N-aryl-2-naphthylamide USP2 inhibitor and medical application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an N-aryl-2-naphthylamide USP2 inhibitor and medical application thereof. The N-aryl-2-naphthylamide provided by the invention has relatively strong USP2 protein inhibitory activity, can effectively inhibit tumor cell proliferation, is simple in synthesis process and relatively low in cost, can be applied to preparation of drugs for treating and / or preventing diseases related to abnormal USP2 activity, has relatively good development potential, and can be used for preparing drugs for treating and / or preventing diseases related to abnormal USP2 activity. And a selectable range is provided for medicines for treating USP2 related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry, and particularly relates to an N-aryl-2-naphthamide USP2 inhibitor and a medical use thereof. BACKGROUND

[0002] Ubiquitin-specific protease 2 (USP2) is a deubiquitinase belonging to the ubiquitin-specific protease family, which can regulate various cellular processes, including cell cycle, DNA repair and signal transduction, by removing ubiquitin chains from substrate proteins. Studies have shown that USP2 is highly expressed in various cancers (such as prostate cancer, glioblastoma and colon cancer), and promotes the occurrence and development of tumors by stabilizing oncogenic proteins (such as MDM2, fatty acid synthase). In addition, USP2 is also closely related to neurodegenerative diseases and inflammatory responses. Therefore, the development of efficient USP2 inhibitors is of great significance for the treatment of related diseases.

[0003] At present, some USP2 inhibitors have been reported, including small molecule compounds Beta-Lapachone, ML364, LCAHA, ZCL-910, 6-TG, STD1D, COH29, etc. (Int. J. Mol. Sci. 2021, 22, 4546). However, the chemical structures of existing inhibitors have limited diversity, and the synthesis routes of some compounds are complex and have low yield, which limits their further development and application; and most patients will eventually develop resistance to these inhibitors after long-term use, therefore, it is a technical problem to be solved to develop USP2 inhibitors with novel structures. SUMMARY

[0004] The purpose of the present application is to provide a novel USP2 inhibitor with N-aryl-2-naphthamide as the parent nucleus, which has the characteristics of novel structure, simple synthesis and significant activity, and can effectively inhibit the enzyme activity of USP2.

[0005] The first aspect of the present application provides a compound represented by general formula (I), a pharmaceutically acceptable salt, isotope, racemate, optically active isomer, polymorph or mixture thereof: ; wherein: R1 is selected from hydrogen atom, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkoxy, C 3-6 cycloalkyl, haloalkyl, haloalkoxy, hydroxyl, hydroxyalkyl, cyano, amino, heterocyclyl, aryl and heteroaryl; R2 is selected from hydrogen atom, halogen, C 1-6 alkyl, C2-6 alkenyl, C 1-6 alkoxy, C 3-6 cycloalkyl, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, heterocyclyl, aryl and heteroaryl; R3is selected from the group consisting of: hydrogen atom, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkoxy, C 3-6 cycloalkyl, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, heterocyclyl, aryl and heteroaryl; Linker1is absent or selected from the group consisting of ; wherein X is selected from CH2, O, S, NH, carbonyl, sulfoxide, sulfone, carbamoyl, formamido, sulfonamido, ester; p and q are each independently selected from any integer from 0 to 4; Linker2is absent or selected from the group consisting of ; wherein Y is selected from CH2, O, S, NH, carbonyl, sulfoxide, sulfone, carbamoyl, formamido, sulfonamido, ester; m and n are each independently selected from any integer from 0 to 4.

[0006] Preferably, said heteroaryl is pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl, imidazolyl, pyrazolyl, thienyl, furanyl, thiazolyl, oxazolyl, triazolyl, piperidinyl or piperazinyl.

[0007] Preferably, said R1is selected from C 1-6 alkyl, C 1-6 alkoxy, C 3-6 cycloalkyl, heterocyclyl, aryl and heteroaryl; wherein said C 1-6 alkyl, C 1-6 alkoxy, C 3-6 cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of: halogen, C 1-6 alkyl, C 1-6 alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, C 3-6 cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0008] Preferably, said R2is selected from C 1-6 alkyl, C 1-6 alkoxy, C 3-6 cycloalkyl, heterocyclyl, aryl and heteroaryl; wherein said C 1-6 alkyl, C 1-6 alkoxy, C 3-6 cycloalkyl, heterocyclyl, aryl and heteroaryl are each independently optionally substituted with one or more substituents selected from the group consisting of: halogen, C 1-6 alkyl, C 1-6Alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, C 3-6 It is substituted by one or more substituents among cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0009] Preferably, R3 is selected from C 1-6 Alkyl, C1 -6 Alkoxy, C 3-6 Cycloalkyl, heterocyclic, aryl, and heteroaryl; wherein, the C 1-6 Alkyl, C1 -6 Alkoxy, C 3-6 Cycloalkyl, heterocyclic, aryl, and heteroaryl groups are each independently and optionally halogenated, C 1-6 Alkyl, C 1-6 Alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, C 3-6 It is substituted by one or more substituents among cycloalkyl, heterocyclic, aryl, and heteroaryl.

