WO2022199587A1 - Pyrimidine heterocyclic compound and application thereof - Google Patents

Abstract

A pyrimidine heterocyclic compound and an application thereof, in particular, relating to a compound as represented by formula (II) or a pharmaceutically acceptable salt thereof.

Description

Pyrimido-heterocyclic compounds and their applications

The present invention claims the following priority:

CN202110312693.8, application date: March 24, 2021;

CN202111127361.9, application date: September 18, 2021;

CN202210108554.8, application date: January 28, 2022;

CN202210179950.X, application date: February 25, 2022.

technical field

The present invention relates to a series of pyrimido-heterocyclic compounds and their applications, in particular to the compounds represented by formula (II) and their pharmaceutically acceptable salts.

Background technique

The KRAS gene is one of the most frequently mutated oncogenes in human cancers. In addition to directly promoting the proliferation and survival of tumor cells, KRAS gene mutations can also have an impact on the tumor microenvironment. In human cancers, the mutation occurs in nearly 90% of pancreatic cancers, 30%-40% of colon cancers, and 15%-20% of lung cancers. Additionally, 80% of oncogenic mutations occurred at codon 12, with the most common mutations including: p.G12D (41%), p.G12V (28%) and p.G12C (14%).

Activation of KRAS can control a variety of cellular functions, such as cell proliferation, apoptosis, metabolism, and more, through a variety of effector proteins. Direct inhibition of KRAS oncoprotein cells has been a long-standing pursuit in precision oncology. Currently, small molecules that directly target KRAS mutations are mainly concentrated in the field of KRAS ^G12C . The discovery and clinical development of some small molecule KRAS ^G12C inhibitors, such as Amgen's AMG510 and Mirati Therapeutics' MRTX849, herald the arrival of a new era of precision oncology. However, so far no KRAS ^G12D small molecule has entered the clinical research stage, and KRAS ^G12D- mutated tumor patients have not benefited from precision medicine. Therefore, research in the field of KRAS ^G12D small molecules is urgently needed.

SUMMARY OF THE INVENTION

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

in,

Ring A is selected from

T ₁ is selected from CH ₂ , NH and O;

T ₂ is selected from CH and N;

T ₃ and T ₄ are independently selected from CH ₂ and NH;

m, n, p and x are each independently selected from 0, 1 or 2;

r, v and w are each independently selected from 1 or 2;

q and u are independently selected from 1, 2 or 3;

each R ₁ is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, CH ₃ and CF ₃ ;

E ₁ and E ₃ are each independently selected from a bond, O, S, Se, NH and _{CH 2} , each independently optionally substituted with 1 or 2 _Ra ;

E ₂ is selected from -C(O)-, -C(O)-(CH ₂ ) _g - and -(CH ₂ ) _h -, when g and h are not selected from 0, the -C(O)- (CH ₂ ) _g - and -(CH ₂ ) _h - are each independently optionally substituted with 1, 2 or 3 R _b ;

X ₁ and X ₂ are each independently selected from N and CR ₄ ;

L ₁ is selected from a bond and CH ₂ optionally substituted with 1 or ₂ R _c ;

R ₂ is selected from 5-10 membered heterocycloalkyl optionally substituted with 1, 2 or 3 R _d ;

R ₃ is selected from C _6-10 aryl and 5-10 membered heteroaryl, said C _6-10 aryl and 5-10 membered heteroaryl respectively independently optionally replaced by 1, 2, 3, 4 or 5 R _e substitutions;

R ₄ is selected from H, F, Cl, Br, I, OH, NH ₂ , CN, COOH, C(=O)NH ₂ , C _1-3 alkyl, C _1-3 alkoxy, C _2-4 Alkenyl and C _2-4 alkynyl, the C(=O)NH ₂ , C _1-3 alkyl, C _1-3 alkoxy, C _2-4 alkenyl and C _2-4 alkynyl are each independently optionally substituted with 1, 2 or 3 _Rf ;

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

f, g and h are independently selected from 0, 1 and 2;

each R _a is independently selected from H and CH ₃ ;

each of R _b and R _c is independently selected from H, F, Cl, Br, I, OH, NH ₂ and CN;

Each R _d is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN and -OCO-C _1-3 alkylamino;

Each R _e is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, COOH, C(=O)NH ₂ , C _1-6 alkyl, C _1-3 alkoxy, C _{1-3 alkylamino, C 1-3 alkylthio ,} C _2-4 alkenyl, C _2-4 alkynyl and C _3-5 cycloalkyl, the C(=O)NH ₂ , C _{1- 6} alkyl, C _1-3 alkoxy, C _1-3 alkylamino, C _1-3 alkylthio, C _2-4 alkenyl, C _2-4 alkynyl and C _3-5 cycloalkyl independently optionally substituted with 1, 2 or 3 R;

each _{Rf is} independently selected from H, F, Cl, Br, I, OH, _NH2 , CN and _CH3 ;

Each R is independently selected from H, F, Cl, Br, I and _CH3 .

In some aspects of the invention, the ring A is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the ring A is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the structural fragment

selected from

R ₁ , e and other variables are as defined herein.

In some aspects of the invention, the structural fragment

selected from

Other variables are as defined in the present invention. In some aspects of the invention, the E ₂ is selected from C(O), C(O)CH ₂ , CH ₂ and CH ₂ CH ₂ , and other variables are as defined herein.

In some aspects of the invention, the structural fragment

selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the structural fragment

selected from

Other variables are as defined in the present invention.

_In some aspects of the present invention, said R4 is selected from H, F, _CH3 and _CF3 , and other variables are as defined herein.

In some aspects of the present invention, the X ₁ is selected from N, CH and C(CF ₃ ), and other variables are as defined herein.

In some aspects of the present invention, the X ₂ is selected from N, CH and CF, and other variables are as defined herein.

In some aspects of the present invention, the R _d is selected from H, F and -OC(=O)-N(CH ₃ ) ₂ , other variables are as defined herein.

In some aspects of the present invention, the R _d is selected from F, and other variables are as defined herein.

In some aspects of the invention, the R ₂ is selected from tetrahydropyrrolyl and hexahydro-1H-pyrrolizinyl, and said tetrahydropyrrolyl and hexahydro-1H-pyrrolizinyl are optionally separated by 1, 2 or 3 R _d substitutions, R _d and other variables are as defined herein.

In some aspects of the invention, the R ₂ is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₂ is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the structural unit

selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the structural unit

selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the _Re is selected from H, F, Cl, Br, I, OH, _NH2 , CN, _CH3 , _CH2CH3 , CH( _{CH3 ) 2} , _OCH3 , OCH _2CH3 , OCH( _CH3 ) ₂ , _NCH3 , _NCH2CH3 , N( _CH3 ) ₂ , _SCH3 , _SCH2CH3 , SCH _{(CH3)2 , -C≡CH , cyclopropyl} and Cyclobutyl, the _CH3 , _CH2CH3 , CH( _CH3 ) ₂ , _OCH3 , _OCH2CH3 , OCH( _{CH3 ) 2} , _NCH3 , _NCH2CH3 , N _{( CH3 ) 2} , _SCH3 , SCH2CH3, SCH( _{CH3 ) 2} , -C≡CH, cyclopropyl and cyclobutyl are optionally substituted with 1, ₂ or 3 R, R and other variables as defined herein.

In some aspects of the present invention, the _Re is selected from H, F, Cl, OH, CF ₃ , CH ₂ CH ₃ , CH(CH ₃ ) ₂ , OCF ₃ , SCH ₃ , -C≡CH,

Other variables are as defined in the present invention.

In some aspects of the invention, the _Re is selected from H, F, _Cl , OH, _NH2 , _CH3 , _CF3 , _CH2CH3 , CH( _{CH3 ) 2} , _OCH3 , OCF3, SCH ₃ , SCF ₃ , -C≡CH,

Other variables are as defined in the present invention.

In some aspects of the invention, the _R is selected from phenyl, naphthyl, and indazolyl, optionally surrounded by 1, 2, 3, 4, or 5 R _e Substitution, _Re and other variables are as defined herein.

In some aspects of the invention, the R ₃ is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₃ is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₃ is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₃ is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the structural unit

selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the structural unit

selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the structural unit

selected from

R4 and other variables are as defined in the present invention _.

In some aspects of the invention, the structural unit

selected from

_R4 is selected from H, F, _CH3 and _CF3 , other variables are as defined in the present invention.

In some aspects of the invention, the compound or a pharmaceutically acceptable salt thereof, the compound is selected from,

wherein, R ₂ and R ₃ are as defined in the present invention.

In some aspects of the invention, the compound or a pharmaceutically acceptable salt thereof, the compound is selected from,

wherein R ₃ and R _d are as defined in the present invention.

