Protein degradation targeting chimera and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a protein degradation targeting chimera and application thereof. The protein degradation targeting chimera has a structure as shown in a formula (I), and the definition of each group in the formula is shown in the specification. The compound shown in the formula (I) has better inhibitory activity on CDK2. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a protein degradation targeting chimera and an application thereof in inhibiting CDK activity. Background Art

[0002] In the field of biomedicine, proteins, as key molecules in cells, are involved in many life activities. Protein dysfunction or abnormal expression is often associated with a variety of diseases, including but not limited to cancer, neurodegenerative diseases and genetic diseases. Traditional small molecule drugs or biological macromolecule drugs mainly work by inhibiting or activating the activity of specific proteins. However, for some difficult-to-drug targets, these methods often have limited effects.

[0003] In recent years, the emergence of protein degradation targeted chimera (PROTAC) technology has provided a new idea for solving this problem. PROTAC is a bifunctional molecule consisting of three main parts: a ligand that specifically binds to the target protein (i.e., target protein), a ligand that specifically binds to the E3 ubiquitin ligase, and a linker that connects the two parts. This design enables PROTAC to bind to the target protein and the E3 ubiquitin ligase at the same time to form a ternary complex, thereby marking 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 many technical challenges in practical applications. First, designing PROTAC molecules with high specificity and affinity requires a deep understanding of the structure and function of the target protein. Secondly, the design of the linker is crucial to the stability and bioavailability of PROTAC. In addition, the cell permeability, metabolic stability and potential side effects of PROTAC are also problems that need to be overcome.

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

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

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

[0008]

[0009] Where R1 is C 3-6 Cycloalkyl;

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

[0011] E3 indicates E3 ubiquitin ligase ligand;

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

[0013]

[0014] in, Indicates that the connection site is located at Any atom on the monocyclic ring where it 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, R4 and the carbon atom to which they are attached together 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] in, Indicates that the connection site is located at Any atom on the single ring.

[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] Another aspect of the present disclosure provides a pharmaceutical composition, which includes the aforementioned compound or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

[0035] Another aspect of the present disclosure provides a 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] In another aspect of the present disclosure, there is provided a use of the aforementioned compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating a CDK-mediated disease.

[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 The degradation activity of the compound on CDK2, CDK4 and CDK6 in MDA-MB-231 cells at different concentrations is shown, and the action time is 16 hours. DETAILED DESCRIPTION

[0041] I. Definitions

[0042] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0043] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0044] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is pharmaceutically acceptable and has the pharmacological activity of the parent compound. Such salts include: acid addition salts formed with inorganic acids or organic acids, such as nitric acid, phosphoric acid, carbonic acid, etc.; organic acids such as propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, gluconic acid, stearic acid, muconic acid, etc.; or salts formed when the acidic protons present on the parent compound are replaced by metal ions, such as alkali metal ions or alkaline earth metal ions; or coordination compounds formed with organic bases, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc. The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. In general, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are prepared by reacting 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 the form of prodrugs. Prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to be converted to the compounds of the invention. Additionally, prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an in vivo environment.

[0045] The compounds of the present invention may exist in the form of stereoisomers, and therefore encompass all possible stereoisomer 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 of the present invention may also exist in the form of any combination or any mixture of the aforementioned stereoisomers, such as mesomorphs, racemates, equal 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 of the present invention contain olefin double bonds, unless otherwise specified, they include cis-isomers and trans-isomers, and any combination thereof. The atropisomers of the present invention are stereoisomers of axial or planar chirality generated based on restricted intramolecular rotation.

[0046] As mentioned above, the present invention provides compounds represented by the above-mentioned structures, or their tautomers, cis-trans isomers, mesomorphs, racemates, enantiomers, diastereomers, atropisomers or mixtures thereof, wherein "their mixtures" include any form of mixtures between any of the aforementioned stereoisomers (e.g., tautomers, cis-trans isomers, enantiomers, diastereomers, atropisomers) and / or mixtures (mesomorphs, 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 enantiomers and mixtures of diastereomers, mixtures of atropisomers and mixtures of diastereomers, etc.

