Ponatinib derivatives, processes for their preparation and uses thereof

Abstract

The application provides a derivative of plinabulin with the structure of formula (I), which is used for anti-tumor, preventing chemotherapy-induced neutropenia and preventing chemotherapy-induced myelosuppression. In the field of cancer treatment, the two derivatives provided by the application have lower toxic side effects under the similar therapeutic effect of plinabulin, have stronger therapeutic effect and higher therapeutic index under the similar toxic side effects, have similar ability of preventing neutropenia and lower toxic side effects compared with plinabulin, have better therapeutic effect than the combination of the commercialized white blood cell increasing drug Neulasta and plinabulin, and can significantly increase the number of various blood cells when combined with a chemotherapy drug. The derivatives provided by the application have wide application prospects in anti-tumor treatment, prevention of neutropenia and prevention of myelosuppression.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical technology, in particular to a plinabulin derivative, a preparation method and application thereof. BACKGROUND

[0002] Plinabulin developed by Beyondspring Company is a diketopiperazine drug synthesized on the basis of natural product structure isolated from marine pyripyropene. Plinabulin has anti-tumor activity and the ability to prevent chemotherapy-induced neutropenia: as an anti-tumor drug, an international multi-center phase III clinical trial has been completed, and all the research endpoints have been reached; as a white blood cell increasing drug, a phase III clinical experiment has been completed, and a new drug marketing application has been submitted in China and the United States.

[0003] However, in the anti-tumor phase III clinical trial, the first dose of plinabulin is close to the maximum tolerated dose (MTD), and if the vomiting level is greater than one, it is reduced to 2 / 3 of the initial dose from the second administration. In the white blood cell increasing drug clinical experiment, the initial administration dose is directly reduced. It can be seen that plinabulin has a large toxic side effect, and the high dose administration is limited, which affects the treatment effect. Therefore, reducing the toxic side effect of plinabulin and improving the therapeutic index will benefit more cancer patients.

[0004] It has been reported that plinabulin and tubulin are mainly combined through hydrogen bonds formed by nitrogen and oxygen atoms on the imidazole ring and the diketopiperazine ring with the colchicine binding domain, and there is no binding site in the benzene ring region, so the modification is selected in the benzene ring region.

[0005] At the same time, based on the three ring structures of plinabulin, various derivatives are constructed. The modification of the benzene ring region is mostly one or more simple substituent group modifications, such as halogen atoms, hydroxyl groups, amino groups, carboxyl groups, etc. Two types of new structure derivatives of formula (I) are designed in the present application, that is, the imidazole ring, the diketopiperazine ring and the four substituent groups in the benzene ring region of plinabulin are further modified. Two derivatives in which the four substituent groups are all hydrogen atoms are taken as representatives, and their anti-tumor activity, the ability to prevent chemotherapy-induced neutropenia and chemotherapy-induced myelosuppression are studied, expecting to reduce the toxic side effect and improve the therapeutic index. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to provide a plinabulin derivative. The plinabulin derivative provided by the present application can significantly reduce the toxic side effect and has a higher therapeutic index compared with plinabulin under similar therapeutic effects.

[0007] The present application provides a plinabulin derivative of formula (I),

[0008]

[0009] wherein R2, R3, R4 are independently selected from hydrogen, hydrocarbyl, thiohydrocarbyl, oxyhydrocarbyl, halogen, nitro, amino, hydroxyl, carboxyl, ester, amide or a combination of the above functional groups;

[0010] R1 and R5 are independently selected from H or a group of formula (a), and R1 and R5 are different.

[0011] Preferably, the two types of plinabulin derivatives of formula (I) are selected from the group consisting of formula (I-1) or formula (I-2):

[0012]

[0013] The present application provides a preparation method of the plinabulin derivative of formula (I) according to any one of the above technical solutions, comprising the following steps:

[0014] Under the catalysis of cesium carbonate, (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione and benzene dicarboxaldehyde are reacted in N,N-dimethylformamide to obtain the product.

[0015] Preferably, the benzene dicarboxaldehyde is p-benzene dicarboxaldehyde containing R2-R5 functional groups or m-benzene dicarboxaldehyde containing R1-R4 functional groups.

[0016] The present application provides an application of the plinabulin derivative of formula (I) according to any one of the above technical solutions in the preparation of an anti-tumor drug.

[0017] Preferably, the tumor includes one or more of nasal cavity and sinus malignant tumor, nasopharyngeal carcinoma, oral cavity cancer, laryngeal cancer, intracranial tumor, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, large intestine cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullar cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, testicular malignant tumor, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytic cancer, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma and multiple myeloma.

[0018] The present application provides an application of the plinabulin derivative of formula (I) according to any one of the above technical solutions in the preparation of a drug for treating side effects of chemotherapy; the side effects of chemotherapy include neutropenia and myelosuppression.

[0019] Preferably, the drug causing neutropenia is selected from the group consisting of docetaxel, paclitaxel, taxane, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, doxorubicin, epirubicin, daunorubicin, valrubicin, and pharmaceutically acceptable salts thereof.

[0020] The drug causing myelosuppression is selected from the group consisting of docetaxel, paclitaxel, taxane, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, doxorubicin, epirubicin, daunorubicin, valrubicin, fluorouracil, and pharmaceutically acceptable salts thereof.

[0021] Preferably, the drug is a tablet, capsule, granule, oral solution, sustained-release preparation, controlled-release preparation, nano-preparation, or injection.

[0022] The present application provides a drug comprising the above technical solution of any one of the above technical solutions.

[0023] Compared with the prior art, the present application provides a plinabulin derivative of formula (I). The application is used for anti-tumor, preventing chemotherapy-induced neutropenia, and preventing chemotherapy-induced myelosuppression. 1) In the field of cancer treatment: the two derivatives provided by the present application have lower toxic side effects under similar therapeutic effects to plinabulin; under similar toxic side effects, the therapeutic effects are stronger and the therapeutic index is higher; 2) In the aspect of preventing chemotherapy-induced neutropenia: the derivatives have similar abilities to prevent neutropenia to plinabulin, and have lower toxic side effects; when combined with the commercialized white blood cell increasing drug Neulasta, the therapeutic effect is better than that of Neulasta alone and the combination of plinabulin; 3) In the aspect of preventing chemotherapy-induced myelosuppression: when combined with the chemotherapy drug, the derivatives can significantly increase the number of various blood cells (platelets, red blood cells, lymphocytes, neutrophils, and white blood cells). The derivatives provided by the present application have broad application prospects in the aspects of anti-tumor treatment, prevention of neutropenia, and prevention of myelosuppression. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The compound a is obtained in Example 1 of the present application 1 H NMR, 13 C NMR and ESI mass spectrum.

[0025] Figure 2 The compound b is obtained in Example 2 of the present application 1 H NMR, 13 C NMR and ESI mass spectrum.