[0010] Preferably, X or Y is selected from CH2, NH, carbamoyl, formamide, and sulfonamide; wherein the H atom in CH2, NH, carbamoyl, formamide, and sulfonamide is independently and optionally replaced by alkyl, halogen, amino, nitro, cyano, hydroxyl, hydroxyalkyl, alkoxy, cycloalkyl, heterocyclic, aryl, and heteroaryl groups.

[0011] A second aspect of the present invention is to provide a method for preparing a compound, a pharmaceutically acceptable salt thereof, an isotope thereof, a racemic mixture thereof, an optically active isomer thereof, a polymorph thereof, or a mixture thereof, characterized in that, [the method involves] [preparing a compound]. and 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in an organic solvent and reacted with stirring overnight. After the reaction was completed, the mixture was washed with 1 M dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine. The organic phases were combined, and the organic solvent was removed by rotary evaporation under vacuum. The purified product was then obtained. ; Among them, the 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and The molar ratio of the organic solvent is 1:(2.0~4.0):(1.0~2.0):(1.0~1.5), the organic solvent is dichloromethane, the stirring temperature is 20~30℃, the stirring time is 12~24 h, and the purification is performed by silica gel column chromatography. Or Dissolve triethylamine in an organic solvent and slowly add it under ice bath conditions. The mixture was stirred and reacted overnight. After the reaction was complete, the mixture was washed with saturated sodium bicarbonate solution and saturated brine. The organic phases were combined, and the organic solvent was removed by rotary evaporation under vacuum. The purified product was then obtained. ; Among them, the Triethylamine and The molar ratio of the solvents is 1:(1.5-3.0):(1.0-2.0), the organic solvent is dichloromethane, the stirring temperature is 20-30 °C, the stirring time is 12-24 h, and the purification is performed by silica gel column chromatography.

[0012] A third aspect of the invention is to provide the use of a compound, a pharmaceutically acceptable salt thereof, an isotope thereof, a racemic mixture thereof, an optically active isomer thereof, a polymorph thereof, or a mixture thereof in the preparation of an inhibitor, a medicament thereof, or a pharmaceutical composition for the prevention, treatment, or adjunctive treatment of diseases related to USP2 activity or expression levels.

[0013] Preferably, the diseases associated with USP2 activity or expression are neurodegenerative diseases, inflammatory responses, colorectal cancer, prostate cancer, or glioblastoma.

[0014] Preferably, the pharmaceutical composition comprises a compound, a pharmaceutically acceptable salt thereof, an isotope thereof, a racemic mixture thereof, an optically active isomer thereof, a polymorph thereof, or a mixture thereof as an active ingredient and a pharmaceutically acceptable excipient; the dosage form of the pharmaceutical composition is a tablet, capsule, sustained-release tablet or capsule, controlled-release tablet or capsule, granule, powder, syrup, oral liquid, or injection.

[0015] The present invention has the following beneficial effects: The N-aryl-2-naphthylcarboxamide derivatives provided by this invention have strong USP2 protein inhibitory activity, which can effectively inhibit tumor cell proliferation. Moreover, the synthesis process is simple and the cost is low. They can be used in the preparation of drugs for the treatment and / or prevention of diseases related to abnormal USP2 activity, and have good development potential, providing a range of options for drugs to treat USP2-related diseases. Detailed Implementation

[0016] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0017] Example 1 Synthesis of compound A-1 (N-(3-butamidophenyl)-3-methoxy-2-naphthylcarboxamide)

[0018] (a) Synthesis of intermediate I-1 Boc20 (0.57 g, 2.61 mmol) was added slowly under stirring and the reaction was left to proceed overnight at room temperature. After the reaction was completed, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and the solvent was evaporated under vacuum. The intermediate 1-1 (0.66 g, 61.2%) was obtained by column chromatography (PE / EA = 3 / 1).

[0019] 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.00 (s, 1H), 6.87-6.79 (m, 2H), 6.53(ddd, J = 8.0, 2.1, 1.0 Hz, 1H), 6.16 (ddd, J = 8.0, 2.3, 1.0 Hz, 1H), 1.45(s, 9H) ppm. (B) Synthesis of intermediate 1-2 Intermediate 1-1 (0.50 g, 2.40 mmol) was dissolved in 15 mL of DCM, n- butyric acid (243 mL, 2.64 mmol) and EDCI (1.15 g, 6.00 mmol) were added and the reaction was left to proceed overnight at room temperature. After the reaction was completed, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and the solvent was evaporated under vacuum. The intermediate 1-2 (0.40 g, 59.3%) was obtained by column chromatography (PE / EA = 4 / 1).

