CN113087724B

CN113087724B - Isothiazolopyrimidinone compounds, pharmaceutical compositions containing the same and uses thereof

Info

Publication number: CN113087724B
Application number: CN202010019057.1A
Authority: CN
Inventors: 陈寿军; 宋帅; 蒋小玲; 汪小蓓; 田强; 宋宏梅; 薛彤彤; 王晶翼
Original assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Current assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Priority date: 2020-01-08
Filing date: 2020-01-08
Publication date: 2024-01-19
Anticipated expiration: 2040-01-08
Also published as: CN113087724A

Abstract

The invention belongs to the field of pharmaceutical chemistry, and relates to an isothiazolopyrimidinone compound, a pharmaceutical composition containing the isothiazolopyrimidinone compound and application of the isothiazolopyrimidinone compound. Specifically, the invention provides a compound with a structure shown in a formula I, which shows good UPS7 inhibition activity, can be used as a high-efficiency UPS7 inhibitor and has anti-tumor activity.

Description

Isothiazolopyrimidinone compounds, pharmaceutical compositions containing the same and uses thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and relates to an isothiazolopyrimidinone compound with UPS7 inhibition activity, a pharmaceutical composition containing the isothiazolopyrimidinone compound and medical application of the isothiazolopyrimidinone compound.

Background

Ubiquitin-protease system (UPS) is a basic physiological regulatory process in cells, and proteins are degraded by proteases after being ubiquitinated and modified through a series of cascade reactions. Abnormalities in UPS are closely related to diseases such as tumors, neurodegenerative diseases, viral infections, and the like. Drugs are currently developed mainly for five classes of targets, protease, E1 activating enzyme, E2 binding enzyme, E3 ligase, deubiquitinases (DUBs) in UPS systems.

Deubiquitinase is capable of specifically cleaving an isopeptide bond formed between a glycine residue at the carbon terminus of ubiquitin and a target protein, allowing ubiquitin to be detached from the target protein, so that the target protein is protected from degradation, relocation, activation, or the like.

Currently there are nearly 100 DUBs in humans, of which ubiquitin-specific proteases (ubiquitin-specific proteases, USPs) are the largest family members of DUBs, including about 85 members, an isopeptidase belonging to the family of cysteine proteases (Wu and Kumar, journal of Medicinal Chemistry,2018, 61:422-443). It has now been found that over 40 members of the USPs family have been associated with the development and progression of tumors.

USP7 is located in the nucleus and is a key deubiquitinase in UPS, capable of specifically cleaving the isopeptide bond formed between the carbon end of ubiquitin and the target protein, allowing ubiquitin to be detached from the target protein, allowing the target protein to be protected from degradation, relocation, activation, etc. (Turnbull and Ioannidis, nature,2017,550,481-486).

USP7 plays an important role in the wide distribution of human tissues, in neural development, cell cycle regulation, epigenetic regulation, DNA damage repair, and immune response. There have been studies showing that USP7 is overexpressed in hepatocellular carcinoma, multiple myeloma, colon cancer, lung cancer, prostate cancer, bladder cancer, and the like cancer cells, and that this overexpression is directly related to tumor invasion and poor prognosis. (Pozhidaeva and Bezsonova, DNA Repair,2019,76,30-39)

USP7 is a rich substrate and most are proteins associated with Cell cycle regulation, immune response, apoptosis, and repair of DNA damage, such as MDM2, p53, ERCC6, foxp3, PTEN, FOXO4, etc. (Chauhan and Tian, cancer Cell,2012,22,345-358). MDM2 is overexpressed in some tumor cells, USP7 protects MDM2 from ubiquitination, and MDM2 promotes ubiquitination and degradation of p53 protein after binding to p53 protein, promoting tumor growth.

USP7 may also exert a tumorigenic effect by directly modulating the expression of tumor suppressor proteins (e.g., p53, PTEN, FOXO4, p114ARF, p16INK 4) and tumor promoter proteins (e.g., N-MYC, REST), up-regulating the expression of tumor-associated factors (e.g., HIF-1), and modulating tumor-associated signaling pathways (e.g., SHH signaling pathway, wnt/β -catenin signaling pathway, androgen receptor signaling pathway, DNA damage repair signaling pathway) (Zhou and Wang, medicinal Chemistry,2018,14,3-18).

In addition, USP7 also plays a role in tumor immune surveillance escape by modulating Treg cell upstream signaling molecules (such as transcription factor FOXP3 and epigenetic regulatory factor Tip 60), up-regulating Treg cell activity (Wang and Wu, PLoS One,2017,12,1-23), and inhibiting Teff cell (cd8+ T cell) activity.

Development of inhibitors of USP7 is one of the hot spots in the field of tumor research. Currently, no drug is marketed worldwide against the USP7 target, and all compounds under investigation are in preclinical research.

Although the companies of hybrid SA, forma Therapeutics, inc., les Laboratoires Servier, almac Discovery Limited have all made corresponding researches on USP7 inhibitors and related patent publications, there is still a need in the art for new USP7 inhibitors, particularly USP7 inhibitors having high activity and other excellent properties.

Disclosure of Invention

Problems to be solved by the invention

Through a great deal of research, the invention unexpectedly discovers an isothiazolopyrimidinone compound and a corresponding preparation method thereof, the compound can obviously inhibit the activity of UPS7, and the compound can be used as a UPS7 inhibitor for treating diseases mediated at least in part by UPS7, especially tumor diseases.

Solution for solving the problem

In a first aspect, the present invention provides a compound having the structure of formula I or a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite or prodrug thereof,

wherein the method comprises the steps of

R ¹ Is C _6-10 Aryl, 5-to 10-membered heteroaryl or

When R is ¹ Is C _6-10 Aryl or 5-to 10-membered heteroaryl, C _6-10 Aryl or 5-10 membered heteroaryl optionally substituted with one to more R ⁴ Substitution;

if present, each R ⁴ Each independently selected from hydrogen, halogen, cyano, C _1-6 Haloalkyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, -OR ^a 、-NR ^b R ^c 、-C _1-6 alkylene-NR ^b R ^c 、-C(＝O)R ⁵ 、-C(＝O)OR ⁶ 、-C(＝O)NR ^b R ^c 、-S(＝O) _q R ⁷ 、-S(＝O) _q NR ^b R ^c 、-O-(C _2-6 alkylene-O) _t -R ^a 、-O-C _2-6 alkylene-NR ^b R ^c and-NR ^a -C _2-6 alkylene-NR ^b R ^c ；

q is selected from 1 and 2, t is selected from 1, 2, 3 and 4;

when R is ¹ Is thatWhen the A ring and the B ring are each independently selected from C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl and 5-10 membered heteroaryl;

if present, each R ⁰ Each independently of the otherIs selected from hydrogen, halogen, cyano, C _1-6 Alkyl and C _1-6 A haloalkyl group;

r is selected from 0, 1, 2 and 3;

if present, each R ³ Each independently selected from hydrogen, oxo, halogen, cyano, C _1-6 Alkyl, C _1-6 Haloalkyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl, 5-10 membered heteroaryl, -OR ^a 、-NR ^b R ^c 、-C(＝O)R ⁵ 、-C(＝O)OR ⁶ 、-C(＝O)NR ^b R ^c 、-O-C _2-6 alkylene-NR ^b R ^c and-NR ^a -C _2-6 alkylene-NR ^b R ^c Wherein C _1-6 Alkyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl or 5-to 10-membered heteroaryl optionally substituted with one or more substituents selected from hydrogen, halogen, cyano, C _1-6 Alkyl, hydroxy, amino, -N (C) _1-6 Alkyl group ₂ and-NH (C) _1-6 Alkyl);

m is selected from 0, 1, 2, 3, 4, 5, 6, 7 and 8;

if present, R ^a 、R ^b And R is ^c Each independently selected from hydrogen, C _1-6 Alkyl, C _1-6 Haloalkyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl, -C (=o) R, a 5-10 membered heteroaryl ⁵ 、-S(＝O) _q R ⁷ Wherein C _1-6 Alkyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl or 5-to 10-membered heteroaryl optionally substituted with one or more substituents selected from hydrogen, halogen, hydroxy, amino, cyano, C _1-6 Alkyl, C _1-6 Haloalkyl and C _3-8 Substituents of cycloalkyl groups; or alternatively

If present, R ^b And R is ^c Together with the nitrogen atom to which it is attached, form a 3-6 membered ring;

if present, R ⁵ 、R ⁶ And R is ⁷ Each independently selected from hydrogen, C _1-6 Alkyl and C _3-8 Cycloalkyl group, wherein C _1-6 Alkyl or C _3-8 Cycloalkyl radicalsOptionally substituted with one or more substituents selected from hydrogen, halogen, cyano, amino and hydroxy;

R ² selected from-C (=O) R ⁸ 、-C(＝O)OR ⁹ 、-C(＝O)NR ^e R ^f 、-S(＝O) _q R ¹⁰ and-S (=o) _q NR ^e R ^f ；

If present, R ⁸ 、R ⁹ 、R ¹⁰ 、R ^e And R is ^f Each independently selected from hydrogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl and 5-10 membered heteroaryl, wherein C _1-6 Alkyl, C _2-6 Alkenyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl or 5-10 membered heteroaryl optionally substituted with one to more R ¹¹ Substitution; or alternatively

If present, R ^e And R is ^f Together with the nitrogen atom to which they are attached form a 3-6 membered ring, wherein the 3-6 membered ring is optionally substituted with one or more R ¹¹ Substitution;

if present, each R ¹¹ Each independently selected from hydrogen, cyano, halogen, hydroxy, -O (C) _1-6 Alkyl) -NR ^g R ^h 、C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl and 5-10 membered heteroaryl, wherein C _1-6 Alkyl, C _2-6 Alkenyl, C _2-6 Alkynyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl or 5-to 10-membered heteroaryl optionally substituted with one or more substituents selected from hydrogen, halogen, cyano, C _1-6 Alkyl, C _1-6 Haloalkyl, C _6-10 Substituents for aryl and 5-10 membered heteroaryl;

if present, R ^g And R is ^h Each independently selected from hydrogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _3-8 Cycloalkyl and 3-8 membered heterocycloalkyl.

In a second aspect, the present invention provides a specific compound having the structure of formula I, comprising:

(1) 6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(1A) 6- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(2) 6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) -3- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(2A) 6- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -3- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(3) 3- (4-fluorophenyl) -6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(3A) (R) -3- (4-fluorophenyl) -6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(4) 3- (4-chlorophenyl) -6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(4A) (R) -3- (4-chlorophenyl) -6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(5) 3- (2-amino-2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(6) 6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(7) 6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(8) 3- (7-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(9) 3- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(10) 6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(11) 3- (7-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(12) 3- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(13) 6- ((1- (3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(14) 6- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(15) 6- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(16) 3- (6-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(17) 3- (6-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(18) 3- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(19) 3- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(20) 3- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3- (4-fluorophenyl) propionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(21) 3- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3- (4-fluorophenyl) propionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(22) 6- ((1- (3- (4-chlorophenyl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -3- (4-hydroxyphenyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(23) 3- (6-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3- (4-chlorophenyl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(24) 3- (4- (aminomethyl) phenyl) -6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(25) 6- ((1- (3- (4-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (4-hydroxyphenyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(26) 3- (6-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(27) 3- (4- (aminomethyl) phenyl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(28) 3- (6-chloro-1-oxo-2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(29) 6- ((1- (3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (6-chloro-2-oxo-2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(30) 6- ((1- (3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (6-chloro-3-oxo-2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(31) 3- (7-chloroindolin-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(32) 3- (7-chloroisoindolin-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(33) 6- ((1- (3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (4-chloroindolin-6-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(34) 6- ((1- (3- (4-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(35) 3- (7-chloro-3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(36) 3- (6-chloro-3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(37) 3- (4-chloro-3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(38) 3- (4-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(39) 3- (7-chloro-2- (dimethylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3- (4-fluorophenyl) propionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(40) 3- (7-chloro-3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3- (4-fluorophenyl) propionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(41) 3- (6-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(42) 3- (4-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one;

(43) 6- ((1- (3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one; and

(44) 3- (6-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3- (4-fluorophenyl) propionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one.

In a third aspect, the present invention provides a pharmaceutical composition comprising a compound having the structure of formula I above, or a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite, or prodrug thereof, and one or more pharmaceutically acceptable carriers.

In a fourth aspect, the present invention provides a kit comprising:

a) A compound having the structure of formula I above or a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite, or prodrug thereof, or a pharmaceutical composition as described above;

b) Optionally package and/or instructions.

In a fifth aspect, the present invention provides a compound having the structure of formula I, or a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite, or prodrug thereof, as described above, or a pharmaceutical composition, or kit, as described above, for use as a UPS7 inhibitor, for use in the prevention and/or treatment of a disease or disorder (particularly cancer) mediated at least in part by UPS 7.

In a sixth aspect, the present invention provides the use of a compound having the structure of formula I, or a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite, or prodrug thereof, or a pharmaceutical composition, or kit, as described above, as a UPS7 inhibitor.

In a seventh aspect, the present invention provides the use of a compound having the structure of formula I, or a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite, or prodrug thereof, or a pharmaceutical composition, or kit, as described above, for the manufacture of a medicament for the prevention and/or treatment of a disease or disorder mediated at least in part by UPS7, particularly cancer.

In an eighth aspect, the invention provides a method for preventing and/or treating a disease or disorder (particularly cancer) mediated at least in part by UPS7, comprising the steps of: a prophylactically and/or therapeutically effective amount of a compound having the structure of formula I as described above, or a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite or prodrug thereof, or a pharmaceutical composition as described above, or a kit as described above, is administered to a subject in need thereof.

ADVANTAGEOUS EFFECTS OF INVENTION

The invention provides an isothiazolopyrimidinone compound with a novel structure, which can be used as a high-efficiency UPS7 inhibitor and has anti-tumor activity. In addition, the synthesis method of the compound is mild, is simple and feasible to operate, and is suitable for industrial mass production.

Detailed Description

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described herein. It will also be understood that, unless defined otherwise, all terms used herein have the same meaning or intent as commonly understood by one of ordinary skill in the art. Although the meaning or intent of the terms used herein will be readily understood by those skilled in the art, in order to better explain the technical solution of the present invention, the following description is provided.

[ definition of terms ]

The terms "comprising," "including," "having," "involving," or any other variation thereof, are intended to cover a non-exclusive or open-ended inclusion. For example, a composition, method, or apparatus that comprises a list of elements is not necessarily limited to only those elements explicitly listed, but may also include other elements not explicitly listed or inherent to such composition, method, or apparatus.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the invention which are substantially non-toxic to organisms. Pharmaceutically acceptable salts generally include, but are not limited to, salts formed from the compounds of the present invention by reaction with pharmaceutically acceptable inorganic/organic acids or inorganic/organic bases, such salts also being referred to as acid addition salts or base addition salts.

The term "pharmaceutically acceptable ester" refers to an ester that is substantially non-toxic to an organism in which it is hydrolyzed to form a compound of the invention or a salt thereof. Pharmaceutically acceptable esters generally include, but are not limited to, esters of the compounds of the present invention with pharmaceutically acceptable carboxylic or sulfonic acids, such esters also being referred to as carboxylic or sulfonic acid esters.

The term "isomer" refers to a compound that has the same molecular weight due to the same number and type of atoms, but differs in the spatial arrangement or configuration of the atoms.

The term "stereoisomer" (or "optical isomer") refers to a stable isomer that has a perpendicular plane of asymmetry due to at least one chiral factor (including chiral center, chiral axis, chiral plane, etc.), thereby enabling rotation of plane polarized light. The present invention also includes stereoisomers and mixtures thereof, due to the presence of asymmetric centers and other chemical structures which may lead to stereoisomers. Since the compounds of the present invention (or pharmaceutically acceptable salts thereof) include asymmetric carbon atoms, they can exist as single stereoisomers, racemates, mixtures of enantiomers and diastereomers. In general, these compounds can be prepared in the form of racemates. However, if desired, such compounds can be prepared or isolated to give pure stereoisomers, i.e., single enantiomers or diastereomers, or mixtures enriched in single stereoisomers (purity. Gtoreq.98%,. Gtoreq.95%,. Gtoreq.93%,. Gtoreq.90%,. Gtoreq.88%,. Gtoreq.85% or. Gtoreq.80%). As described below, individual stereoisomers of the compounds are prepared synthetically from optically active starting materials containing the desired chiral centers or by preparation of mixtures of enantiomeric products followed by separation or resolution, e.g., conversion to mixtures of diastereomers followed by separation or recrystallization, chromatography, use of chiral resolving agents, or direct separation of enantiomers on chiral chromatographic columns. Starting compounds having specific stereochemistry are either commercially available or prepared according to the methods described below and resolved by methods well known in the art. The term "enantiomer" refers to a pair of stereoisomers that have non-overlapping mirror images of each other. The term "diastereoisomer" or "diastereomer" refers to optical isomers that do not form mirror images of each other. The term "racemic mixture" or "racemate" refers to a mixture containing equal parts of a single enantiomer (i.e., an equimolar mixture of the two R and S enantiomers). The term "non-racemic mixture" refers to a mixture containing unequal portions of individual enantiomers. All stereoisomeric forms of the compounds of the invention are within the scope of the invention unless otherwise indicated.

