2-acylamino-N-arylbenzamide derivative, preparation thereof and application of 2-acylamino-N-arylbenzamide derivative in antitumor drugs

Abstract

The invention relates to a 2-acylamino-N-aryl benzamide derivative, a preparation method thereof and an application of the 2-acylamino-N-aryl benzamide derivative in antitumor drugs. According to the invention, 2-acylamino-N-aryl benzamide is used as a parent nucleus to prepare the compound, and the compound has remarkable USP2 inhibitory activity and can be used for treating diseases related to abnormal USP2 activity. The compound has the characteristics of novel structure, simplicity and convenience in synthesis, remarkable activity and the like, can effectively inhibit the enzymatic activity of USP2, and shows a good anti-tumor effect in various cancer models. The compound provided by the invention provides a new solution for the treatment of USP2 related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicinal chemistry, and particularly to a 2-amido-N-arylbenzamide derivative, its preparation, and its application in anti-tumor drugs. Background Art

[0002] Ubiquitin-specific protease 2 (USP2) is a deubiquitinating enzyme belonging to the ubiquitin-specific protease family. It can regulate various cellular processes, including the cell cycle, DNA repair, and signal transduction, by removing ubiquitin chains from substrate proteins. Studies have shown that USP2 is involved in multiple cancers (such as breast cancer, ovarian cancer, colorectal cancer, prostate cancer, bladder cancer, glioblastoma, liver cancer, lymphoma, gastric cancer, lung cancer, renal parenchymal cancer, hematological malignancies [1] ), and promotes tumorigenesis and development by stabilizing oncoproteins (such as MDM2, fatty acid synthase). In addition, USP2 is also closely related to neurodegenerative diseases and inflammatory responses. Therefore, the development of highly efficient and selective USP2 inhibitors is of great significance for the treatment of related diseases.

[0003] Currently, some USP2 inhibitors have been reported, including the small molecule compound Beta-Lapachone [2] , ML364 [3] , LCAHA [4] , ZCL-910 [5] , 6-TG [6] , STD1D [7] , COH29 [8] and so on. For example, ML364 is a known small molecule inhibitor of USP2, but it has poor selectivity and also has strong inhibitory activity against other deubiquitinating enzymes such as USP7, which may lead to potential side effects. In addition, the chemical structure diversity of existing inhibitors is limited, and the synthetic routes of some compounds are complex and the yields are low, which limits their further development and application.

[0004] Therefore, the development of USP2 inhibitors with novel structures, high selectivity, and good drug-likeness remains a difficult point in current research. Summary of the Invention

[0005] The object of the present invention is to provide a 2 - amido - N - arylbenzamide derivative, its preparation method and its application in anti - tumor drugs. The compounds of the present invention are prepared using 2 - amido - N - arylbenzamide as the parent nucleus, which have significant USP2 inhibitory activity and can be used to treat diseases related to abnormal USP2 activity. These compounds have the characteristics of novel structure, simple synthesis and significant activity, can effectively inhibit the enzyme activity of USP2, and show good anti - tumor effects in various cancer models. The compounds of the present invention provide a new solution for the treatment of USP2 - related diseases.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] A 2 - amido - N - arylbenzamide derivative, the molecular formula of the derivative is:

[0008]

[0009] Wherein, R1 is selected from any one of a hydrogen atom, a halogen, a hydroxyl group, a methoxy group, a methylthio group, an alkyl group, a nitro group or a trifluoromethyl group;

[0010] R2 is selected from any one of an alkane, an alicyclic ring, a substituted alicyclic ring, a phenyl group, a substituted phenyl group, a fused - ring group, a heterocyclic group or a substituted heterocyclic group;

[0011] R3 is selected from any one of a hydrogen atom, a halogen, a hydroxyl group, a methoxy group, a methylthio group, an alkyl group, a nitro group or a trifluoromethyl group;

[0012] R4 is selected from any one of an alkane, an alicyclic ring, a substituted alicyclic ring, a phenyl group, a substituted phenyl group, a fused - ring group, a heterocyclic group or a substituted heterocyclic group;

[0013] Ring A and ring B are a substituted or unsubstituted 5 - 6 - membered aromatic ring system, a substituted or unsubstituted 5 - 6 - membered heterocyclic ring system, a substituted or unsubstituted 5 - 6 - membered aromatic ring, a substituted or unsubstituted 5 - 6 - membered aromatic heterocycle, or a substituted or unsubstituted 5 - 6 - membered heterocycle (including fully or partially unsaturated heterocycles).

[0014] Furthermore, the fused - ring group in R2 and R4 includes naphthyl, the heterocyclic ring in the heterocyclic group and heterocyclic substituents in R2 and R4 is selected from any one of furan, thiophene or pyrrole, and the substituents in the substituted phenyl group and substituted heterocyclic group in R2 and R4 are selected from any one of a halogen, a hydroxyl group, a methoxy group, a methylthio group, an alkyl group, a nitro group or a trifluoromethyl group.

[0015] Still further, the above - mentioned 2 - amido - N - arylbenzamide derivative is selected from the following compounds:

[0016] 2-Benzamido-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-1915), 2-(cyclopropanecarboxamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-2000), 2-(cyclopentanecarboxamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-2002), N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-2-(3-methylbenzamido)-4-(trifluoromethyl)benzamide (ZCL-2004), 2-(3-chlorobenzamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-2013), N-(2-((5-(cyclopropylcarbamoyl)-2-fluorophenyl)carbamoyl)-5-(trifluoromethyl)phenyl)-3,4,5-trimethoxybenzamide (ZCL-2021), N-(2-((5-(cyclopropanecarboxamido)-2-fluorophenyl)carbamoyl)-5-(trifluoromethyl)phenyl)thiophene-3-carboxamide (ZCL-2033).

