CN112047933A

CN112047933A - Quinazolinone USP7 inhibitor and preparation method and application thereof

Info

Publication number: CN112047933A
Application number: CN202011104532.1A
Authority: CN
Inventors: 刘宏民; 李鹏; 王志茹; 郑一超; 刘瀛
Original assignee: Zhengzhou University
Current assignee: Zhengzhou University
Priority date: 2020-10-15
Filing date: 2020-10-15
Publication date: 2020-12-08
Anticipated expiration: 2040-10-15
Also published as: CN112047933B

Abstract

The invention discloses a quinazolinone USP7 inhibitor which has a structure shown in a general formula I:

the inhibitor has good inhibition effect on ubiquitin-specific protease 7, can be used for preparing a medicament for inhibiting ubiquitin-specific protease 7, has good inhibition activity on gastric cancer cell lines, particularly MGC-803 cell lines, provides a lead compound structure for anticancer medicaments, and has good application prospect. The invention also provides a preparation method of the quinazolinone USP7 inhibitor, which is simple in preparation method, mild in reaction condition, less in by-product, high in reaction yield and convenient for batch production and commercial application.

Description

Quinazolinone USP7 inhibitor and preparation method and application thereof

Technical Field

The invention relates to the field of pharmaceutical chemistry, and in particular relates to a quinazolinone USP7 inhibitor, and a preparation method and application thereof.

Background

The process of covalent binding of ubiquitin to a target protein is called ubiquitination, which is a post-translational modification process of proteins that primarily regulates the stability, function, and intracellular localization of target proteins. In eukaryotic cells, selective degradation of many short cyclins is achieved by the ubiquitin-proteasome pathway. In recent years, more and more studies have shown that dysregulation of this pathway can cause a number of diseases, such as immune diseases, neurological diseases, and tumors.

Deubiquitinase is an important protease in the ubiquitin-proteasome pathway, which can deubiquitinate ubiquitinated proteins and reverse the fate of the proteins for degradation by the proteasome. The human genome encodes nearly 100 deubiquitinating enzymes, which can be divided into 5 major classes: ubiquitin C-terminal hydrolase (UCH), ubiquitin-specific protease (USP/UBP), Machado-Joseph disease protein domain protease (MJD), JAMM metalloprotease (JAB1/MPN/Mov34 metalloenzyme). USP7 belongs to ubiquitin-specific protease, which plays an important role in maintaining normal cell metabolism, including participating in the regulation of the expression level of tumor suppressor p53 by p53-MDM2-USP7 pathway, influencing the distribution of FOXO4(Forkhead box O4) between nucleus and cytoplasm by deubiquitinating, and losing tumor suppression by removing PTEN (Phosphatase and tensin homologue deleted in chromosome 10) from nucleus, etc. USP7 is widely involved in important pathways in cells, so its abnormal expression often causes many diseases, especially has important role in tumorigenesis. Under pathological conditions, USP7 is highly expressed in cancer cells, and can selectively de-ubiquitinate MDM2(Mouse double minute2) and p53, while the main de-ubiquitination object is MDM2, which enhances the activity of MDM2 as E3 ligase, causes the enhancement of ubiquitination of p53, and finally leads to the degradation of p53 by proteasome to lose tumor inhibition effect. Antagonizing the activity of USP7 and indirectly enhancing the antitumor effect of p53 provides a brand new idea for treating cancer.

In recent years, more and more research has been conducted around USP7, but most of them illustrate the role of USP7 in the development of disease, and there are few reports on USP7 inhibitors. The USP7 inhibitors reported in the literature are roughly classified into two covalent bonding type inhibitors and non-covalent type inhibitors, and their molecular skeletons are mainly classified into substituted thiophene derivatives, acridine derivatives, quinazolinone derivatives, indolopyrazine derivatives and 2-amino-4-ethylpyridine derivatives, and the activities of these compounds are mostly in micromolar order. So far, no drug which targets USP7 appears on the market, so that the development of the USP7 inhibitor has wide prospect.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a novel quinazolinone USP7 inhibitor which can be used as an anti-tumor drug targeting ubiquitin-specific protease 7

The invention also aims to provide a preparation method of the quinazolinone USP7 inhibitor, which is simple in preparation method, mild in reaction condition and less in by-product.

The invention also aims to provide application of the quinazolinone USP7 inhibitor.

One of the purposes of the invention is realized by adopting the following technical scheme:

quinazolinone USP7 inhibitor has a structure shown in a general formula I:

in the general formula I, R₁Is selected from

One of (1);

in the general formula I, R₂Is selected from：

One kind of (1).

Further, the quinazolinone USP7 inhibitor is selected from the following compounds:

the second purpose of the invention is realized by adopting the following technical scheme:

the preparation method of the quinazolinone USP7 inhibitor comprises the following steps:

(1) synthesis of Compound 3: adding N-Boc-piperidone and trimethyl sulfoxide iodide into a solvent, stirring in an ice bath for reaction under the action of an alkaline substance, and performing post-treatment after the reaction to obtain a compound 3; further, the post-treatment process comprises the processes of washing with water, extracting, drying, filtering, concentrating, column chromatography and the like, wherein the used solvent is one of N, N-dimethylformamide, dimethyl sulfoxide and ethanol. The base is one of triethylamine, potassium tert-butoxide, sodium hydride and sodium hydroxide.

(2) Synthesis of Compound 5: dissolving compound 4 in formamide, reacting under heating for a period of time, cooling, adding compound 3 and alkali, reacting for a period of time, and post-treating to obtain compound 5, wherein R₃Is Cl or Br; further, the method can be used for preparing a novel materialHeating to 130-200 deg.C, maintaining for 12h, cooling to 60-100 deg.C, adding anhydrous potassium carbonate, and maintaining for 12 h.

(3) Synthesis of compound 8: adding acid into the compound 5, stirring for a period of time at room temperature, removing redundant acid after the reaction is finished, adding the compound 6, and carrying out condensation reaction under a HATU/TEA system to obtain a compound 8; further, the acid is one of trifluoroacetic acid, concentrated hydrochloric acid and concentrated sulfuric acid.

Further, the solvent used in the HATU/TEA system is one of N, N-dimethylformamide, dimethylsulfoxide and tetrahydrofuran. The alkali is one of triethylamine, potassium carbonate, sodium hydroxide and potassium tert-butoxide.

(4) Synthesis of compounds of general formula I: taking the compound 8 and the compound 7 to carry out Buchwald-Hartwig coupling reaction to obtain the product with the general formula I. Further, the catalytic system is cuprous iodide/ligand/potassium phosphate, and the ligand is one of 2-hydroxyphenyl morpholinomethanone, L-proline and 1, 10-phenanthroline. The alkali is one of potassium carbonate, potassium phosphate, sodium hydroxide and potassium tert-butoxide. The solvent is one of N, N-dimethylformamide, dimethyl sulfoxide, ethanol and toluene.

The third purpose of the invention is realized by adopting the following technical scheme:

the quinazolone USP7 inhibitor is applied to preparing medicines for inhibiting ubiquitin-specific protease 7.

Furthermore, the quinazolinone USP7 inhibitor is applied to preparation of anti-tumor drugs targeting ubiquitin-specific protease 7.

Furthermore, the anti-tumor drug is an anti-gastric cancer drug.

Compared with the prior art, the invention has the beneficial effects that: the invention provides a quinazolinone ubiquitin-like specific protease (USP7) inhibitor, which can be used for preparing a medicament for inhibiting ubiquitin specific protease 7, has a certain degree of inhibition effect on a gastric cancer cell line, particularly an MGC-803 cell line, shows anti-tumor activity, provides a lead compound structure for an anti-cancer medicament, and has a good application prospect. The invention also provides a preparation method of the quinazolinone USP7 inhibitor, which is simple in preparation method, mild in reaction condition, less in by-product, high in reaction yield and convenient for batch production and commercial application.

Drawings

FIG. 1 is a schematic diagram of the synthetic routes of compounds I-1 to I-39 in examples 1 to 39 of the present invention;

FIG. 2 is a graph showing the effect of Compound I-29 on the expression level of p53-MDM2-USP7 pathway-related protein in a gastric cancer cell line MGC-803, wherein Compound I-32 is a negative control compound and Compound I-1 is a positive control compound;

FIG. 3 shows the effect of compounds I-35, I-36 and I-39 on the expression level of p53-MDM2-USP7 pathway and its downstream related proteins in a gastric cancer cell line BGC-823.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.

The intermediate compound 8 related to the embodiment of the invention corresponds to structural formulas I-1 to I-26, and is shown in Table 1, and the synthetic route is shown in figure 1.

TABLE 1

The structural formulas of representative compounds I-27 to I-39 with the general formula I related to the embodiment of the invention are shown in the table 2, and the synthetic route is shown in the figure 1.

TABLE 2

Example 1

Compound (I)

The synthesis process of (A) is as follows:

(1) synthesis of Compound 3: dissolving trimethyl sulfoxide iodide (3.094g,14.06mmol,1.4eqv) in 15mL of anhydrous DMSO, adding sodium hydride (0.562g,14.06mmol,1.4eqv, 60% in kerosene) in three portions under ice bath condition, stirring at 0 deg.C for 30 min, adding N-Boc-4-piperidone (2.0g,10.04mmol,1.0eqv), and stirring at the temperature for 4 hr; 40mL of water was added, extracted with ethyl acetate (3X 90mL), washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product, which was subjected to column chromatography (PE: EA: 10:1, then PE: EA: 5:1) to give compound 3(1.285g, white waxy solid, yield 60%).¹H NMR(400MHz,Chloroform-d)3.71(dt,J＝9.9,4.9Hz,2H),3.43(ddd,J＝13.3,9.5,3.7Hz,2H),2.69(s,2H),1.80(ddd,J＝13.8,9.4,4.5Hz,2H),1.47(s,11H).¹³C NMR(101MHz,Chloroform-d)154.79,79.73,77.28,57.16,53.74,42.49,28.44.

