CN114591334B

CN114591334B - Dihydropyrazolopyrimidinone derivatives

Info

Publication number: CN114591334B
Application number: CN202111465345.0A
Authority: CN
Inventors: 刘斌
Original assignee: Xuanzhu Pharma Co Ltd
Current assignee: Shandong Xuanzhu Pharma Co Ltd
Priority date: 2020-12-04
Filing date: 2021-12-03
Publication date: 2023-10-20
Anticipated expiration: 2041-12-03
Also published as: CN114591334A

Abstract

The invention belongs to the technical field of medicines, and in particular relates to a dihydropyrazolopyrimidinone derivative compound serving as a Wee1 kinase inhibitor, a pharmaceutically acceptable salt or stereoisomer thereof, a pharmaceutical composition and a preparation containing the compound, the pharmaceutically acceptable salt or stereoisomer thereof, a method for preparing the compound, the pharmaceutically acceptable salt or stereoisomer thereof, and application of the compound, the pharmaceutically acceptable salt or stereoisomer thereof.

Description

Dihydropyrazolopyrimidinone derivatives

Technical Field

The invention belongs to the technical field of medicines, and in particular relates to a dihydropyrazolopyrimidinone derivative compound, pharmaceutically acceptable salts thereof and stereoisomers thereof, a pharmaceutical composition and a preparation containing the compound, the pharmaceutically acceptable salts thereof and stereoisomers thereof, a method for preparing the compound, the pharmaceutically acceptable salts thereof and stereoisomers thereof, and application of the compound, the pharmaceutically acceptable salts thereof and stereoisomers thereof.

Background

Cancer is a malignant disease which is difficult to treat worldwide, and has high treatment difficulty and high death rate. Recent global tumor statistics in 2018 show that up to now there are estimated 1819 thousand new cases of cancer and 960 ten thousand cases of cancer death worldwide. The national cancer center published the latest national cancer statistics at month 1 of 2019. About 392.9 thousands of malignant tumors occur annually, with an average of over 1 million diagnosed with cancer per day, and 7.5 diagnosed with cancer per minute. Cancer has become a major disease affecting the health of residents in China, and cancer prevention and treatment are also facing a serious form.

Currently, radiation therapy and chemotherapy are the most effective means of treating cancer in addition to surgical resection, while radiation therapy is the most effective non-surgical treatment for malignant tumors. Both radiation and considerable amounts of anticancer drugs can cause DNA damage. After DNA damage, a series of cellular reactions such as damaged DNA repair can be initiated to improve the survival of tumor cells, which is also one of the mechanisms of tumor cells against chemoradiotherapy. Damaged DNA, if not timely and intact, tumor cells can cause cell death due to apoptosis or/and mitotic disorders. Therefore, the sensitivity of cancer cells to radiotherapy and chemotherapy can be improved and the proliferation of cells can be inhibited as long as the repair of the DNA damage is inhibited.

Wee1 protein kinase is a member of the serine/threonine protein kinase family, and was originally isolated by Nurse et al in merozoite cells (S.pombe). In humans Wee1 contains 647 amino acids with a molecular weight of 96 kDa. In the path of DNA single-chain damage repair, wee1 is positioned at the downstream of an ATR signal path, after the ATR signal path is activated, CHK1 is phosphorylated, and the activated CHK1 activates Wee1 and simultaneously inhibits CDC25 (the phosphorylation of CDK1/Cyclin B complex is released, the activity of the CDK1/Cyclin B complex for regulating and controlling the cell cycle is recovered), so that CDK1/Cyclin B complex is phosphorylated, CDK1/Cyclin B complex is made to enter an inactive state, the cell cycle is blocked in G2/M phase, and time is gained for DNA damage repair; in addition, weel can regulate repair of DNA double strand breaks that occur during DNA replication by phosphorylating CDK2, blocking the S phase of the cell cycle.

Wee1 acts primarily at the G2/M phase checkpoint throughout the DDR (DNA damage repair) pathway. For p53 mutated tumor cells, due to the presence of the G1/S checkpoint defect itself, the G2/M phase checkpoint is more relied on to repair DNA damage, and Wee1 inhibitors are more sensitive to p53 mutated tumor cells in terms of mechanism. In addition, the Wee1 inhibitor can be combined with other targets on the DDR channel, for example, tumor cells have ATM defects/mutation, and can be combined with ATR inhibitors, so that the damage repair of DNA can be blocked more completely, and the synthetic lethal effect is realized.

In conclusion, the Wee1 kinase inhibitor not only can synergistically enhance the effects of radiotherapy and chemotherapy and effectively inhibit the growth of tumors, but also can reduce the damage to normal cells and reduce side effects. At present, the research of the target drug is still in the clinical experimental stage, and no drug is marketed yet. Therefore, the development of a high-efficiency Wee1 kinase inhibitor has important clinical significance, and has wide market prospect in single use or combined with other medicine applications.

Disclosure of Invention

The invention aims to provide a dihydropyrazolopyrimidinone derivative compound with novel structure and inhibitory activity on Wee 1. Furthermore, the compounds can be used for inhibiting the activity of Wee1 kinase, thereby enhancing the immunity of organisms to tumors. Furthermore, the compounds are useful in the treatment of one or more diseases mediated by Wee1, particularly cancer. The compound has good inhibition effect on various cancer cells, and has higher exposure and better in vivo efficacy in organisms.

The technical scheme of the invention is as follows:

in one aspect, the present invention provides a compound of the following formula (I), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof,

wherein, the liquid crystal display device comprises a liquid crystal display device,

X ¹ 、X ² 、X ³ are each independently selected from-C (R) ² ) -or-N-;

X ⁴ 、X ⁵ each independently selected from-CH-or-N-;

each L is independently selected from the group consisting of-C (R ³ )(R ⁴ )-、-O-、-N(R ⁵ ) -or-S-;

ring a is selected from 3-10 membered cycloalkyl optionally substituted with 1-4Q 1, 3-10 membered heterocyclyl, 6-10 membered aryl or 5-10 membered heteroaryl;

ring B is selected from 3-10 membered cycloalkyl optionally substituted with 1-4Q 2, 3-10 membered heterocyclyl, 6-10 membered aryl or 5-10 membered heteroaryl;

ring C is selected from a 5-15 membered bridged ring group, a 5-10 membered spiro ring group, a 5-15 membered bridged ring group or a 5-10 membered spiro ring group optionally substituted with 1-4Q 3 groups, wherein the ring-forming carbon atoms in said 5-15 membered bridged ring group, 5-10 membered spiro ring group, 5-15 membered bridged ring group or 5-10 membered spiro ring group may be optionally substituted with oxygen to form a carbonyl group;

each Q1 is independently selected from halogen, hydroxy, amino, nitro, cyano, carboxy, or a group optionally substituted with 1-3 substituents: c (C) _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylthio, said substituents each being independently selected from halogen, hydroxy, amino, nitro, cyano, carboxy, - (CH) ₂ ) _m -3-10 membered cycloalkyl, - (CH) ₂ ) _m -3-10 membered heterocycloalkyl, - (CH) ₂ ) _m -5-to 10-membered heteroaryl or- (CH) ₂ ) _m -6-10 membered aryl;

each Q2, each Q3 is independently selected from halogen, hydroxy, amino, nitro, cyano, carboxy, C _1-6 Alkyl, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylthio, halo C _1-6 Alkoxy, halo C _1-6 Alkylthio, hydroxy C _1-6 Alkoxy, hydroxy C _1-6 Alkylthio, amino C _1-6 Alkoxy, amino C _1-6 Alkylthio, - (CH) ₂ ) _m -3-10 membered cycloalkyl, - (CH) ₂ ) _m -3-10 membered heterocycloalkyl, - (CH) ₂ ) _m -5-to 10-membered heteroaryl or- (CH) ₂ ) _m -6-10 membered aryl;

R ¹ selected from C optionally substituted by substituents _1-6 Alkyl, C _2-6 Alkenyl or C _2-6 Alkynyl groups, the substituents each being independently selected from halogen, hydroxy, amino, carboxy, cyano, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, halogenated C _1-6 Alkoxy or C _1-6 An alkylcarbonyl group;

R ² 、R ³ 、R ⁴ 、R ⁵ independently selected from hydrogen, halogen, hydroxy, amino, carboxy, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, halogenated C _1-6 Alkoxy or C _1-6 An alkylcarbonyl group;

m, n are each independently selected from 0, 1, 2 or 3.

In certain embodiments, the compound, pharmaceutically acceptable salt thereof, or stereoisomer thereof, wherein,

ring a is selected from 3-8 membered monocyclic cycloalkyl optionally substituted with 1-4Q 1, 3-8 membered mono heterocyclyl, phenyl or 5-8 membered mono heteroaryl;

ring B is selected from phenyl optionally substituted with 1-4Q 2 or 5-8 membered mono heteroaryl;

each Q2 is independently selected from halogen, hydroxy, amino, nitro, cyano, carboxyl, C _1-6 Alkyl, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylthio, halo C _1-6 Alkoxy, halo C _1-6 Alkylthio, hydroxy C _1-6 Alkoxy, hydroxy C _1-6 Alkylthio, amino C _1-6 Alkoxy, amino C _1-6 Alkylthio;

ring C is selected from a 5-15 membered bridged heterocyclyl or a 5-10 membered spiroheterocyclyl optionally substituted with 1-4Q 3, wherein the ring-forming carbon atoms in said 5-15 membered bridged heterocyclyl or 5-10 membered spiroheterocyclyl may be optionally substituted with oxygen to form a carbonyl group;

each Q3 is independently selected from halogen, hydroxy, amino, nitro, cyano, carboxyl, C _1-6 Alkyl, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylthio, halo C _1-6 Alkoxy, halo C _1-6 Alkylthio, hydroxy C _1-6 Alkoxy, hydroxy C _1-6 Alkylthio, amino C _1-6 Alkoxy or amino C _1-6 Alkylthio, - (CH) ₂ ) _m -3-10 membered cycloalkyl, - (CH) ₂ ) _m -3-10 membered heterocycloalkyl, - (CH) ₂ ) _m -5-to 10-membered heteroaryl or- (CH) ₂ ) _m -6-10 membered aryl;

m and n are each independently selected from 0, 1 or 2.

In certain embodiments, ring a is selected from phenyl optionally substituted with 1-3Q 1 or a 5-6 membered nitrogen containing mono heteroaryl;

in certain embodiments, ring a is selected from phenyl substituted with 1Q 1 or a 5-6 membered nitrogen containing mono heteroaryl;

preferably, ring a is selected from phenyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl substituted with 1Q 1; q1 is selected from halogen, hydroxy, amino, nitro, cyano, carboxy or optionally substituted with 1-3 substituents of the following groups: c (C) _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylthio, said substituents each being independently selected from halogen, hydroxy, amino, nitro, cyano, carboxy, - (CH) ₂ ) _m -3-10 membered cycloalkyl, - (CH) ₂ ) _m -3-10 membered heterocycloalkyl, - (CH) ₂ ) _m -5-to 10-membered heteroaryl or- (CH) ₂ ) _m -6-10 membered aryl.

In certain embodiments, ring a is selected from phenyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, or pyridazinyl substituted with 1Q 1; q1 is selected from halogen, hydroxy, amino, nitro, cyano, carboxy or optionally substituted with 1-3 substituents of the following groups: c (C) _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylthio, said substituents each being independently selected from halogen, hydroxy, amino, nitro, cyano, carboxy, - (CH) ₂ ) _m -3-6 membered cycloalkyl, - (CH) ₂ ) _m -3-6 membered heterocycloalkyl, - (CH) ₂ ) _m -5-6 membered heteroaryl or- (CH) ₂ ) _m -6-8 membered aryl.

In certain embodiments, ring A is selected from pyridinyl, pyrimidinyl substituted with 1Q 1A pyridyl, pyrazinyl or pyridazinyl group; q1 is selected from halogen, hydroxy, amino, nitro, cyano, carboxy or optionally substituted with 1-3 substituents of the following groups: c (C) _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylthio, the substituents are each independently selected from halogen, hydroxy, amino, nitro, cyano or carboxyl.

In certain embodiments, ring B is selected from phenyl optionally substituted with 1 or 2Q 2 or a 5-6 membered mono heteroaryl; each Q2 is independently selected from halogen, hydroxy, amino, nitro, cyano, C _1-6 Alkyl, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkoxy, hydroxy C _1-6 Alkoxy or amino C _1-6 An alkoxy group.

In certain embodiments, ring B is selected from phenyl, pyridinyl, pyrazinyl, or pyridazinyl optionally substituted with 1 or 2Q 2; each Q2 is independently selected from halogen, hydroxy, amino, nitro, cyano, C _1-6 Alkyl, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkoxy, hydroxy C _1-6 Alkoxy or amino C _1-6 An alkoxy group.

In certain embodiments, ring B is selected from phenyl, pyridinyl, pyrazinyl, or pyridazinyl optionally substituted with 1 or 2Q 2; each Q2 is independently selected from fluorine, chlorine, bromine, iodine, cyano, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, monofluoromethyl, difluoromethyl, trifluoromethyl, methoxy, ethoxy, propoxy, isopropoxy, monofluoromethoxy, difluoromethoxy or trifluoromethoxy.

ring B is selected from phenyl optionally substituted with 1 or 2Q 2 or 5-6 membered mono heteroaryl; each Q2 is independently selected from halogen, hydroxy, amino, nitro, cyano, C _1-6 Alkyl, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkoxy, hydroxy C _1-6 Alkoxy or amino C _1-6 An alkoxy group.