The term "tautomer" (or "tautomeric form") refers to structural isomers having different energies that can be converted to each other by a low energy barrier. If tautomerism is possible (e.g., in solution), chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (or proton transfer tautomers) include, but are not limited to, interconversions by proton transfer, such as keto-enol isomerisation, imine-enamine isomerisation, amide-imine alcohol isomerisation, and the like. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

The term "polymorph" (or "polymorphic form") refers to a solid crystalline form of a compound or complex. The polymorphs of a molecule can be obtained by a number of known methods by a person skilled in the art. Such methods include, but are not limited to, melt recrystallization, melt cooling, solvent recrystallization, desolvation, rapid evaporation, rapid cooling, slow cooling, vapor diffusion, and sublimation. In addition, polymorphs can be detected, classified and identified using well known techniques including, but not limited to, differential Scanning Calorimetry (DSC), thermogravimetric analysis (TGA), X-ray powder diffraction (XRPD), single crystal X-ray diffraction (SCXRD), solid state Nuclear Magnetic Resonance (NMR), infrared spectroscopy (IR), raman spectroscopy, scanning Electron Microscopy (SEM), and the like.

The term "solvate" refers to a substance formed by the association of a compound of the invention (or a pharmaceutically acceptable salt thereof) with at least one solvent molecule by non-covalent intermolecular forces. Common solvates include, but are not limited to, hydrates (including hemihydrate, monohydrate, dihydrate, trihydrate, and the like), ethanolates, acetonates, and the like.

The term "nitroxide" refers to compounds formed by oxidation of nitrogen atoms in tertiary amines or nitrogen (aromatic) containing heterocyclic structures. For example, since the compound of formula I has a piperidine ring structure, the nitrogen atom therein may be further oxidized, and thus a nitrogen oxide may be formed.

The term "isotopic label" refers to a derivative compound from which a specific atom in a compound of the present invention is replaced by its isotopic atom. Unless otherwise indicated, the compounds of the invention include various isotopes of H, C, N, O, F, P, S, cl, e.g ² H(D)、 ³ H(T)、 ¹³ C、 ¹⁴ C、 ¹⁵ N、 ¹⁷ O、 ¹⁸ O、 ¹⁸ F、 ³¹ P、 ³² P、 ³⁵ S、 ³⁶ S and ³⁷ cl. For example, the number of the cells to be processed, ¹² c can be covered by ¹² C、 ¹³ C or ¹⁴ C is substituted; ¹ h can be covered by ² H (D, deuterium) or ³ H (T, tritium) substitution; ¹⁶ o can be used ¹⁸ O substitution, etc. The invention includes isotopically-labeled compounds obtained by substitution of any atom in the structure with its isotope.

The term "metabolite" refers to a derivative compound of the present invention which is formed after metabolism. For further information on metabolism see Goodman and Gilman's The Pharmacological Basis of Therapeutics (9 ^th ed.)[M],McGraw-Hill International Editions,1996。

The term "prodrug" refers to a derivative compound that is capable of providing a compound of the invention directly or indirectly after administration to a subject. Particularly preferred derivative compounds or prodrugs are compounds that, when administered to an individual, may increase the bioavailability of the compounds of the invention (e.g., are more readily absorbed into the blood) or promote delivery of the parent compound to the site of action (e.g., the lymphatic system). All prodrug forms of the compounds of the invention are within the scope of the invention unless otherwise indicated, and the various prodrug forms are well known in the art.

The term "independently" means that at least two groups (or ring systems) present in the structure that are the same or similar in value range may have the same or different meanings in the particular case. For example, substituent X and substituent Y are each independently hydrogen, halogen, hydroxy, cyano, alkyl or aryl, then when substituent X is hydrogen, substituent Y may be either hydrogen or halogen, hydroxy, cyano, alkyl or aryl; similarly, when the substituent Y is hydrogen, the substituent X may be either hydrogen or halogen, hydroxy, cyano, alkyl or aryl.

The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) and iodine (I).

The term "alkyl" refers to a straight or branched aliphatic hydrocarbon group. For example, the term "C" as used in the present invention _1-6 Alkyl "means an alkyl group having 1 to 6 carbon atoms (e.g., methyl, ethyl,N-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, etc.), optionally substituted with one or more substituents described herein (such as when substituted with halogen, the group is "haloalkyl", e.g., -CF ₃ 、-C ₂ F ₅ 、-CHF ₂ 、-CH ₂ F、-CH ₂ CF ₃ 、-CH ₂ Cl、-CH ₂ CH ₂ CF ₃ Etc.).

The term "cycloalkyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic hydrocarbon group (e.g., monocyclic cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and the like; or bicyclic cycloalkyl including fused, bridged or spiro rings, such as bicyclo [1.1.1 ]]Amyl, bicyclo [2.2.1]Heptyl, etc.), optionally substituted with one or more substituents described herein. For example, the term "C" as used in the present invention _3-8 Cycloalkyl "refers to cycloalkyl having 3 to 8 carbon atoms, optionally substituted with one or more substituents described herein (e.g., methyl substituted cyclopropyl).

The term "alkoxy" refers to an "alkyl" or "cycloalkyl" group, as defined above, attached to the parent molecular moiety through an oxygen atom (e.g., C _1-6 Alkoxy, C _3-8 Cycloalkoxy, and the like, including methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, cyclobutoxy, n-pentoxy, isopentoxy, n-hexoxy, or isomers thereof).

The term "heterocycloalkyl" refers to a saturated or partially saturated, monocyclic or polycyclic (such as bicyclic) non-aromatic group having one or more carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) and one or more (e.g., 2, 3, or 4) groups each independently selected from C (=o), O, S, N, NH, S (=o), and S (=o) ₂ As ring atoms. If the valence requirements are met, the heterocycloalkyl group can be attached to the parent molecular moiety through any one of the ring atoms. For example, the present inventionThe term "3-8 membered heterocycloalkyl" as used in the present specification means a heterocycloalkyl group having 3 to 8 ring atoms (for example, ethylene oxide (oxyalkyl), aziridinyl (aziridinyl), azetidinyl (azetidinyl), oxetanyl (oxyanyl), tetrahydrofuranyl (tetrahydrofuranyl), dioxolyl (dioxanyl), pyrrolidinyl (pyrrosinyl), pyrrolidone group (pyrrosinyl), imidazolidinyl (imidozolidinyl), pyrazolidinyl (pyrrosidinyl), pyrrolidinyl (pyrrosidinyl), tetrahydropyranyl (tetrahydropyranyl), piperidinyl (piperidinyl), piperazinyl (piperizinyl), morpholinyl (morpholinyl), thiomorpholinyl (thiomorpholinyl), dithianyl (dithianyl) or trithianyl (thiatri)).

The term "aryl" refers to a monocyclic or fused polycyclic aromatic hydrocarbon group having a conjugated pi-electron system. For example, the term "C" as used in the present invention _6-10 Aryl "refers to aryl groups having 6 to 10 carbon atoms (e.g., phenyl, naphthyl, etc.) optionally substituted with one or more substituents as described herein (e.g., with C _1-6 Alkyl (methyl, ethyl) or halogen (fluoro, chloro, bromo).

The term "heteroaryl" refers to a monocyclic or fused polycyclic aromatic group having a conjugated pi-electron system with one or more carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 9, or 10 carbon atoms) and one or more heteroatoms or groups of heteroatoms (e.g., 2, 3, or 4) each independently selected from O, S, N and NH as ring atoms. If the valence requirements are met, the heterocycloalkyl group can be attached to the parent molecular moiety through any one of the ring atoms. For example, the term "5-10 membered heteroaryl" as used in the present invention refers to heteroaryl groups having 5 to 10 ring atoms (e.g., thienyl (thienyl), furyl (furyl), pyrrolyl (pyrroyl), oxazolyl (oxazolyl), thiazolyl (thiazolyl), imidazolyl (imidazolyl), pyrazolyl (pyrazoyl), isoxazolyl (isoxazolyl), isothiazolyl (isothiazolyl), oxadiazolyl (oxazoyl), triazolyl (triazolyl), thiadiazolyl (thiadiazozolyl), pyridyl (pyridinyl), pyridazinyl (pyridazinyl), pyrimidinyl (pyrimidyl), pyrazinyl (pyrazoyl), triazinyl (triazinyl) or benzo derivatives thereof), which are optionally substituted One or more substituents described herein (e.g., substituted with C _1-6 Alkyl (methyl, ethyl) or halogen (fluoro, chloro, bromo).

The term "alkenyl" refers to a straight or branched aliphatic hydrocarbon group having at least one carbon-carbon double bond, typically having 2 to 15 carbon atoms. For example, the term "C" as used in the present invention _2-6 Alkenyl "refers to alkenyl groups having 2 to 6 carbon atoms (e.g., ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, n-octenyl, n-decenyl, and the like).

The term "alkynyl" refers to a straight or branched aliphatic hydrocarbon group having at least one carbon-carbon triple bond, typically having 2 to 18 carbon atoms. For example, the term "C" as used in the present invention _2-6 Alkynyl "refers to alkynyl groups having 2 to 6 carbon atoms (e.g., ethynyl, 2-propynyl, 2-butynyl, 1, 3-butadiynyl, and the like).

Alkenyl or alkynyl groups in the present invention may be unsubstituted or substituted with one or more substituents described herein, each independently selected from the group consisting of halogen, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, alkoxy, aryloxy, alkoxyalkyl, alkylthio, amino, alkylamino, dialkylamino, cycloalkylamino, alkanoyloxy, aroyloxy, cycloalkyloxy, carboxy and alkoxycarbonyl.

The term "amino" refers to "-NH- ₂ "groups" which are unsubstituted or substituted by one or more of the substituents described in the present invention (e.g. -NH. Times. ₂ 、-NHCH ₃ 、-N(CH ₃ ) ₂ 、-NH(C _1-3 Alkyl), -N (C) _1-3 Alkyl group ₂ Etc.). The term "amino" as used herein may also be protected by an amino protecting group unless otherwise indicated. Suitable amino protecting groups include acetyl (Ac), t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), and the like.

The term "cyano" refers to the "-CN" group.

The term "hydroxy" refers to an "-OH" group.

The term "oxo" refers to the "=o" group. Oxo groups are generally present on carbon atoms which, in addition to them, are attached to two other atoms, groups of atoms (radicals) or structural fragments, which together form a carbonyl group. The term "carbonyl" refers to a "- (c=o) -" group.

The term "substituted" means that one or more (e.g., 1, 2, 3, or 4) atoms (e.g., hydrogen atoms) or groups of atoms (e.g., triflate groups) in the specified group are replaced with other atoms or groups of atoms, provided that the specified group meets the valence requirements in the current case and forms a stable compound after substitution. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. If a substituent is described as "optionally substituted with …," the substituent may be unsubstituted or substituted. If a first substituent is described as being optionally substituted with one or more of the second list of substituents, one or more hydrogen atoms in the first substituent may be replaced, or unsubstituted, by one or more of the second list of substituents, either alone or each independently.

When the bond of a substituent is shown as a bond through the ring connecting two atoms, then such substituent may be connected to any ring-forming atom in the ring.

The term "one or more" means 1 or more than 1, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, under reasonable conditions.

[ Compounds of the general formula ]

The present invention provides a compound of formula I or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite or prodrug thereof,

wherein the method comprises the steps of

R ¹ Is C _6-10 Aryl, 5-to 10-membered heteroaryl or

q is selected from 1 and 2, t is selected from 1, 2, 3 and 4;

if present, each R ⁰ Each independently selected from hydrogen, halogen, cyano, C _1-6 Alkyl and C _1-6 A haloalkyl group;

r is selected from 0, 1, 2 and 3;

if present, each R ³ Each independently selected from hydrogen, oxo, halogen, cyano, C _1-6 Alkyl, C _1-6 Haloalkyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl, 5-10 membered heteroaryl, -OR ^a 、-NR ^b R ^c 、-C(＝O)R ⁵ 、-C(＝O)OR ⁶ 、-C(＝O)NR ^b R ^c 、-O-C _2-6 alkylene-NR ^b R ^c and-NR ^a -C _2-6 alkylene-NR ^b R ^c Which is provided withMiddle C _1-6 Alkyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl or 5-to 10-membered heteroaryl optionally substituted with one or more substituents selected from hydrogen, halogen, cyano, C _1-6 Alkyl, hydroxy, amino, -N (C) _1-6 Alkyl group ₂ and-NH (C) _1-6 Alkyl);

m is selected from 0, 1, 2, 3, 4, 5, 6, 7 and 8;

if present, R ⁵ 、R ⁶ And R is ⁷ Each independently selected from hydrogen, C _1-6 Alkyl and C _3-8 Cycloalkyl group, wherein C _1-6 Alkyl or C _3-8 Cycloalkyl is optionally substituted with one or more substituents selected from hydrogen, halogen, cyano, amino and hydroxy;

If present, R ⁸ 、R ⁹ 、R ¹⁰ 、R ^e And R is ^f Each independently selected from hydrogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, C _6-10 Aryl and 5-10 membered heteroaryl, wherein C _1-6 Alkyl, C _2-6 Alkenyl, C _3-8 NaphtheneRadicals, 3-to 8-membered heterocycloalkyl, C _6-10 Aryl or 5-10 membered heteroaryl optionally substituted with one to more R ¹¹ Substitution; or alternatively

In some embodiments, R in the above-described compounds of formula I ¹ Is C _6-10 Aryl or 5-10 membered heteroaryl, optionally substituted with one or more R ⁴ Substitution; if present, each R ⁴ Each independently selected from hydrogen, halogen, cyano, -OR ^a 、-NR ^b R ^c and-C _1-6 alkylene-NR ^b R ^c ；R ^a 、R ^b 、R ^c And R is ² Is defined as before.

In some preferred embodiments, R in the above-described compounds of formula I ¹ Is C _6-10 Aryl, optionally substituted with one or more R ⁴ Substitution; if present, each R ⁴ Each independently selected from hydrogen, halogen, -OR ^a and-C _1-6 alkylene-NR ^b R ^c ；R ^a 、R ^b 、R ^c And R is ² Is defined as before.

In some more preferred embodiments, R in the above-described compounds of formula I ¹ Is phenyl, optionally substituted with one or more R ⁴ Substitution; if present, each R ⁴ Each independently selected from hydrogen, fluorine, chlorine, hydroxyl and aminomethyl; r is R ² Is defined as before.

In some more preferred embodiments, R in the above-described compounds of formula I ¹ Selected from the group consisting of 4-fluorophenyl, 4-chlorophenyl, 4-hydroxyphenyl and 4- (aminomethyl) phenyl; r is R ² Is defined as before.

In some embodiments, R in the above-described compounds of formula I ¹ Is thatWherein ring A is C _6-10 Aryl or 5-10 membered heteroaryl; each R is ⁰ Each independently selected from hydrogen, halogen, C _1-6 Alkyl and C _1-6 A haloalkyl group; r is selected from 1, 2 and 3; b ring, R ³ M and R ² Is defined as before.

In some preferred embodiments, R in the above-described compounds of formula I ¹ Is thatWherein ring A is phenyl; each R is ⁰ Each independently selected from hydrogen, fluorine and chlorine; r is selected from 1, 2 and 3; b ring, R ³ M and R ² Is defined as before.

In some embodiments, R in the above-described compounds of formula I ¹ Is thatWherein ring B is C _3-8 Cycloalkyl or 3-8 membered heterocycloalkyl; each R is ³ Each independently selected from hydrogen, oxo, C _1-6 Alkyl, C _1-6 Haloalkyl, C _3-8 Cycloalkyl, 3-8 membered heterocycloalkyl, -OH, -NH ₂ 、-NH(C _1-6 Alkyl) and-N (C) _1-6 Alkyl group ₂ The method comprises the steps of carrying out a first treatment on the surface of the m is selected from 1,2 and 3; ring A, R ⁰ R and R ² Is defined as before.

In some preferred embodiments, R in the above-described compounds of formula I ¹ Is thatWherein ring B is C _3-8 Cycloalkyl; each R is ³ Each independently selected from hydrogen, oxo, C _1-6 Alkyl, -NH ₂ 、-NH(C _1-6 Alkyl) and-N (C) _1-6 Alkyl group ₂ The method comprises the steps of carrying out a first treatment on the surface of the m is selected from 1, 2 and 3; ring A, R ⁰ R and R ² Is defined as before.

In some more preferred embodiments, R in the above-described compounds of formula I ¹ Is thatWherein ring B is C _4-6 Cycloalkyl; each R is ³ Each independently selected from hydrogen, oxo, methyl, -NH ₂ 、-NHCH ₃ and-N (CH) ₃ ) ₂ The method comprises the steps of carrying out a first treatment on the surface of the m is selected from 1, 2 and 3; ring A, R ⁰ R and R ² Is defined as before. />

In some more preferred embodiments, R in the above-described compounds of formula I ¹ Is thatWherein ring B is cyclopentyl; each R is ³ Each independently selected from hydrogen, oxo, methyl, -NH ₂ 、-NHCH ₃ and-N (CH) ₃ ) ₂ The method comprises the steps of carrying out a first treatment on the surface of the m is selected from 1, 2 and 3; ring A, R ⁰ R and R ² Is defined as before.

