CN117946135A - Heterocyclic compound, pharmaceutical composition and application thereof - Google Patents

Abstract

The present invention discloses a heterocyclic compound which can be used as a KRAS inhibitor. The compound of the present invention is an effective, safer KRAS inhibitor with better pharmacokinetic properties and can meet clinical needs.

Description

Heterocyclic compounds, pharmaceutical compositions and applications thereof

Technical Field

The invention relates to a heterocyclic compound, a pharmaceutical composition and application thereof.

Background technique

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), of which 85% of RAS-driven cancers are caused by KRAS subtype mutations. 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, and the most common mutations include: p.G12D (41%), p.G12V (28%), and p.G12C (14%).

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. After receiving external signals, the upstream cell surface receptors such as EGFR (ErbBl), HER2 (ErbB2), ErbB3 and ErbB4 will transmit the signal to the downstream through the RAS protein. When the KRAS protein is not activated, it is tightly bound to GDP (guanine nucleotide diphosphate). After being activated by guanine nucleotide exchange factors such as SOSl, it binds to GTP (guanine nucleotide triphosphate) and becomes a kinase active state. After the KRAS gene mutates, it can independently transmit growth and proliferation signals to the downstream pathways without relying on upstream growth factor receptor signals, causing uncontrolled cell growth and tumor progression. At the same time, whether the KRAS gene is mutated is also an important indicator of tumor prognosis. Statistical results show that among the subtypes of KRAS, KRAS is also a common submutation, 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 tumor treatment. Therefore, it is necessary to develop more effective, safer, and better pharmacokinetic KRAS inhibitors to meet clinical needs.

Summary of the invention

The technical problem to be solved by the present invention is to provide a heterocyclic compound, a pharmaceutical composition and applications thereof in view of the defect that the existing pan-KRAS inhibitors have a relatively single structure. The compound of the present invention has a novel structure and good activity and selectivity.

The present invention solves the above technical problems through the following technical solutions.

The present invention provides a heterocyclic compound as shown in Formula I, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, or a solvate thereof (referring to the aforementioned heterocyclic compound as shown in Formula I, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof):

Wherein, _W1 is independently selected from CR ^W1 or N;

Wherein, _W2 is independently selected from CR ^W2 or N;

Wherein, _W3 is independently selected from CR ^W3 or N;

wherein R ^W1 , R ^W2 , and R ^W3 are each independently selected from hydrogen, deuterium, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ cycloalkyl, halogenated C ₁ -C ₆ alkyl, nitro, hydroxyl, and NR ^a R ^b ;

wherein R ¹ , R ^1′ , R ² , R ^2′ , R ³ , and R ^3′ each independently represent hydrogen, halogen, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, hydroxy C ₁ -C ₆ alkyl, or halogenated C ₁ -C ₆ alkyl;

Wherein, Rx represents hydrogen, C ₁ -C ₆ alkyl;

Wherein, Cy represents a 3-10 membered heterocycle, a 6-10 membered aryl group or a 5-10 membered heteroaryl group; and the Cy may be arbitrarily substituted by 0, 1 or 2 substituents selected from halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ cycloalkyl, halogenated C ₁ -C ₆ alkyl, nitro, -(C ₀ -C ₆ )hydroxy, -(C ₀ -C ₆ )NR ^a R ^b , -(C ₀ -C ₆ )C(O)NR ^a R ^b , -(C ₀ -C ₆ )C(O)OR ^a , -(C ₀ -C ₆ )OC(O)R ^a ; wherein ^Ra and R ^b each independently represent hydrogen or C ₁ -C ₆ alkyl.

In a preferred technical solution of the present invention, W ₁ and W ₂ are selected from N.

In a preferred technical solution of the present invention, W ₃ is selected from CH.

In the preferred technical solution of the present invention, wherein Cy represents Wherein, R ₄ , R _4' , R ₅ , R _5' , R ₆ , R _6' , R ₇ , R _7' , R ₈ each independently represent hydrogen or C ₁ -C ₆ alkyl; or R ₄ , R _4' pair, R ₅ , R _5' pair, R ₆ , R _6' pair, R ₇ , R _7' pair, respectively, together with the carbon atom to which they are connected, form a 3-6 membered ring.

