Deubiquitinating enzyme USP28 small-molecule inhibitor as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicinal chemistry, and discloses a deubiquitinating enzyme USP28 small-molecule inhibitor and a preparation method and application thereof, the general formula of the deubiquitinating enzyme USP28 small-molecule inhibitor is as follows: in the formula, A ring is selected from substituted or non-substituted C5-6 aromatic ring; an indole ring; 2, 4, 5-benzothiazole ring; and a substituted or non-substituted C5-6 aromatic heterocyclic ring, wherein a heteroatom is selected from N, O or S. R1 is selected from hydrogen, nitryl, hydroxyl and halogen; the compound is selected from the group consisting of phenyl, phenyl, phenyl, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, phenylamino, r < 2 > is selected from tetrahydroisoquinolyl or tetrahydroisoquinolyl substituted by one or more halogens or methyl groups, phenyl-N, N-dimethyl methylamino, N-methyl phenyl methylamino, methyl tetrahydropyridoxyl, N, N-dimethyl methylaniline or N, N-dimethyl naphthyl amino or N, N-dimethyl halogenated anilino, R < 2 > is selected from tetrahydroisoquinolyl or tetrahydroisoquinolyl substituted by one or more halogens or methyl groups, phenyl-N, N-dimethyl methylamino, N-methyl phenyl methylamino, methyl tetrahydropyridoxyl, N, N-dimethyl methyl anilino or N, N-dimethyl naphthyl amino or N, N-dimethyl halogenated anilino. The compound shows good selective inhibitory activity on USP28 enzyme, and has a good application prospect when being used as an active component for preparing USP28 inhibitor drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a small molecule inhibitor of the deubiquitinating enzyme USP28, its preparation method, and its application. Background Technology

[0002] The USP protein family, or ubiquitin-specific protease (USP) family, also known as the UBP (ubiquitin-specific processing enzymes) family, is a large and structurally diverse class of deubiquitinating enzymes involved in a variety of biological functions. These include: regulating protein stability: By removing ubiquitin chains from proteins, USP proteins prevent protein degradation by the proteasome, thereby increasing protein stability within the cell. For example, in cell cycle regulation, USP proteins can stabilize some key cell cycle regulatory proteins, ensuring the normal progression of the cell cycle. Participating in cell signal transduction: USP proteins can regulate the activity and stability of signaling molecules, influencing intracellular signal transduction pathways. For instance, in the WNT signaling pathway, USP proteins can stabilize β-catenin protein through deubiquitination, thereby enhancing WNT signal transduction and promoting cell proliferation and differentiation. Maintaining ubiquitin homeostasis: USP proteins are crucial for maintaining intracellular free ubiquitin levels. They can recycle and reuse ubiquitin molecules, ensuring the normal functioning of the intracellular ubiquitin system and indirectly affecting many cellular processes that depend on ubiquitination modification.

[0003] USP28 (ubiquitin-specific protease) plays a crucial role in various cancers and is overexpressed in many human tumors, including head and neck cancers, gastric cancer, liver cancer, ovarian cancer, endometrial cancer, prostate cancer, thyroid cancer, kidney cancer, lymphoma, neuroblastoma, and osteosarcoma. On one hand, it can stabilize some oncogene proteins, such as c-Myc and LSD1, promoting cancer cell proliferation and survival. USP28 also regulates DNA damage response (DDR) in cancer cells by deubiquitination and stabilization of checkpoint kinase 2 (CHK2), tp53-binding protein 1 (TP53BP1), and claspn (CLSPN), thereby preventing apoptosis; however, its exact function in establishing cell cycle arrest to promote DNA repair remains under investigation. On the other hand, high expression of USP28 is associated with poor tumor prognosis. Therefore, targeting USP28 inhibitors holds promise as a novel strategy for treating cancer and hematological diseases.

[0004] Currently, USP28 has become a potential target for cancer treatment, and some inhibitors targeting USP28 are under development. However, due to the structural similarity between USP28 and USP25, the specificity of the inhibitors has become a challenge. In addition to cancer, USP28 may also be related to the occurrence and development of other diseases.

[0005] Research on USP28 inhibitors has become a hot topic in new drug development, with increasing research into their enzyme and crystal structures. This indicates that USP28 inhibitors hold promise as effective anticancer drugs with novel mechanisms of action. To date, although USP28 inhibitors have been discovered or developed, and many highly active compounds have entered Phase I clinical trials, most of these molecules exhibit poor selectivity. Therefore, there is still a lack of effective selective inhibitors targeting USP28 in clinical trials, highlighting the significant potential for their development. Summary of the Invention

[0006] Targeting USP28, this invention aims to provide compounds with good activity that can be used as USP28 inhibitors for the prevention or treatment of diseases in humans or mammals related to USP28 activity.

[0007] To achieve the objectives of this invention, a novel selective inhibitor of USP28 is prepared, which is a compound as shown in general formula I or a pharmaceutically acceptable salt, solvate, hydrate, active metabolite, polymorph, ester, isotopic compound, stereoisomer or prodrug thereof.

[0008] In the formula: ring A is selected from substituted or unsubstituted C5-6 aromatic rings; indole rings; 4,5-benzothiazole rings; substituted or unsubstituted rings. The substituted C5-6 aromatic heterocycle has heteroatoms selected from N, O, or S. R1 is selected from hydrogen, nitro, hydroxyl, halogen; aniline, acetylaminopiperidinyl, aminotetrahydropyranyl, benzylamino or sulfonylmethylpiperazinyl substituted with one or more halogens or methyl groups. R2 is selected from tetrahydroisoquinolinyl or tetrahydroisoquinolinyl, phenyl-N,N-dimethylmethylamino, N-methylphenylmethylamino, methyltetrahydropyridindolyl, N,N-dimethylmethylaniline or N,N-dimethylnaphthylamino or N,N-dimethylhaloaniline.

[0009] Preferably, the A-ring structure is selected from benzene rings, benzene rings substituted with one or more halogens, benzene rings monosubstituted with nitro or C1-3 alkyl groups; pyridine rings, furan rings, and unsaturated six-membered rings containing two nitrogen atoms;

[0010] The A-ring structure is more preferably, but not limited to, one of the following structures:

[0011] Preferably, the R1 group is selected from hydrogen, nitro, hydroxyl, halogen; aniline, acetylaminopiperidinyl, aminotetrahydropyranyl or benzylamino groups substituted with one or more halogens or methyl groups;

[0012] The R1 group is more preferably selected from, but not limited to, one of the following structures: hydrogen,

[0013] Preferably, R2 is selected from tetrahydroisoquinoline, phenyl-N,N-dimethylmethylamino, N-methylphenylmethylamino, methyltetrahydropyridindolyl, N,N-dimethylmethylaniline, or N,N-dimethylnaphthylamino or N,N-dimethylhaloaniline.

