Bifunctional chimera heterocyclic compound for targeted degradation of androgen receptor and application of bifunctional chimera heterocyclic compound

Abstract

The invention relates to a difunctional chimera heterocyclic compound for targeted degradation of an androgen receptor and application of the difunctional chimera heterocyclic compound, and particularly provides a compound shown in a formula (I), or an isotope compound of the compound, or an optical isomer of the compound, or a tautomer of the compound, or a pharmaceutically acceptable salt of the compound, or a prodrug of the compound, or a solvate of the compound, aRB is an androgen receptor recognition / combination part, L is a linking part, and U is a ubiquitin protease recognition / combination part; the three parts are connected through chemical bonds. The compound provided by the invention can degrade androgen receptors in prostate cancer cells in a targeted manner and inhibit proliferation of the prostate cancer cells, and also shows good metabolic stability and pharmacokinetic properties. The compound provided by the invention has a good application prospect in preparation of a protein degradation targeting chimera of an androgen receptor and in preparation of drugs for treating related diseases (including prostatic cancer, breast cancer and Kenildie disease) regulated and controlled by the androgen receptor. # imgabs0 #

Description

[0001] This application is a divisional application for the invention patent with application number 202010301601.1, application date April 16, 2024, and invention name “A class of bifunctional chimeric heterocyclic compounds targeting androgen receptor degradation and their uses”. Technical Field

[0002] The present invention belongs to the field of medicine, and in particular, relates to a class of bifunctional chimeric heterocyclic compounds for targeting androgen receptor degradation and uses thereof. Background Art

[0003] With the continuous growth and aging of the global population, the incidence of prostate cancer continues to increase. Currently, the main treatment method is androgen deprivation therapy. Androgen receptor (AR) belongs to the nuclear receptor family and is a ligand-dependent transcription factor. Abnormal regulation of the AR signaling pathway plays an important role in the occurrence and development of prostate cancer. Studies have shown that castration-resistant prostate cancer (CRPC) still depends on the role of AR. The androgen receptor contains 918 amino acids and has similar structures and functions to other nuclear receptors. It consists of three important domains, namely the DNA binding domain (DBD), the ligand binding domain (LBD) and the nitrogen-terminal binding domain (NTD). The DBD and LBD are connected by a hinge region. The LBD at the carbon end of AR is the site where AR binds to the ligand, which determines the specificity of the ligand binding to AR. The ligand binds to the LBD to activate AR. Two transcriptional activation domains have been identified in AR, namely activation function 1 (AF1) in the NTD domain and highly conserved hydrophobic pocket activation function 2 (AF2) in the LBD domain. Before 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 that can induce the degradation of target proteins. PROTACs are bifunctional molecules, including a small molecule compound that can bind to a target protein (protein of interest, POI), a linker group is introduced at a suitable position, and then connected to a small molecule compound that can bind to a ubiquitin protease. The resulting small molecule probe can bind to the target protein and the ubiquitin protease at the same time, thereby promoting the ubiquitination of the target protein. The multi-ubiquitinated protein can be recognized and degraded by the proteasome.

[0005] Using the PROTACs strategy, we prepared a protein degradation targeted chimera that can target and recognize / bind to the androgen receptor. It can regulate the level of androgen receptor through the intracellular ubiquitin-proteasome degradation system and induce androgen receptor degradation, thereby achieving the effect of treating prostate cancer and other related diseases regulated by the androgen receptor.

[0006] Therefore, developing a bifunctional chimeric molecule that can target and bind to the androgen receptor and effectively degrade the androgen receptor has good application prospects in the treatment of related diseases regulated by the androgen receptor. Summary of the invention

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

[0008] The present invention provides a compound represented by formula (I), or an isotopic compound thereof, or an optical isomer thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof, or a prodrug thereof, or a solvate thereof:

[0009]

[0010] Among them, 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 connected by chemical bonds;

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

[0012]

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

[0014] R w3 For cyano,

[0015] R w4 Selected from H, halogen, C 1-6 Alkyl or its halogenated product or its deuterated product, C 1-6 Alkoxy or its halogenated product or its deuterated product; said L is selected from the structure 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 is CF3; or, the halogen is chlorine or bromine.

[0019] Further, the ARB is selected from the following structures:

[0020]

[0021]

[0022] Furthermore, 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-mentioned compound, or its isotope compound, or its optical isomer, or its tautomer, or its pharmaceutically acceptable salt, or its prodrug, or its solvate in the preparation of androgen receptor protein degradation targeted chimera.

[0028] Furthermore, the protein degradation targeting chimera can target recognition and / or bind to androgen receptor.

[0029] Furthermore, the protein degradation targeting chimera is capable of degrading and / or down-regulating androgen receptor.

[0030] Furthermore, the protein degradation targeting chimera is an active ingredient of a drug for treating related diseases regulated by androgen receptor.

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

[0032] The present invention also provides a drug for treating diseases regulated by androgen receptors, wherein the drug is a preparation prepared with the above-mentioned compound, or its isotope compound, or its optical isomer, or its tautomer, or its pharmaceutically acceptable salt, or its prodrug, or its solvate as an active ingredient and a pharmaceutically acceptable excipient.

[0033] Experiments have shown that the compound of formula (I) provided by the present invention can target androgen receptors in prostate cancer cells and inhibit the proliferation of prostate cancer cells, while also showing good metabolic stability and pharmacokinetic properties. The compound of the present invention has good application prospects in the preparation of protein degradation targeted chimeras of androgen receptors, and in the preparation of drugs for treating related diseases regulated by androgen receptors (including prostate cancer, breast cancer, and Kennedy's disease).

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

[0035] "Isotope compound" refers to a compound in which one or more atoms in a compound are replaced by their corresponding isotopes. For example, a compound in which one or more hydrogens (H) are replaced by deuterium (D) or tritium (T); for example, a compound in which one or more carbons (C) are replaced by 12 Carbon 11 or carbon 13 The compound obtained after replacement.

[0036] "Pharmaceutically acceptable" means that a carrier, vehicle, diluent, excipient, and / or formed salt is generally chemically or physically compatible with the other ingredients that constitute a pharmaceutical dosage form and physiologically compatible with the receptor.

[0037] "Salt" refers to 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 (inner salts), and also includes quaternary ammonium salts, such as alkylammonium salts. These salts can be directly obtained in the final separation and purification of the compound. It can also be obtained by mixing the compound, or its stereoisomer, with a certain amount of acid or base appropriately (e.g., equivalent). These salts may form a precipitate in the solution and be collected by filtering, or be recovered after solvent evaporation, or be obtained by freeze drying after reaction in an aqueous medium. The salt described in the present invention can be a hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

[0038] “Solvate thereof” refers to a 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 the present invention, ARB is the androgen receptor recognition / binding portion and functions as a ligand of the androgen receptor in the compounds.

[0040] There are many ways to name the specific compounds of the present invention: (1) numerical numbering; (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)ureido)phenyl)amino)pentyl)oxy)acetamido)-3,3-dimethylbutanoyl)-5-(((S)-1-(4-(4-methylthiazol-5-yl))phenyl)ethyl)carbamoyl)pyrrolidin-3-yl acetate. Although the naming methods are different, each specific compound of the present invention can be uniquely identified based on its structure.

[0041] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0042] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. DETAILED DESCRIPTION

[0043] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are 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-tetramethylcyclobutyloxy)-2-chlorobenzonitrile

[0047]

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

[0049] The compound 2-methylnicotinate methyl ester (30.0 g, 199.0 mmol) was dissolved in dichloromethane (500 mL), and then m-chloroperbenzoic acid (68.7 g, 398.0 mmol) was added in batches. Stir at room temperature overnight, filter, and wash the filter cake with dichloromethane. Add 5% sodium sulfite solution (200 mL) to the filtrate and stir for 30 minutes, separate the liquids, and extract with dichloromethane (100 mL×3). The filtrates were combined, dried over anhydrous sodium sulfate, spin-dried, and purified by silica gel column chromatography (dichloromethane: methanol = 100: 1 to 20: 1) to obtain a white solid compound 3-(methoxycarbonyl)-2-methylpyridine-1-oxo (18 g, 107.8 mmol), with a yield of 54%. MS: calcd.forC8H9NO3[M+H] + :168.0; found:168.1.

[0050] 2. Compound 6-chloro-2-methylnicotinate

[0051] Compound 3-(methoxycarbonyl)-2-methylpyridine-1-oxo (36.5 g, 218.6 mmol) was added to phosphorus oxychloride (300 mL) in batches under an ice bath. Then the temperature was slowly raised and refluxed for 3 hours. The reaction solution was decompressed to remove most of the solvent, diluted with ethyl acetate (500 mL), and then 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, spin-dried, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 to 5:1) to obtain a white solid 6-chloro-2-methylnicotinate (8 g, 43.2 mmol). Yield: 20%. MS: calcd.forC8H8ClNO2[M+H] + :186.0; found:186.1.