In some preferred embodiments, R in the above-described compounds of formula I ¹ Is thatWherein ring B is 3-8 membered heterocycloalkyl; each R is ³ Each independently selected from hydrogen, oxo, C _1-6 Alkyl, -NH ₂ 、-NH(C _1-6 Alkyl) and-N (C) _1-6 Alkyl group ₂ The method comprises the steps of carrying out a first treatment on the surface of the m is selected from 1, 2 and 3; ring A, R ⁰ R and R ² Is defined as before.

In some more preferred embodiments, R in the above-described compounds of formula I ¹ Is thatWherein ring B is a 4-6 membered heterocycloalkyl; r is R ³ Is hydrogen; ring A, R ⁰ R and R ² Is defined as before.

In some more preferred embodiments, R in the above-described compounds of formula I ¹ Is thatWherein ring B is pyrrolidine; r is R ³ Is hydrogen; ring A, R ⁰ R and R ² Is defined as before.

In some more preferred embodiments, R in the above-described compounds of formula I ¹ Selected from the group consisting of

/>

R ² Is defined as before.

In some embodiments, R in the above-described compounds of formula I ² is-C (=O) R ⁸ or-C (=O) NR ^e R ^f The method comprises the steps of carrying out a first treatment on the surface of the If present, R ⁸ 、R ^e And R is ^f Each independently selected from hydrogen, C _1-6 Alkyl, C _2-6 Alkenyl, C _3-8 Cycloalkyl and 3-8 membered heterocycloalkyl, wherein C _1-6 Alkyl, C _2-6 Alkenyl, C _3-8 Cycloalkyl or 3-8 membered heterocycloalkyl optionally substituted with one or more R ¹¹ Substitution;or, if present, R ^e And R is ^f Together with the nitrogen atom to which they are attached form a 3-6 membered ring, wherein the 3-6 membered ring is optionally substituted with one or more R ¹¹ Substitution; if present, each R ¹¹ Each independently selected from halogen, C _1-6 Alkyl, C _3-8 Cycloalkyl, C _6-10 Aryl and 5-10 membered heteroaryl, wherein C _6-10 Aryl or 5-to 10-membered heteroaryl optionally substituted with one or more substituents selected from hydrogen, halogen, C _1-6 Alkyl and C _1-6 A substituent of a haloalkyl group; r is R ¹ Is defined as before.

In some preferred embodiments, R in the above-described compounds of formula I ² is-C (=O) R ⁸ ；R ⁸ Selected from C _1-6 Alkyl, C _2-6 Alkenyl, C _3-8 Cycloalkyl and 3-8 membered heterocycloalkyl, wherein C _1-6 Alkyl, C _2-6 Alkenyl, C _3-8 Cycloalkyl or 3-8 membered heterocycloalkyl optionally substituted with one or more R ¹¹ Substitution; if present, each R ¹¹ Each independently selected from halogen, C _1-6 Alkyl, C _3-8 Cycloalkyl, C _6-10 Aryl and 5-10 membered heteroaryl, wherein C _1-6 Alkyl, C _3-8 Cycloalkyl, C _6-10 Aryl or 5-10 membered heteroaryl optionally substituted with one or more substituents selected from hydrogen, halogen and C _1-6 Substituent substitution of alkyl; r is R ¹ Is defined as before.

In some more preferred embodiments, R in the above-described compounds of formula I ² is-C (=O) R ⁸ ；R ⁸ Is C _1-6 Alkyl optionally substituted with one or more R ¹¹ Substitution; if present, each R ¹¹ Each independently selected from fluorine, chlorine, cyclopropyl, phenyl and pyrazolyl, wherein phenyl or pyrazolyl is optionally substituted with one or more substituents selected from hydrogen, fluorine, chlorine and methyl; r is R ¹ Is defined as before.

In some more preferred embodiments, R in the above-described compounds of formula I ² is-C (=O) R ⁸ ；R ⁸ Selected from the following groups:

in addition, the invention also provides the following compounds or pharmaceutically acceptable salts, esters, solvates, stereoisomers, tautomers, polymorphs, nitrogen oxides, isotopic labels, metabolites or prodrugs thereof, the structures and names of which are shown in the following table:

/>

[ preparation method ]

The invention provides a preparation method of the compound shown in the formula I.

The preparation method comprises the following steps:

1) Carrying out halogenation reaction on the compound of the formula I-a to obtain a compound of the formula I-b;

2) The compound of the formula I-b and the compound of the formula I-g are subjected to coupling reaction to obtain a compound of the formula I-c;

3) The compound of the formula I-c is subjected to ring closure reaction to obtain a compound of the formula I-d;

4) The compound of the formula I-d and the compound of the formula I-h are subjected to substitution reaction to obtain a compound of the formula I-e;

5) Deprotection reaction of the compound of the formula I-e to obtain a compound of the formula I-f;

6) The compound of the formula I-f and the compound of the formula I-j undergo condensation reaction to obtain the compound of the formula I;

wherein R is ¹ 、R ² Is as defined above; x is selected from chlorine and bromine; r is R ¹² Selected from hydroxyl and halogen, preferably hydroxyl; r is R ¹³ Selected from boric acid groups, quilt C _1-6 Alkyl-substituted boric acid groups and methyl-substituted 1,3, 2-dioxaborolan-2-yl groups; r is R ¹⁵ Selected from C _1-6 Alkyl oxycarbonyl, C _1-6 Haloalkyloxycarbonyl, - (c=o) O-C _1-6 alkylene-Si (C) _1-6 Alkyl group ₃ 、-(C＝O)O-C _1-6 alkylene-C _2-6 Alkenyl, benzyloxycarbonyl (Cbz), benzyl (Bz) and 9-fluorenylmethoxycarbonyl (Fmoc), preferably t-butyloxycarbonyl (Boc) and 2- (trimethylsilyl) ethoxycarbonyl (Teoc).

In some embodiments, the halogenation reaction in step 1) of the above preparation process is carried out in a solvent selected from the group consisting of water, N-Dimethylformamide (DMF) and Tetrahydrofuran (THF), preferably THF.

In some embodiments, the halogenation reaction in step 1) of the above preparation process is carried out in the presence of a halogenating agent selected from the group consisting of bromine, N-bromosuccinimide, N-chlorosuccinimide, preferably N-bromosuccinimide.

In some embodiments, the halogenation reaction in step 1) of the above preparation process is carried out at a temperature of from 0 to 200 ℃, preferably at a temperature of from 0 to 50 ℃.

In some embodiments, the coupling reaction in step 2) of the above preparation method is performed in a solvent selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane, chloroform, 1, 2-dichloroethane, etc.), methanol, ethanol, DMF, acetonitrile, ethers (e.g., ethylene glycol dimethyl ether, THF, dioxane), aromatic hydrocarbons (e.g., toluene, benzene, xylene), water, and any combination thereof, preferably dioxane/water and toluene/water.

In some embodiments, the coupling reaction in step 2) of the above preparation process is carried out in basic conditions, the base being an organic or inorganic base; preferably, the organic base is selected from triethylamine, N-Diisopropylethylamine (DIPEA), N-methylmorpholine (NMM), sodium t-butoxide, potassium acetate and sodium acetate, and the inorganic base is selected from potassium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate and potassium dihydrogen phosphate; more preferably, the base is selected from potassium carbonate, potassium phosphate, potassium acetate and sodium bicarbonate.

In some embodiments, the coupling reaction in step 2) of the above preparation method is carried out in the presence of a catalyst selected from Pd (PPh ₃ ) ₄ 、Pd(OAc) ₂ 、Pd ₂ (dba) ₃ 、Pd(PPh ₃ ) ₂ Cl ₂ 、Pd(PPh ₃ ) ₂ Cl ₂ ·CH ₂ Cl ₂ 、Pd(dppf)Cl ₂ And Pd (amphos) Cl ₂ Pd (PPh) is preferred ₃ ) ₄ And Pd (dppf) Cl ₂ 。

In some embodiments, the coupling reaction in step 2) of the above preparation process is carried out at a temperature of 0-200 ℃, preferably at a temperature of 50-150 ℃.

In some embodiments, the ring closure reaction in step 3) of the above-described preparation method is performed in a solvent selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane, chloroform, 1, 2-dichloroethane, etc.), methanol, n-butanol, ethanol, DMF, acetonitrile, ethers (e.g., ethylene glycol dimethyl ether, THF, dioxane), aromatic hydrocarbons (e.g., toluene, benzene, xylene), water, and any combination thereof, preferably n-butanol.

In some embodiments, the ring closure reaction in step 3) of the above preparation process is performed in the presence of an organic reagent selected from the group consisting of formamidine acetate, formamide, trimethyl orthoformate, triethyl orthoformate and ammonium formate, preferably formamidine acetate.

In some embodiments, the ring closure reaction in step 3) of the above described preparation process is carried out at a temperature of from 0 to 200 ℃, preferably at a temperature of from 80 to 150 ℃.

In some embodiments, the substitution reaction in step 4) of the above preparation method is performed in an organic solvent selected from the group consisting of dimethyl sulfoxide (DMSO), DMF, N-Dimethylacetamide (DMA), alcohols (e.g., methanol, ethanol, isopropanol, t-butanol, etc.), ethers (e.g., anisole, diethyl ether, THF, dioxane, etc.), halogenated hydrocarbons (e.g., dichloromethane, chloroform, carbon tetrachloride, etc.), and acetonitrile, preferably DMSO and DMF.

In some embodiments, the substitution reaction in step 4) of the above preparation process is carried out in the presence of a base selected from triethylamine, DIPEA, pyridine, NMM, p-Dimethylaminopyridine (DMAP), sodium acetate, potassium acetate, ammonium acetate, sodium tert-butoxide, potassium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate, potassium hydroxide and sodium hydroxide, preferably potassium carbonate, cesium carbonate and pyridine.

In some embodiments, the substitution reaction in step 4) of the above preparation process is carried out at a temperature of from 0 to 200 ℃, preferably at a temperature of from 50 to 150 ℃.

In some embodiments, the deprotection reaction in step 5) of the above preparation method is carried out in a solvent selected from DMF, THF, acetonitrile and water, preferably THF.

In some embodiments, the deprotection reaction in step 5) of the above preparation process is carried out in the presence of an organic reagent selected from the group consisting of quaternary ammonium salts, potassium fluoride and cesium fluoride, preferably tetrabutylammonium fluoride.

In some embodiments, the deprotection reaction in step 5) of the above preparation method is carried out at a temperature of 0 to 200 ℃, preferably at a temperature of 0 to 100 ℃.

In some embodiments, the condensation reaction in step 6) of the above preparation method is performed in an organic solvent selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane, chloroform, 1, 2-dichloroethane, etc.), nitriles (e.g., acetonitrile, etc.), N-methylpyrrolidone, DMF, DMA, dioxane, DMSO, and any combination thereof, preferably dichloromethane and DMF.

In some embodiments, the condensation reaction in step 6) of the above preparation process is carried out in the presence of a condensing agent selected from ECF, IPCF, HATU, HBTU, EEDQ, DEPC, DCC, DIC, EDC, BOP, pyAOP and PyBOP, preferably HATU and EDC.

In some embodiments, the condensation reaction in step 6) of the above-described preparation method is carried out in the presence of a base, which is an organic base or an inorganic base; preferably, the organic base is selected from triethylamine, DIPEA, NMM and DMAP, and the inorganic base is selected from sodium hydride, sodium hydroxide, sodium carbonate and potassium carbonate; more preferably, the base is DIPEA.

In some embodiments, the condensation reaction in step 6) of the above described preparation process is carried out at a temperature of from 0 to 100 ℃, preferably at a temperature of from 15 to 50 ℃.

The invention provides another preparation method of the compound of the formula I.

The preparation method comprises the following steps:

1) The compound of the formula I-u and the compound of the formula I-y are subjected to substitution reaction to obtain the compound of the formula I-v;

2) Carrying out halogenation reaction and deprotection reaction on the compound of the formula I-v to obtain a compound of the formula I-w;

3) The compound of the formula I-w and the compound of the formula I-j undergo condensation reaction to obtain a compound of the formula I-x;

4) Coupling reaction is carried out on the compound of the formula I-x and the compound of the formula I-g to obtain the compound of the formula I;

Wherein R is ¹ 、R ² 、R ¹² 、R ¹³ 、R ¹⁵ X is as defined above.

In some embodiments, the substitution reaction in step 1) of the above preparation method is performed in an organic solvent selected from DMSO, DMF, DMA, alcohols (e.g., methanol, ethanol, isopropanol, t-butanol, etc.), ethers (e.g., anisole, diethyl ether, THF, dioxane, etc.), halogenated hydrocarbons (e.g., dichloromethane, chloroform, carbon tetrachloride, etc.), and acetonitrile, preferably DMSO and DMF.

In some embodiments, the substitution reaction in step 1) of the above preparation process is carried out in the presence of a base selected from triethylamine, DIPEA, pyridine, NMM, DMAP, sodium acetate, potassium acetate, ammonium acetate, sodium tert-butoxide, potassium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate, potassium hydroxide and sodium hydroxide, preferably potassium carbonate, cesium carbonate and pyridine.

In some embodiments, the substitution reaction in step 1) of the above preparation process is carried out at a temperature of 0 to 200 ℃, preferably at a temperature of 50 to 150 ℃.

In some embodiments, the halogenation reaction in step 2) of the above preparation method is carried out in a solvent selected from the group consisting of water, DMF and THF, preferably water and DMF.

In some embodiments, the halogenation reaction in step 2) of the above preparation process is carried out in the presence of a halogenating agent selected from the group consisting of bromine, N-bromosuccinimide and N-chlorosuccinimide, preferably N-bromosuccinimide.

In some embodiments, the halogenation (and deprotection) reaction in step 2) of the above preparation process is carried out in the presence of an acid, which is an organic or inorganic acid, preferably sulfuric acid or hydrochloric acid.

In some embodiments, the halogenation (and deprotection) reaction in step 2) of the above preparation process is carried out at a temperature of 0-200 ℃, preferably at a temperature of 0-100 ℃.

In some embodiments, the condensation reaction in step 3) of the above-described preparation method is performed in an organic solvent selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane, chloroform, 1, 2-dichloroethane, etc.), nitriles (e.g., acetonitrile, etc.), N-methylpyrrolidone, DMF, DMA, dioxane, DMSO, and any combination thereof, preferably dichloromethane and DMF.

In some embodiments, the condensation reaction in step 3) of the above preparation process is carried out in the presence of a condensing agent selected from ECF, IPCF, HATU, HBTU, EEDQ, DEPC, DCC, DIC, EDC, BOP, pyAOP and PyBOP, preferably HATU and EDC.

In some embodiments, the condensation reaction in step 3) of the above-described preparation method is carried out in the presence of a base, which is an organic base or an inorganic base; preferably, the organic base is selected from triethylamine, DIPEA, NMM and DMAP, and the inorganic base is selected from sodium hydride, sodium hydroxide, sodium carbonate and potassium carbonate; more preferably, the base is DIPEA.

In some embodiments, the condensation reaction in step 3) of the above described preparation process is carried out at a temperature of from 0 to 100 ℃, preferably at a temperature of from 15 to 50 ℃.

In some embodiments, the coupling reaction in step 4) of the above preparation method is performed in a solvent selected from the group consisting of halogenated hydrocarbons (e.g., dichloromethane, chloroform, 1, 2-dichloroethane, etc.), methanol, ethanol, DMF, acetonitrile, ethers (e.g., ethylene glycol dimethyl ether, tetrahydrofuran, dioxane, etc.), aromatic hydrocarbons (e.g., toluene, benzene, xylene, etc.), water, and any combination thereof, preferably dioxane/water and toluene/water.

In some embodiments, the coupling reaction in step 4) of the above preparation process is carried out in the presence of a base, which is an organic or inorganic base; preferably, the organic base is selected from triethylamine, DIPEA, NMM, sodium t-butoxide, potassium acetate and sodium acetate, and the inorganic base is selected from potassium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate and potassium dihydrogen phosphate; more preferably, the base is selected from potassium carbonate, potassium phosphate, potassium acetate and sodium bicarbonate.

In some embodiments, the coupling reaction in step 4) of the above preparation method is carried out in the presence of a catalyst selected from Pd (PPh ₃ ) ₄ 、Pd(OAc) ₂ 、Pd ₂ (dba) ₃ 、Pd(PPh ₃ ) ₂ Cl ₂ 、Pd(PPh ₃ ) ₂ Cl ₂ ·CH ₂ Cl ₂ 、Pd(dppf)Cl ₂ And Pd (amphos) Cl ₂ Pd (PPh) is preferred ₃ ) ₄ And Pd (dppf) Cl ₂ 。

In some embodiments, the coupling reaction in step 4) of the above preparation process is carried out at a temperature of 0-200 ℃, preferably at a temperature of 50-150 ℃.

