A protein degradation agent, and a method of preparing and use thereof

Abstract

The present application provides a protein degrading agent, a preparation method and use thereof. The protein degrading agent of the present application comprises: at least one moiety capable of binding to LC3; and, at least one moiety capable of binding to CDK; and a linking moiety for covalently linking each of the moiety capable of binding to LC3 to the moiety capable of binding to CDK independently. The protein degrading agent of the present application exhibits excellent anti-tumor activity, can down-regulate the amount of various CDKs in cells, and provides a new paradigm for the treatment, prevention or improvement of diseases or conditions in which CDKs play a role.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a coumarin and CDK inhibitor linked protein degrader and a preparation method thereof, and use thereof in the preparation of a drug for preventing or treating a CDK-related disease. BACKGROUND

[0002] Targeted protein degradation is a very promising biological mechanism research tool and treatment method. By directly degrading target proteins, the problem of drug resistance caused by mutations in target proteins can be overcome. In the past two decades, the PROTAC technology has made great progress, and several PROTAC candidate drugs have entered clinical research. However, the PROTAC technology still faces problems such as insufficient drugability of ubiquitin ligase ligands and dependence on the ubiquitin proteasome system, but many substances in cells are not substrates for proteasome clearance.

[0003] Therefore, there is still a need to develop new protein degraders for degrading target proteins. SUMMARY

[0004] The inventors of the present application took CDK9 distributed in the cytoplasm as an example, connected the ligand of CDK9 at the 5,7 positions of 4-phenylcoumarin through different connection modes, and also investigated the substituents at the 4 position of coumarin, obtaining a series of compounds with degradation effect on CDK9. It is clear that CDK9 can be autophagy degraded by recruiting LC3B, and excellent antitumor effect is shown. The present application first provides that coumarin structure connected with CDK9 inhibitor compounds can autophagy degrade CDK9, show antitumor activity, and it can be speculated that cytoplasmic protein binding compounds connected with this type of coumarin structure can also degrade proteins by recruiting LC3B, produce biological effects, and treat and prevent cytoplasmic protein related diseases. Thus, the present application is completed.

[0005] One of the objects of the present application is to provide a protein degrader.

[0006] The second object of the present application is to provide a preparation method of a protein degrader.

[0007] The third object of the present application is to provide a pharmaceutical composition comprising a protein degrader.

[0008] The fourth object of the present application is to provide a use of a protein degrader in the preparation of a drug for treating, preventing or improving a CDK-related disease.

[0009] In order to achieve the above-mentioned objects of the present application, the following technical solutions are adopted:

[0010] The present application provides, in a first aspect, a protein degrader or a pharmaceutically acceptable salt thereof, comprising:

[0011] at least one moiety that can bind to LC3; and,

[0012] at least one moiety that can bind to CDK;

[0013] a linker for covalently linking each of the moiety that can bind to LC3 independently to the moiety that can bind to CDK.

[0014] 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 ubiquitously present in mammalian tissues and cultured cells and is a key component of autophagy, a recycling system of eukaryotic cells. It is incorporated into the inner and outer membranes of autophagosomes during autophagosome biogenesis. Therefore, LC3 is a specific marker of autophagy, especially autophagosome formation.

[0015] CDK refers to cyclin-dependent kinase, a family of serine / threonine kinases.

[0016] The structure of the protein degrader of the present application can be represented as:

[0017] LCBM-Linker-POIL;

[0018] wherein: LCBM represents a moiety that can bind to LC3 (LC3 Binding Moiety, abbreviated as LCBM moiety); Linker represents a covalent linking moiety; and POIL represents a moiety that can bind to CDK (Protein Of Interest Ligand, abbreviated as POIL moiety).

[0019] The LCBM moiety and the POIL moiety can each independently be selected from a small molecule compound.

[0020] In some embodiments, the LCBM moiety and the POIL moiety each independently have a molecular weight of about 100 to about 2,000, preferably about 100 to about 1,000, for example about 100 to about 900, about 100 to about 800, about 100 to about 700, about 100 to about 600, about 100 to about 500.

[0021] The LCBM moiety can be linked to one or more POIL moieties, and vice versa. In the case where there is more than one POIL moiety, the POIL moieties can each be independently selected, and can be the same or different between individual POIL moieties. In some embodiments, there are multiple POIL moieties for the same target. Even for the same target, multiple POIL moieties, which can be the same or each different, can be used in one molecule as desired. When more than one POIL moiety is used, the linkers used can each be independently selected as well.

[0022] LCBM moiety

[0023] This moiety represents a moiety that can bind to LC3, and refers to a moiety that has affinity for LC3 protein.

[0024] In some embodiments, the LCBM moiety is a coumarin compound, preferably a compound of Formula (1), or a pharmaceutically acceptable salt, stereoisomer, solvate, polymorph, tautomer, isotopologue, metabolite, or prodrug thereof,

[0025]

[0026] wherein:

[0027] Y is O or S;

[0028] R 1 and R 2 are each independently selected from H and R a ;

[0029] R a are each independently selected from hydrogen, C 1-6 alkyl, haloC 1-6 alkyl, C 3-6 cycloalkyl, 3-7 membered heterocyclyl, C 6-10 aryl, 3-7 membered heteroaryl, C 1-6 alkoxy, haloC 1-6 alkoxy, amino; heterocyclyl or aryl optionally substituted with one or more selected from halogen, -NO2, -CN, C 1-6 alkyl, -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -COOH, -C(=O)O(C 1-6 alkyl), -C(=O)NH(C 1-6 alkyl), -C(=O)N(C 1-6 alkyl)2, -OC(=O)(C 1-6 alkyl), -NHC(=O)(C1-6 alkyl), -C(=O)(C 1-6 alkyl) substituted with one substituent selected from the group consisting of -OH, -O(C

[0030] R 3 , R 4 , R 5 and R 6 are each independently selected from the group consisting of H and R b ;

[0031] R b is at each occurrence independently selected from the group consisting of halogen, -NO2, -CN, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, -OR 7 , -SR 7 , -NR 7 R 8 , -C(=O)OR 7 , -C(=O)NR 7 R 8 , -OC(=O)R 7 , -NC(=O)R 7 R 8 , -C(=O)R 7 , -S(=O)2OR 7 , -S(=O)2R 7 , -S(=O)2NR 7 R 8 , -OS(=O)2R 7 , -NS(=O)2R 7 R 8 , -S(=O)R 7 , wherein said alkyl, alkenyl or alkynyl is optionally substituted with one or more substituents selected from the group consisting of halogen, -NO2, -CN, -OH, -O(C 1-6 alkyl), -O(C 3-6 cycloalkyl), -O(C 1-4 alkylene-C 3-6 cycloalkyl), -O(3-7 membered heterocyclyl), -O(C 1-4 alkylene)-(3-7 membered heterocyclyl), -SH, -S(C 1-6 alkyl), -S(C 3-6 cycloalkyl), -S(C 1-4 alkylene-C 3-6 cycloalkyl), -S(3-7 membered heterocyclyl), -S(C 1-4 alkylene)-(3-7 membered heterocyclyl), -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -NH(C 3-6cycloalkyl), -N(C 3-6 cycloalkyl), -N(C 1-4 alkylene-C 3-6 cycloalkyl), -N(C 1-4 alkylene-C 3-6 cycloalkyl), -N(C 1-4 alkylene-3-7 membered heterocyclyl), -N(C 1-4 alkylene-3-7 membered heterocyclyl), =0, -COOH, and C 1-6 alkyl;

[0032] R 7 and R 8 are each independently selected at each occurrence from H, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, C 3-6 cycloalkyl-C 1-4 alkyl, 3-7 membered heterocyclyl, 3-7 membered heterocyclyl-C 1-4 alkyl, C 6-10 aryl-C 1-4 alkyl, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, or aryl is optionally substituted with one or more substituents selected from halogen, -NO2, -CN, C 1-6 alkyl, -OH, -O(C 1-6 alkyl), -NH2, -NH(C 1-6 alkyl), -N(C 1-6 alkyl)2, -COOH, -C(=O)O(C 1-6 alkyl), -C(=O)NH(C 1-6 alkyl), -C(=O)N(C 1-6 alkyl)2, -OC(=O)(C 1-6 alkyl), -NHC(=O)(C 1-6 alkyl), -C(=O)(C 1-6 alkyl;

[0033] the heterocyclyl or heteroaryl contains 1-4 heteroatoms selected from N, O, and S.

[0034] In some embodiments, the compound of formula (1) is selected from the following compounds of formula II:

[0035]

[0036] wherein, R 3 and R 5 are each independently selected from H and OH;

[0037] R 2 selected from hydrogen, C 1-6 alkyl, haloC 1-6 alkyl, C 6-10 aryl, haloC 6-10 aryl, oxoC 6-10 aryl, 3-7 membered heteroaryl; preferably selected from hydrogen, C 1-3 alkyl, haloC 1-3 alkyl, phenyl, halo-phenyl, oxo-phenyl, pyridyl.

[0038] In some embodiments, the compound of formula (1) is selected from the following compounds:

[0039]

[0040] POIL moiety

[0041] The moiety indicates a moiety that can bind to CDK, which means a moiety that can interact with CDK, i.e. the target of the POIL moiety is CDK.

[0042] 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.

[0043] In some embodiments, the POIL moiety binds to CDK9.

[0044] The POIL moiety can be a marketed or literature reported CDK probe, CDK inhibitor, including but not limited to the following compounds:

[0045]

[0046] Linker

[0047] The linker is a linking moiety for connecting the LCBM moiety and the POIL moiety, which can be a chemical bond or a group.

[0048] The linker can be rigid or flexible. In some preferred embodiments, the linker is flexible.

[0049] In some embodiments, the linker is a chemical bond.