In certain embodiments, ring C is selected from a 5-15 membered nitrogen containing bridged heterocyclyl or a 5-10 membered nitrogen containing spiro heterocyclyl optionally substituted with 1-4Q 3, wherein the ring-forming carbon atoms in said 5-15 membered nitrogen containing bridged heterocyclyl or 5-10 membered nitrogen containing spiro heterocyclyl may be optionally substituted with oxygen to form carbonyl;

Each Q3 is independently selected from halogen, hydroxy, amino, nitro, cyano, carboxyl, C _1-6 Alkyl, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylthio, halo C _1-6 Alkoxy, halo C _1-6 Alkylthio, hydroxy C _1-6 Alkoxy, hydroxy C _1-6 Alkylthio, amino C _1-6 Alkoxy or amino C _1-6 Alkylthio groups.

In certain embodiments, ring C is selected from a 7-9 membered nitrogen containing bridged heterocyclyl or a 7-9 membered nitrogen containing spiro heterocyclyl optionally substituted with 1-2Q 3, wherein the ring forming carbon atoms in said 7-9 membered nitrogen containing bridged heterocyclyl or 7-9 membered nitrogen containing spiro heterocyclyl may be optionally substituted with oxygen to form carbonyl;

each Q3 is independently selected from halogen, hydroxy, amino, nitro, cyano, carboxyl, C _1-6 Alkyl, halogenated C _1-6 Alkyl, hydroxy C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkoxy or hydroxy C _1-6 An alkoxy group.

In certain embodiments, ring C is selected from a 7-8 membered nitrogen containing bridged heterocyclyl or a 7-8 membered nitrogen containing spiro heterocyclyl optionally substituted with 1-2Q 3, wherein the ring-forming carbon atoms in said 7-8 membered nitrogen containing bridged heterocyclyl or 7-8 membered nitrogen containing spiro heterocyclyl may be optionally substituted with oxygen to form carbonyl;

each Q3 is independently selected from halogen, hydroxy, amino, nitro, cyano, carboxyl, C _1-6 Alkyl, halogenated C _1-6 Alkyl, hydroxy C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkoxy, hydroxy C _1-6 Alkoxy or 3-4 membered cycloalkyl.

In certain embodiments, ring C is selected from a 7-8 membered, 1-2 nitrogen containing bridged heterocyclyl optionally substituted with 1-2Q 3 groups or a 7-8 membered, 1-2 nitrogen containing spiroheterocyclyl wherein the ring forming carbon atoms in the bridged heterocyclyl or spiroheterocyclyl groups may be optionally substituted with oxygen to form a carbonyl group;

In certain embodiments, ring C is selected from a 7-8 membered 2 nitrogen containing bridged heterocyclyl optionally substituted with 1-2Q 3 or a 7-8 membered 2 nitrogen containing spiroheterocyclyl wherein the ring forming carbon atoms in the bridged heterocyclyl or spiroheterocyclyl may be optionally substituted with oxygen to form a carbonyl group;

In certain embodiments, ring C is selected from the following groups optionally substituted with 1-2Q 3:

In certain embodiments, ring C is selected from a 7-9 membered nitrogen containing bridged heterocyclyl optionally substituted with 1-2Q 3, wherein the ring forming carbon atoms in said 7-9 membered nitrogen containing bridged heterocyclyl may be optionally substituted with oxygen to form carbonyl;

each Q3 is independently selected from halogen, hydroxy, amino, nitro, cyano, carboxyl, C _1-6 Alkyl, halogenated C _1-6 Alkyl, hydroxy C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkoxy, hydroxy C _1-6 Alkoxy or 3-4 membered cycloalkyl. In certain embodiments, ring C is selected from a 7-8 membered, 1-2 nitrogen containing, bridged heterocyclyl optionally substituted with 1-2Q 3, wherein the ring-forming carbon atoms in said bridged heterocyclyl may be optionally substituted with oxygen to form carbonyl;

In certain embodiments, ring C is selected from the following groups optionally substituted with 1-2Q 3: 2-azabicyclo [2.2.1] heptanyl, 7-azabicyclo [2.2.1] heptanyl, 3-azabicyclo [3.2.1] octanyl, 8-azabicyclo [3.2.1] octanyl, 2-azabicyclo [2.2.2] octanyl, 2, 5-diazabicyclo [2.2.1] heptanyl, 3, 6-diazabicyclo [3.1.1] heptanyl, 3, 8-diazabicyclo [3.2.1] octanyl, 2-oxa-5-azabicyclo [2.2.1] heptanyl, 8-oxa-3-azabicyclo [3.2.1] octanyl, 3, 8-diazabicyclo [3.2.1] oct-6-enyl or 3, 9-diazabicyclo [3.1 ] nonanyl, wherein the carbon atoms may optionally be substituted to form a carbonyl group;

In certain embodiments, ring C is selected from the following groups optionally substituted with 1-2Q 3: 2-azabicyclo [2.2.1] heptyl, 7-azabicyclo [2.2.1] heptyl, 3-azabicyclo [3.2.1] octyl, 8-azabicyclo [3.2.1] octyl, 2-azabicyclo [2.2.2] octyl, 2, 5-diazabicyclo [2.2.1] heptyl, 3, 6-diazabicyclo [3.1.1] heptyl or 3, 8-diazabicyclo [3.2.1] octyl, wherein the groups are optionally substituted with oxygen to form carbonyl groups;

each Q3 is independently selected from fluorine, chlorine, hydroxy, amino, nitro, cyano, carboxy, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, isopropoxy, hydroxymethyl, hydroxyethyl, hydroxyisopropyl, trifluoromethyl, trifluoromethoxy, or cyclopropyl.

In certain embodiments, ring C is selected from the following groups optionally substituted with 1-2Q 3: 2, 5-diazabicyclo [2.2.2] octanyl, 2, 5-diazabicyclo [2.2.1] heptanyl, 3, 6-diazabicyclo [3.1.1] heptanyl or 3, 8-diazabicyclo [3.2.1] octanyl, wherein the ring carbon atoms of the groups may optionally be substituted with oxygen to form carbonyl groups;

In certain embodiments, ring C is selected from a 7-9 membered nitrogen containing spiroheterocyclyl optionally substituted with 1-2Q 3, wherein the ring-forming carbon atoms in said 7-9 membered nitrogen containing spiroheterocyclyl may be optionally substituted with oxygen to form a carbonyl group;

In certain embodiments, ring C is selected from a 7-8 membered 1-2 nitrogen containing spiroheterocyclyl optionally substituted with 1-2Q 3, wherein the ring-forming carbon atoms in said spiroheterocyclyl may be optionally substituted with oxygen to form carbonyl;

In certain embodiments, ring C is selected from the following groups optionally substituted with 1-2Q 3: 5-azaspiro [2.4] heptyl, 2-azaspiro [3.3] heptyl, 2-azaspiro [3.5] nonyl, 2, 6-diazaspiro [3.3] heptyl, 2, 7-diazaspiro [3.5] nonyl, 2, 5-diazaspiro [3.4] octyl, 2-oxa-6-azaspiro [3.3] heptyl, 6-oxa-2-azaspiro [3.4] octyl, 6-azaspiro [3.4] octyl, 2-azaspiro [4.4] nonyl, 2-oxa-7-azaspiro [4.4] nonanyl, 6-azaspiro [3.4] oct-7-enyl, 2-oxa-6-azaspiro [3.4] oct-7-enyl or 2-azaspiro [4.4] nonenyl, wherein the carbon atoms may be optionally substituted by carbonyl groups;

In certain embodiments, ring C is selected from the following groups optionally substituted with 1-2Q 3: 5-azaspiro [2.4] heptyl, 2-azaspiro [3.3] heptyl, 2-azaspiro [3.5] nonyl, 2, 6-diazaspiro [3.3] heptyl, 2, 7-diazaspiro [3.5] nonyl, 2, 5-diazaspiro [3.4] octyl, 2-oxa-6-azaspiro [3.3] heptyl, 6-oxa-2-azaspiro [3.4] octyl, 6-azaspiro [3.4] octyl, 2-azaspiro [4.4] nonyl, wherein the groups are optionally substituted with oxygen to form a carbonyl group on a ring carbon atom;

In certain embodiments, ring C is selected from the following groups optionally substituted with 1-2Q 3: 2, 6-diazaspiro [3.3] heptyl, 2, 7-diazaspiro [3.5] nonyl, 2, 5-diazaspiro [3.4] octyl, wherein the ring carbon atoms of the groups may optionally be substituted with oxygen to form carbonyl groups;

each Q3 is independently selected from fluorine, chlorine, hydroxy, amino, nitro, cyano, carboxy, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, isopropoxy, hydroxymethyl, hydroxyethyl, hydroxyisopropyl, trifluoromethyl, trifluoromethoxy, or cyclopropyl. In certain embodiments, ring C is selected from the following groups optionally substituted with 1-2Q 3: 2-azabicyclo [2.2.1] heptyl, 7-azabicyclo [2.2.1] heptyl, 3-azabicyclo [3.2.1] octyl, 8-azabicyclo [3.2.1] octyl, 2-azabicyclo [2.2.2] octyl, 2, 5-diazabicyclo [2.2.1] heptyl, 3, 6-diazabicyclo [3.1.1] heptyl, 3, 8-diazabicyclo [3.2.1] octyl, 2-oxa-5-azabicyclo [2.2.1] heptyl, 8-oxa-3-azabicyclo [3.2.1] octyl, 3, 8-diazabicyclo [3.2.1] oct-6-alkenyl, 3, 9-diazabicyclo [3.1 ] nonyl, 5-azaspiro [2.4] heptyl, 2-azaspiro [3.3] heptyl, 2-azaspiro [3.5] nonyl, 2, 6-diazaspiro [3.3] heptyl, 2, 7-diazaspiro [3.5] nonyl, 2, 5-diazaspiro [3.4] octyl, 2-oxa-6-azaspiro [3.3] heptyl, 6-oxa-2-azaspiro [3.4] octyl, 6-azaspiro [3.4] octyl, 2-azaspiro [4.4] nonyl, 2-oxa-7-azaspiro [4.4] nonanyl, 6-azaspiro [3.4] oct-7-enyl, 2-oxa-6-azaspiro [3.4] oct-7-enyl or 2-azaspiro [4.4] nonenyl, the ring-forming carbon atoms of the groups may optionally be substituted with oxygen to form carbonyl groups;

Each Q3 is independently selected from halogen, hydroxy, amino, nitro, cyano, carboxyl, C _1-6 Alkyl, haloC _1-6 Alkyl, hydroxy C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkoxy or hydroxy C _1-6 An alkoxy group.

In certain embodiments, R ¹ Selected from C optionally substituted by substituents _2-4 Alkenyl groups, the substituents each being independently selected from halogen, hydroxy, amino, carboxy, cyano, C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkyl or halo C _1-6 An alkoxy group.

In certain embodiments, R ¹ Selected from-CH optionally substituted by substituents ₂ -CH＝CH ₂ The substituents are each independently selected from halogen, hydroxy, amino, carboxy, cyano, C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkyl or halo C _1-6 An alkoxy group.

In certain embodiments, R ¹ Selected from-CH ₂ -CH＝CH ₂ 。

In certain embodiments, a compound of the invention, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein X ¹ 、X ² 、X ³ Are each independently selected from-C (R) ² ) -or-N-;

X ⁴ 、X ⁵ each independently selected from-CH-or-N-;

R ² 、R ³ 、R ⁴ 、R ⁵ are independently selected from hydrogen, C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl or halo C _1-6 An alkoxy group;

R ¹ selected from C optionally substituted by substituents _2-6 Alkenyl groups, the substituents each being independently selected from halogen, hydroxy, amino, carboxy, cyano, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxyRadical C _1-6 Alkyl, amino C _1-6 Alkyl or halo C _1-6 An alkoxy group.

In certain embodiments, R ² 、R ³ 、R ⁴ 、R ⁵ Each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, isopropoxy, monofluoromethyl, difluoromethyl, trifluoromethyl or trifluoromethoxy.

In certain embodiments, X ¹ 、X ² 、X ³ Are each independently selected from-C (R) ² ) -or-N-;

X ⁴ 、X ⁵ each independently selected from-CH-or-N-;

R ² 、R ³ 、R ⁴ 、R ⁵ each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, isopropoxy, monofluoromethyl, difluoromethyl, trifluoromethyl or trifluoromethoxy;

R ¹ selected from C optionally substituted by substituents _2-6 Alkenyl groups, the substituents each being independently selected from halogen, hydroxy, amino, carboxy, cyano, C _1-6 Alkyl, C _1-6 Alkoxy, C _1-6 Alkylamino, di (C) _1-6 Alkyl) amino, halo C _1-6 Alkyl, hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl or halo C _1-6 An alkoxy group.

In certain embodiments, R ¹ Selected from C optionally substituted by substituents _2-4 Alkenyl, preferably propen-3-yl, optionally substituted with substituents independently selected from halogen, hydroxy, amino, carboxy, cyano, C _1-6 Alkyl, C _1-6 Alkoxy, halo C _1-6 Alkyl or halo C _1-6 An alkoxy group.