[ pharmaceutical composition ]

The term "pharmaceutical composition" refers to a composition that can be used as a medicament comprising a pharmaceutically active ingredient (API) (or therapeutic agent) and optionally one or more pharmaceutically acceptable carriers, in order to facilitate administration to an organism, facilitate absorption of the active ingredient, and thus exert biological activity. The term "pharmaceutically acceptable carrier" refers to an adjuvant that is administered with a therapeutic agent and which is, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and/or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers that can be used in the present invention include (but are not limited to): a) Diluents such as water, fish oil, triglycerides, hydrogenated or partially hydrogenated vegetable oils (e.g. corn oil, olive oil, sunflower oil, safflower oil, etc.), docosahexaenoic acid or esters thereof, omega-3 fatty acids or derivatives thereof, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glucose, glycine, or mixtures thereof; b) Lubricants, for example, silica, talc, stearic acid or salts thereof (e.g., magnesium salts, calcium salts, etc.), sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, polyethylene glycol, or mixtures thereof; c) Binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose, magnesium carbonate, sugars (e.g., dextrose, lactose, and the like), corn sweeteners, natural and synthetic gums (e.g., acacia, sodium alginate, and the like), waxes, polyvinylpyrrolidone, or mixtures thereof; d) Disintegrants, for example, starch, agar, methylcellulose, bentonite, xanthan gum, alginic acid or a salt thereof (such as sodium salt), effervescent agents, or mixtures thereof; e) Absorbents, colorants, flavors, and sweeteners; f) Emulsifying or dispersing agents, such as caprylic/capric polyethylene glycol glycerides, polyethylene glycol glycerides oleate, glyceryl oleate, diethylene glycol monoethyl, or other acceptable emulsifying agents: and/or g) an agent that enhances absorption of the compound, such as cyclodextrin, hydroxypropyl cyclodextrin, polyethylene glycol 200, polyethylene glycol 400, or mixtures thereof.

The present invention provides a pharmaceutical composition comprising a compound of formula I above or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite, or prodrug thereof.

In some embodiments, the above pharmaceutical compositions further comprise one or more pharmaceutically acceptable carriers.

The above pharmaceutical composition may act systematically and/or locally. For this purpose, they may be administered by any suitable route, for example by parenteral, topical, intravenous, oral, subcutaneous, intra-arterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal, intramuscular, inhalation route or by all means well known to those skilled in the art of medicine. The above pharmaceutical composition may be administered in combination with at least one other therapeutic agent having therapeutic effects on the disease or condition.

The above route of administration may be accomplished by suitable dosage forms. Dosage forms useful in the present invention include (but are not limited to): tablets, capsules, troches, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups and the like.

The above pharmaceutical composition may comprise 0.01mg to 1000mg of at least one compound of formula I above or a pharmaceutically acceptable salt, ester, solvate (e.g. hydrate), stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite or prodrug thereof.

The present invention also provides a process for the preparation of the above pharmaceutical composition or a corresponding formulation thereof, which comprises combining at least one compound of formula I as described above or a pharmaceutically acceptable salt, ester, solvate (e.g. hydrate), stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite or prodrug thereof with one or more pharmaceutically acceptable carriers.

[ medicine box ]

The term "kit" refers to a combination product comprising a therapeutic agent, optionally other therapeutic agents, and optionally packaging and/or instructions.

The present invention provides a kit comprising:

a) A compound of formula I above or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite, or prodrug thereof, or a pharmaceutical composition of the above;

b) Optionally package and/or instructions.

The above kit may comprise 0.01mg to 1000mg of at least one compound of formula I above or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite or prodrug thereof.

The invention also provides a method for preparing the above-mentioned kit, which comprises combining the above-mentioned compound of formula I or a pharmaceutically acceptable salt, ester, solvate (e.g. hydrate), stereoisomer, tautomer, polymorph, oxynitride, isotopic label, metabolite or prodrug thereof, or the above-mentioned pharmaceutical composition, with optional package and/or instructions.

[ medical use ]

The compound of the invention can show stronger inhibition effect on UPS7, IC ₅₀ Most values can be below 100nM, and individual values even below 10nM, and can be used as UPS7 inhibitors. Accordingly, the present invention provides the use of a compound of formula I, or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite, or prodrug thereof, or a pharmaceutical composition, or kit, as described above, as a UPS7 inhibitor.

In addition, the application also provides the use of the compound of the formula I or pharmaceutically acceptable salt, ester, solvate (such as hydrate), stereoisomer, tautomer, polymorph, nitrogen oxide, isotope label, metabolite or prodrug thereof, or the pharmaceutical composition or the kit, in the preparation of medicaments for preventing and/or treating diseases or symptoms (particularly cancers) mediated at least in part by UPS 7.

The term "disease or condition mediated at least in part by UPS 7" refers to a disease in which the pathogenesis includes at least a portion of the factors associated with UPS7, including, but not limited to, cancer, neurodegenerative diseases (e.g., alzheimer's disease, parkinson's disease, etc.), diabetes, bone and joint diseases, arthritic conditions, osteoporosis, immune conditions, cardiovascular diseases, ischemic diseases, viral infections and diseases, bacterial infections and diseases, and the like.

[ method of treatment ]

The present invention provides a method for preventing and/or treating a disease (particularly cancer) mediated at least in part by UPS7, comprising the steps of: a prophylactically and/or therapeutically effective amount of a compound of formula I above or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, polymorph, nitroxide, isotopic label, metabolite, or prodrug thereof, or a pharmaceutical composition, or kit, as described above, is administered to a subject in need thereof.

The term "effective amount" refers to a dose capable of eliciting a biological or medical response from a cell, tissue, organ or organism (e.g., an individual) and sufficient to achieve a desired prophylactic and/or therapeutic effect. The dosing regimen may be adjusted to provide the optimal response. For example, it may be administered in a single dose, it may be administered in divided doses over time, or it may be administered after a proportional decrease or increase in dose depending on the actual situation. It will be appreciated that the particular dosage regimen for any particular individual will be adjusted according to the needs and the discretion of the attendant. In addition, a distinction is also made between prophylactic and therapeutic applications. In prophylactic applications, relatively low doses are typically administered at relatively long intervals over a long period of time. In therapeutic applications, relatively high doses are typically administered at relatively short intervals until the progression of the disease is delayed or stopped, preferably until the individual exhibits a partial or complete improvement in symptoms.

In the present invention, suitable in vitro or in vivo assays are performed to determine the efficacy of the compounds, pharmaceutical compositions and/or kits and whether the administration is suitable for treating a disease or condition in an individual. Examples of such assays are described in the non-limiting examples below. Generally, an effective amount of a compound sufficient to achieve a prophylactic and/or therapeutic effect is from about 0.001 mg/kg body weight/day to about 10,000 mg/kg body weight/day. In suitable cases, the effective amount is from about 0.01 mg/kg body weight/day to about 1,000 mg/kg body weight/day. In more general terms, the effective amount is about 0.01 to 1,000 mg/kg body weight per day, every two or three days, preferably about 0.1 to 500 mg/kg body weight. An exemplary treatment regimen is once every two days or once weekly or monthly.

The term "treatment" refers to the alleviation or elimination of a disease or condition for which it is intended. A subject is indicated to have been successfully "treated" if the subject has received a therapeutic amount of a compound of the invention, or a pharmaceutically acceptable form thereof, or a pharmaceutical composition of the invention, at least one indicator and symptom of which exhibits observable and/or detectable remission and/or improvement. It is understood that treatment includes not only complete treatment, but also less than complete treatment, but achieves some biologically or medically relevant results. In particular, "treatment" means that a compound of the invention or a pharmaceutically acceptable form thereof or a pharmaceutical composition of the invention may achieve at least one of the following effects: (1) Preventing disease in animals that may be predisposed to the disease but have not undergone or displayed disease pathology or symptomology; (2) Inhibiting the disease (i.e., preventing further development of pathology and/or symptomology) in an animal experiencing or exhibiting disease pathology or symptomology; (3) Disease is ameliorated (i.e., pathology and/or symptomology is reversed) in an animal that is experiencing or exhibiting pathology or symptomology of the disease.

The term "administering" refers to the process of applying a pharmaceutically active ingredient (such as a compound of the present invention) or a pharmaceutical composition comprising a pharmaceutically active ingredient (e.g., a pharmaceutical composition of the present invention) to a subject or a cell, tissue, organ, biological fluid, etc. thereof, such that the pharmaceutically active ingredient or pharmaceutical composition is in contact with the subject or a cell, tissue, organ, biological fluid, etc. Common modes of administration include, but are not limited to, oral administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, ocular administration, nasal administration, sublingual administration, rectal administration, vaginal administration, and the like.

The term "in need thereof" refers to a judgment of a physician or other caregiver as to the need of an individual or as to the impending benefit from the prevention and/or treatment process based on various factors of the physician or other caregiver in their area of expertise.

The term "individual" (or subject) refers to a human or non-human animal. The subject of the present invention includes subjects (patients) suffering from diseases and/or disorders and normal subjects. Non-human animals of the present invention include all vertebrates, such as non-mammals, e.g., birds, amphibians, reptiles, etc., and mammals, e.g., non-human primates, domestic animals, and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

In order to make the objects and technical solutions of the present invention more apparent, embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention.

The reagents or apparatus used in the examples were conventional products commercially available without the manufacturer's knowledge. Those not specifying the specific conditions were carried out according to the conventional conditions or the conditions recommended by the manufacturer. The term "room temperature" as used herein refers to 20 ℃ ± 5 ℃. As used herein, the term "about" when used in reference to a particular value or range of values is intended to encompass the value or range of values as well as ranges of errors that are acceptable to those skilled in the art of the value or range of values, such as, for example, ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, etc.

In the conventional synthesis methods and examples of intermediate synthesis, the meanings of the abbreviations are shown in the following table.

The structures of the compounds described in the examples below were prepared by nuclear magnetic resonance ¹ H-NMR) and/or Mass Spectrometry (MS).

Nuclear magnetic resonance ¹ H-NMR) using Bruker 400MHz NMR, the solvent was deuterated methanol (CD) ₃ OD), deuterated chloroform (CDCl) ₃ ) Hexadeuterated dimethyl sulfoxide (DMSO-d) ₆ ) The internal standard substance is Tetramethylsilane (TMS).

Abbreviations in Nuclear Magnetic Resonance (NMR) data in the following examples represent the following meanings:

s: single peak (single), d: dual peak (doubelet), t: triplet (triplet), q: quartet (quaternion), dd: double doublet (double), qd: four doublets (quatet doubelet), ddd: double doublet (double double doublet), ddt: double triplet (double double triplet), dddd: double peak (double double double doublet), m: multiple peaks (multiplet), br: broad peak (broad), J: coupling constant, hz: hertz, delta: chemical shift.

All chemical shift (delta) values are given in parts per million (ppm).

The Mass Spectrum (MS) measuring instrument uses an Agilent 6120B mass spectrometer, and the ion source is an electrospray ion source (ESI).

The examples of the present invention were purified by preparative high performance liquid chromatography (Prep-HPLC) using the methods shown below.

Method A:

chromatographic column: waters SunFire Prep C18 OBD (5 μm, 19X 150 mm)

Mobile phase a: acetonitrile; mobile phase B: water (containing 0.05wt% formic acid)

Time [ min]	Mobile phase A [%]	Mobile phase B [%]	Flow Rate [ mL/min]
				0.00	10.0	90.0	28
16.00	90.0	10.0	28

Method B:

chromatographic column: waters SunFire Prep C18 OBD (5 μm, 19X 150 mm)

Time [ min]	Mobile phase A [%]	Mobile phase B [%]	Flow Rate [ mL/min]
				0.00	30.0	70.0	24
16.00	90.0	10.0	24

Method C:

chromatographic column: waters SunFire Prep C18 OBD (5 μm, 19X 150 mm)

Time [ min]	Mobile phase A [%]	Mobile phase B [%]	Flow Rate [ mL/min]
				0.00	40.0	60.0	28
16.00	90.0	10.0	28

Method D:

chromatographic column: waters SunFire Prep C18 OBD (5 μm, 19X 150 mm)

Mobile phase a: acetonitrile; mobile phase B: water (containing 0.05wt% trifluoroacetic acid)

Method E:

chromatographic column: waters SunFire Prep C18 OBD (5 μm, 19X 150 mm)

Mobile phase a: acetonitrile; mobile phase B: water (containing 0.05wt% ammonium bicarbonate)

[ preparation and identification of Compounds ]

Embodiment one: synthesis of 6- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 1A).

Step 1): synthesis of N-methyl-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydro-1H-inden-1-amine (Compound 1-2):

Compound 1-1 (2.00 g,6.59 mmol) was dissolved in methanol (80 mL) and a methylamine/ethanol solution (7 mL,33 wt%) was added and reacted at room temperature for 12h. Formic acid (0.80 mL,21.08 mmol) was added to adjust pH.apprxeq.5, and sodium cyanoborohydride (1.24 g,19.76 mmol) was added and the reaction was continued at room temperature for 12h. After concentrating the reaction mixture under reduced pressure, suction filtration and concentration of the filtrate under reduced pressure gave the formate of the title compound (2.08 g) which was used directly in the next reaction.

ESI-MS:m/z 274.3[M+H] ⁺ 。

Step 2): synthesis of tert-butyl N-methyl-N- (5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydro-1H-inden-1-yl) carbamate (Compound 1-3):

compound 1-2 (2.03 g,6.36 mmol) was dissolved in tetrahydrofuran (40 mL), followed by addition of saturated aqueous sodium hydrogencarbonate (6.36 mmol,40 mL) and di-tert-butyl dicarbonate (1.67 g,7.63 mmol) and reaction at room temperature for 6h. The reaction solution was extracted with ethyl acetate (100 ml×3), the organic phases were combined, the organic phase was washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, and after concentration under reduced pressure, the residue was separated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=9/1 (v/v)), to give the title compound (0.87 g).

ESI-MS:m/z 318.2[M-56+H] ⁺ 。

Step 3): synthesis of methyl 4-amino-5-bromoisothiazole-3-carboxylate (Compounds 1-5):

Compounds 1-4 (100 mg,0.63 mmol) were dissolved in tetrahydrofuran (7 mL) and N-bromosuccinimide (124 mg,0.70 mmol) was added and reacted at room temperature for 0.1h. After concentration under reduced pressure, purification was performed by preparative thin layer chromatography (eluent: petroleum ether/ethyl acetate=10/1 (v/v)) to give the title compound (130 mg).

ESI-MS:m/z 236.9,238.9[M+H] ⁺ 。

Step 4): synthesis of methyl 4-amino-5- (1- (N-t-butoxycarbonyl-N-methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazole-3-carboxylate (Compounds 1-6):

compounds 1-5 (120 mg,0.51 mmol), compounds 1-3 (283 mg,0.76 mmol), tetrakis (triphenylphosphine) palladium (29 mg,0.03 mmol) and sodium bicarbonate (85 mg,1.01 mmol) were placed in a reaction flask at room temperature, toluene (12 mL), ethanol (2.4 mL) and water (2.4 mL) were added and reacted at 100℃for 10h. After cooling to room temperature, the title compound (80 mg) was obtained by direct preparation and purification by thin layer separation (eluent: petroleum ether/ethyl acetate=5/1 (v/v)).

ESI-MS:m/z 404.1[M+H] ⁺ 。

Step 5): synthesis of tert-butyl N-methyl-N- (5- (7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-1-yl) carbamate (Compounds 1-7):

compounds 1-6 (100 mg,0.24 mmol) were dissolved in n-butanol (5 mL) at room temperature, formamidine acetate (499 mg,4.8 mmol) was added and reacted at 120℃for 6h. After cooling, an aqueous sodium hydrogencarbonate solution (5 mL) was added to the reaction mixture, which was extracted with ethyl acetate (10 mL. Times.10), and the organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a crude title compound (90 mg).

ESI-MS:m/z 399.2[M+H] ⁺ 。

Step 6): synthesis of tert-butyl N- (5- (6- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamate (Compounds 1-8):

compounds 1-7 (5 mg,0.01 mmol), (R) -3-phenyl-1- (1-oxa-6-azaspiro [2.5] oct-6-yl) -1-butanone (methods of synthesis reference WO 2018073602) (7 mg,0.03 mmol) and pyridine (5 mg,0.06 mmol) were dissolved in dimethyl sulfoxide (2.5 mL) at room temperature and reacted at 80℃for 18h. After cooling to room temperature, the reaction was quenched with water (5 mL), the reaction mixture was extracted with ethyl acetate (5 ml×3), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by preparative thin layer chromatography (eluent: petroleum ether/ethyl acetate=1/1 (v/v)) to give the title compound (6 mg).

ESI-MS:m/z 658.3[M+H] ⁺ 。

Step 7): synthesis of 6- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

compounds 1-8 (6 mg,0.01 mmol) were dissolved in hydrochloric acid/ethyl acetate (3 mL, 4M) at room temperature and reacted for 1h at room temperature. The reaction solution was concentrated under reduced pressure, and purified by preparative high performance liquid chromatography (method A), and the preparation solution was freeze-dried to give formate salt of the objective compound (2 mg).

¹ H-NMR(400MHz,DMSO-d ₆ ):δ8.20(d,J＝10.0Hz,1H),7.99(s,1H),7.95(d,J＝7.6Hz,1H),7.53(d,J＝8.0Hz,1H),7.32-7.21(m,4H),7.19-7.11(m,1H),4.94(brs,1H),4.16(t,J＝6.8Hz,1H),4.10-3.95(m,2H),3.93(s,1H),3.75-3.60(m,1H),3.28-3.11(m,3H),3.07-2.96(m,1H),2.92-2.79(m,2H),2.70-2.53(m,2H),2.37(s,3H),2.40-2.30(m,1H),1.90-1.77(m,1H),1.65-1.25(m,4H),1.20(d,J＝7.2Hz,3H)。

ESI-MS:m/z 558.3[M+H] ⁺ 。

Embodiment two: synthesis of 6- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -3- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 2A).