In the preferred technical solution of the present invention, wherein Cy represents

In the preferred technical solution of the present invention, wherein Cy represents Further, the Cy is substituted with 0, 1, or 2 substituents selected from halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ cycloalkyl, halogenated C ₁ -C ₆ alkyl, nitro, -(C ₀ -C ₆ )hydroxy, -(C ₀ -C ₆ )NR ^a R ^b , -(C ₀ -C ₆ )C(O)NR ^a R ^b , -(C ₀ -C ₆ )C(O)OR ^a , or -(C ₀ -C ₆ )OC(O)R ^a .

In the preferred technical solution of the present invention, wherein Cy represents

In a preferred technical solution of the present invention, R ₁ and R _1′ represent H or CH ₃ .

In a preferred technical solution of the present invention, R ₂ and R _2' represent H or CH ₃ .

In a preferred technical solution of the present invention, R ₃ and R _3' represent H or CH ₃ .

Specifically, the present invention provides a compound having the following structure:

The term "pharmaceutically acceptable" means that salts, solvents, excipients, etc. are generally non-toxic, safe, and suitable for use by patients. The "patient" is preferably a mammal, more preferably a human.

The term "pharmaceutically acceptable salt" refers to a salt prepared from a compound of the present invention and a relatively nontoxic, pharmaceutically acceptable acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting a neutral form of such compound with a sufficient amount of a pharmaceutically acceptable base 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 compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting a neutral form of such compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acid includes an inorganic acid, and the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like. The pharmaceutically acceptable acid includes organic acids, including but not limited to acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, bitartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthoic 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).

The term "solvate" refers to a substance formed by the combination of a compound of the present invention and a stoichiometric or non-stoichiometric solvent. The solvent molecules in the solvate may exist in an ordered or non-ordered arrangement. The solvent includes, but is not limited to, water, methanol, ethanol, etc.

The terms "compound", "pharmaceutically acceptable salt", "solvate" and "solvate of a pharmaceutically acceptable salt" may exist in the form of a single stereoisomer or a mixture thereof (e.g., a racemate) if stereoisomers exist. The term "stereoisomer" refers to cis-trans isomers or optical isomers. These stereoisomers can be separated, purified and enriched by asymmetric synthesis methods or chiral separation methods (including but not limited to thin layer chromatography, rotary chromatography, column chromatography, gas chromatography, high pressure liquid chromatography, etc.), and can also be obtained by chiral separation by 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 relative to all stereoisomers of the compound is not less than 95%.

The terms "compound", "pharmaceutically acceptable salt", "solvate" and "solvate of a pharmaceutically acceptable salt" may exist as a single tautomer or a mixture thereof if tautomers exist, preferably in a form in which the more stable tautomer is the main tautomer.

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

The term "alkyl" refers to a linear or branched, saturated, monovalent hydrocarbon group having a specified number of carbon atoms (e.g., C1-C6). Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, etc.

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 ways

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

NMR measurements were performed using a Bruker AVANCE-400 nuclear magnetic spectrometer. The solvent used for the measurements was indicated in the spectrum analysis.

MS was determined using Agilent 1200-G1956A/1200-6110A/1200-6140A/1260-6125B/Prime-6125B/1260-6120 LC/MS, SHIMADZU 20A-2010/20A-2020 LC/MS, and Waters ACQ-QDA LC/MS.

HPLC analysis was performed using a SHIMADZU 20A high performance liquid chromatograph.

SFC analysis was performed using Waters UPCC with PDA Detector and QDa Detector, Waters UPC ² with PDA detector, Agilent 1260 with DAD detector, Shimadzu LC-20AB with PDA detector, and Shimadzu LC-20AD with PDA detector.

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

The SFC separation was performed using The Berger MG II, MG III, Sepiatec's Prep SFC 100 system, Waters Prep 80Q SFC SYSTEM, Prep 150AP SFC SYSTEM, Prep 200SFC SYSTEM, and Prep350SFC SYSTEM.

