N-pyridine piperazine compound as well as preparation method and application thereof

Abstract

The invention discloses an N-pyridine piperazine compound as well as a preparation method and application thereof, and belongs to the technical field of chemical pharmacy. The structure of the imidazopyridine compound is as shown in formula I: (I), R is-OH,-H,-OR1 or-NHR2, and R1 and R2 are respectively and independently selected from hydrogen, C1-C6 alkyl, C1-C8 cycloalkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl. The novel N-pyridine piperazine compound prepared by the invention is simple and feasible in preparation process, and pharmacological experiments find that the N-pyridine piperazine compound provided by the invention shows USP5 enzymatic inhibitory activity and inhibitory activity to various tumor cells, is more sensitive to breast cancer cells, and can be used for preparing the novel N-pyridine piperazine compound. The compound has good plasma stability, liver microsome metabolism stability and oral bioavailability, shows efficient anti-breast cancer activity in vivo and in vitro, and is suitable for development of anti-breast cancer drugs.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceutical technology, and specifically relates to the preparation and application of a USP5 inhibitor compound. Background Technology

[0002] The ubiquitin-proteasome system (UPS) is a core mechanism for regulating protein homeostasis in eukaryotic cells. Deubiquitinating enzymes (DUBs), as important components of this system, maintain target protein stability and participate in the fine regulation of various signaling pathways by reversing the ubiquitination modification of substrate proteins. Currently, approximately 100 DUB-encoding genes have been identified in the human genome, belonging to five families: USP, UCH, OTU, MJD, and JAMM / MPN+. The USP family has the largest number of members and exhibits abnormally high expression in various malignant tumors, making it an important target for anti-tumor drug development. Studies have confirmed that small-molecule inhibitors targeting DUBs such as USP7 and UCHL1 have shown good efficacy in hematological malignancies and neurodegenerative diseases, with some already in clinical trials. For example, the invention patent with publication number CN105705504A discloses a deubiquitinase inhibitor and its usage method, providing compounds that can inhibit DUBs and related strategies, which can be used to treat pathogenic infections, inhibit cell proliferation, reduce cell survival rate or inhibit tumor metastasis, and have application prospects in the treatment of neurodegenerative diseases and their symptoms.

[0003] USP5 is a widely expressed member of the USP family, traditionally believed to primarily participate in the cleavage and recycling of free polyubiquitin chains. In recent years, increasing research has revealed significantly elevated USP5 expression levels in various solid tumors, including breast cancer, colorectal cancer, and glioblastoma, and its close association with poor prognosis. For example, patent CN118203576A discloses a combined anti-tumor application of a USP5 inhibitor and a PD-1 antibody. This study, using the YTH domain as a key region for YTHDF1 protein ubiquitination modification, screened and obtained the deubiquitinating enzyme USP5 inhibitor WP1130, demonstrating that it negatively regulates PD-L1 expression levels by interfering with YTHDF1 protein homeostasis.

[0004] Although the important role of USP5 in tumorigenesis and development is gradually being recognized, there are currently no approved inhibitors for USP5, nor are there any candidate drugs that have entered the clinical stage. Existing small molecule compounds (such as G9 and Spautin-1) generally suffer from poor metabolic stability and low bioavailability, which severely limits their further application as chemical probes or therapeutic drugs.

[0005] Therefore, developing a class of novel small molecule compounds with good metabolic stability and bioavailability that can efficiently target USP5 and induce tumor cell death is of great scientific significance and clinical application value. Summary of the Invention

[0006] This invention addresses the technical problems of poor metabolic stability and low bioavailability of existing USP5 inhibitors by proposing an N-pyridine piperazine compound, its preparation method, and its application.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0008] This invention provides an N-pyridinepiperazine compound, the structure of which is shown in general formula (I):

[0009] (I),

[0010] The R is -OH, -H, -OR1 or -NHR2, wherein R1 and R2 are independently selected from hydrogen, C1-C6 alkyl, C1-C8 cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

[0011] Preferably, R is selected from any of the following structures:

[0012]

[0013] This invention provides a method for preparing the aforementioned N-pyridinepiperazine compound, the preparation process of which is as follows:

[0014] ;

[0015] Includes the following steps:

[0016] (1) Compound S1, compound S2, triethylamine, and 4-dimethylaminopyridine were dissolved in dichloromethane and stirred to react. After the reaction was complete, the resulting organic phase was washed, dried, filtered, concentrated, and purified to obtain compound M1; the structural formula of compound S1 is as follows: The structural formula of compound S2 is as follows: ;

[0017] (2) Compound M1, compound S3, and potassium carbonate obtained in step (1) were dissolved in DMF, heated and stirred to carry out the reaction, cooled, diluted, extracted, washed, dried, filtered, concentrated, and purified to obtain N-pyridinepiperazine compound T1; the structural formula of compound M1 is as follows: The structural formula of compound S3 is as follows: ;

[0018] (3) After dissolving the N-pyridinepiperazine compound T1 obtained in step (2) and LiOH·H2O in a solvent, the reaction was stirred. After the reaction was complete, methanol was removed by rotary evaporation. After adjusting the pH of the residue, it was filtered, washed and dried to obtain N-pyridinepiperazine compound T2.

[0019] (4) The N-pyridinepiperazine compound T2, amine compound, HATU and DIPEA obtained in step (3) were dissolved in DMF and stirred under nitrogen protection. After the reaction was complete, the mixture was diluted and extracted. Then, after washing, drying, filtering, concentrating and purifying, N-pyridinepiperazine compound T3-18 was obtained.

[0020] In step (1), the molar ratio of compound S1, compound S2, triethylamine, and 4-dimethylaminopyridine is 1:1-1.5:1.5-3:0.1-0.25, and the initial concentration of compound S1 is 0.15-0.5 M; the stirring time is 8-24 h; the reaction is monitored by TLC until compound S1 disappears, and the developing solvent is petroleum ether / ethyl acetate = 3 / 1; the washing is performed sequentially with saturated sodium bicarbonate solution and water; the drying is performed using anhydrous sodium sulfate; and the purification is performed by silica gel column chromatography with petroleum ether / ethyl acetate gradient as the eluent, with an initial ratio of 5:1.

