A class of bifunctional chimeric heterocyclic compounds that target androgen receptor degradation and their applications

Abstract

This invention relates to a class of bifunctional chimeric heterocyclic compounds that target androgen receptor degradation and their uses. Specifically, it provides compounds of formula (I), or their isotopic compounds, optical isomers, tautomers, pharmaceutically acceptable salts, prodrugs, or solvates, wherein ARB is the androgen receptor recognition / binding moiety, L is the linking moiety, and U is the ubiquitin protease recognition / binding moiety; these three moieties are linked by chemical bonds. The compounds provided by this invention can target and degrade androgen receptors in prostate cancer cells and inhibit the proliferation of prostate cancer cells, while also exhibiting good metabolic stability and pharmacokinetic properties. The compounds of this invention show promising applications in the preparation of androgen receptor protein degradation-targeting chimeras and in the preparation of drugs for treating androgen receptor-regulated diseases (including prostate cancer, breast cancer, and Kennedy's disease).

Description

[0001] This application is a divisional application of the invention patent with application number 202010301601.1, application date April 16, 2024, entitled "A class of bifunctional chimeric heterocyclic compounds that target and degrade androgen receptors and their uses". Technical Field

[0002] This invention belongs to the field of medicine. Specifically, this invention relates to a class of bifunctional chimeric heterocyclic compounds that target and degrade androgen receptors and their uses. Background Technology

[0003] With a continuously growing and aging global population, the incidence of prostate cancer continues to rise, and the main treatment currently is androgen deprivation therapy. The androgen receptor (AR) belongs to the nuclear receptor family and is a class of ligand-dependent transcription factors. Aberrant regulation of the AR signaling pathway plays a crucial role in the occurrence and development of prostate cancer; studies have shown that castration-resistant prostate cancer (CRPC) still depends on AR. The androgen receptor contains 918 amino acids and shares a similar structure and function with other nuclear receptors. It consists of three important domains: the DNA-binding domain (DBD), the ligand-binding domain (LBD), and the N-terminal domain (NTD). The DBD and LBD are connected by a hinge region. The LBD, located at the C-terminus of the AR, is the site where AR binds to ligands, determining the specificity of ligand binding to AR. Ligand binding to the LBD activates AR. Two transcriptional activation regions have been identified in AR: activation function 1 (AF1) in the NTD domain and highly conserved hydrophobic pocket activation function 2 (AF2) in the LBD domain. Prior to 2010, docetaxel-based chemotherapy was the only treatment that could prolong the survival of patients with metastatic CRPC.

[0004] Protein degradation-targeting chimeras (PROTACs) have attracted widespread attention as small molecules capable of inducing the degradation of target proteins. PROTACs are bifunctional molecules comprising a small molecule compound that binds to the target protein (POI), with a linker group introduced at an appropriate position, and then linked to a small molecule compound that binds to a ubiquitin protease. The resulting small probe can simultaneously bind to both the target protein and the ubiquitin protease, thereby promoting the ubiquitination of the target protein. The multiubiquitinated protein can then be recognized and degraded by the proteasome.

[0005] Using the PROTACs strategy, a protein degradation-targeting chimera capable of targeting and recognizing / binding to androgen receptors was prepared. This chimera can regulate androgen receptor levels through the intracellular ubiquitin-proteasome degradation system, inducing androgen receptor degradation, thereby achieving therapeutic effects on androgen receptor-regulated diseases such as prostate cancer.

[0006] Therefore, developing a bifunctional chimeric molecule that can target and bind to androgen receptors and effectively degrade androgen receptors has promising applications in treating diseases regulated by androgen receptors. Summary of the Invention

[0007] The purpose of this invention is to provide a protein degradation targeting chimera with stronger binding ability to androgen receptors and higher activity in degrading androgen receptors.

[0008] This invention provides compounds of formula (I), or isotopic compounds thereof, or optical isomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof, or prodrugs thereof, or solvates thereof:

[0009]

[0010] ARB is the androgen receptor recognition / binding part, L is the linking part, and U is the ubiquitin protease recognition / binding part; these three parts are linked by chemical bonds.

[0011] The ARB is selected from the following structure:

[0012]

[0013] R w1 R w2 R w5 R w6 For H,

[0014] R w3 It is cyano.

[0015] R w4 Selected from H, halogen, C 1-6 Alkyl groups or their halogenated or deuterated forms, C 1-6 Alkyl group or its halogenated or deuterated form; the L is selected from the structures shown below: The U is selected from the structure of the following formula (XB-1):

[0016]

[0017] Among them, R g3 Selected from H, halogen.

[0018] Furthermore, R w4 The halogen is CF3; or, the halogen is chlorine or bromine.

[0019] Furthermore, the ARB is selected from the structure of the following formula:

[0020]

[0021]

[0022] Furthermore, the U is selected from the following structures:

[0023]

[0024] Furthermore, the compound is selected from one of the following compounds:

[0025]

[0026]

[0027] The present invention also provides the use of the above-described compounds, or isotopic compounds thereof, or optical isomers thereof, or tautomers thereof, or pharmaceutically acceptable salts thereof, or prodrugs thereof, or solvates thereof, in the preparation of protein degradation-targeting chimeras of androgen receptors.

[0028] Furthermore, the protein degradation targeting chimera is capable of targeting and / or binding to androgen receptors.

[0029] Furthermore, the protein degradation targeting chimera can degrade and / or downregulate androgen receptors.

[0030] Furthermore, the protein degradation-targeting chimera is an active ingredient in drugs for treating diseases regulated by androgen receptors.

[0031] Furthermore, the diseases mentioned are selected from prostate cancer, breast cancer, and Kennedy's disease.

[0032] The present invention also provides a medicament for treating diseases regulated by androgen receptors, wherein the medicament is a formulation prepared by adding pharmaceutically acceptable excipients to the above-mentioned compound, or its isotopic compound, or its optical isomer, or its tautomer, or its pharmaceutically acceptable salt, or its prodrug, or its solvate as the active ingredient.

[0033] Experiments have demonstrated that the compound represented by formula (I) provided by this invention can target and degrade androgen receptors in prostate cancer cells and inhibit their proliferation, while also exhibiting good metabolic stability and pharmacokinetic properties. The compound of this invention shows promising application potential in the preparation of androgen receptor protein degradation-targeting chimeras and in the development of drugs for treating androgen receptor-regulated diseases (including prostate cancer, breast cancer, and Kennedy's disease).

[0034] Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

[0035] "Isotopic compounds" refer to compounds obtained by replacing one or more atoms of a compound with their corresponding isotopes. For example, compounds obtained by replacing one or more hydrogen atoms (H) with deuterium (D) or tritium (T); or compounds obtained by replacing one or more carbon atoms with deuterium (D) or tritium (T). 12 Carbon 11 or carbon 13 The compound obtained after substitution.

[0036] "Pharmaceutical acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with receptors.

[0037] "Salt" is an acidic and / or basic salt formed by a compound or its stereoisomer with an inorganic and / or organic acid and / or base, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compound. Alternatively, they can be obtained by mixing the compound, or its stereoisomer, with an appropriate amount (e.g., equimolar amounts) of an acid or base. These salts may form a precipitate in solution and be collected by filtration, or be recovered after solvent evaporation, or be prepared by freeze-drying after reaction in an aqueous medium. The salts described in this invention can be hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluoric acids, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, or trifluoroacetates of the compound.

[0038] "Solvate" refers to the solvate formed by the compound of the present invention and a solvent, wherein the solvent includes (but is not limited to): water, ethanol, methanol, isopropanol, propylene glycol, tetrahydrofuran, and dichloromethane.

[0039] In the compounds of this invention, ARB is the androgen receptor recognition / binding moiety, which acts as a ligand for the androgen receptor in the compound.

[0040] There are several ways to name the specific compounds of this invention: (1) numerical designation; (2) compound naming, such as (3R,5S)-1-((S)-2-(2-((5-((4-(3-((1R,3R)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)ureo)phenyl)amino)pentyl)oxy)acetamido)-3,3-dimethylbutyryl)-5-(((S)-1-(4-(4-methylthiazol-5-yl))phenyl)ethyl)carbamoyl)pyrrolidine-3-ylacetate. Although the naming methods are different, each specific compound of this invention can be uniquely identified based on its structure.

[0041] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0042] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Detailed Implementation

[0043] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0044] First, synthesize the intermediate:

[0045]

[0046] SM-A-1:4-((1r,3r)-3-(2-bromo-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile

[0047]

[0048] 1. Compound 3-(methoxycarbonyl)-2-methylpyridine-1-oxo derivative

[0049] 2-Methylnicotinate (30.0 g, 199.0 mmol) was dissolved in dichloromethane (500 mL), followed by the addition of m-chloroperoxybenzoic acid (68.7 g, 398.0 mmol) in portions. The mixture was stirred overnight at room temperature, filtered, and the filter cake was washed with dichloromethane. The filtrate was added to 5% sodium sulfite solution (200 mL) and stirred for 30 minutes. The mixture was separated and extracted with dichloromethane (100 mL × 3). The filtrates were combined, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (dichloromethane:methanol = 100:1 to 20:1) to give a white solid compound 3-(methoxycarbonyl)-2-methylpyridine-1-oxocyanate (18 g, 107.8 mmol), yield: 54%. MS: calcd for C8H9NO3[M+H] + :168.0; found:168.1.

[0050] 2. Compound 6-chloro-2-methylnicotinic acid methyl ester

[0051] Compound 3-(methoxycarbonyl)-2-methylpyridine-1-oxoform (36.5 g, 218.6 mmol) was added in portions to phosphorus oxychloride (300 mL) under ice bath conditions. The mixture was then slowly refluxed for 3 hours. Most of the solvent was removed from the reaction mixture under reduced pressure. The solution was diluted with ethyl acetate (500 mL), washed with 10% Na₂CO₃ aqueous solution (100 mL × 3), and then washed with saturated brine (100 mL × 3). The organic layer was dried over anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 5:1) to give a white solid, methyl 6-chloro-2-methylnicotinate (8 g, 43.2 mmol). Yield: 20%. MS: calcd. for C₈H₈ClNO₂[M+H] + :186.0; found:186.1.

[0052] 3. Compound 6-bromo-2-methylnicotinic acid methyl ester

[0053] 6-Chloro-2-methylnicotinic acid methyl ester (8.0 g, 43.2 mmol) was dissolved in acetonitrile (80 mL), followed by the addition of trimethylbromosilane (19.8 g, 129.7 mmol). The reaction mixture was heated under reflux overnight. The solvent was removed from the reaction mixture under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1 to 10:1) to give a yellow solid of 6-bromo-2-methylnicotinic acid methyl ester (7.0 g, 30.6 mmol). Yield: 71%. MS: calcd for C8H8BrNO2[M+H] + :230.0; found:230.1.

[0054] 4. Compound 2-(bromomethyl)-6-bromonicotinic acid methyl ester

[0055] 6-Bromo-2-methylnicotinic acid methyl ester (7.0 g, 30.6 mmol) and N-bromosuccinimide (8.16 g, 45.9 mmol) were added to carbon tetrachloride (100 mL), followed by the addition of azobisisobutyronitrile (566 mg, 3.06 mmol). The reaction mixture was refluxed overnight. The solvent was removed from the reaction mixture under reduced pressure, and the residue was subjected to silica gel column chromatography (petroleum ether:ethyl acetate = 30:1 to 10:1) to give crude 2-(bromomethyl)-6-bromonicotinic acid methyl ester (A-1-5).

[0056] The crude product was dissolved in dichloromethane (60 mL), and N,N-diisopropylethylamine (2.36 g, 18.3 mmol) was added. Diethyl phosphite (1.21 g, 8.76 mmol) was added dropwise in an ice bath, and the mixture was stirred overnight at room temperature. The reaction solution was diluted with water (100 mL), extracted with dichloromethane (40 mL × 3), washed with saturated brine (50 mL × 3), and the combined organic phases were dried over anhydrous sodium sulfate. The solutions were then evaporated to dryness and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1 to 10:1) to give a white solid methyl 2-(bromomethyl)-6-bromonicotinic acid (6.0 g, 19.5 mmol), yield: 64%. MS: calcd. for C8H7Br2NO2[M+H] + :307.9; found:308.1.

[0057] 5. Compound 3-carbonyl-2,2,4,4-tetramethylcyclobutanone oxime

[0058] Hydroxylamine hydrochloride (26.0 g, 374.5 mmol) was dissolved in water (40 mL) and ethanol (250 mL), followed by the addition of 1,3-tetramethylcyclobutanedione (50 g, 356.7 mmol) and sodium acetate (29.3 g, 356.7 mmol). The reaction mixture was heated under reflux for 2 hours. Ethanol and water were removed by rotary evaporation. The residue was added to toluene (300 mL) and refluxed for 3 hours, followed by hot filtration. The filter cake was washed with toluene (100 mL). The filtrate was evaporated to dryness to give a white solid, 3-carbonyl-2,2,4,4-tetramethylcyclobutane oxime (32.0 g, 206.5 mmol), yield: 59%. MS: calcd.forC8H 13 NO2[M+H] + :156.1; found:156.1.