(2) Synthesis of Compound 5: suspending 2-amino-4-chlorobenzoic acid (1.0g,5.83mmol,1.0eqv) in 10mL formamide, heating to 150 deg.C, stirring for 12 hr, cooling to 80 deg.C, adding compound 3(1.367g,6.41mmol,1.1eqv) and anhydrous potassium carbonate (2.417g,17.49mmol,3.0 eqv) in sequence) After the reaction was completed, 60mL of water was added, and the mixture was extracted with ethyl acetate (3X 90mL), washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to obtain a crude product, and 20mL of a mixed solvent of petroleum ether and ethyl acetate (volume ratio: 1) was added, and the mixture was stirred at room temperature for 30 minutes to obtain a large amount of white solid, which was then subjected to suction filtration and dried at room temperature to obtain Compound 5(1.922g, white solid, yield 83.7%).¹H NMR(400MHz,DMSO-d₆)8.29(s,1H),8.16(d,J＝8.6Hz,1H),7.75(d,J＝2.1Hz,1H),7.58(dd,J＝8.6,2.0Hz,1H),4.93(s,1H),4.00(s,2H),3.66(d,J＝13.1Hz,2H),3.05(s,2H),1.48(td,J＝12.4,10.9,4.2Hz,2H),1.39(s,11H).¹³C NMR(101MHz,DMSO-d₆)160.16,153.79,150.39,148.98,138.87,128.42,127.14,126.19,120.32,78.47,69.13,53.77,34.28,28.05.

(3) Synthesis of Compound I-1: dissolving compound 5(2.0g,5.08mmol,1.0eqv) in 6mL of trifluoroacetic acid, stirring at room temperature for 4 hours, after the reaction is finished, evaporating the trifluoroacetic acid under reduced pressure, adding 10mL of DMF, and then sequentially adding 3-phenylpropionic acid (1.144g,7.62mmol,1.5eqv), triethylamine (1.028g,10.16mmol,2.0eqv), HATU (3.863g,10.16mmol,2.0eqv), and stirring at room temperature for 5 hours after the addition is finished; adding 50mL of water, extracting with ethyl acetate, washing with saturated brine, drying with anhydrous magnesium sulfate, filtering, concentrating to obtain a crude product, adding 5mL of ethyl acetate, standing overnight, filtering, concentrating the filtrate, and performing column chromatography (PE: EA: 2:1, then PE: EA: 1:2) to obtain a compound I-1(1.731g, colorless oily substance, yield 80%).¹H NMR(400MHz,DMSO-d₆)8.30(s,1H),8.16(d,J＝8.7Hz,1H),7.74(s,1H),7.57(d,J＝8.7Hz,1H),7.25(d,J＝9.3Hz,4H),7.16(t,J＝7.4Hz,1H),4.98(s,1H),4.09(d,J＝13.0Hz,1H),3.99(s,2H),3.63(d,J＝13.6Hz,1H),3.23(t,J＝12.2Hz,1H),2.92(t,J＝11.6Hz,1H),2.81(t,J＝7.8Hz,2H),2.71–2.57(m,2H),2.51(s,1H),1.43(d,J＝15.6Hz,4H),1.27(s,0H).¹³C NMR(101MHz,DMSO-d₆)169.56,160.12,150.32,148.97,141.42,138.87,128.39,128.34,128.14,127.10,126.19,125.76,120.32,69.26,53.79,40.80,36.94,34.89,34.20,33.87,30.87.

HRMS(ESI):Calcd.C₂₃H₂₄ClN₃O₃,[M+H]⁺,m/z:426.1506,found:426.1568。

Example 2

Compound (I)

The synthesis process of (A) is as follows:

wherein the procedures of steps (1) to (3) are the same as in example 1;

the same procedures used in example 1 were repeated except for replacing 3-phenylpropionic acid with benzoic acid to give Compound I-2 (colorless oil, yield 85%).¹H NMR(400MHz,DMSO-d₆)8.31(s,1H),8.16(d,J＝8.6Hz,1H),7.75(d,J＝2.1Hz,1H),7.57(dd,J＝8.5,2.1Hz,1H),7.47–7.42(m,3H),7.39(dd,J＝6.7,3.0Hz,2H),5.05(s,1H),4.21(s,1H),4.04(s,2H),3.27(s,1H),3.15(s,1H),1.63(s,2H),1.54(s,1H),1.36(d,J＝13.6Hz,1H).¹³C NMR(101MHz,DMSO-d₆)168.90,160.18,150.38,148.98,138.87,136.30,129.27,128.41,128.34,127.12,126.62,126.19,120.34,69.40,53.85.HRMS(ESI):Calcd.C₂₁H₂₀ClN₃O₃,[M+H]⁺,m/z:398.1193,found:398.1262。

Example 3

Compound (I)

The synthesis process of (A) is as follows:

wherein the procedures of steps (1) to (3) are the same as in example 1;

the same procedures used in example 1 were repeated except for replacing 3-phenylpropionic acid with 2-pyrrolecarboxylic acid to give compound I-3 (brown solid, yield 45%). Melting point: 116.8 to 116.9 ℃.¹H NMR(400MHz,DMSO-d₆)11.41(s,1H),8.32(s,1H),8.17(d,J＝8.6Hz,1H),7.76(d,J＝2.2Hz,1H),7.58(dd,J＝8.6,2.1Hz,1H),6.87(s,1H),6.46(s,1H),6.10(q,J＝2.8Hz,1H),5.04(s,1H),4.20–3.94(m,4H),3.30(s,1H),1.72–1.55(m,2H),1.48(d,J＝13.4Hz,2H).¹³C NMR(101MHz,DMSO-d₆)161.39,160.18,150.39,148.99,138.89,128.44,127.15,126.21,124.37,120.82,120.34,111.34,108.22,69.50,53.81,34.75.HRMS(ESI):Calcd.C₁₉H₁₉ClN₄O₃,[M+H]⁺,m/z:387.1146,found:387.1208。

Example 4

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with pyridine-2-carboxylic acid, and the rest was the same as in example 1. The product was a brown solid in 31% yield. Melting point: 124.3-124.6 ℃.¹H NMR(400MHz,DMSO-d₆)8.59(d,J＝4.8Hz,1H),8.31(s,1H),8.16(d,J＝8.6Hz,1H),7.98–7.88(m,1H),7.75(d,J＝2.2Hz,1H),7.61–7.52(m,2H),7.47(dd,J＝7.5,5.1Hz,1H),5.06(s,1H),4.23(d,J＝13.0Hz,1H),4.09(d,J＝13.7Hz,1H),4.01(d,J＝13.7Hz,1H),3.48(d,J＝13.6Hz,1H),3.32–3.05(m,2H),1.72–1.51(m,3H),1.37(d,J＝13.6Hz,1H),1.25–1.14(m,0H).¹³C NMR(101MHz,DMSO-d₆)166.59,160.17,154.35,150.37,148.97,148.37,138.89,137.28,128.42,127.15,126.20,124.37,122.77,120.32,69.39,53.83,42.40,37.40,34.90,34.24.HRMS(ESI):Calcd.C₂₀H₁₉ClN₄O₃,[M+H]⁺,m/z:399.1146,found:399.1223。

Example 5

Compound (I)

The difference between the synthesis process of (a) and example 1 is: the 3-phenylpropionic acid was replaced with nicotinic acid, and the rest was the same as in example 1. The product was a brown solid in 40% yield. Melting point: 145.5-145.7 ℃.¹H NMR(400MHz,DMSO-d₆)8.67–8.59(m,2H),8.30(s,1H),8.16(d,J＝8.6Hz,1H),7.84(d,J＝7.9Hz,1H),7.75(d,J＝2.1Hz,1H),7.58(dd,J＝8.6,2.1Hz,1H),7.48(dd,J＝7.7,5.0Hz,1H),5.06(s,1H),4.23(d,J＝12.8Hz,1H),4.04(s,2H),3.14(t,J＝12.1Hz,1H),1.66(d,J＝12.4Hz,2H),1.53(d,J＝13.6Hz,1H),1.38(d,J＝13.5Hz,1H).¹³C NMR(101MHz,DMSO-d₆)166.61,160.18,150.38,150.23,148.98,147.38,138.87,134.52,132.07,128.42,127.13,126.19,123.48,120.35,69.35,53.87.HRMS(ESI):Calcd.C₂₀H₁₉ClN₄O₃,[M+H]⁺,m/z:399.1146,found:399.1218。

Example 6

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with 3, 4-dimethoxybenzoic acid, and the rest was the same as in example 1. The product was a white solid in 65% yield. Melting point: 250.4-250.7 ℃.¹H NMR(400MHz,DMSO-d₆)8.30(s,1H),8.16(d,J＝8.6Hz,1H),7.75(d,J＝2.1Hz,1H),7.58(dd,J＝8.6,2.1Hz,1H),6.97(d,J＝8.8Hz,3H),5.02(s,1H),4.04(s,3H),3.78(d,J＝4.6Hz,6H),3.58(d,J＝22.9Hz,1H),3.21(s,2H),1.78–1.55(m,2H),1.42(d,J＝18.6Hz,2H).¹³C NMR(101MHz,DMSO-d₆)168.89,160.18,150.38,149.63,148.98,148.37,138.87,128.41,128.36,127.13,126.19,120.34,119.57,111.07,110.83,69.45,55.53,55.50,53.86.HRMS(ESI):Calcd.C₂₃H₂₄ClN₃O₅,[M+H]⁺,m/z:458.1404,found:458.1477。