Flash column chromatography separation was performed using a Biotage IsoleraOne flash preparative chromatograph.

The thin layer chromatography silica gel plate used was GF254 acrylic adhesive silica gel plate produced by Anhui Liangchen Silicon Source Material Co., Ltd. The specification of the silica gel plate used in thin layer chromatography (TLC) was 0.25 mm, and the specification of the thin layer chromatography separation and purification product was 0.5 mm.

Pressurized hydrogenation reaction hydride bottles and hydrogen gas cylinders.

Microwave reactions were performed using a Biotage Initiator+ microwave synthesizer.

The glove box is customized by DELLIX.

Example 1: 2-(1-(2-((R)-2'-amino-3'-cyano-5,6-dihydro-4H,5'H,7'H-spiro[benzo[d]isoxazole-7,4'-thieno[2,3-c]pyran]-3-yl)-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-4-yl)-1H-pyrazol-3-yl)-N-isopropylacetamide

Step 1: Under nitrogen protection, add 4-oxotetrahydro-2H-pyran-3-carboxylic acid methyl ester (20.00 g, 126.46 mmol) to a solution of 1,1,3,3,-tetramethylguanidine (29.13 g, 252.92 mmol) in toluene (36 mL) and water (1.00 mL, 55.64 mmol), and rinse with toluene (2 mL). Then add allyl acetate (16.46 g, 164.40 mmol) and rinse with toluene (2 mL). Cool to 10-15 ° C, replace with nitrogen for protection. Add a toluene (2 mL) solution of (1S, 2S)-(-)-1,2-diaminocyclohexane-N,N-bis(2-diphenylphosphobenzoyl) (0.28 g, 0.40 mmol) and rinse with toluene (2 mL). Then add a toluene (2 mL) solution of allylpalladium (II) chloride dimer (0.06 g, 0.18 mmol) and rinse with toluene (2 mL). Stir the mixture at 10-15°C for 8 hours. Add saturated ammonium chloride (40 mL) to the solution at 10°C, stir and separate the liquids. Extract the solution once with toluene (40 mL). Wash the combined organic layers with water (40 mL), dry with sodium sulfate, filter, and concentrate the filtrate to obtain a yellow oily crude compound (S)-3-allyl-4-oxotetrahydro-2H-pyran-3-carboxylic acid methyl ester (30 g). LCMS (ESI): [M+H] ⁺ = 199.1.

Step 2: In an ice bath, trimethylsilyl chloride (54.36 mL, 428.82 mmol) was added dropwise to a solution of (S)-3-allyl-4-oxotetrahydro-2H-pyran-3-carboxylic acid methyl ester (34.00 g, 171.53 mmol) in ethylene glycol (136 mL), with the temperature maintained below 20°C. The mixture was stirred at 25°C for 12 hours. A solution of sodium hydroxide (17.84 g, 445.98 mmol) in water (136 mL) was added to the solution at 0°C, with the temperature maintained below 20°C. Toluene (140 mL) was added, stirred and separated. The aqueous phase was extracted once with toluene (50 mL). The combined organic layer was washed with water (70 mL*2), dried over sodium sulfate, filtered, and the filtrate was concentrated to obtain a yellow oily compound (S)-6-allyl-1,4,8-trioxaspiro[4.5]decane-6-carboxylic acid methyl ester (34 g, 140.50 mmol, yield 82%). LCMS (ESI): [M+H] ⁺ = 243.1.

