WO2022171013A1 - Tetrahydroquinazoline compound - Google Patents

Abstract

Disclosed in the present invention is a tetrahydroquinazoline compound. Specifically, disclosed is a compound represented by formula (II) and a pharmacologically acceptable salt thereof.

Description

Tetrahydroquinazoline compounds

The present invention claims the following priority:

CN202110182328.X, application date: February 9, 2021;

CN202110251657.5, application date: March 8, 2021;

CN202110518250.4, application date: May 12, 2021.

technical field

The present invention relates to a class of tetrahydroquinazoline compounds, in particular to a compound represented by formula (II) or a pharmaceutically acceptable salt thereof.

Background technique

RAS oncogene mutations are the most common activating mutations in human cancers, occurring in 30% of human tumors. The RAS gene family includes three subtypes (KRAS, HRAS, and NRAS), and KRAS subtype mutations account for 85% of RAS-driven cancers. KRAS mutations are commonly found in solid tumors such as lung adenocarcinoma, pancreatic ductal carcinoma, and colorectal cancer. In KRAS mutant tumors, the most common mutations included: p.G12D (41%), p.G12V (28%), and p.G12C (14%).

KRAS is a murine sarcoma virus oncogene and an important member of the RAS protein. KRAS is like a molecular switch, which can control the path of regulating cell growth when it is normal; after KRAS gene mutation, it can independently transmit growth and proliferation signals to downstream pathways independently of upstream growth factor receptor signals, resulting in uncontrolled cell growth and tumor progression. At the same time, whether the KRAS gene has mutation is also an important indicator of tumor prognosis.

Currently, small molecules that directly target KRAS mutations are mainly concentrated in the field of KRAS ^G12C . Among them, Amgen's AMG510 has been launched, and Mirati Therapeutics' MRTX849 has shown good therapeutic effects on KRAS ^G12C- mutated tumor patients in clinical studies. But 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.

SUMMARY OF THE INVENTION

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

in,

is selected from single bond or double bond;

T ₁ is selected from CR ₇ R ₈ , NR ₉ and O;

when

is selected from single bond, T is selected from _CH and N;

when

is selected from _double bonds, and T is selected from C;

L ₁ is selected from -CH ₂ - and a bond;

R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently selected from H and C _1-3 alkyl optionally substituted with ₁ , 2 or 3 _Ra ;

R ₆ is selected from C _6-10 aryl and 5-10 membered heteroaryl, the C _6-10 aryl and 5-10 membered heteroaryl are optionally surrounded by 1, 2, 3, 4 or 5 R _b replace;

R ₇ and R ₈ are each independently selected from H, CH ₃ and NH ₂ ;

R ₉ is selected from H and CH ₃ ;

R ₁₀ is selected from H, C _1-3 alkyl, C _1-3 alkoxy and cyclopropyl, the C _1-3 alkyl, C _1-3 alkoxy and cyclopropyl are optionally replaced by 1, 2 or 3 R _c substitutions;

R ₁₁ is selected from 4-8 membered heterocycloalkyl and

The 4-8 membered heterocycloalkyl and

optionally substituted with 1, 2 or 3 R _d ; structural unit

selected from 5-6 membered heterocycloalkenyl;

requirement is,

1) R ₁ and R ₂ form a ring with the connected atoms, making the structural unit

form

2) Alternatively, R ₁ and R ₂ form a ring with the connected atoms, making the structural unit

form

3) Alternatively, R ₁ and R ₄ form a ring with the connected atoms, making the structural unit

form

4) Alternatively, R ₄ and R ₅ form a ring with the connected atoms, making the structural unit

form

5) Alternatively, R ₂ and R ₇ form tetrahydropyrrolidinyl with the attached atoms;

6) Alternatively, R ₂ and R ₃ and the connected atom form a C _3-5 membered cycloalkyl;

7) Alternatively, R ₇ and R ₈ form a 4-5 membered heterocycloalkyl with the attached atom;

m is selected from 0, 1 or 2;

n is selected from 0, 1 or 2;

p is selected from 1 or 2;

q is selected from 1, 2 or 3;

r is selected from 1 or 2;

s is selected from 1, 2 or 3;

each R _a is independently selected from F, Cl, Br and I;

Each R _b is independently selected from F, Cl, Br, I, OH, NH ₂ , CN, C _1-3 alkyl, C _1-3 alkoxy, C _2-3 alkynyl, C _2-3 alkene and C _3-5 cycloalkyl, the C _1-3 alkyl, C _1-3 alkoxy, C _2-3 alkynyl, C _2-3 alkenyl and C _3-5 cycloalkyl are optional replaced by 1, 2 or 3 Rs;

each R _c is independently selected from F, Cl, Br and I;

Each R _d is independently selected from H, F, Cl, Br, OH, CN, C _1-3 alkyl, C _1-3 alkoxy and -C _1-3 alkyl-O-CO-C _{1- 3} alkylamino;

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

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

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 R ₂ and R ₇ form with the attached atoms

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₂ and R ₃ form with the attached atoms

Other variables are as defined in the present invention.

In some aspects of the invention, the _R7 and _R8 form with the attached atom

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 present invention, each R _b is independently selected from F, Cl, Br, I, OH, NH ₂ , CN, CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , - CH=CH ₂ , -CH ₂ -CH=CH ₂ and -C≡CH, said CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , -CH=CH ₂ , -CH ₂ -CH=CH ₂ and -C≡CH are optionally substituted with 1, 2 or 3 R, other variables are as defined in the present invention.

In some aspects of the present invention, each R _b is independently selected from F, OH, NH ₂ , CH ₃ , CF ₃ , CH ₂ CH ₃ and -C≡CH, and other variables are as defined herein.

In some embodiments of the present invention, the R ₆ is selected from phenyl, pyridyl, naphthyl, indolyl and indazolyl, and said phenyl, pyridyl, naphthyl, indolyl and indazolyl are any Optionally substituted with 1, 2, 3, 4 or 5 R _b , other variables are as defined in the present invention.

In some embodiments of the present invention, said R ₆ is selected from phenyl, naphthyl, indolyl and indazolyl, and said phenyl, naphthyl, indolyl and indazolyl are optionally substituted by 1, 2, 3, 4 or 5 R _b substitutions and other variables are as defined in the present invention.

In some aspects of the invention, the R ₆ is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₆ is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₆ is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₆ is selected from

Other variables are as defined in the present invention.

In some embodiments of the present invention, said R ₁₀ is selected from H, CH ₃ , OCH ₃ and cyclopropyl, said CH ₃ , OCH ₃ and cyclopropyl are optionally substituted with 1, 2 or 3 R _c , Other variables are as defined in the present invention.

In some aspects of the present invention, the R ₁₀ is selected from H and CH ₃ , and other variables are as defined herein.

In some aspects of the invention, each R _d is independently selected from H, F, Cl, Br, OH, CN, CH ₃ , CH ₂ CH ₃ , CH ₂ CF ₃ , OCH ₃ , OCF ₃ and

Other variables are as defined in the present invention.

In some embodiments of the present invention, said R ₁₁ is selected from tetrahydropyrrolyl and hexahydro-1H-pyrrolizinyl, said tetrahydropyrrolyl and hexahydro-1H-pyrrolizinyl being separated by 1, 2 or 3 R _d substitutions, other variables are as defined in the present invention.

