CN113087718A

CN113087718A - Thienopyrimidinone compounds and medical application thereof

Info

Publication number: CN113087718A
Application number: CN202010022368.3A
Authority: CN
Inventors: 陈寿军; 宋帅; 蒋小玲; 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: 2020-01-09
Filing date: 2020-01-09
Publication date: 2021-07-09
Anticipated expiration: 2040-01-09
Also published as: CN113087718B

Abstract

The invention relates to thienopyrimidinone compounds and medical application thereof. The invention belongs to the field of medicines, and particularly relates to a compound shown in a formula I, and a pharmaceutically acceptable salt, an ester, a solvate, a stereoisomer, a tautomer, a prodrug, any crystal form, a metabolite or a mixture thereof. The invention also relates to medical application of the compound shown in the formula I, and pharmaceutically acceptable salts, esters, solvates, stereoisomers, tautomers, prodrugs, any crystal forms, metabolites or mixtures thereof. The compounds of the present invention can be used to inhibit the activity of deubiquitinase USP7 and can also be used to prevent or treat diseases or disorders (e.g. cancer) associated with USP7 modulation.

Description

Thienopyrimidinone compounds and medical application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a thienopyrimidinone compound and a medical application of the compound.

Background

Ubiquitin-proteasome system (UPS) is a basic physiological regulatory process in cells, and proteins are ubiquitinated and modified by proteases and degraded by proteases through a series of cascade reactions. The abnormality of UPS is closely related to tumor, neurodegenerative disease, virus infection and other diseases. At present, medicines are mainly developed aiming at five types of targets of protease, E1 activating enzyme, E2 binding enzyme, E3 ligase and Deubiquitinases (DUBs) in a UPS system.

The deubiquitinase can specifically cut off isopeptide bonds formed between glycine residues at the carbon terminal of ubiquitin and target proteins, so that ubiquitin is separated from the target proteins, and the target proteins are prevented from being degraded, relocated or activated.

There are currently approximately 100 kinds of DUBs in humans, of which ubiquitin-specific proteases (USPs) are the largest family members of DUBs, including about 85 members, and belong to an isopeptidase in the cysteine protease family^[1]. More than 40 family members of USPs have been found to be associated with tumor development and progression.

USP7 is a key deubiquitinase in UPS, and can specifically cut off isopeptide bond formed between ubiquitin carbon terminal and target protein, and make ubiquitin separate from target protein, so that target protein is protected from degradation, relocation or activation^[2]。

USP7 has a wide distribution of human tissues and plays a major role in neural development, cell cycle regulation, epigenetic regulation, DNA damage repair, and immune response. It has been shown that USP7 is overexpressed in hepatocellular carcinoma, multiple myeloma, colon cancer, lung cancer, prostate cancer, bladder cancer and other cancer cells, and this overexpression is directly related to tumor invasion and poor prognosis^[3]。

USP7 has abundant substrate species, and most of them are proteins related to cell cycle regulation, immune response, apoptosis and DNA damage repair, such as MDM2, p53, ERCC6, Foxp3, PTEN and FOXO4^[4]. In some tumor cells MDM2 is overexpressed, USP7 protects MDM2 from ubiquitination, and MDM2, after binding to p53 protein, promotes ubiquitination and degradation of p53 protein, promoting tumor growth.

USP7 can also exert tumor promotion effect by directly regulating the expression of tumor suppressor proteins (e.g., p53, PTEN, FOXO4, p114ARF, p16INK4) and tumor promoting proteins (e.g., N-MYC, REST), up-regulating the expression of tumor associated factors (e.g., HIF-1), and regulating tumor associated signaling pathways (e.g., SHH signaling pathway, Wnt/β -catenin signaling pathway, androgen receptor signaling pathway, DNA damage repair signaling pathway)^[5]。

In addition, USP7 also can regulate upstream signal molecules of Treg cells (such as transcription factor FOXP3 and epigenetic regulatory factor Tip60), up-regulate the activity of the Treg cells, and inhibit the activity of Teff cells (CD8+ T cells), thereby playing a role in tumor immune surveillance escape^[6]。

The development of inhibitors of USP7 is one of the hotspots in the field of tumor research. Currently, no drugs are on the market worldwide for the USP7 target, and the compounds under investigation are all in the preclinical stage of study.

Although companies such as hybrids SA, Forma Therapeutics, inc, Les laboratories server, Almac Discovery Limited have corresponding research on USP7 inhibitors and related patent publications, there is still a great need in the art for new USP7 inhibitors, particularly USP7 inhibitors with high activity and other excellent properties.

Disclosure of Invention

Through a great deal of research, the invention surprisingly discovers the thienopyrimidinone compound which has stronger inhibition effect on the activity of deubiquitinase USP7 and can be used for preventing or treating diseases or symptoms (such as cancer) related to the regulation of USP 7. On the basis, the invention also provides the medical application of the thienopyrimidinone compound.

A first aspect of the present invention relates to a compound of formula I or a pharmaceutically acceptable salt, ester, solvate (e.g., hydrate), stereoisomer, tautomer, prodrug, any crystalline form, metabolite, or mixture thereof:

wherein,

R¹selected from hydrogen, C_1-6Alkyl radical, C_6-10Aryl, 5-to 10-membered heteroaryl,

Halogen and cyano, wherein, said C_1-6Alkyl radical, C_6-10Aryl and 5-to 10-memberedHeteroaryl is each independently optionally substituted with one or more of the following: hydrogen, halogen, cyano, -OR^a、-NR^bR^c、 -C_1-6alkylene-NR^bR^c、C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, optionally halogenated C_1-6Alkyl, -C (O) R⁶、-COOR⁷、-C(O)NR^bR^c、-S(O)_qR⁸、-S(O)_qNR^bR^c、- O-C_2-6alkylene-NR^bR^c、-NR^a-C_2-6alkylene-NR^bR^c；

R²Selected from hydrogen, halogen, cyano, C optionally halogenated_1-6Alkyl radical, C_3-8A cycloalkyl group;

R³selected from the group consisting of-C (O) R⁹、-COOR¹⁰、-C(O)NR^eR^f、-S(O)_qR¹¹and-S (O)_qNR^eR^f；

Ring A and ring B are each independently selected from C_3-8Cycloalkyl radical, C_3-8Cycloalkenyl, 3-8 membered heterocycloalkyl, 3-8 membered heterocycloalkenyl, C_6-10Aryl and 5-10 membered heteroaryl;

in which R radicals are identical or different radicals and m radicals R⁴The groups are the same or different;

r is selected from hydrogen, optionally halogenated C_1-6Alkyl, halogen and cyano;

R⁴selected from hydrogen, oxo, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl, 5-10 membered heteroaryl, -OR^a、-NR^bR^cHalogen, cyano, -C (O) R⁶、-COOR⁷、-C(O)NR^bR^c、-O-C_2-6alkylene-NR^bR^cand-NR^d- C_2-6alkylene-NR^bR^cWherein, the C is_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen, cyano, C_1-6Alkyl, hydroxy, amino, -N (C)_1-6Alkyl radical)₂、-NH(C_1-6Alkyl groups);

R^a、R^b、R^cand R^dEach independently selected from hydrogen and C_1-6Alkyl radical, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl, 5-10 membered heteroaryl, -C (O) R⁶and-S (O)_qR⁸Wherein, the C is_1-6Alkyl radical, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen, hydroxy, amino, cyano, C optionally halogenated_1-6Alkyl radical, C_3-8A cycloalkyl group; or, R^b、R^cForm a 3-6 membered heterocyclic group with the nitrogen atom to which they are attached;

R⁶、R⁷and R⁸Each independently selected from hydrogen and C_1-6Alkyl and C_3-8Cycloalkyl, wherein said C_1-6Alkyl and C_3-8Cycloalkyl is each independently optionally substituted with one or more of the following: hydrogen, halogen, cyano, amino, hydroxy;

R⁹、R¹⁰、R¹¹、R^eand R^fEach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl, 5-10 membered heteroaryl, or, R^e、 R^fForm a 3-6 membered heterocyclic group with the nitrogen atom to which they are attached; wherein, the C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl, 5-10 membered heteroaryl, and 3-6 membered heterocycle are each independently optionally substituted with one or more substituents as follows: hydrogen, cyano, halogen、-OH、C_1-6Alkoxy, -NR^gR^h、C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl, 5-10 membered heteroaryl, and, C in said substituents_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen, cyano, optionally halogenated C_1-6Alkyl radical, C_6-10Aryl, 5-10 membered heteroaryl;

R^g、R^heach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl;

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

q is selected from 1 and 2;

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

In the formula I of the invention, the compound of formula I,

represents: the groups on the ring A can be substituted by R R groups, and the R groups can be the same or different; the radicals on the B ring may be substituted by m R⁴Substituted by radicals m R⁴The groups may be the same or different.

In certain embodiments of the first aspect of the present invention, R¹Is selected from C_1-6Alkyl radical, C_6-10Aryl and

wherein, the C_1-6Alkyl and C_6-10Each aryl is independently optionally substituted with one or more substituents as follows: hydrogen, halogen, cyano, -OR^a、-NR^bR^c、-C_1-6alkylene-NR^bR^c。

wherein, the C_1-6Alkyl and C_6-10Each aryl group is independently optionally substituted with one or more of the following: hydrogen, halogen, -OR^a、-C_1-6alkylene-NR^bR^c；

Ring A and ring B are each independently selected from C_6-10Aryl, 5-10 membered heteroaryl, 3-8 membered heterocycloalkenyl, 3-8 membered heterocycloalkyl, C_3-8Cycloalkenyl radical and C_3-8A cycloalkyl group;

r is selected from hydrogen, optionally halogenated C_1-6Alkyl, halogen and cyano (preferably, R is selected from hydrogen and halogen);

r is selected from 0, 1 and 2;

R⁴selected from hydrogen, -NR^bR^c、C_1-6Alkyl and-OR^a(ii) a m is selected from 0, 1 and 2;

R^a、R^band R^cEach independently selected from hydrogen and C_1-6An alkyl group.

wherein, the C_1-6Alkyl and C_6-10Each aryl group is independently optionally substituted with one or more of the following: hydrogen, hydroxy, NH₂-C_1-6Alkylene-, halogen, amino, cyano;

R⁴selected from hydrogen, amino, C_1-6alkyl-NH-, (C)_1-6Alkyl radical)₂N-, optionally substitutedHalogenated C_1-6Alkyl, oxo, C_3-8Cycloalkyl, hydroxy and cyano;

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

r is selected from 0, 1 and 2.

wherein, the C_6-10Aryl is optionally substituted with one or more of the following: hydrogen, hydroxy, NH₂-C_1-6Alkylene-, halogen, amino, cyano;

ring A and ring B are each independently selected from C_6-10Aryl, 5-10 membered heteroaryl, 3-8 membered heterocycloalkenyl, 3-8 membered heterocycloalkyl, C_3-8Cycloalkenyl radical and C_3-8Cycloalkyl (preferably, ring A and ring B are each independently selected from C_6-10Aryl, 5-10 membered heteroaryl, 3-8 membered heterocycloalkyl and C_3-8Cycloalkyl groups);

r is selected from 0, 1 and 2;

R⁴selected from hydrogen, amino, C_1-6alkyl-NH-, (C)_1-6Alkyl radical)₂N-、C_1-6Alkyl and hydroxy;

m is selected from 0, 1 and 2.

wherein, the C_6-10Aryl is optionally substituted with one or more of the following: hydroxy, NH₂-C_1-6Alkylene-, halogen;

ring A and ring B are each independently selected from C_6-10Aryl, 5-10 membered heteroaryl, 3-8 membered heterocycloalkenyl, 3-8 membered heterocycloalkyl, C_3-8Cycloalkenyl groupAnd C_3-8Cycloalkyl (preferably, ring A and ring B are each independently selected from C_6-10Aryl, 5-10 membered heteroaryl, 3-8 membered heterocycloalkyl and C_3-8Cycloalkyl groups);

r is selected from 0, 1 and 2;

m is selected from 0, 1 and 2.

ring A is C_6-10An aryl group;

ring B is selected from 5-10 membered heteroaryl, 3-8 membered heterocycloalkenyl, 3-8 membered heterocycloalkyl, C_3-8Cycloalkenyl radical and C_3-8A cycloalkyl group.

the ring A is phenyl;

ring B is selected from pyrazolyl, 3-pyrrolinyl, pyrrolidinyl, cyclopentenyl and cyclopentyl, preferably ring B is selected from pyrazolyl, pyrrolidinyl and cyclopentyl;

r is selected from hydrogen and halogen;

r is selected from 0, 1 and 2;

m is selected from 0, 1 and 2.

In certain embodiments of the first aspect of the present invention, R¹Selected from methyl, phenyl and

wherein the phenyl is optionally substituted with one or more of the following: halogen, hydroxy and NH₂-CH₂-；

The ring A is phenyl;

ring B is selected from pyrazolyl, 3-pyrrolinyl, pyrrolidinyl, cyclopentenyl and cyclopentyl;

r is selected from hydrogen and halogen, preferably R is selected from hydrogen, F and Cl;

r is selected from 0 and 1;

R⁴selected from hydrogen, amino, CH₃-NH-、(CH₃)₂N-, methyl and hydroxy;

m is selected from 0, 1 and 2.

In certain embodiments of the first aspect of the present invention, R¹Phenyl optionally substituted with one or more substituents selected from: hydrogen, fluorine, chlorine, -OH, -CH₂NH₂。

In certain embodiments of the first aspect of the present invention, R¹Is composed of

Wherein, A ring is selected from C_6-10Aryl and 5-10 membered heteroaryl; r is selected from hydrogen, optionally halogenated C_1-6Alkyl and halogen; r is selected from 1,2 and 3.

Wherein ring A is C_6-10An aryl group; r is selected from hydrogen, optionally halogenated C_1-6Alkyl radicals andhalogen; r is selected from 1,2 and 3.

Wherein ring A is C_6-10An aryl group; r is selected from hydrogen and halogen; r is selected from 1,2 and 3.

Wherein, ring A is benzene ring; r is selected from hydrogen, fluorine and chlorine; r is selected from 1 and 2.

Wherein, B ring is selected from C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, and 5-10 membered heteroaryl; r⁴Selected from hydrogen, optionally halogenated C_1-6Alkyl, oxo, -OH, -NH₂、-NH(C_1-6Alkyl), -N (C)_1-6Alkyl radical)₂、 C_3-8Cycloalkyl and cyano; m is selected from 1,2 and 3.

Wherein, B ring is selected from C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, and 5-10 membered heteroaryl; r⁴Selected from hydrogen, optionally halogenated C_1-6Alkyl, -OH, -NH₂、-NH(C_1-6Alkyl) and-N (C)_1-6Alkyl radical)₂(ii) a m is selected from 1,2 and 3.

Wherein, B ring is selected from C_4-6Cycloalkyl, 4-6 membered heterocycloalkyl, and 5-10 membered heteroaryl; r⁴Selected from hydrogen, methyl,- NH₂、-NHCH₃-OH and-N (CH)₃)₂(ii) a m is selected from 1 and 2.

Wherein ring B is selected from pyrazolyl, 3-pyrrolinyl, pyrrolidinyl, cyclopentenyl and cyclopentyl; r⁴Selected from hydrogen, methyl, -NH₂、-NHCH₃-OH and-N (CH)₃)₂(ii) a m is selected from 1 and 2.

Wherein, ring B is pyrrolidinyl; r⁴Selected from hydrogen, methyl, -NH₂、-NHCH₃-OH and-N (CH)₃)₂(ii) a m is selected from 1 and 2.

In certain embodiments of the first aspect of the present invention, R¹Selected from methyl,

In certain embodiments of the first aspect of the present invention, R¹Is selected from C_6-10Aryl and

wherein, the C_6-10Aryl is optionally substituted with one or more of the following: halogen and-C_1-6alkylene-NR^bR^c；

Ring A and ring B are each independently selected from C_6-10Aryl, 3-8 membered heterocycloalkenyl, 3-8 membered heterocycloalkyl, C_3-8Cycloalkenyl radical and C_3-8A cycloalkyl group;

r is selected from hydrogen and halogen; r is selected from 0, 1 and 2;

wherein, the C_6-10Aryl is optionally substituted with one or more of the following: halogen and NH₂-C_1-6Alkylene-;

r is selected from hydrogen and halogen; r is selected from 0, 1 and 2;

R⁴selected from hydrogen, amino, C_1-6alkyl-NH-, (C)_1-6Alkyl radical)₂N-、C_1-6Alkyl and hydroxy; m is selected from 0, 1 and 2.

In certain embodiments of the first aspect of the present invention, R¹Selected from phenyl and

wherein the phenyl is optionally substituted with one or more of the following: halogen andNH₂-CH₂-；

the ring A is phenyl;

ring B is selected from the group consisting of 3-pyrrolinyl, pyrrolidinyl, cyclopentenyl and cyclopentanyl;

r is selected from hydrogen and halogen; r is selected from 0 and 1;

R⁴selected from hydrogen, amino, CH₃-NH-、(CH₃)₂N-, methyl and hydroxy; m is selected from 0, 1 and 2.

In certain embodiments of the first aspect of the present invention, R¹Is selected from

In certain embodiments of the first aspect of the present invention, R²Selected from hydrogen, halogen, optionally halogenated C_1-6Alkyl and C_3-8A cycloalkyl group.

In certain embodiments of the first aspect of the present invention, R²Selected from hydrogen, fluorine, methyl, trifluoromethyl; preferably, R²Is hydrogen.

In certain embodiments of the first aspect of the present invention, R³Selected from the group consisting of-C (O) R⁹and-C (O) NR^eR^f； R⁹、R^e、R^fEach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl and 3-8 membered heterocycloalkyl, wherein said C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl and 3-8 membered heterocycloalkyl are each independently optionally substituted with one or more substituents as follows: halogen, optionally halogenated C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_6-10Aryl and 5-10 membered heteroaryl; and, said C_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen, optionally halogenated C_1-6An alkyl group.

In certain embodiments of the first aspect of the present invention, R³is-C (O) R⁹；R⁹Selected from hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl and 3-8 membered heterocycloalkyl, wherein said C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl and 3-8 membered heterocycloalkyl are each independently optionally substituted with one or more substituents as follows: halogen, optionally halogenated C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_6-10Aryl and 5-10 membered heteroaryl; and, said C_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen, optionally halogenated C_1-6An alkyl group.

In certain embodiments of the first aspect of the present invention, R³is-C (O) R⁹；R⁹Selected from hydrogen, C_1-6Alkyl radical, C_2-6Alkenyl and C_3-8Cycloalkyl, wherein said C_1-6Alkyl radical, C_2-6Alkenyl and C_3-8Cycloalkyl is each independently optionally substituted with one or more substituents as follows: halogen, C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_6-10Aryl and 5-10 membered heteroaryl; and, C in the substituents_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen, C_1-6An alkyl group.

In certain embodiments of the first aspect of the present invention, R³is-C (O) R⁹；R⁹Selected from hydrogen, C_1-6Alkyl and C_2-6Alkenyl, wherein said C_1-6Alkyl and C_2-6Each alkenyl group is independently optionally substituted with one or more substituents as follows: halogen, C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_6-10Aryl and 5-10 membered heteroaryl; and, C in the substituents_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen、C_1-6An alkyl group.

In certain embodiments of the first aspect of the present invention, R³is-C (O) R⁹；R⁹Is C optionally substituted by one or more substituents_1-6Alkyl groups: halogen, optionally halogenated C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_6-10Aryl, 5-to 10-membered heteroaryl, wherein, C is_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted by a group selected from hydrogen, halogen and optionally halogenated C_1-6One or more groups in the alkyl group are substituted.

In certain embodiments of the first aspect of the present invention, R³is-C (O) R⁹；R⁹Is C optionally substituted by one or more substituents_1-6Alkyl groups: optionally halogenated C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_6-10Aryl, 5-to 10-membered heteroaryl, wherein, C is_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more halogens.

In certain embodiments of the first aspect of the present invention, R³is-C (O) R⁹；R⁹Is C optionally substituted by one or more substituents_1-6Alkyl groups: methyl, difluoromethyl, trifluoromethyl, cyclopropyl, phenyl optionally halogenated (e.g. phenyl substituted with a fluoro), pyrazolyl optionally halogenated (e.g. pyrazolyl substituted with a fluoro or chloro).

In certain embodiments of the first aspect of the present invention, R³Is C (O) R⁹，R⁹Selected from the following groups:

a second aspect of the present invention relates to a compound of formula ii, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite, or mixture thereof:

wherein,

R¹、R²is as defined in the first aspect of the invention;

R¹³is O;

R¹⁴is selected from C_6-10Aryl and 5-8 membered heteroaryl, wherein, C_6-10Aryl and 5-8 membered heteroaryl are each independently optionally substituted with one or more substituents as follows: hydrogen, halogen, hydroxy, C_1-6Alkyl radical, C_1-6Alkoxy, amino, cyano;

R¹⁵selected from H, C_1-6Alkyl and C_3-8Cycloalkyl radicals, in which C_1-6Alkyl and C_3-8Cycloalkyl is each independently optionally substituted with one or more substituents as follows: halogen, hydroxy, C_1-6Alkyl radical, C_1-6Alkoxy, amino, cyano;

n is selected from 1,2, 3,4, 5 and 6; preferably, n is selected from 1,2 and 3, more preferably n is 1.

In some embodiments of the second aspect of the present invention, R¹⁴Is selected from C_6-10Aryl and 5-8 membered heteroaryl, wherein, C_6-10Aryl and 5-8 membered heteroaryl are each independently optionally substituted with one or more halogens.