[0020] 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.80 (s, 1H), 9.31 (s, 1H), 7.77 (t, J =2.0 Hz, 1H), 7.29 (dt, J = 8.0, 1.4 Hz, 1H), 7.11 (t, J = 8.1 Hz, 1H), 7.00(ddd, J = 8.3, 2.2, 1.1 Hz, 1H), 2.25 (t, J = 7.3 Hz, 2H), 1.59 (q, J = 7.3Hz, 2H), 1.47 (s, 9H), 0.90 (t,J = 7.4 Hz, 3H) ppm. (Three) synthesis of intermediate I-3 Intermediate I-2 (0.45 g, 1.62 mmol) was dissolved in 10 mL of DCM, 3 mL of TFA was added, and the reaction was carried out at room temperature overnight. After the reaction was completed, saturated sodium bicarbonate solution was added dropwise, the pH of the reaction solution was adjusted to 7.0, water was added for extraction, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, rotary evaporated under vacuum, and purified by column chromatography (PE / EA = 3 / 1) to obtain intermediate I-3 (0.27 g, 95.4%).

[0021] 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.53 (s, 1H), 6.94 (t, J = 2.1 Hz, 1H),6.88 (t, J = 7.9 Hz, 1H), 6.67 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 6.23 (ddd, J = 7.9, 2.3, 1.0 Hz, 1H), 5.03 (s, 2H), 2.23 (t, J = 7.3 Hz, 2H), 1.59 (q, J =7.4 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H) ppm. (Four) synthesis of compound A-1 Intermediate I-3 (0.27 g, 1.51 mmol), 3-methoxy-2-naphthoic acid (0.34 g, 1.67 mmol), EDCI (1.16 g, 6.04 mmol), and DMAP (0.28 g, 2.27 mmol) were dissolved in 15 mL of DCM, and the reaction was carried out at room temperature overnight. After the reaction was completed, 1 M dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine were sequentially washed, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, rotary evaporated under vacuum, and purified by column chromatography (PE / EA = 3 / 1) to obtain compound A-1 (353 mg, 64.5%).

[0022] 1 H NMR (400 MHz, DMSO- d 6 ): δ10.28 (s, 1H), 9.91 (s, 1H), 8.13 (s,1H), 8.08 (t, J = 2.0 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 7.89 (d, J = 8.2 Hz,1H), 7.55 (ddd, J = 8.2, 6.8, 1.3 Hz, 1H), 7.49 (s, 1H), 7.42 (ddd, J = 8.2,6.9, 1.3 Hz, 2H), 7.36 (dt, J = 8.4, 1.2 Hz, 1H), 7.24 (t, J = 8.1 Hz, 1H),3.97 (s, 3H), 2.29 (t, J = 7.3 Hz, 2H), 1.62 (q, J = 7.3 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H) ppm; HRMS (ESI): [M + H] + C 22 H 23 N2O3 calcd 363.1709, found363.1709.

[0023] Example 2 Synthesis of Compound A-2 (N-(5-butyramidyl-2-methylphenyl)-3-methoxy-2-naphthamide) Using a similar synthetic procedure to that of Example 1, intermediate I-3 was replaced with N N-(3-amino-4-methylphenyl)butyramide to produce Compound A-2. The final test results are as follows: 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.87 (d, J =10.0 Hz, 2H), 8.40 (s, 1H), 8.07 (d, J = 2.2 Hz, 1H), 7.98 (d, J = 8.2 Hz,1H), 7.87 (d, J = 8.2 Hz, 1H), 7.58-7.50 (m, 2H), 7.45-7.37 (m, 2H), 7.14 (d,J = 8.3 Hz, 1H), 4.04 (s, 3H), 2.31-2.19 (m, 5H), 1.58 (q, J = 7.4 Hz, 2H),0.89 (t, J = 7.4 Hz, 3H) ppm; HRMS (ESI): [M + H] + C 23 H 25 N2O3 calcd 377.1865,found 377.1868.

[0024] Example 3 Synthesis of Compound A-3 (N-(5-butyramidyl-2-methoxyphenyl)-3-methoxy-2-naphthamide) Using a similar synthetic procedure as in Example 1, intermediate I-3 was replaced with N N-(3-amino-4-methoxyphenyl)butyramide to produce Compound A-3. The final test results are as follows: 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.71 (s,1H), 9.80 (s, 1H), 8.67 (s, 1H), 8.61 (d, J = 2.5 Hz, 1H), 8.01 (d, J = 8.1Hz, 1H), 7.89 (dd, J = 8.3, 1.1 Hz, 1H), 7.61-7.55 (m, 2H), 7.49 (dd, J =8.8, 2.6 Hz, 1H), 7.42 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H), 7.00 (d, J = 8.9 Hz,1H), 4.14 (s, 3H), 3.91 (s, 3H), 2.24 (t, J = 7.3 Hz, 2H), 1.59 (q, J = 7.4Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H) ppm; HRMS (ESI): [M + H] + C 23 H 25 N2O4 calcd393.1814, found 393.1815.

