quinazoline compounds

Abstract

The quinazoline compounds and pharmaceutically acceptable salts thereof, in particular, relate to compounds of formula (II) and pharmaceutically acceptable salts thereof.

Description

[0001] This application claims priority to:

[0002] CN202010723467.4, filed on July 24, 2020;

[0003] CN202110653818.3, filed on June 11, 2021. TECHNICAL FIELD

[0004] The present application relates to a class of quinazoline compounds and pharmaceutically acceptable salts thereof, and the use of the compound or its pharmaceutically acceptable salt in the preparation of a drug for treating cancer. BACKGROUND

[0005] Mutations of RAS protein occur in about 20% to 30% of cancer cases, and are the most common mutant oncogenes in pancreatic cancer, colorectal cancer and non-small cell lung cancer. RAS protein acts as a molecular switch, which is in an activated state when combined with GTP and in an inactivated state when combined with GDP. Mutations of RAS protein reduce its ability to hydrolyze GTP, keeping the molecular switch in an active GTP binding state, driving unchecked oncogenic downstream signaling, such as through the RAS-RAF-MEK-ERK pathway and the RAS-P13K-PDK1-AKT pathway, to promote the survival and proliferation of cancer cells.

[0006] SOS1 protein is a class of guanine nucleotide exchange factors (GEF), and studies have shown that this class of proteins can catalyze the binding of KRAS to GTP, thereby promoting the activation of KRAS. Therefore, targeting the inhibition of SOS1 protein, the interaction of SOS1-KRAS, regardless of the mutation of KRAS, becomes a potential tumor treatment target. In addition, studies have shown that combined MEK inhibition can lead to deep pathway arrest and tumor regression in vivo, and is a potential method for treating most KRAS-driven cancers. SUMMARY

[0007] The present application provides a compound of formula (II) or a pharmaceutically acceptable salt thereof,

[0008]

[0009] wherein,

[0010] ring A is selected from phenyl and 5-membered heteroaryl;

[0011] R1is selected from H and NH2;

[0012] R2is selected from C 1-3 alkyl, said C 1-3 alkyl is optionally substituted with 1, 2, 3, 4, or 5 R a substituents;

[0013] R3 is selected from H and F;

[0014] R4 is selected from C 3-8 Cycloalkyl and 3-8 membered heterocycloalkyl, the C 3-8 Cycloalkyl and 3-8 membered heterocycloalkyl are each independently optionally substituted by 1, 2, 3 or 4 R b replace;

[0015] R5 is selected from H, Cl and CH3;

[0016] R a Each is independently selected from D, F, Cl, Br, I, OH, CN, NH2, CH3, -CH2NH2, -NHCH3, -N(CH3)2, -OCH3, -COOH, -COOCH3 and cyclopropyl;

[0017] R b are independently selected from H, F, Cl, Br, I, OH, CN, NH2, C 1-3 Alkyl, -CH2NH2, -NHCH3, -N(CH3)2, C 1-3 Alkoxy, -COOH, -COO-C 1-3 Alkyl, -C(=O)-C 1-3 Alkyl, -S(=O)2-C 1-3 Alkyl and cyclopropyl, the C 1-3 Alkyl, C 1-3 Alkoxy and cyclopropyl are each independently optionally substituted with 1, 2 or 3 R;

[0018] R is each independently selected from F, Cl, Br, I, OH, CN, NH2, -OCH3 and -COOH.

[0019] The present invention also provides a compound of formula (I) or a pharmaceutically acceptable salt thereof,

[0020]

[0021] in,

[0022] R1 is selected from H and NH2;

[0023] R2 is selected from C 1-3 Alkyl, the C 1-3 The alkyl group is optionally substituted with 1, 2, 3, 4 or 5 R a replace;

[0024] R3 is selected from H and F;

[0025] R4 is selected from C 3-8 Cycloalkyl and 3-8 membered heterocycloalkyl, the C 3-8cycloalkyl and 3-8 membered heterocycloalkyl are each independently optionally substituted with 1, 2, 3, or 4 R b substituted;

[0026] R a are each independently selected from the group consisting of D, F, Cl, Br, I, OH, CN, NH2, CH3, -CH2NH2, -NHCH3, -N(CH3)2, -OCH3, -COOH, -COOCH3, and cyclopropyl;

[0027] R b are each independently selected from the group consisting of H, F, Cl, Br, I, OH, CN, NH2, C 1-3 alkyl, -CH2NH2, -NHCH3, -N(CH3)2, -OCH3, -COOH, -COOCH3, and cyclopropyl, C 1-3 alkyl and cyclopropyl are each independently optionally substituted with 1, 2, or 3 R;

[0028] R is independently selected from the group consisting of F, Cl, Br, I, OH, CN, NH2, and -COOH.

[0029] In some embodiments of the application, the above R1 is selected from NH2, and the other variables are as defined in the application.

[0030] In some embodiments of the application, the above R1 is selected from H, and the other variables are as defined in the application.

[0031] In some embodiments of the application, the above R2 is selected from CF3, and the other variables are as defined in the application.

[0032] In some embodiments of the application, the above R2 is selected from CF3, CHF2, and the other variables are as defined in the application.

[0033] In some embodiments of the application, the above R4 is selected from cyclopropyl, bicyclo[2.2.2]octane, 5-6 membered heterocycloalkyl, and 8 membered heterocycloalkyl, each of which is independently optionally substituted with 1, 2, 3, or 4 R b , and the other variables are as defined in the application.

[0034] In some embodiments of the application, the above R4 is selected from cyclopropyl, bicyclo[2.2.2]octane, and 5-6 membered heterocycloalkyl, each of which is independently optionally substituted with 1, 2, 3, or 4 R b , and the other variables are as defined in the application.

[0035] In some embodiments of the application, the above R4 is selected from the are each independently optionally substituted with 1, 2, 3, or 4 R b substituted, and the other variables are as defined herein.

[0036] In some embodiments of the application, the above R4is selected from the are each independently optionally substituted with 1, 2, 3, or 4 R b substituted, and the other variables are as defined herein.

[0037] In some embodiments of the application, the above R4is selected from the are each independently optionally substituted with 1, 2, 3, or 4 R b substituted, and the other variables are as defined herein.

[0038] In some embodiments of the application, the above R b are each independently selected from H, F, Cl, Br, I, OH, CN, NH2, CH3, -CH2CH3, -CH2NH2, -NHCH3, -N(CH3)2, -OCH3, -COOH, -COOCH3, and the other variables are as defined herein.

[0039] In some embodiments of the application, the above R b are each independently selected from H, F, Cl, Br, I, OH, CN, NH2, CH3, -CH2CH3, -CH2NH2, -NHCH3, -N(CH3)2, -OCH3, -COOH, -COOCH3,

[0040] In some embodiments of the application, the above R4is selected from and the other variables are as defined herein.

[0041] In some embodiments of the application, the above R4is selected from and the other variables are as defined herein.

[0042] In some embodiments of the application, the above R4is selected from and the other variables are as defined herein.

[0043] In some embodiments of the application, ring A is selected from phenyl and thienyl, and the other variables are as defined in the application.

[0044] In some embodiments of the application, the compound is selected from

[0045]

[0046] wherein ring A, R1, R2, R3, R4, R5, and R b as defined in the application.

[0047] In some embodiments of the application, the compound is selected from

[0048] wherein R1, R2, R3, R b and R5 are as defined in the application.

[0049] In some embodiments of the application, the compound is selected from

[0050] wherein R1, R2, R3, and R5 are as defined in the application.

[0051] In some embodiments of the application, the compound is selected from

[0052]

[0053] wherein R b as defined in the application.

[0054] The application also provides a use of the above compound, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for inhibiting SOS1 protein.

[0055]

[0056] In some embodiments of the application, the compound is selected from

[0057]

[0058]

[0059]

[0060] The application also provides a use of the above compound, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for inhibiting SOS1 protein.

[0061] The application also provides a use of the above compound, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating lung cancer, pancreatic cancer, and / or rectal cancer.

[0062] Some technical solutions of the present invention are obtained by freely combining the above variables.

[0063] Technical Effects

[0064] The compounds of the present invention have good KRAS (G12C)-SOS1 binding inhibitory activity.

[0065] Definition and Description

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

[0072] 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.

[0073] 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.

[0074] Unless otherwise indicated, "(+)" indicates dextrorotatory, "(-)" indicates levorotatory, and "(±)" indicates racemic.

[0075] 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 key 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 or straight dashed key

[0076] 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 998%, or greater than or equal to 99.9%.

[0077] 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%.

[0078] 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).

[0079] Unless otherwise specified, D in the present invention refers to deuterium ( 2 H).

[0080] 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.

[0081] 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 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.

[0082] 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 feasibility.

[0083] 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.

[0084] When the substituents listed do not specify through which atom they are connected to the substituted group, such substituents can be bonded through any atom thereof. For example, a pyridyl substituent can be connected to the substituted group through any carbon atom on the pyridine ring.

[0085] 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 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; Indicates that any linkable site on the piperidinyl group can be connected to other groups via 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.

[0086] 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.

[0087] 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). 1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), and the like.

[0088] Unless otherwise specified, the term “C 1-3 "Alkoxy" refers to those alkyl groups containing 1 to 3 carbon atoms which are attached to the rest of the molecule via an oxygen atom. The C 1-3 Alkoxy groups include C 1-2 、C 2-3 , C3 and C2 alkoxy, etc. 1-3 Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.

[0089] Unless otherwise specified, the term "5-membered heteroaryl" and "5-membered heteroaromatic ring" are used interchangeably in the present invention. The term "5-membered heteroaryl" refers to a monocyclic group with a conjugated π electron system consisting of 5 ring atoms, wherein 1, 2, 3 or 4 ring atoms are heteroatoms independently selected from O, S and N, and the rest are carbon atoms. The nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O) p , p is 1 or 2). The 5-membered heteroaryl group may be attached to the rest of the molecule via a heteroatom or carbon atom. Examples of the 5-membered heteroaryl group include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2 H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl and 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazolyl and 5-thiazolyl, etc.), furyl (including 2-furyl and 3-furyl, etc.), thienyl (including 2-thienyl and 3-thienyl, etc.). Unless otherwise specified, “C 3-8 "Cycloalkyl" refers to a saturated cyclic hydrocarbon group consisting of 3 to 8 carbon atoms, including monocyclic and bicyclic systems, wherein the bicyclic system includes spirocyclic, fused and bridged rings. 3-8 Cycloalkyl groups include C 3-6 、C 3-5 、C 4-8、C 4-6 、C 4-5 、C 5-8 or C 5-6 Cycloalkyl, etc.; it may be monovalent, divalent or polyvalent. 3-8 Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, [2.2.2]bicyclooctane, and the like.

[0090] Unless otherwise specified, the term "3-8 membered heterocycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group consisting of 3 to 8 ring atoms, 1, 2, 3 or 4 of which are heteroatoms independently selected from O, S and N, and the rest are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the carbon, nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., C(=O), NO and S(O)p, p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein the bicyclic ring system includes spirocyclic, fused and bridged rings. In addition, with respect to the "3-8 membered heterocycloalkyl", heteroatoms may occupy the position at which the heterocycloalkyl is connected to the rest of the molecule. The 3-8 membered heterocycloalkyl includes 3-6 membered, 3-5 membered, 4-6 membered, 5-6 membered, 4 membered, 5 membered and 6 membered heterocycloalkyl, etc. Examples of 3-8 membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl, homopiperidinyl or dioxepanyl, etc.

[0091] Unless otherwise specified, the term "5-6 membered heterocycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group consisting of 5 to 6 ring atoms, 1, 2, 3 or 4 of which are heteroatoms independently selected from O, S and N, and the rest are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the carbon, nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., C(=O), NO and S(O)p, p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein the bicyclic ring system includes spirocyclic, annelated and bridged rings. In addition, with respect to the "5-6 membered heterocycloalkyl", heteroatoms may occupy the position at which the heterocycloalkyl is connected to the rest of the molecule. The 5-6 membered heterocycloalkyl includes 5-membered and 6-membered heterocycloalkyl. Examples of 5-6 membered heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl, 1,2-oxazinyl, 1,2-thiazinyl, hexahydropyridazinyl, homopiperazinyl or homopiperidinyl, etc.

[0092] 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 9-12 Similarly, 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.

[0093] 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.

[0094] 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 the compounds, 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 on the cultured single crystal using a Bruker D8 venture diffractometer, using CuKα radiation as the light source and scanning mode: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.

[0095] The solvent used in the present invention is commercially available.

[0096] The present invention uses the following abbreviations: DMSO stands for dimethyl sulfoxide; DMF stands for N,N-dimethylformamide; DCM stands for dichloromethane; dioxane or 1,4-dioxane stands for 1,4-dioxane; Ts stands for 4-methylbenzylsulfonyl; Boc stands for tert-butyloxycarbonyl, which is a protecting group for amino; Cbz stands for benzyloxycarbonyl, which is a protecting group for amino; CO2 stands for carbon dioxide; Bar stands for bar, which is a unit of pressure; LC-MS stands for liquid chromatography-mass spectrometry; HPLC stands for liquid chromatography; mg stands for milligram; μg stands for microgram; ng stands for nanogram; μL stands for microliter; mL stands for milliliter; mm stands for millimeter; μm stands for micrometer; mM stands for micromole / liter, μM stands for micromole / liter; nM stands for sodium mole / liter; h stands for hour; min stands for minute; HPLC stands for high performance liquid chromatography.

[0097] Compounds are named according to the conventional nomenclature in the art or using Software naming, commercially available compounds use supplier catalog names. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 Tumor growth curve.

[0099] Figure 2 The figure is a curve of weight changes of the test animals. DETAILED DESCRIPTION

[0100] 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.

[0101] Intermediate AA

[0102]

[0103] Step A: Compound A (2.3 g, 11.37 mmol) was dissolved in methanol (10 mL), followed by the addition of tetrahydropyran-4-amine (AA-1) (1 g, 9.89 mmol). The reaction mixture was stirred at 25°C for 12 hours, followed by the addition of sodium methoxide (1.86 g, 11.37 mmol) and continued stirring at 25°C for 2 hours. The reaction was quenched with concentrated hydrochloric acid (12 mol / L, 2 mL), filtered, and the filter cake was washed with water (10 mL). The filter cake was collected and dried under reduced pressure to yield Intermediate AA-2. 1 H NMR (400MHz, DMSO-d6) δ = 10.81 (s, 1H), 8.26 (s, 1H), 5.72 (s, 1H), 4.81 (tt, J = 3.9, 12.0Hz, 1H), 3.97 (d d,J=3.8,11.1Hz,2H),3.81(s,3H),3.50-3.39(m,2H),1.90-1.88(m,2H),1.72(dd,J=2.0,11.9Hz,2H). LC-MS(ESI)m / z: 254.3[M+H] + .