[0047] As used herein, the "-" symbol contained in a substituent in each group indicates a bond to another group or structure.

[0048] As used herein, the term "alkyl" refers to a straight or branched chain saturated aliphatic hydrocarbon group containing 1 to 20 carbon atoms. 1- "C6 alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms. Specific examples of alkyl groups 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.

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

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

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

[0052] The compound shown in formula (I) of the present invention can be prepared using a synthesis method known in the art or using a method known in the art in combination with the method described in the present invention. The solvent, temperature and other reaction conditions given in the present invention are exemplary and can be changed according to methods well known in the art. The embodiment compounds described in the present invention can be synthesized according to the methods described in the embodiments using appropriate starting materials according to their specific structures, or can be synthesized using methods similar to those described in the embodiments. The starting materials used to synthesize the embodiment compounds of the present invention can be prepared by known synthesis methods or similar methods described in the literature or obtained from commercial sources. The embodiment compounds can be further resolved by methods well known in the art, such as crystallization, chromatography, etc. to obtain their stereoisomers as needed, and the resolution conditions are easily obtained by those skilled in the art through conventional means or limited experiments.

[0053] The term "pharmaceutically acceptable carrier" refers to any preparation or carrier medium representative of carriers that can deliver an effective amount of active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or subject, including water, oil, vegetables and minerals, cream base, lotion base, ointment base, etc. These bases include suspending agents, viscosity increasing agents, transdermal enhancers, etc. Their preparations are well known to those skilled in the field of cosmetics or topical medicine.

[0054] In an embodiment of the present invention, the pharmaceutical composition can be administered in any of the following ways: oral, spray inhalation, rectal, nasal, buccal, topical, parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or by means of an explanted reservoir. When administered orally, the compound of the present invention can be prepared into any oral acceptable preparation form, including but not limited to tablets, capsules, aqueous solutions or aqueous suspensions. The carriers used in tablets generally include lactose and corn starch, and lubricants such as magnesium stearate may also be added. The diluents used in capsule preparations generally include lactose and dry corn starch. Aqueous suspension preparations are usually mixed with active ingredients and suitable emulsifiers and suspending agents. If necessary, some sweeteners, aromatics or coloring agents may also be added to the above oral preparation forms.

[0055] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a non-toxic drug or medicament that can achieve the desired effect. In embodiments of the present invention, when a patient is treated according to the present invention, the amount of a given drug depends on many factors, such as a specific dosing regimen, a disease or condition type and its severity, the uniqueness of the subject or host to be treated (e.g., body weight), but, according to specific surrounding conditions, including, for example, a specific drug, route of administration, the condition to be treated, and the subject or host to be treated, the dosage can be routinely determined by methods known in the art. Usually, with respect to the dosage used for adult treatment, the dosage is typically in the range of 0.02-5000 mg / day, such as about 1-1500 mg / day. The desired dose can be conveniently expressed as a dose, or a dose administered simultaneously (or in a short period of time) or in a suitable interval, such as two, three, four, or more doses per day. It will be appreciated by those skilled in the art that, although the above-mentioned dosage range is given, the specific effective amount can be appropriately adjusted according to the patient's condition and in conjunction with the physician's diagnosis.

[0056] The term "T 1 / 2 " refers to the time required for the concentration of the prototype drug in the in vitro liver microsome incubation system to drop to 1 / 2 of the initial concentration.

[0057] II. Embodiment

[0058] In order to make the purpose, 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 regarded as limiting the present invention. All other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0059] Before further describing the embodiments of the present invention in detail, 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 subject to the following interpretations.

