WO2024255845A1 - Protein degradation agent, and pharmaceutical composition and use thereof - Google Patents

Abstract

A protein degradation agent, and a pharmaceutical composition and the use thereof. The structure as shown in formula (A) is used as an LC3B recruitment compound, and a protein degradation agent is obtained by means of connecting the structure as shown in formula (A), particularly a 2,4-quinazolinedione structure with a ligand of a target protein, which enable the autophagy degradation of the target protein, generate a biological effect, and facilitate the treatment and prevention of related diseases. The protein degradation agent can be used for treating, preventing and/or improving diseases associated with CDK, EZH2, or RAS.

Description

A class of protein degrading agents and pharmaceutical compositions and uses thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application No. 202310710665.0, filed on June 14, 2023, entitled “A class of protein degraders, pharmaceutical compositions and uses thereof”, the entire contents of which are incorporated herein by reference in their entirety to the same extent as if fully set forth herein.

Technical Field

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.

Technical Background

Targeted protein degradation is a very promising tool for biological mechanism research and treatment. By directly degrading the target protein, 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. In the past two decades, PROTAC technology has made great progress, and several PROTAC drug candidates have entered clinical research. However, 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.

Therefore, there is still a need to develop novel protein degradation technologies for degrading target proteins.

Summary of the invention

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. Similarly, 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. Therefore, 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.

In view of this, 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.

In order to achieve the above-mentioned purpose of the present invention, the following technical scheme is adopted:

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:

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); and

A linker is used to covalently link the LCBM part to the POIL part independently.

In the present invention, 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.

In a specific embodiment, 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-Linker-POIL(1);

in:

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.

In some embodiments, 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. When there are more than one POIL part, the POIL parts can be selected independently, and the POIL parts can be the same or different. In some embodiments, there are multiple POIL parts for the same target. Even for the same target, multiple identical or different POIL parts can be used in one molecule as needed. When more than one POIL part is used, the linkers used can also be selected independently.

LCBM part

This portion means a portion that can bind to LC3 and refers to a portion that has affinity for the LC3 protein.

In some embodiments, the LCBM moiety is of formula (A),

in:

^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 or substituted 5-15 membered heteroaryl C1-C6 alkyl, wherein the substitution means that one or more hydrogen atoms in the defined group are replaced by one or more substituents selected from halogen, amino, hydroxyl, C1-C3 alkyl (e.g., methyl), C1-C3 alkoxy (e.g., methoxy), and trifluoromethyl;

Indicates connecting to the linker from here.

In some embodiments, in formula (A), 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 ), hydroxyl (-OH).

In some embodiments, in formula (A), ^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 or substituted 5-10 membered heteroarylC1-C6 alkyl, wherein the substitution means that one or more hydrogen atoms in the defined group are replaced by one or more substituents selected from halogen, amino, hydroxyl, C1-C3 alkyl (e.g. methyl), C1-C3 alkoxy (e.g. methoxy), and trifluoromethyl.

Herein, the heteroaryl group and the heterocyclic group may contain 1 to 4, for example 1 to 2, heteroatoms selected from N, O and S.

In some embodiments, the structure represented by formula (A) is selected from the structure represented by the following formula (A-1):

Wherein, 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. methyl), C1-C3 alkoxy (e.g. methoxy), trifluoromethyl; preferably, 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.

In some embodiments, the structure represented by formula (A) is selected from the following structures:

POIL part

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.

In some embodiments, the POIL moiety is capable of binding to CDK9.

In some embodiments, the POIL moiety is capable of binding to CDK7.

In some embodiments, the POIL moiety is capable of binding to CDK2.

In some embodiments, the POIL moiety is capable of binding to CDK2, CDK4, and CDK6 simultaneously.

In some embodiments, the POIL moiety is capable of binding to CDK5.

In some embodiments, the POIL moiety is capable of binding to EZH2.

In some embodiments, the POIL moiety is capable of binding to EZH2, EED, SUZ12, and EZH1 simultaneously.

In some embodiments, the POIL moiety is capable of binding to RAS.

In some embodiments, 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

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.

In some embodiments, the linker is a chemical bond. Herein, the linker is a chemical bond, which means that the LCBM part and the POIL part are directly connected.

In other embodiments, 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. 2, 3, 4, 5, 6) 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.

In some embodiments, the Linker is selected from:

-(CH ₂ ) _n -, -(CH ₂ CH ₂ O) _m -(CH ₂ ) _n -, -CO-(CH ₂ ) _n -,

wherein each 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.

In some embodiments, the linker is selected from the following structures:

In some embodiments, 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. Those skilled in the art can select a suitable reaction for the connection according to the structures of the three parts.

In some embodiments, the protein degrading agent is selected from any of the following structures:

wherein each 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.

In some embodiments, the protein degrader is a degrader of CDK9, cyclin T1 and/or CDK9-cyclin T1 complex, selected from the following structures:

In some embodiments, 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:

In some embodiments, 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:

The definitions of terms in this invention are as follows:

As used herein, "halogen" may be fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

As used herein, the term "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.

As used herein, the term "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. Specific examples of alkoxy 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.

As used herein, the term "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.

As used herein, the term "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.

As used herein, the term "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.

As used herein, 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". Specific examples include 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, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl, quinolyl, isoquinolyl, purinyl, imidazopyridinyl, imidazopyrimidinyl, imidazopyrazinyl, imidazopyridazinyl, imidazotriazinyl, pyrazolopyridinyl, pyrazolopyrimidinyl, pyrazolopyrazinyl, pyrazolotriazinyl, pyrrolopyridinyl, pyrrolopyrimidinyl, pyrrolopyridazinyl, pyrrolopyrazinyl and pyrrolotriazinyl, etc., preferably pyrazolyl, pyridinyl, pyridazinyl, pyrazinyl, pyrimidinyl.

As used herein, "pharmaceutically acceptable 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, I) alkali metal salts, alkaline earth metal salts, ammonium salts of compounds; preferably, the alkali metal includes sodium, potassium, lithium, cesium, and the alkaline earth metal includes magnesium, calcium, strontium. 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; preferably, the trialkylamine includes trimethylamine, triethylamine, N,N-diisopropylethylamine; preferably, 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. As used herein, "isomers" refer to different compounds having the same molecular formula but differing in the arrangement and configuration of the atoms. As used herein, "stereoisomers" include diastereomers, enantiomers and racemates, geometric isomers, conformational isomers (including rotational isomers and atropisomers).

In another aspect, 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.

In some embodiments, 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. For example, 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. However, 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.

In another aspect, the present invention provides 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. In addition, 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. 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). , 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) and base wax (e.g., cocoa butter, white petrolatum or polyethylene glycol).

The dosage regimen can be adjusted to provide the best desired response. For example, when the drug is administered in the form of an injection, a single push, a bolus injection, and/or a continuous infusion, etc., can be administered. For example, 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. For example, several divided doses can be administered over time, or the dose can be proportionally reduced or increased depending on the treatment situation. It should be noted that the dosage value can vary with the type and severity of the condition to be alleviated, and can include single or multiple doses. Generally, 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. For example, 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 or corresponding pathway research.

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.

The above-mentioned 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.; Allergies, pain, fever; 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. ; and complications associated with 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 allograft rejection; bone resorption diseases; osteoporosis; multiple sclerosis; cancer: such as leukemia, lymphoma, colorectal cancer, brain cancer, bone cancer, tumors of epithelial cell origin (epithelial cancer), basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, oral cancer, esophageal cancer, small Intestinal cancer, stomach cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer, squamous cell and/or basal cell carcinoma, prostate cancer, renal cell carcinoma and other cancers known to affect epithelial cells throughout the body, chronic myeloid leukemia (CML), acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL); Central nervous system diseases: including central nervous system diseases with inflammatory or apoptotic components, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury and peripheral neuropathy, or B-cell lymphoma, etc.

In addition, by regulating the amount of intracellular CDK, EZH2, or RAS through LC3-mediated autophagic degradation by the protein degraders of the present application (such as those described herein), 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 has the following beneficial effects:

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.

The present invention has been described in detail above, but the above embodiments are only illustrative in nature and are not intended to limit the present invention. In addition, this article is not limited by any theory described in the above prior art or invention content or the following examples.

BRIEF DESCRIPTION OF THE DRAWINGS

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 1μM compounds Y1, Y2, Y3, Y4, and Y5.

FIG2A shows that representative compounds Y2 and Y3 induce the binding of CDK9 and LC3B, and 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 (0.1μM, 1μM, 10μM); Figure 4B shows the degradation effect of compound Y67 on CDK7 and the corresponding cyclin H at a concentration of 10μM; 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 10μM.

FIG5A shows the degradation effects of compounds Y47 and Y50 on EZH2, EED, SUZ12 complex and EZH1 at a concentration of 10 μM; FIG5B shows the degradation effects of compounds Y50, Y87-Y89 on EZH2, EED, SUZ12 complex and EZH1 at a concentration of 10 μM.

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.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with examples. It should be noted that the following examples are provided for illustrative purposes only and do not constitute a limitation on the scope of protection claimed for the present invention.

Unless otherwise specified, the raw materials, reagents, methods, etc. used in the examples are conventional raw materials, reagents, and methods in the art. Dosage and method.

In the following examples, the nuclear magnetic resonance hydrogen spectrum was recorded by a Bruker AMX-400 nuclear magnetic resonance instrument, and the unit of chemical shift δ 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. and China Pharmaceutical (Group) Shanghai Chemical Reagent Co., Ltd., respectively. Unless otherwise specified, all solvents are analytically pure reagents, and the reagents used are purchased from Sinopharm Chemical Reagent Co., Ltd. Iodine, ultraviolet fluorescence and other methods were used for color development. The organic solvent was removed by evaporation under reduced pressure in a rotary evaporator.

Example 1 Synthesis of protein degradation agent Y1

first step

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. The acetonitrile was removed by vortexing and the mixture was dissolved in ethyl acetate. The ethyl acetate phase was then washed twice with 1 M hydrochloric acid and once with water, dried over anhydrous sodium sulfate, and the ethyl acetate was removed by vortexing. The mixture was slurried with ether to obtain Y1a (white solid, 1.5 g, yield 77.8%). ^1H NMR(400MHz,Chloroform-d)δ12.90(s,1H),8.12(d,J＝8.5Hz,1H),7.58–7.51(m,2H),7.36–7 .24(m,4H),7.20(d,J＝1.8Hz,1H),5.46(s,2H),4.27(q,J＝7.1Hz,2H),1.39(t,J＝7.1Hz,3H).

Step 2

Dissolve 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%). ¹ H NMR (400 MHz, Chloroform-d) δ9.27 (s, 1H), 8.09 (d, J = 8.5 Hz, 1H), 7.53 (d, J = 7.4 Hz, 2H), 7.37–7.30 (m, 3H), 7.21 (dd, J = 8.6, 1.8 Hz, 1H), 7.07 (d, J = 1.8 Hz, 1H), 5.26 (s, 2H).

Step 3

Y1b (50 mg, 0.17 mmol) was dissolved in 1 mL of DMF, and 1,2-dibromoethane (60 μL, 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%). ¹ H NMR (400 MHz, Chloroform-d) δ8.21 (d, J = 8.4 Hz, 1H), 7.55-7.49 (m, 2H), 7.36-7.23 (m, 5H), 5.26 (s, 2H), 4.47 (t, J = 7.5 Hz, 2H), 3.61 (t, J = 7.5 Hz, 2H).

Step 4

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 μL, 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%). ^1H NMR(400MHz,Chloroform-d)δ11.40(s,1H),8.19(d,J＝8.5Hz,1H),7.55–7.47( m,2H),7.39(s,1H),7.36–7.30(m,3H),7.28–7.21(m,2H),6.61(s,1H),5.27(s ,2H),4.26(t,J＝6.9Hz,2H),3.99(s,2H),3.09(d,J＝11.1Hz,2H),2.69(t,J＝6. 9Hz,2H),2.45–2.34(m,1H),2.26–2.15(m,2H),1.94–1.80(m,4H),1.26(s,9H).

Example 2 Synthesis of protein degradation agent Y2

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%). ^1H NMR(400MHz,Chloroform-d)δ11.30(s,1H),8.18(d,J＝8.5Hz,1H),7.56–7.50(m, 2H),7.44(d,J＝1.8Hz,1H),7.36(s,1H),7.35–7.30(m,2H),7.29–7.25(m,1H),7. 22(dd,J＝8.5,1.7Hz,1H),6.61(s,1H),5.27(s,2H),4.18(t,J＝7.2Hz,2H),3.97( s,2H),3.02(d,J=10.5Hz,2H),2.51–2.39(m,3H),2.03–1.88(m,8H),1.26(s,9H).

Example 3 Synthesis of protein degradation agent Y3

The synthesis method was the same as that in Example 1, except that 1,2-dibromoethane was replaced with 1,4-dibromobutane to obtain protein degradation agent Y3 (white solid, yield 38.3%). ¹ H NMR (400 MHz, Chloroform-d) δ11.57 (s, 1H), 8.19 (d, J = 8.5 Hz, 1H), 7.54–7.46 (m, 2H), 7.36 (s, 1H), 7.34–7.25 (m, 4H), 7.22 (dd, J = 8.4, 1.7 Hz, 1H), 6.61 (s, 1H), 5.27 (s, 2H),4.18–4.11(m,2H),3.98(s,2H),3.04(d,J＝11.1Hz,2H),2.56–2.39(m,3H),2.18 –2.03(m,2H),2.01–1.90(m,4H),1.84–1.73(m,2H),1.72–1.62(m,2H),1.26(s,9H).

Example 4 Synthesis of protein degradation agent Y4

The synthesis method was the same as that in Example 1, except that 1,2-dibromoethane was replaced with 1,5-dibromopentane to obtain protein degradation agent Y4 (white solid, yield 47.0%). ¹ H NMR (400 MHz, Chloroform-d) δ11.26 (s, 1H), 8.19 (d, J = 8.4 Hz, 1H), 7.54–7.49 (m, 2H), 7.36 (s, 1H), 7.35–7.26 (m, 3H), 7.23 (dd, J = 8.4, 1.7 Hz, 1H), 7.17 (d, J = 1.8 Hz, 1H), 6.61 (s, 1H), 5.27 (s ,2H),4.13–4.06(m,2H),3.98(s,2H),3.03(d,J＝11.3Hz,2H),2.47–2.36(m,3H),2.14–2.00(m ,2H),1.98–1.87(m,4H),1.81–1.71(m,2H),1.68–1.56(m,2H),1.51–1.38(m,2H),1.26(s,9H).