[0050] In other embodiments, the Linker is a straight-chain, branched, or cyclic structure comprising 1-30, preferably 2-16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) carbon atoms, wherein each carbon atom may optionally be replaced by one or more (e.g., 2, 3, 4, 5, 6), particularly a heteroatom; wherein the heteroatom is selected from oxygen, sulfur, nitrogen, and phosphorus, preferably oxygen, sulfur, or nitrogen, more preferably oxygen or nitrogen, particularly oxygen. Each carbon atom may also optionally be replaced by -C(=O)-, -C(=S)-, -S(=O)-, -SO2-, and a 3- to 6-membered ring having 0 to 4 heteroatoms, wherein the heteroatom is selected from oxygen, sulfur, nitrogen, and phosphorus.

[0051] In some implementations, the Linker includes the following structure:

[0052]

[0053] The site for covalent connection can be any suitable site.

[0054] In some embodiments, the carbon atoms of the LCBM portion are covalently linked to the linker; in other embodiments, the heteroatoms of the LCBM portion are covalently linked to the linker. Similarly, the POIL portion can also be covalently linked to the linker via carbon atoms or heteroatoms. The heteroatoms are selected from oxygen, sulfur, nitrogen, and phosphorus. Those skilled in the art can select appropriate reactions for linking based on the structures of these three portions.

[0055] In some embodiments, the protein degrading agent is selected from any of the following structures: I-1 to I-6

[0056]

[0057] Where n is 1, 2, 3, 4, 5 or 6; m is 1, 2 or 3;

[0058] R 2 R 3 and R 5 The definition is the same as in Formula II.

[0059] In some embodiments, the protein degrading agent is selected from the following structures:

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] The terms in the present invention are defined as follows:

[0066] The "halogen" can be fluorine, chlorine, bromine or iodine.

[0067] The "C 1-6 The "alkyl" means a chain alkyl group having 1 to 6 carbon atoms; specific examples thereof can include methyl, ethyl, propyl, n-propyl, isopropyl, butyl, n-butyl, isobutyl, t-butyl, 1-methyl-butyl, 1-ethyl-butyl, pentyl, n-pentyl, isopentyl, neopentyl, t-pentyl, hexyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 4-methyl-2-pentyl, 3,3-dimethylbutyl, 2-ethylbutyl, and the like, but not limited thereto.

[0068] The "halo-C 1-6 The "halo-C

[0069] The "C 2-6 The "C

[0070] The "C 1-6 The "alkoxy" means a RO- group, wherein R is a C1-C6 alkyl group as described above. Specific examples of the alkyl group include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, t-butoxy, sec-butoxy, n-pentoxy, isopentoxy, neopentoxy, n-hexoxy, isohexoxy, 3-methylpentoxy, 3,3-dimethylbutoxy, 2-ethylbutoxy, and the like.

[0071] The "halo-C 1-6 The "halo-C

[0072] The "C 3-6"Cycloalkyl" refers to a fully saturated cyclic hydrocarbon compound group containing 3 to 6 carbon atoms, and specific examples thereof include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.

[0073] The "3-7 membered heterocyclic group" refers to a 3-7 membered ring alkyl group containing 1 to 4 heteroatoms selected from nitrogen, oxygen, sulfur in the ring, and specific examples thereof include oxirane, tetrahydroimidazole, tetrahydrofuran, etc.

[0074] The "C 6-10 "Aryl" refers to a monocyclic or polycyclic aromatic group having 6 to 10 carbon atoms; and specific examples thereof include phenyl, naphthyl.

[0075] The "3-7 membered heteroaryl" refers to a 3-7 membered aromatic group containing 1 to 4 heteroatoms selected from nitrogen, oxygen, sulfur in the ring, and specific examples thereof include pyridine, pyridazine, pyrimidine, etc.

[0076] "Pharmaceutically acceptable salt" includes anionic salts and cationic salts of the compound of formula (I), for example, a salt of the compound of formula (1) with an acid or a base; for example, an inorganic acid or an organic acid salt 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 an inorganic base or an organic base salt of the compound of formula (1); for example, an alkali metal salt, an alkaline earth metal salt, an ammonium salt of the compound of formula (I); preferably, the alkali metal includes sodium, potassium, lithium, cesium, the alkaline earth metal includes magnesium, calcium, strontium, for example, the organic base includes trialkylamine, pyridine, quinoline, piperidine, imidazole, methylpyridine, dimethylaminopyridine, dimethylphenylamine, 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.

[0077] The second aspect of the present application provides a method for producing a protein degrading agent, the method comprising the step of linking a moiety that can bind to LC3 and a moiety that can bind to CDK by covalent bonding.

[0078] The reaction type for achieving the linkage includes a nucleophilic substitution reaction, a mitsunobu reaction, a condensation reaction, etc., but is not limited thereto, and any suitable reaction capable of linking a moiety that can bind to LC3 and a moiety that can bind to CDK by covalent bonding can be used.

[0079] In some embodiments, the pharmaceutically acceptable salt of the protein degradation agent can be prepared by reacting the protein degradation agent with the corresponding acid in an alcohol or ethyl acetate or 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 obtain the corresponding hydrochloride salt of the protein degradation agent. However, the present application is not limited thereto, and those skilled in the art can use any suitable salt formation method according to the properties of the protein degradation agent.

[0080] The third aspect of the present application provides a pharmaceutical composition comprising one or more of the above-mentioned protein degradation agents or pharmaceutically acceptable salts thereof in a therapeutically effective amount, and optionally a pharmaceutically acceptable carrier.

[0081] The pharmaceutically acceptable carrier refers to the conventional pharmaceutical carrier in the pharmaceutical field, for example, diluents such as water, fillers such as starch, sucrose, binders such as cellulose derivatives, alginate, gelatin, polyvinylpyrrolidone, humectants 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. In addition, other adjuvants such as flavoring agents and sweetening agents can also be added to the above-mentioned pharmaceutical composition.

[0082] The protein degrading agent or the composition thereof of the present application can be orally or parenterally administered to a patient in the form of a conventional preparation, such as a capsule, microcapsule, tablet, granule, powder, lozenge, pill, suppository, injection, suspension, syrup, patch, cream, lotion, ointment, gel, spray, solution, and emulsion. The suitable preparation can be prepared by a commonly employed method using a conventional organic or inorganic additive, such as an excipient (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate or calcium carbonate), a binder (e.g., cellulose, methylcellulose, hydroxymethylcellulose, polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, acacia, polyethylene glycol, sucrose or starch), a disintegrant (e.g., starch, carboxymethylcellulose, hydroxypropyl starch, low-substituted hydroxypropyl cellulose, sodium bicarbonate, calcium phosphate or calcium citrate), a lubricant (e.g., magnesium stearate, light anhydrous silicic acid, talc or sodium lauryl sulfate), a flavoring agent (e.g., citric acid, menthol, glycine or orange powder), a preservative (e.g., sodium benzoate, sodium bisulfite, nipagin or nipasol), a stabilizer (e.g., citric acid, sodium citrate or acetic acid), a suspending agent (e.g., methylcellulose, polyvinylpyrrolidone or aluminum stearate), a dispersant (e.g., hydroxypropyl methylcellulose), a diluent (e.g., water), and a base wax (e.g., cocoa butter, white vaseline or polyethylene glycol).

[0083] The dosage regimen will be adjusted to provide the optimum desired response. For example, a single bolus, infusion, and / or continuous infusion can be administered when the drug is administered in the form of an injection. For example, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For example, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is noted that dosage values can vary with the type and severity of the condition to be alleviated. It is to be understood that the use of treatments described or recommended herein are within the duty of those skilled in the art and that the dosages and schedules of administration are not likely to be critical. Generally, the dose employed will be changed depending on factors, such as: the age, sex and general health condition of the patient to be treated; the frequency of the treatment and the nature of the desired effect; the extent of the tissue damage; the duration of the symptoms; and other variables that can be adjusted by the individual physician. It is further understood that, for any particular individual, specific dose regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Amounts and administration regimens for the pharmaceutical compositions can be readily determined by those of ordinary skill in the clinical arts. For example, the protein degrading agent or the composition thereof of the present application can be administered in divided doses from 4 times per day to once every 7 days, in an amount of, for example, 0.01 to 1000 mg per dose. The desired dose can be administered in one or more administrations, as required by the clinical situation. The pharmaceutical compositions according to the present application can also be provided in unit dosage form.

[0084] Cyclin-dependent kinases (CDKs) of the serine / threonine kinase family are the main driving force of cell cycle regulation mechanisms, which can promote the synthesis of cellular DNA and mitosis by phosphorylating key substrates (Siemeister et al., Mol. Cancer Ther. 2012; 11(10): 2265-2273.). CDK9 plays an important role in the transcriptional regulation of the apoptosis regulator Mcl-1 and the proto-oncogene MYC, which are associated with the proliferation and survival of tumor cells (Rahaman et al., Endocrine-Related Cancer. 2016; 23(12): T211-T226.). CDK9 signaling is overactive in a variety of hematological malignancies and solid tumors. Inhibition of CDK9 can cause cancer cell growth inhibition and induction of apoptosis, which makes CDK9 a potential and attractive target for cancer therapy (Lee et al., Expert Opinion on Investigational Drugs. 2019; 28(11): 989-1001.), in addition, CDK9 has also been shown to play an important role in the pathological processes of viral replication, inflammatory response and cardiovascular disease.

[0085] Therefore, the protein degrader of the present application can be used for treating, preventing or ameliorating a CDK-related disease or disorder.

[0086] Therefore, the fourth aspect of the present application provides the use of the above-mentioned protein degrader in the preparation of a medicament for treating, preventing or ameliorating a CDK-related disease or disorder.

