CN110294742B

CN110294742B - Fused ring ASK1 inhibitor and application thereof

Info

Publication number: CN110294742B
Application number: CN201810790218.XA
Authority: CN
Inventors: 王听中; 陈博; 朱鹏; 刘斌
Original assignee: Xuanzhu Pharma Co Ltd
Current assignee: Shandong Xuanzhu Pharma Co Ltd
Priority date: 2018-03-21
Filing date: 2018-07-18
Publication date: 2023-01-31
Anticipated expiration: 2038-07-18
Also published as: CN110294742A

Abstract

The invention relates to a fused ring ASK1 inhibitor and application thereof, in particular to a compound shown in a general formula (I), and pharmaceutically acceptable salt, ester or a stereoisomer thereof. The invention also relates to a preparation method of the compound, a pharmaceutical preparation containing the compound and a pharmaceutical composition. The compound can effectively inhibit the amino acid phosphorylation of ASK1 and inhibit the activation of ASK 1; thus, can treat and/or prevent ASK 1-mediated diseases and related diseases.

Description

Fused 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 (ASK 1) is a member of the MAPKKK family, and is an oligomer-like compound in which C-helical regions are linked, and N-helical regions are linked with thioredoxin (Trx), which 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: thr 845) of ASK1 is subjected to autophosphorylation, so that MAPKK (such as MAPKK3/MAPKK6, MAPKK4/MAPKK 7) 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.

Currently, only GS-4997 of Gilead is in clinical stage as the 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 the content of the first and second substances,

X、X ¹ 、X ² 、X ³ 、X ⁴ 、X ⁵ each independently selected from C and CR ⁸ Or N;

R ¹ selected from optionally substituted by one or more Q ¹ Substituted C _1-10 Alkyl radical, C _2-10 Alkenyl radical, C _1-10 Alkoxy, 3-to 8-membered cycloalkyl, C _2-10 Alkynyl, aryl, heteroaryl or heterocyclyl;

each Q ¹ Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C _1-6 Alkyl, halo C _1-6 Alkoxy radical, C _1-6 Alkylamino radical, di (C) _1-6 Alkyl) amino, 3-to 8-membered cycloalkyl, 3-to 8-membered heterocyclyl, phenyl, phenoxy, -O-R ⁸ 、-C(O)-R ⁸ 、-OC(O)-R ⁸ 、-C(O)-O-R ⁸ 、-N(R ⁸ )-C(O)-O-R ⁹ 、-N(R ⁸ )-C(O)-R ⁹ 、-N(R ⁸ )-C(O)-N(R ⁸ )(R ⁹ ) or-C (O) -N (R) ⁸ )(R ⁹ ) In which C is _1-6 Alkyl, 3-8 membered cycloalkyl, 3-8 membered heterocyclyl, phenyl, phenoxy optionally substituted with one or more C _1-6 Alkyl, 3-8 membered cycloalkyl, C _1-6 Alkoxy, hydroxy, amino, carboxy or halogen substitution;

or R ¹ And X ¹ Together with the N to which it is attached, form a heterocyclyl, aryl or heteroaryl group, which is optionally substituted with one or more alkyl, hydroxy or halogen;

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxyl and C _1-6 Alkyl radical, C _1-6 Alkoxy, halo C _1-6 Alkyl, halo C _1-6 Alkoxy, hydroxy C _1-6 Alkyl, hydroxy C _1-6 Alkoxy radical, C _1-6 Alkylamino radical, di (C) _1-6 Alkyl) amino, C _1-6 Alkylthio radical, C _1-6 Alkylcarbonyl group, C _1-6 Alkoxy radical C _1-6 Alkyl radical, C _1-6 Alkylcarbonyloxy, C _1-6 Alkylsulfonyl radical, C _1-6 Alkylaminosulfonyl, di (C) _1-6 Alkyl) aminosulfonyl,C _1-6 Alkylsulfonylamino or C _1-6 An alkylsulfonyloxy group;

R ³ selected from absent, hydrogen, halogen, C _1-6 Alkyl, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl or C _1-6 An alkylcarbonyl group;

n is selected from 1 or 2, when n is selected from 2, R ³ May be the same or different;

R ⁵ selected from optionally substituted by one or more Q ² Substituted C _1-6 Alkyl radical, C _1-6 Alkylamino radical, C _1-6 Alkoxy, aryl, heteroaryl, cycloalkyl or heterocyclyl;

each Q ² Is independently selected from C _1-6 Alkyl, 3-8 membered cycloalkyl, 3-8 membered cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, halogen, cyano, nitro, haloC _1-6 Alkoxy radical, C _1-6 Alkylamino, oxo, -O-R ⁸ 、-O-C(O)-R ⁸ 、-O-C(O)-N(R ⁸ )(R ⁹ )、-S-R ⁸ 、-N(R ⁸ )(R ⁹ )、-S(＝O)-R ⁸ 、-S(＝O) ₂ R ⁸ 、-O-S(＝O) ₂ R ⁸ 、-S(＝O) ₂ -N(R ⁸ )(R ⁹ )、-S(＝O) ₂ -OR ⁸ 、-N(R ⁸ )-C(O)-R ⁹ 、-N(R ⁸ )-C(O)-O-R ⁹ 、-N(R ⁸ )-C(O)-N(R ⁸ )(R ⁹ )、-C(O)-R ⁸ 、-C(O)-O-R ⁸ 、-C(O)-N(R ⁸ )-R ⁹ or-N (R) ⁸ )-S(＝O) ₂ -R ⁹ Wherein said C is _1-6 The 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 _1-6 Alkyl or-O-R ⁸ Substitution;

each R ⁸ And R ⁹ Each independently selected from hydrogen and C _1-6 Alkyl, 3-8 membered cycloalkyl, or when R is ⁸ And R ⁹ When attached to the same N atom, R ⁸ And R ⁹ Together with the N atom to which they are attached form 3-1A 2-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,

R ¹ selected from optionally substituted by one or more Q ¹ Substituted C _1-6 Alkyl radical, C _2-6 Alkenyl radical, C _1-6 Alkoxy, 3-to 8-membered cycloalkyl, C _2-6 Alkynyl, 6-14 membered aryl, 5-14 membered heteroaryl, or 3-12 membered heterocyclyl;

each Q ¹ Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C _1-6 Alkyl, halo C _1-6 Alkoxy radical, C ₁ - ₆ Alkylamino radical, di (C) _1-6 Alkyl) amino, 3-to 8-membered cycloalkyl, heterocyclyl, phenyl, phenoxy, -O-R ⁸ 、-C(O)-R ⁸ 、-OC(O)-R ⁸ 、-C(O)-O-R ⁸ 、-N(R ⁸ )-C(O)-O-R ⁹ 、-N(R ⁸ )-C(O)-R ⁹ 、-N(R ⁸ )-C(O)-N(R ⁸ )(R ⁹ ) or-C (O) -N (R) ⁸ )(R ⁹ ) In which C is _1-6 Alkyl, 3-8 membered cycloalkyl, heterocyclyl, phenyl, phenoxy optionally substituted by one or more C _1-6 Alkyl, 3-8 membered cycloalkyl, C _1-6 Alkoxy, hydroxy, amino, carboxy or halogen substitution;

or R ¹ And X ¹ Bonded 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 _1-6 Alkyl, hydroxy or halogen;

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxylBase, C _1-6 Alkyl radical, C _1-6 Alkoxy, halo C _1-6 Alkyl, halo C _1-6 Alkoxy, hydroxy C _1-6 Alkyl, hydroxy C _1-6 Alkoxy radical, C _1-6 An alkylamino group;

n is selected from 1 or 2, and when n is selected from 2, R ³ May be the same or different;

R ⁵ selected from optionally substituted by one or more Q ² Substituted C _1-6 Alkyl radical, C _1-6 Alkylamino radical, C _1-6 Alkoxy, 6-14 membered aryl, 5-14 membered heteroaryl, 3-12 membered cycloalkyl or 3-12 membered heterocyclyl;

each Q ² Independently selected from C _1-6 Alkyl, 3-8 membered cycloalkyl C _1-6 Alkyl, 6-14 membered aryl C _1-6 Alkyl, 5-14 membered heteroaryl C _1-6 Alkyl, 3-12 membered heterocyclic group C _1-6 Alkyl, halogen, cyano, nitro, oxo, -O-R ⁸ 、-O-C(O)-R ⁸ 、-O-C(O)-N(R ⁸ )(R ⁹ )、-S-R ⁸ 、-N(R ⁸ )(R ⁹ )、-S(＝O)-R ⁸ 、-S(＝O) ₂ R ⁸ 、-O-S(＝O) ₂ R ⁸ 、-S(＝O) ₂ -N(R ⁸ )(R ⁹ )、-S(＝O) ₂ -OR ⁸ 、-N(R ⁸ )-C(O)-R ⁹ 、-N(R ⁸ )-C(O)-O-R ⁹ 、-N(R ⁸ )-C(O)-N(R ⁸ )(R ⁹ )、-C(O)-R ⁸ 、-C(O)-O-R ⁸ 、-C(O)-N(R ⁸ )-R ⁹ Or N (R) ⁸ )-S(＝O) ₂ -R ⁹ Wherein said C is _1-6 Alkyl, 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 _1-6 Alkyl or-O-R ⁸ Substitution;