[0021] In step (2), the molar ratio of compound M1, compound S3, and potassium carbonate is 1:1-1.5:2-4, and the initial concentration of compound M1 is 0.12-0.2M; the heating and stirring temperature is 50-90℃, and the time is 8-24 h; the reaction is monitored by TLC (DCM:MeOH = 20:1) until compound M1 disappears; the drying is performed using anhydrous sodium sulfate, and the purification is performed using silica gel column chromatography with a DCM / MeOH gradient as the eluent, starting at a ratio of 50:1.

[0022] In step (3), the molar ratio of N-pyridinepiperazine compound T1 to LiOH·H2O is 1:3-10, and the initial concentration of N-pyridinepiperazine compound T1 is 0.1-0.25M; the solvent is a mixture of methanol and water; the stirring time is 0.5-3h, and the reaction is monitored by HPLC until N-pyridinepiperazine compound T1 is completely hydrolyzed; the pH is adjusted to 3-4 using hydrochloric acid solution with a concentration of 0.5-2M; and the drying is vacuum drying.

[0023] In step (4), the molar ratio of N-pyridinepiperazine compound T2, amine compound, HATU, and DIPEA is 1:1-1.5:1.2-2:1.5-3, and the initial concentration of N-pyridinepiperazine compound T2 is 0.1-0.4 M; the amine compound is a substituted or unsubstituted amine, and the substituted or substituted amine is selected from: substituted or unsubstituted C3-C8 cycloalkylamines, substituted or unsubstituted aromatic amines, and substituted or unsubstituted heteroaromatic amines; the stirring time is 6-18 h, and the reaction is monitored by TLC or LC-MS until N-pyridinepiperazine compound T2 is completely converted; the washing is performed sequentially with saturated NaHCO3 solution and water, the drying is performed using anhydrous sodium sulfate, and the purification is performed by silica gel column chromatography with a DCM / MeOH gradient containing 0.1% NH4OH;

[0024] The amine compound is selected from any one of cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, aniline, 4-aminopyridine, 4-cyanoaniline, 4-nitroaniline, 4-methylaniline, 4-fluoroaniline, 4-chloroaniline, 4-bromoaniline, 1-naphthylamine, 4-fluoro-1-naphthylamine, 8-fluoro-1-naphthylamine, and 5-fluoro-1-naphthylamine.

[0025] This invention provides the use of the aforementioned N-pyridinepiperazine compound or a pharmaceutically acceptable salt thereof in the preparation of a drug that inhibits deubiquitinase USP5.

[0026] This invention provides the use of the aforementioned N-pyridinepiperazine compound or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention or treatment of cancers associated with the deubiquitinase USP5;

[0027] The cancers are preferably leukemia, lymphoma, lung cancer, colorectal cancer, liver cancer, stomach cancer, pancreatic cancer, cervical cancer, and neuroblastoma.

[0028] The present invention provides a pharmaceutical composition comprising the imidazopyridine compound or a pharmaceutically acceptable salt thereof, and further comprising a pharmaceutically acceptable excipient; said excipient being selected from at least one of gum arabic, syrup, lanolin and starch.

[0029] The excipient is stable, has no incompatibility with the active ingredient, does not produce side effects, does not affect efficacy, is not easily deformed, cracked, moldy, or infested by insects at room temperature, is harmless to the human body, has no physiological effects, does not produce chemical or physical reactions with the active ingredient, and does not affect the content determination of the active ingredient.

[0030] The beneficial effects of this invention are:

[0031] The N-pyridinepiperazine compound or its pharmaceutically acceptable salt provided by this invention can target USP5 and induce tumor cell death, and exhibits good metabolic stability and bioavailability, which has significant scientific and clinical translational value. It provides new perspectives and ideas for the development of novel small molecule drugs against breast cancer. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The curve of intravenous administration of N-pyridinepiperazine compound T13.

[0034] Figure 2 The curve is for oral administration of N-pyridinepiperazine compound T13. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] A method for preparing an N-pyridinepiperazine compound T1 is shown in the following flowchart:

[0038] ;

[0039] The specific steps are as follows:

[0040] (1) Add S1 (ethyl 4-amino-3-chlorobenzoate, 5.0 mmol), S2 (4-isopropylbenzenesulfonyl chloride, 5.5 mmol), triethylamine (TEA, 7.5 mmol), and 4-dimethylaminopyridine (DMAP, 0.5 mmol) to a 100 mL round-bottom flask, dissolved in dichloromethane (DCM, 30 mL). Stir the reaction at room temperature for 12 h, and monitor by TLC (petroleum ether: ethyl acetate = 3:1) until S1 is completely consumed. After the reaction, wash the organic phase successively with saturated sodium bicarbonate solution (20 mL × 2) and water (20 mL), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by silica gel column chromatography (eluent: petroleum ether / ethyl acetate gradient, initial ratio 5:1) to obtain a white solid as compound M1, with a yield of 85%. Characterize compound M1, and the results are as follows:

[0041] 1 H NMR (400 MHz, CDCl3): δ 7.98 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 2.0Hz, 1H), 7.52-7.48 (m, 1H), 7.35 (d, J = 8.4 Hz, 2H), 7.28 (dd, J = 8.4, 2.0Hz, 1H), 4.38 (q, J = 7.2 Hz, 2H), 3.05 (sept, J = 6.8 Hz, 1H), 1.38 (t, J =7.2 Hz, 3H), 1.25 (d, J = 6.8 Hz, 6H).

[0042] MS (ESI+): m / z [M+H] + = 398.1.

