WO2024083120A1

WO2024083120A1 - Benzylaminoquinoline compound and preparation method therefor

Info

Publication number: WO2024083120A1
Application number: PCT/CN2023/124976
Authority: WO
Inventors: 贺海鹰; 李鹏; 代天资; 师凯旋; 陈曙辉
Original assignee: 南京明德新药研发有限公司
Priority date: 2022-10-18
Filing date: 2023-10-17
Publication date: 2024-04-25

Abstract

Disclosed in the present invention are a benzylaminoquinoline compound and a preparation method therefor. The present invention specifically relates to a compound represented by formula (II), a stereoisomer thereof and a pharmaceutically acceptable salt thereof.

Description

Benzylaminoquinoline compounds and preparation methods thereof

This application claims the following priority:

CN202211276424.1, October 18, 2022;

CN202211497271.3, November 25, 2022.

Technical Field

The present invention relates to a class of benzylaminoquinoline compounds and a preparation method thereof, and in particular to a compound represented by formula (II), a stereoisomer thereof and a pharmaceutically acceptable salt thereof.

Background technique

RAS protein is a guanine nucleoside binding protein with guanosine triphosphate hydrolase (GTPase) activity, mainly including three subtypes, KRAS, NRAS and HRAS. As a binary molecular switch controlled by GDP/GTP cycle, RAS protein can cycle between active GTP-bound state (GTP-RAS) and inactive GDP-bound state (GDP-RAS). This cycle has important regulatory functions in cells and is closely related to cell proliferation, survival, metabolism, migration, immunity and growth.

SOS1 (Son of Sevenless 1) is a type of GEF that regulates the GDP/GTP cycle of RAS proteins. After the cell surface receptor is activated and binds to intracellular Grb2, Grb2 recruits SOS1 to the cell membrane, and then SOS1 catalyzes RAS-GDP/GTP exchange, thereby activating downstream signaling pathways. Small molecule SOS1 inhibitors that bind to the catalytic site can block the binding of SOS1 to RAS proteins, thereby effectively reducing the abnormal activation of RAS downstream signaling pathways in cancer cells and playing a role in treating cancer. Currently, the only SOS1 small molecule inhibitor, BI-1701963 (WO2018115380, WO2019122129) developed by Boehringer Ingelheim, has entered Phase I clinical trials. The SOS1 inhibitor developed by Bayer (WO2018172250, WO2019201848) is still in the preclinical research stage. In recent years, studies have shown that drugs in the RAS pathway are prone to drug resistance in clinical applications. Part of the reason for drug resistance is that the inhibition of ERK phosphorylation will negatively feedback and activate the upstream RAS pathway. This negative feedback regulation mechanism is closely related to SOS1. Therefore, the development of SOS1 small molecule inhibitors has broad application prospects.

AMG-510 is a potent, orally bioavailable, selective KRAS G12C covalent inhibitor developed by Amgen for the treatment of locally advanced or metastatic non-small cell lung cancer carrying KRAS G12C mutations. Its structure is shown below:

Summary of the invention

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

in,

T is selected from

R ₁ is selected from H, F, Cl, Br, I, -OH, -NH ₂ , -CN and C _1-4 alkyl, wherein the C _1-4 alkyl is optionally substituted with 1, 2, 3 or 4 _Ra ;

R ₂ is selected from H, F, Cl, Br, I, -OH, -NH ₂ , -CN and C _1-4 alkyl, wherein the C _1-4 alkyl is optionally substituted with 1, 2, 3 or 4 R _b ;

R ₃ is selected from H, F, Cl, Br, I, -OH, -NH ₂ , -CN and C _1-4 alkyl, wherein the C _1-4 alkyl is optionally substituted with 1, 2, 3 or 4 R _c ;

Each _Ra is independently selected from F, Cl, Br, I, -OH, _-NH2 , -CN, -COOH, =O and _C1-3 alkyl;

Each R _b is independently selected from F, Cl, Br, I, -OH, -NH ₂ , -CN, -COOH, =O and C _1-3 alkyl;

Each R _c is independently selected from F, Cl, Br, I, -OH, -NH ₂ , -CN, -COOH, =O and C _1-3 alkyl.

in,

T is selected from

_R1 is selected from H, F, Cl and Br;

R ₂ is selected from C _1-4 alkyl, wherein the C _1-4 alkyl is optionally substituted with 1, 2, 3 or 4 R _b ;

_R3 is selected from H, F, Cl and Br;

Each R _b is independently selected from F and -OH.

In some embodiments of the present invention, the above compound has a structure shown in formula (II-1):

wherein T, R ₁ , R ₂ and R ₃ are as defined in the present invention;

The carbon atom with "*" is a chiral carbon atom, which exists in the form of a single enantiomer (R) or (S) or in the form enriched in one enantiomer.

wherein T, R ₁ , R ₂ and R ₃ are as defined in the present invention.

In some embodiments of the present invention, each R _b mentioned above is independently selected from F and -OH, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₁ is selected from H, F, Cl, Br and -NH ₂ , and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₁ is selected from H, and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from H, F, Cl, Br, -CN, -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ and -CH ₂ CH(CH ₃ ) ₂ , wherein said -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ and -CH ₂ CH(CH ₃ ) ₂ are each independently optionally substituted by 1, 2, 3 or 4 R _b , and R _b and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from -CH ₃ and -CH ₂ CH(CH ₃ ) ₂ , wherein said -CH ₃ and -CH ₂ CH(CH ₃ ) ₂ are independently optionally substituted by 1, 2, 3 or 4 R _b , and R _b and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from -CH ₂ CH(CH ₃ ) ₂ , wherein the -CH ₂ CH(CH ₃ ) ₂ is optionally substituted by 1, 2, 3 or 4 R _b , and R _b and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from H, F, Cl, Br, -CN, R _b and other variables are as defined herein.

