Preparation method and application of tubulin inhibitor pralnacasan isomer impurity

Abstract

This invention discloses a method for preparing an isomer of the microtubule inhibitor punabulin and its application, belonging to the field of medicinal chemistry. The method involves irradiating (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione until the reaction reaches equilibrium. After recrystallization and purification in a mixed solvent, the impurity with the 6Z-converted 6E configuration is obtained. This impurity is crucial in the quality research of punabulin, a drug for treating antitumor diseases and neutropenia, and based on its relevant properties, it can be used as an impurity control.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology and relates to a method for preparing (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione, an isomer of the microtubule inhibitor punabulin, and its application. Background Technology

[0002] Plinabulin is a diketopiperazine-based microtubule inhibitor synthesized based on the structure of Phenylahistin, a natural product isolated from marine Aspergillus pyrophorus, through structure-activity relationship studies. Plinabulin is a microtubule inhibitor that acts at the site between α and β microtubule subunits, its site of action being similar to that of colchicine, but not located in the groove of the colchicine binding site. Phase I clinical trials have shown that plinabulin possesses favorable pharmacodynamic, pharmacokinetic, and safety properties. Combined use of plinabulin and docetaxel significantly improves the bioavailability of plinabulin without interference between the two. The recommended optimal dose for Phase II clinical trials is 30 mg / m². 2 Plinabulin with 75mg / m 2 Docetaxel, used in combination with other drugs, is used to treat non-small cell lung cancer. A global multicenter phase III clinical trial was initiated in China in 2015. Currently, Wanchun Pharmaceutical, the company developing the drug, is actively pursuing dual NDA applications for ponabulin in both China and the US. Meanwhile, ponabulin has shown good efficacy in treating neutropenia (CIN) in clinical trials and received Breakthrough Therapy designation from both the China NMPA and the US FDA in September 2020.

[0003] The original patent WO2005 / 07790 describes the synthesis process of Punabulin. During the final condensation reaction, a double-bonded isomer is generated at the benzene ring position. This isomer impurity affects the purity of the product. Since the amount of this isomer impurity generated is not large, it is difficult to obtain a high-purity, large-quantity isomer impurity as a reference standard. Furthermore, because the isomer impurity has a similar structure and polarity to the product molecule and poor solubility, silica gel thin-layer chromatography reveals that the isomer tails and its Rf value overlaps with the product, making separation by silica gel column chromatography impossible. However, quality studies require close attention to impurity research. Therefore, preparing a large-scale isomer impurity reference standard is a key point in impurity research and quality analysis of this product. The method of this invention effectively solves this problem. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione, an isomer of punabulin, and its application. Specifically, it relates to a method for preparing and purifying high-purity impurities, yielding products with a purity greater than 95%, and even compounds with a purity of over 99.0%, which can be used as a reference standard for the study of punabulin impurities.

[0005] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0006] Punabulin isomer impurity (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzyl)-2,5-piperazinedion I and its pharmaceutically acceptable salts, characterized in that impurity I has the following structure:

[0007]

[0008] This invention also provides a method for preparing the isomer impurity of punabulin (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazindione I, comprising the following steps: using (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazindione as a raw material, the reaction is carried out in organic solvent A under irradiation with a light source of 200-450 nm. After the reaction reaches equilibrium, the solution is concentrated to obtain a yellow solid, which is then recrystallized with water using organic solvent B to obtain (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazindione.

[0009] Furthermore, in the above technical solution, organic solvent A is selected from one or more of methanol, ethanol, acetonitrile, acetone, tetrahydrofuran, dioxane, DMF, or DMA.

[0010] Furthermore, in the above technical solution, organic solvent A is selected from acetone, tetrahydrofuran, or a mixture of both.

[0011] Furthermore, in the above technical solution, the raw materials are prepared in an organic solvent to a concentration of 4.0-50.0 mg / mL.

[0012] Furthermore, in the above technical solution, the concentration is 4.0-30.0 mg / mL.

