Proteolysis-Targeting Chimeras and Their Applications

Abstract

The present disclosure belongs to the field of pharmaceutical technologies, and particularly relates to a proteolysis-targeting chimera and its application. The proteolysis-targeting chimera has a structure shown in formula (I), and the definitions of each group in the formula are detailed in the specification. The compound of formula (I) has better inhibitory activity against CDK2.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a proteolysis targeting chimera and its application for inhibiting CDK activity. Background Art

[0002] In the field of biomedicine, proteins, as key molecules within cells, are involved in numerous life activities. Protein dysfunction or abnormal expression is often associated with various diseases, including but not limited to cancer, neurodegenerative diseases, and genetic diseases. Traditional small molecule drugs or biologic drugs mainly exert their effects by inhibiting or activating the activity of specific proteins. However, for some intractable drug targets, these methods often have limited efficacy.

[0003] In recent years, the emergence of proteolysis targeting chimera (PROTAC) technology has provided a new approach to address this problem. A PROTAC is a bifunctional molecule composed of three main parts: a ligand that specifically binds to a target protein (i.e., the target protein), a ligand that specifically binds to an E3 ubiquitin ligase, and a linker that connects these two parts. This design enables a PROTAC to bind to both the target protein and the E3 ubiquitin ligase simultaneously, forming a ternary complex, thereby labeling the target protein for ubiquitination and ultimately degrading it through the 26S proteasome pathway.

[0004] Although PROTAC technology has broad application prospects in theory, it still faces multiple technical challenges in practical applications. First, designing PROTAC molecules with high specificity and affinity requires in-depth understanding of the structure and function of the target protein. Second, the design of the linker is crucial for the stability and bioavailability of PROTACs. In addition, the cell permeability, metabolic stability, and potential side effects of PROTACs are also issues that need to be overcome.

[0005] PROTAC technology has demonstrated great therapeutic potential because it can target proteins that are difficult to act on by traditional small molecule drugs. Summary of the Invention

[0006] In response to the above challenges, the present disclosure aims to propose a novel PROTAC design strategy to optimize the degradation efficiency and selectivity of PROTACs by optimizing the selection of ligands, the length and chemical structure of the linker.

[0007] In one aspect of the present disclosure, there is provided a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0008]

[0009] wherein, R1 is C 3-6 cycloalkyl;

[0010] R2 is hydrogen or C 1-6 alkyl;

[0011] E3 represents an E3 ubiquitin ligase ligand;

[0012] The E3 ubiquitin ligase ligand has the structure shown in the following formula (II):

[0013]

[0014] wherein, represents that the linking site is at any atom of the monocyclic ring where is located; in formula (II),

[0015] R3 is selected from halogen and C 1-6 alkoxy;

[0016] R4 is selected from hydrogen and C 1-3 alkyl;

[0017] or R3 and R4 together with the carbon atom to which they are attached form a 5- or 6-membered partially unsaturated ring. In some embodiments, R1 is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0018] In some embodiments, R1 is cyclopentyl.

[0019] In some embodiments, R2 is hydrogen.

[0020] In some embodiments, R2 is C 1-6 alkyl.

[0021] In some embodiments, R2 is C 1-3 alkyl.

[0022] In some embodiments, R2 is methyl, ethyl, propyl or isopropyl.

[0023] In some embodiments, R2 is methyl, ethyl or isopropyl.

[0024] In some embodiments, R1 is cyclopentyl and R2 is methyl.

[0025] In some embodiments, the compound of formula (II) has the structure shown in the following formula (IIA):

[0026]

[0027] wherein, R3 is selected from halogen and C 1-6 alkoxy; R4 is hydrogen.

[0028] In some embodiments, the compound of formula (II) has the structure shown in the following formula (IIB):

[0029]

[0030] Among them, indicates that the linking site is located at any atom on the single ring where it is located.

[0031] In some embodiments, the E3 ubiquitin ligase ligand is selected from

[0032] In some embodiments, the compound is selected from the following compounds:

[0033]

[0034] On the other hand, the present disclosure provides a pharmaceutical composition comprising the aforementioned compound or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0035] On the other hand, the present disclosure provides the use of the aforementioned compound or a pharmaceutically acceptable salt thereof in the preparation of a CDK inhibitor, wherein the CDK inhibitor is a CDK2, CDK4, and / or CDK6 inhibitor.

[0036] In some embodiments, the CDK inhibitor is a CDK2 inhibitor.

[0037] On yet another aspect, the present disclosure provides the use of the aforementioned compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating CDK-mediated diseases.

[0038] In some embodiments, the CDK-mediated disease is cancer.

[0039] In some embodiments, the CDK-mediated disease is breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Shows the degradation activities of the compound on CDK2, CDK4, and CDK6 in MDA-MB-231 cells at different action concentrations, with an action time of 16 hours. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0041] I. Definitions

[0042] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. And the relevant terms and laboratory operation steps used herein are all widely used terms and conventional steps in the corresponding fields. At the same time, for a better understanding of the present invention, the definitions and explanations of relevant terms are provided below.

[0043] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including their grammatical equivalents, are generally to be understood as open-ended and non-limiting; for example, other unrecited elements or steps are not excluded.

[0044] As used herein, the term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention that are pharmaceutically acceptable and have the pharmacological activity of the parent compound. Such salts include: acid addition salts formed with inorganic acids or with organic acids, such as nitric acid, phosphoric acid, carbonic acid, etc. for the inorganic acids; and such as propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, gluconic acid, stearic acid, mucic acid, etc. for the organic acids; or salts formed when an acidic proton present on the parent compound is replaced by a metal ion, such as an alkali metal ion or an alkaline earth metal ion; or coordination compounds formed with organic bases, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucosamine, etc. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing acidic or basic groups by conventional chemical methods. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of both, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid. In addition to the salt form, the compounds provided by the present invention also exist in prodrug forms. The prodrugs of the compounds described herein are readily chemically changed under physiological conditions to convert into the compounds of the present invention. Further, the prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in the in vivo environment.