In certain embodiments, X ¹ 、X ² Each independently selected from-N-.

In certain embodiments, X ³ is-CH-.

In certain embodiments, X ⁴ 、X ⁵ Each independently-N-.

In certain embodiments, the compounds of the present invention, pharmaceutically acceptable salts thereof, or stereoisomers thereof, have the structure shown in formula (II):

wherein, ring A, ring B, ring C, Q, Q2, Q3, L, R ³ 、R ⁴ 、R ⁵ 、R ¹ The definition of m and n is as described above.

In certain embodiments, the compounds of the present invention, pharmaceutically acceptable salts thereof, or stereoisomers thereof, have the structure shown in formula (III):

wherein, ring A, ring B, ring C, Q, Q2, Q3, L, R ³ 、R ⁴ 、R ⁵ The definition of m and n is as described above.

Wherein ring a is selected from pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl substituted with 1Q 1; q1 is selected from fluorine, chlorine, bromine, hydroxyl, amino, nitro, cyano or the following optionally substituted with 1-3 substituents: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, each of said substituents being independently selected from fluorine, chlorine, bromine, hydroxy, amino, nitroRadical, cyano, carboxyl, - (CH) ₂ ) _m -cyclopropane, - (CH) ₂ ) _m -cyclobutyl, - (CH) ₂ ) _m Cyclopentylalkyl radicals or- (CH) ₂ ) _m -a cyclohexenyl group;

ring B is phenyl or pyridinyl;

each L is independently selected from the group consisting of-C (R ³ )(R ⁴ ) -or-N (R) ⁵ )-；R ³ 、R ⁴ 、R ⁵ Each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, isopropoxy, monofluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl, monofluoromethoxy, difluoromethoxy or trifluoromethoxy;

m is selected from 0, 1 or 2; n is 1.

In certain embodiments, the compounds of the present invention, pharmaceutically acceptable salts thereof, or stereoisomers thereof, wherein,

ring a is selected from pyridinyl, pyrimidinyl, pyrazinyl or pyridazinyl substituted with 1Q 1; q1 is selected from fluorine, chlorine, bromine, hydroxyl, amino, nitro, cyano or the following optionally substituted with 1-3 substituents: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, propoxy, isopropoxy, each of said substituents being independently selected from fluoro, chloro, bromo, hydroxy, amino, nitro, cyano, carboxy, - (CH) ₂ ) _m -cyclopropane, - (CH) ₂ ) _m -cyclobutyl, - (CH) ₂ ) _m Cyclopentylalkyl radicals or- (CH) ₂ ) _m -a cyclohexenyl group;

ring B is selected from phenyl, pyridyl, pyrimidinyl, pyrazinyl or pyridazinyl.

In certain embodiments, ring B is phenyl or pyridinyl.

In certain embodiments, each L is independently selected from the group consisting of-C (R ³ )(R ⁴ ) -or-N (R) ⁵ )-；R ³ 、R ⁴ 、R ⁵ Are each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, isopropoxy, monofluoromethyl, difluoromethylTrifluoromethyl, trifluoroethyl, monofluoromethoxy, difluoromethoxy or trifluoromethoxy.

In certain embodiments, each L is independently selected from the group consisting of-N (R ⁵ )-；R ⁵ Each independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, methoxy, trifluoromethyl or trifluoromethoxy; preferably, L is NH.

In certain embodiments, m is selected from 0, 1, or 2.

In certain embodiments, n is selected from 0, 1, or 2; preferably, n is 1.

The technical schemes in the invention can be mutually combined to form new technical schemes, and the formed new technical schemes are also included in the scope of the invention.

In certain embodiments, the compound of the foregoing general formula (I), general formula (II) or general formula (III), a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, is selected from the following compounds:

The invention also provides a pharmaceutical composition comprising a compound of the general formula (I), the general formula (II) or the general formula (III), a pharmaceutically acceptable salt or a stereoisomer thereof, and one or more pharmaceutically acceptable carriers and/or diluents; the pharmaceutical composition can be prepared into any clinically or pharmaceutically acceptable dosage form, such as tablets, capsules, pills, granules, solutions, suspensions, syrups, injections (including injections, sterile powders for injection and concentrated solutions for injection), suppositories, inhalants or sprays and the like.

In certain embodiments of the present invention, the above-described pharmaceutical formulations may be administered orally, parenterally, rectally, or pulmonary, etc., to a patient or subject in need of such treatment. For oral administration, the pharmaceutical composition may be formulated into oral preparations, for example, into conventional oral solid preparations such as tablets, capsules, pills, granules, etc.; can also be made into oral liquid preparation such as oral solution, oral suspension, syrup, etc. When the composition is formulated into oral preparations, suitable fillers, binders, disintegrants, lubricants, etc. may be added. For parenteral administration, the pharmaceutical preparations may also be formulated as injections, including injectable solutions, injectable sterile powders and injectable concentrated solutions. When the injection is prepared, the conventional method in the existing pharmaceutical field can be adopted for production, and when the injection is prepared, no additive can be added, and the proper additive can be added according to the property of the medicine. For rectal administration, the pharmaceutical composition may be formulated as suppositories and the like. For pulmonary administration, the pharmaceutical composition may be formulated as an inhalant or spray, etc.

The pharmaceutically acceptable carrier and/or diluent useful in the pharmaceutical composition or pharmaceutical formulation of the present invention may be any conventional carrier and/or diluent in the pharmaceutical formulation arts, and the choice of the particular carrier and/or diluent will depend on the mode of administration or type and state of disease for the particular patient being treated. The preparation of suitable pharmaceutical compositions for specific modes of administration is well within the knowledge of those skilled in the pharmaceutical arts.

In a further aspect, the invention also relates to the use of a compound of the aforementioned general formula (I), general formula (II) or general formula (III), a pharmaceutically acceptable salt thereof or a stereoisomer thereof for the manufacture of a medicament for the prevention and/or treatment of diseases and related disorders mediated by Wee1, which medicament may be used in combination with one or more other medicaments for the prevention or treatment of diseases and related disorders mediated by Wee 1. The disease and related conditions are selected from cancers, including carcinoma in situ and metastatic cancers, or benign tumors. Further, the cancers include, but are not limited to, lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, thyroid cancer, female genital tract cancer, lymphoma, neurofibromas, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, leukemia, glioma or sarcoma, and the like.

Furthermore, the application also relates to the use of pharmaceutical preparations containing the compounds of the general formula (I), the general formula (II) or the general formula (III), the pharmaceutically acceptable salts or the stereoisomers thereof for preparing medicaments which can be used in combination with one or more medicaments for treating and/or preventing diseases and related conditions mediated by Wee 1.

In another aspect, the application relates to a medicament comprising a compound of the aforementioned general formula (I), general formula (II) or general formula (III), a pharmaceutically acceptable salt thereof or a stereoisomer thereof, for administration alone or in combination with one or more second therapeutically active agents for use in combination with the Wee1 inhibitor compounds of the present application in the treatment and/or prophylaxis of diseases and related conditions mediated by Wee 1. Thus, in certain embodiments, the pharmaceutical composition further comprises one or more second therapeutically active agents. In certain embodiments, the second therapeutically active agent is selected from the group consisting of anticancer agents including mitotic inhibitors, alkylating agents, antimetabolites, antisense DNA or RNA, antitumor antibiotics, growth factor inhibitors, signaling inhibitors, cell cycle inhibitors, enzyme inhibitors, retinoid receptor modulators, proteasome inhibitors, topoisomerase inhibitors, biological response modifiers, hormonal agents, angiogenesis inhibitors, cytostatic agents, targeting antibodies, HMG-CoA reductase inhibitors, and prenyl protein transferase inhibitors.

In certain embodiments, the individual components to be combined (e.g., a compound of the invention, a pharmaceutically acceptable salt thereof, a stereoisomer thereof, and a second therapeutically active agent) can be administered simultaneously or sequentially and separately administered in sequence. For example, the second therapeutically active agent may be administered before, simultaneously with, or after administration of the compound of the invention, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof. Furthermore, the individual components to be combined can also be administered jointly in the form of the same formulation or in the form of separate different formulations.

In another aspect, the invention also provides a method of treating Wee1 mediated diseases and related conditions, comprising administering to a patient in need thereof an effective amount of a compound of formula (I), a pharmaceutically acceptable salt or stereoisomer thereof, a formulation or a pharmaceutical composition thereof; the Wee1 mediated disease and related conditions are as defined above.

By "effective amount" is meant an amount of a drug capable of alleviating, delaying, inhibiting or curing a condition in a subject. The size of the dose administered is determined by the mode of administration of the drug, the pharmacokinetics of the agent, the severity of the disease, the individual sign (sex, weight, height, age) of the subject, etc.

The medicine containing the compound shown in the general formula (I), the general formula (II) or the general formula (III), the pharmaceutically acceptable salt or the stereoisomer thereof can also be used as a radiotherapy sensitizer by combining with radiotherapy to improve the treatment effect of the radiotherapy.

In another aspect, the compounds of the present invention may also be used as sensitizers for other anticancer agents in the field of cancer treatment. The anticancer agent is as described previously. The sensitizer means a drug that additively or synergistically enhances the therapeutic effects of radiation and/or chemotherapy by being used in combination with such radiation and/or chemotherapy using an anticancer agent in the field of cancer treatment.

[ detailed description of the invention ]

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, however, for a better understanding of the present invention, the following definitions of some terms are provided. When the definition and interpretation of terms provided by the present invention are not identical to the meanings commonly understood by those skilled in the art, the definition and interpretation of terms provided by the present invention is in control.

"halogen" as used herein refers to a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

"C" as described in the present invention _1-6 Alkyl "means straight or branched chain containing 1 to 6 carbonsAlkyl radicals of atoms, including, for example, "C _1-4 Alkyl "," C _1-3 Alkyl "," C _1-2 Alkyl "," C _2-6 Alkyl "," C _2-5 Alkyl "," C _2-4 Alkyl "," C _2-3 Alkyl "," C _3-6 Alkyl "," C _3-5 Alkyl "," C _3-4 Alkyl ", and the like, specific examples include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3-dimethylbutyl, 2-dimethylbutyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 1, 2-dimethylpropyl, and the like. "C" as described in the present invention _1-4 Alkyl "means C _1-6 Specific examples of the alkyl group include 1 to 4 carbon atoms.

"C" as described in the present invention _1-6 Alkoxy "means" C _1-6 alkyl-O- ", said" C _1-6 Alkyl "is as defined above. "C" as described in the present invention _1-4 Alkoxy "means" C _1-4 alkyl-O- ", said" C _1-4 Alkyl "is as defined above.

"C" as described in the present invention _1-6 Alkylthio "means" C _1-6 alkyl-S- ", described as" C _1-6 Alkyl "is as defined above. "C" as described in the present invention _1-4 Alkylthio "means" C _1-4 alkyl-S- ", described as" C _1-4 Alkyl "is as defined above.

The invention relates to a hydroxy C _1-6 Alkyl, amino C _1-6 Alkyl, halogenated C _1-6 Alkyl "means C _1-6 One or more hydrogens in the alkyl group are substituted with one or more hydroxy, amino, or halogen groups, respectively. C (C) _1-6 Alkyl is as defined above

The invention relates to the hydroxy C _1-6 Alkoxy, amino C _1-6 Alkoxy, halo C _1-6 Alkoxy "means" C _1-6 One or more hydrogens in the alkoxy group "are takenOne or more hydroxy, amino or halogen.

The invention relates to the hydroxy C _1-6 Alkylthio, amino C _1-6 Alkylthio, halo C _1-6 Alkylthio "means" C _1-6 One or more hydrogens in the alkylthio group "are substituted with one or more hydroxy, amino, or halogen groups.

"C" as described in the present invention _1-6 Alkylamino, di (C) _1-6 Alkyl) amino "means C respectively _1-6 alkyl-NH-,

"C" as described in the present invention _2-6 Alkenyl "means a straight-chain, branched or cyclic alkenyl group having at least one double bond and having 2 to 6 carbon atoms, and includes, for example," C _2-4 Alkenyl ", and the like. Examples include, but are not limited to: 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.

"C" as described in the present invention _2-6 Alkynyl "refers to straight or branched chain alkynyl groups containing at least one triple bond and having 2 to 6 carbon atoms and includes, for example," C _2-4 Alkynyl ", and the like. Examples include, but are not limited to: ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3-hexynyl, 5-methyl-2-hexynyl and the like.

The term "3-to 10-membered cycloalkyl" as used herein is meant to include "3-to 8-membered monocycloalkyl" and "8-to 10-membered condensed ring alkyl".