Example 7

Compound (I)

The difference between the synthesis process of (a) and example 1 is: the 3-phenylpropionic acid was replaced with 2-phenoxyacetic acid, and the rest was the same as in example 1. The product was a colorless oil in 85% yield.¹H NMR(400MHz,DMSO-d₆)8.31(s,1H),8.17(d,J＝8.6Hz,1H),7.76(d,J＝2.1Hz,1H),7.58(dd,J＝8.7,2.1Hz,1H),7.27(t,J＝7.8Hz,2H),6.91(d,J＝8.4Hz,3H),5.05(s,1H),4.86–4.73(m,2H),4.02(q,J＝13.9Hz,3H),3.64(d,J＝13.6Hz,1H),3.30(t,J＝12.4Hz,1H),2.99(t,J＝11.7Hz,1H),1.66(t,J＝11.1Hz,1H),1.46(q,J＝10.6,8.6Hz,3H).¹³C NMR(101MHz,DMSO-d₆)165.55,160.16,158.03,150.37,148.98,138.90,129.30,128.42,127.17,126.22,120.73,120.33,114.51,69.26,65.87,53.78,37.18,34.80,34.16.HRMS(ESI):Calcd.C₂₂H₂₂ClN₃O₄,[M+Na]⁺,m/z:450.1299,found:450.1184。

Example 8

Compound (I)

The difference between the synthesis process of (a) and example 1 is: the 3-phenylpropionic acid was replaced with 2-nitrobenzoic acid, and the rest was the same as in example 1. The product was a brown solid in 22% yield. Melting point: 162.1-162.2 ℃.¹H NMR(400MHz,DMSO-d₆)8.30(s,1H),8.17(dd,J＝14.2,8.4Hz,2H),7.84(t,J＝7.7Hz,1H),7.75(d,J＝2.0Hz,1H),7.69(t,J＝7.9Hz,1H),7.58(dd,J＝8.6,2.1Hz,1H),5.08(s,1H),4.21(s,1H),4.07(d,J＝13.6Hz,1H),4.01(d,J＝13.8Hz,1H),3.21(s,2H),3.20–3.13(m,1H),1.67(s,1H),1.56(d,J＝13.6Hz,2H),1.34(d,J＝13.6Hz,1H).¹³C NMR(101MHz,DMSO-d₆)165.06,160.17,150.37,148.98,138.90,134.80,132.68,130.08,128.41,127.90,127.16,126.21,124.67,120.32,69.36,53.80,36.91,34.18,33.77.HRMS(ESI):Calcd.C₂₁H₁₉ClN₄O₅，[M+Na]⁺,m/z:465.1044,found:465.0940。

Example 9

Compound (I)

The difference between the synthesis process of (a) and example 1 is: the same procedure as in example 1 was repeated except that 3-phenylpropionic acid was replaced with 3-phenyl-3-methylpropionic acid. The product was a white solid in 50% yield. Melting point: 157.2-157.4 ℃.¹H NMR(600MHz,DMSO-d6)8.28(d,J＝15.3Hz,1H),8.16(d,J＝8.5Hz,1H),7.81–7.73(m,1H),7.59(dd,J＝8.5,2.1Hz,1H),7.33–7.21(m,5H),7.16(ddd,J＝8.7,6.2,2.5Hz,1H),4.95(d,J＝7.2Hz,1H),4.13–4.00(m,1H),4.00–3.87(m,1H),3.66(t,J＝14.1Hz,1H),3.27–3.11(m,2H),2.98–2.81(m,1H),2.67–2.56(m,2H),1.48–1.28(m,3H),1.26–1.13(m,5H).¹³C NMR(151MHz,DMSO-d6):22.25,22.35,22.55,34.67,34.81,35.38,35.51,36.39,36.49,36.71,37.39,38.72,40.70,41.42,41.53,44.05,54.28,69.71,69.77,120.85,126.41,126.45,126.73,127.18,127.36,127.40,127.68,128.67,128.70,128.74,128.94,139.41,147.06,147.18,149.50,150.89,160.62,165.08,169.60,173.38.HRMS(ESI):Calcd.C₂₄H₂₆ClN₃O₃,[M+Na]⁺,m/z:462.1663,found:462.1562。

Example 10

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with 3-bromobenzoic acid, and the rest was the same as in example 1. The product was a colorless oil in 65% yield.¹H NMR(400MHz,DMSO-d₆)8.29(s,1H),8.16(d,J＝8.6Hz,1H),7.75(d,J＝2.1Hz,1H),7.71–7.53(m,3H),7.40(d,J＝4.9Hz,2H),5.03(s,1H),4.18(d,J＝12.1Hz,1H),4.03(s,2H),3.27(s,1H),3.15(d,J＝26.0Hz,1H),1.65(d,J＝12.8Hz,2H),1.50(d,J＝12.7Hz,1H),1.37(s,1H).¹³C NMR(101MHz,DMSO-d₆)167.14,160.18,150.40,148.99,138.87,138.65,132.09,130.63,129.29,128.44,127.14,126.20,125.55,121.66,120.35,69.37,53.85,43.02,34.67。HRMS(ESI):Calcd.C₂₁H₁₉BrClN₃O₃，[M+2+H]⁺,m/z:478.0298,found:478.0350.

Example 11

Compound (I)

The difference between the synthesis process of (a) and example 1 is: the procedure of example 1 was repeated except for replacing 2-amino-4-chlorobenzoic acid with 2-amino-4-bromobenzoic acid. The product was a colorless oil in 90% yield.¹H NMR(400MHz,DMSO-d₆)8.28(s,1H),8.08(d,J＝8.7Hz,1H),7.90(s,1H),7.70(d,J＝8.6Hz,1H),7.31–7.13(m,5H),4.96(s,1H),4.09(d,J＝12.9Hz,1H),3.98(s,1H),3.63(d,J＝13.6Hz,1H),3.22(t,J＝12.4Hz,1H),2.97–2.87(m,1H),2.81(t,J＝7.8Hz,2H),2.69(d,J＝3.4Hz,2H),2.66–2.57(m,2H),1.41(q,J＝15.3,13.6Hz,4H).¹³C NMR(101MHz,DMSO-d₆)169.57,160.25,150.27,149.03,141.43,129.89,129.29,128.39,128.35,128.19,128.15,127.82,125.76,120.62,69.26,53.83,40.80,38.20,36.94,34.89,34.21,33.86,30.86.HRMS(ESI):Calcd.C₂₃H₂₄BrN₃O₃,[M+H]⁺,m/z:470.1001,found:470.1061。

Example 12

Compound (I)

The difference between the synthesis process of (a) and example 1 is: the 3-phenylpropionic acid was replaced with 4, 5-dimethoxy-2-nitrobenzoic acid, and the rest was the same as in example 1. The product was a white solid in 55% yield. Melting point: 138.4-138.6 ℃.¹H NMR(400MHz,DMSO-d₆)8.29(s,1H),8.16(d,J＝8.6Hz,1H),7.75(d,J＝2.1Hz,1H),7.58(dd,J＝8.6,2.1Hz,1H),6.60(s,1H),6.36(s,1H),4.97(s,1H),4.02(s,2H),3.82–3.73(m,2H),3.70(s,3H),3.63(s,3H),3.19(t,J＝11.8Hz,2H),1.68–1.56(m,2H),1.42(d,J＝13.3Hz,2H).¹³C NMR(101MHz,DMSO-d₆)168.73,160.17,150.85,150.41,148.99,141.32,139.54,138.86,128.43,127.13,126.19,120.35,112.83,110.05,100.18,69.47,56.41,55.14,53.81,34.61.HRMS(ESI):Calcd.C₂₃H₂₃ClN₄O₇,[M+H]⁺,m/z:503.1255,found:503.1535.

Example 13

Compound (I)

The difference between the synthesis process of (a) and example 1 is: the same procedures used in example 1 were repeated except for replacing 3-phenylpropionic acid with 2-amino-4-chlorobenzoic acid. The product was a brown solid in 26.7% yield. Melting point: 132.1-132.2 ℃.¹H NMR(400MHz,Chloroform-d)8.22(d,J＝8.6Hz,1H),8.09(s,1H),7.71(d,J＝2.1Hz,1H),7.48(dd,J＝8.5,2.0Hz,1H),6.97(d,J＝8.1Hz,1H),6.86–6.59(m,2H),4.09(s,5H),3.49(s,1H),3.32(d,J＝12.9Hz,3H),1.80–1.37(m,4H).¹³C NMR(101MHz,Chloroform-d)169.27,162.23,148.98,148.23,146.94,141.12,136.43,128.99,128.38,128.30,127.09,120.11,117.71,117.55,116.40,70.55,56.29.HRMS(ESI):Calcd.C₂₁H₂₀Cl₂N₄O₃,[M+H]⁺,m/z:447.0912,found:447.0978。

Example 14

Chemical combinationArticle (A)

The difference between the synthesis process of (a) and example 1 is: the 3-phenylpropionic acid was replaced with 2-amino-4-bromobenzoic acid, and the rest was the same as in example 1. The product was a brown solid in 24.8% yield. Melting point: 133.8-134.0 ℃.¹H NMR(400MHz,Chloroform-d)8.23(d,J＝8.5Hz,1H),8.04(d,J＝28.7Hz,1H),7.73(s,1H),7.49(d,J＝8.6Hz,1H),7.06–6.76(m,3H),4.12(d,J＝9.6Hz,5H),3.35(s,4H),1.65(s,5H).¹³C NMR(101MHz,Chloroform-d)169.32,162.35,148.97,148.12,147.10,129.13,128.40,127.14,124.67,120.44,120.11,119.38,118.16,70.60.HRMS(ESI):Calcd.C₂₁H₂₀BrClN₄O₃,[M+2+H]⁺,m/z:493.0407,found:493.0450.