[0045] The compounds described in the present invention may exist in the form of stereoisomers and thus cover all possible stereoisomeric forms, including but not limited to cis-trans isomers, tautomers, enantiomers, diastereomers, atropisomers (or may also be referred to as rotational isomers), etc. The compounds described in the present invention may also exist in any combination or any mixture of the foregoing stereoisomers, such as meso forms, racemic forms, equimolar mixtures of atropisomers, etc. For example, a single enantiomer, a single diastereomer or a mixture thereof, or a single atropisomer or a mixture thereof. When the compounds described in the present invention contain an olefinic double bond, unless otherwise specified, it includes cis isomers and trans isomers, and any combination thereof. The atropisomers of the present invention are stereoisomers with axial or planar chirality resulting from restricted intramolecular rotation.

[0046] As described above, the present invention provides the compounds shown in the above various structures, or their tautomers, cis-trans isomers, mesomers, racemates, enantiomers, diastereomers, atropisomers, or mixtures thereof, wherein the "mixture thereof" includes any form of mixing between any of the aforementioned stereoisomers (e.g., tautomers, cis-trans isomers, enantiomers, diastereomers, atropisomers) and / or mixtures (mesomers, racemates), such as mixtures of cis-trans isomers, mixtures of enantiomers and diastereomers, mixtures of diastereomers, mixtures of atropisomers, or mixtures of cis-trans isomers and racemates, mixtures of mixtures of enantiomers and diastereomers, mixtures of mixtures of atropisomers and diastereomers, etc.

[0047] As used herein, the "-" symbol contained in the substituents of each group represents a bond connecting to other groups or structures.

[0048] As used herein, the term "alkyl" refers to a straight-chain or branched-chain saturated aliphatic hydrocarbon group containing 1 to 20 carbon atoms. The term "C 1- C6 alkyl" refers to a straight-chain or branched-chain alkyl having 1 to 6 carbon atoms. Specific examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and various branched isomers thereof, etc.

[0049] As used herein, the term "alkoxy" refers to a group having an -O-alkyl structure, wherein the alkyl is defined as above.

[0050] As used herein, the term "substituted" or "substitution" means that any one or more hydrogen atoms on a specific atom are replaced by substituents.

[0051] When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted with 0 - 2 R's, the group may optionally be substituted with up to two R's, and R in each case has independent options. In addition, combinations of substituents and / or their variants are only permitted if such combinations result in stable compounds.

[0052] The compounds represented by formula (I) of the present invention can be prepared using synthetic methods known in the art or by combining methods known in the art with the methods described in the present invention. The solvents, temperatures, and other reaction conditions provided in the present invention are exemplary and can be varied according to methods well known in the art. The exemplified compounds of the present invention can be synthesized according to their specific structures using appropriate starting materials according to the methods described in the examples, or can also be synthesized using methods similar to those described in the examples. The starting materials used for synthesizing the exemplified compounds of the present invention can be prepared by known synthetic methods or methods similar to those described in the literature, or obtained from commercial sources. The exemplified compounds can be further resolved into their stereoisomers by methods well known in the art, such as crystallization, chromatography, etc., and the resolution conditions can be easily obtained by those skilled in the art through conventional means or limited experiments.

[0053] The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and is non-toxic and has no side effects on the host or subject. Representative carriers include water, oils, vegetables and minerals, paste bases, lotion bases, ointment bases, etc. These bases include suspending agents, thickening agents, transdermal promoters, etc. Their formulations are well known to those skilled in the art of the cosmetics field or the topical drug field.

[0054] In the embodiments of the present invention, the pharmaceutical composition can be administered in any of the following ways: orally, by spray inhalation, rectally, nasally, buccally, topically, parenterally, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intracardiac, intracranial injection or infusion, or by means of an implantable reservoir. When administered orally, the compounds of the present invention can be made into any orally acceptable formulation form, including but not limited to tablets, capsules, aqueous solutions or aqueous suspensions. The carriers used for tablets generally include lactose and corn starch, and lubricants such as magnesium stearate can also be added. The diluents used for capsule formulations generally include lactose and dried corn starch. Aqueous suspension formulations usually involve mixing the active ingredient with suitable emulsifying agents and suspending agents. If necessary, some sweeteners, flavoring agents or coloring agents can also be added to the above oral formulation forms.

[0055] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a drug or agent that is non-toxic but capable of achieving the desired effect. In embodiments of the present invention, when treating a patient according to the present invention, the amount of a given drug depends on many factors, such as the specific dosing regimen, the type and severity of the disease or disorder, the uniqueness of the subject or host to be treated (e.g., body weight). However, depending on the specific circumstances, including, for example, the specific drug employed, the route of administration, the disorder being treated, and the subject or host being treated, the dosage can be routinely determined by methods known in the art. Generally, for adult therapeutic use, the dosage typically ranges from 0.02 - 5000 mg / day, for example, about 1 - 1500 mg / day. The required dosage can conveniently be presented as a single dose, or as divided doses administered simultaneously (or within a short period) or at appropriate intervals, for example, two, three, four or more divided doses per day. Those skilled in the art will understand that, although the above dosage ranges are given, the specific effective amount can be appropriately adjusted according to the patient's condition in combination with the physician's diagnosis.

[0056] The term "T 1 / 2 " refers to the time required for the concentration of the parent drug in an in vitro hepatic microsomal incubation system to decrease to 1 / 2 of the initial concentration.

[0057] II. Examples

[0058] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below. The described embodiments should not be construed as limiting the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0059] Before further elaborating on the embodiments of the present invention, the nouns and terms involved in the embodiments of the present invention are described. The nouns and terms involved in the embodiments of the present invention are applicable to the following explanations.

[0060] The raw materials and equipment used in the specific embodiments of the present disclosure are known products and are obtained by purchasing commercially available products.

[0061] Preparation Example 1: Synthesis of Intermediate 1

[0062]

[0063] Step 1: Synthesis of 2-chloro-8-cyclopentyl-6-(1-ethoxyvinyl)-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one

[0064] Dissolve 6-bromo-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one (20.0 g, 58.7 mmol), tributyl(1-ethoxyvinyl)tin (21.2 g, 58.7 mmol) and tetrakis(triphenylphosphine)palladium(0) (2.0 g) in toluene (200 ml), heat to 115 °C under argon protection and stir for 24 hours. After concentrating the reaction mixture under reduced pressure, purify it by silica gel column (PE:EA = 15:1) to obtain 14.2 g of the target compound.