Step 3: Under nitrogen protection, 9-boranebicyclo[3.3.1]nonane (0.5M tetrahydrofuran solution, 307mL, 153.55mmol) was cooled to 0-5°C, (S)-6-allyl-1,4,8-trioxaspiro[4.5]decane-6-carboxylic acid methyl ester (31.00g, 127.96mmol) was added, and rinsed with tetrahydrofuran (30mL). The mixture was warmed to 25°C and stirred at 25°C for 2 hours. The reaction solution was cooled to -40°C, methyl 2-chloroacetate (26.66g, 245.68mmol) was added, and then lithium bis(trimethylsilyl)amide (1M tetrahydrofuran solution, 422mL, 422.26mmol) was added dropwise. The solution was stirred at 25°C for 16 hours. The solution was concentrated to remove half of the tetrahydrofuran solvent. At 25°C, ethanol (125 mL) and sodium hydroxide (5.12 g, 127.96 mmol) in water (83 mL) were added to the solution. The solution was stirred at 70°C for 16 hours. Water (500 mL) was added for dilution and extracted with n-heptane (500 mL*3). The combined organic layer was washed twice with water (500 mL), dried over sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by flash column chromatography (silica gel, 0-25% gradient of tetrahydrofuran/petroleum ether) to obtain a yellow oily compound (R)-1,4,13-trioxabisiro[4.0.5 ⁶ .4 ⁵ ]pentadecan-7-one (10 g, 44.20 mmol, yield 35%). LCMS (ESI): [M+H] ⁺ = 227.1.

Step 4 and Step 5: Under nitrogen protection, lithium bis(trimethylsilyl)amide (1M tetrahydrofuran solution, 49 mL, 48.61 mmol) was cooled to 0-5°C, (R)-1,4,13-trioxabisiro[4.0.5 ⁶ .4 ⁵ ]pentadecan-7-one (10.00 g, 44.19 mmol) was added below 5°C, and rinsed with tetrahydrofuran (15.00 mL). After stirring at 0-5°C for 30 minutes, diethyl oxalate (7.24 mL, 53.03 mmol) was added at 5°C. The mixture was heated to 25°C and stirred for 12 hours. Hydrogen chloride (2M dioxane solution) was added to the reaction solution to adjust the pH to 6-7, and the resulting suspension was directly used for the next step reaction. LCMS (ESI): [M+H] ⁺ = 327.0. At 25°C, hydroxylamine hydrochloride (3.22 g, 46.41 mmol) was added to the above suspension, and the reaction solution was stirred at 70°C for 16 hours. The reaction solution was diluted with water (50 mL), extracted with ethyl acetate (50 mL*3), and the combined organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a brown oily crude compound (R)-5,5',6,6'-tetrahydro-2'H,4H-bisspiro[benzo[d]isoxazole-7,3'-pyran-4',2"-[1,3]dioxolane]-3-carboxylic acid ethyl ester (15.00 g, 37.11 mmol, yield 84%). LCMS (ESI): [M+H] ⁺ = 324.0.

Step 6: Pyridine p-toluenesulfonate (23.32 g, 92.78 mmol) and water (150 mL) were added to a solution of (R)-5,5',6,6'-tetrahydro-2'H,4H-bispiro[benzo[d]isoxazole-7,3'-pyran-4',2"-[1,3]dioxolane]-3-carboxylic acid ethyl ester (15.00 g, 46.39 mmol) in acetone (150 mL) at 25°C, and the reaction solution was stirred at 65°C for 12 hours. The solution was concentrated to remove acetone and washed with ethyl acetate ( 300mL*3) extraction. The combined organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by flash column chromatography (silica gel, 0-50% gradient of tetrahydrofuran/petroleum ether) to obtain a yellow oily compound (R)-4'-oxo-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-3-carboxylic acid ethyl ester (3.50g, 12.53mmol, yield 27%). LCMS (ESI): [M+H] ⁺ = 280.1.

Step 7: At 25°C, add ammonia water (6.27 mL) to a solution of (R)-4'-oxo-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-3-carboxylic acid ethyl ester (1.50 g, 5.37 mmol) in ethanol (6.00 mL) and stir for 16 hours. Concentrate the solution, dilute with water (10 mL), and extract with ethyl acetate (10 mL*3). Dry the combined organic layer with anhydrous sodium sulfate, filter, and concentrate the filtrate to obtain a brown solid compound (R)-4'-oxo-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-3-carboxamide (2.00 g, 5.19 mmol, yield 97%). LCMS (ESI): [M+H] ⁺ = 251.0.