Example 5 Synthesis of protein degradation agent Y5

The synthesis method is the same as that in Example 1, except that 1,2-dibromoethane is replaced with triethylene glycol bis-p-toluenesulfonate to obtain protein degradation agent Y5 (white solid, yield 69.8%). ¹ H NMR (400 MHz, Chloroform-d) δ11.64 (s, 1H), 8.15 (d, J = 8.4 Hz, 1H), 7.53–7.48 (m, 3H), 7.35 (s, 1H), 7.34–7.24 (m, 3H), 7.20 (dd, J = 8.5, 1.7 Hz, 1H), 6.60 (s, 1H), 5.31–5.30 (m, 1H), 5.26 (s, 2H), 4.30 (t, J＝5.5Hz,2H),3.97(s,2H),3.82(t,J＝5.5Hz,2H),3.64–3.60(m,2H),3.59–3.53(m,4H),3.03–2.96( m,2H),2.56(t,J＝5.8Hz,2H),2.42–2.31(m,1H),2.12–2.02(m,2H),1.97–1.79(m,4H),1.25(s,9H).

Example 6 Synthesis of protein degradation agent Y6

first step

4-Chloroisocyanic anhydride (500 mg, 2.53 mmol) and aniline (231 μL, 2.53 mmol) were dissolved in 13 mL of dichloromethane, refluxed for 30 minutes, N,N′-carbonyldiimidazole (CDI, 451 mg, 2.78 mmol) was added, refluxed for 3 hours, dichloromethane was dried by spin drying, ethanol was added, slurried, and filtered to obtain Y6a (white solid, 230 mg, yield 33.3%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.70 (s, 1H), 7.94 (d, J＝8.4 Hz, 1H), 7.52–7.40 (m, 3H), 7.35–7.22 (m,4H).

Step 2

Y6a (230 mg, 0.84 mmol) and 1,2-dibromoethane (364 μL, 4.22 mmol) were dissolved in 4 mL DMF, potassium carbonate (234 mg, 1.69 mmol) was added, and 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%). ¹ H NMR (400 MHz, Chloroform-d) δ8.23 (d, J＝8.4 Hz, 1H), 7.59–7.46 (m, 3H), 7.37–7.26 (m, 4H), 4.51 (t, J＝7.4 Hz, 2H), 3.67 (t, J＝7.4 Hz, 2H).

Step 3

Y6b (85 mg, 0.22 mmol) was dissolved in 1 mL of DMF, and SNS-032 (85 mg, 0.22 mmol) and DIPEA (111 μL, 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)δ10.98(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. 44(s,1H),4.19–4.08(m,2H),3.81(s,2H),2.99–2.88(m,2H),2.63–2.52(m ,2H),2.30–2.17(m,1H),2.11–1.98(m,2H),1.80–1.66(m,4H),1.09(s,9H).

Example 7 Synthesis of protein degradation agent Y7

The synthesis method was the same as that in Example 6, except that aniline was replaced with phenylethylamine to obtain protein degradation agent Y7 (white solid, yield 35.5%). ^1H NMR(400MHz,Chloroform-d)δ10.75(s,1H),8.19(d,J＝8.4Hz,1H),7.38(s,1H),7.36–7.30(m,5H),7.27–7.22(m,2H),6.61(s,1H),4.36–4.20(m ,4H),3.98(s,2H),3.13(d,J＝10.9Hz,2H),3.03–2.96(m,2H),2.75–2.64 (m,2H),2.43(s,1H),2.31–2.19(m,2H),2.01–1.86(m,4H),1.27(s,9H).

Example 8 Synthesis of protein degradation agent Y8

The synthesis method was the same as that in Example 6, except that aniline was replaced with 4-methoxybenzylamine to obtain protein degradation agent Y8 (white solid, yield 35.1%). ¹ H NMR (400 MHz, Chloroform-d) δ11.36 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.53–7.46 (m, 2H), 7.38 (s, 1H), 7.32–7.30 (m, 1H), 7.22 (dd, J = 8.4, 1.7 Hz, 1H), 6.91–6.83 (m, 2H),6.61(s,1H),5.21(s,2H),4.31–4.21(m,2H),3.99(s,2H),3.79(s,3H),3.11(d,J＝11.0Hz ,2H),2.73–2.66(m,2H),2.47–2.35(m,1H),2.28–2.16(m,2H),1.96–1.84(m,4H),1.26(s,9H).

Example 9 Synthesis of protein degradation agent Y9

The synthesis method was the same as that of Example 6, except that aniline was replaced with 4-(trifluoromethyl)benzylamine to obtain protein degradation agent Y9 (white solid, yield 30.1%). ^1H NMR(400MHz,Chloroform-d)δ11.10(s,1H),8.19(d,J＝8.5Hz,1H),7.65–7.57( m,4H),7.36(s,1H),7.34(s,1H),7.26(dd,J＝8.5,1.7Hz,1H),6.61(s,1H),5.3 2(s,2H),4.26(t,J＝7.0Hz,2H),3.99(s,2H),3.10(d,J＝11.1Hz,2H),2.74–2.6 6(m,2H),2.47–2.35(m,1H),2.27–2.15(m,2H),1.95–1.83(m,4H),1.26(s,9H).

Example 10 Synthesis of protein degradation agent Y10

The synthesis method was the same as in Example 6, except that aniline was replaced with 4-methylaminopyridine to obtain protein degradation agent Y10 (white solid, yield 11.0%). ¹ H NMR (400 MHz, Methanol-d ₄ )δ8.53–8.48(m,2H),8.17(d,J＝8.5Hz,1H),7.64(d,J＝1.8Hz,1H),7.46–7.42 (m,2H),7.36(dd,J＝8.5,1.7Hz,1H),7.33(s,1H),6.70(s,1H),5.30(s,2H),4 .37(t,J＝6.7Hz,2H),4.01(s,2H),3.13(d,J＝11.7Hz,2H),2.72(t,J＝6.7Hz,2 H), 2.54–2.42 (m, 1H), 2.20 (t, J = 11.2Hz, 2H), 1.90–1.72 (m, 4H), 1.25 (s, 9H).

Example 11 Synthesis of protein degradation agent Y11

The synthesis method was the same as that in Example 6, except that aniline was replaced with 3-aminomethylpyridine to obtain protein degradation agent Y11 (white solid, yield 11.4%). ¹ H NMR (400 MHz, Methanol-d ₄ )δ8.69(d, J＝2.1 Hz, 1H),8.46(d, J＝4.9 Hz, 1H),8.18(d, J＝8.5 Hz, 1H),7.96(d, J＝7.8 Hz, 1H),7.61(d, J＝1.7 Hz, 1H),7.44(dd, J＝8.0,5.0 Hz, 1H),7.37–7.32(m, 2H),6.69(s, 1H),5.29(s, 2H),4.36(t, J＝6.6 Hz, 2H),4.01(s, 2H), 3.10(d,J＝11.4Hz,2H),2.70(t,J＝6.6Hz,2H),2.53–2.42(m,1H),2.19(t,J＝11.6Hz,2H),1.87–1.81(m,2H),1.79–1.68(m,2H),1.26(s,9H).

Example 12 Synthesis of protein degradation agent Y12

The synthesis method was the same as that in Example 6, except that aniline was replaced with 2-aminomethylpyridine to obtain degradation agent Y12 (white solid, yield 21.2%). ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.21(s,1H),8.46–8.41(m,1H),8.06(d,J＝8.4Hz,1H),7.75(td,J＝7.7,1.8Hz,1H),7.71(d,J＝1.8Hz,1H),7.41–7.35(m,2H),7.31(d,J＝7.9Hz,1H),7.26–7.21(m,1H),6.73(s,1H),5.25 (s,2H),4.26(t,J＝6.3Hz,2H),4.06(s,2H),2.98(d,J＝10.9Hz,2H),2.57(t,J＝6.3Hz,2H),2.4 8–2.38(m,1H),2.01(t,J＝11.4Hz,2H),1.72(d,J＝12.5Hz,2H),1.57–1.45(m,2H),1.17(s,9H).

Example 13 Synthesis of protein degradation agent Y13

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%).

Example 14 Synthesis of protein degradation agent Y14

The synthesis method of Y14a is the same as that of Example 6, except that aniline in the first step is replaced by 2,4-dimethoxybenzylamine to obtain Y14a (white solid, yield 46.4%). ¹ H NMR (400 MHz, Chloroform-d) δ10.64 (s, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.37 (s, 1H), 7.33 (d, J = 1.7 Hz, 1H), 7.24 (dd, J = 8.4, 1.7 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.61 (s, 1H), 6.47 (d, J = 2.4 Hz, 1H), 6.43 (dd, J = 8.4 ,2.4Hz,1H),5.26(s,2H),4.28(t,J＝6.8Hz,2H),3.98(s,2H),3.85(s,3H),3.78(s,3H),3.09(d,J＝11. 3Hz, 2H), 2.71 (t, J = 6.8Hz, 2H), 2.44–2.32 (m, 1H), 2.25–2.15 (m, 2H), 1.94–1.81 (m, 4H), 1.26 (s, 9H).

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%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.48–11.52(m,2H),7.98(d,J＝8.4Hz,1H),7.60(d,J＝1.8Hz,1H),7.3 8(s,1H),7.31(dd,J＝8.4,1.7Hz,1H),6.73(s,1H),4.16(t,J＝6.4Hz,2H) ,4.05(s,2H),2.99(d,J＝11.0Hz,2H),2.56–2.52(m,2H),2.47–2.40(m,1 H),2.07–1.97(m,2H),1.79–1.70(m,2H),1.61–1.48(m,2H),1.17(s,9H).

Example 15 Synthesis of protein degradation agent Y15

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%). ¹ H NMR (400 MHz, Chloroform-d) δ11.42 (s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.38 (s, 1H), 7.35-7.31 (m, 1H), 7.23 (dd, J = 8.5, 1.7 Hz, 1H), 6.61 (s, 1H), 4.30-4.23 (m, 2H), 4.15 ( q,J＝7.0Hz,2H),3.99(s,2H),3.13(d,J＝11.1Hz,2H),2.75–2.67(m,2H),2.50–2.3 9(m,1H),2.30–2.18(m,2H),1.97–1.86(m,4H),1.30(t,J=7.1Hz,3H),1.26(s,9H).

Example 16 Synthesis of protein degradation agent Y16

The synthesis method was the same as that in Example 6, except that aniline was replaced with tert-amylamine to obtain degradation agent Y16 (white solid, yield 36.2%). ^1H NMR(400MHz,Chloroform-d)δ11.50(s,1H),8.17(d,J＝8.4Hz,1H),7.38(s,1H) ,7.32(d,J＝1.8Hz,1H),7.22(dd,J＝8.5,1.7Hz,1H),6.61(s,1H),4.26(t,J＝7.0 Hz,2H),4.04(s,2H),3.98(s,2H),3.19–3.08(m,2H),2.69(t,J＝6.9Hz,2H),2.4 7–2.37(m,1H),2.28–2.14(m,2H),1.96–1.84(m,4H),1.26(s,9H),0.99(s,9H).

Example 17 Synthesis of protein degradation agent Y17

The synthesis method is the same as that of Example 6, except that aniline is replaced by N,N-dimethylethylenediamine to obtain degradation agent Y17 (white solid, yield 19.0%). ¹ H NMR (400MHz, Methanol-d ₄ )δ8.15(d,J＝8.5Hz,1H),7.60(d,J＝1.7Hz, 1H),7.35–7.29(m,2H),6.70(s,1H),4.34(t,J＝6.9Hz,2H),4.25(t,J＝6.8Hz,2H),4.00(s,2H),3.18–3.11(m, 2H),2.71(t,J＝6.8Hz,4H),2.55–2.45(m,1H),2.38(s,6H),2.26–2.17(m,2H),1.93–1.75(m,4H),1.26(s,9H).

Example 18 Synthesis of protein degradation agent Y18

The synthesis method was the same as that in Example 6, except that aniline was replaced with cyclohexylmethylamine to obtain degradation agent Y18 (white solid, yield 39.2%). ^1H NMR(400MHz,Chloroform-d)δ8.18(d,J＝8.5Hz,1H),7.38(s,1H),7.32(d,J＝1.8H z,1H),7.23(dd,J＝8.5,1.7Hz,1H),6.61(s,1H),4.26(t,J＝7.0Hz,2H),4.00–3.94 (m,4H),3.12(d,J＝11.2Hz,2H),2.70(t,J＝7.0Hz,2H),2.47–2.36(m,1H),2.28–2 .19(m,2H),1.96–1.81(m,5H),1.77–1.59(m,6H),1.27(s,9H),1.15–1.03(m,2H).

Example 19 Synthesis of protein degradation agent Y19

The synthesis method was the same as that of Example 6, except that aniline was replaced with (1-methyl-4-piperidinyl)methylamine to obtain degradation agent Y19 (white solid, yield 4.0%). ^1H NMR(400MHz,Chloroform-d)δ8.17(d,J＝8.4Hz,1H),7.35(s,1H),7.30(d,J＝1.7Hz,1H),7.23(dd,J＝8.4,1.7Hz,1H),6.60(s,1H),4.26(t, J＝6.6Hz,2H),4.04(d,J＝6.9Hz,2H),3.97(s,2H),3.14–3.00(m,4H),2.51–2.35(m,4H),2.25–2.13(m,4H),1.96–1.57(m,9H),1.25(s,9H).

Example 20 Synthesis of protein degradation agent Y20

The synthesis method was the same as that of Example 6, except that 4-chloroisatoic anhydride was replaced by isatoic anhydride, and aniline was replaced by benzylamine to obtain degradation agent Y20 (white solid, yield 24.3%). ^1H NMR(400MHz,Chloroform-d)δ11.66(s,1H),8.27(dd,J＝7.9,1.6Hz,1H),7.74–7 .66(m,1H),7.54–7.49(m,2H),7.38(s,1H),7.35–7.22(m,5H),6.61(s,1H),5.32 –5.28(m,2H),4.31(t,J＝7.2Hz,2H),3.99(s,2H),3.13–3.05(m,2H),2.70(t,J＝7 .2Hz,2H),2.47–2.35(m,1H),2.25–2.17(m,2H),1.93–1.82(m,4H),1.25(s,9H).

Example 21 Synthesis of protein degradation agent Y21

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%). ¹ H NMR (400 MHz, Chloroform-d) δ10.58 (s, 1H), 8.27 (dd, J = 7.9, 1.6 Hz, 1H), 7.73–7.66 (m, 1H), 7.38 (s, 1H), 7.32–7.24 (m, 5H), 6.61 (s, 1H), 4.31 (t, J = 7.4 Hz, 2H), 4.18 (q, J = 7. 0Hz,2H),3.98(s,2H),3.13(d,J＝11.4Hz,2H),2.71(t,J＝7.4Hz,2H),2.47–2.37(m,1H ), 2.23(dt,J＝11.0,6.0Hz,2H),1.98–1.85(m,4H),1.32(t,J＝7.0Hz,3H),1.27(s,9H).

