Piperazine bridge substituted heterocyclic pyrimidine compound

Abstract

The invention discloses a piperazine bridge substituted heterocyclic pyrimidine compound or a pharmaceutically acceptable salt thereof, and particularly discloses a compound as shown in a formula (III-1) or a pharmaceutically acceptable salt thereof. # imgabs0 #

Description

Piperazine bridge substituted heterocyclic pyrimidine compounds

[0001] The present invention claims the following priority:

[0002] Application number: CN202210822828.X, filing date: July 12, 2022;

[0003] Application number: CN202211262711.7, filing date: October 14, 2022;

[0004] Application number: CN202211407253.1, filing date: November 10, 2022;

[0005] Application number: CN202310041534.8, filing date: January 11, 2023;

[0006] Application number: CN202310041762.5, filing date: January 12, 2023;

[0007] Application number: CN202310066868.0, filing date: January 19, 2023;

[0008] Application number: CN202310800516.3, application date: June 30, 2023. Technical Field

[0009] The present invention relates to a class of piperazine bridge-substituted heterocyclic pyrimidine compounds or pharmaceutically acceptable salts thereof, and specifically to a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof. Background Art

[0010] Cancers caused by mutations in NRAS, HRAS, and KRAS in the RAS family account for nearly a quarter of all human cancers, making it one of the most common gene mutations associated with cancer. It covers almost all types of cancer and causes 1 million deaths worldwide each year. Among them, KRAS is the most common oncogene (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). Depending on the specific mutations present, G12C, G12D, and G12R are the most common KRAS mutations in patients. In addition, there are G12A, G12S, G12V, etc.

[0011] RAS (Rat Sarcoma) family proteins are widely expressed in various eukaryotic organisms and have two forms: an inactive form bound to GDP (guanosine diphosphate) and an activated form bound to GTP (guanosine triphosphate). By switching between these two forms, RAS proteins regulate multiple downstream pathways, including RAF-MEK-ERK, PI3K / Akt / mTOR, thereby influencing cell growth and differentiation, as well as tumor development and progression.

[0012] Because mutant KRAS has a high affinity for guanosine triphosphate (GTP), coupled with difficult-to-target factors such as a small catalytic site and smooth protein surface, the development of small molecule inhibitors has been challenging, contributing to the legendary "undruggable" status of KRAS. With Mirati's breakthrough in developing a non-covalent inhibitor of KRAS G12D, tumors harboring KRAS G12D mutations are gradually entering the field of precision medicine.

[0013] Summary of the Invention

[0014] The present invention provides a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof,

[0015] in,

[0016] X is selected from CH, C-Rx, N and N + -O - Preferably, X is selected from N and N + -O - ; Rx is selected from F, Cl, Br;

[0017] R1 is selected from described are independently optionally replaced by 1, 2, 3 or 4 R a replace;

[0018] R3 is selected from H and D;

[0019] Each R a are independently selected from F, Cl, Br, I, OH, NH2, C 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Halogenated alkyl, C 2-4 Alkenyl, C 2-4 Alkynyl, -C 1-3 Alkyl-cyclopropyl and cyclopropyl, the 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 with 1, 2 or 3 R;

[0020] Each R is independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

[0021] The present invention provides a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof,

[0022] in,

[0023] X is selected from CH, N and N + -O - Preferably, X is selected from N and N + -O - ;

[0024] R1 is selected from described are independently optionally replaced by 1, 2, 3 or 4 R a replace;

[0025] R3 is selected from H and D;

[0026] Each R a are 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, 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 with 1, 2 or 3 R;

[0027] Each R is independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

[0028] The present invention also provides a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof,

[0029] in,

[0030] X is selected from N and N + -O - ;

[0031] R1 is selected from described are independently optionally replaced by 1, 2, 3 or 4 R a replace;

[0032] R3 is selected from H and D;

[0033] Each R a are 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, 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 with 1, 2 or 3 R;

[0034] Each R is independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

[0035] The present invention also provides a compound represented by formula (III-2) or a pharmaceutically acceptable salt thereof,

[0036] in,

[0037] 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 R a replace;

[0038] R2 is selected from described Each independently optionally replaced by 1, 2 or 3 R c replace;

[0039] R3 is selected from H and D;

[0040] Each R a are 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, 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 with 1, 2 or 3 R;

[0041] R b is selected from H, CN, CH3 and OCH3;

[0042] Each R c Each independently selected from F, Cl, Br, I, CH2F, CHF2, CF3 and CH2CF3;

[0043] Each R is independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

[0044] The present invention also provides a compound represented by formula (II) or a pharmaceutically acceptable salt thereof,

[0045] in,

[0046] 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 R a replace;

[0047] R2 is selected from described Each independently optionally substituted by 1, 2 or 3 R c replace;

[0048] R3 is selected from H and D;

[0049] Each R a are 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, 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 with 1, 2 or 3 R;

[0050] R b is selected from the group consisting of H, CN, CH3 and OCH3;

[0051] Each R c Each independently selected from F, Cl, Br, I, CH2F, CHF2, CF3 and CH2CF3;

[0052] Each R is independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

[0053] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of:

[0054] wherein X, R1 and R3 are as defined in the present invention.

[0055] In some embodiments of the present invention, each R a Each independently selected from F, Cl, OH, NH2, CH3, CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH(CH3)2, and cyclopropyl, said CH3, CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH(CH3)2, and cyclopropyl are each independently optionally substituted with 1, 2 or 3 R, and other variables are as defined herein.

[0056] In some embodiments of the present invention, each R a Each independently selected from F, Cl, OH, NH2, CH3, CHF2, CF3, CH2CF3, CH(CH3)CF3, OCH3, OCF3, Other variables are as defined in the present invention.

[0057] In some embodiments of the present invention, the R1 is selected from Other variables are as defined in the present invention.

[0058] In some embodiments of the present invention, the R1 is selected from described are independently optionally replaced by 1, 2, 3 or 4 R a Substitution, other variables are as defined in the present invention.

[0059] In some embodiments of the present invention, the R1 is selected from Other variables are as defined in the present invention.

[0060] In some embodiments of the present invention, the R1 is selected from Other variables are as defined in the present invention.

[0061] In some embodiments of the present invention, the R1 is selected from Other variables are as defined in the present invention.

[0062] In some embodiments of the present invention, the R2 is selected from Other variables are as defined in the present invention.

[0063] In some embodiments of the present invention, the R2 is selected from Other variables are as defined in the present invention.

[0064] In some embodiments of the present invention, the R2 is selected from Other variables are as defined in the present invention.

[0065] In some embodiments of the present invention, R3 is selected from H and D, and other variables are as defined herein.

[0066] In some embodiments of the present invention, X is selected from N, and other variables are as defined herein.

[0067] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from

[0068] wherein R1, R2 and R3 are as defined in the present invention.

[0069] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of:

[0070] wherein R1, R2 and R3 are as defined in the present invention;

[0071] The condition is that R2 is not

[0072] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of:

[0073] wherein R1 and R3 are as defined in the present invention.

[0074] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of:

[0075] wherein R1 and R3 are as defined in the present invention.

[0076] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of:

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

[0078] In Examples 2 and 3 of the present invention, compound 3 was prepared from compound 2-4A by the steps described in Examples 2 and 3, wherein the product corresponding to the smaller Rf value was compound 2-4A, and the product corresponding to the larger Rf value was its isomer.

[0079] In Examples 2 and 3 of the present invention, compound 3 was prepared from compound 2-4A by the steps described in Examples 2 and 3, wherein the Rf value of compound 2-4A was 0.5 and the Rf value of its isomer was 0.55 after two developments by TLC thin layer chromatography (petroleum ether:acetone = 5:1).

[0080] In Examples 2 and 3 of the present invention, compound 3 was prepared from compound 2-4A by the steps described in Examples 2 and 3. LCMS (chromatographic column: Agilent Poroshell 120EC-C18 2.7 μm 3.0*30 mm, mobile phase: A: water (0.037% formic acid)-B: acetonitrile (0.0187% formic acid); B: 5%-95%) showed that the retention time of 2-4A was 0.776 min, and the retention time of its isomer was 0.801 min.

[0081] In Example 3 of the present invention, compound 3 was prepared from compound 3-2A by the steps described in Example 3, wherein 3-2A was analyzed by SFC (chromatographic column: Cellulose 2 100 mm*4.6 mm, 3 μm, mobile phase: [A (supercritical carbon dioxide), B (methanol (0.05% diethanolamine))]; B: 40%) with a retention time of 4.964 min and a retention time of 8.382 min for its enantiomer.

[0082] In Example 3 of the present invention, compound 3 was analyzed by SFC (chromatographic column: Chiralcel OJ-3 100*4.6 mm, 3 μm; mobile phase: A (supercritical carbon dioxide) and B (ethanol, containing 0.05% diethylamine); gradient: B%: 40%-40%) with a retention time of 3.761 min.

[0083] The present invention also provides the compound and pharmaceutically acceptable salts thereof,

[0084] Compound 3 was analyzed by SFC (chromatographic column: Chiralcel OJ-3 100*4.6 mm, 3 μm; mobile phase: A (supercritical carbon dioxide) and B (ethanol, containing 0.05% diethylamine); gradient: B%: 40%-40%), showing a retention time of 3.761 min.

[0085] The present invention also provides the compound and pharmaceutically acceptable salts thereof,

[0086] Among them, compound 3 was analyzed by chiral HPLC (chromatographic column: FLM Chiral NQ, 150*4.6mm, 3μm; mobile phase: A: (n-hexane) and B: (ethanol, containing 0.2% diethylamine, v / v); gradient: B%: isocratic 30%; elution time: 60min; column temperature: 35°C) and showed a retention time of 17.387min.

[0087] In some embodiments of the present invention, the three stereoisomers of compound 3 were analyzed by chiral HPLC (chromatographic column: FLM Chiral NQ, 150*4.6mm, 3μm; mobile phase: A: (n-hexane) and B: (ethanol, containing 0.2% diethylamine, v / v); gradient: B%: isocratic 30%; elution time: 60min; column temperature: 35°C) to show retention times of 22.587min, 26.205min and 44.765min.

[0088] In one technical solution of the invention, the present invention provides compound 7A2 or a pharmaceutically acceptable salt thereof; the compound 7A2 has a chemical structure selected from the following structures, and has a retention time of 2.236 min under analytical SFC conditions (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).

[0089] The present invention also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof:

[0090] The present invention provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof:

[0091] In some embodiments of the present invention, the compound or a pharmaceutically acceptable salt thereof is selected from

[0092] The present invention provides the compound or a pharmaceutically acceptable salt thereof for use in the preparation of a method for treating KRAS G12D Application of drugs in solid tumors with mutations.

[0093] The present invention provides a pharmaceutical composition comprising a compound represented by any one of the general chemical 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.

[0094] The present invention provides the use of the compound represented by any one of the general chemical 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 preparation of a drug for treating solid tumors with KRAS G12D mutation; preferably, the solid tumor is selected from colon cancer and pancreatic cancer.

[0095] The present invention also provides use of the aforementioned pharmaceutical composition in a drug for treating solid tumors with KRAS G12D mutation; preferably, the solid tumor is selected from colon cancer and pancreatic cancer.

[0096] The present invention also provides the following synthesis method:

[0097] Synthesis Route 1:

[0098] Synthesis route 2:

[0099] Synthesis route 3:

[0100] wherein R3 is selected from H, F and Cl; R 1a Selected from R1 is selected from Synthesis Route 4:

[0101] wherein R3 is selected from H, F and Cl; R 1a Selected from R1 is selected from

[0102] The present invention also provides the following test method:

[0103] Test Method 1. KRAS G12D Inhibitory activity test

[0104] 1. Experimental purpose:

[0105] Through the TR-FRET method, the KRAS inhibitors were screened. G12D Compounds that bind to GTP.

[0106] 2. Consumables and instruments:

[0107] Table 1. Consumables and instruments

[0108] 3. Reagent Preparation:

[0109] a. Storage of reagents:

[0110] 1) KRAS nucleotide exchange buffer

[0111] Prepare 1 L of solution by adding 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. Sterilize by filtration and store at 4°C.

[0112] 2) KRAS assay buffer

[0113] Prepare 1 L of solution by adding 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. Sterilize by filtration and store at 4°C.

[0114] 3) KRAS / Bodipy GDP / Tb-SA mixture

[0115] Take 9.5 μL of 95 μM KRAS G12D Protein was mixed with 440.5 μL KRAS nucleotide exchange buffer, incubated at room temperature for 1 hour, and then mixed with 8.4 μL 17.9 μM Tb-SA, 1.8 μL 5 mM Bodipy GDP, and 9539.8 μL KRAS assay buffer to make a 1 L solution. After mixing, it was allowed to stand at room temperature for 6 hours and stored at -80 °C.

[0116] b. Experimental reagents:

[0117] 1) KRAS enzyme solution

[0118] Take 73.3 μL of KRAS / Bodipy GDP / Tb-SA mixture and 2126.7 μL of KRAS assay buffer to prepare 2200 μL solution.

[0119] 2) SOS / GTP mixture

[0120] Take 1.59 μL of 166 μM SOS protein, 198 μL of 100 mM GTP, and 2000.41 μL of KRAS assay buffer to prepare a 2200 μL solution.

[0121] 4. Experimental process:

[0122] 1) The concentration of the control compound stock solution is 1 mM, and the concentration of the test compound stock solution is 10 mM. Transfer 9 μL of the control compound and the test compound to a 384-LDV plate;

[0123] 2) Use Bravo to make 10-point 3-fold dilutions of the compounds on the LDV plate;

[0124] 3) Use ECHO to transfer 9 nL of the compound on the LDV plate to the experimental plate;

[0125] 4) Use a Dragonfly automated pipette to add 3 μL of a 3 nM Kras / 0.5 nM TB-SA / 30 nM BodipyGDP mixture and 3 μL of Ras buffer to each well of the assay plate. Centrifuge the plate at 1000 rpm / min for 1 minute.

[0126] 5) Incubate the assay plate at room temperature for 1 hour;

[0127] 6) Use a Dragonfly automated pipette to add 3 μL of a 120 nM SOS / 9 mM GTP mixture to each well of the assay plate and centrifuge the plate at 1000 rpm / min for 1 minute.

[0128] 7) Incubate the assay plate at room temperature for 1 hour;

[0129] 8) Use Envision to read the plate and record the data;

[0130] 9) Use Excel and Xlfit to analyze data and calculate the IC value of the test compound 50 .

