Benzyl aminoquinoline compound and preparation method thereof

Abstract

The invention discloses a benzylaminoquinoline compound and a preparation method thereof, and particularly relates to a compound as shown in a formula (II), and a stereoisomer and pharmaceutically acceptable salt thereof. # imgabs0 #

Description

Benzylaminoquinoline compounds and preparation methods thereof

[0001] This application claims priority to:

[0002] CN202211276424.1, October 18, 2022;

[0003] CN202211497271.3, November 25, 2022. Technical Field

[0004] The present invention relates to a class of benzylaminoquinoline compounds and a preparation method thereof, and particularly to a compound represented by formula (II), its stereoisomers and pharmaceutically acceptable salts thereof. Background Art

[0005] RAS proteins are guanine nucleoside-binding proteins with guanosine triphosphate hydrolase (GTPase) activity. They primarily consist of three isoforms: KRAS, NRAS, and HRAS. As binary molecular switches controlled by the GDP / GTP cycle, RAS proteins can cycle between an active, GTP-bound state (GTP-RAS) and an inactive, GDP-bound state (GDP-RAS). This cycle plays a crucial regulatory role in cells and is closely associated with cell proliferation, survival, metabolism, migration, immunity, and growth.

[0006] 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. SOS1 then 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. Recent studies have suggested that drugs targeting the RAS pathway are prone to developing resistance during clinical use. Some of this resistance stems from the negative feedback loop that occurs when ERK phosphorylation is inhibited, activating the upstream RAS pathway. This negative feedback loop is closely linked to SOS1. Therefore, the development of small molecule inhibitors of SOS1 holds great promise.

[0007] 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:

[0008] Summary of the Invention

[0009] The present invention provides a compound represented by formula (II), a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0010] in,

[0011] T is selected from

[0012] R1 is selected from H, F, Cl, Br, I, -OH, -NH2, -CN and C 1-4 Alkyl, wherein the C 1-4 The alkyl group is optionally substituted with 1, 2, 3 or 4 R a replace;

[0013] R2 is selected from H, F, Cl, Br, I, -OH, -NH2, -CN and C 1-4 Alkyl, wherein the C 1-4 The alkyl group is optionally substituted with 1, 2, 3 or 4 R b replace;

[0014] R3 is selected from H, F, Cl, Br, I, -OH, -NH2, -CN and C 1-4 Alkyl, wherein the C 1-4 The alkyl group is optionally substituted with 1, 2, 3 or 4 R c replace;

[0015] Each R a are independently selected from F, Cl, Br, I, -OH, -NH2, -CN, -COOH, =O and C 1-3 alkyl;

[0016] Each R b are independently selected from F, Cl, Br, I, -OH, -NH2, -CN, -COOH, =O and C 1-3 alkyl;

[0017] Each R c are independently selected from F, Cl, Br, I, -OH, -NH2, -CN, -COOH, =O and C 1-3 alkyl.

[0018] The present invention provides a compound represented by formula (II), a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0019] in,

[0020] T is selected from

[0021] R1 is selected from H, F, Cl and Br;

[0022] R2 is selected from C 1-4 Alkyl, wherein the C 1-4 The alkyl group is optionally substituted with 1, 2, 3 or 4 R b replace;

[0023] R3 is selected from H, F, Cl and Br;

[0024] Each R b are independently selected from F and -OH.

[0025] In some embodiments of the present invention, the above compound has a structure represented by formula (II-1):

[0026] wherein T, R1, R2 and R3 are as defined in the present invention;

[0027] The carbon atom marked with "*" is a chiral carbon atom, existing in the form of a single enantiomer (R) or (S) or in the form enriched in one enantiomer.

[0028] In some embodiments of the present invention, the above compound has a structure represented by formula (II-1):

[0029] wherein T, R1, R2 and R3 are as defined in the present invention.

[0030] In some embodiments of the present invention, the above compound has a structure represented by formula (II-1):

[0031] wherein T, R1, R2 and R3 are as defined in the present invention.

[0032] In some embodiments of the present invention, the above R b are independently selected from F and -OH, and other variables are as defined in the present invention.

[0033] In some embodiments of the present invention, the above R1 is selected from H, F, Cl, Br and -NH2, and other variables are as defined in the present invention.

[0034] In some embodiments of the present invention, the above R1 is selected from H, and other variables are as defined in the present invention.

[0035] In some embodiments of the present invention, the above R2 is selected from H, F, Cl, Br, -CN, -CH3, -CH2CH3, -CH(CH3)2 and -CH2CH(CH3)2, wherein the -CH3, -CH2CH3, -CH(CH3)2 and -CH2CH(CH3)2 are independently optionally replaced by 1, 2, 3 or 4 R b Replacement, R b and other variables are as defined in the present invention.