In some aspects of the invention, the compound or a pharmaceutically acceptable salt thereof, the compound is selected from,

in,

R _d is selected from F, Cl, Br, I, OH, NH ₂ , CN and -OCO-C _1-3 alkylamino;

R ₃ is as defined in the present invention.

In some aspects of the invention, the compound or a pharmaceutically acceptable salt thereof, the compound is selected from,

in,

R _d is selected from F, Cl, Br, I, OH, NH ₂ , CN and -OCO-C _1-3 alkylamino;

_R is as defined in the present invention;

The carbon atoms with "*" and "#" are chiral carbon atoms and exist as (R) or (S) single enantiomer or enriched in one enantiomer.

The present invention provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof,

in,

Ring A is selected from

T ₁ is selected from CH ₂ , NH and O;

T ₂ is selected from CH and N;

T ₃ and T ₄ are independently selected from CH ₂ and NH;

m, n, p and x are each independently selected from 0, 1 or 2;

r, v and w are each independently selected from 1 or 2;

q, s and u are each independently selected from 1, 2 or 3;

each R ₁ is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, CH ₃ and CF ₃ ;

E ₁ and E ₃ are each independently selected from a bond, O, S, Se, NR _a and CH ₂ , said CH ₂ being optionally substituted with 1 or 2 _Ra ;

E ₂ is selected from -C(O)-, -C(O)-(CH ₂ ) _g - and -(CH ₂ ) _h -, when g and h are not selected from 0, the -C(O)- (CH ₂ ) _g - and -(CH ₂ ) _h - are optionally substituted with 1, 2 or 3 R _b ;

X ₁ and X ₂ are each independently selected from N and CR ₄ ;

L ₁ is selected from a bond and CH ₂ optionally substituted with 1 or ₂ R _c ;

R ₂ is selected from 5-10 membered heterocycloalkyl optionally substituted with 1, 2 or 3 R _d ;

R ₃ is selected from C _6-10 aryl and 5-10 membered heteroaryl, the C _6-10 aryl and 5-10 membered heteroaryl are optionally replaced by 1, 2, 3, 4 or 5 R _e replace;

R ₄ is selected from H, F, Cl, Br, I, OH, NH ₂ , CN, COOH, C(=O)NH ₂ , C _1-3 alkyl, C _1-3 alkoxy, C _2-4 Alkenyl and C _2-4 alkynyl, the C(=O)NH ₂ , C _1-3 alkyl, C _1-3 alkoxy, C _2-4 alkenyl and C _2-4 alkynyl are optional replaced by 1, 2 or 3 R _f ;

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

f, g and h are independently selected from 0, 1 and 2;

each R _a is independently selected from H and CH ₃ ;

each of R _b and R _c is independently selected from H, F, Cl, Br, I, OH, NH ₂ and CN;

Each R _d is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN and -OCO-C _1-3 alkylamino;

Each R _e is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, COOH, C(=O)NH ₂ , C _1-6 alkyl, C _1-3 alkoxy, C _{1-3 alkylamino, C 1-3 alkylthio ,} C _2-4 alkenyl, C _2-4 alkynyl and C _3-5 cycloalkyl, the C(=O)NH ₂ , C _{1- 6} alkyl, C _1-3 alkoxy, C _1-3 alkylamino, C _1-3 alkylthio, C _2-4 alkenyl, C _2-4 alkynyl and C _3-5 cycloalkyl optional replaced by 1, 2 or 3 Rs;

each _{Rf is} independently selected from H, F, Cl, Br, I, OH, _NH2 , CN and _CH3 ;

Each R is independently selected from H, F, Cl, Br, I and _CH3 .

In some aspects of the invention, the structural fragment

selected from

Other variables are as defined in the present invention.

There are still some solutions of the present invention which are obtained by any combination of the above variables.

The present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof, which compound is selected from,

In some aspects of the invention, the compound or a pharmaceutically acceptable salt thereof, the compound is selected from,

In some aspects of the invention, the compound or a pharmaceutically acceptable salt thereof, the compound is selected from,

The present invention also provides following synthetic method:

method 1:

Method 2:

wherein R _3p is selected from

R ₃ is selected from

Method 3:

wherein R _3p is selected from

R ₃ is selected from

The present invention also provides the following test methods:

Test method 1. KRAS ^G12D inhibitory activity test

1. Objective: To screen out compounds that can effectively inhibit the binding of KRAS to GTP by the method of TR-FRET.

2. See Table 1 for information on consumables and instruments.

Table 1 Consumables and Instrument Information

3. Reagent preparation

a. Storage reagents:

1) KRAS Nucleotide Exchange Buffer

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

2) KRAS assay buffer

Take 20mL of 1000mM HEPES, 10mL of 1000mM magnesium chloride, 30mL of 5M sodium chloride, 100% 0.05mL Tween 20, and 939.95mL of water to prepare a 1L solution, sterilized by filtration, and stored at 4°C.

3) KRAS/Bodipy GDP/Tb-SA mixture

Take 9.5μL of 95μM KRAS protein, mixed with 440.5μL of KRAS nucleotide exchange buffer, incubated at room temperature for 1 hour, mixed with 8.4μL of 17.9μM Tb-SA, 1.8μL of 5mM Bodipy GDP, and 9539.8μL of KRAS experimental buffer to prepare 1L The solution, after mixing, was allowed to stand at room temperature for 6 hours and stored at -80°C.

b. Experimental reagents:

1) KRAS enzyme solution

Take 73.3 μL KRAS/Bodipy GDP/Tb-SA mixture and 2126.7 μL KRAS experimental buffer to prepare a 2200 μL solution.

2) SOS/GTP mixture

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

4. Experimental process

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

2) Use Bravo to make 10:3 dilutions of the compounds on the LDV plate;

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

4) Add 3 μL of 3nM Kras/0.5nM TB-SA/30nM BodipyGDP mixture and 3 μL of Ras buffer to each well of the experimental plate in turn using a Dragonfly automatic sampler, and centrifuge the experimental plate for 1 minute at 1000rpm/min;

5) Incubate the experimental plate at room temperature for 1 hour;

6) Add 3 μL of 120nM SOS/9mM GTP mixture to each well of the experimental plate using the Dragonfly automatic sampler, and centrifuge the experimental plate for 1 minute at 1000rpm/min;

7) Incubate the experimental plate for 1 hour at room temperature;

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

9) Use Excel and Xlfit for data analysis to calculate the IC ₅₀ of the test compound.

Test Method 2. Anticellular Proliferative Effects of Compounds in Tumor Cell Lines AsPC-1 and GP2D

Research purposes: In this experiment, the effect of the compounds on inhibiting cell proliferation was studied by detecting the effect of the compounds on the in vitro cell viability in the tumor cell lines AsPC-1 and GP2D.

The experimental materials are shown in Table 2.

Table 2 Experimental materials

cell line	tumor type	Growth characteristics	Cultivation method
AsPC-1	Pancreatic cancer	adherent growth	RPMI 1640+10% FBS
GP2D	colon cancer	adherent growth	DMEM+10%FBS+2mM L-glutamine

Ultra Low Cluster-96 well plate (Corning-7007)

Greiner CELLSTAR 96-well plate (#655090)

Promega CellTiter-Glo 3D Luminescence Cell Viability Assay Kit (Promega-G9683)

2104-10 EnVision Plate Reader, PerkinElmer

RPMI 1640, DMEM, PBS (phosphate buffered saline), FBS (fetal bovine serum), Antibiotic-antimycotic (antibiotic-antifungal), L-glutamine (L-glutamine), DMSO (dimethyl sulfoxide)

Experimental methods and steps

(1) Cell culture

The tumor cell lines were cultured in a 37°C, 5% CO2 incubator according to the culture conditions indicated in the culture method. Periodically passaged, cells in logarithmic growth phase were taken for plating.

(2) Cell plating

Cells were stained with trypan blue and viable cells were counted. The AsPC-1 cell concentration was adjusted to 7000 cells per well, and the GP2D cell concentration was adjusted to 8000 cells per well.

135 μL of cell suspension was added to each well of the ULA culture plate, and the same volume of culture medium without cells was added to the blank control air.

Immediately after plating, the ULA plates were centrifuged at 1000 rpm for 10 minutes at room temperature. NOTE: After centrifugation, be careful not to cause unnecessary shaking. The plates were incubated overnight in an incubator at 37°C, 5% _CO2 , and 100% relative humidity.

Preparation of 10X compound working solution and compound treatment of cells (the first day): After preparing 10X compound working solution (DMSO 10X working solution), add 15 μL of 10X compound working solution to ULA culture plate respectively, and add 15 μL of 10X compound working solution to the vehicle control and blank control. Add 15 μL of DMSO-cell culture medium mixture to it. Return the 96-well cell plate to the incubator for 120 hours. Cell spheroidization was observed every day until the end of the experiment.