[0043] (2) Add M1 (3.0 mmol), S3 (1-(2-pyridyl)piperazine, 3.6 mmol), and potassium carbonate (K2CO3, 9.0 mmol) to a 50 mL round-bottom flask, dissolved in N,N-dimethylformamide (DMF, 20 mL). Heat to 80 °C and stir for 12 h. Monitor by TLC (DCM:MeOH = 20:1) until M1 disappears. Cool to room temperature, dilute with water (50 mL), and extract with ethyl acetate (30 mL × 3). Combine the organic phases, wash with water (30 mL × 2), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography (eluent: DCM / MeOH gradient, initial ratio 50:1) to obtain a pale yellow oily substance, N-pyridylpiperazine compound T1, with a yield of 72%. Characterize N-pyridylpiperazine compound T1, and the results are as follows:

[0044] 1 H NMR (400 MHz, CDCl3): δ 8.55 (ddd, J = 4.8, 1.8, 0.8 Hz, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.60 (dt, J = 7.8, 1.8 Hz, 1H), 7.45–7.35 (m, 3H), 7.25(d, J = 8.4 Hz, 2H), 6.85 (ddd, J = 7.2, 4.8, 1.2 Hz, 1H), 4.35 (q, J = 7.2Hz, 2H), 3.85–3.75 (m, 4H), 3.05 (sept, J = 6.8 Hz, 1H), 2.65–2.55 (m, 4H),1.35 (t, J = 7.2 Hz, 3H), 1.25 (d, J = 6.8 Hz, 6H).

[0045] MS (ESI+): m / z [M+H] + = 523.2.

[0046] Example 2

[0047] A method for preparing an N-pyridinepiperazine compound T2, the preparation process is as follows:

[0048] ;

[0049] The specific steps are as follows:

[0050] (1) Add S1 (ethyl 4-amino-3-chlorobenzoate, 5.0 mmol), S2 (4-isopropylbenzenesulfonyl chloride, 5.5 mmol), triethylamine (TEA, 7.5 mmol), and 4-dimethylaminopyridine (DMAP, 0.5 mmol) to a 100 mL round-bottom flask, dissolved in dichloromethane (DCM, 30 mL). Stir the reaction at room temperature for 12 h, and monitor by TLC (petroleum ether: ethyl acetate = 3:1) until S1 is completely consumed. After the reaction is complete, wash the organic phase successively with saturated sodium bicarbonate solution (20 mL × 2) and water (20 mL), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by silica gel column chromatography (eluent: petroleum ether / ethyl acetate gradient, initial ratio 5:1) to give a white solid as compound M1, with a yield of 85%.

[0051] (2) Add M1 (3.0 mmol), S3 (1-(2-pyridyl)piperazine, 3.6 mmol), and potassium carbonate (K2CO3, 9.0 mmol) to a 50 mL round-bottom flask, dissolved in N,N-dimethylformamide (DMF, 20 mL). Heat to 80 °C and stir for 12 h. Monitor by TLC (DCM:MeOH = 20:1) until M1 disappears. Cool to room temperature, dilute with water (50 mL), and extract with ethyl acetate (30 mL × 3). Combine the organic phases, wash with water (30 mL × 2), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography (eluent: DCM / MeOH gradient, initial ratio 50:1) to obtain a pale yellow oily substance, N-pyridylpiperazine compound T1, with a yield of 72%.

[0052] (3) Add N-pyridinepiperazine compound T1 (2.0 mmol) and lithium hydroxide monohydrate (LiOH·H2O, 6.0 mmol) to a 50 mL round-bottom flask, dissolved in a mixed solvent of methanol (MeOH, 10 mL) and water (H2O, 5 mL). Stir the reaction at room temperature for 2 h, and monitor by HPLC until T1 is completely hydrolyzed. Remove methanol by rotary evaporation, adjust the pH of the residue to 3 with 1M HCl, and a white precipitate will form. Filter, wash with cold water, and dry under vacuum to obtain a white solid, which is N-pyridinepiperazine compound T2, with a yield of 95%. Characterize N-pyridinepiperazine compound T2, and the detection results are as follows:

[0053] 1H NMR (400 MHz, DMSO-d6): δ 12.80 (br s, 1H, COOH), 8.50 (ddd, J =4.8, 1.8, 0.8 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.55 (dt, J = 7.8, 1.8 Hz,1H), 7.40–7.30 (m, 3H), 7.20 (d, J = 8.4 Hz, 2H), 6.80 (ddd, J = 7.2, 4.8,1.2 Hz, 1H), 3.75–3.65 (m, 4H), 3.00 (sept, J = 6.8 Hz, 1H), 2.55–2.45 (m,4H), 1.20 (d, J = 6.8 Hz, 6H).

[0054] MS (ESI-): m / z [MH] - = 493.1.

[0055] Example 3

[0056] A method for preparing an N-pyridinepiperazine compound T3-18, the preparation process is as follows:

[0057] ;

[0058] The specific steps are as follows:

[0059] (1) Add S1 (ethyl 4-amino-3-chlorobenzoate, 5.0 mmol), S2 (4-isopropylbenzenesulfonyl chloride, 5.5 mmol), triethylamine (TEA, 7.5 mmol), and 4-dimethylaminopyridine (DMAP, 0.5 mmol) to a 100 mL round-bottom flask, dissolved in dichloromethane (DCM, 30 mL). Stir the reaction at room temperature for 12 h, and monitor by TLC (petroleum ether: ethyl acetate = 3:1) until S1 is completely consumed. After the reaction is complete, wash the organic phase successively with saturated sodium bicarbonate solution (20 mL × 2) and water (20 mL), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by silica gel column chromatography (eluent: petroleum ether / ethyl acetate gradient, initial ratio 5:1) to give a white solid as compound M1, with a yield of 85%.

