WRN inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a WRN inhibitor as well as a preparation method and application thereof. The WRN inhibitor can be used for treating cancers.

Description

[0001] Priority information

[0002] This invention claims priority to application numbers PCT / CN2024 / 125550, filed on October 17, 2024, and PCT / CN2025 / 071078, filed on January 7, 2025. The full text of the aforementioned patent applications is incorporated herein by reference. Technical Field

[0003] This invention relates to a WRN inhibitor, its preparation method, and its application. Background Technology

[0004] The loss of DNA mismatch repair is a common event in cancer development. Genomic damage caused by defects in mismatch repair mechanisms (dMMR) is called microsatellite instability (MSI). MSI is common in colorectal cancer, endometrial cancer, ovarian cancer, gastric cancer, and other types of cancer. Mutations or silencing of MMR genes (including MLH1, MSH2, MSH6, and PMS2) impair a cell's ability to repair DNA mismatches. MSI can be assessed by molecular testing of five microsatellites, including two single nucleotides (BAT25 and BAT26) and three dinucleotides (D2S123, D5S346, and D17S250). If two or more microsatellite markers show instability, the tumor is labeled as MSI-high (MSI-H); if only one microsatellite marker shows instability, the tumor is labeled as MSI-low (MSI-L); and if none of the five microsatellite markers show instability, the tumor is labeled as MS-stable (MSS).

[0005] Werner syndrome RecQ helicase (WRN) comprises an exonuclease domain and an ATP-dependent helicase domain. Located in the cell nucleus, it unwinds double-stranded DNA, particularly its secondary structures, during DNA replication, damage, and repair. WRN helicase activity has been shown to be crucial for the survival of mismatch repair deficient cells (MSI cells). Studies have shown that dinucleotide TA repeat sequences are selectively unstable in MSI cells and undergo massive amplification. These amplified TA repeat sequences form secondary DNA structures that require WRN for unwinding. In the absence of WRN proteins or inhibition of their helicase activity, amplified TA repeat sequences in MSI cells are susceptible to nuclease cleavage and chromosome breakage. Therefore, inhibiting WRN is a promising therapeutic strategy for treating mismatch repair deficient cancers, and there is an urgent need to develop novel compounds with WRN inhibitory activity. Summary of the Invention

[0006] This disclosure provides a compound or a pharmaceutically acceptable salt thereof. The structure of the compound is shown below:

[0007] , or .

[0008] This disclosure provides a compound. The structure of the compound is shown below:

[0009] , or .

[0010] This disclosure provides a compound or a pharmaceutically acceptable salt thereof. The compound is selected from one of the compounds in the following table:

[0011]

[0012]

[0013] .

[0014] This disclosure provides a compound. The compound is selected from one of the compounds in the following table:

[0015]

[0016]

[0017] .

[0018] This disclosure also provides a pharmaceutical composition comprising the aforementioned compound or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients.

[0019] This disclosure also provides the use of substance Z in the preparation of medicaments for treating and / or preventing cancer, wherein substance Z is the aforementioned compound or a pharmaceutically acceptable salt thereof, or a combination of the aforementioned medicaments.

[0020] According to embodiments of this disclosure, the cancer is endometrial cancer of the uterine body, colonic adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, esophageal cancer, breast cancer, renal clear cell carcinoma, prostate cancer, or ovarian serous cystadenocarcinoma.

[0021] The term "pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for patient use) acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, calcium, aluminum, magnesium, bismuth, and ammonium salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochlorides, acetates, trifluoroacetates, sulfates, and mesylates. See Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl, 2002) for details.

[0022] The term "pharmaceutical excipients" refers to the excipients and additives used in the production of pharmaceuticals and the dispensing of prescriptions. It includes all substances contained in pharmaceutical preparations, excluding the active ingredient. See the Pharmacopoeia of the People's Republic of China (2020 edition) or Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009) for details.

[0023] The term “treatment” refers to any of the following: (1) alleviating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that triggers the disease; or (3) slowing the development of one or more biological manifestations of a disease.

[0024] The term "prevention" refers to reducing the risk of developing a disease.

[0025] The term "patient" refers to any animal that has received or is about to receive treatment, preferably a mammal, and most preferably a human. Mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans.

[0026] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0027] The reagents and raw materials used in this invention are all commercially available. Detailed Implementation

[0028] 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.

[0029] Example B02

[0030]

[0031]

[0032] At room temperature, acetonitrile (39.5 g, 240 mmol) of methyl 2-chloro-3-oxovalerate (240 mmol) was reacted with acetonitrile (... In a 400 mL solution, triethylamine (TEA, 100 mL, 720 mmol) and 1-tert-butyloxycarbonylpiperazine (44.7 g, 240 mmol) were added. The reaction mixture was stirred overnight at 60 °C, then concentrated and dissolved in ethyl acetate (EtOAc, 1000 mL). The solution was washed with saturated brine (500 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by silica gel column chromatography to give compound BO2-1 (50.1 g). Liquid chromatography-mass spectrometry (LC-MS) showed that its quasi-molecular ion peak... The value is 315.2. LCMS[M+H] + 315.2.

[0033] Polyphosphoric acid (10 g, 122 mmol) was added to a mixture of compound BO2-1 (10.11 g, 32.2 mmol) and 3-bromo-1H-1,2,4-triazol-5-amine (5.24 g, 32.2 mmol) in ethanol (100 mL). The mixture was stirred at 100 °C for 16 hours, cooled to room temperature, and then triethylamine (TEA, 22.41 mL, 161 mmol) and di-tert-butyl carbonate (... 22.40 mL (96 mmol) was stirred at room temperature for 2 hours. The reaction solution was poured into water (300 mL) and extracted with ethyl acetate (EtOAc, 300 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give compound BO2-2 (9.4 g). 1 H NMR (400 MHz, CDCl3) δ 4.21 - 3.95(m, 2H), 3.71 - 3.52 (m, 2H), 3.15 - 2.85 (m, 4H), 2.69 - 2.54 (m, 2H), 1.49(s, 9H), 1.35 (t, J = 7.6 Hz, 3H). LCMS [M+H-tBu] + : 371.0 / 373.0.

[0034] Compound BO2-2 (8.2 g, 19.19 mmol) was reacted with potassium carbonate ( 2-(trimethylsilyl)ethoxymethyl chloride (SEM-Cl, 5.1 mL, 28.8 mmol) was added to an N,N-dimethylformamide (DMF, 80 mL) solution (5.30 g, 38.4 mmol). The mixture was stirred at 25 °C for 16 hours, the reaction was quenched with water (300 mL), and extracted with ethyl acetate (200 mL × 2). The organic phase was washed successively with water (200 mL × 3) and saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give compound BO2-3 (6.3 g). 1 H NMR (400MHz, CDCl3) δ 5.66 (s, 2H), 4.23 - 3.92 (m, 2H), 3.73 - 3.56 (m, 4H), 3.09(q, J = 7.4 Hz, 2H), 2.67 - 2.53 (m, 2H), 1.49 (s, 9H), 1.28 (t, J = 7.4 Hz,3H), 1.00 - 0.94 (m, 2H), 0.00 (s, 9H). LCMS [M+H-tBu] + : 501.2 / 503.2.

