CN117177980A

CN117177980A - Ten-binary macrocyclic compounds

Info

Publication number: CN117177980A
Application number: CN202280027673.9A
Authority: CN
Inventors: 张杨; 付志飞; 孙继奎; 陈健; 黎健; 陈曙辉
Original assignee: Shanghai Qilu Pharmaceutical Research and Development Centre Ltd
Current assignee: Shanghai Qilu Pharmaceutical Research and Development Centre Ltd
Priority date: 2021-05-06
Filing date: 2022-05-06
Publication date: 2023-12-05
Also published as: TW202246287A; WO2022233316A1

Abstract

A series of twelve-membered macrocyclic compounds are disclosed, specifically compounds of formula (III) and pharmaceutically acceptable salts thereof.

Description

Ten-binary macrocyclic compounds

The present invention claims the following priorities:

CN202110492226.8, filing date: 2021, 05, 06;

CN202110502109.5, filing date: 2021, 05, 08;

CN202110545538.0, filing date: 2021, 05, 19;

CN202110738841.2, filing date: 2021, 06, 30;

CN202110825905.2, filing date: 2021, 07, 21;

CN202111131764.0, filing date: 2021, 09, 26;

CN202111162653.6, filing date: 2021, 09, 30;

CN202111389719.5, filing date: 2021, 11, 22;

cn202210452057.X, filing date: 2022, 04, 26.

Technical Field

The invention relates to a series of twelve-membered macrocyclic compounds, in particular to a compound shown in a formula (III) and pharmaceutically acceptable salts thereof.

Background

RAS proteins are products expressed by the RAS gene. RAS proteins can bind to guanylic acid phosphate (GTP) or guanylic acid dinucleotide phosphate (GDP), and the active state of the RAS proteins has an effect on growth, differentiation, cytoskeleton, protein trafficking and secretion of cells, etc., and its activity is regulated by binding to GTP or GDP: when the RAS protein binds to GDP, it is in a dormant state, i.e., an "inactive" state; when stimulated with upstream specific cell growth factors, RAS proteins are induced to exchange GDP for GTP, which is then referred to as the "activated" state. RAS proteins that bind to GTP are able to activate downstream proteins for signaling. RAS proteins themselves have weak GTP hydrolysis activity, and are capable of hydrolyzing GTP to GDP. In this way a transition from the active state to the inactive state can be achieved. GAP (GTPase activating proteins, GTP hydrolase activated protein) is also required to participate in this hydrolysis process. It can interact with RAS proteins, greatly promoting their ability to hydrolyze GTP to GDP. Mutations in the RAS protein affect its interaction with GAP, i.e., its ability to hydrolyze GTP to GDP, leaving it in an activated state at all times. The activated RAS proteins continue to give downstream protein growth signals, ultimately leading to continued growth and differentiation of the cells, ultimately producing tumors. Among the subfamilies closely related to various cancers are mainly the KRAS Teng Da murine sarcoma viral oncogene homolog (KRAS), the Harv murine sarcoma viral oncogene Homolog (HRAS), and the neuroblastoma murine sarcoma viral oncogene homolog (NRAS). It was found that about 30% of human tumors harbor some mutated RAS genes, most pronounced with KRAS mutations accounting for 86% of all RAS mutations. For KRAS mutations, the most common mutations occur at residues 12 glycine (G12), 13 glycine (G13) and 61 glutamine (Q61), with the G12 mutation accounting for 83%.

The G12C mutation is a relatively common subtype of KRAS gene mutation, which refers to the mutation of glycine No. 12 to cysteine. KRAS G12C mutations are most common in lung cancer, and KRAS G12C mutations account for about 10% of all lung cancer patients, as estimated from data reported in literature (Nat Rev Drug Discov 2014; 13:828-851).

Document J Med chem 2020 Jan 9;6 3 (1): 52-65 report AMG510 (structure below) and KRAS ^G12C Eutectic structure of protein (ID: 6 IOM). According to literature reports, AMG510 binds to KRAS ^G12C In the SwichII pocket of the protein, propenyl and Cys12 are added to form a covalent bond, carbonyl and Lys16 form a hydrogen bond, pyrimidopyridine as a parent nucleus and Tyr96 form pi-pi effects respectively, isopropyl picoline and the parent nucleus are inserted into a hydrophobic pocket at a dihedral angle of 86.8 DEG, fluorophenol and the parent nucleus are inserted into the hydrophobic pocket at a dihedral angle of 58.8 DEG, and simultaneously phenolic hydroxyl and Arg68 form a hydrogen bond. (see FIGS. 1 and 2, maestro 2017-2, pymol 1.8.6).

Disclosure of Invention

The invention provides a compound shown as a formula (III) or pharmaceutically acceptable salt thereof,

wherein,

T ₁ 、T ₂ and T ₃ Each independently selected from CH and N;

T ₄ selected from CR ₆ And N;

ring A is selected from piperazinyl,

R ₁ Selected from the group consisting ofThe saidOptionally by 1, 2 or 3R _a Substitution;

each R is ₂ Are independently selected from H and C _1-3 Alkyl, said C _1-3 Alkyl is optionally substituted with 1, 2 or 3R _b Substitution;

R ₃ selected from C _1-6 Alkyl, said C _1-6 Alkyl is optionally substituted with 1, 2 or 3R _b Substitution;

R ₄ selected from H, F, cl, br and I;

R ₅ selected from H and F;

R ₆ selected from H, F, cl and CN;

m is selected from 0, 1, 2 and 3;

each R is _a Are respectively and independently selected from H, F, cl, br, I, CN, C _1-3 Alkyl, -C (O) OC _1-3 Alkyl, -C (O) NHC _1-3 An alkyl group and a cyclobutenyl group,

the C is _1-3 Alkyl and cyclobutenyl are optionally substituted with 1, 2 or 3R;

each R is _b Each independently selected from H, F, cl, br, I and CN;

each R is independently selected from F, OCH ₃ 、N(CH ₃ ) ₂ 、NH ₂ And morpholinyl;

provided that the conditions are that,

1) When T is ₄ Selected from N, ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofWhen R is _a Selected from CN, R ₁ Quilt R _a Substitution formationOr alternatively

2) When T is ₄ Selected from N, ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofWhen in use, theIs covered by 1, 2 or 3R _a Substituted, R _a Selected from C _1-3 Alkyl, -C (O) OC _1-3 Alkyl, -C (O) NHC _1-3 Alkyl and cyclobutenyl groups, the C _1-3 Alkyl and cyclobutenyl are optionally substituted with 1, 2 or 3R, each R being independently selected from F, OCH ₃ 、N(CH ₃ ) ₂ 、NH ₂ And morpholinyl.

In some aspects of the invention, each R _a Are respectively and independently selected from H, F, cl, br, I, CN, CH ₃ 、CH ₂ CH ₃ 、CH(CH ₃ ) ₂ 、-C(O)OCH ₃ 、-C(O)NHCH ₃ And cyclobutenyl groups, the CH ₃ 、CH ₂ CH ₃ 、CH(CH ₃ ) ₂ 、-C(O)OCH ₃ 、-C(O)NHCH ₃ And cyclobutenyl is optionally substituted with 1, 2 or 3R, the other variables being as defined herein.

In some aspects of the invention, each R _a Are respectively and independently selected from H, F, CN, CHF ₂ 、CH ₂ F、CH ₂ OCH ₃ 、CH ₂ N(CH ₃ ) ₂ 、CH ₂ NH ₂ 、-C(O)OCH ₃ 、-C(O)NHCH ₃ 、 The other variables are as defined herein.

In some aspects of the invention, the R ₁ Selected from the group consisting of The other variables are as defined herein.

In some embodiments of the invention, the compound or a drug thereofA pharmaceutically acceptable salt, wherein T ₄ Selected from N, ring A is selected from piperazinyl, R ₁ Selected from the group consisting of The other variables are as defined herein.

In some aspects of the invention, each R ₂ Are independently selected from H and CH ₃ The other variables are as defined herein.

In some aspects of the invention, the R ₃ Selected from CH (CH) ₃ ) ₂ The other variables are as defined herein.

In some aspects of the invention, the R ₄ Selected from F and Cl, and the other variables are as defined herein.

In some aspects of the invention, the building blocksSelected from the group consisting of The other variables are as defined herein.

wherein,

T ₁ 、T ₂ and T ₃ Each independently selected from CH and N;

T ₄ selected from CR ₆ And N;

ring A is selected from piperazinyl,

R ₄ selected from H, F, cl, br and I;

R ₅ selected from H and F;

R ₆ selected from H, F, cl and CN;

m is selected from 0, 1, 2 and 3;

each R is _b Each independently selected from H, F, cl, br, I and CN;

provided that the conditions are that,

2) When T is ₄ Selected from N, ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofWhen in use, theOptionally by 1, 2 or 3R _a Substituted, R _a Selected from C _1-3 Alkyl, -C (O) OC _1-3 Alkyl, -C (O) NHC _1-3 Alkyl and cyclobutenyl groups, the C _1-3 Alkyl and cyclobutenyl are optionally substituted with 1, 2 or 3R, each R being independently selected from F, OCH ₃ 、N(CH ₃ ) ₂ 、NH ₂ And morpholinyl.

In some embodiments of the invention, the compound or pharmaceutically acceptable salt thereof, wherein T ₄ Selected from N, ring A is selected from piperazinyl, R ₁ Selected from the group consisting of The other variables are as defined herein.

In some aspects of the invention, the building blocksSelected from the group consisting of

The other variables are as defined herein.

wherein,

T ₁ 、T ₂ and T ₃ Are respectively and independently selected fromCH and N;

T ₄ selected from CR ₆ And N;

ring A is selected from piperazinyl,

R ₄ selected from H, F, cl, br and I;

R ₅ selected from H and F;

R ₆ selected from H, F, cl and CN;

m is selected from 0, 1, 2 and 3;

each R is _a Are respectively and independently selected from H, F, cl, br, I, CN, C _1-3 Alkyl, -C (O) OC _1-3 Alkyl, -C (O) NHC _1-3 Alkyl and cyclobutenyl groups, the C _1-3 Alkyl and cyclobutenyl are optionally substituted with 1, 2 or 3R;

each R is _b Each independently selected from H, F, cl, br, I and CN;

provided that the conditions are that,

1) When T is ₄ Selected from N, R when ring A is selected from piperazinyl ₁ Selected from the group consisting ofThe saidOptionally by 1, 2 or 3R _a Substitution; or alternatively

2) When T is ₄ Selected from N, R when ring A is selected from piperazinyl ₁ Selected from the group consisting ofThe saidIs covered by 1, 2 or 3C _1-3 Alkyl, -C (O) OC _1-3 Alkyl, -C (O) NHC _1-3 Alkyl and cyclobutenyl groups, the C _1-3 Alkyl is covered by 1, 2 or 3F, OCH ₃ 、NH ₂ And morpholinyl substitution; or alternatively

3) When T is ₄ Selected from CR ₆ Ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofWhen R is ₆ Selected from F, cl and CN.

In some embodiments of the invention, the compound or pharmaceutically acceptable salt thereof, wherein, when T ₄ Selected from N, R when ring A is selected from piperazinyl ₁ Selected from the group consisting of The other variables are as defined herein.

wherein,

T ₁ 、T ₂ and T ₃ Each independently selected from CH and N;

T ₄ selected from CR ₆ And N;

ring A is selected from piperazinyl,

R ₄ selected from H, F, cl, br and I;

R ₅ selected from H and F;

R ₆ selected from H, F, cl and CN;

m is selected from 0, 1, 2 and 3;

Each R is _b Each independently selected from H, F, cl, br, I and CN;

provided that the conditions are that,

1) When T is ₄ Selected from N, ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofWhen in use, theOptionally by 1, 2 or 3R _a Substitution;

2) When T is ₄ Selected from N, ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofWhen in use, theIs covered by 1, 2 or 3C _1-3 Alkyl, -C (O) OC _1-3 Alkyl, -C (O) NHC _1-3 Alkyl and cyclobutenyl groups, the C _1-3 Alkyl is covered by 1, 2 or 3F, OCH ₃ 、NH ₂ And morpholinyl substitution.

In some aspects of the invention, the R ₄ Selected from F, and the other variables are as defined herein.

The invention provides a compound shown as a formula (II) or pharmaceutically acceptable salt thereof,

wherein,

T ₁ 、T ₂ and T ₃ Each independently selected from CH and N;

ring A is selected from piperazinyl,

R ₂ selected from H and C _1-3 Alkyl, said C _1-3 Alkyl is optionally substituted with 1, 2 or 3R _b Substitution;

R ₄ selected from H, F, cl, br and I;

R ₅ selected from H and F;

m is selected from 0, 1, 2 and 3;

each R is _a Are independently selected from H, F, cl,Br、I、CN、C _1-3 Alkyl, -C (O) OC _1-3 Alkyl, -C (O) NHC _1-3 Alkyl and cyclobutenyl groups, the C _1-3 Alkyl and cyclobutenyl are optionally substituted with 1, 2 or 3R;

each R is _b Each independently selected from H, F, cl, br, I and CN;

provided that the conditions are that,

1) When ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofWhen in use, theOptionally by 1, 2 or 3R _a Substitution;

2) When ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofWhen in use, the Is covered by 1, 2 or 3C _1-3 Alkyl, -C (O) OC _1-3 Alkyl, -C (O) NHC _1- ₃ Alkyl and cyclobutenyl groups, the C _1-3 Alkyl is covered by 1, 2 or 3F, OCH ₃ 、NH ₂ And morpholinyl substitution.

In some aspects of the invention, the R ₂ Selected from H and CH ₃ The other variables are as defined herein.

The other variables are as defined herein.

The invention provides a compound shown as a formula (I) or pharmaceutically acceptable salt thereof,

Wherein,

ring A is selected from piperazinyl and

R ₄ selected from H, F, cl, br and I;

m is selected from 0, 1, 2 and 3;

each R is _a Are respectively and independently selected from H, F, cl, br, I, CN, C _1-3 Alkyl, -C (O) OC _1-3 Alkyl and-C (O) NHC _1-3 Alkyl, said C _1-3 Alkyl is optionally substituted with 1, 2 or 3F;

each R is _b Each independently selected from H, F, cl, br, I and CN;

with the proviso that when ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofThe said Optionally by 1, 2 or 3R _a And (3) substitution.

In some aspects of the invention, each R _a Are respectively and independently selected from H, F, cl, br, I, CN, CH ₃ 、CH ₂ CH ₃ And CH (CH) ₃ ) ₂ The CH is ₃ 、CH ₂ CH ₃ And CH (CH) ₃ ) ₂ Optionally substituted with 1, 2 or 3F, with the other variables being as defined herein.

In some aspects of the invention, each R _a Are respectively and independently selected from H, F, CN, CH ₂ F、-C(O)OCH ₃ and-C (O) NHCH ₃ The other variables are as defined herein.

In some embodiments of the invention, the compound or pharmaceutically acceptable salt thereof is selected from the group consisting of

Wherein,

R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ and m is as defined herein.

Still other embodiments of the present invention are derived from any combination of the variables described above.

The present invention also provides the following compounds or pharmaceutically acceptable salts thereof,

The invention also provides application of the compound or pharmaceutically acceptable salt thereof in preparing a medicament for treating tumors.

In some aspects of the invention, the neoplasm refers to KRAS ^G12C Mutation-related tumors.

The invention also provides the following synthesis method:

method 1:

method 2:

method 3:

method 4:

method 5:

method 6:

the invention also provides the following test method:

method 1: MIA-PA-CA-2 cell experiments

Experimental materials:

DMEM medium, fetal bovine serum from Biosera and horse serum from Gibco. CellTiter-Glo (cell viability chemiluminescent detection reagent) reagent was purchased from Promega. MIA-PA-CA-2 cell line was purchased from Nanjac, bai Biotechnology Co. EnVision Multi-tag Analyzer (Perkinelmer).

The experimental method comprises the following steps:

MIA-PA-CA-2 cells were seeded in white 96-well plates, 80. Mu.L of cell suspension per well, containing 1000 MIA-PA-CA-2 cells. Cell plates were placed in a carbon dioxide incubator overnight for culture.

