2, 5-diketopiperazine compound with anti-tumor effect and application of 2, 5-diketopiperazine compound in preparation of medicine for treating and / or preventing neutropenia

Abstract

The invention discloses a 2, 5-diketopiperazine compound with an anti-tumor effect and application of the 2, 5-diketopiperazine compound in preparation of a medicine for treating and / or preventing neutropenia, and the 2, 5-diketopiperazine compound is novel in structure, has good activity and water solubility, and can be used for preparing medicines for treating and / or preventing neutropenia. The defects of poor water solubility of the ponabulin, unfavorable patent medicine and difficult clinical administration can be overcome. The 2, 5-diketopiperazine compound as shown in the formula (I) or the pharmaceutically acceptable salt thereof can be used for treating and / or preventing neutropenia and improving toxic and side effects of clinical tumor treatment, and has an anti-tumor effect at the same time. The invention also discloses application of drug combination of the compound and a granulocyte colony stimulating factor (G-CSF) biological agent in drugs for treating and / or preventing neutropenia. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of chemistry and pharmaceutical technology, and specifically relates to a 2,5-diketopiperazine compound with anti-tumor efficacy and an application thereof in preparing a drug for treating and / or preventing neutropenia. Background Art

[0002] Neutropenia is a common hematological toxicity during chemotherapy. Accurate assessment, prevention and treatment are extremely critical for the recovery of cancer patients. Chemotherapy is one of the main treatments for malignant tumors. 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 therapy, which was developed by Amgen in the United States (trade name: Neulasta) and is currently the main drug for clinical treatment of CIN; for people with high fever risk, global sales have reached US$8 billion. However, given its mechanism of action, it has limited effects in the first week after clinical chemotherapy and unavoidable toxic and side effects. Therefore, there is a huge unmet medical need for the clinical treatment of CIN.

[0003] Plinabulin is a natural product derived from the marine fungus Aspergillus pyrocystis, which is a new type of 2,5-diketopiperazine (DKP) heterocyclic compound. It is a colchicine-type 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 stage of 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, as an effective antigen presenting cell (APC) inducer (by activating dendritic cell maturation), Plinabulin's lasting anti-cancer effect is related to its T cell activation effect. Further studies have shown that Plinabulin can also increase the growth of white blood cells by activating the release of cytokines. Due to the good effect of Plinabulin in treating neutropenia, a new drug NDA has been applied for.

[0004] The chemical structure of Plinabulin is as follows. Its molecular formula is C 19 H 20 N 4 O 2, molecular weight 336.39, CAS No. 714272-27-2, it has good stability but poor water solubility.

[0005]

[0006] Patent CN105705148 discloses a Plinabulin preparation, which contains 40% polyethylene glycol-15 hydroxystearate HS-15 and 60% propylene glycol, with a drug concentration of 4 mg / mL. However, it is understood that HS-15 is not an absolutely safe excipient. The former marketed product Rolapitant fat emulsion The dosage of HS-15 is 4.4%, and the single dosage is 4.07 g based on a single dose of 92.5 ml / bottle, which is close to the preparation disclosed in CN105705148. However, VARUBI-lipid emulsion has been withdrawn from the market due to severe allergic reactions after it was put on the market.

[0007] The difficulty in drug development due to poor water solubility is an existing technical barrier. The current existing technologies have been modified based on the structure of Plinabulin, but the problem of poor water solubility of Plinabulin has not been solved. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the defect of the prior art that there is a lack of effective drugs for treating neutropenia, and to provide a 2,5-diketopiperazine compound with anti-tumor efficacy and its use in the preparation of drugs for treating and / or preventing neutropenia. The 2,5-diketopiperazine compound provided by the present invention has the advantages of significantly improving the toxic and side effects of clinical tumor treatment and having anti-tumor efficacy.

[0009] The present invention solves the above technical problems through the following technical solutions:

[0010] The present invention provides a 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof having anti-tumor efficacy, the structural formula of which is shown in formula (I):

[0011]

[0012] Where:

[0013] n=0-3; m=0-3; x=0-5;

[0014] R 1 is a mono- or poly-substituted group selected from hydrogen, deuterium, halogen, C 1 -C 8 Alkyl, C 1 -C 8 Alkenyl, C 1 -C 8 Alkynyl, C substituted by one or more halogen1 -C 8 Alkyl, C 1 -C 8 Alkoxy, C substituted by one or more halogen 1 -C 8 alkoxy, benzoyl, benzoyl substituted by one or more halogens, phenoxy, phenoxy substituted by one or more halogens, carboxyl, cyano, hydroxy, nitro or methylthienyl;

[0015] L is C 1 -C 8 Heteroalkylene, with one or more R 2 Substituted C 1 -C 8 Heteroalkylene, C 1 -C 8 Alkylene or one or more R 2 Substituted C 1 -C 8 Alkylene;

[0016] R 2 C 1 -C 8 Alkyl or C 3 -C 10 Cycloalkyl;

[0017] R 3 , R 4 , R 6 and R 7 For hydrogen, deuterium, C 1 -C 8 Alkyl, with one or more R 3-1 Substituted C 1 -C 8 Alkyl, C 1 -C 8 Alkenyl, C 1 -C 8 Alkynyl, C 1 -C 8 Alkoxy, C 3 -C 10 Cycloalkyl, C 6 -C 10 Aryl or 3-10 membered heterocycloalkyl; wherein R 3-1 OR 3-1-1 , where R 3-1-1 C 1 -C 8 Alkyl, C 1 -C 8 Alkenyl, C 1 -C 8 Alkynyl or one or more C 6 -C10 Aryl substituted C 1 -C 8 Alkyl, C 1 -C 8 Alkenyl or C 1 -C 8 Alkynyl;

[0018] Z is O, S, SO, SO 2 or N(R 5 );

[0019] R 5 For hydrogen, deuterium, C 1 -C 8 Alkyl, C 1 -C 8 Alkenyl, C 1 -C 8 Alkynyl, with one or more R 5-1 Substituted C 6 -C 10 Aryl, benzyl, 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 is halogen; R 5-2 For hydrogen, C 1 -C 8 Alkoxy, benzyloxy, one or more R 5-2 Substituted benzyloxy, C 1 -C 8 Alkyl, C 3 -C 10 Cycloalkyl, C 2 -C 8 Alkenyl, C 6 -C 10 Aryl or NR 5-3-1 R 5-3-2 ; R 5-3 C 1 -C 8 Alkyl, C 1 -C 8 Alkenyl, C 1 -C 8 Alkynyl or C 6 -C 10 Aryl; wherein R 5 -3-1 and R 5-3-2 For hydrogen, C 1 -C 8 Alkyl, C 1 -C 8 Alkenyl, C 1 -C8 Alkynyl or C 3 -C 10 Cycloalkyl; R 5-4 C 1 -C 8 Alkyl or C 6 -C 10 Aryl.

[0020] Furthermore, the C of L 1 -C 8 The heteroatom in the heteroalkylene group is 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 heterocycloalkyl are selected from one or more of N, O and S, and the number of heteroatoms is 1-5.

[0021] Furthermore, in the structural formula:

[0022] n=0-1; m=0-1; x=0-2;

[0023] R 1 is a mono- or poly-substituted group selected from hydrogen, deuterium, vinyl, ethynyl, methyl, trifluoromethoxy, methoxy, halogen, cyano or benzoyl;

[0024] L is

[0025] R 2 is methyl, ethyl, isopropyl, tert-butyl or cyclopropyl;

[0026] R 3 , R 4 , R 6 and R 7 is hydrogen, deuterium, methyl or ethyl, 3-1 substituted methyl, cyclohexyl, or pyridyl; wherein R 3 -1 OR 3-1-1 , where R 3-1-1 is methyl, ethyl, n-propyl or n-butyl;

[0027] Z is O, S, SO, SO 2 or N(R 5 );

[0028] R 5 is hydrogen, deuterium, methyl, ethyl, n-propyl, isopropyl, n-butyl or 2-methylpropyl, C 1 -C 8 Alkenyl, C 1 -C8 Alkynyl, with one or more R 5-1 Substituted phenyl, benzyl, 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 is halogen; R 5-2 For hydrogen, C 1 -C 8 Alkoxy, benzyloxy, one or more R 5-2 Substituted benzyloxy, methyl, ethyl, n-propyl, isopropyl or n-butyl, cyclopentyl or cyclohexyl, C 2 -C 8 Alkenyl, C 6 -C 10 Aryl or NR 5-3- 1 R 5-3-2 ; R 5-3 C 1 -C 8 Alkyl, C 1 -C 8 Alkenyl, C 1 -C 8 Alkynyl or C 6 -C 10 Aryl; wherein R 5-3-1 and R 5-3-2 For hydrogen, C 1 -C 8 Alkyl, C 1 -C 8 Alkenyl, C 1 -C 8 Alkynyl or C 3 -C 6 Cycloalkyl; R 5-4 C 1 -C 8 Alkyl or C 6 -C 10 Aryl.

[0029] Furthermore, the 2,5-diketopiperazine compounds are specifically compounds P1 to P42 and pharmaceutically acceptable salts thereof, and the structural formulas are:

[0030]

[0031]

[0032]

[0033]

[0034]

[0035] The present invention provides a pharmaceutical composition, which contains the 2,5-diketopiperazine compound or its stereoisomers, tautomers, racemates, pharmaceutically acceptable salts, and pharmaceutically acceptable carriers and pharmaceutical excipients.

[0036] Furthermore, its dosage forms include tablets, capsules, pills, injections, and external preparations.

[0037] The present invention provides the use of the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition as a microtubule protein inhibitor for inhibiting microtubule protein.

[0038] The present invention provides the use of the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing a drug for treating tumors.

[0039] The present invention provides the use of the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition in combination with a chemotherapeutic agent to prepare a drug for treating tumors.

[0040] Furthermore, the chemotherapeutic agent is docetaxel.

[0041] The present invention provides the use of the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof having anti-tumor efficacy in preparing a drug for treating and / or preventing neutropenia.

[0042] Furthermore, 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.

[0043] Furthermore, the pharmaceutically acceptable salt of the 2,5-diketopiperazine compound represented by formula (I) is hydrochloride or methanesulfonate.

[0044] Furthermore, the neutropenia is caused by the administration of chemotherapy or by the administration of radiation therapy during the treatment of cancer.

[0045] Furthermore, the cancer includes liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer and prostate cancer.

[0046] Furthermore, the chemotherapy comprises administering a chemotherapeutic agent or a chemotherapeutic combination.

[0047] Further, the chemotherapeutic agent is docetaxel, paclitaxel or cyclophosphamide; the chemotherapeutic 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". Among them, docetaxel, paclitaxel, cyclophosphamide and the like are used alone, "docetaxel, doxorubicin and cyclophosphamide (TAC)", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide" or "docetaxel and cyclophosphamide (TC)" and the like are used in combination.

[0048] Furthermore, when the neutropenia is induced by the administration of chemotherapy, a single dose of the 2,5-diketopiperazine compound represented by formula (I) or a pharmaceutically acceptable salt thereof may be administered alone during the chemotherapy cycle.

[0049] Furthermore, when the neutropenia is induced by the administration of chemotherapy, the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof is administered after the administration of the chemotherapeutic agent or chemotherapeutic composition.

[0050] Furthermore, in each treatment cycle, the dosage of the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof is less than 80 mg / kg.

[0051] Furthermore, the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof is administered within 24 hours after administration of the chemotherapeutic agent or chemotherapeutic composition.

[0052] Further, the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof is administered within 12 hours after administration of the chemotherapeutic agent or chemotherapeutic composition, for example, 0.5 hours later.

[0053] Furthermore, when the neutropenia is induced by the administration of "docetaxel, doxorubicin and cyclophosphamide", "docetaxel, paclitaxel, vinblastine, doxorubicin and cyclophosphamide" or "docetaxel and cyclophosphamide", the dosage of the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof as shown in formula (I) in each treatment cycle is less than 80 mg / kg.

[0054] 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 a 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".

[0055] Furthermore, the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof can be administered by any suitable route in the art, including oral administration, injection (eg, intravenous, intramuscular, subcutaneous), etc., for example, administration by intravenous infusion.

[0056] Furthermore, the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof can be administered according to the body weight of the individual, and a non-limiting example range can be 0.5-80 mg / kg (single dose), for example 1-65 mg / kg (single dose). Specifically, the administration dose can be 6 mg / kg, 12 mg / kg by intravenous injection, and / or 27 mg / kg or 64 mg / kg by oral administration.

[0057] Furthermore, the above dosage of the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof can be administered at least once a week, for example, once every 7 days.

[0058] Furthermore, the method comprises simultaneously administering a 2,5-diketopiperazine compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0059] Furthermore, when the individual in need suffers from liver cancer, pancreatic cancer, lung cancer, breast cancer, colon cancer or prostate cancer, the method includes identifying a patient suffering from 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.

