Kras inhibitor compounds with macrocyclic structures

Abstract

This invention discloses a heterocyclic compound that can be used as a KRAS inhibitor. The compound of this invention is an effective, safer, and more pharmacokinetic KRAS inhibitor that can meet clinical needs.

Description

Technical Field

[0001] This invention relates to a heterocyclic compound, a pharmaceutical composition, and its application. Background Technology

[0002] RAS oncogene mutations are the most common activating mutations in human cancers, occurring in 30% of human tumors. The RAS gene family includes three subtypes (KRAS, HRAS, and NRAS), with 85% of RAS-driven cancers caused by mutations in the KRAS subtype. KRAS mutations are common in solid tumors such as lung adenocarcinoma, pancreatic ductal carcinoma, and colorectal cancer. In KRAS-mutant tumors, 80% of oncogenic mutations occur at codon 12, with the most common mutations including p.G12D (41%), p.G12V (28%), and p.G12C (14%).

[0003] The full name of the KRAS gene is Kirsten rat sarcoma viral oncogene homolog. KRAS plays a pivotal role in the signal regulation of cell growth. Upstream cell surface receptors such as EGFR (ErbB1), HER2 (ErbB2), ErbB3, and ErbB4, upon receiving external signals, transmit these signals downstream via the KRAS protein. When unactivated, the KRAS protein is tightly bound to GDP (guanine diphosphate). Upon activation by guanine nucleotide exchange factors such as SOS1, it binds to GTP (guanine triphosphate), becoming a kinase-active state. Mutations in the KRAS gene can lead to uncontrolled cell growth and tumor progression, independently transmitting growth and proliferation signals downstream, regardless of upstream growth factor receptor signals. Furthermore, the presence or absence of KRAS gene mutations is an important indicator of tumor prognosis. Statistical results show that KRAS is also a common submutation among KRAS subtypes, accounting for 12% in colorectal cancer, 36% in pancreatic cancer, and 4% in non-small cell lung cancer. Therefore, it is very necessary to develop a new KRAS inhibitor, which has great potential to become a new treatment in the field of cancer treatment. Thus, it is necessary to develop more effective, safer, and better pharmacokinetic KRAS inhibitors to meet clinical needs. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiency of the relatively simple structure of existing pan-KRAS inhibitors, and to provide a heterocyclic compound, a pharmaceutical composition and its application. The compound of the present invention has a novel structure and good activity and selectivity.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0006] This invention provides a heterocyclic compound of Formula I, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof (referring to the aforementioned heterocyclic compound of Formula I, its pharmaceutically acceptable salt thereof, or its stereoisomer thereof):

[0007]

[0008] Among them, W1 is independently selected from CR W1 Or N;

[0009] Among them, W2 is independently selected from CR W2 Or N;

[0010] Among them, W3 is independently selected from CR W3 Or N;

[0011] Among them, R W1 R W2 R W3 Each is independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, C1-C6 alkoxy, cyano, C1-C6 cycloalkyl, halogenated C1-C6 alkyl, nitro, hydroxyl, NR a R b ;

[0012] Among them, R1, R 1’ R2, R 2’ R3, R 3’ Each can be independently represented as hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy C1-C6 alkyl, or halo-C1-C6 alkyl.

[0013] Where X1 represents non-existent or -CR L R L’ -;

[0014] Where X2 represents -(CR) S R S’ ) n -;

[0015] Among them, R L R L’ Each can be independently represented as hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy C1-C6 alkyl, or halo-C1-C6 alkyl.

[0016] Among them, R S R S’ Each can independently represent hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy C1-C6 alkyl, or halo-C1-C6 alkyl; or arbitrarily CR S R S’ It can be replaced by O, NH, or N(CH3);

[0017] Where n represents an integer selected from 1, 2, 3, 4, 5, and 6;

[0018] Among them, X 3 It represents O, NH, N(CH3) or -CR T R T’ -;

[0019] Among them, R T R T’ Each can be independently represented as hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy C1-C6 alkyl, or halo-C1-C6 alkyl.

[0020] Among them, R a R b Each can be used independently to represent hydrogen or C1-C6 alkyl groups.

[0021] In a preferred embodiment of the present invention, W1 and W2 are selected from N.

[0022] In a preferred embodiment of the present invention, W3 is selected from CH.

[0023] In the preferred embodiment of the present invention, X1 represents non-existence.

[0024] In a preferred embodiment of the present invention, X1 represents CH2.

[0025] In the preferred embodiment of the present invention, X2 represents -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH(CH3)CH2CH2CH2CH2-, -C(CH3)2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2CH2-, -CH2C(CH3)2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH2C(CH3)2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH(CH3)CH2-, -CH2OCH2CH2CH2-, -CH2CH2OCH2CH2-, -CH2CH2CH2OCH2-, -CH2NHCH2CH2CH2-, -CH2CH2NHCH2CH2-.

[0026] In a preferred embodiment of the present invention, X3 represents O or CH2.

[0027] In the preferred embodiment of the present invention, R1, R 1’ It represents H or CH3.

[0028] In the preferred embodiment of the present invention, R2, R 2’ It represents H or CH3.

[0029] In the preferred embodiment of the present invention, R3, R 3’ It represents H or CH3.

[0030] Specifically, the present invention provides the following compounds:

[0031]

[0032] The term "pharmaceutically acceptable" means that the salts, solvents, excipients, etc., are generally non-toxic, safe, and suitable for patient use. The term "patient" preferably refers to a mammal, and more preferably a human.

[0033] The term "pharmaceutically acceptable salt" refers to a salt prepared from the compounds of the present invention with a relatively non-toxic, pharmaceutically acceptable acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting a sufficient amount of a pharmaceutically acceptable base with the neutral form of such compounds in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, and diethanolamine salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a sufficient amount of a pharmaceutically acceptable acid with the neutral form of such compounds in a pure solution or a suitable inert solvent. Pharmaceutically acceptable acids include inorganic acids, including but not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, and sulfuric acid. The pharmaceutically acceptable acids include organic acids, including but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acidic citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentian acid, fumaric acid, gluconic acid, succinic acid, formic acid, ethanesulfonic acid, dihydroxynaphthyl acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthylcarboxylic acid)), amino acids (e.g., glutamic acid, arginine), etc. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. For details, see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977), or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0034] The term "solvate" refers to a substance formed by the combination of the compound of this invention with a stoichiometric or non-stoichiometric solvent. Solvent molecules in a solvate can exist in an ordered or disordered arrangement. The solvents include, but are not limited to, water, methanol, and ethanol.

[0035] The terms "compound," "pharmaceutically acceptable salt," "solvent," and "solvent of a pharmaceutically acceptable salt," if stereoisomers exist, can exist as a single stereoisomer or a mixture thereof (e.g., a racemic mixture). The term "stereoisomer" refers to cis-trans isomers or optical isomers. These stereoisomers can be separated, purified, and enriched by asymmetric synthetic methods or chiral separation methods (including but not limited to thin-layer chromatography, rotational chromatography, column chromatography, gas chromatography, high-performance liquid chromatography, etc.), and can also be obtained by chiral resolution through bonding (chemical bonding, etc.) or salt formation (physical bonding, etc.) with other chiral compounds. The term "single stereoisomer" means that the mass content of one stereoisomer of the compound of the present invention is not less than 95% relative to all stereoisomers of the compound.