Example 15

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with 2, 3-dichlorobenzoic acid, and the rest was the same as in example 1. The product was a brown solid in 17.4% yield. Melting point: 226.3-227.0 ℃.¹H NMR(400MHz,Chloroform-d)8.22(dd,J＝8.5,6.3Hz,1H),8.09(d,J＝13.4Hz,1H),7.82–7.67(m,1H),7.48(d,J＝8.2Hz,2H),7.17(dd,J＝22.9,7.6Hz,1H),4.52(dd,J＝39.2,13.6Hz,1H),4.28–3.94(m,2H),3.52–3.19(m,3H),1.74(d,J＝4.7Hz,3H),1.54(d,J＝12.6Hz,2H).¹³C NMR(101MHz,Chloroform-d)162.50,162.14,148.27,148.07,141.20,133.76,130.94,130.85,128.38,128.31,128.16,127.14,125.62,120.11,70.52,70.42,56.73,56.02,42.86,42.24,37.28,35.89,35.46,34.99.HRMS(ESI):Calcd.C₂₁H₁₈Cl₃N₃O₃,[M+Na]⁺,m/z:488.0414,found:488.0296.

Example 16

Compound (I)

The difference between the synthesis process of (a) and example 1 is: by replacing 3-phenylpropionic acid2-Nitrophenylacetic acid, the rest being the same as in example 1. The product was a white solid in 45% yield. Melting point: 122.0-122.9 ℃.¹H NMR(400MHz,DMSO-d₆)8.32(s,1H),8.17(d,J＝8.5Hz,1H),8.04(dd,J＝8.1,1.3Hz,1H),7.76(d,J＝2.1Hz,1H),7.67(td,J＝7.5,1.4Hz,1H),7.62–7.49(m,2H),7.46(dd,J＝7.7,1.5Hz,1H),5.05(s,1H),4.18(d,J＝16.6Hz,1H),4.11(d,J＝15.0Hz,2H),4.00(td,J＝10.5,8.6,4.4Hz,2H),3.80(dd,J＝14.2,3.8Hz,1H),3.39(td,J＝11.1,5.7Hz,1H),2.99(ddd,J＝13.9,10.6,4.0Hz,1H),1.69(td,J＝12.4,11.3,4.2Hz,1H),1.54–1.39(m,3H).¹³C NMR(101MHz,DMSO-d₆)166.86,160.17,150.37,149.14,149.00,138.90,133.47,133.33,131.92,128.42,128.02,127.16,126.22,124.46,120.33,69.31,53.80,40.99,37.87,37.37,34.86,34.39.HRMS(ESI):Calcd.C₂₂H₂₁ClN₄O₅,[M+Na]⁺,m/z:479.1200,found:479.1081.

Example 17

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with 5-methoxy-2-nitrophenylacetic acid, and the rest was the same as in example 1. The product was a brown solid in 20.6% yield. Melting point: 139.5 to 139.7 ℃.¹H NMR(400MHz,Chloroform-d)8.20(q,J＝8.8,8.3Hz,2H),8.04(s,1H),7.72(d,J＝8.4Hz,1H),7.47(t,J＝8.7Hz,1H),7.01–6.94(m,1H),6.80(d,J＝13.8Hz,1H),4.65(d,J＝13.3Hz,1H),4.29(dd,J＝22.0,14.2Hz,1H),4.14–4.08(m,1H),4.00(d,J＝14.1Hz,0H),3.92(d,J＝10.6Hz,3H),3.55–3.39(m,1H),3.29(s,1H),3.24(d,J＝12.7Hz,1H),1.89(t,J＝12.6Hz,1H),1.80(s,1H),1.72(d,J＝13.5Hz,1H),1.46(d,J＝13.1Hz,1H).¹³C NMR(101MHz,Chloroform-d)164.42,147.99,141.22,138.03,135.49,128.35,127.52,127.18,114.76,112.72,70.64,70.52,57.52,56.27,43.04,37.29,35.27,34.48.HRMS(ESI):Calcd.C₂₂H₂₁ClN₄O₆,[M+Na]⁺,m/z:495.1150,found:495.1026.

Example 18

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with p-benzyloxyphenylacetic acid, and the rest was the same as in example 1. The product was a brown solid in 21.2% yield. Melting point: 222.6-223.0 ℃.¹H NMR(400MHz,Chloroform-d)8.20(q,J＝8.8,8.3Hz,2H),8.04(s,1H),7.72(d,J＝8.4Hz,1H),7.47(t,J＝8.7Hz,1H),7.01–6.94(m,1H),6.80(d,J＝13.8Hz,1H),4.65(d,J＝13.3Hz,1H),4.29(dd,J＝22.0,14.2Hz,1H),4.14–4.08(m,1H),4.00(d,J＝14.1Hz,0H),3.92(d,J＝10.6Hz,3H),3.55–3.39(m,1H),3.29(s,1H),3.24(d,J＝12.7Hz,1H),1.89(t,J＝12.6Hz,1H),1.80(s,1H),1.72(d,J＝13.5Hz,1H),1.46(d,J＝13.1Hz,1H).¹³C NMR(101MHz,Chloroform-d)164.42,147.99,141.22,138.03,135.49,128.35,127.52,127.18,114.76,112.72,70.64,70.52,57.52,56.27,43.04,37.29,35.27,34.48.HRMS(ESI):Calcd.C₂₉H₂₈ClN₃O₄,[M+H]⁺,m/z:518.1768,found:518.1827.

Example 19

Compound (I)

The difference between the synthesis process of (a) and example 1 is: the 3-phenylpropionic acid was replaced with 2- (1-naphthyloxy) acetic acid, and the rest was the same as in example 1. The product was a white solid in 85% yield. Melting point: 212.5-213.0 ℃.¹H NMR(400MHz,DMSO-d₆)8.30(s,1H),8.25–8.19(m,1H),8.16(d,J＝8.6Hz,1H),7.91–7.84(m,1H),7.76(d,J＝2.1Hz,1H),7.59(dd,J＝8.6,2.1Hz,1H),7.53(dq,J＝5.9,3.5,2.0Hz,2H),7.48(d,J＝8.5Hz,1H),7.39(t,J＝8.0Hz,1H),6.91(d,J＝7.7Hz,1H),5.06–4.95(m,3H),4.08(d,J＝13.3Hz,2H),3.99(d,J＝13.7Hz,1H),3.74(d,J＝13.5Hz,1H),3.41–3.30(m,1H),3.03(t,J＝11.8Hz,1H),1.69(t,J＝12.0Hz,1H),1.54(s,1H),1.46(d,J＝13.6Hz,2H).¹³C NMR(101MHz,DMSO-d₆)165.37,160.16,153.43,150.38,148.99,138.90,133.99,128.43,127.40,127.18,126.41,126.22,125.98,125.30,124.85,121.56,120.35,120.20,105.55,69.28,66.39,53.78,37.24,34.86,34.19.HRMS(ESI):Calcd.C₂₆H₂₄ClN₃O₄,[M+Na]⁺,m/z:500.1455,found:500.1348.

Example 20

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with p-phenol formic acid, and the rest was the same as in example 1. The product was a white solid in 29.1% yield. Melting point: 203.8-204.0 ℃.¹H NMR(600MHz,DMSO-d6)8.29(dddd,J＝25.5,23.4,14.7,5.8Hz,4H),8.17(ddt,J＝7.9,4.1,2.0Hz,1H),8.09–7.92(m,2H),7.85–7.70(m,1H),7.48(tdd,J＝8.7,5.4,2.4Hz,1H),7.44–7.36(m,1H),7.02–6.89(m,1H),5.16–4.97(m,1H),4.31–3.95(m,2H),3.11(d,J＝4.7Hz,6H),1.77–1.34(m,3H).¹³C NMR(151MHz,DMSO-d6):38.72,40.66,54.38,69.92,116.20,118.64,119.31,120.88,122.51,122.71,123.23,126.72,127.66,128.78,128.97,131.45,132.18,132.94,133.44,139.40,150.92,160.72.HRMS(ESI):Calcd.C₂₁H₂₀ClN₃O₄,[M+H]⁺,m/z:414.1142,found:414.1233。

Example 21

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with p-nitrobenzoic acid, and the rest was the same as in example 1. The product was a white solid in 25% yield. Melting point: 256.1-256.5 ℃.¹H NMR(400MHz,DMSO-d₆)8.29(d,J＝7.3Hz,3H),8.15(d,J＝8.4Hz,1H),7.75(d,J＝2.1Hz,1H),7.68(d,J＝8.2Hz,2H),7.58(dd,J＝8.5,2.1Hz,1H),5.06(s,1H),4.23(d,J＝13.0Hz,1H),4.04(s,2H),3.31–3.24(m,2H),3.15(t,J＝12.2Hz,1H),1.67(dq,J＝22.9,12.3,10.9Hz,2H),1.53(d,J＝13.7Hz,1H),1.35(d,J＝13.3Hz,1H).¹³C NMR(101MHz,DMSO-d₆)166.93,160.18,150.38,148.99,147.63,142.68,138.88,128.41,127.99,127.15,126.21,123.74,120.35,69.34,53.84,42.89,37.34,34.62,33.94.HRMS(ESI):Calcd.C₂₁H₁₉ClN₄O₅,[M+H]⁺,m/z:443.1044,found:443.1105。