In some embodiments of the present invention, the above R ₂ is selected from R _b and other variables are as defined herein.

In some embodiments of the present invention, the above _R2 is selected from H, F, Other variables are as defined in the present invention.

In some embodiments of the present invention, the above _R2 is selected from H, Other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from Other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₃ is selected from H, F, Cl, Br and -NH ₂ , and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₃ is selected from F, and other variables are as defined in the present invention.

Some other solutions of the present invention are obtained by arbitrarily combining the above variables.

in,

_R3 is selected from H, F, Cl and Br;

Each R _b is independently selected from F and -OH.

Wherein, _R2 and _R3 are as defined in the present invention.

in,

_R3 is selected from H, F, Cl and Br;

Each R _b is independently selected from F and -OH.

The present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

In some embodiments of the present invention, the above-mentioned compound, its stereoisomer or its pharmaceutically acceptable salt, the compound is selected from,

The present invention also provides the use of the above compound, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating KRAS mutant solid tumors.

The present application also provides a method for treating KRAS mutant solid tumors in a subject in need thereof, comprising providing the subject with an effective dose of the above compound, its stereoisomer or a pharmaceutically acceptable salt thereof.

The present invention also provides a biological test method for the above compound:

Test method 1: H358 cell 3D proliferation inhibition activity test

Experimental principle:

In H358 cells with KRAS (G12C) mutation, the KRAS signaling pathway is abnormally activated. Small molecule SOS1 inhibitors inhibit the binding of SOS1 to RAS protein, reduce its GEF activity, and reduce the ratio of activated RAS-GTP. Further downregulation of the phosphorylation level of the MEK/ERK pathway downstream of RAS achieves the effect of inhibiting cell proliferation. Small molecules are co-cultured with H358 cells in 3D space, and then cell readouts are used to indirectly reflect the proliferation inhibitory activity of SOS1 inhibitors on H358 cells.

Experimental Materials:

RPMI1640 medium, fetal bovine serum, penicillin/streptomycin antibiotics were purchased from Vicente, and low melting point agarose was purchased from Sigma. Almar blue reagent was purchased from Invitrogen. NCI-H358 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Nivo multi-label analyzer (PerkinElmer).

experimental method:

H358 cells were seeded in a 96-well U-shaped plate. Low melting point agarose was first prepared into a 2% stock solution. When used, the agarose stock solution was first heated in a microwave oven to completely melt it, and then placed in a 42°C water bath to keep the agarose in a liquid state. The gel was added to the serum-containing culture medium to prepare a gel concentration of 0.6% as the bottom layer gel, and 50 μL was spread into the 96-well U-shaped plate at each well. After the bottom layer gel solidified, 2% gel was added to the cell-containing culture medium to prepare a cell-containing upper layer gel with a gel concentration of 0.4%, and the cell density was 4×10 ⁴ cells/ml. 75 μL was added to each well of the 96-well U-shaped plate with the bottom layer gel, and the cell density was 3000 per well. After the upper layer gel solidified, the cell plate was placed in a carbon dioxide incubator for overnight culture.

On the day of adding the compound, add 85 μL of liquid culture medium to the 96-well U-shaped plate with cells. Use a dispenser to dilute the compound to be tested 3-fold to the 9th concentration, that is, from 6mM to 0.9μM, and set up a double-well experiment. Add 97 μL of culture medium to the middle plate, and then transfer 2.5 μL of the gradient dilution compound per well to the middle plate according to the corresponding position, mix well and transfer 40 μL per well to the cell plate. The concentration range of the compound transferred to the cell plate is 30μM to 4.5nM. The cell plate was placed in a carbon dioxide incubator for 7 days. On the 8th day, the compound to be tested was diluted 3-fold to the ninth concentration, that is, from 6mM to 0.9μM, with a dispenser, and a double-well experiment was set up. Add 198 μL of culture medium to the middle plate, and then transfer 2 μL of the gradient dilution compound per well to the first middle plate according to the corresponding position. Then add 100 μL of culture medium to the second middle plate, take 100 μL of the mixed compound in the first middle plate and add it. After mixing, transfer 40 μL per well to the cell plate. The concentration range of the compound transferred to the cell plate is 30 μM to 4.5 nM. The cell plate is placed in a carbon dioxide incubator and cultured for another 7 days. The cells were incubated for 14 days, and 20 μL of Almar blue detection reagent was added to each well of the cell plate. The plate with dye was placed on a horizontal shaker for 15 minutes, and then the plate was incubated at room temperature for 5 hours to stabilize the luminescent signal. The reading was performed using a multi-label analyzer.

data analysis:

The raw data were converted into inhibition rate using the equation (Sample-Min)/(Max-Min)×100%, and the _IC50 value was obtained by four-parameter curve fitting (obtained using the "log(inhibitor)vs.response--Variable slope" mode in GraphPad Prism).

Experimental conclusion: The compounds of the present invention can inhibit the proliferation of H358 cells under 3D conditions.

