Piperazine-crosslinked substituted heterocyclic pyrimidine compounds

Abstract

Disclosed is a piperazine-crosslinked substituted heterocyclic pyrimidine compound or a pharmaceutically acceptable salt thereof, specifically, a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof. JPEG2025523101000245.jpg4748

Description

Technical Field

[0001] This invention claims the following priorities: CN202210822828.X, with a filing date of July 12, 2022, CN202211262711.7, with a filing date of October 14, 2022, CN202211407253.1, with a filing date of November 10, 2022, CN202310041534.8, with a filing date of January 11, 2023, CN202310041762.5, with a filing date of January 12, 2023, CN202310066868.0, with a filing date of January 19, 2023, CN202310800516.3, with a filing date of June 30, 2023.

[0002] [Technical Field]

[0003] This invention relates to a series of piperazine-bridged substituted heterocyclic pyrimidine compounds or pharmaceutically acceptable salts thereof, specifically to compounds represented by formula (III-1) or pharmaceutically acceptable salts thereof.

Background Art

[0004] NRAS, HRAS, and KRAS mutations in the RAS family cause nearly one-fourth of all human cancers and are one of the most common gene mutations associated with cancer. They cover almost all types of cancers and cause 1 million deaths worldwide every year. Among them, KRAS is the most common cancer gene (85% of all RAS mutations), present in 90% of pancreatic cancers, 30 - 40% of colon cancers, and 15 - 20% of lung cancers (mostly non-small cell lung cancers). Based on the specific mutations present, G12C, G12D, and G12R are the most common KRAS mutations in patients. In addition, there are also G12A, G12S, G12V, etc.

[0005] RAS (Rat Sarcoma) family proteins are widely expressed in various eukaryotes and exist in two expression forms: an inactive GDP (guanosine diphosphate)-bound form and an activated GTP (guanosine triphosphate)-bound form. Through the switching between these two expression forms, RAS proteins control multiple downstream pathways such as RAF-MEK-ERK and PI3K / Akt / mTOR, thereby affecting cell proliferation and differentiation, as well as tumorigenesis and development.

[0006] Mutant KRAS has a high affinity for guanosine triphosphate (GTP) and has factors that make it difficult to target, such as a small catalytic site and a smooth protein surface. Therefore, the development of small molecule inhibitors has always been difficult, and as a result, the legend that KRAS "cannot be drugged" has emerged. Due to the breakthrough in Mirati's KRAS G12D non-covalent inhibitor, KRAS G12D mutant tumors are gradually beginning to enter the field of precision medicine.

Summary of the Invention

[0007] The present invention provides a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof.

[0008]

Chemical Formula

[0009] However, X is selected from CH, C-Rx, N and N + -O - and preferably, X is selected from N and N + -O - Rx is selected from F, Cl, Br,

[0010]

Chemical Formula

[0011] R3 is selected from H and D, Each R aare each independently selected from F, Cl, Br, I, OH, NH2, C 1-3 alkyl, C 1-3 alkoxy, C 1-3 haloalkyl, C 2-4 alkenyl, C 2-4 alkynyl, -C 1-3 alkyl-cyclopropyl and cyclopropyl, and the C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, cyclopropyl and -C 1-3 alkyl-cyclopropyl are each independently optionally substituted by one, two or three Rs, each R is independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

[0012] The present invention provides a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof.

[0013]

Chemical formula

[0014] However, X is selected from CN, N and N + -O - and preferably, X is selected from N and N + -O - and

[0015]

Chemical formula

[0016] R3 is selected from H and D, each R a is independently selected from F, Cl, Br, I, OH, NH2, C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, -C 1-3Selected from alkyl-cyclopropyl and cyclopropyl, said C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, cyclopropyl and -C 1-3 alkyl-cyclopropyl is each independently optionally substituted by one, two or three Rs,

[0017] each R is independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

[0018] The present invention further provides a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof.

[0019]

Chemical formula

[0020] However, X is selected from N and N + -O - and is selected from

[0021]

Chemical formula

[0022] R3 is selected from H and D, each R a is independently selected from F, Cl, Br, I, OH, NH2, C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, -C 1-3 alkyl-cyclopropyl and cyclopropyl, said C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, cyclopropyl and -C 1-3Alkyl-cyclopropyl is each independently optionally substituted by 1, 2 or 3 Rs, each R is each independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

[0023] The present invention further provides a compound represented by formula (III-2) or a pharmaceutically acceptable salt thereof.

[0024]

Chemical formula

[0025] However, R1 is selected from phenyl, pyridyl and naphthyl, and the phenyl, pyridyl and naphthyl are each independently optionally substituted by 1, 2, 3 or 4 Rs a and

[0026]

Chemical formula

[0027] R3 is selected from H and D, each R a is each independently selected from F, Cl, Br, I, OH, NH2, C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, -C 1-3 alkyl-cyclopropyl and cyclopropyl, and the C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, cyclopropyl and -C 1-3 alkyl-cyclopropyl is each independently optionally substituted by 1, 2 or 3 Rs, R b is selected from H, CN, CH3 and OCH3, each R care each independently selected from F, Cl, Br, I, CH2F, CHF2, CF3 and CH2CF3, each R is independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

[0028] The present invention further provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof.

[0029]

Chemical formula

[0030] However, R1 is selected from phenyl, pyridyl and naphthyl, and the phenyl, pyridyl and naphthyl are each independently optionally substituted by 1, 2, 3 or 4 Rs a optionally substituted by,

[0031]

Chemical formula

[0032] R3 is selected from H and D, each R a is independently selected from F, Cl, Br, I, OH, NH2, C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, -C 1-3 alkyl-cyclopropyl and cyclopropyl, and the C 1-3 alkyl, C 1-3 alkoxy, C 2-4 alkenyl, C 2-4 alkynyl, cyclopropyl and -C 1-3 alkyl-cyclopropyl is each independently optionally substituted by 1, 2 or 3 Rs, R b is selected from H, CN, CH3 and OCH3, each R care each independently selected from F, Cl, Br, I, CH2F, CHF2, CF3, and CH2CF3, each R is independently selected from F, Cl, Br, I, CH2F, CHF2, and CF3.

[0033] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formulas.

[0034]

Chemical formula

[0035] provided that X, R1, and R3 are as defined in the present invention.

[0036] In some embodiments of the present invention, each of said Rs a is independently selected from F, Cl, OH, NH2, CH3, CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH(CH3)2,

[0037]

Chemical formula

[0038] and cyclopropyl, and said CH3, CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH(CH3)2,

[0039]

Chemical formula

[0040] and cyclopropyl are each independently optionally substituted by one, two, or three Rs, and the other variables are as defined in the present invention.

[0041] In some embodiments of the present invention, each of said Rs aare each independently selected from F, Cl, OH, NH2, CH3, CHF2, CF3, CH2CF3, CH(CH3)CF3, OCH3, OCF3,

[0042]

Chem.

[0043] and the other variables are as defined in the present invention.

[0044] In some embodiments of the present invention, said R1 is

[0045]

Chem.

[0046] and the other variables are as defined in the present invention.

[0047] In some embodiments of the present invention, said R1 is

[0048]

Chem.

[0049] selected from, and said

[0050]

Chem.

[0051] are each independently optionally substituted by 1, 2, 3 or 4 Rs a and the other variables are as defined in the present invention.

[0052] In some embodiments of the present invention, said R1 is

[0053]

Chem.

[0054] selected from, and other variables are as defined in the present invention.

[0055] In some embodiments of the present invention, said R1 is

[0056]

Chemical formula

[0057] selected from, and other variables are as defined in the present invention.

[0058] In some embodiments of the present invention, said R1 is

[0059]

Chemical formula

[0060] selected from, and other variables are as defined in the present invention.

[0061] In some embodiments of the present invention, said R2 is

[0062]

Chemical formula

[0063] selected from, and other variables are as defined in the present invention.

[0064] In some embodiments of the present invention, said R2 is

[0065]

Chemical formula

[0066] selected from, and other variables are as defined in the present invention.

[0067] In some embodiments of the present invention, said R2 is

[0068]

Chemical formula

[0069] selected from, and other variables are as defined in the present invention.

[0070] In some embodiments of the present invention, said R3 is selected from H and D, and other variables are as defined in the present invention.

[0071] In some embodiments of the present invention, said X is selected from N, and other variables are as defined in the present invention.

[0072] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formulas.

[0073]

Chemical formula

[0074] wherein R1, R2 and R3 are as defined in the present invention.

[0075] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formulas.

[0076]

Chemical formula

[0077] wherein R1, R2 and R3 are as defined in the present invention, and

[0078] the condition is that R2 is

[0079]

Chemical formula

[0080] It is not the case.

[0081] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formulas.

[0082] [Chemical formula]

[0083] However, R1 and R3 are as defined in the present invention.

[0084] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formulas.

[0085] [Chemical formula]

[0086] However, R1 and R3 are as defined in the present invention.

[0087] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formulas.

[0088] [Chemical formula]

[0089] However, R1 and R3 are as defined in the present invention.

[0090] In Examples 2 and 3 of the present invention, Compound 3 was obtained by manufacturing from Compound 2-4A according to the steps described in Examples 2 and 3. Here, after developing twice by TLC thin layer chromatography (petroleum ether: acetone = 5:1), the product corresponding to the smaller Rf value is Compound 2-4A, and the product corresponding to the larger Rf value is its isomer.

[0091] In Examples 2 and 3 of the present invention, Compound 3 was obtained by manufacturing from Compound 2-4A according to the steps described in Examples 2 and 3. Here, after developing twice by TLC thin layer chromatography (petroleum ether: acetone = 5:1), the Rf value of Compound 2-4A is 0.5, and the Rf value of its isomer is 0.55.

[0092] In Examples 2 and 3 of the present invention, Compound 3 was obtained by manufacturing from Compound 2-4A according to the steps described in Examples 2 and 3. Here, by LCMS (chromatography column: Agilent Poroshell 120 EC-C18 2.7um 3.0×30mm, mobile phase: A: water (0.037% formic acid) - B: acetonitrile (0.0187% formic acid); B: 5% - 95%), the retention time of 2-4A is 0.776 minutes, and it is shown that the retention time of its isomer is 0.801 min.

[0093] In Example 3 of the present invention, Compound 3 was obtained by manufacturing from Compound 3-2A according to the steps described in Example 3. Here, by SFC analysis (chromatography column: Cellulose 2 100mm×4.6mm, 3μm, mobile phase: [A (supercritical CO2), B (methanol (0.05% diethanolamine))]; B: 40%), the retention time is 4.964 minutes, and it is shown that the retention time of its enantiomer is 8.382 min.

[0094] In Example 3 of the present invention, Compound 3 has a retention time of 3.761 min as shown by SFC analysis (chromatography column: Chiralcel OJ-3 100×4.6 mm, 3 μm; mobile phases: A (supercritical CO2) and B (ethanol containing 0.05% diethylamine); gradient: B%: 40% to 40%).

[0095] The present invention further provides a compound represented by the following formula and a pharmaceutically acceptable salt thereof.

[0096] [Chemical formula]

[0097] However, Compound 3 has a retention time of 3.761 min as shown by SFC analysis (chromatography column: Chiralcel OJ-3 100×4.6 mm, 3 μm; mobile phases: A (supercritical CO2) and B (ethanol containing 0.05% diethylamine); gradient: B%: 40% to 40%).

[0098] The present invention further provides a compound represented by the following formula and a pharmaceutically acceptable salt thereof.

[0099] [Chemical formula]

[0100] However, Compound 3 has a retention time of 17.387 min as shown by chiral HPLC analysis (chromatography column: FLM Chiral NQ, 150×4.6 mm, 3 μm; mobile phases: A: (n-hexane) and B: (ethanol containing 0.02% diethylamine, v / v); gradient: B%: isocratic 30%, elution time: 60 min, column temperature: 35°C).

[0101] In some embodiments of the present invention, the three stereoisomers of Compound 3 have retention times of 22.587 min, 26.205 min, and 44.765 min as shown by chiral HPLC analysis (chromatographic column: FLM Chiral NQ, 150×4.6 mm, 3 μm; mobile phase: A: (n - hexane) and B: (ethanol containing 0.02% diethylamine, v / v); gradient: B%: isocratic 30%, elution time: 60 min, column temperature: 35°C).

[0102] In one embodiment of the present invention, the present invention provides Compound 7A2 or a pharmaceutically acceptable salt thereof, wherein the Compound 7A2 has any of the following chemical structures, and its retention time under the conditions of analytical SFC (column: ChiraPak IG - 3, 50×4.6 mm, 3 μm; mobile phase: A: (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine), gradient: B%: 5% - 5%, 3 min) is shown to be 2.236 min.

[0103]

Chemical formula

[0104] The present invention further provides a compound represented by the following formula and a pharmaceutically acceptable salt thereof.

[0105]

Chemical formula

[0106]

Chemical formula

[0107]

Chemical formula

[0108]

Chemical formula

[0109] The present invention further provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof.

[0110]

Chemical formula

[0111]

Chemical formula

[0112]

Chemical formula

[0113]

Chemical formula

[0114]

Chemical formula

[0115]

Chemical formula

[0116]

Chemical formula

[0117]

Chemical formula

[0118]

Chemical formula

[0119]

Chemical formula

[0120]

Chem.

[0121]

Chem.

[0122]

Chem.

[0123]

Chem.

[0124]

Chem.

[0125]

Chem.

[0126]

Chem.

[0127] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the following formulas.

[0128]

Chem.

[0129] The present invention provides the use of the compound or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating solid tumors having a KRAS G12D mutation.

[0130] The present invention provides a pharmaceutical composition comprising a compound represented by any one of the general formulas of Formula I, Formula II, Formula III-1, and Formula III-2 of the present invention or a pharmaceutically acceptable salt thereof, and any pharmaceutically acceptable carrier.

[0131] The present invention provides the use of the compound represented by any one of the general formulas of Formula I, Formula II, Formula III-1, and Formula III-2 of the present invention or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating solid tumors having a KRAS G12D mutation, preferably, the solid tumors are selected from colon cancer and pancreatic cancer.

[0132] The present invention further provides the use of the pharmaceutical composition in the manufacture of a medicament for treating solid tumors having a KRAS G12D mutation, preferably, the solid tumors are selected from colon cancer and pancreatic cancer.

[0133] The present invention further provides the following synthesis methods.

[0134] Synthesis Route 1:

[0135]

Chemical formula

[0136] Synthesis Route 2:

[0137]

Chemical formula

[0138] Synthesis Route 3:

[0139]

Chemical formula

[0140] However, R3 is selected from H, F, and Cl, and R 1a is

[0141]

Chem.

[0142] selected from, and R1 is

[0143]

Chem.

[0144] selected from.

[0145] Synthesis Route 4:

[0146]

Chem.

[0147] However, R3 is selected from H, F, and Cl, and R 1a is

[0148]

Chem.

[0149] selected from,

[0150] R1 is

[0151]

Chem.

[0152] selected from.

[0153] The present invention further provides the following test methods.

[0154] Test Method 1. KRAS G12D Inhibitory Activity Test 1. Experimental Purpose: By the TR-FRET method, KRAS G12DThis is for screening compounds that can effectively inhibit the binding to GTP.

[0155] 2. Consumables and equipment:

[0156]

Table 1

[0157] 3. Preparation of reagents: a. Stock reagents: 1) KRAS nucleotide exchange buffer

[0158] Take 20 mL of 1000 mM HEPES, 20 mL of 500 mM EDTA, 10 mL of 5 M sodium chloride, 0.1 mL of 100% Tween 20, and 949.9 mL of water to prepare 1 L of solution, sterilize by filtration, and store at 4°C.

[0159] 2) KRAS experimental buffer Take 20 mL of 1000 mM HEPES, 10 mL of 1000 mM magnesium chloride, 30 mL of 5 M sodium chloride, 0.05 mL of 100% Tween 20, and 939.95 mL of water to prepare 1 L of solution, sterilize by filtration, and store at 4°C.

[0160] 3) KRAS / Bodipy GDP / Tb-SA mixture Take 9.5 μL of KRAS G12D protein at 95 μM, 440.5 μL of KRAS nucleotide exchange buffer, mix, incubate at room temperature for 1 hour, then take 8.4 μL of 17.9 μM Tb-SA, 1.8 μL of 5 mM Bodipy GDP, and 9539.8 μL of KRAS experimental buffer to prepare 1 L of solution, mix, leave at room temperature for 6 hours, and store under -80°C conditions.

[0161] b. Experimental reagents: 1) KRAS enzyme solution 73.3 μL of the KRAS / Bodipy GDP / Tb-SA mixture and 2126.7 μL of the KRAS experimental buffer were taken and made into a 2200 μL solution.

[0162] 2) SOS / GTP mixture 1.59 μL of 166 μM SOS protein, 198 μL of 100 mM GTP, and 2000.41 μL of the KRAS experimental buffer were taken and made into a 2200 μL solution.

[0163] 4. Experimental process: 1) The concentration of the control compound stock solution is 1 mM, and the concentration of the test compound stock solution is 10 mM. 9 μL of the control compound and the test compound were transferred to a 384-LDV plate. 2) Using Bravo, the compounds on the LDV plate were serially diluted 3-fold at 10 points. 3) Using ECHO, 9 nL of the compounds on the LDV plate were transferred to the experimental plate. 4) Using the Dragonfly automatic sampler, 3 μL of 3 nM Kras / 0.5 nM TB-SA / 30 nM BodipyGDP mixture and 3 μL of the Ras buffer were sequentially added to each well of the experimental plate, and centrifuged at 1000 rpm / min for 1 minute. 5) The experimental plate was incubated at room temperature for 1 hour. 6) Using the Dragonfly automatic sampler, 3 μL of 120 nM SOS / 9 mM GTP mixture was added to each well of the experimental plate, and centrifuged at 1000 rpm / min for 1 minute. 7) The experimental plate was incubated at room temperature for 1 hour. 8) Using Envision, the plate was read and the data were recorded. 9) Using Excel and Xlfit, data analysis was performed to calculate the IC 50 of the test compound.

[0164] Test method 2: p-ERK inhibition test of GP2D cells 1. Experimental purpose: To screen for compounds that can effectively inhibit p-ERK in GP2D cells by the HTRF method.

[0165] 2. Experimental process: 1). GP2D cells were seeded in a transparent 96-well cell culture plate with a cell suspension of 80 μL / well, and each well contained 8,000 cells. The cell plate was placed in a carbon dioxide incubator and cultured overnight at 37°C. 2). 2 μL of the compound was taken and added to 78 μL of cell culture medium. After uniform mixing, 20 μL of the compound solution was taken and added to the corresponding wells of the cell plate. The cell plate was returned to the carbon dioxide incubator and continued to be cultured for 1 hour. 3). After the culture was completed, the cell supernatant was discarded, and 50 μL of 1X cell lysate was added to each well and cultured with shaking at room temperature for 30 minutes. 4). The Phospho-ERK1 / 2 Eu Cryptate antibody and the Phospho-ERK1 / 2 d2 antibody were diluted 20-fold with the detection buffer. 5). The cell lysate supernatant was taken at 16 μL / well and placed in a new 384-well white microtiter plate. 2 μL of the Phospho-ERK1 / 2 Eu Cryptate antibody dilution and 2 μL of the Phospho-ERK1 / 2 d2 antibody dilution were added and cultured at room temperature for at least 4 hours. 6). After the culture was completed, the HTRF excitation: 320 nm, emission: 615 nm, 665 nm were read using a multi-label analyzer. 7). The IC 50 of the test compound was calculated.

