CN114437116A

CN114437116A - Heterocyclic compound and preparation method, pharmaceutical composition and application thereof

Info

Publication number: CN114437116A
Application number: CN202111249058.6A
Authority: CN
Inventors: 袁建栋; 方华祥; 黄仰青; 顾家宁; 王晨英; 李宸杰; 吴敬浩
Original assignee: Ganjiang New Area Borui Innovative Medicine Co ltd
Current assignee: Ganjiang New Area Borui Innovative Medicine Co ltd
Priority date: 2020-10-30
Filing date: 2021-10-26
Publication date: 2022-05-06
Also published as: WO2022089389A1

Abstract

The invention belongs to the field of medicinal chemistry, and relates to a heterocyclic compound, a preparation method thereof, a medicinal composition and application thereof. Particularly, the invention relates to a heterocyclic compound shown as a formula I, a pharmaceutical composition containing the heterocyclic compound and application of the heterocyclic compound as an SHP2 inhibitor in the field of medicine. The heterocyclic compound of the invention shows excellent biological activity and can become a drug property, and has great drug development prospect.

Description

Heterocyclic compound and preparation method, pharmaceutical composition and application thereof

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to heterocyclic compounds, a preparation method of the heterocyclic compounds and intermediates of the heterocyclic compounds, a medicinal composition containing the heterocyclic compounds, and application of the heterocyclic compounds and intermediates of the heterocyclic compounds in the field of medicines.

Background

Tyrosine phosphatase SHP2 consists of two N-terminal Src homology 2 domains (N-SH)₂And C-SH₂) And a protein tyrosine phosphatase catalytic domain (PTP). In the basic state, N-SH₂Can bind to PTP to form a ring structure, thereby blocking the binding of PTP to substrate, and inhibiting the enzyme catalytic activity; N-SH when tyrosine of the upstream receptor protein is phosphorylated₂In combination therewith, the PTP catalytic domain is released, thereby exerting phosphatase activity.

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

At the same time, SHP2 is also involved in apoptosis receptor 1(PD1) mediated immune system suppression. After binding of PD-1 to PD-L1 in T cells, large amounts of SHP2 could be recruited in the cells. SHP2 is capable of dephosphorylating an antigen receptor pathway protein within T cells, thereby inhibiting activation of T cells. Thus, inhibition of the activity of SHP2 could reverse immunosuppression in the tumor microenvironment.

As an important class of cell signaling factors, SHP2 mutations are closely associated with a variety of diseases. SHP2 mutations were found in neuroblastoma, AML (4%), breast cancer, NSCLC (10%), lung adenocarcinoma (30%), esophageal cancer, head and neck tumors, melanoma, and gastric cancer.

At present, a plurality of SHP2 allosteric inhibitors enter clinical research stages, such as TNO-155 developed by Novartis, RMC-4630 developed by Revolition Medicine, JAB-3068 developed by Beijing plus Koch, and the like, but no SHP2 inhibitor developed and marketed for treating Noonan syndrome, leopard syndrome, leukemia, neuroblastoma, melanoma, breast cancer, esophageal cancer, head and neck tumors, lung cancer and colon cancer is available. Therefore, the development of a kind of SHP2 inhibitor drug with good drug-forming property is urgently needed.

Disclosure of Invention

Problems to be solved by the invention

The invention aims to provide a brand-new heterocyclic compound used as an SHP2 inhibitor, which shows good inhibitory activity to tumor cells, has good druggability and wide drug development prospect. Moreover, the preparation method of the compounds is simple and is beneficial to industrial production.

Means for solving the problems

In a first aspect, the present invention provides a compound represented by formula I or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof, wherein

X¹、X²And X³Each independently selected from CR₅And N, or absent;

when X is present¹、X²And X³Each independently selected from CR₅And when N is, X⁵And X⁶Each independently selected fromC and N, X⁷Is CR₅Or N;

when X is present¹、X²And X³In the absence of, X⁵、X⁶And X⁷Each independently selected from CR₅And N;

X⁴is C or N;

X⁸is N or NR₅；

R₁、R₂、R₃、R₄、R₆And R₇Each independently selected from hydrogen, halogen, hydroxy, amino, oxo, cyano, C₂-C₈Alkenyl radical, C₂-C₈Alkynyl, aldehyde, carbamoyl, C₁-C₈Alkyl radical, C₁-C₈Heteroalkyl group, C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl, C₁-C₈Alkoxy and C₁-C₃Haloalkoxy, wherein said C₁-C₈Alkyl radical, C₁-C₈Heteroalkyl group, C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl radical, C₁-C₈Alkoxy and C₁-C₃Haloalkoxy is each optionally substituted with one or more R₅Substitution;

if present, each R₅Each independently selected from hydrogen, halogen, hydroxy, amino, cyano, carbamoyl, C₁-C₃Alkyl radical, C₁-C₃Heteroalkyl group, C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl, C₁-C₃Alkoxy and C₁-C₃Haloalkoxy, wherein said C₁-C₃Alkyl radical, C₁-C₃Heteroalkyl group, C₃-C₈Cycloalkyl, C₃-C₈Heterocycloalkyl radical, C₁-C₃Alkoxy and C₁-C₃Haloalkoxy is each optionally substituted with one or more R₈Substitution;

if present, each R₈Each independently selected from hydrogen, halogen, hydroxy, amino and cyano;

a and B are each independently selected from C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl radical, C₆-C₁₀Aryl and C₅-C₁₂A heteroaryl group;

wherein the heteroatoms or groups of heteroatoms in said heteroalkyl, heterocycloalkyl, and heteroaryl groups are each independently selected from-C (═ O) NH-, -N ═ O-, -S-, -C (═ O) O-, -C (═ O) -, -C (═ S) -, -S (═ O)₂-and-NHC (═ O) NH-, the number of heteroatoms or groups of heteroatoms in said heteroalkyl, heterocycloalkyl, and heteroaryl groups each being independently selected from 1,2, and 3;

n is 1,2 or 3.

Specifically, the compound shown in the formula I is a compound shown in a formula I-1 or a formula I-2, wherein

X⁵、X⁶And X⁷Each independently selected from CR₅And N;

X⁸、R₁、R₂、R₃、R₄、R₅、R₆b and n are as defined for formula I.

Preferably, in the compound represented by the formula I-1 or the formula I-2,

X⁵、X⁶and X⁷Each independently selected from CR₅And N;

X⁸is N or NR₅；

R₁Is hydrogen, amino, oxo or C₁-C₈Alkyl radical, wherein said C₁-C₈Alkyl is optionally substituted by one or more R₅Substitution;

R₂is hydrogen;

R₃is hydrogen or hydroxy;

each R₄Each independently selected from hydrogen, halogen and C₁-C₈Alkyl radical, wherein said C₁-C₈Alkyl radical orOptionally substituted by one or more R₅Substitution;

R₆is hydrogen;

if present, each R₅Each independently selected from hydrogen, amino, carbamoyl, C₁-C₃Alkyl and C₁-C₃Heteroalkyl group wherein said C₁-C₃Alkyl radical, C₁-C₃Heteroalkyl group, C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl radical, C₁-C₃Alkoxy and C₁-C₃Haloalkoxy is each optionally substituted with one or more R₈Substitution; preferably, if present, each R is₅Each independently selected from hydrogen, amino, carbamoyl and C₁-C₃Alkyl radical, wherein said C₁-C₃Alkyl is optionally substituted by one or more R₈Substitution;

if present, each R₈Each independently selected from hydrogen and hydroxyl;

b is C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl radical, C₆-C₁₀Aryl and C₅-C₁₂A heteroaryl group;

wherein the heteroatoms or groups of heteroatoms in said heteroalkyl, heterocycloalkyl, and heteroaryl groups are each independently selected from-C (═ O) NH-, -N ═ O-, -S-, -C (═ O) O-, -C (═ O) -, -C (═ S) -, -S (═ O)₂-and-NHC (═ O) NH-, the number of heteroatoms or groups of heteroatoms in said heteroalkyl, heterocycloalkyl, and heteroaryl groups each being independently selected from 1,2, and 3; preferably, the heteroatoms or groups of heteroatoms in the heterocycloalkyl and heteroaryl groups are each independently selected from-C (═ O) NH-, -N ═ O-, -S-, -C (═ O) O-, -C (═ O) -, -C (═ S) -, -S (═ O)₂-and-NHC (═ O) NH-, the number of heteroatoms or groups of heteroatoms in said heterocycloalkyl and heteroaryl groups each independently being selected from 1,2 and 3;

n is 1,2 or 3.

More preferably, in the compound represented by the formula I-1 or the formula I-2,

in the structure of

The fragment is selected from any one of the following fragments:

even more preferably, in the structure

The fragment is selected from any one of the following fragments:

or even more preferably, in the structure

The fragment is selected from any one of the following fragments:

specifically, the compound shown in the formula I is a compound shown in a formula I-3 or a formula I-4, wherein

X¹、X²And X³Each independently selected from CR₅Or N, X⁵And X⁶Each independently selected from C and N, X⁷Is CR₅Or N;

Preferably, in the compound represented by the formula I-3 or the formula I-4,

X⁸is N or NR₅；

R₁Is hydrogen or C₁-C₈Alkyl radical, wherein said C₁-C₈Alkyl is optionally substituted by one or more R₅Substitution;

R₂is hydrogen;

R₃hydrogen or hydroxy;

each R₄Each independently selected from hydrogen, halogen and C₁-C₈Alkyl radical, wherein said C₁-C₈Alkyl is optionally substituted by one or more R₅Substitution;

R₆is hydrogen;

if present, each R₅Each independently selected from hydrogen and C₁-C₃An alkyl group;

b is C₃-C₈Cycloalkyl, C₃-C₈Heterocycloalkyl radical, C₆-C₁₀Aryl and C₅-C₁₂A heteroaryl group;

wherein the heteroatoms or groups of heteroatoms in the heterocycloalkyl and heteroaryl groups are each independently selected from-C (═ O) NH-, -N ═ O-, -S-, -C (═ O) O-, -C (═ O) -, -C (═ S) -, -S (═ O)₂-and-NHC (═ O) NH-, the number of heteroatoms or groups of heteroatoms in said heterocycloalkyl and heteroaryl groups each independently being selected from 1,2 and 3;

n is 1,2 or 3.

More preferably, in the compound represented by the formula I-3 or the formula I-4,

in the structure of

The fragment is selected from any one of the following fragments:

even more preferably, in the structure

The fragment is selected from any one of the following fragments:

further preferably, in the compound represented by the formula I-1, the formula I-2, the formula I-3 or the formula I-4,

in the structure of

The fragment is selected from any one of the following fragments:

even further preferably, in the structure

The fragment is selected from any one of the following fragments:

in a second aspect, the present invention provides specific compounds of formula I, formula I-1, formula I-2, formula I-3 or formula I-4 selected from:

(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 [1,2-a ] pyrimidine-3-carboxamide;

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

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

(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;

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

(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;

(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;

(8) (S) -ethyl 3- (5-amino-5, 7-dihydrospiro [ cyclopenta [ b ] pyridine-6, 4 '-piperidine ] -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-carboxylate;

(9) (S) -3- (5-amino-5, 7-dihydrospiro [ cyclopenta [ b ] pyridine-6, 4 '-piperidine ] -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;

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

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

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

In a third aspect, the present invention provides a process for the preparation of a compound according to formula I, comprising the steps of:

1) reacting the compound A with the compound B to obtain a compound C;

2) reacting the compound C with the compound D to obtain a compound E; and

3) reacting the compound E with the compound F to obtain a target product;

wherein

LG₁And LG₂Each independently selected from chlorine and bromine, LG₃Is chlorine, bromine or hydroxyl;

X¹、X²、X³、X⁴、X⁵、X⁶、X⁷、X⁸、R₁、R₂、R₃、R₄、R₆、R₇a, B and n are as defined for formula I.