In some aspects of the invention, the R ₁₁ is selected from

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₁₁ is selected from

Other variables are as defined in the present invention.

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

in,

is selected from single bond or double bond;

T ₁ is selected from CR ₇ R ₈ and NR ₉ ;

when

is selected from single bond, T is selected from _CH and N;

when

is selected from _double bonds, and T is selected from C;

R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently selected from H and C _1-3 alkyl optionally substituted with ₁ , 2 or 3 _Ra ;

R is selected from phenyl and naphthyl optionally substituted with ₁ , ₂ , 3, 4 or 5 R;

R ₇ and R ₈ are each independently selected from H, CH ₃ and NH ₂ ;

R ₉ is selected from H and CH ₃ ;

R ₁₀ is selected from H, C _1-3 alkyl, C _1-3 alkoxy and cyclopropyl, the C _1-3 alkyl, C _1-3 alkoxy and cyclopropyl are optionally replaced by 1, 2 or 3 R _c substitutions;

R ₁₁ is selected from tetrahydropyrrolyl and hexahydro-1H-pyrrolizinyl substituted with 1, 2 or 3 R _d ;

requirement is,

1) R ₁ and R ₂ form a ring with the connected atoms, making the structural unit

form

2) Or, R ₁ and R ₄ are connected with the atoms to form a ring to make the structural unit

form

3) Alternatively, R ₄ and R ₅ form a ring with the connected atoms, making the structural unit

form

4) Alternatively, R ₂ and R ₇ form tetrahydropyrrolidinyl with the attached atom;

5) Alternatively, R ₂ and R ₃ and the connected atoms form a C _3-5 membered cycloalkyl;

6) Alternatively, R ₇ and R ₈ form a 4-5 membered heterocycloalkyl with the attached atom;

m is selected from 0, 1 or 2;

n is selected from 0, 1 or 2;

p is selected from 1 or 2;

q is selected from 1, 2 or 3;

r is selected from 1 or 2;

s is selected from 1, 2 or 3;

each R _a is independently selected from F, Cl, Br and I;

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

each R _c is independently selected from F, Cl, Br and I;

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

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

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₂ and R ₇ form with the attached atoms

Other variables are as defined in the present invention.

In some aspects of the invention, the R ₂ and R ₃ form with the attached atoms

Other variables are as defined in the present invention.

In some aspects of the invention, the _R7 and _R8 form with the attached atom

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 R ₆ is selected from

Other variables are as defined in the present invention.

In some embodiments of the present invention, said R ₁₀ is selected from H, CH ₃ , OCH ₃ and cyclopropyl, said CH ₃ , OCH ₃ and cyclopropyl are optionally substituted with 1, 2 or 3 R _c , Other variables are as defined in the present invention.

In some aspects of the present invention, the R ₁₀ is selected from H and CH ₃ , and other variables are as defined herein.

In some aspects of the invention, the R ₁₁ is selected from

Other variables are as defined in the present invention.

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

in,

selected from single and double bonds;

T ₁ , T ₂ , R ₆ and R ₁₀ are as defined in the present invention.

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

in,

selected from single and double bonds;

T ₃ is selected from C and N;

R _b1 is absent, or selected from H, F, Cl, Br, I, OH, NH ₂ , CN, C _1-3 alkyl, C _1-3 alkoxy, C _2-3 alkynyl, C _{2- 3} alkenyl and C _3-5 cycloalkyl, the C _1-3 alkyl, C _1-3 alkoxy, C _2-3 alkynyl, C _2-3 alkenyl and C _3-5 cycloalkyl optionally substituted with 1, 2 or 3 R;

R _b2 , R _b3 , R _b4 , R _b5 , R _b6 and R _b7 are each independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, C _1-3 alkyl, C _1-3 Alkoxy, C _2-3 alkynyl, C _2-3 alkenyl and C _3-5 cycloalkyl, the C _1-3 alkyl, C _1-3 alkoxy, C _2-3 alkynyl, C _2-3 alkenyl and C _3-5 cycloalkyl are optionally substituted with 1, 2 or 3 R;

T ₁ , T ₂ , R ₁₀ and R are as defined in the present invention.

In some aspects of the invention, the R _b1 , R _b2 , R _b3 , R _b4 , R _b5 , R _b6 and R _b7 are each independently selected from H, F, Cl, Br, I, OH, NH ₂ , CN, _CH3 , _CH2CH3 , _OCH3 , _OCH2CH3 , -CH= _CH2 , _-CH2 - _{CH = CH2} and _-C≡CH , the _CH3 , _CH2CH3 , _OCH3 , OCH ₂ CH ₃ , -CH=CH ₂ , -CH ₂ -CH=CH ₂ and -C≡CH are optionally substituted with 1, 2 or 3 R, other variables are as defined in the present invention.

In some aspects of the invention, the R _b1 , R _b2 , R _b3 , R _b4 , R _b5 , R _b6 and R _b7 are each independently H, F, OH, NH ₂ , CH ₃ , CH ₂ F, CHF ₂ , _CF3 , _CH2CH3 and _-C≡CH , other variables are as defined in the present invention.

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

in,

selected from single and double bonds;

T ₁ , T ₂ , T ₃ , R ₁₀ , R _b1 , R _b2 , R _b3 , R _b4 , R _b5 , R _b6 and R _b7 are as defined in the present invention.

There are also some solutions of the present invention that are formed by any combination of the above variables.

The present invention also provides a compound represented by the following formula or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of

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 use of the compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diseases related to KRAS ^G12D mutation.

The present invention also provides following synthetic method:

method 1:

in,

z is selected from 0, 1, 2, 3 or 4;

R ₁₀ , R ₁₁ and R _b are as defined in the present invention.

Method 2:

z is selected from 0, 1 or 2;

R ₁₀ , R ₁₁ and R _b are as defined in the present invention.

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

Research purposes

In this experiment, the inhibitory effect of the compound on cell proliferation was studied by detecting the effect of the compound on the cell viability in vitro in the tumor cell lines AsPC-1 and GP2D.

Experimental Materials

Table 1. 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

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.

Cell plating

Cells were stained with trypan blue and viable cells were counted.

Adjust the cell concentration to an appropriate concentration.

Table 2. Cell Density

cell line	Density (per hole)
AsPC-1	7000 cells
GP2D	8000 cells

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 (Day 1)

After preparing the 10X compound working solution (DMSO 10X working solution), add 15 μL of 10X compound working solution to the ULA culture plate respectively, and add 15 μL of DMSO-cell culture solution mixture to the vehicle control and blank control.

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 Assay for Cell Viability (Day 5)

The following steps were carried out 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 2104 EnVision 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 .

Test method 3. Pharmacokinetic study of test compounds by oral and intravenous injection in CD-1 mice

Purpose

The in vivo pharmacokinetics of oral and intravenous compounds in CD-1 mice were tested.