In some embodiments of the second aspect of the present invention, R¹⁴Is selected from C_6-10Aryl and 5-8 membered nitrogen-containing heteroaryl, wherein, C_6-10Aryl and 5-8 membered nitrogen containing heteroaryl are optionally substituted with one or more halogens.

In some embodiments of the second aspect of the present invention, R¹⁴Selected from phenyl and pyrazolyl, wherein the phenyl and pyrazolyl are each independently optionally substituted with one or more halogens.

In some embodiments of the second aspect of the present invention, R¹⁴Selected from phenyl,

In some embodiments of the second aspect of the present invention, R¹⁵Is selected from C_1-6Alkyl and C_3-8Cycloalkyl radicals, in which C_1-6Alkyl is optionally substituted with one or more halogens.

In some embodiments of the second aspect of the present invention, R¹⁵Selected from the group consisting of methyl, difluoromethyl, trifluoromethyl and cyclopropyl.

In some embodiments of the second aspect of the present invention, R¹⁵Selected from the group consisting of methyl, difluoromethyl, and cyclopropyl.

In some embodiments of the second aspect of the present invention, R¹Selected from methyl,

R²Selected from hydrogen, fluoro, methyl and trifluoromethyl;

R¹⁴selected from phenyl,

R¹⁵Selected from methyl, difluoromethyl and cyclopropyl; and is

n is selected from 1,2 and 3.

In certain embodiments of the first aspect of the present invention, the compound is selected from compounds 1 to 44 in the following table.

In some embodiments of the second aspect of the present invention, the compound is selected from the group consisting of compounds 1 to 44, R in the following table¹、R¹⁴And R¹⁵As shown in the table, R²Is H, R¹³Is O, and n is 1.

Any atom in the compounds of the present invention may be replaced by its isotope. For example¹²C can be substituted by its isotopes¹³C or¹⁴C, replacing;¹h can be covered²H (D, deuterium) or³H (T, tritium) substitution, and the like. The invention includes isotopically-labeled compounds in which any atom is replaced by its isotope.

A third aspect of the present invention relates to a pharmaceutical composition comprising a compound according to the first or second aspect of the present invention, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite or mixture thereof, and optionally a pharmaceutically acceptable adjuvant.

In some embodiments of the third aspect of the invention, the pharmaceutical composition is a tablet, pill, capsule, lozenge, hard candy, powder, spray, cream, ointment, suppository, gel, paste, lotion, ointment, aqueous suspension, injectable solution, elixir, or syrup.

In some embodiments of the third aspect of the invention, the pharmaceutical composition may be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intradermally, transdermally, rectally, intracranially, intraperitoneally, intranasally, intramuscularly, or as an inhalant, and may be administered by all forms of use well known to those skilled in the art of medicine. The compound of the invention or the pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite or the mixture thereof can be prepared into various suitable dosage forms according to the administration route.

In some embodiments of the third aspect of the present invention, the pharmaceutical composition further comprises an additional active ingredient that inhibits the activity of deubiquitinase USP7 or an additional active ingredient that prevents or treats cancer.

In some embodiments of the third aspect of the present invention, the pharmaceutical composition further comprises other pharmaceutically active ingredients for the prevention or treatment of diseases or disorders related to modulation of deubiquitinase USP 7.

In some embodiments of the third aspect of the present invention, the disease or disorder associated with modulation of deubiquitinase USP7 is selected from cancer, neurodegenerative diseases (e.g. alzheimer's disease, parkinson's disease), diabetes, bone and joint diseases, arthritic inflammatory disorders, osteoporosis, immune disorders, cardiovascular diseases, ischemic diseases, viral infectious diseases and bacterial infectious diseases.

In some embodiments of the third aspect of the invention, the pharmaceutical composition or suitable dosage form may contain from 0.01mg to 1000mg of a compound of the invention.

The fourth aspect of the present invention also relates to a pharmaceutical product comprising a compound according to the first or second aspect of the present invention, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite or mixture thereof, or a pharmaceutical composition according to the third aspect of the present invention, and optionally a product package (e.g. a product package comprising product instructions).

A fifth aspect of the present invention is directed to a method for inhibiting deubiquitinase USP7 activity in vitro or inhibiting tumor cell proliferation in vitro comprising contacting a subject with an effective amount of a compound of the first or second aspect of the present invention, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite or mixture thereof, or a pharmaceutical composition of the third aspect of the present invention.

A sixth aspect of the present invention relates to the use of a compound according to the first or second aspect of the present invention, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite or mixture thereof, or a pharmaceutical composition according to the third aspect of the present invention, for the manufacture of an inhibitor of deubiquitinase USP7 or for the manufacture of a medicament for the prevention or treatment of a disease or condition associated with modulation of deubiquitinase USP 7.

In some embodiments of the sixth aspect of the present invention, the disease or disorder associated with modulation of deubiquitinase USP7 is selected from cancer, neurodegenerative diseases, diabetes, bone and joint diseases, arthritic inflammatory disorders, osteoporosis, immune disorders, cardiovascular diseases, ischemic diseases, viral infectious diseases and bacterial infectious diseases.

In some embodiments of the sixth aspect of the present invention, the cancer is selected from at least one of hepatocellular carcinoma, multiple myeloma, colon cancer, lung cancer, prostate cancer, and bladder cancer.

A seventh aspect of the present invention relates to a method for preventing or treating a disease or disorder associated with modulation of deubiquitinase USP7, comprising administering to a subject in need thereof an effective amount of a compound according to the first or second aspect of the present invention, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite or mixture thereof or a pharmaceutical composition according to the third aspect of the present invention.

In some embodiments of the seventh aspect of the present invention, the disease or disorder associated with modulation of deubiquitinase USP7 is selected from cancer, neurodegenerative diseases, diabetes, bone and joint diseases, arthritic inflammatory disorders, osteoporosis, immune disorders, cardiovascular diseases, ischemic diseases, viral infectious diseases and bacterial infectious diseases.

In some embodiments of the seventh aspect of the present invention, the cancer is selected from at least one of hepatocellular carcinoma, multiple myeloma, colon cancer, lung cancer, prostate cancer, and bladder cancer.

A compound according to the first or second aspect of the present invention, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite or mixture thereof, or a pharmaceutical composition according to the third aspect of the present invention, for use in inhibiting deubiquitinase USP7 activity, or for use in the prevention or treatment of a disease or condition associated with modulation of deubiquitinase USP 7.

In some embodiments of the first or second aspect of the present invention, the disease or disorder associated with modulation of deubiquitinase USP7 is selected from cancer, neurodegenerative diseases, diabetes, bone and joint diseases, arthritic inflammatory disorders, osteoporosis, immune disorders, cardiovascular diseases, ischemic diseases, viral infectious diseases and bacterial infectious diseases.

In some embodiments of the first or second aspect of the present invention, the cancer is selected from at least one of hepatocellular carcinoma, multiple myeloma, colon cancer, lung cancer, prostate cancer, and bladder cancer.

The compounds of the first or second aspect of the invention may be synthesised using the methods described hereinafter, using known synthetic methods in the field of synthetic organic chemistry, or using known variations thereof. Preferred methods include, but are not limited to, the methods described below.

A process for the preparation of a compound as described in the first or second aspect of the invention which comprises:

the method comprises the following steps: reacting the compound a with a compound e to obtain a compound b;

step two: removing the protecting group PG from the compound b through deprotection reaction to obtain a compound c;

step three: reacting the compound c with the compound f to obtain a compound d;

step four: the compound d is coupled with the compound g to obtain the compound shown in the formula I, namely the compound in the first or second aspect of the invention.

Wherein R is¹、R²、R³Is as defined in the first aspect of the invention; x is selected from halogen and-OTf, preferably, X is chlorine or bromine; PG is selected from C_1-6Alkyl oxycarbonyl, halogen substituted C_1-6Alkyl oxycarbonyl radical, C_2-6Alkenyl-alkoxycarbonyl, Cbz, benzyl and 9-fluorenylmethoxycarbonyl, preferably PG is selected from Boc; r¹⁷Selected from hydroxy and halogen, preferably hydroxy; r¹⁸Selected from boric acid, C_1-6Alkyl substituted boronic acids and pentaoxaboranes substituted with one or more methyl groups.

In some embodiments of the preparation method of the present invention, the reaction in step one is carried out in an organic solvent selected from DMSO, DMF, DMA, alcohols (e.g., methanol, ethanol, isopropanol, tert-butanol), ethers (e.g., anisole, diethyl ether, THF, 1, 4-dioxane, etc.), halogenated hydrocarbons (e.g., dichloromethane, chloroform, carbon tetrachloride) and acetonitrile, preferably DMSO, DMF.

In some embodiments of the preparation method of the present invention, the reaction in step one is carried out in the presence of a base selected from triethylamine, DIPEA, pyridine, NMM, DMAP, sodium acetate, potassium acetate, ammonium acetate, potassium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate, potassium hydroxide, sodium hydroxide, preferably the base is selected from potassium carbonate, cesium carbonate and pyridine.

In some embodiments of the preparation method of the present invention, the reaction in step one is carried out at a temperature of 0 ℃ to 200 ℃, preferably at a temperature of 50 ℃ to 150 ℃.

In some embodiments of the preparation method of the present invention, the deprotection reaction in step two is carried out in a solvent selected from the group consisting of water, DMF, DMA, N-methylpyrrolidone, alcohols (e.g., methanol, ethanol, isopropanol, etc.), ethers (e.g., diethyl ether, THF, 1, 4-dioxane, etc.), halogenated hydrocarbons (e.g., dichloromethane, chloroform, carbon tetrachloride, 1, 2-dichloroethane, etc.), acetonitrile and ethyl acetate, preferably dichloromethane, methanol, tetrahydrofuran.

In some embodiments of the production method of the present invention, the deprotection reaction in the second step is carried out in the presence of an acid selected from trifluoroacetic acid, hydrochloric acid, a hydrochloric acid/1, 4-dioxane solution, a hydrochloric acid/ethyl acetate solution, hydrobromic acid.

In some embodiments of the preparation method of the present invention, the deprotection reaction in step two is performed at a temperature of 0 ℃ to 50 ℃, preferably at a temperature of 0 ℃ to 25 ℃.

In some embodiments of the preparation method of the present invention, the condensation reaction in step three is carried out 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, 1, 4-dioxane, DMSO, and any combination thereof, preferably dichloromethane, DMF.

In some embodiments of the preparation method of the present invention, the condensation reaction in step three is carried out in the presence of a condensing agent selected from the group consisting of ethyl chloroformate, isopropyl chloroformate, HATU, HBTU, EEDQ, DEPC, DCC, DIC, EDC, BOP, PyAOP and PyBOP, preferably HATU, EDC.

In some embodiments of the preparation process of the present invention, the condensation reaction in step three is carried out in the presence of a base selected from triethylamine, DIPEA, NMM and DMAP, preferably DIPEA.

In some embodiments of the preparation method of the present invention, the condensation reaction in step three is performed at a temperature of 0 ℃ to 100 ℃, preferably at a temperature of 15 ℃ to 50 ℃.

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

In some embodiments of the preparation method of the present invention, the coupling reaction in step four 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, sodium tert-butoxide, potassium acetate, sodium acetate, and the inorganic base is selected from potassium carbonate, sodium bicarbonate, cesium carbonate, potassium phosphate, potassium dihydrogen phosphate; preferably, the inorganic base is selected from potassium carbonate, potassium phosphate, potassium acetate, sodium carbonate, sodium bicarbonate.

In some embodiments of the preparation method of the present invention, the coupling reaction of step four is carried out under the catalysis of a catalyst selected from palladium tetratriphenylphosphine, palladium acetate, Pd₂(dba)₃、 Pd(PPh₃)₂Cl₂、Pd(PPh₃)₂Cl₂Dichloromethane complex, Pd (dppf) Cl₂And Pd (Amphos) Cl₂Preferably palladium tetratriphenylphosphine, Pd (dppf) Cl₂、Pd(amphos)Cl₂。

In some embodiments of the preparation method of the present invention, the coupling reaction in step four is performed at a temperature of 0 ℃ to 200 ℃, preferably at a temperature of 50 ℃ to 150 ℃.

The present invention encompasses all possible crystalline forms or polymorphs of the compounds of the present invention, which may be single polymorphs or mixtures of polymorphs in any ratio.

The compounds of the present invention or pharmaceutically acceptable salts thereof may also form solvates, such as, for example, alcoholates and the like.

The compounds of the invention may also be prodrugs or forms which release the active ingredient or ingredients upon metabolic changes in the body. The selection and preparation of suitable prodrug derivatives is well known to those skilled in the art.

The compounds of the invention may also be in chemically protected form, the protecting group being protected on an active group (e.g. an amino group) of the compound, which protecting group is metabolised in vivo to release the active ingredient. The selection and preparation of suitable chemically protected forms is well known to those skilled in the art.

It will also be appreciated that certain compounds of the invention may be present in free form for use in therapy or, where appropriate, in the form of a pharmaceutically acceptable derivative thereof. According to the present invention, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, solvates, metabolites or prodrugs, which upon administration to a patient in need thereof are capable of providing, directly or indirectly, a compound of the present invention or a metabolite or residue thereof.

The compounds of the invention may be present in the form of hydrates or solvates, wherein the compounds of the invention comprise as structural element of the crystal lattice of the compound a polar solvent, such as in particular water, methanol or ethanol. The amount of polar solvent, particularly water, may be present in stoichiometric or non-stoichiometric proportions.

Also included within the scope of the present invention are metabolites of the compounds of the present invention, i.e., compounds that are formed in vivo upon administration of a drug.

In the present invention, unless otherwise specified, wherein:

the term "alkyl" is defined as a straight or branched chain saturated aliphatic hydrocarbon group. For example, the term "C_1-6Alkyl "refers to a straight or branched chain group having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, or n-hexyl), which is optionally substituted with one or more (such as 1 to 3) suitable substituents such as halogen (when the group is referred to as" haloalkyl ", e.g., -CF)₃、-C₂F₅、-CHF₂、-CH₂F、-CH₂CF₃、 -CH₂Cl or-CH₂CH₂CF₃Etc.).

The term "cycloalkyl" refers to a saturated monocyclic or bicyclic hydrocarbon group such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl. For example, as used hereinUse, the term "C_3-8Cycloalkyl "refers to a saturated monocyclic or bicyclic hydrocarbon group having 3 to 8 ring-forming carbon atoms (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl) which is optionally substituted with one or more (such as 1 to 3) suitable substituents, for example, methyl-substituted cyclopropyl.

The term "cycloalkenyl" refers to an unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring containing at least one carbon-carbon double bond. For example, as used herein, the term "C_3-8Cycloalkenyl "refers to an unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring having at least one carbon-carbon double bond having 3 to 8 carbon atoms, optionally substituted with one or more (such as 1 to 3) suitable substituents, for example methyl-substituted cyclobutenyl.

The term "alkoxy" means an "alkyl" or "cycloalkyl" group, as defined above, appended to the parent molecular moiety through an oxygen atom, e.g., C_1-6Alkoxy radical, C_1-3Alkoxy or C_3-8Cycloalkoxy is, for example, methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, cyclobutoxy, pentoxy, isopentoxy or n-hexoxy, or isomers thereof.

The term "heterocycloalkyl" refers to a saturated monocyclic or bicyclic group having 2,3, 4,5, 6, 7, 8 or 9 carbon atoms in the ring and one or more (e.g., one, two, three or four) selected from C (═ O), O, S, N, S (═ O), S (═ O)₂A heteroatom-containing group of (a); the heterocycloalkyl group may be attached to the rest of the molecule through any of the carbon atoms or a heteroatom (if present). For example, as used herein, the terms 3-8 membered heterocycloalkyl, 5-6 membered heterocycloalkyl, or 3-7 membered heterocycloalkyl are those having 3-8, 5-6, or 3-7 ring atoms in the ring and containing at least one heteroatom (which may be the same or different) therein (e.g., oxygen, nitrogen, or sulfur). Such as, but not limited to, oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, pyrrolidoneImidazolidinyl, pyrazolidinyl, pyrrolinyl, tetrahydropyranyl.

The term "heterocycloalkenyl" refers to an unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) group containing at least one carbon-carbon double bond, having 2,3, 4,5, 6, 7, 8 or 9 carbon atoms in the ring and one or more (e.g., one, two, three or four) selected from C (═ O), O, S, N, S (═ O), S (═ O)₂A heteroatom-containing group of (a); the heterocycloalkenyl group may be attached to the rest of the molecule through any of the carbon atoms or a heteroatom (if present). For example, as used herein, the term 3-8 membered heterocycloalkenyl is a compound having 3-8 ring atoms in the ring and at least one heteroatom (which may be the same or different) contained therein (the heteroatom being, for example, oxygen, nitrogen or sulfur). Such as dioxolyl (dioxolinyl).

The term "aryl" refers to an all-carbon monocyclic or fused ring polycyclic aromatic group having a conjugated pi-electron system. For example, as used herein, the term "C_6-10Aromatic ring "means an aromatic group containing 6 to 10 carbon atoms, such as phenyl or naphthyl. Aryl is optionally substituted with 1 or more (such as 1 to 3) suitable substituents, for example with alkyl (-CH)₃、-C₂H₅) Halogen (F, Cl, Br).

The term "heteroaryl" refers to a monocyclic, bicyclic or tricyclic aromatic ring system having 5, 6, 8, 9, 10, 11, 12, 13 or 14 ring atoms, for example having 5 to 10 ring atoms, in particular 1 or 2 or 3 or 4 or 5 or 6 or 9 or 10 carbon atoms, and which comprises at least one heteroatom which may be identical or different (said heteroatom being, for example, oxygen, nitrogen or sulfur), and which, in addition, in each case may be benzofused. For example, as used herein, the term "5-10 membered heteroaryl" means a monocyclic, bicyclic, or tricyclic aromatic ring system having 5-10 ring atoms and containing at least one heteroatom (which may be the same or different) therein (e.g., oxygen, nitrogen, or sulfur). In particular, heteroaryl is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolylThiadiazolyl, and the like, and benzo derivatives thereof; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and benzo derivatives thereof. Heteroaryl is optionally substituted with 1 or more (such as 1 to 3) suitable substituents, for example with alkyl (-CH)₃、-C₂H₅) Halogen (F, Cl, Br). As used herein, the term "halo" or "halogen" group is defined to include F, Cl, Br, or I.

The term "halogen" refers to fluorine, chlorine, bromine, iodine.

The term "C_1-6Alkylene "means a divalent radical remaining from a C1-6 alkyl group minus one hydrogen atom, wherein" C "is_1-6Alkyl groups "are as defined above. For example, C1-4 alkylene, C1-2 alkylene, C1 alkylene, C2 alkylene, C3 alkylene, C4 alkylene, C5 alkylene, C6 alkylene.

The term "mercapto" is also known as mercapto or thiol and is of the formula-SH.

The carbon and nitrogen atoms in the term "cyano" are connected by a triple bond, and have the formula-CN.

The term "nitro" refers to the residue of nitric acid molecule after removal of one hydroxyl group, and has the formula-NO₂。

The term "alkenyl" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and having one hydrogen atom replaced by a bond. The alkenyl group can be a straight or branched chain alkenyl group and contains from about 2 to about 15 carbon atoms. E.g. "C" herein_2-6Alkenyl "is alkenyl containing 2 to 6 carbon atoms. Non-limiting examples of alkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, octenyl and decenyl. The alkenyl group may be unsubstituted alkenyl or alkenyl substituted with one or more substituents which may be the same or different, each substituent being independently selected from halogen, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxy, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH₂-NH (alkyl), -N (alkyl)₂-NH (cycloalkyl), -O-C (O) -alkyl, -O-C (O) -aryl, -O-C (O) -cycloalkyl, -C (O) OH and-C (O) O-alkyl.

The term "alkynyl" refers to a hydrocarbyl group having one or more C ≡ C triple bonds. The alkynyl group has, but is not limited to, 2 to 18 carbon atoms, for example it has 2 to 10 carbon atoms, for example 2 to 6 carbon atoms. "lower alkynyl" herein refers to alkynyl groups of lesser carbon number, e.g. having 2 to 8 carbon atoms, e.g. 2 to 6 carbon atoms, e.g. 2 to 4 carbon atoms. Examples of alkynyl groups herein include, but are not limited to, ethynyl, 2-propynyl, 2-butynyl, and 1, 3-butadiynyl, and the like. When a numerical range occurs for alkynyl as defined herein, for example "C_2-6Alkynyl "refers to an alkynyl group that can be composed of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms, and alkynyl groups herein also encompass instances where no numerical range is specified.

The term "amino" refers to substituted or unsubstituted "-NH₂". For example, representative amino groups include-NH₂、-NHCH₃、-N(CH₃)₂、-NHC_1-3Alkyl, -N (C)_1-3Alkyl radical)₂And the like. Unless otherwise indicated, compounds of the present invention containing an amino moiety may include protected derivatives thereof. Suitable protecting groups for the amino moiety include acetyl, t-butyloxycarbonyl, benzyloxycarbonyl and the like.

The term "substituted" means that one or more (e.g., one, two, three, or four) hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the current circumstances is not exceeded and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

The term "optionally substituted with …" means optionally substituted or partially substituted with a specified group, radical or group of atoms or unsubstituted.

When a group is described as "optionally substituted with one or more substituents," the group may be (1) unsubstituted or (2) substituted. If a carbon on a group is described as optionally substituted with one or more substituents, one or more hydrogens on the carbon (to the extent of any hydrogen present) may be individually and/or collectively substituted or unsubstituted with an independently selected substituent. If a nitrogen on a group is described as optionally substituted with one or more substituents, then one or more hydrogens on the nitrogen (to the extent any hydrogen present) may each be substituted with an independently selected substituent or unsubstituted.

