WO2020156242A1

WO2020156242A1 - Shp2 inhibitor and application thereof

Info

Publication number: WO2020156242A1
Application number: PCT/CN2020/072773
Authority: WO
Inventors: 吴颢; 吴文茂; 陈忠研; 李玲; 朱林强; 张展; 吴云飞; 林远望; 冯东杰; 赵新涛; 余军; 束庆玉; 程见洪; 韩晗; 郭晶; 兰宏; 王家炳; 丁列明
Original assignee: 贝达药业股份有限公司
Priority date: 2019-01-31
Filing date: 2020-01-17
Publication date: 2020-08-06
Also published as: CN113316574A; CN113316574B; TW202214589A

Abstract

A compound used as a Src homologyregion 2-containing protein tyrosine phosphatase 2 (SHP2) inhibitor (as represented by formula I), and a pharmaceutical composition thereof and a preparation method therefor, as well as use of same in the treatment of SHP2-mediated diseases. The compound acts by participating in a number of processes, such as cell proliferation regulation, apoptosis, migration, and angiogenesis.

Description

SHP2 inhibitor and its application

Technical field

The present invention relates to a series of compounds as inhibitors of Src homology region 2 (Src homology region 2-containing protein tyrosine phosphatase 2, SHP2), and preparation methods and pharmaceutical compositions thereof. The present invention also relates to the use of the above-mentioned compound or its pharmaceutical composition in the treatment of SHP2-mediated diseases.

Background technique

Src homology region 2 protein tyrosine phosphatase 2 (Src homology region 2-containing protein tyrosine phosphatase 2, SHP2) is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 gene, and PTPN11 is the first The discovered proto-oncogene encoding tyrosine kinase (Chan R J et al.PTPN11 is the first identified proto-oncogene that encodes a tyrosine phosphatase.Blood, 2007,109:862-867), and the encoded SHP2 protein contains N-terminal SHP2 domain (N-SHP2), C-terminal SHP2 domain (C-SHP2), protein phosphatase catalytic domain (PTP), two C-terminal tyrosine residues (Y542 and Y580) and one rich Proline (Pro) motif.

In recent years, research mainly believes that the Ras/ERK pathway is the most important signal transduction pathway for SHP2, and its mechanism (Dance M et al. The molecular functions of Shp2 in the RAS/mitogen-activated protein kinase (ERK1/2) pathway. Cell Signal, 2008, 20:453-459) roughly: After the growth factor receptor is activated, its tyrosine residues undergo autophosphorylation, which is the combination of Grb2 and SHP2 (adapter protein containing SH2 domain) phosphotyrosine Area SH2 provides stop points. The binding of Grb2 to phosphorylated growth factor receptors results in the accumulation of SOS protein in the cell membrane. SOS, as a guanine nucleotide exchange factor (GEF), can catalyze the conversion of membrane-bound protein Ras from inactive Ras-GDP to active Ras-GTP. Ras-GTP further connects with downstream signaling systems to activate Ser/Thr kinase Raf1, etc., and then activate ERK under the action of the regulatory kinase MEK. After activation of ERK, it directly acts on target molecules in the cytoplasm or transfers to the nucleus to regulate genes Transcription to make cells proliferate or differentiate. This process may also be affected by SHP2 binding protein and substrate (SHP substrate-1, SHPS-1), Ras-GTPase activating protein (Ras-GAP) and other members of Src.

SHP2 protein not only regulates the Ras/ERK signaling pathway, it is also reported to regulate multiple signaling pathways such as JAK-STAT3, NF-κB, PI3K/Akt, RHO, and NFAT, thereby regulating cell proliferation, differentiation, migration, and apoptosis Features.

SHP2 has been proved to be related to many diseases, Tartaglia et al. (Tartaglia M et al. Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome. Nat Genet, 2001, 29:465-468) found that about 50% Patients with Noonan syndrome have missense mutations of PTPN11. In addition, studies have found that PTPN11 mutation is an important cause of JMML and a variety of leukemias (Tartaglia M et al. Nat Genet, 2003, 34: 148-150; Loh ML et al. Blood, 2004, 103: 2325-2331; Tartaglia M et al. Br J Haematol, 2005, 129: 333-339; Xu R et al. Blood, 2005, 106: 3142-3149.). With the deepening of research on PTPN11/SHP2, it has been found that it is related to the occurrence of lung cancer, gastric cancer, colon cancer, melanoma, thyroid cancer and other cancers (Tang Chunlan et al. Chinese Journal of Lung Cancer, 2010, 13: 98- 101; Higuchi M et al. Cancer Sci, 2004, 95: 442-447; Bentiers-Al j M et al. Cancer Res, 2004, 64: 8816-8820; Martinelli S et al. Cancer Genet Cytogenet, 2006, 166: 124-129.).

Therefore, SHP2 inhibitors have received more and more attention as potential treatments. There are a variety of SHP2 inhibitors currently under development. Novartis's TNO155 entered phase I clinical trials for the treatment of solid tumors in 2017. JAB-3068 designed and developed by Jacos entered a phase I clinical trial for the treatment of solid tumors in April 2018. The RMC-4630 developed by Revolution conducted its first human clinical trial in August 2018. At present, there is no marketed product for this target at home and abroad. Therefore, the development of small molecule drugs that can target and inhibit the activity of SHP2 and provide patients with safer and more effective SHP2 inhibitors have important research significance.

Summary of the invention

The present invention relates to a compound used as an inhibitor of protein tyrosine phosphatase 2 (SHP2) containing Src homology region 2. The compound of the present invention has a general structure as shown in Formula I or a pharmaceutically acceptable salt, tautomer, solvate, chelate, non-covalent complex or prodrug thereof,

among them,

R ₁ is selected from hydrogen, hydroxy, C _1-8 alkyl, C _1-8 alkyl-containing substituent group, C _1-8 alkoxy, C _1-8 alkoxy group-containing substituent group, C _2-8 Alkenyl, substituted C _2-8 alkenyl, C _2-8 alkynyl or substituted C _2-8 alkynyl;

R ₂ is selected from hydrogen, C _1-4 alkyl group or a substituted group containing C _1-4 alkyl;

R ₃ is selected from hydrogen, _{amino, -C (O) NH 2,} -C≡N, hydroxy, C _1-8 alkyl, C _1-8 alkyl-containing substituent group, C _1-8 alkoxy or an C _1-8 alkoxy of the substituent;

R ₄ is selected from hydrogen, halo, amino, amido, -C≡N, carboxyl, hydroxyl, hydroxymethyl, C _1-8 alkyl, C _1-8 alkyl-containing substituent group, C _1-8 alkoxy group, a substituted group containing C _1-8 alkoxy, C _2-8 alkenyl group, a substituted group containing C _2-8 alkenyl, C _2-8 alkynyl group or a substituted group containing C _2-8 alkynyl;

A _{1 is} arbitrarily selected from CR ₅ or N;

A _{2 is} arbitrarily selected from CR ₆ or N;

A _{3 is} arbitrarily selected from CR ₇ or N;

U is arbitrarily selected from C(R ₈ ) ₂ , O or NR ₉ ;

Wherein, R ₅ , R ₆ , R ₇ , R ₈ or R _{9 are} independently selected from hydrogen, halogen, amino, C _1-8 alkyl, substituted C _1-8 alkyl, C _1-8 alkoxy group, a substituted group containing C _1-8 alkoxy, C _2-8 alkenyl group, a substituted group containing C _2-8 alkenyl, C _2-8 alkynyl group or a substituted group containing C _2-8 alkynyl;

Ring A is optionally selected from a C _6-10 aryl group or a C _5-10 heteroaryl group, the C _5-10 heteroaryl group contains one or two N or S heteroatoms;

Rx is optionally selected from hydrogen, halogen, amino, substituted amino, sulfonyl, C _1-8 alkyl, substituted C _1-8 alkyl, C _1-8 alkoxy, substituted C _1-8 alkoxy, C _3-8 cycloalkyl or substituted C _3-8 cycloalkyl;

n is 0, 1, 2, 3, or 4.

Preferably, A _{3 in} formula I is N. Unexpectedly, when A ₃ is N instead of CR ₇ , not only the SHP2 inhibitory activity can be maintained, but hERG can be significantly improved.

In some embodiments, R ₁ in Formula I is selected from C _1-3 alkyl.

In some embodiments, R ₁ in Formula I is methyl.

In some embodiments, R ₂ in Formula I is selected from hydrogen or C _1-3 alkyl.

In some embodiments, R ₂ in Formula I is hydrogen.

In some embodiments, R ₃ in Formula I is selected from hydrogen, amino, or C _1-3 alkyl.

In some embodiments, R ₃ in Formula I is hydrogen or amino.

In some embodiments, R ₄ of Formula I is selected from hydrogen, halo, amino, C _1-3 alkyl group or an unsubstituted C _1-3 alkyl.

In some embodiments, R ₄ in Formula I is hydrogen, fluorine or chlorine.

In some embodiments, R ₄ in Formula I is hydrogen or chlorine.

In some embodiments, A ₁ in formula I is selected from CR ₅ or N, wherein R _{5 is} selected from halogen or halogen-substituted C _1-3 alkyl.

In some embodiments, A ₁ in formula I is selected from CR ₅ or N, wherein R _{5 is} selected from F, Cl or trifluoromethyl.

In some embodiments, A ₁ in formula I is selected from CR ₅ or N, wherein R _{5 is} selected from Cl or trifluoromethyl.

In some embodiments, A ₂ in formula I is selected from CR ₆ or N, wherein R _{6 is} selected from amino or C _1-3 alkoxy.

In some embodiments, A ₂ in formula I is selected from CR ₆ or N, wherein R _{6 is} selected from amino or methoxy.

In some embodiments, A ₃ in Formula I is selected from CH or N.

In some embodiments, U in Formula I is selected from CH ₂ or O.

In some embodiments, ring A in formula I is selected from phenyl or C _5-6 heteroaryl, and the C _5-10 heteroaryl contains one or two N or S heteroatoms.

In some embodiments, ring A in formula I is selected from phenyl,

In some embodiments, the formula I

Arbitrarily selected from

In some embodiments, the formula I

Arbitrarily selected from

The present invention further provides some preferred technical solutions of the compound represented by formula I. For example, the compound of the present invention has the general structure shown in Formula II or a pharmaceutically acceptable salt, tautomer, solvate, chelate, non-covalent complex or prodrug thereof,

among them,

R _{1 is} selected from C _1-8 alkyl;

R _{2 is} selected from hydrogen;

R _{3 is} selected from H or amino;

R _{4 is} selected from hydrogen and halogen;

A _{1 is} selected from CR ₅ ;

A _{2 is} selected from CR ₆ ;

U is selected from C(R ₈ ) ₂ ;

_{_{Wherein, R 5, R 6, R}} 8 are independently selected from hydrogen, halo, C _1-8 alkyl, substituted group containing C _1-8 alkyl;

Ring A is optionally selected from C _6-10 aryl groups.

In some embodiments, the substituent-containing C _1-8 alkyl group may be a halogenated C _1-8 alkyl group, such as trifluoromethyl.

In some embodiments, ring A in formula II is optionally substituted with hydrogen or C _1-8 alkoxy.

In some embodiments, ring A in formula II is optionally substituted with hydrogen or methoxy.

In some embodiments, R ₁ in Formula II is methyl.

In some embodiments, R ₄ in Formula II is hydrogen.

In some embodiments, U in Formula II is selected from CH ₂ .

In some embodiments, R ₅ in Formula II is selected from Cl.

In some embodiments, R ₆ in Formula II is selected from NH ₂ .

In some embodiments, ring A in formula II is selected from phenyl.

The present invention further provides a compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

(1) (S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino-3-chloro (Pyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(2) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-methoxy-1,3-dihydrospiro(indene -2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(3) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-chloro-1,3-dihydrospiro(indene-2 ,4'-Piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(4)(S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((3-chloro-2-methyl (Oxypyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(5)(S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2,3-dichlorobenzene (B)thio)-3-methylpyrimidin-4(3H)-one;

(6)(S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-3-methyl-5-((2- (Trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one;

(7) (S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((6-amino-3-chloro (Pyridin-2-yl)thio)-3-methylpyrimidin-4(3H)-one;

(8) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-4-bromo-1,3-dihydrospiro(indene-2 ,4'-Piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(9)(S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-(trifluoromethyl)-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(10) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-4-methoxy-1,3-dihydrospiro(indene -2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(11) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(5-amino-5,7-dihydrospiro[cyclopenta[b ]Pyridine-6,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(12) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-5,6-dimethoxy-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(13)(S)-2-(4-Amino-2-chloro-4,6-dihydrospiro[cyclopenta[d]thiazole-5,4'-piperidine]-1'-yl) -5-((2-amino-3-chloropyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(14) (S)-2-(5-Amino-2-methoxy-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'-piperidine]-1'- Yl)-5-((2-amino-3-chloropyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(15) (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2,3 -Dichlorophenyl)thio)-3-methylpyrimidin-4(3H)-one;

(16) (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino -3-Chloropyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(17) (S)-6-amino-2-(1-amino-6-fluoro-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5- ((2-Aminopyrimidin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(18)(S)-6-amino-5-((2-amino-3-chloropyridine-4-yl)thio)-2-(1-amino-6-fluoro-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(19) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-5-fluoro-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(20) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-7-fluoro-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(24)(S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino-3-fluoro Pyridin-4-yl)thio)-3-methylpyrimidine-4(3H)-one;

(25) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-4,7-difluoro-1,3- Dihydrospiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(26) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-chloro-1,3-dihydrospiro [Indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one; or

(27) (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino Pyrimidine-4-yl)thio)-3-methylpyrimidin-4(3H)-one.

