RU2811975C1 - Condensed aza-heterocyclic amide compound and its use - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to a compound of formula (I) or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt, where X is selected from a chemical bond, -O- and -CH₂-; Y is N, Y₁ is C, Y₂ is -CH-, Y₃ is N, and Y₄ is -CH-; X₂ is selected from the chemical bond, -(CH₂)_p - and -NH-, where p is 1; means that a chemical bond is absent or present; R is selected from phenyl or pyridinyl, wherein each phenyl or pyridinyl is unsubstituted or substituted with at least one substituent selected from R₁; each R₁, if present, is independently selected from hydrogen, halogen, -OH, amino, -NHC_1-6 alkyl, -N(C_1-6alkyl)₂, cyano, C_1-6 alkyl C_1-6 alkoxi and -SC_{1- 6} alkyl; R₂ is selected from H, halogen, hydroxyl, amino; R₃ represents H; ring A is selected from heterocycloalkyl containing from 4 to 6 ring atoms, 6-8 membered heterobridged cyclyl, 8-10 membered heterofused cyclyl or 7-10 membered heterospirocyclyl, the heteroatoms in the heterocycloalkyl, heterobridged cyclyl, heterofused cyclyl and heterospirocyclyl being independently selected from O and N, and the number of heteroatoms selected from 1, 2, 3 or 4; R₄ is at any substitutable position on ring A and is independently selected from -H, -OH, halogen, -CN, substituted or unsubstituted C_1-6 alkyl, -(CH₂)_m -OH, -(CH₂)_m - COOH, -(CH₂)_m -CO-NH₂, -CO-(CH₂)_m -NH₂, -CO-CR_4aR_4b-OH and -CO-R_4b; R_4a is selected from hydrogen and unsubstituted or substituted C_1-4 alkyl; R_4b is selected from H, unsubstituted or substituted C_1-6 alkyl and unsubstituted or substituted C_3-6 cycloalkyl, the substituent is independently selected from -OH, -NH₂ and halogen, and the number of substituents is selected from 1, 2 and 3; n is selected from 1, 2, 3 and 4. The invention also relates to a pharmaceutical composition having inhibitory activity against TRK, based on these compounds.

I

EFFECT: obtaining new compounds that have a strong inhibitory effect on various cells containing TRK mutations and tumors, have good safety and can be used in medicine as medicinal products for the treatment of hyperproliferative diseases.

22 cl, 1 dwg, 6 tbl, 47 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims priority based on Chinese Patent Application No. 202010411386.0, filed on May 15, 2020, entitled “PYRAZOLOPYRIMIDINE AMIDE COMPOUND AND ITS USE,” the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the field of pharmaceuticals and, in particular, relates to a fused aza-heterocyclic amide compound having kinase inhibitory activity, or a tautomer, stereoisomer, optical isomer, solvate, isotopic derivative, N-oxide, prodrug or pharmaceutically acceptable salt thereof. compounds, and a pharmaceutical composition containing them, and their use as a medicament for the prevention and/or treatment of TRC-mediated diseases.

BACKGROUND OF THE ART

Tropomyosin-linked kinase or tropomyosin receptor kinase (TRK) is a type of nerve growth factor receptor whose family consists of three highly homologous subtypes TRKA, TRKB and TRKC, encoded by the neurotrophic receptor tyrosine kinase type 1 (NTRK1), NTRK2 and NTRK3 genes, respectively . When TRK receptor protein binds to its corresponding ligand, various physiological functions can be achieved by activating downstream signaling pathways such as the RAS/MAPK pathway, PLCγ pathway, and PI3K pathway. TRK family proteins are generally predominantly expressed in neural tissues, are involved in the differentiation and survival of nerve cells and the formation of axons and dendrites, and also play important roles in embryonic development and the maintenance of normal functions of the nervous system.

TRK kinase is activated in malignancies through various mechanisms, mainly structural rearrangements and altered expression. For example, the NTRK gene, encoding TRK kinase, rearranges with other genes to form a fusion oncogene that causes changes in the structure and expression of TRK kinase and is no longer regulated or controlled by nerve growth factor ligand, but is constitutively activated, and therefore promotes formation and development of tumors. In addition, the gene sequencing result also shows that TRK kinase has a close relationship with the occurrence, metastasis and deterioration of various tumors and is expressed in various tumors such as non-small cell lung cancer, colorectal cancer, melanoma, gallbladder cancer, thyroid cancer, malignant glioma and the like.

Currently, the first generation TRK inhibitors Larotrectinib (LOXO-101) and Entrectinib (RXDX-101) were approved by the US Food and Drug Administration (FDA) in 2018 and 2019, respectively. Larotrectinib is a potent oral and selective tropomyosin receptor kinase inhibitor whose efficacy data were published at the American Society of Clinical Oncology (ASCO) meeting back in June 2017. Of a total of 55 patients enrolled in the phase I and phase II clinical trials, The 46 patients evaluated had an overall response rate (ORR) of 78%. Entrecteinib is a potent inhibitor of TRK, ROS1 and ALK proteins and can cross the blood-brain barrier, with an ORR of 79% in the 24 patients evaluated in a phase I clinical trial.

Like other targeted drugs, TRK inhibitors also face drug resistance issues. A mutation in the NTRK kinase domain can cause a change in the conformation of the TRK family protein kinase domain or ATP binding affinity, thereby affecting the binding of TRK inhibitors to targets, and the types of mutations are G595R, G639R, G667C and the like. In order to solve the problem of drug resistance of first-generation TRK inhibitors, second-generation TRK inhibitors such as LOXO-195, TPX-005 and the like have been studied.

BRIEF DESCRIPTION OF THE INVENTION

One of the objects of the present invention is to provide a type of fused aza-heterocyclic amide compound with excellent TRK inhibition activity (wild type and mutant type) and a novel structure, or a tautomer, stereoisomer, solvate, isotopic derivative, N-oxide, prodrug or pharmaceutically acceptable thereof. salt.

Another object of the present invention is to provide a type of fused aza-heterocyclic amide compound with better activity of inhibiting TK-mutant tumor cells compared to a known compound and a new structure, or a tautomer, stereoisomer, solvate, isotopic derivative, N-oxide, prodrug or pharmaceutically acceptable salt.

Another object of the present invention is to provide a type of fused aza-heterocyclic amide compound with better TRK inhibition activity (wild type and mutant type) and TRK mutant tumor cell inhibition activity compared to a known compound and a new structure, or a tautomer, stereoisomer, solvate thereof, an isotopic derivative, N-oxide, prodrug or pharmaceutically acceptable salt.

Another object of the present invention is to provide a type of fused aza-heterocyclic amide compound with better inhibitory activity against TRK mutation tumor cells and antitumor activity in vivo compared to a known compound, or a tautomer, stereoisomer, solvate, isotopic derivative, N-oxide, thereof, a prodrug or a pharmaceutically acceptable salt.

Another object of the present invention is to provide a type of fused aza-heterocyclic amide compound with better inhibitory activity against TRK mutation tumor cells and antitumor activity in vivo, as well as better safety compared to a known compound, or a tautomer, stereoisomer, solvate, isotopic derivative thereof , N-oxide, prodrug or pharmaceutically acceptable salt.

Another object of the present invention is to provide a type of fused aza-heterocyclic amide compound with better TRK inhibition activity (wild type and mutant type), TRK mutation tumor cell inhibitory activity and in vivo antitumor activity, and better safety compared to the known compound, or a tautomer, stereoisomer, solvate, isotopic derivative, N-oxide, prodrug or pharmaceutically acceptable salt thereof.

In particular, the present description provides a compound of formula (I) or a tautomer, stereoisomer, optical isomer, solvate, isotopic derivative, N-oxide, prodrug or pharmaceutically acceptable salt thereof,

where X is selected from: chemical bond, -O-, -S-, -NH- and -CH ₂ -;

Y, Y ₁ , Y ₂ , Y ₃ and Y ₄ are each independently selected from: -CH-, N and C;

X ₂ is selected from: chemical bond, -(CH ₂ ) _p - and -NH-, where p is 1, 2, 3 or 4;

means that a chemical bond is absent or present;

R is selected from: C _5-12 aryl or heteroaryl, wherein each aryl or heteroaryl is unsubstituted or substituted with at least one substituent selected from R ₁ ;

Each R ₁ , if present, is independently selected from hydrogen, halogen, -OH, amino, -NHC _1-6 alkyl, -N(C _1-6 alkyl) ₂ , cyano, CI _-2 alkyl, unsubstituted or substituted at least one R _1a , C _1-6 alkoxy, unsubstituted or substituted by at least one R _1a , C _3-6 cycloalkyl, unsubstituted or substituted by at least one R _1a , C _3-6 cycloalkoxy, unsubstituted or substituted by at least one one R _1a , and -SC _1-6 alkyl;

R _1a , if present, is each independently selected from C _1-6 alkyl, C _1-6 alkoxy, nitro, halogen, -OH, amino, -NHC _1-6 alkyl, -N(C _1-6 alkyl) ₂ and cyano;

R ₂ is selected from: H, halogen, hydroxyl, amino and substituted or unsubstituted C _1-6 alkyl, where "substituted" means substituted with 1, 2 or 3 substituents selected from halogen and hydroxyl;

R ₃ is selected from: H, halogen, -OH, amino, C _1-6 alkyl and C _1-6 alkoxy;

ring A is selected from: cycloalkyl, heterocycloalkyl, bridged cyclyl, heterobridged cyclyl, fused cyclyl, heterofused cyclyl, spirocyclyl and heterospirocyclyl, wherein the heteroatoms in the heterocycloalkyl, heterobridged cyclyl, heterofused cyclyl and heterospirocyclyl are independently selected from O, S and N, and the number of heteroatoms is selected from 1, 2, 3 and 4;

R ₄ is at any substitutable position on ring A and is independently selected from: -H, -OH, halogen, -CN, oxo, substituted or unsubstituted C _1-6 alkyl, -(CH ₂ ) _m -OH, -(CH ₂ ) _m -COOH, -(CH ₂ ) _m -CO-NH ₂ , -CO-(CH ₂ ) _m -NH ₂ , -CO-CR _4a R _4b -OH and -CO-R _4b ; where oxo means that two H atoms of the same substitution site are replaced by the same O atom to form a double bond; m is selected from 1, 2, 3 and 4;

R _4a is selected from hydrogen and unsubstituted or substituted with _I-4 alkyl;

R _4b is selected from H, unsubstituted or substituted C _1-6 alkyl and unsubstituted or substituted C _3-6 cycloalkyl, the substituent is independently selected from -OH, -NH ₂ and halogen, and the number of substituents is selected from 1, 2 and 3;

n is selected from 1, 2, 3 and 4.

In one embodiment, the present invention provides a compound of formula (I) or a tautomer, stereoisomer, optical isomer, solvate, isotopic derivative, N-oxide, prodrug or pharmaceutically acceptable salt thereof, wherein R is selected from: C _5-9 aryl and heteroaryl, wherein each aryl or heteroaryl is unsubstituted or substituted with at least one substituent selected from R ₁ ; or R is selected from: C _5-6 aryl and heteroaryl, wherein each aryl or heteroaryl is unsubstituted or substituted with at least one substituent selected from R ₁ .

In one embodiment, said compound has the structure of formula (I-1) or formula (I-2):

or

where R ₁ , R ₂ , R ₃ , R ₄ , X, X ₂ , Y, Y ₁ , Y ₂ , Y ₃ , Y ₄ , ring A and n are as described for the compound of formula (I) of the present invention, and X ₁ is selected from: -CH- and N. In one embodiment of the present invention, said compound has the structure of formula (IA):

where R ₁ , R ₂ , R ₃ , R ₄ , X, X ₁ , X ₂ , Y, Y ₁ , Y ₂ , Y ₃ , Y ₄ , ring A and n are as described for the compound of formula (I- 1) or formula (I-2) of the present invention.

In one embodiment, the compound has the structure of formula (I-A-1a), formula (I-A-1b) or formula (I-A-1c):

where R ₁ , R ₂ , R ₃ , R ₄ , X, X ₁ , X ₂ , ring A and n are as described for the compound of formula (I-1) or formula (I-2) of the present invention.

In one embodiment, the compound has the structure of formula (I-B):

where R ₁ , R ₂ , R ₃ , R ₄ , X ₁ , X ₂ , Y , Y ₁ , Y ₂ , Y ₃ , Y ₄ , ring A and n are as described for the compound of formula (I-1) or formula (I-2) of the present invention.

In one embodiment, the compound has the structure of formula (I-B-1a), formula (I-B-1b), or formula (I-B-1c):

where R ₁ , R ₂ , R ₃ , R ₄ , X ₁ , X ₂ , ring A and n are as described for the compound of formula (I-1) or formula (I-2) of the present invention.

In one embodiment, the compound has the structure of formula (I-C):

where R ₁ , R ₂ , R ₃ , R ₄ , X ₁ , X ₂ , Y, Y ₁ , Y ₂ , Y ₃ , Y ₄ , ring A and n are as described for the compound of formula (I-1) or formula (I-2) of the present invention. In one embodiment, the compound has a structure of formula (I-C-1a), formula (IC-1b) or formula (I-C-1c):

where R ₁ , R ₂ , R ₃ , R ₄ , X ₁ , X ₂ , ring A and n are as described for the compound of formula (I-1) or formula (I-2) of the present invention.