[0131] Test method 2. GP2D cell p-ERK inhibition test

[0132] 1. Experimental purpose:

[0133] Using the HTRF method, compounds that can effectively inhibit p-ERK in GP2D cells were screened.

[0134] 2. Experimental process:

[0135] 1) GP2D cells were seeded in a transparent 96-well cell culture plate, with 80 μL of cell suspension per well (8,000 cells per well). The plate was placed in a CO2 incubator and incubated overnight at 37°C.

[0136] 2) Add 2 μL of compound to 78 μL of cell culture medium, mix thoroughly, and then add 20 μL of compound solution to the corresponding wells of the cell plate. Return the cell plate to the CO2 incubator and continue incubation for 1 hour.

[0137] 3) After incubation, discard the cell supernatant and add 50 μL of 1X cell lysis buffer to each well. Incubate at room temperature with shaking for 30 minutes.

[0138] 4) Dilute Phospho-ERK1 / 2 Eu Cryptate antibody and Phospho-ERK1 / 2d2 antibody 20-fold using detection buffer;

[0139] 5) Transfer 16 μL of cell lysate supernatant to each well of a new 384-well white microplate, then add 2 μL of Phospho-ERK1 / 2 Eu Cryptate antibody dilution and 2 μL of Phospho-ERK1 / 2 d2 antibody dilution, and incubate at room temperature for at least 4 hours.

[0140] 6) After incubation, use a multi-label analyzer to read HTRF excitation: 320nm, emission: 615nm, 665nm;

[0141] 7). Calculate the IC of the test compound 50 .

[0142] Test Method 3. GP2D 3D CTG Experiment

[0143] 1. Experimental purpose:

[0144] This experiment aims to verify the inhibitory effect of the compound of the present invention on the proliferation of GP2D human colon cancer cells with KRAS G12D mutation.

[0145] 2. Experimental Materials:

[0146] Cell line GP2D, DMEM medium, penicillin / streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. 3D Cell Viability Assay (3D cell viability chemiluminescence detection reagent) was purchased from Promega.

[0147] 3. Experimental methods:

[0148] GP2D cells were seeded in a 96-well U-bottom cell culture plate, with 80 μL of cell suspension per well, containing 2,000 GP2D cells. The cell plate was cultured overnight in a CO2 incubator. The test compound was diluted 5-fold using a pipette to the eighth concentration, from 200 μM to 2.56 nM, in duplicate wells. 78 μL of culture medium was added to the middle plate, and then 2 μL of the serially diluted compound was transferred to each well of the middle plate according to the corresponding position. After mixing, 20 μL of each well was transferred to the cell plate. The concentration of the compound transferred to the cell plate ranged from 1 μM to 0.0128 nM. The cell plate was cultured in a CO2 incubator for 5 days. After the incubation of the cell plate with the compound added, 100 μL of cell viability chemiluminescent detection reagent was added to each well of the cell plate and incubated at room temperature for 10 minutes to stabilize the luminescent signal. The data were read using a multi-label analyzer.

[0149] 4. Data Analysis:

[0150] The raw data were converted into inhibition rate, IC, using the equation (Sample-Min) / (Max-Min)*100%. 50 The value can be obtained by four-parameter curve fitting (obtained in "log(inhibitor)vs.response--Variable slope" mode in GraphPad Prism).

[0151] Test Method 4. In vivo pharmacokinetic experiments

[0152] 1. Experimental purpose:

[0153] The purpose of this experiment is to investigate the pharmacokinetic characteristics of the compound of the present invention in SD mice after oral and intravenous administration.

[0154] 2. Experimental methods:

[0155] The test compound was mixed with a 10% dimethyl sulfoxide / 60% polyethylene glycol 400 / 30% aqueous solution, vortexed and sonicated to prepare a clear solution of approximately 1 mg / mL, which was filtered through a microporous membrane and then used for further preparation. Male SD mice aged 7 to 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 approximately 30 mg / kg. Whole blood was collected over a certain period of time to prepare plasma, and the drug concentration was analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA).

[0156] Test Method 5. In vivo pharmacodynamics experiment

[0157] 1. Experimental purpose:

[0158] In vivo pharmacodynamic study of human colon cancer GP2D cells in a Balb / c nude mouse model with subcutaneous transplantation of tumors.

[0159] 2. Experimental methods:

[0160] Cell culture: Human colon cancer GP2D cells were cultured as monolayers in DMEM / F12 medium supplemented with 20% fetal bovine serum and 1% double-antibody in a 37°C incubator with 5% CO2. Cells were routinely digested and passaged twice a week using trypsin-EDTA. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and resuspended in an appropriate amount of PBS. Matrigel was added at a 1:1 ratio to obtain a cell density of 25 x 10 6 cells / mL of cell suspension.

[0161] Cell inoculation: 0.2 mL (5 × 10 6 cells / mouse) Mia PaCa-2 cells (with Matrigel, volume ratio of 1:1) were subcutaneously inoculated into the right back of each mouse.

[0162] Experimental operation: The average tumor volume reached about 190mm 3 The mice were randomly divided into groups according to the tumor volume, with 6 mice in each group. The blank group was given 0 dose, and the test groups were given 30 mg / kg and 100 mg / kg doses, respectively. The administration volume was 10 μL / g, and the drugs were administered orally for 22 days, twice a day.

[0163] 3. Tumor Measurement and Experimental Indicators:

[0164] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0165] The anti-tumor efficacy of the compound was evaluated using 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). Relative tumor volume (RTV) was calculated based on the tumor measurement results using the formula RTV = V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., D0), V t It is the average tumor volume at a certain measurement. TRTV and CRTV data are taken on the same day.

[0166] TGI (%) reflects the rate of tumor growth inhibition. TGI (%) = [1 - (average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the start of drug administration) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the start of treatment)] × 100%.

[0167] Technical Effects

[0168] The compounds of the present invention and KRAS G12D Protein has good binding effect and can significantly inhibit KRAS G12D enzyme, GP2D cell p-ERK, the compounds of the present invention have an effect on KRAS G12D The mutated cells have good cell proliferation inhibitory activity and excellent tumor suppression effect. In addition, the compound of the present invention has good pharmacokinetic characteristics.

[0169] Related definitions

[0170] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0171] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0172] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, which are prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases, having specific substituents. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the compounds with a sufficient amount of base in neat solution or a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the compounds with a sufficient amount of acid in neat solution or a suitable inert solvent. Certain specific compounds of the present invention contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0173] Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of the two.

[0174] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0175] Unless otherwise noted, the retention times of compounds in chiral HPLC (chiral high-performance liquid chromatography) and SFC (supercritical fluid chromatography) analyses may vary due to factors such as the instrument used. For any particular compound, the retention time of the compound may be subject to measurement error. Therefore, this error should be taken into account when determining the retention time of each compound, and any error within this error is within the scope of this application.

[0176] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C). For example, deuterated drugs can be formed by replacing hydrogen with heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of this invention.

[0177] The terms "optional" or "optionally" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0178] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or unsubstituted, and unless otherwise specified, the type and number of substituents can be any on the basis of chemical achievable.

[0179] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and / or their variants are permissible only if such combinations result in stable compounds.

[0180] When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0181] When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0182] When the listed linking groups do not specify their linking direction, their linking direction is arbitrary, for example, The connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of reading from left to right to form Combinations of linkers, substituents, and / or variations thereof are permissible only if such combinations result in stable compounds.

[0183] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the chemical bond connection mode is non-positional and there are H atoms at the connectable sites, when the chemical bond is connected, the number of H atoms at the site will decrease accordingly with the number of connected chemical bonds, and become a group with a corresponding valence. The chemical bond connecting the site to other groups can be a straight solid bond. Straight dotted key or wavy lines For example, the straight solid bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group; The straight dashed bond in the group indicates that the two ends of the nitrogen atom in the group are connected to other groups; The wavy line in the phenyl group indicates that it is connected to other groups through the carbon atoms at positions 1 and 2 in the phenyl group; Indicates that any linkable site on the piperidinyl group can be connected to other groups through a chemical bond, including at least In these four connection methods, even if an H atom is drawn on -N-, Still includes For groups connected in this way, when one chemical bond is connected, the H at that site will be reduced by one and become a corresponding monovalent piperidine group.

[0184] Unless otherwise specified, use a solid wedge key. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed key Indicate the relative configuration of stereocenters with a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Indicates a straight solid bond or straight dashed key For example, Use straight solid key and straight dashed key Indicates the relative configuration of the stereocenter, representing mixture. represent mixture of represent

[0185] Certain compounds of the present invention may exist as atropisomers, which are conformational isomers that occur when rotation about a single bond in a molecule is prevented or greatly slowed due to steric interactions with other parts of the molecule. The compounds disclosed herein include all atropisomers, either pure individual atropisomers, or enriched in one atropisomer, or nonspecific mixtures of each. If the rotational potential about the single bond is high enough and the interconversion between conformations is slow enough, separation of the isomers may be permitted. For example, and is a pair of atropisomers, in which the phenyl Indicates that the side is facing outward. Indicates that the three-dimensional orientation of this side is inward.

[0186] Unless otherwise specified, when a compound contains a double bond structure, such as a carbon-carbon double bond, a carbon-nitrogen double bond, and a nitrogen-nitrogen double bond, and each atom on the double bond is connected to two different substituents (in a double bond containing a nitrogen atom, a lone pair of electrons on the nitrogen atom is regarded as a substituent to which it is connected), if the atoms on the double bond in the compound and their substituents are separated by a denoted by , it represents the (Z) isomer, (E) isomer or a mixture of both isomers of the compound.

[0187] Unless otherwise specified, Cn-n+m or C n -C n+m Any specific case including n to n+m carbons, such as C 1-12 Including C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 、C 11 , and C 12 , also includes any range from n to n+m, such as C 1-12 Including 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 Similarly, n-membered to n+m-membered means that the number of atoms in the ring is n to n+m, for example, a 3-12-membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, an 11-membered ring, and a 12-membered ring, and also includes any range from n to n+m, for example, a 3-12-membered ring includes a 3-6-membered ring, a 3-9-membered ring, a 5-6-membered ring, a 5-7-membered ring, a 6-7-membered ring, a 6-8-membered ring, and a 6-10-membered ring, etc.

[0188] Unless otherwise specified, the term “C 1-3 "Alkyl" is used to represent a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. 1-3 Alkyl groups include C 1-2 and C 2-3 Alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). 1- Examples of 3-alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0189]

[0026] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0190] Unless otherwise specified, the term “C 1-3 "Alkoxy" refers to those alkyl groups containing 1 to 3 carbon atoms which are attached to the rest of the molecule via an oxygen atom. The C 1-3 Alkoxy groups include C 1-2 、C 2-3 , C3 and C2 alkoxy, etc. 1-3 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

[0191] Unless otherwise specified, “C 2-4 "Alkenyl" is used to refer to a linear or branched hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon double bond, which may be located at any position of the group. 2-4 Alkenyl groups include C 2-3 , C4, C3 and C2 alkenyl, etc.; the C 2-4 Alkenyl groups can be monovalent, divalent, or polyvalent. 2-4 Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, butadienyl, and the like.

[0192] Unless otherwise specified, “C 2-4 "Alkynyl" is used to represent a linear or branched hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon triple bond, which may be located at any position of the group. 2-4 Alkynyl groups include C 2-3 , C4, C3 and C2 alkynyl, etc. It can be monovalent, divalent or polyvalent. 2-4 Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, and the like.

[0193] Unless otherwise specified, in Formula III of the present invention, when X is N + -O - When "N + -O - ” is used to represent “N(→O)”, which means N oxide.

[0194] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0195] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is performed by collecting diffraction intensity data from a cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation and a φ / ω scanning mode. After collecting relevant data, the crystal structure can be further analyzed using a direct method (Shelxs97) to confirm the absolute configuration.

[0196] The solvents used in the present invention are commercially available. Boc represents tert-butyl carbonate; 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-diazobisspiro[5.4.0]undec-7-ene; the second-generation Grubbs catalyst represents the compound with CAS number 246047-72-3; the ratio of eluent in column chromatography represents the volume ratio; and M in the concentration represents mol / L.

[0197] Compounds are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names. BRIEF DESCRIPTION OF THE DRAWINGS

[0198] Figure 1. Compound A and KRAS G12D Protein binding pattern diagram;

[0199] Figure 2. Compound B and KRAS G12D Protein binding pattern diagram;

[0200] Figure 3. Compound C and KRAS G12D Protein binding pattern diagram;

[0201] Figure 4. Compound D and KRAS G12D Protein binding pattern diagram;

[0202] Figure 5. Compound E and KRAS G12D Protein binding pattern diagram;

[0203] Figure 6. Compound F and KRAS G12D Protein binding pattern diagram;

[0204] Figure 7. Compound G and KRAS G12D Protein binding pattern diagram;

[0205] Figure 8. Compound H and KRAS G12D Protein binding pattern diagram;

[0206] Figure 9. Compound I and KRAS G12D Protein binding pattern diagram;

[0207] Figure 10. Compound J and KRAS G12D Protein binding pattern diagram;

[0208] Figure 11. Compound K and KRASG12D Diagram of the protein binding pattern. DETAILED DESCRIPTION

[0209] The present invention is described in detail below by way of examples, but is not intended to limit the present invention in any way. While the present invention has been described in detail herein, and specific embodiments thereof have been disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0210] Calculation Example 1

[0211] The molecular docking process was performed using Maestro ( Glide SP in version 2017-2 [1] The crystal structure of KRAS_G12C from the PDB database PDB:6UT0 was selected, and Cys12 was simulated and mutated to Asp12. After energy optimization, it was used as the docking template. To prepare the protein, Maestro [2] The protein preparation wizard module was used to add hydrogen atoms and the OPLS3 force field was used. For ligand preparation, the three-dimensional structure of the molecule was generated using LigPrep and energy minimization was performed. [3] , the confgen module is used to sample the small molecule conformations. The ligand of 6UT0 is used as the center of mass to generate the A cube docking grid was created. Reference compounds were placed during the molecular docking process. The interaction types between the protein receptor and the ligand were analyzed. Based on the calculated docking score and binding mode, reasonable docking conformations were selected and saved, as shown in Figures 1 to 11.

[0212] [1]Glide, LLC, New York, NY, 2017.

