2,5-diketopiperazine compound having Anti-tumor effect and use thereof in preparation of drug for treating and / or preventing neutropenia

Abstract

Disclosed in the present invention are a 2,5-diketopiperazine compound having an anti-tumor effect and use thereof in the preparation of a drug for treating and / or preventing neutropenia. The 2,5-diketopiperazine compound has a novel structure, has good activity and water solubility, and can overcome the defects that the poor water solubility of plinabulin is not conducive to drug preparation and leads to difficulty in clinical administration. The 2,5-diketopiperazine compound represented by formula (I) of the present invention or a pharmaceutically acceptable salt thereof can be used for treating and / or preventing neutropenia and improving toxic and side effects in clinical tumor treatment, and meanwhile, has an anti-tumor effect. Also disclosed in the present invention is use of such compounds in combination with a granulocyte colony-stimulating factor (G-CSF) biological formulation in a drug for treating and / or preventing neutropenia.

Description

2,5-diketopiperazine compound with anti-tumor efficacy and its application in the preparation of a drug for treating and / or preventing neutropenia TECHNICAL FIELD

[0001] The present application belongs to the field of chemistry and medicine, and particularly relates to a 2,5-diketopiperazine compound with anti-tumor efficacy and its application in the preparation of a drug for treating and / or preventing neutropenia. BACKGROUND

[0002] Neutropenia is a common hematologic toxicity during chemotherapy, and accurate assessment, prevention and treatment are crucial for the prognosis of tumor patients. Chemotherapy is one of the main treatment methods for malignant tumors, and myelosuppression is the most common dose-limiting toxicity of chemotherapy, among which neutropenia is very common. Neutropenia is a common and potentially life-threatening complication of cytotoxic myelosuppressive chemotherapy. Studies have shown that individuals with neutropenia are more susceptible to infection, and complications are life-threatening. The main method for treating neutropenia is granulocyte colony-stimulating factor (G-CSF) drug treatment, which is developed by Amgen Company in the United States (trade name: Neulasta), and is currently the main drug for clinical treatment of CIN. The global sales amount reaches 8 billion US dollars for the high-risk fever population. However, due to its mechanism of action, it has the disadvantages of limited effect in the first week after clinical chemotherapy, inevitable side effects, and the like. Therefore, there is a huge unmet medical need for the clinical treatment of CIN.

[0003] Plinabulin is derived from the natural product Phenylahistin isolated from the marine fungus Aspergillus fumigatus, and is a new type of 2,5-diketopiperazine (DKP) heterocyclic compound. It is a colchicine microtubule inhibitor that prevents microtubule assembly by affecting the dynamic cycle of microtubule depolymerization-polymerization, thereby interfering with cell division, causing cells to stop in the early mitosis, and inducing cell death. Recent studies have found that plinabulin is also a differentiation immune and stem cell regulator, as well as a guanine nucleotide exchange factor (GEF-H1) activator, which can target and change the tumor microenvironment and destroy tumor blood vessels through multiple mechanisms of action. In addition, plinabulin is an effective antigen-presenting cell (APC) inducer (by activating dendritic cell maturation), and its persistent anti-cancer effect is related to its T cell activation. Further studies have shown that plinabulin can also protect against increasing white blood cell growth by activating the release of cytokines. Due to the good effect of plinabulin in treating neutropenia, a new drug NDA has been applied.

[0004] The chemical structural formula of plinabulin is as follows, and its molecular formula is C19 H 20 N4O2, molecular weight 336.39, CAS number 714272-27-2, which has good stability but poor water solubility.

[0005] Patent CN105705148 discloses a formulation of plinabulin, which contains 40% polyethylene glycol-15 hydroxystearate HS-15 and 60% propylene glycol, and the drug concentration is 4 mg / mL. However, it is known that HS-15 is not an absolutely safe excipient. The former marketed product, rolapitant fat emulsion The amount of HS-15 in the fat emulsion is 4.4%, and the single dose is 92.5 ml / bottle, and the single dose is 4.07 g, which is close to the formulation disclosed in CN105705148. However, VARUBI fat emulsion has been withdrawn from the market due to serious allergic reactions after marketing.

[0006] The difficulty in preparing a medicine due to poor water solubility is a technical barrier existing in the prior art. The prior art has been modified on the basis of the structure of plinabulin, but the problem of poor water solubility of plinabulin has not been solved. SUMMARY

[0007] The technical problem to be solved by the present application is to overcome the defect that there is a lack of drugs for effectively treating neutropenia in the prior art, and to provide a 2,5-diketopiperazine compound having an anti-tumor effect and an application thereof in the preparation of a drug for treating and / or preventing neutropenia. The 2,5-diketopiperazine compound provided by the present application has obvious effects in improving the toxic and side effects of clinical tumor treatment, and has an anti-tumor effect.

[0008] The present application solves the above technical problems by the following technical solutions:

[0009] The present application provides a 2,5-diketopiperazine compound having an anti-tumor effect or a pharmaceutically acceptable salt thereof, the structural formula of which is shown as formula (I):

[0010] In the formula:

[0011] n = 0-3; m = 0-3; x = 0-5;

[0012] R 1 is a monosubstituted or polysubstituted group selected from hydrogen, deuterium, halogen, C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl, C1-C8 alkyl substituted by one or more halogens, C1-C8 alkoxy, C1-C8 alkoxy substituted by one or more halogens, benzoyl, benzoyl substituted by one or more halogens, phenoxy, phenoxy substituted by one or more halogens, carboxyl, cyano, hydroxyl, nitro or methylthiophenyl.

[0013] L is C1-C8 heteroalkylene, substituted C1-C8 heteroalkylene, C1-C8 alkylene, or substituted C1-C8 alkylene; 2 substituted C1-C8 heteroalkylene, C1-C8 alkylene, or substituted C1-C8 alkylene; 2 substituted C1-C8 alkylene;

[0014] R 2 is C1-C8 alkyl or C3-C 10 cycloalkyl;

[0015] R 3 , R 4 , R 6 , and R 7 is hydrogen, deuterium, C1-C8 alkyl, substituted C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl, C1-C8 alkoxy, C3-C 3-1 cycloalkyl, C6-C 10 aryl, or 3-10 membered heterocycloalkyl; wherein R 10 is O-R 3-1 , wherein R 3-1-1 is C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl, or C1-C8 alkyl substituted with one or more C6-C 3-1-1 aryls; 10 substituted C1-C8 alkyl, C1-C8 alkenyl, or C1-C8 alkynyl;

[0016] Z is O, S, SO, SO2, or N(R 5 );

[0017] R 5 is hydrogen, deuterium, C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl, substituted C6-C 5-1 aryl, benzyl, substituted benzyl, -C(=O)-R 10 , or -S(=O)2-R 5-2 ; wherein R 5-3 is halogen; R 5-4 is hydrogen, C1-C8 alkoxy, benzyloxy, substituted benzyloxy, C1-C8 alkyl, C3-C 5-1 cycloalkyl, C2-C8 alkenyl, C6-C 5-2 aryl, or NR 5-2 R 10 ; R 10 is C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl, or C6-C 5-3-1 aryl; wherein R 5-3-2 and R 5-3 are the same or different; 10 substituted C1-C8 alkyl, C1-C8 alkenyl, or C1-C8 alkynyl; 5-3-1 substituted C1-C8 alkyl, C1-C8 alkenyl, or C1-C8 alkynyl.5-3-2 C1-C8alkyl, C1-C8alkenyl, C1-C8alkynyl, or C3-C 10 cycloalkyl; R 5-4 is C1-C8alkyl or C6-C 10 aryl.

[0018] Further, the heteroatom in the C1-C8heteroalkylene of L is selected from one or more of N, O, S, and Se, the number of heteroatoms being from 1 to 4. 3 , R 4 , R 6 and R 7 the heteroatom in the 3-10 membered heterocycloalkyl of R

[0019] Further, in the structural formula:

[0020] n = 0-1; m = 0-1; x = 0-2;

[0021] R 1 is a mono- or poly-substituted group selected from hydrogen, deuterium, vinyl, ethynyl, methyl, trifluoromethoxy, methoxy, halogen, cyano, or benzoyl;

[0022] L is

[0023] R 2 is methyl, ethyl, isopropyl, t-butyl, or cyclopropyl;

[0024] R 3 , R 4 , R 6 and R 7 is hydrogen, deuterium, methyl, or ethyl, methyl substituted with R 3-1 , cyclohexyl, or pyridyl; wherein R 3-1 is O-R 3-1-1 , wherein R 3-1-1 is methyl, ethyl, n-propyl, or n-butyl;

[0025] Z is O, S, SO, SO2, or N(R 5 );

[0026] R 5 is hydrogen, deuterium, methyl, ethyl, n-propyl, isopropyl, n-butyl, or 2-methylpropyl, C1-C8alkenyl, C1-C8alkynyl, phenyl substituted with one or more R 5-1 , benzyl substituted with one or more R 5-2 , -C(=O)-R 5-3 , or -S(=O)2-R 5-4 ; wherein R5-1 halogen; R 5-2 hydrogen, C1-C8alkoxy, benzyloxy, benzyloxy substituted by one or more R 5-2 substituted benzyloxy, methyl, ethyl, n-propyl, i-propyl or n-butyl, cyclopentyl or cyclohexyl, C2-C8alkenyl, C6-C 10 aryl or NR 5-3-1 R 5-3-2 ; R 5-3 C1-C8alkyl, C1-C8alkenyl, C1-C8alkynyl or C6-C 10 aryl; wherein, R 5-3-1 and R 5-3-2 hydrogen, C1-C8alkyl, C1-C8alkenyl, C1-C8alkynyl or C3-C6cycloalkyl; R 5-4 C1-C8alkyl or C6-C 10 aryl.

[0027] Further, the 2,5-diketopiperazine compound is specifically compounds P1-P42 and pharmaceutically acceptable salts thereof, and the structural formulae are respectively:

[0028] The application provides a pharmaceutical composition containing the 2,5-diketopiperazine compound or a stereoisomer, a tautomer, a racemate, a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier and a pharmaceutical excipient.

[0029] Further, the dosage form includes tablets, capsules, pills, injections, and external preparations.

[0030] The application provides application of the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition as a microtubule inhibitor to inhibit microtubulin.

[0031] The application provides application of the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparation of a drug for treating tumors.

[0032] The application provides application of the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparation of a drug for treating tumors in combination with a chemotherapeutic agent.

[0033] Further, the chemotherapeutic agent is docetaxel.

[0034] The application provides application of the 2,5-diketopiperazine compound or its pharmaceutically acceptable salt with anti-tumor efficacy in preparation of a drug for treating and / or preventing neutropenia.

[0035] Further, the pharmaceutically acceptable salt of the compound as shown in formula (I) is a salt formed by the compound as shown in formula (I) and a pharmaceutically acceptable acid in a molar ratio of 1:2 or 1:3.

[0036] Further, the pharmaceutically acceptable salt of the 2,5-diketopiperazine compound as shown in formula (I) is a hydrochloride or a methanesulfonate.

[0037] Further, the neutropenia is caused in the process of treating cancer by administering chemotherapy or by administering radiotherapy.

[0038] Further, the cancer includes liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer and prostate cancer.

[0039] Further, the chemotherapy administers a chemical therapeutic agent or administers a chemical therapeutic composition.

[0040] Further, the chemical therapeutic agent is docetaxel, paclitaxel or cyclophosphamide; the chemical therapeutic composition is a combination of "docetaxel, doxorubicin and cyclophosphamide", a combination of "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide" or a combination of "docetaxel and cyclophosphamide". Wherein, the single use of docetaxel, paclitaxel, cyclophosphamide and the like, the combination use of "docetaxel, doxorubicin and cyclophosphamide (TAC)", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide" or "docetaxel and cyclophosphamide (TC)".

[0041] Further, when the neutropenia is induced by administering chemotherapy, a single dose of the 2,5-diketopiperazine compound as shown in formula (I) or its pharmaceutically acceptable salt can be administered alone in a chemotherapy cycle.

[0042] Further, when the neutropenia is induced by administering chemotherapy, the 2,5-diketopiperazine compound or its pharmaceutically acceptable salt is administered after administering the chemical therapeutic agent or the chemical therapeutic composition.

[0043] Further, the administration dose of the 2,5-diketopiperazine compound or its pharmaceutically acceptable salt in each treatment cycle is less than 80 mg / kg.

[0044] Further, the 2,5-diketopiperazine compound or its pharmaceutically acceptable salt is administered within 24 hours after administering the chemical therapeutic agent or the chemical therapeutic composition.

[0045] Further, the 2,5-diketopiperazine compound or the pharmaceutically acceptable salt thereof is administered within 12 hours after the administration of the chemotherapeutic agent or the chemotherapeutic composition, for example, within 0.5 hours after the administration.

[0046] Further, when the neutropenia is induced by the administration of "docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide", or "docetaxel and cyclophosphamide", the administration dose of the 2,5-diketopiperazine compound or the pharmaceutically acceptable salt thereof as shown in formula (I) is less than 80 mg / kg per treatment cycle.

[0047] Further, when the neutropenia is induced by the administration of "docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide", or "docetaxel and cyclophosphamide", the method comprises administering the 2,5-diketopiperazine compound or the pharmaceutically acceptable salt thereof within 24 hours after the administration of "docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide", or "docetaxel and cyclophosphamide".

[0048] Further, the 2,5-diketopiperazine compound or the pharmaceutically acceptable salt thereof can be administered by any suitable route in the art, including oral, injection (e.g. intravenous, intramuscular, subcutaneous), etc., for example, by intravenous infusion.

[0049] Further, the 2,5-diketopiperazine compound or the pharmaceutically acceptable salt thereof can be administered according to the body weight of the individual, and the administration dose can range from 0.5 to 80 mg / kg (for a single dose), for example, 1 to 65 mg / kg (for a single dose), specifically, the administration dose can be 6 mg / kg, 12 mg / kg for intravenous injection, and / or 27 mg / kg or 64 mg / kg for oral administration.

[0050] Further, the above-mentioned dose of the 2,5-diketopiperazine compound or the pharmaceutically acceptable salt thereof can be administered at least once a week, for example, once every 7 days.

[0051] Further, the method comprises simultaneously administering the 2,5-diketopiperazine compound or the pharmaceutically acceptable salt thereof as shown in formula (I).

[0052] Further, when the individual in need thereof has liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer or prostate cancer, the method comprises identifying a patient having the liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer or prostate cancer; and administering a 2,5-diketopiperazine compound as shown in formula (I) or a pharmaceutically acceptable salt thereof at a dose of about 0.5 mg / kg to 80 mg / kg.

[0053] The present application also provides a pharmaceutical composition comprising about 0.5 mg to 80 mg of a 2,5-diketopiperazine compound as shown in formula (I) or a pharmaceutically acceptable salt thereof.

[0054] In some embodiments, the pharmaceutical composition comprises about 1 mg to 65 mg of a 2,5-diketopiperazine compound as shown in formula (I) or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0056] The present application provides a pharmaceutical composition comprising a 2,5-diketopiperazine compound as shown in formula (I) or a pharmaceutically acceptable salt thereof having anti-tumor efficacy and a granulocyte colony-stimulating factor (G-CSF) drug.

[0057] The pharmaceutical composition comprises a single dose of 0.5 mg to 80 mg of a 2,5-diketopiperazine compound as shown in formula (I) or a pharmaceutically acceptable salt thereof having anti-tumor efficacy.

[0058] The present application provides use of the pharmaceutical composition in the preparation of a drug for treating and / or preventing neutropenia.

[0059] Compared with the prior art, the present application has the advantages and beneficial effects that the 2,5-diketopiperazine compound as shown in formula (I) or a pharmaceutically acceptable salt thereof is used for treating and / or preventing neutropenia, improving the side effects of clinical tumor treatment, and having anti-tumor efficacy. The 2,5-diketopiperazine compound pharmaceutically acceptable salt has good water solubility, and can be administered by intravenous injection in physiological saline / 5% glucose solution or oral administration. The present application also discloses use of such a compound alone or in combination with a granulocyte colony-stimulating factor (G-CSF) biological agent in a drug for treating and / or preventing neutropenia. BRIEF DESCRIPTION OF DRAWINGS

[0060] Figure 1 is a curve of the body weight change of each group of animals during the test period of different dose series of compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0061] Figure 2-1 is a plot of the change in tumor volume of each group of animals during the test period for different dose series of compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0062] Figure 2-2 is a plot of the change in RTV of each group of animals during the test period for different dose series of compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0063] Figure 2-3 is a plot of the change in T / C% of each group of animals during the test period for different dose series of compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0064] Figure 3-1 is a plot of the tumor weight distribution of each group of animals at the end of the test for different dose series of compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0065] Figure 3-2 is a plot of the tumor inhibition rate of each group of animals at the end of the test for different dose series of compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0066] Figure 4 is a plot of the change in body weight of rats induced by intraperitoneal injection of cyclophosphamide for different salt types and administration series of compounds (P32, P34, P36, P37).

[0067] Figure 5 is a plot of the change in neutrophil of rats induced by intraperitoneal injection of cyclophosphamide for different salt types and administration series of compounds (P32, P34, P36, P37).

[0068] Figure 6 is a plot of the change in white blood cell count of rats induced by intraperitoneal injection of cyclophosphamide for different salt types and administration series of compounds (P32, P34, P36, P37).

[0069] Figure 7 is a plot of the change in body weight of rats induced by intraperitoneal injection of cyclophosphamide for series of compounds (P29-0, P30-0, P31-0, P32-0, P32-1). * P≤0.05, normal control group vs model control group; # P≤0.05, ## P≤0.01, model control group vs each treatment group.

[0070] Figure 8 is a plot of the change in neutrophil of rats induced by intraperitoneal injection of cyclophosphamide for series of compounds (P29-0, P30-0, P31-0, P32-0, P32-1). * P≤0.05, ** P≤0.01, *** P≤0.001, normal control group vs model control group; # P≤0.05,## P < 0.01, model control vs each treatment group. P < 0.05, P < 0.01, P < 0.001, P32-0 group vs each treatment group.

[0071] Figure 9 is the effect of a series of compounds (P29-0, P30-0, P31-0, P32-0, P32-1) on the number of white blood cells in rats induced by intraperitoneal injection of cyclophosphamide; ** P < 0.01, *** P < 0.001, normal control vs model control group; # P < 0.05, ## P < 0.01, ### P < 0.001, model control vs each treatment group; P < 0.05, P < 0.01, P < 0.001, P32-0 group vs each treatment group.

[0072] Figure 10 is the effect of a series of compounds (P5-0, P10-0, P11-0, P12-0, P32-0) on the body weight of rats induced by intraperitoneal injection of cyclophosphamide; * P < 0.05, normal control vs model control group; # P < 0.05, ## P < 0.01, model control vs each treatment group.

[0073] Figure 11 is the effect of a series of compounds (P5-0, P10-0, P11-0, P12-0, P32-0) on the number of neutrophils in rats induced by intraperitoneal injection of cyclophosphamide; * P < 0.05, ** P < 0.01, *** P < 0.001, normal control vs model control group; # P < 0.05, ## P < 0.01, model control vs each treatment group; P < 0.05, P < 0.01, P32-0 group vs each treatment group.