Step 8: To a solution of (R)-4'-oxo-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-3-carboxamide (2000 mg, 7.99 mmol) with pyridine (1548 uL, 19.18 mmol) and acetonitrile (5400.00 uL) was added trifluoroacetic anhydride (1352 uL, 9.59 mmol) at 0°C, and the mixture was stirred at 0°C for 5 minutes. Ice water (12 mL) was added, and the mixture was extracted with ethyl acetate (10 mL*3). The organic phase was dried over sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by flash column chromatography (silica gel, 0-30% gradient of tetrahydrofuran/petroleum ether) to obtain a yellow oily compound (R)-4'-oxo-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-3-carbonitrile (900 mg, 3.88 mmol, yield 48%). LCMS (ESI): [M+H] ⁺ = 233.0.

Step 9: At 25°C, sodium methoxide (30% methanol solution, 155.07 mg, 0.86 mmol) was added to a solution of (R)-4'-oxo-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-3-carbonitrile (800 mg, 3.44 mmol) in methanol (4000 uL), and the mixture was stirred at 25°C for 2 hours, and ammonium chloride (203 mg, 3.79 mmol) was added, and the mixture was stirred at 25°C for 16 hours. The suspension was filtered, and the filtrate was concentrated to obtain a yellow solid compound (R)-4'-oxo-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-3-carboxamidine (1200 mg, 3.37 mmol, yield 98%). LCMS (ESI): [M+H] ⁺ = 250.0.

Step 10: To a solution of (R)-4'-oxo-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-3-carboximidamide (1200 mg, 4.81 mmol) in dimethylformamide (6000 uL) was added 1.8-diazabicyclo[5.4.0]undec-7-ene (1512 uL, 10.11 mmol) and diethyl malonate (731 uL, 4.81 mmol) at 0°C, and the mixture was stirred at 90°C for 16 hours. Ice water (8 ml) was added and the pH was adjusted to 3 with 1M hydrochloric acid. The reaction solution was extracted with ethyl acetate (8 mL*3), the organic phase was dried over sodium sulfate, filtered, and the filtrate was concentrated to obtain a brown solid compound (R)-3-(4,6-dihydroxypyrimidin-2-yl)-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-4'-one (990 mg, 3.12 mmol, yield 65%). LCMS (ESI): [M+H] ⁺ = 318.0.

Step 11: At 0°C, dissolve (R)-3-(4,6-dihydroxypyrimidin-2-yl)-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-4'-one (1110 mg, 3.50 mmol) in phosphorus oxychloride (3261 uL, 34.98 mmol), stir at 0°C for 5 minutes, then add diisopropylethylamine (1215 uL, 7.35 mmol) and stir at 80°C for 3 hours. The solution was poured into ice water (10 mL), extracted with ethyl acetate (10 mL*3), the combined organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated to obtain a brown oily compound (R)-3-(4,6-dichloropyrimidin-2-yl)-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-4'-one (2320 mg, 3.47 mmol, yield 99%). LCMS (ESI): [M+H] ⁺ = 353.9.

Step 12: To a solution of (R)-3-(4,6-dichloropyrimidin-2-yl)-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-4'-one (600 mg, 0.90 mmol) and diisopropylethylamine (445 uL, 2.69 mmol) in acetonitrile (6000 uL) was added (S)-((S)-1-methylpyrrolidin-2-yl)ethan-1-ol (232 mg, 1.80 mmol) at 25°C. The mixture was stirred at 25°C for 16 hours. The reaction product was a yellow solution. The mixture was concentrated, diluted with water (2 ml), extracted with ethyl acetate (2 ml * 3), the combined organic layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by flash column chromatography (silica gel, 0-20% gradient methanol/dichloromethane) to give a brown oily compound (R)-3-(4-chloro-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-2-yl)-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-4'-one (140 mg, 313.2 umol, yield 35%). LCMS (ESI): [M+H] ⁺ = 447.0.

