor, RAS probe, RAS inhibitor that has been marketed or reported in the literature, including but not limited to the following compounds:
  • Linker is a linking part used to connect the LCBM part and the POIL part, which can be a chemical bond or a group.
  • the linker can be rigid or flexible. In some preferred embodiments, the linker is flexible.
  • the linker is a chemical bond.
  • the linker is a chemical bond, which means that the LCBM part and the POIL part are directly connected.
  • Linker comprises 1-50, preferably 2-16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16), further preferably a straight or branched alkylene or saturated cyclic alkyl structure having 2-8 (e.g. 2, 3, 4, 5, 6) carbon atoms, in which 1 or more (e.g.
  • carbon atoms in particular 1 or 2 carbon atoms are optionally replaced by heteroatoms selected from O, S, NR a , PR a , preferably O, S or NR a , more preferably O or NR a , in particular O, wherein Ra is H or C1-C3 alkyl; or, in which 1 or more carbon atoms, in particular 1-6, more particularly 1 or 2 carbon atoms are optionally replaced by -C( ⁇ O)-, -C( ⁇ S)-, -S( ⁇ O)-, -SO 2 - or a 3- to 6-membered ring having 0 to 4 heteroatoms selected from O, S, N, P.
  • the Linker is selected from:
  • n is independently an integer selected from 1-20 (e.g., n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), preferably an integer from 1 to 16, an integer from 1 to 10, or an integer from 1 to 8;
  • Each m is independently an integer of 1-10 (for example, m can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably an integer of 1-6, an integer of 1-2;
  • p is an integer of 0-10, preferably an integer of 0-6, and more preferably an integer of 0-2.
  • the linker is selected from the following structures:
  • the POIL part is covalently linked to the linker via a carbon atom or a heteroatom.
  • the heteroatom is selected from oxygen, sulfur, nitrogen, and phosphorus.
  • the protein degrading agent is selected from any of the following structures:
  • n is independently an integer of 1-16 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16), preferably an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, 8);
  • Each m is independently an integer of 1-6 (e.g., 1, 2, 3, 4, 5 or 6), preferably 1 or 2;
  • R 1 and R 2 are as defined above.
  • the protein degrader is a degrader of CDK9, cyclin T1 and/or CDK9-cyclin T1 complex, selected from the following structures:
  • the protein degrading agent is a degrading agent for one or more of CDK2, cyclin A2, cyclin E1, CDK4, CDK5, CDK6, cyclin D1, CDK7, CDK9 or cyclin T1, selected from the following structures:
  • the protein degrader is a degrader of one or more of EZH2, EED, SUZ12, EZH1, CDK2, CDK4, CDK6, CDK7, cyclin H, cyclin A2, cyclin E1, cyclin D1 or RAS, selected from the following structures:
  • halogen may be fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
  • C1-C20 alkyl alone or as part of a composite group refers to a straight or branched alkyl group having 1 to 20 carbon atoms, such as "C1-C10 alkyl”, “C1-C6 alkyl”, “C1-C4 alkyl”, “C1-C3 alkyl”, etc.
  • Specific examples thereof may include methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, tert-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, tert-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl and the like, but are not limited thereto.
  • C1-C20 alkoxy refers to a RO-group, wherein R is a C1-C20 alkyl group as described above, for example, "C1-C10 alkoxy”, “C1-C6 alkoxy”, “C1-C4 alkoxy”, “C1-C3 alkoxy”, etc.
  • alkoxy examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentoxy, isopentoxy, neopentoxy, n-hexyl, isohexyl, 3-methylpentoxy, 3,3-dimethylbutoxy, 2-ethylbutoxy, and the like.
  • C3-C16 cycloalkyl alone or as part of a composite group refers to a fully saturated cyclic hydrocarbon compound group containing 3-16 carbon atoms, such as “C3-C10 cycloalkyl”, “C3-C7 cycloalkyl”, “C3-C6 cycloalkyl”, “C4-C6 cycloalkyl”, etc., and specific examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
  • 3-15 membered heterocyclyl alone or as part of a composite group refers to a 3-15 membered cycloalkyl group containing 1 to 4, for example 1 to 3, 1 to 2 heteroatoms selected from nitrogen, oxygen, and sulfur in the ring, and specific examples thereof include ethylene oxide, tetrahydroimidazole, tetrahydrofuran, and the like.
  • C6-C14 aryl alone or as part of a composite group refers to a monocyclic or polycyclic aromatic group having 6 to 14 carbon atoms, such as "C6-C10 aryl", examples being phenyl and naphthyl, preferably phenyl.
  • the term "5-15 membered heteroaryl” alone or as part of a composite group refers to a monocyclic or polycyclic (e.g., bicyclic or tricyclic) aromatic ring or aromatic group having 5-15 atoms in the ring and 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur in the ring, preferably a "5-10 membered heteroaryl ring", “5-6 membered heteroaryl ring”, or "4-5 membered heteroaryl”.
  • pyrrolyl furanyl, thienyl, imidazolyl, pyrazolyl, oxazolyl, pyridin-2-yl, pyridin-3-yl, pyridin-4-yl, pyrimidin-4-yl, pyrimidin-5-yl, pyrazin-2-yl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzooxazolyl, benzo oxazolyl, benzo oxazolyl, benzisox
  • salts include anionic salts and cationic salts of the compound of formula (I), such as salts of the compound of formula (1) with an acid or a base; for example, inorganic acid or organic acid salts of the compound of formula (1); preferably, the inorganic acid includes hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid, perchloric acid; preferably, the organic acid includes formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, citric acid, tartaric acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, glutamic acid, pamoic acid; or inorganic base or organic base salts of the compound of formula (1); for example,
  • the organic base includes trialkylamine, pyridine, quinoline, piperidine, imidazole, picoline, dimethylaminopyridine, dimethylaniline, N-alkylmorpholine, 1,5-diazabicyclo[4.3.0]nonene-5, 1,8-diazabicyclo[5.4.0]undecene-7, 1,4-diazabicyclo[2.2.2]octane;
  • the trialkylamine includes trimethylamine, triethylamine, N,N-diisopropylethylamine;
  • the N-alkylmorpholine includes N-methylmorpholine.