Experimental Test Method 2: Compound Pharmacokinetic Evaluation

Experimental Materials:

CD-1 mice (male, 7-9 weeks old, Shanghai Slack)

Experimental operation:

The pharmacokinetic characteristics of the compounds after intravenous and oral administration in rodents were tested using standard protocols. In the experiment, the candidate compounds were prepared into clear solutions and given to mice as a single intravenous injection and oral administration. The intravenous and oral solvents were a mixed solvent of 10% dimethyl sulfoxide and 90% of a 10% hydroxypropyl β-cyclodextrin aqueous solution. Four female CD-1 mice were used in this project. Two mice were intravenously injected with the drug at a dose of 1 mg/kg, and plasma samples were collected at 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, and 12 h after administration. The other two mice were orally gavaged with the drug at a dose of 2 mg/kg, and plasma samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 12 h after administration. The samples were stirred at 3,200 x g at 4°C for 10 minutes, and the supernatant was separated to obtain plasma samples. A 20-fold volume of methanol solution containing an internal standard was added to precipitate the protein, and the samples were stirred at 12,000 x g for 15 minutes. The supernatant was centrifuged at 4°C and 50 μL was transferred to a 96-well plate for a second centrifugation. The supernatant was sampled and quantitatively analyzed for blood drug concentrations using LC-MS/MS analysis methods, and pharmacokinetic parameters such as peak concentration (C _max ), clearance (CL), half-life (T _1/2 ), tissue distribution (Vdss), and area under the drug-time curve (AUC _0-last ), bioavailability (F), etc.

Experimental conclusion: The compounds of the present invention have good pharmacokinetic properties, including good oral bioavailability, oral exposure, half-life and clearance rate.

Experimental test method 3: In vivo efficacy evaluation of compounds in Miapaca2 nude mouse transplant tumor model

Cell culture:

Human pancreatic cancer cells (ATCC) were cultured in vitro in an adherent monolayer in DMEM medium with 10% fetal bovine serum at 37°C and 5% CO _2. They were routinely digested and passaged with trypsin-EDTA two to three times a week. When the cell saturation reached 80%–90% and the number reached the required level, the cells were harvested, counted, and inoculated.

Experimental Animals:

Balb/c nude mice, female, 6-7 weeks old, were purchased from Shanghai Xipu-Bikai Experimental Animal Co., Ltd.

Model preparation:

0.2 mL (5×10 ⁶ cells) of Miapaca2 cells (with matrix gel, volume ratio of 1:1) were subcutaneously inoculated into the right back of each mouse. When the average tumor volume reached 118 mm ³ , group dosing began.

Tumor measurements and experimental parameters:

The tumor diameter was measured with a vernier caliper twice a week, and the tumor volume was measured in cubic millimeters and calculated by the following formula: V = 0.5a × b ² , where a and b are the long diameter and short diameter of the tumor, respectively. The anti-tumor efficacy of the test compound was evaluated by using TGI (%). TGI (%) reflects the tumor growth inhibition rate. TGI (%) = [1-(average tumor volume at the end of administration of a treatment group - average tumor volume at the beginning of administration of the treatment group) / (average tumor volume at the end of treatment of the solvent control group - average tumor volume at the beginning of treatment of the solvent control group)] × 100%.

Experimental conclusion: The compound of the present invention combined with AMG-510 showed excellent tumor inhibition effect in the Miapaca2 nude mouse transplant tumor model.

Technical Effects

The compound of the present invention has good KRAS (G12C)-SOS1 binding inhibitory activity, and has significant inhibitory activity on DLD-1 cells and KRAS (G12C) mutated H358 cells; safety experimental studies have found that the compound of the present invention has no obvious inhibitory effect on the hERG potassium channel, has a low risk of cardiac toxicity, and thus obtains excellent activity in inhibiting tumor growth.

Definition and Description

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 to be uncertain or unclear in the absence of a special definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient.

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

The term "pharmaceutically acceptable salt" refers to salts of compounds of the invention, prepared from compounds of the invention having specific substituents with relatively nontoxic acids or bases. When the compounds of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or 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 invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as 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, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, and 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 basic and acidic functional groups, and thus can be converted into any base or acid addition salt.

Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

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

Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

Unless otherwise indicated, the term "cis-trans isomers" or "geometric isomers" arises from the inability of a double bond or single bond forming a ring carbon atom to rotate freely.

Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

Unless otherwise indicated, "(+)" indicates dextrorotatory, "(-)" indicates levorotatory, and "(±)" indicates racemic.

Unless otherwise specified, the key is a solid wedge. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond. and straight dashed key To indicate the relative configuration of a stereocenter, use a wavy line Denotes a solid wedge bond or dotted wedge key Or use a wavy line Represents a straight solid bond and straight dashed key

The compounds of the present invention may exist in specific forms. Unless otherwise indicated, the term "tautomer" or "tautomeric form" means that at room temperature, different functional group isomers are in dynamic equilibrium and can quickly convert to each other. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also called prototropic tautomers) include interconversions by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence isomers (valence tautomers) include interconversions by the reorganization of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between two tautomers of pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer 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 term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or 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 and 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 diastereomeric mixture is separated and the auxiliary group is cleaved to provide the 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, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (for example, a carbamate is generated from an amine).

The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( ^3H ), iodine-125 ( ^125I ) or C-14 ( ^14C ). For another example, deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic effects, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in 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 where it does not.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups.