[0017] Their structures are shown as follows:

[0018]

[0019] Furthermore, the 2-amido-N-arylbenzamide derivatives include

[0020] isotope compounds, racemates, optically active isomers, polymorphs, salts or mixtures thereof.

[0021] Still further, the salts include salts formed with sodium, potassium, lithium or calcium elements, or salts formed with organic amines, pyridine or alkaloids, or salts formed with hydrochloric acid, hydrobromic acid, hydrofluoric acid, nitric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, sulfonic acid or tartaric acid.

[0022] In addition, the present invention also provides a preparation method of 2-amido-N-arylbenzamide derivatives, and the specific steps are as follows:

[0023] S1. After mixing the compound with the formula 1 structure and TEA with DCM, add the compound with the formula 2 structure, and after the reaction is completed, purify to obtain the compound with the formula 3 structure;

[0024] S2. Dissolve the compound with the formula 3 structure obtained in step S1 in a mixed solution of ethanol and water, add iron powder and NH4Cl, heat, and after the reaction is completed, purify to obtain the compound with the formula 4 structure;

[0025] S3. Mix the compound with the structural formula of Formula 4 and TEA obtained in Step S2 with DCM, add the compound with the structural formula of Formula 5, and after the reaction is completed, purify to obtain the compound with the structural formula of Formula 6;

[0026] S4. Dissolve the compound with the structural formula of Formula 6 obtained in Step S3 in a mixed solution of ethanol and water, add iron powder and NH4Cl, heat, and after the reaction is completed, purify to obtain the compound with the structural formula of Formula 7;

[0027] S5. Mix the compound with the structural formula of Formula 7 obtained in Step S4 and TEA with THF, add the compound with the structural formula of Formula 8, and after the reaction is completed, purify to obtain 2-amido-N-arylbenzamide derivatives;

[0028] Among them, the compounds with the structural formula of Formula 1 to the compounds with the structural formula of Formula 8 are as follows:

[0029]

[0030] Among them, R1 is selected from any one of a hydrogen atom, a halogen, a hydroxyl group, a methoxy group, a methylthio group, an alkyl group, a nitro group, or a trifluoromethyl group;

[0031] R2 is selected from any one of an alkane, an alicyclic ring, a substituted alicyclic ring, a phenyl group, a substituted phenyl group, a fused ring group, a heterocyclic group, or a substituted heterocyclic group;

[0032] R3 is selected from any one of a hydrogen atom, a halogen, a hydroxyl group, a methoxy group, a methylthio group, an alkyl group, a nitro group, or a trifluoromethyl group;

[0033] R4 is selected from any one of an alkane, an alicyclic ring, a substituted alicyclic ring, a phenyl group, a substituted phenyl group, a fused ring group, a heterocyclic group, or a substituted heterocyclic group;

[0034] Ring A and ring B are a substituted or unsubstituted 5-6 membered aromatic ring system, a substituted or unsubstituted 5-6 membered heterocyclic ring system, a substituted or unsubstituted 5-6 membered aromatic ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, or a substituted or unsubstituted 5-6 membered heterocycle (including fully or partially unsaturated heterocycles).

[0035] Furthermore, in Step S1, the molar ratio of the compound with the structural formula of Formula 1, TEA, and the compound with the structural formula of Formula 2 is 1:(1.5 - 3):(1.5 - 2), and DCM is used as the solvent.

[0036] Furthermore, in Step S2, the molar ratio of the compound with the structural formula of Formula 3, iron powder, and NH4Cl is 1:(5 - 10):(5 - 10), ethanol and water are used as the solvent, and their volume ratio is 4:1.

[0037] Further, in step S3, the molar ratio of the compound with the structural formula of Formula 4, TEA, and the compound with the structural formula of Formula 5 is 1:(1.5 - 3):(1.5 - 2), and DCM is used as the solvent.

[0038] Further, in step S4, the molar ratio of the compound with the structural formula of Formula 6, iron powder, and NH4Cl is 1:(5 - 10):(5 - 10), ethanol and water are used as the solvents, and their volume ratio is 4:1.

[0039] Further, in step S5, the molar ratio of the compound with the structural formula of Formula 7, TEA, and the compound with the structural formula of Formula 8 is 1:(1.5 - 3):(1.5 - 2), and THF is used as the solvent.

[0040] In addition, the present invention also provides an application of a 2 - amido - N - arylbenzamide derivative in the preparation of an anti - tumor drug related to USP2.

[0041] Further, the dosage form of the drug is a capsule, granule, pill, powder, injection, syrup, paste, emulsion, solution, suspension or tincture.

[0042] Further, the drug also includes excipients, and the excipients are selected from any one or more of a vehicle, filler, solubilizer, binder, humectant, disintegrant, slow - releasing agent, absorption accelerator, adsorbent, diluent, solubilizer, emulsifier, lubricant, wetting agent, suspending agent, flavoring agent or perfume.