R ⁸ and R ⁹ Each independently selected from hydrogen and C _1-6 Alkyl, 3-8 membered cycloalkyl, or when R is ⁸ And R ⁹ When attached to the same N atom, R ⁸ And R ⁹ Together 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 the content of the first and second substances,

X ² 、X ³ 、X ⁴ 、X ⁵ at least one is selected from N;

R ¹ selected from optionally substituted by one or more Q ¹ Substituted C _1-6 Alkyl radical, C _2-6 Alkenyl radical, C _1-6 Alkoxy, 3-to 8-membered cycloalkyl, C _2-6 Alkynyl, 6-12 membered aryl, 5-12 membered heteroaryl or 3-10 membered heterocyclyl;

each Q ¹ Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C _1-6 Alkyl, halo C _1-6 Alkoxy radical, C _1-6 Alkylamino radical, di (C) _1-6 Alkyl) amino, 3-to 8-membered cycloalkyl, 3-to 8-membered heterocyclyl, phenyl, phenoxy, -O-R ⁸ 、-C(O)-R ⁸ 、-OC(O)-R ⁸ 、-C(O)-O-R ⁸ 、-N(R ⁸ )-C(O)-O-R ⁹ 、-N(R ⁸ )-C(O)-R ⁹ 、-N(R ⁸ )-C(O)-N(R ⁸ )(R ⁹ ) or-C (O) -N (R) ⁸ )(R ⁹ )；

Or R ¹ And X ¹ Together with the N to which it is 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 _1-6 Alkyl, hydroxy or halogen substitution;

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxyl and C _1-6 Alkyl radical, C _1-6 Alkoxy, halo C _1-6 Alkyl, halo C _1-6 Alkoxy, hydroxy C _1-6 Alkyl, hydroxy C _1-6 Alkoxy radical, C _1-6 An alkylamino group;

R ⁵ selected from optionally substituted by one or more Q ² Substituted C _1-6 Alkyl radical, C _1-6 Alkylamino radical, C _1-6 Alkoxy, 6-to 10-membered aryl, 5-to 12-membered heteroaryl, 3-to 10-membered cycloalkyl or 3-to 10-membered heterocyclyl;

each Q ² Independently selected from C _1-6 Alkyl, 3-8 membered cycloalkyl C _1-6 Alkyl, 6-14 membered aryl C _1-6 Alkyl, 5-14 membered heteroaryl C _1-6 Alkyl, 3-12 membered heterocyclic group C _1-6 Alkyl, halogen, cyano, nitro, halogeno C _1-6 Alkoxy, oxo, -O-R ⁸ 、-O-C(O)-R ⁸ 、-O-C(O)-N(R ⁸ )(R ⁹ )、-S-R ⁸ 、-N(R ⁸ )(R ⁹ )、-S(＝O)-R ⁸ 、-S(＝O) ₂ R ⁸ 、-O-S(＝O) ₂ R ⁸ 、-S(＝O) ₂ -N(R ⁸ )(R ⁹ )、-S(＝O) ₂ -OR ⁸ 、-N(R ⁸ )-C(O)-R ⁹ 、-N(R ⁸ )-C(O)-O-R ⁹ 、-N(R ⁸ )-C(O)-N(R ⁸ )(R ⁹ )、-C(O)-R ⁸ 、-C(O)-O-R ⁸ 、-C(O)-N(R ⁸ )-R ⁹ or-N (R) ⁸ )-S(＝O) ₂ -R ⁹ Wherein said C is _1-6 Alkyl, 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 _1-6 Alkyl or-O-R ⁸ Substitution;

each R ⁸ And R ⁹ Each independently selected from hydrogen and C _1-6 Alkyl, 3-8 membered cycloalkyl, or when R is ⁸ And R ⁹ When attached to the same N atom, R ⁸ And R ⁹ Together with the N atom to which they are attached form a 3-12 membered heterocyclic ring;

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 the content of the first and second substances,

X、X ¹ each independently selected from C and CR ⁸ Or N;

R ¹ selected from optionally substituted by one or more Q ¹ Substituted C _1-6 Alkyl radical, C _2-6 Alkenyl radical, C _1-6 Alkoxy, 3-6 membered cycloalkyl, 6-10 membered aryl, 5-10 membered heteroaryl or 3-8 membered heterocyclyl;

Or R ¹ And X ¹ Together with the N to which it is 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 by one or more C _1-6 Alkyl, hydroxyOr halogen substitution;

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxyl and C _1-6 Alkyl radical, C _1-6 Alkoxy, halo C _1-6 Alkyl, halo C _1-6 Alkoxy, hydroxy C _1-6 Alkyl, hydroxy C _1-6 Alkoxy or C _1-6 An alkylamino group;

R ⁵ selected from optionally substituted by one or more Q ² Substituted C _1-6 Alkyl radical, C _1-6 Alkylamino radical, C _1-6 Alkoxy, 6-to 8-membered aryl, 5-to 10-membered heteroaryl, 3-to 8-membered cycloalkyl or 3-to 8-membered heterocyclyl;

each Q ² Independently selected from C _1-6 Alkyl, 3-8 membered cycloalkyl C _1-6 Alkyl, 3-8 membered heterocyclic group C _1-6 Alkyl, halo C _1-6 Alkoxy, halogen, cyano, nitro, -O-R ⁸ 、-O-C(O)-R ⁸ 、-O-C(O)-N(R ⁸ )(R ⁹ )、-S-R ⁸ 、-N(R ⁸ )(R ⁹ )、-S(＝O)-R ⁸ 、-S(＝O) ₂ R ⁸ 、-O-S(＝O) ₂ R ⁸ 、-S(＝O) ₂ -N(R ⁸ )(R ⁹ )、-S(＝O) ₂ -OR ⁸ 、-N(R ⁸ )-C(O)-R ⁹ 、-N(R ⁸ )-C(O)-O-R ⁹ 、-N(R ⁸ )-C(O)-N(R ⁸ )(R ⁹ )、-C(O)-R ⁸ 、-C(O)-O-R ⁸ 、-C(O)-N(R ⁸ )-R ⁹ or-N (R) ⁸ )-S(＝O) ₂ -R ⁹ Wherein said C _1-6 Alkyl, 3-8 membered cycloalkyl, 3-8 membered heterocyclyl is further optionally substituted with one or more halogen, hydroxy, amino, cyano, nitro, carboxy, oxo, C _1-6 Alkyl or-O-R ⁸ Substitution;

each R ⁸ And R ⁹ Each independently selected from hydrogen or C _1-6 An 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 the content of the first and second substances,

R ¹ selected from the group consisting of optionally substituted by 1 to 3Q ¹ Substituted C _1-6 Alkyl radical, C _1-6 Alkoxy, 3-6 membered cycloalkyl or 3-8 membered heterocyclyl, each Q ¹ Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C _1-6 Alkyl radical, C _1-6 Alkoxy radical, C _1-6 Alkyl carbonyl, C _1-6 Alkoxycarbonyl group, C _1-6 Alkylamido, aminoacyl, C _1-6 Alkylaminoacyl, di (C) _1-6 Alkyl) aminoacyl, halo C _1-6 Alkoxy radical, C _1-6 Alkylamino radical, di (C) _1-6 Alkyl) amino or 3-8 membered cycloalkyl;

R ³ selected from hydrogen, halogen, C _1-6 Alkyl, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl or C _1-6 An alkylcarbonyl group;

R ⁵ selected from the group consisting of optionally substituted by 1 to 3Q ² Substituted C _1-6 Alkyl radical, C _1-6 Alkylamino radical, C _1-6 Alkoxy, phenyl, 5-8 membered heteroaryl, or 3-6 membered heterocyclyl;

each Q ² Independently selected from C _1-6 Alkyl radical, C _1-6 Alkoxy, 3-6 membered cycloalkyl C _1-6 Alkyl, 3-6 membered heterocyclic group C _1-6 Alkyl, halogen, cyano, hydroxy, amino, nitro, halogeno C _1-6 Alkoxy radical, C _1-6 Alkylamino radical, C _1-6 Alkylcarbonyl group, C _1-6 Alkylcarbonyloxy, C _1-6 Alkyl mercapto group, C _1-6 Alkylsulfonyl radical, C _1-6 Alkyloxycarbonyl, C _1-6 Alkylamido radical, C _1-6 Alkylaminoacyl, C _1-6 Alkylsulfonylamino, wherein said C _1-6 Alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocyclyl is further optionally substituted by 1 to 3 halogen, hydroxy, amino, cyano, nitro, carboxy, C _1-6 Alkyl 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, the first and the second end of the pipe are connected with each other,

R ¹ selected from the group consisting of optionally substituted by 1 to 3Q ¹ Substituted C _1-6 Alkyl radical, C _1-6 Alkoxy, 3-6 membered cycloalkyl, each Q ¹ Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C _1-6 Alkyl radical, C _1-6 Alkoxy radical, C _1-6 Alkoxycarbonyl, aminoacyl, C _1-6 Alkylaminoacyl, di (C) _1-6 Alkyl) aminoacyl, C _1-6 Alkylamino or di (C) _1-6 Alkyl) amino;

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, halogen, hydroxyRadical, amino, cyano, nitro, carboxyl, C _1-6 Alkyl radical, C _1-6 Alkoxy, halo C _1-6 Alkyl or halo C _1-6 An alkoxy group;

R ⁵ selected from the group consisting of optionally substituted 1 to 3Q ² Substituted C _1-6 Alkyl radical, C _1-6 Alkylamino radical, C _1-6 Alkoxy, phenyl or 5-8 membered heteroaryl;

each Q ² Independently selected from C _1-6 Alkyl radical, C _1-6 Alkoxy, 3-6 membered cycloalkyl C _1-6 Alkyl, 3-6 membered heterocyclyl, -6 membered heterocyclyl C _1-6 Alkyl, halogen, hydroxy or amino, wherein said C _1-6 Alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocyclyl is further optionally substituted by 1 to 3 halogen, hydroxy, amino, cyano, nitro, carboxy, C _1-6 Alkyl substitution;

selected from single bonds or double bonds.