CellTiter-Glo Luminescence Cell Viability Assay (Day 5): The following steps were performed according to the instructions of Promega CellTiter-Glo 3D Luminescence Cell Viability Assay Kit (Promega#G9683). Add 150 μL (equivalent to the volume of cell culture medium in each well) of CellTiter-Glo 3D reagent to each well. Wrap the cell plate in aluminum foil to protect from light. Shake the plate on an orbital shaker for 5 minutes. Mix the air mixture carefully by pipetting up and down 10 times. Make sure the spheroids are sufficiently detached before proceeding to the next step. The solution in the ULA plate was then transferred to a black bottom plate (#655090) and left at room temperature for 25 minutes to stabilize the luminescent signal. Luminescent signals were detected on a 2104EnVision plate reader.

data analysis:

The inhibition rate (IR) of the tested compound was calculated by the following formula: IR(%)=(1−(RLU compound−RLU blank control)/(RLU vehicle control−RLU blank control))*100%. The inhibition rates of different concentrations of compounds were calculated in Excel, and then the GraphPad Prism software was used to plot the inhibition curves and calculate the relevant parameters, including the minimum inhibition rate, the maximum inhibition rate and IC ₅₀ .

technical effect

The compound of the present invention has a good binding effect with KRAS ^G12D protein, can effectively inhibit p-ERK of GP2D cells, and has significant cell proliferation inhibitory activity on KRAS ^G12D mutant cells. In addition, the compounds of the present invention have good in vivo pharmacokinetic properties and significant antitumor effects.

Definition and Explanation

Unless otherwise specified, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered indeterminate or unclear without specific definitions, but should be understood in its ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient. As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue , without excessive toxicity, irritation, allergic reactions or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from compounds with specific substituents discovered by the present invention and relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral forms of such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts including, for example, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, and methanesulfonic acids; also include salts of amino acids such as arginine, etc. , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and thus can be converted into either base or acid addition salts.

The pharmaceutically acceptable salts of the present invention can be synthesized from the acid or base containing parent compound by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.

In addition to salt forms, the compounds provided herein also exist in prodrug forms. Prodrugs of the compounds described herein are readily chemically altered under physiological conditions to convert to the compounds of the present invention. Furthermore, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an in vivo environment.

Certain compounds of the present invention may exist in unsolvated as well as solvated forms, including hydrated forms. In general, solvated and unsolvated forms are equivalent and are intended to be included within the scope of the present invention.

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

Unless otherwise indicated, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of each other.

Unless otherwise specified, the terms "cis-trans isomer" or "geometric isomer" result from the inability to rotate freely due to double bonds or single bonds to ring carbon atoms.

Unless otherwise indicated, the term "diastereomer" refers to a stereoisomer in which the molecule has two or more chiral centers and the molecules are in a non-mirror-image relationship.

Unless otherwise specified, "(D)" or "(+)" means dextrorotatory, "(L)" or "(-)" means levorotatory, and "(DL)" or "(±)" means racemic.

Use solid wedge keys unless otherwise specified

and wedge-dotted keys

Indicate the absolute configuration of a stereocenter, using a straight solid key

and straight dashed keys

Indicate the relative configuration of the stereocenter, with a wavy line

Represents a solid wedge key

or wedge-dotted key

or with wavy lines

Represents a straight solid key

and straight dashed keys

Unless otherwise specified, when there is a double bond structure in the compound, such as carbon-carbon double bond, carbon-nitrogen double bond and nitrogen-nitrogen double bond, and each atom on the double bond has two different substituents attached (including nitrogen atom) In the double bond of the compound, a lone pair of electrons on the nitrogen atom is regarded as a substituent for its connection), if the atom on the double bond in the compound and its substituent are

represents the (Z) isomer, (E) isomer or a mixture of both isomers of the compound.

The compounds of the present invention may exist in particular. Unless otherwise specified, the term "tautomer" or "tautomeric form" refers to isomers of different functional groups that are in dynamic equilibrium and are rapidly interconverted at room temperature. A chemical equilibrium of tautomers can be achieved if tautomers are possible (eg, in solution). For example, proton tautomers (also called prototropic tautomers) include interconversions by migration of protons, such as keto-enol isomerization and imine-ene Amine isomerization. Valence tautomers include interconversions by recombination of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise indicated, the terms "enriched in one isomer", "enriched in isomers", "enriched in one enantiomer" or "enriched in one enantiomer" refer to one of the isomers or pairs The enantiomer content is less than 100%, and the isomer or enantiomer content is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, the terms "isomeric excess" or "enantiomeric excess" refer to the difference between two isomers or relative percentages of two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80% .

Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art The diastereoisomers were resolved and the pure enantiomers recovered. In addition, separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate). The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, compounds can be labeled with radioisotopes, such as tritium ( ³ H), iodine-125 ( ¹²⁵ I) or C-14 ( ¹⁴ C). For another example, deuterated drugs can be formed by replacing hydrogen with deuterium, and the bonds formed by deuterium and carbon are stronger than those formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All transformations of the isotopic composition of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention. "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 in which it does not.

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

When any variable (eg, 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 0-2 Rs, the group may optionally be substituted with up to two Rs, with independent options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

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

When one of the variables is selected from a single bond, it means that the two groups connected to it are directly connected, for example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.

When a substituent is vacant, it means that the substituent does not exist. For example, when X in A-X is vacant, it means that the structure is actually A. When the listed substituents do not indicate through which atom it is attached to the substituted group, such substituents may be bonded through any of its atoms, for example, pyridyl as a substituent may be through any one of the pyridine ring The carbon atom is attached to the substituted group.

When the listed linking group does not indicate its direction of attachment, the direction of attachment is arbitrary, for example,

The linking group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right.

It is also possible to connect ring A and ring B in the opposite direction to the reading order from left to right.

Combinations of the linking groups, substituents and/or variants thereof are permissible only if such combinations result in stable compounds.

Unless otherwise specified, the term "aromatic ring" is a cyclic group having a conjugated pi-electron system, the atoms of which are covered by a cloud of delocalized pi-electrons. In the structural formula, when it conforms to the valence state and covalent bonding rules, it can be written in the form of alternating single and double bonds, or it can be written as

represents the delocalized π electron cloud. For example, the structural formula

The structures represented are all the same, the structural formula

The structures represented are all the same. It can be a monocyclic or fused polycyclic ring system, wherein each ring is aromatic. Unless otherwise specified, the ring optionally contains 0, 1 or more heteroatoms independently selected from O, S and N.

Unless otherwise specified, _Cn-n+m or _Cn - _Cn+m includes any particular instance of n to n+ _m carbons, eg _C1-12 includes _C1 , _C2 , C3, C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , and C ₁₂ , also including any range from n to n+ _m , eg C _1-12 includes 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.; in the same way, n yuan to n +m-membered means that the number of atoms in the ring is from 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, also including any one range from n to n+m, for example, 3-12-membered ring includes 3-6 membered ring, 3-9 membered ring, 5-6 membered ring ring, 5-7 membered ring, 6-7 membered ring, 6-8 membered ring, and 6-10 membered ring, etc.

Unless otherwise specified, the term "C _1-6 alkyl" is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 6 carbon atoms. The C _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 and the like; it can be Is monovalent (eg methyl), divalent (eg methylene) or polyvalent (eg methine). Examples of C _1-6 alkyl 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), hexyl, etc.

Unless otherwise specified, the term "C _1-3 alkyl" is used to denote a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. The C _1-3 alkyl group includes C _1-2 and C _2-3 alkyl groups, etc.; it can be monovalent (eg methyl), divalent (eg methylene) or multivalent (eg methine) . Examples of _C1-3 alkyl groups include, but are not limited to, _methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

Unless otherwise specified, the term " _C1-3alkoxy " refers to those alkyl groups containing 1 to 3 carbon atoms attached to the remainder of the molecule through an oxygen atom. The C _1-3 alkoxy group includes C _1-2 , C _2-3 , C ₃ and C ₂ alkoxy and the like. Examples of C _1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

Unless otherwise specified, the term "C _1-3 alkylamino" refers to those alkyl groups containing 1 to 3 carbon atoms attached to the remainder of the molecule through an amino group. The C _1-3 alkylamino groups include C _1-2 , C ₃ and C ₂ alkylamino groups and the like. Examples of C _1-3 alkylamino include, but are not limited to, -NHCH ₃ , -N(CH ₃ ) ₂ , -NHCH ₂ CH ₃ , -N(CH ₃ )CH ₂ CH ₃ , -NHCH ₂ CH ₂ CH ₃ , - NHCH ₂ (CH ₃ ) ₂ and the like.

Unless otherwise specified, the term "C _1-3 alkylthio" refers to those alkyl groups containing 1 to 3 carbon atoms attached to the remainder of the molecule through a sulfur atom. The C _1-3 alkylthio group includes C _1-3 , C _1-2 and C ₃ alkylthio groups and the like. Examples of _C1-3 alkylthio groups include, but are not limited to, _-SCH3 , _-SCH2CH3 , _-SCH2CH2CH3 , _{-SCH2 ( CH3 ) 2 ,} and the like.