Example 22

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with cinnamic acid, and the rest was the same as in example 1. The product was a white solid in 26% yield. Melting point: 209.4-209.8 ℃.¹H NMR(400MHz,DMSO-d₆)8.31(s,1H),8.16(d,J＝8.6Hz,1H),7.76(d,J＝2.1Hz,1H),7.74–7.67(m,2H),7.59(dd,J＝8.6,2.1Hz,1H),7.47(d,J＝15.4Hz,1H),7.44–7.32(m,3H),7.28(d,J＝15.4Hz,1H),5.03(s,1H),4.17(d,J＝12.8Hz,1H),4.13–3.94(m,3H),3.39(t,J＝12.3Hz,1H),3.07(t,J＝11.6Hz,1H),1.59(s,1H),1.54(d,J＝12.0Hz,1H),1.47(d,J＝12.8Hz,2H).¹³C NMR(101MHz,DMSO-d₆)164.27,160.17,150.39,149.00,141.26,138.90,135.18,129.39,128.68,128.40,127.95,127.17,126.23,120.34,118.44,69.40,53.84,41.06,37.67,35.47,34.43.HRMS(ESI):Calcd.C₂₃H₂₂ClN₃O₃,[M+H]⁺,m/z:424.1350,found:424.1416.

Example 23

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with p-fluorophenylacetic acid, and the rest was the same as in example 1. The product was a white solid in 42.7% yield. Melting point: 202.3-202.7 ℃.¹H NMR(400MHz,DMSO-d₆)8.28(s,1H),8.16(d,J＝8.6Hz,1H),7.75(d,J＝2.1Hz,1H),7.58(dd,J＝8.6,2.1Hz,1H),7.29–7.20(m,2H),7.16–7.05(m,2H),4.98(s,1H),4.08–3.99(m,2H),3.94(d,J＝13.7Hz,1H),3.76–3.63(m,3H),3.27(ddd,J＝13.9,10.9,3.2Hz,1H),2.97(ddd,J＝13.5,10.4,3.9Hz,1H),1.43(q,J＝8.1,4.3Hz,3H),1.35(d,J＝13.7Hz,1H).¹³C NMR(101MHz,DMSO-d₆)168.47,160.13,150.36,148.97,138.89,132.23,132.20,130.83,130.75,128.41,127.16,126.21,120.31,114.97,114.76,69.21,53.70,41.28,38.48,37.14,34.88,34.23。HRMS(ESI):Calcd.C₂₂H₂₁ClFN₃O₃,[M+H]⁺,m/z:430.1255,found:430.1322.

Example 24

Compound (I)

The difference between the synthesis process of (a) and example 1 is: the 3-phenylpropionic acid was replaced with 2- (1-naphthyl) acetic acid, and the rest was the same as in example 1. The product was a white solid in 31.7% yield. Melting point: 223.4-223.7 ℃.¹H NMR(400MHz,DMSO-d₆)8.30(s,1H),8.16(d,J＝8.5Hz,1H),7.99–7.87(m,2H),7.81(d,J＝8.2Hz,1H),7.78–7.70(m,1H),7.58(dd,J＝8.6,2.1Hz,1H),7.55–7.46(m,2H),7.43(dd,J＝8.2,7.0Hz,1H),7.41–7.29(m,1H),5.02(s,1H),4.16(s,2H),4.07(td,J＝13.1,7.5Hz,2H),3.98(d,J＝13.8Hz,1H),3.91–3.76(m,1H),3.42–3.26(m,1H),3.09–2.98(m,1H),1.64–1.50(m,1H),1.46(s,2H),1.45–1.38(m,1H).¹³C NMR(101MHz,DMSO-d₆)168.53,160.15,150.38,148.99,138.90,133.27,132.75,132.02,128.43,128.30,127.16,126.94,126.91,126.22,125.85,125.55,125.39,124.22,120.33,69.27,53.75,41.36,37.18,37.15,34.95,34.32.HRMS(ESI):Calcd.C₂₆H₂₄ClN₃O₃,[M+H]⁺,m/z:462.1506,found:462.1570。

Example 25

Compound (I)

The difference between the synthesis process of (a) and example 1 is: 3-phenylpropionic acid was replaced with p-trifluoromethylphenylacetic acid, and the rest was the same as in example 1. The product was a white solid in 45% yield. Melting point: 222.3-222.7 ℃.¹H NMR(400MHz,DMSO-d₆)8.29(s,1H),8.16(d,J＝8.6Hz,1H),7.75(d,J＝2.1Hz,1H),7.66(d,J＝8.1Hz,2H),7.58(dd,J＝8.6,2.1Hz,1H),7.44(d,J＝8.0Hz,2H),5.00(s,1H),4.11–4.02(m,2H),3.95(d,J＝13.7Hz,1H),3.83(d,J＝3.3Hz,2H),3.79–3.70(m,1H),3.30(ddd,J＝14.0,11.1,3.1Hz,1H),2.99(ddd,J＝13.3,10.7,3.7Hz,1H),1.58–1.35(m,4H).¹³C NMR(101MHz,DMSO-d₆)167.93,160.14,150.36,148.97,141.13,138.89,130.01,128.40,127.15,126.21,124.97,124.93,124.89,124.85,120.31,69.22,53.71,41.27,37.19,34.88,34.22.HRMS(ESI):Calcd.C₂₃H₂₁ClF₃N₃O₃,[M+H]⁺,m/z:480.1224,found:480.1287.

Example 26

Compound (I)

The difference between the synthesis process of (a) and example 1 is: the 3-phenylpropionic acid was replaced with 4-benzyloxybenzoic acid, and the rest was the same as in example 1. The product was a white solid in 65% yield. Melting point: 132.2-132.7 ℃.¹H NMR(400MHz,DMSO-d₆)8.31(s,1H),8.16(d,J＝8.6Hz,1H),7.75(d,J＝2.1Hz,1H),7.57(dd,J＝8.6,2.1Hz,1H),7.51–7.25(m,7H),7.06(d,J＝8.2Hz,2H),5.14(s,2H),5.03(s,1H),4.04(s,3H),3.32–3.02(m,2H),2.70(d,J＝2.3Hz,1H),1.61(d,J＝12.6Hz,2H),1.45(s,2H).¹³C NMR(101MHz,DMSO-d₆)168.86,160.18,159.15,150.36,148.98,138.88,136.76,128.75,128.43,127.88,127.70,127.12,126.19,120.34,114.40,69.43,69.30,53.87.HRMS(ESI):Calcd.C₂₈H₂₆ClN₃O₄,[M+H]⁺,m/z:504.1612,found:504.1674.

Example 27

Compound (I)

The synthesis process of (A) is as follows: taking the compound I-11(0.2g,0.43mmol,1.0eqv), 2-furanmethanamine (0.125g,1.29mmol,3.0eqv), cuprous iodide (0.004g,0.022mmol,0.05eqv), 2-hydroxyphenyl morpholinone (0.018g,0.086mmol,0.2eqv) and potassium phosphate (0.183g,0.86mmol,2.0eqv), placing in a 20mL glass test tube, sealing with a rubber plug, N-N₂Replacing for 3 times, adding 1mL of anhydrous DMF, and after the addition is finished, N₂Replacing for 3 times, and keeping the temperature at 90 ℃ for 18 hours; adding 20mL of water, extracting with ethyl acetate (3X 60mL), washing with saturated saline, drying with anhydrous magnesium sulfate, filtering, concentrating to obtain crude product, and performing column chromatography (PE: EA 2:1, then PE: EA 1:2)Compound I-27 was obtained (0.044g, brown solid, yield 21.3%). Melting point: 197.5-197.6 ℃.¹H NMR(400MHz,DMSO-d₆)8.07(s,1H),7.82(d,J＝8.8Hz,1H),7.59(s,1H),7.25(d,J＝8.6Hz,4H),7.19–7.09(m,2H),6.86(dd,J＝8.8,2.2Hz,1H),6.66(d,J＝2.3Hz,1H),6.44–6.38(m,1H),6.34(d,J＝3.2Hz,1H),4.90(s,1H),4.37(d,J＝5.7Hz,1H),4.07(s,1H),4.00–3.79(m,2H),3.60(s,1H),3.22(s,1H),2.92(s,1H),2.79(t,J＝7.9Hz,2H),2.71–2.56(m,2H),1.52–1.27(m,5H).¹³C NMR(101MHz,DMSO-d₆)169.58,169.55,153.13,152.25,150.07,148.82,142.22,141.44,128.36,128.16,127.17,125.78,110.63,110.39,107.29,69.27,40.83,36.98,34.90,34.24,33.86,30.86.HRMS(ESI):Calcd.C₂₈H₃₀N₄O₄,[M+H]⁺,m/z:487.2267,found:487.2327.

