2,5-piperazinediones with antitumor activity, and preparation method and application thereof

Abstract

This invention discloses a class of 2,5-piperazine dione compounds with antitumor activity, their preparation methods, and applications. The structural formula of the compound is shown in Formula (I). The structure-activity relationship of the molecular structure is optimized by introducing multiple substituents into its core skeleton, significantly enhancing its selective targeting ability to tumor cells. This invention also discloses the application of this class of compounds in the preparation of drugs for treating or preventing cancer, particularly in the treatment of lung cancer, liver cancer, pancreatic cancer, breast cancer, and colon cancer. The compounds provided by this invention have innovative molecular structures and broad-spectrum antitumor activity, showing promise for further development of highly active, low-toxicity antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a class of 2,5-piperazindione compounds with antitumor activity, their preparation methods, and applications. Background Technology

[0002] Plinabulin is a diketopiperazine-based microtubule inhibitor synthesized by Hayashi et al. based on the structure of the natural product (-)-Phenylahistin isolated from marine Aspergillus pyrolyticus and its structure-activity relationship study. The Plinabulin action site is located between the α- and β-microtubule subunits, close to the colchicine action site, but not within the groove of the colchicine binding site.

[0003] Early mechanistic studies showed that plinabulin, as a vascular endothelial agent (VDA), disrupts tumor blood supply by targeting tumor vascular endothelial cell microtubules. It received Phase I clinical trial approval from the US FDA in 2006 for patients with advanced solid tumors or lymphoma. In 2019, the immune mechanism of plinabulin (activation of dendritic cells) was published in *Cell Reports*. This was a significant achievement resulting from six years of research by the Wanchun Pharmaceuticals research team in collaboration with Harvard Medical School and the University of Basel Medical School. This study demonstrated that plinabulin is an original drug based on a novel mechanism and a novel target (first-in-class). In June 2020, Wanchun Pharmaceuticals announced that the interim analysis of the PROTECTIVE-2 (Study 106) Phase III clinical trial of plinabulin for chemotherapy-induced severe neutropenia met its primary endpoint. Meanwhile, due to its good efficacy against CIN, Plinabulin received Breakthrough Therapy designation from both the Chinese NMPA and the US FDA in September 2020. However, it failed to gain market approval due to insufficient data from the 106 trial. In September 2024, the results of the DUBLIN-3 Phase III trial were released. Compared with docetaxel, the combination of Plinabulin / docetaxel significantly improved overall survival (OS HR = 0.81), progression-free survival (PFS HR = 0.79), objective response rate (ORR 14% vs 9%), 2-year and 3-year OS rates (median OS extension of 2.6 months in the non-squamous subgroup (11.4 vs 8.8 months), and OS benefit of 4.8 months in patients with ≥4 treatment cycles), and reduced grade 4 neutropenia by 82% (p<0.0001). The results showed that the combination group had better anti-cancer efficacy, which was clinically and statistically significant.

[0004] Although Plinabulin has shown some efficacy in clinical trials, further clinical trials involving its combination with other drugs are still needed. Therefore, in-depth research into its derivatization and structure-activity relationship is expected to yield microtubule inhibitors that possess high activity, microtubule-specific recognition, metabolic stability, and high safety. Summary of the Invention

[0005] The purpose of this invention is to provide a class of 2,5-piperazindione compounds with antitumor activity, their preparation methods, and applications. Based on the piperazindione parent structure of Plinabulin, this invention optimizes the structure by altering or extending the benzene ring and tert-butylimidazole regions, resulting in the preparation of a series of compounds. In vitro cell experiments revealed that some of these compounds exhibit superior inhibitory activity against different tumor cells compared to Plinabulin, providing a reference for the development of novel microtubule inhibitors and holding significant importance for future clinical applications.

[0006] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0007] In one aspect, this invention provides a compound having the structure shown in general formula (I), its isomers, or its pharmaceutically acceptable salts:

[0008]

[0009] R1 is independently selected from any one of hydrogen atom, halogen atom, alkyl, alkoxy or amino atom;

[0010] R2 is any one of hydrogen atom, halogen atom, alkyl, alkenyl, alkynyl, alkoxy, amino, amide or acyloxy group;

[0011] R3 is selected from any one of quinoline, pyrazine, pyrimidine, pyridine, benzopyrimidine, indole, alkoxy-substituted quinoline, alkoxy-substituted pyrazine, alkoxy-substituted pyrimidine, or alkoxy-substituted pyridine.

[0012] Preferably, R1 is a hydrogen atom, F, or a methoxy group.

[0013] Preferably, R2 is F or methoxy.

[0014] Preferably, R3 is quinoline, pyrazine, pyrimidine, pyridine, alkoxy-substituted pyrazine, or alkoxy-substituted pyridine.

[0015] Preferably, the alkoxy-substituted pyrazine or alkoxy-substituted pyridine is

[0016] Particularly preferred, the compound is one of the compounds AC-01, AC-02, AC-03, AC-04, AC-05, AC-06, AC-07, AC-08, AC-09, AC-10, AC-11, AC-12, AC-13, AC-14, AC-15, AC-16, AC-17, AC-18, AC-19, AC-20, LN-21, AC-22, AC-23, AC-24, AC-25, or AC-26 described in the embodiments of the present invention.

[0017] On the other hand, the present invention provides a pharmaceutical composition comprising a therapeutically effective dose of any of the compounds described above, their isomers and / or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.

[0018] In another aspect, the present invention also provides the use of any of the above-described compounds, their isomers, or their pharmaceutically acceptable salts or pharmaceutical compositions in the preparation of medicaments for the prevention and / or treatment of cancer.

[0019] Preferably, the cancer is lung cancer, liver cancer, pancreatic cancer, breast cancer, or colon cancer.

[0020] In another aspect, the present invention provides a method for synthesizing compounds having the structure shown in formula (I), by means of the following general reaction formula:

[0021]

[0022] R1 is independently selected from any one of hydrogen atom, halogen atom, alkyl, alkoxy or amino atom;

[0023] R2 is any one of hydrogen atom, halogen atom, alkyl, alkenyl, alkynyl, alkoxy, amino, amide or acyloxy group;

[0024] R3 is selected from any one of quinoline, pyrazine, pyrimidine, pyridine, benzopyrimidine, indole, alkoxy-substituted quinoline, alkoxy-substituted pyrazine, alkoxy-substituted pyrimidine, or alkoxy-substituted pyridine;

[0025] Synthesis methods include:

[0026] 1) Synthesize or provide piperazine dione condensation intermediate A or C;

[0027] 2) Synthesize or provide intermediate aldehydes B or D;

[0028] 3) The condensation intermediate A or C of 2,5-piperazinide dione is reacted with intermediate aldehyde B or D via an Aldol condensation reaction to obtain a compound having the structure shown in formula (I).

[0029] Compared with existing technologies, the advantages and technical effects of this invention are as follows: The compounds provided by this invention are novel compounds obtained by the inventors through structural and synthetic route design and chemical synthesis, and have not been reported in the literature. Some compounds have better antitumor activity than Plinabulin. The compounds provided by this invention have improved lipophilicity, salt solubility, and bioavailability compared to Plinabulin, and have good development prospects. Attached Figure Description

[0030] Figure 1 The results are from the immunofluorescence assays of compounds AC-15 and Plinabulin.

[0031] Figure 2 The results are from the immunofluorescence assays of compounds AC-15 and Plinabulin.

[0032] Figure 3 The results show the cell cycle arrest effects of compounds AC-15 and Plinabulin on NCI-H460 lung cancer cells. Detailed Implementation

[0033] The methods and techniques of this invention are generally performed according to conventional methods known in the art, unless otherwise stated. The nomenclature, experimental methods, and techniques related to chemistry, biology, pharmacology, and pharmaceutical chemistry described herein are known and commonly used in the art. Chemical synthesis methods, chemical analysis methods, pharmaceutical preparation methods, formulation and administration methods, and patient treatment methods all employ standard techniques.

[0034] Unless otherwise stated, the scientific and technical terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. However, the following terms shall have the following definitions:

[0035] The term "isomer" includes conformational isomers, optical isomers (such as enantiomers and diastereomers), and geometric isomers (such as cis-trans isomers).

[0036] The term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt of a compound of formula (I), preferably an acid addition salt of the compound. Preferred examples of pharmaceutically acceptable salts are acid addition salts of pharmaceutically acceptable inorganic or organic acids, such as hydrohalic acids, sulfuric acid, phosphoric acid, or aliphatic or aromatic carboxylic acids or sulfonic acids, such as acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, or naphthalenesulfonic acid.