In one embodiment, the present invention provides a compound (compound of formula (I), compound of formula (I-1), compound of formula (I-2), compound of formula (IA), compound of formula (I-A-1a), compound of formula ( IA-1b), compound of formula (I-A-1c), compound of formula (IB), compound of formula (I-B-1a), compound of formula (IB-1b), compound of formula (I-B-1c), compound of formula (IC), compound of formula (I-C-1a), compound of formula (IC-1b), compound of formula (IC-1c)) or its tautomer, stereoisomer, optical isomer, solvate, isotopic derivative, N-oxide, a prodrug or pharmaceutically acceptable salt, wherein each R ₁ , if present, is independently selected from hydrogen, halogen, -OH, amino, cyano, C _1-6 alkyl, unsubstituted or substituted by at least one R _1a , C _1-6 alkoxy , unsubstituted or substituted by at least one R _1a , C _3-6 cycloalkyl, unsubstituted or substituted by at least one R _1a , and C _3-6 cycloalkoxy, unsubstituted or substituted by at least one R _1a ; or, each R ₁ , if present, is independently selected from: hydrogen, halogen, -OH, amino, cyano, C _1-6 alkyl, unsubstituted or substituted by at least one R _1a , and C _1-6 alkoxy, unsubstituted or substituted with at least one R _1a ; or, each R ₁ , if present, is independently selected from: hydrogen, halogen, -OH, amino, C _1-6 alkyl and C _1-6 alkoxy; or, each R ₁ , if present, is independently selected from: hydrogen, halogen, -OH, amino, C _1-3 alkyl and C _1-3 alkoxy; or, each R ₁ , if present, is independently selected from: hydrogen, halogen, -OH, amino, methyl, ethyl, methoxy and ethoxy; or, each R ₁ , if present, is independently selected from: hydrogen, F, Cl, -OH, methyl and methoxy; or, each R ₁ , if present, is independently selected from: F, -OH, methyl and methoxy. In a particular embodiment of the present invention, each R ₁ a, if present, is independently selected from C _1-3 alkyl, C _1-3 alkoxy, nitro, halogen, -OH, amino, -NHC _1-6 alkyl, -N(C _1-6 alkyl) ₂ and cyano; or R _1a is selected from halogen, -OH, amino, -NHC _1-3 alkyl, -N(C _1-3 alkyl) ₂ and cyano; or R _1a is selected from halogen, -OH, amino and cyano; or R _1a is selected from F, Cl and -OH.

In one embodiment, the present invention provides a compound (compound of formula (I), compound of formula (I-1), compound of formula (I-2), compound of formula (IA), compound of formula (I-A-1a), compound of formula ( IA-1b), compound of formula (I-A-1c), compound of formula (IB), compound of formula (I-B-1a), compound of formula (IB-1b), compound of formula (I-B-1c), compound formula (IC), compound of formula (I-C-1a), compound of formula (IC-1b), compound of formula (IC-1 c)) or tautomer, stereoisomer, optical isomer, solvate, isotopic derivative, N-oxide, a prodrug or pharmaceutically acceptable salt wherein R ₂ is selected from: H, F, OH and substituted or unsubstituted C _1-6 alkyl, where "substituted" means substituted with 1, 2 or 3 substituents selected from halogen and hydroxyl; or R ₂ is selected from H, F, -OH and C _1-6 alkyl; or R ₂ is selected from H, F, -OH and C _1-3 alkyl; or R ₂ is selected from H and F.

In one embodiment, the present invention provides a compound (compound of formula (I), compound of formula (I-1), compound of formula (I-2), compound of formula (IA), compound of formula (I-A-1a), compound of formula ( IA-1b), compound of formula (I-A-1 c), compound of formula (IB), compound of formula (I-B-1a), compound of formula (IB-1b), compound of formula (I-B-1 c) , compound of formula (IC), compound of formula (I-C-1a), compound of formula (IC-1b), compound of formula (IC-1 c)) or tautomer, stereoisomer, optical isomer, solvate, isotopic derivative, nitric oxide , a prodrug, or a pharmaceutically acceptable salt, wherein X ₁ is selected from -CH- and N, and R ₁ is selected from F and -OCH ₂ .

In one embodiment, X ₁ is selected from -CH-, and R ₁ is selected from F; or X ₁ is selected from N, and R ₁ is selected from -OSH ₃ .

In one embodiment, the compound has the structure of formula (I-D):

where R ₃ , R ₄ , X ₂ , Y, Y ₁ , Y ₂ , Y ₃ , Y ₄ , ring A and n are as described for the compound of formula (I) of the present invention.

In one embodiment, the compound has the structure of formula (I-D-a), formula (I-D-b), or formula (I-D-c):

where R ₃ , R ₄ , X ₂ , ring A and n are as described for the compound of formula (I) of the present invention.

In one embodiment, the present invention provides a compound (compound of formula (I), compound of formula (I-1), compound of formula (I-2), compound of formula (IA), compound of formula (I-A-1a), compound of formula ( IA-1b), compound of formula (I-A-1 c), compound of formula (IB), compound of formula (I-B-1a), compound of formula (IB-1b), compound of formula (I-B-1 c) , compound of formula (IC), compound of formula (I-C-1a), compound of formula (IC-1b), compound of formula (IC-1 c), compound of formula (ID), compound of formula (IDa), compound of formula (IDb ), a compound of formula (IDc)) or a tautomer, stereoisomer, optical isomer, solvate, isotopic derivative, N-oxide, prodrug or pharmaceutically acceptable salt thereof, wherein R ₃ is selected from: H, halogen, -OH, amino, C _{1- 3} alkyl and C _1-3 alkoxy; or R ₃ selected from: H, F, Cl, -OH, methyl and methoxy; or R ₃ is selected from: H and F. In one embodiment, the compound has the structure of formula (ID-1a), formula (ID-1b) or formula (ID-1c):

where R ₄ , X ₂ , ring A and n are as described for the compound of formula (I) of the present invention.

In one embodiment, the compound has the structure of formula (I-C-1a-1):

where R ₄ , X ₂ , ring A and n are as described for the compound of formula (I) of the present invention.

In one embodiment, the compound has the structure of formula (I-C-1a-2):

where R ₄ , X ₂ , ring A and n are as described for the compound of formula (I) of the present invention.

In one embodiment, the present invention provides a compound (compound of formula (I), compound of formula (I-1), compound of formula (I-2), compound of formula (IA), compound of formula (I-A-1a), compound of formula ( IA-1b), compound of formula (I-A-1 c), compound of formula (IB), compound of formula (I-B-1a), compound of formula (IB-1b), compound of formula (I-B-1 c) , compound of formula (IC), compound of formula (I-C-1a), compound of formula (IC-1b), compound of formula (IC-1 c), compound of formula (ID), compound of formula (IDa), compound of formula (IDb ), compound of formula (IDc), compound of formula (ID-la), compound of formula (ID-1b), compound of formula (ID-1c), compound of formula (I-C-1a-1), compound of formula (I-C -1a-2), the same below) or a tautomer, stereoisomer, optical isomer, solvate, isotopic derivative, N-oxide, prodrug or pharmaceutically acceptable salt thereof, wherein ring A is selected from: C _3-6 cycloalkyl, 4-6 -membered heterocycloalkyl, C _6-8 bridged cyclyl, 6-8 membered heterobridged cyclyl, C _8-10 fused cyclyl, 8-10 membered heterofused cyclyl, C7-12 monospirocyclyl and 7-12 membered heteromonospirocyclyl; or ring A is selected from: 4-6 membered heterocycloalkyl, 6-8 membered heterobridged cyclyl, 8-10 membered heterofused cyclyl and 7-12 membered heteromonospirocyclyl; wherein the heteroatoms in heterocycloalkyl, heterobridged cyclyl, heterofused cyclyl and heteromonospirocyclyl are independently selected from O, S and N, and the number of heteroatoms is selected from 1, 2, 3 and 4; or ring A is selected from the following structures:

or ring A is selected from the following structures:

or ring A is selected from the following structures:

or ring A is selected from the following structures:

or ring A is selected from the following structures:

In one embodiment, the present invention provides a compound (compound of formula (I), compound of formula (I-1), compound of formula (I-2), compound of formula (IA), compound of formula (I-A-1a), compound of formula ( IA-1b), compound of formula (I-A-1 c), compound of formula (IB), compound of formula (I-B-1a), compound of formula (IB-1b), compound of formula (I-B-1 c) , compound of formula (IC), compound of formula (I-C-1a), compound of formula (IC-1b), compound of formula (IC-1 c), compound of formula (ID), compound of formula (IDa), compound of formula (IDb ), compound of formula (IDc), compound of formula (ID-1a), compound of formula (ID-1b), compound of formula (ID-1c), compound of formula (I-C-1a-1) and compound of formula (I-C -1a-2)) or a tautomer, stereoisomer, optical isomer, solvate, isotopic derivative, N-oxide, prodrug or pharmaceutically acceptable salt thereof, wherein _R4 is independently selected from: -H, -OH, halogen, -CN, oxo, substituted or unsubstituted C _1-6 alkyl, -COOH, -CONH ₂ , -CO-CR _4a R _4b -OH and -CO-R _4b ; or R ₄ is independently selected from: -H, -OH, halogen, -CN, oxo, substituted or unsubstituted C _1-6 alkyl, -CO-CR _4a R _4b -OH and -CO-R _4b ; or, R ₄ is independently selected from: -H, -OH, halogen, substituted or unsubstituted C _1-6 alkyl, -CO-CR _4a R _4b -OH and -CO-R _4b ; or, R ₄ is independently selected from: -H, -OH, halogen, substituted or unsubstituted C _1-3 alkyl, -CO-CR _4a R _4b -OH and -CO-R _4b ; or, R ₄ is independently selected from: -H, -OH, substituted or unsubstituted C _1-3 alkyl, -CO-CR _4a R _4b -OH and -CO-R _4b ; or, R ₄ is independently selected from: -CO-CR _4a R _4b -OH and -CO-R _4b ; where oxo means that two H atoms of the same substitution center are replaced by the same O atom to form a double bond; R _4a is selected from hydrogen and unsubstituted or substituted C _1-4 alkyl; R _4b is selected from unsubstituted or substituted C _1-6 alkyl and unsubstituted or substituted C _3-6 cycloalkyl, the substituent is independently selected from -OH, -NH ₂ and halogen, and the number of substituents is selected from 1, 2 and 3; or the substituent to be substituted is independently selected from -OH and F, and the number of substituents is selected from 1, 2 and 3.

In one embodiment, the present description provides a compound and/or its tautomer, optical isomer, solvate, isotopic derivative or pharmaceutically acceptable salt, wherein said compound has the structure of formula (I-A-1a):

where X is selected from: chemical bond, -O-, -S-, -NH- and -CH ₂ -;

X ₁ selected from: -CH- and N;

X ₂ is selected from: chemical bond, -(CH ₂ ) _p - and -NH-, where p is 1, 2, 3 or 4;

R ₁ is selected from: H, halogen, -OH, amino, C _1-6 alkyl and C _1-6 alkoxy;

R ₂ is selected from: H, F, OH and substituted or unsubstituted C _1-6 alkyl, where "substituted" means substituted with 1, 2 or 3 substituents selected from halogen and hydroxyl;

R ₃ is selected from: H, halogen, -OH, amino, C _1-6 alkyl and C _1-6 alkoxy; ring A is selected from: cycloalkyl, heterocycloalkyl, bridged cyclyl, heterobridged cyclyl, fused cyclyl, heterofused cyclyl, spirocyclyl and heterospirocyclyl, wherein the heteroatoms in the heterocycloalkyl, heterobridged cyclyl, heterofused cyclyl and heterospirocyclyl are independently selected from O, S and N, and the number of heteroatoms is selected from 1, 2, 3 and 4;

R ₄ is at any substitutable position on ring A and is independently selected from: -H, -OH, halogen, -CN, oxo, substituted or unsubstituted C _1-6 alkyl, -(CH ₂ ) _m -OH, -(CH ₂ ) _m -COOH, -(CH ₂ ) _m -CO-NH ₂ , -CO-(CH ₂ ) _m -NH ₂ , -CO-CR _4a R _4b -OH and -CO-R _4b ; where oxo means that two H atoms of the same substitution center are replaced by the same O atom to form a double bond; m is selected from 1, 2, 3 and 4; R _4a is selected from hydrogen and unsubstituted or substituted C _1-4 alkyl; R _4b is selected from H, unsubstituted or substituted C _1-6 alkyl and unsubstituted or substituted C _3-6 cycloalkyl, the substituent is independently selected from -OH, -NH ₂ and halogen, and the number of substituents is selected from 1, 2 and 3; n is selected from 1, 2, 3 and 4.

In one embodiment, the present disclosure provides a compound, or a tautomer, optical isomer, solvate, isotopic derivative, or pharmaceutically acceptable salt thereof, wherein said compound has the structure represented by formula (I-B-1a):

where R ₁ , R ₂ , R ₃ , R ₄ , X ₁ , X ₂ , ring A and n are as described for the compound of formula (I-A-1a) of the present invention.

In one embodiment, the present disclosure provides a compound, or a tautomer, optical isomer, solvate, isotopic derivative, or pharmaceutically acceptable salt thereof, wherein said compound has the structure represented by formula (I-D-1a):

where R ₁ , R ₃ , R ₄ , X ₁ , X ₂ , ring A and n are as described for the compound of formula (I-A-1a) of the present invention.

In one embodiment, provided herein is a compound, or a tautomer, optical isomer, solvate, isotopic derivative, or pharmaceutically acceptable salt thereof, wherein said compound has the structure represented by formula (I-C-1a):

where R ₁ , R ₃ , R ₄ , X ₁ , X ₂ , ring A and n are as described for the compound of formula (I-A-1a) of the present invention.

In one embodiment, the present disclosure provides a compound, or a tautomer, optical isomer, solvate, isotopic derivative, or pharmaceutically acceptable salt thereof, wherein said compound has the structure represented by the formula (I-C-1a-1):

where R ₄ , X ₂ , ring A and n are as described for the compound of formula (I-A-1a) of the present invention.

In one embodiment, provided herein is a compound, or a tautomer, optical isomer, solvate, isotopic derivative, or pharmaceutically acceptable salt thereof, wherein said compound has the structure represented by the formula (I-C-1a-2):

where R ₄ , X ₂ , ring A and n are as described for the compound of formula (I-A-1a) of the present invention.

In one embodiment, the present invention provides a compound or a tautomer, optical isomer, solvate, isotopic derivative, or pharmaceutically acceptable salt thereof, wherein ring A is selected from: _C3-6 cycloalkyl, 4-6 membered heterocycloalkyl, _C6-8 bridged cyclyl, 6-8 membered heterobridged cyclyl, C _6-10 fused cyclyl, 8-10 membered heterofused cyclyl, C _7-12 monospirocyclyl and 7-12 membered heteromonospirocyclyl, where the heteroatoms in the heterocycloalkyl, heterobridged cyclyl, heterofused cyclyl and heteromonospirocyclyl are independently selected of O, S and N, and the number of heteroatoms is selected from 1, 2 and 3.