R ¹ selected from the group consisting of optionally substituted by 1 to 2Q ¹ Substituted C _1-4 Alkyl radical, C _1-4 Alkoxy, 3-6 membered cycloalkyl, each Q ¹ Independently selected from halogen, hydroxy, amino, C _1-4 Alkyl radical, C _1-4 Alkoxy radical, C _1-4 Alkoxycarbonyl, aminoacyl, C _1-4 Alkylaminoacyl, di (C) _1-4 Alkyl) aminoacyl, C _1-4 Alkylamino or di (C) _1-4 Alkyl) amino;

R ² 、R ⁴ 、R ⁶ 、R ⁷ are each independently selected from hydrogen, halogenHydroxy, amino, C _1-4 Alkyl radical, C _1-4 Alkoxy, halo C _1-4 Alkyl or halo C _1-4 An alkoxy group;

R ³ selected from hydrogen, halogen, C _1-4 Alkyl, halo C _1-4 Alkyl, hydroxy C _1-4 Alkyl or C _1-4 An alkylcarbonyl group;

R ⁵ selected from the group consisting of optionally substituted 1 to 2Q ² Substituted C _1-4 Alkyl radical, C _1-4 Alkylamino radical, C _1-4 Alkoxy, phenyl or 5-6 membered heteroaryl;

each Q ² Is independently selected from C _1-4 Alkyl radical, C _1-4 Alkoxy, 3-6 membered cycloalkyl C _1-4 Alkyl, 3-6 membered heterocyclic group C _1-4 Alkyl, halogen, hydroxy or amino, wherein said C _1-4 Alkyl, 3-6 membered cycloalkyl or 3-6 membered heterocyclyl is further optionally substituted by 1 to 3, preferably 1 to 2, halogens, hydroxy, amino, C _1-4 Alkyl substitution;

selected from single bonds or double bonds.

wherein the content of the first and second substances,

R ¹ selected from the group consisting of optionally substituted by 1 to 2Q ¹ Substituted 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 Q being a substituent ¹ Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxy, methyl, ethyl, methoxy,Ethoxy, propoxy, isopropoxy, methoxycarbonyl, ethoxycarbonyl, methylamino, ethylamino, dimethylamino, diethylamino, aminoacyl, methylamyl, ethylamyl, dimethylaminyl, diethylamino;

R ² 、R ⁴ 、R ⁶ 、R ⁷ each 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;

R ³ selected from hydrogen, fluoro, bromo, chloro, iodo, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, trifluoromethyl, hydroxymethyl, hydroxyethyl, hydroxypropyl or formyl;

R ⁵ selected from the group consisting of optionally substituted 1 to 2Q ² Substituted phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, tetrazolyl, pyridyl or pyrimidinyl;

each Q ² Independently selected 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 said methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl are further optionally substituted by 1 to 3, preferably 1 to 2, halogens, hydroxy, piperidinyl, piperazinylAmino, cyano, nitro, carboxyl, 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 the content of the first and second substances,

R ¹ selected from the group consisting of optionally substituted by 1 to 3Q ¹ Substituted C _1-6 Alkyl radical, C _1-6 Alkoxy or 3-6 membered cycloalkyl, each Q ¹ Independently selected from halogen, cyano, hydroxy, amino, nitro, carboxyl, C _1-6 Alkyl radical, C _1-6 Alkoxy, aminoacyl, C _1-6 Alkylaminoacyl, di (C) _1-6 Alkyl) aminoacyl, C _1-6 Alkylamino or di (C) _1-6 Alkyl) amino;

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, halogen, hydroxyl, amino, cyano, nitro, carboxyl and C _1-6 Alkyl radical, C _1-6 Alkoxy, halo C _1-6 Alkyl or halo C _1-6 An alkoxy group;

R ⁵ selected from the group consisting of optionally substituted 1 to 3Q ² Substituted phenyl or 5-8 membered heteroaryl;

each Q ² Independently selected from C _1-6 Alkyl radical, C _1-6 Alkoxy, 3-6 membered cycloalkyl C _1-6 Alkyl, 3-6 membered heterocyclyl or 3-6 membered heterocyclyl C _1-6 Alkyl, wherein said 3-6 membered cycloalkyl or 3-6 membered heterocyclyl is further optionally substituted with 1 to 3 halogens, hydroxy, amino, cyano, nitro, carboxy, C _1-6 Alkyl 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,

R ¹ selected from the group consisting of optionally substituted 1 to 3Q ¹ Substituted C _1-4 Alkyl radical, C _1-4 Alkoxy or 3-6 membered cycloalkyl, each Q ¹ Independently selected from halogen, hydroxy, amino, C _1-4 Alkyl radical, C _1-4 Alkoxy, aminoacyl, C _1-4 Alkylaminoacyl, di (C) _1-4 Alkyl) aminoacyl, C _1-4 Alkylamino or di (C) _1-4 Alkyl) amino;

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, halogen, hydroxyl, amino, C _1-4 Alkyl radical, C _1-4 Alkoxy, halo C _1-4 Alkyl or halo C _1-4 An alkoxy group;

R ⁵ selected from the group consisting of optionally substituted by 1 to 3Q ² Substituted phenyl or 5-6 membered heteroaryl;

each Q ² Independently selected from C _1-4 Alkyl radical, C _1-4 Alkoxy, 3-to 6-membered cycloalkyl C _1-4 Alkyl, 3-6 membered heterocyclyl or 3-6 membered heterocyclyl C _1-4 Alkyl, wherein said 3-6 membered cycloalkyl or 3-6 membered heterocyclyl is further optionally substituted with 1 to 3 halogens, hydroxy, amino, C _1-4 And (3) alkyl 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,

R ¹ selected from the group consisting of optionally substituted by 1 to 2Q ¹ Substituted methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, the substituents Q ¹ Selected 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, and diamidoAn ethylacyl group; (ii) a

R ² 、R ⁴ 、R ⁶ 、R ⁷ Each 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;

R ⁵ selected from the group consisting of optionally substituted by 1 to 2Q ² Substituted phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, tetrazolylpyridinyl or pyrimidinyl;

each Q ² Independently selected 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 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 to 2 halogens, hydroxy, amino, cyano, nitro, carboxy, 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 general formula (V),

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, halogen, hydroxyl, ammoniaRadical, cyano, nitro, carboxyl, C _1-6 Alkyl radical, C _1-6 Alkoxy, halo C _1-6 Alkyl or halo C _1-6 An alkoxy group;

R ⁵ selected from optionally substituted by one or more Q ² Substituted phenyl or 5-8 membered heteroaryl;

each Q ² Independently selected from C _1-6 Alkyl radical, C _1-6 Alkoxy, 3-6 membered cycloalkyl C _1-6 Alkyl, 3-6 membered heterocyclyl or 3-6 membered heterocyclyl C _1-6 Alkyl, 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,

R ⁵ selected from optionally substituted by one or more Q ² Substituted phenyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolylOxazolyl, thiazolyl, triazolyl, oxadiazolyl, tetrazolyl, pyridyl or pyrimidinyl;

each Q ² Selected 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 said cyclopropyl, cyclopropylmethyl, cyclobutyl, cyclobutylmethyl, cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl, oxetanyl, azetidinyl, tetrahydrofuryl, tetrahydrothienyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, thiazolidinyl, piperidinyl, piperazinyl are further optionally substituted with one or more of halo, hydroxy, amino, cyano, nitro, carboxy, methyl, ethyl, propyl or isopropyl.

The selection of any substituent in any embodiment described herein may be combined with each other, and the combined technical solution is still included in the scope of the 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 leaving groups, such as halogen.

The "functional group transformation reaction" can be achieved by using 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 art, 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, such as those used for the synthesis of intermediates, can be synthesized according to similar procedures and methods described in handbooks of organic chemistry, which are not intended to limit the present invention.