In the above preparation method, the compound A and the compound B may be in the presence of a catalyst (e.g., a palladium catalyst such as P)d₂(dba)₃) Ligands (e.g. xanthphos) and bases (e.g. organic bases such as DIEA; as well as in the presence of an inorganic base, such as cesium carbonate) to give compound C; compound C and compound D can be reacted (e.g., condensation reaction) in the presence of a base and a condensing agent (e.g., HATU, HBTU, EDCT/HOBT, etc.) to give compound E; compound E and compound F are reacted in the presence of a base (e.g., an organic base such as DIEA; and an inorganic base such as potassium carbonate, cesium carbonate, sodium tert-butoxide, etc.) to provide the compound of formula I.

The reaction solvent used in the steps of the above-mentioned production method of the present invention is not particularly limited, and any solvent capable of dissolving the starting materials to some extent and not inhibiting the reaction (e.g., DMF, DMSO, NMP, etc.) is included in the scope of the present invention. In addition, many similar modifications or equivalents in the art, or corresponding combinations of solvents, are contemplated as being within the scope of the present invention.

In a fourth aspect, the present invention provides a pharmaceutical composition, which comprises a compound 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, and at least one pharmaceutically acceptable excipient.

In a fifth aspect, the present invention provides a compound 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 same, for use as an inhibitor of SHP2 or for use in the prevention and/or treatment of a disease or condition associated with abnormal SHP2 activity.

In a sixth aspect, the invention provides a compound shown as 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 containing the same, for use in preparing a medicament for preventing and/or treating a disease or disorder associated with abnormal SHP2 activity.

In a seventh aspect, the present invention provides a method for preventing and/or treating a disease or disorder associated with abnormal SHP2 activity, comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of a compound 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.

Preferably, in the above pharmaceutical use, 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 tumors, gastric cancer, anaplastic large cell lymphoma and glioblastoma, preferably non-small cell lung cancer, esophageal cancer and head and neck tumors.

ADVANTAGEOUS EFFECTS OF INVENTION

The heterocyclic compound is a novel allosteric inhibitor and can achieve the aim of inhibiting the activity of SHP2 by combining with a non-catalytic region of SHP2 and locking a basic state with weak activity of SHP 2. The heterocyclic compound overcomes the defects of poor selectivity and druggability and the like of a PTP catalytic region inhibitor, shows excellent biological activity and druggability, and has a wide drug development prospect.

In addition, in an evaluation system such as an SHP2 enzyme activity inhibition experiment, a phosphorylated protein kinase (p-ERK) cell experiment, an NCI-H358 cell antiproliferation experiment, an MV-4-11 cell antiproliferation experiment and the like under the same conditions, compared with the known compounds RMC4550 and TNO155, the compound of the invention shows more excellent pharmacological activity and pharmacological properties.

Detailed Description

General terms and definitions

Unless stated to the contrary, the terms used in the present invention have the following meanings.

"alkyl" refers to a saturated aliphatic hydrocarbon group including straight and branched chain groups of 1 to 20 carbon atoms, such as straight 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 invention, "alkyl" may be a monovalent, divalent or trivalent group. Non-limiting examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, various branched chain isomers thereof, and the like. Non-limiting examples also include, but are not limited to, methylene, methine, ethylene, ethylidene, propylidene, butylidene, and various branched chain isomers thereof. In addition, in the present invention, "alkyl" may be optionally substituted or unsubstituted.

"heteroalkyl" refers to a saturated aliphatic hydrocarbon group including straight and branched chains of 1 to 20 carbon atoms, such as straight and branched chains 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, interrupted by and connected through one or more (e.g., 1,2, 3, etc.) heteroatoms or groups of heteroatoms selected from-C (═ O) NH-, -N ═ O-, -S-, -C (═ O) O-, -C (═ O) -, -C (═ S) -, -S (═ O)₂-and-NHC (═ O) NH-. In the present invention, "heteroalkyl" may be a monovalent, divalent or trivalent group. Non-limiting examples include, but are not limited to, C interrupted by-C (═ O) O —₁-C₃Heteroalkyl, e.g. carboxy (C)₁Heteroalkyl group), methoxycarbonyl group (C)₂Heteroalkyl group), ethoxycarbonyl group (C)₃Heteroalkyl), or the like, or C interrupted by-C (═ O) NH —₁-C₃Heteroalkyl radicals, e.g. carbamoyl (C)₁Heteroalkyl group), methylcarbamoyl group (C)₂Heteroalkyl group), ethylcarbamoyl group (C)₃Heteroalkyl) and the like. In addition, in the present invention, "heteroalkyl" may be optionally substituted or unsubstituted.

"cycloalkyl" refers to a saturated or partially unsaturated, monocyclic or polycyclic aliphatic hydrocarbon group comprising 3 to 12 ring atoms, which may be, for example, 3 to 12, 3 to 10, or 3 to 6 ring atoms (i.e., a 3 to 6 membered ring). 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 invention, "cycloalkyl" may be optionally substituted or unsubstituted.

"Heterocycloalkyl" means a saturated or partially unsaturated, monocyclic or polycyclic aliphatic hydrocarbon group containing 3 to 20 ring atoms, which may be, for example, 3 to 16, 3 to 12, 3 to 10 or 3 to 6 ring atoms, wherein one or more of the ring atoms is selected from nitrogen, oxygen or S (O)_m(wherein m is 0, 1 or 2) and the remaining ring atoms are carbon. Preferably the heterocycloalkyl group comprises 3 to 12 ring atoms of which 1 to 4 ring atoms are heteroatoms, more preferably 3 to 10 ring atoms, most preferably 5 or 6 ring atoms of which 1 to 4, preferably 1 to 3, more preferably 1 to 2 are heteroatoms. Non-limiting examples of monocyclic heterocycloalkyl 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, spiro or bridged heterocycloalkyl groups.

"halogen" means fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "a heterocyclic group optionally substituted with an alkyl" means that an alkyl may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl and the heterocyclic group is not substituted with an alkyl.

By "substituted" is meant that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in the group are independently substituted with a corresponding number of substituents.

"pharmaceutically acceptable salt" refers to a salt prepared from a compound of the present invention with a relatively nontoxic acid or base.

"pharmaceutical composition" refers to a pharmaceutically acceptable composition comprising one or more compounds of formula I or pharmaceutically acceptable forms thereof (e.g., salts, hydrates, solvates, stereoisomers, tautomers, metabolites, prodrugs, etc.), as well as other components (e.g., pharmaceutically acceptable excipients).

In the present invention, "pharmaceutically acceptable auxiliary materials" refer to auxiliary materials widely used in the field of pharmaceutical production. The main purpose of the use of adjuvants is to provide a pharmaceutical composition that is safe to use, stable in nature and/or has a specific functionality, and to provide a method for dissolving the active ingredient at a desired rate or for promoting an efficient absorption of the active ingredient in the body of the subject to whom it is administered, after administration of the drug to the subject. Pharmaceutically acceptable excipients may be inert fillers or may be functional ingredients that provide some function to the pharmaceutical composition (e.g., stabilize the overall pH of the composition or prevent degradation of the active ingredients in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, adhesives, disintegrating agents, lubricants, antiadherents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffering agents, chelating agents, preservatives, coloring agents, flavoring agents, sweetening agents, and the like.

The pharmaceutical compositions of the present invention may be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, levigating, encapsulating, entrapping and/or lyophilizing processes.

In the present invention, the pharmaceutical composition is used for the purpose of promoting administration to a living body, facilitating absorption of an active ingredient, and exerting biological activity. The pharmaceutical compositions of the present invention may be administered in any form, including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (solid oral, liquid oral), rectal, inhalation, implant, topical (e.g., ocular) administration, and the like. Non-limiting examples of oral solid formulations include, but are not limited to, powders, capsules, lozenges, granules, tablets, and the like. Non-limiting examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, and the like. Non-limiting examples of formulations for topical administration include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum formulations. Non-limiting examples of parenteral formulations include, but are not limited to, injectable solutions, injectable dry powders, injectable suspensions, injectable emulsions, and the like. The pharmaceutical compositions of the present invention may also be formulated as controlled release or delayed release dosage forms (e.g., liposomes or microspheres).

Preferably, the compound of the present invention or the pharmaceutical composition comprising the same is administered to an individual in need thereof by oral or intravenous administration. Other routes of administration may also be applicable and even preferred depending on the particular circumstances of the subject. For example, for patients who are forgetful or have irritability to orally administered drugs, transdermal administration would be a very important mode of administration. In the present invention, the route of administration can be varied or adjusted in any suitable manner to meet the needs of the nature of the drug, the convenience of the patient and the medical staff, and other relevant factors.

The compound or the pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof or the pharmaceutical composition containing the compound or the pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof have excellent SHP2 enzyme activity and cell proliferation inhibition activity, can be used as an SHP2 inhibitor, is used for preventing and/or treating diseases or symptoms related to the abnormal SHP2 activity (or caused by abnormal mutation of SHP 2), and has good clinical application and medical application. Preferably, non-limiting examples of diseases or disorders associated with aberrant activity of SHP2 (or caused by aberrant mutation of SHP 2) include, but are not limited to, noonan syndrome, leopard syndrome, leukemias (e.g., juvenile myelomonocytic leukemia, acute myelogenous leukemia), neuroblastoma, melanoma, breast cancer, esophageal cancer, lung cancer, colon cancer, head and neck tumors, gastric cancer, anaplastic large cell lymphoma, glioblastoma, and the like, preferably non-small cell lung cancer, esophageal cancer, and head and neck tumors.

The technical solutions of the present invention will be illustrated below with reference to specific examples, which are provided to further illustrate the present invention and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made in the specific embodiments of the present invention without departing from the spirit and scope of the invention.

The preparation of the compounds of the present invention may be accomplished by synthetic methods well known to those skilled in the art, including but not limited to the specific embodiments listed below, embodiments formed by combinations with other chemical synthetic methods, and equivalents known to those skilled in the art, with preferred embodiments including but not limited to the examples of the present invention. Known starting materials for use in the present invention may be synthesized by methods known in the art or purchased by conventional commercial means (e.g., from Shaosha remote chemical technology, Beijing coupling technology, etc.). Unless otherwise specified, the reaction was carried out under an argon atmosphere or a nitrogen atmosphere. The hydrogenation reaction was usually evacuated and charged with hydrogen and repeated 3 times. The reaction temperature is room temperature and the temperature range is 20-30 ℃. Monitoring of the progress of the reaction can be accomplished by synthetic methods well known to those skilled in the art, including but not limited to Thin Layer Chromatography (TLC). Thin layer chromatography silica gel plates using Qingdao ocean GF254 silica gel plates, developer systems include but are not limited to A: dichloromethane and methanol systems; b: petroleum ether and ethyl acetate, the volume ratio of the solvent can be adjusted according to the polarity of the compound.