Example 22 Synthesis of protein degradation agent Y22

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%). ^1H NMR(400MHz,Chloroform-d)δ10.17(s,1H),7.57–7.51(m,3H),7.37(s,1H),7.36–7.29(m,3H),7.27–7.21(m,2H),6.61(s,1H),5.28(s,2H),4.31( t,J＝7.1Hz,2H),3.98(s,2H),3.07(d,J＝11.2Hz,2H),2.68(t,J＝7.0Hz,2 H),2.42–2.32(m,1H),2.25–2.13(m,2H),1.95–1.77(m,4H),1.27(s,9H).

Example 23 Synthesis of protein degradation agent Y23

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%). ¹ H NMR (400 MHz, Chloroform-d) δ9.92 (s, 1H), 8.24 (d, J = 2.5 Hz, 1H), 7.64 (dd, J = 8.9, 2.6 Hz, 1H), 7.53–7.49 (m, 2H), 7.36 (s, 1H), 7.35–7.31 (m, 2H), 7.27–7.22 (m, 1H), 6.6 1(s,1H),5.28(s,2H),4.32–4.23(m,2H),3.98(s,2H),3.07(d,J＝11.1Hz,2H),2.74 –2.63(m,2H),2.41–2.31(m,1H),2.27–2.14(m,2H),1.96–1.77(m,4H),1.27(s,9H).

Example 24 Synthesis of protein degradation agent Y24

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%). ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.15(s,1H),8.10(dd,J＝7.8,1.7Hz,1H),7.89(dd,J＝7.9,1.6Hz,1H),7. 38(s,1H),7.37–7.28(m,5H),7.24–7.19(m,1H),6.72(s,1H),5.12(s,2H),4 .58(t,J＝6.2Hz,2H),4.06(s,2H),2.61–2.51(m,2H),2.37–2.25(m,1H),1.8 9(t,J＝11.3Hz,2H),1.57(d,J＝12.6Hz,2H),1.28–1.20(m,2H),1.17(s,9H).

Example 25 Synthesis of protein degradation agent Y25

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%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.22(s,1H),7.68(t,J＝8.5Hz,1H),7.39(s,1H),7.34–7.28(m,4H),7.25–7.19(m,1H),7.05(d,J＝8.6Hz,1H),6.89(d,J＝8.5Hz,1H),6.72(s,1H),5.09(s,2H),4.24(t,J＝6. 7Hz,2H),4.06(s,2H),3.85(s,3H),2.97(d,J＝10.8Hz,2H),2.56(t,J＝6.7Hz,2H),2.48– 2.40(m,1H),2.01(t,J＝11.3Hz,2H),1.77–1.68(m,2H),1.62–1.49(m,2H),1.17(s,9H).

Example 26 Synthesis of protein degradation agent Y26

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 )δ12.22(s,1H),7.54–7.47(m,2H),7.43–7.38(m,2H),7.34–7.30(m,4H),7. 27–7.20(m,1H),6.72(s,1H),5.16(s,2H),4.25(t,J＝6.6Hz,2H),4.06(s,2H) ,3.83(s,3H),2.97(d,J＝10.9Hz,2H),2.56(t,J＝6.6Hz,2H),2.48–2.39(m,1H ),2.05–1.95(m,2H),1.72(d,J＝12.6Hz,2H),1.59–1.48(m,2H),1.17(s,9H).

Example 27 Synthesis of protein degradation agent Y27

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%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.22(s,1H),8.00(d,J＝9.4Hz,1H),7.39(s,1H),7.35–7.28(m,4H),7.26–7.19(m,1H),6.95–6.89(m,2H),6.72(s,1H),5.13(s,2H),4.27(t,J＝6.5Hz,2H),4.06( s,2H),3.93(s,3H),2.99(d,J＝10.8Hz,2H),2.58(t,J＝6.6Hz,2H),2.47–2.39(m,1 H),2.01(t,J＝11.2Hz,2H),1.73(d,J＝12.6Hz,2H),1.63–1.46(m,2H),1.17(s,9H).

Example 28 Synthesis of protein degradation agent Y28

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%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ12.19 (s, 1H), 7.71 (dd, J＝7.8, 1.5 Hz, 1H), 7.47 (dd, J＝8.3, 1.5 Hz, 1H), 7.39 (s, 1H), 7.35–7.26 (m, 6H), 7.25–7.19 (m, 1H), 6.72 (s, 1H), 5.13 (s, 2H), 4.48 (t, J＝6.7 Hz,2H),4.06(s,2H),3.93(s,3H),2.79(d,J＝10.9Hz,2H),2.58–2.52(m,2H),2.44– 2.33(m,1H),2.02–1.93(m,2H),1.72–1.64(m,2H),1.50–1.37(m,2H),1.18(s,9H).

Example 29 Synthesis of protein degradation agent Y29

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%). ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.22(s,1H),7.96(d,J＝8.0Hz,1H),7.39(s,1H),7.37(s,1H),7.34–7.28(m ,4H),7.26–7.21(m,1H),7.14(d,J＝8.2Hz,1H),6.72(s,1H),5.14(s,2H),4.28– 4.22(m,3H),4.06(s,2H),2.99(d,J＝10.9Hz,2H),2.60–2.55(m,2H),2.47–2.4 5(m,4H),2.06–1.97(m,2H),1.76–1.69(m,2H),1.59–1.48(m,2H),1.17(s,9H).

Example 30 Synthesis of protein degradation agent Y30

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%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ12.21 (s, 1H), 7.97 (d, J＝8.4 Hz, 1H), 7.81 (d, J＝1.7 Hz, 1H), 7.49 (dd, J＝8.4, 1.6 Hz, 1H), 7.39 (s, 1H), 7.35–7.31 (m, 4H), 7.27–7.20 (m, 1H), 6.72 (s, 1H), 5.13 (s, 2H), 4 .27(t,J＝6.2Hz,2H),4.06(s,2H),2.97(d,J＝11.1Hz,2H),2.56(t,J＝6.1Hz,2H),2.4 7–2.39(m,1H),2.04–1.95(m,2H),1.76–1.67(m,2H),1.59–1.46(m,2H),1.17(s,9H).

Example 31 Synthesis of protein degradation agent Y31

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%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ12.22 (s, 1H), 8.17–8.10 (m, 1H), 7.49–7.43 (m, 1H), 7.39 (s, 1H), 7.34–7.30 (m, 4H), 7.26–7.21 (m, 1H), 7.19–7.14 (m, 1H), 6.72 (s, 1H), 5.14 (s, 2H), 4.29– 4.20(m,2H),4.06(s,2H),2.98(d,J＝11.2Hz,2H),2.59–2.54(m,2H),2.36–2.3 1(m,1H),2.05–1.95(m,2H),1.75–1.67(m,2H),1.56–1.45(m,2H),1.17(s,9H).

Example 32 Synthesis of protein degradation agent Y32

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%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.21(s,1H),8.33–8.28(m,2H),8.06(dd,J＝8.7,1.9Hz,1H),7.39(s,1 H),7.37–7.30(m,4H),7.28–7.21(m,1H),6.72(s,1H),5.16(s,2H),4.34( t,J＝6.2Hz,2H),4.06(s,2H),2.99(d,J＝10.8Hz,2H),2.62(t,J＝6.1Hz,2H ),2.47–2.39(m,1H),2.09–1.97(m,2H),1.72(d,J＝12.7Hz,2H),1.60–1.45 (m,2H),1.17(s,9H).

Example 33 Synthesis of protein degradation agent Y33

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%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.21(s,1H),8.03(d,J＝8.4Hz,1H),7.66(d,J＝1.8Hz,1H),7.37(s,1H) ,7.31(dd,J＝8.4,1.6Hz,1H),6.70(s,1H),4.10(t,J＝7.0Hz,2H),4.03(s, 2H),3.95(q,J＝7.0Hz,2H),2.85(d,J＝10.9Hz,2H),2.46–2.38(m,1H),2.3 6–2.27(m,2H),1.90–1.69(m,6H),1.65–1.53(m,2H),1.18–1.10(m,12H).

Example 34 Synthesis of protein degradation agent Y34

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%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.22 (s, 1H), 8.04 (d, J = 8.3 Hz, 1H), 7.75 (s, 1H), 7.51–7.27 (m, 6H), 6.70 (s, 1H), 4.11 (t, J = 7.0 Hz, 2H), 4.03 (s, 2H), 2.88 (d, J = 10.9 Hz, 2H), 2.46–2.40 (m, 1H), 2.39–2.32 (m, 2H), 1.92–1.72 (m, 6H), 1.65 (t, J = 11.8 Hz, 2H), 1.15 (s, 9H).

Example 35 Synthesis of protein degradation agent Y35

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. ¹ H NMR (400MHz, DMSO-d ₆ )δ12.23(s,1H),8.53–8.45(m,2H),8.07(d,J＝8.5Hz,1H),7.75(d,J＝1.8 Hz,1H),7.42–7.35(m,2H),7.32–7.25(m,2H),6.72(s,1H),5.15(s,2H),4 .14(t,J＝6.9Hz,2H),4.06(s,2H),2.87(d,J＝10.2Hz,2H),2.47–2.40(m,1 H),2.37–2.31(m,2H),1.92–1.71(m,6H),1.68–1.54(m,2H),1.17(s,9H).

Example 36 Synthesis of protein degradation agent Y36

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%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.24 (s, 1H), 8.51–8.43 (m, 2H), 7.69 (t, J = 8.5 Hz, 1H), 7.39 (s, 1H), 7.27–7.22 (m, 2H), 7.14 (d, J = 8.6 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.73 (s, 1H), 5.10 (s, 2H), 4 .13(t,J＝7.3Hz,2H),4.06(s,2H),3.85(s,3H),2.87(d,J＝11.1Hz,2H),2.47–2.41 (m,1H),2.36(t,J＝6.8Hz,2H),1.92–1.70(m,6H),1.66–1.53(m,2H),1.18(s,9H).

Example 37 Synthesis of protein degradation agent Y37

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%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.73 (s, 1H), 8.53–8.42 (m, 2H), 7.95 (d, J=8.5 Hz, 1H), 7.30–7.26 (m, 3H), 7.24 (d, J=2.0 Hz, 1H), 5.09 (s, 2H).

first step

Y37a (500 mg, 1.74 mmol), methyl 4-hydroxycyclohexanecarboxylate (550 mg, 3.48 mmol), triphenylphosphine (912 mg, 3.48 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran, diisopropyl azodicarboxylate (DIAD, 0.7 mL, 3.48 mmol) was added dropwise in an ice bath, then the temperature was raised to room temperature and stirred overnight. 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.

Step 2

The mixture obtained in the previous step was dissolved in 3 mL of methanol, and 3 mL of water and lithium hydroxide monohydrate (245 mg, 5.84 mmol) were added. The mixture was stirred at room temperature overnight to obtain 200 mg of Y37c. ¹ H NMR (400MHz, DMSO-d ₆ )δ12.09(s,1H),8.54–8.41(m,2H),8.09(d,J＝8.5Hz,1H),7.54(d,J＝2.0Hz,1H),7.43(dd,J＝8.5,2.1Hz,1H),7.28–7. 19(m,2H),5.38–5.31(m,1H),5.24(s,2H),2.32–2.20(m,1H),1.86–1.77(m,2H),1.69–1.54(m,4H),1.43–1.27(m,2H).

Step 3

SNS-032 (50 mg, 0.13 mmol), Y37c (60 mg, 0.14 mmol), -(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 75 mg, 0.20 mmol), DIPEA (70 μL, 0.39 mmol) were dissolved in 2 mL of dichloromethane, stirred at room temperature for 2 hours, and subjected to silica gel column chromatography to obtain degradation agent Y37 (white solid, 80 mg, yield 85.7%). ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.33(s,1H),8.53–8.47(m,2H),8.09(d,J＝8.5Hz,1H),7.54(d,J＝2.0Hz,1H),7.43(dd,J＝8.5 ,2.1Hz,1H),7.40(s,1H),7.29–7.26(m,2H),6.72(s,1H),5.49–5.39(m,1H),5.27–5.19(m,2H), 4.42(d,J＝12.9Hz,1H),4.06(s,2H),3.99(d,J＝13.6Hz,1H),3.04(t,J＝12.7Hz,1H),2.82–2.64( m,2H),2.63–2.53(m,1H),1.98–1.78(m,4H),1.76–1.62(m,2H),1.57–1.32(m,6H),1.18(s,9H).

Example 38 Synthesis of protein degradation agent Y38

first step

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%).

Step 2

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. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ11.64 (s, 1H), 8.51 (dd, J=4.3, 1.4 Hz, 1H), 7.71–7.59 (m, 2H), 7.37–7.21 (m, 5H), 5.11 (s, 2H).

Step 3

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%). ¹ H NMR (400MHz, DMSO-d ₆ )δ8.57(d,J＝4.3Hz,1H),8.03(d,J＝8.6Hz,1H),7.79(dd,J＝8.7,4.4Hz,1H),7.39–7. 22(m,5H),5.16(s,2H),4.22(t,J＝7.2Hz,2H),3.67–3.63(m,2H),2.21–2.12(m,2H).

Step 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 μL, 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%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.22(s,1H),8.62–8.47(m,1H),8.25–8.03(m,1H),7.88–7.67(m, 1H),7.49–7.19(m,6H),6.81–6.66(m,1H),5.17(s,2H),4.14(t,J＝7.2 Hz,2H),4.08–4.04(m,2H),2.82(d,J＝10.9Hz,2H),2.47–2.38(m,1H), 2.38–2.31(m,2H),1.89–1.68(m,6H),1.63–1.45(m,2H),1.18(s,9H).

Example 39 Synthesis of protein degradation agent Y39

The synthesis method was similar to that of Example 38, except that 3-aminopyridine-2-carboxylic acid was replaced with 4-aminopyridine-3-carboxylic acid to obtain degradation agent Y39 (white solid, yield 27.2%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.22(s,1H),9.10(s,1H),8.73(d,J＝5.9Hz,1H),7.58(d,J＝6.1Hz,1H),7.47–7.13(m,6H),6.73(s,1H),5.14(s,2H), 4.17–4.09(m,2H),4.06(s,2H),2.86–2.76(m,2H),2.45–2.30(m,3H),1.89–1.66(m,6H),1.59–1.42(m,2H),1.18(s,9H).

Example 40 Synthesis of protein degradation agent Y40

The synthesis method was similar to that of Example 38, except that 3-aminopyridine-2-carboxylic acid was replaced with 3-aminoisonicotinic acid to obtain degradation agent Y40 (white solid, yield 16.9%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.22(s,1H),9.07(s,1H),8.53(d,J＝5.0Hz,1H),7.93(d,J＝5.0Hz,1H),7.39(s,1H),7.37–7.22(m,5H),6.73(s,1H),5.16(s,2H),4.24(t ,J＝7.1Hz,2H),4.06(s,2H),2.82(d,J＝10.9Hz,2H),2.46–2.32(m,3H) ,1.89–1.79(m,4H),1.75–1.67(m,2H),1.59–1.45(m,2H),1.18(s,9H).