The above is contrary toR in the equation ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ 、R ⁷ 、n、X、X ¹ 、X ² 、X ³ 、X ⁴ 、X ⁵ As 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; r is ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ 、R ⁷ N 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 angina pectoris, arrhythmia, intermittent claudication, myocardial infarction, heart failure or recurrent ischemia. Therapeutically active agents suitable for the combined 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 including but 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; 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 conditions 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 prepared into oral preparations, appropriate fillers, binders, disintegrating agents, lubricants and the like 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 appropriate 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 can be made into inhalant or 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 used 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 ASK1 mediated disease and related disorders 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 and renal 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), the pharmaceutically acceptable salt, ester or stereoisomer thereof, the pharmaceutical preparation or the pharmaceutical composition for 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), pharmaceutically acceptable salt, ester or 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

The term "halogen" as used herein refers to 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 into

S 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 halogen. "halogen" is as defined above.

Said "C" of the present invention _1-10 Alkyl "means a straight-chain or branched alkyl group derived from a hydrocarbon having 1 to 10 carbon atoms by partially removing one hydrogen atom, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl and 1-methylbutyl2-methylpropyl, and the like. Said "C" is _1-6 Alkyl "or" C _1-4 Alkyl "refers to the above examples containing 1 to 6 or 1 to 4 carbon atoms.

"C" according to the invention _2-10 Alkynyl "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 _2-6 Alkynyl "refers to the above examples containing 1-6 carbon atoms.

"C" according to the invention _2-10 Alkenyl "means an alkenyl group of 2 to 10 carbon atoms having a carbon-carbon double bond, from which one hydrogen-derived straight chain or branched chain is partially removed, such as vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1, 3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1, 3-pentadienyl, 1, 4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1, 4-hexadienyl, cyclopentenyl, 1, 3-cyclopentadienyl, cyclohexenyl, 1, 4-cyclohexadienyl and the like. Said "C _1-6 Alkyl "or" C _1-4 Alkyl "refers to the above examples containing 1 to 6 or 1 to 4 carbon atoms.

"C" according to the invention _1-10 Alkoxy "means" C "as defined hereinbefore _1-10 Alkyl "radicals attached to the parent body via an oxygen atom, i.e." C _1-10 alkyl-O- "groups such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy, and the like. Said "C _1-6 Alkoxy "refers to the above examples containing 1 to 6 carbon atoms, i.e." C _1-6 An alkyl-O- "group.

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

As used herein, the term "halo C _1-6 Alkyl group "," halogeno C _1-6 Alkoxy "means C of the group _1-6 The 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 cyclic 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 cycloalkyl group, a 7-10 membered acyclic cycloalkyl group, an 8-9 membered acyclic cycloalkyl 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 can be 6-12 membered bridged cyclic group, 7-11 membered bridged cyclic group, and examples thereof 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 which can be formed, preferably monocyclic cycloalkyl.

The "heterocyclic group" according to the present invention means a non-aromatic cyclic group in which at least one ring carbon atom is replaced by a hetero atom selected from O, S, N, preferably 1 to 3 hetero atoms, and includes carbon atom, nitrogen atom and sulfur atom which may be oxo substituted.

"Heterocyclyl" refers to a monocyclic heterocyclic group or fused heterocyclic 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 and the like. Examples of mono-heterocyclic groups include, but are not limited to, aziridinyl, oxacyclopropane, thietane, azetidinyl, oxetanyl, thietane, 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, 2,5-dihydrooxazolyl, 2,3-dihydrooxazolyl, 3,4-dihydro-2H-pyrrolyl, 2,3-dihydro-1H-pyrrolyl, 2,5-dihydro-1H-imidazolyl, 4,5-dihydro-1H-pyrazolyl, 4,5-dihydro-3H-pyrazolyl, 4, 5-dihydrothiazolyl, 2H-pyranyl, 4H-pyranyl, 2H-thiopyranyl, 4H-thiopyranyl, 2,3,4,5-tetrahydropyridinyl, 1, 2-isoxazolyl, 1, 4-isoxazolyl, or 6H-1, 3-oxazinyl, and the like.

Fused heterocyclic rings include heterocyclo, spiro, bridged heterocyclic groups, which may be saturated, partially saturated or unsaturated, but are not aromatic. The heterocyclic group may be 6-to 12-membered fused ring group, 7-to 11-membered fused ring group, 6-to 10-membered fused ring group, 6-to 12-membered saturated fused ring group, 7-to 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-dihydrobenzofuran-3-yl, indolin-1-yl, indolin-2-yl, indolin-3-yl, 2, 3-dihydrobenzothien-2-yl, octahydro-1H-indolyl, octahydrobenzofuran-yl.

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 heterocyclic group, and examples thereof 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 described 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 also the case where a carbon atom, a sulfur atom, a nitrogen atom is oxo, e.g. a carbon atom by C (O), S (O) ₂ And (4) replacing. Heteroaryl radicalIncluding monoheteroaryl and fused heteroaryl, which 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 ] benzo][1,2,5]Oxadiazoles and 6, 7-dihydro [ c ]][1,2,5]Oxadiazol-4 (5H) onyl.

The 5-to 14-membered heteroaryl, 5-to 12-membered heteroaryl, and 5-to 10-membered heteroaryl of the present invention include, unless otherwise specified, monocyclic and fused ring structures capable of being 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 ASK 1-mediated diseases and related diseases;

(2) The compound, the pharmaceutically acceptable salt, the pharmaceutically acceptable ester or the stereoisomer thereof have 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, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection 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 Pd ₂ (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 invention

Represents that the compound is a single compound with an unidentified cis-trans configuration or a mixture of cis-trans compounds.

Preparation examples

EXAMPLE 1 preparation of 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 (Compound 1)

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

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

Preparation of 1.2, 6-bromopyridine carbohydrazide

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

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

6-Bromopyridinecarboxhydrazide (4.00g, 18.5 mmol) was added to DMF-DMA (15 mL). The reaction was stirred at 80 ℃ for 16h, concentrated, added with n-heptane (100 mL) and ethyl acetate (10 mL), stirred for 1h, filtered with suction, and the solid dried to give the title compound (5.00 g, 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 (18.5 mL) were added to acetonitrile (74 mL), isopropylamine (7.60mL, 92.25mmol) was added dropwise, warmed to 95 ℃ for 16 hours, concentrated, and purified by silica gel column chromatography (PE: EA = 1) to give the objective compound (4.65 g, 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 (190 mg) were added to methanol (50 mL), and hydrogenation was carried out for 3 hours, followed by suction filtration and concentration of the filtrate to obtain the objective compound (1.57 g, yield 98.7%).

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) was added to toluene (20 mL), stirred at 95 ℃ for 6 hours, concentrated, and purified by silica gel column chromatography (PE: EA = 1) to obtain the objective compound (1.50 g, yield 83.0%).

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 (5 mL) for use.

Acetic anhydride (2.49g, 24.4 mmol) was added dropwise to formic acid (1 mL) 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 (100 mL) saturated sodium chloride solution (80 mL), and the organic phase was concentrated to 30mL and used directly in the next step.

Preparation of 8, 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 (20 mL), rotary evaporated to no DCM, and ammonium acetate (1.88g, 24.4 mmol) was added. The reaction was carried out at 110 ℃ for 16 hours. Adjusting pH to 8-9 with saturated sodium hydroxide solution, adding EA (200 mL) for extraction, washing organic phase with saturated sodium chloride solution (150 mL), drying with anhydrous sodium sulfate, and concentrating to obtain solid. EA (20 mL) was added to the solid, stirred for 0.5h, filtered with suction, and the solid was dried to give the title compound (1.40 g, 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), pd ₂ (dba) ₃ (92mg, 0.10 mmol) and X-antphos (116mg, 0.20 mmol) were added to 1, 4-dioxane (300 mL), and the reaction was stirred at 80 ℃ under nitrogen for 16 hours, concentrated and purified by silica gel column chromatography (DCM: meOH =20Compound (150 mg, 17.3% yield).

The molecular formula is as follows: c ₂₅ H ₂₅ N ₇ Molecular weight of O: 439.2 LC-MS (M/e): 440.1 (M + H) ⁺ )

¹ H NMR(CD ₃ OD):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 2 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 (Compound 2)

Preparation of 1,2- (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, 9.6 mmol), 7-nitro-3, 4-dihydroisoquinolin-1 (2H) -one (1.84g, 9.6 mmol), cesium carbonate (6.26g, 19.2mmol), pd ₂ (dba) ₃ (440mg, 0.48mmol) and X-antphos (556 mg, 0.96mmol) were added to 1, 4-dioxane (100 mL), and the reaction was stirred at 80 ℃ under nitrogen for 16 hours, concentrated and purified by silica gel column chromatography (PE: EA = 1) to obtain the objective compound (450 mg, yield 12.5%).

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 (10 mL) and methanol (10 mL), hydrogenated for 2 hours, filtered with suction, and the filtrate was concentrated to give the title compound (400 mg, 96.2% yield).

2.3 preparation 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

7-amino-2- (6- (4-isopropyl-4H-1, 2, 4-triazol-3-yl) pyridin-2-yl) isoquinolin-1 (2H) -one (345mg, 1.0 mmol), 2-bromo-1-cyclopropylethan-1-one (196mg, 1.2 mmol), DIEA (284mg, 2.2 mmol) was added to toluene (10 mL), stirred at 95 ℃ for 16 hours, concentrated, and purified by silica gel column chromatography (DCM: meOH = 20.