  • the compounds of the present invention may contain chiral centers and as such may exist in different isomeric forms.
  • isomers refer to different compounds having the same molecular formula but differing in the arrangement and configuration of the atoms.
  • stereoisomers include diastereomers, enantiomers and racemates, geometric isomers, conformational isomers (including rotational isomers and atropisomers).
  • the present invention provides a method for preparing a protein degrader, which comprises a method for synthesizing an LC3 binding portion and a step of linking the portion that can bind to LC3 with the portion that can bind to CDK, EZH2 and RAS by covalent linkage.
  • the types of reactions to achieve the connection include nucleophilic substitution reaction, mitsunobu reaction, condensation reaction, etc., but are not limited thereto.
  • a pharmaceutically acceptable salt of a protein degrading agent can be prepared by dissolving the protein degrading agent in a phase
  • the protein degradation agent can be prepared by reacting in an alcohol solution saturated with an acid, an ethyl acetate solution or a dioxane solution.
  • the protein degradation agent is dissolved in a methanol solution saturated with hydrogen chloride, stirred at room temperature for 30 minutes, and the solvent is evaporated to dryness to obtain the hydrochloride of the corresponding protein degradation agent.
  • the present invention is not limited thereto, and those skilled in the art can adopt any suitable salt-forming method according to the properties of the protein degradation agent.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of one or more of the above-mentioned protein degraders or their pharmaceutically acceptable salts, stereoisomers, solvates, polymorphs, tautomers, isotopic compounds, metabolites or prodrugs, and an optional pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier refers to conventional drug carriers in the pharmaceutical field, such as: diluents, such as water, etc.; fillers, such as starch, sucrose, etc.; binders, such as cellulose derivatives, alginates, gelatin, polyvinyl pyrrolidone; wetting agents, such as glycerol; disintegrants, such as agar, calcium carbonate and sodium bicarbonate; absorption promoters, such as quaternary ammonium compounds; surfactants, such as cetyl alcohol; adsorption carriers, such as kaolin and soap clay; lubricants, such as talc, calcium stearate and magnesium stearate and polyethylene glycol, etc.
  • other adjuvants such as flavoring agents and sweeteners, etc., may also be added to the above-mentioned pharmaceutical composition.
  • the protein degradation agent of the present invention or its composition can be administered orally or parenterally to a patient in the form of conventional preparations, such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions and emulsions.
  • conventional preparations such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, injections, suspensions, syrups, patches, creams, lotions, ointments, gels, sprays, solutions and emulsions.
  • Suitable preparations can be prepared by commonly used methods using conventional organic or inorganic additives, such as excipients (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate), binders (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose or starch), disintegrants (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low-substituted hydroxypropyl cellulose, sodium bicarbonate).
  • excipients e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate
  • binders e.g., cellulose, methylcellulose, hydroxymethylcellulose, poly
  • calcium phosphate or calcium citrate calcium phosphate or calcium citrate
  • lubricants e.g., magnesium stearate, light anhydrous silicic acid, talc or sodium lauryl sulfate
  • flavoring agents e.g., citric acid, menthol, glycine or orange powder
  • preservatives e.g., sodium benzoate, sodium bisulfite, methylparaben or propylparaben
  • stabilizers e.g., citric acid, sodium citrate or acetic acid
  • suspending agents e.g., methylcellulose, polyvinylpyrrolidone or aluminum stearate
  • dispersants e.g., hydroxypropyl methylcellulose
  • diluents e.g., water
  • base wax e.g., cocoa butter, white petrolatum or polyethylene glycol
  • the dosage regimen can be adjusted to provide the best desired response.
  • a single push, a bolus injection, and/or a continuous infusion, etc. can be administered.
  • several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation.
  • several divided doses can be administered over time, or the dose can be proportionally reduced or increased depending on the treatment situation.
  • the dosage value can vary with the type and severity of the condition to be alleviated, and can include single or multiple doses.
  • the dosage of treatment varies, depending on considerations such as: the age, sex, and general health of the patient to be treated; the frequency of treatment and the nature of the desired effect; the degree of tissue damage; the duration of symptoms; and other variables that can be adjusted by individual physicians. It is further understood that for any particular individual, the specific dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering the composition or supervising the administration of the composition.
  • the dosage and administration regimen of the pharmaceutical composition can be easily determined by a person of ordinary skill in the clinical field.
  • the protein degradation agent or composition of the present invention can be administered in divided doses 4 times a day to once every 7 days, and the dosage can be, for example, 0.01 to 1000 mg/time.
  • the required dose can be administered once or multiple times to obtain the desired effect. Results
  • the pharmaceutical compositions according to the invention may also be provided in unit dosage form.
  • Another aspect of the present invention provides the use of the above-mentioned protein degrading agent or its pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug, or pharmaceutical composition in the preparation of a product for degrading one or more proteins in CDK9, cyclin T1, CDK9-cyclin T1 complex, EZH2, EED, SUZ12, EZH1, CDK2, CDK4, CDK5, CDK6, CDK7, cyclin H, cyclin A2, cyclin E1, cyclin D1 or RAS, especially for simultaneously degrading CDK9 and cyclin T1, EZH2, EED, SUZ12 and EZH1, CDK2, CDK4 and CDK6, CDK5, and cyclin A2, cyclin E1 and cyclin D1.
  • the product can be used as a drug, and can also be used in preclinical or laboratory research, for example, as a reagent for related protein degradation
  • Another aspect of the present invention provides use of the above-mentioned protein degrading agent or pharmaceutical composition in the preparation of a drug for treating, preventing and/or improving CDK or EZH2 or RAS related diseases or conditions.