[0166] Test method 3: GP2D 3D CTG experiment 1. Experimental purpose: The purpose of this experiment is to verify the growth inhibitory effect of the compound of the present invention on GP2D human colon cancer cells having the KRAS G12D mutation.

[0167] 2. Experimental materials: The cell line GP2D, DMEM medium, and penicillin / streptomycin antibiotics were purchased from Wisent, and fetal bovine serum was purchased from Biosera. The CellTiter-Glo® 3D Cell Viability Assay (chemiluminescent detection reagent for 3D cell viability) reagent was purchased from Promega.

[0168] 3. Experimental method: GP2D cells were seeded into a 96-well U-bottom cell culture plate, with 80 μL of cell suspension in each well, such that each well contained 2000 GP2D cells. The cell plate was cultured overnight in a carbon dioxide incubator. The test compound was diluted 5-fold at 8 concentrations with a pipette, i.e., diluted from 200 μM to 2.56 nM, and two wells were set up under the same conditions. 78 μL of medium was added to the middle plate, and then, according to the corresponding positions, 2 μL / well of the serially diluted compound was transferred to the middle plate. After uniform mixing, 20 μL / well was transferred to the cell plate. The concentration range of the compound transferred to the cell plate was 1 μM to 0.0128 nM. The cell plate was placed in a carbon dioxide incubator and cultured for 5 days. After culturing the cell plate with the added compound, 100 μL of the cell viability chemiluminescent detection reagent was added to each well of the cell plate and cultured at room temperature for 10 minutes to stabilize the luminescence signal. Data was read using a multimode microplate reader.

[0169] 4. Data analysis: The raw data was converted to the inhibition rate using the equation (Sample - Min) / (Max - Min)×100%, and the IC 50 value was obtained by curve fitting using four parameters (obtained with the log(inhibitor) vs. response - Variable slope motor in GraphPad Prism).

[0170] Test method 4: In vivo pharmacokinetic experiment 1. Experimental purpose:

[0171] The purpose of this experiment is to investigate the pharmacokinetic properties of the compound of the present invention administered orally and intravenously to SD mice.

[0172] 2. Experimental method: The test compound was mixed with 10% dimethyl sulfoxide / 60% polyethylene glycol 400 / 30% aqueous solution, vortexed and sonicated to produce a clear solution of about 1 mg / mL, and filtered through a microporous membrane for use. Male SD mice aged 7 - 10 weeks were selected, and the candidate compound solution was administered intravenously at a dose of 3 mg / kg. The candidate compound solution was administered orally at a dose of about 30 mg / kg. Whole blood was collected at predetermined times to prepare plasma, the drug concentration was analyzed by LC-MS / MS method, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA).

[0173] Test method 5: In vivo pharmacodynamics experiment

[0174] 1. Experimental purpose: This is for the in vivo pharmacodynamics study of a Balb / c nude mouse tumor model with human colon cancer GP2D cells subcutaneously transplanted into nude mice.

[0175] 2. Experimental method: Cell culture: Human colon cancer GP2D cells were cultured in vitro in monolayers. The culture conditions were DMEM / F12 medium supplemented with 20% fetal bovine serum and 1% double antibody, and cultured in a 5% CO2 incubator at 37°C. Trypsin-EDTA was used for routine digestion twice a week for subculture. When the cell confluence reached 80% - 90% and the required number was reached, the cells were collected, counted, resuspended in an appropriate amount of PBS, and Matrigel was added at a ratio of 1:1 to obtain a cell suspension with a cell density of 25×10 6 cells / mL.

[0176] Cell inoculation: 0.2 mL (5×10 6 cells / mouse) of Mia PaCa-2 cells (added with Matrigel, volume ratio 1:1) were subcutaneously inoculated into the right back of each mouse.

[0177] Experimental operation: When the average tumor volume reached approximately 190 mm 3 Six animals in each group were randomly divided according to the tumor volume. The dosage of the blank group was 0, and the dosages of the test groups were 30 mg / kg and 100 mg / kg respectively. The dosage was 10 μL / g, and oral administration was carried out twice a day for 22 days.

[0178] 3. Tumor measurement and experimental indicators: The diameter of the tumor was measured with vernier calipers twice a week. The calculation formula for tumor volume is: V = 0.5a × b 2 where a and b represent the major axis and minor axis of the tumor respectively.

[0179] The antitumor effect of the compound is evaluated by TGI(%) or relative tumor growth rate T / C(%). Relative tumor growth rate T / C(%) = TRTV / CRTV × 100% (TRTV: RTV of the treatment group, CRTV: RTV of the negative control group). According to the tumor measurement results, the relative tumor volume (RTV) was calculated, and the calculation formula is RTV = V t / V0, where V0 is the average tumor volume measured at the time of dividing the groups for administration (i.e., D0), and V t is the average tumor volume at a specific measurement time, and TRTV takes the data on the same day as CRTV.

[0180] TGI(%) reflects the tumor growth inhibition rate. TGI(%) = [1 - (average tumor volume at the end of administration of a specific treatment group - average tumor volume at the start of administration of the treatment group) / (average tumor volume at the end of treatment of the solvent control group - average tumor volume at the start of treatment of the solvent control group)] × 100%.

[0181] [Technical effects] The compound of the present invention has a good binding effect with KRAS G12D protein, can significantly inhibit the KRAS G12D enzyme and p-ERK of GP2D cells. The compound of the present invention has good cell growth inhibition activity against KRAS G12D mutant cells and has excellent tumor inhibitory effects. Furthermore, the compound of the present invention has more excellent pharmacokinetic properties.

[0182] [Definitions and Explanations] Unless otherwise explained, the following terms and collocations used in this specification have the following meanings. If a particular term or collocation is not specially defined, it should be understood as having its ordinary definition and not being uncertain or unclear. When a trade name appears in this specification, it refers to the corresponding product or its active ingredient.

[0183] As used herein, the term "pharmaceutically acceptable" pertains to those compounds, materials, compositions and / or dosage forms which are within the scope of sound medical judgment, suitable for contact with human and animal tissues, have little toxicity, irritation, allergic reaction or other problems or complications, and meet a reasonable benefit / risk ratio.

[0184] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, which is produced with an acid or base that is relatively non-toxic compared to the compound having the specific substituents found in the present invention. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting these compounds with a sufficient amount of a base in a single solution or in a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting these compounds with a sufficient amount of an acid in a single solution or in a suitable inert solvent. Some specific compounds of the present invention contain both basic and acidic functional groups and can thus be converted into any base addition salt or acid addition salt.

[0185] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing acid groups or basic groups by conventional methods. Usually, the methods for producing such salts involve reacting these compounds in the form of free acids or bases with a stoichiometric amount of a suitable base or acid in water or an organic solvent, or a mixture of both.

[0186] The compounds of the present invention may exist in the form of specific geometric isomers or stereoisomers. The present invention contemplates all such compounds and includes cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, e.g., mixtures rich in enantiomers or diastereomers, and all these mixtures are included within the scope of the present invention. Substituents such as alkyl may have other asymmetric carbon atoms. All these isomers and mixtures thereof are included within the scope of the present invention.

[0187] Unless otherwise defined, in chiral HPLC (chiral high performance liquid chromatography) analysis and SFC (supercritical fluid chromatography) analysis, the retention time of a compound may vary due to factors such as the measuring instrument. For any particular compound, there may be measurement errors in the retention time of the compound. Therefore, it is necessary to consider this error when determining each compound, and this error is also within the scope of this disclosure.

[0188] The compounds of the present invention may contain non-natural proportions of atomic isotopes in one or more atoms constituting the compound. For example, the compound can be labeled with a radioactive isotope such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C). Also, for example, hydrogen can be replaced with deuterium to form a deuterated drug, and the bond formed by deuterium and carbon is stronger than the bond formed by normal hydrogen and carbon. Compared with the non-deuterated drug, the deuterated drug has the advantages of reduced toxic side effects, increased drug stability, improved therapeutic effect, and extended biological half-life of the drug. The conversion of the isotope composition of the compounds of the present invention is included within the scope of the present invention regardless of whether it is radioactive.

[0189] The terms "optional" or "optionally" may appear depending on the matters or circumstances described hereinafter, but do not necessarily appear, and the description means that it includes the cases where the matters or circumstances described therein occur and the cases where they do not occur.

[0190] The term "substituted" refers to the substitution of any one or more hydrogen atoms in a specific atom by a substituent. If the specific valence state is normal and the compound after substitution is stable, the substituent may include deuterium and variants of hydrogen. When the substituent is a ketone (i.e., =O), a ketone substitution meaning the substitution of two hydrogen atoms does not occur in an aromatic group. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise defined, the type and number of substituents are arbitrary as long as they are chemically feasible.

[0191] When any of the variables (e.g., R) appears one or more times in the composition or structure of a compound, its definition is independent in any case. Therefore, for example, when one group is substituted by 0 to 2 Rs, the above group is optionally substituted by 2 or fewer Rs, and in any case, R has independent options. Also, combinations of substituents and / or their variants are allowed only when such combinations result in stable compounds.

[0192] When the number of linking groups is 0, for example, -(CRR)0- means that the linking group is a single bond.

[0193] When one of the variables is a single bond, the two groups linked thereby are directly linked. For example, when L in A-L-Z represents a single bond, this structure actually represents A-Z.

[0194] When the listed linking groups do not specify the linking direction, the linking direction is arbitrary. For example,

[0195]

Chemical formula

[0196] In the formula, the linking group L is -M-W-. At this time, -M-W- is linked to ring A and ring B in the same direction as the reading order from left to right and

[0197]

Chemical formula

[0198] can form, and can also be linked to ring A and ring B in the direction opposite to the reading order from left to right and

[0199]

Chemical formula

[0200] can also form. The combinations of the above linking groups, substituents and / or their variants are only allowed when such combinations result in stable compounds.

[0201] Unless otherwise specified, when a group has one or more bondable sites, any one or more sites of the group may be linked to other groups by chemical bonds. When the linking mode of the chemical bond is delocalized and there is an H atom at the bondable site, when the chemical bond is formed, the number of H atoms at the site decreases to a group with a corresponding valence according to the number of the formed chemical bonds. The chemical bond by which the site binds to other groups

[0202]

Chemical formula

[0203] can be represented by. For example, the straight solid line bond of -OCH3 means that it is linked to other groups through the oxygen atom of the group.

[0204]

Chemical formula

[0205] The straight dashed line connection in it means that it is bonded to other groups through both ends of the nitrogen atom in the group.

[0206]

Chemical formula

[0207] The wavy line in it means that it is bonded to other groups through the carbon atoms at positions 1 and 2 of the phenyl group.

[0208]

Chemical formula

[0209] means that any bondable site of the piperidinyl group can be bonded to another group through one chemical bond connection, and at least

[0210]

Chemical formula

[0211] includes four bonding forms of, and even if an H atom is depicted on -N,

[0212]

Chemical formula

[0213] contains

[0214]

Chemical formula

[0215] groups with bonding forms such as, but when one chemical bond is connected, the H at that site decreases by one to become the corresponding monovalent piperidine group.

[0216] Unless otherwise stated,

[0217] [Chemistry]

[0218] represents the absolute configuration of one stereocenter

[0219] [Chemistry]

[0220] represents the relative configuration of the stereocenter

[0221] [Chemistry]

[0222] or

[0223] [Chemistry]

[0224] represents.

[0225] For example,

[0226] [Chemistry]

[0227] is

[0228] [Chemistry]

[0229] represents the relative configuration of the stereocenter

[0230] [Chemistry]

[0231] represents a mixture with

[0232]

Chem.

[0233] is

[0234]

Chem.

[0235] represents a mixture with

[0236]

Chem.

[0237] is

[0238]

Chem.

[0239] represents a mixture with

[0240] Certain compounds of the present invention may exist as atropisomers, which are conformational isomers that occur when the rotation around a single bond within the molecule is hindered or significantly decelerated due to the spatial interaction of other parts of the molecule. The compounds disclosed in the present invention include pure individual block isomers, or atropisomers with one being enriched, or all atropisomers as non-specific mixtures thereof. When the rotational potential energy around a single bond is sufficiently high and the interconversion between conformational structures is sufficiently slow, the separation of the isomers becomes possible. For example,

[0241]

Chem.

[0242] is a pair of atropisomers, where the phenyl

[0243]

Chem.

[0244] represents that the direction of the solid on this side is outward,

[0245]

Chem.

[0246] represents that the direction of the solid on this side is inward.

[0247] Unless otherwise defined, when a double bond structure such as a carbon-carbon double bond, a carbon-nitrogen double bond, and a nitrogen-nitrogen double bond exists in a compound, and two different substituents are linked to each atom in the double bond (in a double bond containing a nitrogen atom, a pair of lone electron pairs on the nitrogen atom is regarded as one substituent linked thereto), between the atom on the double bond of the compound and its substituent

[0248]

Chem.

[0249] is represented by, it means the (Z)-isomer, (E)-isomer, or a mixture of the two isomers of the compound.

[0250] Unless otherwise defined, C n~n+m or C n ~C n+m includes any one specific form of n to n + m carbons. For example, C 1-12 is C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , and C 12 , and also includes any one range of n to n + m. For example, C 1-12 is C 1-3 , C 1-6 , C 1-9, C 3-6 , C 3-9 , C 3-12 , C 6-9 , C 6-12 , and C 9-12 etc. are included. Similarly, n-membered to n + m-membered represent that the number of atoms in the ring is n to n + m. For example, a 3- to 12-membered ring includes a 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring, and 12-membered ring, and also includes any one range among n to n + m. For example, a 3- to 12-membered ring includes a 3- to 6-membered ring, 3- to 9-membered ring, 5- to 6-membered ring, 5- to 7-membered ring, 6- to 7-membered ring, 6- to 8-membered ring, and 6- to 10-membered ring etc.

[0251] Unless otherwise defined, the term "C 1-3 alkyl" represents a saturated hydrocarbon group composed of 1 to 3 carbon atoms in a straight or branched chain. The C 1-3 alkyl includes C 1-2 and C 2-3 alkyl etc., which may be monovalent (e.g., methyl), divalent (e.g., methylene), and polyvalent (e.g., methine). Examples of C 1-3 alkyl include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.

[0252] Unless otherwise defined, the term "halogenated" or "halo" means a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom, either by itself or as part of another substituent.

[0253] Unless otherwise defined, the term "C 1-3 alkoxy" represents an alkyl group containing 1 to 3 carbon atoms linked to the rest of the molecule through an oxygen atom. The C 1-3 alkoxy includes C 1-2 , C 2-3 , C3, and C2 alkoxy etc. Examples of C 1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy or isopropoxy), etc.

[0254] Unless otherwise defined, "C 2-4 alkenyl" represents a saturated hydrocarbon group composed of 2 to 4 carbon atoms containing at least one carbon-carbon double bond, which may be linear or branched, and the carbon-carbon double bond may be at any position in the group. The C 1-4 alkenyl includes C 2-3 , C4, C3, and C2 alkenyl, etc., and the C 2-4 alkenyl may be monovalent, divalent, or polyvalent. Examples of C 2-4 alkenyl include, but are not limited to, vinyl, propenyl, butenyl, butadienyl, etc.

[0255] Unless otherwise defined, "C 2-4 alkynyl" represents a saturated hydrocarbon group composed of 2 to 4 carbon atoms containing at least one carbon-carbon triple bond, which may be linear or branched, and the carbon-carbon triple bond may be at any position in the group. The C 2-4 alkynyl includes C 2-3 , C4, C3, and C2 alkynyl, etc. It may be monovalent, divalent, or polyvalent. Examples of C 2-4 alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, etc.

[0256] Unless otherwise defined, in Formula III of the present invention, when X is N + -O - , "N + -O - " is used to represent "N(→O)" and represents the oxide of N.

[0257] The compounds of the present invention can be prepared by various synthetic methods well-known to those skilled in the art, including the specific embodiments listed below, embodiments combined with other chemical synthesis methods, and equivalent alternative methods well-known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples of the present invention.

[0258] The structure of the compounds of the present invention can be confirmed by conventional methods well-known to those skilled in the art. When the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by the conventional technical means of those skilled in the art. For example, for single crystal X-ray diffraction (SXRD), the cultured single crystal is collected by a Bruker D8 venture diffractometer, the light source is CuKα radiation, the scanning method is φ / ω scanning. After collecting the relevant data, the direct method (Shelxs97) can be used for crystal structure analysis to confirm the absolute configuration.

[0259] All solvents used in the present invention can be obtained from commercial products. Boc represents tert-butoxycarbonyl, Fmoc represents 9-fluorenylmethoxycarbonyl, TIPS represents triisopropylsilyl, PMB represents p-methoxybenzyl, Tf represents trifluoromethanesulfonyl, DCE represents dichloroethane, THF represents tetrahydrofuran, H2O represents water, FA represents formic acid, ACN represents acetonitrile, PE represents petroleum ether, EA represents ethyl acetate, DEA represents diethanolamine, IPA represents isopropanol, DBU represents 1,8-diazabicyclo[5.4.0]undec-7-ene, the second-generation Grubbs catalyst represents a compound with a CAS number of 246047-72-3, the ratio of the eluent in column chromatography represents the volume ratio, and the concentration of M represents mol / L.

[0260] Compounds are named according to the usual naming principles in this field or by ChemDraw (registered trademark) software. For commercially available compounds, the names in the manufacturer's catalog are used.

Brief Description of the Drawings

[0261]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

DETAILED DESCRIPTION OF THE INVENTION

[0262] Hereinafter, the present invention will be specifically described by way of examples, which are not meant to be limiting of the present invention. The present invention is described in detail herein, and specific embodiments thereof are also disclosed. It is obvious to those skilled in the art that various changes and modifications can be made in the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0263] Calculation Example 1

[0264]

CHEM.

[0265] The molecular docking process was carried out using Glide SP of Maestro (Schrodinger version 2017-2) [1]And it was executed using the default settings. The crystal structure of KRAS_G12C in the PDB database PDB: 6UT0 was selected, simulated so that Cys12 mutates to Asp12, and after energy optimization, it was used as a docking template. To produce the protein, hydrogen atoms were added using the protein production wizard module of Maestro [2] and the OPLS3 force field was used. In the production of the ligand, LigPrep was used to generate the three-dimensional structure of the molecule and energy minimization [3] was performed, and the confgen module was used to sample the conformations of small molecules. Using the ligand of 6UT0 as the center of mass, a docking lattice of a cube with sides of 25 Å × 25 Å × 25 Å was produced. A reference compound was placed in the molecular docking process. The types of interactions between the protein receptor and the ligand were analyzed, and according to the calculated docking score and binding mode, reasonable docking conformations were selected and saved as shown in Figures 1 to 11.