Preferably, the present invention further provides a compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

The present invention also provides a pharmaceutical composition characterized by comprising a therapeutically effective amount of at least one compound represented by formula I or formula II and at least one pharmaceutically acceptable excipient.

The present invention further provides a pharmaceutical composition, characterized in that the mass percentage of the compound represented by structural formula I and pharmaceutically acceptable excipients is 0.0001:1-10.

The invention provides the application of the compound or pharmaceutical composition represented by structural formula I in the preparation of medicines.

The present invention further provides a preferred technical solution for the application:

Preferably, the application is an application for preparing a medicine for treating, preventing, delaying or preventing cancer, cancer metastasis, cardiovascular disease, immune disease, fibrosis or eye disease.

Preferably, the application is an application for preparing a medicine for treating diseases mediated by SHP2.

Preferably, the disease is cancer.

Preferably, the cancer is selected from Noonan syndrome, leopard spot syndrome, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, head and neck squamous cell carcinoma, acute myeloid leukemia, breast cancer, esophageal tumor , Lung cancer, colon cancer, head cancer, gastric cancer, lymphoma, glioblastoma, gastric cancer, pancreatic cancer or a combination thereof.

Preferably, the application is an application for preparing an SHP2 inhibitor.

The present invention also provides a method for treating and/or preventing diseases mediated by SHP2, which comprises administering a therapeutically effective amount of at least any one compound or pharmaceutical composition represented by structural formula I to a subject.

Preferably, in the above method, the SHP2-mediated disease is cancer.

The present invention also provides a method for treating cancer, which comprises administering a therapeutically effective amount of at least any one compound or pharmaceutical composition represented by structural formula I to a subject.

Preferably, in the above method, the cancer is Noonan syndrome, leopard spot syndrome, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, head and neck squamous cell carcinoma, acute myeloid leukemia, breast Cancer, esophageal tumor, lung cancer, colon cancer, head cancer, stomach cancer, lymphoma, glioblastoma, gastric cancer, pancreatic cancer, or a combination thereof.

Preferably, in the above method, the treatment target is a human.

Unless otherwise specified, the terms used in the present invention have the following meanings:

The term "alkyl" includes straight, branched or cyclic saturated alkyl groups. For example, alkyl includes but is not limited to methyl, ethyl, propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, 3 -(2-Methyl)butyl, 2-pentyl, 2-methylbutyl, neopentyl, cyclopentyl, n-hexyl, 2-hexyl, 2-methylpentyl and cyclohexyl and other similar groups group. Similar, "C _1-8 alkyl""C_1-8" means comprising 7 or 8 carbon atoms, a straight chain, branched chain or cyclic Formally arranged groups.

"Alkenyl" and "alkynyl" include linear, branched or cyclic alkenyl and alkynyl groups. Similarly, "C _2-8 alkenyl" and "C _2-8 alkynyl" refer to a linear, branched or cyclic arrangement containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms的alkenyl or alkynyl.

The term "alkoxy" refers to the oxyether form of the aforementioned linear, branched or cyclic alkyl group.

The term "aryl" refers to an unsubstituted or substituted monocyclic or polycyclic aromatic group including carbon atoms. It is preferably a 6 to 10 membered monocyclic or bicyclic aromatic group. Preferably it is phenyl and naphthyl. Most preferred is phenyl.

The term "heteroaryl" refers to a monovalent heteroatom group formed by removing a hydrogen atom from a carbon atom of a parent heteroaromatic ring system. Heteroaryl groups include: 5- to 7-membered aromatic, monocyclic, including at least one heteroatom selected from N, O or S, for example, 1 to 4 heteroatoms, or preferably 1 to 3 heteroatoms, ring The other atoms on the above are carbon; the polyheteroaryl ring includes at least one heteroatom selected from N, O or S, for example, 1 to 4 heteroatoms, or preferably 1 to 3 heteroatoms, other atoms in the ring Is carbon, and at least one of the heteroatoms is on the aromatic ring. Particularly preferred heteroaryl groups are C _3-10 heteroaryl groups, including but not limited to, pyrrolyl, furyl, thienyl, pyridyl, pyranyl, pyrazolyl, pyrimidinyl, pyridazinyl, Pyrazinyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, triazole, indolyl, benzofuranyl, benzothiazolyl, benzimidazolyl, benzopyrazolyl, benzene And similar groups such as triazolyl, carbazolyl, quinolinyl, isoquinolinyl, purinyl and the like.

However, in any case, the heteroaryl group and the aryl group will not cross or contain each other. Therefore, according to the above definition, if at least one all-carbon aromatic ring is fused with a heterocyclic group, the result is a heteroaryl group instead of an aryl group.

"Cycloalkyl" refers to a cyclic group that is saturated or unsaturated but not aromatic. Depending on the specific level of saturation, the terms "cycloalkyl", "cycloalkenyl" or "cycloalkynyl" are used respectively. Representative cycloalkyl groups include, but are not limited to, cyclopropane, cyclobutane, cyclopentane, cyclohexane, or cyclohexene and similar groups. Specifically, the cycloalkyl group may be a C _3-10 cycloalkyl group, such as a C _3-6 cycloalkyl group.

"Heterocyclic group" refers to a saturated or unsaturated but not aromatic cyclic group, and one or more of the carbon atoms (and the connected hydrogen atoms) can be respectively the same or different hetero Atom and the corresponding attached hydrogen atom are replaced. Representative heteroatoms that replace carbon atoms include, but are not limited to, N, P, O, S, and Si. When a specific degree of saturation needs to be described, the terms "heterocycloalkyl" or "heterocycloalkenyl" are used respectively. Representative heterocyclic groups include but are not limited to epoxy compounds, imidazolidine, morpholine, piperazine, piperidine, pyrazolidine, pyrrolidine, quinuclidine, tetrahydrofuran or tetrahydropyran and similar groups . Substituent heterocyclic groups also include ring systems substituted with at least one oxygen-containing (=O) or oxide (-O-) substituent, such as piperidine-nitrogen-oxide, morpholinyl-nitrogen- Oxide, 1-oxo-1-thiomorpholinyl and 1-dioxo-1-thiomorpholinyl.

However, in any case, the heterocycloalkyl group and the cycloalkyl group will not cross or contain each other. Therefore, according to the above definition, if at least one all-carbocyclic ring is fused with a heterocycloalkyl to form a di-, poly- or spiro-ring, it will still be defined as a heterocycloalkyl.

In addition, if a heteroaryl group is fused with a heterocyclic group to form a di-, poly- or spiro-ring, it will be defined as a heterocyclic group instead of a heteroaryl group.

"Halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I). Preferred halogen refers to fluorine, chlorine and bromine.

"Halo" refers to a fluoro, chloro, bromo or iodo group. The preferred halo groups refer to fluoro and chloro.

"Substitution" means that one or more hydrogen atoms in a group are replaced by the same or different substituents. Representative substituents include, but are not limited to, halogen, amino, oxo, carbonyl, alkyl, alkoxy, aryl, cycloalkyl, heterocyclyl, and heteroaryl. In some embodiments, substituents include, but are not limited to halo, amino, methyl, -CH _3, trifluoromethyl, -CH _3.

Whenever the term "alkyl" or "aryl" or its prefixes appear in the name of a substituent (such as aralkyl, or dialkylamino), it shall be the same as the aforementioned "alkyl" and "aryl""The definition defines the substituents. The specified number of carbon atoms (such as C _1-6 ) will independently represent the number of carbon atoms in an alkyl moiety or in an alkyl moiety (where the alkyl group is the prefix stem) in a larger substituent.

The "compound" of the present invention includes the compound represented by formula I, and all pharmaceutically acceptable forms thereof. These pharmaceutically acceptable forms include salts, solvates, non-covalent complexes, chelates or their prodrugs, or any mixture of all the above forms.

The "pharmaceutically acceptable" refers to those that are well known for use in animals, especially those that can be used in humans.

The term "composition" in the present invention includes a product containing a specific quantity of a specific component, and also includes any product directly or indirectly obtained from a specific quantity of a specific component. Therefore, a pharmaceutical composition including the compound of the present invention as an active ingredient and a method for preparing the compound are the content of the present invention.

"Therapeutically effective amount" means that when a compound is administered to a subject to treat and prevent and/or inhibit at least one clinical symptom of a disease, condition, symptom, indication, and/or discomfort, it is sufficient for the disease, condition, A dose that produces a certain effect in the treatment of symptoms, indications or discomfort. The specific "effective therapeutic dose" may vary according to the compound, the route of administration, the age of the patient, the weight of the patient, the type of disease or discomfort to be treated, the symptoms and severity, etc. Whenever possible, an appropriate dose may be obvious to those skilled in the art, or it may be determined by conventional experimental methods.

The compounds provided by the present invention may exist in the form of "pharmaceutically acceptable salts". In terms of pharmaceutical applications, the salt of the compound provided by the present invention refers to a non-toxic pharmaceutically acceptable salt. The form of the pharmaceutically acceptable salt includes a pharmaceutically acceptable acid/anion or base/cation salt. Pharmaceutically acceptable acid/anionic salts generally exist in the form of protonation of basic nitrogen with inorganic or organic acids. Typical organic or inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, perchloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, succinic acid, maleic acid, fumaric acid, malic acid , Tartaric acid, citric acid, α-ketoglutaric acid, hippuric acid, benzoic acid, mandelic acid, methanesulfonic acid, isethionic acid, benzenesulfonic acid, oxalic acid, pamoic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid Acid, cyclohexylamine sulfonic acid, salicylic acid, saccharic acid or trifluoroacetic acid. Pharmaceutically acceptable base/cation salts, including, but not limited to, aluminum salt, calcium salt, chloroprocaine salt, choline, diethanolamine salt, ethylenediamine salt, lithium salt, magnesium salt, potassium salt, sodium Salt and zinc salt.

The prodrug of the compound of the present invention is included in the protection scope of the present invention. Generally, the prodrug is a functional derivative that is easily converted into a desired compound in vivo. Therefore, the term "administration" involved in the treatment method provided by the present invention includes the administration of the compound disclosed in the present invention, or although it is not clearly disclosed but can be transformed into the compound disclosed in the present invention in vivo after administration to the subject. disease. The conventional methods for selecting and preparing suitable prodrug derivatives have been recorded in books such as "Design of Prodrugs" (Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985).

Obviously, the definition of any substituent or variable at a specific position in one molecule is irrelevant to the definition of any substituent or variable at a specific position in other molecules. It is easy to understand that the compounds of the present invention can be selected according to the prior art of the subject to select suitable substituents or substitution forms to provide chemically stable and easy preparation and synthesis using the prior art of the subject or the method described in the present invention.

When the compound represented by Formula I and its pharmaceutically acceptable salt are in the form of a solvate or polymorph, the present invention includes any possible solvate and polymorph. The type of solvent that forms the solvate is not particularly limited, as long as the solvent is pharmacologically acceptable. For example, water, ethanol, propanol, acetone and similar solvents can be used.

The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic base or acid. When the compound provided by the present invention is an acid, its corresponding salt can be prepared from pharmaceutically acceptable non-toxic bases, including inorganic bases and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper (ic and ous), iron, ferrous, lithium, magnesium, manganese (ic and ous), potassium, sodium, zinc and the like. In particular, salts of ammonium, calcium, magnesium, potassium, and sodium are preferred. Non-toxic organic bases that can be derivatized into pharmaceutically acceptable salts include primary, secondary and tertiary amines, as well as cyclic amines and amines containing substituents, such as naturally occurring and synthetic amines containing substituents. Other pharmaceutically acceptable non-toxic organic bases capable of forming salts, including ion exchange resins and arginine, betaine, caffeine, choline, N',N'-dibenzylethylene diamine, diethylamine, 2 -Diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucosamine, glucosamine, histidine, haamine, isopropylamine , Lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc.

When the compound provided by the present invention is a base, the corresponding salt can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids and organic acids. Such acids include, for example, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, isethionic acid, formic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, Hydrochloric acid, lactic acid, maleic acid, malic acid, mandelic acid, α-ketoglutaric acid, hippuric acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, p-toluenesulfonate Acid etc. Preferably, malic acid, citric acid, hydrobromic acid, hydrochloric acid, methanesulfonic acid, maleic acid, phosphoric acid, sulfuric acid and tartaric acid. More preferably, phosphoric acid, hydrochloric acid and malic acid. Since the compound represented by formula I will be used as a pharmaceutical, it is preferable to use a substantially pure form, for example, at least 60% purity, more suitably at least 75% purity, particularly suitably at least 98% purity (% is a weight ratio).