  • Another aspect of the present invention provides use of the above-mentioned protein degrading agent in treating, preventing and/or improving CDK or EZH2 or RAS related diseases or conditions.
  • CDK or EZH2 or RAS related diseases or disorders include but are not limited to the following:
  • Inflammation such as arthritis, rheumatoid arthritis, spondyloarthropathies, gouty arthritis, osteoarthritis, juvenile arthritis and other arthritis diseases
  • Skin-related diseases such as psoriasis, eczema, burns, dermatitis, neuritis, etc.
  • Lung diseases such as lung inflammation, adult respiratory distress syndrome, pulmonary sarcoidosis, asthma, silicosis, chronic inflammatory lung disease, and chronic obstructive pulmonary disease (COPD);
  • Cardiovascular diseases such as arteriosclerosis, myocardial infarction (including post-myocardial infarction indications), thrombosis, congestive heart failure, cardiac reperfusion injury, etc.
  • hypertension and/or heart failure such as vascular organ damage, restenosis, cardiomyopathy; stroke, including ischemic and hemorrhagic stroke; reperfusion injury; local ischemia, including stroke and cerebral local ischemia, as well as local ischemia caused by heart/coronary artery bypass, neurodegenerative diseases, liver disease or nephritis; gastrointestinal disorders: such as inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, ulcerative colitis, ulcerative disease, gastric ulcer, viral and bacterial infections, etc.; sepsis: including septic shock, Gram-negative sepsis, etc.; malaria; meningitis; HIV infection ; opportunistic infections; cachexia secondary to infection or malignancy; cachexia secondary to acquired immunodeficiency syndrome (AIDS), AIDS, ARC (AIDS-related syndrome); myalgia caused by herpes virus infection; influenza; autoimmune diseases; graft-versus-host reaction and allo
  • the present invention also provides a new paradigm for treating, preventing, or ameliorating diseases or disorders in which CDK, EZH2, or RAS plays a role.
  • the protein degrading agent or pharmaceutical composition provided by the present invention can also be used in combination with other therapeutic agents for treating or preventing tumors.
  • the present invention provides a series of small molecules containing 2,4-quinazolinediones that have a recruitment effect on LC3, and obtains a series of protein degraders that have a degradation effect on CDK9 and cyclin T1 or EZH2, EED, SUZ12 and EZH1 or CDK2/4/6 and cyclin A2/E1/D1 or RAS or CDK2 or CDK7 and cyclin H.
  • These degraders have excellent anti-tumor activity, and the present invention also provides a means for degrading protein complexes.
  • Figure 1 shows the Western Blot experimental results of representative compounds inducing CDK9 and Cyclin T1 protein degradation and downregulating Mcl-1 protein, where A represents WSU-DLCL2 cells treated with 100nM compounds Y33, Y35, Y41, Y44, and Y45, and B represents WSU-DLCL2 cells treated with 1 ⁇ M compounds Y1, Y2, Y3, Y4, and Y5.
  • FIG2A shows that representative compounds Y2 and Y3 induce the binding of CDK9 and LC3B
  • FIG2B shows that the degradation effect of compound Y35 can be inhibited by the late autophagy inhibitor bafilomycin A1 (BafA1), indicating the autophagy-lysosomal degradation pathway.
  • Figure 3 shows the Western Blot experimental results showing that compounds Y44 and Y82-Y86 induced CDK9 and Cyclin T1 protein degradation and downregulated Mcl-1 protein at a concentration of 100 nM.
  • Figure 4A shows the degradation effect of compound Y53 on CDK2 at a series of concentrations (0.1 ⁇ M, 1 ⁇ M, 10 ⁇ M);
  • Figure 4B shows the degradation effect of compound Y67 on CDK7 and the corresponding cyclin H at a concentration of 10 ⁇ M;
  • Figure 4C shows the degradation effect of compound Y62 on CDK2 and its corresponding cyclin A2, cyclin E1 and CDK6 and its corresponding cyclin D1;
  • Figure 4D shows the degradation effect of Y59, Y60, and Y61 on CDK2/4/6 and the corresponding cyclin A2/E1/D1 at a concentration of 10 ⁇ M.
  • FIG5A shows the degradation effects of compounds Y47 and Y50 on EZH2, EED, SUZ12 complex and EZH1 at a concentration of 10 ⁇ M
  • FIG5B shows the degradation effects of compounds Y50, Y87-Y89 on EZH2, EED, SUZ12 complex and EZH1 at a concentration of 10 ⁇ M.
  • Figure 6 shows the degradation effect of compound Y69 on Ras protein, which is time- and concentration-dependent. It can also downregulate the levels of related proteins pERK T202/Y204 and ERK.
  • Figure 7 shows the Western Blot experimental results of representative compounds inducing degradation of CDK2, cyclin A2, cyclin E1, CDK4, CDK5, CDK6, cyclin D1, CDK7, CDK9 and Cyclin T1 proteins, where A represents WSU-DLCL2 cells treated with 100 nM compounds Y83, Y95 and Y96, and B represents WSU-DLCL2 cells treated with 100 nM compounds Y84 and Y100-Y106.
  • the raw materials, reagents, methods, etc. used in the examples are conventional raw materials, reagents, and methods in the art. Dosage and method.
  • the nuclear magnetic resonance hydrogen spectrum was recorded by a Bruker AMX-400 nuclear magnetic resonance instrument, and the unit of chemical shift ⁇ was ppm. Unless otherwise specified, all reaction solvents were purified according to conventional methods.
  • Silica gel (200-300 mesh) for column chromatography was produced by Qingdao Ocean Chemical Branch.
  • Thin layer chromatography used GF254 high-efficiency plate, which was produced by Yantai Chemical Research Institute.
  • the preparative thin layer chromatography plate was prepared by the Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and the stationary phase was prepared by GF254 (HG/T2354-92) silica gel and sodium carboxymethyl cellulose (800-1200), which were produced by Qingdao Ocean Chemical Co., Ltd.