The term "optionally substituted" means that the group may be substituted or unsubstituted, and unless otherwise specified, the type and number of the substituents may be any on the basis of what is chemically feasible.

When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, and each occurrence of R is an independent choice. In addition, combinations of substituents and/or variants thereof are permitted 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 it connects 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 substituent does not specify which atom it is connected to the substituted group through, the substituent can be bonded through any atom of it. For example, pyridyl as a substituent can be connected to the substituted group through any carbon atom on the pyridine ring.

When the linking group is listed without specifying its linking direction, its linking direction is arbitrary, for example, The connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of the reading order from left to right to form Combinations of linkers, substituents, and/or variations thereof are permissible only if such combinations result in stable compounds.

Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the chemical bond connection mode is non-positional and there are H atoms at the connectable sites, when the chemical bonds are connected, the number of H atoms at the site will decrease accordingly with the number of connected chemical bonds to become a group with a corresponding valence. The chemical bond connecting the site to other groups can be a straight solid bond. Straight dotted key or wavy line For example, the straight solid bond in -OCH ₃ indicates that it is connected to other groups through the oxygen atom in the group; The straight dashed bond in the group indicates that the two ends of the nitrogen atom in the group are connected to other groups; The wavy line in the phenyl group indicates that it is connected to other groups through the carbon atoms at positions 1 and 2 in the phenyl group. It means that any connectable site on the piperidine group can be connected to other groups through one chemical bond, including at least These four connection methods, even if the H atom is drawn on -N-, Still includes For groups connected in this way, when one chemical bond is connected, the H at that site will be reduced by one and become a corresponding monovalent piperidine group.

When the chemical bond of a substituent intersects the chemical bond connecting two atoms on a ring, it means that the substituent can form a bond with any atom on the ring. When the atom to which a substituent is attached is not specified, the substituent can form a bond with any atom. If the atom to which the substituent is attached is in a bicyclic or tricyclic ring system, it means that the substituent can form a bond with any atom of any ring in the system. Combinations of substituents and/or variables are allowed only if the combination results in a stable compound. For example, the structural unit It means that it can be substituted at any position on the cyclohexyl group or the cyclopentyl group.

Unless otherwise specified, the number of atoms in a ring is generally defined as the ring member number, for example, "5-7 membered ring" refers to a "ring" having 5-7 atoms arranged around it.

Unless otherwise specified, _Cn-n+m or _Cn - _Cn+m includes any specific case of n to n+m carbon atoms, for example, _C1-12 includes _C1 , _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 , and _C12 , and also includes any range from n to n+m, for example, _C1-12 includes _C1-3 , _C1-6 , _C1-9 , _C3-6 , _C3-9 , _C3-12 , _C6-9 , _C6-12 , and C13 _. _9-12 , etc.; similarly, n-membered to n+m-membered means that the number of atoms in the ring is n to n+m, for example, 3-12-membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring, and 12-membered ring, and also includes any 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, 5-7-membered ring, 6-7-membered ring, 6-8-membered ring, and 6-10-membered ring, etc.

Unless otherwise specified, the term "alkyl" by itself or as part of another substituent refers to a straight or branched saturated hydrocarbon group. The alkyl group may be a C _1-6 alkyl group or a C _1-3 alkyl group. The alkyl group may be optionally substituted with one or more of the following groups: oxo, hydroxy, amino, nitro, halogen, cyano, alkenyl, alkynyl, alkoxy, haloalkoxy, alkylamino, dialkylamino, haloalkylamino, halodialkylamino, cycloalkyl, cycloalkyloxy, heterocyclyl, heterocyclyloxy, heterocycloalkyl, heterocycloalkyloxy, heteroaryl, heteroaryloxy, aryl or aryloxy.

Unless otherwise specified, the term "C _1-4 alkyl" is used to represent a straight or branched saturated hydrocarbon group consisting of 1 to 4 carbon atoms. The C _1-4 alkyl group includes C _1-2 , C _1-3 and C _2-3 alkyl groups, etc.; they can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of C _1-4 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), etc.

Unless otherwise specified, the term "C _1-3 alkyl" is used to represent a straight or branched 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 (such as methyl), divalent (such as methylene) or polyvalent (such as methine). Examples of C _1-3 alkyl _groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.

The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (e.g., a nucleophilic substitution reaction). For example, representative leaving groups include trifluoromethanesulfonate; chlorine, bromine, iodine; sulfonate groups, such as mesylate, tosylate, p-brosylate, p-toluenesulfonate, etc.; acyloxy groups, such as acetoxy, trifluoroacetoxy, etc.

The term "protecting group" includes, but is not limited to, an "amino protecting group", a "hydroxy protecting group" or a "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, such as alkanoyl (e.g., acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tert-butyloxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-bis-(4'-methoxyphenyl)methoxycarbon ...oc), 1,1-bis-(4'-methoxyphenyl)methoxycarbonyl (Boc), 1,1-bis-(4'-methoxyphenyl)methoxycarbonyl ( The term "hydroxy protecting group" refers to a protecting group suitable for preventing side reactions of the hydroxyl group. Representative hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (such as acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS) and the like.

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

The structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention relates to the absolute configuration of the compounds, the absolute configuration can be confirmed by conventional technical means in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8venture diffractometer, the light source is CuKα radiation, and the scanning mode is φ/ω scanning. After collecting relevant data, the direct method (Shelxs97) is further used to analyze the crystal structure, and the absolute configuration can be confirmed.