[0087] The CDK-related disease or disorder includes, but is not limited to, inflammation, arthritis, rheumatoid arthritis, spondyloarthropathies, gouty arthritis, osteoarthritis, juvenile arthritis and other arthritic conditions, systemic lupus erythematosus (SLE), skin-related disorders, psoriasis, eczema, burns, dermatitis, neuroinflammation, allergy, pain, neuropathic pain, fever, lung diseases, lung inflammation, adult respiratory distress syndrome, pulmonary sarcoidosis, asthma, silicosis, chronic lung inflammatory diseases, and chronic obstructive pulmonary disease (COPD), cardiovascular diseases, arteriosclerosis, myocardial infarction (including post-myocardial infarction indications), thrombosis, congestive heart failure, cardiac reperfusion injury, and complications associated with hypertension and / or heart failure, such as vascular organ damage, restenosis, cardiomyopathy, stroke, including ischemic and hemorrhagic stroke, reperfusion injury, renal reperfusion injury, ischemia, including stroke and cerebral ischemia, and ischemia caused by heart / coronary bypass, neurodegenerative diseases, liver diseases, and renal inflammation, gastrointestinal disorders, inflammatory bowel disease, Crohn's disease, gastritis, irritable bowel syndrome, ulcerative colitis, ulcerative disease, gastric ulcer, viral and bacterial infections, sepsis, septic shock, gram-negative sepsis, malaria, meningitis, HIV infection, opportunistic infections, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), AIDS, ARC (AIDS related complex), pneumonia, herpes virus, myalgia due to infection, influenza, autoimmune diseases, graft vs. host reactions and allograft rejection, treatment of bone resorption diseases, osteoporosis, multiple sclerosis, cancer, leukemia, lymphoma, colorectal cancer, brain cancer, bone cancer, tumors of epithelial cell origin (carcinomas), basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, oral cavity cancer, esophageal cancer, small bowel cancer, stomach cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer, skin cancer, squamous and / or basal cell carcinoma, prostate cancer, renal cell carcinoma, and other known cancers affecting the entire epithelium of the body, chronic myelogenous leukemia (CML), acute myelogenous leukemia (AML) and acute promyelocytic leukemia (APL), angiogenesis, including neoplasia, metastasis, central nervous system diseases, central nervous system diseases with an inflammatory or apoptotic component, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury and peripheral neuropathy, or B-cell lymphoma. In addition, modulating the amount of CDK within a cell by LC3-mediated autophagic degradation with the protein degraders of the application (e.g., those described herein) also provides a new paradigm for treating, preventing, or ameliorating diseases or disorders in which CDK plays a role.

[0088] The protein degraders provided by the application, or the pharmaceutical compositions of the application, can also be used in combination with other therapeutic agents for the treatment or prevention of tumors.

[0089] The present application has the following beneficial effects:

[0090] The present application provides a series of coumarin small molecules with recruitment effect on LC3, obtains a series of protein degradation agents with degradation effect on CDK9, and the degradation agents exhibit excellent antitumor activity; in addition, the amount of other CDKs in cells can also be down-regulated by means of the degradation agents provided herein, which provides a new paradigm for the treatment, prevention or improvement of diseases or disorders in which CDKs play a role.

[0091] The present application has been described in detail above, but the above-mentioned embodiments are only illustrative in nature and are not intended to limit the present application. In addition, the present application is not limited by any theory described in the foregoing prior art or summary of the application or in the following examples. BRIEF DESCRIPTION OF DRAWINGS

[0092] Figure 1 Representative compounds induce CDK9 degradation and down-regulate Mcl-1 protein, wherein A is compound 2, 3, 4, 5, 6, 7 treated U-2932 cells, B is compound 3, 9, 10 treated U-2932 cells, C is compound 14, 15, 16, 17, 18, 19 treated U-2932 cells, D is compound 20, 16, 21, 22 treated U-2932 cells.

[0093] Figure 2 Representative compounds induce CDK9 and LC3B binding, wherein A is compound 10 and 16 treated U-2932 cells, B is compound 10 and 16 treated 293T cells.

[0094] Figure 3 Representative compounds have degradation effect on other CDKs, wherein A is the degradation effect of compound 10 on other CDKs, B is the degradation effect of compound 16 on other CDKs. DETAILED DESCRIPTION

[0095] The present application will be further described below in conjunction with examples, and it should be noted that the following examples are provided only for illustrative purposes and do not constitute a limitation on the scope of protection required by the present application.

[0096] Unless otherwise specified, the raw materials, reagents, methods, etc. used in the examples are conventional raw materials, reagents, methods in the art.

[0097] In the following examples, the nuclear magnetic resonance hydrogen spectrum is recorded by Bruker AMX-400 type nuclear magnetic resonance instrument, and the unit of chemical shift δ is ppm. Unless otherwise specified, all reaction solvents are purified according to the conventional method. The silica gel (200-300 mesh) used for column chromatography is produced by Qingdao Marine Chemical Factory. Thin layer chromatography uses GF254 high efficiency plate, which is produced by Yantai Chemical Institute. The preparative thin layer chromatography plate is prepared by Shanghai Institute of Materia Medica, Chinese Academy of Sciences, and the stationary phase is prepared by GF254 (HG / T2354-92) silica gel and sodium carboxymethyl cellulose (800-1200), which are produced by Qingdao Marine Chemical Co., Ltd. and China Pharmaceutical (Group) Shanghai Chemical Reagent Co., Ltd. respectively. Unless otherwise specified, all solvents are analytical reagents, and the reagents used are purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. Iodine, ultraviolet fluorescence and other methods are used for color development. The organic solvent is removed by rotary evaporation under reduced pressure.

[0098] Synthesis of protein degrading agent 1 of example 1

[0099]

[0100] First step

[0101] 1-1 (150.0 mg, 0.59 mmol), 1-2 (82.0 mg, 0.59 mmol), triphenylphosphine (309.5 mg, 1.18 mmol) were dissolved in 3 ml of anhydrous tetrahydrofuran, protected by N2, 0 ℃ dropwise added DIAD (0.23 ml, 1.18 mmol), after dropwise addition, heated at 40 ℃ for 8 h, spin dry tetrahydrofuran, add dichloromethane, dry with anhydrous sodium sulfate, then separate by silica gel column chromatography (PE / EA = 4 / 1) to obtain 1-3 (white solid, 114.7 mg, yield 52%). 1 H NMR (400 MHz, Methanol-d4) δ 7.47-7.42 (m, 3H), 7.37-7.31 (m, 2H), 6.45 (d, J = 2.2 Hz, 1H), 6.32 (d, J = 2.2 Hz, 1H), 5.86 (s, 1H), 3.85 (t, J = 5.5 Hz, 2H), 2.79 (t, J = 6.7 Hz, 2H), 1.73-1.66 (m, 2H).

[0102] Second step

[0103] Dissolve 1-3 (114.7 mg, 0.31 mmol), SNS-032 (104.7 mg, 0.28 mmol) in 2 ml DMF, add DIPEA (152 μL, 0.92 mmol), heat at 60 °C overnight, add 10 ml ethyl acetate, wash with water 4 times, saturated sodium chloride solution once, dry over anhydrous sodium sulfate, silica gel column chromatography (DCM / MeOH = 94 / 6), to obtain proteolysis agent 1 (white solid, 54.9 mg, yield 29%). 1 H NMR (400 MHz, Methanol-d4) δ 7.46 - 7.42 (m, 3H), 7.38 - 7.29 (m, 3H), 6.69 (s, 1H), 6.43 (d, J = 2.2 Hz, 1H), 6.31 (d, J = 2.2 Hz, 1H), 5.85 (s, 1H), 4.00 (s, 2H), 3.79 (t, J = 6.0 Hz, 2H), 2.88 - 2.82 (m, 2H), 2.53 - 2.43 (m, 1H), 2.07 - 1.91 (m, 4H), 1.89 - 1.72 (m, 4H), 1.44 - 1.36 (m, 2H), 1.26 (s, 9H).

[0104] Example 2 Synthesis of proteolysis agent 2

[0105]

[0106] First step

[0107] Add aniline (2.16 g, 23.19 mmol) to 11.5 ml 50% aqueous solution of HBF4 at 0 °C, dissolve sodium nitrite (1.76 g, 25.51 mmol) in 3.6 ml water, slowly drop into the reaction system, stir at 0 °C for 1 h. Filter, wash once with 50% aqueous solution of HBF4, once with ethanol, and three times with ether to obtain 2-1 (crude product, 3.14 g, 71%). 1 H NMR (400 MHz, Deuterium Oxide) δ 8.52 - 8.38 (m, 2H), 8.25 - 8.09 (m, 1H), 7.92 - 7.73 (m, 2H).

[0108] Second step

[0109] To a solution of 2-2 (300.0 mg, 1.54 mmol) in 9 ml of methanol was added calcium carbonate (154.6 mg, 1.54 mmol), palladium acetate (34.7 mg, 0.15 mmol), 2-1 (593.0 mg, 3.09 mmol), and the mixture was stirred at 60 °C overnight. After the reaction was completed by TLC, the methanol was removed, and the mixture was washed with ethyl acetate and water twice, and the salt was washed with silica gel column chromatography (PE / EA = 70 / 30) to give 2-3 (white solid, 132.5 mg, 36.2%). 1 H NMR (400 MHz, Chloroform-d) δ 7.60 - 7.54 (m, 3H), 7.51 - 7.38 (m, 3H), 7.03 - 6.96 (m, 1H), 6.74 - 6.67 (m, 1H), 6.18 - 6.13 (m, 1H), 5.41 - 5.37 (m, 1H).

[0110] Third step

[0111] To a solution of 2-3 (132.5 mg, 0.56 mmol), 2-4 (154.6 mg, 1.11 mmol), and triphenylphosphine (291.8 mg, 1.11 mmol) in 3 ml of anhydrous tetrahydrofuran was added DIAD (0.22 ml, 1.11 mmol) dropwise at 0 °C under N2protection. After the addition was completed, the mixture was stirred at room temperature overnight. The tetrahydrofuran was removed, dichloromethane was added, and the mixture was dried over anhydrous sodium sulfate and separated by silica gel column chromatography (PE / EA = 4 / 1) to give 2-5 (yellowish white solid, 92.3 mg, 46.2%). 1 H NMR (400 MHz, Chloroform-d) δ 7.52 - 7.46 (m, 1H), 7.44 - 7.40 (m, 3H), 7.34 - 7.29 (m, 2H), 7.06 (dd, J = 8.4, 1.0 Hz, 1H), 6.70 (dd, J = 8.4, 1.0 Hz, 1H), 6.17 (s, 1H), 3.88 (t, J = 5.5 Hz, 2H), 2.79 (t, J = 6.4 Hz, 2H), 1.73 - 1.64 (m, 2H).