[0065] Step 2: Synthesis of 6-acetyl-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one

[0066] Dissolve the product of the previous step (14.2 g) in tetrahydrofuran (200 mL), add 20 ml of hydrochloric acid (6 M), stir at room temperature for 1 hour, and LCMS shows that the reaction is complete. Pour the reaction mixture into water, extract with ethyl acetate, dry and concentrate the organic phase, and purify it by silica gel column (PE:EA = 15:1) to obtain the target compound (6.9 g, yield 53.1%).

[0067] 1 H NMR (400 MHz, CDCl3) δ: 8.86 (s, 1H), 5.89 - 5.84 (m, 1H), 2.55 (m, 1H), 8.64 (s, 3H), 2.41 (m, 3H), 2.25 - 2.19 (m, 2H), 2.14 - 2.11 (m, 2H), 1.95 - 1.92 (m, 2H), 1.71 - 1.67 (m, 2H)

[0068] Step 3: Synthesis of tert-butyl 4-(4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)phenyl)piperazine-1-carboxylate

[0069] Dissolve 6-acetyl-2-chloro-8-cyclopentyl-5-methylpyrido[2,3-d]pyrimidin-7(8H)-one (6.9 g, 22.6 mmol), tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (7.5 g, 27.1 mmol), and DIEA (5.8 g, 45.2 mmol) in DMSO (50 ml), stir at 100 °C for 5 hours, and LCMS shows that the reaction is complete. Pour the reaction mixture into water, extract with ethyl acetate, concentrate the organic phase under reduced pressure and then purify it by silica gel column to obtain 12.9 g of the target product as a yellow solid, with a yield of 96.3%.

[0070] LC-MS (ESI) m / z: 547.3 [M+H] + .

[0071] Step 4: Synthesis of 6-acetyl-8-cyclopentyl-5-methyl-2-((4-(piperazin-1-yl)phenyl)amino)pyrido[2,3-d]pyrimidin-7(8H)-one

[0072] Dissolve tert-butyl 4-(4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)phenyl)piperazine-1-carboxylate (12.9 g, 22.5 mmol) in dichloromethane (100 ml), add dioxane hydrochloride solution (28.6 ml, 114.3 mmol), stir at room temperature overnight. LCMS shows that the reaction is complete. Concentrate the reaction solution under reduced pressure to remove the solvent completely to obtain 12.6 g of the hydrochloride yellow solid of the target product, with a yield of 100%.

[0073] LC-MS (ESI) m / z: 447.2 [M+H] + , RT = 0.703 min.

[0074] 1H NMR (400 MHz, DMSO) δ: 10.18 - 10.03 (m, 1H), 9.66 - 9.52 (m, 2H), 8.95 (s, 1H), 7.73 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 5.92 - 5.76 (m, 1H), 3.49 - 3.39 (m, 4H), 3.33 - 3.22 (m, 4H), 2.42 (s, 3H), 2.31 (s, 3H), 2.27 - 2.16 (m, 2H), 1.94 - 1.74 (m, 4H), 1.65 - 1.50 (m, 2H).

[0075] Preparation Example 2: Synthesis of Intermediate 2

[0076]

[0077] Step 1: Synthesis of methyl 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-fluorobenzoate

[0078] A mixed system of 4-(dimethoxymethyl)piperidine (5.5 g, 34.9 mmol), methyl 2,4-difluorobenzoate (4 g, 23.3 mmol) and potassium carbonate (6.4 g, 46.6 mmol) in DMSO (40 mL) was purged with nitrogen three times, heated to 80 °C and stirred for 2 hours. Cool the reaction solution to room temperature, add water (300 mL), extract twice with ethyl acetate (200 mL), combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, filter, and the residue obtained by rotary evaporation under reduced pressure was separated and purified by flash silica gel column chromatography (petroleum ether - ethyl acetate, 95 - 90%) to obtain 3.5 g of the title compound as a white solid, with a yield of 49%.

[0079] LCMS: Rt = 2.551 min, MS(ESI) m / z = 312.0 [M+H] + .

[0080] 1H NMR (400 MHz, DMSO-d6) δ 7.86 - 7.76 ppm (m, 1H), 6.63 (dt, J = 9.4, 2.6 Hz, 1H), 6.51 (dt, J = 14.8, 2.6 Hz, 1H), 4.20 - 4.01 (m, 1H), 3.93 - 3.84 (m, 5H), 3.39 (q, J = 1.3 Hz, 6H), 3.02 - 2.71 (m, 2H), 1.86 (dt, J = 10.7, 3.5 Hz, 3H), 1.74 - 1.55 (m, 1H), 1.52 - 1.23 (m, 2H)

[0081] Step 2: Synthesis of 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-fluorobenzoic acid

[0082] An aqueous solution (60 mL) of lithium hydroxide monohydrate (7.9 g, 329.8 mmol) was slowly added to a methanol solution (60 mL) of methyl 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-fluorobenzoate (25.4 g, 82.5 mmol). The system was heated to 50 °C and stirred for 1 h. The reaction solution was rotary evaporated under reduced pressure to remove methanol, diluted with water (60 ml), and hydrochloric acid aqueous solution (1 mol / L) was added dropwise with stirring until a large amount of precipitate was formed, pH was about 6. The precipitate was filtered, washed with water, and dried to obtain 20 g of the title compound as a white solid, with a yield of 87%.

[0083] LC-MS: Rt = 2.391 min, MS(ESI) m / z = 298.0 [M+H] + .

[0084] 1 H-NMR (400 MHz, DMSO-d6) δ 10.93 ppm (s, 1H), 9.82 (d, J = 7.0 Hz, 1H), 7.97 - 7.76 (m, 1H), 7.29 - 7.08 (m, 1H), 6.99 (td, J = 8.3, 2.3 Hz, 1H), 4.85 - 4.68 (m, 1H), 4.50 (s, 1H), 3.16 (s, 2H), 2.88 - 2.62 (m, 3H), 2.24 (d, J = 12.6 Hz, 1H), 2.12 - 1.91 (m, 1H), 1.77 (d, J = 11.5 Hz, 2H), 1.45 (d, J = 12.3 Hz, 3H), 1.27 (d, J = 17.1 Hz, 1H).