[0213] [2]Maestro, LLC, New York, NY, 2017.

[0214] [3]LigPrep, LLC, New York, NY, 2017.

[0215] Conclusion: The compounds of the present invention have a strong affinity for KRAS G12D There is a better combination.

[0216] Synthesis of intermediates Int-3A and Int-3B

[0217] Step 1:

[0218] Under nitrogen protection, 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 allowed to proceed at -40°C for 0.5 hours. 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 the intermediate Int3-2. MS m / z: 488.9, 490.9 [M+1] + .

[0219] Step 2:

[0220] Under nitrogen, 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 reacted at 25°C for 15 hours. 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 the 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] + .

[0221] Step 3:

[0222] Intermediate Int3-3 was separated by preparative SFC (chromatographic column: REGIS (S,S) WHELK-O1 (250mm*25mm, 10μm); mobile phases: A (supercritical CO2) and B (isopropanol containing 0.1% ammonia); gradient: B%: 60%-60%, 10 min) to obtain intermediates Int-3A and Int-3B. Intermediate Int-3A: SFC analysis method: chromatographic column: REGIS (S,S) WHELK-O1 (50mm*4.6mm, 3.5μm); mobile phases: A (supercritical CO2) and B (isopropanol containing 0.1% isopropylamine); gradient: B%: 5%-5%, retention time: 1.780 min, ee value: 95.37%. 1H NMR (400MHz, CDCl3) δ = 7.32 (dd, J = 9.2, 2.0Hz, 1H), 5.02 (s, 1H), 4.94 (s, 1H), 4.38-4.2 5(m,4H),4.24-4.17(m,1H),3.66-3.48(m,3H),3.34-3.20(m,2H),2.93(d,J=17.2Hz,1 H),2.75(dd,J=8.8,4.0Hz,1H),2.47(d,J=18.4Hz,1H),1.95(t,J=5.6Hz,2H),1.89-1. 75(m,3H),1.60-1.55(m,2H),1.52(s,9H),0.75(d,J=3.2Hz,1H),0.52-0.45(m,1H).MS m / z:618.0[M+1] + .

[0223] Intermediate Int-3B: SFC analysis method: chromatographic column REGIS (S,S) WHELK-O1 (50mm*4.6mm, 3.5μm), mobile phase: A (supercritical CO2) and B (isopropanol, containing 0.1% isopropylamine); gradient: B%: 5%-5%, retention time of 1.914min, ee value of 96.62%. 1 H NMR (400MHz, CDCl3) δ = 7.33 (dd, J = 9.2, 2.0Hz, 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=4.4Hz,1H),0.89-0.65(m,1H),0.63-0.40(m,1H).MS m / z:618.0[M+1] + .

[0224] Synthesis of intermediate Int-4

[0225] Step 1:

[0226] Int4-1 (100 g, 397.67 mmol) was dissolved in acetic acid (300 mL) and cooled to 0°C. Concentrated sulfuric acid (390.03 g, 3.98 mol) was then added dropwise. A solution of sodium nitrite (54.87 g, 795.34 mmol) in water (50 mL) was then added dropwise. The mixture was allowed to react at 0°C for 1 hour, at which point the suspension became clear. A solution of potassium iodide (132.03 g, 795.34 mmol) in water (50 mL) was then added dropwise. The mixture was allowed to react at 0°C for 1 hour. 5000 mL of water was added and stirred for 10 minutes. The mixture was then filtered and rinsed five times with water. 500 mL of saturated sodium thiosulfate solution was added and stirred overnight. The mixture was then filtered again and washed five times with water (500 mL x 4). The filter cake was the intermediate Int4-2. 1 H NMR (400MHz, CDCl3) δ = 8.37 (d, J = 2.0 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H).

[0227] Step 2:

[0228] Intermediate Int4-2 (126.15 g, 348.15 mmol) was dissolved in ethanol (500 mL) and water (200 mL). Iron powder (38.88 g, 696.29 mmol) and ammonium chloride (37.25 g, 696.29 mmol) were then added and reacted at 80°C for 2 hours. The mixture was filtered, concentrated under reduced pressure, and extracted with ethyl acetate (500 mL x 2). The mixture was washed with saturated brine (500 mL x 6), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. The crude product was then purified by a rapid column chromatography (eluent: ethyl acetate / petroleum ether, ethyl acetate ratio 0-20%) to obtain intermediate Int4-3. MS (ESI) m / z: 331.8, 333.8 [M+1] + .

[0229] Step 3:

[0230] 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 stirred at 0°C for 0.5 hours. Intermediate Int4-3A (65.03 g, 415.21 mmol) was then added and the temperature was slowly raised to 25°C for 2 hours. 1000 mL of water was slowly added to the reaction solution to completely precipitate the product. The product was then filtered and the filter cake was rinsed with (250 mL*4) of water. The filter cake was then dried under vacuum at 50°C to obtain intermediate Int4-4. 1H NMR (400MHz, CDCl3) δ = 7.09 (d, J = 8.53Hz, 4H), 6.94-6.97 (m, 1H), 6.88 (d, J = 8.53Hz, 4H), 6.80 (d, J = 2.76Hz, 1H), 4.50 (s, 4H), 3.81 (s, 6H).

[0231] Step 4:

[0232] Intermediate Int4-4 (78.66 g, 137.36 mmol) was dissolved in N,N-dimethylformamide (280 mL). Cuprous iodide (130.80 g, 686.80 mmol) was added. After replacing the nitrogen atmosphere, the mixture was heated to 100°C. Intermediate 5-10A (211.11 g, 1.10 mol) was added and allowed to react for 0.5 hours. 500 mL of ethyl acetate and 500 mL of water were added, and the mixture was separated. The organic phase was washed five times with (1 L * 5) volumes of water and then with (1 L * 3) volumes of saturated brine. The aqueous phase was extracted again with 1 L of ethyl acetate, and the organic phase was separated again. The organic phase was washed with (1 L * 5) volumes of water and then with (1 L * 3) volumes of saturated sodium chloride solution. All organic phases were concentrated under reduced pressure to obtain a crude product. 300 mL of methanol was added to the crude product, stirred, and filtered. The filter cake was intermediate Int4-5. 1 H NMR (400MHz, CDCl3) δ = 7.05-7.13 (m, 4H), 6.96 (d, J = 3.01Hz, 1H), 6.84-6.92 (m, 4H), 6.80 (d, J = 2.76Hz, 1H), 4.50 (s, 4H), 3.81 (s, 6H).

[0233] Step 5:

[0234] Intermediate Int4-5 (0.098 g, 190.38 μmol), bis(pinacol boronate) (483.44 mg, 1.90 mmol), and potassium acetate (56.05 mg, 571.14 μmol) were added to anhydrous dioxane (2.5 mL), and the atmosphere was purged with nitrogen three times. 1,1-Bis(diphenylphosphino)diphenylferric palladium chloride (41.79 mg, 57.11 μmol) was added, and the atmosphere was again purged with nitrogen three times. The mixture was then heated to 110°C and reacted for 20 hours. 50 mL of water was added, and the mixture was extracted with 150 mL of ethyl acetate. The organic phase was separated and washed with 3 (50 mL) of saturated brine. The mixture was concentrated under reduced pressure to obtain the crude product, which was then separated by flash column chromatography (eluent: ethyl acetate: petroleum ether: 0-10%) to obtain intermediate Int-4. MS (ESI) m / z: 562.2 [M+1] + .

[0235] Synthesis of intermediate Int-5

[0236] Int5-1 (3 g, 13.37 mmol), pinacol borate (5.09 g, 20.05 mmol), and potassium acetate (2.62 g, 26.73 mmol) were dissolved in N,N-dimethylformamide (30 mL). The atmosphere was purged with nitrogen three times. 1,1-Bis(diphenylphosphino)ferrocenepalladium(II) dichloromethane complex (1.09 g, 1.34 mmol) was added, and the mixture was heated to 100°C and stirred for 6 hours. The reaction mixture was poured into 150 mL of water and extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed with saturated brine (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 to 3:1) to afford Int-5. 1 H NMR (400MHz, CDCl3) δ7.05 (dd, J=4.0, 2.8Hz, 1H), 6.87 (dd, J=7.6, 2.4Hz, 1H), 3.16-4.46 (m, 2H), 1.38 (s, 12H).

[0237] Example 1

[0238] Step 1: Preparation of intermediate 1-1A-2

[0239] Intermediate 1-1A-1 (120 g, 709 mmol) was dissolved in tert-butanol (1200 mL) and water (1200 mL). Potassium osmate dihydrate (10.4 g, 28.3 mmol) and N-methylmorpholine oxide (249 g, 2.13 mol) were then added sequentially. The reaction mixture was stirred at 45°C for 16 hours. The mixture was concentrated under reduced pressure to remove excess solvent, extracted with ethyl acetate (500 mL*2), and washed with saturated sulfite 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 the 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 (400MHz, CDCl3) δ4.23 (t, J = 3.6Hz, 2H), 3.55-3.58 (m, 2H), 3.35-3.32 (m, 2H), 2.87-2.83 (m, 2H), 1.45 (s, 9H).

[0240] Step 2: Preparation of Intermediate 1-1A-3

[0241] 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 dichloromethane (100 mL) was added and stirred for 0.5 hours. The organic phase was then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. Methyl tert-butyl ether (200 mL) was added at 25 ° C and stirred for 10 minutes, filtered and concentrated under reduced pressure to obtain a crude intermediate 1-1A-3.

[0242] Step 3: Preparation of Intermediate 1-1A-4

[0243] 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. The reaction system was then raised to 25 ° C and stirred for 16 hours. Saturated ammonium chloride solution (1000 mL) was added at 10 ° C to quench the reaction system and 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 give a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 1 / 0 to 0 / 1) to give intermediate 1-1A-4. 1 H NMR (400MHz, CDCl3) δ5.82-5.89(m,2H),5.32(t,J=11.6Hz,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=8.8Hz,1H),1.48(s,9H).

[0244] Step 4: Preparation of Intermediate 1-1A-5

[0245] Intermediate 1-1A-4 (80.0 g, 310 mmol) was dissolved in dichloromethane (1000 mL). The reaction system was then 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. The mixture 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 the 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. 1H NMR (400MHz, CDCl3) δ8.37(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).

[0246] Step 5: Preparation of Intermediate 1-1A-6

[0247] Intermediate 1-1A-5A (32.1 g, 238 mmol) was dissolved in DCE (850 mL), followed by the addition of 1,5-cyclooctadiene iridium chloride dimer (12.3 g, 18.3 mmol). 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 was heated to 25°C and stirred for 16 hours. The excess solvent was removed by concentration under reduced pressure to obtain the 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-6. 1 H NMR (400MHz, CDCl3) δ7.52-7.55(m,2H),7.30(t,J=7.2Hz,2H),7.22(t,J=7.2Hz,1H),5.94-6.03(m,2H), 5.10(t,J=19.2Hz,2H), 4.99(d,J=10.4Hz,2H), 3.51-3.61(m,4H), 3.33(t,J=13.6Hz,2H), 1.48(s,15H).

[0248] Step 6: Preparation of Intermediate 1-1A-7

[0249] 1-1A-6 (36.0 g, 50.4 mmol) was dissolved in toluene (900 mL), followed by the addition of the second-generation Grubbs catalyst (2.14 g, 2.52 mmol). The reaction system was heated to 125°C and stirred for 16 hours. The mixture was filtered, the filter cake was discarded, and the filtrate was concentrated under reduced pressure to obtain the 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] + . 1H NMR (400MHz, CDCl3) δ7.60(t,J=1.2Hz,2H),7.31(t,J=7.2Hz,2H),7.22(s,1H),5.96(t,J=9.2Hz, 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 = 6.0Hz, 6H).

[0250] Step 7: Preparation of Intermediate 1-1A-8

[0251] Intermediate 1-1A-7 (26.8 g, 81.6 mmol) was dissolved in methanol (201 mL), followed by the addition of hydrogen chloride / methanol (4 M, 67.3 mL). The reaction mixture was heated to 35°C and stirred for 16 hours. The pH of the reaction mixture was adjusted to 12, and the mixture was extracted with ethyl acetate (30.0 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford 1-1A-8. MS: m / z = 229.2, [M+1] + . 1 H NMR (400MHz, CDCl3) δ7.62(t,J=7.2Hz,2H),7.31(t,J=7.6Hz,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).

[0252] Step 8: Preparation of Intermediate 1-1A-9

[0253] Intermediate 1-1A-8 (18.6 g, 79.4 mmol) was dissolved in THF (190 mL), followed by the addition of 9-fluorenylmethyl chloroformate (20.5 g, 79.4 mmol) and sodium carbonate (25.2 g, 238.2 mmol). The mixture was stirred at 0°C for 1 hour. The mixture was extracted with ethyl acetate (50.0 mL*2) and 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 yield Intermediate 1-1A-9. MS: m / z = 451.3, [M+1] + . 1H NMR (400MHz, CDCl3) δ7.76 (d, J = 13.6Hz, 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=2Hz,2H),3.55-3.59(m,2H),3.15-3.23(m,2H),1.27(d,J=2.4Hz,6H).

[0254] Step 9: Preparation of Intermediate 1-1A-10

[0255] 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 hours. Water (20.0 mL) was added and the pH was adjusted to 9. Dichloromethane (20.0 mL) was then added for extraction. The mixture was 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 hours to obtain the trifluoroacetate salt of 1-1A-10. MS: m / z = 333.1, [M+1] + . 1 H NMR (400MHz, CDCl3) δ7.77(d,J=7.6Hz,2H),7.56(d,J=7.2Hz,2H),7.41(t,J=7.6Hz,2H),7.33(t,J=6Hz,2H),6.18-6.27(m, 2H), 4.38-4.42 (m, 2H), 4.23 (s, 1H), 3.88 (d, J = 2.0Hz, 2H), 3.82 (d, J = 2.4Hz, 1H), 3.72 (d, J = 2.0Hz, 1H), 3.21-3.60 (m, 2H).

[0256] Step 10: Preparation of Intermediate 1-1A-11

[0257] The trifluoroacetic acid salt of intermediate 1-1A-10 (1.00 g, 2.92 mmol) was dissolved in tetrahydrofuran (10.0 mL), followed by the addition of di-tert-butyl dicarbonate (764 mg, 3.50 mmol) and triethylamine (885 mg, 8.75 mmol) and 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] + .