The test compounds were diluted 5-fold to the 8 th concentration, i.e. from 2mM to 26nM, using a row gun and a double multiplex well experiment was set up. 78. Mu.L of medium was added to the intermediate plate, and 2. Mu.L of the gradient diluted compound per well was transferred to the intermediate plate at the corresponding position, and 20. Mu.L of the gradient diluted compound per well was transferred to the cell plate after mixing. The concentration of compound transferred into the cell plate ranged from 10. Mu.M to 0.13nM. The cell plates were placed in a carbon dioxide incubator for 3 days. A cell plate was also prepared and the signal value read on the day of dosing as the maximum value (Max value in the following equation) was used in the data analysis. To this plate, 50. Mu.L of cell viability chemiluminescent detection reagent was added per well and incubated at room temperature for 10 minutes to stabilize the luminescent signal. Multiple marker analyzer readings were used.

Data analysis:

raw data was converted to inhibition, IC, using the equation (sample-min)/(max-min) ×100% for inhibition ₅₀ The values of (a) can be obtained by curve fitting four parameters (in the "log (inhibitor)/response-variable domain" mode in GraphPad Prism software).

Method 2: in vivo pharmacokinetic studies

Pharmacokinetic study of SD mice orally and intravenously injected test compounds

The tested compound is mixed with 10% dimethyl sulfoxide/60% polyethylene glycol 400/30% aqueous solution, vortex and ultrasonic are carried out, 1mg/mL clarified solution is prepared, and the filtration of a microporous filter membrane is carried out for later use. Male SD mice 7 to 10 weeks old were selected and the candidate compound solution was given by intravenous injection at a dose of 2-3mg/kg. The candidate compound solution was orally administered at a dose of 10mg/kg. Whole blood was collected for a certain period of time, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and drug substitution parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

Method 3: in vivo pharmacodynamic studies

In vivo pharmacodynamics study of human pancreatic cancer Mia PaCa-2 cell Nude mice subcutaneously transplanted tumor Balb/c Nude mouse model

1. Cell culture and tumor tissue preparation

Cell culture: human pancreatic cancer Mia PaCa-2 cells (ATCC-CRL-1420) are subjected to in vitro monolayer culture under the condition that 20% fetal bovine serum, 1% double antibody and 5% carbon dioxide incubator at 37 ℃ are added into DMEM/F12 culture medium. Passaging was performed twice a week with conventional digestion treatments with pancreatin-EDTA. When the cell saturation is 80% -90% and the number reaches the requirement, collecting the cells, counting, re-suspending in a proper amount of PBS, adding matrigel 1:1, obtaining the cell density of 25 x 10 ⁶ cell suspension of cells/mL.

Cell inoculation: 0.2mL (5X 10) ⁶ cell/mouse) Mia PaCa-2 cells (matrigel added, volume ratio 1:1) were inoculated subcutaneously on the right back of each mouse, tumor average volume reached 190mm ³ At the time, the tumor volumes are randomly grouped, the administration dose of each group is 0 in a blank group, the administration doses of the test group are 5mg/kg, 10mg/kg and 30mg/kg respectively, the administration volume is 10 mu L/g, and the oral administration is carried out for 21 days once a day.

2. Tumor measurement and experimental index

Tumor diameters were measured twice weekly with vernier calipers. The calculation formula of the tumor volume is: v=0.5a×b ² A and b represent the major and minor diameters of the tumor, respectively.

The tumor-inhibiting effect of the compound was evaluated by TGI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%) =trtv/crtv×100% (TRTV: treatment group RTV; CRTV: negative control group RTV). The relative tumor volume (relative tumor volume, RTV) was calculated from the results of the tumor measurements, with the calculation formula rtv=vt/V0, where V0 is the average tumor volume measured at the time of group administration (i.e., D0), vt is the average tumor volume at a certain measurement, and TRTV and CRTV take the same day data.

TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [ (1- (mean tumor volume at the end of the treatment group administration-mean tumor volume at the beginning of the treatment group administration))/(mean tumor volume at the end of the treatment with solvent control group-mean tumor volume at the beginning of the treatment with solvent control group) ]x100%.

Method 4: in vivo pharmacodynamic studies

In vivo pharmacodynamics study of human non-small cell lung cancer NCI-H358 cell Nude mice subcutaneously transplanted tumor Balb/c Nude mouse model

1. Cell culture and tumor tissue preparation

Cell culture: human non-small cell lung cancer NCI-H358 is cultured by adding 20% foetal calf serum, 1% double antibody and 5% carbon dioxide incubator at 37deg.C into DMEM/F12 culture medium. Passaging was performed twice a week with conventional digestion treatments with pancreatin-EDTA. When the cell saturation is 80% -90% and the number reaches the requirement, collecting cells, counting, re-suspending in PBS, adding matrigel 1:1 to obtain cell density of 25X10 ⁶ cell suspension of cells/mL.

Cell inoculation: 0.2mL (5X 10) ⁶ cells/mouse) NCI-H358 cells (matrigel added with volume ratio of 1:1) were inoculated subcutaneously on the right back of each mouse, and the average tumor volume reached 100-150mm ³ At the time, 8 animals are randomly grouped according to tumor volume, the administration dose of a blank group is 0, the administration doses of a test group are 1.5mg/kg, 5mg/kg and 15mg/kg respectively, the administration volume is 10 mu L/g, and the administration is orally carried out for 28 days once a day.

2. Tumor measurement and experimental index

Technical effects

Pairs of KRAS of the present Compounds ^G12C The target has an inhibiting effect, and shows better inhibiting activity on proliferation of MIA-PA-CA-2 and NCI-H358 cells. Has better inhibition effect on the tumor growth of MiaPaCa-2 xenograft and NCI-H358 xenograft, and the compound has excellent pharmacokinetic property. The compound has good plasma stability, whole blood stability and GSH phosphate buffer solution stability. The compound provided by the invention has no PXR positive activation and no CYP induced activation. The compound has obvious tumor inhibiting effect and no obvious intolerance.

Correlation definition

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular term or phrase, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

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

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention prepared from the compounds of the present invention which have the specified substituents found herein with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts may be obtained by contacting such compounds with a sufficient amount of base in pure solution or in a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, the acid addition salts may be obtained by contacting such compounds with a sufficient amount of acid in pure solution or in a suitable inert solvent. Certain specific compounds of the invention contain basic and acidic functionalities that can be converted to either base or acid addition salts.

Pharmaceutically acceptable salts of the invention can be synthesized from the parent compound containing an acid or base by conventional chemical methods. In general, the preparation of such salts is as follows: prepared via reaction of these compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.

The compounds of the 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 invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of the present invention.

The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more of the atoms comprising the compounds. For example, compounds can be labeled with radioisotopes, such as tritium @, for example ³ H) Iodine-125% ¹²⁵ I) Or C-14% ¹⁴ C) A. The invention relates to a method for producing a fibre-reinforced plastic composite For example, deuterium can be substituted for hydrogen to form a deuterated drug, and the bond between deuterium and carbon is stronger than the bond between normal hydrogen and carbon, so that the deuterated drug has the advantages of reducing toxic and side effects, increasing the stability of the drug, enhancing the curative effect, prolonging the biological half-life of the drug and the like compared with the non-deuterated drug. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

The term "optional" or "optionally" means 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.

The term "substituted" means that any one or more hydrogen atoms on a particular atom is substituted with a substituent, which may include deuterium and variants of hydrogen, provided that the valence 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 substituted. Oxygen substitution does not occur on the aromatic group.

The term "optionally substituted" means that the substituents may or may not be substituted, and the types and numbers of substituents may be arbitrary on the basis that they can be chemically achieved unless otherwise specified.

When any variable (e.g., R) occurs more than once in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0 to 2R, the group may optionally be substituted with up to two R's, and R's in each case have independent options. Furthermore, combinations of substituents and/or variants thereof are only permissible if such combinations result in stable compounds.

When the number of one linking group is 0, such as- (CRR) _n -it is meant that the linking group is a single bond.

When one of the variables is selected from a single bond, the two groups to which it is attached are indicated as being directly linked, e.g., when L in A-L-Z represents a single bond, it is indicated that the structure is actually A-Z.

When the exemplified linking group does not indicate its linking direction, its linking direction is arbitrary, for example,the linking group L is-M-W-, in which case-M-W-may be a group formed by linking the rings A and B in the same direction as the reading order from left to rightThe ring A and the ring B may be connected in a direction opposite to the reading order from left to rightCombinations of such linking groups, substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

Unless otherwise specified, when a group has one or more bondable sites, any one or more of the sites of the group may be bonded to other groups by chemical bonds. When the connection mode of the chemical bond is not positioned and the H atoms exist in the connectable site, the number of the H atoms of the site can be correspondingly reduced to be changed into the corresponding valence group along with the number of the connected chemical bond when the chemical bond is connected. The chemical bond of the site and other groups can be a straight solid line bondStraight dotted line keyOr wave linesAnd (3) representing. For example, a straight solid bond in-OCH 3 indicates that it is attached to other groups through an oxygen atom in that group;the straight dashed bonds in (a) represent the attachment to other groups through both ends of the nitrogen atom in the group;the wavy line means that the carbon atoms at positions 1 and 2 in the phenyl group are attached to other groups;it means that any of the ligatable sites on the piperidinyl group may be attached to other groups by 1 chemical bond, including at least These 4 connection modes, even though H atom is drawn on-N-, areStill includeThe group of this linkage is only when 1 chemical bond is linked, the H at this site will be correspondingly reduced by 1 to the corresponding monovalent piperidinyl group.

Unless otherwise specified, the term "C _1-6 Alkyl "is used to denote a straight or branched saturated hydrocarbon group consisting of 1 to 6 carbon atoms. The C is _1-6 Alkyl includes C _1-5 、C _1-4 、C _1-3 、C _1-2 、C _2-6 、C _2-4 、C ₆ And C ₅ Alkyl groups, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C (C) _1-6 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl)Radical), hexyl radical, and the like.

Unless otherwise specified, the term "C _1-3 Alkyl "is used to denote a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C is _1-3 Alkyl includes C _1-2 And C _2-3 Alkyl groups, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C (C) _1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise specified, C _n-n+m Or C _n -C _n+m Comprising any one of the specific cases of n to n+m carbons, e.g. C _1-12 Comprises C ₁ 、C ₂ 、C ₃ 、C ₄ 、C ₅ 、C ₆ 、C ₇ 、C ₈ 、C ₉ 、C ₁₀ 、C ₁₁ And C ₁₂ Also included is any one of the ranges n to n+m, e.g. C _1-12 Comprises C _1- ₃ 、C _1-6 、C _1-9 、C _3-6 、C _3-9 、C _3-12 、C _6-9 、C _6-12 And C _9-12 Etc.; similarly, n-membered to n+m-membered means that the number of atoms on the ring is n to n+m, for example, 3-12 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring, and 12-membered ring, and any one of n to n+m is also included, for example, 3-12-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring, 5-7-membered ring, 6-8-membered ring, 6-10-membered ring, and the like.

The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments set forth below, embodiments formed by combining with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention.

The compound of the invention has a three-dimensional configuration and can be determined by single crystal diffraction, optical rotation, CD and other test methods.

The compounds of the present invention may be structured by conventional methods well known to those skilled in the art, and if the present invention relates to the absolute configuration of a compound, the absolute configuration may be confirmed by conventional means in the art. For example, single crystal X-ray diffraction (SXRD), the grown single crystal is collected from diffraction intensity data using a Bruker D8 vent diffractometer, and the light source is cukα radiation, scanning:after scanning and collecting the relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by a direct method (Shellxs 97).

Description of the drawings:

FIG. 1 AMG510 and KRAS ^G12C Binding mode of protein (active conformation: 6 IOM);

FIG. 2 AMG510 and KARS ^G12C Is a combined mode 2D map of (2);

FIG. 3 is a graph of the superposition of the low-energy conformation of Compound A and the active conformation of AMG 510;

FIG. 4 is a diagram of the superposition of the low-energy conformation of Compound B and the active conformation of AMG 510;

FIG. 5 is a diagram of the superposition of the low-energy conformation of Compound C and the active conformation of AMG 510;

FIG. 6 is a diagram of the superposition of the low-energy conformation of Compound D and the active conformation of AMG 510;

FIG. 7 is a graph of the superposition of the low-energy conformation of Compound E with the active conformation of AMG 510;

FIG. 8 is a graph of tumor volumes studied in the Nude mice model of subcutaneously transplanted tumor Balb/c in Nude mice with Mia PaCa-2 cells of human pancreatic cancer;

FIG. 9 is a graph showing the change in body weight of a model study of Nude mice with tumor Balb/c Nude transplanted subcutaneously in Nude mice with Mia PaCa-2 cell of human pancreatic cancer.

Detailed Description

The present invention is described in detail below by way of examples, but is not meant to be limiting in any way. The present invention has been described in detail herein, and specific embodiments thereof are also 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 invention without departing from the spirit and scope of the invention.

Calculation example 1

The low energy conformation of the AMG510 was calculated by the Macromodel module of the schrodinger Maestro software. To lock the active conformation of AMG510 and further reduce its rotational energy barrier, we have cyclized the fluorophenol and isopropylmethylpyridine fragments of AMG510 via a linker chain to obtain a series of different macrocyclic molecules with the fluorophenol and isopropylmethylpyridine fragments linked in a chain, and explored these macrocyclic molecules with AMG510 and KRAS ^G12C Binding differences between active conformations in protein co-crystals. AMG510 and KRAS ^G12C The binding pattern of the proteins is shown in FIG. 1, AMG510 and KARS ^G12C The binding pattern 2D of compounds a-E is shown in fig. 2 and the superposition of the low energy conformation of compounds a-E with the active conformation of AMG510 is shown in fig. 3-8.

Conclusion: the compound has high coincidence degree with the active conformation of AMG 510.

Example 1

The synthetic route is as follows:

step 1: synthesis of Compound 001-3

Into a 5.0 liter three-necked flask dried in advance, the compound 001-1 (300 g,1.84mol,1.00 eq), 001-2 (isopropenyl potassium trifluoroborate, 300g,2.03mol,1.10 eq), potassium carbonate (3831 g,2.76mol,1.5 eq), 1, 4-dioxane (3.0 liter) and water (0.3 liter) were charged. After the addition was completed, nitrogen was replaced three times. 1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (16.40 g,22.41mmol,1.22e-2 eq) was then added to the system. After the completion of the addition, the reaction system was stirred at 90℃for 12 hours. The system was concentrated. Water (1.2L) and ethyl acetate (2.0L) were added to the system. And (5) standing and separating the system. The aqueous phase was extracted with ethyl acetate (1.0 l,1 time). The organic phases were combined, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated. The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate=8:1 to 5:1) to give compound 001-3.

LCMS：MS m/z：169.0[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝7.91-7.83(m，1H)，7.11-7.04(m，1H)，5.53-5.45(m，1H)，5.34-5.24(m，1H)，4.48-4.21(m，2H)，2.14(s，3H)。

Step 2: synthesis of Compound 001-5

To a pre-washed 3.0 liter single-necked flask, 001-3 (119.3 g,707.49mmol,1.00 eq), 001-4 (300.05 g,1.41mol,2.0 eq), potassium phosphate (300.36 g,1.41mol,2.0 eq), 2-methyltetrahydrofuran (1200 mL) and water (300 mL) were added. After the addition was completed, nitrogen was replaced three times. 1,1' -Didiphenylphosphino ferrocene palladium dichloride (46.11 g,70.75mmol,0.1 eq) was then added to the system. After the addition was completed, nitrogen was replaced three times. The reaction was stirred at 76 ℃ (system temperature) for 12 hours. The reaction system was cooled to room temperature, diluted with 1.5l of water and 2.0 l of ethyl acetate, and the organic phase was collected after separation, and the aqueous phase was extracted with ethyl acetate (1.0 l,2 times). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a residue. The crude product was purified by flash column chromatography (petroleum ether: ethyl acetate=10:1 to 4:1) to give compound 001-5.