The isolation and purification of the compounds of the present invention can be accomplished 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), and the like. Column chromatography typically uses Qingdao ocean 200-: dichloromethane and methanol systems; b: the volume ratio of the petroleum ether to the ethyl acetate system can be adjusted according to the polarity of the compound, and a small amount of acidic or alkaline tailing-preventing agent can also be added for adjustment. The HPLC profile was determined using an Agilent1200DAD HPLC chromatograph (column: Sunfire C18,150X 4.6mm,5 μm) or a Waters 2695-2996HPLC chromatograph (column: Gimini C18,150X 4.6mm,5 μm).

Structural identification of the compounds of the invention can be accomplished by methods well known to those skilled in the art, including but not limited to Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS), and the like. NMR spectrum is measured by Bruker AVANCE-400 or Varian Oxford-300 nuclear magnetic instrument, and the measuring solvent is deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDC 1)₃) Or deuterated methanol (CD)₃OD), internal standard Tetramethylsilane (TMS), chemical shift at 10^-6(ppm). MS spectra were measured using an Agilent SQD (ESI) mass spectrometer (model: 6110) or Shimadzu SQD (ESI) mass spectrometer (model: 2020).

Synthesis of intermediates

Synthesis of intermediate Q1

The first step is as follows: synthesis of Q1-2

Compound Q1-1(100g,495mmol) was added to DME (1000ml), after cooling to-10 deg.C, isobutyl chloroformate (67.6g,495mmol) and 4-methylmorpholine (50g,495mmol) were added and reacted at room temperature for 5h, after TLC monitoring the reaction was completed, filtration was carried out, the solid was washed with DME (250ml) filtration, then adding the filtrate into a 2L three-necked bottle, adding sodium borohydride (37.6g,990mmol) to process the filtrate, stirring for 30min at room temperature, slowly dropwise adding methanol (250ml), continuing to react for 3h at room temperature, monitoring by TLC to complete the reaction, spin-drying the reaction solution, water (1500ml) was added and the aqueous phase was extracted with DCM (300 ml. times.3), after combining the organic phases, the organic phases were washed successively with water and saturated sodium chloride solution, and the organic phases were dried and spin dried to give compound Q1-2(86.5g, white solid) in 93% yield. The crude product was used in the next reaction without purification.

The second step is that: synthesis of Q1-3

Compound Q1-2(25g,133mmol) was added to DCM (250ml), TEA (26.9g,266mmol) was added, and MsCl (18.3g,156mmol) was added dropwise under ice bath, followed by reaction at room temperature for 1 h. TLC showed the end of the reaction, the reaction was diluted with DCM, washed successively with water and saturated sodium chloride solution, the organic phase was dried and concentrated to dryness, and the residue was chromatographed on silica gel (eluent: V)_{Petroleum ether}:V_{Ethyl acetate}Purification was carried out at 3:1 to 1:1) to obtain compound Q1-3(33.6g, a colorless transparent liquid) in a yield of 95%.

MS(ESI):m/z 266[M+H]⁺。

The third step: synthesis of Q1-4

Adding N-tert-butyloxycarbonyl-4-cyanopiperidine (28g,130mmol) into THF (300ml), cooling to-78 deg.C, slowly adding LDA (75ml,150mmol) of 2.0M dropwise, continuing to react at-78 deg.C for 1.5h after completion of dropwise addition, adding THF solution (150ml) of compound Q1-3(26.6,100mmol), continuing to react at-78 deg.C for 3h after completion of dropwise addition, after TLC showed completion of reaction, adding saturated ammonium chloride solution (50ml) dropwise to quench the reaction, adding saturated sodium chloride solution (500ml), separating out the organic phase, extracting the aqueous phase with ethyl acetate (150 ml. times.3), combining the organic phases, drying with anhydrous sodium sulfate, filtering, concentrating by spin drying, and passing the residue through a silica gel column (eluent: V)_{Petroleum ether}:V_{Ethyl acetate}Purification from 10:1 to 1:1) gave compound Q1-4(25.7g, white solid) in 67.6% yield.

MS(ESI):m/z 380[M+H]⁺。

The fourth step: synthesis of Q1-5

Compound Q1-4(25g,65.8mmol) was added to a mixed solvent of DMA (200ml) and water (20ml), and triethylamine (33.2g,329mmol) and Pd (amphos) as a catalyst were further added₂(4.6g,6.6mmol, CAS:887919-35-9), under nitrogen at 130 ℃ for 4 h. TLC showed the reaction was complete, the reaction was cooled and diluted with water (800mL), the aqueous phase was extracted with ethyl acetate (200 mL. times.3), the organic phases were combined, washed with saturated brine (200 mL. times.2), dried over anhydrous sodium sulfate, filtered, concentrated by rotary drying, and the residue was chromatographed on silica gel (eluent: V)_{Petroleum ether}:V_{Ethyl acetate}＝3:1～1:1) to give compound Q1-5(14.6g, white solid) in 73% yield.

MS(ESI):m/z 303[M+H]⁺。

The fifth step: synthesis of Q1-6

Compound Q1-5(10g,33mol) was added to tetraethyl titanate (100mL), followed by addition of (R) - (+) -tert-butylsulfinamide (4.8g,40mL), warming to 90 ℃ for 3h, TLC showed completion of the reaction, cooling to room temperature, slowly adding the reaction solution to ice water (500mL), extraction of the aqueous phase with dichloromethane (150 mL. times.3), combination of the organic phases, washing with saturated brine (100 mL. times.2), drying over anhydrous sodium sulfate, filtration, spin-drying concentration, and chromatography of the residue on silica gel (eluent: V)_{Petroleum ether}:V_{Ethyl acetate}Purification 3: 1-1: 1) gave compound Q1-6(12g, yellow solid) in 89.6% yield.

MS(ESI):m/z 406[M+H]⁺。

And a sixth step: synthesis of Q1-7

Adding compound Q1-6(10g,24.6mmol) into tetrahydrofuran (100mL), cooling to-78 deg.C, slowly adding DIBAL-H (30mL,30mmol,1M toluene solution) dropwise, continuing to react at-78 deg.C for 0.5H, after TLC shows that the reaction is completed, adding saturated Rochelle salt solution (300mL) at-50 deg.C to quench the reaction, stirring at room temperature for 30min, extracting the aqueous phase with ethyl acetate (150 mL. times.3), combining the organic phases, washing with saturated brine (100 mL. times.2), drying with anhydrous sodium sulfate, filtering, concentrating by spin drying, passing the residue through silica gel chromatography column (eluent: V)_{Petroleum ether}:V_{Ethyl acetate}Purification was carried out 3:1 to 1:1) to obtain compound Q1-7(8.6g, white solid) with a yield of 85.3%.

MS(ESI):m/z 408[M+H]⁺。

The seventh step: synthesis of intermediate Q1

Compound Q1-7(5g,12.2mmol) was added to ethyl acetate (50ml) and 4M HCl/ethyl acetate solution (25ml) was added and the reaction was allowed to proceed at room temperature for 1h, after TLC showed completion of the reaction, filtration was carried out and the solid was washed with ethyl acetate and dried to give intermediate Q1 as the hydrochloride salt (3.2g, white solid) in 94% yield.

MS(ESI):m/z 204[M+H]⁺。

Synthesis of intermediate Q2

The first step is as follows: synthesis of Q2-2

Compound Q2-1(205mg,1mmol) was dissolved in dioxane (5ml), and methyl 3-mercaptopropionate (180mg,1.5mmol), Pd were added₂(dba)₃(22.73mg,0.025mmol,0.05 equiv.), Xantphos (14.36mg,0.025mmol) and DIEA (387mg,3 mmol). After warming to 90 ℃ under nitrogen and stirring for 1 hour, the reaction mixture is concentrated by spin-drying and the residue is chromatographed on silica gel (eluent: V)_{Petroleum ether}:V_{Ethyl acetate}Purification from 3:1 to 1:1) gave compound Q2-2(201mg, pale yellow liquid) in 83% yield.

MS(ESI):m/z 246[M+H]⁺。

The second step is that: synthesis of intermediate Q2

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

MS(ESI):m/z 182[M+H]⁺。

Synthesis of intermediate Q3

The first step is as follows: synthesis of Q3-2

Compound Q3-1(10g,106mmol) was added to xylene (100ml), followed by addition of triethyl methanetricarboxylate (48g,222mmol, CAS:6279-86-3), after the addition was complete, heating to 140 ℃ for reaction for 3h, after TLC showed the reaction was complete, cooling to room temperature, solid precipitated, filtered, the filter cake was washed with ether and dried to give compound Q3-2(16.2g, a pale yellow solid) in 65% yield. The crude product was used in the next reaction without purification.

MS(ESI):m/z 235[M+H]⁺。

The second step is that: synthesis of intermediate Q3-3

Compound Q3-2(5g,21mmol) was added to a mixed solvent of methanol (40ml) and water (10ml), hydrogen balloon was fitted over, air was displaced, and after hydrogen gas was introduced, reaction was carried out for 3h, TLC showed that after the reaction was completed, filtration was carried out, the filter cake was washed with methanol (50ml), and the filtrate was dried by spinning to give compound Q3-3(4.3g, pale yellow solid) in 92% yield. The crude product was used in the next reaction without purification.

MS(ESI):m/z 239[M+H]⁺。

The third step: synthesis of intermediate Q3

Compound Q3-3(5g,21mmol) was added to a mixed solvent of methanol (50ml) and water (10ml), followed by lithium hydroxide monohydrate (1.7g,42mmol), stirred at room temperature for 2h, TLC showed the reaction to complete, pH was adjusted to 3-4 with 1M hydrochloric acid, a large amount of solid precipitated, filtered, and the cake was dried to give intermediate Q3(3.98g, white solid) in 90% yield. The crude product was used in the next reaction without purification.

MS(ESI):m/z 211[M+H]⁺。

Synthesis of intermediate Q4

The first step is as follows: synthesis of Q4-1

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

MS(ESI):m/z 438.2[M+H]⁺。

The second step: synthesis Q4

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

Synthesis of intermediate Q5

The first step is as follows: synthesis of Q5-2

A mixture of the compound 2-aminopyridine (4.35g, 46.3mmol) and diethyl ethoxymethylenemalonate (10.00g, 46.3mmol, cas:87-13-8) was heated at 120 ℃ for 1 hour. After cooling to room temperature and further cooling to 5 ℃, the mixture was slurried with iced ethanol, filtered, and the solid was washed with iced ethanol and dried to give compound Q5-2(9.90g, yellow solid) in 81% yield.