[0074] Figure 12 is the effect of a series of compounds (P5-0, P10-0, P11-0, P12-0, P32-0) on the number of white blood cells in rats induced by intraperitoneal injection of cyclophosphamide; * P < 0.05, *** P < 0.001, normal control vs model control group; # P < 0.05, ## P < 0.01, model control vs each treatment group; P < 0.05, P < 0.01, P < 0.001, P32-0 treatment group vs each treatment group.

[0075] Figure 13 is the effect of a series of compounds (P32-0, P38-0, P39-0, P40-0, P41-0, P42) on body weight changes in rats induced by intraperitoneal injection of cyclophosphamide; # P<0.05, model control group vs each treatment group.

[0076] Figure 14 is the effect of a series of compounds (P32-0, P38-0, P39-0, P40-0, P41-0, P42) on neutrophil changes in rats induced by intraperitoneal injection of cyclophosphamide; * P<0.05, ** P<0.01, *** P<0.001, normal control group vs model control group; # P<0.05, ## P<0.01, ### P<0.001, model control group vs each treatment group.

[0077] Figure 15 is the effect of a series of compounds (P32-0, P38-0, P39-0, P40-0, P41-0, P42) on white blood cell count changes in rats induced by intraperitoneal injection of cyclophosphamide; * P<0.05, *** P<0.001, normal control group vs model control group; # P<0.05, ## P<0.01, model control group vs each treatment group.

[0078] Figure 16. Effect of combination of compound P32-0 and G-CSF on body weight changes in rats induced by cyclophosphamide, # P≤0.05, ## P≤0.01, model control group vs each administration group; ∧∧ P≤0.01, single drug group vs combination administration group.

[0079] Figure 17. Effect of combination of compound P32-0 and G-CSF on neutrophil changes in rats induced by cyclophosphamide, * P≤0.05, ** P≤0.01, *** P≤0.001, normal control group vs model control group; # P≤0.05, ## P≤0.01, ### P≤0.001, model control group vs each treatment group; ∧ P≤0.05, ∧∧ P≤0.01, single drug group vs combination administration group or plinabulin treatment group.

[0080] Figure 18. Effect of Compound P32-0 in combination with G-CSF on the number of white blood cells in cyclophosphamide-induced rats. * P < 0.05, *** P < 0.001, normal control vs. model control; # P < 0.05, ## P < 0.01, ### P < 0.001, model control vs. each treatment group; ∧ P < 0.05, ∧∧ P < 0.01, single agent vs. combination or plinabulin treatment. DETAILED DESCRIPTION

[0081] DEFINITIONS

[0082] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail. Unless otherwise indicated, terms used herein have the same meaning as they would to one of ordinary skill in the art in the field of the disclosure.

[0083] As used herein, "individual" refers to a human or non-human mammal, such as a dog, cat, mouse, rat, cow, sheep, pig, goat, non-human primate, or bird, such as a chicken, and any other vertebrate or invertebrate.

[0084] As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent that is effective, to a certain extent, in alleviating one or more symptoms of a disease or condition or reducing the likelihood of its onset, and includes curing the disease or condition.

[0085] The term "prevention" refers to treatment of an individual who has not yet shown symptoms of a disease or condition, but who is predisposed or otherwise at risk of developing the particular disease or condition, whereby the treatment reduces the likelihood of the patient developing the disease or condition.

[0086] The term "treatment" refers to treatment of an individual already suffering from a disease or condition.

[0087] The term "pharmaceutically acceptable salt" refers to a salt of a compound that retains the biological effectiveness and properties of the compound and that is useful in medicine and is not biologically or otherwise undesirable. In many cases, the compounds disclosed herein are capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids as well as organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable salts can also be formed using inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, alkali metal bases, such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are ammonium, potassium, sodium, calcium, and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0088] The positive progress effect of the present application is that the 2,5-diketopiperazine compounds or pharmaceutically acceptable salts thereof as shown in formula (I) can be used for treating and / or preventing neutropenia, especially for treating neutropenia caused by chemotherapeutic drugs such as docetaxel, doxorubicin, and cyclophosphamide (TAC) or docetaxel and cyclophosphamide (TC), and improving the side effects of clinical tumor treatment. In the experiment with ponatinib as the control group, the treatment effect of the part of 2,5-diketopiperazine compounds or pharmaceutically acceptable salts thereof on neutropenia is better than that of ponatinib. After the part of compounds are salified, they have good water solubility, can be dissolved in the clinically commonly used normal saline / 5% glucose aqueous solution for intravenous injection, and have good development prospects.

[0089] The technical solutions of the present application are further described in detail in combination with the following specific examples. In the following examples, the experimental methods used are conventional methods, and the materials, reagents, and the like used can be purchased from chemical or biological reagent companies, unless otherwise specified.

[0090] The compounds involved in the following examples are specifically shown as follows:

[0091] Example 1: Synthesis of compound P1

[0092] Step 1:

[0093] Into a 50 ml dry round-bottom flask, 2 (298 mg, 1.08 mmol), 1 (355 mg, 2.17 mmol), cesium carbonate (708 mg, 2.17 mmol), potassium iodide (180 mg, 1.08 mmol), 4A molecular sieves (500 mg), N,N-dimethylformamide (6 ml) were added successively, evacuated, and protected by nitrogen. The reaction was stirred at 70 °C for 20 h. After the reaction, the reaction solution was transferred into a 100 ml single-neck flask, washed with ethanol, concentrated under reduced pressure, redissolved with EtOH:DCM = 1:5, suction filtered, the filter cake was washed with EtOH:DCM = 1:5, and concentrated under reduced pressure to obtain 348 mg of brownish solid crude product 3 with a yield of 80.00%. Without purification, it was directly used in the next step.

[0094] Step 2:

[0095] Into a 1 L dry single-neck flask, 4 (14.2 g, 0.10 mol), 5 (33.5 g, 0.11 mmol), and toluene (200 ml) were added. The reaction system was refluxed for 17 h and then concentrated. The crude product was purified by column chromatography to obtain yellow oil 6 (10.9 g, yield: 65%). ESI-MS: m / z = 169 (M+H).

[0096] Step 3:

[0097] Into a 25 ml dry brown round-bottom flask, 3 (351 mg, 0.87 mmol), 6 (190 mg, 1.13 mmol), cesium carbonate (425 mg, 1.30 mmol), anhydrous sodium sulfate (247 mg, 1.74 mmol), N,N-dimethylformamide (6 ml) were added successively, evacuated, and protected by nitrogen. The reaction was stirred at 45 °C for 18 h. LC-MS was used to monitor the reaction. After the reaction, the reaction solution was transferred into a 100 ml single-neck flask, washed with ethanol, concentrated under reduced pressure, and column chromatography to obtain the target product P1 with a yield of 17.52%. ESI-MS: m / z = 512 (M+H).

[0098] The synthesis steps of compounds P2-P4 refer to compound P1.

[0099] Example 2: Synthesis of compound P5

[0100] Step 1, Preparation of 3-fluorocinnamaldehyde 8

[0101] Into a 250 mL three-necked flask, 3-fluorobenzaldehyde 7 (2.00 g, 16.11 mmol) was added, 70 mL of toluene was added, (formylmethylene)triphenylphosphonium 5 (5.39 g, 17.72 mmol) was added, and the temperature was raised to 120 °C for 48 h. The reaction was stopped. The reaction solution was concentrated to sand, and column chromatography (PE-PE:EA = 100:1) was performed to obtain 710 mg of pure product 7, with a yield of 29%. MS (ESI): m / z 151.1 [M+H] + .

[0102] Step 2, Preparation of compound P5 (3Z,6Z)-3-fluorophenylallylidene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0103] Into a 50 mL dry round-bottom flask, (Z)-1-acetyl-3-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione 3 (1.59 g, 3.94 mmol), cesium carbonate (1.93 g, 5.91 mmol), sodium sulfate (1.12 g, 7.88 mmol), N,N-dimethylformamide (20 ml), and 3-fluorocinnamaldehyde 8 (0.71 g, 4.73 mmol) were sequentially added. The reaction was carried out under nitrogen protection in a 50 °C oil bath for 24 h. The reaction was monitored until completion. The reaction solution was added dropwise into 200 mL ice water, and yellow solid was precipitated. After filtration, the filter cake was dried to obtain 280 mg of crude product as a yellow solid. After column chromatography, 63 mg was obtained, with a yield of 3.2%. MS (ESI): m / z 494.3 [M+H] + .

[0104] Step 3, Preparation of P5 hydrochloride:

[0105] Into a 25 mL single-necked dry round-bottom flask, P5 (50.0 mg, 0.10 mmol), methanol (5 mL), and hydrogen chloride in methanol solution (4 mol / L, 2 mL) were sequentially added. The reaction was carried out at 0 °C for 2 h, with light protection throughout. After the reaction was completed, the reaction solution was concentrated, a small amount of MeOH was added to just dissolve the product, and the solution was added dropwise into 10 mL ethyl acetate. After the solid was precipitated, it was filtered and dried to obtain 48 mg of light yellow solid.

[0106] 1H NMR (600 MHz, DMSO-d6) δ 11.86 (s, 1H), 11.32 (s, 1H), 10.89 (s, 1H), 8.03 (s, 1H), 7.81 (dd, J = 15.2, 12.3 Hz, 1H), 7.49 (d, J = 10.7 Hz, 1H), 7.44 - 7.40 (m, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.13-7.10 (m, 1H), 6.91 (d, J = 15.2 Hz, 1H), 6.70 (s, 1H), 6.53 (d, J = 12.2 Hz, 1H), 4.16 (t, J = 7.2 Hz, 2H), 3.95 (d, J = 12.5 Hz, 2H), 3.84-3.80 (m, 2H), 3.40-3.39 (m, 2H), 3.26-3.22 (m, 1H), 3.14-3.10 (m, 2H), 3.09 - 3.00 (m, 2H), 2.19-2.15 (m, 2H), 1.35 (d, J = 7.1 Hz, 6H).

[0107] The synthesis of compounds P6-P9 is described with reference to compound P5.

[0108] Example 3: Synthesis of compound P10

[0109] Step 1, Preparation of 3-cyanocinnamaldehyde 10:

[0110] 3-cyanobenzaldehyde 9 (2.00 g, 15.25 mmol) was added to a 250 mL three-necked flask, 70 mL of toluene was added, (formylmethylene)triphenylphosphonium 5 (5.10 g, 16.71 mmol) was added, and the temperature was raised to 120 degrees for 48 hours. The reaction was stopped. The reaction liquid was concentrated to sand, and column chromatography was used to obtain 820 mg of pure product with a yield of 34%. MS (ESI): m / z 158.1 [M+H] + .

[0111] Step 2, Preparation of compound P10 (3Z,6Z)-3-cyanophenylallylidene-6-((5- isopropyl-1-(-3-morpholino)propylimidazol-4-yl)methylene)piperazine-2,5-dione:

[0112] Into a 100 mL dry round bottom flask, add (Z)-1 -acetyl-3-((5-isopropyl-1 -(3- morpholinopropyl)imidazol-4-yl)methylene)piperazine-2,5-dione 3 (1.75 g, 4.35 mmol), cesium carbonate (2.12 g, 6.52 mmol), sodium sulfate (1.23 g, 8.70 mmol), N,N- dimethylformamide (30 ml), 3-cyanocinnamaldehyde 10 (0.82 g, 5.22 mmol) in sequence, evacuate, protect with nitrogen, put into 50 degree oil bath, avoid light, react for 24 h. Monitor the reaction to completion, drop the reaction into 200 mL ice water, there is yellow solid precipitated, filter, dry the filter cake to get the crude product as yellow solid 470 mg, column chromatography to get 130 mg, HPLC purity 99%, yield 6.0%. m.p = 207-209 °C.

[0113] 1 H NMR (600 MHz, DMSO-d6) δ 11.96 (s, 1H), 10.87 (s, 1H), 7.88 (s, 1H), 7.84-7.74 (m, 2H), 7.57 (d, J = 7.0 Hz, 1H), 7.42-7.35 (m, 2H), 6.88 (d, J = 15.2 Hz, 1H), 6.71 (s, 1H), 6.53 (d, J = 12.1 Hz, 1H), 4.05 (t, J = 7.1 Hz, 2H), 3.58 (t, J = 4.3 Hz, 4H), 3.26-3.21 (m, 1H), 2.38-2.30 (m, 4H), 2.25 (t, J = 6.7 Hz, 2H), 1.89-1.80 (m, 2H), 1.34 (d, J = 7.1 Hz, 6H). MS (ESI): m / z 501.3 [M+H] + .

[0114] Step 3, preparation of P10 hydrochloride salt:

[0115] Into a 25 mL single-neck dry round-bottom flask, add P10 (70.0 mg, 0.14 mmol), methanol (5 mL), drop hydrogen chloride methanol solution (4 mol / L, 3 mL) at 0 degrees, avoid light throughout, after adding, naturally rise to room temperature, react for 2 h, concentrate the reaction liquid, add a small amount of MeOH to just dissolve the product, and drop into 10 mL ethyl acetate, precipitate solid, stand for 20 minutes, then filter, dry the filter cake to get light yellow solid 71 mg.

[0116] 1H NMR (600 MHz, DMSO-d6) δ 11.85 (s, 1H), 11.18 (s, 1H), 10.91 (s, 1H), 8.02 (s, 1H), 7.81 (dd, J = 15.5, 12.5 Hz, 2H), 7.57 (d, J = 6.9 Hz, 1H), 7.41 - 7.35 (m, 2H), 6.89 (d, J = 15.2 Hz, 1H), 6.70 (s, 1H), 6.53 (d, J = 12.2 Hz, 1H), 4.16 (t, J = 7.1 Hz, 2H), 3.95 (d, J = 12.5 Hz, 2H), 3.85 - 3.77 (m, 2H), 3.45-3.41 (m, 2H), 3.27-3.20 (m, 1H), 3.14-3.10 (m, 2H), 3.09-3.01 (m, 2H), 2.19-2.15 (m, 2H), 1.35 (d, J = 7.1 Hz, 6H). m.p = 242-245 °C.

[0117] Example 4: Synthesis of compound P11

[0118] Step 1, Preparation of 3-vinylcinnamaldehyde 12:

[0119] 3-vinylbenzaldehyde 11 (2.00 g, 15.13 mmol) was added to a 250 mL three-necked flask, 70 mL of toluene was added, (formylmethylene)triphenylphosphonium 5 (5.06 g, 16.64 mmol) was added, and the temperature was raised to 120 degrees for 48 h. The reaction was stopped. The reaction liquid was concentrated to sand, column chromatography (PE-PE:EA = 100:1) to obtain 430 mg of pure product, yield 18%. MS (ESI): m / z 159.1 [M+H] + .

[0120] Step 2, Preparation of compound P11 (3Z,6Z)-3-vinylphenylallylidene-6-((5-isopropyl-1-(-3-morpholino)propylimidazol-4-yl)methylene)piperazine-2,5-dione:

[0121] Take 50 mL dry round-bottom flask, add (Z)-1 -acetyl-3-((5-isopropyl-1 -(3- morpholinopropyl)imidazol-4-yl)methylene)piperazine-2,5-dione 3 (0.92 g, 2.27 mmol), cesium carbonate (1.11 g, 3.41 mmol), sodium sulfate (0.64 g, 4.54 mmol), N,N- dimethylformamide (20 ml), 3-vinylcinnamaldehyde 12 (0.43 g, 2.72 mmol) in turn, exhaust, nitrogen protection, placed in 50 degree oil bath, avoid light reaction for 24 h. Monitor the reaction is complete, the reaction liquid drop into 200 mL ice water, there is a yellow solid precipitate, filter, filter cake after dry to get crude product as a yellow solid 220 mg, column chromatography to get 22 mg, yield 2.0%. MS (ESI): m / z 502.3 [M+H] + .

[0122] Example 5: Synthesis of compound P12

[0123] Step 1, preparation of 3-ethynylcinnamaldehyde 14:

[0124] 3-ethynylbenzaldehyde 13 (2.00 g, 15.37 mmol) was added to a 250 mL three-necked flask, 70 mL of toluene was added, (formylmethylene)triphenylphosphonium 5 (5.15 g, 16.91 mmol) was added, and the temperature was raised to 120 degrees for 48 h. The reaction was stopped. The reaction liquid was concentrated to sand, column chromatography (PE-PE:EA=100:1) to get pure product 560 mg, yield 23%. MS (ESI): m / z 157.1 [M+H] + .

[0125] Step 2, preparation of compound P12 (PLN-20-2-BB) (3Z,6Z)-3-ethynylphenyl alkenyl-6-((5-isopropyl-1-(-3-morpholinopropyl)imidazol-4-yl)methylene)piperazine-2,5-dione:

[0126] Into a 50 mL dry round-bottom flask, add (Z)-1-acetyl-3-((5-isopropyl-1-(3- morpholinopropylimidazol-4-yl)methylene)piperazine-2,5-dione 3 (1.20 g, 2.98 mmol), cesium carbonate (1.46 g, 4.47 mmol), sodium sulfate (0.85 g, 5.96 mmol), N,N- dimethylformamide (20 ml), 3-ethynylcinnamaldehyde 14 (0.56 g, 3.58 mmol) under vacuum, nitrogen protection, and place in a 50-degree oil bath. React for 24 h in the dark. Monitor the reaction until complete. Add the reaction solution dropwise into 200 mL ice water. A yellow solid precipitates. Filter the solid, and dry the filter cake to obtain a yellow solid 210 mg as a crude product. Column chromatography yields 46 mg, with a yield of 3.1%. MS (ESI): m / z 500.3 [M+H] + .

[0127] 1 H NMR (600 MHz, DMSO-d6) δ 11.97 (s, 1H), 10.90 (s, 1H), 7.90 (s, 1H), 7.85-7.80 (m, 2H), 7.58 (d, J = 7.0 Hz, 1H), 7.42-7.37 (m, 2H), 6.90 (d, J = 15.2 Hz, 1H), 6.71 (s, 1H), 6.53 (d, J = 12.1 Hz, 1H), 4.27 (s, 1H), 4.05 (t, J = 7.1 Hz, 2H), 3.59 (t, J = 4.2 Hz, 4H), 3.26-3.21 (m, 1H), 2.34 (s, 4H), 2.26 (t, J = 6.7 Hz, 2H), 1.88-1.81 (m, 2H), 1.35 (d, J = 7.1 Hz, 6H).

[0128] The synthesis of compound P13 is described with reference to compound P12.

[0129] Example 6: Synthesis of compound P14

[0130] Step 1:

[0131] Take 50 ml dry round-bottom flask, add 15 (326 mg, 1.08 mmol), 1-chloro-3- morpholinyl propane 1 (355 mg, 2.17 mmol), cesium carbonate (708 mg, 2.17 mmol), potassium iodide (180 mg, 1.08 mmol), 4A molecular sieve (500 mg), DMF (6 ml), exhaust, nitrogen protection, placed in 70 ℃ oil bath, stirring reaction 20 h. After the reaction, the reaction liquid is transferred to a 100 ml single-neck flask, washed with ethanol, concentrated under reduced pressure, redissolved with EtOH:DCM = 1:5, suction filtration, filter cake washed with EtOH:DCM = 1:5, concentrated under reduced pressure, and 301 mg of brown solid crude product 16 was obtained with a yield of 65.00%. Directly into the next step.