[0104] Step B: Intermediate AA-2 (800 mg, 3.16 mmol) was dissolved in acetonitrile (10 ml), followed by the addition of triethylamine (479.47 mg, 4.74 mmol) and p-toluenesulfonyl chloride (632.35 mg, 3.32 mmol). The reaction mixture was stirred at 25 degrees Celsius for 2 hours. The mixture was filtered and the filter cake was washed with water (10 ml). The filter cake was collected and vacuum dried to obtain intermediate AA-3, which was used directly in the next step. 1H NMR (400MHz, DMSO-d6) δ=8.32(s,1H),7.82(d,J=8.4Hz,2H),7.51(d,J=8.1Hz,2H),6.01(s,1H),4.77(tt,J=3.8,12.0H z,1H),3.96(dd,J=3.9,11.3Hz,2H),3.66(s,3H),3.46(s,2H),2.44(s,3H),1.93-1.91(m,2H),1.73(d,J=10.0Hz,2H). LC-MS(ESI)m / z: 408.2[M+H] + .

[0105] Step C: Intermediate AA-3 (250 mg, 613.59 μmol), acetamide (26.24 mg, 613.59 μmol), and potassium phosphate (143.27 mg, 674.95 μmol) were added to 1,4-dioxane (2 mL). Under nitrogen, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (35.50 mg, 61.36 μmol) and bis-bis[(1,2,3)-1-phenyl-2-propene]dipalladium(II) (16.08 mg, 30.68 μmol) were added. After nitrogen replacement three times, the reaction mixture was stirred at 110°C for 2 hours. After cooling to room temperature, the mixture was concentrated under pressure, and the residue was purified by thin-layer chromatography (developing solvent: ethyl acetate / petroleum ether = 1 / 1) to obtain Intermediate AA-4. 1 H NMR (400MHz, CDCl3) δ = 10.80 (s, 1H), 8.26 (s, 1H), 7.74 (s, 1H), 5.12 (tt, J = 4.6, 11.7Hz, 1H), 4.11(dd,J=4.2,11.4Hz,2H),3.89(s,3H),3.63-3.48(m,2H),2.21(s,3H),1.89-1.79(m,4H). LC-MS(ESI)m / z: 295.1[M+H] + .

[0106] Step D: The intermediate AA-4 crude product (60 mg) was added to a methanol solution of ammonia (7 mol / L, 1 ml), and then the mixture was stirred at room temperature for 72 hours in a sealed state. After concentration under reduced pressure, the residue was dissolved in methanol (3 ml), and then an aqueous sodium hydroxide solution (1 mol / L, 2 ml) was added. The reaction mixture was stirred at 50 degrees Celsius for 30 minutes. After natural cooling, the mixture was diluted with water (10 ml) and then extracted with dichloromethane (40 ml / time, 5 times). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give the intermediate AA. 1H NMR (400MHz, DMSO-d6) δ = 11.83 (s, 1H), 8.52 (s, 1H), 6.20 (s, 1H), 5.05-4.86 (m, 1H), 4.00 (d d,J=3.7,11.4Hz,2H),3.54-3.40(m,2H),2.25(s,3H),1.97-1.95(m,2H),1.83-1.77(m,2H). LC-MS(ESI)m / z: 262.2[M+H] + .

[0107] Intermediate AB

[0108]

[0109]

[0110] The preparation of intermediate AB refers to the preparation process of intermediate AA, except that tetrahydropyran-4-amine (AA-1) in step A is replaced by 3-methyltetrahydrofuran-3-amine (AB-1). 1 H NMR (400MHz, CD3OD) δ = 8.63 (s, 1H), 6.34 (s, 1H), 4.44 (d, J = 9.4Hz, 1H), 4.05-3.95 (m, 3H), 2.63-2.50 (m, 2H), 2.35 (s, 3H), 1.68 (s, 3H). LC-MS(ESI)m / z.262.3[M+H] + .

[0111] Intermediate AC

[0112]

[0113] The preparation of intermediate AC refers to the preparation process of intermediate AA, except that tetrahydropyran-4-amine (AA-1) in step A is replaced by 1-(fluoromethyl)cyclopropaneamine (AC-1). 1 H NMR (400MHz, DMSO-d6) δ = 11.83 (br s, 1H), 8.35 (s, 1H), 6.16 (s, 1H), 4.62 (s, 1H), 4.50 (s, 1H), 2.23 (s, 3H), 1.30-1.23 (m, 4H). LC-MS(ESI)m / z: 250.1[M+H] + .

[0114] Intermediate AD

[0115]

[0116] The preparation of intermediate AD refers to the preparation process of intermediate AA, except that tetrahydropyran-4-amine (AA-1) in step A is replaced by 4-aminobicyclo[2.2.2]octan-1-ol (AD-1). 1 H NMR (400MHz, CD3OD) δ = 8.64 (s, 1H), 6.28 (s, 1H), 2.50-2.46 (m, 6H), 2.33 (s, 3H), 1.94-1.78 (m, 6H). LC-MS(ESI)m / z: 302.1[M+H] + .

[0117] Intermediate AE

[0118]

[0119] The preparation of intermediate AE refers to the preparation process of intermediate AA, except that tetrahydropyran-4-amine (AA-1) in step A is replaced by 1-methylcyclopropaneamine (AE-1). 1 H NMR(400MHz, CDCl3)δ=10.40-10.12(m,1H),8.52(s,1H),6.45(s,1H),2.36(s ,3H),1.52(s,3H),1.05(dd,J=2.7,11.6Hz,4H).LC-MS(ESI)m / z:232.2[M+H] + .

[0120] Intermediate AF

[0121]

[0122] Step A: AF-1 (22.3 g, 220.57 mmol) and phthalic anhydride (32.67 g, 220.57 mmol) were added to glacial acetic acid (200 mL). The reaction mixture was stirred at 110°C for 3 hours. After concentration under reduced pressure to remove the acetic acid, water (200 mL) was added and stirred at 15°C for 1 hour. The filter cake was collected by filtration and dried under reduced pressure to provide AF-2. 1 H NMR (400MHz, DMSO-d6) δ = 13.60-12.40 (m, 1H), 7.95-7.82 (m, 4H), 1.66-1.59 (m, 2H), 1.47-1.40 (m, 2H).

[0123] Step B: AF-2 (10 g, 43.25 mmol) and N,N-dimethylformamide (158.07 mg, 2.16 mmol) were added to toluene (30 mL) at room temperature, followed by the slow addition of thionyl chloride (5.40 g, 45.41 mmol). After the addition was complete, the reaction mixture was stirred at 110°C for 3 hours. The solvent was removed by concentration under reduced pressure to afford AF-3, which was used directly in the next reaction.

[0124] Step C: AF-3 (20.5 g, 82.12 mmol) and 2,6-lutidine (10.56 g, 98.54 mmol) were added to tetrahydrofuran (200 mL), followed by the addition of dry palladium on carbon (10%, 1.02 g). The reaction mixture was stirred under 45 psi of hydrogen at 30°C for 20 hours. Additional palladium on carbon (10%, 1.02 g) was then added, and stirring continued under 45 psi of hydrogen at 30°C for 24 hours. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V (petroleum ether / ethyl acetate = 10 / 1 to 3 / 1) + 20% dichloromethane) to yield AF-4. 1 H NMR (400MHz, CDCl3) δ = 8.88 (s, 1H), 7.89-7.82 (m, 2H), 7.77-7.71 (m, 2H), 1.86-1.79 (m, 2H), 1.72-1.65 (m, 2H).

[0125] Step D: Dissolve AF-4 (7.54 g, 35.04 mmol) in dichloromethane (45 mL), followed by the slow dropwise addition of a solution of bis(2-methoxyethyl)aminosulfur trifluoride (18.60 g, 84.09 mmol) in dichloromethane (20 mL). After the addition, the reaction mixture was stirred at 20°C for 48 hours. The reaction was quenched with saturated aqueous sodium carbonate (100 mL) at 10°C and stirred for 1 hour. Water (100 mL) was then added, and the mixture was extracted with dichloromethane (100 mL / extraction, 3 times). The combined organic phases were dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: v / v petroleum ether / ethyl acetate = 15 / 1 to 10 / 1) to afford AF-5. 1 H NMR (400MHz, CDCl3) δ=7.88 (dd, J=3.2, 5.6Hz, 2H), 7.76 (dd, J=3.2, 5.6Hz, 2H), 6.07-5.69 (m, 1H), 1.47-1.41 (m, 2H), 1.27-1.20 (m, 2H).

[0126] Step E: Add AF-5 (7.1 g, 29.93 mmol) to 2-(2-aminoethylamino)ethanol (21.30 mL). Stir the reaction mixture at 80°C for 2 hours. Add methanol (150 mL) and distill at 90°C under atmospheric pressure. Cool the fraction in a dry ice bath. Add a methanolic solution of hydrogen chloride (4 mol / L, 15 mL) to the distillate. Concentrate under reduced pressure to yield AF-6. 1 H NMR (400MHz, CD3OD) δ = 6.04-5.71 (m, 1H), 1.24 (s, 4H).

[0127] Step F: AF-6 (4 g, 27.86 mmol) and compound A (6.76 g, 33.43 mmol) were added to methanol (40 mL), followed by the addition of a methanolic solution of sodium methoxide (25%, 5.42 g). The reaction mixture was stirred at 10°C for 17 hours, followed by the addition of a methanolic solution of sodium methoxide (25%, 7.22 g) and continued stirring for 2 hours. The methanol was removed by concentration under reduced pressure, and the residue was acidified to pH 6-7 with concentrated hydrochloric acid (12 mol / L). The mixture was diluted with water (100 mL) and extracted with ethyl acetate (100 mL / extraction, 3 times). The combined organic phases were dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was stirred in tert-butyl methyl ether (100 mL) for 1 hour, filtered, and the filter cake collected to provide AF-7. 1 H NMR (400MHz, DMSO-d6δ=10.93(s,1H),8.09(s,1H),6.32-5.98(m,1H),5.68(s,1H),3.80(s,3H),1.41-1.23(m,4H).

[0128] Step G: AF-7 (5.17 g, 19.95 mmol) and 4-methylbenzenesulfonyl chloride (4.58 g, 24.02 mmol) were added to acetonitrile (50 mL), followed by the addition of triethylamine (3.03 g, 29.92 mmol). The reaction mixture was stirred at 20°C for 2 hours. After dilution with water (50 mL), the mixture was filtered. The filter cake was washed with water (30 mL / wash, 3 times) and dried under vacuum to afford AF-8. 1 H NMR (400MHz, DMSO-d6) δ = 8.15 (s, 1H), 7.84 (d, J = 8.4Hz, 2H), 7.52 (d, J = 8.4Hz, 2H), 6.31-5.98 (m, 2H), 3.67 (s, 3H), 2.44 (s, 3H), 1.43-1.30 (m, 4H).

[0129] Step H: AF-8 (7.41 g, 17.92 mmol), acetamide (1.06 g, 17.92 mmol), potassium phosphate (4.19 g, 19.72 mmol), bis-bis[(1,2,3)-1-phenyl-2-propene]dipalladium(II) (187.90 mg, 358.50 μmol), and Xantphos (414.87 mg, 717.00 μmol) were added to 1,4-dioxane (35 mL) in one portion. The reaction mixture was stirred at 115°C under nitrogen for 12 hours. After cooling, the reaction mixture was diluted with water (100 mL) and extracted with dichloromethane (100 mL / extraction, 3 times). The combined organic phases were dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was separated and purified by silica gel column chromatography (eluent: v / v dichloromethane / ethyl acetate = 10 / 1 to 5 / 1) to obtain AF-9. 1 H NMR (400MHz, CDCl3) δ = 10.79 (br s,1H),8.23(s,1H),7.69(s,1H),6.40-6.03(m,1H),3.89(s,3H),2.21(s,3H),1.57-1.50(m,2H),1.19-1.11(m,2H).

[0130] Step I: AF-9 (4.22 g, 14.05 mmol) was added to a solution of ammonia in methanol (7 mol / L, 34 mL). The reaction mixture was stirred at 15°C for 3 days. After concentration under reduced pressure, aqueous sodium hydroxide solution (1 mol / L, 30 mL) was added to the residue, followed by stirring at 50°C for 30 minutes. After cooling, the solution was washed with tert-butyl methyl ether (70 mL / wash, 3 times). The aqueous phase was acidified with concentrated hydrochloric acid (12 mol / L), adjusted to a pH of approximately 4, filtered, and the filter cake collected and dried under reduced pressure to yield Intermediate AF. 1 H NMR (400MHz, DMSO-d6) δ = 11.85 (br s, 1H), 8.36 (s, 1H), 6.38-6.05 (m, 2H), 2.24 (s, 3H), 1.50-1.29 (m, 4H).

[0131] Intermediate AG

[0132]

[0133] Step A: Under nitrogen, AG-1 (3.00 g, 14.00 mmol) and Intermediate A (3.40 g, 16.80 mmol) were dissolved in methanol (30 mL). The reaction mixture was stirred at 20°C for 12 hours. A methanol solution of sodium methoxide (3.63 g, 16.80 mmol, 25% by mass) was then added, and the reaction mixture was stirred at 20°C for another 12 hours. The methanol was removed by concentration under reduced pressure, and the residue was diluted with water (100 mL) and washed twice with methyl tert-butyl ether (100 mL / wash). The aqueous phase was adjusted to pH 6-7 with hydrochloric acid (1 mol / L), filtered, and the filtrate was concentrated under reduced pressure to yield AG-2. 1 H NMR (400MHz, CDCl3) δ = 10.43 (s, 1H), 8.32-8.22 (m, 1H), 5.88 (s, 1H), 3.94-3.90 (m, 3H), 3.30 (ddd, J = 3.4, 9.8, 13.7Hz, 2H), 1.72 (s, 3H), 1.66 (br s,5H),1.46(s,9H).

[0134] Step B: AG-2 (3.15 g, 8.60 mmol) and p-toluenesulfonyl chloride (1.97 g, 10.32 mmol) were added to acetonitrile (32 ml), followed by triethylamine (1.30 g, 12.90 mmol). The reaction mixture was stirred at 20 degrees Celsius for 2 hours. After concentration under reduced pressure, the residue was diluted with water (50 ml) and stirred at 20 degrees Celsius for another 2 hours. After filtration, the filter cake was dried under reduced pressure to obtain AG-3. 1 H NMR (400MHz, CDCl3) δ=8.25-8.31(m,1H),7.72-7.84(m,2H),7.31(d,J=8.13Hz,2H),5.95-6.01(m,1H),3.61-3.73(m,2H) ,3.15-3.25(m,2H),2.40(s,3H),2.17-2.27(m,2H),2.05-2.16(m,2H),1.59-1.63(m,3H),1.57(s,3H),1.34-1.42(m,9H).