In one embodiment, the present invention provides a compound or a tautomer, optical isomer, solvate, isotopic derivative, or pharmaceutically acceptable salt thereof, wherein ring A is selected from the following structures:

In one embodiment, the present invention provides a compound or a tautomer, optical isomer, solvate, isotopic derivative, or pharmaceutically acceptable salt thereof, wherein R ₄ is independently selected from: -H, -OH, halogen, -CN, oxo, substituted or unsubstituted C _{1 -6} alkyl, -COOH, -CONH ₂ , -CO-CR _4a R _4b -OH and -CO-R _4b , where oxo means that two H atoms of the same substitution center are replaced by the same O atom to form double bond; R _4a is selected from hydrogen and unsubstituted or substituted C _1-4 alkyl; R _4b is selected from unsubstituted or substituted C _1-6 alkyl and unsubstituted or substituted C _3-6 cycloalkyl, the substituent is independently selected from -OH, -NH ₂ and halogen, and the number of substituents is selected from 1, 2 and 3.

In one embodiment, the present invention provides a compound, or a tautomer, optical isomer, solvate, isotopic derivative, or pharmaceutically acceptable salt thereof, wherein said compound has the following structures:

In another aspect, the present specification further provides a pharmaceutical composition comprising a compound or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt thereof disclosed herein.

In another aspect, the present specification further provides a pharmaceutical composition comprising a compound or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt thereof disclosed herein, and a pharmaceutically acceptable excipient.

In another aspect, the present specification further provides the use of a compound or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt thereof disclosed herein, or a pharmaceutical composition disclosed herein, to produce a medicament for the prevention and/or treatment of a TRK-mediated disease.

In one embodiment, the disease is selected from a painful disease, a cell proliferative disease, an inflammatory disease, a neurodegenerative disease, and an infectious disease.

In one embodiment, the disease is mediated by one, two or three of TRKA, TRKB and TRKC.

In one embodiment, the disease relates to dysregulation of the NTRK gene, TRK protein, or expression, activity, or level thereof; preferably, it refers to a fusion, amplification, rearrangement, mutation or high expression of the NTRK gene; and further preferably it refers to a fusion or mutation of the NTRK gene. In another aspect, the present specification further provides a compound, or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt disclosed herein, or a pharmaceutical composition disclosed herein, for use in preventing and/or treating TRK-mediated disease. In another aspect, the present disclosure further provides a method of preventing and/or treating a TRK-mediated disease, comprising: administering to a patient a compound or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt thereof described in herein, or a pharmaceutical composition described herein.

In another aspect, the present specification further provides the use of a compound, or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt thereof described herein, or a pharmaceutical composition described herein, as medicine.

In one embodiment, the drug is used to treat a disease mediated by TRK, ALK, ROS1, or a combination thereof. In one embodiment, the medicament is used to treat a painful disease, a cell proliferative disease, an inflammatory disease, a neurodegenerative disease, or an infectious disease. In another aspect, the present specification further provides the use of a compound or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt thereof described herein, or a pharmaceutical composition described herein, to produce a TRK kinase inhibitor, an ALK kinase inhibitor, or a ROS1 kinase inhibitor.

In one embodiment, the inhibitor is used to treat a disease mediated by TRK, ALK, ROS 1, or a combination thereof.

In one embodiment, the inhibitor is used to treat a painful disease, a cell proliferative disease, an inflammatory disease, a neurodegenerative disease, or an infectious disease. In another aspect, the present specification further provides the use of a compound or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt thereof described herein, or a pharmaceutical composition described herein, to produce a medicament for the treatment of a painful disease, a cell proliferative disease, an inflammatory disease, a neurodegenerative disease or an infectious disease.

In one embodiment, the painful disease, cell proliferative disease, inflammatory disease, neurodegenerative disease, or infectious disease relates to dysregulation of an NTRK gene, TRK protein, or expression, activity, or level thereof; preferably they relate to fusion, amplification, rearrangement, mutation or high expression of the NTRK gene; and further preferably they relate to a fusion or mutation of the NTRK gene.

In one embodiment, the painful disease, cell proliferative disease, inflammatory disease, neurodegenerative disease, or infectious disease relates to dysregulation of the ROS1 gene, ROS1 protein, or expression, activity, or level thereof; preferably they relate to fusion, amplification, rearrangement, mutation or high expression of the ROS1 gene; and furthermore preferably they relate to a fusion or mutation of the ROS1 gene.

In one embodiment, the painful disease, cell proliferative disease, inflammatory disease, neurodegenerative disease, or infectious disease refers to dysregulation of a gene, protein, or expression, activity, or level of TRK, ALK, ROS1, or a combination thereof; preferably they relate to fusion, amplification, rearrangement, mutation or high expression of the NTRK, ALK, ROS 1 gene or a combination thereof; and further preferably they refer to a fusion or mutation of the NTRK, ALK, ROS 1 gene or a combination thereof.

In one embodiment, the cell proliferative disease is a tumor or cancer.

In one embodiment, the tumor or cancer is a solid tumor and a hematologic tumor, preferably a solid tumor. In one embodiment of the invention, the tumor or cancer is hematologic malignancy, lung cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, brain glioma, colorectal cancer, melanoma, head and neck cancer, gallbladder cancer, thyroid cancer glands, glioblastoma, stomach cancer, neuroblastoma or salivary gland cancer; preferably the lung cancer is non-small cell lung cancer.

In another aspect, the present specification further provides a compound or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt thereof described herein, or a pharmaceutical composition described herein, for use in treating pain, cell proliferative disease, inflammation, neurodegenerative disease or infectious disease.

In another aspect, the present disclosure further provides a method of treating a disease, comprising: administering to a patient a compound or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt thereof as described herein, or a pharmaceutical composition, as described herein, wherein the disease is pain, a cell proliferative disease, inflammation, a neurodegenerative disease, or an infectious disease.

In one embodiment, the compound, or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative, or pharmaceutically acceptable salt thereof described herein, or a pharmaceutical composition described herein, is used in combination with another one, two or more drugs having the effect of treating a cell proliferative disease.

In another aspect, the present specification further provides the use of a compound or a tautomer, optical isomer, stereoisomer, solvate, N-oxide, prodrug, isotopic derivative or pharmaceutically acceptable salt thereof described herein, or a pharmaceutical composition described herein, to produce a medicament for the prevention and/or treatment of a disease mediated by TRK, ALK, ROS 1 or a combination thereof.

In one embodiment, the disease is selected from a painful disease, a cell proliferative disease, an inflammatory disease, a neurodegenerative disease, and an infectious disease.

In one embodiment, the disease refers to dysregulation of a gene, protein, or expression, activity, or level of TRK, ALK, ROS 1, or a combination thereof.

In one embodiment, the disease refers to a fusion, amplification, rearrangement, mutation, or high expression of an NTRK, ALK, ROS1 gene, or a combination thereof; preferably it refers to a fusion or mutation of the NTRK, ALK, ROS1 gene or a combination thereof.

In another aspect, the present disclosure further provides a method of preventing and/or treating a disease mediated by TRK, ALK, ROS1, or a combination thereof, comprising: administering to a patient a compound or a tautomer, stereoisomer, optical isomer, solvate, N-oxide, prodrug, isotopic derivative thereof or a pharmaceutically acceptable salt as described herein, or a pharmaceutical composition as described herein.

Definitions

Unless otherwise specified, the following terms used herein have the following definitions.

The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, that is, a linear or branched group containing from 1 to 20 carbon atoms, preferably containing from 1 to 10 carbon atoms (i.e., C _1-10 alkyl), additionally, preferably containing from 1 to 8 carbon atoms (C _1-8 alkyl), and more preferably containing from 1 to 6 carbon atoms (i.e., C _1-6 alkyl), for example, "C _1-6 alkyl" means, that the group is alkyl and the number of carbon atoms in the carbon chain is from 1 to 6 (namely, 1, 2, 3, 4, 5 or 6). Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2 ,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, n-heptyl, n-octyl and the like.

The term "carbocyclyl" or "carbocycle" refers to a monovalent or multivalent, saturated or partially unsaturated monocyclic, bicyclic or tricyclic system containing from 3 to 12 carbon atoms, where the monocyclic, bicyclic or tricyclic system does not contain an aromatic ring. Carbon bicyclyl includes bridged cyclyl, spirocyclyl, fused cyclyl and the like. Bridged cyclyl means that any two rings have two atoms in common, which may or may not be directly bonded.

The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group containing a specified number of carbon atoms, preferably containing from 3 to 12 carbon atoms (i.e., C _3-12 cycloalkyl), more preferably containing from 3 to 10 carbon atoms ( C _3-10 cycloalkyl), and further preferably containing from 3 to 6 carbon atoms (C _3-6 cycloalkyl), from 4 to 6 carbon atoms (C _4-6 cycloalkyl) or from 5 to 6 carbon atoms (C _{5- 6} cycloalkyl). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, 2-ethylcyclopentyl, dimethylcyclobutyl and the like.

The term "alkoxy" refers to -O-alkyl, where alkyl is as defined above, i.e. alkyl contains from 1 to 20 carbon atoms, preferably from 1 to 10 carbon atoms, more preferably from 1 to 8 carbon atoms, and further more preferably from 1 to 6 carbon atoms (namely 1, 2, 3, 4, 5 or 6). Typical examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy and the like. The term "halogen" or "halo" refers to F, Cl, Br and I.

The term "haloalkyl" means that one, two or more hydrogen atoms or all of the hydrogen atoms in alkyl, as defined above, are replaced by halogen. Typical examples of haloalkyl include CCl ₃ , CF 3 , CHCl ₂ , CH ₂ Cl , CH ₂ Br , CH ₂ I , CH ₂ CF ₃ , CF ₂ CF ₃ and the like.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic hydrocarbon substituent and has a non-aromatic structure containing from 3 to 20 ring atoms, where 1, 2, 3 or more ring atoms are selected from N, O and S, and the rest the ring atoms are C. The heterocyclyl preferably contains 3 to 12 ring atoms (C _3-12 heterocyclyl), further preferably 3 to 10 ring atoms (C 3-10 heterocyclyl), or 3 to 8 ring atoms (C _3-10 heterocyclyl), or 3 to 8 ring atoms (C 3-10 heterocyclyl). C _3-8 heterocyclyl), or from 3 to 6 atoms in the ring (C _3-6 heterocyclyl), or from 4 to 6 atoms in the ring (C _4-6 heterocyclyl), or from 5 to 6 atoms in the ring (C _{5 -6} heterocyclyl). The number of heteroatoms is preferably I-4, more preferably I-3 (ie 1, 2 or 3). Examples of monocyclic heterocyclyl include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyranyl and the like. Polycyclic heterocyclyl includes heterocyclyls such as heterospirocyclyl, heterofused cyclyl, heterobridged cyclyl and the like.

The term “heterocycloalkyl” refers to a saturated “heterocycloalkyl” as defined above, containing from 3 to 20 ring atoms, where 1, 2, 3 or more ring atoms are selected from N, O and S, and the remaining ring atoms are C Heterocycloalkyl preferably contains from 3 to 12 ring atoms (C _3-12 heterocycloalkyl), further preferably from 3 to 10 ring atoms (C _3-10 heterocycloalkyl), or from 3 to 8 ring atoms (C _3-8 heterocycloalkyl ), or from 3 to 7 ring atoms (C _3-7 heterocycloalkyl), or from 3 to 6 ring atoms (C _3-6 heterocycloalkyl), or from 4 to 6 ring atoms (C _4-6 heterocycloalkyl), or from 5 to 6 ring atoms (C _5-6 heterocycloalkyl). The number of heteroatoms is preferably I-4, more preferably I-3 (ie 1, 2 or 3). Examples of heterocycloalkyl include azacyclopropyl, oxacyclopropyl, thiocyclopropyl, azacyclobutyl, oxacyclobutyl, thiocyclobutyl, pyrrolidinyl, tetrahydrofuranyl, oxocyclohexane, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dioxanyl, dithiacyclohexyl, oxazolidinyl, thiaz olidinyl, pyrazolidinyl, imidazolidinyl and the like.

The term "aryl" refers to monocyclic, bicyclic and tricyclic aromatic carbocyclic systems containing from 6 to 16 carbon atoms, or from 6 to 14 carbon atoms, or from 6 to 12 carbon atoms, or from 6 to 10 carbon atoms, preferably from 6 up to 10 carbon atoms, and the term "aryl" is used interchangeably with the term "aromatic ring". Examples of the aryl group may include, but are not limited to, phenyl, naphthyl, anthryl, phenanthryl, pyrenyl or the like.

The term "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system containing a 5-12 membered structure, or preferably a 5-10 membered structure, or a 5-8 membered structure, and more preferably a 5-6 membered structure, wherein 1, 2, 3 or more atoms in the ring are heteroatoms, and the remaining atoms are carbon, the heteroatoms are independently selected from O, N and S, and the number of heteroatoms is preferably 1, 2 or 3. Examples of heteroaryl include, but are not limited to, furanyl , thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiadiazolyl, triazinyl, phthalazinyl, quinolinyl, isoquinolinyl, pteridinyl, purinyl, indole yl, isoindolyl, indazolyl , benzofuranyl, benzothienyl, benzopyridyl, benzopyrimidinyl, benzopyrazinyl, benzimidazolyl, benzophthalizinyl, pyrrolo[2,3-6]pyridyl, imidazo[1,2-a]pyridyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5 -a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [ 1,2,4]triazolo[1,5-a]pyridinyl, etc.

The term "pharmaceutically acceptable salt" refers to a salt that, within the bounds of sound medical judgment, is suitable for use in contact with the tissues of mammals, especially humans, without undue toxicity, irritation, allergic reaction, or the like, and has a reasonable benefit/risk ratio.

The term "salt" includes salt derived from inorganic acids and also includes salt derived from organic acids. If the compound of the present invention is acidic, pharmaceutically acceptable non-toxic basic salts include those derived from inorganic and organic bases.

The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including configurational isomers and conformational isomers, where configurational isomers include geometric isomers (or cis-trans isomers) and optical isomers (including enantiomers and diastereoisomers).