[0258] Step 11: Preparation of Intermediate 1-1A

[0259] Intermediate 1-1A-11 (5.69 g, 12.59 mmol) was dissolved in ethanol (60.0 mL), followed by the addition of dimethylamine (34.4 g, 251.8 mmol). The reaction system was stirred at 25°C for 3 hours. The mixture 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 of the aqueous phase was adjusted to 9, filtered, and 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 (400MHz, CDCl3) δ6.22 (d, J = 10Hz, 2H), 4.40 (d, J = 38.8Hz, 2H), 2.89-3.01 (m, 2H), 2.40 (d, J = 13.2Hz, 2H), 1.49 (s, 9H).

[0260] Step 12: Preparation of Intermediate 1-3A-2

[0261] Intermediate 1-3A-1 (4 g, 17.02 mmol) was dissolved in AcOH (30 mL) and cooled to 0°C in an ice bath. Concentrated sulfuric acid (17.03 g, 170.21 mmol) was then added dropwise, followed by a solution of sodium nitrite (1.76 g, 25.53 mmol) in water (5 mL). Stirring was continued for 0.25 hours, followed by a solution of potassium iodide (4.24 g, 25.53 mmol) in water (5 mL). The mixture was moved to 25°C and reacted for 0.5 hours. Water (50 mL) was added to the reaction solution, which was filtered. The solid was washed with saturated sodium thiosulfate solution (2 x 40 mL) and 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.

[0262] Step 13: Preparation of Intermediate 1-3A-3

[0263] Intermediate 1-3A-2 (2.85 g, 8.24 mmol) and cuprous iodide (3.14 g, 16.48 mmol) were dissolved in N,N-dimethylformamide (45 mL). Methyl fluorosulfonyldifluoroacetate (6.33 g, 32.96 mmol) was then added and the mixture was allowed to react at 80°C for 1 hour. The reaction was continued for 0.75 hours. The reaction solution was cooled to 25°C, the insoluble matter was filtered off, and the mixture was rinsed with ethyl acetate (100 mL), washed with water (3 x 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.

[0264] Step 14: Preparation of Intermediate 1-3A-4

[0265] 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). The temperature was raised to 60°C and the reaction mixture was reacted for 2.5 hours. Ethyl acetate (80 mL) was added to the reaction mixture to dilute it. Insoluble matter was filtered off, and the mixture was washed with water (2 x 50 mL). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain Intermediate 1-3A-4. MS: m / z = 257.9, [M+1] + .

[0266] Step 15: Preparation of Intermediate 1-3A

[0267] Intermediate 1-3A-4 (1.4 g, 5.43 mmol), pinacol diboron (2.07 g, 8.14 mmol, 1.5 eq), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride dichloromethane complex (443.12 mg, 542.61 μmol), and potassium acetate (1.60 g, 16.28 mmol) were dissolved in dioxane (30 mL) and reacted at 85°C under nitrogen for 16 hours. The reaction solution was cooled to 25°C, the insoluble matter was filtered off, the solution was washed with ethyl acetate (50 mL), and the reaction mixture was 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] + .

[0268] Step 16: Preparation of Intermediate 1-2

[0269] Intermediate 1-1 (900 mg, 3.56 mmol) was dissolved in dichloromethane (10 mL) and cooled to 0°C. N,N-diisopropylethylamine (1.38 g, 10.69 mmol) and 1-1A (749.60 mg, 3.56 mmol) were then added sequentially. The mixture was reacted at 0°C for 1 hour. The mixture was then concentrated under reduced pressure to obtain crude product 1-2. MS: m / z = 426.0, [M+1] + .

[0270] Step 17: Preparation of Intermediate 1-3

[0271] Intermediate 1-2 (220 mg, 516.10 μmol) and 1-2A were dissolved in acetonitrile (10 mL), followed by the addition of N,N-diisopropylethylamine (200.10 mg, 1.55 mmol). The reaction mixture was heated to 80°C and stirred for 16 hours. The reaction mixture was concentrated under reduced pressure to remove most of the solvent, followed by the addition of ethyl acetate (10 mL) and water (5 mL), followed by extraction, drying over anhydrous sodium sulfate, and concentration. The crude product was isolated by silica gel column chromatography (PE / EA = 1 / 1 to DCM / MeOH = 20 / 1) to afford Intermediate 1-3. MS: m / z = 549.1, [M+1] + .

[0272] Step 18: Preparation of Intermediate 1-4

[0273] Intermediate 1-3A (0.2 g, 364.29 μmol), Intermediate 1-3 (222.27 mg, 728.58 μmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium 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 under nitrogen for 17 hours. The reaction solution was cooled to 25°C, the insoluble matter was filtered off, the solution was 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] + .

[0274] Step 19: Preparation of Compound 1

[0275] 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 continued for 0.5 hours. The reaction solution was concentrated to dryness, dissolved in dichloromethane (2 mL), and solid sodium bicarbonate (0.5 g) was added and stirred thoroughly. Ethyl acetate (5 mL) was added and stirred for 5 minutes. The insoluble matter was filtered off and concentrated. The crude product was separated by preparative HPLC (column: Boston Green ODS 150*30mm*5μm; flowability: [water (formic acid)-acetonitrile]; acetonitrile %: 10%-40%, 6 min) to obtain compound 1. MS (ESI) m / z: 592.3 [M+1] + . 1 H NMR (400MHz, CD3OD) δ9.01(s,1H),6.58(d,J=14Hz,1H),6.41(s,1H),6.34(s,2H),5.56(d,J=51Hz,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).

[0276] Example 2

[0277] Step 1: Preparation of intermediate 2-2

[0278] Intermediate 2-1 (50 g, 418.09 mmol) was dissolved in dichloromethane (500 mL), and triethylamine (84.61 g, 836.17 mmol) was added, followed by di-tert-butyl dicarbonate (100.37 g, 459.90 mmol). The mixture was allowed to react at 25°C for 16 hours. 100 mL of water was added to the reaction mixture, and the layers were 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 to 50 / 1) to obtain intermediate 2-2. MS: m / z = 206.1, [M+Na] + .

[0279] Step 2: Preparation of intermediate 2-3

[0280] Intermediate 2-2 was dissolved in anhydrous tetrahydrofuran (25 g, 136.43 mmol) (350 mL). 3,7-Dipropyl-3,7-diazabicyclo[3.3.1]nonane (37.31 g, 177.36 mmol) was added and cooled to -65°C. Sec-butyllithium (1.3 M, 157.42 mL) was slowly added dropwise. After reacting for 1 hour, methyl chloroformate (15.73 g, 166.44 mmol) was added dropwise. The mixture was reacted at -65°C for 2 hours. The reaction mixture was quenched by the addition of saturated ammonium chloride (20 mL) and extracted with ethyl acetate (300 mL x 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] + .

[0281] Step 3: Preparation of Intermediate 2-4A

[0282] Intermediate 2-3 (12 g, 49.73 mmol) was dissolved in THF (120 mL), and 3-chloro-2-chloromethylpropene (24.87 g, 198.94 mmol) was added. The mixture was cooled to -40°C, and lithium bistrimethylsilylamide (1 M, 99.47 mL) was slowly added dropwise. The temperature was gradually raised to 20°C and the reaction was allowed to react for 2 hours. The reaction solution was quenched by the dropwise addition of saturated ammonium chloride (20 mL) and extracted with ethyl acetate (150 mL x 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 to 10 / 1) to obtain Intermediate 2-4A. TLC (petroleum ether:acetone = 5:1) was performed twice. 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 on LCMS (column: Agilent Poroshell 120EC-C18 2.7 μm 3.0*30 mm, 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] + .

[0283] Step 4: Preparation of Intermediate 2-5

[0284] Intermediate 2-4A (3.6 g, 10.92 mmol) was dissolved in hydrogen chloride / ethyl acetate (4 M, 27.29 mL) and allowed to react at 20°C for 2 hours. The reaction solution was concentrated under reduced pressure to obtain the crude intermediate 2-5, which was used directly in the next step without purification. MS: m / z = 230.1, [M+H]+ .

[0285] Step 5: Preparation of Intermediate 2-6

[0286] 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 added. The mixture was allowed to react at 20°C for 2 hours. Dichloromethane (80 mL) was added to the reaction solution, filtered, and concentrated. The crude product was separated by silica gel column chromatography (PE / EA = 10 / 1-5 / 1) to obtain Intermediate 2-6. MS: m / z = 194.1, [M+H] + .

[0287] Step 6: Preparation of Intermediate 2-7

[0288] Intermediate 2-6 (1.6 g, 8.28 mmol) was dissolved in anhydrous tetrahydrofuran (20 mL), and lithium aluminum tetrahydride (628.51 mg, 16.56

[0289] mmol) and reacted at 0°C for 2 hours. The reaction solution was diluted with ethyl acetate (10 mL) and water (0.63 mL) dropwise, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude intermediate 2-7, which was used directly in the next step without purification. MS: m / z = 166.1 [M+H] + .

[0290] Step 7: Preparation of Intermediate 2-8

[0291] 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). Sodium tert-butoxide (473.45 mg, 4.93 mmol) was added and 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, and 10 mL of water was added to quench the mixture. The phases 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 / 1-2 / 1) to obtain Intermediate 2-8. MS: m / z = 555.1, [M+H] + .

[0292] Step 8: Preparation of Intermediates 2-9A and 2-9B

[0293] Intermediate 2-8 (0.1 g, 180.17 μmol, 1 eq) and Intermediate 1-3A (82.45 mg, 270.26 μmol) were dissolved in dioxane (2 mL) and water (0.5 mL). [n-Butyldi(1-adamantyl)phosphine](2-amino-1,1'-biphenyl-2-yl)palladium(II) methanesulfonate (13.12 mg, 18.02 μmol) and cesium carbonate (146.75 mg, 450.42 μmol) were added and reacted at 90°C for 16 hours. The reaction mixture was diluted with ethyl acetate (30 mL), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by silica gel column chromatography (PE / EA = 1 / 50-1 / 100) and then separated by preparative SFC (chromatographic 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 intermediates 2-9A and 2-9B. The retention time of intermediate 2-9A in analytical SFC (chromatographic column: (S,S) Whelk-01 100×4.6 mm ID, 5.0 μm, mobile phase: A: supercritical carbon dioxide, 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 analytical SFC (chromatographic column: (S,S) Whelk-01 100×4.6 mm ID, 5.0 μm, mobile phase: A: supercritical carbon dioxide, 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%.

[0294] Step 9: Preparation of compound 2A

[0295] 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 added. The mixture was allowed to react at 20°C for 2 hours. The reaction mixture was diluted with dichloromethane (20 mL), and saturated sodium bicarbonate (10 mL) was added. The phases were separated, and the aqueous phase was extracted with dichloromethane (10 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by preparative HPLC (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] + . 1H NMR(400MHz,CD3OD)δ9.07(s,1H),8.52(br s,1H),6.59(br d,J=14.05Hz,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=11.04Hz,1H),4.27(br s,2H),4.08(br d,J=15.31Hz,1H),3.94(br d,J=11.04Hz,2H),3.62(br d,J=15.81Hz,1H),3.53(d,J=10.54Hz,1H),3.00-3.14(m,2H),2.75(br d,J=16.31Hz,1H),1.86-1.98(m,1H),1.79(td,J=3.67,6.96Hz,1H),0.69-0.85(m,2H).

[0296] Step 10: Preparation of compound 2B

[0297] 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 added. The mixture was allowed to react at 20°C for 1 hour. The reaction mixture was diluted with dichloromethane (20 mL), and saturated sodium bicarbonate (10 mL) was added. The phases were separated, and 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 (column: O-Welch C18 150*30mm*5μm; 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] + . 1H NMR (400MHz, CD3OD) δ9.06 (s, 1H), 8.52 (br s, 1H), 6.59 (br d, J = 13.80Hz, 1H), 6.43 (s, 1H), 6.33 (s, 2H), 5.16 (br d,J=10.29Hz,2H),4.58-4.72(m,3H),4.48(d,J=11.29Hz,1H),4.25(br s,2H),4.07(br d,J=15.56Hz,1H),3.94(br d,J=11.04Hz,2H),3.61(br d,J=15.81Hz,1H),3.51(d,J=10.54Hz,1H),2.99-3.12(m,2H),2.74(br d,J=16.06Hz,1H),1.88-1.95(m,1H),1.72-1.85(m,1H),0.69-0.81(m,2H).

[0298] Example 3

[0299] Step 1: Preparation of intermediate 3-1

[0300] Intermediate 1-1 (25 g, 99.03 mmol) was dissolved in dichloromethane (200 mL) and cooled to -30°C. N,N-diisopropylethylamine (38.40 g, 297.08 mmol) and 1-1B (21.02 g, 99.03 mmol) were then added sequentially. The mixture was reacted at -30°C for 2 hours. The mixture was then concentrated under reduced pressure to obtain crude product 3-1. MS: m / z = 428.1 [M+1] + .

[0301] Step 2: Preparation of Intermediate 3-2A

[0302] Intermediate 3-1 (23 g, 53.70 mmol) and intermediate 2-7 (9.76 g, 59.07 mmol) were dissolved in anhydrous toluene (300 mL). Sodium tert-butoxide (13.93 g, 145.00 mmol) was slowly added at 0°C. The mixture was reacted at 0°C for 0.5 hour and then at 20°C for 0.5 hour. The reaction solution was diluted with 200 mL of ethyl acetate, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to afford crude product 3-2. The crude product was purified by preparative SFC (column: DAICEL CHIRALCEL OD (250 mm x 50 mm, 10 μm); mobile phase: [A (supercritical carbon dioxide), B (ethanol containing 0.1% ammonia)]; B: 40%) to afford products 3-2A and 3-2B. The retention time of 3-2A under the conditions of SFC analysis (chromatographic column: Cellulose 2 100mm*4.6mm, 3μm) mobile phase: [A (supercritical carbon dioxide), B (MeOH (0.05% DEA))]; B: 40%) was 4.964min, and the ee value was 95.7%. 1 H NMR(400MHz,CD3OD)δ8.76(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=9.5Hz,1H),2.78(br d,J=16.8Hz,1H),2.71(dd,J=4.0,9.6Hz,1H),2.45(br d,J=16.8Hz,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=4.2Hz,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%.