[0037] Examples of compounds of the present invention include:

[0038]

[0039]

[0040]

[0041] Unless otherwise specified, the compounds of this invention are generally obtained by synthesis using the following general reaction formula:

[0042]

[0043] R1 is independently selected from any one of hydrogen atom, halogen atom, alkyl, alkoxy or amino atom;

[0044] R2 is any one of hydrogen atom, halogen atom, alkyl, alkenyl, alkynyl, alkoxy, amino, amide or acyloxy group;

[0045] R3 is selected from any one of quinoline, pyrazine, pyrimidine, pyridine, benzopyrimidine, indole, alkoxy-substituted quinoline, alkoxy-substituted pyrazine, alkoxy-substituted pyrimidine, or alkoxy-substituted pyridine;

[0046] Synthesis methods include:

[0047] 1) Synthesize or provide piperazine dione condensation intermediate A or C;

[0048] 2) Synthesize or provide intermediate aldehydes B or D;

[0049] 3) The condensation intermediate A or C of 2,5-piperazinide dione is reacted with intermediate aldehyde B or D via an Aldol condensation reaction to obtain a compound having the structure shown in formula (I).

[0050] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0051] Example 1: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((quinolin-2-yl)methylene)piperazine-2,5-dione (AC-01)

[0052]

[0053] a) Synthesis of 3-(4-fluorophenoxy)benzaldehyde 3

[0054]

[0055] Take a dry 500mL three-necked round-bottom flask and add the weighed 4-fluorophenol (reaction 1 above) (10.00g, 89.2mmol), 3-formylphenylboronic acid (reaction 2 above) (20.10g, 134mmol), DCM (200mL), TEA (2.71g, 28.8mmol), and copper acetate (16.20g, 89.2mmol). Simultaneously, add 1g of... Molecular sieves were used to replace the oxygen atmosphere, and the mixture was stirred overnight at room temperature. TLC (PE:EA = 1:1) was used to monitor the complete conversion of reactant 1. Diatomaceous earth was used as a filter aid. The filtrate was washed with water, concentrated under reduced pressure, dissolved in 6V petroleum ether by heating, and crystallized at 5°C. After filtration and drying, 12.68g of white waxy blocky solid was obtained, which is reaction 3 in the above reaction formula, with a yield of 65.74%. 1 H NMR (400MHz, CDCl3) δ9.95 (s, 1H), 7.61–7.57 (m, 1H), 7.50 (t, J = 7.9Hz, 1H), 7.41 (dd,J=2.9,1.6Hz,1H),7.28–7.23(m,1H),7.12–6.98(m,4H).MS(ESI)m / z:[M+H] + Calcd for C 13 H 10 FO2:217.07, Found:217.27.

[0056] Synthesis of (b)(Z)-1-acetyl-3-((quinolin-2-yl)methylene)piperazine-2,5-dione

[0057]

[0058] Take a 25 mL single-necked flask, add quinoline-2-carboxaldehyde (reaction 4 above) (314 mg, 2 mmol), N,N'-diacetylpiperazine-2,5-dione (reaction 5 above) (791.8 mg, 4 mmol), then add 4 mL of LDM solvent, stir until dissolved, add cesium carbonate (976.4 mg, 3 mmol), and stir overnight at 45 °C. During process control, TLC showed that starting material 4 no longer transformed. Pour the reaction solution into ice water (30 mL), and obvious solid precipitates. Keep stirring at 5 °C for 15 min, filter, wash the filter cake twice with water (20 mL × 2), then slurry with 10 mL of EA, filter, and dry to obtain 275 mg of yellowish-brown (Z)-1-acetyl-3-((quinoline-2-yl)methylene)piperazine-2,5-dione, i.e., reaction 6 above, with a yield of 46.61%. 1 H NMR (400MHz, DMSO-d6) δ13.06 (s, 1H), 8.48 (d, J = 8.4Hz, 1H), 8.01 (t, J = 7.4Hz, 2H), 7.8 4(dd,J=15.4,8.1Hz,2H),7.66(t,J=7.4Hz,1H),7.03(s,1H),4.40(s,2H),2.55(s,3H).

[0059] Synthesis of (c)(3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((quinolin-2-yl)methylene)piperazine-2,5-dione (AC-01)

[0060]

[0061] Take a dry 25 mL brown reaction flask, add intermediate 6 (148 mg, 0.5 mmol), 3-(4-fluorophenoxy)benzaldehyde (3 in the above reaction formula) (119.2 mg, 0.55 mmol), and add 3 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (244 mg, 0.75 mmol), protect under nitrogen atmosphere, and react at 50 °C for 16 h with stirring. TLC monitoring showed that starting material 6 was basically completely converted. Add the reaction solution to 20 mL of ice water and stir. A brownish-yellow solid precipitates out. Filter and purify by reversed-phase C18 column chromatography to obtain 95 mg of pale yellow solid, which is compound AC-01, with a yield of 41.99%. 1 H NMR (400MHz, CF3COOD) δ9.18(d,J=8.6Hz,1H),8.45–8.26(m,5H),8.20–8.07(m,1H),7.67–7.51(m,3H),7.32(d,J=7.6Hz,1H),7.24–7.13(m,5H). 13 C NMR(101MHz,CF3COOD)δ159.13,158.80,151.60,151.57,148.30,147.68,138.60,136.98,132.45,131.23,131.14,129.10,129.06,129 .01,128.67,125.69,123.39,121.82,121.28,121.19,119.81,119.66,117.91,116.43,116.19,105.65,100.00.HRMS(ESI)m / z:[M+Na] + Calcd for C 27 H 18 FN3O3:474.1224,Found:474.1225.

[0062] Example 2: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((quinolin-3-yl)methylene)piperazine-2,5-dione (AC-02)

[0063]

[0064] Synthesis of (a)(Z)-1-acetyl-3-(3-(4-fluorophenoxy)benzylmethyl)piperazine-2,5-one

[0065]

[0066] Take a 50 mL single-necked brown bottle, add 3-(4-fluorophenoxy)benzaldehyde (3 in the above reaction formula) (3 g, 13.9 mmol), N,N'-diacetylpiperazine-2,5-dione (5 in the above reaction formula) (5.5 g, 25.8 mmol), then add 15 mL of DMF solvent, stir until dissolved, add cesium carbonate (6.78 g, 20.8 mmol), replace with nitrogen for protection, and observe that the reaction solution turns brown. Stir overnight at 40 °C in the dark. The process was controlled by TLC, which showed that the conversion of raw material 3 was basically complete. The reaction solution was poured into ice water (100 mL), and a pale yellow solid precipitated out. The mixture was stirred at 5 °C for 15 min, filtered, and the filter cake was washed twice with water (50 mL × 2) and twice with petroleum ether (20 mL × 2). Then it was slurried with 10 mL of ethyl acetate, filtered, and dried to obtain 2.61 g of pale yellow (Z)-1-acetyl-3-(3-(4-fluorophenoxy)benzylmethyl)piperazine-2,5-one, which is the 7 in the above reaction formula, with a yield of 53.09%. 1 H NMR(500MHz,DMSO-d6)δ10.41(s,1H),7.43(t,J=7.9Hz,1H),7.32(d,J=8.0Hz,1H),7.26–7 .19(m,3H),7.11(ddd,J=6.8,5.4,3.2Hz,2H),6.95–6.90(m,2H),4.35(s,2H),2.49(s,3H).

[0067] Synthesis of (b) (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((quinolin-3-yl)methylene)piperazine-2,5-dione (AC-02)

[0068]

[0069] Take a dry 25 mL brown reaction flask, add intermediate 7 (177 mg, 0.5 mmol), quinoline-3-carboxaldehyde (8 in the above reaction formula) (94.2 mg, 0.6 mmol), and add 4 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (244 mg, 0.75 mmol), and protect from light with nitrogen. Stir the reaction at 50 °C for 16 h. TLC monitoring showed that starting material 7 was basically completely converted. Add the reaction solution to 20 mL of ice water and stir. A pale yellow solid precipitates out. Filter and purify by reversed-phase C18 column chromatography to obtain 124 mg of pale yellow solid, which is compound AC-02, with a yield of 55%. 1 H NMR(400MHz,CF3COOD)δ9.28(s,1H),9.19(s,1H),8.45–8.28(m,3H),8.12(t,J=7.6Hz,1H),7.51( t,J=8.1Hz,1H),7.48(s,1H),7.43(s,1H),7.25(d,J=7.7Hz,1H),7.16(s,1H),7.12–7.03(m,6H). 13 C NMR(101MHz,CF3COOD)δ161.37,161.07,153.88,153.85,149.67,146.32,139.44,139.21,134.94,133.90,133.36,131.98,131.89,13 1.79,129.03,126.43,125.78,125.50,123.54,123.46,122.15,121.65,120.07,118.69,118.45,113.72,102.30.HRMS(ESI)m / z:[M+H] + Calcd for C 27 H 18 FN3O3:452.1405,Found:452.1405.