[0085] Step 3: Synthesis of 4-(4-(Dimethoxymethyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)-2-fluorobenzamide

[0086] 2-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (32.3 g, 85.5 mmol) was added in one portion to a solution of 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-fluorobenzoic acid (17 g, 57 mmol), 3-aminopiperidine-2,6-dione hydrochloride (9.35 g, 57 mmol), and diisopropylethylamine (22.1 g, 171 mmol) in dimethylformamide (1700 mL). The mixture was stirred at room temperature for 1 h. Water (1000 mL) was added to the mixture, and the mixture was washed with ethyl acetate (200 mL). The filtrate was filtered to obtain a filter cake, which was the relatively pure target compound. The filter cake was slurried with DCM:EA = 1:10 to obtain 17 g of a pale yellow solid of the title compound with a yield of 73%.

[0087] LC-MS: Rt = 1.12 min, MS(ESI) m / z = 408.3 [M+H] + .

[0088] 1 1H-NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 8.01 (t, J = 7.3 Hz, 1H), 7.62 (t, J = 9.1 Hz, 1H), 6.95 - 6.67 (m, 2H), 4.83 - 4.67 (m, 1H), 4.07 (d, J = 6.9 Hz, 1H), 3.90 (dd, J = 13.2, 3.4 Hz, 2H), 3.27 (s, 6H), 2.78 (tdd, J = 13.5, 9.6, 4.1 Hz, 3H), 2.19 - 1.93 (m, 2H), 1.83 (dtt, J = 11.4, 7.3, 3.6 Hz, 1H), 1.68 (dd, J = 13.4, 3.7 Hz, 2H), 1.26 (qd, J = 12.4, 4.1 Hz, 2H).

[0089] Step 4: Synthesis of 2,2,2-Trifluoroacetate of N-(2,6-Dioxopiperidin-3-yl)-2-fluoro-4-(4-formylpiperidin-1-yl)benzamide

[0090] Trifluoroacetic acid (17 mL) was added dropwise to a dichloromethane solution (170 mL) of 4-(4-(dimethoxymethyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)-2-fluorobenzamide (17 g, 2.9 mmol). The system was stirred at room temperature for 4 hours. The reaction solution was rotary evaporated under reduced pressure to obtain a residue, and the pH was adjusted to weakly basic with saturated sodium bicarbonate solution. Then, it was extracted once with 1.5 L of DCM, and the organic phase was washed with water 3 times. After rotary evaporation to dryness, 12 g of the title compound in the form of a white solid was obtained, with a yield of 79%.

[0091] LCMS: Rt = 0.967 min, MS(ESI) m / z = 362.0 [M+H] + .

[0092] 1 1H-NMR (400 MHz, DMSO-d6) δ = 10.85 ppm (s, 1H), 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.62 (s, 1H), 8.03 (t, J = 7.2 Hz, 1H), 7.63 (t, J = 9.1 Hz, 1H), 6.97 - 6.64 (m, 2H), 4.73 (ddd, J = 12.7, 7.7, 5.4 Hz, 1H), 3.02 (ddd, J = 13.5, 10.9, 3.0 Hz, 2H), 2.78 (ddd, J = 17.2, 13.3, 5.6 Hz, 1H), 2.59 (dtd, J = 12.2, 8.2, 7.3, 4.0 Hz, 1H), 2.19 - 1.97 (m, 2H), 1.90 (dt, J = 12.4, 3.8 Hz, 2H), 1.54 (dtd, J = 14.4, 10.8, 3.9 Hz, 2H).

[0093] Preparation Example 3: Synthesis of Intermediate 3

[0094]

[0095] Step 1: Preparation of methyl 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-methoxybenzoate

[0096] Palladium acetate (640 mg, 2.86 mmol), 1,1'-binaphthalene-2,2'-bis(diphenylphosphine) (3810 mg, 6.12 mmol), and cesium carbonate (26.59 g, 81.61 mmol) were added to a mixed solution of methyl 4-bromo-2-methoxybenzoate (10.00 g, 40.81 mmol) and 4-(dimethoxymethyl)piperidine (7.15 g, 44.89 mmol) in 1,4-dioxane (150 mL). The mixture was purged with argon three times and stirred at 100 °C for 8 hours under argon protection. After the reaction was completed, the mixture was filtered through diatomaceous earth and washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the resulting crude product was separated by normal-phase silica gel column chromatography (ethyl acetate / petroleum ether = 1 / 10 - 3 / 1) to obtain 8.0 g of a pale yellow solid crude product of the title compound, with a yield of 60.63%.

[0097] LC-MS: MS(ESI) m / z = 324.2 [M+H] + .

[0098] Step 2: Preparation of 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-methoxybenzoic acid

[0099] Methyl 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-methoxybenzoate (8.0 g, 24.74 mmol) was dissolved in 40 mL of tetrahydrofuran and 40 mL of methanol. An aqueous solution of lithium hydroxide (4.15 g, 98.95 mmol) in 40 mL was added, and the mixture was stirred at room temperature for 16 hours. After the reaction was completed, the solvent was removed under reduced pressure, 100 mL of water was added again, and the mixture was extracted twice with ethyl acetate (60 mL * 2). The aqueous phase was adjusted to pH 4 - 5 with 1N hydrochloric acid, and a large amount of solid precipitated. The solid was filtered to obtain 6.0 g of a pale yellow solid of the title compound, with a yield of 78%.

[0100] LCMS: MS(ESI) m / z = 310.2 [M+H] + .

[0101] Step 3: Preparation of (S)-4-(4-(dimethoxymethyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)-2-methoxybenzamide

[0102] Under an ice bath, (S)-3-aminopiperidine-2,6-dione hydrochloride (3.35 g, 20.37 mmol) and diisopropylethylamine (7.52 g, 58.19 mmol) were added to a mixed solution of 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-methoxybenzoic acid (6.0 g, 19.40 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.85 g, 23.27 mmol) in N,N-dimethylformamide (30 mL) and dichloromethane (120 mL). The system was stirred at 25 °C for 16 hours. Dichloromethane and diisopropylethylamine were removed by rotary evaporation under reduced pressure. The reaction solution was added dropwise to 200 mL of water with stirring. After stirring for 1 hour, filtration was carried out. The filter cake was washed with water and dried by suction, and then rotary evaporation was carried out under reduced pressure to obtain 7.8 g of a white solid of the title compound with a yield of 95.87%.