[0266] [Chemical formula]

[0267] Conclusion: The compounds of the present invention have good binding properties with KRAS G12D and

[0268] Synthesis of intermediates Int-3A and Int-3B

[0269] [Chemical formula]

[0270] Step 1: Under the protection of nitrogen gas, Int3-1 (3 g, 9.56 mmol) and 1-1B (1.83 g, 8.60 mmol) were dissolved in dichloromethane (30 mL), triethylamine (2.90 g, 28.67 mmol) was added at -40 °C, and the reaction was carried out at -40 °C for 0.5 h. Water (20 mL) was added to the reaction system, and the aqueous phase was extracted with dichloromethane (20 mL × 4) and separated. The organic phase was washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (mobile phase: petroleum ether:ethyl acetate = 20:1 to 1.5:1) to obtain intermediate Int3-2. MS m / z: 488.9, 490.9 [M+1] + 。

[0271] Step 2: Under the protection of nitrogen gas, intermediate Int3-2 (0.8 g, 1.63 mmol) and intermediate 2-7 (269.91 mg, 1.63 mmol) were dissolved in N,N-dimethylformamide (8 mL) and tetrahydrofuran (8 mL), cesium carbonate (1.33 g, 4.08 mmol) and triethylenediamine (54.97 mg, 490.06 μmol) were added, and the reaction was carried out at 25 °C for 15 h. Water (10 mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (10 mL × 5) and separated. The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (mobile phase: petroleum ether:ethyl acetate = 25:1 to 1:1) to obtain intermediate Int3-3. MS m / z: 618.0 [M+1] + 。

[0272] Step 3: Intermediate Int3-3 was separated by preparative SFC (chromatography column: REGIS (S,S) WHELK-O1 (250 mm × 25 mm, 10 μm); mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% aqueous ammonia): gradient: B%: 60% to 60%, 10 min) to obtain intermediates Int-3A and Int-3B.

[0273] Intermediate Int-3A: SFC analysis method: Chromatography column REGIS (S,S) WHELK-O1 (50 mm × 4.6 mm, 3.5 μm), mobile phases: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B%: 5% - 5%, retention time was 1.780 min, and the ee value was 95.37%. 1 H NMR (400 MHz, CDC13) δ = 7.32 (dd, J = 9.2, 2.0 Hz, 1H), 5.02 (s, 1H), 4.94 (s, 1H), 4.38 - 4.25 (m, 4H), 4.24 - 4.17 (m,1H), 3.66 - 3.48 (m, 3H), 3.34 - 3.20 (m, 2H), 2.93 (d, J = 17.2 Hz, 1H), 2.75 (dd, J = 8.8, 4.0 Hz, 1H), 2.47 (d, J =18.4 Hz, 1H), 1.95 (t, J = 5.6 Hz, 2H), 1.89 - 1.75 (m, 3H), 1.60 - 1.55 (m, 2H), 1.52 (s, 9H), 0.75 (d, J = 3.2 Hz,1H), 0.52 - 0.45 (m, 1H). MS m / z: 618.0 [M+1] + 。

[0274] Intermediate Int-3B: SFC analysis method: Chromatography column REGIS (S,S) WHELK-O1 (50 mm × 4.6 mm, 3.5 μm), mobile phases: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B%: 5% - 5%, retention time was 1.914 min, and the ee value was 96.62%. 11H NMR (400 MHz, CDCl3) δ = 7.33 (dd, J = 9.2, 2.0 Hz, 1H), 5.18 - 4.85 (m, 2H), 4.52 - 4.09 (m, 6H), 3.73 - 3.44 (m, 3H), 3.43 - 3.10 (m, 2H), 2.88 - 2.66 (m, 1H), 2.62 - 2.36 (m, 1H), 2.02 - 1.67 (m, 6H), 1.52 (s, 9H), 1.27 (d, J = 4.4 Hz, 1H), 0.89 - 0.65 (m, 1H), 0.63 - 0.40 (m, 1H). MS m / z: 618.0 [M+1] + 。

[0275] Synthesis of Intermediate Int-4

[0276]

Chemical Structure

[0277] Step 1: Dissolve Int4-1 (100 g, 397.67 mmol) in acetic acid (300 mL), cool to 0 °C, then add concentrated sulfuric acid (390.03 g, 3.98 mol). Next, add a solution of sodium nitrite (54.87 g, 795.34 mmol) in water (50 mL) drop by drop and react at 0 °C for 1 hour. At this time, the suspension becomes transparent. Then add a solution of potassium iodide (132.03 g, 795.34 mmol) in water (50 mL) drop by drop and react at 0 °C for 1 hour. Add 5000 mL of water, stir for 10 minutes, perform suction filtration, wash 5 times with water, add 500 mL of saturated aqueous sodium thiosulfate solution, and stir overnight. Then, perform suction filtration again, wash 5 times with water (500 mL × 4), perform suction filtration, and the cake was Intermediate Int4-2. 1 1H NMR (400 MHz, CDCl3) δ = 8.37 (d, J = 2.0 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H).

[0278] Step 2: Intermediate Int4-2 (126.15 g, 348.15 mmol) was dissolved in ethanol (500 mL) and water (200 mL). Next, iron powder (38.88 g, 696.29 mmol) and ammonium chloride (37.25 g, 696.29 mmol) were added, and the mixture was reacted at 80 °C for 2 hours. It was subjected to suction filtration, the filtrate was concentrated under reduced pressure, extracted with ethyl acetate (500 mL × 2), washed with saturated brine (500 mL × 6), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was subjected to flash column chromatography (eluent: ethyl acetate / petroleum ether, ratio of ethyl acetate: 0 - 20%) to obtain Intermediate Int4-3. MS (ESI) m / z: 331.8, 333.8 [M+1] + 。

[0279] Step 3: Intermediate Int4-3 (46 g, 138.40 mmol) was dissolved in N,N-dimethylformamide (300 mL). Sodium hydride (16.61 g, 415.21 mmol, 60%) was added at 0 °C, and the mixture was stirred at 0 °C for 0.5 hour. Further, Intermediate Int4-3A (65.03 g, 415.21 mmol) was added, and the temperature was slowly raised to 25 °C and reacted for 2 hours. 1000 mL of water was slowly added to the reaction solution to completely precipitate it. It was subjected to suction filtration, the cake was washed with water (250 mL × 4), and the cake was dried under vacuum at 50 °C to obtain Intermediate Int4-4. 1 H NMR(400MHz,CDCl3) δ =7.09(d,J=8.53Hz,4H),6.94-0.97(m,1H),6.88 (d, J=8.53 Hz,4H), 6.80(d, J=2.76 Hz, 1H), 4.50(S,4H), 3.81 (S,6H).

[0280] Step 4: The intermediate Int4-4 (78.66 g, 137.36 mmol) was dissolved in N,N-dimethylformamide (280 mL), copper(I) iodide (130.80 g, 686.80 mmol) was added, and after purging with nitrogen gas, the mixture was heated to 100 °C. Further, the intermediate 5-10A (211.11 g, 1.10 mol) was added and reacted for 0.5 h. 500 mL of ethyl acetate was added, followed by 500 mL of water, and the layers were separated. The organic phase was washed five times with water (1 L×5), and further washed with saturated brine (1 L×3). The aqueous phase was extracted again with 1 L of ethyl acetate, and after separation, the organic phase was washed with water (1 L×5) and saturated sodium chloride solution (1 L×3). All the organic phases were concentrated under reduced pressure to obtain a crude product. 300 mL of methanol was added to the obtained crude product, and the mixture was stirred and suction filtered. The cake was the intermediate Int4-5. 1 H NMR (400 MHz, CDCI3) δ = 7.05 - 7.13 (m, 4H), 6.96 (d, J = 3.01 Hz, 1H), 6.84 - 6.92 (m, 4H), 6.80 (d, J = 2.76 Hz, 1H), 4.50 (s, 4H), 3.81 (s, 6H).

[0281] Step 5: The intermediate Int4-5 (0.098 g, 190.38 μmol), bis(pinacolato)diboron (483.44 mg, 1.90 mmol), and potassium acetate (56.05 mg, 571.14 μmol) were added to anhydrous dioxane (2.5 mL), and then purged three times with nitrogen gas. 1,1'-Bis(diphenylphosphino)ferrocenedichloropalladium(II) (41.79 mg, 57.11 μmol) was added, and the mixture was purged three times again with nitrogen gas, and then heated to 110 °C and reacted for 20 h. 50 mL of water was added, and the mixture was extracted with 150 mL of ethyl acetate. After separation, the organic phase was taken and washed with saturated brine (50 mL×3), and concentrated under reduced pressure to obtain a crude product. The crude product was separated by flash column chromatography (eluent: ethyl acetate: petroleum ether: 0 - 10%) to obtain the intermediate Int-4. MS (ESI) m / z: 562.2 [M+1] + 。

[0282] Synthesis of Intermediate Int-5

[0283] [Chemical Formula]

[0284] Int5-1 (3 g, 13.37 mmol), bis(pinacolato)diboron (5.09 g, 20.05 mmol) and potassium acetate (2.62 g, 26.73 mmol) were dissolved in N,N-dimethylformamide (30 mL), replaced with nitrogen gas three times, and 1,1'-bis(diphenylphosphino)ferrocene palladium(II) dichloride dichloromethane adduct (1.09 g, 1.34 mmol) was added. The mixture was heated to 100 °C and stirred for 6 hours. The reaction solution was poured into 150 mL of water and extracted with ethyl acetate (20 mL × 3). The organic phases were combined, washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 to 3:1) to obtain Int-5. 1 1H NMR (400 MHz, CDCI3) δ 7.05 (dd, J = 4.0, 2.8 Hz, 1H), 6.87 (dd, J = 7.6, 2.4 Hz, 1H), 3.16 - 4.46 (m, 2H), 1.38 (s, 12H).

[0285] Example 1

[0286] [Chemical Formula]

[0287] [Chemical Formula]

[0288] [Chemical Formula]

[0289] Step 1: Preparation of Intermediate 1-1A-2 Intermediate 1-1A-1 (120 g, 709 mmol) was dissolved in tert-butanol (1200 mL) and water (1200 mL). Then, potassium osmate(VI) dihydrate (10.4 g, 28.3 mmol) and 4-methylmorpholine N-oxide (249 g, 2.13 mol) were sequentially added. The reaction mixture was stirred at 45 °C for 16 hours. It was concentrated under reduced pressure to remove the excess solvent, extracted with ethyl acetate (500 mL × 2), and washed with saturated sulfurous acid solution (1000 mL). The combined organic layers were washed with saturated brine (500 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain 1-1A-2. 1 H NMR (400 MHz, CDCl3) δ 4.23 (t, J = 3.6 Hz, 2H), 3.55 - 3.58 (m, 2H), 3.35 - 3.32 (m, 2H), 2.87 - 2.83 (m, 2H), 1.45 (s, 9H).

[0290] Step 2: Preparation of Intermediate 1-1A-3 Intermediate 1-1A-2 (107 g, 526 mmol) was dissolved in dichloromethane (1700 mL), cooled to 0 °C, and then iodobenzene diacetate (254 g, 789 mmol) was added. The reaction system was transferred to 25 °C and stirred for 3 hours. Saturated sodium bicarbonate solution (500 mL) was added to quench the reaction system, and further dichloromethane (100 mL) was added and stirred for 0.5 hours. Then, the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. At 25 °C, tert-butyl methyl ether (200 mL) was added, stirred for 10 minutes, filtered, and concentrated under reduced pressure to obtain the crude product Intermediate 1-1A-3.

[0291] Step 3: Preparation of Intermediate 1-1A-4 Intermediate 1-1A-3 (200 g) was dissolved in tetrahydrofuran (600 mL), cooled to -78 °C, and then vinylmagnesium bromide (1 M, 1.79 L) was added to the reaction system. Next, the reaction system was warmed to 25 °C and stirred for 16 hours. A saturated ammonium chloride solution (1000 mL) was added at 10 °C to quench the reaction system, and the mixture was extracted with ethyl acetate (500 mL). The organic phase was washed with saturated brine (500 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain Intermediate 1-1A-4. 1 H NMR (400 MHz, CDCl3) δ 5.82-5.89 (m, 2H), 5.32 (t, J = 11.6 Hz, 2H), 5.16-5.19 (m, 2H), 4.45 (s, 2H), 3.60-3.70 (m, 1H), 3.37 (s, 2H), 3.25 (s, 1H), 2.95 (d, J = 8.8 Hz, 1H), 1.48 (s, 9H).

[0292] Step 4: Preparation of Intermediate 1-1A-5 Intermediate 1-1A-4 (80.0 g, 310 mmol) was dissolved in dichloromethane (1000 mL), and then the reaction system was transferred to 0 °C and DBU (23.6 g, 155 mmol) and 2,2,2-trichloroacetonitrile (269 g, 1.87 mol) were added. The reaction system was transferred to 25 °C and stirred for 16 hours. It was concentrated under reduced pressure, and the residue was extracted with ethyl acetate (500 mL × 2), washed with water (100 mL × 2), washed with saturated brine (100 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to obtain Intermediate 1-1A-5. 1 H NMR (400 MHz, CDCl3) δ 8.37 (s, 2H), 5.81-5.87 (m, 2H), 5.45 (s, 2H), 5.39-5.43 (m, 2H), 5.25-5.30 (m, 2H), 3.61-3.81 (m, 4H), 1.48 (s, 9H).

[0293] Step 5: Production of Intermediate 1-1A-6 Intermediate 1-1A-5A (32.1 g, 238 mmol) was dissolved in DCE (850 mL), and then chloro(1,5-cyclooctadiene)iridium(I) dimer (12.3 g, 18.3 mmol) was added. The reaction system was cooled to 0 °C, and then Intermediate 1-1A-5 (100 g, 183.1 mmol) was dissolved in DCE (1.00 L) and transferred to the above reaction system. The reaction system was warmed to 25 °C and stirred for 16 hours. It was concentrated under reduced pressure to remove the excess solvent, and a crude product was obtained. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1, petroleum ether / ethyl acetate = 10:1) to obtain Intermediate 1-1A-6. 1 H NMR (400 MHz, CDCl3) δ 7.52-7.55 (m, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.22 (t, J = 7.2 Hz, 1H), 5.94-6.03 (m, 2H), 5.10 (t, J = 19.2 Hz, 2H), 4.99 (d, J = 10.4 Hz, 2H), 3.51-3.61 (m, 4H), 3.33 (t, J = 13.6 Hz, 2H), 1.48 (s, 15H).

[0294] Step 6: Production of Intermediate 1-1A-7 1-1A-6 (36.0 g, 50.4 mmol) was dissolved in toluene (900 mL), and then the second-generation Grubbs catalyst (2.14 g, 2.52 mmol) was added. The reaction system was warmed to 125 °C and stirred for 16 hours. It was filtered, the cake was discarded, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1, petroleum ether / ethyl acetate = 10:1) to obtain Intermediate 1-1A-7. MS: m / z = 329.2, [M+1] + 。 11H NMR (400 MHz, CDCl3) δ 7.60 (t, J = 1.2 Hz, 2H), 7.31 (t, J = 7.2 Hz, 2H), 7.22 (s, 1H), 5.96 (t, J = 9.2 Hz, 2H), 3.60 - 3.65 (m, 2H), 3.46 - 3.53 (m, 2H), 3.09 - 3.14 (m, 2H), 1.42 (s, 9H), 1.25 (d, J = 6.0 Hz, 6H).

[0295] Step 7: Preparation of Intermediate 1-1A-8 Intermediate 1-1A-7 (26.8 g, 81.6 mmol) was dissolved in methanol (201 mL), and then hydrogen chloride / methanol (4 M, 67.3 mL) was added. The reaction system was heated to 35 °C and stirred for 16 h. The pH value of the reaction mixture was adjusted to 12, extracted with ethyl acetate (30.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 1-1A-8. MS: m / z = 229.2, [M+1] + . 1 1H NMR (400 MHz, CDCl3) δ 7.62 (t, J = 7.2 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.21 (s, 1H), 6.01 (s, 2H), 3.42 (s, 2H), 2.89 - 2.93 (m, 2H), 2.30 - 2.34 (m, 2H), 1.23 (s, 6H).

[0296] Step 8: Preparation of Intermediate 1-1A-9 Intermediate 1-1A-8 (18.6 g, 79.4 mmol) was dissolved in THF (190 mL), and then 9-fluorenylmethyl chloroformate (20.5 g, 79.4 mmol) and sodium carbonate (25.2 g, 238.2 mmol) were added. The mixture was stirred at 0 °C for 1 h. Extracted with ethyl acetate (50.0 mL × 2), washed with water (200.0 mL). The organic phases were combined, washed with saturated brine (150.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain Intermediate 1-1A-9. MS: m / z = 451.3, [M+1] + .1 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 13.6 Hz, 2H), 7.54 - 7.61 (m, 4H), 7.24 - 7.40 (m, 7H), 5.93 - 6.01 (m, 2H), 4.34 - 4.40 (m, 2H), 4.21 (s, 1H), 3.70 (t, J = 2 Hz, 2H), 3.55 - 3.59 (m, 2H), 3.15 - 3.23 (m, 2H), 1.27 (d, J = 2.4 Hz, 6H).

[0297] Step 9: Preparation of Intermediate 1 - 1A - 10 Intermediate 1 - 1A - 9 (9.52 g, 21.1 mmol) was dissolved in trifluoroacetic acid (192 mL), heated to 75 °C and stirred for 16 h. Water (20.0 mL) was added, the pH was adjusted to 9, dichloromethane (20.0 mL) was further added for extraction, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was stirred with n - heptane (6 mL) at 25 °C for 2 h to obtain the trifluoroacetate salt of 1 - 1A - 10. MS: m / z = 333.1, [M+1] + . 1 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 7.2 Hz, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.33 (t, J = 6 Hz, 2H), 6.18 - 6.27 (m, 2H), 4.38 - 4.42 (m, 2H), 4.23 (s, 1H), 3.88 (d, J = 2.0 Hz, 2H), 3.82 (d, J = 2.4 Hz, 1H), 3.72 (d, J = 2.0 Hz, 1H), 3.21 - 3.60 (m, 2H).

[0298] Step 10: Preparation of Intermediate 1 - 1A - 11 The trifluoroacetate salt of intermediate 1-1A-10 (1.00 g, 2.92 mmol) was dissolved in tetrahydrofuran (10.0 mL). Next, di-tert-butyl dicarbonate (764 mg, 3.50 mmol) and triethylamine (885 mg, 8.75 mmol) were sequentially added, and the mixture was stirred at 25 °C for 1 hour. Ethyl acetate (10.0 mL × 2) and water (10.0 mL) were added for extraction. The organic phases were combined, washed with saturated brine (15.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1, petroleum ether:ethyl acetate = 3:1) to obtain intermediate 1-1A-11. MS: m / z = 433.2, [M+1] + .

[0299] Step 11: Production of intermediate 1-1A Intermediate 1-1A-11 (5.69 g, 12.59 mmol) was dissolved in ethanol (60.0 mL). Next, dimethylamine (34.4 g, 251.8 mmol) was added. The reaction system was stirred at 25 °C for 3 hours. It was directly concentrated under reduced pressure, and the residue was extracted with ethyl acetate (40.0 mL) and 10% citric acid (40.0 mL). The pH value of the aqueous phase was adjusted to 9, filtered, extracted with ethyl acetate (40.0 mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain intermediate 1-1A. MS: m / z = 211.2, [M+1] + . 1 H NMR (400 MHz, CDCl3) δ 6.22 (d, J = 10 Hz, 2H), 4.40 (d, J = 38.8 Hz, 2H), 2.89 - 3.01 (m, 2H), 2.40 (d, J = 13.2 Hz, 2H), 1.49 (s, 9H).