[0070] Example 3: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((quinolin-8-yl)methylene)piperazine-2,5-dione (AC-03)

[0071]

[0072] Take a dry 25 mL brown reaction flask, add intermediate 7 (177 mg, 0.5 mmol), quinoline-8-carboxaldehyde (9 in the above reaction formula) (94.3 mg, 0.6 mmol), and add 4 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (244 mg, 0.75 mmol), and protect from light with nitrogen. Stir the reaction at 50 °C for 16 h. TLC monitoring showed that starting material 7 was basically completely converted. Add the reaction solution to 20 mL of ice water and stir. A pale yellow solid precipitates out. Filter and purify by silica gel column chromatography to obtain 107 mg of pale yellow solid, which is compound AC-03, with a yield of 47.45%. 1 HNMR(400MHz, DMSO-d6), δ13.49(s,1H),10.37(s,1H),9.00(dd,J=4.1,1.5Hz,1H),8.55(dd,J=8.3,1.3Hz,1H),8.02(dd,J=15.6,7.6Hz,2H),7.77– 7.61(m,2H),7.42(t,J=7.9Hz,1H),7.30(d,J=7.9Hz,1H),7.23(dd,J=16. 5,7.8Hz,4H),7.16–7.10(m,2H),6.92(dd,J=8.1,1.8Hz,1H),6.75(s,1H). 13 CNMR(101MHz,DMSO-d6)δ159.39,157.98,157.07,156.91,152.62,152.60,148.71,144.00,138.49,135.22,134.90,132.61,130.28,129.5 0,129.04,127.21,126.75,124.52,122.04,120.61,120.52,118.95,118.93,117.83,116.67,116.44,113.62,113.06.HRMS(ESI)m / z:[M+H] + Calcd for C 27 H 18 FN3O3:452.1405,Found:452.1405.

[0073] Example 4: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((quinolin-7-yl)methylene)piperazine-2,5-dione (AC-04)

[0074]

[0075] Take a dry 25 mL brown reaction flask, add intermediate 7 (177 mg, 0.5 mmol), quinoline-7-carboxaldehyde (10 in the above reaction formula) (94.2 mg, 0.6 mmol), and add 4 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (244 mg, 0.75 mmol), and protect from light with nitrogen. Stir the reaction at 50 °C for 18 h. TLC monitoring showed that starting material 7 was basically completely converted. Add the reaction solution to 20 mL of ice water and stir. A pale yellow solid precipitates out. Filter and purify by silica gel column chromatography to obtain 135 mg of pale yellow solid, which is compound AC-04, with a yield of 59.86%. 1 H NMR(400MHz,CF3COOD)δ9.24(d,J=8.3Hz,1H),9.19(d,J=5.5Hz,1H),8.56(s,1H),8.46(d,J=8.6Hz,1H),8.25–8.18(m,1H) ,8.07(d,J=8.6Hz,1H),7.59(s,1H),7.58–7.53(m,1H),7.47(s,1H),7.30(d,J=7.6Hz,1H),7.20(s,1H),7.17–7.09(m,6H). 13 C NMR(101MHz,CF3COOD)δ161.38,161.08,153.89,153.87,150.40,146.32,142.57,140.21,135.03,133.85,133.36,132.58,131.77,13 0.42,126.16,125.88,125.49,124.38,123.56,123.47,122.23,121.62,120.06,119.22,118.69,118.45,117.63.HRMS(ESI)m / z:[M+H] + Calcd for C 27 H 18 FN3O3:452.1405,Found:452.1402.

[0076] Example 5: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((quinolin-4-yl)methylene)piperazine-2,5-dione (AC-05)

[0077]

[0078] Take a dry 25 mL brown reaction flask, add intermediate 7 (177 mg, 0.5 mmol), quinoline-4-carboxaldehyde (11 in the above reaction formula) (102 mg, 0.65 mmol), and add 4 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (244 mg, 0.75 mmol), protect under nitrogen atmosphere, and react at 50 °C for 18 h with stirring. TLC monitoring showed that starting material 7 was basically completely converted. Add the reaction solution to ice water (20 mL) and stir. Flocculent solid precipitates out. Filter and purify by silica gel column chromatography to obtain 78 mg of pale yellow solid, which is compound AC-05, with a yield of 34.6%. 1 H NMR(400MHz,CF3COOD)δ9.17–9.03(m,1H),8.44–8.33(m,2H),8.32–8.23(m,1H),8.19–8.12(m,1H),8.12–8.04(m,1H),7. 81–7.71(m,1H),7.49(td,J=8.2,3.4Hz,1H),7.44–7.39(m,1H),7.27–7.19(m,1H),7.18–7.12(m,1H),7.12–6.98(m,7H). 13 C NMR(101MHz,CF3COOD)δ161.46,161.12,154.14,153.91,153.88,145.53,140.21,138.96,134.89,133.96,133.52,133.44,130.36,12 8.09,126.96,125.74,125.56,124.13,123.61,123.52,123.47,123.22,121.76,120.12,118.74,118.50,113.25.HRMS(ESI)m / z:[M+H] + Calcd for C 27 H 18 FN3O3:452.1405,Found:452.1407.

[0079] Example 6: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((quinolin-5-yl)methylene)piperazine-2,5-dione (AC-06)

[0080]

[0081] Take a dry 25 mL brown reaction flask, add intermediate 7 (177 mg, 0.5 mmol), quinoline-5-carboxaldehyde (12 in the above reaction formula) (102.1 mg, 0.65 mmol), and add 4 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (244 mg, 0.75 mmol), and protect from light with nitrogen. Stir the reaction at 50 °C for 18 h. TLC monitoring showed that starting material 7 was basically completely converted. Add the reaction solution to ice water (20 mL) and stir. Flocculent solid precipitates out. Filter and purify by silica gel column chromatography to obtain 111 mg of pale yellow solid, which is compound AC-06, with a yield of 49.22%. 1 H NMR(400MHz,CF3COOD)δ9.28(d,J=8.3Hz,1H),9.14(dd,J=5.5,1.3Hz,1H),8.52(s,1H),8.41(d,J=8.9Hz,1H),8.23–8.11 (m,2H),7.51(d,J=6.2Hz,1H),7.49(t,J=8.0Hz,1H),7.39(s,1H),7.23(d,J=7.8Hz,1H),7.13(s,1H),7.09–7.02(m,6H). 13 C NMR(101MHz,CF3COOD)δ162.84,161.44,161.12,153.99,153.96,151.36,146.43,139.86,139.28,137.35,135.14,133.42,132.24,13 1.41,129.67,126.04,125.87,125.53,124.47,123.65,123.62,123.53,121.62,120.15,119.85,118.75,118.52.HRMS(ESI)m / z:[M+H] + Calcd for C 27 H 18 FN3O3:452.1405,Found:452.1406.

[0082] Example 7: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((pyrimidin-5-yl)methylene)piperazine-2,5-dione (AC-07)

[0083]

[0084] Take a dry 25 mL brown reaction flask, add intermediate 7 (177 mg, 0.5 mmol), pyrimidine-5-carboxaldehyde (13 in the above reaction formula) (70.2 mg, 0.65 mmol), and add 4 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (244 mg, 0.75 mmol), and protect from light with nitrogen. Stir the reaction at 50 °C for 18 h. TLC monitoring showed that starting material 7 was basically completely converted. Add the reaction solution to ice water (20 mL) and stir. Flocculent solid precipitates out. Filter and purify by silica gel column chromatography to obtain 99.6 mg of pale yellow solid, which is compound AC-07, with a yield of 49.55%. 1 H NMR(400MHz,CF3COOD)δ9.75(s,1H),9.67–9.48(m,2H),7.52–7.34(m,2H),7.24–7.13(m,2H),7.11–6.96(m,6H). 13 CNMR(101MHz,CF3COOD)δ163.02,161.39,161.08,159.92,159.64,153.89,153.86,152.72,134.83,133.84,133.38 ,133.35,127.04,125.61,125.54,123.55,123.46,121.77,120.06,118.70,118.46,108.50.HRMS(ESI)m / z:[M+Na] + Calcd for C 22 H 15 FN4O3:425.1020,Found:425.1019.