Example 28

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the product was a brown oil obtained in 36.9% yield by substituting 2-furanmethylamine with 2-phenylethylamine and the remainder was the same as in example 27.¹H NMR(400MHz,DMSO-d₆)8.08(d,J＝6.0Hz,1H),7.82(d,J＝8.8Hz,1H),7.30(s,2H),7.36–7.26(m,1H),7.28–7.16(m,5H),7.17(d,J＝6.5Hz,1H),6.85–6.73(m,1H),6.59(d,J＝2.4Hz,1H),4.93(d,J＝16.0Hz,1H),4.06(d,J＝12.7Hz,1H),3.94(dd,J＝30.2,5.7Hz,2H),3.62(d,J＝13.9Hz,1H),3.38(d,J＝6.8Hz,1H),3.22(t,J＝12.4Hz,1H),2.89(q,J＝9.7,7.7Hz,3H),2.80(t,J＝7.8Hz,2H),2.75–2.53(m,2H),1.47–1.39(m,1H),1.37(q,J＝13.6,10.6Hz,3H).¹³C NMR(101MHz,DMSO-d₆)169.58,169.55,160.23,153.43,150.25,148.82,141.44,139.47,128.69,128.36,128.32,128.16,127.28,126.10,125.77,114.38,110.20,104.01,69.28,53.84,53.24,43.94,40.85,36.99,34.91,34.38,34.26,33.86,30.86.HRMS(ESI):Calcd.C₃₁H₃₄N₄O₃,[M+H]⁺,m/z:511.2631,found:511.2695.

Example 29

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the product was a brown solid with a yield of 55.6% by replacing 2-furanmethylamine with 1- (2-aminoethyl) pyrrolidine and using the same procedure as in example 27. Melting point: 105.8-106.1 ℃.¹H NMR(400MHz,DMSO-d₆)8.09(s,1H),7.81(d,J＝8.8Hz,1H),7.30–7.12(m,5H),6.82(d,J＝8.9Hz,1H),6.62(d,J＝5.7Hz,1H),6.57(s,1H),4.94(s,1H),4.06(d,J＝13.0Hz,1H),3.97–3.84(m,2H),3.70–3.58(m,5H),3.24(dq,J＝24.2,5.7,5.1Hz,3H),2.99–2.87(m,1H),2.80(t,J＝7.7Hz,2H),2.73–2.61(m,2H),2.60(d,J＝8.5Hz,1H),2.57–2.45(m,2H),1.91(s,0H),1.71(d,J＝5.8Hz,4H),1.38(tt,J＝18.1,13.3,9.2Hz,4H).¹³C NMR(101MHz,DMSO-d₆)169.56,153.48,150.23,148.81,141.43,128.35,128.16,127.21,125.77,114.46,110.17,103.91,69.28,53.98,53.60,53.20,41.34,40.86,37.00,34.91,34.24,33.86,30.86,23.06.HRMS(ESI):Calcd.C₂₉H₃₇N₅O₃,[M+H]⁺,m/z:504.2896,found:504.2959.

Example 30

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the same operation as in example 27 was repeated except for replacing 2-furanmethylamine with o-methoxybenzylamine, and the product was a brown solid, which was obtained in 33.2% yield. Melting point: 103.9-104.1 ℃.¹H NMR(400MHz,DMSO-d₆)8.14–8.01(m,1H),7.81(d,J＝8.8Hz,1H),7.25(d,J＝10.0Hz,6H),7.15(dd,J＝13.5,6.3Hz,2H),7.03(d,J＝8.2Hz,1H),6.95–6.80(m,2H),6.46(s,1H),4.92(d,J＝29.3Hz,1H),4.33(d,J＝5.8Hz,1H),4.14–3.74(m,6H),3.61(d,J＝11.9Hz,1H),3.21(t,J＝12.3Hz,1H),2.99–2.86(m,1H),2.85–2.71(m,3H),2.70–2.56(m,2H),1.53–1.28(m,5H).¹³C NMR(101MHz,DMSO-d₆)169.54,160.20,156.93,153.53,150.30,150.13,148.79,141.44,129.92,129.30,128.35,128.16,128.12,127.68,127.20,126.17,125.77,120.17,114.54,110.67,110.32,104.25,69.26,55.37,53.83,53.24,40.84,36.97,34.90,34.24,33.86,30.86.HRMS(ESI):Calcd.C₃₁H₃₄N₄O₄,[M+H]⁺,m/z:527.2580,found:527.2649。

Example 31

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the same operation as in example 27 was repeated except for replacing 2-furanmethanamine with p-chlorobenzylamine, and the product was a brown solid, which was obtained in 18.4% yield. Melting point: 95.9-96.5 ℃.¹H NMR(400MHz,DMSO-d₆)8.14–8.00(m,1H),7.82(d,J＝8.8Hz,1H),7.45–7.10(m,9H),6.85(dd,J＝8.9,2.2Hz,1H),6.49(d,J＝2.3Hz,1H),4.92(d,J＝29.9Hz,1H),4.40(d,J＝5.9Hz,1H),4.14–3.80(m,3H),3.60(d,J＝11.9Hz,1H),3.23(d,J＝12.6Hz,1H),2.92(dd,J＝16.1,6.4Hz,1H),2.80(q,J＝6.7Hz,2H),2.69–2.54(m,2H),1.52–1.29(m,4H).¹³C NMR(101MHz,DMSO-d₆)169.54,160.19,153.21,150.08,148.82,141.44,138.30,131.31,129.31,128.91,128.35,128.16,127.25,125.77,114.71,110.58,104.62,69.26,53.83,53.24,45.14,40.83,36.97,34.90,34.24,33.86,30.86.HRMS(ESI):Calcd.C₃₀H₃₁ClN₄O₃,[M+H]⁺,m/z:531.2085,found:531.2157。

Example 32

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the same operation as in example 27 was repeated except for replacing 2-furanmethanamine with o-chlorobenzylamine, and the product was a brown solid, which was obtained in 15.8% yield. Melting point: 113.1-113.5 ℃.¹H NMR(400MHz,DMSO-d₆)8.32–8.13(m,1H),8.11–7.95(m,1H),7.93–7.79(m,1H),7.52–7.06(m,9H),6.86(t,J＝8.7Hz,1H),6.48(d,J＝22.8Hz,1H),4.92(d,J＝27.8Hz,1H),4.43(dd,J＝25.2,5.8Hz,1H),4.15–3.81(m,3H),3.71–3.50(m,1H),3.23(d,J＝12.4Hz,1H),3.01–2.86(m,1H),2.85–2.69(m,2H),2.59(q,J＝9.1,8.3Hz,2H),1.51–1.16(m,5H).¹³C NMR(101MHz,DMSO-d₆)169.58,169.55,160.18,153.13,150.12,148.90,141.44,135.89,132.38,129.36,128.75,128.35,128.16,127.34,127.25,125.77,114.59,104.41,69.26,53.26,43.79,40.82,36.97,34.90,34.24,33.86,30.86.HRMS(ESI):C alcd.C₃₀H₃₁ClN₄O₃,[M+H]⁺,m/z:531.2085,found:531.2145。

Example 33

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the product was a brown solid in 55.6% yield by replacing 2-furanmethanamine with 1- (2-aminoethyl) piperidine, which was the same as in example 27. Melting point: 139.0 to 140.0 ℃.¹H NMR(400MHz,DMSO-d₆)8.07(s,1H),7.80(d,J＝8.9Hz,1H),7.30–7.19(m,3H),7.16(t,J＝7.2Hz,1H),6.81(d,J＝8.9Hz,1H),6.57–6.47(m,1H),4.05(d,J＝12.9Hz,1H),3.96–3.83(m,2H),3.62(d,J＝14.1Hz,1H),3.21(p,J＝6.5,5.6Hz,2H),2.91(d,J＝12.7Hz,1H),2.79(t,J＝7.7Hz,2H),2.60(q,J＝8.1Hz,1H),2.47(d,J＝6.7Hz,1H),2.39(s,3H),2.32(s,1H),1.87(s,2H),1.50(h,J＝8.4,7.1Hz,3H),1.39(dt,J＝11.2,5.1Hz,5H),1.34–1.21(m,1H).¹³C NMR(101MHz,DMSO-d₆)169.56,160.23,153.58,150.24,148.81,141.44,128.35,128.16,127.19,125.77,110.12,69.27,57.07,54.17,53.21,40.85,34.91,34.26,33.86,30.86,25.52,24.02.HRMS(ESI):Calcd.C₃₀H₃₉N₅O3,[M+H]⁺,m/z:518.3053,found:518.3119。

Example 34

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the 2-furanmethanamine was replaced with ethanolamine, and the rest was the same as in example 27, and the product was a brown solid with a yield of 55.6%. Melting point: 151.7-151.9 ℃.¹H NMR(600MHz,DMSO-d6)8.07(s,1H),7.80(d,J＝8.8Hz,1H),7.33–7.21(m,4H),7.21–7.10(m,1H),6.82(dd,J＝8.9,2.3Hz,1H),6.65(t,J＝5.6Hz,1H),6.57(d,J＝2.3Hz,1H),4.91(s,1H),4.78(t,J＝5.5Hz,1H),4.13–4.01(m,1H),3.90(q,J＝13.8Hz,2H),3.70–3.54(m,3H),3.29–3.15(m,3H),2.93(ddd,J＝13.2,10.4,3.9Hz,1H),2.80(t,J＝8.1Hz,2H),2.69–2.55(m,2H),1.52–1.30(m,4H).¹³C NMR(151MHz,DMSO-d6):14.56,31.36,34.37,34.76,35.41,37.50,41.36,45.57,53.73,59.75,69.79,104.40,110.58,126.29,127.69,128.68,128.88,141.96,149.30,150.76,154.28,160.74,70.08.HRMS(ESI):Calcd.C₂₅H₃₀N₄O₄,[M+H]⁺,m/z:451.2267,found:451.2348。

