WO2024022375A1 - Plinabulin derivative, preparation method therefor, and use thereof - Google Patents

Abstract

The present invention provides a plinabulin derivative with a structure represented by formula (I). Also provided is use of the plinabulin derivative for resisting tumors, preventing chemotherapy-induced neutropenia, and preventing chemotherapy-induced myelosuppression. In the field of cancer treatment, the two derivatives provided in the present invention can cause fewer toxic and side effects while having similar therapeutic effects as plinabulin and have stronger therapeutic effects and higher therapeutic indexes while causing similar toxic and side effects as plinabulin. In the aspect of preventing chemotherapy-induced neutropenia, the two derivatives have similar ability to prevent neutropenia as plinabulin, with fewer toxic and side effects. In combination with a commercialized leucocyte-promoting drug mecapegfilgrastim, the therapeutic effect of the two derivatives is more excellent than that of the groups of mecapegfilgrastim alone and in combination with plinabulin. In the aspect of preventing chemotherapy-induced myelosuppression, the number of various blood cells can be remarkably increased in the groups of the two derivatives in combination with a chemotherapy drug. The derivative provided in the present invention has wide application prospects in anti-tumor treatment, prevention of neutropenia, and prevention of myelosuppression, and other such aspects.

Description

Plinabulin derivatives and preparation methods and applications thereof

This application claims priority to the Chinese patent application submitted to the China Patent Office on July 29, 2022, with the application number 202210909894.0 and the invention name "Plinabulin Derivatives and Preparation Methods and Applications thereof", the entire content of which is incorporated by reference. incorporated in this application.

Technical field

The present invention relates to the field of pharmaceutical technology, and in particular to a plinabulin derivative and its preparation method and application.

Background technique

Plinabulin, developed by Beyondspring, is a diketopiperazine drug synthesized based on the structure of a natural product isolated from marine pyrozoin and through structure-activity relationship studies. Plinabulin has anti-tumor activity and the ability to prevent chemotherapy-induced neutropenia: as an anti-tumor drug, it has completed an international multi-center phase III clinical trial and reached all study endpoints; as a white drug, it has completed phase III Clinical trials and submission of new drug marketing applications in China and the United States.

However, in anti-tumor phase III clinical trials, the dose of the first dose of Plinabulin was close to its maximum tolerated dose (MTD). If the vomiting grade was greater than grade one, the second dose was adjusted down to the initial dose. 2/3. In the clinical trials of Shengbaiyao, the initial dosage was directly lowered. It can be seen that Plinabulin has major toxic and side effects, and its high-dose administration is limited, which affects the therapeutic effect. Therefore, reducing the toxic side effects of Plinabulin and improving its therapeutic index will benefit more cancer patients.

It has been reported that Plinabulin binds to tubulin mainly through the formation of hydrogen bonds between the nitrogen and oxygen atoms on the imidazole ring and diketopiperazine ring and the colchicine binding domain, and has no binding in the benzene ring region. site, so the present invention chooses to modify the benzene ring region.

At the same time, various derivatives were constructed based on the three ring structures of plinabulin. Among them, the modification of the benzene ring region is mostly carried out by modifying one or more simple substituent groups, such as: halogen atoms, hydroxyl groups, amino groups, carboxyl groups, etc. The present invention designs two types of new structural derivatives of formula (I), namely, the phenabulin benzene ring region is continuously modified with an imidazole ring, a diketopiperazine ring and four substituent groups. Two derivatives with four substituent groups all being hydrogen atoms were used to study their anti-tumor activity and prevention of chemotherapy-induced neutrophils. The ability to reduce cytopenias and prevent chemotherapy-induced bone marrow suppression is expected to reduce toxic side effects and improve the therapeutic index.

Contents of the invention

In view of this, the technical problem to be solved by the present invention is to provide a Plinabulin derivative. Compared with Plinabulin, the Plinabulin derivative provided by the invention can significantly reduce the toxic and side effects under similar therapeutic effects. , with a higher therapeutic index.

The invention provides a plinabulin derivative with a structure of formula (I),

Among them, R ₂ , R ₃ and R ₄ are independently selected from hydrogen, hydrocarbyl, thiohydrocarbyl, oxyhydrocarbyl, halogenated hydrocarbyl, halogen, nitro, amino, hydroxyl, carboxyl, ester group, amide group or the above functional groups. combination;

R ₁ and R ₅ are independently selected from H or a group represented by formula (a), and R ₁ and R ₅ are different.

Preferably, the two types of plinabulin derivatives of the formula (I) are selected from the structures represented by formula (I-1) or formula (I-2):

The present invention provides a method for preparing a plinabulin derivative of the formula (I) according to any one of the above technical solutions, which includes the following steps:

Catalyzed by cesium carbonate, (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione and benzene It is obtained by reacting diformaldehyde in N,N-dimethylformamide.

Preferably, the phthalaldehyde is terephthalaldehyde containing R ₂ to R ₅ functional groups or isophthalaldehyde containing R ₁ to R ₄ functional groups.

The present invention provides the use of the plinabulin derivative of the formula (I) described in any of the above technical solutions in the preparation of anti-tumor drugs.

Preferably, the tumors include malignant tumors of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, oral cavity cancer, laryngeal cancer, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, stomach cancer, colorectal cancer, sigmoid colon and rectal cancer. , liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, renal cancer, prostate cancer, bladder cancer, testicular malignant tumors, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytic carcinoma, rhabdomyosarcoma carcinoma , one or more of synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma and multiple myeloma.

The present invention provides the use of the Plinabulin derivative with the structure of formula (I) described in the above technical solution in the preparation of drugs for treating side effects of chemotherapy; the side effects of chemotherapy include neutropenia and bone marrow suppression.

Preferably, the drug causing neutropenia is selected from the group consisting of docetaxel, paclitaxel, taxane, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, Protecan, irinotecan, doxorubicin, epirubicin, daunorubicin, valrubicin and their pharmaceutically acceptable salts;

The drugs that cause bone marrow suppression are selected from the group consisting of docetaxel, paclitaxel, taxanes, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, alfa Mycin, epirubicin, daunorubicin, valrubicin, fluorouracil and their pharmaceutically acceptable salts.

Preferably, the drug is a tablet, capsule, granule, oral liquid, sustained release preparation, controlled release preparation, nano preparation or injection.

The present invention provides a medicine, including a plinabulin derivative of the formula (I) described in any one of the above technical solutions.

Compared with the prior art, the present invention provides a plinabulin derivative with a structure of formula (I). For anti-tumor, prevention of chemotherapy-induced neutropenia and prevention of chemotherapy-induced bone marrow suppression. 1) In the field of cancer treatment: the two derivatives provided by the present invention can have similar therapeutic effects to Plinabulin, but with lower toxic and side effects; under the conditions of similar toxic and side effects, the therapeutic effect is stronger and the therapeutic index is higher; 2 ) In terms of preventing chemotherapy-induced neutropenia: it has a similar ability to prevent neutropenia as Plinabulin, and has lower toxic and side effects; it can be used in combination with the commercial white drug thiopefilgrastim , the therapeutic effect is better than that of the combination of thiotropin alone and plinabulin; 3) In terms of preventing chemotherapy-induced bone marrow suppression: the combination with chemotherapy drugs can significantly increase the number of various blood cells (platelets, red blood cells, lymphocytes, granulocytes and leukocytes). The derivatives provided by the invention have broad application prospects in anti-tumor treatment, prevention of neutropenia and prevention of bone marrow suppression.