Experimental procedure

Test compounds were mixed with 5% DMSO + 95% (10% HP-β-CD) in water, vortexed and sonicated to prepare 0.5 mg/mL clear solution (intravenous) or 3 mg/mL clear solution (oral), microporous After filtration through the filter membrane, it is ready for use. Male SD mice aged 7 to 10 weeks were selected, and the candidate compound solution was administered intravenously at a dose of about 2 mg/kg. Candidate compound solutions are administered orally at a dose of approximately 30 mg/kg. Whole blood was collected for a certain period of time, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and pharmacokinetic parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

Test Method 4. In Vivo Pharmacodynamic Studies

experimental method:

To establish a human colon cancer GP2D cell subcutaneous xenograft tumor Balb/c nude mouse model, 0.2 mL (2 × 10 ⁶ ) GP2D cells (plus Matrigel, volume ratio of 1:1) were subcutaneously inoculated into each mouse. On the right back, when the average tumor volume reached 149mm ³ , group administration was started, with 6 mice in each group. On the day of the experiment, the animals were given the corresponding drugs according to the groups. The first group G1 was set as a negative control group, which was given 5% DMSO+95% (10% HP-β-CD) by gavage alone, and the second group G2 to the fourth group G4 were orally given the test compound.

During the experimental period, the body weight and tumor size of the animals were measured twice a week, while the clinical symptoms of the animals were observed and recorded every day, and the last weighed animal body weight was referenced for each administration.

Measurement of tumor The length (a) and width (b) were determined with digital vernier calipers, and the formula for tumor volume (Tumor volume, TV) was: TV=a×b ² /2.

technical effect

The compound of the present invention has good binding effect and inhibitory effect on KRAS ^G12D protein, has good cell proliferation inhibitory activity on KRAS ^G12D mutant cells, has significant GP2D cell p-ERK inhibitory effect, can inhibit tumor growth, and has relatively Good pharmacokinetic properties.

Related Definitions

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 unless specifically defined, 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 which, 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 compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting 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 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.

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 mixtures thereof and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which belong to this within the scope of the 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.

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 bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated 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.

The terms "optional" or "optionally" mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where said event or circumstance occurs and instances 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 thereto 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 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, when a group has one or more attachable sites, any one or more sites in the group can be linked to other groups by chemical bonds. When the connection method of the chemical bond is not positioned and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of chemical bonds connected to the corresponding valence. the group. The chemical bond connecting the site to other groups can be represented by straight solid line bonds

straight dotted key

or wavy lines

express. For example, a straight solid bond in -OCH ₃ indicates that it is connected to other groups through the oxygen atom in this group;

The straight dashed bond in the group indicates that it is connected to other groups through the two ends of the nitrogen atom in the group;

The wavy line in the phenyl group indicates that it is connected to other groups through the 1 and 2 carbon atoms in the phenyl group;

Indicates that any linkable site on the piperidinyl group can be connected to other groups through a chemical bond, including at least

These 4 connection methods, even if the H atom is drawn on -N-, but

still includes

The group in this connection method is only that when one chemical bond is connected, the H at the site will be correspondingly reduced by one to become the corresponding monovalent piperidinyl group.

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

or straight dashed key

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

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

Unless otherwise specified, the term "4-5 membered heterocycloalkyl" by itself or in combination with other terms denotes a saturated monocyclic group consisting of 4 to 5 ring atoms, 1, 2, 3 or 4 ring atoms, respectively 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). Furthermore, with respect to the "4-5 membered heterocycloalkyl", a heteroatom may occupy the position of attachment of the heterocycloalkyl to the remainder of the molecule. The 4-5 membered heterocycloalkyl includes 4 and 5 membered heterocycloalkyl. Examples of 4-5 membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothienyl ( Including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.) or tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.) 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 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 heteroaryl groups, and the like. 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.) .

Unless otherwise specified, the term "4-8 membered heterocycloalkyl" by itself or in combination with other terms denotes a saturated cyclic group consisting of 4 to 8 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 and bicyclic ring systems, wherein bicyclic ring systems include spiro, paracyclic and bridged rings. Additionally, with respect to the "4-8 membered heterocycloalkyl", a heteroatom may occupy the position of attachment of the heterocycloalkyl to the remainder of the molecule. The 4-8 membered heterocycloalkyl includes 4-6 membered, 5-6 membered, 4 membered, 5 membered and 6 membered heterocycloalkyl and the like. Examples of 4-8 membered heterocycloalkyl include, but are not limited to, azetidinyl, oxetanyl, thietanyl, 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-piperidyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), Dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperyl pyridyl or dioxepanyl, etc.

Unless otherwise specified, the term "5-6 membered heterocycloalkenyl" by itself or in combination with other terms respectively denotes a partially unsaturated cyclic group consisting of 5 to 6 ring atoms containing at least one carbon-carbon double bond , whose 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may optionally be Oxidation (ie NO and S(O) _p , p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein bicyclic ring systems include spiro, paracyclic and bridged rings, any ring of this system is non-aromatic. Furthermore, in the case of the "5-6 membered heterocycloalkenyl", a heteroatom may occupy the position of attachment of the heterocycloalkenyl to the rest of the molecule. The 5-6 membered heterocyclenyl includes 5-membered and 6-membered heterocyclenyl and the like. Examples of 5-6 membered heterocycloalkenyl include but are not limited to

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.

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 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 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 and KRAS ^G12D protein;

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

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

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

Figure 5. Binding pattern of compound E and KRAS ^G12D protein;

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

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

Figure 8. Binding pattern map of Compound H and 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 present invention has been described in detail herein, and specific embodiments thereof have also been disclosed. For those skilled in the art, 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. will be obvious.

Calculation example 1

The present invention also provides that the compound or its pharmaceutically acceptable molecular docking process is prepared by using Maestro (

Glide SP[1] and default options in version 2017-2). The crystal structure PDB: 6UT0 of KRAS_G12C in the PDB database was selected, and Cys12 was simulated and mutated to Asp12. After energy optimization, it was used as the docking template. To prepare the protein, hydrogen atoms were added using the Protein Preparation Wizard module of Maestro [2] and the OPLS3 force field was used. For ligand preparation, the 3D 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 6UT0 as the centroid, the side length is

The cube docking grid. Place reference compounds during molecular docking. Analyze the interaction type of protein receptor and ligand, analyze the interaction type of protein receptor and ligand, and then select and save a reasonable docking conformation according to the calculated docking scrore and binding mode, as shown in Figure 1 to Figure 8 .

[1] Glide,

LLC, New York, NY, 2017.

[2] Maestro,

LLC, New York, NY, 2017.

[3] LigPrep,

LLC, New York, NY, 2017.

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

Example 1

Step 1: Synthesis of Compounds 1-2

Compound 1-1 (10 g, 44.83 mmol, 1 eq) was added to anhydrous dichloromethane (60 mL), imidazole ((9.16 g, 134.49 mmol, 3 eq) and tert-butyldimethylsilyl chloride (10.14 g, 67.24mmol, 8.24mL, 1.5eq), react at 20°C for 1 hour. The reaction solution was washed with 50mL saturated sodium bicarbonate solution, 50mL*2 saturated ammonium chloride, 50mL saturated brine, and dried over anhydrous sodium sulfate. After filtration and concentration, compound 1-2 was obtained. ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.18-8.09 (m, 1H), 7.73-7.58 (m, 1H), 7.50-7.31 (m, 3H), 7.14- 7.15 (m, 1H), 1.00 (s, 9H), 0.24 (s, 6H), MS m/z=336.9 [M+H] ⁺ .

Step 2: Synthesis of Compounds 1-3

Compound 1-2 (11g, 32.61mmol, 1eq) and double pinacol boronate (25.06g, 97.83mmol, 3eq) were added to 1,4-dioxane (100mL), potassium acetate ( 9.60g, 97.83mmol, 3eq), replaced with nitrogen three times, added [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (2.66g, 3.26mmol, 0.1eq), 80°C The reaction was carried out under nitrogen protection for 3 hours, the reaction solution was directly concentrated, then dissolved in 50 mL of dichloromethane, washed with 30 mL*2 of water, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column (petroleum ether:ethyl acetate) Ester = 100:0-10:1) to give compound 1-3. MS m/z=385.2 [M+H] ⁺ .