[0036] The terms “compound,” “pharmaceutical acceptable salt,” “solvent,” and “solvent of pharmaceutically acceptable salt” may exist as a single tautomer or a mixture thereof, preferably as the more stable tautomer.

[0037] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0038] The term "alkyl" refers to a saturated monovalent hydrocarbon group that has a specified number of carbon atoms (e.g., C1 to C6), is straight-chain or branched. Alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

[0039] The term "cycloalkyl" refers to a cyclic, saturated monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C3 to C6). Cycloalkyl groups include, but are not limited to: wait. Detailed Implementation

[0040] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0041] The NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer. The solvent used for the measurements is noted in the spectral analysis.

[0042] MS measurements were performed using Agilent 1200-G1956A / 1200-6110A / 1200-6140A / 1260-6125B / Prime-6125B / 1260-6120 LC-MS / MS systems, SHIMADZU 20A-2010 / 20A-2020 LC-MS / MS systems, and Waters ACQ-QDA LC-MS / MS systems.

[0043] HPLC analysis was performed using a SHIMADZU 20A high-performance liquid chromatograph.

[0044] SFC analysis was performed using a Waters UPCC with PDA Detector and QDa Detector ultra-high performance phase chromatography system. 2 Ultra-high performance liquid chromatograph with PDA detector, Agilent 1260 with DAD detector, Shimadzu LC-20AB with PDA detector, Shimadzu LC-20AD with PDA detector.

[0045] Preparative HPLC separation was performed using a Shimadzu LC-20AP pump, Shimadzu LH-40 Liquid Handler, Shimadzu SPD-20A Detector, Gilson GX-281 Liquid Handler, Gilson 322 pump, and Gilson 156UV Detector preparative chromatograph.

[0046] SFC separation uses The Berger MG II, MG III, Sepiatec's Prep SFC 100system, Waters Prep 80Q SFC SYSTEM, Prep 150AP SFC SYSTEM, Prep 200SFC SYSTEM, and Prep350SFC SYSTEM.

[0047] Rapid column chromatography separation was performed using the Biotage IsoleraOne rapid preparative chromatograph.

[0048] The silica gel plates used for thin-layer chromatography are GF254 acrylic adhesive silica gel plates from Anhui Liangchen Silicon Source Materials Co., Ltd. The silica gel plates used in thin-layer chromatography (TLC) are 0.25 mm in diameter, and the silica gel plates used for thin-layer chromatography separation and purification are 0.5 mm in diameter.

[0049] Pressurized hydrogenation reaction hydrogenation bottle and hydrogen cylinder.

[0050] The microwave reaction was performed using a Biotage Initiator+ microwave synthesizer.

[0051] The glove box uses a custom-made DELLIX glove box.

[0052] Example 1: (4S)-2-amino-3'-((5 2 S,4S,E)-4-methyl-12-oxo-1 1 H-3-oxa-11-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-2 2 -yl)-5',6,6',7-tetrahydro-4'H,5H-spiro[benzo[b]thiophene-4,7'-benzo[d]isoxazole]-3-carboxynitrile

[0053] Example 2: (4R)-2-amino-3'-((5 2 S,4S,E)-4-methyl-12-oxo-1 1 H-3-oxa-11-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-2 2 -yl)-5',6,6',7-tetrahydro-4'H,5H-spiro[benzo[b]thiophene-4,7'-benzo[d]isoxazole]-3-carboxynitrile

[0054]

[0055] Step 1: Under nitrogen protection, ethyl 2-cyclohexanone carboxylate (50.00 g, 293.75 mmol) was added to a solution of 1,1,3,3-tetramethylguanidine (67.67 g, 587.51 mmol) in toluene (90 mL) and water (1.46 mL, 80.78 mmol), and rinsed with toluene (5 mL). Then, allyl acetate (38.23 g, 381.88 mmol) was added, and rinsed with toluene (5 mL). The reaction solution was cooled to 10–15 °C and protected with nitrogen purging. A solution of (1S,2S)-(-)-1,2-diaminocyclohexane-N,N-bis(2-diphenylphosphobenzoyl) (0.41 g, 0.59 mmol) in toluene (5 mL) was added, and rinsed with toluene (5 mL). Then, a toluene (5 mL) solution of allyl palladium(II) chloride dimer (94.0 mg, 0.26 mmol) was added, and the mixture was rinsed with toluene (5 mL). The mixture was stirred at 10–15 °C for 4 hours, and then at 25 °C for 12 hours. At 10 °C, saturated ammonium chloride (100 mL) was added to the solution, and the mixture was stirred and separated. The aqueous phase was extracted once more with toluene (90 mL). The combined organic layers were washed with water (150 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated to give a yellow, oily crude compound (R)-1-allyl-2-oxocyclohexane-1-carboxylic acid ethyl ester (70 g). LCMS (ESI): [M+H] + =211.1.

[0056] Step 2: Under ice bath conditions, trimethylsilane chloride (105.50 g, 832.23 mmol) was added dropwise to a solution of ethyl (R)-1-allyl-2-oxocyclohexane-1-carboxylate (70.00 g, 332.91 mmol) in ethylene glycol (280 mL), while maintaining the temperature below 20 °C. The mixture was stirred at 25 °C for 12 hours. The reaction solution was cooled to 0 °C, and a solution of sodium hydroxide (34.62 g, 865.55 mmol) in water (280 mL) was added, while maintaining the temperature below 20 °C. Toluene (300 mL) was added, and the mixture was stirred and separated. The aqueous phase was then extracted once more with toluene (100 mL). The combined organic layers were washed twice with water (140 mL), dried with sodium sulfate, filtered, and the filtrate was concentrated to give a yellow oily compound (R)-6-allyl-1,4-dioxaspiro[4.5]dec-6-carboxylic acid ethyl ester (76 g, 275.59 mmol, yield 90%). 1H NMR (400MHz, CDCl3) δppm 5.76-5.57(m,1H),5.15-4.95(m,2H),4.17(ttd,J=3.7,7.1,10.7Hz,2H),4.04-3.85(m,4H),2.83(dd,J=6.5,13.9Hz,1H),2.35(br d,J=8.2Hz,1H),2.03(ddd,J=4.0,9.8,13.9Hz,1H),1.76-1.43(m,7H),1.28(t,J=7.1Hz,3H).