[0014] The R2 is more preferably selected from, but not limited to, one of the following structures:

[0015] The use of any of the compounds described above in the preparation of medicaments for the prevention or treatment of USP28-mediated diseases, including but not limited to cancer, hematologic disorders, or muscle-related diseases.

[0016] The cancers mentioned include, but are not limited to, bladder cancer, head and neck cancer, stomach cancer, liver cancer, ovarian cancer, endometrial cancer, prostate cancer, thyroid cancer, kidney cancer, lymphoma, neuroblastoma, and osteosarcoma.

[0017] The synthetic route of the USP28 inhibitor includes the following steps:

[0018] (a) Compound II reacts with a compound containing an amino group R2 to give compound III; (b) Compound III is deprotected by removing the Boc protecting group in a solvent to give compound IV; (c) Compound IV is condensed with an amide of a carboxylic acid A ring to give V; (d) Compound V is coupled with a compound containing an amino group R1 to generate target compound I. The specific preparation method is as follows: a) Compound (III) is obtained by reacting an epoxy compound (II) with an amino-containing R2 group compound under solvent and alkaline conditions; wherein the solvent used is tetrahydrofuran, N,N-dimethylformamide, methanol or ethanol; the base used in the reaction is K2CO3, Na2CO3, Cs2CO3 or NaOH; the reaction temperature is preferably 50-100℃; and the reaction time is preferably 30 minutes to 6 hours. b) The 4-hydroxypiperidine compound III obtained in step a) is deprotected by the Boc protecting group under solvent and acidic conditions to generate 4-hydroxypiperidine compound (IV); the solvent used is dichloromethane, ethyl acetate, methanol, dioxane, tetrahydrofuran, N,N-dimethylformamide or toluene; the acid used is trifluoroacetic acid or hydrochloric acid. c) Using a carboxylic acid compound containing an A ring and compound IV obtained in step b) as raw materials, a reaction is carried out under alkaline conditions using a condensing agent in a solvent to generate the corresponding amide compound (V); wherein the solvent used is tetrahydrofuran, N,N-dimethylformamide, chloroform, dichloromethane or toluene, and the reaction can be carried out in a single solvent or a mixture of the above solvents; the condensing agent used is EDCI (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), CDI (1,1'-carbonyldiimidazole), HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) or DIC (N,N'-diisopropylcarbodiimide); the base used in the reaction is sodium bicarbonate, potassium carbonate, triethylamine or pyridine; the reaction temperature is preferably 0-80℃; the reaction time is preferably 30 min-12 h. d) Under solvent and alkaline conditions, the compound (V) obtained in step c) reacts with amino compounds containing different R1 groups under a palladium catalyst to obtain compound (I); the solvent used in the reaction is dimethyl sulfoxide, tetrahydrofuran, N,N-dimethylformamide, chloroform, dichloromethane or toluene; the palladium catalyst used is tetratetraphenylphosphine palladium, palladium acetate, diphenylphosphine ferrocene palladium dichloride, dichloroditriphenylphosphine palladium, chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium (II) or palladium on carbon; the base used in the reaction is Na2CO3, K2CO3, Cs2CO3, potassium tert-butoxide or sodium tert-butoxide; the molar ratio of compound VI:palladium catalyst:base is 1:0.005 to 1.0:1.05; the preferred reaction temperature is 30℃ to 120℃; the preferred reaction time is 5h to 48h.

[0019] Each product obtained in the reaction route can be obtained using conventional separation techniques, including but not limited to filtration, distillation, crystallization, and chromatographic separation. The starting materials required for synthesis can be synthesized in-house or purchased from commercial sources. The compounds described in this invention can be synthesized to obtain single optical isomers or mixtures of optical isomers.

[0020] Advantages of this invention: The synthetic route is simple, and the synthesized compounds exhibit good inhibitory activity against the USP28 enzyme. Biological activity evaluation, particularly compounds 1, 2, 3, and 4, shows similar inhibitory activity to the positive control compound AZ1. These compounds can be used as active ingredients and as USP28 inhibitors in the preparation of drugs for diseases including, but not limited to, cancer, hematological disorders, and muscle-related diseases. Detailed Implementation