[0043] Further, the tumors include breast cancer, ovarian cancer, colorectal cancer, prostate cancer, bladder cancer, glioblastoma, liver cancer, lymphoma, gastric cancer, lung cancer, renal parenchymal cancer and hematological malignancies, etc.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0045] (1) The 2 - amido - N - arylbenzamide derivative described in the present invention has strong USP2 protein inhibitory activity;

[0046] (2) It can be used to prepare USP2 inhibitor drugs, especially for the treatment of cancers (such as colon cancer, prostate cancer, glioblastoma);

[0047] (3) The preparation method is simple, does not involve very cumbersome steps, and is easy to separate and purify. Description of the Drawings

[0048] Figure 1Synthetic route diagram of compound ZCL-1915 prepared in Example 1, where (a) is cyclopropanecarbonyl chloride, TEA, DCM, rt; (b) is Fe, NH4Cl, H2O, C2H5OH, 80 °C; (c) is 2-nitrobenzoyl chloride, TEA, DCM, rt; (d) is Fe, NH4Cl, H2O, C2H5OH, 80 °C; (e) is 4-methylbenzoyl chloride, THF, rt.

[0049] Figure 2 Synthetic route diagram of 2-amido-N-arylbenzamide derivatives, where (a) is Acylchloride, TEA, DCM, rt; (b) is Fe, NH4Cl, H2O, C2H5OH, 80 °C; (c) is Acyl chloride, TEA, DCM, rt; (d) is Fe, NH4Cl, H2O, C2H5OH, 80 °C; (e) is Acyl chloride, TEA, THF, rt. Detailed implementation manners

[0050] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0051] Some embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0052] Example 1

[0053] This example provides a 2-benzamide-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-1915), and its structural formula is shown as follows:

[0054]

[0055] In addition, referring to Figure 1 , this example also provides a preparation method of 2-benzamide-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-1915), and the specific steps are as follows:

[0056] S1. Prepare N-(4-fluoro-3-nitrophenyl)cyclopropanecarboxamide with the following structural formula

[0057]

[0058] 4-Fluoro-3-nitroaniline (5.0 g, 32.03 mmol) and triethylamine (5.34 mL, 38.43 mmol) were dissolved in 50 mL of DCM and stirred for 20 min in an ice bath. Subsequently, cyclopropylcarbonyl chloride (3.30 mL, 48.04 mmol) was added dropwise. After the addition was complete, the ice bath was removed, and the reaction was stirred overnight at room temperature under inert gas protection. After the reaction was completed, the reaction mixture was adjusted to pH 8 with saturated NaCO3, and then extracted three times with an appropriate amount of ethyl acetate (50 mL). The organic phase was collected, dried over anhydrous NaSO4, filtered, and the ethyl acetate was evaporated in vacuo to obtain 7.147 g of an orange-yellow solid compound with a yield of 99.54%.

[0059] 1 H NMR (400 MHz, DMSO-d6) δ 10.68 (s, 1H), 8.53 (dd, J = 7.1, 2.7 Hz, 1H), 7.90–7.77 (m, 1H), 7.61–7.46 (m, 1H), 1.75 (p, J = 6.5 Hz, 1H), 0.88–0.77 (m, 4H).

[0060] S2. Preparation of N-(3-amino-4-fluorophenyl)cyclopropanecarboxamide with the following structural formula

[0061]

[0062] Compound N-(4-fluoro-3-nitrophenyl)cyclopropanecarboxamide (5 g, 22.3 mmol) was dissolved in a mixed solution of 160 mL of ethanol and 40 mL of water. Iron powder (12.46 g, 223 mmol) and NH4Cl (11.93 g, 223 mmol) were added, and the mixture was heated to 80 °C and reacted overnight. After the reaction was completed, the residue in the reaction solution was removed by filtration. After evaporation, it was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was removed in vacuo. The product was separated by column chromatography to obtain 3.79 g of a white solid compound with a yield of 86.8%.

[0063] 1 H NMR (400 MHz, DMSO-d6) δ 9.90 (s, 1H), 7.09 (dd, J = 8.5, 2.6 Hz, 1H), 6.85 (dd, J = 11.3, 8.7 Hz, 1H), 6.72–6.62 (m, 1H), 5.09 (s, 2H), 1.72 (p, 1H), 0.87–0.66 (m, 4H).

[0064] S3. Preparation of N-(5-(Cyclopropanecarboxamido)-2-fluorophenyl)-2-nitro-4-(trifluoromethyl)benzamide with the following structural formula

[0065]

[0066] Dissolve compound N-(3-amino-4-fluorophenyl)cyclopropanecarboxamide (2 g, 10.3 mmol) and TEA (2.863 mL, 20.6 mmol) in 50 mL of DCM. Slowly add 2-nitro-4-trifluoromethylbenzoyl chloride (3.917 g, 15.5 mmol) dropwise under an ice bath. Raise the temperature to room temperature and react overnight. After the reaction is completed, wash successively with saturated sodium bicarbonate solution and saturated brine. Combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate under vacuum, and purify by column chromatography (PE / EA = 3 / 1) to obtain 2.502 g of a white solid compound with a yield of 59.1%.

[0067] 1 H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 10.35 (s, 1H), 8.52 (s, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.21 (dd, J = 7.1, 2.7 Hz, 1H), 8.01 (d, J = 7.9 Hz, 1H), 7.57–7.50 (m, 1H), 7.28–7.18 (m, 1H), 1.77 (p, 1H), 0.84–0.74 (m, 4H).

[0068] S4. Preparation of 2-Amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide with the following structural formula

[0069]

[0070] Dissolve compound N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-2-nitro-4-(trifluoromethyl)benzamide (2.3 g, 5.59 mmol) in a mixed solution of 40 mL of ethanol and 10 mL of water. Add iron powder (1.562 g, 28 mmol) and NH4Cl (1.496 g, 28 mmol), and heat to 80 °C and react overnight. After the reaction is completed, filter to remove the residue in the reaction solution. Concentrate and then extract with ethyl acetate (10 mL × 3). Combine the organic phases, dry with anhydrous sodium sulfate, filter, remove the solvent under vacuum, and separate by column chromatography (PE:EA = 3:1) to obtain 1.63 g of a white solid compound with a yield of 76.5%.