[0059] 6. Compound 3-hydroxy-2,2,4,4-tetramethylcyclobutanone oxime

[0060] 3-Carbonyl-2,2,4,4-Tetramethylcyclobutanone oxime (110.9 g, 714.6 mmol) was dissolved in isopropanol (715 mL), followed by the addition of sodium borohydride (18.9 g, 500.2 mmol) in portions. The reaction mixture was stirred overnight at room temperature. The reaction mixture was quenched at 5 °C with sodium hydroxide (2000 mL). The mixture was then extracted with ethyl acetate (700 mL × 3), and the combined organic phases were dried over sodium sulfate and evaporated to dryness to give a white solid, 3-hydroxy-2,2,4,4-tetramethylcyclobutanone oxime (100.8 g, 642.0 mmol), yield: 90%.

[0061] MS:calcd.for C8H 15 NO2[M+H] + :158.1; found:158.1.

[0062] 7. Compound 3-aminotetramethylcyclobutanone

[0063] 3-Hydroxy-2,2,4,4-Tetramethylcyclobutanone oxime (A-1-7) (35.1 g, 223.3 mmol) was dissolved in tetrahydrofuran (300 mL), and then nickel-aluminum alloy (76.5 g, 893.3 mmol) was added under nitrogen protection. The reaction mixture was refluxed for 30 minutes, and then 15% sodium hydroxide (300 mL) was added while maintaining reflux. After the addition was complete, reflux was continued for 2 hours. After the reaction was complete, the mixture was filtered, and the filter cake was washed with tetrahydrofuran (100 mL × 3). The filtrate was extracted with ethyl acetate (200 mL × 3), and the combined organic phases were washed with saturated brine (200 mL × 3), dried over sodium sulfate, and evaporated to dryness to give a pale yellow solid, 3-aminotetramethylcyclobutanone (24.5 g, 171.3 mmol), yield: 77%. MS: calcd. for C8H 17 NO[M+H] + :144.1; found:144.1.

[0064] 8. Compound 3-hydroxy-2,2,4,4-tetramethylcyclobutylcarbamate tert-butyl ester

[0065] 3-Aminotetramethylcyclobutanone (26.9 g, 188.1 mmol) was dissolved in dichloromethane (500 mL), followed by the addition of di-tert-butyl dicarbonate (41.4 g, 190.0 mmol) and triethylamine (38.0 g, 376.2 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with water (300 mL) and extracted with dichloromethane (100 mL × 3). The combined organic phases were washed with saturated brine (200 mL × 3), dried over sodium sulfate, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 1:1) to give a pale yellow solid, 3-hydroxy-2,2,4,4-tetramethylcyclobutylcarbamate (30.1 g, 123.9 mmol), yield: 66%.

[0066] MS:calcd.for C8H 17 NO[M+H] + :244.2; found:244.2.

[0067] 9. Compound (1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutylcarbamate tert-butyl ester

[0068] 25.0 g (102.9 mmol) of 3-hydroxy-2,2,4,4-tetramethylcyclobutylcarbamate tert-butyl ester was dissolved in tetrahydrofuran (250 mL). Sodium hydride (60% in mineral oil) (8.23 g, 205.8 mmol) was added in portions under ice bath conditions, and the mixture was stirred for 30 minutes under ice bath conditions. Then, 17.6 g (113.2 mmol) of 2-chloro-4-fluorobenzonitrile (50 mL) was dissolved in tetrahydrofuran and slowly added dropwise to the reaction mixture. The reaction mixture was heated to 60 °C and reacted for 3 hours. The reaction mixture was quenched with water (300 mL) and extracted with ethyl acetate (100 mL × 3). The organic phases were washed with saturated brine (200 mL × 3), dried over sodium sulfate, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 50:1 to 30:1) to give a white solid (1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutylcarbamate tert-butyl ester (15.0 g, 39.7 mmol), yield: 39%. MS: calcd. for C 20 H 27 ClN2O3[M+H] + :379.2; found:279.2.

[0069] 10. Compound 4-((1r,3r)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile trifluoroacetate: (1r,3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutylcarbamate tert-butyl ester (5.0 g, 13.2 mmol) was dissolved in dichloromethane (50 mL), and trifluoroacetic acid (50 mL) was added under ice bath conditions. The reaction mixture was stirred at room temperature for 1 hour, and then evaporated to dryness to obtain crude 4-((1r,3r)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile trifluoroacetate, which was directly used in the next reaction. MS: calcd.for C 17 H 19 ClF3N2O2[M+H] + :376.1; found:279.1.

[0070] 11. Compound 4-((1r,3r)-3-(2-bromo-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile

[0071] 4-((1r,3r)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile trifluoroacetate (13.2 mmol, crude product derived from the previous step) was dissolved in acetonitrile (240 mL), followed by the addition of N,N-diisopropylethylamine (8.51 g, 66.0 mmol) and methyl 2-(bromomethyl)-6-bromonicotinic acid (4.0 g, 13.2 mmol). The reaction was also heated under reflux overnight. After the reaction mixture was evaporated to dryness, toluene (50 mL) was added and refluxed overnight. The reaction mixture was cooled and filtered, the filter cake was washed with toluene, and the filter cake was dried to give the target compound as a white solid (3.95 g, 8.3 mmol). Two-step yield: 63%. MS: calcd.for C 22 H 21 BrClN3O2[M+H] + :474.0; found:474.0.

[0072] Other lactam intermediates were synthesized using a method similar to the route described above.

[0073] The following amide intermediates were synthesized using methods described in the literature (US20180099940, US20170327469) or similar methods.

[0074]

[0075] The following SM-E intermediate compounds were synthesized using methods described in the literature (US20180099940, US20170327469) or similar methods.

[0076]

[0077] Intermediate SM-L-1: 4-ethynyl-1,4'-dipiperidine-1'-carboxylic acid tert-butyl ester

[0078]

[0079] 1. Compound 4-(p-Toluenesulfonyloxy)piperidine-1-carboxylic acid tert-butyl ester

[0080] 10.0 g (49.7 mmol) of 4-hydroxypiperidine-1-carboxylic acid tert-butyl ester was dissolved in dichloromethane, followed by the addition of p-toluenesulfonyl chloride (10.8 g, 56.6 mmol), triethylamine (7.5 g, 74.5 mmol), and 4-dimethylaminopyridine (183 mg, 1.5 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with water (100 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 3), dried over sodium sulfate, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 3:1) to give a white solid, 13.2 g (37.2 mmol), in 83% yield.

[0081] MS:calcd.forC 17 H 25 NO5S[M+H] + :356.1; found:356.1.

[0082] 2. Synthesis of compound 4-ethynyl-1,4'-dipiperidine-1'-carboxylic acid tert-butyl ester (SM-L-1)

[0083] 4-(p-Toluenesulfonyloxy)piperidine-1-carboxylic acid tert-butyl ester (1.2 g, 3.4 mmol) was dissolved in acetonitrile, followed by the addition of 4-acetylenide piperidine hydrochloride (490 mg, 3.4 mmol), potassium carbonate (1.0 g, 7.5 mmol), and potassium iodide (113 mg, 0.68 mmol). The reaction mixture was heated under reflux for 36 hours. The reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL × 3), dried over sodium sulfate, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 to pure ethyl acetate) to give a white solid 4-acetylenyl-1,4'-dipiperidine-1'-carboxylic acid tert-butyl ester (0.6 g, 2.1 mmol). Yield:

[0084] 60%. MS:calcd.forC 17 H 28 N₂O₂[M+H] + :293.1; found:293.1.

[0085] SM-L-3 was prepared using a method similar to that used to synthesize SM-L-1.

[0086] Intermediate SM-L-2: 3-(4-ethynylpiperidin-1-yl)azacyclobutane-1-carboxylic acid tert-butyl ester

[0087]

[0088] 4-Ethynepiperidine hydrochloride (2.0 g, 13.8 mmol) and 3-carboxylic acid tert-butyl ester 3-azacyclobutanone (2.35 g, 13.8 mmol)

[0089] Dissolve 1,2-dichloroethane (40 mL), then add acetic acid (1 mL), and finally add sodium triacetoxyborohydride (5.85 g, 27.6 mmol). Stir the reaction mixture overnight at room temperature. Dilute the reaction mixture with water (50 mL) and extract with dichloromethane (30 mL × 3). Organic phase

[0090] The samples were washed with saturated brine (30 mL × 3), dried over sodium sulfate, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 to pure ethyl acetate) to give a colorless oily substance, tert-butyl 3-(4-ethynylpiperidin-1-yl)azacyclobutane-1-carboxylic acid (1.0 g,

[0091] 3.8 mmol), yield: 27%. MS: calcd.forC 15 H 24 N₂O₂[M+H] + :265.2; found:265.2.

[0092] The synthesis of intermediates SM-L-4, SM-L-8, SM-L-9 and SM-L-10 is similar to that of SM-L-2.

[0093]

[0094] Intermediate SM-L-11: 4-(2-methyl-3-yn-2-yl)piperazine-1-carboxylic acid tert-butyl ester

[0095]

[0096] Piperazine-1-carboxylic acid tert-butyl ester (3.3 g, 17.7 mmol), triethylamine (3.57 g, 35.4 mmol), and 3-chloro-3-methylbutyne (1.8 g, 17.7 mmol) were dissolved in tetrahydrofuran (30 mL), followed by the addition of cuprous chloride (179 mg, 1.8 mmol). The reaction mixture was stirred at room temperature for 10 minutes. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 3), dried over sodium sulfate, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 2:1) to give a colorless oily substance, 4-(2-methyl-3-yn-2-yl)piperazine-1-carboxylic acid tert-butyl ester (3.0 g, 11.9 mmol), yield: 67%. MS: calcd.

[0097] forC 14 H 24 N₂O₂[M+H] + :253.2; found:253.2.

[0098] Intermediate SM-L-13: 4-(2-methyl-3-yn-2-ylamino)piperidine-1-carboxylic acid tert-butyl ester (

[0099]

[0100] 4-Aminopiperidine-1-carboxylic acid tert-butyl ester (2.6 g, 13.0 mmol), triethylamine (1.51 g, 15.0 mmol), and cuprous bromide (144 mg, 1.0 mmol) were added to acetonitrile (30 mL), followed by dropwise addition of a 10 mL solution of 3-chloro-3-methylbutyne (1.0 g, 10.0 mmol) in acetonitrile. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was then diluted with 100 mL of water and 50 mL of ethyl acetate.

[0101] 3) Extraction. The combined organic phases were washed with saturated brine (50 mL × 3), dried over sodium sulfate, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 2:1) to give a colorless oily tert-butyl 4-(2-methyl-3-yn-2-ylamino)piperidine-1-carboxylic acid (0.6 g, 2.3 mmol), yield: 22%. MS: calcd.forC 15 H 26 N₂O₂[M+H] + :267.2; found:267.2.

[0102] Intermediate SM-L-14: 4-(4-ethynylphenyl)piperazine-1-carboxylic acid tert-butyl ester

[0103]

[0104] 1. Compound 4-(4-formylphenyl)piperazine-1-carboxylic acid tert-butyl ester

[0105] 4-Fluorobenzaldehyde (5.0 g, 40.3 mmol) and piperazine-1-carboxylic acid tert-butyl ester (7.5 g, 40.3 mmol) were dissolved in N,N-dimethylformamide (100 mL), followed by the addition of potassium carbonate (11.2 g, 80.6 mmol). The reaction mixture was stirred at 110 °C for 2 days. The reaction mixture was diluted with water (150 mL) and extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine (100 mL × 3), dried over sodium sulfate, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 5:1) to give a white solid 4-(4-formylphenyl)piperazine-1-carboxylic acid tert-butyl ester (7.1 g, 24.5 mmol), yield: 61%.

[0106] MS:calcd.forC 16 H 23 O2N3[M+H] + :290.2; found:290.0.

[0107] 2. Compound 4-(4-ethynylphenyl)piperazine-1-carboxylic acid tert-butyl ester

[0108] 4-(4-formylphenyl)piperazine-1-carboxylic acid tert-butyl ester (B-14-1) (2.9 g, 10.0 mmol) and dimethyl 1-diazo-2-oxopropylphosphonate (3.8 g, 20.0 mmol) were dissolved in methanol (50 mL), and potassium carbonate (5.5 g, 40 mmol) was added in an ice bath. The reaction mixture was slowly heated to room temperature overnight. The reaction mixture was evaporated to dryness and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 50:1 to 15:1) to give a white solid 4-(4-ethynylphenyl)piperazine-1-carboxylic acid tert-butyl ester (1.5 g, 5.2 mmol), yield: 54%. MS: calcd.

[0109] forC 17 H 23 O2N2[M+H] + :287.2; found:287.2.