[0132] Step 2:

[0133] Take 25 ml dry brown round-bottom flask, add 16 (359 mg, 0.87 mmol), 6 (190 mg, 1.13 mmol), cesium carbonate (425 mg, 1.30 mmol), anhydrous sodium sulfate (247 mg, 1.74 mmol), N, N-dimethylformamide (6 ml), exhaust, nitrogen protection, placed in 45 ℃ oil bath, stirring reaction 18 h. LC-MS monitoring reaction, after the reaction, the reaction liquid is transferred to a 100 ml single-neck flask, washed with ethanol, concentrated under reduced pressure, column chromatography to obtain the target product P14 with a yield of 19%. ESI-MS: m / z = 538 (M+H).

[0134] The synthesis steps of compounds P15-P25 refer to compound P14.

[0135] Example 7: Synthesis of compound P26

[0136] Take 25 ml dry brown round-bottom flask, add 3 (351 mg, 0.87 mmol), 17 (168 mg, 1.13 mmol), cesium carbonate (425 mg, 1.30 mmol), anhydrous sodium sulfate (247 mg, 1.74 mmol), N, N-dimethylformamide (6 ml), exhaust, nitrogen protection, placed in 45 ℃ oil bath, stirring reaction 18 h. LC-MS monitoring reaction, after the reaction, the reaction liquid is transferred to a 100 ml single-neck flask, washed with ethanol, concentrated under reduced pressure, column chromatography to obtain the target product P-26 with a yield of 18%. ESI-MS: m / z = 493 (M+H).

[0137] The synthesis steps of compounds P27-P28 refer to compound P26.

[0138] Example 8

[0139] Step 1, Preparation of compound P29 (3Z, 6Z)-3-cyanophenyl methylene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2, 5-dione:

[0140] Into a 50 mL dry round-bottom flask, add (Z)-1-acetyl-3-((5-isopropyl-1-(3- morpholinyl)propylimidazol-4-yl)methylene)piperazine-2, 5-dione 3 (1.50 g, 3.62 mmol), cesium carbonate (1.77 g, 5.43 mmol), sodium sulfate (1.03 g, 7.24 mmol), N, N- dimethylformamide (20 ml), 3-cyanobenzaldehyde 18 (0.57 g, 4.34 mmol) in sequence, evacuate, protect with nitrogen, put into 50-degree oil bath, avoid light, react for 24 h. Monitor the reaction to completion, drop the reaction into 200 mL ice water, precipitate yellow solid, filter, dry the filter cake to obtain 690 mg of crude product as yellow solid, column chromatography to obtain 240 mg, yield 14%.

[0141] 1 H NMR (600 MHz, DMSO-d6) δ 12.07 (s, 1H), 10.56 (s, 1H), 7.99 (s, 1H), 7.92 (s, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 6.75 (d, J = 4.0 Hz, 2H), 4.07 (t, J = 7.1 Hz, 2H), 3.60 (t, J = 4.4 Hz, 4H), 3.27-3.21 (m, 1H), 2.35 (s, 4H), 2.26 (t, J = 6.7 Hz, 2H), 1.89-1.82 (m, 2H), 1.35 (d, J = 7.1 Hz, 6H). m.p: 237.6-239.6. M.W [M+H] + : 475.54.

[0142] Step 2, Preparation of P29 hydrochloride:

[0143] Into a 25 mL single-neck dry round-bottom flask, add P29 (100.0 mg, 0.21 mmol), methanol (5 mL), drop hydrogen chloride methanol solution (4 mol / L, 5 mL) at 0 degrees, avoid light throughout, after adding, naturally rise to room temperature, react for 2 h, concentrate the reaction liquid, add a small amount of MeOH to just dissolve the product, and drop into 10 mL ethyl acetate, precipitate solid, stand for 20 min, filter, dry the filter cake to obtain 101 mg of light yellow solid.

[0144] 1H NMR (600 MHz, DMSO-d6) δ 11.90 (s, 1H), 11.56 (s, 1H), 10.60 (s, 1H), 8.11 (s, 1H), 7.98 (s, 1H), 7.79-7.75 (m, 2H), 7.59 (t, J = 7.8 Hz, 1H), 6.75 (s, 1H), 6.71 (s, 1H), 4.18 (t, J = 7.2 Hz, 2H), 3.98-3.93 (m, 2H), 3.88 - 3.83 (m, 2H), 3.47 - 3.36 (m, 2H), 3.27-3.21 (m, 1H), 3.15-3.11 (m, 2H), 3.07-3.02 (m, 2H), 2.23-2.18 (m, 2H), 1.35 (d, J = 7.1 Hz, 6H). m.p: 268.6-270.6 °C.

[0145] Example 9:

[0146] Step 1, Preparation of compound P30 (3Z, 6Z)-3-vinylphenylmethylene-6-((5-isopropyl-1-(-3-morpholino)propylimidazol-4-yl)methylene)piperazine-2, 5-dione:

[0147] Into a 50 mL dry round-bottom flask, add (Z)-1-acetyl-3-((5-isopropyl-1-(3- morpholino)propylimidazol-4-yl)methylene)piperazine-2, 5-dione 3 (1.50 g, 3.62 mmol), cesium carbonate (1.77 g, 5.43 mmol), sodium sulfate (1.03 g, 7.24 mmol), N,N- dimethylformamide (20 ml), 3-vinylbenzaldehyde 11 (0.56 g, 4.34 mmol) in turn, evacuate, protect with nitrogen, place in a 50-degree oil bath, and react in the dark for 24 h. Monitor the reaction until completion, drop the reaction into 200 mL ice water, and precipitate a yellow solid. Filter, and obtain a yellow solid 640 mg of crude after drying the filter cake. Obtain 210 mg after column chromatography, with a yield of 12%.

[0148] 1H NMR (600 MHz, DMSO-d6) δ 12.00 (s, 1H), 10.27 (s, 1H), 7.89 (s, 1H), 7.59 (s, 1H), 7.45-7.43 (m, 2H), 7.41-7.37 (m, 1H), 6.80-6.73 (m, 2H), 6.71 (s, 1H), 5.89 (d, J = 17.6 Hz, 1H), 5.31 (d, J = 11.2 Hz, 1H), 4.05 (t, J = 6.8 Hz, 2H), 3.65-3.52 (m, 4H), 3.27-3.21 (m, 1H), 2.33 (s, 4H), 2.25 (t, J = 6.7 Hz, 2H), 1.88-1.80 (m, 2H), 1.34 (d, J = 7.1 Hz, 6H). m.p: 219.0-221.0. M.W [M+H] + : 476.59.

[0149] Step 2, preparation of P30 hydrochloride salt:

[0150] Take 25 mL single-port dry round-bottom flask, add P30 (100.0 mg, 0.21 mmol), methanol (5 mL) in turn, drop hydrogen chloride methanol solution (4 mol / L, 5 mL) at 0 degrees, avoid light throughout, add natural room temperature after adding, react 2 h, add a small amount of MeOH to just dissolve the product, and drop into 10 mL EA, precipitate solid, stand for 20 min, filter, dry the filter cake, get light yellow solid 98 mg.

[0151] 1 H NMR (600 MHz, DMSO-d6) δ 11.82 (s, 1H), 11.55 (s, 1H), 10.25 (s, 1H), 8.14 (s, 1H), 7.59 (s, 1H), 7.48-7.37 (m, 3H), 6.81-6.74 (m, 2H), 6.69 (s, 1H), 5.90 (d, J = 17.7 Hz, 1H), 5.76 (s, 1H), 5.32 (d, J = 11.5 Hz, 1H), 4.18 (t, J = 7.2 Hz, 2H), 3.96-3.94 (m, 2H), 3.89-3.83 (m, 2H), 3.46-3.37 (m, 2H), 3.27-3.21 (m, 1H), 3.17-3.09 (m, 2H), 3.09-2.99 (m, 2H), 2.23-2.18 (m, 2H), 1.34 (d, J = 7.1 Hz, 6H). m.p: 260.2-262.2 °C.

[0152] Example 10

[0153] Preparation of compound P31 (3Z, 6Z)-3-ethynylphenylmethylene-6-((5-isopropyl-1-(-3- morpholinopropylimidazol-4-yl)methylene)piperazine-2, 5-dione:

[0154] Into a 50 mL dry round bottom flask, (Z)-1-acetyl-3-((5-isopropyl-1-(3-morpholinopropylimidazol-4- yl)methylene)piperazine-2, 5-dione 3 (1.50 g, 3.62 mmol), cesium carbonate (1.77 g, 5.43 mmol), sodium sulfate (1.03 g, 7.24 mmol), N,N-dimethylformamide (20 ml), 3-ethynylbenzaldehyde 13 (0.56 g, 4.34 mmol) were added successively, evacuated, protected by nitrogen, placed in a 50-degree oil bath, and reacted in the dark for 24 h. After the reaction was completed, the reaction solution was dropped into 200 mL of ice water, and a yellow solid was precipitated. After filtration, the filter cake was dried to obtain 580 mg of a yellow solid as a crude product. After column chromatography, 160 mg was obtained, with a yield of 9%.

[0155] 1H NMR (600 MHz, DMSO-d6) δ 12.01 (s, 1H), 7.90 (s, 1H), 7.66 (s, 1H), 7.57-7.54 (m, 1H), 7.46-7.36 (m, 2H), 6.71 (d, J = 6.4 Hz, 2H), 4.25 (s, 1H), 4.06 (t, J = 6.8 Hz, 2H), 3.65-3.52 (m, 4H), 3.27-3.21 (m, 1H), 2.35 (s, 4H), 2.26 (t, J = 6.7 Hz, 2H), 1.90-1.82 (m, 2H), 1.35 (d, J = 7.1 Hz, 6H). m.p: 246.5-248.5 °C. M.W [M+H]+: 474.57.

[0156] Example 11

[0157] Step 1, Preparation of compound P32 (3Z, 6Z)-3-fluorophenylmethylene-6-((5-isopropyl-1-(-3- morpholinopropylimidazol-4-yl)methylene)piperazine-2, 5-dione:

[0158] Into a 25 ml dry brown round bottom flask, add (Z)-1-acetyl-3-((5-isopropyl-1- (3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione 3 (0.73 g, 1.81 mmol), 3-fluorobenzaldehyde 7 (0.22 g, 1.81 mmol), cesium carbonate (0.88 g, 2.71 mmol), anhydrous sodium sulfate (0.51 g, 3.62 mmol), N,N-dimethylformamide (8 ml), evacuate, nitrogen protection, put into 45 °C oil bath, stir for 20 h. Monitor the reaction by LC-MS, after the reaction is completed, drop the reaction solution into 4 °C cold water (80 ml), suction filter, wash the filter cake with cold water, dry, dissolve in methanol and dichloromethane (1:3), filter, concentrate under reduced pressure, dry, chromatograph, concentrate under reduced pressure, dry, methanol ultrasonic slurry, put into -30 °C refrigerator, stand for 20 h, suction filter, wash the filter cake with cold methanol, dry at 50 °C under vacuum, to obtain the target product 301.7 mg, yield 35.66%.

[0159] 1 H NMR (600 MHz, DMSO-d6) δ 12.02 (s, 1H), 10.27 (s, 1H), 7.88 (d, J = 5.34 Hz, 1H), 7.46-7.40 (m, 1H), 7.36 (d, J = 10.26 Hz, 1H), 7.34 (dd, J = 9.02, 19.44 Hz, 1H), 7.13 (td, J = 2.37, 8.57 Hz, 1H), 6.73 (s, 1H), 6.71 (s, 1H), 4.04 (t, J = 7.13 Hz, 2H), 3.57 (t, J = 4.44 Hz, 4H), 3.24 (dt, J = 7.10, 14.24 Hz, 1H), 2.26 (dd, J = 24.07, 30.84 Hz, 6H), 1.84 (p, J = 6.86 Hz, 2H), 1.33 (d, J = 7.13 Hz, 6H). MS (ESI): m / z 468.18 [M+H] + Mp: 208-210 °C.

[0160] Step 2, preparation of P32-0 hydrochloride salt

[0161] (3Z,6Z)-3-(3-fluorophenyl)methylene-6-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4- yl)methylene)piperazine-2,5-dione, hydrochloride salt

[0162] Into a 100 mL single-necked dry round-bottom flask, (3Z,6Z)-3-(3- fluorophenyl)methylene-6-((5-isopropyl-1-(3-morpholinopropyl)imidazol-4- yl)methylene)piperazine-2,5-dione (200.0 mg, 0.43 mmol), methanol (10 mL), hydrogen chloride in methanol (4 mol / L, 15 mL) were added successively at 0 °C, and the whole process was protected from light. After addition, the reaction was allowed to warm to room temperature and stirred for 2 h. The reaction solution was concentrated, a small amount of MeOH was added to just dissolve the product, and the solution was added dropwise to 15 mL of ethyl acetate. The solid was precipitated, stirred for 10 min, and then filtered and dried. The filter cake was collected to give a light yellow solid 218.0 mg in 94% yield.

[0163] 1H NMR (600 MHz, DMSO-d6) δ 11.84 (s, 1H), 10.36 (s, 1H), 8.15 (s, 1H), 7.49 - 7.40 (m, 1H), 7.37 (d, J = 10.2 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.17 - 7.14 (m, 1H), 6.75 (s, 1H), 6.69 (s, 1H), 4.19 (t, J = 7.2 Hz, 2H), 3.96 - 3.93 (m, 2H), 3.86 (t, J = 11.5 Hz, 2H), 3.42 (d, J = 12.2 Hz, 2H), 3.28 - 3.21 (m, 1H), 3.14 - 3.10 (m, 2H), 3.08 - 3.02 (m, 2H), 2.23 - 2.18 (m, 2H), 1.34 (d, J = 7.1 Hz, 6H).

[0164] Step 3, preparation of P32-1 methanesulfonate salt

[0165] (3Z,6Z)-3-(3-Fluorophenyl)methylene-6-((5-isopropyl-1-(3-morpholinopropyl)imidazol-4-yl)methylene)piperazine-2,5-dione, methanesulfonate salt (P32-1)

[0166] Into a 100 mL single-necked dry round-bottom flask, P32 (200.0 mg, 0.43 mmol), methanol (10 mL), methanesulfonic acid (102.8 mg, 1.07 mmol) were added successively at 0 °C, and the whole process was protected from light. After addition, the reaction was allowed to warm to room temperature and stirred for 2 h. The reaction solution was concentrated, a small amount of MeOH was added to just dissolve the product, and the solution was added dropwise to 15 mL of ethyl acetate. The solid was precipitated, stirred for 10 min, and then filtered and dried. The filter cake was collected to give a light yellow solid 268.0 mg in 95% yield.

[0167] 1H NMR (600 MHz, DMSO-d6) δ 11.76 (s, 1H), 10.39 (s, 1H), 8.18 (s, 1H), 7.47-7.43 (m, 1H), 7.38-7.35 (m, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.17-7.14 (m, 1H), 6.76 (s, 1H), 6.68 (s, 1H), 4.16 (t, J = 7.3 Hz, 2H), 4.01-3.98 (m, 2H), 3.69 (t, J = 11.6 Hz, 2H), 3.46 (d, J = 12.2 Hz, 2H), 3.26-3.19 (m, 3H), 3.14-3.06 (m, 2H), 2.42 (s, 6H), 2.16-2.11 (m, 2H), 1.34 (d, J = 7.1 Hz, 6H).

[0168] Example 12

[0169] Preparation of compound P33 (3Z,6Z)-3-fluorophenylmethylene-6-((5-isopropyl-1-(-3- morpholinopropylimidazol-4-yl)allylidene)piperazine-2,5-dione:

[0170] Into a 25 mL dry round-bottom flask, add (Z)-1-acetyl-3-((5-isopropyl-1-(3- morpholinopropylimidazol-4-yl)allylidene)piperazine-2,5-dione 16 (481 mg, 1.12 mmol), cesium carbonate (547 mg, 1.68 mmol), sodium sulfate (318 mg, 2.24 mmol), N,N- dimethylformamide (50 ml), 3-fluorobenzaldehyde 7 (166 mg, 1.34 mmol) under nitrogen protection, and place in a 50-degree oil bath, and react for 24 h in the dark. Monitor the reaction until completion, and then drop the reaction solution into 50 mL ice water. Filter and dry to obtain a yellow solid, [M+H]+494.7.

[0171] Example 13: Preparation of P38-0 (3Z,6Z)-3-(fluorophenylmethylene-6-((5-isopropyl-1-(3- piperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione hydrochloride

[0172] Step 1, Preparation of 1-tert-butoxycarbonyl-4-(3-ethanesulfonylpropyl)piperazine (IM8):

[0173] Into a 100 mL three-necked flask, was placed SM7, 1-tert-butoxycarbonyl-4-(3- hydroxypropan) piperazine (2.00 g, 7.18 mmol), 30 mL DCM, TEA (1.24 g, 12.27 mmol), and the temperature was controlled at about 0 °C. Then, EsCl (1.26 g, 9.82 mmol) was added dropwise, and the temperature was allowed to rise to room temperature. The reaction was allowed to proceed for 2 h. After the reaction was completed, 50 mL of water was added, and the mixture was separated. The aqueous phase was extracted with 30 mL of DCM, and the combined organic phase was washed with 50 mL of water and 50 mL of saturated brine, dried, and concentrated to obtain a brown oil, IM8, 1-tert-butoxycarbonyl-4-(3- ethanesulfonylpropyl) piperazine (2.74 g, 99% yield), which was directly used in the next step.

[0174] Step 2, (Z)-1-acetyl-3-((5-isopropyl-1-(3-1-tert-butoxycarbonylpiperazinyl) propyl- imidazol-4-yl)methyl)piperazine-2,5-dione (IM9) preparation:

[0175] Into a 250 mL dry round-bottom flask was sequentially added A005, (Z)-1-acetyl-3-((5- isopropyl-1H-imidazol-4-yl)methyl)piperazine-2,5-dione (1.50 g, 5.43 mmol), Cs2CO3 (3.54 g, 10.86 mmol), KI (0.90 g, 5.43 mmol), DMF (50 mL), IM8, 1-tert-butoxycarbonyl-4-(3- ethanesulfonylpropyl) piperazine (2.74 g, 8.15 mmol), and the reaction was carried out at 70 °C for 36 h in the dark. After the reaction was completed, 100 mL of water was added to the reaction solution, and 50 mL of DCM was added to extract the aqueous phase. The combined organic phase was washed with 2*50 mL of water and 50 mL of saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain a brownish oil, 2.73 g, 99% yield, which was directly used in the next step without purification.