Geometric isomers may be present in the compound disclosed herein. A compound of the present invention may contain a carbon-carbon double bond or a carbon-nitrogen double bond in the E or Z configuration, wherein the term "E" refers to the next successive substituent on the opposite side of the carbon-carbon or carbon-nitrogen double bond, and the term "Z" represents the next-in-order substituent on the same side of a carbon-carbon or carbon-nitrogen double bond (as determined using Cahn-mgold Prelog priority rules). The compound of the present invention may also be present as mixtures of the "E" and "Z" isomers. Substituents around cycloalkyl or heterocycloalkyl are referred to as cis or t/ad configurations. Optical isomers refer to substances that have completely identical molecular structures and similar physicochemical properties, but different optical rotation. A compound of the present invention may contain asymmetrically substituted carbon atoms in the R or S configuration, where the terms "R" and "S" are as defined in the IUPAC 1974 Recommendations for section E, Fundamental Stereochemistry, Pure Appl. Chem. (1976) 45, 13-10. Compounds with asymmetrically substituted carbon atoms (with equal numbers of R and S configurations) are racemic at those carbon atoms. The presence of an excess of atoms with one configuration (relative to another) allows the configuration to be present in higher amounts, preferably in an excess of about 85% to 90%, more preferably in an excess of about 95% to 99%, and even more preferably in an excess of more than about 99%. Accordingly, the present invention includes racemic mixtures, relative and absolute optical isomers, and mixtures of relative and absolute optical isomers.

The term "N-oxide" refers to the N-oxide formed by the oxidation of one or more nitrogen atoms when the compound contains multiple amino functional groups. Specific examples of N-oxides are N-oxides of tertiary amines or N-oxides of the nitrogen atom of a nitrogen-containing heterocycle.

The term "solvate" refers to the associated compound formed by one or more solvent molecules binding to the compound of the present invention.

The term "tautomer" refers to structural isomers having different energies that are interconvertible through a lower energy barrier. If a tautomer is possible (for example, in solution), then chemical equilibrium of the tautomer can be achieved. For example, a protic tautomer (also known as a prototropic tautomer) involves interconversion via proton migration, such as keto-enol isomerization and imine-enamine isomerization. A valence tautomer involves interconversion by recombination of some of the bonding electrons. Unless otherwise indicated, all tautomeric forms of the compounds described herein are included within the scope of the present invention.

The term “isotopic derivative” means that a compound of the present invention may be present in an isotopically labeled or enriched form containing one or more atoms whose atomic mass or mass number differs from the atomic number of the largest number of atoms found in nature. The isotope may be a radioactive or non-radioactive isotope. Isotopes of atoms such as hydrogen, carbon, phosphorus, sulfur, fluorine, chlorine and iodine include, but are not limited to: ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ^l8 O, ³² P, ³⁵ S, ¹⁸ F , ³⁶ Cl and ¹²⁵ I. A compound containing other isotopes of these and/or other atoms is within the scope of the present invention.

In another embodiment, the isotopically labeled compounds contain isotopes of deuterium ( ^2H ), tritium ( ^3H ), or ^14C . The isotopically labeled compounds of the present invention can be prepared using conventional methods well known to those skilled in the art. In this regard, relevant references include: Lizondo, J et al., Drugs Fut, 21(11), 1116 (1996); Brickner, S. J. et al., J Med Chem, 39(3), 673 (1996); Mallesham, B et al., Org Lett, 5(7), 963 (2003).

Isotope-containing compounds have been used in pharmaceutical research to study the metabolic fate of compounds in vivo by assessing the mechanism of action and metabolic pathway of a non-isotopically labeled parent compound (Blake et al., J. Pharm. Sci. 64, 3, 367-391 (1975)) . Such metabolic studies are important for the development of safe and effective therapeutics, either because the active compound administered to the patient in vivo or because metabolites derived from the parent compound are found to be toxic or carcinogenic (Kushner et al., Can. J. Physiol. Pharmacol, 77, 79-88 (1999); Foster et al., Advances in Drug Research Vol. 14, pp. 2-36, Academic press, London, 1985; Kato et al., J. Labeled Comp. Radiopharmaceut, 36 (10):927-932 (1995); Kushner et al., Can. J. Physiol. Pharmacol, 11, 19-88 (1999)).

In addition, drugs containing a non-radioactive active isotope, such as deuterated drugs, called "heavy drugs", can be used to treat related diseases and conditions. Increasing the amount of isotope present in the above compound beyond its natural abundance is called enrichment. Examples of fortification amounts include from about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 21, 25, 29, 33, 37, 42, 46, 50, 54, 58, 63, 67, 71, 75, 79, 84, 88, 92, 96 to about 100 mol%. Any possible center in a molecular structure can be replaced by an isotope to produce an isotopic derivative. For example, any possible center in the molecule can be replaced with deuterium ( ^2H ) to produce a derivative in deuterated form.

When stable isotopically labeled drugs are used, the physicochemical properties of the drugs, such as pKa and lipid solubility, may be altered. If isotopic substitution affects regions involved in ligand-receptor interactions, then these effects and changes can influence the pharmacodynamic response of drug molecules. Although some physical properties of stable isotope-labeled molecules are different from those of unlabeled molecules, the chemical and biological properties are the same, the important difference is that any bond between a heavy isotope and another atom is stronger than the same bond between a light isotope and that atom from -due to the increased mass of the heavy isotope. Accordingly, the inclusion of isotopes at the site of metabolic or enzymatic conversion can potentially slow down the reaction and may alter the pharmacokinetic properties or effects compared to non-isotopic compounds.

The term "prodrug" is a derivative of an active drug that is intended to improve some specified undesirable physical or biological properties. Physical properties are often related to solubility (too much or too little solubility in lipids or water) or stability, while problematic biological properties include too rapid metabolism or poor bioavailability, which may themselves be related to physicochemical properties.

Prodrugs are generally prepared as follows: a) formation of esters, half-esters, carbonates, nitrates, amides, hydroxamic acids, carbamates, imines, Mannich bases, phosphates, phosphate esters and enamines of active drugs, b) functionalization of drugs with azo, glycoside, peptide and ether functional groups, c) the use of aminal, semi-aminal, polymeric, salt, complex, phosphoramide, acetal, hemiacetal and ketal forms of drugs. See, for example, Andrejus Korolkovas, Essentials of Medicinal Chemistry, John Wiley-Interscience Publications, John Wiley and Sons, New York (1988), pp. 97-118, the contents of which are incorporated herein by reference in their entirety. Esters can be prepared from hydroxyl or carboxyl containing substrates using conventional methods known to those skilled in the art. A typical reaction of these compounds is the replacement of one heteroatom by another atom. Amides can be prepared in a similar manner from substrates containing amino or carboxyl. Esters can also react with amines or ammonia to form amides. /Another way to make an amide is to heat a carboxylic acid and an amine together.

The term "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" includes, without limitation, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, coloring agent, coloring agent, flavor enhancer, surfactant, humectant, a dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier that is approved by the US Food and Drug Administration, National Medical Devices Administration, and the like for acceptable use in humans or pets.

The term "tumor" includes benign tumors, malignant tumors and borderline tumors, with malignant tumors also collectively referred to as cancer.

As used herein, the term “prevention” refers to a compound or drug that, when used to treat a disease or condition (eg, cancer), can reduce the frequency or delay the onset of symptoms of a medical condition in a subject compared to a subject who has not been administered the compound or drug (eg, a combination product as stated herein).

The term “treatment” as used herein refers to alleviating, reducing, or eliminating a symptom of a disease or condition, alleviating an underlying metabolic symptom, and slowing down a disease or symptom, e.g., stopping the progression of a disease or condition, alleviating a disease or condition causing regression of a disease or condition , alleviation of a disorder caused by a disease or condition, or cessation of a symptom of a disease or condition.

The term “cell proliferative disease” as used herein refers to a condition in which the rate of growth of a population of cells is lower or higher than the expected rate for a given physiological condition.

The following terms are used in the present invention:

DCM: dichloromethane; DIPEA: diisopropylethylamine; DMF: N,N-dimethylformamide; EA: ethyl acetate; NBS: N-bromosuccinimide; PE: petroleum ether; DMSO: dimethyl sulfoxide; TBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate; BOP: benzotriazol-1-yloxytris-(dimethylamino)-phosphonium hexafluorophosphate; ATP: adenosine 5'-triphosphate; DTT: 1,4-dithiothreitol; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-bromide sodium.

Based on the knowledge of medicinal chemistry, the N-oxide, isotopic derivative, stereoisomer, optical isomer, solvate, prodrug and the like of the compounds of the present invention can also exhibit an in vitro and in vivo effect similar to that of the compound of the present invention.

Positive effect of the present invention:

The present invention relates to a compound with a novel structure, and an in vitro kinase activity inhibition test shows that: the compound of the present invention exhibits excellent inhibitory activity against various kinases (e.g. TRK, ALK, ROS1) and their mutants, in particular against TRK and their mutant forms; an in vitro cytostatic activity test shows that: the compound of the present invention has a stronger inhibitory effect on various TRK mutant cells and has an IC ₅₀ of less than 10 nM, preferably less than 5 nM and more preferably less than 1 nM on the inhibitory activity of 6 types of cells; the results of the in vivo tumor inhibition test show that: compared with the control compound, the compound of the present invention has better in vivo antitumor effect, better tolerability and higher drug ability; an in vivo mechanism of action investigation test shows that: the compound of the present invention can inhibit TRK in tumor tissues, further effectively inhibit the phosphorylation of PLCγ and AKT, and inhibit the growth of tumor tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a Western blot showing the time-dependent inhibitory effect of Compound No. 4 of the present invention on phosphorylation of downstream target proteins in tumor tissues of TRKA-G595R tumor resistance mutant mice.

DETAILED DESCRIPTION

The starting materials, reaction reagents, catalysts or solvents used in the following specific embodiments of the invention are commercially available or can be prepared by conventional methods known in the art.

The present invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are intended to illustrate the present invention only and not to limit the scope of the present invention. The unspecified test procedures in the following examples are generally carried out under normal conditions or under the conditions recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

Preparation of Example A: Preparation of ethyl 6-chloroimidazo[1,2-A]pyridazine-3-carboxylate

A solution of ethyl (E)-3-ethoxyacrylate (5.00 g, 34.7 mmol, 1 eq.) in 1,4-dioxane (50 ml) and water (50 ml) was cooled to -10°C and added in portions NBS (6.80 g, 38.15 mmol, 1.1 eq.) was warmed to room temperature and allowed to react for 2 hours. 6-Chloro-3-aminopyridazine (4.5 g, 34.7 mmol, 1 eq.) was added to the reaction system, heated to 80°C and allowed to react for 1.5 h, and cooled to room temperature. The reaction system was then concentrated under reduced pressure to obtain a residue. Water (100 ml) and EA (100 ml) were added to the residue, mixed and separated. The aqueous phase was extracted with ethyl acetate (20 ml x 2). The organic phases were combined, washed with H ₂ O (50 ml) and brine (50 ml), concentrated under reduced pressure to remove the organic solvent. The residue was separated by silica gel column chromatography (eluent: n-hexane:EA = 5:1 to 1:1, v/v) to give ethyl 6-chloroimidazo[1,2-b]pyridazine -3-carboxylate (5.2 g, 59.75% yield). (ER, m/z): 225.91 [M+H] ⁺ .

Preparation Example of Intermediate 1: (R)-5-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (Intermediate A1)

Step a: mixed solution of (R)-2-(2,5-difluorophenyl)-pyrrolidine (5.0 g, 27.292 mmol), ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (6, 14 g, 27.292 mmol), n-butanol (70 ml) and diisopropylamine (6.9 g, 68.230 mmol) were refluxed and stirred at 100°C for 4 hours, concentrated under reduced pressure to give an orange viscous solid . Anhydrous ether was added to the mixture and stirred to precipitate a large amount of solid. Suction filtration was performed to obtain crude ethyl (R)-5-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.224 g) . The resulting crude product was directly used in the next step without purification. (ER, m/z): 373.02 [M+H] ⁺ .

Step b: crude ethyl (R)-5-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.224 g, 16.714 mmol) dissolved in absolute ethanol (40 ml) and stirred at 75°C until the mixture became clear. An aqueous solution (40 mL) of LiOH (2.805 g, 66.856 mmol) was then added to the reaction system and stirred at 75° C. for 3 hours. After cooling to room temperature, the reaction system was concentrated under reduced pressure to remove absolute ethanol. A 1 N aqueous HCl solution was slowly added dropwise to the reaction system to adjust the pH to 3-4. A large amount of white solid precipitated. The reaction system was stirred at room temperature for 30 minutes, and then filtered with suction. The filter residue was washed with a small amount of purified water. The filter cake was collected, air dried and weighed to give (R)-5-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid ( 5.64 g, 98%), (ER, m/z): 345.02 [M+H] ⁺ .

Example of preparation of intermediate 2: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]-pyrimidine-3-carboxylic acid ( intermediate connection A2)

Step a: mixed solution of (2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine (5.826 g, 28.958 mmol), ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate ( 6.534 g, 28.958 mmol), n-butanol (50 ml) and diisopropylamine (8.790 g, 86.874 mmol) were allowed to react at 100°C for 4 hours. The reaction system was concentrated under reduced pressure to give crude ethyl-5-((2R ,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate.

The crude product was directly used in the next step without purification. (ER, m/z): 391.05 [M+H] ⁺ .

Step b: crude ethyl 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate was dissolved in absolute ethanol (50 ml). The reaction system was stirred at 75°C until the system became clear. An aqueous solution (50 mL) of LiOH (4.86 g, 115.832 mmol) was then added to the reaction system and stirred at 75° C. for 5 hours. After cooling to room temperature, the reaction system was concentrated under reduced pressure to remove absolute ethanol. A 1 N aqueous HCl solution was slowly added dropwise to the reaction system to adjust the pH to 3-4. A large amount of white solid precipitated. The reaction system was stirred at room temperature for 30 minutes, and then filtered with suction. The filter residue was washed with a small amount of purified water. The filter cake was collected, dried and weighed to give a white powdery solid, 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine -3-carboxylic acid (9.9 g). The filtrate was extracted with ethyl acetate (EA) (2 x 50 ml). The organic phases were combined, washed with water (2×50 ml) and saturated aqueous NaCl (50 ml), then dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting mixture was purified by column chromatography (PE:EA = 4:1 to 2:1, v/v) to provide a white powdery solid, 5-((2R,4S)-2-(2,5-difluorophenyl )-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (386 mg). Taken together, the pure product 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (10.286 g) was obtained , 98%). (ER, m/z): 363.04 [M+H] ⁺ .