Step 1): synthesis of N- (5-bromo-2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamic acid tert-butyl ester (Compound 2-2):

compound 2-1 (700 mg,2.24 mmol) was dissolved in DMF (14 mL), cooled to 0deg.C, sodium hydride (135 mg,3.36mmol,60% purity) was added and the reaction was incubated for 1h. Methyl iodide (382 mg,2.69 mmol) was added thereto, and the mixture was slowly warmed to room temperature and reacted for 16 hours. The reaction solution was quenched with saturated aqueous ammonium chloride (100 mL), extracted with ethyl acetate (50 mL. Times.3), and the organic phases were combined, washed with saturated brine (50 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the title compound (600 mg) which was used directly in the next reaction.

ESI-MS:m/z 270.0,272.0[M-56+H] ⁺ 。

Step 2): synthesis of tert-butyl N-methyl-N- (5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydro-1H-inden-2-yl) carbamate (Compound 2-3):

compound 2-2 (200 mg,0.61 mmol), pinacol diboronate (187 mg,0.74 mmol) and potassium acetate (181 mg,1.84 mmol) were placed in 1, 4-dioxane (5 mL), nitrogen was purged 3 times, and Pd (dppf) Cl was added ₂ (22 mg,0.03 mmol) and nitrogen substitution 3 times. And under the nitrogen atmosphere, the temperature is raised to 80 ℃ for reaction for 16h. After cooling to room temperature, the reaction solution was concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10/1 (v/v)) to give the title compound (200 mg).

ESI-MS:m/z 318.2[M-56+H] ⁺ 。

Step 3): synthesis of methyl 4-amino-5- (2- (N-t-butoxycarbonyl-N-methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazole-3-carboxylate (Compound 2-4):

according to the procedure of step 4) in example one, using compounds 1 to 5 (100 mg,0.42 mmol) and compounds 2 to 3 (189 mg,0.51 mmol) as reaction materials, purification by preparative thin layer chromatography (eluent: petroleum ether/ethyl acetate=5/1 (v/v)) to give the title compound (61 mg).

ESI-MS:m/z 404.1[M+H] ⁺ 。

Step 4): synthesis of tert-butyl N-methyl-N- (5- (7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-yl) carbamate (Compound 2-5):

according to the procedure of step 5) in example one, using compounds 2-4 (68 mg,0.17 mmol) as reaction starting material, the title compound (67 mg) was obtained and was used directly in the next step.

ESI-MS:m/z 399.2[M+H] ⁺ 。

Step 5): synthesis of tert-butyl N- (5- (6- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamate (Compound 2-6)

According to the procedure of step 6) in example one, using crude compound 2-5 (22 mg,55.21 mmol) and (R) -3-phenyl-1- (1-oxa-6-azaspiro [2.5] oct-6-yl) -1-butanone (21 mg,0.08 mmol) as reaction materials, the crude title compound (36 mg) was obtained and used directly in the next step.

ESI-MS:m/z 658.3[M+H] ⁺ 。

Step 6): synthesis of 6- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -3- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 7) in example I, crude products (36 mg,54.73 mmol) of the compounds 2 to 6 are used as reaction raw materials, and the prepared solution is subjected to preparative high performance liquid chromatography separation and purification (method A) and freeze-drying to obtain formate (11 mg) of the target compound.

¹ H-NMR(400MHz,DMSO-d ₆ ):δ8.20(d,J＝8.0Hz,1H),7.97(s,1H),7.94(d,J＝8.0Hz,1H),7.41(d,J＝8.0Hz,1H),7.30-7.22(m,4H),7.19-7.11(m,1H),4.91(brs,1H),4.11-3.91(m,4H),3.64-3.61(m,2H),3.26-3.11(m,5H),2.91-2.82(m,3H),2.63-2.56(m,1H),2.41(s,3H),1.46-1.23(m,4H),1.21(d,J＝8.0Hz,3H)。

ESI-MS:m/z 557.9[M+H] ⁺ 。

Embodiment III: synthesis of (R) -3- (4-fluorophenyl) -6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 3A).

Step 1): synthesis of methyl 4-amino-5- (4-fluorophenyl) isothiazole-3-carboxylate (Compound 3-1):

according to the procedure of step 4) in example one, starting from 4-fluorobenzeneboronic acid (20 mg,0.14 mmol) and compounds 1 to 5 (34 mg,0.14 mmol), the reaction mixture was purified by column chromatography over silica gel (eluent: petroleum ether/ethyl acetate=5/1 (v/v)) to give the title compound (15 mg).

ESI-MS:m/z 253.0[M+H] ⁺ 。

Step 2): synthesis of 3- (4-fluorophenyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 3-2):

according to the procedure of step 5) in example one, starting from compound 3-1 (13 mg,0.05 mmol), the title compound (8 mg) was obtained.

ESI-MS:m/z 247.8[M+H] ⁺ 。

Step 3): synthesis of (R) -3- (4-fluorophenyl) -6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 6) in example one, 3-2 (3 mg,0.01 mmol) and (R) -3-phenyl-1- (1-oxa-6-azaspiro [2.5] oct-6-yl) -1-butanone (6 mg,0.02 mmol) were used as starting materials, and the preparation was separated and purified by preparative high performance liquid chromatography (method B), and the title compound (5 mg) was obtained by freeze-drying the preparation.

¹ H-NMR(400MHz,DMSO-d ₆ ):δ8.26-8.15(m,3H),7.48-7.40(m,2H),7.32-7.22(m,4H),7.19-7.11(m,1H),4.92(d,J＝3.2Hz,1H),4.12-3.95(m,2H),3.94(s,1H),3.75-3.60(m,1H),3.28-3.10(m,2H),2.92-2.80(m,1H),2.66-2.53(m,2H),1.47-1.23(m,4H),1.20(d,J＝6.8Hz,3H)。

ESI-MS:m/z 506.8[M+H] ⁺ 。

Embodiment four: synthesis of (R) -3- (4-chlorophenyl) -6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 4A).

Step 1): synthesis of 4-amino-5- (4-chlorophenyl) isothiazole-3-carboxylic acid (Compound 4-1):

according to the procedure of step 4) in example one, using 4-chlorobenzeneboronic acid (145 mg,0.93 mmol) and compounds 1-2 (200 mg,0.84 mmol) as starting materials, the reaction solution was suction-filtered under reduced pressure, and the filter cake was washed with water and ethyl acetate and the filter cake was collected to give the title compound (200 mg).

ESI-MS:m/z 254.8,256.8[M+H] ⁺ 。

Step 2): synthesis of methyl 4-amino-5- (4-chlorophenyl) isothiazole-3-carboxylate (Compound 4-2):

compound 4-1 (220 mg,0.86 mmol) was placed in methanol (5 mL) at room temperature, thionyl chloride (514 mg,4.30 mmol) was added dropwise, and the mixture was stirred at 80℃for 5h. The reaction solution was filtered under reduced pressure, and the filtrate was concentrated under reduced pressure to give the title compound (150 mg).

ESI-MS:m/z 268.8,270.8[M+H] ⁺ 。

Step 3): synthesis of 3- (4-chlorophenyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 4-3):

according to the procedure of step 5) in example one, using compound 4-2 (130 mg,0.48 mmol) as a starting material, the reaction solution was added to an aqueous sodium hydrogencarbonate solution, and extracted with ethyl acetate (30 ml×3), the organic phase was dried over anhydrous sodium sulfate, and after concentrating under reduced pressure, the crude product was obtained, which was washed with an organic solvent (petroleum ether/ethyl acetate=5/1 (v/v)) to give the title compound (100 mg).

ESI-MS:m/z 263.8,265.8[M+H] ⁺ 。

Step 4): synthesis of (R) -3- (4-chlorophenyl) -6- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 6) in example one, starting from compound 4-3 (30 mg,0.11 mmol) and (R) -3-phenyl-1- (1-oxa-6-azaspiro [2.5] oct-6-yl) -1-butanone (59 mg,0.23 mmol), the preparation was separated and purified by preparative high performance liquid chromatography (method C), and the preparation was freeze-dried to give the title compound (26 mg).

¹ H-NMR(400MHz,DMSO-d ₆ ):δ8.23(d,J＝10.0Hz,1H),8.18(d,J＝8.0Hz,2H),7.66(d,J＝8.0Hz,2H),7.36-7.21(m,4H),7.20-7.12(m,1H),4.93(s,1H),4.15-3.89(m,3H),3.75-3.58(m,1H),3.27-3.11(m,2H),2.95-2.79(m,1H),2.67-2.53(m,2H),1.60-1.25(m,4H),1.21(d,J＝6.4Hz,3H)。

ESI-MS:m/z 522.7,524.7[M+H] ⁺ 。

Fifth embodiment: synthesis of 3- (2-amino-2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 5).

Step 1): synthesis of 2- (trimethylsilyl) ethyl 1-oxa-6-azaspiro [2.5] octane-6-carboxylate (Compound 5-2):

trimethylsulfonium iodide (5.24 g,25.68 mmol) was dissolved in DMSO (20 mL) at room temperature, sodium hydride (740 mg,18.49mmol,60% purity) was added, and after stirring at room temperature for 1h, a solution of Compound 5-1 (2.5 g,10.27 mmol) in DMSO (20 mL) was added dropwise, and the reaction was continued for 2h. After cooling to room temperature, the reaction was quenched by addition of an aqueous ammonium chloride solution (40 mL), the reaction solution was extracted with ethyl acetate (30 ml×3), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=4/1 (v/v)) to give the title compound (2.40 g).

¹ H-NMR(400MHz,CDCl ₃ ):δ4.25-4.12(m,2H),3.87-3.67(m,2H),3.52-3.38(m,2H),2.70(s,2H),1.91-1.72(m,2H),1.50-1.40(m,2H),1.06-0.96(m,2H),0.05(s,9H)。

Step 2): synthesis of methyl 4-amino-5- (2- (tert-Ding Yangtan ylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazole-3-carboxylate (Compound 5-3):

according to the procedure of step 4) in example one, starting from compounds 1-5 (300 mg,1.30 mmol) and 5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydro-1H-inden-2-ylcarbamic acid tert-butyl ester (545 mg,1.50 mmol), the product was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=1/1 (v/v)) to give the title compound (200 mg).

ESI-MS:m/z 390.1[M+H] ⁺ 。

Step 3): synthesis of tert-butyl 5- (7-oxo-6, 7-dihydrothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-ylcarbamate (compound 5-4):

according to the procedure of step 5) in example one, starting from compound 5-3 (200 mg,0.51 mmol). The reaction solution was added to water (5 mL), extracted with ethyl acetate (10 mL), and the organic phase was concentrated under reduced pressure, and the obtained crude product was slurried with petroleum ether (20 mL), filtered, and washed with petroleum ether (20 mL) to give the title compound (150 mg).

ESI-MS:m/z 385.1[M+H] ⁺ 。

Step 4): synthesis of 2- (trimethylsilyl) ethyl 4- ((3- (2-tert-Ding Yangtan ylamino) -2, 3-dihydro-1H-inden-5-yl) -7-oxoisothiazolo [4,3-d ] pyrimidin-6 (7H) -yl) methyl) -4-hydroxypiperidine-1-carboxylate (Compound 5-5):

compound 5-4 (150 mg,0.39 mmol) and compound 5-2 (120 mg,0.47 mmol) were dissolved in DMF (2 mL) under nitrogen atmosphere, potassium carbonate (108 mg,0.78 mmol) was added, the reaction was reacted at 80℃for 20h, the reaction solution was diluted with ethyl acetate, washed with water (20 mL. Times.3), the organic phase was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane/methanol=92/8 (v/v)) to give the title compound (80 mg).

ESI-MS:m/z 642.2[M+H] ⁺ 。

Step 5): synthesis of tert-butyl 5- (6- ((4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-ylcarbamate (compound 5-6):

Compounds 5-5 (80 mg,0.12 mmol) were dissolved in THF (2 mL) at room temperature, TBAF (118 mg,0.37 mmol) was added and reacted at 70℃for 2h. To the reaction solution was added ethyl acetate (10 mL), which was washed with saturated aqueous sodium hydrogencarbonate (50 ml×2), the aqueous phase was extracted with dichloromethane (50 ml×10), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol=3/1 (v/v)) to give the title compound (34 mg).

ESI-MS:m/z 497.9[M+H] ⁺ 。

Step 6): synthesis of tert-butyl 5- (6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-ylcarbamate (Compound 5-7):

compounds 5-6 (35 mg,0.07 mmol) and 4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyric acid (18 mg,0.08 mmol) were dissolved in dichloromethane (1 mL), HATU (28 mg,0.07 mmol) and DIPEA (27 mg,0.21 mmol) were added and reacted at room temperature for 1H, the reaction was concentrated under reduced pressure and the residue was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol=15/1 (v/v)) to give the title compound (20 mg).

ESI-MS:m/z 688.1[M+H] ⁺ 。

Step 7): synthesis of 3- (2-amino-2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (the title compound):

According to the procedure of step 7) in example I, using compounds 5-7 (23 mg,33.44 mmol) as a starting material, the preparation was purified by preparative high performance liquid chromatography (method A), and diluted hydrochloric acid (0.1 mL, 1M) was added to the preparation and lyophilized to give the hydrochloride (4 mg) of the objective compound.

¹ H-NMR(400MHz,CD ₃ OD):δ8.19(d,J＝8.0Hz,1H),8.04(s,1H),7.89(d,J＝8.0Hz,1H),7.63-7.57(m,1H),7.43(d,J＝8.0Hz,1H),6.12(td,J _H-F ＝54.0Hz,J _H-H ＝4.0Hz,1H),5.91-5.83(m,1H),4.99-4.94(m,1H),4.25-4.12(m,4H),3.88-3.75(m,1H),3.57-3.37(m,4H),3.20-3.00(m,3H),2.98-2.84(m,1H),1.79-1.49(m,4H)。

ESI-MS:m/z 588.3[M+H] ⁺ 。

Example six: synthesis of 6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 6).

Step 1): synthesis of 2- (trimethylsilyl) ethyl 4- ((3- (2- (N-t-butoxycarbonyl-N-methylamino) -2, 3-dihydro-1H-inden-5-yl) -7-oxoisothiazolo [4,3-d ] pyrimidin-6 (7H) -yl) methyl) -4-hydroxypiperidine-1-carboxylate (Compound 6-1):

according to the procedure of step 4) in example five, starting from compound 2-5 (304 mg,0.76 mmol) and compound 5-2 (589 mg,2.3 mmol). After cooling the reaction to room temperature, it was quenched with dilute hydrochloric acid (10 mL,1 m), extracted with ethyl acetate (10 ml×3), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=1/2 (v/v)) to give the title compound (230 mg).

ESI-MS:m/z 656.3[M+H] ⁺ 。

Step 2): synthesis of tert-butyl N- (5- (6- ((4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamate (Compound 6-2):

according to the procedure of step 5) in example five, using compound 6-1 (210 mg,0.32 mmol) as a starting material, a saturated aqueous sodium hydrogencarbonate solution (10 mL) was added to the reaction solution, extraction was performed with ethyl acetate (10 mL. Times.10), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound (160 mg) which was directly used in the next step.

ESI-MS:m/z 512.3[M+H] ⁺ 。

Step 3): synthesis of tert-butyl N- (5- (6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazol [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamate (Compound 6-3):

according to the procedure of step 6) in example five, starting from 4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyric acid (33 mg,0.16 mmol) and crude compound 6-2 (80 mg,0.16 mmol). The reaction was quenched by the addition of saturated aqueous ammonium chloride (10 mL), the reaction solution was extracted with ethyl acetate (10 mL. Times.5), and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the title compound (89 mg). Directly used in the next step.

ESI-MS:m/z 702.3[M+H] ⁺ 。

Step 4): synthesis of 6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -3- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (the title compound):

according to the procedure of step 7) in example I, using crude product (80 mg,114 mmol) of Compound 6-3 as a starting material, the preparation was purified by preparative high performance liquid chromatography (method A) and the preparation was lyophilized to give formate salt of the objective compound (4 mg).

¹ H-NMR(400MHz,DMSO-d ₆ ):δ8.20(d,J＝4.0Hz,1H),7.96(s,1H),7.93(d,J＝8.0Hz,1H),7.84-7.78(m,1H),7.40(d,J＝8.0Hz,1H),6.26(td,J _H-F ＝54.0Hz,J _H-H ＝4.0Hz,1H),6.02-5.96(m,1H),5.06-4.92(m,2H),4.08-3.96(m,3H),3.73-3.66(m,1H),3.58-3.53(m,2H),3.24-3.10(m,4H),2.95-2.76(m,4H),2.37(s,3H),1.58-1.33(m,4H)。

ESI-MS:m/z 602.3[M+H] ⁺ 。

Embodiment seven: synthesis of 6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 7).