[0060] (2) Add M1 (3.0 mmol), S3 (1-(2-pyridyl)piperazine, 3.6 mmol), and potassium carbonate (K2CO3, 9.0 mmol) to a 50 mL round-bottom flask, dissolved in N,N-dimethylformamide (DMF, 20 mL). Heat to 80 °C and stir for 12 h. Monitor by TLC (DCM:MeOH = 20:1) until M1 disappears. Cool to room temperature, dilute with water (50 mL), and extract with ethyl acetate (30 mL × 3). Combine the organic phases, wash with water (30 mL × 2), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography (eluent: DCM / MeOH gradient, initial ratio 50:1) to obtain a pale yellow oily substance, N-pyridylpiperazine compound T1, with a yield of 72%.

[0061] (3) Add N-pyridinepiperazine compound T1 (2.0 mmol) and lithium hydroxide monohydrate (LiOH·H2O, 6.0 mmol) to a 50 mL round-bottom flask, dissolved in a mixed solvent of methanol (MeOH, 10 mL) and water (H2O, 5 mL). Stir the reaction at room temperature for 2 h, and monitor with HPLC until T1 is completely hydrolyzed. Remove methanol by rotary evaporation, adjust the pH of the residue to 3 with 1M HCl, and a white precipitate will precipitate. Filter, wash with cold water, and dry under vacuum to obtain a white solid, N-pyridinepiperazine compound T2, with a yield of 95%.

[0062] (4) Add N-pyridinepiperazine compound T2 (0.5 mmol), cyclopropylamine (0.6 mmol), HATU (0.6 mmol), and DIPEA (0.75 mmol) to a 25 mL round-bottom flask, and dissolve in DMF (5 mL). Under nitrogen protection, stir at room temperature for 12 h, monitoring by TLC or LC-MS until T2 is completely converted. After the reaction is complete, dilute with water (20 mL) and extract with ethyl acetate (15 mL × 3). Combine the organic phases, wash successively with saturated NaHCO3 solution (15 mL) and water (15 mL), dry to anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography (eluent: DCM / MeOH gradient, containing 0.1% NH4OH) to obtain N-pyridinepiperazine compound T3. Its chemical structure is:

[0063] ;

[0064] The N-pyridinepiperazine compound T3 was characterized, and the detection results are as follows:

[0065] 1H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.10 (dd, J = 4.5, 1.7Hz, 1H), 7.88 – 7.66 (m, 4H), 7.66 – 7.44 (m, 2H), 7.33 (dd, J = 9.2, 0.7 Hz,2H), 7.16 (d, J = 8.1 Hz, 1H), 6.96 – 6.51 (m, 2H), 3.86 (ddd, J = 19.2, 7.0,4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 3.03 – 2.68 (m, 2H), 1.27(d, J = 6.6 Hz, 6H), 0.95 – 0.45 (m, 4H).

[0066] MS (ESI-): m / z [MH] - = 520.2.

[0067] Replacing cyclopropylamine with cyclobutylamine in step (4) yields N-pyridinepiperazine compound T4. Its chemical structural formula is:

[0068] ;

[0069] The results of the detection of N-pyridinepiperazine compound T4 are as follows:

[0070] 1H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.10 (dd, J = 4.5, 1.7Hz, 1H), 7.94 – 7.44 (m, 6H), 7.33 (dd, J = 9.2, 0.7 Hz, 2H), 7.16 (d, J =8.1 Hz, 1H), 6.77 (dd, J = 7.1, 1.5 Hz, 1H), 6.68 (ddd, J = 7.3, 4.4, 1.5 Hz, 1H), 4.17 (dt, J = 7.1, 4.2 Hz, 1H), 3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H),3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.94 – 2.85 (m, 1H), 2.06 – 1.82 (m,2H), 1.68 (tdd, J = 8.2, 4.2, 1.2 Hz, 3H), 1.27 (d, J = 6.6 Hz, 6H).

[0071] MS (ESI-): m / z [MH] - = 534.2.

[0072] Replacing cyclopropylamine with cyclopentylamine in step (4) yields N-pyridinepiperazine compound T5. Its chemical structural formula is:

[0073] ;

[0074] The results of the detection of N-pyridinepiperazine compound T5 are as follows:

[0075] 1H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.10 (dd, J = 4.5, 1.7Hz, 1H), 7.89 – 7.70 (m, 3H), 7.69 – 7.45 (m, 3H), 7.36 – 7.30 (m, 2H), 7.16(d, J = 8.1 Hz, 1H), 7.02 – 6.50 (m, 2H), 4.18 – 4.11 (m, 1H), 3.86 (ddd, J =19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.94 – 2.85(m, 1H), 1.94 – 1.83 (m, 2H), 1.71 – 1.47 (m, 6H), 1.27 (d, J = 6.6 Hz, 6H).

[0076] MS (ESI-): m / z [MH] - = 548.3.

[0077] Replacing cyclopropylamine with cyclohexylamine in step (4) yields N-pyridinepiperazine compound T6. Its chemical structural formula is:

[0078] ;

[0079] The results of the detection of N-pyridinepiperazine compound T6 are as follows:

[0080] 1 H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.10 (dd, J = 4.5, 1.7Hz, 1H), 7.93 – 7.68 (m, 3H), 7.64 – 7.28 (m, 5H), 7.16 (d, J = 8.1 Hz, 1H), 6.89 – 6.60 (m, 2H), 3.93 – 3.81 (m, 5H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.94 – 2.85 (m, 1H), 1.89 – 1.34 (m, 10H), 1.27 (d, J = 6.6 Hz, 6H).

[0081] MS (ESI-): m / z [MH] - = 562.3.

[0082] Replacing cyclopropylamine with aniline in step (4) yields N-pyridinepiperazine compound T7. Its chemical structural formula is:

[0083] ;

[0084] The detection results of N-pyridinepiperazine compound T7 are as follows:

[0085] 1 H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.94 (s, 1H), 8.10 (dd, J = 4.5, 1.7 Hz, 1H), 7.91 – 7.63 (m, 5H), 7.60 – 7.41 (m, 2H), 7.41 –6.94 (m, 6H), 6.85 – 6.57 (m, 2H), 3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.95 – 2.84 (m, 1H), 1.27 (d, J = 6.6Hz, 6H).