[0071] 11H NMR (400 MHz, DMSO-d6) δ 10.29 (s, 1H), 10.06 (s, 1H), 7.90–7.81 (m, 2H), 7.48–7.40 (m, 1H), 7.20 (t, 1H), 7.11 (s, 1H), 6.86 (dd, J = 8.3, 1.4 Hz, 2H), 6.75 (s, 2H), 1.75 (p, J = 6.5 Hz, 1H), 0.83–0.73 (m, 4H).

[0072] S5. Preparation of 2-benzamido-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide with the following structural formula

[0073]

[0074] Dissolve the compound 2-amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (0.20 g, 0.52 mmol) and TEA (219 μL, 1.57 mmol) in 15 mL of THF. Slowly add benzoyl chloride (120 μL, 1.04 mmol) under an ice bath. Raise the temperature to room temperature and react overnight. After the reaction is completed, wash with saturated sodium bicarbonate solution and saturated brine in sequence. Combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate under vacuum, and purify by column chromatography (PE / EA = 2 / 1) to obtain 86 mg of a white solid compound with a yield of 56.5%.

[0075] 1 1H NMR (400 MHz, DMSO-d6): δ 11.87 (s, 1H), 10.67 (s, 1H), 10.34 (s, 1H), 8.89 (s, 1H), 8.18 (d, J = 8.2 Hz, 1H), 7.91 (d, J = 7.3 Hz, 3H), 7.70 - 7.61 (m, 2H), 7.58 (t, J = 7.1 Hz, 2H), 7.49 (dt, J = 9.1, 3.5 Hz, 1H), 7.25 (t, J = 9.5 Hz, 1H), 1.76 (p, J = 6.2 Hz, 1H), 0.82 - 0.76 (m, 4H) ppm.

[0076] 1313C NMR (176 MHz, DMSO-d6): δ 172.13, 166.96, 165.57, 152.59, 151.21, 139.98, 136.14, 134.38, 132.88, 132.51, 130.91, 129.48, 127.62, 125.50, 125.06, 124.98, 123.33, 120.19, 118.33, 117.99, 117.91, 116.41, 116.30, 14.96, 7.70 ppm; HRMS (ESI): [M+Na] + C 25 H 19 F4N3NaO3 calcd 508.1260, found 508.1263; HPLC: purity 98.5%, retention time 15.2 min.

[0077] Example 2

[0078] This example provides a 2-(cyclopropanecarboxamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-2000), and its structural formula is as follows:

[0079]

[0080] In addition, this example also provides a preparation method of 2-(cyclopropanecarboxamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-2000). The compound ZCL-2000 was prepared with reference to the preparation of the ZCL-1915 compound in Example 1. Among them, the benzoyl chloride in step S5 of the preparation route of ZCL-1915 was replaced with cyclopropylcarbonyl chloride. 2-Amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (0.10 g, 0.26 mmol) and TEA (73 μL, 0.52 mmol) were dissolved in 15 mL of THF, and cyclopropylcarbonyl chloride (48 μL, 0.52 mmol) was slowly added thereto under an ice bath. The mixture was warmed to room temperature and reacted overnight. After the reaction was completed, it was washed successively with saturated sodium bicarbonate solution and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography (PE / EA = 2 / 1) to obtain 18.9 mg of a white solid compound, with a yield of 32.1%.

[0081] 11H NMR (400 MHz, DMSO-d6): δ 10.89 (s, 1H), 10.45 (s, 1H), 10.32 (s, 1H), 8.54 (s, 1H), 7.99 (t, J = 7.2 Hz, 2H), 7.58 (d, J = 8.6 Hz, 1H), 7.53–7.42 (m, 1H), 7.23 (t, J = 9.6 Hz, 1H), 1.77 (p, J = 6.1 Hz, 2H), 0.86–0.81 (m, 4H), 0.81–0.77 (m, 4H) ppm.

[0082] 13 13C NMR (101 MHz, DMSO-d6): δ 172.84, 172.17, 166.37, 152.72, 150.30, 138.98, 136.01, 132.23, 130.66, 126.97, 125.42, 122.75, 119.93, 118.44, 117.89, 117.46, 116.21, 15.75, 14.91, 8.31, 7.67 ppm; HRMS (ESI): [M+Na] + C 22 H 19 F4N3O3Na calcd 472.1255, found 472.1258; HPLC: purity 95.4%, retention time 10.8 min.

[0083] Example 3

[0084] This example provides a 2-(cyclopentanecarboxamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-2002), and its structural formula is shown as follows:

[0085]

[0086] In addition, this embodiment also provides a preparation method of 2-(cyclopentanecarboxamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-2002). The compound ZCL-2002 is prepared by a method similar to that of ZCL-1915 to obtain 2-amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide. Dissolve 2-amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (0.10 g, 0.26 mmol) and TEA (73 μL, 0.52 mmol) in 15 mL of THF, and slowly add cyclopropanecarbonyl chloride (63 μL, 0.52 mmol) under an ice bath. Raise the temperature to room temperature and react overnight. After the reaction is completed, wash with saturated sodium bicarbonate solution and saturated brine in sequence, combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate under vacuum, and purify by column chromatography (PE / EA = 2 / 1) to obtain 31.7 mg of a white solid compound, with a yield of 50.6%.