[0025] Example 4 Synthesis of Compound A-4 (N-(5-butyramidyl-2-chlorophenyl)-3-methoxy-2- naphthamide) Using a similar synthetic procedure as in Example 1, intermediate I-3 was replaced with N- (3-amino-4-chlorophenyl)butyramide to produce Compound A-4. The final test results are as follows: 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.85 (s, 1H), 8.90 (s, 1H), 8.38-8.34 (m, 1H), 7.93-7.86 (m, 1H), 7.73-7.67 (m, 1H), 7.59 (ddd, J = 8.1, 7.0, 1.5 Hz, 1H), 7.53 (d, J = 2.2 Hz, 1H), 7.47-7.42 (m, 2H), 7.40 (d, J = 8.3 Hz, 1H), 7.31 (s, 1H), 3.88 (s, 3H), 2.25 (t, J = 6.2 Hz, 2H), 1.72-1.59 (m, 2H), 0.97 (t, J = 7.6 Hz, 3H).

[0026] Example 5 Synthesis of Compound A-5 (N-(5-butyramidyl-2-fluorophenyl)-3-methoxy-2- naphthamide) Using a similar synthetic procedure as in Example 1, intermediate I-3 was replaced with N (3-amino-4-fluorophenyl)butyramide to produce Compound A-5. The final test results are as follows: 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.31 (s, 1H), 10.01 (s, 1H), 8.48 (s, 1H), 8.43 (dd, J = 7.3, 2.6 Hz, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.92 (d, J = 8.2 Hz, 1H), 7.62-7.57 (m, 2H), 7.52 (ddd, J =8.9, 4.5, 2.7 Hz, 1H), 7.45 (ddd, J= 8.2, 6.8, 1.2 Hz, 1H), 7.24 (dd, J = 8.2, 6.8, 1.2 Hz, 1H), 7.24 (dd, J = 8.2, 6.8, 1.2 Hz, 1H), 7.24 (dd, J = 8.2, 6.8, 1.2 Hz, 1H), 7.24 (dd, J = 8.2, 6.8, 1.2 Hz, 1H), 7.24 (dd, + C 22 H 22 FN2O3 calcd 381.1614, found 381.1614.

[0027] Example 6 Synthesis of Compound A-6 (N-(2-fluoro-5-(2,2,2-trifluoroacetamido)phenyl)-3-methoxy-2-naphthamide) Using a similar synthetic procedure to Example 1, intermediate I-3 was replaced with N N-(2-fluoro-5-(2,2,2-trifluoroacetamido)phenyl)-3-methoxy-2-naphthamide, Compound A-6 was prepared. The final test results are as follows: 1 H NMR (400 MHz, DMSO- d 6): δ 11.42 (s, 1H), 10.42 (s, 1H), 8.67-8.56 (m, 1H), 8.49 (s, 1H), 8.03 (d, J = 8.2, 6.8, 1.2 Hz, 1H), 7.24 (dd, J = 8.2, 6.8, 1.2 Hz, 1H), 7.24 (dd, J = 8.2, 6.8, 1.2 Hz, 1H), 7.24 (dd, + C 20 H 15 F4N2O3 calcd 407.1019,found 407.1021.

[0028] Example 7 Synthesis of Compound A-7 (N-(5-(2-cyclopropylacetamido)-2-fluorophenyl)-3-methoxy-2-naphthamide) Using a similar synthetic procedure to Example 1, intermediate I-3 was replaced with N- (3-amino-4-fluorophenyl)-2-cyclopropylacetamide, Compound A-7 was prepared. The final test results are as follows: 1 H NMR (400 MHz, DMSO- d 6): δ 10.31 (s, 1H), 9.95 (s, 1H), 8.48 (s, 1H), 8.43 (dd, J = 7.3, 2.6 Hz, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.61-7.56 (m, 2H), 7.53 (ddd, J =9.0, 4.5, 2.8 Hz, 1H), 7.45 (ddd, J = 8.1, 6.8, 1.2 Hz, 1H), 7.25 (dd, J =10.6, 8.9 Hz, 1H), 4.07 (s, 3H), 2.21 (d, J = 7.0 Hz, 2H), 1.12-1.03 (m, 1H),0.52-0.46 (m, 2H), 0.21 (q, J = 4.9 Hz, 2H) ppm; HRMS (ESI): [M + H] + C 23 H 22 FN2O3 calcd 393.1614, found 393.1617.