Example 35

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the same procedures used in example 27 were repeated except for replacing 2-furanmethanamine with N, N-dimethylethylenediamine to give a pale yellow oil in a yield of 34%.¹H NMR(600MHz,DMSO-d6)8.09(s,1H),7.81(d,J＝8.8Hz,1H),7.32–7.20(m,4H),7.19–7.11(m,1H),6.83(dd,J＝8.8,2.3Hz,1H),6.60–6.46(m,2H),5.02(s,1H),4.06(dt,J＝13.0,4.5Hz,1H),3.90(q,J＝13.8Hz,2H),3.63(ddt,J＝13.8,8.8,5.1Hz,1H),3.29–3.17(m,3H),3.12(s,1H),2.93(ddd,J＝13.4,10.3,4.2Hz,1H),2.80(t,J＝8.1Hz,2H),2.70–2.56(m,2H),2.46(t,J＝6.6Hz,2H),2.19(s,5H),1.54–1.28(m,4H).¹³C NMR(151MHz,DMSO-d6):31.37,34.37,34.76,35.41,37.50,40.93,41.36,45.75,53.69,57.94,69.76,104.38,110.63,114.98,126.29,127.68,128.68,128.87,141.96,149.34,150.76,154.08,160.74,170.08.HRMS(ESI):Calcd.C₂₇H₃₅N₅O₃,[M+H]⁺,m/z:478.2740,found:478.2814。

Example 36

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the procedure was as in example 27 except for replacing 2-furanmethanamine with 1- (2-aminoethyl) pyrrolidine and replacing compound I-11 with I-9 to give a brown solid in a yield of 32.65%. Melting point: 173.5-173.7 ℃.¹H NMR(600MHz,DMSO-d6)8.06(d,J＝16.2Hz,1H),7.81(d,J＝8.8Hz,1H),7.29–7.22(m,4H),7.15(td,J＝6.1,3.1Hz,1H),6.82(dd,J＝8.8,2.3Hz,1H),6.59(td,J＝5.5,1.7Hz,1H),6.56(d,J＝2.3Hz,1H),4.90(d,J＝8.3Hz,1H),4.06–3.94(m,2H),3.86(d,J＝20.7Hz,2H),3.69–3.61(m,1H),3.21(dq,J＝42.7,7.7,7.0Hz,4H),2.89(td,J＝10.2,9.3,5.5Hz,1H),2.66–2.55(m,4H),2.55–2.47(m,3H),1.70(dq,J＝6.6,3.2Hz,4H),1.50(ddd,J＝13.4,11.2,4.4Hz,1H),1.38(dt,J＝15.2,4.3Hz,1H),1.31(dddd,J＝18.7,15.3,10.0,4.1Hz,2H),1.20(dd,J＝6.9,2.7Hz,4H).¹³C NMR(151MHz,DMSO-d6):22.37,22.55,23.61,34.73,34.88,35.41,35.52,36.50,36.68,37.45,40.69,40.73,41.49,41.59,42.11,53.71,54.17,54.66,69.74,69.79,104.37,110.60,110.63,126.39,126.44,127.37,127.71,128.67,128.70,147.06,147.18,149.31,150.76,154.06,160.71,160.75,169.55,169.57.HRMS(ESI):Calc d.C₃₀H₃₉N₅O₃,[M+H]⁺,m/z:518.3053,found:518.3120。

Example 37

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the same procedures used in example 27 were repeated except for replacing 2-furanmethylamine with 2-phenylethylamine and replacing the compound I-11 with I-9, and the product was a brown solid with a yield of 7.66%. Melting point: 167.3-167.9 ℃.¹H NMR(600MHz,DMSO-d6)8.07(d,J＝16.2Hz,1H),7.82(d,J＝8.7Hz,1H),7.39–7.18(m,8H),7.15(ddt,J＝11.2,6.0,2.6Hz,1H),6.88–6.72(m,2H),6.59(d,J＝2.2Hz,1H),4.91(d,J＝8.3Hz,1H),4.08–3.92(m,1H),3.86(d,J＝21.3Hz,1H),3.65(td,J＝13.6,6.9Hz,1H),3.38(dt,J＝7.9,5.9Hz,2H),3.28–3.09(m,2H),2.89(t,J＝7.4Hz,3H),2.70–2.55(m,2H),2.54–2.48(m,3H),1.44–1.25(m,3H),1.20(dd,J＝7.0,2.5Hz,3H).¹³C NMR(151MHz,DMSO-d6):22.37,22.55,34.73,34.88,35.40,35.52,36.50,36.69,37.45,40.69,40.73,41.49,41.59,44.46,53.71,69.74,69.79,104.53,110.70,110.73,126.39,126.44,126.62,127.37,127.78,128.67,128.70,128.73,128.83,129.21,139.99,147.06,147.19,149.33,150.77,153.94,160.72,160.76,169.58.HRMS(ESI):Calcd.C₃₂H₃₆N₄O₃,[M+H]⁺,m/z:525.2787,found:525.2868。

Example 38

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the compound I-11 was replaced with I-9 and the residue was the same as in example 27 to give a brown solid in a yield of 12.66%. Melting point: 103.1-104.0 ℃.¹H NMR(600MHz,DMSO-d6)8.06(d,J＝16.2Hz,1H),7.87–7.79(m,1H),7.60(dd,J＝2.0,0.8Hz,1H),7.34–7.22(m,4H),7.15(dtd,J＝12.0,5.7,2.2Hz,2H),6.86(dd,J＝8.9,2.3Hz,1H),6.66(d,J＝2.3Hz,1H),6.40(dd,J＝3.3,1.8Hz,1H),6.35(dd,J＝3.2,0.9Hz,1H),4.96–4.83(m,1H),4.38(d,J＝5.9Hz,2H),4.11–3.90(m,2H),3.85(d,J＝21.2Hz,1H),3.64(t,J＝13.7Hz,1H),3.28–3.08(m,2H),2.88(tt,J＝10.4,3.9Hz,1H),2.66–2.55(m,1H),1.44–1.24(m,3H),1.23–1.14(m,3H).¹³C NMR(151MHz,DMSO-d6):14.56,17.69,22.37,22.55,30.58,34.71,34.86,35.40,35.51,36.49,36.69,37.44,40.68,40.72,41.48,41.58,48.95,53.73,69.73,69.78,105.10,107.80,110.90,111.13,111.16,115.11,126.39,126.45,127.37,127.68,128.67,128.70,142.74,147.05,147.18,149.32,150.59,152.77,153.65,160.71,160.75,169.56,169.58.HRMS(ESI):Calcd.C₂₉H₃₂N₄O₄,[M+H]⁺,m/z:501.2424,found:501.2491。

Example 39

Compound (I)

The difference between the synthesis procedure of (1) and example 27 is: the same procedures used in example 27 were repeated except for replacing 2-furanmethylamine with N, N-dimethylethylenediamine and replacing the compound I-11 with I-9 to give a white solid in a yield of 58%. Melting point: 108.7 to 109.0 ℃.¹H NMR(600MHz,DMSO-d6)8.07(d,J＝16.3Hz,1H),7.81(d,J＝8.8Hz,1H),7.29–7.22(m,4H),7.15(td,J＝6.2,3.1Hz,1H),6.83(dd,J＝8.9,2.3Hz,1H),6.56(d,J＝2.3Hz,1H),6.51(td,J＝5.3,1.8Hz,1H),4.96(s,1H),4.05–3.94(m,2H),3.86(d,J＝21.5Hz,2H),3.68–3.61(m,1H),3.27–3.11(m,4H),2.92–2.85(m,1H),2.65–2.55(m,2H),2.54–2.43(m,2H),2.19(s,5H),1.49(ddd,J＝13.4,11.2,4.3Hz,1H),1.41–1.34(m,1H),1.36–1.31(m,1H),1.28(dd,J＝17.2,3.1Hz,1H),1.20(dd,J＝7.0,2.6Hz,4H).¹³C NMR(151MHz,DMSO-d6):22.37,22.55,34.73,34.87,35.40,35.52,36.50,36.69,37.45,40.69,40.73,40.93,41.49,41.59,45.74,53.69,57.95,69.72,69.77,104.40,110.62,110.65,114.98,126.39,126.44,127.37,127.68,128.67,128.70,147.05,147.18,149.32,150.75,154.07,160.72,160.76,169.56,169.58.HRMS(ES I):Calcd.C₂₈H₃₇N₅O₃,[M+H]⁺,m/z:492.2896,found:492.2960。

Test example 1

USP7 inhibitory activity assay: the experiment was divided into three groups, compound, blank and control, each group was treated as follows:

group of compounds: the compounds used were the compounds synthesized in examples 1 to 39, 7.8. mu.L of Buffer (50mM Hepes pH7.5, 0.01% Triton X-100), 1. mu.L of USP7 recombinant protein, 0.2. mu.L of compound (stock solution concentration 2.5mM, 8 concentrations diluted in two-fold ratio when IC50 value was measured) were added to each well of 384-well plate, incubation was carried out at 25 ℃ for 10min, 1. mu.L of substrate was further added thereto, incubation was carried out at 37 ℃ for 75min, 10. mu.L of each assay reagent was added to each well after incubation, incubation was carried out at 25 ℃ for 1h, and the activity of the compound was judged by detecting the fluorescence value at 665nm/615nm using a microplate reader.