[0135] Step C: AG-3 (1.3 g, 2.50 mmol), acetamide (147.50 mg, 2.50 mmol), potassium phosphate (583.07 mg, 2.75 mmol), and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (288.98 mg, 499.43 μmol) were added to 1,4-dioxane (10 mL). After nitrogen replacement three times, bis(phenylpropenylpalladium chloride) (130.88 mg, 249.71 μmol) was added. The reaction mixture was stirred at 110°C for 4 hours. After cooling naturally, the mixture was diluted with water (30 mL) and extracted with dichloromethane (30 mL / extraction, 2 times). The organic phase was washed once with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V dichloromethane / petroleum ether = 10 / 1 to 3 / 1) to obtain AG-4. LC-MS (ESI) m / z: 408.3 [M+H] + .

[0136] Step D: AG-4 (0.67 g, 1.64 mmol) was added to ammonia methanol solution (7 mol / L, 50 mL) and stirred at 20°C for 3 days. After concentration under reduced pressure, the residue was diluted with sodium hydroxide solution (1 mol / L, 20 mL) and washed with methyl tert-butyl ether (30 mL / wash, 3 times). The aqueous phase was adjusted to pH 4 with concentrated hydrochloric acid and filtered under reduced pressure. The filter cake was collected and dried under reduced pressure to yield intermediate AG. 1 H NMR (400MHz, CDCl3) δ = 8.64 (s, 1H), 6.60-6.49 (m, 1H), 3.95-3.74 (m, 2H), 3.36-3.26 (m,2H),2.49(s,3H),2.40-2.20(m,4H),1.85-1.77(m,3H),1.75(s,1H),1.48(s,9H).

[0137] Intermediate AH

[0138]

[0139] Step A: AH-1 (500 mg, 2.5 mmol) and Intermediate A (605.64 mg, 3.0 mmol) were added to methanol (5 mL), followed by a methanolic solution of sodium methoxide (539.49 mg, 3.0 mmol, 30% by mass). The reaction mixture was stirred at 10-20°C for 12 hours, then adjusted to pH 4-5 with hydrochloric acid (2 mol / L) and extracted with dichloromethane (50 mL / extraction, two extractions). The combined organic phases were washed once with brine (20 mL) and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to yield AH-2.1 H NMR (400MHz, CDCl3) δ = 10.51 (s, 1H), 7.97 (s, 1H), 5.88 (s, 1H), 3.95-3.86 (m, 1H), 3.83 (s, 3H), 3.81-3.7 4(m,1H),3.73-3.67(m,1H),3.65-3.60(m,1H),2.21(m,1H),2.08-1.96(m,1H),1.42(s,9H),1.24(s,3H).

[0140] Step B: AH-2 (880 mg, 2.5 mmol) was added to acetonitrile (10 mL) at 0-10°C, followed by triethylamine (379.05 mg, 12.90 mmol) and p-toluenesulfonyl chloride (499.91 mg, 2.62 mmol). The reaction mixture was stirred at 20-30°C for 1 hour, then extracted once with dichloromethane (50 mL). The organic phase was washed with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: v / v dichloromethane / petroleum ether = 10 / 1 to 2 / 1) to obtain AH-3. 1 H NMR (400MHz, CDCl3) δ = 8.13 (br s,1H),7.85(d,J=8.4Hz,2H),7.38(d,J=8.2Hz,2H),6.14(s,1H),5.50-5.36(m,1H),4.20-4.05(m,1H),3.89-3.77 (m,4H),3.50-3.37(m,1H),2.48(s,3H),2.36-2.26(m,1H),2.13-2.06(m,1H),1.48(s,9H),1.31(d,J=5.0Hz,3H).

[0141] Step C: AH-3 (900 mg, 1.78 mmol) was added to 1,4-dioxane (10 mL). Acetamide (110.19 mg, 1.87 mmol), potassium phosphate (414.84 mg, 1.95 mmol), bis(phenylpropenylpalladium chloride) (93.12 mg, 177.67 μmol), and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (205.60 mg, 355.33 μmol) were then added sequentially under nitrogen. The reaction mixture was stirred at 110°C for 2 hours. After cooling, it was diluted with ethyl acetate (50 mL). After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: v / v / v petroleum ether / ethyl acetate / ethanol = 30 / 3 / 1 to 9 / 3 / 1) to provide AH-4. 1H NMR (400MHz, CDCl3) δ = 10.81 (s, 1H), 8.17 (s, 1H), 7.76 (s, 1H), 5.55-5.44 (m, 1H), 4.17-3.99 (m, 1H), 3.90-3.74 (m ,4H),3.70-3.42(m,1H),2.35-2.25(m,1H),2.23-2.19(m,3H),2.15-2.02(m,1H),1.48(s,9H),1.38-1.27(m,3H).

[0142] Step D: AH-4 (510 mg, 1.30 mmol) was added to a methanolic ammonia solution (7 mol / L, 50 mL) and stirred at 20-30°C for 12 hours. After concentration under reduced pressure, the residue was purified by preparative HPLC (column: Welch Ultimate XB-CN, 250 mm × 50 mm × 10 μm; mobile phase: phase A: n-hexane; phase B: 0.1% ethanolic ammonia solution, 5% to 45%; 15 minutes) to obtain intermediate AH. 1 H NMR (400MHz, CDCl3) δ = 11.81 (br s,1H),8.49-8.26(m,1H),6.18(s,1H),5.38-5.20(m,1H),3.98(m,1H),3.71(m,1H),3.56(m ,1H),2.57-2.53(m,1H),2.24(s,3H),2.07-1.92(m,1H),1.40(s,9H),1.21(d,J=5.6Hz,3H).

[0143] Intermediate AI

[0144]

[0145] Step A: AI-1 (2 g, 9.38 mmol), acetic acid (563.29 mg, 9.38 mmol) and ammonium formate (22.36 g, 354.56 mmol) were added to methanol (500 mL), followed by sodium cyanoborohydride (1.18 g, 18.76 mmol). The reaction mixture was stirred at 20-30°C for 72 hours. After concentration under reduced pressure, ethyl acetate (200 mL) was added to dissolve the residue. The solution was washed with saturated brine (50 mL / wash, 2 times) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain AI-2. 1H NMR (400MHz, CDCl3) δ = 4.12-3.88 (m, 1H), 3.76 (td, J = 3.2, 10.8Hz, 1H), 3.62-3.44 (m, 1H), 3.36- 3.23(m,1H),2.34-2.12(m,2H),1.78(td,J=9.8,13.0Hz,2H),1.46(s,9H),1.28(d,J=6.4Hz,3H).

[0146] Step B: AI-2 (1.4 g, 6.53 mmol) and Intermediate A (1.58 g, 7.84 mmol) were added to methanol (20 mL), followed by a methanolic solution of sodium methoxide (1.69 g, 7.84 mmol, 25% by mass). The reaction mixture was stirred at 10-20°C for 12 hours. The pH was adjusted to 4-5 with hydrochloric acid (2 mol / L), and the mixture was extracted with dichloromethane (50 mL / extraction, 2 times). The combined organic phases were washed once with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to yield AI-3. 1 H NMR (400MHz, CDCl3) δ=10.47-10.41(m,1H),8.01(s,1H),5.93-5.82(m,1H),4.96-4.81(m,1H),3.91-3.78(m,4H),3.72- 3.59(m,1H),3.52-3.38(m,1H),2.29-2.19(m,1H),1.92-1.83(m,1H),1.81-1.69(m,2H),1.41(s,9H),1.27-1.23(m,3H).

[0147] Step C: To a solution of AI-3 (1.55 g, 4.23 mmol) in acetonitrile (20 ml) at 0-10 degrees Celsius, triethylamine (642.09 mg, 6.35 mmol) and p-toluenesulfonyl chloride (887.15 mg, 4.65 mmol) were added. The reaction mixture was stirred at 20-30 degrees Celsius for 1 hour and then extracted with dichloromethane (50 ml). The organic phase was washed once with saturated brine (20 ml) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: v / v dichloromethane / petroleum ether = 10 / 1 to 2 / 1) to obtain AI-4. 1H NMR (400 MHz, CDC13) δ = 8.21-8.16 (m, 1H), 7.89-7.81 (m, 2H), 7.40 (d, J = 8.2 Hz, 2H), 6.16-6.05 (m, 1H), 5.27-4.84 (m, 1H), 3.98-3.72 (m, 5H), 3.54-3.39 (m, 1H), 2.49 (s, 3H), 2.40-2.26 (m, 1H), 2.01-1.80 (m, 2H), 1.63-1.57 (m, 1H), 1.50 (s, 9H), 1.33-1.28 (m, 3H).

[0148] Step D: AI-4 (1.77 g, 3.4 mmol), acetamide (210.87 mg, 3.57 mmol), potassium phosphate (793.87 mg, 3.74 mmol), bis (phenylallyl) palladium chloride (178.20 mg, 340.00 μmol) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (393.46 mg, 679.99 μmol) were added into 1,4-dioxane (20 mL) under nitrogen protection. The reaction mixture was stirred at 110 °C for 2 h, and then diluted with ethyl acetate (50 mL) after natural cooling. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluent: V / V / V petroleum ether / ethyl acetate / ethanol - 30 / 10 / 1 to 9 / 3 / 1) to give AI-5. 1 H NMR (400 MHz, CDC13) δ = 8.21-8.16 (m, 1H), 7.89-7.81 (m, 2H), 7.40 (d, J = 8.2 Hz, 2H), 6.16-6.05 (m, 1H), 5.27-4.84 (m, 1H), 3.98-3.72 (m, 5H), 3.54-3.39 (m, 1H), 2.49 (s, 3H), 2.40-2.26 (m, 1H), 2.01-1.80 (m, 2H), 1.63-1.57 (m, 1H), 1.50 (s, 9H), 1.33-1.28 (m, 3H).

[0149] Step E: AI-5 (710 mg, 1.74 mmol) was added into the solution of ammonium hydroxide (7 mol / L, 20 mL) and stirred at 20-30 °C for 12 h. After concentration under reduced pressure, the residue was purified by preparative HPLC (column: Welch Ultimate XB-SiOH, 250 mm x 50 mm x 10 μm; mobile phase: A phase: n-hexane: B phase: 0.1% ammonia in ethanol, 1% to 30%: 15 min) to give intermediate AI. 1H NMR (400MHz, CDCl3) δ = 11.81 (br s,1H),8.69-8.47(m,1H),6.24-6.07(m,1H),5.19-4.66(m,1H),3.94-3.82(m,1H),3.67(ddd,J=2.4,7.2,13.8Hz,1H),3.47 -3.38(m,1H),2.27-2.12(m,5H),1.86(ddd,J=2.8,5.8,12.4Hz,1H),1.72-1.56(m,1H),1.43(s,9H),1.19(d,J=6.4Hz,3H).

[0150] Intermediate AJ

[0151]

[0152] Step A: AJ-1 (0.5 g, 2.5 mmol) and Intermediate A (606.15 mg, 3 mmol) were dissolved in methanol (6 mL). Sodium methoxide (620.41 mg, 2.87 mmol) was then added. The reaction mixture was stirred at 25°C for 12 hours. After the methanol was removed by concentration under reduced pressure, water (30 mL) was added to the residue, which was then washed with methyl tert-butyl ether (40 mL). The aqueous phase was adjusted to pH 5-6 with 1 M hydrochloric acid. After filtration, the filter cake was concentrated under reduced pressure to afford AJ-2. 1 H NMR (400MHz, CDCl3) δ = 8.13 (s, 1H), 6.02-5.86 (m, 1H), 5.93 (s, 1H), 4.30 (m, 1H), 3 .93(s,3H),3.69-3.37(m,3H),2.35(m,1H),1.78(m,1H),1.63(s,3H),1.48(s,9H).

[0153] Step B: Dissolve AJ-2 (470 mg, 1.33 mmol) in acetonitrile (6 mL), then add p-toluenesulfonyl chloride (254.28 mg, 1.33 mmol) and triethylamine (202.45 mg, 2.0 mmol). The reaction mixture is stirred at 25°C for 5 hours. Extraction is performed with dichloromethane (60 mL). The organic phase is washed with water (20 mL / times, 2 washes), then with saturated brine (20 mL / times, 2 washes), dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure. The residue is purified by silica gel column chromatography (eluent: v / v petroleum ether / ethyl acetate = 5 / 1 to 1 / 1) to obtain AJ-3.

[0154] Step C: AJ-3 (0.5 g, 987.02 μmol), acetamide (69.96 mg, 1.18 mmol), potassium phosphate (230.47 mg, 1.09 mmol) and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (114.22 mg, 197.41 μmol) were added into 1,4-dioxane (2 mL) successively, and after nitrogen replacement for 3 times, bis(phenylallyl) palladium chloride (51.73 mg, 98.70 μmol) was added. The reaction mixture was stirred at 110 °C for 2 h. After natural cooling, water (30 mL) was added for dilution, and then extracted with dichloromethane (30 mL / time, twice). The organic phase was combined, washed with saturated brine (30 mL) once, dried over anhydrous sodium sulfate, filtered, and then the filtrate was concentrated under reduced pressure to give AJ-4. 1 H NMR (400 MHz, DMSO-d6) δ = 11.84 (s, 1H), 8.43 (s, 1H), 6.14 (d, J = 4.25 Hz, 1H), 3.49-3.40 (m, 1H), 3.43 (d, J = 11.63 Hz, 3H), 2.25 (s, 2H), 1.53 (d, J = 4.88 Hz, 3H), 1.42 (s, 12H).

[0155] Step D: AJ-4 (250 mg, 635.43 μmol) was dissolved in ammonium methanol solution (7 mol / L, 1.82 mL) and stirred at 20 °C for 3 days. After concentration under reduced pressure, the residue was diluted with sodium hydroxide solution (1 mol / L, 30 mL), and then washed with methyl tert-butyl ether (30 mL / time, three times). The aqueous phase was adjusted to pH about 4 by adding dilute hydrochloric acid (2 mol / L), and then extracted with dichloromethane (30 mL / time, twice). The organic phase was combined, washed with saturated brine (30 mL) once, dried over anhydrous sodium sulfate, filtered, and then the filtrate was concentrated under reduced pressure to give intermediate AJ. 1 H NMR (400 MHz, DMSO-d6) δ = 11.84 (s, 1H), 8.43 (s, 1H), 6.14 (d, J = 4.25 Hz, 1H), 3.49-3.40 (m, 1H), 3.43 (d, J = 11.63 Hz, 3H), 2.25 (s, 2H), 1.53 (d, J = 4.88 Hz, 3H), 1.42 (s, 12H).