[0060] The present invention also provides a pharmaceutical composition comprising about 0.5 mg to 80 mg of a 2,5-diketopiperazine compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0061] In some embodiments, the pharmaceutical composition comprises about 1 mg to 65 mg of the 2,5-diketopiperazine compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0062] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0063] The present invention provides a pharmaceutical composition, which comprises a 2,5-diketopiperazine compound having anti-tumor efficacy as shown in formula (I) or a pharmaceutically acceptable salt thereof and a granulocyte colony stimulating factor (G-CSF) drug.

[0064] The pharmaceutical composition comprises a single dose of 0.5 mg to 80 mg of a 2,5-diketopiperazine compound with anti-tumor efficacy as shown in formula (I) or a pharmaceutically acceptable salt thereof.

[0065] The present invention provides application of the pharmaceutical composition in preparing a drug for treating and / or preventing neutropenia.

[0066] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: the 2,5-diketopiperazine compound or its pharmaceutically acceptable salt as shown in formula (I) of the present invention is used to treat and / or prevent neutropenia, improve the toxic and side effects of clinical tumor treatment, and have anti-tumor efficacy. The pharmaceutically acceptable salt of the 2,5-diketopiperazine compound has good water solubility and can be dissolved in physiological saline / 5% glucose aqueous solution for intravenous administration or oral administration. The present invention also discloses the use of such compounds alone or in combination with granulocyte colony stimulating factor drug (G-CSF) biological preparations in drugs for the treatment and / or prevention of neutropenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the curve of body weight changes of animals in each group during the test period of different doses of series compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0068] Figure 2-1 This is the curve of tumor volume change of each group of animals during the test period of different doses of series compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0069] Figure 2-2 This is the RTV change curve of each group of animals during the test period of different doses of series compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0070] Figure 2-3 This is the T / C% change curve of each group of animals during the test period of different doses of series compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0071] Figure 3-1 The figure shows the tumor weight distribution of each group of animals at the end point of the experiment with different doses of the compound series (P32-0, P34-0, P35-0, P36-0, P37-0).

[0072] Figure 3-2 It is the tumor inhibition rate of each group of animals at the test endpoint of different doses of series compounds (P32-0, P34-0, P35-0, P36-0, P37-0).

[0073] Figure 4 The present invention is the effect of different salt forms and administration series of compounds (P32, P34, P36, P37) on the body weight changes of rats induced by intraperitoneal injection of cyclophosphamide.

[0074] Figure 5 The present invention is to investigate the effects of different salt forms and administration series of compounds (P32, P34, P36, P37) on the changes of neutrophils in rats induced by intraperitoneal injection of cyclophosphamide.

[0075] Figure 6 The present invention is the effect of different salt forms and administration series of compounds (P32, P34, P36, P37) on the changes of leukocyte count in rats induced by intraperitoneal injection of cyclophosphamide.

[0076] Figure 7 It is a series of compounds (P29-0, P30-0, P31-0, P32-0, P32-1) on the changes in body weight of rats induced by intraperitoneal injection of cyclophosphamide; * P ≤ 0.05, normal control group vs model control group; # P≤0.05, ## P≤0.01, model control group vs each treatment group.

[0077] Figure 8 It is a series of compounds (P29-0, P30-0, P31-0, P32-0, P32-1) on the changes of rat neutrophils 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, model control group vs each treatment group. ^ P≤0.05, ^^ P≤0.01, ^^^ P≤0.001, P32-0 group vs each treatment group.

[0078] Fig. 9 It is a series of compounds (P29-0, P30-0, P31-0, P32-0, P32-1) that affect the changes in the number of leukocytes in rats induced by intraperitoneal injection of cyclophosphamide; ** 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, ^^^P≤0.001, P32-0 group vs each treatment group.

[0079] Fig.10 It is the effect of a series of compounds (P5-0, P10-0, P11-0, P12-0, P32-0) on the changes in body weight of rats induced by intraperitoneal injection of cyclophosphamide; * P ≤ 0.05, normal control group vs model control group; # P≤0.05, ## P≤0.01, model control group vs each treatment group.

[0080] Fig.11 It is a series of compounds (P5-0, P10-0, P11-0, P12-0, P32-0) on the changes of rat neutrophils 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, model control group vs each treatment group; ^ P≤0.05, ^^ P≤0.01, P32-0 group vs each treatment group.

[0081] Fig.12 It is the effect of a series of compounds (P5-0, P10-0, P11-0, P12-0, P32-0) on the changes in the number of leukocytes 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; ^ P≤0.05, ^^ P≤0.01, ^^^ P≤0.001, P32-0 treatment group vs. all treatment groups.

[0082] Fig.13 It is a series of compounds (P32-0, P38-0, P39-0, P40-0, P41-0, P42) that affect the changes in body weight of rats induced by intraperitoneal injection of cyclophosphamide; # P<0.05, model control group vs each treatment group.

[0083] Fig.14 It is a series of compounds (P32-0, P38-0, P39-0, P40-0, P41-0, P42) on the changes of rat neutrophils 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.

[0084] Fig.15 It is a series of compounds (P32-0, P38-0, P39-0, P40-0, P41-0, P42) that affect the changes in the number of leukocytes 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.

[0085] Fig.16 Effect of compound P32-0 combined with G-CSF on cyclophosphamide-induced changes in rat body weight. # P≤0.05, ## P≤0.01, model control group vs each drug-treated group; ∧∧ P≤0.01, single drug group vs combination drug group.

[0086] Fig.17 . The effect of compound P32-0 combined with G-CSF on the changes of neutrophils 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-drug group or plinabulin-treated group.

[0087] Fig.18 .The effect of compound P32-0 combined with G-CSF on the changes in leukocyte count in rats induced by cyclophosphamide. * P≤0.05, *** 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-drug group or plinabulin-treated group. DETAILED DESCRIPTION

[0088] definition

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. All patents, applications, published applications and other publications are incorporated herein by reference in their entirety. If there are multiple definitions for a term in this article, the definition in this section shall prevail unless otherwise stated.

[0090] As used herein, "subject" 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, as well as any other vertebrate or invertebrate.

[0091] As used herein, "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent that is effective to alleviate one or more symptoms of a disease or condition to an extent or to reduce the likelihood of its onset, and includes curing the disease or condition.

[0092] The term "preventing" or "preventing" refers to treating an individual who does not yet display symptoms of a disease or condition, but who is susceptible to or at risk for a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition.

[0093] The term "treatment" refers to the treatment of a subject already suffering from a disease or condition.

[0094] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of a compound, and its use in medicine 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 an amino group and / or a carboxyl group or a group similar thereto. Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. 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, etc. Inorganic and organic bases can also be used to form pharmaceutically acceptable salts. Inorganic bases from which salts can be derived include, for example, bases containing sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; ammonium, potassium, sodium, calcium, and magnesium salts are particularly preferred. 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, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.

[0095] The positive and progressive effects of the present invention are: the 2,5-diketopiperazine compounds or pharmaceutically acceptable salts thereof as shown in formula (I) can be used to treat and / or prevent neutropenia, especially for treating neutropenia caused by chemotherapeutic drugs such as docetaxel, adriamycin and cyclophosphamide (TAC) or docetaxel and cyclophosphamide (TC), and improving the toxic and side effects of clinical tumor treatment. Experiments were conducted with Plinabulin as the control group, and the therapeutic effects of some of the 2,5-diketopiperazine compounds or pharmaceutically acceptable salts thereof on neutropenia were better than those of Plinabulin. Some of the compounds have good water solubility after being salted, and can be dissolved in physiological saline / 5% glucose aqueous solution commonly used in clinical practice and then administered intravenously, and have good development prospects.

[0096] The technical solution of the present invention is further described in detail with reference to the following specific examples. In the following examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used can be purchased from chemical or biological reagent companies.

[0097] The compounds involved in the following examples are specifically as follows:

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] Example 1: Synthesis of Compound P1

[0108] Step 1:

[0109]

[0110] Take a 50 ml dry round-bottom flask and add (Z)-1-acetyl-3-((5-isopropyl-1H-imidazol-4-yl)methylene)piperazine-2,5-dione 2 (298 mg, 1.08 mmol), 1-chloro-3-morpholinylpropane 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) in sequence. Exhaust the air, protect with nitrogen, place in an oil bath at 70 ° C, and stir to react for 20 hours. After the reaction, the reaction solution was transferred into a 100 ml single-mouth bottle, washed with ethanol, concentrated under reduced pressure, redissolved with EtOH:DCM=1:5, filtered, the filter cake was washed with EtOH:DCM=1:5, and concentrated under reduced pressure to obtain 348 mg of brown solid crude product 3, with a yield of 80.00%. It was directly used for the next step without purification.

[0111] Step 2:

[0112]

[0113] 3,5-difluorobenzaldehyde 4 (14.2 g, 0.10 mol), 2-(triphenyl-15-phosphonylidene)acetaldehyde 5 (33.5 g, 0.11 mmol) and toluene (200 ml) were added to a 1 L dry single-necked flask. The reaction system was refluxed for 17 hours and then concentrated. The crude product was purified by column chromatography to obtain a yellow oil 6 (10.9 g, yield: 65%). ESI-MS: m / z = 169 (M+H).

[0114] Step 3:

[0115]

[0116] Take a 25ml dry brown round-bottom flask, add 3 (351mg, 0.87mmol), 6 (190mg, 1.13mmol), cesium carbonate (425mg, 1.30mmol), anhydrous sodium sulfate (247mg, 1.74mmol), N,N-dimethylformamide (6ml) in sequence, exhaust, protect with nitrogen, place in a 45℃ oil bath, stir and react for 18h. LC-MS monitors the reaction. After the reaction, transfer the reaction solution into a 100ml single-mouth bottle, wash with ethanol, concentrate under reduced pressure, and obtain the target product P1 by column chromatography with a yield of 17.52%. ESI-MS: m / z=512 (M+H).

[0117] The synthesis steps of compounds P2 to P4 refer to compound P1.

[0118] Example 2: Synthesis of Compound P5

[0119] Step 1, Preparation of 3-fluorocinnamaldehyde 8

[0120]

[0121] 3-Fluorobenzaldehyde 7 (2.00 g, 16.11 mmol) was added to a 250 mL three-necked flask, 70 mL of toluene was added, and (formylmethylene)triphenylphosphine 5 (5.39 g, 17.72 mmol) was added. After the addition, the temperature was raised to 120 degrees and the reaction was continued for 48 hours. The reaction was stopped. The reaction solution was concentrated and sanded, and column chromatography (PE-PE:EA=100:1) was performed to obtain 710 mg of pure product with a yield of 29%. MS (ESI): m / z151.1[M+H] + .

[0122] Step 2: Preparation of compound P5 (3Z, 6Z)-3-fluorophenylallylidene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0123]

[0124] Take a 50mL dry round-bottom flask and add (Z)-1-acetyl-3-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione 3 (1.59g, 3.94mmol), cesium carbonate (1.93g, 5.91mmol), sodium sulfate (1.12g, 7.88mmol), N,N-dimethylformamide (20ml), 3-fluorocinnamaldehyde 8 (0.71g, 4.73mmol) in sequence. Exhaust, nitrogen protection, place in a 50 degree oil bath, and react for 24h in the dark. Monitor the reaction completion, drop the reaction solution into 200mL ice water, and a yellow solid precipitates. Filter, drain the filter cake to obtain a crude product of 280mg of yellow solid, and obtain 63mg after column chromatography, with a yield of 3.2%. MS (ESI): m / z 494.3 [M+H] + .

[0125] Step 3, preparation of P5 hydrochloride:

[0126] Take a 25mL single-necked dry round-bottom flask, add P5 (50.0mg, 0.10mmol) and methanol (5mL) in sequence, and add a methanol solution of hydrogen chloride (4mol / L, 2mL) dropwise at 0 degrees. Protect from light throughout the process. After the addition, warm to room temperature and react for 2h. Concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and add it dropwise to 10mL of ethyl acetate. Filter after the solid precipitates, and dry the filter cake to obtain 48mg of light yellow solid.

[0127] 1 HNMR (600 MHz, DMSO-d 6 )δ11.86(s,1H),11.32(s,1H),10.89(s,1H),8.03(s,1H),7.81(dd,J=15.2,12.3Hz,1H),7.49(d,J=10. 7Hz,1H),7.44–7.40(m,1H),7.38(d,J=7.7Hz,1H),7.13-7.10(m,1H),6.91(d,J=15.2Hz,1H),6.70(s,1H ),6.53(d,J=12.2Hz,1H),4.16(t,J=7.2Hz,2H),3.95(d,J=12.5Hz,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=7.1Hz,6H).

[0128] The synthesis steps of compounds P6 to P9 refer to compound P5.

[0129] Example 3: Synthesis of Compound P10

[0130] Step 1, preparation of 3-cyanocinnamaldehyde 10:

[0131]

[0132] 3-Cyanobenzaldehyde 9 (2.00 g, 15.25 mmol) was added to a 250 mL three-necked flask, 70 mL of toluene was added, and (formylmethylene)triphenylphosphine 5 (5.10 g, 16.71 mmol) was added. After the addition, the temperature was raised to 120 degrees and the reaction was continued for 48 hours. The reaction was stopped. The reaction solution was concentrated and sanded, and 820 mg of pure product was obtained by column chromatography, with a yield of 34%. MS (ESI): m / z 158.1 [M+H] + .