[0035] At -78°C, potassium hexamethyldisilamide (KHMDS, 1 mol / L THF solution, 2.246 mL, 2.246 mmol) was added to a tetrahydrofuran (THF, 10 mL) solution of compound BO2-3 (501 mg, 0.899 mmol). The mixture was stirred at -78°C for 2 hours, followed by the addition of a tetrahydrofuran (0.5 mL) solution of 3-iodopropene (604 mg, 3.59 mmol), and stirring was continued at -78°C for 4 hours. The mixture was then diluted with a saturated aqueous solution of ammonium chloride (…). The reaction was quenched with ethyl acetate (30 mL × 2), and extracted with ethyl acetate (30 mL × 2). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to obtain compound B02-4 (293 mg). 1H NMR (400MHz, CDCl3) δ 5.82 - 5.60 (m, 3H), 5.14 - 4.97 (m, 2H), 4.20 - 3.91 (m, 2H), 3.80 - 3.59 (m, 4H), 3.39 - 3.14 (m, 1H), 3.11 - 2.91 (m, 2H), 2.82 - 2.54(m, 4H), 1.51 - 1.43 (m, 12H), 0.99 - 0.92 (m, 2H), 0.00 (s, 9H). LCMS [M+H-tBu] + : 541.2 / 543.2.

[0036] At 0 °C, m-chloroperoxybenzoic acid (m-CPBA, 11.12 g, 64.4 mmol) was added to a solution of compound BO2-4 (7.7 g, 12.88 mmol) in dichloromethane (DCM, 150 mL). The mixture was stirred at room temperature for 16 hours, filtered, and the residue was washed with dichloromethane (200 mL). The filtrate and washings were combined and then rinsed sequentially with a saturated potassium carbonate solution (…). The sample was washed with 200 mL of saturated saline solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The filtrate was then purified by silica gel column chromatography to obtain compound BO2-5 (4.2 g). LCMS [M+H-tBu] + : 557.2 / 559.2.

[0037] Compound BO2-5 (1.01 g, 1.646 mmol) was reacted with N,N-dimethylformamide (DMF, 10 mL) / water ( Potassium fluoride (0.478 g, 8.23 ​​mmol) was added to a mixed solution of 0.2 mL. The mixture was stirred at 60 °C for 16 hours, the reaction was quenched with water (50 mL), and extracted with ethyl acetate (30 mL × 2). The organic phases were combined, washed successively with water (30 mL × 2) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give compound BO2-6 (370 mg). LCMS [M + H - tBu] + : 427.0 / 429.0.

[0038] At 0 °C, sodium bicarbonate (12.17 g, 7.24 mmol), potassium bromide (KBr, 8.62 mg, 0.072 mmol), 2,2,6,6-tetramethylpiperidine-1-oxygen radical (TEMPO, 1.131 mg, 7.24 μmol), trioctylmethylammonium chloride (TOMAC, 0.015 g, 0.036 mmol), and sodium hypochlorite (2.457 mL, 1.810 mmol) were added to a dichloromethane (9 mL) solution of compound BO2-6 (0.35 g, 0.724 mmol). The mixture was stirred at 0°C for 16 hours, diluted with dichloromethane (20 mL), and stirred vigorously for 2 minutes before separating the organic phase. The aqueous phase was extracted with dichloromethane (20 mL), and the organic phases were combined and washed with sodium bicarbonate aqueous solution (10 mL). The pH was then adjusted to 3 with 5 mol / L hydrochloric acid (HCl), and the organic phase was separated again. The aqueous phase was extracted with dichloromethane (30 mL). All organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The filtrate was purified by silica gel column chromatography to give compound BO2-7 (220 mg). LCMS [M+H-Boc] + : 397.0 / 399.0.

[0039] To a solution of compound BO2-7 (1.4 g, 2.81 mmol) and 2-chloro-4-trifluoromethylaniline (0.826 g, 4.22 mmol) in dichloromethane (5 mL), triethylamine (3.14 mL, 22.52 mmol) was added, followed by polyphosphoric acid (T4P, 10.14 g, 14.07 mmol). The mixture was stirred at room temperature for 16 hours, diluted with dichloromethane (50 mL), washed successively with water (30 mL) and saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain compound BO2-8 (1.2 g). LCMS [M+H-Boc] + : 574.0 / 576.0.

[0040] Potassium phosphate was added to a solution of compound BO2-8 (485 mg, 0.719 mmol) and 2-(2,5,6,7-tetrahydroxamono-4-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (193 mg, 0.862 mmol) in 1,4-dioxane (6 mL). 2.16 mL (2.156 mmol) and XPhos Pd G2 catalyst (30.4 mg, 0.036 mmol). The mixture was stirred at 80 °C for 5 hours under a nitrogen atmosphere. After filtration and concentration, the filtrate was purified by preparative thin-layer chromatography (Pre-TLC, developing solvent: methanol / dichloromethane = 1 / 20) to give compounds B02-9-1 (lower spot in TLC, 51 mg) and B02-9-2 (upper spot in TLC, 62 mg). LCMS [M+H-tBu] + 636.4.

[0041] Compound B02-9-1 (73 mg) was further separated by supercritical fluid chromatography (SFC) to obtain B02-9-A (33 mg) and B02-9-B (23 mg). B02-9-A: SFC retention time 1.785 min; B02-9-B: SFC retention time 3.023 min; SFC analytical conditions: column was Daicel CHIRALPAK® OD column (100 mm × 3.0 mm × 3.0 μm); mobile phase A was supercritical carbon dioxide (… The mobile phase B was methanol containing 0.1% diethylamine (DEA); the proportion of mobile phase B was 20%; the running time was 8 minutes; the flow rate was 1.5 mL / min; and the column temperature was 35℃.

[0042] Compound B02-9-2 (86 mg) was further separated by supercritical fluid chromatography (SCLC) to obtain B02-9-C (42 mg) and B02-9-D (37 mg). B02-9-C: SCLC retention time 1.012 min; B02-9-D: SCLC retention time 3.505 min; SCLC analytical conditions: column was a Daicel CHIRALPAK® OD column (250 mm × 3.0 mm × 3.0 μm); mobile phase A was supercritical carbon dioxide, mobile phase B was methanol containing 0.1% diethylamine; mobile phase B ratio was 30%; run time was 8 min; flow rate was 1.5 mL / min; column temperature was 35℃.