[0026] Figure 3 The compound c is obtained in Example 3 of the present application 1H NMR and ESI mass spectrum;

[0027] Figure 4 H NMR of the resulting example compound d from Example 4 of the present invention 1 H NMR;

[0028] Figure 5 H NMR of the resulting example compound e from Example 5 of the present invention 1 H NMR;

[0029] Figure 6 H NMR of the resulting example compound f from Example 6 of the present invention 1 H NMR;

[0030] Figure 7 H NMR of the resulting example compound g from Example 7 of the present invention 1 H NMR;

[0031] Figure 8 H NMR of the resulting example compound h from Example 8 of the present invention 1 H NMR;

[0032] Figure 9 H NMR of the resulting example compound i from Example 9 of the present invention 1 H NMR;

[0033] Figure 10 H NMR of the resulting example compound j from Example 10 of the present invention 1 H NMR;

[0034] Figure 11 H NMR of the resulting example compound k from Example 11 of the present invention 1 H NMR;

[0035] Figure 12 H NMR of the resulting example compound 1 from Example 12 of the present invention 1 H NMR;

[0036] Figure 13 H NMR of the resulting example compound m from Example 13 of the present invention 1 H NMR;

[0037] Figure 14 H NMR of the resulting example compound n from Example 14 of the present invention 1 H NMR;

[0038] Figure 15 H NMR of the resulting example compound o from Example 15 of the present invention 1 H NMR;

[0039] Figure 16 H NMR of the resulting example TPAL from Example 16 of the present invention1 H NMR, 13 C NMR, ESI spectrum and high performance liquid chromatogram;

[0040] Figure 17 TPAL, MPAL and Plinabulin microtubule depolymerization experiment (A) and inhibition of microtubule formation experiment (B) for the TPAL obtained in Example 18 of the present application; 1 H NMR, 13 C NMR, ESI spectrum and high performance liquid chromatogram;

[0041] Figure 18 TPAL, MPAL and Plinabulin microtubule depolymerization experiment (A) and inhibition of microtubule formation experiment (B) for the TPAL obtained in Example 18 of the present application;

[0042] Figure 19 TPAL, MPAL and Plinabulin cytotoxicity experiment on 4T1 and HUVECs cells for the TPAL obtained in Example 19 of the present application;

[0043] Figure 20 TPAL and Plinabulin tail vein injection in normal Balb / c mice for the TPAL obtained in Example 20 of the present application, the change of mice weight;

[0044] Figure 21 TPAL, MPAL and Plinabulin treatment of 4T1 tumor-bearing mice for the TPAL obtained in Example 21 of the present application, the change of tumor volume (A) and mice weight (B) of subcutaneous tumor-bearing mice;

[0045] Figure 22 TPAL and Plinabulin treatment of CT26 tumor-bearing mice for the TPAL obtained in Example 22 of the present application, the change of tumor volume (A) and mice weight (B) of subcutaneous tumor-bearing mice;

[0046] Figure 23 TPAL and Plinabulin treatment of H22 tumor-bearing mice for the TPAL obtained in Example 23 of the present application, the change of tumor volume (A) and mice weight (B) of subcutaneous tumor-bearing mice;

[0047] Figure 24 TPAL and Plinabulin treatment of 4T1 tumor-bearing mice for the TPAL obtained in Example 24 of the present application, the change of tumor volume (A) and mice weight (B) of subcutaneous tumor-bearing mice;

[0048] Figure 25Treatment timeline (A), body weight change (B) and absolute neutrophil count (ANC) change (C) after TPAL, MPAL and Plinabulin were administered to prevent doxorubicin-induced neutropenia in Example 25 of the present application;

[0049] Figure 26 Treatment timeline (A), absolute neutrophil count (B) and body weight change (C) after TPAL and Sulfofilgrastim were administered to prevent doxorubicin-induced neutropenia in Example 26 of the present application;

[0050] Figure 27 TPAL was administered to prevent 5-fluorouracil-induced myelosuppression in Example 27 of the present application, and the changes in various blood cells over time (A) and the absolute values of various blood cells in each group on a specific day (B) are shown. DETAILED DESCRIPTION

[0051] The present application provides a Plinabulin derivative, its preparation method and application. Those skilled in the art can refer to the content herein, and appropriately improve the process parameters to achieve. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they all belong to the scope protected by the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0052] Plinabulin is a microtubulin inhibitor, which binds near the colchicine binding site of microtubulin, causing dysfunction of the tumor vasculature. It has a wide anti-tumor spectrum and is used for head and neck squamous cell carcinoma, ovarian cancer, embryonic cancer, seminoma, lung cancer, thyroid cancer, lymphosarcoma and reticulum cell sarcoma, etc.

[0053] The present application provides a Plinabulin derivative of formula (I),

[0054]

[0055] wherein R2, R3, R4 are independently selected from hydrogen, hydrocarbyl, thiohydrocarbyl, oxyhydrocarbyl, halohydrocarbyl, halogen, nitro, amino, hydroxyl, carboxyl, ester, amido or a combination of the above functional groups;

[0056] R1 and R5 are independently selected from H or a group represented by formula (a), and R1 and R5 are different.

[0057] In the present application, the two types of Plinabulin derivatives of formula (I) are selected from the structures represented by formula (I-1) or formula (I-2):

[0058]

[0059] According to the systematic nomenclature, the compound name is: (3Z, 3'Z, 6Z, 6'Z)-6,6'-(1,4-phenylenebis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione), abbreviated as: TPAL.

[0060]

[0061]

[0062] According to the systematic nomenclature, the compound name is: (3Z, 3'Z, 6Z, 6'Z)-6,6'-(1,3-phenylenebis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione), abbreviated as: MPAL.

[0063]

[0064] The present application does not limit the source of raw materials, which can be commercially available or prepared according to the methods disclosed by those skilled in the art.

[0065] The plinabulin derivative described in the present application can destroy the microtubule structure that has been formed and inhibit the formation of microtubules.

[0066] The present application provides a preparation method of the plinabulin derivative of formula (I) according to any one of the above technical solutions, comprising the following steps:

[0067] Under the catalysis of cesium carbonate, (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione and benzene dicarboxaldehyde are reacted in N,N-dimethylformamide, and then the product is obtained.

[0068] The basic catalyst described in the present application can be potassium tert-butoxide, DBU, cesium carbonate and other common basic catalysts, and the most preferred is cesium carbonate.

[0069] The molar ratio of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione and benzene dicarboxaldehyde is 5:1 to 2:1, and the most preferred is 3:1.

[0070] Preferably, the benzene dicarboxaldehyde is p-benzene dicarboxaldehyde containing R2-R5 functional groups or m-benzene dicarboxaldehyde containing R1-R4 functional groups.

[0071] The application provides application of the ponatinib derivative with the structure of formula (I) in any one of the above technical solutions in preparation of an anti-tumor drug.

[0072] The tumor includes one or more of nasal cavity and sinus malignant tumor, nasopharyngeal carcinoma, oral cavity cancer, laryngeal cancer, intracranial tumor, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, large intestine cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, testicular malignant tumor, penis cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytic cancer, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma and multiple myeloma.

[0073] The drug is a tablet, a capsule, a granule, an oral liquid, a sustained-release preparation, a controlled-release preparation, a nano preparation or an injection.

[0074] The application provides an anti-tumor drug, which comprises the ponatinib derivative with the structure of formula (I) in any one of the above technical solutions.

[0075] The anti-tumor drug provided by the application comprises the derivative, a salt, a hydrate, a crystal form, an enantiomer, an isomer, a metabolite, a prodrug and a pharmaceutically acceptable excipient of any one of the above technical solutions.

[0076] The application provides application of the ponatinib derivative with the structure of formula (I) in any one of the above technical solutions in preparation of a drug for treating a side effect of chemotherapy.

[0077] The neutropenia includes one or more of a first chemotherapy combination of a chemical therapeutic agent or is induced by administration of radiotherapy.

[0078] Specifically, the drug is administered 1 minute to 24 hours before or after administration of the chemical therapy.

[0079] When the application is used in experiments on rats, the administration dose is preferably 1 mg / kg-100 mg / kg; most preferably, the dose is 3.75 mg / kg.

[0080] The drug of the present application can be used alone or in combination with commercialized white blood cell increasing drugs to improve the effect of preventing chemotherapy-induced neutropenia. Commercialized white blood cell increasing drugs include but are not limited to filgrastim, pegfilgrastim, lenograstim, etc. Preferably, the drug is used in combination with lenograstim to increase the level of neutrophils.

[0081] The myelosuppression of the present application is induced by administration of a first chemotherapy combination comprising one or more chemotherapy drugs or by administration of radiotherapy. The chemotherapy drugs include but are not limited to docetaxel, paclitaxel, taxane, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, doxorubicin, epirubicin, daunorubicin, valrubicin, fluorouracil, etc. and pharmaceutically acceptable salts thereof.