Example 41 Synthesis of protein degradation agent Y41

The synthesis method was similar to that of Example 38, except that 3-aminopyridine-2-carboxylic acid was replaced with 2-aminonicotinic acid to obtain degradation agent Y41 (white solid, yield 23.4%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.20(s,1H),8.77(dd,J＝4.8,1.9Hz,1H),8.43(dd,J＝7.8,1.9Hz,1H),7.41–7.22(m,7H),6.72(s,1H),5.15(s,2H),4.32(t,J＝7.2 Hz,2H),4.06(s,2H),2.81(d,J＝10.9Hz,2H),2.45–2.29(m,3H),1.89–1.74(m,4H),1.72–1.65(m,2H),1.52–1.39(m,2H),1.18(s,9H).

Example 42 Synthesis of protein degradation agent Y42

The synthesis method is as described in Example 38, except that 3-aminopyridine-2-carboxylic acid is replaced with 4-aminopyrimidine-5-carboxylic acid to obtain degradation agent Y42 (white solid, yield 7.9%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.20 (s, 1H), 9.22 (d, J = 7.1 Hz, 2H), 7.41-7.36 (m, 3H), 7.34-7.23 (m, 3H), 6.73 (s, 1H), 5.13 (s, 2H), 4.26 (t, J = 7.1 Hz, 2H), 4.06(s,2H),2.79(d,J＝10.9Hz,2H),2.44–2.30(m,3H),1.88–1.73(m,4H),1.71–1.63(m,2H),1.47–1.33(m,2H),1.18(s,9H).

Example 43 Synthesis of protein degradation agent Y43

The synthesis method was similar to that of Example 38, except that 3-aminopyridine-2-carboxylic acid was replaced with 3-aminopyrazine-2-carboxylic acid to obtain degradation agent Y43 (white solid, yield 6.1%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.21(s,1H),8.81(d,J＝2.4Hz,1H),8.64(d,J＝2.3Hz,1H),7.42–7.36(m,3H),7.34–7.23(m,3H),6.72(s,1H),5.17(s,2H),4.27(t,J＝ 7.2Hz,2H),4.06(s,2H),2.81(d,J＝10.9Hz,2H),2.44–2.30(m,3H),1.88–1.76(m,4H),1.72–1.65(m,2H),1.52–1.39(m,2H),1.18(s,9H).

Example 44 Synthesis of protein degradation agent Y44

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%). ¹ H NMR (400 MHz, DMSO-d ₆ )δ12.20(s,1H),8.75(dd,J＝4.8,1.8Hz,1H),8.41(dd,J＝7.8,1.9Hz,1H),7. 38(s,1H),7.35(dd,J＝7.7,4.8Hz,1H),6.73(s,1H),4.32(t,J＝7.2Hz,2H),4. 06(s,2H),4.00(q,J＝7.0Hz,2H),2.84(d,J＝10.9Hz,2H),2.46–2.33(m,3H), 1.89–1.77(m,4H),1.76–1.65(m,2H),1.54–1.39(m,2H),1.23–1.16(m,12H).

Example 45 Synthesis of protein degradation agent Y45

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%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.20 (s, 1H), 8.80 (dd, J = 4.8, 1.9 Hz, 1H), 8.51–8.49 (m, 2H), 8.44 (dd, J = 7.7, 1.9 Hz, 1H), 7.42–7.37 (m, 2H), 7.35–7.31 (m, 2H), 6.73 (s, 1H), 5.17 (s, 2H), 4.33 (t, J = 7.2 Hz, 2H), 4.06 (s, 2H), 2.83 (d, J＝11.0Hz,2H),2.47–2.32(m,3H),1.90–1.77(m,4H),1.74–1.67(m,2H),1.55–1.42(m,2H),1.19(s,9H).

Example 46 Synthesis of protein degradation agent Y46

The synthesis of Y46a was based on Y38a. In the first step, 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%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.95 (s, 1H), 8.63 (dd, J = 4.7, 1.9 Hz, 1H), 8.31 (dd, J = 7.8, 1.9 Hz, 1H), 7.28 (dd, J = 7.8, 4.8 Hz, 1H), 3.93 (q, J = 7.0 Hz, 2H), 1.16 (t, J = 7.0 Hz, 3H).

first step

Y46a (500 mg, 2.62 mmol), 1,2-ethylene glycol (974 mg, 15.69 mmol), and triphenylphosphine (1.4 g, 5.23 mmol) were dissolved in 10 mL of anhydrous tetrahydrofuran, and DIAD was added dropwise under ice bath. After the addition was complete, the mixture was heated to room temperature and stirred overnight, and then subjected to silica gel column chromatography to obtain a mixture of Y46b and triphenylphosphine (colorless oil, 881 mg).

Step 2

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%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.68 (s, 1H), 8.68 (dd, J=4.8, 1.9 Hz, 1H), 8.45 (dd, J=7.8, 1.9 Hz, 1H), 7.39 (dd, J=7.8, 4.8 Hz, 1H), 5.12 (s, 2H), 3.99 (q, J=7.1 Hz, 2H), 1.18 (t, J=7.1 Hz, 3H).

Step 3

Y46c (13 mg, 0.06 mmol) and C24a (20 mg, 0.04 mmol) were dissolved in 1 mL of dichloroethane (DCE), and one drop of acetic acid was added. After stirring at room temperature for 30 minutes, sodium triacetoxyborohydride (24 mg, 0.11 mmol) was added, and the mixture was stirred at room temperature for 30 minutes. 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%). ^1H NMR(400MHz,Chloroform-d)δ8.67(dd,J＝4.7,1.9Hz,1H),8.52–8.47(m,2H),8.39(s,1H),7.83–7.75(m,2H),7.72–7.70(m,1H),7.61(s,1H) ),7.22(dd,J＝7.7,4.7Hz,1H),6.72(d,J＝8.8Hz,1H),6.03(s,1H),4.96–4.85(m,1H),4.65(d,J＝5.9Hz,2H),4.60(t,J＝6.8Hz,2H),4.17(q, J＝7.0Hz,2H),3.62–3.56(m,4H),2.82(t,J＝6.8Hz,2H),2.79–2.74(m,4H ),2.25(s,3H),2.08(s,3H),1.62(d,J＝6.6Hz,6H),1.31(t,J＝7.0Hz,3H).

Example 47 Synthesis of protein degradation agent Y47

The synthesis method was similar to Example 46, except that 1,2-ethylene glycol was replaced with 1,3-propylene glycol to obtain degradation agent Y47 (white solid, yield 56.7%). ¹ H NMR (400 MHz, Chloroform-d) δ12.25 (s, 1H), 8.68–8.65 (m, 1H), 8.50–8.46 (m, 2H), 8.41 (s, 1H), 7.97 (t, J = 5.9 Hz, 1H), 7.78 (dd, J = 8.8, 2.6 Hz, 1H), 7.73 (s, 1H), 7.60 (s, 1H), 7.24–7.19 (m, 1H), 6.69 (d, J = 8.9 Hz, 1H), 5.9 4(s,1H),4.96–4.83(m,1H),4.66(d,J＝5.8Hz,2H),4.49–4.42(m,2H),4.23–4.10(m,2H),3.61–3.54(m,4H), 2.62–2.54(m,6H),2.43(s,3H),2.18(s,3H),2.05–1.98(m,2H),1.61(d,J＝6.6Hz,6H),1.32(t,J＝7.0Hz,3H).

Example 48 Synthesis of protein degradation agent Y48

The synthesis method refers to Example 46, and 1,2-ethylene glycol is replaced with 1,4-butanediol to obtain degradation agent Y48 (white solid, yield 33.5%). ¹ H NMR (400 MHz, Chloroform-d) δ12.44 (s, 1H), 8.66 (dd, J = 4.8, 2.0 Hz, 1H), 8.53–8.46 (m, 2H), 8.42 (s, 1H), 8.00 (t, J = 5.8 Hz, 1H), 7.79 (dd, J = 8.8, 2.6 Hz, 1H), 7.74 (d, J = 1.3 Hz, 1H), 7.60 (s, 1H), 7.21 (dd, J = 7.8, 4.8 Hz, 1H), 6.70 (d, J = 8.9 Hz, 1H), 5.95 (s, 1 H),4.96–4.81(m,1H),4.66(d,J＝5.8Hz,2H),4.46–4.35(m,2H),4.21–4.12(m,2H),3.66–3.57(m,4H),2.64–2.58(m,4H),2.51 (t,J＝7.6Hz,2H),2.43(s,3H),2.18(s,3H),1.87–1.77(m,2H),1.74–1.63(m,2H),1.60(d,J＝6.6Hz,6H),1.31(t,J＝7.1Hz,3H).

Example 49 Synthesis of protein degradation agent Y49

The synthesis method refers to Example 46, and 1,2-ethylene glycol is replaced with 1,5-pentanediol to obtain degradation agent Y49 (white solid, yield 60.0%). ¹ H NMR (400 MHz, Chloroform-d) δ8.66 (dd, J = 4.8, 1.9 Hz, 1H), 8.51 (d, J = 2.5 Hz, 1H), 8.48 (dd, J = 7.8, 1.9 Hz, 1H), 8.40 (s, 1H), 7.84 (t, J = 6.0 Hz, 1H), 7.80 (dd, J = 8.9, 2.5 Hz, 1H), 7.72 (d, J = 1.2 Hz, 1H), 7.60 (s, 1H), 7.21 (dd, J = 7.7, 4.8 Hz, 1H), 6.72 (d, J = 8.8 Hz, 1H), 6.0 1(s,1H),4.99–4.83(m,1H),4.65(d,J＝5.8Hz,2H),4.43–4.29(m,2H),4.16(q,J＝7.0Hz,2H),3.69–3.61(m,4H),2.69–2.62(m,4H),2 .51–2.44(m,5H),2.23(s,3H),1.84–1.74(m,2H),1.72–1.64(m,2H),1.61(d,J＝6.6Hz,6H),1.53–1.43(m,2H),1.31(t,J＝7.1Hz,3H).

Example 50 Synthesis of protein degradation agent Y50

The synthesis method is as described in Example 46, except that 1,2-ethylene glycol is replaced with diethylene glycol to obtain degradation agent Y50 (white solid, yield 65.7%). ¹ H NMR (400 MHz, Chloroform-d) δ8.65 (dd, J = 4.8, 2.0 Hz, 1H), 8.51–8.46 (m, 2H), 8.41 (s, 1H), 7.91 (t, J = 5.9 Hz, 1H), 7.80 (dd, J = 8.8, 2.6 Hz, 1H), 7.73 (d, J = 1.2 Hz, 1H), 7.60 (s, 1H), 7.22 (dd, J = 7.8, 4.7 Hz, 1H), 6.69 (d, J = 8.9 Hz, 1H), 5.99(s,1H),4.97–4.83(m,1H),4.70–4.56(m,4H),4.16(q,J＝7.0Hz,2H),3.84(t,J＝5.9Hz,2H),3.73(t,J＝5.4Hz, 2H),3.61–3.55(m,4H),2.66–2.56(m,6H),2.45(s,3H),2.21(s,3H),1.61(d,J＝6.6Hz,6H),1.30(t,J＝7.0Hz,3H).

Example 51 Synthesis of protein degradation agent Y51

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%). ¹ H NMR (400 MHz, Chloroform-d) δ8.51 (d, J=2.5 Hz, 1H), 8.42 (s, 1H), 8.18 (d, J=8.4 Hz, 1H), 7.91 (t, J=6.0 Hz, 1H), 7.81 (dd, J=8.8,2.6 Hz, 1H), 7.74 (s, 1H), 7.61 (s, 1H), 7.47 (d, J=1.7 Hz, 1H), 7.23 (dd,J＝8.5,1.6Hz,1H),6.73(d,J＝8.8Hz,1H),5.99(s,1H),4.98–4.81(m, 1H),4.67(d,J＝5.9Hz,2H),4.23(t,J＝7.1Hz,2H),4.16(q,J＝7.1Hz,2H),3. 75–3.66(m,4H),2.72–2.65(m,4H),2.59(t,J＝6.6Hz,2H),2.46(s,3H),2. 22(s,3H),2.06–2.00(m,2H),1.62(d,J＝6.6Hz,6H),1.31(t,J＝7.0Hz,3H).

Example 52 Synthesis of protein degradation agent Y52

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%). ¹ H NMR (400 MHz, Chloroform-d) δ 8.62–8.55 (m, 2H), 8.51 (s, 1H), 8.43 (s, 1H), 8.20 (d, J = 8.5 Hz, 1H), 7.89–7.77 (m, 2H), 7.71 (s, 1H), 7.61 (s, 1H), 7.51 (s, 1H), 7.40–7.34 (m, 2H), 6.74 (d, J = 8.9 Hz, 1H) ,5.99(s,1H),5.27(s,2H),4.97–4.83(m,1H),4.66(d,J＝5.8Hz,2H),4.25(t,J＝7.1Hz,2H),3.80–3 .65(m,4H),2.79–2.55(m,6H),2.46(s,3H),2.24(s,3H),2.10–2.01(m,2H),1.62(d,J＝6.6Hz,6H).

Example 53 Synthesis of protein degradation agent Y53

The synthesis method refers to Example 46, C24a is replaced by SY-5609a, 1,2-ethylene glycol is replaced by 1,3-propylene glycol, and degradation agent Y53 (white solid, yield 70.7%) is obtained. ¹ H NMR (400 MHz, Chloroform-d) δ 11.86 (d, J = 21.9 Hz, 1H), 8.73-8.41 (m, 4H), 8.06 (d, J = 26.7 Hz, 1H), 7.66-7.41 (m, 1H), 7.23-7.11 (m, 1H), 6.48-6.21 (m, 1H), 4.57-4.37 (m, 2H), 4.33-4.23 (m, 1H), 4.20-4.10 (m, 2H), 2.76-2.42 (m,4H),2.17–2.07(m,6H),2.03–1.56(m,6H),1.35–1.21(m,5H).

Example 54 Synthesis of protein degradation agent Y54

The synthesis method refers to Example 46, C24a is replaced by NUV-422a, 1,2-ethylene glycol is replaced by 1,3-propylene glycol, and degradation agent Y54 (white solid, yield 72.6%) is obtained. ¹ H NMR (400MHz, Chloroform-d) δ8.71-8.62 (m, 2H), 8.53-8.46 (m, 2H), 8.29 (d, J = 8.6 Hz, 1H), 8.24 (d, J = 2.3 Hz, 1H), 7.89 (t, J = 1.6 Hz, 1H), 7.70 (dd, J = 10.8, 1.8 Hz, 1H), 7.56 (dd, J = 8.7, 2.4 Hz, 1H), 7.21 (dd, J = 7.8, 4. 7Hz,1H),4.99–4.86(m,1H),4.67(s,2H),4.46(t,J＝7.3Hz,2H),4.17(q,J＝7.0Hz,2H),3.09(d,J＝11.0Hz, 2H),2.65–2.43(m,3H),2.15–1.97(m,4H),1.88–1.69(m,4H),1.61(d,J＝7.0Hz,6H),1.31(t,J＝7.0Hz,3H).