When a bond of a substituent is shown through a bond connecting two atoms in a ring, then such substituent may be bonded to any ring atom in the substitutable ring.

The term "stereoisomer" denotes an isomer formed as a result of at least one asymmetric center. In compounds having one or more (e.g., one, two, three, or four) asymmetric centers, they can give rise to racemates, racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Certain individual molecules may also exist as geometric isomers (cis/trans). Similarly, the compounds of the invention may exist as mixtures of two or more structurally different forms (commonly referred to as tautomers) in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, and the like. It is understood that the scope of this application encompasses all such isomers or mixtures thereof in any ratio (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

The term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other components comprising the formulation and/or the mammal being treated therewith.

The term "pharmaceutically acceptable salts" includes conventional salts with pharmaceutically acceptable inorganic or organic acids or bases.

For a review of suitable Salts, see Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present invention are known to those skilled in the art.

The term "pharmaceutical composition" includes products comprising a therapeutically effective amount of a compound of the invention, as well as any product which results, directly or indirectly, from combination of compounds of the invention.

The term "effective amount" refers to an amount sufficient to achieve a desired therapeutic effect, e.g., to achieve alleviation of symptoms associated with the disease being treated.

The term "treatment" refers to any administration of a compound of the invention, including:

(1) preventing disease development in animals that may be predisposed to disease but have not yet experienced or exhibited disease pathology or symptomology;

(2) inhibiting disease (i.e., arresting further development of pathology and/or symptomatology) in an animal experiencing or exhibiting disease pathology or symptomatology; or

(3) Ameliorating the disease (i.e., reversing the pathology and/or symptomatology) in an animal experiencing or exhibiting disease pathology or symptomatology.

"treatment" is intended to reduce or eliminate the disease state or condition for which it is directed. A subject is successfully "treated" if the subject receives a therapeutic amount of a compound, an optical isomer thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof according to the methods described herein, and the subject exhibits an observable and/or detectable decrease or improvement in one or more of the indications and symptoms of the subject. It is also understood that treatment of the disease state or condition described includes not only complete treatment, but also less than complete treatment, but achieves some biologically or medically relevant result.

The term "room temperature" means 20 ℃. + -. 5 ℃.

The term "about" when used to modify a value or range of values means that the value or range of values and the range of errors acceptable to those skilled in the art, for example, the range of errors is 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, etc.

The invention achieves the following beneficial effects:

the compound or the pharmaceutical composition has strong inhibition effect on the activity of deubiquitinase USP7, and can be used for preventing or treating diseases or symptoms related to USP7 regulation, particularly cancer.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available on the market.

In the conventional syntheses as well as in the examples and intermediate syntheses, the meanings of the abbreviations are shown in the following table.

The structures of the compounds described in the following examples were determined by nuclear magnetic resonance (¹H NMR) or Mass Spectrometry (MS), and the instruments and operating conditions for nuclear magnetic resonance, mass spectrometry are as follows:

(1) nuclear magnetic resonance (¹H NMR) was conducted using a Bruker 400MHz NMR spectrometer. The solvent is deuterium-substituted Methanol (Methanol-d)₄) Deuterated chloroform (CDCl)₃) Hexadeuterio dimethyl sulfoxide (DMSO-d)₆) Deuterated water (D)₂O). The internal standard substance is Tetramethylsilane (TMS).

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

s: unimodal (singlets); d: doublets (doublt); t: triplet (triplet); q: quartet (quartet); dd: doublet (doubledoublet); qd: quartet doublt; ddd: double doublet (double doublet); ddt: double double triplet (double double triple); dddd: double double double doublet (double double double doublet); m: multiplet (multiplex); br: broad peak (broad); δ: chemical shift; j: a coupling constant; hz: hertz.

All delta values are expressed in ppm.

(2) The Mass Spectrometer (MS) was an Agilent (ESI) mass spectrometer model Agilent 6120B.

The preparative thin-layer chromatography used in the following examples was carried out using a thin-layer chromatography silica gel plate (thickness 1mm, specification 200 × 200mm, binder sodium carboxymethylcellulose) produced by nicotiana xinuo chemical co., ltd) and detection apparatus ZF-20D dark box type ultraviolet analyzer with detection wavelength of 254nm and 365 nm.

The following examples used preparative hplc apparatus and operating conditions:

the method A comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

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

the method B comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

the method C comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

the method D comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

the method E comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

method F:

a chromatographic column: waters Xbridge Prep C18 OBD 5 μm 19X 150 mm;

method G:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

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

the method I comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

method J:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

method K:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

the method L comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

method M:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

the method N comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

method O:

a chromatographic column: waters Xbridge Prep C18 OBD 5 μm 19X 150 mm;

the method P comprises the following steps:

a chromatographic column: waters SunAire Prep C18 OBD 5 μm 19X 150 mm;

example one (R) -3- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) -7- (1H-indazole-6-) Yl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 1)

The method comprises the following steps: synthesis of (R) -7-bromo-3- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 1-2)

At room temperature, compound 1-1(0.13g, 0.56mmol), (R) -3-phenyl-1- (1-oxa-6-aza-spiro [2.5 ]]Oct-6-yl) butanone (synthetic method reference WO2018073602) (0.15g, 0.56mmol) was dissolved in DMF (4mL), potassium carbonate (0.16g, 1.13mmol) was added, and the reaction solution was heated to 80 ℃ for reaction for 8 h. Cooling the reaction solution to room temperature, separating and purifying by preparative high performance liquid chromatography (method D), and freezingDrying gave the title compound, 0.14 g. ESI-MS (M/z) 490.1,492.1[ M + H]⁺。

Step two: synthesis of (R) -3- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) -7- (1H-indazol-6-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 1)

Compound 1-2(40mg, 0.08mmol), (1H-indazol-6-yl) boronic acid (16mg, 0.10mmol), and potassium carbonate (31mg, 0.24mmol) were sequentially added to a reaction flask at room temperature, followed by addition of 1, 4-dioxane (2mL) and water (0.5mL), and finally palladium tetratriphenylphosphine (8.0mg, 0.008mmol), and nitrogen substitution three times. The mixture is heated to 80 ℃ under the protection of nitrogen and stirred for reaction for 4 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, purified by preparative high performance liquid chromatography (method D), and lyophilized to give the title compound, 32 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ13.18(s,1H)，8.51(s,1H),8.45(s, 1H),8.12-8.04(m,2H),7.84(d,J＝8.0Hz,1H),7.69(d,J＝8.0Hz,1H), 7.29-7.10(m,5H),4.91(d,J＝4.0Hz,1H),4.09-3.85(m,3H),3.72-3.61 (m,1H),3.26-3.12(m,2H),2.94-2.81(m,1H),2.66-2.53(m,2H),1.59-1.27 (m,4H),1.24-1.16(m,3H).ESI-MS(m/z):528.3[M+H]⁺。

example two (R) -3- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) -7- (4-hydroxybenzene Yl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 2)

Compound 1-2(30mg, 0.06mmol), 4-hydroxyphenylboronic acid (20mg, 0.09mmol), and potassium carbonate (25mg, 0.18mmol) were added sequentially to a reaction flask at room temperature, followed by 1, 4-dioxane (4mL) and water (0.8 mL). Finally, tetrakistriphenylphosphine palladium (7.1mg, 0.006mmol) was added and the nitrogen replaced three times. The mixture is heated to 80 ℃ under the protection of nitrogen and stirred for reaction for 4 hours. The reaction solution was cooled to room temperature, concentrated under reduced pressure, purified by preparative high performance liquid chromatography (method D), and lyophilized to give the title compound, 20 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ9.74(s,1H),8.34(s,1H),7.97(d,J ＝10.4Hz,1H),7.91-7.83(m,2H),7.28-7.22(m,4H),7.20-7.09(m,1H), 6.91-6.82(m,2H),4.89(d,J＝4.8Hz,1H),4.10-3.99(m,1H),3.99-3.80 (m,2H),3.70-3.60(m,1H),3.28-3.09(m,2H),2.87-2.81(m,1H),2.71-2.52 (m,2H),1.57-1.29(m,3H),1.25-1.16(m,4H).ESI-MS(m/z): 504.2[M+H]⁺。

EXAMPLE III (R) -7- (4- (aminomethyl) phenyl) -3- ((4-hydroxy-1- (3-phenylbutyryl) piperidine-4-carboxylic acid) Yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 3)

The method comprises the following steps: synthesis of tert-butyl (R) -4- (3- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl-4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) benzylcarbamate (Compound 3-1)

According to the procedure described in example two, using compounds 1-2(20mg, 0.04mmol) and 4- (N-t-butoxycarbonylaminomethyl) phenylboronic acid (15mg, 0.06mmol) as starting materials, instead of compounds 1-2(30mg, 0.06mmol) and 4-hydroxyphenylboronic acid, respectively, a reaction was carried out, and the reaction solution was concentrated under reduced pressure to give the crude product of the title compound, 21mg, which was used directly in the next reaction. ESI-MS (M/z) 617.3[ M + H]⁺。

Step two: synthesis of (R) -7- (4- (aminomethyl) phenyl) -3- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 3)

To the crude compound 3-1(21mg, 0.03mmol) was added a hydrochloric acid/1, 4-dioxane solution (5mL, hydrochloric acid concentration 4mol/L) and reacted at room temperature for 1 h. Preparative high performance liquid chromatography (method F) was performed by adding dilute hydrochloric acid (0.5mL, 1mol/L) to the preparation and freeze-drying to give the hydrochloride salt of the title compound, 10 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.53(s,1H),8.37(s,3H),8.17-8.02 (m,3H),7.58(d,J＝8.4Hz,2H),7.29-7.25(m,4H),7.17-7.12(m,1H), 4.10-3.86(m,5H),3.71-3.62(m,2H),3.26-3.12(m,2H),2.91-2.84(m,1H), 2.69-2.54(m,2H),1.58-1.23(m,4H),1.20(d,J＝6.2Hz,3H).ESI-MS (m/z):517.3[M+H]⁺。

example tetrakis 7- (1-amino-2, 3-dihydro-1H-inden-5-yl) -3- ((4-hydroxy-1- ((R) -3-phenylbutanoyl) Yl) piperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 4)

The method comprises the following steps: synthesis of tert-butyl (5- (3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-1-yl) carbamate (Compound 4-1)

According to the procedure described in example step two, using compound 1-2(40mg, 0.11 mmol) and tert-butyl 5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydro-1H-inden-1-yl) carbamate (46mg, 0.09mmol) as reaction raw materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, a reaction was carried out, the reaction solution was concentrated under reduced pressure, and separated and purified by silica gel column chromatography (eluent: dichloromethane/methanol-15/1 (v/v)), to give the title compound, 45 mg. ESI-MS (M/z) 643.4[ M + H]⁺。

Step two: synthesis of 7- (1-amino-2, 3-dihydro-1H-inden-5-yl) -3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 4)

According to the procedure described in the third example, using compound 4-1(30mg, 0.05mmol) as the starting material in place of compound 3-1, preparative high performance liquid chromatography was performed to separate and purify the reaction mixture (method C), and the resulting solution was concentrated under reduced pressure and lyophilized to give the title compound as a formate salt, 20 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.47(s,1H),8.43(s,1H),8.02(d,J ＝12.0Hz,1H),7.97(s,1H),7.92(d,J＝8.0Hz,1H),7.58(d,J＝8.0Hz, 1H),7.30-7.21(m,4H),7.19-7.11(m,1H),4.59(t,J＝6.8Hz,1H),4.10- 3.98(m,1H),3.94(d,J＝8.0Hz,1H),3.88(d,J＝14.4Hz,1H),3.82(d,J ＝14.0Hz,1H),3.30-3.01(m,3H),2.98-2.78(m,2H),2.72-2.55(m,2H), 2.49-2.42(m,1H),2.00-1.85(m,1H),1.46-1.10(m,4H),1.21(dd,J＝6.8, 4.0Hz,3H).ESI-MS(m/z):543.3[M+H]⁺。

example five 3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -7- (1- (methylaminomethane) Yl) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 5)

The method comprises the following steps: synthesis of N-methyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1H-inden-1-amine (Compound 5-2)

Dissolving the compound 5-1(2.00g, 6.59mmol) in methanol (80mL), adding an ethanol solution of methylamine (7mL, 33% wt), reacting at room temperature for 12h, adding formic acid (0.80mL, 21.08mmol) to adjust the pH value to be approximately equal to 5, adding sodium cyanoborohydride (1.24g, 19.76mmol), continuing to react at room temperature for 12h, concentrating the reaction solution under reduced pressure, filtering, concentrating the filtrate under reduced pressure to obtain a crude formate of the title compound, 2.08g, and directly using the crude formate in the next reaction. ESI-MS (M/z) 274.3[ M + H]⁺。

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

The crude formate salt of Compound 5-2 (2.03g, 6.36mmol) was dissolved in tetrahydrofuran (40 mL), and then saturated aqueous sodium bicarbonate (6.36mmol, 40mL) and di-tert-butyl dicarbonate (1.67g, 7.63mmol) were added in this order to react at room temperature for 6 h. The reaction was extracted with ethyl acetate (3X 100mL) and the organics combinedThe organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and then separated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 9/1(v/v)) to obtain the title compound (0.87 g). ESI-MS (M/z) 318.2[ M-56+ H]⁺。

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

According to the procedure described in example two, compound 5-3(57mg, 0.15mmol) was used as a starting material in place of 4-hydroxyphenylboronic acid for reaction, and the reaction solution was purified by preparative high performance liquid chromatography (method E), followed by lyophilization to give the title compound, 60 mg. ESI-MS (M/z) 657.4[ M + H]⁺。

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

According to the procedure described in example step two, using compound 5-4(45mg, 0.07mmol) as a starting material in place of compound 3-1, preparative high performance liquid chromatography separation and purification was performed (method C), and dilute hydrochloric acid (0.5mL, 1mol/L) was added to the preparation, followed by lyophilization to give the hydrochloride of the title compound, 40 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ9.45(br,1H),9.30(br,1H),8.52(s, 1H),8.09(d,J＝10.3Hz,1H),8.04(s,1H),7.98(d,J＝8.2Hz,1H),7.78 (d,J＝8.0Hz,1H),7.33-7.20(m,4H),7.20-7.09(m,1H),4.72(br,1H), 4.09-3.83(m,3H),3.72-3.59(m,1H),3.30-3.11(m,3H),3.02-2.83(m,2H), 2.68-2.53(m,5H),2.49-2.39(m,2H),2.26-2.16(m,1H),1.58-1.22(m,4H), 1.20(d,J＝5.9Hz,3H).ESI-MS(m/z):557.4[M+H]⁺。

EXAMPLE six 7- (2-amino-2, 3-dihydro-1H-inden-5-yl) -3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) Yl) piperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 6)

The method comprises the following steps: synthesis of tert-butyl (5- (3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) carbamate (Compound 6-1)

Following the procedure described in example step two, using tert-butyl 5- ((4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1H-inden-2-yl) carbamate (33mg, 0.09mmol) as the starting material for the reaction in place of (1H-indazol-6-yl) boronic acid, the reaction was concentrated under reduced pressure to give the crude title compound, 39mg, which was used directly in the next reaction. ESI-MS (M/z) 643.3[ M + H]⁺。

Step two: synthesis of 7- (2-amino-2, 3-dihydro-1H-inden-5-yl) -3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 6)

According to the procedure described in example step two, a reaction was carried out using the crude compound 6-1 (39mg, 0.06mmol) as a reaction starting material in place of compound 3-1, the reaction solution was purified by preparative liquid phase separation (method F), and dilute hydrochloric acid (0.5mL, 1mol/L) was added to the preparative solution, which was lyophilized to give the hydrochloride of the title compound, 21 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.46(s,1H),8.39(d,J＝5.6Hz, 3H),8.10(d,J＝10.4Hz,1H),7.95(s,1H),7.93-7.88(m,1H),7.38(d,J＝ 8.0Hz,1H),7.29-7.25(m,4H),7.20-7.11(m,1H),4.11-3.81(m,4H),3.73- 3.59(m,1H),3.33(td,J＝16.5,7.6Hz,2H),3.27-3.13(m,2H),3.06(td,J ＝16.3,5.3Hz,2H),2.91-2.84(m,1H),2.69-2.53(m,2H),1.58-1.22(m, 4H),1.20(d,J＝6.7Hz,3H).ESI-MS(m/z):543.3[M+H]⁺。

EXAMPLE seven 3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -7- (2- (methylamino) Yl) -2, 3-dihydro-1H-inden-5-yl) thiopheneAnd [3,4-d ]]Synthesis of pyrimidin-4 (3H) -one (Compound 7)

The method comprises the following steps: synthesis of tert-butyl 5-bromo-2, 3-dihydro-1H-indene-2-methylcarbamate (Compound 7-2)

Compound 7-1(700mg, 2.24mmol) was weighed out and dissolved in DMF (14mL), cooled to 0 deg.C, sodium hydride (135mg, 3.36mmol, 60% purity) was added and the reaction was maintained at that temperature for 1 h. Further, iodomethane (382mg, 2.69mmol) was added thereto, and the mixture was slowly warmed to room temperature to react for 16 hours. The reaction was quenched with saturated aqueous ammonium chloride (100mL), extracted with ethyl acetate (3 × 50mL), and the organic phases combined and washed with saturated brine (3 × 50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated to give the title compound, 600mg, which was used directly in the next reaction. ESI-MS (M/z) 270.0,272.0[ M-56+ H]⁺。

Step two: synthesis of tert-butyl (5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1H-inden-2-yl) -methylcarbamate (Compound 7-3)

Compound 7-2(200mg, 0.61mmol), pinacol ester diboronate (187mg, 0.74mmol) and potassium acetate (181mg, 1.84mmol) were weighed out, added to 1, 4-dioxane (5mL), replaced with nitrogen 3 times, and Pd (dppf) Cl was added₂(22mg, 0.03mmol) was replaced with nitrogen three times, and the temperature was raised to 80 ℃ under a nitrogen atmosphere to react for 16 hours. The reaction mixture was cooled to room temperature, and then 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 three: synthesis of tert-butyl (5- (3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 7-4)

According to the procedure described in example step two, compound 7-3(23mg, 0.06mmol) was used as a starting material in place of (1H-indazol-6-yl) boronic acid to carry out a reaction, and the reaction solution was passed throughPreparative thin layer chromatography separation and purification (developing solvent: dichloromethane/methanol-15/1 (v/v)) afforded the title compound, 10 mg. ESI-MS (M/z) 657.3[ M + H]⁺。

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

According to the procedure described in example step two, using compound 7-4(10mg, 0.02mmol) as a starting material in place of compound 3-1, a reaction was performed, the reaction solution was separated and purified by preparative high performance liquid chromatography (method C), and dilute hydrochloric acid (0.5mL, 1mol/L) was added to the preparation solution, followed by lyophilization to give the hydrochloride of the title compound, 8 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ9.47-9.21(m,2H),8.46(s,1H),8.09 (d,J＝10.4Hz,1H),7.95(s,1H),7.90(dd,J＝7.9,1.7Hz,1H),7.39(t,J ＝8.2Hz,1H),7.32-7.21(m,4H),7.19-7.12(m,1H),4.12-3.79(m,4H), 3.66(t,J＝11.7Hz,1H),3.34(td,J＝16.1,7.8Hz,2H),3.28-3.07(m,4H), 2.86(t,J＝11.2Hz,1H),2.59(m,4H),2.48-2.52(m,1H),1.57-1.22(m, 4H),1.20(dd,J＝7.0,1.7Hz,3H).ESI-MS(m/z):557.3[M+H]⁺。

example eight 7- (2- (dimethylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((4-hydroxy-1- ((R) -3-benzene) Butyryl) piperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 8)

The method comprises the following steps: synthesis of 5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -1H-inden-2 (3H) -one (Compound 8-2)

According to the procedure described in example seven, step two, using compound 8-1(1.00g, 4.74 mmol) as a starting material in place of compound 7-2, reaction was carried out, separation and purification by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1(v/v)) to give the title compound,1.10g。ESI-MS(m/z):259.1[M+H]⁺。

step two: synthesis of 2- ((dimethylamino) -2, 3-dihydro-1H-inden-5-yl) boronic acid (Compound 8-3)

To methanol (5mL) was added compound 8-2(100mg, 0.39mmol) and dimethylamine in methanol (5mL, 2M), and after 20h reaction at room temperature, sodium borohydride (43mg, 1.16mmol) was added and the reaction was continued for 2 h. The reaction solution was purified by preparative high performance liquid chromatography (method C), and lyophilized to give the title compound as formate salt, 10 mg. ESI-MS (M/z):206.2[ M + H]⁺。

Step three: synthesis of 7- (2- (dimethylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 8)

According to the procedure described in example step two, the reaction was carried out using the formate salt of compound 8-3 (10mg, 0.05mmol) as a starting material instead of (1H-indazol-6-yl) boronic acid, and the reaction mixture was purified by preparative high performance liquid chromatography (method C), followed by lyophilization to give the title compound as formate salt, 11 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.43(s,1H),8.21(s,1H),8.01(d,J ＝10.3Hz,1H),7.87(s,1H),7.80(d,J＝7.8Hz,1H),7.31-7.21(m,5H), 7.17-7.12(m,1H),4.92(s,2H),4.08-4.01(m,1H),4.00-3.89(m,1H),3.88- 3.83(m,1H),3.69-3.63(m,1H),3.27-3.14(m,2H),3.10-3.00(m,2H), 2.90-2.76(m,3H),2.66-2.56(m,2H),2.22(s,6H),1.56-1.26(m,4H),1.20 (d,J＝6.8Hz,3H).ESI-MS(m/z):571.3[M+H]⁺。

example nine (R) -3- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) -7- (isoindoline- 5-yl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 9)