MS(ESI):m/z 265.2[M+H]⁺。

The second step is that: synthesis of Q5-3

Compound Q5-2(13g, 5.9mmol) was added to diphenyl ether (100ml), which was then heated to 280 ℃ and refluxed for 2 hours, and after completion of the TLC reaction, the reaction mixture was cooled to room temperature, and n-hexane (500ml) was added thereto to obtain a large amount of solid precipitate. The precipitated solid was filtered, washed with n-hexane and dried to obtain compound Q5-3(10g, yellow solid) in a yield of 93.1%.

MS(ESI):m/z 219.2[M+H]⁺。

The third step: synthesis Q5

Compound Q5-3(5g,23mmol) was added to a mixed solvent of methanol (40ml) and water (10ml), hydrogen balloon was fitted over, air was displaced, hydrogen was introduced and the reaction was carried out for 3h, TLC showed the end of the reaction, filtration was carried out, the filter cake was washed with methanol (50ml), and the filtrate was dried by spinning to give compound Q5(4.6g, pale yellow solid) in 92% yield. The crude product was used in the next reaction without purification.

Synthesis of intermediate Q6

The first step is as follows: synthesis of Q6-2

Compound Q2-1(205mg,1mmol) was dissolved in dioxane (5ml), and methyl 3-mercaptopropionate (180mg,1.5mmol), Pd were added₂(dba)₃(22.73mg,0.025mmol,0.05 equiv.), Xantphos (14.36mg,0.025mmol) and DIEA (387mg,3 mmol). After warming to 90 ℃ under nitrogen and stirring for 1 hour, the reaction mixture is concentrated by spin-drying and the residue is chromatographed on silica gel (eluent: V)_{Petroleum ether}:V_{Acetic acid ethyl ester}Purification 3: 1-1: 1) gave compound Q2-2(201mg, light yellow liquid) in 83% yield.

MS(ESI):m/z 246[M+H]⁺。

The second step is that: synthesis of intermediate Q6

MS(ESI):m/z 182[M+H]⁺。

Synthesis of intermediate Q7

The first step is as follows: synthesis of Q7-2

Compound Q2-1(205mg,1mmol) was dissolved in dioxane (5ml), and methyl 3-mercaptopropionate (180mg,1.5mmol), Pd were added₂(dba)₃(22.73mg,0.025mmol,0.05 eq.), Xantphos (14.36mg,0.025mmol) and DIEA (387mg,3 mmol). After warming to 90 ℃ under nitrogen and stirring for 1 hour, the reaction mixture is concentrated by spin-drying and the residue is chromatographed on silica gel (eluent: V)_{Petroleum ether}:V_{Ethyl acetate}Purification 3: 1-1: 1) gave compound Q2-2(201mg, light yellow liquid) in 83% yield.

MS(ESI):m/z 246[M+H]⁺。

The second step is that: synthesis of intermediate Q7

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

MS(ESI):m/z 182[M+H]⁺。

Example 1: preparation of Compound 1

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 1B

Compound 1A (104mg,0.5mmol) was dissolved in dioxane (5ml), and intermediate Q2(182mg,1mmol) and Pd were added₂(dba)₃(22.73mg,0.025mmol), Xantphos (14.36mg,0.025mmol) and DIEA (190mg,1.5 mmol). After warming to 90 ℃ under nitrogen and stirring for 1h, the reaction mixture is concentrated by spin-drying and the residue is chromatographed on silica gel (eluent: V)_{Methylene dichloride}:V_MethanolPurification 50: 1-10: 1) gave compound 1B (107mg, light yellow solid) in 75% yield.

MS(ESI):m/z 287[M+H]⁺。

The second step is that: synthesis of Compound 1C

Compound 1B (100mg,0.35mmol), DIEA (154mg,1.2mmol), intermediate Q3(147mg,0.7mmol) and HATU (456mg,1.2mmol) were added to DMF and stirred at room temperature for 12h, TLC showed the completion of the reaction, the reaction solution was diluted with dichloromethane (10ml), the organic phase was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was collected, concentrated under reduced pressure, and the residue was chromatographed on silica gel (eluent: V)_{Methylene dichloride}:V_MethanolPurification at 50: 1-10: 1) gave compound 1B (137mg, light yellow solid) in 82% yield.

MS(ESI):m/z 479[M+H]⁺。

The third step: synthesis of Compound 1

Compound 1C (100mg,0.21mmol), intermediate Q1(71mg,0.35mmol) and DIEA (82mg,0.63mmol) were dissolved in dimethyl sulfoxide (5ml) and reacted at 90 ℃ for 1.5 h. After the reaction was completed, ethyl acetate (20ml) and water (40ml) were added, the aqueous phase was extracted with ethyl acetate (20 ml. times.3), the organic phases were combined, washed with saturated sodium oxide solution (100ml), dried over anhydrous sodium sulfate, filtered, the filtrate was collected, concentrated under reduced pressure, and the residue was subjected to silica gel chromatography column (eluent: V)_{Methylene dichloride}:V_MethanolPurification at 50: 1-10: 1) gave compound 1(32mg, light yellow solid).

MS(ESI):m/z 646[M+H]⁺。

¹H-NMR(400MHz,DMSO-d₆):δ12.26(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)。

Example 2: preparation of Compound 2

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 2B

Compound 2A (1.0g,3.6mmol), compound Q1(808mg,4.0mmol) and DIEA (4.6g, 36mmol) were added to MeCN (2240mL) and the resulting solution was stirred at 80 ℃ for 2 hours. TLC showed the reaction was complete. The mixture was cooled to 25 ℃ and Boc₂O (1.6g, 7.2mmol) was added to the solution. The reaction was stirred at 50 ℃ for 2 hours. TLC showed the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 1/2, V/V) to give compound 2B (953mg, pale yellow solid) in 48.4% yield.

MS(ESI):m/z 547.1[M+H]⁺。

The second step is that: synthesis of Compound 2C

Compound 2C (900mg,1.6mmol) was dissolved in NMP (10ml), and intermediate Q4(895mg,2.4mmol) and Pd were added₂(dba)₃(145mg,0.16mmol), Xantphos (92mg,0.16mmol) and DIEA (619mg,4.8 mmol). After warming to 120 ℃ under nitrogen and stirring for 1h, after completion of the TLC reaction, the reaction mixture was added to 75ml of saturated sodium chloride solution, extracted with EtOAc (25 ml. times.3), the organic phases were combined, washed with saturated sodium chloride solution (100ml), dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and the residue was purified by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 2C (553mg, light yellow solid), 45% yield.

MS(ESI):m/z 770.2[M+H]⁺。

The third step: synthesis of Compound 2

Compound 2C (500mg,0.65mmol) was added to ethyl acetate (5ml) and 4M HCl in ethyl acetate (10ml) was added and reacted at room temperature for 1h, after TLC showed completion of the reaction, filtered, the solid was washed with ethyl acetate and dried to give the hydrochloride salt of compound 2 (400mg, white solid) in 94% yield.

MS(ESI):m/z 670.2[M+H]⁺。

¹H NMR(400MHz,DMSO)δ12.28(s,1H),9.13-8.86(m,3H),8.67(d,J＝5.6Hz,1H),8.43-8.27(m,2H),8.22-8.16(m,1H),8.07(d,J＝25.2Hz,2H),7.66-7.57(m,1H),7.21(t,J＝8.0Hz,1H),6.71(d,J＝8.0Hz,1H),4.62-4.58(m,1H),4.16-4.13(m,1H),4.07-4.02(m,1H),3.88(t,J＝6.0Hz,2H),3.55-3.48(m,3H),3.30-3.24(m,1H),2.90(t,J＝6.4Hz,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＝13.6Hz,1H)。

Example 3: preparation of Compound 3

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 3B

Compound 3A (6g,24.80mmol) was dissolved in DMF (50mL) and a solution of DIEA (8.66mL,49.61mmol) and aminoacetaldehyde dimethyl acetal (2.90g, 27.28mmol) in DMF was added dropwise at 0 deg.C and allowed to warm to room temperature for 2 h. TLC showed the reaction was complete, added water and extracted with ethyl acetate, 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 (vol.%)) to give compound 3B (7.4g, pale yellow solid) in 91% yield.

MS(ESI):m/z310[M+H]⁺。

The second step is that: synthesis of Compound 3C

Compound 3B (5g,16.10mmol) was slowly added in portions to concentrated sulfuric acid (15mL) while cooling on ice, and the temperature was raised to 75 ℃ for reaction for 2 h. TLC indicated the reaction was complete, the reaction was cooled to 0 deg.c and 5N aqueous sodium hydroxide was added dropwise to adjust the pH to 6, a yellow solid precipitated, which was filtered and dried to afford compound 3C (2.77g, yellow solid) in 71% yield.

MS(ESI):m/z 228[M+H]⁺。

The third step: synthesis of Compound 3D

Compound 3C (2.77g,12.15mmol) was added to POCl₃DIEA (4.24mL,24.29mmol) was added dropwise at 0 ℃ to 30mL, and the temperature was raised to 115 ℃ for 15 min. TLC showed the reaction was complete, the reaction was spin dried, ethyl acetate was added and poured slowly into water, the pH was adjusted to neutral with saturated aqueous sodium bicarbonate solution, extracted, 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 ═ 3:1 (vol.%)) to give compound 3D (1.17g, pale yellow solid) in 38% yield.

MS(ESI):m/z 246[M+H]⁺。

The fourth step: synthesis of Compound 3E

Compound 3D (886mg,3.6mmol), compound Q1(808mg,4.0mmol) and DIEA (4.6g, 36mmol) were added to MeCN (40mL) and the resulting solution was stirred at 80 ℃ for 2 h. TLC showed the reaction was complete. The mixture was cooled to 25 ℃ and Boc₂O (1.6g, 7.2mmol) was added to the solution. The reaction was stirred at 50 ℃ for 2 hours. TLC showed the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 1/2, V/V) to give compound 3E (786mg, pale yellow solid) in 38.9% yield.

MS(ESI):m/z 561.1[M+H]⁺。

The fifth step: synthesis of Compound 3F

Compound 3E (700mg,1.24mmol) was dissolved in NMP (10ml), and intermediate Q4(895mg,2.4mmol), Pd were added₂(dba)₃(145mg,0.16mmol), Xantphos (92mg,0.16mmol) and DIEA (619mg,4.8 mmol). After warming to 120 ℃ under nitrogen and stirring for 1h, after the TLC reaction was complete, the reaction mixture was added to 75ml of saturated sodium chloride solution and extracted with EtOAc (25 ml. times.3), the organic phases were combined, washed with saturated sodium chloride solution (100ml), dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and the residue was purified by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 3F (340mg, light yellow solid), 35% yieldAnd (4) rate.

MS(ESI):m/z 784.2[M+H]⁺。

And a sixth step: synthesis of Compound 3

Compound 3F (300mg,0.38mmol) was added to ethyl acetate (5ml) and 4M HCl in ethyl acetate (10ml) was added and reacted at room temperature for 1h, after TLC showed completion of the reaction, filtered, the solid was washed with ethyl acetate and dried to give the hydrochloride salt of compound 3 (239mg, white solid) in 92% yield.