Description of drawings

Figure 1 shows the ¹ H NMR, ¹³ C NMR and ESI mass spectra of implementation compound a obtained in Example 1 of the present invention;

Figure 2 shows the ¹ H NMR, ¹³ C NMR and ESI mass spectra of implementation compound b obtained in Example 2 of the present invention;

Figure 3 is the ¹ H NMR and ESI mass spectra of the implementation compound c obtained in Example 3 of the present invention;

Figure 4 is ¹ H NMR of implementation compound d obtained in Example 4 of the present invention;

Figure 5 is ¹ H NMR of the implementation compound e obtained in Example 5 of the present invention;

Figure 6 is ¹ H NMR of the implementation compound f obtained in Example 6 of the present invention;

Figure 7 is ¹ H NMR of implementation compound g obtained in Example 7 of the present invention;

Figure 8 is ¹ H NMR of implementation compound h obtained in Example 8 of the present invention;

Figure 9 is ¹ H NMR of implementation compound i obtained in Example 9 of the present invention;

Figure 10 is ¹ H NMR of compound j obtained in Example 10 of the present invention;

Figure 11 is ¹ H NMR of compound k obtained in Example 11 of the present invention;

Figure 12 is ¹ H NMR of the implementation compound 1 obtained in Example 12 of the present invention;

Figure 13 is ¹ H NMR of the implementation compound m obtained in Example 13 of the present invention;

Figure 14 is ¹ H NMR of compound n obtained in Example 14 of the present invention;

Figure 15 is ¹ H NMR of the implementation compound o obtained in Example 15 of the present invention;

Figure 16 is the ¹ H NMR, ¹³ C NMR, ESI spectrum and high performance liquid chromatogram of TPAL obtained in Example 16 of the present invention;

Figure 17 shows the ¹ H NMR, ¹³ C NMR, ESI spectra and high performance liquid chromatogram of MPAL obtained in Example 17 of the present invention;

Figure 18 shows the microtubule depolymerization experiment (A) and microtubule formation inhibition experiment (B) of TPAL, MPAL and Plinabulin obtained in Example 18 of the present invention;

Figure 19 shows the cytotoxicity experiment of TPAL, MPAL and Plinabulin on 4T1 and HUVECs cells obtained in Example 19 of the present invention;

Figure 20 shows the body weight changes of normal Balb/c mice after TPAL and Plinabulin obtained in Example 20 of the present invention were injected into normal Balb/c mice through the tail vein;

Figure 21 shows the tumor volume changes (A) and mouse body weight changes (B) in subcutaneous tumor-bearing mice after TPAL, MPAL and Plinabulin were used to treat 4T1 tumor-bearing mice in Example 21 of the present invention;

Figure 22 shows the tumor volume changes (A) and mouse body weight changes (B) in subcutaneous tumor-bearing mice after treatment of CT26 tumor-bearing mice with TPAL and Plinabulin obtained in Example 22 of the present invention;

Figure 23 shows the tumor volume changes (A) and mouse body weight changes (B) in subcutaneous tumor-bearing mice after H22 tumor-bearing mice were treated with TPAL and Plinabulin in Example 23 of the present invention;

Figure 24 shows the tumor volume (A) and mouse body weight changes (B) of subcutaneous tumor-bearing mice after TPAL and Plinabulin were used to treat 4T1 tumor-bearing mice in Example 24 of the present invention;

Figure 25 shows the treatment timeline (A), body weight changes (B) and neutrophils in each group after using TPAL, MPAL and Plinabulin to prevent doxorubicin-induced neutropenia obtained in Example 25 of the present invention. Absolute count ANC changes (C);

Figure 26 shows the treatment timeline (A), absolute neutrophil count ( B) and weight changes in each group (C);

Figure 27 shows the implementation of TPAL obtained in Example 27 of the present invention to prevent 5-fluorouracil-induced bone marrow suppression, the changes in various blood cells over time (A) and the absolute values of blood cells in each group on a specific day (B).

Detailed ways

The present invention provides a plinabulin derivative and its preparation method and application. Persons skilled in the art can learn from the content of this article and appropriately improve the process parameters for implementation. It should be pointed out in particular that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the protection scope of the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the present invention. Invent technology.

Plinabulin is a tubulin inhibitor that binds near the colchicine-binding site of tubulin and causes tumor vasculature to malfunction. It has a wide anti-tumor spectrum and is used for head and neck squamous cell carcinoma; ovarian cancer; blastoma; seminoma; lung cancer; thyroid cancer; lymphosarcoma and reticulum cell sarcoma.

The invention provides a plinabulin derivative with a structure of formula (I),

Among them, R ₂ , R ₃ and R ₄ are independently selected from hydrogen, hydrocarbyl, thiohydrocarbyl, oxyhydrocarbyl, halogenated hydrocarbyl, halogen, nitro, amino, hydroxyl, carboxyl, ester group, amide group or the above functional groups. combination;

R ₁ and R ₅ are independently selected from H or a group represented by formula (a), and R ₁ and R ₅ are different.

In the present invention, the two types of plinabulin derivatives of the formula (I) structure are selected from the structures represented by formula (I-1) or formula (I-2):

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

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

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

The plinabulin derivative of the present invention can destroy the formed microtubule structure and inhibit the formation of microtubules.

The present invention provides a method for preparing a plinabulin derivative of the formula (I) according to any one of the above technical solutions, which includes the following steps:

Catalyzed by cesium carbonate, (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione and benzene It is obtained by reacting diformaldehyde in N,N-dimethylformamide.

The alkaline catalyst described in the present invention can be common alkaline catalysts such as potassium tert-butoxide, DBU, cesium carbonate, etc., with cesium carbonate being the most preferred.

The (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione and phthalaldehyde are fed The molar ratio is 5:1 to 2:1, and the most preferred is 3:1.

Preferably, the phthalaldehyde is terephthalaldehyde containing R ₂ to R ₅ functional groups or isophthalaldehyde containing R ₁ to R ₄ functional groups.

The present invention provides the use of the plinabulin derivative of the formula (I) described in any of the above technical solutions in the preparation of anti-tumor drugs.

The tumors described in the present invention include malignant tumors of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, oral cavity cancer, laryngeal cancer, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, colorectal cancer, sigmoid colon and rectal cancer, Liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, renal cancer, prostate cancer, bladder cancer, testicular malignant tumors, penile cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytic carcinoma, rhabdomyosarcoma, One or more of synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma, and multiple myeloma.