[0082] Specifically, the drug is administered 1 minute to 24 hours before or after administration of the chemotherapy. In the experiment on rats, the dose is preferably 1 mg / kg to 150 mg / kg; most preferably, the dose is 3.75 mg / kg.

[0083] The drug of the present application can be used alone or in combination with commercialized myelosuppression preventing drugs to increase the number of various blood cells.

[0084] Specifically, the drug is in the form of a tablet, a capsule, a granule, an oral solution, a sustained-release preparation, a controlled-release preparation, a nano-preparation or an injection.

[0085] The present application provides a drug comprising the ponatinib derivative of formula (I) according to any one of the above technical solutions.

[0086] The present application provides a drug for treating chemotherapy-induced neutropenia, comprising the ponatinib derivative of formula (I) according to any one of the above technical solutions.

[0087] The present application provides a drug for treating chemotherapy-induced myelosuppression, comprising the ponatinib derivative of formula (I) according to any one of the above technical solutions.

[0088] The dose of the TPAL of the present application is preferably 1 to 150 mg / kg; more preferably, 30 to 120 mg / kg; most preferably, 1 to 30 mg / kg in rats and mice.

[0089] The present application creatively synthesizes various ponatinib derivatives. TPAL and MPAL are representatives thereof, which have the ability to prevent chemotherapy-induced neutropenia and chemotherapy-induced myelosuppression, and have a higher therapeutic index than ponatinib.

[0090] The application is preferably administered at a dose of 60mg / kg to 100mg / kg to mice.

[0091] The application provides two types of plinabulin derivatives of formula (I). The application takes TPAL and MPAL as representatives, in which four substituents are hydrogen atoms, and studies the application in inhibiting tumor growth, preventing chemotherapy-induced neutropenia and preventing chemotherapy-induced myelosuppression. In terms of inhibiting tumor growth, the toxic side effects are lower when the therapeutic effect is similar to plinabulin, and the therapeutic effect is better when the toxic side effects are similar. In terms of preventing neutropenia, the ability to prevent chemotherapy-induced neutropenia is similar to plinabulin, and the toxic side effects are lower. When combined with the commercial white blood cell increasing drug Neulasta, the therapeutic effect is significantly better than that of the Neulasta single drug group and the plinabulin combination group. In terms of preventing chemotherapy-induced myelosuppression, the number of various types of blood cells is significantly increased compared to the single drug of the chemotherapy drug. The plinabulin derivatives provided by the application have a higher therapeutic index in inhibiting tumor growth, preventing chemotherapy-induced neutropenia and preventing chemotherapy-induced myelosuppression, and have a broad application prospect.

[0092] The present application obtains a plurality of Plinabulin bidentate derivatives by chemical synthesis, and mainly selects TPAL and MPAL as representatives to perform cell and animal experiments. (1) Microtubule depolymerization and microtubule formation inhibition experiments find that TPAL and MPAL can both destroy the formed microtubule structure and inhibit microtubule formation, and have the characteristics of a vascular blocking agent. (2) Cell experiments find that the toxicity of TPAL and MPAL to 4T1 and HUVECs cells is lower than that of Plinabulin. (3) In vivo experiments, on a normal Balb / c mouse model, the side effects of TPAL are lower than those of Plinabulin. (4) On a 4T1 model, the tumor inhibition ability of TPAL and MPAL is similar to that of Plinabulin, and the side effects are significantly lower than those of Plinabulin; compared with Plinabulin, when the side effects are similar, the treatment effect of TPAL is significantly enhanced, and the therapeutic index is higher. (5) On a CT26 model, the treatment effect of TPAL is similar to that of Plinabulin, and the side effects are lower, and the therapeutic index is higher. (6) On an H22 model, the treatment effect of TPAL is similar to that of Plinabulin, and the side effects are lower, and the therapeutic index is higher. (7) In an adriamycin-chemotherapied normal rat model, compared with Plinabulin, when the effect of preventing granulocytopenia is similar, the side effects of TPAL and MPAL do not superimpose with the chemotherapeutic drugs, and the therapeutic index is higher. (8) In an adriamycin-chemotherapied normal rat model, the effects of TPAL and Plinabulin combined with commercialized Sulfofilgrastim in preventing granulocytopenia are investigated, and compared with the Sulfofilgrastim single-drug group and the Plinabulin combination group, the treatment effects of the TPAL combination group are significantly improved. (9) In a 5-fluorouracil-chemotherapied normal rat model, the effect of TPAL in preventing myelosuppression is investigated. TPAL can significantly improve various blood cells, and plays a role in preventing myelosuppression. The present application takes the low side effects of Plinabulin derivatives as a breakthrough point, and in the case of similar treatment effects, the side effects are lower, and in the case of similar side effects, the treatment effect is better, and the therapeutic index is higher, which provides a new drug selection for clinical treatment of cancer and prevention of chemotherapy-induced neutropenia, and has a good application prospect.

[0093] In order to further illustrate the present application, the following embodiments are used to describe the Plinabulin derivative, the preparation method and application thereof provided by the present application in detail.

[0094] Example 1 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-dimethoxy-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound a)

[0095] The specific preparation process includes the following steps:

[0096] (1) Preparation of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione

[0097] 1 g (6.5 mmol) of 5-(tert-butyl)-1H-imidazole-4-carboxaldehyde was added to anhydrous 7 mL of DMF, 2.59 g (13 mmol) of N,N-diacetylpiperazine-2,5-dione was added, nitrogen protection was performed three times, 3.19 g (9.8 mmol) of cesium carbonate was added, nitrogen protection was performed three times, the reaction was stirred at room temperature for 20 h in the dark. The reaction liquid was poured into (100 mL) ice water, suction filtration was performed, the filter cake was washed with water (100 mL*2) and petroleum ether: ethyl acetate = 8:1 (90 mL) in sequence, ultrasonic dispersion was performed with ethanol and dichloromethane, the insoluble matter was filtered off, water was removed under reduced pressure, anhydrous ethanol was used to bring water, and then ethyl acetate (50 mL) was used to slurry. 0.89 g of brownish red solid was obtained as the (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione, and the yield was 46.9%.

[0098] 1 H NMR (300 MHz, DMSO-d6) δ 12.38 (s, 1H), 12.02 (s, 1H), 7.86 (d, J = 0.9 Hz, 1H), 7.04 (s, 1H), 4.30 (s, 2H), 2.51 (d, J = 1.8 Hz, 3H), 1.39 (s, 9H).

[0099] (2) Preparation of Compound a

[0100] Into 290 mg (1 mmol) of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione in anhydrous DMF, 64.04 mg (0.33 mmol) of 2,5-dimethoxybenzene-1,4-dicarboxaldehyde was added, and the mixture was degassed three times under nitrogen protection. Then, 108.27 mg (0.33 mmol) of cesium carbonate was added, and the mixture was degassed three times under nitrogen protection. The reaction was heated to 80 °C, and stirred for 48 h in the dark. The reaction solution was poured into 100 mL of ice water, and filtered. The filter cake was washed with water (100 mL*2), petroleum ether: ethyl acetate = 8:1 (90 mL), and then dispersed with ethanol and dichloromethane under ultrasonic. The insoluble matter was filtered off, and the filtrate was distilled under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate, and placed at -30 °C overnight. The mixture was filtered, and the filter cake was washed with ethyl acetate (5 mL) to obtain 114.35 mg of a yellow powder solid.