Step 3: Synthesis of Compounds 1-4

Under nitrogen protection, compound 1-3 (8 g, 20.81 mmol, 1 eq) compound 1-3A (3.44 g, 31.22 mmol, 1.5 eq) was added to 1,4-dioxane (50 mL) and water (10 mL), The nitrogen was replaced three times, and (1,5-cyclooctadiene) chlororhodium (I) dimer (513.10 mg, 1.04 mmol, 0.05 eq) and anhydrous potassium phosphate (1.10 g, 5.20 mmol, 0.25 eq) were added, 20 ℃ reacted for 0.5 hours, combined with compound 1-3 (5g) for treatment, concentrated the reaction solution, added 20 mL of dichloromethane, washed with 20 mL*2 of water, dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was purified by column (Petroleum ether:ethyl acetate=100:0-5:1) to obtain compound 1-4. ¹ H NMR (400 MHz, CDCl ₃ ) δ=7.99 (d, J=8.0 Hz, 1H), 7.73 (d, J=8.0 Hz, 1H), 7.48-7.34 (m, 2H), 7.12 (s, 1H) , 7.06(s, 1H), 3.55-3.50(m, 1H), 2.70-2.47(m, 4H), 2.38-2.18(m, 2H), 1.81-1.63(m, 1H), 1.05(s, 9H) , 0.85 (d, J=6.4 Hz, 3H), 0.27 (s, 6H), MS m/z=369.2 [M+H] ⁺ .

Step 4: Synthesis of Compounds 1-5

Compound 1-4 (1.5 g, 4.07 mmol, 1 eq) was added to anhydrous tetrahydrofuran (20 mL), replaced with nitrogen three times, cooled to -60°C, and a solution of lithium bis(trimethylsilyl)amide ( 1M, 8.14mL, 2eq), after 1 hour of reaction, ethyl cyanoformate (806.50mg, 8.14mmol, 798.52μL, 2eq) was added dropwise, and the reaction was continued for 0.5 hours. The reaction solution was slowly poured into 20 mL of saturated ammonium chloride, extracted with 10 mL of ethyl acetate, washed with 30 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product, which was purified by column (petroleum ether:ethyl acetate) Ester = 10:1-3:1) purification to give compound 1-5. ¹ H NMR (400 MHz, CDCl ₃ ) δ=12.25 (s, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.73 (d, J=8.0 Hz, 1H), 7.48-7.32 (m, 2H) , 7.16-7.09(m, 1H), 7.00-6.93(m, 1H), 4.32-4.21(m, 2H), 3.58-3.44(m, 1H), 2.76-2.63(m, 1H), 2.60-2.47( m, 2H), 2.17-2.09 (m, 2H), 1.38-1.32 (m, 3H), 1.04 (s, 9H), 0.87 (d, J=5.6Hz, 3H), 0.24 (s, 6H). MS m/z=441.2[M+H] ⁺

Step 5: Synthesis of Compounds 1-6

Compound 1-5 (0.3g, 680.82μmol, 1eq) was added to ethanol (3mL) and water (0.6mL), sodium bicarbonate (571.96mg, 6.81mmol, 264.79μL, 10eq) and 2-methylisocarbonate were added Thiourea sulfate (947.61 mg, 3.40 mmol, 5 eq) was reacted at 50°C for 4 hours. The reaction solution was spin-dried, 10 mL of water was added, extracted with 5 mL*2 of ethyl acetate, the organic phases were combined, washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain compound 1-6. MS m/z=467.2 [M+H] ⁺ .

Step 6: Synthesis of Compounds 1-7

Compound 1-6 (1.1 g, 2.36 mmol, 1 eq) was added to anhydrous dichloromethane (15 mL), cooled to 0 °C, N,N-diisopropylethylenediamine (609.22 mg, 4.71 mmol, 821.05μL, 2eq) and trifluoromethanesulfonic anhydride (797.98mg, 2.83mmol, 466.65μL, 1.2eq), react for 0.5 hours, wash the reaction solution with 20mL*2 saturated ammonium chloride, dry and filter over anhydrous sodium sulfate After concentration, the crude product was purified by column (petroleum ether:ethyl acetate=100:0-5:1) to obtain compound 1-7. ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.02-7.97 (m, 1H), 7.75 (d, J=8.0 Hz, 1H), 7.50-7.33 (m, 2H), 7.15 (d, J=2.0 Hz, 1H), 6.96-6.88(m, 1H), 3.78-3.59(m, 1H), 3.40-3.24(m, 1H), 3.16-2.90(m, 2H), 2.62-2.45(m, 4H), 2.42- 2.28 (m, 1H), 1.05-0.94 (m, 12H), 0.29-0.16 (m, 6H). MS m/z=599.2 [M+H] ⁺ .

Step 7: Synthesis of Compounds 1-8

Compound 1-7 (0.16 g, 267.21 μmol, 1 eq) was added to N,N-dimethylformamide (2 mL), and N,N-isopropylethylenediamine (69.07 mg, 534.43 μmol, 93.09 μL) was added , 2eq) and compound 1-7A (79.76mg, 320.66μmol, 1.2eq, HCl), react at 50°C for 0.5 hours, add 5mL of methyl tert-butyl ether to the reaction solution, use 5mL*2 of saturated ammonium chloride After washing, washing with 10 mL of water and 10 mL of saturated brine, drying, filtration and concentration over anhydrous sodium sulfate to obtain compound 1-8. MS m/z=661.3 [M+H] ⁺ .

Step 8: Synthesis of Compounds 1-9

Compound 1-8 (0.18g, 272.32μmol, 1eq) was added to anhydrous dichloromethane (4mL), m-chloroperoxybenzoic acid (66.35mg, 326.79μmol, 85% content, 1.2eq) was added, 20°C React for 0.5 hours, add 10 mL of 10% sodium thiosulfate solution to the reaction solution, extract with 5 mL*2 of anhydrous dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate and concentrate, and the crude product is purified by column (petroleum ether:ethyl acetate=10:1-0:1) to give compound 1-9. ¹ H NMR (400 MHz, CDCl ₃ ) δ=8.12-7.98 (m, 1H), 7.75 (d, J=8.0 Hz, 1H), 7.50-7.34 (m, 2H), 7.19-7.10 (m, 1H), 6.99-6.87(m, 1H), 4.48-4.19(m, 3H), 3.78-3.64(m, 2H), 3.58-3.37(m, 2H), 3.21-3.04(m, 2H), 2.94-2.83(m , 3H), 2.80-2.70(m, 1H), 2.63-2.47(m, 1H), 2.30-2.12(m, 1H), 2.02-1.90(m, 3H), 1.80-1.65(m, 1H), 1.50 (s, 9H), 1.00 (s, 9H), 0.94 (d, J=8.0 Hz, 3H), 0.21 (s, 6H). MS m/z=677.4 [M+H] ⁺ .

