CN110294742A

CN110294742A - And ring class ASK1 inhibitor and its application

Info

Publication number: CN110294742A
Application number: CN201810790218.XA
Authority: CN
Inventors: 王听中; 陈博; 朱鹏; 刘斌
Original assignee: Shandong Xuanzhu Pharma Co Ltd
Current assignee: Shandong Xuanzhu Pharma Co Ltd
Priority date: 2018-03-21
Filing date: 2018-07-18
Publication date: 2019-10-01
Anticipated expiration: 2038-07-18
Also published as: CN110294742B

Abstract

The present invention relates to simultaneously ring class ASK1 inhibitor and its applications, and in particular to logical formula (I) compound represented, its pharmaceutically acceptable salt, ester or its stereoisomer.The invention further relates to the preparation method of the compound, the pharmaceutical preparation comprising the compound and pharmaceutical compositions.The compound of the present invention can effectively inhibit the amino acid phosphorylation of ASK1, inhibit the activation of ASK1；Therefore it can treat and/or prevent the disease and related disease of ASK1 mediation.

Description

Fused ring ASK1 inhibitor and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a compound shown as a formula (I), a pharmaceutically acceptable salt, an ester or a stereoisomer thereof, a pharmaceutical composition and a preparation containing the compound, the pharmaceutically acceptable salt, the ester or the stereoisomer thereof, a method for preparing the compound, the pharmaceutically acceptable salt, the ester or the stereoisomer thereof, and application of the compound, the pharmaceutically acceptable salt, the ester or the stereoisomer thereof in preparation of medicines for treating and/or preventing ASK 1-mediated diseases and related diseases.

Background

Mitogen-activated protein kinases (MAPKs) exist as sequentially activated MAPKKKs, MAPKKs, MAPKs. Environmental signals affect MAPKKK, which in turn phosphorylates MAPKK, phosphorylates specific MAPK, which then mediates cellular responses, including cell growth, differentiation, apoptosis and inflammation, by phosphorylating cellular substrates.

Apoptosis signal-regulating kinase 1(ASK1) is a member of the MAPKKK family, and is an oligomer-like compound linked to the C-helical region, the N-helical region is linked to thioredoxin (Trx), and Trx inhibits activation of ASK 1. Under the stimulation conditions of oxidative stress, endoplasmic reticulum pressure, intramolecular calcium concentration, GPCR signal and the like, thioredoxin of ASK1 is dissociated, and the self amino acid (human: Thr838, mouse: Thr845) of ASK1 is subjected to autophosphorylation, so that MAPKK (such as MAPKK3/MAPKK6, MAPKK4/MAPKK7) is phosphorylated; p38 and JNK downstream were subsequently phosphorylated and activated. Activation of this pathway is closely related to tumor growth, metabolic diseases and neurological diseases.

The ASK1 inhibitor has the action mechanism of inhibiting the phosphorylation of ASK1 and blocking the activation of downstream channels, thereby achieving the treatment effect on tumor diseases, metabolic diseases and neurodegenerative diseases.

At present, only GS-4997 of Gilead is in clinical stage as ASK1 inhibitor researched internationally, PhaseIII (stage III) is being developed, PhaseII (stage II) research on diabetic nephropathy, pulmonary hypertension and nonalcoholic steatohepatitis is completed, and the effectiveness and safety of the target are proved through the verification of PhaseII.

Disclosure of Invention

The invention aims to provide a fused ring ASK1 inhibitor and application thereof. The specific technical scheme is as follows:

the invention firstly provides a compound shown in a general formula (I), and pharmaceutically acceptable salt, ester or stereoisomer thereof:

wherein,

X、X¹、X²、X³、X⁴、X⁵each independently selected from C, CR⁸Or N;

R¹selected from optionally substituted by one or more Q¹Substituted C_1-10Alkyl radical, C_2-10Alkenyl radical, C_1-10Alkoxy, 3-to 8-membered cycloalkyl, C_2-10Alkynyl, aryl, heteroaryl or heterocyclyl;

each Q¹Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, 3-to 8-membered cycloalkyl, 3-to 8-membered heterocyclyl, phenyl, phenoxy, -O-R⁸、-C(O)-R⁸、-OC(O)-R⁸、-C(O)-O-R⁸、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹) or-C (O) -N (R)⁸)(R⁹) In which C is_1-6Alkyl, 3-8 membered cycloalkyl, 3-8 membered heterocyclyl, phenyl, phenoxy optionally substituted with one or more C_1-6Alkyl, 3-8 membered cycloalkyl, C_1-6Alkoxy, hydroxy, amino, carboxy or halogen substitution;

or R¹And X¹Together with the N to which they are attached form a heterocyclic, aryl or heteroaryl groupSaid heterocyclyl, aryl or heteroaryl being optionally substituted with one or more alkyl, hydroxy or halogen;

R²、R⁴、R⁶、R⁷each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxyl and C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy, hydroxy C_1-6Alkyl, hydroxy C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, C_1-6Alkylthio radical, C_1-6Alkylcarbonyl group, C_1-6Alkoxy radical C_1-6Alkyl radical, C_1-6Alkylcarbonyloxy, C_1-6Alkylsulfonyl radical, C_1-6Alkylaminosulfonyl, di (C)_1-6Alkyl) aminosulfonyl, C_1-6Alkylsulfonylamino or C_1-6An alkylsulfonyloxy group;

R³selected from absent, hydrogen, halogen, C_1-6Alkyl, halo C_1-6Alkyl, hydroxy C_1-6Alkyl or C_1-6An alkylcarbonyl group;

n is selected from 1 or 2, and when n is selected from 2, R³May be the same or different;

R⁵selected from optionally substituted by one or more Q²Substituted C_1-6Alkyl radical, C_1-6Alkylamino radical, C_1-6Alkoxy, aryl, heteroaryl, cycloalkyl or heterocyclyl;

each Q²Independently selected from C_1-6Alkyl, 3-8 membered cycloalkyl, 3-8 membered cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, haloC_1-6Alkoxy radical, C_1-6Alkylamino, oxo, -O-R⁸、-O-C(O)-R⁸、-O-C(O)-N(R⁸)(R⁹)、-S-R⁸、-N(R⁸)(R⁹)、-S(＝O)-R⁸、-S(＝O)₂R⁸、-O-S(＝O)₂R⁸、-S(＝O)₂-N(R⁸)(R⁹)、-S(＝O)₂-OR⁸、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹)、-C(O)-R⁸、-C(O)-O-R⁸、-C(O)-N(R⁸)-R⁹or-N (R)⁸)-S(＝O)₂-R⁹Wherein said C is_1-6The alkyl, 3-8 membered cycloalkyl, aryl, heteroaryl or heterocyclyl is further optionally substituted with one or more halogen, hydroxy, amino, cyano, nitro, carboxy, oxo, C_1-6Alkyl or-O-R⁸Substitution;

each R⁸And R⁹Each independently selected from hydrogen and C_1-6Alkyl, 3-8 membered cycloalkyl, or when R is⁸And R⁹When attached to the same N atom, R⁸And R⁹Together with the N atom to which they are attached form a 3-12 membered heterocyclic ring;

selected from single bonds or double bonds.

Some embodiments of the present invention relate to compounds of the foregoing general formula (I), a pharmaceutically acceptable salt, ester, or stereoisomer thereof, wherein,

X、X¹、X²、X³、X⁴、X⁵each independently selected from C, CR⁸Or N;

R¹selected from optionally substituted by one or more Q¹Substituted C_1-6Alkyl radical, C_2-6Alkenyl radical, C_1-6Alkoxy, 3-to 8-membered cycloalkyl, C_2-6Alkynyl, 6-14 membered aryl, 5-14 membered heteroaryl or 3-12 membered heterocyclyl;

each Q¹Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C_1-6Alkyl, halo C_1-6Alkoxy radical, C₁-₆Alkylamino radical, di (C)_1-6Alkyl) amino, 3-to 8-membered cycloalkyl, heterocyclyl, phenyl, phenoxy, -O-R⁸、-C(O)-R⁸、-OC(O)-R⁸、-C(O)-O-R⁸、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹) or-C (O) -N (R)⁸)(R⁹) In which C is_1-6Alkyl, 3-8 membered cycloalkyl, heterocyclyl, phenyl, phenoxy optionally substituted by one or more C_1-6Alkyl, 3-8 membered cycloalkyl, C_1-6Alkoxy, hydroxy, amino, carboxy or halogen substitution;

or R¹And X¹Bonded to form a 5-12 membered heterocyclyl, 6-12 membered aryl or 5-12 membered heteroaryl, said 5-12 membered heterocyclyl, 6-12 membered aryl or 5-12 membered heteroaryl being optionally substituted with one or more C_1-6Alkyl, hydroxy or halogen substitution;

R²、R⁴、R⁶、R⁷each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxyl and C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy, hydroxy C_1-6Alkyl, hydroxy C_1-6Alkoxy radical, C_1-6An alkylamino group;

R⁵selected from optionally substituted by one or more Q²Substituted C_1-6Alkyl radical, C_1-6Alkylamino radical, C_1-6Alkoxy, 6-14 membered aryl, 5-14 membered heteroaryl, 3-12 membered cycloalkyl or 3-12 membered heterocyclyl;

each Q²Independently selected from C_1-6Alkyl, 3-8 membered cycloalkyl C_1-6Alkyl, 6-14 membered aryl C_1-6Alkyl, 5-14 membered heteroaryl C_1-6Alkyl, 3-12 membered heterocyclic group C_1-6Alkyl, halogen, cyano, nitro, oxo, -O-R⁸、-O-C(O)-R⁸、-O-C(O)-N(R⁸)(R⁹)、-S-R⁸、-N(R⁸)(R⁹)、-S(＝O)-R⁸、-S(＝O)₂R⁸、-O-S(＝O)₂R⁸、-S(＝O)₂-N(R⁸)(R⁹)、-S(＝O)₂-OR⁸、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹)、-C(O)-R⁸、-C(O)-O-R⁸、-C(O)-N(R⁸)-R⁹Or N (R)⁸)-S(＝O)₂-R⁹Wherein said C is_1-6Alkyl, 3-8 membered cycloalkyl, 6-14 membered aryl, 5-14 membered heteroaryl or 3-12 membered heterocyclyl is further optionally substituted with one or more halogen, hydroxy, amino, cyano, nitro, carboxy, oxo, C_1-6Alkyl or-O-R⁸Substitution;

R⁸and R⁹Each independently selected from hydrogen and C_1-6Alkyl, 3-8 membered cycloalkyl, or when R is⁸And R⁹When attached to the same N atom, R⁸And R⁹Together with the N atom to which they are attached form a 3-12 membered heterocyclic ring;

selected from single bonds or double bonds.

Some embodiments of the present invention relate to a compound described by the foregoing general formula (I), a pharmaceutically acceptable salt, ester, or stereoisomer thereof: wherein,

X²、X³、X⁴、X⁵at least one is selected from N;

R¹selected from optionally substituted by one or more Q¹Substituted C_1-6Alkyl radical, C_2-6Alkenyl radical, C_1-6Alkoxy, 3-to 8-membered cycloalkyl, C_2-6Alkynyl, 6-12 membered aryl, 5-12 membered heteroaryl or 3-10 membered heterocyclyl;

each Q¹Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, 3-to 8-membered cycloalkyl, 3-to 8-membered heterocyclyl, phenyl,Phenoxy, -O-R⁸、-C(O)-R⁸、-OC(O)-R⁸、-C(O)-O-R⁸、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹) or-C (O) -N (R)⁸)(R⁹)；

Or R¹And X¹Together with the N to which they are attached form a 5-10 membered heterocyclyl, 6-12 membered aryl or 5-10 membered heteroaryl, said 5-10 membered heterocyclyl, 6-12 membered aryl or 5-10 membered heteroaryl being optionally substituted by one or more C_1-6Alkyl, hydroxy or halogen substitution;

R⁵selected from optionally substituted by one or more Q²Substituted C_1-6Alkyl radical, C_1-6Alkylamino radical, C_1-6Alkoxy, 6-to 10-membered aryl, 5-to 12-membered heteroaryl, 3-to 10-membered cycloalkyl or 3-to 10-membered heterocyclyl;

each Q²Independently selected from C_1-6Alkyl, 3-8 membered cycloalkyl C_1-6Alkyl, 6-14 membered aryl C_1-6Alkyl, 5-14 membered heteroaryl C_1-6Alkyl, 3-12 membered heterocyclic group C_1-6Alkyl, halogen, cyano, nitro, halogeno C_1-6Alkoxy, oxo, -O-R⁸、-O-C(O)-R⁸、-O-C(O)-N(R⁸)(R⁹)、-S-R⁸、-N(R⁸)(R⁹)、-S(＝O)-R⁸、-S(＝O)₂R⁸、-O-S(＝O)₂R⁸、-S(＝O)₂-N(R⁸)(R⁹)、-S(＝O)₂-OR⁸、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹)、-C(O)-R⁸、-C(O)-O-R⁸、-C(O)-N(R⁸)-R⁹or-N (R)⁸)-S(＝O)₂-R⁹Wherein said C is_1-6Alkyl, 3-8 membered cycloalkyl, 6-14 membered aryl, 5-14 membered heteroaryl or 3-12 membered heterocyclyl is further optionally substituted with one or more halogen, hydroxy, amino, cyano, nitro, carboxy, oxo, C_1-6Alkyl or-O-R⁸Substitution;

selected from single bonds or double bonds.

In some embodiments of the invention, the aforementioned compound, a pharmaceutically acceptable salt thereof, an ester thereof, or a stereoisomer thereof, has a structure represented by formula (II),

wherein,

X、X¹each independently selected from C, CR⁸Or N;

R¹selected from optionally substituted by one or more Q¹Substituted C_1-6Alkyl radical, C_2-6Alkenyl radical, C_1-6Alkoxy, 3-6 membered cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl or 3-8 membered heterocyclyl;

each Q¹Independently selected from halogen, cyano, hydroxy, amino,Nitro, carboxyl, C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, 3-to 8-membered cycloalkyl, 3-to 8-membered heterocyclyl, phenyl, phenoxy, -O-R⁸、-C(O)-R⁸、-OC(O)-R⁸、-C(O)-O-R⁸、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹) or-C (O) -N (R)⁸)(R⁹)；

Or R¹And X¹Together with the N to which they are attached, form a 5-8 membered heterocyclyl, 6-10 membered aryl or 5-8 membered heteroaryl, said 5-8 membered heterocyclyl, 6-10 membered aryl or 5-8 membered heteroaryl being optionally substituted with one or more C_1-6Alkyl, hydroxy or halogen substitution;

R²、R⁴、R⁶、R⁷each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxyl and C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy, hydroxy C_1-6Alkyl, hydroxy C_1-6Alkoxy or C_1-6An alkylamino group;

R⁵selected from optionally substituted by one or more Q²Substituted C_1-6Alkyl radical, C_1-6Alkylamino radical, C_1-6Alkoxy, 6-to 8-membered aryl, 5-to 10-membered heteroaryl, 3-to 8-membered cycloalkyl or 3-to 8-membered heterocyclyl;

each Q²Independently selected from C_1-6Alkyl, 3-8 membered cycloalkyl C_1-6Alkyl, 3-8 membered heterocyclic group C_1-6Alkyl, halo C_1-6Alkoxy, halogen, cyano, nitro, -O-R⁸、-O-C(O)-R⁸、-O-C(O)-N(R⁸)(R⁹)、-S-R⁸、-N(R⁸)(R⁹)、-S(＝O)-R⁸、-S(＝O)₂R⁸、-O-S(＝O)₂R⁸、-S(＝O)₂-N(R⁸)(R⁹)、-S(＝O)₂-OR⁸、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹)、-C(O)-R⁸、-C(O)-O-R⁸、-C(O)-N(R⁸)-R⁹or-N (R)⁸)-S(＝O)₂-R⁹Wherein said C is_1-6Alkyl, 3-8 membered cycloalkyl, 3-8 membered heterocyclyl is further optionally substituted with one or more halogen, hydroxy, amino, cyano, nitro, carboxy, oxo, C_1-6Alkyl or-O-R⁸Substitution;

each R⁸And R⁹Each independently selected from hydrogen or C_1-6An alkyl group;

selected from single bonds or double bonds.

Some embodiments of the invention relate to the aforementioned compound, a pharmaceutically acceptable salt, ester, or stereoisomer thereof, having a structure represented by formula (III),

wherein,

R¹selected from the group consisting of optionally substituted 1-3Q¹Substituted C_1-6Alkyl radical, C_1-6Alkoxy, 3-6 membered cycloalkyl or 3-8 membered heterocyclyl, each Q¹Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylcarbonyl group, C_1-6Alkoxycarbonyl group, C_1-6Alkylamido, aminoacyl, C_1-6Alkylaminoacyl, di (C)_1-6Alkyl) aminoacyl, halo C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino or 3-8 membered ringAn alkyl group;

R³selected from hydrogen, halogen, C_1-6Alkyl, halo C_1-6Alkyl, hydroxy C_1-6Alkyl or C_1-6An alkylcarbonyl group;

R⁵selected from the group consisting of optionally substituted 1-3Q²Substituted C_1-6Alkyl radical, C_1-6Alkylamino radical, C_1-6Alkoxy, phenyl, 5-8 membered heteroaryl, or 3-6 membered heterocyclyl;

each Q²Independently selected from C_1-6Alkyl radical, C_1-6Alkoxy, 3-6 membered cycloalkyl C_1-6Alkyl, 3-6 membered heterocyclic group C_1-6Alkyl, halogen, cyano, hydroxy, amino, nitro, haloC_1-6Alkoxy radical, C_1-6Alkylamino radical, C_1-6Alkylcarbonyl group, C_1-6Alkylcarbonyloxy, C_1-6Alkyl mercapto group, C_1-6Alkylsulfonyl radical, C_1-6Alkyloxycarbonyl, C_1-6Alkylamido radical, C_1-6Alkylaminoacyl, C_1-6Alkylsulfonylamino, wherein said C_1-6The alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocyclyl is further optionally substituted with 1 to 3 halogen, hydroxy, amino, cyano, nitro, carboxy, C_1-6Alkyl substitution;

selected from single bonds or double bonds.