[0057] Step 3: Under nitrogen protection, 9-boronbicyclo[3.3.1]nonane (0.5 M tetrahydrofuran solution, 84.0 mL, 41.99 mmol) was cooled to 0-5 °C, and (R)-6-allyl-1,4-dioxaspiro[4.5]dec-6-carboxylic acid ethyl ester (8.90 g, 35.00 mmol) was added, followed by rinsing with tetrahydrofuran (4.2 mL). The mixture was heated to 25 °C and stirred at 25 °C for 2 hours. The reaction solution was cooled to -40 °C, and 2-chloroacetic acid methyl ester (7.29 g, 67.19 mmol) was added. Below -40 °C, bis(trimethylsilyl)aminolithium (1 M tetrahydrofuran solution, 115.48 mL, 115.48 mmol) was added dropwise. The solution was stirred at 25 °C for 16 hours. The mixture was concentrated to half its volume at 35°C, and an ethanol (33.6 mL) and sodium hydroxide (1.40 g, 35.00 mmol) solution in water (22.4 mL) were added. The solution was stirred at 70°C for 16 hours. The mixture was concentrated to remove the organic solvent, diluted with water (30 mL), and extracted with n-heptane (150 mL * 2). The combined organic layers were washed with water (30 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by rapid column chromatography (silica gel, 0-10% gradient tetrahydrofuran / petroleum ether) to give a yellow oily compound (R)-1,4-dioxabispyrospirol [4.0.5]. 6 0.4 5 Pentadecane-7-one (5.2 g, 23.21 mmol, yield 66%). LCMS (ESI): [M+H] + =225.1. 1H NMR (400MHz, CDCl3) δppm 4.02-3.82(m,4H),2.52-2.42(m,1H),2.40-2.31(m,1H),2.30-2.20(m,1H),2.14-2.03(m,1H),2.02-1.9 4(m,1H),1.92-1.87(m,1H),1.81-1.71(m,4H),1.64-1.57(m,2H),1.53-1.47(m,3H),1.27-1.18(m,1H).

[0058] Steps 4 and 5: Under nitrogen protection, bis(trimethylsilyl)aminolithium (1M tetrahydrofuran solution, 73.6 mL, 73.60 mmol) was cooled to 0-5°C, and (R)-1,4-dioxabisspirol [4.0.5] was added below 5°C. 6 0.4 5 Pentadecane-7-one (15.00 g, 66.87 mmol) was rinsed with tetrahydrofuran (15.00 mL) and stirred at 0–5 °C for 30 minutes. Diethyl oxalate (10.96 mL, 80.25 mmol) was then added at 5 °C. The mixture was heated to 25 °C and stirred for 12 hours. Hydrogen chloride (2 M dioxane solution, 73.6 mL, 147.20 mmol) was added to the reaction mixture to adjust the pH to 6–7. The resulting suspension was used directly in the next reaction step. Hydroxylamine hydrochloride (4.88 g, 70.21 mmol) was added to the above suspension at 25 °C. The reaction mixture was stirred at 70 °C for 16 hours. The solution was concentrated to remove tetrahydrofuran, and water (100 mL) was added. The mixture was extracted with ethyl acetate (100 mL * 3). The combined organic layers were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a yellow, oily crude compound (S)-5,6-dihydro-4H-bis(benzo[d]isoxazole-7,1'-cyclohexane-2',2”-[1,3]dioxolane]-3-carboxylic acid ethyl ester (24.00 g). LCMS (ESI): [M+H] + =322.0.

[0059] Step 6: At 25°C, add pyridine p-toluenesulfonate (37.54 g, 149.36 mmol) and water (240 mL) to a solution of (S)-5,6-dihydro-4H-bis(benzo[d]isoxazole-7,1'-cyclohexane-2',2”-[1,3]dioxolane]-3-carboxylic acid ethyl ester (24.00 g, 74.68 mmol) in acetone (240 mL). Stir the reaction mixture at 70°C for 16 hours. [The solution is then...] The acetone was removed by concentration, and the mixture was extracted with ethyl acetate (150 mL * 3). The combined organic layers were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a brown, oily crude compound, (S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxylic acid ethyl ester (18.60 g, 67.15 mmol, yield 90%). LCMS (ESI): [M+H] + =278.0.

[0060] Step 7: At 25°C, ammonia (77.1 mL) was added to an ethanol (77.00 mL) solution of ethyl (S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxylic acid (18.30 g, 65.99 mmol), and the mixture was stirred for 16 hours. The solution was concentrated to remove some of the ethanol, diluted with water (80 mL), extracted with ethyl acetate (150 mL * 3), and the combined organic layers were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a brown, oily crude compound (S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxamide (13.50 g, 54.44 mmol, yield 82%). LCMS (ESI): [M+H] + =249.0.

[0061] Step 8: At 0°C, trifluoroacetic anhydride (12.26 mL, 87.00 mmol) was added to a solution of (S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxamide (13.50 g, 54.37 mmol) in pyridine (15.80 mL, 195.75 mmol) and acetonitrile (36.00 mL). The reaction solution was stirred at 0°C for 5 minutes. Add ice water (108 mL), extract with ethyl acetate (100 mL * 3), dry the combined organic phases with anhydrous sodium sulfate, filter, concentrate the filtrate, and purify the crude product by rapid column chromatography (silica gel, 0-35% gradient tetrahydrofuran / petroleum ether) to give a yellow solid compound (S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxynitrile (7.00 g, 3.88 mmol, yield 56%). LCMS (ESI): [M+H] + =231.0. 1 H NMR (400MHz, DMSO-d6) δppm 2.85-2.73(m,1H),2.62-2.39(m,3H),2.35-2.18(m,2H),2.08-1.92(m,2H),1.92-1.73(m,5H),1.55-1.41(m,1H).

[0062] Steps 9 and 10: At 25°C, sodium methoxide (30% methanol solution, 1.56 g, 8.69 mmol) was added to a methanol (48 mL) solution of (S)-2'-oxy-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxynitrile (8.00 g, 34.74 mmol). The reaction mixture was stirred at 25°C for 2 hours. Then, ammonium chloride (2.04 g, 38.22 mmol) was added, and the reaction mixture was stirred at 25°C for 16 hours. The suspension was filtered, and the filtrate was concentrated to obtain a yellow solid. LCMS(ESI): [M+H] +=248.1. The above compound was dissolved in N,N-dimethylformamide (40 mL), and 1,8-diazabicyclo[5.4.0]undecane-7-ene (10.91 mL, 72.95 mmol) and diethyl malonate (5.27 mL, 34.74 mmol) were added at 0 °C. The reaction mixture was stirred at 90 °C for 16 hours. Ice water (120 mL) was added, and 1 M hydrochloric acid was added at 0 °C to adjust the pH to 3-4. The solid product was filtered off, washed with water (40 mL), and dried under vacuum to give a gray solid compound (S)-3-(4,6-dihydroxypyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (8.00 g, 25.40 mmol, yield 73%). LCMS (ESI): [M+H] + =316.0.

[0063] Step 11: At 0°C, (S)-3-(4,6-dihydroxypyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (5.6 g, 17.76 mmol) was dissolved in phosphorus oxychloride (16.55 mL, 177.59 mmol). The mixture was stirred at 0°C for 5 minutes, and then diisopropylethylamine (6.46 mL, 39.07 mmol) was added. The reaction mixture was stirred at 80°C for 3 hours. The solution was cooled and poured into ice water (560 mL). The solid product was filtered off, washed with water (50 mL), and dried under vacuum to give a gray solid compound (S)-3-(4,6-dichloropyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (5.2 g, 3.47 mmol, yield 83%). LCMS (ESI): [M+H] + =352.0.