[0052] 3. Compound 6-bromo-2-methylnicotinate

[0053] The compound 6-chloro-2-methylnicotinate (8.0 g, 43.2 mmol) was dissolved in acetonitrile (80 mL), and then trimethylsilyl bromide (19.8 g, 129.7 mmol) was added, and the reaction solution was heated under reflux overnight. The reaction solution was decompressed to remove the solvent, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30:1 to 10:1) to obtain a yellow solid 6-bromo-2-methylnicotinate (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-bromonicotinate

[0055] Compound 6-bromo-2-methylnicotinate (7.0 g, 30.6 mmol) and N-bromosuccinimide (8.16 g, 45.9 mmol) were added to carbon tetrachloride (100 mL), followed by azobisisobutyronitrile (566 mg, 3.06 mmol), and the reaction solution was refluxed overnight. The reaction solution was decompressed to remove the solvent, and the residue was chromatographed on a silica gel column (petroleum ether: ethyl acetate = 30:1 to 10:1) to obtain a crude product of 2-(bromomethyl)-6-bromonicotinate (A-1-5).

[0056] The crude product was dissolved in dichloromethane (60 mL), 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 stirred at room temperature overnight. The reaction solution was diluted with water (100 mL), extracted with dichloromethane (40 mL × 3), washed with saturated brine (50 mL × 3), the organic phases were combined and dried with anhydrous sodium sulfate, spin-dried, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30:1 to 10:1) to obtain a white solid 2-(bromomethyl)-6-bromonicotinoic acid methyl ester (6.0 g, 19.5 mmol), yield: 64%. MS: calcd.forC8H7Br2NO2[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), and then 1,3-tetramethylcyclobutanedione (50 g, 356.7 mmol) and sodium acetate (29.3 g, 356.7 mmol) were added. The reaction solution was heated to reflux for 2 hours. Ethanol and water were removed by rotary evaporation. Toluene (300 mL) was added to the residue and refluxed for 3 hours, then hot filtered, and the filter cake was washed with toluene (100 mL). The filtrate was dried to obtain 3-carbonyl-2,2,4,4-tetramethylcyclobutane oxime (32.0 g, 206.5 mmol) as a white solid, with a yield of 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), and then sodium borohydride (18.9 g, 500.2 mmol) was added in batches. The reaction solution was stirred at room temperature overnight. The reaction solution was controlled at 5°C and quenched with sodium hydroxide (2000 mL). Then it was extracted with ethyl acetate (700 mL × 3), the organic phases were combined and dried over sodium sulfate, and the white solid 3-hydroxy-2,2,4,4-tetramethylcyclobutanone oxime (100.8 g, 642.0 mmol) was obtained by spin drying, with a yield of 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. After the reaction solution was refluxed for 30 minutes, 15% sodium hydroxide (300 mL) was added and kept in reflux state. After the addition was completed, reflux was continued for 2 hours. After the reaction was completed, the filter cake was washed with tetrahydrofuran (100 mL × 3), and the filtrate was extracted with ethyl acetate (200 mL × 3). The organic phase was combined and washed with saturated brine (200 mL × 3), dried with sodium sulfate, and spin-dried to obtain a light yellow solid 3-aminotetramethylcyclobutanone (24.5 g, 171.3 mmol), with a yield of 77%. MS: calcd. for C8H 17 NO[M+H] + :144.1; found:144.1.

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

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

[0068] Dissolve tert-butyl 3-hydroxy-2,2,4,4-tetramethylcyclobutylcarbamate (25.0 g, 102.9 mmol) in tetrahydrofuran (250 mL), add sodium hydride (60% in mineral oil) (8.23 g, 205.8 mmol) in batches under ice bath and stir in ice bath for 30 minutes, then dissolve 2-chloro-4-fluorobenzonitrile (17.6 g, 113.2 mmol) in tetrahydrofuran (50 mL) and slowly add dropwise to the reaction. Heat the reaction solution to 60 ° C for 3 hours. Quench the reaction solution with water (300 mL) and extract with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine (200 mL × 3), dried with sodium sulfate, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 50:1 to 30:1) to obtain a white solid (1r, 3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutylcarbamic acid 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 Dissolve (1r, 3r)-3-(3-chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcyclobutylcarbamic acid tert-butyl ester (5.0 g, 13.2 mmol) in dichloromethane (50 mL), and add trifluoroacetic acid (50 mL) under ice bath. The reaction solution was stirred at room temperature for 1 hour, and the crude product of 4-((1r, 3r)-3-amino-2,2,4,4-tetramethylcyclobutoxy)-2-chlorobenzonitrile trifluoroacetate was obtained by spin drying, which was directly used in the next step. 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-tetramethylcyclobutyloxy)-2-chlorobenzonitrile

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

[0072] Other lactam intermediates were synthesized by methods similar to the above route.

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

[0074]

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

[0076]

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

[0078]

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

[0080] Dissolve tert-butyl 4-hydroxypiperidine-1-carboxylate (10.0 g, 49.7 mmol) in dichloromethane, then add 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 solution was stirred at room temperature overnight. The reaction solution was diluted with water (100 mL) and extracted with dichloromethane (50 mL × 3). The organic phases were combined and washed with saturated brine (50 mL × 3), dried with sodium sulfate, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10: 1 to 3: 1) to obtain white solid tert-butyl 4-(p-toluenesulfonyloxy)piperidine-1-carboxylate (13.2 g, 37.2 mmol), yield: 83%.

[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] Dissolve tert-butyl 4-(p-methylbenzenesulfonyloxy)piperidine-1-carboxylate (1.2 g, 3.4 mmol) in acetonitrile, then add 4-ethynylpiperidine hydrochloride (490 mg, 3.4 mmol), potassium carbonate (1.0 g, 7.5 mmol) and potassium iodide (113 mg, 0.68 mmol). The reaction solution was heated to reflux for 36 hours. The reaction solution was diluted with water (50 mL) and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (20 mL × 3), dried with sodium sulfate, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10: 1 to pure ethyl acetate) to obtain a white solid tert-butyl 4-ethynyl-1,4'-bipiperidine-1'-carboxylate (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 by a method similar to that of SM-L-1:

[0086] Intermediate SM-L-2: tert-butyl 3-(4-ethynylpiperidin-1-yl)azetidine-1-carboxylate

[0087]

[0088] 4-Ethynylpiperidine hydrochloride (2.0 g, 13.8 mmol) and tert-butyl 3-carboxylate 3-azetidinone (2.35 g, 13.8 mmol) were added.

[0089] Dissolve in 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 at room temperature overnight. Dilute the reaction mixture with water (50 mL) and extract with dichloromethane (30 mL × 3). Organic phase

[0090] The combined solution was 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)azetidine-1-carboxylate (1.0 g,

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

[0092] The syntheses of intermediates SM-L-4, SM-L-8, SM-L-9 and SM-L-10 were similar to those of SM-L-2.

[0093]

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

[0095]

[0096] Dissolve tert-butyl piperazine-1-carboxylate (3.3 g, 17.7 mmol), triethylamine (3.57 g, 35.4 mmol) and 3-chloro-3-methylbutyne (1.8 g, 17.7 mmol) in tetrahydrofuran (30 mL), and then add cuprous chloride (179 mg, 1.8 mmol). The reaction solution was stirred at room temperature for 10 minutes. The reaction solution was diluted with water (100 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined and washed with saturated brine (50 mL × 3), dried with sodium sulfate, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1 to 2:1) to obtain a colorless oil tert-butyl 4-(2-methyl-3-alkyn-2-yl)piperazine-1-carboxylate (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: tert-butyl 4-(2-methyl-3-yn-2-ylamino)piperidine-1-carboxylate (

[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), and then a solution of 3-chloro-3-methylbutyne (1.0 g, 10.0 mmol) in acetonitrile (10 mL) was added dropwise to the reaction solution. The reaction solution was stirred at room temperature for 1 hour. The reaction solution was diluted with water (100 mL), and ethyl acetate (50 mL ×

[0101] 3) Extraction. The organic phases were combined, 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 obtain a colorless oily substance, tert-butyl 4-(2-methyl-3-alkyn-2-ylamino)piperidine-1-carboxylate (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: tert-butyl 4-(4-ethynylphenyl)piperazine-1-carboxylate

[0103]

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

[0105] 4-Fluorobenzaldehyde (5.0 g, 40.3 mmol) and tert-butyl piperazine-1-carboxylate (7.5 g, 40.3 mmol) were dissolved in N,N-dimethylformamide (100 mL), and potassium carbonate (11.2 g, 80.6 mmol) was added. The reaction solution was stirred at 110 ° C for 2 days. The reaction solution was diluted with water (150 mL) and extracted with ethyl acetate (100 mL × 3). The organic phases were combined and washed with saturated brine (100 mL × 3), dried with sodium sulfate, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20: 1 to 5: 1) to obtain white solid tert-butyl 4-(4-formylphenyl) piperazine-1-carboxylate (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] Dissolve tert-butyl 4-(4-formylphenyl)piperazine-1-carboxylate (B-14-1) (2.9 g, 10.0 mmol) and dimethyl 1-diazo-2-oxopropylphosphonate (3.8 g, 20.0 mmol) in methanol (50 mL), and add potassium carbonate (5.5 g, 40 mmol) in an ice bath. The reaction solution is slowly heated to room temperature overnight. The reaction solution is spin-dried and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 50:1 to 15:1) to obtain white solid tert-butyl 4-(4-ethynylphenyl)piperazine-1-carboxylate (1.5 g, 5.2 mmol), yield: 54%. MS: calcd.