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 (5 mL) for use.

Acetic anhydride (335mg, 3.28mmol) was added dropwise to formic acid (1 mL) at 0 ℃ and stirring was continued at 0 ℃. 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 (20 mL) saturated sodium chloride solution (15 mL) 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 (0.82 mmol) obtained in the previous step was dissolved in glacial acetic acid (6 mL), 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 (50 mL) was added and water (40 mL) was extracted, the organic phase was concentrated, and after silica gel column chromatography (DCM: meOH = 20).

The molecular formula is as follows: c ₂₅ H ₂₃ N ₇ O; molecular weight: 437.2; LC-MS (M/e): 438.3 (M + H) ⁺ )。

¹ H NMR(CD ₃ OD):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 1- (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 (7.0 g,35.9 mmol) was dissolved in 1-tert-butoxy-N, N' -tetramethylmethanediamine (9.4g, 53.9mmol), reacted at 115 ℃ for 2 hours, a solid precipitated, filtered, and the filter cake was slurry-washed with a mixed solvent of petroleum ether and ethyl acetate (60 mL, petroleum ether: ethyl acetate = 6), filtered, and the filter cake was dried to obtain the objective product (8.1 g, 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 (80 mL) solution, heated to 125 ℃, reacted for 2.5 hours, a solid precipitated, cooled to 25 ℃, filtered, and the cake was then slurry-washed with a mixed solvent of petroleum ether and ethyl acetate (100 mL, petroleum ether: ethyl acetate = = 2), filtered, and the cake was dried to obtain the objective product (9.0 g, 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 (8.4 g,24.7 mmol), zinc powder (8.1 g,123.8 mmol), ammonium chloride (7.9g, 147.7 mmol) were added to methanol (100 mL) and water (100 mL) and reacted at 80 ℃ for 4 hours, filtered, the filtrate was added with water (100 mL) and 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 (7.4 g, 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 (15 mL), tetrahydrofuran (15 mL) and dichloromethane (30 mL), heated to 80 ℃, reacted for 5 hours, spin-dried, and column chromatography was performed on the residue (petroleum ether: ethyl acetate =5: 1-1) to obtain the objective compound (3.6 g, yield: 57.0%).

2-1.5 preparation of N- (2-cyclopropyl-2-oxoethyl) -N- (2, 4-dimethoxybenzyl) -1-oxo-1, 2-dihydroisoquinolin-7-yl) carboxamide

Acetic anhydride (5.2 g,12.7 mmol) was dropped into formic acid (30 mL) at 0 ℃ to react for 0.5 hour, a dichloromethane (30 mL) solution of 7- ((2-cyclopropyl-2-oxoethyl) amino) -2- (2, 4-dimethoxybenzyl) isoquinolin-1 (2H) -one (5.0 g,12.7 mmol) was dropped into the reaction system to react at 0 ℃ for 16 hours, pH =7 was adjusted with 1N sodium hydroxide solution, dichloromethane (400 mL. Times.2) was extracted, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and spin-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, 4-dimethoxybenzyl) -1-oxo-1, 2-dihydroisoquinolin-7-yl) carboxamide (5.0 g, crude) and ammonium acetate (7.5g, 97.3 mmol) were dissolved in acetic acid (30 mL), heated to 105 deg.C, reacted for 16 hours, 0 deg.C, adjusted to pH =7 with sodium hydroxide solution (2M), extracted with dichloromethane (150 mL. Times.3), the organic phases combined, 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 (5.1 g, crude), dissolved in trifluoroacetic acid (30 mL), heated to 90 ℃ for 3H, concentrated, slurried with ethyl acetate (50 mL), filtered, and the solid dried to give the title compound (2.2 g, 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 diphenylphosphine-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 (70 mL) and reacted at 105 ℃ for 16 hours under nitrogen protection. Concentration and reverse phase column chromatography (acetonitrile/water = 0-50%) gave crude product, which was slurried with methanol (5 mL) and methyl tert-butyl ether (20 mL), filtered and the filter cake was dried to give the title compound (400 mg, 27.1% yield).

Molecular formula C ₂₅ H ₂₃ N ₇ O molecular weight 437.51 LC-MS (M/e): 438.2 (M + H) ⁺ )

¹ H-NMR(400MHz,CDCl ₃ )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 3 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 (Compound 3)

Preparation of 3, 2-methyl-4, 5-dinitrobenzoic acid

2-methyl-4-nitrobenzoic acid (10.0 g,55.2 mmol) was dissolved in concentrated sulfuric acid (60 mL), a solution of potassium nitrate (8.4 g,83.2 mmol) in concentrated sulfuric acid (40 mL) was added dropwise to the reaction mixture, poured into ice water at 25 ℃ for 16 hours to precipitate a solid, and vacuum-dried to give the desired compound 9g, yield: 72 percent.

Preparation of 3.2, 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 (200 mL), heated to 70 ℃ for 3 hours, adjusted to pH =3-4 with 2N hydrochloric acid, and a solid was precipitated, filtered, and vacuum-dried to obtain the objective compound (7 g, 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 (100 mL), thionyl chloride (8.5g, 71.4 mmol) 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) to obtain the objective compound (6.0 g, 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) was heated to 115 ℃ for 3 hours, cooled to 25 ℃, added with a mixed solvent (50 mL) of petroleum ether: ethyl acetate = 6.

Preparation of 5, 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) was dissolved in toluene (100 mL), heated to 110 ℃, reacted for 16 hours, the solvent was dried by spinning, a mixed solvent of petroleum ether: ethyl acetate =2:1 (80 mL) was added, filtered, and the solid was dried in vacuo to obtain 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 (5.5 g,14.9 mmol) was dissolved in trifluoroacetic acid (50 mL), heated to 85 deg.C for 3 hours, the solvent was dried, methanol (50 mL) was added, dried, ethyl acetate (60 mL) was added, filtered, and the solid was dried under vacuum to give the title compound (3.1 g, 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 (100 mL) was heated to 110 ℃ for 16 hours, methanol: dichloromethane =9 (100 mL), filtered, and the solid was dried in vacuum to obtain 3.0g of the objective compound with a yield of 66.7%.

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, 1.7 mmol) was dissolved in methanol (30 mL), hydrazine monohydrate solution (3 mL) was added, heated to 65 ℃ for 16H, spun dry and the residue 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) pyridinecarbohydrazide (crude, 1.7 mmol), N, N-dimethylformamide dimethyl acetal (20 mL), 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, 1.7 mmol) was dissolved in a mixed solvent of acetic acid (1 mL) and acetonitrile (4 mL), isopropylamine (0.5g, 8.5 mmol) was added, heated to 70 ℃ for 16 hours, and spin-dried, and column chromatography (dichloromethane: methanol =20: 1) was performed on the residue to obtain the objective compound (0.48 g, 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 (300 mL), stannous chloride (1.1g, 5.8mmol) and hydrochloric acid (1 mL) 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) to obtain the objective compound (0.25 g, 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 (30 mL), N-diisopropylethylamine (0.17g, 1.3mmol) was added, heated to 95 ℃ for 16 hours, spun dry, and the residue was column-chromatographed (dichloromethane: methanol = 30) to give the objective compound (95 mg, 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 (3 mL), 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, 0.21 mmol) was dissolved in acetic acid (5 mL), ammonium acetate (160mg, 2.1mmol) was added, heated to 110 ℃, for 16 hours, spin-dried, sodium bicarbonate solution (10 mL) was added to adjust pH =8-9, dichloromethane (20 mL × 3) was extracted, the organic phases were combined, TLC separation (dichloromethane: methanol =20 1) was prepared to obtain the objective compound 46mg, yield: 47.4 percent.

Molecular formula C ₂₆ H ₂₅ N ₇ O ₂ Molecular weight 467.53 LC-MS (M/e): 468.2 (M + H) ⁺ )

¹ H-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 4 preparation 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 (Compound 4)

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

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, 4 mmol) was dissolved in acetonitrile (30 mL), and water (0.5 mL) was added to conduct a reflux reaction for 5 hours. The reaction mixture was concentrated, water (100 mL) was added, methylene chloride was washed three times (100 mL × 3), the aqueous phase was concentrated, a mixed solvent of methylene chloride and methanol (methylene chloride: methanol = 20) was added to the residue, suction filtration was performed, 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 (10 mL), 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 yielded 100mg of the objective compound in 18.6% yield in two steps.

The molecular formula is as follows: c ₁₅ H ₁₂ FN ₃ O molecular weight: 269.3 LC-MS (M/e): 270.1 (M + H)

¹ H-NMR(400MHz,CD ₃ OD)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).