The method comprises the following steps: synthesis of tert-butyl (R) -5- (3- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) isoindoline-2-carboxylate (Compound 9-1)

According to the procedure described in example step two, tert-butyl 5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) isoindoline-2-carboxylate (32mg, 0.09mmol) was used as a reaction raw material in place of (1H-indazol-6-yl) boronic acid, and the reaction mixture was separated and purified by preparative thin layer chromatography (developing solvent: dichloromethane/methanol-10/1 (v/v)) to give the title compound, 16 mg. ESI-MS (M/z) 629.3[ M + H]⁺。

Step two: synthesis of (R) -3- ((4-hydroxy-1- (3-phenylbutyryl) piperidin-4-yl) methyl) -7- (isoindolin-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 9)

According to the procedure described in example seven, step two, compound 9-1(16mg, 0.03mmol) was used as the starting material in place of compound 7-2 for reaction, the reaction mixture was purified by preparative high performance liquid chromatography (method C), and dilute hydrochloric acid (0.5mL, 1mol/L) was added to the preparation, followed by lyophilization to give the hydrochloride of the title compound, 12 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ9.72(t,J＝5.8Hz,2H),8.52(s,1H), 8.11(s,1H),8.07(d,J＝8Hz,2H),7.51(d,J＝8.1Hz,1H),7.25(p,J＝ 3.1Hz,4H),7.15(dt,J＝5.4,2.6Hz,1H),4.93(s,1H),4.58(t,J＝5.6Hz, 2H),4.54(t,J＝5.6Hz,2H),4.03(t,J＝8.4Hz,1H),3.98-3.83(m,2H), 3.65(s,1H),3.28-3.13(m,2H),2.87(t,J＝11.4Hz,1H),2.68-2.53(m,2H), 1.58-1.24(m,4H),1.20(dd,J＝6.9,1.6Hz,3H).ESI-MS(m/z): 529.3[M+H]⁺。

example ten 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidine-4- Yl) methyl) -7- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Pyrimidin-4 (3H) -one (compounds) 10) Synthesis of (2)

The method comprises the following steps: synthesis of tert-butyl 4- ((7-bromo-4-oxothieno [3,4-d ] pyrimidin-3 (4H) -yl) methyl) -4-hydroxypiperidine-1-carboxylate (Compound 10-1)

Following the procedure described in example step one, 1-oxa-6-aza-spiro [2.5 ] was utilized]Octylene-6-carboxylic acid tert-butyl ester (1.38g, 6.49mmol) as reaction starting material instead of compound (R) -3-phenyl-1- (1-oxa-6-aza-spiro [2.5 ]]Oct-6-yl) butanone was reacted, the reaction solution was poured into water (100mL), the solid precipitated, filtered off, and the filter cake was washed with petroleum ether (3X 10mL) to give the title compound, 1.45 g. ESI-MS (M/z) 388.0,390.0[ M-56+ H]⁺。

Step two: synthesis of 7-bromo-3- ((4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 10-2)

Compound 10-1(200mg, 0.45mmol) was dissolved in dichloromethane (2mL), and a hydrochloric acid/ethyl acetate solution (2mL, hydrochloric acid concentration 4mol/L) was added dropwise and reacted at room temperature for 2 hours. After concentration of the reaction solution under reduced pressure, the hydrochloride of the title compound (150 mg) was obtained. Used in the next reaction without purification. ESI-MS (M/z) 344.0,346.0[ M + H]⁺。

Step three: synthesis of 7-bromo-3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 10-3)

To a solution of 4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyric acid (102mg, 0.49mmol) in dichloromethane (5mL), HATU (178mg, 0.47mmol) and DIPEA (173mg, 1.34mmol) were sequentially added, and after a reaction time of 5 minutes, the hydrochloride (0.17g, 0.45mmol) of the compound 10-2 was added, and after a reaction time of 2 hours at room temperature, the reaction mixture was concentrated under reduced pressure, and subjected to preparative hplc separation and purification (method B), followed by lyophilization, to obtain the title compound of 127 mg. ESI-MS (M/z) 534.0,536.0[ M + H]⁺。

Step four: synthesis of tert-butyl (5- (3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl-4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-1-yl) (methyl) carbamate (Compound 10-4)

According to the embodimentIn the procedure described in the second step, compound 10-3(60mg, 0.07mmol) and compound 5-3(50mg, 0.13mmol) were reacted with each other as starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, followed by separation and purification by silica gel column chromatography (eluent: dichloromethane/methanol-19/1 (v/v)) to give the title compound, 80 mg. ESI-MS (M/z): 701.2[ M + H]⁺。

Step five: synthesis of 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 10)

According to the procedure described in example twelve step II, a reaction was carried out using compound 10-4(80mg, 0.05mmol) as a starting material in place of compound 10-1, the reaction mixture was separated and purified by preparative high performance liquid chromatography (method C), and dilute hydrochloric acid (0.5mL, 1mol/L) was added to the preparation, followed by lyophilization to give the hydrochloride of the title compound, 48 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ9.56(s,1H),9.41(s,1H),8.51(d,J ＝1.6Hz,1H),8.13(d,J＝3.9Hz,1H),8.04(d,J＝1.6Hz,1H),7.98(dd, J＝8.0,1.7Hz,1H),7.85-7.78(m,2H),6.26(td,J_H-F＝55.2Hz,J_H-H＝3.8 Hz,1H),6.05-5.95(m,1H),4.78-4.98(m,1H),4.08-3.88(m,3H),3.69(d, J＝13.6Hz,1H),3.35-3.15(m,3H),3.0-2.84(m,3H),2.57(t,J＝5.3Hz, 3H),2.50-2.42(m,2H),2.28-2.15(m,1H),1.65-1.30(m,4H).ESI-MS (m/z):601.2[M+H]⁺。

EXAMPLE eleven 3- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -7- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 11)

The method comprises the following steps: synthesis of tert-butyl 4-hydroxy-4- ((4-oxo-7- (1-oxo-2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-3 (4H) -yl) methyl) piperidine-1-carboxylate (Compound 11-1)

According to the procedure described in example step two, using compound 10-1(378mg, 0.85mmol) and 5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1H-inden-1-one (329mg, 1.28mmol) as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, reaction was carried out, and separation and purification by silica gel column chromatography (eluent: ethyl acetate/methanol 25/1(v/v)) were carried out to give the title compound, 420 mg. ESI-MS (M/z) 496.1[ M + H]⁺。

Step two: synthesis of 3- ((4-hydroxypiperidin-4-yl) methyl) -7- (1-oxo-2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 11-2)

According to the procedure described in example step two, compound 11-1(420mg, 0.85mmol) was used as a starting material in place of compound 3-1 for reaction, and the reaction mixture was concentrated under reduced pressure, ethyl acetate was added, stirred at room temperature, and filtered by suction to give the hydrochloride of the title compound as 360 mg. ESI-MS (M/z) 396.1[ M + H]⁺。

Step three: synthesis of 3- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -7- (1-oxo-2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 11-3)

4, 4-difluoro-3-phenylbutyric acid (15mg, 0.07mmol) was weighed out and dissolved in DMF (1mL), and HATU (28mg, 0.07mmol), compound 11-2 hydrochloride (30mg, 0.07mmol) and DIPEA (27mg, 0.21mmol) were added in this order to react at room temperature for 1 h. The reaction was purified by preparative high performance liquid chromatography (method D), and lyophilized to give the title compound, 34 mg. ESI-MS (M/z) 578.2[ M + H]⁺。

Step four: synthesis of 3- ((1- (4, 4-difluoro-3-phenylbutyryl) -4-hydroxypiperidin-4-yl) methyl) -7- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 11)

Compound 11-3(16mg, 0.03mmol) was dispersed in methanol (5mL) and dichloromethane (1mL), and a solution of methylamine in ethanol (26mg, 0.83mmol) was added and reacted at room temperature for 16 h. Sodium cyanoborohydride (5mg, 0.08mmol) was added, glacial acetic acid was added dropwise to adjust pH approximately 5, and the reaction was continued at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure, purified by preparative high performance liquid chromatography (method J), and lyophilized to give the title compound as the trifluoroacetate salt, 11 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.90-8.72(m,2H),8.56-8.49(m, 1H),8.11-7.96(m,3H),7.71-7.62(m,1H),7.35-7.25(m,5H),6.23(td,J_H- F＝56.6Hz,J_H-H＝3.9Hz,1H),4.92(s,1H),4.82-4.72(m,1H),4.06-3.92 (m,2H),3.87(s,1H),3.76-3.58(m,2H),3.25-3.08(m,2H),3.01-2.87(m, 3H),2.86-2.75(m,1H),2.69-2.62(m,3H),2.23-2.12(m,1H),1.61-1.14(m, 5H).ESI-MS(m/z):593.2[M+H]⁺。

example twelve (3- (1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidine-4- Yl) methyl) -7- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Pyrimidin-4 (3H) -one (compounds) 12) Synthesis of (2)

The method comprises the following steps: synthesis of tert-butyl (5- (3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 12-1)

According to the procedure described in example step two, compound 10-3(20mg, 0.04mmol) and compound 7-3(21mg, 0.06mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution was concentrated under reduced pressure and then subjected to preparative thin layer chromatography for separation and purification (eluent: dichloromethane/methanol-20/1 (v/v)) to give the title compound, 14 mg. ESI-MS (M/z):701.3[ M + H]⁺。

Step two: synthesis of (3- (1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 12)

According to the procedure described in example twelve step II, compound 12-1(14mg, 0.02mmol) was used as a starting material in place of compound 10-1 to carry out a reaction, the reaction mixture was concentrated under reduced pressure, and then separated and purified by preparative high performance liquid chromatography (method A), to which dilute hydrochloric acid (0.5mL, 1mol/L) was added, followed by lyophilization to give the hydrochloride of the title compound, 5 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ9.03(t,J＝7.0Hz,2H),8.46(d,J ＝1.5Hz,1H),8.05(d,J＝4.3Hz,1H),7.96(s,1H),7.91(dd,J＝7.8,1.7 Hz,1H),7.81(dt,J＝4.1,2.5Hz,1H),7.38(d,J＝8.0Hz,1H),6.26(td, J_H-_F＝56.0Hz,J_H-H＝4.0H z,1H),5.98(dd,J＝5.9,2.5Hz,1H),5.09-4.86 (m,2H),4.01(p,J＝6.3Hz,2H),3.93(d,J＝10.1Hz,2H),3.69(d,J＝ 13.6Hz,1H),3.39-3.31(m,2H),3.31-3.20(m,2H),3.13(td,J＝17.3,6.1 Hz,2H),2.98-2.83(m,2H),2.62(t,J＝5.4Hz,3H),1.66-1.50(m,1H), 1.50-1.29(m,3H).ESI-MS(m/z)：601.2[M+H]⁺。

EXAMPLE thirteen 3- ((4-hydroxy-1- (4,4, 4-trifluoro-3-phenylbutyryl) piperidin-4-yl) methyl) -7- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 13)

The method comprises the following steps: synthesis of 7-bromo-3- ((4-hydroxy-1- (4,4, 4-trifluoro-3-phenylbutyryl) piperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 13-1)

According to the procedure described in example step eleven, using hydrochloride of compound 10-2 (200mg, 0.53mmol) and 4,4, 4-trifluoro-3-phenylbutyric acid (138mg, 0.63mmol) as reaction raw materials in place of hydrochloride of compound 11-2 and 4, 4-difluoro-3-phenylbutyric acid, respectively, a reaction was carried out, the reaction solution was poured into water (20mL), extracted with ethyl acetate (3X 15mL), backwashed with a saturated aqueous sodium chloride solution, and dried over anhydrous sodium sulfateThe filtrate was concentrated under reduced pressure with suction to give a crude product, which was purified by silica gel column chromatography (eluent: dichloromethane/ethyl acetate-1/1 (v/v)) to give the title compound, 250 mg. ESI-MS (M/z) 544.0,546.0[ M + H]⁺。

Step two: synthesis of tert-butyl 5- ((3- ((4-hydroxy-1- (4,4, 4-trifluoro-3-phenylbutanyl) piperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) carbamate (Compound 13-2)

According to the procedure described in example step two, compound 13-1(30mg, 0.06mmol) and compound 7-3(31mg, 0.08mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution was concentrated under reduced pressure and then subjected to preparative thin layer chromatography for separation and purification (developing solvent: dichloromethane/ethyl acetate 3/1(v/v)) to give the title compound, 30 mg. ESI-MS (M/z) 711.3[ M + H]⁺。

Step three: synthesis of 3- ((4-hydroxy-1- (4,4, 4-trifluoro-3-phenylbutyryl) piperidin-4-yl) methyl) -7- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 13)

According to the procedure described in example twelve step II, compound 13-2(30mg, 0.04mmol) was used as a starting material in place of compound 10-1 to carry out a reaction, the reaction mixture was concentrated under reduced pressure, and then purified by preparative high performance liquid chromatography (method C), to which dilute hydrochloric acid (0.5mL, 1mol/L) was added and freeze-dried to give the hydrochloride of the title compound, 21 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ9.22(s,2H),8.46(s,1H),8.06(d,J ＝8.8Hz,1H),7.96(s,1H),7.90(d,J＝8.0Hz,1H),7.43-7.26(m,6H), 4.95(s,1H),4.17-4.06(m,1H),4.02-3.92(m,3H),3.87(d,J＝9.0,2.1Hz, 1H),3.82-3.76(m,1H),3.40-3.29(m,2H),3.28-3.21(m,1H),3.21-3.09(m, 3H),3.01-2.79(m,2H),2.61(t,J＝5.4Hz,3H),1.66-1.14(m,4H).ESI- MS(m/z):611.3[M+H]⁺。

example fourteen 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidine-4- Yl) methyl) -7- (4-fluorophenyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 14)

The method comprises the following steps: synthesis of tert-butyl 4- ((7- (4-fluorobenzene) -4-oxothieno [3,4-d ] pyrimidin-3 (4H) -yl) methyl) -4-hydroxypiperidine-1-carboxylate (Compound 14-1)

4-Fluorophenylboronic acid (619mg, 4.42mmol) and the compound 10-1(1.31 g, 2.95mmol) were dissolved in water (4mL) and 1, 4-dioxane (20mL) at room temperature, potassium carbonate (1.22g, 8.84mmol) and tetrakistriphenylphosphine palladium (341mg, 0.30mmol) were added in that order, nitrogen was replaced three times, and then the mixture was heated to 80 ℃ for 14 hours. The reaction solution was cooled to room temperature, and concentrated under reduced pressure to give a crude product, which was separated and purified by silica gel column chromatography (eluent: dichloromethane/ethyl acetate-2/1 (v/v)) to give the title compound (1.30 g). ESI-MS (M/z) 460.2[ M + H]⁺。

Step two: synthesis of 7- (4-fluorophenyl) -3- ((4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 14-2)

Compound 14-1(1.30g, 2.83mmol) was dissolved in hydrochloric acid/1, 4-dioxane solution (20mL, 4mol/L) at room temperature and reacted for 1h at room temperature. The reaction was filtered with suction and the filter cake was washed with ethyl acetate (2X 30mL) and the filter cake was collected and dried to give the hydrochloride salt of the title compound, 1.10 g. ESI-MS (M/z) 360.1[ M + H]⁺。

Step three: synthesis of 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (4-fluorophenyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 14)

The hydrochloride of compound 14-2 (20mg, 0.05mmol), 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyric acid (12mg, 0.06mmol) and HATU (20mg, 0.05mmol) were weighed out and dissolved in dichloromethane (3mL) at room temperature, DIPEA (20mg, 0.15mmol) was added, and the reaction was carried out at room temperature for 4 hours. The reaction mixture was concentrated under reduced pressure, purified by preparative high performance liquid chromatography (method D), concentrated under reduced pressure, and lyophilized to give the title compound, 19 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.49(d,J＝1.4Hz,1H),8.19-8.06 (m,2H),8.04(d,J＝4.6Hz,1H),7.81(dd,J＝4.3,2.4Hz,1H),7.41-7.27 (m,2H),6.12-6.39(td,J_H-F＝56.0Hz,J_H-H＝3.6Hz,1H),5.98(ddd,J＝ 6.0,2.6,1.5Hz,1H),4.99(d,J＝12.7Hz,1H),4.95(d,J＝2.7Hz,1H), 4.09-3.89(m,3H),3.68(d,J＝13.6Hz,1H),3.31-3.19(m,2H),2.90(ddd, J＝19.1,16.7,4.2Hz,2H),1.64-1.50(m,1H),1.50-1.31(m,3H).ESI-MS (m/z):550.1[M+H]⁺。

example fifteen (3- (1- (4, 4' -difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidine- 4-yl) methyl) - (4-fluorophenyl) thieno [3,4-d]Pyrimidin-4 (3H) -one-A (compound 14-A) and- (3- (1- (4, 4-) Difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) - (4-fluorophenyl) thieno [3,4- d]Synthesis of pyrimidin-4 (3H) -one-B (compound 14-B)

Compound 14(300mg, 0.55mmol) was isolated by chiral high performance liquid chromatography to give compound 14-A (135mg) and compound 14-B (112 mg). Chiral high performance liquid chromatography conditions for compound 14-A and compound 14-B were as follows: chiral column: CHIRALPAK AY-3(AY30CD-TJ004) (0.46cm I.D.. times.15 cm L); sample introduction amount: 2 mu L of the solution; mobile phase: EtOH 100%; flow rate: 0.5 mL/min; wavelength: UV 214 nm; temperature: 35 ℃; high performance liquid chromatography equipment: shimadzu LC-20AD CP-HPLC-05; wherein the retention time of compound 14-a is: 5.383min, the retention time of compound 14-B was 8.216 min.

The structure is characterized as follows:

compound 14-a:¹H NMR(400MHz,DMSO-d₆)δ8.49(d,J＝1.4Hz, 1H),8.15-8.07(m,2H),8.04(d,J＝4.4Hz,1H),7.81(dt,J＝4.8,2.8Hz, 1H),7.38-7.28(m,2H),6.25(td,J_H-F＝54.8Hz,J_H-H＝2.8Hz,1H),5.98 (dt,J＝6.0,2.0Hz,1H),5.01-4.95(m,2H),4.04-3.98(m,1H),3.95-3.90 (m,2H),3.68(d,J＝13.6Hz,1H),3.30-3.20(m,2H),2.98-2.84(m,2H), 1.67-1.50(m,1H),1.49-1.32(m,3H).ESI-MS(m/z):550.0[M+H]⁺。

compound 14-B:¹H NMR(400MHz,DMSO-d₆)δ8.49(d,J＝1.4Hz, 1H),8.16-8.07(m,2H),8.04(d,J＝4.4Hz,1H),7.81(dt,J＝4.8,2.8Hz, 1H),7.39-7.28(m,2H),6.26(td,J_H-F＝54.8Hz,J_H-H＝2.6Hz,1H),5.98 (dt,J＝6.0,2.0Hz,1H),5.03-4.95(m,2H),4.04-3.98(m,1H),3.95-3.90 (m,2H),3.68(d,J＝13.6Hz,1H),3.30-3.20(m,2H),2.96-2.85(m,2H), 1.67-1.51(m,1H),1.48-1.32(m,3H).ESI-MS(m/z):550.0[M+H]⁺。

example sixteen 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidine-4- Yl) methyl) -7-methylthioeno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 15)

The method comprises the following steps: synthesis of tert-butyl 4-hydroxy-4- ((7-methyl-4-oxothieno [3,4-d ] pyrimidin-3 (4H) -yl) methyl) piperidine-1-carboxylate (Compound 15-1)

According to the procedure described in example step two, compound 10-1(100mg, 0.23mmol) and trimethylcyclotriboroxane (113mg, 0.90mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction mixture was concentrated under reduced pressure and then separated and purified by silica gel column chromatography (eluent: dichloromethane/methanol 13/1(v/v)) to give the title compound, 72 mg. ESI-MS (M/z) 380.1[ M + H]⁺。

Step two: synthesis of 3- ((4-hydroxypiperidin-4-yl) methyl) -7-methylthioeno [3,4-d ] pyrimidin-4 (3H) -one (compound 15-2)

According to the operation described in example step two, a reaction was carried out using compound 15-1(72mg, 0.19 mmol) as a starting material in place of compound 10-1, and the reaction mixture was subjected to reduced pressureAfter concentration, the hydrochloride salt of the title compound was obtained, 60 mg. Used in the next reaction without purification. ESI-MS (M/z) 280.1[ M + H]⁺。

Step three: synthesis of 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7-methylthioeno [3,4-d ] pyrimidin-4 (3H) -one (Compound 15)

According to the procedure described in step three, eleventh example, using hydrochloride of compound 15-2 (20mg, 0.06mmol) and 4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyric acid (15mg, 0.07mmol) as reaction raw materials in place of hydrochloride of compound 11-2 and 4, 4-difluoro-3-phenylbutyric acid, respectively, the reaction solution was separated and purified by preparative high performance liquid chromatography (method C), and freeze-dried to give the title compound, 10 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.22(d,J＝1.8Hz,1H),7.93(d,J ＝4.7Hz,1H),7.80(q,J＝2.8Hz,1H),6.40-6.10(m,1H),5.98(dd,J＝5.9,2.5Hz,1H),5.10-4.95(m,1H),4.05-3.95(m,1H),3.92-3.85(m,2H), 3.71-3.62(m,1H),3.33-3.17(m,2H),2.97-2.80(m,2H),2.60(s,3H),1.63- 1.28(m,4H).ESI-MS(m/z):470.1[M+H]⁺。

example seventeen 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4- Yl) methyl) -7- (2-methyl-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Pyrimidin-4 (3H) -ones (Compound 16) Synthesis

The method comprises the following steps: synthesis of tert-butyl (5-bromo-2-methyl-2, 3-dihydro-1H-inden-2-yl) carbamate (Compound 16-2)

Compound 16-1(30mg, 0.13mmol) was suspended in dichloromethane (1.5 mL) at room temperature, DIPEA (34mg, 0.27mmol, 44uL) and di-tert-butyl dicarbonate (29mg, 0.13mmol, 30uL) were added, and the reaction was stirred at room temperature for 5 h. Concentrating under reduced pressure to obtain crude title compound, 40mg。ESI-MS(m/z):270.2,272.2[M-56+H]⁺。

Step two: synthesis of tert-butyl (5-bromo-2-methyl-2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 16-3)

According to the procedure described in example seven, step one, using compound 16-2(40mg, 0.12 mmol) as a reaction starting material in place of compound 7-1, a reaction was carried out, followed by preparative thin layer chromatography separation and purification (developing solvent: petroleum ether/ethyl acetate-5/1 (v/v)) to give the title compound, 29 mg. ESI-MS (M/z) 240.0,242.0[ M-100+ H]⁺。

Step three: synthesis of methyl (2-methyl-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1 h-inden-2-yl) carbamic acid tert-butyl ester (Compound 16-4)

According to the procedure described in example seven, step two, a reaction was carried out using compound 16-3(34mg, 0.10mmol) as a reaction starting material in place of compound 7-2, and separation and purification by preparative thin layer chromatography (developing solvent: petroleum ether/ethyl acetate-10/1 (v/v)) was carried out to give the title compound, 18 mg. ESI-MS (M/z) 288.2[ M-100+ H]⁺。

Step four: synthesis of tert-butyl (5- (3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2-methyl-2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 16-5)

According to the procedure described in example step two, using 10-3(20mg, 0.04mmol) and compound 16-4(18mg, 0.05mmol) and as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, reaction was carried out, followed by preparative thin layer chromatography separation and purification (developing solvent: dichloromethane/methanol 20/1(v/v)) to give the title compound, 26 mg. ESI-MS (M/z) 715.3[ M + H]⁺。

Step five: 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (2-methyl-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Synthesis of Compound 16)

Compound 16-5(27mg, 0.04mmol) was dissolved in ethyl acetate (2mL) at room temperature, a solution of hydrochloric acid in ethyl acetate (2mL, 4mol/L) was added, and the reaction was stirred at 20 ℃ for 3 h. Concentrating under reduced pressure, adding ethyl acetate to precipitate solid, vacuum filtering, washing filter cake with methyl tert-butyl ether, and drying. After dissolution in water, freeze-dried to give the hydrochloride salt of the title compound, 23 mg.

