CN111646995B

CN111646995B - 4-amino-pyrimidoazenitrogen heterocycle-phenylurea derivative and preparation method and application thereof

Info

Publication number: CN111646995B
Application number: CN202010123847.4A
Authority: CN
Inventors: 陈俐娟
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2019-03-04
Filing date: 2020-02-27
Publication date: 2023-03-21
Anticipated expiration: 2040-02-27
Also published as: CN111646995A

Abstract

The invention belongs to the field of chemical medicine, and particularly relates to a 4-amino-pyrimidoazepine-phenylurea derivative, and a preparation method and application thereof. The invention provides a 4-amino-pyrimidoazepine-phenylurea derivative, the structural formula of which is shown in formula I. In addition, the invention also provides a preparation method and application of the 4-amino-pyrimidoazepine-phenylurea derivative. The 4-amino-pyrimidoazepine-phenylurea derivative provided by the invention can be used as a FLT3 kinase inhibitor, has a good effect, and provides a new choice for preparing antitumor drugs.

Description

4-amino-pyrimidoazenitrogen heterocycle-phenylurea derivative and preparation method and application thereof

Technical Field

The invention belongs to the field of chemical medicine, and particularly relates to a 4-amino-pyrimidoazepine-phenylurea derivative, and a preparation method and application thereof.

Background

Leukemia is an abnormal clonal disease of hematopoietic stem cells, is arrested in different stages of cell development and malignant proliferation because of the loss of the ability of the leukemia cells to differentiate into mature functional blood cells, and the leukemia cells are proliferated and accumulated in large quantity in bone marrow and other hematopoietic tissues and infiltrate into other organs and tissues, so that normal hematopoiesis is inhibited, and symptoms such as anemia, hemorrhage, infection, infiltration of various organs and the like are clinically shown. Leukemia belongs to cell heterogeneous malignant tumor, has various types, complex etiology and different clinical manifestations, and some leukemias have the characteristics of quick onset, high mortality, short survival period, easy relapse, poor prognosis and extremely difficult cure.

Protein kinases, the family of receptor protein tyrosine kinases in particular, which catalyze the phosphorylation of hydroxyl groups on tyrosine, serine and threonine residues of proteins, play an important role as growth factor receptors in the control of many signal transduction pathways responsible for cellular functions, such as cell cycle, cell growth, cell differentiation and cell death. Dysregulation of receptor tyrosine kinases is often found in diseases such as proliferative disorders, inflammatory disorders, and immune system disorders.

FLT3 (Fms-like tyrosine kinase 3) is an Fms-like tyrosine kinase 3, which belongs to a member of the type III receptor tyrosine kinase (RTK III) family together with c-Kit, c-Fms and PDGFR, and the structure of FLT3 includes an extracellular domain consisting of 5 immunoglobulin-like molecules, a transmembrane domain and an intracellular tyrosine kinase domain. FLT3 is expressed predominantly on the cell surface of normal hematopoietic stem and progenitor cells, and its ligand is expressed predominantly in bone marrow stromal cells. When ligand is combined with the membrane outer structure domain of FLT3, the ligand promotes dimerization of FLT3 receptor, and simultaneously, the tyrosine kinase domain in the cell membrane generates autophosphorylation, and a series of downstream signal transduction pathways, such as Ras/MAPK, PI3K/Akt/mTOR and STAT5, are activated, so that the proliferation and differentiation of cells are regulated. FLT3 mutation usually leads to its abnormal activation, and in the absence of ligand binding, autophosphorylation activates downstream signaling pathways, leading to abnormal proliferation of hematopoietic cells and lymphocytes, leading to various hematological malignancies.

It has been demonstrated that there are two major activating mutations for FLT 3: point mutations in the activation loop in the internal tandem repeat (ITD) and kinase domain (TKD). FLT3-ITD mutations occur in about 25% of acute myeloid leukemia patients and are associated with some adverse prognosis, while FLT3-TKD mutations occur in about 5% of acute myeloid leukemia patients.

Numerous studies have shown that FLT3 mutation is one of the most common molecular genetic abnormalities and poor prognosis factors in AML, and its transduction involves multiple signaling pathways, making FLT3 an ideal drug target. In previous studies, more than 20 compounds have shown inhibition of FLT3 tyrosine kinase. Many have entered clinical trials. These small molecule tyrosinase inhibitors are mostly heterocyclic purine analogs, are ATP analogs, or intermediates with a structure similar to tyrosine covalently bound to ATP. FLT3 receptor is still expressed, but the ATP pathway is blocked, thus autophosphorylation and sustained phosphorylation of the substrate is terminated, blocking the signaling pathway of FLT3-ITD dependent cell lines, exerting a cytotoxic effect.

At present, research on FLT3-ITD and FLT3-TKD inhibitors belongs to a hotspot of drug development, a single-target FLT3 inhibitor AC220 is currently subjected to clinical three-stage tests and is expected to be on the market, but drug resistance in the later treatment stage becomes a problem to be solved urgently. Therefore, the development of inhibitors effective against both mutations is the direction of development.

Disclosure of Invention

In order to solve the problems, the invention provides a 4-amino-pyrimidoazepine-phenylurea derivative, the structural formula of which is shown as formula I:

wherein X is N or C;

R ₁ is-H, -OH, halogen, C1-C10 alkoxy, C1-C10 haloalkoxy,

C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl,

Or substituted or unsubstituted C1-C10 alkyl; the substituent of the substituted C1-C10 alkyl is-H,

-CN, -OH, phenyl, halogen, C1-C8 alkoxy, C3-C8 cycloalkyl, C1-C8 carbonyl or substituted or unsubstituted 3-to 6-membered heterocycloalkyl; the heteroatom of the 3-6 membered heterocycloalkyl is N, O or S, and the number of the heteroatoms is 1-3; the substituent of the substituted 3-to 6-membered heterocycloalkyl is-H, -OH, halogen, C1-C8 oxycarbonyl, C1-C8 alkyl, C1-C8 alkoxy or

R ₄ is-H, -OH, halogen, C1-C8 alkyl, C1-C8 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C8 alkyl;

R ₂ is-H, -OH, halogen, C1-C10 alkyl, C3-C10 cycloalkyl, C1-C10 haloalkyl, amino-substituted C1-C10 alkyl, benzyl, substituted or unsubstituted C5-C10 aryl or substituted or unsubstituted 5-to 10-membered heteroaryl; the heteroatom of the 5-to 10-membered heteroaryl is N, O or S, and the number of the heteroatoms is 1-3; the substituent of the substituted C5-C10 aryl or 5-10 membered heteroaryl is-H, halogen or-NH ₂ C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl, C1-C8 alkoxy, C1-C8 haloalkoxy, -OH or C1-C8 carbonyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C10 alkyl.

As a preferred embodiment of the present invention, X is N or C; r ₁ is-H, -OH, halogen, C1-C8 alkoxy, C1-C8 haloalkoxy,

C2-C8 alkenyl, C2-C8 alkynylC3-C8 cycloalkyl,

Or substituted or unsubstituted C1-C8 alkyl; the substituent of the substituted C1-C8 alkyl is-H,

-CN, -OH, phenyl, halogen, C1-C6 alkoxy, C3-C6 cycloalkyl, C1-C6 carbonyl or substituted or unsubstituted 3-to 6-membered heterocycloalkyl; the heteroatom of the 3-6 membered heterocycloalkyl is N, O or S, and the number of the heteroatoms is 1-3; the substituent of the substituted 3-to 6-membered heterocycloalkyl is-H, -OH, halogen, C1-C6 oxycarbonyl, C1-C6 alkyl, C1-C6 alkoxy or

R ₄ is-H, -OH, halogen, C1-C6 alkyl, C1-C6 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C6 alkyl;

R ₂ is-H, -OH, halogen, C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl, amino-substituted C1-C8 alkyl, benzyl, substituted or unsubstituted C5-C8 aryl, or substituted or unsubstituted 5-to 8-membered heteroaryl; the heteroatom of the 5-to 8-membered heteroaryl is N, O or S, and the number of the heteroatoms is 1-3; the substituent of the substituted C5-C8 aryl or 5-8 membered heteroaryl is-H, halogen or-NH ₂ C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -OH or C1-C6 carbonyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C8 alkyl.

Further, as a preferable embodiment of the present invention, X is N or C; r ₁ is-H, -OH, halogen, C1-C8 alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy,

C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl,

Or substituted C1-C6 alkyl; the substituent of the substituted C1-C6 alkyl is-H,

-CN, -OH, phenyl, halogen, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl or substituted or unsubstituted 3-to 6-membered heterocycloalkyl; the heteroatom of the 3-6 membered heterocycloalkyl is N, O or S, and the number of the heteroatoms is 1-3; the substituent of the substituted 3-to 6-membered heterocycloalkyl is-H, -OH, halogen, C1-C4 oxycarbonyl, C1-C4 alkyl, C1-C4 alkoxy or

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl;

R ₂ is-H, -OH, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, amino-substituted C1-C6 alkyl, benzyl, substituted or unsubstituted C5-C6 aryl or substituted or unsubstituted 5-to 6-membered heteroaryl; the heteroatom of the 5-6 membered heteroaryl is N, O or S, and the number of the heteroatoms is 1-3; the substituent of the substituted C5-C6 aryl or 5-6 membered heteroaryl is-H, halogen or-NH ₂ C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, -OH or C1-C4 carbonyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Preferably, the 4-amino-pyrimidoazepine-phenylurea derivative has X being N or C; r ₁ is-H, C1-C8 alkyl,

C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl or substituted C1-C4 alkyl; the substituent of the substituted C1-C4 alkyl is-H,

-CN, -OH, phenyl, halogen, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl or substituted or unsubstituted 5-to 6-membered heterocycloalkyl; the heteroatom of the 5-6 membered heterocycloalkyl is N or O, and the number of the heteroatoms is 1-2; the substituent for substituting the 5-to 6-membered heterocycloalkyl is-H, C1-C4 oxycarbonyl, C1-C4 alkyl, C1-C4 alkoxy or

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Further, X is N or C; r ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, halogen, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl,

Or

R ₄ is-H, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl; r ₁₁ is-H, C1-C4 oxycarbonyl, C1-C4 alkyl, C1-C4 alkoxy or

R ₂ is-H, -OH, halogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 haloalkyl, amino-substituted C1-C6 alkyl, benzyl, substituted or unsubstituted C5-C6 aryl or substituted or unsubstituted 5-to 6-membered heteroaryl; the 5-6 membered heteroaryl has heteroatoms of N, O or S, and the number of the heteroatoms is 1-3; the substituent of the substituted C5-C6 aryl or 5-6 membered heteroaryl is-H, halogen or-NH ₂ C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, -OH or C1-C4 carbonyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Further, X is N or C; r is ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, halogen, methoxy, ethoxy,

Formyl, acetyl,

R ₄ is-H, C1-C4 alkyl, tert-butyloxy or

R ₅ ～R ₁₀ independently-H, methyl or ethyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Most preferably, X is N or C; r is ₁ is-H, C1-C8 alkyl,

T-butyloxycarbonyl group, C2-C6 alkenyl group, C2-C6 alkynyl group,

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Preferably, the 4-amino-pyrimidoazepine-phenylurea derivative has X being N or C; r is ₂ Is benzyl, C3-C6 cycloalkyl, substituted or unsubstituted C5-C6 aryl or substituted or unsubstituted 5-to 6-membered heteroaryl; the heteroatom of the 5-6 membered heteroaryl is N or O, and the number of the heteroatoms is 1-2; the substituent of the substituted C5-C6 aryl or 5-6 membered heteroaryl is-H, halogen or-NH ₂ C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, -OH or C1-C4 carbonyl;

R ₁ is-H, C1-C8 alkyl,

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Further, X is N or C; r ₂ Is benzyl, C3-C6 cycloalkyl,

R ₁₂ ～R ₁₉ Independently is-H, halogen, -NH ₂ C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, -OH or C1-C4 carbonyl;

R ₁ is-H, C1-C8 alkyl,

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Further, X is N or C; r ₂ Is benzyl, C3-C6 cycloalkyl,

R ₁₆ ～R ₁₉ Independently is-H, halogen, -NH ₂ C1-C4 alkyl, C1-C4 haloalkyl, -OH or C1-C4 carbonyl;

R ₁ is-H, C1-C8 alkyl,

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Still further, X is N or C; r ₂ Is benzyl, C3-C6 cycloalkyl,

R ₁₆ ～R ₁₉ Independently is-H, -F, -Cl, -Br, -CF ₃ Methyl or acetyl;

R ₁ is-H, C1-C8 alkyl,

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Most preferably, X is N or C; r ₂ Is composed of

R ₁ is-H, C1-C8 alkyl,

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Preferably, the 4-amino-pyrimidoazepine-phenylurea derivative has X being N or C; r ₃ is-H, halogen, -OH or C1-C4 alkyl; r ₂ Is benzyl, C3-C6 cycloalkyl, substituted or unsubstituted C5-C6 aryl or substituted or unsubstituted 5-to 6-membered heteroaryl; the heteroatom of the 5-6 membered heteroaryl is N or O, and the number of the heteroatoms is 1-2; the substituent of the substituted C5-C6 aryl or 5-6 membered heteroaryl is-H, halogen or-NH ₂ C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, -OH or C1-C4 carbonyl;

R ₁ is-H, C1-C8 alkyl,

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl.

Further, X is N or C; r ₃ is-H, halogen or C1-C4 alkyl; r ₂ Is benzyl, C3-C6 cycloalkyl, substituted or unsubstituted C5-C6 aryl or substituted or unsubstituted 5-to 6-membered heteroaryl; the heteroatom of the 5-6 membered heteroaryl is N or O, and the number of the heteroatoms is 1-2; the substituent of the substituted C5-C6 aryl or 5-6 membered heteroaryl is-H, halogen or-NH ₂ C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, -OH or C1-C4 carbonyl;

R ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, halogenC1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl or substituted or unsubstituted 5-to 6-membered heterocycloalkyl; the heteroatom of the 5-6 membered heterocycloalkyl is N or O, and the number of the heteroatoms is 1-2; the substituent for substituting the 5-to 6-membered heterocycloalkyl is-H, C1-C4 oxycarbonyl, C1-C4 alkyl, C1-C4 alkoxy or

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl.