Some embodiments of the present invention relate to a compound described by the foregoing general formula (III), a pharmaceutically acceptable salt, ester, or stereoisomer thereof,

wherein,

R¹selected from the group consisting of optionally substituted 1-3Q¹Substituted C_1-6Alkyl radical, C_1-6Alkoxy, 3-6 membered cycloalkyl, each Q¹Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkoxycarbonyl, aminoacyl, C_1-6Alkylaminoacyl, di (C)_1-6Alkyl) aminoacyl, C_1-6Alkylamino or di (C)_1-6Alkyl) amino;

R²、R⁴、R⁶、R⁷each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxyl and C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl or halo C_1-6An alkoxy group;

R⁵selected from the group consisting of optionally substituted 1-3Q²Substituted C_1-6Alkyl radical, C_1-6Alkylamino radical, C_1-6Alkoxy, phenyl or 5-8 membered heteroaryl;

each Q²Independently selected from C_1-6Alkyl radical, C_1-6Alkoxy, 3-6 membered cycloalkyl C_1-6Alkyl, 3-6 membered heterocyclyl, -6 membered heterocyclyl C_1-6Alkyl, halogen, hydroxy or amino, wherein said C_1-6The alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocyclyl is further optionally substituted with 1 to 3 halogen, hydroxy, amino, cyano, nitro, carboxy, C_1-6Alkyl substitution;

selected from single bonds or double bonds.

R¹selected from optionally 1-2Q¹Substituted C_1-4Alkyl radical, C_1-4Alkoxy, 3-6 membered cycloalkyl, each Q¹Independently selected from halogen, hydroxy, amino, C_1-4Alkyl radical, C_1-4Alkoxy radical, C_1-4Alkoxycarbonyl, aminoacyl, C_1-4Alkylaminoacyl, di (C)_1-4Alkyl) aminoacyl, C_1-4Alkylamino or di (C)_1-4Alkyl) amino;

R²、R⁴、R⁶、R⁷each independently selected from hydrogen, halogen, hydroxy, amino, C_1-4Alkyl radical, C_1-4Alkoxy, halo C_1-4Alkyl or halo C_1-4An alkoxy group;

R³selected from hydrogen, halogen, C_1-4Alkyl, halo C_1-4Alkyl, hydroxy C_1-4Alkyl or C_1-4An alkylcarbonyl group;

R⁵selected from optionally 1-2Q²Substituted C_1-4Alkyl radical, C_1-4Alkylamino radical, C_1-4Alkoxy, phenyl or 5-6 membered heteroaryl;

each Q²Independently selected from C_1-4Alkyl radical, C_1-4Alkoxy, 3-6 membered cycloalkyl C_1-4Alkyl, 3-6 membered heterocyclic group C_1-4Alkyl, halogen, hydroxy or amino, wherein said C_1-4The alkyl group, 3-6 membered cycloalkyl group or 3-6 membered heterocyclic group is further optionally substituted by 1 to 3, preferably 1 to 2, halogens, hydroxyl group, amino group, C_1-4Alkyl substitution;

selected from single bonds or double bonds.

wherein,

R¹selected from optionally 1-2Q¹Substituted methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, or morpholinyl, each Q¹Independently selected from the group consisting of halogen, cyano, hydroxy, amino, nitro, carboxy, methyl, ethyl, methoxy, ethoxy, propoxy, isopropoxy, methoxycarbonyl, ethoxycarbonyl, methylamino, ethylamino, dimethylamino, diethylamino, aminoacyl, methylaminoacyl, ethylamyl, dimethylaminyl, diethylamino;

R²、R⁴、R⁶、R⁷each independently selected from hydrogen, fluoro, bromo, chloro, iodo, hydroxy, amino, cyano, nitro, carboxy, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, trifluoromethyl, trifluoroethyl, difluoromethoxy or trifluoromethoxy;

R³selected from hydrogen, fluoro, bromo, chloro, iodo, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, hydroxypropyl or formyl;

R⁵selected from optionally 1-2Q²Substituted phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, tetrazolyl, pyridyl or pyrimidinyl;

each Q²Independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, methoxy, cyclopropyl, cyclopropylmethylGroup, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, wherein said methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl are further optionally substituted by 1 to 3, preferably 1 to 2, halogens, hydroxy, amino, cyano, nitro, carboxy, methyl, ethyl, propyl, isopropyl;

selected from single bonds or double bonds.

In some embodiments of the invention, the aforementioned compound, a pharmaceutically acceptable salt thereof, an ester thereof, or a stereoisomer thereof, has a structure represented by formula (IV),

wherein,

R¹selected from the group consisting of optionally substituted 1-3Q¹Substituted C_1-6Alkyl radical, C_1-6Alkoxy or 3-6 membered cycloalkyl, each Q¹Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C_1-6Alkyl radical, C_1-6Alkoxy, aminoacyl, C_1-6Alkylaminoacyl, di (C)_1-6Alkyl) aminoacyl, C_1-6Alkylamino or di (C)_1-6Alkyl) amino;

R²、R⁴、R⁶、R⁷each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxyl and C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl or halogenGeneration C_1-6An alkoxy group;

R⁵selected from the group consisting of optionally substituted 1-3Q²Substituted phenyl or 5-8 membered heteroaryl;

each Q²Independently selected from C_1-6Alkyl radical, C_1-6Alkoxy, 3-6 membered cycloalkyl C_1-6Alkyl, 3-6 membered heterocyclyl or 3-6 membered heterocyclyl C_1-6Alkyl, wherein the 3-6 membered cycloalkyl or 3-6 membered heterocyclyl is further optionally substituted with 1 to 3 halogens, hydroxy, amino, cyano, nitro, carboxy, C_1-6Alkyl substitution.

In some embodiments, the present invention relates to a compound having a structure represented by the general formula (IV), a pharmaceutically acceptable salt thereof, an ester thereof, or a stereoisomer thereof,

R¹selected from the group consisting of optionally substituted 1-3Q¹Substituted C_1-4Alkyl radical, C_1-4Alkoxy or 3-6 membered cycloalkyl, each Q¹Independently selected from halogen, hydroxy, amino, C_1-4Alkyl radical, C_1-4Alkoxy, aminoacyl, C_1-4Alkylaminoacyl, di (C)_1-4Alkyl) aminoacyl, C_1-4Alkylamino or di (C)_1-4Alkyl) amino;

R⁵selected from the group consisting of optionally substituted 1-3Q²Substituted phenyl or 5-6 membered heteroaryl;

each Q²Independently selected from C_1-4Alkyl radical, C_1-4Alkoxy, 3-6 membered cycloalkyl C_1-4Alkyl, 3-6 membered heterocyclyl or 3-6 membered heterocyclyl C_1-4Alkyl, wherein the 3-6 membered cycloalkyl or 3-6 membered heterocyclyl is further optionally substituted with 1-3 halogens, hydroxy, amino, C_1-4Alkyl substitution.

Some embodiments of the present invention relate to a compound described by the foregoing general formula (IV), a pharmaceutically acceptable salt, ester, or stereoisomer thereof,

wherein,

R¹selected from optionally 1-2Q¹Substituted methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, said substituents Q¹Selected from the group consisting of halogen, cyano, hydroxy, amino, nitro, carboxy, methyl, ethyl, methoxy, ethoxy, propoxy, isopropoxy, methylamino, ethylamino, dimethylamino, diethylamino, aminoacyl, methylaminoacyl, ethylaminol, dimethylaminyl, diethylaminoacyl; (ii) a

R²、R⁴、R⁶、R⁷Each independently selected from hydrogen, fluoro, bromo, chloro, iodo, hydroxy, amino, cyano, nitro, carboxy, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, trifluoromethyl, trifluoroethyl, or trifluoromethoxy;

R⁵selected from optionally 1-2Q²Substituted phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, tetrazolylpyridinyl or pyrimidinyl;

each Q²Independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, methoxy, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, wherein said cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl is further optionally substituted with 1 to 2 halogens, hydroxy, amino, cyano, methoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, thiazolidinyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, further optionally substituted with 1 to 2 halogens, hydroxy, amino, cyano, or a pharmaceutically acceptable saltNitro, carboxyl, methyl, ethyl, propyl or isopropyl.

In some embodiments of the invention, the aforementioned compound, a pharmaceutically acceptable salt thereof, an ester thereof, or a stereoisomer thereof, has a structure represented by formula (V),

wherein,

R⁵selected from optionally substituted by one or more Q²Substituted phenyl or 5-8 membered heteroaryl;

each Q²Independently selected from C_1-6Alkyl radical, C_1-6Alkoxy, 3-6 membered cycloalkyl C_1-6Alkyl, 3-6 membered heterocyclyl or 3-6 membered heterocyclyl C_1-6Alkyl, wherein the 3-6 membered cycloalkyl or 3-6 membered heterocyclyl is further optionally substituted with one or more halo, hydroxy, amino, cyano, nitro, carboxy.

Some embodiments of the present invention relate to a compound of the foregoing general formula (V), a pharmaceutically acceptable salt, ester, or stereoisomer thereof,

wherein,

R²、R⁴、R⁶、R⁷each independently selected from hydrogen, fluorine, bromine, chlorine, iodine, hydroxyl, amino, cyano, nitro, carboxyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy and ethylOxy, propoxy, isopropoxy, tert-butoxy, trifluoromethyl, trifluoroethyl or trifluoromethoxy;

R⁵selected from optionally substituted by one or more Q²Substituted phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, tetrazolyl, pyridyl or pyrimidinyl;

each Q²Selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, methoxy, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, wherein the cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl are further optionally substituted with one or more halogen, hydroxy, amino, cyano, nitro, carboxy, methyl, ethyl, propyl or isopropyl groups.

The selection of any substituent in any embodiment described in the present invention can be combined with each other, and the combined technical scheme is still included in the protection scope of the present invention.

In some embodiments of the present invention, the structure of a compound of formula (I), (II), (III), (IV) or (V), a pharmaceutically acceptable salt, ester or stereoisomer thereof, is shown in table 1:

TABLE 1

The term "pharmaceutically acceptable salts" as used herein refers to pharmaceutically acceptable acid and base addition salts, which may be prepared from inorganic and organic acids; addition salts of pharmaceutically acceptable bases can be prepared from organic or inorganic bases.

The "ester" as referred to herein means a pharmaceutically acceptable ester, particularly an ester which is hydrolyzed in vivo and includes an ester which is easily decomposed in the human body to leave the parent compound (the compound represented by the general formula (I), (II), (III), (IV) or (V)) or a salt thereof. In some embodiments of the invention, wherein the pharmaceutically acceptable ester comprises: derived from pharmaceutically acceptable aliphatic carboxylic acids and phosphoric acid (salts).

"stereoisomer" of a compound represented by the general formula (I), (II), (III), (IV) or (V) of the present invention means that an enantiomer is produced when asymmetric carbon atoms are present in the compound represented by the general formula (I), (II), (III), (IV) or (V); when the compound has a carbon-carbon double bond or a cyclic structure, cis-trans isomers can be generated; tautomers can arise when a compound is present as a ketone or oxime, and in some embodiments of the invention, stereoisomers include, but are not limited to: enantiomers, diastereomers, racemates, cis-trans isomers, tautomers, geometrical isomers, epimers and mixtures thereof.

The present invention also provides a process for the preparation of a compound of the general formula (I) as defined above, wherein the process comprises:

(1) the preparation method comprises the following steps of reacting a compound shown as a general formula (I-1) with a compound shown as a general formula (I-2) to obtain a compound shown as a general formula (I);

(2) the second preparation method comprises the step of reacting the compound shown in the general formula (I-3) with the compound shown in the general formula (I-2) to obtain a compound shown in the general formula (I-4); the nitro group in the compound shown in the general formula (I-4) is subjected to functional group conversion reaction to obtain a compound shown in the general formula (I);

further, the present invention provides a method for preparing the compound represented by the aforementioned general formula (III), wherein the method comprises:

(1) the preparation method comprises the following steps of reacting a compound shown as a general formula (III-1) with a compound shown as a general formula (III-2) to obtain a compound shown as a general formula (III);

(2) the second preparation method comprises the step of reacting the compound shown in the general formula (III-3) with the compound shown in the general formula (III-2) to obtain a compound shown in the general formula (III-4); the nitro group in the compound shown in the general formula (III-4) is subjected to functional group conversion reaction to obtain a compound shown in the general formula (III);

wherein substituent a is selected from a leaving group, such as halogen.

The "functional group transformation reaction" can be achieved by known chemical synthesis methods, including but not limited to substitution reaction, addition reaction, elimination reaction, dehydration reaction, hydrolysis reaction, oxidation reaction, esterification reaction, etc., which are well known to those skilled in the chemical field, and the present invention is not limited thereto; in the specific implementation process, the reaction can be realized through one-step reaction or multi-step reaction.

In addition, some necessary starting materials, for example, materials for synthesizing intermediates, can be synthesized according to similar procedures and methods described in the handbook of organic chemistry, and the present invention is not limited thereto.

R in the above reaction equation¹、R²、R³、R⁴、R⁵、R⁶、R⁷、n、X、X¹、X²、X³、X⁴、X⁵As defined hereinbefore, for example, in any one of the general formulae (I) to (V).

The invention also provides intermediates useful in the synthesis of compounds of formula (III) having the following structural formula:

wherein substituent a is selected from a leaving group, such as halogen; r¹、R²、R³、R⁴、R⁵、R⁶、R⁷N is as defined above.

The invention also provides a pharmaceutical composition which contains the compound shown in the general formula (I), (II), (III), (IV) or (V), pharmaceutically acceptable salt, ester or stereoisomer thereof, and optionally one or more second therapeutic active agents. The second therapeutically active agent may be used in combination with ASK1 for the alleviation or treatment of related disorders mediated thereby, such as cardiovascular disorders, pulmonary disorders, metabolic disorders, peripheral vascular disorders, gastrointestinal disorders;

the cardiovascular diseases include, but are not limited to, angina pectoris, arrhythmia, intermittent claudication, myocardial infarction, heart failure, or recurrent ischemia. Therapeutically active agents suitable for the combination treatment of cardiovascular-related diseases include, but are not limited to, anti-angina agents, anti-heart failure agents, anti-thrombotic agents, anti-arrhythmic agents, anti-hypertensive agents, and lipid-lowering agents. Anti-angina agents include, but are not limited to, beta-blockers, calcium channel blockers, and nitrates and the like; agents used to treat heart failure include diuretics, ACE inhibitors, vasodilators, cardiac glycosides, and the like; antithrombotic agents including but not limited to platelet inhibitors, anticoagulants, and thrombolytic agents, and the like; antiarrhythmic agents include, but are not limited to, sodium channel blockers, beta-blockers, calcium channel blockers, agents that extend the action potential time course, and adenosine, among others. Antihypertensive agents include, but are not limited to, alpha-1-adrenergic antagonists, beta-adrenergic antagonists, central alpha-adrenergic receptor agonists, combined alpha/beta-adrenergic antagonists, adrenergic neuron blockers, central nervous system acting antihypertensive agents, anti-angiotensin II agents, ACE inhibitors, angiotensin-II receptor antagonists, calcium channel blockers, vasodilators, potassium channel activators and the like; lipid lowering agents include, but are not limited to, statins, fibrates, nicotinic acids, bile acid sequestrants, cholesterol absorption inhibition, and the like.

The lung disease refers to any disease related to the lung, including but not limited to asthma, chronic obstructive pulmonary disease, bronchitis and emphysema. Therapeutically active agents suitable for the combination treatment of pulmonary diseases include, but are not limited to, bronchodilators, including β 2 agonists, anticholinergics, corticosteroids, and electrolyte supplements.

The metabolic diseases include but are not limited to diabetes, metabolic syndrome, dyslipidemia, obesity, glucose intolerance, hypertension, high serum cholesterol, higher triglycerides and the like. Active agents suitable for the combined treatment of metabolic disorders include, but are not limited to, antihypertensive agents and lipid-lowering agents.

Such peripheral vascular disease includes, but is not limited to, peripheral arterial disease.

The gastrointestinal diseases include but are not limited to gastroesophageal reflux disease, inflammatory bowel disease, gastroenteritis, gastritis, peptic ulcer disease, pancreatitis and the like. Suitable agents for the combined treatment of gastrointestinal disorders include, but are not limited to, proton pump inhibitors, H2 blockers, prostaglandins, aluminum sulfate, and antacids, among others.

Patients with acute cardiovascular disease are treated with ASK1 inhibitors, and the associated condition may be treated with antibiotics, analgesics, and/or anxiolytics.

In some embodiments of the invention, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers and/or diluents.

The invention also provides a pharmaceutical preparation which contains the compound shown in the general formula (I), (II), (III), (IV) or (V), the pharmaceutically acceptable salt, ester or stereoisomer thereof, and one or more pharmaceutical carriers and/or diluents; the pharmaceutical preparation is any clinically or pharmaceutically acceptable dosage form.

In some embodiments of the invention, the pharmaceutical formulations described above may be administered to a patient or subject in need of such treatment by oral, parenteral, rectal, or pulmonary administration, and the like. For oral administration, the pharmaceutical composition can be prepared into oral preparations, for example, conventional oral solid preparations such as tablets, capsules, pills, granules and the like; it can also be made into oral liquid, such as oral solution, oral suspension, syrup, etc. When the composition is formulated into oral preparations, appropriate filler, binder, disintegrating agent, lubricant, etc. can be added. For parenteral administration, the pharmaceutical preparations can also be prepared into injections, including injections, sterile powders for injection, and concentrated solutions for injection. The injection can be prepared by conventional method in the existing pharmaceutical field, and can be prepared without adding additives or adding suitable additives according to the properties of the medicine. For rectal administration, the pharmaceutical composition may be formulated as a suppository or the like. For pulmonary administration, the pharmaceutical composition may be formulated as an inhalant or a spray.

The pharmaceutically acceptable carrier and/or diluent useful in the pharmaceutical composition or pharmaceutical formulation of the present invention may be any conventional carrier and/or diluent in the art of pharmaceutical formulation, and the selection of a particular carrier and/or diluent will depend on the mode of administration or the type and state of the disease used to treat a particular patient. The preparation of suitable pharmaceutical compositions for a particular mode of administration is well within the knowledge of those skilled in the pharmaceutical art. For example, the pharmaceutically acceptable carrier and/or diluent may include solvents, diluents, dispersing agents, suspending agents, surfactants, isotonic agents, thickening agents, emulsifiers, binders, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, ion exchangers, release agents, coating agents, flavors, antioxidants, and the like, which are conventional in the pharmaceutical art. If necessary, a flavor, a preservative, a sweetener and the like may be further added to the pharmaceutical composition.

In one embodiment of the present invention, the aforementioned pharmaceutical formulation may further comprise one or more second therapeutically active agents, said second therapeutically active agents being as described hereinbefore.

The invention also provides application of the compound shown in the general formula (I), (II), (III), (IV) or (V), pharmaceutically acceptable salt, ester or stereoisomer thereof, the pharmaceutical preparation or the pharmaceutical composition in preparing medicines for treating and/or preventing ASK 1-mediated diseases and related diseases.