[0029] Example 8 Synthesis of Compound A-8 (N-(5-(cyclopropanecarboxamido)-2- fluorophenyl)-3-methoxy-2-naphthamide)

[0030] (I) Synthesis of Intermediate I-4 Intermediate I-4 (0.34 g, 99.2%) was obtained by dissolving 4-fluoro-3-nitroaniline (0.30 g, 1.53 mmol) and TEA (349 mL, 2.51 mmol) in 12 mL of DCM, slowly adding cyclopropanecarbonyl chloride (1 M in DCM, 2.3 mL, 2.30 mmol) dropwise under ice bath, and reacting at room temperature overnight. After the reaction was completed, the organic phase was washed successively with saturated sodium bicarbonate solution and saturated brine, dried with anhydrous sodium sulfate, filtered, rotary evaporated under vacuum, and separated and purified by column chromatography (DCM) to obtain intermediate I-4 (0.34 g, 99.2%).

[0031] 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.67 (s, 1H), 8.53 (dd, J = 6.9, 2.7 Hz,1H), 7.86 (ddd, J = 9.1, 4.0, 2.7 Hz, 1H), 7.54 (dd, J = 11.2, 9.1 Hz, 1H),1.76 (tt, J = 7.3, 5.2 Hz, 1H), 0.89-0.81 (m, 4H) ppm. (II) Synthesis of intermediate I-5 Intermediate I-4 (0.22 g, 0.98 mmol) was dissolved in a mixture of 10 mL of ethanol and 2.5 mL of water, iron powder (0.55 g, 9.81 mmol) and NH4CI (0.53 g, 9.81 mmol) were added, and the mixture was heated to 80 o C and reacted overnight. After the reaction was completed, the residue in the reaction solution was removed by filtration, and after drying by spinning, ethyl acetate (10 mL x 3) was added for extraction, the organic phase was combined, dried with anhydrous sodium sulfate, filtered, and the solvent was removed under vacuum. Compound I-5 (0.18 g, 94.8%) was obtained by column chromatography (DCM).

[0032] 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.91 (s, 1H), 7.09 (dd, J = 8.5, 2.6 Hz,1H), 6.85 (dd, J = 11.3, 8.7 Hz, 1H), 6.67 (ddd, J = 8.7, 4.0, 2.6 Hz, 1H),5.11 (s, 2H), 1.72 (tt, J = 7.2, 5.2 Hz, 1H), 0.80-0.69 (m, 4H) ppm. (III) Synthesis of compound A-8 Intermediate I-5 (0.12 g, 0.78 mmol) and TEA (215 mL, 1.56 mmol) were dissolved in 16 mL of DCM, 3-methoxy-2-naphthoyl chloride (0.2 g, 1.16 mmol) was added under ice bath, slowly warmed to room temperature and reacted overnight. After the reaction was completed, it was washed with saturated sodium bicarbonate solution and saturated brine solution in turn, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, rotary evaporated under vacuum, and separated and purified by column chromatography (PE / EA = 3 / 1) to obtain compound A-8 (107 mg, 36.6%) as a white solid.

[0033] 1 H NMR (400 MHz, DMSO- d 6 ): δ 10.32 (d, J = 2.7 Hz, 2H), 8.48 (s, 1H),8.43 (dd, J = 7.1, 2.6 Hz, 1H), 8.02 (d, J = 8.2 Hz, 1H), 7.91 (d, J = 8.2Hz, 1H), 7.63-7.55 (m, 2H), 7.51 (ddd, J = 9.1, 4.5, 2.7 Hz, 1H), 7.45 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.24 (dd, J = 10.6, 8.9 Hz, 1H), 4.07 (s, 3H),1.79 (tt, J = 7.4, 5.2 Hz, 1H), 0.85-0.76 (m, 4H) ppm; HRMS (ESI): [M + H] + C 22 H 20 FN2O3 calcd 379.1458, found 379.1459.

[0034] Example 9 Synthesis of compound A-9 (N-(5-benzoylamido-2-fluorophenyl)-3-methoxy-2-naphthamide) Using a similar synthetic method as in Example 8, intermediate I-5 was replaced by N-(3-amino-4-fluorophenyl)benzamide to prepare compound A-9. The final test results are as follows: 1 H NMR (400 MHz, DMSO- d6 ): δ 10.40 (s,1H), 10.35 (s, 1H), 8.64 (dd, J = 7.3, 2.6 Hz, 1H), 8.49 (s, 1H), 8.01 (dd, J = 15.8, 7.6 Hz, 3H), 7.92 (d, J = 8.3 Hz, 1H), 7.67 (ddd, J = 9.0, 4.4, 2.7Hz, 1H), 7.63-7.52 (m, 5H), 7.45 (t, J = 7.8 Hz, 1H), 7.32 (t, J = 9.7 Hz,1H), 4.08 (s, 3H) ppm; HRMS (ESI): [M + H] + C 25 H 20 FN2O3 calcd 415.1458, found415.1460.