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

[0110] Intermediate SM-L-18: tert-butyl 4-((1s, 3s)-3-ethynylcyclobutyl)piperazine-1-carboxylate (SM-L-18Q) and tert-butyl 4-((1r, 3r)-3-ethynylcyclobutyl)piperazine-1-carboxylate (SM-L-18H)

[0111]

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

[0113] Dissolve methyl 3-oxocyclobutanecarboxylate (5.12 g, 40.0 mmol) and tert-butyl piperazine-1-carboxylate (8.9 g, 48.0 mmol) in 1,2-dichloroethane (100 mL), then add acetic acid (4.8 g, 80.0 mmol), and finally add sodium triacetoxyborohydride (21.2 g, 100 mmol). Stir the reaction solution at room temperature overnight. Dilute the reaction solution with water (100 mL) and extract with dichloromethane (50 mL × 3). The organic phases are combined, washed with saturated brine (50 mL × 3), dried with sodium sulfate, and chromatographed on a silica gel column (petroleum ether: ethyl acetate =

[0114] 10:1 to pure ethyl acetate) to give a white solid tert-butyl 4-(3-(methoxycarbonyl)cyclobutyl)piperazine-1-carboxylate (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] Dissolve tert-butyl 4-(3-(methoxycarbonyl)cyclobutyl)piperazine-1-carboxylate (6.0 g, 20.8 mmol) in tetrahydrofuran (120 mL), and add lithium aluminum tetrahydride (4.8 g, 80.0 mmol) in batches in an ice bath. The reaction solution was stirred in an ice bath for 1 hour. The reaction solution was diluted with tetrahydrofuran (100 mL), kept in an ice bath, and then water (4.8 mL) was added dropwise and stirred for 10 minutes, followed by the addition of 15% sodium hydroxide solution (4.8 mL) and continued stirring for 10 minutes, followed by the addition of water (14.4 mL) and continued stirring for 20 minutes, and finally anhydrous magnesium sulfate was added and stirred for 20 minutes. The reaction mixture was filtered, and the filtrate was dried to obtain a colorless oily substance tert-butyl 4-(3-(hydroxymethyl)cyclobutyl)piperazine-1-carboxylate (4.4 g, 14.8 mmol), with a yield of 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] Dissolve tert-butyl 4-(3-(hydroxymethyl)cyclobutyl)piperazine-1-carboxylate (4.0 g, 14.8 mmol) in dichloromethane (80 mL)

[0120] Then add 1,1',1'-(3-oxo-1λ5-1,2-phenyliodo-1(3H)-ylidene)acetoacetate (9.4 g, 22.2 mmol). The reaction solution is stirred at room temperature for 24 hours. Then filter and dry the filtrate to obtain tert-butyl 4-(3-formylcyclobutyl)piperazine-1-carboxylate.

[0121] The crude ester (5.3 g) was used directly in the next step. 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 tert-butyl 3-ethynylcyclobutyl)piperazine-1-carboxylate (SM-L-18H)

[0123] Dissolve tert-butyl 4-(3-formylcyclobutyl)piperazine-1-carboxylate (14.8 mmol, crude product from the previous step) and dimethyl 1-diazo-2-oxopropylphosphonate (5.68 g, 29.6 mmol) in methanol (100 mL), add potassium carbonate (10.2 g, 74.0

[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 organic phases were combined, washed with saturated brine (80 mL × 3), dried with sodium sulfate, and chromatographed on a silica gel column (petroleum ether: ethyl acetate =

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

[0126] found: 265.4. At the same time, off-white solid 4-((1r, 3r)-3-ethynylcyclobutyl)piperazine-1-carboxylic acid tert-butyl ester (SM-L-18H) (0.36 g, 1.4 mmol) was obtained, with a two-step yield of 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 tert-butyl 4-(4-(methoxycarbonyl)cyclohexyl)piperazine-1-carboxylate

[0131] Methyl 4-oxocyclohexylcarboxylate (10.0 g, 64.0 mmol) and tert-butyl piperazine-1-carboxylate (13.0 g, 70.0 mmol) were dissolved in 1,2-dichloroethane (200 mL), followed by acetic acid (7.6 g, 128.0 mmol), and finally sodium triacetoxyborohydride (34.0 g, 160 mmol). The reaction solution was stirred at room temperature overnight. The reaction solution was diluted with water (200 mL) and extracted with dichloromethane (100 mL×3). The organic phases were combined, 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 obtain tert-butyl 4-(4-(methoxycarbonyl)cyclohexyl)piperazine-1-carboxylate (9.6 g, 29.4

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

[0133] 2. Synthesis of tert-butyl 4-((1r,4r)-4-(hydroxymethyl)cyclohexyl)piperazine-1-carboxylate

[0134] Dissolve tert-butyl 4-(4-(methoxycarbonyl)cyclohexyl)piperazine-1-carboxylate (9.6 g, 29.4 mmol) in tetrahydrofuran (200 mL), and add lithium aluminum tetrahydride (2.23 g, 58.8 mmol) in batches in an ice bath. The reaction solution was stirred in an ice bath for 1 hour. The reaction solution was diluted with tetrahydrofuran (100 mL), kept in an ice bath, and then water (2.3 mL) was added dropwise and stirred for 10 minutes, followed by the addition of 15% sodium hydroxide solution (2.3 mL) and continued stirring for 10 minutes, followed by the addition of water (6.9 mL) and continued stirring for 20 minutes, and finally anhydrous magnesium sulfate was added and stirred for 20 minutes. The reaction mixture was filtered, the filtrate was spin-dried, and silica gel column chromatography (petroleum ether: ethyl acetate = 1:1

[0135] to ethyl acetate) to obtain a light yellow oily substance 4-((1r,4r)-4-(hydroxymethyl)cyclohexyl)piperazine-1-carboxylic acid tert-butyl ester (1.2 g, 4.0 mmol), yield: 14%. The configuration was determined by referring 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] At the same time, a light yellow oily substance tert-butyl 4-((1s,4s)-4-(hydroxymethyl)cyclohexyl)piperazine-1-carboxylate (3.0 g, 10.0

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

[0140] 3. Synthesis of tert-butyl 4-((1r,4r)-4-(formyl)cyclohexyl)piperazine-1-carboxylate

[0141] Dissolve tert-butyl 4-((1r,4r)-4-(hydroxymethyl)cyclohexyl)piperazine-1-carboxylate (1.2 g, 4.0 mmol) in dichloromethane (50 mL)

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

[0144] 4. Synthesis of Compound 4-((1r, 3r)-4-ethynylcyclohexyl)piperazine-1-carboxylic acid tert-butyl ester

[0145] Dissolve tert-butyl 4-((1r,4r)-4-(formyl)cyclohexyl)piperazine-1-carboxylate (4.0mmol, crude product from the previous step) and dimethyl 1-diazo-2-oxopropylphosphonate (7.2g, 6.0mmol) in methanol (50mL), and add potassium carbonate (1.1g, 8.0mmol) in an ice bath. Slowly warm the reaction solution to room temperature overnight. Dilute the reaction solution with water (50mL), add ethyl acetate (50mL×

[0146] 3) Extraction. The organic phases were combined, 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 obtain a light yellow solid 4-((1r, 3r)-3-ethynylcyclohexyl)piperazine-1-carboxylic acid tert-butyl ester (0.67 g, 2.3 mmol), with a two-step yield of 57%. MS: calcd.forC 17 H 28 N2O2[M+H] + :293.2; found:

[0147] 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).