In some embodiments of the invention, the ASK 1-mediated and related diseases are selected from cardiovascular diseases including, but not limited to, pulmonary hypertension, multiple sclerosis, atherosclerosis, angina pectoris, intermittent claudication syndrome, myocardial infarction, heart failure; selected from pulmonary diseases including but not limited to asthma, chronic obstructive pulmonary disease, bronchitis, and emphysema; selected from metabolic disorders including but not limited to diabetes and its complications, metabolic syndrome, dyslipidemia, obesity, glucose intolerance, hypertension, high serum cholesterol and higher triglycerides; hepatobiliary kidney diseases, including but not limited to diabetic nephropathy, non-alcoholic fatty liver disease, bile acid disorders, primary sclerosing cholangitis, cholesterol stones, fatty liver, cirrhosis, hepatitis, liver failure, cholestasis, cholelithiasis; hyperproliferative diseases include, but are not limited to, gastric cancer, liver cancer, polyposis, colon cancer, breast cancer, pancreatic cancer, esophageal cancer; other diseases, including neurodegenerative diseases, fibrosis-related diseases, stroke, inflammatory bowel diseases, autoimmune disorders.

The invention also provides the application of the compound shown in the general formula (I), (II), (III), (IV) or (V), the pharmaceutically acceptable salt, ester or stereoisomer thereof, the pharmaceutical preparation or the pharmaceutical composition in treating and/or preventing diseases.

The invention also provides the compound shown in the general formula (I), (II), (III), (IV) or (V), and pharmaceutically acceptable salt, ester or stereoisomer thereof, the pharmaceutical preparation or the pharmaceutical composition thereof, and the application thereof in treating and/or preventing ASK1 mediated diseases and related diseases; the ASK1 mediated diseases and related diseases are as described above.

The invention also provides a method for treating diseases, which comprises the step of administering a therapeutically effective amount of the compound shown in the general formula (I), (II), (III), (IV) or (V), the pharmaceutically acceptable salt, the ester or the stereoisomer thereof, the pharmaceutical preparation or the pharmaceutical composition to a patient in need thereof, wherein the diseases are ASK1 mediated diseases and related diseases; the ASK1 mediated diseases and related diseases are as described above.

Definition of

"halogen" as referred to herein means fluorine, chlorine, bromine and iodine, preferably fluorine and chlorine.

The ' oxo ' in the invention means that any C atom in the substituent structure can be oxidized into ' -C (O) -; if containing heteroatoms, the heteroatoms may form oxides, e.g.Can be oxidized intoS is optionally oxidized to S (O) or S (O)₂。

"halo" as used herein means that any one of the substituents may be substituted by one or more of the same or different halogens. "halogen" is as defined above.

Said "C" of the present invention_1-10Alkyl "refers to a straight or branched chain alkyl group derived from a hydrocarbon containing 1 to 10 carbon atoms with one hydrogen removed from the hydrocarbon moiety, e.g., methyl, ethyl, n-propyl, isopropylN-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, and 1-methyl-2-methylpropyl, and the like. Said "C_1-6Alkyl "or" C_1-4Alkyl "refers to the above examples containing 1 to 6 or 1 to 4 carbon atoms.

"C" according to the invention_2-10Alkynyl "refers to a straight or branched chain alkynyl group having 2 to 10 carbon atoms containing a carbon-carbon triple bond with one hydrogen-derived removed from the alkynyl moiety, such as ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, and the like. Said "C_2-6Alkynyl "refers to the above examples containing 1-6 carbon atoms.

"C" according to the invention_2-10Alkenyl "means a straight chain or branched alkenyl group derived from an olefin of 2 to 10 carbon atoms having a carbon-carbon double bond by removing one hydrogen atom, such as vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1, 3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1, 3-pentadienyl, 1, 4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1, 4-hexadienyl, cyclopentenyl, 1, 3-cyclopentadienyl, cyclohexenyl, 1, 4-cyclohexadienyl and the like. Said "C_1-6Alkyl "or" C_1-4Alkyl "refers to the above examples containing 1 to 6 or 1 to 4 carbon atoms.

"C" according to the invention_1-10Alkoxy "means" C "as defined hereinbefore_1-10Alkyl "radicals attached to the parent body via an oxygen atom, i.e." C_1-10alkyl-O- "groups such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Said "C_1-6Alkoxy "refers to the above examples containing 1 to 6 carbon atoms, i.e." C_1-6An alkyl-O- "group.

And "C_1-10Alkoxy radicalSimilarly, the term "C" as used herein_1-6Alkylamino "means C_1-6alkyl-NH-; "phenoxy" means phenyl-O-; "hydroxy group C_1-6Alkyl "means C_1-6Each hydrogen on the alkyl group may be substituted with a hydroxyl group; "hydroxy group C_1-6Alkoxy "means a hydroxy group C_1-6alkyl-O-; ' two (C)_1-6Alkyl) amino "means (C)_1-6Alkyl radical)₂-N-, wherein each C_1-6The alkyl groups may be the same or different; "C_1-6Alkylthio "means C_1-6alkyl-S-; "C_1-6Alkylcarbonyl "means C_1-6alkyl-C (O) -; "C_1-6Alkoxy radical C_1-6Alkyl "means C_1-6alkyl-O-C_1-6An alkyl group; "C_1-6Alkylcarbonyloxy "means C_1-6alkyl-C (O) -O-; "C_1-6Alkylsulfonyl "means C_1-6alkyl-S (O)₂-；“C_1-6Alkylaminosulfonyl "refers to C_1-6alkyl-NH-S (O)₂-; ' two (C)_1-6Alkyl) aminosulfonyl "means (C)_1-6Alkyl radical)₂-N-S(O)₂-, each of which is C_1-6The alkyl groups may be the same or different; "C_1-6Alkylsulfonamido "means C_1-6alkyl-S (O)₂-NH-；“C_1-6Alkylsulfonyloxy "means C_1-6alkyl-S (O)₂-O-。

As used herein, the term "halo C_1-6Alkyl group "," halogeno C_1-6Alkoxy "means C of the group_1-6The hydrogen in the alkyl group is substituted with one or more halogens; the "halogen" is as defined above.

The "cycloalkyl", "aryl", "heterocyclyl" and "heteroaryl" in the present invention include a monocyclic ring system and a fused ring system (bicyclic ring system or polycyclic ring system), wherein monocyclic ring means that the ring is present in the form of only one ring, and the fused ring means a polycyclic ring structure formed by connecting two or more rings in parallel, spiro or bridged manner. The fused ring refers to a fused ring structure formed by two or more ring structures sharing two adjacent ring atoms with each other (i.e., sharing one bond). The bridged ring refers to a fused ring structure formed by two or more cyclic structures sharing two non-adjacent ring atoms with each other. The spiro ring refers to a fused ring structure formed by two or more cyclic structures sharing one ring atom with each other. The cycloalkyl group, aryl group, heterocyclic group and heteroaryl group defined by the number of atoms in the present invention include, unless otherwise specified, monocyclic and fused ring structures which can be formed.

The "cycloalkyl" as used herein refers to a monocyclic cycloalkyl, bicyclic cycloalkyl system or polycyclic cycloalkyl system which is saturated or partially saturated and which is not aromatic. The monocyclic cycloalkyl group may be a 3-to 10-membered cycloalkyl group, a 5-to 10-membered cycloalkyl group, a 3-to 8-membered cycloalkyl group, a 3-to 6-membered cycloalkyl group, a 5-to 8-membered cycloalkyl group and the like, preferably a 3-to 8-membered saturated cycloalkyl group, a 3-to 6-membered saturated cycloalkyl group, a 5-to 6-membered saturated cycloalkyl group, examples include, but are not limited to: cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclooctane, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1, 4-cyclohexadienyl, cycloheptenyl, 1, 4-cycloheptadienyl, cyclooctenyl, 1, 5-cyclooctadienyl, and the like. The acyclic cycloalkyl group may be a 6-12 membered acyclic heterocycloalkyl group, a 7-10 membered acyclic heterocycloalkyl group, an 8-9 membered acyclic heterocycloalkyl group, representative examples of which include, but are not limited to, bicyclo [3.1.1]Heptane, bicyclo [2.2.1]Heptane, bicyclo [2.2.2]Octane, bicyclo [3.2.2]Nonane, bicyclo [3.3.1]Nonanes and bicyclo [4.2.1]Nonane. Spirocyclic cycloalkyl groups, which may be 6-12-membered spirocyclic groups, 7-11-membered spirocyclic groups, and the like, examples thereof include, but are not limited to: the bridged cycloalkyl group may be 6-12 bridge ring group, 7-11 bridge ring group, examples of which include but are not limited to:

the "3-to 8-membered cycloalkyl" as referred to herein includes, unless otherwise specified, monocyclic and fused ring structures, preferably monocyclic cycloalkyl, which can be formed.

The term "heterocyclic group" as used herein means a non-aromatic cyclic group in which at least one ring carbon atom is replaced by a heteroatom selected from O, S, N, preferably 1 to 3 heteroatoms, and includes carbon, nitrogen and sulfur atoms which may be substituted by oxo.

"Heterocyclyl" refers to a monocyclic heterocyclic group or fused heterocyclic group system that is saturated or partially saturated and has no aromatic character. The monoheterocyclic group may be a 3-12-membered heterocyclic group, a 3-10-membered heterocyclic group, a 3-8-membered saturated heterocyclic group, a 3-6-membered heterocyclic group, a 4-7-membered heterocyclic group, a 5-6-membered oxygen-containing heterocyclic group, a 5-6-membered nitrogen-containing heterocyclic group, a 5-6-membered saturated oxygen-containing heterocyclic group, a 5-6-membered saturated nitrogen-containing heterocyclic group or the like. Examples of mono-heterocyclic groups include, but are not limited to, aziridinyl, oxacyclopropane, thiiranyl, azetidinyl, oxetanyl, thietanyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydrothienyl, imidazolidinyl, pyrazolidinyl, 1, 2-oxazolidinyl, 1, 3-oxazolidinyl, 1, 2-thiazolidinyl, 1, 3-thiazolidinyl, tetrahydro-2H-pyranyl, tetrahydro-2H-thiopyranyl, piperidinyl, piperazinyl, morpholinyl, 1, 4-dioxanyl, 1, 4-oxathianyl; examples of partially saturated heteromonocyclic groups include, but are not limited to, 4, 5-dihydroisoxazolyl, 4, 5-dihydrooxazolyl, 2, 3-dihydrooxazolyl, 3, 4-dihydro-2H-pyrrolyl, 2, 3-dihydro-1H-pyrrolyl, 2, 5-dihydro-1H-imidazolyl, 4, 5-dihydro-1H-pyrazolyl, 4, 5-dihydro-3H-pyrazolyl, 4, 5-dihydrothiazolyl, 2H-pyranyl, 4H-pyranyl, 2H-thiopyranyl, 4H-thiopyranyl, 2,3,4, 5-tetrahydropyridinyl, 2, 5-dihydropyranyl, 2H-pyranyl, 4H-thiopyranyl, 2,3,4, 5-tetrahydropyridinyl, and the like, 1, 2-isooxazinyl, 1, 4-isooxazinyl, or 6H-1, 3-oxazinyl, and the like.

Fused heterocycles include heterocyclo, spiroheterocyclyl, bridged heterocyclo, and may be saturated, partially saturated, or unsaturated, but are not aromatic. The heterocyclic group may be a 6-12 membered fused ring group, a 7-11 membered fused ring group, a 6-10 membered fused ring group, a 6-12 membered saturated fused ring group, a 7-11 membered saturated fused ring group, examples of which include, but are not limited to: 3-azabicyclo [3.10] hexanyl, 3, 6-diazabicyclo [3.2.0] heptane, 3, 8-diazabicyclo [4.2.0] octanyl, 3, 7-diazabicyclo [4.2.0] octanyl, octahydropyrrolo [3,4-c ] pyrrole, octahydropyrrolo [3,4-b ] [1,4] oxazinyl, octahydro-1H-pyrrolo [3,4-c ] pyridine, 2, 3-dihydrobenzofuran-2-yl, 2, 3-dihydrobenzofuranyl-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl, 2, 3-dihydrobenzothien-2-yl, octahydro-1H-indolyl, substituted octahydro-1H-indolyl, Octahydrobenzofuranyl.

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

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

the 3-to 10-membered heterocyclic group and the 3-to 8-membered heterocyclic group in the present invention include, unless otherwise specified, monocyclic and fused ring structures which can be formed, and a monocyclic heterocyclic group is preferable.

The aryl group refers to an aromatic cyclic group, and comprises a monocyclic ring system, a bicyclic ring system or a polycyclic ring system, and can be 6-14-membered aryl, 6-12-membered aryl, 6-10-membered aryl, 6-8-membered monocyclic aryl and 8-14-membered fused ring aryl, wherein representative examples of the 6-8-membered monocyclic aryl include but are not limited to phenyl and cyclooctenyl; representative examples of 8-14 membered fused ring aryl groups include, but are not limited to, pentalene, naphthalene, phenanthrene.

The term "heteroaryl" as used herein refers to an aromatic cyclic group wherein at least one ring carbon atom is replaced by a heteroatom selected from O, S, N, preferably 1-3 heteroatoms, including the case where carbon, sulfur and nitrogen atoms are oxo, e.g. carbon by C (O), S (O)₂And (4) replacing. Heteroaryl includes monoheteroaryl and fused heteroaryl, and may be 5-14 membered heteroaryl, 5-12 membered heteroaryl, 5-10 membered heteroaryl, 5-8 membered heteroaryl, 5-6 membered heteroaryl, representative examples of monoheteroaryl include, but are not limited to, furyl, imidazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, oxazolyl, isoxazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl and triazinyl. Fused heteroaryl refers to a bicyclic or polycyclic ring system fused to a phenyl, cycloalkyl, cycloalkenyl, heterocyclyl, or heteroaryl group. The fused heteroaryl group may be an 8-12 membered heteroaryl group, an 8-10 membered heteroaryl group, a 9-10 membered heteroaryl group, representative examples include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzooxadiazolyl, benzothiazolyl, cinnolinyl, 5, 6-dihydroquinolin-2-yl, 5, 6-dihydroisoquinolin-1-yl, indazolyl, indolyl, isoquinolyl, naphthyridinyl, purinyl, quinolyl, 5,6,7, 8-tetrahydroquinolin-2-yl, 5,6,7, 8-tetrahydroquinolyl, 5,6,7, 8-tetrahydroquinolin-4-yl, 5,6,7, 8-tetrahydroisoquinolin-1-yl, 4,5,6, 7-tetrahydro [ c ] do [ c ] yl, and the like][1,2,5]Oxadiazoles and 6, 7-dihydro [ c ]][1,2,5]Oxadiazol-4 (5H) onyl.

The 5-to 14-membered heteroaryl group, the 5-to 12-membered heteroaryl group, and the 5-to 10-membered heteroaryl group according to the present invention include, unless otherwise specified, monocyclic and fused ring structures which can be formed.

In the present invention, "optionally substituted" includes both cases of "substituted" and "unsubstituted".

A "therapeutically effective amount" as referred to herein, refers to an amount of a compound, pharmaceutical formulation, pharmaceutical composition as described above which, when administered to a patient, is capable of at least alleviating the symptoms of the condition in the patient. The actual amount comprising a "therapeutically effective amount" will vary depending on a variety of circumstances, including but not limited to the particular condition being treated, the severity of the condition, the physical and health of the patient, and the route of administration. The appropriate amount can be readily determined by the skilled medical practitioner using methods known in the medical arts.

Advantageous effects of the invention

(1) The compound, the pharmaceutically acceptable salt, the ester or the stereoisomer thereof has excellent ASK1 inhibitory activity, and can treat and/or prevent ASK1 mediated diseases and related diseases;

(2) the compound, the pharmaceutically acceptable salt, the pharmaceutically acceptable ester or the stereoisomer thereof has good pharmacokinetic property, more lasting effect and high bioavailability;

(3) the compound of the invention has simple preparation process, high medicine purity, stable quality and easy large-scale industrial production.

Detailed Description

The technical solutions of the present invention will be described below with reference to specific embodiments, and the described embodiments are only a part of embodiments of the present invention, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The abbreviations used in the following experiments have the following meanings:

PE: petroleum ether EA: ethyl acetate DCM: dichloromethane MeOH: methanol

DMF-DMA: n, N-dimethylformamide dimethyl acetal DIEA: n, N-diisopropylethylamine

THF: tetrahydrofuran Pd₂(dba)₃: tris (dibenzylideneacetone) dipalladium

X-antiphos: (4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene)

X-phos: 2-dicyclohexylphosphine-2 ', 4 ', 6 ' -triisopropylbiphenyl

EDCI: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride

HOBt: 1-hydroxybenzotriazoles

The double bond structure appearing in the structure of the compound of the present inventionRepresents that the compound is a single compound with an unidentified cis-trans configuration or a mixture of cis-trans compounds.

Preparation examples

EXAMPLE 17 preparation of- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one (Compound 1)

1.1 preparation of 7-nitro-3, 4-dihydroisoquinolin-1 (2H) -one

Potassium nitrate (3.71g,36.7mmol) was added to concentrated sulfuric acid (27mL), and 3, 4-dihydroisoquinolin-1 (2H) -one (4.50g,30.6mmol) was added thereto at 0 ℃ to react for 2 hours. The reaction solution was poured into ice water, filtered under suction, and the obtained solid was washed with water and dried to obtain the objective compound (4.00g, yield 68.0%).

Preparation of 1.2, 6-bromopyridine carbohydrazide

Methyl 6-bromopicolinate (5.00g,23.1mmol) was dissolved in methanol (100mL), and hydrazine hydrate (12.47g,249.1mmol) was added to the solution to react for 2 hours. After concentration, ethyl acetate (10mL) and methyl t-butyl ether (100mL) were added, followed by suction filtration and drying of the solid to obtain the objective compound (4.00g, yield 80.0%).

1.3 preparation of (E) -N' - (6-bromopicolinoyl) -N, N-dimethylformylhydrazone amide

6-Bromopyridinecarboxhydrazide (4.00g,18.5mmol) was added to DMF-DMA (15 mL). The reaction was stirred at 80 ℃ for 16 hours, concentrated, added with n-heptane (100mL) and ethyl acetate (10mL), stirred for 1 hour, filtered with suction, and the solid was dried to give the title compound (5.00g, 99.6% yield).