[0060] The raw materials and equipment used in the specific embodiments of the present disclosure are all known products, which 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] 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 tetrakistriphenylphosphine palladium (2.0 g) were dissolved in toluene (200 ml), heated to 115°C under argon protection and stirred for 24 hours. The reaction solution was concentrated under reduced pressure and purified 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] The product of the previous step (14.2 g) was dissolved in tetrahydrofuran (200 mL), and 20 ml of hydrochloric acid (6 M) was added. The mixture was stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was dried and concentrated, and purified by silica gel column (PE:EA=15:1) to obtain the target compound (6.9 g, yield 53.1%).

[0067] 1 H NMR (400MHz, CDCl3) δ: 8.86 (s, 1H), 5.89-5.84 (m, 1H), 2.55 (m, 1H), 8.64 (s, 3H), 2.4 1(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] 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), DIEA (5.8 g, 45.2 mmol) were dissolved in DMSO (50 ml), stirred at 100°C for 5 hours, and LCMS showed that the reaction was complete. The reaction solution was poured into water, extracted with ethyl acetate, and the organic phase was concentrated under reduced pressure and purified 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] 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) was dissolved in dichloromethane (100 ml), and dioxane hydrochloride solution (28.6 ml, 114.3 mmol) was added. The mixture was stirred at room temperature overnight. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to remove the solvent to obtain 12.6 g of yellow solid hydrochloride of the target product with a yield of 100%.

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

[0074] 1H NMR (400MHz, DMSO) δ: 10.18-10.03 (m, 1H), 9.66-9.52 (m, 2H), 8.95 (s, 1H), 7.73 (d, J = 8.4Hz, 2H), 7.13 (d, J = 8.8Hz, 2H), 5.92-5.7 6(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 mixture 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 replaced with nitrogen three times, heated to 80°C and stirred for 2 hours. The reaction solution was cooled to room temperature, water (300 mL) was added, and extracted twice with ethyl acetate (200 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the residue obtained by vacuum rotary evaporation was separated and purified by rapid 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=2.551min, MS(ESI)m / z=312.0[M+H] + .

[0080] 1H NMR(400MHz,DMSO-d6)δ7.86-7.76ppm(m,1H),6.63(dt,J=9.4,2.6Hz,1H),6.51(dt,J=14.8,2.6Hz,1H),4.20-4.01(m,1H),3 .93-3.84(m,5H),3.39(q,J=1.3Hz,6H),3.02-2.71(m,2H),1.86(dt,J=10.7,3.5Hz,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] The aqueous solution (60 mL) of lithium hydroxide monohydrate (7.9 g, 329.8 mmol) was slowly added to the methanol solution (60 mL) of methyl 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-fluorobenzoate (25.4 g, 82.5 mmol), and the system was heated to 50°C and stirred for 1 hour. The reaction solution was evaporated under reduced pressure to remove methanol, diluted with water (60 ml), and diluted hydrochloric acid aqueous solution (1 mol / L) was added dropwise under stirring until a large amount of precipitation was precipitated, the pH was about 6, and filtered, the filter cake was 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=2.391min, MS(ESI)m / z=298.0[M+H] + .

[0084] 1 H-NMR (400MHz, DMSO-d6) δ10.93ppm (s, 1H), 9.82 (d, J = 7.0Hz, 1H), 7.97-7.76 (m,1H),7.29-7.08(m,1H),6.99(td,J=8.3,2.3Hz,1H),4.85-4.68(m,1H),4.5 0(s,1H),3.16(s,2H),2.88-2.62(m,3H),2.24(d,J=12.6Hz,1H),2.12-1.91( m,1H),1.77(d,J=11.5Hz,2H),1.45(d,J=12.3Hz,3H),1.27(d,J=17.1Hz,1H).

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

[0086] 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (32.3 g, 85.5 mmol) was added to a dimethylformamide solution (1700 mL) 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) at once, and the system was stirred at room temperature for 1 hour. Water (1000 mL) was added to the system, washed with ethyl acetate (200 mL), and the filter cake was filtered to obtain a relatively pure target compound. DCM:EA=1:10 was used for slurrying to obtain 17 g of the light yellow solid title compound with a yield of 73%.