LCMS：MS m/z：219.1[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝7.99(d，J＝5.2Hz，1H)，7.77(d，J＝16.0Hz，1H)，7.08(d，J＝5.2Hz，1H)，6.47(d，J＝15.6Hz，1H)，5.50(s，1H)，5.28(s，1H)，4.31(br s，2H)，3.81(s，3H)，2.15(s，3H)。

Step 3: synthesis of Compound 001-6

Palladium on carbon (17 g,10% strength) was added to a 2.0 liter hydrogenation flask replaced with argon. After the addition was completed, tetrahydrofuran (1.5L), compounds 001-5 (85 g,389.46mmol,1 eq) and sodium carbonate (41.28 g,389.46mmol,1.0 eq) were added to the system. After the addition was completed, the system was replaced with hydrogen 3 times. The hydrogen atmosphere of the system is at 25 ℃ and the pressure: 15psi, and stirred for 1.0 hour. The reaction was filtered through celite and the filter cake was washed with ethyl acetate (200 ml,3 times). The combined filtrates were concentrated directly to compounds 001-6.

LCMS：MS m/z：223.1[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝7.95(d，J＝4.8Hz，1H)，6.78(d，J＝5.2Hz，1H)，3.90-3.77(m，2H)，3.67(s，3H)，3.08-2.97(m，1H)，2.80(t，J＝7.2Hz，2H)，2.66(t，J＝7.2Hz，2H)，1.28(s，3H)，1.27(s，3H)。

Step 4: synthesis of Compounds 001-7

To a 3.0L three-necked flask dried in advance, the compound 001-6 (150 g,674.82mmol,1 eq) and tetrahydrofuran (1.5L) were added. Then, the temperature of the system was lowered to 0 to 5℃and lithium borohydride (29.29 g,1.34mol,1.99 eq) was added to the system in portions. After the addition was completed, the temperature was returned to 25℃and reacted at this temperature for 1.0 hour. The system was slowly added to a stirred saturated ammonium chloride 4.0 liter quench. 1.5L of ethyl acetate was added for dilution, and the organic phase was collected after separation and the aqueous phase was extracted with ethyl acetate (1.0L, 2 times). The combined organic phases were washed with saturated brine (300 mL,1 time), dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the compound 001-7, which was used in the next step without purification.

LCMS：MS m/z：195.1[M+1] ⁺ 。

Step 5: synthesis of Compounds 001-8

To a 3.0L three-necked flask dried in advance was added compound 001-7 (130 g,669.16mmol,1.00 eq), imidazole (68.33 g,1.00mol,1.5 eq) and methylene chloride (2.00L), and after the addition was completed, the system was cooled to 0 to 10℃and t-butyldimethylchlorosilane (102.63 g,680.94mmol,83.44mL, 1.02eq) methylene chloride (500 mL) was slowly added dropwise. After the addition was completed, the temperature of the system was slowly returned to 25℃and reacted at this temperature for 12 hours. 300mL of saturated ammonium chloride was carefully added to the reaction system to quench the reaction. The system was left to stand, the organic phase was collected after separation, and the aqueous phase was extracted with dichloromethane (500 mL,1 time). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a residue. The crude product was purified by flash column chromatography (petroleum ether: ethyl acetate=10:1 to 6:1). To obtain the compound 001-8.

LCMS：MS m/z：309.2[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝7.93(d，J＝4.8Hz，1H)，6.81(d，J＝4.8Hz，1H)，3.87(br s，2H)，3.63(t，J＝5.6Hz，2H)，3.01-2.95(m，1H)，2.59(t，J＝7.2Hz，2H)，1.85-1.72(m，2H)，1.30(s，3H)，1.28(s，3H)，0.90(s，9H)，0.06(s，6H)。

Step 6: synthesis of Compounds 001-10

To a previously dried 3.0L single-necked flask were added 001-9 (70 g,334.92mmol,1.00 eq) and methylene chloride (0.7L) under nitrogen. After the sample was dissolved, oxalyl chloride (70 g,551.50mmol,48.28mL,1.65 eq) was slowly added dropwise to the system while maintaining the temperature of the system at 20 to 25 ℃. After the addition, the temperature of the system is raised to 40-45 ℃ and the reaction is carried out for 5 hours. The reaction system was concentrated to give a yellow compound which was directly put into the next step without purification.

To a 3.0L single-necked flask containing the above yellow compound was added methylene chloride (0.7L) under nitrogen. After the sample was dissolved, the system temperature was cooled to 0℃and a solution of compound 001-8 (108.49 g,351.64mmol,1.05 eq) in methylene chloride (0.28L) was slowly added dropwise to the system while maintaining the temperature at 0 ℃. After the addition was completed, the temperature of the system was recovered to 25℃and the system was reacted at 25℃for 12 hours. The reaction system was concentrated directly. The crude product was purified by flash column chromatography (petroleum ether: ethyl acetate=5:1 to 1:1) to give compound 001-10.

LCMS：MS m/z：543.1[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝10.13(s，1H)，9.71(s，1H)，8.46(d，J＝5.2Hz，1H)，7.82(d，J＝6.8Hz，1H)，7.06(d，J＝4.8Hz，1H)，3.62(t，J＝6.0Hz，2H)，3.27-3.12(m，1H)，2.68-2.57(m，2H)，1.81-1.72(m，2H)，1.21(s，3H)，1.19(s，3H)，0.84(s，9H)，0.00(s，6H)。

Step 7: synthesis of Compounds 001-11

To a 3.0L three-necked flask dried in advance, the compound 001-10 (139 g,255.73mmol,1 eq), 18-crown-6 (33.80 g,127.87mmol,0.5 eq) and tetrahydrofuran (1.39L) were added under nitrogen. After the solution became clear, the system was cooled to 0 to 5℃and potassium hexamethyldisilazide (1M, 460.32mL,1.8 eq) was slowly added dropwise to the system. After the addition was completed, the system was warmed to 25℃and stirred at this temperature for 12 hours. 2 parallel reactions were post-treated and purified. Saturated ammonium chloride (1.0L) was added to each reaction system to quench the reaction. The 2 individuals were combined and extracted. 1.0L of water and 1.0L of ethyl acetate are added into the system for dilution, and the organic phase is collected after standing and liquid separation. The aqueous phase was extracted with ethyl acetate (600 mL,1 time). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a residue. The crude product was purified by flash column chromatography (petroleum ether: ethyl acetate=5:1 to 2:1) to give compound 001-11.

LCMS：MS m/z：507.2[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝9.24(br s，1H)，8.64(d，J＝5.2Hz，1H)，8.23(d，J＝6.8Hz，1H)，7.18(d，J＝4.8Hz，1H)，3.52(t，J＝6.0Hz，2H)，2.71-2.62(m，1H)，2.41(t，J＝8.4Hz，2H)，1.74-1.61(m，2H)，1.21(d，J＝6.8Hz，3H)，1.09(d，J＝6.8Hz，3H)，0.79-0.73(m，9H)，-0.11--0.05(m，6H)。

Step 8: synthesis of Compounds 001-12

To a 3.0 liter three-necked flask, which had been dried in advance, were added, under nitrogen, 001-11 (91.85 g,181.14mmol,1.00 eq), diisopropylethylamine (37.46 g,289.82mmol,50.48mL,1.6 eq) and tetrahydrofuran (920 mL). After the addition was completed, the temperature of the system was lowered to 0 to 5℃and phosphorus oxychloride (41.66 g,271.71mmol,25.25mL,1.5 eq) was slowly added to the system. After the addition, the system was slowly warmed to 40℃and stirred at this temperature for 2.0 hours to give the compound 001-12. The reaction solution was directly used in the next step

Step 9: synthesis of Compounds 001-14

A reaction system containing the compounds 001-12 (95.19 g,181.14mmol,1.00 eq) was cooled to 0-10℃and maintained under nitrogen, and a solution of diisopropylethylamine (58.52 g,452.84mmol,78.87mL,2.5 eq) and a pre-dissolved compound 001-13 (72.55 g,362.27mmol,2.0 eq) in tetrahydrofuran (210 mL) was added to the system. After the addition was complete, the system was stirred at 25℃for 12.0 hours. 2 parallel reactions were combined. The system temperature is controlled to be 20-25 ℃, and 1.0 liter of saturated sodium chloride is added into the system to quench the reaction. And (5) standing and separating the system. The aqueous phase was extracted with ethyl acetate (0.5L, 2 times). The organic phases were combined, washed with brine (0.5 l,1 times), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to give crude intermediate compound which was used in the next step without purification.

LCMS：MS m/z：689.4[M+1] ⁺ 。

To a 1.0L single-necked flask, under nitrogen protection, the above intermediate compound, acetic acid (349.65 g,5.82mol,333mL,36.16 eq), tetrahydrofuran (111 mL) and water (111 mL) were added. After the addition was complete, the system was stirred at 25℃for 12.0 hours. 500mL of water was added to the reaction system. Cooled to 0-5 ℃, and the pH value of the system is regulated to 7-8 by 5.0M sodium hydroxide (-5.0L). And (5) standing and separating the system. The aqueous phase was extracted with ethyl acetate (1.0L, 3 times). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by flash column chromatography (dichloromethane: methanol=40:1 to 20:1) to give compound 001-14.

LCMS：MS m/z：575.2[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.59(d，J＝4.8Hz，1H)，7.76(dd，J＝2.0，8.0Hz，1H)，7.17(d，J＝5.2Hz，1H)，4.99-4.58(m，1H)，4.42-3.78(m，3H)，3.74-3.39(m，3H)，3.36-2.92(m，3H)，2.63-2.47(m，1H)，2.45-2.28(m，2H)，1.52-1.37(m，13H)，1.18(dd，J＝4.0，6.8Hz，3H)，1.06(t，J＝6.4Hz，3H)。

Step 10: synthesis of Compounds 001-16

Compounds 001-14 (50 g,76.30mmol,87.76% purity, 1 eq), 001-15 (14.28 g,91.56mmol,1.2 eq) and anhydrous potassium phosphate (32.39 g,152.60mmol,2.0 eq) were dissolved in 2-methyltetrahydrofuran (250 mL) and water (100 mL), nitrogen was replaced three times, and [1, 1-bis (di-t-butylphosphino) ferrocene ] palladium (II) dichloride (2.49 g,3.81mmol,0.05 eq) was added and the reaction was heated to 75℃and stirred for 12 hours. The reaction solution was cooled to room temperature, filtered, and the filter cake was washed with 50mL of 2-methyltetrahydrofuran. The white filter cake was collected and concentrated under reduced pressure to remove the solvent. The white solid was dried in a vacuum oven at 45℃for 20 hours to give the compound 001-16.

LCMS：MS(ESI)m/z：651.4[M+1] ⁺ 。

SFC analysis method (column: chiralpak IC-3, 50X 4.6mm I.D.,3 μm; mobile phase: A (CO) ₂ ) And B (EtOH, containing 0.1% isopropylamine); gradient: b% = 5-50-5%, 3.0min; flow rate: 3.4mL/min; wavelength: 220nm; pressure: 180 psi, rt=1.321 min, chiral isomer excess 95.58%. ¹ H NMR(400MHz，CD ₃ OD)δppm 8.44(d，J＝4.8Hz，1H)，8.19(d，J＝8.8Hz，1H)，7.29(d，J＝4.8，1H)，7.19(q，J＝7.6Hz，1H)，6.61(d，J＝8.4Hz，1H)，6.51(t，J＝8.8Hz，1H)，5.00(br s，1H)，4.40(br d，J＝13.6Hz，1H)，4.07-4.20(m，1H)，3.99(br d，J＝13.2Hz，1H)，3.71-3.84(m，1H)，3.35-3.50(m，3H)，3.15-3.25(m，1H)，2.68-2.86(m，1H)，2.41(br t，J＝7.6Hz，2H)，1.60-1.78(m，2H)，1.44-1.58(m，12H)，1.19(d，J＝6.8Hz，3H)，1.02(d，J＝6.8Hz，3H)。

Step 11: synthesis of Compounds 001-17

Compounds 001-16 (5.00 g,7.68mmol,1 eq) and tributylphosphine oxide (2.64 g,13.06mmol,3.22mL,1.7 eq) were dissolved in anhydrous dichloromethane (130 mL), replaced with nitrogen three times, cooled to 0deg.C and 1, 1-azodicarbonyl dipiperidine (3.30 g,13.06mmol,1.7 eq) was added in portions. After the addition, stirring was carried out at 15℃for 12 hours. The reaction solution was concentrated under reduced pressure. The crude product was slurried with 150mL of methyl tert-butyl ether, filtered, and the filtrate concentrated under reduced pressure to give the crude compound 001-17.

LCMS：m/z(ESI)＝633.3[M+H] ⁺ 。

Step 12: synthesis of Compounds 001-18

Compounds 001-17 (9.1 g,14.38mmol,1 eq) were dissolved in anhydrous dichloromethane (45 mL), trifluoroacetic acid (15 mL) was added and stirred for 5 hours at 15deg.C. The reaction mixture was concentrated under reduced pressure, the crude product was slurried with methyl tert-butyl ether ((100 mL 4)), filtered, and the solid was collected, dissolved in 100mL of dichloromethane, and 100mL of saturated aqueous sodium bicarbonate solution was added, and the aqueous phase was extracted with dichloromethane ((40 mL 2)) the organic phases were combined, washed with saturated brine ((40 mL)), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 001-18.

LCMS：m/z(ESI)＝533.4[M+H] ⁺ 。

Step 13: synthesis of Compound 001

To a predried single vial was added 001-18 (213 mg, 399.94. Mu. Mol,1 eq), (E) -4-fluorobut-2-enoic acid (49.95 mg, 479.93. Mu. Mol,1.2 eq), methylene chloride (2 mL), diisopropylethylamine (129.22 mg, 999.84. Mu. Mol, 174.15. Mu.L, 2.5 eq) cooled to 0deg.C, O- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (187.04 mg, 491.92. Mu. Mol,1.23 eq) was added and stirred at 20deg.C for 16 hours. The reaction solution was concentrated to dryness under reduced pressure, methanol (2 mL) was added, filtered, and the filtrate was separated and purified by high performance liquid chromatography (column: phenomenex C18 x 40mm x 3 μm; mobile phase: [ water (ammonium bicarbonate) -acetonitrile ];: acetonitrile%: 25% -55%,8 min). Compound 001 was obtained.

LCMS：m/z(ESI)＝619[M+H] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δppm 8.60(d，J＝5.00Hz，1H)，7.83(d，J＝8.50Hz，1H)，7.40(td，J＝8.32，6.75Hz，1H)，7.15(d，J＝5.13Hz，1H)，6.98-7.13(m，1H)，6.94(d，J＝8.25Hz，1H)，6.87(t，J＝8.63Hz，1H)，6.55-6.75(m，1H)，5.05-5.28(m，2H)，4.53-4.99(m，3H)，4.34-4.45(m，1H)，3.89-4.15(m，1H)，3.42-3.84(m，3H)，3.13-3.36(m，1H)，2.98(dt，J＝13.38，6.69Hz，1H)，2.40-2.71(m，2H)，2.11-2.37(m，2H)，1.65-1.77(m，3H)，1.34(d，J＝6.75Hz，3H)，1.08(d，J＝6.75Hz，3H)。

SFC analysis method (column: chiralcel OD-3, 50X 4.6mm I.D.,3 μm; mobile phase: A (CO) ₂ ) And B (EtOH, containing 0.1% isopropylamine); gradient: b% = 5-50-5%, 3.0min; flow rate: 3.4mL/min; wavelength: 220nm; pressure: 180 psi, rt=1.332 min, chiral isomer excess 96.66%.

Example 2

Step 1: synthesis of Compound 002-1

Compounds 001-17 (3 g,4.74mmol,1 eq) were dissolved in 4N methanolic hydrochloric acid (30 mL) and stirred at 20℃for 12 hours. The reaction mixture was concentrated under reduced pressure, and ethyl acetate (30 mL) and saturated aqueous sodium hydrogencarbonate solution (30 mL) were added to separate the solutions. The aqueous phase was extracted with ethyl acetate (15 ml x 2) the organic phases were combined, dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. The crude product is separated and purified by silica gel column chromatography (gradient eluent is dichloromethane: methanol=50:1-10:1) to obtain the compound 002-1.