[0043] To a solution of compound BO2-9-A (33 mg, 0.048 mmol) in dichloromethane (1 mL), a hydrochloric acid / dioxane solution (1.0 mL) was added. The mixture was stirred at room temperature for 2 hours and concentrated to obtain crude compound BO2-10-A (24 mg). To a solution of BO2-10-A (24 mg, 0.038 mmol) and 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (8.8 mg, 0.057 mmol) in N,N-dimethylformamide (0.5 mL), N,N-diisopropylethylamine (DIEA, 24.7 mg, 0.191 mmol), 1-hydroxybenzotriazole (HOBt, 11.7 mg, 0.076 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 14.6 mg, 0.076 mmol) were added. Under nitrogen protection, the mixture was stirred at 25°C for 16 hours, concentrated, and purified by preparative high-performance liquid chromatography (prep-HPLC) to obtain compound BO2-A (3.99 mg). LCMS [M+H] + .: 728.4. 1 H NMR (400 MHz, CD3OD) δ 8.57 (s, 1H), 8.08 (d, J =8.6 Hz, 1H), 7.82 (s, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.04 - 6.87 (m, 1H),5.68 - 5.50 (m, 1H), 4.84 - 4.77 (m, 2H), 4.34 - 4.27 (m, 2H), 3.97 - 3.75(m, 4H), 3.42 - 3.32 (m, 2H), 3.29 - 3.25 (m, 2H), 3.04 - 2.84 (m, 3H), 2.79- 2.69 (m, 1H), 2.57 - 2.40 (m, 4H), 2.02 - 1.86 (m, 2H), 1.62 (d, J = 6.9Hz, 3H).

[0044] Starting from compound BO2-9-B, compound BO2-B (1.01 mg) was prepared using the same method as for BO2-A. LCMS [M+H] + 728.4. 1H NMR (400 MHz, CD3OD) δ 8.47 (s, 1H),8.08 (d, J = 8.6 Hz, 1H), 7.82 (s, 1H), 7.62 (d, J = 8.7 Hz, 1H), 7.04 - 6.89(m, 1H), 5.67 - 5.52 (m, 1H), 4.84 - 4.69 (m, 2H), 4.35 - 4.26 (m, 2H), 3.95 - 3.76 (m, 4H), 3.43 - 3.33 (m, 2H), 3.29 - 3.19 (m, 2H), 3.02 - 2.85 (m,3H), 2.78 - 2.71 (m, 1H), 2.57 - 2.36 (m, 4H), 1.99 - 1.89 (m, 2H), 1.62 (d,J = 6.8 Hz, 3H).

[0045] Starting from compound BO2-9-C, compound BO2-C (3.28 mg) was prepared using the same method as for BO2-A. LCMS [M+H] + 728.2. 1 H NMR (400 MHz, CD3OD) δ 8.55 (s, 1H), 8.07 (d, J = 8.3 Hz, 1H), 7.82 (s, 1H), 7.64 (d, J = 9.2 Hz, 1H), 7.10 - 6.83 (m,1H), 5.58 - 5.50 (m, 1H), 4.80 - 4.64 (m, 2H), 4.37 - 4.28 (m, 2H), 3.97 -3.79 (m, 4H), 3.43 - 3.36 (m, 2H), 3.24 - 3.18 (m, 2H), 3.08 - 2.88 (m, 4H),2.52 (s, 3H), 2.32 - 2.24 (m, 1H), 2.02 - 1.90 (m, 2H), 1.53 (d, J = 7.2 Hz, 3H).

[0046] Starting from compound BO2-9-D, compound BO2-D (5.02 mg) was prepared using the same method as for BO2-A. LCMS [M+H] + 728.2. 1H NMR (400 MHz, CD3OD) δ 8.55 (s, 1H),8.07 (d, J = 8.4 Hz, 1H), 7.82 (s, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.03 - 6.93(m, 1H), 5.56 - 5.51 (m, 1H), 4.76 - 4.63 (m, 2H), 4.37 - 4.29 (m, 2H), 3.97 - 3.76 (m, 4H), 3.45 - 3.39 (m, 2H), 3.20 - 3.15 (m, 2H), 3.06 - 2.89 (m,4H), 2.52 (s, 3H), 2.34 - 2.22 (m, 1H), 2.03 - 1.89 (m, 2H), 1.53 (d, J = 7.3Hz, 3H).

[0047] Example B05

[0048]

[0049]

[0050] Under a nitrogen atmosphere and at 0°C, 1,5-pentanediol (20 g, 192 mmol) was added dropwise to a suspension of sodium hydride (NaH, 9.22 g, 230 mmol, 60%) in tetrahydrofuran (THF, 600 mL). After stirring for 0.5 hours, a solution of 3-bromopropene (23.23 g, 192 mmol) in tetrahydrofuran (20 mL) was added dropwise at 0°C, and the mixture was then stirred at room temperature for 16 hours. The solution was then mixed with saturated ammonium chloride solution (…). The reaction was quenched with ethyl acetate (200 mL × 3), and extracted with ethyl acetate (200 mL × 3). The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to obtain compound B05-1 (13.17 g). 1 H NMR (400 MHz, CDCl3) δ 5.96 - 5.87(m, 1H), 5.30 - 5.24 (m, 1H), 5.19 - 5.15 (m, 1H), 3.96 -3.98 (m, 2H), 3.65(t, J = 6.4 Hz, 2H), 3.44 (t, J = 6.4 Hz, 2H), 1.63 -1.58 (m, 4H), 1.50 -1.39 (m, 2H).

[0051] Under a nitrogen atmosphere at -78°C, dimethyl sulfoxide (DMSO, 19.4 mL, 274 mmol) was added to a solution of oxalyl chloride (12.0 mL, 137 mmol) in dichloromethane (DCM, 550 mL). After stirring for 20 minutes, a solution of BO5-1 (13.17 g, 91 mmol) in dichloromethane (200 mL) was added dropwise over 20 minutes, and stirring was continued at -78°C for 1 hour. Triethylamine (TEA, 63.6 mL, 457 mmol) was then added, and stirring was carried out at 0°C for 0.5 hours. 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 13.8 mL, 91 mmol) and Eschenmoser's salt (33.8 g, 183 mmol) were then added. The mixture was stirred at room temperature for 16 hours, poured into water (300 mL), and extracted with dichloromethane (200 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to obtain compound B05-2 (12.3 g). 1 H NMR (400 MHz, CDCl3) δ 9.55 (s, 1H), 6.28 (s, 1H), 6.02 (s, 1H), 5.96 -586 (m, 1H), 5.30 -5.24 (m, 1H), 5.19 -5.15 (m, 1H), 3.97-3.95 (m, 2H), 3.44 (t, J = 6.4 Hz, 2H), 2.36 - 2.32 (m, 2H), 1.81 - 1.73 (m,2H).