[0086] MS (ESI-): m / z [MH] - = 556.2.

[0087] Replacing cyclopropylamine with 4-aminopyridine in step (4) yields N-pyridinepiperazine compound T8. Its chemical structural formula is:

[0088] ;

[0089] The detection results of N-pyridinepiperazine compound T8 are as follows:

[0090] 1H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 9.33 (s, 1H), 8.43 –8.38 (m, 2H), 8.10 (dd, J = 4.5, 1.7 Hz, 1H), 7.96 – 7.68 (m, 5H), 7.61 –7.38 (m, 2H), 7.38 – 7.04 (m, 3H), 6.83 – 6.51 (m, 2H), 3.86 (ddd, J = 19.2,7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.95 – 2.84 (m, 1H),1.27 (d, J = 6.6 Hz, 6H).

[0091] MS (ESI-): m / z [MH] - = 557.2.

[0092] Replacing cyclopropylamine with 4-cyanoaniline in step (4) yields N-pyridinepiperazine compound T9. Its chemical structural formula is:

[0093] ;

[0094] The detection results of N-pyridinepiperazine compound T9 are as follows:

[0095] 1 H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.55 (s, 1H), 8.10 (dd, J = 4.5, 1.7 Hz, 1H), 7.95 – 7.63 (m, 7H), 7.60 – 7.40 (m, 2H), 7.36 –7.00 (m, 3H), 6.94 – 6.50 (m, 2H), 3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.94 – 2.85 (m, 1H), 1.27 (d, J = 6.6Hz, 6H).

[0096] MS (ESI-): m / z [MH] - = 581.2.

[0097] Replacing cyclopropylamine with 4-nitroaniline in step (4) yields N-pyridinepiperazine compound T10. Its chemical structural formula is:

[0098] ;

[0099] The results of the detection of N-pyridinepiperazine compound T10 are as follows:

[0100] 1 H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.69 (s, 1H), 8.27 –8.21 (m, 2H), 8.10 (dd, J = 4.5, 1.7 Hz, 1H), 8.03 – 7.97 (m, 2H), 7.89 –7.68 (m, 3H), 7.66 – 7.42 (m, 2H), 7.36 – 7.30 (m, 2H), 7.20 (d, J = 7.9 Hz,1H), 6.77 (dd, J = 7.1, 1.5 Hz, 1H), 6.68 (ddd, J = 7.3, 4.4, 1.5 Hz, 1H),3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.95 – 2.84 (m, 1H), 1.27 (d, J = 6.6 Hz, 6H).

[0101] MS (ESI-): m / z [MH] - = 600.2.

[0102] Replacing cyclopropylamine with 4-methylaniline in step (4) yields N-pyridinepiperazine compound T11. Its chemical structural formula is:

[0103] ;

[0104] The results of the detection of N-pyridinepiperazine compound T11 are as follows:

[0105] 1H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.55 (s, 1H), 8.10 (dd, J = 4.5, 1.7 Hz, 1H), 7.95 – 7.68 (m, 3H), 7.64 – 7.40 (m, 4H), 7.40 –7.02 (m, 5H), 6.89 – 6.60 (m, 2H), 3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.95 – 2.84 (m, 1H), 2.51 – 2.21 (m,3H), 1.27 (d, J = 6.6 Hz, 6H).

[0106] MS (ESI-): m / z [MH] - = 570.3.

[0107] Replacing cyclopropylamine with 4-fluoroaniline in step (4) yields N-pyridinepiperazine compound T12. Its chemical structural formula is:

[0108] ;

[0109] The results of the detection of N-pyridinepiperazine compound T12 are as follows:

[0110] 1 H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.56 (s, 1H), 8.10 (dd, J = 4.5, 1.7 Hz, 1H), 7.97 – 7.70 (m, 3H), 7.68 – 7.41 (m, 4H), 7.38 –7.02 (m, 5H), 6.89 – 6.59 (m, 2H), 3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.95 – 2.84 (m, 1H), 1.27 (d, J = 6.6Hz, 6H).

[0111] MS (ESI-): m / z [MH] - = 574.2.

[0112] Replacing cyclopropylamine with 4-chloroaniline in step (4) yields N-pyridinepiperazine compound T13. Its chemical structural formula is:

[0113] ;

[0114] The results of the detection of N-pyridinepiperazine compound T13 are as follows:

[0115] 1 H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.56 (s, 1H), 8.10 (dd, J = 4.5, 1.7 Hz, 1H), 7.97 – 7.70 (m, 5H), 7.68 – 7.41 (m, 2H), 7.38 –7.02 (m, 5H), 6.89 – 6.59 (m, 2H), 3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.95 – 2.84 (m, 1H), 1.27 (d, J = 6.6Hz, 6H).

[0116] MS (ESI-): m / z [MH] - = 590.2.

[0117] Replacing cyclopropylamine with 4-bromoaniline in step (4) yields N-pyridinepiperazine compound T14. Its chemical structural formula is:

[0118] ;

[0119] The results of the detection of N-pyridinepiperazine compound T14 are as follows:

[0120] 1H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.55 (s, 1H), 8.10 (dd, J = 4.5, 1.7 Hz, 1H), 8.01 – 7.41 (m, 9H), 7.39 – 7.08 (m, 3H), 6.88 –6.46 (m, 2H), 3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4,7.0, 4.2 Hz, 4H), 2.95 – 2.84 (m, 1H), 1.27 (d, J = 6.6 Hz, 6H).

[0121] MS (ESI-): m / z [MH] - = 634.1.