[0156] Intermediate AK

[0157]

[0158] Step A: To a saturated aqueous solution of sodium bicarbonate (100 mL) of AK-1 (2.50 g, 16.28 mmol) was added a solution of triphosgene (4.02 g, 13.56 mmol) in toluene (50 mL). After the addition was complete, the reaction mixture was stirred at 25 °C for 12 h. The solution was concentrated under reduced pressure. To the residue was added methanol (100 mL) and ethyl acetate (100 mL) and stirred well. After filtration, the filtrate was concentrated under reduced pressure to give AK-2. 1 H NMR (DMSO-d6, 400 MHz) δ = 4.94 (br s, 1H), 4.50 (t, J = 8.4 Hz, 1H), 4.27 (br s, 1H), 4.10-4.00 (m, 2H), 3.41 (s, 1H), 2.99 (dd, J = 4.0, 12.0 Hz, 1H), 2.12 (ddd, J = 5.2, 8.4, 13.6 Hz, 1H), 1.60-1.50 (m, 1H).

[0159] Step B: AK-2 (2.30 g, 16.07 mmol) was dissolved in dichloromethane (60 mL) followed by the addition of triethylamine (4.88 g, 48.20 mmol), p-toluenesulfonyl chloride (3.37 g, 17.67 mmol) and 4-dimethylaminopyridine (98.15 mg, 0.80 mmol). The reaction mixture was stirred at 25 °C for 12 h, diluted with water (40 mL) and extracted with dichloromethane (50 mL / portion, twice). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give AK-3. 1 H NMR (DMSO-d6, 400 MHz) δ = 4.94 (br s, 1H), 4.50 (t, J = 8.4 Hz, 1H), 4.27 (br s, 1H), 4.10-4.00 (m, 2H), 3.41 (s, 1H), 2.99 (dd, J = 4.0, 12.0 Hz, 1H), 2.12 (ddd, J = 5.2, 8.4, 13.6 Hz, 1H), 1.60-1.50 (m, 1H).

[0160] Step C: AK-3 (3.50 g, 11.77 mmol) was dissolved in N,N-dimethylformamide (60 mL) followed by the addition of sodium azide (2.12 g, 32.61 mmol). The reaction mixture was stirred at 80 °C for 1 h, diluted with saturated sodium carbonate (100 mL) and extracted with ethyl acetate (50 mL / portion, twice). The organic phases were combined and used directly in the next step.

[0161] Step D: To a solution of AK-4 (1.6 g, 11.26 mmol) in ethyl acetate (100 mL) was added methanol (30 mL). After purging with nitrogen, palladium on carbon (0.20 g, 10% loading) was added. The reaction mixture was stirred under an atmosphere of hydrogen at 25 °C for 12 h, then filtered. The filtrate was concentrated under reduced pressure, and the residue was diluted with ethyl acetate (100 mL) and stirred at 30 °C for 30 min. The mixture was filtered again, and the filtrate was concentrated under reduced pressure to give AK-5. 1 H NMR (400 MHz, DMSO-d6) δ = 4.47 (t, J = 8.2 Hz, 1H), 4.22 - 4.06 (m, 2H), 3.64 - 3.53 (m, 2H), 2.73 - 2.61 (m, 1H), 1.80 - 1.65 (m, 2H), 1.56 (ddd, J = 5.8, 8.8, 12.6 Hz, 1H), 1.03 (t, J = 7.2 Hz, 1H).

[0162] Step E: AK-5 (1.6 g, 11.26 mmol) and intermediate A (2.5 g, 12.38 mmol) were dissolved in methanol (20 mL) and stirred for 3 h, then sodium methoxide (30% pure, 2.23 g, 12.38 mmol) was added. After the addition was complete, the reaction mixture was stirred at 30 °C for 12 h. The reaction was diluted with water (100 mL) and washed with ethyl acetate (50 mL x 2). The aqueous phase was adjusted to pH ~ 4 with hydrochloric acid (1 mole / liter) and extracted with ethyl acetate (100 mL x 3). The combined organic phase was washed with saturated brine (200 mL) once and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give AK-6.

[0163] Step F: AK-6 (2.4 g, 8.16 mmol) was dissolved in acetonitrile (60 mL), then p-toluenesulfonyl chloride (1.71 g, 8.97 mmol) and triethylamine (1.24 g, 12.23 mmol, 1.7 mL) were added. The reaction mixture was stirred at 30 °C for 14 h. After concentration under reduced pressure, methyl tert-butyl ether (40 mL) and water (40 mL) were added to the residue and stirred for 30 min. After filtration, the filter cake was collected and dried under reduced pressure to give AK-7. 1H NMR (400MHz, CDCl3) δ = 8.16 (s, 1H), 7.88 (d, J = 8.4Hz, 2H), 7.41 (d, J = 8.4Hz, 2H), 6.17 (s, 1H), 4.96-4.82 (m, 1H), 4.66-4.50 (m, 2H) ,4.31-4.18(m,2H),3.91-3.83(m,3H),3.43(dd,J=5.6,12.7Hz,1H),2.51(s,3H),2.47-2.35(m,1H),2.10(td,J=9.6,13.8Hz,1H).

[0164] Step G: Under nitrogen, AK-7 (250 mg, 557.48 μmol), acetamide (32.93 mg, 557.48 μmol), potassium phosphate (130.17 mg, 613.23 μmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (9.68 mg, 16.72 μmol), and bis(phenylpropenylpalladium chloride) (4.38 mg, 8.36 μmol) were added sequentially to 1,4-dioxane (2 mL). The reaction mixture was stirred at 110°C for 24 hours. After cooling, the mixture was diluted with water (40 mL) and extracted with ethyl acetate (30 mL / extraction, 2 times). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate) to obtain AK-8. 1 H NMR (400MHz, CDCl3) δ = 10.81 (br s,1H),8.21(s,1H),7.76(s,1H),4.81-4.70(m,1H),4.68-4.53(m,2H),4.26-4.11(m,2H),3.92 (s,3H),3.58-3.41(m,1H),2.48(ddd,J=2.4,6.6,13.6Hz,1H),2.25(s,3H),2.12-1.96(m,1H).

[0165] Step H: AK-8 (110 mg, 635.43 μmol) was dissolved in ammonia methanol solution (7 mol / L, 20 ml) and stirred at 20 degrees Celsius for 3 days. After concentration under reduced pressure, the residue was diluted with sodium hydroxide solution (1 mol / L, 30 ml) and washed with methyl tert-butyl ether (30 ml / times, three times). The aqueous phase was adjusted to a pH of approximately 4 by adding dilute hydrochloric acid (2 mol / L) and then extracted with dichloromethane (30 ml / times, two times). The organic phases were combined, washed once with saturated brine (30 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain intermediate AK. LC-MS (ESI) m / z: 303.1 [M+H]+ .

[0166] Intermediate AL

[0167]

[0168] Step A: Dissolve AL-1 (15 g, 68.42 mmol) in dichloromethane (100 mL), followed by the addition of tetraethyl titanate (46.82 g, 205.26 mmol, 42.56 mL). The reaction mixture is stirred at 30°C for 12 hours. Saturated aqueous sodium bicarbonate (200 mL) is slowly added to quench the reaction and stirring is continued for 30 minutes. Filter under reduced pressure, and wash the filter cake with dichloromethane (100 mL / wash, 2 times). Separate the organic phase and concentrate under reduced pressure. The residue is purified by silica gel column chromatography (eluent: v / v petroleum ether / ethyl acetate = 10 / 1 to 3 / 1) to obtain AL-2.

[0169] Step B: AL-2 (3.36 g, 10.41 mmol) and difluoromethylsulfonylbenzene (2 g, 10.41 mmol) were added to tetrahydrofuran (30 ml), cooled to -78 degrees Celsius under nitrogen, and then lithium bis(trimethylsilyl)amide (1 mol / L, 20.81 ml) was added dropwise. After the addition was complete, the reaction mixture was stirred at -78 degrees Celsius for 1 hour. Water (50 ml) was added to quench the reaction and extracted with ethyl acetate (50 ml / time, 2 extractions). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V petroleum ether / ethyl acetate = 1 / 1) to obtain AL-3. LC-MS (ESI) m / z: 515.1 [M+H] + .

[0170] Step C: AL-3 (500 mg, 971.62 μmol), sodium acetate (6.38 g, 77.73 mmol), and acetic acid (10 mL) were added to N,N-dimethylformamide (10 mL), followed by the addition of magnesium powder (472.30 mg, 19.43 mmol) in portions. After the addition, the reaction mixture was stirred at 25°C for 4 hours. After filtration, the filtrate was adjusted to a pH of approximately 7 with saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate (30 mL / extraction, 2 times). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to yield crude AL-4, which was used directly in the next reaction.

[0171] Step D: Dissolve crude AL-4 (188 mg) in tetrahydrofuran (2 mL), then add dioxane hydrochloride solution (4 mol / L, 250 μL). The reaction mixture is stirred at 25°C for 1 hour. After pressure concentration, the residue is washed once with methyl tert-butyl ether (2 mL) and once with petroleum ether (2 mL). After drying under reduced pressure, AL-5 is obtained. 1 H NMR (400MHz, DMSO-d6) δ = 9.29 (s, 2H), 7.51-7.27 (m, 5H), 6.68-6.25 (m, 1H), 5.17-5.05 (m, 2H), 3.76-3.47 (m, 4H), 2.38-2.06 (m, 2H).

[0172] Step E: AL-5 (100 mg, 326 μmol), Intermediate A (75.79 mg, 374.92 μmol), and sodium methoxide (25% purity, 70.45 mg, 326 μmol) were added to methanol (3 mL). The reaction mixture was stirred at 25°C for 12 hours. Water (20 mL) was added to dilute the reaction solution and then washed with ethyl acetate (20 mL / wash, 2 times). The aqueous phase was adjusted to a pH of approximately 4 with 1 M hydrochloric acid and then extracted with ethyl acetate (20 mL / wash, 3 times). The combined organic phases were washed once with saturated brine (20 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to yield AL-6.

[0173] Step F: Dissolve AL-6 (250 mg, 591.88 μmol) in acetonitrile (5 mL), then add p-toluenesulfonyl chloride (124.13 mg, 651.07 μmol) and triethylamine (119.40 mg, 1.18 mmol). The reaction mixture is stirred at 30°C for 14 hours. Dilute with water (20 mL) and extract with ethyl acetate (15 mL / times, 2 extractions). The organic phases are combined and dried over anhydrous sodium sulfate. After filtration, the filtrate is concentrated under pressure. The residue is purified by thin layer chromatography (developing solvent: v / v petroleum ether / ethyl acetate = 2 / 1) to obtain AL-7.

[0174] Step G: Under nitrogen, AL-7 (300 mg, 520.32 μmol), acetamide (30.74 mg, 520.32 μmol), potassium phosphate (132.54 mg, 624.38 μmol), 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (15.06 mg, 26.02 μmol), and bis(phenylpropenylpalladium chloride) (13.64 mg, 26.02 μmol) were added sequentially to 1,4-dioxane (3 mL). The reaction mixture was stirred at 110°C for 24 hours. After cooling, the mixture was diluted with water (40 mL) and extracted with ethyl acetate (30 mL / extraction, twice). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography (developing solvent: ethyl acetate) to yield AL-8. LC-MS (ESI) m / z: 464.2 [M+H] + .

[0175] Step H: AL-8 (160 mg, 345.25 μmol) was dissolved in methanolic ammonia (7 mol / L, 10 mL) and stirred at 20°C for 18 hours. After concentration under reduced pressure, the residue was diluted with sodium hydroxide solution (1 mol / L, 20 mL) and washed with methyl tert-butyl ether (30 mL / times, three times). The aqueous phase was adjusted to a pH of approximately 4 by adding dilute hydrochloric acid (2 mol / L) and extracted with dichloromethane (30 mL / times, two times). The organic phases were combined, washed once with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain AL-9. LC-MS (ESI) m / z: 431.2 [M+H] + .

[0176] Step I: Dissolve AL-9 (120 mg, 278.81 μmol) in dichloromethane (5 mL), followed by the addition of hydrobromic acid in acetic acid (250 μL). The reaction mixture was stirred at 25°C for 5 hours and quenched by the addition of methanol (1 mL). After concentration under reduced pressure, the residue was washed with petroleum ether (5 mL / wash, 2 times). After drying under reduced pressure, AL-10 was obtained.

[0177] Step J: Acetyl chloride (15.90 mg, 202.52 μmol) was dissolved in tetrahydrofuran (0.5 mL) and used as such. AL-10 (60 mg, 202.52 μmol) and triethylamine (40.91 mg, 405.04 μmol) were added to tetrahydrofuran (3 mL) and the resulting solution was cooled to 0 °C. The acetyl chloride solution in tetrahydrofuran was added dropwise. After the addition was complete, the reaction mixture was stirred at 0 °C for 2 h. The reaction was diluted with saturated aqueous sodium bicarbonate (5 mL) and extracted with ethyl acetate (15 mL / twice). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography (eluent: ethyl acetate) to give intermediate AL. LC-MS (ESI) m / z: 339.1 [M+H] + .

[0178] Intermediate B

[0179]

[0180] Step A: To a solution of B-l (3.00 g, 13.69 mmol) in tetrahydrofuran (120 mL) was added cesium carbonate (4.46 g, 13.69 mmol) and (±)-tert-butylsulfinamide (1.66 g, 13.69 mmol). The reaction mixture was stirred at 20 °C for 0.5 h, then quenched by the addition of water (100 mL) and extracted with ethyl acetate (100 mL / twice). After separation, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give B-2 as a crude product, which was used directly in the next step.

[0181] Step B: To a solution of B-2 (4.40 g, 13.65 mmol) in tetrahydrofuran (100 mL) was added vinylmagnesium bromide (1 M, 16.38 mL, 16.38 mmol) dropwise at -78 °C. After the addition was complete, the reaction mixture was stirred at -78 °C for 1 h, then allowed to warm to 0 °C naturally, quenched by the addition of saturated aqueous ammonium chloride (100 mL), and extracted with ethyl acetate (100 mL / twice). After separation, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 5 / 1 ~ 3 / 1) to give B-3.

[0182] Step C: To a solution of B-3 (3.70 g, 10.56 mmol) in tetrahydrofuran (30 mL) was added hydrogen chloride in 1,4-dioxane (4 M, 7.92 mL). The mixture was stirred at 20 °C for 3 h, then filtered through a Buchner funnel with the addition of petroleum ether (60 mL). The collected filter cake was dried under vacuum to give B-4 as a crude product, which was used directly in the next step. 1HNMR (400 MHz, DMSO-d6) δ = 9.16-8.81 (m, 3H), 8.75 (s, 1H), 8.55 (s, 1H), 8.44 (br s, 1H), 6.22-6.06 (m, 1H), 5.53-5.28 (m, 3H).