[0036] In some embodiments of the present invention, the above R2 is selected from -CH3 and -CH2CH(CH3)2, wherein the -CH3 and -CH2CH(CH3)2 are independently optionally replaced by 1, 2, 3 or 4 R b Replacement, R b and other variables are as defined in the present invention.

[0037] In some embodiments of the present invention, the above R2 is selected from -CH2CH(CH3)2, wherein said and -CH2CH(CH3)2 are optionally replaced by 1, 2, 3 or 4 R b Replacement, R b and other variables are as defined in the present invention.

[0038] In some embodiments of the present invention, the above R2 is selected from H, F, Cl, Br, -CN, R b and other variables are as defined in the present invention.

[0039] In some embodiments of the present invention, the above R2 is selected from R b and other variables are as defined in the present invention.

[0040] In some embodiments of the present invention, the above R2 is selected from R b and other variables are as defined in the present invention.

[0041] In some embodiments of the present invention, the above R2 is selected from H, F, Other variables are as defined in the present invention.

[0042] In some embodiments of the present invention, the above R2 is selected from H, Other variables are as defined in the present invention.

[0043] In some embodiments of the present invention, the above R2 is selected from Other variables are as defined in the present invention.

[0044] In some embodiments of the present invention, the above R3 is selected from H, F, Cl, Br and -NH2, and other variables are as defined in the present invention.

[0045] In some embodiments of the present invention, the above R3 is selected from F, and other variables are as defined in the present invention.

[0046] Some other solutions of the present invention are obtained by arbitrarily combining the above variables.

[0047] The present invention provides a compound represented by formula (II), a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0048] in,

[0049] R2 is selected from C 1-4 Alkyl, wherein the C 1-4 The alkyl group is optionally substituted with 1, 2, 3 or 4 R b replace;

[0050] R3 is selected from H, F, Cl and Br;

[0051] Each R b are independently selected from F and -OH.

[0052] The present invention provides a compound represented by formula (II), a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0053] wherein R2 and R3 are as defined in the present invention.

[0054] The present invention provides a compound represented by formula (II), a stereoisomer thereof or a pharmaceutically acceptable salt thereof,

[0055] in,

[0056] R2 is selected from C 1-4 Alkyl, wherein the C 1-4 The alkyl group is optionally substituted with 1, 2, 3 or 4 R b replace;

[0057] R3 is selected from H, F, Cl and Br;

[0058] Each R b are independently selected from F and -OH.

[0059] The present invention provides a compound of the following formula or a pharmaceutically acceptable salt thereof:

[0060] In some embodiments of the present invention, the above-mentioned compound, its stereoisomer or pharmaceutically acceptable salt thereof is selected from the group consisting of:

[0061] The present invention also provides the use of the above-mentioned compound, its stereoisomers or pharmaceutically acceptable salts in the preparation of drugs for treating KRAS mutant solid tumors.

[0062] 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-mentioned compound, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0063] The present invention also provides a biological test method for the above compound:

[0064] Test method 1: H358 cell 3D proliferation inhibition activity test

[0065] Experimental principle:

[0066] In H358 cells harboring the KRAS (G12C) mutation, the KRAS signaling pathway is abnormally activated. Small molecule SOS1 inhibitors inhibit SOS1 binding to RAS proteins, reducing its GEF activity and decreasing the ratio of activated RAS-GTP. This further downregulates phosphorylation of the MEK / ERK pathway downstream of RAS, thereby inhibiting cell proliferation. Small molecules were co-cultured with H358 cells in 3D space, and cellular readouts were used to indirectly assess the inhibitory activity of SOS1 inhibitors on H358 cell proliferation.

[0067] Experimental Materials:

[0068] RPMI1640 medium, fetal bovine serum, and 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).

[0069] Experimental methods:

[0070] 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 each well of the 96-well U-shaped plate. After the bottom layer gel solidified, 2% gel was added to the cell-containing culture medium to prepare a cell-containing top layer gel with a gel concentration of 0.4%. The cell density was 4×10 4 Cells / ml were added to a 96-well U-shaped plate covered with bottom layer gel at a density of 3000 cells per well. After the top layer gel solidified, the cell plate was placed in a CO2 incubator for overnight culture.