The medicines are tablets, capsules, granules, oral liquids, sustained-release preparations, controlled-release preparations, sodium preparations or injections.

The present invention provides an anti-tumor drug, including a plinabulin derivative of the formula (I) described in any one of the above technical solutions.

The anti-tumor drugs provided by the present invention include any derivatives of the above technical solutions, their salts, hydrates, crystal forms, enantiomers, isomers, metabolites, prodrugs and pharmaceutically acceptable excipients.

The present invention provides the use of the Plinabulin derivative with the structure of formula (I) described in the above technical solution in the preparation of drugs for treating side effects of chemotherapy; the side effects of chemotherapy include neutropenia and bone marrow suppression.

The neutropenia is induced by a first chemotherapeutic combination of one or more chemotherapeutic agents or by the administration of radiation therapy. Chemotherapy drugs include but are not limited to: docetaxel, paclitaxel, taxanes, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, adriamycin epirubicin, daunorubicin, valrubicin, etc. and their pharmaceutically acceptable salts.

Specifically, the drug is administered 1 minute to 24 hours before or 1 minute to 24 hours after chemotherapy.

When the present invention is conducted on rats, the dosage is preferably 1 mg/kg to 100 mg/kg; the most preferable dosage is 3.75 mg/kg.

The medicines of the present invention can be used alone or in combination with commercial whitening drugs to improve the effect of preventing chemotherapy-induced neutropenia. Commercial white drugs include but are not limited to: filgrastim, pegfilgrastim, thio-pegfilgrastim, etc. Preferably, combination with thiopegfilgrastim increases neutrophil levels.

Myelosuppression of the present invention comprises a first chemotherapeutic combination of one or more chemotherapeutic agents or is induced by the administration of radiation therapy. Chemotherapy drugs include but are not limited to: docetaxel, paclitaxel, taxanes, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, adriamycin cerebrospinal fluid, epirubicin, daunorubicin, valrubicin, fluorouracil, etc. and their pharmaceutically acceptable salts.

Specifically, the drug is administered 1 minute to 24 hours before or 1 minute to 24 hours after chemotherapy. When the present invention is conducted on rats, the dosage is preferably 1 mg/kg to 150 mg/kg. mg/kg; most preferably, the dose is 3.75 mg/kg.

The drugs described in the present invention can be administered alone or in combination with commercial preventive myelosuppressive drugs to increase the number of various blood cells.

Specifically, the drug is a tablet, capsule, granule, oral liquid, sustained-release preparation, controlled-release preparation, nano-preparation or injection.

The present invention provides a medicine, including a plinabulin derivative of the formula (I) described in any one of the above technical solutions.

The invention provides a drug for treating chemotherapy-induced neutropenia, which includes a plinabulin derivative with a structure of formula (I) described in any one of the above technical solutions.

The present invention is a drug for treating chemotherapy-induced myelosuppression, which is characterized in that it includes a plinabulin derivative with the structure of formula (I) described in the above technical solution.

The dosage of TPAL of the present invention in rats and mice is preferably 1 to 150 mg/kg; more preferably 30 to 120 mg/kg; most preferably 1 to 30 mg/kg.

The present invention creatively synthesizes a variety of plinabulin derivatives. TPAL and MPAL, as representatives, have the ability to fight tumors, prevent chemotherapy-induced neutropenia, and prevent chemotherapy-induced myelosuppression. Compared with Plinabulin, they have a higher therapeutic index.

When conducting experiments on mice in the present invention, the dosage is preferably 60 mg/kg to 100 mg/kg; preferably, the dosage is administered through the tail vein.

The present invention provides two types of plinabulin derivatives of formula (I). The present invention takes TPAL and MPAL when all four substituents are hydrogen atoms as representatives, and studies the application in inhibiting tumor growth, preventing chemotherapy-induced neutropenia, and preventing chemotherapy-induced bone marrow suppression. In terms of inhibiting tumor growth, compared with Plinabulin, the toxic and side effects are lower when the therapeutic effect is similar; when the toxic and side effects are similar, the therapeutic effect is better. In terms of preventing agranulocytosis, it has a similar ability to prevent chemotherapy-induced neutropenia as Plinabulin, and has lower toxic side effects; when combined with the commercial whitening drug Thiopegfilgrastim, the therapeutic effect is significantly better than Thiopectin. The drug group and the Plinabulin combination group. In terms of preventing chemotherapy-induced bone marrow suppression, it can significantly increase the number of various blood cells compared with chemotherapy alone. The plinabulin derivative provided by the invention has a higher therapeutic index in terms of inhibiting tumor growth, preventing neutropenia induced by chemotherapy drugs, and preventing chemotherapy-induced bone marrow suppression, and has broad application prospects.

The present invention obtains a variety of bidentate derivatives of Plinabulin through chemical synthesis, and mainly selects TPAL and MPAL as representatives for cell and animal experiments. (1) Microtubule depolymerization and inhibition of microtubule formation experiments found that both TPAL and MPAL can destroy the formed microtubule structure and inhibit microtubule formation, and have the properties of blood vessel blocking agents. (2) Cell experiments found that TPAL and MPAL were less toxic to 4T1 and HUVECs cells than Plinabulin. (3) In vivo experiments, in the normal Balb/c mouse model, TPAL has lower toxic and side effects than Plinabulin. (4) In the 4T1 model, TPAL and MPAL have similar tumor inhibitory capabilities to Plinabulin, and their toxic and side effects are significantly lower than Plinabulin. Compared with Plinabulin, TPAL has significantly enhanced therapeutic effects and a higher therapeutic index when its toxic and side effects are similar. (5) On the CT26 model, the therapeutic effect of TPAL is equivalent to that of Plinabulin, with lower toxic and side effects and a higher therapeutic index. (6) On the H22 model, the therapeutic effect of TPAL is close to that of Plinabulin, with lower toxic and side effects and a higher therapeutic index. (7) In the normal rat model treated with doxorubicin chemotherapy, compared with Plinabulin, TPAL and MPAL had similar effects in preventing granule deficiency, but the toxic and side effects did not overlap with the chemotherapy drugs, and the therapeutic index was higher. (8) In the normal rat model treated with doxorubicin chemotherapy, the effect of TPAL and Plinabulin combined with commercial thiopegfilgrastim in preventing granulocyte deficiency was examined. Compared with the combination group, the therapeutic effects were significantly improved. (9) In the normal rat model treated with 5-fluorouracil chemotherapy, the effect of TPAL on preventing myelosuppression was investigated. TPAL can significantly increase various types of blood cells and prevent bone marrow suppression. The present invention takes the low toxic and side effects of plinabulin derivatives as the starting point. When the therapeutic effects are similar, the toxic and side effects are lower. When the toxic and side effects are similar, the therapeutic effect is better, and has a higher therapeutic index, which is beneficial to cancer and prevention. The clinical treatment of neutropenia induced by chemotherapy drugs provides new drug options with good application prospects.

In order to further illustrate the present invention, a Plinabulin derivative provided by the present invention and its preparation method and application are described in detail below in conjunction with the examples.