[0303] Step 3: Synthesis of Intermediate 3-4

[0304] Dissolve raw material 3-3 (2 g, 5.58 mmol) in dichloromethane (40 mL). Add N,N-diisopropylethylamine (4.33 g, 33.47 mmol) at 0-10°C under an ice bath, followed by the dropwise addition of trifluoromethanesulfonic anhydride (6.30 g, 22.31 mmol). Continue the reaction for 1.5 hours. Add water (20 mL) to the reaction mixture and stir thoroughly. The aqueous phase is separated, and the organic phase is dried over anhydrous sodium sulfate and concentrated. The crude product is isolated by column chromatography (mobile phase: petroleum ether:ethyl acetate = 10:1 to 2:1) to afford intermediate 3-4.

[0305] Step 4: Synthesis of Intermediate 3-5

[0306] Intermediate 3-4 (3 g, 4.82 mmol), benzophenone imine (1.75 g, 9.64 mmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (557.57 mg, 963.63 μmol), and cesium carbonate (4.71 g, 14.45 mmol) were dissolved in toluene (60 mL). Tris(dibenzylideneindeneacetone)dipalladium (441.21 mg, 481.82 μmol) was added and reacted at 100°C under nitrogen for 2 hours. The reaction solution was cooled to 25°C, the insoluble material was filtered off, and the majority of the toluene was removed. The solution was diluted with ethyl acetate (30 mL), washed with water (20 mL), dried over anhydrous sodium sulfate, and concentrated to obtain the 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.

[0307] Step 5: Synthesis of Intermediates 3-6

[0308] Intermediate 3-5 (3.1 g, 4.74 mmol), pinacol diboron (2.41 g, 9.48 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (693.88 mg, 948.30 μmol), and potassium acetate (1.40 g, 14.22 mmol) were dissolved in toluene (60 mL) and reacted at 110°C under nitrogen for 18 hours. The reaction solution was cooled to 25°C, the insoluble material was filtered off, and the mixture was 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] + .

[0309] Step 6: Synthesis of Intermediates 3-7

[0310] Intermediate 3-2A (11.3 g, 20.29 mmol) was added to water (60 mL) and anhydrous dioxane (240 mL). Then, [n-butyldi(1-adamantyl)phosphine](2-amino-1,1'-biphenyl-2-yl)palladium(II) methanesulfonate (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. The mixture was reacted at 87°C for 2 hours. The reaction mixture was diluted with ethyl acetate (200 mL), washed with saturated brine (40 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the 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] + .

[0311] Step 7: Synthesis of Intermediates 3-8

[0312] Intermediate 3-7 (8.4 g, 8.18 mmol) was added to ethyl acetate (30 mL) and water (10 mL), followed by the addition of hydrochloric acid / ethyl acetate (4 M, 62.37 mL) and allowed to react at 20°C for 2 hours. Water (30 mL) was added to the reaction solution, and the organic phase was washed with water (30 mL x 2). The aqueous phases were combined and the pH was adjusted to 7-8 with saturated sodium bicarbonate. The product was extracted with ethyl acetate (80 mL x 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 used directly in the next step. MS (ESI) m / z: 762.3 [M+1] + .

[0313] Step 8: Synthesis of compound 3

[0314] Intermediate 3-8 (6.2 g, 8.14 mmol) was added to acetonitrile (70 mL) and tetramethylammonium fluoride (2.29 g, 13.83 mmol). The mixture was reacted at 60°C for 1 hour. The reaction mixture was diluted with 100 mL of ethyl acetate, washed with 30 mL of saturated sodium bicarbonate, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography (mobile phase: dichloromethane (10% ammonia in methanol):methanol = 20:1 to 10:1) to obtain compound 3. SFC analysis (column: Chiralcel OJ-3 100*4.6 mm, 3 μm; mobile phase: A (supercritical carbon dioxide) and B (ethanol containing 0.05% diethylamine); gradient: B%:40%-40%) revealed a retention time of 3.761 min and an ee of 97.34%. Chiral HPLC analysis (column: FLM Chiral NQ, 150 x 4.6 mm, 3 μm; mobile phase: A: (n-hexane) and B: (ethanol containing 0.2% diethylamine, v / v); gradient: B%: isocratic to 30%; elution time: 60 min; column temperature: 35°C) showed a retention time of 17.387 min. MS (ESI) m / z: 606.3 [M+1] + , 1 H NMR (400MHz, CD3OD) δ=9.01(s,1H),7.76(dd,J=5.6,9.2Hz,1H),7.28-7.19(m,2H),7.15(d,J=2.3Hz,1H),5.08(br s,1H),5.01(br s,1H),4.70-4.54(m,3H),4.43(dd,J=7.3,10.0Hz,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=9.5Hz,1H),2.93(br d,J=17.3Hz,1H),2.82(dd,J=4.0,9.3Hz,1H),2.56(br d,J=17.1Hz,1H),1.91-1.76(m,5H),1.64(td,J=3.6,6.9Hz,1H),0.72(q,J=4.0Hz,1H),0.61-0.53(m,1H).

[0315] Example 4

[0316] Step 1: Synthesis of intermediate 4-1

[0317] Intermediate 3-2A (150 mg, 269.27 μmol), Intermediate 1-3A (86.58 mg, 269.27 μmol), [n-butyldi(1-adamantyl)phosphine](2-amino-1,1'-biphenyl-2-yl)palladium(II) methanesulfonate (98.05 mg, 134.64 μmol), and cesium carbonate (263.20 mg, 807.82 μmol) were dissolved in dioxane (8 mL) and purged with nitrogen three times. Water (0.5 mL) was then added and the mixture was reacted at 80°C under nitrogen for 6 hours. The insoluble material was removed by filtration, washed with water (10 mL), extracted with ethyl acetate (30 mL x 2), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the organic solvent to obtain a crude product. 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] + .

[0318] Step 2: Synthesis of compound 4

[0319] Intermediate 4-1 (22 mg, 30.1 μmol) was dissolved in dichloromethane (5 mL), followed by the addition of trifluoroacetic acid (1.5 mL) and the reaction was allowed to proceed at 25°C for 3 hours. Saturated sodium bicarbonate solution was added to the system until the pH reached 8, followed by extraction with dichloromethane (5 mL x 2). The mixture was dried over anhydrous sodium sulfate and concentrated under reduced pressure to remove the organic solvent to afford the crude product. The crude product was separated by prep-HPLC (column: O-Welch C18 150 x 30 mm x 5 μm; mobile phase: [A: water (0.5% formic acid) - B: acetonitrile]; B%: 8%-48%, 8 min) to afford the formate salt of compound 4. MS (ESI) m / z: 616.2 [M+1] + . 1 H NMR (400MHz, CD3OD) δ=9.06(s,1H),8.51(br s,1H),6.94(s,1H),6.52(s,1H),5.16(br d,J=10.79Hz,2H),4.71–4.80(m,2H),4.67(br d,J=10.79Hz,1H),4.53(br d,J=11.04Hz,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).

[0320] Example 5

[0321] Step 1: Synthesis of compound 5-2

[0322] Under nitrogen protection, compound 5-1 (21 g, 143.30 mmol) was dissolved in acetonitrile (210 mL), and N-iodosuccinimide (38.69 g, 171.96 mmol) and p-toluenesulfonic acid monohydrate (1.36 g, 7.16 mmol) were added. The reaction was allowed to proceed 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 organic phases were combined and washed sequentially with saturated sodium sulfite 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] + .

[0323] Step 2: Synthesis of compound 5-3

[0324] Compound 5-2 (39.3 g) was dissolved in ethanol (435 mL), and triethylamine (43.79 g, 432.75 mmol) and dichlorobistriphenylphosphine palladium (10.12 g, 14.42 mmol) were added. The mixture was replaced with CO three times, and then reacted at 80°C under a CO atmosphere (50 Psi) for 47 hours. The mixture was concentrated under reduced pressure, and ethyl acetate (300 mL) and 0.3 M hydrochloric acid (300 mL) were added. Solids precipitated and were filtered to obtain a filter cake. The filtrate was separated, and 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 filter cake and organic phase were combined and concentrated under reduced pressure to obtain crude product 5-3. 1 H NMR (400MHz, CDCl3) δ8.54 (s, 1H), 4.39 (q, J = 7.1Hz, 2H), 1.41 (t, J = 7.1Hz, 3H).

[0325] Step 3: Synthesis of compound 5-4

[0326] Under nitrogen, compound 5-3 (41.3 g, 188.92 mmol) was dissolved in tetrahydrofuran (240 mL). Methanol (80 mL) and water (80 mL) were added, and lithium hydroxide monohydrate (23.78 g, 566.76 mmol) was added in batches. The mixture was then reacted at 20°C for 5 hours. The reaction system was concentrated under reduced pressure, and water (100 mL) was added to the reaction system. The pH of the aqueous phase was adjusted to 2 using 4M hydrochloric acid. Solids precipitated and were filtered. The filter cake was dissolved in ethanol (200 mL), stirred at 20°C for 16 hours, filtered, and dried under reduced pressure to obtain the 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 filter cake was dried under reduced pressure to obtain compound 5-4. 1 H NMR (400MHz, DMSO-d6) δ13.95-13.08(m,1H),8.35(s,1H),7.62(br s,2H).

[0327] Step 4: Synthesis of compound 5-5

[0328] Under nitrogen, compound 5-4 (17.8 g, 93.41 mmol) was dissolved in phosphorus oxychloride (234.25 g, 1.53 mol) and reacted at 95°C for 3 hours. The reaction system was concentrated under reduced pressure, tetrahydrofuran (360 mL) was added, and then thiocyanamide (21.33 g, 280.23 mmol) was added portionwise at 20°C. The reaction was then allowed to react 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 x 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 the 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 filter cake was dried under reduced pressure to obtain compound 5-5. 1 H NMR (400MHz, DMSO-d6) δ13.33(br s,1H),12.91(s,1H),8.64(s,1H).

[0329] Step 5: Synthesis of Compound 5-6

[0330] Under nitrogen protection, 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, followed by slow dropwise addition of iodomethane (4.72 g, 33.22 mmol) at 20°C. The mixture was allowed to react at 20°C for 5 hours. Ice water (300 mL) was added to the reaction system, and a solid precipitated. The solid was filtered, and the filter cake was washed with ice water (100 mL) and dried under reduced pressure to obtain compound 5-6. 1H NMR (400MHz, DMSO-d6) δ13.27(br s,1H),8.81(s,1H),2.61(s,3H).

[0331] Step 6: Synthesis of Compounds 5-7

[0332] Under nitrogen, 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 the mixture was reacted 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 x 2) and the layers were separated. The aqueous phase became turbid upon addition of ethyl acetate (200 mL), and the filtrate was filtered through celite, and the filtrate was separated. The aqueous phase was extracted with ethyl acetate (200 mL x 2) and the layers were separated. All organic phases were combined, 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 solvent: petroleum ether: ethyl acetate = 20:1 to 1:5, dichloromethane: methanol = 10:1) to obtain compound 5-7. 1 H NMR (400MHz, DMSO-d6) δ13.19(br s,1H),9.03(s,1H),6.92(dd,J=2.6,7.3Hz,1H),6.70(dd,J=2.7,4.9Hz,1H),5.71(s,2H),2.62(s,3H).

[0333] Step 7: Synthesis of Compounds 5-8

[0334] Under nitrogen, compound 5-7 (2.1 g, 5.92 mmol) was dissolved in ethanol (63 mL). Silver sulfate (2.21 g, 7.10 mmol) and elemental iodine (1.58 g, 6.22 mmol) were added at 0°C. The temperature was then slowly raised to 10°C and the reaction mixture was allowed to react for 2 hours. Saturated sodium sulfite solution (70 mL) and ethyl acetate (100 mL) were added to the reaction system, filtered, and the layers separated. The aqueous phase was extracted with ethyl acetate (100 mL x 3) and the layers separated. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography (developing solvent: petroleum ether:ethyl acetate = 20:1 to 1:5) to obtain compound 5-8. 1H NMR (400MHz, CDCl3) δ10.47 (br s, 1H), 9.34 (s, 1H), 7.10 (d, J = 8.1Hz, 1H), 2.78 (s, 3H).

[0335] Step 8: Synthesis of Compounds 5-9

[0336] To a solution of compound 5-8 (0.33 g, 686.56 μmol) in N,N-dimethylformamide (5 mL) in an ice-water bath at 0°C under nitrogen was added sodium hydroxide (96.11 mg, 2.40 mmol). After stirring for 30 minutes, 4-methoxybenzyl chloride (236.55 mg, 1.51 mmol) was added dropwise. The resulting mixture was naturally warmed to 25°C and stirred for 14 hours. To the reaction solution were added 20 mL of saturated aqueous ammonium chloride and 2 x 20 mL of ethyl acetate, and stirred for 10 minutes. The aqueous phase was removed, and the organic phase was concentrated under reduced pressure to obtain crude compound 5-9.

[0337] Step 9: Synthesis of Compounds 5-10

[0338] Under nitrogen, 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-yloxytripyrrolidinyl hexafluorophosphate (95.28 mg, 183.09 μmol) was added at 10°C and allowed to react for 1 hour. Compound 1-1B (38.87 mg, 183.09 μmol) was then added and allowed to react at 10°C for 17 hours. Water (10 mL) was added to the reaction system and the layers were separated. The aqueous phase was extracted with dichloromethane (10 mL x 3). 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 the crude product. The crude product was separated by column chromatography (developing solvent: ethyl acetate / petroleum ether = 4.0% to 20.0%) to obtain compound 5-10. MS (ESI) m / z: 915.0 [M+1] + .

[0339] Step 10: Synthesis of Compounds 5-11

[0340] Under nitrogen, compound 5-10 (0.15 g, 163.89 μmol) and cuprous iodide (156.07 mg, 819.47 μmol) were dissolved in N,N-dimethylformamide (3.75 mL), and compound 5-10A (314.86 mg, 1.64 mmol) was added. The mixture was reacted at 80°C for 2 hours. Water (10 mL) and ethyl acetate (20 mL) were slowly added, and a solid precipitated, which was filtered. The filtrate was separated, and the aqueous phase was extracted with ethyl acetate (10 mL x 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 column chromatography (developing solvent: ethyl acetate / petroleum ether = 4.0% to 20.0%) to obtain compound 5-11. MS (ESI) m / z: 857.1 [M+1] + .