MS(ESI):m/z 684.2[M+H]⁺。

¹H NMR(400MHz,DMSO)δ12.28(s,1H),9.31(s,1H),8.55(d,J＝4.8Hz,1H),8.49-8.33(m,2H),8.14(d,J＝8.4Hz,1H),7.93(d,J＝7.2Hz,1H),7.35(dd,J＝7.6,4.8Hz,1H),7.28-6.96(m,3H),6.54(d,J＝8.4Hz,1H),4.53(d,J＝4.8Hz,1H),4.23(d,J＝13.6Hz,1H),4.14(d,J＝14.4Hz,1H),3.89(t,J＝5.6Hz,2H),3.60-3.54(m,2H),3.28(d,J＝16.8Hz,1H),3.14(d,J＝16.8Hz,1H),2.89(t,J＝6.5Hz,2H),2.52(s,3H),2.05-1.87(m,3H),1.86-1.69(m,3H),1.62(d,J＝12.8Hz,1H),1.28-1.21(m,1H)。

Example 4: preparation of Compound 4

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 4B

Compound 4A (10g,71mmol) was dissolved in THF (100ml) and NIS (17.6g, 78mmol) was added slowly in portions at room temperature and reacted overnight at room temperature. Reaction completion was checked by LCMS and the reaction was added to 500ml of water to precipitate a large amount of solid which was filtered and washed with dichloromethane and dried to give compound 4B (16.5g, pale yellow solid) in 87% yield.

MS(ESI):m/z 268.1[M+H]⁺。

The second step is that: synthesis of Compound 4C

Compound Q1(1.0g,4.9mmol) was added to DMF (10ml) followed by Compound 4B (2.62g,9.8mmol) and BOP (4.3g,9.8mmol), after which DBU (2.2g,14.7mmol) was added slowly at room temperature and reacted overnight at room temperature after which Boc was added₂O (1.6g, 7.2mmol) was added to the solution. The reaction was stirred at 50 ℃ for 2 hours. TLC showed the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography over silica gel (eluent: petroleum ether/ethyl acetate 1/2, V/V) to give compound 4C (1.1g, a pale yellow solid) in 42.3% yield.

MS(ESI):m/z 553.1[M+H]⁺。

The third step: synthesis of Compound 4D

Compound 4C (830mg,1.5mmol) was dissolved in NMP (10ml), and intermediate Q4(895mg,2.4mmol) and Pd were added₂(dba)₃(145mg,0.16mmol), Xantphos (92mg,0.16mmol) and DIEA (619mg,4.8 mmol). After warming to 120 ℃ under nitrogen and stirring for 1h, after completion of the TLC reaction, the reaction mixture was added to 75ml of saturated sodium chloride solution, extracted with EtOAc (25 ml. times.3), the organic phases were combined, washed with saturated sodium chloride solution (100ml), dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and the residue was purified by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 4D (384mg, light yellow solid), 33% yield.

MS(ESI):m/z 776.2[M+H]⁺。

The fourth step: synthesis of Compound 4

Compound 4D (350mg,0.45mmol) was added to ethyl acetate (5ml) and 4M HCl/ethyl acetate solution (10ml) was added and reacted at room temperature for 1h, TLC showed completion of the reaction, filtered, the solid was washed with ethyl acetate and dried to give the hydrochloride salt of compound 4 (276mg, white solid) in 91% yield.

MS(ESI):m/z 676.2[M+H]⁺。

Example 5: preparation of Compound 5

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 5A

Compound 1B (500mg,1.75mmol) and compound Q5(388mg,1.75mmol) were added to NMP (10ml) and then warmed to 120 ℃ under nitrogen and stirred for 1h, after the TLC reaction was complete, the reaction mixture was added to 75ml of saturated sodium chloride solution and extracted with EtOAc (25ml × 3), the organic phases were combined, washed with saturated sodium chloride solution (100ml), dried over anhydrous sodium sulfate, spin-dried and concentrated, and the residue was purified by silica gel column chromatography (eluent, PE: EA 3:1 (volume ratio)) to give compound 5A (339mg, light yellow solid) in 42% yield.

MS(ESI):m/z 463.2[M+H]⁺。

The third step: synthesis of Compound 5

Compound 5A (300mg,0.64mmol), compound Q1(156mg,0.77mmol) and DIEA (826mg, 6.4mmol) were added to MeCN (10mL) and the resulting solution was stirred at 80 ℃ for 2 h. TLC showed the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 5(106mg, light yellow solid), 26.2% yield.

MS(ESI):m/z 630.2[M+H]⁺。

¹H NMR(400MHz,DMSO)δ11.28(s,1H),8.53(d,J＝4.2Hz,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＝34.4,12.4Hz,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)。

Example 6: preparation of Compound 6

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 6A

Compound 1A (207mg,1.0mmol) was dissolved in dioxane (5ml), and intermediate Q6(322mg,2mmol) and Pd were added₂(dba)₃(45mg,0.05mmol), Xantphos (28mg,0.05mmol) and DIEA (380mg,3.0 mmol). After warming to 90 ℃ under nitrogen and stirring for 1h, the reaction mixture is concentrated by spin-drying and the residue is chromatographed on silica gel (eluent: V)_{Methylene dichloride}:V_MethanolPurification 50: 1-10: 1) gave compound 6A (173mg, light yellow solid) in 65% yield.

MS(ESI):m/z 267.2[M+H]⁺。

The second step is that: synthesis of Compound 6B

Compound 6A (100mg,0.37mmol), DIEA (154mg,1.2mmol), intermediate Q3(147mg,0.7mmol) and HATU (456mg,1.2mmol) were added to DMF and stirred at room temperature for 12h, TLC showed the completion of the reaction, the reaction solution was diluted with dichloromethane (10ml), the organic phase was washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was collected, concentrated under reduced pressure, and the residue was chromatographed on silica gel (eluent: V)_{Methylene dichloride}:V_MethanolPurification 50: 1-10: 1) gave compound 6B (138mg, light yellow solid) in 81.5% yield.

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

The third step: synthesis of Compound 6

Compound 6B (100mg,0.22mmol), intermediate Q1(71mg,0.35mmol) and DIEA (82mg,0.63mmol) were dissolved in dimethyl sulfoxide (5ml) and reacted at 90 ℃ for 1.5 h. After completion of the reaction, ethyl acetate (20ml) and water (40ml) were added to the reaction solution, and the aqueous phase was extracted with acetic acidExtraction with ethyl ester (20 ml. times.3), combination of the organic phases, washing with saturated sodium oxide solution (100ml), drying over anhydrous sodium sulfate, filtration, collection of the filtrate, concentration under reduced pressure, and separation and purification of the residue by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 6(30.7mg, light yellow solid), 22.3% yield.

MS(ESI):m/z 626.2[M+H]⁺。

Example 7: preparation of Compound 7

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 7A

Compound 1A (207mg,1.0mmol) was dissolved in dioxane (5ml), and intermediate Q7(358mg,2mmol) and Pd were added₂(dba)₃(45mg,0.05mmol), Xantphos (28mg,0.05mmol) and DIEA (380mg,3.0 mmol). After warming to 90 ℃ under nitrogen and stirring for 1h, the reaction mixture was concentrated by rotary drying and the residue was purified on silica gel chromatography column (eluent dichloromethane: methanol 50: 1-10: 1, V: V) to give compound 7A (194mg, light yellow solid) in 63.5% yield.

MS(ESI):m/z 305.1[M+H]⁺。

The second step is that: synthesis of Compound 7B

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

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

The third step: synthesis of Compound 7

Compound 7B (110mg,0.22mmol), intermediate Q1(71mg,0.35mmol) and DIEA (82mg,0.63mmol) were dissolved in dimethyl sulfoxide (5ml) and reacted at 90 ℃ for 1.5 h. After the reaction was completed, ethyl acetate (20ml) and water (40ml) were added, the aqueous phase was extracted with ethyl acetate (20 ml. times.3), the organic phases were combined, washed with saturated sodium oxide solution (100ml), dried over anhydrous sodium sulfate, filtered, the filtrate was collected, concentrated under reduced pressure, and the residue was separated and purified by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% of B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 7(37.2mg, light yellow solid), 26.2% yield.

MS(ESI):m/z 644.2[M+H]⁺。

¹H NMR(400MHz,DMSO)δ12.87-12.69(m,1H),8.44(d,J＝4.4Hz,1H),8.03-7.93(m,1H),7.89-7.76(m,2H),7.69-7.62(m,2H),7.26(dd,J＝7.6,5.2Hz,1H),6.30-6.03(m,3H),4.24(t,J＝17.6Hz,2H),4.12(s,2H),3.82-3.68(m,2H),2.96(d,J＝16.8Hz,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＝14.0Hz,1H),1.41(d,J＝12.4Hz,1H)。

Example 8: preparation of Compound 8

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 8B

Compound Q1(1.0g,4.9mmol) was added to DMF (10ml) followed by Compound 8A (2.45g,9.8mmol) and BOP (4.3g,9.8mmol), after which DBU (2.2g,14.7mmol) was added slowly at room temperature followed by room temperatureReacting overnight, and after the reaction is finished, Boc₂O (1.6g, 7.2mmol) was added to the solution. The reaction was stirred at 50 ℃ for 2 hours. TLC showed the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 1/2, V/V) to give compound 8B (1.15g, a pale yellow solid) in 45.2% yield.

MS(ESI):m/z 518.1[M+H]⁺。

The second step is that: synthesis of Compound 8C

Compound 8B (829mg,1.6mmol) was dissolved in NMP (10ml), and intermediate Q4(895mg,2.4mmol), Pd were added₂(dba)₃(145mg,0.16mmol), Xantphos (92mg,0.16mmol) and DIEA (619mg,4.8 mmol). After warming to 120 ℃ under nitrogen and stirring for 1h, after completion of the TLC reaction, the reaction mixture was added to 75ml of saturated sodium chloride solution, extracted with EtOAc (25 ml. times.3), the organic phases were combined, washed with saturated sodium chloride solution (100ml), dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and the residue was purified by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 8C (412mg, light yellow solid), 32.6% yield.

MS(ESI):m/z 789.2[M+H]⁺。

The third step: synthesis of Compound 8

Compound 8C (354mg,0.45mmol) was added to ethyl acetate (5ml) and 4M HCl/ethyl acetate solution (10ml) was added and reacted at room temperature for 1h, TLC showed the reaction was complete, filtered, the solid was washed with ethyl acetate and dried to give the hydrochloride salt of compound 8 (283mg, white solid) in 91.5% yield.

MS(ESI):m/z 689.2[M+H]⁺。

Example 9: preparation of Compound 9

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 9A

Compound 8C (1g,1.3mmol) was added to a mixed solvent of methanol (5.0ml) and water (10.0ml) followed by lithium hydroxide monohydrate (170mg,4.2mmol) and stirred at room temperature for 2h, after TLC indicated completion of the reaction, pH was adjusted to 3-4 with 1M hydrochloric acid and a large amount of solid precipitated, filtered and the filter cake was dried to give intermediate 9A (907mg, off-white solid) in 90% yield. The crude product was used in the next reaction without purification.