The pharmaceutical composition provided by the present invention includes the compound represented by formula I (or a pharmaceutically acceptable salt thereof) as an active component, a pharmaceutically acceptable excipient and other optional therapeutic components or adjuvants. Although in any given case, the most suitable way of administering the active ingredient depends on the particular subject to be administered, the nature of the subject and the severity of the disease, the pharmaceutical composition of the present invention includes oral, rectal, topical and Pharmaceutical composition for parenteral (including subcutaneous administration, intramuscular injection, intravenous administration) administration. The pharmaceutical composition of the present invention can be conveniently prepared in a unit dosage form known in the art and prepared by any preparation method known in the pharmaceutical field.

In fact, according to conventional drug mixing technology, the compound of formula I, or prodrug, or metabolite, or pharmaceutically acceptable salt of the present invention can be combined with drugs as the active component, and mixed with a drug carrier to form a drug combination Things. The pharmaceutical carrier can take various forms, depending on the desired mode of administration, for example, oral or injection (including intravenous injection). Therefore, the pharmaceutical composition of the present invention may take the form of a separate unit suitable for oral administration, such as a capsule, cachet or tablet containing a predetermined dose of the active ingredient. Further, the pharmaceutical composition of the present invention may take the form of powder, granule, solution, aqueous suspension, non-aqueous liquid, oil-in-water emulsion, or water-in-oil emulsion. In addition, in addition to the common dosage forms mentioned above, the compound represented by Formula I or a pharmaceutically acceptable salt thereof can also be administered by a controlled release method and/or a delivery device. The pharmaceutical composition of the present invention can be prepared by any pharmaceutical method. Generally, this method includes the step of associating the active ingredient with the carrier constituting one or more necessary ingredients. In general, the pharmaceutical composition is prepared by uniformly and intimately mixing the active ingredient with a liquid carrier or a finely divided solid carrier or a mixture of both. In addition, the product can be easily prepared into the desired appearance.

Therefore, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier and a compound represented by formula I, or a pharmaceutically acceptable salt thereof. The compound represented by formula I, or a pharmaceutically acceptable salt thereof, and one or more other compounds having therapeutic activity in combination are also included in the pharmaceutical composition of the present invention.

The drug carrier used in the present invention can be, for example, a solid carrier, a liquid carrier or a gas carrier. Examples of solid carriers include lactose, gypsum powder, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, mannitol, sorbitol, microcrystalline cellulose, inorganic salts , Starch, pregelatinized starch, powdered sugar, dextrin, etc. Examples of liquid carriers include syrup, peanut oil, olive oil and water. Examples of gas carriers include carbon dioxide and nitrogen. When preparing oral pharmaceutical preparations, any convenient pharmaceutical medium can be used. For example, water, ethylene glycol, oils, alcohols, flavor enhancers, preservatives, coloring agents, etc. can be used for oral liquid preparations such as suspensions, elixirs and solutions; and carriers, such as starches, sugars, Microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrants, etc. can be used in oral solid preparations such as powders, capsules and tablets. In view of ease of administration, tablets and capsules are preferred for oral preparations. Alternatively, standard aqueous or non-aqueous formulation techniques can be used for tablet coating.

Tablets containing the compound or pharmaceutical composition of the present invention can be prepared by, optionally, mixing, compression or molding with one or more auxiliary components or adjuvants. The active ingredient is in a free-flowing form such as powder or granules, mixed with lubricants, inert diluents, surface active or dispersing agents, and compressed in a suitable machine to obtain compressed tablets. The powdered compound or pharmaceutical composition is wetted with an inert liquid diluent, and then molded in a suitable machine to form a molded tablet. Preferably, each tablet contains about 0.01 mg to 5 g of active ingredient, and each cachet or capsule contains about 0.1 mg to 0.5 g of active ingredient. For example, a dosage form intended for oral administration to humans contains about 0.1 mg to about 0.5 g of the active ingredient, compounded with a suitable and convenient metering auxiliary material, which accounts for about 5% to 99.99% of the total pharmaceutical composition . The unit dosage form generally contains about 0.1mg to about 0.5g of effective ingredients, typically 0.1mg, 0.2mg, 0.5mg, 1mg, 2mg, 2.5mg, 5mg, 10mg, 25mg, 50mg, 100mg, 200mg, 300mg, 400mg Or 500mg.

The pharmaceutical composition suitable for parenteral administration provided by the present invention can be prepared as an aqueous solution or suspension by adding active components into water. Suitable surfactants such as sodium lauryl sulfate, polysorbate-80 (Tween-80), polyoxyethylene hydrogenated castor oil, and poloxamer may be included. In glycerol, liquid polyethylene glycol, and their mixtures in oil, dispersion systems can also be prepared. Furthermore, a preservative may also be included in the pharmaceutical composition of the present invention to prevent the growth of harmful microorganisms.

The present invention provides pharmaceutical compositions suitable for injection use, including sterile aqueous solutions or dispersion systems. Further, the above-mentioned pharmaceutical composition can be prepared in the form of a sterile powder that can be used for immediate preparation of sterile injection. In any case, the final injection form must be sterile, and for easy injection, it must be easy to flow. In addition, the pharmaceutical composition must be stable during preparation and storage. Therefore, preservation against contamination by microorganisms such as bacteria and fungi is preferred. The carrier can be a solvent or dispersion medium, for example, water, ethanol, polyol (such as glycerol, propylene glycol, liquid polyethylene glycol), vegetable oil, and suitable mixtures thereof.

The pharmaceutical composition provided by the present invention may be in a form suitable for topical administration, for example, aerosol, emulsion, ointment, lotion, dusting, or other similar dosage forms. Further, the pharmaceutical composition provided by the present invention can be in a form suitable for use in a transdermal drug delivery device. These preparations can be prepared using the compound represented by formula I of the present invention, or a pharmaceutically acceptable salt thereof, through conventional processing methods. As an example, an emulsion or ointment is prepared by adding a hydrophilic material and water (the total amount of the two is about 5 wt% to 50 wt% of the compound) to prepare a cream or ointment with the desired consistency.

The pharmaceutical composition provided by the present invention can be made into a form suitable for rectal administration with a solid as a carrier. Suppositories in which the mixture forms a unit dose are the most preferred dosage form. Suitable excipients include cocoa butter and other materials commonly used in the art. Suppositories can be conveniently prepared. First, the pharmaceutical composition is mixed with softened or melted excipients, then cooled and molded.

In addition to the above-mentioned carrier components, the above-mentioned pharmaceutical preparations may also include, as appropriate, one or more additional adjuvant components, such as diluents, buffers, flavoring agents, binders, surfactants, and additives. Thickeners, lubricants, preservatives (including antioxidants), etc. Further, other adjuvants may also include penetration enhancers that regulate the isotonic pressure of the drug and blood. The pharmaceutical composition containing the compound represented by Formula I, or a pharmaceutically acceptable salt thereof, can also be prepared in the form of a powder or a concentrated solution.

detailed description

In order to make the above content clearer and clearer, the present invention will use the following embodiments to further illustrate the technical solution of the present invention. The following examples are only used to illustrate specific implementations of the present invention, so that those skilled in the art can understand the present invention, but are not used to limit the protection scope of the present invention. In the specific embodiments of the present invention, technical means or methods that are not specifically described are conventional technical means or methods in the art.

Unless otherwise stated, all parts and percentages in the present invention are calculated by weight, and all temperatures refer to degrees Celsius.

The following abbreviations are used in the examples:

ACE-Cl: 1-chloroethyl chloroformate;

(Boc) ₂ O: di-tert-butyl dicarbonate;

BOP: Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate;

DBU: 1,8-diazabicycloundec-7-ene;

DCE: 1,2-dichloroethane;

DCM: dichloromethane;

DIPEA or DIEA: N,N-diisopropylethylamine;

DMAc: N,N-dimethylacetamide;

DMF: N,N-dimethylformamide;

DMSO: dimethyl sulfoxide;

EA or EtOAc: ethyl acetate;

EGTA: ethylene glycol diaminoethyl ether tetraacetic acid;

EtOH: ethanol;

EtONa: sodium ethoxide;

h, hr or hrs: hour;

Hex: n-hexane;

HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;

LC-MS or LCMS: liquid chromatography-mass spectrometry;

LDA: lithium diisopropylamide;

MeCN: Acetonitrile;

MeOH: methanol;

MeONa: Sodium methoxide;

min or mins: minutes;

MsCl: methanesulfonyl chloride;

NEt ₃ : Triethylamine

NBS: N-bromosuccinimide;

NMP: N-methyl-2-pyrrolidone;

Pd ₂ (dba) ₃ : Tris(dibenzylideneacetone)dipalladium;

Pd(OAc) ₂ : Palladium(II) acetate;

PE: petroleum ether;

PPA: polyphosphoric acid;

rt or RT: room temperature;

TFA: trifluoroacetic acid;

THF: Tetrahydrofuran;

Ti(OEt) ₄ : Tetraethyl titanate;

TLC: thin layer chromatography;

TMEDA: Tetramethylethylenediamine; and

xantphos: 4,5-bisdiphenylphosphine-9,9-dimethylxanthene.

Preparation of intermediate compound M1:

Step 1: Preparation of compound M1-3

15.00 g of compound M1-1 and 7.08 g of compound M1-2 were dissolved in 150 mL of dioxane, and 198 mg of Pd(OAc) ₂ , 1.70 g of Xantphos and 15.00 g of DIEA were added. Replace with nitrogen for three times. Under the protection of nitrogen, the reaction was raised to 85°C and stirred for 12 hrs. TLC detected that the reaction was complete, the reaction solution was filtered, the filter cake was washed with DCM (50 mL×2), the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 20.00 g of compound M1-3.

Step 2: Preparation of compound M1

20.00g of compound M1-3 was dissolved in 200mL THF, EtONa (35mL, 20% EtOH solution) was added dropwise at -30°C, and the reaction was stirred at RT for 3hrs. TLC detected that the reaction was complete, concentrated under reduced pressure, added 200 mL of DCM and stirred for 30 mins, the reaction solution was filtered, and the filter cake was washed with DCM (50 mL×2) to obtain 15.00 g of compound M1 as a solid.

Preparation of intermediate compound M2:

Step 1: Preparation of compound M2-2

1.00 g of compound M2-1 was dissolved in 10 mL of DMSO, and MeONa in MeOH solution (15 mL, 0.5 M) was added, and then reacted at 70° C. for 1 hr. TLC detected the completion of the reaction. The reaction solution was poured into 30mL water, extracted with EtOAc (40mL×3), combined the organic phases, washed with 50mL saturated NaCl, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by column chromatography. 1.10g of compound M2-2 as a pale yellow oil.

Step 2: Preparation of compound M2-3

Dissolve compound 1.10g M2-2 in 15mL dioxane, add 491mg compound M1-2, 46mg Xantphos, 35mg Pd(OAc) ₂ , 1.05g DIEA, replace the mixture with nitrogen 3 times, heat to 90℃ to react for 5hrs . TLC detected that the reaction was complete, the reaction solution was cooled to room temperature, filtered, the filter cake was washed with EtOAc (5 mL×3), the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 1.03 g of light yellow solid M2-3.

Step 3: Preparation of compound M2

Dissolve 1.03g of compound M2-3 in 10mL of anhydrous THF, lower the temperature to -30°C, slowly add EtONa's EtOH solution (2mL, 20%) to the above solution, stir and react at -30°C for 30mins, and slowly rise to room temperature Stir the reaction for 2hrs. TLC detected that the reaction was complete, the reaction solution was concentrated under reduced pressure, and the residue was slurried by adding 20 mL DCM for 30 mins, filtered, the filter cake was washed with DCM (5 mL×3), and the filter cake was vacuum-dried to obtain 990 mg of brown solid M2.

Preparation of intermediate compound M3:

Step 1: Preparation of compound M3-2

Dissolve 3.00g of compound M3-1 and 1.60g of compound M1-2 in 30mL of dioxane, add 243mg of Pd ₂ (dba) ₃ , 384g of Xantphos and 3.40g of DIPEA, replace with nitrogen three times, raise the reaction solution to 110°C and stir. Reaction 3hrs. TLC detected that the reaction was complete, the reaction solution was filtered, the filter cake was washed with DCM (30 mL×2), the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 4.80 g of compound M3-2.

Step 2: Preparation of compound M3

4.80g of compound M3-2 was dissolved in 50mL THF, EtONa (7.4mL, 20% EtOH solution) was added dropwise at -30°C, and the reaction was stirred at RT for 2hrs. TLC detected the completion of the reaction, concentrated under reduced pressure, added 50mL DCM and stirred for 30mins, the reaction solution was filtered, the filter cake was washed with DCM (10mL×2), and drained to obtain 3.60g of compound M3.