[0133] Step 2, preparation of compound P10 (3Z, 6Z)-3-cyanophenylallylidene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione:

[0134]

[0135] Take a 100mL dry round-bottom flask and add (Z)-1-acetyl-3-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione 3 (1.75g, 4.35mol), cesium carbonate (2.12g, 6.52mmol), sodium sulfate (1.23g, 8.70mmol), N,N-dimethylformamide (30ml), 3-cyanocinnamaldehyde 10 (0.82g, 5.22mmol) in sequence. Exhaust, nitrogen protection, place in a 50 degree oil bath, avoid light and react for 24h. Monitor the reaction completion, drop the reaction solution into 200mL ice water, and a yellow solid precipitates. Filter, drain the filter cake to obtain a crude product of 470mg yellow solid, and obtain 130mg after column chromatography, HPLC purity 99%, yield 6.0%. mp = 207-209℃.

[0136] 1 HNMR (600 MHz, DMSO-d 6)δ11.96(s,1H),10.87(s,1H),7.88(s,1H),7.84-7.74(m,2H),7.57(d,J=7.0Hz,1H) ,7.42-7.35(m,2H),6.88(d,J=15.2Hz,1H),6.71(s,1H),6.53(d,J=12.1Hz,1H),4.0 5(t,J=7.1Hz,2H),3.58(t,J=4.3Hz,4H),3.26-3.21(m,1H),2.38-2.30(m,4H),2.25 (t,J=6.7Hz,2H),1.89-1.80(m,2H),1.34(d,J=7.1Hz,6H).MS(ESI):m / z501.3[M+H] + .

[0137] Step 3, preparation of P10 hydrochloride:

[0138] Take a 25mL single-necked dry round-bottom flask, add P10 (70.0mg, 0.14mmol) and methanol (5mL) in sequence, and add a methanol solution of hydrogen chloride (4mol / L, 3mL) dropwise at 0 degrees. Protect from light throughout the process. After the addition, warm to room temperature and react for 2h. Concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and add it dropwise to 10mL of ethyl acetate to precipitate a solid. Let it stand for 20 minutes and then filter. Take the filter cake and dry it to obtain 71mg of light yellow solid.

[0139] 1 HNMR (600 MHz, DMSO-d 6 )δ11.85(s,1H),11.18(s,1H),10.91(s,1H),8.02(s,1H),7.81(dd,J=15.5,12.5Hz,2H),7.57(d ,J=6.9Hz,1H),7.41–7.35(m,2H),6.89(d,J=15.2Hz,1H),6.70(s,1H),6.53(d,J=12.2Hz,1H),4. 16(t,J=7.1Hz,2H),3.95(d,J=12.5Hz,2H),3.85–3.77(m,2H),3.45-3.41(m,2H),3.27-3.20(m,1 H),3.14-3.10(m,2H),3.09-3.01(m,2H),2.19-2.15(m,2H),1.35(d,J=7.1Hz,6H).mp=242-245℃.

[0140] Example 4: Synthesis of Compound P11

[0141] Step 1, preparation of 3-vinylcinnamaldehyde 12:

[0142]

[0143] 3-vinylbenzaldehyde 11 (2.00 g, 15.13 mmol) was added to a 250 mL three-necked flask, 70 mL of toluene was added, and (formylmethylene)triphenylphosphine 5 (5.06 g, 16.64 mmol) was added. After the addition, the temperature was raised to 120 degrees and the reaction was continued for 48 hours. The reaction was stopped. The reaction solution was concentrated and sanded, and column chromatography (PE-PE:EA=100:1) was performed to obtain 430 mg of pure product with a yield of 18%. MS (ESI): m / z159.1[M+H] + .

[0144] Step 2, preparation of compound P11 (3Z, 6Z)-3-vinylphenylallylidene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione:

[0145]

[0146] Take a 50mL dry round-bottom flask and add (Z)-1-acetyl-3-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione 3 (0.92g, 2.27mol), cesium carbonate (1.11g, 3.41mmol), sodium sulfate (0.64g, 4.54mmol), N,N-dimethylformamide (20ml), 3-vinylcinnamaldehyde 12 (0.43g, 2.72mmol) in sequence. Exhaust, nitrogen protection, place in a 50 degree oil bath, and avoid light for 24h. Monitor the reaction completion, drop the reaction solution into 200mL ice water, and a yellow solid precipitates. Filter, drain the filter cake to obtain a crude product of 220mg of yellow solid, and obtain 22mg after column chromatography, with a yield of 2.0%. MS (ESI): m / z 502.3 [M+H] + .

[0147] Example 5: Synthesis of Compound P12

[0148] Step 1, preparation of 3-ethynylcinnamaldehyde 14:

[0149]

[0150] 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) triphenylphosphine 5 (5.15 g, 16.91 mmol) was added, and the temperature was raised to 120 degrees for 48 hours after the addition, and the reaction was stopped. The reaction solution was concentrated and sanded, and column chromatography (PE-PE: EA = 100: 1) was performed to obtain 560 mg of pure product, with a yield of 23%. MS (ESI): m / z 157.1 [M+H] + .

[0151] Step 2, preparation of compound P12 (PLN-20-2-BB) (3Z, 6Z)-3-ethynylphenylallylidene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione:

[0152]

[0153] Take a 50mL dry round-bottom flask and add (Z)-1-acetyl-3-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione 3 (1.20g, 2.98mmol), cesium carbonate (1.46g, 4.47mmol), sodium sulfate (0.85g, 5.96mmol), N,N-dimethylformamide (20ml), 3-ethynylcinnamaldehyde 14 (0.56g, 3.58mmol) in sequence. Exhaust, nitrogen protection, place in a 50 degree oil bath, and react for 24h in the dark. Monitor the reaction completion, drop the reaction solution into 200mL ice water, and a yellow solid precipitates. Filter, drain the filter cake to obtain a crude product of 210mg of yellow solid, and obtain 46mg after column chromatography, with a yield of 3.1%. MS (ESI): m / z 500.3 [M+H] + .

[0154] 1 HNMR (600 MHz, DMSO-d 6 )δ11.97(s,1H),10.90(s,1H),7.90(s,1H),7.85-7.80(m,2H),7.58(d,J=7.0Hz ,1H),7.42-7.37(m,2H),6.90(d,J=15.2Hz,1H),6.71(s,1H),6.53(d,J=12.1Hz ,1H),4.27(s,1H),4.05(t,J=7.1Hz,2H),3.59(t,J=4.2Hz,4H),3.26-3.21(m,1 H), 2.34 (s, 4H), 2.26 (t, J = 6.7Hz, 2H), 1.88-1.81 (m, 2H), 1.35 (d, J = 7.1Hz, 6H).

[0155] The synthesis steps of compound P13 refer to compound P12.

[0156] Example 6: Synthesis of Compound P14

[0157] Step 1:

[0158]

[0159] Take a 50ml dry round-bottom flask, add 15 (326mg, 1.08mmol), 1-chloro-3-morpholinylpropane 1 (355mg, 2.17mmol), cesium carbonate (708mg, 2.17mmol), potassium iodide (180mg, 1.08mmol), 4A molecular sieve (500mg), DMF (6ml) in sequence, exhaust, protect with nitrogen, place in 70℃ oil bath, stir and react for 20h. After the reaction, transfer the reaction solution into a 100ml single-mouth bottle, wash with ethanol, concentrate under reduced pressure, redissolve with EtOH:DCM=1:5, filter, wash the filter cake with EtOH:DCM=1:5, and concentrate under reduced pressure to obtain 301mg brown solid crude product 16, with a yield of 65.00%. Directly use it for the next step.

[0160] Step 2:

[0161]

[0162] Take a 25ml dry brown round-bottom flask, add 16 (359mg, 0.87mmol), 6 (190mg, 1.13mmol), cesium carbonate (425mg, 1.30mmol), anhydrous sodium sulfate (247mg, 1.74mmol), N,N-dimethylformamide (6ml) in sequence, exhaust, protect with nitrogen, place in a 45℃ oil bath, stir and react for 18h. LC-MS monitors the reaction. After the reaction, transfer the reaction solution into a 100ml single-mouth bottle, wash with ethanol, concentrate under reduced pressure, and obtain the target product P14 by column chromatography with a yield of 19%. ESI-MS: m / z=538 (M+H).

[0163] The synthesis steps of compounds P15 to P25 refer to compound P14.

[0164] Example 7: Synthesis of Compound P26

[0165]

[0166] Take a 25ml dry brown round-bottom flask, add 3 (351mg, 0.87mmol), 17 (168mg, 1.13mmol), cesium carbonate (425mg, 1.30mmol), anhydrous sodium sulfate (247mg, 1.74mmol), N,N-dimethylformamide (6ml) in sequence, exhaust, protect with nitrogen, place in a 45℃ oil bath, stir and react for 18h. LC-MS monitors the reaction. After the reaction, transfer the reaction solution into a 100ml single-mouth bottle, wash with ethanol, concentrate under reduced pressure, and obtain the target product P-26 by column chromatography with a yield of 18%. ESI-MS: m / z=493 (M+H).

[0167] The synthesis steps of compounds P27-P28 refer to compound P26.

[0168] Example 8

[0169] Step 1, preparation of compound P29 (3Z, 6Z)-3-cyanophenylmethylene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione:

[0170]

[0171] Take a 50mL dry round-bottom flask and add (Z)-1-acetyl-3-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione 3 (1.50g, 3.62mol), cesium carbonate (1.77g, 5.43mmol), sodium sulfate (1.03g, 7.24mmol), N,N-dimethylformamide (20ml), 3-cyanobenzaldehyde 18 (0.57g, 4.34mmol) in sequence. Exhaust, nitrogen protection, place in a 50 degree oil bath, avoid light and react for 24h. Monitor the reaction completion, drop the reaction solution into 200mL ice water, and a yellow solid precipitates. Filter, drain the filter cake to obtain a crude product of 690mg yellow solid, and obtain 240mg after column chromatography, with a yield of 14%.

[0172] 1 HNMR (600 MHz, DMSO-d 6)δ12.07(s,1H),10.56(s,1H),7.99(s,1H),7.92(s,1H),7.79(d,J=7.9Hz,1H) ,7.76(d,J=7.7Hz,1H),7.60(t,J=7.8Hz,1H),6.75(d,J=4.0Hz,2H),4.07(t,J =7.1Hz,2H),3.60(t,J=4.4Hz,4H),3.27-3.21(m,1H),2.35(s,4H),2.26(t,J= 6.7Hz,2H),1.89-1.82(m,2H),1.35(d,J=7.1Hz,6H).mp:237.6-239.6.MW[M+H] + :475.54.

[0173] Step 2, preparation of P29 hydrochloride:

[0174] Take a 25mL single-necked dry round-bottom flask, add P29 (100.0mg, 0.21mmol), methanol (5mL) in sequence, and add a methanol solution of hydrogen chloride (4mol / L, 5mL) dropwise at 0 degrees. Protect from light throughout the process. After the addition, warm to room temperature and react for 2h. Concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and add it dropwise to 10mL of ethyl acetate to precipitate a solid. Let it stand for 20 minutes and then filter. Take the filter cake and dry it to obtain 101mg of light yellow solid.

[0175] 1 HNMR (600 MHz, DMSO-d 6 )δ11.90(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=7.8Hz,1H),6.75(s,1H),6.71(s,1H),4.18(t,J=7.2Hz,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=7.1Hz,6H).mp:268.6-270.6℃.

[0176] Embodiment 9:

[0177] Step 1, preparation of compound P30 (3Z, 6Z)-3-vinylphenylmethylene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione:

[0178]

[0179] Take a 50mL dry round-bottom flask and add (Z)-1-acetyl-3-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione 3 (1.50g, 3.62mol), cesium carbonate (1.77g, 5.43mmol), sodium sulfate (1.03g, 7.24mmol), N,N-dimethylformamide (20ml), 3-vinylbenzaldehyde 11 (0.56g, 4.34mmol) in sequence. Exhaust, nitrogen protection, place in a 50 degree oil bath, avoid light and react for 24h. Monitor the reaction completion, drop the reaction solution into 200mL ice water, and a yellow solid precipitates. Filter, drain the filter cake to obtain a crude product of 640mg yellow solid, and obtain 210mg after column chromatography, with a yield of 12%.

[0180] 1 HNMR (600 MHz, DMSO-d 6 )δ12.00(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=17.6Hz,1H),5.31(d,J=11.2Hz ,1H),4.05(t,J=6.8Hz,2H),3.65-3.52(m,4H),3.27-3.21(m,1H),2.33(s,4H),2.25 (t,J=6.7Hz,2H),1.88-1.80(m,2H),1.34(d,J=7.1Hz,6H).mp:219.0-221.0.MW[M+H] + :476.59.