Example 55 Synthesis of protein degradation agent Y55

The synthesis method refers to Example 46, C24a is replaced by NUV-422a, 1,2-ethylene glycol is replaced by 1,5-pentanediol, and degradation agent Y55 (white solid, yield 60.7%) is obtained. ¹ H NMR (400MHz, Chloroform-d) δ8.71 (s, 1H), 8.67 (dd, J = 4.8, 2.0 Hz, 1H), 8.51-8.45 (m, 2H), 8.29 (d, J = 8.6 Hz, 1H), 8.27 (d, J = 2.3 Hz, 1H), 7.89 (t, J = 1.6 Hz, 1H), 7.70 (dd, J = 10.8, 1.8 Hz, 1H), 7.59 (dd, J = 8.7, 2.4 Hz, 1H), 7.21 (dd, J = 7.7, 4.8 Hz, 1H), 7.98-7. .87(m,1H),4.67(s,2H),4.42–4.33(m,2H),4.16(q,J＝7.0Hz,2H),3.14(d,J＝11.1Hz,2H),2.60–2.52(m,1H),2.46(t,J＝7.9Hz ,2H),2.17–2.07(m,2H),1.92–1.75(m,6H),1.72–1.65(m,2H),1.61(d,J＝7.0Hz,6H),1.52–1.42(m,2H),1.31(t,J＝7.1Hz,3H).

Example 56 Synthesis of protein degradation agent Y56

The synthesis method refers to Example 46, C24a is replaced by NUV-422a, 1,2-ethylene glycol is replaced by diethylene glycol, and degradation agent Y56 (white solid, yield 52.8%) is obtained. ¹ H NMR (400MHz, Chloroform-d) δ8.68-8.61 (m, 2H), 8.51-8.44 (m, 2H), 8.29 (d, J = 8.6 Hz, 1H), 8.24 (d, J = 2.3 Hz, 1H), 7.90 (t, J = 1.6 Hz, 1H), 7.70 (dd, J = 10.9, 1.9 Hz, 1H), 7.56 (dd, J = 8.6, 2.4 Hz, 1H), 7.21 (dd, J = 7.7, 4.8 Hz, 1H), 4.99-4.86 (m, 1H), 4.67 (s,2H),4.64(t,J＝5.9Hz,2H),4.16(q,J＝7.1Hz,2H),3.85(t,J＝5.9Hz,2H),3.73(t,J＝5.6Hz,2H),3.07(d,J＝11.2Hz,2H),2 .62(t,J＝5.7Hz,2H),2.53–2.43(m,1H),2.16–2.07(m,2H),1.84–1.75(m,4H),1.61(d,J＝7.0Hz,6H),1.31(t,J＝7.0Hz,3H).

Example 57 Synthesis of protein degradation agent Y57

The synthesis method refers to Example 46, C24a is replaced by NUV-422a, 1,2-ethylene glycol is replaced by triethylene glycol, and degradation agent Y57 (white solid, yield 61.8%) is obtained. ¹ H NMR (400MHz, Chloroform-d) δ8.72 (s, 1H), 8.65–8.61 (m, 1H), 8.50–8.43 (m, 2H), 8.29 (d, J = 8.6 Hz, 1H), 8.26–8.23 (m, 1H), 7.89 (s, 1H), 7.74–7.67 (m, 1H), 7.62–7.55 (m, 1H), 7.21 (dd, J = 7.7, 4.7 Hz, 1H), 4.99–4.86 (m, 1H), 4.67 (s, 2H), 4.62(t,J＝6.2Hz,2H),4.15(q,J＝7.1Hz,2H),3.85(t,J＝6.2Hz,2H),3.77–3.55(m,6H),3.14(d,J＝11.3Hz,2H),2.67(t ,J＝5.9Hz,2H),2.58–2.47(m,1H),2.26–2.17(m,2H),1.93–1.78(m,4H),1.61(d,J＝7.0Hz,6H),1.30(t,J＝7.1Hz,3H).

Example 58 Synthesis of protein degradation agent Y58

The synthesis method refers to Example 46, C24a is replaced by NUV-422a, 1,2-ethylene glycol is replaced by 1,8-octanediol, and degradation agent Y58 (white solid, yield 61.8%) is obtained. ¹ H NMR (400MHz, Chloroform-d) δ8.76 (s, 1H), 8.66 (dd, J = 4.7, 2.0 Hz, 1H), 8.51–8.44 (m, 2H), 8.33–8.25 (m, 2H), 7.89 (t, J = 1.6 Hz, 1H), 7.70 (dd, J = 10.8, 1.8 Hz, 1H), 7.59 (dd, J = 8.7, 2.4 Hz, 1H), 7.20 (dd, J = 7.7, 4.7 Hz, 1H), 4.98– 4.85(m,1H),4.67(s,2H),4.40–4.29(m,2H),4.16(q,J＝7.0Hz,2H),3.17(d,J＝11.2Hz,2H),2.61–2.50(m,1H),2. 49–2.41(m,2H),2.20–2.10(m,2H),1.97–1.84(m,4H),1.79–1.69(m,2H),1.64–1.55(m,8H),1.46–1.24(m,11H).

Example 59 Synthesis of protein degradation agent Y59

The synthesis method is as described in Example 46, except that C24a is replaced by NUV-422b, and 1,2-ethylene glycol is replaced by 1,3-propylene glycol to obtain degradation agent Y59 (white solid, yield 41.1%). ¹ H NMR (400 MHz, Chloroform-d) δ8.66 (dd, J = 4.8, 1.9 Hz, 1H), 8.57 (s, 1H), 8.48 (dd, J = 7.8, 1.9 Hz, 1H), 8.39 (d, J = 3.8 Hz, 1H), 8.33 (d, J = 8.7 Hz, 1H), 8.23 (d, J = 2.3 Hz, 1H), 7.55 (dd, J = 8.7, 2.4 Hz, 1H), 7.47 (s, 1H), 7.31 (dd, J = 11.3, 1.8 Hz, 1H) ,7.21(dd,J＝7.7,4.7Hz,1H),4.45(t,J＝7.3Hz,2H),4.38(t,J＝4.4Hz,2H),4.25–4.13(m,3H),3.33(t,J＝4.4Hz,2H),3. 14–3.03(m,2H),2.62–2.43(m,3H),2.13–1.96(m,4H),1.88–1.67(m,4H),1.31(t,J＝7.0Hz,3H),1.26(d,J＝6.5Hz,6H).

Example 60 Synthesis of protein degradation agent Y60

The synthesis method is as described in Example 46, except that C24a is replaced by NUV-422b, and 1,2-ethylene glycol is replaced by 1,5-pentanediol to obtain degradation agent Y60 (white solid, yield 64.6%). ¹ H NMR (400 MHz, Chloroform-d) δ8.68 (dd, J = 4.8, 1.9 Hz, 1H), 8.49 (dd, J = 7.7, 1.9 Hz, 1H), 8.41-8.33 (m, 2H), 8.26-8.08 (m, 2H), 7.61 (d, J = 8.7 Hz, 1H), 7.48 (s, 1H), 7.34-7.30 (m,1H),7.22(dd,J＝7.8,4.8Hz,1H),4.44–4.34(m,4H),4.28–4.09(m,3H),3.49–3.08 (m,3H),2.84–2.18(m,4H),2.06–1.58(m,8H),1.56–1.41(m,2H),1.35–1.25(m,11H).

Example 61 Synthesis of protein degradation agent Y61

The synthesis method is as described in Example 46, except that C24a is replaced by NUV-422b, and 1,2-ethylene glycol is replaced by diethylene glycol to obtain degradation agent Y61 (white solid, yield 59.5%). ¹ H NMR (400 MHz, Chloroform-d) δ8.70–8.64 (m, 1H), 8.49 (d, J = 7.5 Hz, 1H), 8.37 (d, J = 3.8 Hz, 1H), 8.34 (d, J = 8.7 Hz, 1H), 8.20–8.17 (m, 1H), 8.10–8.03 (m, 1H), 7.56 (d, J = 8.9 Hz, 1H), 7.48 (s, 1H), 7.32 (d, J = 11.7 Hz, 1H), 7.22 (dd, J = 7.9, 4.8 Hz, 1H). z,1H),4.65(t,J＝5.9Hz,2H),4.40(t,J＝4.4Hz,2H),4.29–4.09(m,3H),3.86(t,J＝5.9Hz,2H),3.82–3.70(m,1H),3.34 (t,J＝4.4Hz,2H),3.18–3.00(m,1H),2.57(d,J＝51.1Hz,2H),2.24–2.01(m,2H),1.94–1.59(m,4H),1.36–1.15(m,12H).

Example 62 Synthesis of protein degradation agent Y62

The synthesis method was similar to that of Example 46, except that C24a was replaced by NUV-422b, and 1,2-ethylene glycol was replaced by triethylene glycol to obtain degradation agent Y62 (white solid, yield 65.2%). ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.92(s,1H),8.76–8.72(m,1H),8.62(d,J＝3.9Hz,1H),8.40(dd,J＝7.8,1.9Hz,1H),8.20–8.17(m,1H),8.12(d,J＝8 .7Hz,1H),7.61(d,J＝8.7Hz,1H),7.49(s,1H),7.36(dd,J＝7.7,4.8Hz,1H),7.19(d,J＝12.0Hz,1H),4.44(t,J＝6.5Hz, 2H),4.31(t,J＝4.2Hz,2H),4.20–4.11(m,1H),3.98(q,J＝7.4Hz,2H),3.69(t,J＝6.5Hz,2H),3.60–3.54(m,2H),3.52– 3.44(m,4H),3.33–3.29(m,2H),2.96(s,1H),2.46(s,1H),2.10–1.97(m,2H),1.82–1.56(m,3H),1.23–1.12(m,13H).

Example 63 Synthesis of protein degradation agent Y63

The synthesis method refers to Example 46, C24a is replaced by NUV-422b, 1,2-ethylene glycol is replaced by 1,8-octanediol, and degradation agent Y63 (white solid, yield 60.9%) is obtained. ¹ H NMR (400MHz, Chloroform-d) δ9.26 (s, 1H), 8.65 (dd, J = 4.8, 1.9 Hz, 1H), 8.45 (dd, J = 7.8, 1.9 Hz, 1H), 8.42 (d, J = 3.8 Hz, 1H), 8.37-8.31 (m, 2H), 7.57 (dd, J = 8.6, 2.4 Hz, 1H), 7.44 (s, 1H), 7.32-7.24 (m, 1H), 7.18 (dd, J = 7.8, 4.7 Hz, 1H z,1H),4.39–4.27(m,4H),4.23–4.07(m,3H),3.34–3.22(m,4H),2.67–2.48(m,3H),2.38–2.24(m,2H),2.14–1.9 9(m,2H),1.95–1.84(m,2H),1.80–1.59(m,4H),1.43–1.32(m,8H),1.29(t,J＝7.1Hz,3H),1.25(d,J＝6.5Hz,6H).

Example 64 Synthesis of protein degradation agent Y64

The synthesis method was similar to that of Example 46, except that C24a was replaced by SY-5102, and 1,2-ethylene glycol was replaced by 1,3-propylene glycol to obtain degradation agent Y64 (white solid, yield 79.9%). ^1H NMR(400MHz,Chloroform-d)δ11.64(s,1H),8.80–8.53(m,3H),8.45(d,J＝7.6Hz,1H),7.83(d,J＝33.5Hz,1H),7.27–7.09(m,2H),6.37(d ,J＝58.6Hz,1H),4.56–4.22(m,3H),4.14(q,J＝7.0Hz,2H),2.73–2.23(m,12H),2.02–1.91(m,2H),1.89–1.58(m,4H),1.33–1.22(m,3H).

Example 65 Synthesis of protein degradation agent Y65

The synthesis method was similar to that of Example 46, except that C24a was replaced by SY-5102, and 1,2-ethylene glycol was replaced by 1,5-pentanediol to obtain degradation agent Y65 (white solid, yield 55.7%). ^1H NMR(400MHz,Chloroform-d)δ11.13(s,1H),8.76–8.53(m,3H),8.46(dd,J＝7.8,1.9Hz,1H),7.88(d,J＝27.1Hz,1H),7.27–7.15(m,2 H),6.15(s,1H),4.41–4.28(m,3H),4.21–4.08(m,3H),2.59–2.53(m,3H),2.46–2.39(m,3H),1.94–1.40(m,11H),1.33–1.22(m,7H).

Example 66 Synthesis of protein degradation agent Y66

The synthesis method refers to Example 46, C24a is replaced by SY-5102, 1,2-ethylene glycol is replaced by diethylene glycol, and degradation agent Y66 (white solid, yield 6.3%) is obtained. ¹ H NMR (400MHz, Chloroform-d) δ10.36 (s, 1H), 8.79-8.50 (m, 3H), 8.43 (s, 1H), 7.91 (d, J = 29.9 Hz, 1H), 7.27-7.24 (m, 1H), 7.20-7.12 (m, 1H), 6.15-6.05 (m, 1H), 4.66-4.57 (m, 2H), 4.30-4.20 (m, 1H), 4.19-4.06 (m, 2H), 3.89-3.76 (m, 2H), 3.74–3.61(m,2H),2.78–2.52(m,8H),2.45(s,3H),2.40–2.32(m,1H),1.84–1.64(m,3H),1.62–1.54(m,1H),1.30–1.25(m,3H).

Example 67 Synthesis of protein degradation agent Y67

The synthesis method is as described in Example 46, C24a is replaced by SY-5102, 1,2-ethylene glycol is replaced by triethylene glycol, and degradation agent Y67 (white solid, yield 41.2%) is obtained. ¹ H NMR (400MHz, Chloroform-d) δ10.96 (s, 1H), 8.75–8.51 (m, 3H), 8.45 (d, J＝7.9 Hz, 1H), 7.87 (d, J＝31.6 Hz, 1H), 7.25 (d, J＝8.0 Hz, 1H), 7.18 (dd, J＝7.7, 4.8 Hz, 1H), 6.18 (s, 1H), 4.59(t,J＝6.0Hz,2H),4.29(s,1H),4.18–4.08(m,2H),3.82(t,J＝6.2Hz,2H),3.72–3.6 5(m,3H),3.62–3.52(m,3H),2.81–2.37(m,12H),1.88–1.58(m,4H),1.34–1.22(m,3H).