Unless otherwise specified, "C _2-4 alkenyl" is used to denote a straight or branched chain hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon double bond, a carbon-carbon double bond can be located anywhere in the group. The C _2-4 alkenyl group includes C _2-3 , C ₄ , C ₃ and C ₂ alkenyl groups, etc.; the C _2-4 alkenyl group may be monovalent, divalent or multivalent. Examples of C _2-4 alkenyl groups include, but are not limited to, vinyl, propenyl, butenyl, butadienyl, and the like.

Unless otherwise specified, "C _2-4 alkynyl" is used to denote a straight or branched chain hydrocarbon group consisting of 2 to 4 carbon atoms containing at least one carbon-carbon triple bond, a carbon-carbon triple bond can be located anywhere in the group. The C _2-4 alkynyl groups include C _2-3 , C ₄ , C ₃ and C ₂ alkynyl groups and the like. It can be monovalent, bivalent or multivalent. Examples of _C2-4alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, and the like.

Unless otherwise specified, "C _3-5 cycloalkyl" means a saturated cyclic hydrocarbon group consisting of 3 to 5 carbon atoms, which is a monocyclic ring system, said C _3-5 cycloalkyl including C _{3 -4} and C _4-5 cycloalkyl, etc.; it may be monovalent, divalent or polyvalent. Examples of _C3-5 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and the like.

Unless otherwise specified, the term "5-10 membered heterocycloalkyl" by itself or in combination with other terms denotes a saturated cyclic group consisting of 5 to 10 ring atoms, respectively, of which 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S, and N, and the remainder are carbon atoms, where the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (ie, NO and S(O) _p , p is 1 or 2). It includes monocyclic, bicyclic and tricyclic rings, wherein bicyclic and tricyclic rings include spiro, paracyclic and bridged rings. Furthermore, with respect to the "5-10 membered heterocycloalkyl", a heteroatom may occupy the position of attachment of the heterocycloalkyl to the remainder of the molecule. The 5-10-membered heterocycloalkyl includes 5-6-membered, 5-membered, 6-membered, 7-membered, 8-membered, 9-membered, 10-membered, and the like. Examples of 5-10 membered heterocycloalkyl include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.) , tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1 -piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazole Alkyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperidinyl or dioxepanyl and the like.

Unless otherwise specified, the terms "C _6-10 aryl ring" and "C _6-10 aryl group" can be used interchangeably in the present invention, and the term "C _6-10 aryl ring" or "C _6-10 aryl group" means by A cyclic hydrocarbon group composed of 6 to 10 carbon atoms with a conjugated π-electron system, which may be a monocyclic, fused bicyclic or fused tricyclic system, wherein each ring is aromatic. It may be monovalent, divalent or polyvalent, and _C6-10 aryl groups include _C6-9 , _C9 , _C10 and _C6 aryl groups and the like. Examples of _C6-10 aryl groups include, but are not limited to, phenyl, naphthyl (including 1-naphthyl and 2-naphthyl, and the like).

Unless otherwise specified, the terms "5-10-membered heteroaryl ring" and "5-10-membered heteroaryl" can be used interchangeably in the present invention, and the term "5-10-membered heteroaryl" refers to a ring consisting of 5 to 10 rings. A cyclic group composed of atoms with a conjugated π-electron system, wherein 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms. It can be a monocyclic, fused bicyclic or fused tricyclic ring system, wherein each ring is aromatic. Where the nitrogen atom is optionally quaternized, the nitrogen and sulfur heteroatoms may be optionally oxidized (ie, NO and S(O) _p , p is 1 or 2). A 5-10 membered heteroaryl group can be attached to the rest of the molecule through a heteroatom or a carbon atom. The 5-10-membered heteroaryl groups include 5-8-membered, 5-7-membered, 5-6-membered, 5- and 6-membered, 7-membered, 8-membered, 9-membered, 10-membered and other heteroaryl groups. Examples of the 5-10 membered heteroaryl group include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrrolyl, etc.) azolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5- oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1, 2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl and 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl , 4-thiazolyl and 5-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), thienyl (including 2-thienyl and 3-thienyl, etc.), pyridyl (including 2- -pyridyl, 3-pyridyl and 4-pyridyl, etc.), pyrazinyl, pyrimidinyl (including 2-pyrimidinyl and 4-pyrimidinyl, etc.), benzothiazolyl (including 5-benzothiazolyl, etc.) , purinyl, benzimidazolyl (including 2-benzimidazolyl, etc.), benzoxazolyl, indolyl (including 5-indolyl, etc.), isoquinolinyl (including 1-isoquinolinyl, etc.) and 5-isoquinolinyl, etc.), quinoxalinyl (including 2-quinoxalinyl and 5-quinoxalinyl, etc.) or quinolinyl (including 3-quinolinyl and 6-quinolinyl, etc.) .

The term "protecting group" includes, but is not limited to, "amino protecting group", "hydroxy protecting group" or "thiol protecting group". The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to: formyl; acyl groups, such as alkanoyl groups (eg, acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl groups, such as tert-butoxycarbonyl (Boc) ; Arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); Arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-di -(4'-Methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing hydroxyl side reactions. Representative hydroxy protecting groups include, but are not limited to: alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (eg acetyl); arylmethyl groups such as benzyl (Bn), p-methyl Oxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyl Dimethylsilyl (TBS) and the like.

The structure of the compound of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction method (SXRD), the cultured single crystal is collected by Bruker D8 venture diffractometer, the light source is CuKα radiation, and the scanning mode is:

After scanning and collecting relevant data, the crystal structure was further analyzed by the direct method (Shelxs97), and the absolute configuration could be confirmed.

The following abbreviations are used in the present invention: TBDPS: tert-butyltriphenylsilyl; MOM: methoxymethyl; TIPS: tri-tert-butylsilyl; hr: hours; min: minutes.

The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments enumerated below, embodiments formed in combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent to alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention.

The solvent used in the present invention is commercially available. Compounds are named according to conventional nomenclature in the art or are used

Software naming, commercially available compounds use supplier catalog names.

Description of drawings

Figure 1. Binding pattern of Compound A to KRAS ^G12D protein.

Figure 2. Binding pattern of compound B to KRAS ^G12D protein.

Figure 3. Binding pattern of compound C to KRAS ^G12D protein.

Figure 4. Binding pattern of compound D to KRAS ^G12D protein.

Figure 5. Binding pattern of Compound E to KRAS ^G12D protein.

Figure 6. Binding pattern of compound F to KRAS ^G12D protein.

Figure 7. Binding pattern of compound G to KRAS ^G12D protein.

Figure 8. Binding pattern of Compound H to KRAS ^G12D protein.

Figure 9. Binding profile of Compound I to KRAS ^G12D protein.

Figure 10. Binding pattern of Compound J to KRAS ^G12D protein.

Detailed ways

The present invention will be described in detail by the following examples, but it does not mean any unfavorable limitation of the present invention. The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments enumerated below, embodiments formed in combination with other chemical synthesis methods, and those well known to those skilled in the art Equivalent to alternatives, preferred embodiments include, but are not limited to, the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention.

Calculation example 1

The molecular docking process was performed by using Maestro (

Glide SP ^[1] and default options in version 2020-2). For the preparation of the protein model, select the crystal structure PDB: 5V6S of KRAS_G12C in the PDB database, mutate Cys12 to Asp12, use the protein preparation wizard module of Maestro ^[2] to add hydrogen atoms, and use the OPLS3 force field after energy optimization, as Docking template. For ligand preparation, the three-dimensional structure of the molecule was generated using LigPrep and energy minimization was performed ^[3] , and the small molecule conformation was sampled using the confgen module. Using the ligand of 5V6S as the centroid, the side length of the docking template was generated as

The cube docking grid of , and the related molecules were docked using this grid file. Then, according to the calculated docking scrore and binding mode, a reasonable docking conformation was selected and saved, and Pymol was used to generate a binding mode map. Binding patterns of compounds A to J to KRAS ^G12D protein are shown in Figures 1 to 10 .

[1]Glide,

LLC, New York, NY, 2020.

[2] Maestro,

LLC, New York, NY, 2020.

[3]LigPrep,

LLC, New York, NY, 2020.

[4] Pymol,

LLC, New York, NY, 2020.

Conclusion: The compound of the present invention has good binding with KRAS ^G12D .

