A method for preparing a CDK2 / 4 / 6 protein hydrolysis targeting chimera, a pharmaceutical composition and its uses

Abstract

This invention discloses a CDK2 / 4 / 6 protein hydrolysis targeting chimera, its preparation method, pharmaceutical composition, and uses. The chemical structure of the CDK2 / 4 / 6 protein hydrolysis targeting chimera is shown in Formula I. The CDK2 / 4 / 6 protein hydrolysis targeting chimera or its isomers, pharmaceutically acceptable salts, solvates, prodrugs, deuterated compounds, or polymorphs can induce the selective degradation of CDK2, CDK4, and CDK6 through ubiquitin-proteasome, thereby reducing the phosphorylation level of RB1, inhibiting the release of transcription factor E2F, blocking the cell progression from G1 phase to S phase, and reducing the cell proliferation of cancer cell lines. Compared with inhibitors, it is more efficient and less toxic, and can be used to treat a variety of cancers.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a CDK2 / 4 / 6 protein hydrolysis targeting chimera, its preparation method, pharmaceutical composition, and uses. Background Technology

[0002] Cancer remains a major public health problem worldwide and a leading contributor to the global disease burden. Lung cancer is the most common cancer globally, followed by breast cancer, colorectal cancer, prostate cancer, and stomach cancer.

[0003] Cyclin-dependent kinases (CDKs) and cyclin chaperones synergistically regulate cell cycle progression and transcription, ensuring the orderly conduct of cell cycle events. CDK2, CDK4, and CDK6 are involved in the crucial transition from G1 to S phase, playing a key role in the occurrence and development of various cancers. In the development of drugs targeting CDKs, the most advanced area is the development of CDK4 / 6 inhibitors. Currently, selective CDK4 / 6 inhibitors such as Palbociclib (PD-0332991), Ribociclib (LEE011), and Abemaciclib (LY2835219) have been approved for the treatment of breast cancer. However, natural and acquired resistance to CDK4 / 6 inhibitors remains a challenging problem in clinical practice. Increased activity of the Cyclin-E-CDK2 complex is one of the reasons for CDK4 / 6 inhibitor resistance. Studies have shown that the combination of CDK2 inhibitors and CDK4 / 6 inhibitors is effective against cell lines resistant to CDK4 / 6 inhibitors due to Cyclin E overexpression. As a result, a series of targeted inhibitors against CDK2 / 4 / 6 have been developed.

[0004] Proteolytic targeting chimeras (PROTACs), as a novel drug development technology, are characterized by high efficiency, high selectivity, targeting of untreatable targets, and the ability to overcome acquired drug resistance. A PROTAC consists of a linker and ligands at both ends: one ligand recruits and binds to the target protein (POI), while the other recruits and binds to an E3 ubiquitin ligase. The PROTAC simultaneously binds to the POI and the ligase, inducing POI ubiquitination. The POI is then degraded by the ubiquitin-proteasome system (UPS), after which the PROTAC is released and enters the next round of POI degradation. Compared to traditional small molecule inhibitors, PROTAC molecules have the following advantages: 1. They have the potential to target untreatable targets of small molecule inhibitors; 2. They overcome drug resistance caused by mutations by clearing pathology-related proteins; 3. Their catalytic mechanism and event-driven pharmacology allow for high efficiency at low doses; 4. They achieve high selectivity through protein-protein interactions between the target protein and the E3 ubiquitin ligase.

[0005] Recent studies have shown that designing PROTAC molecules targeting CDK4 / 6 can overcome CDK4 / 6 inhibitor resistance caused by CDK6 overexpression. Therefore, based on the unique mechanism of action of PROTAC degraders, the development of CDK2 / 4 / 6 PROTAC molecules has a better application prospect than CDK2 / 4 / 6 inhibitors.

[0006] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to provide a CDK2 / 4 / 6 protein hydrolysis targeting chimera, its preparation method, pharmaceutical composition, and uses. By inducing selective degradation of CDK2, CDK4, and CDK6 through ubiquitin-proteasome, the phosphorylation level of RB1 is reduced, thereby inhibiting the release of transcription factor E2F, blocking the cell progression from G1 phase to S phase, and reducing cell proliferation in cancer cell lines. Compared with inhibitors, it is more efficient and less toxic, and can be used to treat a variety of cancers.

[0008] To achieve the above objectives, the present invention provides a CDK2 / 4 / 6 protein hydrolysis targeting chimera or its isomers, pharmaceutically acceptable salts, solvates, prodrugs, deuterated compounds, or polymorphs, wherein the chemical structure of the chimera is shown in Formula I:

[0009]

[0010] R1 is selected from any of the following structures: hydrogen, deuterium, cyano, halogen, amino, hydroxyl, nitro, CHF2, CF3, C(O)R a C(O)NHR a C(O)N(R) a 2. In the presence of C1-C6 alkyl, C1-C6 deuterated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, 3-8 membered cycloalkyl, C6-C10 aryl or 5-10 membered heteroaryl, or at least one H in the amino, C1-C6 alkyl, C1-C6 deuterated alkyl, C2-C6 alkenyl, C1-C6 alkoxy, 3-8 membered cycloalkyl, C6-C10 aryl or 5-10 membered heteroaryl groups is optionally substituted with Ra;

[0011] Wherein, the R a The group is selected from deuterium, nitro, cyano, hydroxyl, amino, trifluoromethyl, halogen, C1-C6 alkyl or C1-C6 deuterated alkyl, or at least one H in the C1-C6 alkyl and C1-C6 deuterated alkyl groups is optionally substituted with a halogen;

[0012] R2 is selected from hydrogen, halogen, C1-C6 alkyl or C1-C6 deuterated alkyl, wherein at least one H in the C1-C6 alkyl group is optionally substituted with a halogen;

[0013] R3 is selected from hydrogen, C1-C8 alkyl, C1-C8 alkoxy, 3-7 membered cycloalkyl, C3-C8 alkenyl, 5-8 membered cycloalkenyl, 6-10 membered aryl, 5-10 membered heteroaryl, or at least one H from the aryl, C1-C8 alkyl, C1-C8 alkoxy, 3-7 membered cycloalkyl, C3-C8 alkenyl, 5-8 membered cycloalkenyl, 6-10 membered aryl, 5-10 membered heteroaryl groups optionally replaced by R. a replace;

[0014] R4 is selected from any of the following structures:

[0015]

[0016] Where m and f are each an integer from 1 to 4, n is an integer from 0 to 14, W and Q are each selected from CH or N, and L is selected from O or S;

[0017] Y is selected from -S(O)2-, -SO-, -S(O)2NR e -, -S-, -CH2- or -C(O)-; where R eThe hydrogen atom is selected from hydrogen, deuterium, C1-C6 alkyl, C1-C6 deuterated alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C6-C10 aryl or 5-10 heteroaryl, or at least one H atom selected from the C1-C6 alkyl, C1-C6 deuterated alkyl, C2-C6 alkenyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C6-C10 aryl or 5-10 heteroaryl groups is optionally replaced by R. a replace;

[0018] R5 is selected from any of the following structures:

[0019] Where f1, f3, and m3 are each an integer from 1 to 4, and W1 and Q1 are each independently selected from CH or N;

[0020] Linker can be selected from any of the following structures:

[0021]

[0022] f2 and m1 are each an integer from 1 to 4, j is an integer from 0 to 14, n1 is an integer from 1 to 14, W2 and Q2 are each independently selected from CH or N, and L1 is selected from O or S.

[0023] The E3 ubiquitin ligase ligand is selected from any of the following structures:

[0024]

[0025] In formula i, ring A is selected from any of the following structures:

[0026]

[0027] Where R b and R f Each of the following structures is independently selected: hydrogen, alkyl, alkoxy, halogen, haloalkyl, haloalkoxy, or cyano, or R b and R f The combination forms -C=O;

[0028] R c R d Each of the following structures is independently selected: hydrogen, alkyl, alkoxy, halogen, haloalkyl, haloalkoxy, or cyano;

[0029] G is selected from a single covalent bond, -O-, -C(=O)-, or -NH-;

[0030] The C-ring is selected from the following structures: m2 is an integer from 1 to 4;

[0031] R h Selected from hydrogen or alkyl;

[0032] In formula ii, W3 is selected from CH or N;

[0033] Z is selected from a single covalent bond connecting the W and B rings, -CH2-, -NH-, -O-, or -NHC(O)-, with the NH end of -NHC(O)- connected to W;

[0034] X is selected from a single covalent bond connecting the Linker and E3 ubiquitin ligase, alkylene, -O-, -(O-alkylene)-, -(alkylene-O)-, -NH-, -N(alkyl)-, -C(O)NH-;

[0035] The B ring is selected from C6-C10 monocyclic aryl, C5-C10 monocyclic heteroaryl, C9-C10 fused bicyclic heteroaryl, or at least one H in the C6-C10 monocyclic aryl, C5-C10 monocyclic heteroaryl, or C9-C10 fused bicyclic heteroaryl group, and is optionally replaced by R. a replace.

[0036] The terminology used in this invention is explained as follows:

[0037] The term "deuterated" as used in this invention refers to the substitution of one or more hydrogen atoms in the mentioned group by deuterium atoms;

[0038] As used in this invention, the term "halogen" refers to fluorine, chlorine, bromine, or iodine;

[0039] As used in this invention, the term "alkyl" refers to a branched or straight-chain saturated aliphatic hydrocarbon group having a specific number of carbon atoms. For example, the definition of "C1-C6" in "C1-C6 alkyl" includes saturated aliphatic hydrocarbon groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a straight or branched manner. Specifically, "C1-C6 alkyl" includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl, and hexyl.

[0040] As used in this invention, the term "alkylene" refers to a straight-chain or branched divalent saturated hydrocarbon group composed of carbon and hydrogen.

[0041] As used in this invention, the term "alkoxy" refers to a straight-chain or branched alkoxy group with the structural formula alkyl-O-. Optionally, the alkyl portion of the alkoxy group may contain 1-10 carbon atoms. Representative examples of "alkoxy" include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentooxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, etc. The term "C1-C6 alkoxy" refers to a straight-chain or branched alkoxy group containing 1 to 6 carbon atoms.

[0042] As used in this invention, the term "heteroaryl" refers to a 5- to 10-membered monocyclic, bicyclic, or polycyclic group containing at least one aromatic ring having one or more (e.g., 1 to 6, 1 to 5, 1 to 4, or 1 to 3) heteroatoms independently selected from oxygen, nitrogen, and sulfur. Bicyclic or polycyclic heteroaryls include bicyclic, tricyclic, or tetracyclic heteroaryls, wherein one ring is an aromatic ring having one or more heteroatoms independently selected from O, S, and N, and the other rings may be saturated, partially unsaturated, or aromatic rings and may be carbocyclic or contain one or more heteroatoms independently selected from O, S, and N;

[0043] As used in this invention, the term "aryl" refers to a monovalent aromatic hydrocarbon group comprising 5 to 20 (e.g., 5 to 14, 5 to 15, 5 to 10, 5 to 9, 5 to 8, 6 to 15, or 6 to 10) carbon atoms and optionally comprising one or more fused rings, such as phenyl, thioyl, or fluorenyl. Optionally, in this disclosure, the "aryl" is a substituted aryl group;

[0044] As used in this invention, the term "cycloalkyl" refers to a monocyclic, bicyclic, tricyclic, or polycyclic hydrocarbon group that is saturated or partially unsaturated (i.e. has one or more double bonds, but is not fully conjugated) and has 3 to 8 carbon atoms in some embodiments.

[0045] In some embodiments, a polymorphic form of the chimera of the present invention or a salt of the compound disclosed herein is also provided. The salt of the chimera of the present invention can be a pharmaceutically acceptable salt, including but not limited to hydrohalides (including hydrochlorides and hydrobroms), sulfates, citrates / citric acid salts, maleates, methanesulfonates, lactates, lactobionates, L-tartrates, fumarates, L-malates, α-ketoglutarate, hippurate, D-glucuronide, D-glucose, α-D-glucohepanoate, glycolates, mucilages, L-ascorbate, orotate, picrates, glycinates, alanineates, arginineates, cinnamates, laurates, sebacic acid salts, benzenesulfonates, methanesulfonates, ethanesulfonates, ethanedisulfonates, formates, acetates, 2, 2-Dichloroacetate, trimethylacetate, propionate, valerate, palmitate, triphenylacetate, 2-ethyl-succinate, iodate, nicotinate, L-pyroglutamate, L-proline, ferulic acid, 2-hydroxyethanesulfonate, nitrate, gentianate, cholate, salicylate, terephthalate, glutarate, adipate, stearate, oleate, undecenoate, dodecyl sulfonate, phosphate, thiocyanate, dihydrophosphate, pyrophosphate, metaphosphate, oxalate, malonate, benzoate, mandelate, succinate, pyruvate, pyruvate, p-chlorobenzenesulfonate, 1,5-naphthalenedisulfonate, 3-hydroxy-2-naphthalenecarboxate, 1-hydroxy-2-naphthalenecarboxate, 2-naphthalenesulfonate, trifluoroacetate, hydroxyacetate, or p-toluenesulfonate, etc.

[0046] In some embodiments, the chimera of the present invention can exist in the form of a non-solvent or a solvate, such as a pharmaceutically acceptable solvent like water or ethanol.

[0047] In some embodiments, the disclosed compounds can be prepared as prodrugs or prodrugs. Prodrugs can be converted into parent drugs in the body to exert their effects.

[0048] In some embodiments, isotopically labeled compounds of this disclosure are also provided, examples of which include deuterium (D or...). 2 H).

[0049] Preferably, R1 is selected from any one of the following structures: C(O)R a C(O)NHR a or C(O)N(R) a )2; wherein, the R a R2 is selected from C1-C6 alkyl groups; R3 is selected from 3-7 membered cycloalkyl groups; R4 is selected from... Where m and f are each an independent integer from 1 to 4, W is selected from CH or N; Y is selected from -S(O)2-; R5 is selected from any of the following structures:

[0050] Where f1, f3, and m3 are each an integer from 1 to 4, and W1 and Q1 are each independently selected from CH or N;

[0051] The Linker is selected from any of the following structures: Where f2 and m1 are each an integer from 1 to 4, j is an integer from 0 to 14, and W2 and Q2 are each independently selected from CH or N.

[0052] Preferably, the chemical structure of the chimera is shown in Formula II:

[0053]

[0054] The Linker is selected from any of the following structures: f2 and m1 are each an integer from 1 to 4, j is an integer from 0 to 14, and W2 and Q2 are each independently selected from CH or N;

[0055] R5 is selected from any of the following structures:

[0056] Where f2 and m1 are each an integer from 1 to 4, j is an integer from 0 to 14, and W2 and Q2 are each independently selected from CH or N.

[0057] Preferably, the Linker is selected from any of the following structures:

[0058]

[0059] Where j is an integer from 0 to 14, and W2 and Q2 are each independently selected from CH or N;

[0060] R5 is selected from any of the following structures:

[0061] Among them, f1, f3, m3 are each an integer from 1 to 4, and W1 and Q1 are each independently selected from CH or N.

[0062] More preferably, in the Linker, j is an integer from 0 to 1, and W2 and Q2 are each independently selected from CH or N; in R5, f1 is 1, f3 is 2, m3 is 2, and W1 and Q1 are each independently selected from CH or N.

[0063] Preferably, the E3 ubiquitin ligase ligand is selected from any one of the following structures:

[0064]

[0065] Preferably, the chemical structure of the chimera is selected from any one of the following structures:

[0066]

[0067]

[0068] A second objective of this invention is to provide a method for preparing the CDK2 / 4 / 6 protein hydrolysis-targeting chimera or its isomers, pharmaceutically acceptable salts, solvates, prodrugs, deuterated compounds, or polymorphs, wherein the method for preparing the chimera comprises:

[0069]

[0070] The chimera is obtained by reacting pyrido[2,3-d]pyrimidine-7(8H)-one intermediate 1 with the chemical structure shown in Formula III, intermediate 2 containing E3 ubiquitin ligase ligand and Linker structure, and DIEA in an organic solvent with or without HATU.