[0052] Under a nitrogen atmosphere and at 0°C, a Grubbs II catalyst (1.652 g, 1.945 mmol) was added to a solution of B05-2 (3 g, 19.45 mmol) in dichloromethane (1000 mL). The reaction mixture was stirred at 40°C for 16 hours, concentrated, and purified by silica gel column chromatography to give compound B05-3 (811 mg). 1H NMR (400MHz, CDCl3) δ 9.55 (s, 1H), 6.28 (s, 1H), 6.02 (s, 1H), 5.96 -586 (m, 1H), 5.30 -5.24 (m, 1H), 5.19 -5.15 (m, 1H), 3.97 -3.95 (m, 2H), 3.44 (t, J = 6.4Hz, 2H), 2.36 - 2.32 (m, 2H), 1.81 - 1.73 (m, 2H).

[0053] At 0°C, titanium (IV) isopropoxide (16.3 mL, 55.7 mmol) and titanium tetrachloride (18.9 mL, 172 mmol) were added to dichloromethane (500 mL) using a syringe. After stirring for 10 minutes, N,N-diisopropylethylamine (DIEA, 41.2 mL, 236 mmol) was added, and the resulting brown solution was stirred for 10 minutes. Then, (S)-4-benzyl-3-propionyloxazolidine-2-one (50.01 g, 214 mmol) was added. The resulting deep red solution was stirred at 0°C for 1 hour, and tert-butyl acrylate (41.2 g, 322 mmol) was added dropwise using a syringe. The solution gradually turned brown, and after stirring at 0°C for 4 hours, a saturated sodium bicarbonate aqueous solution was added. The organic phase was separated, and the aqueous phase was extracted with dichloromethane (500 mL). The organic phases were combined, washed with saturated brine (200 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to obtain compound B05-4 (67.5 g). 1 H NMR(400 MHz, CDCl3) δ 7.36-7.21 (m, 5H), 4.73 - 4.61 (m, 1H), 4.24 - 4.14 (m,2H), 3.82 - 3.71 (m, 1H), 3.33 (dd, J = 13.3, 3.3 Hz, 1H), 2.74 (dd, J =13.3, 9.8 Hz, 1H), 2.36 - 2.22 (m, 2H), 2.15 - 2.04 (m, 1H), 1.85 - 1.72 (m,1H), 1.45 (s, 9H), 1.20 (d, J = 6.8 Hz, 3H). LCMS [M+Na] + 384.2.

[0054] At 0°C, hydrogen peroxide was added to a mixed solution of tetrahydrofuran (250 mL) and water (83 mL) containing B05-4 (25 g, 48.4 mmol). 29.7 mL, 291 mmol), followed by the addition of a 30 mL solution of lithium hydroxide monohydrate (4.06 g, 97 mmol). After stirring for 1 hour, the solution was incubated at 0°C with 10% sodium sulfite ( The reaction was quenched by solution. The mixture was concentrated to remove tetrahydrofuran, and 2 mol / L sodium hydroxide solution (6 mL) was added. The aqueous phase was extracted with dichloromethane (100 mL × 2), cooled to 0°C, and the pH was adjusted to 1-2 with 2 mol / L hydrochloric acid (HCl), followed by extraction with ethyl acetate (100 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give compound B05-5 (10.3 g). 1 H NMR (400 MHz, CDCl3) δ 8.99 (s, 1H), 4.33 - 4.25 (m, 1H), 4.11 - 3.97 (m, 2H), 3.76 - 3.50 (m, 2H), 3.32 (s, 9H), 3.02 (d, J = 6.9 Hz,3H).

[0055] Under a nitrogen atmosphere, 1,1'-carbonyldiimidazole (CDI, 24.06 g, 148 mmol) was added to a 200 mL solution of tetrahydrofuran containing B05-5 (20.01 g, 99 mmol), and the mixture was stirred at room temperature for 2 hours to obtain mixture A. Magnesium chloride was added to a 200 mL solution of tetrahydrofuran containing potassium 3-methoxy-3-oxopropionate (30.9 g, 198 mmol). Mixtures B were prepared by stirring 18.84 g (198 mmol) of 4-dimethylaminopyridine (DMAP, 1.209 g, 9.89 mmol) and 55.2 mL (396 mmol) of triethylamine at room temperature under a nitrogen atmosphere for 2 hours. Mixture B was then added to mixture A and stirred overnight at 80 °C under a nitrogen atmosphere. The reaction mixture was filtered, the filtrate was concentrated, and purified by silica gel column chromatography to give compound B05-6 (23.2 g). 1H NMR (400 MHz, CDCl3) δ 3.73 (s, 3H), 3.52 (s, 2H), 2.78 - 2.61 (m, 1H), 2.27 - 2.17 (m, 2H), 2.06 - 1.92 (m, 1H), 1.67 - 1.61(m, 1H), 1.45 - 1.43 (m, 9H), 1.13 (d, J = 7.9 Hz, 3H). LCMS [M+H-tBu] + :203.0.

[0056] Under a nitrogen atmosphere, sodium methoxide (13.96 g, 78 mmol) was added to a methanol (MeOH, 200 mL) solution of aminoguanidine hydrochloride (21.42 g, 194 mmol). After stirring at room temperature for 15 minutes, B05-6 (10.01 g, 38.8 mmol) was added, and the mixture was stirred at room temperature under a nitrogen atmosphere for 16 hours. The reaction mixture was diluted with ethyl acetate (100 mL) and water (100 mL), and extracted with ethyl acetate (300 mL × 2). The organic phases were combined, washed with saturated brine (200 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give compound B05-7 (3.88 g). 1 H NMR (400 MHz, CDCl3) δ 6.03 (s, 1H), 5.77(s, 1H), 4.58 (s, 2H), 2.50 - 2.39 (m, 1H), 2.17 (t, J = 7.8 Hz, 2H), 1.87-1.84 (d, J = 7.5 Hz, 1H), 1.77 - 1.69 (m, 1H), 1.44 (s, 9H), 1.16 (d, J =10.2 Hz, 3H). LCMS [M+H] + : 283.2.