Step 13: To a solution of (R)-3-(4-chloro-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-2-yl)-5,5',6,6'-tetrahydro-2'H,4H,4'H-spiro[benzo[d]isoxazole-7,3'-pyran]-4'-one (130.0 mg, 0.29 mmol) in ethanol (1500 uL) was added sulfur (14.9 mg, 0.47 mmol) and ammonium acetate (35.9 mg, 0.47 mmol) at 25°C, and the reaction solution was stirred at 60°C for 15 minutes. Then, a solution of malononitrile (31.7 mg, 0.48 mmol) in ethanol (200 uL) was slowly added. The solution was stirred at 80°C for 16 hours. The reaction mixture was diluted with water (2 mL) and extracted with ethyl acetate (2 mL*3). The combined organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by flash column chromatography (silica gel, 0-10% gradient methanol/dichloromethane) to give a brown solid compound (R)-2'-amino-3-(4-chloro-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H,5'H,7'H-spiro[benzo[d]isoxazole-7,4'-thieno[2,3-c]pyran]-3'-carbonitrile (105 mg, 199.22 umol, yield 68%). LCMS (ESI): [M+H] ⁺ =527.1.

Step 14: To a solution of (R)-2'-amino-3-(4-chloro-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H,5'H,7'H-spiro[benzo[d]isoxazole-7,4'-thieno[2,3-c]pyran]-3'-carbonitrile (90 mg, 0.17 mmol) and cesium carbonate (167 mg, 0.51 mmol) in dimethyl sulfoxide (1000 uL) was added N-isopropyl-2-(1H-pyrazol-3-yl)acetamide (purity 60%, 95 mg, 0.34 mmol) at 25°C. The mixture was stirred at 90°C for 2 hours. The reaction was a yellow solution. The reaction solution was filtered, and the filtrate was purified by preparative HPLC to give a yellow solid compound 2-(1-(2-((R)-2'-amino-3'-cyano-5,6-dihydro-4H,5'H,7'H-spiro[benzo[d]isoxazole-7,4'-thieno[2,3-c]pyran]-3-yl)-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-4-yl)-1H-pyrazol-3-yl)-N-isopropylacetamide (3.97 mg, 6.03 umol, yield 4%). LCMS (ESI): [M+H] ⁺ = 658.1. 1HNMR (400 MHz, CD ₃ OD) δ ppm 8.73-8.71 (m, 1H), 7.23-7.22 (m, 1H), 6.53-6.51 (m, 1H), 5.48-5.42 (m, 1H), 4.68-4.64 (m, 2H), 4.15-4.10 (m, 1H), 4.04-3.95 (m, 1H), 3.91-3.83 (m, 1H), 3.62-3.60 (m, 2H), 3.16-3.07 (m, 1H), 2.93-2.77 (m, 2H), 2.60-2.51 (m, 3H), 2.48-2.35 (m, 2H), 2.14-1.75 (m, 8H), 1.44-1.40 (m, 3H), 1.16 (d, J = 6.6 Hz, 6H).

Example 2: (R)-2'-amino-3-(4-(4-methyl-4,7-diazaspiro[2.5]octan-7-yl)-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H,5'H,7'H-spiro[benzo[d]isoxazole-7,4'-thieno[2,3-c]pyran]-3'-carbonitrile

Example 3: (S)-2'-amino-3-(4-(4-methyl-4,7-diazaspiro[2.5]octan-7-yl)-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H,5'H,7'H-spiro[benzo[d]isoxazole-7,4'-thieno[2,3-c]pyran]-3'-carbonitrile

Example 2 and Example 3 were prepared by referring to the synthesis method of Example 1: 4-methyl-4,7-diazaspiro[2.5]octane hydrochloride (67 mg, 0.34 mmol) was added to a solution of (R)-2'-amino-3-(4-chloro-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H,5'H,7'H-spiro[benzo[d]isoxazole-7,4'-thieno[2,3-c]pyran]-3'-carbonitrile (161 mg, 0.31 mmol) and diisopropylethylamine (504 uL, 3.05 mmol) in dimethyl sulfoxide (2000 uL) at 25°C. The mixture was stirred at 90°C for 2 hours. The reaction solution was filtered and the filtrate was purified by preparative HPLC to obtain the product as a yellow solid compound (21 mg).