The structure is characterized as follows:

¹H NMR(400MHz,D₂O)δ8.14(d,J＝4.8Hz,1H),7.81(d,J＝18.8 Hz,1H),7.60-7.44(m,3H),7.28(d,J＝3.6Hz,1H),6.10(td,J_H-F＝54.4 Hz,J_H-H＝3.2Hz,1H),5.84(ddd,J＝44.4,5.6,2.8Hz,1H),5.00-4.80(m, 1H),4.11-3.65(m,4H),3.44-3.18(m,4H),3.17-3.06(m,2H),3.04-2.80(m, 2H),2.64(s,3H),1.73-1.54(m,1H),1.50(s,3H),1.48-1.18(m,3H).ESI- MS(m/z):615.2[M+H]⁺。

EXAMPLE eighteen 3- ((1- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutanoyl) -4-hydroxypiperidin-4-yl) Methyl) -7- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Pyrimidin-4 (3H) -one (compound 17) Synthesis of (2)

The method comprises the following steps: synthesis of 3- ((1- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (1-oxo-2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 17-1)

According to the procedure described in example step eleven, a reaction was carried out using 3- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutyric acid (30mg, 0.69mmol) as a starting material in place of 4, 4-difluoro-3-phenylbutyric acid, water (5mL) was added to the reaction solution, ethyl acetate (3 × 5mL) was extracted, the organic phase was washed with saturated brine (5mL), dried over anhydrous sodium sulfate, and concentrated to give the crude product of the title compound, 41 mg. ESI-MS (M/z) 601.7,603.7[ M + H]⁺。

Step two: synthesis of 3- ((1- (3-chloro-1H-pyrazol-1-yl) -4, 4-difluorobutanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 17)

The crude compound 17-1 (41mg) was dissolved in methanol (10mL) at room temperature, methylamine/ethanol solution (4mL, 33% wt) was added and stirred at room temperature for 18 h. After the reaction solution was concentrated, the concentrate was dissolved in methanol (10mL), and sodium cyanoborohydride (21mg, 0.034 mmol) and acetic acid (20mg, 0.034 mmol) were added thereto, followed by stirring at room temperature for 2 hours. The reaction was concentrated and purified by preparative high performance liquid chromatography (method I), freeze-dried to give the title compound as formate salt, 10 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.45(d,J=2.4Hz,1H)，8.03(d, J=4.4Hz,1H),7.97-7.90(m,2H),7.82(d,J=8.0Hz,1H),7.46(d,J= 8.0Hz,1H),6.34(d,J=2.4Hz,1H),6.29(t,J_H-F=56.0Hz,1H),5.10(br, 1H),4.96(br,1H),4.22(t,J=6.4Hz,1H),4.06-3.97(m,1H),3.96-3.89 (m,2H),3.72-3.65(m,1H),3.07-2.79(m,6H),2.43-2.33(m,4H),1.92- 1.82(m,1H),1.66-1.31(m,5H)；ESI-MS(m/z):616.7,618.7[M+H]⁺。

example nineteen 3- ((1- (3-chloro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 18)

The method comprises the following steps: synthesis of 3- ((1- (3-chloro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (1-oxo-2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 18-1)

According to the procedure described in example step eleven, a reaction was carried out using 3- (3-chloro-1H-pyrazol-1-yl) butyric acid (30mg, 0.69mmol) as a starting material in place of 4, 4-difluoro-3-phenylbutyric acid, the reaction mixture was added with water (5mL), extracted with ethyl acetate (3X 5mL), and the organic phase was washed with saturated brine (5mL), dried over anhydrous sodium sulfate, and concentrated to give the crude product of the title compound as 39 mg. ESI-MS (M/z) 565.8,567.8[ M + H]⁺。

Step two: synthesis of 3- ((1- (3-chloro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 18)

According to the procedure described in example eighteen step two, a reaction was carried out using the crude compound 18-1 (39 mg) as a starting material in place of the crude compound 17-1, the reaction mixture was subjected to preparative high performance liquid chromatography for separation and purification (method I), and the prepared solution was concentrated under reduced pressure and lyophilized to give the title compound as the formate salt, 14 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.45(d,J＝2.4Hz,1H),8.34(s,1 H),8.03(d,J＝5.2Hz,1H),7.92(s,1H),7.87(d,J＝8.0Hz,1H),7.80(dd, J＝3.6,2.4Hz,1H),7.46(d,J＝8.0Hz,1H),6.23(t,J＝2.0Hz,1H),4.95 (brs,1H),4.79-4.68(m,1H),4.21(t,J＝6.8Hz,1H),4.08-3.99(m,1H), 3.96-3.88(m,2H),3.67-3.61(m,1H),3.30-3.11(m,1H),3.03-2.91(m,2H), 2.90-2.78(m,2H),2.77-2.65(m,1H),3.39(s,3H),2.42-2.33(m,1H),1.93- 1.82(m,1H),1.47-1.38(m,7H)；ESI-MS(m/z):580.8,582.8[M+H]⁺。

example twenty 3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -7- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 19)

The method comprises the following steps: synthesis of 7-bromo-3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 19-1)

According to the procedure described in example thirteen steps, a reaction was carried out using 3-cyclopropyl-3-phenylpropionic acid (150 mg, 0.79mmol) as a starting material in place of 4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyric acid compound, and separation and purification by silica gel column chromatography (eluent: ethyl acetate ═ 100%) To give the title compound, 270 mg. ESI-MS (M/z) 516.0,518.0[ M + H]⁺。

Step two: synthesis of tert-butyl 5- ((3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-1-yl) (methyl) carbamate (Compound 19-2)

According to the procedure described in example step two, compound 19-1(30mg, 0.06mmol) and compound 5-3(26mg, 0.07mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution was concentrated under reduced pressure to prepare a title compound, 35mg, which was isolated and purified by thin layer chromatography (developing solvent: ethyl acetate ═ 100%). ESI-MS (M/z) 683.3[ M + H]⁺。

Step three: synthesis of 3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -7- (1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 19)

At room temperature, compound 19-2(35mg, 0.05mmol) was weighed into a reaction flask, ethyl acetate hydrochloride solution (2mL, 4mol/L) was added, and the reaction was stirred at room temperature for 1 h. The solvent was removed by concentration under reduced pressure and purified by preparative high performance liquid chromatography (method C), and the preparation was added with dilute hydrochloric acid (0.5mL, 1mol/L) and lyophilized to give the hydrochloride of the title compound, 17 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)：δ8.81-8.17(m,2H),7.94-7.67(m, 3H),7.38-7.15(m,5H),4.99-4.93(m,1H),4.87-4.78(m,1H),4.27-3.84(m, 3H),3.74(t,J＝14.8Hz,1H),3.29-3.23(m,1H),3.20-2.91(m,3H),2.88- 2.71(m,4H),2.70-2.60(m,1H),2.37-2.22(m,2H),1.73-1.52(m,1H), 1.51-1.41(m,1H),1.40-1.31(m,1H),1.31-1.18(m,1H),1.18-0.70(m,1H), 0.69-0.56(m,1H),0.48-0.38(m,1H),0.37-0.26(m,1H),0.16-0.07(m,1H). ESI-MS(m/z):583.4[M+H]⁺。

example twenty one 3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -7- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Pyrimidine-4 (3)H) Synthesis of (Compound 20) Ketone

The method comprises the following steps: synthesis of tert-butyl 5- (3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 20-1)

According to the procedure described in example step two, compound 19-1(30mg, 0.06mmol) and compound 7-3(26mg, 0.07mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution was concentrated under reduced pressure to prepare a title compound, 35mg, which was isolated and purified by thin layer chromatography (developing solvent: ethyl acetate ═ 100%). ESI-MS (M/z) 683.3[ M + H]⁺。

Step two: synthesis of 3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -7- (2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 20)

According to the procedure described in example twenty, step three, compound 20-1(35mg, 0.05mmol) was used as a starting material in place of compound 19-2 to carry out a reaction, the reaction mixture was concentrated under reduced pressure and purified by preparative high performance liquid chromatography (method C), and the preparation was lyophilized with dilute hydrochloric acid (0.5mL, 1mol/L) to give the hydrochloride of the title compound, 21 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)：δ8.73-8.56(m,2H),7.73(s,1H), 7.63(d,J＝8.4Hz,1H),7.48(d,J＝7.8Hz,1H),7.41-7.13(m,5H),4.31- 4.02(m,3H),4.02-3.89(m,1H),3.75(t,J＝14.0Hz,1H),3.57-3.46(m, 2H),3.37-3.31(m,1H),3.28-3.18(m,2H),3.08-2.89(m,2H),2.88-2.69(m, 4H),2.33-2.23(m,1H),1.73-1.53(m,1H),1.50-1.41(m,1H),1.37-1.30(m, 1H),1.28-1.17(m,1H),1.16-0.70(m,1H),0.67-0.59(m,1H),0.47-0.38(m, 1H),0.36-0.27(m,1H),0.15-0.07(m,1H).ESI-MS(m/z):583.3[M+H]⁺。

example twenty two 7- (4-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-) 3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Pyrimidin-4 (3H) -ones Synthesis of (Compound 21)

The method comprises the following steps: synthesis of 7- (4-chloro-1-oxo-2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 21-1)

At room temperature, 5-bromo-4-chloro-2, 3-dihydro-1H-inden-1-one (60mg, 0.24mmol), pinacol diboron (68mg, 0.27mmol) and [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride (18mg, 0.02mmol) and potassium acetate (48mg, 0.49mmol) were placed in 1, 4-dioxane (5mL), the reaction system was replaced with nitrogen three times, and the temperature was raised to 80 ℃ for reaction for 16 h. The reaction mixture was cooled to room temperature, and then compound 10-3(40mg, 0.07mmol), potassium carbonate (31mg, 0.22mmol), tetrakis (triphenylphosphine) palladium (9mg, 0.007mmol) and water (1mL) were added thereto, and the mixture was replaced with nitrogen three times, and the temperature was raised to 100 ℃ to react for 4 hours. Cooled to room temperature, concentrated under reduced pressure and purified by preparative thin layer chromatography (developing solvent: ethyl acetate ═ 100%) to give the title compound, 25 mg. ESI-MS (M/z) 620.1,622.1[ M + H]⁺。

Step two: synthesis of 7- (4-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 21)

Compound 21-1(25mg, 0.04mmol) was weighed into a reaction flask at room temperature, methylamine/ethanol solution (2mL, 33% wt) was added, and the reaction was stirred at room temperature for 5 h. The solvent was removed by concentration under reduced pressure, dissolved in methanol (2mL), and stirred at room temperature for 2h with sodium borohydride (3mg, 0.08 mmol). The reaction solution was purified by preparative high performance liquid chromatography (method J), and lyophilized to give the title compound, trifluoroacetate salt, 8 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)：δ8.54(d,J＝1.4Hz,1H),7.99 (d,J＝7.8Hz,1H),7.67-7.51(m,3H),6.12(td,J_H-F＝55.4Hz,J_H-H＝4.4 Hz,1H),5.91-5.84(m,1H),4.99-4.90(m,2H),4.19-4.09(m,1H),4.05(d, J＝3.4Hz,1H),4.00(d,J＝1.8Hz,1H),3.84-3.75(m,1H),3.49-3.36(m, 2H),3.29-3.22(m,1H),3.18-3.10(m,1H),3.10-3.00(m,1H),2.96-2.83(m, 1H),2.79(s,3H),2.73-2.64(m,1H),2.38-2.29(m,1H),1.77-1.46(m,4H). ESI-MS(m/z):635.2,637.2[M+H]⁺。

example twenty three 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidine- 4-yl) methyl) -7- (4-fluoro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Pyrimidin-4 (3H) -ones Synthesis of (Compound 22)

The method comprises the following steps: synthesis of 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (4-fluoro-1-oxo-2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 22-1)

According to the procedure described in example twenty-two-step one, a reaction was carried out using 5-bromo-4-fluoro-2, 3-dihydro-1H-inden-1-one (100mg, 0.44mmol) as a reaction starting material in place of 5-bromo-4-chloro-2, 3-dihydro-1H-inden-1-one, and the reaction mixture was concentrated under reduced pressure and separated and purified by preparative thin layer chromatography (developing solvent: ethyl acetate ═ 100%) to give the title compound, 35 mg. ESI-MS (M/z): 604.2[ M + H]⁺。

Step two: synthesis of 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (4-fluoro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 22)

According to the procedure described in example twenty-second step, compound 22-1(35mg, 0.06mmol) was used as a starting material in place of compound 21-1 for reaction, and the reaction mixture was subjected to preparative high performance liquid chromatography for separation and purification (method C), followed by lyophilization to give the title compound as formate salt, 28 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)：δ8.54(s,1H),8.49(d,J＝1.6Hz, 1H),8.13(t,J＝7.2Hz,1H),8.03(d,J＝7.8Hz,1H),7.59(dt,J＝5.2,2.4 Hz,1H),7.43(d,J＝7.8Hz,1H),6.12(td,J_H-F＝55.4Hz,J_H-H＝4.4Hz, 1H),5.91-5.84(m,1H),5.01-4.93(m,1H),4.70(dd,J＝7.8,4.4Hz,1H), 4.19-4.09(m,1H),4.05(d,J＝3.6Hz,1H),4.00(s,1H),3.84-3.75(m,1H), 3.48-3.36(m,2H),3.26-3.17(m,1H),3.12-3.02(m,2H),2.96-2.83(m,1H), 2.69(s,3H),2.67-2.58(m,1H),2.30-2.21(m,1H),1.76-1.47(m,4H).ESI- MS(m/z):619.2[M+H]⁺。

example twenty-four 7- (4-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl- 3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 23)

The method comprises the following steps: synthesis of tert-butyl (5-bromo-4-chloro-2, 3-dihydro-1H-inden-1-yl) (methyl) carbamate (Compound 23-2)

Compound 23-1(100mg, 0.41mmol) was dissolved in methanol (2mL) at room temperature, methylamine/ethanol solution (2mL, 33% wt) was added and reacted at room temperature for 16h, the reaction solution was dried, the residue was dissolved in methanol (2mL), sodium borohydride (30mg, 0.81mmol) was added and reacted at room temperature for 1h, followed by di-tert-butyl dicarbonate (444mg, 2.04mmol) and reaction continued at room temperature for 1 h. The solvent was drained under reduced pressure and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 10/1(v/v)) to give the title compound, 130 mg. ESI-MS (M/z):304.3,306.3[ M-56+ H]⁺。

Step two: synthesis of tert-butyl 4-chloro-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -2, 3-dihydro-1H-inden-1-yl) (methyl) carbamate (Compound 23-3)

According to the procedure described in example seven, step two, a reaction was carried out using compound 23-2(130mg, 0.36mmol) as a starting material in place of compound 7-2, and the reaction mixture was concentrated and purified by preparative thin layer chromatography (developing solvent: petroleum ether/ethyl acetate-15/1 (v/v)) to give the title compound, 100 mg. ESI-MS (M/z) 352.2,354.2[ M-56+ H]⁺。

Step three: synthesis of tert-butyl 4-chloro-5- (3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-1-yl) (methyl) carbamate (Compound 23-4)

According to the procedure described in example step two, using compound 19-1(20mg, 0.04mmol) and compound 23-4(17mg, 0.04mmol) as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, the reaction solution was concentrated under reduced pressure and subjected to preparative thin layer chromatography purification (developing solvent: ethyl acetate/methanol 60/1(v/v)) to give the title compound, 15 mg. ESI-MS (M/z) 717.3,719.3[ M + H]⁺。

Step four: synthesis of 7- (4-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 23)

According to the procedure described in example twenty, step three, compound 23-4(15mg, 0.02mmol) was used as the starting material in place of compound 19-2 for reaction, and the reaction mixture was concentrated under reduced pressure, purified by preparative high performance liquid chromatography (method K), and lyophilized to give the title compound as formate salt, 8 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)δ8.48(d,J＝7.0Hz,1H),7.92(s, 0.4H),7.86(s,0.6H),7.55-7.39(m,2H),7.32-7.08(m,5H),4.65-4.54(m, 1H),4.22-3.59(m,4H),3.30(s,1H),3.20-2.76(m,5H),2.65-2.51(m,1H), 2.58(s,3H),2.30-2.08(m,2H),1.64-1.04(m,5H),0.63-0.49(m,1H),0.43- 0.29(m,2H),0.31-0.19(m,1H),0.14-0.02(m,1H).ESI-MS(m/z):617.3, 619.3[M+H]⁺。

example twenty-five 7- (6-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-) 3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Pyrimidin-4 (3H) -ones (Compound 24) Synthesis

The method comprises the following steps: synthesis of 6-chloro-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1H-inden-1-one (Compound 24-2)

At room temperature, compound 24-1(100mg, 0.41mmol), pinacol diboron (207mg, 0.25mmol) and [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride (15mg, 0.02mmol) and potassium acetate (120mg, 1.22mmol) were dissolved in 1, 4-dioxane (2mL), replaced with nitrogen three times, and the reaction was warmed to 80 ℃ and stirred for 16 h. The reaction was filtered and concentrated to give the crude title compound, 119 mg. ESI-MS (M/z) 293.1,295.1[ M + H]⁺。

Step two: synthesis of 7- (6-chloro-1-oxo-2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 24-3)

According to the procedure described in example step two, compound 10-3(35mg, 0.07mmol) and the crude product of compound 24-2 (23mg) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution was filtered and concentrated to give the crude product of the title compound, 41 mg. ESI-MS (M/z) 620.0,622.0[ M + H]⁺。

Step three: synthesis of 7- (6-chloro-1-oxo-2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 24)

According to the procedure described in example twenty-second step, a reaction was carried out using the crude product (41mg) of compound 24-3 as a starting material in place of compound 21-1, and the reaction mixture was subjected to preparative high performance liquid chromatography for purification (method J), followed by lyophilization to give the title compound, trifluoroacetate salt, 4 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)δ8.54(s,1H),7.99(d,J＝8.0Hz, 1H),7.75(s,1H),7.62(s,1H),7.61-7.56(m,1H),6.11(td,J_H-F＝55.2Hz, J_H-H＝4.4Hz,1H),5.91-5.84(m,1H),5.00-4.90(m,1H),4.85-4.79(m,1H), 4.19-4.09(m,1H),4.07-3.98(m,2H),3.85-3.75(m,1H),3.45-3.35(m,2H), 3.23-3.15(m,1H),3.09-3.05(m,1H),2.96-2.84(m,1H),2.79(s,3H),2.71- 2.63(m,1H),2.34-2.26(m,1H),1.79-1.45(m,4H).ESI-MS(m/z):635.2, 637.2[M+H]⁺。

example ceryl-7- (6-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-) 3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 25)

The method comprises the following steps: synthesis of 7- (6-chloro-1-oxo-2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 25-1)