"3-8 membered monocycloalkyl" as used herein refers to a saturated or partially saturated and non-aromatic monocycloalkyl group containing 3-8 carbon atoms, and includes "3-8 membered saturated monocycloalkyl" and "3-8 membered partially saturated monocycloalkyl"; preferred are "3-4 membered monocycloalkyl", "3-5 membered monocycloalkyl", "3-6 membered monocycloalkyl", "3-7 membered monocycloalkyl", "4-5 membered monocycloalkyl", "4-6 membered monocycloalkyl", "4-7 membered monocycloalkyl", "4-8 membered monocycloalkyl", "5-6 membered monocycloalkyl", "5-7 membered monocycloalkyl", "5-8 membered monocycloalkyl", "6-7 membered monocycloalkyl", "6-8 membered monocycloalkyl", "7-8 membered monocycloalkyl", "3-6 membered saturated monocycloalkyl", "5-8 membered saturated monocycloalkyl", "5-7 membered saturated monocycloalkyl", "5-6 membered saturated monocycloalkyl", and the like. Specific examples of the "3-8 membered saturated monocyclic alkyl group" include, but are not limited to: cyclopropane (cyclopropyl), cyclobutane (cyclobutyl), cyclopentane (cyclopentyl), cyclohexane (cyclohexyl), cycloheptane (cycloheptyl), cyclooctyl (cyclooctyl), and the like; specific examples of the "3-8 membered partially saturated monocyclic alkyl group" include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohex-1, 3-diene, cyclohex-1, 4-diene, cycloheptenyl, cyclohepta-1, 3-dienyl, cyclohepta-1, 4-dienyl, cyclohepta-1, 3, 5-trienyl, cyclooctenyl, cycloocta-1, 3-dienyl, cycloocta-1, 4-dienyl, cycloocta-1, 5-dienyl, cycloocta-1, 3, 5-trienyl, cyclooctatetraenyl and the like.

The term "8-to 10-membered condensed ring group" as used herein refers to a saturated or partially saturated, non-aromatic cyclic group containing 8 to 10 ring atoms formed by sharing two or more adjacent carbon atoms with each other by two or more cyclic structures, wherein one ring of the condensed rings may be an aromatic ring, but the whole condensed ring does not have aromaticity; including "8-9 membered fused ring group", "9-10 membered fused ring group", etc., the manner of fusion may be: 5-6 membered cycloalkyl, benzo 5-6 membered saturated cycloalkyl, and the like. Examples include, but are not limited to: bicyclo [3.1.0] hexyl, bicyclo [4.1.0] heptyl, bicyclo [2.2.0] hexyl, bicyclo [3.2.0] heptyl, bicyclo [4.2.0] octyl, octahydropentalenyl, octahydro-1H-indenyl, decahydronaphthyl, decatetrahydrophenanthryl, bicyclo [3.1.0] hex-2-enyl, bicyclo [4.1.0] hept-3-enyl, bicyclo [3.2.0] hept-3-enyl, bicyclo [4.2.0] oct-3-enyl, 1,2,3 a-tetrahydropentalenyl, 2, 3a,4,7 a-hexahydro-1H-indenyl, 1,2,3, 4a,5,6, 4 a-octahydronaphthyl, 1,2,4a,5,6,8 a-hexahydronaphthyl, 1,2,3,4, 6,7,8, 10-benzopentalenyl, benzocyclohexyl, benzopentalenyl, and the like.

The "3-10 membered heterocyclic group" as used herein includes "3-8 membered single heterocyclic group" and "8-10 membered condensed heterocyclic group".

"3-8 membered mono-heterocyclic group" as used herein means a saturated or partially saturated and non-aromatic monocyclic ring group containing at least one heteroatom (e.g., containing 1,2, 3,4 or 5) and having 3 to 8 ring atoms, the heteroatom being a nitrogen atom, an oxygen atom and/or a sulfur atom, optionally, a ring atom in the ring structure (e.g., carbon atom, nitrogen atom or sulfur atom) may be oxo. The "3-8 membered monocyclic group" described in the present invention includes "3-8 membered saturated monocyclic group" and "3-8 membered partially saturated monocyclic group". Preferably, the "3-8 membered mono-heterocyclic group" according to the present invention contains 1-3 heteroatoms; preferably, the "3-8 membered mono-heterocyclic group" according to the present invention contains 1-2 hetero atoms, and the hetero atoms are selected from nitrogen atoms and/or oxygen atoms; preferably, the "3-8 membered mono-heterocyclic group" according to the present invention contains 1 nitrogen atom. The "3-8 membered mono-heterocyclic group" is preferably "3-7 membered mono-heterocyclic group", "3-6 membered mono-heterocyclic group", "4-7 membered mono-heterocyclic group", "4-6 membered mono-heterocyclic group", "6-8 membered mono-heterocyclic group", "5-7 membered mono-heterocyclic group", "5-6 membered mono-heterocyclic group", "3-6 membered saturated mono-heterocyclic group", "5-6 membered saturated mono-heterocyclic group", "3-6 membered nitrogen-containing mono-heterocyclic group", "3-6 membered saturated nitrogen-containing mono-heterocyclic group", "5-6 membered saturated nitrogen-containing mono-heterocyclic group", or the like. For example, containing only 1 or 2 nitrogen atoms, or containing one nitrogen atom and 1 or 2 other heteroatoms (e.g., oxygen and/or sulfur atoms). Specific examples of "3-8 membered mono-heterocyclyl" include, but are not limited to: aziridinyl, 2H-aziridinyl, diazabicycloalkyl, 3H-diazapropenyl, azetidinyl, 1, 4-dioxanyl, 1, 3-dioxolanyl, 1, 4-dioxadienyl, tetrahydrofuranyl, dihydropyrrole, pyrrolidinyl, imidazolidinyl, 4, 5-dihydroimidazolyl, pyrazolidinyl, 4, 5-dihydropyrazolyl, 2, 5-dihydrothienyl, tetrahydrothienyl, 4, 5-dihydrothiazolyl, thiazolidinyl, piperidinyl, tetrahydropyridinyl, piperidonyl, tetrahydropyridinonyl, dihydropyridinonyl, piperazinyl, morpholinyl, 4, 5-dihydro-oxazolyl, 4, 5-dihydro-isoxazolyl, 2, 3-dihydro-isoxazolyl, oxazolidinyl, 2H-1, 2-oxazinyl, 4H-1, 2-oxazinyl, 6H-1, 2-oxazinyl, 4H-1, 3-oxazinyl, 6H-1, 3-oxazinyl, 4H-1, 4-oxazinyl, 4H-1, 3-thiazinyl, 6H-1, 3-thiazinyl, 2H-pyranyl, 2H-pyran-2-onyl, 3, 4-dihydro-2H-pyranyl, and the like.

The "8-10 membered fused heterocyclic group" as used herein refers to a saturated or partially saturated, non-aromatic cyclic group containing 8-10 ring atoms and at least one ring atom being a heteroatom, formed by two or more cyclic structures sharing two adjacent atoms with each other, one of which may be an aromatic ring but the whole of which is not aromatic, and the heteroatom is a nitrogen atom, an oxygen atom and/or a sulfur atom, optionally, a ring atom (e.g., a carbon atom, a nitrogen atom or a sulfur atom) in the cyclic structure may be oxo-substituted, including, but not limited to, "8-9 membered fused heterocyclic group", "9-10 membered fused heterocyclic group" and the like, and the manner of the fusion may be 5-6 membered heterocyclic group and 5-6 membered heterocyclic group, 5-6 membered cycloalkyl, benzo 5-6 membered saturated heterocyclic group, 5-6 membered heteroaryl and 5-6 membered saturated heterocyclic group; a 5-6 membered heteroaryl group as defined hereinbefore; specific examples of the "8-to 10-membered fused heterocyclic group" include, but are not limited to: pyrrolidinyl, cyclopentylazacyclopropyl, pyrrolidinyl-cyclobutyl, pyrrolidinyl-pyrrolidinyl, pyrrolidinyl-piperidinyl, pyrrolidinyl-piperazinyl, pyrrolidinyl-pentalinyl, piperidinyl-pentalinyl, benzopyrrolidinyl, benzocyclopentyl, benzocyclohexyl, benzotetrahydrofuranyl, benzopyrrolidinyl, benzimidazolidinyl, benzoxazolidinyl, benzothiazolidinyl, benzisoxazolidinyl, benzisothiazolidinyl, benzopiperidinyl, benzomorpholinyl, benzopiperazinyl, benzopyranyl, pyridocyclopentyl, pyridocyclohexyl, benzofuranyl-pentalinyl, and benzopiperidinyl-pentalinyl pyridotetrahydrofuranyl, pyridopyrrolidinyl, pyridoimidazolidinyl, pyridooxazolidinyl, pyridothiazolidinyl, pyridoisoxazolidinyl, pyridoisothiazolidinyl, pyridopiperidinyl, pyridomorpholinyl, pyridopiperazinyl, pyridotetrahydropyranyl, pyrimidocyclopentyl, pyrimidocyclohexyl, pyrimidoethylphenyl, pyrimidopyrrolidinyl, pyrimidoimidazolidinyl, pyrimidooxazolidinyl, pyrimidoidinyl, pyrimidoidithiazolidinyl, pyrimidopiperidyl, pyrimidomomorpholinyl, pyrimidopiperidinyl, pyrimidoetetrahydropyranyl; tetrahydroimidazo [4,5-c ] pyridinyl, 3, 4-dihydroquinazolinyl, 1, 2-dihydroquinoxalinyl, benzo [ d ] [1,3] dioxolyl, 2H-chromene-2-onyl, 4H-chromene, 4H-chromen-4-onyl, 4H-1, 3-benzoxazolyl, 4, 6-dihydro-1H-furo [3,4-d ] imidazolyl, 3a,4,6 a-tetrahydro-1H-furo [3,4-d ] imidazolyl, 4, 6-dihydro-1H-thieno [3,4-d ] imidazolyl, 4, 6-dihydro-1H-pyrrolo [3,4-d ] imidazolyl, octahydro-benzo [ d ] imidazolyl, decahydroquinolinyl, hexahydrothienoimidazoyl, hexahydrofuroimidazoyl, 4,5,6, 7-tetrahydro-1H-benzo [3,4-d ] imidazolyl, octahydro-pyrrolo [3,4-d ] pyrrolyl, and the like.

The term "benzocyclopentyl" refers to its structure(also known as 2, 3-dihydro-1H-indenyl); the term "benzopyrrolidine" includes the structure +.>Etc.; the term "pyridotetrahydrofuranyl" includes structures thereof Specific examples of other "defined other fused heterocyclic groups" defined hereinbefore have a cyclic structure similar thereto.

The "6-10 membered aryl" described herein includes "6-8 membered monocyclic aryl" and "8-10 membered condensed ring aryl".

"6-8 membered monocyclic aryl" as used herein refers to monocyclic aryl groups containing 6-8 ring carbon atoms, examples of which include, but are not limited to: phenyl, cyclooctatetraenyl, and the like; phenyl is preferred.

The term "8-to 10-membered condensed ring aryl" as used herein refers to an unsaturated, aromatic cyclic group containing 8 to 10 ring carbon atoms, preferably "9-to 10-membered condensed ring aryl", which is formed by sharing two or more adjacent atoms with each other by two or more cyclic structures, and specific examples thereof are naphthyl and the like.

The "5-to 10-membered heteroaryl" as used herein includes "5-to 8-membered single heteroaryl" and "8-to 10-membered fused heteroaryl".

"5-8 membered mono-heteroaryl" as used herein refers to an aromatic monocyclic ring group containing 5-8 ring atoms, at least one of which is a heteroatom, such as nitrogen, oxygen or sulfur. Optionally, a ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be oxo. "5-8 membered mono-heteroaryl" includes, for example, "5-7 membered mono-heteroaryl", "5-6 membered nitrogen containing mono-heteroaryl", "6 membered nitrogen containing mono-heteroaryl", etc., wherein the heteroatom in the "nitrogen containing heteroaryl" contains at least one nitrogen atom, e.g., contains only 1 or 2 nitrogen atoms, or contains one nitrogen atom and 1 or 2 other heteroatoms (e.g., oxygen and/or sulfur atoms), or contains 2 nitrogen atoms and 1 or 2 other heteroatoms (e.g., oxygen and/or sulfur atoms). Specific examples of "5-8 membered monocyclic heteroaryl" include, but are not limited to, furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazolyl, pyrazolyl, 1,2, 3-triazolyl, 1,2, 4-triazolyl, 1,2, 3-oxadiazolyl, 1,2, 4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3, 4-oxadiazolyl, pyridyl, 2-pyridonyl, 4-pyridonyl, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2, 3-triazinyl, 1,3, 5-triazinyl, 1,2,4, 5-tetrazinyl, azepanyl, 1, 3-diazinoheptenyl, azocyclotetraenyl and the like. The "5-6 membered single heteroaryl" refers to a specific example in which 5-8 membered heteroaryl contains 5-6 ring atoms.

The "8-to 10-membered fused heteroaryl group" as used herein refers to an unsaturated aromatic ring structure containing 8 to 10 ring atoms (at least one of which is a heteroatom such as a nitrogen atom, an oxygen atom or a sulfur atom) formed by two or more ring structures sharing two adjacent atoms with each other. Optionally, a ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be oxo. Including "9-10 membered fused heteroaryl", "8-9 membered fused heteroaryl", and the like, which may be fused in such a manner as to be benzo 5-6 membered heteroaryl, 5-6 membered heteroaryl and 5-6 membered heteroaryl, and the like; specific examples include, but are not limited to: pyrrolopyrroles, pyrrolofurans, pyrazolopyrroles, pyrazolothiophenes, furanthiophenes, pyrazolooxazoles, benzofuranyl, benzisofuranyl, benzothienyl, indolyl, isoindolyl, benzoxazolyl, benzimidazolyl, indazolyl, benzotriazolyl, quinolinyl, 2-quinolinonyl, 4-quinolinonyl, 1-isoquinolonyl, isoquinolinyl, acridinyl, phenanthridinyl, benzopyridazinyl, phthalazinyl, quinazolinyl, quinoxalinyl, purinyl, naphthyridinyl, and the like.