LCMS：m/z(ESI)＝451.1[M+H] ⁺ 。

¹ H NMR(400MHz，CD ₃ Cl)δppm 9.29(s，1H)，8.61(d，J＝4.8Hz，1H)，8.30(d，J＝8.0Hz，1H)，7.31-7.45(m，1H)，7.14(d，J＝5.2Hz，1H)，6.76-6.97(m，2H)，4.32-4.45(m，1H)，3.54-3.70(m，1H)，2.87-3.01(m，1H)，2.60-2.71(m，2H)，2.16-2.32(m，2H)，1.33(d，J＝6.8Hz，3H)，0.99(d，J＝6.8Hz，3H)。

Step 2: synthesis of Compound 002-2

Compound 002-1 (2.1 g,4.66mmol,1 eq) and N, N-diisopropylethylamine (964.08 mg,7.46mmol,1.30mL,1.6 eq) were dissolved in anhydrous tetrahydrofuran (21 mL), replaced with nitrogen three times, and phosphorus oxychloride (1.07 g,6.99mmol, 649.87. Mu.L, 1.5 eq) was added dropwise. After the addition, the mixture was heated to 50℃and stirred for 1 hour to give Compound 002-2. The product was calculated on a theoretical basis without treatment and used directly in the next step.

Step 3: synthesis of Compound 002-4

To a tetrahydrofuran solution of compound 002-2 (2.19 g,4.67mmol,1 eq) was added N, N-diisopropylethylamine (1.51 g,11.68mmol,2.03mL,2.5 eq), replaced with nitrogen three times, cooled to 0℃and a solution of compound 002-3 (1.20 g,5.60mmol,1.2 eq) in anhydrous tetrahydrofuran (10 mL) was added dropwise. After the dripping is finished, naturally heating to 20 ℃, and stirring for 12 hours. The reaction was poured into 100mL of saturated brine and extracted with ethyl acetate (15 mL x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product is separated by silica gel column chromatography and purified (gradient eluent is dichloromethane: methanol=50:1-10:1) to obtain the compound 002-4.

LCMS：m/z(ESI)＝647.4[M+H] ⁺ 。

¹ H NMR(400MHz，CD ₃ Cl)δppm 8.56(d，J＝5.2Hz，1H)，7.79-7.89(m，1H)，7.29-7.42(m，1H)，7.13(d，J＝4.8Hz，1H)，6.90(d，J＝8.4Hz，1H)，6.82(t，J＝8.8Hz，1H)，4.61-4.73(m，1H)，4.32-4.56(m，2H)，3.78-4.00(m，1H)，3.56-3.70(m，2H)，3.21-3.47(m，2H)，2.86-3.02(m，1H)，2.39-2.67(m，2H)，2.10-2.33(m，2H)，1.36-1.64(m，15H)，1.29(d，J＝6.8Hz，3H)，1.07(d，J＝6.8Hz，3H)。

Step 4: synthesis of Compound 002-5

Compound 002-4 (3.2 g,4.95mmol,1 eq) was dissolved in anhydrous dichloromethane (30 mL), trifluoroacetic acid (10 mL) was added and stirred at 25℃for 2 hours. The reaction was concentrated under reduced pressure, 20mL of dichloromethane and 20mL of water were added, the pH was adjusted to 8 with saturated aqueous sodium bicarbonate, the solution was separated and the aqueous phase was extracted with dichloromethane (15 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product is separated and purified by silica gel column chromatography (gradient eluent is dichloromethane: methanol=30:1-5:1) to obtain compound 002-5.

LCMS：m/z(ESI)＝547.4[M+H] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δppm 8.55(d，J＝5.2Hz，1H)，7.87(d，J＝8.8Hz，1H)，7.31-7.46(m，1H)，7.11(d，J＝4.8Hz，1H)，6.91(d，J＝8.4Hz，1H)，6.82(t，J＝8.8Hz，1H)，4.52-4.68(m，1H)，4.32-4.46(m，2H)，3.30-3.79(m，5H)，2.81-3.00(m，2H)，2.56-2.66(m，1H)，2.44-2.54(m，1H)，2.13-2.30(m，2H)，1.68(d，J＝6.8Hz，3H)，1.42(d，J＝6.4Hz，3H)，1.29(d，J＝6.8Hz，3H)，1.06(d，J＝6.8Hz，3H)。

Step 5: synthesis of Compound 002

Compound 002-5 (100 mg, 182.95. Mu. Mol,1 eq) and (E) -4-fluorobut-2-enoic acid (57.12 mg, 548.84. Mu. Mol,3 eq) were dissolved in methylene chloride (4 mL) under nitrogen, then N, N-diisopropylethylamine (141.86 mg,1.10mmol, 191.19. Mu.L, 6 eq) was added, O- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (104.34 mg, 274.42. Mu. Mol,1.5 eq) was added at 0℃and the temperature was raised to 20℃for reaction for 2 hours. To the reaction system was added a saturated sodium bicarbonate solution (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane (20 ml x 3) and separated. The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product was purified by high performance liquid chromatography (column Waters Xbridge Prep OBD C: 150 x 40mm x 10 μm; mobile phase: a (acetonitrile) and B (water, 0.04% ammonium bicarbonate; gradient: B%:40% -60%,8 min) to give compound 002.

LCMS：MS m/z：633.7[M+1] ⁺ .

¹ H NMR(400MHz，CD ₃ OD)δ＝8.49(d，J＝5.2Hz，1H)，8.13(dd，J＝8.8，12.0Hz，1H)，7.49-7.35(m，2H)，7.05(d，J＝8.4Hz，1H)，7.01-6.89(m，1H)，6.89-6.84(m，1H)，6.80(s，1H)，5.19(m，1H)，5.10-4.74(m，2H)，4.60(s，2H)，4.49-4.38(m，2H)，3.92-3.76(m，1H)，3.68(m，1H)，3.60-3.52(m，1H)，3.06-2.96(m，1H)，2.58-2.35(m，3H)，2.22-2.08(m，1H)，1.60(m，3H)，1.51-1.41(m，3H)，1.25(d，J＝6.8Hz，3H)，1.02(dd，J＝3.2，6.8Hz，3H)。

SFC analysis method (column: chiralcel OD-3, 50X 4.6mm I.D.,3 μm; mobile phase: A (CO) ₂ ) And B (MeOH, 0.1% isopropylamine); gradient: b% = 5-50-5%, 3.0min; flow rate: 3.4mL/min; wavelength: 220nm; pressure: 180 psi, rt= 1.256min, chiral isomer excess 99.72%.

Example 3

Step 1: synthesis of Compound 002-2

Compound 002-1 (0.8 g,1.78mmol,1 eq) was dissolved in tetrahydrofuran (12 mL), N-diisopropylethylamine (688.61 mg,5.33mmol, 928.05. Mu.L, 3 eq) was added, followed by phosphorus oxychloride (680.81 mg,4.44mmol, 412.61. Mu.L, 2.5 eq) and reacted at 40℃for 3 hours under nitrogen. Compound 002-2 was obtained, and the reaction system was used directly in the next step.

Step 2: synthesis of Compound 003-2

Compound 002-2 (0.8 g,1.71mmol,1 eq) was dissolved in tetrahydrofuran (13 mL), N-diisopropylethylamine (1.10 g,8.53mmol,1.49mL,5 eq) was added, and then compound 003-1 (596.07 mg,2.22mmol,1.3eq, HCl) was added at 0deg.C and reacted for 15 hours at 20deg.C. N, N-diisopropylethylamine (1.10 g,8.53mmol,1.49mL,5 eq) was added and reacted at 20℃for 15 hours. The reaction was carried out at 50℃for 15 hours because of incomplete reaction. N, N-diisopropylethylamine (1.10 g,5 eq) was added and reacted at 70℃for 30 hours. The reaction was added to ice water (50 mL), the aqueous phase was extracted with ethyl acetate (50 mL x 3) and the solution was separated. The organic phases were combined, washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (mobile phase: petroleum ether: ethyl acetate=10:1 to 1:3, dichloromethane: methanol=40:1 to 30:1) to give compound 003-2.

LCMS：MS m/z：665.2[M+1] ⁺ .

¹ H NMR(400MHz，CDCl ₃ )δ＝8.55(dd，J＝5.2，2.8Hz，1H)，7.81-7.71(m，1H)，7.41-7.32(m，6H)，7.09(d，J＝5.2Hz，1H)，6.89(d，J＝8.4Hz，1H)，6.82(t，J＝8.8Hz，1H)，5.60(s，1H)，5.21(s，2H)，4.88-4.82(m，2H)，4.45-4.34(m，2H)，4.12-4.10(m，1H)，3.77-3.71(m，1H)，3.65-3.59(m，1H)，2.96-2.86(m，1H)，2.71-2.50(m，3H)，2.27-2.12(m，3H)，1.33(d，J＝6.8Hz，3H)，0.96(dd，J＝10.8，6.8Hz，3H)。

Step 3: synthesis of Compound 003-3

Compound 003-2 (0.6 g, 902.66. Mu. Mol,1 eq) was dissolved in tetrahydrofuran (30 mL), palladium on carbon (0.5 g, 902.66. Mu. Mol,10% content, 1.00 eq) was added under nitrogen, hydrogen was replaced three times, and then reacted under a hydrogen atmosphere (15 Psi) at 25℃for 8 hours. The reaction system is directly filtered, and the filtrate is decompressed and concentrated to obtain the compound 003-3, which can be directly used for the next reaction without purification.

LCMS：MS m/z：531.1[M+1] ⁺ .

¹ H NMR(400MHz，CDCl ₃ )δ＝8.55(dd，J＝5.2，2.0Hz，1H)，7.92-7.85(m，1H)，7.35(q，J＝8.0Hz，1H)，7.10(d，J＝5.2Hz，1H)，6.89(d，J＝8.4Hz，1H)，6.82(t，J＝8.8Hz，1H)，5.63-5.57(m，1H)，4.96-4.80(m，1H)，4.67-4.59(m，1H)，4.37-4.35(m，1H)，3.77-3.74(m，2H)，3.67-3.62(m，1H)，3.59-3.49(m，1H)，2.93-2.84(m，1H)，2.61-2.51(m，3H)，2.27-2.14(m，2H)，1.88-1.84(m，2H)，1.32(dd，J＝6.4，2.0Hz，3H)，0.96(dd，J＝9.6，6.8Hz，3H)。

Step 4: synthesis of Compounds 003 and 004

Compound 003-3 (0.1 g, 188.48. Mu. Mol,1 eq) was dissolved in methylene chloride (5 mL), N-diisopropylethylamine (121.79 mg, 942.39. Mu. Mol, 164.14. Mu.L, 5 eq) was added, and then acryloyl chloride (34.12 mg, 376.96. Mu. Mol, 30.74. Mu.L, 2 eq) was added, followed by reaction at 60℃for 10 minutes under nitrogen. To the reaction system was added a saturated sodium bicarbonate solution (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane (50 ml x 3) and separated. The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the crude product. The crude product is separated by high performance liquid chromatography (chromatographic column: phenomnex Luna 80 x 30mm x 3 μm; mobile phase: A (acetonitrile) and B (water, 0.04% hydrochloric acid; gradient: B%:5% -35%,8 min), purified to obtain the pure product. The pure product was further resolved by SFC (column: REGIS (S, S) WHELK-O1 (250 mm. Times.25 mm,10 μm), mobile phase: A (CO) ₂ ) And B (methanol, 0.1% ammonia); gradient: b% = 60% -60%,7 min) to give compound 003 and compound 004, respectively.

Compound 003:

LCMS：MS m/z：585.2[M+1] ⁺ .

¹ H NMR(400MHz，CDCl ₃ )δ＝8.56(d，J＝5.2Hz，1H)，7.81(d，J＝8.2Hz，1H)，7.36(q，J＝8.0Hz，1H)，7.13(d，J＝5.2Hz，1H)，6.90(d，J＝8.4Hz，1H)，6.82(t，J＝8.8Hz，1H)，6.62-6.55(m，1H)，6.51-6.46(m，1H)，5.83(dd，J＝9.6，2.0Hz，1H)，5.58(t，J＝5.6Hz，1H)，5.09(s，1H)，5.01-4.95(m，1H)，4.46(d，J＝8.0Hz，1H)，4.37-4.35(m，1H)，4.16(t，J＝10.0Hz，1H)，3.98-3.91(m，1H)，3.64-3.58(m，1H)，2.96-2.70(m，2H)，2.63-2.53(m，2H)，2.32-2.14(m，3H)，1.35(d，J＝6.8Hz，3H)，0.97(d，J＝6.8Hz，3H)。

SFC (column (S, S) -WHELK-O1,3.5 μm,0.46 cmid. Times.5 cmL; mobile phase: A (CO) ₂ ) And B (MeOH, 0.1% isopropylamine); gradient: b% = 40-40%, 4min; flow rate: 4mL/min; wavelength: 220nm; pressure: 124bar, rt=1.237 min, chiral isomer excess 100%.

Compound 004:

LCMS：MS m/z：585.2[M+1] ⁺ .

¹ H NMR(400MHz，CDCl ₃ )δ＝8.63(d，J＝2.4Hz，1H)，7.79(d，J＝8.4Hz，1H)，7.37(q，J＝8.0Hz，1H)，7.24(s，1H)，6.90(d，J＝8.4Hz，1H)，6.83(t，J＝8.8Hz，1H)，6.62-6.40(m，2H)，5.83(dd，J＝10.0，2.0Hz，1H)，5.60(t，J＝4.8Hz，1H)，5.09(s，1H)，4.98-4.95(m，1H)，4.48(d，J＝8.0Hz，1H)，4.38(d，J＝7.6Hz，1H)，4.16(t，J＝9.6Hz，1H)，3.96-3.89(m，1H)，3.60(t，J＝10.0Hz，1H)，3.00-2.93(m，1H)，2.79(dd，J＝13.6，5.2Hz，1H)，2.68-2.60(m，2H)，2.29-2.20(m，3H)，1.41(d，J＝6.4Hz，3H)，1.06(d，J＝6.4Hz，3H)。

SFC (column (S, S) -WHELK-O1,3.5 μm,0.46 cmid. Times.5 cmL; mobile phase: A (CO) ₂ ) And B (MeOH, 0.1% isopropylamine); gradient: b% = 40-40%, 4min; flow rate: 4mL/min; wavelength: 220nm; pressure: 124bar, rt= 1.995min, chiral isomer excess 99.80%.

Example 4

Step 1: synthesis of Compound 005

To a previously dried single-necked flask was added 001-18 (150 mg, 281.65. Mu. Mol,1 eq), 005-1 (41.01 mg, 422.47. Mu. Mol,1.5 eq), dichloromethane (3 mL), diisopropylethylamine (72.80 mg, 563.29. Mu. Mol, 98.12. Mu.L, 2 eq), cooled to 0℃and O- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (128.51 mg, 337.98. Mu. Mol,1.2 eq) was added, and the mixture was stirred for 1 hour after natural tempering to 20 ℃. Concentrating the reaction solution under reduced pressure to dryness, adding acetonitrile (2 mL) for dissolution, and separating and purifying the crude product by high performance liquid chromatography (chromatographic column: waters Xbridge BEH C, 25mm, 5 μm; mobile phase: water (ammonium bicarbonate) -acetonitrile; acetonitrile%: 25% -55%,10 min) to obtain the compound 005.

LCMS：m/z(ESI)＝612[M+H] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ -d)δppm 8.59(d，J＝4.88Hz，1H)7.78(d，J＝8.38Hz，1H)7.31-7.42(m，2H)7.11-7.26(m，1H)6.80-6.95(m，2H)6.62(dd，J＝15.76，5.00Hz，1H)4.47-4.95(m，3H)4.30-4.43(m，1H)3.13-4.00(m，5H)2.87-3.04(m，1H)2.36-2.70(m，2H)2.09-2.33(m，2H)1.61-1.69(m，3H)1.33(br d，J＝6.63Hz，3H)1.06(br d，J＝6.75Hz，3H).

SFC analysis method (column: chiralcel OD-3, 50X 4.6mm I.D.,3 μm; mobile phase: A (CO) ₂ ) And B (MeOH, 0.1% isopropylamine); gradient: b% = 5-50-5%, 3.0min; flow rate: 3.4mL/min; wavelength: 220nm; pressure: 180 psi, rt= 1.405min, chiral isomer excess 100%.

