CN109721620B

CN109721620B - HPK1 inhibitors and uses thereof

Info

Publication number: CN109721620B
Application number: CN201811172437.8A
Authority: CN
Inventors: 吴永谦
Original assignee: Yaojie Ankang Nanjing Technology Co ltd
Current assignee: Yaojie Ankang Nanjing Technology Co ltd
Priority date: 2017-10-27
Filing date: 2018-10-09
Publication date: 2022-05-13
Anticipated expiration: 2038-10-09
Also published as: CN109721620A

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an HPK1 inhibitor compound shown in a formula (I) or pharmaceutically acceptable salts and stereoisomers thereof, and also relates to a pharmaceutical preparation, a pharmaceutical composition and application of the compounds. X₁、X₂、X₃、X₄、X₅、X₆、X₇、X₈、X₉、R₁、R₂、R₃As defined in the specification. The compounds of the invention are useful in the preparation of medicamentsTreating or preventing diseases related to HPK1 mediated diseases.

Description

HPK1 inhibitors and uses thereof

Technical Field

The invention belongs to the technical field of medicines, and relates to an HPK1 inhibitor compound shown in a general formula (I), or pharmaceutically acceptable salts and stereoisomers thereof, and application thereof.

Background

T Cell Receptor (TCR) -mediated T cell activation plays a crucial role in thymic T cell development, T cell subset differentiation, and effector T cell function. The TCR specifically recognizes an antigen peptide (peptide) presented on MHC on the surface of an antigen-presenting cell, and converts extracellular antigen peptide into a signal that can be delivered to the inside of the cell through recognition by MHC (major histocompatibility complex). Among these, MHC molecules on the surface of antigen-presenting cells include MHC class II and MHC class I molecules, which are specifically recognized by the corresponding co-receptor CD4 and CD8 molecules on the surface of CD4+ and CD8+ T cells, respectively, and subsequently cause activation of downstream signaling pathways. Typical intracellular signals for TCR activation include signaling pathways such as MAPK (mitogen-activated protein kinases), PKC (protein kinase C), and calceium (calcium ion). Activation of these signals ultimately activates specific gene expression of the T cell, causing proliferation of the cell and differentiation of the T cell into an effector T cell.

HPK1, also known as MAP4K1, is a serine/threonine kinase that is a member of the MAP4K family. Besides, there are 5 members in the family, including MAP3K2, MAP4K3, MAP4K4, MAP4K5, MAP4K 6. HPK1 can bind to many linker proteins, such as SLP-76 family, CARD11, HIS, HIP-55, GRB2 family, LAT, CRK family, etc., and activate JNK/SAPK signaling pathway of hematopoietic stem cells, thereby negatively regulating TCR pathways. The biological effect of MAP4K3, also known as GLK kinase, is exactly opposite to that of HPK 1. GLK may facilitate activation of the TCR pathway by binding to downstream adaptor proteins. Therefore we need to screen for inhibitors of HPK1 that are selective for GLK.

The main processes involved in the regulation of TCR by HPK1 are (1) binding of TCR to extracellular antigen via MHC, thereby activating the TCR pathway to signal downstream adaptor molecules; (2) joint protein tyrosine kinase Lck and Zap70 activated SLP76, which in turn phosphorylates HPK 1; (3) activated HPK1 will in turn phosphorylate the receptor protein SLP-76; (4) phosphorylation reaction of SLP-76 provides multiple protein binding sites for 14-3-3(TCR pathway inhibitory protein) receptor protein to form a complex; (5) the SLP-76 phosphorylated complex is involved in the down-regulation of the Erk signaling pathway and is coupled to the ubiquitination degradation process of SLP76, resulting in a decrease in TCR signaling pathway and T cell proliferation. In conclusion, HPK1 can negatively regulate TCR signaling pathway, and thus HPK1 can be a new regulatory mechanism of T cell-mediated immune response, and becomes a hot spot for development of new immune anti-tumor.

Because of its important role in immunity, HPK1 inhibitors play an important role in malignant solid tumors or hematological cancers (e.g., acute myelogenous leukemia, urothelial cancer, breast cancer, colon cancer, lung cancer, pancreatic cancer, melanoma), autoimmune diseases (e.g., systemic lupus erythematosus, psoriatic arthritis), and inflammatory responses.

At present, aiming at the HPK1 target, no medicine is on the market, in order to better meet the huge clinical requirement, the aim is to develop the HPK1 inhibitor with high selectivity and high activity, and the invention provides a selective HPK1 inhibitor with a novel structure, which has good physicochemical property and patent medicine property.

Disclosure of Invention

The invention aims to provide an HPK1 kinase inhibitor compound, which has good kinase selectivity, inhibitory activity on HPK1 and good drug property and can be used for treating or preventing related diseases mediated by HPK 1.

The technical scheme adopted by the invention is as follows:

a compound represented by the general formula (I) or a pharmaceutically acceptable salt, stereoisomer thereof:

wherein, X₁、X₂、X₃、X₄Are each independently selected from CR₄Or N;

X₅selected from the group consisting of CR₄Or N;

X₆selected from the group consisting of CR₄R₅、NR₆O or S;

X₇、X₈、X₉are each independently selected from CR₄、CR₄R₅、N、NR₆O or S;

R₄、R₅at each occurrence, is independently selected from hydrogen, hydroxy, amino, carboxy, cyano, nitro, a halogen atom, or C optionally substituted with a substituent_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino, halogeno C_1-6Alkyl, halo C_1-6Alkoxy radical, C_2-8Alkenyl radical, C_2-8Alkynyl, 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl, said substituents selected from hydroxy, amino, carboxy, cyano, nitro, halogen, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6An alkylsulfonylamino group;

R₆at each occurrence, is independently selected from hydrogen, carboxyl, cyano, halogen atom, or C optionally substituted by a substituent_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino, halogeno C_1-6Alkyl, halo C_1-6Alkoxy radical, C_2-8Alkenyl radical, C_2-8Alkynyl, 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl, said substituents selected from hydroxy, amino, carboxy, cyano, nitro, halogen, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6An alkylsulfonylamino group;

R₁selected from hydrogen, -NRaRb, -ORc, -SO₂-NRaRb、-NRa-SO₂-Rd, CO-NRaRb, -NRa-CO-Rd, cyano, nitro, halogen atom, C_1-6An alkyl group;

ra is selected from hydrogen, hydroxyl, amino, carboxyl, cyano, nitro, halogen atom, or C optionally substituted by substituent_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino, halogeno C_1-6Alkyl, halo C_1-6Alkoxy radical, C_2-8Alkenyl radical, C_2-8Alkynyl, C_1-6Alkylsulfonyl radical, C_1-6Alkylcarbonyl group, C_1-6Alkylthio, said substituent being selected from the group consisting of hydroxy, amino, carboxy, cyano, nitro, halogen, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6Alkylsulfonylamino, 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl;

rb is selected from hydrogen or C_1-6An alkyl group;

rc is selected from hydrogen, hydroxyl, amino, carboxyl, cyano, nitro, halogen atom, or C optionally substituted by substituent_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino, halogeno C_1-6Alkyl, halo C_1-6Alkoxy radical, C_2-8Alkenyl radical, C_2-8Alkynyl, C_1-6Alkylsulfonyl radical, C_1-6Alkylcarbonyl group, C_1-6Alkylthio, -Y₁-Q₁The substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical,C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6Alkylsulfonylamino, 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl;

rd is selected from hydrogen, or C optionally substituted by substituents_1-6Alkyl, the substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6Alkylsulfonylamino, 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl;

R₂selected from hydrogen, hydroxy, amino, carboxyl, cyano, nitro, halogen atoms, or C optionally substituted by substituents_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino, halogeno C_1-6Alkyl, halo C_1-6Alkoxy radical, C_2-8Alkenyl radical, C_2-8Alkynyl, C_1-6Alkylsulfonyl radical, C_1-6Alkylcarbonyl group, C_1-6Alkylthio, -Y₂-Q₂The substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6Alkylsulfonylamino, 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl;

Y₁and Y₂Are each independently selected from the group consisting of a bond, - (CH)₂)n、-C(O)-、-SO_2-or-NH-C (O) -;

Q₁、Q₂each independently selected from 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl optionally substituted with a substituent selected from hydroxy, amino, carboxyRadical, cyano radical, nitro radical, halogen atom, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6Alkylsulfonylamino, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_2-8Alkenyl radical, C_2-8Alkynyl, C_1-6Alkylsulfonyl radical, C_1-6Alkylcarbonyl group, C_1-6Alkylthio, 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl;

n is 1 to 4;

R₃selected from hydrogen or C_1-6An alkyl group.

Provided that when X is₁、X₂、X₃、X₄Are respectively CR₄，

X₅Is selected from the group consisting of N,

X₆selected from NR₆，

X₇、X₈、X₉One of which is S and the other two are CR₄，

R₁When Ra and Rb are each independently hydrogen for-NRaRb,

R₂is-Y₂-Q₂，Y₂Is a bond, Q₂Is composed of

In another preferred embodiment, the compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer thereof,

X₅is selected from N;

X₆selected from the group consisting of CR₄R₅、NR₆O or S;

R₄、R₅、R₆at each occurrence, R₄、R₅、R₆Each independently selected from hydrogen, halogen, cyano, C_1-6An alkyl group;

R₁selected from hydrogen, -NRaRb, -NRa-CO-Rd, -NRa-SO₂-Rd、C_1-6Alkyl, -ORc;

ra is selected from hydrogen and C_1-6Alkyl, cyano;

rb, Rc and Rd are respectively and independently selected from hydrogen and C_1-6An alkyl group;

R₂selected from hydrogen, C_1-6Alkyl or-Y₂-Q₂；

Y₂Is a bond;

Q₂selected from the group consisting of_1-6Alkyl or 3-12 membered cycloalkyl substituted 3-12 membered heterocyclyl;

R₃selected from hydrogen or C_1-6An alkyl group;

provided that when X is₁、X₂、X₃、X₄Are respectively CR₄，

X₅Is selected from the group consisting of N,

X₆selected from NR₆，

X₇、X₈、X₉One of which is S and the other two are each independently CR₄，

R₁When Ra and Rb are each independently hydrogen for-NRaRb,

R₂is-Y₂-Q₂，Y₂Is a bond, Q₂Is composed of

In another preferred embodiment, the compound shown in the formula (I) or the pharmaceutically acceptable salt and the stereoisomer thereof have a structure shown in a general formula (II),

wherein, X₁、X₂、X₃、X₄Are each independently selected from CR₄Or N, and X₁、X₂、X₃、X₄At least one of which is N;

X₆selected from the group consisting of CR₄R₅、NR₆O or S;

R₄、R₅at each occurrence, is independently selected from hydrogen, hydroxy, amino, carboxy, cyano, nitro, a halogen atom, or C optionally substituted with a substituent_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino, halogeno C_1-6Alkyl, halo C_1-6Alkoxy, 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl, said substituents selected from: hydroxy, amino, carboxyl, cyano, nitro, halogen, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂An amino group;

R₆at each occurrence, is independently selected from hydrogen, carboxyl, cyano, halogen atom, or C optionally substituted by a substituent_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino, halogeno C_1-6Alkyl, halo C_1-6Alkoxy, 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl, said substituents selected from hydroxy, amino, carboxy, cyano, nitro, halogen, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6Alkyl sulfonamidesA group;

R₁selected from-NRaRb, -ORc, -NRa-SO₂-Rd、-NRa-CO-Rd、C_1-6An alkyl group;

ra is selected from hydrogen, cyano, or C optionally substituted by a substituent_1-6Alkyl radical, C_1-6Alkylsulfonyl radical, C_1-6Alkylcarbonyl, said substituent being selected from the group consisting of hydroxy, amino, carboxy, cyano, nitro, halogen, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6An alkylsulfonylamino group;

rb is selected from hydrogen;

rc is selected from hydrogen, hydroxyl, amino, carboxyl, cyano, nitro, halogen atom, or C optionally substituted by substituent_1-6Alkyl, -Y₁-Q₁The substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6An alkylsulfonylamino group;

rd is selected from hydrogen, or C optionally substituted by substituents_1-6Alkyl, the substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6An alkylsulfonylamino group;

Y₁selected from a bond or- (CH)₂)n-；

Q₁Selected from 3-12 membered cycloalkyl optionally substituted by a substituent selected from the group consisting of hydroxy, amino, carboxy, cyano, nitro, a halogen atom, C, a heterocyclic group, a 5-6 membered heteroaryl, phenyl_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6Alkylsulfonylamino, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylsulfonyl radical, C_1-6Alkylthio, 3-8 membered cycloalkyl, aryl, 4-6 membered heterocyclyl, 5-10 membered heteroaryl;

R₂selected from hydrogen, hydroxy, amino, carboxyl, cyano, nitro, halogen atoms, or C optionally substituted by substituents_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino, halogeno C_1-6Alkyl, halo C_1-6Alkoxy, -Y₂-Q₂The substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6An alkylsulfonylamino group;

Y₂selected from a bond or- (CH)₂)n-，

Q₂Selected from 3-12 membered heterocyclic group optionally substituted by a substituent selected from hydroxyl group, amino group, carboxyl group, cyano group, nitro group, halogen atom, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6Alkylsulfonylamino, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylsulfonyl radical, C_1-6Alkylthio, 3-8 membered cycloalkyl, aryl, 4-6 membered heterocyclyl, 5-10 membered heteroaryl;

n is 1 to 4;

R₃selected from hydrogen or C_1-6An alkyl group.

In another embodiment, Q₂Is a 4-8 membered heteromonocyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spiroheterocyclic group, 6-12 membered heterocyclo group, 5-6 membered heteroaryl group, 9-10 membered heteroaryl group, more optionally substituted with a substituentFurther preferred are a 4-to 8-membered saturated nitrogen-containing heteromonocyclic group, a 7-to 10-membered saturated nitrogen-containing bridged heterocyclic group, a 7-to 10-membered saturated nitrogen-containing spiroheterocyclic group, and a 7-to 10-membered saturated nitrogen-containing heterocyclic group, which are optionally substituted by a substituent as defined above.

In another embodiment, X₇Is S, X₈Is CR₄，X₉Is CR₄Or N;

in another embodiment, X₇Is CR₄，X₈Is S, X₉Is CR₄；

In another embodiment, X₇Is CR₄，X₈Is CR₄，X₉Is S.

In another preferred embodiment, the compound represented by the formula (II) or a pharmaceutically acceptable salt, stereoisomer thereof,

X₆selected from the group consisting of CR₄R₅、NR₆O or S;

R₄、R₅、R₆at each occurrence, R₄、R₅、R₆Each independently selected from hydrogen and C_1-6An alkyl group;

R₁selected from-NRaRb, -ORc, C_1-6An alkyl group;

ra, Rb and Rc are respectively and independently selected from hydrogen or C_1-6An alkyl group;

R₂selected from hydrogen, C_1-6Alkyl or-Y₂-Q₂；

Y₂Is a key, and is provided with a plurality of keys,

Q₂selected from the group consisting of_1-6Alkyl, 3-to 8-membered cycloalkyl substituted 3-to 12-membered heterocyclyl;

R₃selected from hydrogen or C_1-6An alkyl group.