Blank group: the blank group and the compound group are distinguished as follows: the USP7 recombinant protein was replaced with an equal amount of Buffer and the compound was replaced with an equal amount of DMSO, all the remaining being the same.

Control group: the differences between the control and compound groups were: the compound was replaced with an equal amount of DMSO, and the rest was the same.

The inhibition and IC50 were calculated as:

inhibition rate ═ 100% (compound group fluorescence value-control group fluorescence value)/(blank group fluorescence value-control group fluorescence value).

IC50 values were calculated using IBM SPSS software. The results are shown in Table 3.

TABLE 3

As can be seen from Table 3, the compounds I-1 to I-26 as intermediates have generally lower inhibition rate to USP7 and poorer inhibition activity, the compounds I-27 to I-39 have better inhibition activity to USP7, and the inhibition rate of 50 mu M inhibition rate of some compounds such as I-27, I-28, I-29, I-34, I-35, I-36, I-38, I-39 and the like reaches more than 80 percent, which shows that the invention connects substituent groups R on the basis of the intermediate compounds I-1 to I-26₁The inhibitory activity of the compound on USP7 is obviously improved.

Test example 2

The USP7 inhibitory activity of the compounds was determined by the Ub-AMC method: this test method was the same as "test example 1", and compound I-1 was used as a positive control compound, and a portion of the compounds with better activity was retested using a commercial USP7 inhibitor test kit, with the results shown in table 4. The detailed information of the kit is as follows: name: USP7 inhibition screening assay kit, manufacturer: BPS Bioscience, model: catalog #79256, specification: the specific operation method of Size 96reactions is as follows:

(1) to "5 XUSP 7 Assay Buffer" was added 13. mu.l of 0.5M DTT and diluted with distilled water to "1 XUSP 7 Assay Buffer" for use.

(2) "Ub-AMC Substrate" was diluted 400-fold with "1X USP7 Assay Buffer". At the time of the Test, the Test results were classified into "inhibitor group" (Test inhibitor), "Positive control group" (Positive control), "Blank group" (Blank). A96-well plate was taken, and 20. mu.l of diluted "Ub-AMC Substrate" was added to each well of each group.

(3) After the Test compound was dissolved in DMSO, 5. mu.l of the solution was added to the "Test inhibitor" group. The "Positive Control" and "Blank" groups were supplemented with 5. mu.l DMSO.

(4) To the "Blank" group was added 25. mu.l of "1 × USP7 Assay Buffer".

(5) Sample tubes containing USP7 enzyme were thawed on ice. Note that the remaining unused USP7 should be stored quickly in a-80 ℃ refrigerator and avoid repeated freeze-thaw processes.

(6) USP7 enzyme was diluted to 0.4 ng/. mu.l with "1 XUSP 7 Assay Buffer". The specific sample loading is shown in Table 4.

TABLE 4

(7) 25. mu.l of the diluted USP7 enzyme was added to the "Test inhibitor" group and the "Positive control" group, followed by incubation on a shaker at room temperature for 30 minutes.

(8) The fluorescence intensity of the treated 96-well plate containing the test sample was read immediately on a microplate reader (excitation: 360nm, emission: 460nm) and the values of the "Blank" set were subtracted from all readings, as shown in table 5.

TABLE 5

Number of Compounds	IC₅₀/μM
		I-1 (Positive control Compound)	36.95
I-29	5.048
		I-35	10.17
I-36	0.489
		I-39	0.595

As can be seen from Table 5, the linkage R is compared with the positive control compound I-1₁After the compounds are combined, the inhibitory activity of the compounds I-29, I-35, I-36 and I-39 on USP7 is obviously improved. Meanwhile, the Ub-AMC method is a general method for screening USP7 inhibitor, and the experimental results also demonstrate the inhibitory activity of the compound of the present invention against USP 7.

Test example 3

Test compounds for tumor cell inhibitory activity: the experimental objects are gastric cancer cell lines MGC-803 and BGC-823, logarithmic phase cells are collected, cell suspension concentration is adjusted, cell count is carried out, the cells are inoculated to a 96-well plate according to cell morphology, size and relative cell number of experimental time, the marginal hole is filled with sterile PBS, and 5% CO is added₂And incubating overnight at 37 ℃, adding compounds to be detected with final concentrations of 0.78125 mu M, 1.5625 mu M, 3.125 mu M, 6.25 mu M, 12.5 mu M, 25 mu M, 50 mu M and 100 mu M (concentration gradient is reasonably selected according to drug conditions), setting 3 duplicate wells for each drug concentration, adding 20 mu L of MTT solution (final concentration is 0.5g/L) to each well after culturing for a certain time, stopping culturing after incubating for 4 hours at 37 ℃, carefully sucking and discarding culture supernatant, adding 200 mu L of dimethyl sulfoxide (DMSO) to each well, placing on a shaking bed, shaking at low speed for 10min, fully dissolving crystals, measuring absorbance (A) at 490/570nm by using a microplate reader, and setting a zero-adjusting group and a control group.

The inhibition ratio ═ 100% (control absorbance value-dosing absorbance value)/(control absorbance-zero absorbance). Cell growth Inhibition Rate (IR) was calculated and half inhibitory concentration (IC50) was calculated using IBM SPSS software. In Table 6, the inhibition ratio and IC under 50. mu.M condition₅₀The value is obtained.

TABLE 6

As can be seen from Table 6, in the selected partial representative compounds, the intermediate compounds I-1 to I-11 have less ideal inhibitory effects on two cell lines, the compounds I-14, I-19, I-20, I-28, I-29 and I-32 have certain inhibitory effects on MGC-803 and BGC-823, and have obvious inhibitory effects on MGC-803, wherein the inhibitory effects on the compounds I-20, I-28, I-32 and the like are obvious, and the antitumor activities of the compounds can be further researched.

Test example 4

The influence of the compounds I-29, I-35, I-36 and I-39 on the expression levels of related proteins and downstream substrates p21 in the human gastric cancer cell line p53-MDM2-USP7 pathway is determined by a Western blotting experiment method: the experimental objects are gastric cancer cell lines MGC-803 and BGC-823, the compounds are dissolved in DMSO to prepare mother liquor, and the mother liquor is diluted to a proper concentration by adopting a complete culture medium before use; the experimental materials also include RMPI1640 medium; fetal bovine serum; a 60mm petri dish; USP7(ab10893, p21(CST2946T, GAPDH (GoodHere No. ab-M001) antibody.

Taking cell with living cell proportion more than 90%, performing experiment, digesting cell, adding appropriate amount of cell into 60mm dish, 37 deg.C, and 5% CO₂Culturing in incubator for 24 hr, diluting with complete culture medium to desired concentration, and setting solvent control group at 37 deg.C and 5% CO₂After 24 hours of incubation in an incubator, cells were digested and cell pellets were collected, washed twice with phosphate buffer, lysed using RIPA lysate on ice for 30 minutes, centrifuged at 12000rpm for 10 minutes, the supernatant was collected, and then quantified by BCA, and denatured by 6 x Loading buffer at 100 ℃ for 10 minutes. Finally, Western blotting is carried out to detect the expression levels of p53, p21, MDM2, USP7 and GAPDH. The results are shown in FIGS. 2 and 3. FIG. 2 is a graph showing the effect of compound I-29 on the expression level of p53-MDM2-USP7 pathway-related protein in a gastric cancer cell line MGC-803, wherein compound I-32 is a negative control compound and compound I-1 is a positive control compound. FIG. 3 shows the results of the comparison of the compounds I-35, I-36 and I-39 against the gastric cancer cell line BInfluence of the expression levels of p53-MDM2-USP7 pathway and its downstream related proteins in GC-823. From FIGS. 2 and 3, it can be seen that the compounds of the present invention induce the expression of downstream target genes in the gastric cancer cells MGC-803 and BGC-823 in a concentration-dependent manner.

The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims

1. The quinazolone USP7 inhibitor is characterized by having a structure shown as a general formula I:

in the general formula I, R₁Is selected from

One of (1);

in the general formula I, R₂Selected from:

one kind of (1).

2. The quinazolinone USP7 inhibitor according to claim 1, characterized in that it is selected from the following compounds:

3. the process for preparing a quinazolinone USP7 inhibitor according to claim 1, comprising the steps of:

(1) synthesis of Compound 3: adding N-Boc-piperidone and trimethyl sulfoxide iodide into a solvent, stirring in an ice bath for reaction under the action of an alkaline substance, and performing post-treatment after the reaction to obtain a compound 3;

(2) synthesis of Compound 5: dissolving compound 4 in formamide, reacting under heating for a period of time, cooling, adding compound 3 and alkali, reacting for a period of time, and post-treating to obtain compound 5, wherein R₃Is Cl or Br;

(3) synthesis of compound 8: adding acid into the compound 5, stirring for a period of time at room temperature, removing redundant acid after the reaction is finished, adding the compound 6, and carrying out condensation reaction under a HATU/TEA system to obtain a compound 8;

(4) synthesis of compounds of general formula I: taking the compound 8 and the compound 7 to carry out Buchwald-Hartwig coupling reaction to obtain the product with the general formula I.

4. The use of the quinazolinone USP7 inhibitor according to claim 1 in the preparation of a medicament for inhibiting ubiquitin-specific protease 7.

5. The use of the quinazolinone USP7 inhibitor according to claim 4, in the preparation of anti-tumor drugs targeting ubiquitin-specific protease 7.

6. The use of the quinazolinone USP7 inhibitor according to claim 5, wherein said antineoplastic agent is an anti-gastric cancer agent.