[0110] Intermediates SM-L-18: 4-((1s,3s)-3-ethynylcyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (SM-L-18Q) and 4-((1r,3r)-3-ethynylcyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (SM-L-18H)

[0111]

[0112] 1. Compound 4-(3-(methoxycarbonyl)cyclobutyl)piperazine-1-carboxylic acid tert-butyl ester

[0113] Methyl 3-oxocyclobutane carboxylate (5.12 g, 40.0 mmol) and piperazine-1-carboxylate tert-butyl ester (8.9 g, 48.0 mmol) were dissolved in 1,2-dichloroethane (100 mL), followed by the addition of acetic acid (4.8 g, 80.0 mmol), and finally sodium triacetoxyborohydride (21.2 g, 100 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with water (100 mL) and extracted with dichloromethane (50 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 3), dried over sodium sulfate, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 100 mL / mL).

[0114] Purified with ethyl acetate (10:1 to pure ethyl acetate) to give a white solid tert-butyl 4-(3-(methoxycarbonyl)cyclobutyl)piperazine-1-carboxylic acid (6.0 g, 20.8 mmol), yield: 52%. MS: calcd.forC 15 H 26 N₂O₄[M+H] + :299.2; found:299.4.

[0115] 2. Compound 4-(3-(hydroxymethyl)cyclobutyl)piperazine-1-carboxylic acid tert-butyl ester

[0116] 4-(3-(methoxycarbonyl)cyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (6.0 g, 20.8 mmol) was dissolved in tetrahydrofuran (120 mL), and lithium aluminum tetrahydrogenate (4.8 g, 80.0 mmol) was added in portions while in an ice bath. The reaction mixture was stirred in an ice bath for 1 hour. The reaction mixture was diluted with tetrahydrofuran (100 mL), kept in an ice bath, and then water (4.8 mL) was added dropwise with stirring for 10 minutes. Then, 15% sodium hydroxide solution (4.8 mL) was added with stirring for another 10 minutes, followed by water (14.4 mL) with stirring for another 20 minutes. Finally, anhydrous magnesium sulfate was added with stirring for another 20 minutes. The reaction mixture was filtered, and the filtrate was evaporated to dryness to give a colorless oily product, 4-(3-(hydroxymethyl)cyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (4.4 g, 14.8 mmol), yield: 78%. MS: calcd.forC 14 H 26 N₂O₃[M+H] + :

[0117] 271.2; found: 271.4.

[0118] 3. Compound 4-(3-formylcyclobutyl)piperazine-1-carboxylic acid tert-butyl ester

[0119] 4-(3-(hydroxymethyl)cyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (4.0 g, 14.8 mmol) was dissolved in dichloromethane (80 mL).

[0120] Then, 1,1',1'-(3-oxo-1λ5-1,2-phenyliodo-1(3H)-yrolone) acetoacetate (9.4 g, 22.2 mmol) was added. The reaction mixture was stirred at room temperature for 24 hours. The mixture was then filtered, and the filtrate was evaporated to dryness to give 4-(3-formylcyclobutyl)piperazine-1-carboxylic acid tert-butyl

[0121] The crude ester (5.3 g) was used directly in the next reaction. MS: calcd.forC 14 H 24 N₂O₃[M+H] + :269.2; found:269.4.4. Compounds 4-((1s,3s)-3-ethynylcyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (SM-L-18Q) and 4-((1r,3r)

[0122] Synthesis of 3-ethynylcyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (SM-L-18H)

[0123] 4-(3-formylcyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (14.8 mmol, crude product from the previous step) and 1-diazo-2-oxopropylphosphonate dimethyl ester (5.68 g, 29.6 mmol) were dissolved in methanol (100 mL), and potassium carbonate (10.2 g, 74.0 mmol) was added in an ice bath.

[0124] mmol). The reaction solution was slowly heated to room temperature overnight. The reaction solution was diluted with water (200 mL) and extracted with ethyl acetate (80 mL × 3). The combined organic phases were washed with saturated brine (80 mL × 3), dried over sodium sulfate, and subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 1000 mmol / L).

[0125] Purification at a ratio of 20:1 to 12:1 yielded a white solid, 4-((1s,3s)-3-ethynylcyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (SM-L-18Q) (0.84 g, 3.2 mmol), with a two-step yield of 16%. MS: calcd.forC 15 H 24 N₂O₂[M+H] + 265.2;

[0126] Found: 265.4. A white solid, 4-((1r,3r)-3-ethynylcyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (SM-L-18H) (0.36 g, 1.4 mmol), was also obtained. Two-step yield: 7%. MS: calcd.forC 15 H24 N₂O₂[M+H] + 265.2;

[0127] Found: 265.4.

[0128] Synthesis of intermediate SM-L-5

[0129]

[0130] 1. Synthesis of compound 4-(4-(methoxycarbonyl)cyclohexyl)piperazine-1-carboxylic acid tert-butyl ester

[0131] Methyl 4-oxocyclohexylcarboxylate (10.0 g, 64.0 mmol) and piperazine-1-carboxylate tert-butyl ester (13.0 g, 70.0 mmol) were dissolved in 1,2-dichloroethane (200 mL), followed by the addition of acetic acid (7.6 g, 128.0 mmol), and finally sodium triacetoxyborohydride (34.0 g, 160 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was diluted with water (200 mL) and extracted with dichloromethane (100 mL × 3). The combined organic phases were washed with saturated brine (50 mL × 3), dried over sodium sulfate, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to give a white solid 4-(4-(methoxycarbonyl)cyclohexyl)piperazine-1-carboxylate tert-butyl ester (9.6 g, 29.4 mmol).

[0132] mmol), yield: 46%. MS: calcd.forC 17 H 30 N₂O₄[M+H] + :327.2; found:327.4.

[0133] 2. Synthesis of compound 4-((1r,4r)-4-(hydroxymethyl)cyclohexyl)piperazine-1-carboxylic acid tert-butyl ester

[0134] 4-(4-(methoxycarbonyl)cyclohexyl)piperazine-1-carboxylic acid tert-butyl ester (9.6 g, 29.4 mmol) was dissolved in tetrahydrofuran (200 mL), and lithium aluminum tetrahydrogenate (2.23 g, 58.8 mmol) was added in portions while in an ice bath. The reaction mixture was stirred in an ice bath for 1 hour. The reaction mixture was diluted with tetrahydrofuran (100 mL), kept in an ice bath, and then water (2.3 mL) was added dropwise with stirring for 10 minutes. Then, 15% sodium hydroxide solution (2.3 mL) was added with stirring for another 10 minutes, followed by water (6.9 mL) with stirring for another 20 minutes. Finally, anhydrous magnesium sulfate was added with stirring for another 20 minutes. The reaction mixture was filtered, the filtrate was evaporated to dryness, and then subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 1:1).

[0135] Purified to ethyl acetate, the product yielded a pale yellow oil, 4-((1r,4r)-4-(hydroxymethyl)cyclohexyl)piperazine-1-carboxylic acid tert-butyl ester (1.2 g, 4.0 mmol), in 14% yield. The configuration was determined with reference to patent WO2012145361. MS: calcd.forC 14 H 26 N2O3[M+

[0136] H] + :299.2; found:299.4. 1 HNMR(400MHz, CDCl3): δ3.46–3.33(m,6H),2.51–2.38(m,4H),

[0137] 2.28–2.17(m,1H),1.91–1.84(m,4H),1.62–1.48(m,1H),1.42(s,9H),1.27–1.13(m,3H),1.02–0.88(m,2H).

[0138] Simultaneously, a light yellow oily substance, 4-((1S,4S)-4-(hydroxymethyl)cyclohexyl)piperazine-1-carboxylic acid tert-butyl ester (3.0 g, 10.0 g), was obtained.

[0139] mmol), yield: 34%. MS: calcd.forC 14 H 26 N₂O₃[M+H] + :299.2; found:299.4.

[0140] 3. Synthesis of compound 4-((1r,4r)-4-(formyl)cyclohexyl)piperazine-1-carboxylic acid tert-butyl ester

[0141] 4-((1r,4r)-4-(hydroxymethyl)cyclohexyl)piperazine-1-carboxylic acid tert-butyl ester (1.2 g, 4.0 mmol) was dissolved in dichloromethane (50 mL).

[0142] Then, 1,1',1'-(3-oxo-1λ5-1,2-phenyliodo-1(3H)-yrolone) acetoacetate (2.0 g, 4.8 mmol) was added. The reaction mixture was stirred at room temperature for 24 hours. The mixture was then filtered, and the filtrate was evaporated to dryness to give crude 4-((1r,4r)-4-(formyl)cyclohexyl)piperazine-1-carboxylic acid tert-butyl ester (1.4 g), which was used directly in the next step. MS: calcd.forC 16 H 28 N₂O₃[M+H] + :297.2; found:297.4.

[0143] 4. Synthesis of compound 4-((1r,3r)-4-ethynylcyclohexyl)piperazine-1-carboxylic acid tert-butyl ester

[0144] 4-((1r,4r)-4-(formyl)cyclohexyl)piperazine-1-carboxylic acid tert-butyl ester (4.0 mmol, crude product derived from Method 1 in the previous step) and 1-diazo-2-oxopropylphosphonate dimethyl ester (7.2 g, 6.0 mmol) were dissolved in methanol (50 mL), and potassium carbonate (1.1 g, 8.0 mmol) was added in an ice bath. The reaction solution was slowly heated to room temperature overnight. The reaction solution was diluted with water (50 mL) and ethyl acetate (50 mL ×

[0145] 3) Extraction. The combined organic phases were washed with saturated brine (50 mL × 3), dried over sodium sulfate, and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1 to 2:1) to give a pale yellow solid 4-((1r,3r)-3-ethynylcyclohexyl)piperazine-1-carboxylic acid tert-butyl ester (0.67 g, 2.3 mmol), two-step yield: 57%. MS: calcd.forC 17 H 28 N₂O₂[M+H] + :293.2;found:

[0146] 293.2. 1 HNMR (400MHz, CDCl3): δ3.46–3.36(m,4H),2.53–2.44(m,4H),2.35–2.23(m,1H),2.22–2.11(m ,1H),2.09–2.00(m,3H),1.93–1.83(m,2H),1.45(s,9H),1.43–1.34(m,2H),1.28–1.20(m,2H).

[0147] The synthesis of intermediates SM-L-6, SM-L-7, SM-L-15, SM-L-16, SM-L-17, SM-L-18, SM-L-19, SM-L-20, SM-L-21, SM-L-22, SM-L-23 and SM-L-24 is similar to that of SM-L-18.

[0148]

[0149] The following are specific examples of the preparation of compounds provided by this invention: 131: Synthesis of N-((1r,4r)-4-(4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0150]

[0151] LC / MS(ESI+)calcdforC 41 H 41 ClN9O6 + (M+H + )m / z,800.4;found800.3.

[0152] 1 HNMR (400MHz, DMSO-d6) δ11.08(s,1H),8.59(d,J=4.4Hz,1H),7.84(d,J=4.8Hz,1H),7.84(d,J=4.4Hz,2H),7.64(d,J =4.4Hz,1H),7.33(d,J=4.8Hz,1H),7.13(d,J=4.4Hz,2H),6.78(s,1H),6.65(d,J=4.0Hz,1H),5.05(dd,J=9.2,3.6Hz ,1H),4.49-4.45(m,3H),3.85(m,1H),3.75(s,4H),3.35(s,5H),3.17(s,1H),3.00(t,J=8.0Hz,1H),2.91-2.85(m,1H ),2.36-2.36(m,3H),2.16-2.10(m,4H),2.00-1.99(m,1H),1.77-1.77(m,5H),1.65-1.62(m,2H),1.55-1.46(m,2H),

[0153] 1.23-1.23(m,3H),1.14-1.03(m,3H).

[0154] Synthesis of 132:N-((1r,4r)-4-(4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxapiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0155]

[0156] LC / MS(ESI+)calcdforC 41 H 41 ClN9O6 + (M+H + )m / z,818.4;found818.3.

[0157] 1HNMR (400MHz, DMSO-d6) δ11.09(s,1H),8.58(d,J=4.4Hz,1H),7.80(d,J=4.8Hz,1H),7.74(d,J=4.4Hz,2H),7.60(d,J=6.0H z,1H),7.33(d,J=4.8Hz,1H),7.13(d,J=4.4Hz,2H),6.89(d,J=2.4Hz,1H),5.06(dd,J=9.2,4.0Hz,1H),4.51-4.46(m,3H), 3.87(m,1H),3.76(s,4H),3.38(s,5H),3.16(s,1H),3.02(t,J=8.0Hz,1H),2.93-2.86(m,1H),2.38-2.35(m,3H),2.15-2.0 8(m,4H),2.03-1.99(m,1H),1.77-1.77(m,5H),1.66-1.61(m,2H),1.57-1.49(m,2H),1.25-1.25(m,3H),1.16-1.03(m,3H).