The second step: synthesis of Compound 9

Compound 9A (900mg,1.16mmol) was added to ethyl acetate (5ml) and 4M HCl/ethyl acetate solution (10ml) was added and reacted at room temperature for 1h, after TLC showed completion of the reaction, the reaction mixture was added to 75ml saturated sodium bicarbonate solution and then diluted with dichloromethane: methanol (5: 1, V: V) extraction (25 ml. times.3), combining the organic phases, washing with saturated sodium chloride solution (100ml), drying over anhydrous sodium sulfate, spin-drying and concentration, and separation and purification of the residue by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 9(123mg, white solid), 15.7% yield.

MS(ESI):m/z 675.1[M+H]⁺。

¹H NMR(400MHz,DMSO)δ12.30(s,1H),8.39-8.32(m,2H),8.26-8.19(m,1H),7.56(d,J＝7.2Hz,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＝9.2Hz,1H),3.95-3.73(m,5H),3.50(s,1H),3.12(d,J＝16.8Hz,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)。

Example 10: preparation of Compound 10

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 10A

Compound 9A (900mg,1.16mmol) was added to a DMF (10ml) solution followed by HATU (661mg,1.74mmol) and DIEA (300mg,2.32mmol), stirred at room temperature for half an hour, then ammonium chloride (320mg,5.8mmol) was added, then reacted at room temperature overnight, TLC showed the reaction was complete, added to 75ml saturated sodium bicarbonate solution then diluted with dichloromethane: methanol (5: 1, V: V) extraction (25 ml. times.3), combining the organic phases, washing with saturated sodium chloride solution (100ml), drying over anhydrous sodium sulfate, spin-drying and concentration, and separation and purification of the residue by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 10A (303mg, white solid), 33.8% yield.

MS(ESI):m/z 774.1[M+H]⁺。

The second step is that: synthesis of Compound 10

Compound 10A (300mg,0.38mmol) was added to ethyl acetate (5ml) and 4M HCl/ethyl acetate solution (10ml) was added and reacted at room temperature for 1h, TLC showed the reaction was complete, filtered, the solid was washed with ethyl acetate and dried to give the hydrochloride salt of compound 10 (232mg, white solid) in 90.5% yield.

MS(ESI):m/z 674.2[M+H]⁺。

¹H NMR(400MHz,MeOD)δ8.74-8.62(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＝11.6Hz,1H),1.69(d,J＝11.65Hz,1H),1.64-1.54(m,1H)。

Example 11: preparation of Compound 11

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 11B

Compound Q1(1.0g,4.9mmol) was added to DMF (10ml) followed by Compound 11A (2.74g,9.8mmol) and BOP (4.3g,9.8mmol), after which DBU (2.2g,14.7mmol) was added slowly at room temperature and reacted overnight at room temperature after which Boc was added₂O (1.6g, 7.2mmol) was added to the solution. The reaction was stirred at 50 ℃ for 2 hours. TLC showed the reaction was complete. The mixture was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate 1/2, V/V) to give compound 11B (1.2g, a pale yellow solid) in 45.2% yield.

MS(ESI):m/z 546.2[M+H]⁺。

The second step is that: synthesis of Compound 11C

Compound 11B (872mg,1.6mmol) was dissolved in NMP (10ml), and intermediate Q4(895mg,2.4mmol), Pd were added₂(dba)₃(145mg,0.16mmol), Xantphos (92mg,0.16mmol) and DIEA (619mg,4.8 mmol). After warming to 120 ℃ under nitrogen and stirring for 1h, after completion of the TLC reaction, the reaction mixture was added to 75ml of saturated sodium chloride solution, extracted with EtOAc (25 ml. times.3), the organic phases were combined, washed with saturated sodium chloride solution (100ml), dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and the residue was purified by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 11C (398mg, light yellow solid), 30.5% yield.

MS(ESI):m/z 817.2[M+H]⁺。

The third step: synthesis of Compound 11

Compound 11C (367mg,0.45mmol) was added to ethyl acetate (5ml) and 4M HCl/ethyl acetate solution (10ml) was added and reacted at room temperature for 1h, TLC showed the reaction was complete, filtered, the solid was washed with ethyl acetate and dried to give the hydrochloride salt of compound 11 (295mg, white solid) in 91.5% yield.

MS(ESI):m/z 717.2[M+H]⁺。

¹H NMR(400MHz,DMSO)δ12.26(s,1H),8.76(s,2H),8.60(d,J＝4.4Hz,1H),8.21(dd,J＝24.4,7.6Hz,2H),7.47(dd,J＝7.2,5.6Hz,1H),7.43-7.34(m,1H),7.30(t,J＝8.0Hz,1H),6.69(d,J＝8.0Hz,1H),4.56-4.46(m,1H),4.30(q,J＝7.2Hz,2H),3.97(d,J＝13.2Hz,1H),3.89-3.84(m,2H),3.80(s,1H),3.36-3.23(m,3H),3.15(d,J＝17.2Hz,1H),2.89(t,J＝6.4Hz,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＝13.2Hz,1H),1.26(t,J＝7.2Hz,3H)。

Example 12: preparation of Compound 12

The specific synthetic route is as follows:

the first step is as follows: synthesis of Compound 12A

The second step is that: synthesis of Compound 12B

Compound 9A (900mg,1.16mmol) was added to a DMF (10ml) solution followed by HATU (661mg,1.74mmol) and DIEA (300mg,2.32mmol), stirred at room temperature for half an hour, added with ammonium chloride (320mg,5.8mmol) and reacted at room temperature overnight, after TLC showed the reaction was complete, the reaction was added to 75ml of saturated sodium bicarbonate solution and then quenched with di-HClMethyl chloride: methanol (5: 1, V: V) extraction (25 ml. times.3), combining the organic phases, washing with saturated sodium chloride solution (100ml), drying over anhydrous sodium sulfate, spin-drying and concentration, and separation and purification of the residue by preparative high performance liquid chromatography (Welch Xtrimate C)₁₈150 x 30mm x 5 μ M; conditions are as follows: 36-67% of B (A: water (0.05% ammonia), B: acetonitrile); flow rate: 25ml/min) to give compound 10A (303mg, white solid), 33.8% yield.

MS(ESI):m/z 774.1[M+H]⁺。

The third step: synthesis of Compound 12

MS(ESI):m/z 717.2[M+H]⁺。

¹H NMR(400MHz,DMSO)δ12.25(s,1H),8.63-8.50(m,3H),8.21(d,J＝8.4Hz,1H),8.05(d,J＝7.6Hz,1H),7.84(s,1H),7.56(s,1H),7.40(dd,J＝7.6,5.2Hz,1H),7.29(t,J＝8.0Hz,1H),6.66(d,J＝8.0Hz,1H),4.52-4.48(m,1H),4.07(d,J＝13.2Hz,1H),3.96(d,J＝13.2Hz,1H),3.86(t,J＝6.0Hz,2H),3.74-3.64(m,2H),3.28-3.25(m,1H),3.11(d,J＝17.2Hz,1H),2.88(t,J＝6.4Hz,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＝11.6Hz,1H),1.53(d,J＝13.2Hz,1H).

Experimental example 1: antiproliferative activity assay of Compounds of the invention against NCI-H358 cells

Experimental materials:

DMEM medium, penicillin/streptomycin antibiotics were purchased from weissent. Fetal bovine serum was purchased from Biosera. 3DCellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega. The H358 cell line was purchased from Biotech, Inc., Bai, Kyoto. Envision multi-label analyzer (PerkinElmer).

The experimental method comprises the following steps:

h358 cells were seeded in ultra-low adsorption 96-well U-plates, containing 3000H 358 cells per well, with 80 μ L of cell suspension. The cell plates were placed in a carbon dioxide incubator overnight.

The test compounds were diluted 3-fold with a rifling to the 8 th concentration, i.e., from 0.2mM to 91.44nM, setting up a double well experiment. Add 78. mu.L of medium to the intermediate plate, transfer 2. mu.L of each well of the gradient dilution compound to the intermediate plate according to the corresponding position, mix well and transfer 20. mu.L of each well to the cell plate. The concentration of compound transferred to the cell plate ranged from 1. mu.M to 0.457 nM. The cell plates were placed in a carbon dioxide incubator for 5 days.

The chemiluminescent detection reagent was added to the cell plate at a cell viability rate of 100. mu.L and incubated for 10 minutes at room temperature to stabilize the luminescent signal. Reading with a multi-label analyzer.

And (3) data analysis:

the original data was converted to inhibition rate, IC, using the equation (Sample-Min)/(Max-Min) × 100%₅₀The values of (A) can be obtained by curve fitting of four parameters (obtained in the GraphPad Prism "log (inhibitor)" vs. response- -Variable slope "mode). Table 1 provides the inhibitory activity of the compounds of the present invention on H358 cell proliferation.

TABLE 1 data on the anti-cell proliferation Activity of the Compounds of the Invention (IC)₅₀)

Compound numbering	NCI-H358 anti-cell proliferation Activity, IC₅₀(unit: 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

As can be seen from the experimental results in Table 1, the compounds of the present invention have excellent activity in inhibiting the proliferation of the lung cancer cell line NCI-H358. The activity of part of the compounds exceeds that of TNO-155 by 40 times. Shows extremely important antitumor potential.

Experimental example 2: antiproliferative activity of Compounds of the invention against MV-4-11 cells

Experimental materials:

IMDM medium, fetal bovine serum, penicillin/streptomycin antibiotics were purchased from Promega (Madison, Wis.). The MV-4-11 cell line was purchased from the cell bank of Chinese academy of sciences. Envision multi-label analyzer (PerkinElmer).

The experimental method comprises the following steps:

MV-4-11 cells were seeded in white 96-well plates in 80. mu.L cell suspension per well, containing 6000 MV-4-11 cells. The cell plate was placed in a carbon dioxide incubator overnight.

The test compounds were diluted 3-fold with a calandria to the 9 th concentration, i.e. from 2mM to 304nM, setting up a duplicate well experiment. Add 78. mu.L of medium to the intermediate plate, transfer 2. mu.L of each well of the gradient dilution compound to the intermediate plate according to the corresponding position, mix well and transfer 20. mu.L of each well to the cell plate. The concentration of compound transferred to the cell plate ranged from 10. mu.M to 1.52 nM. The cell plates were incubated in a carbon dioxide incubator for 3 days.

25uL of Promega CellTiter-Glo reagent per well was added to the cell plate and incubated at room temperature for 10 minutes to stabilize the luminescence signal. Readings were taken using a PerkinElmer Envision multi-label analyzer.

And (3) data analysis:

the original data was converted to inhibition rate, IC, using the equation (Sample-Min)/(Max-Min) × 100%₅₀The values of (A) can be obtained by curve fitting of four parameters (obtained in the GraphPad Prism "log (inhibitor)" vs. response- -Variable slope "mode). Table 2 provides the inhibitory activity of the compounds of the present invention on MV-4-11 cell proliferation.

TABLE 2 anti-cell proliferation Activity data (IC) for the Compounds of the invention₅₀)

As can be seen from the experimental results in Table 2, the compounds of the present invention have excellent activity in inhibiting MV-4-11 cell proliferation. The activity of compound 1 was 10 times greater than that of TNO-155. Shows extremely important antitumor potential.