  • Methyl 2-amino-4-chlorobenzoate (1.0 g, 5.39 mmol) was dissolved in 27 mL of anhydrous acetonitrile, and ethoxycarbonyl isothiocyanate (0.8 mL, 6.47 mmol) was added. The mixture was stirred at room temperature overnight. After the raw material was consumed, benzylamine (0.9 mL, 8.08 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 2.1 g, 10.78 mmol) were added. The mixture was stirred at room temperature for 24 hours.
  • EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • Y1a (1.5 g, 4.19 mmol) in 51 ml methanol, add 8 mL of 1 M sodium hydroxide solution, reflux for 2 hours, remove the methanol by rotation, add dichloromethane, wash the dichloromethane layer once with water, once with saturated sodium chloride solution, dry with anhydrous sodium sulfate, and chromatograph on a silica gel column to obtain Y1b (white solid, 150 mg, yield 12.5%).
  • Y1b (50 mg, 0.17 mmol) was dissolved in 1 mL of DMF, and 1,2-dibromoethane (60 ⁇ L, 0.70 mmol) was added. Potassium carbonate (48 mg, 0.35 mmol), stirred at room temperature overnight, added ethyl acetate and water, washed the ethyl acetate layer with water 4 times, washed once with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and chromatographed on a silica gel column to obtain Y1c (white solid, 35 mg, yield 50.7%).
  • Y1c 49 mg, 0.12 mmol was dissolved in 1 mL of DMF, and SNS-032 (47 mg, 0.12 mmol) and diisopropylethylamine (DIPEA, 62 ⁇ L, 0.37 mmol) were added. The mixture was stirred at room temperature overnight. After the raw material was completely consumed, ethyl acetate and water were added. The ethyl acetate layer was washed 4 times with water and once with a saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain Y1 (white solid, 20 mg, yield 23.2%).
  • DIPEA diisopropylethylamine
  • the synthesis method was the same as that in Example 1, except that 1,2-dibromoethane was replaced with 1,3-dibromopropane to obtain protein degradation agent Y2 (white solid, yield 63.4%).
  • Y6a 230 mg, 0.84 mmol
  • 1,2-dibromoethane 364 ⁇ L, 4.22 mmol
  • potassium carbonate 234 mg, 1.69 mmol
  • the mixture was stirred at room temperature overnight.
  • Ethyl acetate and water were added, and the ethyl acetate layer was washed with water 4 times, washed with saturated sodium chloride once, dried over anhydrous sodium sulfate, and chromatographed on a silica gel column to obtain compound Y6b (white solid, 163 mg, yield 50.7%).
  • Y6b (85 mg, 0.22 mmol) was dissolved in 1 mL of DMF, and SNS-032 (85 mg, 0.22 mmol) and DIPEA (111 ⁇ L, 0.67 mmol) were added. The mixture was stirred at 60° C. overnight to obtain compound Y6 (white solid, 58 mg, yield 38.1%).
  • 1H NMR(400MHz,Chloroform-d) ⁇ 10.98(s,1H),8.05(d,J 8.5,2.9Hz,1H),7.41–7.34(m,2H),7.34–7.28(m,1H),7.26–7.17(m,2H),7.15–7.06(m,4H),6.
  • the synthesis method was the same as that of Example 6, except that aniline was replaced with 4-(aminomethyl)-1-methylpyrazole to obtain degradation agent Y13 (white solid, yield 10.0%).
  • Y14a (95 mg, 0.13 mmol) was dissolved in 1 mL of trifluoroacetic acid, refluxed for 24 h, and water was added to precipitate a white solid. The solid was filtered to obtain Y14 (white solid, 50 mg, yield 65.7%).
  • the synthesis method is the same as that of Example 6, except that aniline in the first step is replaced with ethylamine (2M, THF) to obtain compound Y15a, and then the second and third steps are continued to obtain degradation agent Y15 (white solid, yield 20.0%).
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by isatoic anhydride, and aniline is replaced by ethylamine (2M, THF) to obtain degradation agent Y21 (white solid, yield 34.2%).
  • the synthesis method was the same as that of Example 6, except that 4-chloroisatoic anhydride was replaced with 5-chloro-1H-benzo[d][1,3]oxazine-2,4-dione and aniline was replaced with benzylamine to obtain degradation agent Y22 (white solid, yield 13.4%).
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 5-chloroisatoic anhydride, and aniline is replaced by benzylamine to obtain degradation agent Y23 (white solid, yield 12.6%).
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 3-chloroisatoic anhydride, and aniline is replaced by benzylamine to obtain degradation agent Y24 (white solid, yield 14.2%).
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 6-methoxy-1H-benzo[d][1,3]oxazine-2,4-dione, and aniline is replaced by benzylamine to obtain degradation agent Y25 (white solid, yield 19.3%).
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 5-methoxyisatoic anhydride, and aniline is replaced by benzylamine to obtain degradation agent Y26 (white solid, yield 31.4%).
  • 1 H NMR 400 MHz, DMSO-d 6 ) ⁇ 12.22(s,1H),7.54–7.47(m,2H),7.43–7.38(m,2H),7.34–7.30(m,4H),7.
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 7-methoxy-1H-benzo[D][1,3]oxazine-2,4-dione, and aniline is replaced by benzylamine to obtain degradation agent Y27 (white solid, yield 28.8%).
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 3-methoxyisatoic anhydride, and aniline is replaced by benzylamine to obtain degradation agent Y28 (white solid, yield 32.1%).
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 4-methylisatoic anhydride, and aniline is replaced by benzylamine to obtain degradation agent Y29 (white solid, yield 11.3%).
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 4-bromoisatoic anhydride, and aniline is replaced by benzylamine to obtain degradation agent Y30 (white solid, yield 27.6%).
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 4-fluoroisatoic anhydride, and aniline is replaced by benzylamine to obtain degradation agent Y31 (white solid, yield 20.1%).