[0064] Step 12: Sodium hydroxide (60% concentration, 204.4 mg, 5.11 mmol) was added to a 30 mL solution of tert-butyl(S)-2-((S)-1-hydroxyethyl)pyrrolidine-1-carboxylic acid ester (916.9 mg, 4.26 mmol) in tetrahydrofuran at 0 °C. The mixture was stirred at 0 °C for 0.5 h, and then (S)-3-(4,6-dichloropyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (1.5 g, 4.26 mmol) was added. The reaction mixture was stirred at 0 °C for 1 h, and then at 25 °C for 1 h. The reaction solution was cooled to 0°C and quenched with water (20 ml). Extraction was performed with ethyl acetate (30 ml x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by rapid column chromatography (silica gel, 0-30% gradient tetrahydrofuran / petroleum ether) to obtain a colorless oily compound, tert-butyl(S)-2-((S)-1-((6-chloro-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazol-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)oxy)ethyl)pyrrolidine-1-carboxylic acid ester (1255.0 mg, 3.57 mmol, yield 55%). LCMS (ESI): [M+H] + =531.3.

[0065] Step 13: Add hydrogen chloride (2M dioxane solution, 14.12mL, 28.25mmol) to a solution of tert-butyl(S)-2-((S)-1-((6-chloro-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazol-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)oxy)ethyl)pyrrolidine-1-carboxylic acid ester (1.0g, 1.88mmol) in dichloromethane (20mL). The reaction mixture was stirred at 25°C for 2 hours, then concentrated to give a white solid crude compound (S)-3-(4-chloro-6-((S)-1-((S)-pyrrolidine-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (0.94 g). LCMS (ESI): [M+H] + =431.0.

[0066] Step 14: At -78°C, dimethyl sulfoxide (1.39 mL, 19.60 mmol) was added to a solution of oxaloyl chloride (0.91 mL, 10.63 mmol) in dichloromethane (36 mL). The reaction mixture was stirred at -78°C for 15 minutes, and then a solution of (5-hydroxypentyl)carbamate tert-butyl ester (1.8 g, 8.85 mmol) in dichloromethane (18 mL) was added dropwise. The reaction mixture was stirred at -78°C for 45 minutes, and then triethylamine (6.17 mL, 44.46 mmol) was added, and the temperature was raised to 25°C. The reaction mixture was filtered, diluted with water (30 mL), extracted with dichloromethane (30 mL x 3), the combined organic layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a brown, oily crude compound (5-oxopentyl)carbamate tert-butyl ester (2.1 g). LCMS (ESI): [M + Na] + =224.0.

[0067] Step 15: Add sodium borohydride acetate (1032.8 mg, 4.87 mmol) to a solution of (S)-3-(4-chloro-6-((S)-1-((S)-pyrrolidine-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (700.0 mg, 1.62 mmol) and (5-oxopentyl)carbamate tert-butyl ester (980.8 mg, 4.87 mmol) in dichloromethane (7 mL). Stir the reaction mixture at 25 °C for 1 hour. Dilute with water (10 ml), extract with dichloromethane (10 ml * 3), combine the organic layers, dry with anhydrous sodium sulfate, filter, concentrate the filtrate, and purify the crude product by rapid column chromatography (silica gel, 0-10% gradient methanol / dichloromethane) to obtain a white solid compound (5-((S)-2-((S)-1-((6-chloro-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)oxy)ethyl)pyrrolidine-1-yl)pentyl)tert-butyl carbamate (620.0 mg, 1.0 mmol, yield 62%). LCMS (ESI): [M+H) + =616.4.

[0068] Step 16: Add cesium carbonate (1744.9 mg, 5.36 mmol) to a solution of (5-((S)-2-((S)-1-((6-chloro-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazol-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)oxy)ethyl)pyrrolidine-1-yl)pentyl)carbamate tert-butyl ester (220.0 mg, 0.36 mmol) and 1H-pyrazole-3-carboxylic acid (200.1 mg, 1.79 mmol) in dimethyl sulfoxide (3.3 mL). Stir the reaction mixture at 80 °C for 20 minutes. Filter the solution and wash the solid with dimethyl sulfoxide (1.5 mL). The filtrate was adjusted to pH 5-6 by adding concentrated hydrochloric acid (approximately 150 μL). The mixture was purified by rapid column chromatography (C18, 0-75% methanol / water gradient) to give a yellow solid compound 1-(6-((S)-1-((S)-1-(5-((tert-butoxycarbonyl)amino)pentyl)pyrrolidine-2-yl)ethoxy)-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)-1H-pyrazole-3-carboxylic acid (190.0 mg, 0.27 mmol, yield 77%). LCMS (ESI): [M+H] + =692.2.

[0069] Step 17: Add hydrogen chloride (2M dioxane solution, 4.2mL, 8.46mmol) to a solution of 1-(6-((S)-1-((S)-1-(5-((tert-butoxycarbonyl)amino)pentyl)pyrrolidine-2-yl)ethoxy)-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)-1H-pyrazole-3-carboxylic acid (390.0mg, 0.56mmol) in dichloromethane (8mL). The reaction was stirred at 25°C for 1 hour, then concentrated under reduced pressure to give a white solid crude compound 1-(6-((S)-1-((S)-1-(5-aminopentyl)pyrrolidine-2-yl)ethoxy)-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)-1H-pyrazole-3-carboxylic acid (410 mg). LCMS (ESI): [M+H] + =592.3.

[0070] Step 18: Add tri-n-butylcyclic phosphoric anhydride (1.13 g, 2.08 mmol) to a solution of 1-(6-((S)-1-((S)-1-(5-aminopentyl)pyrrolidine-2-yl)ethoxy)-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)-1H-pyrazole-3-carboxylic acid (410.0 mg, 0.69 mmol) and diisopropylethylamine (2.24 mL, 13.52 mmol) in dichloromethane (16 mL), and stir at 25 °C for 16 hours. Quench the reaction with water (20 mL), extract with dichloromethane (20 mL * 3), dry the combined organic phases with anhydrous sodium sulfate, filter, and evaporate the filtrate to dryness. A yellow solid crude compound (5 2 S,4S,E)-4-methyl-2 2 -((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)-1 1 H-3-oxa-11-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-12-one (420.0 mg). LCMS (ESI): [M+H] + =574.1.

[0071] Step 19: Place (5 2 S,4S,E)-4-methyl-2 2 -((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)-1 1 H-3-oxa-11-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-12-one (100 mg, 0.17 mmol), sulfur (8.9 mg, 0.28 mmol), and ammonium acetate (21.5 mg, 0.28 mmol) were added to ethanol (2.0 mL), and the mixture was stirred at 60 °C for 15 min. Then, malononitrile (18 μL, 0.29 mmol) was slowly added, and the reaction was carried out at 80 °C with stirring for 3 h. The residue was purified by rapid column chromatography (C18, 0-85% methanol / water gradient) to give the product as a yellow solid compound (50 mg).