Further, X is N or C; r is ₃ is-H or halogen; r ₂ Is benzyl, C3-C6 cycloalkyl, substituted or unsubstituted C5-C6 aryl or substituted or unsubstituted 5-to 6-membered heteroaryl; the heteroatom of the 5-6 membered heteroaryl is N or O, and the number of the heteroatoms is 1-2; the substituent of the substituted C5-C6 aryl or 5-6 membered heteroaryl is-H, halogen or-NH ₂ C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, -OH or C1-C4 carbonyl;

R ₁ is-H, C1-C8 alkyl,

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl.

Most preferably, X is N or C; r ₃ is-H, -F, -Cl or-Br; r ₂ Is benzyl, C3-C6 cycloalkyl, substituted or unsubstituted C5-C6 aryl or substituted or unsubstituted 5-to 6-membered heteroaryl; the heteroatom of the 5-6 membered heteroaryl is N or O, and the number of the heteroatoms is 1-2; the substituent of the substituted C5-C6 aryl or 5-6 membered heteroaryl is-H, halogen or-NH ₂ C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, -OH or C1-C4 carbonyl;

R ₁ is-H, C1-C8 alkyl,

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl.

AsIn the preferable technical scheme of the invention, X is N or C in the 4-amino-pyrimidoazepine-phenylurea derivative; r ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, halogen, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl,

R ₄ is-H, C1-C4 alkyl, C1-C4 alkoxy or

R ₂ Is benzyl, C3-C6 cycloalkyl,

R ₃ is-H, halogen or C1-C4 alkyl.

Preferably, X is N or C; r ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, halogen methoxy, ethoxy,

Formyl, acetyl,

R ₄ is-H, C1-C4 alkyl, tert-butyloxy or

R ₅ ～R ₁₀ independently-H, methyl or ethyl;

R ₂ is benzyl, C3-C6 cycloalkyl,

R ₃ is-H or halogen.

Most preferably, X is N or C; r is ₁ is-H, C1-C8 alkyl,

T-butyloxycarbonyl, C2-C6 alkenyl, C2-C6 alkynyl,

R ₂ Is composed of

R ₃ is-H, -F, -Cl or-Br.

As a preferred embodiment of the present invention, when R is the above-mentioned 4-amino-pyrimidoazepine-phenylurea derivative ₂ Is composed of

And the structure is shown as formula II:

wherein X is N or C; r ₁ is-H, -OH, halogen, C1-C10 alkoxy, C1-C10 haloalkoxy,

C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl,

R ₄ is-H, -OH, halogen, C1-C8 alkyl, C1-C8 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C8 alkyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C10 alkyl.

Preferably, X is N or C; r ₁ is-H, -OH, halogen, C1-C8 alkoxy, C1-C8 haloalkoxy,

C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl,

R ₄ is-H, -OH, halogen, C1-C6 alkyl, C1-C6 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C6 alkyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C8 alkyl.

Further preferably, X is N or C; r ₁ is-H, -OH, halogen, C1-C8 alkyl, C1-C6 alkoxy, C1-C6 haloalkoxy,

C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl,

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C6 alkyl.

Further, X is N or C; r ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, halogen, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl or substituted or unsubstituted 5-to 6-membered heterocycloalkyl; the heteroatom of the 5-6 membered heterocycloalkyl is N or O, and the number of the heteroatoms is 1-2; the substituent for substituting the 5-to 6-membered heterocycloalkyl is-H, C1-C4 oxycarbonyl, C1-C4 alkylC1-C4 alkoxy or

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl;

R ₃ is-H, -OH, halogen or C1-C4 alkyl.

Further, X is N or C; r ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, halogen, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl,

R ₄ is-H, C1-C4 alkyl, C1-C4 alkoxy or

R ₃ is-H, -OH or halogen.

More preferably, X is N or C; r is ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, halogen methoxy, ethoxy,

Formyl, acetyl,

R ₄ is-H, C1-C4 alkyl, tert-butyloxy or

R ₅ ～R ₁₀ Independently is-H, methyl or ethyl;

R ₃ is-H or halogen.

Most preferably, X is N or C; r ₁ is-H, C1-C8 alkyl,

T-butyloxycarbonyl group, C2-C6 alkenyl group, C2-C6 alkynyl group,

R ₃ is-H, -F, -Cl or-Br.

As a preferred embodiment of the present invention, the above 4-amino-pyrimidoazepine-phenylurea derivative has the structure wherein R is ₂ Is composed of

R ₃ When the structure is-H, the structure is shown as formula III:

C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl,

R ₄ is-H, -OH, halogen, C1-C8 alkyl, C1-C8 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C8 alkyl.

C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 ringAn alkyl group,

R ₄ is-H, -OH, halogen, C1-C6 alkyl, C1-C6 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C6 alkyl.

C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl,

-CN, -OH, phenyl, halogen, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl or substituted or unsubstituted 3-to 6-membered heterocycloalkyl; the heteroatom of the 3-6 membered heterocycloalkyl is N, O or S, and the number of the heteroatoms is 1-3; the substituent of the substituted 3-to 6-membered heterocycloalkyl is-H, -OH and halogenAn element, a C1-C4 oxycarbonyl group, a C1-C4 alkyl group, a C1-C4 alkoxy group or

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl.

Further, X is N or C; r ₁ is-H, C1-C8 alkyl,

R ₄ is-H, -OH, halogen, C1-C4 alkyl, C1-C4 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C4 alkyl.

Further, X is N or C; r ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, halogen, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl,

R ₄ is-H, C1-C4 alkyl, C1-C4 alkoxy or

More preferably, X is N or C; r is ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, halogen methoxy, ethoxy,

Formyl, acetyl,

R ₄ is-H, C1-C4 alkyl, tert-butyloxy or

R ₅ ～R ₁₀ independently-H, methyl or ethyl.

Most preferably, X is N or C; r ₁ is-H, C1-C8 alkyl,

T-butyloxycarbonyl group, C2-C6 alkenyl group, C2-C6 alkynyl group,

R ₃ is-H, X is C, R ₁ Is composed of

Then, the structure is shown as formula IV:

wherein R is ₁₆ ～R ₁₉ Independently is-H, halogen, -NH ₂ C1-C4 alkyl, C3-C6 cycloalkyl, C1-C4 haloalkyl, C1-C4 alkoxy, C1-C4 haloalkoxy, -OH or C1-C4 carbonyl.

Preferably, R ₁₆ ～R ₁₉ Independently is-H, halogen, -NH ₂ C1-C4 alkyl, C1-C4 haloalkyl, -OH or C1-C4 carbonyl.

Most preferably, R ₁₆ ～R ₁₉ Independently is-H, -F, -Cl, -Br, -CF ₃ Methyl or acetyl.

The 4-amino-pyrimidoazenitrogen heterocycle-phenylurea derivative has the following structural formula:

the invention also provides a synthesis method of the 4-amino-pyrimidoazenitrogen heterocycle-phenylurea derivative, which mainly adopts the following synthesis route:

the synthesis method of the compound comprises the following steps: the structure of the compound of the general formula (I) is divided into a part A and a part B, wherein the part A is an amine intermediate (formula V) compound, and the part B is an active urea intermediate (formula VI) compound or an isocyanate intermediate.

The first scheme is as follows: the synthesis method of the part A amine intermediate (formula V) compound is as follows:

the synthesis method of the pyrimido pyrrole intermediate is shown as the following scheme:

step a: ammoniation: taking 4-chloro-pyrrolopyrimidine (commercially available) as a starting material SM1, adding ammonia water (g/mL is approximately equal to 5-7 times) into a high-pressure kettle under the pressure of 10-15 atmospheric pressures, reacting for 4 hours, cooling, filtering to obtain an intermediate M1, and drying without further purification;

step b: iodination: dissolving the intermediate M1 in a solvent at room temperature, adding N-iodosuccinimide (NIS) (1.5 times of equivalent) to react for 1 hour, concentrating the solution, adding 30-40 times of water to pulp, adding 0.5 equivalent of sodium thiosulfate, filtering to obtain an intermediate M2, and drying without further purification; the solvent is tetrahydrofuran, DMF or acetonitrile, etc.;

step c: and (4) Boc protection: dissolving the intermediate M2 in a solvent, adding 2 equivalents of organic base, adding 1 equivalent of Boc anhydride (such as di-tert-butyl dicarbonate) and reacting at 50-60 ℃ for 2 hours, concentrating the reaction solution after the reaction is finished, pulping with 30-40 times of water, filtering to obtain an intermediate M3, and drying without further purification; the solvent is acetonitrile, DMF or dioxane, etc.; the organic base is triethylamine, DIEA or pyridine and the like;

step d: suzuki reaction: mixing the intermediate M3 with boric acid/boric acid pinacol ester, alkali, catalyst and the like, adding a solvent, reacting under the protection of nitrogen at the reaction temperature of 70-90 ℃ for 2-4 hours, treating, and simplifyingObtaining an intermediate M4 after single treatment, and further purifying; the alkali is inorganic alkali such as potassium carbonate, sodium carbonate, potassium bicarbonate or sodium bicarbonate; the catalyst is (dppf) PdCl ₂ Or tetrakis (triphenylphosphine) palladium; the solvent is a mixed solvent of dioxane, ethanol and water or a mixed solvent of toluene, ethanol and water;

step e: removing Boc: dissolving the intermediate M4 in a solvent at room temperature, adding strong acid, adjusting the pH of the system to be about 1, adjusting the pH to be about 13 by using inorganic base after the reaction is finished, extracting by using dichloromethane, concentrating an organic phase, pulping by using diethyl ether, filtering to obtain an intermediate M5, and drying without further purification; the solvent is water or ethanol and the like; the strong acid is inorganic strong acid such as sulfuric acid, methanesulfonic acid or hydrochloric acid; the inorganic alkali is potassium hydroxide or sodium hydroxide and the like;

step f: and (3) substitution: dissolving the intermediate M5 in a solvent, adding 1.2 equivalents of halide, reacting at 70-80 ℃ for 1 hour, filtering the reaction solution to remove inorganic salts to obtain a solution of the intermediate M6 (the compound of the formula V), and carrying out no further purification; the solvent is acetonitrile, toluene, dioxane or DMF.

The synthesis method of the part B amine intermediate (formula VI) compound is as follows:

dissolving triphosgene in a solvent, adding 1 equivalent of amine raw material, adding organic base, reacting at 60-65 ℃ for 4-5 hours, filtering to remove solid residues after the reaction is finished, and concentrating the filtrate to obtain an intermediate (a compound shown in a formula VI); the solvent is tetrahydrofuran and the like; the organic base is triethylamine or DIEA and the like.

Then, dissolving the intermediate compound (formula V) in a solvent, adding 1 equivalent of the intermediate compound (formula VI), reacting at 70-80 ℃ for 1 hour, precipitating a large amount of solid, and filtering to obtain a filter cake, namely the final urea compound, namely the compound of the general formula (I); the solvent is acetonitrile, dioxane, toluene or DMF, etc.;

C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl,

R ₄ is-H, -OH, halogen, C1-C8 alkyl, C1-C8 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C8 alkyl;

R ₂ is-H, -OH, halogen, C1-C10 alkyl, C3-C10 cycloalkyl, C1-C10 haloalkyl, amino-substituted C1-C10 alkyl, benzyl, substituted or unsubstituted C5-C10 aryl or substituted or unsubstituted 5-to 10-membered heteroaryl; the heteroatom of the 5-to 10-membered heteroaryl is N, O or S, and the number of the heteroatoms is 1-3; the substituent of the substituted C5-C10 aryl or 5-10 membered heteroaryl is-H, halogen or-NH ₂ C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkylC1-C8 alkoxy, C1-C8 haloalkoxy, -OH or C1-C8 carbonyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C10 alkyl.

Scheme II:

the synthesis of the intermediate compound (formula V) is the same as the first scheme, the intermediate compound (formula V) is dissolved in a solvent, 1 equivalent of isocyanate intermediate is added, the reaction is carried out for 1 hour at 70-80 ℃, a large amount of solid is separated out, and a filter cake after filtration is the final urea compound, namely the compound of the general formula (I); the solvent is acetonitrile, dioxane, toluene or DMF, etc.;

C2-C10 alkenyl, C2-C10 alkynyl, C3-C10 cycloalkyl,

R ₄ is-H, -OH, halogen, C1-C8 alkyl, C1-C8 alkoxy or

R ₅ ～R ₁₀ Independently is-H or C1-C8 alkyl;

R ₂ is-H, -OH, halogen, C1-C10 alkyl, C3-C10 cycloalkyl, C1-C10 haloalkyl, amino-substituted C1-C10 alkyl, benzyl, substituted or unsubstituted C5-C10 aryl or substituted or unsubstituted 5-to 10-membered heteroaryl; the heteroatom of the 5-to 10-membered heteroaryl is N, O or S, and the number of the heteroatoms is 1 to 3; the substituent of the substituted C5-C10 aryl or 5-10 membered heteroaryl is-H, halogen or-NH ₂ C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 haloalkyl, C1-C8 alkoxy, C1-C8 haloalkoxy, -OH or C1-C8 carbonyl;

R ₃ is-H, -OH, halogen, -NH ₂ Or C1-C10 alkyl.

The invention also provides the 4-amino-pyrimidoazenitrogen heterocyclic-phenylurea derivative, including tautomers, stereoisomers, mixtures of all proportions thereof and isotopically substituted compounds thereof.

The invention also provides pharmaceutically acceptable salts of the 4-amino-pyrimidoazepine-phenylurea derivatives. Wherein the salt with an acid is obtained by reacting the free base of the parent compound with an inorganic or organic acid. The inorganic acid includes hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid, perchloric acid and the like. The organic acid includes acetic acid, propionic acid, acrylic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, hydroxybenzoic acid, γ -hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, mandelic acid, succinic acid, malonic acid, or the like.

The term "pharmaceutically acceptable" as used herein, means that which, within the scope of sound medical judgment, is suitable for use in contact with the tissues of human beings and other mammals without undue toxicity, irritation, allergic response and the like, and which, when administered to a recipient, provides, directly or indirectly, a compound of the invention or a prodrug of the compound.

The invention also provides pharmaceutically acceptable hydrates of the 4-amino-pyrimidoazenitrogen heterocyclic-phenylurea derivatives. The term "hydrate" refers to a compound that further binds stoichiometric or non-stoichiometric water by non-covalent intermolecular forces.

The invention also provides pharmaceutically acceptable polymorphic substances of the 4-amino-pyrimidoazepine-phenylurea derivatives. The term "polymorph" denotes a solid crystalline form of a compound or a complex thereof, which can be characterized by physical means, such as x-ray powder diffraction patterns or infrared spectroscopy.