[0300] Step 12: Production of intermediate 1-3A-2 Intermediate 1-3A-1 (4 g, 17.02 mmol) was dissolved in AcOH (30 mL), cooled to 0 °C in an ice bath, then concentrated sulfuric acid (17.03 g, 170.21 mmol) was added, followed by dropwise addition of an aqueous solution (5 mL) of sodium nitrite (1.76 g, 25.53 mmol). Stirring was continued for 0.25 h, then an aqueous solution (5 mL) of potassium iodide (4.24 g, 25.53 mmol) was added dropwise, and the reaction was carried out at 25 °C for 0.5 h. Water (50 mL) was added to the reaction solution, and the mixture was filtered. The solid was washed with a saturated sodium thiosulfate solution (2 × 40 mL) and then with water (40 mL). The solid was dissolved in dichloromethane (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain Intermediate 1-3A-2.

[0301] Step 13: Preparation of Intermediate 1-3A-3 Intermediate 1-3A-2 (2.85 g, 8.24 mmol) and copper(I) iodide (3.14 g, 16.48 mmol) were dissolved in N,N-dimethylformamide (45 mL), then methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (6.33 g, 32.96 mmol) was added, and the reaction was carried out at 80 °C for 1 h. The reaction was continued for 0.75 h. The reaction solution was cooled to 25 °C, filtered to remove insolubles, washed with ethyl acetate (100 mL), washed with water (3 × 200 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 30 / 1 to 20 / 1) to obtain Intermediate 1-3A-3.

[0302] Step 14: Preparation of Intermediate 1-3A-4 Intermediate 1-3A-3 (1.66 g, 5.76 mmol), iron powder (1.13 g, 20.17 mmol), and ammonium chloride (1.54 g, 28.82 mmol) were dissolved in ethanol (20 mL) and water (10 mL), heated to 60 °C, and reacted for 2.5 h. Ethyl acetate (80 mL) was added to the reaction solution for dilution, and the mixture was filtered to remove insolubles, washed with water (2 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain Intermediate 1-3A-4. MS: m / z = 257.9, [M+1] + 。

[0303] Step 15: Production of Intermediate 1-3A Intermediate 1-3A-4 (1.4 g, 5.43 mmol), bis(pinacolato)diboron (2.07 g, 8.14 mmol, 1.5 eq), [1,1'-bis(diphenylphosphino)ferrocene palladium(II) dichloride dichloromethane complex (443.12 mg, 542.61 μmol), potassium acetate (1.60 g, 16.28 mmol) were dissolved in dioxane (30 mL), and reacted at 85°C for 16 hours under the protection of nitrogen gas. The reaction solution was cooled to 25°C, filtered to remove insolubles, washed with ethyl acetate (50 mL), and concentrated. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 20 / 1 to 10 / 1) to obtain Intermediate 1-3A. MS: m / z = 306.0, [M+1] + 。

[0304] Step 16: Production of Intermediate 1-2 Intermediate 1-1 (900 mg, 3.56 mmol) was dissolved in dichloromethane (10 mL), cooled to 0°C, and then N,N-diisopropylethylamine (1.38 g, 10.69 mmol) and 1-1A (749.60 mg, 3.56 mmol) were added sequentially, and reacted at 0°C for 1 hour. It was directly concentrated under reduced pressure to obtain the crude product 1-2. MS: m / z = 426.0, [M+1] + 。

[0305] Step 17: Production of Intermediate 1-3 Intermediate 1-2 (220 mg, 516.10 μmol) and 1-2A were dissolved in acetonitrile (10 mL), then N,N-diisopropylethylamine (200.10 mg, 1.55 mmol) was added, and the reaction system was heated to 80°C and stirred for 16 hours. The reaction solution was concentrated under reduced pressure to remove most of the solvent, ethyl acetate (10 mL) was added, washed with water (5 mL) and extracted, dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by silica gel column chromatography (PE / EA = 1 / 1 to DCM / MeOH = 20 / 1) to obtain Intermediate 1-3. MS: m / z = 549.1, [M+1]+ .

[0306] Step 18: Production of Intermediate 1-4 Intermediate 1-3A (0.2 g, 364.29 μmol), Intermediate 1-3 (222.27 mg, 728.58 μmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride dichloromethane complex (29.75 mg, 36.43 μmol), and cesium carbonate (356.08 mg, 1.09 mmol) were dissolved in dioxane (5 mL) and water (1.25 mL), and reacted at 90°C for 17 hours under the protection of nitrogen gas. The reaction solution was cooled to 25°C, filtered to remove insoluble matters, washed with ethyl acetate (50 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by silica gel column chromatography (DCM / MeOH = 50 / 1 to 20 / 1) to obtain Intermediate 1-4. MS: m / z = 692.2, [M+1] + .

[0307] Step 19: Production of Compound 1 Intermediate 1-4 (92 mg, 133.01 μmol) was dissolved in dichloromethane (4 mL), trifluoroacetic acid (1.54 g, 13.51 mmol, 1 mL) was added at 20°C, and the reaction was continued for 0.5 hour. The reaction solution was concentrated to dryness, dissolved in dichloromethane (2 mL), solid sodium hydrogen carbonate (0.5 g) was added and stirred well, then ethyl acetate (5 mL) was added and stirring was continued for 5 minutes. The insoluble matters were removed by filtration and the solution was concentrated. The crude product was separated by preparative HPLC (chromatography column: Boston Green ODS 150×30 mm×5 μm; mobile phase: [water (formic acid)-acetonitrile]; acetonitrile %: 10% - 40%, 6 min) to obtain Compound 1. MS (ESI) m / z: 592.3 [M+1] + . 11H NMR (400 MHz, CD3OD) δ 9.01 (s, 1H), 6.58 (d, J = 14 Hz, 1H), 6.41 (s, 1H), 6.34 (s, 2H), 5.56 (d, J = 51 Hz, 1H), 4.79 - 4.75 (m, 2H), 4.62 - 4.61 (m, 2H), 4.72 (s, 2H), 4.03 - 3.82 (m, 5H), 3.31 - 3.30 (m, 1H), 2.59 - 2.54 (m, 2H), 2.38 - 2.29 (m, 4H).

[0308] Example 2

[0309]

Chem.

[0310]

Chem.

[0311]

Chem.

[0312] Step 1: Preparation of Intermediate 2-2 Intermediate 2-1 (50 g, 418.09 mmol) was dissolved in dichloromethane (500 mL), triethylamine (84.61 g, 836.17 mmol) was added, and then di-tert-butyl dicarbonate (100.37 g, 459.90 mmol) was added. The reaction was carried out at 25 °C for 16 h. 100 mL of water was added to the reaction solution, and the mixture was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by silica gel column chromatography (PE / EA = 100 / 1 - 50 / 1) to obtain Intermediate 2-2. MS: m / z = 206.1, [M+Na] + .

[0313] Step 2: Preparation of Intermediate 2-3 Intermediate 2-2 (25 g, 136.43 mmol) was dissolved in anhydrous tetrahydrofuran (350 mL), 3,7-dipropyl-3,7-diazabicyclo[3.3.1]nonane (37.31 g, 177.36 mmol) was added, and the mixture was cooled to -65 °C. Further, sec-butyllithium (1.3 M, 157.42 mL) was slowly added dropwise, and after reacting for 1 hour, methyl chloroformate (15.73 g, 166.44 mmol) was added dropwise, and the reaction was carried out at -65 °C for 2 hours. Saturated ammonium chloride (20 mL) was added dropwise to the reaction solution for quenching, and the mixture was extracted with ethyl acetate (300 mL × 2). The organic phases were combined, washed with saturated brine (150 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by silica gel column chromatography (PE / EA = 50 / 1 to 25 / 1) to obtain Intermediate 2-3. MS: m / z = 186.0 [M-tBu+H] + .

[0314] Step 3: Preparation of Intermediate 2-4A Intermediate 2-3 (12 g, 49.73 mmol) was dissolved in THF (120 mL), and 2-chloromethyl-3-chloropropene (24.87 g, 198.94 mmol) was further added. The mixture was cooled to -40 °C, and lithium bis(trimethylsilyl)amide (1 M, 99.47 mL) was slowly added dropwise. The temperature was gradually raised to 20 °C and reacted for 2 hours. Saturated ammonium chloride (20 mL) was added dropwise to the reaction solution for quenching, and the mixture was extracted with ethyl acetate (150 mL × 2). The organic phases were combined, washed with saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by silica gel column chromatography (PE / EA = 20 / 1 - 10 / 1) to obtain Intermediate 2-4A. After developing twice by TLC thin-layer chromatography (petroleum ether:acetone = 5:1), the Rf value of 2-4A was 0.5, and the Rf value of its isomer was 0.55. The retention time of 2-4A by LCMS (chromatography column: Agilent Poroshell 120 EC-C18 2.7um 3.0×30mm, mobile phase: A: H2O (0.037% FA) - B: ACN (0.0187% FA); B: 5% - 95%) was 0.776 min, and the retention time of its isomer was 0.801 min. MS: m / z = 274.0 [M-tBu+H] + .

[0315] Step 4: Preparation of Intermediate 2-5 Intermediate 2-4A (3.6 g, 10.92 mmol) was dissolved in hydrogen chloride / ethyl acetate (4 M, 27.29 mL) and reacted at 20 °C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the crude product Intermediate 2-5, which was directly charged into the next step without purification. MS: m / z = 230.1, [M+H] + .

[0316] Step 5: Preparation of Intermediate 2-6 Intermediate 2-5 (2.9 g, 10.90 mmol) was dissolved in methanol (100 mL), and potassium carbonate (4.52 g, 32.69 mmol) was further added. The mixture was reacted at 20 °C for 2 hours. Dichloromethane (80 mL) was added to the reaction solution, followed by filtration and concentration. The crude product was separated by silica gel column chromatography (PE / EA = 10 / 1 to 5 / 1) to obtain Intermediate 2-6. MS: m / z = 194.1, [M+H] + 。

[0317] Step 6: Preparation of Intermediate 2-7 Intermediate 2-6 (1.6 g, 8.28 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), and lithium aluminum hydride (628.51 mg, 16.56 mmol) was further added. The mixture was reacted at 0 °C for 2 hours. Ethyl acetate (10 mL) was added dropwise to the reaction solution for dilution, followed by the dropwise addition of water (0.63 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product Intermediate 2-7, which was directly charged into the next step without purification. MS: m / z = 166.1 [M+H] + 。

[0318] Step 7: Preparation of Intermediate 2-8 Intermediate 1-2 (700 mg, 1.64 mmol) and Intermediate 2-7 (407.00 mg, 2.46 mmol) were dissolved in anhydrous toluene (20 mL), and sodium tert-butoxide (473.45 mg, 4.93 mmol) was further added. The mixture was reacted at 0 °C for 2 hours, and then at 20 °C for 2 hours. Ethyl acetate (50 mL) was added to the reaction solution, followed by the addition of 10 mL of water for quenching. The layers were separated, and the organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was separated by silica gel column chromatography (PE / EA = 5 / 1 to 2 / 1) to obtain Intermediate 2-8. MS: m / z = 555.1, [M + H] + 。

[0319] Step 8: Preparation of Intermediates 2-9A and 2-9B Intermediate 2-8 (0.1 g, 180.17 μmol, 1 eq), Intermediate 1-3A (82.45 mg, 270.26 μmol) were dissolved in dioxane (2 mL) and water (0.5 mL), and further methanesulfonato(diadamantyl-n-butylphosphino)-2'-amino-1,1'-biphenyl-2-yl)palladium(II) (13.12 mg, 18.02 μmol), cesium carbonate (146.75 mg, 450.42 μmol) were added, and the reaction was carried out at 90 °C for 16 hours. Ethyl acetate (30 mL) was added to the reaction solution for dilution, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was subjected to silica gel column chromatography (PE / EA = 1 / 50~1 / 100), and then separated by SFC (chromatography column: REGIS(S,S)WHELK-O1(250 mm×25 mm, 10 μm); mobile phase: A: CO2, B: [0.1% NH3H2O IPA]; B%: 45%~45%) to obtain Intermediate 2-9A and 2-9B. Here, the retention time of Intermediate 2-9A in SFC (column chromatography: (S,S)Whelk-01 100×4.6 mm I.D., 5.0 μm, mobile phase: A: supercritical CO2, B: IPA(0.05% DEA), gradient: 40% B, flow rate: 2.5 mL / min) was 3.276 min, and the ee value was 98.3%. The retention time of Compound 2-9B in SFC (chromatography column: (S,S)Whelk-01 100×4.6 mm I.D., 5.0 μm, mobile phase: A: supercritical CO2, B: IPA(0.05% DEA), gradient: 40% B, flow rate: 2.5 mL / min) was 3.879 min, and the ee value was 96.4%.

[0320] Step 9: Preparation of Compound 2A Intermediate 2-9A (30 mg, 43.00 μmol) was dissolved in dichloromethane (1.5 mL), and trifluoroacetic acid (147.08 mg, 1.29 mmol) was further added. The reaction was carried out at 20 °C for 2 hours. The reaction solution was diluted with dichloromethane (20 mL), saturated sodium bicarbonate (10 mL) was added, the layers were separated, the aqueous phase was extracted with dichloromethane (10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by preparative HPLC (chromatography column: O-Welch C18 150×30 mm×5 μm; mobile phase: [H2O(FA)-ACN]; B%: 1% - 41%, 10 min) to obtain the formate salt of Compound 2A. LCMS: MS (ESI) m / z: 598.2 [M+H] + 。 1 H NMR (400 MHz, CD3OD) δ 9.07 (s, 1H), 8.52 (br s, 1H), 6.59 (br d, J = 14.05 Hz, 1H), 6.38 - 6.48 (m, 1H), 6.33 (s, 2H), 5.10 - 5.26 (m, 2H), 4.59 - 4.72 (m, 3H), 4.49 (d, J = 11.04 Hz, 1H), 4.27 (br s, 2H), 4.08 (br d, J = 15.31 Hz, 1H), 3.94 (br d, J = 11.04 Hz, 2H), 3.62 (br d, J = 15.81 Hz, 1H), 3.53 (d, J = 10.54 Hz, 1H), 3.00 - 3.14 (m, 2H), 2.75 (br d, J = 16.31 Hz, 1H), 1.86 - 1.98 (m, 1H), 1.79 (td, J = 3.67, 6.96 Hz, 1H), 0.69 - 0.85 (m, 2H).

[0321] Step 10: Preparation of Compound 2B Intermediate 2-9B (31 mg, 44.43 μmol) was dissolved in dichloromethane (1.5 mL), and trifluoroacetic acid (42.31 mg, 371.05 μmol) was further added. The reaction was carried out at 20 °C for 1 hour. The reaction solution was diluted with dichloromethane (20 mL), saturated sodium bicarbonate (10 mL) was added, the layers were separated, the aqueous phase was extracted with dichloromethane (10 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by preparative HPLC (chromatography column: O-Welch C18 150×30 mm×5 μm; mobile phase: [H2O(FA)-ACN]; B%: 1% - 41%, 12 min) to obtain the formate salt of compound 2B. LCMS: MS (ESI) m / z: 598.2 [M+H] + 。 1 H NMR (400 MHz, CD3OD) δ 9.06 (s, 1H), 8.52 (br s, 1H), 6.59 (br d, J = 13.80 Hz, 1H), 6.43 (s, 1H), 6.33 (s, 2H), 5.16 (br d, J = 10.29 Hz, 2H), 4.58 - 4.72 (m, 3H), 4.48 (d, J = 11.29 Hz, 1H), 4.25 (br s, 2H), 4.07 (br d, J = 15.56 Hz, 1H), 3.94 (br d, J = 11.04 Hz, 2H), 3.61 (br d, J = 15.81 Hz, 1H), 3.51 (d, J = 10.54 Hz, 1H), 2.99 - 3.12 (m, 2H), 2.74 (br d, J = 16.06 Hz, 1H), 1.88 - 1.95 (m, 1H), 1.72 - 1.85 (m, 1H), 0.69 - 0.81 (m, 2H).

[0322] Example 3

[0323]

Chemical formula

[0324]

Chemical formula

[0325]

Chem.

[0326] Step 1: Preparation of Intermediate 3-1 Dissolve Intermediate 1-1 (25 g, 99.03 mmol) in dichloromethane (200 mL), cool to -30 °C, then sequentially add N,N-diisopropylethylamine (38.40 g, 297.08 mmol) and 1-1B (21.02 g, 99.03 mmol), and react at -30 °C for 2 hours. Directly concentrate under reduced pressure to obtain crude product 3-1. MS: m / z = 428.1 [M+1] + .

[0327] Step 2: Preparation of Intermediate 3-2A Dissolve Intermediate 3-1 (23 g, 53.70 mmol) and Intermediate 2-7 (9.76 g, 59.07 mmol) in anhydrous toluene (300 mL), slowly add sodium tert-butoxide (13.93 g, 145.00 mmol) at 0 °C, react at 0 °C for 0.5 hour, and further react at 20 °C for 0.5 hour. Add 200 mL of ethyl acetate to the reaction solution for dilution, wash with saturated brine (100 mL), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain crude product 3-2. Purify the crude product by SFC (chromatography column: DAICEL CHIRALCEL OD (250 mm × 50 mm, 10 μm); mobile phase: [A (supercritical CO2), B (ethanol containing 0.1% aqueous ammonia)]; B: 40%) to obtain products 3-2A and 3-2B. 3-2A has a retention time of 4.964 min and an ee value of 95.7% under the conditions of SFC (chromatography column: Cellulose 2 100 mm × 4.6 mm, 3 μm) mobile phase: [A (supercritical CO2), B (MeOH (0.05% DEA))]; B: 40%). 11H NMR (400 MHz, CD3OD) δ 8.76 (s, 1H), 4.98 - 4.87 (m, 2H), 4.51 - 4.48 (m, 3H), 4.34 - 4.29 (m, 1H), 4.27 (br s, 2H), 4.23 - 4.18 (m, 1H), 3.61 - 3.58 (m, 3H), 3.12 (d, J = 9.5 Hz, 1H), 2.78 (br d, J = 16.8 Hz, 1H), 2.71 (dd, J = 4.0, 9.6 Hz, 1H), 2.45 (br d, J = 16.8 Hz, 1H), 1.83 - 1.72 (m, 2H), 1.75 - 1.62 (m, 3H), 1.55 - 1.51 (m, 1H), 1.42 (s, 9H), 0.60 (q, J = 4.2 Hz, 1H), 0.47 - 0.42 (m, 1H). MS: m / z = 557.2 [M+1] + Under the same conditions, the retention time of 3-2B was 8.382 min, and the ee value was 96.5%.