[0148] The syntheses 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 were similar to SM-L-18.

[0149]

[0150] The following is an example of the preparation of a specific compound provided by the present invention: 131: Synthesis of N-((1r,4r)-4-(4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0151]

[0152] LC / MS (ESI+) calculation for C 41 H 41 ClN9O6 + (M+H + )m / z,800.4; found800.3.

[0153] 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),

[0154] 1.23-1.23(m,3H),1.14-1.03(m,3H).

[0155] 132: Synthesis of N-((1r,4r)-4-(4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxapiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0156]

[0157] LC / MS (ESI+) calculation for C 41 H 41 ClN9O6 + (M+H + )m / z,818.4; found818.3.

[0158] 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).

[0159] 135: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyrimidine-2-carboxamide

[0160]

[0161] Step 1: Add 2-(2,6-dioxopiperidin-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) to 10 mL DMSO. Heat to 135 °C and stir to react overnight. Cool to room temperature, add ethyl acetate and water to extract, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, and remove the solvent by rotary evaporation to obtain a crude product, which is separated and purified by Pre-TLC. Obtain the compound tert-butyl 2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoline-5-yl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (530 mg). Yield: 55%. LC / MS (ESI+) calculation for C 25 H 30 N4O6([M+H] + )m / z482.22; found483.1.

[0162] Step 2: Dissolve the compound tert-butyl 2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (530 mg, 1.09 mmol) in 10 mL of dichloromethane, add 4 mL of trifluoroacetic acid, and stir at room temperature for 3 h. TLC monitors the complete consumption of the raw materials, and removes the excess trifluoroacetic acid and solvent by rotary evaporation, and removes the residual trifluoroacetic acid by multiple rotary stripping with dichloromethane. 10 mL of dichloromethane and 3 mL of water were added again, and the system was then moved to an ice-water bath for cooling and stirring. A saturated sodium bicarbonate solution was then added dropwise to the system to adjust the pH of the system to about 9. After completion, the system was allowed to stand for stratification. The aqueous phase was washed with dichloromethane (3 mL*3). The organic layers were combined and then washed with brine, dried over anhydrous sodium sulfate, and the crude product was separated and purified by column chromatography to obtain the compound 2-(2,6-dioxopiperidin-3-yl)-5-(2,7-diazaspiro[3.5]non-2-yl)isoindole-1,3-dione (342 mg). Yield: 82%. LC / MS (ESI+) calculated for C 20 H 20 N4O4([M+H] + )m / z382.16; found383.1.

[0163] Step 3: Add NaH (360 mg, 9.0 mmol) to 5 mL DMF, then place the system in an ice-water bath to cool and stir, then add (1r, 4r)-4-aminocyclohexane-1-ol (455 mg, 3.0 mmol), stir for 10 min, and then add 2-chloro-4-fluorobenzonitrile (460 mg, 3 mmol). Naturally return to temperature and stir to react overnight. TLC monitoring shows that the raw material is consumed, ethyl acetate and water are added for extraction, the organic layer is washed with saturated brine, dried over anhydrous sodium sulfate, spin-dried, and separated and purified by Pre-TLC. Compound 4-((1r, 4r)-4-aminocyclohexyl)oxy)-2-chlorobenzonitrile (279 mg) is obtained. Yield: 62%. LC / MS (ESI+) calculated for C 13 H 15 ClN2O([M+H] + )m / z250.73; found251.1.

[0164] Step 4: Add compound 5-chloropyrimidine-2-carboxylic acid (79 mg, 0.50 mmol) and HATU (304 mg, 0.80 mmol) to 5 mL of dichloromethane, then place the system in an ice-water bath to cool and stir, add diisopropylethylamine (194 mg, 1.50 mmol) after 10 min, and then add 4-((1r, 4r)-4-aminocyclohexyl)oxy)-2-chlorobenzonitrile (150 mg, 0.50 mmol). After completion, remove the ice bath and stir at room temperature overnight. TLC monitoring shows that the raw material is consumed, add dichloromethane and water to extract, wash the organic layer with saturated brine, dry over anhydrous sodium sulfate, spin dry, and separate and purify by Pre-TLC. Obtain compound 5-chloro-N-((1r, 4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyrimidine-2-carboxamide (185 mg). Yield: 92%. LC / MS (ESI+) calculation for C 18 H 16 Cl2N4O2([M+H] + )m / z391.25; found391.1.

[0165] Step 5: Add 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) to 8 mL of dioxane. Heat to 115°C, reflux and stir to react overnight. Cool to room temperature, add ethyl acetate and water to extract, wash the organic phase with 0.05N HCl, then wash with saturated brine, dry over anhydrous sodium sulfate, and remove the solvent by rotary evaporation to obtain a crude product, which is separated and purified by Pre-TLC. Obtain compound 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+) calculation for C 24 H 28 ClN5O3([M+H] + )m / z469.97; found470.1.

[0166] 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, add dichloromethane (5 mL), and stir at room temperature to dissolve and clarify. Then place the system in an ice-water bath to cool and stir. When the temperature in the system drops to about 0°C, add Dess-Martin periodinant (72 mg, 0.17 mmol) to the system. After completion, allow the system to naturally warm to room temperature and stir to react. After 2 hours, TLC shows that the raw material is basically consumed, and the reaction is stopped. The system was filtered using diatomaceous earth, the filter cake was rinsed with dichloromethane in small amounts for multiple times, the filtrates were combined, and the solvent was removed by rotary evaporation to obtain a crude product, which was then separated and purified by Pre-TLC to obtain the compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-formylpiperidin-1-yl)pyrimidine-2-carboxamide (30 mg). Yield: 58%. LC / MS (ESI+) calculated for C 24 H 26 ClN5O3([M+H] + )m / z467.95; found468.1.

[0167] Step 7: Weigh N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-formylpiperidin-1-yl)pyrimidine-2-carboxamide (30 mg, 0.06 mmol) in a 25 mL single-necked round-bottom flask, add dichloromethane (3 mL), and stir at room temperature to dissolve and clarify. Then add 2-(2,6-dioxopiperidin-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. Then, add sodium triacetylborohydride (38 mg, 0.18 mmol) to the system, and let the system stir at room temperature for reaction. After 4 h, take a sample and spot plate, and monitor the complete consumption of raw materials by TLC. Stop stirring, add dichloromethane (15mL) and water (15mL) to the system, stir vigorously, and then stand to separate. The aqueous layer is back-extracted with dichloromethane (10mL*3), and the organic phases are combined, washed with water (10mL*2) and saturated brine (15mL) in sequence, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation to obtain a crude product, which is then separated and purified by Pre-TLC to obtain the compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-((6-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)-2,6-diazaspiro[3.4]octan-2-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide (21mg). 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.

[0168] 136: N-((1r,4r)-4-((6-cyano-5-(trifluoromethyl)pyridin-3-yl)oxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0169]

[0170] LC / MS(ESI+)forC 44 H 48 F3N 10 O6([M+H] + )m / z869.

[0171] 148: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-((2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyrimidine-2-carboxamide

[0172]

[0173] Step 1: Add compound 5-bromopyrimidine-2-carboxylic acid (0.80 g, 3.94 mmol) and HATU (2.41 g, 6.22 mmol) to 30 mL of dichloromethane, then place the system in an ice-water bath to cool and stir, add diisopropylethylamine (1.03 g, 7.97 mmol) after 10 min, and then add 4-((1r, 4r)-4-aminocyclohexyl)oxy)-2-chlorobenzonitrile (1.01 g, 3.99 mmol). After completion, remove the ice bath, stir at room temperature and react overnight. TLC monitoring shows that the raw material is consumed, add dichloromethane and water to extract, wash the organic layer with saturated brine, dry over anhydrous sodium sulfate, spin dry, and separate and purify by Pre-TLC. Obtain compound 5-bromo-N-((1r, 4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyrimidine-2-carboxamide (1.09 g). Yield: 63.5%. LC / MS (ESI+) calculated for C 18 H 16 BrClN4O2([M+H] + )m / z 434.01; found 435.1.