[0112] Fourth step

[0113] To a solution of 2-5 (92.3 mg, 0.26 mmol) and SNS-032 (88.0 mg, 0.23 mmol) in 2 ml of DMF was added DIPEA (127 μL, 0.77 mmol), and the mixture was heated at 60 °C overnight. After the reaction was completed by TLC, 10 ml of ethyl acetate was added, and the mixture was washed with water four times and saturated sodium chloride solution once, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography (DCM / MeOH = 94 / 6) to give proteolysis agent 2 (white solid, 14.9 mg, 9% yield).1 H NMR (400 MHz, Methanol-d4) δ 7.61 - 7.56 (m, 1H), 7.49 - 7.43 (m, 3H), 7.40 - 7.35 (m, 2H), 7.33 (s, 1H), 7.07 - 7.02 (m, 1H), 6.92 - 6.87 (m, 1H), 6.69 (s, 1H), 6.13 (s, 1H), 4.00 (s, 2H), 3.86 (t, J = 6.1 Hz, 2H), 2.89 - 2.78 (m, 2H), 2.51 - 2.41 (m, 1H), 2.00 - 1.91 (m, 4H), 1.88-1.74 (m, 4H), 1.45 - 1.34 (m, 2H), 1.26 (s, 9H).

[0114] Synthesis of proteolysis agent 3

[0115]

[0116] First step

[0117] Dissolve 3-1 (400.0 mg, 2.08 mmol) in 5 ml DMF, add 1,3-dibromopropane (634 μL, 6.24 mmol), potassium carbonate (345.2 mg, 2.50 mmol), stir at room temperature for 6 h, add ethyl acetate, wash with water 4 times, saturated brine once, dry over anhydrous sodium sulfate, and purify by silica gel column chromatography to obtain 3-2 (white solid, 65.7 mg, yield 10%). 1 H NMR (400 MHz, Methanol-d4) δ 7.61 - 7.56 (m, 1H), 7.49 - 7.43 (m, 3H), 7.40 - 7.35 (m, 2H), 7.33 (s, 1H), 7.07 - 7.02 (m, 1H), 6.92 - 6.87 (m, 1H), 6.69 (s, 1H), 6.13 (s, 1H), 4.00 (s, 2H), 3.86 (t, J = 6.1 Hz, 2H), 2.89 - 2.78 (m, 2H), 2.51 - 2.41 (m, 1H), 2.00 - 1.91 (m, 4H), 1.88-1.74 (m, 4H), 1.45 - 1.34 (m, 2H), 1.26 (s, 9H).

[0118] Second step

[0119] Synthesis method is the same as the fourth step of Example 2, replace 2-5 with 3-2 to obtain proteolysis agent 3 (white solid, yield 20%). 1H NMR (400 MHz, DMSO-d6) δ 12.26 (s, 1H), 10.71 (s, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 6.39 - 6.26 (m, 2H), 5.92 (s, 1H), 4.07 - 4.00 (m, 4H), 2.99 - 2.86 (m, 2H), 2.50 - 2.38 (m, 6H), 1.98 - 1.88 (m, 4H), 1.82 - 1.73 (m, 2H), 1.69 - 1.54 (m, 2H), 1.18 (s, 9H).

[0120] Synthesis of protein degrader 4, Example 4

[0121]

[0122] First step

[0123] Dissolve resorcinol 4-1 (2.0 g, 15.86 mmol) in 30 ml trifluoroacetic acid, add 10 ml 4-2 (3.4 g, 16.33 mmol) solution in trifluoroacetic acid dropwise at 0 °C, and stir overnight after the addition is complete. Remove the trifluoroacetic acid by distillation under reduced pressure, add 40 ml dichloromethane, and filter to obtain 4-3 (off-white solid, 367.6 mg, 8%). 1 H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 10.12 (s, 1H), 7.32 - 7.26 (m, 2H), 6.96 - 6.90 (m, 2H), 6.26 (d, J = 2.3 Hz, 1H), 6.18 (d, J = 2.4 Hz, 1H), 5.74 (s, 1H), 3.80 (s, 3H).

[0124] Second step

[0125] Synthesize compound 4-4 (white solid, yield 45%) in the same way as the synthesis of compound 2-5, except that 4-3 replaces 2-3. 1 H NMR (400 MHz, Chloroform-d) δ 7.27 - 7.22 (m, 2H), 6.96 - 6.92 (m, 3H), 6.73 (d, J = 2.3 Hz, 1H), 6.28 (d, J = 2.4 Hz, 1H), 5.99 (s, 1H), 3.89 (s, 3H), 3.86 (t, J = 5.6 Hz, 2H), 2.81 (t, J = 6.6 Hz, 2H), 1.83 - 1.75 (m, 2H).

[0126] Third step

[0127] Proteolysis inhibitor 4 was synthesized in the same manner as proteolysis inhibitor 2, except 4-4 was used instead of 2-5 (white solid, yield 30%). 1 H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.63 (s, 1H), 7.39 (s, 1H), 7.29 - 7.23 (m, 2H), 6.99 - 6.93 (m, 2H), 6.73 (s, 1H), 6.40 (d, J = 2.2 Hz, 1H), 6.29 (d, J = 2.3 Hz, 1H), 5.78 (s, 1H), 4.06 (s, 2H), 3.81 (s, 3H), 3.73 (t, J = 5.9 Hz, 2H), 2.72 - 2.64 (m, 2H), 2.47 - 2.36 (m, 1H), 1.82 - 1.67 (m, 6H), 1.62 - 1.48 (m, 2H), 1.32 (s, 2H), 1.18 (s, 9H).

[0128] Example 5 Synthesis of proteolysis inhibitor 5

[0129]

[0130] Proteolysis inhibitor 5 was synthesized in the same manner as Example 4, except 4-2 was replaced with 4-fluorobenzoyl acetic acid ethyl ester (white solid, yield 30%). 1 H NMR (400 MHz, Methanol-d4) δ 7.41 - 7.36 (m, 2H), 7.34 (s, 1H), 7.22 - 7.16 (m, 2H), 6.70 (s, 1H), 6.43 (d, J = 2.2 Hz, 1H), 6.33 (d, J = 2.2 Hz, 1H), 5.87 (s, 1H), 4.00 (s, 2H), 3.82 (t, J = 6.0 Hz, 2H), 2.91 - 2.83 (m, 2H), 2.54 - 2.44 (m, 1H), 2.05 - 1.96 (m, 4H), 1.90 - 1.73 (m, 4H), 1.52 - 1.43 (m, 2H), 1.26 (s, 9H).

[0131] Example 6 Synthesis of proteolysis inhibitor 6

[0132]

[0133] Proteolysis inhibitor 6 was synthesized in the same manner as Example 4, except 4-2 was replaced with isonicotinyl acetic acid ethyl ester (white solid, yield 19%). 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.82 (s, 1H), 8.62 - 8.59 (m, 2H), 7.38 (s, 1H), 7.37 - 7.35 (m, 2H), 6.72 (s, 1H), 6.43 (d, J = 2.2 Hz, 1H), 6.30 (d, J = 2.3 Hz, 1H), 5.87 (s, 1H), 4.05 (s, 2H), 3.73 (t, J = 6.1 Hz, 2H), 2.74 - 2.66 (m, 2H), 2.47 - 2.37 (m, 1H), 1.85 - 1.68 (m, 6H), 1.61 - 1.48 (m, 2H), 1.18 (s, 9H).

[0134] Example 7 Synthesis of proteolysis agent 7

[0135]

[0136] The synthesis method refers to Example 3, and 3-1 is replaced with 5,7-dihydroxycoumarin to obtain proteolysis agent 7 (white solid, yield 25%). 1 H NMR (400 MHz, Methanol-d4) δ 8.13 - 8.08 (m, 1H), 7.34 (s, 1H), 6.70 (s, 1H), 6.38 - 6.30 (m, 2H), 6.13 - 6.09 (m, 1H), 4.15 (t, J = 6.1 Hz, 2H), 4.00 (s, 2H), 3.15 - 3.07 (m, 2H), 2.69 - 2.62 (m, 2H), 2.59 - 2.48 (m, 1H), 2.23 - 2.06 (m, 4H), 1.95 - 1.81 (m, 4H), 1.26 (s, 9H).

[0137] Example 8 Synthesis of proteolysis agent 8

[0138]

[0139] The synthesis method refers to Example 3, and 1,3-dibromopropane is replaced with 1,2-dibromoethane to obtain proteolysis agent 8 (white solid, yield 17%). 1H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.54 (s, 1H), 7.39 (s, 1H), 6.72 (s, 1H), 6.37 (d, J = 2.2 Hz, 1H), 6.31 (d, J = 2.1 Hz, 1H), 5.93 (d, J = 1.3 Hz, 1H), 4.13 - 4.08 (m, 2H), 4.05 (s, 2H), 3.00 - 2.93 (m, 2H), 2.77 - 2.72 (m, 2H), 2.55 - 2.53 (m, 3H), 2.48 - 2.44 (m, 1H), 2.10 - 2.01 (m, 2H), 1.82 - 1.73 (m, 2H), 1.67 - 1.54 (m, 2H), 1.17 (s, 9H).

[0140] Example 9 Synthesis of proteolysis agent 9

[0141]

[0142] The synthesis was performed according to Example 3, replacing 1,3-dibromopropane with 1,4-dibromobutane, to give proteolysis agent 9 (white solid, yield 32%). 1 H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.54 (s, 1H), 7.39 (s, 1H), 6.72 (s, 1H), 6.37 (d, J = 2.2 Hz, 1H), 6.31 (d, J = 2.1 Hz, 1H), 5.93 (d, J = 1.3 Hz, 1H), 4.13 - 4.08 (m, 2H), 4.05 (s, 2H), 3.00 - 2.93 (m, 2H), 2.77 - 2.72 (m, 2H), 2.55 - 2.53 (m, 3H), 2.48 - 2.44 (m, 1H), 2.10 - 2.01 (m, 2H), 1.82 - 1.73 (m, 2H), 1.67 - 1.54 (m, 2H), 1.17 (s, 9H).