[0085] Example 8: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((1H-indol-3-yl)methylene)piperazine-2,5-dione (AC-08)

[0086]

[0087] Take a dry 25 mL brown reaction flask, add intermediate 7 (177 mg, 0.5 mmol), 1H-indole-3-carboxaldehyde (14 in the above reaction formula) (108.8 mg, 0.75 mmol), and add 4 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (244 mg, 0.75 mmol), protect under nitrogen atmosphere, and react at 80 °C for 18 h with stirring. TLC monitoring showed that starting material 7 was basically completely converted. Add the reaction solution to ice water (20 mL) and stir. Obvious flocculent solid precipitates out. Filter and purify by reversed-phase C18 column chromatography to obtain 10.6 mg of pale yellow solid, which is compound AC-08, with a yield of 4.83%. HRMS (ESI) m / z: [M+Na] + Calcd for C 26 H 18 FN3O3:462.1224,Found:462.1222.

[0088] Example 9: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((1H-indol-5-yl)methylene)piperazine-2,5-dione (AC-09)

[0089]

[0090] Take a dry 25 mL brown reaction flask, add intermediate 7 (177 mg, 0.5 mmol), 1H-indole-5-carboxaldehyde (15 in the above reaction formula) (87 mg, 0.6 mmol), and add 4 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (244 mg, 0.75 mmol), and protect from light with nitrogen. Stir the reaction at 80 °C for 18 h. TLC monitoring showed that starting material 7 was basically completely converted. Add the reaction solution to ice water (20 mL) and stir. Flocculent solid precipitates out. Filter and purify by reversed-phase C18 column chromatography to obtain 26.6 mg of pale yellow solid, which is compound AC-09, with a yield of 12.12%. 1 H NMR (400MHz, DMSO-d6) δ11.25 (s, 1H), 10.12 (brs, J = 83.1Hz, 2H), 8.51–8.12 (m, 1H), 7.91–7. 75(m,1H),7.58–7.02(m,6H),7.00–6.82(m,3H),6.83–6.70(m,2H),6.65(s,1H),6.50(s,1H). 13C NMR(101MHz,DMSO-d6)δ158.29,157.60,157.53,153.13,145.58,130.81,127.90,125.23,125.06,125.01,124.90,124.29 ,123.56,122.24,122.18,121.14,121.05,117.20,116.96,114.00,113.65,112.32,102.32,100.00.HRMS(ESI)m / z:[M+Na] + Calcd for C 26 H 18 FN3O3:462.1224,Found:462.1226.

[0091] Example 10: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-hydroxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-10)

[0092]

[0093] Synthesis of a)(Z)-1-acetyl-3-((5-hydroxypyridin-2-yl)methylene)piperazine-25-dione 17

[0094]

[0095] Take a 50 mL single-necked flask, add 5-hydroxypyridine-2-carboxaldehyde (reaction 16 in the above formula) (123 mg, 1 mmol), N,N'-diacetylpiperazine-2,5-dione (396 mg, 2 mmol), then add 3 mL of LDM solvent, stir until dissolved, add cesium carbonate (488.3 mg, 1.5 mmol), and stir overnight at 45 °C. During process control, TLC showed that starting material 16 was essentially completely converted. Pour the reaction solution into ice water (20 mL), and a yellowish-brown solid precipitated. Filter, wash the filter cake twice with water (50 mL × 2), then slurry with 15 mL of ethyl acetate, filter, and dry to obtain 127 mg of yellowish-brown solid, which is reaction 17 in the above formula, with a yield of 48.66%.

[0096] b) Synthesis of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-hydroxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-11)

[0097]

[0098] Take a dry 25 mL brown reaction flask, add intermediate 17 (105 mg, 0.4 mmol), 3-(4-fluorophenoxy)benzaldehyde (3 in the above reaction formula) (104 mg, 0.48 mmol), and add 3 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (196 mg, 0.6 mmol), protect under nitrogen atmosphere, and react at 50 °C for 14 h with stirring. TLC monitoring showed that starting material 17 was basically completely converted. Add the reaction solution to 20 mL of ice water and stir. A pale yellow solid precipitates out. Filter and purify by reversed-phase C18 column chromatography to obtain 29.7 mg of pale yellow solid, which is compound AC-10, with a yield of 17.7%. 1 H NMR (400MHz, DMSO-d6) δ12.33(s,1H),8.28(s,1H),7.52(d,J=8.3Hz,1H),7.41(t,1H), 7.34–7.17(m,6H),7.17–7.07(m,2H),6.91(d,J=6.7Hz,1H),6.77(s,1H),6.65(s,1H). 13 C NMR(101MHz,DMSO-d6)δ157.11,157.07,156.54,153.52,152.61,145.34,137.23,135.06,130.30,127.97, 127.84,127.02,124.55,123.64,120.62,120.53,119.02,117.90,116.67,116.44,114.03,108.71,99.48. 19 F NMR(376MHz,DMSO-d6)δ-121.20.HRMS(ESI)m / z:[M+Na] + Calcd forC 23 H 16 FN3O4:440.1017,Found:440.1012.

[0099] Example 11: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-methoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-11)

[0100]

[0101] Take a dry 25 mL brown reaction flask, add intermediate 7 (177 mg, 0.5 mmol), 5-methoxypyridine-2-carboxaldehyde (18 in the above reaction formula) (82.2 mg, 0.6 mmol), and add 4 mL of dry DMF to dissolve. Under thorough stirring, add Cs₂CO₃ (244 mg, 0.75 mmol), protect under nitrogen atmosphere, and react at 50 °C for 16 h with stirring. TLC monitoring showed that starting material 7 was basically completely converted. Add the reaction solution to ice water (20 mL) and stir. A yellow solid precipitates. Let stand at 5 °C for 3 h, filter, and purify by column chromatography to obtain 96.7 mg of a pale yellow solid, which is compound AC-11, with a yield of 44.87%. HRMS (ESI) m / z: [M+H] + Calcd for C 24 H 18 FN3O4:432.1354,Found:432.1347.

[0102] Example 12: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-ethoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-12)

[0103]

[0104] a) Synthesis of 5-ethoxypyridine-2-aldehyde 20

[0105]

[0106] Take a 25 mL dry round-bottom flask and add 16 mg (123 mg, 1.0 mmol) of 5-hydroxypyridine-2-carboxaldehyde, 156 mg (1.0 mmol) of iodoethane, 138 mg (1.0 mmol) of potassium carbonate, and 5 mL of DMF in sequence. Under nitrogen protection, place the flask in an oil bath at 100 °C and stir for 1 h. The reaction was confirmed to be complete by LC-MS. Heating was stopped and the flask was cooled. Water (30 mL) was added to the reaction solution, and the mixture was extracted three times with EA (50 mL × 3). The organic phase was washed with saturated NaCl aqueous solution (50 mL) and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure. The filtrate was purified by silica gel column chromatography (EA:PE = 1:1) to obtain 98.8 mg of a pale yellow oil, which is 20 in the above reaction formula, with a yield of 65.42%. 1 H NMR (400MHz, CDCl3) δ9.98 (s, 1H), 8.41 (s, 1H), 7.95 (d, J = 8.6Hz, 1H), 7.27 (dd, J =2.7,2.8Hz,1H),4.30(t,J=6.7Hz,2H),4.13(t,J=6.6Hz,3H).MS(ESI)m / z:[M+H]+ Calcd for C8H9NO2:152.07,Found:151.35.

[0107] Synthesis of (b) (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-ethoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-12)

[0108]

[0109] (Z)-1-acetyl-3-(3-(p-fluorophenoxy)methylene)piperazin-2,5-dione 7 (85 mg, 0.24 mmol) and 5-ethoxypyridine-2-aldehyde (20 in the above reaction formula) (43.6 mg, 0.288 mmol) were placed in a single-necked flask, dissolved in 3 mL of DMF, and finally Cs₂CO₃ (117 mg, 0.36 mmol) was added. The mixture was protected under nitrogen atmosphere and kept in the dark. The mixture was stirred at 50 °C for 24 h, and TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 20 mL of ice water and stirred. A pale yellow solid precipitated out. After filtration, the solid was purified by column chromatography to obtain 32.2 mg of the pale yellow solid, which was compound AC-12, with a yield of 30.13%. HRMS (ESI) m / z: [M+H] + Calcd for C 25 H 20 FN3O4:446.1511,Found:446.1515.