[0057] Add ferric chloride (NMP) to a solution of B05-7 (3.2 g, 11.33 mmol) in N-methylpyrrolidone (NMP, 8 mL). Compound B05-8 (1.3 g) was reacted with an NMP solution of B05-3 (1 g / mL, 3.68 g, 22.67 mmol). The reaction was stirred at 50°C in air for 48 hours, quenched with water, and extracted with ethyl acetate (200 mL × 2). The organic phases were combined, washed with water (50 mL × 2) and saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by C-18 column chromatography to give compound B05-8 (1.3 g). LCMS [M+H] + : 389.2.

[0058] Under a nitrogen atmosphere at -78°C, lithium bis(trimethylsilyl)amino (LiHMDS, 1 mol / L THF solution, 10.19 mL, 10.19 mmol) was added to a 20 mL solution of tetrahydrofuran (1.32 g, 3.40 mmol) of B05-8. After stirring at -78°C for 2 hours, N-bromosuccinimide (NBS, 0.907 g, 5.10 mmol) was added, and stirring was continued for another 2 hours. The solution was then diluted with 50 mL of saturated ammonium chloride aqueous solution and 10% sodium thiosulfate. The reaction was quenched with an aqueous solution (50 mL), and extracted with ethyl acetate (100 mL × 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give compound B05-9 (780 mg). LCMS [M+H] + : 465.2 / 467.2.

[0059] To a solution of B05-9 (640 mg, 1.375 mmol) in acetonitrile (ACN, 6 mL), tert-butyl (1S,6S)-2,5-diazabicyclo[4.2.0]octane-2-carboxylic acid (876 mg, 4.13 mmol) and potassium acetate (KOAc, 405 mg, 4.13 mmol) were added. The mixture was stirred at 90 °C for 96 hours, concentrated, and purified by silica gel column chromatography to give compound B05-10 (591 mg). LCMS [M+H- t Bu] + : 541.2.

[0060] B05-10 (591 mg, 0.990 mmol) was mixed with trifluoroacetic acid (TFA, 3 mL) and stirred at room temperature for 16 hours. After concentration, it was dissolved in dichloromethane (5 mL), and triethylamine (0.21 mL, 1.486 mmol) and di-tert-butyl carbonate (TFA, 3 mL) were added. (0.35 mL, 1.486 mmol). The concentrated mixture was purified by C-18 column chromatography to give compound B05-11 (440 mg). LCMS [M+H-tBu] + : 485.2.

[0061] Under a nitrogen atmosphere, polyphosphoric acid (T4P, 1.1 mL, 1.276 mmol, 50% ethyl acetate solution) was added to a pyridine (2 mL) solution of B05-11 (115 mg, 0.213 mmol) and 2-chloro-4-trifluoromethylaniline (166 mg, 0.851 mmol). The mixture was stirred at 25 °C for 16 hours, the reaction was quenched with water (20 mL), and extracted with ethyl acetate (30 mL × 2). The organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The filtrate was purified by preparative thin-layer chromatography (pre-TLC, developing solvent: methanol / dichloromethane = 1 / 20) to give compounds B05-12-1 (lower spot in TLC, 29 mg) and B05-12-2 (upper spot in TLC, 42 mg).

[0062] B05-12-1 (100 mg) was separated by preparative supercritical fluid chromatography (prep-SFC) to obtain B05-12-A (49 mg) and B05-12-B (48 mg). The retention time of B05-12-A by SFC was 2.153 min; the retention time of B05-12-B by SFC was 2.792 min. The SFC analytical conditions were as follows: column: CHIRALCEL® IB column (100 mm × 3.0 mm × 3.0 μm); mobile phase A: supercritical carbon dioxide. The mobile phase B was methanol containing 0.1% diethylamine (DEA); the proportion of mobile phase B was 20%; the run time was 7 minutes; the flow rate was 1.5 mL / min; and the column temperature was 35℃.

[0063] B05-12-2 (300 mg) was separated by preparative supercritical fluid chromatography to obtain B05-12-C (49 mg) and B05-12-D (235 mg). LCMS: The retention time was 662.4. B05-12-C: Supercritical fluid chromatography retention time was 1.064 min; B05-12-D: Supercritical fluid chromatography retention time was 2.631 min; Supercritical fluid chromatography analysis conditions: Column was a CHIRALCEL® IB column (100 mm × 3.0 mm × 3.0 μm); Mobile phase A was supercritical carbon dioxide, and mobile phase B contained 0.

[0064] Compound B05-12-A (49 mg, 0.068 mmol) was dissolved in a mixture of 1,4-dioxane in 4 mol / L hydrochloric acid (1.5 mL) and dichloromethane (DCM, 1.5 mL), and stirred at room temperature for 2 hours. The mixture was concentrated and dissolved in N,N-dimethylformamide (DMF, 0.5 mL), followed by the addition of 5-hydroxy-6-methylpyrimidine-4-carboxylic acid (21.8 mg, 0.141 mmol), N,N-diisopropylethylamine (DIEA, 0.04 mL, 0.226 mmol), 1-hydroxybenzotriazole (HOBt, 17.3 mg, 0.113 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 21.7 mg, 0.113 mmol). The mixture was stirred at 25°C for 16 hours and purified by preparative high-performance liquid chromatography (prep-HPLC) to obtain compound B05-A (3.03 mg). LCMS [M+H] + : 754.1. 1 H NMR (400 MHz, CD3OD) δ 8.52 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.81 (s, 1H), 7.61 (d, J =7.7 Hz, 1H), 7.07 - 6.90 (m, 1H), 5.64 - 5.52 (m, 1H), 4.80 - 4.55 (m, 1H), 4.40 - 4.22 (m, 2H), 3.97 - 3.43 (m, 8H), 3.00 - 2.90 (m, 2H), 2.78 - 2.68(m, 1H), 2.52 - 2.40 (m, 4H), 2.01- 1.91 (m, 2H), 1.88 - 1.25 (m, 7H).

[0065] Starting from compound B05-12-B, compound B05-B (24.24 mg) was prepared using the same method as that used to prepare B05-A. LCMS [M+H]+ : 754.1. 1 H NMR (400 MHz, CD3OD) δ 8.52 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.88 - 7.74 (m, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.07- 6.90 (m, 1H), 5.64 - 5.52 (m, 1H), 4.80 - 4.55 (m, 1H), 4.40 - 4.22 (m,2H), 3.97 - 3.43 (m, 8H), 3.00 - 2.90 (m, 2H), 2.78 - 2.68 (m, 1H), 2.52 -2.40 (m, 4H), 2.01 - 1.91 (m, 2H), 1.88 - 1.25 (m, 7H).