[0143] Example 10 Synthesis of proteolysis agent 10

[0144]

[0145] The synthesis was performed according to Example 3, replacing 1,3-dibromopropane with 1,5-dibromopentane, to give proteolysis agent 10 (white solid, yield 20%). 1H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 10.52 (s, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 6.34 (d, J = 2.3 Hz, 1H), 6.30 (d, J = 2.2 Hz, 1H), 5.94 - 5.90 (m, 1H), 4.05 (s, 2H), 4.01 (t, J = 6.2 Hz, 2H), 2.94 - 2.85 (m, 2H), 2.48 - 2.40 (m, 1H), 2.34 - 2.25 (m, 2H), 1.92 - 1.72 (m, 6H), 1.66 - 1.56 (m, 2H), 1.54 - 1.42 (m, 4H), 1.18 (s, 9H).

[0146] Example 11 Synthesis of Protein Degrader 11

[0147]

[0148] The synthesis was performed according to Example 3 by replacing 1,3-dibromopropane with 2,2'-dibromo-diethyl ether to give Protein Degrader 11 (white solid, yield 31%). 1 H NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 10.52 (s, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 6.34 (d, J = 2.3 Hz, 1H), 6.30 (d, J = 2.2 Hz, 1H), 5.94 - 5.90 (m, 1H), 4.05 (s, 2H), 4.01 (t, J = 6.2 Hz, 2H), 2.94 - 2.85 (m, 2H), 2.48 - 2.40 (m, 1H), 2.34 - 2.25 (m, 2H), 1.92 - 1.72 (m, 6H), 1.66 - 1.56 (m, 2H), 1.54 - 1.42 (m, 4H), 1.18 (s, 9H).

[0149] Example 12 Synthesis of Protein Degrader 12

[0150]

[0151] The synthesis was performed according to Example 3 by replacing 3-1 with 5,7- dihydroxycoumarin and 1,3-dibromopropane with 1,5-dibromopentane to give Protein Degrader 12 (white solid, yield 17%). 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 7.97 (d, J = 9.7 Hz, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 6.36 - 6.34 (m, 1H), 6.32 - 6.30 (m, 1H), 6.12 (d, J = 9.7 Hz, 1H), 4.09 - 4.02 (m, 4H), 2.96 - 2.86 (m, 2H), 2.48 - 2.39 (m, 1H), 2.35 - 2.23 (m, 2H), 1.94 - 1.83 (m, 2H), 1.82 - 1.72 (m, 4H), 1.66 - 1.56 (m, 2H), 1.55 - 1.40 (m, 4H), 1.17 (s, 9H).

[0152] Synthesis of protein degrader 13

[0153]

[0154] First step

[0155] Dissolve 2,4-dihydroxybenzaldehyde (2.0 g, 14.48 mmol) in 36 ml dichloromethane, add methoxyformylmethylene triphenylphosphonium (5.8 g, 17.38 mmol), stir at room temperature overnight, concentrate under reduced pressure, and purify by silica gel column chromatography to obtain 13-1 (white solid, 1.4 g, yield 50%). 1 H NMR (400 MHz, Methanol-d4) δ 7.90 (d, J = 16.0 Hz, 1H), 7.38 - 7.33 (m, 1H), 6.43 (d, J = 16.0 Hz, 1H), 6.35 - 6.31 (m, 2H), 3.76 (s, 3H).

[0156] Second step

[0157] Synthesis method according to the second step of Example 2, replace 2-2 with 13-1 to obtain compound 13-2 (black green solid, yield 7%)

[0158] Third step

[0159] Synthesis method according to the third step of Example 2, replace 2-3 with 13-2 to obtain compound 13-3 (yellow solid, yield 49%). 1H NMR (400 MHz, Chloroform-d) δ 7.57 - 7.51 (m, 3H), 7.49 - 7.44 (m, 2H), 7.41 (d, J = 8.9 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 6.82 (dd, J = 8.9, 2.5 Hz, 1H), 6.24 (s, 1H), 4.21 (t, J = 5.8 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.38 (p, J = 6.1 Hz, 2H).

[0160] Fourth Step

[0161] The synthetic method refers to Example 2 Fourth Step, replace 2-5 with 13-3 to obtain protease inhibitor 13 (white solid, yield 21%). 1 H NMR (400 MHz, Chloroform-d) δ 7.57 - 7.51 (m, 3H), 7.49 - 7.44 (m, 2H), 7.41 (d, J = 8.9 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 6.82 (dd, J = 8.9, 2.5 Hz, 1H), 6.24 (s, 1H), 4.21 (t, J = 5.8 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.38 (p, J = 6.1 Hz, 2H).

[0162] Example 14 Synthesis of Protease Inhibitor 14

[0163]

[0164] First Step

[0165] Dissolve 1-1 (1.0 g, 3.93 mmol) in 10 ml dichloromethane, add acetic anhydride (2.2 ml, 23.60 mmol), DMAP (48.1 mg, 0.39 mmol), stir at room temperature for 4 h, wash twice with water, once with saturated brine, dry over anhydrous sodium sulfate, and separate by silica gel column chromatography. Obtain 14-1 (white solid, 1.3 g, yield 98%). 1 H NMR (400 MHz, Chloroform-d) δ 7.57 - 7.51 (m, 3H), 7.49 - 7.44 (m, 2H), 7.41 (d, J = 8.9 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 6.82 (dd, J = 8.9, 2.5 Hz, 1H), 6.24 (s, 1H), 4.21 (t, J = 5.8 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.38 (p, J = 6.1 Hz, 2H).

[0166] Second step

[0167] Compound 14-1 (600.0 mg, 1.77 mmol) was dissolved in 2 ml of dichloromethane, 1 ml of methanol, 1 ml of water, stirred at room temperature for 4 hours, the solvent was removed by reduced pressure distillation, 10 ml of ethyl acetate was added, washed once with water, once with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography. Compound 14-2 (white solid, 217.3 mg, yield 41%) was obtained. 1 H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 7.53-7.46 (m, 3H), 7.35-7.31 (m, 2H), 6.77 (d, J = 2.4 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 5.98 (s, 1H), 1.27 (s, 3H).

[0168] Third step

[0169] Compound 14-2 (217.3 mg, 0.73 mmol) was dissolved in 3 ml of anhydrous DMF, 1,3-dibromopropane (0.37 ml, 3.67 mmol), anhydrous potassium carbonate (152.1 mg, 1.10 mmol) were added, stirred at room temperature for 6 h, 10 ml of ethyl acetate was added, washed 4 times with water, once with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain compound 14-3 (colorless oil, 200.1 mg, 65%). 1 H NMR (400 MHz, Chloroform-d) δ 7.51-7.45 (m, 3H), 7.36-7.31 (m, 2H), 6.88-6.86 (m, 1H), 6.52-6.49 (m, 1H), 6.10-6.08 (m, 1H), 4.20 (t, J = 5.7 Hz, 2H), 3.61 (t, J = 6.2 Hz, 2H), 2.40-2.34 (m, 2H), 1.35 (s, 3H).

[0170] Fourth step

[0171] Compound 14-3 (200.1 mg, 0.48 mmol) was dissolved in 2 ml of DMF, 2 ml of water was added, potassium carbonate (198.8 mg, 1.44 mmol) was added, stirred at room temperature for 2 h, 10 ml of ethyl acetate was added, washed 4 times with water, once with saturated brine, dried over anhydrous sodium sulfate, and separated by silica gel column chromatography to obtain compound 14-4 (white solid, 44.2 mg, yield 25%). 1H NMR (400 MHz, Methanol-d4) δ 7.41 - 7.33 (m, 5H), 6.54 (d, J = 2.5 Hz, 1H), 6.28 (d, J = 2.4 Hz, 1H), 5.90 (s, 1H), 4.19 (t, J = 5.9 Hz, 2H), 3.65 (t, J = 6.5 Hz, 2H), 2.34 (p, J = 6.2 Hz, 2H).

[0172] Fifth step

[0173] The synthetic method was referred to Example 2 fourth step, and 2-5 was replaced by 14-4 to obtain protease inhibitor 14 (white solid, yield 29%). 1 H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.31 (s, 1H), 7.44 - 7.32 (m, 6H), 6.72 (s, 1H), 6.52 (d, J = 2.4 Hz, 1H), 6.22 (d, J = 2.4 Hz, 1H), 5.84 (s, 1H), 4.10 - 4.01 (m, 4H), 2.98 - 2.84 (m, 2H), 2.48 - 2.37 (m, 3H), 1.97 - 1.84 (m, 4H), 1.81 - 1.71 (m, 2H), 1.70 - 1.54 (m, 2H), 1.18 (s, 9H).

[0174] Example 15 Synthesis of protease inhibitor 15

[0175]

[0176] The synthetic method was referred to Example 14, and 1-1 was replaced by 5,7- dihydroxycoumarin to obtain protease inhibitor 15 (white solid, yield 23%). 1 H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.31 (s, 1H), 7.44 - 7.32 (m, 6H), 6.72 (s, 1H), 6.52 (d, J = 2.4 Hz, 1H), 6.22 (d, J = 2.4 Hz, 1H), 5.84 (s, 1H), 4.10 - 4.01 (m, 4H), 2.98 - 2.84 (m, 2H), 2.48 - 2.37 (m, 3H), 1.97 - 1.84 (m, 4H), 1.81 - 1.71 (m, 2H), 1.70 - 1.54 (m, 2H), 1.18 (s, 9H).

[0177] Example 16 Synthesis of protease inhibitor 16

[0178]

[0179] The synthesis method refers to Example 14, and 1-1 is replaced by 5,7-dihydroxy-4- methylcoumarin to obtain protein degrader 16 (white solid, yield 21%). 1 H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.69 (s, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 6.41 (d, J = 2.4 Hz, 1H), 6.32 (d, J = 2.4 Hz, 1H), 5.96 - 5.93 (m, 1H), 4.08 - 3.99 (m, 4H), 2.97 - 2.86 (m, 2H), 2.48 - 2.37 (m, 3H), 1.96 - 1.84 (m, 4H), 1.80 - 1.72 (m, 2H), 1.69 - 1.54 (m, 2H), 1.18 (s, 9H).