[0071] A third object of the present invention is to provide a method for preparing pyrido[2,3-d]pyrimidine-7(8H)-one intermediate 1, wherein the chemical structure of pyrido[2,3-d]pyrimidine-7(8H)-one intermediate 1 is shown in Formula IV or Formula V, and the method for preparing pyrido[2,3-d]pyrimidine-7(8H)-one intermediate 1 as shown in Formula IV comprises:

[0072]

[0073] Step 1: Synthesis of 2-chloro-8-cyclopentyl-6-(1-ethoxyvinyl)-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one

[0074] Under an inert atmosphere, 6-bromo-2-chloro-8-cyclopentyl-5-methylpyridano[2,3-d]pyrimidin-7(8H)-one was reacted with tributyl(1-ethoxyethylene)tin at 45 °C in N-methylpyrrolidone under the action of di(tri-tert-butylphosphine)palladium to obtain 2-chloro-8-cyclopentyl-6-(1-ethoxyethylene)-5-methylpyridano[2,3-d]pyrimidin-7(8H)-one;

[0075] Step 2: Synthesis of 6-acetyl-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one

[0076] 2-Chloro-8-cyclopentyl-6-(1-ethoxyvinyl)-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one was placed in THF, and an aqueous hydrochloric acid solution was added. The reaction was stirred at room temperature to obtain 6-acetyl-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one.

[0077] Step 3: Synthesis of tert-butyl 4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-carboxylate

[0078] 6-Acetyl-2-chloro-8-cyclopentyl-5-methylpyridino[2,3-d]pyrimidin-7(8H)-one was reacted with 1-tert-butoxycarbonyl-4-aminopiperidine in DMF and stirred at 80°C under the action of DIEA to obtain 4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridino[2,3-d]pyrimidin-2-yl)amino)piperidine-1-carboxylic acid tert-butyl ester;

[0079] Step 4: Synthesis of 6-acetyl-8-cyclopentyl-5-methyl-2-(piperidin-4-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one

[0080] 4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridino[2,3-d]pyrimidin-2-yl)amino)piperidin-1-carboxylic acid tert-butyl ester and dioxane hydrochloride solution were reacted with stirring at room temperature to obtain 6-acetyl-8-cyclopentyl-5-methyl-2-(piperidin-4-ylamino)pyridino[2,3-d]pyrimidin-7(8H)-one;

[0081] Step 5: Synthesis of pyrido[2,3-d]pyrimidine-7(8H)-one intermediate 1 as shown in Formula IV

[0082] 6-Acetyl-8-cyclopentyl-5-methyl-2-(piperidin-4-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one was reacted with the compound shown in formula d in an ice bath under the action of DIEA in DCM to obtain pyrido[2,3-d]pyrimidin-7(8H)-one intermediate 1 shown in formula IV;

[0083] The method for preparing pyrido[2,3-d]pyrimidine-7(8H)-one intermediate 1 as shown in Formula V includes:

[0084]

[0085] Step 1: Synthesis of tert-butyl 4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridino[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)piperidin-1-carboxylate

[0086] 6-Acetyl-8-cyclopentyl-5-methyl-2-(piperidin-4-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one was reacted with 4-chlorothioylpiperidinic acid butyl ester at room temperature in anhydrous DCM under the action of DIEA and in an ice bath with stirring at room temperature to obtain 4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)piperidin-1-carboxylic acid tert-butyl ester;

[0087] Step 2: Synthesis of pyrido[2,3-d]pyrimidine-7(8H)-one intermediate 1 as shown in Formula V

[0088] 4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)piperidin-1-carboxylic acid tert-butyl ester and HCl·dioxane solution were reacted with the mixture at room temperature to obtain pyrido[2,3-d]pyrimidin-7(8H)-one intermediate 1 as shown in Formula V.

[0089] A fourth object of the present invention is to provide a pharmaceutical composition comprising the CDK2 / 4 / 6 protein hydrolysis targeting chimera or isomer thereof, a pharmaceutically acceptable salt, a solvate, a prodrug, a deuterated compound or a polymorph thereof.

[0090] In some embodiments, the pharmaceutical compositions disclosed herein comprise a chimeric compound of Formula I of the present invention or a pharmaceutically acceptable salt, solvate, isotope-enriched analog, polymorph, prodrug, stereoisomer (including enantiomers), or mixture of stereoisomers as an active ingredient, and at least one pharmaceutically acceptable carrier.

[0091] In some implementations, pharmaceutically acceptable carriers include, but are not limited to, fillers, stabilizers, dispersants, suspending agents, diluents, excipients, thickeners, colorants, solvents, or encapsulating materials. The carrier must be compatible with other components of the formulation (including compounds useful in this invention) and harmless to the patient; the carrier must be "acceptable." Some examples of pharmaceutically acceptable carrier materials include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered xanthophyll; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glyceryl sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactant phosphate buffer solutions; polyoxyethylene, polyvinylpyrrolidone, polyacrylamide, poloxamer; and other non-toxic compatible substances used in pharmaceutical formulations.

[0092] The pharmaceutical composition of the present invention further comprises at least one second therapeutic agent, such as an anticancer agent. The second therapeutic agent can be used in combination with the chimera of Formula I described in the present invention to treat the diseases or conditions described in the present invention. The second therapeutic agent includes, but is not limited to, chemotherapeutic agents, immunotherapeutic agents, gene therapy agents, etc.

[0093] A fifth object of the present invention is to provide the use of the CDK2 / 4 / 6 protein hydrolysis targeting chimera or isomer thereof, pharmaceutically acceptable salt, solvate, prodrug, deuterated or polymorph thereof, or the pharmaceutical composition thereof, in the preparation of a medicament for the prevention and / or treatment of cancer.

[0094] The pharmaceutical compositions comprising a chimera of Formula I or a pharmaceutically acceptable salt thereof as an active ingredient, as described in this invention, may be prepared into suitable formulations, such as sprays, patches, tablets (e.g., conventional tablets, dispersible tablets, orally disintegrating tablets), capsules (e.g., soft capsules, hard capsules, enteric-coated capsules), sugar-coated pills, lozenges, granules, powders for injection, suppositories, or liquid formulations (e.g., suspensions, such as aqueous or oily suspensions; emulsions or syrups, or conventional injectable formulations, such as injectable solutions, such as sterile injectable solutions formulated according to methods known in the art using water, Ringer's solution, or isotonic sodium chloride solution as a carrier or solvent) or lyophilized compositions, according to suitable routes of administration (including but not limited to nasal administration, inhalation, topical administration, oral administration, oral mucosal administration, rectal administration, pleural administration, peritoneal administration, vaginal administration, intramuscular administration, subcutaneous administration, transdermal administration, epidural administration, intrathecal administration, and intravenous administration). Those skilled in the art can also prepare the chimera of the present invention into conventional, dispersible, chewable, or orally disintegrating or rapidly dissolving formulations, or sustained-release capsules or controlled-release capsules, as needed.

[0095] The chimera of Formula I described in this invention, or its pharmaceutically acceptable salt, solvate, isotope-enriched analog, polymorph, prodrug, stereoisomer (including enantiomers), or mixture of stereoisomers, can be used as a pharmaceutical agent. In particular, the chimera of Formula I described in this invention, or its pharmaceutically acceptable salt, solvate, isotope-enriched analog, polymorph, prodrug, stereoisomer (including enantiomers), or mixture of stereoisomers, can be used to prepare medicaments for treating and / or preventing diseases or conditions selected from tumors or cancer.

[0096] In some implementations, the diseases or conditions mentioned include, but are not limited to: myeloma, including multiple myeloma, plasma cell myeloma, smoldering myeloma, and smoldering multiple myeloma; transplant-related cancers; neutropenia; leukemia, including chronic myeloid leukemia, B-cell chronic lymphocytic leukemia, leukemia-associated anemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), monocytic leukemia, myeloid monocytic leukemia, acute lymphoblastic leukemia (ALL), acute T-lymphoblastic leukemia, and T-lymphoblastic leukemia. Chronic lymphocytic leukemia (CLL); lymphomas, including diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, anaplastic lymphoma, anaplastic large cell lymphoma, immunoblastic T-cell lymphoma, CD20-positive lymphoma, mantle cell lymphoma, follicular lymphoma (FL), Burkitts lymphoma, marginal zone lymphoma (MZL), primary lymphoma, B-cell lymphoma, relapsed B-cell non-Hodgkin's lymphoma, relapsed diffuse large B-cell lymphoma, relapsed mediastinal (thymic) large B-cell lymphoma, primary mediastinal (thymic) large B-cell lymphoma. Large B-cell lymphoma, relapsed-transformed non-Hodgkin lymphoma, refractory B-cell non-Hodgkin lymphoma, refractory diffuse large B-cell lymphoma, refractory primary mediastinal (thymic) large B-cell lymphoma, refractory-transformed non-Hodgkin lymphoma; thyroid cancer; melanoma; lung cancer, including adenocarcinoma, squamous cell carcinoma, non-small cell lung cancer, and small cell lung cancer; inflammatory myofibroblastic tumor; colorectal cancer; bowel cancer; glioma; glioblastoma; glioma; peripheral neuroepithelial tumor; ovarian cancer; bronchial cancer; prostate cancer; breast cancer, including triple-negative breast cancer. Patients with incidental breast cancer, ductal carcinoma, and Cowden's disease; pancreatic cancer; central nervous system cancers; neuroblastoma; extramedullary plasmacytoma; plasmacytoma; gastric cancer; gastrointestinal stromal tumors; esophageal cancer; colorectal adenocarcinoma; esophageal squamous cell carcinoma; liver cancer; renal cell carcinoma; bladder cancer; endometrial cancer; head and neck cancer; brain cancer; oral cancer; sarcomas, including rhabdomyosarcoma, various adipose-derived tumors, Ewing sarcoma / primitive neuroectodermal tumors (Ewing / PNETS), and leiomyosarcoma; urothelial carcinoma; basal cell carcinoma; oral squamous cell carcinoma; bile duct carcinoma; bone cancer; cervical cancer; and skin cancer.

[0097] Preferably, the disease or condition is selected from non-small cell lung cancer and breast cancer.

[0098] The Formula I chimera, as the active ingredient, is contained in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a subject a therapeutically effective amount for the indication requiring treatment without causing serious toxicity in the treated subject. The dosage of the active compound for all diseases or conditions mentioned in this invention is, for example, about 25 mg / kg of test body weight / day.

[0099] The chimera of Formula I of the present invention or a pharmaceutically acceptable salt thereof may be conveniently administered in any suitable formulation, the specifications of which include, but are not limited to, less than 1 mg, 1 mg to 3000 mg, 5 mg to 1000 mg, for example 5 to 500 mg, 25 to 250 mg of active ingredient per unit dosage form.

[0100] The "therapeutic effective dose" of the chimeric compound of this invention depends on a variety of factors, including the activity of the compound used, the metabolic stability and duration of action of the compound, the patient's age, sex, and weight, the patient's overall medical condition, the route and timing of administration, the excretion rate, combination therapy, and the progression of the disease or condition in the treated patient. Those skilled in the art can determine the appropriate dose based on these and other factors. It should be understood that the choice of use of one or more active compounds and / or compositions and their dosages depends on the individual's fundamental circumstances (generally, the individual's condition should be optimized for effectiveness).

[0101] The patients or subjects of the above treatment refer to animals, such as mammals, including but not limited to primates (such as humans), cattle, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice, etc.

[0102] The CDK2 / 4 / 6 protein hydrolysis-targeting chimera, its preparation method, pharmaceutical composition, and uses of the present invention have the following advantages:

[0103] The chimera of this invention induces the selective degradation of CDK2, CDK4, and CDK6 through ubiquitin-proteasome, thereby reducing the phosphorylation level of RB1, inhibiting the release of transcription factor E2F, blocking the cell progression from G1 phase to S phase, reducing cell proliferation in cancer cell lines, and promoting cancer cell apoptosis. It has been well validated at the cellular, organoid, and animal model levels. Compared with inhibitors, it is more efficient and less toxic, and can be used to treat a variety of cancers. Attached Figure Description

[0104] Figure 1 Results of the degradation ability of the PROTAC drug prepared for the present invention on CDK2, 4, and 6 in A549 cells and its effect on RB1 phosphorylation level.

[0105] Figure 2 Results of the degradation ability of the PROTAC drug prepared for the present invention on CDK2, 4, and 6 in NCI-H1975 cells and its effect on RB1 phosphorylation level.

[0106] Figure 3 Results of the degradation ability of the PROTAC drug prepared for the present invention on CDK2, 4, and 6 in NCI-H1299 cells and its effect on RB1 phosphorylation level.

[0107] Figure 4 The results show the time-varying effects of compounds WYY-03-127, WYY-03-120, WYY-03-185, and WYY-03-186 of the present invention on the degradation ability of CDK2, 4, and 6 in A549 cells and on the phosphorylation level of RB1.

[0108] Figure 5 The results show the effects of compounds WYY-03-127 and 3600 on the degradation of CDK2, 4, and 6 in A549 cells and their influence on RB1 phosphorylation levels as a function of concentration.

[0109] Figure 6 The results show the inhibitory effects of proteasome inhibitors MLN4924, Thalidomide, and MG132 on the ubiquitination degradation ability of compound WYY-03-127.

[0110] Figure 7 The results of the clonogenic assay show the inhibitory effect of compounds WYY-03-127 and WYY-03-120 on the proliferation of A549 cells.

[0111] Figure 8 The results show the effects of intraperitoneal injection of the compound on tumor volume (A) and mouse body weight (B) in an A549 tumor cell mouse model. Detailed Implementation

[0112] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0113] In this invention, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used for simplicity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible secondary ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0114] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0115] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention includes, but is not limited to, these embodiments.

[0116] The structures of the following compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). Nuclear magnetic resonance 1 H NMR was measured using a Bruker AVANCE NEO 600 (600 MHz) and a Bruker Biospin AV400 (400 MHz). 13 C NMR was measured using a Bruker AVANCE NEO 600 (600 MHz). 1 H NMR and 13 C NMR chemical shift is in ppm, coupling constant is in Hz, and tetramethylsilane is an internal standard; 1 The related representation of peak types in H NMR: s, singlet; d, doublet; t, triplet; m, multiplet.

[0117] The abbreviations used in the following embodiments are as follows:

[0118] NMP: N-methylpyrrolidone; Pd(t-Bu3P)2: bis(tri-tert-butylphosphine)palladium; EA: ethyl acetate; THF: tetrahydrofuran; DMF: N,N-dimethylformamide; DIEA: N,N-diisopropylethylamine; dioxane: dioxane; MeOH: methanol; DCM: dichloromethane; Pd(dppf)Cl2: 1,1-bis(diphenylphosphine)diferro-palladium dichloride; EtOH: ethanol; Pd(dppf)C l2·DCM: [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride dichloromethane complex; TBAB: tetrabutylammonium bromide; HOAc: acetic acid; Tol: toluene; BINAP: 1,1'-binaphthyl-2,2'-bisdiphenylphosphine; DMSO: dimethyl sulfoxide; Pd-PEPPSI-IPent(Cl): [1,3-bis(2,6-di-3-pentylphenyl)imidazol-2-yl](3-chloropyridinyl)palladium dichloride(II).

[0119] The solvent and reagent treatments involved in the following examples are as follows:

[0120] The solvents used in the reaction, such as EtOH, DMSO, DMF, EtOH, MeOH, EA, and other reaction substrates and reagents, can all be obtained commercially. The water used in the reaction is preparative deionized water.

[0121] It should be noted that: unless specific conditions are specified in the examples, they should be performed under conventional conditions or conditions recommended by the manufacturer. All starting materials or reagents used in each scheme can be purchased commercially or prepared using methods known to those skilled in the art.