[0158] 135: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]non-7-yl)methyl)piperidin-1-yl)pyrimidine-2-carboxamide

[0159]

[0160] Step 1: 2-(2,6-dioxadiazin-3-yl)-5-fluoroisoindoline-1,3-dione (553 mg, 2.0 mmol), tert-butyl 2,7-diazaspiro[3.5]nonane-7-carboxylate (453 mg, 2.0 mmol), and diisopropylethylamine (1.3 g, 10.0 mmol) were added to 10 mL of DMSO. The mixture was heated to 135 °C and stirred overnight. After cooling to room temperature, the mixture was extracted with ethyl acetate and water. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was purified by Pre-TLC. The compound tert-butyl 2-(2-(2,6-dioxadiazin-3-yl)-1,3-dioxadiazindoline-5-yl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (530 mg) was obtained. Yield: 55%. LC / MS(ESI+)calcdforC 25 H 30 N4O6([M+H)) + )m / z482.22; found483.1.

[0161] Step 2: 530 mg (1.09 mmol) of tert-butyl 2-(2-(2,6-dioxadiazin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]nonane-7-carboxylic acid ester was dissolved in 10 mL of dichloromethane, and 4 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 3 h. TLC monitoring showed that the starting material was completely consumed. Excess trifluoroacetic acid and solvent were removed by rotary evaporation, and residual trifluoroacetic acid was removed by repeated rotary evaporation with dichloromethane. Add 10 mL of dichloromethane and 3 mL of water again, then transfer the system to an ice-water bath to cool and stir. Next, add saturated sodium bicarbonate solution dropwise to adjust the pH of the system to approximately 9. After completion, allow the layers to separate. Wash the aqueous phase with dichloromethane (3 mL * 3), combine the organic layers, wash with brine, dry with anhydrous sodium sulfate, and purify the crude product by column chromatography to obtain compound 2-(2,6-dioxadiazin-3-yl)-5-(2,7-diazaspiro[3.5]non-2-yl)isoindole-1,3-dione (342 mg). Yield: 82%. LC / MS (ESI+) calcd for C 20 H 20 N4O4([M+H)) + )m / z382.16; found383.1.

[0162] Step 3: NaH (360 mg, 9.0 mmol) was added to 5 mL of LDM, and the system was cooled and stirred in an ice-water bath. Then (1r,4r)-4-aminocyclohexane-1-ol (455 mg, 3.0 mmol) was added, and the mixture was stirred for 10 min. Then 2-chloro-4-fluorobenzonitrile (460 mg, 3 mmol) was added. The mixture was allowed to return to room temperature and stirred overnight. After the starting material was consumed by TLC, ethyl acetate and water were added for extraction. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness. Pre-TLC purification was performed to obtain compound 4-((1r,4r)-4-aminocyclohexyl)oxy)-2-chlorobenzonitrile (279 mg). Yield: 62%. LC / MS (ESI+) calcd for C 13 H 15 ClN2O([M+H)) + )m / z250.73; found251.1.

[0163] Step 4: 5-Chloroprene-2-carboxylic acid (79 mg, 0.50 mmol) and HATU (304 mg, 0.80 mmol) were added to 5 mL of dichloromethane. The system was then cooled and stirred in an ice-water bath. After 10 min, diisopropylethylamine (194 mg, 1.50 mmol) was added, followed by 4-((1r,4r)-4-aminocyclohexyl)oxy)-2-chlorobenzonitrile (150 mg, 0.50 mmol). Afterward, the ice bath was removed, and the reaction was stirred overnight at room temperature. TLC monitoring showed that the starting material was consumed. Dichloromethane and water were added for extraction. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness. Pre-TLC purification yielded 5-chloro-N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyrimidine-2-carboxamide (185 mg). Yield: 92%. LC / MS(ESI+)calcdforC 18 H 16 Cl2N4O2([M+H) + )m / z391.25; found391.1.

[0164] Step 5: 5-Chloro-N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyrimidine-2-carboxamide (185 mg, 0.46 mmol), piperidin-4-ylmethanol (260 mg, 0.92 mmol), and diisopropylethylamine (297 mg, 2.30 mmol) were added to 8 mL of dioxane. The mixture was heated to 115 °C and refluxed with stirring overnight. After cooling to room temperature, the mixture was extracted with ethyl acetate and water. The organic phase was washed with 0.05 NHCl, then with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. Pre-TLC purification yielded N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-(hydroxymethyl)piperidin-1-yl)pyrimidine-2-carboxamide (109 mg). Yield: 37%. LC / MS(ESI+)calcdforC 24 H 28 ClN5O3([M+H) + )m / z469.97; found470.1.

[0165] Step 6: Place N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-(hydroxymethyl)piperidin-1-yl)pyrimidine-2-carboxamide (50 mg, 0.11 mmol) in a 25 mL single-necked round-bottom flask, and add 5 mL of dichloromethane. Stir at room temperature until dissolved and clear. Then, place the system in an ice-water bath to cool and stir. When the temperature of the system drops to about 0 °C, add Dys-Martin oxidant (72 mg, 0.17 mmol). After completion, allow the system to naturally warm to room temperature while stirring. After 2 hours, TLC showed that the starting material was almost completely consumed, and the reaction was stopped. The system was filtered using diatomaceous earth. The filter cake was washed repeatedly with small amounts of dichloromethane. The filtrates were combined, and the solvent was removed by rotary evaporation to obtain the crude product. Pre-TLC purification yielded N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-formylpiperidin-1-yl)pyrimidine-2-carboxamide (30 mg). Yield: 58%. LC / MS (ESI+) calcd for C 24 H 26 ClN5O3([M+H) + )m / z467.95; found468.1.

[0166] Step 7: Weigh N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-formylpiperidin-1-yl)pyrimidin-2-carboxamide (30 mg, 0.06 mmol) into a 25 mL single-necked round-bottom flask, add dichloromethane (3 mL), and stir at room temperature until dissolved and clear. Then add 2-(2,6-dioxadipinidin-3-yl)-5-(2,7-diazaspiro[3.5]non-2-yl)isoindole-1,3-dione (24 mg, 0.06 mmol) to the system and stir at room temperature for 15 min. Afterward, add sodium triacetylborohydride (38 mg, 0.18 mmol) to the system and stir at room temperature. After 4 h, take a sample and spot it on a TLC plate to monitor the complete consumption of the starting material. Stop stirring, add dichloromethane (15 mL) and water (15 mL) to the system, stir vigorously, and then allow to stand for separation. The aqueous layer is back-extracted with dichloromethane (10 mL * 3), the organic phases are combined, and washed successively with water (10 mL * 2), saturated brine (15 mL), dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation to obtain the crude product. This crude product is then purified by Pre-TLC to obtain compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-((6-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)-2,6-diazaspiro[3,4]octane-2-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide (21 mg). Yield: 39%. 1HNMR (400MHz, DMSO-d6) δ8.51(s,2H),8.33(d,J=8.1Hz,1H),7.86(d,J=8.7Hz,1H),7.63(d,J=8.1Hz,1H),7. 38(s,1H),7.13(d,J=8.4Hz,1H),6.78(s,1H),6.65(d,J=8.2Hz,1H),5.05(dd,J=12.8,5.3Hz,1H),4.53(s,1 H),3.97(d,J=11.3Hz,3H),2.87(t,J=11.9Hz,3H),2.60(s,2H),2.32(s,4H),2.12(s,4H),1.99(d,J=5.1Hz, 1H), 1.88 (s, 2H), 1.79 (s, 2H), 1.67-1.42 (m, 6H), 1.23 (s, 4H), 1.16 (d, J = 10.9Hz, 3H), 0.84 (d, J = 7.3Hz, 2H). LC / MS(ESI+)calcdforC 44 H 48 ClN9O6([M+H)) + )m / z834.38; found834.3.

[0167] 136: N-((1r,4r)-4-((6-cyano-5-(trifluoromethyl)pyridin-3-yl)oxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonane-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0168]

[0169] LC / MS (ESI+) for C 44 H 48 F3N 10 O6([M+H] + )m / z869.

[0170] 148: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-((2-(2,6-dioxopiridine-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]non-7-yl)methyl)piperidin-1-yl)pyrimidin-2-carboxamide

[0171]

[0172] Step 1: 5-Bromopyrimidine-2-carboxylic acid (0.80 g, 3.94 mmol) and HATU (2.41 g, 6.22 mmol) were added to 30 mL of dichloromethane. The system was then cooled and stirred in an ice-water bath. After 10 min, diisopropylethylamine (1.03 g, 7.97 mmol) was added, followed by 4-((1r,4r)-4-aminocyclohexyl)oxy)-2-chlorobenzonitrile (1.01 g, 3.99 mmol). Afterward, the ice bath was removed, and the reaction was stirred overnight at room temperature. TLC monitoring showed that the starting material was consumed. Dichloromethane and water were added for extraction. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness. Pre-TLC purification yielded 5-bromo-N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyrimidine-2-carboxamide (1.09 g). Yield: 63.5%. LC / MS (ESI+) calcd for C 18 H 16 BrClN4O2([M+H] + )m / z 434.01; found435.1.

[0173] Step 2: 5-Bromo-N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyrimidine-2-carboxamide (435 mg, 1.0 mmol), piperidin-4-ylmethanol (116 mg, 1.0 mmol), palladium acetate (15 mg, 0.05 mmol), diisopropylethylamine (35 mg, 0.05 mmol), and cesium carbonate (652 mg, 2.0 mmol) were added to 10 mL of dioxane. The mixture was heated to 110 °C under nitrogen protection and stirred under reflux overnight. After cooling to room temperature, the mixture was extracted with ethyl acetate and water. The organic phase was washed with 0.05 NHCl, then with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. Pre-TLC purification was then performed. The compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-(hydroxymethyl)piperidin-1-yl)pyrimidin-2-carboxamide (191 mg) was obtained. Yield: 41%. LC / MS (ESI+) calcd for C 24 H 28 ClN5O3([M+H) + )m / z 469.97; found470.1.

[0174] Step 3: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-(hydroxymethyl)piperidin-1-yl)pyrimidine-2-carboxamide (50 mg, 0.11 mmol) was placed in a 25 mL single-necked round-bottom flask, and dichloromethane (5 mL) was added. The mixture was stirred at room temperature until dissolved and clear. The system was then placed in an ice-water bath to cool and stir. When the temperature of the system dropped to approximately 0 °C, Dys-Martin oxidant (72 mg, 0.17 mmol) was added. After this step, the system was allowed to warm naturally to room temperature with stirring. After 2 hours, TLC showed that the starting material was almost completely consumed, and the reaction was stopped. The system was filtered using diatomaceous earth. The filter cake was washed repeatedly with small amounts of dichloromethane. The filtrates were combined, and the solvent was removed by rotary evaporation to obtain the crude product. Pre-TLC purification yielded N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-formylpiperidin-1-yl)pyrimidine-2-carboxamide (30 mg). Yield: 58%. LC / MS (ESI+) calcd for C 24 H 26 ClN5O3([M+H) + )m / z 467.95; found468.1.

[0175] Step 4: Weigh N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-formylpiperidin-1-yl)pyrimidin-2-carboxamide (30 mg, 0.06 mmol) into a 25 mL single-necked round-bottom flask, add dichloromethane (3 mL), and stir at room temperature until dissolved and clear. Then add 2-(2,6-dioxadipinidin-3-yl)-5-(2,7-diazaspiro[3.5]non-2-yl)isoindole-1,3-dione (24 mg, 0.06 mmol) to the system and stir at room temperature for 15 min. Afterward, add sodium triacetylborohydride (38 mg, 0.18 mmol) to the system and stir at room temperature. After 4 h, take a sample and spot it on a TLC plate to monitor the complete consumption of the starting materials. Stop stirring, add dichloromethane (15 mL) and water (15 mL) to the system, stir vigorously, and then allow to stand for separation. The aqueous layer is back-extracted with dichloromethane (10 mL * 3), the organic phases are combined, and washed successively with water (10 mL * 2), saturated brine (15 mL), dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation to obtain the crude product. This crude product is then purified by Pre-TLC to obtain compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-((2-(2,6-dioxopiridine-3-yl)-6-fluoro-1,3-dioxoisoindol-5-yl)-2,7-diazaspiro[3.5]non-7-yl)methyl)piperidin-1-yl)pyrimidine-2-carboxamide (21 mg). Yield: 39%. 1HNMR (400MHz, DMSO-d6) δ8.51(s,2H),8.32(d,J=8.1Hz,1H),7.87(d,J=8.6Hz,1H),7.67(d,J=8.0Hz,1H),7.60(d,J =10.7Hz,1H),7.38(s,1H),7.29(d,J=7.9Hz,1H),7.23(d,J=7.4Hz,1H),7.13(d,J=9.5Hz,1H),6.90(d,J=7.7Hz,1H ),6.85(s,1H),5.07(s,1H),4.53(s,1H),3.96(s,4H),2.86(d,J=12.7Hz,5H),2.64(d,J=28.8Hz,2H),2.33(s,4H), 2.12(s,5H),2.05-1.96(m,2H),1.87(s,3H),1.63-1.48(m,6H),1.20-1.11(m,4H),0.85(s,2H).LC / MS(ESI+)calcd for C 44 H 47 ClFN9O6([M+H)) + )m / z 852.37; found 852.3.