[0176] Step 3, P38 (3Z,6Z)-3-(fluorobenzylidene-6-((5-isopropyl-1-(3-1-tert-butoxycarbonylpiperazinyl) propylimidazol-4-yl)methyl)piperazine-2,5-dione

[0177] Take 25 ml dry brown round-bottom flask, add IM9, (Z)-1-acetyl-3-((5-isopropyl-1- (3-1-tert-butoxycarbonylpiperazinyl) propyl-imidazol-4-yl) methylene) piperazine-2,5- dione (2.73 g, 5.43 mmol), 3-fluorobenzaldehyde 7 (0.67 g, 5.43 mmol), cesium carbonate (2.66 g, 8.15 mmol), anhydrous sodium sulfate (1.54 g, 10.86 mmol), DMF (30 ml), exhaust, nitrogen protection, placed in 45 °C oil bath, stirred for 20 h. LC-MS monitoring reaction, reaction, drop the reaction liquid into 4 °C cold water (80 ml), suction filtration, dry the filter cake, and column chromatography to obtain 820 mg of the target product with a purity of 98% and a yield of 27%. M.W[M+H] + :567.51

[0178] Step 4, preparation of P38-0 (3Z, 6Z)-3-(fluorobenzylidene-6-((5-isopropyl-1- (3-piperazinyl) propylimidazol-4-yl) methylene) piperazine-2, 5-dione hydrochloride

[0179] Take 250 mL single-port dry round-bottom flask, add P38 (700.0 mg, 1.23 mmol), MeOH (60 mL), drop HCl / MeOH (4 mol / L, 50 mL) at 0 degrees, avoid light throughout, add naturally to room temperature for 2 h, concentrate the reaction liquid, add a small amount of MeOH to just dissolve the product, and drop into 100 mL EA, precipitate the solid, stand for 30 min, filter, dry the filter cake, and obtain 633 mg of light yellow solid with a HPLC purity of 98% and a yield of 95%.

[0180] 1 H NMR (600 MHz, DMSO) δ 12.11 (s, 1H), 11.89 (s, 1H), 10.37 (s, 1H), 10.04-9.63 (m, 2H), 8.08 (s, 1H), 7.48-7.44 (m, 1H), 7.39-7.37 (m, 1H), 7.36-7.32 (m, 1H), 7.18-7.14 (m, 1H), 6.76 (s, 1H), 6.72 (s, 1H), 4.19 (t, J = 7.1 Hz, 2H), 3.71 (s, 2H), 3.58-3.44 (m, 4H), 3.31 (s, 2H), 3.29-3.24 (m, 1H), 3.23-3.19 (m, 2H), 2.22-2.17 (m, 2H), 1.36 (d, J = 7.1 Hz, 6H).

[0181] Example 14: Preparation of P39 (3Z, 6Z)-3-fluorophenylmethylene-6-((5-isopropyl-1- (3-1-oxoethoxypiperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0182] Step 1,

[0183] Take 250 mL dry round-bottom flask, at 0 degrees, add P38, (Z)-1-acetyl-3-((5-isopropyl-1- (3-piperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione (0.55 g, 1.02 mmol), DCM (50 ml), TEA (0.52 g, 5.10 mmol), SM8, oxalyl chloride monoethyl ester (0.28 g, 2.04 mmol) in turn, and react at room temperature for 24 h in the dark. Monitor the reaction to completion, and concentrate the reaction liquid to column chromatography to obtain 338 mg, HPLC purity 98%, yield 58%.

[0184] 1 H NMR (600 MHz, DMSO) δ 12.04 (s, 1H), 10.32 (s, 1H), 7.92 (s, 1H), 7.49-7.42 (m, 1H), 7.38 (d, J = 10.2 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.18-7.14 (m, 1H), 6.75 (s, 1H), 6.73 (s, 1H), 4.30 (q, J = 7.1 Hz, 2H), 4.08 (t, J = 7.1 Hz, 2H), 3.52 (s, 2H), 3.38 (s, 2H), 3.30-3.21 (m, 1H), 2.48-2.25 (m, 6H), 1.91-1.82 (m, 2H), 1.35 (d, J = 7.1 Hz, 6H), 1.28 (t, J = 7.1 Hz, 3H). M. W [M+H] + : 567.58

[0185] Step 2, synthesis of P39-0

[0186] Take 25 mL single-port dry round-bottom flask, add P39 (80 mg, 0.14 mmol), MeOH (10 mL), and drop HCl / MeOH (4 mol / L, 5 mL) at 0 degrees, and react for 2 h at room temperature in the dark. Concentrate the reaction liquid, add a small amount of MeOH to just dissolve the product, and drop into 100 mL EA. The solid is precipitated, and after standing for 30 min, filter and dry the filter cake to obtain 76 mg of light yellow solid, HPLC purity 98%, yield 85%.

[0187] Example 15: Preparation of P40 (3Z, 6Z)-3-fluorophenylmethylene-6-((5-isopropyl- 1 -(3-1 -acetic acid piperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0188] Step 1,

[0189] P39 (0.25 g, 0.44 mmol) was added to a 100 mL single-neck flask, 50 mL MeCN, 2 mL H2O, TEA (0.18 g, 1.76 mmol), LiBr (0.23 g, 2.64 mmol) were added, and the reaction was warmed to 40 °C for 48 h. The reaction was stopped, and the reaction solution was concentrated and column chromatography to obtain 47 mg of pure product, purity 97%, yield 20%.

[0190] 1 H NMR (600 MHz, DMSO) δ 12.04 (s, 1H), 10.32 (s, 1H), 7.92 (s, 1H), 7.49-7.42 (m, 1H), 7.38 (d, J = 10.2 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.18-7.14 (m, 1H), 6.75 (s, 1H), 6.73 (s, 1H), 4.08 (t, J = 7.2 Hz, 2H), 3.46 (s, 4H), 3.30-3.21 (m, 1H), 2.48-2.35 (m, 6H), 1.93-1.85 (m, 2H), 1.35 (d, J = 7.1 Hz, 6H). M.W [M+H] + : 539.48

[0191] Step 2, Synthesis of P40-0

[0192] A 25 mL single-neck dry round-bottom flask was charged with P40 (36 mg, 0.07 mmol), MeOH (5 mL), and HCl / MeOH (4 mol / L, 3 mL) was added dropwise at 0 °C, and the reaction was allowed to warm to room temperature for 2 h. The reaction solution was concentrated, a small amount of MeOH was added to just dissolve the product, and the solution was added dropwise to 20 mL EA. The solid was precipitated, and after standing for 30 min, it was filtered and dried to obtain 30 mg of a light yellow solid, HPLC purity 97%, yield 75%.

[0193] Example 16: Preparation of P41 (3Z, 6Z)-3-fluorophenylmethylene-6-((5-isopropyl- 1 -(3-N-methylpiperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0194] Step 1, Preparation of 1-(3-ethanesulfonylpropyl)-4-methylpiperazine (IM11)

[0195] Into a 100 mL three-necked flask, SM9, 1-(3-hydroxypropan)-4-methylpiperazine (2.00 g, 12.64 mmol) was added, 30 mL DCM was added, TEA (1.92 g, 18.96 mmol) was added, temperature was controlled at about 0 degrees, EsCl (1.95 g, 15.17 mmol) was added dropwise, and after addition, it was naturally warmed to room temperature for 2 h of reaction. After the reaction was completed, 50 mL water was added, and the liquid was separated. The water phase was extracted with 30 mL DCM, and the organic phases were combined. 50 mL water and 50 mL saturated brine were added respectively for washing, drying, and concentration to obtain brown oil compound IM11, 1-(3-ethanesulfonylpropyl)-4-methylpiperazine 2.96 g, with a yield of 94%, which was directly used in the next step.

[0196] Step 2, Preparation of (Z)-1-acetyl-3-((5-isopropyl-1-(3-N-methylpiperazinyl)propyl-imidazol-4-yl)methyl)piperazine-2,5-dione (IM12)

[0197] Into a 100 mL dry round-bottom flask, A005, (Z)-1-acetyl-3-((5-isopropyl-1H-imidazol-4-yl)methyl)piperazine-2,5-dione (0.50 g, 1.81 mmol), Cs2CO3 (1.18 g, 3.62 mmol), KI (0.30 g, 1.81 mmol), DMF (20 mL), IM11, 1-(3-ethanesulfonylpropyl)-4-methylpiperazine (0.68 g, 2.72 mmol) were sequentially added, and the reaction was carried out at 70 degrees for 36 h in the dark. After the reaction was completed, 80 mL water was added to the reaction liquid, and 30 mL DCM was extracted. The water phase was further extracted with 30 mL DCM, and the organic phases were combined. 2*50 mL water and 50 mL saturated brine were added respectively for washing. The organic phase was dried with anhydrous sodium sulfate, and then concentrated to obtain 0.75 g of crude product in the form of brownish oil, with a yield of 99%. The product was directly used in the next step without purification.

[0198] Step 3, P41 (3Z,6Z)-3-fluorobenzylidene-6-((5-isopropyl-1-(3-N-methylpiperazinyl)propyl-imidazol-4-yl)methyl)piperazine-2,5-dione

[0199] Into a 25 mL dry brown round-bottom flask, IM12, (Z)-1-acetyl-3-((5-isopropyl-1- (3-N-methylpiperazinyl)propyl-imidazol-4-yl)methylene)piperazine-2,5-dione (0.75 g, 1.81 mmol), 3-fluorobenzaldehyde 7 (0.22 g, 1.81 mmol), cesium carbonate (0.88 g, 2.71 mmol), anhydrous sodium sulfate (0.51 g, 3.62 mmol), DMF (8 mL), exhaust, nitrogen protection, placed in 45 °C oil bath, stirred for 20 h. Monitor the reaction by LC-MS, and after the reaction, drop the reaction solution into 4 °C cold water (80 mL), suction filter, dry the filter cake, and column chromatography to obtain 180 mg of the target product with a purity of 98% and a yield of 20%.

[0200] 1 H NMR (600 MHz, DMSO-d6) δ 11.94 (s, 1H), 10.38 (s, 1H), 8.00 (s, 1H), 7.48-7.44 (m, 1H), 7.38 (d, J = 10.2 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.18-7.15 (m, 1H), 6.78 (s, 1H), 6.72 (s, 1H), 4.18 (t, J = 6.3 Hz, 2H), 3.71-3.62 (m, 4H), 3.44 (s, 4H), 3.31-3.10 (m, 2H), 2.82 (s, 3H), 2.24-2.14 (m, 2H), 1.34 (d, J = 6.92 Hz, 6H) M.W [M+H] + : 481.39

[0201] Step 4, Preparation of P41-0 (3Z, 6Z)-3-fluorobenzylidene-6-((5-isopropyl-1- (3-N-methylpiperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione hydrochloride:

[0202] Into a 25 mL single-port dry round-bottom flask, add P41 (40 mg, 0.08 mmol), MeOH (5 mL), and drop HCl / MeOH (4 mol / L, 3 mL) at 0 °C, avoid light throughout, and naturally raise the temperature to room temperature after addition. After 2 h of reaction, concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and drop into 20 mL EA. The solid is precipitated, and after standing for 30 min, filter the solid. Dry the filter cake to obtain 41 mg of a yellowish solid with a HPLC purity of 97% and a yield of 90%.

[0203] Example 17: P42 (3Z, 6Z)-3-fluorobenzylidene-6-((5-isopropyl-1-(3-1,1- dioxidothiomorpholino)propylimidazol-4-yl)methyl)piperazine-2,5-dione

[0204] Step 1, Preparation of 4-(3-ethanesulfonylpropyl)thiomorpholine 1,1-dioxide (IM13)

[0205] Into a 100 mL three-necked flask, SM10, 4-(3-hydroxypropyl)thiomorpholine 1,1-dioxide (20.00 g, 0.14 mol) was added, 30 mL DCM was added, TEA (1.55 g, 15.26 mmol) was added, and the temperature was controlled at about 0 degrees. EsCl (1.60 g, 12.42 mmol) was added dropwise, and the temperature was allowed to rise to room temperature naturally for 2 h of reaction. After the reaction was completed, 50 mL of water was added, and the liquid was separated. The aqueous phase was extracted with 30 mL of DCM, and the organic phases were combined. 50 mL of water and 50 mL of saturated brine were added, respectively, and washed, dried, and concentrated to obtain brown oil compound IM13, 4-(3-ethanesulfonylpropyl)thiomorpholine 1,1-dioxide 2.80 g, yield 95%, which was directly used in the next step.

[0206] Step 2, Preparation of (Z)-1-acetyl-3-((5-isopropyl-1-(3-1,1-dioxidothiomorpholino)propyl- imidazol-4-yl)methyl)piperazine-2,5-dione (IM14)

[0207] Into a 100 mL dry round-bottom flask, A005, (Z)-1-acetyl-3-((5-isopropyl-1H-imidazol-4- yl)methyl)piperazine-2,5-dione (0.50 g, 1.81 mmol), Cs2CO3 (1.18 g, 3.62 mmol), KI (0.30 g, 1.81 mmol), DMF (20 mL), IM13, 4-(3-ethanesulfonylpropyl)thiomorpholine 1,1-dioxide (0.78 g, 2.72 mmol) were added in sequence, and the reaction was carried out at 70 degrees for 36 h in the dark. After the reaction was completed, 80 mL of water was added to the reaction solution, and 30 mL of DCM was added for extraction. The aqueous phase was further extracted with 30 mL of DCM, and the organic phases were combined. 2*50 mL of water and 50 mL of saturated brine were added, respectively, and washed. The organic phase was dried with anhydrous sodium sulfate and concentrated to obtain 0.82 g of crude product as a brownish oil, with a yield of 99%. The product was directly used in the next step without purification.

[0208] Step 3, P42 (3Z, 6Z)-3-fluorobenzylidene-6-((5-isopropyl-1-(3-1,1-dioxidothiomorpholino)propylimidazol-4-yl)methyl)piperazine-2,5-dione

[0209] Into a 25ml dry brown round bottom flask, IM14, (Z)-1-acetyl-3-((5-isopropyl-1-(3-1,1-dioxidothiomorpholinyl)propyl-imidazol-4-yl)methylene)piperazine-2,5-dione (0.82g, 1.81mmol), 3-fluorobenzaldehyde 7 (0.22g, 1.81mmol), cesium carbonate (0.88g, 2.71mmol), anhydrous sodium sulfate (0.51g, 3.62mmol), DMF (8ml), evacuate, nitrogen protection, put into 45°C oil bath, stir the reaction for 20h. LC-MS monitoring reaction, after the reaction, the reaction liquid is dropped into 4°C cold water (80ml), suction filtration, the filter cake is dried and column chromatography to obtain 60mg of the target product with a purity of 97% and a yield of 6%. M.W[M+H] + :516.43

[0210] Example 18: Titration of hydrochloride salt of series of compounds

[0211] 1) Titration of P5-0 hydrochloride salt:

[0212] Take 14.9mg P5-0 into a conical flask, add 20ml water, calibrate with sodium hydroxide with a concentration of c(NaOH) = 0.02012mmol / mL, use 2.63ml of sodium hydroxide standard solution, calculate n(HCl) = 0.02012*2.63 = 0.0529mmol, n(P5-0) = 14.9 / 566.50 = 0.02630mmol, n(HCl) / n(P5-0) = 2.01.

[0213] 2) Titration of P10-0 hydrochloride salt:

[0214] Take 13.2mg P10-0 into a conical flask, add 20ml water, calibrate with sodium hydroxide with a concentration of c(NaOH) = 0.02012mmol / mL, use 2.43ml of sodium hydroxide standard solution, calculate n(HCl) = 0.02012*2.63 = 0.0489mmol, n(P10-0) = 14.9 / 566.50 = 0.02630mmol, n(HCl) / n(P10-0) = 1.86.

[0215] 3) P29-0 hydrochloride titration: take 8.6 mg P29-0 into a conical flask, add 20 mL water, calibrate with sodium hydroxide of concentration c(NaOH) = 0.02012 mmol / mL, use 1.55 mL sodium hydroxide standard solution, calculate n(HCl) = 0.02012 * 1.55 = 0.0312 mmol, n(P29-0) = 8.6 / 573.52 = 0.0150 mmol, n(HCl) / n(P29-0) = 2.08.

[0216] 4) P30-0 hydrochloride titration: take 10.3 mg P30-0 into a conical flask, add 20 mL water, calibrate with sodium hydroxide of concentration c(NaOH) = 0.02012 mmol / mL, use 1.84 mL sodium hydroxide standard solution, calculate n(HCl) = 0.02012 * 1.84 = 0.0370 mmol, n(P30-0) = 10.3 / 574.55 = 0.0179 mmol, n(HCl) / n(P30-0) = 2.07.

[0217] 5) P38-0 hydrochloride titration: take 15.7 mg P38-0 into a conical flask, add 20 mL water, calibrate with sodium hydroxide of concentration c(NaOH) = 0.02007 mmol / mL, use 3.96 mL sodium hydroxide standard solution, calculate n(HCl) = 0.02007 * 3.96 = 0.0796 mmol, n(P38-0) = 15.7 / 575.95 = 0.0273 mmol, n(HCl) / n(P38-0) = 2.92.

[0218] 6) P39-0 hydrochloride titration: take 12.2 mg P39-0 into a conical flask, add 20 mL water, calibrate with sodium hydroxide of concentration c(NaOH) = 0.02007 mmol / mL, use 1.86 mL sodium hydroxide standard solution, calculate n(HCl) = 0.02007 * 1.86 = 0.0371 mmol, n(P39-0) = 12.2 / 639.55 = 0.0190 mmol, n(HCl) / n(P39-0) = 1.96

[0219] 7) P40-0 hydrochloride titration: 6.9 mg P40-0 was taken into a conical flask, 20 mL water was added, and it was calibrated with sodium hydroxide solution with a concentration of c(NaOH) = 0.02007 mmol / mL. 1.63 mL of the sodium hydroxide standard solution was used, and the calculation was as follows: n(HCl) = 0.02007 * 1.63 = 0.0327 mmol, n(P40-0) = 6.9 / 611.50 = 0.0113 mmol, n(HCl) / n(P40-0) = 2.90. This indicated that one molecule of carboxylic acid of the compound itself was removed, and the molecule combined with 2 molecules of hydrochloric acid.

[0220] 8) P41-0 hydrochloride titration: 19.3 mg P41-0 was taken into a conical flask, 20 mL water was added, and it was calibrated with sodium hydroxide solution with a concentration of c(NaOH) = 0.02007 mmol / mL. 4.82 mL of the sodium hydroxide standard solution was used, and the calculation was as follows: n(HCl) = 0.02007 * 4.82 = 0.0967 mmol, n(P41-0) = 19.3 / 589.96 = 0.0327 mmol, n(HCl) / n(P41-0) = 2.96.

[0221] Example 19: Water solubility experiment of the series of compounds

[0222] The hydrochloride or mesylate salt of the compound was dissolved in pure water at 24°C or 27°C, and the solubility of the compound was determined. The results are shown in Table 1, which shows that the compound has good water solubility after salification.

[0223] Table 1: Results of water solubility experiment of the compound

[0224] Example 20: Cell proliferation inhibition experiment of the series of compounds

[0225] The tumor cells in the logarithmic growth phase (NCI-H460, BxPC-3, HT-29, HCCLM3, HepG2) were digested, centrifuged, and the supernatant was removed. The cells were resuspended by adding fresh culture medium containing 10% fetal bovine serum, counted using a cell counting plate, and 100 μL of the medium (containing cells) was added to the 96-well plate according to the standard of 2000-6000 cells per well, and the 96-well plate was filled with PBS around the four sides. The plate was incubated in an incubator (37°C, 5% CO2) for 24 h. After the cells were completely adhered, the test samples and the positive control Plinabulin were diluted to different concentrations using fresh culture medium, and the drug was added in concentration gradients. Six different concentration gradients were set, 100 μL per well, and three replicate wells were set for each concentration. Incubation was carried out in a 37°C incubator with 5% CO2 for 72 h. After 20 μL of MTT with a concentration of 5 mg / mL was added to each well and placed in the incubator for 4 h, the MTT was discarded, 100 μL of DMSO was added to each well, and the plate was placed on a 96-well plate shaker to completely dissolve the purple formazan. Then, the absorbance (OD value) was detected using a microplate reader at a wavelength of 490 nm. The experiment also required setting a zero adjustment hole (blank culture medium, MTT, DMSO). The cell inhibition rate = 1- (drug group OD value-zero adjustment group OD value) / (blank group OD value-zero adjustment group OD value) x 100%. The experimental data were processed using Excel and Origin 8.5 software to calculate the IC 50 values. The experiment was repeated 3 times, and the average value was taken.