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 4-nitroisatoic anhydride, and aniline is replaced by benzylamine to obtain degradation agent Y32 (white solid, yield 25.6%).
  • the synthesis method was the same as that of Example 6, except that aniline was replaced by ethylamine (2M in THF) and 1,2-dibromoethane was replaced by 1,3-dibromoethane to obtain degradation agent Y33 (white solid, yield 33.4%).
  • the synthesis method is the same as that of Example 6, except that 1,2-dibromoethane is replaced by 1,3-dibromoethane to obtain degradation agent Y34 (white solid, yield 39.1%).
  • the synthesis method is the same as that of Example 6, except that aniline is replaced by 4-methylaminopyridine and 1,2-dibromoethane is replaced by 1,3-dibromoethane to obtain The degradation agent Y35 (white solid, yield 26.0%) was obtained.
  • the synthesis method is the same as that of Example 6, except that 4-chloroisatoic anhydride is replaced by 5-methoxy-[1,3]benzoxazine-2,4-dione, aniline is replaced by 4-methylaminopyridine, and 1,2-dibromoethane is replaced by 1,3-dibromoethane to obtain degradation agent Y36 (white solid, yield 38.9%).
  • Y37a The synthesis method of Y37a is the same as that of Y6a in Example 6, except that aniline in the first step is replaced with 4-methylaminopyridine to obtain Y37a (white solid, yield 15.9%).
  • Y37a 500 mg, 1.74 mmol
  • methyl 4-hydroxycyclohexanecarboxylate 550 mg, 3.48 mmol
  • triphenylphosphine 912 mg, 3.48 mmol
  • the tetrahydrofuran was spin-dried and subjected to silica gel column chromatography to obtain 500 mg of a mixture of Y37b and triphenylphosphine as a colorless oil.
  • 3-Aminopyridine-2-carboxylic acid (5.0 g, 36.20 mmol) and benzylamine (5.9 mL, 54.30 mmol) were dissolved in 90 mL of dichloromethane, and DIPEA (18.9 mL, 108.60 mmol) and HATU (17.9 g, 47.06 mmol) were added. The mixture was stirred at room temperature overnight and purified by silica gel column chromatography to obtain Y38a (light yellow oil, 5.6 g, yield 68.6%).
  • Y38a (5.6 g, 24.82 mmol) was dissolved in 62 mL of acetonitrile, and Boc 2 O (6.5 g, 29.8 mmol) and p-dimethylaminopyridine (DMAP, 303 mg, 2.48 mmol) were added. The mixture was stirred at room temperature overnight. After Y38a was completely consumed, Y38b (3.2 g, white solid, yield 51.3%) was obtained by suction filtration.
  • Boc 2 O 6.5 g, 29.8 mmol
  • DMAP p-dimethylaminopyridine
  • Y38b (300 mg, 1.18 mmol) was dissolved in 4 mL of DMF, 1,3-dibromopropane (0.7 mL, 7.11 mmol) and potassium carbonate (327 mg, 2.37 mmol) were added, and the mixture was stirred at room temperature overnight. Ethyl acetate and water were added, and the ethyl acetate layer was washed 4 times with water and once with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography to obtain Y38c (white solid, 392 mg, yield 88.4%).
  • Y38c (100 mg, 0.27 mmol) was dissolved in 2 mL of DMF, and SNS-032 (91.5 mg, 0.24 mmol) and DIPEA (140 ⁇ L, 0.80 mmol) were added. The mixture was stirred at 60° C. overnight. Ethyl acetate and water were added. The ethyl acetate layer was washed four times with water and once with a saturated sodium chloride aqueous solution. The mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography to obtain Y38 (white solid, 112 mg, yield 63.5%).
  • Example 38 The synthesis method was similar to that of Example 38, except that 3-aminopyridine-2-carboxylic acid was replaced by 2-aminonicotinic acid, and benzylamine was replaced by ethylamine hydrochloride to obtain degradation agent Y44 (white solid, yield 4.6%).
  • the synthesis method is as described in Example 38, except that 3-aminopyridine-2-carboxylic acid is replaced by 2-aminonicotinic acid, and benzylamine is replaced by 4-methylaminopyridine to obtain degradation agent Y45 (white solid, yield 6.7%).
  • Y46a The synthesis of Y46a was based on Y38a.
  • 3-aminopyridine-2-carboxylic acid was replaced with 2-aminonicotinic acid and benzylamine was replaced with ethylamine hydrochloride to obtain Y46a (white solid, yield 66.0%).
  • Y46a 500 mg, 2.62 mmol
  • 1,2-ethylene glycol 974 mg, 15.69 mmol
  • triphenylphosphine 1.4 g, 5.23 mmol
  • Y46b obtained in the previous step was dissolved in 12 mL of dichloromethane, and Dess-Martin periodinane (1.44 g, 3.40 mmol) was added under ice bath, stirred at 0°C for 5 hours, and subjected to silica gel column chromatography to obtain Y46c (white solid, 305 mg, yield 34.9%).
  • Y46c 13 mg, 0.06 mmol
  • C24a 20 mg, 0.04 mmol
  • DCE dichloroethane
  • one drop of acetic acid was added.
  • sodium triacetoxyborohydride 24 mg, 0.11 mmol
  • Dichloromethane and water were added, and the dichloromethane layer was washed twice with water and once with a saturated sodium chloride aqueous solution. The mixture was dried over anhydrous sodium sulfate and purified by silica gel column chromatography to obtain degradation agent Y46 (white solid, 17 mg, yield 60.6%).
  • the synthesis method is as in Example 46, except that Y46a is replaced by 7-chloro-3-ethylquinazoline-2,4(1H,3H)-dione, and 1,2-ethylene glycol is replaced by 1,3-propylene glycol to obtain degradation agent Y51 (white solid, yield 30.0%).
  • the synthesis method is as described in Example 46, except that Y46a is replaced by 7-chloro-3-(pyridin-4-ylmethyl)quinazoline-2,4(1H,3H)-dione, and 1,2-ethylene glycol is replaced by 1,3-propylene glycol to obtain degradation agent Y52 (white solid, yield 34.5%).