[0021] The invention can be better understood through the following examples. However, those skilled in the art will readily understand that the embodiments described are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0022] Example 1: Synthesis of Compound 1 (1) Synthesis of tert-butyl 4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypyridine-1-carboxylate Tetrahydroisoquinoline (4.67 g, 35.1 mmol) and tert-butyl 1-oxo-6-azaspiro[2.5]octane-6-carboxylic acid (5 g, 23.4 mmol) were added to a flask, 50 ml of anhydrous ethanol was added, the mixture was dissolved by sonication, potassium carbonate was added, the mixture was reacted at 80 °C for 6 h, concentrated by rotary evaporation, and separated by column chromatography (petroleum ether: ethyl acetate = 1:1) to give the compound as a colorless viscous oily liquid (5.34 g, yield 66%). HNMR (400MHz, CDCl3) δ7.09(dd,J=6.7,2.1Hz,3H),6.95(d,J=6.5Hz,1H),3.86(s,2H),3.77(s,2H),3.34(dd,J=2 2.7, 20.1Hz, 1H), 3.15 (s, 2H), 2.87 (s, 4H), 2.45 (s, 2H), 1.54 (d, J = 12.5Hz, 3H), 1.45 (s, 9H), 1.44-1.40 (m, 1H). (2) Synthesis of 4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)piperidine-4-ol 2-hydrochloride Compound B (6.5 g, 19 mmol) was dissolved in 10 mL of methanol, and 5 mL of 4 mol / L hydrochloric acid-methanol solution was added with stirring at room temperature. After the reaction was completed by TLC monitoring, the solvent was evaporated to dryness under reduced pressure, and the mixture was slurried with a mixture of anhydrous ethanol and ethyl acetate to give compound C, a white solid, yield: 98%. 1H NMR (400MHz, DMSO-d6) δ10.58(s,1H),9.34(s,2H),7.36-7.11(m,4H),5.99(s,1H),4.63(d,J=15.2Hz,1H),4.48(dd,J=15.4,5.2Hz,1H),3 .85-3.70(m,1H),3.53(d,J=6.5Hz,1H),3.42-3.34(m,1H),3.28(dd,J=18.5,5.3Hz,2H),3.19-2.99(m,5H),1.96(dt,J=24.1,12.4Hz,4H). (3) Synthesis of (6-bromopyridin-2-yl)(4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-yl) methyl ketone. The starting material 6-bromo-2-pyridinecarboxylic acid (200 mg, 1.0 mmol) and the condensing agent CDI (1,1'-carbonyldiimidazole) (258 mg, 1.59 mmol) were dissolved in 5 mL of dichloromethane. Triethylamine (1 mL, 7.25 mmol) was added, and the mixture was activated at room temperature with stirring for 30 min. Then, compound C (510 mg, 1.60 mmol) was added to the reaction system, and the reaction was continued with stirring at room temperature. After the reaction was completed as monitored by TLC, 20 mL of water was added to the system, and the mixture was extracted with DCM (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The solution was purified by silica gel column chromatography (PE / EA = 1 / 2, v / v) to give compound D as a white solid, with a yield of 39%. 1HNMR (400MHz, Acetone-d6) δ8.49-8.38 (m, 1H), 7.59 (s, 1H), 7.39 (dd, J = 4.9 ,1.3Hz,1H),7.14-7.03(m,3H),7.03-6.92(m,1H),4.33(d,J=12.8Hz,1H),3. 79(s,2H),3.51-3.39(m,2H),3.21(td,J=12.5,3.1Hz,1H),2.89(td,J=8.2,3 .4Hz,4H),2.56(s,2H),1.76-1.56(m,4H).LC-MS(ESI+)m / z[M+H]+calculated for C21H24BrN3O2:430.11, found 430.27. (4) 1-(4-((6-(4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-carbonyl)pyridin-2-yl)amino)piperidin-1-yl)acetone The compound D (75 mg, 0.175 mmol), 1-acetyl-4-aminopiperidine (CAS No. 160357-94-8) (50 mg, 0.35 mmol), sodium tert-butoxide (34 mg, 0.35 mmol), and chloro(2-dicyclohexylphosphino-2',4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) (20 mg, 0.027 mmol) were simultaneously added to a 25 mL round-bottom flask. 5 mL of toluene was added, and the mixture was degassed by sonication. After replacing the air in the flask with N2 four times, the reaction system was placed in an oil bath at 85 °C and stirred for 12 h. After the reaction was complete, the system was cooled to room temperature, filtered through a diatomaceous earth liner, and the filter cake was washed with 5 mL of toluene. The filtrates were combined and evaporated to dryness under reduced pressure. Preparative liquid chromatography (Waters 2545 prep LC, 2489 UV, 2707 autosampler, C18 19*150 mm column; mobile phase A: ultrapure water with 0.1% v / v trifluoroacetic acid, B: methanol) was performed to obtain the target final product 1, which was lyophilized to give a white solid with a yield of 26%. 1H NMR (400MHz, MeOD) δ7.44(dd,J=8.4,7.2Hz,1H),7.11-7.03(m,3H),6.97(d,J=7.7Hz,1H),6.67(dd,J=7.1,3.3Hz,1H),6.55-6.49(m,1H), 4.36(d,J=13.4Hz,2H),4.07-3.96(m,1H),3.77(q,J=15.1Hz,4H),3.41(t,J=12.4Hz,1H),3.29-3.19(m,1H),3.19-3.07(m,1H),2.95(dd,J =10.4,3.8Hz,1H),2.88(s,2H),2.87-2.82(m,1H),2.77(dd,J=18.0,6.8Hz,1H),2.56(d,J=2.5Hz,2H),2.04(d,J=21.0Hz,4H),1.96(d,J=1 3.4Hz,1H),1.83(ddd,J=15.4,7.6,4.0Hz,1H),1.78-1.70(m,2H),1.70-1.59(m,1H),1.46-1.27(m,2H).HRMS(ESI+)m / z[M+H]+calculated forC28H37N5O3:492.29747; found 492.29645.

[0023] Example 2 (4-Chloro-3-nitrophenyl)(4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-yl)methyl ketone Compound 2: The synthesis process is the same as in Example 1, except that the raw material 6-bromo-2-pyridinecarboxylic acid in step (3) is replaced with 4-chloro-3-nitrobenzoic acid to obtain Compound 2, a pale yellow solid with a yield of 77%. 1H NMR (400MHz, MeOD) δ8.01(d,J=1.8Hz,1H),7.74(d,J=8.3Hz,1H),7.66(dd,J=8.3,1.9Hz,1H),7.11-7.03(m,3H),7.01-6.95(m, 1H),4.36(d,J=12.3Hz,1H),3.77(s,2H),3.48(d,J=10.8Hz,2H),3.30(s,1H),2.88(s,4H),2.54(s,2H),1.79-1.56(m,4H).13C NMR (100MHz, Acetone-d6) δ166.80,148.95,138.26,136.40,135.26,132.76,132.74,129.37,127.2 4,127.18,126.79,126.29,125.07,70.36,68.74,58.99,54.21,44.64,39.00,36.72,35.87,30.07.

[0024] Example 3 (4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-yl)(3-hydroxyphenyl)methyl ketone Compound 3: The synthesis process was the same as in Example 1, except that the raw material 6-bromo-2-pyridinecarboxylic acid described in step (3) was replaced with 3-hydroxybenzoic acid to obtain compound 3, a white solid with a yield of 27%. ¹H NMR (400MHz, CDCl₃) δ 7.20 (d, J = 8.5Hz, 2H), 7.15-7.07 (m, 3H), 6.98 (d, J = 6.4Hz, 1H), 6.77 (d, J = 8.3Hz, 2H), 4.39 (s, 1H), 3.84 (s, 2H), 3.65 (s, 1H), 3.43 (s, 1H), 3.23 (s, 1H), 2.93 (dd, J = 17.5, 4.8Hz, 4H), 2.58 (s, 2H), 1.62 (m, 4H). ¹³C NMR(100MHz,DMSO-d6)δ169.09,158.44,135.45,134.18,128.90,128.36,126.65,126.27,12 5.78,125.31,114.78,69.74,67.41,57.57,52.58,28.76.LC-MS(ESI+)m / z[M+H]+calculated for C22H26N2O3:367.20, found 367.28.