[0176] 149: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]non-7-yl)methyl)piperidin-1-yl)pyrimidine-2-carboxamide

[0177]

[0178] 1HNMR (400MHz, DMSO-d6) δ8.51(s,2H),8.33(d,J=8.1Hz,1H),7.86(d,J=8.7Hz,1H),7.63(d,J=8.1Hz,1H),7.38(s, 1H),7.13(d,J=8.4Hz,1H),6.78(s,1H),6.65(d,J=8.2Hz,1H),5.05(dd,J=12.8,5.3Hz,1H),4.53(s,1H),3.97(d, J=11.3Hz,3H),2.87(t,J=11.9Hz,3H),2.60(s,2H),2.32(s,4H),2.12(s,4H),1.99(d,J=5.1Hz,1H),1.88(s,2H), 1.79(s,2H),1.67-1.42(m,6H),1.23(s,4H),1.16(d,J=10.9Hz,3H),0.84(d,J=7.3Hz,2H).LC / MS(ESI+)calcdforC 44 H 48 ClN9O6([M+H)) + )m / z834.38; found834.3.

[0179] 151: N-((1r,4r)-4-(4-cyano-3-(trifluoromethyl)phenoxy)cyclohexyl)-6-(4-((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]non-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0180]

[0181] 1HNMR (400MHz, DMSO-d6) δ11.09(s,1H),8.61(d,J=8.3Hz,1H),8.07(d,J=8.4Hz,1H),7.80(d,J=9.5Hz,1H),7.63(d,J=8.3Hz,1H),7.49(s,1H) ,7.33(d,J=9.7Hz,1H),6.78(d,J=1.7Hz,1H),6.70-6.60(m,1H),5.05(dd,J=12.8,5.4Hz,1H),4.63(s,2H),4.47(d,J=12.7Hz,3H),3.87(d,J =8.3Hz,2H),3.75(s,4H),3.00(t,J=11.8Hz,2H),2.88(dd,J=15.4,10.0Hz,1H),2.32(s,3H),2.12(d,J=7.0Hz,4H),2.05-1.95(m,1H),1.89( s,3H),1.82(s,3H),1.65(dd,J=24.3,11.0Hz,3H),1.60-1.46(m,3H),1.29-1.17(m,2H),0.86(dd,J=18.3,7.5Hz,1H).LC / MS(ESI+)calcdforC 45 H 48 F3N9O6([M+H) + )m / z867.93; found868.4.

[0182] 152: N-((1r,4r)-4-(4-cyano-3-(trifluoromethyl)phenoxy)cyclohexyl)-6-(4-((2-(2,6-dioxopiridine-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]non-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0183]

[0184] 1HNMR (400MHz, DMSO-d6) δ8.61(d,J=8.4Hz,1H),8.07(d,J=8.4Hz,1H),7.80(d,J=9.5Hz,1H),7.59(d,J=11.2Hz,1H),7.51(s,1H),7 .33(d,J=9.8Hz,1H),6.90(d,J=7.7Hz,1H),5.06(dd,J=12.9,5.5Hz,1H),4.63(s,1H),4.47(d,J=12.3Hz,2H),3.00(t,J=11.6Hz,2H ),2.87(t,J=13.0Hz,1H),2.58(d,J=17.8Hz,1H),2.30(s,3H),2.12(d,J=6.8Hz,4H),2.06-1.96(m,1H),1.90(d,J=10.2Hz,3H),1.7 7(s,5H),1.68(s,5H),1.59-1.46(m,3H),1.23(s,2H),1.06(dd,J=17.3,10.3Hz,3H),0.84(d,J=7.5Hz,1H).LC / MS(ESI+)calcdforC 45 H 47 F4N9O6([M+H) + )m / z885.92; found886.4.

[0185] 153: N-((1r,4r)-4-(4-cyano-3-(methoxy-d3)phenoxy)cyclohexyl)-6-(4-((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]non-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0186]

[0187] 1HNMR (400MHz, DMSO-d6) δ11.08(s,1H),8.58(d,J=8.3Hz,1H),7.81(t,J=8.5Hz,1H),7.68-7.56(m,2H),7.32(d,J=9.7Hz,1H),6.77(s,1H ),6.74-6.69(m,2H),6.65(d,J=8.3Hz,1H),5.76(s,1H),5.05(dd,J=12.9,5.4Hz,1H),4.47(d,J=11.7Hz,3H),3.85(s,1H),3.16(s,1H),3 .00(t,J=11.8Hz,2H),2.94-2.81(m,1H),2.63-2.53(m,2H),2.32(s,4H),2.12(d,J=6.9Hz,4H),2.04-1.97(m,1H),1.89(s,3H),1.76(s,6 H),1.64(dd,J=23.7,11.0Hz,2H),1.56-1.47(m,2H),1.23(s,2H),1.09(dd,J=22.4,9.9Hz,2H),0.84(d,J=6.8Hz,1H).LC / MS(ESI+)calcd forC 45 H 48 D3N9O7([M+H)) + )m / z832.98; found833.3.

[0188] 154: N-((1r,4r)-4-(4-cyano-3-ethoxyphenoxy)cyclohexyl)-6-(4-((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]non-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0189]

[0190] 1HNMR(400MHz,DMSO-d6)δ11.08(s,1H),8.58(d,J=8.3Hz,1H),7.80(d,J=9.6Hz,1 H),7.66-7.56(m,2H),7.32(d,J=9.8Hz,1H),6.78(d,J=1.8Hz,1H),6.70(dd,J=4. 5,2.4Hz,2H),6.65(dd,J=8.4,1.9Hz,1H),5.76(s,1H),5.05(dd,J=12.8,5.4Hz,1 H),4.53-4.42(m,3H),4.17(q,J=7.0Hz,2H),3.85(d,J=8.2Hz,1H),3.75(s,4H),3 .16(s,1H),3.00(t,J=11.8Hz,2H),2.94-2.81(m,1H),2.57(dd,J=18.9,5.0Hz,2H ),2.32(s,3H),2.12(d,J=6.5Hz,4H),2.04-1.96(m,1H),1.89(s,3H),1.76(s,4H) ,1.63(dd,J=24.3,10.8Hz,2H),1.50(dd,J=23.3,10.4Hz,2H),1.35(t,J=7.0Hz,3 H),1.23(s,1H),1.17-1.01(m,2H),0.84(d,J=6.9Hz,1H).LC / MS(ESI+)calcdforC 46 H 53 N9O7([M+H)) + )m / z843.99; found844.4.

[0191] 155: N-((1r,4r)-4-(4-cyano-3-(2-methoxyethoxy)phenoxy)cyclohexyl)-6-(4-((2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]non-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0192]

[0193] 1HNMR(400MHz,DMSO-d6)δ11.07(s,1H),8.59(d,J=8.1Hz,1H),7.80(d,J=9.5H z,1H),7.62(dd,J=12.7,8.5Hz,2H),7.33(d,J=9.5Hz,1H),6.76(d,J=9.6Hz, 2H),6.71(d,J=8.6Hz,1H),6.65(d,J=8.5Hz,1H),5.76(s,2H),5.05(dd,J=12 .8,5.4Hz,1H),4.47(d,J=10.1Hz,3H),4.30-4.22(m,2H),3.85(s,1H),3.75( s,4H),3.71-3.65(m,2H),3.30(s,2H),3.17(d,J=5.3Hz,1H),3.00(t,J=11.7 Hz,2H),2.86(d,J=10.9Hz,1H),2.59(s,2H),2.33(s,4H),2.12(s,4H),1.99( s,1H),1.89(d,J=9.7Hz,3H),1.79(d,J=21.6Hz,6H),1.63(d,J=12.9Hz,2H), 1.50(d,J=12.8Hz,2H),1.17-1.01(m,3H),0.85(s,1H).LC / MS(ESI+)calcdfor C 47 H 55 N9O8([M+H)) + )m / z874.01; found875.0.

[0194] 158: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-((2-(2,6-dioxopiridine-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]non-7-yl)methyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0195] LC / MS (ESI+) Calcd for C 43 H 46 FClN 10 O6(M+H + )m / z,853.3;found853.3. 1HNMR (400MHz, CDCl3) δ8.81 (s, 1H), 8.05 (s, 1H), 7.57 (dd, J = 8.4, 2.5Hz, 2H), 7.36 (d, J = 10.9Hz, 1H),7.00(d,J=2.4Hz,1H),6.87–6.79(m,2H),4.91(dd,J=12.3,5.3Hz,2H),4.31(dd,J=12.2,8.3 Hz,1H),4.10–4.01(m,1H),3.89(d,J=1.9Hz,4H),3.05(s,2H),2.94–2.65(m,4H),2.40(s,3H),2 .23–2.15(m,5H),1.90–1.85(m,7H),1.69(d,J=12.4Hz,3H),1.46(d,J=12.7Hz,3H),1.25(s,3H).

[0196] 197:N-((1r,4r)-4-(4-cyano-3-(methoxy-d3)phenoxy)cyclohexyl)-3-(4-((2-(2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]non-7-yl)methyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0197]

[0198] LC / MS(ESI+)calcdforC 44 H 48 D3N 10 O7(M+H + )m / z,834.4;found835.

[0199] 230:N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0200]

[0201] 1. Compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyridazine-3-carboxamide

[0202] Compound 6-chloro-N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyridazin-3-carboxamide (1.00 g, 2.56 mmol), 4-hydroxymethylpiperidine (883 mg, 7.67 mmol), potassium carbonate (1.77 g, 12.78 mmol), and 12 mL of DMF were added sequentially to the reactor. Under nitrogen protection, the reaction was carried out at 80 °C for 3 hours, and the reaction endpoint was determined by TLC. After confirming the completion of the reaction, the reaction solution was extracted three times with water and ethyl acetate. The ethyl acetate layers were combined and washed three times with saturated brine. The solutions were dried and evaporated to dryness. Column chromatography was used to separate the solid compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyridazin-3-carboxamide (1.09 g, 2.32 mmol), yield: 91%.

[0203] LC / MS(ESI+)calcdforC 24 H 27 ClN5O3(M+H + )m / z,470.2;found,470.2.

[0204] 2. Compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-formylpiperidin-1-yl)pyridazine-3-carboxamide

[0205] Compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyridazin-3-carboxamide (95 mg, 0.20 mmol) was dissolved in 2 mL of LCM. After the solution was cleared, Dess-Martin reagent (128 mg, 0.30 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction endpoint was confirmed by TLC. After the reaction was completed, the filter cake was washed with dichloromethane, and the filtrate was extracted with sodium sulfite solution. The residue was dried and evaporated to dryness to obtain a pale yellow solid. The crude product was directly added to the next reaction.

[0206] LC / MS(ESI+)calcdforC 24 H 25 ClN5O3(M+H + )m / z,467.2; found,467.9.

[0207] 3. Synthesis of compound 7-((1-(6-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)carbamoyl)pyridazin-3-yl)piperidin-4-yl)methyl)-2,7-diazaspirocyclic[3.5]nonane-2-carboxylic acid tert-butyl ester

[0208] Crude N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-formylpiperidin-1-yl)pyridazine-3-carboxamide was dissolved in 2 mL of 1,2-dichloroethane, followed by the addition of tert-butyl 2,7-diazaspiro[3.5]nonane-2-carboxylic acid (55 mg, 0.24 mmol) and glacial acetic acid (24 mg, 0.40 mmol). The mixture was stirred at room temperature for 30 minutes, followed by the addition of sodium triacetylborohydride (128 mg, 0.61 mmol), and the reaction was carried out at room temperature for 2 hours. The mixture was extracted with DCM and water, the organic layer was washed with brine, dried over anhydrous sodium sulfate, evaporated to dryness, and purified by silica gel column chromatography. The compound 7-((1-(6-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)carbamoyl)pyridazin-3-yl)piperidin-4-yl)methyl)-2,7-diazaspirocyclo[3.5]nonane-2-carboxylic acid tert-butyl ester (113.0 mg, 0.17 mmol) was obtained. Yield: 85.0%.

[0209] LC / MS(ESI+)calcdforC 36 H 47 ClN7O4(M+H + )m / z,678.4; found,678.0.

[0210] 4. Synthesis of compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0211] The compound tert-butyl 7-((1-(6-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)carbamoyl)pyridazin-3-yl)piperidin-4-yl)methyl)-2,7-diazaspirocyclo[3.5]nonane-2-carboxylic acid tert-butyl ester (50 mg, 0.074 mmol) was dissolved in 2 mL CH2Cl2 and 2 mL trifluoroacetic acid, and stirred at room temperature for 2 h. The solvent was evaporated to dryness, and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (24 mg, 0.088 mmol), 1 mL DMSO, and DIEA (95 mg, 0.737 mmol) were added. Under nitrogen protection, the mixture was stirred overnight at 120 °C. TLC confirmed the reaction was complete. The reaction solution was extracted three times with water and ethyl acetate. The ethyl acetate layers were combined and washed three times with saturated brine. After drying, the solution was evaporated to dryness and separated by column chromatography to obtain a yellow solid compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide (17 mg, 0.020 mmol). Yield: 27%. LC / MS (ESI+) calcd for C 45 H 48 ClN8O6(M+H + )m / z,834.3;found,834.3.