Preparation of intermediate compound M4:

Step 1: Preparation of compound M4-3

300 mg of compound M4-1 and 174 mg of compound M4-2 were dissolved in 10 mL of dioxane, and 4 mg of Pd(OAc) ₂ , 34 mg of Xantphos and 300 mg of DIEA were added. Replace with nitrogen for three times. Under the protection of nitrogen, the reaction was raised to 85°C and stirred for 12 hrs. TLC detected that the reaction was complete, the reaction solution was filtered, the filter cake was washed with DCM (10 mL×2), the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 266 mg of compound M4-3.

Step 2: Preparation of compound M4

266 mg of compound M4-3 was dissolved in 5 mL of THF, EtONa (0.47 mL, 20% EtOH solution) was added dropwise at -30°C, and the reaction was stirred at RT for 3 hrs. TLC detected that the reaction was complete, concentrated under reduced pressure, added 15 mL of DCM and stirred for 30 mins, the reaction solution was filtered, and the filter cake was washed with DCM (20 mL×2) to obtain 182 mg of compound M4 as a solid.

Preparation of intermediate compound M5:

Step 1: Preparation of compound M5-3

Under nitrogen protection, dissolve 25.00g of compound M5-1 in 200mL of DMF, cool to 0°C, add 22.70g NaH in batches, keep at 0°C for 1hr, and then slowly add 54.96g of compound M5-2 dropwise to the reaction solution After dropping, react at 0°C for 1 hr, and then heat to 60°C to continue the reaction for 1 hr. The reaction solution was cooled to 0°C, quenched with 500 mL ice water, extracted with EtOAc (500 mL×3), combined the organic phases, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was passed through the column Analyze and purify to obtain 29.00g of brown oil M5-3.

Step 2: Preparation of compound M5-5

29.00g of compound M5-3 was dissolved in 50 mL of Ti(OEt) ₄ , 34.99g of compound M5-4 was added, and then heated to 90°C for 12 hrs. TCL detected that the reaction was complete. Pour the reaction solution into 500 mL ice water, add 300 mL EtOAc and stir for 1 hr, extract with EtOAc (300 mL×3), combine the organic phases, and wash the organic phase with saturated brine (100 mL×4), anhydrous sulfuric acid It was dried with sodium and concentrated under reduced pressure to obtain 39.00 g of crude compound M5-5 as a brown oil.

Step 3: Preparation of compound M5-6

Under the protection of nitrogen, 48.00g of compound M5-5 was dissolved in 500mL of anhydrous THF, the temperature was lowered to -20°C, 6.73g of NaHB ₄ was slowly added, and then the temperature was raised to RT and stirred for 2hrs. After the reaction, the reaction solution was cooled to 0°C, quenched with 300 mL of water, extracted with EtOAc (300 mL×3), combined the organic phases, washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was passed through a column Chromatographic purification yielded 25.40 g of compound M5-6 as a brown oil.

Step 4: Preparation of compound M5

10.00 g of compound M5-6 was dissolved in 100 mL of DCM solution, 28.04 g of TFA solution was added dropwise, and then reacted at RT for 1 hr. The reaction solution was cooled to 0°C, quenched with 100 mL saturated aqueous NaHCO ₃ , extracted with EtOAc:THF=3:1 (100 mL×3), combined the organic phases, washed with saturated brine, dried over anhydrous sodium sulfate, and reduced pressure It was concentrated to obtain 7.64 g of a brown solid, the crude compound M5, which was directly used in the next reaction.

¹ H NMR(500MHz, DMSO-d ₆ ): δ7.26-7.16(m,4H), 5.50(d,J=10.0Hz,1H), 4.30(d,J=10.0Hz,1H), 3.04(d ,J = 16.0Hz, 1H), 2.87-2.80 (m, 2H), 2.67-2.58 (m, 3H), 1.88-1.82 (m, 1H), 1.59-1.53 (m, 1H), 1.37-1.34 (m ,1H),1.21(s,9H),1.12-1.09(m,1H).

Preparation of intermediate compound M6:

Step 1: Preparation of compound M6-3

10.00 g of compound M6-1 and 19.50 g of compound M6-2 were dissolved in 100 mL of MeCN, and 26.20 g of K ₂ CO _{3 was added} . The reaction was raised to 90°C and stirred for 3hrs. TLC detected that the reaction was complete, the reaction solution was filtered, the filter cake was washed with EtOAc (50 mL×2), the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 5.90 g of compound M6-3.

Step 2: Preparation of compound M6-4

1.50 g of compound M6-3 was dissolved in 15 mL of toluene, 1.1 mL of PBr _{3 was} added dropwise, the reaction solution was raised to 105° C. and stirred for reaction for 12 hrs. TLC detects the completion of the reaction, concentrated under reduced pressure, added 15 mL of water, adjusted to pH=9 with NaOH solution, extracted with EtOAc (30 mL×3), combined the organic phases, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 1.50 g of compound M6- 4.

Step 3: Preparation of compound M6-6

Dissolve 450 mg of compound M6-5 in 6 mL DMF, add 271 mg of NaH in batches at 0°C, stir and react at 60°C for 1 hr under nitrogen protection, then add 1.20 g of compound M6-4 and stir at 60°C for 1 hr. TLC detected the completion of the reaction. The reaction was quenched by adding 30 mL of water, and extracted with EtOAc (25 mL×2) and water (30 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography. 160 mg of compound M6-6.

Step 4: Preparation of compound M6-7

160 mg of compound M6-6 was dissolved in 2 mL of DCE at 0° C., 155 mg of ACE-Cl was added dropwise, and the reaction was stirred at RT for 2 hrs. TLC detected that the reaction was complete, concentrated under reduced pressure, 4mL MeOH was added, and the reaction was raised to 80°C and stirred for 3hrs. The reaction was completed by TLC detection, concentrated under reduced pressure, 4 mL DCM, 242 mg (B _OC ) ₂ O and 239 mg DIEA were added, and the reaction was stirred at RT for 12 hrs. TLC detected that the reaction was complete, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 25 mg of compound M6-7.

Step 5: Preparation of compound M6

The procedure for preparing compound M6 from compound M6-7 is similar to the procedure from compound M5-3 to compound M5. Preparation of intermediate compound M7:

Step 1: Preparation of compound M7-3

Dissolve 4.00g of compound M7-1 in 50mL of anhydrous THF, replace with nitrogen three times, cool to -78°C, slowly add LDA solution in THF (11.70mL, 2.0M) dropwise, and then react at -78°C for 1 hr to convert compound M7 -2 in THF (10 mL) was slowly added dropwise to the above reaction solution, reacted at -78°C for 30 mins, and slowly raised to room temperature for 2 hrs. TLC detected the completion of the reaction. The reaction was quenched with 30 mL saturated NH ₄ Cl solution, 50 mL water was added, and EtOAc was added for extraction (60 mL×3). The organic phases were combined, washed with 50 mL saturated NaCl, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified by column chromatography to obtain 4.30 g of colorless oil M7-3.

Step 2: Preparation of compound M7-4

4.30g of compound M7-3 was dissolved in THF/MeOH (40mL/40mL), NaOH aqueous solution (20mL, 2.4N) was added, and the reaction was heated to 80°C for 18hrs. TLC detected the completion of the reaction, the reaction solution was cooled to room temperature, concentrated under reduced pressure to remove the organic solvent, the residue was adjusted to pH 3-4 with concentrated hydrochloric acid, filtered, the filter cake was washed with water (10 mL×3), and the filter cake was vacuum dried 3.40 g of compound M7-4 was obtained as a white solid.

Step 3: Preparation of compound M7-5

3.40g of compound M7-4 was dissolved in 40mL PPA, and the temperature was raised to 120°C to react for 2hrs. TLC detected that the reaction was complete. The reaction solution was slowly added dropwise to 200 mL crushed ice, adjusted to pH 9-10 with 2.4N NaOH aqueous solution, 4.40 g (B _OC ) ₂ O was added, and the reaction was stirred at RT for 18 hrs. TLC detected that the reaction was complete, extracted with EtOAc (100 mL×3), combined the organic phases, washed with 100 mL saturated NaCl, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 2.40 g of white solid M7-5.

Step 4: Preparation of compound M7

The procedure for preparing compound M7 from compound M7-5 is similar to the procedure from compound M5-3 to compound M5. Preparation of intermediate compound M8:

Step 1: Preparation of compound M8-2

10.00 g of compound M8-1 was dissolved in 100 mL of MeOH, 2.0 mL of concentrated sulfuric acid was added, and the reaction was heated at 70° C. for 3 hrs. After the reaction is complete, spin off the solvent, add 20 mL of water, adjust to pH=9 with saturated aqueous Na ₂ CO ₃ , extract with EtOAc (100 mL×3), combine the organic phases, dry with anhydrous sodium sulfate, and concentrate under reduced pressure. Purification by column chromatography yielded 9.23 g of white solid, compound M8-2.

Step 2: Preparation of compound M8-3

Dissolve 9.23 g of compound M8-2 in 150 mL of MeOH, cool to 0° C. in an ice bath, add 6.97 g of NaHB _{4 in} batches, and naturally increase the temperature to RT for 5 hrs. After the reaction is complete, add 20mL saturated NH ₄ Cl solution, spin dry the solvent, add EtOAc (100mL×3) for extraction, combine the organic phases, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and purify the residue by column chromatography to obtain 6.40g Color liquid, compound M8-3.

Step 3: Preparation of compound M8-4

Under nitrogen protection, dissolve 3.00g of compound M8-3 in 50mL of dichloromethane, cool the reaction solution to -15°C, add 2.81mL of NEt ₃ , and then add 1.04mL of MsCl solution dropwise. After the addition, the temperature is raised to 0 React at ℃ for 1hr. After the reaction was completed, a water layer was added, the organic phase was washed with 20 mL of saturated brine, the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 4.05 g of white solid compound M8-4.

Step 4: Preparation of compound M8-6

Under the protection of nitrogen, dissolve 3.39g of compound M8-5 in 20mL of anhydrous THF, lower the temperature to -50°C, and add 1.71g LDA solution dropwise. After dripping, keep the temperature at -50°C and react for 1hr. A solution of 3.00 g of compound M8-4 in anhydrous THF (10 mL) was added dropwise, after the dropping, the temperature was raised to RT and reacted for 1 hr. After completion of the reaction, 50 mL brine was added, extracted with EtOAc (50 mL×3), the organic phases were combined, the organic phase was dried with sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 4.61 g of white solid compound M8-6.

Step 5: Preparation of compound M8-7

Dissolve 4.61g of compound M8-6 in 8mL of water and 40mL of MeOH, and add 2.07g of NaOH. The temperature was raised to 65°C and stirred overnight. After the reaction is complete, add 30 mL of water, spin dry the solvent methanol, adjust the concentrate to pH=6 with 2N hydrochloric acid, extract with EtOAc (50 mL×3), combine the organic phases, dry with anhydrous sodium sulfate, concentrate under reduced pressure, and pass the residue through a column Chromatographic purification yielded 3.98 g of white solid, compound M8-7.

Step 6: Preparation of compound M8-8

Under the protection of nitrogen, 3.98g of compound M8-7 was dissolved in 20mL of anhydrous THF, the reaction solution was cooled to -15°C, NaH (60%, 0.42g) was added in batches, and the reaction was kept at -15°C for 1 hr, and then cooled to At -60°C, n-butyllithium (1.6M, 7.8 mL) was added dropwise, and the reaction was incubated for 1 hr. After the reaction was completed, 50 mL of water was added, extracted with EtOAc (50 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 2.36 g of a pale yellow solid, namely compound M8-8.

Step 7: Preparation of compound M8

The procedure for preparing compound M8 from compound M8-8 is similar to the procedure from compound M5-3 to compound M5. Preparation of intermediate compound M9:

Step 1: Preparation of compound M9-3

Under nitrogen protection, 2.83 g of compound M9-2 was dissolved in 50 mL of anhydrous THF, the temperature was lowered to -78° C., and a THF/Hex solution of LDA (2M, 6 mL) was added dropwise. Incubate at -78°C for 1 hr, then slowly drop 1.69 g of compound M9-1 in THF (3 mL) into the reaction solution, and react at -78°C for 1 hr. The reaction solution was quenched with 50 mL saturated brine, extracted with EtOAc (30 mL×2), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 1.44 g of a pale yellow oil compound M9- 3.

Step 2: Preparation of compound M9-4

Under nitrogen protection, 900 mg of compound M9-3 was dissolved in 50 mL of anhydrous THF, and the temperature was lowered to -78°C. A THF/Hex solution of LDA (2M, 3 mL) was added dropwise. The reaction was incubated at -78°C for 1 hr. The reaction solution was quenched with 50 mL saturated brine, extracted with EtOAc (30 mL×2), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 300 mg of light yellow solid compound M9-4.

Step 3: Preparation of compound M9

The procedure for preparing compound M9 from compound M9-4 is similar to the procedure from compound M5-3 to compound M5.

Through different reaction starting materials and suitable reagents, for example, the starting material for the synthesis of M11 is 6-methoxy-1-indanone, the intermediate compound M10 in Table 1 is prepared by a method similar to the foregoing intermediate 5-9 -M14.