The product was separated by SFC (column: DAICEL CHIRALPAK IG (250 mm*30 mm, 10 um); mobile phase: phase A is carbon dioxide, phase B is 0.1% ammonia water/ethanol; phase B is maintained at 60%; flow rate: 80 ml/min), and the two stereoisomers obtained were purified again by preparative HPLC to obtain two stereoisomers Example 2 and Example 3.

Example 2: (S)-2'-amino-3-(4-(4-methyl-4,7-diazaspiro[2.5]octan-7-yl)-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H,5'H,7'H-spiro[benzo[d]isoxazole-7,4'-thieno[2,3-c]pyran]-3'-carbonitrile. LCMS (ESI): [M+H] ⁺ = 617.3; SFC analysis (column: Chiralpak IG-3 50×4.6 mm ID, 3 um; mobile phase: phase A is carbon dioxide, phase B is 0.05% diethylamine/ethanol; gradient: maintain 40% phase B; flow rate: 4 ml/min): chiral column peak position is 0.831 min; ¹ H NMR (400 MHz, CD ₃ OD) δ ppm 8.60-8.43(m,1H),6.19-6.12(m,1H),5.36-5.25(m,1H),4.64(s,2H),4.13-4.06(m,1H),3.88-3.75(m,3H),3.72-3.64(m,2H),3.59-3.52(m,1H),3.20-3.06(m,2H),3.05-2.98( m, 2H), 2.94-2.86 (m, 3H), 2.82-2.70 (m, 1H), 2.69-2.62 (m, 1H), 2.54-2.45 (m, 3H), 2.43-2.29 (m, 2H), 2.23-1.82 (m, 6H), 1.48-1.40 (m, 3H), 0.80-0.72 (m, 2H), 0.66-0.58 (m, 2H)

Example 3: (R)-2'-amino-3-(4-(4-methyl-4,7-diazaspiro[2.5]octan-7-yl)-6-((S)-1-((S)-1-methylpyrrolidin-2-yl)ethoxy)pyrimidin-2-yl)-5,6-dihydro-4H,5'H,7'H-spiro[benzo[d]isoxazole-7,4'-thieno[2,3-c]pyran]-3'-carbonitrile. LCMS (ESI): [M+H] ⁺ = 617.3; SFC analysis (column: Chiralpak IG-3 50×4.6 mm ID, 3 um; mobile phase: phase A is carbon dioxide, phase B is 0.05% diethylamine/ethanol; gradient: maintain 40% phase B; flow rate: 4 ml/min): chiral column peak position is 1.373 min; ¹ H NMR (400 MHz, CD ₃ OD) δ ppm 8.61-8.48 (m, 1H), 6.13-6.06 (m, 1H), 5.36-5.26 (m, 1H), 4.67-4.63 (m, 2H), 4.13-4.06 (m, 1H), 3.87-3.73 (m, 3H), 3.67-3.59 (m, 2H), 3.53-3.44 (m, 1H), 3.29-3.22 (m, 1H), 3.16-3.06 (m, 1H), 3.04-2.98 (m, 2H), 2.95-2.85 (m, 1H), 2.81-2.70 (m, 4H), 2.52-2.45 (m, 3H), 2.41-2.33 (m, 1H), 2.29-2.17 (m, 1H), 2.10-1.79 (m, 6H), 1.44-1.36 (m, 3H), 0.79-0.72 (m, 2H), 0.66-0.59 (m, 2H).

Effect Example 1: 3D Cell Proliferation Experiment

3D proliferation assay of AsPC-1 cells

The diluted test compound was added to a 384-well low-adsorption cell culture plate using a nanoliter pipetting system (LABCYTE, P-0200). After the cells were inoculated, the culture plate was placed in a 37°C, 5% CO ₂ constant temperature incubator. After the compound and cells were incubated for 5 days, 3D reagent, use Envision multifunctional microplate reader to read the luminescence value (the light signal is proportional to the amount of ATP in the system, and the content of ATP directly represents the number of living cells in the system). Finally, use XLFIT software to obtain the IC50 (half-maximal inhibitory concentration) of the compound using a nonlinear fitting formula.