Step 9: Synthesis of Compounds 1-10

Compound 1-9A (28.22mg, 177.26μmol, 1.2eq) was added to anhydrous tetrahydrofuran (5mL), cooled to -15°C, sodium tert-butoxide (21.29mg, 221.57μmol, 1.5eq) was added under nitrogen protection, After the reaction for 10 minutes, a solution of compound 1-9 (0.1 g, 147.71 μmol, 1 eq) in anhydrous tetrahydrofuran (1 mL) was added, and the reaction was continued for 0.5 hours. washed with ammonium, washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain compound 1-10. MS m/z=772.5[M+H] ⁺

Step 10: Synthesis of Compounds 1A & 1B

Compound 1-10 (90 mg, 116.57 μmol, 1 eq) was added to hydrochloric acid/ethyl acetate (4 M, 10 mL), reacted at 20°C for 9.5 hours, the reaction solution was filtered, and the filter cake was rinsed with 2 mL*2 of ethyl acetate After spin-drying, carry out pre-HPLC separation and purification (column: Phenomenex Luna 80*30mm*3μm; mobile phase: [water (0.04% hydrochloric acid)-acetonitrile]; (acetonitrile)%: 1%-30%, 8min) to obtain The hydrochloride salt of compound 1. ¹ H NMR (400 MHz, CD ₃ OD) δ=8.09 (d, J=8.4 Hz, 1H), 7.69 (d, J=8.4 Hz, 1H), 7.45-7.21 (m, 2H), 7.11-6.99 (m , 2H), 5.70-5.60(m, 1H), 5.55-5.45(m, 1H), 4.64-4.41(m, 3H), 4.20(d, J=14.4Hz, 2H), 4.08-4.00(m, 2H) ), 3.94-3.80(m, 2H), 3.79-3.60(m, 2H), 3.58-3.34(m, 2H), 3.30-3.06(m, 2H), 3.05-2.91(m, 1H), 2.88-2.73 (m, 1H), 2.72-2.65 (m, 1H), 2.65-2.48 (m, 2H), 2.46-2.24 (m, 4H), 2.24-2.01 (m, 4H), 0.96 (d, J=6.6Hz) , 2H). MS m/z=558.3[M+H] ⁺

The hydrochloride salt of compound 1 was chiral resolved according to SFC (column: DAICEL CHIRALPAK IE (250 mm*30 mm, 10 μm); mobile phase: [heptane-ethanol (0.1% NH ₃ H ₂ O)]; ethanol (0.1%) _{NH3H2O )} %: 50%-50%, 10 min) to give compound 1A to give compound 1B.

Analysis and Characterization of Compound 1A:

SFC analysis method (column: Chiralpak IE-3, 50 x 4.6 mm ID, 3 μm; mobile phase: A (hexane) and B (ethanol/acetonitrile (4:1) with 0.1% isopropylamine); gradient: A: B=50:50, 5min; flow rate: 1.3mL/min; wavelength: 220nm; column temperature: 30℃, Rt=1.67min, chiral isomer excess 100%. MS m/z=558.3[M+H] ⁺ .

Analysis and Characterization of Compound 1B:

SFC analysis method (column: Chiralpak IE-3, 50 x 4.6 mm ID, 3 μm; mobile phase: A (hexane) and B (ethanol/acetonitrile (4:1) with 0.1% isopropylamine); gradient: A: B=50:50, 5min; flow rate: 1.3mL/min; wavelength: 220nm; column temperature: 30℃, Rt=2.471min, chiral isomer excess 99.57%. MS m/z=558.3[M+H] ⁺ .

Example 2

Step 1: Synthesis of Compound 2-2

Compound 2-1 (50g, 399.54mmol, 1eq), potassium carbonate (138.05g, 998.86mmol, 2.5eq) and potassium iodide (66.32g, 399.54mmol, 1eq) were added to N-methylpyrrolidone (500mL), slowly p-Methoxybenzyl chloride (128.27 g, 819.06 mmol, 111.54 mL, 2.05 eq) was added dropwise, the system was exothermic to 60°C, and the reaction was carried out for 0.5 hour. The reaction solution was poured into 1L of water, then 500mL of methyl tert-butyl ether was added and stirred, the organic phase was collected after separation, washed with saturated brine (1L*2), dried over anhydrous sodium sulfate, and 200mL of petroleum ether was added to the crude product, Beat for 3 hours, filter, wash the filter cake with petroleum ether (50 mL*2), spin the filter cake to dry to obtain compound 2-2. MS m/z=366.2 [M+H] ⁺ .

Step 2: Synthesis of Compounds 2-3

2,2,6,6-Tetramethylpiperidine (108.23g, 766.20mmol, 130.08mL, 4eq) was added to anhydrous tetrahydrofuran (700mL), replaced with nitrogen three times, cooled to -5°C, dropped under nitrogen protection Add n-butyllithium (2.5M, 306.48mL, 4eq), react at -5-0°C for 10 minutes, cool down to -60°C, add compound 2-2 (70g, 191.55mmol, 1eq) in anhydrous tetrahydrofuran (70mL) ) solution, reacted for 0.5 hours, added triisopropyl borate (43.23 g, 229.86 mmol, 52.85 mL, 1.2 eq) dropwise, and reacted for 10 minutes. The reaction solution was slowly poured into 1 L of saturated ammonium chloride, extracted with 300 mL of ethyl acetate, washed with 800 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product, which was purified by column chromatography (petroleum ether:acetic acid) ethyl ester = 10:1-1:1) purification to give compound 2-3. MS m/z=410.1 [M+H] ⁺ , ¹ H NMR (400 MHz, CDCl ₃ ) δ=7.24-4.20 (m, 5H), 6.89-6.85 (m, 5H), 4.23-4.20 (m, 4H) , 3.83 (s, 6H), 2.24 (s, 3H).

Step 3: Synthesis of Compounds 2-4

Under nitrogen protection, compound 2-3 (7 g, 17.10 mmol, 1 eq) and compound 1-3A (2.83 g, 25.66 mmol, 1.5 eq) were added to 1,4 dioxane (70 mL), and nitrogen was replaced three times. (1,5-Cyclooctadiene) rhodium chloride (I) dimer (421.69 mg, 855.21 μmol, 0.05 eq), potassium hydroxide (395.18 mg, 5.99 mmol, 85% content, 0.35 eq) in water ( 14mL) solution, reacted at 20°C for 15 minutes, added 100mL of water to the reaction solution, extracted with ethyl acetate (50mL*2), combined the organic phases, washed with 100mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column (petroleum ether:ethyl acetate=100:0-3:1) to obtain compound 2-4. MS m/z=476.3 [M+H] ⁺ .

Step 4: Synthesis of Compounds 2-5

Compound 2-4 (4.5g, 9.46mmol, 1eq) was added to acetonitrile (45mL), iodosuccinimide (2.34g, 10.41mmol, 1.1eq) was added at 15°C, trifluoroacetic acid ( 107.88 mg, 946.19 μmol, 70.05 μL, 0.1 eq), reacted at 15°C for 20 minutes, concentrated the reaction solution, added 30 mL of dichloromethane, washed with 30 mL of water, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (Petroleum ether:ethyl acetate=100:0-1:1) to obtain compound 2-5. MS m/z=602.1 [M+H] ⁺ .