[0181] Step 2, preparation of P30 hydrochloride:

[0182] Take a 25mL single-necked dry round-bottom flask, add P30 (100.0mg, 0.21mmol), methanol (5mL) in sequence, and add a methanol solution of hydrogen chloride (4mol / L, 5mL) dropwise at 0 degrees. Protect from light throughout the process. After the addition, warm to room temperature and react for 2h. Concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and add it dropwise to 10mL EA. Solid precipitates, let stand for 20min, then filter, take the filter cake and dry it to obtain 98mg of light yellow solid.

[0183] 1 HNMR (600 MHz, DMSO-d 6 )δ11.82(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=17.7Hz,1H),5.76(s,1H),5.32(d,J=11.5Hz,1H),4.18(t ,J=7.2Hz,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),3.1 7-3.09(m,2H),3.09-2.99(m,2H),2.23-2.18(m,2H),1.34(d,J=7.1Hz,6H).mp:260.2-262.2℃.

[0184] Example 10

[0185] Preparation of compound P31 (3Z, 6Z)-3-ethynylphenylmethylene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione:

[0186]

[0187] Take a 50mL dry round-bottom flask and add (Z)-1-acetyl-3-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione 3 (1.50g, 3.62mol), cesium carbonate (1.77g, 5.43mmol), sodium sulfate (1.03g, 7.24mmol), N,N-dimethylformamide (20ml), 3-ethynylbenzaldehyde 13 (0.56g, 4.34mmol) in sequence. Exhaust, nitrogen protection, place in a 50 degree oil bath, avoid light and react for 24h. Monitor the reaction completion, drop the reaction solution into 200mL ice water, and a yellow solid precipitates. Filter, drain the filter cake to obtain a crude product of 580mg of yellow solid, and obtain 160mg after column chromatography, with a yield of 9%.

[0188] 1HNMR(600MHz,DMSO-d6)δ12.01(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=6.4Hz,2H),4.25(s,1H),4.06(t,J=6.8Hz, 2H),3.65-3.52(m,4H),3.27-3.21(m,1H),2.35(s,4H),2.26(t,J=6.7Hz,2H), 1.90-1.82(m,2H),1.35(d,J=7.1Hz,6H).mp:246.5-248.5℃.MW[M+H]+:474.57.

[0189] Embodiment 11

[0190] Step 1, preparation of compound P32 (3Z, 6Z)-3-fluorophenylmethylene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione:

[0191]

[0192] Take a 25 ml dry brown round-bottom flask and 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) in sequence. Exhaust the air, protect with nitrogen, place in a 45 ° C oil bath, and stir to react for 20 hours. The reaction was monitored by LC-MS. After the reaction, the reaction solution was dropped into 4°C cold water (80 ml), filtered with suction, the filter cake was washed with cold water, dried, dissolved with methanol and dichloromethane (1:3), filtered, concentrated under reduced pressure, dried, chromatographed, concentrated under reduced pressure, dried, ultrasonically slurried with methanol, placed in a -30°C refrigerator and allowed to stand, filtered with suction, the filter cake was washed with cold methanol, and dried in vacuo at 50°C to obtain 301.7 mg of the target product with a yield of 35.66%.

[0193] 1 HNMR (600 MHz, DMSO-d 6 )δ12.02(s,1H),10.27(s,1H),7.88(d,J=5.34Hz,1H),7.46-7.40(m,1H),7.36(d,J=10.26Hz ,1H),7.34(dd,J=9.02,19.44Hz,1H),7.13(td,J=2.37,8.57Hz,1H),6.73(s,1H),6.71(s,1H) ,4.04(t,J=7.13Hz,2H),3.57(t,J=4.44Hz,4H),3.24(dt,J=7.10,14.24Hz,1H),2.26(dd,J=2 4.07,30.84Hz,6H),1.84(p,J=6.86Hz,2H),1.33(d,J=7.13Hz,6H).MS(ESI):m / z468.18[M+H] + .Mp:208-210℃.

[0194] Step 2, preparation of P32-0 hydrochloride

[0195] (3Z,6Z)-3-(3-fluorophenyl)methylene-6-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione, hydrochloride

[0196]

[0197] Take a 100mL single-necked dry round-bottom flask, add (3Z,6Z)-3-(3-fluorophenyl)methylene-6-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione (200.0mg, 0.43mmol) and methanol (10mL) in sequence, and add a methanol solution of hydrogen chloride (4mol / L, 15mL) dropwise at 0 degrees. Protect from light throughout the process. After the addition, warm to room temperature and react for 2h. Concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and add it dropwise to 15mL of ethyl acetate to precipitate a solid. Stir for 10 minutes and filter and dry. Take the filter cake to obtain 218.0mg of a light yellow solid with a yield of 94%.

[0198] 1HNMR(600MHz,DMSO-d6)δ11.84(s,1H),10.36(s,1H),8.15(s,1H),7.49–7.40(m,1H),7.37 (d,J=10.2Hz,1H),7.34(d,J=7.8Hz,1H),7.17-7.14(m,1H),6.75(s,1H),6.69(s,1H),4.19 (t,J=7.2Hz,2H),3.96-3.93(m,2H),3.86(t,J=11.5Hz,2H),3.42(d,J=12.2Hz,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=7.1Hz,6H).

[0199] Step 3, Preparation of P32-1 Methanesulfonate

[0200] (3Z,6Z)-3-(3-fluorophenyl)methylene-6-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione, methanesulfonate (P32-1)

[0201]

[0202] Take a 100mL single-necked dry round-bottom flask and add P32 (200.0mg, 0.43mmol), methanol (10mL), and methanesulfonic acid (102.8mg, 1.07mmol) in sequence. Protect from light throughout the process. After the addition, warm to room temperature and react for 2h. Concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and add dropwise to 15mL of ethyl acetate to precipitate a solid. Stir for 10 minutes, then filter and dry. Take the filter cake to obtain 268.0mg of a light yellow solid with a yield of 95%.

[0203] 1HNMR(600MHz,DMSO-d6)δ11.76(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=7.8Hz,1H),7.17-7.14(m,1H),6.76(s,1H),6.68(s,1H),4.1 6(t,J=7.3Hz,2H),4.01-3.98(m,2H),3.69(t,J=11.6Hz,2H),3.46(d,J=12.2Hz,2H),3.2 6-3.19(m,3H),3.14-3.06(m,2H),2.42(s,6H),2.16-2.11(m,2H),1.34(d,J=7.1Hz,6H).

[0204] Example 12

[0205] Preparation of compound P33 (3Z, 6Z)-3-fluorophenylmethylene-6-((5-isopropyl-1-(-3-morpholinyl)propylimidazol-4-yl)allylidene)piperazine-2,5-dione:

[0206]

[0207] Take a 25mL dry round-bottom flask and add (Z)-1-acetyl-3-((5-isopropyl-1-(3-morpholinyl)propylimidazol-4-yl)allylidene)piperazine-2,5-dione 16 (481mg, 1.12mmol), cesium carbonate (547mg, 1.68mmol), sodium sulfate (318mg, 2.24mmol), N,N-dimethylformamide (50ml), 3-fluorobenzaldehyde 7 (166mg, 1.34mmol) in sequence. Exhaust, protect with nitrogen, place in a 50 degree oil bath, and react for 24h in the dark. Monitor the reaction to be complete, drop the reaction solution into 50mL ice water, filter and dry to obtain a yellow solid, [M+H]+ is 494.7.

[0208] Example 13: Preparation of P38-0(3Z,6Z)-3-(fluorobenzylidene-6-((5-isopropyl-1-(3-piperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione hydrochloride

[0209] Step 1, preparation of 1-tert-butyloxycarbonyl-4-(3-ethylsulfonylpropyl)piperazine (IM8):

[0210]

[0211] SM7, 1-tert-butyloxycarbonyl-4-(3-hydroxypropane)piperazine (2.00 g, 7.18 mmol) was added to a 100 mL three-necked flask, 30 mL of DCM was added, TEA (1.24 g, 12.27 mmol) was added, the temperature was controlled at about 0 degrees, EsCl (1.26 g, 9.82 mmol) was added dropwise, and the temperature was naturally raised to room temperature for 2 hours after the addition, and the reaction was complete. 50 mL of water was added, the liquid was separated, 30 mL of DCM was added to the aqueous phase for extraction, the organic phases were combined, 50 mL of water was added respectively, 50 mL of saturated brine was washed, dried, and concentrated to obtain a brown oily compound IM8, 1-tert-butyloxycarbonyl-4-(3-ethylsulfonylpropyl)piperazine 2.74 g, with a yield of 99%, which was directly put into the next step.

[0212] Step 2, preparation of (Z)-1-acetyl-3-((5-isopropyl-1-(3-1-tert-butyloxycarbonylpiperazinyl)propyl-imidazol-4-yl)methylene)piperazine-2,5-dione (IM9):

[0213]

[0214] A 250 mL dry round-bottom flask was charged with A005, (Z)-1-acetyl-3-((5-isopropyl-1H-imidazol-4-yl)methylene)piperazine-2,5-dione (1.50 g, 5.43 mmol), Cs 2 CO 3 (3.54g, 10.86mmol), KI (0.90g, 5.43mmol), DMF (50mL), IM8, 1-tert-butyloxycarbonyl-4-(3-ethylsulfonylpropyl)piperazine (2.74g, 8.15mmol), react at 70 degrees in the dark for 36h. After the reaction, add 100mL of water to the reaction solution, extract with 50mL of DCM, continue to add 50mL of DCM to the aqueous phase, extract, combine the organic phases, wash with 2*50mL of water and 50mL of saturated brine respectively, dry the organic phase with anhydrous sodium sulfate and concentrate to obtain 2.73g of crude product, which is a brown oil with a yield of 99%, and directly use it for the next step without purification.

[0215] Step 3, P38 (3Z, 6Z)-3-(fluorobenzylidene-6-((5-isopropyl-1-(3-1-tert-butyloxycarbonylpiperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0216]

[0217] Take a 25ml dry brown round-bottom flask, add IM9, (Z)-1-acetyl-3-((5-isopropyl-1-(3-1-tert-butyloxycarbonylpiperazinyl)propyl-imidazol-4-yl)methylene)piperazine-2,5-dione (2.73g, 5.43mmol), 3-fluorobenzaldehyde 7 (0.67g, 5.43mmol), cesium carbonate (2.66g, 8.15mmol), anhydrous sodium sulfate (1.54g, 10.86mmol), DMF (30ml), exhaust, nitrogen protection, place in a 45℃ oil bath, stir and react for 20h. LC-MS monitors the reaction. After the reaction, the reaction solution is dropped into 4℃ cold water (80ml), filtered, and the filter cake is dried and then column chromatographed to obtain 820mg of the target product with a purity of 98% and a yield of 27%. MW[M+H] + :567.51

[0218] 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

[0219]

[0220] Take a 250mL single-necked dry round-bottom flask, add P38 (700.0mg, 1.23mmol), MeOH (60mL) in sequence, and add HCl / MeOH (4mol / L, 50mL) dropwise at 0 degrees. Protect from light throughout the process. After the addition, naturally warm to room temperature and react for 2h. Concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and add it dropwise to 100mL EA. Precipitate solid, let it stand for 30min, then filter, take the filter cake and dry it to obtain 633mg of light yellow solid, HPLC purity 98%, yield 95%.

[0221] 1 HNMR(600MHz,DMSO)δ12.11(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=7.1Hz,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=7.1Hz,6H).

[0222] Example 14: Preparation of P39 (3Z, 6Z)-3-fluorophenylmethylene-6-((5-isopropyl-1-(3-1-glyoxyethylpiperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0223] Step 1

[0224]

[0225] Take a 250mL dry round-bottom flask, add P38, (Z)-1-acetyl-3-((5-isopropyl-1-(3-piperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione (0.55g, 1.02mmol), DCM (50ml), TEA (0.52g, 5.10mmol), SM8, ethyl oxalyl chloride (0.28g, 2.04mmol) in sequence at 0 degrees, and react at room temperature in the dark for 24h. Monitor the reaction until complete, concentrate the reaction solution and column chromatography to obtain 338mg, HPLC purity 98%, yield 58%.

[0226] 1 HNMR(600MHz,DMSO)δ12.04(s,1H),10.32(s,1H),7.92(s,1H),7.49-7.42(m,1H),7.38( d,J=10.2Hz,1H),7.35(d,J=8.1Hz,1H),7.18-7.14(m,1H),6.75(s,1H),6.73(s,1H),4.3 0(q,J=7.1Hz,2H),4.08(t,J=7.1Hz,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=7.1Hz,6H),1.28(t,J=7.1Hz,3H).MW[M+H] + :567.58

[0227] Step 2: Synthesis of P39-0

[0228]

[0229] Take a 25mL single-necked dry round-bottom flask, add P39 (80mg, 0.14mmol), MeOH (10mL) in sequence, add HCl / MeOH (4mol / L, 5mL) dropwise at 0 degrees, protect from light throughout the process, and naturally warm to room temperature to react for 2h after the addition. Concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and add it dropwise to 100mL EA to precipitate a solid. After standing for 30min, filter and take the filter cake to dry to obtain 76mg of light yellow solid with HPLC purity of 98% and yield of 85%.