In another preferred embodiment, the compound shown in the formula (II) or the pharmaceutically acceptable salt and the stereoisomer thereof have a structure shown in a general formula (III),

X₉are each independently selected from CR₄Or N;

R₄at each occurrence, is independently selected from hydrogen, hydroxy, amino, carboxy, cyano, nitro, halogen atom, C_1-6Alkyl radical, C_1-6An alkoxy group;

ra is selected from hydrogen, or C optionally substituted by substituents_1-6Alkyl, the substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6An alkoxy group;

rb is selected from hydrogen;

rc is selected from hydrogen, or C optionally substituted by substituents_1-6Alkyl, -Y₁-Q₁The substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6An alkoxy group;

rd is selected from hydrogen, or C optionally substituted by substituents_1-6Alkyl, the substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6An alkoxy group;

Y₁selected from a bond or- (CH)₂)n；

Q₁Selected from 3-8 membered cycloalkyl optionally substituted by a substituent, 3-8 membered heterocyclyl,the substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen atom and C_1-6Alkyl radical, C_1-6An alkoxy group;

R₂is selected from-Y₂-Q₂；

Y₂Selected from a bond or- (CH)₂)n-，

Q₂Selected from the group consisting of 4-8 membered heteromonocyclic group, 6-12 membered spiroheterocyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered heterocyclic group and 6-12 membered heterocyclic group optionally substituted with a substituent selected from the group consisting of hydroxyl group, amino group, carboxyl group, cyano group, nitro group, halogen atom, C_1-6Alkyl radical, C_1-6Alkoxy, 3-6 membered cycloalkyl;

n is 1 to 4.

In another embodiment, Q2 is selected from 4-8 membered saturated heteromonocyclic group, 7-10 membered saturated spiroheterocyclic group, 7-10 membered saturated bridged heterocyclic group, optionally substituted with a substituent;

in another embodiment, Q2 is selected from the group consisting of a 4-8 membered saturated nitrogen-containing heteromonocyclic group, a 7-10 membered saturated nitrogen-containing spiroheterocyclic group, and a 7-10 membered saturated nitrogen-containing bridged heterocyclic group, optionally substituted with a substituent.

In another embodiment, Q2 is selected from piperazinyl, morpholinyl, piperidinyl, optionally substituted with substituents as defined above.

In another preferred embodiment, the compound represented by the formula (III) or a pharmaceutically acceptable salt, stereoisomer thereof,

X₉are each independently selected from CR₄Or N;

R₄at each occurrence, is independently selected from hydrogen, C_1-6An alkyl group;

R₁selected from-NRaRb, -ORc, C_1-6An alkyl group;

ra, Rb and Rc are independently selected from hydrogen or C_1-6An alkyl group;

R₂is selected from-Y₂-Q₂；

Y₂Is selected from a bond;

Q₂selected from the group consisting of 4-8 membered heteromonocyclic group, 6-12 membered spiroheterocyclic group, 6-12 membered bridged heterocyclic group optionally substituted with a substituent selected from the group consisting of C_1-6Alkyl, 3-6 membered cycloalkyl.

wherein,

X₁、X₂、X₃are respectively CR₄，X₄Is N;

X₉are each independently selected from CR₄Or N;

R₄at each occurrence, independently selected from hydrogen, C_1-6An alkyl group;

R₁selected from-NRaRb, ORc, C_1-6An alkyl group;

ra, Rb and Rc are respectively and independently selected from hydrogen and C_1-6An alkyl group;

R₂selected from hydrogen, C_1-6Alkyl or-Y₂-Q₂；

Y₂Is selected from the group consisting of a bond,

Q₂selected from the group consisting of pyrrolidinyl, piperazinyl, morpholinyl, piperidinyl, azepinyl, optionally substituted,

The substituent is selected from C_1-6Alkyl radical, C_1-6An alkoxy group.

wherein,

X₁、X₂、X₃are respectively CR₄，X₄Is N;

X₉are each independently selected from CR₄Or N;

R₁selected from-NRaRb, -ORc, C_1-6An alkyl group;

ra, Rb and Rc are independently selected from hydrogen or C_1-6An alkyl group;

R₂selected from hydrogen, C_1-6Alkyl or-Y₂-Q₂；

Y₂Is selected from the group consisting of a bond,

The substituent is selected from cyclopropyl and C_1-6An alkyl group.

In another preferred embodiment, the compound shown in the formula (I) or the pharmaceutically acceptable salt and the stereoisomer thereof have a structure shown in a general formula (IV),

ra is selected from hydrogen, cyano, or C optionally substituted by a substituent_1-6Alkyl, the substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6An alkoxy group;

rb is selected from hydrogen;

rc is selected from hydrogen, or C optionally substituted by a substituent_1-6Alkyl, -Y₁-Q₁The substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6An alkoxy group;

Y₁selected from a bond or- (CH)₂)n-；

Q₁Selected from 3-8 membered cycloalkyl groups optionally substituted with a substituent selected from the group consisting of a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group, a halogen atom, C_1-6Alkyl radical, C_1-6An alkoxy group;

R₂is selected from-Y₂-Q₂；

Y₂Selected from a bond or- (CH)₂)n-，

Q₂Selected from the group consisting of 4-8 membered heteromonocyclic group, 6-12 membered spiroheterocyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered heterocyclic group, and 6-12 membered heterocyclic group optionally substituted with a substituent selected from the group consisting of hydroxyl group, amino group, carboxyl group, cyano groupNitro group, halogen atom, C_1-6Alkyl radical, C_1-6Alkoxy, 3-6 membered cycloalkyl;

n is 1 to 4.

In another embodiment, Q2 is piperazinyl, morpholinyl, piperidinyl, optionally substituted with substituents as defined above.

In another preferred embodiment, the compound shown in the formula (I) or the pharmaceutically acceptable salt and the stereoisomer thereof have a structure shown in a general formula (V),

rb is selected from hydrogen;

rc is selected from hydrogen, or C optionally substituted by substituents_1-6Alkyl, -Y₁-Q₁Said substituent groupSelected from hydroxy, amino, carboxyl, cyano, nitro, halogen, C_1-6Alkyl radical, C_1-6An alkoxy group;

Y₁selected from a bond or- (CH)₂)n-；

R₂is selected from-Y₂-Q₂；

Y₂Selected from a bond or- (CH)₂)n-，

n is 1 to 4.

in another embodiment, Q2 is selected from 4-8 membered saturated nitrogen-containing heteromonocyclic group, 7-10 membered saturated nitrogen-containing spiroheterocyclic group, 7-10 membered saturated nitrogen-containing bridged heterocyclic group, optionally substituted with a substituent;

In another preferred embodiment, the compound shown in the formula (I) or the pharmaceutically acceptable salt and the stereoisomer thereof have a structure shown in a general formula (VI),

wherein, X₁、X₂、X₃Are each independently selected from CR₄Or N;

X₆selected from the group consisting of CR₄R₅、NR₆O or S;

X₇、X₉are each independently selected from CR₄、CR₄R₅、N、NR₆O or S;

R₄、R₅at each occurrence, is independently selected from hydrogen, hydroxy, amino, carboxy, cyano, nitro, a halogen atom, or C optionally substituted with a substituent_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino, halogeno C_1-6Alkyl, halo C_1-6Alkoxy radical, C_2-8Alkenyl radical, C_2-8Alkynyl, 3-8 membered cycloalkyl, 3-8 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl, said substituents selected from: hydroxy, amino, carboxyl, cyano, nitro, halogen, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6An alkylsulfonylamino group;

R₆at each occurrence, is selected from a hydrogen, carboxyl, cyano, halogen atom, or C optionally substituted by a substituent_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino, halogeno C_1-6Alkyl, halo C_1-6Alkoxy radical, C_2-8Alkenyl radical, C_2-8Alkynyl, 3-12 membered cycloalkyl, 3-12 membered cycloalkenyl, aryl, 3-12 membered heterocyclyl, 5-10 membered heteroaryl, said substituents selected from hydroxy, amino, carboxy, cyano, nitro, halogen, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6AlkylcarbonylaminesBase, C_1-6An alkylsulfonylamino group;

R₁selected from hydrogen, -NRaRb, -ORc, -NRa-SO₂-Rd, -NRa-CO-Rd, cyano, nitro, halogen atom, C_1-6An alkyl group;

rb is selected from hydrogen;

Y₁selected from a bond or- (CH)₂)n-；

Q₁Selected from 3-8 membered cycloalkyl optionally substituted by a substituent selected from the group consisting of hydroxy, amino, carboxy, cyano, nitro, a halogen atom, C, 5-6 membered heterocyclyl, 5-6 membered heteroaryl, phenyl_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6Alkylsulfonylamino, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylsulfonyl radical, C_1-6Alkylthio, 3-8 membered cycloalkyl, aryl, 4-6 membered heterocyclyl, 5-10 membered heteroaryl;

Y₂selected from a bond or- (CH)₂)n-，

Q₂Is selected from 3-12 membered heterocyclic group optionally substituted by a substituent selected from the group consisting of hydroxyl group, amino group, carboxyl group, cyano group, nitro group, halogen atom, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, (C)_1-6Alkyl radical)₂Amino group, C_1-6Alkylcarbonylamino, C_1-6Alkylsulfonylamino, halo C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylsulfonyl radical, C_1-6Alkylthio, 3-8 membered cycloalkyl, aryl, 4-6 membered heterocyclyl, 5-10 membered heteroaryl;

n is 1 to 4.

In another embodiment, Q₂Is a 4-8 membered heteromonocyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered spiroheterocyclic group, 6-12 membered heterocyclic group, 5-6 membered heteroaryl group, 9-10 membered heteroaryl group, which is optionally substituted, more preferably optionally substitutedSubstituted 4-8 membered saturated nitrogen-containing heteromonocyclic group, 7-10 membered saturated nitrogen-containing bridged heterocyclic group, 7-10 membered saturated nitrogen-containing spiro heterocyclic group, and 7-10 membered saturated nitrogen-containing heterocyclic group, wherein the substituents are as defined above.

In another preferred embodiment, the compound shown in the formula (VI) or the pharmaceutically acceptable salt and the stereoisomer thereof have a structure shown in a general formula (VII),

wherein, X₁、X₂、X₃Are each independently selected from CR₄Or N;

X₆selected from NR₆；

R₆At each occurrence, is independently selected from hydrogen, cyano, C_1-6Alkyl radical, C_1-6An alkoxy group;

R₁selected from-NRaRb, -ORc, -NRa-SO₂-Rd or-NRa-CO-Rd;

rb is selected from hydrogen;

rd is selected from hydrogen, or C optionally substituted by substituents_1-6Alkyl, the substituent is selected from hydroxyl, amino, carboxyl, cyano, nitro, halogen and C_1-6Alkyl radical, C_1-6An alkoxy group; y is₁Selected from a bond or- (CH)₂)n-；

R₂is selected from-Y₂-Q₂；

Y₂Is selected from the group consisting of a bond,

Q₂selected from the group consisting of 4-8 membered heteromonocyclic group, 6-12 membered spiroheterocyclic group, 6-12 membered bridged heterocyclic group, 6-12 membered heterocyclic group and 6-12 membered heterocyclic group optionally substituted with a substituent selected from the group consisting of hydroxyl group, amino group, carboxyl group, cyano group, nitro group, halogen atom, C_1-6Alkyl radical, C_1-6An alkoxy group;

n is 1 to 4.

In another preferred embodiment, the compound represented by the formula (VII) or a pharmaceutically acceptable salt, stereoisomer thereof,

wherein, X₁、X₂、X₃Are each independently selected from CR₄Or N;

X₆selected from NR₆；

R₆At each occurrence, independently selected from hydrogen, C_1-6An alkyl group;

R₁selected from-NRaRb, -ORc, C_1-6An alkyl group;

R₂selected from hydrogen, C_1-6Alkyl, -Y₂-Q₂；

Y₂Is selected from a bond;

Q₂selected from pyrrolidinyl, piperazinyl, morpholinyl, piperidinyl, azepanyl optionally substitutedA base,

The substituent is selected from cyclopropyl and C_1-6An alkyl group.

wherein, X₁、X₂、X₃、X₄Are respectively CR₄；

X₅Is selected from N;

X₆selected from NR₆；

X₇、X₈、X₉One of which is S and the other two are CR₄；

R₄、R₆At each occurrence, independently selected from hydrogen, C_1-6An alkyl group;

R₁selected from hydrogen, -NRa-SO₂-Rd、-CO-NRaRb、-NRa-CO-Rd、C_1-6An alkyl group;

ra, Rb, Rc and Rd are respectively and independently selected from hydrogen or C_1-6An alkyl group;

R₂selected from hydrogen, C_1-6Alkyl, -Y₂-Q₂；

Y₂Is selected from a bond;

Q₂selected from the group consisting of_1-6An alkyl-substituted 3-12 membered heterocyclic group,

preferably, selected from optionally substituted C_1-6An alkyl-substituted 4-to 8-membered heteromonocyclic group,

more preferably, Q₂Selected from the group consisting of:

R₃selected from hydrogen or C_1-6An alkyl group;

provided that when X is₁、X₂、X₃、X₄Are respectively CR₄，

X₅Is selected from the group consisting of N,

X₆selected from NR₆，

X₇、X₈、X₉One of which is S and the other two are CR₄，

R₁When Ra and Rb are each independently hydrogen for-NRaRb,

R₂is-Y₂-Q₂，Y₂Is a bond, Q₂Is composed of

A compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer thereof, wherein the compound is:

TABLE 1

The invention also claims a pharmaceutical composition of any compound of the invention or pharmaceutically acceptable salts and stereoisomers thereof, which can be optionally prepared into any pharmaceutically acceptable pharmaceutical preparation by containing one or more pharmaceutically acceptable carriers.

In one embodiment of the present invention, the aforementioned pharmaceutical composition may further comprise one or more second therapeutically active agents. The second therapeutically active agent is an antimetabolite, a growth factor inhibitor, a filamentation classification inhibitor, an antineoplastic hormone, an alkylating agent, a metal, a topoisomerase inhibitor, a hormonal agent, an immunomodulator, a tumor suppressor, a cancer vaccine, an immune checkpoint or an antibody or small molecule drug associated with tumor immunotherapy.

The invention also claims a pharmaceutical preparation of any compound of the invention or pharmaceutically acceptable salt, stereoisomer thereof, which is characterized by comprising one or more pharmaceutical carriers.

The pharmaceutical carrier of the present invention may be one or more solid or liquid filler or gel materials suitable for human use. The pharmaceutically acceptable carrier is preferably of sufficient purity and sufficiently low toxicity, and is compatible with the active ingredients of the present invention without significantly diminishing the efficacy of the active ingredient. For example, the pharmaceutically acceptable carrier may be a filler, a binder, a disintegrant, a lubricant, an aqueous solvent or a non-aqueous solvent, and the like.

The pharmaceutical preparation of the present invention may be prepared into any pharmaceutically acceptable dosage form, and administered to a patient or subject in need of such treatment by any suitable administration, for example, oral, parenteral, rectal, or pulmonary administration. For oral administration, it can be made into tablet, capsule, pill, granule, etc. For parenteral administration, it can be made into injection, injectable sterile powder, etc.