Synthesis of 3,7- (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 (10 mL) and reacted at 110 ℃ for 15 hours under nitrogen protection. The reaction solution was concentrated, water (100 mL) was added, methylene chloride was extracted three times (100 mL × 3), the organic phase was concentrated and dried, and the residue was subjected to silica gel column chromatography (methylene chloride: methanol = 20) 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: c ₂₅ H ₂₂ FN ₇ O molecular weight: 455.5 LC-MS (M/e): 456.2 (M + H)

¹ H-NMR(400MHz,CD ₃ OD)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 5 preparation of 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 (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, 31.6 mmol), tetramethylethylenediamine (90mg, 0.79mmol) and cuprous iodide (0.15g, 0.79mmol) were added to toluene (150 mL), stirred under nitrogen, heated to 100 ℃ and reacted for 16 hours. Cooled to 20 ℃, concentrated to dryness, washed with water (200 mL), filtered, the filter cake washed with methanol and dried to give the title compound (1.6 g, 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), and dichloro (pentamethylcyclopentadienyl) rhodium dimer (0.15g, 0.24mmol) were dissolved in 1, 2-dichloroethane (100 mL), and the reaction was stirred for 16 hours with heating to 80 ℃ under nitrogen protection. 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, concentration of the filtrate, purification by silica gel column chromatography (ethyl acetate: petroleum ether = 1) gave the objective compound (0.25 g, yield: 16.0%) and recovered methyl 6- (7-nitro-1-oxoisoquinolin-2 (1H) -yl) picolinate (1.3 g).

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 (10 mL), triethylamine (1 mL) and Pd/C (0.3 g) 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 resulting crude product by silica gel column chromatography (dichloromethane: methanol = 20) gave the target compound (0.38 g, 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, 2.4 mmol) were dissolved in toluene (10 mL), 2-bromo-1-cyclopropyleth-1-one (0.24g, 1.5 mmol) was added, nitrogen protected, heated to 95 ℃ and stirred for 16 hours, concentrated, and the resulting crude product was purified by silica gel column chromatography (dichloromethane: methanol = 20) to give the objective compound (0.15 g, yield 32.0%), and methyl 6- (7-amino-3-methyl-1-oxoisoquinolin-2 (1H) -yl) picolinate (0.22 g) 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 (5 mL), reacted with stirring 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 reacted with stirring 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 (0.3 g) was dissolved in acetic acid, ammonium acetate (0.3 g, 3.89mmol) was added, the mixture was heated to 110 ℃ under nitrogen protection, stirred for reaction for 16 hours, concentrated, and the crude product was isolated and purified by medium pressure preparative isolation to give the title compound (0.2 g, 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, 0.5 mmol) was dissolved in methanol (5 mL), 85% hydrazine hydrate (2 mL) was added, and the reaction was stirred at 20 ℃ for 16 hours. 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 ℃. And concentrating, and washing the obtained crude product with methyl tert-butyl ether to obtain a crude product (92 mg) of the target compound.

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-dimethylcarbohydrazide (92 mg) was dissolved in a mixed solvent of acetic acid (1 mL) and acetonitrile (4 mL), isopropylamine (60mg, 1mmol) was added and the mixture was heated to 95 ℃ for 16 hours. Concentration and purification of the resulting crude product under high pressure were carried out to obtain the objective compound (11 mg, three-step yield: 4.9%).

The molecular formula is as follows: c ₂₆ H ₂₅ N ₇ O molecular weight: 451.5 LC-MS (M/e): 452.3 (M + H) ⁺ )

¹ H NMR(CD ₃ OD):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 6 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 (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, 13.5 mmol), 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 (30 mL) and reacted at 35 ℃ for 40 hours. Dichloromethane (20 mL) was added by evaporation and the title compound (1.68 g, 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, 19.6 mmol), ammonium chloride (610mg, 11.4 mmol) and water (6 mL) were added to isopropanol (24 mL), the reaction was stirred at 80 ℃ for 16 hours, methanol (20 mL) and dichloromethane (20 mL) 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 (1.55 g) was added to triethyl orthoformate (20 mL), reacted at 143 ℃ for 2 hours, cooled to precipitate a solid, and filtered by suction to give 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 (1.75 g), 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 (15 mL), reacted for 16 hours at 100 ℃ under nitrogen, concentrated to dryness, and subjected to silica gel column chromatography (eluent methanol: dichloromethane =1 25) to obtain the target compound (950 mg, total yield in three steps: 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, 2.1 mmol), trifluoroacetic acid (2 mL) was added to dichloromethane (6 mL), 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 (555 mg, 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 (18 mL), reacted at 90 ℃ for 6 hours, concentrated to dryness, and passed through a silica gel column (eluent methanol: dichloromethane = 1) to give the title compound (145 mg, 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 (1 mL), added dropwise to formic acid (2 mL) 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 (2 mL), 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 (15 mL), and the organic phase evaporated to dryness to give a 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 (5 mL), reacted at 110 ℃ for 20H, pH =7-8 adjusted with sodium hydroxide solution, dichloromethane (15 mL) was added for extraction, the organic phase was evaporated to dryness, TLC plates (developing solvent methanol: dichloromethane = 1) were dried to give the target compound (37 mg, total yield in two steps: 24.8%).

The molecular formula is as follows: c ₂₄ H ₂₂ N ₈ O molecular weight: 438.5 LC-MS (M/e): 439.2 (M + H) ⁺ )

¹ H-NMR(CDCl ₃ )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 7 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 (Compound 7)

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

Methyl 2-methyl-5-nitrobenzoate (7.0 g,35.9 mmol) was dissolved in 1-tert-butoxy-N, N, N ', N' -tetramethylmethanediamine (9.4 g,53.9 mmol), and the reaction was heated to 115 ℃ for 2 hours. Solid is separated out and filtered, and filter cakes are prepared by petroleum ether: ethyl acetate (6.

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

Methyl 2- (2- (dimethylamino) vinyl) -5-nitrobenzoate (8.1g, 32.4 mmol), 2, 4-dimethoxybenzylamine (7.6g, 45.4 mmol) were dissolved in toluene (80 mL), heated to 125 ℃ for 2.5 hours, solid precipitated, cooled to 25 ℃, filtered, and the filter cake was washed with petroleum ether: ethyl acetate (2.

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) and ammonium chloride (7.9g, 147.7mmol) were added to methanol (100 mL) and water (100 mL) in this order and reacted at 80 ℃ for 4 hours. After filtration, the filtrate was extracted with methylene chloride (300 mL. Times.3) by adding water (100 mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain 7.4g of the objective product with a yield of 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 (30 mL), tetrahydrofuran (15 mL), and toluene (15 mL) in this order, and reacted at 80 ℃ for 5 hours. Concentration and purification of the residue by a silica gel column (petroleum ether: ethyl acetate =5: 1-1.

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

Acetic anhydride (5.2 g,50.9 mmol) was added dropwise to formic acid (30 mL) 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, 12.7 mmol) in methylene chloride (30 mL) was added dropwise to the reaction system, followed by reaction at 0 ℃ for 16 hours. pH =7 adjusted with NaOH (1M), extracted with dichloromethane (400 mL methane), combined organic phases, dried over anhydrous sodium sulfate, and concentrated to give crude 5.0g.

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

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

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 (5.1 g, crude) was dissolved in trifluoroacetic acid (30 mL) and reacted at 90 ℃ for 3 hours. Concentration, slurrying of the residue with ethyl acetate (50 mL), 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), pd ₂ (dba) ₃ (46mg, 0.05mmol) and Xantphos (29mg, 0.05mmol) were added in this order to dioxane (50 mL) and reacted at 120 ℃ for 16 hours. Concentration and purification by reverse phase C18 column (water/methanol =10% -35%) gave the target product (50 mg, yield: 13.4%).

Molecular formula C ₂₆ H ₁₉ N ₇ O molecular weight 445.49 LC-MS (M/e): 446.2 (M + H) ⁺ )

¹ H-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 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 (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-diazotized bicyclo 2.2.2 octane bis (tetrafluoroborate) salt (709mg, 2mmol) were dissolved in acetonitrile (10 mL), water (1 mL) was added, and reaction was carried out 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 (122 mg, 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, 0.4 mmol), triethylsilane (1 mL), and trifluoroacetic acid (1 mL) were added to a microwave tube and reacted with microwaves at 70 ℃ for 10 hours. After the reaction was completed, the residue was purified by silica gel column chromatography (methanol: dichloromethane =1 20) to obtain the objective compound (24 mg, yield 20.8%). Preparation of 3,7- (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-dicyclohexylphosphorus-2, 4, 6-triisopropylbiphenyl (4 mg, 20%), palladium acetate (2 mg, 10%) and cesium carbonate (54mg, 0.166mmol) were dissolved in 1, 4-dioxane (5 mL) 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).

The molecular formula is as follows: c ₂₅ H ₂₃ F ₂ N ₇ O molecular weight: 475.5 LC-MS (M/e): 238.5 (M + H/2), 475.9 (M + H)

¹ H-NMR(400MHz,CDCl ₃ )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 9 preparation of (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 (Compound 9)

9.1 Preparation of 7-bromo-5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one

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

9.2 Preparation of 2- ((benzyloxy) methyl) -7-bromo-5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one

7-bromo-5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one (2g, 8.2 mmol) was dissolved in DMF (20 mL), sodium hydrogen (656 mg,16.4 mmol) was added, and after stirring for one hour ((chloromethoxy) methyl) benzene (1.9g, 12.2 mmol) was added, and 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 (50 mL × 2), the organic phases were combined, concentrated, and the residue was subjected to column chromatography (petroleum ether: ethyl acetate = 10) to obtain the product (2.3 g, yield 76.7%).