[0110] Example 13: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-n-propoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-13)

[0111]

[0112] a) Preparation of 5-propoxypyridine-2-carboxaldehyde 22

[0113]

[0114] 100 mg (0.81 mmol) of 5-hydroxypyridine-2-carboxaldehyde (reaction 16 above) and 138 mg (0.81 mmol) of n-iodopropane (reaction 21 above) were used as the starting materials for this step of the reaction. Potassium carbonate (112 mg, 0.81 mmol) and DMF (4 mL) were added. Under nitrogen protection, the mixture was placed in an oil bath at 100 °C and stirred for 1 h. The reaction was confirmed to be complete by LC-MS. Heating was stopped and the mixture was cooled. Water (30 mL) was added to the reaction solution, and the mixture was extracted three times with EA (50 mL × 3). The organic phase was washed with saturated NaCl aqueous solution (50 mL) and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure. The filtrate was purified by silica gel column chromatography to obtain 101 mg of a pale yellow oil, which is reaction 22 above, with a yield of 75.48%. 1 H NMR (400MHz, CDCl3) δ9.99(s,1H),8.43(s,1H),7.95(d,J=8.6Hz,1H),7.29(d,J=2.8Hz,1 H),4.06(t,J=6.5Hz,2H),1.88(h,J=7.0Hz,2H),1.07(t,J=7.4Hz,3H).MS(ESI)m / z:[M+H] + Calcd for C9H 11 NO2:166.09, Found 165.67.

[0115] b) Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-n-propoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-13)

[0116]

[0117] Intermediate 7 (85 mg, 0.24 mmol) and 5-propoxypyridine-2-aldehyde (reaction 22 above) (47.6 mg, 0.288 mmol) were placed in a single-necked flask, dissolved in 3 mL of DMF, and finally Cs₂CO₃ (117 mg, 0.36 mmol) were added. The mixture was protected from light by nitrogen and stirred at 50 °C for 24 h. TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 20 mL of ice water and stirred, resulting in the precipitation of a pale yellow solid. After filtration, the solid was purified by column chromatography to obtain 29 mg of the pale yellow solid, which was compound AC-13, with a yield of 26.31%. HRMS (ESI) m / z: [M+H] + CalcdforC 26 H 22 FN3O4:460.1667,Found:460.1659.

[0118] Example 14: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-isobutoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-14)

[0119]

[0120] a) Preparation of 5-isobutoxypyridine-2-carboxaldehyde 16

[0121] 123 mg (1.0 mmol) of 5-hydroxypyridine-2-carboxaldehyde (reaction 16 in the above formula) and iodoisobutane (reaction 23 in the above formula) (184 mg, 1.0 mmol) were used as the starting materials for this step of the reaction. Potassium carbonate (138 mg, 1.0 mmol) and DMF (4 mL) were added. Under nitrogen protection, the mixture was placed in an oil bath at 100 °C and stirred for 1 h. The reaction was confirmed to be complete by LC-MS. Heating was stopped and the mixture was cooled. Water (30 mL) was added to the reaction solution, and the mixture was extracted three times with EA (50 mL × 3). The organic phase was washed with saturated NaCl aqueous solution (50 mL) and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated under reduced pressure. The filtrate was purified by silica gel column chromatography to obtain 124 mg of a pale yellow oil, which is reaction 24 in the above formula, with a yield of 69.18%. This oil was used directly in the next step of the reaction. MS (ESI) m / z: [M+H] + Calcd forC 10 H 13 NO2:180.10, Found 180.19.

[0122] b) Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-isobutoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-14)

[0123]

[0124] Intermediate 7 (122 mg, 0.34 mmol) and 5-isobutoxypyridine-2-aldehyde (reaction 24 above) (74 mg, 0.41 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (168 mg, 0.52 mmol) were added. The mixture was protected under nitrogen atmosphere and kept in the dark. The mixture was stirred at 50 °C for 24 h. TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 30 mL of ice water and stirred. A pale yellow solid precipitated out. After filtration, the solid was purified by column chromatography to obtain 51 mg of the pale yellow solid, which was compound AC-14, with a yield of 31.28%. HRMS (ESI) m / z: [M+H] + Calcd for C 27 H 24FN3O4:474.1824,Found:4674.1827.

[0125] Example 15: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-15)

[0126]

[0127] a) Synthesis of 5-n-butoxypyridine-2-carboxaldehyde 26

[0128]

[0129] Weigh and add 2.0 g (24.4 mmol) of 5-hydroxypyridine-2-carboxaldehyde (reaction 16 in the above formula), 4.93 g (26.8 mmol) of 1-iodobutane (reaction 25 in the above formula), 3.37 g (24.4 mmol) of K₂CO₃, and 20 mL of DMF to a 50 mL dry single-necked flask. Under nitrogen protection, stir the mixture at 100 °C for 1 h. TLC monitoring showed that reactant 16 was essentially completely converted. Stop the reaction and cool to room temperature. Add 50 mL of water to the reaction mixture, and extract twice with EA (100 mL × 2). Wash the combined organic phases with saturated NaCl aqueous solution, and back-extract the aqueous phase twice with EA (100 mL × 2). Purge the organic phase under reduced pressure with silica gel, and purify by silica gel column chromatography to obtain 2.85 g of a pale yellow oil, which is reaction 26 in the above formula, with a yield of 65.26%. 1 HNMR(400MHz, CDCl3)δ9.99(s,1H),8.42(d,J=2.8Hz,1H),7.95(d,J=8.1Hz,1H),7.31–7.26(m,1 H), 4.10 (t, J = 6.4Hz, 2H), 1.98–1.72 (m, 2H), 1.52 (td, J = 15.0, 7.5Hz, 2H), 1.00 (t, J = 7.4Hz, 3H).

[0130] Synthesis of (b) (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-15)

[0131]

[0132] Intermediate 7 (177 mg, 0.5 mmol) and 5-n-butoxypyridine-2-aldehyde (reaction 26 above) (108 mg, 0.6 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (244 mg, 0.75 mmol) were added. The mixture was protected from light by nitrogen and stirred at 50 °C for 18 h. TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 30 mL of ice water and stirred, resulting in the precipitation of a pale yellow solid. After filtration, the solid was purified by column chromatography to obtain 157 mg of the pale yellow solid, yielding compound AC-15, with a yield of 66.38%. MP: 289-291 °C. 1 H NMR (400MHz, DMSO-d6) δ12.31(s,1H),10.34(s,1H),8.46(d,J=3.0Hz,1H),7.64(d,J=8.6Hz, 1H),7.51(dd,J=8.8,3.0Hz,1H),7.42(t,J=7.9Hz,1H),7.32–7.27(m,1H),7.27–7.21(m,2H) ,7.20(d,J=2.1Hz,1H),7.15–7.09(m,1H),6.92(ddd,J=8.2,2.4,0.5Hz,1H),6.78(s,1H),6. 70(s,1H),4.11(t,J=6.5Hz,2H),1.79–1.65(m,2H),1.53–1.35(m,2H),0.94(t,J=7.4Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ159.40,157.06,156.98,156.57,153.89,152.59,152.56,146.89,136.86,134.98,130.30,128.59,127.65,1 24.56,122.53,120.62,120.53,119.02,117.95,116.66,116.43,114.26,108.19,68.02,30.50,18.61,13.63.HRMS(ESI)m / z:[M+Na] + Calcd forC 27 H 24 FN3O4:496.1643,Found:496.1641.

[0133] Example 16: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((3-hydroxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-16)

[0134]

[0135] Intermediate 7 (88 mg, 0.248 mmol) and 3-hydroxypyridine-2-carboxaldehyde (reaction 26 above) (37 mg, 0.298 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (121 mg, 0.372 mmol) were added. The mixture was protected from light by nitrogen and stirred at 50 °C for 18 h. TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 30 mL of ice water and stirred, resulting in the precipitation of a pale yellow solid. After filtration, the solid was purified by column chromatography to obtain 32 mg of the pale yellow solid, which was compound AC-16, with a yield of 30.87%. HRMS (ESI) m / z: [M+Na] + CalcdforC 23 H 16 FN3O4:440.1017,Found:440.1017.

[0136] Example 17: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((3-methoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-17)

[0137]

[0138] Intermediate 7 (88 mg, 0.248 mmol) and 3-methoxypyridine-2-carboxaldehyde (reaction 28 above) (41 mg, 0.298 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (121 mg, 0.372 mmol) were added. The mixture was protected from light by nitrogen and stirred at 50 °C for 18 h. TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 30 mL of ice water and stirred, resulting in the precipitation of a pale yellow solid. After filtration, the solid was purified by column chromatography to obtain 34 mg of the pale yellow solid, which was compound AC-17, with a yield of 31.73%. HRMS (ESI) m / z: [M+H] + CalcdforC 24 H 18 FN3O4:432.1354,Found:432.1358.