[0328] Step 3: Synthesis of Intermediate 3-4 The raw material 3-3 (2 g, 5.58 mmol) was dissolved in dichloromethane (40 mL). Under an ice bath at 0 - 10 °C, N,N-diisopropylethylamine (4.33 g, 33.47 mmol) was added. Next, trifluoromethanesulfonic anhydride (6.30 g, 22.31 mmol) was added dropwise, and the reaction was continued for 1.5 h. Water (20 mL) was added to the reaction solution and stirred well. The aqueous phase was separated, and the organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was separated by column chromatography (mobile phase: petroleum ether:ethyl acetate = 10:1 - 2:1) to obtain Intermediate 3-4.

[0329] Step 4: Synthesis of Intermediate 3-5 Intermediate 3-4 (3g, 4.82 mmol), benzophenone imine (1.75 g, 9.64 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (557.57 mg, 963.63 μmol), cesium carbonate (4.71 g, 14.45 mmol) were dissolved in toluene (60 mL). Next, tris(dibenzylideneacetone)dipalladium (441.21 mg, 481.82 μmol) was added, and the reaction was carried out at 100 °C for 2 hours under the protection of nitrogen gas. The reaction solution was cooled to 25 °C, filtered to remove insoluble matters, concentrated to remove most of the toluene, diluted with ethyl acetate (30 mL), washed with water (20 mL), dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. The crude product was separated by column chromatography (mobile phase: petroleum ether:ethyl acetate = 10:1 to 1:1) to obtain Intermediate 3-5.

[0330] Step 5: Synthesis of Intermediate 3-6 Intermediate 3-5 (3.1 g, 4.74 mmol), bis(pinacolato)diboron (2.41 g, 9.48 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (693.88 mg, 948.30 μmol), potassium acetate (1.40 g, 14.22 mmol) were dissolved in toluene (60 mL), and the reaction was carried out at 110 °C for 18 hours under the protection of nitrogen gas. The reaction solution was cooled to 25 °C, filtered to remove insoluble matters, extracted with ethyl acetate (50 mL), washed with water (50 mL), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by column chromatography (mobile phase: petroleum ether:ethyl acetate = 10:1 to 2:1) to obtain Intermediate 3-6. MS (ESI) m / z: 468.2 [M+H2O-Ph2CO+1] + 。

[0331] Step 6: Synthesis of Intermediate 3-7 Intermediate 3-2A (11.3 g, 20.29 mmol) was added to water (60 mL) and anhydrous dioxane (240 mL), and further methanesulfonato(diadamantyl-n-butylphosphino)-2'-amino-1,1'-biphenyl-2-yl)palladium(II) (1.48 g, 2.03 mmol), intermediate 3-6 (15.38 g, 24.34 mmol), and cesium carbonate (13.22 g, 40.57 mmol) were added, and the mixture was reacted at 87 °C for 2 hours. Ethyl acetate (200 mL) was added to the reaction solution for dilution, and it was washed with saturated brine (40 mL × 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (mobile phase: dichloromethane:methanol = 50:1 to 20:1) to obtain intermediate 3-7. MS (ESI) m / z: 862.4 [M+H2O-Ph2CO+1] + .

[0332] Step 7: Synthesis of intermediate 3-8 Intermediate 3-7 (8.4 g, 8.18 mmol) was added to ethyl acetate (30 mL) and water (10 mL), and further hydrochloric acid / ethyl acetate (4 M, 62.37 mL) was added, and the mixture was reacted at 20 °C for 2 hours. Water (30 mL) was added to the reaction solution, the organic phase was washed with water (30 mL × 2), the aqueous phases were combined, the pH was adjusted to 7 - 8 with saturated sodium hydrogen carbonate, and it was extracted with ethyl acetate (80 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude product 3-8, which was directly used in the next step. MS (ESI) m / z: 762.3 [M+1] + .

[0333] Step 8: Synthesis of compound 3 Intermediate 3-8 (6.2 g, 8.14 mmol) was added to acetonitrile (70 mL), and further tetrabutylammonium fluoride (2.29 g, 13.83 mmol) was added, and the reaction was carried out at 60 °C for 1 hour. 100 mL of ethyl acetate was added to the reaction solution for dilution, washed with 30 mL of saturated sodium bicarbonate, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (mobile phase: dichloromethane (methanol solution of 10% ammonia gas): methanol = 20:1 to 10:1) to obtain Compound 3. The SFC analysis (chromatography column: Chiralcel OJ-3 100×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (ethanol, 0.05% diethylamine); gradient: B%: 40% to 40%) showed a retention time of 3.761 min and an ee value of 97.34%. The chiral HPLC analysis (chromatography column: FLM Chiral NQ, 150×4.6 mm, 3 μm, mobile phase: A: (n-hexane) and B: (ethanol, containing 0.2% diethylamine, v / v), gradient: B%: isocratic 30%, elution time: 60 min, column temperature: 35 °C) showed that the retention time was 17.387 min. MS (ESI) m / z: 606.3 [M+1] + , 11H NMR (400 MHz, CD3OD) δ = 9.01 (s, 1H), 7.76 (dd, J = 5.6, 9.2 Hz, 1H), 7.28 - 7.19 (m, 2H), 7.15 (d, J = 2.3 Hz, 1H), 5.08 (br s, 1H), 5.01 (br s, 1H), 4.70 - 4.54 (m, 3H), 4.43 (dd, J = 7.3, 10.0 Hz, 1H), 4.32 (dd, J = 6.3, 10.3 Hz, 1H), 3.78 - 3.65 (m, 5H), 3.39 - 3.34 (m, 1H), 3.23 (d, J = 9.5 Hz, 1H), 2.93 (br d, J = 17.3 Hz, 1H), 2.82 (dd, J = 4.0, 9.3 Hz, 1H), 2.56 (br d, J = 17.1 Hz, 1H), 1.91 - 1.76 (m, 5H), 1.64 (td, J = 3.6, 6.9 Hz, 1H), 0.72 (q, J = 4.0 Hz, 1H), 0.61 - 0.53 (m, 1H).

[0334] Example 4

[0335]

Chemical Structure

[0336] Step 1: Synthesis of Intermediate 4-1 Intermediate 3-2A (150 mg, 269.27 μmol), Intermediate 1-3A (86.58 mg, 269.27 μmol), methanesulfonato(diadamantyl-n-butylphosphino)-2’-amino-1,1’-biphenyl-2-yl)palladium(II) (98.05 mg, 134.64 μmol), and cesium carbonate (263.20 mg, 807.82 μmol) were dissolved in dioxane (8 mL), purged with nitrogen gas three times, then water (0.5 mL) was added, and the reaction was carried out at 80 °C for 6 hours under the protection of nitrogen gas. Filtration was performed to remove insoluble substances, washed with water (10 mL), extracted with ethyl acetate (30 mL × 2), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the organic solvent to obtain a crude product, and the crude product was purified by column chromatography (mobile phase: dichloromethane:methanol = 100:1 to 50:1) to obtain Intermediate 4-1. MS (ESI) m / z: 716.3 [M+1] + 。

[0337] Step 2: Synthesis of Compound 4 Intermediate 4-1 (22 mg, 30.1 μmol) was dissolved in dichloromethane (5 mL), then trifluoroacetic acid (1.5 mL) was added, and the reaction was carried out at 25 °C for 3 hours. After adding saturated sodium bicarbonate solution to the reaction system to adjust the pH to 8, dichloromethane (5 mL × 2) was added for extraction, dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the organic solvent to obtain a crude product. The crude product was separated by prep-HPLC (chromatography column: O-Welch C18 150×30 mm×5 μm; mobile phase: [A: water (0.5% formic acid) - B: acetonitrile]; B%: 8% - 48%, 8 min) to obtain the formate salt of Compound 4. MS (ESI) m / z: 616.2 [M+1] + 。 11H NMR (400 MHz, CD3OD) δ = 9.06 (s, 1H), 8.51 (br s, 1H), 6.94 (s, 1H), 6.52 (s, 1H), 5.16 (br d, J=10.79 Hz, 2H), 4.71-4.80 (m, 2H), 4.67 (br d, J=10.79 Hz, 1H), 4.53 (br d, J=11.04 Hz, 1H), 4.02-4.10 (m, 2H), 3.84-3.90 (m, 2H), 3.59-3.63 (m, 1H), 3.50-3.53 (m, 1H), 3.03-3.09 (m, 2H), 2.73-2.78 (m, 1H), 1.96-2.09 (m, 4H), 1.90-1.93 (m, 1H), 1.75-1.81 (m, 1H), 0.87-0.97 (m, 1H), 0.68-0.83 (m, 2H).

[0338] Example 5

[0339]

Chem.

[0340]

Chem.

[0341] Step 1: Synthesis of Compound 5-2 Under the protection of nitrogen gas, Compound 5-1 (21 g, 143.30 mmol) was dissolved in acetonitrile (210 mL), N-iodosuccinimide (38.69 g, 171.96 mmol) and p-toluenesulfonic acid monohydrate (1.36 g, 7.16 mmol) were added, and the reaction was carried out at 75 °C for 5 hours. Water (150 mL) was slowly added, and the aqueous phase was extracted with ethyl acetate (150 mL × 3) and separated. The combined organic phases were washed successively with saturated sodium bisulfite solution (15 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 5-2. MS (ESI) m / z: 272.9 [M+1] + .

[0342] Step 2: Synthesis of Compound 5-3 Compound 5-2 (39.3 g) was dissolved in ethanol (435 mL), and triethylamine (43.79 g, 432.75 mmol) and bis(triphenylphosphine)palladium(II) dichloride (10.12 g, 14.42 mmol) were added. The mixture was purged with CO three times, and then reacted at 80 °C for 47 hours under a CO atmosphere (50 Psi). The reaction mixture was concentrated under reduced pressure, ethyl acetate (300 mL) and 0.3 M hydrochloric acid (300 mL) were added, a solid precipitated, and the cake was obtained by filtration. The filtrate was separated, the aqueous phase was extracted with ethyl acetate (200 mL × 2) and separated. The organic phases were combined, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and the cake was combined with the organic phase and concentrated under reduced pressure to obtain crude product 5-3. 1 H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 4.39 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H).

[0343] Step 3: Synthesis of Compound 5-4 Under the protection of nitrogen gas, compound 5-3 (41.3 g, 188.92 mmol) was dissolved in tetrahydrofuran (240 mL), methanol (80 mL) and water (80 mL) were added, lithium hydroxide monohydrate (23.78 g, 566.76 mmol) was added in one batch, and then the reaction was carried out at 20 °C for 5 hours. The reaction system was concentrated under reduced pressure, water (100 mL) was added to the reaction system, the pH of the aqueous phase was adjusted to 2 with 4 M hydrochloric acid, a solid precipitated, and filtration was carried out. The cake was dissolved in ethanol (200 mL), stirred at 20 °C for 16 hours, filtered, and the cake was dried under reduced pressure to obtain a crude product. The crude product was dissolved in petroleum ether (20 mL) and ethyl acetate (30 mL), stirred at 20 °C for 1 hour, filtered, and the cake was dried under reduced pressure to obtain compound 5-4. 1 H NMR (400 MHz, DMSO-d6) δ 13.95 - 13.08 (m, 1H), 8.35 (s, 1H), 7.62 (br s, 2H).

[0344] Step 4: Synthesis of Compound 5-5 Under the protection of nitrogen gas, Compound 5-4 (17.8 g, 93.41 mmol) was dissolved in phosphorus oxychloride (234.25 g, 1.53 mol), and then reacted at 95 °C for 3 hours. The reaction system was concentrated under reduced pressure. After adding tetrahydrofuran (360 mL), ammonium thiocyanate (21.33 g, 280.23 mmol) was added in one batch at 20 °C, and then reacted at 40 °C for 16 hours. Water (300 mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (300 mL × 3) and separated. The organic phases were combined, washed with saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. Ethyl acetate (40 mL) and petroleum ether (20 mL) were added to the crude product, stirred at 20 °C for 0.5 hour, filtered, and the cake was dried under reduced pressure to obtain Compound 5-5. 1 H NMR (400 MHz, DMSO-d6) δ 13.33 (br s, 1H), 12.91 (s, 1H), 8.64 (s, 1H).

[0345] Step 5: Synthesis of Compound 5-6 Under the protection of nitrogen gas, Compound 5-5 (8.55 g, 36.91 mmol) was dissolved in N,N-dimethylformamide (140 mL), sodium methoxide (2.09 g, 38.76 mmol) was added at 20 °C, and then iodomethane (4.72 g, 33.22 mmol) was slowly added dropwise at 20 °C, followed by reaction at 20 °C for 5 hours. Ice water (300 mL) was added to the reaction system, a solid precipitated, was filtered, the cake was washed with ice water (100 mL), and the cake was dried under reduced pressure to obtain Compound 5-6. 1 H NMR (400 MHz, DMSO-d6) δ 13.27 (br s, 1H), 8.81 (s, 1H), 2.61 (s, 3H).

[0346] Step 6: Synthesis of Compound 5-7 Under the protection of nitrogen gas, compound 5-6 (3.5 g, 14.25 mmol) and compound 5-6A (3.98 g, 14.67 mmol) were dissolved in dioxane (70 mL), water (7 mL) and ethanol (14 mL), potassium phosphate (9.07 g, 42.74 mmol) and chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II) (1.68 g, 2.14 mmol) were added, and then the reaction was carried out at 105 °C for 1.5 hours. Water (200 mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (200 mL × 2) and separated. Ethyl acetate (200 mL) was added to the aqueous phase to make it turbid, and it was filtered through diatomaceous earth, and the filtrate was separated. The aqueous phase was extracted with ethyl acetate (200 mL × 2) and separated. All the organic phases were combined, the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (developing agent: petroleum ether:ethyl acetate = 20:1~1:5, dichloromethane:methanol = 10:1) to obtain compound 5-7. 1 H NMR (400 MHz, DMSO-d6) δ 13.19 (br s, 1H), 9.03 (s, 1H), 6.92 (dd, J = 2.6, 7.3 Hz, 1H), 6.70 (dd, J = 2.7, 4.9 Hz, 1H), 5.71 (s, 2H), 2.62 (s, 3H).

[0347] Step 7: Synthesis of compound 5-8 Under the protection of nitrogen gas, compound 5-7 (2.1 g, 5.92 mmol) was dissolved in ethanol (63 mL), silver sulfate (2.21 g, 7.10 mmol) and iodine (1.58 g, 6.22 mmol) were added at 0 °C, and then the temperature was slowly raised to 10 °C and reacted for 2 hours. A saturated sodium sulfite solution (70 mL) and ethyl acetate (100 mL) were added to the reaction system, filtered and separated. The aqueous phase was extracted with ethyl acetate (100 mL × 3) and separated. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (developing agent: petroleum ether:ethyl acetate = 20:1 to 1:5) to obtain compound 5-8. 1 H NMR (400 MHz, CDCl3) δ 10.47 (br s, 1H), 9.34 (s, 1H), 7.10 (d, J = 8.1 Hz, 1H), 2.78 (s, 3H).

[0348] Step 8: Synthesis of compound 5-9 Under the protection of nitrogen gas at 0 °C in an ice-water bath, sodium hydride (96.11 mg, 2.40 mmol) was added to a solution of compound 5-8 (0.33 g, 686.56 μmol) in N,N-dimethylformamide (5 mL), and after stirring for 30 minutes, 4-methoxybenzyl chloride (236.55 mg, 1.51 mmol) was added dropwise. The resulting mixture was allowed to warm to 25 °C naturally and stirred for 14 hours. 20 mL of saturated ammonium chloride aqueous solution and ethyl acetate (2 × 20 mL) were added to the reaction solution and stirred for 10 minutes. The aqueous phase was removed, and the organic phase was concentrated under reduced pressure to obtain the crude product compound 5-9.

[0349] Step 9: Synthesis of compound 5-10 Under the protection of nitrogen gas, compound 5-9 (0.11 g) and N,N-diisopropylethylamine (59.16 mg, 457.73 μmol) were dissolved in tetrahydrofuran (2.7 mL). 1H-benzotriazol-1-yloxytris(1-pyrrolidinyl)phosphonium hexafluorophosphate (95.28 mg, 183.09 μmol) was added at 10 °C, and the reaction was carried out at 10 °C for 1 hour. Next, compound 1-1B (38.87 mg, 183.09 μmol) was added, and the reaction was carried out at 10 °C for 17 hours. Water (10 mL) was added to the reaction system and separated. The aqueous phase was extracted with dichloromethane (10 mL × 3), separated, the organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (eluent: ethyl acetate / petroleum ether = 4.0% - 20.0%) to obtain compound 5-10. MS (ESI) m / z: 915.0 [M+1] + 。

[0350] Step 10: Synthesis of compound 5-11 Under the protection of nitrogen gas, compound 5-10 (0.15 g, 163.89 μmol) and copper(I) iodide (156.07 mg, 819.47 μmol) were dissolved in N,N-dimethylformamide (3.75 mL). Compound 5-10A (314.86 mg, 1.64 mmol) was added, and the reaction was carried out at 80 °C for 2 hours. Water (10 mL) and ethyl acetate (20 mL) were slowly added to precipitate a solid, which was then filtered. The filtrate was separated, and the aqueous phase was extracted with ethyl acetate (10 mL × 3) and separated. The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated by column chromatography (eluent: ethyl acetate / petroleum ether = 4.0% - 20.0%) to obtain compound 5-11. MS (ESI) m / z: 857.1 [M+1] + 。

[0351] Step 11: Synthesis of compound 5-12 Under the protection of nitrogen gas, compound 5-11 (0.06 g, 69.98 μmol) was dissolved in tetrahydrofuran (1.2 mL) and water (0.4 mL). Potassium peroxymonosulfate (82.38 mg, 489.89 μmol) was added at 0 °C, and the temperature was slowly raised to 10 °C and reacted for 4 hours. Potassium peroxymonosulfate (35.31 mg, 209.95 μmol) was added and reacted at 10 °C for 2 hours. Saturated sodium bisulfite solution (5 mL) was slowly added, and the aqueous phase was extracted with ethyl acetate (10 mL × 3) and separated. The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 5-12. MS (ESI) m / z: 889.2 [M+1] + ; MS (ESI) m / z: 873.2 [M+1] + 。

[0352] Step 12: Synthesis of compound 5-13 Under the protection of nitrogen gas, compound 5-12 (0.05 g, 56.22 μmol) and compound 2-7 (13.93 mg, 84.33 μmol) were dissolved in tetrahydrofuran (1.5 mL). Sodium tert-butoxide (10.81 mg, 112.44 μmol) was added at 0 °C, and the temperature was slowly raised to 10 °C and reacted for 3 hours. Water (5 mL) and ethyl acetate (5 mL) were added to the reaction system, filtered, and the filtrate was separated. The aqueous phase was extracted with ethyl acetate (5 mL × 3) and separated. The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 5-13. MS (ESI) m / z: 974.3 [M+1] + 。

[0353] Step 13: Synthesis of compound 5 Under the protection of nitrogen gas, compound 5-13 (0.1 g, 102.62 μmol) was dissolved in dichloromethane (2.5 mL), trifluoroacetic acid (468.04 mg, 4.10 mmol) was added, and the reaction was carried out at 10 °C for 6 hours. The reaction system was concentrated under reduced pressure to obtain a crude product. The crude product was separated by preparative HPLC twice to obtain compound 5. HPLC production method 1: Chromatography column: Phenomenex Luna 75×30 mm×3 μm; Mobile phase A: water (0.04% hydrochloric acid), Mobile phase B: acetonitrile; Execution gradient: B%: 1% - 43%, executed for 8 min. HPLC production method 2: Chromatography column: Waters Xbridge BEH 100×30 mm×10 μm; Mobile phase A: water (10 mM ammonium bicarbonate), Mobile phase B: acetonitrile; Execution gradient: B%: 30% - 60%, executed for 8 min. MS (ESI) m / z: 634.2 [M+1] + .