[0122] I. Synthetic method of pyrido[2,3-d]pyrimidine-7(8H)-one intermediate

[0123] Example 1: 6-Acetyl-2-((1-((4-(bromomethyl)phenyl)sulfonyl)piperidin-4-yl)amino)-8-cyclopentyl-5-methylpyridino[2,3-d]pyrimidin-7(8H)-one

[0124]

[0125] Step 1: Synthesis of 2-chloro-8-cyclopentyl-6-(1-ethoxyvinyl)-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one

[0126] Weigh 20.0 g (58.3 mmol, 1.0 eq) of 6-bromo-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one into a 1000 mL round-bottom flask, dissolve it in N-methylpyrrolidone (300 mL), then add tributyl(1-ethoxyethylene)tin (25 mL, 75.8 mmol, 1.3 eq), followed by the addition of di(tri-tert-butylphosphine)palladium (1.49 g, 2.9 mmol, 0.05 eq) under N2 protection, and heat to 45 °C for 24 hours. After the reaction was complete as shown by TLC and LCMS, saturated potassium fluoride solution (600 mL) was added and stirred overnight to quench the reaction. The reaction mixture was extracted using EA (300 mL × 3). The organic phase was washed with saturated NaCl solution (300 mL), dried with anhydrous Na2SO4, and EA was removed by vacuum distillation. The yellow oily liquid was then separated by column chromatography (5%–9% EA in PE) with a yield of 78.4%. 1 H NMR(600MHz, DMSO-d6)δ9.08(s,1H),5.73(tt,J=9.7,7.7Hz,1H),4.52–4.50(m,1H),4.11(d,J=2. 4Hz, 1H), 3.85 (q, J=7.0Hz, 2H), 2.44 (s, 3H), 2.15–1.59 (m, 8H), 1.25 (t, J=7.0Hz, 3H); LCMS (ESI) C 17 H 20 ClN3O2 + [M+H] + =334.23.

[0127] Step 2: Synthesis of 6-acetyl-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one

[0128] Weigh 15.25 g (45.7 mmol, 1.0 eq) of compound 2-chloro-8-cyclopentyl-6-(1-ethoxyvinyl)-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one into a 500 mL round-bottom flask. Dissolve the compound in 150 mL of THF, then add 150 mL of 1 mol / L hydrochloric acid aqueous solution. Stir at room temperature for 1 hour. After the reaction was completed as monitored by TLC and LCMS, the reaction was quenched with saturated NaHCO3 solution. The reaction solution was extracted with EA (200 mL × 3). The organic phase was washed with saturated NaCl solution (400 mL), dried over anhydrous Na2SO4, and EA was removed by vacuum distillation. The target product was purified by column chromatography to obtain a pale yellow solid with a yield of 54.7%. 1 HNMR(400MHz,DMSO-d6)δ9.23(s,1H),5.84–5.75(m,1H),2.50(s,3H),2.41(s,3H),2.10–1.52(m,8H); LCMS(ESI)C 15 H 16 ClN3O2 + [M+H] + =306.14.

[0129] Step 3: Synthesis of tert-butyl 4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidine-1-carboxylate

[0130] Compounds 6-acetyl-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one (3.00 g, 9.8 mmol, 1.0 eq) and 1-tert-butoxycarbonyl-4-aminopiperidine (3.94 g, 19.7 mmol, 2.0 eq) were weighed separately and placed in a 250 mL round-bottom flask. 60 mL of DMF was added to dissolve the compound, followed by the addition of DIEA (5.1 mL, 29.5 mmol, 3.0 eq). The mixture was stirred in an oil bath at 80 °C for 4 hours. After the reaction was confirmed by TLC and LCMS, water (160 mL) was added to quench the reaction. The reaction solution was extracted with EA (300 mL × 3). The organic phase was washed with saturated NaCl solution (300 mL), dried over anhydrous Na₂SO₄, and EA was removed by vacuum distillation. The product was then separated by column chromatography to obtain a white powder with a yield of 84.5%. 1H NMR(600MHz,DMSO-d6)δ8.80(d,J=27.4Hz,1H),5.95–5.69(m,1H),4.09–3.99(m,1H),3.98–3.89(m,2H),2.96–2.75(m,2H),2.40(s,3H),2 .25(s,3H),2.20–2.12(m,1H),2.00–1.90(m,2H),1.89–1.81(m,2H),1.79–1.71(m,2H),1.68–1.53(m,2H),1.46–1.33(m,12H); LCMS(ESI)C 25 H 35 N5O4 + [M+H] + =470.28.

[0131] Step 4: Synthesis of 6-acetyl-8-cyclopentyl-5-methyl-2-(piperidin-4-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one

[0132] 2.58 g (5.49 mmol, 1 eq) of tert-butyl 4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-carboxylic acid was weighed into a 50 mL round-bottom flask, and 20 mL (4 mol / L) of dioxane hydrochloride solution was added. The mixture was stirred at room temperature for 3 hours. After the reaction was completed as indicated by LC-MS, the solvent was removed under reduced pressure, and MeOH was added. The mixture was then distilled three times under reduced pressure to remove residual hydrochloric acid. The product was dried under vacuum to obtain a white solid powder with a yield of 95.3%. 1 H NMR(400MHz,DMSO-d6)δ8.83(s,1H),5.92(s,1H),3.56–3.54(m,1H),2.99-3 .16(m,2H),2.40–2.21(m,5H),2.26(s,3H),2.21–1.49(m,12H); LCMS(ESI)C 20 H 27 N5O2 + [M+H] + =370.51.

[0133] Step 5: 6-Acetyl-2-((1-((4-(bromomethyl)phenyl)sulfonyl)piperidin-4-yl)amino)-8-cyclopentyl-5-methylpyridino[2,3-d]pyrimidin-7(8H)-one

[0134] 500 mg, 1.23 mmol, 1.0 eq) of 6-acetyl-8-cyclopentyl-5-methyl-2-(piperidin-4-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one was weighed into a 25 mL flask. 10 mL of DCM was added, followed by DIEA (1.1 mL, 6.15 mmol, 5.0 eq). Then, 332 mg, 1.23 mmol, 1.0 eq of 4-bromomethylbenzenesulfonyl chloride was added in portions in an ice bath. The reaction was carried out in an ice bath for 30 min. After the reaction was completed, the solvent was removed by vacuum distillation, and the crude product was dried under vacuum to obtain 632 mg of a white powder, which was the target compound. 1 H NMR(400MHz, DMSO-d6)δ8.76(s,1H),7.78(d,J=8.2Hz,2H),7.72(d,J=7.7Hz,2H),5.92–5.60(m,1H),4.88(s,2H),3.9 0–3.64(m,1H),3.64–3.48(m,2H),2.48–2.41(m,2H),2.38(s,3H),2.28–2.08(m,5H),1.97–1.44(m,10H); LCMS(ESI)C 27 H 32 BrN5O4S + [M+H] + =602.08.

[0135] Example 2: 6-Acetyl-8-cyclopentyl-5-methyl-2-((1-(piperidin-4-ylsulfonyl)piperidin-4-yl)amino)pyrido[2,3-d]pyrimidine-7(8H)-one hydrochloride

[0136]

[0137]

[0138] Step 1: Synthesis of tert-butyl 4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridino[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)piperidin-1-carboxylate

[0139] 1.00 g (2.46 mmol, 1.0 eq) of 6-acetyl-8-cyclopentyl-5-methyl-2-(piperidin-4-ylamino)pyrido[2,3-d]pyrimidin-7(8H)-one was weighed into a round-bottom flask. 16 mL of ultradry DCM was added, followed by 2.2 mL (12.30 mmol, 5.0 eq) of DIEA. 4-chlorothioylpiperidinic acid butyl ester (698 mg, 2.46 mmol, 1.0 eq) was added in portions under ice bath conditions. The mixture was then stirred at room temperature for 3 hours. After the reaction was complete, the solution was evaporated to dryness and purified by column chromatography to obtain a white powder in 84.2% yield. 1 HNMR(600MHz,Chloroform-d)δ8.62(s,1H),5.89–5.73(m,1H),5.31–5.07(m,1H),4.38–3.94(m,3H),3.92–2.96(m ,6H),2.78–2.60(m,2H),2.52(s,3H),2.32(s,5H),2.17–1.77(m,8H),1.69–1.58(m,4H),1.46(s,9H); LCMS(ESI)C 30 H 44 N6O6S + [M+H] + =617.16.

[0140] Step 2: Synthesis of 6-acetyl-8-cyclopentyl-5-methyl-2-((1-(piperidin-4-ylsulfonyl)piperidin-4-yl)amino)pyrido[2,3-d]pyrimidine-7(8H)-one hydrochloride

[0141] 840 mg, 1.36 mmol, 1.0 eq of 4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)piperidin-1-carboxylic acid tert-butyl ester was weighed into a round-bottom flask, and 2 mL of HCl·dioxane solution was added. The mixture was stirred at room temperature for 3 hours. After the reaction was completed as indicated by LCMS, the mixture was evaporated to dryness and then freeze-dried to obtain a white solid powder, which was the target compound. 1HNMR(600MHz,DMSO-d6)δ8.84(s,1H),6.13–6.02(m,1H),5.83(tt,J=10.6,2.7Hz,1H),3.68(dt,J=13.0,4.1Hz,2H),3.54(tt,J=10.6,3.3Hz ,1H),3.34–3.22(m,2H),3.14–3.07(m,2H),2.95–2.85(m,2H),2.41(s,3H),2.26(s,5H),2.14–2.08(m,2H),2.00–1.53(m,12H); LCMS(ESI)C 25 H 36 N6O4S + [M+H] + =517.57.

[0142] II. Synthesis methods of E3 ubiquitin ligase ligands

[0143] Example 3: 3-(6-(piperazin-1-yl)pyridin-3-yl)piperidine-2,6-dione hydrochloride

[0144]

[0145]

[0146] Step 1: Synthesis of tert-butyl 4-(2',6'-bis(benzyloxy)-[3,3'-bipyridine]-6-yl)piperazine-1-carboxylate

[0147] Weigh 500 mg (1.28 mmol, 1.2 eq) of 6-(4-Boc-1-piperazinyl)pyridine-3-boronic acid pinacol ester, 2,6-bis(benzyloxy)-3-bromopyridine (395 mg, 1.07 mol, 1.0 eq), and 295 mg (2.14 mmol, 1.0 eq) of K₂CO₃ into a 25 mL round-bottom flask. Add 10 mL of ultradry dioxane and 1.5 mL of water. After purging with N₂ three times, add 39 mg (54 μmol, 0.05 eq) of Pd(dppf)Cl₂ catalyst. Pd(dppf)Cl₂ is then added, and the mixture is purged with N₂ three more times. The mixture is heated under reflux in an oil bath at 95 °C overnight. After LCMS monitoring showed that the reaction was complete, the reaction solution was cooled to room temperature and filtered. The filter cake was thoroughly washed with EA, and the filtrate was evaporated to dryness. Purification by column chromatography yielded a white solid in 78.6%. 1H NMR (400MHz, DMSO-d6) δ8.32–8.30(m,1H),7.77–7.71(m,2H),7.45–7.30(m,10H),6.86(d,J=8.8Hz,1H),6. 53(d,J=8.0Hz,1H),5.38(d,J=10.9Hz,4H),3.51–3.46(m,4H),3.43–3.38(m,4H),1.42(s,9H); LCMS(ESI)C 33 H 36 N4O4 + [M+H] + =553.39.

[0148] Step 2: Synthesis of tert-butyl 4-(5-(2,6-dioxopiperidin-3-yl)pyridin-2-yl)piperazine-1-carboxylate

[0149] 556 mg, 1.01 mmol, 1.0 eq of tert-butyl 4-(2',6'-bis(benzyloxy)-[3,3'-bipyridine]-6-yl)piperazine-1-carboxylate (15 mL) was weighed into a 50 mL round-bottom flask. 15 mL of EtOH and 15 mL of EA were added, followed by the addition of 10% palladium on carbon (500 mg of 55% water). The mixture was purged with H2 three times and stirred at room temperature for 16 hours. After TLC and LCMS showed that the reaction was complete, the reaction solution was filtered, the filter cake was washed with EA, and the filtrate was evaporated under reduced pressure. The filtrate was purified by column chromatography to obtain a pale pink solid with a yield of 63.5%. 1 H NMR (400MHz, DMSO-d6) δ10.82(s,1H),7.97(d,J=2.4Hz,1H),7.42(dd,J=8.8,2.5Hz,1H),6.82(d,J=8.7Hz,1H),3.75(dd,J=12.2,4.9Hz,1H) ,3.48–3.43(m,4H),3.42–3.37(m,4H),2.73–2.62(m,1H),2.55–2.51(m,1H),2.23–2.13(m,1H),2.00–1.93(m,1H),1.42(s,9H); LCMS(ESI)C 19 H 26 N4O4 + [M+H] + =375.19.

[0150] Step 3: Synthesis of 3-(6-(piperazin-1-yl)pyridin-3-yl)piperidine-2,6-dione hydrochloride

[0151] The synthesis of compound 4-(5-(2,6-dioxadiazin-3-yl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl ester (240 mg, 641 mmol, 1.0 eq) was weighed into a 25 mL round-bottom flask, and then dioxane hydrochloride solution (3 mL, 4 mol / L) and DCM (4 mL) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was completed as indicated by LCMS, the solvent was removed by rotary evaporation under reduced pressure. MeOH was added and evaporated three times to remove excess hydrochloric acid. The crude product was obtained by vacuum drying, which is the target compound. 1 H NMR (400MHz, DMSO-d6) δ10.93(s,1H),7.99(d,J=2.2Hz,1H),7.88(dd,J=9.3,2.3Hz,1H),7.30(d,J=9.2Hz,1H),3.96–3.93(m,4H ),3.57–3.55(m,1H),3.24–3.21(m,4H),2.73–2.64(m,1H),2.60–2.55(m,1H),2.33–2.22(m,1H),2.00–1.92(m,1H); LCMS(ESI)C 14 H 18 N4O2 + [M+H] + =275.35.

[0152] Example 4: 3-(3-(piperazin-1-yl)phenyl)piperidine-2,6-dione hydrochloride

[0153]

[0154] Step 1: 4-(3-(2,6-bis(benzyloxy)pyridin-3-yl)phenyl)piperazine-1-carboxylic acid tert-butyl ester

[0155] Weigh 1-BOC-4-(3-bromophenyl)piperazine (500 mg, 1.47 mmol, 1.0 eq), 2,6-bis(benzyloxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxoboropentane-2-yl)pyridine (673 mg, 1.61 mmol, 1.1 eq), and K₂CO₃ (672 mg, 161 mmol, 1.1 eq) into a 25 mL round-bottom flask, add water (2 mL) and ultra-dry dioxane (8 mL). The system is purged with N₂ three times. After adding Pd(dppf)Cl₂·DCM (122 mg, 0.15 mmol, 0.1 eq), the N₂ is purged three more times. The system is stirred overnight at 110 °C. After the reaction was complete as shown by TLC and LCMS, the phase was quenched with water (12 mL), extracted with EA (8 mL × 3), washed with saturated NaCl solution (10 mL × 2), dried with anhydrous Na2SO4, evaporated to dryness, and purified by column chromatography to obtain a white solid with a yield of 72.7%. 1 H NMR(600MHz,DMSO-d6)δ7.75(d,J=8.1Hz,1H),7.48–7.29(m,10H),7.25–7.21(m,1H),7.12–7.10(m,1H),6.96(dd,J=7.6,1.5Hz,1H),6.8 6(dd,J=8.2,2.5Hz,1H),6.55(d,J=8.0Hz,1H),5.40(s,2H),5.37(s,2H),3.41–3.38(m,4H),3.02–2.92(m,4H),1.43(s,9H); LCMS(ESI)C 34 H 37 N3O4 + [M+H] + =552.15.