[0341] Step 11: Synthesis of Compounds 5-12

[0342] Under nitrogen protection, compound 5-11 (0.06 g, 69.98 μmol) was dissolved in tetrahydrofuran (1.2 mL) and water (0.4 mL). Potassium monopersulfate (82.38 mg, 489.89 μmol) was added at 0°C, and the temperature was slowly raised to 10°C for 4 hours. Potassium monopersulfate (35.31 mg, 209.95 μmol) was added and the reaction was continued at 10°C for 2 hours. Saturated sodium sulfite 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] + .

[0343] Step 12: Synthesis of Compounds 5-13

[0344] Under nitrogen protection, 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 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] + .

[0345] Step 13: Synthesis of compound 5

[0346] Under nitrogen, compound 5-13 (0.1 g, 102.62 μmol) was dissolved in dichloromethane (2.5 mL), and trifluoroacetic acid (468.04 mg, 4.10 mmol) was added. The reaction was allowed to react 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 two preparative HPLC chromatography cycles to yield compound 5. HPLC preparative method 1: Column: Phenomenex Luna 75*30 mm*3 μm; Mobile phase A: Water (0.04% hydrochloric acid), Mobile phase B: acetonitrile; Gradient: B%: 1%-43% over 8 min. HPLC preparative method 2: Column: Waters Xbridge BEH100*30 mm*10 μm; Mobile phase A: Water (10 mM ammonium bicarbonate), Mobile phase B: acetonitrile; Gradient: B%: 30%-60% over 8 min. MS (ESI) m / z: 634.2 [M+1] + .

[0347] Example 6

[0348] Step 1:

[0349] Under nitrogen protection, intermediates 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 (2-dicyclohexylphosphino-2′,6′-dimethoxy-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium II chloride (23.30 mg, 32.34 μmol) were added, and the mixture was reacted 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 the crude product. The crude product was separated and purified by HPLC (column: Phenomenex luna C18 100*40mm*5μm; 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] + .

[0350] Referring to step 1, intermediate Int-3B was used as the starting material to obtain compound 6-1B. MS m / z: 973.1 [M+1] + .

[0351] Step 2:

[0352] Compound 6-1A (0.2 g, 205.45 μmol) was separated and purified by preparative SFC (chromatographic column: ChiralPak IH, 250*30 mm, 10 μm; mobile phase: A (supercritical CO2) and B (isopropanol, containing 0.1% ammonia water); gradient: B%: 60%-60%, 10 min) to obtain compounds 6-1A1 and 6-1A2, respectively.

[0353] Compound 6-1A1: The retention time under analytical SFC (column: ChiralPak IH-3, 50*4.6mm, 3μm; mobile phase: A (supercritical CO2) and B (isopropanol, containing 0.1% isopropylamine); gradient: B%: 5%-5%, 3min) conditions was 1.463min, and the ee value was 97.49%. 1 H NMR (400MHz, CDCl3) δ = 7.30 (s, 1H), 7.10 (d, J = 8.8Hz, 4H), 6.94 (d, J = 2.0Hz, 1H), 6.88 (d, J = 8.8Hz, 4H), 6.52 (d, J=2.0Hz,1H),5.25(s,2H),5.04(d,J=11.2Hz,1H),4.69(d,J=10.8Hz,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=15.6Hz ,2H),3.56-3.43(m,1H),3.36(d,J=17.6Hz,1H),3.19-3.08(m,1H),2.77(d,J=17.6Hz,1 H),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] + .

[0354] Compound 6-1A2: The retention time under analytical SFC (column: ChiralPak IH-3, 50*4.6mm, 3μm; mobile phase: A (supercritical CO2) and B (isopropanol, containing 0.1% isopropylamine); gradient: B%: 5%-5%, 3min) conditions was 1.983min, and the ee value was 95.66%. 1H NMR (400MHz, CDCl3) δ = 7.30 (s, 1H), 7.10 (d, J = 8.8Hz, 4H), 6.94 (d, J = 2.4Hz, 1H), 6.88 (d, J = 8.4Hz, 4H) ,6.51(d,J=2.4Hz,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] + .

[0355] Step 3:

[0356] 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% ammonia water); gradient: B%: 40%-40%, 11 min) to obtain compound 6-1B1 and compound 6-1B2, respectively.

[0357] Compound 6-1B1: The retention time under analytical SFC (column: ChiralPak IH-3, 50*4.6mm, 3μm; mobile phase: A (supercritical CO2) and B (isopropanol, containing 0.1% isopropylamine); gradient: B%: 5%-5%, 3min) conditions was 1.330min, and the ee value was 94.45%. 1H NMR (400MHz, CDCl3) δ = 7.26 (s, 1H), 7.10 (d, J = 8.4Hz, 4H), 6.94 (d, J = 2.0Hz, 1H), 6.88 (d, J = 8.4Hz, 4 H),6.51(d,J=2.4Hz,1H),5.01(s,1H),4.93(s,1H),4.54(s,4H),4.42-4.16(m,6H),3.81(s,6H),3.6 5-3.50(m,3H),3.33-3.19(m,2H),2.94(d,J=16.0Hz,1H),2.75(d,J=6.4Hz,1H),2.46(dd,J=15.6,2. 0Hz,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] + .

[0358] Compound 6-1B2: The retention time under analytical SFC (column: ChiralPak IH-3, 50*4.6mm, 3μm; mobile phase: A (supercritical CO2) and B (isopropanol, containing 0.1% isopropylamine); gradient: B%: 5%-5%, 3min) conditions was 1.495min, and the ee value was 98.01%. 1 H NMR (400MHz, CDCl3) δ = 7.25 (s, 1H), 7.10 (d, J = 8.4Hz, 4H), 6.93 (s, 1H), 6.88 (d, J = 8.4Hz, 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=8.8Hz,1H),3.81(s,6H),3 .64-3.49(m,3H),3.33-3.19(m,2H),2.95(d,J=18.8Hz,1H),2.75(d,J=4.8Hz,1H),2.46(d,J=16.4Hz ,1H),1.99-1.89(m,2H),1.88-1.72(m,4H),1.52(s,9H),0.74(d,J=1.6Hz,1H),0.53-0.43(m,1H).MS m / z:973.1[M+1] + .

[0359] Step 4:

[0360] Compound 6A1:

[0361] Under nitrogen, compound 6-1A1 (0.07 g, 71.91 μmol) was dissolved in dichloromethane (3.5 mL) and trifluoroacetic acid (1.07 g, 9.42 mmol) was added. The mixture was then reacted 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 high-performance liquid chromatography (HPLC 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 (400MHz, CDCl3) δ = 7.31 (d, J = 9.6Hz, 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=11.6Hz,2H),3.37-3.23(m,2H),2 .98(d,J=16.8Hz,1H),2.77(dd,J=8.4,2.8Hz,1H),2.47(d,J=16.4Hz,1H),2.23-2.04(m,2H),1. 91-1.81(m,4H),1.56(s,1H),1.28(d,J=10.8Hz,1H),0.89(t,J=7.2Hz,1H),0.57-0.44(m,1H).MS m / z:633.1[M+1] + .

[0362] Compound 6A2:

[0363] Under nitrogen, compound 6-1A2 (0.07 g, 71.91 μmol) was dissolved in dichloromethane (3.5 mL) and trifluoroacetic acid (1.07 g, 9.42 mmol) was added. The mixture was then reacted 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 high-performance liquid chromatography (HPLC 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. 1H NMR (400MHz, CDCl3) δ = 7.30 (d, J = 10.0Hz, 1H), 6.87 (s, 1H), 6.42 (s, 1H), 5.02 ( s,1H),4.93(s,1H),4.42(d,J=12.0Hz,1H),4.36-4.26(m,2H),4.21(d,J=9.2H z,3H),3.65(s,2H),3.61-3.54(m,1H),3.47(d,J=12.4Hz,1H),3.35-3.23(m,2 H),2.95(d,J=16.8Hz,1H),2.84-2.72(m,1H),2.46(d,J=16.4Hz,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] + .

[0364] Step 5:

[0365] Compound 6B1:

[0366] Under nitrogen, compound 6-1B1 (0.06 g, 61.64 μmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (921.00 mg, 8.08 mmol) was added. The mixture was then reacted at 20°C for 2 hours. The mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated by HPLC (column: Waters Xbridge Prep OBD C18 150*40 mm*10 μm; mobile phase: A (acetonitrile) and B (water containing 0.05% ammonia + 10 mM ammonium bicarbonate); gradient: B%: 30%-70%) to obtain compound 6B1. 1H NMR (400MHz, CDCl3) δ = 7.31 (d, J = 9.6Hz, 1H), 6.87 (s, 1H), 6.42 (s, 1H), 5.01 (s, 1H), 4.92 (s, 1H), 4.42 (d, J = 12.8Hz ,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=12.4Hz,1H),3.45(d, J=12.4Hz,1H),3.32-3.21(m,2H),2.95(d,J=16.8Hz,1H),2.75(dd,J=8.8,3.6Hz,1H),2.46(d,J=17.2Hz,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=4.0Hz,1H),0.53-0.44(m,1H).MS m / z:633.2[M+1] + .

[0367] Compound 6B2:

[0368] Under nitrogen, compound 6-1B2 (0.06 g, 61.64 μmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (921.00 mg, 8.08 mmol) was added. The mixture was then reacted at 20°C for 2 hours. The mixture was concentrated under reduced pressure to obtain a crude product. The crude product was separated by HPLC (column: Waters Xbridge Prep OBD C18 150*40 mm*10 μm; mobile phase: A (acetonitrile) and B (water containing 0.05% ammonia + 10 mM ammonium bicarbonate); gradient: B%: 25%-65%) to obtain compound 6B2. 1 H NMR(400MHz, 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=10.8Hz ,2H),3.29(d,J=11.2Hz,2H),2.98(d,J=15.6Hz,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]+ .

[0369] Example 7

[0370] Step 1:

[0371] Under nitrogen protection, 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 [(di(1-adamantyl)-N-butylphosphine)-2-(2-aminobiphenyl)palladium(II) chloride (32.43 mg, 48.50 μmol) were added, and then 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 the crude product. The crude product was separated and purified by high performance liquid chromatography (chromatographic column: Phenomenex luna C18 250*50mm*10μm; mobile phase: A (acetonitrile) and B (water, containing 0.04% hydrochloric acid); gradient: B%: 60%-80%, 10min) to obtain compound 7-1A. MS m / z: 879.3 [MC 13 H 10 O] + .

[0372] Step 2:

[0373] Under nitrogen, 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 [(di(1-adamantyl)-N-butylphosphino)-2-(2-aminobiphenyl)palladium(II) chloride) (32.43 mg, 48.50 μmol) were added, and 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 x 3) and separated. The organic phases were combined, washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was separated and purified by column chromatography (mobile phase: petroleum ether:ethyl acetate = 50:1 to 1:1) to obtain compound 7-1B. MS m / z:522.4[M / 2+1] + .

[0374] Step 3:

[0375] Under nitrogen protection, 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 the mixture was reacted at 20°C for 2 hours. The mixture was concentrated under reduced pressure to obtain crude compound 7-2A. MS m / z: 779.6 [M+1] + .

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

[0377] Step 4:

[0378] Under nitrogen, compound 7-2A (0.4 g, 143.58 μmol) was dissolved in N,N-dimethylformamide (4 mL), and cesium fluoride (2.18 g, 14.36 mmol) and sodium carbonate (91.31 mg, 861.50 μmol) were added. The mixture was reacted 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 x 3). The layers were 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*30mm*3μm; 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 (250mm*30mm, 10μm); mobile phase: A (supercritical CO2) and B (ethanol, containing 0.1% ammonia water); gradient: B%: 50%-50%, 11min) to obtain compound 7A1 and compound 7A2, respectively.

[0379] Compound 7A1: The retention time under analytical SFC conditions (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) was 1.693 min, and the ee value was 97.92%. 1H NMR (400MHz, CDCl3) δ = 7.67 (dd, J = 8.8, 5.6Hz, 1H), 7.28 (s, 1H), 7.22 (t, J = 8.8Hz, 1H), 7.12 (d, J = 2.0Hz, 1H), 6.97 (d, J = 2.0Hz, 1H),5.03(s,1H),4.95(s,1H),4.41(d,J=12.4Hz,1H),4.35(d,J=10.4Hz,2H),4.23(d,J=10.0Hz,1H),4.04-3.80(m,2H),3.66(s ,3H),3.55(d,J=12.4Hz,1H),3.51(d,J=12.0Hz,1H),3.31(d,J=4.4Hz,1H),3.28(s,1H),3.01(d,J=16.8Hz,1H),2.82-2.74(m,2 H),2.48(d,J=16.8Hz,1H),1.90-1.86(m,5H),1.60-1.53(m,1H),1.32-1.19(m,1H),0.80(q,J=4.4Hz,1H),0.56-0.48(m,1H).MS m / z:623.2[M+1] + Compound 7A2: 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) showed a retention time of 2.236 min and an ee value of 98.26%. 1 H NMR(400MHz, CDCl3)δ=7.67(dd,J=8.8,6.0Hz,1H),7.28(s,1H),7.22(t,J=8.8Hz,1H),7.25-7.19(m,1H),7.12(d,J =2.0Hz,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=10.8Hz,2H),3.32(s,1H),3.29(d,J=5.2Hz,1H),2.99(d,J=16.4Hz,1H),2.83-2.73(m,2H),2.49(d,J=16 .8Hz,1H),1.94-1.86(m,5H),1.63-1.53(m,1H),1.30-1.11(m,1H),0.82(d,J=4.0Hz,1H),0.53(q,J=8.0Hz,1H).MS m / z:623.2[M+1]+ .

[0380] Step 5:

[0381] Under nitrogen, compound 7-2B (0.55 g, 198.24 μmol) was dissolved in N,N-dimethylformamide (5 mL), and cesium fluoride (3.01 g, 19.82 mmol) and sodium carbonate (126.07 mg, 1.19 mmol) were added. The mixture was reacted 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 x 3). The layers were 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*30mm*3μm; 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 (250mm*30mm, 10μm); mobile phase: A (supercritical CO2) and B (ethanol, containing 0.1% ammonia water); gradient: B%: 55%-55%, 7min) to obtain compound 7B1 and compound 7B2, respectively.