The invention also claims the use of the compounds of the invention in medicaments for the treatment or prevention of diseases related to HPK1 mediated thereby. HPK1 has negative feedback regulation effect in T cell mediated signal path, so that the inhibitor of HPK1 can be used as an antitumor drug for treating cancer or non-cancerous proliferative diseases, wherein said disease comprises lung cancer, squamous cell carcinoma, bladder cancer, stomach cancer, ovarian cancer, peritoneal cancer, breast cancer, ductal carcinoma of the breast, head and neck cancer, endometrial cancer, uterine corpus cancer, rectal cancer, liver cancer, kidney cancer, renal pelvis cancer, esophageal adenocarcinoma, glioma, prostate cancer, thyroid cancer, cancer of the female reproductive system, carcinoma in situ, lymphoma, neurofibromatosis, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, oral cancer, pharyngeal cancer, multiple myeloma, leukemia, non-Hodgkin's lymphoma, villous adenoma of the large intestine, melanoma, cell tumor, and sarcoma, myelodysplastic syndrome.

Detailed Description

The "halogen" in the present invention means fluorine, chlorine, bromine, iodine, etc., and preferably fluorine atom, chlorine atom.

The term "halo" as used herein means that any hydrogen atom in a substituent may be substituted by one or more halogen atoms which may be the same or different. "halogen" is as defined above.

"C" according to the invention_1-6The "alkyl group" means a straight chain or branched alkyl group derived from a hydrocarbon moiety having 1 to 6 carbon atoms by removing one hydrogen atom, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 1-methyl-2-methylpropyl, and the like. Said "C_1-4Alkyl "refers to the above examples containing 1 to 4 carbon atoms.

"C" according to the invention_2-8The "alkenyl group" means a straight chain or branched chain derived from an olefin having 2 to 8 carbon atoms containing a carbon-carbon double bond with one hydrogen atom removed, such as vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1, 3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1, 3-pentadienyl, 1, 4-pentadienyl, 1-hexenyl, 1, 4-hexadienyl.

"C" according to the invention_2-8Alkynyl refers to straight-chain or branched alkynyl derived from an alkynyl containing 2-8 carbon atoms with a carbon-carbon triple bond and one hydrogen atom removed, such as ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, and the like.

"C" according to the invention_1-6Alkoxy radical"means" C "as defined hereinbefore_1-6Alkyl "radicals" attached to the parent molecule via an oxygen atom, i.e. "C_1-6alkyl-O- "groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Said "C_1-4Alkoxy "refers to the above examples containing 1 to 4 carbon atoms, i.e." C_1-4An alkyl-O- "group.

"C" of the invention_1-6Alkylamino "," (C)_1-6Alkyl radical)₂Amino group and C_1-6Alkylcarbonylamino group and C_1-6Alkylsulfonylamino group and "C_1-6Alkylaminocarbonyl "," (C)_1-6Alkyl radical)₂Amino-carbonyl group and C_1-6Alkoxy-carbonyl group and C_1-6Alkylsulfonyl group "," C_1-6Alkylthio group "," C_1-6Alkylcarbonyl "each denotes C_1-6alkyl-NH-, (C)_1-6Alkyl) (C_1-6Alkyl) N-, C_1-6alkyl-C (O) -NH-, C_1-6alkyl-S (O)₂-NH₂-、C_1-6alkyl-NH-C (O) -, (C)_1-6Alkyl) (C_1-6Alkyl) N-C (O) -, C_1-6alkyl-O-C (O) -, C_1-6alkyl-S (O)₂-、C_1-6alkyl-S-, C_1-6alkyl-C (O) -; said "C_1-6Alkyl "is as defined above, preferably" C_1-4Alkyl groups ".

The "condensed ring" in the present invention refers to a polycyclic structure formed by connecting two or more cyclic structures in a parallel, spiro or bridged manner. The fused ring refers to a fused ring structure formed by two or more ring structures sharing two adjacent ring atoms with each other (i.e., sharing one bond). The bridged ring refers to a condensed ring structure formed by two or more ring-assembled structures sharing two non-adjacent ring atoms with each other. The spiro ring refers to a fused ring structure formed by two or more cyclic structures sharing one ring atom with each other.

The "3-to 12-membered cycloalkenyl" as used herein includes, unless otherwise specified, all monocyclic and fused rings (including fused rings in the form of a parallel, spiro or bridge) which may be formed, for example, 3-to 8-membered cycloalkenes, 7-to 11-membered spirocycloalkenes, 7-to 11-membered benzocycloalkenes, 6-to 11-membered bridged cycloalkenes, and the like.

The "3-12 membered cycloalkyl" group of the present invention may be a monocyclic, bicyclic, or polycyclic cycloalkyl system (also referred to as fused ring system). Unless otherwise specified, all monocyclic, fused ring (including fused in the form of a parallel, spiro, or bridge) forms that may be formed are included. Monocyclic ring systems are cycloalkyl groups containing 3 to 8 carbon atoms. Examples of 3-8 membered cycloalkyl groups include, but are not limited to: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, etc. Fused ring cycloalkyl includes fused ring cycloalkyl, bridged cycloalkyl, spirocycloalkyl. The acyclic cycloalkyl group may be a 6-11 membered acyclic heterocycloalkyl group, a 7-10 membered acyclic heterocycloalkyl group, representative examples of which include, but are not limited to, bicyclo [3.1.1]Heptane, bicyclo [2.2.1]Heptane, bicyclo [2.2.2]Octane, bicyclo [3.2.2]Nonane, bicyclo [3.3.1]Nonanes and bicyclo [4.2.1]Nonane. The spiro group may be 7-12-membered spiro ring group, 7-11-membered spiro ring group, and examples thereof include, but are not limited to:

the bridge ring group may be a 6-11-membered bridge ring group, a 7-10-membered bridge ring group, examples of which include, but are not limited to:

the "heterocyclic group" as used herein means a non-aromatic cyclic group in which at least one ring carbon atom of 3 to 12 members is replaced with a hetero atom selected from O, S, N, preferably 1 to 3 hetero atoms, and includes carbon atoms, nitrogen atoms and sulfur atoms which may be substituted with oxo groups.

"3-12 membered heterocyclyl" means a monocyclic heterocyclyl, bicyclic heterocyclyl system, or polycyclic heterocyclyl system (also referred to as fused ring systems), including saturated, partially saturated heterocyclyl groups, but excluding aromatic rings. All monocyclic, fused ring (including fused in the form of a parallel, spiro, bridge), saturated, partially saturated situations are included, where possible, unless otherwise specified.

The monoheterocyclic group may be a 3-to 8-membered heterocyclic group, a 3-to 8-membered saturated heterocyclic group, a 3-to 6-membered heterocyclic group, a 4-to 7-membered heterocyclic group, a 5-to 6-membered oxygen-containing heterocyclic group, a 3-to 8-membered nitrogen-containing heterocyclic group, a 5-to 6-membered saturated heterocyclic group, a 4-to 8-membered monoheterocyclic group, a 4-to 8-membered saturated monoheterocyclic group, etc. A "3-8" membered saturated heterocyclic group, examples of which include, but are not limited to, aziridinyl, oxetanyl, thietanyl, azetidinyl, oxetanyl, thietanyl, tetrahydrofuryl, tetrahydrothienyl, imidazolidinyl, pyrazolidinyl, 1, 2-oxazolidinyl, 1, 3-oxazolidinyl, 1, 2-thiazolidinyl, 1, 3-thiazolidinyl, tetrahydro-2H-pyranyl, tetrahydro-2H-thiopyranyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, azepinyl, 1, 4-dioxanyl, 1, 4-oxathietanyl; "3-8" membered partially saturated heterocyclic group, examples of which include, but are not limited to, 4, 5-dihydroisoxazolyl, 4, 5-dihydrooxazolyl, 2, 3-dihydrooxazolyl, 3, 4-dihydro-2H-pyrrolyl, 2, 3-dihydro-1H-pyrrolyl, 2, 5-dihydro-1H-imidazolyl, 4, 5-dihydro-1H-pyrazolyl, 4, 5-dihydro-3H-pyrazolyl, 4, 5-dihydrothiazolyl, 2H-pyranyl, 4H-pyranyl, 2H-thiopyranyl, 4H-thiopyranyl, 2,3,4, 5-tetrahydropyridinyl, 1, 2-isooxazinyl, 1, 4-isooxazinyl or 6H-1, 3-oxazinyl and the like. Fused heterocycles include heterocyclo, spiroheterocyclyl, bridged heterocyclo, and may be saturated, partially saturated, or unsaturated, but are not aromatic. Fused heterocyclyl is a 5-6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5-6 membered monocyclic cycloalkyl, a 5-6 membered monocyclic cycloalkenyl, a 5-6 membered monocyclic heterocyclyl or a 5-6 membered monocyclic heteroaryl. The heterocyclic group may be 6-12 membered fused ring group, 7-10 membered fused ring group, 6-12 membered saturated fused ring group, representative examples include but are not limited to: 3-azabicyclo [3.1.0]Hexane radical, 3, 6-diazabicyclo [3.2.0]Heptylalkyl, 3, 8-diazabicyclo [4.2.0]Octyl, 3, 7-diazabicyclo [4.2.0 ]]Octyl radical, octahydropyrrolo[3,4-c]Pyrrolyl, octahydropyrrolo [3,4-b ]]Pyrrolyl, octahydropyrrolo [3,4-b ]][1,4]Oxazinyl, octahydro-1H-pyrrolo [3,4-c]Pyridyl, 2, 3-dihydrobenzofuran-2-yl, 2, 3-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin 3-yl, 2, 3-dihydrobenzothien-2-yl, octahydro-1H-indolyl, octahydrobenzofuranyl. The spiro heterocyclic group may be a 6-12 membered spiro heterocyclic group, a 7-11 membered spiro heterocyclic group, a 6-12 membered saturated spiro heterocyclic group, a 7-10 membered saturated spiro heterocyclic group, examples of which include, but are not limited to:

the bridged heterocyclic group may be a 6-12 membered bridged heterocyclic group, a 7-11 membered bridged heterocyclic group, a 6-12 membered saturated bridged cyclic group, a 7-11 membered saturated bridged heterocyclic group, examples of which include, but are not limited to:

the "aryl" in the present invention means a cyclic aromatic group having 6 to 14 carbon atoms, and includes phenyl, naphthalene, phenanthrene, and the like.

The term "5-to 10-membered heteroaryl" as used herein means an aromatic cyclic group wherein at least one ring carbon atom is replaced by a heteroatom selected from O, S, N, preferably 1 to 3 heteroatoms, and includes cases wherein a carbon atom and a sulfur atom are replaced by oxo, for example, a carbon atom is replaced by C (O), a sulfur atom is replaced by S (O), S (O)₂And (4) replacing. Heteroaryl includes both mono-and fused heteroaryl groups, including all monocyclic, fused, wholly aromatic, and partially aromatic moieties that may be formed, unless otherwise specified. The monoheteroaryl group may be a 5-7 membered heteroaryl group, a 5-6 membered heteroaryl group, examples of which include, but are not limited to, furyl, imidazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridyl, pyridazineAryl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thienyl, triazolyl and triazinyl. In certain embodiments, fused heteroaryl refers to a group formed by fusing a monocyclic heteroaryl ring to a phenyl, cycloalkenyl, heteroaryl, cycloalkyl, heterocyclyl group, the fused heteroaryl group can be an 8-12 membered fused heteroaryl, a 9-10 membered fused heteroaryl, examples include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzooxadiazolyl, benzothiadiazolyl, benzothiazolyl, cinnolinyl, 5, 6-dihydroquinolin-2-yl, 5, 6-dihydroisoquinolin-1-yl, furopyridinyl, indazolyl, indolyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, quinolinyl, 5,6,7, 8-tetrahydroquinolin-2-yl, 5,6,7, 8-tetrahydroquinolinyl, 5,6,7, 8-tetrahydroquinolin-4-yl, 5,6,7, 8-tetrahydroisoquinolin-1-yl, thienopyridinyl, 4,5,6, 7-tetrahydro [ c ] o][1,2,5]Oxadiazolyl and 6, 7-dihydro [ c ]][1,2,5]Oxadiazol-4 (5H) onyl.

The "pharmaceutically acceptable salt" of the present invention refers to a pharmaceutically acceptable acid and base addition salt or a solvate thereof. Such pharmaceutically acceptable salts include salts of acids such as: hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, sulfurous acid, formic acid, toluenesulfonic acid, methanesulfonic acid, nitric acid, benzoic acid, citric acid, tartaric acid, maleic acid, hydroiodic acid, alkanoic acids (such as acetic acid, HOOC- (CH)₂) n-COOH (wherein n is 0 to 4)), and the like. Salts of bases: sodium, potassium, calcium, ammonium salts and the like. The person skilled in the art is aware of a number of non-toxic pharmaceutically acceptable addition salts.

"stereoisomers" of the compounds of formula (I) according to the invention mean that enantiomers are formed when asymmetric carbon atoms are present in the compounds of formula (I); when the compound has a carbon-carbon double bond or a cyclic structure, cis-trans isomers can be generated; tautomers can occur when ketones or oximes are present in the compounds, and all enantiomers, diastereomers, racemates, cis-trans isomers, tautomers, geometric isomers, epimers and mixtures thereof of the compounds of formula (I) are included within the scope of the present invention.

Detailed Description

Preparation example of intermediate: synthesis of 2-aminothiophene-3-carbonitrile

Step 1: synthesis of 2-aminothiophene-3-carbonitrile

The starting material 1, 4-dithiane-2, 5-diol (100g,0.66mol,1.0eq) was dissolved in methanol (500mL), malononitrile (86.86g,1.32mol,2.0eq) and triethylamine (20mL) were added and the reaction was carried out at 70 ℃ for 30 minutes, TLC detected complete, the reaction was concentrated and the crude product was purified by silica gel column chromatography (PE: EA. RTM. 4:1) to give a yellow solid (80g, yield: 98%).

EXAMPLE 1 preparation of the intermediate ethyl 2- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) acetate

Step 1: synthesis of 5- (4-methylpiperazin-1-yl) -2-nitroaniline

5-chloro-2-nitroaniline (30g,0.19mol,1equiv) was dissolved in anhydrous NMP (40mL), triethylamine (58mL,0.42mol,2.2eq) was added at room temperature, stirring was carried out for 30 minutes, 1-methylpiperazine (18mL,0.21mol,1.1eq) was slowly added dropwise, and the mixture was heated to 160 ℃ for reaction for 16 hours. After completion of the TLC monitoring reaction, water (1L) was added, filtered, and the filter cake was washed with ether and dried to give 5- (4-methylpiperazin-1-yl) -2-nitroaniline (23g, yield: 56%).

Step 2: synthesis of ethyl 2- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) acetate

5- (4-Methylpiperazin-1-yl) -2-nitroaniline (16g,67.8mmol,1.0eq) was dissolved in absolute ethanol (320mL), 10% Pd/C (4.8g) was added, hydrogen was charged, and the reaction was stirred until completion. Ethyl 3-ethoxy-3-iminopropionate hydrochloride (31.7g,162mmol,2.4eq) was added under nitrogen and heated to 50 ℃ for 3 hours. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, the resulting residue was dissolved in methylene chloride (500mL), neutralized with aqueous ammonia, washed with water (2X 100mL), the organic phase was concentrated, and the crude product was purified by silica gel column chromatography (EA elution) to give ethyl 2- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) acetate (3.23g, yield: 15.7%) as a white solid.