[0025] Example 4 (2-Bromopyridin-4-yl)(4-(((3-fluorobenzyl)(methyl)amino)methyl)-4-hydroxyperidin-1-yl)methyl ketone Compound 4: The synthesis process was the same as in Example 1, except that the raw material 6-bromo-2-pyridinecarboxylic acid in step (3) was replaced with 2-bromoisonicotinic acid; tetrahydroisoquinoline was replaced with N-methyl-3-fluorobenzylamine, yielding a white solid with a yield of 90%. ¹H NMR (400MHz, DMSO-d6) δ 7.62 (d, J = 7.8Hz, 2H), 7.33 (d, J = 7.9Hz, 2H), 7.09 (d, J = 3.1Hz, 3H), 7.00 (s, 1H), 4.39 (s, 1H), 4.16 (s, 1H), 3.70 (s, 2H), 3.18 (s, 2H), 2.80 (s, 4H), 2.45 (s, 2H), 1.58 (s, 4H). ¹³C NMR(100MHz,DMSO-d6)δ167.76,135.58,135.44,134.18,131.34,128.89,128.36,126.28 ,125.79,125.32,122.54,69.65,67.40,57.57,52.57,43.35,37.73,35.19,34.47,28.74.

[0026] Example 5 (4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-yl)(4-nitrophenyl)methyl ketone Compound 5: The synthesis process was the same as in Example 1, except that the starting material 6-bromo-2-pyridinecarboxylic acid in step (3) was replaced with 4-nitrobenzoic acid, yielding compound 5, a white solid with a yield of 85%. ¹H NMR (400MHz, CDCl₃) δ 8.27 (d, J = 8.7Hz, 2H), 7.57 (d, J = 8.7Hz, 2H), 7.18-7.09 (m, 3H), 7.02-6.95 (m, 1H), 4.54 (d, J = 12.7Hz, 1H), 3.82 (s, 2H), 3.55-3.38 (m, 2H), 3.26 2.92(d,J=3.9Hz,4H),2.55(d,J=2.5Hz,2H),1.75(d,J=13.2Hz,1H),1.69-1.54( m,2H),1.47(td,J=12.6,4.5Hz,1H).13CNMR(100MHz,CDCl3)δ167.75,148.13,142 .47,134.59,133.85,128.62,127.80,126.36,126.28,125.65,123.75,68.69,67. 42,58.25,53.56,43.75,38.31,35.46,29.06.LC-MS(ESI+)m / z[M+H]+calculated for C22H25N3O4:396.19, found 396.22.

[0027] Example 6 1-(4-((4-(4-(benzyl(methyl)amino)methyl)-4-hydroxypiperidin-1-carbonyl)pyridin-2-yl)amino)piperidin-1-yl)acetoone Compound 6: The synthesis process was the same as in Example 1, except that the R2 group was replaced with N-methylbenzylamine instead of tetrahydroisoquinoline, yielding compound 6, a white solid, with a yield of 32%. ¹H NMR (400 MHz, Acetone-d6) δ 8.08–8.00 (m, 1H), 7.41–7.29 (m, 4H), 7.25 (t, J = 7.2 Hz, 1H), 6.48–6.42 (m, 2H), 5.91 (d, J = 7.5 Hz, 1H), 4.39 (d, J = 13.2 Hz, 1H), 4.23 (d, J = 12.4 Hz, 1H), 4.11–4.00 (m, 1H), 3.88 (d, J = 13.7 Hz, 1H), 3.65 (s, 2H), 3.41 (d,J=11.1Hz,2H),3.22(ddd,J=32.9,15.4,6.7Hz,2H),2.86-2.75(m,2H),2.51(s,2H),2.30(s,3H),2.09(s,1H),2.02 (s,3H),1.98(d,J=5.4Hz,1H),1.67(d,J=12.8Hz,1H),1.58(d,J=13.8Hz,3H),1.49-1.41(m,1H),1.37-1.31(m,1H).13C NMR (100MHz, Acetone-d6) δ168.67,168.55,159.38,159.34,149.05,146.51,129.77,129.06,127.83,110.23,106.41, 70.59,68.29,65.09,48.77,45.77,45.27,44.12,40.86,33.45,32.65,30.60,21.46.HRMS(ESI+)m / z[M+H]+calculated for C27H37N5O3:480.29747; found 480.29666.

[0028] Example 7 1-(4-((3-(4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-carbonyl)-5-fluorophenyl)amino)piperidin-1-yl)ethyl-1-one Compound 7: The synthesis process was the same as in Example 1, except that the starting material 6-bromo-2-pyridinecarboxylic acid in step (3) was replaced with 3-bromo-5-fluorobenzoic acid to obtain compound 7, with a yield of 19%. ¹H NMR (400MHz, MeOD) δ 7.12-7.03 (m, 3H), 7.02-6.95 (m, 1H), 6.46-6.39 (m, 2H), 6.32-6.26 (m, 1H), 4.42-4.27 (m, 2H), 3.87 (dd, J = 13.9, 1.8Hz, 1H), 3.77 (s, 2H), 3.54 (dd, J = 1 6.6,12.2Hz,2H),3.42(t,J=10.2Hz,1H),3.25(t,J=10.9Hz,2H),2.94-2.84(m,5H),2 .54(s,2H),2.10(s,3H),2.08-1.96(m,2H),1.76-1.56(m,4H),1.44-1.27(m,2H).13C NMR(100MHz,MeOD)δ171.74,171.48,166.56,151.29(d,J=11.4Hz),139.62(d,J =9.7Hz),136.38,135.41,129.58,127.47,127.18,126.64,107.65,101.77,101. 53,101.23,100.98,71.41,69.03,59.39,54.60,50.35,46.29,45.03,41.57,39. 36,36.60,35.84,33.19,32.54,30.08,21.22.HRMS(ESI+)m / z[M+H]+calculated forC29H37FN4O3:509.29279; found 509.29172.