Step 5: Synthesis of Compounds 2-6

Compound 2-5 (1.7 g, 2.83 mmol, 1 eq) and methyl fluorosulfonyldifluoroacetate (2.71 g, 14.13 mmol, 1.80 mL, 5 eq) were added to N,N-dimethylformamide (20 mL) , cuprous iodide (1.08g, 5.65mmol, 2eq) was added under nitrogen protection, and the reaction was carried out at 100°C for 2 hours. The reaction solution was poured into 30 mL of water, extracted with 20 mL of methyl tert-butyl ether, the organic phase was washed with saturated brine (20 mL*2), dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether:ethyl acetate=100:0-3:1) to obtain compound 2-6. MS m/z=544.3 [M+H] ⁺ .

Step 6: Synthesis of Compounds 2-7

Compound 2-6 (0.65g, 1.20mmol, 1eq) was added to anhydrous tetrahydrofuran (10mL), cooled to -60°C, replaced with nitrogen three times, and lithium bistrimethylsilylamide (1M) was added dropwise under nitrogen protection. , 2.39mL, 2eq), react at -60°C for 30 minutes, add ethyl cyanoformate (236.97mg, 2.39mmol, 234.62μL, 2eq), react for 30 minutes, add the reaction solution to 15mL of water, use 10mL of formazan extracted with ethyl tert-butyl ether, washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated, and the crude product was purified by column chromatography (petroleum ether:ethyl acetate=100:0-1:1) to obtain compound 2-7. MS m/z=616.2 [M+H] ⁺ .

Step 7: Synthesis of Compounds 2-8

Compound 2-7 (0.55g, 893.36μmol, 1eq) was added to ethanol (6mL) and water (1.2mL), sodium bicarbonate (1.50g, 17.87mmol, 694.92μL, 20eq) and methylisothiourea were added Sulfate (2.49g, 8.93mmol, 10eq) was reacted at 60°C for 10 hours, the reaction solution was diluted with 10mL of ethyl acetate, washed with 10mL of water, then washed with 10mL of saturated brine, dried over anhydrous sodium sulfate and filtered After concentration, the crude product was purified by column chromatography (petroleum ether:ethyl acetate=100:0-1:1) to obtain compound 2-8. MS m/z=642.2 [M+H] ⁺ .

Step 8: Synthesis of Compounds 2-9

Compound 2-8 (0.25g, 389.58μmol, 1eq) was added to anhydrous dichloromethane (5mL), cooled to 0°C, N,N-diisopropylethylenediamine (151.05mg, 1.17mmol, 203.57μL, 3eq) and trifluoromethanesulfonic anhydride (164.87mg, 584.37μmol, 96.42μL, 1.5eq), react for 0.5 hours. 5 mL of water was added to the reaction solution, extracted with 3 mL of dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (petroleum ether:ethyl acetate=100:0-1:1) to obtain compound 2- 9. MS m/z=774.2 [M+H] ⁺ .

Step 9: Synthesis of Compounds 2-10

Compound 2-9 (0.07g, 90.47μmol, 1eq) was added to N,N-dimethylformamide (1.5mL), and N,N-diisopropylethylenediamine (23.38mg, 180.93μmol, 31.51 g) was added. μL, 2eq) and compound 1-7A (23.05mg, 108.56μmol, 1.2eq) were reacted at 50°C for 0.5 hours, 4mL of methyl tert-butyl ether was added to the reaction solution, and 5mL of water and 5mL of saturated chlorinated After successively washing with ammonium and 5 mL of saturated brine, drying over anhydrous sodium sulfate, filtration, and concentration, compound 2-10 was obtained. MS m/z=836.4 [M+H] ⁺ .

Step 10: Synthesis of Compounds 2-11

Compound 2-10 (0.06g, 71.77μmol, 1eq) was added to anhydrous dichloromethane (3mL), m-chloroperoxybenzoic acid (17.49mg, 86.13μmol, 85% content, 1.2eq) was added, 20°C The reaction was carried out for 0.5 hours. 5mL of 10% sodium thiosulfate solution was added to the reaction solution, extracted with DCM (5mL*2), the organic phases were combined, dried over anhydrous sodium sulfate and concentrated, and the crude product was purified by column chromatography (petroleum ether:ethyl acetate=10: 1-0:1) to give compound 2-11. MS m/z=868.4 [M+H] ⁺ .

Step 11: Synthesis of Compounds 2-12

Compound 1-9A (6.60mg, 41.48μmol, 3eq) was added to anhydrous tetrahydrofuran (1mL), cooled to -15°C, sodium tert-butoxide (3.99mg, 41.48μmol, 3eq) was added under nitrogen protection, and the reaction was carried out for 10 After 2 minutes, compound 2-11 (12 mg, 13.83 μmol, 1 eq) in anhydrous tetrahydrofuran (0.5 mL) was added, and the reaction was continued for 1 hour. 3 mL of ethyl acetate was added to the reaction solution, and washed with saturated ammonium chloride (3 mL*2). , washed with 5 mL of saturated brine, dried over anhydrous sodium sulfate and concentrated to obtain compound 2-12. MS m/z=947.4 [M+H] ⁺ .

Step 12: Hydrochloride Salt Synthesis of Compound 2

Compound 2-12 (15 mg, 15.84 μmol, 1 eq) was added to anhydrous dichloromethane (2 mL), trifluoroacetic acid (0.2 mL) was added, and the reaction was carried out at 25° C. for 5 hours. 5mL of water was added to the reaction solution, extracted with ethyl acetate (3mL*2), the aqueous phase was adjusted to pH 8 with saturated sodium bicarbonate solution, extracted with ethyl acetate (5mL*2), and the organic phases were combined. The crude product was separated and purified by high performance liquid chromatography [(chromatographic column: Phenomenex Luna 80*30mm*3μm; mobile phase A: water (0.05% hydrochloric acid), mobile phase B: acetonitrile; running gradient: B : 1%-25%; running time: 8 min)] to obtain the hydrochloride salt of compound 2. MS m/z=607.3 [M+H] ⁺ .

Example 3

Step 1: Synthesis of compound 3-2

Compound 3-1 (25g, 58.50mmol, 1eq), lithium chloride (12.40g, 292.51mmol, 5.99mL, 5eq), tris(dibenzylideneacetone)palladium (5.36g, 5.85mmol, 0.1eq), Tricyclohexylphosphine (3.28g, 11.70mmol, 3.79mL, 0.2eq) was dissolved in dioxane 250mL), then hexabutylditin (67.87g, 117.00mmol, 58.51mL, 2eq) was added, and nitrogen was replaced twice , and stirred at 115 °C for 1 hour. The reaction solution was lowered to 25°C, 400 mL of water was added to the reaction solution, extracted with ethyl acetate (200 mL*2), the organic phases were combined, washed with saturated brine (200 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated. Neutral alumina was purified by column (petroleum ether:ethyl acetate=10:1) to obtain compound 3-2.

Step 2: Synthesis of Compounds 3-3

Compound 3-2 (21.5g, 33.73mmol, 1eq) and compound 1-3A (5.57g, 50.59mmol, 1.5eq) were dissolved in THF (200mL), and then (1,5-cyclooctadiene)rhodium chloride was added (I) Dimer (1.66 g, 3.37 mmol, 0.1 eq), stirred at 60°C for 1 hour. The reaction solution was directly concentrated and purified by column (petroleum ether:ethyl acetate=10:1) to obtain compound 3-3. MS m/z=459.3 [M+H] ⁺ .