Example 5

Step 1: synthesis of Compound 006

To a previously dried single-necked flask, 002-5 (200 mg, 365.89. Mu. Mol,1 eq), 005-1 (53.28 mg, 548.84. Mu. Mol,1.5 eq), methylene chloride (4 mL), diisopropylethylamine (94.58 mg, 731.78. Mu. Mol, 127.46. Mu.L, 2 eq) were added, the temperature was lowered to 0℃and O- (7-azobenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate (166.95 mg, 439.07. Mu. Mol,1.2 eq) was added, followed by stirring for 1 hour at 20℃by natural tempering. The reaction solution is concentrated to dryness under reduced pressure, acetonitrile (2 mL) is added for dissolution, filtration is carried out, and the crude product is separated and purified by high performance liquid chromatography (chromatographic column: waters Xbridge BEH C: 100 x 25mm x 5 μm; mobile phase: water (ammonium bicarbonate) -acetonitrile;: acetonitrile%: 30% -60%,10 min) to obtain the compound 006.

LCMS：m/z(ESI)＝626[M+H] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δppm 8.59(br d，J＝4.88Hz，1H)7.83(dd，J＝18.26，8.50Hz，1H)7.31-7.42(m，1H)7.13-7.25(m，2H)6.79-6.95(m，2H)6.61(dd，J＝15.70，5.69Hz，1H)4.78-5.21(m，2H)4.26-4.54(m，2H)3.56-3.86(m，3H)3.22-3.37(m，1H)2.85-3.02(m，1H)2.39-2.71(m，2H)2.06-2.35(m，2H)1.59-1.68(m，3H)1.55(br s，3H)1.31(br s，3H)1.09(brt，J＝6.75Hz，3H)。

SFC analysis method (column: chiralcel OD-3, 50X 4.6mm I.D.,3 μm; mobile phase: A (CO) ₂ ) And B (MeOH, 0.1% isopropylamine); gradient: b% = 5-50-5%, 3.0min; flow rate: 3.4mL/min; wavelength: 220nm; pressure: 180 psi, rt=1.323 min, chiral isomer excess 96.88%.

Example 6

The synthetic route is as follows:

step 1: synthesis of Compound 007-1

Cyanoacetic acid (3.64 g,42.78mmol,6 eq) was dissolved in dichloromethane (35 mL), oxalyl chloride (8.15 g,64.17mmol,5.62mL,9 eq) was added, and then N, N-dimethylformamide (104.23 mg,1.43mmol, 109.72. Mu.L, 0.2 eq) was added to react at 25℃for 3 hours. After the reaction is finished, the reaction system is directly decompressed and concentrated to obtain the intermediate state of the acyl chloride. Compounds 001-8 (2.2 g,7.13mmol,1 eq) were dissolved in dichloromethane (40 mL), triethylamine (2.89 g,28.52mmol,3.97mL,4 eq) was added, the acid chloride intermediate was slowly added at 0deg.C, and then slowly warmed to 25deg.C for 2 hours. Water (50 mL) was added to the reaction system, and the mixture was separated. The aqueous phase was extracted with dichloromethane (30 ml x 2) and separated. The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was separated by column chromatography (mobile phase: petroleum ether: ethyl acetate=20:1 to 1:1) and purified to give compound 007-1.

LCMS：MS m/z：376.1[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.47(d，J＝4.8Hz，1H)，7.86(s，1H)，7.05(d，J＝4.8Hz，1H)，3.65(t，J＝5.6Hz，2H)，3.58(s，2H)，3.19-3.12(m，1H)，2.65(t，J＝7.2Hz，2H)，1.83-1.76(m，2H)，1.26(s，3H)，1.24(s，3H)，0.89(s，9H)，0.07(s，6H)。

Step 2: synthesis of Compound 007-2

Compound 007-1 (2.1 g,5.59mmol,1 eq) was dissolved in tetrahydrofuran (30 mL) under nitrogen and tetramethyl ammonium fluoride (10.42 g,111.83mmol,20 eq) was added and reacted at 20℃for 23 hours. To the reaction system was added water (50 mL), the aqueous phase was extracted with ethyl acetate (100 mL x 3) and separated. The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 007-2, which was used in the next reaction without purification.

LCMS：MS m/z：262.1[M+1] ⁺ 。

¹ H NMR(400MHz，CD ₃ OD)δ＝8.37(d，J＝5.2Hz，1H)，7.23(d，J＝4.8Hz，1H)，3.86-3.84(m，1H)，3.59(t，J＝6.0Hz，2H)，3.28-3.19(m，2H)，2.67(t，J＝8.0Hz，2H)，1.83-1.76(m，2H)，1.24(s，3H)，1.22(s，3H)。

Step 3: synthesis of Compound 007-3

Compound 007-2 (1.5 g,5.74mmol,1 eq) was dissolved in dichloromethane (45 mL) and imidazole (1.17 g,17.22mmol,3 eq) and t-butyldiphenylchlorosilane (2.37 g,8.61mmol,2.21mL,1.5 eq) were added at 0deg.C and reacted for 6 hours at 20deg.C. To the reaction system was added water (100 mL), the aqueous phase was extracted with dichloromethane (100 mL x 3) and separated. The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was separated by column chromatography (mobile phase: petroleum ether: ethyl acetate=30:1 to 1:1) and purified to give compound 007-3.

LCMS：MS m/z：500.2[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.47(d，J＝4.8Hz，1H)，7.66-7.64(m，4H)，7.48-7.38(m，7H)，7.03(d，J＝4.8Hz，1H)，3.74(t，J＝6.0Hz，2H)，3.36(s，2H)，3.18-3.12(m，1H)，2.70(t，J＝7.6Hz，2H)，1.88-1.82(m，2H)，1.27(s，3H)，1.25(s，3H)，1.09(s，9H)。

Step 4: synthesis of Compound 007-5

Compound 007-4 (961.46 mg,4.58mmol,1.3 eq) was dissolved in thionyl chloride (10.48 g,88.05mmol,6.39mL,25 eq) and reacted at 80℃for 2 hours under nitrogen. After the reaction is finished, the reaction system is directly decompressed and concentrated to obtain the intermediate state of the acyl chloride. Compound 007-3 (1.76 g,3.52mmol,1 eq) was dissolved in tetrahydrofuran (30 mL), sodium tert-butoxide (676.93 mg,7.04mmol,2 eq) was added at 0deg.C, stirred for 0.5 hours at 0deg.C, then a solution of acid chloride intermediate in tetrahydrofuran (24 mL) was slowly added at 0deg.C, then slowly warmed to 25deg.C for 3 hours. To the reaction system was added water (50 mL), the aqueous phase was extracted with ethyl acetate (100 mL x 2) and separated. The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was isolated by column chromatography (mobile phase: petroleum ether: ethyl acetate=50:1 to 1:1, dichloromethane: methanol=80:1 to 30:1) and purified to give compound 007-5.

LCMS：MS m/z：691.1[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝11.90(br s，1H)，8.49(s，1H)，7.64-7.62(m，4H)，7.53(d，J＝6.8Hz，1H)，7.47-7.36(m，8H)，3.74(t，J＝6.0Hz，2H)，3.50-3.42(m，1H)，3.01(t，J＝7.6Hz，2H)，1.91-1.85(m，2H)，1.09(s，4H)，1.06(s，11H)。

Step 5: synthesis of Compound 007-6

Compound 007-5 (0.91 g,1.32mmol,1 eq) was dissolved in tetrahydrofuran (35 mL), sodium hydride (263.10 mg,6.58mmol,60% strength, 5 eq) was added slowly at 0deg.C, and then reacted at 50deg.C for 4 hours under nitrogen. Saturated ammonium chloride solution (50 mL) was added to the reaction system to quench the reaction, and the aqueous phase was extracted with ethyl acetate (3×100 mL) and separated. The organic phases were combined, washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was isolated by column chromatography (mobile phase: dichloromethane: methanol=40:1 to 8:1) and purified to give compound 007-6.

LCMS：MS m/z：655.2[M+1] ⁺ 。

¹ H NMR(400MHz，CD ₃ OD)δ＝8.51(d，J＝5.2Hz，1H)，8.22(d，J＝8.0Hz，1H)，7.53-7.49(m，4H)，7.43-7.32(m，7H)，3.64-3.55(m，2H)，2.79-2.72(m，1H)，2.58-2.46(m，2H)，1.81-1.66(m，2H)，1.23(d，J＝6.8Hz，3H)，1.07(d，J＝6.8Hz，3H)，0.88(s，9H)。

Step 6: synthesis of Compound 007-7

Compound 007-6 (1.17 g,1.79mmol,1 eq) was dissolved in tetrahydrofuran (18 mL), N-diisopropylethylamine (692.32 mg,5.36mmol, 933.05. Mu.L, 3 eq) was added, followed by phosphorus oxychloride (684.48 mg,4.46mmol, 414.83. Mu.L, 2.5 eq) and reacted at 40℃for 3 hours to give the crude compound 007-7 which was directly used in the next reaction.

Step 7: synthesis of Compound 007-8

Compound 007-7 (1.1 g,1.63mmol,1 eq) was dissolved in tetrahydrofuran (17.2 mL), N-diisopropylethylamine (844.11 mg,6.53mmol,1.14mL,4 eq) was added, and then compound 001-13 (425.12 mg,2.12mmol,1.3 eq) was added at 0deg.C and reacted for 16 hours at 20deg.C. The reaction was added to water (50 mL), the aqueous phase was extracted with ethyl acetate (50 mL x 3) and the solution was separated. The organic phases were combined, washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was isolated by column chromatography (mobile phase: dichloromethane: methanol=200:1 to 12:1) and purified to give compound 007-8.

LCMS：MS m/z：837.3[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.63-8.59(m，1H)，7.81-7.72(m，1H)，7.58-7.52(m，4H)，7.44-7.33(m，6H)，7.17-7.13(m，1H)，4.37-3.76(m，4H)，3.66-3.56(m，3H)，3.22-3.10(m，1H)，2.97-2.76(m，1H)，2.60-2.45(m，1H)，2.41-2.26(m，2H)，1.81-1.64(m，2H)，1.61(s，3H)，1.52-1.46(m，9H)，1.24-1.19(m，3H)，1.03(d，J＝6.8Hz，3H)，0.95-0.92(m，9H)。

Step 8: synthesis of Compound 007-9

Compound 007-8 (0.69 g, 823.89. Mu. Mol,1 eq) and compound 001-15 (256.92 mg,1.65mmol,2 eq) were dissolved in 1, 4-dioxane (14 mL) and water (1.4 mL), sodium carbonate (261.97 mg,2.47mmol,3 eq) and tetrakis (triphenylphosphine) palladium (95.21 mg, 82.39. Mu. Mol,0.1 eq) were added and reacted at 80℃for 1 hour under nitrogen. To the reaction system was added water (50 mL), the aqueous phase was extracted with ethyl acetate (60 mL x 3) and separated. The organic phases were combined, washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was separated by column chromatography (mobile phase: petroleum ether: ethyl acetate=20:1 to 2:1) and purified to give compound 007-9.

LCMS：MS m/z：913.3[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝9.18(d，J＝8.8Hz，1H)，8.69(d，J＝5.2Hz，1H)，7.92(t，J＝6.8Hz，1H)，7.58(d， J＝7.6Hz，1H)，7.55-7.50(m，4H)，7.41-7.33(m，6H)，7.21(dd，J＝12.4，4.8Hz，1H)，6.72-6.68(m，2H)，4.22-4.07(m，3H)，3.65-3.58(m，3H)，3.28-3.06(m，2H)，2.74-2.62(m，1H)，2.52-2.40(m，2H)，2.34(t，J＝8.0Hz，1H)，1.86-1.66(m，2H)，1.53(s，9H)，1.38(d，J＝6.4Hz，2H)，1.31(s，1H)，1.24-1.20(m，3H)，1.03(dd，J＝14.4，6.4Hz，3H)，0.91(d，J＝7.2Hz，9H)。

Step 9: synthesis of Compound 007-10

Compound 007-9 (0.34 g, 372.34. Mu. Mol,1 eq) was dissolved in tetrahydrofuran (10 mL) under nitrogen, and tetramethyl ammonium fluoride (1.21 g,13.03mmol,35 eq) was added and reacted at 20℃for 23 hours. To the reaction system was added water (10 mL), the aqueous phase was extracted with ethyl acetate (10 mL x 3) and the solution was separated. The organic phases were combined, washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give crude compound 007-10, which was used in the next reaction without purification.

LCMS：MS m/z：675.2[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝9.26-9.04(m，1H)，8.70(t，J＝4.8Hz，1H)，8.00(d，J＝9.2Hz，1H)，7.60-7.50(m，1H)，7.30-7.29(m，1H)，6.72-6.67(m，2H)，4.30-4.18(m，2H)，4.16-3.99(m，2H)，3.65-3.49(m，3H)，3.47-3.41(m，1H)，3.30-3.23(m，2H)，2.76-2.62(m，1H)，2.48(t，J＝7.2Hz，1H)，2.32(t，J＝7.6Hz，1H)，1.84-1.76(m，2H)，1.54(s，9H)，1.45-1.43(m，6H)，1.25-1.20(m，3H)。

Step 10: synthesis of Compound 007-11

Compound 007-10 (0.34 g, 503.90. Mu. Mol,1 eq) was dissolved in tetrahydrofuran (21 mL), triphenylphosphine (462.59 mg,1.76mmol,3.5 eq) was added, diethyl azodicarboxylate (307.16 mg,1.76mmol, 320.62. Mu.L, 3.5 eq) was added at 20℃and reacted for 2 hours at 20 ℃. To the reaction was added water (20 mL), the aqueous phase was extracted with ethyl acetate (30 mL x 3) and separated. The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was separated by column chromatography (mobile phase: petroleum ether: ethyl acetate=20:1 to 2:1) and purified to give compound 007-11.

LCMS：MS m/z：657.2[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.59(t，J＝4.4Hz，1H)，7.97-7.89(m，1H)，7.39-7.34(m，1H)，7.12(d，J＝5.2Hz，1H)，6.89(t，J＝6.8Hz，1H)，6.86-6.80(m，1H)，4.37(d，J＝8.8Hz，1H)，4.32-4.18(m，2H)，4.09-4.03(m，1H)，4.01-3.90(m，1H)，3.71-3.56(m，2H)，3.52-3.29(m，2H)，2.81-2.69(m，1H)，2.64-2.52(m，1H)，2.48-2.33(m，1H)，2.39-2.15(m，2H)，1.53(s，9H)，1.42(t，J＝6.4Hz，3H)，1.31(dd，J＝6.8，3.2Hz，3H)，1.00-0.88(m，3H)。

Step 11: synthesis of trifluoroacetate salt of Compound 007-12

Compound 007-11 (0.33 g, 402.00. Mu. Mol,1 eq) was dissolved in dichloromethane (13 mL) under nitrogen, trifluoroacetic acid (2.29 g,20.10mmol,1.49mL,50 eq) was added and reacted at 20℃for 2 hours. The reaction system was directly concentrated under reduced pressure to give the trifluoroacetate salt of compound 007-12, which was directly used in the next reaction without purification.

LCMS：MS m/z：557.2[M+1] ⁺ 。

Step 12: synthesis of Compounds 007 and 008

The trifluoroacetate salt of Compound 007-12 (0.5 g, 351.33. Mu. Mol,1 eq) was dissolved in methylene chloride (12 mL), N-diisopropylethylamine (681.08 mg,5.27mmol, 917.90. Mu.L, 15 eq) was added, and then acryloyl chloride (63.60 mg, 702.65. Mu. Mol, 57.29. Mu.L, 2 eq) was added and reacted at-60℃for 10 minutes under nitrogen. To the reaction system was added a saturated sodium bicarbonate solution (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane (50 ml x 3) and separated. Combining the organic phases The mixture was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a crude product. The crude product was separated by high performance liquid chromatography (column Waters Xbridge BEH C, 250, 50mM, 10 μm; mobile phase A (acetonitrile) and B (water, 10mM ammonium bicarbonate; gradient: B%:40% -70%,10 min), and further by SFC (column DAICEL CHIRALCEL OJ (250 mM, 30mM,10 μm); mobile phase A (CO) ₂ ) And B (ethanol, 0.1% ammonia); gradient: b% = 37% -37%,18 min) resolution gives compound 007 and compound 008, respectively.