The invention also provides a pharmaceutically acceptable pharmaceutical composition of the 4-amino-pyrimidoazepine-phenylurea derivative, which is prepared by adding pharmaceutically acceptable auxiliary components into the 4-amino-pyrimidoazepine-phenylurea derivative shown in formulas I to IV or salt or hydrate thereof. The auxiliary component is cyclodextrin, arginine or meglumine. The cyclodextrin is selected from alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and (C) _1-4 Alkyl) -alpha-cyclodextrin, (C) _1-4 Alkyl) -beta-cyclodextrin, (C) _1-4 Alkyl) -gamma-cyclodextrin, (hydroxy-C) _1-4 Alkyl) -alpha-cyclodextrin, (hydroxy-C) _1-4 Alkyl) -beta-cyclodextrin, (hydroxy-C) _1-4 Alkyl) -gamma-cyclodextrin, (carboxy-C) _1-4 Alkyl) -alpha-cyclodextrin, (carboxy-C) _1-4 Alkyl) -beta-cyclodextrin, (carboxy-C) _1-4 Alkyl) -gamma-cyclodextrin, saccharide ethers of alpha-cyclodextrin, saccharide ethers of beta-cyclodextrin, saccharide ethers of gamma-cyclodextrin, sulfobutyl ethers of alpha-cyclodextrin, sulfobutyl ethers of beta-cyclodextrin and sulfobutyl ethers of gamma-cyclodextrin. The auxiliary components also comprise a pharmaceutically acceptable carrier, adjuvant or vehicle. Can be used in pharmaceutically acceptable pharmaceutical composition, such as ion exchanger, aluminum oxide, aluminum stearate, and lecithin; buffer substances include phosphate, glycine, arginine, sorbic acid, and the like.

The pharmaceutical composition may be in liquid form or solid form. Wherein the liquid form may be an aqueous solution. The solid form may be in the form of a powder, granules, tablets or lyophilized powder. The pharmaceutical composition further comprises water for injection, saline solution, aqueous glucose solution, saline for injection/infusion, glucose for injection/infusion, grignard solution or Grignard solution containing lactate.

The invention also provides application of the 4-amino-pyrimidoazepine-phenylurea derivatives, salts, hydrates or pharmaceutical compositions shown in the formulas I to IV in preparation of FLT3 kinase inhibitors; in particular a mutant FLT3 kinase; especially FLT3/ITD mutant kinase.

The invention also provides application of the 4-amino-pyrimido nitrogen heterocyclic-phenylurea derivatives, salts, hydrates or pharmaceutical compositions shown in the formulas I to IV in preparation of medicines for treating cell proliferation disorder diseases.

The invention also provides application of the 4-amino-pyrimidoazepine-phenylurea derivatives, salts, hydrates or pharmaceutical compositions shown in the formulas I to IV in preparing medicaments for treating tumors.

Further, the tumor is a solid tumor and/or a hematological tumor.

Still further, the solid tumor includes lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic lymphoma, lymphoplasmacytic lymphoma, ovarian cancer, breast cancer, prostate cancer, bladder cancer, kidney cancer, esophageal cancer, neck cancer, pancreatic cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, thyroid cancer, brain cancer, lymphatic cancer, epidermal hyperproliferation, psoriasis, and/or prostatic hyperplasia.

Still further, the hematologic neoplasm comprises: acute myeloid leukemia, chronic myeloid leukemia, myeloma, acute lymphocytic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, chronic neutrophilic leukemia, acute undifferentiated cell leukemia, myelodysplastic syndrome, myelodysplasia, multiple myeloma, and/or myelosarcoma.

The invention also provides application of the 4-amino-pyrimidoazenitrogen heterocycle-phenylurea derivatives, salts, hydrates or pharmaceutical compositions shown in the formulas I to IV in preparation of oral or intravenous injection preparations. The oral or intravenous injection preparation at least comprises a 4-amino-pyrimidoazepine-phenylurea derivative, salt, hydrate or pharmaceutical composition shown in formulas I-IV and any excipient and/or adjuvant.

The 4-amino-pyrimidoazepine-phenylurea derivative provided by the invention can be used as a FLT3 kinase inhibitor, has a good effect, and provides a new choice for preparing antitumor drugs.

Drawings

FIG. 1MV4-11 verifies target access;

FIG. 2Molm-13 verifies the target pathway;

FIG. 3Molm-13 in vivo pharmacodynamic model;

FIG. 4MV4-11 in vivo pharmacodynamic model;

FIG. 5 survival study of NCG mice.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments in the form of examples, but the present invention is not limited thereto.

In the examples, the reaction temperature is, without particular indication, room temperature, i.e.20-30 ℃.

Example 1:1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (4-fluorophenyl) urea compound CLJ-1

Step a: preparation of M1 intermediate

Raw materials SM1 (20g, 130mmol) and ammonia water (150mL, 25% -28% industrial ammonia water) are weighed and added into a high-pressure reaction kettle, the system is sealed, a thermometer is inserted, the temperature is set to be 130 ℃, and after 4 hours of reaction, the temperature is cooled to room temperature. The reaction suspension is directly filtered, and the filter cake is washed by ether to obtain an intermediate M1. ¹ H NMR(400MHz,DMSO)δ:11.45(s,1H),8.03(s,1H),7.06(d,J＝3.2Hz,1H),6.88(s,2H),6.51(d,J＝3.3Hz,1H).

Step b: preparation of M2 intermediate

Intermediate M1 (1lg, 112mmol) from the previous step was added to 200mL of tetrahydrofuran at room temperature, and NIS (37.8g, 168mmol) was added in portions and reacted for 1h. The reaction suspension was evaporated under reduced pressure to give a solid mixture, 300mL of water was added, sodium thiosulfate (8.8g, 56mmol) was added with stirring, the mixture turned from brown to yellow, stirring was continued for 0.5h, the mixture was filtered, and the filter cake was washed with ether to give intermediate M2. ¹ H NMR(400MHz,DMSO)δ:11.97(s,1H),8.07(s,1H),7.36(s,1H),6.56(s,2H).

Step c: preparation of M3 intermediate

Intermediate M2 (28g, 107.7mmol), potassium carbonate (29.7g, 215.4mmol) and Boc anhydride (25.8g, 118.5mmol) in the previous step were added to 500mL of acetonitrile, and the resulting mixture was heated to 60 ℃ and reacted for 2 hours. And filtering the reaction liquid, discarding a filter cake, distilling the mother liquor under reduced pressure to obtain a mixture solid, adding 500mL of water for pulping, continuously stirring for 0.5h, filtering, draining the filter cake, transferring to a vacuum drying oven, and drying at 60 ℃ for 5h to obtain an intermediate M3. ¹ H NMR(400MHz,DMSO)δ:8.28(s,1H),7.73(s,1H),6.87(s,2H),1.62(s,10H).

Step d: preparation of M4 intermediate

Intermediate M3 (9.56g, 26.6 mmol) in the previous step, p-aminobenzoate (5.5g, 31.8mmol), potassium carbonate (7.3g, 53.1mmol) and dppf (Pd) ₂ Cl ₂ ) The mixture was charged into a 250mL three-necked flask, and dioxane/ethanol/water =7 (120 mL total) was added as a solvent, and after replacing nitrogen three times, the mixture was transferred into an oil bath at 80 ℃ for reaction for 2h. After the reaction is finished, concentrating the reaction solution to be dry, mixing the sample, and separating the mixture by a column to obtain an intermediate M4. ¹ H NMR(400MHz,DMSO)δ:9.57(s,1H),9.02(s,1H),8.28(s,1H),7.59(d,J＝8.6Hz,2H),7.47(s,1H),7.44(d,J＝8.5Hz,2H),6.52(s,1H),6.23(s,2H),1.61(s,9H),1.31(s,9H).

Step e: preparation of M5 intermediate

Intermediate M4 (5g, 15.4 mmol) in the previous step was added to 50mL of methylene chloride, and concentrated hydrochloric acid (10mL, 36-38% concentrated hydrochloric acid) was added to complete the reaction for 1 hour. 200mL of methylene chloride and 100mL of water were added to adjust the pH of the mixture to basic pH (about pH 10), and the organic phase was retained by extractionAfter spin-drying, the intermediate M5 is obtained. ¹ H NMR(400MHz,DMSO)δ:11.57(s,1H),8.07(s,1H),7.12(d,J＝8.3Hz,2H),7.03(s,1H),6.66(d,J＝8.3Hz,2H),5.94(s,2H),5.16(s,2H).

Step f: preparation of intermediate M6

The intermediate M5 (225mg, 1mmol) obtained in the previous step, cesium carbonate (650mg, 2mmol) and N- (2-chloroethyl) morpholine hydrochloride (223mg, 1.2mmol) were added to 20mL of acetonitrile, and the mixture was heated to 80 ℃ for 1 hour. The reaction solution was spin dried and extracted with dichloromethane to obtain the purer intermediate M6. ¹ H NMR(400MHz,CDCl ₃ ) δ 8.68 (s, 1H), 7.25 (d, J =8.4hz, 4H), 7.13 (s, 1H), 6.80 (d, J =8.4hz, 2h), 4.44 (s, 2H), 3.72 (s, 4H), 2.87 (s, 2H), 2.59 (s, 4H). Step g: preparation of the end product CLJ-1

The intermediate M6 (338mg, 1mmol) in the previous step was added to 20mL of methylene chloride, 4-fluorophenylisocyanate (137mg, 1mmol) was added, and after 0.5 hour of reaction, a solid precipitated out, and the filter cake was rinsed with diethyl ether after filtration to obtain a high-purity final product CLJ-1. ¹ H NMR(400MHz,DMSO)δ:9.56(s,1H),9.47(s,1H),8.54(s,1H),7.71(s,1H),7.62(d,J＝8.6Hz,2H),7.52–7.45(m,2H),7.40(d,J＝8.6Hz,2H),7.16–7.10(m,2H),4.75(t,J＝6.5Hz,2H),3.89(s,4H),3.65(t,J＝6.4Hz,4H).HRMS(ESI),m/z:476.2214[M+H] ⁺ .

EXAMPLE 2 CLJ-2- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (2-chlorophenyl) urea Compound

The phenyl 4-fluoroisocyanate was replaced with 2-chlorophenyl isocyanate in step g in the same manner as in the synthesis of example 1 to give CLJ-2 as a final product. ¹ H NMR(400MHz,DMSO)δ:11.47(s,1H),10.10(s,1H),8.59(s,1H),8.55(s,1H),8.15(dd,J＝8.3,1.5Hz,1H),7.72(s,1H),7.66(d,J＝8.6Hz,2H),7.46(dd,J＝8.0,1.5Hz,1H),7.42(d,J＝8.6Hz,2H),7.34–7.28(m,1H),7.07–7.01(m,1H),4.75(t,J＝6.5Hz,2H),3.89(s,4H),3.65(t,J＝6.4Hz,3H).HRMS(ESI),m/z:492.1922[M+H] ⁺ .

EXAMPLE 3 1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3-benzylurea Compound CLJ-3

The phenyl 4-fluoroisocyanate was replaced with benzyl isocyanate in step g in the same manner as in the synthesis of example 1 to give the final product CLJ-3. ¹ H NMR(400MHz,DMSO)δ:11.58(s,1H),9.31(s,1H),8.55(s,1H),7.70(s,1H),7.59(d,J＝8.6Hz,2H),7.37–7.30(m,6H),7.27–7.20(m,1H),7.05(s,1H),4.75(t,J＝6.5Hz,2H),4.32(s,2H),3.87(m,9H),3.65(t,J＝6.4Hz,3H).HRMS(ESI),m/z:472.2455[M+H] ⁺ .

EXAMPLE 4 CLJ-4-Compound of 1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (4-chlorophenyl) urea

The phenyl 4-fluoroisocyanate was replaced with phenyl 4-chloroisocyanate in step g in the same manner as in the synthesis of example 1 to give CLJ-4 as a final product. ¹ H NMR(400MHz,DMSO)δ:8.86(s,1H),8.82(s,1H),8.14(s,1H),7.56(d,J＝8.5Hz,2H),7.53–7.49(m,2H),7.38(d,J＝8.5Hz,2H),7.35–7.30(m,3H),6.05(s,2H),4.28(t,J＝6.5Hz,2H),3.56–3.50(m,4H),2.71(t,J＝6.4Hz,2H),2.46(s,4H).HRMS(ESI),m/z:492.1913[M+H] ⁺ .

EXAMPLE 5 CLJ-5-Compound of 1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-chlorophenyl) urea

The phenyl 4-fluoroisocyanate was replaced with 3-chlorophenyl isocyanate in step g in the same manner as in the synthesis of example 1 to give CLJ-5 as a final product. ¹ H NMR(400MHz,DMSO)δ:9.44(s,1H),9.33(s,1H),8.22(s,1H),7.73(s,1H),7.59(d,J＝8.5Hz,2H),7.43–7.35(m,3H),7.32–7.27(m,2H),7.05–6.98(m,1H),6.39(s,2H),4.49(s,2H),3.73(s,4H),2.98(s,4H).HRMS(ESI),m/z:492.1915[M+H] ⁺ .

EXAMPLE 6 CLJ-6, a 1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (2-fluorophenyl) urea compound

In the same manner as in the synthesis method of example 1, phenyl 4-fluoroisocyanate was replaced with phenyl 2-fluoroisocyanate in step g to obtain CLJ-6 as a final product. ¹ H NMR(400MHz,DMSO)δ:9.23(s,1H),8.62(s,1H),8.16(dd,J＝15.3,6.0Hz,2H),7.57(d,J＝8.4Hz,2H),7.39(d,J＝8.4Hz,2H),7.32(s,1H),7.29–7.20(m,1H),7.15(t,J＝7.6Hz,1H),7.01(dd,J＝12.7,6.5Hz,1H),6.04(s,2H),4.28(t,J＝6.4Hz,2H),3.54(s,4H),2.70(t,J＝6.5Hz,2H),2.45(s,4H).HRMS(ESI),m/z:476.2207[M+H] ⁺ .