Table 1

Example 1: Compound (S)-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino-3 -Chloropyridin-4-yl)sulfanyl)-3-methylpyrimidin-4(3H)-one

Step 1: Preparation of compound 1-2

Dissolve 2.00 g of compound 1-1 in 40 mL of THF, add NaOH (30 mL, 1N), and then react with RT for 2 hrs. TLC detects the completion of the reaction. Pour the reaction solution into 100mL water, adjust the pH to 4-5 with 6N HCl, add EtOAc to extract (100mL×4), combine the organic phases, wash with 50mL saturated NaCl, dry with anhydrous sodium sulfate, reduce After pressure concentration, the residue was slurried with n-hexane to obtain 1.67 g of light yellow solid 1-2.

Step 2: Preparation of compound 1-3

Dissolve 1.28g of compound 1-2 in 13mL DMF, lower the temperature to 0°C, add 3.26g of Cs ₂ CO ₃ , and then add 2.13g of CH ₃ I dropwise, and then heat up to 10°C for 1 hr. TLC detected that the reaction was complete, the reaction solution was cooled to 0°C, 20mL EtOAc and 20mL water were added, the organic phase was separated, the aqueous phase was extracted with 20mL EtOAc, the organic phases were combined, washed with saturated NaCl (5×4mL), and dried over anhydrous sodium sulfate After concentration under reduced pressure, the residue was purified by column chromatography to obtain 635 mg of compound 1-3.

Step 3: Preparation of compound 1-5

Dissolve 359mg compound 1-3 and 448mg compound M5 in 6mL DMAc, add 860mg DIPEA, replace with nitrogen three times, and react in microwave at 120°C for 1hr. TLC detects the completion of the reaction, cools the reaction solution to room temperature, adds 20 mL of water, adds EtOAc (20 mL×3) for extraction, combines the organic phases, washes with saturated NaCl (5 mL×4), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue Purified by column chromatography to obtain 290 mg of compound 1-5.

Step 4: Preparation of compound 1-6

250 mg of compound 1-5 and 127 mg of compound M1 were dissolved in 8 mL of dioxane, 18 mg of CuI, 22 mg of TMEDA and 293 mg of K ₃ PO _{4 were added} , and the mixture was replaced with nitrogen three times, and heated to 100° C. to react for 48 hrs. The reaction solution was cooled to room temperature, filtered, the filter cake was washed with EtOAc (10 mL×3), the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 153 mg of compound 1-6.

Step 5: Preparation of compound 1

29 mg of compound 1-6 was dissolved in 0.5 mL EtOAc and 0.5 mL MeOH, 0.1 mL 2N HCl in MeOH solution was added, and the reaction was carried out at RT for 2 hrs. TLC detects the completion of the reaction, concentrates under reduced pressure, adds NaHCO ₃ to adjust the pH to 7-8, extracts with EtOAc (10 mL×3), combines the organic phases, dried over anhydrous sodium sulfate, and concentrates under reduced pressure. The residue is purified by column chromatography 11.9 mg of compound 1 was obtained.

[M+H ⁺ ]=469.16.

¹ H NMR (500MHz, DMSO-d ₆ ): δ8.12 (s, 1H), 7.65 (d, J = 5.5 Hz, 1H), 7.32-7.31 (m, 1H), 7.20-7.15 (m, 3H) ,6.27(s,2H),6.02(d,J=5.5Hz,1H),3.87(s,1H),3.71-3.65(m,2H),3.41(s,3H),3.21-3.11(m,2H) ),3.04(d,J=15.5Hz,1H),2.63(d,J=15.5Hz,1H),1.92-1.87(m,1H),1.83-1.78(m,1H),1.56-1.54(m, 1H), 1.17-1.14 (m, 1H).

Example 2: Compound (S)-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-methoxy-1,3-dihydrospiro [Indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one

Step 1: Preparation of compound 2-1

215 mg DIEA was added to the dioxane (2 mL) solution of 205 mg of compound M11 and 150 mg of compound 1-3, and stirred at 60°C for 2 hrs. The reaction solution was distilled under reduced pressure to remove the organic solvent, and the crude product was separated and purified by Pre-TLC (PE:EtOAc=1:1) to obtain 180 mg of compound 2-1.

[M+H ⁺ ]:571.10.

Step 2: Preparation of compound 2-2

Under nitrogen protection, 179 mg K ₃ PO ₄ , 11 mg CuI and 13 mg TMEDA were added to a DMSO (2 mL) solution of 160 mg of compound 2-1 and 61 mg of compound M1, replaced with nitrogen three times, and stirred at 100° C. for 12 hrs. The reaction solution was diluted with 20 mL of water, extracted with EtOAc (20 mL×3), and the organic phases were combined. The organic phases were washed with saturated NaCl water, dried over anhydrous sodium sulfate and concentrated. The crude product was separated by Pre-TLC (DCM:MeOH=20:1) After purification, 70 mg of compound 2-2 was obtained.

[M+H ⁺ ]=603.28.

Step 3: Preparation of compound 2

At 0°C, a methanol solution of hydrochloric acid (2M, 0.5 mL) was added dropwise to a solution of 70 mg of compound 2-2 in dioxane (3 mL), and stirred at 25°C for 1 hr. The reaction solution was adjusted to pH 8-9 with saturated aqueous NaHCO ₃ solution, the reaction was quenched, EtOAc:THF=5:1 (10 mL×3) extraction, and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate After concentration, the crude product was separated and purified by Pre-TLC (DCM:MeOH=20:1) and then separated and purified by Pre-HPLC (basic) to obtain 12 mg of compound 2.

[M+H+]=499.17

¹ H NMR(500MHz,DMSO-d6)δ: 8.12(s,1H), 7.65(d,J=5.5Hz,1H), 7.08(d,J=8.0Hz,1H), 6.90(d,J=2.5 Hz, 1H), 6.70 (dd, J = 8.0, 2.5 Hz, 1H), 6.27 (s, 2H), 6.02 (d, J = 5.5 Hz, 1H), 3.82 (s, 1H), 3.73 (s, 3H) ), 3.70–3.62 (m, 2H), 3.40 (s, 3H), 3.21–3.07 (m, 2H), 2.96 (d, J = 15.0 Hz, 1H), 2.53 (d, J = 15.0 Hz, 1H) ,1.93--1.87(m,1H),1.82--1.76(m,1H),1.56-1.53(m,1H),1.14--1.12(m,1H)

Example 3: Compound (S)-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-chloro-1,3-dihydrospiro[indene -2,4'-Piperidine]-1'-yl)-3-Methylpyrimidin-4(3H)-one

Step 1: Preparation of compound 3-1

215 mg DIEA was added to the dioxane (2 mL) solution of 150 mg of compound M10 and 208 mg of compound 1-3, and stirred at 60°C for 2 hrs. The reaction solution was distilled under reduced pressure to remove the organic solvent, and the crude product was separated and purified by Pre-TLC (PE:EtOAc=1:1) to obtain 200 mg of compound 3-1.

[M+H ⁺ ]:575.08.

Step 2: Preparation of compound 3-2

Under nitrogen protection, 200 mg of compound 3-1 and 76.23 mg of compound M1 were dissolved in DMSO (2 mL), and 221.53 mg of K ₃ PO ₄ , 13.25 mg of CuI and 16.17 mg of TMEDA were added, replaced with nitrogen three times, and stirred at 100° C. for 12 hrs. The reaction solution was diluted with 20 mL of water, extracted with EtOAc (20 mL×3), and the organic phases were combined. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The crude product was separated by Pre-TLC (DCM:MeOH=20:1) After purification, 95 mg of compound 3-2 was obtained as a brown solid.

[M+H ⁺ ]=607.21.

Step 3: Preparation of compound 3

At 0°C, a methanol solution of hydrochloric acid (2M, 0.5 mL) was added dropwise to a solution of 95 mg of compound 3-2 in dioxane (3 mL), and stirred at 25°C for 1 hr. The reaction solution was quenched with saturated NaHCO ₃ aqueous solution to adjust the pH to 8-9, extracted with EA:THF=5:1 (10mL×3), combined the organic phases, washed with saturated brine, and dried over anhydrous sodium sulfate After concentration, the crude product was separated and purified by Pre-TLC (DCM:MeOH=20:1) and then separated and purified by Pre-HPLC (basic) to obtain 11 mg of compound 3.

[M+H ⁺ ]:503.13.

Using different reaction starting materials and appropriate reagents, the compound 4-compounds 14 and 24 in Table 2 were prepared by a method similar to the foregoing Examples 1-3.

Table 2

The NMR data of compounds 6, 8, 9, 10, 12 and 24 are as follows:

¹ H NMR (500MHz, DMSO-d ₆ ): δ8.46(d,J=4.4Hz,1H), 8.17(s,1H), 7.60(d,J=8.2Hz,1H), 7.53(dd,J ＝8.2,4.5Hz,1H),7.33(m,1H),7.20-7.15(m,3H),3.90(s,1H),3.75-3.60(m,2H),3.39(s,3H),3.22- 3.13(m,3H),3.05(d,J=15.6Hz,1H), 2.65(d,J=15.5Hz,1H),1.92-1.87(m,1H),1.83-1.79(m,1H),1.57 -1.54(m,1H),1.20-1.17(m,1H). (Compound 6)

¹ H NMR (500MHz, CDCl ₃ ): δ8.11 (s, 1H), 7.72 (d, J = 5.5 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 7.5 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 6.14 (d, J = 5.5 Hz, 1H), 4.85 (s, 2H), 4.08 (s, 1H), 3.67-3.61 (m, 2H) ),3.55(s,3H),3.28-3.22(m,2H),3.11(d,J=16.0Hz,1H),2.73(d,J=16.0Hz,1H),2.03-1.97(m,1H) ,1.93-1.87(m,1H),1.70-1.68(m,1H),1.43-1.40(m,1H). (Compound 8)

¹ H NMR (500MHz, CDCl ₃ ): δ8.10 (s, 1H), 7.74 (d, J = 5.4 Hz, 1H), 7.60 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.32(d,J=7.8Hz,1H),6.18(d,J=5.4Hz,1H),4.08(s,1H),3.71-3.60(m,2H),3.54(s,3H),3.2-3.17 (m, 2H), 3.13 (d, J = 16.0 Hz, 1H), 2.76 (d, J = 16.0 Hz, 1H), 2.03-1.99 (m, 1H), 1.89-1.87 (m, 1H), 1.69- 1.67 (m, 1H), 1.38-1.34 (m, 1H). (Compound 9)

¹ H NMR (500MHz, CDCl ₃ ): δ8.11 (s, 1H), 7.72 (d, J = 5.5 Hz, 1H), 7.24 (t, J = 8.0 Hz, 1H), 6.95 (d, J = 7.5 Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.15 (d, J = 5.5 Hz, 1H), 4.85 (s, 2H), 4.00 (s, 1H), 3.84 (s, 3H), 3.66-3.61(m,2H),3.54(s,3H),3.28-3.19(m,2H),3.05(d,J=16.0Hz,1H), 2.64(d,J=16.0Hz,1H), 2.00 -1.94 (m, 1H), 1.93-1.88 (m, 1H), 1.69-1.66 (m, 1H), 1.45-1.43 (m, 1H). (Compound 10)

¹ H NMR(500MHz, DMSO-d ₆ ): δ8.12(s,1H), 7.65(d,J=5.5Hz,1H), 6.93(s,1H), 6.80(s,1H), 6.28(s ,2H),6.02(d,J=5.0Hz,1H),3.79(s,1H),3.73(s,3H),3.72(s,3H),3.68-3.65(m,2H),3.41(s, 3H), 3.20-3.12 (m, 2H), 2.93 (d, J = 15.5 Hz, 1H), 2.56 (d, J = 15.5 Hz, 1H), 1.88-1.79 (m, 2H), 1.54-1.52 (m ,1H),1.23-1.20(m,1H). (Compound 12)

¹ H NMR(500MHz,DMSO-d ₆ )δ8.12(s,1H), 7.52(d,J=5.3Hz,1H), 7.36-7.31(m,1H), 7.21-7.16(m,3H), 6.16 (s, 2H), 6.05 (t, J = 5.1 Hz, 1H), 3.93 (s, 1H), 3.68-3.64 (m, 2H), 3.40 (s, 3H), 3.20-3.11 (m, 2H) ,3.05(d,J=15.6Hz,1H),2.66(d,J=15.7Hz,1H),1.92-1.78(m,2H),1.56-1.53(m,1H),1.21-1.18(m,1H) ).(Compound 24)

Example 15: Compound (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2 ,3-Dichlorophenyl)thio)-3-methylpyrimidin-4(3H)-one

Step 1: Preparation of compound 15-2

Dissolve 1.50 g of compound 15-1 in 10 mL of anhydrous DMF, add 2.36 g of NBS to the above solution in batches under ice bath, and slowly increase to RT for 4 hrs. LC-MS detected that the reaction was complete, the reaction solution was poured into 100 mL of water, stirred for 30 mins, filtered, the filter cake was washed with water (15 mL×3), and the filter cake was dried to obtain 1.57 g of white solid 15-2.