  • N,N-diisopropylethylamine (1.80 mL, 11.60 mmol) was added to 7-chloro-8-fluoropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione (500 mg, 2.32 mmol) under nitrogen protection, and phosphorus oxychloride (4.96 mL, 53.35 mmol) was added to the system under ice bath, and then the system was placed at 50 °C to react for 3 h.
  • the product of the previous step was added to a single-mouth bottle with 90 mL of 1,4-dioxane, followed by N,N'-carbonyldiimidazole (17.61 g, 108.60 mmol) and DIEA (25.22 mL, 144.80 mmol), and refluxed at 120°C for 16 h.
  • the reaction was complete after TLC monitoring.
  • the product was cooled to room temperature to precipitate crystals, which were filtered and washed with DCM.
  • the filter cake was dried to obtain the product Y46a (white solid, 7.20 g, yield 52%).
  • Y69c 150 mg, 784.56 ⁇ mol
  • potassium carbonate 110.60 mg, 800.25 ⁇ mol
  • 2-bromoethanol 111 ⁇ L, 1.58 mmol
  • water and ethyl acetate were added to the system for extraction.
  • the ethyl acetate was washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, and the product Y69d (white solid, 103 mg, yield 56%) was obtained by column chromatography.
  • Y69d (85.68 mg, 364.24 ⁇ mol) was dissolved in 5 mL of dry tetrahydrofuran, and then sodium hydride (14.57 mg, wt 60%, 364.24 ⁇ mol) was slowly added under ice bath. After 10 min, 5 mL of tetrahydrofuran solution of Y69b (130 mg, 303.53 ⁇ mol) was added, and the mixture was moved to room temperature for 16 h. TLC monitored the complete consumption of Y69b. Water and ethyl acetate were added to the reaction system for extraction, and the mixture was washed with saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate.
  • Y69e was dissolved in 3 mL of toluene, 500 ⁇ L of water was added, potassium phosphate (101 mg, 478.41 ⁇ mol) and ((2-fluoro-6-(methoxymethoxy)-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)ethynyl)triisopropylsilane (90 mg, 175.42 ⁇ mol) were added, and finally methanesulfonic acid (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium (II)) (13.4 mg, 15.95 ⁇ mol) was added. The mixture was ventilated three times, the reaction was monitored by TLC, the mixture was directly spin-dried, and column chromatography was performed to
  • Y69f (52 mg) was dissolved in dry N,N-dimethylformamide, and then cesium fluoride was added. The reaction was allowed to react at room temperature for 1 h. The reaction was monitored by TLC. Water and ethyl acetate were added and extracted three times. The ethyl acetate layer was washed with a saturated sodium chloride aqueous solution and dried over anhydrous sodium sulfate. The organic layer was dried by spin drying. Then, a hydrochloric acid dioxane solution was added thereto in an ice bath under nitrogen protection. The reaction was monitored by TLC. The compound Y69 (yellow solid, 8 mg) was obtained by preparative separation.
  • Y46a 1.0 g, 5.23 mmol
  • methyl 3-hydroxypropionate 816.8 mg, 7.85 mmol
  • triphenylphosphine 2.7 g, 10.46 mmol
  • DIAD 2.0 mL, 10.46 mmol
  • Y82b (1.58 g, 5.69 mmol) was dissolved in 10 mL of methanol, and 10 mL of water and lithium hydroxide monohydrate (716.4 mg, 17.07 mmol) were added. The mixture was stirred at room temperature overnight. The methanol was removed by rotary evaporation and the pH was adjusted to 4 with 1N dilute hydrochloric acid. A white solid was precipitated and Y82c (white solid, 514.1 mg, yield 34.7%) was obtained by suction filtration.
  • KI-ARV-03 (20.0 mg, 0.08 mmol) was dissolved in 1 mL of dichloromethane, and HATU (44.0 mg, 0.12 mmol), DIPEA (0.04 mL, 0.23 mmol), and Y82c (24.4 mg, 0.09 mmol) were added, and the mixture was stirred at room temperature for 3 hours.
  • Y82 (colorless oil, 8 mg, yield 17.6%) was obtained by silica gel column chromatography.
  • Y87b (1.0 g, 3.90 mmol) was dissolved in 20 mL DMF, and K 2 CO 3 (1.1 g, 7.90 mmol) and 2,2'-dibromodiethyl ether (3.0 mL, 23.60 mmol) were added in sequence. The reaction was carried out at room temperature for about 5 hours. After the reaction was complete, water and ethyl acetate were added. The ethyl acetate layer was washed with water 3 times, then washed with saturated brine, and dried over anhydrous sodium sulfate. Silica gel column chromatography gave compound Y87c (oily liquid, 67.0 mg, yield 10.6%).
  • Y87c 136.0 mg, 0.34 mmol was dissolved in 2 mL DMF, and C24a (168.0 mg, 0.34 mmol) and K 2 CO 3 (93.0 mg, 0.67 mmol) were added, and the mixture was reacted at 60°C for about 4 hours. After the reaction was completed as monitored by TLC, the protein degradation agent Y87 (white solid, 7.0 mg, yield 2.6%) was obtained by silica gel column chromatography separation and purification.
  • Y46a (300.0 mg, 1.57 mmol), methyl 4-hydroxycyclohexanecarboxylate (496.5 mg, 3.14 mmol), and triphenylphosphine (823.1 mg, 3.14 mmol) were dissolved in 4 mL of tetrahydrofuran. Under nitrogen protection, DIAD (0.6 mL, 3.14 mmol) was added dropwise at 0°C. After the addition was completed, the mixture was kept at room temperature overnight. The tetrahydrofuran was removed by rotary evaporation, and Y95a (white solid, 455.4 mg, yield 87.6%) was obtained.