[0029] Example 8 (2,4-Difluorophenyl)(4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-yl)methyl ketone Compound 8: The synthesis process was the same as in Example 1, except that the starting material 6-bromo-2-pyridinecarboxylic acid in step (3) was replaced with 2,4-difluorobenzoic acid, yielding compound 8, a white solid with a yield of 23%. ¹H NMR (400MHz, MeOD) δ 7.41 (dd, J = 14.5, 7.6Hz, 1H), 7.11-7.01 (m, 5H), 6.97 (d, J = 5.3Hz, 1H), 4.39 (d, J = 13.1Hz, 1H), 3.75 (s, 2H), 3.51-3.38 (m, 1H), 3.35 (s, 1H), 3.31-3.23 (m, 1H), 2.86 (s, 4H), 2.52 (s, 2H), 1.78-1.56 (m, 4H). ¹³C NMR (100MHz, DMSO-d6) δ162.89,135.41,134.16,130.12,128.36,126.27,125.79,125.32,121.25,112.27,112. 06,104.30,69.60,67.27,57.54,52.56,42.88,37.48,35.21,34.47,28.74.LC-MS(ESI+)m / z[M+H]+calculated for C22H24F2N2O2:387.19, found 387.22.

[0030] Example 9 (4-((benzylamino)methyl)-4-hydroxypiperidin-1-yl)(2-((2-fluorophenyl)amino)pyridin-4-yl)methyl ketone Compound 9: The synthesis process was the same as in Example 1, except that the R1 group was replaced with 2-F-aniline instead of p-acetylaniline, and the R2 group was replaced with benzylamine instead of tetrahydroisoquinoline, to obtain compound 9, a white solid, yield: 16%. ¹H NMR (400 MHz, CDCl₃) δ 11.81 (s, 1H), 9.10 (s, 2H), 7.92 (d, J = 6.1 Hz, 1H), 7.36 (d, J = 6.2 Hz, 6H), 7.06 (d, J = 7.8 Hz, 1H), 7.03–6.96 (m, 3H), 6.74 (d, J = 6.2 Hz, 1H), 4.31 (d, J = 12.8 Hz, 1H), 4.02 (s, 2H), 3.38 (t, J) =12.2Hz,1H),3.27(d,J=11.1Hz,1H),3.07(t,J=12.4Hz,1H),2.88(s,2H),1.80(d,J=13.0Hz,1H),1.7 3(d,J=13.1Hz,1H),1.47(t,J=11.1Hz,1H),1.34(d,J=10.8Hz,1H).LC-MS(ESI+)m / z[M+H]+calculated for C25H27FN4O2:435.22,found435.28.

[0031] Example 10 1-(4-((6-(4-((benzylamino)methyl)-4-hydroxypiperidin-1-carbonyl)pyrimidin-4-yl)amino)piperidin-1-yl)acetoone Compound 10: The synthesis process is the same as in Example 1, except that the raw material 6-bromo-2-pyridinecarboxylic acid in step (3) is replaced with 6-bromopyrimidine-4-carboxylic acid, and the R2 group is replaced with benzylamine instead of tetrahydroisoquinoline, to obtain compound 10, a white solid with a yield of 12%. ¹H NMR (400MHz, CDCl₃) δ 12.10 (s, 1H), 9.22 (s, 2H), 7.99 (d, J = 8.3Hz, 3H), 7.36 (d, J = 7.7Hz, 7H), 7.13 (s, 1H), 6.79 (d, J = 6.0Hz, 1H), 4.33 (d, J = 11.9Hz, 1H), 3.99 (s, 2H), 3.40 (t, J = 12.0Hz, 1H), 3.30 ( d,J=12.6Hz,1H),3.08(t,J=12.3Hz,1H),2.87(s,2H),2.60(s,3H),1.82(d,J=13.8Hz,1H),1.74(d ,J=12.8Hz,1H),1.47(t,J=10.7Hz,1H),1.32(t,J=11.1Hz,1H).LC-MS(ESI+)m / z[M+H]+calculated for C27H30N4O3:459.24, found 459.22.

[0032] Example 11 1-(4-((4-(4-hydroxy-4-)(methyl(4-methylbenzyl)amino)methyl)piperidin-1-carbonyl)pyridin-2-yl)amino)piperidin-1-yl)acetic-1-one Compound 11: The synthesis process is the same as in Example 1, except that the starting material 6-bromo-2-pyridinecarboxylic acid in step (3) is replaced with 2-bromoisonicotinic acid, and R2 is replaced with N-methyl-1-(p-tolyl)methylamine, to obtain compound 11, a white solid, with a yield of 32%. 1H NMR (400MHz, Acetone-d6) δ8.08-8.00(m,1H),7.41-7.29(m,4H),7.25(t,J=7.2Hz,1H),6.48-6.42(m,2H),5.91(d,J=7 .5Hz,1H),4.39(d,J=13.2Hz,1H),4.23(d,J=12.4Hz,1H),4.11-4.00(m,1H),3.88(d,J=13.7Hz,1H),3.65(s,2H),3.41 (d,J=11.1Hz,2H),3.22(ddd,J=32.9,15.4,6.7Hz,2H),2.86-2.75(m,2H),2.51(s,2H),2.30(s,3H),2.09(s,1H),2.02 (s,3H),1.98(d,J=5.4Hz,1H),1.67(d,J=12.8Hz,1H),1.58(d,J=13.8Hz,3H),1.49-1.41(m,1H),1.37-1.31(m,1H).13C NMR (100MHz, Acetone-d6) δ168.67,168.55,159.38,159.34,149.05,146.51,129.77,129.06,127.83,110.23,106.41, 70.59,68.29,65.09,48.77,45.77,45.27,44.12,40.86,33.45,32.65,30.60,21.46.HRMS(ESI+)m / z[M+H]+calculated for C27H37N5O3:480.29747; found 480.29666.

[0033] Example 12 (4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-yl)(4-hydroxyphenyl)methyl ketone Compound 12: The synthesis process is the same as in Example 1, except that the starting material 6-bromo-2-pyridinecarboxylic acid in step (3) is replaced with p-hydroxybenzoic acid. It is a white solid. The yield of Compound 12 is 27%. ¹H NMR (400MHz, CDCl₃) δ 7.20 (d, J = 8.5Hz, 2H), 7.15-7.07 (m, 3H), 6.98 (d, J = 6.4Hz, 1H), 6.77 (d, J = 8.3Hz, 2H), 4.39 (s, 1H), 3.84 (s, 2H), 3.65 (s, 1H), 3.43 (s, 1H), 3.23 (s, 1H), 2.93 (dd, J = 17.5, 4.8Hz, 4H), 2.58 (s, 2H), 1.62 (m, 4H). ¹³C NMR(100MHz,DMSO-d6)δ169.09,158.44,135.45,134.18,128.90,128.36,126.65,126.27,12 5.78,125.31,114.78,69.74,67.41,57.57,52.58,28.76.LC-MS(ESI+)m / z[M+H]+calculated for C22H26N2O3:367.20, found 367.28.