[0071] On the day of compound addition, add 85 μL of liquid culture medium to the 96-well U-shaped plate containing the cells. Use a dispenser to dilute the test compound 3-fold to the ninth concentration, from 6 mM to 0.9 μM, in duplicate. Add 97 μL of culture medium to the middle plate. Transfer 2.5 μL of the serially diluted compound to each well of the middle plate, mixing thoroughly before transferring 40 μL to each well of the cell plate. The concentration of the compound transferred to the cell plate ranges from 30 μM to 4.5 nM. Incubate the cell plate in a CO2 incubator for 7 days. On the eighth day, use a dispenser to dilute the test compound 3-fold to the ninth concentration, from 6 mM to 0.9 μM, in duplicate. Add 198 μL of culture medium to the middle plate. Then, transfer 2 μL of the serially diluted compound per well to the first middle plate, following the corresponding position. Then, add 100 μL of culture medium to the second middle plate, add 100 μL of the mixed compound from the first middle plate, mix thoroughly, and transfer 40 μL per well to the cell plate. The concentration of the compound transferred to the cell plate ranges from 30 μM to 4.5 nM. The cell plate is incubated in a CO2 incubator for an additional 7 days. After incubating the cells with the compound for 14 days, add 20 μL of Almar blue detection reagent per well to the cell plate. The plate with dye is shaken on a horizontal shaker for 15 minutes, then incubated at room temperature for 5 hours to allow the luminescence signal to stabilize. The results are read using a multilabel analyzer.

[0072] Data Analysis:

[0073] The raw data were converted into inhibition rate, IC using the equation (Sample-Min) / (Max-Min)×100%. 50 The value can be obtained by four-parameter curve fitting (obtained in "log(inhibitor)vs.response--Variable slope" mode in GraphPad Prism).

[0074] Experimental conclusion: The compound of the present invention can inhibit the proliferation of H358 cells under 3D conditions.

[0075] Experimental Test Method 2: Compound Pharmacokinetic Evaluation

[0076] Experimental Materials:

[0077] CD-1 mice (male, 7-9 weeks old, Shanghai Slack)

[0078] Experimental operation:

[0079] The pharmacokinetic properties of the compounds were tested in rodents following intravenous and oral administration using a standard protocol. The candidate compounds were formulated as clear solutions and administered to mice as single intravenous and oral injections. The intravenous and oral vehicles were a mixture of 10% dimethyl sulfoxide and 90% 10% hydroxypropyl β-cyclodextrin in water. Four female CD-1 mice were used in this project. Two mice were intravenously injected with 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 hours after administration. The other two mice were orally gavaged with a dose of 2 mg / kg, and plasma samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 12 hours after administration. The samples were stirred at 3,200×g at 4°C for 10 minutes, and the supernatant was separated to obtain plasma samples. 20 times the volume of methanol solution containing internal standard was added to precipitate the protein, stirred at 12,000×g for 15 minutes, and centrifuged at 4°C. 50 μL of the supernatant was transferred to a 96-well plate for a second centrifugation. The blood drug concentration was quantitatively analyzed by LC-MS / MS analysis, and pharmacokinetic parameters such as peak concentration (C max ), clearance (CL), half-life (T 1 / 2 ), tissue distribution (Vdss), area under the drug-time curve (AUC 0-last ), bioavailability (F), etc.

[0080] Experimental conclusion: The compounds of the present invention have good pharmacokinetic properties, including good oral bioavailability, oral exposure, half-life and clearance rate.

[0081] Experimental test method 3: In vivo efficacy evaluation of the compound in the Miapaca2 nude mouse xenograft tumor model

[0082] Cell culture:

[0083] Human pancreatic cancer cells (ATCC) were cultured as adherent monolayers in DMEM supplemented with 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Cells were routinely digested and passaged two to three times weekly using trypsin-EDTA. When cell saturation reached 80%–90% and the desired number of cells was reached, cells were harvested, counted, and plated.

[0084] Experimental animals:

[0085] Balb / c nude mice, female, 6-7 weeks old, were purchased from Shanghai Xipul-Bikai Laboratory Animal Co., Ltd.

[0086] Model preparation:

[0087] 0.2 mL (5 × 10 6Miapaca2 cells (with Matrigel, volume ratio of 1:1) were subcutaneously inoculated on the right back of each mouse, and the average tumor volume reached 118 mm 3 The group dosing started at 14:00.

[0088] Tumor measurements and experimental parameters:

[0089] Tumor diameter was measured twice a week with a vernier caliper, and tumor volume was measured in cubic millimeters and calculated using the following formula: V = 0.5a × b 2 , where a and b are the long and short diameters of the tumor, respectively. The antitumor efficacy of the test compound was evaluated using the TGI (%). TGI (%) reflects the rate of tumor growth inhibition. TGI (%) = [1 – (average tumor volume of a treatment group at the end of dosing – average tumor volume of the treatment group at the start of dosing) / (average tumor volume of the solvent control group at the end of treatment – ​​average tumor volume of the solvent control group at the start of treatment)] × 100%.

[0090] 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.

[0091] Technical Effects

[0092] The compound of the present invention has good KRAS (G12C)-SOS1 binding inhibitory activity, and has significant inhibitory activity against 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 has excellent activity in inhibiting tumor growth.