[0139] Example 18: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((3-ethoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-18)

[0140]

[0141] Intermediate 7 (88 mg, 0.248 mmol) and 3-ethoxypyridine-2-carboxaldehyde (reaction 29 above) (45 mg, 0.298 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (121 mg, 0.372 mmol) were added. The mixture was protected from light by nitrogen and stirred at 50 °C for 20 h. TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 30 mL of ice water and stirred, resulting in the precipitation of a pale yellow solid. After filtration, the solid was purified by column chromatography to obtain 28 mg of the pale yellow solid, which was compound AC-18, with a yield of 25.25%. HRMS (ESI) m / z: [M+H] + Calcdfor C 25 H 20 FN3O4:446.1511,Found:446.1517.

[0142] Example 19: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((3-isopropoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-19)

[0143]

[0144] Intermediate 7 (88 mg, 0.248 mmol) and 3-isopropoxypyridine-2-carboxaldehyde (30 in the above reaction formula) (49 mg, 0.298 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (121 mg, 0.372 mmol) were added. The mixture was protected under nitrogen atmosphere and kept in the dark. The mixture was stirred at 60 °C for 18 h. TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 30 mL of ice water and stirred. A pale yellow solid precipitated out. After filtration, the solid was purified by column chromatography to obtain 41 mg of the pale yellow solid, which was compound AC-19, with a yield of 35.85%. HRMS (ESI) m / z: [M+H] + Calcd for C 26 H 22 FN3O4:460.1667,Found:460.1661.

[0145] Example 20: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((pyrazin-2-yl)methylene)piperazine-2,5-dione (AC-20)

[0146]

[0147] Intermediate 7 (88 mg, 0.248 mmol) and pyrazine-2-carboxaldehyde (31 in the above reaction formula) (33 mg, 0.30 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (121 mg, 0.372 mmol) were added. The mixture was protected from light by nitrogen and stirred at 50 °C for 16 h. TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 30 mL of ice water and stirred, resulting in the precipitation of a pale yellow solid. After filtration, the solid was purified by column chromatography to obtain 24 mg of the pale yellow solid, which was compound AC-20, with a yield of 24.02%. HRMS (ESI) m / z: [M+H] + Calcd forC 22 H 15 FN4O3:403.1201,Found:403.1205.

[0148] Example 21: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((3-methoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-21)

[0149]

[0150] Intermediate 7 (88 mg, 0.248 mmol) and 3-methoxypyrazine-2-carboxaldehyde (32 in the above reaction formula) (42 mg, 0.30 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (121 mg, 0.372 mmol) were added. The mixture was protected from light by nitrogen and stirred at 50 °C for 16 h. TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 30 mL of ice water and stirred, resulting in the precipitation of a pale yellow solid. After filtration, the solid was purified by column chromatography to obtain 40 mg of the pale yellow solid, which was compound AC-21, with a yield of 37.25%. HRMS (ESI) m / z: [M+H] + CalcdforC 23 H 17 FN4O4:433.1307,Found:403.1307.

[0151] Example 22: Preparation of (3Z,6Z)-3-(3-(p-fluorophenoxy)benzylmethyl)-6-((5-methoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-22)

[0152]

[0153] Intermediate 7 (88 mg, 0.248 mmol) and 5-methoxypyrazine-2-carboxaldehyde (reaction 33 above) (42 mg, 0.30 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (121 mg, 0.372 mmol) were added. The mixture was protected from light by nitrogen and stirred at 50 °C for 16 h. TLC monitoring showed that starting material 7 was essentially completely converted. The reaction solution was added to 30 mL of ice water and stirred, resulting in the precipitation of a pale yellow solid. After filtration, the solid was purified by column chromatography to obtain 35 mg of the pale yellow solid, which was compound AC-22, with a yield of 32.59%. HRMS (ESI) m / z: [M+H] + Calcd for C 23 H 17 FN4O4:433.1307,Found:403.1301.

[0154] Example 23: Preparation of (3Z,6Z)-3-(3-(3,4-difluorophenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-23)

[0155]

[0156] Preparation of (a)(Z)-1-acetyl-3-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione 34

[0157]

[0158] Take a 50 mL single-necked flask, add 2.5 g (14.0 mmol) of 5-n-butoxypyridin-2-carboxaldehyde (26 in the above reaction formula), 5.53 g (27.9 mmol) of N,N'-diacetylpiperazine-2,5-dione, and then add 20 mL of DMF solvent. Stir until dissolved, then add cesium carbonate (6.82 g, 20.9 mmol) and stir overnight at 40 °C. During process control, TLC showed that starting material 26 was essentially completely converted. Pour the reaction solution into ice water (200 mL), and a yellowish-brown solid precipitated. Filter, wash the filter cake twice with water (100 mL × 2), then slurry with 30 mL of ethyl acetate, filter, and dry to obtain 2.16 g of yellowish-brown (Z)-1-acetyl-3-((5-butoxypyridin-2-yl)methylene)piperazine-2,5-dione, which is compound 34, with a yield of 48.79%. 1HNMR (400MHz, DMSO-d6) δ12.17(s,1H),8.46(d,J=3.0Hz,1H),7.69(d,J=7.7Hz,1H),7.53(dd,J=8.7,3.0Hz,1H),6.86 (s,1H),4.34(s,2H),4.11(t,J=6.5Hz,2H),2.51(s,3H),1.82–1.65(m,2H),1.53–1.37(m,2H),0.94(t,J=7.4Hz,3H).

[0159] b) Preparation of 3-(3,4-difluorophenoxy)benzaldehyde 36

[0160]

[0161] Weigh out 130 mg (1.0 mmol) of 3,4-difluorophenol (35 in the above reaction formula), 225 mg (1.0 mmol) of 3-formylphenylboronic acid, 10 mL of DCM, 34 mg (0.33 mmol) of TEA, and 182 mg (1.0 mmol) of copper acetate. Simultaneously, add 60 mg of... Molecular sieves, prepared according to the method for compound 3, yielded 146 mg of a white solid, which is 36 in the above reaction formula, with a yield of 62.38%, and was directly used in the next reaction.

[0162] c) Preparation of (3Z,6Z)-3-(3-(3,4-difluorophenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-23)

[0163]

[0164] Intermediate 34 (80 mg, 0.252 mmol) and 3-(3,4-difluorophenoxy)benzaldehyde 36 (71 mg, 0.303 mmol) were dissolved in 4 mL of DMF in a single-necked flask. Finally, Cs₂CO₃ (123 mg, 0.378 mmol) was added. The mixture was reacted under nitrogen atmosphere, protected from light, and stirred at 55 °C for 16 h. The reaction was monitored by TLC until complete. The reaction solution was added to 20 mL of ice water and stirred. An orange-yellow solid precipitated out. After filtration, the solid was purified by column chromatography to obtain 46 mg of a pale yellow solid, which was compound AC-23, with a yield of 37.13%. HRMS (ESI) m / z: [M+H] + Calcd for C 27 H 23 F2N3O4:492.1729,Found:492.1735.

[0165] Example 24: Preparation of (3Z,6Z)-3-(3-(3-fluorophenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-24)

[0166]

[0167] a) Preparation of 3-(3-fluorophenoxy)benzaldehyde 38

[0168]

[0169] Weigh out 112 mg (1.0 mmol) of 3-fluorophenol (37 in the above reaction formula), 225 mg (1.0 mmol) of 3-formylphenylboronic acid (2 in the above reaction formula), 10 mL of DCM, 34 mg (0.33 mmol) of TEA, and 182 mg (1.0 mmol) of copper acetate. Simultaneously, add 60 mg of... Molecular sieves, prepared according to the method of compound 3, yielded 132 mg of white solid, which is 38 in the above reaction formula, with a yield of 61.11%, and were directly used in the next reaction.

[0170] Preparation of (a)(3Z,6Z)-3-(3-(3-fluorophenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-24)

[0171]

[0172] Intermediate 34 (80 mg, 0.252 mmol) and 3-(3-fluorophenoxy)benzaldehyde (38 in the above reaction formula) (66 mg, 0.303 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (123 mg, 0.378 mmol) were added. The mixture was protected under nitrogen atmosphere and kept in the dark. The reaction was stirred at 55 °C for 18 h, and the reaction was monitored by TLC until completion. The reaction solution was added to 20 mL of ice water and stirred. An orange-yellow solid precipitated out. After filtration, the solid was purified by column chromatography to obtain 55 mg of a pale yellow solid, which was compound AC-24, with a yield of 46.17%. HRMS (ESI) m / z: [M+H] + Calcd for C 27 H 24 FN3O4:474.1824,Found:474.1821.