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

The specific preparation process includes the following steps:

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

Add 1g (6.5mmol) 5-(tert-butyl)-1H-imidazole-4-carbaldehyde to anhydrous 7mL DMF, then add 2.59g (13mmol) N,N-diacetylpiperazine-2,5- Diketone, nitrogen protection three times, add 3.19g (9.8 mmol) cesium carbonate, nitrogen protection three times, stir and react for 20 hours at room temperature in the dark. Pour the reaction solution into ice water (100 mL), filter with suction, wash the filter cake with water (100 mL*2), petroleum ether: ethyl acetate = 8:1 (90 mL), ultrasonically disperse with ethanol and dichloromethane, and filter out The insoluble matter was distilled under reduced pressure, mixed with absolute ethanol and water, and then slurried with ethyl acetate (50 mL). 0.89g of a brownish-red solid was obtained as the (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-di Ketone, yield 46.9%.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.38 (s, 1H), 12.02 (s, 1H), 7.86 (d, J = 0.9Hz, 1H), 7.04 (s, 1H), 4.30 (s, 2H), 2.51 (d, J = 1.8Hz, 3H), 1.39 (s, 9H).

(2) Preparation of compound a

290 mg (1 mmol) (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione was added to In water DMF, add 64.04mg (0.33mmol) 2,5-dimethoxybenzene-1,4-dicardehyde under nitrogen protection and exhaust gas three times. Add 108.27mg (0.33mmol) cesium carbonate under nitrogen protection and exhaust gas three times. Procedure The temperature was raised to 80°C and the reaction was stirred for 48 h in the dark. Pour the reaction solution into ice water (100 mL), filter with suction, wash the filter cake with water (100 mL*2), petroleum ether: ethyl acetate = 8:1 (90 mL), ultrasonically disperse with ethanol and dichloromethane, and filter out Insoluble matter, distill under reduced pressure, anhydrous ethanol with water. Disperse in 50 mL of ethyl acetate and let stand at -30°C overnight. After suction filtration, the filter cake was washed with glacial ethyl acetate (5 mL) to obtain 114.35 mg of yellow powder solid.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.36(s,2H),12.27(s,2H),9.98(s,2H),7.87(s,2H),7.15(s,2H),6.84( d, J＝16.0Hz, 4H), 3.86 (s, 6H), 1.39 (s, 18H); ¹³ C NMR (126MHz, DMSO-d ₆ ) 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) ⁺ (the accurate mass of compound a is C ₃₄ H ₃₈ N ₈ O ₆ 654.73). The assignment of each hydrogen atom of compound a and the raw material, carbon spectrum and mass spectrum are shown in Figure 1, which proves that compound a was successfully synthesized.

Example 2(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) Preparation

The specific preparation process includes the following steps:

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

The specific steps are the same as step (1) in Embodiment 1 and will not be described again.

(2) Preparation of compound b

290 mg (1 mmol) (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione was added to In water DMF, add 50.01 mg (0.33 mmol) of 5-hydroxyisophthalaldehyde under nitrogen protection and exhaust gas three times, add 108.27 mg (0.33 mmol) of cesium carbonate under nitrogen protection and exhaust gas three times, program the temperature to 80°C and avoid light, stir and react for 48 hours. . Pour the reaction solution into ice water (100 mL), filter with suction, wash the filter cake with water (100 mL*2), petroleum ether: ethyl acetate = 8:1 (90 mL), ultrasonically disperse with ethanol and dichloromethane, and filter out Insoluble matter, distill under reduced pressure, anhydrous ethanol with water. Disperse in 50 mL of ethyl acetate and let stand at -30°C overnight. After suction filtration, the filter cake was washed with glacial ethyl acetate (5 mL) to obtain 84.78 mg of yellow powder solid.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.34(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 = 18.6Hz, 4H), 6.70 (s, 2H), 1.38 (s, 18H); ¹³ C NMR (126MHz, DMSO-d ₆ ) 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(MH) ^- (The exact mass of compound b is C ₃₂ H ₃₄ N ₈ O ₅ 610.53 ). The assignment of each hydrogen atom of compound b and the raw material, the carbon spectrum and the mass spectrum are shown in Figure 2, which proves that compound b was successfully synthesized.

Example 3(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) Preparation

The specific preparation process includes the following steps:

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

The specific steps are the same as step (1) in Embodiment 1 and will not be described again.

(2) Preparation of compound c

290 mg (1 mmol) (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione was added to In water DMF, add 70.65 mg (0.33 mmol) of 5-bromoisophthalaldehyde under nitrogen protection and exhaust gas three times, add 108.27 mg (0.33 mmol) of cesium carbonate under nitrogen protection and exhaust gas three times, program the temperature to 80°C and avoid light, stir and react for 48 hours. . Pour the reaction solution into ice water (100 mL), filter with suction, wash the filter cake with water (100 mL*2), petroleum ether: ethyl acetate = 8:1 (90 mL), ultrasonically disperse with ethanol and dichloromethane, and filter out Insoluble matter, distill under reduced pressure, anhydrous ethanol with water. Disperse in 50 mL of ethyl acetate and let stand at -30°C overnight. After suction filtration, the filter cake was washed with glacial ethyl acetate (5 mL) to obtain 34.19 mg of yellow powder solid.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.33 (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(MH) ^- (The exact mass of compound c is C ₃₂ H ₃₄ N ₈ O ₅ 672.41 ). The assignment of each hydrogen atom of compound c and the raw material and the mass spectrum are shown in Figure 3, which proves that compound c was successfully synthesized.

Example 4(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) Preparation

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

The specific steps are the same as step (1) in Embodiment 1 and will not be described again.

(2) Preparation of compound d

Replace the raw material 5-bromoisophthalaldehyde in compound c with 2-bromoterephthalaldehyde, and keep the remaining raw materials, feed molar ratio and post-reaction treatment method unchanged.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.36 (s, 4H), 10.47 (s, 2H), 8.11 (s, 2H), 7.89 (d, J = 17.8Hz, 2H), 7.67 (s, 3H),6.87(s,2H),6.70(s,2H),1.38(s,18H).

Example 5(3Z,3'Z,6Z,6'Z)-6,6'-((2,5-dichloro-1,4-phenylene)bis(methanelylidene))bis(3-((5-( Preparation of tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione) (compound e)

Replace the raw material 5-bromoisophthalaldehyde in compound c with 2,5-dichloroterephthalaldehyde, and keep the remaining raw materials, feed molar ratio and post-reaction treatment method unchanged.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.37 (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).

Example 6(3Z,3'Z,6Z,6'Z)-6,6'-((2,5-bis(hexyloxy)-1,4-phenylene)bis(methanelylidene))bis(3-(( Preparation of 5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione) (compound f)

The raw material 5-bromoisophthalaldehyde in compound c is replaced with 2,5-dihexyloxyterephthalaldehyde, and the remaining raw materials, feed molar ratio and post-reaction treatment method remain unchanged.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.36(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＝6.5Hz,4H),1.75(t,J＝7.3Hz,4H),1.38(s,18H),1.27(s,8H), 1.23(s,4H),0.83(d,J=7.0Hz,6H).