Example 68 Synthesis of protein degradation agent Y68

The synthesis method was similar to that of Example 46, except that C24a was replaced by SY-5102, and 1,2-ethylene glycol was replaced by 1,8-octanediol to obtain degradation agent Y68 (white solid, yield 31.6%). ^1H NMR(400MHz,Chloroform-d)δ10.99(s,1H),8.77–8.53(m,3H),8.47(dd,J＝7.8, 1.8Hz,1H),7.88(d,J＝33.3Hz,1H),7.25(d,J＝8.2Hz,1H),7.19(dd,J＝7.8,4.8Hz ,1H),6.12(s,1H),4.33(t,J＝7.8Hz,3H),4.16(q,J＝7.1Hz,2H),2.82–2.28(m,12 H),1.88–1.79(m,2H),1.77–1.64(m,4H),1.55–1.44(m,2H),1.42–1.24(m,11H).

Example 69 Synthesis of protein degradation agent Y69

first step

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. After the reaction, the phosphorus oxychloride was evaporated at 70 °C, diluted with dichloromethane, washed once with ice water, and dried to obtain Y69a (brown block, 585.56 mg, 2.32 mmol), which was directly used for the next step without further purification.

Step 2

To a solution of Y69a in 10 mL of dichloromethane was added dropwise N,N-diisopropylethylamine (DIEA, 2.71 mL, 15.54 mmol) at -40°C, followed by a solution of tert-butyl 3,8-diazabicyclo[3.2.1]octane-3-carboxylate (492 mg, 2.32 mmol) in dichloromethane. The mixture was reacted for 0.5 h after the addition was complete. The reaction was monitored to be complete by TLC. The mixture was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and subjected to column chromatography to obtain Y69b (yellow solid, 695 mg, two-step yield 70%). ¹ H NMR (400MHz, Chloroform-d) δ 8.84 (s, 1H), 4.54 (d, 2H), 4.40 (d, 2H), 3.73 (m, 2H), 1.99 (d, J = 5.9Hz, 2H), 1.67 (d, J = 7.7Hz, 2H), 1.52 (s, 9H).

Step 3

To 2-aminonicotinic acid (10 g, 72.40 mmol), 120 mL of dichloromethane was added, followed by HATU (35.78 g, 94.12 mmol) and DIEA (31.53 mL, 180.99 mmol). After 15 min, ethylamine hydrochloride (7.08 g, 86.88 mmol) was added. The mixture was reacted at room temperature for 16 hours and then spin-dried for the next step.

Step 4

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%). ¹ H NMR (400 MHz, DMSO-d6) δ11.93 (s, 1H), 8.61 (dd, J＝4.8, 1.9 Hz, 1H), 8.30 (dd,J＝7.8,1.9Hz,1H),7.27(dd,J＝7.8,4.8Hz,1H),3.91(q,J＝7.0Hz,2H),1.14(t,J＝7.0Hz,3H).

Step 5

Y69c (150 mg, 784.56 μmol) and potassium carbonate (110.60 mg, 800.25 μmol) were dissolved in 5 mL of N,N-dimethylformamide. After 5 min, 2-bromoethanol (111 μL, 1.58 mmol) was added. After reacting at room temperature for 16 h, 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. ^1H NMR(400MHz, DMSO-d6)δ8.72(dd,J＝4.8,2.0Hz,1H),8.39(dd,J＝7.7,1.9Hz,1H),7.40–7.28(m,1H),4.79( t,J＝6.0Hz,1H),4.34(t,J＝6.7Hz,2H),3.97(q,J＝7.0Hz,2H),3.63(q,J＝6.5Hz,2H),1.17(t,J＝7.0Hz,3H).

Step 6

Under nitrogen protection, Y69d (85.68 mg, 364.24 μmol) was dissolved in 5 mL of dry tetrahydrofuran, and then sodium hydride (14.57 mg, wt 60%, 364.24 μmol) was slowly added under ice bath. After 10 min, 5 mL of tetrahydrofuran solution of Y69b (130 mg, 303.53 μmol) 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. The product Y69e (light yellow solid, 109 mg, yield 57%) was obtained by column chromatography. ^1H NMR (400MHz, DMSO-d6) δ8.86(s,1H),8.61(dd,J＝4.8,1.8Hz,1H),8.33(dd,J＝7.8,1.9Hz,1H),7.29(dd,J＝7.7,4.8Hz,1H),4.71(dd,J＝13.9,4.9Hz,4H),4 .42(d,J＝12.7Hz,2H),4.20(s,2H),3.89(q,J＝7.0Hz,2H),3.57(d,J＝12.6Hz ,2H),1.76(s,2H),1.55(d,J＝7.7Hz,2H),1.46(s,9H),1.05(t,J＝7.0Hz,3H).

Step 7

Under argon protection, Y69e was dissolved in 3 mL of toluene, 500 μL of water was added, potassium phosphate (101 mg, 478.41 μmol) 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 μmol) 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 μmol) 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 obtain compound Y69f (52 mg), which was directly used for the next step without further separation.

Step 8

Under nitrogen protection, 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. ¹ H NMR (400 MHz, Methanol-d4) δ8.97 (s, 1H), 8.58 (dd, J = 4.8, 1.9 Hz, 1H), 8.40 (dd, J = 7.8, 1.9 Hz, 1H), 7.85 (dd, J = 9.2, 5.7 Hz, 1H), 7.35–7.33 (m, 1H), 7.33–7.19 (m, 3H), 4.84 (s, 3 H),4.62(d,J＝16.6Hz,2H),4.01(q,J＝7.0Hz,2H),3.82(s,2H),3.74(d,J＝12.2Hz,2H), 3.34(d,J＝1.0Hz,1H),3.33(s,2H),1.89(dd,J＝22.0,8.9Hz,4H),1.15(t,J＝7.0Hz,3H).

Example 70 Synthesis of protein degradation agent Y82

first step

Y46a (1.0 g, 5.23 mmol), methyl 3-hydroxypropionate (816.8 mg, 7.85 mmol), and triphenylphosphine (2.7 g, 10.46 mmol) were dissolved in 15 mL of anhydrous tetrahydrofuran, and DIAD (2.0 mL, 10.46 mmol) was added dropwise under ice bath. After the addition was complete, the temperature was raised to room temperature overnight, and tetrahydrofuran was removed by rotary evaporation. After silica gel column chromatography, Y82b (white solid, 1.58 g, yield 100%) was obtained. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.74(dd,J＝4.8,1.9Hz,1H),8.41(dd,J＝7.7,1.9Hz,1H),7.37(dd,J＝7.7,4.8Hz,1H),4. 55–4.43(m,2H),3.97(q,J＝7.0Hz,2H),3.59(s,3H),2.75–2.64(m,2H),1.17–1.13(m,3H).

Step 2

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. ¹ H NMR (400MHz, DMSO-d ₆ )δ8.75(dd,J＝4.8,1.9Hz,1H),8.40(dd,J＝7.8,1.9Hz,1H),7.36(dd,J＝7.7,4.8Hz,1H), 4.45(dd,J＝8.7,7.0Hz,2H),3.97(q,J＝7.0Hz,2H),2.68–2.55(m,2H),1.17–1.13(m,3H).

Step 3

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. NMR(400MHz,Chloroform-d)δ8.68(dd,J＝4.8,1.9Hz,1H),8.50(dd,J＝7.8,1.9Hz,1H),7.95(d,J＝2.2Hz,1H ),7.25(dd,J＝7.8,4.8Hz,1H),6.45–6.41(m,2H),6.26(d,J＝6.9Hz,1H),5.82(s,1H),4.68(t,J＝7.1Hz,2H), 4.50–4.38(m,1H),4.26–4.12(m,3H),2.81–2.68(m,4H),2.45–2.36(m,1H),2.34–2.25(m,1H),2.23–2.15(m ,1H),2.14–2.04(m,1H),1.88–1.72(m,3H),1.70–1.55(m,1H),1.31(t,J＝7.1Hz,3H),1.03(t,J＝7.4Hz,3H).

Example 71 Synthesis of protein degradation agent Y83

Synthesis method: Refer to Example 70, replace KI-ARV-03 with compound 754 to obtain compound Y83 (white solid, yield 79.8%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.07 (s, 1H), 8.75 (dd, J = 4.8, 1.9 Hz, 1H), 8.41 (dd, J = 7.7, 1.9 Hz, 1H), 8.23 (s, 1H), 7.97 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.68 (s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.36 (dd, J = 7.7, 4.8 Hz, 1H), 4.49-4.43 (m, 2H), 4.02-3.92 (m, 4H), 3.57- 3.39(m,2H),2.88(s,2H),2.46(t,J＝7.5Hz,2H),1.89(s,2H),1.76(s,2H),1.27(s,6H),1.26–1.15(m,7H).

Example 72 Synthesis of protein degradation agent Y84

Synthesis method: Refer to Example 46, replace C24a with compound 754, replace 1,2-ethylene glycol with 1,3-propylene glycol to obtain compound Y84 (colorless oil, yield 22.9%). ¹ H NMR (400 MHz, Chloroform-d) δ8.95 (s, 1H), 8.80–8.67 (m, 1H), 8.63 (dd, J=4.8, 2.0 Hz, 1H), 8.45 (dd, J=7.8, 1.9 Hz, 1H), 8.11 (s, 1H), 7.97 (s, 1H), 7.21 (dd, J=7.7, 4.8 Hz, 1H), 7.17 (s, 1H), 4.46 (t, J=6 .6Hz,2H),4.12(q,J＝7.0Hz,2H),3.93(s,2H),3.80–3.65(m,1H),2.95(t,J＝7.2Hz,2H),2.92(s,2H) ,2.89–2.81(m,1H),2.29–2.11(m,6H),1.63–1.49(m,2H),1.40–1.29(m,8H),1.27(t,J＝7.0Hz,3H).

Example 73 Synthesis of protein degradation agent Y85

Synthesis method: Refer to Example 70, replace KI-ARV-03 with compound 627 to obtain compound Y85 (white solid, yield 57.3%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.17 (s, 1H), 8.74 (dd, J = 4.8, 1.9 Hz, 1H), 8.40 (dd, J = 7.7, 1.9 Hz, 1H), 8.27 (s, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.52 (s, 1H), 7.44-7.32 (m, 2H), 7.22 (dd, J = 8.4, 6.7 Hz, 1H), 7.08 (dd, J = 11.5, 2.4 Hz, 1H), 6. 89(td,J＝8.4,2.5Hz,1H),4.45(t,J＝7.6Hz,2H),3.98(q,J＝7.0Hz,2H),3.77(s,3H),3.58–3.47(m,1H ),3.46–3.36(m,1H),2.45(t,J＝7.7Hz,2H),1.95–1.84(m,2H),1.80–1.66(m,2H),1.28–1.11(m,7H).

Example 74 Synthesis of protein degradation agent Y86

Synthesis method: Refer to Example 46, replace C24a with compound 627, replace 1,2-ethylene glycol with 1,3-propylene glycol to obtain compound Y86 (white solid, yield 20.2%). ¹ H NMR (400 MHz, Chloroform-d) δ8.89 (s, 1H), 8.67 (dd, J = 4.9, 1.9 Hz, 1H), 8.50 (dd, J = 7.7, 1.9 Hz, 1H), 8.21 (s, 1H), 7.68 (s, 1H), 7.25 (dd, J = 7.8, 4.9 Hz, 1H), 7.13 (dd, J = 8.3, 6.6 Hz, 1H), 6.80-6.71 (m, 3H), 4.49 (t, J = 6.6 Hz, 2H), 4.16 (q,J＝7.0Hz,2H),3.84–3.68(m,4H),2.85(t,J＝6.7Hz,2H),2.79–2.68(m,1H),2.22–2.09(m,6H),1.46–1.34(m,4H),1.30(t,J＝7.4Hz,3H).

Example 75 Synthesis of protein degradation agent Y87

first step

2-Aminonicotinic acid (2.0 g, 14.48 mmol) was dissolved in 36 mL DCM, HATU (7.2 g, 18.82 mmol) and DIPEA (5.0 mL, 180.99 mmol) were added, and 4-methylaminopyridine (1.9 g, 17.38 mmol) was added after 15 minutes. The mixture was stirred at room temperature for 16 hours, and the reaction was complete after TLC monitoring. Y87a was obtained and dried for the next step.

Step 2

The product of the previous step was placed in a single-mouth bottle, 36 mL of 1,4-dioxane was added, and then CDI (2.4 g, 14.50 mmol) and DIPEA (2.5 mL, 14.50 mmol) were added. The mixture was refluxed at 110°C for 16 hours, and the reaction was complete after TLC monitoring. The solid was precipitated after standing and cooling to room temperature, and the filter cake was filtered and washed with DCM. The filter cake was spin-dried to obtain crude Y87b (white solid, 4.4 g). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ12.14 (s, 1H), 8.66 (dd, J＝4.8, 1.8 Hz, 1H), 8.49 (d, 2H), 8.33 (dd, J＝7.8, 1.9 Hz, 1H), 7.33–7.29 (m, 3H), 5.10 (s, 2H).

Step 3

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%). ¹ H NMR (400MHz, DMSO-d ₆ )δ8.78(dd,J＝4.8,1.9Hz,1H),8.50–8.48(m,2H),8.43(dd,J＝7.7,1.9Hz,1H),7.40(dd,J＝7.8,4.8Hz ,1H),7.33–7.30(m,2H),5.16(s,2H),4.47(t,J＝6.3Hz,2H),3.78–3.72(m,4H),3.49(t,J＝5.6Hz,2H).

Step 4

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. ¹ H NMR (400 MHz, Chloroform-d) δ12.30 (s, 1H), 8.67 (dd, J=4.8, 1.9 Hz, 1H), 8.56–8.52 (m, 2H), 8.48 (td, J=3.6, 1.9 Hz, 2H), 8.40(s,1H),7.95(t,J＝5.8Hz,1H),7.76(dd,J＝8.8,2.6Hz,1H),7.71(s,1H),7.58(s,1H) ,7.36–7.33(m,2H),7.23(dd,J＝7.8,4.8Hz,1H),6.65(d,J＝8.9Hz,1H),5.91(s,1H),5.24( s,2H),4.87(p,J＝6.6Hz,1H),4.67–4.60(m,4H),3.82(t,J＝5.8Hz,2H),3.69(t,J＝5.6Hz,2 H),3.58–3.51(m,4H),2.60–2.50(m,6H),2.41(s,3H),2.15(s,3H),1.58(d,J＝6.6Hz,6H).

Example 76 Synthesis of protein degradation agent Y88

Synthesis method: Refer to Example 75, replace 2-aminonicotinic acid with 4-aminopyrimidine-5-carboxylic acid, replace 4-methylaminopyridine with ethylamine hydrochloride, and obtain compound Y88 (white solid, yield 36.7%). ¹ H NMR (400 MHz, Chloroform-d) δ9.28 (s, 1H), 9.11 (s, 1H), 8.47 (d, J = 2.6 Hz, 1H), 8.40 (s, 1H), 8.02 (t, J = 5.8 Hz, 1H), 7.76 (dd, J = 8.9, 2.6 Hz, 1H), 7.73 (s, 1H), 7.58 (s, 1H), 6.65 (d, J = 8.9 Hz, 1H), 5.93 (s, 1H), 4.88 (p, J = 6.6 Hz, 1H), 4 .64(d,J＝5.8Hz,2H),4.56(t,J＝5.7Hz,2H),4.12(q,J＝7.1Hz,2H),3.82(t,J＝5.7Hz,2H),3.69(t,J＝5.5Hz,2H),3. 55(t,J＝5.0Hz,4H),2.59–2.53(m,6H),2.41(s,3H),2.15(s,3H),1.59(s,3H),1.57(s,3H),1.28(t,J＝7.0Hz,3H).