Experimental example 3: inhibition of p-ERK Activity

1. Experimental Material

H358 cells were purchased from tokyo bai biotechnology;

1640 media from Biological industries;

fetal bovine serum was purchased from Biosera;

phosphorylation pERK (phosphorylation site 202/204) assay kit was purchased from Cisbio.

2. Experimental methods

H358 cells were seeded in clear 96-well cell culture plates, 80 μ L cell suspension/well, each well containing 10000H 358 cells. The cell plates were placed in a carbon dioxide incubator and incubated overnight at 37 ℃. The cell supernatant was discarded, 80. mu.L/well of starvation medium (1640+ 0.02% fetal bovine serum + 1% double antibody) was added, and the cell plate was placed in a carbon dioxide incubator and subjected to cell starvation overnight.

Test compounds were diluted to 4mM with 100% DMSO as the first concentration and then 5-fold diluted to the 8 th concentration with a pipette, i.e., from 4mM to 10.24 μ M. Adding 1 mu L of compound into 79 mu L of cell starvation culture medium, uniformly mixing, transferring 20 mu L/hole compound solution to a hole of a corresponding cell plate, and putting the cell plate back to a carbon dioxide incubator to continue incubation for 1h, wherein the concentration of the compound is 10 mu M-0.0256 nM, and the concentration of DMSO is 0.25%;

after the incubation is finished, discarding cell supernatant, adding 50 mu L of cell lysate/hole, and incubating for 30min at room temperature by shaking; diluting the europium cryptate-labeled phosphorylated extracellular regulatory protein kinase antibody and the d 2-labeled phosphorylated extracellular regulatory protein kinase antibody by 20-fold using a detection buffer; adding 16 mu L of cell lysate supernatant into a new 384 white microplate, adding 2 mu L of europium cryptate labeled phosphorylated extracellular regulated protein kinase antibody diluent and 2 mu L d2 labeled phosphorylated extracellular regulated protein kinase antibody diluent, and incubating at normal temperature for 4 h; after the incubation was complete, the homogeneous time-resolved fluorescence HTRF (excitation wavelength 320nm, emission wavelengths 615nm and 665nm) was read using a multi-label analyzer.

3. Data analysis

The raw data was converted to inhibition, IC, using the equation (sample-Min)/(Max-Min) to 100%₅₀Values can be derived by curve fitting with four parameters (e.g., by the "log (inhibitor) vs. pressure- -Variable slope" model in GraphPad Prism). Wherein, Max hole: positive control wells read 1X cell lysate; min hole: negative control wells read 0.25% DMSO well cell lysate.

TABLE 3 inhibition of p-ERK Activity data (IC) for the compounds of the invention₅₀)

Compound numbering	p-ERK inhibitory Activity, IC₅₀(unit: nM)
		TNO-155	85.76
Compound 1	9.53

The experimental results in table 3 show that the compound of the present invention has excellent inhibitory effect on Erk phosphorylation downstream of SHP2 in NCI-H358 cells, and further, the compound of the present invention has a good antitumor prospect.

Experimental example 4: SHP2 enzymological experiment

1. Experimental materials and instruments

Homogeneous full-length SHP2 enzymatic assay kit was purchased from BPS Bioscience;

multiple label analyzers were purchased from Perkin Elmer.

2. The experimental method comprises the following steps:

diluting the 5X detection buffer solution into 1X detection buffer solution by using deionized water, using the detection buffer solution as a preparation, and placing the detection buffer solution on ice for later use after the preparation.

Test compounds were diluted to 100 μ M with 100% DMSO as the first concentration and then diluted 4-fold with a pipette to the 8 th concentration, i.e., from 100 μ M to 6.1 nM. The test compound was diluted with 1 Xbuffer to 10% DMSO in each gradient, 5. mu.L/well was added to the corresponding well, and a double-well assay was set up. Centrifuge at 1000rpm for 1 min.

Adding 18 mu L of prepared reaction mixed solution into each hole, wherein the reaction mixed solution contains 12.25 mu L of deionized water; 5 μ L of 5 Xdetection buffer; 0.25 μ L protein tyrosine phosphatase activating polypeptide (100 μ M); 0.5. mu.L DTT (250 mM). Centrifuge at 1000rpm for 1 min.

SHP2 enzyme was diluted to 0.1 ng/. mu.L with 1 Xdetection buffer, 2. mu.L/well was added to the corresponding well, 2. mu.L of 1 Xdetection buffer, SHP2(0.2ng), was added to the negative control well, this step was done on ice, the reaction was incubated at 25 ℃ for 60min and pre-incubated with compound.

After the compound is preincubated, 25 mu L of substrate working solution containing 19.45 mu L of deionized water is added into each hole; 5 μ L of 5 Xdetection buffer; mu.L of DTT (250mM) and 0.05. mu.L of SHP2 substrate (DiFMUP) (10mM) were allowed to stand at 25 ℃ for 30 min. The final concentration gradient of the compound was 1. mu.M to 0.061nM at this time. After the reaction is finished, a fluorescence value (excitation wavelength is 360nm, and emission wavelength is 460nm) is read by a multi-label analyzer.

The detection method of the background reading value of the compound is as follows: taking 5 mu L of each gradient of a compound to be detected diluted by 100% DMSO, adding 45 mu L of 1X detection buffer solution to dilute by 10 times to prepare 10% DMSO working solution, taking 5 mu L/hole of the compound working solution to a detection plate, then adding 45 mu L of 1X detection buffer solution to dilute by 10 times, wherein the final concentration of DMSO is 1%, centrifuging at 1000rpm for 1 minute, and reading a fluorescence value (the excitation wavelength is 360nm and the emission wavelength is 460nm) by using a multi-label analyzer.

3. Data analysis

Using the data obtained by subtracting the compound background reading from the enzyme reaction original reading as the initial value of the inhibition rate calculation, and converting the initial value into the inhibition rate, IC, by using the equation (sample-Min)/(Max-Min). times.100%₅₀Values can be derived by curve fitting with four parameters (e.g., by the "log (inhibitor) vs. pressure- -Variable slope" model in GraphPad Prism). Wherein, Max hole: positive control well initial values; min hole: negative control wells were initialized.

TABLE 4 SHP2 enzyme Activity inhibition data (IC) for the compounds of the invention₅₀)

Compound number	SHP2 enzyme inhibitory Activity IC₅₀(unit: nM)
		TNO-155	2.6
Compound 1	0.98

As can be seen from the experimental results in Table 4, the compounds of the present invention showed very excellent SHP2 enzyme inhibitory activity, IC, on an enzymatic level₅₀Reaching below 1nM, and having great development and application prospects.

Experimental example 5: hERG potassium channel inhibitor activity assay

1 cell preparation

CHO-hERG cells cultured at 175cm²Culturing in culture flask until cell density reaches 60-80%, removingThe culture was washed once with 7mL PBS and then digested by the addition of 3mL Detachin.

Adding 7mL of culture solution for neutralization after complete digestion, centrifuging, sucking off supernatant, adding 5mL of culture solution for resuspension to ensure cell density of 2-5 × 10⁶/mL。

2 solution formulation see table 5:

TABLE 5 composition of intracellular and extracellular fluids

3 electrophysiological recording procedure

The single cell high negative impedance sealing and whole cell mode forming process is completed automatically by Qpatch instrument, after obtaining whole cell record mode, the cell clamp is prepared at-80 mV, before giving a depolarization stimulation of +40 mV for 5 s, a 50 mSec pre-voltage is given, then repolarization is carried out to-50 mV to maintain for 5 s, and then the voltage returns to-80 mV. This voltage stimulus was applied every 15 seconds, and 2 minutes after recording extracellular fluid was administered for 5 minutes, and then the dosing process was started, with compound concentrations starting from the lowest test concentration, each test concentration being administered for 2.5 minutes, and after all concentrations were administered in succession, 0.1 μ M Cisapride was administered as a positive control compound. At least 3 cells (n.gtoreq.3) were tested per concentration.

4 preparation of Compounds

The 20mM compound stock solution was diluted with the extracellular solution, and 5. mu.L of the 20mM compound stock solution was added to 2495. mu.L of the extracellular solution, diluted 500-fold to 40. mu.M, and then serially diluted 3-fold in the extracellular solution containing 0.2% DMSO in order to obtain the final concentration to be tested.

The highest concentration tested was 40. mu.M, which was in turn 40, 13.33, 4.44, 1.48, 0.49, 0.16. mu.M for 6 concentrations.

The final concentration of DMSO in the test concentration did not exceed 0.2%, and the concentration of DMSO had no effect on the hERG potassium channel.

5 data analysis

Experimental data were analyzed by XLFit software.

6 quality control

Environment: humidity is 20-50%, and temperature is 22-25 DEG C

Reagent: the experimental reagent is purchased from Sigma, and has purity of 98%

The experimental data reported must meet the following criteria:

whole cell sealing impedance >100M omega

Tail current amplitude >300pA

Pharmacological parameters:

the inhibitory effect of multiple concentrations of Cisapride on the hERG channel was set as a positive control.

7 results of the experiment

TABLE 6 inhibition of hERG Current by Compounds of the invention at multiple concentrations

Compound numbering	hERG(μM)
		Compound 1	>40
Compound 2	>40
		Cisapride	0.021

8 conclusion of the experiments, the inhibition of cardiac hERG potassium channel by drugs is the main cause of QT prolongation syndrome caused by drugs. The experimental results show that the compound provided by the embodiment of the invention has no obvious inhibition effect on the cardiac hERG potassium ion channel, and the risk of toxic and side effects on the heart is low.

Experimental example 6: pharmacokinetic experiments

1. Experimental Material

Using the compounds prepared in the above examples, the oral drug was formulated as a 0.3mg/mL clear solution (2% DMSO + 30% PEG300+ 2% Tween80+ 66% H)₂O), intravenous drug was formulated as a 0.2mg/mL clear solution (2% DMSO + 30% PEG300+ 2% Tween80+ 66% H)₂O)。

2. Laboratory animal

Male CD-1 mice or rats, 3 each per group, weighing 27-28g, were provided by Shanghai Si Laike laboratory animal liability, Inc. The test mice are given an environmental adaptation period of 2-4 days before the experiment, fasted for 8-12h before the administration, fed with water after 2h and fed with food after 4 h.

3. Experimental methods

1) After a mouse or a rat fasts but can freely drink water for 12 hours, blank plasma at 0 moment is adopted;

2) taking the mice in the step 1), and orally taking (PO) the compound to be detected for 3 mg/kg; intravenous (IV) administration of 1mg/kg of test compound;

3) continuously taking blood from fundus venous plexus 5min, 15min, 30min, 1h, 2h, 4h, 8h, 10h and 24h after oral administration, placing in an EP tube distributed with heparin, centrifuging at 8000rpm for 5min, taking upper layer plasma, freezing at-20 deg.C, and analyzing by LC-MS/MS;

4) calculating pharmacokinetic parameters by adopting WinNonlin software according to the blood concentration-time data obtained in the step 3), wherein the specific data are shown in a table 7.

TABLE 7 pharmacokinetic data for the compounds of the invention

As shown in Table 7, the compounds of the present invention, administered orally or intravenously to mice or rats, exhibited higher exposure in the plasma of animals and low clearance, and could be administered orally.