[0156] Step 2: 4-(3-(2,6-dioxopiperidin-3-yl)phenyl)piperazine-1-carboxylic acid tert-butyl ester

[0157] 590 mg, 1.07 mmol, 1.0 eq of tert-butyl piperazine-1-carboxylate (2,6-bis(benzyloxy)pyridin-3-yl)phenyl)piperazine-1-carboxylate were weighed and placed in a 25 mL round-bottom flask. EA (5 mL), EtOH (5 mL), and ultra-dry THF (5 mL) were added, followed by the addition of 10% Pd / C (200 mg) to remove H2 three times. The mixture was stirred at room temperature for 16 hours. After the reaction was confirmed to be complete by TLC and LCMS, the reaction solution was filtered and the filter cake was washed with EA. The filtrate was then distilled under reduced pressure to obtain approximately 400 mg of crude white solid. 1H NMR (600MHz, DMSO-d6) δ10.79(s,1H),7.19–7.15(m,1H),6.85–6.81(m,2H),6.66–6.64(m,1H),3.81–3.71(m,1H),3.48–3.41( LCMS(ESI)C 20 H 27 N3O4 + [M+H] + =374.24.

[0158] Step 3: 3-(3-(piperazin-1-yl)phenyl)piperidine-2,6-dione hydrochloride

[0159] 400 mg of compound 4-(3-(2,6-dioxadiazin-3-yl)phenyl)piperazine-1-carboxylic acid tert-butyl ester was weighed into a 25 mL round-bottom flask, DCM (4 mL) was added, followed by HCl·dioxane solution (4 mL). The reaction was stirred at room temperature for 2 hours. After the reaction was complete as indicated by LCMS, the solvent was removed by vacuum distillation, and an appropriate amount of MeOH was added and the mixture was evaporated to dryness. This process was repeated three times. The crude product was then dried under vacuum to obtain a white powder, which is the target compound. 1 H NMR (400MHz, DMSO-d6) δ10.82(s,1H),7.25–7.17(m,1H),6.97–6.84(m,2H),6.72(d,J=7.5Hz,1H),3.40–3.31(m ,4H),3.24–3.16(m,4H),2.72–2.59(m,1H),2.50–2.43(m,1H),2.27–2.13(m,1H),2.07–1.96(m,1H); LCMS(ESI)C 15 H 19 N3O2 + [M+H] + =274.13.

[0160] Example 5: 1-(7-fluoro-1-methyl-6-(piperidin-4-yl)-1H-indazol-3-yl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride

[0161]

[0162] Step 1: Synthesis of 6-bromo-5-fluoro-1-methyl-1H-indolezol-3-amine

[0163] Weigh 7.70 g (91.6 mmol, 4.0 eq) of NaHCO3 into a 25 mL round-bottom flask, add 4 mL of water, and then add methylhydrazine sulfate (9.90 g, 68.7 mmol, 3.0 eq). Stir for half an hour to release the methylhydrazine. Then add 2 mL of EtOH to precipitate the salt in the system, filter, and transfer the filtrate to a 100 mL pressure-resistant bottle. Add 35 mL of EtOH (5.00 g, 22.9 mmol, 1.0 eq) to the pressure-resistant bottle, and stir the reaction system at 80 °C for 16 hours. After the reaction is completed by LCMS and TLC monitoring, cool to room temperature, add 60 mL of water, filter, wash the filter cake with an appropriate amount of water, collect the filter residue, evaporate to dryness, and vacuum dry to obtain a crude product as a white solid. 1 H NMR (400MHz, DMSO-d6) δ7.83 (d, J = 5.6Hz, 1H), 7.64 (d, J = 8.9Hz, 1H), 5.53 (s, 2H), 3.73 (s, 3H); LCMS (ESI) C8H7BrFN3 + [M+H] + =244.16.

[0164] Step 2: Synthesis of 3-((6-bromo-5-fluoro-1-methyl-1H-indazol-3-yl)amino)propionic acid

[0165] 2.50 g (10.2 mmol, 1.0 eq) of 6-bromo-5-fluoro-1-methyl-1H-indolezol-3-amine was weighed into a 100 mL round-bottom flask. 25 mL of freshly prepared 2M dilute hydrochloric acid solution was added, followed by tetrabutylammonium bromide (330 mg, 1.0 mmol, 0.1 eq) and acrylic acid (1.0 mL, 15.3 mmol, 1.5 eq). The reaction mixture was heated to 100 °C and stirred overnight. Afterward, the reaction mixture was cooled to room temperature, and the pH was adjusted to 8 with saturated NaHCO3 solution, then to 5 with acetic acid. The mixture was then extracted with EA (20 mL × 3), washed with saturated NaCl solution (50 mL × 1), and dried over anhydrous Na2SO4. The resulting crude product was obtained by rotary evaporation and yielded 2.7 g of a brown oily substance. 1 H NMR (400MHz, DMSO-d6) δ7.82(d,J=5.6Hz,1H),7.65(d,J=9.0Hz,1H),6.23(s,1H),3.75(s,3H),3.47–3.43(m,2H),2.61–2.56(m,2H); LCMS(ESI)C 11 H 11 BrFN3O2 + [M+H] + =316.11.

[0166] Step 3: Synthesis of 1-(6-bromo-7-fluoro-1-methyl-1H-indazol-3-yl)dihydropyrimidine-2,4(1H,3H)-dione

[0167] Weigh 2.70 g (8.5 mmol, 1.0 eq) of compound 3-((6-bromo-5-fluoro-1-methyl-1H-indazol-3-yl)amino)propionic acid into a 100 mL round-bottom flask, add 25 mL of AcOH to form a suspension, then add sodium isocyanate (1.11 g, 17.0 mmol, 2.0 eq), and then heat the reaction solution to 60 °C and stir for 16 hours. Then add concentrated hydrochloric acid (25 mL) and stir at 60 °C for 3 hours. After that, cool the reaction solution to room temperature, add an appropriate amount of water to dilute, filter, wash the filter cake with water, collect the filter residue, evaporate to dryness and then vacuum dry to obtain a white solid crude product. 1 HNMR(400MHz, DMSO-d6)δ8.16(d,J=5.6Hz,1H),7.61(d,J=9.1Hz,1H),3.99(s,3H),3.95–3.88(m,2H),2.78–2.73(m,2H); LCMS(ESI)C 12 H 10 BrFN4O2 + [M+H] + =341.24.

[0168] Step 4: Synthesis of tert-butyl 4-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-7-fluoro-1-methyl-1H-indazol-6-yl)-3,6-dihydropyridine-1(2H)-carboxylate

[0169] Weigh 1-(6-bromo-7-fluoro-1-methyl-1H-indazol-3-yl)dihydropyrimidine-2,4(1H,3H)-dione (1.50 g, 4.4 mmol, 1.0 eq), Na₂CO₃ (2.19 g, 13.2 mmol, 3.0 eq), and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (1.36 g, 4.4 mmol, 1.0 eq) into a 100 mL round-bottom flask, add ultra-dry dioxane (30 mL) and water (5 mL). Under a N₂ atmosphere, add Pd(dppf)Cl₂·DCM (359 mg, 440 μmol, 0.1 eq), then replace the N₂ three times, and heat to 80 °C with stirring overnight. After the reaction was confirmed to be complete by LCMS and TLC, the reaction solution was filtered, the filter cake was washed with an appropriate amount of EA, the filtrate was collected, concentrated, washed with water (20 mL × 1) and saturated NaCl solution (30 mL × 1), dried with anhydrous Na2SO4, purified by column chromatography after rotary evaporation, and dried under vacuum to obtain a white powder with a yield of 48%.1 H NMR (400MHz, DMSO-d6) δ10.60(s,1H),7.60(d,J=6.1Hz,1H),7.42(d,J=11.4Hz,1H),6.05(s,1H),4.03(t,J =4.5Hz,2H),4.00(s,3H),3.93–3.88(m,2H),3.61–3.53(m,2H),2.79–2.72(m,2H),1.44(s,9H); LCMS(ESI)C 22 H 26 FN5O4 + [M+H] + =444.16.

[0170] Step 5: Synthesis of tert-butyl 4-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-7-fluoro-1-methyl-1H-indazol-6-yl)piperidine-1-carboxylate

[0171] 483 mg (1.09 mmol, 1.0 eq) of tert-butyl 4-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-7-fluoro-1-methyl-1H-indazol-6-yl)-3,6-dihydropyridine-1(2H)-carboxylic acid was weighed into a 100 mL round-bottom flask. 20 mL of MeOH and 30 mL of EA were added, followed by 552 mg of 10% Pd / C. The mixture was purged with H2 three times, and the reaction was stirred at room temperature for 16 hours. After the reaction was complete as shown by TLC and LCMS, the reaction mixture was filtered, the filter cake was washed with EA, and the filtrate was collected and evaporated to dryness to obtain 280 mg of crude product. 1 H NMR (600MHz, DMSO-d6) δ7.59(d,J=5.9Hz,1H),7.37(d,J=10.8Hz,1H),3.99(s,3H),3.92–3. 86(m,2H),3.10–2.97(m,1H),2.78–2.70(m,2H),1.84–1.58(m,4H),1.42(s,9H); LCMS(ESI)C 22 H 28 FN5O4 + [M+H] + =446.13.

[0172] Step 6: Synthesis of 1-(7-fluoro-1-methyl-6-(piperidin-4-yl)-1H-indazol-3-yl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride

[0173] 280 mg (629 μmol, 1.0 eq) of tert-butyl 4-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-7-fluoro-1-methyl-1H-indazol-6-yl)piperidin-1-carboxylic acid was weighed into a 25 mL round-bottom flask. 4 mL of DCM was added, followed by 1 mL of HCl·dioxane solution. The mixture was stirred at room temperature for 3 hours. LC-MS showed complete reaction. The solvent was then removed by rotary evaporation, and the mixture was slurried with methyl tert-butyl ether. After filtration, the mixture was rotary evaporated and dried under vacuum to obtain 228 mg of a white solid powder, which was the target compound. 1 H NMR (400MHz, DMSO-d6) δ10.60(s,1H),7.50(d,J=5.9Hz,1H),7.42(d,J=11.0Hz,1H),4.01(s,3H),3.94 –3.86(m,2H),3.25–3.14(m,1H),3.12–2.99(m,2H),2.79–2.71(m,2H),2.09–1.95(m,4H); LCMS(ESI)C 17 H 20 FN5O2 + [M+H] + =346.18.

[0174] Example 6: 1-(2-methoxy-5-(piperazine-1-formyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride

[0175]

[0176] Step 1: Synthesis of 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoic acid

[0177] 4.50 g (27 mmol, 1.0 eq) of 3-amino-4-methoxybenzoic acid was weighed and placed in a 250 mL round-bottom flask. Toluene (50 mL) was added, followed by acrylic acid (2.78 mL, 41 mmol, 1.5 eq). The mixture was stirred at 100 °C for 24 hours. LC-MS showed that the substrate was completely consumed. The reaction mixture was cooled to room temperature, and urea (8.10 g, 135 mmol, 5.0 eq) and AcOH (50 mL) were added. The mixture was stirred at room temperature for 24 hours. TLC showed that the reaction was complete. The reaction mixture was evaporated to dryness to remove the solvent, and purified by column chromatography to obtain a brown solid with a yield of 15.3%. 1HNMR (400MHz, DMSO-d6) δ10.37(s,1H),9.95(s,1H),7.91(dd,J=8.6,2.2Hz,1H),7.82(d,J=2. 2Hz,1H),7.20(d,J=8.8Hz,1H),3.87(s,3H),3.61–3.57(m,2H),2.46–2.40(m,2H); LCMS(ESI)C 12 H 12 N2O5 + [M+H] + =265.25.

[0178] Step 2: Synthesis of tert-butyl 4-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperazine-1-carboxylate

[0179] Compounds 3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoic acid (250 mg, 946 μmol, 1.0 eq), HATU (360 mg, 946 μmol, 1.0 eq), and N-BOC-piperazine (211 mg, 1.14 mmol, 1.2 eq) were weighed and placed in a 50 mL round-bottom flask. DMF (10 mL) and DIEA (494 μL, 2.84 mmol, 3.0 eq) were added, and the mixture was stirred at room temperature for 4 hours. LC-MS and TLC showed that the reaction was complete. The reaction was quenched with water (30 mL), extracted with EA (10 mL × 3), and the organic phase was washed with saturated NaCl solution (20 mL × 2). The mixture was dried over anhydrous Na2SO4 and then evaporated to dryness to obtain 159 mg of crude product. 1 H NMR (400MHz, DMSO-d6) δ10.35(s,1H),7.41(dd,J=8.5,2.2Hz,1H),7.37(d,J=2.2Hz,1H),7.17(d,J=8.5 Hz,1H),3.85(s,3H),3.64–3.42(m,8H),2.91–2.85(m,2H),2.73–2.65(m,2H),1.41(s,9H); LCMS(ESI)C 21 H 28 N4O6 + [M+H] + =433.09.

[0180] Step 3: Synthesis of 1-(2-methoxy-5-(piperazin-1-formyl)phenyl)dihydropyrimidine-2,4(1H,3H)-dione hydrochloride

[0181] 159 mg (368 μmol, 1.0 eq) of 4-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-4-methoxybenzoyl)piperazine-1-carboxylic acid tert-butyl ester was weighed into a round-bottom flask, and HCl·dioxane solution was added. The mixture was stirred at room temperature for 3 hours. After the reaction was completed as indicated by LCMS, the solvent was evaporated and the product was dried under vacuum to obtain 126 mg of crude product as a white solid, which is the target compound. 1 H NMR (400MHz, DMSO-d6) δ10.39(s,1H),7.46(dd,J=8.5,2.2Hz,1H),7.43(d,J=2.1Hz,1H),7.18(d,J=8.6Hz,1H),3.85 (s,3H),3.77–3.67(m,4H),3.59–3.57(m,2H),3.25–3.20(m,1H),2.94–2.85(m,3H),2.72–2.64(m,2H).; LCMS(ESI)C 16 H 20 N4O4 + [M+H] + =333.27.

[0182] Example 7: N-(2,6-dioxopiperidin-3-yl)-2-fluoro-4-(piperazin-1-yl)benzamide hydrochloride

[0183]

[0184] Step 1: Synthesis of tert-butyl 4-(3-fluoro-4-(methoxycarbonyl)phenyl)piperazine-1-carboxylate

[0185] Weigh 500 mg (2.15 mmol, 1.0 eq) of 4-bromo-2-fluoroacetophenone, 602 mg (3.23 mmol, 1.5 eq) of N-BOC-piperazine, 736 mg (2.26 mmol, 1.05 eq) of cesium carbonate, and 133 mg (220 μmol, 0.1 eq) of BINAP into a 25 mL round-bottom flask, and add 10 mL of toluene. Under a nitrogen atmosphere, add 24 mg (110 μmol, 0.05 eq) of palladium acetate, purging the mixture three times, and heat at 60 °C for 20 hours. After LC-MS and TLC showed complete substrate consumption, filter and concentrate the reaction mixture, and purify by column chromatography to obtain a white solid in 90.9% yield. 1 H NMR (400MHz, DMSO-d6) δ7.71 (dd, J=7.9, 0.5Hz, 1H), 6.82–6.72 (m, 2H), 3.76 (s, 3H), 3.45–3.40 (m, 4H), 1.41 (s, 9H); LCMS (ESI) C17 H 23 FN2O4 + [M+H] + =339.27.

[0186] Step 2: Synthesis of 4-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-2-fluorobenzoic acid

[0187] 630 mg, 1.86 mmol, 1.0 eq of tert-butyl 4-(3-fluoro-4-(methoxycarbonyl)phenyl)piperazine-1-carboxylate was weighed into a round-bottom flask. MeOH (6 mL) was added, followed by 2 M NaOH solution (6 mL). The mixture was heated and stirred at 50 °C for 5 hours. After the reaction was completed as indicated by LC-MS, an appropriate amount of 1 M dilute hydrochloric acid solution was added to adjust the pH to 5. The solid in the mixture was filtered, the filter cake was washed with water, the filter residue was collected, and the residue was evaporated to dryness to obtain 550 mg of crude product as a white solid powder. 1 H NMR (400MHz, DMSO-d6) δ7.69 (dd, J=7.9, 0.5Hz, 1H), 6.85–6.64 (m, 2H), 3.48–3.39 (m, 8H), 1.41 (s, 9H); LCMS (ESI) C 16 H 21 FN2O4 + [M+H] + =324.19.