¹H NMR(400MHz,DMSO-d₆)δ(ppm):12.08-12.01(1H,m),7.39-7.29(1H,m),6.94-6.87(2H,m),4.14(1H,q,J＝7.2Hz),3.90(2H,s),3.09-3.07(4H,m),2.51-2.47(4H,m),2.23(3H,s),1.21(3H,t,J＝7.2Hz).

EXAMPLE 2 preparation of intermediate 5-aminothiazole-4-carbonitrile

Step 1: synthesis of 5-bromothiazole-4-carboxamide

Ethyl 5-bromothiazole-4-carboxylate (10g,42.3mmol,1.0eq) was added to aqueous ammonia (100mL) and stirred at room temperature overnight. After completion of the TLC detection, water (100mL) was added, extraction was performed with ethyl acetate (100 mL. times.6), the organic phases were combined, washed with saturated brine (100mL), dried and concentrated to give the product (7.0g crude) which was used in the next step without purification.

Step 2: synthesis of 5-bromothiazole-4-carbonitrile

5-bromothiazole-4-carboxamide (7g crude, 33.8mmol,1.0eq) was added to toluene (70mL), phosphorus oxychloride (10.4g,67.6mmol,2.0eq) was added dropwise, and after the dropwise addition was complete, the reaction was stirred under reflux for 2 h. TLC detection reaction was complete, cooled to room temperature, poured into ice water (100mL), extracted with ethyl acetate (50 mL. times.3), washed with saturated sodium bicarbonate (50 mL. times.2), washed with brine (50 mL. times.2), dried, and concentrated to give 5-bromothiazole-4-carbonitrile (5.6g crude) which was used in the next step without purification.

And 3, step 3: synthesis of 5- ((diphenylmethylene) amino) thiazole-4-carbonitrile

5-bromothiazole-4-carbonitrile (4.6g,24.3mmol,1.0eq), benzophenone imine (6.6g,36.5mmol,1.5eq), cesium carbonate (17.5g,53.5mmol,2.2eq), Pd₂(dba)₃(2.3g,2.43mmol,0.1eq) and Xantphos (2.99g,5.1mmol,0.21eq) were added to toluene (115mL), the mixture was purged with nitrogen 3 times, and the reaction was stirred at 80 ℃ overnight. After completion of the TLC detection reaction, it was cooled to room temperature, filtered under suction, the filter cake was washed with ethyl acetate (50 mL. times.3), the filtrate was concentrated, and the crude product was subjected to silica gel column chromatography to give a yellow solid (3.9g, yield: 30%).

And 4, step 4: synthesis of 5-aminothiazole-4-carbonitrile

5- ((diphenylmethylene) amino) thiazole-4-carbonitrile (4.2g,14.5mmol,1.0eq) was added to tetrahydrofuran (16.8mL) followed by 4mol/L hydrochloric acid (16.8mL,67.2mmol,4.6eq) and the reaction stirred at 25 ℃ for 3 h. TLC detection of the reaction was complete, water (50mL) was added, the pH was adjusted to 8-9 with saturated sodium bicarbonate at 0 deg.C, extracted with ethyl acetate (50 mL. times.3), washed with brine (50 mL. times.3), dried, concentrated, and the crude product was purified by silica gel column chromatography to give a yellow solid (1.3g, yield: 76%).

¹H NMR(300MHz,DMSO-d₆)δ(ppm):8.06(s,1H),7.38(s,2H).

Example 34 Synthesis of amino-5- (5- (4-methylpiperazin-1-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one (Compound 1)

Step 1: preparation of 6- (4-methylpiperazin-1-yl) -3-nitropyridin-2-amine

6-chloro-3-nitropyridin-2-amine (5g,0.029mol,1eq) was dissolved in anhydrous DMF (40mL), potassium carbonate (8.8g,0.064mol,2.2eq) was added at room temperature, stirring was carried out for 30 minutes, N-methylpiperazine (2.7mL,0.032mol,1.1eq) was slowly added dropwise, the temperature was raised to 150 ℃ and stirring was carried out for 16 hours. After TLC to monitor the reaction was complete, water (1L) was added, filtered and the resulting yellow solid was collected. Washed with ether and dried in air to give 6- (4-methylpiperazin-1-yl) -3-nitropyridin-2-amine (6g, crude) which was used in the next step without purification.

Step 2: preparation of ethyl 2- (5- (4-methylpiperazin-1-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) acetate

6- (4-Methylpiperazin-1-yl) -3-nitropyridin-2-amine (6g, crude, 1.0eq) was dissolved in absolute ethanol (100mL) and 10% Pd/C (0.6g) was added. Hydrogen is filled in and stirred until the reaction is finished. Ethyl 3-ethoxy-3-iminopropionate hydrochloride (11.8g,61mmol,2.4eq) was added under nitrogen and heated to 80 ℃ for 3 hours. Incomplete reaction, suction filtration, concentration, addition of toluene and DMF, heating reflux and complete conversion. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was dissolved in methylene chloride (100mL), neutralized with aqueous ammonia, washed with water (2X 50mL), the organic phase was concentrated, and the crude product was purified by silica gel column chromatography (eluent: EA) to give ethyl 2- (5- (4-methylpiperazin-1-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) acetate (2.1g, two-step total yield: 26.5%).

¹H NMR(400MHz,CDCl₃)δppm:7.77(1H,d,J＝8.8Hz),6.65(1H,d,J＝8.8Hz),4.25(1H,q,J＝7.2Hz),4.01(2H,s),3.64-3.60(4H,m),2.63-2.60(4H,m),2.40(3H,s),1.31(3H,t,J＝7.2Hz).MS:304.1[M+H]⁺

And step 3: preparation of 4-amino-5- (5- (4-methylpiperazin-1-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one

Dissolving ethyl 2- (5- (4-methylpiperazin-1-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) acetate (200mg,0.659mmol,1.0eq) and 2-aminothiophene-3-carbonitrile (82mg,0.659mmol,1.0eq) in THF (3mL), heating to 40 ℃, dropwise adding LDA (1.6mL,3.296mmol,5eq), stirring for 2 hours, heating to 60 ℃, continuing to react for 2 hours, cooling the reaction solution to room temperature, slowly adding saturated ammonium chloride solution, stirring for a while, EA extracting (50mL × 3), combining organic phases, drying with anhydrous sodium sulfate, suction-filtering, concentrating the filtrate under reduced pressure to obtain a solid crude product, adding an appropriate amount of EA and DCM, pulping with a small amount of methanol, suction-filtering, washing the filter cake with DCM, suction-drying, and drying under reduced pressure to obtain 4-amino-5- (5- (4-methylpiperazin-1-yl) 5-amino-5-methyl piperazine-2-yl -yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one (67.97mg, yield 27.1%).

¹HNMR(400MHz,DMSO)δ(ppm):12.74-12.79(d,1H),12.16(s,1H),10.33-10.54(d,1H),8.15(s,1H),7.86-7.88(d,1H),7.61-7.62(d,1H),7.18-7.21(t,1H),6.80-6.85(t,1H),3.73-3.82(d,4H),3.15(s,4H),2.68-2.71(d,3H).

Molecular formula C₁₈H₁₉N₇OS, molecular weight 381.46, LC-MS (Pos, M/z) ═ 382.3[ M + H ]⁺].

Example 47 Synthesis of amino-6- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) thiazolo [5,4-b ] pyridin-5 (4H) -one (Compound 2)

Step 1 Synthesis of 7-amino-6- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) thiazolo [5,4-b ] pyridin-5 (4H) -one

Ethyl 2- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) acetate (150.0mg,0.495mmol,1.0eq) and 5-aminothiazole-4-carbonitrile (62.1mg,0.495mmol,1.0eq) were dissolved in tetrahydrofuran (4.5mL), stirred under nitrogen at 40 ℃ for 10min, lithium diisopropylamide (1.7mL,3.465mmol,7.0eq) was slowly added, and stirred at 40 ℃ overnight. TLC detects the reaction is complete, saturated ammonium chloride aqueous solution (5mL) is added for quenching, ethyl acetate (5mL × 3) is extracted, anhydrous sodium sulfate is dried, suction filtration is carried out, filtrate is concentrated, and the crude product is purified by silica gel column chromatography (DCM: MeOH: 30: 1-5: 1) to obtain 7-amino-6- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) thiazolo [5,4-b ] pyridin-5 (4H) -one as a red solid (10.0mg, yield: 5.3%).

¹H NMR(400MHz,DMSO-d₆)δ(ppm):12.50-12.48(d,1H),12.17-12.13(s,1H),10.63-10.42(m,1H),8.88(s,1H),8.65(s,1H),8.12-8.00(t,2H),7.53-7.44(m,1H),7.22-7.12(d,1H),6.92-6.89(d,1H),3.17-3.11(t,4H),2.27(s,3H).

Molecular formula C₁₈H₁₉N₇OS molecular weight 381.46 LC-MS (Pos, M/z) ═ 382.21[ M + H ]]⁺.

EXAMPLE 54 Synthesis of amino-5- (5- (piperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride (hydrochloride of Compound 16)

Step 1: synthesis of 6-amino-5-nitro-3 ',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -carboxylic acid tert-butyl ester

Tert-butyl (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 6-dihydropyridine-1 (2H) -carboxylate (23.32g,74.9mmol,1.3eq) and 6-chloro-3-nitropyridin-2-amine (10g,57.6mmol,1.0eq) were added to dioxane (500mL), tetrabutylammonium bromide (1.86g,7.5mmol,0.1eq), [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium dichloromethane complex (2.38g,3.74mmol,0.05eq), sodium carbonate (15.3g,187.3mmol,2.5eq) and water (48mL) were added and the reaction was stirred at 80 ℃ under nitrogen overnight. After completion of the reaction, it was cooled to room temperature, filtered, the solid was washed with ethyl acetate (200 mL. times.3), the filtrate was concentrated, the residue was added with water (500mL), extracted with ethyl acetate (200 mL. times.3), the organic phases were combined, washed with brine (200 mL. times.3), dried, filtered, concentrated, and the crude product was purified by silica gel column chromatography to give the product as a yellow solid (12g, yield: 65%).

Step 2: synthesis of 4- (5, 6-diaminopyridin-2-yl) piperidine-1-carboxylic acid tert-butyl ester

Tert-butyl 6-amino-5-nitro-3 ',6' -dihydro- [2,4' -bipyridine ] -1' (2' H) -carboxylate (12g,37.5mmol) was added to absolute ethanol (240mL), palladium on carbon (6g, 10%) was added, and after the reaction was completed, hydrogenation was performed at room temperature overnight, HPLC, TLC detection was performed, suction filtration was performed, the solid was washed with ethanol (50 mL. times.3), and the filtrate was concentrated to give tert-butyl 4- (5, 6-diaminopyridin-2-yl) piperidine-1-carboxylate (9.5g, crude) as a black solid, which was used in the next step without purification.

And step 3: synthesis of tert-butyl 4- (2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperidine-1-carboxylate

Tert-butyl 4- (5, 6-diaminopyridin-2-yl) piperidine-1-carboxylate (5g,17.1mol,1.0eq) was added to ethanol (100mL) followed by ethyl 3-ethoxy-3-iminopropionate hydrochloride (6.7g,34.2mmol,2.0eq) and refluxed overnight. After completion of the HPLC and LC-MS detection reactions, concentration was carried out, water (25mL) was added to the residue, the pH was adjusted to 9-10 with aqueous ammonia at 0 ℃, ethyl acetate was extracted (20 mL. times.5), the organic phase was dried, filtered, concentrated, and the crude product was purified by silica gel column chromatography (EA elution) to give a white solid (1.4g, yield: 21%).

Molecular formula C₂₀H₂₈N₄O₄Molecular weight 388.47 LC-MS (Pos, M/z) ═ 389.1[ M + H ]]⁺.

¹HNMR(300MHz,DMSO-d₆)δ(ppm):12.8-12.5(br,1H),7.88-7.78(m,1H),7.12(d,J＝8.1Hz,1H),4.13-3.99(m,4H),2.91-2.88(m,3H),1.84(m,2H),1.64(m,2H),1.41(s,9H),1.19(t,J＝6.6Hz,3H).

And 4, step 4: synthesis of tert-butyl 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperidine-1-carboxylate

Reacting 4- (2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4, 5-b)]Pyridin-5-yl) piperidine-1-carboxylic acid tert-butyl ester (100.0mg,0.257mmol,1.0eq), 2-aminothiophene-3-carbonitrile (31.9mg,0.257mmol,1.0eq) were dissolved in tetrahydrofuran (2.0mL), N₂Stirring for 20min at 40 ℃ under protection, slowly dropwise adding lithium bis (trimethylsilyl) amide (2.57mL,2.574mmol,10.0eq), stirring for 2h at 40 ℃, and then heating to 80 ℃ for reaction for 2 h. Adding saturated ammonium chloride solution (5mL) for quenching, extracting by ethyl acetate (5mL multiplied by 3), drying by anhydrous sodium sulfate, filtering, concentrating the filtrate, and purifying the crude product by silica gel column chromatography (DCM: MeOH 150: 1-40: 1) to obtain a yellow solid product, namely tert-butyl 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] thieno [2,3-b ]]Pyridin-5-yl) -3H-imidazo [4,5-b]Pyridin-5-yl) piperidine-1-carboxylic acid ester (40.0mg, yield: 40%).

And 5: synthesis of 4-amino-5- (5- (piperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride

Tert-butyl 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperidine-1-carboxylate (40.0mg,0.0857mmol,1.0eq) was dissolved in ethanol (1.0mL), and a solution of hydrogen chloride in ethanol (1.0mL) was added overnight at room temperature. The reaction was completed by TLC, and a small amount of water (about 0.5mL) was added to the reaction solution, which was then subjected to suction filtration to give 4-amino-5- (5- (piperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride as a yellow solid (7.88mg, yield: 19.7%).

¹HNMR(400MHz,DMSO-d₆)δ(ppm):13.47(s,1H),12.37(s,1H),10.21(s,2H),9.11-8.95(m,2H),8.65(s,1H),8.31-8.30(m,1H),7.68-7.67(m,1H),7.34-7.24(m,2H),3.51-3.30(m,4H),3.09-3.01(m,2H),2.17-2.03(m,4H).

Molecular formula C₁₈H₁₈N₆OS molecular weight 366.44 LC-MS (Pos, M/z) ═ 367.06[ M + H ]]⁺.

Example 6 Synthesis of N- (5- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) -6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-4-yl) acetamide (Compound 17)

Step 1 Synthesis of 4-amino-5- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one

Ethyl 2- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) acetate (866mg,2.864mmol,1.0eq) and 2-aminothiophene-3-carbonitrile (355.6mg,2.864mmol,1.0eq) were dissolved in THF, warmed to 40 ℃ under nitrogen, lithium diisopropylamide (2mol/L,7.134mL,14.32mmol,5.0eq) was slowly added dropwise over 40min, the reaction was continued for 2H, and LC-MS monitored for completion. The reaction was added to saturated aqueous ammonium chloride (50mL), extracted with EA (50 mL. times.3), the organic phases combined, washed with water (30mL), the organic phase dried, concentrated, and the crude product slurried with DCM (1mL) and filtered to give the product (301mg, 27.6% yield).