[0087] 1 H NMR (400 MHz, DMSO-d6): δ 10.76 (s, 1H), 10.48 (s, 1H), 10.32 (s, 1H), 8.65 (s, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.95 (dd, J = 7.0, 2.6 Hz, 1H), 7.58 (dd, J = 8.2, 1.9 Hz, 1H), 7.54–7.43 (m, 1H), 7.24 (t, J = 9.6 Hz, 1H), 2.80 (p, J = 8.0 Hz, 1H), 1.90–1.48 (m, 9H), 0.84–0.75 (m, 4H) ppm.

[0088] 13 C NMR (101 MHz, DMSO-d6): δ 175.25, 172.17, 166.51, 152.85, 150.43, 139.44, 136.05, 136.02, 132.38, 132.06, 130.66, 126.26, 125.30, 122.76, 119.80, 118.04, 117.58, 116.30, 46.50, 30.12, 25.88, 14.92, 7.68 ppm; HRMS (ESI): [M+Na] + C 24 H 23 F4N3O3Na calcd 500.1568, found 500.1565; HPLC: purity 96.9%, retention time 11.5 min.

[0089] Example 4

[0090] This example provides N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-2-(3-methylbenzamido)-4-(trifluoromethyl)benzamide (ZCL-2004), and its structural formula is shown as follows:

[0091]

[0092] In addition, this example also provides a preparation method of N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-2-(3-methylbenzamido)-4-(trifluoromethyl)benzamide (ZCL-2004). Compound ZCL-2004 was prepared by a method similar to that of ZCL-1915 to obtain 2-amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-methoxybenzamide. 2-Amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (0.10 g, 0.26 mmol) and TEA (73 μL, 0.52 mmol) were dissolved in 15 mL of THF, and 3-methylbenzoyl chloride (69 μL, 0.52 mmol) was slowly added thereto under an ice bath. The mixture was warmed to room temperature and reacted overnight. After the reaction was completed, it was washed successively with saturated sodium bicarbonate solution and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified by column chromatography (PE / EA = 2 / 1) to obtain 7.2 mg of a white solid compound, with a yield of 11.0%.

[0093] 1 H NMR (400 MHz, DMSO-d6): δ 11.74 (s, 1H), 10.64 (s, 1H), 10.33 (s, 1H), 8.84 (s, 1H), 8.16 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 5.8 Hz, 1H), 7.73 (s, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.52–7.39 (m, 3H), 7.24 (t, J = 9.6 Hz, 1H), 2.38 (s, 3H), 1.77 (p, J = 11.5, 5.3 Hz, 1H), 0.79 (d, J = 6.2 Hz, 4H) ppm.

[0094] 1313C NMR (101 MHz, DMSO-d6): δ 172.19, 166.90, 165.78, 161.19, 153.19, 150.64, 139.82, 138.86, 136.11, 134.42, 133.48, 130.86, 129.33, 128.34, 125.90, 125.46, 125.13, 120.23, 118.34, 118.16, 117.88, 116.42, 116.21, 21.43, 14.94, 7.69 ppm; HRMS (ESI): [M+Na] + C 26 H 21 F4N3O3Na calcd 522.1411, found 522.1410; HPLC: purity 98.6%, retention time 12.7 min.

[0095] Example 5

[0096] This example provides a 2-(3-chlorobenzamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-2013), and its structural formula is shown as follows:

[0097]

[0098] In addition, this example also provides a preparation method of 2-(3-chlorobenzamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (ZCL-2013). The compound ZCL-2013 was prepared by a method similar to that of ZCL-1915 to obtain 2-amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide. 2-Amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (0.10 g, 0.26 mmol) and TEA (73 μL, 0.52 mmol) were dissolved in 15 mL of THF, and 3-chlorobenzoyl chloride (67 μL, 0.52 mmol) was slowly added thereto under an ice bath. The reaction was allowed to proceed overnight at room temperature. After the reaction was completed, it was washed successively with saturated sodium bicarbonate solution and saturated brine. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified by column chromatography (PE / EA = 2 / 1) to obtain 11.3 mg of a white solid compound, with a yield of 16.6%.

[0099] 11H NMR (400 MHz, DMSO-d6): δ 11.68 (s, 1H), 10.59 (s, 1H), 10.31 (s, 1H), 8.69 (s, 1H), 8.14 (d, J = 8.2 Hz, 1H), 7.98–7.89 (m, 2H), 7.84 (d, J = 7.8 Hz, 1H), 7.70 (d, J = 8.1 Hz, 2H), 7.60 (t, J = 7.9 Hz, 1H), 7.50–7.44 (m, 1H), 7.23 (t, J = 9.6 Hz, 1H), 1.76 (p, J = 6.3 Hz, 1H), 0.84–0.75 (m, 4H) ppm.

[0100] 13 13C NMR (101 MHz, DMSO-d6): δ 172.14, 166.64, 164.35, 152.94, 150.52, 139.18, 136.50, 136.10, 134.15, 132.57, 131.38, 130.91, 127.77, 127.23, 126.23, 125.43, 125.21, 122.72, 120.79, 118.85, 118.19, 117.74, 116.16, 14.93, 7.68 ppm; HRMS (ESI): [M+Na] + C 25 H 18 ClF4N3O3Na calcd 542.0865, found 542.0862; HPLC: purity 95.1%, retention time 12.9 min.