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

Replace the raw material 5-bromoisophthalaldehyde in compound c with 2,3,5,6-tetramethylterephthalaldehyde, and keep the remaining raw materials, feed molar ratio and post-reaction treatment method unchanged.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.35 (s, 4H), 8.65 (s, 2H), 7.88 (s, 2H), 6.87 (d, J = 5.7Hz, 4H), 2.09 (s, 12H),1.38(s,18H).

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

The raw material 5-bromoisophthalaldehyde in compound c is replaced with 2,5-bis(octyloxy)terephthalaldehyde, and the remaining raw materials, feed molar ratio and post-reaction treatment method remain unchanged.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.35(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＝6.9Hz,2H),1.73(d,J＝7.5Hz,4H),1.38(s,18H), 1.27–1.11(m,20H),0.88–0.73(m,6H).

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

Replace the raw material 5-bromoisophthaldialdehyde in compound c with 2,5-bis(prop-2-yn-1-yloxy)terephthalaldehyde, and maintain the remaining raw materials, feed molar ratio and post-reaction treatment method constant.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.33 (d, J = 19.5Hz, 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 = 2.4Hz, 4H), 3.62 (t, J = 2.4Hz, 2H), 1.39 (s, 18H).

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

The raw material 5-bromoisophthalaldehyde in compound c is replaced with 2,5-bis(heptyloxy)terephthalaldehyde, and the remaining raw materials, feed molar ratio and post-reaction treatment method remain unchanged.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.36(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＝6.6Hz,4H),1.74(s,4H),1.38(s,18H),1.23(s,16H),0.88–0.74(m ,6H).

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

The raw material 5-bromoisophthalaldehyde in compound c is replaced with 2,5-divinylterephthalaldehyde, and the remaining raw materials, feed molar ratio and post-reaction treatment method remain unchanged.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.35 (d, J = 15.9 Hz, 4H), 7.87 (s, 2H), 7.38 (s, 6H), 7.05 (s, 4H), 6.97 (s, 2H),1.38(s,18H).

Example 12(3Z,3'Z,6Z,6'Z)-6,6'-((2,5-bis(docosyloxy)-1,4-phenylene)bis(methanelylidene))bis(3-(( Preparation of 5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione) (compound 1)

The raw material 5-bromoisophthalaldehyde in compound c is replaced with 2,5-docosyloxy-1,4-terephthalaldehyde, and the remaining raw materials, feed molar ratio and post-reaction treatment method remain unchanged.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.35(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＝6.6Hz ,6H).

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

Replace the raw material 5-bromoisophthalaldehyde in compound c with 2,5-2F-1,4-terephthalaldehyde, and keep the remaining raw materials, feed molar ratio and post-reaction treatment method unchanged.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.36 (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＝2.0Hz,18H).

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

Replace the raw material 5-bromoisophthalaldehyde in compound c with 2,5-bis(2-methoxyethoxy)-terephthalaldehyde, and keep the remaining raw materials, feed molar ratio and post-reaction treatment method unchanged. .

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.35(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＝12.7,2.2Hz,6H),1.39(d,J＝2.1Hz,18H).

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

Replace the raw material 5-bromoisophthalaldehyde in compound c with 2,5-dipropoxy-terephthalaldehyde, and keep the remaining raw materials, the molar ratio of the feed, and the post-reaction treatment method unchanged.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.35(s,2H),12.27(s,2H),9.93(s,2H),7.87(s,2H),7.14(s,2H),6.83( d,J＝18.3Hz,4H),4.04(t,J＝6.5Hz,4H),1.78(q,J＝6.9Hz,4H),1.39(s,18H),0.98(t,J＝7.4Hz, 6H).

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

The specific preparation process includes the following steps:

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

The specific steps are the same as step (1) in Embodiment 1 and will not be described again.

(2) Preparation of TPAL

290 mg (1 mmol) (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione was added to In water DMF, add 44.71 mg (0.33 mmol) of terephthalaldehyde for three times of nitrogen protection and exhaust, add 108.27 mg (0.33 mmol) of cesium carbonate for three times of nitrogen protection, and program the temperature to 80°C to avoid light and stir for 48 hours. Pour the reaction solution into ice water (100 mL), filter with suction, wash the filter cake with water (100 mL*2), petroleum ether: ethyl acetate = 8:1 (90 mL), ultrasonically disperse with ethanol and dichloromethane, and filter out Insoluble matter, distill under reduced pressure, anhydrous ethanol with water. Disperse in 50 mL of ethyl acetate and let stand at -30°C overnight. After suction filtration, the filter cake was washed with glacial ethyl acetate (5 mL) to obtain 178.35 mg of yellow powder solid with a yield of 90.9%.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.32 (d, J = 21.6Hz, 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(126MHz,DMSO-d ₆ )δ158.30,157.01,141.23,135.17,133.69,131.49,130.31,127.72,124.50,114.14,105.99 , 32.70,31.41; MS(ESI)m/z 593.5(MH) ^- (The exact mass of TPAL is C ₃₂ H ₃₄ N ₈ O ₄ 594.5). The retention time t _R =5.68min was measured by high performance liquid chromatography. The individual hydrogen sums of the compound TPAL The chemical shift assignment of carbon, mass spectrum and high performance liquid chromatography spectrum are shown in Figure 16, proving that TPAL was successfully synthesized.

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

The specific preparation process includes the following steps:

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

The specific steps are the same as step (1) in Embodiment 1 and will not be described again.

(2) Preparation of MPAL

290 mg (1 mmol) (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione was added to In water DMF, add 44.71 mg (0.33 mmol) of isophthalaldehyde, and exhaust gas three times under nitrogen protection. Add 108.27 mg (0.33 mmol) of cesium carbonate, exhaust gas three times under nitrogen protection, and program the temperature to 80°C to avoid light and stir for 48 hours. Pour the reaction solution into ice water (100 mL), filter with suction, wash the filter cake with water (100 mL*2), petroleum ether: ethyl acetate = 8:1 (90 mL), ultrasonically disperse with ethanol and dichloromethane, and filter out Insoluble matter, distill under reduced pressure, anhydrous ethanol with water. Disperse in 50 mL of ethyl acetate and let stand at -30°C overnight. After suction filtration, the filter cake was washed with glacial ethyl acetate (5 mL) to obtain 190.78 mg of yellow powder solid with a yield of 97.2%.

¹ H NMR (300MHz, DMSO-d ₆ ) δ12.32 (d, J = 18.5 Hz, 4H), 10.30 (s, 2H), 7.87 (s, 2H), 7.64 (s, 1H), 7.42 (q, J＝5.4Hz, 3H), 6.86 (s, 2H), 6.80 (s, 2H), 1.39 (s, 18H); ¹³ C NMR (126MHz, DMSO-d ₆ ) 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(MH) ^- (MP AL accurate mass is C ₃₂ H ₃₄ N ₈ O ₄ 594.5). The retention time t _R =5.19min was measured by high performance liquid chromatography. The chemical shift assignments, mass spectra and high performance liquid chromatograms of each hydrogen and carbon of compound MPAL are shown in Figure 17, which proves that MPAL was successfully synthesized.