[0035] Example 10 Synthesis of Compound A-10 (N-(5-(cyclopropylcarbamoyl)-2- fluorophenyl)-3-methoxy-2-naphthamide) Using a synthetic method analogous to Example 8, intermediate I-5 was replaced with 3-amino-4-fluoro- N phenylbenzamide to produce Compound A-10. The final test results are as follows: 1 H NMR (400 MHz, DMSO- d 6): δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ 10.37 (s,1H), 8.57 (dd, J = 7.5, 2.2 Hz, 1H), 8.53 (d, J = 4.2 Hz, 1H), 8.48 (s, 1H),8.03 (d, J = 8.2 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.67 (ddd, J = 8.6, 4.8,2.2 Hz, 1H), 7.62-7.55 (m, 2H), 7.47-7.37 (m, 2H), 4.07 (s, 3H), 2.86 (td, J = 7.3, 3.8 Hz, 1H), 0.70 (td,J = 7.1, 4.6 Hz, 2H), 0.58 (dd, J = 4.8, 2.2Hz, 2H) ppm; HRMS (ESI): [M + H] + C 22 H 20 FN2O3 calcd 379.1458, found 379.1462. The structures of the compounds obtained in Examples 1-10 above are shown in Table 1 below, respectively: Table 1 Structures and names of the compounds of Examples 1-10

[0036]

[0037]

[0038] Example 11 Test of the inhibitory activity of the compounds on ubiquitin-specific protease 2 (USP2) I. Construction of USP2 plasmid First, the PCR product of USP2a was obtained from the Addgene plasmid Flag-HA-USP2 using USP2a primers. The pET28a vector was digested with Hind III and Xho I restriction enzymes, and the USP2a gene was loaded with T4 ligase. The ligation product was transformed into DH5a, and the correctly sequenced plasmid was picked out, and then the USP2cc primers were used to mutate it into a USP2 catalytic core plasmid (USP2cc) using the TOYOBO KOD Plus Mutagenesis Kit. The PCR product was digested with Dpn I restriction enzyme, and then ligated with T4 ligase. The plasmid was transformed into DH5a, and the correctly sequenced USP2cc plasmid was obtained.

[0039] II. Construction of UBA52 plasmid First, the PCR was performed using UBA52 primers with NB4-cDNA as the template. The pGEX-6P-1 vector was digested with BamH I and Xho I, and the PCR product was digested with Dpn I, and then ligated with T4 ligase. The plasmid was transformed into DH5a, and the correctly sequenced UBA52 plasmid was obtained.

[0040] III. Expression and purification of USP2 The recombinant plasmid pET28a-USP2cc (catalytic core, residues 258-605) was transformed into E. coli strain BL21 (DE3) selected with kanamycin. A colony was inoculated into 10 mL of LB-broth containing 100 μg / mL of kanamycin and incubated at 37 °C overnight; the overnight culture was inoculated at a 1:100 ratio by volume into 500 mL of LB-broth containing 100 μg / mL of kanamycin and incubated at 37 °C until the OD600 value reached 0.6-0.8. The recombinant protein expression was induced with 0.5 mM IPTG for 5 hours at 37 °C. The bacteria were separated by centrifugation at 6000 g for 10 minutes and the pellet was resuspended in lysis buffer [50 mM Na2HPO4 / NaH2PO4 (pH 7.4), 300 mM NaCl, 10% glycerol, 10 mM β-mercaptoethanol, and 10 mM imidazole] and then disrupted by sonication. The free cell extract was bound to Ni-NTA resin and extensively washed with wash buffer. The protein was eluted with elution buffer containing 150 mM imidazole. The protein was concentrated with ultracentrifuge tubes and its purity was analyzed by SDS-PAGE and Coomassie blue staining.

[0041] IV. Expression and purification of UBA52 The GEX-6P-1-UbA52 plasmid was transformed into E. coli strain BL21 (DE3) and selected with ampicillin. Similar procedures to USP2 were followed. The protein pellet was resuspended in lysis buffer [50 mM Tris-HCl (pH 7.0), 300 mM NaCl, EDTA 5 mM, DTT 4 mM] and disrupted by sonication. The protein supernatant was supplemented with 0.1% Triton X-100 and incubated with glutathione-agarose beads for 2 hours at 4 °C and then washed twice with wash buffer containing 0.1% Triton X-100 [50 mM Tris-HCl (pH 7.0), 1 M NaCl, EDTA 5 mM, DTT 4 mM] and once with wash buffer without Triton X-100. The purified protein was eluted with elution buffer [1 M Tris-HCl (pH 8.0), 100 mM NaCl, reduced glutathione 20 mM].