[0183] Step D: B-4 (2.50 g, 10.15 mmol) was dissolved in ethanol (50 mL), and iron powder (2.84 g, 50.77 mmol), ammonium chloride (2.72 g, 50.77 mmol) and water (10 mL) were added successively. The reaction mixture was stirred at 60 °C for 3 h. After natural cooling to room temperature, the reaction was quenched by the addition of aqueous sodium carbonate solution (100 mL), and extracted with ethyl acetate (100 mL x 2). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate B, which was used directly in the next step. LC-MS (ESI) m / z: 217.2 [M+H] + .

[0184] Intermediate C

[0185]

[0186]

[0187] Step A: To a solution of C-1 (10.00 g, 45.46 mmol) in tetrahydrofuran (100 mL) was added cesium carbonate (14.87 g, 45.64 mmol) and (R)-tert-butylsulfinamide (5.53 g, 45.64 mmol). The reaction mixture was stirred at 20 °C for 0.5 h, then quenched by the addition of water (100 mL), and extracted with ethyl acetate (200 mL x 2). The combined organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give C-2 as a crude product, which was used directly in the next step.

[0188] Step B: To a solution of C-2 (14.90 g, 46.23 mmol) in tetrahydrofuran (300 mL) was added vinylmagnesium bromide (1 M, 55.48 mL, 55.48 mmol) dropwise at -78 °C. After the addition, the reaction mixture was stirred at -78 °C for 1 h, then allowed to warm to 0 °C naturally, quenched by the addition of saturated aqueous ammonium chloride solution (100 mL), and extracted with ethyl acetate (200 mL x 2). The combined organic phase was washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether / ethyl acetate = 5 / 1 ~ 3 / 1) to give C-3. 1H NMR (400 MHz, CDC13) δ = 8.37 (s, 2H), 7.89 (s, 1H), 5.90-5.87 (m, 1H), 5.47-5.26 (m, 2H), 5.08 (dd, J = 4.2, 7.2 Hz, 1H), 3.55 (d, J = 3.9 Hz, 1H), 1.21 (s, 9H).

[0189] Step C: To a solution of C-3 (6.20 g, 17.70 mmol) in tetrahydrofuran (100 mL) was added hydrogen chloride in 1,4-dioxane (4 mol / L, 8.85 mL). The mixture was stirred at 20 °C for 1 h, then petroleum ether (20 mL) was added, and the mixture was filtered through a Buchner funnel. The collected filter cake was dried under vacuum to give C-4 as a crude product, which was used directly in the next step.

[0190] Step D: C-4 (4.3 g, 15.21 mmol) was dissolved in ethanol (100 mL), and iron powder (4.25 g, 76.07 mmol), ammonium chloride (4.07 g, 76.07 mmol), and water (10 mL) were added sequentially. The reaction mixture was stirred at 60 °C for 3 h. After natural cooling to room temperature, the reaction mixture was quenched with aqueous sodium carbonate solution (100 mL) and extracted with 300 mL of ethyl acetate three times (100 mL each time). The combined organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give intermediate C, which was used directly in the next step. 1 H NMR (400 MHz, CDC13) δ = 8.37 (s, 2H), 7.89 (s, 1H), 5.90-5.87 (m, 1H), 5.47-5.26 (m, 2H), 5.08 (dd, J = 4.2, 7.2 Hz, 1H), 3.55 (d, J = 3.9 Hz, 1H), 1.21 (s, 9H). + .

[0191] Intermediate D

[0192]

[0193] Step A: Compound D-1 (25 g, 130.13 mmol) was added to concentrated sulfuric acid (40 mL), followed by dropwise addition of fuming nitric acid (19.87 g, 315.33 mmol) at 0 °C. After the addition was completed, the reaction mixture was allowed to warm to 20 °C and stirring was continued for 2 h, then slowly poured into ice water (200 mL) and extracted with dichloromethane (100 mL / times, 3 times). The combined organic phase was washed with 1 M aqueous sodium hydroxide solution (100 mL) once, then the aqueous phase was extracted with dichloromethane (50 mL / times, 2 times). All the organic phases were combined and concentrated under reduced pressure to remove the solvent to give compound D-2, which was used directly in the next step. 1 H NMR (400 MHz, CDC13) δ = 10.43 (s, 1H), 8.96 (dd, J = 2.8, 5.6 Hz, 1H), 8.78 (dd, J = 2.4, 5.6 Hz, 1H).

[0194] Step B: D-2 (26 g, 109.65 mmol) was added to tetrahydrofuran (200 mL), followed by addition of (R)-tert-butylsulfinamide (13.29 g, 109.65 mmol) and cesium carbonate (35.73 g, 109.65 mmol). The reaction mixture was stirred at 2 °C for 30 min. After dilution with water (300 mL), it was extracted with ethyl acetate (300 mL / times, 2 times). The combined organic phase was washed with saturated brine (300 mL / times, 1 time) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography on silica gel (eluent: V / V, petroleum ether / ethyl acetate = 10 / 1 ~ 5 / 1) to give D-3. 1 H NMR (400 MHz, CDC13) δ = 9.08 (dd, J = 2.8, 5.4 Hz, 1H), 8.95 (s, 1H), 8.68 (dd, J = 2.8, 5.6 Hz, 1H), 1.34 (s, 9H).

[0195] Step C: To a solution of dimethylzinc solution (1 M, 101.38 mL) was added vinylmagnesium bromide solution (1 M, 88.16 mL) slowly at 20 °C under nitrogen protection. After the addition, the mixture was stirred for 3 h. Then, the reaction mixture was cooled to -78 °C and a solution of D-3 (15 g, 44.08 mmol) in THF (100 mL) was added slowly. After the addition, the mixture was stirred at -78 °C for 30 min. The reaction was quenched with saturated NH4CI solution (200 mL) and extracted with ethyl acetate (300 mL x 2). The combined organic phase was washed with saturated brine (300 mL x 1) and dried over anhydrous Na2S04. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: ethyl acetate / petroleum ether = 5 / 1 ~ 3 / 1, V / V) to give D-4. 1 H NMR (400 MHz, CDC13) δ = 8.53 (ddd, J = 2.8, 5.6, 18.0 Hz, 2H), 5.98 (ddd, J = 6.8, 10.0, 17.0 Hz, 1H), 5.50-5.30 (m, 3H), 3.70 (d, J = 5.6 Hz, 1H), 1.29 (s, 9H). LC-MS (ESI) m / z: 369.1 [M+H] + .

[0196] Step D: To a solution of D-4 (2.5 g, 6.79 mmol) in THF (20 mL) was added hydrogen chloride solution in 1,4-dioxane (4 M, 3.4 mL). The reaction mixture was stirred at 20 °C for 1 h. After dilution with petroleum ether (80 mL), the filter cake was collected by filtration to give D-5, which was used directly in the next step.

[0197] Step E: D-5 (2 g, 7.57 mmol) was added to a mixture of ethanol (50 mL) and water (10 mL), followed by the addition of iron powder (2.11 g, 37.85 mmol) and ammonium chloride (2.02 g, 37.85 mmol). After the reaction system was replaced with nitrogen for 3 times, it was stirred at 60 °C for 3 h. After cooling, the reaction mixture was filtered. The filtrate was neutralized with saturated Na2C03 aqueous solution (30 mL) and extracted with ethyl acetate (30 mL x 2). The combined organic phase was dried over anhydrous Na2S04. After filtration, the filtrate was concentrated under reduced pressure to give intermediate D.

[0198] Intermediate E

[0199]

[0200] Step A: E-1 (6.42 g, 30.57 mmol) was added to dimethylsulfoxide (50 mL), followed by potassium fluoride (3.55 g, 61.14 mmol) and copper iodide (11.64 g, 61.14 mmol), and finally (trimethylsilyl)ethyl difluoroacetate (12 g, 61.14 mmol). The reaction mixture was stirred at 60 °C for 15 h under nitrogen. After cooling to room temperature, the reaction mixture was diluted with tert-butyl methyl ether (200 mL) and water (300 mL) and stirred for 10 min, then filtered. The filter cake was washed with tert-butyl methyl ether (30 mL each, 3 times). The filtrate was combined and the organic phase was separated. The organic phase was washed with water (300 mL) and saturated brine (300 mL) each once, and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether) to give E-2. 1 H NMR (400 MHz, CDC13) δ = 7.75-7.65 (m, 1H), 7.44-7.38 (m, 1H), 7.28 (s, 1H), 4.36 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H).

[0201] Step B: E-2 (3.8 g, 18.43 mmol) was added to a mixture of tetrahydrofuran (30 mL) and ethanol (5 mL), and cooled to 0 °C. Sodium borohydride (1.40 g, 36.86 mmol) was added. After the addition was completed, the reaction mixture was stirred at 15-20 °C for 2 h. The reaction was quenched with saturated aqueous ammonium chloride solution (50 mL), followed by dilution with water (50 mL) and extraction with tert-butyl methyl ether (50 mL each, 2 times). The combined organic phase was washed with saturated brine (50 mL each, 2 times) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V petroleum ether / ethyl acetate = 10 / 1) to give E-3.

[0202] Step C: E-3 (2.37 g, 14.44 mmol) was added to anhydrous dichloromethane (30 mL), followed by the addition of imidazole (1.08 g, 15.88 mmol) and 4-dimethylaminopyridine (176.36 mg, 1.44 mmol) successively. With stirring, tert-butyldiphenylsilyl chloride (4.17 g, 15.16 mmol) was added dropwise slowly. After the addition was completed, the reaction mixture was stirred at 10-15 °C for 16 h. The reaction mixture was diluted with water (100 mL) and the organic phase was washed with saturated brine (100 mL) once and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: V / V petroleum ether / ethyl acetate = 25 / 1) to give E-4. 1H NMR (400MHz, CDCl3) δ = 7.61-7.55 (m, 5H), 7.45-7.36 (m, 7H), 7.18 (dd, J = 1.2, 5.0Hz, 1H), 3.99 (t, J = 12.0Hz, 2H), 1.02 (s, 9H).

[0203] Step D: Under nitrogen protection, E-4 (1.5 g, 3.73 mmol) was added to anhydrous tetrahydrofuran (20 ml). After cooling to minus 65 degrees Celsius, lithium diisopropylamide solution (1 mol / L, 6.71 ml) was slowly added dropwise. After the addition, the reaction mixture was stirred at minus 65 degrees Celsius for 1 hour. N,N-dimethylformamide (1.63 g, 22.36 mmol) was then added, and stirring was continued at minus 65 degrees Celsius for 30 minutes. Water (40 ml) was added to dilute the reaction solution and extracted with tert-butyl methyl ether (30 ml / time, extraction 2 times). The combined organic phase was washed once with saturated brine (50 ml) and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to obtain the crude product of E-5, which was directly used in the next reaction.

[0204] Step E: E-5 (1.6 g, 3.72 mmol) was added to tetrahydrofuran (20 ml), followed by the addition of (R)-tert-butylsulfenamide (495.41 mg, 4.09 mmol) and cesium carbonate (1.33 g, 4.09 mmol). The reaction mixture was stirred at 10-15 degrees Celsius for 16 hours. The reaction solution was diluted with water (40 ml) and extracted with tert-butyl methyl ether (30 ml / time, 2 extractions). The combined organic phases were washed once with saturated brine (40 ml) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was separated and purified by thin layer chromatography (developing solvent: V / V, petroleum ether / ethyl acetate = 20 / 1) to obtain E-6. 1 H NMR (400MHz, CDCl3) δ = 8.62 (d, J = 0.6Hz, 1H), 7.75 (s, 1H), 7.56 (td, J = 1.4, 8.0Hz, 4H), 7.52 (d, J = 1 .4Hz,1H),7.48-7.42(m,2H),7.40-7.36(m,4H),3.99(t,J=11.8Hz,2H),1.27(s,9H),1.02(s,9H). LC-MS(ESI)m / z: 534.2[M+H] + .

[0205] Step F: Under nitrogen protection, E-6 (2.6 g, 4.87 mmol) was added to anhydrous tetrahydrofuran (50 ml). The solution was cooled to -60 to -65 °C, and vinylmagnesium bromide solution (1 M, 9.74 ml) was added dropwise slowly. After the addition, the reaction mixture was stirred at -65 °C for 10 minutes, and then was allowed to warm to 0 °C. The stirring was continued for 50 minutes. The reaction was quenched with saturated aqueous ammonium chloride solution (50 ml) slowly, and then diluted with water (60 ml). The mixture was extracted with t-butyl methyl ether (60 ml x 2). The combined organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography (eluent: V / V, petroleum ether / ethyl acetate = 5 / 1) to give E-7.

[0206] Step G: E-7 (930 mg, 1.66 mmol) was added to tetrahydrofuran (5 ml), followed by the addition of hydrogen chloride solution in 1,4-dioxane (4 M, 1.24 ml). The reaction mixture was stirred at 10-15 °C for 2 hours. Petroleum ether (40 ml) was added to dilute the mixture, and the stirring was continued at 10-15 °C for 30 minutes. After filtration, the filter cake was dried under reduced pressure to give intermediate E. 1 H NMR (400 MHz, CD3OD) δ = 7.80 (s, 1H), 7.58 (d, J = 6.6 Hz, 4H), 7.52-7.31 (m, 8H), 6.17 (ddd, J = 6.8, 10.4, 17.2 Hz, 1H), 5.58-5.49 (m, 2H), 5.29 (d, J = 6.8 Hz, 1H), 4.03 (t, J = 12.2 Hz, 2H), 1.01 (s, 9H).

[0207] Intermediate F

[0208]

[0209] Step A: To a solution of F-1 (10.00 g, 49.26 mmol) in dichloromethane (100 ml) was added diethylamine sulfide (15.88 g, 98.52 mmol) dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 1 hour, and then saturated aqueous sodium bicarbonate solution was added slowly to pH = 8. The mixture was extracted with dichloromethane (50 ml x 3), and the combined organic phase was dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure to give F-2 as a crude product, which was used directly in the next step.

[0210] Step B: To a solution of F-2 (5.00 g, 22.22 mmol) in tetrahydrofuran (50 mL) at -78°C under nitrogen was added n-butyllithium (2.5 mol / L, 13.33 mL, 33.33 mmol) dropwise. After the addition, the reaction mixture was stirred at -78°C for 1 hour. N,N-dimethylformamide (3.25 g, 44.44 mmol) was then added and stirred at -78°C for another 1 hour. The reaction mixture was poured into dilute hydrochloric acid (1 mol / L, 100 mL) and extracted with ethyl acetate (30 mL / extraction, three times). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford crude F-3, which was used directly in the next reaction.

[0211] Step C: To a solution of F-3 (3.20 g, 18.38 mmol) in tetrahydrofuran (60 mL) were added cesium carbonate (5.99 g, 18.38 mmol) and (R)-tert-butylsulfenamide (2.23 g, 18.38 mmol). The reaction mixture was stirred at 0°C for 1 hour, then diluted with water (200 mL) and extracted with ethyl acetate (100 mL / extraction, 2 times). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Phenomenex luna C18, 250 × 50 mm × 10 μm; mobile phase: phase A: 0.225% formic acid in water; phase B: acetonitrile, 40-70%; 18 minutes) to afford F-4. 1 H NMR (400MHz, DMSO-d6) δ = 8.72 (s, 1H), 8.19 (t, J = 7.0Hz, 1H), 7.89 (t, J = 7.0Hz, 1H), 7.52 (t, J = 7.8Hz, 1H), 7.48-7.14 (m, 1H), 1.21 (s, 9H).

[0212] Step D: At 20 degrees Celsius, under nitrogen protection, vinyl magnesium bromide solution (1 mol / L, 3.61 mmol) was added dropwise to a dimethyl zinc toluene solution (1 mol / L, 4.15 mmol). After the addition, the reaction mixture was stirred at 20 degrees Celsius for 4 hours, then cooled to -78 degrees Celsius, and then a solution of F-4 (0.50 g, 1.80 mmol) in tetrahydrofuran (10 ml) was slowly added dropwise. After completion, stirring was continued at -78 degrees Celsius for 0.5 hours. The reaction mixture was quenched with saturated aqueous ammonium chloride (200 ml) and then extracted with ethyl acetate (300 ml / time, extracted twice). The combined organic phases were washed once with saturated sodium chloride (300 ml / time) and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by column silica gel chromatography (eluent: V / V, petroleum ether / ethyl acetate = 5 / 1 to 3 / 1).

[0213] Compound 1 was obtained by the reaction of intermediate A and B. 1 H NMR (400 MHz, DMSO-d6) δ = 7.70 (t, J = 7.0 Hz, 1H), 7.56 (t, J = 7.0 Hz, 1H), 7.42-7.07 (m, 2H), 6.04-5.95 (m, 1H), 5.35-5.08 (m, 3H), 1.13 (s, 9H).

[0214] Step E: To a solution of F-5 (0.40 g, 1.31 mmol) in tetrahydrofuran (5 mL) was added hydrogen chloride in 1,4-dioxane (4 mol / L, 0.98 mL). After the mixture was stirred at 15-20 °C for 2 hours, petroleum ether (40 mL) was added and the stirring was continued for 30 minutes. After filtration, the collected filter cake was dried under reduced pressure to give intermediate F.

[0215] Compound 1

[0216]

[0217] To a solution of intermediate AA (20 mg, 76.55 μmol), hexachlorotriphosphazene (29.27 mg, 84.20 μmol) and potassium phosphate (40.62 mg, 191.37 μmol) in acetonitrile (2 mL) was added intermediate B (16.55 mg, 76.55 μmol) and stirred at 20-25 °C for 1 hour. After the solvent was removed by concentration under reduced pressure, the residue was separated and purified by preparative HPLC (column: Unisil 3-100 C18 Ultra 150 x 50 mm x 3 μm, mobile phase: phase A: 0.225% trifluoroacetic acid in water; phase B: acetonitrile, 20% to 40% in 10 minutes) to give compound 1. 1 H NMR (400 MHz, CD3OD) δ = 9.09 (s, 1H), 6.93 (d, J = 6.5 Hz, 2H), 6.85 (s, 1H), 6.42 (s, 1H), 6.24-6.12 (m, 2H), 5.44-5.22 (m, 3H), 4.12 (d, J = 11.8 Hz, 2H), 3.63 (t, J = 11.8 Hz, 2H), 2.39 (s, 3H), 2.14-2.04 (m, 2H), 1.93 (d, J = 8.6 Hz, 2H). LC-MS (ESI) m / z: 460.4 [M+H] + .

[0218] Compound 2

[0219]

[0220]

[0221] Compound 2 was purified by preparative HPLC (Separation condition: Column: Unisil 3-100 C18 Ultra 150 x 50 mm x 3 pm; Mobile phase: A phase: 0.225% formic acid in water; B phase: acetonitrile, 18%~38%, 10 min) according to the preparation procedure of Reference Example 1, with intermediate AA replaced by intermediate AB.

[0222] Compound 2 was purified by preparative HPLC (Separation condition: Column: Unisil 3-100 C18 Ultra 150 x 50 mm x 3 pm; Mobile phase: A phase: 0.225% formic acid in water; B phase: acetonitrile, 18%~38%, 10 min) according to the preparation procedure of Reference Example 1, with intermediate AA replaced by intermediate AB. 1 H NMR (400 MHz, DMSO-d6) d = 9.00 (s, 1H), 8.87 (s, 1H), 8.28 (s, 1H), 6.84-6.73 (m, 3H), 6.22-6.07 (m, 3H), 5.63 (br s, 2H), 5.36-5.26 (m, 2H), 4.36 (dd, J = 4.9, 9.0 Hz, 1H), 3.96-3.82 (m, 3H), 2.26 (s, 3H), 1.59 (s, 3H). LC-MS (ESI) m / z: 460.3 [M+H] + .

[0223] Compound 3

[0224]

[0225] Compound 3 was purified by preparative HPLC (Separation condition: Column: Phenomenex luna C18 150 x 25 mm x 10 pm; Mobile phase: A phase: 0.225% formic acid in water; B phase: acetonitrile, 12%~42%, 10 min) according to the preparation procedure of Reference Example 1, with intermediate AA replaced by intermediate AC.

[0226] Compound 3 was purified by preparative HPLC (Separation condition: Column: Phenomenex luna C18 150 x 25 mm x 10 pm; Mobile phase: A phase: 0.225% formic acid in water; B phase: acetonitrile, 12%~42%, 10 min) according to the preparation procedure of Reference Example 1, with intermediate AA replaced by intermediate AC. 1 H NMR (400 MHz, DMSO-d6) d = 9.21 (s, 2H), 6.90-6.64 (m, 3H), 6.29-6.02 (m, 3H), 5.90-5.52 (m, 2H), 5.42-5.22 (m, 2H), 4.92-4.37 (m, 2H), 2.28 (s, 3H), 1.31 (br s, 4H). LC-MS (ESI) m / z: 448.1 [M+H] + .

[0227] Compound 4

[0228]

[0229] Compound 4 was purified by preparative HPLC (Separation condition: Column: Phenomenex luna C18 150 x 25 mm x 10 pm; Mobile phase: A phase: 0.225% formic acid in water; B phase: acetonitrile, 12%~42%, 10 min) according to the preparation procedure of Reference Example 1, with intermediate AA replaced by intermediate AD.

[0230] Compound 4 was isolated and purified by preparative HPLC (separation conditions: column: Phenomenex luna C18 150×25 mm×10 μm; mobile phase: phase A: 0.225% formic acid aqueous solution; phase B: acetonitrile, 12%-42%, 10 minutes). 1 H NMR (400MHz, DMSO-d6) δ = 8.95-9.07 (m, 1H), 8.73 (s, 1H), 6.83-6.73 (m, 3H), 6.20-6.10 (m, 2H), 6.03 (s, 1H), 5.63 (br s, 2H), 5.35-5.24 (m, 2H), 3.16 (s, 1H), 2.42-2.35 (m, 6H), 2.24 (s, 3H), 1.75-1.68 (m, 6H). LC-MS(ESI)m / z500.1[M+H] + .

[0231] Compound 5

[0232]

[0233] The preparation of compound 5 followed the preparation process of Example 1, except that intermediate AA was replaced by intermediate AE, and intermediate B was replaced by intermediate C.

[0234] Compound 5 was isolated and purified by preparative HPLC (separation conditions: column: Unisil 3-100 C18 Ultra 150×50 mm×3 μm; mobile phase: phase A: 0.225% formic acid aqueous solution; phase B: acetonitrile, 18%-38%, 10 minutes). 1 H NMR (400MHz, CD3OD) δ = 9.32 (s, 1H), 6.99-6.92 (m.2H), 6.88 (s, 1H), 6.39-6.14 (m, 3H), 5.42-5.27 (m, 2H), 2.45 (s, 3H), 1.60 (s, 3H), 1.24 (t, J=3.1Hz, 2H), 1.17-1.05 (m, 2H). LC-MS(ESI)m / z: 430.2[M+H + .

[0235] Compound 6

[0236]

[0237] Intermediate AF (60 mg, 224.52 μmol) was added to acetonitrile (5 mL), followed by potassium phosphate (119.15 mg, 561.31 μmol) and hexachlorotriphosphazene (78.06 mg, 224.52 μmol). The reaction mixture was stirred at 15°C for 1 hour. Intermediate D (52.58 mg, 224.52 μmol) was then added and stirring continued for 1 hour. Aqueous ammonia (25%, 1 mL) was then added and stirred for 1 hour. Saturated aqueous potassium carbonate (30 mL) was then added and stirred for 16 hours. The mixture was extracted with ethyl acetate (20 mL / extraction, 2 times). The organic phases were combined and concentrated under reduced pressure. The residue was separated and purified by preparative HPLC (chromatographic column: Unisil 3-100 C18 Ultra 150×50 mm×3 μm; mobile phase: phase A: 0.225% formic acid aqueous solution; phase B: acetonitrile, 13%-43%; 10 minutes) to obtain compound 6. 1 H NMR (400MHz, DMSO-d6) δ=9.35-9.06 (m, 2H), 6.83 (dd, J=2.6, 5.6Hz, 1H), 6.78 (dd, J=2.8, 5.4Hz , 1H), 6.51-6.10(m, 4H), 5.64-5.37(m, 2H), 5.36-5.18(m, 2H), 2.25(s, 3H), 1.56-1.27(m, 4H). LC-MS(ESI)m / z: 484.2[M+H + .

[0238] Compound 7

[0239]

[0240] Step A: Intermediate AF (200 mg, 748.42 μmol) was added to acetonitrile (10 mL), followed by potassium phosphate (397.16 mg, 1.87 mmol) and hexachlorotriphosphazene (260.19 mg, 748.42 μmol). The reaction mixture was stirred at 15°C for 1 hour. Intermediate E (369.80 mg, 448.42 μmol) was then added and stirring continued for 1 hour. Aqueous ammonia (25%, 1 mL) was then added and stirred for 1 hour. Saturated aqueous potassium carbonate (30 mL) was then added and stirred for 16 hours. The mixture was extracted with ethyl acetate (30 mL / extraction, 2 times). The organic phases were combined and concentrated under reduced pressure. The residue was separated and purified by preparative HPLC (chromatographic column: Welch Ultimate XB-SiOH 250×70×10 μm; mobile phase: phase A: n-hexane; phase B: 0.1% ethanolic ammonia solution, 1%-35%; 20 minutes) to obtain 7-1.

[0241] Step B: 7-1 (360 mg, 509.28 μmol) was added to methanol (10 mL), followed by the addition of ammonium fluoride (188.63 mg, 5.09 mmol). The reaction mixture was stirred at 70°C for 2 hours. After concentration under reduced pressure, the residue was purified by preparative HPLC (chromatographic column: Waters Xbridge 150 × 25 mm × 5 μm; mobile phase: phase A: aqueous ammonia; phase B: acetonitrile; 22%-52%; 9 minutes) to afford compound 7. 1 H NMR (400MHz, CD3OD) δ=9.12 (s, 1H), 7.67-7.52 (m, 1H), 7.18 (s, 1H), 6.51 (dd, J=1.0, 6.4Hz, 1H), 6.38 (s, 1H), 6.33-6.05 (m, 2H), 5.48-5.34 (m, 2H), 3.90 (t, J=13.2Hz, 2H), 2.42 (s, 3H), 1.66-1.27 (m, 4H). LC-MS(ESI)m / z: 469.1[M+H] + .

[0242] Compound 8

[0243]

[0244]

[0245] Intermediate AF (700 mg, 2.62 mmol), hexachlorotriphosphazene (867.31 mg, 2.50 mmol), and potassium phosphate (1.32 g, 6.24 mmol) were added to acetonitrile (20 ml) and stirred at 155°C for 1 hour. Intermediate F (593 mg, 2.50 mmol) was then added and stirring continued for 1 hour. After concentration under reduced pressure to remove the solvent, the residue was separated and purified by preparative HPLC (column: Waters Xbridge BEH C18 250×50 mm×10 μm; mobile phase: phase A: aqueous ammonia; phase B: acetonitrile, 35%-65%; 20 minutes) to obtain compound 8. 1 H NMR (400MHz, CD3OD) δ = 9.21 (s, 1H), 7.70-7.50 (m, 2H), 7.39-7.25 (m, 1H), 7.20-6 .84 (m, 1H), 6.56-5.95 (m, 4H), 5.42-5.20 (m, 2H), 2.33 (s, 3H), 1.69-1.31 (m, 4H). LC-MS(ESI)m / z: 451.2[M+H] + .

[0246] Compound 9

[0247]

[0248] Step A: Intermediate AG (270 mg, 114 mmol), hexachlorocyclotriphosphazene (394.99 mg, 1.14 mmol), and potassium phosphate (602.92 mg, 2.84 mmol) were added to acetonitrile (10 mL). The reaction mixture was stirred at 20°C for 1 hour, followed by the addition of Intermediate F (446.67 mg, 1.19 mmol), and stirring was continued at 20°C for 2 hours. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch Ultimate XB-SiOH, 250 mm × 70 mm × 3 μm; mobile phase: phase A: n-hexane; phase B: 0.1% ammonia in ethanol, 1% to 35%; 20 minutes) to afford 9-1. 1 HNMR (400MHz, CDCl3) δ = 9.02-8.91 (m, 1H), 7.76-7.51 (m, 2H), 7.33 (t, J = 7.8Hz, 1H), 7.20-6.84(m, 1H), 6.55-6.43(m, 1H), 6.36-6.30(m, 1H), 5.47-5.23(m, 2H), 4.61(br s, 1H), 3.90-3.79 (m, 2H), 3.63 (d, J=7.2Hz, 2H), 2.58-2.42 (m, 2H), 2.38 (s, 3H), 1.84 (s, 2H), 1.55-1.38 (m, 9H), 1.20 (s, 3H).

[0249] Step B: 9-1 (156 mg, 279.77 μmol) was dissolved in 1,4-dioxane (3 mL), followed by the addition of a dioxane hydrochloride solution (4 mol / L, 1 mL). The reaction mixture was stirred at 40°C for 2 hours and then concentrated under reduced pressure to afford 9-2. LC-MS (ESI) m / z: 458.0 [M+H] + .

[0250] Step C: Acetic acid (7.29 mg, 121.47 μmol), N,N-diisopropylethylamine (52.33 mg, 404.90 μmol), and 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (46.19 mg, 121.47 μmol) were added to N,N-dimethylformamide (4 mL). The reaction mixture was stirred at 20°C for 15 minutes, and then 9-2 (40 mg, 80.98 μmol) was added. The reaction mixture was stirred at 20°C for another hour. After concentration under reduced pressure, the residue was purified by preparative HPLC (Phenomenex Synergi C18 column, 150 mm × 25 mm × 10 μm; mobile phase: phase A: 0.225% formic acid in water; phase B: 10% to 10% acetonitrile; 10 minutes) to yield compound 9. 1 H NMR (400MHz, CDCl3) δ = 9.22-9.06 (m, 1H), 8.84 (s, 1H), 7.72-7.64 (m, 1H), 7.60-7.52 (m, 1H), 7.39-7.09 (m, 2H), 6.44-6.32 (m, 1H), 6.23 (ddd, J=6.1, 10.4, 16.9Hz, 1H), 6.07 (s, 1H), 5.36 -5.13 (m, 2H), 4.06-3.90 (m, 1H), 3.61 (dd, J=5.2, 13.6Hz, 1H), 3.19 (td, J=6.6, 13.6Hz, 2H), 2.64-2.53 (m, 1H), 2.34-2.23 (m, 3H), 2.19 (d, J=1.6Hz, 3H), 2.00 (s, 3H), 1.79-1.68 (m, 3H). LC-MS(ESI)m / z: 500.2[M+H] + .

[0251] Compound 10

[0252]

[0253] 9-2 (75 mg, 151.84 μmol) and 2,6-lutidine (56.94 mg, 531.43 μmol) were added to dichloromethane (1 ml), and the mixture was cooled to 0 degrees Celsius, followed by the addition of methyl chloroformate (12.91 mg, 136.65 μmol). After the addition, the reaction mixture was stirred at 20 degrees Celsius for 1 hour. After cooling to 0 degrees Celsius again, water (20 ml) was added to quench the reaction, and then extracted with ethyl acetate (20 ml / time, twice). The combined organic phase was washed once with water (20 ml), once with saturated brine (20 ml), and then dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (chromatographic column: Unisil 3-100 C18 Ultra, 150 mm×50 mm×3 μm; mobile phase: phase A: 0.225% formic acid aqueous solution; phase B: 13%-43% acetonitrile; 15 minutes) to give compound 10. 1 H NMR (400MHz, DMSO-d6) δ = 9.23-8.96 (m, 1H), 8.87-8.77 (m, 1H), 7.74-7.51 (m, 2H), 7.40-7.08 (m, 2H), 6.46-6 .35(m, 1H), 6.30-6.16(m, 1H), 6.11-6.03(m, 1H), 5.38-5.12(m, 2H), 3.77-3.70(m, 2H), 3.59(s, 3H), 3.28(br s, 2H), 2.46-2.37 (m, 2H), 2.31-2.22 (m, 2H), 2.19 (s, 3H), 1.73 (s, 3H). LC-MS (ESI) m / z: 516.2 [M+H] + .

[0254] Compound 11

[0255]

[0256] Step A: Intermediate AH (200 mg, 554.93 μmol), potassium phosphate (294.48 mg, 1.39 mmol), and hexachlorocyclotriphosphazene (192.93 mg, 554.93 μmol) were added to acetonitrile (3 mL). The reaction mixture was stirred at 20-30°C for 1 hour, followed by the addition of Intermediate F (145.07 mg, 610.42 μmol) and continued stirring for 12 hours. Aqueous ammonia (25% by mass, 0.5 mL) was then added and stirred for 1 hour. Aqueous potassium carbonate (20% by mass, 20 mL) was then added and stirred for another hour. The mixture was extracted once with dichloromethane (50 mL). The organic phase was washed once with saturated brine (10 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography (developing solvent: v / v dichloromethane / methanol = 10 / 1) to yield 11-1. LC-MS (ESI) m / z: 544.3 [M+H] + .

[0257] Step B: 11-1 (110 mg, 202.36 μmol) was added to 1,4-dioxane (3 mL), followed by a dioxane hydrochloride solution (4 mol / L, 1 mL). The reaction mixture was stirred at 30°C for 2 hours. After concentration under reduced pressure, 11-2 was obtained. LC-MS (ESI) m / z: 444.2 [M+H] + .

[0258] Step C: Acetic acid (18.77 mg, 312.55 μmol), 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (118.84 mg, 312.55 μmol) and N,N-diisopropylethylamine (107.72 mg, 833.46 μmol) were added to N,N-dimethylformamide (1 ml), and the reaction mixture was stirred at 20-30°C for 15 minutes. Then, 11-2 (100 mg, 208.37 μmol) was added, and stirring was continued at 20-30°C for 0.5 hours. After concentration under reduced pressure, the residue was purified by preparative HPLC (chromatographic column: Phenomenex Synergi C18, 150 mm×25 mm×10 μm; mobile phase: phase A: 0.225% formic acid aqueous solution; phase B: 13%-46% acetonitrile; 11 min) to obtain 11. 1H NMR (400MHz, MeOD) δ = 9.24 (m, 1H), 7.68 (t, J = 7.2Hz, 1H), 7.61 (t, J = 7.0Hz, 1H), 7.43-7.31 (m, 1H), 7 .21-6.86 (m, 1H), 6.54 (m, 1H), 6.41-6.23 (m, 2H), 5.86-5.67 (m, 1H), 5.44 (d, J=10.4Hz, 1H), 5.33 (d , J=17.4Hz, 1H), 4.56-4.41(m, 1H), 4.28-3.99(m, 1H), 3.77-3.59(m, 1H), 2.73-2.62(m, 1H), 2.47(d , J=2.8Hz, 3H), 2.34-2.20(m, 1H), 2.19-2.03(m, 3H), 1.43-1.33(m, 3H). LC-MS (ESI) m / z: 486.3[M+H] + .

[0259] Compound 12

[0260]

[0261] Step A: Intermediate AI (200 mg, 534.14 μmol), potassium phosphate (283.46 mg, 1.34 mmol), and hexachlorocyclotriphosphazene (185.70 mg, 554.93 μmol) were added to acetonitrile (7 mL). The reaction mixture was stirred at 20-30°C for 1 hour. Intermediate F (139.63 mg, 587.55 μmol) was then added and stirring continued for 12 hours. Aqueous ammonia (25% by mass, 0.5 mL) was then added and stirred for 1 hour. Aqueous potassium carbonate (20% by mass, 20 mL) was then added and stirring continued for 1 hour. After extraction with dichloromethane (50 mL), the organic phase was washed with saturated brine (10 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by thin-layer chromatography (developing solvent: v / v dichloromethane / methanol = 10 / 1) to yield compound 12-1. 1H NMR (400MHz, CDCl3) δ=8.61-8.42 (m, 1H), 7.44 (td, J=6.6, 12.9Hz, 2H), 7.16--7.05 (m, 1H), 6.98-6.64 (m, 1H), 6.49-6.38 (m, 1H), 6.37-6.23 (m, 1H), 6.16-5.99 (m, 1H), 5.29-5.07 (m, 2H), 5.01-4.82 (m, 1H), 3.84-3.69 (m, 1H), 3.64-3.53 (m, 1H), 3.28 (ddd, J=6.0, 8.6, 14.4Hz, 1H), 2.37-2.29 (m, 3H), 2.24-2.08 (m, 1H), 1.90-1.82 (m, 1H), 1.80-1.71 (m, 1H), 1.66-1.57 (m, 1H), 1.42-1.34 (m, 9H), 1.18-1.10 (m, 3H). LC-MS(ESI)m / z: 558.3[M+H] + .

[0262] Step B: Compound 12-1 (200 mg, 358.68 μmol) was added to 1,4-dioxane (3 mL), followed by a dioxane hydrochloride solution (4 mol / L, 1 mL). The reaction mixture was stirred at 30°C for 2 hours. The resulting mixture was concentrated under reduced pressure to afford compound 12-2. LC-MS (ESI) m / z: 458.2 [M+H] + .

[0263] Step C: Acetic acid (32.28 mg, 537.50 μmol), 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (204.37 mg, 537.50 μmol), and N,N-diisopropylethylamine (185.24 mg, 1.43 mmol) were added to N,N-dimethylformamide (1 mL). The reaction mixture was stirred at 20-30°C for 15 minutes. 12-2 (177 mg, 358.33 μmol) was then added, and stirring was continued for 0.5 hours. After concentration under reduced pressure, the residue was purified by preparative HPLC (Phenomenex Synergi C18 column, 150 mm × 25 mm × 10 μm; mobile phase: phase A: 0.225% formic acid in water; phase B: 15%-54% acetonitrile; 13 minutes) to provide compound 12. 1H NMR (400MHz, MeOD) δ = 9.33-9.20 (m, 1H), 7.76-7.68 (m, 1H), 7.67-7.57 (m, 1H), 7.42-7 .31(m, 1H), 7.18-6.87(m, 1H), 6.59-6.50(m, 1H), 6.39-6.24(m, 2H), 5.51-5.41(m, 1H ), 5.385.27(m, 1H), 5.06-4.91(m, 3H), 4.44-4.28(m, 1H), 2.54-2.44(s, 3H), 2.35-2. 22(m, 1H), 2.22-2.14(m, 4H), 2.09-1.92(m, 1H), 1.85-1.72(m, 1H), 1.50-1.29(m, 3H). LC-MS(ESI)m / z: 500.3[M+H] + .

[0264] Compound 13

[0265]

[0266]

[0267] Step A: Intermediate AJ (146 mg, 405.10 μmol) was dissolved in acetonitrile (4 mL), and hexachlorocyclotriphosphazene (140.84 mg, 405.10 μmol) and potassium phosphate (257.97 mg, 1.22 mmol) were added. The reaction mixture was stirred at 25°C for 1 hour, followed by the addition of Intermediate F (81.50 mg, 405.10 μmol) and continued stirring at 25°C for 1 hour. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (column: Welch Ultimate XB-SiOH, 250 mm × 50 mm × 10 μm; mobile phase: phase A: n-hexane; phase B: 0.1% ammonia in ethanol, 15% to 55%; 15 min) to afford 13-1. LC-MS (ESI) m / z: 544.3 [M+H] + .

[0268] Step B: Dissolve 13-1 (50 mg, 91.98 μmol) in 1,4-dioxane (4 mL), and then add dioxane hydrochloride solution (4 mol / L, 1 mL). The reaction mixture is stirred at 40°C for 1 hour. After concentration under reduced pressure, 13-2 is obtained. LC-MS (ESI) m / z: 444.2 [M+H] + .

[0269] Step C: Acetic acid (8.26 mg, 137.52 μmol), N,N-diisopropylethylamine (47.4 mg, 366.72 μmol), and 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (52.29 mg, 137.52 μmol) were added to N,N-dimethylformamide (2 mL). The reaction mixture was stirred at 25°C for 15 minutes, followed by the addition of 13-2 (44 mg, 91.68 μmol). After complete addition, stirring was continued at 25°C for 1 hour. After concentration under reduced pressure, the residue was purified by preparative HPLC (Phenomenex Synergi C18 column, 150 mm × 50 mm × 3 μm; mobile phase: phase A: 0.225% formic acid in water; phase B: 8% to 38% acetonitrile; 15 minutes) to yield compound 13. 1 H NMR (400MHz, DMSO-d6) δ = 9.19-9.02 (m, 1H), 8.87 (d, J = 15.6Hz, 1H), 7.73-7.54 (m, 2H), 7.40-7.10(m, 2H), 6.46-6.34(m, 1H), 6.31-6.16(m, 1H), 6.10(d, J=3.4Hz, 1H) , 5.34 (dd, J=10.8Hz, 1H), 5.20 (dd, J=17.0Hz, 1H), 4.50-4.40 (m, 1H), 3.73-3.50 ( m, 4H), 2.80-2.70 (m, 1H), 2.27-2.18 (m, 3H), 2.02-1, 93 (m, 3H), 1.63-1.52 (m, 3H). LC-MS(ESI)m / z: 486.2[M+H] + .

[0270] Compound 14

[0271]

[0272] Intermediate AK (50 mg, 165.41 μmol) was dissolved in acetonitrile (5 ml), and hexachlorocyclotriphosphazene (60.38 mg, 173.68 μmol) and potassium phosphate (140.44 mg, 661.63 μmol) were added. The reaction mixture was stirred at 30 degrees Celsius for 3 hours, and then intermediate F (39.31 mg, 165.41 μmol) was added and stirred at 30 degrees Celsius for 12 hours. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography (developing solvent: ethyl acetate) to obtain compound 14. LC-MS (ESI) m / z: 486.2 [M+H] + .

[0273] Compound 15

[0274]

[0275] Intermediate AL (50 mg, 147.79 μmol) was dissolved in acetonitrile (5 ml), and hexachlorocyclotriphosphazene (51.38 mg, 147.79 μmol) and potassium phosphate (94.11 mg, 443.37 μmol) were added. The reaction mixture was stirred at 30 degrees Celsius for 3 hours, and then intermediate F (35.10 mg, 147.79 μmol) was added and stirred at 30 degrees Celsius for 12 hours. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by thin layer chromatography (developing solvent: ethyl acetate) to obtain compound 15. LC-MS (ESI) m / z: 522.2 [M+H] + .

[0276] Biological tests:

[0277] Experimental Example 1. KRAS (G12C) and SOS1 binding experiment

[0278] 1. Experimental materials:

[0279] KRAS (G12C) protein was expressed and purified by Wuhan Pujian Biotechnology Co., Ltd., SOS1 exchange domin (564-1049) protein (Human recombinant) was purchased from Cytoskeleton, and Mab Antibody 6HIS-XL665 and Mab Antibody GST-Eu cryptate were purchased from Cisbio. A multifunctional microplate reader Nivo5 was purchased from PerkinElmer.

[0280] 2. Preparation of 1× buffer:

[0281] The buffer solution in this experiment was prepared and used immediately.

[0282] Preparation method: 4-hydroxyethylpiperazineethanesulfonic acid (Hepes): 5mM; sodium chloride (NaCl): 150mM; ethylenediaminetetraacetic acid (EDTA): 10mM; CO-630 (Igepal): 0.0025%; potassium fluoride (KF): 100mM; dithiothreitol (DTT): 1mM; bovine serum albumin (BSA): 0.05%.

[0283] 3. Experimental methods:

[0284] 1) The test compound was diluted 5-fold with DMSO using a dispenser to the eighth concentration, i.e., from 1 mM to 0.064 μM.

[0285] 2) Dilute the test compound to a 2% DMSO working solution in 1× buffer. Add 5 μL / well to the corresponding wells, with a concentration gradient from 20 μM to 0.00128 nM. Set up a duplicate experiment. Centrifuge at 1000 rpm for 1 minute.

[0286] 3) Prepare a mixed working solution of KRAS (G12C) (200 nM) and Mab Anti GST-Eu cryptate (1 ng / μL) in 1× buffer, incubate the mixed working solution at 25°C for 5 minutes, and add 2.5 μL / well to the corresponding wells.