Compound 007: LCMS: MS m/z:611.2[ M+1 ]] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.60(d，J＝4.8Hz，1H)，7.96(d，J＝9.2Hz，1H)，7.38(q，J＝8.0Hz，1H)，7.16(d，J＝4.8Hz，1H)，6.90(d，J＝8.0Hz，1H)，6.85(t，J＝8.8Hz，1H)，6.75-6.52(m，1H)，6.42(dd，J＝16.4，1.2Hz，1H)，5.84(d，J＝10.0Hz，1H)，4.46-4.27(m，3H)，4.14-3.95(m，2H)，3.79-3.53(m，4H)，2.74-2.67(m，1H)，2.66-2.59(m，1H)，2.57-2.49(m，1H)，2.31-2.15(m，2H)，1.43(d，J＝6.4Hz，3H)，1.32(d，J＝6.8Hz，3H)，0.91(d，J＝6.8Hz，3H)。

SFC (column: chiralcel OJ-3,3 μm,0.46cm id. Times.5 cm L; mobile phase: A (CO) ₂ ) And B (EtOH, containing 0.1% isopropylamine); gradient: b% = 5-50%, 3min; flow rate: 3.4mL/min; wavelength: 220nm; pressure: 124bar, rt= 1.278min, chiral isomer excess 100%.

Compound 008: LCMS: MS m/z:611.2[ M+1 ]] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.59(d，J＝5.2Hz，1H)，7.90(d，J＝6.8Hz，1H)，7.37(q，J＝7.6Hz，1H)，7.12(d，J＝5.2Hz，1H)，6.89(d，J＝8.4Hz，1H)，6.83(t，J＝8.8Hz，1H)，6.73-6.56(m，1H)，6.43(dd，J＝16.4，1.2Hz，1H)，5.84(d，J＝11.2Hz，1H)，4.67-4.52(m，1H)，4.44-4.36(m，2H)，4.27-3.99(m，2H)，3.65-3.60(m，1H)，3.58-3.26(m，3H)，2.79-2.71(m，1H)，2.58-2.52(m，1H)，2.43-2.35(m，1H)，2.24-2.15(m，2H)，1.41(d，J＝6.8Hz，3H)，1.31(d，J＝6.8Hz，3H)，0.98(d，J＝6.8Hz，3H)。

SFC (column: chiralcel OJ-3,3 μm,0.46cm id. Times.5 cm L; mobile phase: A (CO) ₂ ) And B (EtOH, containing 0.1% isopropylamine); gradient: b% = 5-50%, 3min; flow rate: 3.4mL/min; wavelength: 220nm; pressure: 124bar, rt=1.501 min, chiral isomer excess 98.48%.

Example 7

The synthetic route is as follows:

step 1: synthesis of Compound 009-2

To a previously dried single-necked flask, compound 009-1 (20 g,121.96mmol,1 eq), potassium isopropenyl trifluoroborate 001-2 (21.66 g,146.35mmol,1.2 eq), sodium carbonate (38.78 g,365.87mmol,3 eq), dioxane (200 mL), water (50 mL), 1-bis (diphenylphosphorus) ferrocene palladium chloride (4.46 g,6.10mmol,0.05 eq) were charged with nitrogen and stirred at 100℃for 5 hours. The reaction solution was concentrated under reduced pressure, methyl tert-butyl ether (200 mL) was added, and the mixture was filtered, and the cake was rinsed thoroughly with methyl tert-butyl ether until no product remained. The filtrate was extracted with methyl tert-butyl ether (200 mL x 3), the combined organic phases were collected and added to a saturated aqueous sodium chloride solution (40 mL), the separated organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate=4:0-4:1) to give compound 009-2.

LCMS：MS m/z：170.1[M+H] ⁺ 。

Step 2: synthesis of Compound 009-3

To a predried single-necked flask was added compound 009-2 (5.15 g,30.36mmol,1 eq), 001-4 (9.01 g,42.51mmol,1.4 eq), dioxane (60 mL), water (15 mL), potassium phosphate (12.89 g,60.73mmol,2 eq), and [1, 1-bis (di-t-butylphosphino) ferrocene ] palladium dichloride (1.98 g,3.04mmol,0.1 eq) were added with sufficient nitrogen substitution and stirred at 100℃for 12 hours. After concentrating the reaction solution under reduced pressure, adding methyl tert-butyl ether (60 mL), stirring thoroughly, filtering, eluting the filter cake with methyl tert-butyl ether until no product residue, collecting the filtrate, washing the organic phase of the filtrate with saturated saline (50 mL), separating the organic phase, adding anhydrous sodium sulfate, drying, filtering, concentrating the filtrate under reduced pressure, and separating and purifying the crude product by silica gel column chromatography (petroleum ether: ethyl acetate=9:0-9:1) to obtain a compound 009-3.

LCMS：MS m/z：220.1[M+H] ⁺ 。

Step 3: synthesis of Compound 009-4

After sufficient argon displacement, palladium on carbon (750 mg,10% content), tetrahydrofuran (30 mL), compound 009-3 (3 g,13.68mmol,1 eq) was added to a single vial which had been previously dried, and stirred for 2 hours at 20℃under a 15psi hydrogen atmosphere. The combined system was filtered through celite and the filter cake was rinsed thoroughly with ethyl acetate (500 mL) until no product remained, the filtrate was collected and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate=7:0-7:3) to give compound 009-4.

LCMS：MS m/z：224.2[M+H]+。

¹ H NMR(400MHz，CDCl3)δ＝8.57(s，1H)3.70(s，3H)3.02(dt，J＝13.52，6.73Hz，1H)2.88-2.96(m，4H)1.30(d，J＝6.78Hz，6H)。

Step 4: synthesis of Compound 009-5

Two reactions were set up in parallel: to a previously dried three-necked flask, lithium aluminum hydride (169.99 mg,4.48mmol,2 eq) and tetrahydrofuran (5 mL) were added, the temperature was lowered to 0℃and a solution of compound 009-4 (500 mg,2.24mmol,1 eq) in tetrahydrofuran (1 mL) was added, and the mixture was stirred for 1 hour after the addition was warmed to 20℃naturally. To the two reactions were added water (0.17 mL), 15% aqueous sodium hydroxide solution (0.17 mL), water (0.51 mL), dried over anhydrous sodium sulfate, stirred well, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate=1:0-1:1) to give compound 009-5.

LCMS：MS m/z：196.2[M+H] ⁺ 。

Step 5: synthesis of Compound 009-6

To a predried single-necked flask was added compound 009-5 (323 mg,1.65mmol,1 eq), dichloromethane (4 mL), imidazole (337.84 mg,4.96mmol,3 eq), cooled to 0deg.C, a solution of t-butyldimethylchlorosilane (498.65 mg,3.31mmol, 405.40. Mu.L, 2 eq) in dichloromethane (1 mL) was added, and after addition, immediately transferred to 20deg.C and stirred for 2 hours. Water (15 mL) and dichloromethane (10 mL x 3) were added to extract, the combined organic phases were collected, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate=4:0-4:1) to give compound 009-6.

LCMS：MS m/z：310.2[M+H] ⁺ 。

Step 6: synthesis of Compound 009-7

To a predried single-necked flask was added 001-9 (345 mg,1.65mmol,1 eq), methylene chloride (4 mL), oxalyl chloride (419.04 mg,3.30mmol, 288.99. Mu.L, 2 eq) and stirred at 45℃for 4 hours. The two batches of reaction solution were concentrated to dryness under reduced pressure to give an isocyanate-reactive intermediate compound, which was directly subjected to the next step.

To a reaction flask previously charged with the above isocyanate-reactive intermediate compound was replaced with nitrogen, methylene chloride (1 mL) was added under nitrogen atmosphere, cooled to 0℃and a solution of compound 009-6 (357.65 mg,1.16mmol,0.7 eq) in methylene chloride (1 mL) was added, and the mixture was naturally warmed to 20℃and stirred for 1 hour. Saturated aqueous sodium bicarbonate (20 mL) was added to the reaction mixture immediately and quenched, and after stirring thoroughly at 20 ℃, extracted with dichloromethane (10 mL x 3), the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated under reduced pressure to give crude compound 009-7.

LCMS：MS m/z：545.9[M+1] ⁺ 。

Step 7: synthesis of Compound 009-8

To a single vial previously dried was added compound 009-7 (500 mg, 918.24. Mu. Mol,1 eq), tetrahydrofuran (10 mL), cooled to 0℃and then a solution of potassium bis (trimethylsilyl) amide in tetrahydrofuran (1M, 1.84mL,2 eq) was added, and after the addition was completed, the ice bath was withdrawn and stirred for 1 hour at 20 ℃. Saturated aqueous ammonium chloride (30 mL) was added to the system, ethyl acetate (20 mL x 3) was added for extraction, the combined organic phases were collected, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated under reduced pressure, and the crude product was purified by column chromatography over silica gel (petroleum ether: ethyl acetate=7:0-7:3) to give compound 009-8.

LCMS：MS m/z：508.2[M+H] ⁺ 。

Step 8: synthesis of Compound 009-9

To a reaction flask dried in advance was added compound 009-8 (100 mg, 196.83. Mu. Mol,1 eq), tetrahydrofuran (1.2 mL), diisopropylethylamine (76.32 mg, 590.48. Mu. Mol, 102.85. Mu.L, 3 eq), phosphorus oxychloride (69.41 mg, 452.70. Mu. Mol, 42.07. Mu.L, 2.3 eq), and stirred at 45℃for 2 hours to give compound 009-9, and the reaction solution was used directly in the next step.

Step 9: synthesis of Compound 009-10

Diisopropylethylamine (50.88 mg, 393.66. Mu. Mol, 68.57. Mu.L, 2 eq) was added to a reaction flask previously charged with compound 009-9 (103.63 mg, 196.83. Mu. Mol,1 eq), cooled to 0deg.C, then tetrahydrofuran (0.5 mL) was added to compound 002-3 (50.62 mg, 236.20. Mu. Mol,1.2 eq), kept at 0deg.C under stirring for 5 minutes, a sufficient amount of saturated aqueous sodium bicarbonate solution (10 mL) was added to quench the combined system, ethyl acetate (10 mL. Times.4) was added for extraction, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give crude compound 009-10, which was used directly in the next reaction.

LCMS：MS m/z：704.6[M+1] ⁺ 。

Step 10: synthesis of Compound 009-11

To a previously dried single-necked flask, compound 009-10 (200 mg, 283.95. Mu. Mol,1 eq), tetrahydrofuran (4 mL), acetic acid (12 mL), water (4 mL) and stirring at 20℃for 20 minutes were added. The combined system was added with saturated aqueous sodium bicarbonate to adjust pH to 8, extracted with ethyl acetate (20 ml x 3), the organic phase was collected, dried over anhydrous sodium sulfate, filtered, the filtrate concentrated under reduced pressure, and the crude product was purified by column chromatography over silica gel (petroleum ether: ethyl acetate=100:0-0:100) to give compound 009-11.

LCMS：MS m/z：590.3[M+1] ⁺ 。

Step 11: synthesis of Compound 009-12

To a previously dried single-necked flask, 009-11 (223 mg, 377.91. Mu. Mol,1 eq), 001-15 (88.39 mg, 566.86. Mu. Mol,1.5 eq), dioxane (4 mL), water (1 mL), potassium phosphate (160.43 mg, 755.82. Mu. Mol,2 eq), and [1, 1-bis (di-t-butylphosphine) ferrocene ] palladium dichloride (24.63 mg, 37.79. Mu. Mol,0.1 eq) were added after sufficient nitrogen substitution, and stirred at 100℃for 3 hours. Saturated aqueous sodium chloride (10 mL) was added to the system, extracted with ethyl acetate (10 mL x 3), the organic phase was collected, dried over anhydrous sodium sulfate, filtered, the filtrate concentrated under reduced pressure, and the crude product was purified by column chromatography over silica gel (petroleum ether: ethyl acetate=100:0-0:100) to give compound 009-12.

LCMS：MS m/z：666.6[M+1] ⁺ 。

Step 12: synthesis of Compound 009-13

To a previously dried single-necked flask was added compound 009-12 (248 mg, 372.52. Mu. Mol,1 eq), dichloromethane (5 mL), n-tributylphosphine (296.95 mg,1.47mmol,3.94 eq), 1-azodicarbonyl dipiperidine (371.27 mg,1.47mmol,3.95 eq) and stirred at 20℃for 18 hours. Water (10 mL) was added, dichloromethane (10 mL. Times.2) was extracted, the combined organic phases were collected, dried over anhydrous sodium sulfate, filtered, the filtrate was added to silica gel (0.5 g) and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate=7:0-7:3) to give compound 009-13.

LCMS：MS m/z：648.4[M+1] ⁺ 。

Step 13: synthesis of Compound 009-14

To a previously dried single-necked flask was added compound 009-13 (87mg, 1.34mmol,1 eq), methylene chloride (8 mL), trifluoroacetic acid (3.06 g,26.86mmol,1.99mL,20 eq) and stirred at 20℃for 16 hours. Saturated aqueous sodium bicarbonate (10 mL) was added to the reaction to adjust the pH to 8, dichloromethane (5 mL x 3) was extracted, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, the filtrate concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (petroleum ether: ethyl acetate=1:1-0:1) to give compound 009-14.

LCMS：MS m/z：548.3[M+1] ⁺ 。

Step 14: synthesis of Compounds 009 and 010

To a predried single vial was added compound 009-14 (100 mg, 182.62. Mu. Mol,1 eq), (E) -4-fluorobut-2-enoic acid (28.51 mg, 273.92. Mu. Mol,1.5 eq), dichloromethane (2.5 mL), diisopropylethylamine (47.20 mg, 365.23. Mu. Mol, 63.62. Mu.L, 2 eq), cooled to 0deg.C, and O- (7-azabenzotriazol-1-YL) -N, N, N, N-tetramethylurea hexafluorophosphine salt (83.32 mg, 219.14. Mu. Mol,1.2 eq) was added, and transferred to 20deg.C for stirring for 1 hour. Water (10 mL) was added to the reaction solution, dichloromethane (10 mL. Times.3) was added, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by thin layer chromatography on silica gel plate (developer, dichloromethane: methanol=40:1) to give compounds 009 and 010.

Compound 009: LCMS: MS m/z:634.4[ M+1 ]] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝9.10(s，1H)，7.92-7.82(m，1H)，7.42-7.34(m，1H)，7.11-6.96(m，1H)，6.93(d，J＝8.5Hz，1H)，6.83(t，J＝8.7Hz，1H)，6.71-6.52(m，1H)，5.20(br d，J＝9.1Hz，1H)，5.11-5.07(m，1H)，4.97-4.77(m，2H)，4.57-4.37(m，3H)，3.78-3.59(m，3H)，2.99-2.89(m，1H)，2.64-2.59(m，2H)，1.63(br d，J＝6.8Hz，2H)，1.55-1.50(m，6H)，1.31(dd，J＝2.5，6.8Hz，3H)，1.07(t，J＝6.8Hz，3H)。

SFC analysis method (column: chiralpak AD-3, 50X 4.6mm I.D.,3 μm; mobile phase: A (CO) ₂ ) And B (EtOH, containing 0.1% iso)Propylamine); gradient: b% = 5-50%, 3min; flow rate: 3.4mL/min; wavelength: 220nm; pressure: 180 psi, rt=1.182 min, chiral isomer excess 93.9%.

Compound 010: LCMS: MS m/z:634.4[ M+1 ]] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝9.10(s，1H)，7.85-7.76(m，1H)，7.43-7.34(m，1H)，7.07-6.98(m，1H)，6.94(d，J＝8.4Hz，1H)，6.83(t，J＝8.6Hz，1H)，6.66-6.52(m，1H)，5.20(br d，J＝2.3Hz，1H)，5.11-5.06(m，1H)，4.47-4.34(m，2H)，4.17-3.91(m，3H)，3.78-3.58(m，3H)，3.01-2.87(m，1H)，2.67-2.60(m，2H)，1.43(d，J＝7.0Hz，2H)，1.40-1.33(m，3H)，1.31(d，J＝6.6Hz，3H)，1.26-1.21(m，3H)，1.02(d，J＝6.8Hz，3H)。

SFC analysis method (column: chiralpak AD-3, 50X 4.6mm I.D.,3 μm; mobile phase: A (CO) ₂ ) And B (EtOH, containing 0.1% isopropylamine); gradient: b% = 5-50%, 3min; flow rate: 3.4mL/min; wavelength: 220nm; pressure: 180 psi, rt=1.398 min, chiral isomer excess 98.62%.