[0188] Step 3: Synthesis of tert-butyl 4-(4-((2,6-dioxopiperidin-3-yl)carbamoyl)-3-fluorophenyl)piperazine-1-carboxylate

[0189] Weigh 550 mg (1.70 mmol, 1.0 eq) of 4-(4-(tert-butyloxycarbonyl)piperazin-1-yl)-2-fluorobenzoic acid and HATU (646 mg, 1.70 mmol, 1.0 eq) into a round-bottom flask, add 6 mL of DMF and stir at room temperature for 5 minutes. Then add 280 mg (1.70 mmol, 1.0 eq) of 3-aminopiperidine-2,6-dione hydrochloride and DIEA (841 μL, 5.10 mmol, 3.0 eq) and stir at room temperature for 3 hours. After the reaction is complete as shown by LCMS, add an appropriate amount of water to the reaction solution, filter, wash the filter cake with water, collect the filter residue, and evaporate to dryness to obtain 700 mg of crude product as a white solid. 1H NMR (400MHz, DMSO-d6) δ10.86(s,1H),8.10(dd,J=8.0,0.5Hz,1H),7.64(dd,J=8.0,1.3Hz,1H),6.88–6.76(m,2H),4.73(ddd, J=12.6,7.9,5.7Hz,1H),3.47–3.42(m,4H),3.32–3.28(m,4H),2.82–2.52(m,2H),2.17–1.98(m,2H),1.43(s,9H); LCMS(ESI)C 21 H 27 FN4O5 + [M+H] + =435.16.

[0190] Step 4: Synthesis of N-(2,6-dioxopiperidin-3-yl)-2-fluoro-4-(piperazin-1-yl)benzamide hydrochloride

[0191] 700 mg (1.61 mmol, 1.0 eq) of tert-butyl 4-(4-((2,6-dioxadiazin-3-yl)carbamoyl)-3-fluorophenyl)piperazine-1-carboxylate was weighed into a round-bottom flask, and DCM (5 mL) and HCl·dioxane solution (5 mL) were added. The mixture was stirred at room temperature for 3 hours. LC-MS showed that the substrate was completely consumed. The solvent was evaporated, and the crude product was dried under vacuum to obtain a white powder, which was the target compound. 1 HNMR (400MHz, DMSO-d6) δ10.85(s,1H),9.29(s,2H),8.15(dd,J=8.0,0.5Hz,1H),7.66(dd,J=8.0,1.3Hz,1H),6.87(dd,J=1.3,0.5Hz,1H),4.73(dd, J=7.8,5.4Hz,1H),3.58–3.51(m,4H),3.22–3.13(m,4H),2.77(ddd,J=17. 2,13.3,5.6Hz,1H),2.53(d,J=3.9Hz,1H),2.18–1.96(m,2H); LCMS(ESI)C 16 H 19 FN4O3 + [M+H] + =335.15.

[0192] Example 8: 3-((3-fluoro-4-(piperazin-1-yl)phenyl)amino)piperidine-2,6-dione hydrochloride

[0193]

[0194]

[0195] Step 1: Synthesis of tert-butyl 4-(4-((2,6-dioxopiperidin-3-yl)amino)-2-fluorophenyl)piperazine-1-carboxylate

[0196] Weigh 500 mg (1.69 mmol, 1.0 eq) of tert-butyl (1-(4-amino-2-fluorophenyl)piperidin-4-yl)carbamate and 427 mg (5.08 mmol, 1.0 eq) of 3-bromopiperidin-2,6-dione into a round-bottom flask, dissolve in DMF (8 mL), then add NaHCO3 (1.42 g, 16.9 mmol, 10.0 eq), and stir at 80 °C for 12 hours. After the reaction is complete, cool to room temperature, add water (25 mL), extract with EA (12 mL × 3), wash the organic phase with saturated NaCl solution, dry with anhydrous Na2SO4, evaporate to dryness, and purify by column chromatography to obtain a green solid in 88.9% yield. 1 H NMR (400MHz, DMSO-d6) δ10.77(s,1H),6.83(dd,J=9.9,8.7Hz,1H),6.51(dd,J=14.9,2.6Hz,1H),6.41(dd,J=8.7,2.6Hz,1H),4.26(dd,J=7.7,4.8H z,1H),3.45–3.39(m,4H),2.81–2.75(m,4H),2.74–2.67(m,1H),2.60–2. 52(m,1H),2.16–2.02(m,1H),1.93–1.78(m,1H),1.41(s,9H); LCMS(ESI)C 20 H 27 FN4O4 + [M+H] + =407.31.

[0197] Step 2: Synthesis of 3-((3-fluoro-4-(piperazin-1-yl)phenyl)amino)piperidine-2,6-dione hydrochloride. 610 mg, 1.56 mmol, 1.0 eq of tert-butyl piperazine-1-carboxylate (2,6-dioxopiperidine-3-yl)amino)-2-fluorophenyl)piperazin-1-carboxylate (tert-butyl) was weighed into a round-bottom flask. 5 mL of HCl·dioxane solution and 5 mL of DCM were added. The mixture was stirred at room temperature for 3 hours. After the reaction was completed by LCMS monitoring, the solvent was evaporated, and the mixture was dried under vacuum to obtain a pale green crude powder, which is the target compound. 1HNMR(400MHz, DMSO-d6)δ10.83(s,1H),6.98–6.89(m,1H),6.60(dd,J=14.9,2.6Hz,1H),6.51(dd,J=8.7,2.6Hz,1H),4.35–4.29(m, 1H),3.24–3.18(m,4H),3.16–3.09(m,4H),2.79–2.68(m,1H),2.62–2.54(m,1H),2.12–2.02(m,1H),1.94–1.81(m,1H); LCMS(ESI)C 15 H 19 FN4O2 + [M+H] + =307.28.

[0198] Example 9: 3-(1-oxo-5-(piperazin-1-yl)isoindoline-2-yl)piperidine-2,6-dione hydrochloride

[0199]

[0200]

[0201] Step 1: Synthesis of tert-butyl 4-(3-cyano-4-(methoxycarbonyl)phenyl)piperazine-1-carboxylate

[0202] Methyl 2-cyanomethyl-4-fluorobenzoate (5.00 g, 27.91 mmol, 1.0 eq) and tert-butyl piperazine-1-carboxylate (6.20 g, 33.49 mmol, 1.2 eq) were weighed into a 250 mL round-bottom flask. DMSO (100 mL) was added to dissolve the methyl 2-cyanomethyl-4-fluorobenzoate, followed by the addition of DIEA (15 mL, 84 mmol, 3.0 eq). The reaction was stirred overnight at 120 °C. LCMS and TLC monitoring showed that the reaction was complete. Water (100 mL) was added to quench the reaction, and the mixture was extracted with EA (100 mL × 3). The organic phase was washed with saturated NaCl solution (200 mL × 2), dried over anhydrous Na2SO4, and then evaporated to dryness. The product was purified by column chromatography with a yield of 80.5%. 1 H NMR(400MHz, DMSO-d6)δ7.90(d,J=9.0Hz,1H),7.41(d,J=2.7Hz,1H),7.21(dd, J=9.1,2.7Hz,1H),3.82(s,3H),3.42(d,J=2.8Hz,8H),1.41(s,9H); LCMS(ESI)C 18 H 23 N3O4 + [M+H] + =346.07.

[0203] Step 2: Synthesis of tert-butyl 4-(3-aldehyde-4-(methoxycarbonyl)phenyl)piperazine-1-carboxylate

[0204] 7.00 g (20.27 mmol, 1.0 eq) of tert-butyl 4-(3-cyano-4-(methoxycarbonyl)phenyl)piperazine-1-carboxylate was weighed and added to a round-bottom flask, followed by water (35 mL), pyridine (27 mL), and AcOH (3 mL). The system was purged with N2 three times before and after the addition of Raney nickel (20 mL). The reaction was carried out overnight at 50 °C. LC-MS showed complete reaction. The reaction solution was diluted with EA (50 mL), filtered, and evaporated to dryness to obtain approximately 7.1 g of crude product. LC-MS (ESI) C 18 H 24 N2O5 + [M+H] + =349.18.

[0205] Step 3: Synthesis of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindoline-5-yl)piperazine-1-carboxylate

[0206] 7.1 g (20.38 mmol, 1.0 eq) of tert-butyl 4-(3-aldehyde-4-(methoxycarbonyl)phenyl)piperazine-1-carboxylate and 5.0 g (30.57 mmol, 1.5 eq) of 3-amino-2,6-piperidinidone hydrochloride were weighed into a round-bottom flask and dissolved in 140 mL of DCM. Then, 7.1 mL (40.76 mmol, 2.0 eq) of DIEA and 3 mL of AcOH were added. After stirring at room temperature for 2 hours, sodium cyanoborohydride (1.9 g, 30.57 mmol, 1.5 eq) was added, and the reaction was allowed to proceed overnight at room temperature. LC-MS showed that the reaction was complete. The reaction was quenched with 150 mL of saturated NaHCO3 solution, extracted with DCM (40 mL × 3), washed with an appropriate amount of water, washed with saturated NaCl solution, dried over anhydrous Na2SO4, and column chromatography was performed to give a white solid in 17.2% yield. 1 H NMR (400MHz, DMSO-d6) δ10.97(s,1H),7.55(d,J=8.4Hz,1H),7.08(d,J=8.9Hz,2H),5.06(dd,J=13.3,5.1Hz,1H),4.40–4.15(m ,2H),3.54–3.45(m,4H),3.33–3.24(m,4H),3.01–2.54(m,2H),2.46–2.30(m,1H),2.01–1.92(m,1H),1.44(s,9H); LCMS(ESI)C 22 H 28N4O5 + [M+H] + =429.10;

[0207] Step 4: Synthesis of 3-(1-oxo-5-(piperazin-1-yl)isoindoline-2-yl)piperidine-2,6-dione hydrochloride

[0208] 1.40 g (3.84 mmol, 1.0 eq) of tert-butyl 4-(2-(2,6-dioxopiridine-3-yl)-1-oxoisoindoline-5-yl)piperazine-1-carboxylate was weighed and added to a round-bottom flask. 15 mL of HCl·dioxane solution was added. After stirring at room temperature for 3 hours, LC-MS showed that the reaction was complete. The mixture was evaporated to dryness and freeze-dried to obtain a pale green solid crude product, which is the target compound, about 1.21 g. 1 H NMR (600MHz, DMSO-d6) δ10.95(s,1H),7.57(d,J=8.4Hz,1H),7.15(d,J=2.2Hz,1H),7.12(dd,J=8.6,2.3Hz,1H),5.05(dd,J=13.3,5.1 Hz,1H),4.40–4.19(m,2H),3.55–3.52(m,4H),3.22–3.18(m,4H),2.94–2.59(m,2H),2.42–2.32(m,1H),1.99–1.92(m,1H); LCMS(ESI)C 17 H 20 N4O3 + [M+H] + =329.21.

[0209] Example 10: 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione hydrochloride

[0210]

[0211] Step 1: Synthesis of tert-butyl piperazine-1-carboxylate (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-4-yl)piperazine-1-carboxylate

[0212] 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (1.00 g, 3.62 mmol, 1.0 eq) and piperazine-1-carboxylic acid tert-butyl ester (670 mg, 3.62 mmol, 1.0 eq) were weighed into a round-bottom flask, DMSO (16 mL) was added, followed by DIEA (1.8 mL, 10.86 mmol, 3.0 eq). The reaction was stirred at 90 °C for 4 hours. After LCMS showed that the reaction was complete, water (40 mL) was added to quench the reaction, and the mixture was extracted with EA (20 mL × 3). The organic phase was then washed with saturated NaCl solution, dried over anhydrous Na2SO4, and evaporated to dryness. Column chromatography yielded a yellow solid with a yield of 63.8%. 1 H NMR (400MHz, DMSO-d6) δ11.11(s,1H),7.73–7.69(m,1H),7.41–7.32(m,2H),5.15–5.07(m,1H),3.50(s,4 LCMS(ESI)C 22 H 26 N4O6 + [M+H] + =443.33.

[0213] Step 2: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)isoindoline-1,3-dione hydrochloride

[0214] 1.02 g (2.30 mmol, 1.0 eq) of compound 4-(2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-4-yl)piperazine-1-carboxylic acid tert-butyl ester was weighed into a flask, HCl·dioxane solution (10 mL) was added, the reaction was carried out for 2 hours, the mixture was evaporated to dryness, and then dried under vacuum to obtain an orange-yellow powder, which is the target compound. 1 HNMR(400MHz,DMSO-d6)δ11.13(s,1H),7.79–7.72(m,1H),7.48–7.39(m,2H),5.14–5.08(m,1H),3.53 –3.47(m,4H),3.30–3.23(m,4H),2.94–2.79(m,1H),2.64–2.52(m,2H),2.07–1.98(m,1H); LCMS(ESI)C 17 H 18 N4O4 + [M+H] + =344.29.

[0215] Example 11: 2-(1-(4-((2,6-dioxopiperidin-3-yl)amino)phenyl)-4-hydroxypiperidin-4-yl)acetic acid

[0216]

[0217] Step 1: Synthesis of tert-butyl 2-(4-hydroxy-1-(4-nitrophenyl)piperidin-4-yl)acetate

[0218] 1.88 g (8.73 mmol, 1.0 eq) of 2-(4-hydroxypiperidin-4-yl)AcOH tert-butyl ester (hydrochloride) was weighed into a round-bottom flask, and DMF (30 mL) was added. Then, p-fluoronitrobenzene (1.0 mL, 9.60 mmol, 1.1 eq) and DIEA (4.4 mL, 26.20 mmol, 3.0 eq) were added. The reaction was stirred overnight at 100 °C. After the reaction was complete as shown by LC-MS, the mixture was quenched with water (90 mL), extracted with EA (30 mL × 3), washed with saturated NaCl solution (50 mL × 2), dried over anhydrous Na₂SO₄, and subjected to column chromatography after rotary evaporation to obtain a yellow solid product in 88.8% yield. 1 H NMR(400MHz,DMSO-d6)δ8.02(d,J=9.1Hz,2H),7.01(d,J=9.1Hz,2H),4.75(s,1H) ,3.80(d,J=13.3Hz,2H),2.34(s,2H),1.72–1.59(m,4H),1.34(s,9H); LCMS(ESI)C 17 H 24 N2O5 + [M+H] + =337.44.

[0219] Step 2: Synthesis of tert-butyl 2-(1-(4-aminophenyl)-4-hydroxypiperidin-4-yl)acetate

[0220] 2.61 g (7.76 mmol, 1.0 eq) of 2-(4-hydroxy-1-(4-nitrophenyl)piperidin-4-yl)acetic acid tert-butyl ester was weighed into a round-bottom flask, dissolved in 25 mL of EA, and then 10% Pd / C (200 mg) was added. The mixture was purged with H2 three times and stirred overnight at room temperature. LC-MS showed that the reaction was complete. The reaction solution was diluted with an appropriate amount of EA, filtered, and evaporated to dryness to obtain a pale pink crude solid of approximately 2.18 g. 1HNMR(400MHz,DMSO-d6)δ6.71–6.63(m,2H),6.50–6.42(m,2H),4.53(s,2H),4.45(s,1H),3. 06–2.97(m,2H),2.89–2.78(m,2H),2.31(s,2H),1.77–1.53(m,4H),1.39(s,9H); LCMS(ESI)C 17 H 26 N2O3 + [M+H] + =307.10.