Examples of preparation of intermediates 1a-2a:

Referring to the process steps of Preparation Examples of Intermediates 1 and 2, Intermediates A1a to A2a were prepared by using the following respective starting materials and preparation methods:

Preparation Example of Intermediate 3: 5-(3-(2,5-difluorophenyl)morpholinyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (Intermediate A3)

Step a: Under nitrogen atmosphere, trimethylsilyl cyanide (39.66 g, 0.4 mol) was added dropwise to a solution (30 ml) of 2,5-difluorobenzaldehyde (28.4 g, 0.2 mol) in 7 mol/L NH ₃ /CH ₃ OH at 0°C. The reaction system was stirred at room temperature overnight after the dropwise addition was completed. The reaction liquid was concentrated under reduced pressure and the residue was purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH=50:1, v/v) to obtain 2-amino-2-(2,5-difluorophenyl )-acetonitrile (21.92 g, 65.2%). (ER, m/z): 169[M+H] ⁺ .

Step b: 2-amino-2-(2,5-difluorophenyl)-acetonitrile (21.92 g, 130 mmol) was added to 2 mol/L aqueous NaOH solution (130 ml). The reaction system was refluxed for 6 hours and then cooled to 0°C. The pH of the mixture was adjusted to 3 with concentrated hydrochloric acid and then concentrated under reduced pressure. The residue was dissolved in tetrahydrofuran (200 ml), stirred for 30 minutes and filtered. The filtrate was dried over anhydrous sodium sulfate overnight, filtered and concentrated under reduced pressure to obtain crude 2-amino-2-(2,5-difluorophenyl)-acetic acid (20.78 g), which was used directly next stages without purification. (ER, m/z): 186.02[M-N] ^- .

Step c: Under a nitrogen atmosphere, a solution of 2-amino-2-(2,5-difluorophenyl)-acetic acid (20.78 g, 111 mmol) in tetrahydrofuran (600 ml) was cooled to -10°C to -5°C, lithium aluminum hydride (10.53 g, 278 mmol) was added portionwise and allowed to react at room temperature overnight after addition was complete. A saturated ammonium chloride solution (500 ml) was added to the reaction liquid to quench the reaction and filtered through a pad of celite. The filtrate was separated. The aqueous phase was extracted with ethyl acetate (2 x 100 ml). The organic phases were combined, dried over anhydrous sodium sulfate and filtered; and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 10:1, v/v) to obtain 2-amino-2-(2,5-difluorophenyl)-ethanol (7.177 g, 32% two-stage yield). (ER, m/z): 174.02[M+H] ⁺ .

Step d: Under nitrogen atmosphere, chloroacetyl chloride (5.62 g, 49.736 mmol) was added to a solution of 2-amino-2-(2,5-difluorophenyl)-ethanol (7.177 g, 41.447 mmol) and triethylamine (8.388 g, 82.894 mmol) in tetrahydrofuran (200 ml) at 0°C. Maintaining the specified temperature, the reaction system was stirred for 30 minutes. 60% NaH (4.974 g, 124.341 mmol) was added to the reaction system in portions. The reaction mixture was allowed to react at room temperature for 2 hours after completion of the addition, and was quenched with saturated ammonium chloride solution (100 ml). The aqueous phase was extracted with ethyl acetate (2 x 50 ml). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 80:1 to 20:1, v/v) to give 5-(2,5-difluorophenyl)-morpholine-3- he (4.532 g, 51.3%). (ER, m/z): 214.01 [M+H] ⁺ .

Step e: under a nitrogen atmosphere, lithium aluminum hydride (3.229 g, 85.849 mmol) was added in portions to a solution of 5-(2,5-difluorophenyl)-morpholin-3-one (4.532 g, 21.271 mmol) in tetrahydrofuran (100 ml) at 0° WITH. The reaction system was allowed to react for 2 hours at 50°C after completion of the addition and then cooled to 0°C. The mixture was quenched with saturated ammonium chloride (90 ml) and concentrated under reduced pressure. The aqueous phase was extracted with ethyl acetate (EA) (3 × 50 ml). The organic phases were combined, washed with water (50 ml) and brine (50 ml), then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 50:1 to 25:1, v/v), resulting in 3-(2,5-difluorophenyl)-morpholine (3, 40 g, 80.3%). (ER, m/z): 200.03[M+H] ⁺ .

Step f: mixed solution of 3-(2,5-difluorophenyl)-morpholine (3.40 g, 17.068 mmol), ethyl 5-chloropyrazolo[1,5-a]pyrimidine-3-carboxylate (3.851 g, 17.068 mmol), n-butanol (50 ml) and diisopropylamine (5.181 g, 51.204 mmol) were left to react at 100°C overnight. The reaction system was concentrated under reduced pressure. EA (100 ml) and water (100 ml) were added to the residue, and the mixture was stirred. The aqueous phase was extracted with ethyl acetate (2 x 50 ml). The organic phases were combined, washed with water (100 ml) and brine (50 ml), then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (p-hexane:ethyl acetate = 50:1 to 10:1, v/v) to give ethyl 5-(3-(2,5-difluorophenyl)-morpholinyl )-pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.066 g, 91.5%). (ER, m/z): 389.05[M+H] ⁺ .

Step g: To a solution of ethyl 5-(3-(2,5-difluorophenyl)morpholinyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate (6.066 g, 15.623 mmol) in ethanol (60 ml) was added an aqueous solution (60 ml) of LiOH (2.622 g, 62.492 mmol), and the mixture was heated to 75° C. to react overnight. The reaction system was cooled to room temperature and concentrated under reduced pressure. The aqueous phase was adjusted to pH=2-3 with 1 N aqueous hydrochloric acid and extracted with ethyl acetate (EA) (3 x 60 ml). The organic phases were combined, washed with water (100 ml) and brine (50 ml), then dried over anhydrous Na ₂ SO ₄ , filtered, concentrated under reduced pressure and purified by column chromatography (PE:EA = 4:1 to 1 :1, v/v) to give 5-(3-(2,5-difluorophenyl)-morpholinyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (5.324 g, 94.6%) . (ER, m/z): 361.09[M+H] ⁺ .

Example of preparation of intermediate 4: 5-(3-(5-fluoro-2-methoxypyridin-3-yl)-morpholino)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (intermediate A4)

According to steps a-f of the preparation of intermediate, Example 3, ethyl 5-(3-(5-fluoro-2-methoxypyridin-3-yl)-morpholinyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate was prepared from 5-fluoro-2-methoxy-3-pyridinecarbaldehyde.

To a solution of ethyl 5-(3-(5-fluoro-2-methoxypyridin-3-yl)-morpholinyl)-pyrazolo[1,5-a]pyrimidine-3-carboxylate (5.00 g, 12.46 mmol) in absolute ethanol (50 ml), an aqueous solution (50 ml) of LiOH (2.091 g, 49.827 mmol) was added and the mixture was allowed to react at 75°C overnight. The reaction system was cooled to room temperature and concentrated under reduced pressure. The aqueous phase was brought to pH=2-3 with an aqueous solution of hydrochloric acid 1 N and extracted with ethyl acetate (EA) (3 × 50 ml). The organic phases were combined, washed with water (50 ml) and brine (50 ml), then dried over anhydrous Na ₂ SO ₄ , filtered, concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA = 4:1 to 1:1, v/v) to give 5-(3-(5-fluoro-2-methoxypyridin-3-yl)- morpholino)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (4.277 g, 92%). (ER, m/z): 374.02[M+H] ⁺ .

Example of preparation of intermediate 5: (R)-5-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-N-(4-(piperazin-1-yl)-phenyl)-pyrazolo[1, 5-a]pyrimidine-3-carboxamide (intermediate A5)

Step a: 1-Boc-4-(4-aminophenyl)-piperazine (850 mg, 3.067 mmol) was added to a solution of (R)-5-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)- pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (880 mg, 2.556 mmol) and TBTU (985 mg, 3.067 mmol) in anhydrous DMF (10 ml) followed by dropwise addition of DIPEA (991 mg, 7.668 mmol) at 0°C. The reaction system was left to react at room temperature overnight. Water (50 ml) was added to the reaction liquid and stirred. A solid precipitated. The mixture was filtered under reduced pressure to obtain a filter cake, which was dried in a vacuum oven to give (R)-tert-butyl-4-(4-(5-(2-(2,5-difluorophenyl)-pyrrolidine- 1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxamido)-phenyl)-piperazine-1-carboxylate (1.450 g, 94%). (ER, m/z): 604.52[M+H] ⁺ .

Step b: to (R)-tert-butyl-4-(4-(5-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidin-3- carboxamido)-phenyl)-piperazine-1-carboxylate (1.45 g, 2.402 mmol) was added DCM and CF ₃ COOH (12 ml, 3/1, v/v), and the reaction system was stirred at room temperature for 2.5 h and concentrated under reduced pressure. Water (12 ml) and EA (6 ml) were added to the residue, followed by aqueous ammonia to adjust the pH to 9. The solid precipitated with stirring. The mixture was filtered under reduced pressure to obtain a filter cake, which was washed with a small amount of water, dried and weighed to obtain (R)-5-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-N-(4 -(piperazin-1-yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (999 mg). (ER, m/z): 504.13 [M+H] ⁺ .

Examples of preparation of intermediates 5a and 6-9:

Referring to the process steps in the Preparation Example of Intermediate 5, Intermediates A5a and A6-A9 were prepared by adopting the following respective starting materials and preparation methods:

Preparation example of intermediate 10: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperazin-1-yl)-phenyl)- pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate A10)

Step a: 1-Boc-4-(4-aminophenyl)-piperazine (918 mg, 3.312 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1 -yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (intermediate A2, 1000 mg, 2.76 mmol) and TBTU (1063 mg, 3.312 mmol) in anhydrous DM FA (10 ml) followed by adding dropwise DIPEA (1284 mg, 9.936 mmol) at 0°C; and the reaction system was left to react at room temperature overnight. Water (50 ml) was added to the reaction liquid and stirred. A solid precipitated. The mixture was filtered under reduced pressure to obtain a filter cake, which was dried in a vacuum oven to obtain tert-butyl-4-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine -1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxamido)-phenylpiperazine-1-carboxylate (1320 mg, 77%) (ER, m/z): 622.09[M+H] ⁺ .

Step b: to tert-butyl-4-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a] DCM and CF ₃ COOH (12 mL, 3/1, v/v) were added to pyrimidine-3-carboxamido)-phenylpiperazine-1-carboxylate (1.320 g, 2.125 mmol) and the reaction system was stirred at room temperature for 4 hours and concentrated under reduced pressure. Water (80 ml) and ethyl acetate (10 ml) were added to the residue, followed by aqueous ammonia to adjust the pH to 9. The solid precipitated with stirring. The mixture was filtered under reduced pressure, resulting in a precipitate of filter, which was washed with a small amount of water, dried and weighed, resulting in 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-( piperazin-1-yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (907 mg, 82%), (ER, m/z): 522.09 [M+H] ⁺ .

Example of preparation of intermediate 10a:

Referring to the process steps of the Preparation Example of Intermediate 10, Intermediate A10a was prepared using the following respective starting materials and preparation methods:

Preparation example of intermediate 11: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperidin-4-yl)-phenyl) -pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate A11)

Step a: I-Boc-4-(4-aminophenyl)-piperidine (458 mg, 1.656 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1 -yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (intermediate A2, 500 mg, 1.380 mmol) and TBTU (532 mg, 1.656 mmol) in anhydrous DMF (5 ml), then added dropwise DIPEA (535 mg, 4.140 mmol) at 0°C; and the reaction system was left to react at room temperature for 18 hours overnight. Water (50 ml) was added to the reaction liquid and stirred. A solid precipitated. The mixture was filtered under reduced pressure to obtain a filter cake, which was dried in a vacuum oven to obtain tert-butyl-4-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine -1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxamido)-phenylpiperidine-1-carboxylate (627 mg, 73%) (ER, m/z): 621.15 [M+H] ⁺ .

Step b: to tert-butyl-4-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a] DCM and CF ₃ COOH (8 mL, 3/1, v/v) were added to pyrimidine-3-carboxamido)-phenylpiperidine-1-carboxylate (627 mg, 1.101 mmol) and the reaction liquid was stirred at room temperature for 4 h, and concentrated under reduced pressure.Water (80 ml) and EA (50 ml) were added to the residue, followed by the addition of aqueous ammonia to adjust pH=9. The aqueous phase was extracted with EA (55 ml x 2), and the combined extracted phase was washed with H ₂ O (20 ml) and brine (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, and the resulting residue was purified by silica gel column chromatography. Specifically, the eluent was collected with 10% (v/v) MeOH-DCM after the less polar impurities were washed out with 3% (v/v) MeOH-DCM, and the eluent was concentrated to give 5-((2R,4S)-2 -(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperidin-4-yl)-phenyl)-pyrazolo[1,5-a]pyrimidin-3-carboxamide (437 mg , 76%). (ER, m/z): 521.14 [M+H] ⁺ .