[0354] Example 6

[0355] [Chemical formula]

[0356] [Chemical formula]

[0357] [Chemical formula]

[0358] Step 1: Under the protection of nitrogen gas, intermediate Int-3A (0.2 g, 323.36 μmol) and Int-4 (181.67 mg, 323.36 μmol) were dissolved in tetrahydrofuran (8 mL) and water (2 mL), potassium phosphate (137.28 mg, 646.71 μmol) and chloro(2-dicyclohexylphosphino-2’,6’-dimethoxy-1,1’-biphenyl)[2-(2’-amino-1,1’-biphenyl)]palladium(II) (23.30 mg, 32.34 μmol) were added, and then the reaction was carried out at 50 °C for 2 hours. Water (5 mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (8 mL × 3) and separated. The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by HPLC (chromatography column: Phenomenex luna C18 100×40 mm×5 μm; mobile phase: A (acetonitrile) and B (water containing 0.04% hydrochloric acid); gradient: B%: 50% - 80%, 8 min) to obtain compound 6-1A. MS m / z: 973.1 [M+1] + 。

[0359] Referring to Step 1, intermediate Int-3B was used as a raw material to obtain compound 6-1B. MS m / z: 973.1 [M+1] + 。

[0360] Step 2: Compound 6-1A (0.2 g, 205.45 μmol) was separated and purified by preparative SFC (chromatography column: ChiralPak IH, 250×30 mm, 10 μm; mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% aqueous ammonia); gradient: B%: 60% - 60%, 10 min) to obtain compounds 6-1A1 and 6-1A2, respectively.

[0361] Compound 6-1A1: Under the conditions of analytical SFC (column: ChiralPak IH-3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B%: 5% - 5%, 3 min), the retention time was 1.463 min and the ee value was 97.49%. 1 H NMR (400 MHz, CDCl3) δ = 7.30 (s, 1H), 7.10 (d, J = 8.8 Hz, 4H), 6.94 (d, J = 2.0 Hz, 1H), 6.88 (d, J = 8.8 Hz, 4H), 6.52 (d, J = 2.0 Hz, 1H), 5.25 (s, 2H), 5.04 (d, J = 11.2 Hz, 1H), 4.69 (d, J = 10.8 Hz, 1H), 4.55 (s, 4H), 4.52 - 4.44 (m, 2H), 4.44 - 4.32 (m, 3H), 4.12 - 4.02 (m, 1H), 3.81 (s, 6H), 3.63 (d, J = 15.6 Hz, 2H), 3.56 - 3.43 (m, 1H), 3.36 (d, J = 17.6 Hz, 1H), 3.19 - 3.08 (m, 1H), 2.77 (d, J = 17.6 Hz, 1H), 2.07 - 1.87 (m, 4H), 1.85 - 1.77 (m, 2H), 1.51 (s, 9H), 1.26 (s, 1H), 0.91 - 0.84 (m, 1H). MS m / z: 973.1 [M+1] + 。

[0362] Compound 6-1A2: Under the conditions of analytical SFC (column: ChiralPak IH-3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B%: 5% - 5%, 3 min), the retention time was 1.983 min and the ee value was 95.66%. 11H NMR (400 MHz, CDCl3) δ = 7.30 (s, 1H), 7.10 (d, J = 8.8 Hz, 4H), 6.94 (d, J = 2.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 4H), 6.51 (d, J = 2.4 Hz, 1H), 5.22 (s, 2H), 5.06 - 4.83 (m, 1H), 4.77 - 4.62 (m, 1H), 4.54 (s, 4H), 4.49 - 4.24 (m, 5H), 4.13 - 3.94 (m, 1H), 3.81 (s, 6H), 3.70 - 3.46 (m, 3H), 3.39 - 3.23 (m, 1H), 3.18 - 3.02 (m, 1H), 2.80 - 2.64 (m, 1H), 2.09 - 1.89 (m, 4H), 1.85 - 1.76 (m, 2H), 1.52 (s, 9H), 1.32 - 1.25 (m, 1H), 0.90 - 0.75 (m, 1H). MS m / z: 973.1 [M+1] + 。

[0363] Step 3: Compound 6 - 1B (0.2 g, 205.45 μmol) was separated and purified by preparative SFC (column: ChiralPak IH, 250×30 mm, 10 μm; mobile phase: A (supercritical CO2) and B (methanol containing 0.1% aqueous ammonia); gradient: B%: 40% - 40%, 11 min) to obtain Compound 6 - 1B1 and Compound 6 - 1B2, respectively.

[0364] Compound 6 - 1B1: Under the conditions of analytical SFC (column: ChiralPak IH - 3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B%: 5% - 5%, 3 min), the retention time was 1.330 min and the ee value was 94.45%. 11H NMR (400 MHz, CDCl3) δ = 7.26 (s, 1H), 7.10 (d, J = 8.4 Hz, 4H), 6.94 (d, J = 2.0 Hz, 1H), 6.88 (d, J = 8.4 Hz, 4H), 6.51 (d, J = 2.4 Hz, 1H), 5.01 (s, 1H), 4.93 (s, 1H), 4.54 (s, 4H), 4.42 - 4.16 (m, 6H), 3.81 (s, 6H), 3.65 - 3.50 (m, 3H), 3.33 - 3.19 (m, 2H), 2.94 (d, J = 16.0 Hz, 1H), 2.75 (d, J = 6.4 Hz, 1H), 2.46 (dd, J = 15.6, 2.0 Hz, 1H), 2.00 - 1.92 (m, 2H), 1.92 - 1.72 (m, 4H), 1.52 (s, 9H), 0.78 - 0.69 (m, 1H), 0.54 - 0.42 (m, 1H). MS m / z: 973.1 [M+1] + 。

[0365] Compound 6 - 1B2: Under the conditions of analytical SFC (column: ChiralPak IH - 3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B%: 5% - 5%, 3 min), the retention time was 1.495 min and the ee value was 98.01%. 1 1H NMR (400 MHz, CDCl3) δ = 7.25 (s, 1H), 7.10 (d, J = 8.4 Hz, 4H), 6.93 (s, 1H), 6.88 (d, J = 8.4 Hz, 4H), 6.51 (s, 1H), 5.01 (s, 1H), 4.92 (s, 1H), 4.53 (s, 4H), 4.39 - 4.24 (m, 5H), 4.17 (d, J = 8.8 Hz, 1H), 3.81 (s, 6H), 3.64 - 3.49 (m, 3H), 3.33 - 3.19 (m, 2H), 2.95 (d, J = 18.8 Hz, 1H), 2.75 (d, J = 4.8 Hz, 1H), 2.46 (d, J = 16.4 Hz, 1H), 1.99 - 1.89 (m, 2H), 1.88 - 1.72 (m, 4H), 1.52 (s, 9H), 0.74 (d, J = 1.6 Hz, 1H), 0.53 - 0.43 (m, 1H). MS m / z: 973.1 [M+1] + 。

[0366] Step 4: Compound 6A1: Under the protection of nitrogen gas, Compound 6-1A1 (0.07 g, 71.91 μmol) was dissolved in dichloromethane (3.5 mL), trifluoroacetic acid (1.07 g, 9.42 mmol) was added, and then the reaction was carried out at 20 °C for 3 hours. The mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by HPLC (chromatography column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: A (acetonitrile) and B (water containing 10 mM ammonium bicarbonate); gradient: B%: 35% - 70%) to obtain Compound 6A1. 1 H NMR (400 MHz, CDCl3) δ = 7.31 (d, J = 9.6 Hz, 1H), 6.87 (s, 1H), 6.42 (s, 1H), 5.03 (s, 1H), 4.94 (s, 1H), 4.44 - 4.26 (m, 3H), 4.26 - 4.11 (m, 3H), 3.66 (s, 2H), 3.53 (t, J = 11.6 Hz, 2H), 3.37 - 3.23 (m, 2H), 2.98 (d, J = 16.8 Hz, 1H), 2.77 (dd, J = 8.4, 2.8 Hz, 1H), 2.47 (d, J = 16.4 Hz, 1H), 2.23 - 2.04 (m, 2H), 1.91 - 1.81 (m, 4H), 1.56 (s, 1H), 1.28 (d, J = 10.8 Hz, 1H), 0.89 (t, J = 7.2 Hz, 1H), 0.57 - 0.44 (m, 1H). MS m / z: 633.1 [M+1] + 。

[0367] Compound 6A2: Under the protection of nitrogen gas, Compound 6-1A2 (0.07 g, 71.91 μmol) was dissolved in dichloromethane (3.5 mL), trifluoroacetic acid (1.07 g, 9.42 mmol) was added, and then the reaction was carried out at 20 °C for 3 hours. The mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by HPLC (chromatography column: Waters Xbridge BEH C18 100×30 mm×10 μm; mobile phase: A (acetonitrile) and B (water containing 10 mM ammonium bicarbonate); gradient: B%: 35% - 70%) to obtain Compound 6A2. 11H NMR (400 MHz, CDCl3) δ = 7.30 (d, J = 10.0 Hz, 1H), 6.87 (s, 1H), 6.42 (s, 1H), 5.02 (s, 1H), 4.93 (s, 1H), 4.42 (d, J = 12.0 Hz, 1H), 4.36 - 4.26 (m, 2H), 4.21 (d, J = 9.2 Hz, 3H), 3.65 (s, 2H), 3.61 - 3.54 (m, 1H), 3.47 (d, J = 12.4 Hz, 1H), 3.35 - 3.23 (m, 2H), 2.95 (d, J = 16.8 Hz, 1H), 2.84 - 2.72 (m, 1H), 2.46 (d, J = 16.4 Hz, 1H), 2.11 (br s, 2H), 1.92 - 1.80 (m, 4H), 1.63 - 1.51 (m, 1H), 1.40 - 1.19 (m, 1H), 0.85 - 0.70 (m, 1H), 0.60 - 0.42 (m, 1H). MS m / z: 633.2 [M+1] + 。

[0368] Step 5: Compound 6B1: Under the protection of nitrogen gas, compound 6 - 1B1 (0.06 g, 61.64 μmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (921.00 mg, 8.08 mmol) was added, and then the reaction was carried out at 20 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by HPLC (chromatographic column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: A (acetonitrile) and B (water containing 0.05% aqueous ammonia + 10 mM ammonium bicarbonate); gradient: B%: 30% - 70%) to obtain compound 6B1. 11H NMR (400 MHz, CDCl3) δ = 7.31 (d, J = 9.6 Hz, 1H), 6.87 (s, 1H), 6.42 (s, 1H), 5.01 (s, 1H), 4.92 (s, 1H), 4.42 (d, J = 12.8 Hz, 1H), 4.33 - 4.24 (m, 2H), 4.22 - 4.17 (m, 1H), 4.14 (s, 2H), 3.63 (s, 2H), 3.58 (s, 1H), 3.54 (d, J = 12.4 Hz, 1H), 3.45 (d, J = 12.4 Hz, 1H), 3.32 - 3.21 (m, 2H), 2.95 (d, J = 16.8 Hz, 1H), 2.75 (dd, J = 8.8, 3.6 Hz, 1H), 2.46 (d, J = 17.2 Hz, 1H), 1.92 - 1.86 (m, 2H), 1.85 - 1.78 (m, 3H), 1.58 - 1.52 (m, 1H), 1.35 - 1.20 (m, 1H), 0.74 (q, J = 4.0 Hz, 1H), 0.53 - 0.44 (m, 1H). MS m / z: 633.2 [M+1] + 。

[0369] Compound 6B2: Under the protection of nitrogen gas, compound 6 - 1B2 (0.06 g, 61.64 μmol) was dissolved in dichloromethane (3 mL), trifluoroacetic acid (921.00 mg, 8.08 mmol) was added, and then the reaction was carried out at 20 °C for 2 hours. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by HPLC (chromatographic column: Waters Xbridge Prep OBD C18 150×40 mm×10 μm; mobile phase: A (acetonitrile) and B (water, 0.05% aqueous ammonia + 10 mM ammonium bicarbonate); gradient: B%: 25% - 65%) to obtain compound 6B2. 11H NMR (400 MHz, CDCl3) δ = 7.35 - 7.27 (m, 1H), 6.88 (s, 1H), 6.43 (s, 1H), 5.03 (s, 1H), 4.95 (s, 1H), 4.46 - 4.26 (m, 3H), 4.26 - 4.06 (m, 3H), 3.66 (s, 3H), 3.53 (t, J = 10.8 Hz, 2H), 3.29 (d, J = 11.2 Hz, 2H), 2.98 (d, J = 15.6 Hz, 1H), 2.78 (s, 1H), 2.48 (d, J = 14.8 Hz, 1H), 1.85 (s, 5H), 1.57 (s, 1H), 1.38 - 1.22 (m, 1H), 0.79 (s, 1H), 0.51 (s, 1H). MS m / z: 633.2 [M+1] + 。

[0370] Example 7

[0371]

Chem.

[0372]

Chem.

[0373] Step 1: Under the protection of nitrogen gas, intermediate Int-3A (0.2 g, 323.36 μmol) and intermediate 3-6 (265.54 mg, 420.36 μmol) were dissolved in toluene (2 mL) and water (0.4 mL), potassium phosphate (137.28 mg, 646.71 μmol) and chloro[(di(1-adamantyl)-N-butylphosphine)-2-(2-aminobiphenyl)]palladium(II) (32.43 mg, 48.50 μmol) were added, and then the mixture was reacted at 90 °C for 6 hours. Water (7 mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (7 mL × 3) and separated. The organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by HPLC (chromatography column: Phenomenex luna C18 250×50 mm×10 μm; mobile phase: A (acetonitrile) and B (water containing 0.04% hydrochloric acid); gradient: B%: 60% - 80%, 10 min) to obtain compound 7-1A. MS m / z: 879.3[M-C 13 H 10 O] + 。

[0374] Step 2: Under the protection of nitrogen gas, intermediate Int-3B (0.2 g, 323.36 μmol) and intermediate 3-6 (265.54 mg, 420.36 μmol) were dissolved in toluene (2 mL) and water (0.4 mL), potassium phosphate (137.28 mg, 646.71 μmol) and chloro[(di(1-adamantyl)-N-butylphosphine)-2-(2-aminobiphenyl)]palladium(II) (32.43 mg, 48.50 μmol) were added, and then the mixture was reacted at 90 °C for 4 hours. Water (7 mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (7 mL × 3) and separated. The organic phases were combined, the combined organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated and purified by column chromatography (mobile phase: petroleum ether:ethyl acetate = 50:1 - 1:1) to obtain compound 7-1B. MS m / z: 522.4[M / 2+1] + 。

[0375] Step 3: Under the protection of nitrogen gas, 7-1A (0.19 g, 216.12 μmol) was dissolved in dichloromethane (7.5 mL), trifluoroacetic acid (3.46 g, 30.36 mmol) was added, and then the reaction was carried out at 20 °C for 2 hours. It was concentrated under reduced pressure to obtain the crude product compound 7-2A. MS m / z: 779.6 [M+1] + 。

[0376] Referring to Step 3, using 7-1B as the raw material, compound 7-2B was obtained. MS m / z: 779.6 [M+1]+.

[0377] Step 4: Under the protection of nitrogen gas, compound 7-2A (0.4 g, 143.58 μmol) was dissolved in N,N-dimethylformamide (4 mL), cesium fluoride (2.18 g, 14.36 mmol) and sodium carbonate (91.31 mg, 861.50 μmol) were added, and the reaction was carried out at 20 °C for 15 hours. Water (6 mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (7 mL×3) and separated. The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated by HPLC (chromatographic column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: A (acetonitrile) and B (water, containing 0.04% formic acid); gradient: B%: 15%~55%), and further separated and purified by preparative SFC (column: DAICEL CHIRALPAK IG (250 mm×30 mm, 10 μm); mobile phase: A (supercritical CO2) and B (ethanol, containing 0.1% aqueous ammonia); gradient: B%: 50%~50%, 11 min) to obtain compound 7A1 and compound 7A2 respectively.

[0378] Compound 7A1: Under the conditions of analytical SFC (column: ChiralPak IG-3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (isopropanol, containing 0.1% isopropylamine); gradient: B%: 5%~5%, 3 min), the retention time was 1.693 min and the ee value was 97.92%. 11H NMR (400 MHz, CDCl3) δ = 7.67 (dd, J = 8.8, 5.6 Hz, 1H), 7.28 (s, 1H), 7.22 (t, J = 8.8 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 6.97 (d, J = 2.0 Hz, 1H), 5.03 (s, 1H), 4.95 (s, 1H), 4.41 (d, J = 12.4 Hz, 1H), 4.35 (d, J = 10.4 Hz, 2H), 4.23 (d, J = 10.0 Hz, 1H), 4.04 - 3.80 (m, 2H), 3.66 (s, 3H), 3.55 (d, J = 12.4 Hz, 1H), 3.51 (d, J = 12.0 Hz, 1H), 3.31 (d, J = 4.4 Hz, 1H), 3.28 (s, 1H), 3.01 (d, J = 16.8 Hz, 1H), 2.82 - 2.74 (m, 2H), 2.48 (d, J = 16.8 Hz, 1H), 1.90 - 1.86 (m, 5H), 1.60 - 1.53 (m, 1H), 1.32 - 1.19 (m, 1H), 0.80 (q, J = 4.4 Hz, 1H), 0.56 - 0.48 (m, 1H). MS m / z: 623.2 [M+1]+. Compound 7A2: Under the conditions of analytical SFC (column: ChiralPak IG-3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B%: 5% - 5%, 3 min), the retention time was 2.236 min and the ee value was 98.26%. 11H NMR (400 MHz, CDCl3) δ = 7.67 (dd, J = 8.8, 6.0 Hz, 1H), 7.28 (s, 1H), 7.22 (t, J = 8.8 Hz, 1H), 7.25 - 7.19 (m, 1H), 7.12 (d, J = 2.0 Hz, 1H), 5.03 (s, 1H), 4.95 (s, 1H), 4.44 - 4.30 (m, 3H), 4.29 - 4.23 (m, 1H), 4.07 - 3.83 (m, 2H), 3.77 - 3.65 (m, 3H), 3.57 (t, J = 10.8 Hz, 2H), 3.32 (s, 1H), 3.29 (d, J = 5.2 Hz, 1H), 2.99 (d, J = 16.4 Hz, 1H), 2.83 - 2.73 (m, 2H), 2.49 (d, J = 16.8 Hz, 1H), 1.94 - 1.86 (m, 5H), 1.63 - 1.53 (m, 1H), 1.30 - 1.11 (m, 1H), 0.82 (d, J = 4.0 Hz, 1H), 0.53 (q, J = 8.0 Hz, 1H). MS m / z: 623.2 [M+1] + 。

[0379] Step 5: Under the protection of nitrogen gas, compound 7 - 2B (0.55 g, 198.24 μmol) was dissolved in N,N - dimethylformamide (5 mL), cesium fluoride (3.01 g, 19.82 mmol) and sodium carbonate (126.07 mg, 1.19 mmol) were added, and the reaction was carried out at 20 °C for 15 hours. Water (6 mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (7 mL × 3) and separated. The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was separated by HPLC (chromatographic column: Phenomenex Luna C18 75×30 mm×3 μm; mobile phase: A (acetonitrile) and B (water containing 0.04% formic acid); gradient: B%: 15% - 55%), and further separated and purified by preparative SFC (column: DAICEL CHIRALPAK IG (250 mm×30 mm, 10 μm); mobile phase: A (supercritical CO2) and B (ethanol containing 0.1% aqueous ammonia); gradient: B%: 55% - 55%, 7 min) to obtain compound 7B1 and compound 7B2 respectively.