Reference Example 1 Synthesis of Intermediate A

first step

Dimethyl sulfate (31.7 g, 251.33 mmol, 23.83 mL, 1.44 eq) was added to compound A-1 (25 g, 174.65 mmol, 1 eq), and the mixture was stirred in an oil bath at 60° C. for 19 hours. After the reaction solution was cooled to room temperature, it was added dropwise to triethylamine (120 mL), stirred for 1 hour, water (300 mL) was added, extracted with ethyl acetate (300 mL*3), all organic phases were combined and dried over anhydrous sodium sulfate , filtered, and the filtrate was spin-dried to obtain compound A-2, which was directly sent to the next step without purification.

¹ H NMR (400MHz, CDCl ₃ )δ=2.13-2.24(m,1H), 2.28-2.39(m,1H), 2.44-2.66(m,2H), 3.73(s,3H), 3.85(s,3H) ), 4.49-4.55 (m, 1H).

second step

Compound A-2 (44 g, 279.96 mmol, 1 eq) and methyl 2-nitroacetate (44.28 g, 371.85 mmol, 1.33 eq) were mixed, and then stirred in an oil bath at 60° C. for 40 hours. After the reaction solution was cooled to room temperature, the crude product was purified by column chromatography (petroleum ether:dichloromethane=1:1～0:1, followed by petroleum ether:ethyl acetate=3:1) to obtain compound A-3.

¹ H NMR (400 MHz, CDCl ₃ ) δ = 2.26-2.37 (m, 1H), 2.44-2.55 (m, 1H), 3.15-3.44 (m, 2H), 3.80-3.87 (m, 6H), 4.62 (ddd , J=17.38, 9.10, 5.65Hz, 1H), 9.42-10.02 (m, 1H).

third step

Wet palladium carbon (containing palladium 10%, water 50%) was added to a solution of compound A-3 (18.00 g, 73.71 mmol, 1 eq) in anhydrous methanol (250 mL), replaced with hydrogen three times, under H ₂ (45 psi) ) in an oil bath at 60°C for 2 days. The reaction solution was cooled to room temperature, filtered, and the filtrate was spin-dried to obtain compound A-4.

¹ H NMR (400MHz, CDCl ₃ ) δ=1.29-2.30(m, 9H), 3.71-3.75(m, 3H), 3.76-3.81(m, 6H), 4.02-4.13(m, 2H), 4.41(d) , J=4.13Hz, 1H), 6.11-6.38 (m, 1H).

the fourth step

Lithium aluminum hydride (12.5 g, 329.38 mmol, 6.16 eq) was added to the reaction solution of compound A-4 (9.85 g, 53.48 mmol, 1 eq) in anhydrous tetrahydrofuran (200 mL) at 0 °C, and gradually warmed to room temperature (20 °C) stirring for 16hr. After the reaction, water (12.5 mL) was added dropwise with stirring at low temperature, 15% sodium hydroxide solution (12.5 mL) was added dropwise, and then water (25 mL) was added dropwise. At this moment the reaction system will become a sandy solid, add tetrahydrofuran (200mL), stir at room temperature for 1hr, filter directly, take the filtrate; add tetrahydrofuran (500mL*3) to the filter residue and stir for 1hr, filter, take the filtrate, combine the above filtrate and spin dry to obtain Compound A-5.

¹ H NMR (400 MHz, CDCl ₃ ) δ = 0.94-2.44 (m, 6H), 2.47-3.05 (m, 2H), 3.22-4.03 (m, 3H).

the fifth step

Combine tert-butyldiphenylchlorosilane (39.43g, 143.46mmol, 36.85mL, 1.5eq), imidazole (13.02g, 191.28mmol, 2eq) and 4-dimethylaminopyridine (2.34g, 19.14mmol, 0.2eq) It was added to a solution of compound A-5 (13.6 g, 95.64 mmol, 1 eq) in anhydrous dichloromethane (200 mL), and stirred at room temperature (25° C.) for 20 hr. After the reaction, the reaction solution was washed with saturated ammonium chloride (100 mL*3), the organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was spin-dried. The crude product was purified by column chromatography (petroleum ether:ethyl acetate=10:1, followed by dichloromethane:methanol=10:1) to obtain compound A-6.

MS (ESI) m/z: 381.1 [M+H] ⁺ ; ¹ H NMR (400 MHz, CDCl ₃ ) δ=1.00-1.10 (m, 9H), 1.87-2.02 (m, 3H), 2.07-2.22 (m ,1H),2.59(br d,J＝12.80Hz,1H),2.72-2.89(m,1H),3.15-3.30(m,1H),3.39-3.60(m,3H),3.64-3.84(m, 1H), 3.86-3.99 (m, 1H), 7.35-7.47 (m, 6H), 7.59-7.73 (m, 4H).

Step 6

Triethylamine (16.54g, 163.43mmol, 22.75mL, 5eq) and di-tert-butyl carbonate (6.42g, 29.42mmol, 6.76mL, 0.9eq) were added to compound A-6 (12.44g, 32.69mmol) at 0°C , 1 eq) in anhydrous DCM (150 mL), stirred at 0 °C for 2 hr. After the reaction was completed, saturated ammonium chloride solution (80mL*3) was added to the reaction solution to quench the reaction, the layers were separated, the organic phase was taken, washed with saturated brine (80mL), dried with anhydrous sodium sulfate, filtered, and the filtrate was Spin dry. The crude product was purified by column chromatography (petroleum ether:tetrahydrofuran=10:1～8:1) to obtain Intermediate A. MS (ESI) m/z: 481.1 [M+H] ⁺ ; ¹ H NMR (400 MHz, CDCl ₃ ) δ=1.02-1.11 (m, 9H), 1.25-1.51 (m, 9H), 1.57-2.04 (m , 4H), 2.38-3.17(m, 3H), 3.37-3.78(m, 2H), 4.01-4.69(m, 2H), 7.35-7.47(m, 6H), 7.59-7.73(m, 4H).

Example 1

first step

To a solution of compound 1-1 (18.77 g, 115.15 mmol) in N,N-dimethylformamide (100 mL) and acetonitrile (100 mL) was added (1-chloromethyl-4-fluoro-1,4-diazo Heterobicyclo[2.2.2]octane bis(tetrafluoroborate) salt (48.95 g, 138.18 mmol), the reaction was stirred at 80 ° C for 1 hour. After the reaction was complete, 500 mL of water was added to the reaction solution, and extracted twice with ethyl acetate , 100 mL each time, the organic phase was washed twice with water, 100 mL each time, and then washed with 200 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. After preparative chromatography column (column: Phenomenex luna C18 (250* 70 mm, 10 microns); mobile phase: [water (formic acid)-acetonitrile]; gradient: acetonitrile %: 24%-54%, 24 min) purification gave compound 1-2. MS (ESI) m/z: 181.1 [M+H] ⁺ .

second step

To a solution of compound 1-2 (8 g, 44.20 mmol) in acetonitrile (80 mL), N-iodosuccinimide (14.92 g, 66.30 mmol) and p-toluenesulfonic acid monohydrate (420.38 mg, 2.21 mmol) were added to react The solution was stirred at 70°C for 1 hour. After the reaction was completed, 10 mL of saturated sodium sulfite and 20 mL of water were added, extracted twice with 20 mL of ethyl acetate, the combined organic phases were washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain the compound 1-3. MS (ESI) m/z: 306.9 [M+H] ⁺ .

third step

To compound 1-3 (13.5 g, 43.99 mmol) in N,N-dimethylacetamide (100 mL), cuprous cyanide (5.91 g, 65.98 mmol) was added, and the reaction was stirred at 140° C. under nitrogen protection for 2 hours . After the reaction was completed, the reaction solution was added with 400 mL of water, 200 mL of ethyl acetate, filtered, and the filtrate was extracted twice with 100 mL of ethyl acetate. The organic layers were combined, washed twice with 100 mL of water, and then with 100 mL of saturated aqueous sodium chloride solution. Washed once, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain compound 1-4. MS (ESI) m/z: 205.9 [M+H] ⁺ .

the fourth step

Compound 1-4 (11.5 g, 55.82 mmol) was added to concentrated sulfuric acid (30 mL), and the reaction solution was stirred at 70° C. for 2 hours. After the reaction was completed, the reaction solution was added to 300 mL of ice water, and the pH was adjusted to 7-8 with saturated potassium carbonate, and extracted twice with 200 mL of ethyl acetate. The combined ethyl acetate phases were washed with 200 mL of saturated brine, Dry over anhydrous sodium sulfate, filter, and concentrate to dryness under reduced pressure to obtain compound 1-5. MS(ESI) m/z: 224.0 [M+H] ⁺ .

the fifth step

To a solution of compound 1-5 (5.09 g, 22.72 mmol) in N,N-dimethylformamide (70 mL), potassium carbonate (12.56 g, 90.88 mmol), carbonyldiimidazole (14.74 g, 90.88 mmol) were added, The reaction was stirred at 90°C for 14 hours. After the reaction was completed, the reaction solution was added to 200 mL of water, and the pH was adjusted to 6-7 with 1N hydrochloric acid, and extracted twice with 200 mL of ethyl acetate. The combined ethyl acetate phase was washed with 200 mL of water and 200 mL of saturated brine. , dried over anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, added 5 mL of ethyl acetate, 25 mL of petroleum ether, beat and stirred at 20°C for 30 minutes, filtered, and the filter cake was concentrated to dryness under reduced pressure to obtain compound 1-6. MS(ESI) m/z: 249.8 [M+H] ⁺ .