[0072] 100 mg of the product was separated by SFC (column: DAICEL CHIRALPAK AS (250 mm * 30 mm, 10 μm); mobile phase: phase A is carbon dioxide, phase B is 0.1% ammonia / isopropanol; phase B is maintained at 45%; flow rate: 150 mL / min) to obtain two stereoisomers, Example 1 and Example 2.

[0073] Example 1: (4R)-2-amino-3'-((5 2 S,4S,E)-4-methyl-12-oxo-1 1 H-3-oxa-11-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-2 2 5',6,6',7-Tetrahydro-4'H,5H-spiro[benzo[b]thiophene-4,7'-benzo[d]isoxazole]-3-carboxynitrile (white solid, 2.0 mg). LCMS (ESI): [M+H] + =654.3; SFC analysis (column: Chiralpak AS-3 100×4.6mm ID, 3um; mobile phase: phase A is carbon dioxide, phase B is 0.05% diethylamine / isopropanol; gradient: maintain 50% phase B; flow rate: 2.5 mL / min): chiral column elution position is 2.051 min; 1 H NMR (400MHz, CD3OD) δppm 8.83-8.68(m,1H),7.81(s,1H),7.06-6.85(m,1H),5.04(br dd,J=2.3,6.3Hz,1H),3.61-3.51(m,1H),3.24-3.12(m,4H),2.84(ddd,J=5.4,11.4,16.8 Hz,1H),2.73-2.58(m,3H),2.33-2.24(m,1H),2.15-1.65(m,19H),1.51(d,J=6.1Hz,3H).

[0074] Example 2: (4S)-2-amino-3'-((5 2 S,4S,E)-4-methyl-12-oxo-1 1 H-3-oxa-11-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-2 2 5',6,6',7-Tetrahydro-4'H,5H-spiro[benzo[b]thiophene-4,7'-benzo[d]isoxazole]-3-carboxynitrile (54.0 mg, 0.08 mmol, yield 24%). LCMS (ESI): [M+H] + =654.2; SFC analysis (column: Chiralpak AS-3 100×4.6mm ID, 3um; mobile phase: phase A is carbon dioxide, phase B is 0.05% diethylamine / isopropanol; gradient: maintain 50% phase B; flow rate: 2.5 mL / min): chiral column peak position is 2.699 min; 1H NMR (400MHz, CD3OD) δppm 8.72(d,J=2.8Hz,1H),7.78(s,1H),7.05-6.86(m,1H),5.04(br dd,J=2.2,6.2Hz,1H),3.54(br dd,J=4.9,12.9Hz,1H),3.26-3.08(m,4H),2.90-2.80(m,1H),2.75-2.54(m,3H),2.37-2.25(m,1H),2.17-1.55(m,19H),1.50(d,J=6.3Hz,3H).

[0075] Example 3: (4S)-2-amino-3′-((5 2 S,4S,Z)-4-methyl-11-oxo-1 1 H-3-oxa-10-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-2 2 -yl)-5′,6,6′,7-tetrahydro-4′H,5H-spiro[benzo[b]thiophene-4,7′-benzo[d]isoxazole]-3-carboxynitrile

[0076] Example 4: (4R)-2-amino-3′-((5 2 S,4S,Z)-4-methyl-11-oxo-1 1 H-3-oxa-10-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-2 2 -yl)-5′,6,6′,7-tetrahydro-4′H,5H-spiro[benzo[b]thiophene-4,7′-benzo[d]isoxazole]-3-carboxynitrile

[0077]

[0078] Step 1: Under nitrogen protection, ethyl 2-cyclohexanone carboxylate (50.00 g, 293.75 mmol) was added to a solution of 1,1,3,3-tetramethylguanidine (67.67 g, 587.51 mmol) in toluene (90 mL) and water (1.46 mL, 80.78 mmol), and rinsed with toluene (5 mL). Then, allyl acetate (38.23 g, 381.88 mmol) was added, and rinsed with toluene (5 mL). The reaction solution was cooled to 10–15 °C and protected with nitrogen purging. A solution of (1S,2S)-(-)-1,2-diaminocyclohexane-N,N-bis(2-diphenylphosphobenzoyl) (0.41 g, 0.59 mmol) in toluene (5 mL) was added, and rinsed with toluene (5 mL). Then, a toluene (5 mL) solution of allyl palladium(II) chloride dimer (94.0 mg, 0.26 mmol) was added, and the mixture was rinsed with toluene (5 mL). The mixture was stirred at 10–15 °C for 4 hours, and then at 25 °C for 12 hours. At 10 °C, saturated ammonium chloride (100 mL) was added to the solution, and the mixture was stirred and separated. The aqueous phase was extracted once more with toluene (90 mL). The combined organic layers were washed with water (150 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated to give a yellow, oily crude compound (R)-1-allyl-2-oxocyclohexane-1-carboxylic acid ethyl ester (70 g). LCMS (ESI): [M+H] + =211.1.

[0079] Step 2: Under ice bath conditions, trimethylsilane chloride (105.50 g, 832.23 mmol) was added dropwise to a solution of ethyl (R)-1-allyl-2-oxocyclohexane-1-carboxylate (70.00 g, 332.91 mmol) in ethylene glycol (280 mL), while maintaining the temperature below 20 °C. The mixture was stirred at 25 °C for 12 hours. The reaction solution was cooled to 0 °C, and a solution of sodium hydroxide (34.62 g, 865.55 mmol) in water (280 mL) was added, while maintaining the temperature below 20 °C. Toluene (300 mL) was added, and the mixture was stirred and separated. The aqueous phase was then extracted once more with toluene (100 mL). The combined organic layers were washed twice with water (140 mL), dried with sodium sulfate, filtered, and the filtrate was concentrated to give a yellow oily compound (R)-6-allyl-1,4-dioxaspiro[4.5]dec-6-carboxylic acid ethyl ester (76 g, 275.59 mmol, yield 90%). 1H NMR (400MHz, CDCl3) δppm 5.76-5.57(m,1H),5.15-4.95(m,2H),4.17(ttd,J=3.7,7.1,10.7Hz,2H),4.04-3.85(m,4H),2.83(dd,J=6.5,13.9Hz,1H),2.35(br d,J=8.2Hz,1H),2.03(ddd,J=4.0,9.8,13.9Hz,1H),1.76-1.43(m,7H),1.28(t,J=7.1Hz,3H).