[0066] Starting from compound B05-12-C, compound B05-C (23.84 mg) was prepared using the same method as for B05-A. LCMS [M+H] + : 754.3. 1 H NMR (400 MHz, CD3OD) δ 8.57 (s, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 1.5 Hz, 1H), 7.62 (dd, J = 8.6, 1.7Hz, 1H), 7.01 - 6.92 (m, 1H), 5.65 - 5.53 (m, 1H), 4.84 - 4.51 (m, 1H), 4.36 - 4.27 (m, 2H), 4.05 - 3.40 (m, 8H), 2.99 - 2.88 (m, 2H), 2.80 - 2.69 (m,1H), 2.52 (s, 3H), 2.50 - 2.38 (m, 1H), 2.03 - 1.90 (m, 2H), 1.88 - 1.21 (m,7H).

[0067] Starting from compound B05-12-D, compound B05-D (37.13 mg) was prepared using the same method as for B05-A. LCMS [M+H] + : 754.3. 1H NMR (400 MHz, CD3OD) δ 8.58 (s, 1H),8.07 (d, J = 8.5 Hz, 1H), 7.90 - 7.78 (m, 1H), 7.71 - 7.58 (m, 1H), 6.98 (t,J = 4.7 Hz, 1H), 5.59 - 5.49 (m, 1H), 4.83 - 4.57 (m, 1H), 4.32 (d, J = 4.6Hz, 2H), 3.94 - 3.47 (m, 8H), 3.13 - 3.03 (m, 1H), 2.99 - 2.91 (m, 2H), 2.51(s, 3H), 2.31 - 2.22 (m, 1H), 2.00 - 1.92 (m, 2H), 1.83 - 1.26 (m, 7H).

[0068] Example B06

[0069]

[0070] Under a nitrogen atmosphere and at 0°C, tetrahydro-2H-pyran-2-one (50 g, 499 mmol) was reacted with titanium isopropoxide (IV). In a stirred solution of anhydrous tetrahydrofuran (THF, 1000 mL) containing 3 mol / L (146 mL, 499 mmol), ethyl magnesium bromide (EtMgBr, 308 mL, 1049 mmol) was added dropwise over 1 hour. The reaction mixture was allowed to warm to room temperature and stirred for 4 hours, followed by the addition of saturated ammonium chloride aqueous solution (500 mL). The aqueous phase was extracted with ethyl acetate (EtOAc, 1 L × 3), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give compound B06-1 (27.4 g). 1 H NMR (400 MHz, CDCl3) δ 3.66 - 3.62 (m, 2H), 1.55 - 1.16 (m, 4H), 0.94 - 0.78 (m, 2H), 0.77 - 0.64 (m, 2H), 0.51 - 0.33 (m, 2H). LCMS[M+H] + 131.0

[0071] At 0 °C, tert-butyldimethylchlorosilane (TBSCl, 31.7 g, 210 mmol) was added in portions to a stirred solution of B06-1 (27.4 g, 210 mmol) and imidazole (28.7 g, 421 mmol) in dichloromethane (DCM, 1300 mL). The mixture was stirred at room temperature for 16 hours, the reaction was quenched with water (300 mL), and extracted with dichloromethane (1000 mL × 2). The organic phases were combined, washed with saturated brine (800 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give compound B06-2 (45.1 g). 1 H NMR (400 MHz, CDCl3) δ 3.67 - 3.62 (m,2H), 1.57 (s, 6H), 0.89 (s, 9H), 0.76 - 0.70 (m, 2H), 0.47 - 0.41 (m, 2H),0.05 (s, 6H)

[0072] Under a nitrogen atmosphere and at -40°C, a solution of 10.1 g (41.3 mmol) of B06-2 in tetrahydrofuran (300 mL) was added to potassium hexamethyldisilamide (KHMDS, 1 mol / L THF solution, 50 mL, 50 mmol), and the mixture was kept at this temperature for 1 hour. Subsequently, an anhydrous tetrahydrofuran solution of allyl bromide (18 mL, 207 mmol) was added dropwise at -40°C (20 mL). After stirring for 2 hours under a nitrogen atmosphere and at -40°C, the reaction was quenched with an ice-bath cooled saturated ammonium chloride solution (200 mL), and the mixture was extracted with ethyl acetate (300 mL × 3). The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to give compound B06-3 (35.0 g). 1 H NMR (400 MHz, CDCl3) δ 6.00 - 5.80 (m, 1H), 5.28 - 5.18 (m, 1H), 5.16 - 5.05 (m, 1H), 4.04- 3.84 (m, 2H), 3.68 - 3.58 (m, 2H), 1.58 - 1.53 (m, 6H), 0.89 (s, 9H), 0.81- 0.74 (m, 2H), 0.45 - 0.36 (m, 2H), 0.04 (s, 6H).

[0073] At 0 °C, tetrabutylammonium fluoride (TBAF, 1 mol / L THF solution, 356 mL, 356 mmol) was added to a tetrahydrofuran (500 mL) solution of B06-3 (50.8 g, 178.4 mmol). The reaction mixture was stirred at room temperature for 2 hours, concentrated, and purified by silica gel column chromatography to give compound B06-4 (17.1 g). 1 H NMR (400 MHz, CDCl3) δ 5.98 - 5.78 (m, 1H), 5.29 - 5.16 (m, 1H), 5.14 - 5.08 (m, 1H), 4.02- 3.88 (m, 2H), 3.72 - 3.56 (m, 2H), 1.65 - 1.56 (m, 6H), 0.82 - 0.75 (m, 2H), 0.44 - 0.35 (m, 2H).

[0074] Under a nitrogen atmosphere at -78°C, dimethyl sulfoxide (DMSO, 6.3 mL, 88 mmol) was added dropwise to a solution of oxalyl chloride (3.9 mL, 44.1 mmol) in dichloromethane (150 mL). After stirring at -78°C for 20 minutes, a solution of BO6-4 in dichloromethane (50 mL) was added dropwise over 20 minutes, and stirring was continued at -78°C for 1 hour. Triethylamine (TEA, 20.47 mL, 147 mmol) was then added, and the mixture was stirred at 0°C for 0.5 hours. Finally, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 4.4 mL, 29.4 mmol) and Eschenmoser's salt (10.9 g, 58.7 mmol) were added. The mixture was stirred at room temperature for 16 hours, poured into water (300 mL), and extracted with dichloromethane (100 mL × 2). The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and purified by silica gel column chromatography to give compound B06-5 (3.1 g). 1H NMR (400 MHz, CDCl3) δ 9.55 (s, 1H), 6.30 (d, J = 0.8 Hz, 1H), 6.00 (s, 1H), 5.97 - 5.84 (m, 1H), 5.29 - 5.21 (m,1H), 5.15 - 5.09 (m, 1H), 4.05 - 3.89 (m, 2H), 2.54 - 2.42 (m, 2H), 1.73 -1.65 (m, 2H), 0.85 - 0.74 (m, 2H), 0.48 - 0.34 (m, 2H).