[0101] Example 6

[0102] This example provides N-(2-((5-(cyclopropylcarbamoyl)-2-fluorophenyl)carbamoyl)-5-(trifluoromethyl)phenyl)-3,4,5-trimethoxybenzamide (ZCL-2021), and its structural formula is shown as follows:

[0103]

[0104] In addition, this embodiment also provides a preparation method of N-(2-((5-(cyclopropylcarbamoyl)-2-fluorophenyl)carbamoyl)-5-(trifluoromethyl)phenyl)-3,4,5-trimethoxybenzamide (ZCL-2021). Compound ZCL-2021 was prepared by a method similar to that of ZCL-1915 to obtain 2-amino-N-(5-(cyclopropanecarboxamide)-2-fluorophenyl)-4-(trifluoromethyl)benzamide. 2-Amino-N-(5-(cyclopropanecarboxamide)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (0.20 g, 0.52 mmol) and TEA (146 μL, 1.04 mmol) were dissolved in 15 mL of THF, and 3,4,5-trimethoxybenzoyl chloride (195 μL, 1.04 mmol) was slowly added thereto under an ice bath. The mixture was warmed to room temperature and reacted overnight. After the reaction was completed, it was washed successively with saturated sodium bicarbonate solution and saturated brine, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under vacuum, and purified by column chromatography (PE / EA = 2 / 1) to obtain 41.8 mg of a white solid compound with a yield of 55.4%.

[0105] 1 H NMR (400 MHz, DMSO-d6): δ11.56 (s, 1H), 10.57 (s, 1H), 10.31 (s, 1H), 8.69 (s, 1H), 8.12 (d, J = 8.2 Hz, 1H), 8.05 (dd, J = 7.1, 2.6 Hz, 1H), 7.67 (dd, J = 8.3, 1.9 Hz, 1H), 7.52–7.33 (m, 1H), 7.23 (s, 3H), 3.82 (s, 6H), 3.73 (s, 3H), 1.76 (p, J = 6.3 Hz, 1H), 0.79 (d, 4H) ppm.

[0106] 13 C NMR (101 MHz, DMSO-d6): δ172.20, 166.68, 165.29, 153.36, 152.92, 150.50, 141.30, 139.27, 135.99, 135.96, 130.71, 129.71, 127.31, 125.44, 125.15, 125.03, 122.73, 120.58, 118.61, 118.26, 117.82, 116.24, 116.04, 105.32, 60.65, 56.45, 14.89, 7.66 ppm; HRMS (ESI): [M+Na] + C 28 H 25F4N3O6Na, calculated 598.1577, found 598.1573; HPLC: purity 97.3%, retention time 12.1 min.

[0107] Example 7

[0108] This example provides N-(2-((5-(cyclopropanecarboxamido)-2-fluorophenyl)carbamoyl)-5-(trifluoromethyl)phenyl)thiophene-3-carboxamide (ZCL-2033), and its structural formula is as follows:

[0109]

[0110] In addition, this example also provides a preparation method of N-(2-((5-(cyclopropanecarboxamido)-2-fluorophenyl)carbamoyl)-5-(trifluoromethyl)phenyl)thiophene-3-carboxamide (ZCL-2033). Compound ZCL-2033 is prepared by a method similar to that of ZCL-1915 to obtain 2-amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide. Dissolve 2-amino-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide (0.20 g, 0.52 mmol) and TEA (146 μL, 1.04 mmol) in 15 mL of THF, and slowly add 3-thiophenecarbonyl chloride (108 μL, 1.04 mmol) under an ice bath. Raise the temperature to room temperature and react overnight. After the reaction is completed, wash with saturated sodium bicarbonate solution and saturated brine in sequence, combine the organic phases, dry with anhydrous sodium sulfate, filter, spin dry under vacuum, and separate and purify by column chromatography (PE / EA = 2 / 1) to obtain 43.4 mg of a white solid compound, with a yield of 33.7%.

[0111] 1 1H NMR (400 MHz, DMSO-d6): δ 11.67 (s, 1H), 10.64 (s, 1H), 10.33 (s, 1H), 8.81 (d, J = 0.8 Hz, 1H), 8.24 (dd, J = 2.9, 1.3 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 7.92 (dd, J = 6.9, 2.5 Hz, 1H), 7.72–7.67 (m, 1H), 7.67–7.62 (m, 1H), 7.52–7.45 (m, 2H), 7.29–7.20 (m, 1H), 1.77 (p, J = 5.7 Hz, 1H), 0.85–0.70 (m, 4H) ppm.

[0112] 1313C NMR(101MHz,DMSO-d6):δ172.19,166.94,161.20,153.15,150.73,139.83,137.54,136.08,132.73,131.04,130.85,128.65,126.59,125.43,125.07,124.94,120.06,118.24,118.35,116.43,116.23,14.95,7.70ppm;HRMS(ESI):[M+Na] + C 23 H 17 F4N3O3SNa calcd 514.0819,found 514.0817;HPLC:purity 97.8%,retention time 11.8min.

[0113] Comparative Example 1

[0114] This comparative example provides a standard inhibitor of ubiquitin - specific peptidase ML364, purchased from Shanghai TargetMol Biotechnology Co., Ltd.

[0115] Example 9

[0116] This example provides a method for testing USP2 inhibitory activity and anti - tumor activity, and the specific steps are as follows:

[0117] Based on the previously reported method [5] , USP2 and UbA52 proteins were respectively expressed and purified, and a USP2 - UbA52 hydrolysis test system based on SDS - PAGE was established for the in vitro USP2 inhibitory activity test of the compounds prepared in Comparative Example 1 and Examples 1 - 7. The results are shown in Table 1.

[0118] Table 1 In vitro USP2 inhibitory activity results of the compounds prepared in Examples 1 - 7

[0119]

[0120] It can be seen from Table 1 that the compounds prepared in Example 2 and Example 6 have good USP2 inhibitory activity.