[0101] 1 H NMR (300 MHz, DMSO-d6) δ 12.36 (s, 2H), 12.27 (s, 2H), 9.98 (s, 2H), 7.87 (s, 2H), 7.15 (s, 2H), 6.84 (d, J = 16.0 Hz, 4H), 3.86 (s, 6H), 1.39 (s, 18H); 13 C NMR (126 MHz, DMSO-d6) δ 157.91, 156.91, 151.26, 141.16, 135.17, 131.51, 127.76, 124.58, 123.64, 114.35, 110.52, 105.94, 56.85, 32.70, 31.41; MS (ESI) m / z 677.73 (M+Na) + (Exact mass of compound a: C 34 H 38 N8O6 654.73). The attribution of each hydrogen atom, the carbon spectrum, and the mass spectrum of compound a and the starting material are shown in Figure 1 , which proves that compound a is successfully synthesized.

[0102] Example 2 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((5-hydroxy-1,3-phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione) (compound b)

[0103] The specific preparation process includes the following steps:

[0104] (1) Preparation of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione

[0105] The specific steps are the same as step (1) in Example 1, which will not be repeated here.

[0106] (2) Preparation of compound b

[0107] Into 290 mg (1 mmol) of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione, 50.01 mg (0.33 mmol) of 5-hydroxyisophthalaldehyde was added, and the mixture was degassed three times under nitrogen protection. Then, 108.27 mg (0.33 mmol) of cesium carbonate was added, and the mixture was degassed three times under nitrogen protection. The temperature was raised to 80°C, and the reaction was stirred in the dark for 48 h. The reaction solution was poured into 100 mL of ice water, and the mixture was filtered under suction. The filter cake was washed with water (100 mL*2) and petroleum ether: ethyl acetate = 8:1 (90 mL) in sequence, and then dispersed with ethanol and dichloromethane under ultrasonic. The insoluble matter was filtered off, and the filtrate was distilled under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate, and the mixture was left to stand at -30°C overnight. The mixture was filtered under suction, and the filter cake was washed with 5 mL of ethyl acetate to obtain 84.78 mg of a yellow powder solid.

[0108] 1 H NMR (300 MHz, DMSO-d6) δ 12.34 (s, 2H), 12.27 (s, 2H), 10.13 (s, 2H), 9.72 (s, 1H), 7.86 (s, 2H), 7.07 (s, 1H), 6.82 (d, J = 18.6 Hz, 4H), 6.70 (s, 2H), 1.38 (s, 18H); 13 C NMR (126 MHz, DMSO-d6) δ 158.31, 158.25, 156.97, 141.13, 135.51, 135.14, 131.48, 127.79, 124.54, 121.45, 116.43, 114.68, 105.87, 32.70, 31.42; MS (ESI) m / z 609.53 (M-H) - (Exact mass of compound b: C 32 H 34 N8O5 610.53). The attribution of each hydrogen atom, the carbon spectrum, and the mass spectrum of compound b and the starting material are shown in Figure 2 Figures 1 and 2, respectively, which prove that compound b is successfully synthesized.

[0109] Example 3 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((5-bromo-1,3-phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione) (Compound c)

[0110] The specific preparation process includes the following steps:

[0111] (1) Preparation of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione

[0112] The specific steps are the same as step (1) in Example 1, which will not be repeated here.

[0113] (2) Preparation of Compound c

[0114] Into 290 mg (1 mmol) of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione in anhydrous DMF, 70.65 mg (0.33 mmol) of 5-bromoisophthalaldehyde was added, and the air was discharged three times. Cesium carbonate 108.27 mg (0.33 mmol) was added, and the air was discharged three times. The temperature was programmed to 80°C, and the reaction was stirred in the dark for 48 h. The reaction solution was poured into (100 mL) ice water, and the filter cake was washed with water (100 mL*2), petroleum ether: ethyl acetate = 8:1 (90 mL) in sequence, and then dispersed with ethanol and dichloromethane under ultrasonic, and the insoluble matter was filtered off. The filtrate was distilled under reduced pressure, and anhydrous ethanol was used to remove water. 50 mL of ethyl acetate was dispersed, and the mixture was placed at -30°C overnight. The filter cake was washed with ice ethyl acetate (5 mL) to obtain 34.19 mg of yellow powder solid.

[0115] 1 H NMR (300 MHz, DMSO-d6) δ 12.33 (d, J = 14.5 Hz, 4H), 10.48 (s, 2H), 7.87 (s, 2H), 7.58 (d, J = 4.9 Hz, 3H), 6.87 (s, 2H), 6.74 (s, 2H), 1.39 (s, 18H); MS (ESI) m / z 671.41 (M-H) - (Compound c has an accurate mass of C 32 H 34 N8O5 672.41). The assignment of each hydrogen atom of Compound c and the mass spectrum are shown in Figure 3 Fig. 1, which proves that Compound c is successfully synthesized.

[0116] Example 4 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2-bromo-1,4-phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione) (Compound d)

[0117] (1) Preparation of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione

[0118] The specific procedure is the same as Step (1) in Example 1, which is not repeated here.

[0119] (2) Preparation of Compound d

[0120] The raw material 5-bromoisophthalaldehyde in Compound c is replaced by 2-bromoparadimaldehyde, and the rest of the raw materials, the molar ratio of the feed, and the post-reaction treatment method remain unchanged.

[0121] 1 H NMR (300 MHz, DMSO-d6) δ 12.36 (s, 4H), 10.47 (s, 2H), 8.11 (s, 2H), 7.89 (d, J = 17.8 Hz, 2H), 7.67 (s, 3H), 6.87 (s, 2H), 6.70 (s, 2H), 1.38 (s, 18H).

[0122] Example 5 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-dichloro-1,4-phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione) (Compound e)

[0123] The raw material 5-bromoisophthalaldehyde in Compound c is replaced by 2,5-dichloroparadimaldehyde, and the rest of the raw materials, the molar ratio of the feed, and the post-reaction treatment method remain unchanged.

[0124] 1 H NMR (300 MHz, DMSO-d6) δ 12.37 (d, J = 8.1 Hz, 4H), 10.61 (s, 2H), 7.88 (s, 2H), 7.72 (s, 2H), 6.89 (s, 2H), 6.67 (s, 2H), 1.39 (s, 18H).

[0125] Example 6 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-bis(hexyloxy)-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound f)

[0126] The raw material 5-bromoisophthalaldehyde in compound c was replaced by 2,5- dihexyloxy-p-phenylenedimethanal, and the rest of the raw materials, the molar ratio of the feed, and the post-reaction treatment method were kept unchanged.

[0127] 1 H NMR (300 MHz, DMSO-d6) δ 12.36 (s, 2H), 12.26 (s, 2H), 9.86 (s, 2H), 7.87 (s, 2H), 7.13 (s, 2H), 6.86 (s, 2H), 6.79 (s, 2H), 4.07 (d, J = 6.5 Hz, 4H), 1.75 (t, J = 7.3 Hz, 4H), 1.38 (s, 18H), 1.27 (s, 8H), 1.23 (s, 4H), 0.83 (d, J = 7.0 Hz, 6H).

[0128] Example 7 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,3,5,6-tetramethyl-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound g)

[0129] The raw material 5-bromoisophthalaldehyde in compound c was replaced by 2,3,5,6- tetramethyl-p-phenylenedimethanal, and the rest of the raw materials, the molar ratio of the feed, and the post-reaction treatment method were kept unchanged.

[0130] 1 H NMR (300 MHz, DMSO-d6) δ 12.35 (s, 4H), 8.65 (s, 2H), 7.88 (s, 2H), 6.87 (d, J = 5.7 Hz, 4H), 2.09 (s, 12H), 1.38 (s, 18H).

[0131] Example 8 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-bis(octyloxy)-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound h)

[0132] The starting material 5-bromoisophthaldehyde in compound c was replaced by 2,5-bis(octyloxy)benzene-1,4-dicarboxyaldehyde, and the rest of the starting materials, the molar ratio of the feed, and the post-reaction treatment method were kept unchanged.