Step 2: Preparation of compound 15-4

1.57g of compound 15-2 and 2.56g of compound 15-3 were added to 20mL of dioxane, 0.27g of CuI, 0.33g of TMEDA and 4.54g of K ₃ PO _{4 were added} , the mixture was replaced with nitrogen three times, and the mixture was heated at 100°C for 12 hrs. The reaction solution was cooled to room temperature, 50 mL MeOH was added and filtered, the filter cake was washed with MeOH (10 mL×5), the filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography to obtain 0.50 g of white solid 15-4.

Step 3: Preparation of compound 15-5

200mg of compound 15-4 and 288mg of compound M5 were dissolved in 5mL DMF, 832mg BOP, 480mg DBU were added, and the reaction was stirred at RT for 4hrs. TLC detected the completion of the reaction. The reaction solution was added to 20 mL of water, extracted with EtOAc (20 mL×3), combined the organic phases, washed with saturated NaCl (20 mL×3), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was subjected to column chromatography It was purified to obtain 69 mg of compound 15-5.

Step 4: Preparation of compound 15

69 mg of compound 15-5 was dissolved in 2 mL of MeOH, 0.2 mL of 2N HCl in methanol was added, and the reaction was carried out at RT for 2 hrs. TLC detects the completion of the reaction, concentrates under reduced pressure, adds saturated aqueous NaHCO ₃ to adjust the pH to 7-8, extracts with EtOAc (10 mL×3), combines the organic phases, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was subjected to column chromatography 44 mg of compound 15 was obtained by purification.

[M+H ⁺ ]:502.10.

¹ H NMR (500MHz, DMSO-d ₆ ): δ7.35-7.1 (m, 2H), 7.22-7.15 (m, 4H), 6.72-6.70 (m, 1H), 3.89 (s, 1H), 3.57- 3.51 (m, 2H), 3.29 (s, 3H), 3.09-2.99 (m, 2H), 2.64 (d, J = 15.0 Hz, 1H), 2.53 (d, J = 15.0 Hz, 1H), 1.89-1.86 (m, 1H), 1.81-1.76 (m, 1H), 1.55-1.52 (m, 1H), 1.17-1.14 (m, 1H).

Example 16: Compound (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2 Preparation of -amino-3-chloropyridin-4-yl)thio)-3-methylpyrimidine-4(3H)-one:

Step 1: Preparation of compound 16-2

0.50g compound 16-1 and 1.19g compound M5 were dissolved in 15ML anhydrous N,N-dimethylformamide, and 3.13g benzotriazol-1-yloxy tris (two (Methylamino)phosphonium hexafluorophosphate, 3.78g DBU, stirred and reacted at room temperature for 18 hours. The reaction was completed by LCMS, 25mL of water was added, and then extracted with DCM (20mL×3), the organic layers were combined, washed with saturated brine (20mL×3), dried with anhydrous sodium sulfate and desolventized, and purified by column chromatography (DCM :MeOH=95%-92%) to obtain 0.59 g of compound 16-2.

Step 2: Preparation of compound 16-3

Under the protection of nitrogen, 0.59 g of compound 16-2 was dissolved in 10 mL of anhydrous N,N-dimethylformamide, and under ice bath, 0.39 g of N-iodosuccinimide was added to the above reaction solution. TLC detected that the reaction was complete, 15 mL of water was added, and then extracted with DCM (20 mL×3). The organic layers were combined, washed with saturated brine (10 mL×3), dried with anhydrous sodium sulfate, and the solution was removed, and purified by column chromatography (DCM :MeOH=95%-92%) to obtain 0.42 g of compound 16-3.

Step 3: Preparation of compound 16-4

300 mg of compound 16-3 and 198 mg of compound M1 were dissolved in 5 mL of 1,4-dioxane, 25 mg TMEDA, 344 mg K ₃ PO ₄ and 21 mg CuI were added, and the reaction was stirred at 95° C. for 24 hrs under nitrogen protection. LCMS detected that the reaction was complete, the reaction solution was filtered, concentrated under reduced pressure, and the residue was purified with a preparation plate to obtain 77 mg of white solid compound 16-4.

Step 4: Preparation of compound 16

77 mg of compound 16-4 was dissolved in 3 mL of dioxane, and methanol solution of hydrochloric acid (2N, 0.3 mL) was added dropwise, and the reaction was followed by TLC. After the reaction was completed, the solvent was removed under reduced pressure, and after solidification with a small amount of n-hexane, the n-hexane was poured out, 2.0 mL of water was added to dissolve the solid, and then saturated sodium bicarbonate aqueous solution was added dropwise to adjust pH=9-10, and the solid was precipitated. After filtration, the filter cake was washed with a small amount of water, and dried under vacuum to obtain 33.6 mg of compound 16 as a white solid.

[M+H ⁺ ]=484.17.

¹ H NMR(500MHz,DMSO-d6)δ7.62(d,J=5.4Hz,1H), 7.31(d,J=6.9Hz,1H), 7.19–7.13(m,3H), 6.15(s,2H) ), 5.97(d,J=5.4Hz,1H), 3.86(s,1H), 3.56–3.49(m,2H), 3.25(s,3H), 3.10–2.98(m,3H), 2.61(d, J = 15.6 Hz, 1H), 1.91–1.81 (m, 1H), 1.82–1.76 (m, 1H), 1.54–1.52 (m, 1H), 1.19–1.12 (m, 1H).

Example 17: Preparation of compound 17:

Step 1: Preparation of compound 17-2

Under nitrogen protection, 7.30g compound 17-1 and 6.77g compound M1-2 were dissolved in 100mL dioxane, 1.55g Pd ₂ (dba) ₃ , 1.95g Xantphos and 14.57g DIEA were added, and the temperature was raised to 100°C for reaction 16hrs. TLC detected that the reaction was complete, the reaction solution was concentrated, 100 mL ethyl acetate and 50 mL water layer were added, the organic phase was separated, and the water phase was extracted with 50 mL ethyl acetate. 50 mL (PE:EA=4:1) was beaten to obtain 8.5 g of compound 17-2 as a pale yellow solid, which was directly used in the next step.

Step 2: Preparation of compound 17-3

8.5 g of compound 17-2 was dissolved in 30 mL of dichloromethane, EtONa (16.27 g, 20% EtOH solution) was added dropwise at RT, and the reaction was stirred at RT for 0.5 hrs. TLC detected the completion of the reaction, concentrated under reduced pressure, added 10 mL of dichloromethane and stirred for 30 mins, the reaction solution was filtered, and the filter cake was washed with dichloromethane (15 mL×2) to obtain 4g of compound 17-3 as an off-white solid, which was used directly in the next step .

Step 3: Preparation of compound 17-4

Under the protection of nitrogen, 44 mg of compound 18-2 and 23 mg of compound 17-3 were dissolved in 2 mL of dioxane, 5.8 mg CuI, 7.1 mg TMEDA and 48.8 mg K ₃ PO _{4 were added} , and the temperature was raised to 100° C. to react for 48 hrs. TLC detected that the reaction was complete, concentrated, and the residue was purified by preparative thin-layer chromatography to obtain 8.5 mg of white compound 17-4.

Step 3: Preparation of compound 17

Under a nitrogen atmosphere, dissolve 8.5 mg of compound 17-4 in 1.5 mL of dioxane and 0.5 mL of MeOH, add hydrochloric acid and methanol solution (2N, 0.3 mL), stir and react at RT for 1 hrs. TLC detects that the reaction is complete and the reaction solution is reduced. Pressure concentration. Add H ₂ O (2 mL) to the residue to dissolve, adjust the solution to pH=8 with saturated NaHCO ₃ , solid precipitate, filter, wash the filter cake with H ₂ O (2 mL), take the filter cake and vacuum dry to obtain 2.8 mg of off-white solid compound 17.

[M+H ⁺ ]=469.25.

¹ H NMR (500MHz, DMSO-d ₆ ) δ 7.88 (d, J = 5.4 Hz, 1H), 7.21-7.18 (m, 1H), 7.10 (d, J = 9.0 Hz, 1H), 6.97-6.94 ( m,1H),6.54(s,2H),6.04(d,J=5.4Hz,1H),3.88(s,1H),3.56–3.46(m,2H),3.28(s,3H),3.07–2.93 (m,3H),2.63(d,J=15.6Hz,1H),1.92–1.87(m,1H),1.80–1.75(m,1H)1.56–1.51(m,1H),1.14–1.12(m, 1H).

Preparation of Intermediate Compound 18-2:

The synthesis of compound 18-2 refers to the preparation method of compound 16-3.

Preparation of intermediate compound 19-6:

Step 1: Preparation of compound 19-3

Dissolve 33.00g of compound 19-1 in 400mL dry THF, replace with nitrogen three times, cool to -78°C, slowly add LDA solution in THF (64.00mL, 2.0M) dropwise, and then react at -78°C for 1 hr. -2 in THF (50 mL) was slowly added dropwise to the above reaction solution, reacted at -78°C for 30 mins, and slowly raised to room temperature for 18 hrs. The reaction was completed by TLC detection. The reaction was quenched with 200 mL saturated NH ₄ Cl solution, 150 mL water was added, and EtOAc was added for extraction (200 mL×3). The organic phases were combined, washed with 200 mL saturated NaCl, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was purified by column chromatography to obtain 34.31 g of colorless oil 19-3.

Step 2: Preparation of compound 19-4

34.31g of compound 19-3 was dissolved in dioxane/MeOH (400mL/160mL), NaOH aqueous solution (80mL, 6N) was added, and the reaction was heated to 100°C for 18hrs. The reaction was completed by TLC detection, the reaction solution was cooled to room temperature, concentrated under reduced pressure to remove the organic solvent, the residue was adjusted to pH 3-4 with concentrated hydrochloric acid, filtered, the filter cake was washed with water (80 mL×3), and the filter cake was vacuum dried 28.00 g of compound 19-4 was obtained as a white solid.

Step 3: Preparation of compound 19-5

28.00 g of compound 19-4 was dissolved in 200 mL of PPA, and the temperature was raised to 120° C. for 0.5 hrs. TLC detected that the reaction was complete. The reaction solution was slowly added dropwise to 400 mL crushed ice, adjusted to pH 9-10 with 2.4N NaOH aqueous solution, 23.30 g (Boc) ₂ O was added, and the reaction was stirred at RT for 1 hrs. TLC detected the completion of the reaction, extracted with EtOAc (150 mL×3), combined the organic phases, washed with 200 mL saturated NaCl, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 8.50 g of white solid 19-5 and 900mg white solid 20-1.

Step 4: Preparation of compound 19-6

The procedure for preparing compound 19-6 from compound 19-5 is similar to the procedure from compound M5-3 to compound M5.

The procedure for preparing compound 20-2 from compound 20-1 is similar to the procedure from compound M5-3 to compound M5

Using different reaction starting materials and appropriate reagents, the compound 18-compound 20 and compound 25-compound 27 in Table 4 were prepared by a method similar to that in the foregoing Example 16.

Table 4

The NMR data of compound 18-compound 20 and compound 25-compound 27 are as follows:

¹ H NMR(500MHz,DMSO-d6)δ7.62(d,J=5.4Hz,1H), 7.22-7.19(m,1H), 7.11(d,J=9.0Hz,1H), 6.99-6.95(m ,1H),6.15(s,2H),5.97(d,J=5.4Hz,1H),3.91(s,1H),3.57-3.50(m,2H),3.28(s,3H),3.03-2.97( m,3H), 2.60(d,J=15.6Hz,1H),1.92-1.88(m,1H),1.80-1.76(m1H),1.56-1.54(m,1H),1.16-1.14(m,1H) .(Compound 18)

¹ H NMR(500MHz,DMSO-d6)δ7.62(d,J=5.4Hz,1H), 7.31(d,J=6.9Hz,1H), 7.02–6.96(m,2H), 6.15(s,2H) ),5.97(d,J=5.4Hz,1H),3.82(s,1H),3.55–3.49(m,2H), 3.28(s,3H),3.06–2.97(m,3H), 2.61(d, J = 16.1 Hz, 1H), 1.89–1.83 (m, 1H), 1.81–1.76 (m, 1H), 1.55–1.52 (m, 1H), 1.16–1.13 (m, 1H). (Compound 19)

¹ H NMR (500MHz, DMSO-d6) δ 7.63 (d, J = 5.2 Hz, 1H), 7.22 (d, J = 7.0 Hz, 1H), 7.05-7.03 (m, 1H), 6.96-6.93 (m ,1H), 6.16(s,2H),5.97(d,J=5.2Hz,1H),4.08(s,1H),3.49–3.39(m,2H),3.28(s,3H),3.19–3.08( m, 3H), 2.76 (d, J = 16.0 Hz, 1H), 2.02–1.90 (m, 2H), 1.62–1.59 (m, 1H), 1.44–1.41 (m, 1H). (Compound 20)

¹ H NMR(500MHz,DMSO-d6)δ7.62(d,J=5.4Hz,1H), 7.08–7.02(m,2H), 5.97(d,J=5.4Hz,1H), 4.10(s,1H) ), 3.51–3.38(m,2H), 3.28(s,3H), 3.20–3.12(m,2H), 2.94(d,J=15.6Hz,1H), 2.80(d,J=15.6Hz,1H) , 2.03–1.88(m,1H), 1.62–1.57(m,1H), 1.47–1.44(m,1H), 1.24–1.19(m,1H). (Compound 25)

¹ H NMR(500MHz,DMSO-d6)δ7.62(d,J=5.3Hz,1H), 7.33(s,1H), 7.21-7.17(m,2H), 6.14(s,2H), 5.97(d ,J=5.3Hz,1H),3.87(s,1H),3.56–3.49(m,2H), 3.28(s,3H),3.06–2.96(m,3H),2.58(d,J=15.7Hz, 1H), 1.92–1.87(m,1H), 1.79–1.74(m,1H), 1.56–1.53(m,1H), 1.12–1.09(m,1H). (Compound 26)

¹ H NMR(500MHz,DMSO-d ₆ )δ7.88(d,J=5.2Hz,1H), 7.32(d,J=6.9Hz,1H), 7.19-7.13(m,3H), 6.53(s, 2H), 6.05(d, J=5.3Hz, 1H), 3.87(s, 1H), 3.55-3.51(m, 2H), 3.29(s, 3H), 3.09–2.96(m, 3H), 2.61(d ,J=15.7Hz,1H),1.91-1.86(m,1H),1.81-1.76(m,1H),1.54-1.52(m,1H),1.16-1.13(m,1H). (Compound 27)

Control example

According to the method described in EXAMPLE 48 in WO2018172984, the following comparative examples were prepared.