[0180] Example 17 Synthesis of protein degrader 17

[0181]

[0182] The synthesis method refers to Example 14, and 1-1 is replaced by 5,7-dihydroxy-4- methylcoumarin to obtain protein degrader 16 (white solid, yield 21%). 1 H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.69 (s, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 6.41 (d, J = 2.4 Hz, 1H), 6.32 (d, J = 2.4 Hz, 1H), 5.96 - 5.93 (m, 1H), 4.08 - 3.99 (m, 4H), 2.97 - 2.86 (m, 2H), 2.48 - 2.37 (m, 3H), 1.96 - 1.84 (m, 4H), 1.80 - 1.72 (m, 2H), 1.69 - 1.54 (m, 2H), 1.18 (s, 9H).

[0183] Example 18 Synthesis of protein degrader 18

[0184]

[0185] The synthesis method refers to Example 14, and 1-1 is replaced by 5,7-dihydroxy-4- methylcoumarin to obtain protein degrader 16 (white solid, yield 21%). 1H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.35 (s, 1H), 7.44 - 7.36 (m, 3H), 7.25 - 7.17 (m, 2H), 6.72 (s, 1H), 6.52 (d, J = 2.4 Hz, 1H), 6.23 (d, J = 2.4 Hz, 1H), 5.87 (s, 1H), 4.09 - 4.01 (m, 4H), 2.99 - 2.86 (m, 2H), 2.49 - 2.37 (m, 3H), 1.97 - 1.83 (m, 4H), 1.83 - 1.72 (m, 2H), 1.69 - 1.54 (m, 2H), 1.18 (s, 9H).

[0186] Example 19 Synthesis of proteolysis agent 19

[0187]

[0188] The synthesis method refers to Example 14, and 1-1 is replaced by 4-(4- methoxyphenyl)-5,7-dihydroxy-2H-l-benzopyran-2-one to obtain proteolysis agent 19 (white solid, yield 36%). 1 H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.35 (s, 1H), 7.44 - 7.36 (m, 3H), 7.25 - 7.17 (m, 2H), 6.72 (s, 1H), 6.52 (d, J = 2.4 Hz, 1H), 6.23 (d, J = 2.4 Hz, 1H), 5.87 (s, 1H), 4.09 - 4.01 (m, 4H), 2.99 - 2.86 (m, 2H), 2.49 - 2.37 (m, 3H), 1.97 - 1.83 (m, 4H), 1.83 - 1.72 (m, 2H), 1.69 - 1.54 (m, 2H), 1.18 (s, 9H).

[0189] Example 20 Synthesis of proteolysis agent 20

[0190]

[0191] The synthesis method refers to Example 14, and 1-1 is replaced by 5,7- dihydroxy-4-methylcoumarin and 1,3-dibromopropane is replaced by 1,2- dibromoethane to obtain proteolysis agent 20 (white solid, yield 42%). 1H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.70 (s, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 6.45 (d, J = 2.4 Hz, 1H), 6.32 (d, J = 2.5 Hz, 1H), 5.95 - 5.93 (m, 1H), 4.11 (t, J = 5.7 Hz, 2H), 4.05 (s, 2H), 3.02 - 2.93 (m, 2H), 2.69 (t, J = 5.7 Hz, 2H), 2.52 (s, 3H), 2.48 - 2.42 (m, 1H), 2.11 - 2.01 (m, 2H), 1.81 - 1.71 (m, 2H), 1.68 - 1.55 (m, 2H), 1.17 (s, 9H).

[0192] Example 21 Synthesis of proteolysis agent 21

[0193]

[0194] The synthesis method of Example 14 was followed, replacing 1-1 with 5,7-dihydroxy-4- methylcoumarin and 1,3-dibromopropane with 1,4-dibromobutane to obtain proteolysis agent 21 (white solid, yield 37%). 1 H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.70 (s, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 6.45 (d, J = 2.4 Hz, 1H), 6.32 (d, J = 2.5 Hz, 1H), 5.95 - 5.93 (m, 1H), 4.11 (t, J = 5.7 Hz, 2H), 4.05 (s, 2H), 3.02 - 2.93 (m, 2H), 2.69 (t, J = 5.7 Hz, 2H), 2.52 (s, 3H), 2.48 - 2.42 (m, 1H), 2.11 - 2.01 (m, 2H), 1.81 - 1.71 (m, 2H), 1.68 - 1.55 (m, 2H), 1.17 (s, 9H).

[0195] Example 22 Synthesis of proteolysis agent 22

[0196]

[0197] The synthesis method of Example 14 was followed, replacing 1-1 with 5,7-dihydroxy-4- methylcoumarin and 1,3-dibromopropane with 1,5-dibromopentane to obtain proteolysis agent 22 (white solid, yield 39%). 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.70 (s, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 6.42 (d, J = 2.4 Hz, 1H), 6.31 (d, J = 2.5 Hz, 1H), 5.96 - 5.92 (m, 1H), 4.05 (s, 2H), 3.99 (t, J = 6.5 Hz, 2H), 2.94 - 2.86 (m, 2H), 2.48 - 2.40 (m, 1H), 2.32 - 2.24 (m, 2H), 1.93 - 1.82 (m, 2H), 1.81 - 1.68 (m, 4H), 1.66 - 1.54 (m, 2H), 1.52 - 1.35 (m, 4H), 1.18 (s, 9H).

[0198] Example 23 Synthesis of proteolysis agent 23

[0199]

[0200] The synthesis method of Example 14 was followed, replacing 1-1 with 5,7-dihydroxy-4- methylcoumarin and 1,3-dibromopropane with 2,2'-dibromo-diethyl ether, to obtain proteolysis agent 23 (white solid, yield 30%). 1 H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.70 (s, 1H), 7.38 (s, 1H), 6.72 (s, 1H), 6.42 (d, J = 2.4 Hz, 1H), 6.31 (d, J = 2.5 Hz, 1H), 5.96 - 5.92 (m, 1H), 4.05 (s, 2H), 3.99 (t, J = 6.5 Hz, 2H), 2.94 - 2.86 (m, 2H), 2.48 - 2.40 (m, 1H), 2.32 - 2.24 (m, 2H), 1.93 - 1.82 (m, 2H), 1.81 - 1.68 (m, 4H), 1.66 - 1.54 (m, 2H), 1.52 - 1.35 (m, 4H), 1.18 (s, 9H).

[0201] Example 24 Synthesis of proteolysis agent 24

[0202]

[0203] First step

[0204] Into a three-necked flask, 24-1 (1.0 g, 2.23 mmol), K2HPO4(1.2 g, 6.70 mmol), X-Phos (212.9 mg, 0.45 mmol), Pd2(dba)3(204.5 mg, 0.22 mmol) were placed under nitrogen protection, 20 ml 1,4-dioxane, 4 ml water were added, then 24-2 (702.6 mg, 2.68 mmol) was added, stirred at 85 °C overnight, TLC monitored the reaction was complete, cooled to room temperature, 40 ml dichloromethane, 40 ml water were added, extracted with dichloromethane, saturated brine was washed, dried over anhydrous sodium sulfate, silica gel column chromatography, to give compound 24-3 (306.0 mg, white solid, yield 27%). 1 H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.23 (s, 1H), 7.97 (s, 1H), 7.67 (s, 1H), 7.46 - 7.39 (m, 1H), 6.76 (d, J = 7.9 Hz, 1H), 3.94 (s, 2H), 3.48 - 3.38 (m, 1H), 3.28 - 3.19 (m, 1H), 2.88 (s, 2H), 1.92 - 1.85 (m, 2H), 1.82 - 1.76 (m, 2H), 1.38 (s, 9H), 1.27 (s, 6H), 1.25 - 1.23 (m, 2H), 1.23 - 1.21 (m, 2H).

[0205] Second step

[0206] 24-3 (50 mg, 0.10 mmol) was dissolved in 1 ml dichloromethane, 0.25 ml 4M hydrochloric acid dioxane solution was added, stirred at room temperature for 1 h, the solvent was rotary evaporated, to give 24-4 (46.3 mg, white solid, yield 98%). 1 H NMR (400 MHz, DMSO-d6) δ 9.08 (s, 1H), 8.23 (s, 1H), 7.97 (s, 1H), 7.67 (s, 1H), 7.46 - 7.39 (m, 1H), 6.76 (d, J = 7.9 Hz, 1H), 3.94 (s, 2H), 3.48 - 3.38 (m, 1H), 3.28 - 3.19 (m, 1H), 2.88 (s, 2H), 1.92 - 1.85 (m, 2H), 1.82 - 1.76 (m, 2H), 1.38 (s, 9H), 1.27 (s, 6H), 1.25 - 1.23 (m, 2H), 1.23 - 1.21 (m, 2H).

[0207] Third step

[0208] To a solution of 3-1 (1.0 g, 5.20 mmol) in 17 ml DMF, 24-5 (2.0 g, 10.41 mmol), potassium carbonate (1.4 g, 10.41 mmol) was added and stirred at room temperature for 6 h to give 24-6 (325.0 mg, white solid, yield 20%). 1 H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 6.34 (d, J = 2.3 Hz, 1H), 6.26 (d, J = 2.4 Hz, 1H), 5.86 - 5.85 (m, 1H), 4.01 (t, J = 5.9 Hz, 2H), 3.60 (s, 3H), 2.49 (d, J = 1.2 Hz, 3H), 2.41 (t, J = 7.1 Hz, 2H), 1.84 - 1.76 (m, 2H), 1.75 - 1.68 (m, 2H).