The term "5-to 10-membered spirocyclic group" as used herein means a cyclic structure having 5 to 10 ring carbon atoms formed by sharing one carbon atom with two or more cyclic structures. Optionally, carbon atoms in the cyclic structure may be oxidized. "5-10 membered spirocyclic group" includes, for example, "4-10 membered spirocyclic group", "6-10 membered spirocyclic group", "7-10 membered spirocyclic group", "6-9 membered spirocyclic group", "7-9 membered spirocyclic group", "9-10 membered spirocyclic group" and the like. Specific examples include, but are not limited to:

Etc. The "7-to 9-membered spirocyclic group" means a specific example in which 7 to 9 ring atoms are contained in the 5-to 10-membered spirocyclic group.

The term "5-15 membered bridged ring radical" as used herein refers to a cyclic structure containing 5 to 15 ring carbon atoms formed by two or more cyclic structures sharing two non-adjacent carbon atoms with each other. Optionally, carbon atoms in the cyclic structure may be oxo. "5-15 membered bridged ring radical" includes, for example, "5-11 membered bridged ring radical", "6-11 membered bridged ring radical", "5-10 membered bridged ring radical", "7-10 membered bridged ring radical", "6-9 membered bridged ring radical", "7-9 membered bridged ring radical", "9-10 membered bridged ring radical", and the like. Specific examples include, but are not limited to:/>

etc. The term "7-to 9-membered bridged ring radical" means a specific example in which 7 to 9 ring atoms are contained in the 5-to 15-membered bridged ring radical.

The "5-10 membered spiroheterocyclic group" as used herein refers to a cyclic structure containing 5 to 10 ring atoms (at least one of which is a heteroatom such as nitrogen atom, oxygen atom or sulfur atom) formed by two or more cyclic structures sharing one ring atom with each other, and includes "5-10 membered saturated spiroheterocyclic group" and "5-10 membered partially saturated spiroheterocyclic group". Optionally, a ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be oxidized. "5-10 membered spiroheterocyclyl" includes, for example, "6-11 membered spiroheterocyclyl", "6-9 membered spiroheterocyclyl", "7-9 membered spiroheterocyclyl", "9-10 membered spiroheterocyclyl", "5-10 membered nitrogen containing spiroheterocyclyl", "7-9 membered nitrogen containing spiroheterocyclyl", "8-9 membered nitrogen containing spiroheterocyclyl", "7-9 membered nitrogen containing saturated spiroheterocyclyl", "8-9 membered nitrogen containing saturated spiroheterocyclyl", etc. . Specific examples include, but are not limited to: etc.

The "5-15 membered bridged heterocyclic group" as used herein refers to a cyclic structure containing 5 to 15 ring atoms (at least one of which is a heteroatom such as nitrogen atom, oxygen atom or sulfur atom) formed by two or more cyclic structures sharing two non-adjacent ring atoms with each other, and includes "5-15 membered saturated bridged heterocyclic group" and "5-15 membered partially saturated bridged heterocyclic group". Optionally, a ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be oxidized. "5-15 membered bridged heterocyclic group" includes, for example, "5-10 membered bridged heterocyclic group", "6-11 membered bridged heterocyclic group", "6-9 membered bridged heterocyclic group", "6-10 membered bridged heterocyclic group", "7-9 membered nitrogen-containing bridged heterocyclic group", "7-8 membered nitrogen-containing bridged heterocyclic group", "5-9 membered nitrogen-containing bridged heterocyclic group", "5-15 membered nitrogen-containing bridged heterocyclic group", "5-10 membered bridged heterocyclic group", "7-9 membered nitrogen-containing saturated bridged heterocyclic group", etc. Specific examples include, but are not limited to:/> etc.

The term "carbon atom, nitrogen atom or sulfur atom is oxo" as used herein means that a c= O, N = O, S =o or SO is formed ₂ Is a structure of (a).

"optionally substituted" as used herein refers to both cases where one or more hydrogen atoms on the substituted group may be "substituted" or "unsubstituted" with one or more substituents.

The term "pharmaceutically acceptable salt" as used herein refers to the acidic functional groups present in the compound (e.g., -COOH, -OH, -SO) ₃ H, etc.) with suitable inorganic or organic cations (bases), including salts with alkali metals or alkaline earth metals, ammonium salts, and salts with nitrogen-containing organic bases; and salts of basic functional groups (e.g., -NH2, etc.) present in the compounds with suitable inorganic or organic anions (acids), including salts with inorganic or organic acids (e.g., carboxylic acids, etc.).

"isomers" as used herein refers to compounds of the present invention which contain one or more asymmetric centers and are therefore useful as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. The compounds of the present invention may have asymmetric centers that each independently produce two optical isomers. The scope of the present invention includes all possible optical isomers and mixtures thereof. The compounds of the present invention, if they contain olefinic double bonds, include cis-isomers and trans-isomers unless specified otherwise. The compounds described herein may exist in tautomeric (one of the functional group isomers) forms having different points of attachment of hydrogen through displacement of one or more double bonds, for example, the keto and enol forms thereof are keto-enol tautomers. The compounds of the invention contain spiro structures, which are affected by the steric structure of the ring, and substituents on the ring may be present on both sides of the ring to form the opposite cis (cis) and trans (trans) isomers. Each tautomer and mixtures thereof are included within the scope of the present invention. Enantiomers, diastereomers, racemates, meso, cis-trans isomers, tautomers, geometric isomers, epimers, mixtures thereof and the like of all compounds are included within the scope of the present invention.

The compounds of the invention may be prepared by enantiospecific synthesis or resolution from mixtures of enantiomers to give the individual enantiomers. Conventional resolution techniques include formation of a salt of the free base of each enantiomer of an enantiomer pair using an optically active acid (followed by fractional crystallization and free base regeneration), formation of an acid form salt of each enantiomer of an enantiomer pair using an optically active amine (followed by fractional crystallization and free acid regeneration), formation of an ester or amide of each enantiomer of an enantiomer pair using an optically pure acid, amine or alcohol (followed by chromatographic separation and chiral auxiliary removal) or resolution of mixtures of enantiomers of the starting material or end product using various well known chromatographic methods.

When the stereochemistry of a disclosed compound is named or depicted by structure, the named or depicted stereoisomer is at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 99 wt%, or 99.9 wt% pure relative to the other stereoisomers. When a single isomer is named or depicted by structure, the depicted or named enantiomer is at least 60% by weight, 70% by weight, 80% by weight, 90% by weight, 99% by weight, or 99.9% by weight pure. Optical purity wt% is the ratio of the weight of an enantiomer to the weight of the enantiomer plus the weight of its optical isomer.

The term "dosage form" as used herein refers to a form of a medicament formulated for clinical use, including, but not limited to, powders, tablets, granules, capsules, solutions, emulsions, suspensions, injections (including injectable solutions, injectable sterile powders and injectable concentrated solutions), sprays, aerosols, powder sprays, lotions, liniments, ointments, plasters, pastes, patches, gargles or suppositories, more preferably powders, tablets, granules, capsules, solutions, injections, ointments, gargles or suppositories.

Advantageous effects of the invention

1. The compound, the pharmaceutically acceptable salt or the stereoisomer thereof has excellent Wee1 activity inhibition effect, good pharmacokinetic property in organisms, lasting effect and high exposure and bioavailability, and can treat and/or prevent diseases mediated by Wee 1.

2. The compound, the pharmaceutically acceptable salt or the stereoisomer thereof has better treatment effect on Wee1 mediated cancers.

3. The compound, the pharmaceutically acceptable salt or the stereoisomer thereof can synergistically enhance the effect of radiotherapy and chemotherapy, effectively inhibit the growth of tumors, and can reduce the damage to normal cells and reduce side effects.

4. The compound has the advantages of simple preparation process, high purity of the medicine, stable quality and easy mass industrial production.

Detailed description of the preferred embodiments

The technical scheme of the present invention will be described in detail below with reference to specific embodiments, but the scope of the subject matter of the present invention should not be construed as being limited to the following examples. All techniques implemented based on the above description of the invention are within the scope of the invention.

Example 1: preparation of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- ((4- (8-methyl-3, 8-diazabicyclo [3.2.1] oct-3-yl) phenyl) amino) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (Compound 1)

Preparation of ethyl 4- (2-allyl-2- (tert-butoxycarbonyl) hydrazino) -2- (methylthio) pyrimidine-5-carboxylate

Tert-butyl 1-allylhydrazine-1-carboxylate (0.37 g,21.5 mmol) and ethyl 4-chloro-2- (methylthio) pyrimidine-5-carboxylate (0.5 g,21.5 mmol) were dissolved in tetrahydrofuran (100.0 mL), N-diisopropylethylamine (0.69 g,53.8 mmol) was added, reacted at 70℃for 16h, the solvent was dried, and column chromatography (ethyl acetate/petroleum ether=25%) afforded the product (0.62 g, 78.5%).

Preparation of ethyl 4- (2-allylhydrazino) -3- (methylthio) -1,2, 4-triazine-6-carboxylate

Ethyl 4- (2-allyl-2- (tert-butoxycarbonyl) hydrazino) -2- (methylthio) pyrimidine-5-carboxylate (0.62 g,16.8 mmol) was dissolved in trifluoroacetic acid (50.0 mL) and reacted at 70 ℃ for 1 hour, and the crude product was obtained by spin-drying and used directly in the next step.

Preparation of 2-allyl-6- (methylthio) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one

Ethyl 4- (2-allylhydrazino) -3- (methylthio) -1,2, 4-triazine-6-carboxylate (crude product of the above step) was dissolved in 70.0mL of ethanol, aqueous sodium hydroxide (25.6 mL, 178 mmol) was added, the reaction was carried out at 20℃for 30 minutes, 3N diluted hydrochloric acid was added to adjust pH to 4, dichloromethane and 5% methanol (50.0 mL) were extracted, and the organic phase was dried by spinning to give 4.0g of crude product.

4.preparation of 2- (6-bromopyridin-2-yl) propan-2-ol

Methyl 6-bromopyridine carboxylate (5.0 g,23.3 mmol) was dissolved in 150mL tetrahydrofuran, methyl magnesium bromide solution (16.0 mL,100.0 mmol) was added at 0deg.C, the mixture was allowed to react at 25deg.C for 2h, quenched with saturated ammonium chloride solution, extracted with ethyl acetate, and concentrated column chromatography gave 4.5g in 90% yield.

Preparation of 2-allyl-1- (6- (2- (hydroxy-prop-2-yl) pyridin-2-yl) -6- (methylthio) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one

2-allyl-6- (methylthio) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (crude from the previous step) was dissolved in 1, 4-dioxane (150 mL), cuprous iodide (3.4 g,18.0 mmol), potassium carbonate (3.5 g,25.2 mmol), N, N' -dimethylethylenediamine (3.2 g,36.0 mmol), 2- (6-bromopyridin-2-yl) propan-2-ol (5.1 g,23.4 mmol), reacted at 95℃for 16H, concentrated column chromatography (ethyl acetate/petroleum ether=50%) gave 4.0g of the product in three total yields: 66.61%.

6. Preparation of (1R, 5S) -3- (4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester

3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester (0.5 g,2.4 mmol), dissolved in N, N-dimethylacetamide (10.0 mL), 1-fluoro-4-nitrobenzene (0.33 g,2.4 mmol), N, N-diisopropylethylamine (0.9 g,7.2 mmol) were added and reacted at 90℃for 16h, concentrated column chromatography (ethyl acetate/petroleum ether=25%) afforded the product (0.33 g, 41.3%).

7. Preparation of (1R, 5S) -3- (4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] octane

(1R, 5S) -3- (4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] octane-8-carboxylic acid tert-butyl ester (0.15 g,0.45 mmol), dissolved in 10.0mL of dichloromethane, was added 3.0mL of trifluoroacetic acid and reacted at room temperature for 1h, and dried by spinning to give 190.0mg of crude product.

8. Preparation of (1R, 5S) -8-methyl-3- (4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] octane

(1R, 5S) -3- (4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] octane (crude product of the above step) was dissolved in 12.0mL of N, N-dimethylformamide, sodium triacetoxyborohydride (0.3 g,1.6 mmol) was added, the mixture was reacted at 20℃for 3 hours, the reaction was quenched with 10.0mL of water, extracted with methylene chloride, and concentrated to give 0.27g.

Preparation of 9.4- ((1R, 5S) -8-methyl-3, 8-diazabicyclo [3.2.1] oct-3-yl) aniline

(1R, 5S) -8-methyl-3- (4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] octane (0.25 g,1.0 mmol) was dissolved in 6.0mL methanol, and 0.2g palladium on carbon (10%) was reacted for 1h and concentrated to give 0.23g crude product.