Pharmacological test

Example A: SHP2 allosteric inhibition enzyme activity assay

SHP2 is allosterically activated by the binding of a bis-tyrosyl-phosphorylated peptide to its Src homology 2 (SH2) domain. The subsequent activation step results in the release of the SHP2 auto-inhibitory interface, which in turn activates the SHP2 protein tyrosine phosphatase (PTP) and can be used for substrate recognition and reaction catalysis. The surrogate DiFMUP was used to monitor the catalytic activity of SHP2 in the rapid fluorescence assay format.

experiment procedure:

(1) Compound preparation:

The compound of the present invention (10mM stock solution) was diluted to an appropriate multiple with 100% DMSO. The final test concentration of the compound of the present invention was 10μM, 3.3333μM, 1.1111μM, 0.3704μM, 0.1235μM, 0.0412μM, 0.0137μM, 0.0046μM, 0.0015μM , 0.00μM;

(2) Prepare enzyme reaction working solution:

In a 96-well black polystyrene plate (flat bottom, low flange, non-binding surface) (PE, Cat#6005270) at room temperature, use a final reaction volume of 50 μL and the following assay buffer conditions for phosphatase activity detection: 60mM HEPES, 75mM NaCl, 75mM KCl, 0.05% BRIJ-35, 1mM EDTA, 5mM DTT.

(3) Enzyme catalytic reaction and data monitoring:

Take 5 μL of the compound of the present invention and add it to the corresponding 96-well plate, and set the blank test well without adding the compound or enzyme and only adding buffer. Put SHP2 Activating Peptide (IRS1_pY1172(dPEG8)pY1222) on ice to melt, add 25uM to each well, then add 0.2ng SHP2 protein sample to the corresponding well plate, and incubate at room temperature for 1 hour. The substitution substrate DiFMUP (Invitrogen, Cat#D6567) was added to the reaction, and the reaction was carried out at room temperature for 2 hours. The fluorescence signal was monitored by a microplate reader (Envision, Perki Elmer) with excitation wavelength and emission wavelength of 340nm and 450nm, respectively.

(4) Data analysis:

Calculation formula:

Inhibition rate%=[1-(Conversion_ _sample -Conversion_ _min )/(Conversion_ _max -Conversion_ _min )]×100%

Among them: Conversion_sample is the conversion rate reading of the sample; Conversion_min is the average value of the blank control well, representing the conversion rate reading of the wells without enzyme activity; Conversion_max is the average value of the positive control well ratio, representing the conversion rate reading of the wells without compound inhibition. The analysis software GraphPad Prism log (inhibitor) vs. response-Variable slope was used to fit the dose-response curve, and the IC ₅₀ value of the compound to the enzyme activity was calculated.

The IC ₅₀ data of some examples are shown in Table 3.

table 3

The compound of the present invention has an allosteric inhibitory effect on SHP2 phosphatase.

Example B: Cell Proliferation Test

An in vitro cell assay was used to evaluate the effects of the compounds of the present invention on the proliferation of lung squamous cell carcinoma KYSE-520 cells and leukemia cells MV-4-11 cells. The detection method used in the experiment is the CELL TITER-GLO (CTG) luminescence method, which can detect the number of living cells by quantitatively measuring ATP. Because ATP participates in a variety of enzymatic reactions in organisms, it is an indicator of living cell metabolism. Its content directly reflects the number and cell state of cells. During the experiment, CellTiter-Glo ^TM reagent was added to the cell culture medium to measure the luminescence value. The value is directly proportional to the amount of ATP, and ATP is positively related to the number of living cells, so cell viability can be inspected by detecting ATP content.

experiment procedure:

(1) Cell plating:

Take a bottle of KYSE-520 cells in logarithmic growth phase, digest the resuspended cells, count them, adjust the cell density and inoculate them into a 96-well plate, inoculate 1000 cells per well, and place the well plate at 37℃, 5% CO ₂ After culturing in an incubator for 24hrs, adding the compound of the present invention for treatment;

Take a bottle of MV-4-11 cells in logarithmic growth phase, digest and resuspend the cells, count them, adjust the cell density and inoculate them in a 96-well plate, inoculate 4000 cells per well, and place the plate at 37°C, 5% CO After culturing in the incubator ₂ for 24 hrs, the compound of the present invention was added for treatment.

(2) Cell compound treatment:

Prepare an appropriate amount of the compound of the present invention for cell treatment, and the final concentration of the compound from high to low is 1000nM, 333.3nM, 111.1nM, 37.04nM, 12.35nM, 4.115nM, 1.372nM, 0.4572nM, 0.1524nM, 0nM. Enter 37°C, 5% CO ₂ incubator for 120hrs. Only adding medium without adding cell wells is set as the zero adjustment group; the compound concentration of 0 nM group is the blank group.

(3) CTG detection:

After cell culture for 120hrs, add 50μL to each well

Luminescent Cell Viability Assay solution, gently shake for 2mins, continue to incubate at room temperature for 10mins, and read the detection value of each well on the multifunctional microplate reader.

(4) Data analysis:

Calculate the inhibition rate based on the luminous value reading,

Inhibition rate%=(1-(administration group value-zero adjustment group value)/(blank group value-zero adjustment group value)*100

GraphPad Prism the log (inhibitor) vs.response-Variable slope fitting dose-response curve and calculate IC ₅₀ of compounds to inhibit cell proliferation. The experimental data of some example compounds are shown in Table 5.

table 5

The compound of the present invention has a good inhibitory effect on the proliferation of KYSE-520 cells and the proliferation of MV-4-11 cells.

Example C: Inhibition test of hERG potassium ion channel

The whole cell patch clamp technique was used to detect the blocking effect of the test compound on the hERG channel.

Cell culture

The HEK293 cell line stably expressing the hERG potassium channel was cultured in DMEM medium containing 10% fetal bovine serum and 0.8 mg/mL G418 at a culture temperature of 37°C and a carbon dioxide concentration of 5%.

Cell passage: Remove the old medium and wash once with PBS, then add 1mL TrypLE ^TM Express solution and incubate at 37°C for 0.5 minutes. When the cells detach from the bottom of the dish, add 5 mL of complete medium pre-warmed at 37°C. The cell suspension was gently pipetted to separate the aggregated cells. The cell suspension was transferred to a sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes to collect the cells. To expand or maintain the culture, inoculate the cells in a 6 cm cell culture dish, each cell culture dish, the amount of cells inoculated is 2.5*105 cells (final volume: 5mL).

In order to maintain the electrophysiological activity of cells, the cell density must not exceed 80%.

For patch clamp detection, the cells were separated with TrypLE ^TM Express before the experiment, and 3*103 cells were spread on a cover glass and cultured in a 24-well plate (final volume: 500 μL). After 18 hours, the experiment was performed.

Intracellular fluid

Extracellular fluid: 140mM NaCl, 3.5mM KCl, 1mM MgCl ₂ ·6H ₂ O, 2mM CaCl ₂ , 10mM D-glucose, 10mM HEPES, 1.25mM NaH ₂ PO ₄ , NaOH to adjust pH=7.4.

Intracellular fluid: 20mM KCl, 115mM K-Aspartic, 1mM MgCl ₂ ·6H ₂ O, 5mM EGTA, 10mM HEPES, 2mM Na ₂ -ATP, KOH to adjust pH=7.2.

Compound formulation

Dilute the test compound stock solution with extracellular fluid to prepare a 10 μM working solution, or a gradient dilution to a solution of 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM.

The solubility of the test compound was visually checked, and the test compound was completely dissolved without visible precipitation.

Cisapride (positive control)

The weighed 1.2mg cisap must be prepared into a 10mM stock solution using 243μL DMSO.

The cisapride stock solution was diluted successively with DMSO at a 10-fold dilution from high to low to a 10μM dilution.

Dilute the 10 μM cisapride diluent with extracellular fluid to prepare a 10 nM working solution.

Visually inspecting the solubility of cisapride, cisapride was completely dissolved without visible precipitation.

Experimental method reference:

The whole cell patch clamp voltage stimulation scheme for recording the whole cell hERG potassium current is as follows: when the whole cell seal is formed, the cell membrane voltage is clamped to -80mV. The clamping voltage is depolarized from -80mV to -50mV for 0.5 seconds, then stepped to 30mV for 2.5 seconds, and then quickly restored to -50mV for 4 seconds to stimulate the tail current of the hERG channel. Collect data repeatedly every 10 seconds to observe the effect of the drug on the hERG tail current. A stimulus of -50mV for 0.5 seconds was used as leakage current detection. The experimental data is collected by EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA) software.

The capillary glass tube is drawn into a recording electrode with a microelectrode drawing instrument. Operate the microelectrode manipulator under the inverted microscope to contact the recording electrode on the cell, and apply negative pressure suction to form a GΩ seal. After forming the GΩ seal, perform fast capacitance compensation, and then continue to give negative pressure to suck and break the cell membrane to form a whole-cell recording mode. Then perform slow capacitance compensation and record the film capacitance and series resistance. No leakage compensation is given.

When the hERG current recorded by the whole cell stabilizes, the drug is administered, and each drug concentration acts for 5 minutes (or the current stabilizes). Place the cover glass covered with cells in the recording bath in the inverted microscope. The test compound and the compound-free external fluid flow through the recording bath sequentially by gravity perfusion to act on the cells, and the vacuum pump is used to carry out the liquid during recording. exchange. The current detected by each cell in the compound-free external fluid serves as its own control group. Test multiple cells independently and repeatedly. All electrophysiological experiments were performed at room temperature.

Data quality standards

The following criteria are used to determine whether the data is acceptable:

(1) Series resistance ≤20MΩ

(2) Sealing resistance≥1GΩ

(3) Peak tail current peak ≥400pA

(4) The initial tail current peak is greater than the activation current peak

(5) There is no obvious spontaneous attenuation of tail current (the spontaneous attenuation is less than 5% within 5 minutes)

(6) There is no obvious leakage current under the membrane potential of -80mV (leakage current ≤100pA)

data analysis

First, the current after each drug concentration is normalized to the blank control current

Then calculate the inhibition rate corresponding to each drug concentration

Calculate the mean and standard error for each concentration, and use the following equation to calculate the half inhibitory concentration of each compound:

Use the above equation to perform a non-linear fitting of the dose-dependent effect, where c represents the drug concentration, IC50 is the half-inhibitory concentration, and h represents the Hill coefficient. The curve fitting and IC ₅₀ calculation are done using IGOR software.

See Table 6 for the hERG test results of the example compounds.

Table 6

编号Numbering	抑制率@10μMInhibition rate @10μM	IC ₅₀ IC ₅₀
对照例Control example	95.12％95.12%	1.94μM1.94μM
化合物1Compound 1	73.30％73.30%	5μM5μM
化合物2Compound 2	65.08％65.08%	6.9μM6.9μM
化合物3Compound 3	84.54％84.54%	//
化合物5Compound 5	99.87％99.87%	//
化合物9Compound 9	36.41％36.41%	//
化合物10Compound 10	48.50％48.50%	//
化合物11Compound 11	20.53％20.53%	//
化合物13Compound 13	42.63％42.63%	//
化合物15Compound 15	88.92％88.92%	//
化合物16Compound 16	25.53％25.53%	>30μM>30μM
化合物24Compound 24	54.50％54.50%	13.6μM13.6μM

Note: "/" means not tested.

We unexpectedly found that while the compound of the present invention generally has better inhibitory activity on SHP2, when the position of A ₃ in the general formula I is N instead of C, for example, the specific embodiment of the present invention, compound 1 , Compound 2, Compound 3, Compound 9, Compound 10, Compound 11, Compound 13, Compound 16, Compound 24 have a significant improvement effect on hERG.