Example 18 Experimental results of microtubule depolymerization and inhibition of microtubule formation by Plinabulin, TPAL and MPAL

The vascular blocking effects of Plinabulin, TPAL and MPAL are characterized by microtubule depolymerization and inhibition of formation. Specifically, for the microtubule depolymerization experiment, HUVECs (human umbilical vein endothelial cells) cells were seeded in Matrigel-coated 96-well plates and cultured in a 37°C incubator. After 8 hours, take pictures under a fluorescent inverted microscope to observe the cell morphology and form a tube structure, which is used as the 0 hour picture. Subsequently, medium containing the same concentration of Plinabulin, TPAL and MPAL was added to each well, and culture was continued in the incubator for 6 hours. The same position was searched for and photographed under a microscope. For the microtubule formation inhibition experiment, HUVECs were seeded on Matrigel-coated 96-well plates, then medium containing the same concentration of Plinabulin, TPAL and MPAL was added, cultured in an incubator for 8 hours, and the cell status was observed under a microscope.

Figure 18A shows a microtubule depolymerization experiment. The experimental results show that the tube structure of the PBS group was not destroyed after culture, while the tube structures of Plinabulin, TPAL and MPAL were damaged to varying degrees. Figure 18B In the experiment of inhibiting microtubule formation, the PBS group was able to form tube structures after culture, while no tube-like structures were found in the Plinabulin, TPAL and MPAL groups. Combining the two experiments, it was proved that the synthesized TPAL and MPAL derivatives can effectively destroy and inhibit the formation of microtubules, and have the characteristics of blood vessel blocking agents.

Example 19 Plinabulin, TPAL and MPAL cytotoxicity experimental results

The cytotoxicity of blank solvent, Plinabulin, TPAL and MPAL was characterized by MTT assay. Specifically, 4T1 (mouse triple-negative breast cancer cells) and HUVECs (human umbilical vein endothelial cells) cells were seeded in a 96-well plate (5000 cells/well, 100 μL DMEM) and cultured overnight. The next day, discard the old culture medium in the well plate, add 200 μL of fresh culture medium containing different concentrations of Plinabulin, TPAL and MPAL to each well, and continue culturing in a 37°C cell culture incubator for 24 or 48 hours. After the culture time is reached, add 20 μL MTT solution (5 mg/mL sterile PBS solution). After culturing for 4 hours as needed, discard the medium containing MTT, add 100 μL DMSO, shake thoroughly for 5 minutes, and then use a microplate reader to detect each well of the 96-well plate. The UV absorption value of each hole at 490nm. The calculation formula of cell survival rate (%) is as follows:

Cell survival rate (%) = (A _experimental /A _control ) × 100

Among them, A _experimental and A _control represent the absorption values of the sample well and the control well at 490nm respectively.

As shown in Figure 19, for 4T1 cells, the IC ₅₀ (50% lethal concentration) of Plinabulin at 24 and 48 hours was 60.15±0.27nM and 51.57±0.96nM, respectively. In contrast, even when the concentration of TPAL and MPAL reached 100 nM, the cytotoxicity to 4T1 cells was still very low. For HUVECs cells, Plinabulin has stronger toxic side effects than TPAL and MPAL. Comprehensive analysis shows that at the cellular level, TPAL and MPAL have lower cytotoxicity to tumor cells and normal cells than Plinabulin.

Example 20 Body weight changes in normal Balb/c mice injected with Plinabulin and TPAL via tail vein

The toxicity and side effects of Plinabulin and TPAL were compared through the weight change experiment of normal Balb/c mice. Specifically, Balb/c mice were divided into three groups: 7.5mg/kg Plinabulin, 100mg/kg TPAL and 75mg/kg TPAL, with 5 mice in each group. 7.5mg/kg Plinabulin is close to its maximum tolerated dose (MTD). On the first day, the drug was administered through the tail vein, and the weight changes of the mice in the group were continuously observed.

The ordinate of Figure 20 represents the ratio of the average body weight of the five mice in the group to the average weight before administration on the first day. The change trend shows that the body weight of 7.5mg/kg Plinabulin continued to decrease in the first three days, reaching the lowest value on the third day, with a decrease of more than 5%. In contrast, the weight loss of TPAL at either 75 mg/kg or 100 mg/kg dose was lower than that of Plinabulin, and the time to return to pre-administration body weight was shorter. In other words, even if the dose of TPAL is 100 mg/kg, the toxic side effects are still lower than that of 7.5 mg/kg Plinabulin.

Example 21 Tumor inhibition and body weight changes in 4T1 tumor-bearing mice treated with Plinabulin, TPAL and MPAL

4T1 cells were inoculated subcutaneously in Balb/c mice. When the subcutaneous tumor volume increased to ^150mm3 , the mice were randomly divided into 4 groups: PBS, Plinabulin, TPAL and MPAL, with six mice in each group. Plinabulin is administered as follows: 7.5 mg/kg, administered via tail vein injection on days 1, 3, and 5; TPAL and MPAL are administered as follows: 30 mg/kg, administered via tail vein injection on days 1, 3, and 5 . The conditions of the mice were observed the next day, and the long and short diameters of the mouse tumors were measured with vernier calipers, and the weight of the mice was recorded with a balance. The drug treatment effects of each group were evaluated by calculating the mouse tumor volume and tumor inhibition rate, and the drug safety was evaluated by measuring the weight changes of the mice. Tumor volume and tumor inhibition rate were calculated by the following formula:

Calculation formula of mouse tumor volume: V=(a× ^b2 )/2

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

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

As shown in Figure 21A, compared with the PBS group, Plinabulin, TPAL and MPAL can all inhibit tumor growth. At the end of treatment, the tumor suppression rates (TSR%) of the Plinabulin, TPAL and MPAL groups were 38.6%, 47.7% and 57.3% respectively. The therapeutic effects of the TPAL and MPAL groups were comparable to those of the Plinabulin group. Figure 21B shows the weight change curve of mice. The weight of mice in the Plinabulin group continued to decrease from days 1 to 9, reaching the lowest value on day 9, with a decrease of more than 10%, reflecting obvious toxic and side effects. In contrast, there was no significant decrease in body weight of mice in the TPAL and MPAL groups, proving that TPAL and MPAL have lower toxic side effects than Plinabulin. Considering the efficacy and side effects, the therapeutic index of TPAL and MPAL is higher than that of Plinabulin.