Experimental example 7: in vivo pharmacodynamic experiment 1

1. Purpose of experiment

The in vivo efficacy of the test compounds on a model of human non-small cell lung carcinoma NCI-H358 subcutaneous xenograft tumor was evaluated.

2. Laboratory animal

BALB/nude mouse, female, 6-8 weeks old, weight 18-20 g, total 18, provided by Shanghai Ling Chang laboratory animals Co., Ltd.

3. Experimental methods

NCI-H358 tumor cells were resuspended in PBS to a density of 5X 10⁷one/mL cell suspension was inoculated subcutaneously into the right dorsal aspect of each mouse (0.1mL, 5X 10)⁶/only), wait for tumor growth. The average volume of the tumor reaches about 151mm³At that time, random group administration is started. After dosing, tumor diameters were measured twice weekly using a vernier caliper and tumor volumes were calculated as follows:

V＝0.5a×b²wherein a and b represent the major and minor diameters of the tumor, respectively.

Tumor inhibitory therapeutic utility of the compounds TGI (%) was evaluated, which reflects the tumor growth inhibition rate, and was calculated as follows:

TGI (%) - [ (1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group)/(average tumor volume at the end of treatment of the solvent control group-average tumor volume at the start of treatment of the solvent control group) ] × 100%.

4. Results of the experiment

The correlation results are shown in table 8.

TABLE 8 in vivo efficacy test results

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

5. Conclusion of the experiment

After 35 days of administration, under the condition of the same dose (30mg/kg), the compound has more obvious tumor inhibition effect compared with a positive control TNO-155, the TGI (%) reaches 94.57%, and is obviously superior to 67.72% of the positive control TNO155, which indicates that the compound in the invention shows good in-vivo efficacy on a non-small cell lung cancer NCI-H358 subcutaneous allograft tumor model. As an SHP2 inhibitor, the compound has good anti-tumor application prospect in clinic.

Experimental example 8: in vivo pharmacodynamics experiment 2

1. Purpose of experiment

The in vivo efficacy of test compounds on a human pancreatic cancer MIA-PaCa2 cell subcutaneous xenograft tumor model was evaluated.

2. Laboratory animal

BALB/nude mouse, female, 6-8 weeks old, weight 18-22 g, total 24 mice, supplied by Hua Ke Tech Co., Ltd, Wei Tong Li, Beijing.

3. Experimental methods

MIA-PaCa2 tumor cells were resuspended in PBS to a density of 1X 10⁷one/mL cell suspension, 0.2mL cell suspension was subcutaneously inoculated into the right back of each mouse (matrigel added, volume ratio 1:1) and the tumor growth was awaited. In case of tumorAverage volume reaches about 142mm³At that time, random group administration is started. After dosing, tumor diameters were measured twice weekly using a vernier caliper and tumor volumes were calculated as follows:

Tumor inhibitory therapeutic effect of the compounds TGI (%) was evaluated, which reflects the tumor growth inhibition rate, and was calculated as follows:

4. Results of the experiment

The correlation results are shown in table 9.

TABLE 9 in vivo efficacy test results

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

5. Conclusion of the experiment

22 days after the administration, compared with a positive control TNO-155, the compound has more obvious tumor inhibition effect and obvious dose-effect relationship under the condition of the same dose (30mg/kg), which indicates that the compound in the invention shows good in-vivo efficacy on a human pancreatic cancer MIA-PaCa2 subcutaneous allograft tumor model.

Experimental example 9: pharmacodynamic experiment III

1. Purpose of experiment

The in vivo efficacy of the test compounds on a murine colon cancer MC38 subcutaneous xenograft tumor model was evaluated.

2. Laboratory animal

C57BL/6 mice, female, 6-8 weeks old, weight 18-20 g. A total of 32 animals were provided by Shanghai Ling laboratory animals Co., Ltd.

3. Experimental methods

The MC38 tumor cells were resuspended in PBS to a density of 0.3X 10⁶one/mL cell suspension, 0.1mL cell suspension was subcutaneously inoculated into the right back of each mouse (matrigel added, volume ratio 1:1) and the tumor growth was awaited. The average volume of the tumor reaches about 65mm³At that time, random group administration was started. After dosing, tumor diameters were measured twice weekly using a vernier caliper and tumor volumes were calculated as follows:

4. Results of the experiment

The correlation results are shown in table 10.

TABLE 10 in vivo efficacy test results

5. Conclusion of the experiment

After the administration is started for 18 days, the compound and the PD-1 antibody are combined, compared with a single medicine, the compound can obviously increase the tumor inhibition effect, and has good in-vivo efficacy on a murine colon cancer MC38 subcutaneous xenograft tumor model. The compound of the invention is combined with PD1 monoclonal antibody to show synergistic antitumor effect.

Experimental example 10: pharmacodynamics experiment in vivo

1. Purpose of experiment

The in vivo efficacy of the test compounds was evaluated in a human esophageal carcinoma KYSE-520 subcutaneous allograft tumor model.

2. Laboratory animal

BALB/nude mouse, female, 6-8 weeks old, weight 18-24 g, total 40, supplied by Hua Kongyun, Vantonli, Beijing.

3. Experimental method

KYSE-520 tumor cells were resuspended in PBS to a density of 10X 10⁶one/mL of cell suspension, 0.2mL of cell suspension was subcutaneously inoculated into the right dorsal aspect of each mouse (matrigel added, volume ratio 1:1) and the tumor growth was awaited. The average volume of the tumor reaches about 141mm³At that time, random group administration is started. After dosing, tumor diameters were measured twice weekly using a vernier caliper and tumor volumes were calculated as follows:

4. Results of the experiment

The correlation results are shown in table 11.

TABLE 11 in vivo efficacy test results

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

5. Conclusion of the experiment

After 21 days of administration, under the condition that the dose of the compound 1 is one tenth of the dose of a positive control (3.0mg/kg), the compound 1 has a remarkably better tumor inhibition effect compared with a positive control TNO-155(30mg/kg), and the TGI (%) reaches 91.9% which is obviously better than 82.3% of the positive control TNO 155. In addition, the compound 1 of the invention still shows obvious antitumor effect at lower dose (0.3mg/kg), which indicates that the compound of the invention shows good in-vivo efficacy in a human esophageal cancer KYSE-520 subcutaneous allograft model, and the antitumor effect has a dose-dependent trend.

While embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be taken as limiting the invention. Variations, modifications, substitutions and changes to the above-described embodiments can be made by those skilled in the art within the scope of the present invention without departing from the principle and spirit of the invention, and these variations, modifications, substitutions and changes are intended to be included within the scope of the present invention.

Claims

1. A compound of formula I or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite, or prodrug thereof, wherein

X¹、X²And X³Each independently selected from CR₅And N, or absent;

when X is present¹、X²And X³Each independently selected from CR₅And when N is, X⁵And X⁶Each independently selected from C and N, X⁷Is CR₅Or N;

X⁴is C or N;

X⁸is N or NR₅；

R₁、R₂、R₃、R₄、R₆And R₇Each independently selected from hydrogen, halogen, hydroxy, amino, oxo, cyano, C₂-C₈Alkenyl radical, C₂-C₈Alkynyl, aldehyde, carbamoyl, C₁-C₈Alkyl radical, C₁-C₈Heteroalkyl group, C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl radical, C₁-C₈Alkoxy and C₁-C₃Haloalkoxy, wherein said C₁-C₈Alkyl radical, C₁-C₈Heteroalkyl group, C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl, C₁-C₈Alkoxy and C₁-C₃Haloalkoxy is each optionally substituted with one or more R₅Substitution;

if present, each R₅Each independently selected from hydrogen, halogen, hydroxy, amino, cyano, carbamoyl, C₁-C₃Alkyl radical, C₁-C₃Heteroalkyl group, C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl radical, C₁-C₃Alkoxy and C₁-C₃Haloalkoxy, wherein said C₁-C₃Alkyl radical, C₁-C₃Heteroalkyl group, C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl radical, C₁-C₃Alkoxy and C₁-C₃Haloalkoxy is each optionally substituted with one or more R₈Substitution;

a andeach B is independently selected from C₃-C₈Cycloalkyl radical, C₃-C₈Heterocycloalkyl radical, C₆-C₁₀Aryl and C₅-C₁₂A heteroaryl group;

n is 1,2 or 3.

2. The compound of claim 1, wherein the compound is of formula I-1 or formula I-2, wherein

X⁵、X⁶And X⁷Each independently selected from CR₅And N;

X⁸、R₁、R₂、R₃、R₄、R₅、R₆b and n are as defined in claim 1.

3. The compound of claim 2,

X⁵、X⁶and X⁷Each independently selected from CR₅And N;

X⁸is N or NR₅；

R₂is hydrogen;

R₃is hydrogen or hydroxy;

each R₄Are independent of each otherIs selected from hydrogen, halogen and C₁-C₈Alkyl radical, wherein said C₁-C₈Alkyl is optionally substituted by one or more R₅Substitution;

R₆is hydrogen;

if present, each R₈Each independently selected from hydrogen and hydroxy;

n is 1,2 or 3.

4. A compound according to claim 2 or 3,

in the structure of

The fragment is selected from any one of the following fragments:

preferably, in the structure

The fragment is selected from any one of the following fragments:

or preferably, in the structure

The fragment is selected from any one of the following fragments:

5. the compound of claim 1, wherein the compound is of formula I-3 or formula I-4, wherein

6. The compound of claim 5,

X⁸is N or NR₅；

R₂is hydrogen;

R₃hydrogen or hydroxy;

R₆is hydrogen;

n is 1,2 or 3.

7. The compound of claim 5 or 6,

in the structure of

The fragment is selected from any one of the following fragments:

preferably, in the structure

The fragment is selected from any one of the following fragments:

8. the compound according to any one of claims 2 to 7,

in the structure of

The fragment is selected from any one of the following fragments:

preferably, in the structure

The fragment is selected from any one of the following fragments:

9. the following compounds or pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, metabolites or prodrugs thereof:

10. a process for the preparation of a compound according to claim 1, comprising the steps of:

1) reacting the compound A with the compound B to obtain a compound C;

2) reacting the compound C with the compound D to obtain a compound E; and

3) reacting the compound E with the compound F to obtain a target product;

wherein

X¹、X²、X³、X⁴、X⁵、X⁶、X⁷、X⁸、R₁、R₂、R₃、R₄、R₆、R₇a, B and n are as defined in claim 1.

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite, or prodrug thereof, and at least one pharmaceutically acceptable adjuvant.

12. Use of a compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof, or a pharmaceutical composition according to claim 11, for the manufacture of a medicament for the prevention and/or treatment of a disease or condition associated with abnormal activity of SHP 2;

preferably, the disease or disorder associated with aberrant activity of SHP2 is selected from noonan syndrome, leopard syndrome, leukemia, neuroblastoma, melanoma, breast cancer, esophageal cancer, lung cancer, colon cancer, head and neck tumors, gastric cancer, anaplastic large cell lymphoma, and glioblastoma, preferably non-small cell lung cancer, esophageal cancer, and head and neck tumors.