[0013] Furthermore, in the above technical solution, the wavelength range of the light source is 320-420nm.

[0014] Furthermore, in the above technical solution, the organic solvent B is selected from one or more of acetonitrile, methanol, and dioxane, and the HPLC purity of the product after recrystallization is >95.0%.

[0015] Furthermore, in the above technical solution, the organic solvent B is selected from acetonitrile, and the HPLC purity of the product after recrystallization is >99.0%.

[0016] This invention also provides the application of the isomer of punabulin, impurity I, and its pharmaceutically acceptable salt as impurity reference standards. Because this isomer and its pharmaceutically acceptable salt have high purity, they are suitable for use as impurity reference standards.

[0017] Studies have shown that the antitumor activity of the isomer impurities of punabulin is inferior to that of punabulin itself. However, they have significant advantages as impurity research controls, in process control of preparation, and in safety evaluation during the quality research of punabulin.

[0018] Beneficial effects of the invention

[0019] To address the difficulty in obtaining and purifying isomer impurity reference standards in Plinabulin-related matter research, this invention provides a method for preparing and purifying Plinabulin isomer impurity I and its pharmaceutically acceptable salt. The photo-irradiation method and recrystallization solvent used in this method were obtained through extensive experimental screening and have the advantages of readily available photo-irradiation wavelengths and solvents, simple operation, high yield, and good reproducibility.

[0020] The combination of recrystallization solvents selected by this method can avoid solvent loss caused by multiple crystallizations, and the purity of the product is greater than 95.0%. It can even prepare isomer impurities with a purity of more than 99.0%. It can be used as a reference for impurity research in the quality research process of punabulin, for process control of preparation process and safety evaluation.

[0021] Meanwhile, during the research process of this invention, it was found that increasing the temperature had little effect on the conversion rate of the punabulin isomer impurity. Furthermore, after the light irradiation reached equilibrium, extending the reaction time had little impact on the conversion rate. Additionally, excessively high reaction solution concentrations reduced the conversion rate. Studies have shown that the punabulin isomer impurity has lower antitumor activity than punabulin itself, and therefore has significant advantages as an impurity control standard in punabulin quality research, for process control during preparation, and for safety evaluation. Attached Figure Description

[0022] Figure 1 The HPLC chromatogram of (3Z,6Z)-3-benzylmethyl-6-((5-tert-butyl-1H-imidazol-4-yl)methylene)piperazine-2,5-dione (Plinabulin) in Example 1 is shown below.

[0023] Figure 2 The HPLC chromatogram of (3Z,6E)-3-benzylmethylene-6-((5-tert-butyl-1H-imidazol-4-yl)methylene)piperazine-2,5-dione in Example 2;

[0024] Figure 3 The (3Z,6E)-3-benzylmethyl-6-((5-tert-butyl-1H-imidazol-4-yl)methylene)piperazine-2,5-dione in Example 2 1 H NMR spectrum;

[0025] Figure 4 The (3Z,6E)-3-benzylmethyl-6-((5-tert-butyl-1H-imidazol-4-yl)methylene)piperazine-2,5-dione in Example 2 13 C NMR spectrum;

[0026] Figure 5 The mass spectrum of (3Z,6E)-3-benzylmethyl-6-((5-tert-butyl-1H-imidazol-4-yl)methylene)piperazine-2,5-dione in Example 2. Detailed Implementation

[0027] Example 1

[0028] Preparation of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzyl)-2,5-piperazinedione

[0029]