Step 2 Synthesis of N- (5- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) -6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-4-yl) acetamide

4-amino-5- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one (155mg,0.407mmol,1.0eq) and DIPEA (157.9mg,1.22mmol,3.0eq) were dissolved in DMF (1mL), acetyl chloride (63.9mg,0.815mmol,2.0eq) was added dropwise, followed by reaction at 70 ℃ for 6H with addition of microwave, followed by addition of DIPEA (157.9mg,1.22mmol,3.0eq) and acetyl chloride (63.9mg,0.815mmol,2.0eq) and reaction at 70 ℃ for 6H with addition of microwave. The reaction solution was poured into ice water, DCM was extracted (50mL × 3), the organic phase was washed with saturated brine (30mL), separated, dried, concentrated, and the crude product was purified by preparative thin layer chromatography (methanol: dichloromethane ═ 1:20) to give the product (3.4mg, yield: 2%).

¹H NMR(400MHz,DMSO-d₆)δ(ppm):12.97-12.92(s,1H),12.87-12.74(s,1H),7.58-7.56(s,1H),7.21-7.19(m,2H),7.15-7.13(s,1H),7.03-7.01(s,1H),3.15(d,4H),2.57(d,4H),2.36(s,3H),2.29(s,3H).

Molecular formula C₂₁H₂₂N₆O₂Molecular weight of S422.51 LC-MS (Pos, M/z) ═ 423.25[ M + H ]]⁺.

Example 7: synthesis of N- (5- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) -6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-4-yl) methanesulfonamide (Compound 18)

Step 1: synthesis of N- (5- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) -6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-4-yl) methanesulfonamide

4-amino-5- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one (100mg,0.263mmol,1.0eq) and pyridine (124.7mg,1.576mmol,6.0eq) were dissolved in DMAC (1mL), methanesulfonic anhydride (91.5mg,0.526mmol,2.0eq) was added dropwise, after addition, reaction was carried out at 40 ℃ for 2H, and after addition, methanesulfonic anhydride (91.5mg,0.526mmol,2.0eq) was added further, and after addition, reaction was carried out at 40 ℃ for 2H, further methanesulfonic anhydride (91.5mg,0.526mmol,2.0eq) was added, and after addition, reaction was carried out at 40 ℃ for 12H. The reaction solution was poured into ice water, extracted with DCM (50mL × 3), the organic phases were combined, washed with saturated brine (30mL), dried, concentrated, and the crude product was purified by preparative thin layer chromatography (methanol: dichloromethane ═ 1:20) to give N- (5- (6- (4-methylpiperazin-1-yl) -1H-benzo [ d ] imidazol-2-yl) -6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-4-yl) methanesulfonamide (3.6mg, yield: 2%).

¹H NMR(400MHz,CDCl₃)δ(ppm):10.27-11.25(s,1H),8.13(s,1H),7.75-7.77(s,1H),7.64-7.66(s,1H),7.36-7.37(s,1H),7.22-7.24(s,1H),3.33(d,3H),2.74(d,3H),2.45-2.50(m,3H),2.26-2.28(m,2H),2.22-2.24(m,2H),2.03-2.06(m,2H).

Molecular formula C₂₀H₂₂N₆O₃S₂Molecular weight 458.56 LC-MS (Pos, M/z) ═ 459.07[ M + H ]]⁺.

Example 8: synthesis of 4-amino-5- (5- (1-methylpiperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one (Compound 25)

The method comprises the following steps:

4-amino-5- (5- (piperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride (30.0mg,0.0745mmol,1.0eq) and aqueous formaldehyde (37%) (12.1mg,0.149mmol,2.0eq) were dissolved in methanol (1.5mL) and water (0.5mL), acetic acid (0.1mL) was added and stirred at 0 deg.C for 1H, sodium cyanoborohydride (23.4mg,0.372mmol,5.0eq) was added and stirred at room temperature for 5H, TLC showed completion of the reaction. The reaction mixture was concentrated under reduced pressure, and the crude product was separated by preparative thin layer chromatography (DCM: MeOH: 48:10) to give the product (10.0mg, yield: 33.3%).

¹HNMR(400MHz,DMSO-d₆)δ(ppm):13.00-12.90(s,1H),12.16(s,1H),10.61-10.41(s,1H),8.22(s,1H),7.93-7.91(d,1H),7.64-7.63(d,1H),7.24-7.20(m,1H),7.16-7.06(m,1H),3.52-3.42(m,2H),3.05(m,2H),2.78-2.77(m,1H),2.38-2.28(m,4H),1.91(s,3H).

The molecular formula is as follows: c₁₉H₂₀N₆Molecular weight of OS: 380.47LC-MS (Pos, M/z) ═ 381.14[ M + H [ ]]⁺

EXAMPLE 9 Synthesis of 4-amino-5- (5- (1-cyclopropylpiperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one (Compound 26)

Step 1: synthesis of tert-butyl 4- ((2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperidine-1-carboxylate

The starting material, tert-butyl 4- (5, 6-diaminopyridin-2-yl) piperidine-1-carboxylate (1.7g,5.81mmol,1.0eq) and ethyl 3-ethoxy-3-iminopropionate hydrochloride (3.63g,18.6mmol,3.0eq) were dissolved in ethanol (20mL) and the reaction was allowed to warm to 90 ℃ under nitrogen for 3 hours. LC-MS detects the reaction is complete, the reaction solution is poured into water (50mL) to be quenched, the water phase is extracted by ethyl acetate (50mL multiplied by 2), the organic phase is combined, dried by anhydrous sodium sulfate, filtered, concentrated, and the crude product is purified by silica gel column chromatography (EA) to obtain the product (1.2g, yield: 53%).

Step 2: synthesis of tert-butyl 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridine-5-tert-butyloxycarbonyl) piperidine-1-carboxylate

Tert-butyl 4- ((2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperidine-1-carboxylate (500mg,1.29mmol,1.0eq) and 2-aminothiophene-3-carbonitrile (160mg,1.29mmol) were dissolved in tetrahydrofuran (10mL), warmed to 40 ℃, bis (trimethylsilyl) aminolithium tetrahydrofuran solution (13mL,13mmol,10.0eq) was slowly added, warmed to 80 ℃ and reacted for 2 hours, LC-MS detected reaction completion, the reaction was poured into water (30mL) and quenched, the aqueous phase was extracted with ethyl acetate (30mL × 3), the organic phase was combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was chromatographed over a silica gel column (DCM: 20:1) to give the product (240mg, yield: 40%).

And step 3: synthesis of 4-amino-5- (5- (piperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride

Tert-butyl 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridine-5-tert-butyloxycarbonyl) piperidine-1-carboxylate (240mg,0.51mmol) was dissolved in ethanol (5mL), and a hydrogen chloride ethanol solution (5mL) was added to react at room temperature overnight. The reaction solution became turbid by clarification, a solid precipitated, the reaction was detected by LC-MS to be complete, and the product was concentrated under reduced pressure (250mg, yield: 100%).

And step 3: synthesis of 4-amino-5- (5- (1-cyclopropylpiperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one

Dissolving intermediate 4-amino-5- (5- (piperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride (250mg,0.682 mmol) and (1-ethoxycyclopropoxy) trimethylsilane (594mg,3.41mmol,5.0eq) in methanol (10mL), adding acetic acid (0.5mL) and sodium cyanoborohydride (258mg,4.10mmol,6.0eq), heating to 60 ℃ for 12 hours, leaving LC-MS unreacted, adding N, N-dimethylformamide (1mL), reacting at 70 ℃ for 2 hours, detecting completion of the reaction by LC-MS, pouring the reaction solution into water (30mL), adjusting the pH to 8 with a saturated aqueous sodium bicarbonate solution, extracting with dichloromethane (30 mL. times.3), the organic phases were combined, washed twice with saturated brine, dried, filtered, concentrated and the crude product was washed with dichloromethane to give the product (46mg, yield: 16.6%).

¹HNMR(400MHz,DMSO-d₆)δ(ppm):12.89(s,1H),12.19(s,1H),10.36-10.68(m,1H),8.25(s,1H),7.90-7.92(d,1H),7.63-7.65(d,1H),

7.19-7.21(m,1H),3.08(m,3H),2.56-2.59(m,2H),2.29-2.33(m,2H),1.80-1.85(m,2H),1.65-1.85(m,3H),0.33-0.44(m,4H).

Molecular formula C₂₁H₂₂N₆OS molecular weight 406.51LC-MS (Pos, M/z) ═ 407.32[ M + H ]]⁺

EXAMPLE 10 Synthesis of 4-amino-5- (5- (azepan-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride (hydrochloride of Compound 39)

Step 1: synthesis of 5- (((trifluoromethyl) sulfonyl) oxy) -2,3,4, 7-tetrahydro-1H-azepane-1-carboxylic acid tert-butyl ester

Dissolving the raw material tert-butyl 4-oxocycloheptane-1-carboxylate (5.0g,23.44mmol,1.0eq) in tetrahydrofuran (50mL), cooling to-70 ℃, slowly adding a lithium bis (trimethylsilyl) amido tetrahydrofuran solution (28mL,28.13mmol,1.2eq) to react at-70 ℃ for 0.5h, slowly adding 1,1, 1-trifluoro-N-phenyl-N- ((trifluoromethyl) sulfonyl) methanesulfonamide (10.0g,28.13mmol,1.2eq), naturally heating to room temperature to react for 16 h. TLC (PE: EA ═ 5:1) detected that the reaction was complete, the reaction solution was quenched into ice water (100mL), extracted with ethyl acetate (100mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (PE: EA ═ 5:1) to give the product (4.5g, yield: 56%).

Step 2: synthesis of tert-butyl 5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2,3,4, 7-tetrahydro-1H-azepane-1-carboxylate

The intermediate 5- (((trifluoromethyl) sulfonyl) oxy) -2,3,4, 7-tetrahydro-1H-azepane-1-carboxylic acid tert-butyl ester (4.5g,13.03mmol,1.0eq) and pinacol diboron diboride (3.3g,13.03mmol,1.0eq) were dissolved in 1, 4-dioxane (50mL), potassium acetate (3.84g,39.09mmol,3.0eq), 1 '-bis (diphenylphosphino) ferrocene (722mg,1.303mmol,0.1eq) and 1,1' -bis (diphenylphosphino) ferrocene palladium dichloride (953mg,1.303mmol,0.1eq) were added and the reaction was warmed to 90 ℃ under nitrogen protection for 3 hours. TLC (PE: EA ═ 5:1) detected the reaction was complete, the reaction was quenched into ice water (100mL), extracted with ethyl acetate (100mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (PE: EA ═ 5:1) to give the product (3.2g, yield: 76%).

And step 3: synthesis of tert-butyl 5- (6-amino-5-nitropyridin-2-yl) -2,3,4, 7-tetrahydro-1H-azepane-1-carboxylate

The intermediate 5-chloro-2-nitroaniline (1.74g,10mmol,1.0eq) and 5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2,3,4, 7-tetrahydro-1H-azepane-1-carboxylic acid tert-butyl ester (3.23g,10mmol,1.0eq) were dissolved in 1, 4-dioxane (40mL) and water (10mL), sodium carbonate (3.2g,30mmol,3.0eq), 1' -bis (diphenylphosphino) ferrocene palladium dichloride (732mg,0.1mmol,0.1eq) were added, and the temperature was raised to 90 ℃ under nitrogen protection for 3 hours. TLC (PE: EA ═ 5:1) detected the reaction was complete, the reaction solution was quenched into ice water (100mL), extracted with ethyl acetate (100mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (PE: EA ═ 5:1) to give the product (850mg, yield: 25%).

And 4, step 4: synthesis of 4- (5, 6-diaminopyridin-2-yl) azepane-1-carboxylic acid tert-butyl ester

Intermediate 5- (6-amino-5-nitropyridin-2-yl) -2,3,4, 7-tetrahydro-1H-azepane-1-carboxylic acid tert-butyl ester (850mg,2.54mmol,1.0eq) was dissolved in ethanol (10mL), 10% palladium on carbon (500mg) was added, the reaction was carried out under hydrogen at room temperature for 3 hours, and LC-MS detected that the reaction was complete. The reaction mixture was filtered, concentrated, and the crude product was purified by silica gel column chromatography (DCM: MeOH 10:1) to give a product (650mg, yield: 82%).

And 5: synthesis of tert-butyl 4- (2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) azepane-1-carboxylate

The intermediate 4- (5, 6-diaminopyridin-2-yl) azepane-1-carboxylic acid tert-butyl ester (950mg,3.1mmol,1.0eq) and ethyl 3-ethoxy-3-iminopropionate hydrochloride (1.82g,9.3mmol,3.0eq) were dissolved in ethanol (25mL), warmed to 90 ℃ under nitrogen for 3 hours, TLC (DCM: MeOH 10:1) checked for completion of the reaction, the reaction was quenched by pouring into water (50mL), extracted with ethyl acetate (50mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (DCM: MeOH 10:1) to give the product (540mg, yield: 43%).

Step 6: synthesis of tert-butyl 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) azepane-1-carboxylate

Dissolving intermediate 4- (2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) azepane-1-carboxylic acid tert-butyl ester (540mg,1.33mmol,1.0eq) and 2-aminothiophene-3-carbonitrile (165mg,1.33mmol,1.0eq) in tetrahydrofuran (50mL), heating to 40 ℃, slowly adding bis (trimethylsilyl) aminolithium tetrahydrofuran solution (13.3mL,13.3mmol,10.0eq), heating to 80 ℃ for 2 hours, and detecting by LC-MS that the reaction is complete. The reaction solution was quenched by pouring into water (30mL), extracted with ethyl acetate (30mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (DCM: MeOH ═ 10:1) to give a product (240mg, yield: 38%).

Step 6: synthesis of 4-amino-5- (5- (azepan-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one-hydrochloride

Hydrochloride salt of compound 39

Dissolving the intermediate 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) azepan-1-carboxylic acid tert-butyl ester (240mg,0.5mmol,1.0eq) in ethanol (4mL), slowly adding hydrogen chloride ethanol solution (1mL), heating to 35 ℃ for reaction for 2 hours, allowing the reaction solution to become turbid by clarification, separating out a solid, detecting by LC-MS that the reaction is complete, filtering, rinsing the filter cake twice with acetone, and drying at 60 ℃ under normal pressure to obtain the product (158mg, yield: 76%).

¹HNMR(400MHz,DMSO-d₆)δ(ppm):12.39(s,1H),10.17(s,1H),9.20(s,1H),8.68-8.72(m,1H),8.35(s,1H),7.65-7.67(d,1H),7.39-7.41(s,1H),7.25-7.26(d,1H),3.17-3.40(m,6H),1.97-2.37(m,7H).

Molecular formula C₁₉H₂₀N₆OS molecular weight 380.47LC-MS (Pos, M/z) ═ 381.2[ M + H ]]⁺

EXAMPLE 11 Synthesis of 4-hydroxy-5- (5- (piperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride (hydrochloride of Compound 41)

Step 1: synthesis of tert-butyl 4- (2- (4-hydroxy-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperidine-1-carboxylate

Dissolving the raw materials of tert-butyl 4- ((2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperidine-1-carboxylate (1.0g,2.57mmol,1.0eq) and methyl 2-aminothiophene-3-carboxylate (404mg,2.57mmol,1.0eq) in tetrahydrofuran (20mL), heating to 40 ℃, slowly adding a tetrahydrofuran solution of lithium bis (trimethylsilyl) amide (25.7mL,25.7mmol,10.0eq), reacting for 2 hours, heating to 80 ℃ for 2 hours, detecting the reaction is complete by LC-MS, pouring the reaction solution into water (30mL) for quenching, extracting the aqueous phase with ethyl acetate (30 mL. times.3), combining the organic phases, drying with anhydrous sodium sulfate, filtering, concentrating, purifying by silica gel column chromatography (DCM: MeOH ═ 10:1) to obtain a crude product (200mg, yield 16.6%).