[0382] Compound 7B1: The retention time under 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) conditions was 1.745 min, and the ee value was 98.56%. 1H NMR(400MHz,CDCl3)δ=7.67(dd,J=9.2,6.0Hz,1H),7.30-7.25(m,1H),7.22(t,J=8.8Hz,1H),7.11(d,J=1.6Hz,1H),6.97(d,J=1.6Hz,1H),5.03(s,1H),4.94(s,1H),4.38(t,J=14.0Hz,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=11.6Hz,2H),3.34-3.23(m,2H),2.98(d,J=16.8Hz,1H),2.81-2.73(m,2H),2.48(d,J=16.8Hz,1H),1.92-1.79(m,5H),1.60-1.52(m,1H),1.26(s,1H),0.79(q,J=3.6Hz,1H),0.51(q,J=7.6Hz,1H).MS m / z:623.2[M+1]+。

[0383] Compound 7B2: 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) showed a retention time of 2.343 min and an ee value of 98.62%. NMR (400MHz, CDCl3) δ=7.67(dd,J=8.8,5.6Hz,1H),7.29-7.25(m,1H),7.22(t,J=8.8Hz,1H),7.11(d,J=2.4Hz,1H),6.97(d,J=2.0 Hz,1H),5.03(s,1H),4.95(s,1H),4.41(d,J=12.0Hz,1H),4.36(d,J=10.4Hz,2H),4.23(d,J=10.0Hz,1H),4.07-3.78(m,2H),3.75 -3.63(m,3H),3.59(d,J=12.0Hz,1H),3.54(d,J=12.4Hz,1H),3.31(d,J=3.2Hz,1H),3.28(s,1H),3.00(d,J=16.8Hz,1H),2.82-2. 73(m,2H),2.48(d,J=16.8Hz,1H),1.93-1.80(m,5H),1.61-1.51(m,1H),1.26(s,1H),0.81(q,J=4.0Hz,1H),0.55-0.47(m,1H).MS m / z:623.2[M+1]+.

[0384] Example 8

[0385] Step 1:

[0386] Compound 8-1 (2 g, 6.05 mmol) and 1-1B (1.35 g, 6.36 mmol) were dissolved in dichloromethane (20 mL), replaced with nitrogen three times, and triethylamine (1.84 g, 18.16 mmol) was added dropwise at -40°C. After the addition was complete, the temperature was raised to 25°C and the reaction was allowed to proceed for 3 hours. Pour the mixture into 50 mL of water and separate the liquids. The aqueous phase was extracted with dichloromethane (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 to 3:1) to obtain compound 8-2. MS m / z: 505.0, 507.0 [M+1] + .

[0387] Step 2:

[0388] Dissolve 8-2 (646 mg, 1.28 mmol) and potassium fluoride (1.48 g, 25.52 mmol) in dimethyl sulfoxide (12 mL). Replace the atmosphere with nitrogen three times, heat to 120°C, and stir for 1 hour. Pour the reaction mixture into 60 mL of water and extract with ethyl acetate (10 mL x 3). Combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The crude product is purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 to 5:1) to yield 8-3. 1 H NMR (400MHz, CDCl3) δ7.80 (d, J = 1.6Hz, 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).

[0389] Step 3:

[0390] 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). The atmosphere was purged with nitrogen three times. (2-Dicyclohexylphosphino-2,4,6-triisopropyl-1,1-biphenyl)[2-(2-amino-1,1-biphenyl]methanesulfonate palladium (86.76 mg, 102.50 μmol, 0.1 eq) was added, and the atmosphere was purged with nitrogen three times. The mixture was stirred at 35°C for 3 hours. The reaction mixture was cooled, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 to 4:1) to afford 8-4. 1 H NMR(400MHz, CDCl3) δ7.81(d,J=1.6Hz,1H),6.90(dd,J=7.2,2.4Hz,1H),6.65(dd,J =5.2,2.4Hz,1H),4.29-4.56(m,4H),3.51-3.87(m,2H),1.93-2.03(m,2H),1.77(br d,J=8.0Hz,2H),1.54(s,9H).

[0391] Step 4:

[0392] 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), the atmosphere was replaced with nitrogen three times, and the mixture was heated at 50°C and stirred for 28 hours. The reaction mixture 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 x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 to 3:1) to obtain compound 8-5. 1 H NMR(400MHz, CDCl3) δ7.85(d,J=1.6Hz,1H),7.11(d,J=8.4Hz,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).

[0393] Step 5:

[0394] Sodium hydride (36.69 mg, 917.26 μmol, 60%) was dissolved in tetrahydrofuran (1 mL), the atmosphere was replaced with nitrogen three times, and the mixture was 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 the mixture was stirred for 10 minutes. The mixture was cooled to 0°C, and 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 the mixture was stirred for 4 hours. The reaction mixture was poured into 10 mL of saturated aqueous ammonium chloride and extracted with ethyl acetate (2 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 to 1:2) to afford 8-6. MS m / z: 825.1 [M+1] + .

[0395] Step 6:

[0396] 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). The atmosphere was purged with nitrogen three times. [1,1-bis(diphenylphosphino)ferrocene]palladium dichloride (15.95 mg, 21.80 μmol, 0.1 eq) was added, and the atmosphere was purged with nitrogen three times. The temperature was raised to 80°C and stirred for 60 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by column chromatography (eluent: petroleum ether:ethyl acetate = 1:0 to 1:2) to obtain the crude product. The crude product was separated and purified by preparative SFC (column: DAICEL CHIRALPAK AD (250 mm x 30 mm, 10 μm); mobile phases: A (supercritical carbon dioxide) and B (isopropanol containing 0.1% ammonia); gradient: B% = 45% to 45%) to yield a mixture of 8-7A and 8-7B, 8-7C, and 8-7D, respectively. The retention time of 8-7C by SFC analysis (column: Chiralpak AD-3, 50 × 4.6 mm, 3 μm; mobile phases: A (supercritical carbon dioxide) and B (isopropanol containing 0.1% isopropylamine); gradient: B% = 5% to 5% to 5% over 3.0 min) was 1.468 min, indicating a chiral isomer excess of 97.36%. The retention time of 8-7D in the SFC analysis method (chromatographic column: Chiralpak AD-3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical carbon dioxide) and B (isopropanol, containing 0.1% isopropylamine); gradient: B% = 5%-50%-5%, 3.0 min) was 1.603 min, and the chiral isomer excess was 100%.

[0397] Step 7:

[0398] A mixture of 8-7A and 8-7B (32 mg, 44.84 μmol) was dissolved in dichloromethane (0.6 mL), and trifluoroacetic acid (0.2 mL) was added. The mixture was stirred at 15°C for 0.5 h. Aqueous ammonia was added dropwise to the reaction mixture until the pH reached 9, and the mixture was concentrated under reduced pressure. The crude product was separated and purified by preparative HPLC (Phenomenex Luna 100*30 mm*3 μm column; mobile phase A: water (0.2% formic acid), mobile phase B: acetonitrile; gradient: B%: 1%-30% over 8 min) to obtain the crude product. The crude product was then separated and purified by preparative SFC (DAICEL CHIRALPAK AD column (250 mm*30 mm, 10 μm); mobile phases: A (supercritical carbon dioxide) and B (ethanol containing 0.1% aqueous ammonia); gradient: B% = 50%-50% over 11 min) to obtain 8A and 8B.

[0399] The retention time of 8A in the SFC analysis method (chromatographic column: Chiralpak IG-3, 50×4.6 mm, 3 μm; mobile phase: A (supercritical carbon dioxide) and B (ethanol, containing 0.1% isopropylamine); gradient: B% = 5%-50%-5%, 3.0 min) was 1.727 min, and the chiral isomer excess was 100%. 1H NMR (400MHz, CD3OD) δ: 7.91 (s, 1H), 7.01 (d, J = 8.4Hz, 1H), 4.95-5.10 (m, 2H), 4.47 (br d,J=12.4Hz,2H),4.25-4.42(m,2H),3.56-3.75(m,5H),3.26-3.30(m,1H),3.20(br d,J=9.6Hz,1H),2.91(br d,J=16.4Hz,1H),2.79(br dd,J=9.2,3.6Hz,1H),2.53(br d,J=17.2Hz,1H),1.97(s,3H),1.81(br s, 5H), 1.56-1.70 (m, 1H), 0.70 (q, J = 4.0Hz, 1H), 0.49-0.59 (m, 1H). MS m / z:613.2[M+1] + .

[0400] 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 carbon dioxide) and B (ethanol, containing 0.1% isopropylamine); gradient: B% = 5%-50%-5%, 3.0 min) was 2.216 min, and the chiral isomer excess was 99.22%. 1H NMR (400MHz, CD3OD) δ: 7.92 (d, J = 2.0Hz, 1H), 7.01 (d, J = 8.4Hz, 1H), 4.96-5.10 (m, 2H), 4.47 (br t,J=13.2Hz,2H),4.25-4.41(m,2H),3.57-3.74(m,5H),3.26-3.30(m,1H),3.20(d,J=9.6Hz,1H),2.91(br d,J=16.8Hz,1H),2.79(dd,J=9.6,4.0Hz,1H),2.53(br d,J=16.4Hz,1H),1.76-1.91(m,8H),1.56-1.69(m,1H),0.70(q,J=4.4Hz,1H),0.49-0.59(m,1H). MS m / z:613.2[M+1] + .

[0401] Step 8:

[0402] Dissolve 8-7C in dichloromethane (0.6 mL), add trifluoroacetic acid (0.2 mL), and stir at 15°C for 0.5 hours. Aqueous ammonia was added dropwise to the reaction mixture until the pH reached 9, and the mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC (Phenomenex Luna 100*30mm*3μm column; mobile phase A: water (0.2% formic acid), mobile phase B: acetonitrile; gradient: B%: 1%-30% over 8 min) to afford the formate salt of 8C. 1 H NMR(400MHz,CD3OD)δ8.51(br s,1H),7.94(s,1H),7.02(d,J=8.0Hz,1H),5.09(d,J=16.4Hz,2H),4.50-4.63(m,3H),4.41(d,J=10.8Hz,1H),3.86-4.08(m,3H),3.76(br dd,J=12.8,8.8Hz,2H),3.48(br d,J=15.6Hz,1H),3.34-3.41(m,1H),2.92-3.03(m,2H),2.66(br d,J=16.4Hz,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] + .

[0403] Step 9:

[0404] 8-7D (21 mg, 29.43 μmol) was dissolved in dichloromethane (0.6 mL), and trifluoroacetic acid (0.2 mL) was added. The mixture was stirred at 15°C for 0.5 hours. Aqueous ammonia was added dropwise to the reaction mixture until the pH reached 9, and the mixture was concentrated under reduced pressure. The crude product was purified by preparative HPLC (Phenomenex Luna 100*30 mm*3 μm column; mobile phase A: water (0.2% formic acid), mobile phase B: acetonitrile; gradient: B%: 1%-30% over 8 min) to afford the formate salt of 8D. 1H NMR (400MHz, CD3OD) δ8.53(br s,1H),7.93(s,1H),7.02(d,J=8.0Hz,1H),5.07(br d,J=18.8Hz,2H),4.42-4.65(m,3H),4.37(br d,J=10.8Hz,1H),3.81-3.97(m,3H),3.73(br d,J=12.4Hz,2H),3.43(br d,J=15.6Hz,1H),3.34(br s,1H),2.84-3.03(m,2H),2.62(br d,J=17.2Hz,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] + .

[0405] Experimental Example 1. GP2D cell p-ERK inhibition test

[0406] 1. Purpose

[0407] Using the HTRF method, compounds that can effectively inhibit p-ERK in GP2D cells were screened.

[0408] 2. Experimental Procedure

[0409] 1) GP2D cells were seeded in a transparent 96-well cell culture plate, with 80 μL of cell suspension per well (8,000 cells per well). The plate was placed in a CO2 incubator and incubated overnight at 37°C.

[0410] 2) Add 2 μL of compound to 78 μL of cell culture medium, mix thoroughly, and then add 20 μL of compound solution to the corresponding wells of the cell plate. Return the cell plate to the CO2 incubator and continue incubation for 1 hour.

[0411] 3) After incubation, discard the cell supernatant and add 50 μL of 1X cell lysis buffer to each well. Incubate at room temperature with shaking for 30 minutes.

[0412] 4) Dilute Phospho-ERK1 / 2 Eu Cryptate antibody and Phospho-ERK1 / 2 d2 antibody 20-fold using detection buffer;

[0413] 5) Transfer 16 μL of cell lysate supernatant to each well of a new 384-well white microplate, then add 2 μL of Phospho-ERK1 / 2 Eu Cryptate antibody dilution and 2 μL of Phospho-ERK1 / 2 d2 antibody dilution, and incubate at room temperature for at least 4 hours.

[0414] 6) After incubation, use a multi-label analyzer to read HTRF excitation: 320nm, emission: 615nm, 665nm;

[0415] 7). Calculate the IC of the test compound 50 .

[0416] 3. Experimental Results

[0417] The results are shown in Table 2.

[0418] Table 2 IC of compounds against p-ERK inhibition in GP2D cells 50 value

[0419] Experimental conclusion: The compound of the present invention has a significant GP2D p-ERK inhibitory effect.

[0420] Experimental Example 2. GP2D 3D CTG Experiment

[0421] 1. Experimental purpose:

[0422] This experiment aims to verify the inhibitory effect of the compound of the present invention on the proliferation of GP2D human colon cancer cells with KRAS G12D mutation.

[0423] 2. Experimental Materials:

[0424] The cell line GP2D, DMEM medium were purchased from GIBCO, FBS was purchased from Hyclone, and L-glutamine was purchased from Invitrogen. 96-well plates were purchased from Ultra Low Cluster. 3D Cell Viability Assay (3D cell viability chemiluminescence detection reagent) reagent was purchased from Promega, and 2104 EnVision plate reader was purchased from PerkinElmer.

[0425] 3. Experimental methods:

[0426] GP2D cells were cultured in DMEM + 10% FBS + 2mM L-glutamine at 37°C, 5% CO2 in an incubator. Cells were passaged regularly, and cells in the logarithmic growth phase were plated. GP2D cells were seeded in 96-well U-bottom cell culture plates, with 135μL of cell suspension per well containing 6,000 GP2D cells. The plates were incubated overnight in an incubator at 37°C, 5% CO2, and 100% relative humidity. Test compounds were diluted five-fold using a pipette to the eighth concentration, from 200μM to 2.56nM, in duplicate. 78μL of culture medium was added to the middle plate, and 2μL of the serially diluted compound was transferred to each well of the corresponding position. After mixing, 20μL of the compound was transferred to each well of the cell plate. The concentration of the compound transferred to the cell plate ranged from 1μM to 0.0128nM. The cell plates were incubated in a CO2 incubator for 5 days. After the incubation of the cell plates with the added compounds, 100 μL of cell viability chemiluminescent detection reagent was added to each well of the cell plates and incubated at room temperature for 10 minutes to allow the luminescent signal to stabilize. The cells were read using a multi-label analyzer.