Step 6

Compound 1-6 (4g, 16.00mmol, 1eq.) was dissolved in anhydrous methanol (150mL), then sodium methoxide (1.90g, 35.20mmol, 2.2eq.) was added, and the temperature was raised to 70°C for 12 hours. After completion of the reaction, the mixture was concentrated under reduced pressure, 150 mL of 1N aqueous hydrochloric acid was added, and the mixture was stirred for 10 minutes. After filtration, the filter cake was collected and concentrated under reduced pressure. Compounds 1-7 were obtained. MS (ESI) m/z: 245.9 [M+H] ⁺ .

Step 7

Compound 1-7 (1g, 4.07mmol, 1eq.) was dissolved in phosphorus oxychloride (20mL), and then N,N-diisopropylethylamine (1.48g, 11.48mmol, 2mL, 2.82eq.) was added , raise the temperature to 90 ℃ and react for 1 hour. After the reaction was completed, it was concentrated under reduced pressure, the concentrate was diluted with dichloromethane 80 mL, then poured into an iced 4M aqueous sodium bicarbonate solution (200 mL), washed and separated, the organic phase was collected, and the aqueous phase was extracted with dichloromethane (70 mL× 2), combine the organic phases, concentrate under reduced pressure, and purify by silica gel column (petroleum ether:ethyl acetate=1:0～5:1) to obtain compound 1-8.

Step 8

Compound 1-8 (535mg, 1eq.) and Intermediate A (1.00g, 2.08mmol, 1.1eq.) were sequentially added to dichloromethane (10mL), followed by N,N-diisopropylethylamine ( 734.30mg, 5.68mmol, 989.63μL, 3eq.), the reaction was reacted at 25°C for 12 hours. After the reaction was completed, it was concentrated under reduced pressure. The concentrate was purified by silica gel column (petroleum ether:ethyl acetate=1:0-5:1) to obtain compound 1-9. MS (ESI) m/z: 732.1 [M+H] ⁺ .

Step 9

To compound 1-9 (800 mg, 1.09 mmol, 1 eq.) was dissolved in tetrahydrofuran (5 mL), then sodium methanethiolate (107.36 mg, 1.53 mmol, 97.60 μL, 1.4 eq.) was added, and the reaction was stirred at 25 °C for reaction 1 Hour. After the reaction was completed, 10 mL of water was added, and 20 mL of ethyl acetate (10 mL*2) was added for extraction. The organic phases were combined and concentrated under reduced pressure. The concentrate was purified by silica gel column (petroleum ether:ethyl acetate=1:0～2:1) to obtain compound 1-10. MS (ESI) m/z: 742.2 [M+H] ⁺ .

Step 10

Compound 1-10 (720mg, 969.30μmol, 1eq.) was dissolved in tetrahydrofuran (5mL), then tetrabutylammonium fluoride (1M tetrahydrofuran solution) (1M, 1.07mL, 1.1eq.) was added, and the reaction was carried out at 25°C for 1 Hour. After the reaction was completed, it was concentrated under reduced pressure, and the concentrate was purified by silica gel column (petroleum ether:ethyl acetate=1:0～3:1) to obtain compound 1-11

MS (ESI) m/z: 468.1 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl ₃ )δ=4.94-5.12(d, J=13.20Hz, 1H), 3.96-4.61(m, 5H), 3.09-3.25(m, 1H), 2.59(s, 3H), 1.88-2.12(m, 2H), 1.68-1.86(m, 2H), 1.42-1.54(s, 9H).

Step 11

Compound 1-11 (250mg, 534.25μmol, 1eq.), compound 1-12 (410.73mg, 801.38μmol, 1.5eq.), 1,1-bis(tert-butylphosphino)ferrocene palladium chloride ( 27.48mg, 53.43μmol, 0.1eq.) and sodium carbonate (169.87mg, 1.60mmol, 3eq.) were added to a mixed solution of dioxane (10mL) and water (2mL), and the reaction was stirred at 90°C for 12 hours under nitrogen protection , after the completion of the reaction, concentrated under reduced pressure. The concentrate was purified by silica gel column (petroleum ether:ethyl acetate=1:0～3:1) to obtain compound 1-13. MS (ESI) m/z: 818.4 [M+H] ⁺ .

Step 12

Compound 1-13 (325mg, 397.28μmol, 1eq.) was dissolved in dichloromethane (10mL), then m-chloroperoxybenzoic acid (182.82mg, 794.57μmol, 75% content, 2eq.) was added, and the reaction was carried out at 25°C 20 minutes. After the reaction is completed, filter, and the filtrate is directly wet-loaded through a silica gel column, and purified (petroleum ether:ethyl acetate=1:0-0:1) to obtain compound 1-14. MS (ESI) m/z: 850.4 [M+H] ⁺ .

step thirteen

Compound 1-14 (100mg, 117.64μmol, 1eq.) and compound 1-15 (93.64mg, 588.20μmol, 5eq.) were dissolved in dichloromethane (5mL), then sodium hydride (14.12mg, 352.92μmol, 60% purity, 3eq.), react at 25°C for 10 minutes. After the reaction was completed, 5 mL of 1N aqueous hydrochloric acid was added dropwise to the reaction to quench, and then purified by thin-layer silica gel chromatography (petroleum ether:ethyl acetate=1:1) to obtain compound 1-16. MS (ESI) m/z: 929.4 [M+H] ⁺ .

Step 14

Compound 1-16 (105 mg, 113.01 μmol, 1 eq.) was dissolved in tetrahydrofuran (5 mL), and then tetrabutylammonium fluoride (1 M tetrahydrofuran solution) (1 M, 339.02 μL, 3 eq.) was reacted at 25° C. for 10 minutes. After the completion of the reaction, the reaction solution was purified by thin-layer silica gel chromatography (dichloromethane:ethyl acetate=1:1) to obtain compound 1-17. MS (ESI) m/z: 773.2 [M+H] ⁺ .

Step 15

Compound 1-17 (120 mg, 155.28 μmol, 1 eq.) was dissolved in methanol (3 mL), then a methanolic hydrochloric acid solution (4 M, 2 mL, 51.52 eq.) was added, and the reaction was carried out at 25° C. for 12 minutes. After the reaction was completed, it was concentrated under reduced pressure, the concentrate was added to 10 mL of saturated aqueous sodium bicarbonate solution, extracted with 30 mL of ethyl acetate (10 mL*3), the organic phases were combined and concentrated under reduced pressure. The concentrate was prepared by high performance liquid chromatography (HPLC) (column: Phenomenex C18150*40mm*5μm; mobile phase: [water (0.1% formic acid)-acetonitrile]; gradient: acetonitrile %: 1%-30%, 8min) Purified, and then subjected to supercritical fluid chromatography (SFC) (chiral column: DAICEL Chiralcel OD-3 100*4.6mm I.D., 3 μm); mobile phase: [A: carbon dioxide, B: ethanol (0.05% diethylamine)]; Elution gradient: B: 40%; flow rate: 2.8 mL/min) separation to give compound 1a and compound 1b.

Compound 1a (SFC analysis method: chiral column: Chiralcel OD-3 100*4.6mm ID, 3μm; mobile phase: [A: carbon dioxide, B: ethanol (0.05% diethylamine)] elution gradient: B: 40% ; flow rate: 2.8 mL/min, retention time: 2.239 min, ee=96%): MS (ESI) m/z: 629.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, CD ₃ OD) δ: 7.80- 7.90(m,1H),7.17-7.40(m,3H),5.27-5.50(m,1H),5.04(br d,J＝13.6Hz,1H),4.57-4.69(m,1H),4.28-4.53 (m,3H),4.15(br d,J＝6.4Hz,1H),3.65-3.86(m,2H),3.35-3.62(m,4H),3.10-3.30(m,2H),1.75-2.55( m, 10H).

Compound 1b (SFC analysis method: Chiral column: Chiralcel OD-3 100*4.6 mm ID, 3 μm; Mobile phase: [A: carbon dioxide, B: ethanol (0.05% diethylamine)] elution gradient: B: 40% ; flow rate: 2.8 mL/min, retention time: 3.392 min, ee=99%): MS (ESI) m/z: 629.4 [M+H] ⁺ ; ¹ H NMR (400 MHz, CD ₃ OD) δ: 7.82- 7.86(m, 1H), 7.13-7.38(m, 3H), 5.25-5.45(m, 1H), 5.03-5.06(m, 1H), 4.56-4.64(m, 2H), 4.25-4.50(m, 3H ), 4.15(br d, J＝7.3Hz, 1H), 3.64-3.81(m, 2H), 3.34-3.61(m, 3H), 3.20-3.30(m, 1H), 3.04-3.15(m, 1H) ,1.85-2.40(m,10H).