[0230] 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:

[0231] Step 1

[0232]

[0233] P39 (0.25 g, 0.44 mmol) was added to a 100 mL single-necked bottle, and 50 mL MeCN and 2 mL H 2 O, TEA (0.18 g, 1.76 mmol) and LiBr (0.23 g, 2.64 mmol) were added, and the temperature was raised to 40 degrees for 48 hours, and the reaction was stopped. The reaction solution was concentrated and column chromatography was performed to obtain 47 mg of pure product with a purity of 97% and a yield of 20%.

[0234] 1 HNMR(600MHz,DMSO)δ12.04(s,1H),10.32(s,1H),7.92(s,1H),7.49-7.42 (m,1H),7.38(d,J=10.2Hz,1H),7.35(d,J=8.1Hz,1H),7.18-7.14(m,1H),6 .75(s,1H),6.73(s,1H),4.08(t,J=7.2Hz,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=7.1Hz,6H).MW[M+H] + :539.48

[0235] Step 2: Synthesis of P40-0

[0236]

[0237] Take a 25mL single-necked dry round-bottom flask, add P40 (36mg, 0.07mmol), MeOH (5mL) in sequence, and add HCl / MeOH (4mol / L, 3mL) dropwise at 0 degrees. Protect from light throughout the process. After the addition, warm to room temperature and react for 2h. Concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and add it dropwise to 20mL EA. Precipitate solid, let it stand for 30min, then filter, take the filter cake and dry it to obtain 30mg of light yellow solid with HPLC purity of 97% and yield of 75%.

[0238] Example 16: Preparation of P41(3Z,6Z)-3-fluorobenzylidene-6-((5-isopropyl-1-(3-N-methylpiperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0239] Step 1, preparation of 1-(3-ethylsulfonylpropyl)-4-methylpiperazine (IM11):

[0240]

[0241] SM9, 1-(3-hydroxypropane)-4-methylpiperazine (2.00 g, 12.64 mmol) was added to a 100 mL three-necked flask, 30 mL DCM was added, TEA (1.92 g, 18.96 mmol) was added, the temperature was controlled at about 0 degrees, EsCl (1.95 g, 15.17 mmol) was added dropwise, and the temperature was naturally raised to room temperature for 2 hours after the addition, and the reaction was complete. 50 mL of water was added, the liquid was separated, 30 mL DCM was added to the aqueous phase for extraction, the organic phases were combined, 50 mL of water was added, 50 mL of saturated brine was added, and the mixture was washed with dried. After concentration, a brown oily compound IM11, 1-(3-ethylsulfonylpropyl)-4-methylpiperazine 2.96 g was obtained, with a yield of 94%, which was directly used for the next step.

[0242] Step 2, Preparation of (Z)-1-acetyl-3-((5-isopropyl-1-(3-N-methylpiperazinyl)propyl-imidazol-4-yl)methylene)piperazine-2,5-dione (IM12)

[0243]

[0244] A 100 mL dry round-bottom flask was added with A005, (Z)-1-acetyl-3-((5-isopropyl-1H-imidazol-4-yl)methylene)piperazine-2,5-dione (0.50 g, 1.81 mmol), Cs 2 CO 3(1.18g, 3.62mmol), KI (0.30g, 1.81mmol), DMF (20mL), IM11, 1-(3-ethylsulfonylpropyl)-4-methylpiperazine (0.68g, 2.72mmol), react at 70 degrees in the dark for 36h. After the reaction, add 80mL of water to the reaction solution, extract with 30mL of DCM, continue to add 30mL of DCM to the aqueous phase, combine the organic phases, wash with 2*50mL of water and 50mL of saturated brine respectively, dry the organic phase with anhydrous sodium sulfate and concentrate to obtain 0.75g of crude product, which is a brown oil with a yield of 99%, and directly use it for the next step without purification.

[0245] Step 3, P41 (3Z, 6Z)-3-fluorobenzylidene-6-((5-isopropyl-1-(3-N-methylpiperazinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0246]

[0247] Take a 25ml dry brown round-bottom flask, add IM12, (Z)-1-acetyl-3-((5-isopropyl-1-(3-N-methylpiperazinyl)propyl-imidazol-4-yl)methylene)piperazine-2,5-dione (0.75g, 1.81mmol), 3-fluorobenzaldehyde 7 (0.22g, 1.81mmol), cesium carbonate (0.88g, 2.71mmol), anhydrous sodium sulfate (0.51g, 3.62mmol), DMF (8ml), exhaust, nitrogen protection, place in a 45℃ oil bath, stir and react for 20h. LC-MS monitors the reaction. After the reaction, the reaction solution is dropped into 4℃ cold water (80ml), filtered, and the filter cake is dried and column chromatographed to obtain 180mg of the target product with a purity of 98% and a yield of 20%.

[0248] 1 HNMR (600 MHz, DMSO-d 6 )δ11.94(s,1H),10.38(s,1H),8.00(s,1H),7.48-7.44(m,1H),7.38(d,J =10.2Hz,1H),7.35(d,J=7.8Hz,1H),7.18-7.15(m,1H),6.78(s,1H),6.7 2(s,1H),4.18(t,J=6.3Hz,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=6.92Hz,6H)MW[M+H] + :481.39

[0249] 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:

[0250]

[0251] Take a 25mL single-necked dry round-bottom flask, add P41 (40mg, 0.08mmol), MeOH (5mL) in sequence, and add HCl / MeOH (4mol / L, 3mL) dropwise at 0 degrees. Protect from light throughout the process. After the addition, warm to room temperature and react for 2h. Concentrate the reaction solution, add a small amount of MeOH to just dissolve the product, and add it dropwise to 20mL EA. Precipitate solid, let it stand for 30min, then filter, take the filter cake and dry it to obtain 41mg of light yellow solid with HPLC purity of 97% and yield of 90%.

[0252] Example 17: P42 (3Z, 6Z)-3-fluorobenzylidene-6-((5-isopropyl-1-(3-1,1-dioxidothiomorpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0253] Step 1, preparation of 4-(3-ethylsulfonylpropyl)thiomorpholine 1,1-dioxide (IM13):

[0254]

[0255] SM10,4-(3-hydroxypropyl)thiomorpholine 1,1-dioxide (20.00g, 0.14mol) was added to a 100mL three-necked flask, 30mL DCM was added, TEA (1.55g, 15.26mmol) was added, the temperature was controlled at about 0 degrees, EsCl (1.60g, 12.42mmol) was added dropwise, and the temperature was naturally raised to room temperature for 2h after the addition, and the reaction was complete. 50mL of water was added, the liquid was separated, 30mL of DCM was added to the aqueous phase for extraction, the organic phases were combined, 50mL of water was added respectively, 50mL of saturated brine was washed, dried, and concentrated to obtain a brown oily compound IM13,4-(3-ethylsulfonylpropyl)thiomorpholine 1,1-dioxide 2.80g, the yield was 95%, and it was directly put into the next step.

[0256] Step 2, preparation of (Z)-1-acetyl-3-((5-isopropyl-1-(3-1,1-dioxidothiomorpholinyl)propyl-imidazol-4-yl)methylene)piperazine-2,5-dione (IM14)

[0257]

[0258] A 100 mL dry round-bottom flask was added with A005, (Z)-1-acetyl-3-((5-isopropyl-1H-imidazol-4-yl)methylene)piperazine-2,5-dione (0.50 g, 1.81 mmol), Cs 2 CO 3 (1.18g, 3.62mmol), KI (0.30g, 1.81mmol), DMF (20mL), IM13, 4-(3-ethylsulfonylpropyl)thiomorpholine 1,1-dioxide (0.78g, 2.72mmol), react at 70 degrees in the dark for 36h. After the reaction, add 80mL of water to the reaction solution, extract with 30mL of DCM, continue to add 30mL of DCM to the aqueous phase, combine the organic phases, wash with 2*50mL of water and 50mL of saturated brine respectively, dry the organic phase with anhydrous sodium sulfate and concentrate to obtain 0.82g of crude product, which is a brown oil with a yield of 99%, and directly use it for the next step without purification.

[0259] Step 3, P42 (3Z, 6Z)-3-fluorobenzylidene-6-((5-isopropyl-1-(3-1,1-dioxidothiomorpholinyl)propylimidazol-4-yl)methylene)piperazine-2,5-dione

[0260]

[0261] Take a 25ml dry brown round-bottom flask, add 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), exhaust, nitrogen protection, place in a 45℃ oil bath, stir and react for 20h. LC-MS monitors the reaction. After the reaction, the reaction solution is dropped into 4℃ cold water (80ml), filtered, and the filter cake is dried and then column chromatographed to obtain 60mg of the target product with a purity of 97% and a yield of 6%. MW[M+H] + :516.43

[0262] Example 18: Hydrochloride Titration of a Series of Compounds

[0263] 1) P5-0 hydrochloride titration:

[0264] Take 14.9 mg P5-0 and add it to a conical flask, add 20 mL water, and calibrate with sodium hydroxide with a concentration of c(NaOH) = 0.02012 mmol / mL. Use 2.63 mL of sodium hydroxide standard solution, and calculate n(HCl) = 0.02012*2.63 = 0.0529 mmol, n(P5-0) = 14.9 / 566.50 = 0.02630 mmol, n(HCl) / n(P5-0) = 2.01.

[0265] 2) P10-0 hydrochloride titration:

[0266] Take 13.2mgP10-0 and add it to a conical flask, add 20mL of water, and calibrate it with sodium hydroxide with a concentration of c(NaOH)=0.02012mmol / mL. Use 2.43mL of sodium hydroxide standard solution, and 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.

[0267] 3) P29-0 hydrochloride titration: Take 8.6 mg P29-0 and add it to a conical flask, add 20 mL water, and calibrate with sodium hydroxide with a concentration of c(NaOH) = 0.02012 mmol / mL. Use 1.55 mL of sodium hydroxide standard solution, and 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.

[0268] 4) P30-0 hydrochloride titration: Take 10.3 mg P30-0 and add it to a conical flask, add 20 mL water, and calibrate with sodium hydroxide with a concentration of c(NaOH) = 0.02012 mmol / mL. Use 1.84 mL of sodium hydroxide standard solution, and 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.

[0269] 5) P38-0 hydrochloride titration: Take 15.7 mg P38-0 and add it to a conical flask, add 20 mL water, and calibrate with sodium hydroxide with a concentration of c(NaOH) = 0.02007 mmol / mL. Use 3.96 mL of sodium hydroxide standard solution, and 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.

[0270] 6) P39-0 hydrochloride titration: 12.2 mg P39-0 was added to a conical flask, 20 mL of water was added, and sodium hydroxide with a concentration of c(NaOH) = 0.02007 mmol / mL was used for calibration. 1.86 mL of sodium hydroxide standard solution was used, and n(HCl) = 0.02007*1.86 = 0.0371 mmol, n(P39-0) = 12.2 / 639.55 = 0.0190 mmol, and n(HCl) / n(P39-0) = 1.96

[0271] 7) P40-0 hydrochloride titration: Take 6.9 mg P40-0 and add it to a conical flask, add 20 mL of water, and calibrate with sodium hydroxide with a concentration of c(NaOH)=0.02007 mmol / mL. Use 1.63 mL of sodium hydroxide standard solution, and calculate 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 shows that one molecule of carboxylic acid of the compound itself is removed, and the molecule is combined with 2 molecules of hydrochloric acid.

[0272] 8) P41-0 hydrochloride titration: Take 19.3 mg of P41-0 and add it to a conical flask, add 20 mL of water, calibrate with sodium hydroxide with a concentration of c(NaOH)=0.02007 mmol / mL, use 4.82 mL of sodium hydroxide standard solution, and calculate 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.

[0273] Example 19: Water solubility test of a series of compounds

[0274] The hydrochloride or methanesulfonate of the compound was dissolved in pure water at 24° C. or 27° C. to measure the solubility of the compound. The results are shown in Table 1, indicating that the compound has good water solubility after being salted.

[0275] Table 1: Water solubility test results of compounds

[0276]

[0277]

[0278] Example 20: Cell proliferation inhibition test of a series of compounds

[0279] The tumor cells (NCI-H460, BxPC-3, HT-29, HCCLM3, HepG2) in the logarithmic growth phase were digested and centrifuged. The supernatant was removed and the cells were resuspended in fresh medium containing 10% fetal bovine serum. The cells were counted using a cell counting plate. According to the standard of 2000-6000 cells per well, 100 μL of medium (containing cells) was added to a 96-well plate. At the same time, the 96-well plate was filled with PBS around the wells and placed in an incubator (37°C, 5% CO 2 ) for 24 h. When the cells were completely attached, the test samples and the positive control Plinabulin were diluted to different concentrations using fresh culture medium, and the drugs with concentration gradients were added. Six different concentration gradients were set, 100 μL per well, and 3 replicates were set for each concentration. Incubate at 5% CO 2 , incubated in a 37°C incubator for 72 hours. After adding 20 μL of 5 mg / mL MTT to each well and placing it in the incubator for 4 hours, discard the MTT, add 100 μL of DMSO to each well, place it on a 96-well plate oscillator and oscillate to completely dissolve the purple formazan. Subsequently, use an enzyme reader to detect the absorbance (OD value) at a wavelength of 490 nm. This experiment also requires setting up a zeroing well (blank culture medium, MTT, DMSO). Cell inhibition rate = 1-(OD value of the drug-addition group-OD value of the zeroing group) / (OD value of the blank group-OD value of the zeroing group) × 100%. Use Excel and Origin8.5 software to process the experimental data and calculate the IC 50 The experiment was repeated 3 times and the results were averaged.