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

X: log value of compound concentration

Y: Inhibition rate (%)

Inhibition rate (%) = 100 × (negative control average value - compound reading) / (negative control average value - positive control average value)

Negative control: DMSO

Positive control: Medium only

The inhibitory effects of the compounds of the present invention on 3D proliferation of AGS and AsPC-1 cells are shown in Table 1. (A: IC50 < 100 nM; B: 100 nM to 1 uM; C: 1 uM to 10 uM; D: > 10 uM)

Table 1

Example WT_IC50 AsPC-1_IC50 NCI358_IC50 H727_IC50 1 A B A B 2 B A C D 3 B A C D

Claims (11)

1. A heterocyclic compound as shown in formula I, a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a solvate thereof: Wherein, _W1 is independently selected from CR ^W1 or N; Wherein, _W2 is independently selected from CR ^W2 or N; Wherein, _W3 is independently selected from CR ^W3 or N; wherein R ^W1 , R ^W2 , and R ^W3 are each independently selected from hydrogen, deuterium, halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ cycloalkyl, halogenated C ₁ -C ₆ alkyl, nitro, hydroxyl, and NR ^a R ^b ; wherein R ¹ , R ^1′ , R ² , R ^2′ , R ³ , and R ^3′ each independently represent hydrogen, halogen, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, hydroxy C ₁ -C ₆ alkyl, or halogenated C ₁ -C ₆ alkyl; Wherein, Rx represents hydrogen, C ₁ -C ₆ alkyl; wherein Cy represents a 3-10 membered heterocycle, a 6-10 membered aryl or a 5-10 membered heteroaryl; and the Cy may be arbitrarily substituted by 0, 1 or 2 substituents selected from halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ cycloalkyl, halogenated C ₁ -C ₆ alkyl, nitro, -(C ₀ -C ₆ )hydroxy, -(C ₀ -C ₆ )NR ^a R ^b , -(C ₀ -C ₆ )C(O)NR ^a R ^b , -(C ₀ -C ₆ )C(O)OR ^a , -(C ₀ -C ₆ )OC(O)R ^a ; wherein ^Ra and ^Rb each independently represent hydrogen or _C1 - _C6 alkyl. 2. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, its stereoisomer or its solvate, wherein _W1 and _W2 are selected from N. 3. The heterocyclic compound of formula (I) according to claim 1, its pharmaceutically acceptable salt, its stereoisomer or its solvate, wherein W ₃ is selected from CH. 4. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, its stereoisomer or their solvate, wherein Cy represents Wherein, R ₄ , R _4' , R ₅ , R _5' , R ₆ , R _6' , R ₇ , R _7' , R ₈ each independently represent hydrogen or C ₁ -C ₆ alkyl; or R ₄ , R _4' pair, R ₅ , R _5' pair, R ₆ , R _6' pair, R ₇ , R _7' pair, respectively, together with the carbon atom to which they are connected, form a 3-6 membered ring. 5. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, its stereoisomer or their solvate, wherein Cy represents 6. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, its stereoisomer or their solvate, wherein Cy represents Further, the Cy is substituted with 0, 1, or 2 substituents selected from halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, cyano, C ₁ -C ₆ cycloalkyl, halogenated C ₁ -C ₆ alkyl, nitro, -(C ₀ -C ₆ )hydroxy, -(C ₀ -C ₆ )NR ^a R ^b , -(C ₀ -C ₆ )C(O)NR ^a R ^b , -(C ₀ -C ₆ )C(O)OR ^a , or -(C ₀ -C ₆ )OC(O)R ^a . 7. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, its stereoisomer or their solvate, wherein Cy represents 8. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, its stereoisomer or its solvate, wherein _R1 and R1 _' represent H or _CH3 . 9. The heterocyclic compound of formula I according to claim 1, its pharmaceutically acceptable salt, its stereoisomer or its solvate, wherein _R2 and R2 _' represent H or _CH3 . 10 . The heterocyclic compound of formula I according to claim 1 , its pharmaceutically acceptable salt, its stereoisomer or its solvate, wherein R ₃ and R _3′ represent H or CH ₃ . 11. A compound having the following structure: