Heterocyclic compounds, processes for their preparation, pharmaceutical compositions and uses thereof

Abstract

The present application belongs to the field of pharmaceutical chemistry, and relates to a heterocyclic compound, a preparation method thereof, a pharmaceutical composition and application. In particular, the present application relates to a heterocyclic compound as shown in formula I, a pharmaceutical composition containing the same and application thereof as a SHP2 inhibitor in the medical field. The heterocyclic compound in the present application shows excellent biological activity and drugability, and has great prospects for drug development.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and particularly relates to a class of heterocyclic compounds, a preparation method of the class of compounds and intermediates thereof, a pharmaceutical composition containing the class of compounds, and application thereof in the medical field. BACKGROUND

[0002] Tyrosine phosphatase SHP2 is composed of two N-terminal Src homology 2 domains (N-SH2 and C-SH2) and a protein tyrosine phosphatase catalytic domain (PTP). In the basic state, N-SH2 can bind to PTP to form a ring structure, thereby hindering the binding of PTP to the substrate, so that the catalytic activity of the enzyme is inhibited; when the tyrosine of the upstream receptor protein is phosphorylated, N-SH2 binds to it, and the PTP catalytic domain is released, thereby exerting phosphatase activity.

[0003] At the cellular level, SHP2 participates in multiple tumor cell signaling pathways such as RTK / Ras / MAPK, JAK / STAT and PB3K / Akt through the downstream functional role in the cytoplasm of many receptor tyrosine kinases. Through the regulation of these kinases and signaling pathways, SHP2 is closely related to many important cell life activities, such as cell proliferation, migration, differentiation, death, cytokine regulation and tumor occurrence, etc.

[0004] At the same time, SHP2 also participates in the immune system inhibition mediated by programmed death receptor 1 (PD1). After the PD-1 of T cells binds to PD-L1, a large number of SHP2 can be recruited in the cell. SHP2 can dephosphorylate the intracellular antigen receptor pathway protein of T cells, thereby inhibiting the activation of T cells. Therefore, inhibiting the activity of SHP2 can reverse the immune suppression in the tumor microenvironment.

[0005] As an important cell signaling factor, SHP2 mutation is closely related to a variety of diseases. Studies have found that SHP2 mutations exist in neuroblastoma, AML (4%), breast cancer, NSCLC (10%), lung adenocarcinoma (30%), esophageal cancer, head and neck tumors, melanoma and gastric cancer.

[0006] Currently, there are several SHP2 allosteric inhibitors in clinical research stage, such as TNO-155 developed by Novartis, RMC-4630 developed by Revolution Medicine, and JAB-3068 developed by Beijing Gacos, etc. However, there is no SHP2 inhibitor on the market for the preparation of Noonan syndrome, leopard syndrome, leukemia, neuroblastoma, melanoma, breast cancer, esophageal cancer, head and neck tumor, lung cancer and colon cancer. Therefore, it is urgent to develop a class of SHP2 inhibitor drugs with good drug properties. SUMMARY

[0007] Problem to be solved by the invention

[0008] The present application aims to provide a new class of heterocyclic compounds as SHP2 inhibitors, which exhibit good inhibitory activity on tumor cells and good drug properties, and have broad prospects for drug development. Moreover, the preparation method of the compounds is simple, which is conducive to industrial production.

[0009] Solution for solving the problem

[0010] In a first aspect, the present application provides a compound as shown in formula I or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof, wherein

[0011]

[0012] X 1 , X 2 and X 3 are each independently selected from CR5and N, or absent;

[0013] when X 1 , X 2 and X 3 are each independently selected from CR5and N, X 5 and X 6 are each independently selected from C and N, X 7 is CR5or N;

[0014] when X 1 , X 2 and X 3 are absent, X 5 , X 6 and X 7 are each independently selected from CR5and N;

[0015] X 4 is C or N;

[0016] X 8N or NR5;

[0017] R1, R2, R3, R4, R6, and R7are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, oxo, cyano, C2-C8alkenyl, C2-C8alkynyl, aldehydo, carbamoyl, C1-C8alkyl, C1-C8heteroalkyl, C3-C8cycloalkyl, C3-C8heterocycloalkyl, C1-C8alkoxy, and C1-C3haloalkoxy, wherein each of said C1-C8alkyl, C1-C8heteroalkyl, C3-C8cycloalkyl, C3-C8heterocycloalkyl, C1-C8alkoxy, and C1-C3haloalkoxy is optionally substituted with one or more R5;

[0018] each R5, if present, is each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, cyano, carbamoyl, C1-C3alkyl, C1-C3heteroalkyl, C3-C8cycloalkyl, C3-C8heterocycloalkyl, C1-C3alkoxy, and C1-C3haloalkoxy, wherein each of said C1-C3alkyl, C1-C3heteroalkyl, C3-C8cycloalkyl, C3-C8heterocycloalkyl, C1-C3alkoxy, and C1-C3haloalkoxy is optionally substituted with one or more R8;

[0019] each R8, if present, is each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, and cyano;

[0020] A and B are each independently selected from C3-C8cycloalkyl, C3-C8heterocycloalkyl, C6-C 10 aryl, and C5-C 12 heteroaryl;

[0021] wherein each of the heteroatoms or groups of heteroatoms in said heteroalkyl, heterocycloalkyl, and heteroaryl is independently selected from the group consisting of -C(=O)NH-, -NH-, -N=, -O-, -S-, -C(=O)O-, -C(=O)-, -C(=S)-, -S(=O)-, -S(=O)2-, and -NHC(=O)NH-, the number of each of the heteroatoms or groups of heteroatoms in said heteroalkyl, heterocycloalkyl, and heteroaryl is independently selected from the group consisting of 1, 2, and 3;

[0022] n is 1, 2, or 3.

[0023] In particular, the compound as shown in Formula I is a compound as shown in Formula I-1 or Formula I-2, wherein

[0024]

[0025] X 5 , X 6 , and X 7 are each independently selected from CR5and N;

[0026] X8 R1, R2, R3, R4, R5, R6, B, and n are defined as in Formula I.

[0027] Preferably, in the compound as shown in Formula I-1 or Formula I-2,

[0028] X 5 X 6 X 7 are each independently selected from CR5and N;

[0029] X 8 is N or NR5;

[0030] R1is hydrogen, amino, oxo, or C1-C8alkyl, wherein the C1-C8alkyl is optionally substituted with one or more R5;

[0031] R2is hydrogen;

[0032] R3is hydrogen or hydroxyl;

[0033] each R4is independently selected from hydrogen, halogen, and C1-C8alkyl, wherein the C1-C8alkyl is optionally substituted with one or more R5;

[0034] R6is hydrogen;

[0035] each R5, if present, is independently selected from hydrogen, amino, aminocarbonyl, C1-C3alkyl, and C1-C3heteroalkyl, wherein the C1-C3alkyl, C1-C3heteroalkyl, C3-C8cycloalkyl, C3-C8heterocycloalkyl, C1-C3alkoxy, and C1-C3haloalkoxy are each optionally substituted with one or more R8; preferably, each R5, if present, is independently selected from hydrogen, amino, aminocarbonyl, and C1-C3alkyl, wherein the C1-C3alkyl is optionally substituted with one or more R8;

[0036] each R8, if present, is independently selected from hydrogen and hydroxyl;

[0037] B is C3-C8cycloalkyl, C3-C8heterocycloalkyl, C6-C 10 aryl, and C5-C 12 heteroaryl;

[0038] wherein the heteroatoms or groups of heteroatoms in the heteroalkyl, heterocycloalkyl and heteroaryl are each independently selected from the group consisting of -C(=0)NH-, -NH-, -N=, -0-, -S-, -C(=0)0-, -C(=0)-, -C(=S)-, -S(=0)-, -S(=0)2- and -NHC(=0)NH-, the number of heteroatoms or groups of heteroatoms in the heteroalkyl, heterocycloalkyl and heteroaryl is each independently selected from 1, 2 and 3; preferably, the heteroatoms or groups of heteroatoms in the heterocycloalkyl and heteroaryl are each independently selected from the group consisting of -C(=0)NH-, -NH-, -N=, -0-, -S-, -C(=0)0-, -C(=0)-, -C(=S)-, -S(=0)-, -S(=0)2- and -NHC(=0)NH-, the number of heteroatoms or groups of heteroatoms in the heterocycloalkyl and heteroaryl is each independently selected from 1, 2 and 3;

[0039] n is 1, 2 or 3.

[0040] More preferably, in the compound according to Formula I-1 or Formula I-2,

[0041] in the structure the fragment is selected from any one of the following fragments:

[0042]

[0043] Even more preferably, in the structure the fragment is selected from any one of the following fragments:

[0044]

[0045] or even more preferably, in the structure the fragment is selected from any one of the following fragments:

[0046]

[0047] In particular, the compound according to Formula I is a compound according to Formula I-3 or Formula I-4, wherein

[0048]

[0049] X 1 , X 2 and X 3 are each independently selected from CR5or N, X 5 and X 6 are each independently selected from C and N, X 7 is CR5or N;

[0050] X 8R1, R2, R3, R4, R5, R6, B and n are defined as in formula I.

[0051] Preferably, in the compound according to formula I-3 or I-4,

[0052] X 1 X 2 X 3 each independently is selected from CR5or N, X 5 X 6 each independently is selected from C and N, X 7 is CR5or N;

[0053] X 8 is N or NR5;

[0054] R1is hydrogen or C1-C8alkyl, wherein said C1-C8alkyl is optionally substituted with one or more R5;

[0055] R2is hydrogen;

[0056] R3is hydrogen or hydroxyl;

[0057] each R4is independently selected from hydrogen, halogen and C1-C8alkyl, wherein said C1-C8alkyl is optionally substituted with one or more R5;

[0058] R6is hydrogen;

[0059] each R5, if present, is independently selected from hydrogen and C1-C3alkyl;

[0060] B is C3-C8cycloalkyl, C3-C8heterocycloalkyl, C6-C 10 aryl and C5-C 12 heteroaryl;

[0061] wherein the heteroatoms or heteroatom groups in said heterocycloalkyl and heteroaryl are each independently selected from -C(=O)NH-, -NH-, -N=, -O-, -S-, -C(=O)O-, -C(=O)-, -C(=S)-, -S(=O)-, -S(=O)2- and -NHC(=O)NH-, the number of heteroatoms or heteroatom groups in said heterocycloalkyl and heteroaryl is each independently selected from 1, 2 and 3;

[0062] n is 1, 2 or 3.

[0063] More preferably, in the compound according to formula I-3 or I-4,

[0064] the fragment in the structure is selected from any one of the following fragments:

[0065]

[0066] Even more preferably, the structure is the fragment is selected from any one of the following fragments:

[0067]

[0068] Further preferably, in the compound of formula I-1, formula I-2, formula I-3, or formula I-4,

[0069] the structure is the fragment is selected from any one of the following fragments:

[0070]

[0071] Even further preferably, the structure is the fragment is selected from any one of the following fragments:

[0072]

[0073] In a second aspect, the present application provides specific compounds of formula I, formula I-1, formula I-2, formula I-3, or formula I-4, selected from:

[0074] (1) (S)-N-(3-(3-amino-5-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'- piperidin]-1'-yl)pyrazin-2-ylsulfanyl)-2-chlorophenyl)-2-hydroxy-4-oxo-6,7,8,9- tetrahydro-4H-pyrido[l,2-a]pyrimidine-3-carboxamide;

[0075] (2) (S)-N-(3-(5-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'-piperidin]-1'- yl)imidazo[l,2-c]pyrimidin-8-ylsulfanyl)-2-chlorophenyl)-2-hydroxy-4-oxo-6,7,8,9- tetrahydro-4H-pyrido[l,2-a]pyrimidine-3-carboxamide;

[0076] (3) (S)-N-(3-(5-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'-piperidin]-1'- yl)-7-methylimidazo[l,2-c]pyrimidin-8-ylsulfanyl)-2-chlorophenyl)-2-hydroxy-4-oxo- 6,7,8,9-tetrahydro-4H-pyrido[l,2-a]pyrimidine-3-carboxamide;

[0077] (4) (S)-N-(3-(4-amino-2-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'- piperidin]-1'-yl)-1-methyl-6-oxo-1,6-dihydropyrimidin-5-ylsulfanyl)-2-chlorophenyl)-2- hydroxy-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxamide;

[0078] (5) (S)-N-(3-(3-amino-5-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'- piperidin]-1'-yl)pyrazin-2-ylsulfanyl)-2-chlorophenyl)-4-oxo-6,7,8,9-tetrahydro-4H- pyrido[1,2-a]pyrimidine-3-carboxamide;

[0079] (6) (S)-N-(3-(3-amino-5-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'- piperidin]-1'-yl)pyrazin-2-ylsulfanyl)-2-methylphenyl)-2-hydroxy-4-oxo-6,7,8,9- tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxamide;

[0080] (7) (S)-N-(3-(3-amino-5-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'- piperidin]-1'-yl)pyrazin-2-ylsulfanyl)-5-fluoro-2-methylphenyl)-2-hydroxy-4-oxo-6,7,8,9- tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxamide;

[0081] (8) (S)-3-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'-piperidin]-1'-yl)-6-(2- chloro-3-(2-hydroxy-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxamido) phenylsulfanyl)pyrazine-2-carboxylic acid ethyl ester;

[0082] (9) (S)-3-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'-piperidin]-1'-yl)-6-(2- chloro-3-(2-hydroxy-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carboxamido) phenylsulfanyl)pyrazine-2-carboxylic acid;

[0083] (10) (S)-N-(3-(5-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'- piperidin]-1'-yl)-6-carbamoylpyrazin-2-ylsulfanyl)-2-chlorophenyl)-2-hydroxy-4- oxo-6,7,8,9-tetrahydro-4H-pyrido[l,2-a]pyrimidine-3-carboxamide;

[0084] (11) (S)-3-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'-piperidin]-1'- yl)-6-(2-chloro-3-(2-hydroxy-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[l,2-a]pyrimidine- 3-carboxamido)phenylsulfanyl)-5-methylpyrazine-2-carboxylic acid ethyl ester; and

[0085] (12) (S)-N-(3-(5-(5-amino-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'-piperidin]- 1'-yl)-6-carbamoyl-3-methylpyrazin-2-ylsulfanyl)-2-chlorophenyl)-2-hydroxy-4- oxo-6,7,8,9-tetrahydro-4H-pyrido[l,2-a]pyrimidine-3-carboxamide.

[0086] In a third aspect, the present application provides a method for preparing a compound of Formula I, comprising the following steps:

[0087] 1) reacting compound A with compound B to obtain compound C;

[0088]

[0089] 2) reacting compound C with compound D to obtain compound E; and

[0090]

[0091] 3) reacting compound E with compound F to obtain the target product;

[0092]

[0093] wherein

[0094] LG1and LG2are each independently selected from the group consisting of chlorine and bromine, and LG3is chlorine, bromine or hydroxyl;

[0095] X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8R1, R2, R3, R4, R6, R7, A, B and n are defined as in Formula I.

[0096] In the above preparation method, compound A and compound B can be reacted in the presence of a catalyst (e.g., a palladium catalyst such as Pd2(dba)3), a ligand (e.g., Xantphos), and a base (e.g., an organic base such as DIEA; or an inorganic base such as cesium carbonate) to obtain compound C; compound C and compound D can be reacted (e.g., a condensation reaction) in the presence of a base and a condensing agent (e.g., HATU, HBTU, EDCT / HOBT, etc.) to obtain compound E; and compound E and compound F can be reacted in the presence of a base (e.g., an organic base such as DIEA; or an inorganic base such as potassium carbonate, cesium carbonate, sodium tert-butoxide, etc.) to obtain a compound of Formula I.

[0097] The present application does not have a particular limitation on the reaction solvent used in each step of the above preparation method, and any solvent (e.g., DMF, DMSO, NMP, etc.) that can dissolve the starting material to some extent and does not inhibit the reaction is included in the scope of the present application. In addition, many similar modifications or equivalent replacements or corresponding solvent combinations in the art are considered to be included in the scope of the present application.

[0098] In a fourth aspect, the present application provides a pharmaceutical composition comprising a compound of Formula I, Formula I-1, Formula I-2, Formula I-3, or Formula I-4, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite, or prodrug thereof, and at least one pharmaceutically acceptable excipient.

[0099] In a fifth aspect, the present application provides a compound of Formula I, Formula I-1, Formula I-2, Formula I-3, or Formula I-4, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite, or prodrug thereof, or a pharmaceutical composition comprising the same, for use as a SHP2 inhibitor or for use in the prevention and / or treatment of a disease or disorder associated with abnormal SHP2 activity.

[0100] In a sixth aspect, the present application provides the use of a compound of Formula I, Formula I-1, Formula I-2, Formula I-3, or Formula I-4, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite, or prodrug thereof, or a pharmaceutical composition comprising the same, in the preparation of a medicament for the prevention and / or treatment of a disease or disorder associated with abnormal SHP2 activity.

[0101] In a seventh aspect, the present application provides a method for preventing and / or treating a disease or disorder associated with abnormal SHP2 activity, comprising administering to an individual in need thereof a prophylactically and / or therapeutically effective amount of a compound as represented by Formula I, Formula I-1, Formula I-2, Formula I-3, or Formula I-4, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite, or prodrug thereof, or a pharmaceutical composition comprising the same.

[0102] Preferably, in the above medical uses, the disease or disorder associated with abnormal SHP2 activity is selected from Noonan syndrome, Leopard syndrome, leukemia, neuroblastoma, melanoma, breast cancer, esophageal cancer, lung cancer, colon cancer, head and neck tumor, gastric cancer, anaplastic large cell lymphoma, and glioblastoma, preferably non-small cell lung cancer, esophageal cancer, and head and neck tumor.

[0103] Effects of the invention

[0104] The heterocyclic compound in the present application is a novel allosteric inhibitor, which can inhibit the activity of SHP2 by binding to the non-catalytic region of SHP2 and "locking" the weakly active basal state of SHP2, thereby achieving the purpose of inhibiting the activity of SHP2. The heterocyclic compound in the present application overcomes the shortcomings of poor selectivity and drugability of PTP catalytic region inhibitors, and exhibits excellent biological activity and drugability, and has great prospects for drug development.

[0105] In addition, compared with known compounds RMC4550 and TNO155, the compound of the present application exhibits superior pharmacological activity and pharmacokinetic properties in the same condition of SHP2 enzyme activity inhibition experiment, phosphorylated protein kinase (p-ERK) cell experiment, NCI-H358 cell anti-proliferation experiment, MV-4-11 cell anti-proliferation experiment, and other evaluation systems. DETAILED DESCRIPTION

[0106] General terms and definitions

[0107] Unless otherwise stated, the terms used in the present application have the following meanings.

[0108] "Alkyl" refers to saturated aliphatic hydrocarbon radicals including straight chain and branched chain groups of 1 to 20 carbon atoms, for example, straight chain and branched chain groups of 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In the present application, "alkyl" can be a monovalent, divalent, or trivalent radical. Non-limiting examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, and various branched isomers thereof, and the like. Non-limiting examples also include, but are not limited to, methylene, methine, ethylene, ethine, propylene, propine, butylene, butine, and various branched isomers thereof. Additionally, in the present application, "alkyl" can be optionally substituted or unsubstituted.

[0109] "Heteroalkyl" refers to saturated aliphatic hydrocarbon radicals including straight chain and branched chain groups of 1 to 20 carbon atoms, for example, straight chain and branched chain groups of 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms, the straight chain or branched chain carbon chain being interrupted and attached by one or more (e.g., 1, 2, 3, etc.) heteroatoms or groups of heteroatoms selected from -C(=O)NH-, -NH-, -N=, -O-, -S-, -C(=O)O-, -C(=O)-, -C(=S)-, -S(=O)-, -S(=O)2-, and -NHC(=O)NH-. In the present application, "heteroalkyl" can be a monovalent, divalent, or trivalent radical. Non-limiting examples include, but are not limited to, C1-C3 heteroalkyl interrupted by -C(=O)O-, for example, carboxy (C1 heteroalkyl), methoxycarbonyl (C2 heteroalkyl), ethoxycarbonyl (C3 heteroalkyl), and the like, or C1-C3 heteroalkyl interrupted by -C(=O)NH-, for example, carbamoyl (C1 heteroalkyl), methylcarbamoyl (C2 heteroalkyl), ethylcarbamoyl (C3 heteroalkyl), and the like. Additionally, in the present application, "heteroalkyl" can be optionally substituted or unsubstituted.

[0110] "Cycloalkyl" refers to saturated or partially unsaturated, monocyclic or polycyclic aliphatic hydrocarbon groups including 3 to 12 ring atoms, for example, can be 3 to 12, 3 to 10, or 3 to 6 ring atoms (i.e., 3 to 6 membered rings). Non-limiting examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. In the present application, "cycloalkyl" can be optionally substituted or unsubstituted.

[0111] "Heterocycloalkyl" refers to saturated or partially unsaturated, monocyclic or polycyclic aliphatic hydrocarbon groups including 3 to 20 ring atoms, for example, can be 3 to 16, 3 to 12, 3 to 10, or 3 to 6 ring atoms, wherein one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) m (wherein m is 0, 1, or 2) and the remaining ring atoms are carbon. Preferred heterocycloalkyl groups include 3 to 12 ring atoms, wherein 1 to 4 ring atoms are heteroatoms, more preferably 3 to 10 ring atoms, most preferably 5 or 6 ring atoms, wherein 1 to 4, preferably 1 to 3, more preferably 1 to 2 are heteroatoms. Non-limiting examples of monocyclic heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Non-limiting examples of polycyclic heterocycloalkyl groups include, but are not limited to, spirocyclic or bridged ring heterocycloalkyl groups.

[0112] "Halogen" refers to fluorine, chlorine, bromine, and iodine, preferably fluorine, chlorine, and bromine.

[0113] "Optional" or "optionally" means that the subsequently described event or circumstance can or can not occur, and thus such description includes instances in which the event or circumstance occurs and instances in which it does not. For example, "heterocyclyl optionally substituted with alkyl" means that alkyl can or can not be present, and such description includes instances in which the heterocyclyl group is substituted with alkyl and instances in which the heterocyclyl group is not substituted with alkyl.

[0114] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3, of a group are independently of each other replaced by a corresponding number of substituents.

[0115] "Pharmaceutically acceptable salt" refers to a salt prepared from a compound in the present application with a relatively non-toxic acid or base.

[0116] "Pharmaceutical composition" refers to a composition that is useful in medicine, which comprises one or more compounds as shown in Formula I or a pharmaceutically acceptable form thereof (e.g., salt, hydrate, solvate, stereoisomer, tautomer, metabolite, prodrug, etc.), and other components (e.g., pharmaceutically acceptable excipient).

[0117] In the present application, "pharmaceutically acceptable excipient" refers to an auxiliary material widely used in the field of pharmaceutical production. The main purpose of using an excipient is to provide a pharmaceutical composition that is safe to use, stable in properties, and / or has a specific functionality, and to provide a method by which, after administration of the pharmaceutical to a subject, the active ingredient can be dissolved at a desired rate or facilitate the effective absorption of the active ingredient in the subject to whom the drug is administered. The pharmaceutically acceptable excipient can be an inert filler or a functional ingredient that provides a certain function to the pharmaceutical composition, such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient in the composition. Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifying agents, diluents (or fillers), granulating agents, adhesives, disintegrants, lubricants, anti-adherents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavorants, sweeteners, and the like.

[0118] The pharmaceutical composition in the present application can be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, micronizing, encapsulating, entrapping, and / or lyophilizing processes.

[0119] In the present application, the purpose of using the pharmaceutical composition is to facilitate the administration to an organism, facilitate the absorption of the active ingredient, and thereby exert a biological activity. The pharmaceutical composition of the present application can be administered by any form, including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid preparation, oral liquid preparation), rectal, inhalation, implantation, topical (e.g., ocular) administration, and the like. Non-limiting examples of oral solid preparations include, but are not limited to, powders, capsules, tablets, granules, tablets, and the like. Non-limiting examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, and the like. Non-limiting examples of preparations for topical administration include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serums. Non-limiting examples of parenteral administration preparations include, but are not limited to, solutions for injection, dry powders for injection, suspensions for injection, emulsions for injection, and the like. The pharmaceutical composition of the present application can also be prepared into a controlled release or delayed release dosage form (e.g., a liposome or a microsphere).

[0120] Preferably, the compound in the present application or the pharmaceutical composition comprising the same is administered to an individual in need thereof in a manner of oral or intravenous administration. Depending on the specific condition of the administration subject, even other administration routes can be applied or are preferred. For example, for forgetful or irritable patients to oral drugs, transdermal administration will be a very important administration manner. In the present application, the administration route can be varied or adjusted in any applicable manner to meet the needs of the nature of the drug, the convenience of the patient and medical staff, and other related factors.

[0121] The compound of the present application or its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof or the pharmaceutical composition comprising the same has excellent SHP2 enzyme activity and cell proliferation inhibition activity, can be used as a SHP2 inhibitor for preventing and / or treating diseases or disorders related to abnormal SHP2 activity (or caused by abnormal SHP2 mutation), and has good clinical application and medical use. Preferably, non-limiting examples of diseases or disorders related to abnormal SHP2 activity (or caused by abnormal SHP2 mutation) include but are not limited to Noonan syndrome, leopard syndrome, leukemia (such as juvenile myelomonocytic leukemia, acute myeloid leukemia), neuroblastoma, melanoma, breast cancer, esophageal cancer, lung cancer, colon cancer, head and neck tumor, gastric cancer, anaplastic large cell lymphoma, glioblastoma, etc., preferably non-small cell lung cancer, esophageal cancer and head and neck tumor.

[0122] The technical solutions of the present application will be described below in combination with specific examples, and the following examples are provided to further illustrate the present application, but not to limit the scope of the present application. It will be apparent to those skilled in the art that various changes and improvements can be made to the specific embodiments of the present application without departing from the spirit and scope of the present application.

[0123] The preparation of the compounds of the present application can be achieved by synthetic methods well known to those skilled in the art, including but not limited to the specific embodiments listed below, embodiments formed by a combination of the other chemical synthetic methods well known to those skilled in the art, and equivalent replacements well known to those skilled in the art, and preferred embodiments include but are not limited to the examples of the present application. The known starting materials used in the present application can be synthesized by methods known in the art or purchased by conventional commercial means (for example, from companies such as Shaoyuan Chemical Technology, Beijing Coupling Technology, etc.). Unless otherwise specified, the reactions are carried out under an argon or nitrogen atmosphere. The hydrogenation reaction is usually vacuumed and filled with hydrogen, and the operation is repeated 3 times. The reaction temperature is room temperature, and the temperature range is 20-30°C. The monitoring of the reaction progress can be achieved by synthetic methods well known to those skilled in the art, including but not limited to thin layer chromatography (TLC). The thin layer chromatography silica gel plate uses Qingdao Ocean GF254 silica gel plate, and the developing agent system includes but is not limited to A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system, and the volume ratio of the solvents can be adjusted according to the polarity of the compound.

[0124] The separation and purification of the compounds of the present application can be achieved by synthetic methods well known to those skilled in the art, including but not limited to column chromatography (CC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UPLC), etc. The column chromatography generally uses Qingdao Ocean 200-300 mesh silica gel as the carrier, and the eluent system includes but is not limited to A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system, and the volume ratio of the solvents can be adjusted according to the polarity of the compound, or a small amount of acidic or basic anti-tailing reagent can be added for adjustment. The HPLC spectrum is determined by Agilent 1200DAD HPLC chromatograph (chromatographic column: Sunfire C18, 150x4.6mm, 5μm) or Waters 2695-2996 HPLC chromatograph (chromatographic column: Gimini C18, 150x4.6mm, 5μm).

[0125] The structural identification of the compounds of the present application can be achieved by methods well known to those skilled in the art, including but not limited to nuclear magnetic resonance (NMR), mass spectrometry (MS), etc. The NMR spectrum is determined by Bruker AVANCE-400 or Varian Oxford-300 nuclear magnetic instrument, the determination solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDC13) or deuterated methanol (CD3OD), the internal standard is tetramethylsilane (TMS), and the chemical shift is counted in 10 -6 (ppm). The MS spectrum is determined by Agilent SQD (ESI) mass spectrometer (model: 6110) or Shimadzu SQD (ESI) mass spectrometer (model: 2020).

[0126] Synthesis of intermediates

[0127] Synthesis of intermediate Q1

[0128]

[0129] First step: synthesis of Q1-2

[0130] Compound Q1-1 (100 g, 495 mmol) was added to DME (1000 ml), cooled to -10 °C, then isobutyl chloroformate (67.6 g, 495 mmol) and 4-methylmorpholine (50 g, 495 mmol) were added, and the reaction was carried out at room temperature for 5 h. After the reaction was completed as monitored by TLC, the solid was filtered, washed with DME (250 ml), and then the filtrate was added to a 2 L three-necked flask. The filtrate was treated with sodium borohydride (37.6 g, 990 mmol), stirred at room temperature for 30 min, and then methanol (250 ml) was slowly added dropwise. The reaction was continued at room temperature for 3 h. After the reaction was completed as monitored by TLC, the reaction liquid was rotary evaporated, water (1500 ml) was added, and the aqueous phase was extracted with DCM (300 ml x 3). The combined organic phase was washed with water and saturated sodium chloride solution in turn, and then dried. After rotary evaporation, compound Q1-2 (86.5 g, white solid) was obtained in a yield of 93%. The crude product was used directly in the next step without purification.

[0131] Second step: synthesis of Q1-3

[0132] Compound Q1-2 (25 g, 133 mmol) was added to DCM (250 ml), and then TEA (26.9 g, 266 mmol) was added. MsCl (18.3 g, 156 mmol) was added dropwise under ice bath, and then the reaction was carried out at room temperature for 1 h. After the reaction was completed as monitored by TLC, the reaction liquid was diluted with DCM, washed with water and saturated sodium chloride solution in turn, and then dried. After rotary evaporation, the residue was purified by silica gel column chromatography (eluent: V 石油醚 :V 乙酸乙酯 = 3:1 ~ 1:1) to obtain compound Q1-3 (33.6 g, colorless transparent liquid) in a yield of 95%.

[0133] MS (ESI): m / z 266 [M+H] + .

[0134] Third step: synthesis of Q1-4

[0135] N-tert-butoxycarbonyl-4-cyanopiperidine (28 g, 130 mmol) was added to THF (300 ml), cooled to -78 °C, then slowly added 2.0 M LDA (75 ml, 150 mmol) dropwise, after the dropwise addition was completed, continued to maintain -78 °C for 1.5 h, then added a THF solution (150 ml) of compound Q1-3 (26.6, 100 mmol) dropwise, after the dropwise addition was completed, continued to maintain -78 °C for 3 h, TLC showed that the reaction was completed, then added saturated ammonium chloride solution (50 ml) to quench the reaction, and added saturated sodium chloride solution (500 ml), separated the organic phase, the aqueous phase was extracted with ethyl acetate (150 ml x 3), the combined organic phase was dried over anhydrous sodium sulfate, filtered, concentrated by rotary evaporation, and the residue was purified by silica gel column chromatography (eluent: V 石油醚 :V 乙酸乙酯 = 10:1 ~ 1:1) to obtain compound Q1-4 (25.7 g, white solid), in a yield of 67.6%.

[0136] MS (ESI): m / z 380 [M+H] + .

[0137] Fourth step: synthesis of Q1-5

[0138] Compound Q1-4 (25 g, 65.8 mmol) was added to a mixed solvent of DMA (200 ml) and water (20 ml), then added triethylamine (33.2 g, 329 mmol) and a catalyst Pd(amphos)Cl2 (4.6 g, 6.6 mmol, CAS: 887919-35-9), and reacted at 130 °C for 4 h under nitrogen protection. TLC showed that the reaction was completed, then the reaction liquid was cooled, diluted with water (800 ml), the aqueous phase was extracted with ethyl acetate (200 ml x 3), the combined organic phase was washed with saturated brine (200 ml x 2), dried over anhydrous sodium sulfate, filtered, concentrated by rotary evaporation, and the residue was purified by silica gel column chromatography (eluent: V 石油醚 :V 乙酸乙酯 = 3:1 ~ 1:1) to obtain compound Q1-5 (14.6 g, white solid), in a yield of 73%.

[0139] MS (ESI): m / z 303 [M+H] + .

[0140] Fifth step: synthesis of Q1-6

[0141] Compound Q1-5 (10 g, 33 mmol) was added to tetraethyl titanate (100 ml), then (R)-(+)-tert-butylsulfinamide (4.8 g, 40 ml) was added, and the mixture was warmed to 90°C and reacted for 3h. After TLC showed that the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was slowly added to ice water (500 ml). The aqueous phase was extracted with dichloromethane (150 ml x 3), and the combined organic phases were washed with saturated brine (100 ml x 2), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The residue was purified by silica gel column chromatography (eluent: V(V=3:1~1:1) to obtain compound Q1-6 (12 g, yellow solid) at a yield of 89.6%. 石油醚 :V 乙酸乙酯

[0142] MS (ESI): m / z 406 [M+H] + .

[0143] Step 6: Synthesis of Q1-7

[0144] Compound Q1-6 (10 g, 24.6 mmol) was added to tetrahydrofuran (100 ml), and the mixture was cooled to -78°C. Then, DIBAL-H (30 ml, 30 mmol, 1M solution in toluene) was slowly added dropwise, and the mixture was further reacted at -78°C for 0.5h. After TLC showed that the reaction was complete, saturated Rochelle salt solution (300 ml) was added at -50°C to quench the reaction. The mixture was stirred at room temperature for 30 min, and the aqueous phase was extracted with ethyl acetate (150 ml x 3). The combined organic phases were washed with saturated brine (100 ml x 2), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The residue was purified by silica gel column chromatography (eluent: V(V=3:1~1:1) to obtain compound Q1-7 (8.6 g, white solid) at a yield of 85.3%. 石油醚 :V 乙酸乙酯

[0145] MS (ESI): m / z 408 [M+H] + .

[0146] Step 7: Synthesis of intermediate Q1

[0147] Compound Q1-7 (5 g, 12.2 mmol) was added to ethyl acetate (50 ml), and 4M HCl / ethyl acetate solution (25 ml) was further added. The mixture was reacted at room temperature for 1h. After TLC showed that the reaction was complete, the mixture was filtered, and the solid was washed with ethyl acetate and dried in an oven to obtain the hydrochloride salt of intermediate Q1 (3.2 g, white solid) at a yield of 94%.

[0148] MS (ESI): m / z 204 [M+H] + .

[0149] ​​Synthesis of intermediate Q2

[0150]

[0151] First Step: Synthesis of Q2-2

[0152] Compound Q2-1 (205 mg, 1 mmol) was dissolved in dioxane (5 ml), then 3- mercaptopropionic acid methyl ester (180 mg, 1.5 mmol), Pd2(dba)3(22.73 mg, 0.025 mmol, 0.05 eq), Xantphos (14.36 mg, 0.025 mmol) and DIEA (387 mg, 3 mmol) were added. After stirring at 90 °C for 1 h under nitrogen protection, the reaction mixture was concentrated by rotary evaporation, and the residue was purified by silica gel column chromatography (eluent: V 石油醚 :V 乙酸乙酯 = 3:1 ~ 1:1) to give compound Q2-2 (201 mg, yellowish liquid), 83% yield.

[0153] MS (ESI): m / z 246 [M+H] + .

[0154] Second Step: Synthesis of Intermediate Q2

[0155] Compound Q2-2 (200 mg, 0.81 mmol) was dissolved in THF (2 ml), then cooled to 0 °C, and sodium tert-butoxide (96 mg, 1 mmol) was added. After stirring at 0 °C for 0.5 h, the reaction mixture was diluted with PE, and a large amount of solid was precipitated. The precipitated solid was collected by filtration and washed with ethyl acetate to give intermediate Q2 (130 mg, light yellow solid), 88% yield.

[0156] MS (ESI): m / z 182 [M+H] + .

[0157] Synthesis of intermediate Q3

[0158]

[0159] First Step: Synthesis of Q3-2

[0160] Compound Q3-1 (10 g, 106 mmol) was added to xylene (100 ml), then triethyl methane tricarboxylate (48 g, 222 mmol, CAS:6279-86-3) was added, after the addition was completed, the temperature was raised to 140 °C for 3 h, TLC showed that the reaction was completed, after cooling to room temperature, a solid was precipitated, filtration, the filter cake was washed with diethyl ether, and then dried to obtain compound Q3-2 (16.2 g, light yellow solid), 65% yield. The crude product was used directly in the next step without purification.

[0161] MS (ESI): m / z 235 [M+H] + .

[0162] Second step: synthesis of intermediate Q3-3

[0163] Compound Q3-2 (5 g, 21 mmol) was added to a mixed solvent of methanol (40 ml) and water (10 ml), a hydrogen balloon was covered, after replacing the air with hydrogen, the reaction was carried out for 3 h after hydrogen was passed, TLC showed that the reaction was completed, filtration, the filter cake was washed with methanol (50 ml), and the filtrate was rotary dried to obtain compound Q3-3 (4.3 g, light yellow solid), yield 92%. The crude product was used directly in the next step without purification.

[0164] MS (ESI): m / z 239 [M+H] + .

[0165] Third step: synthesis of intermediate Q3

[0166] Compound Q3-3 (5 g, 21 mmol) was added to a mixed solvent of methanol (50 ml) and water (10 ml), then lithium hydroxide monohydrate (1.7 g, 42 mmol) was added, stirring at room temperature for 2 h, TLC showed that the reaction was completed, the pH value was adjusted to 3-4 with 1M hydrochloric acid, a large amount of solid was precipitated, filtration, the filter cake was dried to obtain intermediate Q3 (3.98 g, white solid), yield 90%. The crude product was used directly in the next step without purification.

[0167] MS (ESI): m / z 211 [M+H] + .

[0168] Synthesis of intermediate Q4

[0169]

[0170] First step: synthesis of Q4-1

[0171] A solution of compound Q2-2 (1.5 g, 6.1 mmol) and compound Q3-3 (1.2 g, 5.0 mmol) in DMF (15 ml) was heated at 160 °C for 2.5 h. After cooling, the reaction mixture was added to 75 ml of saturated sodium chloride solution, then extracted with EtOAc (25 ml x 3), the organic phases were combined, washed with saturated sodium chloride solution (100 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was triturated with MeOH to give compound Q4-1 (196 g, yellow solid) with a yield of 92%.

[0172] MS (ESI): m / z 438.2 [M+H] + .

[0173] Second step: synthesis of Q4

[0174] Compound Q4-1 (500 mg, 1.14 mmol) was dissolved in THF (2 ml), then cooled to 0 °C, and then sodium tert-butoxide (131 mg, 1.37 mmol) was added, stirred at 0 °C for 0.5 h, after TLC showed that the reaction was complete, the reaction mixture was diluted with PE, a large amount of solid was precipitated, the precipitated solid was collected by filtration and washed with ethyl acetate to give intermediate Q4 (370 mg, light yellow solid) with a yield of 87%.

[0175] Synthesis of intermediate Q5

[0176]

[0177] First step: synthesis of Q5-2

[0178] A mixture of compound 2-aminopyridine (4.35 g, 46.3 mmol) and diethyl ethoxymethylmalonate (10.00 g, 46.3 mmol, cas: 87-13-8) was heated at 120 °C for 1 h. After cooling to room temperature, further cooling to 5 °C, the mixture was slurried with ice ethanol, filtered, and the solid was washed with ice ethanol and dried to give compound Q5-2 (9.90 g, yellow solid) with a yield of 81%.

[0179] MS (ESI): m / z 265.2 [M+H] + .

[0180] Second step: synthesis of Q5-3

[0181] Compound Q5-2 (13 g, 5.9 mmol) was added to diphenyl ether (100 ml) and heated to 280 °C and refluxed for 2 h. After completion of the reaction by TLC, the reaction mixture was cooled to room temperature and n-hexane (500 ml) was added to it to obtain a large amount of precipitated solid. The precipitated solid was filtered, washed with n-hexane and dried to obtain compound Q5-3 (10 g, yellow solid) with 93.1% yield.

[0182] MS (ESI): m / z 219.2 [M+H] + .

[0183] Third step: synthesis of Q5

[0184] Compound Q5-3 (5 g, 23 mmol) was added to a mixture of methanol (40 ml) and water (10 ml) and a hydrogen balloon was fitted. After replacing the air with hydrogen, the reaction was carried out for 3 h after which the reaction was completed by TLC. It was filtered, the filter cake was washed with methanol (50 ml) and the filtrate was evaporated to obtain compound Q5 (4.6 g, light yellow solid) with 92% yield. The crude product was used directly in the next step without purification.

[0185] Synthesis of intermediate Q6

[0186]

[0187] First step: synthesis of Q6-2

[0188] Compound Q2-1 (205 mg, 1 mmol) was dissolved in dioxane (5 ml) and then 3- mercaptopropionic acid methyl ester (180 mg, 1.5 mmol), Pd2(dba)3(22.73 mg, 0.025 mmol, 0.05 equivalent), Xantphos (14.36 mg, 0.025 mmol) and DIEA (387 mg, 3 mmol) were added. After warming to 90 °C under nitrogen protection and stirring for 1 h, the reaction mixture was concentrated by evaporation and the residue was purified by silica gel column chromatography (eluent: V 石油醚 :V 乙酸乙酯 = 3:1 ~ 1:1) to obtain compound Q2-2 (201 mg, light yellow liquid) with 83% yield.

[0189] MS (ESI): m / z 246 [M+H] + .

[0190] Second step: synthesis of intermediate Q6

[0191] Compound Q2-2 (200 mg, 0.81 mmol) was dissolved in THF (2 ml), then cooled to 0 °C, and sodium tert-butoxide (96 mg, 1 mmol) was added. The mixture was stirred at 0 °C for 0.5 h. After the reaction was complete as shown by TLC, the reaction mixture was diluted with PE, and a large amount of solid precipitated. The precipitate was collected by filtration and washed with ethyl acetate to give intermediate Q2 (130 mg, pale yellow solid) in 88% yield.

[0192] MS(ESI): m / z 182[M+H] + .

[0193] Synthesis of intermediate Q7

[0194]

[0195] Step 1: Synthesize Q7-2

[0196] Compound Q2-1 (205 mg, 1 mmol) was dissolved in dioxane (5 ml), followed by the addition of methyl 3-mercaptopropionate (180 mg, 1.5 mmol), Pd2(dba)3 (22.73 mg, 0.025 mmol, 0.05 equivalent), Xantphos (14.36 mg, 0.025 mmol), and DIEA (387 mg, 3 mmol). The mixture was heated to 90 °C under nitrogen protection and stirred for 1 hour. The reaction mixture was then concentrated to dryness, and the residue was purified by silica gel column chromatography (eluent: V). 石油醚 :V 乙酸乙酯 The mixture was purified by a ratio of 3:1 to 1:1 to give compound Q2-2 (201 mg, pale yellow liquid) in 83% yield.

[0197] MS(ESI): m / z 246 [M+H] + .

[0198] Step 2: Synthesizing intermediate Q7

[0199] Compound Q7-2 (200 mg, 0.81 mmol) was dissolved in THF (2 ml), then cooled to 0 °C, and sodium tert-butoxide (96 mg, 1 mmol) was added. The mixture was stirred at 0 °C for 0.5 h. After the reaction was complete as shown by TLC, the reaction mixture was diluted with PE, and a large amount of solid precipitated. The precipitate was collected by filtration and washed with ethyl acetate to give intermediate Q2 (130 mg, pale yellow solid) in 88% yield.

[0200] MS(ESI): m / z 182[M+H] + .

[0201] Example 1: Preparation of Compound 1

[0202]

[0203] The specific synthesis route is as follows:

[0204]

[0205] First step: synthesis of compound 1B

[0206] Compound 1A (104 mg, 0.5 mmol) was dissolved in dioxane (5 ml), and intermediate Q2 (182 mg, 1 mmol), Pd2(dba)3(22.73 mg, 0.025 mmol), Xantphos (14.36 mg, 0.025 mmol) and DIEA (190 mg, 1.5 mmol) were added. After being heated to 90°C under nitrogen protection and stirred for 1 h, the reaction mixture was concentrated by rotary evaporation, and the residue was purified by silica gel column chromatography (eluent: V 二氯甲烷 :V 甲醇 = 50:1~10:1) to obtain compound 1B (107 mg, light yellow solid) with a yield of 75%.

[0207] MS (ESI): m / z 287 [M+H] + .

[0208] Second step: synthesis of compound 1C

[0209] Compound 1B (100 mg, 0.35 mmol), DIEA (154 mg, 1.2 mmol), intermediate Q3 (147 mg, 0.7 mmol) and HATU (456 mg, 1.2 mmol) were added to DMF, and stirred at room temperature for 12 h. After TLC showed that the reaction was completed, the reaction solution was diluted with dichloromethane (10 ml), and the organic phase was washed with saturated aqueous sodium chloride solution, 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 二氯甲烷 :V 甲醇 = 50:1~10:1) to obtain compound 1B (137 mg, light yellow solid) with a yield of 82%.

[0210] MS (ESI): m / z 479 [M+H] + .

[0211] Third step: synthesis of compound 1

[0212] Compound 1C (100 mg, 0.21 mmol), intermediate Q1 (71 mg, 0.35 mmol) and DIEA (82 mg, 0.63 mmol) were dissolved in dimethyl sulfoxide (5 ml) and reacted at 90 °C for 1.5 h. After the reaction was completed, ethyl acetate (20 ml) and water (40 ml) were added, the aqueous phase was extracted with ethyl acetate (20 ml x 3), the combined organic phase was washed with saturated sodium oxide solution (100 ml), dried over anhydrous sodium sulfate, filtered, the filtrate was collected and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: V 二氯甲烷 :V 甲醇 = 50:1~10:1) to obtain compound 1 (32 mg, light yellow solid).

[0213] MS (ESI): m / z 646 [M+H] + .

[0214] 1 H-NMR (400MHz, DMSO-d6): δ 12.26 (brs, 1H), 8.54-8.53 (m, 1H), 8.31 (m, 3H), 8.15 (m, 1H), 7.90-7.88 (m, 1H), 7.70 (s, 1H), 7.36-7.21 (m, 1H), 6.47-6.45 (m, 1H), 6.20-6.18 (m, 2H), 4.48-4.67 (m, 1H), 4.35-4.23 (m, 2H), 3.87-3.67 (m, 2H), 3.26-3.13 (m, 4H), 2.90-2.87 (m, 2H), 2.02-1.72 (m, 5H), 1.55-1.45 (m, 3H).

[0215] Example 2: Preparation of compound 2

[0216]

[0217] The specific synthesis route is as follows:

[0218]

[0219] First step: synthesis of compound 2B

[0220] Compound 2A (1.0 g, 3.6 mmol), compound Q1 (808 mg, 4.0 mmol) and DIEA (4.6 g, 36 mmol) were added into MeCN (2240 mL), then the resulting solution was stirred at 80 °C for 2 h. TLC showed the reaction was completed. The mixture was cooled to 25 °C, and Boc20 (1.6 g, 7.2 mmol) was added into the solution. The reaction was stirred at 50 °C for 2 h. TLC showed the reaction was completed. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 2, V / V) to give compound 2B (953 mg, light yellow solid), 48.4% yield.

[0221] MS (ESI): m / z 547.1 [M+H] + .

[0222] Second step: synthesis of compound 2C

[0223] Compound 2C (900 mg, 1.6 mmol) was dissolved in NMP (10 ml), and intermediate Q4 (895 mg, 2.4 mmol), Pd2(dba)3 (145 mg, 0.16 mmol), Xantphos (92 mg, 0.16 mmol) and DIEA (619 mg, 4.8 mmol) were added. After being heated to 120 °C and stirred for 1 h under nitrogen protection, the reaction was completed by TLC, then the reaction mixture was added to 75 ml saturated sodium chloride solution, and extracted with EtOAc (25 ml x 3), the organic phase was combined, washed with saturated sodium chloride solution (100 ml), dried over anhydrous sodium sulfate, rotary concentrated, and the residue was separated and purified by preparative high performance liquid chromatography (Welch Xtrimate C 18 150*30mm*5μM; condition: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min), to give compound 2C (553 mg, light yellow solid), 45% yield.

[0224] MS (ESI): m / z 770.2 [M+H] + .

[0225] Third step: synthesis of compound 2

[0226] Compound 2C (500 mg, 0.65 mmol) was added to ethyl acetate (5 ml), and 4M HCl / ethyl acetate solution (10 ml) was added, and the reaction was carried out at room temperature for 1 h. After TLC showed that the reaction was completed, it was filtered, the solid was washed with ethyl acetate, and dried to give the hydrochloride salt of compound 2 (400 mg, white solid), yield 94%.

[0227] MS (ESI): m / z 670.2 [M+H] + .

[0228] 1 H NMR (400 MHz, DMSO) δ 12.28 (s, 1H), 9.13-8.86 (m, 3H), 8.67 (d, J = 5.6 Hz, 1H), 8.43-8.27 (m, 2H), 8.22-8.16 (m, 1H), 8.07 (d, J = 25.2 Hz, 2H), 7.66-7.57 (m, 1H), 7.21 (t, J = 8.0 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 4.62-4.58 (m, 1H), 4.16-4.13 (m, 1H), 4.07-4.02 (m, 1H), 3.88 (t, J = 6.0 Hz, 2H), 3.55-3.48 (m, 3H), 3.30-3.24 (m, 1H), 2.90 (t, J = 6.4 Hz, 2H), 2.18-2.09 (m, 1H), 2.08-1.99 (m, 1H), 1.95-1.88 (m, 2H), 1.87-1.77 (m, 3H), 1.70 (d, J = 13.6 Hz, 1H).

[0229] Example 3: Preparation of compound 3

[0230]

[0231] The specific synthesis route is as follows:

[0232]

[0233] First step: synthesis of compound 3B

[0234] Compound 3A (6 g, 24.80 mmol) was dissolved in DMF (50 mL), and a solution of DIEA (8.66 mL, 49.61 mmol) and aminoacetaldehyde dimethyl acetal (2.90 g, 27.28 mmol) in DMF was added dropwise at 0 °C, and the reaction was allowed to proceed at room temperature for 2 h. After TLC showed that the reaction was complete, it was added to water and extracted with ethyl acetate, and the organic phase was washed with brine, dried over anhydrous sodium sulfate, and the residue was purified by silica gel column chromatography (eluent: PE:EA = 5:1 (by volume)) to obtain compound 3B (7.4 g, light yellow solid) with a yield of 91%.

[0235] MS (ESI): m / z 670.2 [M+H] + .

[0236] Second step: synthesis of compound 3C

[0237] Compound 3B (5 g, 16.10 mmol) was slowly added into concentrated sulfuric acid (15 mL) in an ice bath, and the temperature was raised to 75 °C for 2 h. TLC showed that the reaction was completed. The reaction solution was cooled to 0 °C, and 5N aqueous sodium hydroxide solution was added dropwise to adjust the pH to 6. Yellow solid precipitated, which was filtered and dried to obtain compound 3C (2.77 g, yellow solid), with a yield of 71%.

[0238] MS (ESI): m / z 228 [M+H] + .

[0239] Step 3: Synthesis of compound 3D

[0240] Compound 3C (2.77 g, 12.15 mmol) was added to POCl3 (30 mL), and DIEA (4.24 mL, 24.29 mmol) was added dropwise at 0 °C. The temperature was raised to 115 °C for 15 min. After TLC showed that the reaction was completed, the reaction solution was rotary evaporated, ethyl acetate was added and slowly poured into water, and the pH was adjusted to neutral with saturated aqueous sodium bicarbonate solution. The organic phase was extracted with brine, dried over anhydrous sodium sulfate, and the residue was purified by silica gel column chromatography (eluent, PE:EA = 3:1 (by volume)) to obtain compound 3D (1.17 g, light yellow solid), with a yield of 38%.

[0241] MS (ESI): m / z 246 [M+H] + .

[0242] Step 4: Synthesis of compound 3E

[0243] Compound 3D (886 mg, 3.6 mmol), compound Q1 (808 mg, 4.0 mmol) and DIEA (4.6 g, 36 mmol) were added to MeCN (40 mL), and the resulting solution was stirred at 80 °C for 2 hours. TLC showed that the reaction was completed. The mixture was cooled to 25 °C, and Boc2O (1.6 g, 7.2 mmol) was added to the solution. The reaction was stirred at 50 °C for 2 hours. TLC showed that the reaction was completed. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 2, V / V) to obtain compound 3E (786 mg, light yellow solid), with a yield of 38.9%.

[0244] MS (ESI): m / z 561.1 [M+H] + .

[0245] Step 5: Synthesis of compound 3F

[0246] Compound 3E (700 mg, 1.24 mmol) was dissolved in NMP (10 ml), and intermediate Q4 (895 mg, 2.4 mmol), Pd2(dba)3(145 mg, 0.16 mmol), Xantphos (92 mg, 0.16 mmol) and DIEA (619 mg, 4.8 mmol) were added. After heating to 120 °C under nitrogen for 1 h, the reaction was complete by TLC, the reaction mixture was added to 75 ml saturated sodium chloride solution, and then extracted with EtOAc (25 ml x 3), the organic phases were combined, washed with saturated sodium chloride solution (100 ml), dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and the residue was purified by preparative high performance liquid chromatography (Welch Xtrilate C18 ODS3 30 mm x 150 mm x 5 μm; conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min) to give compound 3F (340 mg, yellowish solid), 35% yield. 18 150*30 mm x 5 μm; conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min) to give compound 3F (340 mg, yellowish solid), 35% yield.

[0247] MS (ESI): m / z 784.2 [M+H] + .

[0248] Step 6: Synthesis of compound 3

[0249] Compound 3F (300 mg, 0.38 mmol) was added to ethyl acetate (5 ml), and 4M HCl / ethyl acetate solution (10 ml) was added, and the reaction was allowed to proceed at room temperature for 1 h. After the reaction was complete by TLC, it was filtered, the solid was washed with ethyl acetate, and dried in an oven to give the hydrochloride salt of compound 3 (239 mg, white solid), 92% yield.

[0250] MS (ESI): m / z 684.2 [M+H] + .

[0251] 1H NMR (400 MHz, DMSO) δ 12.28 (s, 1H), 9.31 (s, 1H), 8.55 (d, J = 4.8 Hz, 1H), 8.49 - 8.33 (m, 2H), 8.14 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 7.2 Hz, 1H), 7.35 (dd, J = 7.6, 4.8 Hz, 1H), 7.28 - 6.96 (m, 3H), 6.54 (d, J = 8.4 Hz, 1H), 4.53 (d, J = 4.8 Hz, 1H), 4.23 (d, J = 13.6 Hz, 1H), 4.14 (d, J = 14.4 Hz, 1H), 3.89 (t, J = 5.6 Hz, 2H), 3.60 - 3.54 (m, 2H), 3.28 (d, J = 16.8 Hz, 1H), 3.14 (d, J = 16.8 Hz, 1H), 2.89 (t, J = 6.5 Hz, 2H), 2.52 (s, 3H), 2.05 - 1.87 (m, 3H), 1.86 - 1.69 (m, 3H), 1.62 (d, J = 12.8 Hz, 1H), 1.28 - 1.21 (m, 1H).

[0252] Example 4: Preparation of compound 4

[0253]

[0254] The specific synthesis route is as follows:

[0255]

[0256] First step: synthesis of compound 4B

[0257] Compound 4A (10 g, 71 mmol) was dissolved in THF (100 ml), and NIS (17.6 g, 78 mmol) was slowly added in batches at room temperature, and the reaction was carried out at room temperature overnight. LCMS detection showed that the reaction was complete, and the reaction liquid was added to 500 ml of water, a large amount of solid was precipitated, and the obtained solid was washed with dichloromethane and dried to obtain compound 4B (16.5 g, light yellow solid), with a yield of 87%.

[0258] MS (ESI): m / z 268.1 [M+H] + .

[0259] Second step: synthesis of compound 4C

[0260] Compound Ql (1.0 g, 4.9 mmol) was added to DMF (10 ml), then compound 4B (2.62 g, 9.8 mmol) and BOP (4.3 g, 9.8 mmol) were added, after addition, DBU (2.2 g, 14.7 mmol) was slowly added at room temperature, then the reaction was carried out at room temperature overnight, after the reaction was completed, Boc20 (1.6 g, 7.2 mmol) was added to the solution. The reaction was stirred at 50 °C for 2 h. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 2, V / V) to obtain compound 4C (1.1 g, light yellow solid), 42.3% yield.

[0261] MS (ESI): m / z 553.1 [M+H] + .

[0262] Step 3: Synthesis of compound 4D

[0263] Compound 4C (830 mg, 1.5 mmol) was dissolved in NMP (10 ml), and intermediate Q4 (895 mg, 2.4 mmol), Pd2(dba)3 (145 mg, 0.16 mmol), Xantphos (92 mg, 0.16 mmol) and DIEA (619 mg, 4.8 mmol) were added. After being heated to 120 °C and stirred for 1 h under nitrogen protection, the reaction was completed by TLC, then the reaction mixture was added to 75 ml of saturated sodium chloride solution, then extracted with EtOAc (25 ml x 3), the organic phase was combined, washed with saturated sodium chloride solution (100 ml), dried over anhydrous sodium sulfate, rotary evaporated and concentrated, and the residue was separated and purified by preparative high performance liquid chromatography (Welch Xtrimate C 18 150*30mm*5μM; condition: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min), to obtain compound 4D (384 mg, light yellow solid), 33% yield.

[0264] MS (ESI): m / z 776.2 [M+H] + .

[0265] Step 4: Synthesis of compound 4

[0266] Compound 4D (350 mg, 0.45 mmol) was added to ethyl acetate (5 ml), then 4M HCl / ethyl acetate solution (10 ml) was added, and the reaction was carried out at room temperature for 1 h, after the reaction was completed by TLC, filtration, the solid was washed with ethyl acetate, and dried to obtain the hydrochloride salt of compound 4 (276 mg, white solid), yield 91%.

[0267] MS (ESI): m / z 676.2 [M+H] + .

[0268] Example 5: Preparation of compound 5

[0269]

[0270] The specific synthesis route is as follows:

[0271]

[0272] First step: synthesis of compound 5A

[0273] Compound 1B (500 mg, 1.75 mmol) and compound Q5 (388 mg, 1.75 mmol) were added to NMP (10 ml), then heated to 120°C under nitrogen protection and stirred for 1 h, after TLC reaction was completed, the reaction mixture was added to 75 ml saturated sodium chloride solution, then extracted with EtOAc (25 ml x 3), the combined organic phase was washed with saturated sodium chloride solution (100 ml), dried over anhydrous sodium sulfate, rotary evaporated to concentrate, the residue was purified by silica gel column chromatography (eluent, PE:EA = 3:1 (volume ratio)), to obtain compound 5A (339 mg, light yellow solid), yield 42%.

[0274] MS (ESI): m / z 463.2 [M+H] + .

[0275] Third step: synthesis of compound 5

[0276] Compound 5A (300 mg, 0.64 mmol), compound Q1 (156 mg, 0.77 mmol) and DIEA (826 mg, 6.4 mmol) were added to MeCN (10 mL), then the resulting solution was stirred at 80°C for 2 hours. TLC showed that the reaction was completed. The mixture was concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (Welch Xtrimate C 18 150*30mm*5μM; condition: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min), to obtain compound 5 (106 mg, light yellow solid), 26.2% yield.

[0277] MS (ESI): m / z 630.2 [M+H] + .

[0278] 1H NMR (400 MHz, DMSO) δ 11.28 (s, 1H), 8.53 (d, J = 4.2 Hz, 1H), 8.43 - 8.30 (m, 2H), 7.94 - 7.85 (m, 1H), 7.72 - 7.63 (m, 1H), 7.54 - 7.45 (m, 1H), 7.37 - 7.29 (m, 1H), 7.26 - 7.10 (m, 2H), 6.55 - 6.43 (m, 1H), 6.34 - 6.25 (m, 1H), 6.13 (s, 1H), 4.47 (s, 1H), 4.28 (dd, J = 34.4, 12.4 Hz, 2H), 3.46 (s, 1H), 3.27 - 3.15 (m, 3H), 3.15 - 3.00 (m, 2H), 2.93 - 2.77 (m, 2H), 2.05 - 1.83 (m, 2H), 1.83 - 1.64 (m, 3H), 1.62 - 1.46 (m, 2H), 1.42 - 1.32 (m, 1H).

[0279] Example 6: Preparation of compound 6

[0280]

[0281] The specific synthesis route is as follows:

[0282]

[0283] First step: synthesis of compound 6A

[0284] Compound 1A (207 mg, 1.0 mmol) was dissolved in dioxane (5 ml), and intermediate Q6 (322 mg, 2 mmol), Pd2(dba)3(45 mg, 0.05 mmol), Xantphos (28 mg, 0.05 mmol) and DIEA (380 mg, 3.0 mmol) were added. After being heated to 90°C under nitrogen protection and stirred for 1 h, the reaction mixture was concentrated by rotary evaporation, and the residue was purified by silica gel column chromatography (eluent: V 二氯甲烷 :V 甲醇 = 50:1 ~ 10:1) to obtain compound 6A (173 mg, light yellow solid) with a yield of 65%.

[0285] MS (ESI): m / z 267.2 [M+H] + .

[0286] Second step: synthesis of compound 6B

[0287] Compound 6A (100 mg, 0.37 mmol), DIEA (154 mg, 1.2 mmol), intermediate Q3 (147 mg, 0.7 mmol) and HATU (456 mg, 1.2 mmol) were added into DMF, stirred at room temperature for 12 h, after TLC showed the reaction was completed, the reaction solution was diluted with dichloromethane (10 ml), the organic phase was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was collected and concentrated under reduced pressure, the residue was purified by silica gel column chromatography (eluent: V 二氯甲烷 :V 甲醇 = 50:1 ~ 10:1) to give compound 6B (138 mg, light yellow solid), 81.5% yield.

[0288] MS (ESI): m / z 459.2 [M+H]

[0289] Step 3: Synthesis of compound 6

[0290] Compound 6B (100 mg, 0.22 mmol), intermediate Q1 (71 mg, 0.35 mmol) and DIEA (82 mg, 0.63 mmol) were dissolved in dimethyl sulfoxide (5 ml) and reacted at 90°C for 1.5 h. After the reaction was completed, ethyl acetate (20 ml) and water (40 ml) were added, the aqueous phase was extracted with ethyl acetate (20 ml x 3), the combined organic phase was washed with saturated sodium oxide solution (100 ml), dried over anhydrous sodium sulfate, filtered, the filtrate was collected and concentrated under reduced pressure, and the residue was separated and purified by preparative high performance liquid chromatography (Welch Xtrimate C 18 150*30mm*5μM; conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min) to give compound 6 (30.7 mg, light yellow solid), 22.3% yield.

[0291] MS (ESI): m / z 626.2 [M+H] + .

[0292] Example 7: Preparation of compound 7

[0293]

[0294] The specific synthesis route is as follows:

[0295]

[0296] Step 1: Synthesis of compound 7A

[0297] Compound 1A (207 mg, 1.0 mmol) was dissolved in dioxane (5 ml), and intermediate Q7 (358 mg, 2 mmol), Pd2(dba)3(45 mg, 0.05 mmol), Xantphos (28 mg, 0.05 mmol) and DIEA (380 mg, 3.0 mmol) were added. After being heated to 90 °C under nitrogen protection and stirred for 1 h, the reaction mixture was concentrated by rotary evaporation, and the residue was purified by silica gel column chromatography (eluent, dichloromethane:methanol = 50:1 ~ 10:1, V:V) to obtain compound 7A (194 mg, yellowish solid), 63.5% yield.

[0298] MS (ESI): m / z 305.1 [M+H] + .

[0299] Second step: synthesis of compound 7B

[0300] Compound 7A (106 mg, 0.35 mmol), DIEA (154 mg, 1.2 mmol), intermediate Q3 (147 mg, 0.7 mmol) and HATU (456 mg, 1.2 mmol) were added to DMF, and stirred at room temperature for 12 h. After TLC showed that the reaction was completed, the reaction solution was diluted with dichloromethane (10 ml), and the organic phase was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent, dichloromethane:methanol = 50:1 ~ 10:1, V:V) to obtain compound 7B (145 mg, yellowish solid), 83.6% yield.

[0301] MS (ESI): m / z 497.2 [M+H]

[0302] Third step: synthesis of compound 7

[0303] Compound 7B (110 mg, 0.22 mmol), intermediate Q1 (71 mg, 0.35 mmol) and DIEA (82 mg, 0.63 mmol) were dissolved in dimethyl sulfoxide (5 ml), and reacted at 90 °C for 1.5 h. After the reaction was completed, ethyl acetate (20 ml) and water (40 ml) were added, the aqueous phase was extracted with ethyl acetate (20 ml x 3), the combined organic phase was washed with saturated sodium oxide solution (100 ml), dried over anhydrous sodium sulfate, filtered, and the filtrate was collected and concentrated under reduced pressure. The residue was separated and purified by preparative high performance liquid chromatography (Welch Xtrimate C 18 150*30mm*5μM; condition: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min) to obtain compound 7 (37.2 mg, yellowish solid), 26.2% yield.

[0304] MS (ESI): m / z 644.2 [M+H] + .

[0305] 1 H NMR (400 MHz, DMSO) δ 12.87 - 12.69 (m, 1H), 8.44 (d, J = 4.4 Hz, 1H), 8.03 - 7.93 (m, 1H), 7.89 - 7.76 (m, 2H), 7.69 - 7.62 (m, 2H), 7.26 (dd, J = 7.6, 5.2 Hz, 1H), 6.30 - 6.03 (m, 3H), 4.24 (t, J = 17.6 Hz, 2H), 4.12 (s, 2H), 3.82 - 3.68 (m, 2H), 2.96 (d, J = 16.8 Hz, 1H), 2.72 - 2.62 (m, 2H), 2.56 - 2.52 (m, 2H), 2.36 - 2.22 (m, 3H), 1.89 - 1.66 (m, 6H), 1.54 (t, J = 14.0 Hz, 1H), 1.41 (d, J = 12.4 Hz, 1H).

[0306] Example 8: Preparation of compound 8

[0307]

[0308] The specific synthesis route is as follows:

[0309]

[0310] First step: synthesis of compound 8B

[0311] Compound Q1 (1.0 g, 4.9 mmol) was added to DMF (10 ml), then compound 8A (2.45 g, 9.8 mmol) and BOP (4.3 g, 9.8 mmol) were added, after adding, DBU (2.2 g, 14.7 mmol) was slowly added at room temperature, then the reaction was carried out at room temperature overnight, after the reaction was completed, Boc2O (1.6 g, 7.2 mmol) was added to the solution. The reaction was stirred at 50°C for 2 hours. TLC showed that the reaction was completed. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 2, V / V) to obtain compound 8B (1.15 g, light yellow solid), 45.2% yield.

[0312] MS (ESI): m / z 518.1 [M+H] + .

[0313] Second step: synthesis of compound 8C

[0314] Compound 8B (829 mg, 1.6 mmol) was dissolved in NMP (10 ml), and intermediate Q4 (895 mg, 2.4 mmol), Pd2(dba)3(145 mg, 0.16 mmol), Xantphos (92 mg, 0.16 mmol) and DIEA (619 mg, 4.8 mmol) were added. After stirring for 1 h under nitrogen at 120 °C, the reaction was completed by TLC, the reaction mixture was added to 75 ml saturated sodium chloride solution, and then extracted with EtOAc (25 ml x 3), the organic phase was combined, washed with saturated sodium chloride solution (100 ml), dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The residue was separated and purified by preparative high performance liquid chromatography (Welch Xtrimate C18 ODS3 30*30mm*5μM; conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min) to give compound 8C (412 mg, light yellow solid), 32.6% yield. 18 150*30mm*5μM; conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min), to give compound 8C (412 mg, light yellow solid), 32.6% yield.

[0315] MS (ESI): m / z 789.2 [M+H] + .

[0316] Third step: synthesis of compound 8

[0317] Compound 8C (354 mg, 0.45 mmol) was added to ethyl acetate (5 ml), and 4M HCl / ethyl acetate solution (10 ml) was added. After reaction at room temperature for 1 h, the reaction was completed by TLC, and then filtered. The solid was washed with ethyl acetate and dried in an oven to give the hydrochloride salt of compound 8 (283 mg, white solid), 91.5% yield.

[0318] MS (ESI): m / z 689.2 [M+H] + .

[0319] Example 9: preparation of compound 9

[0320]

[0321] The specific synthesis route is as follows:

[0322]

[0323] First step: synthesis of compound 9A

[0324] Compound 8C (1 g, 1.3 mmol) was added to a mixture solvent of methanol (5.0 ml) and water (10.0 ml), then lithium hydroxide monohydrate (170 mg, 4.2 mmol) was added, stirred at room temperature for 2 h, TLC showed that the reaction was completed, then the pH value was adjusted to 3-4 with 1M hydrochloric acid, a large amount of solid was precipitated, filtered, and the filter cake was dried to obtain intermediate 9A (907 mg, off-white solid) with a yield of 90%. The crude product was used directly in the next step without purification.

[0325] Second step: synthesis of compound 9

[0326] Compound 9A (900 mg, 1.16 mmol) was added to ethyl acetate (5 ml), then 4M HCl / ethyl acetate solution (10 ml) was added, and the reaction was carried out at room temperature for 1 h. After TLC showed that the reaction was completed, the reaction mixture was added to 75 ml of saturated sodium bicarbonate solution, and then extracted with dichloromethane:methanol (5:1, V:V) (25 ml x 3). The combined organic phase was washed with saturated sodium chloride solution (100 ml), dried over anhydrous sodium sulfate, rotary evaporated and concentrated, and the residue was separated and purified by preparative high performance liquid chromatography (Welch Xtrimate C 18 150*30mm*5μM; Conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min) to obtain compound 9 (123 mg, white solid) with a yield of 15.7%.

[0327] MS (ESI): m / z 675.1 [M+H] + .

[0328] 1 H NMR (400 MHz, DMSO) δ 12.30 (s, 1H), 8.39-8.32 (m, 2H), 8.26-8.19 (m, 1H), 7.56 (d, J = 7.2 Hz, 1H), 7.39-7.28 (m, 2H), 7.27-7.16 (m, 2H), 6.86-6.73 (m, 2H), 6.53 (s, 1H), 4.86 (d, J = 9.2 Hz, 1H), 3.95-3.73 (m, 5H), 3.50 (s, 1H), 3.12 (d, J = 16.8 Hz, 1H), 2.92-2.84 (m, 2H), 2.76-2.72 (m, 1H), 2.04-1.85 (m, 3H), 1.85-1.74 (m, 2H), 1.74-1.57 (m, 3H).

[0329] Example 10: Preparation of compound 10

[0330]

[0331] The specific synthesis route is as follows:

[0332]

[0333] First step: synthesis of compound 10A

[0334] Compound 9A (900 mg, 1.16 mmol) was added to a solution of DMF (10 ml), then HATU (661 mg, 1.74 mmol) and DIEA (300 mg, 2.32 mmol) were added, after stirring at room temperature for half an hour, ammonium chloride (320 mg, 5.8 mmol) was added, then the reaction was carried out at room temperature overnight, after TLC showed that the reaction was completed, the reaction liquid was added to 75 ml of saturated sodium bicarbonate solution, then extracted with dichloromethane:methanol (5:1, V:V) (25 ml x 3), the combined organic phase was washed with saturated sodium chloride solution (100 ml), dried over anhydrous sodium sulfate, rotary evaporated and concentrated, and the residue was separated and purified by preparative high performance liquid chromatography (Welch Xtrimate C 18 150*30mm*5μM; Conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min), to obtain compound 10A (303 mg, white solid), 33.8% yield.

[0335] MS (ESI): m / z 774.1 [M+H] + .

[0336] Second step: synthesis of compound 10

[0337] Compound 10A (300 mg, 0.38 mmol) was added to ethyl acetate (5 ml), then 4M HCl / ethyl acetate solution (10 ml) was added, and the reaction was carried out at room temperature for 1 h, after TLC showed that the reaction was completed, it was filtered, the solid was washed with ethyl acetate, and dried in an oven to obtain the hydrochloride salt of compound 10 (232 mg, white solid), yield 90.5%.

[0338] MS (ESI): m / z 674.2 [M+H] + .

[0339] 1H NMR (400 MHz, MeOD) δ 8.74-8.62 (m, 1H), 8.44-8.29 (m, 1H), 8.23-8.13 (m, 1H), 7.78 (s, 1H), 7.71-7.57 (m, 1H), 7.36-7.19 (m, 1H), 7.14-7.03 (m, 1H), 4.64 (s, 1H), 4.15-3.95 (m, 3H), 3.77-3.72 (m, 2H), 3.66-3.59 (m, 3H), 3.52-3.40 (m, 1H), 3.12-3.012 (m, 1H), 2.13-1.90 (m, 5H), 1.81 (d, J = 11.6 Hz, 1H), 1.69 (d, J = 11.65 Hz, 1H), 1.64-1.54 (m, 1H).

[0340] Example 11: Preparation of compound 11

[0341]

[0342] The specific synthesis route is as follows:

[0343]

[0344] First step: synthesis of compound 11B

[0345] Compound Ql (1.0 g, 4.9 mmol) was added to DMF (10 ml), then compound 11A (2.74 g, 9.8 mmol) and BOP (4.3 g, 9.8 mmol) were added, after addition, DBU (2.2 g, 14.7 mmol) was slowly added at room temperature, then the reaction was carried out at room temperature overnight, after the reaction was completed, Boc20 (1.6 g, 7.2 mmol) was added to the solution. The reaction was stirred at 50 °C for 2 hours. TLC showed that the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1 / 2, V / V) to obtain compound 11B (1.2 g, light yellow solid), 45.2% yield.

[0346] MS (ESI): m / z 546.2 [M+H] + .

[0347] Second step: synthesis of compound 11C

[0348] Compound 11B (872 mg, 1.6 mmol) was dissolved in NMP (10 ml), and intermediate Q4 (895 mg, 2.4 mmol), Pd2(dba)3(145 mg, 0.16 mmol), Xantphos (92 mg, 0.16 mmol) and DIEA (619 mg, 4.8 mmol) were added. After heating to 120 °C under nitrogen protection and stirring for 1 h, the reaction was completed by TLC, the reaction mixture was added to 75 ml saturated sodium chloride solution, and then extracted with EtOAc (25 ml x 3), the organic phase was combined, washed with saturated sodium chloride solution (100 ml), dried over anhydrous sodium sulfate, rotary evaporated and concentrated, and the residue was separated and purified by preparative high performance liquid chromatography (Welch Xtrilate C18 ODS3 150*30 mm*5 μm; conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min) to give compound 11C (398 mg, light yellow solid), 30.5% yield. 18 150*30 mm*5 μm; conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min) to give compound 11C (398 mg, light yellow solid), 30.5% yield.

[0349] MS (ESI): m / z 817.2 [M+H] + .

[0350] Step 3: Synthesis of compound 11

[0351] Compound 11C (367 mg, 0.45 mmol) was added to ethyl acetate (5 ml), and 4M HCl / ethyl acetate solution (10 ml) was added, and the reaction was carried out at room temperature for 1 h, TLC showed that the reaction was completed, then it was filtered, the solid was washed with ethyl acetate, and dried in an oven to give the hydrochloride salt of compound 11 (295 mg, white solid), 91.5% yield.

[0352] MS (ESI): m / z 717.2 [M+H] + .

[0353] 1H NMR (400 MHz, DMSO) δ 12.26 (s, 1H), 8.76 (s, 2H), 8.60 (d, J = 4.4 Hz, 1H), 8.21 (dd, J = 24.4, 7.6 Hz, 2H), 7.47 (dd, J = 7.2, 5.6 Hz, 1H), 7.43 - 7.34 (m, 1H), 7.30 (t, J = 8.0 Hz, 1H), 6.69 (d, J = 8.0 Hz, 1H), 4.56 - 4.46 (m, 1H), 4.30 (q, J = 7.2 Hz, 2H), 3.97 (d, J = 13.2 Hz, 1H), 3.89 - 3.84 (m, 2H), 3.80 (s, 1H), 3.36 - 3.23 (m, 3H), 3.15 (d, J = 17.2 Hz, 1H), 2.89 (t, J = 6.4 Hz, 2H), 2.45 (s, 3H), 1.96 - 1.87 (m, 2H), 1.87 - 1.76 (m, 3H), 1.75 - 1.64 (m, 2H), 1.56 (d, J = 13.2 Hz, 1H), 1.26 (t, J = 7.2 Hz, 3H).

[0354] Example 12: Preparation of compound 12

[0355]

[0356] The specific synthesis route is as follows:

[0357]

[0358] First step: synthesis of compound 12A

[0359] Compound 8C (1 g, 1.3 mmol) was added to a mixed solvent of methanol (5.0 ml) and water (10.0 ml), then lithium hydroxide monohydrate (170 mg, 4.2 mmol) was added, stirred at room temperature for 2 h, TLC showed that the reaction was completed, the pH value was adjusted to 3-4 with 1M hydrochloric acid, a large amount of solid was precipitated, filtered, the filter cake was dried to obtain intermediate 9A (907 mg, off-white solid), the yield was 90%. The crude product was not purified and was directly used in the next step reaction.

[0360] Second step: synthesis of compound 12B

[0361] Compound 9A (900 mg, 1.16 mmol) was added to a solution of DMF (10 ml), followed by HATU (661 mg, 1.74 mmol) and DIEA (300 mg, 2.32 mmol), after stirring at room temperature for half an hour, ammonium chloride (320 mg, 5.8 mmol) was added, then the reaction was allowed to proceed at room temperature overnight, after TLC showed that the reaction was complete, the reaction solution was added to 75 ml of saturated sodium bicarbonate solution, then extracted with dichloromethane:methanol (5:1, V:V) (25 ml x 3), the combined organic phase was washed with saturated sodium chloride solution (100 ml), dried over anhydrous sodium sulfate, rotary evaporated and concentrated, the residue was separated and purified by preparative high performance liquid chromatography (Welch Xtrimate C18 ODS3 30*30mm*5μM; conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min), to obtain compound 10A (303 mg, white solid), 33.8% yield. 18 150*30mm*5μM; conditions: 36-67% B (A: water (0.05% ammonia water), B: acetonitrile); flow rate: 25 ml / min), to obtain compound 10A (303 mg, white solid), 33.8% yield.

[0362] MS (ESI): m / z 774.1 [M+H] + .

[0363] Step 3: Synthesis of compound 12

[0364] Compound 11C (367 mg, 0.45 mmol) was added to ethyl acetate (5 ml), followed by 4M HCl / ethyl acetate solution (10 ml), and the reaction was allowed to proceed at room temperature for 1 h, after TLC showed that the reaction was complete, it was filtered, the solid was washed with ethyl acetate and oven dried to obtain the hydrochloride salt of compound 11 (295 mg, white solid), yield 91.5%.

[0365] MS (ESI): m / z 717.2 [M+H] + .

[0366] 1H NMR (400 MHz, DMSO) δ 12.25 (s, 1H), 8.63-8.50 (m, 3H), 8.21 (d, J = 8.4 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.84 (s, 1H), 7.56 (s, 1H), 7.40 (dd, J = 7.6, 5.2 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 6.66 (d, J = 8.0 Hz, 1H), 4.52-4.48 (m, 1H), 4.07 (d, J = 13.2 Hz, 1H), 3.96 (d, J = 13.2 Hz, 1H), 3.86 (t, J = 6.0 Hz, 2H), 3.74-3.64 (m, 2H), 3.28-3.25 (m, 1H), 3.11 (d, J = 17.2 Hz, 1H), 2.88 (t, J = 6.4 Hz, 2H), 2.43 (s, 3H), 2.02-1.96 (m, 1H), 1.94-1.85 (m, 2H), 1.85-1.75 (m, 3H), 1.63 (d, J = 11.6 Hz, 1H), 1.53 (d, J = 13.2 Hz, 1H).

[0367] Experimental Example 1: Anti-proliferative activity test of the compounds of the application against NCI-H358 cells

[0368] Experimental materials:

[0369] DMEM medium, penicillin / streptomycin antibiotics were purchased from Vincents. Fetal bovine serum was purchased from Biosera. 3D CellTiter-Glo (Cell Viability Chemiluminescent Assay) reagent was purchased from Promega. H358 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision multi-label analyzer (PerkinElmer).

[0370] Experimental method:

[0371] H358 cells were seeded in ultra-low attachment 96-well U-shaped plates, 80 μL of cell suspension per well, containing 3000 H358 cells. The cell plate was placed in a carbon dioxide incubator overnight.

[0372] The test compound was diluted 3 times with a row gun to the 8th concentration, i.e. from 0.2 mM to 91.44 nM, and a double-replicate well experiment was set up. 78 μL of medium was added to the intermediate plate, and then 2 μL of gradient-diluted compound per well was transferred to the intermediate plate according to the corresponding position, and after mixing, 20 μL per well was transferred to the cell plate. The concentration range of the compound transferred to the cell plate was 1 μM to 0.457 nM. The cell plate was placed in a carbon dioxide incubator for 5 days.

[0373] To the cell plate, add 100 μL of CellTiter-Glo reagent per well and incubate at room temperature for 10 minutes to stabilize the luminescent signal. Read the plate on a Multilabel Reader.

[0374] Data Analysis:

[0375] The raw data is converted to percent inhibition using the equation (Sample-Min) / (Max-Min)*100% and IC 50 values are determined by four parameter curve fitting (using the "log(inhibitor) vs. response - Variable slope" model in GraphPad Prism). Table 1 provides the inhibitory activity of the compounds of the present application against the proliferation of H358 cells.

[0376] Table 1. Anti-cell proliferation activity data (IC 50 )

[0377] Compound No. NCI-H358 anti-proliferative activity, IC 50 (nM) RMC-4550 64 TNO-155 65 Compound 1 1.51 Compound 2 2.4 Compound 3 29.8 Compound 4 56.6 Compound 5 48.9 Compound 6 107 Compound 7 111 Compound 8 3.69 Compound 9 167 Compound 10 2.84 Compound 11 128 Compound 12 2.34

[0378] As can be seen from the results in Table 1, the compounds of the present application have good activity in inhibiting the proliferation of lung cancer cell line NCI-H358. Some of the compounds have an activity that is more than 40 times that of TNO-155. This shows extremely important anti-tumor potential.

[0379] Experimental Example 2: Anti-proliferation activity test of the compounds of the present application against MV-4-11 cells

[0380] Experimental Materials:

[0381] IMDM medium, fetal bovine serum, penicillin / streptomycin antibiotics were purchased from Promega (Madison, WI). The MV-4-11 cell line was purchased from the Chinese Academy of Sciences Cell Bank. The Envision Multilabel Reader (PerkinElmer).

[0382] Experimental Methods:

[0383] MV-4-11 cells were seeded in white 96-well plates, 80 μL of cell suspension per well, containing 6000 MV-4-11 cells. The cell plate was incubated in a carbon dioxide incubator overnight.

[0384] The test compound was diluted 3 times by gun to the 9th concentration, i.e. from 2 mM to 304 nM, and a double-replicate experiment was set up. 78 μL of medium was added to the intermediate plate, and then 2 μL of gradient-diluted compound per well was transferred to the intermediate plate according to the corresponding position, and after mixing, 20 μL per well was transferred to the cell plate. The compound concentration range transferred to the cell plate was 10 μM to 1.52 nM. The cell plate was placed in a carbon dioxide incubator for 3 days.

[0385] 25 uL of Promega CellTiter-Glo reagent was added to each well of the cell plate, and the luminescent signal was stabilized by incubation at room temperature for 10 minutes. A PerkinElmer Envision multi-label analyzer was used for reading.

[0386] Data analysis:

[0387] The raw data was converted into inhibition rate by the equation (Sample-Min) / (Max-Min)*100%, and the value of IC 50 was obtained by four-parameter curve fitting (obtained in the "log(inhibitor) vs. response--Variable slope" mode in GraphPad Prism). Table 2 provides the inhibitory activity of the compounds of the present application on the proliferation of MV-4-11 cells.

[0388] Table 2. Anti-cell proliferation activity data (IC 50 ) of compounds in the present application

[0389]

[0390] As can be seen from the experimental results in Table 2, the compounds in the present application have good activity in inhibiting the proliferation of MV-4-11 cells. The activity of compound 1 is more than 10 times that of TNO-155, showing extremely important anti-tumor potential.

[0391] Experimental Example 3: p-ERK activity inhibition experiment

[0392] 1. Experimental materials

[0393] H358 cells were purchased from Nanjing Kebai Biological Technology;

[0394] 1640 medium was purchased from Biological industries;

[0395] Fetal bovine serum was purchased from Biosera;

[0396] The phosphorylated pERK (phosphorylation site 202 / 204) detection kit was purchased from Cisbio.

[0397] 2. Experimental Methods

[0398] H358 cells were seeded in clear 96-well cell culture plates, 80 μL cell suspension per well, containing 10,000 H358 cells per well. The cell plates were placed in a carbon dioxide incubator and incubated overnight at 37 °C. The cell supernatant was discarded and 80 μL / well of starvation medium (1640 + 0.02% fetal bovine serum + 1% double antibiotic) was added. The cell plates were placed in a carbon dioxide incubator and the cells were starved overnight.

[0399] The test compound was diluted with 100% DMSO to 4 mM as the first concentration, and then diluted 5-fold with a row gun pipettor to the eighth concentration, i.e., from 4 mM to 10.24 μM. 1 μL of the compound was added to 79 μL of the cell starvation medium, mixed, and then 20 μL / well of the compound solution was transferred to the corresponding cell plate well. The cell plate was placed back in the carbon dioxide incubator and incubated for 1 h, at which time the compound concentration was 10 μM to 0.0256 nM and the DMSO concentration was 0.25%.

[0400] After the incubation was completed, the cell supernatant was discarded and 50 μL of cell lysis solution was added per well. The cell plate was shaken at room temperature for 30 min. The europium cryptate-labeled phospho-ERK antibody and the d2-labeled phospho-ERK antibody were diluted 20-fold using the detection buffer. 16 μL of the cell lysis supernatant was added to a new 384 white microplate, and 2 μL of the europium cryptate-labeled phospho-ERK antibody dilution and 2 μL of the d2-labeled phospho-ERK antibody dilution were added. The plate was incubated at room temperature for 4 h. After the incubation was completed, the homogeneous time-resolved fluorescence HTRF (excitation wavelength of 320 nm and emission wavelengths of 615 nm and 665 nm) was read using a multilabel reader.

[0401] 3. Data Analysis

[0402] The raw data was converted to inhibition using the equation (sample - Min) / (Max - Min) x 100%, and the IC 50 values were obtained by four-parameter curve fitting (e.g., by the "log(inhibitor) vs. response - Variable slope" mode in GraphPad Prism). Max well: 1X cell lysis solution for the positive control well; Min well: 0.25% DMSO cell well for the negative control well.

[0403] Table 3. p-ERK activity inhibition data (IC 50 )

[0404] Table 3. p-ERK activity inhibition data (IC 50 )

[0404] Compound No. p-ERK inhibitory activity, IC 50 (nM) TNO-155 85.76 Compound 1 9.53

[0405] As can be seen from the experimental results in Table 3, the compounds in the present application have excellent inhibitory effect on the phosphorylation of SHP2 downstream Erk in NCI-H358 cells, further illustrating that the compounds in the present application have better anti-tumor prospects from the mechanism.

[0406] Experimental Example 4: SHP2 enzymatic experiment

[0407] 1. Experimental materials and instruments

[0408] The homogeneous full-length SHP2 enzymatic experiment kit was purchased from BPS Bioscience;

[0409] The multi-label analyzer was purchased from Perkin Elmer.

[0410] 2. Experimental method:

[0411] The 5X detection buffer was diluted with deionized water to 1X detection buffer, and was prepared and used immediately. After preparation, it was placed on ice for standby.

[0412] The test compound was diluted with 100% DMSO to 100 μM as the first concentration, and then 4-fold dilution was performed to the 8th concentration by using the row gun pipette, i.e. from 100 μM to 6.1 nM. The test compound was diluted with 1X buffer to a working solution containing 10% DMSO, 5 μL / well was added to the corresponding well, and double duplicate well experiment was set. Centrifugation at 1000 rpm for 1 min.

[0413] 18 μL of prepared reaction mixture was added to each well, which contained 12.25 μL of deionized water; 5 μL of 5X detection buffer; 0.25 μL of protein tyrosine phosphatase activating polypeptide (100 μM); 0.5 μL of DTT (250 mM). Centrifugation at 1000 rpm for 1 min.

[0414] SHP2 enzyme was diluted with 1X detection buffer to 0.1 ng / μL, 2 μL / well was added to the corresponding well, 2 μL of 1X detection buffer was added to the negative control well, SHP2 (0.2 ng), and this step was operated on ice. The reaction system was incubated at 25°C for 60 min for compound pre-incubation.

[0415] After the end of the pre-incubation of the compounds, 25 μL of substrate working solution was added to each well, which contained 19.45 μL of deionized water; 5 μL of 5X detection buffer; 0.5 μL of DTT (250 mM) and 0.05 μL of SHP2 substrate (DiFMUP) (10 mM), and the reaction system was placed at 25°C for 30 min. At this time, the final concentration gradient of the compound was 1 μM to 0.061 nM. After the reaction was completed, the fluorescence value was read using a multi-label analyzer (excitation wavelength 360 nm, emission wavelength 460 nm).

[0416] The detection method of the background reading value of the compound is as follows: 5 μL of each gradient of the compound diluted with 100% DMSO was taken into a new compound plate, 45 μL of 1X detection buffer was added for 10-fold dilution, and a working solution of 10% DMSO was prepared, 5 μL / well of the compound working solution was taken into the detection plate, then 45 μL of 1X detection buffer was added for 10-fold dilution, at this time the final concentration of DMSO was 1%, and the fluorescence value was read using a multi-label analyzer (excitation wavelength 360 nm, emission wavelength 460 nm) after centrifugation at 1000 rpm for 1 min.

[0417] 3. Data analysis

[0418] The data obtained after subtracting the background reading value of the compound from the original reading value of the enzyme reaction was used as the initial value for inhibition rate calculation, and the initial value was converted into inhibition rate using the equation (sample-Min) / (Max-Min) x 100%, and the IC 50 value was obtained by four-parameter curve fitting (for example, obtained by "log(inhibitor) vs. response--Variable slope" mode in GraphPad Prism). Among them, Max well: initial value of positive control well; Min well: initial value of negative control well.

[0419] Table 4. SHP2 enzyme activity inhibition data (IC 50 ) of the compounds in the application

[0420] Compound No. SHP2 enzyme inhibitory activity IC 50 (nM) TNO-155 2.6 Compound 1 0.98

[0421] As can be seen from the experimental results in Table 4, the compounds in the application show very excellent SHP2 enzyme inhibition activity at the enzymatic level, with IC 50 reaching below 1 nM, which has great development and application prospects.

[0422] Experimental Example 5: hERG potassium ion channel inhibitor activity test

[0423] 1. Cell preparation

[0424] CHO-hERG cells were cultured in a 175 cm2 When the cell density reached 60-80%, the culture medium was removed and the cells were washed once with 7 mL PBS, then 3 mL Detachin was added to digest the cells.

[0425] After complete digestion, 7 mL culture medium was added to neutralize the cells, then centrifuged, the supernatant was removed and 5 mL culture medium was added to resuspend the cells to ensure the cell density was 2-5 x 10 6 / mL.

[0426] 2. Solution preparation: see Table 5:

[0427] Table 5. Composition of intracellular and extracellular solutions

[0428]

[0429] 3. Electrophysiological recording procedure

[0430] The whole process of single cell high resistance seal formation and whole cell mode formation was automatically completed by the Qpatch instrument. After obtaining the whole cell recording mode, the cell was clamped at -80 mV. Before giving a 5-second +40 mV depolarization stimulus, a 50-millisecond -50 mV pre-voltage was given, then repolarized to -50 mV for 5 seconds, and then returned to -80 mV. This voltage stimulus was applied every 15 seconds, and recording was performed for 2 minutes. Then extracellular recording was performed for 5 minutes, and then the drug administration process began. The compound concentration started from the lowest test concentration, and each test concentration was given for 2.5 minutes. After all concentrations were continuously administered, the positive control compound 0.1 μM Cisapride was administered. At least 3 cells were tested for each concentration (n≥3).

[0431] 4. Compound preparation

[0432] The 20 mM compound stock solution was diluted with extracellular solution. 5 μL of 20 mM compound stock solution was added to 2495 μL of extracellular solution, which was diluted 500 times to 40 μM. Then, 3 times of continuous dilution was performed in extracellular solution containing 0.2% DMSO to obtain the final concentration required for testing.

[0433] The highest test concentration was 40 μM, and the concentrations were 40, 13.33, 4.44, 1.48, 0.49, and 0.16 μM, respectively.

[0434] The DMSO content in the final test concentration was not more than 0.2%, and this concentration of DMSO had no effect on the hERG potassium channel.

[0435] 5. Data analysis

[0436] The experimental data was analyzed by XLFit software.

[0437] 6. Quality control

[0438] Environment: humidity 20-50%, temperature 22-25℃

[0439] Reagents: The experimental reagents used were purchased from Sigma Company, purity >98%

[0440] The experimental data in the report must meet the following standards:

[0441] Whole cell seal impedance >100MΩ

[0442] Tail current amplitude >300pA

[0443] Pharmacological parameters:

[0444] The inhibitory effect of cisapride at multiple concentrations on hERG channels was set as a positive control.

[0445] 7. Experimental results

[0446] Table 6. Inhibition of hERG current by compounds in the present application at multiple concentrations

[0447] Compound No. hERG (μM) Compound 1 >40 Compound 2 >40 Cisapride 0.021

[0448] 8. Experimental conclusion: Inhibition of the hERG potassium ion channel in the heart by drugs is the main cause of drug-induced QT prolongation syndrome. From the experimental results, it can be seen that the compounds of the present application have no significant inhibitory effect on the hERG potassium ion channel in the heart, and the risk of cardiotoxic side effects is low.

[0449] Experimental Example 6: Pharmacokinetic experiment

[0450] 1. Experimental materials

[0451] Using the compounds prepared in the above examples, the oral drug was formulated into a 0.3mg / mL clear solution (2% DMSO+30% PEG300+2% Tween80+66% H2O), and the intravenous drug was formulated into a 0.2mg / mL clear solution (2% DMSO+30% PEG300+2% Tween80+66% H2O).

[0452] 2. Experimental animals

[0453] Male CD-1 mice or rats, 3 in each group, weighing 27-28g, were provided by Shanghai Slek Experimental Animal Responsibility Co., Ltd. The test mice were given an environmental adaptation period of 2-4 days before the experiment, and were fasted for 8-12h before administration, and were given water 2h after administration, and were given food 4h after administration.

[0454] 3. Experimental method

[0455] 1) After mice or rats are fasted but allowed free access to water for 12 h, take the blank plasma at time 0;

[0456] 2) Take the mice in step 1), orally (PO) administer the test compound at 3 mg / kg; intravenously (IV) administer the test compound at 1 mg / kg;

[0457] 3) At 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 10 h and 24 h after oral administration, continuously take blood from the ophthalmic venous plexus, place in an EP tube distributed with heparin, centrifuge at 8000 rpm for 5 min, take the upper plasma, and store at -20°C for LC-MS / MS analysis;

[0458] 4) According to the blood concentration-time data obtained in step 3), calculate the pharmacokinetic parameters using WinNonlin software, and the specific data are shown in Table 7.

[0459] Table 7. Pharmacokinetic data of the compound in the application

[0460]

[0461] As shown in Table 7, after oral or intravenous administration of the compound in the application to mice or rats, there is a high exposure in the plasma of the animals, and the clearance is low, which can be achieved by oral administration.

[0462] Experimental Example 7: In vivo pharmacodynamic experiment one

[0463] 1. Purpose of the experiment

[0464] To evaluate the in vivo efficacy of the test compound on a human non-small cell lung cancer NCI-H358 subcutaneous xenograft tumor model.

[0465] 2. Experimental animals

[0466] BALB / c nude mice, female, 6-8 weeks old, weighing 18-20 grams, a total of 18, provided by Shanghai Sino-British Experimental Animal Technology Co., Ltd.

[0467] 3. Experimental method

[0468] Resuspend NCI-H358 tumor cells in PBS to prepare a cell suspension with a density of 5 x 10 7 / mL, subcutaneously inoculate 0.1 mL (5 x 10 6 / mouse) into the right back of each mouse, and wait for tumor growth. When the average volume of the tumor reaches about 151 mm 3 , start random grouping and dosing. After dosing, measure the tumor diameter twice a week with a vernier caliper, and the formula for calculating the tumor volume is as follows:

[0469] V = 0.5 a x b2 wherein a and b represent the long diameter and the short diameter of the tumor, respectively.

[0470] The antitumor efficacy of the compound was evaluated by TGI (%), which reflects the tumor growth inhibition rate, and was calculated as follows:

[0471] TGI (%) = [(1-(the average tumor volume at the end of administration in a certain treatment group - the average tumor volume at the beginning of administration in the treatment group) / (the average tumor volume at the end of treatment in the solvent control group - the average tumor volume at the beginning of treatment in the solvent control group)] x 100%.

[0472] 4. Experimental results

[0473] The relevant results are shown in Table 8.

[0474] Table 8. Results of in vivo pharmacodynamic experiments

[0475] Group Tumor volume (mm3 3 )(day 35)) TGI (%) Solvent control 784.14±40.83 / TNO-155 (30 mg / kg) 355.37±68.17 67.72 Compound 1 (30 mg / kg) 185.34±20.85 94.57

[0476] 5. Experimental conclusion

[0477] After 35 days of administration, the compound in the present application had a more significant tumor inhibition effect than the positive control TNO-155 at the same dose (30 mg / kg), with a TGI (%) of 94.57%, which was significantly better than the 67.72% of the positive control TNO-155, indicating that the compound in the present application exhibited good in vivo pharmacodynamics on the human non-small cell lung cancer NCI-H358 subcutaneous xenotransplant tumor model. As a SHP2 inhibitor, it has a very good anti-tumor application prospect in the clinic.

[0478] Experimental Example 8: In vivo pharmacodynamic experiment two

[0479] 1. Experimental purpose

[0480] To evaluate the in vivo pharmacodynamics of the test compound on the human pancreatic cancer MIA-PaCa2 cell subcutaneous xenotransplant tumor model.

[0481] 2. Experimental animals

[0482] BALB / c nude mice, female, 6-8 weeks old, weighing 18-22 grams, a total of 24, provided by Beijing VitoLabs Technology Co., Ltd.

[0483] 3. Experimental method

[0484] MIA-PaCa2 tumor cells were resuspended in PBS to prepare a density of 1 x 10 7A cell suspension of 1 cell / mL was subcutaneously inoculated into the right posterior back of each mouse (with added matrix gel, volume ratio 1:1), and tumor growth was observed. The tumors reached an average volume of approximately 142 mm². 3 At that time, randomized grouping for drug administration began. After drug administration, tumor diameter was measured twice weekly using calipers. The tumor volume was calculated using the following formula:

[0485] V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively.

[0486] The antitumor efficacy of the compound was evaluated using TGI (%), which reflects the tumor growth inhibition rate. The calculation is as follows:

[0487] TGI(%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in solvent control group - mean tumor volume at the start of treatment in solvent control group)] × 100%.

[0488] 4. Experimental Results

[0489] The relevant results are shown in Table 9.

[0490] Table 9. Results of in vivo efficacy experiments

[0491] Group Tumor volume (mm3 3 )(day 22)) TGI (%) Solvent control 1560±137 / TNO-155 (30 mg / kg) 868±72 48.8 Compound 1 (3.0 mg / kg) 696±112 60.9 Compound 1 (30 mg / kg) 560±93 70.4

[0492] 5. Experimental Conclusions

[0493] Twenty-two days after the start of administration, at the same dose (30 mg / kg), the compound of the present invention showed a more significant tumor-suppressive effect compared with the positive control TNO-155, and there was a clear dose-response relationship, indicating that the compound of the present invention exhibited good in vivo efficacy in the human pancreatic cancer MIA-PaCa2 subcutaneous allogeneic transplantation tumor model.

[0494] Experiment Example 9: In vivo pharmacodynamics experiment three

[0495] 1. Experimental Objective

[0496] To evaluate the in vivo efficacy of the test compound in a mouse MC38 subcutaneous xenograft tumor model of colon cancer.

[0497] 2. Laboratory animals

[0498] C57BL / 6 mice, female, 6–8 weeks old, weighing 18–20 grams. A total of 32 mice are needed, to be provided by Shanghai Lingchang Laboratory Animal Co., Ltd.

[0499] 3. Experimental Methods

[0500] The MC38 tumor cells were resuspended in PBS to prepare a cell suspension with a density of 0.3 x 10 6 3 When the average tumor volume reached about 65 mm 2 The tumor volume was calculated according to the following formula:

[0501] V = 0.5a x b 2 wherein a and b represent the long diameter and the short diameter of the tumor, respectively.

[0502] The antitumor effect of the compound was evaluated by TGI (%), which reflects the tumor growth inhibition rate and is calculated as follows:

[0503] TGI (%) = [(1-(the average tumor volume at the end of administration in a certain treatment group - the average tumor volume at the beginning of administration in the treatment group) / (the average tumor volume at the end of treatment in the solvent control group - the average tumor volume at the beginning of treatment in the solvent control group)] x 100%.

[0504] 4. Experimental results

[0505] The relevant results are shown in Table 10.

[0506] Table 10. In vivo pharmacodynamic experiment results

[0507]

[0508] 5. Experimental conclusion

[0509] After 18 days of administration, the compound of the present application combined with PD-1 antibody can significantly increase the tumor inhibition effect compared with single drug, and shows good in vivo pharmacodynamics in the mouse colon cancer MC38 subcutaneous xenotransplant tumor model. It is shown that the compound of the present application combined with PD1 monoclonal antibody shows synergistic antitumor effect.

[0510] Experimental Example 10: In vivo pharmacodynamics experiment four

[0511] 1. Experimental purpose

[0512] To evaluate the in vivo pharmacodynamics of the test compound in the human esophageal cancer KYSE-520 subcutaneous xenotransplant tumor model.

[0513] 2. Experimental animals

[0514] BALB / c nude mice, female, 6-8 weeks old, weighing 18-24 grams, a total of 40, provided by Beijing VitoLabs Technology Co., Ltd.

[0515] 3. Experimental method

[0516] KYSE-520 tumor cells were resuspended in PBS to prepare a cell suspension with a density of 10 x 10 6 Each mouse was subcutaneously inoculated with 0.2 mL of the cell suspension in the right back (with Matrigel, volume ratio 1:1) and waited for tumor growth. When the average volume of the tumors reached about 141 mm 3 When the average volume of the tumors reached about 141 mm 2 , the random grouping and dosing were started. After dosing, the tumor diameters were measured twice a week with a vernier caliper, and the tumor volume was calculated according to the following formula:

[0517] V = 0.5a x b 2 , where a and b represent the long diameter and the short diameter of the tumor, respectively.

[0518] The antitumor efficacy of the compound was evaluated by TGI (%), which reflects the tumor growth inhibition rate and is calculated as follows:

[0519] TGI (%) = [(1 - (average tumor volume at the end of dosing in a certain treatment group - average tumor volume at the start of dosing in the treatment group) / (average tumor volume at the end of treatment in the solvent control group - average tumor volume at the start of treatment in the solvent control group)] x 100%.

[0520] 4. Experimental results

[0521] The relevant results are shown in Table 11.

[0522] Table 11. Results of in vivo pharmacodynamic experiments

[0523] Group Tumor volume (mm3 3 )(Day 21) TGI (%) Solvent control 1,059±176 / TNO-155 (30 mg / kg) 304±36 82.3 Compound 1 (0.3 mg / kg) 439±69 67.5 Compound 1 (1.0 mg / kg) 321±45 80.4 Compound 1 (3.0 mg / kg) 216±42 91.9

[0524] 5. Experimental conclusion

[0525] After 21 days of starting dosing, the compound 1 in the present application had a significantly better tumor inhibition effect than the positive control TNO-155 (30 mg / kg) at a dose of one-tenth of the positive control (3.0 mg / kg), with a TGI (%) of 91.9%, which was significantly better than the 82.3% of the positive control TNO-155. In addition, the compound 1 in the present application still showed a significant antitumor effect at a lower dose (0.3 mg / kg), which indicated that the compound in the present application showed good in vivo pharmacodynamic effect in the human esophageal cancer KYSE-520 subcutaneous xenotransplantation model and the antitumor effect had a dose-dependent trend.

[0526] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application. It is to be understood that changes, modifications, substitutions and variations can be made by those skilled in the art without departing from the spirit and scope of the present application, and such changes, modifications, substitutions and variations are to be encompassed within the scope of the present application.

Claims

1. A compound of the following formula: ###0001### or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof.

2. A pharmaceutical composition comprising a compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, and at least one pharmaceutically acceptable excipient.

3. Use of a compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof, or a pharmaceutical composition according to claim 2 for the manufacture of a medicament for the prevention and / or treatment of a disease or disorder associated with abnormal SHP2 activity.

4. The use according to claim 3, wherein the disease or disorder associated with abnormal SHP2 activity is selected from Noonan syndrome, Leopard syndrome, leukemia, neuroblastoma, melanoma, breast cancer, esophageal cancer, lung cancer, colon cancer, head and neck tumor, gastric cancer, anaplastic large cell lymphoma, and glioblastoma.

5. The use according to claim 3, wherein the disease or disorder associated with abnormal SHP2 activity is selected from non-small cell lung cancer, esophageal cancer, and head and neck tumor. ​ ​

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