Example 8

The synthetic route is as follows:

step 1: synthesis of Compound 011-1

Compound 007-6 (1.8 g,2.75mmol,1 eq) was dissolved in tetrahydrofuran (27 mL), N-diisopropylethylamine (1.07 g,8.24mmol,1.44mL,3 eq) was added, followed by phosphorus oxychloride (1.05 g,6.87mmol, 638.21. Mu.L, 2.5 eq) and reacted at 40℃for 3 hours under nitrogen. Compound 011-1 is obtained and the reaction system is used directly in the next step.

Step 2: synthesis of Compound 011-3

Under nitrogen, compound 011-1 (1.8 g,2.67mmol,1 eq) was dissolved in tetrahydrofuran (26 mL), N-diisopropylethylamine (1.38 g,10.68mmol,1.86mL,4 eq) was added, and then compound 011-2 (646.94 mg,3.47mmol,1.3 eq) was added and the mixture was heated to 20℃for reaction for 1 hour. Additional compound 011-2 (248.64 mg,1.34mmol,0.5 eq) was reacted at 20℃for 1 hour. The reaction was added to water (50 mL), the aqueous phase was extracted with ethyl acetate (50 mL x 3) and the solution was separated. The organic phases were combined, washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was purified by column chromatography (dichloromethane: methanol=200:1 to 30:1) to give compound 011-3.

LCMS：MS m/z：823.2[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.62(d，J＝5.2Hz，1H)，7.71(d，J＝8.0Hz，1H)，7.56-7.51(m，4H)，7.43-7.32(m，6H)，7.14(d，J＝5.2Hz，1H)，3.66-3.62(m，4H)，3.49-3.43(m，4H)，3.23-3.18(m，2H)，2.50-2.43(m，1H)，2.42-2.35(m，2H)，1.81-1.66(m，2H)，1.52(s，9H)，1.21(d，J＝6.8Hz，3H)，1.06(d，J＝6.4Hz，3H)，0.94(s，9H)。

Step 3: synthesis of Compound 011-4

Under nitrogen, compound 011-3 (1.92 g,2.33mmol,1 eq) and compound 001-15 (727.08 mg,4.66mmol,2 eq) were dissolved in 1, 4-dioxane (40 mL) and water (4 mL), sodium carbonate (741.38 mg,6.99mmol,3 eq) and tetrakis (triphenylphosphine) palladium (269.43 mg, 233.16. Mu. Mol,0.1 eq) were added and reacted at 80℃for 1 hour. To the reaction system was added water (50 mL), the aqueous phase was extracted with ethyl acetate (60 mL x 3) and separated. The organic phases were combined, washed with saturated brine (60 mL), 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=20:1 to 1:1) to give compound 011-4.

LCMS：MS m/z：899.2[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝9.22(s，1H)，8.69(d，J＝5.2Hz，1H)，7.86(d，J＝9.6Hz，1H)，7.54(t，J＝1.2Hz，1H)，7.52(d，J＝1.2Hz，2H)，7.50(d，J＝1.6Hz，1H)，7.40(d，J＝3.2Hz，1H)，7.38-7.29(m，6H)，7.21(d，J＝5.2Hz，1H)，6.72-6.68(m，2H)，3.69(d，J＝3.6Hz，4H)，3.65-3.59(m，6H)，2.64-2.57(m，1H)，2.44-2.40(m，2H)，1.85-1.77(m，1H)，1.71-1.67(m，1H)，1.53(s，9H)，1.23(d，J＝6.8Hz，3H)，1.02(d，J＝6.4Hz，3H)，0.91(s，9H)。

Step 4: synthesis of Compound 011-5

Under nitrogen, compound 011-4 (1.6 g,1.78mmol,1 eq) was dissolved in tetrahydrofuran (48 mL), and tetramethyl ammonium fluoride (5.80 g,62.28mmol,35 eq) was added and reacted at 20℃for 23 hours. To the reaction was added water (40 mL), the aqueous phase was extracted with ethyl acetate (50 mL x 3) and the solution was separated. The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 011-5, which was used directly in the next reaction without purification.

LCMS：MS m/z：661.2[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.71(d，J＝5.2Hz，1H)，7.97(d，J＝9.6Hz，1H)，7.59-7.52(m，1H)，7.31(d，J＝7.2Hz，1H)，7.26-7.24(m，1H)，6.70-6.65(m，2H)，3.80(s，8H)，3.53(t，J＝5.6Hz，2H)，3.36-3.26(m，1H)，2.65-2.59(m，1H)，2.41(t，J＝7.6Hz，2H)，1.85-1.77(m，2H)，1.53(s，9H)，1.23(d，J＝6.4Hz，3H)，1.03(d，J＝6.8Hz，3H)。

Step 5: synthesis of Compound 011-6

Under nitrogen, 011-5 (1.6 g,2.42mmol,1 eq) was dissolved in tetrahydrofuran (96 mL), triphenylphosphine (2.22 g,8.48mmol,3.5 eq) was added, diethyl azodicarboxylate (1.48 g,8.48mmol,1.54mL,3.5 eq) was added at 20℃and reacted for 1 hour at 20 ℃. To the reaction was added water (20 mL), the aqueous phase was extracted with ethyl acetate (30 mL x 3) and separated. The organic phases were combined, washed with saturated brine (20 mL), 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=25:1 to 1:4) to give compounds 011-6, respectively.

LCMS：MS m/z：643.1[M+1] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.58(d，J＝5.2Hz，1H)，7.88(d，J＝8.8Hz，1H)，7.39-7.33(m，1H)，7.11(d，J＝5.2Hz，1H)，6.88(d，J＝8.4Hz，1H)，6.82(t，J＝8.8Hz，1H)，3.95-3.88(m，2H)，3.85-3.79(m，3H)，3.77-3.60(m，5H)，2.81-2.74(m，1H)，2.58-2.51(m，1H)，2.46-2.38(m，1H)，2.28-2.11(m，2H)，1.53(s，9H)，1.31(d，J＝6.8Hz，3H)，0.96(d，J＝6.8Hz，3H)。

Step 6: synthesis of Compound 011-7

Compound 011-6 (0.5 g, 777.98. Mu. Mol,1 eq) was dissolved in methylene chloride (15 mL) under nitrogen, and trifluoroacetic acid (3.55 g,31.12mmol,2.30mL,40 eq) was added and reacted at 20℃for 1 hour. The reaction system is directly decompressed and concentrated to obtain the compound 011-7, which can be directly used for the next reaction without purification.

LCMS：MS m/z：543.2[M+1] ⁺ 。

Step 7: synthesis of Compounds 011 and 012

Under nitrogen, compound 011-7 (0.96 g, 692.47. Mu. Mol,1 eq) was dissolved in methylene chloride (24 mL), N-diisopropylethylamine (1.34 g,10.39mmol,1.81mL,15 eq) was added, and then acryloyl chloride (125.35 mg,1.38mmol, 112.93. Mu.L, 2 eq) was added and reacted at 60℃for 10 minutes. To the reaction system was added a saturated sodium bicarbonate solution (20 mL), and the mixture was separated. The aqueous phase was extracted with dichloromethane (50 ml x 3) and separated. The organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was separated by high performance liquid chromatography (column WatersXbridge BEH C, 250, 50mM, 10 μm; mobile phase A (acetonitrile) and B (water, 10mM ammonium bicarbonate; gradient B%:35% -55%,10 min), then SFC (column DAICEL CHIRALPAK AD (250, 30mM,10 μm), mobile phase A (CO) ₂ ) Andb (isopropanol, 0.1% ammonia); gradient: b% = 35% -35%,10 min), and purifying to obtain compound 011 and compound 012.

Compound 011: LCMS: MS m/z:597.2[ M+1 ]] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.58(d，J＝5.2Hz，1H)，7.89(d，J＝8.8Hz，1H)，7.36(q，J＝8.4Hz，1H)，7.12(d，J＝5.2Hz，1H)，6.88(d，J＝8.4Hz，1H)，6.82(t，J＝8.8Hz，1H)，6.64(dd，J＝16.8，10.4Hz，1H)，6.40(dd，J＝16.8，1.2Hz，1H)，5.83(dd，J＝10.4，1.2Hz，1H)，4.37(d，J＝8.4Hz，1H)，4.07-3.78(m，6H)，3.75-3.66(m，2H)，3.65-3.59(m，1H)，2.80-2.73(m，1H)，2.60-2.50(m，1H)，2.45-2.34(m，1H)，2.28-2.11(m，2H)，1.30(d，J＝6.8Hz，3H)，0.96(d，J＝6.4Hz，3H)。

SFC analysis (column: chiralpak AD-3,3 μm,0.46cm id. Times.5 cm L; mobile phase: A (CO) ₂ ) And B (IPA, 0.1% isopropyl amine); gradient: b% = 5-50%, 3min; flow rate: 3.4mL/min; wavelength: 220nm; pressure: 124bar, rt=1.383 min, chiral isomer excess 100%.

Compound 012: LCMS: MS m/z:597.2[ M+1 ]] ⁺ 。

¹ H NMR(400MHz，CDCl ₃ )δ＝8.58(d，J＝4.8Hz，1H)，7.89(d，J＝8.8Hz，1H)，7.36(q，J＝8.0Hz，1H)，7.12(d，J＝5.2Hz，1H)，6.88(d，J＝8.0Hz，1H)，6.82(t，J＝8.8Hz，1H)，6.64(dd，J＝16.8，10.4Hz，1H)，6.40(dd，J＝16.4，1.2Hz，1H)，5.83(dd，J＝10.8，1.6Hz，1H)，4.36(d，J＝8.4Hz，1H)，4.10-3.79(m，6H)，3.76-3.67(m，2H)，3.65-3.57(m，1H)，2.82-2.73(m，1H)，2.60-2.50(m，1H)，2.45-2.37(m，1H)，2.28-2.11(m，2H)，1.30(d，J＝6.8Hz，3H)，0.96(d，J＝6.8Hz，3H)。

SFC analysis (column: chiralpak AD-3,3 μm,0.46cm id. Times.5 cm L; mobile phase: A (CO) ₂ ) And B (IPA, 0.1% isopropyl amine); gradient: b% = 5-50%, 3min; flow rate: 3.4mL/min; wavelength of:220nm; pressure: 124bar, rt=1.562 min, chiral isomer excess 96.28%.

Test example 1: MIA-PA-CA-2 cell experiments

Experimental materials:

The experimental method comprises the following steps:

Data analysis:

raw data was converted to inhibition, IC, using the equation (sample-min)/(max-min) ×100% for inhibition ₅₀ The values of (a) can be obtained by curve fitting four parameters (in the "log (inhibitor)/response-variable domain" mode in GraphPad Prism software). Table 1 provides the inhibitory activity of the compounds of the invention on MIA-PA-CA-2 cell proliferation.

TABLE 1 in vitro screening test results for test compounds of the invention

Compounds of formula (I)	MIA-PA-CA-2 IC ₅₀ (nM)
001	19

002	17
005	1.1
006	0.5
009	24
012	18

Conclusion: the compound of the invention has better inhibitory activity on MIA-PA-CA-2 cell proliferation.

Test example 2: in vivo pharmacokinetic studies

The purpose of the experiment is as follows: pharmacokinetic study of SD mice orally and intravenously injected test compounds

Experimental operation: the tested compound is mixed with 10% dimethyl sulfoxide/60% polyethylene glycol 400/30% aqueous solution, vortex and ultrasonic are carried out, 1mg/mL clarified solution is prepared, and the filtration of a microporous filter membrane is carried out for later use. Male SD mice of 7 to 10 weeks of age were selected and given the candidate compound solution by intravenous injection at a dose of about 2mg/kg. The candidate compound solution is administered orally at a dose of about 10mg/kg. Whole blood was collected for a certain period of time, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and drug substitution parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

The experimental results are shown in table 2:

TABLE 2 pharmacokinetic results of test compounds

Conclusion of results: the compound of the invention has excellent total systemic exposure, peak concentration and bioavailability after oral administration, and shows excellent pharmacokinetic properties.

Test example 3: plasma stability study

The purpose of the experiment is as follows: evaluation of plasma stability of test Compounds in CD-1 mice, SD rats, beagle dogs, cynomolgus monkeys, humans, respectively

Experimental operation: thawing frozen blood plasma for 10-20 min, centrifuging at 3220 Xg for 5 min after the blood plasma is completely thawed, and removing suspended substances and sediments. 96 Kong Fuyo plates, designated T0, T10, T30, T60, T120, respectively, were prepared. Corresponding to the addition of 98. Mu.L of mouse, rat, dog, monkey and human blank plasma in the incubation plate, then 2. Mu.L of working solution of compound or control compound was added to the corresponding incubation plate, and two parallel wells were prepared for each sample. All samples were incubated in a 37 ℃ water bath. The final incubation concentrations of compound and control compound bisacodyl, enalapril maleate, procaine and procaine were 2 μm and the final organic phase content was 2.0%. At the end of each incubation time point, the corresponding incubation plate was removed and 400 μl of acetonitrile solution containing 200ng/mL of tolbutamide and la Bei Nuoer was added to each corresponding sample well to precipitate the protein. After all sample plates were pelleted and shaken well, they were centrifuged at 3220 Xg for 20 minutes. 50. Mu.L of the supernatant was diluted with 100. Mu.L of ultrapure water, and all samples were mixed and analyzed by the LC/MS/MS method. The experimental results are shown in table 3.

TABLE 3 plasma stability of test compounds CD-1 mice, SD rats, beagle dogs, cynomolgus monkeys and humans

Conclusion of experiment: the compounds of the invention have good stability in CD-1 mice, SD rats, beagle dogs, cynomolgus monkeys and human plasma.

Test example 4: whole blood stability study

The purpose of the experiment is as follows: assessment of test Compounds stability of test compounds in Whole blood in CD-1 mice, SD rats, beagle dogs, and cynomolgus monkeys, respectively

Experimental operation: on the day of the experiment or the day prior to the experiment, fresh CD-1 mice, SD rats, beagle dogs, cynomolgus monkey whole blood was collected using the anticoagulant EDTA-K2. Before the experiment starts, whole blood is mixed with PBS in a ratio of 1:1 (v:v), and the mixture is placed in a water bath kettle at 37 ℃ for preheating for 10 to 20 minutes. 96 Kong Fuyo plates, designated T0, T30, T60, T240, respectively, were prepared. In the corresponding incubation plates, including T0, T30, T60 and T240 incubation plates, 2 μl of working fluid of compound or control compound was mixed with 98 μl of mouse, rat, canine, monkey and human blank whole blood, and two parallel wells were prepared per sample. All samples were incubated in a 37 ℃ water bath. The final incubation concentration of the compound was 5. Mu.M and the final incubation concentration of the control compound was 2. Mu.M. At the end of each time point incubation, the corresponding incubation plate was removed, 100 μl of ultrapure water was immediately added to the corresponding sample wells, mixed well, and then 800 μl of acetonitrile solution containing 200ng/mL of tolbutamide and pull Bei Nuoer was added to precipitate the protein. After the sample plates were sealed and shaken well, the plates were centrifuged at 4000 rpm for 20 minutes. 150. Mu.L of the supernatant was analyzed by LC/MS/MS method. The experimental results are shown in table 4.

TABLE 4 Whole blood stability of test compounds CD-1 mice, SD rats, beagle dogs, cynomolgus monkeys

Conclusion of experiment: various whole blood stability studies have shown that the compounds of the present invention have good stability in whole blood.