Examples of preparation of intermediates 11A and 12:

Referring to the process steps in Preparation Example 11, intermediates A11a and A12 were prepared by adopting the following respective starting materials and preparation methods:

Preparation example of intermediate 13: 5-(3-(2,5-difluorophenyl)-morpholinyl)-N-(4-(piperazin-1-yl)-phenyl)-pyrazolo[1,5-a]pyrimidin-3- carboxamide (intermediate A13)

Step a: Under nitrogen atmosphere, 1-Boc-4-(4-aminophenyl)-piperazine (3.812 g, 13.756 mmol) was added to a solution of 5-(3-(2,5-difluorophenyl)-morpholino)-pyrazolo[1.5 -a]pyrimidine-3-carboxylic acid (4.127 g, 11.46 mmol) and TBTU (4.417 g, 13.756 mmol) in anhydrous DMF (20 ml), then DIPEA (4.441 g, 34.362 mmol) was added dropwise at 0° C, and the reaction system was left to react at room temperature overnight. EA (100 ml) and water (100 ml) were added to the reaction liquid, stirred and separated. The aqueous phase was extracted with ethyl acetate (2 x 50 ml). The organic phases were combined, washed with water (2 x 50 ml) and brine (50 ml), then dried over anhydrous Na ₂ SO ₄ and filtered. The solvent was concentrated under reduced pressure and the resulting residue was purified by column chromatography (PE:EA = 80:1 to 60:1, v/v) to obtain tert-butyl-4-(4-(5 -(3-(2,5-difluorophenyl)-morpholino)-pyrazolo[1,5-a]pyrimidine-3-carboxamido)-phenyl-tert-butylpiperazine-1-carboxylate (3.924 g, 55.3%). ER, m/z): 620.49 [M+H] ⁺ .

Step b: to a solution of tert-butyl-(4-(5-(3-(2,5-difluorophenyl)-morpholino)-pyrazolo[1,5-a]pyrimidine-3-carboxamido)-phenyl-tert-butylpiperazine- 1-carboxylate (3.924 g, 6.332 mmol) in DCM (30 ml) was added CF ₃ COOH (10 ml), and the reaction system was stirred at room temperature for 4 hours, and concentrated under reduced pressure. To the residue was added water (100 ml ) and EA (100 ml), followed by the addition of aqueous ammonia to adjust pH=9, and separated. The aqueous phase was extracted with ethyl acetate (2 x 50 ml). The organic phases were combined, washed with water (100 ml) and brine (50 ml). , then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.The residue was purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH=50:1 to 10:1, v/v) to give 5 -(3-(2,5-difluorophenyl)-morpholinyl)-N-(4-(piperazin-1-yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (2.978 g, 90. 5%). (ER, m/z): 520.11 [M+H] ⁺ .

An example of the preparation of intermediate 14:

Referring to the process steps of the Preparation Example of Intermediate 13, Intermediate A14 was prepared using the following respective starting materials and preparation methods:

Preparation example of intermediate 15: 5-(3-(5-fluoro-2-methoxypyridin-3-yl)-morpholinyl)-N-(4-(piperazin-1-yl)-phenyl)-pyrazolo[1,5- a]pyrimidine-3-carboxamide (intermediate A15)

Step a: Under nitrogen atmosphere, 1-Boc-4-(4-aminophenyl)-piperazine (3.813 g, 13.747 mmol) was added to a solution of 5-(3-(5-fluoro-2-methoxypyridin-3-yl)-morpholino) -pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (4.277 g, 11.46 mmol) and TBTU (4.414 g, 13.747 mmol) in anhydrous DM FA (20 ml), then DIPEA (4.441 g) was added dropwise , 34.368 mmol) at 0°C, and the reaction system was allowed to react at room temperature overnight. EA (100 ml) and water (100 ml) were added to the reaction liquid, stirred and separated. The aqueous phase was extracted with ethyl acetate (2 x 50 ml). The organic phases were combined, washed with water (2 × 50 ml) and brine (50 ml), then dried over anhydrous Na2S04, filtered, concentrated under reduced pressure. The resulting residue was purified by column chromatography (PE:EA = 100:1 to 50:1, v/v) to obtain tert-butyl-4-(4-(5-(3-(5- fluoro-2-methoxypyridin-3-yl)-morpholino)-pyrazolo[1,5-a]pyrimidine-3-carboxamido)-phenyl)-piperazine-1-carboxylate (4.736 g, 65.3%). (ER, m/z): 633.09[M+H] ⁺ .

Step b: to a solution of tert-butyl-4-(4-(5-(3-(5-fluoro-2-methoxypyridin-3-yl)-morpholino)-pyrazolo[1,5-a]pyrimidine-3-carboxamido )-phenyl)-piperazine-1-carboxylate (4.736 g, 7.485 mmol) in DCM (30 ml) was added CF ₃ COOH (10 ml) and the reaction system was stirred at room temperature for 4 hours and concentrated under reduced pressure. Water (100 ml) and EA (100 ml) were added to the residue, followed by the addition of aqueous ammonia to adjust pH=9, and separated. The aqueous phase was extracted with ethyl acetate (2 x 50 ml). The organic phases were combined, washed with water (100 ml) and brine (50 ml), then dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (CH ₂ Cl ₂ :CH ₃ OH = 50:1 to 10:1, v/v) to obtain 5-(3-(5-fluoro-2-methoxypyridin-3-yl )-morpholinyl)-N-(4-(piperazin-1-yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (3.685 g, 92.5%). (ER, m/z): 533.10 [M+H] ⁺ .

An example of the preparation of intermediate 16:

Referring to the process steps of Preparation Example 15, Intermediate A16 was prepared using the following respective starting materials and preparation methods:

Preparation example of intermediate 17: N ^1- (oxetan-3-yl)-benzene-1,4-diamine (intermediate B1)

Step a: 3-oxetamine (124 mg, 1.701 mmol) was added to a solution of p-fluoroitrobeisole (200 mg, 1.417 mmol) in DMSO (3 ml), followed by the addition of DIPEA (366 mg, 2.834 mmol). The reaction system was stirred at 120°C for 5 hours, water (20 ml) was added and stirred, precipitating a large amount of solid, which was then filtered under reduced pressure, resulting in a filter cake of N-(4-nitrophenyl)-oxetane -3-amine (268 mg, 97%). (ER, m/z): 195.01 [M+H] ⁺ . Step b: The filter cake of M-(4-nitrophenyl)-oxetan-3-amine (268 mg, 1.380 mmol) obtained in the previous step and methanol (15 ml) were added to the reaction flask. 10% Pd/C was added to the reaction mixture, the air was replaced with H ₂ and stirred for 4 hours. Pd/C was removed by suction filtration and the filtrate was concentrated under reduced pressure to give N ¹ -(oxetan-3-yl )-benzene-1,4-diamine as a solid (110 mg). (ER, m/z): 164.92 [M+H] ⁺ .

Examples of preparation of intermediates 18 to 24:

Referring to the process steps in the Preparation Example of Intermediate 17, Intermediates B2-B8 were prepared by adopting the following respective starting materials, p-fluoronitrobenzene, and preparation methods:

Example of preparation of intermediate 25:(R)-6-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]pyrazine-3-carboxylic acid acid (intermediate C1)

Steps a to c: (R)-6-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]pyrazine-3-carboxylic the acid was prepared according to steps 1-3 of Example 1 in patent CN108794484 B. (ER, m/z): 346.02 [M+H] ⁺ .

Preparation Example of Intermediate 26: 6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]pyrazine -3-carboxylic acid (intermediate C2)

Steps a to c: 6-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-[1,2,4]triazolo[4,3-a]pyrazine -3-carboxylic acid was prepared with reference to the steps of Preparation Example of Intermediate 25. (ER, m/z): 364.03 [M+H] ⁺ .

Examples of preparation of intermediates 27 to 30:

Referring to the process steps in the Preparation Example of Intermediate 5, Intermediates C3-C6 were prepared by adopting the following respective starting materials and preparation methods:

Below are examples of compounds according to the present invention.

Example 1: (R)-5-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-R-(4-(4-(2-hydroxyacetyl)-piperazin-1-yl)-phenyl) -pyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 1)

Hydroxyacetic acid (32 mg, 0.417 mmol) was added to a solution of (R)-5-(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-N-(4-(piperazin-1-yl)-phenyl )-pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate A5, 70 mg, 0.139 mmol) and BOP (74 mg, 0.167 mmol) in anhydrous DM FA (3 ml) followed by dropwise addition of DIPEA ( 54 mg, 0.417 mmol) at 0°C, and the reaction system was stirred at room temperature for 4 hours. Water (20 ml) was added to the reaction liquid to mix, and the mixture was extracted with ethyl acetate (EA) (15 ml×2). The combined organic phases were washed with H ₂ O (20 ml) and brine (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography. Specifically, the residue was eluted with 1% (v/v) MeOH-DCM followed by 2% (v/v) MeOH-DCM, and the eluent containing the desired product was collected and concentrated to give (R)-5 -(2-(2,5-difluorophenyl)-pyrrolidin-1-yl)-N-(4-(4-(2-hydroxyacetyl)-piperazin-1-yl)-phenyl)-pyrazolo[1,5-a ]pyrimidine-3-carboxamide (44 mg, 56%). (ER, m/z):562.13 [M+H] ⁺ .

Examples 1a, 1b and 2-3:

Referring to the production methods and operations of Example 1, compounds 1a, 1b and 2-3 were prepared by taking the following materials and intermediate A5, A5a or C3 as starting materials.

Example 4: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(4-(2-hydroxyacetyl)-piperazin-1-yl )-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 4)

Hydroxyacetic acid (306 mg, 4.026 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperazine-4- yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate A10, 700 mg, 1.342 mmol) and BOP (712 mg, 1.610 mmol) in anhydrous DMF (10 ml) followed by addition of drops of DIPEA (520 mg, 4.026 mmol) at 0°C, and the reaction system was stirred at room temperature for 4 hours. Water (80 ml) was added to the reaction liquid to mix, and the mixture was extracted with EA (55 ml × 2). The combined organic phases were washed with H ₂ O (80 ml) and brine (80 ml), respectively. The organic phases were then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography. Specifically, the residue was eluted with 1% (v/v) MeOH-DCM followed by 2% (v/v) MeOH-DCM, and the eluent containing the desired product was collected and concentrated to give 5-((2R ,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(4-(2-hydroxyacetyl)-piperazin-1-yl)-phenyl)-pyrazolo[1 ,5-a]pyrimidine-3-carboxamide (666 mg, 86%). (ER, m/z): 580.14 [M+H] ⁺ . ^1H NMR (600 MHz, DMSO- _d6 ) δ 9.810 (s, 1H), 8.906-8.723 (m, 1H), 8.283-8.229 (m, 1H), 7.623 (s, 1H), 7.343 (s, 1H ), 7.210 (s, 2H), 7.06I-6.842 (m, 4H), 5.711-5.495 (m, 2H), 4.631 (t, J=5.4 Hz, 1H), 4.556-4.548 (m, 1H ), 4.318-4.225 (m, 1H), 4.150 (d, J=5.4 Hz, 2H), 3.637 (s, 2H), 3.513 (s, 2H), 3.124-3.106 (m, 4H), 2.957- 2.912 (m, 1H).

Examples 4a-4b, 5a, 7a-7b and 5-9:

According to the production methods and operations of Example 4, compounds 4a-4b, 5a, 7a-7b and 5-9 were prepared by taking intermediate A10, A10a or C4 and the following materials as starting materials.

Example 10: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-l-yl)-N-(4-(1-(2-hydroxyacetyl)-piperidin-4-yl )-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 10)

Hydroxyacetic acid (438 mg, 5.763 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperidine-4- yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate A11, 1000 mg, 1.921 mmol) and BOP (1274 mg, 2.882 mmol) in anhydrous DMF (10 ml) followed by the addition of drops of DIPEA (745 mg, 5.763 mmol) at 0°C, and the reaction system was stirred at room temperature overnight. Water (100 ml) was added to the reaction liquid for mixing. The mixture was extracted with EA (80 ml x 2). The combined organic phases were washed with H ₂ O (100 ml) and brine (110 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography. Specifically, the residue was eluted with 1% (v/v) MeOH-DCM followed by 2% (v/v) MeOH-DCM. The eluent containing the desired product was collected and concentrated to give 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(1-(2- hydroxyacetyl)-piperidin-4-yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (820 mg, 74%). (ER, m/z): 579.14 [M+H] ⁺ . ^1H NMR (600 MHz, DMSO- _d6 ) δ 9.952 (s, 1H), 9.022-8.753 (m, 1H), 8.355-8.152 (m, 2H), 7.663 (s, 1H), 7.343-7.042 (m , 6H), 5.773-5.552 (m, 2H), 4.529-4.481 (m, 2H), 4.358-4.115 (m, 3H), 3.803-3.779 (m, 1H), 3.640-3.603 (m, 1H), 3.090 -3.049 (m, 1H), 2.968-2.957 (m, 1H), 2.792-2.752 (m, 1H), 2.722-2.682 (m, 1H), 2.241-2.212 (m, 1H), 1.808 (s, 2H) , 1.608-1.589 (m, 1H), 1.492-1.476 (m, 1H). Examples 10A-10B, 11A-11B and 11-17:

According to the production methods and operations of Example 10, compounds 10A-10B, 11A-11B and 11-17 were prepared by taking intermediate A11, A11a or C6 and the following materials as starting materials.

Example 18: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(4-(2-(2-hydroxyacetyl)-piperazin- 1-yl)-methyl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 18)

Hydroxyacetic acid (51 g, 0.672 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperazine-1- ylmethyl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate A12, 120 g, 0.224 mmol) and BOP (149 g, 0.336 mmol) in anhydrous DM FA (12 ml) followed by addition dropwise DIPEA (87 g, 0.672 mmol) at 0°C, and the reaction system was stirred at room temperature for 4 hours. Water (20 ml) was added to the reaction liquid to mix, and the mixture was extracted with ethyl acetate (EA) (10 ml×2). The combined organic phases were washed with H ₂ O (10 ml) and brine (10 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, and specifically, the residue was eluted with 1% (v/v) MeOH-DCM followed by 2% (v/v) MeOH-DCM to obtain 5- ((2R,45)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(4-(2-(2-hydroxyacetyl)-piperazin-1-yl)- methyl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (30 mg, 22%). (ER, m/z): 594.12 [M+H] ⁺ .