[0075] Under a nitrogen atmosphere, a Grubbs II catalyst (1.5 g, 1.72 mmol) was added to a solution of B06-5 (3.1 g, 17.20 mmol) in dichloromethane (1.5 L). The reaction mixture was stirred at 40 °C for 16 hours, concentrated, and purified by silica gel column chromatography to give compound B06-6 (2.1 g). 1 H NMR (400 MHz, CDCl3) δ 9.43 (s, 1H), 6.74-6.66 (m, 1H), 4.44 - 4.33 (m, 2H), 2.64 - 2.57(m, 2H), 1.87 - 1.80 (m, 2H), 0.92 - 0.86 (m, 2H), 0.57 - 0.49 (m, 2H).

[0076] Compounds B06-7-1 (lower spot in thin-layer chromatography, 99 mg) and B06-7-2 (upper spot in thin-layer chromatography, 120 mg) were prepared using the same method as in example B05.

[0077] B06-7-1 (99 mg) was separated by preparative supercritical fluid chromatography (prep-SFC) to obtain B06-7-A (60 mg) and B06-7-B (11 mg). LCMS [M+H- t Bu] + : 688.2.; B06-7-A: Supercritical fluid chromatography (SFC) retention time is 2.249 min; B06-7-B: Supercritical fluid chromatography retention time is 3.060 min; Supercritical fluid chromatography analysis conditions: chromatographic column is CHIRALCEL® IB column (100 mm × 3.0 mm × 3.0 μm); mobile phase A is supercritical carbon dioxide (… The mobile phase B was methanol containing 0.1% diethylamine (DEA); the proportion of mobile phase B was 20%; the run time was 7 minutes; the flow rate was 1.5 mL / min; and the column temperature was 35℃.

[0078] B06-7-2 (120 mg) was separated by preparative supercritical fluid chromatography to yield B06-7-C (21 mg) and B06-7-D (76 mg). LCMS: : 688.2; B06-7-C: Supercritical fluid chromatography retention time is 1.162 min; B06-7-D: Supercritical fluid chromatography retention time is 2.945 min; Supercritical fluid chromatography analysis conditions: chromatographic column is CHIRALCEL®IB column (100 mm × 3.0 mm × 3.0 μm); mobile phase A is supercritical carbon dioxide, mobile phase B is methanol containing 0.1% diethylamine; mobile phase B ratio is 30%, run time is 7 min; flow rate is 1.5 mL / min; column temperature is 35℃.

[0079] Starting from compound B06-7-A, compound B06-A (1.97 mg) was prepared using the same method as that used to prepare B05-A. LCMS [M+H] + 780.3. 1 H NMR (400 MHz, CD3OD) δ 8.56 (s, 1H),8.08 (d, J = 8.5 Hz, 1H), 7.85 - 7.78 (m, 1H), 7.61 (dd, J = 8.3, 1.7 Hz,1H), 7.04 (t, J = 5.2 Hz, 1H), 5.67 - 5.52 (m, 1H), 4.83 - 4.49 (m, 1H), 4.29(t, J = 4.4 Hz, 2H), 3.90 - 3.48 (m, 6H), 3.07 - 2.97 (m, 2H), 2.81 - 2.67(m, 1H), 2.55 - 2.40 (m, 4H), 2.11 - 1.86 (m, 3H), 1.83 - 1.45 (m, 6H), 0.86- 0.81 (m, 2H), 0.60 - 0.51 (m, 2H).

[0080] Starting from compound B06-7-C, compound B06-C (1.97 mg) was prepared using the same method as for B05-A. LCMS [M+H] +780.1. 1 H NMR (400 MHz, CD3OD) δ 8.57 (s, 1H),8.15 - 7.99 (m, 1H), 7.88 - 7.76 (m, 1H), 7.73 - 7.53 (m, 1H), 7.03 (t, J =5.1 Hz, 1H), 5.63 - 5.55 (m, 1H), 4.76 - 4.48 (m, 1H), 4.32 - 4.25 (m, 2H), 4.02 - 3.40 (m, 6H), 3.05 - 2.99 (m, 2H), 2.78 - 2.70 (m, 1H), 2.55 - 2.44(m, 4H), 1.92 - 1.87 (m, 2H), 1.83 - 1.36 (m, 7H), 0.87 - 0.80 (m, 2H), 0.59- 0.50 (m, 2H).

[0081] Starting from compound B06-7-D, compound B06-D (6.72 mg) was prepared using the same method as for B05-A. LCMS [M+H] + 780.3. 1 H NMR (400 MHz, CD3OD) δ 8.57 (s, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.83 (d, J = 1.5 Hz, 1H), 7.67 - 7.62 (m, 1H), 7.05(t, J = 5.1 Hz, 1H), 5.62 - 5.46 (m, 1H), 4.84 - 4.54 (m, 1H), 4.33 - 4.26(m, 2H), 3.98 - 3.50 (m, 6H), 3.12 - 3.00 (m, 3H), 2.51 (s, 3H), 2.31 - 2.21(m, 1H), 1.94 - 1.88 (m, 2H), 1.85 - 1.24 (m, 7H), 0.87 - 0.82 (m, 2H), 0.59- 0.53 (m, 2H).

[0082] Biological test examples

[0083] Unwinding activity test

[0084] The compounds were added to 384-well dilution plates, and each column of compound was serially diluted 1:3 in dimethyl sulfoxide (DMSO) to obtain 10 doses. Using an Echo pipetting system, 0.15 μL of the diluted compound solution from each row was transferred to the 384-well plate to a final DMSO concentration of 1%, with two replicates per column. 5 μL of enzyme working solution was added to the 384-well plate, and the plate was centrifuged at 1000 rpm for 1 minute. A high control group (DMSO containing enzyme) and a low control group (DMSO without enzyme) were also included. After incubation at 25°C for 10 minutes, 5 μL of adenosine triphosphate (ATP) working solution was added, and the plate was centrifuged at 1000 rpm for 1 minute; subsequently, 5 μL of double-stranded DNA (dsDNA) working solution was added, and the plate was centrifuged again at 1000 rpm for 1 minute. The final reaction system contained 0.5 nM enzyme, 10 nM double-stranded DNA, and 50 μM adenosine triphosphate in the assay buffer (containing 1 mM magnesium chloride). After incubation at 25°C for 20 minutes, the fluorescence intensity signals at excitation wavelength (Ex) of 620 nm and emission wavelength (Em) of 685 nm were detected using a BMG multi-mode microplate reader (model: CLARIO Star Plusacu).

[0085] The inhibition rate (% inh) of the compound pores is calculated as follows: Inhibition rate (%) = 100 × (average value of high control group - compound pore value) / (average value of high control group - average value of low control group).