[0174] Step 2: Add 5-bromo-N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyrimidine-2-carboxamide (435mg, 1.0mmol), piperidin-4-ylmethanol (116mg, 1.0mmol), palladium acetate (15mg, 0.05mmol), diisopropylethylamine (35mg, 0.05mmol) and cesium carbonate (652mg, 2.0mmol) to 10mL dioxane. Heat to 110°C under nitrogen protection, reflux and stir to react overnight. Cool to room temperature, add ethyl acetate and water for extraction, wash the organic phase with 0.05NHCl, then with saturated brine, dry over anhydrous sodium sulfate, and remove the solvent by rotary evaporation to obtain a crude product, which is separated and purified by Pre-TLC. The compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-(hydroxymethyl)piperidin-1-yl)pyrimidine-2-carboxamide (191 mg) was obtained. Yield: 41%. LC / MS (ESI+) calculation for C 24 H 28 ClN5O3([M+H] + )m / z 469.97; found 470.1.

[0175] Step 3: 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, add dichloromethane (5 mL), and stir at room temperature to dissolve and clarify. Then place the system in an ice-water bath to cool and stir. When the temperature in the system drops to about 0°C, add Dess-Martin periodinant (72 mg, 0.17 mmol) to the system. After completion, allow the system to naturally warm to room temperature and stir to react. After 2 hours, TLC shows that the raw material is basically consumed, and the reaction is stopped. The system was filtered using diatomaceous earth, and the filter cake was rinsed with dichloromethane in small amounts for multiple times. The filtrates were combined and the solvent was removed by rotary evaporation to obtain a crude product, which was then separated and purified by Pre-TLC to obtain the compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-formylpiperidin-1-yl)pyrimidine-2-carboxamide (30 mg). Yield: 58%. LC / MS (ESI+) calculated for C 24 H 26 ClN5O3([M+H] + )m / z 467.95; found 468.1.

[0176] Step 4: Weigh N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-formylpiperidin-1-yl)pyrimidine-2-carboxamide (30 mg, 0.06 mmol) in a 25 mL single-necked round-bottom flask, add dichloromethane (3 mL), and stir at room temperature to dissolve and clarify. Then add 2-(2,6-dioxopiperidin-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. Then, add sodium triacetylborohydride (38 mg, 0.18 mmol) to the system, and let the system stir at room temperature to react. After 4 h, take a sample and spot plate, and monitor the complete consumption of the raw materials by TLC. Stop stirring, add dichloromethane (15mL) and water (15mL) to the system, stir vigorously, and then stand to separate. The aqueous layer is back-extracted with dichloromethane (10mL*3), and the organic phases are combined, washed with water (10mL*2) and saturated brine (15mL) in turn, dried over anhydrous sodium sulfate, and the solvent is removed by rotary evaporation to obtain a crude product, which is then separated and purified by Pre-TLC to obtain the compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-((2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindole-5-yl)-2,7-diazaspiro[3.5]non-7-yl)methyl)piperidin-1-yl)pyrimidine-2-carboxamide (21mg). 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.

[0177] 149: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyrimidine-2-carboxamide

[0178]

[0179] 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.

[0180] 151: N-((1r,4r)-4-(4-cyano-3-(trifluoromethyl)phenoxy)cyclohexyl)-6-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0181]

[0182] 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.

[0183] 152: N-((1r,4r)-4-(4-cyano-3-(trifluoromethyl)phenoxy)cyclohexyl)-6-(4-((2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0184]

[0185] 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.

[0186] 153: N-((1r,4r)-4-(4-cyano-3-(methoxy-d3)phenoxy)cyclohexyl)-6-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0187]

[0188] 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.

[0189] 154: N-((1r,4r)-4-(4-cyano-3-ethoxyphenoxy)cyclohexyl)-6-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0190]

[0191] 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.

[0192] 155: N-((1r,4r)-4-(4-cyano-3-(2-methoxyethoxy)phenoxy)cyclohexyl)-6-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0193]

[0194] 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.

[0195] 158: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-((2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0196] LC / MS(ESI+)CalcdforC 43 H 46 FCl 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).

[0197] 197: N-((1r,4r)-4-(4-cyano-3-(methoxy-d3)phenoxy)cyclohexyl)-3-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0198]

[0199] LC / MS (ESI+) calculation for C 44 H 48 D3N 10 O7(M+H + )m / z,834.4; found835.

[0200] 230: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0201]

[0202] 1. Compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyridazine-3-carboxamide

[0203] Compound 6-chloro-N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyridazine-3-carboxamide (1.00 g, 2.56 mmol), 4-hydroxymethylpiperidine (883 mg, 7.67 mmol), potassium carbonate (1.77 g, 12.78 mmol) and DMF 12 mL were added to the reactor in sequence, nitrogen was protected, and the reaction was carried out at 80°C for 3 hours. The reaction end point was determined by TLC. After confirming the completion of the reaction, the reaction solution was extracted with water and ethyl acetate for 3 times, the ethyl acetate layers were combined, and washed with saturated brine for 3 times, dried and spin-dried, and separated by column chromatography to obtain a white solid compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyridazine-3-carboxamide (1.09 g, 2.32 mmol), with a yield of 91%.

[0204] LC / MS (ESI+) calculation for C 24 H 27 ClN5O3(M+H + )m / z,470.2;found,470.2.

[0205] 2. Compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-formylpiperidin-1-yl)pyridazine-3-carboxamide

[0206] The compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyridazine-3-carboxamide (95 mg, 0.20 mmol) was dissolved in 2 mL DCM and then Dess-Martin reagent (128 mg, 0.30 mmol) was added. The mixture was stirred at room temperature for 1 hour and the reaction endpoint was confirmed by TLC. After the reaction was completed, the filter cake was filtered and washed with dichloromethane, and the filtrate was extracted with sodium sulfite solution, dried and spin-dried to obtain a light yellow solid, which was directly used for the next step.

[0207] LC / MS (ESI+) calculation for C 24 H 25 ClN5O3(M+H + )m / z,467.2;found,467.9.

[0208] 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-diazaspiro[3.5]nonane-2-carboxylic acid tert-butyl ester

[0209] The crude compound 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, and tert-butyl 2,7-diazaspiro[3.5]nonane-2-carboxylate (55 mg, 0.24 mmol) and glacial acetic acid (24 mg, 0.40 mmol) were added in sequence, stirred at room temperature for 30 minutes, and then sodium triacetylborohydride (128 mg, 0.61 mmol) was added, and reacted at room temperature for 2 hours. DCM and water were added for extraction, and the organic layer was washed with brine, dried over anhydrous sodium sulfate, spin-dried, 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-diazaspiro[3.5]nonane-2-carboxylic acid tert-butyl ester (113.0 mg, 0.17 mmol) was obtained. Yield: 85.0%.

[0210] LC / MS (ESI+) calculation for C 36 H 47 ClN7O4(M+H + )m / z,678.4; found,678.0.

[0211] 4. Synthesis of the compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0212] 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-diazaspiro[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 dried by rotation, and 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindole-1,3-dione (24 mg, 0.088 mmol), DMSO 1 mL, DIEA (95 mg, 0.737 mmol) were added, and the reaction was stirred at 120°C overnight under nitrogen protection. TLC confirmed that the reaction was complete. The reaction solution was extracted with water and ethyl acetate three times. The ethyl acetate layers were combined and washed with saturated brine three times, dried and spun dry, 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-dioxoisoindolin-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+) calculated for C 45 H 48 ClN8O6(M+H + )m / z,834.3; found,834.3.

[0213] 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).

[0214] 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-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0215] LC / MS (ESI+) calculation for C 44 H 47 ClFN9O6(M+H + )m / z,852.3; found,852.3.

[0216] 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).

[0217] 233: Synthesis of N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-5-(4-((2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyrazine-2-carboxamide

[0218]

[0219] LC / MS (ESI+) calculation for C 44 H 47 ClFN9O6(M+H + )m / z,852.3; found851.8.

[0220] 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).

[0221] 241: Synthesis of N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2-,2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyrazine-3-carboxamide

[0222] 1. Compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyrazine-3-carboxamide

[0223] Compound 6-chloro-N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)pyrazine-3-carboxamide (300 mg, 0.77 mmol), 4-hydroxymethylpiperidine (265 mg, 2.30 mmol), potassium carbonate (530 mg, 3.83 mmol) and DMF 5 mL were added to the reactor in sequence, nitrogen was protected, and the reaction was carried out at 80°C for 3 hours. The reaction end point was determined by TLC. After confirming the completion of the reaction, the reaction solution was extracted with water and ethyl acetate for 3 times, the ethyl acetate layers were combined, and washed with saturated brine for 3 times, dried and spin-dried, and separated by column chromatography to obtain a white solid compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyrazine-3-carboxamide (355 mg, 0.76 mmol), with a yield of 98%.

[0224] LC / MS (ESI+) calculation for C 24 H 27 ClN5O3(M+H + )m / z,470.2;found,470.2.