[0280] Table 2: IC values ​​of the compounds for various cell types 50

[0281]

[0282]

[0283] NCI-H460 is a human non-small cell lung cancer cell line, BxPC-3 is a human pancreatic cancer 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.

[0284] The results are shown in Table 2. Some of the new series of 2,5-diketopiperazine compounds designed and synthesized by the present invention, including compounds P29, P30, P31, P32, P34, P35, P36, P37, and their hydrochlorides or methanesulfonates, have significant ability to inhibit the growth of tumor cells in cells such as H460, BxPC-3, HT-29, HCCLM3 and HepG2.

[0285] Example 21: Study on the anti-tumor effect of a series of compounds on the BALB / c nude mouse NCI-H460 subcutaneous transplanted tumor model

[0286] In this example, a subcutaneous transplant tumor model was established using non-small cell lung cancer cells NCI-H460 to evaluate the anti-tumor effects of a series of compounds on the subcutaneous transplant tumor model of human non-small cell lung cancer cells NCI-H460.

[0287] 1 Test materials

[0288] 1.1 Test sample

[0289] The test products are a series of anti-tumor products, including compounds P36-0, P37-0, P34-0, P35-0, and P32-0, all of which are yellow powders, easily soluble in water; stored at 2-8°C, dry, and protected from light; the preparation and use process must be strictly protected from light;

[0290] 1.2 Preparation of drug delivery preparations

[0291] Preparation method: Accurately weigh an appropriate amount of test sample, dissolve it in glucose solution and make up to the required concentration, prepare it on the day of administration, and strictly protect it from light during the preparation and administration process; transfer it at room temperature, and it will be effective on the same day.

[0292] 1.3 Positive Control

[0293] Paclitaxel injection; colorless to light yellow clear viscous liquid; concentration: 5.99 mg / mL; light-shielded, airtight, stored below 25°C;

[0294] 1.4 Preparation of drug delivery preparations

[0295] Preparation method: Dilute paclitaxel injection to 1 mg / mL with normal saline and mix thoroughly; strictly protect from light after preparation; transfer at room temperature, effective on the same day.

[0296] 1.5 Modeling tumor cells

[0297] Human non-small cell lung cancer cells NCI-H460; adherent cells; stored at 2-8°C, valid within 24 hours.

[0298] 2 Experimental animals

[0299] 2.1 Experimental system

[0300] BALB / c nude mice, SPF female; about 4-5 weeks old when purchased, about 5-6 weeks old when the experiment started; weight 12.9-15.9g when purchased, 16.1-18.9g when grouped, individual weight within the range of average weight ±20%;

[0301] Provider: Beijing Weitonglihua Experimental Animal Technology Co., Ltd.; Quality certificate: NO.110011221101329612; Production license number: SCXK(Beijing)2021-0006.

[0302] 2.2 Animal feeding and management

[0303] The mice were allowed to adapt to the environment for 3 days before the experiment, and healthy mice (female mice must not be pregnant) were selected as test animals; the number of animals that passed the quarantine was 110. The breeding environment conditions were based on the national standard of the People's Republic of China GB14925-2010; 19.82-26.33°C; relative humidity 41.19-72.66%; automatic lighting, 12-hour light and dark alternation.

[0304] 2.3 Test methods

[0305] A BALB / c nude mouse subcutaneous transplant tumor model of human non-small lung cancer cell line NCI-H460 was established, and the average screening volume was about 120mm 3 A total of 60 tumor-forming animals were divided into 10 groups, with 6 animals in each group, namely 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), and P32-0 high-dose group (15 mg / kg). The body weight and tumor diameter of the animals were continuously measured during the experiment. The experimental animals were euthanized on D12. The anti-tumor effects of compounds P36-0, P37-0, P34-0, P35-0, and P32-0 were evaluated.

[0306] 3 Model building and grouping

[0307] 3.1 Model establishment

[0308] 3.1.1 Preparation of human non-small cell lung cancer cells NCI-H460

[0309] The culture conditions were 90% RPMI1640 medium containing double antibodies + 10% FBS; the growth conditions were 5% CO 2 +95% air, culture at 37°C; when the cells in the culture flask grow to 85-90% of the flask wall, subculture.

[0310] Centrifuge the cells in the logarithmic growth phase (1000 rpm, 5 min); discard the supernatant, add an appropriate amount of PBS for dilution, and adjust the density of the cell suspension to 5×10 7 Pieces / mL.

[0311] 3.1.2 Establishment of subcutaneous transplant tumor model of human non-small cell lung cancer cell NCI-H460

[0312] Take SPF BALB / c mice that have passed quarantine, disinfect the skin at the injection site, and use a 0.3 mL insulin microsyringe to extract cells with a concentration of 5 × 10 7 0.1 mL of cell suspension was inoculated subcutaneously in the right middle wing of mice, that is, 5.0×10 cells / mL were subcutaneously inoculated in each mouse. 6 The average tumor volume grew to 120 mm 3 Around 2.5 dpi, 60 mice with relatively uniform tumor volumes were randomly divided into groups for drug administration.

[0313] 3.2 Animal grouping

[0314] A total of 10 groups were set up in the experiment, with 6 females in each group; among them, group 1 was the blank control group, group 2 was the paclitaxel positive control group, group 3 was test article group 1 (P36-0 group, 15 mg / kg), group 4 was test article 2 low-dose group (P37-0 low-dose group, 5 mg / kg), group 5 was test article 2 high-dose group (P37-0 high-dose group, 15 mg / kg), group 6 was test article 3 low-dose group (P34-0 low-dose group, 5 mg / kg), group 7 was test article 3 high-dose group (P34-0 high-dose group, 15 mg / kg), group 8 was test article group 4 (P35-0 group, 15 mg / kg), group 9 was test article 5 low-dose group (P32-0 low-dose group, 5 mg / kg), and group 10 was test article 5 high-dose group (P32-0 high-dose group, 15 mg / kg).

[0315] Grouping method: The average tumor volume was taken when it grew to 120 mm 3 The animals were randomly divided into groups. The grouping information is shown in Table 3.

[0316] Table 3 Experimental grouping and dosage design

[0317]

[0318] Note: The first digit of the animal number represents the group, the second letter represents the sex (F for female), and the 3rd, 4th, and 5th digits represent the individual animal number. [1] iv, tail vein injection; [2] q3d: once every 3 days, q3d×4, i.e. once every 3 days, for a total of 4 doses.

[0319] 3.3 Dosage method: Follow the requirements of SOP TEC-02-02 "Common drug administration methods for rodents".

[0320] The blank control group was injected into the tail vein; dosing frequency: q3d×4; dosing volume: 10mL / kg; dosing period: 12 days (once every 3 days, 4 times in total).

[0321] Paclitaxel positive control group was injected into the tail vein; q3d×4; dosage: 10 mg / kg; administration volume: 10 mL / kg; administration period: 12 days (once every 3 days, 4 times in total)

[0322] Test product 1: P36-0, P37-0, P34-0, P35-0, P32-0 were injected into the tail vein; dosing frequency: q3d×4; dosing dose: 15 mg / kg or 5 mg / kg; dosing volume: 10 mL / kg; dosing period: 12 days (once every 3 days, a total of 4 times).

[0323] Note: The day of group administration is defined as the first day of the experiment, recorded as D1, followed by D2 and D3 in sequence.

[0324] 4. Observation and inspection

[0325] 4.1 Clinical observation: Refer to SOPTEC-01-05 "General Clinical Observation of Laboratory Animals".

[0326] During the animal quarantine and modeling period, the general condition of the animals was observed once a day; after administration, the general condition of the animals was observed every day; the observation period for each group of animals was 2 weeks;

[0327] Observation content: including but not limited to the animal's physical signs, general behavior, mental state, glandular secretion, respiratory status, fecal characteristics and survival observation, etc. Record all deaths, symptoms, and the onset, severity, duration, weight changes, etc. of the symptoms.

[0328] 4.2 Body weight measurement: refer to the requirements of SOPTEC-01-10 "Measurement of Body Weight of Laboratory Animals".

[0329] Once each at the time of animal receipt, at the end of quarantine, and before grouping; 3 times a week during the modeling period and drug administration period.

[0330] 4.3 Evaluation of tumor efficacy

[0331] 4.3.1 Tumor volume measurement and evaluation criteria

[0332] The growth of tumors at the inoculation site was observed in the first week after cell inoculation. As tumor growth accelerated, tumor volume was measured once a day and 3 times a week after administration.

[0333] Measurement method: Use a vernier caliper to measure and record the long and short diameters of the tumor and calculate the tumor volume (volume formula V = 1 / 2 × a × b 2 , a is the long diameter, b is the short diameter).

[0334] The relative tumor volume (RTV) and relative tumor proliferation rate (T / C%) were calculated according to the following formula:

[0335] RTV=Vt / V0

[0336] Vt: tumor volume obtained at each tumor measurement; V0: initial tumor volume (before drug administration)

[0337] T / C% = average RTV value of the drug administration group / average RTV value of the negative control group × 100%

[0338] 4.3.2 Tumor weight measurement and evaluation criteria

[0339] After the test, use CO 2 The animals were euthanized and the tumors were removed and weighed.

[0340] Tumor inhibition rate IR TW (%) = (1-TWt / TWc) × 100%, where TWt is the tumor weight of the treatment group and TWc is the tumor weight of the solvent control group

[0341] 5 Statistical analysis

[0342] Descriptive analysis was used for general status and other data.

[0343] Quantitative indicators such as body weight were calculated by group with mean ± standard deviation When the sample size was less than 3, the data of this group were not included in the statistical comparison. The comparison between multiple groups was analyzed according to the following procedures: Levene's test for homogeneity of variance. If there was no statistical significance (P>0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If ANOVA was statistically significant (P<0.05), the LSD method was used for comprehensive comparison. If the variance was unequal (P<0.05), the Kruskal-Wallis test was used, and pairwise comparisons between means were performed.

[0344] SPSS24.0 software was used to perform the above statistical operations.

[0345] 6 Test results

[0346] 6.1 General Observation and Body Weight

[0347] During the observation period of drug administration, the weight of animals in the blank control group remained stable; mice in the paclitaxel positive control group showed body cooling and decreased activity from D11 to D12, and one animal died on D11 (2F001). The weight of the animal continued to decrease before death. The cause of death was considered to be the poor tolerance of individual mice to paclitaxel, which was related to drug toxicity. One mouse (5F006) in the P37-0 high-dose group (15 mg / kg) died on D7 after administration, which was considered to be related to drug toxicity; three mice (3F004-3F006) in the P36-0 group (15 mg / kg) had gray-black tails starting from D5, and two mice (3F004, 3F006) died on D12, and their weight dropped sharply before death, which was considered to be related to drug toxicity; the clinical observation of mice in the other groups was normal.

[0348] Compared with the blank control group, the body weight of mice in the paclitaxel positive control group was significantly decreased on days 8, 10, and 12 (*P<0.05, **P<0.01); the body weight of mice in the P36-0 group (15 mg / kg) was significantly decreased from days 3 to 12 (*P<0.05, **P<0.01, ***P<0.001), and the body weight of mice in the P37-0 high-dose group (15 mg / kg) was significantly decreased from days 3 to 12 (*P<0.05, ***P<0.001).

[0349] Animal weight statistics are shown in Table 4 and Figure 1 .

[0350] Table 4 Statistical data of animal weight in each group (g, X±SD)

[0351]

[0352] Note: *, P<0.05; **, P<0.01; ***, P<0.001 vs model control group.

[0353] 6.2 Evaluation of tumor efficacy

[0354] Tumor volume, RTV, T / C%: During the test period, the tumor volume and RTV of the blank control group maintained a rapid growth trend. At the end of the test (D12), the average tumor volume was (1020.58±507.01) mm 3 Compared with the blank control group, the average tumor volume and RTV of the paclitaxel-positive group were reduced to a certain extent, with the average tumor volume being (628.28±214.79) mm 3 , the relative tumor proliferation rate was 66.20%.

[0355] Compared with the blank control group, the average tumor volume and RTV of the test article P37-0 low-dose and high-dose groups (5 / 15 mg / kg), P32-0 low-dose 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 the P34-0 low-dose group (5 mg / kg) and P35-0 group (15 mg / kg) were reduced to a certain extent, but without statistical significance.

[0356] At the end of the experiment, the average tumor volumes of the test article P36-0 group (15 mg / kg), P37-0 low-dose and high-dose groups (5 / 15 mg / kg), P34-0 low-dose and high-dose groups (5 mg / kg), P35-0 dose group (15 mg / kg), and P32-0 low-dose and high-dose groups (5 / 15 mg / kg) were (113.78±26.36) mm, respectively. 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 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 the P37-0 low- and high-dose groups (5 mg / kg), 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%.