[0103] LC-MS: MS(ESI) m / z = 420.2 [M+H] + .

[0104] Step 4: Preparation of (S)-N-(2,6-dioxopiperidin-3-yl)-4-(4-formylpiperidin-1-yl)-2-methoxybenzamide trifluoroacetate

[0105] (S)-4-(4-(Dimethoxymethyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)-2-methoxybenzamide (7.80 g, 18.59 mmol) was dissolved in 30 mL of dichloromethane, and 30 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was evaporated to dryness. The obtained crude product was separated by a normal-phase silica gel column (methanol / dichloromethane = 1 / 20 - 10 / 1) to obtain 3.90 g of a pale yellow solid of the title compound with a yield of 46%.

[0106] LCMS: MS(ESI) m / z = 374.2 [M+H] + .

[0107] Preparation Example 4: Synthesis of Intermediate 4

[0108]

[0109] Step 1: Preparation of 3-(6-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione

[0110] Sodium acetate (5.4 g, 65.8 mmol) was added to a methanol solution (160 mL) of 3-amino-2,6-piperidinedione hydrochloride (5.69 g, 36.2 mmol). The resulting turbid solution was stirred at 15 °C for 10 minutes, acetic acid (19.76 g, 0.33 mol) was added dropwise, and then methyl 5-bromo-2-formylbenzoate (8 g, 33 mmol) was added. The system was stirred at 15 °C for 20 minutes, and sodium cyanoborohydride (4.13 g, 65.8 mmol) was added in portions. The system was heated to 35 °C and stirred for 16 hours. Water (20 mL) was added dropwise to the reaction solution, and then methanol was removed by rotary evaporation under reduced pressure. Water (500 mL) was added, and the mixture was filtered. The filter cake was dried to obtain 8.3 g of the title compound as a white solid, with a yield of 78%.

[0111] LC-MS: Rt = 1.07 min, MS(ESI) m / z = 323.0 / 325.0 [M+H] + .

[0112] Step 2: Preparation of 3-(6-(4-(dimethoxymethyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione

[0113] (SP-4-1)-[1,3-Bis[2,6-bis(1-propylbutyl)phenyl]-4,5-dichloro-1,3-dihydro-2H-imidazol-2-ylidene]dichloropalladium(II) (3-chloropyridine-κN) (0.3 g, 0.3 mmol) was added to a mixed system of 3-(6-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione (2 g, 6.2 mmol), 4-(dimethoxymethyl)piperidine (1.28 g, 8.1 mmol) and cesium carbonate (6.06 g, 18.6 mmol) in dioxane (40 mL). Nitrogen was displaced three times, and the system was heated to 100 °C and stirred for 3 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate and filtered. The filtrate was concentrated by rotary evaporation under reduced pressure and purified by silica gel column chromatography (methanol / dichloromethane, 0 - 10%) to obtain 0.9 g of the title compound as a yellow solid, with a yield of 35.5%.

[0114] LC-MS: Rt = 0.98 min, MS(ESI) m / z = 402.0 [M+H] + .

[0115] Step 3: Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-3-oxoisoindolin-5-yl)piperidine-4-carbaldehyde trifluoroacetate

[0116] Trifluoroacetic acid (6 mL) was added dropwise to a dichloromethane solution (20 mL) of 3-(6-(4-(dimethoxymethyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (0.9 g, 2.2 mmol), and the system was stirred at room temperature for 2 hours. The reaction solution was rotary evaporated under reduced pressure to remove the solvent, ether (30 mL) was added, sonicated for 10 minutes, filtered, and the filter cake was dried to obtain 0.83 g of the title compound as a gray solid, with a yield of 73%.

[0117] LC-MS: Rt = 0.88 min, MS(ESI) m / z = 356.2 [M+H] + .

[0118] 1 1H-NMR (400 MHz, DMSO) δ ppm 10.98 (s, 1H), 9.64 (s, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.27 (m, 2H), 5.10 (dd, J = 13.3, 5.1 Hz, 1H), 4.34 (d, J = 16.7 Hz, 1H), 4.20 (d, J = 16.8 Hz, 1H), 3.67 (d, J = 12.6 Hz, 2H), 2.91 (m, 3H), 2.56 (m, 2H), 2.37 (m, 1H), 1.97 (dd, J = 12.9, 10.7 Hz, 3H), 1.61 (m, 2H).

[0119] Preparation Example 5: Synthesis of Intermediate 5

[0120]

[0121] Step 1: Preparation of 3-(5-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione

[0122] Sodium acetate (670 mg, 8.2 mmol) was added to a methanol solution (20 mL) of 3-amino-2,6-piperidinedione hydrochloride (0.74 g, 4.5 mmol). The resulting turbid solution was stirred at 15 °C for 10 minutes, glacial acetic acid (2.46 g, 41 mmol) was added dropwise, then methyl 4-bromo-2-formylbenzoate (1 g, 4.1 mmol) was added. The system was stirred at 15 °C for 20 minutes, and sodium cyanoborohydride (0.52 g, 8.2 mmol) was added in portions. The system was heated to 35 °C and stirred for 16 hours. Water (5 mL) was added dropwise to the reaction solution, then methanol was removed by rotary evaporation under reduced pressure. Water (50 mL) was added, filtered, and the filter cake was dried to obtain 1.2 g of the title compound as a white solid, with a yield of 81%.

[0123] LC-MS: Rt = 1.03 min, MS(ESI) m / z = 322.9 / 324.9 [M+H] + .

[0124] Step 2: Preparation of 3-(5-(4-(dimethoxymethyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione

[0125] (SP-4-1)-[1,3-Bis[2,6-bis(1-propylbutyl)phenyl]-4,5-dichloro-1,3-dihydro-2H-imidazol-2-ylidene]dichlorido(3-chloropyridine-κN)palladium (0.3 g, 0.3 mmol) was added to a mixed system of 3-(5-bromo-1-oxoisoindolin-2-yl)piperidine-2,6-dione (2 g, 6.2 mmol), 4-(dimethoxymethyl)piperidine (1.28 g, 8.1 mmol) and cesium carbonate (6.06 g, 18.6 mmol) in dioxane (40 mL). Nitrogen was displaced three times, and the system was heated to 100 °C and stirred for 3 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography (methanol / dichloromethane, 0 - 10%) to obtain 1.5 g of the title compound as a yellow solid, with a yield of 59%.