Example 19: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(oxetan-3-ylamino)-phenyl)-pyrazolo[1 ,5-a]pyrimidine-3-carboxamide (compound 19)

N ^1- (oxetan-3-yl)-benzene-1,4-diamine (105 mg, 0.640 mmol) was added to the solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine -1-yl)-N-(1,2,3,4-tetrahydroisoquinolin-7-yl)-pyrazolo[1,5-a]pyrimidin-3-carboxamide (intermediate A2, 193 mg, 0.534 mmol) and TBTU (205 mg, 0.640 mmol) in anhydrous DM FA (5 ml), then DIPEA (205 mg, 1.602 mmol) was added dropwise at 0°C, and the reaction system was stirred overnight at room temperature for 14 hours. Water (25 ml) was added to the reaction liquid to mix, and the mixture was extracted with EA (15 ml × 2). The combined organic phases were washed with H ₂ O (20 ml) and brine (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, resulting in a residue, which was separated using preparative liquid chromatography (X-Bridge C18 19*150 mm 5 μm, elution system: 5-25% aqueous solution of acetonitrile for gradient elution, 0.05% NH ₄ HCO ₃ for modification), resulting in 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(oxetan-3-ylamino )-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (60 mg, 22.1%). (ER, m/z): 509.12 [M+H] ⁺ .

Examples 19A-19B and 20-24:

According to the preparation methods and operations of Example 19, compounds 19A-19B and 20-24 were prepared by taking the following intermediates as starting materials:

Example 25: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(6-(2-hydroxyacetyl)-3,6-diazabicyclo [3.1.1]hept-3-yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 25)

Step a: tert-butyl-3-(4-aminophenyl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate (442 mg, 1.528 mmol) (intermediate B5) was added to the solution of 5-((2R ,45)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (intermediate A2, 460 mg, 1.270 mmol) and TBTU (491 mg, 1.528 mmol) in anhydrous DMF (5 ml), then DIPEA (494 mg, 3.819 mmol) was added dropwise at 0° C. and the reaction system was stirred overnight at room temperature for 14 hours. Water (100 ml) was added to the reaction liquid and stirred. A large amount of solid precipitated, filtered under reduced pressure and dried to obtain crude tert-butyl-3-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1- yl)-pyrazolo[1,5-a]pyrimidine-3-formamido)-phenyl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate.

Step b: crude tert-butyl-3-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-] obtained above a]pyrimidine-3-formamido)-phenyl)-3,6-diazabicyclo[3.1.1]heptane-6-carboxylate was dissolved in DCM (25 ml). CF ₃ COOH (5 ml) was added to the solution and stirred at room temperature for 3 hours. The reaction liquid was concentrated by rotary evaporation and water was added for dilution, followed by aqueous ammonia to adjust the pH. The solid precipitated, suction filtered and dried to give N-(4-(3,6-diazabicyclo[3.1.1]heptyl-3-yl)phenyl)-5-((2R,4S)-2-(2 ,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide as a solid (450 mg).

Step c: Hydroxyacetic acid (35 mg, 0.450 mmol) was added to a solution of N-(4-(3,6-diazabicyclo[3.1.1]heptyl-3-yl)-phenyl)-5-((2R,4S)- 2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (200 mg, 0.375 mmol) and TBTU (145 mg, 0.450 mmol) in anhydrous DMF (5 ml), then DIPEA (146 mg, 1.125 mmol) was added dropwise at 0°C and the reaction system was stirred at room temperature for 4 hours. Water (30 ml) was added to the reaction liquid for mixing. The mixture was extracted with EA (20 ml x 2). The combined organic phases were washed with H ₂ O (30 ml) and brine (40 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, specifically the residue was eluted with 1% MeOH-DCM followed by 2% MeOH-DCM. The eluent containing the desired product was collected and concentrated to give 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(6-(2- hydroxyacetyl)-3,6-diazabicyclo[3.1.1]hept-3-yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (180 mg, 81%). (ER, m/z): 592.02 [M+H] ⁺ .

Example 26: 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(6-(2-hydroxyacetyl)-2,6-diazaspiro [3.3]heptan-2-yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (compound 26)

Step a: tert-butyl-6-(4-aminophenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate (178 mg, 0.607 mmol) (intermediate B8) was added to the solution of 5-((2R,4S )-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (intermediate A2, 200 mg, 0.552 mmol) and TBTU (217 mg, 0.607 mmol) in anhydrous DM FA (5 ml), then DIPEA (217 mg, 1.656 mmol) was added dropwise at 0°C and the reaction system was stirred overnight at room temperature for 14 hours. Water (30 ml) was added to the reaction liquid and stirred. A large amount of solid precipitated and then filtered under reduced pressure and dried to obtain crude tert-butyl-6-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine- 1-yl)-pyrazolo[1,5-a]pyrimidin-3-formamido)-phenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate as a solid. (ER, m/z): 534.12 [M+H] ⁺ .

Step b: the above crude tert-butyl-6-(4-(5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5- a] pyrimidine-3-formamido)-phenyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate was dissolved in DCM (6 ml). CF ₃ COOH (2 ml) was added to the solution and stirred at room temperature for 3 hours. The reaction liquid was concentrated by rotary evaporation and water was added for dilution, followed by aqueous ammonia to adjust the pH. The solid precipitated, suction filtered and dried to give N-(4-(2,6-diazaspiro[3.3]heptyl-2-yl)phenyl)-5-((2R,4S)-2-(2,5 -difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide as a solid (218 mg).

Step c: Hydroxyacetic acid (37 mg, 0.490 mmol) was added to a solution of N-(4-(2,6-diazaspiro[3.3]heptyl-2-yl)-phenyl)-5-((2R,4S)-2- (2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (218 mg, 0.409 mmol) and TBTU (157 mg, 0.490 mmol) in anhydrous DMF ( 10 ml) followed by dropwise addition of DIPEA (158 mg, 1.226 mmol) at 0°C, and the reaction mixture was stirred at room temperature for 4 hours. Water (30 ml) was added to the reaction liquid to mix, and the mixture was extracted with ethyl acetate (20 ml×2). The combined organic phases were washed with H ₂ O (30 ml) and brine (40 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a residue, which was purified using a preparative plate (DCM:MeOH=15:1) to obtain 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidine-1 -yl)-N-(4-(6-(2-hydroxyacetyl)-2,6-diazaspiro[3.3]heptan-2-yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide ( 51 mg, 94%). (ER, m/z): 592.12 [M+H] ⁺ .

Example 27: 5-(3-(2,5-difluorophenyl)-morpholinyl)-N-(4-(4-(2-hydroxyacetyl)-piperazin-1-yl)-phenyl)-pyrazolo[1,5-a ]pyrimidine-3-carboxamide (compound 27)

Hydroxyacetic acid (584 mg, 7.676 mmol) was added to a solution of 5-((2R,4S)-2-(2,5-difluorophenyl)-4-fluoropyrrolidin-1-yl)-N-(4-(piperidine-4- yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate A13, 997 mg, 1.919 mmol) and BOP (1274 mg, 2.878 mmol) in anhydrous DMF (10 ml), then dropwise DIPEA (744 mg, 5.756 mmol) was added at 0°C and the reaction mixture was stirred at room temperature. Water (100 ml) was added to the reaction liquid to mix, and the mixture was extracted with ethyl acetate (80 ml x 2). The organic phases were combined. The organic phase was washed with WO (100 ml) and brine (110 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM:CH ₃ OH = 100:1 to 50:1, v/v). The eluent containing the desired product was collected and concentrated to give 5-(3-(2,5-difluorophenyl)-morpholinyl)-N-(4-(4-(2-hydroxyacetyl)-piperazin-1-yl)-phenyl) -pyrazolo[1,5-a]pyrimidine-3-carboxamide (609.8 mg, 55%). (ER, m/z): 578.12 [M+H] ⁺ .

Example 28:

According to the production methods and operations of Example 27, compound 28 was prepared by using intermediate A14 and hydroxyacetic acid as raw materials.

Example 29: 5-(3-(5-fluoro-2-methoxypyridin-3-yl)-morpholinyl)-N-(4-(4-(2-hydroxyacetyl)-piperazin-1-yl)-phenyl)-pyrazolo [1,5-a]pyrimidine-3-carboxamide (compound 29)

Hydroxyacetic acid (511 mg, 6.723 mmol) was added to a solution of 5-(3-(5-fluoro-2-methoxypyridin-3-yl)-morpholino)-N-(4-(piperazin-1-yl)-phenyl)- pyrazolo[1,5-a]pyrimidine-3-carboxamide (intermediate A15, 1.022 g, 1.92 mmol) and BOP (1274 mg, 2.878 mmol) in anhydrous DMF (10 ml), then add DIPEA (744) dropwise mg, 5.763 mmol) at 0°C, and the reaction system was stirred at room temperature overnight. Water (100 ml) was added to the reaction liquid to mix, and the mixture was extracted with ethyl acetate (80 ml x 2). The organic phases were combined. The organic phase was washed with H ₂ O (100 ml) and brine (110 ml), dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: DCM:CH ₂ OH = 100:1 to 50:1). The eluent containing the desired product was collected and concentrated to give 5-(3-(5-fluoro-2-methoxypyridin-3-yl)-morpholinyl)-N-(4-(4-(2-hydroxyacetyl)-piperazine-1 -yl)-phenyl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide (737 mg, 65.02%). (ER, m/z): 591.20 [M+H] ⁺ .

Example 30:

According to the production methods and operations of Example 29, compound 30 was prepared by using intermediate A16 and hydroxyacetic acid as raw materials.

Examples 31-47:

Referring to the preparation methods and operations described in Preparation Examples of Intermediates 1-30 and Examples 1-30 and the method described in CN111936500A, compounds 31-47 were prepared using (2R)-2-(2,5-difluorophenyl)- 3-azabicyclo[3.1.0]hexane and each of the indicated intermediates as starting materials.

Examples of obtaining reference compounds I-5:

Compounds D1-D5 were prepared with reference to the production methods and operations in patent documents WO 2019029629A1 and WO 2012034095A1.

Test Example 1: Compound Inhibition of TRK

1. Procedures:

1.1 Kinase reaction:

The compound to be tested with a specific concentration gradient and the enzyme solution were sequentially added to the plate (kinase buffer (1× kinase buffer (Cisbio, cat. no. 62EZBFDD), pH 7.5; 5 mM MgCl ₂ , 1 mM DTT) was added to the wells negative control), and the compound was centrifuged at 1000 rpm for 30 seconds. The plate was hermetically sealed and incubated in an incubator at a constant temperature of 25°C for 30 minutes. A substrate solution containing TK-Sub-biotin (Cisbio, cat. no. 61TKOBL) and ATP (Sigma, cat. no. R0441) was prepared, the substrate solution was added to a 384-well plate and centrifuged at 1000 rpm for 30 seconds. The plate was hermetically sealed and incubated in an incubator at a constant temperature of 25°C for 60 minutes.

1.2 Kinase assay:

TK antibody and XL665 were diluted, mixed and added to the assay plate, and the plate was centrifuged at 1000 rpm for 30 seconds. The plate was hermetically sealed and incubated in an incubator at a constant temperature of 25°C for 60 minutes. The assay plate was placed on an Envision reader for reading, (HTRF 665/615 ratio: signal value at 665 nm/signal value at 615 nm) Inhibition = ( _{well negative control} ratio - _{well compound} ratio) / ( _{well negative control} ratio - _well ratio _{control without enzyme} ) × 100%

1.3 Data analysis and curve fitting

Data processing was carried out in the XLFit excel add-in version 5.4.0.8 to obtain IC ₅₀ values.

1.4 Quality Control Parameters

The reference compound was contained in each plate with an IC ₅₀ within 3 times the value each time.

2. Test results: as shown in Table 1

The results show that: the compounds of the present invention exhibit higher inhibitory activity against various kinases. The inhibitory activity of the compounds of the present invention against TRKA, TRKB, TRKC and TRKC-G696A is superior to or equivalent to that of compounds RXDX-101, LOXO-195 and LOXO-101, while the inhibitory activity of the compounds of the present invention against various mutant drugs stable kinases (G595R, G667C and G623R) significantly exceeds the activity of compounds RXDX-101, LOXO-195 and LOXO-101.

Test Example 2: Compound Inhibition of ALK and ROS1

1. Procedures:

1.1 Kinase reaction:

Compounds were diluted to specific concentrations in DMSO using 4-fold gradients. A specific concentration of compound, enzyme solution, and DMSO were added to a 384-well plate, and the plate was incubated at room temperature for 10 minutes. Fluorescein-labeled peptide and ATP (sigma, cat. no.: A7699-1G, lot no.: 987-65-5) were added to the plate, incubated at 28°C for a certain time and then a solution was added to stop the reaction. The tablet was read.

Inhibition rate for one concentration: inhibition rate = (OD _{well negative control} - OD _{well compound} ) / (OD _{well negative control} - OD _{well control without enzyme} ) × 100%

1.2 Data analysis and curve fitting

Data processing was carried out using XLFit excel add-in version 4.3.1 to obtain IC50 values, and the results are shown in Table 2.

The results show that: many of the compounds disclosed herein exhibit stronger ROS1 kinase inhibitory activity that is significantly superior to that of RXDX-101 and LOXO-101 and superior to that of LOXO-195. the compounds have good inhibitory activity against ALK kinase, which is superior to the activity of LOXO-101 and LOXO-195.

Test Example 3: In vitro Cell Proliferation Inhibitory Activity of the Compounds

1. Cell lines

Source of 6 kinds of cell lines for testing: KYinno Biotechnology (Beijing) Co., Ltd.

Cell type: mouse B cells

Culture medium: RPMI-1640+10%FBS

2. Test method

Cells in logarithmic growth phase were collected and counted using a platelet counter. A cell suspension at a certain density was pipetted evenly and seeded into a 96-well plate with each well containing 100 μL and shaken to ensure that the cells were evenly distributed among the wells. 100 μl of a compound solution with a certain concentration gradient was added to each well and 3 replicate wells were set up for each concentration. The plate was cultured in an incubator in a CO ₂ atmosphere at 37°C for 72 hours. 20 μl of MTT working solution (5 mg/ml) was added to each well, and the plate was placed at 37°C for 4 h and centrifuged at 1000 rpm for 5 min using plate centrifugation. After 180 μL of culture medium was discarded, 150 μL of DMSO was added to the plate and mixed well using a microplate shaker, the bottom of the plate was wiped, and the optical density (OD) at 550 nm was determined using a microplate reader.