Preparation of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- ((4- (8-methyl-3, 8-diazabicyclo [3.2.1] oct-3-yl) phenyl) amino) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one

2-allyl-1- (6- (2- (hydroxy-prop-2-yl) pyridin-2-yl) -6- (methylsulfanyl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (0.29 g,0.81 mmol), dissolved in 3.0mL toluene, 85% m-chloroperoxybenzoic acid (m-CPBA, 0.21g,1.2 mmol) was added and reacted at room temperature for 30 min, N-diisopropylethylamine (0.54 g,4.2 mmol), 4- ((1 r,5 s) -8-methyl-3, 8-diazabicyclo [3.2.1] oct-3-yl) aniline (0.23 g,1.1 mmol), reacted at room temperature for 16H, saturated sodium bicarbonate quenching reaction, ethyl acetate extraction, drying, concentrated reverse phase column chromatography (water/methanol=60%) gave 23.0mg of product with a yield of 5.5%.

Molecular formula C ₂₉ H ₃₄ N ₈ O ₂ Molecular weight 526.0 LC-MS (M/e): 527.0 (M+H) ⁺ )

¹ H-NMR(400MHz,CDCl ₃ )δ:8.81(s,1H),7.84(t,J＝7.8Hz,1H),7.75(d,J＝8.0Hz,1H),7.51-7.39(m,2H),7.32(d,J＝7.6Hz,1H),6.78(d,J＝8.2Hz,2H),5.74-5.65(m,1H),5.03(d,J＝10.1Hz,1H),4.93(d,J＝17.2Hz,1H),4.73(d,J＝6.0Hz,2H),3.96(s,1H),3.34(d,J＝10.5Hz,2H),3.28(s,2H),3.02(d,J＝10.5Hz,2H),2.36(s,3H),2.04-2.03(m,2H),1.79-1.77(m,2H),1.58(s,6H).

Example 2: preparation of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- ((4- (5-methyl-2, 5-diazabicyclo [2.2.2] oct-2-yl) phenyl) amino) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (Compound 2)

Preparation of 1, 2-methyl-5- (4-nitrophenyl) -2, 5-diazabicyclo [2.2.2] octane

2-methyl-2, 5-diazabicyclo [2.2.2] octane (100 mg,0.5 mmol) was dissolved in 10ml of acetonitrile, and 1-fluoro-4-nitrobenzene (141 mg,1.0 mmol) and N, N-diisopropylethylamine (323 mg,2.5 mmol) were added to react at 60℃for 16h. Concentration and purification on a silica gel column (methanol: dichloromethane=1:7) gave 74mg of the title compound in 60.0% yield.

Preparation of 4- (5-methyl-2, 5-diazabicyclo [2.2.2] oct-2-yl) aniline

2-methyl-5- (4-nitrophenyl) -2, 5-diazabicyclo [2.2.2] octane (70 mg,0.28 mmol) was dissolved in methanol (5 mL), and palladium on carbon (40 mg) was added to react at 16℃under a hydrogen atmosphere for 1 hour. Concentration gave crude (72 mg) which was used directly in the next reaction.

Preparation of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- ((4- (5-methyl-2, 5-diazabicyclo [2.2.2] oct-2-yl) phenyl) amino) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one

2-allyl-1- (6- (2- (hydroxy-prop-2-yl) pyridin-2-yl) -6- (methylthio) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (77 mg,0.22 mmol) was dissolved in toluene (10 mL), 62.5% m-CPBA (77 mg,0.28 mmol) was added and reacted for 1 hour at 16℃N, N-diisopropylethylamine (148 mg,1.14 mmol) and 4- (5-methyl-2, 5-diazabicyclo [2.2.2] oct-2-yl) aniline (70 mg,0.32 mmol) were added and reacted for 5 hours at 16℃concentrated and purified by C18 column (water: methanol=3:1) to give 29mg of the title compound in 25.0% yield.

Molecular formula C ₂₉ H ₃₄ N ₈ O ₂ Molecular weight 526.6 LC-MS (M/e): 527.2 (M+H) ⁺ )

¹ H-NMR(400MHz,DMSO-d ₆ )δ:10.12(s,1H),8.79(s,1H),8.03(s,1H),7.76(d,J＝7.6Hz,1H),7.60(d,J＝8.0Hz,1H),7.59-7.52(m,2H),6.64(d,J＝8.4Hz,2H),5.76-5.63(m,1H),5.32(s,1H),5.00(d,J＝10.4Hz,1H),4.85-4.81(m,1H),4.69-4.67(m,2H),3.86(s,1H),3.63(d,J＝9.6Hz,1H),3.23(d,J＝9.2Hz,1H),2.90(d,J＝8.8Hz,1H),2.33(s,3H),2.03-1.96(m,2H),1.83-1.56(m,3H),1.44(s,6H).

Example 3: preparation of 2-allyl-1- (6- (2-hydroxy-prop-2-yl) pyridin-2-yl) -6- (((4- (6-methyl-2, 6-diazaspiro [3.3] hept-2-yl) phenyl) amino) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (Compound 3)

Preparation of 1, 2-methyl-6- (4-nitrophenyl) -2, 6-diazaspiro [3.3] heptane

2-methyl-2, 6-diazaspiro [3.3] heptane dihydrochloride (100 mg,0.54 mmol), p-fluoronitrobenzene (83 mg,0.59 mmol) and N, N-diisopropylethylamine (349 mg,2.7 mmol) were dissolved in acetonitrile (20 mL), reacted at 60℃for 6h, and saturated sodium bicarbonate (3 mL) solution was added, concentrated, and the crude product was purified by silica gel column chromatography (dichloromethane: methanol=10:1) to give the product (100 mg, yield 79.1%).

Preparation of 4- (6-methyl-2, 6-diazaspiro [3.3] hept-2-yl) aniline

2-methyl-6- (4-nitrophenyl) -2, 6-diazaspiro [3.3] heptane (30 mg,0.13 mmol) was dissolved in methanol (10 mL), palladium on carbon (15 mg) was added, and the mixture was reacted at 25℃under a hydrogen atmosphere for 0.5 hour. Filtration and concentration gave crude (28 mg) which was used directly in the next reaction.

Preparation of 2-allyl-1- (6- (2-hydroxy-prop-2-yl) pyridin-2-yl) -6- (((4- (6-methyl-2, 6-diazaspiro [3.3] hept-2-yl) phenyl) amino) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one

2-allyl-1- (6- (2- (hydroxy-prop-2-yl) -6- (methylsulfanyl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (32 mg,0.09 mmol) was dissolved in toluene (7 mL), m-chloroperoxybenzoic acid (20 mg,0.12 mmol) was added and reacted for 0.5H at 25℃was complete.

Molecular formula C ₂₈ H ₃₂ N ₈ O ₂ Molecular weight 512.3 LC-MS (M/e): 513.2 (M+H) ⁺ )

¹ H-NMR(400MHz,CDCl ₃ )δ:8.83(s,1H),7.86(t,J＝8.0Hz,1H),7.76(d,J＝8.0Hz,1H),7.54-7.26(m,4H),6.46(d,J＝8.0Hz,2H),5.76-5.67(m,1H),5.07-4.94(m,2H),4.75(d,J＝6.0Hz,2H),3.96(s,4H),3.42(s,4H),2.35(s,3H),1.60(s,6H).

Example 4: (2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -3-oxo-2, 3-dihydro-1H-pyrazolo [3,4-d ] pyrimidin-6-yl) amino) phenyl) -3-methyl-3, 6-diazabicyclo [3.1.1] heptan-2-one (compound 4)

Preparation of tert-butyl 1, 3-methyl-2-oxo-3, 6-diazabicyclo [3.1.1] heptane-6-carboxylate

2-oxo 3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester (106 mg,0.5 mmol) was dissolved in tetrahydrofuran (10 mL), 60% sodium hydride (40 mg,1 mmol) was added, reacted at 25℃for 2h, methyl iodide (142 mg,1 mmol) was added, reacted at 25℃for 2h, saturated brine (20 mL) was quenched, extracted with ethyl acetate (30 mL), and the organic phase dried over anhydrous sodium sulfate and spun dry to give crude product, which was used directly in the next step.

Preparation of 2, 3-methyl-3, 6-diazabicyclo [3.1.1] hept-2-one hydrochloride

The crude product of the previous step of 3-methyl-2-oxo-3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester is dissolved in 4M dioxane hydrochloride (2 mL), reacted for 16 hours at 20 ℃, and dried by a cyclone to obtain the crude product which is directly used in the next step.

Preparation of 3.3-methyl-6- (4-nitrophenyl) -3, 6-diazabicyclo [3.1.1] hept-2-one

Crude 3-methyl-3, 6-diazabicyclo [3.1.1] hept-2-one hydrochloride, 1-fluoro-4-nitrobenzene (141 mg,1 mmol) and diisopropylethylamine (194 mg,1.5 mmol) were dissolved in DMA (7 mL), reacted at 90℃for 16 hours, quenched with saturated brine (15 mL), extracted with ethyl acetate (30 mL) and the organic phase was prepared in reverse phase (0-40% methanol/water) by spin-dry medium pressure to give the product (40 mg, 32.4%).

Preparation of 6- (4-aminophenyl) -3-methyl-3, 6-diazabicyclo [3.1.1] hept-2-one

3-methyl-6- (4-nitrophenyl) -3, 6-diazabicyclo [3.1.1] heptan-2-one (50 mg,0.2 mmol) was reacted with 10% wet palladium on carbon (5 mg) in methanol (7 mL), hydrogen (15 Psi) at 15℃for 1 hour, and the filtrate was concentrated by suction filtration to give the product (40 mg, 91.0%).

Preparation of 2-allyl-1- (6- (2- (hydroxy prop-2-yl) pyridin-2-yl) -6- (methylsulfinyl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one

2-allyl-1- (6- (2-hydroxy-prop-2-yl) pyridin-2-yl) -6- (methylsulfanyl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (100 mg,0.28 mmol) was reacted with 65% m-chloroperoxybenzoic acid (89 mg,0.34 mmol) in toluene (2 mL) at 15℃for 3 hours, concentrated, and purified by column chromatography (30% tetrahydrofuran/ethyl acetate) to give the product (80 mg, 76.6%).

6.6 preparation of 6- (4- ((2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -3-oxo-2, 3-dihydro-1H-pyrazolo [3,4-d ] pyrimidin-6-yl) amino) phenyl) -3-methyl-3, 6-diazabicyclo [3.1.1] heptan-2-one

2-allyl-1- (6- (2-hydroxy-prop-2-yl) pyridin-2-yl) -6- (methylsulfinyl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (103 mg,0.27 mmol), 6- (4-aminophenyl) -3-methyl-3, 6-diazabicyclo [3.1.1] hept-2-one (40 mg,0.18 mmol) and diisopropylethylamine (48 mg,0.36 mmol) were dissolved in THF (12 mL), reacted at 15℃for 16 hours, dried by spin-on, medium pressure reverse phase preparation (0-40% acetonitrile/water) was purified to give the product (10 mg, 10.3%).

The molecular formula: molecular weight of C28H30N8O 3: 526.6LC-MS (M/e): 527.2 (M+H+)

¹ HNMR(400MHz,CDCl ₃ ):δ:10.11(s,1H),8.82(s,1H),8.10-7.95(m,1H),7.80-7.55(m,2H),7.55-7.45(m,2H),6.60-6.50(m,2H),5.70-5.60(m,1H),5.33(s,1H),5.05-4.95(m,1H),4.85-4.75(m,1H),4.75-4.65(m,2H),4.60-4.50(m,1H),4.10-4.05(m,1H),3.65-3.55(m,1H),3.30-3.20(m,1H),2.80-2.70(m,1H),2.57(m,3H),1.95-1.85(m,1H),1.45(s,6H).

Example 5: preparation of 3- (4- ((2-allyl-1- (6- (2-hydroxypropyl-2-yl) pyridin-2-yl) -3-oxo-2, 3-dihydro-1H-pyrazolo [3,4-d ] pyrimidin-6-yl) amino) phenyl) -6-methyl-3, 6-diazabicyclo [3.1.1] heptan-2-one (Compound 5)

Preparation of tert-butyl 3- (4-nitrophenyl) -2-oxo-3, 6-diazabicyclo [3.1.1] heptane-6-carboxylate

2-oxo 3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester (149 mg,0.70 mmol), p-nitroiodobenzene (262 mg,1.05 mmol), cuprous iodide (27 mg,0.14 mmol), potassium phosphate (149 mg,3.5 mmol) and 1, 10-phenanthroline (25 mg,0.14 mmol) were dissolved in DMF (15 mL), reacted at 110℃for 5 hours, quenched with saturated brine (20 mL), extracted with ethyl acetate (30 mL), dried over anhydrous sodium sulfate in the organic phase, spun-dried, and purified by column chromatography (ethyl acetate/petroleum ether=30%) to give the product (140 mg, 59.8%).

Preparation of 3- (4-nitrophenyl) -3, 6-diazabicyclo [3.1.1] heptane-2-one trifluoroacetate salt

3- (4-nitrophenyl) -2-oxo-3, 6-diazabicyclo [3.1.1] heptane-6-carboxylic acid tert-butyl ester (140 mg,0.42 mmol) was dissolved in dichloromethane (10 mL) and trifluoroacetic acid (5 mL), reacted at 20℃for 1 hour, and dried to give crude product which was used directly in the next step.