  • Y95a (455.4 mg, 1.37 mmol) was dissolved in 2.5 mL of methanol and 2.5 mL of water, and lithium hydroxide (173.0 mg, 4.12 mmol) was added. The mixture was stirred overnight at room temperature, and the organic solvent was removed by rotary evaporation. Water was added to dissolve the mixture, and the pH was adjusted to 4-5 with 1N hydrochloric acid. A white solid was precipitated and filtered to obtain Y95b (white solid, 315.6 mg, yield 72.4%).
  • Y95b (50.0 mg, 0.16 mmol), compound 754 (63.5 mg, 0.16 mmol), HATU (71.9 mg, 0.19 mmol) and DIPEA (0.04 mL, 0.24 mmol) were dissolved in 1 mL of dichloromethane, stirred at room temperature overnight, and subjected to silica gel column chromatography to obtain Y95 (white solid, 57.0 mg, yield 51.5%).
  • Synthesis method Refer to Example 46, replace compound C24a with compound 754, replace 1,2-ethylene glycol with 4-hydroxycyclohexanone, omit the second step of Example 46, i.e., the step of converting the hydroxyl group into the aldehyde group, to obtain compound Y100 (white solid, yield 6.1%).
  • Example 46 C24a is replaced by compound 754, 1,2-ethylene glycol is replaced by 3-(hydroxymethyl)cyclobutan-1-one, and the second step of Example 46, i.e., the step of converting the hydroxyl group to the aldehyde group, is omitted to obtain compound Y102 (white solid, yield 19.3%).
  • Y46a (1.0 g, 5.23 mmol), cis-4-(Boc-amino)cyclohexanol (1.4 g, 6.28 mmol), and triphenylphosphine (2.7 g, 10.46 mmol) were dissolved in 13 mL of anhydrous tetrahydrofuran, and DIAD (2 mL, 10.46 mmol) was added dropwise under ice bath. After the addition was completed, the mixture was allowed to stand at room temperature overnight, and the organic solvent was removed by rotary evaporation. The mixture was subjected to silica gel column chromatography to obtain crude Y105a (white solid, 2.7 g).
  • Y105a (2.7 g, 7.04 mmol) was dissolved in 18 mL of dichloromethane, and 8.8 mL of 4M HCl in ethyl acetate was added. The mixture was stirred at room temperature overnight to obtain Y105b (white solid, 428.3 mg).
  • the solvent was spin-dried, toluene, Y105b (425.0 mg, 1.47 mmol), and triethylamine (0.4 mL, 2.95 mmol) were added, and the reaction was carried out at 100°C for 4 hours, and water was added, and the mixture was extracted with ethyl acetate, and the mixture was spin-dried and subjected to silica gel column chromatography to obtain Y105c (white solid, 550.0 mg, yield 65.6%).
  • Y105c (200.0 mg, 0.35 mmol), 5,5-dimethyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole (110.6 mg, 0.42 mmol), Pd(dppf)Cl 2 (14.4 mg, 0.02 mmol), potassium carbonate (721.5 mg, 0.88 mmol) were dissolved in 2 mL of dioxane and 2 mL of water, and stirred at 60°C overnight.
  • the positive control compounds used were SNS-032 and THAL-SNS-032, and the structures were as follows.
  • CDK can combine with cyclin to form heterodimers, in which CDK is the catalytic subunit and cyclin is the regulatory subunit.
  • CDK is the catalytic subunit
  • cyclin is the regulatory subunit.
  • Polycomb repressive complex 2 is a histone methyltransferase complex that includes three core subunits: EZH2 (or EZH1), EED, and SUZ12. Dysregulation of PRC2 is associated with the occurrence, progression, and poor prognosis of hematological and solid tumors.
  • EZH2 is generally considered to be the main catalytic subunit of the PRC2 complex, which catalyzes the trimethylation modification of histone H3 lysine 27 (H3K27me3) through its SET domain, thereby maintaining the silencing state of downstream target genes.
  • EZH2 is overexpressed or has gain-of-function mutations in many cancers, and the role of EZH2 inhibitors in cancer treatment has been clinically verified.
  • WSU-DLCL2 cells, U-2932 cells or GP2D cells human colon cancer cells
  • Appropriate amount of WSU-DLCL2 cells, U-2932 cells or GP2D cells were plated in 6-well plates, treated with drugs (see the corresponding Figures 1-7 for drugs and drug concentrations), and then centrifuged to collect cells for subsequent experiments.
  • the corresponding amount of 1X loading buffer (ingredients: 50mM Tris-HCI (pH6.8), 2% (W/V) SDS, 0.1% (W/V) BPB (bromophenol blue), 10% (V/V) glycerol, 0.1M ⁇ -mercaptoethanol) was added, and the samples were boiled at 100°C for 20min after lysis.
  • CDK9 is mainly involved in the transcriptional regulation process.
  • the heterodimer composed of CDK9 and cyclin (T1, T2a, T2b, K) participates in the formation of positive transcription elongation factor (P-TEFb).
  • CDK9 has two subtypes (CDK9 42 and CDK9 55), and about 80% of CDK9 binds to cyclin T1.
  • compounds Y44, Y45, Y41, Y35, and Y33 can degrade two subtypes of CDK9, CDK9 55 and CDK9 42, at a concentration of 100 nM, and can also degrade cyclin T1 and downregulate the level of CDK9 downstream protein Mcl-1.
  • the CDK9 inhibitor SNS-032 does not have the effect of protein degradation.
  • the positive control THAL-SNS-032 can degrade CDK9, but cannot degrade cyclin T1 or downregulate the level of Mcl-1.
  • Figure 1B shows that at a concentration of 1 ⁇ M, compounds Y1-Y5 can effectively degrade CDK9 and cyclin T1 and downregulate Mcl-1 levels.
  • Figure 2B shows that at a concentration of 100 nM, compound Y35 can effectively induce the degradation of CDK9 and cyclin T1 and downregulate the level of CDK9 downstream protein Mcl-1; when cells are co-treated with Y35 and the autophagy inhibitor bafilomycin A1 (BafA1), the degradation effect of Y35 on CDK9 and cyclin T1 disappears.