[0357] Table 5 Statistical data of tumor volume of animals in each group (m 3 , X±SD)

[0358]

[0359] Note: *, P<0.05; **, P<0.01; ***, P<0.001; vs. model control group.

[0360] Table 6 Statistical data of relative tumor volume (RTV) of animals in each group (X±SD)

[0361]

[0362] Note: *, P<0.05; **, P<0.01; ***, P<0.001; vs. model control group.

[0363] Table 7 Statistical data of relative tumor proliferation rate T / C (%) of each group of animals

[0364]

[0365] Note: #, indicates T / C%≤40%, and P<0.05 compared with the model control group.

[0366] Tumor weight: at the experimental endpoint, the tumor weights of the blank control group, paclitaxel positive control group, 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 / 15 mg / kg), P35-0 group (15 mg / kg), and P32-0 low and high dose groups (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. Compared with the model control group, the tumor weights of the P36-0 group (15 mg / kg), the P34-0 high-dose group (15 mg / kg), the P37-0 low- and high-dose groups (5 / 15 mg / kg), and the P32-0 low- and high-dose groups (5 / 15 mg / kg) were significantly reduced (*P < 0.05, **P < 0.01, ***P < 0.001). The tumor weights of the other drug-treated groups were reduced to a certain extent, but without statistical significance. The tumor inhibition rates of each medication group were 20.53%, 87.82%, 65.39%, 80.35%, 16.34%, 45.85%, 12.62%, 46.50% and 73.35%, respectively. Among them, the tumor inhibition rates of the P36-0 dosage group (15 mg / kg), the P32-0 high dosage group (15 mg / kg) and the P37-0 low and high dosage groups (5 / 15 mg / kg) were all greater than 60%.

[0367] Table 8 Tumor weight data of each group of animals (g, X±SD)

[0368]

[0369] Note: *, P<0.05; **, P<0.01; ***, P<0.001; vs. model control group.

[0370] Table 9 Statistical data of tumor inhibition rate IR (%) of each group of animals

[0371]

[0372] Note: #, indicates IR (%) ≥ 60%, and P < 0.05 compared with the model control group

[0373] Tumor volume statistics are shown in Tables 5 and Figure 2-1 ; The relative tumor volume RTV value data statistics are shown in Table 6 and Figure 2-2 ; T / C% data statistics are shown in Table 7 and Figure 2-3 The tumor weight and inhibition rate of each group of animals at the end of the experiment are shown in Tables 8 and 9, and Figure 3-1 and Figure 3-2 .

[0374] 10 Conclusion

[0375] Under the experimental conditions, P36-0, P37-0, P34-0, P35-0, and P32-0 all have certain anti-tumor effects; at the same dose, the anti-tumor effects of each drug are ranked as P36-0>P37-0>P32-0>P34-0>P35-0.

[0376] Example 22: Efficacy test 1 of a series of compounds on cyclophosphamide-induced neutropenia in rats

[0377] 1 Experimental Materials and Methods

[0378] 1.1 Cyclophosphamide preparation

[0379] Cyclophosphamide for injection (manufacturer: Baxter Oncology GmbH, batch number: OJ415A, 0.2 g / bottle), white crystals or crystalline powder, stored at 5°C to 25°C.

[0380] Preparation method: Add an appropriate amount of sterile 0.9% sodium chloride injection into the medicine bottle containing the drug powder, shake it gently to fully dissolve and mix it into a solution of corresponding concentration. If the dry powder cannot be completely dissolved immediately, the solution can be allowed to stand for a few minutes until it is completely clear (the entire process must be performed under a sterile environment).

[0381] 1.2 Preparation of series of compounds

[0382] Use an appropriate amount of sterile 5% glucose injection solution to dissolve a series of compounds at a certain concentration, shake gently to fully dissolve and mix, filter and sterilize the samples for tail vein injection, and strictly protect from light during and after the preparation process.

[0383] 1.3 Preparation of Plinabulin

[0384] Solvent: 1,2-propylene glycol and HS-15 (80 mg Plinabulin: 12 mL of 1,2-propylene glycol, 8 mL of HS-15);

[0385] Preparation method: Each preparation shall be made according to the actual required amount and the prescription ratio. The specific preparation process is as follows:

[0386] 1) Under strict light-proof conditions, take the prescribed amount of 1,2-propylene glycol, add Plinabulin to dissolve at 60°C, and keep warm and stir until the API is completely dissolved, and keep warm and stir for 0.25-0.5h;

[0387] 2) Continue to add half of the HS-15 while stirring and keep warm, stir for 10 minutes, and then add half of the HS-15 again;

[0388] 3) Under heat preservation conditions, the drug solution was sterilized by filtering through a 0.22 μm microporous filter and then prepared into a concentrated solution with a concentration of 4 mg / mL;

[0389] 4) Take an appropriate amount of concentrated solution and dilute it to 0.3 mg / mL with 0.9% sodium chloride injection;

[0390] Preparation frequency: Single preparation, injection diluent needs to be prepared on the day of administration, concentrated solution can be prepared once a week;

[0391] Storage conditions and validity period after preparation: Store in a dry place at room temperature away from light, transport to the animal room away from light, and use immediately after preparation;

[0392] Temporary storage conditions and shelf life after preparation and packaging: Administer the injection diluent in a dark place within 1 hour after preparation.

[0393] 1.4 Experimental animals

[0394] SD male rats were used; they were about 6 to 7 weeks old when purchased and about 7 to 8 weeks old when grouped. The individual body weight was within the range of ±20% of the average body weight. They were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2016-0011.

[0395] 1.5 Rearing conditions

[0396] There were 5 mice in each cage, and the temperature was kept at room temperature of 16-26℃ (daily temperature difference ≤4℃), with 12 / 12 hours of light and dark alternation. The mice and rats were fed maintenance feed purchased from Beijing Ke'ao Xieli Feed Co., Ltd., and the animals were free to take feed and drink experimental animal drinking water.

[0397] 1.6 Experimental Instruments

[0398] Fully automatic five-category blood analyzer (Siemens, Germany; ADVIA2120i).

[0399] 2 Test methods and detection indicators

[0400] 2.1 Animal grouping

[0401] The experiment set up 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 uniform neutrophil levels before modeling were randomly divided into groups.

[0402] Table 10. Experimental animal grouping table

[0403]

[0404] 2.2 Dosage design and administration

[0405] After the experiment was divided into groups, except for the normal control group, the animals in the other groups were intraperitoneally injected with cyclophosphamide for modeling, the modeling dose was 50 mg / kg, and the administration volume was 10 mL / kg; 30 minutes after the administration of the modeling drug, the normal control group and the model control group were injected with 5% glucose solution through 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 all injected with 6 mg / kg, and the dosage was injected through the tail vein; the Plinabulin treatment group was injected with 6 mg / kg, and the dosage was injected through the tail vein for 20 minutes; the P34-1 treatment group, the P32-1 treatment group, the P36-1 treatment group, and the P37-1 treatment group were all injected with 15 mg / kg, and the dosage was administered by gavage; the test group was given a single dose. The design is shown in Table 11.

[0406] Table 11. Dosage design table

[0407]

[0408] Note: The day of drug administration is defined as Day 1 (D1).

[0409] 2.3 Detection indicators

[0410] After administration, general condition observations were performed once a day, including but not limited to the injection site, food and water intake, slow movement, skin ulceration, lymphadenopathy, death and other abnormal symptoms of the rats.

[0411] 2.3.1 Weighing before administration, and on D2, D3, D4, D6, D8, D10, D12, D14, and D16 after administration;

[0412] 2.3.2 Food intake measurement time: D1-D2, D2-D3, D6-D7, D13-D14 after administration;

[0413] 2.3.3 Hematological tests

[0414] Testing time: before administration, D2, D3, D4, D6, D8, D10, D12, D14, and D16 after administration;

[0415] Test animals: All surviving animals in each group;

[0416] Blood collection method: about 0.1mL of blood was collected from the tail vein;

[0417] Blood sample processing method: Blood samples for hematological tests were treated with EDTA-K 2 Anticoagulation, the whole blood sample is directly injected for testing, the sample is stored at 15-25℃, and the test is completed within 8 hours;

[0418] 2.3.4 Gross anatomical observation

[0419] Dissection time: Day 16; all surviving animals in each group;

[0420] Anesthesia and dissection methods: The mice were anesthetized with 10% chloral hydrate (400 mg / kg) according to body weight, and blood was collected from the abdominal aorta for euthanasia. Gross dissection was performed, abnormal tissues were observed with the naked eye, the thymus was removed and weighed, and the thymus coefficient (organ weight (mg) per 10 g body weight) was calculated.

[0421] 3 Experimental results

[0422] 3.1 General clinical observations

[0423] After administration, most rats in the P36-0 treatment group had bloody eye and nose secretions, were inactive, had unformed feces, and had contaminated abdominal hair. 6M06, 6M03, and 6M02 were found dead on D6, D8, and D10 of the experiment. Autopsy revealed severe lung hemorrhage, gastric bloating, yellow intestinal contents, and urine retention. Some animals had black livers and spleens, and black or white kidneys. On D6 after administration, most of the P37-0 treatment group had yellowing hair, and 7M04 also had bloody secretions around the eyes. On D12 of the P36-1 treatment group, 10M02 and 10M03 had sparse hair and contaminated abdominal hair. The general clinical observations of the remaining groups were normal.

[0424] 3.2 Body weight (Table 12 and Figure 4 )

[0425] During the period from administration to the end of the experiment, the body weights of rats in the normal control group and the model control group were similar and showed a gradual increase trend; the body weights of rats in the model control group were significantly lower than those in the normal control group on the second day (Table 11, P≤0.05). Compared with the model control group, the body weights of the Plinabulin treatment group were significantly reduced on the third and fourth days (Table 11, P≤0.05); the body weights of the P34-0 treatment group were significantly reduced on the second to sixth days (Table 12, P≤0.05); the body weights of the P36-0 treatment group were significantly reduced on the third to 14th days (Table 11, P≤0.05); the body weights of the P37-0 treatment group were significantly reduced on the third to 12th days (Table 11, P≤0.05); the body weights of the P36-1 treatment group were significantly reduced on the third to sixth days (Table 11, P≤0.05); the body weights of the P37-1 treatment group were significantly reduced on the fourth day (Table 11, P≤0.05). These indicate that these compounds have certain toxic and side effects.

[0426] There was no significant difference in the body weight of rats in the P32-0 treatment group, the P34-1 treatment group, the P32-1 treatment group and the model control group, which showed a gradual increase trend. For detailed data, please see Table 12 and Figure 4 .

[0427] Table 12 Statistical summary of weight changes of rats in each group

[0428]

[0429] ^ 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;

[0430] & 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.

[0431]

[0432] a p <0.05, model control group vs normal control group; b p <0.05, Plinabulin Treatment group vs model control group;

[0433] e p <0.05, P36-1 treatment group vs model control group; f p <0.05, P37-1 treatment group vs model control group

[0434] 3.3 Food intake

[0435] During the experiment, the food intake of the normal control group and the model control group was equivalent. Compared with the model control group, the food intake of animals in each treatment group decreased on D1 and D2. Except for the P36-0 treatment group on D6, the food intake of the other groups was basically equivalent to that of the model control group. All groups resumed normal food intake on the 13th day.

[0436]

[0437] 3.4 Hematological indicators

[0438] 1) Neutrophil count (Neut, ×10 9 cells / L) results (Table 13 and Figure 5 ):

[0439] On the 2nd to 4th day, the neutrophil count in the model control group was significantly lower than that in the normal control group (P≤0.05, P≤0.01); on the 10th day, the neutrophil count in the model control group was significantly higher than that in the normal control group (P≤0.01), and the overall trend was that the neutrophil count decreased after modeling, then increased, and then returned to normal levels.

[0440] Compared with the model control group, the neutrophil counts in the Plinabulin treatment group, P34-0 treatment group, P32-0 treatment group, P36-0 treatment group, and P37-0 treatment group increased significantly on the second day after administration (P≤0.05, P≤0.01, P≤0001); the neutrophil counts in the P36-0 treatment group were significantly higher than those in the model control group on days 6-16 after administration (P≤0.05, P≤0.01, P≤0.001). The neutrophil counts in the P37-0 treatment group were significantly higher than those in the model control group on day 14 after administration (P≤0.05).

[0441] Compared with the Plinabulin treatment group, the neutrophil counts in the P34-0 treatment group, the P36-0 treatment group, and the P37-0 treatment group increased significantly on the second day after administration (P≤0.01, P≤0.001). Please see Table 13 for detailed data.

[0442] Table 13 Statistical summary of neutrophil counts in each group of rats

[0443]

[0444] 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;

[0445] &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.

[0446]

[0447] 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;

[0448] d p <0.05, P32-1 treatment group vs model control group; f p <0.05, P37-1 treatment group vs model control group.