[0122] Replacing cyclopropylamine with 1-naphthylamine in step (4) yields N-pyridinepiperazine compound T15. Its chemical structural formula is:

[0123] ;

[0124] The results of the detection of N-pyridinepiperazine compound T15 are as follows:

[0125] 1 H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 9.65 (s, 1H), 8.10 (dd, J = 4.5, 1.7 Hz, 1H), 7.98 – 7.46 (m, 11H), 7.39 – 7.11 (m, 4H), 6.92 –6.52 (m, 2H), 3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4,7.0, 4.2 Hz, 4H), 2.95 – 2.84 (m, 1H), 1.27 (d, J = 6.6 Hz, 6H).

[0126] MS (ESI-): m / z [MH] - = 606.3.

[0127] Replacing cyclopropylamine with 4-fluoro-1-naphthylamine in step (4) yields N-pyridinepiperazine compound T16. Its chemical structural formula is:

[0128] ;

[0129] The results of the detection of N-pyridinepiperazine compound T16 are as follows:

[0130] 1 H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 9.69 (s, 1H), 8.30 –7.89 (m, 3H), 7.89 – 7.39 (m, 8H), 7.37 – 7.08 (m, 4H), 6.88 – 6.60 (m, 2H), 3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.95 – 2.84 (m, 1H), 1.27 (d, J = 6.6 Hz, 6H).

[0131] MS (ESI-): m / z [MH] - = 624.2.

[0132] Replacing cyclopropylamine with 8-fluoro-1-naphthylamine in step (4) yields N-pyridinepiperazine compound T17. Its chemical structural formula is:

[0133] ;

[0134] The results of the detection of N-pyridinepiperazine compound T17 are as follows:

[0135] 1 H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.10 (dd, J = 4.5, 1.7Hz, 1H), 7.95 – 7.02 (m, 14H), 6.88 – 6.54 (m, 2H), 3.86 (ddd, J = 19.2, 7.0,4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.94 – 2.85 (m, 1H), 1.27(d, J = 6.6 Hz, 6H).

[0136] MS (ESI-): m / z [MH] - = 624.2.

[0137] Replacing cyclopropylamine with 5-fluoro-1-naphthylamine in step (4) yields N-pyridinepiperazine compound T18. Its chemical structural formula is:

[0138] ;

[0139] The results of the detection of N-pyridinepiperazine compound T18 are as follows:

[0140] 1 H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 9.65 (s, 1H), 8.31 –7.95 (m, 2H), 7.94 – 7.66 (m, 5H), 7.66 – 7.07 (m, 8H), 6.94 – 6.43 (m, 2H), 3.86 (ddd, J = 19.2, 7.0, 4.2 Hz, 4H), 3.35 (ddd, J = 10.4, 7.0, 4.2 Hz, 4H), 2.95 – 2.84 (m, 1H), 1.27 (d, J = 6.6 Hz, 6H).

[0141] MS (ESI-): m / z [MH] - = 624.2.

[0142] Example of implementation effect 1

[0143] The USP5 enzymatic inhibitory activity of the N-pyridine piperazine compounds prepared in Examples 1-3 was studied, as follows:

[0144] The in vitro enzyme activity of USP was assessed using the Ub-AMC (C-terminal-7-acylamino-labeled ubiquitin protein-4-methylcoumarin-labeled) method. The direct interaction between the small molecule and deubiquitinating enzymes was evaluated to assess the activity of the small molecule, with commercially available USP5 inhibitors WP1130 and USP5-IN-1 used as positive controls. The fluorescent substrate AMC does not emit fluorescence when bound to the ubiquitin chain. When USP cleaves the fluorescent substrate AMC from the ubiquitin chain, AMC emits fluorescence. The degree of inhibition by USP was analyzed by detecting the fluorescence intensity. Experiments were performed in 96-well black polypropylene microplates (TECAN) at a volume of 200 μL. Fluorescence measurements were performed using a multi-functional microplate detector (TECAN) with an excitation wavelength of 345 nm and an emission wavelength of 445 nm. 2 nM of USP5 was added to each well. 1−858Different concentrations of the compound were incubated at room temperature for 1 hour. Finally, 250 nM Ub-AMC was added, and fluorescence readings were immediately taken. The data were analyzed using GraphPad Prism 8.2.0 software to calculate the IC50. 50 Values. Specific test results are shown in Table 1; IC 50 Less than 1 μM, marked as ++++, IC 50 At 1 μM-5 μM, marked as +++, active IC 50 In the range of 5 μM to 20 μM, marked as ++, IC 50 A value greater than 20 μM was marked as +; the results are shown in Table 1:

[0145] Table 1 USP5 enzyme activity IC 50

[0146]

[0147] As shown in Table 1, compounds T1-T18 all exhibited good USP5 enzyme inhibitory activity. Among them, compounds T3 / T9 / T18 showed USP5 enzyme inhibitory activity comparable to the positive control, while compound T13 showed superior USP5 inhibitory activity compared to the positive control. These compounds hold promise for development as potential small molecule inhibitors of USP5.

[0148] Example 2 of implementation results

[0149] The N-pyridine piperazine compounds prepared in Examples 1-3 were subjected to in vitro cancer cell inhibition, as detailed below:

[0150] The inhibitory effect of N-pyridine piperazine compounds on cancer cells was detected by the MTT assay.