[0086] Using XLfit 5.5.0 software, a nonlinear regression equation was used to fit the half-maximal inhibitory concentration (IC50) of the compound. ).

[0087] ADP-Glo ​​assay

[0088] The compounds were added to 384-well dilution plates, and each column of compound was serially diluted 1:3 in dimethyl sulfoxide (DMSO) to obtain 10 doses. Using an Echo pipetting system, 0.1 μL of the diluted compound solution from each row was transferred to the 384-well assay plate to a final DMSO concentration of 1%, with two replicates per column. 5 μL of enzyme working solution was added to the 384-well assay plate, and the plate was centrifuged at 1000 rpm for 1 minute. A high control group (DMSO containing enzyme) and a low control group (DMSO without enzyme) were also prepared. After incubation at 25°C for 10 minutes, 5 μL of a mixture of adenosine triphosphate (ATP) and single-stranded DNA (ssDNA) was added, and the plate was centrifuged at 1000 rpm for 1 minute. The final reaction system contained 0.1 nM enzyme, 2.5 nM single-stranded DNA, and 15 μM ATP in assay buffer (containing 2 mM magnesium chloride).

[0089] After incubating at 25°C for 60 minutes, add 5 μL of ADP-Glo™ reagent solution and incubate at 25°C for 40 minutes. Then, add 10 μL of kinase detection reagent to each well and continue incubating at 25°C for 40 minutes. Detect the luminescence signal using a BMG multi-mode microplate reader (model: Phera star FSX).

[0090] The formula for calculating the inhibition rate (% inh) of the compound pores is the same as

[00184] : Inhibition rate (%) = 100 × (average value of high control group - compound pore value) / (average value of high control group - average value of low control group)

[0091] Using XLfit 5.5.0 software, a nonlinear regression equation was used to fit the half-maximal inhibitory concentration (IC50) of the compound. ).

[0092] HCT116 cell proliferation inhibition activity

[0093] 1. Experimental Materials

[0094] Table 2-1 Information on Main Reagents and Consumables

[0095]

[0096] Table 2-2 Main Instrument Information

[0097]

[0098] Table 2-3 Solvent Information

[0099]

[0100] Table 2-4 Cell Information

[0101]

[0102] 2. Cell Culture and Processing

[0103] 2.1 Cell Culture

[0104] 1) In this project, HCT116 cells were purchased by Beijing Aisiyipu Biotechnology Co., Ltd. from ATCC.

[0105] 2) The HCT116 cell line was cultured in M'5A complete medium containing 10% fetal bovine serum and 1% PS in an incubator at 37 ℃ and 5% carbon dioxide.

[0106] 3) Cell passage: When the cell confluence reaches a suitable passage density, passage the cells. Transfer the cell suspension to a sterile centrifuge tube, centrifuge at 1000 rpm for 5 minutes to collect the cells, remove the supernatant, resuspend and count the cells, and then transfer them to a new culture medium.

[0107] 4) All operations follow the standard operating procedures for cell culture of Beijing Aisiyipu Biotechnology Co., Ltd.

[0108] 2.2 Cell Test

[0109] 1) Once the cells have grown to the logarithmic growth phase, collect the cell suspension, centrifuge at 1000 rpm for 5 min to collect the cells, and discard the supernatant;

[0110] 2) After resuspending and counting, HCT116 cells were evenly seeded into 96-well plates at a seeding rate of 100 cells / well / 195 μL and cultured at 37°C in a 5% CO2 incubator until the cells adhered.

[0111] 3) Test DAY0 on the second day: Select a board and test the DYA0 reading. See step 5 for specific test operations;

[0112] 4) Drug addition on the second day: Dilute the test substance and positive control compound to 10 mM with DMSO, then perform a 3-fold serial dilution with DMSO, resulting in 9+0 concentration points. Then, perform a second dilution with culture medium, adding 4 μL of drug to 96 μL of culture medium, diluting 25 times. Add 5 μL of the diluted compound to 195 μL of cell plate wells, resulting in a total dilution of 1000 times. The final concentration of the test substance and positive control compound in the wells is: 10 μM, 3-fold serial dilution, 9+0 doses, single well. At this point, the final concentration of DMSO in the wells is 0.1%.

[0113] 5) Place the cell culture plates containing the added drugs in an incubator at 37℃ and 5% CO2 for 7 days;

[0114] 6) On the day of the test, discard 100 μL of supernatant, then add 60 μL of CellCounting-Lite 2.0 Luminescent Cell Viability Assay (2D CTG) reagent to each well of the cell culture plate, shake at 200 rpm for 2 min at room temperature, and incubate for 30 min. The ATP level is detected by reading the BMG.

[0115] Data processing was performed using cell wells containing only DMSO and no drugs as positive controls and cell wells containing only culture medium as negative controls.

[0116] 3. Data Analysis

[0117] Set the negative control reading to 100% inhibition rate and the positive control reading to 0% inhibition rate, and calculate the inhibition rate of each test solution.

[0118]

[0119] Average value of positive control well readings

[0120] Average value of negative control well readings

[0121] The IC of the compound is obtained using the following nonlinear fitting formula. 50 (Half-maximal inhibitory concentration):

[0122]

[0123] X: Compound concentration

[0124] Y: Compound inhibition rate (% inh)

[0125] Z' factor calculation equation:

[0126] Z'=1-3(SD min +SD max ) / (AVE min -AVE max )

[0127] in:

[0128] Min is the negative control reading, and Max is the positive control reading.

[0129] Among them, R_IC50 and A_IC50 were calculated by curve fitting using the negative control reading after seven days of drug incubation as the low control, while R_GI50 and A_GI50 were calculated by curve fitting using the DAY0 reading as the low control.

[0130] HCT116 CTG Experiment

[0131] .

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The structure of the compound is shown below: , or .

2. A compound, characterized in that, The structure of the compound is shown below: , or .

3. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from one of the compounds in the following table: 、 。 4. A compound, characterized in that, The compound is selected from one of the compounds in the following table: 、 、 。 5. A pharmaceutical composition comprising a compound as described in any one of claims 1-4 or a pharmaceutically acceptable salt thereof, and a pharmaceutical excipient.

6. The use of a substance Z in the preparation of a medicament for treating and / or preventing cancer, wherein the substance Z is a compound as described in any one of claims 1-4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in claim 5.

7. The method according to claim 6, characterized in that, The cancers mentioned are endometrial cancer of the uterine body, colon adenocarcinoma, gastric adenocarcinoma, rectal adenocarcinoma, adrenocortical carcinoma, uterine carcinosarcoma, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, esophageal cancer, breast cancer, renal clear cell carcinoma, prostate cancer, or ovarian serous cystadenocarcinoma.

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