With reference to the synthetic steps of Example 1, the following compounds were prepared:

biological test data

Experimental example 1: GP2D CTG test

Experimental purpose: The purpose of this experiment is to verify the proliferation inhibitory effect of the compounds of the present invention on KRAS ^G12D mutant GP2D human colon cancer cells.

Experimental materials: cell line GP2D, DMEM medium, penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera.

3D Cell Viability Assay (3D Cell Viability Chemiluminescence Detection Reagent) reagent was purchased from Promega.

experimental method:

GP2D cells were seeded in a 96-well U-bottom cell culture plate, 80 μL of cell suspension per well, which contained 2000 GP2D cells. Cell plates were incubated overnight in a carbon dioxide incubator. The compounds to be tested were diluted 5-fold to the 8th concentration, that is, from 200 μM to 2.56 nM, and a double-well experiment was set up. Add 78 μL of medium to the middle plate, and then transfer 2 μL of each well of the compound to the middle plate according to the corresponding position. After mixing, transfer 20 μL of each well to the cell plate. Compound concentrations transferred to the cell plate ranged from 1 μM to 0.0128 nM. The cell plates were placed in a carbon dioxide incubator for 5 days. After the incubation of the compound-added cell plate, 100 μL of cell viability chemiluminescence detection reagent was added to the cell plate, and incubated at room temperature for 10 minutes to stabilize the luminescence signal. Read using a multi-label analyzer.

data analysis:

Using the equation (Sample-Min)/(Max-Min)*100% to convert the raw data into inhibition rate, the IC ₅₀ value can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode).

Experimental results: The results are shown in Table 3.

Table 3 Test results of the compounds of the present invention on GP2D CTG cell proliferation inhibition activity

testing sample	GP2D CTG IC 50 (nM)
Compound 1b	8.92

Conclusion: The compound of the present invention has a significant inhibitory effect on the proliferation of GP2D cells.

Experimental example 2: GP2D p-ERK experiment

Objective: To screen out compounds that can effectively inhibit p-ERK in GP2D cells by HTRF method.

experimental method:

1) GP2D cells were seeded in a transparent 96-well cell culture plate, 80 μL of cell suspension per well, each well containing 8000 cells, the cell plate was placed in a carbon dioxide incubator, and incubated at 37°C overnight;

2) Add 2 μL of compound to 78 μL of cell culture medium, after mixing, take 20 μL of compound solution and add it to the corresponding well of the cell plate, put the cell plate back into the carbon dioxide incubator and continue to incubate for 1 hour;

3) After the incubation, discard the cell supernatant and add 50 μL of 1X cell lysis solution to each well, and incubate with shaking at room temperature for 30 minutes;

4) Use detection buffer to dilute Phospho-ERK1/2 Eu Cryptate antibody and Phospho-ERK1/2 d2 antibody by 20 times;

5) Take 16μL of cell lysate supernatant per well into a new 384 white microplate, add 2μL of Phospho-ERK1/2 Eu Cryptate antibody dilution and 2μL of Phospho-ERK1/2 d2 antibody dilution, and incubate at room temperature for at least 4 Hour

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

7) Calculate the IC ₅₀ of the test compound.

Experimental results: The experimental results are shown in Table 4.

Table 4 Test results of the compounds of the present invention on GP2D p-ERK cell inhibitory activity

testing sample	GP2D p-ERK inhibitory activity IC 50 (nM)
Compound 1b	2.11

Conclusion: The compounds of the present invention have a significant inhibitory effect on p-ERK in GP2D cells.

Experimental example 3: AsPC1 CTG experiment

Experimental purpose: The purpose of this experiment is to verify the inhibitory effect of the compounds of the present invention on the proliferation of AsPC-1 cells with KRAS ^G12D mutation.

Experimental Materials:

RPMI-1640 medium, penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (Cell Viability Chemiluminescence Detection Reagent) reagent was purchased from Promega. The AsPC-1 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Nivo Multilabel Analyzer (PerkinElmer).

experimental method:

AsPC-1 cells were seeded in white 96-well plates, 80 μL of cell suspension per well, which contained 3000 AsPC-1 cells. Cell plates were incubated overnight in a carbon dioxide incubator.

The compounds to be tested were diluted 5-fold to the 8th concentration with a spray gun, that is, from 2 mM to 25.6 nM, and a double-well experiment was set up. Add 78 μL of medium to the middle plate, and then transfer 2 μL of each well of the compound to the middle plate according to the corresponding position. After mixing, transfer 20 μL of each well to the cell plate. Compound concentrations transferred to cell plates ranged from 10 [mu]M to 0.128 nM. The cell plates were placed in a carbon dioxide incubator for 6 days. Another cell plate was prepared, and the signal value was read on the day of drug addition as the maximum value (Max value in the following equation) to participate in data analysis. Add 25 μL of cell viability chemiluminescence detection reagent to each well of the cell plate, and incubate at room temperature for 10 minutes to stabilize the luminescence signal. Read using a multi-label analyzer.

Add 25 μL of cell viability chemiluminescence detection reagent per well to the cell plate, and incubate at room temperature for 10 minutes to stabilize the luminescence signal. Read using a multi-label analyzer.

data analysis:

Using the equation (Sample-Min)/(Max-Min)*100% to convert the raw data into inhibition rate, the IC ₅₀ value can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode).

Experimental results: The experimental results are shown in Table 5.

Table 5 Test results of the compounds of the present invention on AsPC-1 CTG cell inhibitory activity

testing sample	AsPC-1 CTG IC 50 (nM)
Compound 1b	45.8

Conclusion: The compounds of the present invention have a significant inhibitory effect on the proliferation of AsPC-1 cells.

Experimental example 4: PANC0403 CTG experiment

Experimental purpose: The purpose of this experiment is to verify the proliferation inhibitory effect of the compounds of the present invention on KRAS ^G12D mutant PANC0403 cells

Experimental Materials:

Cell line PANC0403 was purchased from Nanjing Kebai, RPMI1640 medium was purchased from BI, penicillin/streptomycin antibiotics were purchased from Yuanbi, and fetal bovine serum was purchased from Gibco.

3D Cell Viability Assay (3D Cell Viability Chemiluminescence Detection Reagent) reagent was purchased from Promega. experimental method:

The PANC0403 cells were seeded in a 96-well U-bottom cell culture plate, 80 μL of cell suspension per well, which contained 4000 PANC0403 cells. Cell plates were incubated overnight in a carbon dioxide incubator. The compounds to be tested were diluted 5-fold to the 8th concentration, that is, from 2000 μM to 25.6 nM, and a double-well experiment was set up. Add 78 μL of medium to the middle plate, and then transfer 2 μL of each well of the compound to the middle plate according to the corresponding position. After mixing, transfer 20 μL of each well to the cell plate. Compound concentrations transferred to cell plates ranged from 10 [mu]M to 0.128 nM. The cell plates were placed in a carbon dioxide incubator for 5 days. After the incubation of the cell plate with the compound added, 100 μL of cell viability chemiluminescence detection reagent was added to the cell plate, and incubated at room temperature for 10 minutes to stabilize the luminescence signal. Read using a multi-label analyzer.

data analysis:

Using the equation (Sample-Min)/(Max-Min)*100% to convert the raw data into inhibition rate, the IC ₅₀ value can be obtained by curve fitting with four parameters ("log(inhibitor) vs. response--Variable slope" mode).

Experimental results: The experimental results are shown in Table 6.

Table 6 Test results of the compounds of the present invention on the inhibitory activity of PANC0403 CTG cells

testing sample	PANC0403 CTG inhibitory activity IC 50 (nM)
Compound 1b	54

Conclusion: The compound of the present invention has a significant inhibitory effect on the proliferation of PANC0403 cells.

Experimental Example 5: Pharmacokinetic Data

Experimental purpose: To test the pharmacokinetic data of the compound in CD-1 mice

experimental method:

Weigh 2.08 mg of the test compound into a sample bottle, add 204 μL of Solutol, stir for 3 minutes, add (1000+837) μL of pure water, and stir for 3 minutes to obtain a clear solution. 7-10-week-old male CD-1 mice were selected and given the test compound solution by intraperitoneal injection (IP) at a dose of 10 mg/kg. Whole blood was collected at 0.25, 0.5, 1, 2, 4, 8, and 24 hours, and plasma was prepared. The drug concentration was analyzed by LC-MS/MS method, and the pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

Definition of each parameter: IP: intraperitoneal injection; C _max : the highest blood drug concentration after administration; T _max : the time required to reach the peak drug concentration after administration; T _1/2 : the time required for the blood drug concentration to drop by half T _last : the time of the last detection point; AUC _0-last : the area under the drug-time curve, which refers to the area enclosed by the blood drug concentration curve versus the time axis.