According to the procedure described in example step two, compound 19-1(30mg, 0.06mmol) and compound 24-2(13mg, 0.06mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution was concentrated under reduced pressure and subjected to preparative thin layer chromatography purification (developing solvent: ethyl acetate/methanol 30/1(v/v)) to give the title compound, 20 mg. ESI-MS (M/z) 602.2,604.2[ M + H]⁺。

Step two: synthesis of 7- (6-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 25)

According to the procedure described in example twenty-second step, compound 25-1(20mg, 0.03mmol) was used as a starting material in place of compound 21-1 for reaction, and the reaction mixture was separated and purified by preparative high performance liquid chromatography (method C), followed by lyophilization to give the title compound, 8 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)：δ8.50(d,J＝7.2Hz,1H),7.92 (d,J＝24.4Hz,1H),7.62(s,1H),7.48(s,1H),7.34-7.24(m,4H),7.22-7.15 (m,1H),4.38(t,J＝6.4Hz,1H),4.25-4.11(m,1H),4.00(q,J＝14.0Hz, 1H),3.92-3.76(m,1H),3.75-3.65(m,1H),3.29-3.14(m,1H),3.12-3.03(m, 1H),3.02-2.84(m,3H),2.83-2.64(m,1H),2.53(s,3H),2.52-2.41(m,1H), 2.32-2.23(m,1H),2.11-1.99(m,1H),1.63-1.26(m,4H),1.24-1.11(m,1H), 0.66-0.55(m,1H),0.46-0.36(m,1H),0.35-0.25(m,1H),0.14-0.04(m,1H). ESI-MS(m/z):617.3,619.3[M+H]⁺。

example twenty-seven 7- (7-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-) 3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Pyrimidin-4 (3H) -ones Synthesis of (Compound 26)

The method comprises the following steps: synthesis of 7-chloro-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1H-inden-1-one (Compound 26-2)

According to the procedure described in the twenty-five step one of example, using compound 26-1(20mg, 0.08mmol) as a starting material in place of compound 24-1, a reaction was carried out, and the reaction solution was filtered and concentrated to give the crude title compound (54 mg). ESI-MS (M/z) 293.1,295.1[ M + H]⁺。

Step two: synthesis of 7- (7-chloro-1-oxo-2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 26-3)

According to the procedure described in example step two, compound 10-3(44mg, 0.08mmol) and crude 26-2 (54mg) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution was filtered and concentrated to give crude title compound, 51 mg. ESI-MS (M/z) 620.0,622.0[ M + H]⁺。

Step three: synthesis of 7- (7-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 26)

According to the procedure described in example twenty-second step, a reaction was carried out using the crude product (51mg) of compound 24-3 as a reaction starting material in place of compound 21-1, and the reaction solution was subjected to preparative high performance liquid chromatography purification (method J), followed by lyophilization to give the title compound, trifluoroacetate salt, 6 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)δ8.44(d,J＝1.2Hz,1H),8.20(s, 1H),8.07(d,J＝8.0Hz,1H),7.96(s,1H),7.61-7.56(m,1H),6.11(td,J_H-F＝55.6Hz,J_H-H＝4.4Hz,1H),5.87(td,J＝6.0Hz,2.4Hz,1H),4.97- 4.93(m,2H),4.19-4.09(m,1H),4.05(s,1H),4.01(s,1H),3.85-3.75(m, 1H),3.45-3.35(m,2H),3.18-3.10(m,1H),3.09-3.02(m,1H),2.96-2.82 (m,1H),2.77(s,3H),2.64-2.56(m,1H),2.43-2.35(m,1H),1.79-1.44(m, 4H),1.40-1.34(m,1H)；ESI-MS(m/z):635.2,637.2[M+H]⁺。

example twenty-eight 7- (7-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl- 3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 27)

The method comprises the following steps: synthesis of 7- (7-chloro-1-oxo-2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 27-1)

According to the procedure described in example step two, compound 19-1(40mg, 0.08mmol) and compound 26-2(18mg, 0.09mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution was concentrated under reduced pressure and subjected to preparative thin layer chromatography purification (developing solvent: ethyl acetate/methanol 30/1(v/v)) to give the title compound, 20 mg. ESI-MS (M/z):602.2,604.2[ M + H [)]⁺。

Step two: synthesis of 7- (7-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 27)

Compound 27-1(20mg, 0.03mmol) was weighed into methanol (20mL) and dichloromethane (10mL) at room temperature, methylamine/ethanol solution (2mL, 33% wt) was added, and the reaction was stirred at room temperature for 20 h. The solvent was removed by concentration under reduced pressure, and the mixture was dissolved in methanol (20mL) and methylene chloride (10mL), and sodium borohydride (2mg, 0.07mmol) was added thereto, followed by stirring at room temperature for 1 hour. The reaction was purified by preparative high performance liquid chromatography (method J) using dilute hydrochloric acid (0.5mL, 1mol/L) freeze-dried to give the title compound as the hydrochloride salt, 11 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)：δ8.53(d,J＝8.8Hz,1H),8.27(d, J＝17.6Hz,1H),8.09(s,1H),7.89(s,1H),7.41-7.10(m,5H),4.97(dd,J ＝7.6,1.8Hz,1H),4.26-4.13(m,1H),4.13-4.01(m,1H),3.97-3.82(m,1H), 3.79-3.69(m,1H),3.43-3.31(m,2H),3.20-3.11(m,1H),3.07-2.89(m,2H), 2.88-2.69(m,4H),2.67-2.56(m,1H),2.47-2.39(m,1H),2.32-2.23(m,1H), 1.69-1.30(m,4H),1.25-1.13(m,1H),0.66-0.56(m,1H),0.48-0.36(m,1H), 0.36-0.25(m,1H),0.16-0.05(m,1H).ESI-MS(m/z):617.2,619.2[M+H]⁺。

example twenty-nine-3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidine- 4-yl) methyl) -7- (3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d]Pyrimidin-4 (3H) -ones Object 28) ofSynthesis of

The method comprises the following steps: synthesis of 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (3-oxo-2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 28-1)

According to the procedure described in example step two, compound 10-3(25mg, 0.05mmol) and (3-oxo-2, 3-dihydro-1H-inden-5-yl) boronic acid (14mg, 0.06mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, to carry out a reaction, and the reaction solution was concentrated and purified by preparative thin layer chromatography (developing solvent: ethyl acetate ═ 100%) to give the title compound, 24 mg. ESI-MS (M/z) 586.2[ M + H]⁺。

Step two: synthesis of 3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -7- (3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 28)

According to the procedure described in example twenty-second step, compound 28-1(24mg, 0.04mmol) was used as the starting material in place of compound 21-1 for reaction, and the reaction mixture was purified by preparative high performance liquid chromatography (method C), followed by lyophilization to give the title compound as formate salt, 13 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)：δ8.44(d,J＝1.6Hz,1H),8.34(s, 1H),8.03(d,J＝4.8Hz,1H),8.00(s,1H),7.97(d,J＝7.6Hz,1H),7.81 (dt,J=4.8,2.8Hz,1H),7.35(d,J=7.8Hz,1H),6.32(dd,J_H-F=54.8Hz, J_H-H=3.6Hz,1H),6.01-5.95(m,1H),5.23-4.79(m,2H),4.26(t,J=6.6 Hz,1H),4.06-3.99(m,1H),3.97-3.89(m,2H),3.69(d,J=13.6Hz,1H), 3.31-3.21(m,2H),3.02-2.96(m,1H),2.95-2.85(m,2H),2.85-2.75(m,1H), 2.41(s,3H),2.39-2.32(m,1H),1.93-1.82(m,1H),1.65-1.31(m,4H).ESI- MS(m/z):601.2[M+H]⁺。

example thirty 7-, (7-chloro-3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3-) (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d](meth) acrylic acid esters of pyrimidin-4 (3H) -one Synthesis of Compound 29)

The method comprises the following steps: synthesis of 7- (7-chloro-3-oxo-2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 29-1)

According to the procedure described in example twenty-two-step one, using the compound 6-bromo-4-chloro-2, 3-dihydro-1H-inden-1-one (50mg, 0.20 mmol) as a reaction starting material in place of 5-bromo-4-chloro-2, 3-dihydro-1H-inden-1-one, the reaction solution was concentrated under reduced pressure and separated and purified by preparative thin layer chromatography (developing solvent: ethyl acetate = 100%) to give the title compound, 30 mg. ESI-MS (M/z) 620.2,622.2[ M + H]⁺。

Step two: synthesis of 7- (7-chloro-3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 29)

According to the procedure described in example twenty-second step, compound 29-1 (30mg, 0.05mmol) was used as the starting material in place of compound 21-1 for reaction, and the reaction mixture was purified by preparative high performance liquid chromatography (method C), followed by lyophilization to give the title compound as formate salt, 13 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)：δ8.51(d,J＝1.6Hz,1H),8.29(s, 1H),8.17(d,J＝1.4Hz,1H),8.08(d,J＝4.8Hz,1H),7.87(s,1H),7.81 (q,J＝2.8Hz,1H),6.26(td,J_H-F＝55.2Hz,J_H-H＝4.4Hz,1H),5.98(dd, J＝6.0,2.4Hz,1H),5.15-4.86(m,2H),4.28(t,J＝6.8Hz,1H),4.04-3.98 (m,1H),3.96-3.89(m,2H),3.69(d,J＝13.6Hz,1H),3.31-3.21(m,2H), 3.03-2.87(m,3H),2.86-2.77(m,1H),2.61-2.52(m,1H),2.43-2.32(m,4H), 1.93-1.83(m,1H),1.60-1.30(m,4H).ESI-MS(m/z):635.2,637.2[M+H]⁺。

example thirty one 7- (4-chloro-3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-) 3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Pyrimidin-4 (3H) -ones Synthesis of (Compound 30)

The method comprises the following steps: synthesis of 7-chloro-6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1H-inden-1-one (Compound 30-2)

According to the procedure described in example seven, step two, a reaction was carried out using compound 30-1(450mg,1.83 mmol) as a starting material in place of compound 7-2, the reaction solution was cooled to room temperature, concentrated under reduced pressure, and then separated and purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 6/1(v/v)) to give the title compound, 130 mg. ESI-MS (M/z) 293.1,295.1[ M + H]⁺。

Step two: synthesis of 7- (4-chloro-3-oxo-2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) thieno [3,4-d ] pyrimidin-4 (3H) -one (compound 30-3)

According to the procedure described in example step two, compound 10-3(50mg, 0.09mmol) and compound 30-2(41mg,0.14mmol) were reacted as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, the reaction solution was cooled to room temperature, then filtered through celite, and the filtrate was concentrated under reduced pressure and purified by preparative thin layer chromatography (developing solvent: dichloromethane/methanol 10/1(v/v)) to give the title compound, 25 mg. ESI-MS (M/z) 620.1,622.1[ M + H]⁺。

Step three: synthesis of 7- (4-chloro-3- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 30)

Dissolving compound 30-3(25mg, 0.04mmol) in methylamine/ethanol solution (2mL, 33% wt), reacting at room temperature for 17h, concentrating under reduced pressure, adding methanol (2mL) and sodium borohydride (3mg, 0.08mmol) in sequence, and reacting at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure, purified by preparative high performance liquid chromatography (method G), and lyophilized to give the title compound, trifluoroacetate salt, 8 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)δ8.52(d,J＝1.2Hz,1H),7.97(d, J＝7.7Hz,1H),7.63(d,J＝7.8Hz,1H),7.60-7.53(m,1H),7.44(d,J＝ 7.8Hz,1H),6.09(td,J_H-F＝55.4Hz,J_H-H＝4.4Hz,1H),5.89-5.81(m,1H), 4.97(d,J＝6.7Hz,1H),4.94-4.88(m,1H),4.18-4.04(m,1H),4.00(d,J＝ 16.6Hz,2H),3.82-3.73(m,1H),3.48-3.28(m,4H),3.20-3.07(m,2H), 3.09-2.98(m,1H),2.94-2.81(m,1H),2.77(s,3H),2.67-2.55(m,1H),2.42 (dd,J＝15.1,7.7Hz,1H),1.76-1.42(m,4H).ESI-MS(m/z):635.2, 637.2[M+H]⁺。

example thirty-two 7- (2-chloro-4-fluorophenyl) -3- ((1- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxy Arylpiperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 32)

Under the protection of nitrogen, compound 10-3(40mg, 0.08mmol) is dissolved in 1, 4-dioxane (2mL), and (2-chloro-4-fluorobenzene) boric acid (15mg, 0.08mmol) and water (0.1 mL) are added, after the protection of nitrogen, palladium tetratriphenylphosphine (9mg, 0.007mmol) and sodium bicarbonate (21mg, 0.23mmol) are sequentially added, after the replacement of nitrogen for three times, the mixture is reacted at 85 ℃ for 5 hours, cooled to room temperature, concentrated under reduced pressure, and separated and purified by preparative thin layer chromatography (developing solvent: dichloromethane/methanol-15/1 (v/v)), to give the crude title compound, which was isolated and purified by preparative high performance liquid chromatography (method C), and lyophilized to give the title compound, 20 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)δ8.52(t,J＝1.4Hz,1H),7.97(d, J＝7.2Hz,1H),7.63(dd,J＝8.8,6.0Hz,1H),7.59(dt,J＝6.4,2.4Hz, 1H),7.42(ddd,J＝8.8,2.8,1.2Hz,1H),7.26-7.19(m,1H),6.12(td,J_H-F＝55.4Hz,J_H-H＝4.4Hz,1H),5.87(ddd,J＝10.4,5.6,2.4Hz,1H),5.00- 4.90(m,1H),4.14(t,J＝12.8Hz,1H),4.07-3.97(m,2H),3.84-3.75(m, 1H),3.48-3.35(m,2H),3.10-3.00(m,1H),2.98-2.82(m,1H),1.77-1.47(m, 4H).ESI-MS(m/z):584.1,586.1[M+H]⁺。

example thirty-three 7- (4-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((4-hydroxy-1-) ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (compound 33)

The method comprises the following steps: synthesis of tert-butyl (4-chloro-5- (3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-1-yl) (methyl) carbamate (Compound 33-1)

According to the procedure described in example step two, compound 23-3(30mg, 0.07mmol) was used as a reaction raw material in place of (1H-indazol-6-yl) boronic acid, and the reaction mixture was concentrated under reduced pressure and then separated and purified by preparative thin layer chromatography (developing solvent: dichloromethane/methanol-20/1 (v/v)) to give the title compound, 30 mg. ESI-MS (M/z) 691.3,693.3[ M + H]⁺。

Step two: synthesis of 7- (4-chloro-1- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((4-hydroxy-1- ((R) -3-phenylbutyryl) piperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 33)

According to the procedure described in the seventeenth step five of the example, compound 33-1(30mg, 0.04mmol) was used as a starting material in place of compound 16-5 for reaction, and the reaction mixture was concentrated under reduced pressure to give a reaction solution, which was subjected to preparative high performance liquid chromatography purification (method L), and freeze-drying to give the title compound as formate salt, 18 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.61(s,1H),7.94(d,J＝10.4Hz, 1H),7.48(d,J＝7.8Hz,1H),7.40(d,J＝8.0Hz,1H),7.32-7.22(m,4H), 7.15(m,1H),4.89(brs,1H),4.24(t,J＝6.8Hz,1H),4.09-3.99(m,1H), 3.90(q,J＝14.0Hz,1H),3.88-3.78(m,1H),3.72-3.58(m,1H),3.27-3.10 (m,3H),3.07-2.98(m,1H),2.92-2.78(m,2H),2.69-2.53(m,2H),2.43-2.36 (m,1H),2.35(s,3H),1.95-1.87(m,1H),1.58-1.15(m,4H),1.20(dd,J＝ 7.2,2.0Hz,3H).ESI-MS(m/z):591.3,593.2[M+H]⁺。

example thirty-four 7- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-) 3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Pyrimidin-4 (3H) -ones (Compound 34) Synthesis

The method comprises the following steps: synthesis of 5-bromo-4-chloro-1H-indene (Compound 34-1)

Compound 30-1(2.00g, 8.15mmol) was dissolved in methanol (100mL) at room temperature, and sodium borohydride (0.60g, 16.29mmol) was added in portions and stirred at room temperature for 2 h. The solvent was removed under reduced pressure, diluted hydrochloric acid (20mL, 1mol/L) was added to quench the reaction, and extraction was performed with ethyl acetate, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, the concentrate was dissolved in toluene (100mL), p-toluenesulfonic acid (139mg, 0.81mmol) was added, and the reaction was performed at 120 ℃ for 3 h. The solvent was drained under reduced pressure and purified by silica gel column chromatography (eluent: petroleum ether ═ 100%) to give the title compound, 1.70 g. The structure is characterized as follows:

¹H NMR(400MHz,CDCl₃)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 two: synthesis of 3-bromo-2-chloro-6, 6 a-dihydro-1 aH-indeno [1,2-b ] oxirane (Compound 34-2)

Compound 34-1(1.50g, 6.54mmol) was dissolved in dichloromethane (160 mL) at room temperature, and m-chloroperoxybenzoic acid (3.98g, 19.61mmol) was added and reacted at room temperature for 18 h. The solvent was drained under reduced pressure and purified by silica gel column chromatography (eluent: petroleum ether/dichloromethane ═ 1/1(v/v)) to give the title compound, 900 mg.

The structure is characterized as follows:

¹H NMR(400MHz,CDCl₃)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 three: synthesis of tert-butyl (5-bromo-4-chloro-2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 34-3)

Compound 34-2(900mg, 3.67mmol) was dissolved in toluene (50mL) at room temperature, silica gel (2.70g) was added, and the reaction was carried out at 120 ℃ for 6 h. Filtration and vacuum suction of the filtrate were carried out, the residue was dissolved in methanol (2.6mL), aqueous methylamine (1mL, 40% wt) was added, after stirring for 10min, sodium borohydride (94mg, 2.54mmol) was added, and after 1h reaction at room temperature, di-tert-butyl dicarbonate (3.24g, 14.83mmol) was added and reaction continued at room temperature for 1 h. The solvent was drained under reduced pressure and purified by silica gel column chromatography (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 four: synthesis of tert-butyl (4-chloro-5- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 34-4)

According to the procedure described in example seven, step two, compound 34-3(200mg, 0.65mmol) was used as a reaction starting material in place of compound 7-2, and after concentration, the reaction mixture was separated and purified by preparative thin layer chromatography (developing solvent: petroleum ether/ethyl acetate-60/1 (v/v)) to give the title compound, 80 mg. ESI-MS (M/z): ESI-MS (M/z):352.3,354.3[ M-56+ H]⁺。

The structure is characterized as follows:

¹H NMR(400MHz,CDCl₃)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 five: synthesis of tert-butyl (4-chloro-5- (3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 34-5)

According to the procedure described in example step two, compound 10-3(15mg, 0.03mmol) and compound 34-4(12mg, 0.03mmol) were reacted as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution after concentration under reduced pressure was separated and purified by preparative thin layer chromatography (eluent: ethyl acetate/methanol 60/1(v/v)) to give the title compound, 10 mg. ESI-MS (M/z) 735.3,737.3[ M + H]⁺。

Step six: synthesis of 7- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 34)

According to the procedure described in example twelve step II, compound 34-5(10mg, 0.01mmol) was used as a starting material in place of compound 10-1 for reaction, and the reaction mixture was concentrated under reduced pressure, purified by preparative high performance liquid chromatography (method C), and lyophilized to give the title compound as formate salt, 2 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)δ8.73-8.45(br,1H),8.49(d,J＝ 1.2Hz,1H),7.96(d,J＝7.6Hz,1H),7.59(dt,J＝6.0,2.8Hz,1H),7.43(d, J＝7.6Hz,1H),7.30(d,J＝7.6Hz,1H),6.11(td,J_H-F＝55.2Hz,J_H-H＝ 4.4Hz,1H),5.87(ddd,J＝9.6,6.0,2.8Hz,1H),5.02-4.93(m,1H),4.21- 4.08(m,1H),4.03(d,J＝4.0Hz,1H),3.99(s,1H),3.94-3.85(m,1H),3.84- 3.73(m,1H),3.54-3.34(m,4H),3.18-2.98(m,3H),2.88(ddd,J＝30.0, 16.4,4.0Hz,1H),2.63(s,3H),1.80-1.44(m,4H).ESI-MS(m/z):635.2, 637.2[M+H]⁺。

example thirty-five 7- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-) 3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 35)

The method comprises the following steps: synthesis of tert-butyl (4-chloro-5- (3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 35-1)

According to the procedure described in example step two, compound 19-1(20mg, 0.04mmol) and compound 34-5(16mg, 0.04mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and after concentration under reduced pressure, the reaction solution was separated and purified by preparative thin layer chromatography (developing solvent: ethyl acetate/methanol 60/1(v/v)) to give the title compound, 12 mg. ESI-MS (M/z) 717.3,719.3[ M + H]⁺。

Step two: synthesis of 7- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 35)

According to the procedure described in example twelve step II, compound 35-1(12mg, 0.02mmol) was used as a starting material in place of compound 10-1 for reaction, and the reaction mixture was purified by preparative high performance liquid chromatography (method C), followed by lyophilization to give the title compound as a formate salt, 8 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)δ8.65-8.53(brs,1H),8.50(d,J＝ 7.1Hz,1H),7.96(s,0.4H),7.90(s,0.6H),7.45(d,J＝7.7Hz,1H),7.38- 7.22(m,5H),7.22-7.14(m,1H),4.24-3.96(m,3H),3.93-3.69(m,2H),3.52 (dt,J＝15.5,7.5Hz,2H),3.22-3.10(m,2H),3.10-2.78(m,3H),2.71(s, 3H),2.33-2.24(m,1H),1.65-1.12(m,5H),0.66-0.56(m,1H),0.46-0.36(m, 1H),0.34-0.25(m,1H),0.13-0.04(m,1H).ESI-MS(m/z):617.3, 619.3[M+H]⁺。

example thirty-six 7- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-) 3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Pyrimidin-4 (3H) -ones (Compound 36) Synthesis

The method comprises the following steps: synthesis of 5-bromo-7-chloro-1H-indene (Compound 36-2)

According to the procedure described in example thirty-four step one, compound 36-1(12mg, 0.02mmol) was used as a starting material in place of compound 30-1 for reaction, the reaction solution was concentrated under reduced pressure to remove the solvent, the crude product was washed with petroleum ether, filtered, and the filtrate was collected and concentrated under reduced pressure to give the title compound, 560 mg.