EXAMPLE 7 CLJ-7-4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (4-chloro-3- (trifluoromethyl) phenyl) urea Compound

The phenyl 4-fluoroisocyanate was replaced in step g with 4-chloro-3-trifluoromethyl-phenyl isocyanate in the same manner as the synthesis of example 1 to give the final product CLJ-7. ¹ H NMR(400MHz,DMSO)δ:11.11(s,1H),9.99(s,1H),9.66(s,1H),8.52(s,1H),8.14(d,J＝1.8Hz,1H),7.70(s,1H),7.66–7.59(m,3H),7.41(d,J＝8.6Hz,2H),4.73(t,J＝6.3Hz,2H),3.87(s,4H),3.64(m,3H),3.41–3.13(m,4H).HRMS(ESI),m/z:560.1786[M+H] ⁺ .

EXAMPLE 8 CLJ-8, a 1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (p-tolyl) urea compound

The phenyl 4-fluoroisocyanate was replaced with p-tolylisocyanate in step g in the same manner as in the synthesis of example 1 to give the final product CLJ-8. ¹ H NMR(400MHz,DMSO)δ:9.46(s,1H),9.20(s,1H),8.52(s,1H),7.69(s,1H),7.62(d,J＝8.6Hz,2H),7.39(d,J＝8.6Hz,2H),7.36(d,J＝8.5Hz,2H),7.09(d,J＝8.3Hz,2H),4.73(t,J＝6.5Hz,2H),3.88(s,4H),3.64(t,J＝6.1Hz,3H),3.44–3.11(m,4H),2.25(s,3H).HRMS(ESI),m/z:472.2458[M+H] ⁺ .

EXAMPLE 9 1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-chloro-4-methylphenyl) urea Compound CLJ-9

The phenyl 4-fluoroisocyanate was replaced with 3-chloro-4-methyl-phenyl isocyanate in step g in the same manner as in the synthesis of example 1 to give the final product CLJ-9. ¹ H NMR(400MHz,DMSO)δ:11.40(s,1H),9.66(d,J＝4.5Hz,2H),8.55(s,1H),7.72(s,1H),7.71(d,J＝1.9Hz,1H),7.62(d,J＝8.6Hz,2H),7.40(d,J＝8.6Hz,2H),7.27–7.19(m,2H),4.75(t,J＝6.5Hz,2H),3.89(s,4H),3.66(t,J＝6.5Hz,3H),2.26(s,3H).HRMS(ESI),m/z:506.2066[M+H] ⁺ .

EXAMPLE 10 1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-acetylphenyl) urea Compound CLJ-10

The phenyl 4-fluoroisocyanate was replaced with 3-acetylphenyl isocyanate in step g in the same manner as in the synthesis of example 1 to give CLJ-10 as a final product. ¹ H NMR(400MHz,DMSO)δ:11.39(s,1H),9.70(d,J＝8.8Hz,2H),8.54(s,1H),8.11(t,J＝1.8Hz,1H),7.73–7.68(m,2H),7.65(d,J＝8.6Hz,2H),7.61–7.57(m,1H),7.48–7.42(m,1H),7.41(d,J＝8.6Hz,2H),4.75(t,J＝6.5Hz,2H),3.89(s,4H),3.66(t,J＝6.5Hz,2H),2.57(s,3H).HRMS(ESI),m/z:500.2406[M+H] ⁺ .

EXAMPLE 11 1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3-cyclohexylurea Compound CLJ-11

The phenyl 4-fluoroisocyanate was replaced with cyclohexyl isocyanate in step g in the same manner as in the synthesis of example 1 to give the final product CLJ-11. ¹ H NMR(400MHz,DMSO)δ:8.38(s,1H),8.12(s,1H),7.47(d,J＝8.6Hz,2H),7.30(d,J＝8.6Hz,2H),7.28(s,1H),6.09(d,J＝7.9Hz,1H),4.27(t,J＝6.6Hz,2H),3.56–3.51(m,4H),2.69(t,J＝6.6Hz,2H),2.45(d,J＝4.1Hz,4H),1.81(dd,J＝8.5,3.9Hz,2H),1.67(dd,J＝9.0,4.0Hz,2H),1.59–1.49(m,1H),1.38–1.25(m,2H),1.24–1.12(m,4H).HRMS(ESI),m/z:464.2773[M+H] ⁺ .

EXAMPLE 12 CLJ-12, a 1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (4-bromophenyl) urea compound

The phenyl 4-fluoroisocyanate was replaced in step g with 4-bromobenzene isocyanate in the same manner as the synthesis of example 1 to give the final product CLJ-12. ¹ H NMR(400MHz,DMSO)δ:8.86(s,1H),8.82(s,1H),8.14(s,1H),7.56(d,J＝8.5Hz,2H),7.53–7.49(m,2H),7.38(d,J＝8.5Hz,2H),7.35–7.30(m,3H),6.05(s,2H),4.28(t,J＝6.5Hz,2H),3.56–3.50(m,4H),2.71(t,J＝6.4Hz,2H),2.46(s,4H).HRMS(ESI),m/z:536.1408,538.1390[M+H] ⁺ .

EXAMPLE 13 CLJ-13- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

Preparation of reactive urea intermediate 1, 3-bis (5- (tert-butyl) isoxazol-3-yl) urea

Triphosgene (47.07g, 156.9 mmol) was added to 300mL of tetrahydrofuran, and 3-amino-5-tert-butylisoxazole (20g, 142.65mmol) was dissolved in 100mL of tetrahydrofuran under ice bath, and then added dropwise to the triphosgene solution, and finally triethylamine (39.8mL, 285.3mmol) was added dropwise. The reaction was transferred to a 60 ℃ oil bath and reacted for 5h. After the reaction is completed, the reaction mixture is filtered, the filtrate is retained, and the filtrate is concentrated under reduced pressure to obtain a solid, namely the active urea intermediate. ¹ H NMR(400MHz,DMSO)δ:6.71(s,1H),6.35(s,1H),4.90(s,2H),1.36(s,9H),1.35(s,9H).

Combining steps f and h into a one-pot reaction process

The intermediate M5 (225mg, 1mmol) obtained in the previous step, cesium carbonate (650mg, 2mmol) and N- (2-chloroethyl) morpholine hydrochloride (223mg, 1.2mmol) were added to 20mL of acetonitrile, and the mixture was heated to 80 ℃ to react for 1 hour. The reaction solution was filtered, and the mother liquor was retained. Transferring the mother liquor to 80 ℃, adding an active urea intermediate (306mg, 1mmol), reacting for 0.5h, separating out a large amount of solid, filtering, and leaching with diethyl ether to obtain the high-purity final product CLJ-13. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.91(s,1H),8.14(s,1H),7.56(d,J＝8.3Hz,2H),7.40(d,J＝8.2Hz,2H),7.33(s,1H),6.52(s,1H),6.06(s,2H),4.29(t,J＝6.3Hz,2H),3.54(s,4H),2.71(t,J＝6.4Hz,2H),2.46(s,4H),1.31(s,9H).HRMS(ESI),m/z:505.2672[M+H] ⁺ .

Example 14 1- (4- (4-amino-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-14

Adding the intermediate M5 (225mg, 1mmol) in the previous step into 20mL acetonitrile, heating to 80 ℃ for reaction, adding an active urea intermediate (306mg, 1mmol), reacting for 0.5h, precipitating a large amount of solid, filtering, and leaching with diethyl ether to obtain the high-purity intermediate M5Degree of final product CLJ-14. ¹ H NMR(400MHz,DMSO)δ:11.74(s,1H),9.53(s,1H),8.93(s,1H),8.11(s,1H),7.55(d,J＝8.5Hz,2H),7.40(d,J＝8.5Hz,2H),7.19(d,J＝2.3Hz,1H),6.52(s,1H),5.99(s,2H),1.31(s,9H).HRMS(ESI),m/z:392.1831[M+H] ⁺ .

EXAMPLE 15 CLJ-15-1- (4- (4-amino-7-tert-butoxycarbonyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound

And adding the intermediate M4 (325mg, 1mmol) in the last step into 20mL of acetonitrile, heating to 80 ℃ for reaction, adding an active urea intermediate (306mg, 1mmol), reacting for 0.5h, precipitating a large amount of solid, filtering, and leaching with diethyl ether to obtain the high-purity final product CLJ-15. ¹ H NMR(400MHz,DMSO)δ:9.57(s,1H),9.02(s,1H),8.28(s,1H),7.59(d,J＝8.6Hz,2H),7.47(s,1H),7.44(d,J＝8.5Hz,2H),6.52(s,1H),6.23(s,2H),1.61(s,9H),1.31(s,9H).HRMS(ESI),m/z:592.2353[M+H] ⁺ .

EXAMPLE 16 tert-butyl 4- (2- (4-amino-5- (4- (3- (5- (tert-butyl) isoxazol-3-yl) ureido) phenyl) -7H-pyrrolo [2,3-d ] tert-butylpyrimidin-7-yl) ethyl) piperazine-1-carboxylate Compound CLJ-16

In the same manner as in example 13, N-Boc-2-ethylpiperazine was used in place of N- (2-chloroethyl) morpholine hydrochloride, to give CLJ-16 as a highly pure final product. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.91(s,1H),8.14(s,1H),7.56(d,J＝8.5Hz,2H),7.39(d,J＝8.5Hz,2H),7.33(s,1H),6.52(s,1H),6.06(s,2H),4.29(t,J＝6.5Hz,2H),3.28(s,4H),2.73(t,J＝6.5Hz,2H),2.46–2.37(m,4H),1.39(s,9H),1.31(s,9H).HRMS(ESI),m/z:604.3353[M+H] ⁺ .

EXAMPLE 17 CLJ-17- (4- (4-amino-7- (2-piperazinoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

The compound CLJ-16 (252mg, 0.5mmol) was added to 20mL of methylene chloride, and concentrated hydrochloric acid (10mL, 36-38% concentrated hydrochloric acid) was added to complete the reaction after 1 hour. 200mL of methylene chloride and 100mL of water were added to adjust the pH of the mixture to basic pH (about 10), the organic phase was retained by extraction, and the final product, CLJ-17, was obtained after concentration. ¹ H NMR(400MHz,DMSO)δ:9.58(s,1H),9.01(s,1H),8.14(s,1H),7.57(d,J＝8.5Hz,2H),7.39(d,J＝8.5Hz,2H),7.33(s,1H),6.52(s,1H),6.05(s,2H),4.27(t,J＝6.6Hz,2H),2.75–2.63(m,6H),2.41(s,4H),1.31(s,9H).HRMS(ESI),m/z:504.2829[M+H] ⁺ .

EXAMPLE 18 CLJ-18- (4- (4-amino-7- (3-morpholinopropyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

In the same manner as in example 13, high-purity CLJ-18 was obtained by substituting N- (2-chloroethyl) morpholine hydrochloride with N- (3-chloropropyl) morpholine. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.90(s,1H),8.14(s,1H),7.56(d,J＝8.6Hz,2H),7.40(d,J＝8.6Hz,2H),7.31(s,1H),6.52(s,1H),6.05(s,2H),4.20(t,J＝7.0Hz,2H),3.59–3.51(m,4H),2.29(dd,J＝14.3,7.0Hz,6H),2.02–1.91(m,2H),1.31(s,9H).HRMS(ESI),m/z:519.2825[M+H] ⁺ .

EXAMPLE 19 1- (4- (4-amino-7- (2- (dimethylamino) ethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-19

In the same manner as in the synthesis of example 13, N-dimethylThe N- (2-chloroethyl) morpholine hydrochloride is replaced by the-2-chloroethyl, and the high-purity final product CLJ-19 can be obtained. ¹ H NMR(400MHz,DMSO)δ:9.54(s,1H),8.93(s,1H),8.14(s,1H),7.56(d,J＝8.3Hz,2H),7.39(d,J＝8.3Hz,2H),7.33(s,1H),6.52(s,1H),6.05(s,2H),4.26(t,J＝6.6Hz,2H),2.67(t,J＝6.5Hz,2H),2.20(s,6H),1.31(s,9H).HRMS(ESI),m/z:463.2563[M+H] ⁺ .

EXAMPLE 20 1- (4- (4-amino-7-allyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-20

The high purity final product CLJ-20 can be obtained by replacing N- (2-chloroethyl) morpholine hydrochloride with bromopropene in the same way as the synthesis method of the embodiment 13. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.91(s,1H),8.15(s,1H),7.56(d,J＝8.5Hz,2H),7.40(d,J＝8.4Hz,2H),7.25(s,1H),6.52(s,1H),6.06(m,3H),5.18(d,J＝10.1Hz,1H),5.09(d,J＝17.1Hz,1H),4.81(d,J＝5.4Hz,2H),1.31(s,9H).HRMS(ESI),m/z:432.2149[M+H] ⁺ .

Example 21- (4- (4-amino-7- (cyanomethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-21

High purity final product CLJ-21 was obtained by substituting chloroacetonitrile for N- (2-chloroethyl) morpholine hydrochloride in the same manner as in example 13. ¹ H NMR(400MHz,DMSO)δ:9.54(s,1H),8.94(s,1H),8.24(s,1H),7.58(d,J＝7.4Hz,2H),7.42(d,J＝7.6Hz,2H),7.38(s,1H),6.53(s,1H),6.26(s,2H),5.41(s,2H),1.31(s,9H).HRMS(ESI),m/z:431.1937[M+H] ⁺ .

EXAMPLE 22 1- (4- (4-amino-7-isopropyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-22

In the same manner as in example 13, high-purity CLJ-22 was obtained by substituting 2-iodopropane for N- (2-chloroethyl) morpholine hydrochloride. ¹ H NMR(400MHz,DMSO)δ:9.55(s,1H),8.97(s,1H),8.33(s,1H),7.65(s,1H),7.59(d,J＝8.6Hz,2H),7.43(d,J＝8.5Hz,2H),6.52(s,1H),5.10–4.93(m,1H),1.49(d,J＝6.8Hz,6H),1.31(s,9H).HRMS(ESI),m/z:434.2299[M+H] ⁺ .

EXAMPLE 23 1- (4- (4-amino-7- (4-hydroxybutyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-23

In the same manner as in example 13, 4-iodobutanol was used in place of N- (2-chloroethyl) morpholine hydrochloride to give high purity CLJ-23. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.91(s,1H),8.14(s,1H),7.56(d,J＝8.4Hz,2H),7.40(d,J＝8.4Hz,2H),7.32(s,1H),6.52(s,1H),6.05(s,2H),4.41(t,J＝5.2Hz,1H),4.17(t,J＝7.0Hz,2H),4.09(q,J＝5.2Hz,1H),3.41(dd,J＝11.8,6.2Hz,2H),3.18(d,J＝5.2Hz,2H),1.88–1.76(m,2H),1.46–1.36(m,2H),1.31(s,9H).HRMS(ESI),m/z:464.2409[M+H] ⁺ .