[0042] V. UBA52 hydrolysis experiments USP2 (20 nM) and test compounds were incubated in reaction buffer (50 mM Tris-HCl pH 8.0, 20 mM NaCl, 2 mM DTT, 2 mM CaCl2) at 37 ℃ for 10 minutes. Then 3.6 μL UBA52 (27.78 μM) was added to the reaction buffer and incubated at 37 ℃ for 60 minutes. The final volume of the reaction mixture was 50 μL. The reaction was terminated by adding SDS loading buffer and boiled on a heating block for 5 minutes. The proteins were separated by SDS-PAGE, then stained by Coomassie and destained (after electrophoresis, the gel was rinsed with water, then stained with 0.01% Coomassie blue R250 mixed with 50% methanol (volume fraction) and 10% acetic acid (volume fraction) for 10 minutes. The gel was rinsed with 40% methanol (volume fraction) and 7% acetic acid (volume fraction), then destained in the same solution for 30 minutes. Then the gel was soaked in water for 5 minutes, and after changing the water, it was soaked for another 5 minutes), scanned on an Odyssey infrared scanner (λex = 680 nm and λem = 720 nm), and the UBA52 band was quantitatively determined.

[0043] Six, experimental results of USP2 inhibitory activity The inhibitory activity of the compounds of Examples 1-10 on the enzyme is represented by the half inhibitory concentration (IC 50 ), and the positive control drug is ML364, and the results are shown in Table 2.

[0044] Table 2 Test results of inhibitory activity of compounds of Examples 1-10 on USP2

[0045]

[0046] As can be seen from the table, compounds A-4, A-5, A-7, and A-8 have good inhibitory activity on USP2, and the inhibitory activity is better than that of the positive drug ML364, which is a potent USP2 inhibitor.

[0047] Example 12 Test of inhibitory activity of compound A-8 on the proliferation of human colon cancer cell line in vitro I. Cell culture The human colon cancer cell line HCT116 is from the American Type Culture Collection, and the HCT116 cells are cultured in Dulbecco's modified Eagle's medium (DMEM) complete growth medium (high-sugar DMEM medium supplemented with 10% fetal bovine serum, 100 units / mL penicillin and 100 mg / mL streptomycin), and the cells are cultured in a 37 ℃ incubator containing 5% CO2.

[0048] II. In vitro cell viability evaluation Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded into 96-well plates and incubated for 24 hours, treated with different concentrations of compounds (the highest concentration was 100 μM, 3-fold gradient dilution, a total of 9 concentrations, the specific concentrations were 100, 33.33, 11.11, 3.70, 1.23, 0.41, 0.137, 0.0457, 0.01524 μM) for 72 hours, then 20 μL MTT (5 mg / mL) was added to each well. After 4 hours of culture, the MTT solution was removed, DMSO (150 μL) was added to each well, and then the OD value of each well was determined at 550 nm wavelength using a Thermo Varioskan Flash microplate reader, with a blank group (only adding cell-containing culture solution) and a control group (replacing the drug with culture solution) being set. The cell proliferation inhibition rate was calculated. Each concentration of compound was tested in triplicate. Inhibition rate (%) = (1- experimental group 3-hole OD value average / control group 3-hole OD value average) x 100%. The inhibition rate was taken as the vertical coordinate, and a regression curve was drawn to calculate the sample IC 50 value. The measured data was analyzed using GraphPad Prism 5.

[0049] III. Anti-colon cancer cell activity experiment results The inhibition activity of compound A-8 obtained from Example 8 on human colon cancer cells HCT116 is shown in Table 3 below.

[0050] Table 3 Test results of inhibition activity of compound A-8 on HCT116

[0051] As can be seen from Table 3, compound A-8 has good proliferation inhibition activity on HCT116 cell lines.