[0030] Under nitrogen protection, 5.0 g (32.85 mmol) of 5-(tert-butyl)-1H-imidazol-4-carboxaldehyde and 35 mL of LDM were added to a reaction flask, followed by 13.02 g (65.7 mmol) of N,N-diacetylpiperazine-2,5-dione and 16.05 g (49.28 mmol) of cesium carbonate. The mixture was stirred at room temperature in the dark for 20 h. The reaction solution was poured into 500 mL of ice water, and the filter cake was washed successively with water (100 mL x 2) and petroleum ether / ethyl acetate 8 / 1 (400 mL). The filter cake was ultrasonically dispersed with ethanol and dichloromethane, and the insoluble matter was filtered off. The mixture was concentrated under reduced pressure, and the water was removed by anhydrous ethanol. Ethyl acetate (200 mL) was slurried to give 4.16 g of a brownish-yellow solid (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione, yield 43.62%. 1H NMR(500MHz, DMSO-d6)δ:12.37(s,1H),12.02(s,1H),7.85(s,1H),7.05(s,1H),4.31(s,2H),2.51(s,3H),1.39(s,9H).

[0031] Under nitrogen protection, 4.16 g (14.33 mmol) of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)deuterium)piperazine-2,5-dione and 60 mL of DMF were added to a reaction flask, along with 1.67 g (15.76 mmol) of benzaldehyde and 7.0 g (21.50 mmol) of cesium carbonate. The reaction was carried out at 50 °C in the dark for 8 hours. The reaction solution was poured into 200 mL of ice water, and a yellow solid was produced. The mixture was filtered, and the filter cake was washed with 100 mL of water, slurried with 50 mL of ethyl acetate, filtered, and dried to obtain 4.50 g of yellow solid, with a yield of 84.43%. HPLC analysis at 365 nm showed a purity of 99.74%, of which the isomer (3E,6Z)-3-benzylmethyl-6-(5-tert-butyl-1H-imidazol-4-yl)deuterium)piperazine-2,5-dione accounted for 0.09%. 1 HNMR(500MHz,DMSO-d6)δ:12.31(s,1H),12.22(s,1H),10.00(s,1H),7.84(s,1H),7.52(d, J=8Hz,2H),7.39(t,J=8Hz,2H),7.32(t,J=8Hz,1H),6.86(s,1H),6.73(s,1H),1.37(s,9H).

[0032] HPLC method conditions:

[0033] Instrument: High performance liquid chromatograph equipped with a diode array detector.

[0034] Chromatographic column: YMC-Pack Pro C18 250×4.6mm, 5μm.

[0035] Mobile phase A: Acetonitrile solution.

[0036] Mobile phase B: 1% glacial acetic acid aqueous solution.

[0037] Detection wavelengths: 254nm; 365nm.

[0038] Flow rate: 1.0 mL / min.

[0039] Injection volume: 10 μL.

[0040] Column temperature: 30℃.

[0041] Running time: 40 minutes.

[0042] Mobile phase ratio: Mobile phase A / Mobile phase B = 40 / 60.

[0043] Example 2

[0044] Preparation of (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzyl)-2,5-piperazinedione

[0045]

[0046] In a 500 mL glass reaction flask, 1.0 g of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione (99.74% purity) from Example 1 and 100 mL of tetrahydrofuran were added. The mixture was stirred and dissolved at room temperature, and then stirred for 72 hours under a 365 nm UV lamp (300 W). HPLC monitoring was performed during the reaction. The reaction reached equilibrium when 45% of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione remained. The reaction solution was concentrated under reduced pressure to dryness to obtain 1.03 g of a yellow solid. Various solvents were used to crystallize the solid. Some solvents, such as DMF, DMA, and DMSO, are not conducive to crystallization due to their high solubility; others, such as ethyl acetate, dichloromethane, n-hexane, and methyl tert-butyl ether, have poor solubility and are not suitable for crystallization. Several suitable and readily available solvents were screened, and crystallization purification experiments were conducted, as shown in the table below. During the experiments, it was important to avoid light exposure.