[0427] 4. Data Analysis:

[0428] The raw data were converted into inhibition rate, IC, using the equation (Sample-Min) / (Max-Min)*100%. 50 The value can be obtained by four-parameter curve fitting (obtained in "log(inhibitor)vs.response--Variable slope" mode in GraphPad Prism).

[0429] 5. Experimental results:

[0430] The results are shown in Table 3.

[0431] Table 3 IC values ​​of compounds for anti-proliferation of GP2D cells 50 value

[0432] Experimental conclusion: The compound of the present invention has significant anti-proliferative activity against GP2D cells with KRAS G12D mutation.

[0433] Experimental Example 3. AsPC-1 3D CTG Experiment

[0434] 1. Experimental purpose:

[0435] This experiment aims to verify the inhibitory effect of the compound of the present invention on the proliferation of KRAS G12D mutated AsPC-1 human pancreatic cancer cells.

[0436] 2. Experimental Materials:

[0437] Cell line AsPC-1, RPMI-1640 culture medium were purchased from GIBCO, FBS was purchased from Hyclone. 96-well plates were purchased from Ultra Low Cluster, 3D Cell Viability Assay (3D cell viability chemiluminescence detection reagent) reagent was purchased from Promega, and 2104 EnVision plate reader was purchased from PerkinElmer.

[0438] 3. Experimental methods:

[0439] AsPC-1 cells were cultured in RPMI-1640 with 10% FBS in a 37°C, 5% CO2 incubator. Cells were passaged regularly, and cells in the logarithmic growth phase were plated. AsPC-1 cells were seeded in 96-well U-bottom cell culture plates, with 135 μL of cell suspension per well containing 500 AsPC-1 cells. The plates were incubated overnight in a 37°C, 5% CO2, and 100% relative humidity incubator. Test compounds were diluted 5-fold using a pipette to the eighth concentration, from 200 μM to 2.56 nM, in duplicate. 78 μL of culture medium was added to the middle plate, and 2 μL of the serially diluted compound was transferred to each well of the middle plate according to the corresponding position. After mixing, 20 μL of the compound was transferred to each well of the cell plate. The concentration of the compound transferred to the cell plate ranged from 1 μM to 0.0128 nM. The cell plates were incubated in a CO2 incubator for 7 days. After the incubation of the cell plates with the added compounds, 100 μL of cell viability chemiluminescent detection reagent was added to each well of the cell plates and incubated at room temperature for 10 minutes to allow the luminescent signal to stabilize. The cells were read using a multi-label analyzer.

[0440] 4. Data Analysis:

[0441] The raw data were converted into inhibition rate, IC, using the equation (Sample-Min) / (Max-Min)*100%. 50 The value can be obtained by four-parameter curve fitting (obtained in "log(inhibitor)vs.response--Variable slope" mode in GraphPad Prism).

[0442] 5. Experimental results:

[0443] The results are shown in Table 4.

[0444] Table 4 IC values ​​of compounds for anti-proliferation of AsPC-1 cells 50 value

[0445] Experimental conclusion: The compound of the present invention has significant anti-proliferative activity against KRAS G12D mutated AsPC-1 cells.

[0446] Experimental Example 4. PANC04.03 3D CTG Experiment

[0447] 1. Experimental purpose:

[0448] This experiment aims to verify the inhibitory effect of the compound of the present invention on the proliferation of KRAS G12D mutated PANC04.03 human pancreatic cancer cells.

[0449] 2. Experimental Materials:

[0450] The cell line PANC04.03 and RPMI-1640 culture medium were purchased from GIBCO, FBS was purchased from Hyclone, and human insulin was purchased from Yeasen. 96-well plates were purchased from Ultra Low Cluster, 3D Cell Viability Assay (3D cell viability chemiluminescence detection reagent) reagent was purchased from Promega, and 2104 EnVision plate reader was purchased from PerkinElmer.

[0451] 3. Experimental methods:

[0452] PANC04.03 cells were cultured in RPMI-1640 with 15% FBS and 5 μg / ml human insulin at 37°C and 5% CO₂ in an incubator. Cells were passaged regularly, and cells in the logarithmic growth phase were harvested for plating. PANC04.03 cells were seeded in 96-well U-bottom cell culture plates, with 135 μL of cell suspension per well containing 2,000 PANC04.03 cells. The plates were incubated overnight at 37°C, 5% CO₂, and 100% relative humidity. Test compounds were diluted five-fold using a pipette to the eighth concentration, from 200 μM to 2.56 nM, in duplicate. 78 μL of culture medium was added to the middle plate, and 2 μL of the serially diluted compound was transferred to each well of the corresponding position. After mixing, 20 μL of the compound was transferred to each well of the cell plate. The concentration of the compound transferred to the cell plate ranged from 1 μM to 0.0128 nM. The cell plates were incubated in a CO2 incubator for 7 days. After the incubation period, 100 μL of cell viability chemiluminescent detection reagent was added to each well of the cell plates. The cells were incubated at room temperature for 10 minutes to allow the luminescent signal to stabilize. The cells were then read using a multi-label analyzer.

[0453] 4. Data Analysis:

[0454] The raw data were converted into inhibition rate, IC, using the equation (Sample-Min) / (Max-Min)*100%. 50 The value can be obtained by four-parameter curve fitting (obtained in "log(inhibitor)vs.response--Variable slope" mode in GraphPad Prism).

[0455] 5. Experimental results:

[0456] The results are shown in Table 5.

[0457] Table 5 IC values ​​of compounds for anti-proliferation of PANC04.03 cells 50 value

[0458] Experimental conclusion: The compound of the present invention has significant anti-proliferative activity against PANC04.03 cells with KRAS G12D mutation.

[0459] Experimental Example 5. In vivo pharmacokinetic experiment

[0460] 1. Experimental purpose:

[0461] The purpose of this experiment is to investigate the pharmacokinetic characteristics of the compound of the present invention in CD-1 mice after oral and intravenous administration.

[0462] 2. Experimental methods:

[0463] The test compound was mixed with 10% dimethyl sulfoxide (DMSO) and 90% (10% hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solution), vortexed, and sonicated to prepare clear solutions at 0.6, 3.0, and 10.0 mg / mL, respectively. Male CD-1 mice aged 7 to 10 weeks were administered the candidate compound solution intravenously (iv) at a dose of 3 mg / kg (dosing concentration 0.6 mg / mL). The candidate compound solution was administered orally (po) at a dose of 30 mg / kg (dosing concentration 3.0 mg / mL) or 100 mg / kg (dosing concentration 10.0 mg / mL). Whole blood was collected at specific times and plasma was prepared. Drug concentrations were analyzed by LC-MS / MS, and pharmacokinetic parameters were calculated using Phoenix WinNonlin software (Pharsight, USA).

[0464] 3. Experimental results:

[0465] The results are shown in Table 6.

[0466] Table 6 PK properties of compound 3 in CD-1 mice

[0467] Experimental conclusion: The compound of the present invention has good pharmacokinetic characteristics in mice.

[0468] Experimental Example 6. In vivo pharmacodynamics experiment in GP2D tumor model

[0469] 1. Experimental purpose:

[0470] In vivo pharmacodynamic study of a Balb / c nude mouse model with subcutaneous transplantation of human colon cancer GP2D cells

[0471] 2. Experimental methods:

[0472] Cell Culture: Human colon cancer GP2D cells were cultured as monolayers in DMEM / F12 medium supplemented with 20% fetal bovine serum in a 37°C, 5% CO2 incubator. Cells were routinely digested and passaged twice weekly using trypsin-EDTA. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and resuspended in an appropriate amount of PBS. Matrigel was added at a 1:1 ratio to obtain a cell density of 25 x 10 6 cells / mL of cell suspension.

[0473] Cell inoculation: 0.2 mL (5 × 10 6 cells / mouse) GP2D cells (plus Matrigel, volume ratio of 1:1) were subcutaneously inoculated into the right back of each mouse.

[0474] Experimental operation: The average tumor volume reached 140mm 3 The mice were randomly divided into groups according to the tumor volume, with 6 mice in each group. The blank group was given 0 dose, and the test groups were given doses of 30 mg / kg and 100 mg / kg, respectively. The administration volume was 10 μL / g, and the drugs were administered orally for 28 days, twice a day.

[0475] 3. Tumor Measurement and Experimental Indicators:

[0476] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0477] The anti-tumor efficacy of the compound was evaluated using 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). Relative tumor volume (RTV) was calculated based on the tumor measurement results using the formula RTV = V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., D0), Vt It is the average tumor volume at a certain measurement. TRTV and CRTV data are taken on the same day.

[0478] TGI (%) reflects the rate of tumor growth inhibition. TGI (%) = [1 - (average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the start of drug administration) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the start of treatment)] × 100%.

[0479] 4. Experimental results:

[0480] The experimental results are shown in Table 7.

[0481] Table 7 Pharmacological efficacy results of compound 3 in the GP2D tumor model in vivo

[0482] Experimental conclusion: The compound of the present invention has excellent tumor inhibition effect.

[0483] Experimental Example 7. In vivo pharmacodynamics study in PANC04.03 tumor model

[0484] 1. Experimental purpose:

[0485] In vivo pharmacodynamic study of a Balb / c nude mouse model with subcutaneous transplantation of human pancreatic cancer PANC04.03 cells

[0486] 2. Experimental methods:

[0487] Cell Culture: Human pancreatic cancer PANC04.03 cells were cultured as monolayers in RPMI-1640 supplemented with 15% FBS and 10 units / mL insulin at 37°C in a 5% CO2 incubator. Twice weekly, cells were routinely digested and passaged using trypsin-EDTA. When cell saturation reached 80%-90% and the desired number of cells was reached, cells were harvested, counted, and resuspended in an appropriate amount of PBS to a cell density of 25 x 10 6 cells / mL of cell suspension.

[0488] Cell inoculation: 0.2 mL (5 × 10 6 PANC04.03 cells (cells / mouse) were subcutaneously inoculated into the right back of each mouse.

[0489] Experimental operation: The average tumor volume reached 190mm 3 The mice were randomly divided into groups according to the tumor volume, with 6 mice in each group. The blank group was given 0 dose, and the test groups were given doses of 30 mg / kg, 100 mg / kg, and 150 mg / kg, respectively. The dosage was 10 μL / g, and the drugs were orally administered for 28 days, twice a day.

[0490] 3. Tumor Measurement and Experimental Indicators:

[0491] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively.

[0492] The anti-tumor efficacy of the compound was evaluated using 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). Relative tumor volume (RTV) was calculated based on the tumor measurement results using the formula RTV = V t / V0, where V0 is the average tumor volume measured at the time of group administration (i.e., D0), V t It is the average tumor volume at a certain measurement. TRTV and CRTV data are taken on the same day.

[0493] TGI (%) reflects the rate of tumor growth inhibition. TGI (%) = [1 - (average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the start of drug administration) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the start of treatment)] × 100%.

[0494] 4. Experimental results:

[0495] The experimental results are shown in Table 8.

[0496] Table 8 Pharmacological efficacy results of compound 3 in the mouse PACN04.03 in vivo efficacy model

[0497] Experimental conclusion: The compound of the present invention has excellent tumor inhibition effect.

Claims

1. The present invention provides a compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof, in, X is selected from CH, C-Rx, N and N + -O - Preferably, X is selected from N and N + -O - ; Rx is selected from F, Cl, Br; R1 is selected from described are independently optionally replaced by 1, 2, 3 or 4 R a replace; R3 is selected from H and D; Each R a are independently selected from F, Cl, Br, I, OH, NH2, C 1-3 Alkyl, C 1-3 Alkoxy, C 1-3 Halogenated alkyl, C 2-4 Alkenyl, C 2- 4-Alkynyl, -C 1-3 Alkyl-cyclopropyl and cyclopropyl, 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 with 1, 2 or 3 R; Each R is independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

2. A compound represented by formula (III-1) or a pharmaceutically acceptable salt thereof, in, X is selected from CH, N and N + -O - Preferably, X is selected from N and N + -O - ; R1 is selected from described are independently optionally replaced by 1, 2, 3 or 4 R a replace; R3 is selected from H and D; Each R a are 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, 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 with 1, 2 or 3 R; Each R is independently selected from F, Cl, Br, I, CH2F, CHF2 and CF3.

3. Compounds represented by formula (II) and (III-2) or pharmaceutically acceptable salts thereof, in, 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 R a replace; R2 is selected from described Each independently optionally replaced by 1, 2 or 3 R c replace; R3 is selected from H and D; Each R a are 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, 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 with 1, 2 or 3 R; R b is selected from the group consisting of H, CN, CH3 and OCH3; Each R c 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.

4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: Each R a Each independently selected from F, Cl, OH, NH2, CH3, CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH(CH3)2, -C≡CH, -C≡CCH3, and cyclopropyl, said CH3, CH2CH3, CH(CH3)2, OCH3, OCH2CH3, OCH(CH3)2, -C≡CH, -C≡CCH3, and cyclopropyl are each independently optionally substituted with 1, 2 or 3 R.

5. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: Each R a Each independently selected from F, Cl, OH, NH2, CH3, CHF2, CF3, CH2CF3, CH(CH3)CF3, OCH3, OCF3, -C≡CH, -C≡CCH3, 6. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from 7. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from described are independently optionally replaced by 1, 2, 3 or 4 R a replace.

8. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from 9. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein: R1 is selected from 10. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein: R2 is selected from 11. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein: R2 is selected from 12. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein: X is selected from N.

13. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: in, R1 and R3 are as defined in claim 1.

14. The compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: in, X, R1 and R3 are as defined in claim 1.

15. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of: in, R1 and R3 are as defined in claim 3.

16. The following compound or a pharmaceutically acceptable salt thereof:

17. The compound according to claim 16 or a pharmaceutically acceptable salt thereof, which is selected from, 18. The compound according to claim 17 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

19. The compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof for use in the preparation of a drug for treating KRAS G12D Application of compounds to mutant solid tumors.

Images