EXAMPLE 24 CLJ-24-1- (4- (4-amino-7- (N, N-diethylformyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

In the same manner as in example 13, N-diethylchloroformamide was used in place of N- (2-chloroethyl) morpholine hydrochloride to obtain high-purity CLJ-24. ¹ H NMR(400MHz,DMSO)δ:9.55(s,1H),8.96(s,1H),8.20(s,1H),7.58(d,J＝8.6Hz,2H),7.44(d,J＝8.5Hz,2H),7.41(s,1H),6.53(s,1H),6.25(s,2H),3.39(m,4H),1.31(s,9H),1.21–1.06(m,6H).HRMS(ESI),m/z:491.2493[M+H] ⁺ .

EXAMPLE 25 CLJ-25, a 1- (4- (4-amino-7- (2- (methylsulfonyl) piperazin-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound

The compound CLJ-17 (252mg, 0.5 mmol) was dissolved in 20mL of dichloromethane, triethylamine (61mg, 0.6 mmol) was added, methanesulfonyl chloride (57mg, 0.5 mmol) diluted with dichloromethane was added under ice bath, after 1h of reaction, extraction was performed with dichloromethane and water, and the organic phase was spin-dried to give the purer final product CLJ-25. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.92(s,1H),8.15(s,1H),7.56(d,J＝8.6Hz,2H),7.40(d,J＝8.5Hz,2H),7.34(s,1H),6.52(s,1H),6.05(s,2H),4.30(t,J＝6.4Hz,2H),3.12–3.03(m,4H),2.85(s,3H),2.79(t,J＝6.5Hz,2H),2.62–2.54(m,4H),1.31(s,9H).HRMS(ESI),m/z:582.2610[M+H] ⁺ .

EXAMPLE 26 CLJ-26, a 1- (4- (4-amino-7- (2-methoxyethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound

In the same manner as the synthesis method in example 13, N- (2-chloroethyl) morpholine hydrochloride was replaced with chloroethyl methyl ether to obtain high-purity final product CLJ-26. ¹ H NMR(400MHz,DMSO)δ:9.56(s,1H),8.95(s,1H),8.15(s,1H),7.56(d,J＝8.4Hz,2H),7.39(d,J＝8.4Hz,2H),7.29(s,1H),6.53(s,1H),6.09(s,2H),4.33(t,J＝5.3Hz,2H),3.71(t,J＝5.4Hz,2H),3.26(s,3H),1.31(s,9H).HRMS(ESI),m/z:450.2249[M+H] ⁺ .

EXAMPLE 27 CLJ-27 1- (4- (4-amino-7- (but-2-yn-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

In the same manner as in example 13, 1-bromo-2-butyne was used in place of N- (2-chloroethyl) morpholine hydrochloride to give high purity final product CLJ-27. ¹ H NMR(400MHz,DMSO)δ:9.55(s,1H),8.97(s,1H),8.17(s,1H),7.57(d,J＝8.5Hz,2H),7.41(d,J＝8.4Hz,2H),7.32(s,1H),6.53(s,1H),6.12(s,1H),4.97(d,J＝2.3Hz,2H),1.81(dd,J＝5.3,3.1Hz,3H),1.31(s,9H).HRMS(ESI),m/z:444.2147[M+H] ⁺ .

EXAMPLE 28 CLJ-28-1- (4- (4-amino-7- (pent-2-yn-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

In the same manner as in example 13, 1-bromo-2-pentyne was substituted for N- (2-chloroethyl) morpholine hydrochloride to give high purity final product CLJ-28. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.93(s,1H),8.16(s,1H),7.56(d,J＝8.6Hz,2H),7.40(d,J＝8.5Hz,2H),7.31(s,1H),6.52(s,1H),6.12(s,2H),4.98(t,J＝2.1Hz,2H),2.25–2.16(m,2H),1.30(s,9H),1.06(t,J＝7.5Hz,3H).HRMS(ESI),m/z:458.2299[M+H] ⁺ .

EXAMPLE 29 CLJ-29-1- (4- (4-amino-7- (but-3-yn-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

In the same manner as in example 13, 1-bromo-2-pentyne was substituted for N- (2-chloroethyl) morpholine hydrochloride to give high purity final product CLJ-29. ¹ H NMR(400MHz,DMSO)δ:9.57(s,1H),8.99(s,1H),8.15(s,1H),7.56(d,J＝8.4Hz,2H),7.39(d,J＝8.3Hz,2H),7.32(s,1H),6.52(s,1H),6.05(s,2H),5.82(ddd,J＝23.8,10.3,6.7Hz,1H),5.14–4.93(m,2H),4.24(t,J＝7.0Hz,2H),2.56(dd,J＝13.3,6.5Hz,2H),1.31(s,9H).HRMS(ESI),m/z:446.2302[M+H] ⁺ .

EXAMPLE 30 CLJ-30- (4- (4-amino-7- (2-ethoxyethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

The same synthesis method as that in example 13 is adopted, and bromoethyl ether is used to replace N- (2-chloroethyl) morpholine hydrochloride to obtain the high-purity final product CLJ-30. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.92(s,1H),8.15(s,1H),7.56(d,J＝8.4Hz,2H),7.39(d,J＝8.4Hz,2H),7.29(s,1H),6.52(s,1H),6.06(s,2H),4.32(t,J＝5.4Hz,2H),3.74(t,J＝5.5Hz,2H),3.46(q,J＝6.9Hz,2H),1.31(s,9H).HRMS(ESI),m/z:464.2406[M+H] ⁺ .

EXAMPLE 31 1- (4- (4-amino-7- (3-methylbut-2-en-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-31

In the same manner as in example 13, 3-dimethylallyl bromide was used in place of N- (2-chloroethyl) morpholine hydrochloride to give high-purity CLJ-31 as a final product. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.92(s,1H),8.15(s,1H),7.56(d,J＝8.4Hz,2H),7.39(d,J＝8.4Hz,2H),7.23(s,1H),6.52(s,1H),6.05(s,2H),5.41(t,J＝6.8Hz,1H),4.76(d,J＝7.0Hz,2H),1.81(s,3H),1.72(s,3H),1.31(s,9H).HRMS(ESI),m/z:460.2457[M+H] ⁺ .

EXAMPLE 32 1- (4- (4-amino-7- (2-methylallyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-32

In the same manner as in example 13, 2-methylallyl bromide was used in place of N- (2-chloroethyl) morpholine hydrochloride to give high-purity CLJ-32 as a final product. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.92(s,1H),8.15(s,1H),7.56(d,J＝8.6Hz,2H),7.40(d,J＝8.6Hz,2H),7.20(s,1H),6.52(s,1H),6.09(s,2H),4.87(s,1H),4.73(s,2H),4.61(s,1H),1.68(s,3H),1.31(s,9H).HRMS(ESI),m/z:446.2298[M+H] ⁺ .

EXAMPLE 33 CLJ-33-1- (4- (4-amino-7- (pent-4-en-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

In the same manner as in example 13, 5-bromo-1-pentene was used in place of N- (2-chloroethyl) morpholine hydrochloride to give high-purity CLJ-33 as a final product. ¹ H NMR(400MHz,DMSO)δ:9.77(s,1H),9.58(s,1H),8.48(s,1H),7.68(s,1H),7.61(d,J＝8.6Hz,2H),7.42(d,J＝8.6Hz,2H),6.53(s,1H),5.83(ddt,J＝16.8,10.2,6.4Hz,1H),5.09–4.95(m,2H),4.27(t,J＝7.0Hz,2H),2.05(dd,J＝13.7,6.7Hz,2H),1.99–1.87(m,2H),1.31(s,9H).HRMS(ESI),m/z:460.2458[M+H] ⁺ .

EXAMPLE 34 CLJ-34-1- (4- (4-amino-7- (hex-5-en-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

In the same manner as in example 13, high-purity CLJ-34 was obtained by substituting 5-bromo-1-pentene for N- (2-chloroethyl) morpholine hydrochloride. ¹ H NMR(400MHz,DMSO)δ:9.78(s,1H),9.60(s,1H),8.48(s,1H),7.68(s,1H),7.61(d,J＝8.6Hz,2H),7.41(d,J＝8.6Hz,2H),6.53(s,1H),5.78(ddt,J＝16.9,10.2,6.7Hz,1H),5.05–4.90(m,2H),4.27(t,J＝7.0Hz,2H),2.06(q,J＝7.2Hz,2H),1.83(dt,J＝14.9,7.2Hz,2H),1.41–1.31(m,2H),1.31(s,9H).HRMS(ESI),m/z:474.2618[M+H] ⁺ .

EXAMPLE 35 1- (4- (4-amino-7-methyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-35

The same synthesis method as that in example 13 was used to replace N- (2-chloroethyl) morpholine hydrochloride with methyl iodide to obtain high purity final product CLJ-35. ¹ H NMR(400MHz,DMSO)δ:9.53(s,1H),8.93(s,1H),8.11(s,1H),7.55(d,J＝8.5Hz,2H),7.40(d,J＝8.5Hz,2H),7.19(d,J＝2.3Hz,1H),6.52(s,1H),5.99(s,2H),3.7(s,3H),1.31(s,9H).HRMS(ESI),m/z:406.1992[M+H] ⁺ .

Example 36 1- (4- (4-amino-7-ethyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-36

The same synthesis as in example 13 was carried out using iodoethane instead of N- (2-chloroethyl) morpholine hydrochloride to give high purity final product CLJ-36. ¹ H NMR(400MHz,DMSO)δ:9.78(s,1H),9.61(s,1H),8.48(s,1H),7.69(s,1H),7.61(d,J＝8.6Hz,2H),7.42(d,J＝8.5Hz,2H),6.53(s,1H),4.30(q,J＝7.2Hz,2H),1.43(t,J＝7.2Hz,3H),1.31(s,9H).HRMS(ESI),m/z:420.2142[M+H] ⁺ .

Example 37 1- (4- (4-amino-7-propyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-37

High purity final product CLJ-37 was obtained by substituting iodopropane for N- (2-chloroethyl) morpholine hydrochloride in the same manner as in the synthesis of example 13. ¹ H NMR(400MHz,DMSO)δ:9.80(s,1H),9.66(s,1H),8.48(s,1H),7.68(s,1H),7.61(d,J＝8.5Hz,2H),7.41(d,J＝8.5Hz,2H),6.53(s,1H),4.23(t,J＝7.0Hz,2H),1.90–1.77(m,2H),1.31(s,9H),0.87(t,J＝7.4Hz,3H).HRMS(ESI),m/z:434.2303[M+H] ⁺ .

Example 38 1- (4- (4-amino-7-butyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-38

High purity final product CLJ-38 was obtained by substituting N- (2-chloroethyl) morpholine hydrochloride with bromobutane in the same manner as in the synthesis of example 13. ¹ H NMR(400MHz,DMSO)δ:9.78(s,1H),9.61(s,1H),8.48(s,1H),7.68(s,1H),7.61(d,J＝8.6Hz,2H),7.41(d,J＝8.5Hz,2H),6.53(s,1H),4.26(t,J＝7.1Hz,2H),1.87–1.75(m,2H),1.31(s,9H),1.26(dd,J＝14.9,7.5Hz,2H),0.91(t,J＝7.4Hz,3H).HRMS(ESI),m/z:448.2460[M+H] ⁺ .

Example 39 1- (4- (4-amino-7-cyclopentyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-39

The same synthesis method as that of example 13 was followed, and N- (2-chloroethyl) morpholine hydrochloride was replaced with bromocyclopentane to obtain CLJ-39 as a high-purity final product. ¹ H NMR(400MHz,DMSO)δ:9.78(s,1H),9.62(s,1H),8.47(s,1H),7.73(s,1H),7.61(d,J＝8.6Hz,2H),7.44(d,J＝8.5Hz,2H),6.53(s,1H),5.22–5.08(m,1H),2.16(d,J＝7.9Hz,2H),2.02–1.83(m,4H),1.71(d,J＝7.0Hz,2H),1.31(s,9H).HRMS(ESI),m/z:460.2457[M+H] ⁺ .

Example 40 1- (4- (4-amino-7-hexyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-40

The same synthesis method as that in example 13 was used to replace N- (2-chloroethyl) morpholine hydrochloride with N-bromo-hexane to obtain high-purity final product CLJ-40. ¹ H NMR(400MHz,DMSO)δ:9.71(s,1H),9.40(s,1H),8.46(s,1H),7.67(s,1H),7.61(d,J＝8.6Hz,2H),7.41(d,J＝8.5Hz,2H),6.52(s,1H),4.25(t,J＝7.1Hz,2H),1.88–1.77(m,2H),1.31(s,9H),1.28(m,6H),0.86(t,J＝6.8Hz,3H).HRMS(ESI),m/z:476.2770[M+H] ⁺ .

EXAMPLE 41 1- (4- (4-amino-7-benzyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-41

High purity final product CLJ-41 was obtained by substituting benzyl bromide for N- (2-chloroethyl) morpholine hydrochloride in the same manner as in the synthesis of example 13. ¹ H NMR(400MHz,DMSO)δ:9.71(s,1H),8.49(s,1H),7.74(s,1H),7.60(d,J＝8.6Hz,2H),7.41(d,J＝8.5Hz,2H),7.38–7.27(m,5H),6.52(s,1H),5.49(s,2H),1.31(s,9H).HRMS(ESI),m/z:482.2303[M+H] ⁺ .

Example 42 1- (4- (4-amino-7-propynyl-7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-42

The same synthesis method as that in example 13 is adopted, and N- (2-chloroethyl) morpholine hydrochloride is replaced by propargyl bromide to obtain the high-purity final product CLJ-42. ¹ H NMR(400MHz,DMSO)δ:9.66(s,1H),9.28(s,1H),8.49(s,1H),7.66(s,1H),7.61(d,J＝8.5Hz,2H),7.43(d,J＝8.5Hz,2H),6.52(s,1H),5.15(d,J＝2.3Hz,2H),3.52(t,J＝2.4Hz,2H),1.31(s,9H).HRMS(ESI),m/z:430.1985[M+H] ⁺ .