Step 1): synthesis of 2- (trimethylsilyl) ethyl 4- ((3- (1- (N-t-butoxycarbonyl-N-methylamino) -2, 3-dihydro-1H-inden-5-yl) -7-oxoisothiazolo [4,3-d ] pyrimidin-6 (7H) -yl) methyl) -4-hydroxypiperidine-1-carboxylate (compound 7-1):

according to the procedure of step 4) in example five, starting from compounds 1-7 (100 mg,0.25 mmol) and compound 5-2 (646 mg,2.51 mmol), the product was purified by column chromatography over silica gel (eluent: petroleum ether/ethyl acetate=1/2 (v/v)) to give the title compound (110 mg).

ESI-MS:m/z 656.3[M+H] ⁺ 。

Step 2): synthesis of tert-butyl N- (5- (6- ((4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamate (Compound 7-2):

according to the procedure of step 5) in example five, compound 7-1 (100 mg,0.15 mmol) was used as starting material. The reaction was quenched with saturated aqueous sodium bicarbonate (10 mL), extracted with ethyl acetate (10 ml×10), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was separated and purified by column chromatography on silica gel (eluent: dichloromethane/methanol/triethylamine=1/1/0.001 (v/v/v)), to give the title compound (60 mg).

ESI-MS:m/z 512.3[M+H] ⁺ 。

Step 3): synthesis of tert-butyl N- (5- (6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazol [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamate (Compound 7-3):

according to the procedure of step 6) in example five, using 4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyric acid (27 mg,0.13 mmol) and compound 7-2 (60 mg,0.12 mmol) as starting materials, was isolated and purified by silica gel column chromatography (eluent: ethyl acetate/methanol=60/1 (v/v)) to give the title compound (60 mg).

ESI-MS:m/z 702.3[M+H] ⁺ 。

Step 4): synthesis of 6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (the title compound):

according to the procedure of step 7) in example I, using 7-3 (60 mg,0.09 mmol) as a starting material, the preparation was purified by preparative high performance liquid chromatography (method D) and the preparation was freeze-dried to give the trifluoroacetate salt of the target compound (44 mg).

¹ H-NMR(400MHz,DMSO-d ₆ ):δ8.92(brs,2H),8.23(d,J＝4.0Hz,1H),8.13(s,1H),8.08(d,J＝8.0Hz,1H),7.84-7.78(m,1H),7.76(d,J＝8.0Hz,1H),6.26(td,J _H-F ＝55.2Hz,J _H-H ＝3.6Hz,1H),5.99(dt,J＝6.0,2.4Hz,1H),5.06-4.92(m,2H),4.85-4.75(m,1H),4.10-3.94(m,3H),3.75-3.64(m,1H),3.32-3.12(m,3H),3.06-2.82(m,3H),2.65(s,3H),2.56-2.44(m,1H),2.25-2.12(m,1H),1.65-1.25(m,4H)。

ESI-MS:m/z 602.3[M+H] ⁺ 。

Example eight: synthesis of 3- (7-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (Compound 8).

Step 1): synthesis of N- (5-bromo-7-chloro-2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamic acid tert-butyl ester (Compound 8-2):

compound 8-1 (300 mg,1.2 mmol) and methylamine hydrochloride (248 mg,3.7 mmol) were dissolved in a mixed solvent of tetrahydrofuran (18 mL) and methanol (6 mL) at room temperature, and a methylamine/ethanol solution (0.5 mL,33 wt%) and sodium cyanoborohydride (426 mg,18.33 mmol) were added. And under the protection of nitrogen, heating to 65 ℃ and carrying out reflux reaction for 24 hours. After stopping heating and cooling to room temperature, di-tert-butyl carbonate (53 mg,0.6mL,2.44 mmol) and DIPEA (316 mg,0.4mL,2.44 mmol) were added and the reaction was stirred for 3h. The reaction was quenched with water, extracted with ethyl acetate (30 mL. Times.3), the organic phase was washed successively with saturated aqueous sodium hydrogencarbonate (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the residue was isolated and purified by preparative high performance liquid chromatography (method E), and the preparation was freeze-dried to give the title compound (248 mg).

ESI-MS:m/z 304.0,306.0[M-56+H] ⁺ 。

Step 2): synthesis of tert-butyl N- (7-chloro-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydro-1-hydro-inden-1-yl) -N-methylcarbamate (Compound 8-3):

according to the procedure of step 2) in example two, starting from compound 8-2 (248 mg,0.69 mmol) and pinacol diboronate (218 mg,0.86 mmol), the product was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=90/10 (v/v)) to give the title compound (211 mg).

ESI-MS(m/z):352.1,354.1[M-56+H] ⁺ 。

Step 3): synthesis of isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 8-4):

compounds 1-4 (1.60 g,8.22 mmol) and formamidine acetate (1.73 g,16.65 mmol) were dissolved in absolute ethanol (100 mL) at room temperature and reacted with stirring at 80℃for 16h. After cooling to room temperature, a solid was precipitated, filtered, and the filter cake was washed with ethanol to give the title compound (1.14 g).

ESI-MS:m/z 154.1[M+H] ⁺ 。

Step 4): synthesis of tert-butyl 4-hydroxy-4- ((7-oxoisothiazolo [4,3-d ] pyrimidin-6 (7H) -yl) methyl) piperidine-1-carboxylate (compound 8-5):

according to the procedure of step 6) in example one, using compound 8-4 (0.94 g,6.12 mmol) and tert-butyl 1-oxa-6-azaspiro [2.5] octane-6-carboxylate (6.53 g,30.62 mmol) as starting materials, dilute hydrochloric acid (80 mL,1 m) was added to the reaction solution, and extracted with ethyl acetate (80 ml×3), the organic phases were combined, washed with saturated aqueous copper sulfate (80 mL), saturated brine (80 mL), dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (eluent: dichloromethane/methanol=90/10 (v/v)) to give the title compound (2.23 g).

ESI-MS(m/z):311.0[M-56+H] ⁺ 。

Step 5): synthesis of 3-bromo-6- ((4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 8-6):

compound 8-5 (1.14 g,2.49 mmol) and N-bromosuccinimide (0.53 g,2.99 mmol) were dissolved in water (32 mL) at room temperature, diluted sulfuric acid (18 mL,10 wt%) was added thereto, pH was adjusted to approximately 3, and the reaction mixture was heated to 50℃for 16h. After cooling to room temperature, the reaction solution was neutralized with saturated aqueous sodium bicarbonate and 6M aqueous sodium hydroxide, and the pH was adjusted to approximately 8. The reaction solution was purified by preparative high performance liquid chromatography (method E), and the preparation was freeze-dried to give the title compound (0.48 g).

ESI-MS:m/z 345.0,347.0[M+H] ⁺ 。

Step 6): synthesis of 3-bromo-6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 8-7):

according to the procedure of step 6) in example five, starting from compound 8-6 (100 mg,0.29 mmol) and 4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyric acid (66 mg,0.32 mmol), it was isolated and purified by preparative thin layer chromatography (eluent: dichloromethane/methanol=20/1 (v/v)) to give the title compound (127 mg).

ESI-MS:m/z 535.2,537.2[M+H] ⁺ 。

Step 7): synthesis of tert-butyl N- (7-chloro-5- (6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamate (Compound 8-8):

Compound 8-7 (30 mg,0.06 mmol), compound 8-3 (28 mg,0.07 mmol) and potassium carbonate (24 mg,0.17 mmol) were dissolved in 1, 4-dioxane (2.5 mL) and water (0.5 mL) at room temperature, nitrogen was replaced 3 times, tetrakis (triphenylphosphine) palladium (7 mg,0.01 mmol) was added, nitrogen was replaced three times, and the temperature was raised to 80℃under nitrogen atmosphere to react for 4 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol=20/1 (v/v)) to give the title compound (25 mg).

ESI-MS(m/z)：680.3,682.3[M-56+H] ⁺ 。

Step 8): synthesis of 3- (7-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (the title compound):

according to the procedure of step 7) in example I, using compounds 8 to 8 (25 mg,0.03 mmol) as a starting material, the reaction solution was concentrated under reduced pressure, purified water was added thereto, and then freeze-dried to obtain the hydrochloride (18 mg) of the objective compound.

¹ H-NMR(400MHz,DMSO-d ₆ ):δ9.45-9.32(m,1H),8.97-8.84(m,1H),8.31(d,J＝4.0Hz,1H),8.25(s,1H),8.06(s,1H),7.85-7.79(m,1H),6.26(td,J _H-F ＝54.0Hz,J _H-H ＝4.0Hz,1H),6.01-5.95(m,1H),5.04-4.95(m,1H),4.90-4.82(m,1H),4.11-3.95(m,3H),3.72-3.68(m,1H),3.49-3.39(m,1H),3.33-3.19(m,2H),3.010-3.00(m,1H),2.97-2.84(m,2H),2.63(t,J＝5.2Hz,3H),2.47-2.36(m,2H),1.68-1.55(m,1H),1.54-1.35(m,3H)。

ESI-MS:m/z 636.2[M+H] ⁺ 。

Example nine: synthesis of 3- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (Compound 9).

Step 1): synthesis of 6-bromo-4-chloro-2, 3-dihydro-1H-inden-1-ol (Compound 9-2):

compound 9-1 (600 mg,2.44 mmol) was placed in methanol (4 mL) at room temperature, sodium borohydride (185 mg,4.89 mmol) was added, and the mixture was stirred at room temperature for 2h. After concentrating under reduced pressure, water (30 mL) was added to dilute the mixture, pH was adjusted to about 6 with 1M dilute hydrochloric acid, the mixture was extracted with methylene chloride (30 mL. Times.3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the title compound (605 mg).

¹ H-NMR(400MHz,CDCl ₃ ):δ7.43(s,1H),7.41(s,1H),5.25(t,J＝6.4Hz,1H),3.11-2.96(m,1H),2.82-2.70(m,1H),2.59-2.46(m,1H),2.02-1.94(m,1H)。

Step 2): synthesis of 5-bromo-7-chloro-1H-indene (Compound 9-3):

compound 9-2 (605 mg,2.44 mmol) was dissolved in toluene (5 mL) at room temperature, p-toluenesulfonic acid (42 mg,0.24 mmol) was added, the temperature was raised to 120℃for 3h, the reaction mixture was concentrated under reduced pressure, the crude product was washed with petroleum ether, filtered, and the filtrate was concentrated under reduced pressure to give the title compound (560 mg).

¹ H-NMR(400MHz,CDCl ₃ )：δ7.44(d,J＝1.6Hz,1H),7.33(d,J＝1.5Hz,1H),6.82(dt,J＝5.5,1.9Hz,1H),6.65(dt,J＝5.5,2.0Hz,1H),3.38(t,J＝1.9Hz,2H)。

Step 3): synthesis of 3-bromo-5-chloro-6, 6 a-dihydro-1 aH-indeno [1,2-b ] oxirane (Compound 9-4):

compound 9-3 (560 mg,2.44 mmol) was dissolved in dichloromethane (10 mL) at room temperature, and m-chloroperoxybenzoic acid (1.26 g,7.32 mmol) was added and reacted at room temperature for 16h. Petroleum ether was added to the reaction solution for dilution, filtration, and the filtrate was separated and purified by silica gel column chromatography (eluent: petroleum ether/methylene chloride=2/1 (v/v)), to obtain the title compound (400 mg).

¹ H-NMR(400MHz,CDCl ₃ )：δ7.53(d,J＝1.7Hz,1H),7.42(d,J＝1.7Hz,1H),4.26(dd,J＝2.7,1.4Hz,1H),4.15(t,J＝2.8Hz,1H),3.20(dd,J＝18.6,1.6Hz,1H),2.89(dd,J＝18.6,3.0Hz,1H)。

Step 4): synthesis of 6-bromo-4-chloro-1H-inden-2 (3H) -one (Compound 9-5):

compound 9-4 (400 mg,1.63 mmol) was dissolved in toluene (10 mL) at room temperature, and silica gel (2 g) was added and reacted at 120℃for 6h. The heating was stopped, cooled to room temperature, and the reaction solution was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: petroleum ether/dichloromethane=2/1 (v/v)) to give the title compound (300 mg).

¹ H-NMR(400MHz,CDCl ₃ )：δ7.46(s,1H),7.37(s,1H),3.62(s,2H),3.52(s,2H)。

Step 5): synthesis of N- (6-bromo-4-chloro-2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamic acid tert-butyl ester (Compound 9-6):

9-5 (330 mg,1.34 mmol) was dissolved in methanol (1.5 mL) at room temperature, and a methylamine/water solution (1.5 mL,40 wt%) was added and reacted at room temperature for 5 hours. Sodium borohydride (60 mg,1.61 mmol) was added and the reaction was continued for 1h. Di-tert-butyl dicarbonate (4.40 g,20.15 mmol) was finally added and the reaction continued for 1h. The reaction solution was poured into water, extracted with ethyl acetate (30 ml×3), backwashed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and separated and purified by silica gel column chromatography (eluent: petroleum ether/tetrahydrofuran=30/1 (v/v)) to give the title compound (310 mg).

¹ H-NMR(400MHz,CDCl ₃ )：δ7.32(s,1H),7.24(s,1H),5.26-4.93(m,1H),3.25-3.12(m,2H),3.05-2.87(m,2H),2.72(s,3H),1.46(s,9H)。

Step 6): synthesis of tert-butyl N- (4-chloro-6- (6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-yl-N-methylcarbamate (Compound 9-7):

Compounds 9-6 (30 mg,0.08 mmol), pinacol biborate (25 mg,0.10 mmol), [1,1' -bis (diphenylphosphino) ferrocene ] palladium dichloride (6.09 mg,0.01 mmol) and potassium acetate (16 mg,0.17 mmol) were placed in 1, 4-dioxane (2 mL), nitrogen was replaced three times, and the temperature was raised to 80℃for 16h. After cooling to room temperature, compound 8-8 (30 mg,0.06 mmol), potassium carbonate (23 mg,0.17 mmol), tetrakis (triphenylphosphine) palladium (6 mg,0.01 mmol) and water (0.4 mL) were added, nitrogen was substituted three times, and the temperature was raised to 80℃for reaction for 4 hours. After cooling to room temperature, the crude product was concentrated under reduced pressure, and purified by preparative thin layer chromatography (mobile phase: ethyl acetate/methanol=60/1 (v/v)) to give the title compound (30 mg).

ESI-MS:m/z 736.4,738.4[M+H] ⁺ 。

Step 7): synthesis of 3- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (the title compound):

according to the procedure of step 7) in example I, using compounds 9-7 (40 mg,0.04 mmol) as a starting material, the preparation was purified by preparative high performance liquid chromatography (method A), and diluted hydrochloric acid (0.5 mL, 1M) was added to the preparation and lyophilized to give the hydrochloride (13 mg) of the objective compound.

¹ H-NMR(400MHz,CD ₃ OD)：δ8.23(d,J＝8.0Hz,1H),8.07(d,J＝4.0Hz,1H),7.89(s,1H),7.64-7.55(m,1H),6.12(td,J _H-F ＝54.0,J _H-H ＝4.0Hz,1H),5.91-5.84(m,1H),5.00-4.94(m,1H),4.23-4.04(m,4H),3.85-3.77(m,1H),3.64-3.51(m,2H),3.47-3.36(m,2H),3.36-3.32(m,1H),3.26-3.20(m,1H),3.10-3.01(m,1H),2.98-2.87(m,1H),2.84(s,3H),1.78-1.48(m,4H)。

ESI-MS:m/z 636.3,638.3[M+H] ⁺ 。

Example ten: synthesis of 6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 10).

Step 1): synthesis of tert-butyl N- (5- (6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamate (compound 10-1):

according to the procedure of step 6) in example five, starting from 3-cyclopropyl-3-phenylpropionic acid (8 mg,0.04 mmol) and compound 7-2 (20 mg,0.04 mmol), the mixture was purified by silica gel column chromatography (eluent: ethyl acetate/methanol=60/1 (v/v)) to give the title compound (20 mg).

ESI-MS:m/z 684.3[M+H] ⁺ 。

Step 2): synthesis of 6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 7) in example I, using compound 10-1 (20 mg,0.03 mmol) as a starting material, the preparation was purified by preparative high performance liquid chromatography (method A) and the preparation was lyophilized to give the formate salt of the target compound (15 mg).

¹ H-NMR(400MHz,CD ₃ OD):δ8.15(d,J＝22.4Hz,1H),8.12(s,1H),8.04(d,J＝8.0Hz,1H),7.65(d,J＝8.0Hz,1H),7.45-7.05(m,5H),4.65(dd,J＝7.2,4.4Hz,1H),4.27-4.02(m,2H),3.90(q,J＝14.0Hz,1H),3.80-3.65(m,1H),3.30-2.80(m,6H),2.69(s,3H),2.70-2.52(m,1H),2.35-2.12(m,2H),1.70-1.10(m,5H),0.70-0.55(m,1H),0.50-0.35(m,1H),0.35-0.25(m,1H),0.15-0.04(m,1H)。

ESI-MS:m/z 584.3[M+H] ⁺ 。

Example eleven: synthesis of 3- (7-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 11).

Step 1): synthesis of 3-bromo-6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 11-1):

according to the procedure of step 6) in example five, starting from compound 8-6 (180 mg,0.52 mmol) and 3-cyclopropyl-3-phenylpropionic acid (109 mg,0.57 mmol), the product was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol=20/1 (v/v)) to give the title compound (160 mg).