[0173] Example 25: Preparation of (3Z,6Z)-3-(3-(p-methoxyphenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-25)

[0174]

[0175] a) Preparation of 3-(4-methoxyphenoxy)benzaldehyde 40

[0176]

[0177] Weigh out p-methoxyphenol (39 in the above reaction formula) (124 mg, 1.0 mmol), 3-formylphenylboronic acid (2 in the above reaction formula) (225 mg, 1.0 mmol), DCM (10 mL), TEA (34 mg, 0.33 mmol), and copper acetate (182 mg, 1.0 mmol). Simultaneously, add 60 mg of... Molecular sieves, prepared according to the method of compound 3, yielded 153 mg of white solid, which is 40 in the above reaction formula, with a yield of 67.11%, and were directly used in the next reaction.

[0178] b) Preparation of (3Z,6Z)-3-(3-(p-methoxyphenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-25)

[0179]

[0180] Intermediate (34 in the above reaction formula) (80 mg, 0.252 mmol) and 3-(p-methylphenoxy)benzaldehyde (40 in the above reaction formula) (69 mg, 0.303 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (123 mg, 0.378 mmol) were added. The mixture was protected from light by nitrogen and stirred at 55 °C for 16 h. The reaction was monitored by TLC until completion. The reaction solution was added to 20 mL of ice water and stirred. A brownish-yellow solid precipitated out. After filtration, the solid was purified by column chromatography to obtain 47 mg of yellow solid, which was compound AC-25, with a yield of 38.48%. HRMS (ESI) m / z: [M+H] + Calcd for C 28 H 27 N3O5:486.2023,Found:486.2027.

[0181] Example 26: Preparation of (3Z,6Z)-3-(3-(p-methylphenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-26)

[0182] a) Preparation of 3-(4-methoxyphenoxy)benzaldehyde 42

[0183]

[0184] Weigh out p-phenylphenol (reaction 41 above) (108 mg, 1.0 mmol), 3-formylphenylboronic acid (reaction 2 above) (225 mg, 1.0 mmol), DCM (10 mL), TEA (34 mg, 0.33 mmol), and copper acetate (182 mg, 1.0 mmol). Simultaneously, add 60 mg of... Molecular sieves, prepared according to the method of compound 3, yielded 119 mg of white solid, with a yield of 56.14%, which was directly used in the next reaction.

[0185] b) Preparation of (3Z,6Z)-3-(3-(p-methylphenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione (AC-26)

[0186]

[0187] Intermediate 34 (80 mg, 0.252 mmol) and 3-(p-methylphenoxy)benzaldehyde (42 in the above reaction formula) (65 mg, 0.303 mmol) were placed in a single-necked flask, dissolved in 4 mL of DMF, and finally Cs₂CO₃ (123 mg, 0.378 mmol) were added. The mixture was protected from light by nitrogen and stirred at 55 °C for 16 h. The reaction was monitored by TLC until completion. The reaction solution was added to 20 mL of ice water and stirred. A brownish-yellow solid precipitated out. After filtration, the solid was purified by column chromatography to obtain 55 mg of the yellow solid, which was compound AC-26, with a yield of 46.57%. HRMS (ESI) m / z: [M+H] + Calcd for C 28 H 27 N3O4:470.2074,Found:470.2073.

[0188] Example 27: Assay of the activity of the test compound against NCI-H460 cells

[0189] 1) Preparation of experimental materials

[0190] a. Cell model: Human non-small cell lung cancer cells NCI-H460 were cultured in RPMI-1640 high glucose medium (Gibco) containing 10% fetal bovine serum (Hyclone), supplemented with 2 mM L-glutamine, 100 U / mL penicillin and 100 μg / mL streptomycin, and routinely passaged in a 37℃, 5% CO2 incubator. Cells were then dissociated using 0.25% trypsin-EDTA digestion solution (Gibco).

[0191] b. Compound processing system: Accurately weigh 2.0 mg of the test compound and prepare a 10 mM stock solution using DMSO (from Sigma) as the solvent. Before use, serially dilute with serum-free RPMI-1640 to a final concentration of 100 μM (controlling the DMSO volume fraction ≤0.3%), aliquot, and store at -20℃ for later use.

[0192] c. Test reagents: MTT stock solution: Thiazol blue (from Sigma) was dissolved in PBS (pH 7.4) to prepare a 5 mg / mL solution, which was sterilized by 0.22 μm filter membrane and then aliquoted and frozen.

[0193] 2) Experimental Procedure

[0194] The MTT assay was used to evaluate the inhibitory effect of the compound on the proliferation of tumor cell lines. The specific procedure is as follows:

[0195] a. Standardized cell seeding: Collect cells in the logarithmic growth phase, digest with trypsin, prepare a single-cell suspension, and quantitatively adjust the cell density to 8.0 × 10⁻⁶ using a hemocytometer. 4 180 μL of suspension was seeded per well of a 96-well plate (4.0 × 10⁶ cells / mL). 3 Pre-culture the cells at 37°C for 6 hours to ensure complete cell adhesion.

[0196] b. Drug intervention: Experimental group: fresh culture medium containing a gradient concentration of compounds (6 concentration gradients); Control group: equal volume of culture medium containing 0.3% DMSO. Each group had 3 technical replicates, and the intervention was carried out for 72 hours under dark conditions.

[0197] c. MTT colorimetric analysis and detection: 4 hours before termination of culture, add 20 μL MTT solution (final concentration 0.5 mg / mL) to each well, carefully aspirate the culture medium, and then add 150 μL DMSO with shaking to dissolve the formazan crystals. Measure the OD value at 540 nm using a microplate reader.

[0198] d. Data analysis model:

[0199] Cell proliferation inhibition rate is calculated using the formula:

[0200] Inhibition rate (%) = [(OD 540 Reference Hole - OD 540 Dosage port / OD 540 [Reference Hole] × 100%

[0201] The dose-response curve was fitted using GraphPad Prism software, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ).

[0202] See the table for specific data:

[0203] Table 1. Inhibition of NCI-H460 cell proliferation by the compounds of the present invention.

[0204]

[0205]

[0206] Note: NCI-H460 is a human lung cancer cell line, and punabulin is a positive control. The control group contains DMSO without the sample, and the blank group contains neither DMSO nor the sample.

[0207] Table 1 shows that compounds AC-15, AC-24, and AC-25 exhibited significantly better activity than punabulin in inhibiting the proliferation of NCI-H460 lung cancer cells. Compounds AC-13, AC-17, AC-18, AC-19, AC-21, AC-22, AC-23, and AC-26 also possessed certain antitumor activity.

[0208] Example 28: Testing the biological activity of the test compound on various cell types

[0209] 1) Preparation of experimental materials

[0210] a. Cell models: Human pancreatic cancer cells PANC-1, human colon cancer cells HCT116, human liver cancer cells Hep3B, and breast cancer cells T-47D were cultured in RPMI-1640 high-glucose medium (Gibco) containing 10% fetal bovine serum (Hyclone), supplemented with 2 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin. The cells were routinely passaged in a 37℃, 5% CO2 incubator, and dissociated using 0.25% trypsin-EDTA digestion solution (Gibco).

[0211] b. Compound processing system: Accurately weigh 2.0 mg of the test compound and prepare a 10 mM stock solution using DMSO (from Sigma) as the solvent. Before use, serially dilute with serum-free RPMI-1640 to a final concentration of 100 μM (controlling the DMSO volume fraction ≤0.3%), aliquot, and store at -20℃ for later use.

[0212] c. Test reagents: MTT stock solution: Thiazol blue (from Sigma) was dissolved in PBS (pH 7.4) to prepare a 5 mg / mL solution, which was sterilized by 0.22 μm filter membrane and then aliquoted and frozen.

[0213] 2) Experimental Procedure

[0214] The MTT assay was used to evaluate the inhibitory effect of the compound on the proliferation of tumor cell lines. The specific procedure is as follows:

[0215] a. Standardized cell seeding: Collect cells in the logarithmic growth phase, digest with trypsin, prepare a single-cell suspension, and quantitatively adjust the cell density to 8.0 × 10⁻⁶ using a hemocytometer. 4 180 μL of suspension was seeded per well of a 96-well plate (4.0 × 10⁶ cells / mL). 3 Pre-culture the cells at 37°C for 6 hours to ensure complete cell adhesion.