Example 77 Synthesis of protein degradation agent Y89

Synthesis method: Refer to Example 75, replace 2-aminonicotinic acid with 4-aminopyrimidine-5-carboxylic acid, and replace 4-methylaminopyridine with benzylamine to obtain compound Y89 (white solid, yield 28.5%). ¹ H NMR (400 MHz, Chloroform-d) δ9.31 (s, 1H), 9.12 (s, 1H), 8.49 (d, J = 2.5 Hz, 1H), 8.41 (s, 1H), 8.01 (t, J = 5.8 Hz, 1H), 7.78 (dd, J = 8.9, 2.6 Hz, 1H), 7.74 (d, J = 1.3 Hz, 1H), 7.60 (s, 1H), 7.54-7.51 (m, 1H), 7.51-7.50 (m, 1H), 7.35-7.29 (m, 3H), 6.66 (d, J＝8.8Hz,1H),5.94(s,1H),5.24(s,2H),4.89(p,J＝6.7Hz,1H),4.66(d,J＝5.8Hz,2H),4.58(t,J＝5.7Hz,2H),3.82(t,J＝5. 7Hz,2H),3.68(t,J＝5.6Hz,2H),3.57–3.52(m,4H),2.56–2.50(m,6H),2.43(s,3H),2.16(s,3H),1.61(s,3H),1.59(s,3H).

Example 78 Synthesis of protein degradation agent Y90

Synthesis method: Refer to Example 46, replace ethylene glycol with diethylene glycol, replace C24a with EPZ6438a, and obtain compound Y90 (white solid, yield 60.1%). ¹ H NMR (400MHz, Chloroform-d) δ8.62 (dd, J = 4.8, 1.9 Hz, 1H), 8.44 (dd, J = 7.8, 2.0 Hz, 1H), 7.48-7.42 (m, 2H), 7.32 (d, J = 7.9 Hz, 3H), 7.29 (d, J = 1.8 Hz, 1H), 7.22-7.15 (m, 2H), 5.91 (s, 1H), 4.60 (t, J = 5.9 Hz, 2H), 4.56 (d, J = 5.8 Hz, 2H), 4.13 (q, J = 7.1 Hz, 2H), 3.98-3. 92(m,2H),3.80(t,J＝5.9Hz,2H),3.66(t,J＝5.6Hz,2H),3.52(s,2H),3.32(td,J＝11.3,3.1Hz,2H),3.10(q,J＝7.0Hz,2H),3.06–2. 98(m,1H),2.57–2.46(m,8H),2.40(s,3H),2.35(s,3H),2.12(s,3H),1.76–1.63(m,4H),1.34–1.20(m,5H),0.90(t,J＝6.9Hz,3H).

Example 79 Synthesis of protein degradation agent Y91

The synthesis method was similar to Example 46, except that ethylene glycol was replaced with 1,5-pentanediol and C24a was replaced with EPZ6438a to obtain compound Y91 (white solid, yield 60.0%) ^. NMR(400MHz,Chloroform-d)δ12.57(s,1H),8.63(dd,J＝4.8,2.0Hz,1H),8.44(dd,J＝7.7,2.0Hz,1H),7.46–7.40(m,2H),7.33–7.2 9(m,3H),7.28(d,1H),7.22(t,J＝5.8Hz,1H),7.18(dd,J＝7.7,4.8Hz,1H),5.89(s,1H),4.54(d,J＝5.8Hz,2H),4.36–4.28(m,2H),4. 17–4.11(m,2H),3.96–3.90(m,2H),3.52(s,2H),3.35–3.26(m,2H),3.08(q,J＝7.0Hz,2H),3.04–2.95(m,1H),2.54(s,6H),2.46–2 .39(m,2H),2.38(s,3H),2.33(s,3H),2.09(s,3H),1.78–1.55(m,8H),1.45–1.36(m,2H),1.30–1.23(m,5H),0.88(t,J＝6.9Hz,3H).

Example 80 Synthesis of protein degradation agent Y92

Synthesis method Referring to Example 46, ethylene glycol was replaced with 1,4-butanediol and C24a was replaced with EPZ6438a to obtain compound Y92 (white solid, yield 63.6%). ¹ H NMR (400 MHz, Chloroform-d) δ12.66 (s, 1H), 8.62 (dd, J = 4.7, 1.9 Hz, 1H), 8.44 (dd, J = 7.8, 2.0 Hz, 1H), 7.42 (d, J = 8.2 Hz, 2H), 7.33–7.29 (m, 3H), 7.28–7.26 (m, 1H), 7.21 (t, J = 5.8 Hz, 1H), 7.17 (dd, J = 7.7, 4.8 Hz, 1H), 5.89 (s, 1H), 4.55 (d, J = 5.8 Hz, 2H), 4.38–4.31 (m, 2H), 4.1 2(t,J＝6.9Hz,2H),3.97–3.90(m,2H),3.50(s,2H),3.30(td,J＝11.3,3.0Hz,2H),3.09(q,J＝7.0Hz,2H),3.04–2.96(m,1H),2.49(s,6H), 2.41(d,J＝7.7Hz,2H),2.38(s,3H),2.33(s,3H),2.09(s,3H),1.78–1.55(m,8H),1.28–1.26(m,2H),1.25(s,3H),0.88(t,J＝6.9Hz,3H).

Example 81 Synthesis of protein degradation agent Y93

Synthesis method: Refer to Example 46, replace ethylene glycol with 1,3-propylene glycol, replace C24a with EPZ6438a, and obtain compound Y93 (white solid, yield 59.2%). ¹ H NMR (400 MHz, Chloroform-d) δ8.62 (dd, J = 4.7, 2.0 Hz, 1H), 8.45 (dd, J = 7.7, 2.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.36-7.31 (m, 3H), 7.29 (d, J = 2.1 Hz, 1H), 7.20-7.16 (m, 2H), 5.90 (s, 1H), 4.56 (d, J = 5.8 Hz, 2H), 4.41 (t, J = 7.3 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2 H),3.98–3.92(m,2H),3.53(s,2H),3.32(td,J＝11.2,3.0Hz,2H),3.10(q,J＝7.0Hz,2H),3.05–2.97(m,1H),2.60–2.45(m,8H ),2.40(s,3H),2.35(s,3H),2.11(s,3H),1.97(p,J＝7.2Hz,2H),1.75–1.64(m,4H),1.32–1.24(m,5H),0.89(t,J＝6.9Hz,3H).

Example 82 Synthesis of protein degradation agent Y94

The synthesis method is as shown in Example 46, C24a is replaced with EPZ6438a to obtain compound Y94 (white solid, yield 71.9%). ¹ H NMR (400 MHz, Chloroform-d) δ12.66 (s, 1H), 8.61 (dd, J = 4.8, 2.0 Hz, 1H), 8.44 (dd, J = 7.8, 2.0 Hz, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.34-7.30 (m, 3H), 7.28 (d, 1H), 7.23 (t, J = 5.8 Hz, 1H), 7.18 (dd, J = 7.7, 4.8 Hz, 1H), 5.90 (s, 1H), 4.55 (d, J = 5.8 Hz, 2H), 4.50 (t, J = 7.0 Hz, 2H), 4.13 ( q,J＝7.0Hz,2H),3.96–3.90(m,2H),3.51(s,2H),3.31(td,J＝11.3,3.0Hz,2H),3.09(q,J＝7.0Hz,2H),3.04–2.96(m,1H),2.70(t,J＝7.0H z,2H),2.63(s,4H),2.47(s,4H),2.39(s,3H),2.34(s,3H),2.09(s,3H),1.72–1.60(m,4H),1.28(t,J＝7.0Hz,3H),0.89(t,J＝7.0Hz,3H).

Example 83 Synthesis of protein degradation agent Y95

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%). ¹ H NMR (400MHz, DMSO-d ₆ )δ12.19(s,1H),8.83–8.65(m,1H),8.52–8.24(m,1H),7.48–7.24(m,1H),5.48–5.17(m,1H),4 .05–3.85(m,2H),2.69–2.54(m,2H),2.27–1.99(m,2H),1.80–1.41(m,4H),1.21–1.08(m,3H).

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%). ¹ H NMR (400 MHz, Chloroform-d) δ 8.71–8.57 (m, 2H), 8.51–8.43 (m, 1H), 8.34–8.14 (m, 2H), 7.97 (s, 1H), 7.25–7.14 (m, 1H), 7.04–6.90 (m, 1H), 5.79–5.51 (m, 1H), 4.19–4.09 (m ,2H),4.05–3.94(m,3H),3.87–3.67(m,1H),3.25–3.12(m,1H),3.01–2.81(m,3H),2.2 2–2.12(m,2H),1.88–1.60(m,6H),1.53–1.37(m,8H),1.35(s,6H),1.32–1.26(m,3H).

Example 84 Synthesis of protein degradation agent Y96

Synthesis method: Refer to Example 83, replace 4-hydroxycyclohexanecarboxylic acid methyl ester with cis-3-hydroxycyclobutylcarboxylic acid methyl ester to obtain compound Y96 (white solid, yield 72.5%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.09 (s, 1H), 8.74 (d, J＝4.7 Hz, 1H), 8.39 (d, J＝7.8 Hz, 1H), 8.23 (s, 1H), 7.97 (s, 1H), 7.76 (d, J＝7.7 Hz, 1H), 7.67 (s, 1H), 7.53–7.44 (m, 1H), 7.41–7.30 (m, 1H), 6.23–6.6 .09(m,1H),4.04–3.89(m,4H),3.67–3.44(m,2H),3.21–3.05(m,3H),2.88(s,2H),2 .36(t,J＝10.1Hz,2H),2.00–1.78(m,4H),1.34–1.22(m,10H),1.18(t,J＝7.0Hz,3H).

Example 85 Synthesis of protein degradation agent Y97

Synthesis method: Refer to Example 83, replace 4-hydroxycyclohexanecarboxylic acid methyl ester with trans-3-hydroxycyclobutylcarboxylic acid methyl ester to obtain compound Y97 (white solid, yield 63.7%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.08 (s, 1H), 8.69 (dd, J = 4.7, 1.9 Hz, 1H), 8.38 (dd, J = 7.7, 2.0 Hz, 1H), 8.22 (s, 1H), 7.96 (s, 1H), 7.67 (s, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.45 (d, J = 7.6 Hz, 1H), 7.34 (dd, J = 7. 8,4.8Hz,1H),5.39–5.25(m,1H),4.01–3.90(m,4H),3.64–3.42(m,2H),3.01–2.84(m, 4H), 2.79–2.66 (m, 1H), 2.00–1.73 (m, 4H), 1.33–1.22 (m, 10H), 1.17 (t, J = 7.0Hz, 3H).

Example 86 Synthesis of protein degradation agent Y98

Synthesis method: Refer to Example 83, replace Y46a with Y15a, replace 4-hydroxycyclohexanecarboxylic acid methyl ester with cis-3-hydroxycyclobutylcarboxylic acid methyl ester to obtain compound Y98 (white solid, yield 82.9%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.09 (s, 1H), 8.24 (s, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.97 (s, 1H), 7.83 (d, J = 7.7 Hz, 1H), 7.67 (s, 1H), 7.52 (d, J = 2.0 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.39 (dd, J = 8.4, 2.0 Hz, 1H), 5.53-5.43 ( m,1H),4.09–4.00(m,2H),3.95(s,2H),3.70–3.44(m,3H),3.20–2.97(m,2H),2.89(s,2H),2. 62–2.53(m,2H),2.46–2.36(m,2H),1.98–1.89(m,2H),1.87–1.80(m,2H),1.32–1.18(m,11H).

Example 87 Synthesis of protein degradation agent Y99

Synthesis method: Refer to Example 83, replace Y46a with Y37a, replace 4-hydroxycyclohexanecarboxylic acid methyl ester with cis-3-hydroxycyclobutylcarboxylic acid methyl ester to obtain compound Y99 (white solid, yield 76.1%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.09 (s, 1H), 8.57–8.50 (m, 2H), 8.23 (s, 1H), 8.07 (d, J=8.5 Hz, 1H), 7.97 (s, 1H), 7.78 (d, J=7.8 Hz, 1H), 7.67 (s, 1H), 7.57 (d, J=2.1 Hz, 1H), 7.47 (d, J=8.2 Hz, 1H), 7.43 (dd, J=8.5, 2.1 Hz,1H),7.32–7.27(m,2H),5.49–5.38(m,1H),5.23(s,2H),3.94(s,2H),3.66–3.42(m,2H),2.96–2.82( m,3H),2.50–2.45(m,2H),2.29–2.18(m,2H),1.97–1.88(m,2H),1.85–1.76(m,2H),1.33–1.17(m,10H).

Example 88 Synthesis of protein degradation agent Y100

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%). ¹ H NMR (400MHz, Chloroform-d) δ8.69–8.57 (m, 1H), 8.52–8.41 (m, 1H), 8.15 (s, 1H), 8.06 (s, 1H), 7.49–7.33 (m, 1H), 7.24–7.15 (m, 1H), 5.79–5.36 (m, 1H), 4.17–4.07 (m, 2H), 3 .96(s,2H),3.88–3.74(m,1H),3.31–3.14(m,1H),3.03(s,2H),2.87–2.69(m,1H),2.5 0–2.17(m,6H),2.06–1.62(m,8H),1.49–1.39(m,2H),1.36(s,6H),1.32–1.24(m,3H).

Example 89 Synthesis of protein degradation agent Y101

Synthesis method: With reference to Example 46, C24a was replaced by compound 754, 1,2-ethylene glycol was replaced by 4-hydroxymethylcyclohexanone, and the second step of Example 46, i.e., the step of converting the hydroxyl group into the aldehyde group, was omitted to obtain compound Y101 (white solid, yield 34.8%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.12 (d, J=2.6 Hz, 1H), 8.73 (d, J=4.8 Hz, 1H), 8.41 (dd, J=7.7, 1.9 Hz, 1H), 8.21 (d, J=2.1 Hz, 1H), 7.97 (s, 1H), 7.70 (d, J=2.6 Hz, 1H), 7.54–7.46 (m, 1H), 7.40–7.31 (m, 1H), 4.28 (d, J=7.2 Hz, 1H), 4.14 (d,J＝7.1Hz,1H),4.05–3.96(m,2H),3.96(s,2H),3.54–3.42(m,2H),3.18–2.98(m,2H),2.88(s,2H) ,2.19–1.67(m,10H),1.65–1.55(m,1H),1.52–1.36(m,3H),1.34–1.22(m,8H),1.17(t,J＝7.0Hz,3H).