Step 3: Synthesis of Compounds 3-4

Compound 3-3 (4.8g, 10.47mmol, 1eq) was dissolved in N,N-dimethylformamide (50mL), cooled to 0°C, and then iodosuccinimide (2.59g, 11.51mmol, 1.1 eq), stirring at 5-10°C for 1 hour. 100 mL of water was added to the reaction solution, extracted with ethyl acetate (100 mL*2), the organic phases were combined, washed with saturated brine (60 mL*3), dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column (petroleum ether:ethyl acetate) Ester=10:1) to give compound 3-4. MS m/z=585.2 [M+H] ⁺ .

Step 4: Synthesis of Compounds 3-5

Compound 3-4 (7g, 11.98mmol, 1eq), cuprous iodide (6.84g, 35.93mmol, 3eq), methyl fluorosulfonyldifluoroacetate (11.50g, 59.88mmol, 7.62mL, 5eq) were dissolved in N,N-dimethylformamide (70 mL) was replaced with nitrogen twice, and stirred at 90°C for 3 hours. The reaction solution was filtered, then 150 mL of ethyl acetate was added, washed with saturated brine (100 mL*3), dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column (petroleum ether:ethyl acetate=10:1) to obtain compound 3- 5. MS m/z=527.3 [M+H] ⁺ .

Step 5: Synthesis of Compounds 3-6

Compound 3-5 (2.1 g, 3.99 mmol, 1 eq) was dissolved in tetrahydrofuran (10 mL), cooled to -70°C, then lithium hexamethyldisilazide (1 M, 5.98 mL, 1.5 eq) was added and stirred for 0.5 hour. Ethyl cyanoformate (474.19 mg, 4.79 mmol, 469.49 μL, 1.2 eq) was then added and stirring was continued for 0.5 h. 20 mL of saturated ammonium chloride was added to the reaction solution, followed by extraction with ethyl acetate (30 mL*2). The organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. Column purification (petroleum ether:ethyl acetate=10:1-5:1) gave compound 3-6. MS m/z=599.3 [M+H] ⁺ , 1H NMR (400 MHz, CDCl ₃ ) δ 12.20 (s, 1H), 7.13-7.10 (m, 4H), 6.88-6.84 (m, 4H), 6.18 ( s, 1H), 4.82-4.56(m, 4H), 4.24-4.19(m, 2H), 3.81(s, 6H), 3.08-3.07(m, 1H), 2.73-2.60(m, 1H), 2.47- 2.13 (m, 7H), 1.33-1.31 (m, 3H), 0.77-0.73 (m, 3H).

Step 6: Hydrochloride Salt Synthesis of Compounds 3-7

Compound 3-6 (1.05g, 1.75mmol, 1eq) was dissolved in ethanol (20mL), water (4mL), then methyl isothiourea sulfate (4.88g, 17.54mmol, 10eq), sodium bicarbonate (3.68g) were added g, 43.85 mmol, 1.71 mL, 25 eq), stirring was continued at 50°C for 5 hours. The reaction solution was concentrated, then 30 mL of water was added, extracted with ethyl acetate (30 mL*2), dried over anhydrous sodium sulfate, filtered and concentrated. Column purification (petroleum ether: ethyl acetate = 5: 1-1: 1), and then organic fractionation [(chromatographic column: Phenomenex Luna 80*30mm*3μm; mobile phase A: water (0.025% hydrochloric acid), mobile phase A: water (0.025% hydrochloric acid) Phase B: acetonitrile; run gradient: B: 60%-80%; run time: 8 min)]. The hydrochloride salt of compound 3-7 was obtained. MS m/z=625.3 [M+H] ⁺ .

Step 7: Synthesis of Compounds 3-8

The hydrochloride salt of compound 3-7 (70 mg, ) was dissolved in dichloromethane (5 mL), and then N,N-diisopropylethylamine (28.96 mg, ) and trifluoromethanesulfonic anhydride (47.42 mg) were added, Stirring was continued for 1 hour at 0°C. The reaction solution was diluted with 10 mL of DCM, washed with saturated ammonium chloride (10 mL), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 3-8. MS m/z=757.2 [M+H] ⁺ .

Step 8: Synthesis of Compounds 3-9

Compound 3-8 (50 mg, 66.07 μmol, 1 eq) was dissolved in N,N-dimethylformamide (2 mL), then N,N-diisopropylethylamine (17.08 mg, 132.14 μmol, 23.02 μL) was added, 2eq), 1-7A (21.04mg, 99.10μmol, 1.5eq), stirring was continued for 1 hour at 50°C. To the reaction solution, 15 mL of water was added, extracted with ethyl acetate (15 mL*2), the organic phases were combined, washed with saturated brine (10 mL*3), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 3-9.

Step 9: Synthesis of Compounds 3-10

Compound 3-9 (35 mg, 42.74 μmol, 1 eq) was dissolved in dichloromethane (3 mL), then m-chloroperoxybenzoic acid (8.68 mg, 42.74 μmol, 85% content, 1 eq) was added, and stirring was continued at 10°C for 1 hour . The reaction solution was washed with 5% sodium thiosulfate (10 mL), saturated sodium bicarbonate (10 mL), saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 3-10. MS m/z=851.4 [M+H] ⁺ .

Step 10: Synthesis of Compounds 3-11

Compound 1-9A (11.22 mg, 70.51 μmol, 2 eq) was dissolved in tetrahydrofuran (3 mL), cooled to -15°C, then sodium tert-butoxide (3.39 mg, 35.25 μmol, 1 eq) was added, stirred for 15 min, and then compound 3 was added -10 (30 mg, 35.25 μmol, 1 eq) in THF (0.5 mL) and stirring continued for 30 min. The reaction solution was quenched with saturated ammonium chloride (10 mL), then extracted with ethyl acetate (10 mL*2), washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain compound 3-11. MS m/z=930.7 [M+H] ⁺ .

Step 11: Synthesis of Compounds 3-12

Compound 3-11 (15 mg, 16.13 μmol, 1 eq) was dissolved in dichloromethane (0.3 mL), then trifluoroacetic acid (91.95 mg, 806.38 μmol, 59.71 μL, 50 eq) was added, and the mixture was stirred at 30° C. for 16 hours. The reaction solution was directly concentrated to obtain compound 3-12. MS m/z=710.3 [M+H] ⁺ .

Step 12: Compound 3 Synthesis

Compound 3-12 (11.45 mg, 16.13 μmol, 1 eq) was dissolved in trifluoroacetic acid (3.08 g, 27.01 mmol, 2 mL, 1674.94 eq), and stirred at 75° C. for 6 hours. 10 mL of water and 10 mL of DCM were added to the reaction solution, and the solution was separated. The aqueous phase was adjusted to pH=9 with saturated sodium bicarbonate, and then extracted with ethyl acetate (10 mL*2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain Compound 3. MS m/z=590.3 [M+H] ⁺ .

Experimental example 1. KRAS ^G12D inhibitory activity test

1. Purpose

Through the method of TR-FRET, compounds that can effectively inhibit the binding of KRAS ^G12D mutation to GTP were screened.

2. Consumables and Instruments

Table 3. Consumables and Instruments

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 ^G12D protein, mixed with 440.5μL of KRAS nucleotide exchange buffer, and incubated at room temperature for 1 hour, then mixed with 8.4μL of 17.9μM Tb-SA, 1.8μL of 5mM Bodipy GDP, and 9539.8μL of KRAS experimental buffer. 1L of the solution, after mixing, let it stand at room temperature for 6 hours, and store it 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 procedure

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 a 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.