[0093] Definition and Description

[0094] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be construed as indefinite or unclear unless specifically defined, but rather should be understood in accordance with its ordinary meaning. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0095] The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0096] The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the 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 such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; 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) and organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

[0097] Pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of the two.

[0098] The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are encompassed within the scope of the present invention.

[0099] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0100] Unless otherwise indicated, the term "cis-trans isomers" or "geometric isomers" arises from the inability to rotate freely about double bonds or single bonds forming ring carbon atoms.

[0101] 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.

[0102] Unless otherwise indicated, "(+)" indicates dextrorotatory, "(-)" indicates levorotatory, and "(±)" indicates racemic.

[0103] Unless otherwise specified, use a solid wedge key. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed bond Indicate the relative configuration of stereocenters with a wavy line Indicates a wedge-shaped solid key or dotted wedge key Or use a wavy line Indicates a straight solid bond and straight dashed bond

[0104] The compounds of the present invention may exist in specific forms. Unless otherwise indicated, the term "tautomer" or "tautomeric form" refers to isomers with different functional groups that are in dynamic equilibrium at room temperature and can quickly convert into each other. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be achieved. For example, proton tautomers (also known as prototropic tautomers) include interconversions that occur through proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions that occur through the reorganization of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

[0105] 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 that 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%.

[0106] 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%.

[0107] Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting 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 resolved by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

[0108] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C). For example, deuterated drugs can be formed by replacing hydrogen with heavy hydrogen. The bond between deuterium and carbon is stronger than the bond between ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of this invention.

[0109] 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.

[0110] 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.

[0111] The term "optionally substituted" means that the group may be substituted or not substituted, and unless otherwise specified, the type and number of the substituents may be any based on chemical practicability.

[0112] When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and / or their variants are permissible only if such combinations result in stable compounds.

[0113] When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

[0114] When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a single bond, it means that the structure is actually AZ.

[0115] When a substituent is vacant, it means that the substituent does not exist. For example, when X in AX is vacant, it means that the structure is actually A. When the substituent is listed without specifying which atom it is connected to the substituted group, the substituent can be bonded through any atom of the substituent. For example, a pyridyl substituent can be connected to the substituted group through any carbon atom on the pyridine ring.

[0116] When the listed linking groups do not specify their linking direction, their linking direction is arbitrary, for example, The connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of reading from left to right to form Combinations of linkers, substituents, and / or variations thereof are permissible only if such combinations result in stable compounds.

[0117] Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of the group can be connected to other groups through chemical bonds. When the chemical bond connection mode is non-positional and there are H atoms at the connectable sites, when the chemical bond is connected, the number of H atoms at the site will decrease accordingly with the number of connected chemical bonds, and become a group with a corresponding valence. The chemical bond connecting the site to other groups can be a straight solid bond. Straight dotted key or wavy lines For example, the straight solid bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group; The straight dashed bond in the group indicates that the two ends of the nitrogen atom in the group are connected to other groups; The wavy lines in the phenyl group represent the connection to other groups through the carbon atoms at positions 1 and 2 in the phenyl group. Indicates that any linkable site on the piperidinyl group can be connected to other groups through a chemical bond, including at least In these four connection methods, even if an H atom is drawn on -N-, Still includes For groups connected in this way, when one chemical bond is connected, the H at that site will be reduced by one and become a corresponding monovalent piperidine group.

[0118] 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 or cyclopentyl group.

[0119] Unless otherwise specified, the number of atoms in a ring is generally defined as the number of members of the ring, for example, a "5-7 membered ring" refers to a "ring" having 5-7 atoms arranged around it.

[0120] Unless otherwise specified, C n-n+m or C n -C n+m Any specific case including n to n+m carbons, such as C 1-12 Including C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 、C 11 , and C 12 , also includes any range from n to n+m, such as C 1-12 Including C 1- 3. C 1-6 、C 1-9 、C 3-6 、C 3-9 、C 3-12 、C 6-9 、C 6-12 , and C 9-12Similarly, n-membered to n+m-membered means that the number of atoms in the ring is n to n+m, for example, a 3-12-membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, an 11-membered ring, and a 12-membered ring, and also includes any range from n to n+m, for example, a 3-12-membered ring includes a 3-6-membered ring, a 3-9-membered ring, a 5-6-membered ring, a 5-7-membered ring, a 6-7-membered ring, a 6-8-membered ring, and a 6-10-membered ring, etc.

[0121] Unless otherwise specified, the term "alkyl" by itself or as part of another substituent refers to a straight or branched chain saturated hydrocarbon group. 1-6 Alkyl or C 1-3 Alkyl. The alkyl group is optionally substituted with one or more of the following: 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.