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

[0088] 1 H-NMR (400MHz, DMSO-d6) δ10.85(s,1H),8.01(t,J=7.3Hz,1H),7.62(t,J=9.1Hz,1H) ,6.95-6.67(m,2H),4.83-4.67(m,1H),4.07(d,J=6.9Hz,1H),3.90(dd,J=13.2,3.4H z,2H),3.27(s,6H),2.78(tdd,J=13.5,9.6,4.1Hz,3H),2.19-1.93(m,2H),1.83(dtt ,J=11.4,7.3,3.6Hz,1H),1.68(dd,J=13.4,3.7Hz,2H),1.26(qd,J=12.4,4.1Hz,2H).

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

[0090] Trifluoroacetic acid (17 mL) was added dropwise to a dichloromethane solution (170 mL) of 4-(4-(dimethoxymethyl)piperidin-1-yl)-N-(2,6-dicarbonylpiperidin-3-yl)-2-fluorobenzamide (17 g, 2.9 mmol), and the system was stirred at room temperature for 4 hours. The reaction solution was evaporated under reduced pressure to obtain a residue, and the pH was adjusted to weak alkalinity with saturated sodium bicarbonate solution, and DCM1.5 L was added to extract once, and the organic phase was washed with water 3 times and dried to obtain 12 g of the title compound as an off-white solid, with a yield of 79%.

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

[0092] 1 H-NMR (400MHz, DMSO-d6) δ = 10.85ppm (s, 1H), 1H NMR (400MHz, DMSO-d6) δ10.85(s,1H),9.62(s,1H),8.03(t,J=7.2Hz,1H),7.63(t,J=9. 1Hz,1H),6.97-6.64(m,2H),4.73(ddd,J=12.7,7.7,5.4Hz,1H),3.02(ddd,J=13.5,10.9 ,3.0Hz,2H),2.78(ddd,J=17.2,13.3,5.6Hz,1H),2.59(dtd,J=12.2,8.2,7.3,4.0Hz,1H ),2.19-1.97(m,2H),1.90(dt,J=12.4,3.8Hz,2H),1.54(dtd,J=14.4,10.8,3.9Hz,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'-binaphthyl-2,2'-bisdiphenylphosphine (3810 mg, 6.12 mmol), 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), argon was replaced 3 times, and the mixture was stirred at 100°C for 8 hours under argon protection. After the reaction was completed, diatomaceous earth was used for suction filtration and washed with ethyl acetate. The filtrate was dried and the obtained crude product was separated by a normal silica gel column (ethyl acetate / petroleum ether = 1 / 10 to 3 / 1) to obtain 8.0 g of the crude product of the light yellow solid 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] Dissolve 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-methoxybenzoic acid methyl ester (8.0g, 24.74mmol) in 40mL tetrahydrofuran and 40mL methanol, add 40mL of prepared lithium hydroxide (4.15g, 98.95mmol) aqueous solution, and stir at room temperature for 16 hours. After the reaction is completed, spin dry, add 100mL of water again, extract twice with ethyl acetate (60mL*2), adjust the pH of the aqueous phase to 4-5 with 1N hydrochloric acid, a large amount of solid precipitates, and filter to obtain 6.0g of light yellow solid 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-dicarbonylpiperidin-3-yl)-2-methoxybenzamide

[0102] Under ice bath, (S)-3-aminopiperidine-2,6-dione hydrochloric acid (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), and the system was stirred at 25 ° C for 16 hours. The dichloromethane and diisopropylethylamine were removed by rotary evaporation under reduced pressure, and the reaction solution was added dropwise into 200 mL of water with stirring. After stirring for 1 hour, the mixture was filtered and the filter cake was washed with water. After the filter cake was dried, it was rotary evaporated under reduced pressure to obtain 7.8 g of the title compound as a white solid 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-dicarbonylpiperidin-3-yl)-4-(4-formylpiperidin-1-yl)-2-methoxybenzamide trifluoroacetate