[0080] Step 3: Under nitrogen protection, 9-boronbicyclo[3.3.1]nonane (0.5 M tetrahydrofuran solution, 84.0 mL, 41.99 mmol) was cooled to 0-5 °C, and (R)-6-allyl-1,4-dioxaspiro[4.5]dec-6-carboxylic acid ethyl ester (8.90 g, 35.00 mmol) was added, followed by rinsing with tetrahydrofuran (4.2 mL). The mixture was heated to 25 °C and stirred at 25 °C for 2 hours. The reaction solution was cooled to -40 °C, and 2-chloroacetic acid methyl ester (7.29 g, 67.19 mmol) was added. Below -40 °C, bis(trimethylsilyl)aminolithium (1 M tetrahydrofuran solution, 115.48 mL, 115.48 mmol) was added dropwise. The solution was stirred at 25 °C for 16 hours. The mixture was concentrated to half its volume at 35°C, and an ethanol (33.6 mL) and sodium hydroxide (1.40 g, 35.00 mmol) solution in water (22.4 mL) were added. The solution was stirred at 70°C for 16 hours. The mixture was concentrated to remove the organic solvent, diluted with water (30 mL), and extracted with n-heptane (150 mL * 2). The combined organic layers were washed with water (30 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by rapid column chromatography (silica gel, 0-10% gradient tetrahydrofuran / petroleum ether) to give a yellow oily compound (R)-1,4-dioxabispyrospirol [4.0.5]. 6 0.4 5 Pentadecane-7-one (5.2 g, 23.21 mmol, yield 66%). LCMS (ESI): [M+H] + =225.1. 1H NMR (400MHz, CDCl3) δppm 4.02-3.82(m,4H),2.52-2.42(m,1H),2.40-2.31(m,1H),2.30-2.20(m,1H),2.14-2.03(m,1H),2.02-1.9 4(m,1H),1.92-1.87(m,1H),1.81-1.71(m,4H),1.64-1.57(m,2H),1.53-1.47(m,3H),1.27-1.18(m,1H).

[0081] Steps 4 and 5: Under nitrogen protection, bis(trimethylsilyl)aminolithium (1M tetrahydrofuran solution, 73.6 mL, 73.60 mmol) was cooled to 0-5°C, and (R)-1,4-dioxabisspirol [4.0.5] was added below 5°C. 6 0.4 5 Pentadecane-7-one (15.00 g, 66.87 mmol) was rinsed with tetrahydrofuran (15.00 mL) and stirred at 0–5 °C for 30 minutes. Diethyl oxalate (10.96 mL, 80.25 mmol) was then added at 5 °C. The mixture was heated to 25 °C and stirred for 12 hours. Hydrogen chloride (2 M dioxane solution, 73.6 mL, 147.20 mmol) was added to the reaction mixture to adjust the pH to 6–7. The resulting suspension was used directly in the next reaction step. Hydroxylamine hydrochloride (4.88 g, 70.21 mmol) was added to the above suspension at 25 °C. The reaction mixture was stirred at 70 °C for 16 hours. The solution was concentrated to remove tetrahydrofuran, and water (100 mL) was added. The mixture was extracted with ethyl acetate (100 mL * 3). The combined organic layers were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a yellow, oily crude compound (S)-5,6-dihydro-4H-bis(benzo[d]isoxazole-7,1'-cyclohexane-2',2”-[1,3]dioxolane]-3-carboxylic acid ethyl ester (24.00 g). LCMS (ESI): [M+H] + =322.0.

[0082] Step 6: At 25°C, add pyridine p-toluenesulfonate (37.54 g, 149.36 mmol) and water (240 mL) to a solution of (S)-5,6-dihydro-4H-bis(benzo[d]isoxazole-7,1'-cyclohexane-2',2”-[1,3]dioxolane]-3-carboxylic acid ethyl ester (24.00 g, 74.68 mmol) in acetone (240 mL). Stir the reaction mixture at 70°C for 16 hours. [The solution is then...] The acetone was removed by concentration, and the mixture was extracted with ethyl acetate (150 mL * 3). The combined organic layers were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a brown, oily crude compound, (S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxylic acid ethyl ester (18.60 g, 67.15 mmol, yield 90%). LCMS (ESI): [M+H] + =278.0.

[0083] Step 7: At 25°C, ammonia (77.1 mL) was added to an ethanol (77.00 mL) solution of ethyl (S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxylic acid (18.30 g, 65.99 mmol), and the mixture was stirred for 16 hours. The solution was concentrated to remove some of the ethanol, diluted with water (80 mL), extracted with ethyl acetate (150 mL * 3), and the combined organic layers were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a brown, oily crude compound (S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxamide (13.50 g, 54.44 mmol, yield 82%). LCMS (ESI): [M+H] + =249.0.

[0084] Step 8: At 0°C, trifluoroacetic anhydride (12.26 mL, 87.00 mmol) was added to a solution of (S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxamide (13.50 g, 54.37 mmol) in pyridine (15.80 mL, 195.75 mmol) and acetonitrile (36.00 mL). The reaction solution was stirred at 0°C for 5 minutes. Add ice water (108 mL), extract with ethyl acetate (100 mL * 3), dry the combined organic phases with anhydrous sodium sulfate, filter, concentrate the filtrate, and purify the crude product by rapid column chromatography (silica gel, 0-35% gradient tetrahydrofuran / petroleum ether) to give a yellow solid compound (S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxynitrile (7.00 g, 3.88 mmol, yield 56%). LCMS (ESI): [M+H] + =231.0. 1 H NMR (400MHz, DMSO-d6) δppm 2.85-2.73(m,1H),2.62-2.39(m,3H),2.35-2.18(m,2H),2.08-1.92(m,2H),1.92-1.73(m,5H),1.55-1.41(m,1H).

[0085] Steps 9 and 10: At 25°C, sodium methoxide (30% methanol solution, 1.56 g, 8.69 mmol) was added to a methanol (48 mL) solution of (S)-2'-oxy-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-carboxynitrile (8.00 g, 34.74 mmol). The reaction mixture was stirred at 25°C for 2 hours. Then, ammonium chloride (2.04 g, 38.22 mmol) was added, and the reaction mixture was stirred at 25°C for 16 hours. The suspension was filtered, and the filtrate was concentrated to obtain a yellow solid. LCMS(ESI): [M+H] +=248.1. The above compound was dissolved in N,N-dimethylformamide (40 mL), and 1,8-diazabicyclo[5.4.0]undecane-7-ene (10.91 mL, 72.95 mmol) and diethyl malonate (5.27 mL, 34.74 mmol) were added at 0 °C. The reaction mixture was stirred at 90 °C for 16 hours. Ice water (120 mL) was added, and 1 M hydrochloric acid was added at 0 °C to adjust the pH to 3-4. The solid product was filtered off, washed with water (40 mL), and dried under vacuum to give a gray solid compound (S)-3-(4,6-dihydroxypyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (8.00 g, 25.40 mmol, yield 73%). LCMS (ESI): [M+H] + =316.0.

[0086] Step 11: At 0°C, (S)-3-(4,6-dihydroxypyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (5.6 g, 17.76 mmol) was dissolved in phosphorus oxychloride (16.55 mL, 177.59 mmol). The mixture was stirred at 0°C for 5 minutes, and then diisopropylethylamine (6.46 mL, 39.07 mmol) was added. The reaction mixture was stirred at 80°C for 3 hours. The solution was cooled and poured into ice water (560 mL). The solid product was filtered off, washed with water (50 mL), and dried under vacuum to give a gray solid compound (S)-3-(4,6-dichloropyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (5.2 g, 3.47 mmol, yield 83%). LCMS (ESI): [M+H] + =352.0.