Test example 5: GSH addition stability study

The purpose of the experiment is as follows: assessment of the stability of addition of test compounds in GSH buffer

Experimental operation: 96 Kong Fuyo plates were prepared, designated T0, T60, T120, T240, NGSH, respectively. 1500. Mu.L of GSH-potassium phosphate buffer (final test GSH concentration 5. Mu.M) was added to the corresponding incubation plate, and then working solution of test compound or control compound was added to the corresponding incubation plate, and two parallel wells were prepared for each sample. All samples were incubated in a 37 ℃ water bath. The final incubation concentrations of the test compound and the control compound afatinib, ibrutinib, were 1 μm and 10 μm, respectively. At the end of each incubation time point, 600 μl/well of 200ng/mL of tolbutamide and pull Bei Nuoer in acetonitrile was added as stop solution and frozen in-80 ℃ refrigerator until sample treatment at the last time point was completed. All sample plates were pelleted and shaken for 5 min, and centrifuged at 4000 rpm for 20 min. 50. Mu.L of the supernatant was diluted with 100. Mu.L of ultrapure water, and all samples were mixed and analyzed by the LC/MS/MS method. The experimental results are shown in table 5.

TABLE 5 GSH addition stability of test compounds

Compounds of formula (I)	Test compound content detection at 120min
002	84.3％
009	91.3％

Conclusion of experiment: the compound of the invention has good stability in GSH phosphate buffer solution.

Test example 6: bi-directional permeability and efflux studies

The purpose of the experiment is as follows: the bi-directional permeability and efflux of the test compounds were determined using the MDR1-MDCKII monolayer cell model.

Experimental operation: in the experiment, MDR1-MDCK II cells (11 th generation) are inoculated into a 96-well cell culture plate and used for the transportation experiment after 4-7 days of continuous culture. The test compounds were diluted from stock to 2. Mu.M concentration (DMSO < 1%) with transport buffer (HBSS with 10mM Hepes, pH 7.4) and applied to the top or basolateral side of the cell monolayer. The degree of penetration of the test compounds from A to B or B to A was determined repeatedly with the addition of P-gp inhibitor (GF 120918, 10. Mu.M) and without the addition of P-gp inhibitor (GF 120918, 10. Mu.M), respectively. Digoxin was bi-directionally tested for 10 μm in the presence or absence of 10 μm GF120918, while nadolol and metoprolol were bi-directionally tested for 2 μm in the absence of GF 120918. Plates were incubated in a carbon dioxide incubator at 37.+ -. 1 ℃ with 5% carbon dioxide saturated in humidity for 2.5 hours without shaking. In addition, the jet ratio of each compound was also measured. The test and reference compounds were quantified by LC-MS/MS analysis based on the peak area ratio of analyte/IS. The experimental results are shown in table 6.

TABLE 6 study of the bi-directional permeability and efflux of test compounds

Conclusion of experiment: compound 009 had a moderate permeation rate and efflux ratio.

Test example 7: pregnane X nuclear receptor (PXR) studies

The purpose of the experiment is as follows: determining whether a test compound activates pregnane X nuclear receptor (PXR)

Experimental operation: cell seeding and PXR receptor activation incubation: cryopreserved PXR reporter cells were added to the experimental tubes, 6.4mL of cell resuscitator (final volume 7 mL/tube) was added to each tube, and resuscitated in a 37℃water bath for 5-10 minutes. 200 mu L of cell dispersion liquid is added to each hole of the cell culture plate, no cells are added to a blank hole for multiple detection of living cells, and the inoculated cells are placed in an incubator with saturated humidity of 5% carbon dioxide and 37 ℃ for incubation for 4-6 hours. Dosing solutions of test compound (TA), blank (VC) and yang compound (PC) were prepared and preheated in a 37 ℃ water bath. Cells are retrieved from the incubator and the culture fluid is aspirated from each well in the cell culture plate. 200 μl of the dosing solution of the test compound or control compound was added to the corresponding wells, and a blank control working solution was added to the living cell multiplex assay blank wells, three parallel wells per sample. All samples were incubated in an incubator at 37℃with saturated humidity of 5% carbon dioxide for 22-24 hours.

PXR receptor and living cell multiplex assay (activity): preparation of a living cell multiplex detection reagent, addition of 6.7. Mu.L of 300 Xliving cell multiplex detection substrate to 2mL of a living cell multiplex detection buffer, and storage at room temperature. Cells were removed from the incubator, the dosing solution was aspirated, and washed once with 200 μl of live cell multiplex assay buffer at room temperature. 50 mu L of multiple detection reagent of living cells is added to each well, the plates are tilted left and right for 2-3 times, and incubated for 15 minutes at the dark room temperature.

Preparing a luciferase detection reagent: 2mL of detection substrate was added to 2mL of detection buffer. After 15 minutes incubation, the living cell multiplex assay reagent was aspirated, 100 μl of luciferase assay reagent was added per well and incubated for at least 5 minutes. Fluorescence values (fluorescence relative units at 485nm/535nm, activity) were read in a SpectraMax M4 plate reader. After adding the luciferase detection reagent and incubating for at least 5 minutes, the luminescence (fluorescence relative unit, PXR activation) was read with a SpectraMax M4 plate reader read time of 500 ms/well.

The experimental results are shown in table 7:

TABLE 7 test compound PXR activation

Conclusion of experiment: compound 002 and compound 009 had no PXR positive activation (2-fold threshold for PXR positive activation).

Test example 8: CYP Induction experiment

The purpose of the experiment is as follows: determining whether test compounds induce CYP enzyme overexpression

Experimental operation:

hepatocyte incubation

All incubations were at 37.+ -. 1 ℃ and 5% CO ₂ And in an incubator saturated with humidity.

After the culture system was established, the upper culture medium of the sandwich medium was discarded, 200. Mu.L of the freshly prepared dosing working solution (containing the test sample, positive control and matrix control) preheated to 37℃was added to each cell culture well, and the cell culture plates were placed in an incubator for continuous culture for 24 hours. After 24 hours of incubation, the freshly prepared dosing working fluid was replaced and incubation continued for 24 hours. The entire incubation time was 48 hours. Three replicates were made for each drug concentration and control concentration.

After the cells were incubated with the dosing working solution for 48 hours, the remaining drug solution in the plate was discarded, the wells were washed 2 times with 0.5mL of HBSS solution preheated to 37 ℃ and 100 μl of enzyme-labeled substrate working solution preheated to 37 ℃ was added to each well and incubated for 30 minutes. After 30 minutes incubation, 75.0. Mu.L of supernatant samples were removed from each well and added to 96-well deep well plates containing 150. Mu.L of stop solution. The plate was shaken for 10 minutes, centrifuged at 3220g for 20 minutes at 4℃and the supernatant was diluted 1:4 with an aqueous solution containing 0.1% formic acid. After 10 minutes of sample shaking after dilution, the amount of the metabolite produced was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

After completion of the enzyme activity detection reaction, the remaining supernatant solution was discarded, and the cells were washed with 0.5mL of pre-warmed HBSS. 280. Mu.L of a lysate containing 1% beta-mercaptoethanol was added to each well, the plates were closed, shaken for 10 minutes, and then transferred to a refrigerator at-60℃for storage.

Sample analysis and detection

The concentration of the metabolites of three CYP enzyme substrates (acetaminophen, hydroxyanthrone, and 1' -hydroxy midazolam) in the hepatocytes after protein precipitation was determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Cytotoxicity test

The potential toxicity of the test sample is assessed by the release of Lactate Dehydrogenase (LDH) in the hepatocytes. 100. Mu.L of each of the working solutions for administration after 24 hours and 48 hours of incubation with hepatocytes was taken out, and the concentration of lactate dehydrogenase was measured using a commercial LDH kit. Cell lysis solution was used as experimental positive control and incubation medium was used as blank control.

RNA analysis detection

The sample plate was thawed at room temperature and all samples were transferred to a new 96-well plate. RNA was extracted using a nucleic acid extraction purification device. Samples with more than 10% of the total amount of the samples are randomly extracted at different positions of the sample plate, OD values of 260nm and 280nm are measured by using an ND2000 micro-spectrophotometer, and the purity of the total RNA is judged by calculating the ratio of the two. Reverse transcription to obtain cDNA. The selected genes were quantitatively analyzed in real time by a real-time fluorescent quantitative PCR instrument. The reaction conditions were set as follows: two minutes at 50 ℃; ten minutes at 95 ℃; the following two steps are performed for 40 cycles: fifteen seconds at 95℃and one minute at 60 ℃. Endogenous control 18S rRNA served as an internal standard.

Data analysis

Analysis of enzyme Activity data

Experimental data shows the production of enzyme metabolites of CYP1A2, CYP2B6 and CYP3 A4. The change in enzyme activity is manifested by comparison of the fold induction of the corresponding cytochrome enzyme in the presence or absence of the compound. The method for calculating the induction fold and the induction ratio of the control compound are as follows:

fold induction = enzyme activity in test (or control compound) treated sample/enzyme activity in matrix control treated sample

Induction ratio (%) = (fold induction of test sample-treated sample-1)/(fold induction of control compound-treated sample-1) ×100) to control compound

Analysis of Gene expression data

The program adopts a delta Ct relative quantitative method to compare the difference of gene expression between different treatment groups, and uses 18S rRNA as an internal reference gene to correct the gene expression quantity of each sample. The Ct value of the target gene minus the Ct value of the reference gene is Δct, i.e., ct target gene-ct18s=Δct. The delta Ct value of the treatment group minus the delta Ct value of the matrix control group is then delta Ct, i.e. delta Ct treatment group-delta Ct matrix control group = delta Ct. Finally, statistical analysis was performed by a method of 2- ΔΔct, comparing the fold change between the treatment group and the matrix control group. The induction ratio of the test and control compounds was calculated as follows:

The experimental results are shown in table 8:

TABLE 8 CYP-induced assay of test compounds

Conclusion of experiment: compound 009 did not have CYP-induced activation.

Test example 9: in vivo pharmacodynamics research cell culture and tumor tissue preparation of human pancreatic cancer Mia PaCa-2 cell Nude mice subcutaneously transplanted with tumor Balb/c Nude mouse model

Cell culture: human pancreatic cancer Mia PaCa-2 cells (ATCC-CRL-1420) in vitroMonolayer culture, wherein the culture condition is that 20% of fetal bovine serum, 1% of diabody and 5% of carbon dioxide incubator at 37 ℃ are added into DMEM/F12 culture medium. Passaging was performed twice a week with conventional digestion treatments with pancreatin-EDTA. When the cell saturation is 80% -90% and the number reaches the requirement, collecting cells, counting, re-suspending in PBS, adding matrigel 1:1 to obtain cell density of 25X10 ⁶ cell suspension of cells/mL.

Cell inoculation: 0.2mL (5X 10) ⁶ cells/mouse) MiaPaCa-2 cells (matrigel added, volume ratio 1:1) were inoculated subcutaneously on the right back of each mouse, and the average tumor volume reached 190mm ³ At this time, the dosing was started according to the protocol in table 9, with random groupings based on tumor volumes.

Table 9 experimental animal grouping and dosing regimen

Note that: PO represents oral administration; QD stands for once daily.

Tumor measurement and experimental index

Experimental results

The experimental results are shown in fig. 8 and 9.

The results show that: the tumor volume of the solvent control group tumor-bearing mice reaches 2275mm at 25 days of administration ³ Tumor volume average of test compound 002 (1.5 mg/kg) and compound 002 (5 mg/kg) was 1675mm, respectively ³ And 534 (534) mm (mm) ³ The method comprises the steps of carrying out a first treatment on the surface of the T/C was 74.23% and 23.93%, respectively; TGI was 28.02% and 81.32%, respectively, and significantly inhibited tumor growth at both concentrations (p-values less than 0.001). Tumor volume average of test compound 009 (1.5 mg/kg) and compound 009 (5 mg/kg) was 1325mm, respectively ³ And 506mm ³ The method comprises the steps of carrying out a first treatment on the surface of the T/C was 56.99% and 21.89%, respectively; TGI was 44.37% and 82.59%, respectively, and significantly inhibited tumor growth at both concentrations (p-values less than 0.001). Tumor volume mean of test AMG510 (5 mg/kg) was 963mm ³ T/C is 41.78%, TGI is 61.28%, p values are less than 0.001, the tumor inhibition effect is obvious, the weight of each dosage group of mice is stable, and no obvious intolerance phenomenon exists.

Claims

A compound represented by the formula (III) or a pharmaceutically acceptable salt thereof,

wherein,

T ₁ 、T ₂ and T ₃ Each independently selected from CH and N;

T ₄ selected from CR ₆ And N;

ring A is selected from piperazinyl,

R ₁ Selected from the group consisting ofThe saidOptionally by 1, 2 or 3R _a Substitution;

each R is ₂ Are independently selected from H and C _1-3 Alkyl, said C _1-3 Alkyl is optionally substituted with 1, 2 or 3R _b Substitution;

R ₃ selected from C _1-6 Alkyl, said C _1-6 Alkyl is optionally substituted with 1, 2 or 3R _b Substitution;

R ₄ selected from H, F, cl, br and I;

R ₅ selected from H and F;

R ₆ selected from H, F, cl and CN;

m is selected from 0, 1, 2 and 3;

each R is _a Are respectively and independently selected from H, F, cl, br, I, CN, C _1-3 Alkyl, -C (O) OC _1-3 Alkyl, -C (O) NHC _1-3 Alkyl and cyclobutenyl groups, the C _1-3 Alkyl and cyclobutenyl are optionally substituted with 1, 2 or 3R;

each R is _b Each independently selected from H, F, cl, br, I and CN;

each R is independently selected from F, OCH ₃ 、N(CH ₃ ) ₂ 、NH ₂ And morpholinyl;

provided that the conditions are that,

1) When T is ₄ Selected from N, ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofWhen R is _a Selected from CN, R ₁ Quilt R _a Substitution formationOr 2) when T ₄ Selected from N, ring A is selected from piperazinyl, R ₁ Selected from the group consisting ofWhen in use, theIs covered by 1, 2 or 3R _a Substituted, R _a Selected from C _1-3 Alkyl, -C (O) OC _1-3 Alkyl, -C (O) NHC _1-3 Alkyl and cyclobutenyl groups, the C _1-3 Alkyl and cyclobutenyl are optionally substituted with 1, 2 or 3R, each R being independently selected from F, OCH ₃ 、N(CH ₃ ) ₂ 、NH ₂ And morpholinyl.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein each R _a Are respectively and independently selected from H, F, cl, br, I, CN, CH ₃ 、CH ₂ CH ₃ 、CH(CH ₃ ) ₂ 、-C(O)OCH ₃ 、-C(O)NHCH ₃ And cyclobutenyl groups, the CH ₃ 、CH ₂ CH ₃ 、CH(CH ₃ ) ₂ 、-C(O)OCH ₃ 、-C(O)NHCH ₃ And cyclobutenyl is optionally substituted with 1, 2 or 3R.
A compound according to claim 1 or 2 or a pharmaceutically acceptable thereof Salts, wherein each R _a Are respectively and independently selected from H, F, CN, CHF ₂ 、CH ₂ F、CH ₂ OCH ₃ 、CH ₂ N(CH ₃ ) ₂ 、CH ₂ NH ₂ 、-C(O)OCH ₃ 、-C(O)NHCH ₃ 、
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₁ Selected from the group consisting of
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein T ₄ Selected from N, ring A is selected from piperazinyl, R ₁ Selected from the group consisting of
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein each R ₂ Are independently selected from H and CH ₃ 。
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₃ Selected from CH (CH) ₃ ) ₂ 。
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₄ Selected from F and Cl.
The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the structural unitSelected from the group consisting of
The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein the structural unitSelected from the group consisting of
The compound according to any one of claims 1, 9 or 10, or a pharmaceutically acceptable salt thereof, wherein the structural unitSelected from the group consisting of
The compound according to any one of claims 1, 9 or 10, or a pharmaceutically acceptable salt thereof, wherein the structural unitSelected from the group consisting of
The compound of claim 12, or a pharmaceutically acceptable salt thereof, wherein the structural unit Selected from the group consisting of
A compound according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, selected from

Wherein,

R ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ and m is as defined in any one of claims 1 to 13.
The following compounds or pharmaceutically acceptable salts thereof,
a compound according to claim 15, or a pharmaceutically acceptable salt thereof, selected from the group consisting of
A compound according to claim 16, or a pharmaceutically acceptable salt thereof, selected from the group consisting of
Use of a compound according to any one of claims 1 to 17, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a tumour.
Use according to claim 18, wherein the tumour refers to KRAS ^G12C Mutation-related tumors.