[0209] Fourth step

[0210] To a solution of 24-6 (325.0 mg, 1.06 mmol) in 2 ml tetrahydrofuran, 2 ml water, lithium hydroxide monohydrate (68.4 mg, 1.63 mmol) was added and stirred at room temperature for 3 h. The solvent was evaporated and 4 ml water was added. The pH was adjusted to 4-5 with glacial acetic acid. A large amount of white solid precipitated. Filtration under suction gave 24-7 (132.2 mg, white solid, yield 61%). 1 H NMR (400 MHz, DMSO-d6) δ 6.34 (d, J = 2.3 Hz, 1H), 6.31 (d, J = 2.2 Hz, 1H), 5.94 - 5.92 (m, 1H), 4.01 (t, J = 6.1 Hz, 2H), 2.50 (d, J = 1.2 Hz, 3H), 2.31 (t, J = 7.2 Hz, 2H), 1.85 - 1.76 (m, 2H), 1.73 - 1.63 (m, 2H).

[0211] Fifth step

[0212] To a solution of 24-4 (46.3 mg, 0.10 mmol), 24-7 (28.4 mg, 0.10 mmol), DIPEA (85 μL, 0.49 mmol) in 1 ml dichloromethane, HATU (55.5 mg, 0.15 mmol) was added and stirred at room temperature for 1 h. Silica gel column chromatography gave proteolysis agent 24 (12.9 mg, white solid, yield 20%). 1H NMR (400 MHz, DMSO-d6) δ 10.55 (s, 1H), 9.08 (s, 1H), 8.23 (s, 1H), 7.97 (s, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.68 (s, 1H), 7.47 - 7.42 (m, 1H), 6.34 (d, J = 2.2 Hz, 1H), 6.31 (d, J = 2.1 Hz, 1H), 5.95 - 5.93 (m, 1H), 4.01 (t, J = 6.0 Hz, 2H), 3.94 (s, 2H), 3.58 - 3.51 (m, 1H), 3.49 - 3.43 (m, 1H), 2.88 (s, 2H), 2.12 (t, J = 7.0 Hz, 2H), 1.94 - 1.87 (m, 2H), 1.82 - 1.73 (m, 4H), 1.72 - 1.65 (m, 2H), 1.29 - 1.22 (m, 10H).

[0213] Synthesis of protein degrader 25

[0214]

[0215] First step

[0216] Dissolve 25-1 (2.0 g, 24.07 mmol), 25-2 (4.0 g, 25.27 mmol) in 60 ml glacial acetic acid, reflux for 3 h, cool to room temperature, spin off the solvent, add ethyl acetate, suction filter, to obtain 25-3 (3.6 g, white solid, yield 84%). 1 H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 7.83 (d, J = 2.0 Hz, 1H), 6.11 (d, J = 2.0 Hz, 1H), 5.59 (s, 1H), 2.57 - 2.49 (m, 2H), 1.74 - 1.61 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H).

[0217] Second step

[0218] Dissolve 25-3 (3.6 g, 20.29 mmol) in 50 ml acetonitrile, drop in phosphorus oxychloride (7.6 ml, 81.15 mmol), then add pyridine (2.0 ml, 24.35 mmol), DMAP (123.9 mg, 1.01 mmol), reflux for 3 h, spin off the solvent, add ice water, extract with ethyl acetate, dry over anhydrous sodium sulfate, silica gel column chromatography, to obtain 25-4 (2.2 g, colorless oil, yield 55%). 1HNMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 2.2 Hz, 1H), 6.86 (s, 1H), 6.69 (d, J = 2.2 Hz, 1H), 2.81 (t, J = 7.6, 1.3 Hz, 2H), 1.90 - 1.76 (m, 2H), 1.05 - 0.98 (m, 3H).

[0219] Third Step

[0220] Dissolve 25-4 (235 mg, 1.20 mmol), 25-5 (250.0 mg, 1.25 mmol) in 4 ml acetonitrile, add potassium carbonate (332.0 mg, 2.40 mmol), stir at 60 °C for 3 h, cool, spin off acetonitrile, silica gel column chromatography to obtain 25-6 (342.9 mg, white solid, yield 79%). 1 H NMR (400 MHz, Chloroform-d) δ 7.95 (d, J = 2.2 Hz, 1H), 6.41 (d, J = 2.3 Hz, 1H), 6.22 (d, J = 6.8 Hz, 1H), 5.80 (s, 1H), 4.64 - 4.57 (m, 1H), 4.23 - 4.10 (m, 2H), 2.74 - 2.68 (m, 2H), 2.43 - 2.21 (m, 2H), 2.18 - 2.02 (m, 2H), 1.86 - 1.67 (m, 3H), 1.65 - 1.52 (m, 1H), 1.47 (s, 9H), 1.02 (t, J = 7.3 Hz, 3H).

[0221] Fourth Step

[0222] Dissolve 25-6 (342.9 mg, 0.95 mmol) in 3 ml dichloromethane, add 2.4 ml 4M hydrochloric acid dioxane solution, after TLC monitoring reaction is complete, suction filter to obtain 25-7 (227.8 mg, white solid, yield 92%). 1 H NMR (400 MHz, DMSO-d6) δ 9.96 (d, J = 8.3 Hz, 1H), 8.51 - 8.38 (m, 3H), 8.32 (d, J = 2.2 Hz, 1H), 6.82 (s, 1H), 6.57 (d, J = 2.2 Hz, 1H), 4.90 - 4.77 (m, 1H), 3.79 - 3.69 (m, 1H), 2.83 (t, J = 7.6 Hz, 2H), 2.29 - 2.13 (m, 4H), 1.89 - 1.77 (m, 3H), 1.76 - 1.64 (m, 1H), 0.96 (t, J = 7.3 Hz, 3H).

[0223] Fifth Step

[0224] To a solution of 25-7 (20 mg, 0.07 mmol), 24-7 (19.8 mg, 0.07 mmol), DIPEA (35 μL, 0.20 mmol) in 1 ml of dichloromethane was added HATU (38.6 mg, 0.10 mmol) and stirred at room temperature for 1 h. Silica gel column chromatography gave proteolysis agent 25 (13.1 mg, white solid, yield 36%). 1 H NMR (400 MHz, Chloroform-d) δ 7.93 (d, J = 2.3 Hz, 1H), 6.97 (d, J = 7.1 Hz, 1H), 6.42 (d, J = 2.1 Hz, 1H), 6.39 - 6.35 (m, 2H), 6.31 (d, J = 2.2 Hz, 1H), 5.85 (s, 1H), 5.81 - 5.79 (m, 1H), 4.53 - 4.41 (m, 1H), 4.28 - 4.17 (m, 1H), 3.94 - 3.86 (m, 2H), 3.74 - 3.59 (m, 2H), 3.12 (q, J = 7.4 Hz, 2H), 2.72 - 2.63 (m, 2H), 2.48 - 2.44 (m, 3H), 2.35 - 2.30 (m, 2H), 2.17 - 2.12 (m, 2H), 1.79 - 1.64 (m, 6H), 0.96 (t, J = 7.3 Hz, 3H).

[0225] Example 26 Synthesis of proteolysis agent 26

[0226]

[0227] The synthesis method was referred to Example 1, and 3-bromo-1-propanol was replaced by 2-bromoethanol to obtain proteolysis agent 26 (white solid, yield 45%). 1 H NMR (400 MHz, Chloroform-d) δ 7.93 (d, J = 2.3 Hz, 1H), 6.97 (d, J = 7.1 Hz, 1H), 6.42 (d, J = 2.1 Hz, 1H), 6.39 - 6.35 (m, 2H), 6.31 (d, J = 2.2 Hz, 1H), 5.85 (s, 1H), 5.81 - 5.79 (m, 1H), 4.53 - 4.41 (m, 1H), 4.28 - 4.17 (m, 1H), 3.94 - 3.86 (m, 2H), 3.74 - 3.59 (m, 2H), 3.12 (q, J = 7.4 Hz, 2H), 2.72 - 2.63 (m, 2H), 2.48 - 2.44 (m, 3H), 2.35 - 2.30 (m, 2H), 2.17 - 2.12 (m, 2H), 1.79 - 1.64 (m, 6H), 0.96 (t, J = 7.3 Hz, 3H).

[0228] Example 27 Synthesis of proteolysis agent 27

[0229]

[0230] The synthetic method was referred to Example 1, and 3-bromo-1-propanol was replaced by 4-bromo-1-butanol to obtain proteolysis inhibitor 27 (white solid, yield 22%). 1 H NMR (400 MHz, Chloroform-d) δ 7.38 - 7.29 (m, 4H), 7.25 - 7.20 (m, 2H), 6.59 (s, 1H), 6.23 (d, J = 2.0 Hz, 1H), 6.16 (d, J = 2.2 Hz, 1H), 5.86 (s, 1H), 3.98 (s, 2H), 3.67 - 3.58 (m, 2H), 2.98 - 2.90 (m, 2H), 2.53 - 2.42 (m, 1H), 2.22 - 2.13 (m, 2H), 2.12 - 1.97 (m, 2H), 1.95 - 1.82 (m, 4H), 1.24 (s, 9H), 1.14 - 1.03 (m, 4H).

[0231] Example 28 Synthesis of proteolysis inhibitor 28

[0232]

[0233] The synthetic method was referred to Example 1, and 3-bromo-1-propanol was replaced by 5-bromo-1-pentanol to obtain proteolysis inhibitor 28 (white solid, yield 15%). 1 H NMR (400 MHz, Chloroform-d) δ 7.38 - 7.29 (m, 4H), 7.25 - 7.20 (m, 2H), 6.59 (s, 1H), 6.23 (d, J = 2.0 Hz, 1H), 6.16 (d, J = 2.2 Hz, 1H), 5.86 (s, 1H), 3.98 (s, 2H), 3.67 - 3.58 (m, 2H), 2.98 - 2.90 (m, 2H), 2.53 - 2.42 (m, 1H), 2.22 - 2.13 (m, 2H), 2.12 - 1.97 (m, 2H), 1.95 - 1.82 (m, 4H), 1.24 (s, 9H), 1.14 - 1.03 (m, 4H).