[0380] Compound 7B1: Under the conditions of analytical SFC (column: ChiralPak IG-3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B%: 5% - 5%, 3 min), the retention time was 1.745 min and the ee value was 98.56%. 1 H NMR (400 MHz, CDCl3) δ = 7.67 (dd, J = 9.2, 6.0 Hz, 1H), 7.30 - 7.25 (m, 1H), 7.22 (t, J = 8.8 Hz, 1H), 7.11 (d, J = 1.6 Hz, 1H), 6.97 (d, J = 1.6 Hz, 1H), 5.03 (s, 1H), 4.94 (s, 1H), 4.38 (t, J = 14.0 Hz, 2H), 4.32 - 4.27 (m, 1H), 4.27 - 4.19 (m, 1H), 4.11 - 3.76 (m, 2H), 3.73 - 3.61 (m, 3H), 3.53 (t, J = 11.6 Hz, 2H), 3.34 - 3.23 (m, 2H), 2.98 (d, J = 16.8 Hz, 1H), 2.81 - 2.73 (m, 2H), 2.48 (d, J = 16.8 Hz, 1H), 1.92 - 1.79 (m, 5H), 1.60 - 1.52 (m, 1H), 1.26 (s, 1H), 0.79 (q, J = 3.6 Hz, 1H), 0.51 (q, J = 7.6 Hz, 1H). MS m / z: 623.2 [M + 1] + 。

[0381] Compound 7B2: Under the conditions of analytical SFC (column: ChiralPak IG-3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B%: 5% - 5%, 3 min), the retention time was 2.343 min and the ee value was 98.62%. 11H NMR (400 MHz, CDCl3) δ = 7.67 (dd, J = 8.8, 5.6 Hz, 1H), 7.29 - 7.25 (m, 1H), 7.22 (t, J = 8.8 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 6.97 (d, J = 2.0 Hz, 1H), 5.03 (s, 1H), 4.95 (s, 1H), 4.41 (d, J = 12.0 Hz, 1H), 4.36 (d, J = 10.4 Hz, 2H), 4.23 (d, J = 10.0 Hz, 1H), 4.07 - 3.78 (m, 2H), 3.75 - 3.63 (m, 3H), 3.59 (d, J = 12.0 Hz, 1H), 3.54 (d, J = 12.4 Hz, 1H), 3.31 (d, J = 3.2 Hz, 1H), 3.28 (s, 1H), 3.00 (d, J = 16.8 Hz, 1H), 2.82 - 2.73 (m, 2H), 2.48 (d, J = 16.8 Hz, 1H), 1.93 - 1.80 (m, 5H), 1.61 - 1.51 (m, 1H), 1.26 (s, 1H), 0.81 (q, J = 4.0 Hz, 1H), 0.55 - 0.47 (m, 1H). MS m / z: 623.2 [M+1] + 。

[0382] Example 8

[0383]

Chemical formula

[0384] Step 1: Dissolve compound 8 - 1 (2 g, 6.05 mmol) and 1 - 1B (1.35 g, 6.36 mmol) in dichloromethane (20 mL), displace with nitrogen gas three times, and add triethylamine (1.84 g, 18.16 mmol) dropwise at -40°C. After the addition is complete, warm to 25°C and react for 3 hours. Pour into 50 mL of water and separate. Extract the aqueous phase with dichloromethane (10 mL × 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the crude product by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 - 3:1) to obtain compound 8 - 2. MS m / z: 505.0, 507.0 [M+1] + 。

[0385] Step 2: 8-2 (646 mg, 1.28 mmol) and potassium fluoride (1.48 g, 25.52 mmol) were dissolved in dimethyl sulfoxide (12 mL), replaced with nitrogen gas three times, heated to 120 °C, and stirred for 1 hour. The reaction solution was poured into 60 mL of water and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0~5:1) to obtain 8-3. 1 H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 1.6 Hz, 1H), 4.22 - 4.58 (m, 4H), 3.51 - 3.89 (m, 2H), 1.90 - 2.04 (m, 2H), 1.66 - 1.84 (m, 2H), 1.53 (s, 9H).

[0386] Step 3: Int-5 (333.98 mg, 1.23 mmol), 8-3 (502 mg, 1.03 mmol) and potassium phosphate (435.17 mg, 2.05 mmol) were dissolved in tetrahydrofuran (10 mL) and water (2.5 mL), replaced with nitrogen gas three times, and [methanesulfonato(2-dicyclohexylphosphino-2′,4′,6′-tri-i-propyl-1,1′-biphenyl)(2′-amino-1,1′-biphenyl-2-yl)palladium(II)] (86.76 mg, 102.50 μmol, 0.1 eq) was added. After replacing with nitrogen gas three times, the mixture was stirred at 35 °C for 3 hours. The reaction solution was cooled, filtered, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0~4:1) to obtain 8-4. 1 H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 1.6 Hz, 1H), 6.90 (dd, J = 7.2, 2.4 Hz, 1H), 6.65 (dd, J = 5.2, 2.4 Hz, 1H), 4.29 - 4.56 (m, 4H), 3.51 - 3.87 (m, 2H), 1.93 - 2.03 (m, 2H), 1.77 (br d, J = 8.0 Hz, 2H), 1.54 (s, 9H).

[0387] Step 4: 8-4 (330 mg, 595.25 μmol) and N-iodosuccinimide (200.88 mg, 892.87 μmol) were dissolved in N,N-dimethylformamide (3.3 mL), replaced with nitrogen gas three times, heated to 50 °C, and stirred for 28 hours. The reaction solution was poured into 5 mL of saturated aqueous sodium bicarbonate solution and 5 mL of saturated aqueous sodium thiosulfate solution, and extracted with ethyl acetate (5 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 to 3:1) to obtain compound 8-5. 1 H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 1.6 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 4.30 - 4.63 (m, 4H), 3.52 - 3.91 (m, 2H), 1.94 - 2.05 (m, 2H), 1.72 - 1.88 (m, 2H), 1.54 (s, 9H).

[0388] Step 5: Sodium hydride (36.69 mg, 917.26 μmol, 60%) was dissolved in tetrahydrofuran (1 mL), replaced with nitrogen gas three times, cooled to 0 °C, a solution of 2-7 (151.56 mg, 917.26 μmol) in tetrahydrofuran (1.5 mL) was added, the temperature was raised to 20 °C, and stirred for 10 minutes. It was cooled to 0 °C, a solution of 8-5 (208 mg, 305.75 μmol) in tetrahydrofuran (2.5 mL) was added, the temperature was raised to 20 °C, and stirred for 4 hours. The reaction solution was poured into 10 mL of saturated aqueous ammonium chloride solution and extracted with ethyl acetate (2 mL × 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 to 1:2) to obtain 8-6. MS m / z: 825.1 [M+1] + 。

[0389] Step 6: 8-6 (180 mg, 218.05 μmol), methylboronic acid (39.16 mg, 654.14 μmol) and potassium carbonate (60.27 mg, 436.09 μmol) were dissolved in dioxane (2 mL) and water (0.5 mL), and the mixture was replaced with nitrogen gas three times. [1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (15.95 mg, 21.80 μmol, 0.1 eq) was added, and the mixture was replaced with nitrogen gas three times. The temperature was raised to 80 °C and stirred for 60 hours. The reaction solution was concentrated under reduced pressure. The crude product was separated and purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 to 1:2) to obtain a crude product. The crude product was separated and purified by preparative SFC (chromatography column: DAICEL CHIRALPAK AD (250 mm × 30 mm, 10 μm); mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% aqueous ammonia); gradient: B% = 45% to 45%) to obtain a mixture of 8-7A and 8-7B, 8-7C and 8-7D, respectively. The retention time of 8-7C by the SFC analysis method (chromatography column: Chiralpak AD-3, 50 × 4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B% = 5% to 50% to 5%, 3.0 min) was 1.468 min, and the enantiomeric excess of the chiral isomer was 97.36%. The retention time of 8-7D by the SFC analysis method (chromatography column: Chiralpak AD-3, 50 × 4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B% = 5% to 50% to 5%, 3.0 min) was 1.603 min, and the enantiomeric excess of the chiral isomer was 100%.

[0390] Step 7: A mixture of 8-7A and 8-7B (32 mg, 44.84 μmol) was dissolved in dichloromethane (0.6 mL), trifluoroacetic acid (0.2 mL) was added, and the mixture was stirred at 15 °C for 0.5 h. Ammonia water was added dropwise to the reaction solution to adjust the pH to 9, and the solution was concentrated under reduced pressure. The crude product was separated and purified by preparative HPLC (chromatography column: Phenomenex Luna 100×30 mm×3 μm; mobile phase A: water (0.2% formic acid), mobile phase B: acetonitrile; running gradient: B%: 1% - 30%, run for 8 min) to obtain a crude product. The crude product was separated and purified by preparative SFC (chromatography column: DAICEL CHIRALPAK AD (250 mm×30 mm, 10 μm); mobile phase: A (supercritical CO2) and B (ethanol containing 0.1% ammonia water); gradient: B% = 50% - 50%, run for 11 min) to obtain 8A and 8B.

[0391] The retention time of 8A in the SFC analysis method (chromatography column: Chiralpak IG-3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (ethanol containing 0.1% isopropylamine); gradient: B% = 5% - 50% - 5%, run for 3.0 min) was 1.727 min, and the enantiomeric excess was 100%. 1 1H NMR (400 MHz, CD3OD) δ: 7.91 (s, 1H), 7.01 (d, J = 8.4 Hz, 1H), 4.95 - 5.10 (m, 2H), 4.47 (br d, J = 12.4 Hz, 2H), 4.25 - 4.42 (m, 2H), 3.56 - 3.75 (m, 5H), 3.26 - 3.30 (m, 1H), 3.20 (br d, J = 9.6 Hz, 1H), 2.91 (br d, J = 16.4 Hz, 1H), 2.79 (br dd, J = 9.2, 3.6 Hz, 1H), 2.53 (br d, J = 17.2 Hz, 1H), 1.97 (s, 3H), 1.81 (br s, 5H), 1.56 - 1.70 (m, 1H), 0.70 (q, J = 4.0 Hz, 1H), 0.49 - 0.59 (m, 1H). MS m / z: 613.2 [M+1] + 。

[0392] The retention time of 8B in the SFC analysis method (chromatographic column: Chiralpak IG-3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical CO2) and B (ethanol containing 0.1% isopropylamine); gradient: B% = 5% - 50% - 5%, 3.0 min) was 2.216 min, and the enantiomeric excess was 99.22%. 1 1H NMR (400 MHz, CD3OD) δ: 7.92 (d, J = 2.0 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 4.96 - 5.10 (m, 2H), 4.47 (br t, J = 13.2 Hz, 2H), 4.25 - 4.41 (m, 2H), 3.57 - 3.74 (m, 5H), 3.26 - 3.30 (m, 1H), 3.20 (d, J = 9.6 Hz, 1H), 2.91 (br d, J = 16.8 Hz, 1H), 2.79 (dd, J = 9.6, 4.0 Hz, 1H), 2.53 (br d, J = 16.4 Hz, 1H), 1.76 - 1.91 (m, 8H), 1.56 - 1.69 (m, 1H), 0.70 (q, J = 4.4 Hz, 1H), 0.49 - 0.59 (m, 1H). MS m / z: 613.2 [M+1] + 。

[0393] Step 8: Dissolve 8-7C in dichloromethane (0.6 mL), add trifluoroacetic acid (0.2 mL), and stir at 15 °C for 0.5 h. Add aqueous ammonia dropwise to the reaction solution to adjust the pH to 9, and concentrate under reduced pressure. The crude product was separated and purified by preparative HPLC (chromatographic column: Phenomenex Luna 100×30 mm×3 μm; mobile phase A: water (0.2% formic acid), mobile phase B: acetonitrile; running gradient: B%: 1% - 30%, run for 8 min) to obtain the formate salt of 8C. 11H NMR (400 MHz, CD3OD) δ 8.51 (br s, 1H), 7.94 (s, 1H), 7.02 (d, J = 8.0 Hz, 1H), 5.09 (d, J = 16.4 Hz, 2H), 4.50 - 4.63 (m, 3H), 4.41 (d, J = 10.8 Hz, 1H), 3.86 - 4.08 (m, 3H), 3.76 (br dd, J = 12.8, 8.8 Hz, 2H), 3.48 (br d, J = 15.6 Hz, 1H), 3.34 - 3.41 (m, 1H), 2.92 - 3.03 (m, 2H), 2.66 (br d, J = 16.4 Hz, 1H), 1.93 - 2.07 (m, 7H), 1.81 - 1.92 (m, 1H), 1.66 - 1.76 (m, 1H), 0.71 - 0.78 (m, 1H), 0.60 - 0.69 (m, 1H). MS m / z: 613.2 [M+1] + 。

[0394] Step 9: 8-7D (21 mg, 29.43 μmol) was dissolved in dichloromethane (0.6 mL), trifluoroacetic acid (0.2 mL) was added, and the mixture was stirred at 15 °C for 0.5 h. Ammonia water was added dropwise to the reaction solution to adjust the pH to 9, and the solution was concentrated under reduced pressure. The crude product was separated and purified by preparative HPLC (chromatographic column: Phenomenex Luna 100×30 mm×3 μm; mobile phase A: water (0.2% formic acid), mobile phase B: acetonitrile; running gradient: B%: 1% - 30%, run for 8 min) to obtain the formate salt of 8D. 1 1H NMR (400 MHz, CD3OD) δ 8.53 (br s, 1H), 7.93 (s, 1H), 7.02 (d, J = 8.0 Hz, 1H), 5.07 (br d, J = 18.8 Hz, 2H), 4.42 - 4.65 (m, 3H), 4.37 (br d, J = 10.8 Hz, 1H), 3.81 - 3.97 (m, 3H), 3.73 (br d, J = 12.4 Hz, 2H), 3.43 (br d, J = 15.6 Hz, 1H), 3.34 (br s, 1H), 2.84 - 3.03 (m, 2H), 2.62 (br d, J = 17.2 Hz, 1H), 1.97 (s, 7H), 1.81 - 1.89 (m, 1H), 1.64 - 1.73 (m, 1H), 0.69 - 0.81 (m, 1H), 0.57 - 0.66 (m, 1H). MS m / z: 613.2 [M+1] +。

[0395] Experimental Example 1: p-ERK Inhibition Test of GP2D Cells 1. Purpose To screen for compounds that can effectively inhibit p-ERK of GP2D cells by the HTRF method.

[0396] 2. Experimental Process 1). Seed GP2D cells into a transparent 96-well cell culture plate, add 80 μL / well of cell suspension, with 8000 cells in each well, place the cell plate in a carbon dioxide incubator, and culture overnight at 37°C. 2). Add 2 μL of the compound to 78 μL of cell culture medium, mix uniformly, then add 20 μL of the compound solution to the corresponding wells of the cell plate, and return the cell plate to the carbon dioxide incubator to continue culturing for 1 hour. 3). After the culture is completed, discard the cell supernatant, add 50 μL of 1X cell lysate to each well, and culture with shaking at room temperature for 30 minutes. 4). Dilute the Phospho-ERK1 / 2 Eu Cryptate antibody and Phospho-ERK1 / 2 d2 antibody 20-fold with detection buffer. 5). Take 16 μL / well of the cell lysate supernatant and add it to a new 384-well white microplate, add 2 μL of the Phospho-ERK1 / 2 Eu Cryptate antibody dilution and 2 μL of the Phospho-ERK1 / 2 d2 antibody dilution, and culture at room temperature for at least 4 hours. 6). After the culture is completed, read at HTRF excitation: 320 nm, emission: 615 nm, 665 nm using a multimode microplate reader. 7). Calculate the IC 50 of the test compound.

[0397] 3. Experimental Results The results are as shown in Table 2.

[0398]

Table 2

[0399] Experimental conclusion: The compound of the present invention has a significant GP2D p-ERK inhibitory effect.

[0400] Experimental Example 2. GP2D 3D CTG experiment 1. Experimental purpose: The purpose of this experiment is to verify the growth inhibitory effect of the compound of the present invention on GP2D human colon cancer cells with KRAS G12D mutation.

[0401] 2. Experimental materials: The cell line GP2D, DMEM medium was purchased from GIBCO, FBS was purchased from Hyclone, and L-glutamine was purchased from Invitrogen. The 96-well plate was purchased from Ultra Low Cluster. The CellTiter-Glo® 3D Cell Viability Assay (chemiluminescent detection reagent for 3D cell viability) reagent was purchased from Promega, and the 2104 EnVision plate reader was purchased from PerkinElmer.

[0402] 3. Experimental method: GP2D cells were cultured in an incubator at 37 °C and 5% CO2 according to the culture conditions of DMEM + 10% FBS + 2 mM L-glutamine. They were passaged regularly, and cells in the logarithmic growth phase were collected for plating. GP2D cells were seeded in a 96-well U-bottom cell culture plate, with 135 μL of cell suspension in each well, containing 6000 GP2D cells. The culture plate was cultured overnight in an incubator at 37 °C, 5% CO2, and 100% relative humidity. The test compound was diluted 5-fold at 8 concentrations with a pipette, i.e., diluted from 200 μM to 2.56 nM, and two wells were set up under the same conditions. 78 μL of medium was added to the middle plate, and then 2 μL / well of the gradient-diluted compound was transferred to the middle plate according to the corresponding positions, mixed uniformly, and then 20 μL / well was transferred to the cell plate. The concentration range of the compound transferred to the cell plate was 1 μM to 0.0128 nM. The cell plate was placed in a carbon dioxide incubator and cultured for 5 days. After the culture of the cell plate with the compound was completed, 100 μL of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and cultured at room temperature for 10 minutes to stabilize the luminescence signal. Data was read using a multimode microplate reader.

[0403] 4. Data analysis: The raw data was converted to the inhibition rate using the equation (Sample - Min) / (Max - Min)×100%, and the IC 50 value was obtained by curve fitting using four parameters (obtained with the log(inhibitor) vs. response--Variable slope motor of GraphPad Prism).

[0404] 5. Experimental results: The experimental results were as shown in Table 3.

[0405]

Table 3

[0406] Experimental conclusion: The compound of the present invention has significant anti-proliferative activity against GP2D cells having the KRAS G12D mutation.