Step 2: synthesis of 4-hydroxy-5- (5- (piperidin-4-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride

Hydrochloride salt of Compound 41

The starting material, tert-butyl 4- (2- (4-hydroxy-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperidine-1-carboxylate (200mg,0.43mmol), was dissolved in methanol (10mL), a hydrogen chloride ethanol solution (10mL) was added, and the mixture was heated to 50 ℃ for reaction for 2 hours. The system turns turbid after clarification, solid is separated out, LC-MS detects that the reaction is complete, filtration is carried out, a filter cake is leached twice by dichloromethane, and the product is obtained after vacuum drying at 50 ℃ (139mg, yield: 80%).

¹HNMR(400MHz,DMSO-d₆)δ(ppm):14.30(s,1H),13.29(s,1H),12.01(s,1H),9.07-9.10(m,1H),8.76-8.79(m,1H),8.04-8.06(d,1H),7.29-7.31(d,1H),7.24-7.26(d,1H),7.06-7.08(d,1H),3.37-3.40(m,2H),3.11-3.15(m,1H),2.99-3.10(m,2H),1.96-2.08(m,4H)。

Molecular formula C₁₈H₁₇N₅O₂Molecular weight of S367.43 LC-MS (Pos, M/z) ═ 368.11[ M + H ]]⁺

EXAMPLE 12 Synthesis of 4-amino-5- (5- (piperazin-1-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride (hydrochloride of Compound 47)

Step 1: synthesis of 4- (6-amino-5-nitropyridin-2-yl) piperazine-1-carboxylic acid tert-butyl ester

The starting material 6-chloro-3-nitropyridin-2-amine (5.0g,28.81mmol,1.0eq) was dissolved in N, N-dimethylacetamide (50mL), piperazine-1-carboxylic acid tert-butyl ester (8.05g,43.21mmol,1.5eq) and anhydrous potassium carbonate (15.9g,115.24mmol,4.0eq) were added, the temperature was raised to 80 ℃ for reaction for 3h, TLC monitored for completion of the reaction, concentrated under reduced pressure to give a pale yellow solid, washed with water slurry, filtered, and the filter cake was dried to give the product (8.3g, yield: 90%).

Step 2: synthesis of tert-butyl 4- (5, 6-diaminopyridin-2-yl) piperazine-1-carboxylate

Dissolving the intermediate 4- (6-amino-5-nitropyridin-2-yl) piperazine-1-carboxylic acid tert-butyl ester (1g,3.09mmol,1.0eq) in absolute ethyl alcohol (30mL), adding 10% palladium carbon (500mg), replacing hydrogen, heating to 50 ℃ under hydrogen atmosphere, reacting for 12h, monitoring the reaction by TLC, filtering under the protection of nitrogen, leaching a filter cake with absolute ethyl alcohol, concentrating the filtrate under reduced pressure, adding toluene (20mL), concentrating, repeating for three times, adding absolute ethyl alcohol (20mL), concentrating to obtain a product, and directly using the product in the next reaction without purification.

And step 3: synthesis of tert-butyl 4- (2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperazine-1-carboxylate

Dissolving the product of the last step in absolute ethyl alcohol (20mL), adding 3-ethoxy-3-imino ethyl propionate hydrochloride (1.45g,7.42mmol,2.4eq) under the protection of nitrogen, heating to 50 ℃ for reaction for 3h, detecting complete reaction by LC-MS, cooling to 10 ℃, filtering, leaching a filter cake with ethanol, concentrating the filtrate under reduced pressure, adding dichloromethane (100mL) to dissolve the obtained solid, adjusting the pH value to about 8 with saturated sodium carbonate solution, washing with water (50mL multiplied by 2), separating, drying the organic phase, filtering, concentrating, and washing the obtained solid with methyl tert-butyl ether slurry to obtain the product (475mg, 2-step yield: 39.5%).

And 4, step 4: synthesis of tert-butyl 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperazine-1-carboxylate

Intermediate tert-butyl 4- (2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperazine-1-carboxylate (200mg,0.51mmol,1.0eq) was dissolved in anhydrous tetrahydrofuran (5mL), adding 2-aminothiophene-3-carbonitrile (64.2mg,0.51mmol,1.0eq), heating to 40 ℃ under the protection of nitrogen, dropwise adding a tetrahydrofuran solution (1mol/L,5.1mL,10.0eq) of lithium bis (trimethylsilyl) amide, reacting at 40 ℃ for 2h after addition, heating to 60 ℃ again, reacting for 2h, monitoring the reaction completion by TLC, adjusting the pH value to about 8 by using a saturated ammonium chloride solution, extracting by using dichloromethane (100mL multiplied by 3), separating, combining organic phases, drying, filtering, and concentrating the filtrate under reduced pressure to obtain a product (270mg crude product).

And 5: synthesis of 4-amino-5- (5- (piperazin-1-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one hydrochloride

Hydrochloride salt of Compound 47

Dissolving the intermediate tert-butyl 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) piperazine-1-carboxylate (100mg,0.21mmol,1.0eq) in absolute ethanol (5mL), cooling to 0-10 ℃, adding 30% ethanol hydrogen chloride solution (5mL) dropwise, and stirring at room temperature for 4H. The reaction was monitored by LC-MS for completion, concentrated, ethyl acetate (10mL) was added, concentrated and repeated three times, and the resulting solid was slurry washed with ethyl acetate (5mL), filtered and the filter cake was dried to give the product (63mg, yield: 73%).

¹HNMR(400MHz,DMSO-d₆)δ(ppm):12.23-12.16(s,1H),9.37(s,2H),8.02-8.00(d,1H),7.64-7.63(d,1H),7.21-7.20(d,1H),6.94-6.92(d,1H),3.81(m,4H),3.23(m,4H).

Molecular formula C₁₇H₁₈N₇OSCl molecular weight 367.43 LC-MS (Pos, M/z) ═ 368.26[ M + H ]]⁺.

EXAMPLE 13 Synthesis of 5- (5- (2, 5-diazabicyclo [2.2.2] octan-2-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -4-aminothieno [2,3-b ] pyridin-6 (7H) -one hydrochloride (hydrochloride of Compound 48)

Step 1: synthesis of tert-butyl 5- (6-amino-5-nitropyridin-2-yl) -2, 5-diazabicyclo [2.2.2] octane-2-carboxylate

Dissolving 6-chloro-3-nitropyridine-2-amine (1.5g,8.64mmol,1.0eq) as a raw material in N, N-dimethylacetamide (5mL), adding 2, 5-diazabicyclo [2.2.2] octane-2-carboxylic acid tert-butyl ester (1.8g,8.64mmol,1.0eq) and anhydrous potassium carbonate (4.7g,34.56mmol,4.0eq), heating to 80 ℃ to react for 3 hours, monitoring by TLC for reaction completion, cooling to room temperature, adding water (50mL), extracting with ethyl acetate (100 mL. times.3), separating, combining organic phases, washing with saturated saline (100 mL. times.3), drying, filtering, and concentrating the filtrate under reduced pressure to obtain a product (2.9g, yield: 96.17).

Step 2: synthesis of tert-butyl 5- (5, 6-diaminopyridin-2-yl) -2, 5-diazabicyclo [2.2.2] octane-2-carboxylate

Dissolving an intermediate tert-butyl 5- (6-amino-5-nitropyridin-2-yl) -2, 5-diazabicyclo [2.2.2] octane-2-carboxylate (2.9g,8.3mmol,1.0eq) in absolute ethyl alcohol (30mL), adding 10% palladium carbon (500mg), heating to 50 ℃ under a hydrogen atmosphere for reaction for 12 hours, monitoring the reaction by TLC to be complete, filtering under the protection of nitrogen, leaching a filter cake by using absolute ethyl alcohol, concentrating the filtrate under reduced pressure, adding toluene (50mL), concentrating, repeating for three times, adding absolute ethyl alcohol (50mL), concentrating to obtain a product, and directly putting the product into the next step without purification.

And step 3: synthesis of tert-butyl 5- (2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) -2, 5-diazabicyclo [2.2.2] octane-2-carboxylate:

dissolving the product of the last step in absolute ethyl alcohol (50mL), adding a 3A molecular sieve (2.6g), adding 3-ethoxy-3-imino ethyl propionate hydrochloride (4.0g,20.69mmol,2.4eq) under the protection of nitrogen, heating to 80 ℃ under the protection of nitrogen, reacting for 12h, detecting the reaction completion by LC-MS, filtering while hot, filtering, leaching a filter cake with ethanol, concentrating the filtrate under reduced pressure, adding dichloromethane (100mL) to dissolve the obtained solid, adjusting the pH value to about 8 with saturated sodium carbonate aqueous solution, separating liquid, extracting an aqueous phase with dichloromethane (100mL multiplied by 3), combining organic phases, drying, filtering, concentrating, purifying a crude product by silica gel column chromatography (MeOH: DCM ═ 1:100-1:50), and washing with methyl tert-butyl ether to obtain the product (2g, yield: 55.8%).

And 4, step 4: synthesis of tert-butyl 5- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) -2, 5-diazabicyclo [2.2.2] octane-2-carboxylate

Dissolving an intermediate tert-butyl 5- (2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) -2, 5-diazabicyclo [2.2.2] octane-2-carboxylate (600mg,1.44mmol,1.0eq) in anhydrous tetrahydrofuran (6mL), adding 2-aminothiophene-3-carbonitrile (180.7mg,1.44mmol,1.0eq), heating to 40 ℃ under nitrogen protection, adding a solution of lithium bis (trimethylsilyl) amide in tetrahydrofuran (1mol/L,14.4mL,10.0eq) dropwise, reacting at 40 ℃ for 2H, heating to 60 ℃ for 2H, monitoring by LC-MS for complete reaction, cooling to room temperature, adjusting the pH value to about 8 with a saturated aqueous ammonium chloride solution, extracting with ethyl acetate (100 mL. times.2), separating, mixing the organic phases, drying, filtering, concentrating the filtrate under reduced pressure, washing the obtained solid with methyl tert-butyl ether, filtering, and drying the filter cake to obtain the product (688mg, yield: 96%).

And 5: synthesis of 5- (5- (2, 5-diazabicyclo [2.2.2] octan-2-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -4-aminothieno [2,3-b ] pyridin-6 (7H) -one hydrochloride

Hydrochloride salt of Compound 48

Dissolving the intermediate 5- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) -2, 5-diazabicyclo [2.2.2] octane-2-carboxylic acid tert-butyl ester (200mg,0.40mmol,1.0eq) in absolute ethanol (5mL), cooling to 0-10 ℃, adding 30% ethanol hydrochloride solution (5mL), and stirring at room temperature for 12H. The reaction was monitored by LC-MS for completion, concentrated, ethyl acetate (10mL) was added, the concentration was repeated three times, the resulting solid was then slurry washed with ethyl acetate (10mL), filtered, and the filter cake was dried to give the product (131.2mg, yield: 76.34).

¹HNMR(400MHz,D₂O)δ(ppm):7.56(d,1H),6.80-6.77(d,2H),6.33-6.31(d,1H),4.37(m,1H),4.06(m,1H),3.87-3.85(m,1H),3.64-3.50(m,3H),2.23-2.20(m,2H),2.01-1.98(m,2H).

Molecular formula C₁₉H₂₀N₇OS molecular weight 395.49LC-MS (Neg, M/z) ═ 394.26[ M-H ]]^-.

Example 14: synthesis of 5- (5- (2, 6-diazaspiro [3.3] heptan-2-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -4-aminothieno [2,3-b ] pyridin-6 (7H) -one trifluoroacetate (trifluoroacetate salt of Compound 49)

Step 1: synthesis of tert-butyl 6- (6-amino-5-nitropyridin-2-yl) -2, 6-diazaspiro [3.3] heptane-2-carboxylate

The starting materials 6-chloro-3-nitropyridine-2-amine (1.426g,8.22mmol,2.0eq) and 2, 6-diazaspiro [3.3] heptane-2-carboxylic acid tert-butyl ester hemioxalate (2.0g,4.11mmol,1.0eq) were dissolved in DMF (10mL), potassium carbonate (3.403g,24.66mmol,6.0eq) was added and the reaction was completed under nitrogen protection at 80 ℃ for 12 hours and TLC detection. The reaction solution was poured into water (100mL), stirred for 10 minutes, and then ethyl acetate (100 mL. times.3) was added for extraction, and the organic phase was washed with water (100 mL. times.3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the product (2.7g, yield: 98%).

Step 2: synthesis of tert-butyl 6- (5, 6-diaminopyridin-2-yl) -2, 6-diazaspiro [3.3] heptane-2-carboxylate

Intermediate 6- (6-amino-5-nitropyridin-2-yl) -2, 6-diazaspiro [3.3] heptane-2-carboxylic acid tert-butyl ester (2.52g,7.51mmol,1.0eq) was dissolved in ethanol (20mL), Pd/C (500mg) was added, hydrogen was substituted three times, the reaction was carried out under hydrogen for 12 hours, and LC-MS checked for completion. Suction filtration and concentration under reduced pressure gave the product as a yellow solid (2.29g, yield: 100%).

And step 3: synthesis of tert-butyl 6- (2- (2-ethoxy-2-oxoethyl) -1H-imidazo [4,5-b ] pyridin-5-yl) -2, 6-diazaspiro [3.3] heptane-2-carboxylate

The intermediate tert-butyl 6- (5, 6-diaminopyridin-2-yl) -2, 6-diazaspiro [3.3] heptane-2-carboxylate (2.2g,7.2mmol,1.0eq) was dissolved in ethanol (50mL), 3A molecular sieve (2.0g) was added, stirring was carried out for 10 minutes, ethyl 3-ethoxy-3-iminopropionate hydrochloride (3.382g,17.28mmol,2.4eq) was added, and the reaction was completed by TLC at 80 ℃ under nitrogen. The reaction solution was filtered by suction, the filtrate was concentrated under reduced pressure, the resulting product was poured into water (100mL), the pH was adjusted to 9 with saturated aqueous sodium carbonate, ethyl acetate (100mL × 3) was added and extracted, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (MeOH: DCM ═ 1:50) to give a product (1.5g, yield: 52%).

And 4, step 4: synthesis of tert-butyl 6- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) -2, 6-diazaspiro [3.3] heptane-2-carboxylate

The intermediate, namely, the tert-butyl 6- (2- (2-ethoxy-2-oxoethyl) -1H-imidazo [4,5-b ] pyridin-5-yl) -2, 6-diazaspiro [3.3] heptane-2-carboxylate (1g,2.49mmol,1.0eq) and 2-aminothiophene-3-carbonitrile (309mg,2.49mmol,1.0eq) were dissolved in tetrahydrofuran (20mL), and under the protection of nitrogen, bistrimethylsilyl amino lithium (25mL,24.9mmol,10eq) was added dropwise at 40 ℃ for reaction at 40 ℃ for 2 hours, and the reaction was detected to be complete by LC-MS. The reaction mixture was poured into water (50mL), ethyl acetate (50 mL. times.3) was added for extraction, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the crude product was washed with methyl t-butyl ether syrup to give a product (785mg, yield: 65%).