[0234] Example 29 Synthesis of proteolysis inhibitor 29

[0235]

[0236] The synthetic method was referred to Example 1, and 3-bromo-1-propanol was replaced by 2-(2-bromoethoxy)ethanol to obtain proteolysis inhibitor 29 (white solid, yield 33%). 1H NMR (400 MHz, Methanol-d4) δ 7.49 - 7.39 (m, 3H), 7.37 - 7.30 (m, 3H), 6.69 (s, 1H), 6.43 (d, J = 2.2 Hz, 1H), 6.31 (d, J = 2.3 Hz, 1H), 5.86 (s, 1H), 4.00 (s, 2H), 3.85 (t, J = 4.6 Hz, 2H), 3.10 (t, J = 4.5 Hz, 2H), 3.06 - 2.99 (m, 2H), 2.54 (t, J = 5.6 Hz, 2H), 2.51 - 2.45 (m, 1H), 2.23 - 2.12 (m, 2H), 1.91 - 1.78 (m, 4H), 1.26 (s, 9H).

[0237] Example 30 Synthesis of proteolysis inhibitor 30

[0238]

[0239] The synthesis method of Example 14 was followed, replacing 1,3-dibromopropane with 1,2-dibromoethane to obtain proteolysis inhibitor 30 (white solid, yield 25%). 1 H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 10.30 (s, 1H), 7.44 - 7.31 (m, 6H), 6.72 (s, 1H), 6.56 (d, J = 2.4 Hz, 1H), 6.21 (d, J = 2.5 Hz, 1H), 5.85 (s, 1H), 4.13 (t, J = 5.8 Hz, 2H), 4.05 (s, 2H), 3.02 - 2.93 (m, 2H), 2.70 (t, J = 5.7 Hz, 2H), 2.48 - 2.41 (m, 1H), 2.06 (t, J = 11.4 Hz, 2H), 1.82 - 1.72 (m, 2H), 1.69 - 1.55 (m, 2H), 1.18 (s, 9H).

[0240] Example 31 Synthesis of proteolysis inhibitor 31

[0241]

[0242] The synthesis method of Example 14 was followed, replacing 1,3-dibromopropane with 1,4-dibromobutane to obtain proteolysis inhibitor 31 (white solid, yield 28%). 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.30 (s, 1H), 7.42 - 7.31 (m, 6H), 6.72 (s, 1H), 6.53 (d, J = 2.4 Hz, 1H), 6.20 (d, J = 2.5 Hz, 1H), 5.84 (s, 1H), 4.09 - 4.00 (m, 4H), 2.97 - 2.85 (m, 2H), 2.49 - 2.40 (m, 1H), 2.36 - 2.27 (m, 2H), 1.95 - 1.83 (m, 2H), 1.79 - 1.67 (m, 4H), 1.66 - 1.52 (m, 4H), 1.17 (s, 9H).

[0243] Example 32 Synthesis of proteolysis agent 32

[0244]

[0245] The synthesis method of Example 14 was followed, replacing 1,3-dibromopropane with 1,5-dibromopentane to obtain proteolysis agent 32 (white solid, yield 31%). 1 H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.30 (s, 1H), 7.42 - 7.31 (m, 6H), 6.72 (s, 1H), 6.53 (d, J = 2.4 Hz, 1H), 6.20 (d, J = 2.5 Hz, 1H), 5.84 (s, 1H), 4.09 - 4.00 (m, 4H), 2.97 - 2.85 (m, 2H), 2.49 - 2.40 (m, 1H), 2.36 - 2.27 (m, 2H), 1.95 - 1.83 (m, 2H), 1.79 - 1.67 (m, 4H), 1.66 - 1.52 (m, 4H), 1.17 (s, 9H).

[0246] Example 33 Synthesis of proteolysis agent 33

[0247]

[0248] The synthesis method of Example 14 was followed, replacing 1,3-dibromopropane with 2,2'-dibromo-diethyl ether to obtain proteolysis agent 33 (white solid, yield 39%). 1H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.31 (s, 1H), 7.44 - 7.30 (m, 6H), 6.72 (s, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.21 (d, J = 2.4 Hz, 1H), 5.85 (s, 1H), 4.18 - 4.12 (m, 2H), 4.05 (s, 2H), 3.76 - 3.69 (m, 2H), 3.58 (t, J = 5.9 Hz, 2H), 2.98 - 2.86 (m, 2H), 2.48 - 2.38 (m, 1H), 2.03 - 1.91 (m, 2H), 1.78 - 1.69 (m, 2H), 1.65 - 1.50 (m, 2H), 1.17 (s, 9H).

[0249] Example 34 Synthesis of Protein Degrader 34

[0250]

[0251] The synthesis method of Example 4 was followed, with 4-2 replaced by ethyl trifluoroacetylacetate and 3-bromo-1-propanol replaced by 5-bromo-1-pentanol to give Protein Degrader 34 (white solid, yield 20%). 1 H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 10.31 (s, 1H), 7.44 - 7.30 (m, 6H), 6.72 (s, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.21 (d, J = 2.4 Hz, 1H), 5.85 (s, 1H), 4.18 - 4.12 (m, 2H), 4.05 (s, 2H), 3.76 - 3.69 (m, 2H), 3.58 (t, J = 5.9 Hz, 2H), 2.98 - 2.86 (m, 2H), 2.48 - 2.38 (m, 1H), 2.03 - 1.91 (m, 2H), 1.78 - 1.69 (m, 2H), 1.65 - 1.50 (m, 2H), 1.17 (s, 9H).

[0252] Biological experiments

[0253] Cell IC 50 Determination

[0254] Each well of a 96-well plate was seeded with 10000 cells in 100 μΐ fresh medium. After incubation with compounds for 72 hours, 10 μΐ Cell Counting Kit-8 (CCK8) was added to each well. After incubation at 37 °C for 2 h, the absorbance of each well was measured at 450 nm with SpectraMAX 190 (Molecular Devices) and IC values were calculated with SoftMax Pro. 50

[0255] ​The positive control compounds are SNS-032 and THAL-SNS-032, whose structures are as follows.

[0256]

[0257] The results are shown in Table 1.

[0258] Table 1 Inhibitory activity on related cell lines

[0259]

[0260] Note: IC of compound with * 50 The human lymphoma cell line WSU-DLCL2 was used for determination, and the human lymphoma cell line U-2932 was used for the rest of the compounds.

[0261] Western Blot experiment

[0262] An appropriate amount of human lymphoma cell line U-2932 was seeded in a 6-well plate, and after drug treatment, the cells were centrifuged and collected for subsequent experiments. According to the amount of cells, the corresponding amount of 1X loading buffer was added, and after lysis, the sample was boiled at 100°C for 20 min. The same volume of sample was taken and subjected to SDS-PAGE. After electrophoresis, the protein on the gel was transferred to a nitrocellulose membrane using a KINFEI fast transfer instrument. According to the size of the protein, the corresponding band was cut off, and the TBST containing 5% skim milk was blocked for 1 h. The primary antibody was incubated at 4°C overnight. The excess primary antibody was washed with TBST for 10 min each time for a total of three times. The secondary antibody was incubated at room temperature for 1 h. The excess secondary antibody was washed with TBST for 10 min each time for a total of three times. Finally, the band was developed and photographed using a Bio-Rad colorimeter. The results are shown in Figure 1 and Figure 3 . Figure 1 The CDK9 and Mcl-1 protein levels in U-2932 cells treated with different compounds for 24 h were analyzed. Figure 3 Western Blot detection of U-2932 cells treated with 100 nM concentration of compounds 10 (A) and 16 (B) for 24 hours. The compounds can degrade other CDKs, such as CDK1, CDK4, and CDK6.

[0263] Co-IP

[0264] The drug-treated cells were centrifuged and collected, and then lysed with an appropriate amount of NP-40 lysis solution containing phosphatase and protease inhibitors on ice for 60 min. After centrifugation at 12000g and 4°C for 10 min, the supernatant was quantified by BCA. The adjusted sample was divided into IgG and IP groups and incubated with the corresponding antibody at 4°C overnight. The next day, protein A / G magnetic beads were added and incubated for 4 h. After washing with NP-40 and PBS for three times each, the sample was boiled and subjected to Western Blot.Figure 2 Co-IP experiments demonstrated that compounds induced CDK9 and LC3B binding in U-2932 cells (A) and 293T cells (B) treated with 500 nM of compounds 10 and 16 in the presence of 500 nM Baf Al for 12 hours.

[0265] The above embodiments are only used to illustrate the technical solutions of the present application, but not limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, it is to be understood that modifications and / or substitutions can be made to the technical solutions described in the foregoing embodiments, or some or all of the technical features can be replaced by equivalent features, without departing from the spirit and scope of the present application as defined in the appended claims.

Claims

1. A protein degrading agent or a pharmaceutically acceptable salt thereof, wherein, The protein degrading agent is selected from any one of the following structures I-1 to I-6: wherein n is 1, 2, 3, 4, 5 or 6; and m is 1, 2 or 3. R 3 and R 5 are each independently selected from H and OH; In formulae I-1 and I-2, R 2 is selected from hydrogen, C 1-6 alkyl, haloC 1-6 alkyl; In formulae I-3 and I-6, R 2 is selected from hydrogen, C 1-6 alkyl, haloC 1-6 alkyl, C 6-10 aryl, haloC 6-10 aryl, oxoC 6-10 aryl, 3-7 membered heteroaryl.

2. The protein degrading agent or a pharmaceutically acceptable salt thereof according to claim 1, wherein, In formulae I-1 and I-2, R 2 is selected from hydrogen, C 1-3 alkyl, haloC 1-3 alkyl; In formulae I-3 and I-6, R 2 is selected from hydrogen, C 1-3 alkyl, haloC 1-3 alkyl, phenyl, halo- phenyl, oxo-phenyl, pyridyl.

3. A protein degradation agent, or a pharmaceutically acceptable salt thereof, wherein, The protein degrading agent is selected from any one of the following structures I-1 to I-6: 。 4. A pharmaceutical composition comprising: one or more of the protein degrading agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, and optionally a pharmaceutically acceptable carrier.

5. Use of the protein degrading agent or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the manufacture of a medicament for treating, preventing or ameliorating a CDK-related disease or disorder.

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