[0047]

[0048]

[0049] Methanol, dioxane, and acetonitrile, when used individually, are effective for isomer purification. When mixed with water as a solvent, they can significantly improve the purity of isomer impurities. Acetonitrile showed the best results, with its mixed solvent with water increasing the purity of isomer impurities to over 99.9%, requiring relatively little solvent and achieving a total yield of 31.9%. This compound can be used as an impurity reference standard. 1 HNMR(500MHz,DMSO-d6)δ:12.29(s,1H),12.05(s,1H),10.73(s,1H),7.83(s,1H),7.54(d,J=7 .6Hz,1H),7.28(t,J=7.4Hz,1H),7.23(t,J=7.3Hz,1H),6.79(s,1H),6.52(s,1H),1.38(s,9H); 13CNMR(125MHz,DMSO-d6)δ:157.36,155.40,139.96,134.34,134.28,130.67,130.27 ,127.38,127.32,127.16,123.76,119.72,31.88,30.62; MS(ESI):m / z337.17[M+H] + .

[0050] Example 3

[0051] Preparation of (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzyl)-2,5-piperazinedione

[0052] Weigh 300 mg of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione (99.74% purity) into a 100 mL glass reaction flask, add 50 mL of acetonitrile, stir to dissolve at room temperature, and stir for 72 hours under a 365 nm UV lamp (300 W) in a black, sealed environment. The reaction is monitored by HPLC. The remaining 43% of 1H-imidazol-4-yl)methylene]-6-(benzyl)-2,5-piperazinedione was concentrated under reduced pressure to dryness to obtain a yellow solid. The yellow solid was dissolved in 100 mL of acetonitrile under reflux. While maintaining the temperature, 50 mL of purified water was added dropwise. After the addition was complete, the mixture was kept at room temperature and stirred for 3 hours to allow crystallization. A pale yellow solid precipitated out. The solid was filtered and dried to obtain 93 mg of pale yellow solid, with a yield of 31%. The purity of the product was 99.87% as determined by HPLC.

[0053] Example 4

[0054] Preparation of (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzyl)-2,5-piperazinedione

[0055] Weigh 300 mg of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione (99.74% purity) into a 100 mL glass reaction flask, add 60 mL of acetone, stir to dissolve at room temperature, and stir for 72 hours under a 365 nm UV lamp (300 W) in a black, sealed environment. During the reaction, samples are taken for HPLC monitoring. The remaining 56% of the H-imidazol-4-yl)methylene]-6-(benzyl)-2,5-piperazinedione was concentrated under reduced pressure to dryness, yielding a yellow solid. The yellow solid was dissolved in 100 mL of acetonitrile under reflux. While maintaining the temperature, 50 mL of purified water was added dropwise. After the addition was complete, the mixture was kept in the same position for 10 min, and then allowed to cool naturally to room temperature with stirring for 3 hours to allow crystallization. A pale yellow solid precipitated out. The solid was filtered and dried to obtain 96.5 mg of pale yellow solid, with a yield of 32.1%. The purity of the product was 98.73% as determined by HPLC.

[0056] Example 5

[0057] Preparation of (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzyl)-2,5-piperazinedione

[0058] Weigh 300 mg of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione (99.74% purity) into a 100 mL glass reaction flask. Add 60 mL of 1,4-dioxane and stir to dissolve at room temperature. In a black, sealed environment, stir for 72 hours under 365 nm UV light (300 W). During the reaction, samples are taken for HPLC monitoring. The remaining 52% of the reaction solution was concentrated under reduced pressure to dryness to obtain a yellow solid. The yellow solid was dissolved in 100 mL of acetonitrile under reflux. While maintaining the temperature, 50 mL of purified water was added dropwise. After the addition was complete, the mixture was allowed to cool naturally to room temperature and stirred for 3 hours to allow crystallization. A pale yellow solid precipitated out. The solid was filtered and dried to obtain 100.2 mg of pale yellow solid, with a yield of 33.4%. HPLC analysis showed the product purity to be 98.34%.