The volume used in the present invention is commercially available.

The present invention uses the following abbreviations: Alloc represents allyloxycarbonyl; SEM represents trimethylsilylethoxymethyl; OTs represents 4-toluenesulfonyl; Boc represents tert-butyloxycarbonyl; DCM represents dichloromethane; DIEA represents N,N-diisopropylethylamine; MeI represents iodomethane; PE represents petroleum ether; EA represents ethyl acetate; THF represents tetrahydrofuran; EtOH represents ethanol; MeOH represents methanol; Boc ₂ O represents di-tert-butyl dicarbonate; NH ₄ Cl represents ammonium chloride; T ₃ P represents 1-propylphosphoric acid tricyclic anhydride; Pd/C represents palladium/carbon catalyst; TMSN ₃ represents azidotrimethylsilane; NCS represents N-chlorosuccinimide; HBr represents hydrobromic acid; AcOH represents acetic acid; HATU represents O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DBU represents 1,8-diazabicycloundec-7-ene; FA represents formic acid; ACN represents acetonitrile; TLC represents thin layer chromatography; HPLC represents high pressure liquid chromatography; LCMS represents liquid chromatography-mass spectrometry. DMSO represents dimethyl sulfoxide; DMSO-d ₆ represents deuterated dimethyl sulfoxide; CD ₃ OD represents deuterated methanol; CDCl ₃ represents deuterated chloroform; D ₂ O represents deuterated water.

Compounds are named according to the conventional nomenclature in the art or using The software names were used, and commercially available compounds were named using the supplier's catalog names.

Detailed ways

The present invention is described in detail below by examples, but it is not intended to limit the present invention in any adverse way. The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by the combination thereof with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art, and 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 improvements are made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

Intermediate A

synthetic route:

first step

Compound A-1 (10.0 g, 47.3 mmol) was dissolved in acetonitrile (24 mL), and dry hydrogen chloride gas was introduced into the mixture at 25°C for 0.5 hours. The reaction solution was stirred at 90°C for 3 hours, and the reaction solution was cooled to 25°C, filtered, and the filter cake was collected and dissolved in water (100 mL). The solution was neutralized with 10% sodium bicarbonate solution (100 mL), filtered, and the filter cake was washed with ice water (100 mL) and dried to obtain compound A-2. ¹ H NMR (400 MHz, CD ₃ OD) δ7.55 (s, 1H), 7.07 (s, 1H), 3.98 (s, 3H), 3.95 (s, 3H), 2.45 (s, 3H).

Step 2

Compound A-2 (3.00 g, 13.6 mmol) was dissolved in methanesulfonic acid (15 mL), DL-methionine (2.44 g, 16.4 mmol) was added to the compound, the reaction solution was reacted at 110°C for 12 hours, the reaction solution was quenched with water (90 mL) and sodium hydroxide aqueous solution (2 mol/L, 150 ml), the reaction solution was filtered, the filter cake was collected and vacuum dried to obtain intermediate A. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ7.33 (s, 1H), 7.02 (s, 1H), 3.88 (s, 3H), 2.88 (s, 3H).

Intermediate B

synthetic route:

first step

Dissolve B-2 (3.37 g, 16.6 mmol) in dimethyl sulfoxide (20 mL), add copper powder (1.06 g, 16.6 mmol). The reaction solution was stirred at 25 ° C for 1 hour. Then compound B-1 (2.00 g, 6.65 mmol) was added to the reaction solution, and stirred at 70 ° C for 12 hours. Pour the reaction solution into 20 mL of ice water, add ethyl acetate (20 mL), filter, and extract the filtrate with ethyl acetate (20 mL × 3). The organic phase was washed with saturated brine (20 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate, 1/0～20/1, V/V) to obtain compound B-3. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.75-7.68 (m, 1H), 7.64-7.57 (m, 1H), 7.16 (t, J=8.0 Hz, 1H), 4.38 (m, 2H), 1.34 (t, J=8.0 Hz, 3H).

Step 2

Under nitrogen protection, compound B-3 (677 mg, 1.82 mmol) was dissolved in toluene (10 mL), and methylmagnesium bromide solution (compound B-4) (3M, 2.43 mL) was added at 0 ° C. The reaction solution was stirred at 25 ° C for 2 hours. Saturated ammonium chloride solution (10 mL) was added to quench, and extracted with ethyl acetate (10 mL × 2). The organic phase was washed with saturated brine (10 mL × 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The product was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate, 1/0-10/1, V/V) to obtain compound B-5. ¹ H NMR (400 MHz, CDCl ₃ ) δ7.66 (t, J=6.4 Hz, 1H), 7.51-7.34 (m, 1H), 7.16-6.93 (m, 1H), 2.01 (s, 1H), 1.35 (s, 6H).

third step

Under nitrogen protection, compound B-5 (441 mg, 1.56 mmol) was dissolved in toluene (5 mL), and compound B-6 (1.87 g, 5.19 mmol) and bistriphenylphosphine palladium dichloride (109 mg, 0.16 mmol) were added. The reaction solution was stirred at 120 ° C for 12 hours. Saturated potassium fluoride solution (20 mL) was added to quench, and extracted with ethyl acetate (15 mL × 2). Filtered and concentrated under reduced pressure to obtain compound B-7.