[0407] Experimental Example 3. AsPC-1 3D CTG experiment 1. Experimental purpose: The purpose of this experiment is to verify the growth inhibitory effect of the compound of the present invention on AsPC-1 human pancreatic cancer cells having the KRAS G12D mutation.

[0408] 2. Experimental materials: The cell line AsPC-1 and RPMI-1640 medium were purchased from GIBCO, and FBS was purchased from Hyclone. The 96-well plate was purchased from Ultra Low Cluster, the CellTiter-Glo® 3D Cell Viability Assay (chemiluminescent detection reagent for 3D cell viability) reagent was purchased from Promega, and the 2104 EnVision plate reader was purchased from PerkinElmer.

[0409] 3. Experimental method: AsPC-1 cells were cultured in RPMI-1640 + 10% FBS under the culture conditions of 37°C and 5% CO2 in an incubator. They were passaged regularly, and cells in the logarithmic growth phase were collected for plating. AsPC-1 cells were seeded into a 96-well U-bottom cell culture plate, with 135 μL of cell suspension in each well, such that each well contained 500 AsPC-1 cells. The culture plate was cultured overnight in an incubator at 37°C, 5% CO2, and 100% relative humidity. The test compound was diluted 5-fold at 8 concentrations with a pipette, i.e., diluted from 200 μM to 2.56 nM, and two wells were set up under the same conditions. 78 μL of medium was added to the middle plate, and then 2 μL / well of the serially diluted compound was transferred to the middle plate according to the corresponding positions. After mixing uniformly, 20 μL / well was transferred to the cell plate. The concentration range of the compound transferred to the cell plate was 1 μM to 0.0128 nM. The cell plate was placed in a carbon dioxide incubator and cultured for 7 days. After the culture of the cell plate with the compound was completed, 100 μL of cell viability chemiluminescence detection reagent was added to each well of the cell plate, and cultured at room temperature for 10 minutes to stabilize the luminescence signal. Data was read using a multimode microplate reader.

[0410] 4. Data analysis: The raw data was converted to the inhibition rate using the equation (Sample - Min) / (Max - Min)×100%, and the IC 50 value was obtained by curve fitting using four parameters (obtained with the log(inhibitor) vs. response - Variable slope motor in GraphPad Prism).

[0411] 5. Experimental results: The experimental results were as shown in Table 4.

[0412]

Table 4

[0413] Experimental conclusion: The compound of the present invention has significant anti-proliferative activity against AsPC-1 cells having the KRAS G12D mutation.

[0414] Experimental Example 4: PANC04.03 3D CTG experiment 1. Experimental purpose: The purpose of this experiment is to verify the growth inhibitory effect of the compound of the present invention on PANC04.03 human pancreatic cancer cells having the KRAS G12D mutation.

[0415] 2. Experimental materials: The cell line PANC04.03 and RPMI-1640 medium were purchased from GIBCO, FBS was purchased from Hyclone, and human insulin was purchased from Yeasen. The 96-well plate was purchased from Ultra Low Cluster, the CellTiter-Glo® 3D Cell Viability Assay (chemiluminescent detection reagent for 3D cell viability) reagent was purchased from Promega, and the 2104 EnVision plate reader was purchased from PerkinElmer.

[0416] 3. Experimental method: PANC04.03 cells were cultured in an incubator at 37°C and 5% CO2 according to the culture conditions of RPMI-1640 + 15% FBS + 5 μg / ml human insulin. They were passaged regularly, and cells in the logarithmic growth phase were collected for plating. PANC04.03 cells were seeded into a 96-well U-bottom cell culture plate, with 135 μL of cell suspension in each well, such that each well contained 2000 PANC04.03 cells. The culture plate was cultured overnight in an incubator at 37°C, 5% CO2, and 100% relative humidity. The test compound was diluted 5-fold at 8 concentrations with a pipette, i.e., diluted from 200 μM to 2.56 nM, and two wells were set up under the same conditions. 78 μL of medium was added to the middle plate, and then, according to the corresponding positions, 2 μL / well of the serially diluted compound was transferred to the middle plate. After uniform mixing, 20 μL / well was transferred to the cell plate. The concentration range of the compound transferred to the cell plate was 1 μM to 0.0128 nM. The cell plate was placed in a carbon dioxide incubator and cultured for 7 days. After the culture of the cell plate with the compound was completed, 100 μL of cell viability chemiluminescence detection reagent was added to each well of the cell plate and cultured at room temperature for 10 minutes to stabilize the luminescence signal. Data was read using a multimode microplate reader.

[0417] 4. Data analysis: The raw data was converted to the inhibition rate using the equation (Sample - Min) / (Max - Min)×100%, and the IC 50 value was obtained by curve fitting using four parameters (obtained with the log(inhibitor) vs. response--Variable slope motor in GraphPad Prism).

[0418] 5. Experimental results: The results were as shown in Table 5.

[0419]

Table 5

[0420] Experimental conclusion: The compound of the present invention has significant anti-proliferative activity against PANC04.03 cells with KRAS G12D mutation.

[0421] Experimental Example 5 In vivo pharmacokinetic experiment 1. Experimental purpose: The purpose of this experiment is to investigate the pharmacokinetic properties of the compound of the present invention orally administered and intravenously injected to CD-1 mice.

[0422] 2. Experimental method: The test compound was mixed with 10% dimethyl sulfoxide + 90% (aqueous solution of 10% hydroxypropyl-β-cyclodextrin (HP-β-CD)), vortexed, and sonicated to produce clear solutions of 0.6, 3.0, and 10.0 mg / mL, respectively. Male CD-1 mice aged 7-10 weeks were selected, and the candidate compound solution was intravenously administered (i.v.) at a dose of 3 mg / kg (administration concentration: 0.6 mg / mL). The candidate compound solution was orally administered (p.o.) at a dose of 30 mg / kg (administration concentration: 3.0 mg / mL) or 100 mg / kg (administration concentration: 10.0 mg / mL). Whole blood was collected at predetermined times to prepare plasma, the drug concentration was analyzed by LC-MS / MS method, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA).

[0423] 3. Experimental results: The results are as shown in Table 6.

[0424]

Table 6

[0425] Experimental conclusion: The compound of the present invention has better pharmacokinetic properties in the body of mice.

[0426] Experimental Example 6 In vivo pharmacodynamic experiment of GP2D tumor model 1. Experimental purpose: This is for the in vivo pharmacodynamics study of a Balb / c nude mouse model with subcutaneous transplantation of human colon cancer GP2D cells into nude mice.

[0427] 2. Experimental methods: Cell culture: Human colon cancer GP2D cells were cultured in monolayer in vitro. The culture conditions were adding 20% fetal bovine serum to DMEM / F12 medium and culturing in an incubator at 37°C with 5% CO2. Trypsin-EDTA was used for normal digestion twice a week for subculture. When the cell saturation reached 80% - 90% and the required number was reached, the cells were collected and counted, resuspended in an appropriate amount of PBS, and Matrigel was added at a ratio of 1:1 to obtain a cell suspension with a cell density of 25×10 6 cells / mL.

[0428] Cell inoculation: 0.2 mL (5×10 6 cells / mouse) of GP2D cells (added with Matrigel, volume ratio 1:1) were subcutaneously inoculated into the right back of each mouse.

[0429] Experimental operation: When the average tumor volume reached 140 mm 3 , according to the tumor volume, 6 animals in each group were randomly divided. The dosage of the blank group was 0, and the dosages of the test groups were 30 mg / kg and 100 mg / kg respectively. The administration volume was 10 μL / g, and oral administration was carried out twice a day for 28 days.

[0430] 3. Tumor measurement and experimental indicators: The diameter of the tumor was measured with calipers twice a week. The calculation formula for tumor volume was: V = 0.5a×b 2 , where a and b represent the major axis and minor axis of the tumor respectively.

[0431] The antitumor effect of the compound was evaluated by TGI (%) or relative tumor growth rate T / C (%). Relative tumor growth rate T / C (%) = TRTV / CRTV×100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). According to the tumor measurement results, the relative tumor volume (RTV) was calculated, and the calculation formula was RTV = V t / V0, where V0 is the average tumor volume measured at the time of separate administration of the groups (i.e., D0), and V t is the average tumor volume at a specific measurement time, and TRTV took data on the same day as CRTV.

[0432] TGI(%) reflects the tumor growth inhibition rate. TGI(%) = [(1 - (average tumor volume at the end of administration of a specific treatment group - average tumor volume at the start of administration of that treatment group) / (average tumor volume at the end of treatment of the solvent control group - average tumor volume at the start of treatment of the solvent control group)) × 100%.

[0433] 4. Experimental results: The experimental results are as shown in Table 7.

[0434]

Table 7

[0435] Experimental conclusion: The compound of the present invention has an excellent tumor inhibitory effect.

[0436] Experimental Example 7 In Vivo Pharmacodynamics Experiment of PANC04.03 Tumor Model 1. Experimental purpose: It is for the in vivo pharmacodynamics study of a Balb / c nude mouse model with subcutaneous transplantation of human pancreatic cancer PANC04.03 cells into nude mice.

[0437] 2. Experimental method: Cell culture: Human pancreatic cancer PANC04.03 cells were cultured in vitro in a monolayer. The culture conditions were RPMI - 1640 + 15% FBS + 10 units / mL insulin, cultured in an incubator at 37°C with 5% CO2. Twice a week, trypsin - EDTA was used for normal digestion treatment and sub - culture. When the cell saturation reached 80% - 90% and the required number was reached, the cells were collected, counted, resuspended in an appropriate amount of PBS, and a cell suspension with a cell density of 25×10 6 cells / mL was obtained.

[0438] Cell inoculation: 0.2 mL (5×106 PANC04.03 cells (cells / mice) were subcutaneously inoculated into the right dorsal part of each mouse.

[0439] Experimental operation: When the average tumor volume reached 190 mm 3 animals were randomly divided into groups with 6 animals in each group according to the tumor volume. The dosage of the blank group was 0, and the dosages of the test groups were 30 mg / kg, 100 mg / kg, and 150 mg / kg respectively. The dosage volume was 10 μL / g, and oral administration was carried out twice a day for 28 days.

[0440] 3. Tumor measurement and experimental indicators: The diameter of the tumor was measured with calipers twice a week. The calculation formula for tumor volume was: V = 0.5a × b 2 where a and b represent the long diameter and short diameter of the tumor respectively.

[0441] The antitumor effect of the compound was evaluated by TGI (%) or relative tumor growth rate T / C (%). Relative tumor growth rate T / C (%) = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). According to the tumor measurement results, the relative tumor volume (RTV) was calculated, and the calculation formula was RTV = V t / V0, where V0 is the average tumor volume measured when dividing the groups for administration (i.e., D0), and V t is the average tumor volume at a specific measurement time, and TRTV was taken from the data on the same day as CRTV.

[0442] TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [(1 - (average tumor volume at the end of administration of a specific treatment group - average tumor volume at the start of administration of the said treatment group) / (average tumor volume at the end of treatment of the solvent control group - average tumor volume at the start of treatment of the solvent control group)) × 100%.

[0443] 4. Experimental results: The experimental results are as shown in Table 8.

[0444]

Table 8

[0445] Experimental conclusion: The compound of the present invention has excellent tumor inhibitory effect.

Claims

1. A compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof. 【Chemical 1】 (However, X is CH, C—Rx, N, and N ＋ —O － selected from, preferably, X is selected from N and N ＋ —O － selected from, and Rx is selected from F, Cl, and Br R １ is 【Chemical 2】 selected from, said [Chemical Formula 3] is independently substituted by 1, 2, 3 or 4 Rs each, ａ optionally substituted by R ３ is selected from H and D, Each R ａ is independently selected from F, Cl, Br, I, OH, NH ２ , C １－３ alkyl, C １－３ alkoxy, C １－３ haloalkyl, C ２－４ alkenyl, C ２－４ alkynyl, -C １－３ alkyl-cyclopropyl and cyclopropyl, and the C １－３ alkyl, C １－３ alkoxy, C ２－４ alkenyl, C ２－４ alkynyl, cyclopropyl and -C １－３ alkyl-cyclopropyl are each independently optionally substituted by one, two or three Rs, Each R is independently F, Cl, Br, I, CH ２ F, CHF ２ and CF ３ selected from).

2. A compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof. [Chemical Formula 4] (However, X is selected from CN, N and N ＋ -O － preferably, X is selected from N and N ＋ -O － and R １ is 【Chemical Formula 5】 selected from, said 【Chemical Formula 6】 is independently substituted by one, two, three or four Rs, respectively ａ optionally substituted by R ３ is selected from H and D, Each R ａ is independently selected from F, Cl, Br, I, OH, NH ２ , C １－３ alkyl, C １－３ alkoxy, C ２－４ alkenyl, C ２－４ alkynyl, -C １－３ alkyl-cyclopropyl and cyclopropyl, and the C １－３ alkyl, C １－３ alkoxy, C ２－４ alkenyl, C ２－４ alkynyl, cyclopropyl and -C １－３ alkyl-cyclopropyl are each independently optionally substituted by one, two or three Rs, Each R is independently F, Cl, Br, I, CH ２ F, CHF ２ and CF ３ and is selected from.)

3. A compound represented by formulas (II) and (III-2) or a pharmaceutically acceptable salt thereof. 【Chemical Formula 7】 (However, R １ is selected from phenyl, pyridyl and naphthyl, and the phenyl, pyridyl and naphthyl are each independently optionally substituted by 1, 2, 3 or 4 R ａ groups R ２ is [Chemical 8] selected from, said 【Chemical Formula 9】 is independently optionally substituted by one, two or three Rs each ｃ respectively R ３ is selected from H and D, Each R ａ is independently selected from F, Cl, Br, I, OH, NH ２ , C １－３ alkyl, C １－３ alkoxy, C ２－４ alkenyl, C ２－４ alkynyl, -C １－３ alkyl - cyclopropyl and cyclopropyl, and the C １－３ alkyl, C １－３ alkoxy, C ２－４ alkenyl, C ２－４ alkynyl, cyclopropyl and -C １－３ alkyl - cyclopropyl may each be optionally substituted by one, two or three Rs, R ｂ is selected from H, CN, CH ３ and OCH ３ and is selected from Each R ｃ is independently selected from F, Cl, Br, I, CH ２ F, CHF ２ CF ３ and CH ２ CF ３ and is selected from Each R is independently F, Cl, Br, I, CH ２ F, CHF ２ and CF ３ selected from.)

4. Each R ａ is independently F, Cl, OH, NH ２ , CH ３ , CH ２ CH ３ , CH(CH ３ ), ２ , OCH ３ , OCH ２ CH ３ , OCH(CH ３ ), ２ , 【Chemical Formula 10】 and selected from cyclopropyl, said CH ３ 、CH ２ CH ３ 、CH(CH ３ ) ２ 、OCH ３ 、OCH ２ CH ３ 、OCH(CH ３ ) ２ 、 【Chemical 11】 And cyclopropyl is each independently optionally substituted by one, two or three Rs, the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

5. Each R ａ is independently F, Cl, OH, NH ２ , CH ３ , CHF ２ , CF ３ , CH ２ CF ３ , CH(CH ３ )CF ３ , OCH ３ , OCF ３ , 【Chemical 12】 selected from, the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

6. R １ is 【Chemical Formula 13】 selected from, the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

7. R １ is 【Chemical 14】 selected from, said 【Chemical 15】 which are each independently 1, 2, 3 or 4 Rs ａ The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, which is optionally substituted by

8. R １ is 【Chemical 16】 selected from, the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

9. R １ is 【Chemical 17】 selected from, the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

10. R ２ is 【Chemical Formula 18】 selected from, the compound according to claim 3 or a pharmaceutically acceptable salt thereof.

11. R ２ is 【Chemical Formula 19】 selected from, the compound according to claim 3 or a pharmaceutically acceptable salt thereof.

12. X is selected from N, the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof.

13. The compound is selected from the following formulas, the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof. 【Chemical 20】 (However, R １ and R ３ are as defined in claim 1.)

14. The compound is selected from the following formulas, the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof. 【Chemical 21】 (However, X, R １ and R ３ are as defined in claim 1.)

15. The compound is selected from the following formulas, the compound according to claim 3 or a pharmaceutically acceptable salt thereof. 【Chemical 22】 (However, R １ and R ３ are as defined in claim 3.)

16. A compound represented by the following formula or a pharmaceutically acceptable salt thereof. 【Chemical 23】 【Chemical 24】 【Chemical 25】 【Chemical 26】

17. selected from the following formulas, the compound according to claim 16 or a pharmaceutically acceptable salt thereof. 【Chemical 27】 【Chemical formula 28】 【Chemical formula 29】 【Chemical Formula 30】 【Chemical 31】 【Chemical 32】 【Chemical 33】 【Chemical 34】 【Chemical 35】 【Chemical Formula 36】 【Chemical 37】 【Chemical Formula 38】 【Chemical Formula 39】 【Chemical 40】 【Chemical 41】 【Chemical 42】 【Chemical 43】 【Chemical 44】 【Chemical 45】 【Chemical Formula 46】 【Chemical 47】 【Chemical 48】 【Chemical 49】 【Chemical Formula 50】 【Chemical Formula 51】 【Chemical Formula 52】 【Chemical 53】 【Chemical 54】 【Chemical 55】 【Chemical 56】 【Chemical 57】 【Chemical Formula 58】 【Chemical Formula 59】 【Chemical 60】 【Chemical Formula 61】 【Chemical Formula 62】 【Chemical Formula 63】 【Chemical Formula 64】 【Chemical Formula 65】 【Chemical Formula 66】 【Chemical 67】 【Chemical Formula 68】 【Chemical Formula 69】 【Chemical 70】 【Chemical Formula 71】 【Chemical Formula 72】 【Chemical 73】 【Chemical 74】 【Chemical 75】 【Chemical 76】 【Chemical 77】 【Chemical 78】 【Chemical Formula 79】 【Chemical 80】 【Chemical Formula 81】 【Chemical 82】 【Chemical 83】 【Chemical 84】 【Chemical 85】 【Chemical 86】 【Chemical 87】 【Chemical 88】 【Chemical 89】 【Chemical Formula 90】 【Chemical Formula 91】 【Chemical Formula 92】 【Chemical Formula 93】 【Chemical Formula 94】 【Chemical Formula 95】 【Chemical Formula 96】 【Chemical 97】 【Chemical Formula 98】 【Chemical Formula 99】 【Chemical 100】 【Chemical 101】 【Chemical 102】 【Chemical Formula 103】 【Chemical 104】 【Chemical 105】 【Chemical 106】 【Chemical 107】 【Chemical 108】 【Chemical 109】 【Chemical 110】 【Chemical 111】 【Chemical 112】 【Chemical 113】 【Chemical 114】 【Chemical 115】 【Chemical 116】 【Chemical 117】 【Chemical 118】 【Chemical 119】 【Chemical 120】 【Chemical 121】 【Chemical 122】 【Chemical 123】 。

18. The compound is selected from the following formulas, the compound according to claim 17 or a pharmaceutically acceptable salt thereof. 【Chemical 124】 【Chemical 125】

19. KRAS Ｇ１２Ｄ Use of a compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof in the manufacture of a compound for treating solid tumors having a mutation.

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