[0087] Step 12: Sodium hydroxide (60% concentration, 204.4 mg, 5.11 mmol) was added to a 30 mL solution of tert-butyl(S)-2-((S)-1-hydroxyethyl)pyrrolidine-1-carboxylic acid ester (916.9 mg, 4.26 mmol) in tetrahydrofuran at 0 °C. The mixture was stirred at 0 °C for 0.5 h, and then (S)-3-(4,6-dichloropyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (1.5 g, 4.26 mmol) was added. The reaction mixture was stirred at 0 °C for 1 h, and then at 25 °C for 1 h. The reaction solution was cooled to 0°C and quenched with water (20 ml). Extraction was performed with ethyl acetate (30 ml x 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by rapid column chromatography (silica gel, 0-30% gradient tetrahydrofuran / petroleum ether) to obtain a colorless oily compound, tert-butyl(S)-2-((S)-1-((6-chloro-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazol-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)oxy)ethyl)pyrrolidine-1-carboxylic acid ester (1255.0 mg, 3.57 mmol, yield 55%). LCMS (ESI): [M+H] + =531.3.

[0088] Step 13: Add hydrogen chloride (2M dioxane solution, 14.12mL, 28.25mmol) to a solution of tert-butyl(S)-2-((S)-1-((6-chloro-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazol-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)oxy)ethyl)pyrrolidine-1-carboxylic acid ester (1.0g, 1.88mmol) in dichloromethane (20mL). The reaction mixture was stirred at 25°C for 2 hours, then concentrated to give a white solid crude compound (S)-3-(4-chloro-6-((S)-1-((S)-pyrrolidine-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (0.94 g). LCMS (ESI): [M+H] + =431.0.

[0089] Step 14: At -78°C, dimethyl sulfoxide (1.80 mL, 25.36 mmol) was added to a solution of oxaloyl chloride (1.08 mL, 12.68 mmol) in dichloromethane (60.0 mL), and the reaction mixture was stirred at -78°C for 15 minutes. Then, a solution of (4-hydroxybutyl)carbamate tert-butyl ester (2.00 g, 10.57 mmol) in dichloromethane (40.0 mL) was slowly added, and the reaction mixture was stirred at -78°C for 30 minutes. Next, triethylamine (7.33 mL, 52.84 mmol) was slowly added, and the reaction mixture was stirred at 20°C for 1 hour. Water (100 mL) was added, and the mixture was extracted with dichloromethane (70 mL * 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a yellow, oily crude compound (4-oxobutyl)carbamate tert-butyl ester (2.00 g, 10.68 mmol). 1 H NMR (400MHz, DMSO-d6) δppm 9.65 (s, 1H), 6.77 (m, 1H), 2.91 (q, J = 6.7Hz, 2H), 2.42 (td, J = 7.2, 0.9Hz, 2H), 1.61 (quin, J = 7.1Hz, 2H), 1.37 (s, 9H).

[0090] Step 15: At 25°C, sodium triacetoxyborohydride (1.03 g, 4.87 mmol) was added to a solution of (S)-3-(4-chloro-6-((S)-1-((S)-pyrrolidine-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-2'-one (700.00 mg, 1.62 mmol) and (4-oxobutyl)carbamate tert-butyl ester (912.00 mg, 4.87 mmol) in dichloromethane (7.00 mL). The reaction mixture was stirred at 25°C for 1 hour. Water (10 mL) was added, and the mixture was extracted with dichloromethane (5 mL * 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by rapid column chromatography (silica gel, 0-10% gradient of methanol / dichloromethane) to give a yellow oily compound (4-((S)-2-((S)-1-((6-chloro-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazol-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)oxy)ethyl)pyrrolidine-1-yl)butyl)carbamate (870.0 mg, 1.44 mmol, yield 89%). LCMS (ESI): [M+H) + =602.2.

[0091] Step 16: At 25°C, add cesium carbonate (4.87 g, 14.95 mmol) to a solution of (4-((S)-2-((S)-1-((6-chloro-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)oxy)ethyl)pyrrolidine-1-yl)butyl)carbamate (600.00 mg, 0.99 mmol) and 2-(1H-pyrazole-3-yl)acetic acid (966.0 mg, 4.98 mmol) in dimethyl sulfoxide (10.00 mL). Stir the reaction mixture at 80°C for 20 minutes. Filter the solution, and adjust the pH of the filtrate to 6-7 with concentrated hydrochloric acid (70 μL). The reaction mixture was purified by rapid column chromatography (C18, 0-75% methanol / water gradient) to give a white solid 2-(1-(6-((S)-1-((S)-1-(4-((tert-butoxycarbonyl)amino)butyl)pyrrolidine-2-yl)ethoxy)-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)-1H-pyrazol-3-yl)acetic acid (240.0 mg, 0.35 mmol, yield 35%). LCMS (ESI): [M+H] + =692.2.

[0092] Step 17: At 25°C, add hydrogen chloride (2M dioxane solution, 2.17 mL, 4.34 mmol) to a solution of 2-(1-(6-((S)-1-((S)-1-(4-((tert-butoxycarbonyl)amino)butyl)pyrrolidine-2-yl)ethoxy)-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)-1H-pyrazol-3-yl)acetic acid (200.00 mg, 0.29 mmol) in dichloromethane (4.00 mL). Stir the reaction mixture at 25°C for 1 hour. The reaction solution was concentrated under reduced pressure to give a white solid crude compound 2-(1-(6-((S)-1-((S)-1-(4-aminobutyl)pyrrolidine-2-yl)ethoxy)-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)-1H-pyrazol-3-yl)acetic acid (260.0 mg, 0.44 mmol). LCMS (ESI): [M+H] + =592.3

[0093] Step 18: At 25°C, add tri-n-butylcyclophosphine (50% ethyl acetate solution, 745.00 mg, 1.37 mmol) to a solution of 2-(1-(6-((S)-1-((S)-1-(4-aminobutyl)pyrrolidine-2-yl)ethoxy)-2-((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)pyrimidin-4-yl)-1H-pyrazol-3-yl)acetic acid (270.0 mg, 0.46 mmol) and diisopropylethylamine (3.19 mL, 18.25 mmol) in dichloromethane (45.00 mL). Stir the reaction mixture at 25°C for 12 hours. Add water (50 mL), extract with dichloromethane (40 mL * 2), dry the combined organic phases with anhydrous sodium sulfate, filter, concentrate the filtrate to give a white solid compound (5... 2 S,4S,Z)-4-methyl-2 2 -((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)-1 1 H-3-oxa-10-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-11-one (220.0 mg, 0.44 mmol, yield 84%). LCMS (ESI): [M+H] + =574.3

[0094] Step 19: Place (5 2 S,4S,Z)-4-methyl-2 2 -((S)-2'-oxo-5,6-dihydro-4H-spiro[benzo[d]isoxazole-7,1'-cyclohexane]-3-yl)-1 1 H-3-oxa-10-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-11-one (200.00 mg, 0.35 mmol), sulfur (18.00 mg, 0.56 mmol) and ammonium acetate (43.00 mg, 0.56 mmol) were added to ethanol (4.00 mL). The reaction mixture was stirred at 60 °C for 15 minutes, and then malononitrile (38.00 mg, 0.58 mmol) was slowly added. The reaction mixture was stirred at 80 °C for 4 hours. The reaction solution was purified by rapid column chromatography (C18, 0-80% gradient of methanol / water). The product was separated by SFC (column: DAICEL CHIRALCEL OD (250mm*30mm, 10um); mobile phase: phase A is carbon dioxide, phase B is 0.1% ammonia / ethanol; phase B is maintained at 60%; flow rate: 80 mL / min) to obtain two stereoisomers, Example 3 and Example 4.