[0221] Step 3: Synthesis of tert-butyl 2-(1-(4-((2,6-dioxopiperidin-3-yl)amino)phenyl)-4-hydroxypiperidin-4-yl)acetate

[0222] 2.00 g (6.53 mmol, 1.0 eq) of tert-butyl 2-(1-(4-aminophenyl)-4-hydroxypiperidin-4-yl)acetate, 1.88 g (9.79 mmol, 1.5 eq) of 3-bromopiperidin-2,6-dione, and 1.65 g (19.58 mmol, 3.0 eq) of solid NaHCO3 were weighed into a round-bottom flask. DMF (20 mL) was added to dissolve the mixture. The mixture was stirred at 65 °C for 16 hours. After the reaction was completed, the mixture was quenched with water (60 mL), extracted with EA (35 mL × 3), washed with saturated NaCl solution (60 mL × 2), dried over anhydrous Na2SO4, evaporated to dryness, and column chromatography was performed to obtain a black solid product with a yield of 72.1%. 1 H NMR (400MHz, DMSO-d6) δ10.77(s,1H),6.74(d,J=8.5Hz,2H),6.58(d,J=8.4Hz,2H),4.46(s,1H),4.23–4.12(m,1H),3.11–3. 01(m,2H),2.90–2.85(m,2H),2.60–2.52(m,1H),2.32(s,2H),2.13–2.04(m,1H),1.93–1.53(m,6H),1.39(s,9H); LCMS(ESI)C 22 H 31 N3O5 + [M+H] + =418.18.

[0223] Step 4: Synthesis of 2-(1-(4-((2,6-dioxopiperidin-3-yl)amino)phenyl)-4-hydroxypiperidin-4-yl)acetic acid

[0224] Weigh 1.76 g (5.74 mmol, 1.0 eq) of tert-butyl 2-(1-(4-((2,6-dioxadiazin-3-yl)amino)phenyl)-4-hydroxypiperidin-4-yl)acetate, dissolve it in 4 mL of 1,4-dioxane, and then add 4 mL of HCl·dioxane solution. React overnight at room temperature. After the reaction is complete, evaporate to dryness and freeze dry to obtain a dark green solid, which is the target compound. 1 H NMR(400MHz, DMSO-d6)δ6.76(d,J=2.1Hz,2H),6.75(d,J=2.0Hz,2H),3.61(s,1H),3.34–3.30(m,2H),2.89–2.8 7(m,2H),2.73–2.71(m,2H),2.58–2.54(m,1H),2.32(s,2H),2.08–2.04(m,1H),1.93–1.87(m,4H); LCMS(ESI)C 18 H 23 N3O5 + [M+H] + =362.09.

[0225] Example 12: 1-(5-(2,6-dioxopiperidin-3-yl)pyridin-2-yl)piperidin-4-carboxylic acid

[0226]

[0227]

[0228] Step 1: Synthesis of tert-butyl 1-(5-bromopyridin-2-yl)piperidine-4-carboxylate

[0229] 2-Fluoro-5-bromopyridine (1.7 mL, 17.05 mmol, 1.0 eq) and piperidine-4-carboxylic acid tert-butyl hydrochloride (5.68 g, 25.57 mmol, 1.5 eq) were weighed into a round-bottom flask, dissolved in 40 mL of DMSO, and then DIEA (12.1 mL, 68.18 mmol, 4.0 eq) were added. The mixture was stirred at 90 °C for 24 hours. After the reaction was completed, the mixture was quenched with 120 mL of water, extracted with EA (50 mL × 3), washed with saturated NaCl solution (100 mL × 2), dried over anhydrous Na₂SO₄, evaporated to dryness, and column chromatography was performed to give a white solid in 54.3% yield. 1H NMR (600MHz, DMSO-d6) δ8.14(dd,J=2.7,0.7Hz,1H),7.64(dd,J=9.1,2.6Hz,1H),6.83(dd,J=9.2,0.7Hz,1H),4.15–4.08 (m,2H),2.98–2.87(m,2H),2.47(tt,J=11.1,4.0Hz,1H),1.85–1.77(m,2H),1.50–1.43(m,2H),1.39(s,9H); LCMS(ESI)C 15 H 21 BrN2O2 + [M+H] + =341.18.

[0230] Step 2: Synthesis of tert-butyl 1-(2',6'-bis(benzyloxy)-[3,3'-bipyridine]-6-yl)piperidine-4-carboxylate

[0231] Weigh tert-butyl 1-(5-bromopyridin-2-yl)piperidin-4-carboxylate (3.16 g, 9.26 mmol, 1.0 eq), 2,6-bis(benzyloxy)-3-(4,4,5,5-tetramethyl-1,3,2-dioxoboropentane-2-yl)pyridine (4.25 g, 10.19 mmol, 1.1 eq), and Cs2CO3 (9.05 g, 27.18 mmol, 3.0 eq) into a round-bottom flask. Add ultra-dry 1,4-dioxane (50 mL) and water (10 mL). After purging with N2 three times, add Pd(dppf)Cl2·DCM (113 mg, 0.14 mmol, 0.015 eq), and then purge with N2 three more times. Stir the system at 110 °C for 16 hours. After the reaction was complete, the reaction solution was diluted with an appropriate amount of EA, filtered, evaporated to dryness, and subjected to column chromatography to obtain a yellow solid with a yield of 33.0%. 1 H NMR (600MHz, DMSO-d6) δ8.28(d,J=2.5Hz,1H),7.74–7.69(m,2H),7.45–7.27(m,10H),6.85(d,J=8.9Hz,1H),6.53(d,J=8.1Hz,1H),5.39(s ,2H),5.36(s,2H),4.21–4.16(m,2H),2.96–2.91(m,2H),2.49–2.45(m,1H),1.86–1.79(m,2H),1.53–1.47(m,2H),1.40(s,9H); LCMS(ESI)C 34 H 37 N3O4 + [M+H] + =552.46.

[0232] Step 3: Synthesis of tert-butyl 1-(5-(2,6-dioxopiperidin-3-yl)pyridin-2-yl)piperidine-4-carboxylate

[0233] Weigh 1.69 g (3.06 mmol, 1.0 eq) of 1-(2',6'-bis(benzyloxy)-[3,3'-bipyridine]-6-yl)piperidin-4-carboxylic acid tert-butyl ester into a round-bottom flask, add EA (5 mL), EtOH (5 mL), and THF (10 mL), then add 10% Pd / C (1.5 g). The mixture is purged with H2 three times and stirred overnight at room temperature. After the reaction is complete, filter the solution, wash the filter cake with a mixed solution of DCM and MeOH, collect the filtrate, and evaporate to dryness to obtain a brown, oily crude product. 1 H NMR (600MHz, DMSO-d6) δ7.94(d,J=2.5Hz,1H),7.37(dd,J=8.8,2.5Hz,1H),6.80(d,J=8.9Hz,1H),4.22–4.07(m,2H),3.75–3.71(m,1H),2 .93–2.87(m,2H),2.71–2.60(m,1H),2.21–2.10(m,1H),2.00–1.94(m,1H),1.84–1.79(m,2H),1.51–1.45(m,2H),1.39(s,9H); LCMS(ESI)C 34 H 37 N3O4 + [M+H] + =552.46.

[0234] Step 4: Synthesis of 1-(5-(2,6-dioxopiperidin-3-yl)pyridin-2-yl)piperidin-4-carboxylic acid

[0235] Weigh out tert-butyl 1-(5-(2,6-dioxadiazin-3-yl)pyridin-2-yl)piperidine-4-carboxylate (865 mg, 2.73 mmol, 1.0 eq), add 10 mL of HCl·dioxane solution, stir overnight at room temperature, after the reaction is complete, evaporate to dryness, and then dry under vacuum to obtain a black solid, which is the target compound. 1H NMR (400MHz, DMSO-d6) δ10.91(d,J=2.6Hz,1H),7.90(d,J=2.7Hz,1H),7.77(dd,J=9.2,2.3Hz,1H),7.26(d,J=9.7Hz,1H),4.13–4.07( LCMS(ESI)C 16 H 19 N3O4 + [M+H] + =318.12.

[0236] Example 13: 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-carboxylic acid

[0237]

[0238] Step 1: Synthesis of tert-butyl 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidine-4-carboxylate

[0239] Weigh 1.00 g (3.62 mmol, 1.0 eq) of 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione and 872 mg (4.71 mmol, 1.3 eq) of piperidine-4-carboxylic acid tert-butyl hydrochloride into a round-bottom flask, add 10 mL of DMSO, then add 1.8 mL (10.86 mmol, 3.0 eq) of DIEA, stir at 90 °C for 12 hours. After the reaction is complete, quench with 30 mL of water, extract the reaction solution with 15 mL × 3, wash the organic phase with saturated NaCl solution, dry with anhydrous Na2SO4, evaporate to dryness and then column chromatography to give a yellow solid with a yield of 92.7%. 1H NMR (600MHz, DMSO-d6) δ11.07(s,1H),7.66(d,J=8.5Hz,1H),7.32(d,J=2.4Hz,1 H),7.24(dd,J=8.6,2.4Hz,1H),5.06(dd,J=12.9,5.4Hz,1H),4.00–3.90(m,2H) ,3.11–3.02(m,2H),2.90–2.80(m,1H),2.62–2.58(m,1H),2.58–2.52(m,2H),2. 04–1.97(m,1H),1.90–1.83(m,2H),1.62–1.51(m,2H),1.40(s,9H); LCMS(ESI)C 23 H 27 N3O6 + [M+H] + =442.45.

[0240] Step 2: Synthesis of 1-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidin-4-carboxylic acid

[0241] 1.48 g (3.35 mmol, 1.0 eq) of compound 1-(2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindoline-5-yl)piperidine-4-carboxylic acid tert-butyl ester was weighed into a flask, and 15 mL of HCl·dioxane solution was added. The mixture was stirred overnight at room temperature. After the reaction was completed, the crude product was obtained by rotary evaporation, which yielded the target compound. 1 HNMR (400MHz, DMSO-d6) δ11.08(s,1H),7.66(d,J=8.5Hz,1H),7.33(d,J=2.3Hz,1H),7.24(dd,J=8.6,2.4Hz,1H),5.09–5.03(m, 1H),3.12–3.05(m,2H),2.94–2.81(m,2H),2.56(dq,J=10.5,5.1,4.5Hz,2H),2.06–1.86(m,4H),1.65–1.54(m,2H); LCMS(ESI)C 19 H 19 N3O6 + [M+H] + =386.11.

[0242] Example 14: 2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-5-fluoro-1-methyl-1H-indazol-6-yl)-4-hydroxypiperidin-4-yl)acetic acid

[0243]

[0244] Step 1: Synthesis of tert-butyl 2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-5-fluoro-1-methyl-1H-indazol-6-yl)-4-hydroxypiperidin-4-yl)acetate

[0245] Weigh out 1-(6-bromo-7-fluoro-1-methyl-1H-indazol-3-yl)dihydropyrimidine-2,4(1H,3H)-dione (100 mg, 293 μmol, 1.0 eq), 2-(4-hydroxypiperidin-4-yl)tert-butyl acetate (130 mg, 602 μmol, 2.0 eq), and Cs2CO3 (244 mg, 749 μmol, 2.5 eq) into a pressure-resistant bottle, add ultra-dry 1,4-dioxane (3 mL), and under N2 atmosphere, add Pd-PEPPSI-IPent(Cl) (14 mg, 15 μmol, 0.05 eq) and stir at 110 °C for 16 hours. After the reaction was completed, the mixture was quenched with water (10 mL), extracted with EA (5 mL × 3), washed with saturated NaCl solution (100 mL × 2), dried over anhydrous Na2SO4, and evaporated to dryness. The yellow solid product was obtained by thin-layer chromatography with a yield of 28.8%. 1 H NMR (600MHz, DMSO-d6) δ10.52(s,1H),7.32(d,J=12.8Hz,1H),7.10(d,J=7.1Hz,1H),4.59(s,1H),3.93(s,3H),3 .90–3.86(m,2H),3.17–3.01(m,4H),2.76–2.71(m,2H),2.38(s,2H),1.88–1.68(m,4H),1.42(s,9H); LCMS(ESI)C 23 H 30 FN5O5 + [M+H] + =476.29.

[0246] Step 2: Synthesis of 2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-5-fluoro-1-methyl-1H-indazol-6-yl)-4-hydroxypiperidin-4-yl)acetic acid

[0247] Weigh 40 mg, 190 μmol, 1.0 eq of tert-butyl 2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-5-fluoro-1-methyl-1H-indazol-6-yl)-4-hydroxypiperidin-4-yl)acetate into a round-bottom flask, add 2 mL of HCl·dioxane solution, stir overnight at room temperature, and after the reaction is complete, evaporate to dryness to obtain the crude product, which is the target compound.1 HNMR (400MHz, DMSO-d6) δ10.53(s,1H),7.33(d,J=12.8Hz,1H),7.12(d,J=7.0Hz,1H),3.94(s,3H),3.9 0–3.86(m,2H),3.18–2.99(m,4H),2.76–2.71(m,2H),2.45–2.39(m,2H),1.91–1.70(m,4H); LCMS(ESI)C 19 H 22 FN5O5 + [M+H] + =420.04.

[0248] Example 15: 2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-5-fluoro-1-methyl-1H-indazol-6-yl)piperidin-4-yl)acetic acid

[0249]

[0250] Step 1: Synthesis of tert-butyl 2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-5-fluoro-1-methyl-1H-indazol-6-yl)piperidin-4-yl)acetate

[0251] Weigh out 1-(6-bromo-7-fluoro-1-methyl-1H-indazol-3-yl)dihydropyrimidine-2,4(1H,3H)-dione (100 mg, 293 μmol, 1.0 eq), 4-piperidine AcOH tert-butyl ester (120 mg, 602 μmol, 2.0 eq), and Cs2CO3 (244 mg, 749 μmol, 2.5 eq) into a sealed tube. Then, add ultra-dry 1,4-dioxane (5 mL), replace N2 three times, add catalyst Pd-PEPPSI-IPent(Cl) (14 mg, 15 μmol, 0.05 eq), replace N2 three more times, and stir at 110 °C for 16 hours. After the reaction was completed, the mixture was cooled to room temperature, quenched with water (10 mL), extracted with EA (5 mL × 3), washed with saturated NaCl solution (100 mL × 2), dried with anhydrous Na2SO4, and evaporated to dryness. The yellow solid product was obtained by thin-layer chromatography purification with a yield of 34.4%. 1H NMR (600MHz, DMSO-d6) δ10.52(s,1H),7.33(d,J=12.7Hz,1H),7.09(d,J=7.1Hz,1H),3.93(s,3H),3.91–3.87 (m,2H),3.43–3.39(m,2H),2.75–2.65(m,4H),2.23–2.17(m,2H),1.86–1.74(m,4H),1.42(s,9H); LCMS(ESI)C 23 H 30 FN5O4 + [M+H] + =460.22.

[0252] Step 2: Synthesis of 2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-5-fluoro-1-methyl-1H-indazol-6-yl)piperidin-4-yl)acetic acid

[0253] 45 mg, 95 μmol, 1.0 eq) of tert-butyl 2-(1-(3-(2,4-dioxotetrahydropyrimidin-1(2H)-yl)-5-fluoro-1-methyl-1H-indazol-6-yl)piperidin-4-yl)acetate was weighed into a 25 mL round-bottom flask, and 2 mL of HCl·dioxane solution was added. The mixture was stirred overnight at room temperature. After the reaction was completed as shown by LCMS, the crude product was obtained by rotary evaporation, which is the preparation of the target compound. 1 HNMR(400MHz,DMSO-d6)δ10.53(s,1H),7.34(d,J=12.6Hz,1H),7.12(dd,J=7.1,2.4Hz,1H),3.93(s,3H),3 .91–3.85(m,2H),3.43–3.40(m,2H),2.75–2.67(m,4H),2.36–2.30(m,2H),1.91–1.72(m,4H); LCMS(ESI)C 19 H 22 FN5O4 + [M+H] + =404.07.