EXAMPLE 43 1- (4- (4-amino-7- (2-cyanoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-43

The high purity final product CLJ-43 can be obtained by replacing N- (2-chloroethyl) morpholine hydrochloride with bromopropionitrile in the same way as the synthesis method of the embodiment 13. ¹ H NMR(400MHz,DMSO)δ:9.71(s,1H),9.42(s,1H),8.50(s,1H),7.70(s,1H),7.63(d,J＝8.6Hz,2H),7.42(d,J＝8.5Hz,2H),6.53(s,1H),4.56(t,J＝6.4Hz,2H),3.19(t,J＝6.4Hz,2H),1.31(s,9H).HRMS(ESI),m/z:445.2097[M+H] ⁺ .

Example 44 CLJ-44, a 1- (4- (4-amino-7H-pyrrolo [2,3-d ] pyrimidin-5-) -2-fluorophenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound

Step i: preparation of M7 intermediate

Intermediate M3 (9g, 25mmol), 3-fluoro-4-aminophenylboronic acid pinacol ester (6.5g, 27.5 mmol), potassium carbonate (6.9g, 50mmol) and dppf (PdCl) ₂ ) The mixture was charged into a 250mL three-necked flask, and dioxane/ethanol/water =7 (120 mL total) was added as a solvent, and after replacing nitrogen three times, the mixture was transferred into an oil bath at 80 ℃ for reaction for 2h. After the reaction is finished, concentrating the reaction solution, mixing the reaction solution with a sample, and separating the mixture by a column to obtain an intermediate M7.

Step j: preparation of M8 intermediate

The intermediate M7 (4 g,11.6 mmol) in the previous step was added to 50mL of methylene chloride, concentrated hydrochloric acid (10mL, 36-38% concentrated hydrochloric acid) was added, and the reaction was completed after 1 hour. 200mL of dichloromethane and 100mL of water were added to adjust the pH of the mixture to basic pH (about 10), the organic phase was retained by extraction and dried to give intermediate M8. ¹ H NMR(400MHz,DMSO)δ:11.66(s,1H),8.08(s,1H),7.11(d,J＝2.1Hz,1H),7.07(dd,J＝12.5,1.8Hz,1H),6.98(dd,J＝8.1,1.8Hz,1H),6.85(dd,J＝9.5,8.2Hz,1H),5.99(s,2H),5.21(s,2H).

Step k: preparation of CLJ-44

And adding the intermediate M8 (243mg, 1mmol) in the previous step into 20mL of acetonitrile, adding an active urea intermediate (306mg, 1mmol), reacting for 0.5h, separating out a solid, filtering, and leaching a filter cake by using diethyl ether to obtain the high-purity final product CLJ-44. ¹ H NMR(400MHz,DMSO)δ:13.04(s,1H),10.13(s,1H),9.20(s,1H),8.46(s,1H),8.23(t,J＝8.5Hz,1H),7.61(d,J＝2.3Hz,1H),7.37(dd,J＝12.0,1.7Hz,1H),7.27(d,J＝8.4Hz,1H),6.51(s,2H),1.31(s,9H).HRMS(ESI),m/z:410.1734[M+H] ⁺ .

EXAMPLE 45 1- (4- (4-amino-7-propynyl-7H-pyrrolo [2,3-d ] pyrimidin-5-) -2-fluorophenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-45

Combining steps l and m into a reaction process of a' one-pot method

The intermediate M8 (243mg, 1mmol) obtained in the previous step, cesium carbonate (650mg, 2mmol) and bromopropyne (143mg, 1.2mmol) were added to 20mL of acetonitrile, and the mixture was heated to 80 ℃ to react for 1 hour. The reaction solution was filtered, and the mother liquor was retained. Transferring the mother liquor to 80 ℃, adding an active urea intermediate (306mg, 1mmol), reacting for 0.5h, separating out a large amount of solid, filtering, and leaching with diethyl ether to obtain the high-purity final product CLJ-45. ¹ H NMR(400MHz,DMSO)δ:10.01(s,1H),9.08(s,1H),8.52(s,1H),8.26(t,J＝8.5Hz,1H),7.73(s,1H),7.39(dd,J＝12.0,1.8Hz,1H),7.30–7.25(m,1H),6.51(s,1H),5.16(d,J＝2.4Hz,2H),1.31(s,9H).HRMS(ESI),m/z:448.1891[M+H] ⁺ .

EXAMPLE 46 CLJ-46, a 1- (4- (4-amino-7-allyl-7H-pyrrolo [2,3-d ] pyrimidin-5) -2-fluorophenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound

The high purity final product, CLJ-46, was obtained by substituting bromopropyne for bromopropene in the same manner as in example 45. ¹ H NMR(400MHz,DMSO)δ:9.86(s,1H),8.88(s,1H),8.20(t,J＝8.5Hz,1H),8.16(s,1H),7.33(s,1H),7.33(dd,J＝12.1,1.8Hz,2H),7.26(dd,J＝8.5,1.3Hz,1H),6.51(s,1H),6.19(s,2H),6.05(ddd,J＝22.5,10.6,5.5Hz,1H),5.18(dd,J＝10.2,1.3Hz,1H),5.09(dd,J＝17.1,1.4Hz,1H),4.80(d,J＝5.5Hz,2H),1.31(s,9H).HRMS(ESI),m/z:450.2042[M+H] ⁺ .

EXAMPLE 47 1- (4- (4-amino-7- (but-3-en-1-yl) -7H-pyrrolo [2,3-d ] pyrimidin-5) -2-fluorophenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-47

In the same manner as in example 45, the bromopropyne was replaced with 4-bromo-1-butene, thereby obtaining high-purity final product CLJ-47. ¹ H NMR(400MHz,DMSO)δ:10.05(s,1H),9.12(s,1H),8.49(s,1H),8.25(t,J＝8.5Hz,1H),7.73(s,1H),7.36(dd,J＝12.0,1.7Hz,1H),7.26(d,J＝8.4Hz,1H),6.51(s,1H),5.80(ddt,J＝17.0,10.3,6.7Hz,1H),5.10–4.99(m,2H),4.34(t,J＝7.0Hz,2H),2.61(q,J＝6.8Hz,2H),1.31(s,9H).HRMS(ESI),m/z:464.2212[M+H] ⁺ .

EXAMPLE 48 CLJ-48, a 1- (4- (4-amino-7-methoxyethyl-7H-pyrrolo [2,3-d ] pyrimidin-5) -2-fluorophenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound

In the same manner as the synthesis method in example 45, bromopropyne was replaced with chloroethyl methyl ether, thereby obtaining high-purity final product CLJ-48. ¹ H NMR(400MHz,DMSO)δ:10.10(s,1H),9.16(s,1H),8.51(s,1H),8.25(t,J＝8.5Hz,1H),7.70(s,1H),7.43–7.33(m,1H),7.26(m,1H),6.51(s,1H),4.43(t,J＝5.1Hz,2H),3.76(t,J＝5.3Hz,2H),1.31(s,9H).HRMS(ESI),m/z:468.2159[M+H] ⁺ .

Example 49 1- (4- (4-amino-7-ethoxyethyl-7H-pyrrolo [2,3-d ] pyrimidin-5) -2-fluorophenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-49

The synthesis method of example 45 was followed, and bromopropyne was replaced with bromoethyl ether to obtain high-purity final product CLJ-49. ¹ H NMR(400MHz,DMSO)δ:10.02(s,1H),9.09(s,1H),8.48(s,1H),8.26(t,J＝8.5Hz,1H),7.68(s,1H),7.37(dd,J＝12.0,1.8Hz,1H),7.27(d,J＝8.5Hz,1H),6.51(s,1H),4.42(t,J＝5.3Hz,2H),3.78(t,J＝5.3Hz,2H),3.46(dd,J＝14.0,7.0Hz,2H),1.31(s,9H),1.06(t,J＝7.0Hz,3H).HRMS(ESI),m/z:482.2313[M+H] ⁺ .

EXAMPLE 50 1- (4- (4-amino-7-cyanomethyl-7H-pyrrolo [2,3-d ] pyrimidin-5) -2-fluorophenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-50

In the same manner as in example 45, the bromopropyne was replaced with chloroacetonitrile, whereby CLJ-50, a high-purity final product, was obtained. ¹ H NMR(400MHz,DMSO)δ:10.10(s,1H),9.16(s,1H),8.57(s,1H),8.26(t,J＝8.5Hz,1H),7.72(s,1H),7.39(dd,J＝11.9,1.8Hz,1H),7.28(d,J＝8.4Hz,1H),6.51(s,1H),5.55(s,2H),1.31(s,9H).HRMS(ESI),m/z:449.1848[M+H] ⁺ .

EXAMPLE 51 1- (4- (4-amino-7- (2-cyanoethyl) -7H-pyrrolo [2,3-d ] pyrimidin-5) -2-fluorophenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-51

In the same manner as in the synthesis of example 45, bromopropionitrile was replacedPropyne bromide is adopted to obtain the high-purity final product CLJ-51. ¹ H NMR(400MHz,DMSO)δ:10.05(s,1H),9.12(s,1H),8.54(s,1H),8.28(t,J＝8.5Hz,1H),7.79(s,1H),7.65(s,1H),7.37(dd,J＝11.9,1.8Hz,1H),7.28(d,J＝8.5Hz,1H),6.51(s,1H),4.60–4.51(t,J＝6.5Hz,2H),3.19(t,J＝6.6Hz,2H),1.31(s,9H).HRMS(ESI),m/z:463.2004[M+H] ⁺ .

Example 52 1- (4- (4-amino-7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-52

The synthetic route is shown as follows;

the synthetic route corresponds essentially to example 13, except that the starting material is replaced by 4-chloropyrazolo [2,3-d ]]Pyrimidine is reacted in 6 steps to obtain the final product CLJ-52. ¹ H NMR(400MHz,DMSO)δ:13.80(s,1H),9.35(s,1H),9.14(s,1H),8.50(s,1H),7.53(s,2H),7.24–7.18(m,2H),6.73–6.67(m,2H),6.46(s,1H),1.29(s,9H).HRMS(ESI),m/z:392.1907[M+H] ⁺ .

Example 53 CLJ-53-1- (4- (4-amino-7-allyl-7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound

In line with the route of example 52, except that bromopropene was added in g, and the reactive urea intermediate reacted with the next step was synthesized according to the "one-pot" synthesis to give final product CLJ-53. ¹ H NMR(400MHz,DMSO)δ:9.56(s,1H),9.02(s,1H),8.26(s,1H),7.63(t,J＝7.0Hz,4H),7.00(dd,J＝114.0,63.4Hz,2H),6.53(s,1H),6.10–6.01(m,1H),5.19(d,J＝10.3Hz,1H),5.11(d,J＝17.1Hz,1H),4.97(d,J＝5.5Hz,2H),1.31(s,9H).HRMS(ESI),m/z:433.2099[M+H] ⁺ .

EXAMPLE 54 1- (4- (4-amino-7- (2-morpholinoethyl) -7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound CLJ-54

This compound was synthesized in the same manner as in example 53 except that N- (2-chloroethylmorpholine) hydrochloride was used in place of bromopropene to give the desired end product CLJ-54. ¹ H NMR(400MHz,DMSO)δ9.57(s,1H),9.04(s,1H),8.25(s,1H),7.66–7.63(m,2H),7.63–7.61(m,2H),6.75(s,2H),6.53(s,1H),4.48–4.46(m,2H),3.52–3.48(m,4H),2.83–2.81(m,2H),2.49–2.46(m,4H),1.31(s,9H).HRMS(ESI),m/z:506.2628[M+H] ⁺ .

EXAMPLE 55 CLJ-55- (4- (4-amino-7- (2- (piperidin-1-yl) ethyl) -7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound

This compound was synthesized in the same manner as in example 53 except that N- (2-chloroethylpiperidine) hydrochloride was used in place of bromopropene to obtain the objective end product CLJ-55. ¹ H NMR(400MHz,DMSO)δ：9.58(s,1H),9.05(s,1H),8.25(s,1H),7.66–7.63(m,2H),7.63–7.61(m,2H),6.75(s,2H),6.53(s,1H),4.52–4.48(m,2H),4.44–4.40(m,2H),2.78–2.74(m,2H),2.44–2.38(m,4H),1.46–1.40(m 4H),1.31(s,9H).HRMS(ESI),m/z:504.2829[M+H] ⁺ .

EXAMPLE 56 1- (4- (4-amino-7- (cyclopropylmethyl) -7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound CLJ-56

The synthesis method of the compoundThe same procedure as in example 53, except for replacing bromopropene with 2-chlorocyclopropane, gave the desired final product CLJ-56. ¹ H NMR(400MHz,DMSO)δ:9.56(s,1H),9.02(s,1H),8.25(s,1H),7.66–7.63(m,2H),7.63–7.61(m,2H),6.90(s,2H),6.53(s,1H),5.84–5.76(m,1H),5.05–4.97(m,2H),4.42–4.39(m,2H),2.66–2.61(m,2H),1.31(s,9H).HRMS(ESI),m/z:447.2257[M+H] ⁺ .

Example 57 1- (4- (4-amino-7-propargyl-7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-57

The synthesis of this compound was performed in the same manner as in example 53 except that bromopropyne was used in place of bromopropene to obtain the objective final product, CLJ-57. ¹ H NMR(400MHz,DMSO)δ9.63(s,1H),9.10(s,1H),8.34(s,1H),7.72–7.68(m,4H),6.59(s,1H),5.51(s,2H),5.25(s,2H),3.43(s,1H),1.37(s,9H).HRMS(ESI),m/z:431.1944[M+H] ⁺ .

Example 58 1- (4- (4-amino-7-methyl-7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound CLJ-58

The synthesis of this compound was performed in the same manner as in example 53 except that methyl iodide was used instead of propylene bromide to obtain the desired final product, CLJ-58. ¹ H NMR(400MHz,DMSO)δ:9.56(s,1H),9.05(s,1H),8.26(s,1H),7.66–7.61(m,4H),6.53(s,1H),5.41(s,2H),4.03–3.99(m,3H),1.31(s,9H).HRMS(ESI),m/z:407.1948[M+H] ⁺ .

Example 59 1- (4- (4-amino-7-ethyl-7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-59

The synthesis of this compound was performed in the same manner as in example 53 except that iodoethane was used instead of bromopropene to obtain CLJ-59, the objective final product. ¹ H NMR(400MHz,DMSO)δ:9.58(s,1H),9.05(s,1H),8.26(s,1H),7.66–7.61(m,4H),6.53(s,1H),5.41(s,2H),3.97–3.93(m,2H),1.31(s,9H),1.26–1.22(m,3H).HRMS(ESI),m/z:421.2100[M+H] ⁺ .