The structure is characterized as follows:

¹H NMR(400MHz,CDCl₃)：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 two: synthesis of 3-bromo-5-chloro-6, 6 a-dihydro-1 aH-indeno [1,2-b ] oxirane (Compound 36-3)

According to the procedure described in example thirty-four step two, compound 36-2(560mg, 2.44mmol) was used as a reaction raw material in place of compound 34-1, and separation and purification by silica gel column chromatography (eluent: petroleum ether/dichloromethane 2/1(v/v)) was carried out to give the title compound, 400 mg.

The structure is characterized as follows:

¹H NMR(400MHz,CDCl₃)：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 three: synthesis of tert-butyl (6-bromo-4-chloro-2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 36-4)

According to the procedure described in example thirty-four step three, using compound 36-3(400mg, 1.63mmol) as a reaction starting material in place of compound 34-2, a reaction was carried out, and separation and purification by silica gel column chromatography (eluent: petroleum ether/tetrahydrofuran ═ 30/1(v/v)) were carried out to give the title compound, 310 mg.

The structure is characterized as follows:

¹H NMR(400MHz,CDCl₃)：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 four: synthesis of tert-butyl (4-chloro-6- (3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 36-5)

According to the procedure described in example twenty-two-step one, using compound 36-4(30mg, 0.08mmol) as a starting material for the reaction instead of 5-bromo-4-chloro-2, 3-dihydro-1H-inden-1-one, separation and purification by preparative thin layer chromatography (developing solvent: ethyl acetate/methanol-60/1 (v/v)) was carried out to give the title compound, 40 mg. ESI-MS (M/z) 735.2,737.2[ M + H]⁺。

Step five: synthesis of 7- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 36)

According to the procedure described in example twelve step II, compound 36-5(35mg, 0.05mmol) was used as a starting material in place of compound 10-1 for reaction, the reaction mixture was purified by preparative high performance liquid chromatography (method C), and dilute hydrochloric acid (0.5mL, 1mol/L) was added to the preparation, followed by lyophilization to give the hydrochloride of the title compound, 20 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)：δ8.55(s,1H),8.45(d,J＝5.6Hz, 1H),7.87(s,1H),7.73(s,1H),7.63-7.56(m,1H),6.12(td,J_H-F＝55.2Hz, J_H-H＝4.4Hz,1H),5.92-5.84(m,1H),5.00-4.94(m,1H),4.22-4.04(m,4H), 3.85-3.75(m,1H),3.64-3.53(m,2H),3.48-3.36(m,2H),3.35-3.32(m,1H), 3.26-3.19(m,1H),3.11-3.02(m,1H),2.97-2.84(m,1H),2.80(s,3H),1.79- 1.46(m,4H).ESI-MS(m/z):635.3,637.3[M+H]⁺。

example thirty-seven 7- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-) 3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 37)

The method comprises the following steps: synthesis of tert-butyl (4-chloro-6- (3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 37-1)

According to the procedure described in example twenty-two-step one, using compound 19-1 (38mg, 0.07mmol) and compound 36-4(30mg, 0.08mmol) as reaction starting materials in place of compound 10-3 and 5-bromo-4-chloro-2, 3-dihydro-1H-inden-1-one, respectively, reaction was carried out, and separation and purification by preparative thin layer chromatography (developing solvent: ethyl acetate/methanol-60/1 (v/v)) gave the title compound, 50 mg. ESI-MS (M/z) 717.4,719.4[ M + H]⁺。

Step two: synthesis of 7- (7-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 37)

According to the procedure described in example twelve step II, a reaction was carried out using 37-1(50mg, 0.07mmol) as a starting material in place of 10-1, the reaction mixture was separated and purified by preparative high performance liquid chromatography (method C), and diluted hydrochloric acid (0.5mL, 1mol/L) was added to the preparation, followed by lyophilization to give the hydrochloride of the title compound, 28 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)：δ8.57(d,J＝9.6Hz,1H),8.44 (d,J＝15.2Hz,1H),7.86(s,1H),7.72(s,1H),7.38-7.16(m,5H),4.33-4.12 (m,2H),4.13-4.04(m,1H),3.99-3.85(m,1H),3.81-3.69(m,1H),3.65-3.52 (m,2H),3.36-3.32(m,1H),3.30-3.16(m,2H),3.08-2.70(m,6H),2.34-2.23 (m,1H),1.71-1.27(m,3.5H),1.26-1.13(m,1H),0.82-0.73(m,0.5H),0.67- 0.58(m,1H),0.48-0.38(m,1H),0.36-0.26(m,1H),0.15-0.06(m,1H).ESI- MS(m/z):617.3,619.3[M+H]⁺。

example thirty-eight 7- (6-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-) 3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Pyrimidin-4 (3H) -ones (Compound 38) Synthesis

The method comprises the following steps: synthesis of 5-bromo-6-chloro-2- (hydroxyimino) -2, 3-dihydro-1H-inden-1-one (Compound 38-1)

Compound 24-1(200mg, 0.81mmol) and chlorotrimethylsilane (106 mg, 0.98mmol) were dissolved in methyl tert-butyl ether (20mL) at room temperature, isoamyl nitrite (115mg, 0.98mmol) was slowly added dropwise, and the reaction was stirred at room temperature for 2 h. The reaction was filtered and the filter cake was washed with methyl tert-butyl ether (2X 10mL) to give the crude title compound, 165 mg. ESI-MS (M/z) 274.0,276.0[ M + H]⁺。

Step two: synthesis of 2-amino-5-bromo-6-chloro-2, 3-dihydro-1H-inden-1-ol (Compound 38-2)

Compound 38-1(160mg, 0.58mmol) was dissolved in dry tetrahydrofuran (20mL) at room temperature, sodium borohydride (86mg, 2.33mmol) and boron trifluoride etherate (331mg, 2.33mmol) were added, and the reaction was warmed to 65 ℃ and stirred for 5 h. The reaction was cooled to room temperature and then quenched with ethanol (20mL), concentrated to give a crude product which was purified by preparative high performance liquid chromatography (method C), lyophilized to give the title compound as formate salt, 80 mg. ESI-MS (m/z):262.3,264.3[M+H]⁺。

Step three: synthesis of 5-bromo-6-chloro-2, 3-dihydro-1H-inden-2-amine (Compound 38-3)

The formate salt of Compound 38-2 (67mg, 0.22mmol) was dissolved in 1, 2-dichloroethane (20mL) at room temperature, triethylsilane (6mL) and boron trifluoride etherate (6mL) were added, and the reaction was warmed to 85 ℃ and stirred for 18 h. The reaction was concentrated and the concentrate was purified by preparative high performance liquid chromatography (method C) and lyophilized to give the title compound as formate salt, 43 mg. ESI-MS (M/z) 246.3,248.3[ M + H]⁺。

Step four: synthesis of tert-butyl (5-bromo-6-chloro-2, 3-dihydro-1H-inden-2-yl) carbamate (Compound 38-4)

The formate salt of compound 38-3 (43mg, 0.15mmol) was dissolved in dichloromethane (10mL) at room temperature, triethylamine (30mg, 0.29mmol) and di-tert-butyl dicarbonate (218mg, 0.29mmol) were added, and the mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated, and the concentrate was purified by preparative thin layer chromatography (developing solvent: 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 five: synthesis of tert-butyl (5-bromo-6-chloro-2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (38-5)

According to the procedure described in example seven, step one, a reaction was carried out using compound 38-4(40mg, 0.12 mmol) as a reaction starting material in place of compound 7-1, and the reaction mixture was separated and purified by preparative thin layer chromatography (developing solvent: 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 six: synthesis of tert-butyl (5-chloro-6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 38-6)

According to the procedure described in example seven step two, compound 38-5(40mg, 0.11 mmol) was used as a starting material in place of compound 7-2 for reaction, and the reaction mixture was filtered and concentrated to give the crude title compound, 45 mg. ESI-MS (M/z) 352.2,354.2[ M-56+ H]⁺。

Step seven: synthesis of tert-butyl (5-chloro-6- (3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 38-7)

According to the procedure described in example step two, compound 10-3(9mg, 0.02mmol) and the crude product of compound 38-6 (6mg) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution was filtered and concentrated to give the crude product of the title compound, 9 mg. ESI-MS (M/z) 735.4,737.4[ M + H]⁺。

Step eight: synthesis of 7- (6-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 38)

According to the procedure described in example twelve step II, a reaction was carried out using the crude product (9 mg) of compound 38-7 as a starting material in place of compound 10-1, the reaction mixture was subjected to preparative high performance liquid chromatography for separation and purification (method C), and the preparation was lyophilized to give the title compound as a formate salt, 2 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)δ8.53(s,1H),8.51(s,1H),7.98(d, J＝8.0Hz,1H),7.62-7.57(m,1H),7.50(d,J＝8.0Hz,2H),6.12(td,J_H-F＝55.2Hz,J_H-H＝4.4Hz,1H),5.91-5.83(m,1H),5.01-4.94(m,1H),4.17- 4.09(m,1H),4.07-3.96(m,3H),3.85-3.75(m,1H),3.50-3.35(m,4H), 3.17-3.03(m,3H),2.96-2.87(m,1H),2.72(s,3H),1.68-1.46(m,4H).ESI- MS(m/z):635.2,637.2[M+H]⁺。

example thirty-nine 7- (2-amino-6-chloro-1-hydroxy-2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-bis) Fluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Pyrimidine-4 (3H) - Synthesis of ketone (Compound 39)

The method comprises the following steps: synthesis of tert-butyl (5-bromo-1- ((tert-butoxycarbonyl) oxy) -6-chloro-2, 3-dihydro-1H-inden-2-yl) carbamate (Compound 39-1)

Compound 38-2(20mg, 0.076mmol) was dissolved in dichloromethane (10mL) at room temperature, triethylamine (16mg, 0.23mmol) and di-tert-butyl dicarbonate (50mg, 0.23mmol) were added, and the reaction was carried out at room temperature for 4 hours. The reaction solution was concentrated, and the residue was purified by preparative thin layer chromatography (developing solvent: petroleum ether/ethyl acetate-8/1 (v/v)) to give the title compound, 30 mg. ESI-MS (M/z) 461.1,463.1[ M + H]⁺。

Step two: synthesis of tert-butyl (5-chloro-6- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 39-2)

According to the procedure described in example seven, step two, compound 39-1(30mg, 0.06mmol) was used as a starting material in place of compound 7-2 for reaction, and the reaction mixture was filtered and concentrated to give the crude title compound, 30 mg. ESI-MS (M/z) 509.5,511.5[ M + H]⁺。

Step three: synthesis of tert-butyl (1- ((tert-butoxycarbonyl) oxy) -6-chloro-5- (3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) carbamate (Compound 39-3)

According to the procedure described in example step two, compound 10-3(31mg, 0.06mmol) and the crude product of compound 39-2 (30mg, 0.05mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and the reaction solution was filtered and concentrated to give a concentrate which was separated and purified by preparative thin layer chromatography (developing solvent: dichloromethane/methanol-20/1 (v/v)) to give the title compound, 15 mg. ESI-MS (M/z) 837.2,839.3[ M + H]⁺。

Step four: synthesis of 7- (2-amino-6-chloro-1-hydroxy-2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 39)

According to the procedure described in example twelve step II, a reaction was carried out using compound 39-3(15mg, 0.020 mmol) as a starting material in place of compound 10-1, the reaction mixture was separated and purified by preparative high performance liquid chromatography (method J), and the preparation was lyophilized to give the title compound, trifluoroacetate salt, 6 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄)δ8.53(d,J＝4.0Hz,1H),8.01- 7.96(m,1H),7.64-7.57(m,2H),7.54-7.50(m,1H),6.12(td,J_H-F＝55.2 Hz,J_H-H＝4.4Hz,1H),5.91-5.84(m,1H),5.31(d,J＝4.0Hz,0.3H),5.17 (d,J＝4.0Hz,0.7H),5.01-4.94(m,1H),4.19-4.05(m,4H),3.94(s,3H), 3.85-3.75(m,1H),3.59-3.36(m,4H),3.27-3.13(m,2H),3.10-3.01(s,1H), 2.97-2.84(m,1H),2.80(s,3H),1.77-1.47(m,4H).ESI-MS(m/z):637.3, 639.2[M+H]⁺。

example forty 7- (6-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-3-) Phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Synthesis of pyrimidin-4 (3H) -one (Compound 40)

The method comprises the following steps: synthesis of tert-butyl (5-chloro-6- (3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 40-1)

According to the procedure described in example step two, compound 19-1(30mg, 0.06mmol) and compound 38-6(20mg, 0.05mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and were separated and purified by preparative high performance liquid chromatography (method M) to give the title compound, 20 mg. ESI-MS (M/z) 717.3,719.3[ M + H]⁺。

Step two: synthesis of 7- (6-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-3-phenylpropionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 40)

According to the procedure described in example twelve step II, a reaction was carried out using compound 40-1(20mg, 0.03mmol) as a starting material in place of compound 10-1, the reaction mixture was concentrated under reduced pressure, purified water was added thereto, and freeze-drying was carried out to give the hydrochloride of the title compound, 14 mg.

The structure is characterized as follows:

¹H NMR(400MHz,Methanol-d₄):δ8.56(d,J＝8.0Hz,1H),8.06(d, J＝20.0Hz,1H),7.54(s,1H),7.51(s,1H),7.35-7.14(m,5H),4.24-4.01 (m,3H),3.96-3.72(m,2H),3.55-3.43(m,2H),3.22-3.18(m,1H),3.17-3.11 (m,1H),2.98-2.82(m,2H),2.78(s,3H),2.72-2.64(m,1H),2.33-2.24(m, 1H),1.65-1.54(m,1H),1.50-1.41(m,1H),1.35-1.29(m,2H),1.23-1.15(m, 1H),0.82-0.72(m,1H),0.46-0.36(m,1H),0.35-0.26(m,1H),0.14-0.06(m, 1H).ESI-MS(m/z):617.3,619.3[M+H]⁺。

example forty-one 7- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-) 3- (4-fluorophenyl) propionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Pyrimidin-4 (3H) -one (compound 41) Synthesis of (2)

The method comprises the following steps: synthesis of 7-bromo-3- ((1- (3-cyclopropyl-3- (4-fluorophenyl) propionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 41-2)

According to the procedure described in example thirteen steps, compound 41-1(127mg, 0.61mmol) was used as a reaction raw material in place of compound 4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butyric acid, and after concentration under reduced pressure, the reaction mixture was separated and purified by silica gel column chromatography (eluent: dichloromethane/methanol 19/1(v/v)) to give the title compound, 285 mg. ESI-MS (M/z):534.2, 536.2[ M + H ]]⁺。

Step two: synthesis of tert-butyl (4-chloro-5- (3- ((1- (3-cyclopropyl-3- (4-fluorophenyl) propionyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 41-3)

According to the procedure described in example step two, compound 41-2(26mg, 0.05mmol) and compound 34-4(20mg, 0.05mmol) were used as reaction starting materials in place of compound 1-2 and (1H-indazol-6-yl) boronic acid, respectively, and after concentration under reduced pressure, the reaction mixture was separated and purified by preparative thin layer chromatography (eluent: ethyl acetate/methanol 60/1(v/v)) to give the title compound, 15 mg. ESI-MS (M/z) 735.3,737.3[ M + H]⁺。

Step three: synthesis of 7- (4-chloro-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (3-cyclopropyl-3- (4-fluorophenyl) propionyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 41)

According to the procedure described in example twelve step II, compound 41-3(15mg, 0.02mmol) was used as the starting material in place of compound 10-1 for reaction, and the reaction mixture was purified by preparative high performance liquid chromatography (method C), followed by lyophilization to give the title compound as formate salt, 9 mg.

The structure is characterized as follows:

¹H NMR(400MHz,MeOH-d₄)δ8.55(brs,1H),8.49(d,J＝4.0Hz, 1H),7.94(d,J＝14.8Hz,1H),7.43(d,J＝7.6Hz,1H),7.38-7.18(m,3H), 7.06(t,J＝8.8Hz,1H),6.98(t,J＝8.4Hz,1H),4.24-4.10(m,1H),4.09- 3.82(m,3H),3.82-3.70(m,1H),3.58-3.40(m,2H),3.30-3.02(m,3H), 3.02-2.65(m,3H),2.65(s,3H),2.40-2.20(m,1H),1.70-1.35(m,3H),1.22- 0.95(m,2H),0.66-0.54(m,1H),0.47-0.35(m,1H),0.35-0.23(m,1H), 0.16-0.04(m,1H).ESI-MS(m/z):635.3,637.3[M+H]⁺。

example forty-two 7- (6-chloro-2-methyl-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d]Pyrimidine-4 (3H) Synthesis of the ketones (Compound 42)

The method comprises the following steps: synthesis of 5-bromo-6-chloro-2-methyl-2, 3-dihydro-1H-inden-2-amine (Compound 42-1)

At room temperature, compound 16-1(125mg, 0.46mmol) is weighed and dissolved in hydrochloric acid (10mL, 6mol/L), after stirring and dissolving, N-chlorosuccinimide (64mg, 0.48mmol) is added, and the temperature is raised to 60 ℃ and stirring reaction is carried out for 8 h. The reaction was then cooled to room temperature, adjusted to pH 9 with saturated aqueous sodium bicarbonate, extracted with ethyl acetate (3X 10mL), the organic phases combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The concentrate was purified by preparative high performance liquid chromatography (method N), and lyophilized to give the title compound as trifluoroacetate salt, 66 mg. ESI-MS (M/z) 260.0,262.0[ M + H]⁺。

Step two: synthesis of tert-butyl (5-bromo-6-chloro-2-methyl-2, 3-dihydro-1H-inden-2-yl) carbamate (Compound 42-2)

The trifluoroacetate salt of compound 42-1 (30mg, 0.13mmol) was suspended in dichloromethane (4mL) at room temperature, and N, N-diisopropylethylamine (70mg, 0.54mmol) and di-tert-butyl dicarbonate (68mg, 0.18mmol) were added and reacted at room temperature for 5 h. The reaction solution was concentrated under reduced pressure to give the title compound, 65 mg. ESI-MS (M/z) 304.1,306.1[ M-56+ H]⁺。

Step three: synthesis of tert-butyl (5-bromo-6-chloro-2-methyl-2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (Compound 42-3)

According to the procedure described in the seventh step one of the example, compound 42-2(65mg, 0.16 mmol) was used as a reaction starting material in place of compound 7-1 to carry out a reaction, and the reaction solution was purified by preparative high performance liquid chromatography (method O), followed by lyophilization to give the title compound, 48 mg. ESI-MS (M/z) 274.0,276.0[ M-100+ H]⁺。

Step four: synthesis of tert-butyl (5-chloro-6- (3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) -4-oxo-3, 4-dihydrothieno [3,4-d ] pyrimidin-7-yl) -2-methyl-2, 3-dihydro-1H-inden-2-yl) (methyl) carbamate (compound 42-4)

At room temperature, compound 42-3(63mg, 0.17mmol) and pinacol diboron (64mg, 0.25mmol) were dissolved in 1, 4-dioxane (4mL), potassium acetate (50mg, 0.50mmol) was added, nitrogen was replaced, and Pd (dppf) Cl was added₂(12mg, 0.02mol), the nitrogen was again replaced with 5 times, and the reaction was carried out at 80 ℃ for 12 hours under a nitrogen blanket. The reaction was stopped and the reaction solution was cooled to room temperature. Then, compound 10-3(30mg, 0.06mmol), 1, 4-dioxane (0.5mL), water (0.5mL) and potassium carbonate (23mg, 0.17mmol) were sequentially added thereto, and after replacing nitrogen, Pd (PPh) was added₃)₄(7mg, 0.01 mmol). And reacting for 14 hours at 80 ℃ under the protection of nitrogen. The reaction was stopped, cooled to room temperature, and the reaction solution was purified by preparative thin layer chromatography (developing solvent: dichloromethane/methanol-20/1 (v/v)) to give the title compound, 21 mg. ESI-MS (M/z) 749.3,751.3[ M + H]⁺。

Step five: synthesis of 7- (6-chloro-2-methyl-2- (methylamino) -2, 3-dihydro-1H-inden-5-yl) -3- ((1- (4, 4-difluoro-3- (3-fluoro-1H-pyrazol-1-yl) butanoyl) -4-hydroxypiperidin-4-yl) methyl) thieno [3,4-d ] pyrimidin-4 (3H) -one (Compound 42)

Compound 42-4(21mg, 0.03mmol) was dissolved in ethyl acetate (1mL) at room temperature, and a hydrochloric acid/ethyl acetate solution (2mL, 4mol/L) was added. The reaction was carried out at room temperature for 2 h. The reaction solution was subjected to preparative high performance liquid chromatography purification (method P), and lyophilized to give the title compound as formate salt, 8 mg.