[0034] Example 13 1-(4-((4-(4-hydroxy-4-)((1,3,4,5-tetrahydro-2H-pyridin[4,3-b]indol-2-yl)methyl)piperidin-1-carbonyl)pyridin-2-yl)amino)piperidin-1-yl)acetyl-1-one Compound 13: The synthesis process was the same as in Example 1, except that the raw material 6-bromo-2-pyridinecarboxylic acid in step (3) was replaced with 2-bromoisonicotinic acid, and the R2 tetrahydroisoquinoline substituent was replaced with 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole, to obtain compound 13, a white solid, with a yield of 27%. ¹H NMR (400MHz, MeOD) δ 8.77 (d, J = 1.8Hz, 1H), 8.58 (s, 1H), 8.10 (t, J = 1.9Hz, 1H), 7.50 (d, J = 7.8Hz, 1H), 7.36 (d, J = 8.1Hz, 1H), 7.16 (t, J = 7.5Hz, 1H), 7.06 (t, J = 7.5Hz, 1H), 4.78 (d, J = 14.3Hz, 1H). H),4.61(d,J=14.5Hz,1H),4.44(d,J=11.1Hz,1H),3.90(s,1H),3.70(s,1H),3.56(d,J=12.3H z,2H),3.42(s,1H),3.36(d,J=6.4Hz,2H),3.19(t,J=5.4Hz,2H),1.90(s,1H),1.77(s,3H).13C NMR(100MHz,MeOD)δ167.74,152.82,146.73,138.91,138.42,134.74,127.27,125.44,123.58,121.95,120.71 ,119.06,112.41,106.56,69.00,65.38,54.65,53.13,44.46,38.90,18.68.LC-MS(ESI+)m / z[M+H]+calculated for C23H25BrN4O2:469.12, found 468.91.

[0035] Example 14 1-(4-((4-(4-hydroxy-4-(methyl(naphthyl-1-ylmethyl)amino)methyl)piperidin-1-carbonyl)pyridin-2-yl)amino)piperidin-1-yl)acetoone Compound 14: The synthesis process was the same as in Example 1, except that the starting material 6-bromo-2-pyridinecarboxylic acid in step (3) was replaced with 2-bromoisonicotinic acid, and the R2 tetrahydroisoquinoline substituent was replaced with N,N-dimethyl-1-(naphth-1-yl)methylamine, to obtain compound 14, a white solid, with a yield of 17%. 1H NMR (400MHz, MeOD) δ8.31(d,J=8.5Hz,1H),8.07(d,J=8.2Hz,1H),8.01(d,J=8.0Hz,1H),7.94(d,J=6.5Hz,1H),7.78(d,J=7.0Hz,1H),7.69(t,J= 7.3Hz,1H),7.61(dd,J=14.8,7.2Hz,2H),7.04(s,1H),6.83(d,J=6.5Hz ,1H),5.24(s,1H),4.75(s,1H),4.49(d,J=13.4Hz,1H),4.29(s,1H),3.9 8(d,J=13.7Hz,1H),3.91(dq,J=9.8,3.3Hz,1H),3.52(dd,J=18.3,6.8H z,2H),3.42(s,2H),3.31(s,1H),3.28(d,J=11.9Hz,1H),2.98(s,3H),2. 92-2.82(m,1H),2.13(s,3H),2.11-2.00(m,2H),1.78(s,2H),1.67-1.61 (m,1H),1.59(d,J=2.4Hz,1H),1.57-1.51(m,1H),1.51-1.43(m,1H).13C NMR(100MHz,MeOD)δ171.67,167.15,153.82,135.56,133.63,132.88,132.55,130.34,128.81,127.75,127.03,126.46,124.25,111.21 ,68.85,67.35,60.58,50.63,45.95,44.83,43.86,41.18,38.54,36.39,35.42,32.46,31.81,21.20.LC-MS(ESI+)m / z[M+H]+calculated forC31H39N5O3:530.31, found 530.28.

[0036] Example 15 1-(4-((3-(4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-carbonyl)-5-nitrophenyl)amino)piperidin-1-yl)ethyl-1-one Compound 15: The synthesis process was the same as in Example 1, except that the starting material 6-bromo-2-pyridinecarboxylic acid in step (3) was replaced with 3-bromo-5-nitrobenzoic acid, yielding compound 15, a white solid with a yield of 10%. ¹H NMR (400MHz, CDCl₃) δ 7.47 (s, 1H), 7.41 (d, J = 2.0Hz, 1H), 7.13 (dt, J = 9.8, 4.8Hz, 3H), 6.99 (d, J = 6.2Hz, 1H), 6.90 (s, 1H), 4.52 (d, J = 13.2Hz, 2H), 4.35 (d, J = 7.7Hz, 1H), 3.83 (s, 3H), 3.62-3.46 (m, 3H). 3.24(dd,J=18.3,6.9Hz,2H),2.92(s,4H),2.85(d,J=11.1Hz,1H),2.55(s,2H),2.12(s,3H),2.06(d ,J=11.5Hz,2H),1.74(d,J=12.8Hz,1H),1.65-1.55(m,2H),1.54-1.45(m,1H),1.44-1.33(m,2H).13C NMR (100MHz, CDCl3) δ168.99,168.25,149.20,147.99,138.53,134.59,133.90,128.76,126.49,125.82,116.94,109.59,107.32,68.66 ,67.43,58.39,53.73,49.86,44.98,43.98,40.24,38.52,36.75,35.71,32.38,31.72,29.17,21.48.HRMS(ESI+)m / z[M+H]+calculated for C29H37N5O5:536.28729; found 536.28656.

[0037] Example 16 1-(3-((3-(4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-carbonyl)-2-methylphenyl)amino)piperidin-1-yl)acetic acid-1-one Compound 16: The synthesis process was the same as in Example 1, except that the starting material 6-bromo-2-pyridinecarboxylic acid in step (3) was replaced with 3-bromo-2-methylbenzoic acid, yielding compound 16, a white solid with a yield of 13%. ¹H NMR (400MHz, Acetone-d6) δ 7.17-6.96 (m, 5H), 6.72 (d, J = 8.3Hz, 1H), 6.44 (dd, J = 19.9, 7.3Hz, 1H), 4.42 (t, J = 15.3Hz, 2H), 4.23 (dd, J = 14.9, 7.6Hz, 1H), 3.91 (d, J = 12.8Hz). ,1H),3.69(dd,J=39.8,29.6Hz,3H),3.32-3.15(m,4H),2.82(d,J=36.4Hz,4H),2.56( s,2H),2.13(s,1H),2.01(d,J=13.6Hz,7H),1.77-1.58(m,2H),1.57-1.28(m,4H).13C NMR (100MHz, Acetone-d6) δ170.18,168.68,146.41,139.23,135.60,130.08,128.53,127.40,127.07,126.63,119.02,118.79,114.44,110 .90,60.20,50.58,45.80,45.64,43.08,40.93,33.38,32.77,32.65,30.15,23.69,21.45,14.27,13.93.HRMS(ESI+)m / z[M+H]+calculated for C30H40N4O3:505.31787; found 505.3170.