Although the present invention has been fully described through its embodiments, it is worth noting that various changes and modifications are obvious to those skilled in the art. Such changes and modifications should be included in the scope of the appended claims of the present invention.

Claims

The compound represented by formula I, or a pharmaceutically acceptable salt, tautomer, solvate, chelate, non-covalent complex or prodrug thereof,

among them,

R 1 is selected from hydrogen, hydroxy, C 1-8 alkyl, C 1-8 alkyl-containing substituent group, C 1-8 alkoxy, C 1-8 alkoxy group-containing substituent group, C 2-8 Alkenyl, substituted C 2-8 alkenyl, C 2-8 alkynyl or substituted C 2-8 alkynyl;

R 2 is selected from hydrogen, C 1-4 alkyl group or a substituted group containing C 1-4 alkyl;

R 3 is selected from hydrogen, amino, -C (O) NH 2, -C≡N, hydroxy, C 1-8 alkyl, C 1-8 alkyl-containing substituent group, C 1-8 alkoxy or an C 1-8 alkoxy of the substituent;

R 4 is selected from hydrogen, halo, amino, amido, -C≡N, carboxyl, hydroxyl, hydroxymethyl, C 1-8 alkyl, C 1-8 alkyl-containing substituent group, C 1-8 alkoxy group, a substituted group containing C 1-8 alkoxy, C 2-8 alkenyl group, a substituted group containing C 2-8 alkenyl, C 2-8 alkynyl group or a substituted group containing C 2-8 alkynyl;

A 1 is arbitrarily selected from CR 5 or N;

A 2 is arbitrarily selected from CR 6 or N;

A 3 is arbitrarily selected from CR 7 or N;

U is arbitrarily selected from C(R 8 ) 2 , O or NR 9 ;

Wherein, R 5 , R 6 , R 7 , R 8 or R 9 are independently selected from hydrogen, halogen, amino, C 1-8 alkyl, substituted C 1-8 alkyl, C 1-8 alkoxy group, a substituted group containing C 1-8 alkoxy, C 2-8 alkenyl group, a substituted group containing C 2-8 alkenyl, C 2-8 alkynyl group or a substituted group containing C 2-8 alkynyl;

Ring A is optionally selected from a C 6-10 aryl group or a C 5-10 heteroaryl group, the C 5-10 heteroaryl group contains one or two N or S heteroatoms;

Rx is optionally selected from hydrogen, halogen, amino, substituted amino, sulfonyl, C 1-8 alkyl, substituted C 1-8 alkyl, C 1-8 alkoxy, substituted C 1-8 alkoxy, C 3-8 cycloalkyl or substituted C 3-8 cycloalkyl;

n is 0, 1, 2, 3, or 4.
The compound of claim 1, wherein A 3 is selected from N.
The compound of claim 1 or 2, wherein R 1 is selected from C 1-3 alkyl.
The compound of any one of claims 1-3, wherein R 1 is methyl.
The compound according to any one of claims 1-4, wherein R 2 is selected from hydrogen or C 1-3 alkyl.
The compound of any one of claims 1-5, wherein R 2 is hydrogen.
The compound according to any one of claims 1-6, wherein R 3 is selected from hydrogen, amino or C 1-3 alkyl.
The compound of any one of claims 1-7, wherein R 3 is hydrogen or amino.
The compound according to any one of claims 1-8, wherein, R 4 is selected from hydrogen, halo, amino, C 1-3 alkyl group or an unsubstituted C 1-3 alkyl.
The compound according to any one of claims 1-9, wherein R 4 is hydrogen, fluorine or chlorine.
The compound according to any one of claims 1-10, wherein R 4 is hydrogen or chlorine.
The compound according to any one of claims 1-11, wherein A 1 is selected from CR 5 or N, wherein R 5 is selected from halogen or halogen-substituted C 1-3 alkyl.
The compound according to any one of claims 1-12, wherein A 1 is selected from CR 5 or N, wherein R 5 is selected from F, Cl or trifluoromethyl.
The compound according to any one of claims 1-13, wherein A 1 is selected from CR 5 or N, wherein R 5 is selected from Cl or trifluoromethyl.
The compound according to any one of claims 1-14, wherein A 2 is selected from CR 6 or N, wherein R 6 is selected from amino or C 1-3 alkoxy.
The compound according to any one of claims 1-15, wherein A 2 is selected from CR 6 or N, wherein R 6 is selected from amino or methoxy.
The compound according to any one of claims 1-16, wherein U is selected from CH 2 or O.
The compound according to any one of claims 1-17, wherein the ring A is selected from a phenyl group or a C 5-6 heteroaryl group, and the C 5-10 heteroaryl group contains one or two N or S Heteroatom.
The compound according to any one of claims 1-18, wherein ring A is selected from phenyl,
The compound according to any one of claims 1-19, wherein:
Arbitrarily selected from
The compound according to any one of claims 1-20, wherein:
Arbitrarily selected from
The compound according to claim 1 or a pharmaceutically acceptable salt, tautomer, solvate, chelate, non-covalent complex or prodrug thereof, wherein the compound has formula II The structure shown,

among them,

R 1 is selected from C 1-8 alkyl;

R 2 is selected from hydrogen;

R 3 is selected from H or amino;

R 4 is selected from hydrogen or halogen;

A 1 is selected from CR 5 ;

A 2 is selected from CR 6 ;

U is selected from C(R 8 ) 2 ;

Wherein, R 5, R 6, R 8 are independently selected from hydrogen, halo, C 1-8 alkyl, substituted group containing C 1-8 alkyl;

Ring A is optionally selected from C 6-10 aryl groups.
The compound according to claim 22, wherein ring A is optionally substituted with hydrogen or C 1-8 alkoxy.
The compound according to claim 22 or 23, wherein ring A is optionally substituted with hydrogen or methoxy.
The compound of any one of claims 22-24, wherein R 1 is methyl.
The compound of any one of claims 22-25, wherein R 4 is hydrogen.
The compound according to any one of claims 22-26, wherein U is selected from CH 2 .
The compound according to any one of claims 22-27, wherein R 5 is selected from Cl.
The compound according to any one of claims 22-28, wherein R 6 is selected from NH 2 .
The compound according to any one of claims 22-29, wherein ring A is selected from phenyl.
A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

(1) (S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino-3-chloro (Pyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(2) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-methoxy-1,3-dihydrospiro(indene -2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(3) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-chloro-1,3-dihydrospiro(indene-2 ,4'-Piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(4)(S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((3-chloro-2-methyl (Oxypyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(5)(S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2,3-dichlorobenzene (B)thio)-3-methylpyrimidin-4(3H)-one;

(6)(S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-3-methyl-5-((2- (Trifluoromethyl)pyridin-3-yl)thio)pyrimidin-4(3H)-one;

(7) (S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((6-amino-3-chloro (Pyridin-2-yl)thio)-3-methylpyrimidin-4(3H)-one;

(8) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-4-bromo-1,3-dihydrospiro(indene-2 ,4'-Piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(9)(S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-(trifluoromethyl)-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(10) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-4-methoxy-1,3-dihydrospiro(indene -2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(11) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(5-amino-5,7-dihydrospiro[cyclopenta[b ]Pyridine-6,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(12) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-5,6-dimethoxy-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(13)(S)-2-(4-Amino-2-chloro-4,6-dihydrospiro[cyclopenta[d]thiazole-5,4'-piperidine]-1'-yl) -5-((2-amino-3-chloropyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(14) (S)-2-(5-Amino-2-methoxy-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'-piperidine]-1'- Yl)-5-((2-amino-3-chloropyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(15) (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2,3 -Dichlorophenyl)thio)-3-methylpyrimidin-4(3H)-one;

(16) (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino -3-Chloropyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(17) (S)-6-amino-2-(1-amino-6-fluoro-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5- ((2-Aminopyrimidin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(18)(S)-6-amino-5-((2-amino-3-chloropyridine-4-yl)thio)-2-(1-amino-6-fluoro-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(19) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-5-fluoro-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(20) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-7-fluoro-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(24)(S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino-3-fluoro Pyridin-4-yl)thio)-3-methylpyrimidine-4(3H)-one;

(25) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-4,7-difluoro-1,3- Dihydrospiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(26) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-chloro-1,3-dihydrospiro [Indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one; or

(27) (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino Pyrimidine-4-yl)thio)-3-methylpyrimidin-4(3H)-one.
A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

(1) (S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino-3-chloro (Pyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(2) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-methoxy-1,3-dihydrospiro(indene -2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(3) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-chloro-1,3-dihydrospiro(indene-2 ,4'-Piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(4)(S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((3-chloro-2-methyl (Oxypyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(8) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-4-bromo-1,3-dihydrospiro(indene-2 ,4'-Piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(9)(S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-(trifluoromethyl)-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(10) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-4-methoxy-1,3-dihydrospiro(indene -2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(11) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(5-amino-5,7-dihydrospiro[cyclopenta[b ]Pyridine-6,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(12) (S)-5-((2-Amino-3-chloropyridin-4-yl)thio)-2-(1-amino-5,6-dimethoxy-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(13)(S)-2-(4-Amino-2-chloro-4,6-dihydrospiro[cyclopenta[d]thiazole-5,4'-piperidine]-1'-yl) -5-((2-amino-3-chloropyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(14) (S)-2-(5-Amino-2-methoxy-5,7-dihydrospiro[cyclopenta[b]pyridine-6,4'-piperidine]-1'- Yl)-5-((2-amino-3-chloropyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(16) (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino -3-Chloropyridin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(17) (S)-6-amino-2-(1-amino-6-fluoro-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5- ((2-Aminopyrimidin-4-yl)thio)-3-methylpyrimidin-4(3H)-one;

(18)(S)-6-amino-5-((2-amino-3-chloropyridine-4-yl)thio)-2-(1-amino-6-fluoro-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(19) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-5-fluoro-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(20) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-7-fluoro-1,3-dihydro Spiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(24)(S)-2-(1-Amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino-3-fluoro Pyridin-4-yl)thio)-3-methylpyrimidine-4(3H)-one;

(25) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-4,7-difluoro-1,3- Dihydrospiro[indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one;

(26) (S)-6-amino-5-((2-amino-3-chloropyridin-4-yl)thio)-2-(1-amino-6-chloro-1,3-dihydrospiro [Indene-2,4'-piperidine]-1'-yl)-3-methylpyrimidin-4(3H)-one; or

(27) (S)-6-amino-2-(1-amino-1,3-dihydrospiro[indene-2,4'-piperidine]-1'-yl)-5-((2-amino Pyrimidine-4-yl)thio)-3-methylpyrimidin-4(3H)-one.
A pharmaceutical composition characterized by comprising a therapeutically effective amount of at least one compound according to any one of claims 1-32 and at least one pharmaceutically acceptable excipient.
The pharmaceutical composition according to claim 33, wherein the mass percentage of the compound and pharmaceutically acceptable excipients is 0.0001:1-10.
Use of the compound of any one of claims 1-34 or the pharmaceutical composition of claim 33 or 34 in the preparation of medicines.
The application according to claim 35, wherein the medicine is used to treat, prevent, delay or stop cancer, cancer metastasis, cardiovascular disease, immune disease, fibrosis or eye disease.
Use of the compound of any one of claims 1-34 or the pharmaceutical composition of claim 33 or 34 in the preparation of a medicine for treating diseases mediated by SHP2.
The use according to claim 37, wherein the disease is cancer.
The use according to claim 37 or 38, wherein the cancer is selected from Noonan syndrome, leopard spot syndrome, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, head and neck squamous cell Cancer, acute myeloid leukemia, breast cancer, esophageal tumor, lung cancer, colon cancer, head cancer, gastric cancer, lymphoma, glioblastoma, gastric cancer, pancreatic cancer or a combination thereof.
The use according to claim 39, wherein the drug is used as an SHP2 inhibitor.
A method for treating and/or preventing diseases mediated by SHP2, characterized in that a therapeutically effective amount of the compound according to any one of claims 1-32 or the drug according to claim 33 or 34 is administered to a subject to be treated combination.
The method of claim 41, wherein the SHP2-mediated disease is cancer.
The method according to claim 42, wherein the cancer is selected from Noonan syndrome, leopard spot syndrome, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, head and neck squamous cell carcinoma, Acute myeloid leukemia, breast cancer, esophageal tumor, lung cancer, colon cancer, head cancer, gastric cancer, lymphoma, glioblastoma, gastric cancer, pancreatic cancer or a combination thereof.
A method for treating cancer, comprising administering a therapeutically effective amount of the compound of any one of claims 1-32 or the pharmaceutical composition of claim 33 or 34 to a subject, characterized in that the cancer is Noonan Syndrome, leopard spot syndrome, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, head and neck squamous cell carcinoma, acute myeloid leukemia, breast cancer, esophageal tumor, lung cancer, colon cancer, head cancer, Gastric cancer, lymphoma, glioblastoma, gastric cancer, pancreatic cancer, or a combination thereof.
The method according to any one of claims 41-44, wherein the treatment subject is a human.