[0122] Unless otherwise specified, the term “C 1-4 "Alkyl" is used to represent a straight or branched chain saturated hydrocarbon group consisting of 1 to 4 carbon atoms. 1-4 Alkyl groups include C 1-2 、C 1-3 and C 2-3 Alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). 1-4 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), and the like.

[0123] Unless otherwise specified, the term “C 1-3 "Alkyl" is used to represent a straight or branched chain saturated hydrocarbon group consisting of 1 to 3 carbon atoms. 1-3 Alkyl groups include C 1-2 and C 2-3 Alkyl, etc.; it can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine). 1- Examples of 3-alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0124] 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 methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-toluenesulfonate, etc.; acyloxy groups such as acetoxy and trifluoroacetoxy, etc.

[0125] 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)methyl; silyl, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing side reactions at 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 (e.g., 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.

[0126] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0127] The structures of the compounds of the present invention can be confirmed by conventional methods well known to those skilled in the art. If the present invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed by conventional techniques in the art. For example, single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data from a cultured single crystal using a Bruker D8venture diffractometer, using CuKα radiation and a φ / ω scanning mode. After collecting relevant data, the crystal structure can be further analyzed using a direct method (Shelxs97) to confirm the absolute configuration.

[0128] The volumes used in the present invention are commercially available.

[0129] 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; Boc2O represents di-tert-butyl dicarbonate; NH4Cl represents ammonium chloride; T3P represents 1-propylphosphonium Acid tricyclic anhydride; Pd / C represents palladium / carbon catalyst; TMSN3 represents trimethylsilyl azide; 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-d6 represents deuterated dimethyl sulfoxide; CD3OD represents deuterated methanol; CDCl3 represents deuterated chloroform; and D2O represents deuterated water.

[0130] Compounds are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names. DETAILED DESCRIPTION

[0131] The present invention is described in detail below by examples, but it is not intended to limit the present invention in any 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 combining them with other chemical synthesis methods, and equivalent replacement modes well known to those skilled in the art. Preferred embodiments include but are not limited to the embodiments of the present invention. It will be apparent to those skilled in the art that various changes and modifications will be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

[0132] Intermediate A

[0133] Synthesis route:

[0134] first step

[0135] 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. The reaction solution was cooled to 25°C and filtered. 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 (400MHz, CD3OD) δ7.55(s,1H),7.07(s,1H),3.98(s,3H),3.95(s,3H),2.45(s,3H).

[0136] Step 2

[0137] Compound A-2 (3.00 g, 13.6 mmol) was dissolved in methanesulfonic acid (15 mL), and 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, and the filter cake was collected and vacuum dried to obtain intermediate A. 1 H NMR (400MHz, DMSO-d6) δ7.33(s,1H),7.02(s,1H),3.88(s,3H),2.88(s,3H).

[0138] Intermediate B

[0139] Synthesis route:

[0140] first step

[0141] Dissolve B-2 (3.37 g, 16.6 mmol) in dimethyl sulfoxide (20 mL), and add copper powder (1.06 g, 16.6 mmol). The reaction mixture is stirred at 25°C for 1 hour. Compound B-1 (2.00 g, 6.65 mmol) is then added to the reaction mixture, and the mixture is stirred at 70°C for 12 hours. The reaction mixture is poured into 20 mL of ice water, and ethyl acetate (20 mL) is added. The mixture is filtered, and the filtrate is extracted with ethyl acetate (20 mL x 3). The organic phase is washed with saturated brine (20 mL x 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue is purified by silica gel column chromatography (petroleum ether / ethyl acetate, 1 / 0 to 20 / 1, v / v) to obtain compound B-3. 1H NMR (400MHz, CDCl3) δ7.75-7.68(m,1H),7.64-7.57(m,1H),7.16(t,J=8.0Hz,1H),4.38(m,2H),1.34(t,J=8.0Hz,3H).

[0142] Step 2

[0143] Under nitrogen, compound B-3 (677 mg, 1.82 mmol) was dissolved in toluene (10 mL), and methylmagnesium bromide solution (compound B-4) (3 M, 2.43 mL) was added at 0°C. The reaction mixture was stirred at 25°C for 2 hours. The mixture was quenched by the addition of saturated ammonium chloride solution (10 mL) and extracted with ethyl acetate (10 mL x 2). The organic phase was washed with saturated brine (10 mL x 2), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was isolated and purified by silica gel column chromatography (petroleum ether / ethyl acetate, 1 / 0 to 10 / 1, v / v) to obtain compound B-5. 1 H NMR (400MHz, CDCl3) δ7.66 (t, J = 6.4Hz, 1H), 7.51-7.34 (m, 1H), 7.16-6.93 (m, 1H), 2.01 (s, 1H), 1.35 (s, 6H).