[0038] Example 17 (4-((benzylamino)methyl)-4-hydroxypiperidin-1-yl)(2-(benzylamino)pyridin-4-yl)methyl ketone The synthesis of compound 17 was performed as in Example 1, except that the R1 group was replaced with benzylamine instead of p-acetylaniline, and the R2 group was replaced with benzylamine instead of tetrahydroisoquinoline, yielding compound 17, a white solid, in 15% yield. 1H NMR (400MHz, Acetone) δ8.62(s,1H),8.44(d,J=4.0Hz,1H),7.76(d,J=7.7Hz,1H),7.29(dd ,J=7.7,4.8Hz,1H),7.15–7.07(m,4H),7.00(d,J=5.1Hz,1H),6.72(dd,J=8.1,1.6Hz,1H), 6.66–6.59(m,2H),5.80(s,1H),4.42(d,J=4.4Hz,2H),4.26(s,1H),3.77(s,2H),3.46(s,1 H), 3.34–3.10 (m, 2H), 2.91–2.84 (m, 5H), 2.51 (s, 2H), 1.97 (s, 1H), 1.53 (d, J = 34.9Hz, 4H).

[0039] Example 18 1-(4-((3-(4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-carbonyl)-5-fluorophenyl)amino)piperidin-1-yl)ethyl-1-one Compound 18: The synthesis process is the same as in Example 1, except that the raw material 6-bromo-2-pyridinecarboxylic acid in step (3) is replaced with 3-bromo-5-fluorobenzoic acid to obtain compound 18, a white solid with a yield of 19%. 1H NMR(400MHz,MeOD)δ7.12-7.03(m,3H),7.02-6.95(m,1H),6.46-6.39(m,2H ),6.32-6.26(m,1H),4.42-4.27(m,2H),3.87(dd,J=13.9,1.8Hz,1H),3.77( s,2H),3.54(dd,J=16.6,12.2Hz,2H),3.42(t,J=10.2Hz,1H),3.25(t,J=10. 9Hz,2H),2.94-2.84(m,5H),2.54(s,2H),2.10(s,3H),2.08-1.96(m,2H),86 1.76-1.56(m,4H),1.44-1.27(m,2H).13C NMR(100MHz,MeOD)δ171.74,171.48,166.56,151.29(d,J=11.4Hz),139.62(d,J =9.7Hz),136.38,135.41,129.58,127.47,127.18,126.64,107.65,101.77,101. 53,101.23,100.98,71.41,69.03,59.39,54.60,50.35,46.29,45.03,41.57,39. 36,36.60,35.84,33.19,32.54,30.08,21.22.HRMS(ESI+)m / z[M+H]+calculated for C29H37FN4O3:509.29279; found 509.29172.

[0040] Example 19 (5-Bromofuran-2-yl)(4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-yl)methyl ketone [000643] Compound 19: The synthesis process is as in Example 1, except that the raw material 6-bromo-2-pyridinecarboxylic acid in step (3) is replaced with 5-bromofuran-2-carboxylic acid to obtain compound 19, a white solid, with a yield of 59%. 1H NMR (400MHz, CDCl3) δ7.18-7.08(m,3H),7.02-6.97(m,1H),6.93(d,J=3.5Hz,1H),6.40(d,J=3.5Hz,1H),4.32(s,2H),3.82(s,2H),3.53(s, 1H),3.26(s,2H),2.95-2.86(m,4H),2.52(s,2H),1.69(d,J=12.3Hz,2H),1.58(td,J=12.9,4.7Hz,2H).LC-MS(ESI+)m / z[M+H]+calculated forC20H23BrN2O3:419.10,found419.11.

[0041] Example 20 1-(4-((3-(4-((3,4-dihydroisoquinoline-2(1H)-yl)methyl)-4-hydroxypiperidin-1-carbonyl)-2-fluorophenyl)amino)piperidin-1-yl)ethyl-1-one Compound 20: The synthesis process was the same as in Example 1, except that the starting material 6-bromo-2-pyridinecarboxylic acid in step (3) was replaced with 3-bromo-2-fluorobenzoic acid, yielding compound 20, a white solid, with a yield of 8%. ¹H NMR (400MHz, MeOD) δ 7.11-7.01 (m, 4H), 7.01-6.95 (m, 1H), 6.89 (t, J = 7.8Hz, 1H), 6.52 (s, 1H), 4.42 (dd, J = 23.7, 13.3Hz, 2H), 3.93 (d, J = 12.3Hz, 1H), 3.77 (s, 2H), 3.65- 3.59(m,1H),3.41(s,2H),3.30-3.21(m,2H),2.88(s,4H),2.84(d,J=12.5Hz,1H),2. 54(s,2H),2.11(s,3H),2.10-1.98(m,2H),1.78-1.55(m,4H),1.52-1.33(m,2H).13C NMR(100MHz,MeOD)δ171.50,167.76,137.31,137.19,136.37,135.42,129.56,127.46,127.16,126.62,126.24,126.21,115.29,114.85 ,71.34,68.97,59.35,54.58,50.69,46.53,41.79,39.06,36.56,35.89,33.36,32.71,30.06,21.23.HRMS(ESI+)m / z[M+H]+calculated for C29H37FN4O3:509.29279; found509.29214. Application Example 1: Evaluation of Biological Activity

[0042] Experimental Methods: In this study, the fluorescent group rhodamine (Ub1-72-Leu73-Arg74-Gly75-Gly76-Rho110, ubiquitin-rhodamine 110, Ub-Rho110) was modified with glycine at position 76 of ubiquitin molecules. USP28 / USP25 can cleave the C-terminus of glycine at position 76 of ubiquitin molecules (both purchased from Nanjing Youai Co., Ltd.). The enzyme activity of USP28 can be quantitatively analyzed by measuring the emitted fluorescence intensity using a multi-functional microplate reader with a dichroic mirror (excitation wavelength 485nm, emission wavelength 535nm), and the inhibitory activity of the small molecule compound can be quantitatively calculated.