Preparation of 4, 2-bromo-6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridine

(E) -N' - (6-bromopicolinoyl) -N, N-dimethylformylhydrazone amide (5.00g,18.45mmol) and acetic acid (18.5mL) were added to acetonitrile (74mL), isopropylamine (7.60mL,92.25mmol) was added dropwise, the mixture was heated to 95 ℃ for 16 hours, concentrated, and purified by silica gel column chromatography (PE: EA ═ 1:1) to obtain the objective compound (4.65g, yield: 94.3%).

1.5 preparation of 7-amino-3, 4-dihydroisoquinolin-1 (2H) -one

7-Nitro-3, 4-dihydroisoquinoline-1 (2H) -one (1.88g,9.8mmol) and Pd/C (190mg) were added to methanol (50mL), hydrogenated for 3 hours, filtered with suction, and the filtrate was concentrated to give the title compound (1.57g, 98.7% yield).

Preparation of 6,7- ((2-cyclopropyl-2-oxoethyl) amino) -3, 4-dihydroisoquinolin-1 (2H) -one

7-amino-3, 4-dihydroisoquinolin-1 (2H) -one (1.20g,7.4mmol), 2-bromo-1-cyclopropylethan-1-one (1.32g,8.1mmol), DIEA (1.91g,14.8mmol) were added to toluene (20mL), stirred at 95 ℃ for 6 hours, concentrated, and purified by silica gel column chromatography (PE: EA ═ 1:1) to give the title compound (1.50g, 83.0% yield).

1.7 preparation of N- (2-cyclopropyl-2-oxoethyl) -N- (1-oxo-1, 2,3, 4-tetrahydroisoquinolin-7-yl) carboxamide

7- ((2-cyclopropyl-2-oxoethyl) amino) -3, 4-dihydroisoquinolin-1 (2H) -one (1.50g,6.1mmol) was dissolved in dichloromethane (5mL) for further use.

Acetic anhydride (2.49g,24.4mmol) was added dropwise to formic acid (1mL) at 0 deg.C, and stirring was continued at 0 deg.C after the addition was complete. After 0.5 hours, a solution of 7- ((2-cyclopropyl-2-oxoethyl) amino) -3, 4-dihydroisoquinolin-1 (2H) -one in dichloromethane was slowly added dropwise to the system and reacted for 2 hours. The pH was adjusted to 8 with saturated sodium hydroxide solution, extracted with dichloromethane (100mL) saturated sodium chloride solution (80mL), and the organic phase was concentrated to 30mL and used directly in the next step.

1.8 preparation of 7- (4-cyclopropyl-1H-imidazol-1-yl) -3, 4-dihydroisoquinolin-1 (2H) -one

The crude N- (2-cyclopropyl-2-oxoethyl) -N- (1-oxo-1, 2,3, 4-tetrahydroisoquinolin-7-yl) carboxamide solution obtained in the previous step was added to glacial acetic acid (20mL), rotary evaporated to no DCM, and ammonium acetate (1.88g,24.4mmol) was added. The reaction was carried out at 110 ℃ for 16 hours. Adjusting pH to 8-9 with saturated sodium hydroxide solution, adding EA (200mL) for extraction, washing organic phase with saturated sodium chloride solution (150mL), drying with anhydrous sodium sulfate, and concentrating to obtain solid. EA (20mL) was added to the solid, stirred for 0.5h, filtered with suction, and the solid was dried to give the title compound (1.40g, 90.6% overall yield over two steps).

Preparation of 9, 7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one

7- (4-cyclopropyl-1H-imidazol-1-yl) -3, 4-dihydroisoquinolin-1 (2H) -one (500mg,1.97mmol), 2-bromo-6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridine (579mg,2.17mmol), cesium carbonate (1.28g,3.94mmol), Pd₂(dba)₃(92mg,0.10mmol), X-antiphos (116mg,0.20mmol) was added to 1,4-Dioxane (300mL) was stirred under nitrogen at 80 ℃ for 16h, concentrated and purified by silica gel column chromatography (DCM: MeOH ═ 20:1) to give the title compound (150mg, 17.3% yield).

The molecular formula is as follows: c₂₅H₂₅N₇O molecular weight: 439.2 LC-MS (M/e): 440.1(M + H)⁺)

¹H NMR(CD₃OD):8.85(s,1H),8.18(d,J＝2.4Hz 1H),8.12-8.00(m,3H),8.00-7.93(m,1H),7.78-7.71(m,1H),7.55-7.45(m,1H),7.34(s,1H),5.60-5.45(m,1H),4.38-4.25(m,2H),3.30-3.22(m,2H),1.95-1.80(m,1H),1.65-1.55(m,6H),0.95-0.85(m,2H),0.75-0.65(m,2H).

Example 27 preparation of- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one (Compound 2)

2.1 preparation of 2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -7-nitroisoquinolin-1 (2H) -one

2-bromo-6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridine (2.55g,9.6mmol), 7-nitro-3, 4-dihydroisoquinolin-1 (2H) -one (1.84g,9.6mmol), cesium carbonate (6.26g,19.2mmol), Pd₂(dba)₃(440mg,0.48mmol), X-antphos (556mg,0.96mmol) was added to 1, 4-dioxane (100mL), stirred at 80 ℃ under nitrogen for 16 hours, concentrated, and purified by silica gel column chromatography (PE: EA ═ 1:1) to give the title compound (450mg, 12.5% yield).

2.2 preparation of 7-amino-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one

2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -7-nitroisoquinolin-1 (2H) -one (450mg,1.2mmol) was dissolved in THF (10mL) and methanol (10mL), hydrogenated for 2 hours, filtered with suction, and the filtrate was concentrated to give the title compound (400mg, 96.2% yield).

Preparation of 3,7- ((2-cyclopropyl-2-oxoethyl) amino) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one

7-amino-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one (345mg,1.0mmol), 2-bromo-1-cyclopropyleth-1-one (196mg,1.2mmol), DIEA (284mg,2.2mmol) was added to toluene (10mL), the reaction was stirred at 95 ℃ for 16 hours, concentrated, and purified by silica gel column chromatography (DCM: MeOH ═ 20:1) to give the title compound (350mg, 81.7% yield).

2.4 preparation of N- (2-cyclopropyl-2-oxoethyl) -N- (2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1-oxo-1, 2-dihydroisoquinolin-7-yl) carboxamide

7- ((2-cyclopropyl-2-oxoethyl) amino) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one (350mg,0.82mmol) was dissolved in dichloromethane (5mL) for use.

Acetic anhydride (335mg,3.28mmol) was added dropwise to formic acid (1mL) at 0 deg.C, and stirring was continued at 0 deg.C after the addition was complete. After 0.5 hour, a dichloromethane solution of 7- ((2-cyclopropyl-2-oxoethyl) amino) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one was slowly added dropwise to the system and reacted for 2 hours. The pH was adjusted to 8 with saturated sodium hydroxide solution, extracted with dichloromethane (20mL) saturated sodium chloride solution (15mL) and the organic phase was concentrated and used directly in the next step.

Preparation of 7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one

The crude N- (2-cyclopropyl-2-oxoethyl) -N- (2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -1-oxo-1, 2-dihydroisoquinolin-7-yl) carboxamide (0.82mmol) obtained in the above step was dissolved in glacial acetic acid (6mL), and ammonium acetate (253mg,3.28mmol) was added. The reaction was carried out at 110 ℃ for 16 hours. The pH was adjusted to 8 with saturated sodium hydroxide solution, dichloromethane (50mL) was added and water (40mL) was added for extraction, the organic phase was concentrated, and after silica gel column chromatography (DCM: MeOH ═ 20:1), the title compound (50mg, 14.0% total yield over two steps) was prepared under high pressure.

The molecular formula is as follows: c₂₅H₂₃N₇O; molecular weight: 437.2, respectively; LC-MS (M/e): 438.3(M + H)⁺)。

¹H NMR(CD₃OD):8.90(s,1H),8.45(d,J＝2.4Hz,1H),8.25-8.18(m,2H),8.18-8.10(m,1H),8.10-7.90(m,2H),7.90-7.75(m,2H),7.45-7.43(m,1H),6.86(d,J＝7.6Hz,1H),5.55-5.38(m,1H),2.00-1.89(m,1H),1.65-1.52(m,6H),0.93-0.90(m,2H),0.82-0.71(m,2H).

Example 2 preparation of 17- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one (Compound 2)

Preparation of 2-1.1 methyl 2- (2- (dimethylamino) vinyl) -5-nitrobenzoate

Methyl 2-methyl-5-nitrobenzoate (7.0g,35.9mmol) was dissolved in 1-tert-butoxy-N, N' -tetramethylmethanediamine (9.4g,53.9mmol), reacted at 115 ℃ for 2 hours to precipitate a solid, filtered, the cake was then washed with a mixed solvent of petroleum ether and ethyl acetate (60mL, petroleum ether: ethyl acetate ═ 6:1), filtered, and the cake was dried to obtain the objective product (8.1g, yield: 90.2%).

Preparation of 2-1.2- (2, 4-dimethoxybenzyl) -7-nitroisoquinolin-1 (2H) -one

Methyl 2- (2- (dimethylamino) vinyl) -5-nitrobenzoate (8.19g,32.7mmol) and 2, 4-dimethoxybenzylamine (7.6g,45.4mmol) were added to a toluene (80mL) solution, heated to 125 ℃, reacted for 2.5 hours, a solid was precipitated, cooled to 25 ℃, filtered, and the filter cake was then slurry-washed with a mixed solvent of petroleum ether and ethyl acetate (100mL, petroleum ether: ethyl acetate ═ 2:1), filtered, and the filter cake was dried to obtain the objective product (9.0g, yield: 81.6%).

Preparation of 2-1.3.7-amino-2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one

2- (2, 4-Dimethoxybenzyl) -7-nitroisoquinolin-1 (2H) -one (8.4g,24.7mmol), zinc powder (8.1g,123.8mmol), ammonium chloride (7.9g,147.7mmol) were added to methanol (100mL) and water (100mL), reacted at 80 ℃ for 4 hours, filtered, the filtrate was added with water (100mL), extracted with dichloromethane (300 mL. times.3), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the objective compound (7.4g, yield: 96.6%).

Preparation of 2-1.4.7- ((2-cyclopropyl-2-oxoethyl) amino) -2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one

7-amino-2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one (5.0g,16.1mmol), 2-bromo-1-cyclopropylethyl-1-one (3.1g,19.0mmol), N-diisopropylethylamine (4.2g,32.6mmol) were sequentially added to a mixed solvent of toluene (15mL), tetrahydrofuran (15mL) and dichloromethane (30mL), heated to 80 deg.C, reacted for 5 hours, spin-dried, and the residue was subjected to column chromatography (petroleum ether: ethyl acetate: 5:1-1:1) to obtain the objective compound (3.6g, yield: 57.0%).

Preparation of N- (2-cyclopropyl-2-oxoethyl) -N- (2- (2, 4-dimethoxybenzyl) -1-oxo-1, 2-dihydroisoquinolin-7-yl) carboxamide

Acetic anhydride (5.2g,12.7mmol) was dropped into formic acid (30mL) at 0 ℃ to react for 0.5 hour, a solution of 7- ((2-cyclopropyl-2-oxoethyl) amino) -2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one (5.0g,12.7mmol) in dichloromethane (30mL) was dropped into the reaction system to react at 0 ℃ for 16 hours, PH was adjusted to 7 with 1N sodium hydroxide solution, dichloromethane (400mL × 2) was extracted, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and rotary-dried to obtain a crude product (5.0 g).

Preparation of 2-1.6.7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one

N- (2-cyclopropyl-2-oxoethyl) -N- (2- (2, 4-dimethoxybenzyl) -1-oxo-1, 2-dihydroisoquinolin-7-yl) carboxamide (5.0g, crude) and ammonium acetate (7.5g,97.3mmol) were dissolved in acetic acid (30mL), heated to 105 ℃, reacted for 16 hours, 0 ℃, adjusted pH 7 with sodium hydroxide solution (2M), extracted with dichloromethane (150 mL. times.3), the combined organic phases were dried over anhydrous sodium sulfate, filtered, and spin-dried to give crude (5.1 g).

Preparation of 2-1.7.7- (4-cyclopropyl-1H-imidazol-1-yl) isoquinolin-1 (2H) -one

7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one (5.1g, crude) was dissolved in trifluoroacetic acid (30mL), heated to 90 ℃ for 3 hours, concentrated, slurried with ethyl acetate (50mL), filtered, and the solid was dried to give the title compound (2.2g, three-step yield: 68.9%).

Preparation of 2-1.8.7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one

7- (4-cyclopropyl-1H-imidazol-1-yl) isoquinolin-1 (2H) -one (1.0g,3.98mmol), 2-bromo-6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridine (900mg, 3.37mmol), 4, 5-bis diphenylphosphino-9, 9-dimethylxanthene (129mg, 0.22mmol), tris (dibenzylideneacetone) dipalladium (204mg, 0.22mmol) and cesium carbonate (2.4g, 7.37mmol) were dissolved in 1, 4-dioxane (70mL) and reacted at 105 ℃ for 16 hours under nitrogen. Concentration and reverse phase column chromatography (acetonitrile/water 0-50%) to give crude product, slurried with methanol (5mL) and methyl tert-butyl ether (20mL), filtered and the filter cake dried to give the title compound (400mg, 27.1% yield).

Molecular formula C₂₅H₂₃N₇O molecular weight 437.51 LC-MS (M/e):438.2(M + H)⁺)

¹H-NMR(400MHz,CDCl₃)δ:8.46(s,1H),8.39(s,1H),8.32-8.36(m,1H),8.01-8.07(m,1H),7.86(d,J＝1.2Hz,1H),7.66-7.74(m,3H),7.16(d,J＝0.8Hz,1H),6.68(d,J＝8Hz,1H),5.44-5.51(m,1H),1.91-1.96(m,1H),1.53(d,J＝11.2Hz,6H),0.80-0.93(m,4H).

EXAMPLE 37 preparation of- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxyisoquinolin-1 (2H) -one (Compound 3)

Preparation of 3.1, 2-methyl-4, 5-dinitrobenzoic acid

Dissolving 2-methyl-4-nitrobenzoic acid (10.0g, 55.2mmol) in concentrated sulfuric acid (60mL), adding a solution of potassium nitrate (8.4g, 83.2mmol) in concentrated sulfuric acid (40mL) dropwise to the reaction mixture, pouring into ice water at 25 ℃, for 16 hours, precipitating a solid, and drying in vacuum to obtain the target compound 9g, yield: 72 percent.

Preparation of 3, 4-methoxy-2-methyl-5-nitrobenzoic acid

2-methyl-4, 5-dinitrobenzoic acid (9g,39.8mmol) was added in portions to a solution of potassium hydroxide (11g,196.4mmol) in methanol (200mL), heated to 70 ℃ for 3 hours, adjusted in pH 3-4 with 2N hydrochloric acid, and a solid was precipitated, filtered, and dried in vacuo to give the objective compound (7g, yield: 83.3%).

Preparation of methyl 3, 4-methoxy-2-methyl-5-nitrobenzoate

4-methoxy-2-methyl-5-nitrobenzoic acid (7.0g,33.2mmol) was dissolved in methanol (100mL), thionyl chloride (8.5g,71.4mmol) was added at 0 ℃, heated to 70 ℃, for 16 hours, spin-dried, added with 200mL of ethyl acetate, washed with a saturated sodium bicarbonate solution, spin-dried, and column-chromatographed (petroleum ether: ethyl acetate ═ 8:1) to give the title compound (6.0g, yield: 80%).

3.4 preparation of methyl (E) -2- (2- (dimethylamino) ethenyl) -4-methoxy-5-nitrobenzoate

Methyl 4-methoxy-2-methyl-5-nitrobenzoate (5.0g,22.2mmol), 1-tert-butoxy-N, N' -tetramethylmethanediamine (5.8g,33.3mmol) were heated to 115 ℃ for 3 hours, cooled to 25 ℃, added with a mixed solvent of petroleum ether and ethyl acetate (6: 1) (50mL), filtered, and the solid was dried under vacuum to give the objective compound 5.5g, yield: 88.7%.

Preparation of 2- (2, 4-dimethoxybenzyl) -6-methoxy-7-nitroisoquinolin-1 (2H) -one

Methyl (E) -2- (2- (dimethylamino) vinyl) -4-methoxy-5-nitrobenzoate (5.0g,17.8mmol), 2, 4-dimethoxybenzylamine (4.5g,26.9mmol) were dissolved in toluene (100mL), heated to 110 ℃, reacted for 16 hours, the solvent was dried, a mixed solvent of petroleum ether and ethyl acetate 2:1 (80mL) was added, filtered, and the solid was dried in vacuo to give the objective compound 5.5g, yield: 91.1%.

Preparation of 6, 6-methoxy-7-nitroisoquinolin-1 (2H) -one

2- (2, 4-Dimethoxybenzyl) -6-methoxy-7-nitroisoquinolin-1 (2H) -one (5.5g,14.9mmol) was dissolved in trifluoroacetic acid (50mL), heated to 85 deg.C for 3 hours, the solvent was dried, methanol (50mL) was added, ethyl acetate (60mL) was added, filtered, and the solid was dried under vacuum to give the title compound (3.1g, yield: 93.9%).

3.7 preparation of methyl 6- (6-methoxy-7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinate

6-methoxy-7-nitroisoquinolin-1 (2H) -one (2.8g,12.7mmol), methyl 6-bromopicolinate (4.1g,18.9mmol), cuprous iodide (0.24g,1.26mmol), potassium phosphate (5.4g,25.5mmol), tetramethylethylenediamine (0.3g,2.58mmol), toluene (100mL), heated to 110 deg.C for 16 hours, methanol: a mixed solvent (100mL) of 9:15 methylene chloride was filtered, and the solid was dried in vacuo to give 3.0g of the objective compound in 66.7% yield.

3.8 preparation of 6- (6-methoxy-7-nitro-1-oxoisoquinolin-2 (1H) -yl) pyridinecarbohydrazide

Methyl 6- (6-methoxy-7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinate (600mg,1.7mmol) was dissolved in methanol (30mL), hydrazine monohydrate solution (3mL) was added, heated to 65 ℃ for 16H, spun dry, and the residue was used directly in the next step.

3.9 preparation of (E) -N' - (6- (6-methoxy-7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinoyl) -N, N-dimethylformamidine

6- (6-methoxy-7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinohydrazide (crude, 1.7mmol), N, N-dimethylformamide dimethyl acetal (20mL) was heated to 90 ℃ and reacted for 16 hours. Spin dry and the residue was used directly in the next step.