[0144] Step 3

[0145] Under nitrogen, compound B-5 (441 mg, 1.56 mmol) was dissolved in toluene (5 mL). Compound B-6 (1.87 g, 5.19 mmol) and bistriphenylphosphine palladium dichloride (109 mg, 0.16 mmol) were added. The reaction mixture was stirred at 120°C for 12 hours. The mixture was quenched by the addition of saturated potassium fluoride solution (20 mL) and extracted with ethyl acetate (15 mL x 2). The mixture was filtered and concentrated under reduced pressure to obtain compound B-7.

[0146] Step 4

[0147] Under nitrogen, compound B-7 (425 mg, 1.55 mmol) was dissolved in acetone (10 mL). Hydrochloric acid solution (12 M, 1.03 mL) was added dropwise at 0°C, and the mixture was stirred at 25°C for 1 hour. Saturated sodium bicarbonate solution was added to neutralize the mixture to pH 8, and the mixture was 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 isolated 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, measured value 247.

[0148] Step 5

[0149] 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 mixture was stirred at 80°C for 36 hours. Sodium borohydride (64.0 mg, 1.69 mmol) was then added to the reaction mixture at -5°C, and the reaction was stirred at 25°C for 1 hour. The reaction mixture was poured into 20 mL of ice water, filtered, and the filtrate was extracted with ethyl acetate (5 mL x 2). The organic phase was washed with saturated brine (5 mL x 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was isolated 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.

[0150] Step 6

[0151] Compound B-10 (366 mg, 1.04 mmol) was dissolved in dioxane (2.5 mL), and a 4 M solution of hydrogen chloride in dioxane (1.15 mL) was added. The reaction mixture was stirred at 25°C for 6 hours. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane / methanol, 1 / 0 to 8 / 1, v / v) to obtain the hydrochloride salt of intermediate B. The hydrochloride salt of intermediate B was added to 1 M NaOH (5 mL) and extracted with ethyl acetate (5 mL x 2). The organic phase was washed with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and dried to obtain intermediate B.

[0152] MS-ESI calculated value [M+H] + 248, measured value 248.

[0153] Example 1

[0154] Synthesis route:

[0155] first step

[0156] Intermediate A (300 mg, 2.91 mmol) was dissolved in tetrahydrofuran (5 mL), followed by the addition of compound 1-1 (674 mg, 3.35 mmol) and N,N-diisopropylethylamine (1.13 g, 8.73 mmol). The reaction mixture was stirred at 25°C for 1 hour. The reaction mixture was poured into water (20 mL) and filtered. The filtrate was extracted with ethyl acetate (20 mL x 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield compound 1-2.

[0157] Step 2

[0158] Compound 1-2 (500 mg, 1.35 mmol) was dissolved in N,N-dimethylformamide (5 mL), and triethylamine (940 μL, 6.75 mmol) and compound 1-3 (541 mg, 2.70 mmol) were added dropwise. The mixture was stirred at 25°C for 12 hours. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by 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, measured value 433.

[0159] Step 3

[0160] 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. The mixture was stirred at 25°C for 12 hours. The reaction mixture was filtered and concentrated under reduced pressure. 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, measured value 699.

[0161] Step 4

[0162] 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. 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 purified by 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, measured value 662.

[0163] Step 5

[0164] Compound 1-7 (540 mg, 816 μmol) was dissolved in ethyl acetate (5 mL), and a hydrogen chloride / ethyl acetate solution (4 mol / L, 8.31 mL) was added. The mixture was stirred at 25°C for 1 hour. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative HPLC (column: Phenomenex C18 150 mm × 40 mm × 5 μm; mobile phase: 0.05% aqueous hydrochloric acid-acetonitrile; gradient: acetonitrile 10%-40% over 10 min) to obtain the hydrochloride salt of compound 1. 1 H NMR (400 MHz, CD3OD) 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 calcd. [M+H] + 562, measured value 562.

[0165] Biological activity:

[0166] Experimental Example 1: KRAS(G12C) and SOS1 binding experiment

[0167] Experimental principle:

[0168] Small molecule compounds bind to the catalytic site of SOS1, inhibiting the binding of SOS1 to KRAS(G12C). When the binding of fluorescently labeled SOS1 to fluorescently labeled KRAS(G12C) is inhibited, the emitted fluorescence changes. By measuring this fluorescence change, the ability of small molecules to inhibit the binding of SOS1 to KRAS(G12C) can be tested. A homogeneous time-resolved fluorescence (HTRF) binding assay was used to test the ability of the compounds of the present invention to inhibit the binding of SOS1 to KRAS(G12C).