[0059] Example 6

[0060] Preparation of (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzyl)-2,5-piperazinedione

[0061] Weigh 300 mg of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione (99.74% purity) into a 100 mL glass reaction flask. Add 60 mL of 1,4-dioxane and stir to dissolve at room temperature. In a black, sealed environment, stir for 120 hours under 254 nm UV light (300 W). During the reaction, samples are taken for HPLC monitoring of the reaction (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione. The remaining 61% of the reaction solution was concentrated under reduced pressure to dryness to obtain a yellow solid. The yellow solid was dissolved in 100 mL of acetonitrile under reflux. While maintaining the temperature, 50 mL of purified water was added dropwise. After the addition was completed, the mixture was allowed to cool naturally to room temperature and stirred for 3 hours to crystallize. A pale yellow solid precipitated out. The solid was filtered and dried to obtain 98.3 mg of pale yellow solid, with a yield of 32.8%. The purity of the product was 95.82% as determined by HPLC.

[0062] Example 7

[0063] Reactivity test of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzyl)-2,5-piperazindione under yellow light.

[0064] 50 mg of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione with a purity of 99.74% was weighed and added to a 25 mL glass reaction flask. 10 mL of tetrahydrofuran was added, and the mixture was stirred and dissolved at room temperature. The mixture was then stirred for 72 hours under a 589.0 nm yellow light lamp (300 W) in a black, sealed environment. HPLC analysis of the sample showed that the proportion of (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione was 2.13%, indicating that Plinabulin is sensitive to yellow light, but the conversion rate is low.

[0065] Example 8

[0066] Reactivity test of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazindione under visible light.

[0067] 50 mg of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione with a purity of 99.74% was weighed and added to a 25 mL glass reaction flask. 10 mL of tetrahydrofuran was added, and the mixture was stirred and dissolved at room temperature. The mixture was then stirred for 72 hours under a 300 W fluorescent lamp atmosphere. The sample was analyzed by HPLC. The proportion of (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione was 18.7%, indicating that Plinabulin is sensitive to visible light, but the conversion rate is low.

[0068] Example 9

[0069] Reactivity test of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazindione under light-protected conditions.

[0070] 50 mg of (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione with a purity of 99.74% was weighed and added to a 25 mL glass reaction flask. 10 mL of tetrahydrofuran was added, and the mixture was stirred to dissolve at room temperature. The mixture was stirred for 72 hours in the dark. HPLC analysis showed that the proportion of (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione was 0.10%, indicating that the Plinabulin solution was relatively stable under light-protected conditions and could not be converted into isomers.

[0071] Example 10

[0072] NCI-H460 cell proliferation inhibition assay

[0073] 1) Cell culture and preparation of test compounds

[0074] Human large cell lung cancer cells (NCI-H460) were cultured in RPMI-1640 medium containing 10% heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, 100 U / mL penicillin, and 100 g / mL streptomycin at 37°C in a 5% CO2 cell culture incubator. The medium was changed every two days. After the cells reached 80% confluence, they were trypsinized, passaged, and kept in good logarithmic growth phase. All test samples were dissolved in DMSO.

[0075] 2) Detection method:

[0076] NCI-H460 cells in logarithmic growth phase were seeded at 5000 cells / well (180 μl / well) in 96-well plates and cultured for 24 h. Then, the test sample (final concentration as shown in the table) was added, with three replicates per sample. The amount of DMSO used in the solvent control group was calculated based on the maximum dose used in the test group. After 72 h of drug treatment, cells were fixed in 50% (m / v) ice-cold trichloroacetic acid (TCA) per well. After SRB staining, 150 μl / well of Tris solution was added, and the OD value at 540 nm was measured using a microplate reader. The IC50 of the tested compound on the NCI-H460 cell line was calculated. 50 Values. Test data is as follows:

[0077] Table 1. Inhibition of NCI-H460 cell proliferation by the compounds of the present invention.

[0078]

[0079] Note: NCI-H460 is a human lung cancer cell line, and Plinabulin is a positive control. The control group received no DMSO sample; the blank group received neither DMSO nor the sample.

[0080] Table 1 shows that compound PLN-A has significantly lower activity than Plinabulin in inhibiting the proliferation of NCI-H460 lung cancer cells.