EXAMPLE 60 1- (4- (4-amino-7- (2-cyanoethyl) -7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound CLJ-60

This compound was synthesized in the same manner as in example 53 except that chloropropionitrile was used in place of bromopropene to give the desired final product CLJ-60. ¹ H NMR(400MHz,DMSO)δ9.62(s,1H),9.10(s,1H),8.34(s,1H),7.72–7.66(m,4H),6.59(s,1H),5.67(s,2H),4.66(s,2H),3.22–3.18(m,2H),1.36(s,9H).HRMS(ESI),m/z:446.2056[M+H] ⁺ .

Example 61 1- (4- (4-amino-7-hexyl-7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound CLJ-61

This compound was synthesized in the same manner as in example 53 except that 1-bromohexane was used in place of bromopropene to obtain the objective final product CLJ-61. ¹ H NMR(400MHz,DMSO)δ9.56(s,1H),9.03(s,1H),8.25(s,1H),7.65–7.59(m,4H),6.54(s,1H),5.42(s,2H),4.34–4.31(m,2H),1.84–1.82(m,4H),1.31(s,9H),0.85–0.82(m,7H).HRMS(ESI),m/z:477.2396[M+H] ⁺ .

Example 62 1- (4- (4-amino-7-propyl-7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-62

The synthesis of this compound was performed in the same manner as in example 53 except that 1-bromopropyl was used instead of bromopropene to obtain the desired final product, CLJ-62. ¹ H NMR(400MHz,DMSO)δ:9.56(s,1H),9.02(s,1H),8.25(s,1H),7.64–7.60(m,4H),6.53(s,1H),5.41(s,2H),4.32–4.28(m,2H),1.92–1.84(m,2H),1.31(s,9H),0.89–0.85(m,3H).HRMS(ESI),m/z:435.2256[M+H] ⁺ .

Example 63 1- (4- (4-amino-7-cyclopentyl-7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound CLJ-63

The synthesis of this compound was the same as in example 53 except that bromocyclopentane was substituted for bromopropene to give the desired end product, CLJ-63. ¹ H NMR(400MHz,DMSO)δ9.55(s,1H),9.03(s,1H),8.23(s,1H),7.64–7.60(m,4H),6.72(s,2H),6.53(s,1H),5.28–5.19(m,1H),2.08–2.08(m,4H),1.92–1.87(m,2H),1.74–1.67(m,2H),1.31(s,9H).HRMS(ESI),m/z:461.2407[M+H] ⁺ .

Example 64 CLJ-64- (4- (4-amino-7- (2-ethoxyethyl) -7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

The synthesis of this compound was the same as in example 53 except that bromoethyl ether was used instead of bromopropene to give the desired end product, CLJ-64. ¹ H NMR(400MHz,DMSO)δ:9.57(s,1H),9.05(s,1H),8.25(s,1H),7.67–7.61(m,4H),6.88(s,2H),6.54(s,1H),4.49–4.42(m,4H),1.31(s,9H),1.04–1.00(m,5H).HRMS(ESI),m/z:465.2361[M+H] ⁺ .

EXAMPLE 65 CLJ-65-1- (4- (4-amino-7- (3-methylbut-2-en-1-yl) -7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea Compound

The synthesis of this compound was the same as in example 53 except that 3, 3-dimethylbromopropene was used instead of bromopropene to give the desired end product, CLJ-65. ¹ H NMR(400MHz,DMSO)δ:9.57(s,1H),9.05(s,1H),8.25(s,1H),7.65–7.59(m,4H),6.53(s,1H),5.42(s,2H),4.94–4.88(m,3H),1.82–1.79(m,5H),1.70(s,3H),1.31(s,9H).HRMS(ESI),m/z:461.2407[M+H] ⁺ .

EXAMPLE 66 CLJ-66, a 1- (4- (4-amino-7- (2, 2-dimethoxyethyl) -7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound

The synthesis of this compound was the same as in example 53 except that 2, 2-dimethylbromoethyl was used instead of bromopropene to give the desired end product, CLJ-66. ¹ H NMR(400MHz,DMSO)δ:9.57(s,1H),9.04(s,1H),8.27(s,1H),7.66–7.60(m,4H),6.54(s,1H),5.46(s,2H),4.96–4.93(m,1H),4.44–4.42(m,2H),3.33(s,6H),1.31(s,9H).HRMS(ESI),m/z:481.2307[M+H] ⁺ .

EXAMPLE 67 CLJ-67- (4- (4-amino-7- (4-oxopentyl) -7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (5-tert-butyl-isoxazol-3-yl) urea compound

The synthesis of this compound was the same as in example 53 except that 2, 2-dimethylbromoethyl was used instead of bromopropene to give the desired end product, CLJ-67. ¹ H NMR(400MHz,DMSO)δ:9.57(s,1H),9.05(s,1H),8.21(s,1H),7.66–7.60(m,4H),6.54(s,1H),5.42(s,2H),4.30–4.25(m,2H),2.48–2.42(m,4H),2.05(s,3H),1.31(s,9H).HRMS(ESI),m/z:477.2396[M+H] ⁺ .

EXAMPLE 68 CLJ-68-1- (4- (4-amino-7H-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-tert-butyl-1H-pyrazol-5-yl) urea Compound

Preparation of active urea intermediate 1,3- (3-tert-butyl-1H-pyrazol-5-yl) urea

Triphosgene (4.71g, 15.7 mmol) was added to 50mL of tetrahydrofuran, and 3-amino-5-tert-butylpyrazole (1.99g, 14.3 mmol) was dissolved in 5mL of tetrahydrofuran under ice-cooling, and then added dropwise to the triphosgene solution, and finally triethylamine (4.0 mL,28.5 mmol) was added dropwise. The reaction was transferred to a 60 ℃ oil bath and reacted for 5h. And after the reaction is completed, filtering the reaction mixture, reserving filtrate, concentrating the filtrate under reduced pressure to obtain a solid, and separating the solid by a column to obtain the active urea intermediate. ¹ H NMR(400MHz,DMSO)δ:12.66(s,2H),9.51(s,2H),6.30(s,2H),1.30(s,18H).

And adding the intermediate M5 (225mg, 1mmol) into 20mL of acetonitrile, heating to 80 ℃ for reaction, adding the active urea intermediate (390mg, 1mmol) in the previous step, reacting for 0.5h, precipitating a large amount of solid, filtering, and leaching with diethyl ether to obtain the high-purity final product CLJ-68. ¹ H NMR(400MHz,DMSO)δ:12.64(s,1H),11.54(s,1H),9.52(s,1H),8.86(s,1H),8.11(s,1H),7.80(d,J＝8.5Hz,2H),7.65(d,J＝8.5Hz,2H),7.19(d,J＝2.3Hz,1H),6.54(s,1H),6.01(s,2H),1.31(s,9H).HRMS(ESI),m/z:391.1924[M+H] ⁺ .

EXAMPLE 69 1- (4- (4-amino-7- (2-morpholinoethyl) -pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-tert-butyl-1H-pyrazol-5-yl) urea Compound CLJ-69

The synthesis and implementation of the compoundThe same procedure as in example 54, except that the isoxazole reactive intermediate was replaced with the reactive intermediate of pyrazolylurea, gave the desired final product, CLJ-69. ¹ H NMR(400MHz,DMSO)δ12.00(s,1H),9.46(s,1H),9.05(s,1H),8.24(s,1H),7.61(dd,J＝21.8,8.6Hz,4H),6.78(s,2H),6.02(s,1H),4.46(t,J＝6.6Hz,2H),3.54–3.47(m,4H),2.80(t,J＝6.7Hz,2H),2.46(d,J＝3.8Hz,4H),1.27(s,9H).HRMS(ESI),m/z:505.2811[M+H] ⁺ .

EXAMPLE 70 1- (4- (4-amino-7-ethyl-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-tert-butyl-1H-pyrazol-5-yl) urea Compound CLJ-70

The synthesis of this compound is the same as in example 59 except that the isoxazole reactive intermediate is replaced by the reactive intermediate of pyrazolylurea to give the desired end product CLJ-70. ¹ H NMR(400MHz,DMSO)δ:12.01(s,1H),9.42(s,1H),9.00(s,1H),8.25(s,1H),7.61(dd,J＝18.2,8.6Hz,4H),6.69(s,2H),6.02(s,1H),4.37(dd,J＝14.2,7.0Hz,2H),1.42(t,J＝7.1Hz,3H),1.19(s,9H).HRMS(ESI),m/z:420.2279[M+H] ⁺ .

EXAMPLE 71 1- (4- (4-amino-7- (cyclopropylmethyl) -pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-tert-butyl-1H-pyrazol-5-yl) urea Compound CLJ-71

The synthesis of this compound is the same as in example 56 except that the active intermediate of pyrazolylurea is substituted for the active intermediate of isoxazole to give the desired end product CLJ-71. ¹ H NMR(400MHz,DMSO)δ:12.00(s,1H),9.49(s,1H),9.04(s,1H),8.25(s,1H),7.61(dd,J＝21.9,8.5Hz,4H),6.69(s,2H),6.03(s,1H),5.03(dd,J＝35.8,13.0Hz,2H),4.40(d,J＝7.1Hz,2H),2.63(dd,J＝13.6,6.8Hz,2H),1.19(s,9H),0.84(dd,J＝10.0,7.0Hz,1H).HRMS(ESI),m/z:446.2456[M+H] ⁺ .

Example 72 1- (4- (4-amino-7-propyl-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-tert-butyl-1H-pyrazol-5-yl) urea Compound CLJ-72

The synthesis of this compound is the same as in example 62 except that the active intermediate of pyrazolylurea is substituted for the active intermediate of isoxazole to give the desired end product CLJ-72. ¹ H NMR(400MHz,DMSO)δ:12.00(s,1H),9.43(s,1H),8.98(s,1H),8.25(s,1H),7.61(dd,J＝21.9,8.5Hz,4H),6.02(s,1H),4.29(t,J＝6.2Hz,2H),1.90–1.84(m,2H),1.27(s,9H),0.86(m,3H).HRMS(ESI),m/z:434.2419[M+H] ⁺ .

Example 73 1- (4- (4-amino-7-cyclopentyl-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-tert-butyl-1H-pyrazol-5-yl) urea Compound CLJ-73

The synthesis of this compound is the same as in example 63 except that the active intermediate of pyrazolylurea is substituted for the active intermediate of isoxazole to give the desired end product CLJ-73. ¹ H NMR(400MHz,DMSO)δ12.01(s,1H),9.40(s,1H),8.98(s,1H),8.23(s,1H),7.61(dd,J＝19.4,8.4Hz,4H),6.72(s,2H),6.02(s,1H),5.23(dt,J＝14.8,7.5Hz,1H),2.12–2.03(m,4H),1.90(d,J＝8.5Hz,2H),1.73–1.65(m,2H),1.27(s,9H).HRMS(ESI),m/z:460.2624[M+H] ⁺ .

Example 74 CLJ-74- (4- (4-amino-7-allyl-pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-tert-butyl-1H-pyrazol-5-yl) urea Compound

The synthesis of this compound is the same as in example 53 except that the active intermediate of pyrazolylurea is substituted for the active intermediate of isoxazole to give the desired end product CLJ-74. ¹ H NMR(400MHz,DMSO)δ:12.04(s,1H),9.41(s,1H),9.00(s,1H),8.25(s,1H),7.61(dd,J＝16.8,7.2Hz,4H),,6.72(s,2H),6.11–6.04(m,1H),6.02(s,1H),5.19(d,J＝10.4Hz,1H),5.11(d,J＝17.5Hz,1H),4.97(s,2H),1.27(s,9H).HRMS(ESI),m/z:432.2307[M+H] ⁺ .

EXAMPLE 75 1- (4- (4-amino-7- (2-cyanoethyl) -pyrazolo [2,3-d ] pyrimidin-5-yl) phenyl) -3- (3-tert-butyl-1H-pyrazol-5-yl) urea Compound CLJ-75

The synthesis method of the compound is the same as that of example 60, except that the active intermediate of the pyrazole urea is used for replacing the active intermediate of the isoxazole to obtain the target final product CLJ-75. ¹ H NMR(400MHz,DMSO)δ:12.00(s,1H),9.52(s,1H),9.05(s,1H),8.28(s,1H),7.63(dd,J＝19.1,8.1Hz,4H),6.73(s,2H),6.03(s,1H),4.61(t,J＝6.1Hz,2H),3.17(t,J＝6.0Hz,2H),1.27(s,9H).HRMS(ESI),m/z:445.2244[M+H] ⁺ .

Pharmacodynamic test section

The following representative experiments, without limitation, were used to analyze the biological activity of the compounds of the present invention.

Measuring MV4-11, molm-13 and RS4 by an MTT method; 11 cell proliferation inhibition assay

The test compounds of the invention were tested for their effect on cancer cell viability on MV4-11 and Molm-13 cells, which are human leukemia cell lines, expressing the constitutive FLT3 receptor and containing the FLT3-ITD mutation. If the compound has strong growth inhibition activity on FLT3-ITD expression cells, the compound has obvious effect on FLT3-ITD mutant strains. If the growth inhibitory activity on cells expressing FLT3-WT is poor, the compound has poor effect on FLT3-WT wild type, and the selectivity is better. The FLT3-ITD high-specificity compound AC220 (CAS: 950769-58-1) is selected as a positive control, and is synthesized by the laboratory according to a preparation method of a literature (J.Med.chem.2009, 52, 7808-7816).