[0226] Table 2: IC 50

[0227] NCI-H460 is a human non-small cell lung cancer tumor cell line, BxPC-3 is a human pancreatic cancer tumor cell line, HT-29 is a human colon cancer cell line, HCCLM3 & HepG2 is a human liver cancer cell line. Plinabulin is a positive control.

[0228] The results are shown in Table 2. The newly designed 2,5-diketopiperazine series of compounds, including compounds P29, P30, P31, P32, P34, P35, P36, P37, and their hydrochloride or methanesulfonate, etc., have a significant ability to inhibit the growth of tumor cells against H460, BxPC-3, HT-29, HCCLM3, and HepG2 cells.

[0229] Example 21: Anti-tumor effect of the series of compounds on the NCI-H460 subcutaneous tumor model of BALB / c nude mice

[0230] This example uses non-small cell lung cancer cells NCI-H460 to establish a subcutaneous tumor model and evaluate the anti-tumor effect of the series of compounds on human non-small cell lung cancer cells NCI-H460 subcutaneous tumor model.

[0231] 1 Test material

[0232] 1.1 Test product

[0233] The test product is a series of anti-tumor products. It includes: compounds P36-0, P37-0, P34-0, P35-0, P32-0, all of which are yellow powders and are easily soluble in water; store at 2-8°C, dry and away from light; formulation preparation and use process must be strictly protected from light;

[0234] 1.2 Preparation of administration preparation

[0235] Preparation method: accurately weigh an appropriate amount of test product, dissolve with glucose solution and dilute to the required concentration, prepare on the day of administration, and strictly protect from light during preparation and administration; room temperature transfer, effective on the same day.

[0236] 1.3 Positive control

[0237] Paclitaxel injection; colorless to pale yellow clear viscous liquid; concentration: 5.99 mg / mL; store in the dark, tightly closed, below 25°C;

[0238] 1.4 Preparation of administration preparation

[0239] Preparation method: dilute paclitaxel injection to 1 mg / mL with normal saline and mix well; strictly protect from light after preparation; room temperature transfer, effective on the same day.

[0240] 1.5 Tumor cells for modeling

[0241] Human non-small cell lung cancer cells NCI-H460; adherent cells; store at 2-8°C, effective within 24 hours.

[0242] 2 Experimental animals

[0243] 2.1 Experimental system

[0244] BALB / c nude mice, SPF female; about 4-5 weeks old at the time of purchase, about 5-6 weeks old at the start of the experiment; body weight at the time of purchase 12.9-15.9 g, at the time of grouping 16.1-18.9 g, individual body weight within the average body weight ± 20% range;

[0245] Providing unit: Beijing Vitolink Experimental Animal Technology Co., Ltd.; quality certificate: NO.110011221101329612; production license number: SCXK(Jing)2021-0006.

[0246] 2.2 Animal feeding management

[0247] The mice were acclimated for 3 days before the experiment, and healthy mice (female, non-pregnant) were selected as test animals; the number of animals that passed quarantine was 110. The standard feeding environment conditions refer to the national standard of the People's Republic of China GB14925-2010; 19.82-26.33℃; relative humidity 41.19-72.66%; automatic lighting, 12 hours of light and dark alternation.

[0248] 2.3 Test method

[0249] BALB / c nude mice subcutaneous tumor models of human non-small lung cancer cells NCI-H460 were established, and 60 tumor-bearing animals with an average volume of about 120mm 3 were selected, a total of 10 groups, 6 animals in each group, respectively, blank control group, paclitaxel positive control group (10 mg / kg), P36-0 group (15 mg / kg), P37-0 low-dose group (5 mg / kg), P37-0 high-dose group (15 mg / kg), P34-0 low-dose group (5 mg / kg), P34-0 high-dose group (15 mg / kg), P35-0 group (15 mg / kg), P32-0 low-dose group (5 mg / kg), P32-0 high-dose group (15 mg / kg), the animal body weight and tumor diameter were measured during the experiment, and the animals were euthanized on D12, and the anti-tumor effects of compounds P36-0, P37-0, P34-0, P35-0 and P32-0 were evaluated.

[0250] 3 Model establishment and grouping

[0251] 3.1 Model establishment

[0252] 3.1.1 Preparation of human non-small cell lung cancer cells NCI-H460

[0253] The culture conditions were 90% RPMI1640 medium containing double antibodies + 10% FBS; the growth conditions were 5% CO2+95% air, and the culture was carried out at 37℃; when the cells in the culture bottle grew to 85-90% of the bottle wall, subculture was carried out.

[0254] The cells in the logarithmic growth phase were centrifuged (1000 rpm, 5 min); the supernatant was discarded, and an appropriate amount of PBS was added for dilution, and after counting, the density of the cell suspension was adjusted to 5×10 7 / mL.

[0255] 3.1.2 Establishment of human non-small cell lung cancer cell NCI-H460 subcutaneous tumor model

[0256] SPF level BALB / c mice were taken, the injection site skin of the mice was disinfected, and 0.1 mL of cell suspension with a cell concentration of 5x10 7 6 When the average tumor volume grew to about 120 mm 3 , 60 mice with relatively uniform tumor volume were selected for random grouping and administration.

[0257] 3.2 Animal grouping

[0258] A total of 10 groups were set up, with 6 female mice in each group. Among them, group 1 was the blank control group, group 2 was the paclitaxel positive control group, group 3 was the test product 1 group (P36-0 group, 15 mg / kg), group 4 was the test product 2 low-dose group (P37-0 low-dose group, 5 mg / kg), group 5 was the test product 2 high-dose group (P37-0 high-dose group, 15 mg / kg), group 6 was the test product 3 low-dose group (P34-0 low-dose group, 5 mg / kg), group 7 was the test product 3 high-dose group (P34-0 high-dose group, 15 mg / kg), group 8 was the test product 4 group (P35-0 group, 15 mg / kg), group 9 was the test product 5 low-dose group (P32-0 low-dose group, 5 mg / kg), and group 10 was the test product 5 high-dose group (P32-0 high-dose group, 15 mg / kg).

[0259] Grouping method: Animals with an average tumor volume of about 120 mm 3 were randomly grouped. The grouping information is shown in Table 3.

[0260] Table 3 Experimental grouping and dose design table

[0261] Note: The first digit of the animal number represents the group, the second letter represents the gender (F for female), and the 3rd, 4th and 5th digits represent the individual animal number.[1]i.v, tail vein injection;[2]q3d: once every 3 days, q3d x 4, i.e. once every 3 days for a total of 4 administrations.

[0262] 3.3 Administration method: The requirements of SOP TEC-02-02 "Common administration methods for rodents" were followed.

[0263] The blank control group was injected intravenously; the administration frequency was q3d x 4; the administration volume was 10 mL / kg; and the administration period was 12 days (once every 3 days for a total of 4 times).

[0264] ​Paclitaxel positive control group tail vein injection; q3d x 4; dose: 10 mg / kg; volume: 10 mL / kg; duration: 12 days (once every 3 days, a total of 4 times)

[0265] Test product 1: P36-0, P37-0, P34-0, P35-0, P32-0 tail vein injection; dose frequency: q3d x 4; dose: 15 mg / kg or 5 mg / kg; volume: 10 mL / kg; duration: 12 days (once every 3 days, a total of 4 times).

[0266] Note: The day of administration is defined as the first day of the test, recorded as D1, followed by D2, D3, and so on.

[0267] 4 Observation and examination

[0268] 4.1 Clinical observation: Refer to SOP TEC-01-05 "General clinical observation of experimental animals".

[0269] During the animal quarantine and modeling period, the general state of the animals was observed once a day; after administration, the general state of the animals was observed every day; the observation period for animals in each group was 2 weeks;

[0270] Observation content: including but not limited to the appearance of the animals, general behavior, mental state, gland secretion, respiratory status, fecal characteristics, and survival period observation, etc. All deaths, symptoms, and the onset time, severity, duration of symptoms, body weight changes, etc. were recorded.

[0271] 4.2 Body weight measurement: The requirements of SOP TEC-01-10 "Measurement of body weight of experimental animals" were followed.

[0272] Once at the time of animal reception, at the end of quarantine, and before grouping; 3 times a week during the modeling period and administration period.

[0273] 4.3 Tumor efficacy evaluation

[0274] 4.3.1 Tumor volume measurement and evaluation criteria

[0275] The tumor growth at the inoculation site was observed in the week before inoculation of cells, and as the tumor grew faster, the tumor volume was measured once a day, and 3 times a week after administration.

[0276] Measurement method: The long diameter and short diameter of the tumor were measured and recorded, and the tumor volume was calculated (volume formula V = 1 / 2 x a x b 2 , a is the long diameter, and b is the short diameter).

[0277] The relative tumor volume (RTV) and relative tumor proliferation rate T / C% were calculated according to the following formula:

[0278] RTV = Vt / V0

[0279] Vt: tumor volume obtained in each measurement; V0: initial tumor volume (before administration)

[0280] T / C% = RTV average of administration group / RTV average of negative control group x 100%

[0281] 4.3.2 Tumor weight measurement and evaluation criteria

[0282] After the end of the experiment, the animals were euthanized using CO2. After euthanasia, the tumors were stripped and weighed.

[0283] Tumor inhibition rate IR TW (%) = (1-TWt / TWc) x 100%, where TWt is the tumor weight of the treatment group, and TWc is the tumor weight of the solvent control group

[0284] 5 Statistical analysis

[0285] General state and other data were analyzed by descriptive analysis.

[0286] Body weight and other quantitative indicators were calculated by group as mean ± standard deviation When the sample size is less than 3, the data of this group is not included in statistical comparison. Comparison between multiple groups is analyzed according to the following procedure: Levene's test for homogeneity of variance. If there is no statistical significance (P>0.05), single factor analysis of variance (ANOVA) is used for statistical analysis. If ANOVA has statistical significance (P<0.05), LSD method is used for comprehensive comparison. If the variance is not homogeneous (P<0.05), Kruskal-Wallis test is used, and pairwise comparison between means is performed.

[0287] SPSS 24.0 software was used for the above statistical operations.

[0288] 6 Test results

[0289] 6.1 General state observation and body weight

[0290] During the observation period, the body weight of the blank control group animals remained stable; the paclitaxel positive control group mice showed body cooling and reduced activity from D11 to D12, and one animal died (2F001) on D11. The animal's body weight decreased continuously before death, and the cause of death was considered to be poor individual tolerance of the mice to paclitaxel, which was related to drug toxicity. One mouse (5F006) in the P37-0 high-dose group (15 mg / kg) died after administration on D7, which was considered to be related to drug toxicity. Three mice (3F004-3F006) in the P36-0 group (15 mg / kg) showed gray-black tails from D5, and two mice (3F004, 3F006) died on D12, with a sharp decrease in body weight before death, which was considered to be related to drug toxicity. The mice in the other groups were normal in clinical observation.

[0291] Compared with the blank control group, the body weight of the paclitaxel positive control group mice was significantly reduced on days 8, 10, and 12 (*P<0.05, **P<0.01); the body weight of the mice in the P36-0 group (15 mg / kg) was significantly reduced on days 3 to 12 (*P<0.05, **P<0.01, ***P<0.001), and the body weight of the mice in the P37-0 high-dose group (15 mg / kg) was significantly reduced on days 3 to 12 (*P<0.05, ***P<0.001).

[0292] The animal body weight data are shown in Table 4 and Fig. 1.

[0293] Table 4: Animal body weight data (g, X ± SD) of each group Note: *P<0.05; **P<0.01; ***P<0.001 vs model control group

[0294] 6.2 Tumor efficacy evaluation

[0295] Tumor volume, RTV, T / C%: During the test period, the tumor volume and RTV of the blank control group animals showed a rapid growth trend, and the average tumor volume was (1020.58 ± 507.01) mm 3 at the end of the test (D12). Compared with the blank control group, the average tumor volume and RTV of the paclitaxel positive drug group were reduced to some extent, the average tumor volume was (628.28 ± 214.79) mm 3 , and the relative tumor proliferation rate was 66.20%.

[0296] Compared with the blank control group, the average tumor volume and RTV of P37-0 low and high dose groups (5 / 15 mg / kg), P32-0 low and high dose groups (5 / 15 mg / kg), P36-0 group (15 mg / kg), and P34-0 high dose group (15 mg / kg) were significantly reduced (*P<0.05, **P<0.01, ***P<0.001); the average tumor volume of P34-0 low dose group (5 mg / kg) and P35-0 group (15 mg / kg) was reduced to a certain extent, but there was no statistical significance.

[0297] At the end of the experiment, the average tumor volume of P36-0 group (15 mg / kg), P37-0 low and high dose groups (5 / 15 mg / kg), P34-0 low and high dose groups (5 mg / kg), P35-0 dose group (15 mg / kg), and P32-0 low and high dose groups (5 / 15 mg / kg) was (113.78±26.36) mm 3 , (250.63±60.42) mm 3 , (151.47±48.13) mm 3 , (599.43±160.21) mm 3 , (376.68±137.30) mm 3 , (618.67±122.86) mm 3 , (464.84±169.89) mm 3 , (220.12±42.38) mm 3 , respectively, and the relative tumor proliferation rates were 12.34%, 26.09%, 14.52%, 63.82%, 39.40%, 65.03%, 48.40%, and 22.71%, respectively; the relative tumor proliferation rates (T / C) of P37-0 low and high dose groups (5 mg / kg) and P36-0 group (15 mg / kg), P34-0 high dose group (15 mg / kg), and P32-0 high dose group (15 mg / kg) were all less than 40%.

[0298] Table 5: Statistical data table of tumor volume of animals in each group (mm 3 , X±SD) Note: *, P<0.05; **, P<0.01; ***, P<0.001; vs model control group.

[0299] Table 6: Statistical data table of relative tumor volume (RTV) of animals in each group (X±SD) Note: *, P<0.05; **, P<0.01; ***, P<0.001; vs model control group.

[0300] Table 7 Statistics table of relative tumor growth rate T / C (%) of each group of animals Note: #, T / C%≤40% and P<0.05 compared with the model control group.

[0301] Tumor weight: At the end of the experiment, the tumor weights of the blank control group, the paclitaxel positive control group, the P36-0 group (15 mg / kg), the P37-0 low-high dose group (5 / 15 mg / kg), the P34-0 low-high dose group (5 / 15 mg / kg), the P35-0 group (15 mg / kg), and the P32-0 low-high dose group (5 / 15 mg / kg) were (1.08±0.39) g, (0.86±0.27) g, (0.13±0.04) g, (0.37±0.14) g, (0.21±0.08) g, (0.91±0.21) g, (0.59±0.18) g, (0.95±0.22) g, (0.58±0.22) g, and (0.29±0.06) g, respectively. The tumor weights of the P36-0 group (15 mg / kg), the P34-0 high dose group (15 mg / kg), the P37-0 low-high dose group (5 / 15 mg / kg), and the P32-0 low-high dose group (5 / 15 mg / kg) were significantly reduced compared with the model control group (*P<0.05, **P<0.01, ***P<0.001), and the tumor weights of the other dosing groups were reduced to a certain extent but not statistically significantly. The tumor inhibition rates of the dosing groups were 20.53%, 87.82%, 65.39%, 80.35%, 16.34%, 45.85%, 12.62%, 46.50%, and 73.35%, respectively, among which the tumor inhibition rates of the P36-0 dose group (15 mg / kg), the P32-0 high dose group (15 mg / kg), and the P37-0 low-high dose group (5 / 15 mg / kg) were all greater than 60%.

[0302] Table 8 Tumor weight data table of each group of animals (g, X±SD) Note: *, P<0.05; **, P<0.01; ***, P<0.001; vs model control group.

[0303] Table 9 Statistics table of tumor inhibition rate IR (%) of each group of animals Note: #, IR(%)≥60% and P<0.05 compared with the model control group

[0304] The tumor volume data statistics are shown in Table 5 and FIG. 2-1; the relative tumor volume RTV value data statistics are shown in Table 6 and FIG. 2-2; and the T / C% data statistics are shown in Table 7 and FIG. 2-3. The tumor weights and inhibition rates of each group of animals at the end of the experiment are shown in Table 8 and Table 9, and FIG. 3-1 and FIG. 3-2.

[0305] 10 CONCLUSION

[0306] Under the experimental conditions, P36-0, P37-0, P34-0, P35-0, P32-0 all have certain anti-tumor effect; under the same dose, the anti-tumor effect of each drug is ranked as P36-0>P37-0>P32-0>P34-0>P35-0.

[0307] Example 22: Pharmacodynamic test 1 of the series of compounds on the cyclophosphamide-induced neutropenia model in rats

[0308] 1. Experimental materials and methods

[0309] 1.1 Preparation of cyclophosphamide

[0310] Cyclophosphamide for injection (manufacturer: Baxter Oncology GmbH, batch number: OJ415A, 0.2 g / bottle) is a white crystal or crystalline powder, which is stored at 5-25°C.

[0311] Preparation method: add an appropriate amount of sterile 0.9% sodium chloride injection into the drug bottle containing the drug powder, and gently shake to fully dissolve and mix to form a solution of the corresponding concentration. If the dry powder cannot be completely dissolved immediately, the solution can be left to stand for a few minutes until it is completely clear (the whole process needs to be operated in a sterile environment).

[0312] 1.2 Preparation of the series of compounds

[0313] Dissolve an appropriate amount of sterile 5% glucose injection to obtain a certain concentration of the series of compounds, and gently shake to fully dissolve and mix. The sample for tail vein injection is filtered and sterilized, and the preparation process and after preparation are strictly protected from light.

[0314] 1.3 Preparation of Plinabulin

[0315] Solvent: 1,2 propanediol and HS-15 (80 mg Plinabulin: 12 mL of 1,2 propanediol, 8 mL of HS-15);

[0316] Preparation method: according to the actual use amount, prepare according to the prescription ratio, and the specific preparation process is as follows:

[0317] 1) Under strict light protection conditions, take the prescribed amount of 1,2 propanediol, add Plinabulin at 60°C to dissolve, and keep stirring after the raw material is completely dissolved. Keep stirring for 0.25-0.5 h;

[0318] 2) Continue to keep stirring and add half of the HS-15, stir for 10 min, and then add the other half of the HS-15;

[0319] 3) Under the condition of keeping warm, the drug solution was sterilized by 0.22 μm micropore filter, and then prepared into a concentrated solution with a concentration of 4 mg / mL;

[0320] 4) An appropriate amount of concentrated solution was diluted to 0.3 mg / mL with 0.9% sodium chloride injection;

[0321] Preparation frequency: single preparation, injection diluent needs to be prepared on the day of administration, and the concentrated solution can be prepared once a week;

[0322] Storage conditions and shelf life after preparation: dry storage at room temperature in the dark, avoid light during transportation to the animal house, and use it immediately after preparation;

[0323] Storage conditions and shelf life after preparation and dispensing: injection diluent should be prepared within 1 hour of preparation to avoid light administration.