[0151] MTT colorimetric assay procedure: Inoculate cancer cells in the logarithmic growth phase at a concentration of 5 × 10⁻⁶ cells per milliliter. 4 Cells were seeded at the specified density into 96-well cell culture plates, with the zero well containing cell-free normal medium. After 12 hours, the medium was replaced with different concentration gradients of N-pyridine piperazine compounds (or positive controls WP1130 and USP5-IN-1), and the zero wells were replaced with normal medium. Five replicates were set for each concentration gradient, and the plates were incubated at 37°C in a 5% CO2 incubator. Cell status and growth changes were observed under a microscope after 24 hours. After 48 hours, tetramethylazoazol blue (Kaiji Biotechnology, 5 mg / mL) solution was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator. After 4 hours, the medium was aspirated, and 100 μL of DMSO was added to each well. The absorbance was measured at 570 nm using a microplate reader, and the data were statistically analyzed using Graphpad software to calculate the half-maximal effective concentration (IC50). 50(Unit: μM). The inhibition effect is shown in Table 2 below. IC 50 Less than 1 μM, marked as ++++, IC 50 At 1 μM-5 μM, marked as +++, active IC 50 In the range of 5 μM to 20 μM, marked as ++, IC 50 A value greater than 20 μM is marked as +;

[0152] Table 2 Effects of N-pyridinepiperazine compounds on the proliferation of different cancer cell lines

[0153]

[0154] In summary, compared with positive control compounds WP1130 and USP5-IN-1, these N-pyridine piperazine compounds generally exhibited enhanced antiproliferative activity. Furthermore, they were more sensitive to breast cancer cells, exhibiting extremely low IC50 values. 50 It has value for further in-depth research and possesses broad-spectrum antitumor activity. In particular, the N-pyridinepiperazine compound T13 exhibits the strongest antitumor activity.

[0155] Example of implementation effect 3

[0156] The plasma metabolic stability of the N-pyridine piperazine compound T13 prepared in Example 3 was evaluated, and the details are as follows:

[0157] Plasma metabolic stability was assessed using WP1130 as a positive control.

[0158] The specific method for evaluating plasma metabolic stability is as follows: Weigh a sample (2.00 mg), dissolve it in acetonitrile (1.00 ml), and prepare a standard solution with a concentration of 2.00 mg / mL. After sonication, filter through a 0.22 μL filter membrane. Observe the solution with the naked eye until it is clear and free of residue, then set aside. Dilute the 2 mg / mL standard stock solution sequentially to 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL. Optimal elution time and peak shape of the 2 mg / mL standard were determined by adjusting the mobile phase conditions (MeCN / H2O = 95:5). The peak area of ​​samples at different concentrations was measured, and the linear regression equation of the standard curve was calculated.

[0159] SD rat plasma (0.800 mL) was mixed with 40 μL of a 1 mg / mL standard solution to obtain a plasma sample with a concentration of 0.095 mg / mL. Timing was started after adding the standard solution. Subsequently, at 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h, 50 μL of plasma sample was collected, and 250 μL of acetonitrile precipitant was added. After vortexing, the sample was centrifuged at 12000 rpm (4℃, 12000 rpm, 12 min). The supernatant was filtered through a membrane and then injected for analysis. The sample concentration at different time points was calculated using the linear regression equation of the standard curve. The results are shown in Table 3.

[0160] Table 3 Plasma stability results

[0161]

[0162] The results showed that, compared with the positive control drug WP1130, the N-pyridine piperazine compound T13 has higher plasma stability and is expected to exert better efficacy in vivo.

[0163] Example of implementation effect 4

[0164] The metabolic stability of the N-pyridinepiperazine compound T13 prepared in Example 3 was evaluated by the following experiments:

[0165] The stability of liver microsomal metabolism was assessed using WP1130 as a positive control.

[0166] The specific method for evaluating the metabolic stability of liver microsomes was as follows: Liver microsomes were removed from a -80℃ freezer and preheated for 3 min in a 37℃ water bath with a constant temperature shaker to thaw them before use. Then, a certain amount of NADPH was weighed and dissolved in an appropriate amount of magnesium chloride solution to prepare a 2 mM solution. A mixed solution (excluding NADPH) was prepared according to the composition ratio of the experimental incubation system (3 Mm MgCl2-PB solution, 1 μM compound T13, 0.5 mg / mL liver microsomes), and dispensed in 40 μL tubes. For the 0 min sample, 240 μL of internal standard working precipitant was added, followed by 40 μL of NADPH solution (40 μL of magnesium chloride solution was added to the negative control group). For other samples, 40 μL of NADPH solution was added to initiate the reaction (40 μL of magnesium chloride solution was added to the negative control group). After incubation at 37 °C for 5 min, 15 min, 30 min, and 60 min, 240 μL of precipitant containing internal standard was added. For the positive control group, 40 μL of NADPH solution was added to initiate the reaction, and after incubation at 37 °C for 5 min and 15 min, 240 μL of precipitant containing internal standard was added. All samples were vortexed and centrifuged, and 150 μL of the supernatant was collected. 150 μL of water was added, and the mixture was vortexed and homogenized. LC-MS / MS analysis was then performed. The results are shown in Table 4.

[0167] Table 4. Results of liver microsomal stability

[0168]

[0169] The results showed that compound T13 exhibited moderate clearance efficiency in three species: humans, mice, and rats, and had a lower risk of in vivo metabolic clearance compared to WP1130.

[0170] Example of implementation effect 5

[0171] The bioavailability of the N-pyridinepiperazine compound T13 prepared in Example 3 was evaluated, as follows:

[0172] The in vivo oral bioavailability was evaluated using WP1130 as a positive control.

[0173] The specific method for evaluating oral bioavailability was as follows: After fasting overnight, three rats were administered the drug by gavage (20 mg / kg), and three rats were administered the drug via tail vein (5 mg / kg). Blood samples were then collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 8 h, and placed in pre-heparinized tubes. After gently tapping the tubes several times to thoroughly mix the blood with the heparin sodium, the tubes were centrifuged (4 ℃, 3000 rpm, 10 min) to obtain plasma. 50 μL of plasma sample was taken, and 50 μL of diluent (50% methanol / water) and 250 μL of methanol precipitant were added. The mixture was vortexed and centrifuged (4 ℃, 12000 rpm, 10 min). The supernatant was then sealed through a membrane and sent for LC-MS / MS analysis.