MV4-11 cells (from the American type culture center, culture and breed conservation by cell Bank of the national center for biotherapy, sichuan university) were plated in 96-well plates in a medium of 100. Mu.L IMDM, molm-13 cells (Lai-Mi)From the American type culture center, by national emphasis laboratory cell Bank of biotherapy of Sichuan university) was charged in 96-well culture dishes in a medium of 100. Mu.L RPMI1640 containing 10% fetal bovine serum with 10000-15000 cells per well, the test compound was prepared in 100% DMSO, added to the cells to obtain a concentration of 100nM to 0.032nM (6 concentration points at 5-fold dilution concentration) in culture dishes at 37 ℃ of 5 CO% ₂ And culturing for 72h. RS4;11 cells (from the American type culture center, from national institute of biotherapy, national emphasis laboratory cell bank culture protection, sichuan university) were plated in 96-well plates in a medium of 100 μ L RPMI1640 containing 10% fetal bovine serum, 10000-15000 cells per well, the test compound was prepared in 100% DMSO, added to the cells to obtain a concentration of 1000nM, and the plates were incubated at 37 ℃ for 5% CO ₂ And culturing for 72h. At the end point, 20 μ LMTT (5 mg/mL) was added to each well and the cells were incubated for an additional 1-4 hours. After overnight treatment with 20% SDS, an absorbance value at a wavelength of 570nM was obtained on a spectrophotometer (Molecular Devices, sunnyvale, USA). Calculation of IC Using percent growth compared to untreated control ₅₀ The values and measurement results are shown in Table 1.

TABLE 1 IC inhibition of leukemia cell proliferation by test Compounds ₅₀ Value of

******:0.01-0.1nM；*****:0.1-1nM；****:1-10nM；***:10-100nM；**:100-1000nM；*:>1000nM

The results show that the test compounds of the present invention have better inhibitory activity on the proliferation of MV4-11 and Molm-13 cells, some of which exhibit better antiproliferative activity than AC220, and on RS4;11 is poor in inhibitory activity and is a novel, potential and potential inhibitor for treating FLT3-ITD related diseases.

In vitro kinase inhibition assay

Buffer (8 mM) MOPS, pH 7.0,0.2mM EDTA, and 10mM MnCl were added to one reaction tube ₂ ) A kinase to be tested, a substrate for the kinase to be tested, 10mM magnesium acetate and gamma ^33P ATP solution, and different concentrations of compounds, then MgATP was added to the reaction to start the enzymatic reaction process, and incubated at room temperature for 40 min. Finally, stopping the reaction by using 5 microliter of 3% phosphate buffer solution, titrating 10 microliter of reaction solution onto a Filtermat A membrane, washing the Filtermat A membrane by using 75mM phosphate solution for three times, 5 minutes each time, washing the Filtermat A membrane by using methanol once again, finally drying the Filtermat A membrane and carrying out scintillation counting on the Filtermat A membrane, wherein the scintillation counting value reflects the phosphorylation degree of a substrate, so that the kinase activity inhibition condition can be characterized.

TABLE 2 partial compound FLT3 kinase inhibitor Activity of the present invention

Test compounds	FLT3(IC ₅₀ ,nM)
		CLJ-13	20
CLJ-14	6
		CLJ-20	7
CLJ-21	9
		CLJ-22	20
CLJ-42	13
		CLJ-44	4

The result shows that the compound has better in-vitro enzymology inhibitory activity on FLT 3.

TABLE 3 dissociation constants of the inventive compound CLJ-20 for FLT3 mutant kinase

Type of mutation	Kd,nM
		FLT3 WT	4.37
FLT3(D835V)	7.04
		FLT3(ITD)	20.52
FLT3(ITD,D835V)	41.26
		FLT3(ITD,F691L)	48.47
FLT3(N841I)	2.37
		FLT3(R834Q)	10.15
FLT3(D835H)	5.14
		FLT3(D835Y)	7.78
FLT3(K663Q)	2.48

The results show that CLJ-20 also has good combination effect on various mutations of FLT3 kinase.

Part of tested compounds verify the target effect on Western Blot

The test method comprises the following steps: MV4-11 cells, molm-13 cells, were treated with the indicated concentrations of the compounds. The cells were then harvested and total protein extracted with RIPA lysis buffer (beyond time Co. P0013B, fraction: 50mM Tris, pH 7.4,150mM NaCl,1% Triton X-100,1% sodium deoxycholate, 0.1% SDS, 1). 1mM sodium orthovanadate, sodium fluoride, EDTA and leupeptin). Protein concentration was measured by BCA protein assay (ThermoScientific, USA). An equivalent sample (30. Mu.g protein) was subjected to SDS-PAGE, and then the protein was transferred onto a PVDF membrane (Millipore, USA). After blocking with 5% skim milk for 1 hour at room temperature, the membranes were incubated with the indicated primary antibodies (FLT 3 (Cell signaling technology, 3462S), p-FLT3 (Cell signaling technology, 3464S), STAT5 (Cell signaling technology, 9363S), p-STAT5 (Abcam, AB 32364), ERK (Zen Bioscience, 220003), p-ERK (Zen Bioscience, 340767), β -Actin (Abways, AB 0035)) overnight at 4 ℃ and probed with the appropriate secondary antibody (Abways, F300409) coupled with horseradish peroxidase for 1 hour. The immunoreactive bands were visualized using enhanced chemiluminescence (Millipore, USA). The molecular size of the detected protein was determined by comparison with a relevant protein marker (ThermoScientific, USA).

The experimental results are shown in figures 1 and 2, and the results show that the tested compound CLJ-20 can significantly reduce p-FLT3, p-STAT5 and p-ERK in a dose-dependent manner, and AC220 also shows the same effect, which indicates that the tested compound really influences the phosphorylation of a downstream signal channel by inhibiting the activity of FLT 3.

In vivo pharmacodynamic experiment of compound CLJ-20 on NOD/SCID nude mice

The purpose of this experiment was to examine the in vivo anti-tumor effect of the compounds of the invention using a mouse subcutaneous tumor model using MV4-11 and Molm-13 cell lines as well as developing clinical drug AC220 as a positive control.

The test method comprises the following steps: 6-8 weeks NOD/SCID mice (purchased from Beijing Huafukang Biotech GmbH) were used, as 10 ⁷ The cells with the tumor swelling to 200-500mm are inoculated in the subcutaneous posterior costal region of the mouse at the concentration of 0.1 mL/Molm-13 and MV4-11 cells ³ Thereafter, mice were grouped (6 per group) and dosed with 2% DMSO +2% PEG-400+96% normal saline, experimental groups were grouped as drug solvent control groups, and gavage was performed orally at 200. Mu.l per day, compound CLJ-20 was gavaged orally at a dose of 3mg/kg per day, compound CLJ-20 was gavaged orally at a dose of 10mg/kg per day, and positive drug AC220 was gavaged orally at 3mg/kg per day. The observation indexes are that the body weight of the mice and the long diameter and the short diameter of the tumor are measured once every two days, the volume of the tumor is calculated, and the physiological state of each group of mice is observed.

The experimental results are as follows: the Molm-13 model experiment results are shown in FIG. 3, and the MV4-11 model results are shown in FIG. 4. Experimental results show that the compound CLJ-20 has an obvious in-vivo anti-tumor effect on a Molm-13 model, can obviously inhibit tumor growth under the oral dosage of 3mg/kg, has the tumor inhibition rate of 94 percent, has the tumor inhibition rate of 96 percent at the time of 10mg/kg, has a better anti-tumor effect on AC220, has the tumor inhibition rate of 98 percent, and shows that the CLJ-20 and the AC220 have equivalent in-vivo effects. In MV4-11 model, the tumor-inhibiting rate of the test drug CLJ-20 was as high as 98% at 3mg/kg, and tumor regression was achieved in two mice, 10mg/kg on the eighth day of administration, and AC220 mg/kg group on the eighth day. No adverse reactions such as weight reduction, rash, diarrhea and the like of the mice are found in the administration process. While a slight decrease in body weight was observed in the AC220 group, indicating that CLJ-20 was less toxic over the range of doses administered at the doses tested.

In vivo survival study of compound CLJ-20 in NCG mouse model

The test method comprises the following steps: 7-8 weeks of NCG mice (purchased from Jiangsu Jiejicaokang Biotech, inc.) were used, as per 10 weeks ⁶ When the Molm-13 cells are inoculated in the tail vein of the mouse at a concentration, the cells can be massively proliferated in the blood circulation system of the mouse, so that the mouse has the symptoms of hind limb paralysis and even death. Mice were grouped (8 per group) and dosed with 2% DMSO +2% PEG-400+96% normal saline, experimental groups were drug solvent control groups, gavage was performed orally at 200 μ l per day, compound CLJ-20 was gavaged orally at a dose of 10mg/kg per day for 30 days continuously. The therapeutic effect of CLJ-20 was examined by observing and recording the survival time of mice after model establishment.

The results of the experiment show that the Median Survival Time (MST) of the mice in the solvent control group is 20 days, as shown in fig. 5. The median survival time of the 10mg/kg dose group reaches 46.5 days, is prolonged by 26.5 days compared with the control group, and is sufficiently prolonged by more than one time. This indicates that CLJ-20 has significant tumor growth inhibition effects in AML xenograft in situ models, greatly prolonging survival.

Although the present invention has been described in detail hereinabove by way of general description, specific examples and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the present invention. Accordingly, it is intended that all such modifications and variations be included within the scope of the invention as claimed and not departing from the spirit thereof.

Claims

1.4-amino-pyrimidoazepine-phenylurea derivatives having the structural formula shown in formula I:

wherein X is N or C;

R ₁ is-H, C1-C8 alkyl,

C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl or substituted C1-C4 alkyl; the substituent of the substituted C1-C4 alkyl is

-CN, -OH, phenyl, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl,

R ₄ Is C1-C4 alkoxy or

R ₅ ～R ₆ 、R ₉ ～R ₁₀ Independently is C1-C4 alkyl; r ₁₁ is-H, C1-C4 oxycarbonyl or

R ₂ Is composed of

R ₃ is-H or halogen.

2. 4-amino-pyrimidoazepine-phenylurea derivatives according to claim 1The method is characterized in that: x is N or C; r ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, methoxy, ethoxy,

Formyl, acetyl,

R ₄ Is tert-butyloxy or

R ₅ ～R ₆ 、R ₉ ～R ₁₀ Independently methyl or ethyl;

R ₂ is composed of

R ₃ is-H or halogen.

3. The 4-amino-pyrimidoazetidic-phenylurea derivative according to claim 2, characterized in that: x is N or C; r ₁ is-H, C1-C8 alkyl,

T-butyloxycarbonyl group, C2-C6 alkenyl group, C2-C6 alkynyl group,

R ₂ Is composed of

R ₃ is-H, -F, -Cl or-Br.

4. The 4-amino-pyrimidoazetidic-phenylurea derivative according to claim 1, characterized in that: when R is ₂ Is composed of

And the structure is shown as formula II:

wherein X is N or C; r is ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl,

R ₄ Is C1-C4 alkoxy or

R ₅ ～R ₆ 、R ₉ ～R ₁₀ Independently is C1-C4 alkyl;R ₁₁ is-H, C1-C4 oxycarbonyl or

R ₃ is-H or halogen.

5. The 4-amino-pyrimidoazetidic-phenylurea derivative according to claim 4, characterized in that: x is N or C; r ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, methoxy, ethoxy,

Formyl, acetyl,

R ₄ Is tert-butyloxy or

R ₅ ～R ₆ 、R ₉ ～R ₁₀ Independently methyl or ethyl;

R ₃ is-H or halogen.

6. The 4-amino-pyrimidoazetidic-phenylurea derivative according to claim 5, characterized in that: x is N or C; r ₁ is-H, C1-C8 alkyl,

T-butyloxycarbonyl group, C2-C6 alkenyl group, C2-C6 alkynyl group,

R ₃ is-H, -F, -Cl or-Br.

7. The 4-amino-pyrimidoazetidic-phenylurea derivative according to claim 1, characterized in that: when R is ₂ Is composed of

R ₃ When the structure is-H, the structure is shown as formula III:

wherein X is N or C; r is ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 carbonyl,

R ₄ Is C1-C4 alkoxy or

8. The 4-amino-pyrimidoazetidic-phenylurea derivative according to claim 7, characterized in that: x is N or C; r is ₁ is-H, C1-C8 alkyl,

-CN, -OH, phenyl, methoxy, ethoxy,

Formyl, acetyl,

R ₄ Is tert-butyloxy or

R ₅ ～R ₆ 、R ₉ ～R ₁₀ Independently methyl or ethyl.

9. The 4-amino-pyrimidoazetidic-phenylurea derivative according to claim 8, characterized in that: x is N or C; r ₁ is-H, C1-C8 alkyl,

Tert-butyloxycarbonyl radicalA C2-C6 alkenyl group, a C2-C6 alkynyl group,

4-amino-pyrimidoazetidine-phenylurea derivatives having the following structural formula:

11. a pharmaceutically acceptable salt of a 4-amino-pyrimidoazepine-phenylurea derivative according to any one of claims 1 to 10.

12. A pharmaceutically acceptable hydrate of a 4-amino-pyrimidoazetidi-n-acyclo-phenylurea derivative as claimed in any one of claims 1 to 10.

13. A pharmaceutical composition comprising the 4-amino-pyrimidoazepine-phenylurea derivative according to any one of claims 1 to 10, the salt according to claim 11 or the hydrate according to claim 12 as an active ingredient, together with a pharmaceutically acceptable carrier.

14. Use of a 4-amino-pyrimidoazepine-phenylurea derivative according to any one of claims 1 to 10, a salt according to claim 11, a hydrate according to claim 12 or a pharmaceutical composition according to claim 13 for the preparation of a FLT3 kinase inhibitor.

15. Use according to claim 14, characterized in that: the FLT3 kinase is a mutant FLT3 kinase.

16. Use according to claim 15, characterized in that: the mutant FLT3 kinase is FLT3/ITD mutant kinase.

17. Use of a 4-amino-pyrimidoazepine-phenylurea derivative according to any of claims 1 to 10, a salt according to claim 11, a hydrate according to claim 12 or a pharmaceutical composition according to claim 13 for the preparation of a medicament for the treatment of a tumor.

18. The use of claim 17, wherein the tumor comprises a solid tumor and/or a hematological tumor.

19. The use of claim 18, the solid tumor comprising: lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic lymphoma, lymphoplasmacytic lymphoma, ovarian cancer, breast cancer, prostate cancer, bladder cancer, kidney cancer, esophageal cancer, neck cancer, pancreatic cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, thyroid cancer, brain cancer, lymphatic cancer, epidermal hyperplasia, psoriasis and/or prostatic hyperplasia.

20. The use of claim 18, the hematological neoplasm comprising: acute myeloid leukemia, chronic myeloid leukemia, myeloma, acute lymphocytic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, chronic neutrophilic leukemia, acute undifferentiated cell leukemia, myelodysplastic syndrome, myelodysplasia, multiple myeloma, and/or myelosarcoma.