ESI-MS:m/z 517.2,519.2[M+H] ⁺ 。

Step 2): synthesis of tert-butyl N- (7-chloro-5- (6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamate (Compound 11-2):

according to the procedure of step 7) in example eight, starting from compound 11-1 (30 mg,0.06 mmol) and compound 8-3 (29 mg,0.07 mmol), the product was purified by preparative thin layer chromatography (eluent: dichloromethane/methanol=20/1 (v/v)) to give the title compound (29 mg).

ESI-MS:m/z 662.3,664.3[M-56+H] ⁺ 。

Step 3): synthesis of 3- (7-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 7) in example one, compound 11-2 (29 mg,0.04 mmol) was used as starting material. After concentrating under reduced pressure, purified water was added thereto, and freeze-drying was performed to obtain the hydrochloride (20 mg) of the objective compound.

¹ H-NMR(400MHz,DMSO-d ₆ ):δ9.65-9.53(m,1H),9.10-8.96(m,1H),8.32(d,J＝12.0Hz,1H),8.25(s,1H),8.06(s,1H),7.34-7.20(m,4H),7.19-7.12(m,1H),4.88-4.80(m,1H),4.10-3.97(m,2H),3.94(s,1H),3.78-3.67(m,1H),3.49(dt,J＝16.8,8.8Hz,1H),3.28-3.12(m,1H),3.09-3.00(m,1H),2.87-2.74(m,2H),2.61(t,J＝5.2Hz,3H),2.46-2.38(m,2H),2.36-2.28(m,1H),1.54-1.32(m,3H),1.29-1.20(m,1H),1.16-1.06(m,1H),1.06-0.99(m,1H),0.53-0.45(m,1H),0.33-0.26(m,1H),0.25-0.16(m,1H),0.10-0.02(m,1H)。

ESI-MS:m/z 618.3,620.3[M+H] ⁺ 。

Embodiment twelve: synthesis of 3- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 12).

Step 1): synthesis of tert-butyl N- (4-chloro-6- (6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamate (Compound 12-1):

according to the procedure of step 6) in example nine, starting from compound 11-1 (33 mg,0.06 mmol), pinacol diboronate (25 mg,0.1 mmol) and compound 9-6 (30 mg,0.08 mmol), the product was isolated and purified by preparative thin layer chromatography (eluent: ethyl acetate/methanol=60/1 (v/v)) to give the title compound (30 mg).

ESI-MS:m/z 718.4,720.4[M+H] ⁺ 。

Step 2): synthesis of 3- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 7) in example I, using 12-1 (29.27 mg,0.04 mmol) as a starting material, the preparation was purified by preparative high performance liquid chromatography (method A), diluted hydrochloric acid (0.5 mL, 1M) was added to the preparation, and freeze-dried to give the hydrochloride (15 mg) of the objective compound.

¹ H-NMR(400MHz,CD ₃ OD):δ8.19(d,J＝20.0Hz,1H),8.09(s,1H),7.90(s,1H),7.47-7.07(m,5H),4.35-4.02(m,3H),4.02-3.85(m,1H),3.80-3.69(m,1H),3.65-3.51(m,2H),3.36-3.32(m,1H),3.29-3.14(m,2H),3.05-2.67(m,6H),2.33-2.23(m,1H),1.70-1.29(m,3.5H),1.25-1.13(m,1H),0.82-0.74(m,0.5H),0.66-0.57(m,1H),0.46-0.37(m,1H),0.36-0.26(m,1H),0.15-0.06(m,1H)。

ESI-MS:m/z 618.2,620.2[M+H] ⁺ 。

Embodiment thirteen: synthesis of 6- ((1- (3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 13).

Step 1): synthesis of tert-butyl N- (5- (6- ((1- (3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamate (compound 13-1):

according to the procedure of step 6) in example five, using 3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyric acid (synthesis method reference US 20160185785) (5 mg,0.02 mmol) and compound 7-2 (10 mg,0.02 mmol) as starting materials, the product was isolated and purified by silica gel column chromatography (eluent: ethyl acetate/methanol=60/1 (v/v)) to give the title compound (12 mg).

ESI-MS:m/z 718.3,720.3[M+H] ⁺ 。

Step 2): synthesis of 6- ((1- (3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 7) in example I, using compound 13-1 (10 mg,0.01 mmol) as a starting material, the preparation was purified by preparative high performance liquid chromatography (method D) and the preparation was lyophilized to give the trifluoroacetate salt of the target compound (8 mg).

¹ H-NMR(400MHz,DMSO-d ₆ ):δ8.93(brs,2H),8.23(d,J＝3.2Hz,1H),8.13(s,1H),8.08(d,J＝8.0Hz,1H),7.53(t,J＝2.8Hz,1H),7.76(d,J＝8.8Hz,1H),6.30(td,J _H-F ＝54.8Hz,J _H-H ＝3.6Hz,1H),6.35(t,J＝2.4Hz,1H),5.18-5.03(m,1H),5.02-4.92(m,1H),4.80(s,1H),4.10-3.95(m,3H),3.78-3.64(m,1H),3.32-3.12(m,3H),3.06-2.82(m,3H),2.65(s,3H),2.56-2.44(m,1H),2.25-2.12(m,1H),1.65-1.25(m,4H)。

ESI-MS:m/z 618.3,620.3[M+H] ⁺ 。

Fourteen examples: synthesis of 6- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 14).

Step 1): synthesis of tert-butyl N- (5- (6- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamate (Compound 14-1):

according to the procedure of step 6) in example five, using 4, 4-difluoro-3-phenylbutyric acid (synthetic method reference WO 2018020242) (6 mg,0.03 mmol) and compound 7-2 (15 mg,0.03 mmol) as starting materials, was isolated and purified by silica gel column chromatography (eluent: ethyl acetate/methanol=60/1 (v/v)) to give the title compound (15 mg).

ESI-MS:m/z 694.3[M+H] ⁺ 。

Step 2): synthesis of 6- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 7) in example I, using 14-1 (10 mg,0.01 mmol) as a starting material, the preparation was purified by preparative high performance liquid chromatography (method D) and the preparation was freeze-dried to give the trifluoroacetate salt of the target compound (9 mg).

¹ H-NMR(400MHz,CD ₃ OD):δ8.97(brs,1H),8.91(brs,1H),8.22(d,J＝8.4Hz,1H),8.14(s,1H),8.08(d,J＝8.0Hz,1H),7.76(d,J＝8.0Hz,1H),7.40-7.29(m,4H),7.29-7.21(m,1H),6.24(td,J＝56.4,3.6Hz,1H),4.95(s,1H),4.85-4.75(m,1H),4.10-3.97(m,2H),3.95(s,1H),3.80-3.55(m,2H),3.35-3.12(m,3H),3.05-2.80(m,4H),2.65(t,J＝4.8Hz,1H),2.26-2.10(m,1H),1.65-1.10(m,4H)。

ESI-MS:m/z 594.3[M+H] ⁺ 。

Example fifteen: synthesis of 6- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 15).

Step 1): synthesis of tert-butyl N- (5- (6- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamate (Compound 15-1):

according to the procedure of step 6) in example five, using 4, 4-difluoro-3-phenylbutyric acid (31 mg,0.16 mmol) and compound 6-2 (80 mg,0.16 mmol) as starting materials, the reaction was quenched with saturated aqueous ammonium chloride (10 mL), extracted with ethyl acetate (10 ml×5), the organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the title compound (86 mg).

ESI-MS:m/z 693.8[M+H] ⁺ 。

Step 2): synthesis of 6- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -3- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 7) in example I, using compound 15-1 (41 mg,0.06 mmol) as a starting material, the preparation was purified by preparative high performance liquid chromatography (method A) and the preparation was lyophilized to give the formate salt of the target compound (2 mg).

¹ H-NMR(400MHz,DMSO-d ₆ ):δ8.19(d,J＝4.0Hz,1H),7.95(s,1H),7.93(d,J＝8.0Hz,1H),7.40(d,J＝8.0Hz,1H),7.36-7.22(m,5H),6.24(td,J _H-F ＝55.2Hz,J _H-H ＝3.6Hz,1H),4.95(brs,2H),4.05-3.91(m,3H),3.78-3.58(m,3H),3.55-3.51(m,1H),3.21-3.10(m,3H),2.92-2.73(m,5H),2.36(s,3H),1.48-1.21(m,4H)。

ESI-MS:m/z 594.3[M+H] ⁺ 。

Example sixteen: synthesis of 3- (6-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (Compound 16).

Step 1): synthesis of N- (5-bromo-6-chloro-2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamic acid tert-butyl ester (Compound 16-2):

compound 16-1 (200 mg,0.82 mmol) was dissolved in methanol (2 mL) at room temperature, and a methylamine/ethanol solution (0.5 mL,33 wt%) was added and reacted at room temperature for 16h. The reaction mixture was concentrated under reduced pressure, dissolved in methanol (2 mL), and sodium borohydride (30 mg,0.82 mmol) was added thereto to react at room temperature for 1h. Di-tert-butyl dicarbonate (350 mg,1.64 mmol) was slowly added dropwise and reacted at room temperature for 1h. The reaction was quenched with water (5 mL), extracted with ethyl acetate (5 ml×3), the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=10/1 (v/v)) to give the title compound (240 mg).

ESI-MS:m/z 304.0,306.0[M-56+H] ⁺ 。

Step 2): synthesis of tert-butyl N- (6-chloro-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamate (Compound 16-3):

according to the procedure of step 2) in example two, using compound 16-2 (240 mg,0.66 mmol) as a starting material, purification by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=15/1 (v/v)) to give the title compound (110 mg).

ESI-MS:m/z 352.2,354.2[M-56+H] ⁺ 。

Step 3): synthesis of tert-butyl N- (6-chloro-5- (6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-1-yl) -N-methylcarbamate (Compound 16-4):

according to the procedure of step 4) in example one, using compound 16-3 (42 mg,0.10 mol) and compound 8-7 (45 mg,0.84 mmol) as starting materials, purification by silica gel column chromatography (eluent: dichloromethane/methanol=15/1 (v/v)) to give the title compound (30 mg).

ESI-MS:m/z 736.4,738.3[M+H] ⁺ 。

Step 4): synthesis of 3- (6-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (the title compound):

According to the procedure of step 7) in example I, using 16-4 (30 mg,0.05 mmol) as a starting material, the preparation was purified by preparative high performance liquid chromatography (method A) and the preparation was lyophilized to give the formate salt of the target compound (15 mg).

¹ H-NMR(400MHz,CD ₃ OD):δ8.53(s,1H),8.18(d,J＝7.3Hz,1H),8.02(s,1H),7.76(s,1H),7.60(dt,J＝7.3,2.5Hz,1H),6.12(td,J _H-F ＝55.4Hz,J _H-H ＝4.5Hz,1H),5.88(ddd,J＝13.4,5.8,2.6Hz,1H),5.00-4.92(m,1H)4.65-4.60(m,1H),4.20-4.05(m,3H),3.85-3.75(m,1H),3.48-3.35(m,2H),3.22-3.12(m,1H),3.10-2.87(m,2H),2.90(ddd,J＝28.6,16.4,3.8Hz,1H),2.67(s,3H),2.65-2.55(m,1H),2.24-2.14(m,1H),1.80-1.49(m,4H)。

ESI-MS:m/z 636.3,638.3[M+H] ⁺ 。

Example seventeenth: synthesis of 3- (6-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 17).

Step 1): synthesis of 5-bromo-6-chloro-2- (hydroxyimino) -2, 3-dihydro-1H-inden-1-one (Compound 17-2):

compound 17-1 (200 mg,0.81 mmol) and trimethylchlorosilane (106 mg,0.98 mmol) were dissolved in methyl tert-butyl ether (20 mL) at room temperature, isoamyl nitrite (115 mg,0.98 mmol) was slowly added dropwise and stirred at room temperature for 2h. The reaction solution was filtered, and the cake was washed with methyl tert-butyl ether (10 mL. Times.2) to give a crude product of the title compound (165 mg).

ESI-MS:m/z 274.0,276.0[M+H] ⁺ 。

Step 2): synthesis of 2-amino-5-bromo-6-chloro-2, 3-dihydro-1H-inden-1-ol (Compound 17-3):

compound 17-2 (160 mg,0.58 mmol) was dissolved in dry tetrahydrofuran (20 mL) at room temperature, sodium borohydride (86 mg,2.33 mmol) and boron trifluoride etherate (331 mg,2.33 mmol) were added, and the mixture was heated to 65℃and stirred for 5h. After cooling to room temperature, ethanol (20 mL) was added to quench, and the mixture was concentrated to give a crude product, which was purified by preparative HPLC (method A), and lyophilized to give the formate salt of the title compound (80 mg).

ESI-MS:m/z 262.3,264.3[M+H] ⁺ 。

Step 3): synthesis of 5-bromo-6-chloro-2, 3-dihydro-1H-inden-2-amine (Compound 17-4):

compound 17-3 (67 mg,0.22 mmol) was dissolved in 1, 2-dichloroethane (20 mL) at room temperature, triethylsilane (6 mL) and boron trifluoride etherate (6 mL) were added, and the mixture was heated to 85℃and stirred for 18h. The reaction mixture was concentrated, and the residue was purified by preparative high performance liquid chromatography (method A) and freeze-dried to give the formate of the title compound (43 mg).

ESI-MS:m/z 246.3,248.3[M+H] ⁺ 。

Step 4): synthesis of 5-bromo-6-chloro-2, 3-dihydro-1H-inden-2-ylcarbamic acid tert-butyl ester (Compound 17-5):

compound 17-4 (43 mg,0.15 mmol) was dissolved in dichloromethane (10 mL) at room temperature, triethylamine (30 mg,0.29 mmol) and di-tert-butyl dicarbonate (218 mg,0.29 mmol) were added, and the mixture was stirred at room temperature for 4h. The reaction solution was concentrated, and the residue was purified by preparative thin layer chromatography (eluent: petroleum ether/ethyl acetate=8/1 (v/v)) to give the title compound (40 mg).

ESI-MS:m/z 290.3,292.3[M-56+H] ⁺ 。

Step 5): synthesis of N- (5-bromo-6-chloro-2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamic acid tert-butyl ester (Compound 17-6):

according to the procedure of step 1) in example two, using compound 17-5 (40 mg,0.12 mmol) as a reaction starting material, purification by preparative thin layer chromatography (eluent: petroleum ether/ethyl acetate=8/1 (v/v)) to give the title compound (39 mg).

ESI-MS:m/z 304.3,306.3[M-56+H] ⁺ 。

Step 6): synthesis of tert-butyl N- (5-chloro-6- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamate (Compound 17-7):

according to the procedure of step 2) in example two, using compound 17-6 (40 mg,0.11 mmol) and pinacol diboronate (56 mg,0.22 mmol) as reaction materials, the reaction solution was filtered and concentrated to give the crude product of the title compound (45 mg).

ESI-MS:m/z 352.2,354.2[M-56+H] ⁺ 。

Step 7): synthesis of tert-butyl N- (5-chloro-6- (6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamate (Compound 17-8):

according to the procedure of step 4) in example one, using the crude product of compounds 17-7 (15 mg,0.04 mmol) and compounds 8-7 (20 mg,0.04 mmol) as reaction materials, the reaction solution was filtered and concentrated to give the crude product of the title compound (27 mg).

ESI-MS:m/z 736.3,738.3[M+H] ⁺ 。

Step eight: synthesis of 3- (6-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (the title compound):

according to the procedure of step 7) in example one, crude product (27 mg,0.04 mmol) of compound 38-7 was used as a reaction starting material, and the reaction solution was separated and purified by preparative high performance liquid chromatography (method D), and the preparation was freeze-dried to give trifluoroacetate salt (9 mg) of the title compound.

¹ H-NMR(400MHz,CD ₃ OD):δ8.18(d,J＝8.0Hz,1H),8.01(s,1H),7.63-7.57(m,2H),6.12(td,J _H-F ＝55.2Hz,J _H-H ＝4.4Hz,1H),5.91-5.84(m,1H),5.01-4.94(m,1H),4.17-4.07(m,4H),3.85-3.75(m,1H),3.57-3.48(m,2H),3.45-3.36(m,2H),3.24-3.15(m,2H),3.09-3.01(s,1H),2.97-2.84(m,1H),2.80(s,3H),1.69-1.48(m,4H)。

ESI-MS:m/z 636.3,638.3[M+H] ⁺ 。

Example eighteenth: synthesis of 3- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 18).

Step 1): synthesis of 5-bromo-4-chloro-1H-indene (Compound 18-2):

compound 18-1 (2.00 g,8.15 mmol) was dissolved in methanol (100 mL) at room temperature, sodium borohydride (0.60 g,16.29 mmol) was added in portions and stirred at room temperature for 2h. The solvent was drained under reduced pressure, the reaction was quenched with dilute hydrochloric acid (20 mL,1 mol/L), extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, the filtrate concentrated under reduced pressure, the residue was dissolved in toluene (100 mL), p-toluenesulfonic acid (139 mg,0.81 mmol) was added, and the reaction was carried out at 120℃for 3h. The solvent was drained under reduced pressure and purified by column chromatography on silica gel (eluent: petroleum ether) to give the title compound (1.70 g).