9.3 Preparation of 2- ((benzyloxy) methyl) -7- (4-cyclopropyl-1H-imidazol-1-yl) -5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one

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

9.4 Preparation of 7- (4-cyclopropyl-1H-imidazol-1-yl) -5-fluoro-3, 4-dihydroisoquinolin-1 (2H) -one

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 (20 mL) and reacted at 80 ℃ for 36 hours. Concentration, addition of dichloro (50 mL) methane and sodium bicarbonate solution (50 mL) to the residue, stirring for half an hour for liquid separation, extraction of the aqueous phase with dichloromethane (20 mL × 2), combination of the organic phases, concentration, and column chromatography of the residue (ethyl acetate: methanol = 20) afforded the product (180 mg, 51.1% yield).

9.5 Preparation of 7- (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

Reacting 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 (10 mL) and reacted at 110 ℃ for 16 hours under nitrogen, after which time the reaction was completed, filtered, the filtrate was concentrated, and the residue was separated by TLC to give the product (85 mg, 50.3% yield).

The molecular formula is as follows: c ₂₅ H ₂₄ FN ₇ Molecular weight of O: 457.5 LC-MS (M/e): 458.2 (M + H) ⁺ )

¹ HNMR(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 10 preparation of 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 (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 (10 mL) were added to acetonitrile (40 mL), DL-alaninol (3.76g, 50.0mmol) was added thereto, and the mixture was heated to 95 ℃ to react for 6 hours. The system was adjusted to pH 8 with sodium hydroxide solution, EA (100 mL) and saturated sodium chloride solution (80 mL) were added, the organic phase was dried over anhydrous sodium sulfate, and the solvent was dried to give the product (1.4 g, 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

7- (4-cyclopropyl-1H-imidazol-1-yl) -3, 4-dihydroisoquinolin-1 (2H) -one (200mg, 0.79mmol), 2- (3- (6-bromopyridin-2-yl) -4H-1,24-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) are added into 1, 4-dioxane (20 mL), the mixture is stirred and reacted for 2 hours at 102 ℃ under the protection of nitrogen, the system is filtered by suction, and the filtrate is filtered through C ₁₈ Column chromatography (water: acetonitrile = 6) purified the product (180 mg, 50.1% yield).

The molecular formula is as follows: c ₂₅ H ₂₅ N ₇ O ₂ Molecular weight: 455.5 LC-MS (M/e): 456.3 (M + H) ⁺ )

¹ H-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 11 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 (Compound 14)

11.1 preparation of methyl (3-fluorophenethyl) carbamate

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

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

To methyl (3-fluorophenethyl) carbamate (9.0 g, 46mmol) was slowly added trifluoromethanesulfonic acid (45mL, 510mmol) at 0 ℃ to react at 70 ℃ for 25 hours, the reaction solution was poured into ice water (300 mL), 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 (6.6 g, 65mmol) into concentrated sulfuric acid solution (65 mL) of 6-fluoro-3, 4-dihydroisoquinoline-1 (2H) -ketone (8.26 g, crude product) at 0 ℃, reacting for 20 minutes at 0 ℃, pouring the reaction solution into ice water (200 mL), filtering, washing (100 mL) of filter cake, pulping (20 mL) with methanol, filtering, drying to obtain crude product, and directly carrying out the next reaction.

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

The crude 6-fluoro-7-nitro-3, 4-dihydroisoquinolin-1 (2H) -one (5.0g, 23.8mmol) and iron powder (6.7g, 119mmol) were added to acetic acid (150 mL) and tetrahydrofuran (70 mL) 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, 155.5 mmol) were added in this order to tetrahydrofuran (55 mL) and toluene (250 mL) and reacted at 100 ℃ for 16 hours. Concentration and purification on silica gel column (petroleum ether: ethyl acetate = 3) gave the product (5.6 g, yield: 66%).

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

Preparation of 7, 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 (500 mg, crude), ammonium acetate (586 mg,7.6 mmol) were added to acetic acid (10 mL), reacted at 110 ℃ for 16 hours, cooled to 0 ℃, adjusted to pH =9 with NaOH (2M), extracted with ethyl acetate (500 mL. Times.2), the organic phases combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give crude (247 mg, 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

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 (20 mL) and reacted at 110 deg.C under nitrogen for 16h, after completion of the reaction, the filtrate was filtered and concentratedThe residue was separated by TLC to give the crude product, which was prepared in reverse phase (32 mg, 18.3% yield).

The molecular formula is as follows: c ₂₅ H ₂₄ FN ₇ O ₂ Molecular weight: 473.5 LC-MS (M/e): 474.2 (M + H) ⁺ )

¹ H-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

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 (10 mL), reacted at 110 ℃ for 16 hours under nitrogen, filtered, the filtrate was concentrated, and the residue was purified by TLC (dichloromethane: methanol = 10.

¹ HNMR(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 compounds 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-dicyclohexylphosphorus-2, 4, 6-triisopropylbiphenyl (10mg, 20%), palladium acetate (5mg, 10%) and cesium carbonate (104mg, 0.318mmol) were dissolved in 1, 4-dioxane (10 mL) 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).

The molecular formula is as follows: c ₂₅ H ₂₃ F ₂ N ₇ O ₂ Molecular weight: 491.5 LC-MS (M/e): 246.7 (M + H/2), 492.2 (M + H)

¹ H-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 14 preparation of 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 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, 1.6 mmol), cuprous iodide (75mg, 0.39mmol) and 8-hydroxyquinoline (57mg, 0.39mmol) were added to DMSO (8 mL) in that order, N ₂ The reaction is carried out for 16 hours at the temperature of 100 ℃ under protection. The reaction was poured into water (50 mL), filtered and cake TLC separated (DCM: meOH = 10).

Molecular formula C ₂₅ H ₂₃ N ₇ O ₂ Molecular weight 453.51 LC-MS (M/e) 454.2 (M + H) ⁺ )

¹ H-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, 0.72mmol), (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 to DMSO (8 mL) in that order, and N was ₂ The reaction is carried out for 16 hours at the temperature of 100 ℃ under protection. The reaction mixture was poured into water (50 mL) and filteredAnd the cake was TLC separated (DCM: meOH = 8) to give the product (50 mg, yield: 15.3%).

¹ H-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 by the embodiment of the invention is shown in the table 1.

Experimental materials:

preparing a reagent:

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. vortexing, mixing well to form a homogeneous solution, and allowing the kinase detection substrate to enter the solution for at least 1min.

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. Stock solutions of Staurosporine and compounds 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 invention, to 384 well plates, respectively.

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 was left at 25 ℃ for 90 minutes, 20. Mu.L of ADP-Glo was added ^TM Reagent was incubated for 40 minutes.

5) Incubation for 35 min with 40. Mu.L of Kinase Detection Regent, enSpire ^TM Fluorescence reading of enzyme label instrumentA light value.

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

Lum _LC represents: luminescence signal intensity of Low control (Staursporine 1. Mu.M);

Lum _cpd represents: indicating the intensity of the luminescent signal of the test compound;

curve fitting is carried out by adopting XLFIT software to obtain IC ₅₀ 。

The experimental results are as follows:

TABLE 4 ASK1 inhibitory Activity of the Compounds of the invention

From the experimental results, the compound prepared by the invention can effectively inhibit the amino acid phosphorylation 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 (luc 2P-AP1-RE-Hygro, AP1 for short), pcDNA3.1/pcDNA3.1-hASK 1), followed by incubation at room temperature for 5min.

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) The mixed solution was added to a culture dish containing HEK-293T cells for transfection, and the cells were incubated at 37 ℃ and 5% CO ₂ Incubate in incubator for 16h.

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) Collecting transfected cells, performing cell counting, diluting the cells with a medium to a desired density according to the cell counting result, adding 30. Mu.L of cell suspension (about 10000 cells) to 384-well cell culture plates containing the compound to be detected, placing the cell culture plates at 37 ℃ and 5% CO ₂ Incubate in incubator for 24h.

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, unfreeze

Luciferase reagent，And equilibrating to room temperature;

3) Adding 30 μ L

And (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 value of the values of the ASK1 and AP1 co-transfected multi-well luminescence signals;

Lum _NC : average of values of the AP1 transfected multiwell luminescence signal;

Lum _sample : means for representing the average of the luminescent signal values of the test compound;

2) Curve fitting is carried out by adopting XLFIT software to obtain IC ₅₀ The 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, the compound of the present invention can effectively block the phosphorylation of ASK1 in cells and block the activation of downstream pathways in vitro cell experiments, 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 experiment of the compound 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 (C57 BL/6), 9/administration route/compound, weight 22-26 g/mouse.

Preparing a test solution:

dissolution scheme:

compound 1 IV administration: 2% DMSO +10% by weight of PEG400+88% (28% HP- β -CD)

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

Compound 2 IV administration: 2% of DMSO +10% the percentage of PEG400+88% (weight: HP- β -CD)

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

Compound 3 IV administration: 3% of DMSO +5% the percentage PEG400+92% (weight: HP- β -CD)

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

Preparation of a blank solvent 1:

method for preparing 28-percent HP-beta-CD solution: HP- β -CD (hydroxypropyl betacyclodextrin) (28 g) was weighed and sterilized water for injection (72 mL) was added to obtain a 28% HP- β -CD solution.