[0105] Dissolve (S)-4-(4-(dimethoxymethyl)piperidin-1-yl)-N-(2,6-dicarbonylpiperidin-3-yl)-2-methoxybenzamide (7.80 g, 18.59 mmol) in 30 mL of dichloromethane, add 30 mL of trifluoroacetic acid, and stir at room temperature for 3 hours. After the reaction is completed, the reaction solution is spin-dried, and the obtained crude product is separated by a normal silica gel column (methanol / dichloromethane = 1 / 20 to 10 / 1) to obtain 3.90 g of the light yellow solid 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-piperidindione hydrochloride (5.69 g, 36.2 mmol), and the resulting turbid solution was stirred at 15°C for 10 minutes, glacial acetic acid (19.76 g, 0.33 mol) was added dropwise, and then methyl 5-bromo-2-formylbenzoate (8 g, 33 mmol) was added, and the system was stirred at 15°C for 20 minutes, sodium cyanoborohydride (4.13 g, 65.8 mmol) was added in batches, and the system was heated to 35°C and stirred for 16 hours. Water (20 mL) was added dropwise to the reaction solution, and then the methanol was removed by rotary drying under reduced pressure, and water (500 mL) was added, filtered, and 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=1.07min, 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]dichloro(3-chloropyridine-κN)-palladium (0.3 g, 0.3 mmol) was added to a mixture 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), the nitrogen was replaced three times, 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 dried under reduced pressure and separated and purified by silica gel column chromatography (methanol / dichloromethane, 0-10%) to obtain 0.9 g of the title compound as a yellow solid in a yield of 35.5%.

[0114] LC-MS: Rt=0.98min, 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 decompressed and dried to remove the solvent, and ether (30 mL) was added. Ultrasonic vibration was performed for 10 minutes, and the mixture was 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=0.88min, MS(ESI)m / z=356.2[M+H] + .

[0118] 1 H-NMR(400MHz,DMSO)δppm 10.98(s,1H),9.64(s,1H),7.43(d,J=8.4Hz,1H),7.27(m,2H),5.10(dd,J=13.3,5.1Hz,1H),4.34(d,J=16.7Hz,1H),4.20(d ,J=16.8Hz,1H),3.67(d,J=12.6Hz,2H),2.91(m,3H),2.56(m,2H),2.37(m,1H),1.97(dd,J=12.9,10.7Hz,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-piperidindione hydrochloride (0.74 g, 4.5 mmol), and the resulting turbid solution was stirred at 15°C for 10 minutes, glacial acetic acid (2.46 g, 41 mmol) was added dropwise, and then methyl 4-bromo-2-formylbenzoate (1 g, 4.1 mmol) was added, and the system was stirred at 15°C for 20 minutes, sodium cyanoborohydride (0.52 g, 8.2 mmol) was added in batches, and the system was heated to 35°C and stirred for 16 hours. Water (5 mL) was added dropwise to the reaction solution, and then the methanol was removed by rotary drying under reduced pressure, and 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=1.03min, 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]dichloro(3-chloropyridine-κN)-palladium (0.3 g, 0.3 mmol) was added to a mixture 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), the nitrogen was replaced three times, 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 dried under reduced pressure and separated and purified by silica gel column chromatography (methanol / dichloromethane, 0-10%) to obtain 1.5 g of the title compound as a yellow solid in a yield of 59%.

[0126] LC-MS: Rt=1.17min, 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-carboxaldehyde 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), and the system was stirred at room temperature for 2 hours. The reaction solution was decompressed and dried to remove the solvent, and ether (50 mL) was added. Ultrasonic vibration was performed for 10 minutes, and the mixture was 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)δppm 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),3.7 8(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=0.97min, 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 a solution (3 mL) of N,N-dimethylformamide of the amine compound intermediate 1 (44.7 mg, 0.1 mmol), and the mixture was stirred at room temperature for 5 minutes. Triethylamine (20.2 mg, 0.2 mmol) was added dropwise, and the mixture was stirred at room temperature for 2 hours. Then sodium triacetoxyborohydride (42.4 mg, 0.2 mmol) was added in two batches, and the system was stirred at room temperature for 1 hour. The reaction was completed under TLC monitoring. 15 ml of water was added to the system, and the mixture was extracted with ethyl acetate and n-butanol in turn. 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 in a yield of 92.7%.