[0133] 1 H NMR (300 MHz, DMSO-d6) δ 12.35 (s, 2H), 12.27 (s, 2H), 9.83 (s, 2H), 7.87 (s, 2H), 7.12 (s, 2H), 6.86 (s, 2H), 6.78 (s, 2H), 4.06 (s, 2H), 4.02 (d, J = 6.9 Hz, 2H), 1.73 (d, J = 7.5 Hz, 4H), 1.38 (s, 18H), 1.27 - 1.11 (m, 20H), 0.88 - 0.73 (m, 6H).

[0134] Example 9 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-bis(prop-2-yn-1-yloxy)-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound i)

[0135] The starting material 5-bromoisophthaldehyde in compound c was replaced by 2,5-bis(prop-2-yn-1-yloxy)benzene-1,4-dicarboxyaldehyde, and the rest of the starting materials, the molar ratio of the feed, and the post-reaction treatment method were kept unchanged.

[0136] 1 H NMR (300 MHz, DMSO-d6) δ 12.33 (d, J = 19.5 Hz, 4H), 9.91 (s, 2H), 7.87 (s, 2H), 7.24 (s, 2H), 6.87 (s, 2H), 6.81 (s, 2H), 4.92 (d, J = 2.4 Hz, 4H), 3.62 (t, J = 2.4 Hz, 2H), 1.39 (s, 18H).

[0137] Example 10 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-bis(heptyloxy)-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound j)

[0138] The starting material 5-bromoisophthaldehyde in compound c was replaced by 2,5-bis(heptyloxy)benzene-1,4-dicarboxyaldehyde, and the rest of the starting materials, the molar ratio of the feed, and the post-reaction treatment method were kept unchanged.

[0139] 1 H NMR (300 MHz, DMSO-d6) δ 12.36 (s, 2H), 12.27 (s, 2H), 9.85 (s, 2H), 7.87 (s, 2H), 7.13 (s, 2H), 6.86 (s, 2H), 6.78 (s, 2H), 4.04 (q, J = 6.6 Hz, 4H), 1.74 (s, 4H), 1.38 (s, 18H), 1.23 (s, 16H), 0.88 - 0.74 (m, 6H).

[0140] Example 11 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-divinyl-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound k)

[0141] The starting material 5-bromoisophthaldehyde in compound c was replaced by 2,5-bis(heptyloxy)benzene-1,4-dicarboxyaldehyde, and the rest of the starting materials, the molar ratio of the feed, and the post-reaction treatment method were kept unchanged.

[0142] 1 H NMR (300 MHz, DMSO-d6) δ 12.36 (s, 2H), 12.27 (s, 2H), 9.85 (s, 2H), 7.87 (s, 2H), 7.13 (s, 2H), 6.86 (s, 2H), 6.78 (s, 2H), 4.04 (q, J = 6.6 Hz, 4H), 1.74 (s, 4H), 1.38 (s, 18H), 1.23 (s, 16H), 0.88 - 0.74 (m, 6H).

[0143] Example 12 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-bis(docosyloxy)-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound 1)

[0144] The raw material 5-bromo isophthalaldehyde in compound c was replaced by 2,5- docosyloxy-1,4-p-phenylenedimethanal, and the other raw materials, molar ratio of feeding, and post-reaction treatment method were kept unchanged.

[0145] 1 H NMR (300 MHz, DMSO-d6) δ 12.35 (s, 2H), 12.27 (s, 2H), 9.82 (s, 2H), 7.86 (s, 2H), 7.13 (s, 2H), 6.86 (s, 2H), 6.72 (s, 2H), 4.06 (s, 4H), 1.74 (s, 2H), 1.38 (s, 18H), 1.23 - 1.16 (d, 72H), 0.82 (t, J = 6.6 Hz, 6H).

[0146] Example 13 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-difluoro-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound m)

[0147] The raw material 5-bromo isophthalaldehyde in compound c was replaced by 2,5- 2F-1,4-p-phenylenedimethanal, and the other raw materials, molar ratio of feeding, and post-reaction treatment method were kept unchanged.

[0148] 1 H NMR (300 MHz, DMSO-d6) δ 12.36 (d, J = 6.9 Hz, 4H), 10.40 (s, 2H), 7.88 (s, 2H), 7.51 (t, J = 8.5 Hz, 2H), 6.89 (s, 2H), 6.65 (s, 2H), 1.39 (t, J = 2.0 Hz, 18H).

[0149] Example 14 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-bis(2-methoxyethoxy)-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound n)

[0150] The raw material 5-bromoisophthaldehyde in compound c was replaced by 2,5-bis(2- methoxyethoxy)-p-phenylenedimethanol, and the other raw materials, the molar ratio of the feed, and the post-reaction treatment method were kept unchanged.

[0151] 1 H NMR (300 MHz, DMSO-d6) δ 12.35 (s, 2H), 12.27 (s, 2H), 9.95 (s, 2H), 7.87 (s, 2H), 7.16 (s, 2H), 6.90 - 6.76 (m, 4H), 4.20 (s, 4H), 3.70 (s, 4H), 3.32 (dd, J = 12.7, 2.2 Hz, 6H), 1.39 (d, J = 2.1 Hz, 18H).

[0152] Example 15 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-((2,5-dipropoxy-1,4- phenylene)bis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4- yl)methylene)piperazine-2,5-dione) (Compound o)

[0153] The raw material 5-bromoisophthaldehyde in compound c was replaced by 2,5-dipropoxy- p-phenylenedimethanol, and the other raw materials, the molar ratio of the feed, and the post- reaction treatment method were kept unchanged.

[0154] 1 H NMR (300 MHz, DMSO-d6) δ 12.35 (s, 2H), 12.27 (s, 2H), 9.95 (s, 2H), 7.87 (s, 2H), 7.16 (s, 2H), 6.90 - 6.76 (m, 4H), 4.20 (s, 4H), 3.70 (s, 4H), 3.32 (dd, J = 12.7, 2.2 Hz, 6H), 1.39 (d, J = 2.1 Hz, 18H).

[0155] Example 16 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-(1,4-phenylenebis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione) (TPAL)

[0156] The specific preparation process includes the following steps:

[0157] (1) Preparation of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione

[0158] The specific steps are the same as step (1) in Example 1, which will not be repeated here.

[0159] (2) Preparation of TPAL

[0160] Add 290 mg (1 mmol) of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione to anhydrous DMF, then add 44.71 mg (0.33 mmol) of p-xylylene glycol, and then remove the air three times under nitrogen protection. Add 108.27 mg (0.33 mmol) of cesium carbonate, remove the air three times under nitrogen protection, and program the temperature to 80°C. Stir the reaction under light protection for 48 h. Pour the reaction liquid into (100 mL) ice water, filter, and then wash the filter cake with water (100 mL*2), petroleum ether: ethyl acetate = 8:1 (90 mL), and then ultrasonically disperse with ethanol and dichloromethane. Filter off the insoluble matter, and then distill under reduced pressure with water. Disperse in 50 mL of ethyl acetate, and then stand at -30°C overnight. Filter, and then wash the filter cake with ice ethyl acetate (5 mL) to obtain 178.35 mg of yellow powder solid, with a yield of 90.9%.

[0161] 1 H NMR (300 MHz, DMSO-d6) δ 12.32 (d, J = 21.6 Hz, 4H), 10.06 (s, 2H), 7.87 (s, 2H), 7.58 (s, 4H), 6.87 (s, 2H), 6.77 (s, 2H), 1.39 (s, 18H); 13 C NMR (126 MHz, DMSO-d6) δ 158.30, 157.01, 141.23, 135.17, 133.69, 131.49, 130.31, 127.72, 124.50, 114.14, 105.99, 32.70, 31.41; MS (ESI) m / z 593.5 (M-H) - (TPAL has an accurate mass of C32 H 34 N8O4 594.5). The retention time t R = 5.68 min was measured by HPLC. The chemical shifts of each hydrogen and carbon of compound TPAL, the mass spectrum and the HPLC spectrum are shown in Figure 16 Fig. 1, which prove that TPAL is successfully synthesized.