the fourth step

Under nitrogen protection, compound B-7 (425 mg, 1.55 mmol) was dissolved in acetone (10 mL), and hydrochloric acid solution (12 M, 1.03 mL) was added dropwise at 0 ° C. The mixture was stirred at 25 ° C for 1 hour. Saturated sodium bicarbonate solution was added to neutralize to pH = 8, and extracted with ethyl acetate (10 mL). The organic phase was washed with saturated brine (10 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate, 20/1 to 4/1, V/V) to obtain compound B-8. MS-ESI calculated value [M+H] ⁺ 247, found value 247.

the fifth step

Compound B-8 (346 mg, 1.41 mmol) was dissolved in tetrahydrofuran (5 mL) at 25 °C, and compound B-9 (511 mg, 4.22 mmol) and tetraethoxytitanium (2.00 g, 7.03 mmol) were added. The reaction solution was stirred at 80 °C for 36 hours. Then sodium borohydride (64.0 mg, 1.69 mmol) was added to the reaction solution at -5 °C, and the reaction was stirred at 25 °C for 1 hour. The reaction solution was poured into 20 mL of ice water, filtered, and the filtrate was extracted with ethyl acetate (5 mL × 2). The organic phase was washed with saturated brine (5 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate, 2/1 to 0/1, V/V) to obtain compound B-10. MS-ESI calculated value [M+H] ⁺ 352, measured value 352.

Step 6

Compound B-10 (366 mg, 1.04 mmol) was dissolved in dioxane (2.5 mL), and a solution of hydrogen chloride in dioxane (4 M, 1.15 mL) was added. The reaction solution was stirred at 25 °C for 6 hours. The reaction solution was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (dichloromethane/methanol, 1/0-8/1, V/V) to obtain the hydrochloride of intermediate B. The hydrochloride of intermediate B was added to 1M NaOH (5 mL), extracted with ethyl acetate (5 mL×2), and the organic phase was filtered with anhydrous sodium sulfate, concentrated under reduced pressure, and dried to obtain intermediate B.

MS-ESI calculated value [M+H] ⁺ 248, found value 248.

Example 1

synthetic route:

first step

Intermediate A (300 mg, 2.91 mmol) was dissolved in tetrahydrofuran (5 mL), and then compound 1-1 (674 mg, 3.35 mmol) and N,N-diisopropylethylamine (1.13 g, 8.73 mmol) were added, and the reaction solution was stirred at 25°C for 1 hour. The reaction solution was poured into water (20 mL), filtered, and the filtrate was extracted with ethyl acetate (20 mL×3), and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain compound 1-2.

Step 2

Compound 1-2 (500 mg, 1.35 mmol) was dissolved in N,N-dimethylformamide (5 mL), triethylamine (940 μL, 6.75 mmol) and compound 1-3 (541 mg, 2.70 mmol) were added dropwise, and the mixture was stirred at 25°C for 12 hours. The reaction solution was filtered and concentrated under reduced pressure, and the residue was subjected to silica gel column chromatography (dichloromethane/methanol, 100/1 to 10/1, V/V) to obtain compound 1-4. MS-ESI calculated value [M+H] ⁺ 433, found value 433.

third step

Compound 1-4 (460 mg, 1.06 mmol) was dissolved in dichloromethane (10 mL), and compound 1-5 (387 mg, 1.28 mmol), 4-dimethylaminopyridine (13.0 mg, 106 μmol) and N, N-diisopropylethylamine (556 μL, 3.19 mmol) were added, and the mixture was stirred at 25 ° C for 12 hours. The reaction solution was filtered and concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (dichloromethane/methanol, 100/1 to 10/1, V/V) to obtain compound 1-6. MS-ESI calculated value [M+H] ⁺ 699, found value 699.

the fourth step

Compound 1-6 (620 mg, 887 μmol) was dissolved in dimethyl sulfoxide (10 mL), and intermediate B (219 mg, 887 μmol) and triethylamine (370 μL, 2.66 mmol) were added, and the mixture was stirred at 90 °C for 12 hours. After cooling to room temperature, water (100 mL) was slowly added to the reaction solution, and the mixture was extracted with ethyl acetate (100 mL × 3). The organic phase was washed with saturated brine (100 mL × 5), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was subjected to silica gel column chromatography (dichloromethane/methanol, 100/1 to 10/1, V/V) to obtain compound 1-7. MS-ESI calculated value [M+H] ⁺ 662, found value 662.

the fifth step

Compound 1-7 (540 mg, 816 μmol) was dissolved in ethyl acetate (5 mL), and hydrogen chloride/ethyl acetate solution (4 mol/L, 8.31 mL) was added, and the mixture was stirred at 25°C for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was separated and purified by preparative high performance liquid chromatography (chromatographic column: Phenomenex C18 150 mm×40 mm×5 μm; mobile phase: 0.05% hydrochloric acid aqueous solution-acetonitrile; gradient: acetonitrile 10%-40%, 10 min) to obtain the hydrochloride of compound 1. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.45 (s, 1H), 7.70-7.57 (m, 1H), 7.48-7.37 (m, 1H), 7.29-7.21 (m, 1H), 7.20 (s, 1H), 6.08-5.94 (m, 1H), 4.79-4.14 (m, 2H), 4.04 (s, 3H), 3.66-3.44 (m, 2H), 3.44-3.35 (m, 2H), 3.29-3.15 (m, 1H), 2.62 (s, 3H), 1.74 (d, J = 6.8 Hz, 3H), 1.51 (d, J = 6.0 Hz, 3H), 1.37-1.21 (m, 6H). MS-ESI calculated value [M+H] ⁺ 562, found value 562.