[0121] Example 10

[0122] This example provides a method for testing cell proliferation inhibition, and the specific steps are as follows:

[0123] Using the MTT method, the compounds prepared in Comparative Example 1 and Example 1 were used to treat HCT116 colon cancer cells, and their inhibitory effects on cell proliferation were measured. The results are shown in Table 2:

[0124] Table 2 Inhibitory results of cell proliferation of the compounds prepared in Comparative Example 1 and Example 1

[0125]

[0126] As can be seen from Table 2, the compound prepared in Example 1 of the present invention has good activity of inhibiting cell proliferation.

[0127] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

[0128] The above references are as follows:

[0129] [1]S.Zhang,Y.Guo,S.Zhang,Z.Wang,Y.Zhang,S.Zuo.Targeting the deubiquitinase USP2 for malignant tumor therapy(Review),J.Oncology reports,50(2023).

[0130] [2]S.Ohayon,M.Refua,A.Hendler,A.Aharoni,A.Brik.Harnessing the Oxidation Susceptibility of Deubiquitinases for Inhibition with Small Molecules,J.Angewandte Chemie-International Edition,54(2015),599-603.

[0131] [3] M.I. Davis, R. Pragani, J.T. Fox, M. Shen, K. Parmar, E.F. Gaudiano, L. Liu, C. Tanega, L. McGee, M.D. Hall, C. McKnight, P. Shinn, H. Nelson, D. Chattopadhyay, A.D. D'Andrea, D.S. Auld, L.J. DeLucas, Z. Li, M.B. Boxer, A. Simeonov. Small Molecule Inhibition of the Ubiquitin-specific Protease USP2 Accelerates cyclin D1 Degradation and Leads to Cell Cycle Arrest in Colorectal Cancer and Mantle Cell Lymphoma Models*, J. Journal of Biological Chemistry, 291(2016), 24628-24640.

[0132] [4] K. Magiera, M. Tomala, K. Kubica, V. De Cesare, M. Trost, B.J. Zieba, N. Kachamakova-Trojanowska, M. Les, G. Dubin, T.A. Holak, L. Skalniak. Lithocholic Acid Hydroxyamide Destabilizes Cyclin D1 and Induces G0 / G1 Arrest by Inhibiting Deubiquitinase USP2a, J. Cell Chemical Biology, 24(2017), 458-470.e418.

[0133] [5] Z. Wang, W. Xie, M. Zhu, H. Zhou. Development of a highly reliable assay for ubiquitin-specific protease 2 inhibitors, J. Bioorganic & Medicinal Chemistry Letters, 27(2017), 4015-4018.

[0134] [6]S.J.Chuang,S.C.Cheng,H.C.Tang,C.Y.Sun,C.Y.Chou.6-Thioguanine isanoncompetitive and slow binding inhibitor of human deubiquitinating proteaseUSP2,J.Scientific Reports,8(2018).

[0135] [7]M.D.Tomala,K.Magiera-Mularz,K.Kubica,S.Krzanik,B.Zieba,B.Musielak,M.Pustula,G.M.Popowicz,M.Sattler,G.Dubin,L.Skalniak,T.A.Holak.Identificationof small-molecule inhibitors ofUSP2a,J.European Journal ofMedicinalChemistry,150(2018),261-267.

[0136] [8]M.Y.Zhu,H.Wang,Y.L.Ding,Y.Y.Yang,Z.Xu,L.Shi,N.X.Zhang.Ribonucleotide reductase holoenzyme inhibitor COH29 interacts withdeubiquitinaseubiquitin-specific protease 2 and downregulates its substrateprotein cyclin D1,J.FasebJournal,36(2022).

Claims

1. A 2 - amido - N - arylbenzamide derivative, characterized in that, The molecular formula of the derivative is: Wherein, R1 is selected from any one of a hydrogen atom, a halogen, a hydroxyl group, a methoxy group, a methylthio group, an alkyl group, a nitro group or a trifluoromethyl group; R2 is selected from any one of an alkane, an alicyclic ring, a substituted alicyclic ring, a phenyl group, a substituted phenyl group, a fused ring group, a heterocyclic group or a substituted heterocyclic group; R3 is selected from any one of a hydrogen atom, a halogen, a hydroxyl group, a methoxy group, a methylthio group, an alkyl group, a nitro group or a trifluoromethyl group; R4 is selected from any one of an alkane, an alicyclic ring, a substituted alicyclic ring, a phenyl group, a substituted phenyl group, a fused ring group, a heterocyclic group or a substituted heterocyclic group; Ring A and ring B are a substituted or unsubstituted 5- to 6-membered aromatic ring system, a substituted or unsubstituted 5- to 6-membered heterocyclic ring system, a substituted or unsubstituted 5- to 6-membered aromatic ring, a substituted or unsubstituted 5- to 6-membered aromatic heterocycle, or a substituted or unsubstituted 5- to 6-membered heterocycle.

2. The 2 - amido - N - arylbenzamide derivative according to claim 1, wherein The fused ring groups in R2 and R4 include naphthyl, the heterocycles in the heterocyclic groups and heterocyclic substituents in R2 and R4 are selected from any one of furan, thiophene or pyrrole, and the substituents in the substituted phenyl groups and substituted heterocyclic groups in R2 and R4 are selected from any one of a halogen, a hydroxyl group, a methoxy group, a methylthio group, an alkyl group, a nitro group or a trifluoromethyl group.