[0212] 1HNMR (400MHz, DMSO-d6) δ11.08(s,1H),8.60(d,J=8.2Hz,1H),7.86(d,J=8.8Hz,1H),7.80(d,J=9.6Hz,1H),7.63(d,J=8.3Hz,1H),7.39(d,J=2.3 Hz,1H),7.33(d,J=9.7Hz,1H),7.13(dd,J=8.8,2.4Hz,1H),6.77(s,1H),6.65(dd,J=8.5,1.7Hz,1H),5.05(dd,J=12.8,5.3Hz,1H),4.53(t,J=7. 4Hz,1H),4.47(d,J=13.3Hz,2H),3.85(d,J=8.0Hz,1H),3.74(s,4H),2. 99(t,J=11.8Hz,2H),2.93–2.79(m,1H),2.63–2.51(m,2H),2.31(s,4H), 2.11(s,4H),2.06–1.95(m,1H),1.84(dd,J=40.7,16.8Hz,9H),1.63(dd,J=23.7,10.8Hz,2H),1.50(dd,J=22.8,10.0Hz,2H),1.16–1.01(m,2H).

[0213] 231: Synthesis of N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2-,2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0214] LC / MS(ESI+)calcdforC 44 H 47 ClFN9O6(M+H + )m / z,852.3;found,852.3.

[0215] 1HNMR (400MHz, CDCl3) δ8.10(s,1H),7.97(d,J=9.3Hz,1H),7.88(d,J=8.1Hz,1H),7.56(d,J=8.7Hz,1H),7.36(d,J=10 .8Hz,1H),7.00(d,J=2.4Hz,1H),6.98(d,J=9.8Hz,1H),6.86(dd,J=8.8,2.4Hz,1H),6.81(d,J=7.5Hz,1H),4.91(dd,J =12.2,5.3Hz,1H),4.51(d,J=11.8Hz,2H),4.35–4.27(m,1H),4.05(dd,J=11.5,7.4Hz,1H),3.89(s,4H),3.04(s,2H), 2.92–2.74(m,4H),2.37(s,3H),2.22–2.09(m,8H),1.86(s,4H),1.64(d,J=21.2Hz,4H),1.46(dd,J=20.7,8.5Hz,3H).

[0216] 233: Synthesis of N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-((2-(2,6-dioxopiridine-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]non-7-yl)methyl)piperidin-1-yl)pyrazine-2-carboxamide

[0217]

[0218] LC / MS(ESI+)calcdforC 44 H 47 ClFN9O6(M+H + )m / z,852.3;found851.8.

[0219] 1HNMR (400MHz, CDCl3) δ8.82(s,1H),8.78–8.49(m,1H),7.95(s,1H),7.56(d,J=8.7Hz,1H),7.47(d,J=10.8Hz,1H),7.38(d,J= 6.6Hz,2H),6.99(d,J=2.0Hz,1H),6.85(dd,J=8.7,2.1Hz,1H),4.93(dd,J=12.1,5.3Hz,1H),4.45(d,J=13.2Hz,2H),4.32(d, J=10.0Hz,1H),4.03(d,J=7.6Hz,1H),3.24(s,2H),3.17(s,5H),3.03–2.92(m,2H),2.87(d,J=11.0Hz,1H),2.78(dd,J=23.8, 12.5Hz,2H),2.53(s,2H),2.18(s,5H),2.05–1.84(m,8H),1.66(d,J=10.1Hz,2H),1.52–1.40(m,2H),1.26(t,J=10.7Hz,3H).

[0220] 241: Synthesis of N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2-,2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyrazine-3-carboxamide

[0221] 1. Compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyrazine-3-carboxamide

[0222] Compound 6-chloro-N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyrazin-3-carboxamide (300 mg, 0.77 mmol), 4-hydroxymethylpiperidine (265 mg, 2.30 mmol), potassium carbonate (530 mg, 3.83 mmol), and 5 mL of DMF were sequentially added to the reactor. Under nitrogen protection, the reaction was carried out at 80 °C for 3 hours, and the reaction endpoint was determined by TLC. After confirming the completion of the reaction, the reaction solution was extracted three times with water and ethyl acetate. The ethyl acetate layers were combined and washed three times with saturated brine. The solutions were dried and evaporated to dryness. Column chromatography was used to separate the solid compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyrazin-3-carboxamide (355 mg, 0.76 mmol), yield: 98%.

[0223] LC / MS(ESI+)calcdforC 24 H 27 ClN5O3(M+H + )m / z,470.2;found,470.2.

[0224] 2. Synthesis of compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-((3aR,5R,6aS)-5-((5-(2-(2,6-dioxoperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)hexahydropyrrole[3,4-c]pyrrole-2(1H)-yl)methyl)hexahydrocyclopentan[c]pyrrole-2(1H)-yl)pyrazin-3-carboxamide

[0225] The compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyrazin-3-carboxamide (70 mg, 0.15 mmol) was dissolved in 2 mL of DCM, followed by the addition of DMP (76 mg, 0.18 mmol). The mixture was stirred at room temperature for 2 h. The reaction endpoint was determined by TLC. The reaction mixture was filtered, and the filter cake was washed with DCM. The filtrate was evaporated to dryness to obtain the crude product. The crude product was dissolved in 2-(2,6-dioxadiazin-3-yl)-5-fluoro-6-(hexahydropyrrolo[3,4-c]pyrrolo-2(1H)-yl)isoindol-1,3-dione (68 mg, 0.18 mmol) and 2 mL of LCM / MeOH (1:1). Glacial acetic acid (18 mg, 0.30 mmol) was added and the mixture was stirred at room temperature for 30 min. Then, sodium triacetylborohydride (126 mg, 0.60 mmol) was added and the mixture was stirred at room temperature for 2 h. The reaction endpoint was determined by TLC. A saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted three times with dichloromethane. The extracts were combined, dried, and then evaporated to dryness. Pre-TLC separation yielded a white solid compound, N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-((3aR,5R,6aS)-5-((5-(2-(2,6-dioxoperidin-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)hexahydropyrrole[3,4-c]pyrrole-2(1H)-yl)methyl)hexahydrocyclopentan[c]pyrrole-2(1H)-yl)pyrazin-3-carboxamide (86 mg, 0.10 mmol). Yield: 69%.

[0226] LC / MS(ESI+)calcdforC 45 H 48 ClN8O6(M+H + )m / z,834.3;found,834.3.

[0227] 1HNMR(400MHz, CDCl3)δ8.79(s,1H),7.98(s,1H),7.64(d,J=8.2Hz,1H),7.57(d,J=8 .7Hz,1H),7.48(d,J=8.1Hz,1H),7.01(d,J=2.0Hz,1H),6.86(dd,J=8.7,2.0Hz,1H) ,6.78(s,1H),6.52(d,J=8.3Hz,1H),4.93(dd,J=12.0,5.2Hz,1H),4.93(dd,J=12.0 ,5.2Hz,1H),4.46(d,J=12.8Hz,2H),4.32(t,J=9.9Hz,1H),4.01(d,J=7.6Hz,1H),3. 75(s,4H),3.08–2.96(m,3H),2.82–2.75(m,3H),2.40(s,4H),2.15(dd,J=25.3,8.7 Hz,7H),1.91(d,J=22.6Hz,6H),1.69(dd,J=22.2,10.6Hz,2H),1.48(dd,J=22.2,10. 9Hz, 2H), 1.23(d, J = 18.1Hz, 2H). 267: N-((1r, 4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-2-(4-((2-(2,6-dioxadiazin-3-yl)-1,3-dioxadiazindoline-5-yl)-2,7-diazospiro[3.5]non-7-yl)methyl)piperidin-1-yl)pyrimidin-5-carboxamide

[0228]

[0229] LC / MS(ESI+)calcdfor:C 44 H 48 ClN9O6(M+H + )m / z,834.3;found,834.3.

[0230] 1HNMR (400MHz, DMSO-d6) δ11.08(s,1H),8.74(s,2H),8.11(d,J=7.4Hz,1H),7.86(d,J=8.7Hz,1H),7.63(d,J=8.2Hz,1H),7.38( d,J=2.4Hz,1H),7.14(dd,J=8.8,2.4Hz,1H),6.77(d,J=2.0Hz,1H),6.64(dd,J=8.4,2.1Hz,1H),5.05(dd,J=12.9,5.4Hz,1H),4. 72(d,J=12.8Hz,2H),4.59-4.48(m,1H),3.74(s,5H),3.01-2.81(m,3H),2.55(d,J=6.5Hz,1H),2.31(s,3H),2.11(t,J=6.6Hz,4H ),2.03-1.95(m,2H),1.91(t,J=6.3Hz,2H),1.77(q,J=8.3,5.7Hz,6H),1.56-1.42(m,4H),1.24-1.13(m,2H),1.07-0.93(m,2H).

[0231] 268:N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-2-(4-((2-(2,6-dioxopiridine-3-yl)-6-fluoro-1,3-dioxoisoindoline-5-yl)-2,7-diazospiro[3.5]non-7-yl)methyl)piperidin-1-yl)pyrimidine-5-carboxamide

[0232]

[0233] LC / MS(ESI+)calcdfor:C 44 H 47 ClFN9O6(M+H + )m / z,852.3;found852.3.

[0234] 1HNMR (400MHz, DMSO-d6) δ11.10(s,1H),8.73(s,2H),8.11(d,J=7.5Hz,1H),7.88-7.84(m,1H),7.59(d,J=11.2Hz,1H),7.39(d, J=2.4Hz,1H),7.14(dd,J=8.8,2.5Hz,1H),6.89(d,J=7.6Hz,1H),5.06(dd,J=12.8,5.4Hz,1H),4.71(d,J=12.8Hz,2H),4.54(dd ,J=9.6,4.6Hz,1H),3.94-3.72(m,5H),2.99-2.79(m,3H),2.30(s,3H),2.10(q,J=7.2Hz,4H),2.04-1.97(m,1H),1.95-1.87(m, 2H),1.77(q,J=5.5,4.9Hz,6H),1.57-1.41(m,4H),1.20(d,J=21.9Hz,3H),1.01(q,J=11.4,10.6Hz,2H),0.84(d,J=7.2Hz,1H).

[0235] 274:N-((1r,4r)-4-(3-bromo-4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2-,2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0236]

[0237] LC / MS(ESI+)calcdforC 44 H 47 BrFN9O6(M+H + )m / z880.3; found880.3. 1HNMR (400MHz, DMSO-d6) δ11.08(s,1H),8.58(d,J=8.2Hz,1H),7.81(t,J=9.4Hz,2H),7.63(d,J=8.3Hz,1H),7.49(d,J=2.4Hz,1H),7. 33(d,J=9.7Hz,1H),7.16(dd,J=8.8,2.5Hz,1H),6.77(d,J=2.1Hz,1H),6.64(dd,J=8.4,2.1Hz,1H),5.05(dd,J=12.9,5.4Hz,1H),4. 58-4.41(m,3H),3.90-3.82(m,1H),3.75(s,4H),3.08-2.80(m,4H),2.64-2.53(m,2H),2.21(s,2H),2.14-2.06(m,2H),2.00(ddd,J= 13.0,5.5,3.4Hz,1H),1.89(d,J=11.7Hz,4H),1.81(d,J=11.4Hz,6H),1.71-1.43(m,5H),1.22(s,1H),1.10(dd,J=18.1,6.7Hz,2H).

[0238] 275:N-((1r,4r)-4-(4-cyano-3-methylphenoxy)cyclohexyl)-6-(4-((2-(2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0239]

[0240] LC / MS(ESI+)calcdforC 45 H 51 N9O6(M+H + )m / z814.4;found814.4.

[0241] 1HNMR (400MHz, DMSO-d6) δ11.08(s,1H),8.58(d,J=8.3Hz,1H),7.80(d,J=9.5Hz,1H),7.64(dd,J=8.5,6.7Hz,2H),7.32(d,J=9.7Hz,1H) ,7.04(d,J=2.4Hz,1H),6.93(dd,J=8.6,2.5Hz,1H),6.77(d,J=2.1Hz,1H),6.64(dd,J=8.5,2.1Hz,1H),5.05(dd,J=12.9,5.4Hz,1H),4 .45(t,J=10.9Hz,3H),3.86(d,J=9.0Hz,1H),3.74(s,4H),2.99(t,J=12.4Hz,2H),2.89-2.81(m,1H),2.43(s,3H),2.32(s,3H),2.11(t ,J=9.2Hz,3H),2.00(dd,J=8.9,4.3Hz,1H),1.91(s,6H),1.76(q,J=5.4Hz,5H),1.67-1.44(m,5H),1.22(s,1H),1.10(t,J=11.9Hz,2H).

[0242] 276:N-((1r,4r)-4-(4-cyano-3-methoxyphenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0243]

[0244] LC / MS(ESI+)calcdforC 45 H 51 N9O7(M+H + )m / z830.3; found830.3.