3. Data analysis

Inhibition rate - (OD _{control well} - OD _{test well} ) / (OD _{control well} - OD _{blank well} ) x 100%, and half maximum inhibitory concentration IC ₅₀ values were calculated for each inhibitory concentration rate using SPSS software.

4. Test results: as shown in Table 3:

The results show that: multiple compounds of the present invention exhibit better in vitro inhibitory activity against various wild-type and mutant drug-resistant cell strains, and are significantly superior to RXDX-101, LOXO-195, LOXO-101 and compounds DI-D5 according to the prior art.

Test Example 4: In Vivo Study of the Mechanism of Compounds

1. Test method

1.1 Model preparation:

Mutant drug-resistant Ba/F3 LMNA-NTRKI-G595R logarithmic growth phase cells were collected and resuspended in serum-free culture medium to achieve a cell concentration of 6×10 ⁷ -10×10 ⁷ cells/ml. An equal volume of Matrigel was added to the cell suspension to obtain a final cell concentration of 3×10 ⁷ -5×10 ⁷ cells/ml. The animal model was prepared by subcutaneously inoculating 0.1 ml of tumor cell suspension into the axilla of the forelimb of NuNu mice (female, 4-6 weeks, Beijing Vital River Laboratory Animal Technology Co., Ltd.) in an amount of 3×10 ⁶ -5× 10 ⁶ cells/mouse.

1.2 Grouping:

The maximum and minimum tumor diameters of xenografts in nude mice were measured using calipers, and tumor volume (TV) was calculated using the formula: V = 1/2 × a × b ² , where a and b represent the maximum and minimum diameters of the tumor mass, respectively. Nude mice with appropriate tumor volume were selected, and mice were evenly divided into 7 groups of 3 mice (200-300 mm ³ ) according to tumor volume using the random number method.

1.3 Introduction

Gavage administration was carried out according to the weight of the animal at an administration volume of 10 ml/kg, and compound No. 4 was formulated at the required administration concentration using “3% DMSO + 96% HP-β-CD (0.5 g/ ml)+1% HCl.”

A total of 3 mice were selected in the control group, and tumor tissues were removed and cryopreserved 4 hours after vehicle administration. Compound No. 4 of the present invention was administered to other groups at a dose of 100 mg/kg, and tumor tissues were removed for cryopreservation after 0.25 hours, 1 hour, 4 hours, 8 hours, 12 hours and 24 hours.

1.4 Protein extraction and quantitation

A certain mass of tumor tissue was selected and added to the appropriate volume of protein lysate (RTPA lysate (Thermo Fisher, cat. no. 89900): protease inhibitor (cOmplete, Mini, EDTA-free, EASYpack; Roche, cat. no. 04693159001): phosphatase inhibitor (PhosStop , EASY pack; Roche, cat. no. 04906837001)=8:1:1), homogenized and lysed in an ice bath for 30 min. The mixture was subjected to high-speed centrifugation at low temperature, and then the supernatant was collected for BCA protein quantitation (procedure according to the BCA Protein Quantitation Kit (Qiagen, Cat. No. PA115-01)). Finally, after the protein concentration was adjusted to homogeneity with the lysate, loading buffer was added and the resulting mixture was boiled at 100°C for 10 minutes.

1.5 Western blotting

A 4-20% 10-well preformed gel was taken; the sample loading amount was 100 μg; 140 V electrophoresis was carried out for 1-1.5 hours; wet transfer 300 mA was carried out for 1.5-2 hours; the membrane was blocked with 5% BSA for 2-3 hours; incubated with the primary antibody overnight at 4°C (Trk 1:5000, p-Trk, PLCγ1, p-PLCγ1, AKT, p-AKT, actin 1:1000) and washed the membrane with 0.1% TBST for 5 min 4 times; incubated with secondary antibody at room temperature for 2 h (1:5000), irradiated with ECL and exposed.

2. Test results: as shown in Fig.1

From the analysis results, it can be seen that: over time, the expression of TRK, p-TRK, p-PLCγ1 and p-AKT in Fig. 1 is significantly reduced, which proves that Compound No. 4 of the present invention can significantly reduce the protein level of TRK and p-TRK, and further effectively inhibit the phosphorylation of p-PLCγ1/PLCγ1 and p-AKT/AKT to regulate cell growth and proliferation.

Test Example 5: In Vivo Pharmacodynamic Test of Compounds in an NTRK Mutation Resistant Xenograft Model

Test method

1.1 Model preparation

Cells in logarithmic growth phase were collected and resuspended in serum-free culture medium to achieve a cell concentration of 6×10 ⁷ -10×10 ⁷ cells/ml. An equal volume of Matrigel was added to the cell suspension to obtain a final cell concentration of 3×10 ⁷ -5×10 ⁷ cells/ml. The animal model was prepared by subcutaneously inoculating 0.1 ml of tumor cell suspension into the axilla of the forelimb of NuNu mice (female, 4-6 weeks, Beijing Vital River Laboratory Animal Technology Co., Ltd.) in an amount of 3×10 ⁶ -5× 10 ⁶ cells/mouse.

1.2 Grouping

The maximum and minimum tumor diameters of xenografts in nude mice were measured using calipers, and tumor volume (TV) was calculated using the formula: V = 1/2 × a × b ² , where a and b represent the maximum and minimum diameters of the tumor mass, respectively. Nude mice with the appropriate tumor volume were selected, and the mice were evenly divided into 7 groups of 6 mice (100-200 mm ³ ) according to tumor volume using the random number method.

1.3 Index monitoring

Administration via gastric tube was carried out on the day of grouping according to the weight of the animal with an administration volume of 10 ml/kg, LOXO-195 was prepared as a formulation with 0.5% CMC-Na to obtain the required solution for administration, and compounds No. 4 and No. 10 was prepared as a formulation with “3% DMSO+96% HP-β-CD (0.5 g/ml)+1% HCl” to obtain the required solution for administration. Tumor diameters were measured twice a week and tumor volume was calculated. Specific indices are described below: Animal weight: animals were weighed before dosing each morning, and weight loss greater than 20% was defined as a toxic response to the drug (observed the day after the last dose);

Tumor volume (TV) = V = 1/2 × a × b ² where a and b represent the maximum and minimum diameters of the tumor mass, respectively (observed the day after the last injection);

Relative tumor proliferation rate T/S (%): T/S (%)=TRTV / CRTV × 100% (TRTV: RTV administration group, CRTV: RTV control group); Tumor growth inhibition rate (TGI)=[1 - (Ti - T0) / (Vi - V0)] × 100%. (where Ti represents the average tumor volume of a given administration group on a specific day; T0 represents the average tumor volume of the administration group at the start of administration, Vi represents the average tumor volume of the vehicle control group on a specific day (the same day as Ti) and V0 represents the average tumor volume of the vehicle control group at the beginning of administration);

Tumor inhibition rate: At the end of the test, animals were sacrificed by removing the cervical vertebrae. Tumor masses were peeled off, weighed and photographed, and the tumor inhibition rate was calculated using the formula: tumor inhibition rate = (average tumor weight of the control group - average tumor weight of the injection group) / average tumor weight of the control group × 100%.

Test results

2.1 Model Ba/F3 LMNA-NTRKI-G667C

2.1.1 Effect of drugs on the weight of mice with tumors

The weight of mice in each dosage group of each compound tended to increase, and the increasing trend was more significant than that of the control group. The weight of mice in each dosage group of each compound increases significantly, which may be due to the compound, or because the compound can inhibit tumor growth, so that the mice have a better condition and a significant increase in weight. The results are presented in Table 5.

2.1.2 Effect of drugs on tumor weight and rate of tumor inhibition in tumor-bearing mice

The data results show that, compared with LOXO-195, Compound No. 4 and Compound No. 10 of the present invention exhibit greater tumor growth inhibition at the same administration dose (100 mg/kg); in addition, Compound No. 4 and Compound No. 10 (100 mg/kg) of the present invention also showed better tumor inhibition effect than the LOXO-195 (200 mg/kg) group at a higher dose. The results are presented in Table 5.

2.2 Model Ba/F3 LMNA-NTRK1 -G595R

2.2.1 Effect of drugs on the weight of mice with tumors

The weight of mice in each dosage group of each compound tended to increase, and the increasing trend was more significant than that of the control group. The weight of mice in each dosage group of each compound increases significantly, which may be due to the compound, or because the compound can inhibit tumor growth, so that the mice have a better condition and a significant increase in weight. The results are presented in Table 6.

2.2.2 Effect of drugs on tumor weight and rate of tumor inhibition in tumor-bearing mice

The data results show that compared with LOXO-195 (100 mg/kg), Compound No. 4 and Compound No. 10 of the present invention can achieve significant inhibition of tumor tissue mass at a lower dosage (50 mg/kg) with a tumor mass inhibition rate of more 90%. The results are presented in Table 6.

Claims (41)

1. A compound of formula (I) or a tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt thereof , where X is selected from a chemical bond, -O- and -CH ₂ -; Y is N, Y ₁ is C, Y ₂ is -CH-, Y ₃ is N, and Y ₄ is -CH-; X ₂ is selected from the chemical bond, -(CH ₂ ) _p - and -NH-, where p is 1; means that a chemical bond is absent or present; R is selected from phenyl or pyridinyl, wherein each phenyl or pyridinyl is unsubstituted or substituted with at least one substituent selected from R ₁ ; each R ₁ , if present, is independently selected from hydrogen, halogen, -OH, amino, -NHC _1-6 alkyl, -N(C _1-6 alkyl) ₂ , cyano, C _1-6 alkyl, C _1-6 alkoxy and -SC _1-6 alkyl; R ₂ is selected from: H, halogen, hydroxyl, amino; R ₃ represents H; ring A is selected from heterocycloalkyl containing from 4 to 6 ring atoms, 6-8 membered heterobridged cyclyl, 8-10 membered heterofused cyclyl or 7-10 membered heterospirocyclyl, the heteroatoms in the heterocycloalkyl, heterobridged cyclyl, heterofused cyclyl and heterospirocyclyl being independently selected from O and N, and the number of heteroatoms selected from 1, 2, 3 or 4; R ₄ is at any substitutable position on ring A and is independently selected from -H, -OH, halogen, -CN, substituted or unsubstituted C _1-6 alkyl, -(CH ₂ ) _m -OH, -(CH ₂ ) _m - COOH, -(CH ₂ ) _m -CO-NH ₂ , -CO-(CH ₂ ) _m -NH ₂ , -CO-CR _4a R _4b -OH and -CO-R _4b ; R _4a is selected from hydrogen and unsubstituted or substituted C _1-4 alkyl; R _4b is selected from H, unsubstituted or substituted C _1-6 alkyl and unsubstituted or substituted C _3-6 cycloalkyl, the substituent is independently selected from -OH, -NH ₂ and halogen, and the number of substituents is selected from 1, 2 and 3; n is selected from 1, 2, 3 and 4. 2. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to claim 1, characterized in that said compound has the structure represented by formula (I-1) or formula (I-2): or , where X ₁ is selected from -CH- and N. 3. A compound or a tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt thereof according to claim 1 or 2, characterized in that said compound has the structure represented by formula (I-A): . 4. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-3, characterized in that said compound has the structure of formula (I-B-1a) or formula (I-C-1a): or 5. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-4, wherein R ₁ , if present, is each independently selected from hydrogen, halogen, -OH, amino, C _1-3 alkyl and C _1-3 alkoxy. 6. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-5, characterized in that R ₁ , if present, is each independently selected from hydrogen, F, Cl, -OH, methyl, ethyl, methoxy and ethoxy. 7. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-6, characterized in that R ₂ is selected from H, F, OH. 8. The compound or its tautomer, stereoisomer, optical isomer or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-3, characterized in that said compound has a structure represented by formula (I-D-1a), formula (I-C-1a-1) or formula (I-C-1a-2): or . 9. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-8, characterized in that ring A is selected from the following structures: , , , , , , , , , , , , And . 10. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-9, characterized in that ring A is selected from the following structures: And . 11. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-10, characterized in that R ₄ is independently selected from -H, -OH, substituted or unsubstituted C _1-3 alkyl, -CO-CR _4a R _4b -OH and -CO-R _4b . 12. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-11, characterized in that R ₄ is independently selected from -CO-CR _4a R _4b -OH and -CO-R _4b . 13. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-12, characterized in that R _4a is selected from hydrogen and unsubstituted or substituted C _1-4 alkyl; R _4b is selected from unsubstituted or substituted C _1-6 alkyl or unsubstituted or substituted C _3-6 cycloalkyl; the substituent for substitution is independently selected from -OH and F, and the number of substituents is selected from 1, 2 and 3. 14. The compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-13, characterized in that X ₂ represents a bond. 15. A compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt of claim 1, characterized in that said compound has the following structures: Serial number Structural formulas Serial number Structural formulas 1 2 3 4 5 6 7 8 9 10 eleven 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 thirty 31 34 37 41 44 46 16. A pharmaceutical composition having inhibitory activity against TRK, containing a compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-15 as the sole active compound and a pharmaceutically acceptable excipient. 17. The use of a compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-15 or a pharmaceutical composition according to claim 16 for the preparation of a medicament for the prevention and/or treatment of a disease mediated by TRK, ALK, ROS1 or a combination thereof. 18. The use of a compound or its tautomer, stereoisomer, optical isomer or pharmaceutically acceptable salt according to any one of paragraphs. 1-15 or a pharmaceutical composition according to claim 16 for preparing a medicament for treating a hyperproliferative disease, wherein the hyperproliferative disease is a tumor. 19. Use according to claim 18, characterized in that the tumor is selected from a solid tumor and a hematological tumor. 20. Use according to claim 18, characterized in that the tumor is selected from hematological malignancy, lung cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, brain glioma, colorectal cancer, melanoma, head and neck cancer, cancer gallbladder, thyroid cancer, glioblastoma, stomach cancer, neuroblastoma or salivary gland cancer. 21. Use according to claim 20, characterized in that the lung cancer is non-small cell lung cancer. 22. Application according to any one of paragraphs. 18-21, characterized in that said tumor refers to a fusion, amplification, rearrangement, mutation or high expression of the NTRK, ALK, ROS1 gene or a combination thereof.