[0324] 1.4 Experimental animals

[0325] SD male rats were used; they were about 6-7 weeks old when purchased and about 7-8 weeks old when grouped. Individual body weight was within ±20% of the average body weight. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. with production license number: SCXK (Jing) 2016-0011.

[0326] 1.5 Rearing conditions

[0327] Five rats per cage, rearing temperature 16-26°C (daily temperature difference ≤4°C), 12 / 12 hour light-dark cycle. The rats were fed with maintenance feed purchased from Beijing Kaoshe Lihua Feed Co., Ltd. and allowed to freely ingest feed and drink experimental animal drinking water.

[0328] 1.6 Experimental instruments

[0329] Automatic five-class blood analyzer (Germany Siemens; ADVIA2120i).

[0330] 2 Test method and detection index

[0331] 2.1 Animal grouping

[0332] The test was divided into 11 groups, each with 6 male rats, a total of 66 rats. As shown in Table 10, they were normal control group, model control group, Plinabulin treatment group, P34-0 treatment group, P32-0 treatment group, P36-0 treatment group, P37-0 treatment group, P34-1 treatment group, P32-1 treatment group, P36-1 treatment group, and P37-1 treatment group. Rats with the same level of neutrophil count before modeling were randomly grouped.

[0333] Table 10. Grouping table of experimental animals

[0334] 2.2 Dose design and administration

[0335] After grouping, the animals in each group except the normal control group were intraperitoneally injected with cyclophosphamide for modeling, the modeling dose was 50 mg / kg, and the administration volume was 10 mL / kg; 30 min after administration of the modeling drug, the normal control group and the model control group were injected with 5% glucose solution via the tail vein, the P34-0 treatment group, the P32-0 treatment group, the P36-0 treatment group, and the P37-0 treatment group were administered at a dose of 6 mg / kg via the tail vein; the Plinabulin treatment group was administered at a dose of 6 mg / kg via the tail vein for 20 min; the P34-1 treatment group, the P32-1 treatment group, the P36-1 treatment group, and the P37-1 treatment group were administered at a dose of 15 mg / kg via gavage; the test product groups were administered once. The design is shown in Table 11.

[0336] Table 11. Dose design table Note: The day of administration is defined as the first day of the test, Day 1 (D1).

[0337] 2.3 Detection index

[0338] General state observation was performed once a day after administration: including but not limited to the injection site of the rat, food and water intake, slow movement of the rat, skin ulceration, lymph node enlargement, death, and other abnormal symptoms.

[0339] 2.3.1 Weighing, before administration, D2, D3, D4, D6, D8, D10, D12, D14, D16 after administration;

[0340] 2.3.2 Food intake determination time: D1-D2, D2-D3, D6-D7, D13-D14 after administration;

[0341] 2.3.3 Hematology detection

[0342] Detection time: before administration, D2, D3, D4, D6, D8, D10, D12, D14, D16 after administration;

[0343] Detection animals: all surviving animals in each group;

[0344] Blood sampling method: about 0.1 mL of blood was sampled from the tail vein;

[0345] Blood sample processing method: the blood sample for hematology detection was anticoagulated with EDTA-K2, the blood sample was directly sampled for detection when detected, the sample was stored at 15-25°C, and the detection was completed within 8 hours;

[0346] 2.3.4 Gross anatomical observation

[0347] Dissection time: day 16; all live animals in each group;

[0348] Anesthesia and dissection method: anesthetized with 10% chloral hydrate (400 mg / kg) according to body weight, euthanized by abdominal aortic blood sampling, observed by gross anatomy, observed abnormal tissues by naked eye, took thymus, weighed, and calculated thymus coefficient (organ weight per 10 g body weight (mg)).

[0349] 3 Experimental results

[0350] 3.1 General clinical observation

[0351] Most of the rats in the P36-0 treatment group showed eye and nasal blood-colored secretions, inactivity, unformed feces, and abdominal fur pollution after administration. On D6, D8, and D10 of the test, 6M06, 6M03, and 6M02 were found dead. Dissection found severe pulmonary hemorrhage, gastric distension, intestinal yellow contents, urinary retention, and individual animals with black liver, spleen, and black or white kidney. In the P37-0 treatment group, most of the rats showed yellow hair on D6 after administration, and 7M04 also showed periorbital blood-colored secretions. In the P36-1 treatment group, 10M02 and 10M03 were found to have sparse hair and abdominal fur pollution on D12. The general clinical observation of the remaining groups was normal.

[0352] 3.2 Body weight (Table 12 and Figure 4)

[0353] During the administration to the end of the test, the body weights of the normal control group and the model control group rats were comparable and showed a gradual increasing trend. The body weight of the model control group rats was significantly lower than that of the normal control group on day 2 (Table 11, P≤0.05). Compared with the model control group, the body weight of the Plinabulin treatment group was significantly reduced on days 3 and 4 (Table 11, P≤0.05); the body weight of the P34-0 treatment group was significantly reduced on days 2-6 (Table 12, P≤0.05); the body weight of the P36-0 treatment group was significantly reduced on days 3-14 (Table 11, P≤0.05); the body weight of the P37-0 treatment group was significantly reduced on days 3-12 (Table 11, P≤0.05); the body weight of the P36-1 treatment group was significantly reduced on days 3-6 (Table 11, P≤0.05); and the body weight of the P37-1 treatment group was significantly reduced on day 4 (Table 11, P≤0.05). This suggests that these compounds have certain toxic side effects.

[0354] There was no significant difference in the body weight of the rats in the P32-0 treatment group, P34-1 treatment group, and P32-1 treatment group compared with the model control group, and they showed a gradual increasing trend. For detailed data, please refer to Table 12 and Figure 4.

[0355] Table 12 Statistical summary data of body weight changes of rats in each group *p<0.05, model control vs normal control; *p<0.05, Plinabulin treatment vs model control; # *p<0.05, P34-0 treatment vs model control; *p<0.05, P32-0 treatment vs model control; ● *p<0.05, P36-0 treatment vs model control; ▲ *p<0.05, P37-0 treatment vs model control. a *p<0.05, model control vs normal control; b *p<0.05, Plinabulin treatment vs model control; e *p<0.05, P36-1 treatment vs model control; f *p<0.05, P37-1 treatment vs model control

[0356] 3.3 Food intake

[0357] During the experiment, the food intake of the normal control group and the model control group was comparable; compared with the model control group, the food intake of the animals in each treatment group was reduced on D1 and D2, and on D6, the food intake of the animals in the P36-0 treatment group was comparable to that of the model control group, and on day 13, the food intake of the animals in each group returned to normal.

[0358] 3.4 Hematological parameters

[0359] 1) Neutrophil count (Neut, x 10 9 cells / L) Results (Table 13 and Figure 5):

[0360] On days 2-4, the neutrophil count of the model control group was significantly lower than that of the normal control group (P < 0.05, P < 0.01); on day 10, the neutrophil count of the model control group was significantly higher than that of the normal control group (P < 0.01), and overall, it decreased after modeling, then increased, and then decreased to the normal level.

[0361] Compared with the model control group, the neutrophil count of the Plinabulin treatment group, the P34-0 treatment group, the P32-0 treatment group, the P36-0 treatment group, and the P37-0 treatment group was significantly higher on day 2 after administration (P < 0.05, P < 0.01, P < 0.001); the neutrophil count of the P36-0 treatment group was significantly higher than that of the model control group on days 6-16 after administration (P < 0.05, P < 0.01, P < 0.001). The neutrophil count of the P37-0 treatment group was significantly higher than that of the model control group on day 14 after administration (P < 0.05).

[0362] Compared with the Plinabulin treatment group, the P34-0 treatment group, the P36-0 treatment group, and the P37-0 treatment group had significantly increased neutrophils on day 2 after administration (P≤0.01, P≤0.001). For detailed data, see Table 13.

[0363] Table 13 Statistical summary data of neutrophil counts in rats in each group *p<0.05, model control group vs normal control group; *p<0.05, Plinabulin treatment group vs model control group; * *p<0.05, P34-0 treatment group vs model control group; *p<0.05, P32-0 treatment group vs model control group; ● *p<0.05, P36-0 treatment group vs model control group; ▲ *p<0.05, P37-0 treatment group vs model control group. a *p<0.05, model control group vs normal control group; b *p<0.05, Plinabulin treatment group vs model control group; c *p<0.05, P34-1 treatment group vs model control group; d *p<0.05, P32-1 treatment group vs model control group; f *p<0.05, P37-1 treatment group vs model control group.

[0364] 2) White blood cell count (WBC, x 10 9 cells / L) results (Table 14 and FIG. 6):

[0365] From day 2 to day 8, the model control group had significantly lower white blood cell counts than the normal control group (P≤0.01, P≤0.001), and the overall trend was that the white blood cell counts decreased after modeling and then increased to normal levels.

[0366] Compared with the model control group, the Plinabulin treatment group, the P34-0 treatment group, the P36-0 treatment group, and the P37-0 treatment group had significantly increased white blood cell counts on day 2 after administration (P≤0.05, P≤0.01, P≤0.001), and the P32-0 treatment group had significantly increased white blood cell counts on day 14 (P≤0.05).

[0367] Compared with the Plinabulin treatment group, the P34-0 treatment group, the P36-0 treatment group, and the P37-0 treatment group had significantly increased neutrophils on day 2 after administration (P≤0.05, P≤0.001). For detailed data, see Table 14.

[0368] Table 14 Statistical summary data of white blood cell counts in rats in each group p<0.05, model control vs normal control; * p<0.05, Plinabulin treatment vs model control; # p<0.05, P34-0 treatment vs model control; P32-0 treatment vs model control; ● p<0.05, P36-0 treatment vs model control; ▲ p<0.05, P37-0 treatment vs model control. a p<0.05, model control vs normal control; b p<0.05, Plinabulin treatment vs model control; f p<0.05, P37-1 treatment vs model control.

[0369] 3.5 Thymus Index

[0370] The thymus index of model control rats was significantly lower than that of normal control rats (P≤0.05), and the thymus index of Plinabulin treatment group, P32-0 treatment group and P37-0 treatment group was significantly higher than that of model control group (P≤0.05, P≤0.05).

[0371] Table 15 Statistical summary data of thymus weight and thymus index of rats in each group p<0.05, model control vs normal control; *p<0.05, Plinabulin treatment vs model control; ▲ p<0.05, P37-0 treatment vs model control.

[0372] 4 Summary

[0373] Under the conditions of this test, the model control group had no abnormal general clinical observation after 50 mg / kg cyclophosphamide intraperitoneal modeling, and there was no significant change in body weight and food intake. Four days after modeling, neutrophils and leukocytes were significantly reduced, indicating that the cyclophosphamide-induced neutropenia model was successfully established at this dose.

[0374] During the test, the body weight and food intake of Plinabulin treatment group, P34-0 treatment group, P36-0 treatment group, P37-0 treatment group, P36-1 treatment group and P37-1 treatment group were reduced to a certain extent. Neutrophils and leukocytes of Plinabulin treatment group, P34-0 treatment group, P32-0 treatment group, P36-0 treatment group, P37-0 treatment group and P37-1 treatment group were significantly increased on the 2nd day after administration, suggesting that these compounds had a certain degree of therapeutic effect on neutropenia.

[0375] Example 23: Pharmacodynamic test 2 of the series compounds on the cyclophosphamide-induced neutropenia model in rats

[0376] 1. Experimental materials and methods same as Example 22

[0377] 2. Test methods and detection indexes

[0378] 2.1 Group design

[0379] The test was designed with 8 groups, each with 6 male rats, a total of 48 rats; normal control group, model control group, Plinabulin treatment group, P32-0 treatment group, P32-1 treatment group, P31-0 treatment group, P29-0 treatment group, P30-0 treatment group; rats with the same level of neutrophils before modeling were randomly divided into groups.

[0380] Table 16. Experimental animal grouping table

[0381] 2.2 Dose design

[0382] After grouping, the animals in the normal control group were given intraperitoneal injection of cyclophosphamide for modeling, the modeling dose was 50 mg / kg, and the drug volume was 10 mL / kg; 30 min after the modeling drug was given, the normal control group and the model control group were given 5% glucose solution by tail vein injection; the Plinabulin treatment group was given a dose of 7.5 mg / kg, and the drug was given by tail vein infusion for 20 min; the remaining treatment groups were given a dose of 12 mg / kg, and the drug was given by tail vein injection. The test product groups were given single dose. The design is shown in Table 17.

[0383] Table 17. Dose design table

[0384] 2.3 Drug administration

[0385] Route of administration: tail vein injection; single dose was given 30 min after the modeling drug was given;

[0386] Note: The day of administration is defined as the first day of the test, Day 1 (D1).

[0387] 2.3 Detection indexes

[0388] General state observation was performed once a day after administration: including but not limited to rat injection site, food and water intake, and rat movement retardation, skin ulceration, lymph node enlargement, death, and other abnormal symptoms.

[0389] 2.3.1 Weighing, before administration, D2, D3, D5, D7, D9, D11, D14, D16, D18 after administration;

[0390] 2.3.2 Food consumption measurement time: D1-D2, D2-D3, D6-D7, D13-D14 after dosing;

[0391] Hematological examination was performed once a day before and after dosing. On day 15 after dosing, all rats were dissected, and the thymus of each animal was weighed and the organ coefficient (weight of organ per 10 g of body weight (mg)) was calculated.

[0392] 2.3.3 Hematological examination

[0393] Examination time: before dosing, D1, D2, D3, D5, D7, D8, D9, D11, D12, D14, D16 after dosing; about 0.1 mL of blood was collected from the tail vein;

[0394] Blood sample processing method: the blood sample for hematological examination was anticoagulated with EDTA-K2, and the whole blood sample was directly injected for examination at the time of examination. The sample was stored at 15-25°C, and the examination was completed within 8 hours;

[0395] 2.3.4 Gross anatomical observation

[0396] Dissection time: day 16; all surviving animals in each group;

[0397] Anesthesia and dissection method: anesthesia was performed with 10% chloral hydrate (400 mg / kg) according to body weight, and the animals were euthanized by abdominal aortic blood collection. Gross anatomical observation was performed, the thymus was removed, weighed, and the thymus coefficient (weight of organ per 10 g of body weight (mg)) was calculated.

[0398] 3. Experimental results

[0399] 3.1 Body weight (Table 18 and Figure 7)

[0400] Compared with the normal control group, the body weight of the model control group decreased significantly on D2 (P≤0.05), and was comparable to the normal control group at other times. Compared with the model control group, the body weight of the P32-0 treatment group decreased significantly on D3 and D5 (P≤0.05, P≤0.01), and the body weight of the P32-1 treatment group decreased significantly on D3 (P≤0.05). The body weights of the other treatment groups were not significantly different from those of the model control group, and showed a gradual increasing trend.

[0401] The average body weight of the normal control group increased by 2.42% on D2, while the average body weight of the model control group decreased by 3.87% on D2, decreased by 0.41% on D3, and increased by 3.16% on D5. The average body weight of the P32-0 treatment group decreased by 7.96% and 2.12% on D3 and D5, respectively. The average body weight of the P32-1 treatment group decreased by 6.96% on D3.

[0402] The animal weight statistics are shown in Table 18. The weight growth trend of each group is shown in Figure 7.

[0403] Table 18 Animal weight statistics table (g) * P≤0.05, normal control group vs model control group; # P≤0.05, ## P≤0.01, model control group vs each treatment group.

[0404] 3.2 Food intake

[0405] During the experiment, the model control group, P36-0 treatment group, P29-0 treatment group, and D1 food intake decreased, and D2 returned to normal food intake; the Plinabulin treatment group, P32-0 treatment group, P32-1 treatment group, and P30-0 treatment group D1 and D2 food intake decreased, and D6 each group returned to normal food intake. The statistical data are shown in Table 19,

[0406] Table 19 Animal food intake statistics

[0407] 3.3 Hematology index

[0408] 1) Neutrophil count (Neut, ×10 9 cells / L) results (Table 20 and Figure 8):

[0409] From the 2nd to the 8th day, the neutrophil count of the model control group was significantly lower than that of the normal control group (P≤0.01, P≤0.001); on the 11th day, the neutrophil count of the model control group was significantly higher than that of the normal control group (P≤0.05), and the overall trend was that it rose after modeling, then decreased, then rose again, and then decreased to the normal level.

[0410] Compared with the model control group, the neutrophil count of the Plinabulin treatment group significantly increased on the 1st and 2nd day after administration (P≤0.01), the P32-0 treatment group significantly increased on the 2nd, 11th-14th day after administration (P≤0.05, P≤0.01); the P32-1 treatment group significantly increased on the 2nd, 11th-14th day after administration (P≤0.05, P≤001); the P31-0 treatment group significantly increased on the 2nd and 3rd day after administration (P≤0.05); the P30-0 treatment group significantly increased on the 2nd day after administration (P≤0.05, P≤0.01).

[0411] The animal neutrophil statistics are shown in Table 20. The neutrophil changes of each group are shown in Figure 8

[0412] Table 20 Animal neutrophil statistics (×10 9 cells / L) * P≤0.05, ** P≤0.01, *** P≤0.001, normal control vs model control; # P≤0.05, ## P≤0.01, model control vs each treatment group; P≤0.05, P≤0.01, P≤0.001, P32-0 group vs each treatment group

[0413] 3) White blood cell count (WBC, x 10 9 cells / L) results (Table 21 and Figure 9):

[0414] From day 2 to day 9, the white blood cell count of the model control group was significantly lower than that of the normal control group (P≤0.01, P≤0.001), and overall it rose after modeling, then fell, and then slowly rose to normal level.

[0415] Compared with the model control group, the white blood cell count of the P32-0 treatment group significantly increased on days 1-2 after administration (P≤0.001); the white blood cell count of the P32-1 treatment group significantly increased on days 1-2 and 7 after administration (P≤0.05, P≤0.01); the white blood cell count of the P29-0 treatment group significantly increased on days 8 and 14 after administration (P≤0.05, P≤0.01).

[0416] The animal white blood cell statistical data are shown in Table 21. The white blood cell changes in each group are shown in Figure 9.

[0417] Table 21 Animal white blood cell statistical data (x 10 9 cells / L) ** P≤0.01, *** P≤0.001, normal control vs model control; # P≤0.05, ## P≤0.01, ### P≤0.001, model control vs each treatment group; P≤0.05, P≤0.01, P≤0.001, P32-0 group vs each treatment group.

[0418] 3.4 Thymus weight and thymus coefficient

[0419] There was no significant difference in thymus weight and thymus coefficient among the groups. The animal thymus weight and thymus coefficient statistical data are shown in Table 22.

[0420] Table 22 Animal thymus weight and thymus coefficient statistical data

[0421] 4. Summary

[0422] Under the experimental conditions, the model control group had no abnormal general clinical observation after the intraperitoneal modeling of 50 mg / kg cyclophosphamide, and the body weight and food intake had no obvious change. About one week after modeling, the neutrophils and leukocytes were significantly reduced, indicating that the neutrophil reduction model was successfully established under the dose of cyclophosphamide.

[0423] During the experiment, except for the P29-0 treatment group, the Plinabulin treatment group, the P32-0 treatment group, the P32-1 treatment group, the P31-0 treatment group, and the P30-0 treatment group generally had a significant increase in neutrophils on the 2nd and 3rd day after administration, indicating that these compounds had a certain degree of prevention and treatment effect on neutropenia.