5 Experimental results

The results are shown in Table 4.

Table 4. Compounds of the present invention effectively inhibit the binding of KRASG12D mutation to GTP

Compound number	IC50 (nM)
1B	5

Conclusion: Some compounds of the present invention can effectively inhibit the binding of KRAS ^G12D mutation and GTP.

Experimental example 2. GP2D cell p-ERK inhibition test

1. Purpose

Through the method of HTRF, compounds that can effectively inhibit p-ERK in GP2D cells were screened out.

2. Experimental procedure

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 degrees overnight;

2). Add 2μL of compound to 78μL of cell culture medium. After mixing, take 20μL 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 50μL of 1X cell lysate to each well, and incubate 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 Phospho-ERK1/2 Eu Cryptate antibody dilution and 2μL Phospho-ERK1/2 d2 antibody dilution, incubate at room temperature for at least 4 hours;

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

7). Calculate the IC ₅₀ of the compound to be tested.

3 Experimental results

The results are shown in Table 5.

Table 5. _IC50 values of compounds for p-ERK inhibition in GP2D cells

Compound number	GP2D p-ERK IC 50 (nM)
The hydrochloride salt of compound 2	24.3
Compound 3	13.2

Experimental conclusion: The compound of the present invention has a significant inhibitory effect on p-ERK in GP2D cells.

Claims (22)

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

   

   in,

   

   is selected from single bond or double bond;

   T ₁ is selected from CR ₇ R ₈ , NR ₉ and O;

   when

   

   is selected from single bond, T is selected from _CH and N;

   when

   

   is selected from _double bonds, and T is selected from C;

   L ₁ is selected from -CH ₂ - and a bond;

   R ₁ , R ₂ , R ₃ , R ₄ and R ₅ are each independently selected from H and C _1-3 alkyl optionally substituted with ₁ , 2 or 3 _Ra ;

   R ₆ is selected from C _6-10 aryl and 5-10 membered heteroaryl, the C _6-10 aryl and 5-10 membered heteroaryl are optionally surrounded by 1, 2, 3, 4 or 5 R _b replace;

   R ₇ and R ₈ are each independently selected from H, CH ₃ and NH ₂ ;

   R ₉ is selected from H and CH ₃ ;

   R ₁₀ is selected from H, C _1-3 alkyl, C _1-3 alkoxy and cyclopropyl, the C _1-3 alkyl, C _1-3 alkoxy and cyclopropyl are optionally replaced by 1, 2 or 3 R _c substitutions;

   R ₁₁ is selected from 4-8 membered heterocycloalkyl and

   

   The 4-8 membered heterocycloalkyl and

   

   optionally substituted with 1, 2 or 3 _Rd ;

   Structural units

   

   selected from 5-6 membered heterocycloalkenyl;

   requirement is,

   1) R ₁ and R ₂ form a ring with the connected atoms, making the structural unit

   

   form

   

   2) Alternatively, R ₁ and R ₂ form a ring with the attached atoms, making the structural unit

   

   form

   

   3) Alternatively, R ₁ and R ₄ form a ring with the connected atoms, making the structural unit

   

   form

   

   4) Alternatively, R ₄ and R ₅ form a ring with the connected atoms, making the structural unit

   

   form

   

   5) Alternatively, R ₂ and R ₇ form tetrahydropyrrolidinyl with the attached atoms;

   6) Alternatively, R ₂ and R ₃ and the connected atom form a C _3-5 membered cycloalkyl;

   7) Alternatively, R ₇ and R ₈ form a 4-5 membered heterocycloalkyl with the attached atom;

   m is selected from 0, 1 or 2;

   n is selected from 0, 1 or 2;

   p is selected from 1 or 2;

   q is selected from 1, 2 or 3;

   r is selected from 1 or 2;

   s is selected from 1, 2 or 3;

   each R _a is independently selected from F, Cl, Br and I;

   Each R _b is independently selected from F, Cl, Br, I, OH, NH ₂ , CN, C _1-3 alkyl, C _1-3 alkoxy, C _2-3 alkynyl, C _2-3 alkene and C _3-5 cycloalkyl, the C _1-3 alkyl, C _1-3 alkoxy, C _2-3 alkynyl, C _2-3 alkenyl and C _3-5 cycloalkyl are optional replaced by 1, 2 or 3 Rs;

   each R _c is independently selected from F, Cl, Br and I;

   Each R _d is independently selected from H, F, Cl, Br, OH, CN, C _1-3 alkyl, C _1-3 alkoxy and -C _1-3 alkyl-O-CO-C _{1- 3} alkylamino; each R is independently selected from F, Cl and Br.
2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the structural unit

   

   selected from

   

   
3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the structural unit

   

   selected from

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

   

   selected from

   

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

   

   selected from

   
6. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₂ and R ₇ form with the attached atom

   
7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₂ and R ₃ form with the atoms to which they are attached

   
8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein R ₇ and R ₈ form with the attached atom

   
9. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the structural unit

   

   selected from

   

   
10. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein each R _b is independently selected from F, Cl, Br, I, OH, NH ₂ , CN, CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , -CH=CH ₂ , -CH ₂ -CH=CH ₂ and -C≡CH, the CH ₃ , CH ₂ CH ₃ , OCH ₃ , OCH ₂ CH ₃ , -CH=CH ₂ , _-CH2 -CH= _CH2 and -C≡CH are optionally substituted with 1, 2 or 3 Rs.
11. The compound of claim 10, or a pharmaceutically acceptable salt thereof, wherein each R _b is independently selected from F, OH, NH ₂ , CH ₃ , CF ₃ , CH ₂ CH ₃ and -C≡CH.
12. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₆ is selected from phenyl, pyridyl, naphthyl, indolyl and indazolyl, the phenyl, pyridyl, naphthyl, Indolyl and indazolyl are optionally substituted with 1, 2, 3, 4 or 5 R _b .
13. The compound according to claim 12 or a pharmaceutically acceptable salt thereof, wherein R ₆ is selected from

    

    
14. The compound according to any one of claims 1, 12 or 13, or a pharmaceutically acceptable salt thereof, wherein R ₆ is selected from

    

    
15. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₁₀ is selected from H, CH ₃ , OCH ₃ and cyclopropyl, wherein said CH ₃ , OCH ₃ and cyclopropyl are optionally 2 or 3 R _c substitutions.
16. The compound of claim 1 or 15, or a pharmaceutically acceptable salt thereof, wherein R ₁₀ is selected from H and CH ₃ .
17. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein each R _d is independently selected from H, F, Cl, Br, OH, CN, CH ₃ , CH ₂ CH ₃ , CH ₂ CF ₃ , OCH ₃ , OCF ₃ and

    
18. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R ₁₁ is selected from tetrahydropyrrolyl and hexahydro-1H-pyrrolizinyl, said tetrahydropyrrolyl and hexahydro-1H-pyrrole Rizinyl is substituted with 1, 2 or 3 _Rd .
19. The compound according to any one of claims 1, 17 or 18, or a pharmaceutically acceptable salt thereof, wherein R ₁₁ is selected from

    

    
20. The compound represented by the following formula or its pharmaceutically acceptable salt, its compound is selected from

    
21. The compound of claim 20, or a pharmaceutically acceptable salt thereof, selected from the group consisting of,

    
22. The compound of claim 21, or a pharmaceutically acceptable salt thereof, selected from the group consisting of,

    

    

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