[0225] 2. Synthesis of the compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-((3aR,5R,6aS)-5-((5-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrazine-3-carboxamide

[0226] Compound N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-(4-(hydroxymethyl)piperidin-1-yl)pyrazine-3-carboxamide (70 mg, 0.15 mmol) was added to 2 mL DCM to dissolve, and DMP (76 mg, 0.18 mmol) was added to stir at room temperature for 2 h. The reaction endpoint was determined by TLC, the reaction solution was filtered, the filter cake was rinsed with DCM, and the filtrate was dried to obtain a crude product. The crude product was added with 2-(2,6-dioxopiperidine-3-yl)-5-fluoro-6-(hexahydropyrrolo[3,4-c]pyrrole-2(1H)-yl)isoindole-1,3-dione (68 mg, 0.18 mmol) and 2 mL DCM / MeOH (1:1) to dissolve, and glacial acetic acid (18 mg, 0.30 mmol) was added and stirred at room temperature for 30 min. Subsequently, sodium triacetylborohydride (126 mg, 0.60 mmol) was added and stirred at room temperature for 2 h. The reaction endpoint was determined by TLC, and a saturated sodium bicarbonate aqueous solution was added. The mixture was extracted with dichloromethane for 3 times, dried and spun down, and separated by pre-TLC to obtain an off-white solid compound, N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-6-((3aR,5R,6aS)-5-((5-(2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)hexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)methyl)hexahydrocyclopenta[c]pyrrol-2(1H)-yl)pyrazine-3-carboxamide (86 mg, 0.10 mmol). Yield: 69%.

[0227] LC / MS (ESI+) calculation for C 45 H 48 ClN8O6(M+H + )m / z,834.3; found,834.3.

[0228] 1 HNMR(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. 9 Hz, 2H), 1.23 (d, J=18.1 Hz, 2H). 267: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-2-(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisopropylindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyrimidine-5-carboxamide

[0229]

[0230] LC / MS (ESI+) calculation for: C 44 H 48 ClN9O6(M+H + )m / z,834.3; found,834.3.

[0231] 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).

[0232] 268: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-2-(4-((2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)pyrimidine-5-carboxamide

[0233]

[0234] LC / MS (ESI+) calculation for: C 44 H 47 ClFN9O6(M+H + )m / z,852.3; found852.3.

[0235] 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).

[0236] 274: N-((1r,4r)-4-(3-bromo-4-cyanophenoxy)cyclohexyl)-6-(4-((2-(2-(2-,2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0237]

[0238] LC / MS (ESI+) calculation for C 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).

[0239] 275: N-((1r,4r)-4-(4-cyano-3-methylphenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0240]

[0241] LC / MS (ESI+) calculation for C 45 H 51 N9O6(M+H + )m / z814.4; found814.4.

[0242] 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).

[0243] 276: N-((1r,4r)-4-(4-cyano-3-methoxyphenoxy)cyclohexyl)-6-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonyl-7-yl)methyl)piperidin-1-yl)pyridazine-3-carboxamide

[0244]

[0245] LC / MS (ESI+) calculation for C 45 H 51 N9O7(M+H + )m / z830.3; found830.3.

[0246] 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).

[0247] 279: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0248]

[0249] 1.3-(Methylthio)-1,2,4-triazine-6-carboxylic acid methyl ester

[0250] Using 3-amino-6-bromo-1,2,4-triazine as raw material, it was prepared by the literature method (PCTInt.Appl.,2015182712)

[0251] 3-amino-1,2,4-triazine-6-carboxylic acid methyl ester. Then it was used as a raw material to prepare pale yellow solid 3-(methylthio)-1,2,4-triazine-6-carboxylic acid methyl ester using the literature method (PCTInt.Appl.,2015181539). MS:calcd.forC6H7N3O2S[M+H] + :

[0252] 186.0; found:186.3. 1 HNMR (400MHz, CDCl3): δ8.95(s,1H),4.08(s,3H),2.73(s,3H).

[0253] 2. Methyl 3-(4-(hydroxymethyl)piperidin-1-yl)-1,2,4-triazine-6-carboxylate

[0254] 3-(Methylthio)-1,2,4-triazine-6-carboxylic acid methyl ester (5.0g, 27.00mmol) was dissolved in 200mL of dichloromethane, and m-chloroperbenzoic acid (10.7g, 62.09mmol) was added in batches. Stir at room temperature for 4h. Triethylamine (10.9g, 107.99mmol) was added, and then 4-piperidinemethanol (9.3g, 80.99mmol) was added. Stir at room temperature overnight. Water and dichloromethane were added, and the pH was adjusted to 8-9 with sodium carbonate, extracted, and the organic layer was washed with sodium thiosulfate, dried, spin-dried, and purified by silica gel column. The compound methyl 3-(4-(hydroxymethyl)piperidin-1-yl)-1,2,4-triazine-6-carboxylate (2.0g, 7.93mmol) was obtained. The yield was 29.9%.

[0255] LC / MS (ESI+) calculation for C 11 H 17 N4O3 + ([M+H] + )m / z:253.1;found253.1.

[0256] 3. Synthesis of the compound N-((1r, 4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-hydroxymethyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0257] Methyl 3-(4-(hydroxymethyl)piperidin-1-yl)-1,2,4-triazine-6-carboxylate (100.0 mg, 0.39 mmol), 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. Cooled to room temperature, dried by spin drying, 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

[0258] mmol). Yield: 38.5%. LC / MS (ESI+) calculation for C 23 H 28 ClN6O3 + ([M+H] + )m / z:471.2; found471.1.

[0259] 4. Synthesis of the compound N-((1r, 4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-formylpiperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0260] 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

[0261] mmol). Stir at room temperature for 4 h. Add dichloromethane and water for extraction, wash the organic layer with saturated brine, dry and spin dry.

[0262] 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 with a yield of 100.0%.

[0263] LC / MS (ESI+) calculation for C 23 H 26 ClN6O3 + ([M+H] + )m / z:469.2; found469.3.

[0264] 5. Compound N-((1r, 4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-((2-(2,6-dioxopiperidin-3-yl)

[0265] -1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)-1,2,4-triazine-6-carboxylic acid

[0266] Synthesis of amines

[0267] 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), 2-(2,6-dioxopiperidin-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. Stir at room temperature overnight. Dichloromethane and water were added for extraction, and the organic layer was washed with saturated brine, dried, spin-dried, and purified on a large plate. The target compound (56 mg, 0.06 mmol) was obtained. The yield was 44.9%.

[0268] LC / MS (ESI+) calculation for C 43 H 48 C1N 10 O6([M+H] + )m / z:835.3; found835.3.

[0269] 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

[0270] 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

[0271] –1.63(m,2H),1.44(dd,J=17.2,8.6Hz,2H).

[0272] 417: N-((1r,4r)-4-(3-chloro-4-cyanophenoxy)cyclohexyl)-3-(4-((2-(2,6-dioxopiperidin-3-yl)-6-fluoro-1-oxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)-1,2,4-triazine-6-carboxamide

[0273] LC / MS(ESI+)CalcdforC 43 H 48 FCl 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).

[0274] 419: N-((1r,4r)-4-(3-bromo-4-cyanophenoxy)cyclohexyl)-3-(4-((2-(2-(2,6-dioxopiperidine-3-

[0275] 1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-yl)-1,2,4-triazole-6-carboxamide

[0276]

[0277] LC / MS(ESI+)forC 43 H 48 Bn 10 O6[M+H] + m / z 879.

[0278] 420: N-((1r,4r)-4-(4-cyano-3-trifluoromethyl-phenoxy)cyclohexyl)-3-(4-((2-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)piperidin-1-

[0279] 1,2,4-Triazine-6-carboxamide

[0280]

[0281] LC / MS(ESI+)forC 44 H 48 F3N 10 O6[M+H] + m / z 869.

[0282] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0283] Experimental Example 1 Proliferation Inhibitory Activity of the Compounds of the Invention on Prostate Cancer Cells

[0284] (1) Experimental materials and instruments:

[0285] LNCaP / ARcellline (provided by Sichuan Kangcheng Biotechnology Co., Ltd.)

[0286] Fetal bovine serum FBS (Gibco, Cat. No. 10099-141)

[0287] 0.01MPBS(Biosharp,Cat.No.162262)

[0288] RIPM1640 culture medium (Hyclone, Cat. No. 308090.01)

[0289] Penicillin-Streptomycin (Hyclone, Cat. No. SV30010)

[0290] Cell counting kit-8 kit (Signalway Antibody, Cat. No. CP002)

[0291] Dimethyl sulfoxide (DMSO) (Sigma, Cat. No. D5879)

[0292] Centrifuge Tube, 15 ml (ExcellBio, Cat. No. CS015-0001)

[0293] Cell Culture Dish, (ExcellBio, Cat. No. CS016-0128)

[0294] Cell culture plate 96-well cell culture cluster (Corning, Cat. No. 3599)

[0295] Microplate reader (ThermoMultiskanMK3)

[0296] (2) Experimental methods:

[0297] a. Buffer preparation

[0298] Cell culture medium: RIPM1640 medium, 10% FBS, 1% PenStrep;

[0299] PBS buffer: Dissolve PBS powder in 2L ultrapure water and sterilize.