[0052] The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A compound represented by the general formula (I), a pharmaceutically acceptable salt, isotope, racemate, optically active isomer, polymorph, or mixture thereof: wherein: R1 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R2 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R3 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R4 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R5 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R6 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R7 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R8 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R9 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R10 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R11 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R12 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R13 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R14 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R15 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R16 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R17 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R18 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R19 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R20 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R21 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R22 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R23 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R24 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R25 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R26 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R27 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R28 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R29 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R30 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R31 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R32 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R33 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R34 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R35 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R36 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R37 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R38 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R39 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R40 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R41 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R42 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R43 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R44 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R45 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R46 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R47 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R48 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R49 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R50 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R51 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R52 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R53 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R54 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R55 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R56 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R57 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R58 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R59 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R60 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R61 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R62 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R63 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R64 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R65 is H, alkyl, haloalkyl, cycloalkyl, heterocyclyl, ； ​ R1is selected from the group consisting of: a hydrogen atom, a halogen, C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkoxy, C 3-6 cycloalkyl, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, heterocyclyl, aryl, and heteroaryl; R2is selected from the group consisting of: a hydrogen atom, halogen, C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkoxy, C 3-6 cycloalkyl, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, heterocyclyl, aryl, and heteroaryl; R3is selected from the group consisting of: a hydrogen atom, a halogen, C 1-6 alkyl, C 2-6 alkenyl, C 1-6 alkoxy, C 3-6 cycloalkyl, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, cyano, amino, heterocyclyl, aryl, and heteroaryl; Linker1 is absent or selected from ; wherein X is selected from CH2, O, S, NH, carbonyl, sulfoxide, sulfone, carbamoyl, formamido, sulfonamido, ester; p and q are each independently selected from any integer from 0 to 4; Linker2 is absent or selected from ; wherein Y is selected from CH2, O, S, NH, carbonyl, sulfoxide, sulfone, carbamoyl, formamido, sulfonamido, ester; m and n are each independently selected from any integer from 0 to 4.

2. The compound, pharmaceutically acceptable salt, isotope, racemate, optically active isomer, polymorph, or mixture thereof of claim 1, wherein, ​ 3. The compound, pharmaceutically acceptable salt, isotope, racemate, optically active isomer, polymorph, or mixture thereof of claim 1, wherein, R1is selected from C 1-6 alkyl, C 1-6 alkoxy, C 3-6 cycloalkyl, heterocyclyl, aryl, and heteroaryl; wherein each of said C 1-6 alkyl, C 1-6 alkoxy, C 3-6 cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently optionally substituted with one or more substituents selected from the group consisting of halogen, C 1-6 alkyl, C 1-6 alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, C 3-6 cycloalkyl, heterocyclyl, aryl, and heteroaryl.

4. The compound, pharmaceutically acceptable salt, isotope, racemate, optically active isomer, polymorph, or mixture thereof of claim 1, wherein, R2is selected from C 1-6 alkyl, C 1-6 alkoxy, C 3-6 cycloalkyl, heterocyclyl, aryl, and heteroaryl; wherein the C 1-6 alkyl, C 1-6 alkoxy, C 3-6 cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently optionally substituted with one or more substituents selected from halo, C 1-6 alkyl, C 1-6 alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, C 3-6 cycloalkyl, heterocyclyl, aryl, and heteroaryl.

5. The compound, pharmaceutically acceptable salt, isotope, racemate, optically active isomer, polymorph, or mixture thereof of claim 1, wherein, R3is selected from C 1-6 alkyl, C1 -6 alkoxy, C 3-6 cycloalkyl, heterocyclyl, aryl, and heteroaryl; wherein each of said C 1-6 alkyl, C1 -6 alkoxy, C 3-6 cycloalkyl, heterocyclyl, aryl, and heteroaryl is independently optionally substituted with one or more substituents selected from the group consisting of halo, C 1-6 alkyl, C1 1-6 alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, C 3-6 cycloalkyl, heterocyclyl, aryl, and heteroaryl.

6. The compound of claim 1, a pharmaceutically acceptable salt, isotope, racemate, optically active isomer, polymorph, or mixture thereof, wherein, ​ 7. A method of preparing a compound of claim 1, a pharmaceutically acceptable salt, isotope, racemate, optically active isomer, polymorph, or mixture thereof, characterized in that, The compound of formula (I) is prepared by the following reaction scheme: The compound of formula (I) is prepared by the following reaction scheme: The compound of formula (I) is prepared by the following reaction scheme: The compound of formula (I) is prepared by the following reaction scheme: wherein the , 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and molar ratio of 1: (2.0-4.0): (1.0-2.0): (1.0-1.5), the organic solvent is dichloromethane, the temperature of stirring is 20-30 °C, the time is 12-24 h, and purification is performed using silica gel column chromatography. or will be with triethylamine in an organic solvent, slowly added under ice bath , stirring overnight reaction, after the reaction is finished with saturated sodium bicarbonate solution and saturated brine wash, combined organic phase, rotary evaporation under vacuum to remove the organic solvent, purification, get ; wherein the , triethylamine and molar ratio of 1: (1.5-3.0): (1.0-2.0), the organic solvent is dichloromethane, the temperature of stirring is 20-30 °C, the time is 12-24 h, and purification is performed using silica gel column chromatography. ​ 9. Use according to claim 8, characterized in that, ​ 10. Use according to claim 8, characterized in that, ​

Images