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

[0135] 1H-NMR(400MHz,Chloroform-d)δ8.68(s,1H),8.52(s,1H),7.99-7.91(m,1H),7.49(d,J=8.6Hz,1H),7.41(dd,J=14.4,5.6H z,1H),6.95(d,J=8.6Hz,1H),6.71(dd,J=8.9,2.3Hz,1H),6.52(dd,J=16.6,2.3Hz,1H),5.89-5.79(m,1H),4.79(dd,J=12.3 ,5.9Hz,1H),3.89-3.81(m,2H),3.55(d,J=6.3Hz,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 synthesis method of compound Example 1, intermediate 1 and intermediate 3 were used to prepare compound Example 2 (71 mg, 89%).

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

[0140] 1H NMR (400MHz, Methanol-d4) δ8.75(d,J=1.8Hz,1H),7.96(d,J=8.9Hz,1H),7.51(d,J=3.7Hz,2H),7.00(d,J=8.6Hz,2 H),6.61(dd,J=8.9,2.3Hz,1H),6.46(s,1H),5.91-5.84(m,1H),4.72(dd,J=12.6,5.5Hz,1H),4.02(s,3H),3.91(d, J=12.9Hz,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=12.4Hz,2H).

[0141] Example 3: Preparation of 3-(6-(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 synthesis method of compound Example 1, intermediate 1 and intermediate 4 were used to prepare compound Example 3 (63 mg, 80%).

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

[0145] 1H-NMR(400MHz,Chloroform-d)δ8.70(d,J=2.5Hz,1H),7.50(d,J=8.3Hz,2H),7.42(d,J=2.4Hz,1H),7.34(d,J=8.5Hz, 1H),7.25-7.18(m,1H),7.01-6.91(m,2H),5.91-5.81(m,1H),5.25(dd,J=13.3,5.1Hz,1H),4.42(d,J=15.5Hz,1H),4. 34-4.27(m,1H),3.78(d,J=12.0Hz,2H),3.32-3.21(m,4H),2.92-2.78(m,4H),2.72(s,4H),2.56(d,J=2.3Hz,3H),2.4 4-2.31(m,7H),2.29-2.17(m,2H),2.11(d,J=3.1Hz,3H),1.95(d,J=12.1Hz,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 compound Example 4 (68 mg, 86%).

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

[0150] 1H-NMR(400MHz,Chloroform-d)δ8.68(s,1H),8.46(s,1H),7.72(d,J=8.6Hz,1H),7.49-7.45(m,2H),7.03-6.99(m,1H), 6.97-6.93(m,2H),6.88(d,J=2.1Hz,1H),5.82(q,J=8.9Hz,1H),5.21(dd,J=13.2,5.2Hz,1H),4.41(d,J=15.6Hz,1H),4. 25(d,J=15.6Hz,1H),3.85(d,J=12.6Hz,2H),3.22(t,J=4.7Hz,4H),2.90-2.81(m,4H),2.66(s,4H),2.54(s,3H),2.33( d,J=6.9Hz,7H),2.24-2.16(m,2H)2.09(s,3H),1.92(d,J=11.9Hz,4H),1.87-1.78(m,2H),1.42-1.33(d,J=12.0Hz,2H).