Preparation of 3, 6-methyl-3- (4-nitrophenyl) -3, 6-diazabicyclo [3.1.1] heptane-2-one

3- (4-Nitrophenyl) -3, 6-diazabicyclo [3.1.1] heptane-2-one trifluoroacetate crude product from the previous step, 37% aqueous formaldehyde (3411 mg,4.2 mmol) and sodium borohydride acetate (267 mg,1.26 mmol) were dissolved in DMF (6 mL), reacted for 2 h at 20℃saturated sodium bicarbonate (20 mL) was quenched, extracted with ethyl acetate (30 mL), the organic phase dried over anhydrous sodium sulfate, spun dry and purified by column chromatography (methanol/dichloromethane=4%) to give the product (85 mg, 81.9%).

Preparation of 3- (4-aminophenyl) -6-methyl-3, 6-diazabicyclo [3.1.1] heptane-2-one

6-methyl-3- (4-nitrophenyl) -3, 6-diazabicyclo [3.1.1] heptane-2-one (85 mg,0.34 mmol) was reacted with 10% wet palladium on carbon (8.5 mg) in methanol (12 mL), under an atmosphere of hydrogen (15 Psi) at 15℃for 1 hour, suction filtered, and the filtrate was concentrated to give the product (70 mg, 93.7%).

5.preparation of 3- (4- ((2-allyl-1- (6- (2-hydroxypropyl-2-yl) pyridin-2-yl) -3-oxo-2, 3-dihydro-1H-pyrazolo [3,4-d ] pyrimidin-6-yl) amino) phenyl) -6-methyl-3, 6-diazabicyclo [3.1.1] heptan-2-one

2-allyl-1- (6- (2-hydroxy-prop-2-yl) pyridin-2-yl) -6- (methylsulfinyl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (103 mg,0.27 mmol) and 3- (4-aminophenyl) -6-methyl-3, 6-diazabicyclo [3.1.1] heptan-2-one (48 mg,0.22 mmol) were dissolved in toluene (8 mL) and reacted at 40℃for 16 hours, dried by spinning, medium pressure reverse phase preparation (0-40% acetonitrile/water) to give the product (20 mg, 17.2%).

The molecular formula: c (C) ₂₈ H ₃₀ N ₈ O ₃ Molecular weight: 526.6LC-MS (M/e): 527.2 (M+H) ⁺ )

¹ HNMR(400MHz,CDCl ₃ ):δ:10.38(s,1H),8.91(s,1H),8.15-8.05(m,1H),7.80-7.70(m,3H),7.65-7.55(m,1H),7.50-7.40(m,2H),5.70-5.60(m,1H),5.36(s,1H),5.05-4.95(m,1H),4.85-4.75(m,1H),4.75-4.65(m,2H),3.90-3.70(m,3H),3.55-3.45(m,1H),2.60-2.50(m,1H),2.25(m,3H),1.85-1.80(m,1H),1.45(s,6H).

Example 6: preparation of 2-allyl-6- ((4- (8-cyclopropyl-3, 8-diazabicyclo [3.2.1] oct-3-yl) phenyl) amino) -1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (Compound 6)

Preparation of 1, 8-cyclopropyl-3- (4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] octane

3- (4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] octane trifluoroacetate (2.2 g, crude product) was dissolved in methanol (30 mL), sodium cyanoborohydride (3.8 g,60.5 mmol), acetic acid (5.7 mL,99.6 mmol) and (1-ethoxycyclopropyloxy) trimethylsilane (3.8 g,21.8 mmol) were sequentially added at 0deg.C, the reaction was allowed to proceed for 4h at 60deg.C, LC-MS detection was completed, the reaction solution was diluted with water, dichloromethane/methanol (10:1) mixed solvent was extracted, the organic phase was dried, and spin-dried, and the residue was separated by column chromatography (dichloromethane: methanol=10:1) to give the objective compound (1.5 g).

Preparation of 4- (8-cyclopropyl-3, 8-diazabicyclo [3.2.1] octyl-3-yl) aniline

8-cyclopropyl-3- (4-nitrophenyl) -3, 8-diazabicyclo [3.2.1] octane (0.6 g,2.2 mmol) was dissolved in methanol (20 mL), palladium on carbon (1.0 g) was added, hydrogen was replaced three times, the reaction was carried out at 25℃for 1.5h, LC-MS detection was completed, the reaction solution was filtered, and the filtrate was dried to give a crude product of the objective compound (480 mg) which was directly used in the next step.

Preparation of 2-allyl-6- ((4- (8-cyclopropyl-3, 8-diazabicyclo [3.2.1] oct-3-yl) phenyl) amino) -1- (6- (2-hydroxy-propan-2-yl) pyridin-2-yl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one

2-allyl-1- (6- (2-hydroxy-prop-2-yl) pyridin-2-yl) -6- (methylsulfinyl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (580 mg, crude) was dissolved in toluene (20 mL), 4- (8-cyclopropyl-3, 8-diazabicyclo [3.2.1] oct-3-yl) aniline (400 mg, crude) and DIEA (6278 mg,4.86 mmol) were added, the reaction was completed at 25℃for 14H, LC-MS detected, the reaction solution was dried by spin-drying to give crude product (500 mg) which was then separated by reverse phase column chromatography (C18 column, water: methanol=90:10-40:60) to give the title compound (98 mg, yield 11%).

Molecular formula C ₃₁ H ₃₆ N ₈ O ₂ Molecular weight 552.7LC-MS (M/z): 553.1 (M+H) ⁺ )

¹ H-NMR(400MHz,CD ₃ OD)δ:8.82(s,1H),7.88-7.82(m,3H),7.42-7.25(m,3H),6.80-6.73(m,2H),5.75-5.65(m,1H),5.04(d,1H,J＝10.4),4.95(d,1H,J＝17.2),4.76-4.74(m,2H),4.00(s,1H),3.50-3.46(m,3H),3.38-3.33(m,2H),3.31-2.90(m,2H),2.15-2.00(m,2H),1.93-1.80(m,2H),1.70-1.30(m,6H),0.55-0.45(m,4H).

Example 7: preparation of 2-allyl-1- (6- (2-hydroxy-prop-2-yl) pyridin-2-yl) -6- ((4- (7-methyl-4, 7-diazaspiro [2.5] octyl-4-yl) phenyl) amino) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (compound 8)

Preparation of tert-butyl 4- (4-nitrophenyl) -4, 7-diazaspiro [2.5] octane-7-carboxylate

1-iodo-4-nitrobenzene (2.1 g,8.3 mmol) was dissolved in toluene (40 mL), tert-butyl 4, 7-diazaspiro [2.5] octane-7-carboxylate (1.5 g,7.1 mmol), palladium bis (tri-tert-butylphosphine) (562 mg,1.1 mmol) and sodium tert-butoxide (1.3 g,13.8 mmol) were added, the reaction was allowed to react at 110℃for 4h, LCMS detection was complete, the reaction solution was poured into water, EA extraction, anhydrous sodium sulfate was dried, the organic phase was dried, and silica gel column chromatography (EA: PE=1:3) gave the title compound (1.8 g, yield 76.0%).

Preparation of 2.4- (4-nitrophenyl) -4, 7-diazaspiro [2.5] octane trifluoroacetate salt

4- (4-Nitrophenyl) -4, 7-diazaspiro [2.5] octane-7-carboxylic acid tert-butyl ester (900 mg,2.7 mmol) was dissolved in dichloromethane (8 mL), trifluoroacetic acid (2 mL) was added dropwise at 25℃and reacted at 25℃for 1h, and completion of the reaction was detected by LCMS, and the reaction solution was dried by spin-drying to give a crude product (1.4 g).

Preparation of 3.7-methyl-4- (4-nitrophenyl) -4, 7-diazaspiro [2.5] octane

4- (4-Nitrophenyl) -4, 7-diazaspiro [2.5] octane trifluoroacetate (1.4 g, crude) was dissolved in N, N-dimethylformamide (20 mL), aqueous formaldehyde (37%) (2.2 g,27 mmol) was added, and the mixture was reacted at 25℃for 1h after which time the reaction was quenched by addition of water, the organic phase was extracted with dichloromethane, and the organic phase was concentrated and purified by silica gel column (methanol/dichloromethane=1:10) to give the title compound (650 mg, 97.4% yield over two steps).

Preparation of 4.4- (7-methyl-4, 7-diazaspiro [2.5] oct-4-yl) aniline

7-methyl-4- (4-nitrophenyl) -4, 7-diazaspiro [2.5] octane (650 mg,2.63 mmol) was dissolved in methanol (20 mL), palladium on carbon (300 mg) was added, and the mixture was reacted at 25℃for 1 hour, suction filtration and concentration of the filtrate to give a crude product (600 mg) which was directly used in the next reaction.

Preparation of 2-allyl-1- (6- (2-hydroxy-prop-2-yl) pyridin-2-yl) -6- ((4- (7-methyl-4, 7-diazaspiro [2.5] octyl-4-yl) phenyl) amino) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one

2-allyl-1- (6-2-hydroxypropan-2-yl) pyridin-2-yl) -6- (methylsulfinyl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (440 mg,1.18 mmol) was dissolved in toluene (20 mL) and 4- (7-methyl-4, 7-diazaspiro [2.5] oct-4-yl) aniline (395 mg, crude) and DIEA (458 mg,3.54 mmol) were added and reacted at 25℃for 16H, LCMS detection was complete, the reaction was dried by spin-drying and silica gel column chromatography (MeOH: DCM=1: 10 Crude product was obtained, and the objective compound (15 mg, yield 2.6%) was obtained by beating with ethyl acetate.

Molecular formula C ₂₉ H ₃₄ N ₈ O ₂ Molecular weight 526.6LC-MS (M/e): 527.1 (M+H) ⁺ )

¹ H-NMR(400MHz,MeOD)δ:8.81(s,1H),7.95-8.05(m,1H),7.70-7.80(m,1H),7.62-7.68(m,1H),7.55-7.61(m,2H),7.06-7.09(m,2H),5.65-5.75(m,1H),5.05(d,J＝10.4Hz,1H),4.90-4.96(m,1H),4.80-4.90(m,2H),4.60(s,1H),3.80-4.00(m,2H),2.95-3.10(m,3H),2.81(s,3H),1.59(s,6H),1.10-1.20(m,2H),0.90-1.10(m,2H).

Example 8: preparation of 2-allyl-6- ((4- (4-cyclopropyl-4, 7-diazaspiro [2.5] octane-7-yl) phenyl) amino) -1- (6- (2-hydroxypropyl-2-yl) pyridin-2-yl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (Compound 9)

Preparation of tert-butyl 7- (4-nitrophenyl) -4, 7-diazaspiro [2.5] octane-4-carboxylate

To 1-fluoro-4-nitrobenzene (1.0 g,7.1 mmol) in dimethyl sulfoxide (50.0 mL) were added 4, 7-diazaspiro [2.5] octane-4-carboxylic acid tert-butyl ester (1.7 g,8.0 mmol) and potassium carbonate (2.0 g,14.2 mmol), and the reaction mixture was reacted at 70℃for 5 hours, poured into water, the solid was separated and suction filtered, the filter cake was washed with water, and the filter cake was dried by spinning to give the objective compound (2.4 g, yield: 99.7%).

Preparation of 2.7- (4-nitrophenyl) -4, 7-diazaspiro [2.5] octane

To tert-butyl 7- (4-nitrophenyl) -4, 7-diazaspiro [2.5] octane-4-carboxylate (1.8 g,5.4 mmol) in dichloromethane (12.0 mL) was added trifluoroacetic acid (6.0 mL), and the mixture was reacted at 25℃for 1h, the reaction mixture was concentrated to dryness, diluted with water, pH was adjusted to alkaline with saturated sodium bicarbonate solution, extracted with dichloromethane, the organic phase was dried over anhydrous sodium sulfate, and the concentrated dryness was used directly in the next step.

Preparation of 3, 4-cyclopropyl-7- (4-nitrophenyl) -4, 7-diazaspiro [2.5] octane

To methanol (15.0 mL) of 7- (4-nitrophenyl) -4, 7-diazaspiro [2.5] octane (crude) (500.0 mg,2.1 mmol) was added acetic acid (1.3 g,22.3 mmol), sodium cyanoborohydride (841.5 mg,13.4 mmol) and (1-ethoxycyclopropyloxy) trimethylsilane (836.6 g,4.8 mmol), and the mixture was reacted at 65℃for 5 hours, the system was quenched with water, extracted with dichloromethane, the organic phase was concentrated, and the silica gel column was purified (ethyl acetate/petroleum ether=1:2) to give the title compound (560.0 mg, yield 95.6%).

Preparation of 4- (4-cyclopropyl-4, 7-diazaspiro [2.5] octyl-7-yl) aniline

To methanol (6.0 mL) of 4-cyclopropyl-7- (4-nitrophenyl) -4, 7-diazaspiro [2.5] octane (560.0 mg,2.0 mmol) was added palladium on carbon (200 mg), and the mixture was reacted at 25℃for 1h, suction filtered, and the filtrate was concentrated to give crude product (500.0 mg) which was directly used in the next reaction.