[0169] Experimental Materials:

[0170] 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, and Mab Antibody 6HIS-XL665 and Mab Antibody GST-Eμcryptate were purchased from Cisbio. A multifunctional microplate reader Nivo5 was purchased from PerkinElmer.

[0171] Experimental methods:

[0172] 1X buffer preparation (prepared and used immediately): Hepes: 5mM; NaCl: 150mM; EDTA: 10mM; Igepal: 0.0025%; KF: 100mM; DTT: 1mM; BSA: 0.05%;

[0173] 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.

[0174] Dilute the test compound to a 2% DMSO working solution using 1X buffer. Add 5 μL / well to the corresponding wells, with a concentration gradient from 20 μM to 0.00128 nM. Perform the experiment in duplicate. Centrifuge at 1000 rpm for 1 minute.

[0175] 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.

[0176] A working solution of SOS1 (80 nM) and Mab Antibody 6HIS-XL665 (8 g / μL) was prepared in 1X buffer and added to the corresponding wells at 2.5 μL / well. In blank wells, 2.5 μL of the Mab Antibody 6HIS-XL665 (8 g / μL) dilution was added. The final compound concentration gradient was diluted from 10 μM to 0.64 nM: KRAS (G12C) (500 nM), Mab Antibody GST-Eu cryptate (0.25 ng / μL), SOS1 (20 nM), and Mab Antibody 6HIS-XL665 (2 g / μL). The reaction system was incubated at 25°C for 60 minutes. After completion of the reaction, HTRF was read using a multilabel analyzer.

[0177] Data Analysis:

[0178] The raw data were converted into inhibition rate, IC using the equation (Sample-Min) / (Max-Min)×100%. 50 The value of can be obtained by four-parameter curve fitting (derived using the 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.

[0179] Table 1 IC values ​​of the compounds of the present invention for binding to KRAS (G12C) and SOS1 50 Value test results

[0180] Experimental conclusion: The compound of the present invention has a significant inhibitory effect on the binding of KRAS (G12C) and SOS1.

[0181] Experimental Example 2: DLD-1 cell p-ERK proliferation inhibition activity test

[0182] Experimental Materials:

[0183] DLD-1 cells were purchased from Nanjing Kebai; 1640 culture medium was purchased from Biological Industries; fetal bovine serum was purchased from Biosera; and Advanced Phospho-ERK1 / 2 (THR202 / TYR204) KIT was purchased from Cisbio. See Table 2 for the composition of Advanced Phospho-ERK1 / 2 (THR202 / TYR204) KIT.

[0184] Table 2. Ingredients of Advanced Phospho-ERK1 / 2 (THR202 / TYR204) KIT

[0185] Experimental methods:

[0186] 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 8,000 DLD-1 cells. The cell plate was placed in a CO2 incubator and incubated overnight at 37°C.

[0187] Dilute the test compound to 2 mM with 100% DMSO as the first concentration, then pipette a further 5-fold dilution to the eighth concentration, from 2 mM to 0.026 μM. Add 2 μL of compound to 78 μL of cell starvation medium and mix thoroughly. Then, add 20 μL of compound solution to the corresponding well of the cell plate. Return the cell plate to the CO2 incubator and incubate for another 1 hour. At this point, the compound concentration ranges from 10 μM to 0.128 nM, and the DMSO concentration is 0.5%.

[0188] 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;

[0189] Use detection buffer to dilute the Eu Cryptate-labeled phosphorylated ERK1 / 2 antibody and phosphorylated ERK1 / 2d2 antibody 20 times;

[0190] 16 μL of cell lysate supernatant was transferred to each well of a new 384-well white microplate. 2 μL of Eu Cryptate-labeled phosphorylated ERK1 / 2 antibody dilution and 2 μL of phosphorylated ERK1 / 2d2 antibody dilution were added and incubated at room temperature for 4 hours.

[0191] After the incubation, a multi-label analyzer was used to read the HTRF excitation wavelength: 320 nm, emission wavelength: 615 nm, 665 nm.

[0192] Data Analysis:

[0193] The raw data were converted into inhibition rate, IC, using the equation (Sample-Min) / (Max-Min)*100%. 50 The value can be obtained by four-parameter curve fitting (obtained by log(inhibitor) vs.response--Variable slope mode in GraphPad Prism).

[0194] Max well: Positive control well reading is 1X lysate

[0195] Min well: negative control well reading value is 0.5% DMSO cell well cell lysate

[0196] 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.

[0197] Table 3 IC values ​​of the compounds of the present invention on the proliferation of p-ERK in DLD-1 cells 50 Value test results

[0198] Experimental conclusion: The compound of the present invention has a significant inhibitory effect on the proliferation of p-ERK in DLD-1 cells.