[0162] Example 17 Preparation of (3Z,3'Z,6Z,6'Z)-6,6'-(1,3-phenylenebis(methaneylylidene))bis(3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione) (MPAL)

[0163] The specific preparation process includes the following steps:

[0164] (1) Preparation of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione

[0165] The specific steps are the same as step (1) in Example 1, which will not be repeated here.

[0166] (2) Preparation of MPAL

[0167] Add 290 mg (1 mmol) of (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione to anhydrous DMF, then add 44.71 mg (0.33 mmol) of m-xylylene glyoxal, and then perform three times of nitrogen protection and exhaust. Add 108.27 mg (0.33 mmol) of cesium carbonate, and then perform three times of nitrogen protection and exhaust. Program the temperature to 80°C, and then stir the reaction under light protection for 48 h. Pour the reaction liquid into (100 mL) ice water, and then perform suction filtration. Wash the filter cake with water (100 mL*2) and petroleum ether: ethyl acetate = 8:1 (90 mL) in sequence, and then perform ultrasonic dispersion with ethanol and dichloromethane. Filter out the insoluble substances, and then perform vacuum distillation with anhydrous ethanol. Disperse in 50 mL of ethyl acetate, and then stand at -30°C overnight. Perform suction filtration, and then wash the filter cake with ice ethyl acetate (5 mL) to obtain 190.78 mg of yellow powder solid, with a yield of 97.2%.

[0168] 1H NMR (300 MHz, DMSO-d6) δ 12.32 (d, J = 18.5 Hz, 4H), 10.30 (s, 2H), 7.87 (s, 2H), 7.64 (s, 1H), 7.42 (q, J = 5.4 Hz, 3H), 6.86 (s, 2H), 6.80 (s, 2H), 1.39 (s, 18H); 13 C NMR (126 MHz, DMSO-d6) δ 158.50, 156.90, 141.19, 135.11, 134.33, 131.50, 129.93, 129.77, 127.71, 124.48, 114.57, 106.02, 32.69, 31.41; MS (ESI) m / z 593.5 (M-H) - (MPAL exact mass C 32 H 34 N8O4 594.5). The retention time t R = 5.19 min was measured by high performance liquid chromatography. The chemical shifts of each hydrogen and carbon of compound MPAL, the mass spectrum and the high performance liquid chromatogram are shown in Figure 17 FIG. 1, which prove that MPAL is successfully synthesized.

[0169] Example 18 Plinabulin, TPAL and MPAL microtubule depolymerization experiment and microtubule formation inhibition experiment results

[0170] The vascular blocking effect of Plinabulin, TPAL and MPAL is characterized by microtubule depolymerization and inhibition of formation. Specifically, in the microtubule depolymerization experiment, HUVECs (human umbilical vein endothelial cells) cells are seeded in a Matrigel-coated 96-well plate and placed in a 37°C incubator for culture. After 8 hours, the cell morphology, tube structure formation is observed under a fluorescence inverted microscope and photographed as a 0-hour picture. Then, the same concentration of Plinabulin, TPAL and MPAL-containing medium is added to each well, and the incubator is continued to culture for 6 hours, and the same position is photographed under the microscope. In the microtubule formation inhibition experiment, after HUVECs are seeded in a Matrigel-coated 96-well plate, the same concentration of Plinabulin, TPAL and MPAL-containing medium is immediately added, and the cells are cultured in the incubator for 8 hours, and the cell state is observed under the microscope.

[0171] Figure 18 A is the microtubule depolymerization experiment, and the experimental results show that the tube structure of the PBS group is not destroyed after culture, while the tube structure of Plinabulin, TPAL and MPAL is damaged to varying degrees. Figure 18In the microtubule formation inhibition experiment, PBS group was able to form tube structure after incubation, while no tube structure was found in Plinabulin, TPAL and MPAL groups. In summary, both experiments proved that the synthesized TPAL and MPAL derivatives can effectively destroy and inhibit microtubule formation, with the characteristics of vascular blockers.

[0172] Example 19 Plinabulin, TPAL and MPAL cytotoxicity experiment results

[0173] The cytotoxicity of blank solvent, Plinabulin, TPAL and MPAL was characterized by MTT experiment. Specifically, 4T1 (mouse triple negative breast cancer cells) and HUVECs (human umbilical vein endothelial cells) cells were inoculated in 96-well plates (5000 cells / well, 100 μL DMEM) and incubated overnight. The next day, the old culture medium in the well plate was discarded, and 200 μL of fresh culture medium containing different concentrations of Plinabulin, TPAL and MPAL was added to each well, and the cells were further incubated at 37°C for 24 or 48 h. After the incubation time, 20 μL of MTT solution (5 mg / mL sterile PBS solution) was added, and after 4 h of incubation as needed, the MTT-containing culture medium was discarded, and 100 μL of DMSO was added. After shaking for 5 min, the ultraviolet absorption value of each well of the 96-well plate at 490 nm was detected by a microplate reader. The cell survival rate (%) was calculated as follows:

[0174] Cell survival rate (%) = (A experimental / A control ) x 100

[0175] Wherein, A experimental and A control are the absorption values at 490 nm of the sample well and the control well, respectively.

[0176] As Figure 19 shown, for 4T1 cells, the IC 50 (half lethal concentration) of Plinabulin for 24 and 48 h was 60.15 ± 0.27 nM and 51.57 ± 0.96 nM, respectively. In contrast, even at a concentration of 100 nM, the cytotoxicity of TPAL and MPAL to 4T1 cells was still low. For HUVECs cells, the toxic side effects of Plinabulin were stronger than those of TPAL and MPAL. In summary, at the cellular level, the cytotoxicity of TPAL and MPAL to tumor cells and normal cells was lower than that of Plinabulin.

[0177] Example 20 Plinabulin and TPAL tail vein injection of normal Balb / c mice weight change

[0178] Plinabulin and TPAL were compared in terms of toxicity using a weight change assay in normal Balb / c mice. Specifically, Balb / c mice were divided into three groups (n=5 per group): 7.5 mg / kg Plinabulin, 100 mg / kg TPAL, and 75 mg / kg TPAL. The 7.5 mg / kg Plinabulin dose was close to its maximum tolerated dose (MTD). Administration was via tail vein on day 1, and weight changes within each group were continuously monitored.

[0179] Figure 20 The vertical axis represents the ratio of the average body weight of the five mice in the group to the average body weight before administration on day one. The trend shows that with 7.5 mg / kg Plinabulin, body weight decreased continuously for the first three days, reaching its lowest point on day three, with a decrease exceeding 5%. In contrast, TPAL, at both 75 mg / kg and 100 mg / kg doses, resulted in a smaller decrease in body weight compared to Plinabulin, and the time to return to pre-administration body weight was shorter. This means that even at a dose of 100 mg / kg, the toxic side effects of TPAL were still lower than those of 7.5 mg / kg Plinabulin.

[0180] Example 21: Results of tumor suppression and weight change in 4T1 tumor-bearing mice treated with Plinabulin, TPAL, and MPAL.