Preparation of 2-allyl-6- ((4- (4-cyclopropyl-4, 7-diazaspiro [2.5] octane-7-yl) phenyl) amino) -1- (6- (2-hydroxy-prop-2-yl) pyridin-2-yl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one

To toluene (20.0 mL) of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (methylsulfinyl) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (crude, 500.0mg,1.3 mmol) were added N, N-diisopropylethylamine (840.5 mg,6.5 mmol) and 4- (4-cyclopropyl-4, 7-diazaspiro [2.5] oct-7-yl) aniline (486.8 mg,2.0 mmol), and the mixture was reacted at 25℃for 16H, saturated sodium bicarbonate quenching, dichloromethane extraction, organic phase concentration, silica gel column purification (methanol/dichloromethane=1:20) to give the title compound (8.5 mg).

Molecular formula C ₃₁ H ₃₆ N ₈ O ₂ Molecular weight 552.7 LC-MS (M/e) 523.0 (M+H) ⁺ )

¹ H-NMR(400MHz,CDCl ₃ )δ:10.20-10.05(m,1H),8.81(s,1H),7.95-8.15(m,1H),7.85-7.70(m,1H),7.59-7.61(m,2H),6.90-6.88(m,2H),5.70-5.50(m,2H),5.31(s,1H),5.05-4.95(m,1H),4.80-4.90(m,1H),4.72-4.62(m,2H),3.20-3.10(m,2H),3.05-2.95(m,4H),2.00(s,1H),1.48(s,6H),0.80-0.70(m,2H),0.55-0.40(m,4H),0.32-0.20(m,2H).

Experimental protocol

Exemplary protocols for some of the compounds of the present invention are provided below to demonstrate the advantageous activity and beneficial technical effects of the compounds of the present invention. It should be understood that the following experimental schemes are merely illustrative of the present disclosure and are not intended to limit the scope of the present disclosure.

Experimental example 1 inhibition experiment of cell proliferation Activity by the Compound of the present invention

Test article: the structural formula and the preparation method of part of the compounds are shown in the preparation examples.

Positive control drug: AZD1775, prepared according to the method disclosed in the prior art WO2007126128, has the structure shown below:

experimental method

1. Experimental materials and reagents

Name of the name	Source	Goods number
			DMEM high sugar culture medium	Gibco	11995-065
MEM medium	Gibco	10370-021
			FBS (fetal bovine serum)	Gibco	10099-141C
Horse serum	Solarbio	S9050
			Pyruvic acid sodium salt	Gibco	11360-070
Glutamine	Gibco	35050-061
			Green streptomycin-diabody	Gibco	1507-063
0.25% trypsin	Gibco	25200-072
			10 XDPBS (Du's phosphate buffer)	Gibco	14200-075
96-hole bottom transparent white board	Corning	3610
			Gemcitabine (Guitar)	MCE	HY-B0003
CTG	Promega	G7571

2. Cell culture medium ratio and plating number

3. Experimental procedure

3.1 preparation of Compounds

Test compounds and reference positive control were diluted 3-fold in DMSO at 8 concentrations in 10mM DMSO. Test compounds and reference positive control were formulated with DMSO as 1000-fold solutions at the combined concentrations, i.e., 300 μm,100 μm,30 μm in 10mM DMSO.

A10 mM stock of Gemcitabine (Gemcitabine) was prepared and diluted to 1000-fold the final concentration, i.e., 100. Mu.M, and then diluted with a 3-fold DMSO gradient for 5 concentrations.

3.2 test procedure

Culturing cells according to the recommended conditions of source, removing culture medium, washing with PBS, digesting with 0.25% trypsin, collecting cells after complete culture, re-suspending to appropriate concentration, inoculating into 96-well plate at 37deg.C and 5% CO ₂ The incubator was incubated for 24 hours.

The compound is used singly: compound solution with final concentration of 1000 times, culture medium diluted 100 times, 10 μl/well added, 37 ℃ and 5% CO ₂ Culturing in incubator for 72 hr, and detecting.

Compound and gemcitabine combination: solution of gemcitabine with 1000 times final concentration, medium dilution 100 times, 10 μl/well, 37 ℃ and 5% co ₂ The incubator continues to incubate for 24 hours. Compound solution with final concentration of 1000 times, culture medium diluted 20 times, 5 μl/well, 37 ℃ and 5% CO ₂ The incubator continues to culture for 24 hours and then detects.

The culture plate is placed at room temperature in advance, 60 mu L of CTG detection liquid is added into each hole, the cells are cracked by shaking for 2 minutes on an orbital shaker in a dark place, and after 20 minutes of reaction, the luminescence value is read by a multifunctional enzyme-labeled instrument.

4. Data processing

Analysis of data using GraphPad Prism 8.0 software, fitting data to derive dose-response curves using nonlinear S-curve regression, and calculating IC therefrom ₅₀ Values.

Experimental results

TABLE 1 in vitro cytostatic Activity of Compounds of the invention alone

TABLE 2 in vitro cytostatic Activity of the Compounds of the invention in combination with gemcitabine

Conclusion of the experiment

The single compound has good inhibition effect on A431, SK-MES-1 cell proliferation activity, and has obvious synergistic effect on MIAPaCa2 cell proliferation activity inhibition when being combined with gemcitabine.

Experimental example 2 experiments on the metabolic stability of the Compound of the invention in liver microsomes in different species

Test article: the compound 3 of the invention is self-made, and the chemical name and the preparation method are shown in the preparation examples of the compound.

Positive control drug: AZD1775, prepared according to the methods disclosed in the prior art.

Experimental materials:

mixed liver microsomes of Cyno monkeys were purchased from the rad liver disease research center (Shanghai limited), lot number: SHZS, liver microsomal protein concentration is 20mg.mL < -1 >.

CD-1 mice, SD rats, beagle dogs and human mixed liver microsomes were purchased from corning corporation, and the batch numbers were 9025002 (mice), 0112002 (SD rats), 9259006 (Beagle dogs) and 38295 (humans), respectively, and the liver microsome protein concentration was 20 mg.mL-1.

The experimental initiation factor beta-NADPH is purchased from Solarbio company; phosphate Buffer (PBS) at pH 7.4 was self-made by the present laboratory.

Sample solution preparation:

accurately weighing a proper amount of sample powder, adding a proper amount of dimethyl sulfoxide (DMSO) to dissolve to 1mM, and diluting with methanol to 20 times to 50 μm working solution.

The experimental method comprises the following steps:

TABLE 3 composition of the liver microsome metabolic stability experiment incubation System

The experimental operation steps are as follows:

(1) According to the proportions of "experimental incubation System composition" in Table 3 above, 100mM PBS 5.85mL,20mM MgCl was taken for each compound ₂ Solution 0.585mL and H ₂ O3.57 mL, a mixed solution 1 of the incubation system (without microsomes, test sample and. Beta. -NADPH) was prepared. The experiment is carried out simultaneously with the positive drug verapamil of the experiment incubation system to prove that the activity of the liver microsome enzyme is normal.

(2) Liver microsomes (20 mg protein/mL) were removed from the-80℃refrigerator and pre-incubated for 3min on a 37℃water bath thermostated shaker.

(3) 1.9mL of the incubation system mixed solution 1 was taken for each species of the compound, and 56. Mu.L of microsomes of different species were added to prepare an incubation system mixed solution 2 (containing no test sample and. Beta. -NADPH).

(4) Sample group (containing microsomes and β -NADPH): mu.L of the mixed solution 2 of the incubation system was taken, 14. Mu.L of the working solution of the test sample having a concentration of 50. Mu.M was added, and 70. Mu.L of the working solution of 10mM of beta-NADPH was added. Mixing, and repeating. Sampling time points are 0min,5min,10min,20min,30min and 60min. This sample set was used to evaluate the metabolic stability of compounds via β -NADPH.

(5) Control group (microsome-containing, beta-NADPH-free, water instead of beta-NADPH): 264. Mu.L of the mixed solution 2 of the incubation system was added with 6. Mu.L of the working solution of the test sample having a concentration of 50. Mu.M, and 30. Mu.L of water was added. Mixing, and repeating. Sampling time points were 0min and 60min. The negative control group was used to evaluate whether the compound was present in the liver microsome incubation system for non- β -NADPH mediated metabolism.

(6) At each predetermined time point 50 μl was sampled from the incubation sample tube and added to the stop sample tube (300 μl of cold stop containing internal standard tolbutamide 50ng/mL acetonitrile solution), vortexed, and the reaction stopped.

(7) After vortexing for 10min, centrifugation was carried out for 5min (12000 rpm).

(8) 100 μl of the supernatant was taken, 100 μl of water was added, mixed well by vortexing, and analyzed by LC-MS/MS.

Data analysis:

the ratio of the test sample to the internal standard peak area is converted into the residual percentage by the following formula.

Experimental results:

TABLE 4 results of hepatic microsomal stability of the compounds of the invention

Conclusion of experiment:

the compound has good stability in human, monkey, dog, rat and mouse liver microsomes, and the stability in the mouse liver microsomes is equivalent to that of a control; in the other species of liver microsomes, the stability of the compound of the invention is obviously better than that of the control AZD1775.

Experimental example 3 inhibition experiment of in vitro kinase Activity by the Compounds of the present invention

Test article: the structural formula and the preparation method of part of the compounds are shown in the preparation examples. Abbreviations used in the following experiments represent the following meanings:

DMSO: dimethyl sulfoxide; HEPES: hydroxyethyl piperazine ethanesulfanilic acid.

The experimental method comprises the following steps: compounds were evaluated on WEE1 Kinase in vitro using the ADP-Glo Kinase Assay method.

The experimental steps are as follows:

1. compound dilution:

test compounds (10 mM stock) were diluted 100-fold with 100% DMSO at 1 in 384 dilution plates (labbyte, PP-0200): 3 performing equal ratio dilution of the concentration of the compound to be tested: 100. Mu.M, 33.33. Mu.M, 11.11. Mu.M, 3.7. Mu.M, 1.23. Mu.M, 0.41. Mu.M, 0.137. Mu.M, 0.046. Mu.M, 0.015. Mu.M, 0.005. Mu.M, 0. Mu.M.

2. Transfer 0.1. Mu.L of the test compound with Echo into 384 reaction plates (PE, 6007290), 1000rpm/min, and centrifuge for 1min.

3. Transfer 5. Mu.L of kinase to 384 reaction plates, centrifuge at 1000rpm/min, incubate at 25℃for 15min.

4. Transfer 5. Mu.L of the substrate mixture to 384 reaction plates, centrifuge at 1000rpm/min, incubate at 25℃for 60min. In the reaction system, the final concentrations of the test compounds were 1000nM,333.33nM,111.11nM,37.04nM,12.35nM,4.12nM,1.37nM,0.46nM,0.15nM,0.05nM,0nM. The final DMSO concentration was 1%.

5. Transfer 5. Mu.L ADP-Glo to 384 reaction plates at 1000rpm/min, centrifuge 1min, incubate at 25℃for 60min.

6. Transfer 10. Mu.L of Detection solution to 384 reaction plates at 1000rpm/min, centrifuge 1min, incubate at 25℃for 60min.

7. The RLU (Relative luminescence unit) signal was read using an Envision multifunction reader.

8. Data computation

1) Mean data and Standard Deviation (SD) of DMSO and positive quality control compound AZD-1775 (1000 nM inhibition 100%) were calculated for each plate as high and low controls.

2) Percent inhibition (% inh) =100× (max-compound signal)/(max-min)

"minimum" is the negative control well reading without compound; "maximum" is the positive control well reading plus positive quality control compound AZD-1775 (1000 nM inhibition 100%).

3) Fitting compound IC using nonlinear regression equation ₅₀ 。

Experimental results:

TABLE 5 in vitro enzymatic inhibitory Activity of the Compounds of the invention

Conclusion of experiment:

the compound has good inhibitory activity on WEE 1.

Claims

1. A compound as shown below or a pharmaceutically acceptable salt thereof:

2. a compound as shown below or a pharmaceutically acceptable salt thereof:

3. a compound as shown below or a pharmaceutically acceptable salt thereof:

4. a pharmaceutical formulation comprising a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, in any one of pharmaceutically acceptable dosage forms, comprising one or more pharmaceutically acceptable excipients.

5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, comprising one or more second therapeutically active agents selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, antisense DNA or RNA, antitumor antibiotics, growth factor inhibitors, signaling inhibitors, cell cycle inhibitors, enzyme inhibitors, retinoid receptor modulators, proteasome inhibitors, topoisomerase inhibitors, biological response modifiers, hormonal agents, angiogenesis inhibitors, cytostatic agents, targeting antibodies, HMG-CoA reductase inhibitors and prenyl protein transferase inhibitors.

6. Use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, a pharmaceutical formulation according to claim 4, or a pharmaceutical composition according to claim 5, in the manufacture of a medicament for the treatment and/or prophylaxis of a disease or condition mediated by Wee1 selected from cancer or benign neoplasm selected from lung cancer, squamous cell carcinoma, bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, rectal cancer, liver cancer, kidney cancer, esophageal adenocarcinoma, esophageal squamous cell carcinoma, prostate cancer, thyroid cancer, female genital tract cancer, lymphoma, neurofibromas, bone cancer, skin cancer, brain cancer, colon cancer, testicular cancer, gastrointestinal stromal tumors, mast cell tumors, multiple myeloma, melanoma, leukemia, glioma or sarcoma.

7. Use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, a pharmaceutical formulation according to claim 4, or a pharmaceutical composition according to claim 5, in the manufacture of a medicament for the treatment and/or prophylaxis of a disease or condition mediated by Wee1 selected from small cell lung cancer, non-small cell lung cancer.