[0081] Example 11

[0082] BXPC-3 cell proliferation inhibition assay

[0083] The inhibition rate of the compound of the present invention on the growth of pancreatic cancer BXPC-3 cells was determined by SRB analysis.

[0084] 1) Cell culture and preparation of test compounds

[0085] Human pancreatic cancer cells BXPC-3 were cultured in DMEM medium containing 10% heat-inactivated FBS (fetal bovine serum), 2 mM L-glutamine, 100 U / mL penicillin, and 100 g / mL streptomycin in a 37°C, 5% CO2 cell culture incubator. The medium was changed every two days. After 80% confluence, the cells were trypsinized, passaged, and maintained in a good logarithmic growth phase. All test samples were dissolved in DMSO and sterilized by 0.22 μm filtration.

[0086] 2) Detection method:

[0087] BXPC-3 cells in logarithmic growth phase were seeded at 4,000 cells / well (180 μl / well) in 96-well plates. After 24 h of culture, different concentrations of the test sample (final concentrations of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.12 nM, and 1.56 nM) were added, with four replicates for each concentration. The solvent control group used 0.1% of the maximum dose used in the test group. After 72 h of drug treatment, cells were fixed in 50% (m / v) ice-cold trichloroacetic acid (TCA) in each well. After SRB staining, 150 μl / well of Tris solution was added, and the OD value at 540 nm was measured using a microplate reader. The IC50 of the tested compound on the BXPC-3 cell line was calculated. 50 Value. The test results are as follows:

[0088] Table 2. Inhibition of BXPC-3 cell proliferation by the compounds of the present invention.

[0089]

[0090] Note: BxPC-3 is a human pancreatic cancer cell line. Plinabulin is the positive control. The control group is without DMSO sample. The blank group is without DMSO and sample.

[0091] Table 2 shows that compound PLN-A is significantly less effective than Plinabulin in inhibiting the proliferation of BXPC-3 pancreatic cancer cells.

[0092] The results of the activity studies and preliminary pharmaceutical studies on the inhibitory effects of the compound of this invention on BXPC-3 pancreatic cancer cells and NCI-H460 lung cancer cells revealed that the Plinabulin isomer impurity exhibits significantly lower antitumor activity than Plinabulin itself. Therefore, controlling the content of this isomer impurity and conducting structural confirmation and detailed studies are essential to better ensure the quality of Plinabulin.

[0093] 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 method for preparing (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazinedione, the structure of which is as follows: characterized in that The preparation method includes the following steps: using (3Z,6Z)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazindione as raw material, the reaction is carried out in organic solvent A under irradiation with a light source of 200-450 nm. After the reaction reaches equilibrium, the solution is concentrated to obtain a yellow solid, which is then recrystallized with water using organic solvent B to obtain (3Z,6E)-3-[(5-tert-butyl-1H-imidazol-4-yl)methylene]-6-(benzylmethyl)-2,5-piperazindione.

2. The preparation method according to claim 1, characterized in that: Organic solvent A is selected from one or more of methanol, ethanol, acetonitrile, acetone, tetrahydrofuran, dioxane, DMF, or DMA.

3. The method of claim 1, wherein: Organic solvent A is selected from acetone, tetrahydrofuran, or a mixture of both.

4. The method of claim 1, wherein: The raw materials are prepared in organic solvents at a concentration of 4.0-50.0 mg / mL.

5. The method of claim 1, wherein: The raw materials are prepared in organic solvents at a concentration of 4.0-30.0 mg / mL.

6. The method of claim 1, wherein: The wavelength range of the light source is 320-420nm.

7. The method of claim 1, wherein: Organic solvent B is selected from one or more of acetonitrile, methanol, and dioxane. After recrystallization, the product has an HPLC purity >95.0%.

8. The method of claim 1, wherein: Organic solvent B is selected from acetonitrile, and the HPLC purity of the product after recrystallization is >99.0%.

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