[0151] Test Example 1: Antiproliferation activity of compounds on tumor cells

[0152] MDA-MB-231 cells in the logarithmic growth phase (purchased from the Cell Bank of the Chinese Academy of Sciences) were inoculated in a 96-well plate (NEST, 701001), with 80 μL per well (2 × 10 cells per well). 3 ), place the culture plate in a 37°C incubator containing 5% carbon dioxide and incubate for 4 hours. The test compound was prepared into a 10mM stock solution using dimethyl sulfoxide (DMSO) (Aladdin, D103277). After gradient dilution, a series of working solutions of different concentrations were prepared using complete culture medium. Add 20uL of the working solution to the corresponding wells of the well plate, place the well plate in a 5% carbon dioxide incubator, and take it out after incubation at 37°C for 72h. Add 10μL Cellcounting kit-8 reagent (Mei5bio, MF128-Pro-02) to each well, incubate at room temperature for 90min, and measure the absorbance at 450nm using an enzyme reader. Calculate the cell proliferation inhibition rate, cell proliferation inhibition rate = [1-(A sample well-A blank well) / (A control well-A blank well)]×100%. The inhibition curve was drawn using the four-parameter equation in GraphPad 8.0 software to calculate the IC 50 The results of the inhibitory activity of each compound in the example on MDA-MB-231 cell proliferation are shown in Table 1. Wherein, ++++ represents IC 50 <10nM; +++ means 10nM <IC 50 <100nM; +++ means 100nM <IC50 <500nM.

[0153] Table 1: Proliferation inhibition activity of compounds on MDA-MB-231 cells

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

[0155] Palbociclib structure:

[0156] Test Example 2: Western Blot assay to determine the degradation activity of the compound on CDK2, CDK4 and CDK6 proteins:

[0157] Experimental steps:

[0158] 1) After subculturing MDA-MB-231 cells with cell culture medium, cells with good growth status were inoculated into 12-well plates (NEST, 712001), with 1 mL of culture medium per well and 2*10 cells per well. 5 , and cultured overnight in a 37°C, 5% CO2 cell incubator.

[0159] 2) Prepare the test compound with dimethyl sulfoxide (DMSO) into a 10mM stock solution. Dilute with DMSO again before use, add 1μl of the diluted compound to the cell culture well, and gently shake to mix. Also set up a negative control well (add an equal amount of DMSO) and a positive control well.

[0160] 3) After 16 hours of culture, cells were lysed with RIPA cell lysis buffer (Biyuntian, P0013) (containing 1% Cocktail), protein was extracted, and protein concentration was measured with BCA kit (Thermo, 23227). 5 times concentrated protein loading buffer was added, heated at 100°C for 5 minutes, and then the samples were stored at -20°C.

[0161] 4) SDS-PAGE gel electrophoresis experiment:

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

[0163] b. Loading: After assembling the device, add the Running buffer, add the protein marker and sample 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 and run the sample at a constant voltage of 80V until the pre-stained protein marker is completely separated. Then stop the voltage, discard the electrophoresis plate, and rinse the sample tank with water.

[0165] d. Transfer: Remove the gel in the glass plate, cut off the required part and place it in Trans buffer, assemble the transfer device in the order of sponge-filter paper-gel-activated PVDF membrane-filter paper-sponge, then place the electrophoresis tank in an ice water bath, turn on the power, and transfer the membrane at 300V for 1 hour. After the transfer is completed, quickly place the PVDF membrane in TBS buffer.

[0166] 5) Immunohistochemical hybridization and color development:

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

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

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

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

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

[0172] 6) Result detection: Finally, add ECL color developing solution (Biyuntian, Cat. No. P0018S) for color development, take pictures with an automatic chemiluminescence instrument, collect pictures and analyze them.