Biological activity:

Experimental Example 1: KRAS (G12C) and SOS1 binding experiment

Experimental principle:

Small molecule compounds bind to the catalytic site of SOS1 and inhibit the binding of SOS1 to KRAS (G12C). When the binding of fluorescently labeled SOS1 protein to fluorescently labeled KRAS (G12C) protein is inhibited, the emitted fluorescence changes. By detecting the change in fluorescence, the ability of small molecules to prevent the binding of SOS1 to KRAS (G12C) can be tested. A homogeneous time-resolved fluorescence (HTRF) binding assay is used to detect the ability of the compounds of the present invention to inhibit the binding of SOS1 to KRAS (G12C).

Experimental Materials:

KRAS (G12C) protein was expressed and purified by Wuhan Pujian Biotechnology Co., Ltd., SOS1 exchange domin (564-1049) protein (Hμman recombinant) was purchased from Cytoskeleton, Mab Anti 6HIS-XL665 and Mab Anti GST-Eμcryptate were purchased from Cisbio. Multifunctional microplate reader Nivo5 was purchased from PerkinElmer.

experimental method:

1X buffer preparation (prepared and used immediately): Hepes: 5mM; NaCl: 150mM; EDTA: 10mM; Igepal: 0.0025%; KF: 100mM; DTT: 1mM; BSA: 005%;

The test compound was diluted 5-fold with DMSO using a dispenser to the eighth concentration, that is, from 1 mM to 0.064 μM.

Use 1X buffer to dilute the compound to be tested into a 2% DMSO working solution, add 5 μL/well to the corresponding wells, the corresponding concentration gradient is 20 μM to 0.00128 nM, set up a duplicate well experiment. Centrifuge at 1000 rpm for 1 minute.

Use 1X buffer to prepare a mixed working solution of KRAS (G12C) (200nM) and Mab Anti GST-Eμcryptate (1ng/μL), incubate the mixed working solution at 25°C for 5 minutes, and add 2.5μL/well to the corresponding wells.

Use 1X buffer to prepare a mixed working solution of SOS1 (80nM) and Mab Anti 6HIS-XL665 (8g/μL), 2.5μL/well is added to the corresponding wells, and 2.5μL of Mab Anti 6HIS-XL665 (8g/μL) dilution is added to the Blank well. At this time, the final concentration gradient of the compound is diluted from 10μM to 0.64nM, KRAS (G12C) (500nM), MAb Anti GST-Eu cryptate (0.25ng/μL), SOS1 (20nM), Mab Anti 6HIS-XL665 (2g/μL), and the reaction system is placed at 25℃ for 60 minutes. After the reaction, HTRF is read using a multi-label analyzer.

data analysis:

The raw data were converted into inhibition rate using the equation (Sample-Min)/(Max-Min)×100%, and the IC ₅₀ value was obtained by four-parameter curve fitting (obtained by log(inhibitor) vs.response--Variable slope mode in GraphPad Prism). Table 1 provides the inhibitory activity of the compounds of the present invention on the binding of KRAS (G12C) and SOS1.

Table 1 IC ₅₀ test results of the compounds of the present invention for binding to KRAS (G12C) and SOS1

Experimental conclusion: The compounds of the present invention have a significant inhibitory effect on the binding of KRAS (G12C) and SOS1.

Experimental Example 2: DLD-1 cell p-ERK proliferation inhibition activity test

Experimental Materials:

DLD-1 cells were purchased from Nanjing Kebai; 1640 culture medium was purchased from Biological Industries; fetal bovine serum was purchased from Biosera; Advanced Phospho-ERK1/2 (THR202/TYR204) KIT was purchased from Cisbio. The composition of Advanced Phospho-ERK1/2 (THR202/TYR204) KIT is shown in Table 2.

Table 2. Ingredients of Advanced Phospho-ERK1/2 (THR202/TYR204) KIT

experimental method:

DLD-1 cells were seeded in a transparent 96-well cell culture plate, with 80 μL of cell suspension per well, and each well contained 8000 DLD-1 cells. The cell plate was placed in a carbon dioxide incubator and incubated overnight at 37°C;

The compound to be tested was diluted to 2 mM with 100% DMSO as the first concentration, and then diluted 5 times with a pipette to the eighth concentration, that is, from 2 mM to 0.026 μM. 2 μL of compound was added to 78 μL of cell starvation medium, mixed, and 20 μL of compound solution was added to the corresponding cell plate wells. The cell plate was returned to the carbon dioxide incubator and incubated for 1 hour. At this time, the compound concentration was 10 μM to 0.128 nM, and the DMSO concentration was 0.5%;

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

Use detection buffer to dilute the phosphorylated ERK1/2 antibody and phosphorylated ERK1/2d2 antibody labeled with Eu Cryptate 20 times;

Take 16 μL of cell lysate supernatant per well into a new 384-well white microplate, then add 2 μL of phosphorylated ERK1/2 antibody dilution labeled with Eu Cryptate and 2 μL of phosphorylated ERK1/2d2 antibody dilution, and incubate at room temperature for 4 hours;

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

data analysis:

The raw data were converted into inhibition rate using the equation (Sample-Min)/(Max-Min)*100%, and the _IC50 value was obtained by four-parameter curve fitting (derived by log(inhibitor) vs.response--Variable slope mode in GraphPad Prism).