[0424] Compared with the Plinabulin treatment group, the P32-0 treatment group had a significant increase in neutrophils on the 2nd, 11th or 12th or 14th day after administration, indicating that the compound P32-0 had a better effect on preventing and treating neutropenia.

[0425] Example 24: Efficacy test 3 of the series of compounds on the cyclophosphamide-induced neutrophil reduction model in rats

[0426] 1. Experimental materials and methods are the same as in Example 22.

[0427] 2. Test methods and detection indexes

[0428] 2.1 Group design

[0429] The experiment was divided into 8 groups, each with 6 male rats, a total of 48 rats; the normal control group, the model control group, the Plinabulin treatment group, the P32-0 treatment group, the P5-0 treatment group, the P12-0 treatment group, the P10-0 treatment group, and the P11-0 treatment group; the rats with the same neutrophil level before modeling were randomly divided into groups.

[0430] Table 23: Experimental animal grouping table

[0431] 2.2 Dose design

[0432] After grouping, the animals in the normal control group were given cyclophosphamide intraperitoneally for modeling, the modeling dose was 50 mg / kg, and the administration volume was 10 mL / kg; 30 min after the administration of the modeling drug, the normal control group and the model control group were injected with 5% glucose solution via the tail vein; the Plinabulin treatment group was administered at a dose of 7.5 mg / kg, and the drug was administered by intravenous infusion for 20 min; the remaining treatment groups were administered at a dose of 12 mg / kg, and the drug was administered by intravenous injection. The test product groups were administered once. The design is shown in Table 24.

[0433] Table 24 Dose Design Table

[0434] 2.3 Dosing

[0435] Dosing Route: Tail vein injection; single dose administration 30 min after administration of the model drug;

[0436] Note: The day of dosing is defined as Day 1 (D1) of the experiment.

[0437] 3. Test Indexes

[0438] General state observation was performed once a day after dosing: including but not limited to the injection site of the rats, food and water intake, and the conditions such as movement retardation, skin ulceration, lymph node enlargement, death, and other abnormal symptoms of the rats.

[0439] 3.1 Weighing, before dosing, and on D2, D3, D5, D7, D9, D11, D14, D16 after dosing;

[0440] 3.2 Food intake determination time: D1-D2, D2-D3, D6-D7, D13-D14 after dosing;

[0441] Hematological examination was performed once a day before and after dosing. All rats were dissected on Day 15 after dosing, and the thymus of each animal was taken, weighed, and the organ coefficient (weight of the organ per 10 g of body weight (mg)) was calculated.

[0442] 3.3 Hematological examination

[0443] Examination time: before dosing, and on D1, D2, D3, D5, D7, D9, D11, D12, D14, D16 after dosing; about 0.1 mL of blood was taken from the tail vein;

[0444] Blood sample processing method: the blood sample for hematological examination was anticoagulated with EDTA-K2, and the blood sample was directly injected for examination when the examination was performed, the sample was stored at 15-25°C, and the examination was completed within 8 hours;

[0445] 3.4 Gross anatomical observation

[0446] Dissection time: Day 16; all surviving animals in each group;

[0447] Anesthesia and dissection method: anesthesia was performed with 10% chloral hydrate (400 mg / kg) according to the body weight, and euthanasia was performed by abdominal aortic blood sampling, and gross anatomical observation was performed on the rats, the thymus was taken, weighed, and the thymus coefficient (weight of the organ per 10 g of body weight (mg)) was calculated

[0448] 4. Experimental Results

[0449] 4.1 Body weight (Table 25 and FIG. 10)

[0450] Compared with the normal control group, the model control group was significantly decreased in body weight at D2 and D9 (P≤0.05), and was comparable to the normal control group at other time points. Compared with the model control group, the P32-0 treatment group was significantly decreased in body weight at the first week (P≤0.05, P≤0.01). The other treatment groups showed no significant difference in body weight compared with the model control group, and showed a gradual growth trend.

[0451] The average body weight of the normal control group was increased by 2.06% at D2, and by 21.86% at D9, while the average body weight of the model control group was decreased by 4.39% at D2, increased by 2.40% at D3, by 6.64% at D5, and by 12.02% at D7. The average body weight of the P32-0 treatment group was decreased by 9.62%, 7.84%, and 0.61% at D2, D3, and D5, respectively, and increased by 5.72% at D7.

[0452] The animal body weight statistics are shown in Table 25. The body weight growth trend of each group is shown in Figure 10.

[0453] Table 25: Statistical summary of body weight changes of rats in each group * P≤0.05, normal control group vs model control group; # P≤0.05, ＃＃ P≤0.01, model control group vs each treatment group.

[0454] 4.2 Food intake

[0455] During the experiment, the model control group, the Plinabulin treatment group, the P5-0 treatment group, the P12-0 treatment group, the P10-0 treatment group, and the P11-0 treatment group showed reduced food intake at D1, and normal food intake at D2. The P32-0 treatment group showed reduced food intake at D1 and D2, and normal food intake at D6.

[0456] Table 26: Animal food intake statistics

[0457] 4.3 Hematological indicators

[0458] 1) Neutrophil count (Neut, ×10 9 cells / L) results (Table 27 and Figure 11):

[0459] On day 1, the neutrophil count of the model control group was significantly higher than that of the normal control group (P≤0.001). On days 2-7, the neutrophil count of the model control group was significantly lower than that of the normal control group (P≤0.05, P≤0.01, P≤0.001). On day 12, the neutrophil count of the model control group was significantly higher than that of the normal control group (P≤0.05), and overall showed an upward trend after modeling, followed by a downward trend, and then an upward trend again, and finally returned to the normal level.

[0460] Compared with the model control group, the neutrophil count of the Plinabulin treatment group was significantly increased on day 1 and day 2 (P≤0.05, P≤0.01) and significantly decreased on day 7 (P≤0.05); the neutrophil count of the P32-0 treatment group was significantly increased on day 2, day 3 and day 11 (P≤0.05, P≤0.01); the neutrophil count of the P12-0 treatment group and the P11-0 treatment group was significantly increased on day 2 (P≤0.05).

[0461] The neutrophil count of the animals in each group is shown in Table 27. The neutrophil count in each group is shown in Figure 11.

[0462] Table 27 Summary of the neutrophil count of rats in each group * P≤0.05, ** P≤0.01, *** P≤0.001, normal control group vs model control group; # P≤0.05, ## P≤0.01, model control group vs each treatment group; ∧ P≤0.05, ∧∧ P≤0.01, P32-0 group vs each treatment group.

[0463] 4) White blood cell count (WBC, ×10 9 cells / L) results (Table 28 and Figure 12):

[0464] From day 2 to day 7 and day 11, the white blood cell count of the model control group was significantly lower than that of the normal control group (P≤0.05, P≤0.001), and the overall trend was an increase after modeling, followed by a decrease, and then a slow increase to the normal level.

[0465] Compared with the model control group, the white blood cell count of the Plinabulin treatment group was significantly increased on day 1 and day 2 (P≤0.05, P≤0.01) and significantly decreased on day 7 (P≤0.05); the white blood cell count of the P32-0 group was significantly increased on day 2 (P≤0.01); the white blood cell count of the P12-0 treatment group and the P11-0 treatment group was significantly increased on day 2 (P≤0.01);

[0466] The white blood cell count of the animals in each group is shown in Table 28. The white blood cell count in each group is shown in Figure 12.

[0467] Table 28 Summary of the white blood cell count of rats in each group * P≤0.05, *** P≤0.001, normal control group vs model control group; # P≤0.05,## P < 0.01, model control group vs each treatment group; ∧ P < 0.05, ∧∧ P < 0.01, ∧∧∧ P < 0.001, P32-0 group vs each treatment group.

[0468] 4.4 Thymus weight and thymus coefficient

[0469] Compared with the model control group, the thymus weight of the P5-0 treatment group, the P12-0 treatment group and the P10-0 treatment group decreased significantly (P < 0.05); the thymus coefficient of the P12-0 treatment group and the P10-0 treatment group decreased significantly (P < 0.05).

[0470] The statistical data of the thymus weight and the thymus coefficient of the animals are shown in Table 29.

[0471] Table 29 Statistical data of the thymus weight and the thymus coefficient of the animals # P < 0.05, model control group vs each treatment group.

[0472] 4 Summary

[0473] Under the conditions of the present experiment, the model control group had no abnormality in general clinical observation after modeling with 50 mg / kg cyclophosphamide intraperitoneally, and had no obvious change in body weight and food intake. About one week after modeling, neutrophils and white blood cells decreased significantly, indicating that the neutrophil reduction model induced by cyclophosphamide at this dose was successfully established.

[0474] During the experiment, the neutrophils of the Plinabulin treatment group and the P32-0 treatment group increased significantly on the 2nd and 3rd day after administration, indicating that the compound P32-0 had obvious preventive and therapeutic effects on neutrophil reduction; the compounds P12-0 and P11-0 also showed certain white blood cell increasing effects at a dose of 12 mg / kg.

[0475] Example 25: Efficacy test 4 of the series of compounds on the cyclophosphamide-induced neutrophil reduction model in rats

[0476] 1 Experimental materials and methods

[0477] 1.1 Preparation of the series of compounds

[0478] Dissolve the compounds in an appropriate amount of sterile 5% glucose injection solution, with the concentration of P32-0, P38-0, P39-0 and P41-0 being 1.2 mg / mL; the concentration of P40-0 being 0.8 mg / mL; and the concentration of P42 being 2.4 mg / mL. Shake gently to fully dissolve and mix, filter sterilize through a 0.22 μm filter, and strictly avoid light during the preparation process and after preparation.

[0479] 1.2 Other experimental materials and methods see effect embodiment 22.

[0480] 2 Test method and detection index

[0481] 2.1 Group design

[0482] Test 9 groups, each group of 6, male, a total of 54; Normal control group, model control group, Plinabulin treatment group, P32-0 treatment group, P38-0 treatment group, P39-0 treatment group, P40-0 treatment group, P41-0 group, P42 treatment group; Select the average neutrophil level before modeling Randomly divided into groups.

[0483] Table 30 Experimental animal grouping table

[0484] 2.2 Dose design

[0485] This test group after normal control group, the rest of the animals in each group were given intraperitoneal injection of cyclophosphamide modeling, modeling dose was 50mg / kg, the volume of drug was 10mL / kg.

[0486] 30min after giving the modeling drug, the normal and model control group according to 10mL / kg tail vein injection of 5% glucose solution.

[0487] Plinabulin treatment group 30min after modeling, according to 7.5mg / kg intravenous infusion for 20 minutes single dose.

[0488] P32-0 treatment group, P38-0 treatment group, P39-0 treatment group, P40-0 treatment group, P41-0 treatment group 30min after modeling, respectively, according to 12mg / kg intravenous injection of corresponding compounds, single dose. P42 treatment group 30min after modeling, according to 24mg / kg gavage, single dose.

[0489] Table 31 Dose design table Note: Because P40-0 solubility is poor, the volume of drug is adjusted to 15mL / kg.

[0490] 2.3 Drug administration

[0491] Route of administration: tail vein injection; tail vein infusion; gavage; 30min after giving the modeling drug, single dose; The rate of administration: when the tail vein infusion is slow, it is completed within 20min;

[0492] Note: The day of administration is defined as the first day of the test, Day 1 (D1).

[0493] 2.4 Detection index

[0494] General state observation was performed once a day after administration: including but not limited to injection site, food and water intake, movement retardation, skin ulceration, lymph node enlargement, death and other abnormal symptoms.

[0495] 2.4.1 Weighing, before administration, 2, 3, 7, 11, 14, 16 after administration;

[0496] 2.4.2 Food intake determination time: 1-2, 2-3, 6-7, 13-14 after administration;

[0497] Hematological examination was performed once a day before and after administration. All rats were dissected on the 15th day after administration, and the thymus of each animal was weighed and the organ coefficient (weight of organ per 10 g of body weight (mg)) was calculated.

[0498] 2.4.3 Hematological examination

[0499] Examination time: before administration, D2, D3, D5, D7, D8, D9, D11, D12, D14, D16 after administration; about 0.1 mL of blood was collected from the tail vein;

[0500] Blood sample processing method: the blood sample for hematological examination was anticoagulated with EDTA-K2, and the whole blood sample was directly injected for examination during examination. The sample was stored at 15-25°C, and the examination was completed within 8 hours;

[0501] 2.4.4 Gross anatomical observation

[0502] Dissection time: day 16; all surviving animals in each group;

[0503] Anesthesia and dissection method: anesthesia with 10% chloral hydrate (400 mg / kg) according to body weight, euthanasia by abdominal aortic blood sampling, gross anatomical observation, thymus removal, weighing, and calculation of thymus coefficient (organ weight per 10 g of body weight (mg)).

[0504] 3. Experimental results

[0505] 3.1 Body weight (Table 32 and Figure 13)

[0506] Compared with the model control group, the P32-0 treatment group showed significant weight loss in the first week (P<0.05), and the P38-0 treatment group showed significant weight loss on D2 and D3 (P<0.05). The body weights of the other treatment groups showed no significant difference compared with the model control group, and showed a gradual growth trend.

[0507] The animal body weight statistical data are shown in Table 32. The body weight growth trend of each group is shown in Figure 13.

[0508] Table 32 Summary of body weight change data of rats in each group # P < 0.05, model control vs. each treatment group.

[0509] 3.2 Food intake

[0510] During the experiment, the model control group, P39-0 treatment group, P40-0 treatment group, P41-0 treatment group, P42 treatment group D1 food intake decreased, D2 normal food intake; Plinabulin treatment group, P32-0 treatment group, P38-0 treatment group D1 and D2 food intake decreased, D6 normal food intake.

[0511] Table 33 statistical summary data of food intake changes of rats in each group

[0512] 3.3 Hematology index

[0513] 1) Neutrophil count (Neut, x 10 9 cells / L) results (Table 34 and Figure 14):

[0514] From the 3rd to the 8th day, the neutrophil count of the model control group was significantly lower than that of the normal control group (P < 0.01, P < 0.001); on the 14th and 16th day, the neutrophil count of the model control group was significantly higher than that of the normal control group (P < 0.05), and the overall trend was that it rose after modeling, then fell, then rose again, and then fell to near the normal level.

[0515] Compared with the model control group, the neutrophil count of the Plinabulin treatment group significantly increased on the 2nd day after administration (P < 0.05) and significantly decreased on the 8th day (P < 0.01); the neutrophil count of the P32-0 treatment group significantly increased on the 2nd, 7th-14th day after administration (P < 0.05, P < 0.01, P < 0.001); the neutrophil count of the P38-0 treatment group and the P41-0 treatment group significantly increased on the 2nd day after administration (P < 0.05, P < 0.01); the neutrophil count of the P40-0 treatment group significantly increased on the 7th-14th day.

[0516] Table 34 statistical summary data of neutrophil count of rats in each group

[0517] Table 34 statistical summary data of neutrophil count of rats in each group * P < 0.05, ** P < 0.01, *** P < 0.001, normal control group vs. model control group; # P < 0.05, ## P < 0.01, ### P < 0.001, model control group vs. each treatment group.

[0518] 5) White blood cell count (WBC, x 10 9 Results (Table 35 and Figure 15):

[0519] From day 2 to day 8 and day 11, the white blood cell count of the model control group was significantly lower than that of the normal control group (P<0.05, P<0.001), and generally decreased after modeling, and then slowly increased to the normal level.

[0520] Compared with the model control group, the white blood cell count of the P32-0 treatment group significantly increased on day 2 and day 11-14 after administration (P<0.05); the white blood cell count of the P38-0 treatment group and the P41-0 treatment group significantly increased on day 2 after administration (P<0.05, P<0.01); and the white blood cell count of the P40-0 treatment group significantly increased on day 11-14 after administration.

[0521] The animal white blood cell count data is shown in Table 35. The white blood cell count changes of each group are shown in Figure 15.

[0522] Table 35 Animal white blood cell count data (x 10 9 cells / L) * P<0.05, *** P<0.001, normal control group vs model control group; # P<0.05, ## P<0.01, model control group vs each treatment group.

[0523] 3.4 Thymus weight and thymus coefficient

[0524] Compared with the normal control group, the thymus weight and thymus coefficient of the model control group significantly decreased (P<0.05). There was no significant difference between the other groups.

[0525] The animal thymus weight and thymus coefficient data are shown in Table 36.

[0526] Table 36 Animal thymus weight and thymus coefficient data

[0527] 4. Summary

[0528] Under the conditions of this experiment, the model control group had no abnormal general clinical observation after modeling with 50 mg / kg cyclophosphamide intraperitoneally, and the body weight and food intake did not change significantly. One week after modeling, the neutrophil and white blood cell counts significantly decreased, indicating that the neutrophil reduction model induced by cyclophosphamide at this dose was successfully established.

[0529] During the experiment, the Plinabulin treatment group, the P32-0 treatment group, the P38-0 treatment group and the P41-0 treatment group had a significant increase in neutrophils on the second day after administration, and the P40-0 treatment group had a significant increase in neutrophils on the 7th to 14th day after administration, indicating that these compounds have a certain degree of prevention and treatment effect on neutropenia.

[0530] Example 26: Pharmacodynamic experiment of compound P32-0 combined with G-CSF in the treatment of cyclophosphamide-induced neutropenia model in rats

[0531] 1. Experimental materials and methods

[0532] 1.1 Plinabulin preparation

[0533] Solvent: 1,2 propylene glycol and HS-15 (80 mg of Plinabulin: 12 mL of 1,2 propylene glycol, 8 mL of HS-15)

[0534] Preparation method: According to the actual use amount, the prescription ratio is prepared, and the specific preparation process is as follows:

[0535] 1) Under strict light protection conditions, take the prescription amount of 1,2 propylene glycol, dissolve Plinabulin at 60°C, and keep stirring for 0.25-0.5h after the raw material drug is completely dissolved;

[0536] 2) Continue to keep stirring and add half of the amount of HS-15, stir for 10 min, and then add the other half of the amount of HS-15;

[0537] 3) Under the condition of keeping warm, the liquid medicine is sterilized through a 0.22 μm microporous filter, and a concentrated solution with a concentration of 4 mg / mL is prepared;

[0538] 4) Take an appropriate amount of concentrated solution and dilute it to 0.375 mg / mL with 5% glucose injection;

[0539] Preparation frequency: The concentrated solution is prepared once a week, and is stored at room temperature under light protection. It is transported to the animal house under light protection conditions; the injection diluent needs to be prepared on the day of administration, and is stored at room temperature under light protection. It is transported to the animal house under light protection conditions, and is used immediately after preparation; the injection diluent is prepared within 6 hours after preparation, and is administered under light protection.

[0540] 1.2 Sulfofilgrastim injection (PEG-rhG-CSF) preparation

[0541] Manufacturer: Jiangsu Hengrui Medicine Co., Ltd.; Batch number: 210219CG / 210208CG;

[0542] Characteristics: colorless and clear liquid; specifications: 0.6 mL: 6 mg, pre-filled injection, chlorobutyl rubber piston seal, 1 box / box; 2-8°C, light-protected storage.

[0543] Preparation method: under sterile conditions, the pre-filled injection mother liquor (0.6 mL: 6 mg = 10 mg / mL) is diluted with 0.9% sodium chloride injection to the required concentration of 0.6 mg / mL; 2-8°C, light-protected dry storage, and light-protected transportation to the animal house;

[0544] 1.3 P32-0 Preparation

[0545] Dissolve with an appropriate amount of sterile 5% glucose injection to obtain 0.6, 1.2 mg / mL P32-0, and shake gently to fully dissolve and mix. Filter the sample for tail vein injection, and strictly avoid light during the preparation process and after preparation.