[0245] 1HNMR (400MHz, DMSO-d6) δ11.08(s,1H),8.58(d,J=8.2Hz,1H),7.80(d,J=9.5Hz,1H),7.62(dd,J=10.5,8.7Hz,2H ),7.33(d,J=9.7Hz,1H),6.78(d,J=2.1Hz,1H),6.72(dd,J=6.3,2.4Hz,2H),6.65(dd,J=8.4,2.1Hz,1H),5.05(dd ,J=12.8,5.4Hz,1H),4.49(dd,J=13.3,8.9Hz,3H),3.89(s,4H),3.75(s,4H),3.08-2.80(m,4H),2.12(d,J=12.9H z,5H),2.06-1.96(m,2H),1.90(d,J=7.6Hz,4H),1.78(s,5H),1.67-1.42(m,5H),1.23(s,2H),1.18-1.02(m,2H).

[0246] 279: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-((2-(2-(2,6-dioxopiridine-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspirocyclo[3.5]nonane-7-yl)methyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0247]

[0248] 1,3-(methylthio)-1,2,4-triazine-6-carboxylic acid methyl ester

[0249] It was prepared from 3-amino-6-bromo-1,2,4-triazine using a literature method (PCTInt.Appl., 2015182712).

[0250] 3-Amino-1,2,4-triazine-6-carboxylic acid methyl ester. Then, using this as a starting material, a pale yellow solid of 3-(methylthio)-1,2,4-triazine-6-carboxylic acid methyl ester was prepared by a method described in the literature (PCTInt. Appl., 2015 181539). MS: calcd. for C6H7N3O2S[M+H] + :

[0251] 186.0; found: 186.3. 1 HNMR (400MHz, CDCl3): δ8.95(s,1H),4.08(s,3H),2.73(s,3H).

[0252] 2. Methyl 3-(4-(hydroxymethyl)piperidin-1-yl)-1,2,4-triazine-6-carboxylic acid ester

[0253] Methyl 3-(methylthio)-1,2,4-triazine-6-carboxylic acid ester (5.0 g, 27.00 mmol) was dissolved in 200 mL of dichloromethane, and m-chloroperoxybenzoic acid (10.7 g, 62.09 mmol) was added in portions. The mixture was stirred at room temperature for 4 h. Triethylamine (10.9 g, 107.99 mmol) was added, followed by 4-piperidinemethanol (9.3 g, 80.99 mmol). The mixture was stirred overnight at room temperature. Water and dichloromethane were added, and the pH was adjusted to 8-9 with sodium carbonate. The mixture was extracted, and the organic layer was washed with sodium thiosulfate, dried, evaporated to dryness, and purified by silica gel column chromatography. The compound methyl 3-(4-(hydroxymethyl)piperidin-1-yl)-1,2,4-triazine-6-carboxylic acid ester (2.0 g, 7.93 mmol) was given. Yield: 29.9%.

[0254] LC / MS(ESI+)calcdforC 11 H 17 N4O3 + ([M+H)) + )m / z:253.1;found253.1.

[0255] 3. Synthesis of compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-hydroxymethyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0256] Methyl 3-(4-(hydroxymethyl)piperidin-1-yl)-1,2,4-triazine-6-carboxylic acid ester (100.0 mg, 0.39 mmol) and 4-(((1r,4r)-4-aminocyclohexyl)oxy)-2-chlorobenzonitrile (149.1 mg, 0.59 mmol) were dissolved in 2 mL of methanol and 0.3 mL of triethylamine and heated to reflux for 36 h. After cooling to room temperature, the solution was evaporated to dryness and purified by silica gel column chromatography. The compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-hydroxymethyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide (72.0 mg, 0.15 mmol) was obtained.

[0257] mmol). Yield 38.5%. LC / MS (ESI+) calcd for C 23 H 28 ClN6O3 + ([M+H)) + )m / z:471.2;found471.1.

[0258] 4. Synthesis of compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-formylpiperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0259] N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-hydroxymethyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide (72.0 mg, 0.15 mmol) was dissolved in 5 mL of dichloromethane, and Dess-Martin reagent (86 mg, 0.21 mmol) was added.

[0260] (mmol). Stir at room temperature for 4 h. Add dichloromethane and water for extraction, wash the organic layer with saturated brine, dry, and evaporate to dryness.

[0261] The compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-formylpiperidin-1-yl)-1,2,4-triazine-6-carboxamide (80 mg, 0.15 mmol) was obtained. The yield was 100.0%.

[0262] LC / MS(ESI+)calcdforC 23 H 26 ClN6O3 + ([M+H)) + )m / z:469.2;found469.3.

[0263] 5. Compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-((2-(2,6-dioxopiridine-3-yl)

[0264] -1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]non-7-yl)methyl)piperidin-1-yl)-1,2,4-triazine-6-carboxyl

[0265] Amine Synthesis

[0266] N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-formylpiperidin-1-yl)-1,2,4-triazin-6-carboxamide (80 mg, 0.15 mmol), 2-(2,6-dioxadipinidin-3-yl)-5-(2,7-diazaspiro[3.5]non-2-yl)isoindoline-1,3-dione (57.0 mg, 0.15 mmol) were dissolved in 2 mL of dichloromethane, 1 drop of acetic acid was added, and finally sodium triethoxyborohydride (126.0 mg, 0.60 mmol) was added. The mixture was stirred overnight at room temperature. The mixture was extracted with dichloromethane and water, the organic layer was washed with saturated brine, dried, and evaporated to dryness to prepare a large plate for purification. The target compound (56 mg, 0.06 mmol) was obtained. The yield was 44.9%.

[0267] LC / MS(ESI+)calcdforC 43 H 48 ClN 10 O6([M+H] + )m / z:835.3;found835.3.

[0268] 1 HNMR (400MHz, CDCl3) δ8.82(s,1H),8.15(s,1H),7.65(d,J=8.3Hz,1H),7.57(t,J=8.6

[0269] Hz,2H),7.00(d,J=2.3Hz,1H),6.85(dd,J=8.7,2.4Hz,1H),6.78(d,J=1.9Hz,1H),6.51(dd,J=8.3,1.9Hz,1H),4.94(dd,J=12.2,5.3Hz,3H ),4.31(t,J=9.9Hz,1H),4.13–4.00(m,1H),3.75(s,4H),3.06(s,2H),2.93–2.66(m,4H),2.45(s,3H),2.35–2.07(m,8H),1.96(s,8H),1.72

[0270] –1.63(m,2H),1.44(dd,J=17.2,8.6Hz,2H).

[0271] 417: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-((2-(2,6-dioxadiazin-3-yl)-6-fluoro-1-oxoisoindoline-5-yl)-2,7-diazaspirocyclic[3.5]non-7-yl)methyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0272] LC / MS (ESI+) Calcd for C 43 H 48 FClN 10 O5(M+H + )m / z,839.3;found839.3. 1 HNMR (400MHz, CDCl3) δ8.82(s,1H),8.09(s,1H),7.57(dd,J=8.5,5.4Hz,2H),7.40(d,J=11.3Hz,1H),7.00(d ,J=2.4Hz,1H),6.85(dd,J=8.8,2.4Hz,1H),6.39(d,J=7.6Hz,1H),5.18(dd,J=13.2,5.2Hz,1H),4.33(dd,J= 18.8,12.9Hz,2H),4.10–4.00(m,1H),3.83(s,3H),3.06(s,2H),2.92–2.75(m,2H),2.58(s,3H),2.45–2.27( m,4H),2.25–2.12(m,5H),2.09(s,2H),1.99(s,5H),1.72–1.62(m,2H),1.52–1.41(m,2H),1.32–1.20(m,3H).

[0273] 419:N-((1r,4r)-4-(3-bromo-4-cyanophenoxy)cyclohexyl)-3-(4-((2-(2-(2,6-dioxanil-3-

[0274] (3.5)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonane-7-yl)methyl)piperidin-1-yl)-1,2,4-triazol-6-carboxamide

[0275]

[0276] LC / MS (ESI+) for C 43 H 48 BrN 10 O6[M+H] + m / z879.

[0277] 420:N-((1r,4r)-4-(4-cyano-3-trifluoromethyl-phenoxy)cyclohexyl)-3-(4-((2-(2-(2,6-dioxoperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonane-7-yl)methyl)piperidin-1-

[0278] 1,2,4-triazine-6-carboxamide

[0279]

[0280] LC / MS (ESI+) for C 44 H 48 F3N 10 O6[M+H] + m / z869.

[0281] The following experimental examples demonstrate the beneficial effects of the present invention.

[0282] Experimental Example 1: Inhibitory Activity of the Compounds of the Present Invention on the Proliferation of Prostate Cancer Cells

[0283] (1) Experimental materials and instruments:

[0284] LNCaP / ARcellline (provided by Sichuan Kangcheng Biotechnology Co., Ltd.)

[0285] Fetal bovine serum FBS (Gibco, Cat. No. 10099-141)

[0286] 0.01MPBS(Biosharp,Cat.No.162262)

[0287] RIPM1640 medium (Hyclone, Cat. No. 308090.01)

[0288] Penicillin-Streptomycin (Hyclone, Cat. No. SV30010)

[0289] Cellcountingkit-8 reagent kit (SignalwayAntibody, Cat. No. CP002)

[0290] Dimethyl sulfoxide (DMSO) (Sigma, Cat. No. D5879)

[0291] Centrifuge tube, 15ml (ExcellBio, Cat. No. CS015-0001)

[0292] Cell Culture Dish (ExcellBio, Cat. No. CS016-0128)

[0293] 96-well cell culture cluster (Corning, Cat. No. 3599)

[0294] Microplate reader (Thermo Multiskan MK3)

[0295] (2) Experimental methods:

[0296] a. Buffer preparation

[0297] Cell culture medium: RIPM1640 medium, 10% FBS, 1% PenStrep;

[0298] PBS buffer: Dissolve PBS powder in 2L of ultrapure water and sterilize.

[0299] b. Experimental steps:

[0300] 1) LNCaP / AR cells were passaged in cell culture medium. Cells in good growth condition were seeded into 96-well plates at a density of 80 μL per well, with 1000 cells per well. The cells were then cultured overnight at 37°C in a 5% CO2 cell incubator.

[0301] 2) Prepare a 10 mM stock solution of the test compound using dimethyl sulfoxide (DMSO). Just before use, dilute the solution 3-fold with DMSO, then perform serial dilutions of 3-fold to obtain 9 concentration gradients. Dilute each concentration of the compound 200-fold with culture medium (to ensure the DMSO concentration in the culture system is 0.1%). Perform duplicates for each concentration in two wells. Add 20 μL of the diluted compound to each cell culture well (final concentrations: 10 μM, 3.3 μM, 1.1 μM…), and gently vortex to mix. Also, set up 3 negative control wells with only cells and 3 blank control wells with only culture medium (add 20 μL of culture medium diluted 200-fold to each of the 6 wells).

[0302] c. Result detection:

[0303] 1) After 6 days of culture, add 10 μL CCK-8 to each well and continue culturing for 1 hour in a 37°C, 5% CO2 cell incubator.

[0304] 2) Measure the absorbance (OD value) at 450 nm using a multi-functional microplate reader.

[0305] 3) The data were analyzed using the Dose-response-inhibition equation in GraphPad Prism 5 software to obtain the IC. 50 value.

[0306] (3) Experimental results:

[0307] The compound of this invention can effectively inhibit the proliferation of LNCap / AR cells.

[0308] Experiment Example 2 The effect of the compounds of this invention on androgen receptor (AR) proteins was analyzed using enzyme-linked immunosorbent assay (ELISA). White degradation activity

[0309] Experimental materials:

[0310] Prostate cancer VCaP cells (ATCC, CRL-2876)

[0311] Fetal bovine serum treated with activated charcoal (Gibco, Cat. No. 12676-029)

[0312] Metribolone(R1881)(MacklinBiochemical,CAS.No.965-93-5)

[0313] Phosphate-buffered saline (PBS) 0.01 M PBS (Biosharp, Cat. No. 162262)

[0314] DMEM / HIGHGLUCOSE(Hyclone,Cat.No.SH30243.01)

[0315] Penicillin-Streptomycin(Hyclone,Cat.No.SV30010)

[0316] Dimethyl sulfoxide (DMSO) (Sigma, Cat. No. D5879)

[0317] Centrifuge tube, 15ml (ExcellBio, Cat. No. CS015-0001)

[0318] Cell Culture Dish (ExcellBio, Cat. No. CS016-0128)

[0319] Enzyme-linked immunosorbent assay (ELISA) kit TotalAndrogenReceptorSandwichELISAKit (CST, Cat. No. 12850C)

[0320] Experimental methods:

[0321] 1. Buffer solution preparation

[0322]

[0323] 2. Experimental steps:

[0324] 1) VCaP After passage of cells in cell culture medium, cells in good growth condition were seeded into 96-well plates at 80 μL per well, with 30,000 cells per well. The cells were then cultured at 37°C in a 5% CO2 cell incubator for 3 days until the cells adhered to the plate.