Max well: The reading value of the positive control well is 1X lysate

Min well: negative control well reading value is 0.5% DMSO cell well cell lysate

The results of the inhibitory activity of the compounds of the present invention on p-ERK in DLD-1 cells are shown in Table 3.

Table 3 IC ₅₀ test results of the compounds of the present invention on the proliferation of p-ERK in DLD-1 cells

Experimental conclusion: The compounds of the present invention have a significant inhibitory effect on the proliferation of p-ERK in DLD-1 cells.

Experimental Example 3: Fully automated patch clamp (Qpatch) test of hERG potassium channel action

experimental method:

CHO-hERG cells were cultured in a 175 ^cm2 culture flask. When the cell density grew to 60-80%, the culture medium was removed, and the cells were washed once with 7 mL of PBS (phosphate buffered saline), and then 3 mL of cell dissociation reagent was added for digestion. After complete digestion, 7 mL of culture medium was added for neutralization, and then centrifuged, the supernatant was aspirated, and 5 mL of culture medium was added for re-suspending to ensure that the cell density was 2-5×10 ⁶ /mL.

Dilute the compound stock solution with DMSO, take 10μL of the compound stock solution and add it to 20μL DMSO solution, and dilute it 3 times continuously to 6 DMSO concentrations. Take 4μL of the compound of 6 DMSO concentrations respectively, add it to 396μL of extracellular fluid, dilute it 100 times to 6 intermediate concentrations, then take 80μL of the 6 intermediate concentration compounds respectively, add it to 320μL of extracellular fluid, and dilute it 5 times to the final concentration to be tested. The highest test concentration is 40.00μM, and there are 6 concentrations of 40.00, 13.33, 4.44, 1.48, 0.49, and 0.16μM respectively. The DMSO content in the final test concentration does not exceed 0.2%, and this concentration of DMSO has no effect on the hERG potassium channel. The compound preparation is completed by the Bravo instrument throughout the dilution process.

The Qpatch instrument automatically completes the electrophysiological recording process, single-cell high-impedance sealing and whole-cell pattern formation. After obtaining the whole-cell recording mode, the cell is clamped at -80 mV. Before a 5-second +40 mV depolarizing stimulus is given, a 50-millisecond -50 mV pre-voltage is given, and then repolarizes to -50 mV for 5 seconds, and then returns to -80 mV. This voltage stimulus is applied every 15 seconds. After recording for 2 minutes, the extracellular solution is given for 5 minutes, and then the drug administration process begins. The compound concentration starts from the lowest test concentration, and each test concentration is given for 2.5 minutes. At least 3 cells (n≥3) are tested for each concentration.

data analysis:

The experimental data were analyzed by GraphPad Prism 5.0 software.

The results of the inhibitory activity of the compounds of the present invention on hERG potassium channels are shown in Table 3.

Table 3 IC ₅₀ test results of the compounds of the present invention on hERG potassium channel

Experimental conclusion: The compound of the present invention has no obvious inhibitory effect on hERG potassium channel and has a low risk of cardiac toxicity.

Claims

A compound of formula (II), a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

in,

T is selected from

R 1 is selected from H, F, Cl, Br, I, -OH, -NH 2 , -CN and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1, 2, 3 or 4 Ra ;

R 2 is selected from H, F, Cl, Br, I, -OH, -NH 2 , -CN and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1, 2, 3 or 4 R b ;

R 3 is selected from H, F, Cl, Br, I, -OH, -NH 2 , -CN and C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1, 2, 3 or 4 R c ;

Each Ra is independently selected from F, Cl, Br, I, -OH, -NH2 , -CN, -COOH, =O and C1-3 alkyl;

Each R b is independently selected from F, Cl, Br, I, -OH, -NH 2 , -CN, -COOH, =O and C 1-3 alkyl;

Each R c is independently selected from F, Cl, Br, I, -OH, -NH 2 , -CN, -COOH, =O and C 1-3 alkyl.
The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by formula (II-1):

wherein T, R 1 , R 2 and R 3 are as defined in claim 1;

The carbon atom with "*" is a chiral carbon atom, which exists in the form of a single enantiomer (R) or (S) or in the form enriched in one enantiomer.
The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein each R b is independently selected from F and -OH.
The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein R 1 is selected from H, F, Cl, Br and -NH 2 .
The compound according to claim 1, its stereoisomer or its pharmaceutically acceptable salt, wherein R2 is selected from H, F, Cl, Br, -CN, -CH3 , -CH2CH3 , -CH( CH3 ) 2 and -CH2CH ( CH3 ) 2 , wherein said -CH3 , -CH2CH3 , -CH( CH3 ) 2 and -CH2CH ( CH3 ) 2 are each independently optionally substituted with 1, 2, 3 or 4 Rb .
The compound according to claim 5, its stereoisomer or its pharmaceutically acceptable salt, wherein R2 is selected from H, F,
The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein R 3 is selected from H, F, Cl, Br and -NH 2 .
The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

in,

R 2 is selected from C 1-4 alkyl, wherein the C 1-4 alkyl is optionally substituted with 1, 2, 3 or 4 R b ;

R3 is selected from H, F, Cl and Br;

Each R b is independently selected from F and -OH.
The following compound, its stereoisomer or its pharmaceutically acceptable salt, wherein the compound is selected from:
The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:
Use of the compound according to any one of claims 1 to 9, its stereoisomer or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating KRAS mutant solid tumors.