And 5: synthesis of 5- (5- (2, 6-diazaspiro [3.3] heptan-2-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -4-aminothieno [2,3-b ] pyridin-6 (7H) -one trifluoroacetate

Trifluoroacetic acid salt of compound 49

Intermediate 6- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) -2, 6-diazaspiro [3.3] heptane-2-carboxylic acid tert-butyl ester (200mg,0.42mmol,1.0eq) was dissolved in dichloromethane (5mL), trifluoroacetic acid (5mL) was added at 0 ℃ for 1 hour and the reaction was complete by TLC. The reaction mixture was concentrated under reduced pressure, and the crude product was washed with methylene chloride slurry to give a product (190mg, yield: 92%).

¹H NMR(400MHz,DMSO-d₆)δ(ppm):13.07-13.10(d,1H),12.23(s,1H),10.13-10.27(d,1H),8.71(s,2H),8.20(s,1H),8.00-8.03(d,1H),7.60-7.61(d,1H),7.21-7.22(d,1H),6.38-6.40(d,1H),4.21-4.27(d,8H).

Molecular formula C₁₈H₁₇N₇OS molecular weight 379.44 LC-MS (Pos, M/z) ═ 380.12[ M + H ]]⁺.

EXAMPLE 15 Synthesis of 4-amino-5- (5- (5-methyl-2, 5-diazabicyclo [2.2.2] octan-2-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one (Compound 50)

Step 1: synthesis of 5- (5- (2, 5-diazabicyclo [2.2.2] octan-2-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -4-aminothieno [2,3-b ] pyridin-6 (7H) -one 2,2, 2-trifluoroacetate

Intermediate 5- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) -2, 5-diazabicyclo [2.2.2] octane-2-carboxylic acid tert-butyl ester (200mg,0.40mmol,1.0eq) was dissolved in dichloromethane (5mL), cooled to 0 deg.C, and trifluoroacetic acid (5mL) in dichloromethane (5mL) was added dropwise and stirred at 0 deg.C for 4H. The reaction was monitored by LC-MS for completion, concentrated, dichloromethane (10mL) was added, concentrated and repeated three times to give a red-brown solid, which was then slurry washed with methyl tert-butyl ether (10mL), filtered and the filter cake dried to give the product (190mg, yield: 93.2%).

Step 2: synthesis of 4-amino-5- (5- (5-methyl-2, 5-diazabicyclo [2.2.2] octan-2-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one

Intermediate 5- (5- (2, 5-diazabicyclo [ 2.2.2)]Octane-2-yl) -3H-imidazo [4,5-b]Pyridin-2-yl) -4-aminothieno [2,3-b]Pyridin-6 (7H) -one 2,2, 2-trifluoroacetate salt (108mg,0.21mmol,1.0eq) was dissolved in anhydrous methanol (5mL), acetic acid (12.7mg,0.21mmol,1.0eq) and 37% aqueous formaldehyde (85.1mg,1.05mmol,5.0eq) were added, stirring was carried out at room temperature for 1H, sodium cyanoborohydride (65.9mg,1.05mmol,5.0eq) was added, stirring was carried out at room temperature for 12H, LC-MS was used to monitor completion of the reaction, concentration under reduced pressure was carried out, saturated brine (10mL) and saturated aqueous sodium carbonate (10mL) were added in this order, extraction was carried out with dichloromethane (100 mL. times.3), the organic phases were combined, dried over anhydrous sodium sulfate, filtration was carried out, and the filtrate was concentrated under reduced pressure to give a product (72mg, yield: 80.2%).¹HNMR(400MHz,DMSO-d₆)δ(ppm):12.61(s,1H),8.70(s,1H),10.58-10.34(s,1H),8.04(d,1H),7.86-7.77(d,1H),7.75-7.59(d,1H),7.20-7.17(d,1H),6.47-6.37(d,1H),6.06(s,1H),4.59-4.55(d,1H),3.74-3.71(d,1H),2.93-2.90(d,1H),2.81-2.76(m,2H),2.33-2.30(s,3H),2.27-2.02(d,1H),1.88-1.75(m,2H),1.59-1.50(d,1H).

Molecular formula C₂₀H₂₁N₇OS molecular weight 407.51 LC-MS (Neg, M/z) ═ 408.32[ M + H ]]^-.

Example 16: synthesis of 5- (5- (7-azaspiro [3.5] nonan-7-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -4-aminothieno [2,3-b ] pyridin-6 (7H) -one (Compound 51)

Step 1: synthesis of 3-nitro-6- (7-azaspiro [3.5] nonan-7-yl) pyridin-2-amine

The starting materials 6-chloro-3-nitropyridin-2-amine (3g,17.28mmol,1.0eq) and 7-azaspiro [3.5] nonane hydrochloride (3.35g,20.74mmol,1.4eq) and potassium carbonate (9.54g,69.12mmol,4.0eq) were dissolved in DMAC (20mL) and reacted at 80 ℃ for 12 hours with TLC check for completion. The reaction solution was poured into water (100mL), stirred for 30 minutes, and filtered to give the product (4.2g, yield: 93%).

Step 2: synthesis of 6- (7-azaspiro [3.5] nonan-7-yl) pyridine-2, 3-diamine

Dissolving the intermediate 3-nitro-6- (7-azaspiro [3.5] nonane-7-yl) pyridine-2-amine (1.2g,4.57mmol,1.0eq) in ethanol (10mL), adding Pd/C (500mg), replacing with hydrogen for three times, reacting for 3 hours under hydrogen condition, detecting complete reaction by LC-MS, filtering, and concentrating the filtrate under reduced pressure to obtain the product (1.06g, yield: 100%).

And step 3: synthesis of ethyl 2- (5- (7-azaspiro [3.5] nonan-7-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) acetate

The intermediate 6- (7-azaspiro [3.5] nonan-7-yl) pyridine-2, 3-diamine (1.06g,4.57mmol,1.0eq) was dissolved in ethanol (10mL), 3A molecular sieve (2.0g) was added, stirring was carried out for 10 minutes, and ethyl 3-ethoxy-3-iminopropionate hydrochloride (1.68g,8.6mmol,2.0eq) was added under nitrogen protection and reacted at 50 ℃ for 2 hours with TLC detection of completion of the reaction. The reaction solution was filtered under suction, concentrated under reduced pressure, and the resulting product was poured into water (100mL), adjusted to pH 9 with saturated aqueous sodium carbonate, added with ethyl acetate (100mL × 3) for extraction, dried over anhydrous sodium sulfate as the organic phase, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (MeOH: DCM ═ 1:200) to give the product (400mg, yield: 28%).

And 4, step 4: synthesis of 5- (5- (7-azaspiro [3.5] nonan-7-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -4-aminothieno [2,3-b ] pyridin-6 (7H) -one

The intermediate ethyl 2- (5- (7-azaspiro [3.5] nonan-7-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) acetate (150mg,0.46mmol,1.0eq) and 2-aminothiophene-3-carbonitrile (57mg,0.46mmol,1.0eq) were dissolved in tetrahydrofuran (5mL), under nitrogen protection, bistrimethylsilyl amino lithium (4.6mL,4.6mmol,10eq) was added dropwise at 40 ℃ for 2 hours, and the starting material was left as detected by TLC. And adding 2-aminothiophene-3-carbonitrile (12mg,0.92mmol,0.2eq), heating to 80 ℃ for reaction for 4h, detecting by TLC that the reaction is complete, pouring the reaction solution into water (10mL), adding ethyl acetate (20 mL. times.3) for extraction, drying the organic phase with anhydrous sodium sulfate, filtering, concentrating the filtrate under reduced pressure, and purifying the crude product by preparative thin layer chromatography (MeOH: DCM ═ 1:30) to obtain a product (30mg, yield: 16%).

¹HNMR(400MHz,DMSO-d₆)δ(ppm):12.65(s,1H),12.24(s,0.5H),12.16(s,0.5H),10.59(s,0.5H),10.35(s,0.5H),8.13-8.17(d,1H),7.75-7.79(d,1H),7.63-7.65(d,1H),7.18-7.20(d,1H),6.71-6.76(m,1H),3.42-3.46(m,4H),1.86-1.90(m,2H),1.77-1.79(m,4H),1.62(s,4H).

Molecular formula C₂₁H₂₂N₆OS molecular weight 406.51LC-MS (Pos, M/z) ═ 407.16[ M + H ]]⁺.

EXAMPLE 17 Synthesis of 4-amino-5- (6- (1-methylpiperidin-4-yl) -1H-benzo [ d ] imidazol-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one 2,2, 2-trifluoroacetate (trifluoroacetate salt of Compound 56)

Step 1: synthesis of 4- (3-amino-4-nitrophenyl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester

The starting materials 5-chloro-2-nitroaniline (1.0g,5.8mmol,1.0eq) and 4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (2.0g,6.4mmol,1.1eq) were dissolved in 1, 4-dioxane (20mL) and water (5mL), potassium carbonate (3.2g,23.2mmol,3.0eq) and 1,1' -bis (diphenylphosphino) ferrocene dichloropalladium (212mg,0.29mmol,0.05eq) were added, the reaction was allowed to warm to 90 ℃ under nitrogen for 3 hours, and the reaction was checked for completion by TLC (PE: EA ═ 5: 1). The reaction solution was quenched with ice water (50mL), the aqueous phase was extracted with ethyl acetate (50mL × 2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was purified by silica gel column chromatography (PE: EA ═ 5:1) to give the product (1.3g, yield: 70%).

And 2, step: synthesis of tert-butyl 4- (3, 4-diaminophenyl) piperidine-1-carboxylate

Dissolving the intermediate 4- (3-amino-4-nitrophenyl) -3, 6-dihydropyridine-1 (2H) -carboxylic acid tert-butyl ester (1.3g,4.1mmol,1.0eq) in methanol (10mL), adding 10% wet palladium on carbon (500mg), introducing hydrogen, reacting at room temperature for 3 hours, detecting complete reaction by LC-MS, filtering the reaction solution, and concentrating to obtain a product (1.2g crude product).

And step 3: synthesis of tert-butyl 4- (2- (2-ethoxy-2-oxoethyl) -1H-benzo [ d ] imidazol-6-yl) piperidine-1-carboxylate

Dissolving the intermediate 4- (3, 4-diaminophenyl) piperidine-1-carboxylic acid tert-butyl ester (1.2g crude) and 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride (2.4g,12.36mmol,3.0eq) in ethanol (20mL), heating to 90 ℃ under nitrogen protection, reacting for 3 hours, detecting by LC-MS that the reaction is complete, pouring the reaction solution into water (50mL) to quench, extracting with ethyl acetate (50 mL. times.2), combining the organic phases, drying with anhydrous sodium sulfate, filtering, concentrating, and purifying the crude product by silica gel column chromatography (EA) to obtain the product (1.2g, two-step yield: 75%).

And 4, step 4: synthesis of tert-butyl 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -1H-benzo [ d ] imidazol-6-yl) piperidine-1-carboxylate

The intermediate, tert-butyl 4- (2- (2-ethoxy-2-oxoethyl) -1H-benzo [ d ] imidazol-6-yl) piperidine-1-carboxylate (1.2g,3.1mmol,1.0eq) and 2-aminothiophene-3-carbonitrile (385mg,3.1mmol,1.0eq) were dissolved in tetrahydrofuran (50mL), warmed to 40 ℃, a solution of lithium bis (trimethylsilyl) amide in tetrahydrofuran (31mL,31mmol,10.0eq) was slowly added, warmed to 80 ℃ for 2 hours and the reaction was complete as detected by LC-MS. The reaction was quenched by pouring into water (50mL), extracted with ethyl acetate (50 mL. times.2), the combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was washed with methyl tert-butyl ether to give the product (800mg, 55% yield).

And 5: synthesis of 4-amino-5- (6- (piperidin-4-yl) -1H-benzo [ d ] imidazol-2-yl) thieno [2,3-b ] pyridin-6I (7H) -one hydrochloride

Tert-butyl 4- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -1H-benzo [ d ] imidazol-6-yl) piperidine-1-carboxylate, intermediate (800mg,1.72mmol,1.0eq), was dissolved in ethanol (5mL), and ethanolic hydrogen chloride solution (5mL) was added and reacted at room temperature overnight. The reaction solution turns turbid from clear, solid is separated out, and the LC-MS detects that the reaction is complete. Filtering, drying the filter cake at 60 ℃ under normal pressure to obtain the product (600mg, yield: 87%).

Step 6: synthesis of 4-amino-5- (6- (1-methylpiperidin-4-yl) -1H-benzo [ d ] imidazol-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one 2,2, 2-trifluoroacetate

Trifluoroacetic acid salt of Compound 56

Intermediate 4-amino-5- (6- (piperidin-4-yl) -1H-benzo [ d ] imidazol-2-yl) thieno [2,3-b ] pyridin-6- (7H) -one hydrochloride (300mg,0.75mmol,1.0eq) and 37% aqueous formaldehyde (304mg,3.75mmol,5.0eq) were dissolved in methanol (6mL), stirred for 0.5H, sodium cyanoborohydride (95mg,1.5mmol,2.0eq) was added, the reaction was warmed to 60 ℃ for 2H, LC-MS checked for completion, and the crude product was purified by preparative HPLC (0.1% aqueous trifluoroacetic acid: acetonitrile 70:30) to give the product (182mg, yield: 49%).

¹HNMR(400MHz,DMSO-d₆)δ(ppm):12.14(s,1H),9.54(s,1H),7.59-7.62(m,2H),7.52(m,1H),7.19-7.21(m,1H),7.06-7.09(m,1H),3.53-3.56(m,2H),3.10-3.17(m,2H),2.90-2.94(m,4H),2.05-2.08(m,2H),1.90-1.93(m,2H).

Molecular formula C₂₀H₂₁N₅OS molecular weight 379.48 LC-MS (Pos, M/z) ═ 380.2[ M + H ]]⁺

EXAMPLE 18 Synthesis of 4-amino-5- (5- (8-methyl-8-azabicyclo [3.2.1] octan-3-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one (Compound 57)

Step 1: synthesis of tert-butyl 3- (((trifluoromethyl) sulfonyl) oxy) -8-azabicyclo [3.2.1] oct-2-ene-8-carboxylate

Dissolving a raw material of 3-oxo-8-azabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester (6.0g,26.63mmol,1.0eq) in an anhydrous tetrahydrofuran solution (30mL), cooling to-70 to-60 ℃ under the protection of nitrogen, dropwise adding a tetrahydrofuran solution (1mol/L,32mL,1.2eq) of lithium bis (trimethylsilyl) amide, stirring for 1h at-70 to-60 ℃, dropwise adding a tetrahydrofuran solution (30mL) of N-phenyl bis (trifluoromethanesulfonyl) imide (11.4g,31.96mmol,1.2eq) again, reacting for 3h below 0 ℃, naturally heating to room temperature, and stirring for 12 h. Cooling to 0-10 ℃, adding saturated salt water (50mL) and saturated sodium carbonate aqueous solution (50mL), extracting with ethyl acetate (100mL multiplied by 3), combining organic phases, drying, filtering, and concentrating the filtrate under reduced pressure to obtain the product.