Example 90 Synthesis of protein degradation agent Y102

The synthesis method is as follows: 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%). ¹ H NMR (400 MHz, Chloroform-d) δ 8.95–8.72 (m, 1H), 8.67–8.59 (m, 1H), 8.52–8.43 (m, 1H), 8.16 (d, J = 3.5 Hz, 1H), 8.00 (d, J = 4.4 Hz, 1H), 7.25–7.14 (m, 1H), 7.09–6.90 (m, 1H), 4.54 (d, J = 8.0 Hz, 1H), 4.45 (d,J＝6.3Hz,1H),4.21–4.09(m,2H),3.97(s,2H),3.81–3.67(m,1H),3.40–3.24(m,1H),2.93(s,2H ),2.83–2.63(m,1H),2.55–2.39(m,2H),2.30–2.07(m,7H),1.71–1.43(m,3H),1.41–1.22(m,12H).

Example 91 Synthesis of protein degradation agent Y103

Synthesis method: Refer to Example 46, replace C24a with compound 754, replace 1,2-ethylene glycol with diethylene glycol to obtain compound Y103 (white solid, yield 43.7%). ¹ H NMR (400 MHz, Chloroform-d) δ8.95 (s, 1H), 8.76–8.66 (m, 1H), 8.63 (dd, J=4.8, 1.9 Hz, 1H), 8.46 (dd, J=7.7, 1.9 Hz, 1H), 8.13 (s, 1H), 7.96 (s, 1H), 7.21 (dd, J=7.7, 4.8 Hz, 1H), 7.11 (s, 1H), 4.59 ( t,J＝5.8Hz,2H),4.13(q,J＝7.0Hz,2H),3.94(s,2H),3.83(t,J＝5.8Hz,2H),3.76(t,J＝5.0Hz,2H) ,3.73–3.67(m,1H),2.95–2.88(m,4H),2.75–2.65(m,1H),2.19–2.02(m,4H),1.48–1.21(m,13H).

Example 92 Synthesis of protein degradation agent Y104

Synthesis method: Refer to Example 46, replace C24a with compound 754 to obtain compound Y104 (white solid, yield 68.6%). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ9.13 (s, 1H), 8.75 (d, J = 4.6 Hz, 1H), 8.45 (d, J = 7.7 Hz, 1H), 8.22 (s, 1H), 7.96 (s, 1H), 7.66 (s, 1H), 7.54 (s, 1H), 7.45-7.34 (m, 1H), 4.55-4.40 (m, 2H),4.04–3.96(m,2H),3.94(s,2H),3.20–2.95(m,4H),2.88(s,2H),2.07–1.88(m,4H),1.34–1.12(m,13H).

Example 93 Synthesis of protein degradation agent Y105

first step

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). ¹ H NMR (400MHz, DMSO-d ₆ )δ8.75(dd,J＝4.7,2.0Hz,1H),8.40(dd,J＝7.7,1.9Hz,1H),7.35(dd,J＝7.7,4.7Hz,1H),6.79(d,J＝8.1Hz,1H),5.37–5.21(m,1H),4.93–4.85(m, 1H),3.96(q,J＝7.0Hz,2H),2.64–2.53(m,2H),1.89(d,J＝12.5Hz,2H),1. 66(d,J＝12.3Hz,2H),1.40(s,9H),1.36–1.29(m,2H),1.24–1.21(m,3H).

Step 2

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). ¹ H NMR (400MHz, DMSO-d ₆ )δ8.74(dd,J＝4.7,2.0Hz,1H),8.40(dd,J＝7.7,1.9Hz,1H),7.35(dd,J＝7.7,4.7Hz,1H),5.41–5.22(m,1H),3.96(q ,J＝7.0Hz,2H),2.69–2.53(m,3H),1.95–1.80(m,2H),1.71–1.55(m,2H),1.24–1.18(m,2H),1.16(t,J＝7.1Hz,3H).

Step 3

5-Chloro-4-iodopyridin-2-amine (375.0 mg, 1.47 mmol) was dissolved in anhydrous dichloromethane, and a dichloromethane solution of triphosgene (148.7 mg, 0.50 mmol) was added dropwise under an ice bath, and triethylamine (0.4 mL, 2.95 mmol) was added dropwise, and the reaction was carried out at room temperature until the raw material was completely consumed. 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%). ¹ H NMR (400MHz, DMSO-d ₆ )δ10.15(s,1H),9.18(s,1H),8.76(dd,J＝4.7,2.0Hz,1H),8.41(dd,J＝7.7 ,2.0Hz,1H),8.31(s,1H),8.26(s,1H),7.36(dd,J＝7.7,4.8Hz,1H),5.49–5 .26(m,1H),3.97(q,J＝6.9Hz,2H),3.62–3.48(m,1H),2.74–2.57(m,2H),2 .09–1.99(m,2H),1.76–1.67(m,2H),1.47–1.30(m,2H),1.25–1.10(m,3H).

Step 4

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. After the reaction was completed, water was added to the reaction system, extracted with ethyl acetate, and subjected to silica gel column chromatography to obtain Y105 (white solid, 46.7 mg, yield 23.0%). ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.12(s,1H),8.76(dd,J＝4.8,2.0Hz,1H),8.41(dd,J＝7.7,2.0Hz,1H),8.24(s,1H), 7.98(s,1H),7.70(s,1H),7.51(d,J＝7.5Hz,1H),7.36(dd,J＝7.7,4.7Hz,1H),5.45–5.2 6(m,1H),4.04–3.90(m,4H),3.67–3.54(m,1H),2.90(s,2H),2.76–2.61(m,2H),2.08– 2.01(m,2H),1.77–1.67(m,2H),1.46–1.33(m,2H),1.28(s,6H),1.17(t,J=7.0Hz,3H).

Example 94 Synthesis of protein degradation agent Y106

Synthesis method: Refer to Example 46, replace C24a with compound 754a, replace 1,2-ethylene glycol with 1,3-propylene glycol to obtain compound Y106 (white solid, yield 35.7%). ¹ H NMR (400 MHz, Chloroform-d) δ8.83 (s, 1H), 8.65 (dd, J = 4.8, 1.9 Hz, 1H), 8.46 (dd, J = 7.7, 1.9 Hz, 1H), 8.24 (s, 1H), 8.02 (s, 1H), 7.21 (dd, J = 7.7, 4.8 Hz, 1H), 4.49 (t, J = 6.5 Hz, 2H), 4.17–4.07(m,3H),3.96(s,2H),3.00–2.91(m,4H),2.90–2.83(m,1H),2.31–2.21(m,2H), 2.01–1.90(m,2H),1.84–1.71(m,2H),1.69–1.54(m,2H),1.34(s,6H),1.29–1.25(m,5H).

Biological activity test examples

Test Example 1: Cellular _IC50 Determination

In each well of a 96-well plate, 100 μl of fresh culture medium containing different concentrations (the highest concentration is 10 μM, 3-fold dilution, a total of 8 gradient concentrations) of the compound of the present invention or the positive control drug was inoculated with 100 μl. After the cells were incubated with the compound for 72 hours, 10 μL of Cell Counting Kit-8 (CCK8, purchased from Li Ji Biology) was added to each well, and after incubation at 37°C for 2 hours, the absorbance of each well was measured at 450 nm using SpectraMAX190 (Molecular Devices), and the IC ₅₀ value was calculated using SoftMax Pro. The results are shown in Table 1.

The positive control compounds used were SNS-032 and THAL-SNS-032, and the structures were as follows.

Table 1 Inhibitory activity of compounds on WSU-DLCL2 cells

Test Example 2: Western Blot Experiment

CDK can combine with cyclin to form heterodimers, in which CDK is the catalytic subunit and cyclin is the regulatory subunit. Different cyclin-CDK complexes, through CDK activity, catalyze the phosphorylation of different substrates, thereby achieving the promotion and transformation of different phases of the cell cycle.

Polycomb repressive complex 2 (PRC2) 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.

The above proteins and Ras proteins can be used as targets for tumor treatment. Therefore, this test example studies the degradation effect of the degrading agent of the present invention on CDK9, CDK2, CDK2/4/6, CDK5, CDK7, EZH2, EED, SUZ12, EZH1, Ras and related proteins. The specific method is as follows:

Appropriate amount of WSU-DLCL2 cells, U-2932 cells or GP2D cells (human colon cancer 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. According to the amount of cells, 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 β-mercaptoethanol) was added, and the samples were boiled at 100℃ for 20min after lysis. The same volume of samples was taken and SDS-PAGE electrophoresis was performed. After the electrophoresis, the gel was transferred using a GenScript fast transfer instrument. The protein was transferred to a nitrocellulose membrane, and the corresponding bands were cut according to the size of the protein. The membrane was blocked with TBST containing 5% skim milk powder for 1 hour, and the primary antibody was incubated at 4°C overnight. The excess primary antibody was washed off with TBST for 10 minutes each time for a total of three times, and the secondary antibody was incubated at room temperature for 1 hour, and then the excess secondary antibody was washed off with TBST for 10 minutes each time for a total of three times. Finally, the bands were stained and photographed using a Bio-Rad chromatometer.

The results are shown in Figure 1, Figure 2B, Figure 3, Figure 4, Figure 7 (WSU-DLCL2 cells), and Figure 5A (WSU-DLCL2 cells) and B (U-2932 cells), and Figure 6 (GP2D cells).

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. As shown in Figure 1, A, 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. However, 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 μM, 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 μM. Figure 4C shows that compound Y62 can degrade CDK2 and CDK6 and their corresponding cyclinA2/E1/D1 at a concentration of 10 μM. Figure 4D shows that compounds Y59-Y61 can degrade CDK2/4/6 and their corresponding cyclinA2/E1/D1 at a concentration of 10 μM.

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 μM, 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 μM.

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.

Test Example 3: Co-Immunoprecipitates Experiment

After treatment with the compounds, 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. 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.

The results are shown in Figure 2 A. At a concentration of 1 μM, Y2 and Y3 can increase the interaction between CDK9 and LC3B.

Figure 2 A and B together demonstrate the LC3B-dependent autophagy-lysosomal degradation pathway of this class of compounds.

Claims (10)

A protein degradation agent represented by formula (1) or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof,
LCBM-Linker-POIL(1); in: LCBM represents a part that binds to LC3; it is a structure shown in formula (A),
in: ^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, and n is an integer selected from 0 to 4; Each R ¹ is independently selected from halogen, -CN, -OH, -NH ₂ , -NO ₂ , -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, -NH ₂ , -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 heterocyclo Alkyl C1-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 or substituted 5-15 membered heteroaryl C1-C6 alkyl, wherein the substitution means that one or more hydrogen atoms in the defined group are replaced by one or more substituents selected from halogen, amino, hydroxyl, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl; wherein the heteroaryl and heterocyclic groups contain 1 to 4 heteroatoms selected from N, O and S; Indicates connecting to linker from here; Linker represents a covalently linked moiety; POIL represents the target protein ligand portion that binds to CDK or EZH2 or RAS. The protein degrading agent according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, solvate, A polymorph, tautomer, isotope compound, metabolite or prodrug, characterized in that, in formula (A), Each R ¹ is independently selected from halogen, -CN, -OH, -NH ₂ , -NO ₂ , -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, wherein the substitution means that one or more hydrogen in the defined group is replaced by one or more substituents selected from halogen, -NH ₂ , -OH; and/or R ² 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 or substituted 5-10 membered heteroarylC1-C6 alkyl, wherein the substitution means that one or more hydrogens in the defined group are selected from halogen, -NH ₂ , -OH, C1-C3 alkyl, C1-C3 alkoxy, trifluoromethyl or one or more substituents. The protein degradation agent according to claim 1 or 2, or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof, characterized in that: The structure represented by formula (A) is selected from the structure represented by the following formula (A-1):
Wherein, 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, preferably, n is 0 or 1; Each R ¹ is independently selected from halogen, -NO ₂ , -COOH, unsubstituted or substituted C1-C6 alkyl, unsubstituted or 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, -NH ₂ , -OH; preferably, R ¹ is selected from halogen, -NO ₂ , 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, C1-C3 alkoxy, trifluoromethyl; preferably, 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 protein degradation agent according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof, characterized in that: The structure shown in formula (A) is selected from the following structures:
The protein degrading agent or pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof according to claim 1, characterized in that: POIL represents a target protein ligand portion that binds to CDK, wherein the CDK includes CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CDK10, CDK11, CDK12, CDK13, CDK14, CDK15, CDK16, CDK17, CDK18, CDK19, CDK20, CDK21, preferably CDK9, CDK7, CDK2, CDK4, CDK6; or The POIL part is a CDK probe, a CDK inhibitor, an EZH2 probe, an EZH2 inhibitor, a RAS probe, a RAS inhibitor, including the following compounds:
The protein degrading agent or pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof according to claim 1, characterized in that: Linker is a chemical bond or a linear or branched alkylene or saturated cyclic alkyl structure containing 1-50, preferably 2-16 carbon atoms, wherein one or more carbon atoms, especially 1-6, more especially 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 , especially O, wherein R ^a is H or C1-C3 alkyl; or, wherein one or more carbon atoms, especially 1-6, more especially 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; Preferably, Linker is selected from: -(CH ₂ ) _n -, -(CH ₂ CH ₂ O) _m -(CH ₂ ) _n -, -CO-(CH ₂ ) _n -, wherein each n is independently an integer selected from 1-20, preferably an integer from 1-16, and more preferably an integer from 1-8; Each m is independently an integer of 1-10, preferably an integer of 1-6, more preferably an integer of 1-2; p is an integer of 0-10, preferably an integer of 0-6, more preferably an integer of 0-2; Further preferably, Linker is selected from the following structures:
The protein degrading agent according to any one of claims 1 to 6 or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof, characterized in that the protein degrading agent represented by formula (1) is selected from the following structures:


Wherein, n is an integer of 1-16, preferably an integer of 1-8; m is an integer of 1-6, preferably 1 or 2; R ¹ and R ² are as defined in any one of claims 1-4. The protein degrading agent or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof according to any one of claims 1 to 7, characterized in that the protein degrading agent is selected from the following structures:






A pharmaceutical composition comprising a therapeutically effective amount of a protein degrader selected from any one of claims 1 to 8, or one or more of its pharmaceutically acceptable salts, stereoisomers, solvates, polymorphs, tautomers, isotopic compounds, metabolites or prodrugs, and an optional pharmaceutically acceptable carrier. The protein degrader according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotope compound, metabolite or prodrug thereof, or the pharmaceutical composition according to claim 8 for one or more of the following uses: a. Use in the preparation of products 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; b. Use in the preparation of a medicament for treating, preventing and/or ameliorating a disease or condition related to CDK or EZH2 or RAS.