[0173] Experimental results: The degradation activities of the compounds of Examples 1-4 and the control compound on CDK2, CDK4, and CDK6 at concentrations of 10 nM, 100 nM, and 100 nM were measured by Western Blot. The degradation activities of the compounds are shown in Table 2. Among them, A represents that the degradation percentage of the target protein is greater than 80%; B represents that the degradation percentage of the target protein is between 50% and 80%; C represents that the degradation percentage of the target protein is 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 activity of the example compounds on CDK2, CDK4, and CDK6

[0176]

[0177] Reference compound structure:

[0178]

[0179] Test Example 3: Liver Metabolic Stability Experiment

[0180] Experimental materials and instruments:

[0181] Sources of liver microsomes: human liver microsomes (Corning 452117), CD-1 mouse liver microsomes (XENOTECHM1000); Na2HPO4, KH2PO4, MgCl2; NADPH (Solarbio); AB Sciex Triple Quad 4000 LC-MS instrument.

[0182] Experimental steps:

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

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

[0185] Table 3: Reaction system preparation information

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

[0187] (3) The reaction system was pre-incubated in a 37°C water bath for 10 minutes, and 40 μL of 10 mM NADPH enzyme solution (NADPH solution was dissolved in 100 mM phosphate buffer) was added to the reaction system. The final concentration of the NADPH solution was 1 mM. 40 μL of phosphate buffer was used instead of the NADPH solution as a negative control. The purpose of the negative control was to exclude the influence of the chemical stability of the compound itself.

[0188] (4) 4 μL of 100 μM compounds of Examples 1 and 2 and positive control compound clozapine were added to the reaction system to initiate the reaction. The final concentration of the compounds was 1 μM.

[0189] (5) At 0.5, 15, 30, 45 and 60 minutes after addition, after thorough mixing on a vortex oscillator, 50 μL of the incubation sample was taken out respectively, and the reaction was terminated with 4 times the volume of ice-cold acetonitrile containing the internal standard. The sample was centrifuged at 3220 g for 45 min. After centrifugation, 90 μL of the supernatant was transferred to the injection plate, and 90 μL of ultrapure water was added to mix for LC-MS / MS analysis.

[0190] All data were calculated using EXCEL software, and the peak area was detected by extracting ion spectra. The in vitro half-life (T) of the parent drug was detected by linear fitting of the natural logarithm of the parent drug elimination percentage and time. 1 / 2 ).

[0191] Table 4 In vitro half-life test results

[0192]

[0193]

[0194] It is not difficult to see from the results in Table 2 that the compounds of Examples 1 to 4 of the present disclosure have significant CDK2, CDK4 and CDK6 degradation activities, and the degradation activities of CDK2, CDK4 and CDK6 at a concentration of 100 nM are all greater than 50%, or even greater than 80%. In addition, the proliferation inhibition activity of the compounds of Examples 1-4 on MDA-MB-231 cells is lower than 100 nM, or 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 the human body were 358 min and 178 min, respectively; and the intrinsic clearance rates 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 disclosed embodiment compounds have better liver metabolic stability (Table 4).

[0196] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, in, R1 is C 3-6 Cycloalkyl; R2 is hydrogen or C 1-6 alkyl; E3 indicates E3 ubiquitin ligase ligand; The E3 ubiquitin ligase ligand has a structure shown in the following formula (II): in, Indicates that the connection site is located at Any atom on the single ring; In formula (II), R3 is selected from halogen and C 1-6 Alkoxy; R4 is selected from hydrogen and C 1-3 alkyl; Or R3, R4 and the carbon atom to which they are connected together form a 5- or 6-membered partially unsaturated ring.

2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: R1 is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

3. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein: R2 is methyl, ethyl or isopropyl.

4. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein: R1 is cyclopentyl, and R2 is methyl.

5. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: The compound of formula (II) has the structure shown in the following formula (IIA): Wherein, R3 is selected from halogen and C 1-6 Alkoxy; R4 is hydrogen.

6. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein: The compound of formula (II) has the structure shown in the following formula (IIB): in, Indicates that the connection site is located at Any atom on the single ring.

7. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The E3 ubiquitin ligase ligand is selected from 8. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the following compounds:

9. A pharmaceutical composition comprising the compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.

10. Use of the compound according to any one of claims 1 to 8 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, preferably a CDK2 inhibitor.

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