[0181] 4T1 cells were subcutaneously in Balb / c mice. When the subcutaneous tumor volume increased to 150 mm, the cells were implanted. 3 Mice were randomly divided into four groups: PBS, Plinabulin, TPAL, and MPAL, with six mice in each group. Plinabulin was administered at a dose of 7.5 mg / kg via tail vein injection on days 1, 3, and 5. TPAL and MPAL were administered at a dose of 30 mg / kg via tail vein injection on days 1, 3, and 5. Mice were observed every other day, and the long and short diameters of the tumors were measured using calipers, while mouse weight was recorded. The therapeutic efficacy of each group was evaluated by calculating tumor volume and tumor inhibition rate, and drug safety was evaluated by measuring changes in mouse body weight. Tumor volume and tumor inhibition rate were calculated using the following formula:

[0182] Formula for calculating mouse tumor volume: V=(a×b) 2 ) / 2

[0183] Tumor inhibition rate (TSR,%) = [(Ac-Ax) / Ac] × 100%

[0184] Where a is the long diameter of the tumor, b is the short diameter of the tumor; Ac is the average tumor volume of the control group, and Ax is the average tumor volume of the treatment group.

[0185] Depend onFigure 21 As shown in Figure A, compared with the PBS group, Plinabulin, TPAL, and MPAL all inhibited tumor growth. At the end of treatment, the tumor inhibition rates (TSR%) of the Plinabulin, TPAL, and MPAL groups were 38.6%, 47.7%, and 57.3%, respectively. The treatment effects of the TPAL and MPAL groups were comparable to those of the Plinabulin group. Figure 21 The body weight change curves of mice in group B show that the body weight of mice in the Plinabulin group decreased continuously from day 1 to day 9, reaching its lowest value on day 9, with a decrease of more than 10%, demonstrating significant toxic side effects. In contrast, the body weight of mice in the TPAL and MPAL groups did not decrease significantly, proving that the toxic side effects of TPAL and MPAL are lower than those of Plinabulin. Considering both efficacy and toxic side effects, the therapeutic index of TPAL and MPAL is higher than that of Plinabulin.

[0186] Example 22: Results of tumor suppression and weight change in CT26 tumor-bearing mice treated with Plinabulin and TPAL.

[0187] CT26 cells were subcutaneously injected into Balb / c mice. When the subcutaneous tumor volume increased to 150 mm, the cells were targeted. 3 Mice were randomly divided into three groups: PBS, Plinabulin, and TPAL, with seven mice in each group. Plinabulin was administered at a dose of 7.5 mg / kg via tail vein injection on days 1, 3, and 5; this dose was close to the maximum tolerated dose. TPAL was administered at a dose of 100 mg / kg via tail vein injection on days 1, 3, and 5. Mice were observed every other day, and the long and short diameters of the tumors were measured using calipers, while mouse weight was recorded. The therapeutic efficacy of each group was evaluated by calculating tumor volume and tumor inhibition rate, and drug safety was evaluated by measuring changes in mouse weight. Tumor volume and tumor inhibition rate were calculated using the following formula:

[0188] Formula for calculating mouse tumor volume: V=(a×b) 2 ) / 2

[0189] Tumor inhibition rate (TSR,%) = [(Ac-Ax) / Ac] × 100%

[0190] Where a is the long diameter of the tumor, b is the short diameter of the tumor; Ac is the average tumor volume of the control group, and Ax is the average tumor volume of the treatment group.

[0191] Depend on Figure 22As shown in Figure A, both Plinabulin and TPAL inhibited tumor growth compared to the PBS group. At the end of treatment, the tumor inhibition rates (TSR%) of Plinabulin and TPAL were 52.9% and 57.5%, respectively, with TPAL showing comparable efficacy to the Plinabulin group. Figure 22 The body weight change curves of mice in the PBS and TPAL groups show that the body weight of mice in the Plinabulin group decreased continuously from day 1 to day 7, reaching its lowest value on day 7, with a decrease of more than 10%, which is significantly different from the body weight of the TPAL group, demonstrating obvious toxic side effects. In contrast, the body weight of mice in the PBS and TPAL groups did not decrease significantly, proving that the toxic side effects of TPAL are lower than those of Plinabulin. Considering both therapeutic efficacy and toxic side effects, TPAL has a higher therapeutic index.

[0192] Example 23: Results of tumor suppression and weight change in H22 tumor-bearing mice treated with Plinabulin and TPAL.

[0193] H22 cells were subcutaneously in Balb / c mice. When the subcutaneous tumor volume increased to 150 mm, the cells were targeted. 3 Mice were randomly divided into three groups: PBS, Plinabulin, and TPAL, with seven mice in each group. Plinabulin was administered via tail vein injection at a dose of 7.5 mg / kg on days 1, 3, and 5. TPAL was administered via tail vein injection at a dose of 100 mg / kg on days 1, 3, and 5. Mice were observed every other day, and the long and short diameters of the tumors were measured using calipers, while mouse weight was recorded. The therapeutic efficacy of each group was evaluated by calculating tumor volume and tumor inhibition rate, and drug safety was evaluated by measuring changes in mouse body weight. Tumor volume and tumor inhibition rate were calculated using the following formula:

[0194] Formula for calculating mouse tumor volume: V=(a×b) 2 ) / 2

[0195] Tumor inhibition rate (TSR,%) = [(Ac-Ax) / Ac] × 100%

[0196] Where a is the long diameter of the tumor, b is the short diameter of the tumor; Ac is the average tumor volume of the control group, and Ax is the average tumor volume of the treatment group.

[0197] Depend on Figure 23 As shown in Figure A, both Plinabulin and TPAL inhibited tumor growth compared to the PBS group. At the end of treatment, the tumor inhibition rates (TSR%) of Plinabulin and TPAL were 63.9% and 56.8%, respectively, with the therapeutic effect of TPAL being similar to that of the Plinabulin group. Figure 23The B mouse weight change curve can be seen, Plinabulin group mice weight reached the minimum value on the 5th day, the decrease was more than 10%, there was significant difference in body weight with TPAL group, which reflected obvious side effects. In contrast, the PBS and TPAL two groups of mice had no significant decrease in body weight, which proved that the side effects of TPAL were lower than Plinabulin. Considering the therapeutic effect and side effects, TPAL had higher therapeutic index.

[0198] Example 24 Plinabulin and TPAL treatment of 4T1 tumor-bearing mice tumor inhibition and body weight change results

[0199] 4T1 cells were inoculated subcutaneously in Balb / c mice, when the subcutaneous tumor volume grew to 150mm 3 , the mice were randomly divided into 4 groups: PBS, Plinabulin (high and low dose) and TPAL, seven in each group. The Plinabulin administration method is as follows: 7.5mg / kg (high dose), 3mg / kg (low dose), 1, 3, 5 days by tail vein injection; TPAL administration method is as follows: 100mg / kg, 1, 3, 5 days by tail vein injection. The mice were observed every other day, and the mouse tumor length and width were measured with vernier caliper, and the mouse body weight was recorded with balance. The therapeutic effect of each group of drugs was evaluated by calculating the mouse tumor volume and tumor inhibition rate, and the safety of the drugs was evaluated by measuring the change of mouse body weight. The tumor volume and tumor inhibition rate were calculated by the following formula:

[0200] Mouse tumor volume calculation formula: V = (a x b 2 ) / 2

[0201] Tumor inhibition rate (TSR, %) = [(Ac-Ax) / Ac] x 100%

[0202] Where a is the tumor length, b is the tumor width; Ac is the average tumor volume of the control group, Ax is the average tumor volume of the treatment group.

[0203] From Figure 24 A can be seen, compared with the PBS group, Plinabulin 7.5mg / kg, Plinabulin 3mg / kg and TPAL can inhibit tumor growth. At the end of treatment, the tumor inhibition rate (TSR%) of Plinabulin 7.5mg / kg, Plinabulin 3mg / kg and TPAL groups were 45.6%, 15.6% and 52.8% respectively, the therapeutic effect of TPAL and MPAL groups was equivalent to that of Plinabulin 7.5mg / kg group, and superior to that of Plinabulin 3mg / kg group. Figure 24The body weight change curves of mice in group B show that the body weight of mice in the Plinabulin 7.5 mg / kg group decreased continuously from day 1 to day 5, reaching its lowest value on day 5, with a decrease of more than 10%, demonstrating significant toxic side effects. This proves that TPAL has lower toxic side effects when the therapeutic effect is similar. In contrast, the body weight of mice in the TPAL and Plinabulin 3 mg / kg groups did not decrease significantly, while the therapeutic effect of the TPAL group was significantly improved, proving that the therapeutic effect is better under conditions of similar toxic side effects.