3.10 preparation of 2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxy-7-nitroisoquinolin-1 (2H) -one

(E) -N' - (6- (6-methoxy-7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinoyl) -N, N-dimethylformamidine (crude, 1.7mmol) was dissolved in a mixed solvent of acetic acid (1mL) and acetonitrile (4mL), isopropylamine (0.5g,8.5mmol) was added, heated to 70 ℃ for 16 hours, spin-dried, and column chromatography of the residue (dichloromethane: methanol ═ 20:1) gave the title compound (0.48g, three-step yield: 69.6%).

Preparation of 11, 7-amino-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxyisoquinolin-1 (2H) -one

2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxy-7-nitroisoquinolin-1 (2H) -one (0.48g,1.2mmol) was added to methanol (300mL), stannous chloride (1.1g,5.8mmol) and hydrochloric acid (1mL) were added, heated to 70 ℃ for 16 hours, a saturated sodium bicarbonate solution was added, filtered, the filtrate was spun dry, and the residue was subjected to column chromatography (dichloromethane: methanol ═ 30:1) to give the objective compound (0.25g, yield: 56.8%).

Preparation of 12, 7- ((2-cyclopropyl-2-oxoethyl) amino) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxyisoquinolin-1 (2H) -one

7-amino-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxyiso-1 (2H) -one (0.25g,0.66mmol), 2-bromo-1-cyclopropylethyl-1-one (0.16g,0.98mmol) was dissolved in toluene (30mL), N-diisopropylethylamine (0.17g,1.3mmol) was added, heated to 95 ℃ for 16 hours, spun-dried, and the residue was subjected to column chromatography (dichloromethane: methanol ═ 30:1) to give the objective compound (95mg, yield: 31.7%).

3.13 preparation of N- (2-cyclopropyl-2-oxoethyl) -N- (2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxy-1-oxo-1, 2-dihydroisoquinolin-7-yl) carboxamide

7- ((2-cyclopropyl-2-oxoethyl) amino) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxyisoquinolin-1 (2H) -one (95mg,0.21mmol) was dissolved in acetic acid (3mL), acetic anhydride (43mg,0.42mmol) was added, heated to 25 ℃ for 16H, adjusted to pH 8-9 with 2N sodium hydroxide solution, extracted with dichloromethane (30 mL. times.2), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, spun dry and the residue was used directly in the next step.

3.14 preparation of 7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxyisoquinolin-1 (2H) -one

N- (2-cyclopropyl-2-oxoethyl) -N- (2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -6-methoxy-1-oxo-1, 2-dihydroisoquinolin-7-yl) carboxamide (crude, 0.21mmol) was dissolved in acetic acid (5mL), ammonium acetate (160mg,2.1mmol) was added, heating was carried out to 110 ℃, for 16 hours, spin-dried, sodium bicarbonate solution (10mL) was added to adjust pH to 8-9, dichloromethane (20mL × 3) was extracted, the organic phases were combined, TLC separation (dichloromethane: methanol: 20:1) was carried out to obtain the objective compound 46mg, yield: 47.4 percent.

Molecular formula C₂₆H₂₅N₇O₂Molecular weight 467.53 LC-MS (M/e):468.2(M + H)⁺)

¹H-NMR(400MHz,d-DMSO)δ:8.92(s,1H),8.15-8.19(m,2H),8.11(s,1H),7.95(dd,J＝7.2Hz,2.0Hz,1H),7.86(d,J＝7.6Hz,1H),7.81(s,1H),7.50(s,1H),7.26(s,1H),6.82(d,J＝7.6Hz,1H),5.27-5.30(m,1H),3.97(s,3H),1.82-1.89(m,1H),1.47(d,J＝6.4Hz,6H),0.76-0.81(m,2H),0.67-0.70(m,2H).

Example 47 preparation of- (4-cyclopropyl-1H-imidazol-1-yl) -4-fluoro-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one (Compound 4)

4.1 Synthesis of 7- (4-cyclopropyl-1H-imidazol-1-yl) -4-fluoro-3-hydroxy-3, 4-dihydroisoquinolin-1 (2H) -one

The compound 7- (4-cyclopropyl-1H-imidazol-1-yl) isoquinolin-1 (2H) -one (502mg,2mmol), 1-chloromethyl-4-fluoro-1, 4-diazobicyclo 2.2.2 octane bis (tetrafluoroborate) salt (1.42g, 4mmol) was dissolved in acetonitrile (30mL), and water (0.5mL) was added to conduct a reaction under reflux for 5 hours. The reaction mixture was concentrated, water (100mL) was added thereto, the mixture was washed with dichloromethane three times (100mL × 3), the aqueous phase was concentrated, a mixed solvent of dichloromethane and methanol (dichloromethane: methanol ═ 20:2) was added to the residue, the mixture was filtered with suction, and the filtrate was concentrated for use.

4.2 Synthesis of 7- (4-cyclopropyl-1H-imidazol-1-yl) -4-fluoroisoquinolin-1 (2H) -one

The crude product obtained in the above step was dissolved in methylene chloride (10mL), and methanesulfonic acid (1.25g,13mmol) was added at 0 ℃ to react at 10 ℃ for 3 hours. Concentration and reverse phase column chromatography (0-35% MeOH) of the residue afforded 100mg of the title compound in 18.6% yield over two steps.

The molecular formula is as follows: c₁₅H₁₂FN₃O molecular weight: 269.3 LC-MS (M/e):270.1(M+H)

¹H-NMR(400MHz,CD₃OD)δ:9.40(brs,1H),8.55(s,1H),8.12-8.15(m,1H),8.05-8.08(m,1H),7.92(brs,1H),7.37(d,J＝5.6HZ,1H),2.00-2.10(m,1H),1.13-1.16(m,2H),0.92-0.94(m,2H).

4.3 Synthesis of 7- (4-cyclopropyl-1H-imidazol-1-yl) -4-fluoro-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one

The compound 7- (4-cyclopropyl-1H-imidazol-1-yl) -4-fluoroisoquinolin-1 (2H) -one (100mg,0.37mmol), 2-bromo-6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridine (99mg,0.37mmol), 4, 5-bis diphenylphosphine-9, 9-dimethylxanthene (20mg, 20%), tris (dibenzylideneacetone) dipalladium (10mg, 10%) and cesium carbonate (362mg,1.11mmol) were dissolved in 1, 4-dioxane (10mL) and reacted at 110 ℃ for 15 hours under nitrogen. The reaction mixture was concentrated, water (100mL) was added thereto, methylene chloride was extracted three times (100mL × 3), the organic phase was concentrated and dried, and the residue was subjected to silica gel column chromatography (methylene chloride: methanol ═ 20:1) and reverse phase column chromatography (0-40% methanol) in this order to obtain 15mg of the objective compound with a yield of 8.9%.

The molecular formula is as follows: c₂₅H₂₂FN₇O molecular weight: 455.5 LC-MS (M/e):456.2(M + H)

¹H-NMR(400MHz,CD₃OD)δ:9.37(s,1H),8.92(s,1H),8.70(s,1H),8.19-8.24(m,3H),8.10-8.16(m,3H),7.93(s,1H),5.45-5.49(m,1H),2.00-2.10(m,1H),1.58-1.61(m,6H),1.12-1.21(s,2H),0.91-1.00(s,2H).

Example preparation of 57- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3-methylisoquinolin-1 (2H) -one (Compound 5)

5.1 preparation of methyl 6- (7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinate

7-Nitroisoquinolin-1 (2H) -one (3g,15.8mmol), methyl 6-bromopicolinate (4.4g,20.4mmol), potassium phosphate (6.7g,31.6mmol), tetramethylethylenediamine (90mg,0.79mmol) and cuprous iodide (0.15g,0.79mmol) were added to toluene (150mL) and the reaction stirred at 100 deg.C for 16 hours under nitrogen. Cooled to 20 ℃, concentrated to dryness, washed with water (200mL), filtered, the filter cake washed with methanol and dried to give the title compound (1.6g, yield: 31.2%).

5.2 preparation of methyl 6- (3-methyl-7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinate

Methyl 6- (7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinate (1.5g,4.61mmol), potassium methyltrifluoroborate (1.7g,13.9mmol), silver hexafluoroantimonate (0.32g,0.93mmol), silver oxide (1.6g,6.91mmol), dichloro (pentamethylcyclopentadienyl) rhodium dimer (0.15g,0.24mmol) were dissolved in 1, 2-dichloroethane (100mL), nitrogen protected, heated to 80 ℃ and stirred for reaction for 16 hours. Potassium methyltrifluoroborate (1.7g,13.9mmol), silver hexafluoroantimonate (0.32g,0.93mmol), and dichloro (pentamethylcyclopentadienyl) rhodium dimer (0.15g,0.24mmol) were added, and the reaction was continued with stirring for 16 hours. Filtration and concentration of the filtrate were carried out, and purification by silica gel column chromatography (ethyl acetate: petroleum ether ═ 1:1) was carried out to give the objective compound (0.25g, yield: 16.0%) and methyl 6- (7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinate (1.3g) was recovered.

5.3 preparation of methyl 6- (7-amino-3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinate

Methyl 6- (3-methyl-7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinate (0.63g,1.86mmol) was dissolved in methanol (10mL), triethylamine (1mL) and Pd/C (0.3g) were added, and the reaction was stirred under hydrogen pressure at 20 ℃ for 3 hours. Filtration and concentration of the filtrate, and purification of the crude product by silica gel column chromatography (dichloromethane: methanol ═ 20:1) gave the title compound (0.38g, yield 66.0%).

Preparation of methyl 6- (7- ((2-cyclopropyl-2-oxoethyl) amino) -3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinate

Methyl 6- (7-amino-3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinate (0.37g,1.2mmol) and DIEA (0.31g,2.4mmol) were dissolved in toluene (10mL), 2-bromo-1-cyclopropyleth-1-one (0.24g,1.5mmol) was added, the reaction was stirred under nitrogen protection at 95 ℃ for 16 hours, concentrated, and the resulting crude product was purified by silica gel column chromatography (dichloromethane: methanol ═ 20:1) to give the objective compound (0.15g, yield 32.0%), and methyl 6- (7-amino-3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinate (0.22g) was recovered.

5.5 preparation of methyl 6- (7- (N- (2-cyclopropyl-2-oxoethyl) carboxamido) -3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinate

Acetic anhydride (0.37g,3.62mmol) was dissolved in formic acid (5mL), and the reaction was stirred at 20 ℃ for 30 minutes, methyl 6- (7- ((2-cyclopropyl-2-oxoethyl) amino) -3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinate (0.28g,0.72mmol) was added, and the reaction was stirred at 25 ℃ for 16 hours. Saturated aqueous sodium bicarbonate was added to neutralize the pH to 7. Extraction was performed with ethyl acetate (200 mL. times.5), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the crude target compound (0.3 g).

5.6 preparation of methyl 6- (7- (4-cyclopropyl-1H-imidazol-1-yl) -3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinate

The crude methyl 6- (7- (N- (2-cyclopropyl-2-oxoethyl) carboxamido) -3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinate (0.3g) was dissolved in acetic acid, ammonium acetate (0.3g,3.89mmol) was added, the reaction was stirred under nitrogen at 110 ℃ for 16 hours and concentrated to give the crude product which was isolated and purified by medium pressure to give the title compound (0.2g, 70.0% yield over two steps).

Preparation of 6- (7- (4-cyclopropyl-1H-imidazol-1-yl) -3-methyl-1-oxoisoquinolin-2 (1H) -yl) pyridine carboxylic acid hydrazide

Methyl 6- (7- (4-cyclopropyl-1H-imidazol-1-yl) -3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinate (0.2g,0.5mmol) was dissolved in methanol (5mL), 85% hydrazine hydrate (2mL) was added and the reaction stirred at 20 ℃ for 16H. And concentrating, and directly using the obtained crude product for the next reaction.

Preparation of N' - (6- (7- (4-cyclopropyl-1H-imidazol-1-yl) -3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinoyl) -N, N-dimethylcarbohydrazide

The crude 6- (7- (4-cyclopropyl-1H-imidazol-1-yl) -3-methyl-1-oxoisoquinolin-2 (1H) -yl) pyridine carbohydrazide obtained in the previous step was dissolved in DMF-DMA (5 mL). The reaction was stirred for 16 hours while heating to 80 ℃. Concentration and washing the crude product with methyl tert-butyl ether to give crude target compound (92 mg).

Preparation of 9, 7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3-methylisoquinolin-1 (2H) -one

Crude N' - (6- (7- (4-cyclopropyl-1H-imidazol-1-yl) -3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinoyl) -N, N-dimethylformylhydrazine (92mg) was dissolved in a mixed solvent of acetic acid (1mL) and acetonitrile (4mL), isopropylamine (60mg,1mmol) was added, and the mixture was heated to 95 ℃ for 16 hours. Concentration and the resulting crude product was purified by preparative high pressure to give the objective compound (11mg, three-step yield: 4.9%).

The molecular formula is as follows: c₂₆H₂₅N₇O molecular weight: 451.5 LC-MS (M/e): 452.3(M + H)⁺)

¹H NMR(CD₃OD):8.85(s,1H),8.27-8.34(m,3H),8.13(s,1H),7.94-7.97(m,1H),7.79(d,J＝8.4Hz 1H),7.68(dd,J1＝7.6Hz,J2＝1.2Hz,1H),7.42(s,1H),6.75(s,1H),5.37-5.42(m,1H),2.11(s,3H),1.88-1.95(m,1H),1.45-1.55(m,6H),0.88-0.92(m,2H),0.76-0.80(m,2H)。

Example 66 preparation of (4-cyclopropyl-1H-imidazol-1-yl) -3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) quinazolin-4 (3H) -one (Compound 6)

Preparation of 1, 5-bromo-N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -2-nitrobenzamide

5-bromo-2-nitrobenzoic acid (3.31g,13.5mmol), 6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-amine (3.0g,14.8mmol), EDCI (5.15g,26.9mmol), DMAP (327mg,2.7mmol) were added to pyridine (30mL) and reacted at 35 ℃ for 40 hours. Dichloromethane (20mL) was added by evaporation to dryness, and the title compound (1.68g, yield: 28.9%) was obtained by suction filtration.

Preparation of 2, 2-amino-5-bromo-N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) benzamide

5-bromo-N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -2-nitrobenzamide (1.66g,3.8mmol), iron powder (1.1g,19.6mmol), ammonium chloride (610mg,11.4mmol), and water (6mL) were added to isopropanol (24mL), stirred at 80 ℃ for 16 hours, methanol (20mL), dichloromethane (20mL) were added, the mixture was sonicated, filtered, and the filtrate was evaporated to dryness to give a crude product of the objective compound (2.0 g).

Preparation of 3, 6-bromo-3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) quinazolin-4 (3H) -one

The crude 2-amino-5-bromo-N- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) benzamide (1.55g) was added to triethyl orthoformate (20mL), reacted at 143 ℃ for 2 hours, cooled to precipitate a solid, and filtered to obtain the crude target compound (1.75 g).

Preparation of tert-butyl (3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -4-oxo-3, 4-dihydroquinazolin-6-yl) carbamate

Crude 6-bromo-3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) quinazolin-4 (3H) -one (1.75g), palladium acetate (83mg,0.34mmol), xanthphos (393mg,0.68mmol), cesium carbonate (2.76g,8.5mmol), tert-butyl carbamate (597mg,5.1mmol) were added to 1, 4-dioxane (15mL), reacted under nitrogen at 100 ℃ for 16 hours, concentrated to dryness, and subjected to silica gel column chromatography (eluent methanol: dichloromethane: 1:25) to give the target compound (950mg, three-step overall yield: 55.9%).

Preparation of 5, 6-amino-3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) quinazolin-4 (3H) -one

Tert-butyl (3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -4-oxo-3, 4-dihydroquinazolin-6-yl) carbamate (920mg,2.1mmol), trifluoroacetic acid (2mL) was added to dichloromethane (6mL), reacted at 12 ℃ for 3 hours, concentrated to dryness, and subjected to silica gel column chromatography (eluent methanol: dichloromethane ═ 1:20) to obtain the objective compound (555mg, yield: 76%).

6.6 preparation of 6- ((2-cyclopropyl-2-oxoethyl) amino) -3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) quinazolin-4 (3H) -one

6-amino-3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) quinazolin-4 (3H) -one (480mg,1.38mmol), 1-cyclopropyl-2-bromoethanone (225mg,1.38mmol), DIEA (356mg,2.76mmol) was added to toluene (18mL), reacted at 90 ℃ for 6 hours, concentrated to dryness, and passed through a silica gel column (eluent methanol: dichloromethane ═ 1:25) to give the title compound (145mg, yield: 24.5%).

Preparation of N- (2-cyclopropyl-2-oxoethyl) -N- (3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -4-oxo-3, 4-dihydroquinazolin-6-yl) carboxamide

Acetic anhydride (138mg,1.35mmol) was diluted with dichloromethane (1mL), added dropwise to formic acid (2mL) at 0 ℃, stirred at 0 ℃ for 0.5H, 6- ((2-cyclopropyl-2-oxoethyl) amino) -3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) quinazolin-4 (3H) -one (145mg,0.34mmol) was dissolved in dichloromethane (2mL), added dropwise to the above formic acid solution, reacted at 0 ℃ for 2H, adjusted to pH 8-9 with sodium hydroxide solution, extracted with dichloromethane (15mL), and the organic phase evaporated to dryness to give the crude product.

Preparation of 6- (4-cyclopropyl-1H-imidazol-1-yl) -3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) quinazolin-4 (3H) -one

Crude N- (2-cyclopropyl-2-oxoethyl) -N- (3- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -4-oxo-3, 4-dihydroquinazolin-6-yl) carboxamide, ammonium acetate (104mg,1.35mmol), was added to glacial acetic acid (5mL), reacted at 110 ℃ for 20H, pH 7-8 was adjusted with sodium hydroxide solution, dichloromethane (15mL) was added for extraction, the organic phase was evaporated to dryness, and TLC plates (developing solvent methanol: dichloromethane ═ 1:12) gave the title compound (37mg, total yield in two steps: 24.8%).

The molecular formula is as follows: c₂₄H₂₂N₈O molecular weight: 438.5 LC-MS (M/e): 439.2(M + H)⁺)

¹H-NMR(CDCl₃)δ:8.58(s,1H),8.47-8.45(d,1H),8.41(s,1H),8.35-8.34(d,1H),8.13-8.09(t,1H),7.97-7.95(d,1H),7.91-7.83(m,3H),7.16(s,1H),5.53-5.46(m,1H),1.98-1.90(m,1H),1.58-1.56(m,6H),0.95-0.89(m,2H),0.87-0.84(m,2H).