[0300] b. Experimental steps:

[0301] 1) LNCaP / AR cells were subcultured with cell culture medium, and cells in good growth state were inoculated into 96-well plates, with 80 μL per well and 1000 cells per well, and cultured overnight in a cell incubator at 37° C. and 5% CO 2 .

[0302] 2) Prepare the test compound into a 10mM stock solution with dimethyl sulfoxide (DMSO). Dilute it 3 times with DMSO before use, and then dilute it 3 times to obtain 9 concentration gradients. Then dilute the compound of each concentration 200 times with culture medium (to ensure that the DMSO concentration in the culture system is 0.1%), and repeat 2 wells for each concentration. Take 20μL of the diluted compound and add it to the cell culture well (the final concentration is 10μM, 3.3μM, 1.1μM...), and gently shake to mix. In addition, set up 3 negative control wells with only cells and 3 blank control wells with only culture medium (6 wells each with 20μL of culture medium diluted 200 times DMSO).

[0303] c. Result detection:

[0304] 1) After 6 days of culture, add 10 μL CCK-8 to each well and continue culturing in a 37°C, 5% CO2 cell incubator for 1 hour.

[0305] 2) Measure the absorbance (OD value) at 450 nm using a multifunctional microplate reader.

[0306] 3) The data were analyzed using the dose-response-inhibition equation in GraphPad Prism5 software to obtain the IC 50 value.

[0307] (3) Experimental results:

[0308] The compound of the present invention can effectively inhibit the proliferation of LNCap / AR cells.

[0309] Experimental Example 2 The effects of the compounds of the present invention on androgen receptor (AR) protein were analyzed by enzyme-linked immunosorbent assay (ELISA). Degradation activity of white

[0310] Experimental Materials:

[0311] Prostate cancer VCaP cells (ATCC, CRL-2876)

[0312] Charcoal stripped FBS (Gibco, Cat. No. 12676-029)

[0313] Metribolone(R1881)(MacklinBiochemical,CAS.No.965-93-5)

[0314] Phosphate buffer 0.01 M PBS (Biosharp, Cat. No. 162262)

[0315] DMEM / HIGHGLUCOSE(Hyclone,Cat.No.SH30243.01)

[0316] Penicillin-Streptomycin(Hyclone,Cat.No.SV30010)

[0317] Dimethyl sulfoxide (DMSO) (Sigma, Cat. No. D5879)

[0318] Centrifuge Tube, 15 ml (ExcellBio, Cat. No. CS015-0001)

[0319] Cell Culture Dish, (ExcellBio, Cat. No. CS016-0128)

[0320] ELISA kit TotalAndrogenReceptorSandwichELISAKit (CST, Cat. No. 12850C)

[0321] Experimental methods:

[0322] 1. Buffer Preparation

[0323]

[0324] 2. Experimental steps:

[0325] 1) VCaP After the cells were subcultured with cell culture medium, cells in good growth condition were inoculated into 96-well plates, 80ul per well, 30,000 cells per well, and cultured in a cell incubator at 37°C, 5% CO2 for 3 days until the cells adhered to the wall.

[0326] 2) Prepare the test compound into a 10mM stock solution with dimethyl sulfoxide (DMSO). Dilute with cell culture medium before use, take 20μl of the diluted compound and add it to the cell culture wells, and finally make 8 concentration gradients (300, 100, 10, 3, 1, 0.3, 0.1, 0.03nM), make 1 well for each concentration, and gently shake to mix. In addition, set up negative control wells (only add culture medium) and positive control wells (only add cells and DMSO).

[0327] 3) After the cells were cultured in the incubator for 16 hours, the culture medium was removed and the cells were rinsed once with ice-cold 1X PBS.

[0328] 4) Discard PBS, add 50ul of ice-cold 1X cell lysis buffer to each well of the cell culture plate, incubate on ice for 15 minutes, and then shake to mix.

[0329] 5) Add 115ul of sample diluent to a new 96-well plate, pipette 5ul of cell lysate supernatant from the cell culture plate and add it to the sample diluent, and shake evenly.

[0330] 6) Pipette 100ul of the diluted sample into the ELISA plate, seal the ELISA plate with plastic film and incubate it in a 37°C incubator for 2 hours.

[0331] 7) Gently remove the film, add 200 μl of 1X washing buffer to each well, shake for 5 minutes, pour out, and pat dry. Wash 4 times.

[0332] 8) Add 100 μl of freshly prepared detection antibody (green) to each well of the ELISA plate, seal with plastic film and incubate in a 37°C incubator for 1 hour.

[0333] 9) Gently remove the film, pour out the detection antibody, pat dry, add 200 μl of 1X washing buffer to each well, shake for 5 minutes, pour out, pat dry. Wash 4 times.

[0334] 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 on

[0335] Incubate in a 37°C incubator for 30 minutes.

[0336] 11) Gently remove the film, pour off the secondary antibody, pat dry, add 200 μl of 1X washing buffer to each well, shake for 5 minutes, pour off, and pat dry.

[0337] Wash 4 times.

[0338] 12) Add 100 μl of TMB substrate to each well of the ELISA plate and incubate in a 37°C incubator for 5 minutes.

[0339] 13) Add 100 μl of STOP solution to each well of the ELISA plate and shake gently for a few seconds.

[0340] 3. Read the results

[0341] Wipe the bottom of each well with a lint-free cloth. Read the absorbance at 450 nm within 30 minutes after adding the STOP solution. Use the formula: Remaining AR% = 100*(OD value of the test well - OD value of the blank control well) / (OD value of the positive control well - OD value of the blank control well) and analyze the data using the Dose-response equation in GraphPadPrism5 software to obtain DC 50 , D max value.

[0342] DC 50 It is the concentration of compound that degrades 50% of the target protein. max It is the percentage of the target protein that can be degraded to the maximum extent.

[0343] Experimental results:

[0344] The degradation activity of the compounds of the present invention on androgen receptor (AR) at a concentration of 100 nM is shown in Table 1, wherein % degradation is the percentage of degraded AR. The degradation activity of the compounds of the present invention on androgen receptor (AR) at a concentration gradient is shown in Table 2. In Table 2, DC 50 D is the concentration of compound that degrades androgen receptor by 50%. max is the percentage of androgen receptor (AR) that can be degraded the most.

[0345] It can be seen that the compounds of the present invention can effectively degrade androgen receptors, and most of the compounds have an effect on the DC of androgen receptors. 50 Below 0.3 μM, some even <10 nM; most compounds have an effect on the D max Above 40%, some even >80%.

[0346] Table 1. Degradation activity of compounds on androgen receptor (AR) at 100 nM concentration

[0347] Compound %degradation 197 A

[0348] A:>80%,B:79%-50%,C:49%-25%,D:<25%

[0349] Table 2. Effects of compounds on androgen receptor (AR) degradation in DCs 50 and D max

[0350]

[0351]

[0352] DC 50 :A:<10nM,B:10-100nM,C:0.1-0.3μM,D:>0.3μM

[0353] D max :++++:>80%,+++:80%-60%,++:60%-40%,+:<40%

[0354] Experimental Example 3 Western Blot assay to determine the effect of compounds on androgen receptor (AR) protein of drop

[0355] Deactivation

[0356] Experimental Materials:

[0357] Prostate cancer VCaP cells (ATCC, CRL-2876)

[0358] FBS (Gibco, Cat. No. 10099-141)

[0359] 0.01MPBS(Biosharp,Cat.No.162262)

[0360] Metribolone(R1881)(MacklinBiochemical,CAS.No.965-93-5)

[0361] DMEM / HIGHGLUCOSE(Hyclone,Cat.No.SH30243.01)

[0362] Penicillin-Streptomycin(Hyclone,Cat.No.SV30010)

[0363] Dimethyl sulfoxide (DMSO) (Sigma, Cat. No. D5879)

[0364] Centrifuge tube, 15 ml (ExcellBio, Cat. No. CS015-0001)

[0365] Cell culture plate, (ExcellBio, Cat. No. CS016-0128)

[0366] 6-wellcellculturecluster(Corning,Cat.No.3516)

[0367] Buffer RIPAlysate buffer (Beyotime, Cat. No. P0013B)

[0368] Buffer Protein Loding Buffer (Beyotime, Cat. No. P0015L)

[0369] BCA Protein Assay Kit (Beyotime, Cat. No. P0012)

[0370] SDS-PAGE gel preparation kit (Chengdu Baihe Technology Co., Ltd., Cat. No. PG112)

[0371] Anti-β-ActinrabbitmAb (CST, Cat.No.4970)

[0372] Androgen Receptor (D6F11) XP Rabbit mAb (CST, Cat. No. 5153)

[0373] PeroxidaseAffinipure(HRP)GoatAnti-RabbitIgG(ZenBioscience, Cat.No.511203)

[0374] TBST (Biosharp, Cat. No. BL601A)

[0375] ECL Chemiluminescence Kit ( Beyotime, Cat. No. P0018)

[0376] Experimental methods:

[0377] 1. Buffer Preparation

[0378]

[0379] 2. Experimental steps:

[0380] 1) VCaP After the cells were subcultured with cell culture medium, cells in good growth condition were inoculated into 12-well plates, 1 ml per well, 500,000 cells per well, and cultured overnight in a cell incubator at 37°C and 5% CO2.