[0174] Figure 1 The curve of intravenous administration of N-pyridinepiperazine compound T13. Figure 2 The results show the oral administration curve. Parameter analysis using WinNonlin software revealed that compound T13 has good oral bioavailability (37.1%), while the positive control WP1130 has an oral bioavailability of less than 3%, making it difficult to administer orally.

[0175] Example of implementation effect 6

[0176] The N-pyridinepiperazine compound T13 prepared in Example 3 was evaluated in vivo in animal experiments, as follows:

[0177] The efficacy of the selected N-pyridine piperazine compound T13 against breast cancer in a subcutaneous xenograft model was evaluated. Nu / Nu mice (24 mice) were used, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Specifically, human breast cancer cells MDA-MB-231 were injected into Nu / Nu immunodeficient mice. When the average tumor diameter reached 3 mm, the mice were randomly divided into a control group (6 mice), a low-dose oral administration group of N-pyridine piperazine compound T13 (20 mpk, 6 mice), a high-dose oral administration group of compound T13 (50 mpk, 6 mice), and a positive control group (WP1130 intraperitoneal administration group, 50 mpk, 6 mice). After 12 days, there were no significant fluctuations in body weight in either the treatment group or the control group, and the mice showed no significant abnormalities, indicating good safety of compound T13. The tumor weight and volume in the treatment group were significantly lower than those in the control group. The tumor inhibition rate ((1 - tumor weight in the treatment group / tumor weight in the control group) * 100%) is shown in Table 5.

[0178] Table 5. TGI model of MDA-MB-231 cell xenograft

[0179]

[0180] The results showed that the high-dose group of compound T13 had stronger activity compared with the positive control WP1130.

[0181] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An N-pyridinepiperazine compound, characterized in that: The structure of the compound is shown in general formula I: （Ⅰ）， The R is -OH, -H, -OR1 or -NHR2, wherein R1 and R2 are independently selected from hydrogen, C1-C6 alkyl, C1-C8 cycloalkyl, aryl, substituted aryl, heteroaryl, and substituted heteroaryl.

2. The N-pyridinepiperazine compound according to claim 1, characterized in that: The R is selected from any of the following structures: -OEt,-OH, , , , , , , , , , , , , , , , 。 3. The method for preparing the N-pyridinepiperazine compound according to claim 1 or 2, characterized in that, Includes the following steps: (1) Compound S1, compound S2, triethylamine, and 4-dimethylaminopyridine were dissolved in dichloromethane and stirred to react. After the reaction was complete, the resulting organic phase was washed, dried, filtered, concentrated, and purified to obtain compound M1; the structural formula of compound S1 is as follows: The structural formula of compound S2 is as follows: ; (2) Compound M1, compound S3, and potassium carbonate obtained in step (1) were dissolved in DMF, heated and stirred to carry out the reaction, cooled, diluted, extracted, washed, dried, filtered, concentrated, and purified to obtain N-pyridinepiperazine compound T1; the structural formula of compound M1 is as follows: The structural formula of compound S3 is as follows: ; (3) After dissolving the N-pyridinepiperazine compound T1 obtained in step (2) and LiOH·H2O in a solvent, the mixture was stirred and reacted. After the reaction was complete, the mixture was rotary evaporated. The residue was adjusted to pH, filtered, washed and dried to obtain N-pyridinepiperazine compound T2. (4) The N-pyridinepiperazine compound T2, amine compound, HATU and DIPEA obtained in step (3) are dissolved in DMF and stirred to react. After the reaction is complete, the mixture is diluted and extracted. Then, the N-pyridinepiperazine compound is obtained by washing, drying, filtering, concentrating and purifying.

4. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of compound S1, compound S2, triethylamine and 4-dimethylaminopyridine is 1:1-1.5:1.5-3:0.1-0.25, the initial concentration of compound S1 is 0.15-0.5M, and the stirring time is 8-24h.

5. The preparation method according to claim 4, characterized in that: In step (2), the molar ratio of compound M1, compound S3 and potassium carbonate is 1:1-1.5:2-4, and the initial concentration of compound M1 is 0.12-0.2M; the heating and stirring temperature is 50-90℃ and the time is 8-24h.

6. The preparation method according to claim 5, characterized in that: In step (3), the molar ratio of N-pyridinepiperazine compound T1 to LiOH·H2O is 1:3-10, and the initial concentration of N-pyridinepiperazine compound T1 is 0.1-0.25M; the solvent is a mixture of methanol and water; the stirring time is 0.5-3h; and the pH is adjusted to 3-4 using hydrochloric acid solution with a concentration of 0.5-2M.

7. The preparation method according to claim 6, characterized in that: In step (4), the molar ratio of N-pyridinepiperazine compound T2, amine compound, HATU and DIPEA is 1:1-1.5:1.2-2:1.5-3, and the initial concentration of N-pyridinepiperazine compound T2 is 0.1-0.4M; the amine compound is a substituted or unsubstituted amine, and the substituted or unsubstituted amine is selected from: substituted or unsubstituted C3-C8 cycloalkylamines, substituted or unsubstituted aromatic amines, substituted or unsubstituted heteroaromatic amines; the stirring time is 6-18h.

8. The preparation method according to claim 7, characterized in that: The amine compound in step (4) is selected from any one of cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, aniline, 4-aminopyridine, 4-cyanoaniline, 4-nitroaniline, 4-methylaniline, 4-fluoroaniline, 4-chloroaniline, 4-bromoaniline, 1-naphthylamine, 4-fluoro-1-naphthylamine, 8-fluoro-1-naphthylamine, and 5-fluoro-1-naphthylamine.

9. The use of the N-pyridinepiperazine compound of claims 1-2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting deubiquitinase USP5.

10. A pharmaceutical composition, characterized in that: It includes the N-pyridinepiperazine compound as described in any one of claims 1-2 or a pharmaceutically acceptable salt thereof, and also includes at least one pharmaceutically acceptable excipient.

Images