¹ H-NMR(400MHz,CDCl ₃ ):δ7.35(d,J＝8.0Hz,1H),7.13(d,J＝8.0Hz,1H),6.96-6.89(m,1H),6.57(dt,J＝5.6,2.0Hz,1H),3.38-3.36(m,2H)。

Step 2): synthesis of 3-bromo-2-chloro-6, 6 a-dihydro-1 aH-indeno [1,2-b ] oxirane (Compound 18-3):

according to the procedure of step 3) in example nine, using compound 18-2 (1.50 g,6.54 mmol) as a starting material, the product was purified by column chromatography over silica gel (eluent: petroleum ether/dichloromethane=1/1 (v/v) to give the title compound (900 mg).

¹ H-NMR(400MHz,CDCl ₃ ):δ7.48(d,J＝8.0Hz,1H),6.98(d,J＝8.0Hz,1H),4.50-4.45(m,1H),4.12(s,1H),3.22(d,J＝18.4Hz,1H),6.98(dd,J＝18.4,2.4Hz,1H)。

Step 3): synthesis of N- (5-bromo-4-chloro-2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamic acid tert-butyl ester (Compound 18-4):

compound 18-3 (900 mg,3.67 mmol) was dissolved in toluene (50 mL) at room temperature, silica gel (2.70 g) was added, and the mixture was reacted at 120℃for 6 hours. The filtrate was filtered, dried under reduced pressure, and the residue was dissolved in methanol (2.6 mL), and then methylamine/water solution (1 mL,40 wt%) was added, followed by stirring for 10min, sodium borohydride (94 mg,2.54 mmol), and after 1h of reaction at room temperature, di-tert-butyl dicarbonate (3.24 g,14.83 mmol) was added, and the reaction was continued at room temperature for 1h. The solvent was drained under reduced pressure and purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=30/1 (v/v)) to give the title compound (500 mg).

ESI-MS:m/z 304.3,306.3[M-56+H] ⁺ 。

Step 4): synthesis of tert-butyl N- (4-chloro-5- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamate (Compound 18-5):

according to the procedure of step 2) in example two, using compound 18-4 (200 mg,0.65 mmol) and pinacol diboronate (110 mg,0.43 mmol) as reaction starting materials, purification by preparative thin layer chromatography (eluent: petroleum ether/ethyl acetate=60/1 (v/v)) to give the title compound (80 mg).

¹ H-NMR(400MHz,CDCl ₃ ):δ7.52(d,J＝7.2Hz,1H),7.08(d,J＝7.2Hz,1H),5.08(brs,1H),3.30-3.15(m,2H),2.99(td,J＝15.2,6.0Hz,2H),2.68(s,3H),1.46(s,9H),1.36(s,12H)。

ESI-MS:m/z 352.3,354.3[M-56+H] ⁺ 。

Step 5): synthesis of tert-butyl N- (4-chloro-5- (6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamate (Compound 18-6):

According to the procedure of step 7) in example eight, using compounds 8 to 7 (39 mg,0.07 mmol) and compounds 18 to 5 (30 mg,0.07 mmol) as reaction starting materials, purification by preparative thin layer chromatography (eluent: ethyl acetate/methanol=60/1 (v/v)) to give the title compound (30 mg).

ESI-MS:m/z 735.3,737.3[M+H] ⁺ 。

Step 6) Synthesis of 3- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyryl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 7) in example I, using 18-6 (30 mg,0.04 mmol) as a starting material, the preparation was purified by preparative high performance liquid chromatography (method A), diluted hydrochloric acid (0.1 mL, 3M) was added to the preparation, and freeze-dried to give the hydrochloride (21 mg) of the objective compound.

¹ H-NMR(400MHz,CD ₃ OD):δ8.32(d,J＝6.8Hz,1H),7.91(d,J＝8.0Hz,1H),7.60(dt,J＝8.0,2.8Hz,1H),7.44(d,J＝8.0Hz,1H),7.30(d,J＝7.6Hz,1H),6.13(td,J _H-F ＝55.2Hz,J _H-H ＝4.4Hz,1H),5.87(ddd,J＝15.2,6.0,2.4Hz,1H),4.95-4.86(m,1H),4.21-4.08(m,3H),4.11(s,1H),3.86-3.74(m,1H),3.68-3.56(m,2H),3.49-3.33(m,3H),3.31-3.23(m,1H),3.12-2.99(m,1H),2.95-2.84(m,1H),2.82(s,3H),1.80-1.44(m,4H)。

ESI-MS:m/z 636.3,638.3[M+H] ⁺ 。

Example nineteenth: synthesis of 3- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (compound 19).

Step 1): synthesis of tert-butyl N- (4-chloro-5- (6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -7-oxo-6, 7-dihydroisothiazolo [4,3-d ] pyrimidin-3-yl) -2, 3-dihydro-1H-inden-2-yl) -N-methylcarbamate (Compound 19-1):

According to the procedure of step 7) in example eight, using compound 11-1 (30 mg,0.06 mmol) and compound 18-5 (28 mg,0.07 mmol) as reaction starting materials, purification by preparative thin layer chromatography (eluent: dichloromethane/methanol=20/1 (v/v)) to give the title compound (33 mg).

ESI-MS:m/z 718.3,720.3[M+H] ⁺ 。

Step 2): synthesis of 3- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -6- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) isothiazolo [4,3-d ] pyrimidin-7 (6H) -one (target compound):

according to the procedure of step 7) in example one, using compound 19-1 (33 mg,0.04 mmol) as a starting material, the reaction solution was concentrated under reduced pressure, then purified water was added, and freeze-drying was performed to obtain the hydrochloride (25.00 mg) of the objective compound.

¹ H-NMR(400MHz,DMSO-d ₆ ):δ9.15(s,2H),8.20(d,J＝12.4Hz,1H),7.99(d,J＝8.0Hz,1H),7.48(d,J＝8.0Hz,1H),7.34-7.20(m,4H),7.19-7.09(m,1H),4.95(s,1H),4.16-4.07(m,1H),4.07-3.96(m,2H),3.92(s,1H),3.80-3.67(m,1H),3.53(s,1H),3.44(s,1H),3.34-3.16(m,3H),2.86-2.74(m,2H),2.65(t,J＝5.2Hz,3H),2.35-2.28(m,1H),1.62-1.44(m,1H),1.45-1.20(m,3H),1.19-0.97(m,2H),0.53-0.44(m,1H),0.34-0.26(m,1H),0.25-0.17(m,1H),0.10-0.02(m,1H)。

ESI-MS:m/z 618.3,620.3[M+H] ⁺ 。

[ pharmacological Activity test ]

Test example one: USP7 (protease) inhibition assay of enzymatic activity in vitro.

1. Test system:

the kit comprises: a USP7 inhibitor screening assay kit (BPS catalyst: 79256) comprising:

protease: USP7 His-FLAG-tags enzyme (BPS catalyst: 80395);

a substrate: ub-AMC substrate (BPS catalyst: 81150);

buffer solution: 5 XUSP 7 assay buffer (BPS catalyst: 79274).

2. Test parameters:

USP7 concentration: 3nM;

Ub-AMC concentration: 100nM;

Buffer system: 1.25 XUSP 7 assay buffer; 0.06% bsa;1mM DTT; ddH ₂ O；

Compound and enzyme incubation time: 20min;

enzyme kinetic reaction time: 20min;

parameters of the enzyme-labeled instrument: BMG PHERAstar FS fluorescence enzyme label instrument, excitation wavelength 350nm, emission wavelength 460nm.

3. The test method comprises the following steps:

the test was performed according to the kit instructions, the steps were as follows:

test group: incubating a mixture of a compound to be tested and protease USP7 for 20min at room temperature in a buffer system, adding a substrate Ub-AMC to start a reaction, and reading the fluorescence value of each well in each cycle (1 min) by adopting an enzyme kinetic method, and reading 20 cycles.

Negative group: the test compounds were replaced with 0.2% dmso aqueous solution and the experimental procedure was the same as the test group.

Blank group: the test compounds were replaced with 0.2% dmso aqueous solution and no protease USP7 was added, the experimental procedure was the same as for the test group.

4. And (3) data processing:

the relative inhibition activity of each concentration group was calculated, inhibition ratio (%) =100% - (fluorescence value of test group-fluorescence value of blank group)/(fluorescence value of negative group-fluorescence value of blank group) ×100%. The half maximal Inhibitory Concentration (IC) of the compound was calculated by fitting a curve according to a four parameter model ₅₀ )。

5. Test results:

Inhibition of USP7 activity by the compounds of the present invention was determined as described above and the results are shown in table 1.

TABLE 1 results of USP7 enzyme Activity inhibition assay

Examples numbering	IC ₅₀ (nM)
		1	25.10±10.37
2	15.21±4.70
		5	58.46±5.69
6	66.53±4.33
		7	7.82±1.42
10	30.76±7.84
		13	63.16±8.92
14	22.33±1.99
		15	43.10±2.49
16	84.61±17.67

6. Conclusion:

in the USP7 enzyme activity inhibition test, the compounds of the present invention exhibit a strong inhibitory activity, and in particular, the compounds of examples 1, 2, 7, 10, 14 and 15 have a strong inhibitory activity against USP 7.

Test example two: mm.1s cell proliferation activity inhibition assay.

1. Test system:

and (3) cells: mm.1s (south tokyo, herborist);

the kit comprises: cellTiter-Luminescence cell viability assay kit (Promega).

2. Test parameters:

cell number: 3000 cells/well;

plating medium: mm.1s 1640+10% fbs;

dosing medium: mm.1s 1640+10% fbs;

compound incubation conditions: 37 ℃,5% CO ₂ ；

Incubation time: 5d;

detecting the temperature: room temperature;

BMG PHARMASTAR FS detects chemiluminescence.

3. The test method comprises the following steps:

culturing cells in a medium containing 10% fetal bovine serum, and standing at 37deg.C with 5% CO ₂ Culturing under culture conditions. Appropriate amount of cells were plated into 96-well plates and cultured overnight in an incubator. The following day, complete medium containing pre-diluted compounds was added and incubated for 5d at 37 ℃. On the fifth day, a detection reagent CellTiter- Chemiluminescence was measured for the Relative Luminescence Units (RLU) of each well.

4. And (3) data processing:

CellTiter using cell-free mediumA background value is obtained. Cell viability (%) = (sample RLU-background RLU)/(vehicle RLU-background RLU) ×100%, maximum inhibition (%) = 100% -cell viability _{Maximum concentration of} (%) and the following. The half maximal Inhibitory Concentration (IC) of the compound was calculated by fitting a curve according to a four parameter model ₅₀ )。

5. Test results:

the inhibitory activity of the compounds of the present invention on mm.1s cell proliferation was measured using the method described above, and the results are shown in table 2.

TABLE 2 inhibition of MM.1S cell proliferation activity by Compounds

Examples numbering	MM.1S，IC ₅₀ (nM)
		1	71.77±7.19
2	7.59±0.33
		3	46.61±3.69
4	75.28±22.34
		6	35.06±5.08
12	98.07±12.72
		14	46.92±10.07
15	23.13±3.95

6. Conclusion:

in the mm.1s cell proliferation activity inhibition assay, the compounds of the present invention showed strong cell proliferation inhibition activity, and in particular, the compounds of examples 2, 3, 6, 14 and 15 have extremely strong inhibition activity on mm.1s cell proliferation.

Test example three: biochemical hERG inhibition assay.

1. Test system:

the kit comprises: preconductor ^TM hERG fluorescence polarization assay kit (ThermoFisher Catalog:PV 5365), containing:

compound E4031 as a positive control;

hERG cell membrane;

affinity tracers; and

hERG buffer.

2. Test parameters:

hERG concentration: 1×;

concentration of tracer: 1nM;

incubation time: 2h;

parameters of the enzyme-labeled instrument: BMG PHERAstar FS fluorescence enzyme labelling instrument.

3. The test method comprises the following steps:

test group: the compounds to be tested with different concentrations are added into a microplate containing hERG cell membranes, a tracer with high hERG affinity is added into each well, and after the microplate is incubated for 2 hours at room temperature, the change of fluorescence polarization (excitation wavelength: 540nm; emission wavelength: 590 nm) values is detected by using a multifunctional enzyme-labeled instrument.

Positive control group: the test compound was replaced with 30. Mu.M of Compound E4031, and the experimental procedure was the same as that of the test group.

Blank control group: the test compounds were replaced with hERG buffer and hERG cell membranes were not added, and the experimental procedure was the same as for the test group.

4. And (3) data processing:

based on the data ratio, the percent inhibition (%) of the compounds of the invention against hERG at various concentrations was calculated, and the half Inhibitory Concentration (IC) of the compounds was determined ₅₀ ) Is not limited in terms of the range of (a). Percentage inhibition (%) = (1- (fluorescence polarization value of test compound-fluorescence polarization value of positive control)/(fluorescence polarization value of blank control-fluorescence polarization value of positive control)) ×100%.

5. Test results:

inhibition of hERG by the compounds was determined using the methods described above and the results are shown in table 3.

TABLE 3 hERG inhibition assay results

Examples numbering	IC ₅₀ (μM)
		5	＞10
6	＞10
		7	＞10
9	＞10
		13	＞10
17	＞10
		18	＞10

6. Conclusion:

the test results showed that the compounds of examples 5, 6, 7, 9, 13, 17 and 18 have low affinity for hERG and compete with the affinity tracer for IC ₅₀ Are all greater than 10. Mu.M.

Test example four: biochemical CYP enzyme (cytochrome P450) inhibition assay.

1. Test system:

P450-Glo ^TM CYP1A2 screening System (Promega catalyst: V9770);

P450-Glo ^TM CYP2D6 screening System (Promega catalyst: V9890);

P450-Glo ^TM CYP3A4 screening System (Promega catalyst: V9920).

2. Test instrument:

BMG PHARMASTAR FS detects chemiluminescence.

3. The test method comprises the following steps:

the test was performed according to the kit instructions, respectively, as follows:

3.1 inhibition of CYP1 A2:

test group: the test compounds with different concentrations are added into a microplate, and Luciferin-ME (100 mu M) and K are added into each well ₃ PO ₄ (100 mM) and CYP1A2 (0.01 pmol/. Mu.L), preincubated at room temperature for 10min, then added to the NADPH regeneration system to react at room temperature for 30min, finally added with an equal volume of detection buffer at room temperatureAfter incubation for 20min at room temperature, chemiluminescent detection was performed.

Negative control group: the experimental procedure was the same as for the test group except that no test compound was added.

Blank control group: the experimental procedure was the same as for the test group except that no test compound was added and CYP1A2 was replaced with CYP1A2 membrane (0.01 pmol/. Mu.L).

3.2 inhibition of CYP2D 6:

test group: adding test compounds with different concentrations into microplates, adding Luciferin-ME EGE (3 μm), K into each well ₃ PO ₄ (100 mM) and CYP2D6 (5 nM) were pre-incubated at room temperature for 10min, then reacted at 37℃for 30min with the addition of NADPH regeneration system, and finally incubated at room temperature for 20min with an equal volume of detection buffer for chemiluminescent detection.

Blank control group: the experimental procedure was the same as for the test group except that no test compound was added and CYP2D6 membrane (5 nM) was used instead of CYP2D6.

3.3 inhibition of CYP3 A4:

test group: the test compounds with different concentrations are added into a microplate, and Luciferin-IPA (3 mu M) and K are added into each well ₃ PO ₄ (100 mM) and CYP3A4 (2 nM) were pre-incubated at room temperature for 10min, then added to the NADPH regeneration system to react at room temperature for 30min, and finally added with an equal volume of detection buffer to incubate at room temperature for 20min for chemiluminescent detection.

Blank control group: the experimental procedure was the same as for the test group except that no test compound was added and CYP3A4 was replaced with CYP3A4 membrane (2 nM).

4. And (3) data processing:

percentage inhibition (%) = (1- (chemiluminescent value of test compound concentration group-chemiluminescent value of blank control group)/(chemiluminescent value of negative control group-chemiluminescent value of blank control group)) ×100%.

According to the different concentrations of the compoundInhibition of CYP enzyme by the compound, half inhibition concentration (IC ₅₀ ) Or range.

IC ₅₀ =x× (1-percent inhibition (%))/percent inhibition (%), where X is the compound test concentration.

5. Test results:

inhibition of three CYPs by the compounds of the present invention was determined as described above and the results are shown in table 4.

TABLE 4 CYPs inhibition test results

6. Conclusion:

the above results indicate that the compounds of examples 3, 5, 6, 7, 9 and 18 have no significant inhibition on all 3 major CYP subtypes, indicating that their potential drug interactions are relatively low, with good pharmaceutical properties.

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including all patents, patent applications, journal articles, books, and any other publications) is incorporated herein by reference in its entirety.

Claims

1. A compound having the structure of formula I or a pharmaceutically acceptable salt thereof,

wherein the method comprises the steps of

R ¹ Selected from the group consisting of

R ² is-C (=O) R ⁸ ；R ⁸ Selected from the following groups:

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

R ¹ Selected from the group consisting ofR ² As defined in claim 1.

3. The compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein

R ² is-C (=O) R ⁸ ；R ⁸ Selected from the following groups:

4. the following compounds or pharmaceutically acceptable salts thereof:

5. a pharmaceutical composition comprising a compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

6. A kit, comprising:

a) A compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 5;

b) Optionally package and/or instructions.

7. Use of a compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 5, or a kit according to claim 6, in the manufacture of a medicament for the prevention and/or treatment of a disease or disorder mediated at least in part by UPS 7.