[0381] 2) Prepare the test compound into a 10 mM stock solution with dimethyl sulfoxide (DMSO). Dilute it with DMSO again before use.

[0382] Take 2 μl of the diluted compound and add it to the cell culture well (to ensure that the DMSO concentration in the culture system is 0.1%), repeat 2 wells for each concentration, and gently shake to mix. In addition, set up negative control wells (add the same amount of DMSO) and positive control wells.

[0383] 3) After 16 hours of culture, cells were lysed with RIPA cell lysis buffer, proteins were extracted, and protein concentration was measured using a BCA kit.

[0384] 5-fold concentrated protein loading buffer was heated at 100°C for 5 minutes and then the samples were stored at -20°C.

[0385] 4) 30 μg of protein per well was loaded onto polyacrylamide gel for electrophoresis.

[0386] 5) The proteins were transferred from the polyacrylamide gel to a PVDF membrane and blocked with 5% skim milk at room temperature for 1 hour. The membrane was incubated with primary antibodies (Androgen Receptor (D6F11) XP Rabbit mAb and Anti-β-Actin Rabbit mAb) at 4°C overnight, washed three times with TBST solution for 10 minutes each time, incubated with secondary antibodies (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.

[0387] 3. Result detection:

[0388] Finally, ECL color developing solution was added for color development, and photos were taken with an automatic chemiluminescence instrument to collect and analyze the images.

[0389] Experimental results:

[0390] use Western Blot method was used to measure The degradation activity of the compounds of the present invention on androgen receptor (AR) at a concentration of 100 nM is shown in Table 3, and % degradation is the percentage of degraded AR. It can be seen that the compounds of the present invention can effectively degrade androgen receptor.

[0391] Table 3. AR degradation activity of the compounds of the present invention

[0392] Compound %degradation 158 A 230 A 231 A

[0393] A:>80%,B:79%-50%,C:49%-25%,D:<25%

[0394] Experimental Example 4 Metabolic Stability Experiment of the Compounds of the Invention

[0395] 1. Materials and instruments

[0396] Liquid phase system (Shimadzu), mass spectrometry system (API4000 instrument from AB Inc (Canada) with an ESI interface), chromatographic column (ACE Excel3AQ 30×2.1 mm Column), human liver drug enzyme (Corning, Cat. #452117), phosphate buffer, ultrapure water, MgCl2 solution, NADPH.

[0397] 2. Methods and Results

[0398] 10μl 20mg / ml liver microsomes and 40μl 10mN ADPH were added to the incubation tube. The final concentrations of liver microsomes and NADPH were 0.5mg / mL and 1mM, respectively. At the same time, a control group without NADPH and an equal amount of ultrapure water was prepared. Then 4μl of a control compound (verapamil) with a concentration of 200μM or the compound to be tested was added. The final concentration of the compound was 2μM. 50μl of the reaction solution was taken out at 0, 15, 30, 45 and 60min of incubation, and 4 times the volume of ice acetonitrile was added to terminate the reaction. After the sample was centrifuged for 40min (3220g), 100μl of the supernatant was taken out, and 100μl of ultrapure water was added to the supernatant to mix for LC-MS / MS detection. Finally, the experimental parameters of liver microsome metabolic stability were calculated.

[0399] The experimental results show that the compounds of the present invention have good metabolic stability.

[0400] Experimental Example 5 Pharmacokinetics of the compounds of the present invention

[0401] 1. Experimental materials and instruments:

[0402] LC-20AD high performance liquid chromatography system (Shimadzu, Japan)

[0403] API4000 triple quadrupole mass spectrometer, (Applied Biosystem, USA)

[0404] PhenixWinnolin pharmacokinetic software (Version 6.3, Certara, USA)

[0405] High-speed refrigerated centrifuge (ThermoFisherScientific)

[0406] Analytical balance (Sartorius, SECURA225D-1CN)

[0407] Experimental animals: ICR mice (Chengdu Dashuo Experimental Animal Co., Ltd.)

[0408] DMSO (Sigma)

[0409] CMC-Na (Chengdu Kelon Chemical)

[0410] Heparin (Chengdu Kelon Chemical)

[0411] 2. Experimental methods and results

[0412] Accurately weigh the compound to be tested according to the dosage, add the solvent to the final volume of 10 ml, and mix by ultrasonic vortex. 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°C for concentration determination. 9 healthy adult ICR mice (20-30 g) were fasted overnight (free drinking water) and then gavaged with a volume of 0.2 ml / 10 g; 0.1 ml of blood was collected from the retro-orbital venous plexus before and 0.5, 1, 2, 4, 6, 8, 12, and 24 h after administration, and the plasma was separated by centrifugation at 4°C for 5 min and stored at -20°C for testing. The concentration of the compound to be tested in plasma was then determined by LC / MS / MS.

[0413] The experimental results show that the compounds of the present invention have good pharmacokinetics.

[0414] In summary, the present invention provides a class of compounds shown in formula (I), which can target androgen receptors in prostate cancer cells, inhibit the proliferation of prostate cancer cells, and also show good metabolic stability and pharmacokinetic properties. The compounds of the present invention have good application prospects in the preparation of protein degradation targeted chimeras of androgen receptors, and in the preparation of drugs for treating related diseases regulated by androgen receptors (including prostate cancer, breast cancer, and Kennedy's disease).

Claims

1. A compound represented by formula (I), or an isotopic compound thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof: in, 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 connected by chemical bonds; The ARB is selected from the following structures: R w1 , R w2 , R w5 , R w6 For H, R w3 For cyano, R w4 Selected from H, halogen, C 1-6 Alkyl or its halogenated product or its deuterated product, C 1-6 Alkoxy or its halogenated or deuterated form; The L is selected from the following structures: The U is selected from the structure of the following formula (XB-1): Among them, R g3 Selected from H, halogen.

2. The compound according to claim 1, or its isotopic compound, or its tautomer, or its pharmaceutically acceptable salt, characterized in that: R w4 is CF3; or, the halogen is chlorine or bromine.

3. The compound according to claim 1, or its isotopic compound, or its tautomer, or its pharmaceutically acceptable salt, characterized in that: The ARB is selected from the following structures:

4. The compound according to claim 1, or its isotopic compound, or its tautomer, or its pharmaceutically acceptable salt, characterized in that: The U is selected from the following structures:

5. The compound according to claim 1, or its isotopic compound, or its tautomer, or its pharmaceutically acceptable salt, characterized in that: The compound is selected from one of the following compounds:

6. Use of the compound according to any one of claims 1 to 5, or its isotopic compound, or its tautomer, or its pharmaceutically acceptable salt in the preparation of a protein degradation targeted chimera of androgen receptor.

7. The use according to claim 6, characterized in that: The protein degradation targeting chimera can target recognition and / or bind to androgen receptor; or, the protein degradation targeting chimera can degrade and / or down-regulate androgen receptor.

8. The use according to any one of claims 6-7, characterized in that: The protein degradation targeting chimera is an active ingredient of a drug for treating related diseases regulated by androgen receptor.

9. The use according to claim 8, characterized in that: The disease is selected from prostate cancer, breast cancer, Kennedy's disease.

10. A drug for treating diseases regulated by androgen receptors, characterized in that: The drug is a preparation prepared by using the compound according to any one of claims 1 to 5, or its isotope compound, or its tautomer, or its pharmaceutically acceptable salt as an active ingredient, and adding pharmaceutically acceptable excipients.

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