Preparation of a blank solvent 2:

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

Preparation of a blank solvent 3:

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

The preparation method comprises the following steps:

compound 1:

weighing the compound 1 (1.48 mg), 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 (2.131 mL), 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 (1.51 mg) and SDS (sodium dodecyl sulfate) (4.9 mg) were weighed, added to blank vehicle 3 (4.943 mL), ground, vortexed and mixed to prepare a 0.3mg/mL homogeneous suspension as compound 1C57BL/6 mouse PO dosing solution 2.

Compound 2:

weighing a compound 2 (1.50 mg), 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 (2.05 mL), 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 (1.51 mg) was weighed, added to blank vehicle 2 (4.691 mL), 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 a compound 3 (1.57 mg), adding DMSO (dimethyl sulfoxide) (72.6 mu L), carrying out vortex and ultrasonic dissolution, adding PEG400 (polyethylene glycol 400) (121 mu L), carrying out vortex mixing, finally adding a blank solvent 1 (2.230 mL), carrying out vortex mixing, preparing into a uniform solution with the concentration of 0.6mg/mL, and taking the solution as a compound 3C57BL/6 mouse IV administration liquid medicine 1;

compound 3 (1.50 mg) was weighed, added to blank vehicle 2 (4.620 mL), 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 3C57BL/6 mouse.

Experimental method

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

About 100 μ L of blood was collected from the inner canthus at each time point and placed in the container containing EDTA-K ₂ Centrifuging blood sample at 4 deg.C at 8000 rpm for 6min in anticoagulation tube of anticoagulantTo plasma samples, the plasma was cryopreserved in a-80 ℃ freezer.

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, then adding 100 mu L of water, vortexing and uniformly mixing for 3min, and then analyzing by LC-MS/MS.

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 _inf Area 0 → ∞ under curve when drug is represented

CL stands for clearance

V _ss Representing steady state apparent distribution volume

T _max Representing the time to peak of blood concentration

C _max Representing peak plasma concentration

T _1/2 Represents 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/patient.

Preparing a test solution:

dissolution scheme:

compound IV administration: 5% DMSO +20% by weight of PEG400+75% (28% HP- β -CD)

Compound PO administration: 2% of HPC +0.1%

Preparation of a blank solvent 1:

method for preparing 28% HP-beta-CD solution: HP- β -CD (hydroxypropyl betacyclodextrin) (28 g) was weighed and sterilized water for injection (72 mL) was added to obtain a 28% HP- β -CD solution.

Preparation of a blank solvent 2:

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

The preparation method comprises the following steps:

compound 2:

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

(2) compound 2 (80.37 mg) was weighed, added with blank vehicle 2 (197.7 mL), ground, suspended uniformly, and prepared into a uniform suspension with a concentration of 0.4mg/mL, which was used as compound 2 beagle PO administration solution 2.

Experimental method

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-K ₂ Centrifuging 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, then adding 100 mu L of water, vortexing and uniformly mixing for 3min, and then analyzing by LC-MS/MS.

The experimental results are as follows:

table 8: beagle PK evaluation results (IV)

AUC _inf Area 0 → ∞ under curve when drug is represented

CL stands for clearance

V _ss Representing steady state apparent distribution volume

T _max Representing the peak time of blood drug concentration

C _max Representing peak plasma concentration

T _1/2 Represents 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 are described in detail above. The principles and embodiments of the present invention have been described 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 modifications and variations can be made in the present invention without departing from the principles of the invention, and such modifications and variations are intended to be included within the scope of the claims.

Claims

1. A compound represented by the general formula (III), a pharmaceutically acceptable salt thereof or a stereoisomer thereof,

wherein the content of the first and second substances,

R ¹ selected from the group consisting of optionally substituted by 1 to 3Q ¹ Substituted C _1-6 Alkyl radical, each Q ¹ Independently selected from halogen, hydroxy, amino, C _1-6 Alkyl radical, C _1-6 Alkoxycarbonyl, aminoacyl, C _1-6 Alkylaminoacyl or C _1-6 An alkylamino group;

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, halogen, C _1-6 Alkyl radical, C _1-6 Alkoxy or halo C _1-6 An alkoxy group;

R ³ selected from hydrogen or halogen;

R ⁵ selected from the group consisting of optionally substituted by 1 to 3Q ² A substituted imidazolyl group;

each Q ² Is independently selected from C _1-6 Alkyl, 3-6 membered cycloalkyl wherein said C is _1-6 Alkyl, or 3-6 membered cycloalkyl further optionally substituted with 1 to 3 halogens;

selected from single bonds or double bonds;

and the compound is not:

2. the compound, pharmaceutically acceptable salt thereof, or stereoisomer thereof according to claim 1,

R ¹ selected from the group consisting of optionally substituted by 1 to 2Q ¹ Substituted methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, each Q ¹ Independently selected from the group consisting of halogen, hydroxy, amino, methyl, ethyl, methoxycarbonyl, ethoxycarbonyl, methylamino, ethylamino, aminoacyl, methylamyl, ethylamyl;

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, fluoro, bromo, chloro, iodo, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, difluoromethoxy or trifluoromethoxy;

R ³ selected from hydrogen, fluorine, bromine, chlorine or iodine;

R ⁵ selected from the group consisting of optionally substituted 1 to 2Q ² A substituted imidazolyl group;

each Q ² Independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, cyclopropyl, cyclobutylAlkyl, cyclopentyl, cyclohexyl, wherein said methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl are further optionally substituted by 1 to 3 halogens.

3. A compound represented by the general formula (V), a pharmaceutically acceptable salt thereof or a stereoisomer thereof,

wherein the content of the first and second substances,

R ³ selected from hydrogen or halogen;

R ⁵ selected from the group consisting of optionally substituted 1 to 3Q ² A substituted imidazolyl group;

each Q ² Is independently selected from C _1-6 Alkyl or 3-6 membered cycloalkyl, wherein said 3-6 membered cycloalkyl is further optionally substituted by 1 to 3 halogens.

4. The compound of claim 3, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof,

R ² 、R ⁴ 、R ⁶ 、R ⁷ each independently selected from hydrogen, fluoro, bromo, chloro, iodo, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy or trifluoromethoxy;

R ³ selected from hydrogen, fluorine, bromine, chlorine or iodine;

each Q ² Selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, wherein said cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl are further optionally substituted by 1 to 3 halogens.

5. A compound, pharmaceutically acceptable salt thereof, or stereoisomer thereof according to claim 1 or claim 3, selected from:

6. 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 a general formula (III);

wherein R is ¹ 、R ² 、R ³ 、R ⁴ 、R ⁵ 、R ⁶ 、R ⁷ N are as defined in any of claims 1-2, and substituent a is selected from a leaving group, such as halogen.

7. A pharmaceutical composition comprising a compound of any one of claim 1 or claim 3, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and optionally one or more second therapeutically active agents.

8. The pharmaceutical composition of claim 7, further comprising one or more pharmaceutically acceptable carriers and/or diluents.

9. A pharmaceutical formulation comprising a compound according to any one of claims 1 or 3, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, and one or more pharmaceutically acceptable carriers and/or diluents; the pharmaceutical preparation is any clinically or pharmaceutically acceptable dosage form.

10. The pharmaceutical preparation of claim 9, which is an oral preparation or an injection.

11. The pharmaceutical formulation of claim 10, further comprising one or more second therapeutically active agents.

12. Use of a compound of any one of claim 1 or claim 3, a pharmaceutically acceptable salt or a stereoisomer thereof, or a pharmaceutical composition of claim 7, or a pharmaceutical formulation of claim 9, in the manufacture of a medicament for the treatment and/or prevention of ASK1 mediated diseases and related diseases.

13. The use according to claim 12, wherein the ASK1 mediated diseases and related diseases are selected from cardiovascular diseases, pulmonary diseases, metabolic diseases, diseases of the liver, gallbladder and kidney, hyperproliferative diseases, neurodegenerative diseases, stroke or inflammatory bowel diseases.

14. The use according to claim 13, wherein the cardiovascular disease is selected from pulmonary hypertension, multiple sclerosis, atherosclerosis, angina pectoris, intermittent claudication syndrome, myocardial infarction, heart failure; the pulmonary disease is selected from asthma, chronic obstructive pulmonary disease, bronchitis or emphysema; said metabolic disorder is selected from the group consisting of diabetes and its complications, metabolic syndrome, dyslipidemia, obesity, glucose intolerance, hypertension, elevated serum cholesterol, elevated triglycerides; the hepatobiliary and renal diseases are selected from diabetic nephropathy, non-alcoholic fatty liver disease, bile acid disorder, primary sclerosing cholangitis, cholesterol calculus, fatty liver, liver cirrhosis, hepatitis, liver failure, cholestasis, and cholelithiasis; the hyperproliferative diseases are selected from gastric cancer, liver cancer, polyposis, colon cancer, breast cancer, pancreatic cancer and esophageal cancer.