EXAMPLE 72 preparation of (6- ([1,2,4] triazolo [4,3-a ] pyridin-3-yl) pyridin-2-yl) -7- (4-cyclopropyl-1H-imidazol-1-yl) isoquinolin-1 (2H) -one (Compound 7)

Preparation of methyl 2- (2- (dimethylamino) ethenyl) -5-nitrobenzoate

Methyl 2-methyl-5-nitrobenzoate (7.0g,35.9mmol) was dissolved in 1-tert-butoxy-N, N, N ', N' -tetramethylmethanediamine (9.4g,53.9mmol) and heated to 115 ℃ for 2 hours. Solid is separated out and filtered, and filter cake is treated by petroleum ether: ethyl acetate (6:1,60mL) was slurry washed, filtered, and the filter cake was dried to give 8.1g of the desired product in 90.2% yield.

Preparation of 2,2- (2, 4-dimethoxybenzyl) -7-nitro-1 (2H) -isoquinolinone

Methyl 2- (2- (dimethylamino) vinyl) -5-nitrobenzoate (8.1g,32.4mmol), 2, 4-dimethoxybenzylamine (7.6g,45.4mmol) was dissolved in toluene (80mL), heated to 125 ℃ for 2.5 h to precipitate a solid, cooled to 25 ℃, filtered, and the filter cake was washed with petroleum ether: ethyl acetate (2:1, 100mL) was slurry washed, filtered, and the filter cake was dried to give 9.0g of the desired product in 81.6% yield.

Preparation of 3, 7-amino-2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one

2- (2, 4-Dimethoxybenzyl) -7-nitro-1 (2H) -isoquinolinone (8.4g,24.7mmol), zinc powder (8.1g,123.8mmol), ammonium chloride (7.9g,147.7mmol) were added to methanol (100mL) and water (100mL) in this order and reacted at 80 ℃ for 4 hours. Filtration was carried out, the filtrate was extracted with methylene chloride (300 mL. times.3) by adding water (100mL), the organic phases were combined, dried over anhydrous sodium sulfate, and filtered, and the filtrate was concentrated to obtain 7.4g of the objective product, yield: 96.6%.

Preparation of 4, 7- ((2-cyclopropyl-2-oxoethyl) amino) -2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one

7-amino-2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one (5.0g,16.1mmol), 2-bromo-1-cyclopropylethanone (3.1g,19.0mmol), DIEA (4.2g,32.6mmol) were added to dichloromethane (30mL), tetrahydrofuran (15mL), toluene (15mL) in that order and reacted at 80 ℃ for 5 hours. Concentration and purification of the residue by silica gel column (petroleum ether: ethyl acetate: 5:1-1:1) gave 3.6g of the objective product in 57.0% yield.

7.5 preparation of N- (2-cyclopropyl-2-oxoethyl) -N- (2- (2, 4-dimethoxybenzyl) -1-oxo-1, 2-dihydroisoquinolin-7-yl) carboxamide

Acetic anhydride (5.2g,50.9mmol) was added dropwise to formic acid (30mL) at 0 ℃ for 0.5 hour, and then a solution of 7- ((2-cyclopropyl-2-oxoethyl) amino) -2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one (5.0g,12.7mmol) in methylene chloride (30mL) was added dropwise to the reaction system and reacted at 0 ℃ for 16 hours. The pH was adjusted to 7 with NaOH (1M), extracted with dichloromethane (400mL methane), the organic phases were combined, dried over anhydrous sodium sulfate and concentrated to give crude 5.0 g.

Preparation of 7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one

N- (2-cyclopropyl-2-oxoethyl) -N- (2- (2, 4-dimethoxybenzyl) -1-oxo-1, 2-dihydroisoquinolin-7-yl) carboxamide (5.0g, crude product), ammonium acetate (7.5g,97.3mmol) were added to acetic acid (30mL), reacted at 105 ℃ for 16 hours, cooled to 0 ℃, adjusted to pH 7 with NaOH (2M), extracted with dichloromethane (150mL methane), the organic phases combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude product 5.1 g.

7.7 preparation of 7- (4-cyclopropyl-1H-imidazol-1-yl) isoquinolin-1 (2H) -one

7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one (5.1g, crude) was dissolved in trifluoroacetic acid (30mL) and reacted at 90 ℃ for 3 hours. Concentration, slurrying of the residue with ethyl acetate (50mL), filtration, and drying of the cake to obtain 2.2g of the objective product, in a three-step yield of 68.9%.

Preparation of 2- (6- ([1,2,4] triazolo [4,3-a ] pyridin-3-yl) pyridin-2-yl) -7- (4-cyclopropyl-1H-imidazol-1-yl) isoquinolin-1 (2H) -one

7- (4-cyclopropyl-1H-imidazol-1-yl) isoquinolin-1 (2H) -one (0.21g,0.84mmol), 3- (6-chloropyridin-2-yl) - [1,2, 4%]Triazolo [4,3-a]Pyridine (196mg,0.85mmol), cesium carbonate (0.55g,1.69mmol), Pd₂(dba)₃(46mg,0.05mmol) and Xantphos (29mg,0.05mmol) were added sequentially to dioxane (50mL) and reacted at 120 ℃ for 16 hours. The concentrate was purified by reverse phase C18 column (water/methanol 10% to 35%) to obtain the objective product (50mg, yield: 13.4%).

Molecular formula C₂₆H₁₉N₇O molecular weight 445.49 LC-MS (M/e):446.2(M + H)⁺)

¹H-NMR(400MHz,DMSO-d6)δ:9.47(d,J＝5.6Hz,1H),9.39(s,1H),8.55(s,1H),8.45(d,J＝6.8Hz,1H),8.20-9.30(m,1H),7.93-8.13(m,6H),7.52(s,1H),7.16(s,1H),6.95(d,J＝6.0Hz,1H),1.98(s,1H),0.99(m,2H),0.84(s,2H).

Example 87 preparation of- (4-cyclopropyl-1H-imidazol-1-yl) -4, 4-difluoro-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one (Compound 8)

Preparation of 1, 7- (4-cyclopropyl-1H-imidazol-1-yl) -4, 4-difluoro-3-hydroxy-3, 4-dihydroisoquinolin-1 (2H) -one

The compound 7- (4-cyclopropyl-1H-imidazol-1-yl) -4-fluoroisoquinolin-1 (2H) -one (269mg,1mmol) and 1-chloromethyl-4-fluoro-1, 4-diazobicyclo 2.2.2 octane bis (tetrafluoroborate) salt (709mg,2mmol) were dissolved in acetonitrile (10mL), and water (1mL) was added to react at 60 ℃ for 2 hours. After the reaction was completed, the reaction solution was concentrated, and the residue was prepared under medium pressure (0-50% methanol) to obtain the objective compound (122mg, yield 40.0%).

Preparation of 2, 7- (4-cyclopropyl-1H-imidazol-1-yl) -4, 4-difluoro-3, 4-dihydroisoquinolin-1 (2H) -one

7- (4-cyclopropyl-1H-imidazol-1-yl) -4, 4-difluoro-3-hydroxy-3, 4-dihydroisoquinolin-1 (2H) -one (122mg, 0.4mmol), triethylsilane (1mL), and trifluoroacetic acid (1mL) were added to a microwave tube, and reacted at 70 ℃ for 10 hours with a microwave. After the reaction was completed, the residue was purified by silica gel column chromatography (methanol: dichloromethane ═ 1:20) to obtain the objective compound (24mg, yield 20.8%). Preparation of 7- (4-cyclopropyl-1H-imidazol-1-yl) -4, 4-difluoro-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one

The compound 7- (4-cyclopropyl-1H-imidazol-1-yl) -4, 4-difluoro-3, 4-dihydroisoquinolin-1 (2H) -one (24mg, 0.083mmol), 2-bromo-6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridine (22mg, 0.083mmol), 2-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl (4mg, 20%), palladium acetate (2mg, 10%) and cesium carbonate (54mg, 0.166mmol) were dissolved in 1, 4-dioxane (5mL) and reacted at 110 ℃ for 2 hours under nitrogen atmosphere. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane: methanol ═ 20:1) to give the objective compound (8mg, yield 20.3%).

The molecular formula is as follows: c₂₅H₂₃F₂N₇O molecular weight: 475.5 LC-MS (M/e):238.5(M + H/2),475.9(M + H)

¹H-NMR(400MHz,CDCl₃)δ:8.40(s,1H),8.28(s,1H),8.20(d,J＝7.6Hz),8.12(d,J＝8.0Hz,1H),7.98(t,J＝8.0Hz,1H),7.85-7.93(m,2H),7.72-7.78(m,1H),7.15(s,1H),5.40-5.55(m,1H),4.65(t J＝12.0Hz,2H),1.90-1.98(m,1H),1.58-1.63(m,6H),0.85-0.98(m,4H)。

EXAMPLE 97- (4-cyclopropyl-1H-imidazol-1-yl) -5-fluoro-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one preparation (Compound 9)

9.17-bromo-5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one preparation

6-bromo-4-fluoro-2, 3-dihydro-1H-inden-1-one (5g,21.8mmol) was dissolved in a mixed solution of dichloromethane (60mL) and methanesulfonic acid (30mL), and sodium azide (2.1g,32.3mmol) was added at 0 ℃ to react at 20 ℃ for 2 hours. After the reaction was completed, water was slowly added at 0 ℃ to quench the reaction, then water (100mL) and dichloromethane (50mL) were added, the layers were separated, the aqueous layer was extracted with dichloromethane (50mL × 2), the organic layers were combined, concentrated, and the residue was purified by column chromatography (petroleum ether: ethyl acetate ═ 2:1) to give the product (2.4g, yield 45.3%).

9.22- ((benzyloxy) methyl) -7-bromo-5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one preparation

7-bromo-5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one (2g,8.2mmol) was dissolved in DMF (20mL), sodium hydride (656mg,16.4mmol) was added, and after stirring for one hour ((chloromethoxy) methyl) benzene (1.9g,12.2mmol) was added, and the reaction was carried out at 20 ℃ for 4 hours. After the reaction was completed, 100mL of water and 50mL of ethyl acetate were added, liquid separation was performed, the aqueous phase was extracted with ethyl acetate (50mL × 2), the organic phases were combined, concentrated, and the residue was subjected to column chromatography (petroleum ether: ethyl acetate ═ 10:1) to give the product (2.3g, yield 76.7%).

9.32- ((benzyloxy) methyl) -7- (4-cyclopropyl-1H-imidazol-1-yl) -5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one preparation

Dissolving 2- ((benzyloxy) methyl) -7-bromo-5-fluoro-3, 4-dihydroisoquinoline-1 (2H) -one (1.2g,3.3mmol) and cyclopropylimidazole (356mg,3.3mmol) in DMF (6mL), adding cuprous iodide (125mg,0.66mmol), phenanthroline (118mg,0.66mmol) and potassium carbonate (910mg,6.6mmol), and carrying out microwave reaction at 120 ℃ for 7 hours under the protection of nitrogen. After filtration, water (50mL) and ethyl acetate (50mL) were added to the filtrate, followed by liquid separation, the aqueous phase was extracted with ethyl acetate (50mL × 2), the organic phases were combined, concentrated, and the residue was subjected to column chromatography (petroleum ether: ethyl acetate ═ 2:1) to give the product (680mg, yield 52.6%).

9.47- (4-cyclopropyl-1H-imidazol-1-yl) -5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one preparation

2- ((benzyloxy) methyl) -7- (4-cyclopropyl-1H-imidazol-1-yl) -5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one (500mg,1.3mmol) was dissolved in TFA (20mL) and reacted at 80 ℃ for 36 hours. Concentration, addition of dichloro (50mL) methane and sodium bicarbonate solution (50mL) to the residue, liquid separation with stirring for half an hour, extraction of the aqueous phase with dichloromethane (20mL × 2), combination of the organic phases, concentration, and column chromatography of the residue (ethyl acetate: methanol ═ 20:1) gave the product (180mg, 51.1% yield).

Preparation of 57- (4-cyclopropyl-1H-imidazol-1-yl) -5-fluoro-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one

The reaction mixture was washed with 7- (4-cyclopropyl-1H-imidazol-1-yl) -5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one (100mg,0.37mmol), 2-bromo-6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridine (100mg,0.37mmol), cesium carbonate (241mg,0.74mmol), Pd (OAc)₂(8mg,0.037mmol) and Xphos (35mg,0.073mmol) were added to 1, 4-dioxane (10mL) and reacted at 110 ℃ under nitrogen for 16h, after completion of the reaction, filtration was carried out, the filtrate was concentrated and the residue was isolated by TLC to give the product (85mg, 50.3% yield).

The molecular formula is as follows: c₂₅H₂₄FN₇O molecular weight: 457.5 LC-MS (M/e): 458.2(M + H)⁺)

¹HNMR(400MHz,MeOD)δ:8.86(s,1H),8.11(s,1H),8.07-8.08(m,1H),8.03-8.05(m,2H),7.96-7.99(m,1H),7.67-7.70(m,1H),7.40(s,1H),5.48-5.52(m,1H),4.35(t,J＝6.4Hz,2H),3.23(t,J＝6.4Hz,2H),1.80-1.95(m,1H),1.59(d,J＝6.8Hz,1H),0.86-0.90(m,2H),0.74-0.78(m,2H)

Example 107- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one preparation (Compound 10)

10.1 preparation of 2- (3- (6-bromopyridin-2-yl) -4H-1,2, 4-triazol-4-yl) propan-1-ol

N' - (6-Bromopyridinoyl) -N, N-dimethylformylhydrazone amide (2.7g,10.0mmol) and acetic acid (10mL) were added to acetonitrile (40mL), DL-alaninol (3.76g,50.0mmol) was added, and the mixture was heated to 95 ℃ for reaction at room temperature for 6 hours. The system was adjusted to pH 8 with sodium hydroxide solution, EA (100mL) and saturated sodium chloride solution (80mL) were added, the organic phase was dried over anhydrous sodium sulfate, and the solvent was dried to give the product (1.4g, 49.5% yield).

Preparation of 2, 7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one

Mixing 7- (4-cyclopropyl-1H-imidazol-1-yl) -3, 4-dihydroisoquinolin-1 (2H) -one (200mg,0.79mmol), 2- (3- (6-bromopyridin-2-yl) -4H-1,2, 4-triazol-4-yl) propan-1-ol (223.7mg,0.79mmol), cesium carbonate (514.8mg,1.58mmol), Pd (OAc)₂(17.7mg,0.079mmol) and X-phos (37.7mg,0.079mmol) were added to 1, 4-dioxane (20mL), the reaction was stirred at 102 ℃ for 2 hours under nitrogen, the system was filtered with suction, and the filtrate was filtered through C₁₈Column chromatography (water: acetonitrile ═ 6:4) afforded the product (180mg, 50.1% yield).

The molecular formula is as follows: c₂₅H₂₅N₇O₂Molecular weight: 455.5 LC-MS (M/e): 456.3(M + H)⁺)

¹H-NMR(400MHz,DMSO)δ:8.88(s,1H),8.13-8.05(m,2H),8.05-7.96(m,1H),7.96-7.91(m,2H),7.78-7.71(m,1H),7.55-7.45(m,2H),5.36-5.28(m,1H),5.04-4.98(m,1H),4.23-4.18(m,2H),3.72-3.62(m,2H),1.90-1.78(m,1H),1.47-1.39(m,3H),0.82-0.75(m,2H),0.75-0.68(m,2H)。

Example 117- (4-cyclopropyl-1H-imidazol-1-yl) -6-fluoro-2- (6- (4- (1-hydroxypropan-2 yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one preparation (Compound 14)

11.1 preparation of methyl (3-fluorophenethyl) carbamate

2- (3-fluorophenyl) ethan-1-amine (10.0g,71.8mmol) and triethylamine (13.0mL,93.3mmol) were dissolved in dichloromethane (140mL), methyl chloroformate (6.1mL,79mmol) was added at 0 ℃, reacted at 0 ℃ for 1h, concentrated, and purified on silica gel column (petroleum ether: ethyl acetate ═ 2:1) to give the product (10.0g, yield: 71%).

Preparation of 2, 6-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one

To methyl (3-fluorophenethyl) carbamate (9.0g,46mmol) was slowly added trifluoromethanesulfonic acid (45mL,510mmol) at 0 deg.C, reacted at 70 deg.C for 25 hours, the reaction solution was poured into ice water (300mL), extracted with dichloromethane (100 mL. times.3), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated to give the crude product which was directly subjected to the next reaction.

Preparation of 3, 6-fluoro-7-nitro-3, 4-dihydroisoquinolin-1 (2H) -one

Slowly adding potassium nitrate (6.6g,65mmol) into a concentrated sulfuric acid solution (65mL) of 6-fluoro-3, 4-dihydroisoquinoline-1 (2H) -ketone (8.26g, crude product) at 0 ℃, reacting for 20 minutes at 0 ℃, pouring the reaction solution into ice water (200mL), filtering, washing (100mL) of a filter cake, pulping (20mL) with methanol, and performing suction filtration and drying to obtain the crude product to directly perform the next reaction.

Preparation of 4, 7-amino-6-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one

6-fluoro-7-nitro-3, 4-dihydroisoquinolin-1 (2H) -one crude product (5.0g,23.8mmol) and iron powder (6.7g,119mmol) were added to acetic acid (150mL) and tetrahydrofuran (70mL) and reacted at 70 ℃ for 4 hours. The filtrate was filtered and concentrated on silica gel column to give the product (6.5 g).

Preparation of 5, 7- ((2-cyclopropyl-2-oxoethyl) amino) -6-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one

7-amino-6-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one (5.8g,32.2mmol), 2-bromo-1-cyclopropylethanone (3.7g,38mmol) and DIEA (26.4mL,155.5mmol) were added sequentially to tetrahydrofuran (55mL) and toluene (250mL) and reacted at 100 ℃ for 16 hours. Concentration and silica gel column purification (petroleum ether: ethyl acetate: 3:1) gave the product (5.6g, yield: 66%).

11.6 preparation of N- (2-cyclopropyl-2-oxoethyl) -N- (6-fluoro-1-oxo-1, 2,3, 4-tetrahydroisoquinolin-7-yl) carboxamide

Acetic anhydride (778mg,7.6mmol) was dropped into formic acid (10mL) at 0 ℃ for 0.5 hour, and then a solution of 7- ((2-cyclopropyl-2-oxoethyl) amino) -6-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one (500mg,1.9mmol) in methylene chloride (10mL) was dropped into the reaction system to react at 0 ℃ for 16 hours. The pH was adjusted to 7 with NaOH (1M), dichloromethane (400mL) was extracted, the organic phases were combined, dried over anhydrous sodium sulfate and concentrated to give the crude product which was directly subjected to the next reaction.