[0216] b. Drug intervention: Experimental group: fresh culture medium containing a gradient concentration of compounds (6 concentration gradients); Control group: equal volume of culture medium containing 0.3% DMSO. Each group had 3 technical replicates, and the intervention was carried out for 72 hours under dark conditions.

[0217] c. MTT colorimetric analysis and detection: 4 hours before termination of culture, add 20 μL MTT solution (final concentration 0.5 mg / mL) to each well, carefully aspirate the culture medium, and then add 150 μL DMSO with shaking to dissolve the formazan crystals. Measure the OD value at 540 nm using a microplate reader.

[0218] d. Data analysis model:

[0219] Cell proliferation inhibition rate is calculated using the formula:

[0220] Inhibition rate (%) = [(OD 540 Reference Hole - OD 540 Dosage port / OD 540 [Reference Hole] × 100%

[0221] The dose-response curve was fitted using GraphPadPrism software, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ).

[0222] See Table 2 for specific data:

[0223] Table 2. Inhibition of the proliferation of PANC-1, HCT116, Hep3B and T-47D cells by the compounds of the present invention.

[0224]

[0225] Note: PANC-1 is human pancreatic cancer cells, HCT116 is human colon cancer cells, Hep3B is human liver cancer cells, T-47D is breast cancer cells, and ponabulin is a positive control. The control group contains DMSO without the sample, and the blank group contains neither DMSO nor the sample.

[0226] Table 2 shows that compounds AC-13, AC-15, AC-23, AC-24, AC-25, and AC-26 exhibited significantly better activity than punabulin in inhibiting the proliferation of different tumor cells. Preliminary studies on the solubility and bioavailability of the compounds of this invention also showed that compounds AC-13, AC-15, AC-23, AC-24, AC-25, and AC-26 had better lipophilicity and bioavailability than punabulin.

[0227] Example 29: Microtubule immunofluorescence assay of compound AC-15

[0228] Experimental steps:

[0229] 1) Slide inoculation: Sterile coverslips are placed inside 12-well cell culture plates and NCI-H460 cells are inoculated.

[0230] 2) Drug treatment: After adding the test drug to the logarithmic growth phase cells for 24 hours, the cells were collected, the drug solution was discarded, and the cells were washed twice with pre-cooled PBS (5 min / time).

[0231] 3) Cell fixation and permeabilization: Fix with 4% paraformaldehyde at room temperature for 20 min, then wash three times with PBS (5 min each time). Permeabilize with 0.5% Triton X-100 for 15 min (to enhance antibody permeability), then wash three times with PBS (5 min each time), incubate with 0.1% Tween for 5 min, and then rinse twice with PBS (5 min each time).

[0232] 4) Non-specific binding blocking: Add 5% FBS to cover the glass slide, block in a humidified chamber at 37°C for 60 min, and rinse with PBS to remove excess blocking solution.

[0233] 5) Antibody incubation system: For primary antibody reaction, dilute the primary antibody 1:100 with blocking buffer, incubate 10-20 μL / piece in a humidified chamber at 4°C overnight, and wash three times with PBS; for secondary antibody labeling, dilute the fluorescent secondary antibody 1:100 under light-protected conditions, incubate at 37°C for 60 min, and wash three times with PBS after each antibody incubation (5 min / time).

[0234] 6) Nuclear staining and mounting: Stain cell nuclei with DAPI in the dark, and seal the edges of the slide with nail polish as a mounting medium.

[0235] 7) Microscopic examination and acquisition: Observe the slides with a camera-mounted fluorescence microscope and acquire images.

[0236] Results and Analysis: The core mechanism of action of microtubule inhibitors lies in interfering with the dynamic assembly process of cellular microtubules, thereby affecting microtubule-dependent physiological activities by inhibiting spindle formation during mitosis and cytoskeleton function. In this experiment, lung cancer NCI-H460 cells were used as a model. Plinabulin (14.5 nM) and compound AC-15 (12.5 nM) were treated for 24 h. Cells were then stained with DAPI (blue fluorescent labeling of cell nuclei) and β-tubulin antibody (green fluorescent labeling of microtubules). Merge represents the overlap of DAPI and β-tubulin fluorescence. Figure 1 Lung cancer NCI-H460 cells were treated with Plinabulin (72.5 nM) and its derivative AC-15 (62.5 nM) for 24 h, followed by staining with DAPI (blue fluorescent labeling of cell nuclei) and β-tubulin antibody (green fluorescent labeling of microtubules). Merge represents the overlap of DAPI and β-tubulin fluorescence. Figure 2 ).

[0237] The test compound AC-15 and the control drug Plinabulin were compared at IC5 50 Concentrations (AC-15: 12.5 nM; Plinabulin: 14.5 nM, see...) Figure 1 ) and 5 times IC 50 Concentrations (AC-15: 62.5 nM; Plinabulin: 72.5 nM, see...) Figure 2 The microtubule inhibition effect was observed under these conditions. Fluorescence microscopy (blue-stained nuclei, green-labeled tubulin distribution) showed that untreated cells exhibited typical regular morphology, intact nuclear structure, and uniform fluorescence, with the microtubule network arranged radially in an orderly manner. In the high-concentration treatment group, cell morphology significantly changed to irregular round shapes, nuclear membrane integrity was disrupted, and the radial structure of microtubules disintegrated and its density sharply decreased, showing obvious shrinkage. In the low-concentration group, some nuclear morphology was observed to be disrupted, but no significant changes in microtubule structure were found. The experiment confirmed that both compounds AC-15 and plinabulin significantly reduced microtubule fluorescence intensity, suggesting that both can effectively inhibit the microtubule polymerization process and disrupt its normal network, with the high-concentration group showing a particularly significant effect.

[0238] Example 30: Experiment on the effect of compound AC-15 on cell cycle distribution

[0239] The cell cycle is the cyclical process by which cells complete one division and initiate the next, consisting of G1 phase (pre-DNA replication preparation), S phase (DNA synthesis), G2 phase (pre-division check phase), and M phase (mitosis phase, chromosome separation dependent on spindle traction). Microtubule inhibitors disrupt microtubule homeostasis, interfering with spindle assembly and function, thereby blocking tumor cell proliferation. This experiment uses specific fluorescent staining of DNA with propidium iodide (PI), followed by flow cytometry analysis of DNA content to perform cell cycle analysis. In this experiment, NCI-H460 cells were treated with compounds AC-15 (62.5 nM) and plinabulin (72.5 nM), and the flow cytometry results (e.g., ...) were analyzed. Figure 3 The results showed that both significantly induced G2 / M phase arrest, with the AC-15 group achieving an arrest rate of 74.34%, significantly higher than the 59.49% in the plinabulin group. This indicates that at the same concentration, the derivative AC-15 has a stronger cell cycle arrest effect. This phenomenon is consistent with the structural damage results induced by high drug concentrations in microtubule immunofluorescence, further verifying the mechanism by which the derivative AC-15 interferes with mitotic processes by inhibiting microtubule function.

[0240] Summarizing the results of the activity studies and preliminary pharmaceutical studies on the inhibitory effects of the compounds of this invention on human non-small cell lung cancer cells NCI-H460, human pancreatic cancer cells PANC-1, human colon cancer cells HCT116, human liver cancer cells Hep3B, and breast cancer cells T-47D, it can be found that when R2 in the compound shown in formula (I) of this invention is fluorine-substituted and R3 is a substituted pyridine or a substituted pyrazine, the compound of this invention can have better anticancer activity than Plinabulin. In particular, when 5-butoxypyridine is used, the compound of this invention has better lipophilicity and bioavailability. Among them, compound AC-15 has a stronger inhibitory effect on microtubule polymerization than Plinabulin and has better development prospects.

[0241] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions claimed by the present invention.

Claims

1. A class of 2,5-piperazindione compounds with antitumor activity, characterized in that, The compound is: (3Z,6Z)-3-(3-(p-methoxyphenoxy)benzylmethyl)-6-((5-n-butoxypyridin-2-yl)methylene)piperazine-2,5-dione 2. A pharmaceutical composition, characterized in that, The compound of claim 1 or a pharmaceutically acceptable salt thereof, comprising a therapeutically effective dose, and a pharmaceutically acceptable carrier.

3. The use of the compound of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for the prevention and / or treatment of cancer, characterized in that, The cancer mentioned is lung cancer, liver cancer, pancreatic cancer, breast cancer, or colon cancer.

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