[0546] 2.4 Cyclophosphamide Preparation

[0547] Dissolve cyclophosphamide with an appropriate amount of sterile 0.9% sodium chloride injection, and shake gently to fully dissolve and mix to become 5 mg / mL. Store at room temperature, avoiding light.

[0548] 2.5 Experimental Animals

[0549] SPF grade SD male rats, purchased at about 210-230 g, and grouped at about 280-290 g.

[0550] 2 Test Design and Administration Method

[0551] 2.1 Group Design

[0552] Group design: the test has 9 groups, namely normal control group, model control group, Plinabulin treatment group, G-CSF (filgrastim) 0.25 mg / kg group, G-CSF (filgrastim) 0.5 mg / kg group, P32-0 6 mg / kg group, P32-0 12 mg / kg group, P32-0 6 mg / kg + G-CSF (filgrastim) 0.25 mg / kg group, and P32-0 12 mg / kg + G-CSF (filgrastim) 0.5 mg / kg group; each group has 6 male rats, and a total of 54 rats;

[0553] Grouping method: rats with the same level of neutrophil count before modeling are randomly grouped.

[0554] Table 37. Grouping table of experimental animals

[0555] 2.2 Dose Design

[0556] After grouping, the animals in each group were given cyclophosphamide by intraperitoneal injection for modeling, with a modeling dose of 50 mg / kg and a drug administration volume of 10 mL / kg.

[0557] 30 min after administration of the modeling drug, the normal control group and the model control group were given 5% glucose solution by tail vein injection at a dose of 10 mL / kg.

[0558] The Plinabulin treatment group was given Plinabulin by intravenous infusion at a dose of 7.5 mg / kg for 20 min 30 min after modeling, as a single dose.

[0559] The G-CSF 0.25 mg / kg group and the G-CSF 0.5 mg / kg group were given PEG-rhG-CSF by single subcutaneous injection at a low dose of 0.25 mg / kg and a high dose of 0.5 mg / kg, respectively, 24 h after modeling.

[0560] The P32-0 6 mg / kg group and the P32-0 12 mg / kg group were given P32-0 by intravenous injection at a low dose of 6 mg / kg and a high dose of 12 mg / kg, respectively, 30 min after modeling, as a single dose.

[0561] The P32-0 6 mg / kg+G-CSF 0.25 mg / kg group was given P32-0 by intravenous injection at a dose of 6 mg / kg 30 min after modeling, and PEG-rhG-CSF by single subcutaneous injection at a dose of 0.25 mg / kg 24 h after modeling.

[0562] The P32-0 12 mg / kg+G-CSF 0.5 mg / kg group was given P32-0 by intravenous injection at a dose of 12 mg / kg 30 min after modeling, and PEG-rhG-CSF by single subcutaneous injection at a dose of 0.5 mg / kg 24 h after modeling.

[0563] Table 38. Dose design table

[0564] 3. Experimental results

[0565] 3.1 Body weight

[0566] There was no difference in body weight between the model control group and the normal control group. Compared with the model control group, the P32-0 12 mg / kg group and the P32-0 12 mg / kg+G-CSF 0.5 mg / kg group had significantly reduced body weight on D3 and D5 (P≤0.05, P≤0.01). Compared with the G-CSF 0.5 mg / kg group, the P32-0 12 mg / kg+G-CSF 0.5 mg / kg group had significantly reduced body weight on D3 (P≤0.01). There was no significant difference in body weight between the other groups and the model control group, and the body weight showed a growth trend.

[0567] The animal body weight statistics are shown in Table 39. The body weight growth trend of each group is shown in Figure 16.

[0568] Table 39. Animal body weight statistics table (g)

[0569] 3.2 Food intake

[0570] During the experiment, the food intake of the model control group and each administration group on D1 was reduced, and the higher the dose of P32-0, the lower the food intake. On D2, the food intake of the groups other than the P32-0 12 mg / kg group and the P32-0 12 mg / kg+G-CSF 0.5 mg / kg group basically returned to normal. The P32-0 12 mg / kg group and the P32-0 12 mg / kg+G-CSF 0.5 mg / kg group returned to normal food intake on D6.

[0571] Table 40. Food intake of animals in each group (g / day)

[0572] 3.3 Hematology index

[0573] 1) Neutrophil count (Neut, ×10 9 cells / L) results (Table 41 and Figure 17)

[0574] On days 2-8, the neutrophil count of the model control group was significantly lower than that of the normal control group (P≤0.01, P≤0.001); on days 9-11, the neutrophil count of the model control group was significantly higher than that of the normal control group (P≤0.05, P≤0.01), and the overall trend was that it first decreased after modeling, then increased, and then decreased to the normal level.

[0575] As shown in Table 41 and Figure 17A, compared with the model control group, the neutrophil count of the Plinabulin treatment group was significantly increased on day 2 after administration (P≤0.05), the neutrophil count of the G-CSF 0.25 mg / kg group was significantly increased on days 2-7 and 11 after administration (P≤0.05, P≤0.01), the neutrophil count of the P32-0 6 mg / kg group was significantly increased on day 2 after administration (P≤0.01), and the neutrophil count of the P32-0 6 mg / kg+G-CSF 0.25 mg / kg group was significantly increased on days 2-9 after administration (P≤0.05, P≤0.01). Compared with the P32-0 6 mg / kg group, the neutrophil count of the P32-0 6 mg / kg+G-CSF 0.25 mg / kg group was significantly increased on days 3-8 after administration (P≤0.05, P≤0.01).

[0576] As shown in Figure 17B, compared with the model control group, the neutrophil count of the Plinabulin treatment group was significantly increased on day 2 after administration (P≤0.05), the neutrophil count of the G-CSF 0.5 mg / kg group was significantly increased on days 2-8 and 12 after administration (P≤0.05, P≤0.01, P≤0.001), the neutrophil count of the P32-0 12 mg / kg group was significantly increased on days 2 and 10-12 after administration (P≤0.05, P≤0.01), and the neutrophil count of the P32-0 12 mg / kg+G-CSF 0.5 mg / kg group was significantly increased on days 2-9 and 11-12 after administration (P≤0.05, P≤0.01, P≤0.001). Compared with the G-CSF 0.5 mg / kg group, the neutrophil count of the P32-0 12 mg / kg+G-CSF 0.5 mg / kg group was significantly increased on days 9-10 after administration (P≤0.05). Compared with the P32-0 12 mg / kg group, the neutrophil count of the P32-0 12 mg / kg+G-CSF 0.5 mg / kg group was significantly increased on days 3-8 after administration (P≤0.05, P≤0.01).

[0577] The neutrophil count of animals is shown in Table 41. The change of neutrophil count in each group is shown in Figure 17.

[0578] Table 41. Neutrophil count of animals (×10 9 cells / L)

[0579] 6) White blood cell count (WBC, ×10 9 cells / L) Results (Table 42 and Figure 18)

[0580] From days 2-8, the white blood cell count of the model control group was significantly lower than that of the normal control group (P≤0.05, P≤0.01), and the white blood cell count decreased first and then slowly increased to the normal level.

[0581] As shown in Figure 18A, compared with the model control group, the white blood cells of the G-CSF 0.25 mg / kg group were significantly increased on days 2-3 and 7-8 after administration (P < 0.05, P < 0.01), the white blood cells of the P32-0 6 mg / kg group were significantly decreased on days 3, 5, and 14 after administration (P < 0.05, P < 0.01), and the white blood cells of the P32-0 6 mg / kg + G-CSF 0.25 mg / kg group were significantly increased on days 2-3 and 7-9 after administration (P < 0.05, P < 0.01). Compared with the G-CSF 0.25 mg / kg group, the white blood cells of the P32-0 6 mg / kg + G-CSF 0.25 mg / kg group were significantly increased on days 7-9 after administration (P < 0.05). Compared with the P32-0 6 mg / kg group, the white blood cells of the P32-0 6 mg / kg + G-CSF 0.25 mg / kg group were significantly increased on days 3-9 after administration (P < 0.05, P < 0.01, P < 0.001).

[0582] As shown in Figure 18B, compared with the model control group, the white blood cells of the G-CSF 0.5 mg / kg group were significantly increased on days 2-8 after administration (P < 0.05, P < 0.01, P < 0.001), the white blood cells of the P32-0 12 mg / kg group were significantly decreased on days 3 and 5 after administration (P < 0.05, P < 0.01), and the white blood cells of the P32-0 12 mg / kg + G-CSF 0.5 mg / kg group were significantly increased on days 7-12 after administration (P < 0.05, P < 0.001). Compared with the G-CSF 0.5 mg / kg group, the white blood cells of the P32-0 12 mg / kg + G-CSF 0.5 mg / kg group were significantly increased on days 8-12 after administration (P < 0.05, P < 0.01) and significantly decreased on days 3 and 5 (P < 0.01, P < 0.001). Compared with the P32-0 12 mg / kg group, the white blood cells of the P32-0 12 mg / kg + G-CSF 0.5 mg / kg group were significantly increased on days 5-12 after administration (P < 0.05, P < 0.001).

[0583] The white blood cell data of the animals are shown in Table 42, and the changes in the white blood cells of the various groups are shown in Figure 18.

[0584] Table 42 White blood cell data of animals (x 10 9 cells / L)

[0585] 3.4 Thymus weight and thymus coefficient

[0586] Compared with the model control group, the thymus weight and thymus coefficient of the Plinabulin treatment group, the G-CSF 0.25 mg / kg group, the G-CSF 0.5 mg / kg group, and the P32-0 12 mg / kg+G-CSF 0.5 mg / kg group were significantly reduced (P≤0.05, P≤0.01, P≤0.001). Compared with the G-CSF 0.25 mg / kg group, the thymus weight and thymus coefficient of the P32-0 6 mg / kg+G-CSF 0.25 mg / kg group were significantly increased (P≤0.05). Compared with the Plinabulin treatment group, the thymus weight and thymus coefficient of the P32-0 6 mg / kg group were significantly increased (P≤0.05). There was no significant difference in the thymus weight and thymus coefficient between the other groups.

[0587] The statistical data of the thymus weight and thymus coefficient of the animals are shown in Table 43.

[0588] Table 43 Statistical data of the thymus weight and thymus coefficient of the animals

[0589] 5 Summary

[0590] Under the conditions of the present experiment, the model control group had no abnormal general clinical observation after being modeled with 50 mg / kg cyclophosphamide intraperitoneally, and the body weight and food intake did not change significantly. The neutrophils and leukocytes significantly decreased about one week after modeling, indicating that the neutrophil reduction model induced by cyclophosphamide was successfully established at this dose.

[0591] During the experiment, the neutrophils of the G-CSF low and high dose groups, the P32-0 low and high dose groups, and the combined use groups significantly increased after administration, indicating that the G-CSF, P32-0, G-CSF and P32-0 combined use groups had a certain degree of prevention and treatment effect on neutropenia.

[0592] The above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, the technical solutions recorded in the foregoing examples can still be modified by those of ordinary skill in the art, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed by the present application.

Claims

1. A 2,5-diketopiramate compound or a pharmaceutically acceptable salt thereof with antitumor activity, characterized in that: Its structural formula is shown in equation (I): In the formula: n=0-3; m=0-3; x=0-5; R 1 It is a monosubstituted or polysubstituted group, wherein the group is selected from hydrogen, deuterium, halogen, C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl substituted with one or more halogens, C1-C8 alkoxy, C1-C8 alkoxy substituted with one or more halogens, benzoyl, benzoyl substituted with one or more halogens, phenoxy, phenoxy substituted with one or more halogens, carboxyl, cyano, hydroxyl, nitro or methylthiophene; L is a C1-C8 heteroalkylene group, and is surrounded by one or more R 2 Substituted C1-C8 heteroalkylene, C1-C8 alkylene, or substituted with one or more R 2 Substituted C1-C8 alkylene groups; R 2 It is a C1-C8 alkyl or C3-C 10 cycloalkyl; R 3 R 4 R 6 and R 7 It is hydrogen, deuterium, C1-C8 alkyl, or formed by one or more R 3-1 Substituted C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkoxy, C3-C 10 cycloalkyl, C6-C 10 Aryl or 3-10 membered heterocyclic alkyl; wherein, R 3-1 OR 3-1-1 , where R 3-1-1 It is a C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl, or composed of one or more C6-C groups. 10 Aryl-substituted C1-C8 alkyl, C1-C8 alkenyl or C1-C8 ynyl; Z is O, S, SO, SO2 or N(R) 5 ); R 5 It is hydrogen, deuterium, C1-C8 alkyl, C1-C8 alkenyl, or C1-C8 alkynyl, and is affected by one or more R groups. 5-1 Replacement C6-C 10 aryl, benzyl, and one or more R 5-2 Substituted benzyl, -C(=O)-R 5-3 or -S(=O)2-R 5-4 Among them, R 5-1 It is a halogen; R 5-2 Hydrogen, C1-C8 alkoxy, benzyloxy, or with one or more R 5-2 Substituted benzyloxy, C1-C8 alkyl, C3-C 10 Cycloalkyl, C2-C8 alkenyl, C6-C 10 Aryl or NR 5-3-1 R 5-3-2 ;R 5-3 It is a C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl or C6-C 10 Aryl; wherein, R 5-3-1 and R 5-3-2 It is hydrogen, C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl or C3-C 10 cycloalkyl; R 5-4 It is a C1-C8 alkyl or C6-C 10 Aryl.

2. The 2,5-diketopiramate compound or its pharmaceutically acceptable salt with antitumor efficacy according to claim 1, characterized in that, The heteroatoms in the C1-C8 heteroalkylene group of L are selected from one or more of N, O, S, and Se, and the number of heteroatoms is 1-4; the R 3 R 4 R 6 and R 7 The heteroatoms in the 3-10 membered heterocyclic alkyl groups are selected from one or more of N, O and S, and the number of heteroatoms is 1-5.

3. The 2,5-diketopiramate compound or its pharmaceutically acceptable salt with antitumor efficacy according to claim 2, characterized in that, In the structural formula: n = 0-1; m = 0-1; x = 0-2; R 1 It is a monosubstituted or polysubstituted group, wherein the group is selected from hydrogen, deuterium, vinyl, ethynyl, methyl, trifluoromethoxy, methoxy, halogen, cyano or benzoyl; L is R 2 It can be methyl, ethyl, isopropyl, tert-butyl, or cyclopropyl; R 3 R 4 R 6 and R 7 It is hydrogen, deuterium, methyl or ethyl, or R 3-1 Substituted methyl, cyclohexyl, or pyridyl; wherein, R 3-1 OR 3-1-1 , where R 3-1-1 It is methyl, ethyl, n-propyl or n-butyl; Z is O, S, SO, SO2 or N(R) 5 ); R 5 It is hydrogen, deuterium, methyl, ethyl, n-propyl, isopropyl, n-butyl or 2-methylpropyl, C1-C8 alkenyl, C1-C8 ynyl, and is bonded by one or more R 5-1 Substituted phenyl, benzyl, or substituted with one or more R 5-2 Substituted benzyl, -C(=O)-R 5-3 or -S(=O)2-R 5-4 Among them, R 5-1 It is a halogen; R 5-2 Hydrogen, C1-C8 alkoxy, benzyloxy, or with one or more R 5-2 Substituted benzyloxy, methyl, ethyl, n-propyl, isopropyl or n-butyl, cyclopentyl or cyclohexyl, C2-C8 alkenyl, C6-C 10 Aryl or NR 5-3-1 R 5-3-2 ;R 5-3 It is a C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl or C6-C 10 Aryl; wherein, R 5-3-1 and R 5-3-2 It is hydrogen, C1-C8 alkyl, C1-C8 alkenyl, C1-C8 ynyl or C3-C6 cycloalkyl; R 5-4 It is a C1-C8 alkyl or C6-C 10 Aryl.

4. The 2,5-diketopiramate compound or its pharmaceutically acceptable salt with antitumor efficacy according to claim 2, characterized in that, Their specific structural formulas are as follows:

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the 2,5-diketopiramate compound or its stereoisomers, tautomers, racemates, pharmaceutically acceptable salts, and pharmaceutically acceptable carriers and excipients as described in claim 1, which have antitumor effects.

6. The pharmaceutical composition according to claim 5, characterized in that, Its dosage forms include tablets, capsules, pills, injections, and topical preparations.

7. The use of the 2,5-diketopiramate compound with antitumor activity as described in claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in claim 5, as a microtubule inhibitor for inhibiting microtubules.

8. The use of the 2,5-diketopiramate compound with antitumor efficacy as described in claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in claim 5, in the preparation of a medicament for treating tumors.

9. The use of the 2,5-diketopiramate compound with antitumor efficacy as described in claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition as described in claim 5, in the preparation of a medicament for treating tumors in combination with a chemotherapeutic agent.

10. The application according to claim 9, characterized in that, The chemotherapy agent is docetaxel.

11. The use of the 2,5-diketopiramate compound with antitumor efficacy as described in claim 1, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment and / or prevention of neutropenia.

12. The application according to claim 11, characterized in that, The neutropenia is caused by the administration of chemotherapy or radiation therapy during cancer treatment.

13. The application according to claim 12, characterized in that, The cancers mentioned include liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer, and prostate cancer.

14. The application according to claim 12 or 13, characterized in that, The chemotherapy involves the application of a chemotherapeutic agent or a chemotherapeutic composition.

15. The application according to claim 14, characterized in that, The chemotherapeutic agent is docetaxel, paclitaxel, or cyclophosphamide; the chemotherapeutic composition is a combination of docetaxel, doxorubicin, and cyclophosphamide, a combination of docetaxel, paclitaxel, vincristine, doxorubicin, and cyclophosphamide, or a combination of docetaxel and cyclophosphamide.

16. The application according to claim 11, characterized in that, The 2,5-diketopiramate compound or its pharmaceutically acceptable salt with antitumor efficacy is administered orally or by injection.

17. The application according to claim 16, characterized in that, The 2,5-dikepiperazine compound or a pharmaceutically acceptable salt thereof may be administered alone during a chemotherapy cycle; or the 2,5-dikepiperazine compound or a pharmaceutically acceptable salt thereof may be administered after the administration of the chemotherapeutic agent or chemotherapeutic composition.

18. The application according to claim 17, characterized in that, The 2,5-diketopiramate compound or a pharmaceutically acceptable salt thereof shall be administered within 24 hours after the administration of the chemotherapeutic agent or chemotherapeutic composition.

19. The application according to claim 17, characterized in that, During each treatment cycle, the dosage of the 2,5-diketopiramate compound or a pharmaceutically acceptable salt thereof is less than 80 mg / kg.

20. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a 2,5-diketopiramate compound with antitumor efficacy as described in claim 1, or a pharmaceutically acceptable salt thereof, and a granulocyte colony-stimulating factor drug.

21. The pharmaceutical composition according to claim 20, characterized in that, The pharmaceutical composition comprises a single dose of 0.5 mg to 80 mg of the aforementioned 2,5-diketopiramate compound with antitumor efficacy or a pharmaceutically acceptable salt thereof.

22. Use of the pharmaceutical composition of claim 20 in the preparation of a medicament for treating and / or preventing neutropenia.

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