11.7 preparation of 7- (4-cyclopropyl-1H-imidazol-1-yl) -6-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one

N- (2-cyclopropyl-2-oxoethyl) -N- (6-fluoro-1-oxo-1, 2,3, 4-tetrahydroisoquinolin-7-yl) carboxamide (500mg, crude), ammonium acetate (586mg,7.6mmol) were added to acetic acid (10mL), reacted at 110 ℃ for 16 hours, cooled to 0 ℃, adjusted pH 9 with NaOH (2M), extracted with ethyl acetate (500mL × 2), the organic phases combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude (247mg, 48% yield over two steps).

Preparation of 7- (4-cyclopropyl-1H-imidazol-1-yl) -6-fluoro-2- (6- (4- (1-hydroxypropan-2 yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one

Mixing 7- (4-cyclopropyl-1H-imidazol-1-yl) -6-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one (100mg,0.37mmol), 2- (3- (6-bromopyridin-2-yl) -4H-1,2, 4-triazol-4-yl) propan-1-ol (125mg,0.44mmol), cesium carbonate (240mg,0.74mmol), Pd (OAc)₂(9mg,0.04mmol) and X-phos (38mg,0.08mmol) were added to 1, 4-dioxane (20mL) and reacted at 110 ℃ under nitrogen for 16h, after completion of the reaction, filtration, concentration of the filtrate and TLC separation of the residue to give the crude product which was prepared in reverse phase (32mg, 18.3% yield).

The molecular formula is as follows: c₂₅H₂₄FN₇O₂Molecular weight: 473.5 LC-MS (M/e): 474.2(M + H)⁺)

¹H-NMR(400MHz,MeOD)δ:8.82(s,1H),8.19(d,J＝7.6Hz,1H),7.88-8.02(m,4H),7.41(d,J＝10.8Hz,1H),7.21(s,1H),5.45-5.51(m,1H),4.32(t,J＝6.4Hz,2H),3.85(d,J＝5.2Hz,1H),3.23-3.26(m,2H),1.87-1.93(m,1H),1.58(d,J＝6.8Hz,1H),0.86-0.91(m,2H),0.75-0.68(m,2H)。

EXAMPLE 12 preparation of (R) -7- (4-cyclopropyl-1H-imidazol-1-yl) -6-fluoro-2- (6- (4- (1-hydroxypropan-2-yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one (Compound 15)

Preparation of (R) -7- (4-cyclopropyl-1H-imidazol-1-yl) -6-fluoro-2- (6- (4- (1-hydroxypropan-2-yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one

The reaction mixture was washed with 7- (4-cyclopropyl-1H-imidazol-1-yl) -6-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one (100mg,0.37mmol), (R) -2- (3- (6-bromopyridin-2-yl) -4H-1,2, 4-triazol-4-yl) propan-1-ol (115mg,0.41mmol), cesium carbonate (240mg,0.74mmol), Pd (OAc)₂(8mg,0.04mmol) and Xphos (35mg,0.07mmol) were added to 1, 4-dioxane (10mL) and reacted at 110 ℃ under nitrogen for 16 hours, after completion of the reaction, filtration was carried out, the filtrate was concentrated, and the residue was purified by TLC (dichloromethane: methanol ═ 10:1) to give a compound (42mg, 24% yield).

Molecular formula：C₂₅H₂₄FN₇O₂Molecular weight: 473.5 LC-MS (M/e): 474.2(M + H)⁺)

¹HNMR(400MHz,MeOD)δ:8.83(s,1H),8.20(d,J＝7.6Hz,1H),7.96-7.98(m,3H),7.89(s,1H),7.42(d,J＝10.8Hz,1H),7.22(s,1H),5.45-5.51(m,1H),4.33(t,J＝6.4Hz,2H),3.85(d,J＝5.2Hz,1H),3.23-3.26(m,2H),1.87-1.93(m,1H),1.58(d,J＝6.8Hz,1H),0.86-0.91(m,2H),0.75-0.78(m,2H)。

EXAMPLE 13 preparation of (R) -7- (4-cyclopropyl-1H-imidazol-1-yl) -4, 4-difluoro-2- (6- (4- (1-hydroxypropan-2-yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) -3, 4-dihydroisoquinolin-1 (2H) -one (Compound 20)

13.1 Synthesis of (R) -7- (4-cyclopropyl-1H-imidazol-1-yl) -4, 4-difluoro-2- (6- (4- (1-hydroxypropan-2-yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) -34-dihydroisoquinolin-1 (2H) -one

The compound 7- (4-cyclopropyl-1H-imidazol-1-yl) -4, 4-difluoro-3, 4-dihydroisoquinolin-1 (2H) -one (46mg, 0.159mmol), (R) -2- (3- (6-bromopyridin-2-yl) -4H-1,2, 4-triazol-4-yl) propan-1-ol (45mg, 0.159mmol), 2-dicyclohexylphosphonium-2, 4, 6-triisopropylbiphenyl (10mg, 20%), palladium acetate (5mg, 10%) and cesium carbonate (104mg, 0.318mmol) were dissolved in 1, 4-dioxane (10mL) and reacted at 110 ℃ for 18 hours under nitrogen protection. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (dichloromethane: methanol ═ 15:1) to give the objective compound (8mg, yield 10.2%).

The molecular formula is as follows: c₂₅H₂₃F₂N₇O₂Molecular weight: 491.5 LC-MS (M/e):246.7(M + H/2),492.2(M + H)

¹H-NMR(400MHz,MeOD)δ:8.85(s,1H),8.36(s,1H),8.21(d,J＝1.2Hz,1H),8.06-8.15(m,2H),8.00-8.05(m,2H),7.94-7.98(m,1H),7.48(s,1H),5.45-5.55(m,1H),4.78(t,J＝12.0Hz,2H),3.87(d,J＝4.8Hz,2H),1.89-1.96(m,1H),1.60(d,J＝6.8Hz,3H),0.75-0.95(m,4H)。

Preparation example 147- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4- (1-hydroxypropan-2-yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one preparation (Compound 21)

Preparation of 1, 7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4- (1-hydroxypropan-2-yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one

7- (4-cyclopropyl-1H-imidazol-1-yl) isoquinolin-1 (2H) -one (0.2g,0.79mmol), 2- (3- (6-bromopyridin-2-yl) -4H-1,2, 4-triazol-4-yl) propan-1-ol (225mg,0.79mmol), potassium carbonate (0.21g,1.6mmol), cuprous iodide (75mg,0.39mmol) and 8-hydroxyquinoline (57mg,0.39mmol) were added sequentially to DMSO (8mL), N₂The reaction is carried out for 16 hours at the protection temperature of 100 ℃. The reaction was poured into water (50mL), filtered and the filter cake was TLC separated (DCM: MeOH ═ 10:1) to give the product (80mg, yield: 22.3%).

Molecular formula C₂₅H₂₃N₇O₂Molecular weight 453.51 LC-MS (M/e):454.2(M + H)⁺)

¹H-NMR(400MHz,DMSO)δ:8.82(s,1H),8.35(d,J＝2.0Hz,1H),8.25(s,1H),8.16-8.20(m,2H),8.05-8.08(m,1H),7.97-7.99(dd,J＝2.0Hz,J＝7.2Hz,1H),7.89(d,J＝8.8Hz,1H),7.82(d,J＝7.2Hz,1H),7.64(s,1H),6.86(d,J＝7.6Hz,1H),5.26(q,J＝6.8Hz,1H),5.00(t,J＝5.6Hz,1H),3.67(t,J＝5.6Hz,2H),1.83-1.87(m,1H),1.43(d,J＝6.8Hz,3H),0.78-0.83(m,2H),0.69-0.73(m,2H)。

EXAMPLE 15 preparation of (R) -7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4- (1-hydroxypropan-2-yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one (Compound 22)

15.1 preparation of (R) -7- (4-cyclopropyl-1H-imidazol-1-yl) -2- (6- (4- (1-hydroxypropan-2-yl) -4H-1,2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one

7- (4-cyclopropyl-1H-imidazol-1-yl) isoquinolin-1 (2H) -one (0.18g,0.72 mmo)l), (R) -2- (3- (6-bromopyridin-2-yl) -4H-1,2, 4-triazol-4-yl) propan-1-ol (202mg,0.72mmol), potassium carbonate (0.3g,2.1mmol), cuprous iodide (70mg,0.37mmol) and 8-hydroxyquinoline (54mg,0.37mmol) were added sequentially to DMSO (8mL), N₂The reaction is carried out for 16 hours at the protection temperature of 100 ℃. The reaction was poured into water (50mL), filtered and the filter cake was TLC separated (DCM: MeOH ═ 8:1) to give the product (50mg, yield: 15.3%).

¹H-NMR(400MHz,DMSO)δ:8.71(s,1H),8.35(d,J＝2.0Hz,1H),8.24(s,1H),8.16-8.22(m,2H),8.05-8.08(m,1H),7.96-7.99(dd,J＝1.6Hz,J＝5.1Hz,1H),7.88(d,J＝8.8Hz,1H),7.82(d,J＝7.2Hz,1H),7.64(s,1H),6.86(d,J＝7.6Hz,1H),5.26(q,J＝6.0Hz,1H),5.00(t,J＝5.2Hz,1H),3.67(t,J＝5.6Hz,2H),1.83-1.87(m,1H),1.43(d,J＝6.4Hz,3H),0.78-0.83(m,2H),0.69-0.73(m,2H).

The beneficial effects of the compounds of the present invention are further illustrated below by in vitro enzymology and cytostatic activity experiments and pharmacokinetic experiments, but this should not be understood as the only beneficial effects of the compounds of the present invention.

Biological Experimental example

Experimental example 1: in vitro enzymatic Activity of Compounds of the invention

And (3) testing the sample: the structural formula of the compound synthesized in the embodiment of the invention is shown in table 1.

Experimental materials:

preparing a reagent:

kinase detection buffer (kinase detection buffer)

a. Unfreezing the kinase detection buffer to room temperature, and observing whether a precipitate exists or not;

b. if a precipitate is present, the kinase detection buffer is incubated at 37 ℃ and vortexed for 15min to dissolve the precipitate, or the precipitate is removed from the kinase detection buffer.

Kinase detection reagent

a. Equilibrating kinase detection buffer and kinase detection substrate to room temperature;

b. transferring all volume kinase detection buffers to a kinase detection subsystem to form a kinase detection reagent;

c. vortex, mix well to form a homogeneous solution, and the kinase detection substrate is added to the solution for at least 1 min.

The experimental method comprises the following steps:

1. dilution of Compounds

a. Staurosporine was diluted to 1mM, the compound of the invention to 1mM, using DMSO as a stock solution.

b. Staurosporine and the compound stock solution of the invention were diluted 1000-fold to 1 μ M.

c. Staursporine was diluted 3-fold in gradient to 11 concentrations with a maximum concentration of 1 μ M and the compound of the invention was diluted 3-fold in gradient to 11 concentrations with a maximum concentration of 1 μ M using the TECAN EVO2000 system.

d. Echo550 was used to transfer 20nLStaurosporine, a compound of the present invention, respectively, to 384 well plates.

2. Experiment of enzyme reaction

1) A1.3 Xenzyme reaction system comprising a kinase, a substrate and factors required for the reaction was prepared as shown in Table 2.

2) To the reaction well, 15. mu.L of 1.3 Xenzyme reaction system was added and incubated at room temperature for 30 minutes.

TABLE 2.1.3 Xenzyme reaction System

3) The enzyme reaction was initiated by adding 5. mu.L of 4 × ATP solution (see Table 3) to each well.

TABLE 3.4 XATP solution

4) After the reaction system is placed at 25 ℃ for 90 minutes, add20 μ L ADP-Glo^TMReagent was incubated for 40 minutes.

5) Incubation for 35 min with 40. mu.L of Kinase Detection Regent, EnSpire^TMThe fluorescence was read by a microplate reader.

3. Data analysis

The Remaining enzyme Activity (% Remaining Activity) was calculated using the following formula:

Remaining Activity(％)＝100％×(Lum_cpd-Lum_LC)/(Lum_HC-Lum_LC)

wherein, Lum_HCRepresents: the intensity of a luminescent signal of High control (the reaction system contains DMSO at the same concentration as the compound assay);

Lum_LCrepresents: luminescence signal intensity of Low control (Staursporine 1. mu.M);

Lum_cpdrepresents: indicating the intensity of the luminescent signal of the test compound;

curve fitting is carried out by adopting XLFIT software to obtain IC₅₀。

The experimental results are as follows:

TABLE 4 inhibitory Activity of the Compounds of the invention on ASK1

From the experimental results, the compound prepared by the invention can effectively inhibit the phosphorylation of the amino acid of ASK1 and inhibit the activation of ASK1, and is an effective ASK1 inhibitor.

In addition, the prior art has reported that ASK1 inhibitors have preventive and/or therapeutic effects on ASK 1-mediated diseases and related diseases; namely, the compound shown in the general formula (I), (II), (III), (IV) or (V) can prevent and/or treat the ASK1 mediated diseases and related diseases.

Experimental example 2 in vitro cytological Activity of Compounds of the invention

And (3) testing the sample: the chemical name and the structural formula of the compound synthesized in the embodiment of the invention are shown in table 1.

Experimental materials:

wherein: cell culture medium composition: DMEM + 10% FBS +1 XPS

The experimental method comprises the following steps:

1. transfection

1) 25. mu.L of the transfection reagent Lipofectamine2000 was mixed with 0.7mL of Opti-MEM medium, and 0.7mL of Opti-MEM medium was used to dilute the plasmid DNA (pGL4.44(luc2P-AP1-RE-Hygro, AP1 for short) and pcDNA3.1/pcDNA3.1-hASK1), followed by incubation at room temperature for 5 min.

2) After 5min incubation, the plasmid DNA mixture was added to the transfection reagent Lipo2000 mixture, mixed well and incubated for 20min at room temperature.

3) Adding the mixed solution into a culture dish containing HEK-293T cells for transfection, and placing the culture dish at 37 ℃ and 5% CO₂Incubate in incubator for 16 h.

2. Test Compound dilution and cell plating

1) Diluting the test compound to 10mM stock solution with DMSO, and diluting the stock solution to 10 concentrations with a 3-fold gradient using a TECAN EVO2000 system;

2) transferring 30nL of each concentration of test compound into 384-well cell culture plates using Echo 550;

3) the transfected cells were collected, counted, and according to the result of cell counting, the cells were diluted with a medium to a desired density, and then 30. mu.L of a cell suspension (about 10000 cells) was added to a 384-well cell culture plate containing the compound to be tested, and the cell culture plate was placed at 37 ℃ with 5% CO₂Incubate in incubator for 24 h.

3. Detection of

1) Taking the 384-well cell culture plate of the step 2.3 out of the incubator, and balancing for 15min at room temperature;

2) before the experiment, unfreezeLuciferase reagent, and equilibrating to room temperature;

3) adding 30 μ LAnd (3) incubating the Luciferase reagent to a 384-well plate at room temperature for 10min, and reading the plate by using a microplate reader.

4. Data analysis

1) The Remaining Activity (% Remaining Activity) was calculated using the following formula:

Remaining Activity(％)＝100％×(Lum_sample-Lum_NC)/(Lum_PC-Lum_NC)

wherein, Lum_PC: average values of the luminescence signal values of wells co-transfected with ASK1 and AP 1;

Lum_NC: average of values of the luminescence signal from wells transfected with AP 1;

Lum_sample: means for representing the average of the luminescence signal values of the test compound;

2) curve fitting is carried out by adopting XLFIT software to obtain IC₅₀The value is obtained.

The experimental results are as follows:

TABLE 5 in vitro cytological Activity of Compounds of the invention

As can be seen from the above experimental results, in vitro cell experiments, the compound of the present invention can effectively block the phosphorylation of ASK1 in cells and block the activation of downstream pathways, and thus, the compound of the present invention can be effectively used for treating related diseases mediated by ASK 1.

Experimental example 3: c57BL/6 mouse in vivo pharmacokinetic experiments with Compounds of the invention

And (3) testing the sample: the chemical name and the preparation method of part of the compound of the invention, self-made, are shown in the preparation examples of each compound.

The test animals were: male mice (C57BL/6), 9/route of administration/compound, weight 22-26 g/mouse.

Preparing a test solution:

dissolution scheme:

compound 1 IV administration: 2% DMSO + 10% PEG400+ 88% (28% HP-beta-CD)

Compound 1 PO administration: 0.1% SDS + 0.5% MC

Compound 2 IV administration: 2% DMSO + 10% PEG400+ 88% (28% HP-beta-CD)

Compound 2 PO administration: 2% HPC + 0.1% Tween80

Compound 3 IV administration: 3% DMSO + 5% PEG400+ 92% (28% HP-beta-CD)

Compound 3 PO administration: 2% HPC + 0.1% Tween80

Preparation of a blank solvent 1:

the preparation method of the 28 percent HP-beta-CD solution comprises the following steps: HP- β -CD (hydroxypropyl betacyclodextrin) (28g) was weighed and sterile water for injection (72mL) was added to give a 28% solution of HP- β -CD.

Preparation of a blank solvent 2:

preparation of 2% HPC + 0.1% tween 80: HPC (hydroxypropyl cellulose) (30g) was weighed, slowly added to stirred purified water (1470mL), and Tween80 (1.5mL) was added, stirred until clear.

Preparation of a blank solvent 3:

preparation of 0.5% MC: weighing 5g of MC (methyl cellulose), slowly adding 500mL of stirred purified water, stirring until the mixture is clear and transparent, then adding purified water to a constant volume of 1000mL, and stirring uniformly.

The preparation method comprises the following steps:

compound 1:

weighing the compound 1(1.48mg), adding DMSO (dimethyl sulfoxide) (48.4 mu L), carrying out ultrasonic dissolution, adding PEG400 (polyethylene glycol 400) (242.2 mu L), carrying out vortex mixing, finally adding a blank solvent 1(2.131mL), carrying out vortex mixing, preparing a colorless transparent solution with the concentration of 0.6mg/mL, and taking the solution as compound 1C57BL/6 mouse IV administration liquid medicine 1;

compound 1(1.51mg) and SDS (sodium dodecyl sulfate) (4.9mg) were weighed, added to blank vehicle 3(4.943mL), ground, vortexed and mixed to prepare a homogeneous suspension solution with a concentration of 0.3mg/mL, which was used as PO administration solution 2 of compound 1C57BL/6 mouse.