[0043] Specific steps: First, perform a single-point 25μM initial screening for inhibition rate of the compound: Incubate the prepared compound with protein USP28 / USP25 (final concentration 3.9nM) for 10 minutes. The system composition consists of 25mM Tris and 0.1M NaCl solution at pH 7.5, with a final concentration containing 1mM dithiothreitol (DTT). Finally, add the substrate ubiquitin-rhodamine 110 (final concentration 100nM), wait for 5 minutes, and then rapidly measure the fluorescence signal value. If the inhibition rate is greater than 60%, prepare compound concentration gradients of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.12 μM, 1.56 μM, 0.78 μM, and 0.39 μM. Use a 384 black well plate (384 ProxiPlate, purchased from PE). The reaction system is as follows: first add 9 μL of the prepared protein solution, then add 4 μL of the prepared DTT solution, add 5 μL of the prepared compound to each well, and finally add 1 μL of the prepared substrate, for a final volume of 20 μL. The final compound concentration was reduced to one-quarter of its original concentration. Therefore, the final compound concentrations for each well were 25 μM, 12.5 μM, 6.25 μM, 3.12 μM, 1.56 μM, 0.78 μM, 0.39 μM, 0.195 μM, and 0.097 μM, respectively. Finally, the inhibition rate (IC50) of the small molecule was calculated using GraphPad Prism 6.0 software. 50 This detection method is simple, rapid, and highly sensitive. Furthermore, the positive control compound AZ1 was used, and its inhibition rate (IC50 of AZ1) was determined using the same method. 50 It is 0.858 μM.

[0044] Data analysis: Calculating the inhibition rate using formulas Inhibition rate (%) = (fluorescence intensity of 100% group - fluorescence intensity of compound group) / (fluorescence intensity of 100% group - fluorescence intensity of positive control group) * 100%. The inhibition rate IC50 of small molecules was calculated using GraphPad Prism 6.0 software. 50 . The above experiments tested the 25 μM inhibition rate and IC50 of some compounds of this invention against USP28. 50 The values ​​are shown in Tables 1 and 2. Table 1. Inhibitory activity of the compounds of the present invention against USP28 and USP25 proteins. Table 2. USP28 protein inhibitory activity of representative compounds of the present invention. Note: AZ1 is a positive control.

[0045] The novel scaffold compound 1 obtained in this invention selectively inhibits USP28 without inhibiting USP25 (the single-point inhibition rate of 25 μM is 3.03 times more selective), which is 2.16 times better than the positive control AZ1. This invention expands the new scaffold type of selective inhibitors targeting USP28.

[0046] In summary, the compounds provided by this invention exhibit good enzyme-selective inhibitory activity against USP28, possessing significant development potential. They offer a new scaffold type for the development of novel USP28 inhibitor drugs and provide more options for clinical treatment of anti-tumor drugs.

Claims

1. A small molecule inhibitor of the deubiquitinase USP28, characterized in that, It has the structure shown in general formula (I), In the formula: ring A is selected from one of the following substituents: R1 is selected from one of the following substituents: hydrogen, R2 is selected from one of the following substituents:

2. The small molecule inhibitor of deubiquitinase USP28 as described in claim 1, characterized in that, Choose one of the following compounds.

3. The pharmaceutical application of the small molecule inhibitor of deubiquitinase USP28 as described in claim 1 or 2, characterized in that, Using it as the active ingredient, it can be prepared into drugs for the prevention or treatment of USP28-mediated diseases.

4. The pharmaceutical application of the selective small molecule inhibitor of deubiquitinase USP28 as described in claim 3, characterized in that, It is used in the preparation of drugs for the prevention or treatment of cancer, Fanconi anemia, muscle atrophy, or tumor angiogenesis-related diseases.

5. The pharmaceutical application of the selective small molecule inhibitor of deubiquitinase USP28 as described in claim 4, characterized in that, The cancers mentioned include bladder cancer, pancreatic cancer, breast cancer, colon cancer, lung cancer, glioma, melanoma, head and neck cancer, stomach cancer, liver cancer, ovarian cancer, endometrial cancer, prostate cancer, thyroid cancer, or kidney cancer.

6. A method for preparing a small molecule inhibitor of the deubiquitinating enzyme USP28 as described in claim 1 or 2, characterized in that, This can be achieved using the following method: Wherein, the A ring, R1 and R2 groups are as described in claim 1; a) Compound I-2 is obtained by reacting epoxy compound I-1 with R2 amine compound under solvent and alkaline conditions; the solvent used is tetrahydrofuran, N,N-dimethylformamide, methanol or ethanol; the base used is K2CO3, Na2CO3, CS2CO3 or NaOH; b) The 4-hydroxypiperidine compound I-2 obtained in step a) is deprotected by the Boc protecting group under solvent and acidic conditions to generate 4-hydroxypiperidine compound I-3; the solvent used is dichloromethane, ethyl acetate, methanol, dioxane, tetrahydrofuran, N,N-dimethylformamide or toluene; the acid used is trifluoroacetic acid or hydrochloric acid; c) In a solvent, under alkaline conditions of the condensing agent, the amine compound I-3 obtained in step b) undergoes an acylation reaction with a carboxylic acid A-ring compound to generate amide compound I-4; the solvent used is tetrahydrofuran, N,-dimethylformamide, chloroform, dichloromethane, or toluene; the condensing agent used is T3P, EDC, EDCI, CDI, or HATU; the base used is sodium bicarbonate, potassium carbonate, triethylamine, or pyridine; d) Under solvent and alkaline conditions, compound I-4 obtained from step c) reacts with amino compounds containing different R1 groups in the presence of a palladium catalyst to obtain compound I; the solvent used is dimethyl sulfoxide, tetrahydrofuran, N,-dimethylformamide, chloroform, dichloromethane or toluene; the palladium catalyst used is tetratetraphenylphosphine palladium, palladium acetate, diphenylphosphine ferrocene palladium dichloride, dichloroditriphenylphosphine palladium, chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2-aminoethyl)phenyl)]palladium(II) or palladium on carbon; the base used is Na2CO3, CS2CO3, potassium tert-butoxide or sodium tert-butoxide.

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