[0287] 4) Prepare a working solution of SOS1 (80 nM) and Mab Anti-6HIS-XL665 (8 g / μL) in 1× buffer and add 2.5 μL / well to the corresponding wells. Add 2.5 μL of the Mab Anti-6HIS-XL665 (8 g / μL) dilution to the blank wells. The final compound concentration gradient is now 10 μM to 0.64 nM, KRAS (G12C) (500 nM), MAb Anti-GST-Eucryptate (0.25 ng / μL), SOS1 (20 nM), and Mab Anti-6HIS-XL665 (2 g / μL). Incubate the reaction system at 25°C for 60 minutes. After completion of the reaction, read the HTRF using a multi-label analyzer.

[0288] 4. Data Analysis

[0289] 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 (obtained in GraphPad Prism using the Log(inhibitor) vs. response--Variable slope mode). Table 1 provides the inhibitory activity of the compounds of the present invention on the binding of KRAS (G12C) and SOS1.

[0290] Table 1 Inhibitory activity of the compounds of the invention on the binding of KRAS (G12C) and SOS1

[0291]

[0292]

[0293] Conclusion: The compounds of the present invention have ideal KRAS(G12C)-SOS1 binding inhibitory activity.

[0294] Experimental Example 2. HTRF detection of pERK expression levels in DLD-1 cells

[0295] The phosphorylation level of ERK protein in DLD-1 cells with KRAS G13D mutation was detected by homogeneous time-resolved fluorescence (HTRF). 50 and IC 90 The value was used as an indicator to evaluate the inhibitory activity of the compound on the degree of ERK phosphorylation in the RAS signaling pathway.

[0296] 1. Experimental steps and methods:

[0297] 1) DLD-1 cells (50,000 cells / well) were seeded into a 96-well plate, 90 μl of RPMI-1640 medium (10% fetal bovine serum) was added to each well, and the plate was incubated at 37° C. with 5% carbon dioxide overnight.

[0298] 2) Add 10 μl of the 10× compound working solution as indicated in Table 2 to the cell culture plate. Add 10 μl of the DMSO-cell culture medium mixture to the vehicle control and positive control. The final DMSO concentration is 0.25%. Return the 96-well plate to the incubator and incubate for 1.5 hours.

[0299] Table 2 400× Storage Plate of Compounds of the Invention (μM)

[0300]

[0301] 3) Remove the cell supernatant and immediately add 50 μl of 1× lysis buffer. Incubate at room temperature with shaking for 30 minutes.

[0302] 4) Use a pipette to mix well and transfer 16 μl of cell lysate from the 96-well plate to the HTRF 384-well assay plate.

[0303] 5) Add 4 μl of pre-mixed antibody solution, seal the plate, and incubate at 4°C. Detect fluorescence emission intensity at 665 nm and 620 nm using an HTRF plate reader.

[0304] 2. Data Analysis

[0305] 1) Set the fluorescence wavelength of the Envision instrument to 665 nm and 620 nm for fluorescence quantification;

[0306] 2) Calculate the acceptor and donor emission signals in each well: Ratio = Signal 665nM / Signal 620nM;

[0307] 3) After removing the background value from the raw data, the relative pERK level was calculated by comparing it with the DMSO-treated group;

[0308] 4) IC was calculated based on Log[inhibitor] vs Response-variance slope using GraphPad 8.0 software. 50 Table 3 provides the inhibitory activity of the compounds of the present invention on pERK.

[0309] Table 3 Inhibitory activity of the compounds of the present invention on p-ERK in DLD-1 cells

[0310] Sample <![CDATA[IC 50 (nM)]]> Compound 6 23 Compound 8 37 Compound 9 36 Compound 10 87 Compound 13 26 Compound 15 25

[0311] Conclusion: The compounds of the present invention have significant inhibitory activity on the phosphorylation level of ERK kinase in DLD-1 cells.

[0312] Experimental Example 3. In vitro anti-proliferation experiment of A427 cells (KRAS G12D)

[0313] 1. Experimental materials:

[0314] EMEM 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. A427 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision multi-label analyzer was purchased from PerkinElmer.

[0315] 2. Experimental methods:

[0316] 1) A427 cells were seeded in a 96-well U-shaped plate. Low-melting-point agarose was first prepared into a 2% stock solution. When using, 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. 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 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 a bottom layer of gel at a density of 3000 cells per well. After the top layer of gel solidified, the cell plate was placed in a CO2 incubator for overnight culture.

[0317] 2) On the day of compound addition, 85 μL of liquid medium was added to the 96-well U-bottom plate where cells were plated. The test compound was diluted 3-fold by gun to the 9th concentration, i.e. from 6 mM to 0.9 μM, in duplicate. 97 μL of medium was added to the intermediate plate, and 2.5 μL of each well of the gradient diluted compound was transferred to the intermediate plate according to the corresponding position, mixed and 40 μL of each well was transferred to the cell plate. The concentration of the compound transferred to the cell plate ranged from 30 μM to 4.5 nM. The cell plate was incubated in a CO2incubator for 7 days. On the 8th day, the test compound was diluted 3-fold by gun to the 9th concentration, i.e. from 6 mM to 0.9 μM, in duplicate. 198 μL of medium was added to the intermediate plate, and 2 μL of each well of the gradient diluted compound was transferred to the first intermediate plate according to the corresponding position, 100 μL of medium was added to the second intermediate plate, 100 μL of the mixed compound from the first intermediate plate was added to the second intermediate plate, and 40 μL of each well was transferred to the cell plate after mixing. The concentration of the compound transferred to the cell plate ranged from 30 μM to 4.5 nM. The cell plate was incubated in a CO2incubator for another 7 days. The compound was co-incubated with the cells for 14 days, 20 μL of Almar blue reagent was added to each well of the cell plate, the plate was shaken on a horizontal shaker for 15 minutes, and the plate was incubated at room temperature for 5 hours to stabilize the luminescent signal. The reading was taken by using a multi-label reader.

[0318] 3. Data analysis:

[0319] The original data was converted into inhibition rate by using the equation (Sample-Min) / (Max-Min)χ100%, and the value of IC50was obtained by four-parameter curve fitting (obtained by "log(inhibitor) vs. response - Variable slope" mode in GraphPad Prism). 50 Table 4 provides the inhibitory activity of the compounds of the present application on the proliferation of A427 cells. "A" means that the value of IC50is less than or equal to 100 nM; "B" means that the value of IC50is greater than 100 nM and less than or equal to 500 nM; and "C" means that the value of IC50is greater than 500 nM. 50 50 50

[0320] Table 4 Inhibitory activity of the compounds of the present application on the proliferation of A427 cells

[0321] Sample <![CDATA[IC 50 (nM)]]> Compound 8 A Compound 13 A Compound 15 A

[0322] Conclusion: The compounds of the present application exhibit significant inhibitory activity on the proliferation of A427 cells.

[0323] ​​​Experimental Example 4. Pharmacokinetic Study in Mice

[0324] This study aimed to evaluate the pharmacokinetic behavior of the compound in mice following a single intravenous or oral administration. For intravenous administration, the compound was prepared as a 0.2 mg / mL clear solution in a 5% DMSO / 95% (10% hydroxypropyl-β-cyclodextrin) aqueous solution; for oral administration, the compound was prepared as a 0.3 mg / mL suspension in a 5% DMSO / 95% (10% hydroxypropyl-β-cyclodextrin) aqueous solution.

[0325] Compound concentrations in plasma were determined by high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). Retention times of the compounds and the internal standard (diclofenac), chromatogram acquisition, and chromatogram integration were performed using Analyst (Applied Biosystems) software. Data statistics were performed using Watson LIMS (Thermo Fisher Scientific) or Analyst (Applied Biosystems) software. Analyte concentrations in samples were expressed in ng / mL to three significant figures, and all values ​​expressed as percentages (e.g., % deviation and % coefficient of variation) were rounded to one decimal place. Each calibration curve included at least six concentration levels. Calibration standards were prepared from a different stock solution than the quality control samples. Calibration standards whose calculated concentrations deviated from the labeled value by more than ±15.0% (limit of quantitation exceeding ±20.0%) were rejected from the regression analysis. Rejected calibration standards should have a variance of less than 25%, and each calibration curve included at least six calibration standards that met the acceptance criteria. If the lower and upper limits of quantitation calibration standards need to be rejected, the upper and lower limits of quantitation for the analytical batch will be increased and decreased accordingly.

[0326] Using WinNonlin TM The plasma concentrations were processed using a non-compartmental model using the pharmacokinetic software Version 6.3 (Pharsight, Mountain View, CA). Pharmacokinetic parameters were calculated using the linear-log trapezoidal method. Pharmacokinetic parameters to be calculated included, but were not limited to, (data permitting) T for the IV group. 1 / 2 、Vd ss , CL, AUC 0-24h ; C of PO group max 、T max , AUC 0-24h , oral bioavailability (F%).

[0327] The pharmacokinetic parameters of the present invention in mice at an intravenous injection dose of 1 mg / Kg and an oral gavage dose of 3 mg / Kg are shown in Table 5 below.

[0328] Table 5 Pharmacokinetic parameters of the compounds of the present invention after intravenous injection and oral gavage in mice

[0329]

[0330] Conclusion: The compounds of the present invention exhibited good pharmacokinetic properties in mouse PK studies.

[0331] Experimental Example 5. Evaluation of the anti-tumor effect of the test compound in a BALB / c nude mouse model of subcutaneous xenografts of human pancreatic cancer MIA PaCa-2 cells

[0332] Human pancreatic cancer MIA PaCa-2 cells are a KRAS G12C mutation-dependent cell line. The KRAS G12C mutation leads to sustained KRAS activation, activating downstream signaling pathways such as ERK and AKT, promoting tumor cell growth and proliferation. This maintains the KRAS protein in an inactive state bound to GDP, thereby inhibiting tumor growth.

[0333] BALB / c nude mice were subcutaneously inoculated with MIA PaCa-2 cells to establish a subcutaneous human pancreatic cancer tumor model. Six animals were divided into a test compound group and a vehicle control group. The compounds were administered orally. The vehicle control group received a single dose daily, while the test compound groups received two doses daily, 12 hours apart, for a total of 18 days. Efficacy was evaluated based on relative tumor inhibition (TGI), and safety was assessed based on changes in animal body weight and mortality.

[0334] Female BALB / c nude mice, 6-7 weeks old at the time of tumor cell inoculation, were purchased from Shanghai Bikai Laboratory Animal Co., Ltd. All animals were housed in individually ventilated cages at a constant temperature and humidity of 21-24°C, 40-60% humidity, 10-20 air changes per hour, and a 12-hour light / dark cycle. They were provided with a continuous supply of cobalt-60-irradiated complete mouse pellets, with ad libitum access. Tap water (autoclaved) was also available, and water was provided via a continuous water bottle. The cages were autoclaved polysulfone, 325 mm × 210 mm × 180 mm, and the bedding consisted of autoclaved corn cobs. Five animals were housed per cage. Cage cards were labeled with the IACUC approval number, experiment number, experiment start date, project leader, experimenter, animal source, group, and animal number. Animals were ear-tagged.

[0335] MIA PaCa-2 cells were cultured in DEME medium containing 10% fetal bovine serum and 2.5% HS. MIA PaCa-2 cells were harvested during the exponential growth phase, resuspended in phosphate-buffered saline (PBS) to an appropriate concentration, and mixed with Matrigel at a 1:1 ratio for subcutaneous tumor inoculation in mice. 5×10 6 MIA PaCa-2 cells.

[0336] When the tumor grows to 70-80 mm 3 Around 13 days after cell inoculation, drug administration was started. When the body weight of a single mouse decreased by more than 20%, it was euthanized according to animal welfare.

[0337] After tumor inoculation, routine monitoring included tumor growth and the effects of treatment on the normal behavior of the animals. Specific content included the activity of the experimental animals, food and water intake, weight gain or loss (weight was measured twice a week), eye, coat and other abnormalities. Clinical symptoms observed during the experiment were recorded in the original data. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter). TM The software collects data, including tumor diameter measurements and animal weight. Raw data, measured using a balance and vernier calipers, is directly imported into the software, and any changes to the data are recorded.

[0338] The tumor growth curve is as follows Figure 1 The weight change curve of the test animals is shown in Figure 2 As shown in the experimental results, the compound of the present invention alone has a significant inhibitory effect on tumor proliferation in a BALB / c nude mouse model of subcutaneous xenografts of human pancreatic cancer MIA PaCa-2 cells, and no weight loss was found in the test animals, indicating that the compound of the present invention has good safety after administration.

Claims

1. A compound of formula (II) or a pharmaceutically acceptable salt thereof, in, Ring A is selected from phenyl and thienyl; R1 is selected from H and NH2; R2 is selected from C 1-3 Alkyl, the C 1-3 The alkyl group is optionally substituted with 1, 2 or 3 R a replace; R3 is selected from H and F; R4 is selected from C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl and The C 3-8 Cycloalkyl, 3-8 membered heterocycloalkyl and Each independently optionally substituted by 1, 2 or 3 R b Substitution; the 3-8 membered heterocycloalkyl group is a saturated cyclic group consisting of 3 to 8 ring atoms, 1 or 2 of which are heteroatoms independently selected from O and N, and the rest are carbon atoms; R5 is selected from H, Cl and CH3; R a are independently selected from F and OH; R b are independently selected from H, OH, C 1-3 Alkyl, -COO-C 1-3 Alkyl and -C(=O)-C 1-3 Alkyl, the C 1-3 Alkyl is optionally substituted independently with 1 or 2 R; R is independently selected from F.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R1 is selected from NH2.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R2 is selected from CF3, CHF2 and 4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R4 is selected from cyclopropyl, bicyclo[2.2.2]octyl, 5-6 membered heterocycloalkyl and 8 membered heterocycloalkyl, wherein the cyclopropyl, bicyclo[2.2.2]octyl, 5-6 membered heterocycloalkyl and 8 membered heterocycloalkyl are each independently optionally substituted by 1, 2 or 3 R b replace.

5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein R4 is selected from described Each independently optionally substituted by 1, 2 or 3 R b replace.

6. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R b are independently selected from H, OH, CH3, -CH2F, -CHF2, -COOCH3 and 7. The compound according to claim 5 or 6 or a pharmaceutically acceptable salt thereof, wherein R4 is selected from 8. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein Compounds selected from Among them, ring A, R1, R2, R3, R4, R5 and R b As defined in claim 1.

9. The compound according to claim 8 or a pharmaceutically acceptable salt thereof, wherein Compounds selected from Among them, R1, R2, R3, R b and R5 is as defined in claim 8.

10. A compound or a pharmaceutically acceptable salt thereof, wherein: Compounds selected from 11. The compound according to claim 10 or a pharmaceutically acceptable salt thereof, wherein Compounds selected from 12. Use of the compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting SOS1 protein, wherein the drug is a drug for treating lung cancer, pancreatic cancer and / or rectal cancer.

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