[0126] LC-MS: Rt = 1.17 min, MS(ESI) m / z = 402.0 [M+H] + .

[0127] Step 3: Preparation of 1-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-5-yl)piperidine-4-carbaldehyde trifluoroacetate

[0128] Trifluoroacetic acid (10 mL) was added dropwise to a dichloromethane solution (30 mL) of 3-(5-(4-(dimethoxymethyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione (1.5 g, 3.7 mmol). The system was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure to remove the solvent, ether (50 mL) was added, sonicated for 10 minutes, filtered, and the filter cake was dried to obtain 1.35 g of the title compound as a green solid, with a yield of 70%.

[0129] 1H-NMR(400MHz, DMSO) δ ppm 10.95(s, 1H), 9.62(s, 1H), 7.51(d, J = 8.4Hz, 1H), 7.07(d, J = 10.4Hz, 2H), 5.05(dd, J = 13.3, 5.1Hz, 1H), 4.26(dd, J = 49.5, 16.8Hz, 2H), 3.78(m, 2H), 3.02(m, 2H), 2.90(m, 1H), 2.58(dd, J = 11.0, 4.0Hz, 2H), 2.36(dt, J = 13.3, 8.9Hz, 1H), 1.94(m, 3H), 1.57(qd, J = 11.0, 3.7Hz, 2H).

[0130] LC-MS: Rt = 0.97 min, MS(ESI) m / z = 356.1 [M + H] +

[0131] Example 1: Preparation of 4-(4-((4-(4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)-2-fluorobenzamide

[0132]

[0133] The aldehyde compound intermediate 2 (36.1 mg, 0.1 mmol) was added to the N,N-dimethylformamide solution (3 mL) of the amine compound intermediate 1 (44.7 mg, 0.1 mmol), stirred at room temperature for 5 minutes, triethylamine (20.2 mg, 0.2 mmol) was added dropwise, stirred at room temperature for 2 hours, and then sodium triacetoxyborohydride (42.4 mg, 0.2 mmol) was added in two batches. The system was stirred at room temperature for 1 hour. The reaction was monitored by TLC until completion. 15 ml of water was added to the system, and then extracted successively with ethyl acetate and n-butanol. The organic phases were combined, dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and further purified by preparative TLC (DCM:MeOH = 10:1) to obtain 73.4 mg of the target compound with a yield of 92.7%.

[0134] LC-MS: Rt = 1.608 min, MS(ESI) m / z = 792.4 [M + H] + .

[0135] 1H-NMR (400 MHz, Chloroform-d) δ 8.68 (s, 1H), 8.52 (s, 1H), 7.99 - 7.91 (m, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.41 (dd, J = 14.4, 5.6 Hz, 1H), 6.95 (d, J = 8.6 Hz, 1H), 6.71 (dd, J = 8.9, 2.3 Hz, 1H), 6.52 (dd, J = 16.6, 2.3 Hz, 1H), 5.89 - 5.79 (m, 1H), 4.79 (dd, J = 12.3, 5.9 Hz, 1H), 3.89 - 3.81 (m, 2H), 3.55 (d, J = 6.3 Hz, 1H), 3.30 - 3.18 (m, 4H), 2.98 - 2.81 (m, 4H), 2.80 - 2.76 (m, 2H), 2.75 - 2.61 (m, 4H), 2.54 (s, 2H), 2.35 (s, 3H), 2.32 - 2.26 (m, 2H), 2.10 (s, 3H), 2.04 - 1.89 (m, 4H), 1.89 - 1.79 (m, 4H), 1.37 - 1.30 (m, 2H).

[0136] Example 2: Preparation of (S)-4-(4-((4-(4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-N-(2,6-dioxopiperidin-3-yl)-2-methoxybenzamide

[0137]

[0138] Referring to the synthetic method of Example 1 of the reference compound, the compound of Example 2 (71 mg, 89%) was prepared from Intermediate 1 and Intermediate 3.

[0139] LC-MS: Rt = 1.606 min, MS(ESI) m / z = 804.4 [M + H] + .

[0140] 11H NMR (400 MHz, Methanol-d4) δ 8.75 (d, J = 1.8 Hz, 1H), 7.96 (d, J = 8.9 Hz, 1H), 7.51 (d, J = 3.7 Hz, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.61 (dd, J = 8.9, 2.3 Hz, 1H), 6.46 (s, 1H), 5.91 - 5.84 (m, 1H), 4.72 (dd, J = 12.6, 5.5 Hz, 1H), 4.02 (s, 3H), 3.91 (d, J = 12.9 Hz, 2H), 3.32 - 3.25 (m, 4H), 2.97 - 2.87 (m, 3H), 2.82 - 2.76 (m, 5H), 2.67 - 2.56 (m, 2H), 2.54 (s, 3H), 2.49 - 2.41 (m, 2H), 2.37 (s, 3H), 2.34 - 2.26 (m, 2H), 2.03 (s, 3H), 1.99 - 1.92 (m, 4H), 1.88 - 1.82 (m, 2H), 1.35 (d, J = 12.4 Hz, 2H).

[0141] Example 3: Preparation of 3-(6-(4-((4-(4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione

[0142]

[0143] Referring to the synthetic method of Reference Compound Example 1, Intermediate 1 and Intermediate 4 were used to prepare the compound of Example 3 (63 mg, 80%).

[0144] LC-MS: Rt = 1.594 min, MS(ESI) m / z = 786.4 [M + H] + .

[0145] 1H-NMR (400 MHz, Chloroform-d) δ 8.70 (d, J = 2.5 Hz, 1H), 7.50 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 2.4 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 7.25 - 7.18 (m, 1H), 7.01 - 6.91 (m, 2H), 5.91 - 5.81 (m, 1H), 5.25 (dd, J = 13.3, 5.1 Hz, 1H), 4.42 (d, J = 15.5 Hz, 1H), 4.34 - 4.27 (m, 1H), 3.78 (d, J = 12.0 Hz, 2H), 3.32 - 3.21 (m, 4H), 2.92 - 2.78 (m, 4H), 2.72 (s, 4H), 2.56 (d, J = 2.3 Hz, 3H), 2.44 - 2.31 (m, 7H), 2.29 - 2.17 (m, 2H), 2.11 (d, J = 3.1 Hz, 3H), 1.95 (d, J = 12.1 Hz, 3H), 1.90 - 1.79 (m, 3H), 1.48 - 1.39 (m, 2H).