[0199] Experimental Example 3: Fully Automated Patch Clamp (Qpatch) Test of hERG Potassium Channel Effects

[0200] Experimental methods:

[0201] CHO-hERG cells were cultured at 175 cm 2When the cell density in the culture flask reaches 60-80%, remove the culture medium, wash once with 7 mL PBS (phosphate buffered saline), and then add 3 mL cell dissociation reagent for digestion. After digestion is complete, add 7 mL culture medium to neutralize, then centrifuge, aspirate the supernatant, and add 5 mL culture medium to resuspend to ensure that the cell density is 2-5×10 6 / mL.

[0202] Compound stock solutions were diluted with DMSO. 10 μL of the stock solution was added to 20 μL of DMSO solution and serially diluted 3-fold to six DMSO concentrations. 4 μL of each of the six DMSO concentrations was added to 396 μL of extracellular fluid and diluted 100-fold to six intermediate concentrations. 80 μL of each of the six intermediate concentrations was then added to 320 μL of extracellular fluid and diluted 5-fold to the desired final concentration. The highest concentration tested was 40.00 μM, followed by six concentrations of 40.00, 13.33, 4.44, 1.48, 0.49, and 0.16 μM. The DMSO content in the final test concentration did not exceed 0.2%, as this concentration has no effect on hERG potassium channels. Compound preparation was performed using the Bravo instrument throughout the entire dilution process.

[0203] The Qpatch instrument automatically performed the electrophysiological recording process, including single-cell high-impedance sealing and whole-cell pattern formation. After acquiring the whole-cell recording mode, the cell was clamped at -80 mV. A 50-millisecond pre-voltage of -50 mV was applied before a 5-second depolarizing stimulus of +40 mV. The cell then repolarized to -50 mV for 5 seconds before returning to -80 mV. This voltage stimulus was applied every 15 seconds. After recording for 2 minutes, extracellular solution was added for 5 minutes. Drug administration then began. Compound concentrations were administered for 2.5 minutes at each test concentration, starting with the lowest tested concentration. At least three cells (n ≥ 3) were tested for each concentration.

[0204] Data Analysis:

[0205] The experimental data were analyzed by GraphPad Prism 5.0 software.

[0206] The results of the inhibitory activity of the compounds of the present invention on hERG potassium channels are shown in Table 3.

[0207] Table 3 IC of the compounds of the present invention on hERG potassium channel 50 Value test results

[0208] Experimental conclusion: The compound of the present invention has no obvious inhibitory effect on hERG potassium channel and has a low risk of cardiotoxicity.

Claims

1. A compound of formula (II), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, in, T is selected from R1 is selected from H, F, Cl, Br, I, -OH, -NH2, -CN and C 1-4 Alkyl, wherein the C 1-4 The alkyl group is optionally substituted with 1, 2, 3 or 4 R a replace; R2 is selected from H, F, Cl, Br, I, -OH, -NH2, -CN and C 1-4 Alkyl, wherein the C 1-4 The alkyl group is optionally substituted with 1, 2, 3 or 4 R b replace; R3 is selected from H, F, Cl, Br, I, -OH, -NH2, -CN and C 1-4 Alkyl, wherein the C 1-4 The alkyl group is optionally substituted with 1, 2, 3 or 4 R c replace; Each R a are independently selected from F, Cl, Br, I, -OH, -NH2, -CN, -COOH, =O and C 1-3 alkyl; Each R b are independently selected from F, Cl, Br, I, -OH, -NH2, -CN, -COOH, =O and C 1-3 alkyl; Each R c are independently selected from F, Cl, Br, I, -OH, -NH2, -CN, -COOH, =O and C 1-3 alkyl.

2. The compound according to claim 1, its stereoisomer or pharmaceutically acceptable salt thereof, wherein the compound has the structure represented by formula (II-1): in, T, R1, R2 and R3 are as defined in claim 1; The carbon atoms marked with "*" are chiral carbon atoms, existing in the form of (R) or (S) single enantiomers or in the form enriched in one enantiomer.

3. The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein Each R b are independently selected from F and -OH.

4. The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein R1 is selected from H, F, Cl, Br and -NH2.

5. The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, 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 replaced by 1, 2, 3 or 4 R b replace.

6. The compound according to claim 5, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein R2 is selected from H, F, 7. The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein R3 is selected from H, F, Cl, Br and -NH2.

8. The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: in, R2 is selected from C 1-4 Alkyl, wherein the C 1-4 The alkyl group is optionally substituted with 1, 2, 3 or 4 R b replace; R3 is selected from H, F, Cl and Br; Each R b are independently selected from F and -OH.

9. A compound of the following formula, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

10. The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

11. Use of the compound according to any one of claims 1 to 9, its stereoisomers or pharmaceutically acceptable salts thereof in the preparation of a drug for treating KRAS mutant solid tumors.

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