Experimental results: The experimental results are shown in Table 7:

Table 7 PK test results in plasma of compounds of the present invention

Conclusion: The compounds of the present invention exhibit higher drug exposure, longer half-life, and have good in vivo pharmacokinetic properties.

Experimental Example 6: In vivo efficacy test

Experimental purpose: To investigate the antitumor effect of the compounds of the present invention on the human pancreatic cancer PANC0403 xenograft tumor model

Experimental material: sulfobutylcyclodextrin (supplier: CyDex Pharmaceuticals, KS),

experimental method:

Female Balb/c nude mice were inoculated subcutaneously with the PANC0403 human pancreatic cancer cell line and randomly divided into vehicle control and compound groups (6 animals per group) according to tumor volume and body weight on day 23 after inoculation, and administered as described below. Drug treatment:

Group 1 (vehicle control group): administration started in the afternoon on the day of grouping (the day of administration was the 0th day of administration), and the vehicle was administered by intraperitoneal injection at a dose of 0.1 mL/10 g body weight twice a day. Group 2 (compound group): administration started in the afternoon on the day of grouping (the day of administration was the 0th day of administration), and the compound was administered by intraperitoneal injection at a dose of 5 mg/kg body weight twice a day. The vehicle was 10% Sulfobutylcyclodextrin in 50 mM pH 5.0 citric acid buffer, Sulfobutylcyclodextrin Supplier: CyDex Pharmaceuticals, KS.

The length (a) and width (b) of the tumor were measured, and the tumor volume (T), tumor inhibition rate (TGI) and tumor proliferation rate (T/C) were calculated. The calculation formula is: T=a×b ² /2; TGI(%)=[1-(tumor volume of compound group on the nth day of administration-tumor volume of compound group on the 0th day of administration)/(vehicle control group on the nth day of administration) Tumor volume-tumor volume of vehicle control group on day 0)]×100%; T/C(%)=tumor volume of compound group/tumor volume of control group×100%.

Experimental results: The experimental results are shown in Table 8.

Table 8 Inhibitory effect of compounds of the present invention on PANC0403 xenograft tumor model

Note*: Mean±SEM, n=6.

Conclusion: The compounds of the present invention have significant antitumor effect on human pancreatic cancer PANC0403 xenograft tumor model.

Claims (25)

1. A compound represented by formula (II) or a pharmaceutically acceptable salt thereof,

   

   in,

   Ring A is selected from

   

   T ₁ is selected from CH ₂ , NH and O;

   T ₂ is selected from CH and N;

   T ₃ and T ₄ are independently selected from CH ₂ and NH;

   m, n, p and x are each independently selected from 0, 1 or 2;

   r, v and w are each independently selected from 1 or 2;

   q and u are independently selected from 1, 2 or 3;

   each R ₁ is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, CH ₃ and CF ₃ ;

   E ₁ and E ₃ are each independently selected from a bond, O, S, Se, NH and _{CH 2} , each independently optionally substituted with 1 or 2 _Ra ;

   E ₂ is selected from -C(O)-, -C(O)-(CH ₂ ) _g - and -(CH ₂ ) _h -, when g and h are not selected from 0, the -C(O)- (CH ₂ ) _g - and -(CH ₂ ) _h - are each independently optionally substituted with 1, 2 or 3 R _b ;

   X ₁ and X ₂ are each independently selected from N and CR ₄ ;

   L ₁ is selected from a bond and CH ₂ optionally substituted with 1 or ₂ R _c ;

   R ₂ is selected from 5-10 membered heterocycloalkyl optionally substituted with 1, 2 or 3 R _d ;

   R ₃ is selected from C _6-10 aryl and 5-10 membered heteroaryl, said C _6-10 aryl and 5-10 membered heteroaryl respectively independently optionally replaced by 1, 2, 3, 4 or 5 R _e substitutions;

   R ₄ is selected from H, F, Cl, Br, I, OH, NH ₂ , CN, COOH, C(=O)NH ₂ , C _1-3 alkyl, C _1-3 alkoxy, C _2-4 Alkenyl and C _2-4 alkynyl, the C(=O)NH ₂ , C _1-3 alkyl, C _1-3 alkoxy, C _2-4 alkenyl and C _2-4 alkynyl are each independently optionally substituted with 1, 2 or 3 _Rf ;

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

   f, g and h are independently selected from 0, 1 and 2;

   each R _a is independently selected from H and CH ₃ ;

   each of R _b and R _c is independently selected from H, F, Cl, Br, I, OH, NH ₂ and CN;

   Each R _d is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN and -OCO-C _1-3 alkylamino;

   Each R _e is independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, COOH, C(=O)NH ₂ , C _1-6 alkyl, C _1-3 alkoxy, C _{1-3 alkylamino, C 1-3 alkylthio ,} C _2-4 alkenyl, C _2-4 alkynyl and C _3-5 cycloalkyl, the C(=O)NH ₂ , C _{1- 6} alkyl, C _1-3 alkoxy, C _1-3 alkylamino, C _1-3 alkylthio, C _2-4 alkenyl, C _2-4 alkynyl and C _3-5 cycloalkyl independently optionally substituted with 1, 2 or 3 R;

   each _{Rf is} independently selected from H, F, Cl, Br, I, OH, _NH2 , CN and _CH3 ;

   Each R is independently selected from H, F, Cl, Br, I and _CH3 .
2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R _d is selected from H, F and -OC(=O)-N(CH ₃ ) ₂ .
3. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R _e is selected from H, F, Cl, OH, NH ₂ , CH ₃ , CF ₃ , CH ₂ CH ₃ , CH(CH ₃ ) ₂ , OCH ₃ , OCF ₃ , SCH ₃ , SCF ₃ ,

   
4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Ring A is selected from

   

   
5. The compound according to claim 1 or 4 or a pharmaceutically acceptable salt thereof, wherein the structural fragment

   

   selected from

   
6. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the structural fragment

   

   selected from

   

   
7. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein the structural fragment

   

   selected from

   
8. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X ₁ is selected from N, CH and C(CF ₃ ).
9. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein X ₂ is selected from the group consisting of N, CH and CF.
10. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1, 8 and 9, wherein the structural unit

    

    selected from

    

    R ₄ is selected from H, F, CH ₃ and CF ₃ .
11. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₂ is selected from tetrahydropyrrolyl and hexahydro-1H-pyrrolizinyl, said tetrahydropyrrolyl and hexahydro-1H- Pyrrolizinyl is optionally substituted with 1, 2 or 3 _Rd .
12. The compound of claim 11 or a pharmaceutically acceptable salt thereof, wherein R ₂ is selected from

    

    
13. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1, 11 and 12, wherein the structural unit

    

    selected from

    
14. The compound of claim 13 or a pharmaceutically acceptable salt thereof, wherein the structural unit

    

    selected from

    
15. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₃ is selected from phenyl, naphthyl and indazolyl, said phenyl, naphthyl and indazolyl optionally being replaced by 1, 2 , 3, 4 or 5 R _e substitutions.
16. The compound of claim 15, or a pharmaceutically acceptable salt thereof, wherein _R is selected from

    

    
17. The compound of claim 16, or a pharmaceutically acceptable salt thereof, wherein _R is selected from

    

    
18. The compound of claim 16 or 17, or a pharmaceutically acceptable salt thereof, wherein _R is selected from

    
19. The compound according to any one of claims 1 to 18 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from,

    

    wherein R ₂ and R ₃ are as defined in any one of claims 1-18.
20. The compound of claim 19, or a pharmaceutically acceptable salt thereof, selected from the group consisting of,

    

    wherein _R3 and _Rd are as defined in claim 19.
21. The compound of claim 20, or a pharmaceutically acceptable salt thereof, selected from the group consisting of,

    

    in,

    R _d is selected from F, Cl, Br, I, OH, NH ₂ , CN and -OCO-C _1-3 alkylamino;

    _R3 is as defined in claim 20.
22. The compound of claim 21, or a pharmaceutically acceptable salt thereof, selected from the group consisting of,

    

    in,

    R _d is selected from F, Cl, Br, I, OH, NH ₂ , CN and -OCO-C _1-3 alkylamino;

    _R3 is as defined in claim 21;

    The carbon atoms with "*" and "#" are chiral carbon atoms and exist as (R) or (S) single enantiomer or enriched in one enantiomer.
23. A compound of the following formula, or a pharmaceutically acceptable salt thereof, is selected from the group consisting of,

    
24. The compound according to claim 23 or a pharmaceutically acceptable salt thereof, which compound is selected from,

    
25. According to the compound described in claim 23 or 24 or its pharmaceutically acceptable salt, its compound is selected from,

    

    

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