[0204] Example 25: Plinabulin, TPAL, and MPAL for the prevention of chemotherapy-induced neutropenia

[0205] Cytotoxic chemotherapy drugs suppress the hematopoietic system, impair host protective mechanisms, and limit the amount of chemotherapy that can be tolerated. Chemotherapy-induced neutropenia (CIN) is the most serious hematologic toxicity, associated with life-threatening infection risks and potentially reduced or delayed chemotherapy doses that can affect treatment outcomes. In this embodiment, the ability of Plinabulin, TPAL, and MPAL to prevent CIN is validated.

[0206] like Figure 25 As shown in Figure A, a CIN model was established by injecting doxorubicin hydrochloride (Dox, 10 mg / kg) into the tail vein of normal healthy rats. One hour after chemotherapy, Plinabulin, TPAL, and MPAL were injected, and blood was collected from the orbital cavity to monitor the absolute neutrophil count (ANC). Figure 25 The body weight change curves showed that the body weight of rats in the Dox+Plinabulin group was still more than 10% lower on day 21 compared to day -2, while the Dox+TPAL, Dox+MPAL, and Dox+Vehicle groups showed the same trend, with their body weight returning to normal on day 21. This indicates that the toxic side effects of TPAL and MPAL were significantly lower than those of Plinabulin. Figure 25 C indicates that, compared to the Saline group, 10 mg / kg Dox significantly reduced neutrophils, especially reaching as low as 0.1 neutrophils on day 9, demonstrating the successful establishment of the CIN model. Comprehensive analysis of ANC results from -2 to 21 days showed no significant differences among the Dox+Plinabulin, Dox+TPAL, and Dox+MPAL groups, but all groups showed significantly higher ANC than the Dox+Vehicle group. At day 21, the ANC in all three combination groups recovered to their respective -2 day levels. This suggests that TPAL and MPAL are comparable in efficacy to Plinabulin as white blood cell boosters, and their toxic side effects do not overlap with those of chemotherapy drugs, resulting in a higher therapeutic index.

[0207] Example 26 Plinabulin and TPAL in combination with Mecapegfilgrastim to prevent chemotherapy-induced neutropenia

[0208] Mecapegfilgrastim is a commercialized granulopoietic agent that can immediately increase ANC level after administration, and the ANC level will decrease quickly, usually within 2-5 days. The results of Example 25 show that both Plinabulin and TPAL can sustainably increase ANC for 9-21 days. In this example, Plinabulin and TPAL were combined with Mecapegfilgrastim to study the effect of preventing CIN from -2 to 21 days.

[0209] Figure 26 A is the administration time axis, in which Dox, Plinabulin and TPAL were administered on day 0, and Mecapegfilgrastim was subcutaneously injected on day 1. Figure 26 The body weight change chart B shows that TPAL did not add extra toxic side effects compared with the Dox group, while the toxic side effects of Plinabulin were superimposed on Dox. Figure 26 C is the ANC change of each group. Compared with the Saline group, the Dox group can significantly reduce ANC, which proves that the neutropenia model is successfully constructed. Compared with the Dox group, the Plinabulin and TPAL single-drug groups can significantly increase ANC. Compared with the Mecapegfilgrastim single-drug group, the TPAL combination group can significantly increase ANC. Compared with the Plinabulin combination group, the TPAL combination group can increase the average ANC value by 1.23 times, and there is a significant difference, which proves that the TPAL combination treatment effect is better. Combining the treatment effect and toxic side effect analysis, the TPAL combination group has a higher therapeutic index.

[0210] Example 27 TPAL prevents 5-fluorouracil (5-FU)-induced myelosuppression

[0211] Chemotherapy usually causes myelosuppression, and the degree of myelosuppression caused by different chemotherapy drugs is different. Among them, it has been found that high-dose 5-FU can cause strong myelosuppression. Therefore, 150mg / kg 5-FU was selected to construct the myelosuppression model. Myelosuppression refers to the decrease in the activity of blood cell precursor cells in bone marrow, which is a comprehensive effect on blood cells, mainly including the effects on platelets, red blood cells, lymphocytes, neutrophils and white blood cells. In this experiment, SD rats were divided into control group, 5-FU single drug group and 5-FU+TPAL combined use group, four in each group, and the dose of TPAL was 3.75mg / kg by gavage. TPAL was administered one hour before 5-FU administration, and this day was recorded as day 0. From the sixth day, the rats were taken blood from the eye socket every two days, and the absolute counts of the above five blood cells were counted and statistically analyzed.

[0212] By analyzing various blood cells comprehensively, it was found that Figure 27 A) compared with the Control group, the blood cells in the 5-FU group were significantly reduced, which proved that the myelosuppression model was successfully constructed. Among them, the platelets in the TPAL group were significantly different from those in the 5-FU single drug group from the eighth day, and the significant difference was *** on the eighth day; the red blood cells had significant difference from the tenth day, and the significant difference was ** on the twelfth day; for lymphocytes, neutrophils and white blood cells, the largest significant difference was on the tenth day. Figure 27 B is the absolute count of various blood cells on a certain day, among which the platelets and red blood cells are on the eighth day and the twelfth day, respectively, and the lymphocytes, neutrophils and white blood cells are on the tenth day. In summary, TPAL can comprehensively improve the level of blood cells and prevent chemotherapy-induced myelosuppression.

[0213] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered as the protection scope of the present application.

Claims

1. A plinabulin derivative of the structure of formula (I), ###0001### (I) Formula (I); Formula (a); wherein R2, R3, R4 are independently selected from the group of hydrogen, halogen or a combination of the above functional groups; R1 and R5 are independently selected from H or a group of formula (a), and R1 and R5 are not the same.

2. The derivative according to claim 1, characterized in that, The two types of plinabulin derivatives of the structure of formula (I) are selected from the structure of formula (I-1) or (I-2): ###0002### (I-1) (I-2) Formula (I-1); Formula (I-2).

3. A method for preparing the plinabulin derivative of the structure of formula (I) of any one of claims 1-2, comprising the following steps: (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione and benzene dicarboxaldehyde are reacted in N,N-dimethylformamide under the catalysis of cesium carbonate, and the product is obtained.

4. The production method according to claim 3, characterized by, The benzene dicarboxaldehyde is p-benzene dicarboxaldehyde containing R2-R5 functional groups or m-benzene dicarboxaldehyde containing R1-R4 functional groups.

5. Use of the plinabulin derivative of the structure of formula (I) of any one of claims 1-2 in the preparation of an anti-tumor drug. The tumor is selected from one or more of breast cancer and sigmoid colon cancer.

6. Use of the plinabulin derivative of the structure of formula (I) of any one of claims 1-2 in the preparation of a drug for treating a side effect of chemotherapy, wherein the side effect of chemotherapy is selected from neutropenia and myelosuppression.

7. Use according to claim 6, characterized in that, The drug causing neutropenia is selected from docetaxel, paclitaxel, taxane, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, doxorubicin, epirubicin, daunorubicin, valrubicin and pharmaceutically acceptable salts thereof. The drug causing myelosuppression is selected from docetaxel, paclitaxel, taxane, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, doxorubicin, epirubicin, daunorubicin, valrubicin, fluorouracil and pharmaceutically acceptable salts thereof.

8. Use according to claim 6, characterized in that, The drug is a tablet, a capsule, a granule, an oral solution, a sustained-release preparation, a controlled-release preparation, a nano-preparation or an injection.

9. A drug comprising the plinabulin derivative of the structure of formula (I) of any one of claims 1-2.

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