Example 22 Results of tumor inhibition and body weight changes in CT26 tumor-bearing mice treated with Plinabulin and TPAL

CT26 cells were inoculated subcutaneously in Balb/c mice. When the subcutaneous tumor volume increased to 150 mm ³ , the mice were randomly divided into 3 groups: PBS, Plinabulin and TPAL, with seven mice in each group. Plinabulin is administered as follows: 7.5 mg/kg, administered via tail vein injection on days 1, 3, and 5. This dose is close to the maximum tolerated dose; TPAL is administered as follows: 100 mg/kg, administered on days 1, 3, and 5 Tianjing was administered via tail vein injection. The conditions of the mice were observed the next day, and the long and short diameters of the mouse tumors were measured with vernier calipers, and the weight of the mice was recorded with a balance. The drug treatment effects of each group were evaluated by calculating the mouse tumor volume and tumor inhibition rate, and the drug safety was evaluated by measuring the weight changes of the mice. Tumor volume and tumor inhibition rate were calculated by the following formula:

Calculation formula of mouse tumor volume: V=(a× ^b2 )/2

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

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

As shown in Figure 22A, compared with the PBS group, both Plinabulin and TPAL can inhibit tumor growth. At the end of treatment, the tumor inhibition rates (TSR%) of Plinabulin and TPAL were 52.9% and 57.5% respectively, and the therapeutic effect of TPAL was comparable to that of the Plinabulin group. As can be seen from the mouse body weight change curve in Figure 22B, the body weight of the mice in the Plinabulin group continued to decrease from 1 to 7 days, reaching the lowest value on the 7th day. The decrease was more than 10%, which was significantly different from the body weight of the TPAL group, reflecting obvious toxic and side effects. In contrast, there was no significant decrease in body weight of mice in the PBS and TPAL groups, proving that TPAL has lower toxic side effects than Plinabulin. Taking into account the therapeutic effects and toxic and side effects, TPAL has a higher therapeutic index.

Example 23 Tumor inhibition and body weight changes in H22 tumor-bearing mice treated with Plinabulin and TPAL

H22 cells were inoculated subcutaneously in Balb/c mice. When the subcutaneous tumor volume increased to 150 mm ³ , the mice were randomly divided into 3 groups: PBS, Plinabulin and TPAL, with seven mice in each group. The administration method of Plinabulin is as follows: 7.5 mg/kg, administered via tail vein injection on days 1, 3, and 5; the administration method of TPAL is as follows: 100 mg/kg, administered via tail vein injection on days 1, 3, and 5. The conditions of the mice were observed the next day, and the long and short diameters of the mouse tumors were measured with vernier calipers, and the weight of the mice was recorded with a balance. The drug treatment effects of each group were evaluated by calculating the mouse tumor volume and tumor inhibition rate, and the drug safety was evaluated by measuring the weight changes of the mice. Tumor volume and tumor inhibition rate were calculated by the following formula:

Calculation formula of mouse tumor volume: V=(a× ^b2 )/2

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

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

As shown in Figure 23A, compared with the PBS group, both Plinabulin and TPAL can inhibit tumor growth. At the end of treatment, the tumor inhibition rates (TSR%) of Plinabulin and TPAL were 63.9% and 56.8% respectively. The therapeutic effect of TPAL was close to that of the Plinabulin group. As can be seen from the mouse body weight change curve in Figure 23B, the body weight of the mice in the Plinabulin group reached the lowest value on the 5th day, with a decrease of more than 10%. There was a significant difference from the body weight of the TPAL group, reflecting obvious toxic and side effects. In contrast, there was no significant decrease in body weight of mice in the PBS and TPAL groups, proving that TPAL has lower toxic side effects than Plinabulin. Taking into account the therapeutic effects and toxic and side effects, TPAL has a higher therapeutic index.

Example 24 Tumor inhibition and body weight changes in 4T1 tumor-bearing mice treated with Plinabulin and TPAL

4T1 cells were inoculated subcutaneously in Balb/c mice. When the subcutaneous tumor volume increased to ^150mm3 , the mice were randomly divided into 4 groups: PBS, Plinabulin (high dose and low dose) and TPAL. Seven per group. Plinabulin is administered as follows: 7.5 mg/kg (high dose), 3 mg/kg (low dose), via tail vein injection on days 1, 3, and 5; TPAL is administered as follows: 100 mg/kg, on days 1 and 5. Administer via tail vein injection on days 3 and 5. The conditions of the mice were observed the next day, and the long and short diameters of the mouse tumors were measured with vernier calipers, and the weight of the mice was recorded with a balance. The drug treatment effects of each group were evaluated by calculating the mouse tumor volume and tumor inhibition rate, and the drug safety was evaluated by measuring the weight changes of the mice. Tumor volume and tumor inhibition rate were calculated by the following formula:

Calculation formula of mouse tumor volume: V=(a× ^b2 )/2

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

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

As can be seen from Figure 24A, compared with the PBS group, Plinabulin 7.5mg/kg, Plinabulin 3mg/kg and TPAL can all inhibit tumor growth. At the end of treatment, the tumor inhibition rates (TSR%) of the Plinabulin 7.5mg/kg, Plinabulin 3mg/kg and TPAL groups were 45.6%, 15.6% and 52.8% respectively. The therapeutic effects of the TPAL and MPAL groups were similar to those of Plinabulin 7.5mg/kg. The two groups were equivalent and better than the Plinabulin 3mg/kg group. As can be seen from the mouse body weight change curve in Figure 24B, the body weight of mice in the Plinabulin7.5mg/kg group continued to decrease from 1 to 5 days, reaching the lowest value on the 5th day, with a decrease of more than 10%, reflecting obvious toxic and side effects, proving the therapeutic effect of TPAL. Under similar circumstances, the toxic and side effects are lower. In contrast, the body weight of mice in the TPAL and Plinabulin 3mg/kg groups showed no significant decrease, while the therapeutic effect of the TPAL group was significantly improved, proving that the therapeutic effect is better under the conditions of similar toxic and side effects.

Example 25 Plinabulin, TPAL and MPAL prevent chemotherapy-induced neutropenia

Chemotherapy with cytotoxic chemotherapeutic agents suppresses the hematopoietic system, impairs host protective mechanisms, and limits the dose of chemotherapy that can be tolerated. Chemotherapy-induced neutropenia (CIN) is the most severe hematological toxicity and is associated with the risk of life-threatening infections as well as chemotherapy dose reductions and delays that may affect treatment outcomes. In this example, the ability of Plinabulin, TPAL and MPAL to prevent CIN was verified.

As shown in Figure 25A, normal healthy rats were injected with doxorubicin hydrochloride (Dox, 10 mg/kg) into the tail vein to construct a CIN model. Plinabulin, TPAL and MPAL were injected 1 hour after chemotherapy. Orbital blood was taken to monitor the absolute neutrophil count (ANC). Figure 25B body weight change curve shows that the body weight of rats in the Dox+Plinabulin group still dropped by more than 10% on day 21 compared with day -2, while the change trend of the Dox+TPAL, Dox+MPAL and Dox+Vehicle groups was the same. On day 21 The weight has returned to normal, which shows that the toxic and side effects of TPAL and MPAL are significantly lower than that of Plinabulin. Figure 25C shows that compared with the Saline group, 10 mg/kg Dox can significantly reduce neutrophils, especially on the 9th day, the number of neutrophils was as low as 0.1, proving the successful construction of the CIN model. Comprehensive analysis of ANC results from days 2 to 21 showed that there was no significant difference between the Dox+Plinabulin, Dox+TPAL, and Dox+MPAL groups, but the ANC was significantly higher than that of the Dox+Vehicle group. At 21 days, the ANC of the three combination groups could be restored to Each - 2 days level. This shows that the efficacy of TPAL and MPAL as whitening drugs is equivalent to that of Plinabulin, and the toxic and side effects are not superimposed with chemotherapy drugs, and the therapeutic index is higher.