[0254] Example 16: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione

[0255]

[0256] Step 1: Synthesis of tert-butyl piperazine-1-carboxylate (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazine-1-carboxylate

[0257] 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione (1.00 g, 3.62 mmol, 1.0 eq) and piperazine-1-carboxylic acid tert-butyl ester (670 mg, 3.62 mmol, 1.0 eq) were weighed into a round-bottom flask, DMSO (16 mL) was added, followed by DIEA (1.8 mL, 10.86 mmol, 3.0 eq). The reaction was stirred at 90 °C for 4 hours. After LCMS showed complete reaction, water (40 mL) was added to quench the reaction, and the mixture was extracted with EA (20 mL × 3). The organic phase was then washed with saturated NaCl solution, dried over anhydrous Na2SO4, and evaporated to dryness. Column chromatography yielded a yellow solid with a yield of 72.4%. HRMS (ESI): m / z calcd for C 22 H 26 N4O6[M+H] + 442.1852, found 442.1847.

[0258] Step 2: Synthesis of 2-(2,6-dioxopiperidin-3-yl)-5-(piperazin-1-yl)isoindoline-1,3-dione hydrochloride

[0259] 1.02 g (2.30 mmol, 1.0 eq) of 4-(2-(2,6-dioxopiridin-3-yl)-1,3-dioxoisoindoline-5-yl)piperazine-1-carboxylic acid tert-butyl ester was weighed into a round-bottom flask, and 10 mL of HCl·dioxane solution was added. The mixture was reacted for 2 hours, evaporated to dryness under vacuum, and dried to obtain an orange-yellow powder, which is the target compound. HRMS(ESI): m / z calcd for C 17 H 18 N4O4[M+H] + 342.1328, found 342.1326.

[0260] Example 17: Synthesis of 2-fluoro-N-(1-methyl-2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)benzamide hydrochloride

[0261]

[0262] Step 1: Synthesis of (2,6-dioxopiperidin-3-yl)tert-butyl carbamate

[0263] 3-N-tert-Butoxycarbonylamino-2,6-dioxopiperidine (1 g, 4.38 mmol, 1.0 eq) and K₂CO₃ (1.66 g, 13.14 mmol, 3.0 eq) were weighed into a round-bottom flask, dissolved in DMF (15 mL), and iodomethane (818 μL, 13.14 mmol, 3.0 eq) was slowly added dropwise in an ice bath. The reaction mixture was then refluxed at 50 °C for 3 hours. After the reaction was complete as indicated by LCMS and TLC, water (30 mL) was added to quench the reaction. The mixture was then extracted with EA (10 mL × 3) and DCM (10 mL × 3), dried over anhydrous Na₂SO₄, and evaporated to dryness to obtain 1.01 g of crude product.

[0264] Step 2: Synthesis of 3-amino-1-methyl-2,6-piperidinidone hydrochloride

[0265] Weigh 1.01 g (4.16 mmol, 1.0 eq) of compound (2,6-dioxadiidine-3-yl) tert-butyl carbamate into a round-bottom flask, add DCM (10 mL) to dissolve it, then add HCl·dioxane (4 M, 10 mL). After the reaction is complete as shown by LCMS, evaporate to dryness and dry under vacuum to obtain the crude product, which is a yellow solid.

[0266] Step 3: Synthesis of tert-butyl piperazine-1-carboxylate

[0267] 200 mg (617 μmol, 1.0 eq) of 4-(4-(tert-butyloxycarbonyl)piperazin-1-yl)-2-fluorobenzoic acid was weighed into a round-bottom flask. HATU (235 mg, 617 μmol, 1.0 eq) was added, followed by 3-amino-1-methyl-2,6-piperidinidone hydrochloride (146 mg, 740 μmol, 1.2 eq). After dissolving in 10 mL of DMF, DIEA (322 μL, 1.85 mmol, 3.0 eq) was added. The reaction was stirred at room temperature for 1 hour. After LC-MS and TLC showed completion, the reaction was quenched with 20 mL of water, extracted with EA (5 mL × 3), dried over anhydrous Na₂SO₄, evaporated to dryness, and column chromatography yielded a pale purple solid.

[0268] Step 4: Synthesis of 2-fluoro-N-(1-methyl-2,6-dioxopiperidin-3-yl)-4-(piperazin-1-yl)benzamide hydrochloride

[0269] 200 mg, 446 μmol, 1.0 eq) of 4-(3-fluoro-4-((1-methyl-2,6-dioxadiazin-3-yl)carbamoyl)phenyl)piperazine-1-carboxylic acid tert-butyl ester was weighed into a flask, DCM (2 mL) was added, followed by HCl·dioxane (4 M, 2 mL). The reaction was stirred at room temperature for 1 hour. After the reaction was completed as indicated by LCMS, the reaction solution was evaporated to dryness and then freeze-dried to obtain the crude product, which was a black solid.

[0270] III. Synthesis methods of various CDK2 / 4 / 6 protein hydrolysis targeting chimeras in this invention

[0271] The compounds of Formula I and / or their pharmaceutically acceptable salts can be synthesized using commercially available raw materials using synthetic techniques known in the art. The synthetic methods described below illustrate the preparation methods of most of the compounds. Those skilled in the art can prepare salts, racemates, enantiomers, phosphates, sulfates, hydrochlorides, and prodrug forms of the compounds of Formula I according to conventional techniques in the art.

[0272] Synthesis Method A: Weigh 1.0 eq of pyrido[2,3-d]pyrimidine-7(8H)-one intermediate and 1.5 eq of E3 ubiquitin ligase ligand into a round-bottom flask, add DMF, then add 5.0 eq of DIEA, stir overnight at room temperature, and after the reaction is complete as shown by LCMS, add water to quench the reaction, extract three times with EA, wash the organic phase twice with saturated NaCl solution, dry with anhydrous Na2SO4, evaporate to dryness, purify by preparative thin-layer chromatography, and freeze-dry to obtain the final product.

[0273] Synthesis Method B: Weigh 1.0 eq of pyrido[2,3-d]pyrimidine-7(8H)-one intermediate, HATU (1.0 eq), and E3 ubiquitin ligand (1.0 eq) into a round-bottom flask, add DMF, then add DIEA (5.0 eq), stir at room temperature for 1 hour, after the reaction is complete, quench the reaction solution with water, extract three times with EA, wash the organic phase twice with saturated NaCl solution, dry with anhydrous Na2SO4, evaporate to dryness, purify by preparative thin-layer chromatography, and then vacuum dry to obtain the final product.

[0274] Table 1. Substrates and synthesis methods of various CDK2 / 4 / 6 protein hydrolysis targeting chimeras in this invention.

[0275]

[0276]

[0277]

[0278] Example 18: 3-(6-(4-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridino[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperazin-1-yl)pyridin-3-yl)piperidin-2,6-dione (numbered WYY-03-120)

[0279] Characterization results of WYY-03-120: 1 H NMR(600MHz,Chloroform-d)δ10.07(s,1H),8.61(s,1H),8.03(s,1H),7.72(d,J=7.8Hz,2H),7.54(d,J=8.0Hz,2H),7.31(d,J=8.8Hz,1H), 6.64(d,J=8.9Hz,1H),5.80–5.62(m,1H),3.92–3.48(m,10H),2.76–2 .42(m,11H),2.34–2.14(m,7H),2.11–2.03(m,2H),1.89–1.49(m,8H); 13 C NMR(151MHz,Chloroform-d)δ202.97,174.03,173.34,161.73,160.27,158.93,157.28,155.86,147.57,143.89,137.3 1,129.63,127.86,121.89,107.18,62.42,53.23,47.67,45.10,32.12,30.49,27.93,26.37,25.41,14.03; LCMS(ESI)C 41 H 49 N9O6S + [M+H] + =796.35.

[0280] Example 19: 3-(3-(4-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperazin-1-yl)phenyl)piperidin-2,6-dione (numbered WYY-03-121)

[0281] Characterization results of WYY-03-121: 1H NMR(600MHz,Chloroform-d)δ9.85(s,1H),8.63(s,1H),7.72(d,J=7.9Hz,2H),7.5 4(d,J=7.9Hz,2H),7.25–7.18(m,1H),6.84(dd,J=8.3,2.4Hz,1H),6.76–6.72(m,1 H),6.67(d,J=7.5Hz,1H),5.79–5.67(m,1H),3.90–3.58(m,6H),3.26–3.14(m,4H) ,2.72–2.45(m,11H),2.33–2.16(m,7H),2.08(d,J=13.7Hz,2H),1.92–1.52(m,8H); 13 C NMR(151MHz,Chloroform-d)δ202.87,173.96,173.38,162.63,161.68,160 .19,157.23,155.78,151.65,143.91,142.63,138.20,129.67,129.53,127 .77,119.17,116.01,115.27,106.23,62.25,53.35,53.12,49.00,48.14,4 7.63,44.96,36.54,31.52,30.92,27.85,26.51,25.33,13.96; LCMS(ESI)C 42 H 50 N8O6S + [M+H] + =795.24.

[0282] Example 20: 1-(6-(1-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridino[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperidin-4-yl)-5-fluoro-1-methyl-1H-indazol-3-yl)dihydropyrimidin-2,4(1H,3H)-dione (numbered WYY-03-122)

[0283] Characterization results of WYY-03-122: 1H NMR (600MHz, DMSO-d6) δ10.33(d,J=5.3Hz,1H),8.75(d,J=4.3Hz,1H),7.72(d ,J=7.9Hz,2H),7.61(d,J=7.4Hz,2H),7.41–7.37(m,1H),7.35(d,J=2.1Hz,1H) ,5.94–5.54(m,1H),3.84(s,3H),3.76–3.44(m,11H),2.73–2.64(m,2H),2.40( d,J=25.0Hz,8H),2.23(d,J=6.5Hz,5H),1.99–1.87(m,3H),1.76–1.39(m,8H); 13 CNMR(151MHz,DMSO-d6)δ202.43,170.62,160.95,160.27,158.46,154.78 ,153.64,151.80,142.70,141.43,137.98,137.73,131.58,129.37,127.5 6,115.59,109.40,106.31,104.77,53.61,52.33,51.98,47.16,45.15,43 .94,35.58,31.26,30.93,30.29,27.20,25.45,24.58,13.53; LCMS(ESI)C 44 H 51 FN 10 O6S + [M+H] + =867.25.

[0284] Example 21: 1-(5-(4-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridino[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperazin-1-formyl)-2-methoxyphenyl)dihydropyrimidin-2,4(1H,3H)-dione (numbered WYY-03-123)

[0285] Characterization results of WYY-03-123: 1H NMR(600MHz,DMSO-d6)δ10.33(s,1H),8.75(d,J=4.3Hz,1H),8.03(d,J=7.7Hz,1H), 7.72(d,J=7.9Hz,2H),7.61(d,J=7.4Hz,2H),7.39(dd,J=8.4,2.2Hz,1H),7.35(d,J= 2.1Hz,1H),5.94–5.54(m,1H),3.84(s,3H),3.75–3.43(m,11H),2.73–2.64(m,2H), 2.40(d,J=25.0Hz,8H),2.23(d,J=6.5Hz,5H),1.99–1.87(m,3H),1.76–1.39(m,8H); 13 C NMR(151MHz,DMSO-d6)δ170.85,168.17,161.00,160.30,158.52,155.85,152.14,142.75,133.72,129.54,128.70,128.16,127.87–127 .35(m),112.09,104.81,61.06,56.00,52.36,47.17,45.16,44.32,31.30,31.10,30.60,30.30,27.66,27.22,25.47,24.58; LCMS(ESI)C 43 H 51 N9O8S + [M+H] + =854.22.

[0286] Example 22: 4-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperazin-1-yl)-N-(2,6-dioxopiperidin-3-yl)-2-fluorobenzamide (numbered WYY-03-127)

[0287] Characterization results of WYY-03-127: 1H NMR(600MHz,DMSO-d6)δ10.85(s,1H),8.74(d,J=7.0Hz,1H),8.06–8.02(m,1H),7.7 3(d,J=7.9Hz,2H),7.65–7.59(m,3H),6.81(d,J=9.1Hz,1H),6.76(d,J=15.5Hz,1H), 5.90–5.61(m,1H),4.76–4.70(m,1H),3.90–3.55(m,6H),3.31(d,J=5.3Hz,4H),2.92 –2.70(m,2H),2.38(s,6H),2.27–2.05(m,7H),2.04–1.89(m,4H),1.75–1.42(m,8H); 13 C NMR(151MHz,DMSO-d6)δ202.51,173.04,172.30,163.04,162.18,161.00,160.55,160.30,154.83,154.34,142.75,133.75,131.66,129.52,12 7.58,109.80,104.82,100.77,100.59,61.11,52.27(d,J=39.3Hz),49. 78,47.20,46.78,45.16,31.31,31.01,27.25,24.61,24.16; LCMS(ESI)C 43 H 50 FN9O7S + [M+H] + =856.54.

[0288] Example 23: 3-((4-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperazin-1-yl)-3-fluorophenyl)amino)piperidin-2,6-dione (numbered WYY-03-128)

[0289] Characterization results of WYY-03-128: 1H NMR(600MHz,Chloroform-d)δ9.92(s,1H),8.61(s,1H),7.72(d,J=7.9Hz,2H),7.55(d,J=7.8 Hz,2H),6.89–6.79(m,1H),6.46–6.33(m,2H),5.81–5.64(m,1H),4.65(d,J=4.1Hz,1H),4.04 –3.95(m,1H),3.90–3.79(m,1H),3.72–3.61(m,3H),3.05–2.96(m,3H),2.84–2.72(m,2H),2. 67–2.56(m,5H),2.52–2.43(m,4H),2.34–2.04(m,8H),2.01–1.94(m,1H),1.91–1.49(m,9H); 13 C NMR(151MHz,Chloroform-d)δ202.99,173.08,172.37,161.74,160.24,157.23,156.16,155.90,142.96,132.06,129.79,129.59,127. 85,120.68,109.27,102.58,102.41,62.39,54.92,53.44,52.73,51.25,44.99,31.66,31.24,27.94,25.75,25.41,14.04; LCMS(ESI)C 42 H 50 FN9O6S + [M+H] + =828.43.

[0290] Example 24: 3-(5-(4-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperazin-1-yl)-1-oxoisoindoline-2-yl)piperidin-2,6-dione (numbered WYY-03-129)

[0291] Characterization results of WYY-03-129: 1H NMR(600MHz,Chloroform-d)δ10.10(s,1H),8.59(s,1H),7.71(dd,J=12.3,8.3Hz,3H),7 .54(d,J=8.0Hz,2H),6.96(dd,J=8.8,2.1Hz,1H),6.87(s,1H),5.81–5.63(m,1H),5.22–5 .08(m,1H),4.41–4.21(m,2H),3.90–3.58(m,5H),3.37–3.27(m,4H),2.87–2.74(m,2H), 2.65–2.55(m,5H),2.47(s,3H),2.34–2.22(m,6H),2.19–2.01(m,4H),1.91–1.49(m,8H); 13 C NMR(151MHz,Chloroform-d)δ202.97,172.52,170.79,169.66,161.73,160.23, 157.23,155.86,154.42,143.82(d,J=6.8Hz),142.65,135.04,129.62,127.89, 125.07,122.06,115.61,108.49,106.18,62.30,53.43,52.97,51.83,48.40,47 .17,45.01,31.70(d,J=15.4Hz),31.09,27.93,25.40,23.59,14.03; LCMS(ESI)C 44 H 51 N9O7S + [M+H] + =850.20.