Step 2: synthesis of tert-butyl 3- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -8-azabicyclo [3.2.1] oct-2-ene-8-carboxylate

Dissolving the intermediate obtained in the previous step in anhydrous 1, 4-dioxane (200mL), adding pinacol diboron (8.1g,31.96mmol,1.2eq) and potassium acetate (9.1g,93.22mmol,3.5eq), adding 1,1 '-bis (triphenylphosphine) ferrocene (737.8mg,1.33mmol,0.05eq) and [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (974.3mg,1.33mmol,0.05eq) under nitrogen protection, heating to 100 ℃ under nitrogen protection, reacting for 3h, cooling to room temperature, adding saturated saline (100mL), extracting with ethyl acetate (100 mL. times.3), separating, combining the organic phases, drying, filtering, and concentrating the filtrate under reduced pressure to obtain the product.

And step 3: synthesis of 3- (6-amino-5-nitropyridin-2-yl) -8-azabicyclo [3.2.1] oct-2-ene-8-carboxylic acid tert-butyl ester

The product obtained in the previous step is dissolved in 1, 4-dioxane (200mL), 6-chloro-3-nitropyridine-2-amine (4.6g,26.63mmol,1.0eq), potassium carbonate (14.7g,106.53mmol,4.0eq) and water (60mL) are added, and [1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (974.3mg,1.33mmol,0.05eq) is added under nitrogen protection, and the temperature is raised to 100 ℃ for reaction for 12 h. Cooling to 50 ℃, adding saturated saline solution (100mL), ethyl acetate (100mL) and activated carbon (2g), stirring for 0.5h at 50 ℃, filtering while hot, leaching a filter cake with ethyl acetate, separating liquid, extracting an aqueous phase with ethyl acetate (100mL multiplied by 2), combining organic phases, drying, filtering, concentrating a filtrate under reduced pressure, purifying a crude product by silica gel column chromatography (EA: PE ═ 1:30), washing with methyl tert-butyl ether, filtering, and drying a filter cake to obtain a product (3.5g, the yield of 3 steps: 38%).

And 4, step 4: synthesis of 3- (5, 6-diaminopyridin-2-yl) -8-azabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

Dissolving an intermediate tert-butyl 3- (6-amino-5-nitropyridin-2-yl) -8-azabicyclo [3.2.1] octane-2-ene-8-carboxylate (2g,5.77mmol,1.0eq) in absolute ethyl alcohol (60mL), adding 10% palladium carbon (500mg), heating to 50 ℃ in a hydrogen atmosphere for reaction for 12h, monitoring the reaction by TLC to be complete, filtering under the protection of nitrogen, leaching a filter cake by using absolute ethyl alcohol, concentrating the filtrate under the protection of nitrogen, adding toluene (50mL), concentrating, repeating for three times, adding absolute ethyl alcohol (50mL), concentrating to obtain a product, and directly putting the product into the next step without purification.

And 5: synthesis of tert-butyl 3- (2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) -8-azabicyclo [3.2.1] octane-8-carboxylate

Dissolving the product of the last step in absolute ethyl alcohol (50mL), adding a 3A molecular sieve (1.8g), adding 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride (2.7g,13.86mmol,2.4eq) under nitrogen protection, heating to 80 ℃ under nitrogen protection, reacting for 12h, adding 3-ethoxy-3-iminopropionic acid ethyl ester hydrochloride (2.7g,13.86mmol,2.4eq), reacting for 12h under nitrogen protection at 80 ℃, filtering while hot, leaching a filter cake with ethanol, concentrating the filtrate under reduced pressure, adding dichloromethane (100mL) to dissolve the obtained solid, adjusting the pH value to about 8 with saturated sodium carbonate aqueous solution, separating, extracting the aqueous phase with dichloromethane (100mL x 3), combining the organic phases, drying, filtering, concentrating the filtrate, purifying the crude product by silica gel column chromatography (MeOH: DCM ═ 1:100-1:50) to obtain a product (257mg, yield 10.7%).

Step 6: synthesis of tert-butyl 3- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) -8-azabicyclo [3.2.1] octane-8-carboxylate

Dissolving an intermediate 3- (2- (2-ethoxy-2-oxoethyl) -3H-imidazo [4,5-b ] pyridin-5-yl) -8-azabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester (257mg,0.62mmol,1.0eq) in anhydrous tetrahydrofuran (5mL), adding 2-aminothiophene-3-carbonitrile (77.5mg,0.62mmol,1.0eq), heating to 40 ℃ under the protection of nitrogen, adding a solution of bis (trimethylsilyl) aminolithium in tetrahydrofuran (1mol/L,6.2mL,10.0eq) dropwise, reacting at 40 ℃ for 2H, heating to 60 ℃ for 4H, monitoring by LC-MS for complete reaction, cooling to room temperature, adjusting the pH value to about 8 with a saturated ammonium chloride aqueous solution, extracting with ethyl acetate (50 mL. times.3), the organic phases were combined, dried, filtered, the filtrate was concentrated under reduced pressure, the crude product was washed with methyl tert-butyl ether and petroleum ether (1:1), filtered and the filter cake was dried to give the product (270mg, yield: 88.2%).

And 7: synthesis of 5- (5- (8-azabicyclo [3.2.1] octan-3-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -4-aminothieno [2,3-b ] pyridin-6 (7H) -one 2,2, 2-trifluoroacetate

Dissolving the intermediate 3- (2- (4-amino-6-oxo-6, 7-dihydrothieno [2,3-b ] pyridin-5-yl) -3H-imidazo [4,5-b ] pyridin-5-yl) -8-azabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester (270mg,0.547mmol,1.0eq) in absolute ethanol (4mL), cooling to 0-10 ℃, adding 30% hydrogen chloride ethanol solution (4mL), and stirring at room temperature for 12H after the addition. The reaction was monitored by LC-MS for completion, concentrated, added with ethyl acetate (10mL), concentrated, repeated three times, the resulting solid was further washed with ethyl acetate (10mL), filtered, and the filter cake was purified by preparative HPLC (mass fraction 0.1% trifluoroacetic acid aqueous solution: acetonitrile 70:30) to give the product (64.6mg, yield: 23.3%).

And 8: synthesis of 4-amino-5- (5- (8-methyl-8-azabicyclo [3.2.1] octan-3-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) thieno [2,3-b ] pyridin-6 (7H) -one:

dissolving intermediate 5- (5- (8-azabicyclo [3.2.1] octane-3-yl) -3H-imidazo [4,5-b ] pyridin-2-yl) -4-aminothieno [2,3-b ] pyridin-6 (7H) -one 2,2, 2-trifluoroacetate (61.8mg,0.12mmol,1.0eq) in anhydrous methanol (3mL), adding acetic acid (7.3mg,0.12mmol,1.0eq) and 37% aqueous formaldehyde (49.3mg,0.60mmol,5.0eq), stirring at room temperature for 1H, adding sodium cyanoborohydride (61.1mg,0.97mmol,8.0eq), stirring at room temperature for 1H, monitoring by LC-MS, further adding sodium cyanoborohydride (98.6mg,1.2mmol,10.0eq), concentrating under reduced pressure, purifying the crude product by reverse phase column chromatography (mass fraction 0.1% acetonitrile: 70 mg: 30.5 mg), yield 7%).

¹HNMR(400MHz,DMSO-d₆)δ(ppm):12.98(s,1H),12.21(s,1H),10.55(s,1H),9.49-9.31(s,2H),8.23(s,1H),8.00-7.95(d,1H),7.63-7.60(d,1H),7.45-7.30(d,1H),7.23-7.17(d,1H),3.90(s,3H),3.17(m,1H),3.00-2.97(m,1H),2.89(m,2H),2.73-2.71(m,1H),2.67-2.65(m,2H),2.33-2.29(m,1H),2.12-2.02(m,2H),1.98-1.93(m,1H).

Molecular formula C₂₁H₂₂N₆OS molecular weight 406.51LC-MS (Pos, M/z) ═ 407.04[ M + H ]]⁺.

The present invention can be better understood from the following experimental examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.

Experimental example 1 enzymatic Activity test

Test article: the compounds of the invention, shown in table 1, were prepared by the methods of the examples. The test method comprises the following steps:

(1) preparation of stock solutions of the Compounds

Compounds were dissolved in 100% DMSO to prepare stock solutions with a maximum concentration of 500 μ M.

(2) Preparation of Compound working solutions

Compound stock solutions were diluted to compound final concentrations of 500, 150, 50, 15, 5, 1.5, 0.5, 0.15, 0.05 μ M to compound working solutions (50X).

(3) Preparation of different enzyme reaction solutions

(a) MAP4K1(HPK1) reaction solution

HPK1 enzyme was dissolved in 8mM MOPS (pH 7.0), 0.2mM EDTA, and 0.33mg/mL myelin basic protein to prepare an enzyme solution at a final concentration of 1.7 nM. 10mM magnesium acetate, [ gamma-³³P]ATP constitutes a Mg/ATP mixture, activating the enzymatic reactionShould be used.

Wherein, compounds 1,2, 16, 17 and 18, [ gamma-³³P]-the ATP concentration is 10. mu.M,

compounds 25, 26, 39, 47, 48, 49, 50, 51, 56 and 57, [ gamma- ] of the Mg/ATP mixture³³P]ATP concentration 15. mu.M.

(b)MAP4K3(GLK)

MAP4K3 enzyme was dissolved in 8mM MOPS (pH 7.0), 0.2mM EDTA, 250. mu. M RLGRDKYKTLRQIRQ to prepare an enzyme solution at a final concentration of 5 nM. The final concentration is 10mM magnesium acetate, 45 μ M [ gamma-33P ] -ATP constitutes Mg/ATP mixed liquor, and the enzymatic reaction is activated.

(4) Enzymatic reaction

The compound working solution was added to a 384-well plate to a final concentration of 10000, 3000, 1000, 300, 100, 30, 10, 3, 1nM, and the different enzyme reactions prepared under the above conditions were added, incubated at room temperature for 40min, and then 0.5% phosphoric acid solution was added to terminate the assay. Taking 10 mu L of reaction liquid to drop on P30 filter paper, washing for 4 times by 0.425% phosphoric acid solution, washing once by methanol, placing in a scintillation counter for detection, and replacing compound solution by 2% DMSO to be used as a positive control (Max); high concentration positive control (10 μ M staurosporine for MAP4K1, 100 μ M PKR inhibitor for MAP4K 3) inhibitor was used as a negative control (Min) in place of compound solution.

TABLE 2 enzyme inhibitory Activity of the Compounds of the invention

"-" represents not determined

The experimental results in table 2 show that the compound of the present invention has good inhibitory activity on HPK1 and good selectivity with respect to MAP4K3, which indicates that the compound of the present invention has good clinical application potential in treating diseases mediated by HPK 1.

Experimental example 2 evaluation of human liver microsome stability of Compound of the present invention

Test article: the compounds of the invention shown in Table 1 and Compound A1 of the patent publication No. WO2016205942A1

The purpose is as follows: the compounds of the invention were evaluated for human liver microsomal stability.

The incubation system comprises the following components:

the test steps are as follows:

(1) the liver microsomes (20mg protein/mL) are taken out from a refrigerator at the temperature of-80 ℃, put on a water bath constant temperature oscillator at the temperature of 37 ℃ for pre-incubation for 3min, and melted for standby.

(2) According to the proportion of the experimental incubation system, a mixed solution of the incubation system (without the compound and the beta-NADPH) is prepared and placed on a water bath constant temperature oscillator at 37 ℃ for pre-incubation for 2 min.

3) Control group (without β -NADPH): and (3) respectively adding 30 mu L of water and 30 mu L of compound working solution (10 mu M) into 240 mu L of the mixed solution of the incubation system in the step (2), vortexing for 30s, uniformly mixing, reacting for the total volume of 300 mu L, and performing sample recovery. And putting the mixture into a water bath constant temperature oscillator at 37 ℃ for incubation, and starting timing, wherein sampling time points are 0min and 60 min.

4) Sample set: and (3) adding 70 mu L of beta-NADPH solution (10mM) and 70 mu L of compound working solution (10 mu M) into 560 mu L of the mixed solution in the step (2), reacting for total volume of 700 mu L, vortexing for 30s, mixing uniformly, and performing pore renaturation. Putting into a 37 ℃ water bath constant temperature oscillator for incubation, and starting timing, wherein the sampling time points are 0min,5min,10min,20min,30min and 60min after timing.

(5) After vortexing for 3min, centrifuge at 12000rpm for 5 min.

(6) Taking 50 mu L of supernatant, adding 150 mu L of water, mixing uniformly by vortex, and analyzing by LC/MS/MS sample injection.

And (3) data analysis:

half-life (t) was calculated using the following first order kinetic equation_1/2) And clearance (Cl):

Ct＝C₀*e^–kt

t_1/2＝ln2/k＝0.693/k

Cl_int＝V_d*k

vd 1/protein content in liver microsomes

Note: k is the slope of the logarithm of the remaining amount of compound plotted against time, V_dIs the apparent volume of distribution.

As a result:

and (4) experimental conclusion: compared with the prior art, the compound has lower clearance rate and good stability of liver microsomes.

Claims

1. A compound represented by the general formula (III):

wherein X₁、X₂、X₃Each independently selected from CH;

X₄is selected from N;

X₉selected from the group consisting of CR₄、N；

R₄Each occurrence is independently selected from hydrogen;

R₁is selected from-NRaRb;

ra is selected from hydrogen and C_1-6An alkyl group;

rb is selected from hydrogen or C_1-6An alkyl group;

R₂is selected from-Y₂-Q₂；

Y₂Is selected from a bond;

Q₂selected from the group consisting of piperidinyl, piperazinyl, 6-12 membered spiroheterocyclyl, 6-12 membered bridged heterocyclyl optionally substituted with a substituent, said substituted piperidinyl, piperazinyl, 6-12 membered spiroheterocyclyl, 6-12 membered bridged heterocyclylRadical selected from C_1-6An alkyl group.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof,

wherein, X₁、X₂、X₃Are each independently CH, X₄Is N;

X₉selected from the group consisting of CR₄Or N;

R₄selected from hydrogen;

R₁is selected from-NRaRb;

ra is selected from hydrogen and C_1-6An alkyl group;

rb is selected from hydrogen;

R₂is selected from-Y₂-Q₂；

Y₂Is selected from the group consisting of a bond,

Q₂selected from piperazinyl, piperidinyl, piperazinyl optionally substituted,

The substituted piperazinyl, the piperidinyl,

Is selected from C_1-6An alkyl group.

3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from compounds of the following structures:

4. a pharmaceutical composition comprising a compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof.

5. A pharmaceutical formulation comprising a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, characterised in that it comprises one or more pharmaceutically acceptable carriers.

6. Use of a compound of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment or prevention of a disease associated with HPK1 mediated disease.

7. The use according to claim 6, wherein the HPK 1-mediated related disease is cancer or a non-cancerous proliferative disease, wherein the disease is lung cancer, squamous cell carcinoma, bladder cancer, stomach cancer, ovarian cancer, peritoneal cancer, breast cancer, ductal carcinoma of the breast, head and neck cancer, endometrial cancer, uterine body cancer, rectal cancer, liver cancer, kidney cancer, renal pelvis cancer, esophageal adenocarcinoma, glioma, prostate cancer, thyroid cancer, cancer of the female reproductive system, carcinoma in situ, lymphoma, neurofibromatosis, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumor, oral cancer, pharyngeal cancer, multiple myeloma, leukemia, non-hodgkin's lymphoma, large intestine villous adenoma, melanoma, cytoma and sarcoma, myelodysplastic syndrome.