The structure is characterized as follows:

¹H NMR(400MHz,DMSO-d₆)δ8.60(d,J＝1.2Hz,1H),7.95(d,J ＝4.8Hz,1H),7.84-7.78(m,1H),7.44(d,J＝2.4Hz,2H),6.26(td,J_H-F＝ 55.2Hz,J_H-H＝3.6Hz,1H),5.99(ddd,J＝6.0,2.8,1.2Hz,1H),5.07-4.86 (m,2H),4.06-3.98(m,1H),3.96-3.85(m,2H),3.72-3.65(m,1H),3.30-3.22 (m,2H),3.03(t,J＝16.8Hz,2H),2.97-2.76(m,4H),2.31(s,3H),1.64- 1.33(m,4H),1.26(s,3H).ESI-MS(m/z):649.3,651.3[M+H]⁺。

pharmacological Activity test

Test example 1: USP7 (protease) in vitro enzymatic Activity inhibition assay

The test system comprises:

kit USP7 inhibition Screening Assay Kit, (BPS Catalog:79256) containing:

protease USP7 HisFLAGtags enzyme, (BPS Catalog:80395),

substrate Ub-AMC Substrate, (BPS Catalog:81150),

buffer 5 × USP7 Assay Buffer, (BPS Catalog: 79274).

Testing an instrument:

BMG PHERAstar FS。

test parameters are as follows:

concentration of USP 7: 3 nM; Ub-AMC concentration: 100 nM;

buffer system: 1.25 × USP7 Assay Buffer; 0.06% BSA; 1mM DTT; ddH₂O；

Compound and enzyme incubation time: 20 min;

enzyme kinetic reaction time: 20 min;

fluorescence: the excitation wavelength is 350nm, and the emission wavelength is 460 nm.

Parameters of the microplate reader: BMG PHERAStator FS Fluorescence, excitation wavelength 350nm, emission wavelength 460 nm.

The test method comprises the following steps:

the assay was performed according to the kit instructions, the procedure was as follows,

test group: the mixture of test compound and protease USP7 was incubated for 20min at room temperature in a buffer system, the substrate Ub-AMC was added to initiate the reaction, and the fluorescence value of each well was read for 20 cycles in each cycle (1min) using the enzyme kinetics method.

Negative group: the test compound was replaced with 0.2% DMSO in water and the experimental procedure was the same as for the test group.

Blank group: test compounds were replaced with 0.2% aqueous DMSO and the test was performed in the same manner as the test group without protease USP 7.

Data processing:

the relative inhibitory activity of each concentration group was calculated, and the inhibition rate was 100% - (fluorescence value of test group-fluorescence value of blank group)/(fluorescence value of negative group-fluorescence value of blank group) × 100%. Fitting the curve according to a four parameter modelLine, calculate half maximal Inhibitory Concentration (IC) of compound₅₀)。

And (3) test results:

the inhibition of USP7 activity by the compounds of the present invention was determined according to the above method and the results are shown in table 1.

TABLE 1 results of enzyme activity inhibition test of USP7

And (4) conclusion:

in a USP7 enzyme activity inhibition test, the compound of the invention shows stronger inhibitory activity, and particularly, the compounds of examples 3,4, 6, 7, 8, 10, 17, 18, 23, 31, 39 and 42 have extremely strong inhibitory activity on USP 7.

Test example 2: test for inhibition of MM.1S cell proliferation Activity by Compound

The test system comprises:

cell name/manufacturer: MM.1S/Nanjing Kebai

Kit name/manufacturer:

Luminescent Cell Viability Assay,Promega。

testing an instrument:

BMG PHERAstar FS。

test parameters are as follows:

cell number: 3000 cells/well

Plating a culture medium: MM.1S:1640+ 10% FBS

Adding a culture medium: MM.1S:1640+ 10% FBS

Compound incubation conditions: 37 ℃ and 5% CO₂

Incubation time: 5d

Detecting the temperature: RT (reverse transcription)

Chemiluminescence: luminescent

The test method comprises the following steps:

the cells were cultured in a medium containing 10% fetal bovine serum and placed at 37 ℃ under 5% CO₂Culturing is carried out under culture conditions. Appropriate amount of cells were plated in 96-well plates and cultured overnight in an incubator. The next day, complete medium containing the prediluted compound was added and incubated at 37 ℃ for 5 d. On the fifth day, the detection reagent CellTiter-GLo was added to each well, and Relative Luminescence Units (RLU) of each well were detected by chemiluminescence.

Data processing:

background values were obtained using CellTiter-Glo in cell-free medium. Cell viability rate (sample RLU-background RLU)/(vehicle RLU-background RLU) × 100%, maximum inhibition rate 100% -cell viability rate_{Maximum concentration}The median Inhibitory Concentration (IC) of the compound was calculated by fitting a curve to a four parameter model₅₀)。

And (3) test results:

the inhibitory activity of the compounds on the proliferation of mm.1s cells was measured by the above-described method, and the results are shown in table 2.

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

And (4) conclusion:

in the test for inhibiting the cell growth activity of mm.1s, the compounds of the present invention showed strong cell growth inhibitory activity, and in particular, the compounds 14-B, 36 and 38 in examples 3, 5, 6, 7, 10, 12 and 21 and example 15 showed very strong inhibitory activity against the cell growth of mm.1s.

Test example 3: biochemical hERG inhibition assay

The test system comprises:

the kit comprises: predictor^TMhERG Fluorescence Polarization Assay, (ThermoFisher Catalog: PV5365) containing:

positive control compound E4031;

hERG cell membrane;

an affinity Tracer Tracer; and

hERG buffer solution.

Testing an instrument:

BMG PHERAstar FS。

test parameters are as follows:

hERG concentration: 1 is prepared from

Tracer concentration: 1nM

Incubation time: 2h

Fluorescence polarization: excitation wavelength: 540 nm; emission wavelength: 590nm

The test method comprises the following steps:

the assay was performed according to kit instructions, with the following steps:

test group: the compounds to be detected with different concentrations are added into a microplate containing hERG cell membranes, a Tracer Tracer with high hERG affinity is added into each well, the microplate is incubated for 2 hours at room temperature, and then a multifunctional microplate reader is used for detecting the change of the fluorescence polarization (excitation wavelength: 540 nm; emission wavelength: 590 nm).

Positive control group: the test compound was replaced with 30. mu.M of the positive control compound E4031, and the experimental procedure was the same as in the test group.

Blank control group: the test group was run in the same manner as the test group, with hERG buffer instead of test compound and without hERG cell membrane.

Data processing:

according to the data ratio, the percentage inhibition rate (%) of the compound of the invention to hERG at different concentrations is calculated, and the half Inhibition Concentration (IC) of the compound is judged₅₀) The range of (1).

Percent inhibition (%) × 100 (1- (fluorescence polarization value of test compound-fluorescence polarization value of positive control group)/(fluorescence polarization value of blank control group-fluorescence polarization value of positive control group)) × 100

And (3) test results:

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

TABLE 3 hERG inhibition assay results

Example numbering	IC₅₀(μM)
		10	＞10
12	＞10
		34	＞10
38	＞10
		39	＞10

The test results show that the compounds of examples 10, 12, 34, 38, 39 of the invention have low affinity for hERG and compete with the affinity Tracer Tracer for IC₅₀All are > 10. mu.M.

Test example 4: biochemical CYP enzyme (cytochrome P450) inhibition assay

The test system comprises:

P450-Glo^TM CYP1A2 Screening System,(Promega Catalog:V9770)；

P450-Glo^TM CYP2D6 Screening System,(Promega Catalog:V9890)；

P450-Glo^TM CYP3A4 Screening System,(Promega Catalog:V9920)。

testing an instrument:

BMG PHERAstar FS。

the test method comprises the following steps:

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

4.1 inhibition of CYP1A 2:

test group: adding compounds to be detected with different concentrations (10 mu M/1 mu M) into a microplate, and adding Luciferin-ME (100 mu M) and KPO into each well₄(100mM) and CYP1A2 (0.01 pmol/. mu.L), were preincubated at room temperature for 10min, followed by addition of NADPH regenerating system for reaction at room temperature for 30min, and finally, addition of an equal volume of assay buffer for incubation at room temperature for 20min before chemiluminescence detection.

Negative control group: the experimental procedure was the same as in the test group except that the test compound was not added.

Blank control group: experimental methods were the same as for the test groups except that the test compound was not added and CYP1A2 Membrane (0.01 pmol/. mu.L) was used in place of CYP1A 2.

4.2 inhibition of CYP2D 6:

test group: adding compounds to be detected with different concentrations (10 mu M/1 mu M) into a microplate, and adding Luciferin-ME EGE (30 mu M) and KPO into each well₄(100mM) and CYP2D6 (5nM), preincubated at room temperature for 10min, followed by addition of NADPH regenerating system for reaction at 37 ℃ for 30min, and finally addition of an equal volume of assay buffer for incubation at room temperature for 20min before chemiluminescence detection.

Blank control group: experimental methods were the same as for the test groups except that the test compound was not added and CYP2D6 Membrane (5nM) was used instead of CYP2D 6.

4.3 inhibition of CYP3a 4:

test group: adding compounds to be detected with different concentrations (10 mu M/1 mu M) into a microplate, and adding Luciferin-IPA (3 mu M) and KPO into each well₄(100mM) And CYP3A4(2 nM), preincubated at room temperature for 10min, followed by addition of NADPH regenerating system for reaction at room temperature for 30min, and finally addition of an equal volume of detection buffer for incubation at room temperature for 20min before chemiluminescence detection.

Blank control group: experimental methods were the same as for the test groups except that the test compound was not added and CYP3A4 Membrane (2nM) was used instead of CYP3A 4.

Data processing:

percent inhibition (%) × 100 (1- (chemiluminescence value of test compound concentration group-chemiluminescence value of blank group)/(chemiluminescence value of negative control group-chemiluminescence value of blank group)).

Estimating the median Inhibitory Concentration (IC) of the compound according to the inhibition rate of the compound on CYP enzyme under different concentrations₅₀) Or a range. IC (integrated circuit)₅₀X (1-percent inhibition (%)/percent inhibition (%), where X is the compound concentration tested.

And (3) test results:

the inhibition of three CYPs by the compounds of the present invention was determined according to the above method, and the results are shown in Table 4.

TABLE 4 CYPs inhibition test results

And (4) conclusion:

the results show that the compounds of examples 12 and 17 of the invention have no obvious inhibition effect on 3 main CYP subtypes, and show that the potential drug interaction possibility is relatively low and the compounds have good drug property.

Test example 5: rat Pharmacokinetic (PK) study

Male SD rats were given the compound of the present application by Intravenous (IV) and intragastric (PO) administration, respectively, and pharmacokinetic profiles were examined. The dose of IV and PO is 1mg/kg and 5mg/kg, respectively, the IV solvent is 10% DMSO and 10% Solutol (polyethylene glycol-15 hydroxystearate) 80% of normal saline, and PO as a solvent comprises 10% of DMSO, 10% of Solutol (polyethylene glycol-15 hydroxystearate) and 80% of normal saline. Blood was collected at various time points after IV and PO dosing (0 h before dosing, 0.083, 0.25, 0.5, 1,2, 4, 6, 8 and 24h venous blood samples after dosing; 0h before dosing, 0.25, 0.5, 1,2, 4, 6, 8 and 24h oral blood samples after dosing). EDTA.K is adopted for blood₂Anticoagulated, centrifuged to obtain plasma samples, stored at-80 ℃. Plasma samples were processed for precipitated protein and analyzed by LC-MS/MS. Pharmacokinetic parameters were calculated using WinNonlin 6.3 software using a non-compartmental model, and the results are shown in table 5.

TABLE 5 pharmacokinetic parameters of Compounds administered IV and PO in plasma in rats in vivo

The bioavailability of 14-B in example 15 given orally (PO group) to rats was 32.3% compared to intravenous administration. It is shown that 14-B in example 15 of the present application absorbs better when administered in the vehicle 10% DMSO 10% Solutol (polyethylene glycol-15 hydroxystearate) 80% physiological saline.

Test example 6: mouse Pharmacokinetic (PK) study

Female balb/c mice were given the compound of the present application by Intravenous (IV) and intragastric (PO) administration, respectively, and pharmacokinetic profiles were examined. The dosages of IV and PO were 1mg/kg and 10mg/kg, respectively, with the IV vehicle being 5% DMSO: 5% Solutol (polyethylene glycol-15 hydroxystearate): 90% saline and the PO vehicle being 10% DMSO: 10% Solutol (polyethylene glycol-15 hydroxystearate): 80% saline. Collecting venous blood samples 0h before administration, 0.083, 0.25, 0.5, 1,2, 4, 6, 8 and 24h after administration; collecting 0h before administration, 0.25, 0.5, 1,2, 4, 6, 8 and 24h after administration, and collecting blood with EDTA.K₂Anticoagulated, centrifuged to obtain plasma samples, stored at-80 ℃. Plasma samples were processed for precipitated protein and analyzed by LC-MS/MS. Using WinNonlin 6.3 software, adopting non-softwareThe compartmental model calculated pharmacokinetic parameters and the results are shown in table 6.

TABLE 6 pharmacokinetic parameters of Compounds administered IV and PO in plasma in mice

The bioavailability of 14-B in example 15 given orally (PO group) in mice was 23.1% compared to intravenous administration. It is shown that 14-B in example 15 of the present application absorbs better when administered in the vehicle 10% DMSO 10% Solutol (polyethylene glycol-15 hydroxystearate) 80% physiological saline.

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, including all patents, patent applications, journal articles, books, and any other publications, cited in this application is hereby incorporated by reference in its entirety.

Reference documents:

[1]Wu and Kumar,Journal of Medicinal Chemistry,2018,61:422-443.

[2]Turnbull and Ioannidis,Nature,2017,550,481-486.

[3]Pozhidaeva and Bezsonova,DNA Repair,2019,76,30-39.

[4]Chauhan and Tian,Cancer Cell,2012,22,345-358.

[5]Zhou and Wang,Medicinal Chemistry,2018,14,3-18.

[6]Wang and Wu,PLoS One,2017,12,1-23。

Claims

1. a compound of formula I, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite, or mixture thereof:

wherein,

R¹is hydrogen, C_1-6Alkyl radical, C_6-10Aryl, 5-to 10-membered heteroaryl,

Halogen and cyano, wherein, said C_1-6Alkyl radical, C_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen, cyano, -OR^a、-NR^bR^c、-C_1-6alkylene-NR^bR^c、C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, optionally halogenated C_1-6Alkyl, -C (O) R⁶、-COOR⁷、-C(O)NR^bR^c、-S(O)_qR⁸、-S(O)_qNR^bR^c、-O-C_2-6alkylene-NR^bR^c、-NR^a-C_2-6alkylene-NR^bR^c；

R⁴is selected fromHydrogen, oxo, C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl, 5-10 membered heteroaryl, -OR^a、-NR^bR^cHalogen, cyano, -C (O) R⁶、-COOR⁷、-C(O)NR^bR^c、-O-C_2-6alkylene-NR^bR^cand-NR^d-C_2-6alkylene-NR^bR^cWherein, the C is_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen, cyano, C_1-6Alkyl, hydroxy, amino, -N (C)_1-6Alkyl radical)₂、-NH(C_1-6Alkyl groups);

R⁹、R¹⁰、R¹¹、R^eand R^fEach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl, 5-10 membered heteroaryl, or, R^e、R^fForm a 3-6 membered heterocyclic group with the nitrogen atom to which they are attached; wherein, the C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl, 5-10 membered heteroaryl, and 3-6 membered heterocyclyl are each independently optionally substituted with one or more substituents as follows: hydrogen, cyano, halogen, -OH, C_1-6Alkoxy, -NR^gR^h、C_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl, 5-10 membered heteroaryl, and, C in said substituents_1-6Alkyl radical, C_2-6Alkenyl radical, C_2-6Alkynyl, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl, C_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen, cyano, optionally halogenated C_1-6Alkyl radical, C_6-10Aryl, 5-10 membered heteroaryl;

R^g、R^heach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl and 3-8 membered heterocycloalkyl;

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

q is selected from 1 and 2;

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

2. The compound, pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite, or mixture thereof, according to claim 1, wherein,

R¹is selected from C_1-6Alkyl radical, C_6-10Aryl and

wherein, the C_1-6Alkyl and C_6-10Each aryl group is independently optionally substituted with one or more of the following:hydrogen, halogen, -OR^a、-C_1-6alkylene-NR^bR^c；

r is selected from 0, 1 and 2;

R⁴selected from hydrogen, -NR^bR^c、C_1-6Alkyl and-OR^a；

m is selected from 0, 1 and 2;

R^a、R^band R^cEach independently selected from hydrogen and C_1-6An alkyl group;

preferably, R is selected from hydrogen and halogen.

3. The compound, pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite, or mixture thereof, according to claim 1 or 2, wherein,

R¹is selected from C_1-6Alkyl radical, C_6-10Aryl and

wherein, the C_6-10Aryl is optionally substituted with one or more of the following: hydrogen, hydroxy, NH₂-C_1-6Alkylene-, halogen;

preferably, R⁴Selected from hydrogen, amino, CH₃-NH-、(CH₃)₂N-, methyl and hydroxy.

4. The compound, pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite, or mixture thereof, according to any one of claims 1-3, wherein,

R¹is selected from C_1-6Alkyl radical, C_6-10Aryl and

ring A is C_6-10An aryl group;

5. The compound, pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite, or mixture thereof, according to any one of claims 1-4, wherein,

the ring A is phenyl;

ring B is selected from pyrazolyl, 3-pyrrolinyl, pyrrolidinyl, cyclopentenyl and cyclopentyl.

6. The compound, pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite, or mixture thereof, according to any one of claims 1-5, wherein,

R¹selected from methyl,

Preferably, R¹Selected from methyl,

7. The compound, pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite, or mixture thereof, according to any one of claims 1 to 6, wherein,

R²selected from hydrogen, halogen, optionally halogenated C_1-6Alkyl and C_3-8A cycloalkyl group;

preferably, R²Selected from hydrogen, fluoro, methyl and trifluoromethyl;

more preferably, R²Is hydrogen.

8. The compound, pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite, or mixture thereof, according to any one of claims 1 to 7, wherein,

R³selected from the group consisting of-C (O) R⁹and-C (O) NR^eR^f；R⁹、R^e、R^fEach independently selected from hydrogen and C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl and 3-8 membered heterocycloalkyl, wherein said C_1-6Alkyl radical, C_2-6Alkenyl radical, C_3-8Cycloalkyl, 3-8 membered heterocycloalkyl are each independently optionally substituted with one or more substituents as follows: halogen, optionally halogenated C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_6-10Aryl, 5-to 10-membered heteroAn aryl group; and, C in the substituents_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more of the following: hydrogen, halogen, optionally halogenated C_1-6An alkyl group;

preferably, R³is-C (O) R⁹(ii) a Wherein R is⁹Is C optionally substituted by one or more substituents_1-6Alkyl groups: optionally halogenated C_1-6Alkyl radical, C_3-8Cycloalkyl radical, C_6-10Aryl, 5-to 10-membered heteroaryl, wherein, C is_6-10Aryl and 5-10 membered heteroaryl are each independently optionally substituted with one or more halogens;

more preferably, R³is-C (O) R⁹(ii) a Wherein R is⁹Is C optionally substituted by one or more substituents_1-6Alkyl groups: methyl, difluoromethyl, trifluoromethyl, cyclopropyl, phenyl, halophenyl, halopyrazolyl;

further preferably, R³is-C (O) R⁹Wherein R is⁹Selected from the following groups:

9. the compound of any one of claims 1 to 8, pharmaceutically acceptable salts, esters, solvates, stereoisomers, tautomers, prodrugs, any crystal forms, metabolites or mixtures thereof, wherein the compound is selected from:

10. a pharmaceutical composition comprising a compound of any one of claims 1 to 9, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite, or mixture thereof, and optionally a pharmaceutically acceptable adjuvant;

preferably, the pharmaceutical composition further comprises other effective ingredients for inhibiting the activity of deubiquitinase USP7 or other effective ingredients for preventing or treating diseases or disorders related to modulation of deubiquitinase USP 7;

more preferably, said disease or disorder associated with modulation of deubiquitinase USP7 is selected from the group consisting of cancer, neurodegenerative diseases, diabetes, bone and joint diseases, arthritic inflammatory disorders, osteoporosis, immune disorders, cardiovascular diseases, ischemic diseases, viral infectious diseases and bacterial infectious diseases.

11. A pharmaceutical product comprising a compound of any one of claims 1 to 9, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite or mixture thereof, or a pharmaceutical composition of claim 10, and optionally a product package.

12. Use of a compound of any one of claims 1 to 9, a pharmaceutically acceptable salt, ester, solvate, stereoisomer, tautomer, prodrug, any crystal form, metabolite or mixture thereof, or a pharmaceutical composition of claim 10, for the manufacture of a de-ubiquitinase USP7 inhibitor or for the manufacture of a medicament for the prevention or treatment of a disease or disorder associated with modulation of de-ubiquitinase USP 7;

preferably, said diseases or conditions associated with modulation of deubiquitinase USP7 are selected from cancer, neurodegenerative diseases, diabetes, bone and joint diseases, arthritic inflammatory conditions, osteoporosis, immune conditions, cardiovascular diseases, ischemic diseases, viral infectious diseases and bacterial infectious diseases;

more preferably, the cancer is selected from at least one of hepatocellular carcinoma, multiple myeloma, colon cancer, lung cancer, prostate cancer, and bladder cancer.