[0292] Example 25: 4-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)benzyl)piperazin-1-yl)-2-(2,6-dioxopiridin-3-yl)isoindoline-1,3-dione (numbered WYY-03-133)

[0293] Characterization results of WYY-03-133: 1H NMR(600MHz,DMSO-d6)δ11.08(s,1H),8.73(d,J=9.6Hz,1H),7.80–7.71(m,2H),7.70 –7.58(m,3H),7.34(d,J=7.1Hz,1H),7.31(d,J=8.3Hz,1H),5.94–5.52(m,1H),5.13– 5.00(m,1H),3.87–3.56(m,5H),3.35–3.14(m,4H),2.91–2.82(m,1H),2.66–2.54(m, 5H),2.46–2.30(m,5H),2.22(d,J=8.4Hz,5H),2.07–1.86(m,4H),1.76–1.40(m,8H); 13 C NMR(151MHz,DMSO-d6)δ202.49,172.82,170.00,167.06,166.34,161.02,1 60.28,158.50,154.80,149.61,142.78,135.91,133.67,129.61,127.60,12 3.73,116.57,114.93,105.25,104.84,61.25,52.50,52.03,50.44,48.84, 47.22,45.25,31.29,30.98,30.34,27.21,25.48,24.54,22.09; LCMS(ESI)C 44 H 49 N9O8S + [M+H] + =864.24.

[0294] Example 26: WWZ-01-102

[0295] Characterization results of WWZ-01-102: HRMS(ESI): m / z calcd for C 44 H 49 N9O8S[M+H] + 863.3425, found 863.3423.

[0296] Example 27: WYY-04-065

[0297] Characterization results of WYY-04-065: HRMS(ESI): m / z calcd for C 44 H 52 FN9O7S[M+H] + 870.3772, found 870.3766;1 H NMR(600MHz,Chloroform-d)δ8.56(s,1H),7.95–7.90(m,1H),7.75–7.70(m,2H),7.56–7.52( m,2H),7.52–7.48(m,1H),6.74–6.62(m,1H),6.55–6.44(m,1H),4.76–4.67(m,1H),3.91–3.83 (m,1H),3.63(s,3H),3.35–3.30(m,4H),3.17(s,3H),2.94–2.84(m,2H),2.82–2.76(m,1H),2. 66–2.61(m,1H),2.60–2.56(m,4H),2.52–2.42(m,4H),2.34–2.06(m,8H),1.93–1.53(m,10H). 13 C NMR(151MHz,Chloroform-d)δ202.93,172.21,171.62,163.74,163.33,161. 70,160.44,157.47,155.75,155.10,155.03,143.75,142.39,135.11,132.91 ,132.88,129.52,128.03,127.88,110.32,109.44,100.84,100.65,62.22,52.71,52.01,47.36,38.68,36.57,31.77,31.61,31.51,27.30,24.59,13.98.

[0298] Example 28: 3-((4-(4-(2-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridino[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)piperidin-1-yl)-2-oxoethyl)-4-hydroxypiperidin-1-yl)phenyl)amino)piperidin-2,6-dione (numbered WYY-03-182)

[0299] Characterization results of WYY-03-182: 1H NMR(600MHz,Chloroform-d)δ8.63(s,1H),7.16–6.81(m,2H),6.75–6.39(m,2H),5.86–5.71(m,1H),5.19–4.85(m,1H),4 .64(d,J=79.1Hz,1H),4.21–3.58(m,6H),3.30–2.90(m,8H),2.56–2.39(m,6H),2.38–2.04(m,10H),2.02–1.48(m,16H); 13 C NMR(151MHz,Chloroform-d)δ203.25,173.41,170.86,161.77,160.38,157.74,155.94,142.57,119.84,114.98 ,67.88,59.16,53.53,47.97,44.71,42.44,40.42,36.85,31.72,29.83,28.13,25.71,14.09,1.16; LCMS(ESI)C 43 H 57 N9O8S + [M+H] + =860.25.

[0300] Example 29: 3-(6-(4-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)piperidin-1-yl)piperidin-1-yl)pyridin-3-yl)piperidin-2,6-dione (numbered WYY-03-183)

[0301] Characterization results of WYY-03-183: 1H NMR(600MHz,Chloroform-d)δ9.96(s,1H),8.65(s,1H),8.01(d,J=2.5Hz,1H),7.32(dd,J=8.9,2.5Hz,1 H),6.68(d,J=8.9Hz,1H),5.83–5.76(m,1H),4.76–4.70(m,1H),4.35–4.25(m,2H),4.13–4.00(m,2H),3. 82–3.71(m,2H),3.67–3.61(m,1H),3.18–3.06(m,4H),2.95–2.86(m,4H),2.76–2.69(m,2H),2.68–2.62 (m,1H),2.60–2.53(m,1H),2.50–2.49(m,3H),2.38–2.27(m,6H),2.26–1.93(m,11H),1.72–1.57(m,7H); 13 C NMR(151MHz,Chloroform-d)δ203.02,174.08,173.35,173.18,161.75,160.29,158.54,157.26,155.96,147.20,142. 64,137.54,121.61,107.58,59.48,44.95,40.83,38.74,31.86,31.39,29.64,28.13,26.52,25.63,22.81; LCMS(ESI)C 41 H 53 N9O7S + [M+H] + =816.30.

[0302] Example 30: 5-(4-(4-((4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)piperidin-1-formyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (numbered WYY-03-184)

[0303] Characterization results of WYY-03-184: 1H NMR(600MHz,Chloroform-d)δ10.49(s,1H),8.65(d,J=8.9Hz,1H),7.65(d,J=8.4Hz,1H),7.2 7–7.22(m,1H),7.05(d,J=8.8Hz,1H),5.87–5.74(m,1H),4.99–4.90(m,1H),4.77–4.66(m,1H ),4.14–4.02(m,2H),3.88–3.64(m,3H),3.20–3.10(m,4H),2.95–2.90(m,2H),2.85–2.74(m, 4H),2.50(s,5H),2.38–2.16(m,9H),2.14–2.05(m,5H),2.01–1.96(m,3H),1.75–1.59(m,7H); 13 C NMR(151MHz,Chloroform-d)δ203.07,172.66,169.62,168.14,167.43,161.74,157.30,155.92,155.35,134.43,129.51,125.52,119 .02,118.12,108.73,59.22,55.40,53.54,49.20,47.38,44.42,40.85,40.48,37.90,31.68,28.06,27.09,25.67,22.92; LCMS(ESI)C 44 H 53 N9O9S + [M+H] + =884.56.

[0304] Example 31: 1-6-(4-(2-(4-(4-(4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridino[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)piperidin-1-yl)-2-oxoethyl)-4-hydroxypiperidin-1-yl)-5-fluoro-1-methyl-1H-indolezol-3-yl)dihydropyrimidin-2,4(1H,3H)-dione (numbered WYY-03-185)

[0305] Characterization results of WYY-03-185: 1H NMR(600MHz,Chloroform-d)δ8.71(d,J=11.2Hz,1H),7.36–7.28(m,1H),6.8 3(d,J=6.8Hz,1H),5.91–5.76(m,1H),4.17–4.06(m,3H),4.01–3.92(m,3H), 3.86–3.65(m,2H),3.33–2.99(m,8H),2.93–2.83(m,2H),2.61–2.45(m,6H), 2.44–2.28(m,6H),2.18–2.08(m,3H),2.05–1.90(m,5H),1.88–1.54(m,11H); 13 C NMR(151MHz,Chloroform-d)δ203.02,170.72,161.77,160.27,157.24,155.99,152.26,151.61,142.88,141.11,138.79, 111.20,106.74,97.94,67.98,58.84,53.56,47.18,44.53,42.67,37.43,35.78,31.68,31.32,28.07,26.69; LCMS(ESI)C 44 H 56 FN 11 O8S + [M+H] + =918.26.

[0306] Example 32: 1-6-(4-(2-(4-(4-(4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyridino[2,3-d]pyrimidin-2-yl)amino)piperidin-1-yl)sulfonyl)piperidin-1-yl)-2-oxoethyl)piperidin-1-yl)-5-fluoro-1-methyl-1H-indazol-3-yl)dihydropyrimidin-2,4(1H,3H)-dione (numbered WYY-03-186)

[0307] Characterization results of WYY-03-186: 1H NMR(600MHz,Chloroform-d)δ8.66(d,J=14.5Hz,1H),7.32(d,J=12.2Hz,1H),6.78–6.53(m,1H),5.87–5.75(m,1H),4.13–3.92(m,5H),3.89–3.4 7(m,4H),3.31–2.91(m,6H),2.89–2.68(m,3H),2.63–2.49(m,4H),2.42 –2.20(m,8H),2.18–2.10(m,3H),2.04–1.88(m,5H),1.86–1.47(m,11H); 13 C NMR(151MHz,Chloroform-d)δ169.11,160.62,156.15,154.79,151.99,141.81,137.61,105.82,105.66,58. 09,52.39,50.55,43.46,39.49,34.68,31.69,31.41,30.55,30.18,28.68,26.92,25.66,24.68; LCMS(ESI)C 44 H 56 FN 11 O7S + [M+H] + =920.29.

[0308] IV. Bioactivity Detection Experiment

[0309] Example 33: Western blot detection of the effect of PROTAC drug on RB1 signal

[0310] Cells A549, NCI-H1299, and NCI-H1975 were seeded in 6-well plates (2.5 × 10⁻⁶ cells each). 5 Cells / well), after cell adhesion, 0.5 μM of the PROTAC drugs WYY-03-120, 121, 122, 123, 127, 128, 129, 133, 182, 183, 184, 185, and 186 prepared in the above examples were added, with DMSO added as a control. After drug treatment for 36 h, total cell protein was extracted for Western blot analysis.

[0311] like Figures 1-3 As shown ( Figure 1In the figures, 102 (WWZ-01-102) represents the degradation ability of the PROTAC drug prepared in this embodiment of the invention on CDK2, 4, and 6 in A549, NCI-H1975, and NCI-H1299 cells, as well as the effect on RB1 phosphorylation level. It can be seen that the PROTAC drug prepared in this embodiment of the invention can target and degrade specific proteins (CDK2, 4, and 6). The bands at CDK2, 4, 6, and p-RB1 S807 / S811 (phosphorylation of RB1 protein at serine 807 / serine 811) and p-RB1-780 (phosphorylation of RB1 protein at serine 780) are somewhat weakened or even absent, especially at WYY-03-120, 127, 185, and 186.

[0312] In addition, this experiment also investigated the effects of changes in PROTAC drug concentration (100 nM, 500 nM, 1 μM) over time (0, 5, 8, 12, 24 h) on the degradation capacity of CDK2, 4, and 6, and on the phosphorylation level of RB1. The results are shown in [link to results]. Figure 4 and Figure 5 .like Figure 4 The figure shows the effects of compounds WYY-03-127, WYY-03-120, WYY-03-185, and WYY-03-186 of the present invention on the degradation of CDK2, 4, and 6 in A549 cells and on the phosphorylation level of RB1 over time (compound concentration fixed at 0.5 μM). With prolonged time, the degradation of CDK2, 4, and 6 was significant, while the phosphorylation level of RB1 decreased. Figure 5 As shown, the degradation ability of compounds WYY-03-127 and 3600 on CDK2, 4, and 6 in A549 cells and their effect on RB1 phosphorylation level vary with concentration (positive control is PF-06873600, i.e., 3600 in the figure). The results show that as the concentration increases, the phosphorylation level of RB1 is significantly reduced compared to the positive control.

[0313]

[0314] A549 cells were seeded in 6-well plates. After cell adhesion, one group was pretreated with 0.5 μM protease inhibitor MLN4924 for 2 h, followed by WYY-03-127 (0.5 μM). The other three groups were treated with DMSO, WYY-04-065 (0.5 μM), and WYY-03-127 (0.5 μM), respectively, for 24 h. Total cell protein was then extracted for Western blotting (WB). Figure 6 As shown, the inhibitory effect of the proteasome inhibitor MLN4924 (0.5 μM) on the ubiquitination degradation ability of compound WYY-03-127 is shown (WYY-04-065 is the negative control, i.e., 065 in the figure).

[0315]

[0316] Example 34: Cloning Experiment

[0317] A549 cells were seeded in 6-well plates at a density of 3000 cells / well. After cell attachment, 0.5 μM PROTAC (WYY-03-127 and WYY-03-120, with PF-06873600 and Palbocilib as positive controls, and WYY-04-065 as a negative control) and DMSO as a control were added. The cells were incubated at 37°C for 6–9 days. After removing the culture medium, the cells were fixed with 4% formaldehyde, then stained with 0.1% crystal violet for 20 min, rinsed with water, and air-dried.

[0318] like Figure 7 The results of the clonogenic assay show the inhibitory effects of compounds WYY-03-127 and WYY-03-120 on the proliferation of A549 cells. It can be seen that, compared with the positive control, compounds WYY-03-127 and WYY-03-120 of the present invention inhibit the proliferation of A549 cells more significantly, especially compound WYY-03-127.

[0319] Example 35: In vivo experiments in mice

[0320] Male BALB / c nude mice aged 6–7 weeks were purchased and housed under SPF conditions, allowing them to acclimatize for one week before use. In the tumor formation experiment, the nude mice were divided into three groups of five mice each, and A549 cells (1.5 × 10⁻⁶) were introduced into each group. 6 (1 cell / mouse) was subcutaneously injected into the right axilla of BALB / c nude mice. Once a tumor (average size approximately 50 mm) was detected... 3 Then, administration began. Compounds WYY-03-127 and PF-06873600 (positive control) powder were dissolved in 5% DMSO + 30% PEG300 + 5% Tween-80 + 60% physiological saline. Mice were intraperitoneally injected with WYY-03-127 (25 mg / kg) and PF-06873600 (25 mg / kg) per mouse. The control group received the same volume of the solvent (5% DMSO + 30% PEG300 + 5% Tween-80 + 60% physiological saline). Administration was performed every other day, and tumor size was continuously measured using calipers. The tumor size was determined by V = 0.5 × L (length) × W (width). 2 Calculate the tumor volume and weigh the mouse.

[0321] like Figure 8As shown, the results of intraperitoneal injection of the compound on tumor volume and mouse weight in an A549 tumor cell mouse model are presented (PF-3600 in the figure is PF-06873600; the blank control is the solvent). It can be seen that, compared with the positive control, the compound WYY-03-127 of the present invention can significantly reduce tumor volume, while having little effect on mouse weight.

[0322] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A CDK2 / 4 / 6 protein hydrolysis targeting chimera or pharmaceutically acceptable salt, characterized in that, The chemical structure of the chimera is selected from any one of the following structures: 、 、 、 、 、 、 、 、 、 、 。 2. The method for preparing the CDK2 / 4 / 6 protein hydrolysis targeting chimera or pharmaceutically acceptable salt as described in claim 1, characterized in that, The method for preparing the chimera includes: 、 ； Pyridine with a chemical structure as shown in formula IV or V [2,3- d Pyrimidine-7(8) H Intermediate 1 (-ketone), intermediate 2 (containing E3 ubiquitin ligase ligand and Linker structure), and DIEA are reacted in an organic solvent with or without HATU to obtain the chimera.

3. A pyrido[2,3- d Pyrimidine-7(8) H The method for preparing ketone intermediate 1 is characterized in that, The pyrido[2,3- d Pyrimidine-7(8) H The chemical structure of )-ketone intermediate 1 is shown in Formula IV or Formula V, as shown in Formula IV, pyrido[2,3- d Pyrimidine-7(8) H The preparation method of ketone intermediate 1 includes: ； As shown in formula V, pyrido[2,3- d Pyrimidine-7(8) H The preparation method of ketone intermediate 1 includes: 。 4. A pharmaceutical composition, characterized in that, Contains the CDK2 / 4 / 6 protein hydrolysis targeting chimera as described in claim 1 or a pharmaceutically acceptable salt.

5. Use of the CDK2 / 4 / 6 protein hydrolysis targeting chimera or pharmaceutically acceptable salt as described in claim 1, or the pharmaceutical composition as described in claim 4, in the preparation of a medicament for the prevention and / or treatment of cancer, wherein the cancer is non-small cell lung cancer.

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