Example 26 Combination of Plinabulin and TPAL with Mecapegfilgrastim to prevent chemotherapy-induced neutropenia

Thiopegfilgrastim is a commercialized whitening drug that can increase ANC levels immediately after administration. At the same time, ANC will also decrease quickly. The drug effect is usually 2-5 days. The results of Example 25 show that both Plinabulin and TPAL can continuously increase ANC from 9 to 21 days. This example combines Plinabulin and TPAL with Mecapegfilgrastim to study the effect of preventing CIN from -2 to 21 days.

Figure 26A is a dosing timeline, in which Dox, Plinabulin and TPAL were all administered on day 0, and Mecapegfilgrastim was injected subcutaneously on day 1. Figure 26B shows that the body weight change chart shows that compared with the Dox group, TPAL did not increase additional toxic and side effects, while the toxic and side effects of Plinabulin were superimposed on Dox. Figure 26C shows the changes in ANC in each group. Compared with the Saline group, the Dox group could significantly reduce ANC, proving that the granule deficiency model was successfully constructed. Compared with the Dox group, the two single drug groups, Plinabulin and TPAL, could significantly improve ANC. Compared with Mecapegfilgrastim single drug group, TPAL combination group can significantly improve ANC. Compared with the Plinabulin combination group, the TPAL combination group can increase the average ANC by 1.23 times, and there is a significant difference, proving that the TPAL combination treatment effect is better. Comprehensive analysis of treatment effects and toxic and side effects showed that the TPAL combination group had a higher therapeutic index.

Example 27 TPAL prevents bone marrow suppression caused by 5-fluorouracil (5-FU)

Chemotherapy usually causes bone marrow suppression, and the degree of bone marrow suppression caused by different chemotherapy drugs varies. Among them, studies have shown that high doses of 5-FU can cause strong bone marrow suppression. Therefore, this experiment selected 150 mg/kg 5-FU for the construction of myelosuppression model. Myelosuppression refers to the decrease in the activity of blood cell precursor cells in the bone marrow, which is a comprehensive impact on blood cells, including platelets, red blood cells, lymphocytes, neutrophils and white blood cells. In this experiment, SD rats were divided into three groups: control group, 5-FU single drug group and 5-FU+TPAL combination group, with four rats in each group. The dose of TPAL was 3.75mg/kg by gavage. Give medicine. TPAL administration was performed one hour before 5-FU administration, and this day was recorded as day 0. Starting from the sixth day, blood was collected from the orbit of the rats every two days, and absolute counts and statistical analysis of the above five blood cells were performed.

Through comprehensive analysis of various blood cells, it was found (Figure 27A) that compared with the Control group, various types of blood cells were significantly reduced in the 5-FU group, proving that the bone marrow suppression model was successfully constructed. Among them, there was a significant difference in platelets between the TPAL group and the 5-FU single drug group starting from the eighth day, and the significant difference was *** on the eighth day; there was a significant difference in red blood cells starting from the tenth day, and on the twelfth day Day is **; for lymphocytes, neutrophils, and leukocytes, the most significant differences were at day 10. Figure 27B shows the absolute count values of various blood cells on a certain day, where platelets and red blood cells are on the eighth and twelfth days respectively, while lymphocytes, neutrophils and leukocytes are all on the tenth day. Based on the above data, TPAL can comprehensively increase blood cell levels and prevent chemotherapy-induced bone marrow suppression.

The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (10)

1. A Plinabulin derivative with a structure of formula (I),
   

   Among them, R ₂ , R ₃ and R ₄ are independently selected from hydrogen, hydrocarbyl, thiohydrocarbyl, oxyhydrocarbyl, halogenated hydrocarbyl, halogen, nitro, amino, hydroxyl, carboxyl, ester group, amide group or the above functional groups. combination;

   R ₁ and R ₅ are independently selected from H or a group represented by formula (a), and R ₁ and R ₅ are different.
2. The derivative according to claim 1, characterized in that the two types of plinabulin derivatives of the formula (I) structure are selected from the structures represented by formula (I-1) or formula (I-2):
   
   
3. A method for preparing the Plinabulin derivative of the formula (I) according to any one of claims 1 to 2, comprising the following steps:

   Catalyzed by cesium carbonate, (Z)-1-acetyl-3-((5-(tert-butyl)-1H-imidazol-4-yl)methylene)piperazine-2,5-dione and benzene It is obtained by reacting diformaldehyde in N,N-dimethylformamide.
4. The preparation method according to claim 3, characterized in that the phthalaldehyde is terephthalaldehyde containing R ₂ to R ₅ functional groups or isophthalaldehyde containing R ₁ to R ₄ functional groups.
5. The use of the Plinabulin derivative with the structure of formula (I) according to any one of claims 1 to 2 in the preparation of anti-tumor drugs.
6. The application according to claim 5, wherein the tumors include malignant tumors of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, oral cavity cancer, laryngeal cancer, intracranial tumors, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer , stomach cancer, colorectal cancer, sigmoid colon and rectal cancer, liver cancer, pancreatic cancer and periampullary cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, testicular malignant tumors, penile cancer, cervical cancer, endometrial cancer, ovary One or more of carcinoma, fibrous histiocytic carcinoma, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma, and multiple myeloma.
7. The application of the Plinabulin derivative with the structure of formula (I) according to any one of claims 1 to 2 in the preparation of a drug for treating side effects of chemotherapy; the side effects of chemotherapy include neutropenia and bone marrow suppression.
8. The application according to claim 7, characterized in that the drug causing neutropenia is selected from the group consisting of docetaxel, paclitaxel, taxanes, cyclophosphamide, ifosfamide, cisplatin, carboxyl Platinum, etoposide, gemcitabine, topotecan, irinotecan, doxorubicin, epirubicin, daunorubicin, valrubicin and their pharmaceutically acceptable salts;

   The drugs that cause bone marrow suppression are selected from the group consisting of docetaxel, paclitaxel, taxanes, cyclophosphamide, ifosfamide, cisplatin, carboplatin, etoposide, gemcitabine, topotecan, irinotecan, alfa Mycin, epirubicin, daunorubicin, valrubicin, fluorouracil and their pharmaceutically acceptable salts.
9. The application according to claim 7, characterized in that the drug is a tablet, capsule, granule, oral liquid, sustained release preparation, controlled release preparation, nano preparation or injection.
10. A medicine comprising a plinabulin derivative with a structure of formula (I) according to any one of claims 1 to 2.