[0146] Example 4: Preparation of 3-(5-(4-((4-(4-((6-acetyl-8-cyclopentyl-5-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)phenyl)piperazin-1-yl)methyl)piperidin-1-yl)-1-oxoisoindolin-2-yl)piperidine-2,6-dione

[0147]

[0148] Referring to the synthesis method of Compound Example 1, Intermediate 1 and Intermediate 5 were used to prepare the compound of Example 4 (68 mg, 86%).

[0149] LC-MS: Rt = 1.580 min, MS(ESI) m / z = 786.4 [M + H] + .

[0150] 1H-NMR (400 MHz, Chloroform-d) δ 8.68 (s, 1H), 8.46 (s, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.49 - 7.45 (m, 2H), 7.03 - 6.99 (m, 1H), 6.97 - 6.93 (m, 2H), 6.88 (d, J = 2.1 Hz, 1H), 5.82 (q, J = 8.9 Hz, 1H), 5.21 (dd, J = 13.2, 5.2 Hz, 1H), 4.41 (d, J = 15.6 Hz, 1H), 4.25 (d, J = 15.6 Hz, 1H), 3.85 (d, J = 12.6 Hz, 2H), 3.22 (t, J = 4.7 Hz, 4H), 2.90 - 2.81 (m, 4H), 2.66 (s, 4H), 2.54 (s, 3H), 2.33 (d, J = 6.9 Hz, 7H), 2.24 - 2.16 (m, 2H) 2.09 (s, 3H), 1.92 (d, J = 11.9 Hz, 4H), 1.87 - 1.78 (m, 2H), 1.42 - 1.33 (d, J = 12.0 Hz, 2H).

[0151] Test Example 1: Proliferation Inhibitory Activity of Compounds against Tumor Cells

[0152] MDA-MB-231 cells in the logarithmic growth phase (purchased from the Cell Bank of the Chinese Academy of Sciences) were seeded in a 96-well plate (NEST, 701001), 80 μL per well (the number of cells per well was 2×10 3 ). The culture plate was placed in an incubator at 37 °C containing 5% carbon dioxide and incubated for 4 hours. The compound to be tested was prepared into a 10 mM stock solution with dimethyl sulfoxide (DMSO) (Aladdin, D103277). After gradient dilution, a series of working solutions with different concentrations were prepared with complete medium. 20 μL of the working solution was added to the corresponding wells of the plate, and the plate was taken out after being cultured in a 5% carbon dioxide incubator at 37 °C for 72 h. 10 μL of Cellcounting kit-8 reagent (Mei5bio, MF128-Pro-02) was added to each well, incubated at room temperature for 90 min, and the absorbance was measured at 450 nm with an enzyme-linked immunosorbent assay reader. The cell proliferation inhibition rate was calculated as follows: cell proliferation inhibition rate = [1 - (A sample well - A blank well) / (A control well - A blank well)] × 100%. The inhibition curve was plotted using the four-parameter equation in GraphPad 8.0 software, and the IC 50 value was calculated. The results of the proliferation inhibitory activity of the compounds in the examples against MDA-MB-231 cells are shown in Table 1. Among them, ++++ indicates IC 50 < 10 nM; +++ indicates 10 nM < IC 50 < 100 nM; ++ indicates 100 nM < IC50 <500 nM.

[0153] Table 1: Proliferation Inhibitory Activity of Compounds against MDA-MB-231 Cells

[0154] Serial number <![CDATA[IC 50 (nM)]]> Example 1 +++ Example 2 +++ Example 3 +++ Example 4 ++++ Palbociclib ++

[0155] Structural Formula of Palbociclib:

[0156] Test Example 2: Determination of the Degradation Activity of Compounds against CDK2, CDK4, and CDK6 Proteins by Western Blot

[0157] Experimental Procedure:

[0158] 1) After subculturing MDA-MB-231 cells with cell culture medium, take cells in good growth state and inoculate them into a 12-well plate (NEST, 712001), 1 mL of medium per well, and the number of cells per well is 2×10 5 , and place them in a cell incubator at 37 °C and 5% CO2 for overnight culture.

[0159] 2) Prepare a 10 mM stock solution of the compound to be tested with dimethyl sulfoxide (DMSO). Dilute it with DMSO before use. Take 1 μL of the diluted compound and add it to the cell culture well, and gently shake and mix. Additionally, set up negative control wells (add an equal amount of DMSO) and positive control wells.

[0160] 3) After culturing for 16 hours, lyse the cells with RIPA cell lysis buffer (Beyotime, P0013) (containing 1% Cocktail), extract the protein, and measure the protein concentration with a BCA kit (Thermo, 23227). Add 5× concentrated protein loading buffer, heat the sample at 100 °C for 5 minutes, and then store the sample at -20 °C.

[0161] 4) SDS-PAGE Gel Electrophoresis Experiment:

[0162] a. Gel Preparation: All 7.5%-15% polyacrylamide gels in the present invention are used to separate proteins;

[0163] b. Loading: After assembling the device, add Running buffer, add protein Marker and samples to the loading wells, the loading volume per well is 5-15 μL, and the loading protein amount is 20-30 μg;

[0164] c. Electrophoresis: Connect the power supply, run the sample at a constant voltage of 80 V until the pre-stained protein Marker is completely separated, then stop the voltage, discard the electrophoresis buffer, and rinse the sample tank with water;

[0165] d. Transfer: Remove the gel from the glass plate, cut off the desired portion, and place it in Trans buffer. Assemble the transfer apparatus in the following order: sponge - filter paper - gel - activated PVDF membrane - filter paper - sponge. Place the electrophoresis tank in an ice-water bath, connect the power supply, and transfer the membrane at 300V for 1 hour. After transfer, quickly place the PVDF membrane in TBS buffer.