[0325] 2) Prepare a 10 mM stock solution of the test compound using dimethyl sulfoxide (DMSO). Dilute with cell culture medium immediately before use. Add 20 μL of the diluted compound to each cell culture well, creating eight concentration gradients (300, 100, 10, 3, 1, 0.3, 0.1, 0.03 nM). Perform one well for each concentration and gently vortex to mix. Also, set up negative control wells (culture medium only) and positive control wells (cells and DMSO only).

[0326] 3) After culturing the cells in the incubator for 16 hours, remove the culture medium and rinse the cells once with ice-cold 1XPBS.

[0327] 4) Discard the PBS, add 50 μL of ice-cold 1X cell lysis buffer to each well of the cell culture plate, incubate on ice for 15 minutes, and then vortex to mix.

[0328] 5) Add 115 μL of sample diluent to the new 96-well plate, and add 5 μL of cell lysate supernatant from the cell culture plate to the sample diluent. Shake well.

[0329] 6) Pipette 100 μL of diluted sample into an ELISA plate, seal the ELISA plate with plastic film, and incubate it in a 37°C incubator for 2 hours.

[0330] 7) Gently remove the membrane, add 200 μl of 1X washing buffer to each well, shake for 5 min, discard, and blot dry. Wash 4 times.

[0331] 8) Add 100 μl of freshly prepared detection antibody (green) to each well of the ELISA plate. Seal with plastic film and incubate at 37°C for 1 hour.

[0332] 9) Gently remove the gel membrane, discard the detection antibody, blot dry, add 200 μl of 1X wash buffer to each well, vortex for 5 min, discard, and blot dry. Wash 4 times.

[0333] 10) Add 100 μl of freshly prepared HRP-labeled secondary antibody (red) to each well of the ELISA plate. Seal with plastic film and place in...

[0334] Incubate at 37°C for 30 minutes.

[0335] 11) Gently remove the gel membrane, discard the secondary antibody, pat dry, add 200 μl of 1X wash buffer to each well, shake for 5 min, discard, and pat dry.

[0336] Wash 4 times.

[0337] 12) Add 100 μl of TMB substrate to each well of the ELISA plate and incubate at 37°C for 5 minutes.

[0338] 13) Add 100 μl of STOP solution to each well of the ELISA plate and gently shake for a few seconds.

[0339] 3. Result Reading

[0340] Wipe the bottom of each well with a lint-free cloth. Read the absorbance at 450 nm within 30 minutes after adding STOP solution. Using the formula: Residual AR% = 100 * (Detector well OD value - Blank control OD value) / (Positive control well OD value - Blank control well OD value), the data were analyzed using the Dose-response equation in GraphPadPrism 5 software to obtain the DC value. 50 D max value.

[0341] DC 50 This is the compound concentration required to degrade 50% of the target protein. (D) max It represents the percentage of the target protein that can be degraded to the maximum extent.

[0342] Experimental results:

[0343] The degradation activities of the compounds of the present invention against androgen receptors (AR) at a concentration of 100 nM are shown in Table 1, where % degradation refers to the percentage of AR that has been degraded. The degradation activities of the compounds of the present invention against androgen receptors (AR) at concentration gradients are shown in Table 2. In Table 2, DC... 50 This represents the concentration of the compound required to degrade the androgen receptor by 50%. (D) max The percentage of androgen receptors (ARs) that are most readily degraded.

[0344] It can be seen that the compounds of this invention can effectively degrade androgen receptors, and most of the compounds have an effect on the DCs of androgen receptors. 50 Below 0.3 μM, some even <10 nM; most compounds have an effect on the androgen receptor D... max The percentages are over 40%, and in some cases even exceed 80%.

[0345] Table 1. Degradation activity of compounds on androgen receptor (AR) at 100 nM concentration.

[0346] compound %degradation 197 A

[0347] A:>80%,B:79%-50%,C:49%-25%,D:<25%

[0348] Table 2. The effect of compounds on the degradation of androgen receptor (AR) DCs 50 and D max

[0349]

[0350]

[0351] DC 50 :A:<10nM,B:10-100nM,C:0.1-0.3μM,D:>0.3μM

[0352] D max :++++: >80%, +++: 80% - 60%, ++: 60% - 40%, +: <40%

[0353] Example 3: Western blotting assay to determine the effect of a compound on androgen receptor (AR) protein. of drop

[0354] Deactivation

[0355] Experimental materials:

[0356] Prostate cancer VCaP cells (ATCC, CRL-2876)

[0357] FBS (Gibco, Cat. No. 10099-141)

[0358] 0.01MPBS(Biosharp,Cat.No.162262)

[0359] Metribolone(R1881)(MacklinBiochemical,CAS.No.965-93-5)

[0360] DMEM / HIGHGLUCOSE(Hyclone,Cat.No.SH30243.01)

[0361] Penicillin-Streptomycin(Hyclone,Cat.No.SV30010)

[0362] Dimethyl sulfoxide (DMSO) (Sigma, Cat. No. D5879)

[0363] Centrifuge tube, 15ml (ExcellBio, Cat.No.CS015-0001)

[0364] Cell culture dish (ExcellBio, Cat. No. CS016-0128)

[0365] 6-wellcellculturecluster(Corning,Cat.No.3516)

[0366] Buffer solution RIPAlysate buffer (Beyotime, Cat. No. P0013B)

[0367] Protein Loding Buffer (Beyotime, Cat. No. P0015L)

[0368] BCA Protein Assay Kit (Beyotime, Cat. No. P0012)

[0369] SDS-PAGE Gel Preparation Kit (Chengdu Baihe Technology Co., Ltd., Cat. No. PG112)

[0370] Anti-β-ActinrabbitmAb (CST, Cat.No.4970)

[0371] Androgen receptor (D6F11)XPRabbitmAb (CST, Cat. No. 5153)

[0372] PeroxidaseAffinipure(HRP)GoatAnti-RabbitIgG(ZenBioscience, Cat. No. 511203)

[0373] TBST (Biosharp, Cat. No. BL601A)

[0374] ECL chemiluminescence reagent kit Beyotime, Cat. No. P0018)

[0375] Experimental methods:

[0376] 1. Buffer solution preparation

[0377]

[0378] 2. Experimental steps:

[0379] 1) VCaP After passage of cells in cell culture medium, cells in good growth condition were seeded into 12-well plates at 1 ml per well, with 500,000 cells per well, and cultured overnight at 37°C in a 5% CO2 cell incubator.

[0380] 2) Prepare a 10 mM stock solution of the test compound using dimethyl sulfoxide (DMSO). Dilute with DMSO 3 mM before use.

[0381] Add 2 μl of the diluted compound to each cell culture well (to ensure the DMSO concentration in the culture system is 0.1%). Perform two replicates for each concentration, and gently vortex to mix. Also include negative control wells (with an equal volume of DMSO) and positive control wells.

[0382] 3) After culturing for 16 hours, cells were lysed using RIPA cell lysis buffer, proteins were extracted, and protein concentration was measured using a BCA assay kit.

[0383] The protein loading buffer is 5x concentrated. After heating at 100°C for 5 minutes, the sample is stored at -20°C.

[0384] 4) Load 30 μg of protein per well onto a polyacrylamide gel for electrophoresis.

[0385] 5) The protein was transferred from the polyacrylamide gel to the PVDF membrane, blocked with 5% skim milk at room temperature for 1 hour, incubated overnight at 4°C with primary antibody (Androgen Receptor (D6F11)XPRabbitmAb and Anti-β-ActinRabbitmAb), washed three times with TBST solution for 10 minutes each time, incubated with secondary antibody (horseradish peroxidase-labeled goat anti-mouse IgG) at room temperature for 2 hours, and then washed three times with TBST solution for 10 minutes each time.

[0386] 3. Result Detection:

[0387] Finally, ECL developer was added for color development, and the images were photographed using an automated chemiluminescence analyzer. The images were then collected and analyzed.

[0388] Experimental results:

[0389] use Protein immunoblotting assay (Western Blot) method was used to detect... The degradation activity of the compounds of this invention against androgen receptors (AR) at a concentration of 100 nM is shown in Table 3, where % represents the percentage of AR that has been degraded. It can be seen that the compounds of this invention can effectively degrade androgen receptors.

[0390] Table 3. Degradation activity of the compounds of the present invention against AR

[0391] compound %degradation 158 A 230 A 231 A

[0392] A:>80%,B:79%-50%,C:49%-25%,D:<25%

[0393] Experimental Example 4: Metabolic stability experiment of the compounds of the present invention

[0394] 1. Materials and Instruments

[0395] Liquid chromatography system (Shimadzu), mass spectrometry system (API4000 instrument from AB Inc (Canada) with an ESI interface), chromatographic column (ACE Excel 3AQ 30 × 2.1 mm column), human liver drug-metabolizing enzyme (Corning, Cat. #452117), phosphate buffer, ultrapure water, MgCl2 solution, NADPH.

[0396] 2. Methods and Results

[0397] 10 μl of 20 mg / mL liver microsomes and 40 μl of 10 mM NADPH were added together to an incubation tube. The final concentrations of liver microsomes and NADPH were 0.5 mg / mL and 1 mM, respectively. A control group was prepared with an equal volume of ultrapure water but no NADPH. Then, 4 μl of either the control compound (verapamil) or the test compound at a concentration of 200 μM was added. The final concentration of the compound was 2 μM. At 0, 15, 30, 45, and 60 min of incubation, 50 μl of the reaction solution was taken out and 4 times the volume of ice-cold acetonitrile was added to terminate the reaction. The samples were centrifuged for 40 min (3220 g), and 100 μl of the supernatant was collected. 100 μl of ultrapure water was added to the supernatant and mixed well for LC-MS / MS analysis. Finally, the metabolic stability parameters of liver microsomes were calculated.

[0398] Experimental results show that the compounds of this invention exhibit good metabolic stability.

[0399] Experimental Example 5: Pharmacokinetics of the Compounds of the Invention

[0400] 1. Experimental materials and instruments:

[0401] LC-20AD High Performance Liquid Chromatography System (SHIMADZU Corporation, Japan)

[0402] API4000 triple quadrupole mass spectrometer (Applied Biosystems, USA)

[0403] PhenixWinnolin pharmacokinetic software (Version 6.3, Certara, USA)

[0404] High-speed refrigerated centrifuge (Thermo Fisher Scientific)

[0405] Analytical balance (Sartorius, SECURA 225D-1CN)

[0406] Laboratory animals: ICR mice (Chengdu Dashuo Laboratory Animal Co., Ltd.)

[0407] DMSO (Sigma)

[0408] CMC-Na (Chengdu Kelong Chemical)

[0409] Heparin (Chengdu Kelong Chemical)

[0410] 2. Experimental methods and results

[0411] Accurately weigh the analyte according to the prescribed dosage, add solvent to a final volume of 10 ml, and mix thoroughly by sonication. Prepare a solution with a concentration of 0.5 mg / ml. Take 0.2 ml of the prepared final solution and store it at -20℃ for concentration determination. Nine healthy adult ICR mice (20-30 g) were fasted overnight (with free access to water) and then administered the drug by gavage at a volume of 0.2 ml / 10 g. Blood samples of 0.1 ml were collected from the retro-orbital venous plexus before administration and at 0.5, 1, 2, 4, 6, 8, 12, and 24 h after administration. The plasma was separated by centrifugation at 4℃ for 5 min and stored at -20℃ for analysis. The concentration of the analyte in the plasma was then determined by LC / MS / MS.

[0412] Experimental results show that the compounds of this invention exhibit good pharmacokinetics.

[0413] In summary, this invention provides a class of compounds represented by formula (I) that can target and degrade androgen receptors in prostate cancer cells and inhibit their proliferation, while also exhibiting good metabolic stability and pharmacokinetic properties. The compounds of this invention show promising applications in the preparation of androgen receptor protein degradation-targeting chimeras and in the development of drugs for treating androgen receptor-regulated diseases (including prostate cancer, breast cancer, and Kennedy's disease).

Claims

1. A compound, or an isotope thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, said compound being selected from one of the following:

2. Use of the compound of claim 1, or its isotopic compounds, tautomers, or pharmaceutically acceptable salts thereof, in the preparation of protein degradation-targeting chimeras of androgen receptors.

3. The use according to claim 2, characterized in that: The protein degradation targeting chimera can target and / or bind to androgen receptors; or, the protein degradation targeting chimera can degrade and / or downregulate androgen receptors.

4. The use according to any one of claims 2-3, characterized in that: The protein degradation-targeting chimera is the active ingredient in drugs for treating diseases regulated by androgen receptors.

5. The use according to claim 4, characterized in that: The diseases mentioned are selected from prostate cancer, breast cancer, and Kennedy's disease.

6. A drug for treating diseases regulated by androgen receptors, characterized in that: The drug is a formulation made from the compound of claim 1, or its isotope, its tautomer, or its pharmaceutically acceptable salt as the active ingredient, plus pharmaceutically acceptable excipients.

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