  • Figure 3 shows that at a concentration of 100 nM, compounds Y44, Y82-Y86 can degrade CDK9, among which Y44, Y83 and Y84 can also degrade cyclin T1 while degrading CDK9, thereby downregulating the level of Mcl-1.
  • Figure 4A shows that compared with the CDK2 inhibitor SY-5609a, the degrader Y53 can effectively degrade CDK2.
  • Figure 4B shows that compound Y67 can degrade the levels of CDK7 and cyclin H at a concentration of 10 ⁇ M.
  • Figure 4C shows that compound Y62 can degrade CDK2 and CDK6 and their corresponding cyclinA2/E1/D1 at a concentration of 10 ⁇ M.
  • Figure 4D shows that compounds Y59-Y61 can degrade CDK2/4/6 and their corresponding cyclinA2/E1/D1 at a concentration of 10 ⁇ M.
  • Figure 5A shows that compounds Y47 and Y50 can degrade EZH2, EED, SUZ12, and EZH1, and the effect is stronger than the positive control MS177 (CAS No.: 2225938-86-1, EZH2 small molecule degrader), and the EZH2 inhibitor C24 does not show degradation.
  • Figure 5B shows that at a concentration of 10 ⁇ M, Y50, Y87-Y89 can effectively degrade EZH2, EED, SUZ12, and EZH1, and the effect is stronger than the positive control MS177.
  • FIG6 shows that compound Y69 can degrade Ras and downregulate the level of pERK at a concentration of 10 ⁇ M.
  • Figure 7A shows that at a concentration of 100 nM, compounds Y83, Y95, and Y96 can effectively induce the degradation of multiple proteins in CDK2, cyclin A2, cyclin E1, CDK5, CDK6, cyclin D1, CDK7, CDK9, and cyclin T1.
  • Figure 7B shows that at a concentration of 100 nM, compounds Y84, Y100-Y106 can effectively induce the degradation of multiple proteins in CDK2, cyclin A2, cyclin E1, CDK4, CDK5, CDK6, cyclin D1, CDK7, CDK9, and cyclin T1.
  • WSU-DLCL2 cells were incubated with 5% paraformaldehyde supplemented with phosphatase inhibitors (purchased from Roche, catalog number: 4906845001) and protease inhibitor cocktail (purchased from Roche, catalog number: 04693132001) NP-40 (purchased from Beyotime, catalog number: P0013F) was lysed on ice for 1 hour and centrifuged at 12000g for 10 minutes at 4°C.
  • phosphatase inhibitors purchasedd from Roche, catalog number: 4906845001
  • protease inhibitor cocktail purchased from Roche, catalog number: 04693132001
  • NP-40 purchased from Beyotime, catalog number: P0013F
  • the protein concentration in the supernatant was measured using a BCA protein assay kit (Thermo Scientific, 23225), and equal amounts of protein were incubated with primary antibodies (Normal Rabbit IgG (purchased from Cell Signaling Technology, catalog number: 2729), CDK9 rabbit mAb (purchased from Abclonal, catalog number: A11145) at 4°C overnight, and then protein A/G magnetic beads (purchased from Thermo Scientific, catalog number: 88803) were added and incubated at 4°C for another 4 hours.
  • the immunoprecipitate was washed 3 times with NP-40 and PBS, then boiled with SDS-PAGE loading buffer and subjected to immunoblotting.

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Abstract

L'invention concerne un agent de dégradation de protéine, une composition pharmaceutique et une utilisation associées. La structure telle que représentée dans la formule (A) est utilisée en tant que composé de recrutement de LC3B, et un agent de dégradation de protéine est obtenu au moyen de la liaison de la structure telle que représentée dans la formule (A), en particulier une structure de 2,4-quinazolinedione avec un ligand d'une protéine cible, qui permettent la dégradation de l'autophagie de la protéine cible, génèrent un effet biologique, et facilitent le traitement et la prévention de maladies associées. L'agent de dégradation de protéine peut être utilisé pour traiter, prévenir et/ou améliorer des maladies associées à CDK, EZH2 ou RAS.
PCT/CN2024/099218 2023-06-14 2024-06-14 Agent de dégradation de protéine, composition pharmaceutique et utilisation associées Pending WO2024255845A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190134056A1 (en) * 2017-03-10 2019-05-09 The Trustees Of The Stevens Institute Of Technolog K-ras mutations and antagonists
CN110036004A (zh) * 2016-07-13 2019-07-19 希洛斯医药品股份有限公司 细胞周期蛋白依赖性激酶7(cdk7)的抑制剂
WO2020047487A1 (fr) * 2018-08-31 2020-03-05 The Regents Of The University Of California Méthodes de traitement du cancer à l'aide d'inhibiteurs rorgamma et de statines
US20200231551A1 (en) * 2017-06-26 2020-07-23 University Of Virginia Patent Foundation Compositions and uses thereof
CN115785199A (zh) * 2021-09-10 2023-03-14 润佳（苏州）医药科技有限公司 一种双官能化合物及其用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110036004A (zh) * 2016-07-13 2019-07-19 希洛斯医药品股份有限公司 细胞周期蛋白依赖性激酶7(cdk7)的抑制剂
US20190134056A1 (en) * 2017-03-10 2019-05-09 The Trustees Of The Stevens Institute Of Technolog K-ras mutations and antagonists
US20200231551A1 (en) * 2017-06-26 2020-07-23 University Of Virginia Patent Foundation Compositions and uses thereof
WO2020047487A1 (fr) * 2018-08-31 2020-03-05 The Regents Of The University Of California Méthodes de traitement du cancer à l'aide d'inhibiteurs rorgamma et de statines
CN115785199A (zh) * 2021-09-10 2023-03-14 润佳（苏州）医药科技有限公司 一种双官能化合物及其用途

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