[0166] 5) Immunohistochemical hybridization and color development:

[0167] a. Wash the PVDF membrane with 50 mL of 1× TBS for 5 minutes, then add 50 mL of 5% skim milk blocking buffer and shake at room temperature for 1 hour.

[0168] b. Wash the PVDF membrane three times with 50 mL of 1×TBS / T, each time for 10 min.

[0169] c. Add primary antibodies of appropriate dilutions, including CDK2 (Beyotime, AF1063), CDK4 (Beyotime, AF2515), and CDK6 (Beyotime, AF0114), and incubate overnight at 4°C.

[0170] d. Wash the PVDF membrane three times with 50 mL of 1× TBS / T buffer, each time for 10 min.

[0171] e. Add a secondary antibody, Peroxidase Affinipure (HRP) Goat Anti-Rabbit IgG (Bio-Regene, Cat. BN20601), at an appropriate dilution and incubate at room temperature for 2 h. 6. Wash the membrane three times with 50 mL of 1× TBS / T buffer, 10 min each time. 7. Develop and expose the membrane, using α-actin as an internal control.

[0172] 6) Result detection: Finally, ECL color developing solution (Biyuntian, Cat. No. P0018S) was added for color development, and photos were taken using an automatic chemiluminescence analyzer. The images were collected and analyzed.

[0173] Experimental Results: Western Blot analysis was used to determine the degradation activities of the compounds of Examples 1-4 and the control compound against CDK2, CDK4, and CDK6 at concentrations of 10 nM, 100 nM, and 100 nM. The degradation activities of the compounds are shown in Table 2. A represents a target protein degradation percentage greater than 80%; B represents a target protein degradation percentage between 50% and 80%; and C represents a target protein degradation percentage less than 50%.

[0174] From Table 2 and Figure 1 It is not difficult to see from the results that the compounds of Examples 1-4 all have multi-target degradation effects, and the degradation effect of CDK2 is significantly better than that of the control compound.

[0175] Table 2: Degradation activities of the example compounds against CDK2, CDK4, and CDK6

[0176]

[0177] Structural formula of the control compound:

[0178]

[0179] Test Example 3: Liver metabolic stability experiment

[0180] Experimental materials and instruments:

[0181] Source of liver microsomes: human liver microsomes (Corning 452117), CD-1 mouse liver microsomes (XENOTECH M1000); Na2HPO4, KH2PO4, MgCl2; NADPH (Solarbio); AB Sciex Triple Quad 4000 liquid chromatography-mass spectrometry instrument.

[0182] Experimental procedure:

[0183] (1) Prepare 100 nM phosphate buffer;

[0184] (2) Prepare the reaction system as shown in Table 3 below:

[0185] Table 3: Information on the preparation of the reaction system

[0186] Reagent Stock solution concentration Volume Final concentration Liver microsomes 20mg / mL 10μL 0.5mg / mL Phosphate buffer 100mM 346 μL 100mM

[0187] (3) Incubate the reaction system in a 37°C water bath for 10 minutes. Add 40 μL of 10 mM NADPH enzyme solution (the NADPH solution is dissolved in 100 mM phosphate buffer) to the reaction system. The final concentration of the NADPH solution is 1 mM. Use 40 μL of phosphate buffer instead of the NADPH solution as the negative control. The role of the negative control is to exclude the influence of the chemical stability of the compound itself.

[0188] (4) Add 4 μL of 100 μM example 1 and 2 compounds and the positive control compound clozapine to initiate the reaction in the reaction system. The final concentration of the compound is 1 μM.

[0189] (5) At 0.5, 15, 30, 45, and 60 minutes after addition, after thoroughly mixing with a vortex oscillator, take out 50 μL of the incubation sample respectively, terminate the reaction with 4 times the volume of ice acetonitrile containing an internal standard. The sample is centrifuged at 3220 g for 45 min. After centrifugation, transfer 90 μL of the supernatant to the sample injection plate, add 90 μL of ultrapure water and mix well for LC-MS / MS analysis.

[0190] All data were calculated using EXCEL software. The peak areas were detected by extracting ion chromatograms. The in vitro half-life (T 1 / 2 ) of the parent drug was detected by performing a linear fit of the natural logarithm of the percentage of parent drug elimination versus time.

[0191] Table 4 Results of in vitro half-life detection

[0192]

[0193]

[0194] From the results in Table 2, it is not difficult to see that the compounds of Examples 1 to 4 of the present disclosure have obvious CDK2, CDK4, and CDK6 degradation activities. At a concentration of 100 nM, the degradation activities against CDK2, CDK4, and CDK6 are all greater than 50%, and even greater than 80%. Moreover, the proliferation inhibitory activities of the compounds of Examples 1-4 against MDA-MB-231 cells are lower than 100 nM, and even lower than 10 nM, which is significantly better than the control compound Palbociclib (Table 1).

[0195] In addition, the metabolic half-lives of the compounds of Example 1 and Example 2 in humans reached 358 min and 178 min, respectively; and the intrinsic clearances of the compounds of Example 1 and Example 2 in human liver microsomes were 2.47 and 6.81 mL / min / kg, respectively, indicating that the compounds of the examples of the present disclosure have better hepatic metabolic stability (Table 4).

[0196] The foregoing description of specific exemplary embodiments of the present invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and obviously, many changes and variations are possible in light of the above teaching. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical applications, so that those skilled in the art can implement and utilize various different exemplary embodiments of the present invention as well as various different selections and changes. The scope of the present invention is intended to be defined by the claims and their equivalents.

Claims

1. The following compound or its pharmaceutically acceptable salt: , , and .

2. A pharmaceutical composition, which comprises the compound or its pharmaceutically acceptable salt according to claim 1, and optionally a pharmaceutically acceptable carrier.

3. Use of the compound or its pharmaceutically acceptable salt according to claim 1 in the preparation of a CDK inhibitor, wherein the CDK inhibitor is a CDK2, CDK4 and / or CDK6 inhibitor.

4. The use according to claim 3, wherein The CDK inhibitor is a CDK2 inhibitor.

5. Use of the compound or its pharmaceutically acceptable salt according to claim 1 in the preparation of a medicament for treating CDK-mediated diseases.

6. The use according to claim 5, wherein, The CDK-mediated disease is cancer.

7. The use according to claim 5, wherein, The CDK-mediated disease is breast cancer.

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