[0095] Example 3: (4S)-2-amino-3′-((5 2 S,4S,Z)-4-methyl-11-oxo-1 1 H-3-oxa-10-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-2 2 5′,6,6′,7-Tetrahydro-4′H,5H-spiro[benzo[b]thiophene-4,7′-benzo[d]isoxazole]-3-carboxynitrile (yellow solid, 53.64 mg). SFC analysis (column: Chiralcel OD-3 50×4.6 mm ID, 3 μm; mobile phase: phase A was carbon dioxide, phase B was 0.05% diethylamine / ethanol; gradient: phase B from 5% to 40% in 2 min, holding phase B at 40% for 1.2 min, then phase B at 5% for 0.8 min; flow rate: 4 mL / min): chiral column elution position was 2.372 min; LCMS (ESI): [M+H] + =654.1. 1 H NMR(400MHz,CD3OD)δppm 8.70-8.63(m,1H),7.44-7.38(m,1H),6.51(d,J=2.7Hz,1H),5.02-4.92(m,1H),3.72-3.59(m,3H),3.27-2.99(m,5H),2.79(br s,1H),2.51(br d,J=14.9Hz,4H),2.21-1.85(m,10H),1.81-1.60(m,6H),1.50(br d,J=6.2Hz,3H).

[0096] Example 4: (4R)-2-amino-3′-((5 2 S,4S,Z)-4-methyl-11-oxo-1 1 H-3-oxa-10-aza-2(4,6)-pyrimidin-1(1,3)-pyrazole-5(2,1)-pyrrolidinecyclododecane-2 2 5′,6,6′,7-Tetrahydro-4′H,5H-spiro[benzo[b]thiophene-4,7′-benzo[d]isoxazole]-3-carboxynitrile (yellow solid, 1.78 mg). SFC analysis (column: Chiralcel OD-3 50×4.6 mm ID, 3 μm; mobile phase: phase A was carbon dioxide, phase B was 0.05% diethylamine / ethanol; gradient: phase B from 5% to 40% in 2 min, holding phase B at 40% for 1.2 min, then phase B at 5% for 0.8 min; flow rate: 4 mL / min): chiral column elution position was 2.671 min; LCMS (ESI): [M+H] + =654.3;1 HNMR (400MHz, CD3OD) δppm8.72-8.62(m,1H),7.42(s,1H),6.51(d,J=2.7Hz,1H),4.99(dd,J=8.4, 6.3Hz,1H),3.71-3.61(m,3H),3.28-3.00(m,5H),2.93-2.55(m,5H),2.19-1.61(m,16H),1.54(br d,J=6.1Hz,3H).

[0097] Example 1: 3D cell proliferation experiment

[0098] 3D proliferation experiment of AsPC-1 cells

[0099] The diluted analyte compound was added to 384-well low-adsorption cell culture plates using a nanoliter pipetting system (LABCYTE, P-0200). After cell seeding, the plates were placed in a 37°C, 5% CO2 incubator. After co-incubating the compound with the cells for 5 days, the following steps were taken: The 3D reagent was used, and the luminescence value was read using an Envision multi-functional microplate reader (the light signal is directly proportional to the amount of ATP in the system, and the ATP content directly represents the number of viable cells in the system). Finally, the IC50 (half-maximal inhibitory concentration) of the compound was obtained using a non-linear fitting formula with XLFIT software.

[0100] Y=Bottom+(Top-Bottom) / (1+10^((LogIC50-X)×HillSlope))

[0101] X: Log value of compound concentration

[0102] Y: Inhibition rate (%)

[0103] Inhibition rate (%) = 100 × (Negative control average value - Compound reading) / (Negative control average value - Positive control average value)

[0104] Negative control: DMSO

[0105] Positive control: Medium only

[0106] The inhibitory effects of the compounds of this invention on the 3D proliferation of AGS and AsPC-1 cells are shown in Table 1. (A: IC50 < 100 nM; B: 100 nM ~ 1 μM; C: 1 μM ~ 10 μM; D: > 10 μM)

[0107] Table 1

[0108] Example WT_IC50 AsPC-1_IC50 NCI358_IC50 H727_IC50 1 C C C C 2 D C C C 3 B C B B 4 C C C C

Claims

1. A heterocyclic compound as shown in Formula I, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: Formula I in, W1 is independently selected from CR W1 Or N; Among them, W2 is independently selected from CR W2 Or N; Among them, W3 is independently selected from CR W3 Or N; Among them, R W1 R W2 R W3 Each is independently selected from hydrogen, deuterium, halogen, C1-C6 alkyl, C1-C6 alkoxy, cyano, C1-C6 cycloalkyl, halogenated C1-C6 alkyl, nitro, hydroxyl, NR a R b ; Among them, R1, R 1’ R2, R 2’ R3, R 3’ Each can be independently represented as hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy C1-C6 alkyl, or halo-C1-C6 alkyl. Where X1 represents non-existent or -CR L R L’ -; Where X2 represents -(CR) S R S’ ) n -; Among them, R L R L’ Each can be independently represented as hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy C1-C6 alkyl, or halo-C1-C6 alkyl. Among them, R S R S’ Each can independently represent hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy C1-C6 alkyl, or halo-C1-C6 alkyl; or arbitrarily CR S R S’ It can be replaced by O, NH, or N(CH3); Where n represents an integer selected from 1, 2, 3, 4, 5, and 6; Among them, X 3 It represents O, NH, N(CH3) or -CR T R T’ -; Among them, R T R T’ Each can be independently represented as hydrogen, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, hydroxy C1-C6 alkyl, or halo-C1-C6 alkyl. Among them, R a R b Each can be used independently to represent hydrogen or C1-C6 alkyl groups.

2. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, W1 and W2 are selected from N.

3. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, W3 is selected from CH.

4. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, X1 indicates that it does not exist or CH2.

5. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, X2 represents -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH(CH3)CH2CH2CH2CH2-, -C(CH3)2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2CH2-, -CH2C(CH3)2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2 -, -CH2CH2C(CH3)2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH(CH3)CH2-, -CH2OCH2CH2CH2-, -CH2CH2OCH2CH2-, -CH2CH2CH2OCH2-, -CH2NHCH2CH2CH2-, -CH2CH2NHCH2CH2-.

6. The heterocyclic compound of Formula I according to claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, X3 represents O or CH2.

7. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, R1, R 1’ It represents H or CH3.

8. The heterocyclic compound of Formula I according to claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, R2, R 2’ It represents H or CH3.

9. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, or its stereoisomer, wherein, R3, R 3’ It represents H or CH3.

10. A compound having the following structure: 。

Images