3. A 2 - amido - N - arylbenzamide derivative according to claim 2, characterized in that, The 2-amido-N-arylbenzamide derivatives are selected from the following compounds: 2-Benzamide-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide, 2-(cyclopropanecarboxamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide, 2-(cyclopentanecarboxamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide, N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-2-(3-methylbenzamido)-4-(trifluoromethyl)benzamide, 2-(3-chlorobenzamido)-N-(5-(cyclopropanecarboxamido)-2-fluorophenyl)-4-(trifluoromethyl)benzamide, N-(2-((5-(cyclopropylcarbamoyl)-2-fluorophenyl)carbamoyl)-5-(trifluoromethyl)phenyl)-3,4,5-trimethoxybenzamide, N-(2-((5-(cyclopropanecarboxamido)-2-fluorophenyl)carbamoyl)-5-(trifluoromethyl)phenyl)thiophene-3-carboxamide.

4. A 2 - amido - N - arylbenzamide derivative according to claim 1, characterized in that, The 2-amino-N-arylbenzamide derivatives include isotope compounds, racemates, optically active isomers, polymorphs, salts or mixtures thereof.

5. A 2 - amido - N - arylbenzamide derivative according to claim 4, characterized in that, The salts include salts formed with sodium, potassium, lithium or calcium elements, or salts formed with organic amines, pyridine or alkaloids, or salts formed with hydrochloric acid, hydrobromic acid, hydrofluoric acid, nitric acid, sulfuric acid, phosphoric acid, formic acid, acetic acid, sulfonic acid or tartaric acid.

6. A method for preparing a 2 - amido - N - arylbenzamide derivative according to any one of claims 1 - 5, characterized in that, The specific steps are as follows: S1. After mixing the compound with the structural formula of Formula 1 and TEA with DCM, add the compound with the structural formula of Formula 2, and after the reaction is completed, purify to obtain the compound with the structural formula of Formula 3; S2. Dissolve the compound with the structural formula of Formula 3 obtained in step S1 in a mixed solution of ethanol and water, add iron powder and NH4Cl, heat, and after the reaction is completed, purify to obtain the compound with the structural formula of Formula 4; S3. After mixing the compound with the structural formula of Formula 4 obtained in step S2 and TEA with DCM, add the compound with the structural formula of Formula 5, and after the reaction is completed, purify to obtain the compound with the structural formula of Formula 6; S4. Dissolve the compound with the structural formula of Formula 6 obtained in Step S3 in a mixed solution of ethanol and water, add iron powder and NH4Cl, heat, and after the reaction is completed, purify to obtain the compound with the structural formula of Formula 7; S5. Mix the compound with the structural formula of Formula 7 obtained in Step S4 with TEA and THF, add the compound with the structural formula of Formula 8, and after the reaction is completed, purify to obtain 2-amido-N-arylbenzamide derivatives; Among them, the compounds with the structural formula of Formula 1 to Formula 8 are as follows: Among them, R1 is selected from any one of a hydrogen atom, a halogen, a hydroxyl group, a methoxy group, a methylthio group, an alkyl group, a nitro group, or a trifluoromethyl group; R2 is selected from any one of an alkane, an alicyclic ring, a substituted alicyclic ring, a phenyl group, a substituted phenyl group, a fused ring group, a heterocyclic group, or a substituted heterocyclic group; R3 is selected from any one of a hydrogen atom, a halogen, a hydroxyl group, a methoxy group, a methylthio group, an alkyl group, a nitro group, or a trifluoromethyl group; R4 is selected from any one of an alkane, an alicyclic ring, a substituted alicyclic ring, a phenyl group, a substituted phenyl group, a fused ring group, a heterocyclic group, or a substituted heterocyclic group; Ring A and ring B are a substituted or unsubstituted 5-6 membered aromatic ring system, a substituted or unsubstituted 5-6 membered heterocyclic ring system, a substituted or unsubstituted 5-6 membered aromatic ring, a substituted or unsubstituted 5-6 membered aromatic heterocycle, or a substituted or unsubstituted 5-6 membered heterocycle.

7. The preparation method of a 2-amido-N-arylbenzamide derivative according to claim 6, characterized in that, In Step S1, the molar ratio of the compound with the structural formula of Formula 1, TEA, and the compound with the structural formula of Formula 2 is 1:(1.5 - 3):(1.5 - 2), and DCM is used as the solvent; In Step S2, the molar ratio of the compound with the structural formula of Formula 3, iron powder, and NH4Cl is 1:(5 - 10):(5 - 10), and ethanol and water are used as the solvent, and their volume ratio is 4:1; In Step S3, the molar ratio of the compound with the structural formula of Formula 4, TEA, and the compound with the structural formula of Formula 5 is 1:(1.5 - 3):(1.5 - 2), and DCM is used as the solvent; In Step S4, the molar ratio of the compound with the structural formula of Formula 6, iron powder, and NH4Cl is 1:(5 - 10):(5 - 10), and ethanol and water are used as the solvent, and their volume ratio is 4:1; In Step S5, the molar ratio of the compound with the structural formula of Formula 7, TEA, and the compound with the structural formula of Formula 8 is 1:(1.5 - 3):(1.5 - 2), and THF is used as the solvent.

8. Use of a 2-amido-N-arylbenzamide derivative as described in any one of claims 1 - 5 in the preparation of an anti-tumor drug related to USP2.

9. The application according to claim 8, wherein The dosage form of the drug is a capsule, a granule, a pill, a powder, an injection, a syrup, an ointment, an emulsion, a solution, a suspension, or a tincture.

10. The application according to claim 8, characterized in that, The drug further includes excipients, and the excipients are selected from any one or more of an inerting agent, a filler, a solubilizer, a binder, a humectant, a disintegrant, a slow solvent, an absorption accelerator, an adsorbent, a diluent, a solubilizer, an emulsifier, a lubricant, a wetting agent, a suspending agent, a flavoring agent, or a perfume.

Images