Compound 2:

weighing a compound 2(1.50mg), adding DMSO (dimethyl sulfoxide) (47.0 mu L), carrying out vortex and ultrasonic dissolution, adding PEG400 (polyethylene glycol 400) (233 mu L), carrying out vortex mixing, finally adding a blank solvent 1(2.05mL), carrying out vortex mixing, placing in a constant-temperature water bath kettle at 50 ℃ for heat preservation for 20min, carrying out vortex mixing, preparing into a uniform solution with the concentration of 0.6mg/mL, and taking the solution as a compound 2C57BL/6 mouse IV administration liquid medicine 1;

compound 2(1.51mg) was weighed, added to blank vehicle 2(4.691mL), ground, suspended uniformly, and prepared into a uniform suspension solution with a concentration of 0.3mg/mL, which was used as PO administration solution 2 of compound 2C57BL/6 mouse.

Compound 3:

weighing the compound 3(1.57mg), adding DMSO (dimethyl sulfoxide) (72.6 mu L), carrying out vortex and ultrasonic dissolution, then adding PEG400 (polyethylene glycol 400) (121 mu L), carrying out vortex mixing, finally adding a blank solvent 1(2.230mL), carrying out vortex mixing, preparing into a uniform solution with the concentration of 0.6mg/mL, and taking the solution as compound 3C57BL/6 mouse IV administration liquid medicine 1;

compound 3(1.50mg) was weighed, added to blank vehicle 2(4.620mL), ground, suspended uniformly, and prepared into a uniform suspension solution with a concentration of 0.3mg/mL, which was used as PO administration solution 2 for compound 3C57BL/6 mice.

Experimental methods

Administration:

the test liquid medicine is administered according to the following method:

blood collection:

collecting time points: 0.083, 0.25, 0.5, 1,2,4, 6, 8, 24h after administration

Approximately 100. mu.L of blood was collected from the canthus at each time point and placed in the eye containing EDTA-K₂In the anticoagulant tube of the anticoagulant agent,centrifuging the blood sample at 4 deg.C for 6min at 8000 rpm to obtain plasma sample, and freezing the plasma in-80 deg.C refrigerator.

Plasma sample analysis

Plasma samples of control and compound were analyzed by protein precipitation: taking 20 mu L of plasma, adding 200 mu L of internal standard (acetonitrile solution containing 50ng/mL of tolbutamide), vortexing for 10min, then centrifuging for 20min at 4000 rpm, taking 100 mu L of supernatant, adding 100 mu L of water, vortexing and uniformly mixing for 3min, and then carrying out LC-MS/MS analysis.

The experimental results are as follows:

table 6: c57BL/6 mouse PK evaluation results (IV)

Table 7: c57BL/6 mouse PK evaluation results (PO)

AUC_infArea 0 → ∞ under curve when drug is represented

CL stands for clearance

V_ssRepresenting steady state apparent distribution volume

T_maxRepresenting the time to peak of blood concentration

C_maxRepresenting peak plasma concentration

T_1/2Represents the half-life

F% represents absolute bioavailability

As shown in the experimental results in tables 6 and 7, the compound of the present invention has high exposure and high bioavailability, shows good pharmacokinetic properties, has good clinical application prospects, and is convenient for preparation of various clinical dosage forms.

Experimental example 4 in vivo pharmacokinetic experiment of beagle dog with the Compound of the present invention

And (3) testing the sample: the chemical name and the preparation method of the compound 2 are shown in the preparation examples of the compound.

The test animals were: male beagle dogs, control: 3 per administration route, body weight 8-11kg per body; a compound: 3/administration route, body weight 7-11 kg/body.

Preparing a test solution:

dissolution scheme:

compound IV administration: 5% DMSO + 20% PEG400+ 75% (28% HP-beta-CD)

Compound PO administration: 2% HPC + 0.1% Tween80

Preparation of a blank solvent 1:

Preparation of a blank solvent 2:

The preparation method comprises the following steps:

compound 2:

weighing a compound 2(40.49mg), adding DMSO (dimethyl sulfoxide) (1.99mL), carrying out vortex dissolution, adding PEG400 (polyethylene glycol 400) (7.96mL), carrying out vortex mixing, adding a blank solvent 1(29.85mL), carrying out vortex mixing, and preparing a colorless transparent solution with the concentration of 1mg/mL, wherein the solution is used as a compound beagle IV administration liquid medicine 1;

② weighing the compound 2(80.37mg), adding blank solvent 2(197.7mL), grinding, suspending evenly, preparing even suspension with concentration of 0.4mg/mL, and taking the solution as compound 2 beagle PO administration liquid medicine 2.

Experimental methods

Administration:

the test liquid medicine is administered according to the following method:

blood collection:

collecting time points:

compound IV administration group: 0.083, 0.25, 0.5, 1,2,4, 6, 8, 24 hours post-administration;

compound PO dosing group: 0.167, 0.5, 1,2,4, 6, 8, 24h after administration;

approximately 400 μ L of blood was collected via the forelimb vein at each time point and placed in the presence of EDTA-K₂Centrifuging the blood sample at 8000 rpm at 4 deg.C for 6min in an anticoagulant tube to obtain a plasma sample, and freezing the plasma in a refrigerator at-80 deg.C.

Plasma sample analysis

Plasma samples of compound 2 were analyzed by protein precipitation: taking 20 mu L of plasma, adding 200 mu L of internal standard (acetonitrile solution containing 50ng/mL of tolbutamide), vortexing for 10min, then centrifuging for 20min at 4000 rpm, taking 100 mu L of supernatant, adding 100 mu L of water, vortexing and uniformly mixing for 3min, and then carrying out LC-MS/MS analysis.

The experimental results are as follows:

table 8: beagle PK evaluation results (IV)

AUC_infArea 0 → ∞ under curve when drug is represented

CL stands for clearance

V_ssRepresenting steady state apparent distribution volume

T_maxRepresenting the time to peak of blood concentration

C_maxRepresenting peak plasma concentration

T_1/2Represents the half-life

F% represents absolute bioavailability

As can be seen from the experimental results in tables 8 and 9, after the compound is administered in beagle dogs in IV and PO, the results show that the compound of the present invention has good pharmacokinetic properties, higher exposure, longer half-life, and higher bioavailability when orally administered.

The combined-ring ASK1 inhibitor and the application thereof provided by the invention are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its central concept. It should be noted that it would be apparent to those skilled in the art that various changes and modifications can be made in the invention without departing from the principles of the invention, and such changes and modifications are intended to be covered by the appended claims.

Claims

1. A compound of formula (I), a pharmaceutically acceptable salt, ester or stereoisomer thereof:

wherein, X, X¹、X²、X³、X⁴、X⁵Each independently selected from C, CR⁸Or N;

each Q¹Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, 3-to 8-membered cycloalkyl, 3-to 8-membered heterocyclyl, phenyl, phenoxy, -O-R⁸、-C(O)-R⁸、-OC(O)-R⁸、-C(O)-O-R⁸、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹) or-C (O) -N (R)⁸)(R⁹) In which C is_1-6Alkyl, 3-8 membered cycloalkyl, 3-8 membered heterocyclyl, phenyl, phenoxy optionally substituted by one or more C_1-6Alkyl, 3-8 membered cycloalkyl, C_1-6Alkoxy, hydroxy, amino, carboxy or halogen substitution;

or R¹And X¹In conjunction therewithThe attached N is bonded to form a heterocyclyl, aryl or heteroaryl group, which is optionally substituted with one or more alkyl, hydroxy or halogen;

each Q²Independently selected from C_1-6Alkyl, 3-8 membered cycloalkyl, 3-8 membered cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, haloC_1-6Alkoxy radical, C_1-6Alkylamino, oxo, -O-R⁸、-O-C(O)-R⁸、-O-C(O)-N(R⁸)(R⁹)、-S-R⁸、-N(R⁸)(R⁹)、-S(＝O)-R⁸、-S(＝O)₂R⁸、-O-S(＝O)₂R⁸、-S(＝O)₂-N(R⁸)(R⁹)、-S(＝O)₂-OR⁸、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹)、-C(O)-R⁸、-C(O)-O-R⁸、-C(O)-N(R⁸)-R⁹or-N (R)⁸)-S(＝O)₂-R⁹Wherein said C is_1-6Alkyl, 3-8 membered cycloalkyl, aryl, heteroaryl or heterocyclyl is optionally substituted with one or more halogen, hydroxy, amino, cyano, nitro, carboxy, oxo, C_1-6Alkyl or-O-R⁸Substitution;

selected from single bonds or double bonds.

2. The compound of claim 1, a pharmaceutically acceptable salt, ester, or stereoisomer thereof: wherein,

X²、X³、X⁴、X⁵at least one is selected from N;

each Q¹Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C_1-6Alkyl, halo C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino, 3-to 8-membered cycloalkyl, 3-to 8-membered heterocyclyl, phenyl, phenoxy, -O-R⁸、-C(O)-R⁸、-OC(O)-R⁸、-C(O)-O-R⁸、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹) or-C (O) -N (R)⁸)(R⁹)；

each Q²Independently selected from C_1-6Alkyl, 3-8 membered cycloalkyl C_1-6Alkyl, 6-14 membered aryl C_1-6Alkyl, 5-14 membered heteroaryl C_1-6Alkyl, 3-12 membered heterocyclic group C_1-6Alkyl, halogen, cyano, nitro, halogeno C_1-6Alkoxy, oxo, -O-R⁸、-O-C(O)-R⁸、-O-C(O)-N(R⁸)(R⁹)、-S-R⁸、-N(R⁸)(R⁹)、-S(＝O)-R⁸、-S(＝O)₂R⁸、-O-S(＝O)₂R⁸、-S(＝O)₂-N(R⁸)(R⁹)、-S(＝O)₂-OR⁸、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹)、-C(O)-R⁸、-C(O)-O-R⁸、-C(O)-N(R⁸)-R⁹or-N (R)⁸)-S(＝O)₂-R⁹Wherein said C is_1-6Alkyl, 3-8 membered cycloalkyl, 6-14 membered aryl, 5-14 membered heteroaryl or 3-12 membered heterocyclyl is optionally substituted with one or more halogen, hydroxy, amino, cyano, nitro, carboxy, oxo, C_1-6Alkyl or-O-R⁸And (4) substitution.

3. The compound, pharmaceutically acceptable salt thereof, ester thereof, or stereoisomer thereof according to any one of claims 1 or 2, having a structure represented by general formula (II),

wherein,

X、X¹each independently selected from C, CR⁸Or N;

Or R¹And X¹Together with the N to which they are attached form a 5-8 membered heterocyclyl, 6-10 membered aryl or 5-8 membered heterocychcAryl, said 5-8 membered heterocyclyl, 6-10 membered aryl or 5-8 membered heteroaryl optionally substituted with one or more C_1-6Alkyl, hydroxy or halogen substitution;

R⁵selected from optionally substituted by one or more Q²Substituted C_1-6Alkyl radical, C_1-6Alkylamino radical, C_1-6Alkoxy, 6-8 membered aryl, 5-10 membered heteroaryl, 3-8 membered cycloalkyl or 3-8 membered heterocyclyl;

each Q²Independently selected from C_1-6Alkyl, 3-8 membered cycloalkyl C_1-6Alkyl, 3-8 membered heterocyclic group C_1-6Alkyl, halo C_1-6Alkoxy, halogen, cyano, nitro, -O-R⁸、-O-C(O)-R⁸、-O-C(O)-N(R⁸)(R⁹)、-S-R⁸、-N(R⁸)(R⁹)、-S(＝O)-R⁸、-S(＝O)₂R⁸、-O-S(＝O)₂R⁸、-S(＝O)₂-N(R⁸)(R⁹)、-S(＝O)₂-OR⁸、-N(R⁸)-C(O)-R⁹、-N(R⁸)-C(O)-O-R⁹、-N(R⁸)-C(O)-N(R⁸)(R⁹)、-C(O)-R⁸、-C(O)-O-R⁸、-C(O)-N(R⁸)-R⁹or-N (R)⁸)-S(＝O)₂-R⁹Wherein said C is_1-6Alkyl, 3-8 membered cycloalkyl, 3-8 membered heterocyclyl optionally substituted with one or more halogens,Hydroxy, amino, cyano, nitro, carboxy, oxo, C_1-6Alkyl or-O-R⁸Substitution;

selected from single bonds or double bonds.

4. The compound, pharmaceutically acceptable salt, ester or stereoisomer thereof according to any one of claims 1-3, having a structure represented by formula (III),

wherein,

R¹selected from the group consisting of optionally substituted 1-3Q¹Substituted C_1-6Alkyl radical, C_1-6Alkoxy, 3-6 membered cycloalkyl or 3-8 membered heterocyclyl, each Q¹Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C_1-6Alkyl radical, C_1-6Alkoxy radical, C_1-6Alkylcarbonyl group, C₁-₆Alkoxycarbonyl group, C_1-6Alkylamido, aminoacyl, C_1-6Alkylaminoacyl, di (C)_1-6Alkyl) aminoacyl, halo C_1-6Alkoxy radical, C_1-6Alkylamino radical, di (C)_1-6Alkyl) amino or 3-8 membered cycloalkyl; r²、R⁴、R⁶、R⁷Each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxyl and C_1-6Alkyl radical, C_1-6Alkoxy, halo C_1-6Alkyl, halo C_1-6Alkoxy, hydroxy C_1-6Alkyl, hydroxy C_1-6Alkoxy or C_1-6An alkylamino group;

selected from single bonds or double bonds.

5. The compound of any one of claims 1-4, a pharmaceutically acceptable salt, ester, or stereoisomer thereof,

wherein,

R¹selected from optionally 1-2Q¹Substituted methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, or morpholinyl, each Q¹Independently selected from halogen, cyano, hydroxyAmino, nitro, carboxyl, methyl, ethyl, methoxy, ethoxy, propoxy, isopropoxy, methoxycarbonyl, ethoxycarbonyl, methylamino, ethylamino, dimethylamino, diethylamino, aminoacyl, methylaminoacyl, ethylacylo, dimethylaminyl, ethylacylo;

R²、R⁴、R⁶、R⁷each independently selected from hydrogen, fluoro, bromo, chloro, iodo, hydroxy, amino, cyano, nitro, carboxy, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, trifluoromethyl, trifluoroethyl, difluoromethoxy, or trifluoromethoxy;

each Q²Independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, methoxy, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, wherein said methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl are further optionally further coated with1-3 halogen, hydroxyl, amino, cyano, nitro, carboxyl, methyl, ethyl, propyl and isopropyl.

6. The compound of any one of claims 1-3, a pharmaceutically acceptable salt, ester, or stereoisomer thereof, having a structure according to formula (IV):

wherein,

7. The compound of claim 6, a pharmaceutically acceptable salt, ester, or stereoisomer thereof,

wherein,

each Q²Independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, methoxy, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, wherein said cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl are further optionally substituted with 1-2 halogens, hydroxy, amino, cyano, nitro, carboxy, methyl, ethyl, propyl or isopropyl.

8. The compound of any one of claims 1-3, a pharmaceutically acceptable salt, ester, or stereoisomer thereof, having a structure represented by formula (V):

wherein,

9. The compound of claim 8, a pharmaceutically acceptable salt, ester or stereoisomer thereof,

wherein,

R²、R⁴、R⁶、R⁷each independently selected from hydrogen, fluorine, bromine, chlorine, iodine, hydroxyl, amino, cyano, nitro, carboxyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, hydroxyl, carboxyl, amino, cyano, amino, cyano, nitro, carboxyl, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy,tert-butoxy, trifluoromethyl, trifluoroethyl, or trifluoromethoxy;

10. The compound of any one of claims 1-9, a pharmaceutically acceptable salt, ester, or stereoisomer thereof, selected from:

。

11. a process for the preparation of a compound of formula (I), wherein the process comprises:

(1) reacting a compound shown as a general formula (I-1) with a compound shown as a general formula (I-2) to obtain a compound shown as a general formula (I);

or (2) reacting the compound represented by the general formula (I-3) with the compound represented by the general formula (I-2) to obtain a compound represented by the general formula (I-4); the nitro group in the compound shown in the general formula (I-4) is subjected to functional group conversion reaction to obtain a compound shown in the general formula (I);

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、n、X、X¹、X²、X³、X⁴、X⁵as defined in any of claims 1 to 10, substituent a is selected from leaving groups, such as halogen.

12. A method for synthesizing a compound of formula (III), wherein the method comprises:

(1) reacting the compound shown in the general formula (III-1) with the compound shown in the general formula (III-2) to obtain a compound shown in the general formula (III);

or (2) reacting the compound shown in the general formula (III-3) with the compound shown in the general formula (III-2) to obtain a compound shown in a general formula (III-4); the nitro group in the compound shown in the general formula (III-4) is subjected to functional group conversion reaction to obtain a compound shown in the general formula (III);

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷N is as defined in any one of claims 4 to 10, and substituent a is selected from a leaving group, such as halogen.

13. A compound of the formula having the formula:

14. A pharmaceutical composition comprising a compound of any one of claims 1-10, a pharmaceutically acceptable salt, ester, or stereoisomer thereof, and optionally one or more second therapeutically active agents; preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers and/or diluents.

15. A pharmaceutical formulation comprising a compound according to any one of claims 1 to 10, a pharmaceutically acceptable salt, ester or stereoisomer thereof and one or more pharmaceutically acceptable carriers and/or diluents; the pharmaceutical preparation is any clinically or pharmaceutically acceptable dosage form, preferably an oral preparation or an injection; more preferably, the pharmaceutical formulation further comprises one or more second therapeutically active agents.

16. Use of a compound of any one of claims 1-10, a pharmaceutically acceptable salt, ester or stereoisomer thereof, or a pharmaceutical composition of claim 14, or a pharmaceutical formulation of claim 15, in the manufacture of a medicament for the treatment and/or prevention of ASK 1-mediated diseases and related disorders.

17. The use of claim 16, said ASK1 mediated and related diseases are selected from cardiovascular diseases including but not limited to pulmonary hypertension, multiple sclerosis, atherosclerosis, angina pectoris, intermittent claudication syndrome, myocardial infarction, heart failure; selected from pulmonary diseases including but not limited to asthma, chronic obstructive pulmonary disease, bronchitis, and emphysema; selected from metabolic disorders including but not limited to diabetes and its complications, metabolic syndrome, dyslipidemia, obesity, glucose intolerance, hypertension, high serum cholesterol and higher triglycerides; hepatobiliary kidney diseases, including but not limited to diabetic nephropathy, non-alcoholic fatty liver disease, bile acid disorders, primary sclerosing cholangitis, cholesterol stones, fatty liver, cirrhosis, hepatitis, liver failure, cholestasis, cholelithiasis; hyperproliferative diseases include, but are not limited to, gastric cancer, liver cancer, polyposis, colon cancer, breast cancer, pancreatic cancer, esophageal cancer; other diseases, including neurodegenerative diseases, fibrosis-related diseases, stroke, inflammatory bowel diseases, autoimmune disorders.