[0449] 2) White blood cell count (WBC, ×10 9 cells / L) results (Table 14 and Figure 6 ):

[0450] From the 2nd to the 8th day, the white blood cell count in the model control group was significantly lower than that in the normal control group (P≤0.01, P≤0.001), and the overall trend was a decrease after modeling and then an increase to the normal level.

[0451] Compared with the model control group, the white blood cell counts in the Plinabulin treatment group, P34-0 treatment group, P36-0 treatment group, and P37-0 treatment group were significantly increased on the second day after administration (P≤0.05, P≤0.01, P≤0.001), and the white blood cell count in the P32-0 treatment group was significantly increased on the 14th day (P≤0.05).

[0452] Compared with the Plinabulin treatment group, the neutrophil counts in the P34-0 treatment group, the P36-0 treatment group, and the P37-0 treatment group increased significantly on the second day after administration (P≤0.05, P≤0.001). Please see Table 14 for detailed data.

[0453] Table 14 Summary of statistical data on white blood cell counts of rats in each group

[0454]

[0455] 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;

[0456] & 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.

[0457]

[0458] a p <0.05, model control group vs normal control group; b p <0.05, Plinabulin Treatment group vs model control group; f p <0.05, P37-1 treatment group vs model control group.

[0459] 3.5 Thymus Index

[0460] The thymus index of rats in the model control group was significantly lower than that in the normal control group (P≤0.05), while the thymus index of rats in the Plinabulin treatment group, P32-0 treatment group and P37-0 treatment group was significantly higher than that in the model control group (P≤0.05, P≤0.05).

[0461] Table 15 Statistical summary of thymus weight and thymus index of rats in each group

[0462]

[0463] ^ p<0.05, model control group vs normal control group; *p<0.05, Plinabuli n Treatment group vs model control group; ▲ p<0.05, P37-0 treatment group vs model control group.

[0464] 4 Summary

[0465] Under the conditions of this experiment, after the model control group was modeled with 50 mg / kg cyclophosphamide intraperitoneally, there were no abnormalities in general clinical observations, and there were no significant changes in body weight and food intake. About 4 days after modeling, the neutrophil and white blood cell counts decreased significantly, indicating that the cyclophosphamide-induced neutropenia model was successfully established at this dose.

[0466] During the experiment, the body weight and food intake of the Plinabulin treatment group, P34-0 treatment group, P36-0 treatment group, P37-0 treatment group, P36-1 treatment group, and P37-1 treatment group decreased to a certain extent. The neutrophils and white blood cells of the Plinabulin treatment group, P34-0 treatment group, P32-0 treatment group, P36-0 treatment group, P37-0 treatment group, and P37-1 treatment group increased significantly on the second day after administration, indicating that these compounds have a certain degree of therapeutic effect on neutropenia.

[0467] Example 23: Efficacy test of a series of compounds on cyclophosphamide-induced neutropenia in rats 2

[0468] 1 Experimental materials and methods are the same as in Example 22

[0469] 2 Test methods and detection indicators

[0470] 2.1 Group design

[0471] The experiment set up 8 groups, each with 6 male rats, for a total of 48 rats; they were 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, and P30-0 treatment group; rats with uniform neutrophil levels before modeling were selected and randomly divided into groups.

[0472] Table 16. Experimental animal grouping table

[0473]

[0474] 2.2 Dosage design

[0475] After the experiment was divided into groups, except for the normal control group, all other groups of animals were intraperitoneally injected with cyclophosphamide for modeling, with a modeling dose of 50 mg / kg and a dosing volume of 10 mL / kg; 30 minutes after the modeling drug was administered, 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 with a dose of 7.5 mg / kg, which was administered via the tail vein infusion for 20 minutes; the other treatment groups were administered with a dose of 12 mg / kg, which was injected via the tail vein. All the test groups were given a single dose. The design is shown in Table 17.

[0476] Table 17. Dosage design table

[0477]

[0478] 2.3 Administration

[0479] Administration route: Tail vein injection; single administration 30 minutes after the modeling drug is administered;

[0480] Note: The day of drug administration is defined as Day 1 (D1).

[0481] 2.3 Detection indicators

[0482] After administration, general condition observations were performed once a day, including but not limited to the injection site, food and water intake, slow movement, skin ulceration, lymphadenopathy, death and other abnormal symptoms of the rats.

[0483] 2.3.1 Weighing before administration, and on D2, D3, D5, D7, D9, D11, D14, D16, and D18 after administration;

[0484] 2.3.2 Food intake measurement time: D1-D2, D2-D3, D6-D7, D13-D14 after administration;

[0485] Hematological tests were performed once a day before and after administration. On the 15th day after administration, all rats were dissected, and the thymus of each animal was taken, weighed, and its organ coefficient (weight of the organ per 10 g body weight (mg)) was calculated.

[0486] 2.3.3 Hematological tests

[0487] Detection time: before administration, after administration, D1, D2, D3, D5, D7, D8, D9, D11, D12, D14, D16; about 0.1 mL of blood was collected from the tail vein;

[0488] Blood sample processing method: Blood samples for hematological tests were treated with EDTA-K 2 Anticoagulation, the whole blood sample is directly injected for testing, the sample is stored at 15-25℃, and the test is completed within 8 hours;

[0489] 2.3.4 Gross anatomical observation

[0490] Dissection time: Day 16; all surviving animals in each group;

[0491] Anesthesia and dissection method: The rats were anesthetized with 10% chloral hydrate (400 mg / kg) according to body weight, and blood was collected from the abdominal aorta for euthanasia. The rats were subjected to gross dissection observation, and the thymus was removed and weighed to calculate the thymus coefficient (organ weight (mg) per 10 g body weight).

[0492] 3 Experimental results

[0493] 3.1 Body weight (Table 18 and Figure 7 )

[0494] Compared with the normal control group, the body weight of the model control group decreased significantly on D2 (P≤0.05), and the rest of the time was comparable to that of the normal control group. 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). There was no significant difference in body weight between the other treatment groups and the model control group, and there was a gradual increase trend.

[0495] 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.

[0496] The statistical data of animal weight are shown in Table 18. The weight growth trend of each group is shown in Figure 7 .

[0497] Table 18 Statistical data of animal weight (g)

[0498]

[0499] *P≤0.05, normal control group vs model control group; # P≤0.05, ## P≤0.01, model control group vs each treatment group.

[0500] 3.2 Food intake

[0501] During the experiment, the food intake of the model control group, P36-0 treatment group, and P29-0 treatment group decreased on D1, and returned to normal on D2; the food intake of the Plinabulin treatment group, P32-0 treatment group, P32-1 treatment group, and P30-0 treatment group decreased on D1 and D2, and returned to normal on D6. Statistical data are shown in Appendix 19.

[0502] Table 19 Statistical data on animal food intake

[0503]

[0504] 3.3 Hematological indicators

[0505] 1) Neutrophil count (Neut, ×10 9 cells / L) results (Table 20 and Figure 8 ):

[0506] On the 2nd to 8th day, the neutrophil count in the model control group was significantly lower than that in the normal control group (P≤0.01, P≤0.001); on the 11th day, the neutrophil count in the model control group was significantly higher than that in the normal control group (P≤0.05), and the overall trend was that the neutrophil count increased after modeling, then decreased, then increased, and then returned to normal levels.

[0507] Compared with the model control group, the neutrophil count in the Plinabulin treatment group increased significantly on the 1st and 2nd days after administration (P≤0.01); the neutrophil count in the P32-0 treatment group increased significantly on the 2nd and 11-14th days after administration (P≤0.05, P≤0.01); the neutrophil count in the P32-1 treatment group increased significantly on the 2nd and 11-14th days after administration (P≤0.05, P≤001); the neutrophil count in the P31-0 treatment group increased significantly on the 2nd and 3rd days after administration (P≤0.05,); and the neutrophil count in the P30-0 treatment group increased significantly on the 2nd day after administration (P≤0.05, P≤0.01).

[0508] The statistical data of animal neutrophils are shown in Table 20. The changes of neutrophils in each group are shown in Figure 8

[0509] Table 20 Animal neutrophil statistics (×10 9 cells / L)

[0510]

[0511] * 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, ^^^ P≤0.001, P32-0 group vs each treatment group

[0512] 3) White blood cell count (WBC, ×10 9 cells / L) results (Table 21 and Fig. 9 ):

[0513] From the 2nd to the 9th day, the white blood cell count in the model control group was significantly lower than that in the normal control group (P≤0.01, P≤0.001). Overall, it increased after modeling, then decreased, and then slowly increased to the normal level.

[0514] Compared with the model control group, the white blood cell count in the P32-0 treatment group increased significantly on days 1-2 after administration (P≤0.001); the white blood cell count in the P32-1 treatment group increased significantly on days 1-2 and 7 after administration (P≤0.05, P≤0.01); the white blood cell count in the P29-0 treatment group increased significantly on days 8 and 14 after administration (P≤0.05, P≤0.01).

[0515] The statistical data of animal leukocytes are shown in Table 21. The changes of leukocytes in each group are shown in Fig. 9 .

[0516] Table 21 Animal leukocyte statistics (×10 9 cells / L)

[0517]

[0518] ** 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 line; ^ P≤0.05, ^^ P≤0.01, ^^^ P≤0.001, P32-0 group vs each treatment group.

[0519] 3.4 Thymus weight and thymus coefficient

[0520] There was no significant difference in thymus weight and thymus coefficient among the groups. The statistical data of animal thymus weight and thymus coefficient are shown in Appendix 22.

[0521] Table 22 Statistical data of animal thymus weight and thymus coefficient

[0522] average value Thymus weight (g) Thymus index (mg / 10 g) Normal control group 0.5359 0.1405 Model control group 0.4298 0.1161 <![CDATA[ Plinabulin Treatment Group]]> 0.4271 0.1150 P32-0 treatment group 0.4571 0.1251 P32-1 treatment group 0.5179 0.1374 P31-0 treatment group 0.4683 0.1259 P29-0 treatment group 0.4532 0.1230 P30-0 treatment group 0.5067 0.1331

[0523] 4. Summary

[0524] Under the conditions of this experiment, after the model control group was modeled with 50 mg / kg cyclophosphamide intraperitoneally, there was no abnormality in general clinical observation, and there was no significant change in body weight and food intake. About one week after modeling, the neutrophil and white blood cell counts decreased significantly, indicating that the cyclophosphamide-induced neutropenia model was successfully established at this dose.

[0525] During the experiment, except for the P29-0 treatment group, the neutrophil counts of 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 increased significantly on the 2nd and 3rd days after administration, indicating that these compounds have a certain degree of preventive and therapeutic effects on neutropenia.

[0526] Compared with the Plinabulin treatment group, the neutrophil count in the P32-0 treatment group was significantly increased on days 2, 11, 12 or 14 after administration, indicating that compound P32-0 has better efficacy in preventing and treating neutropenia.

[0527] Example 24: Efficacy test of a series of compounds on cyclophosphamide-induced neutropenia in rats model 3

[0528] 1 Experimental materials and methods are the same as Example 22.

[0529] 2 Test methods and detection indicators

[0530] 2.1 Group design

[0531] The experiment set up 8 groups, each with 6 male rats, for a total of 48 rats; they were normal control group, model control group, Plinabulin treatment group, P32-0 treatment group, P5-0 treatment group, P12-0 treatment group, P10-0 treatment group, and P11-0 treatment group; rats with uniform neutrophil levels before modeling were selected and randomly divided into groups.

[0532] Table 23 Experimental animal grouping table

[0533]

[0534] 2.2 Dosage design

[0535] After the experiment was divided into groups, except for the normal control group, all other groups of animals were intraperitoneally injected with cyclophosphamide for modeling, with a modeling dose of 50 mg / kg and a dosing volume of 10 mL / kg; 30 minutes after the modeling drug was administered, 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 with a dose of 7.5 mg / kg, which was administered via the tail vein infusion for 20 minutes; the other treatment groups were administered with a dose of 12 mg / kg, which was injected via the tail vein. All the test groups were given a single dose. The design is shown in Table 24.

[0536] Table 24 Dosage design table

[0537]

[0538] 2.3 Administration

[0539] Administration route: Tail vein injection; single administration 30 minutes after the modeling drug is administered;

[0540] Note: The day of drug administration is defined as Day 1 (D1).

[0541] 3. Detection indicators

[0542] After administration, general condition observations were performed once a day, including but not limited to the injection site, food and water intake, slow movement, skin ulceration, lymphadenopathy, death and other abnormal symptoms of the rats.

[0543] 3.1 Weigh before administration, and on D2, D3, D5, D7, D9, D11, D14, and D16 after administration;

[0544] 3.2 Food intake measurement time: D1-D2, D2-D3, D6-D7, D13-D14 after administration;

[0545] Hematological tests were performed once a day before and after administration. On the 15th day after administration, all rats were dissected, and the thymus of each animal was taken, weighed, and its organ coefficient (weight of the organ per 10 g body weight (mg)) was calculated.

[0546] 3.3 Hematological tests

[0547] Detection time: before administration, and on D1, D2, D3, D5, D7, D9, D11, D12, D14, and D16 after administration; about 0.1 mL of blood was collected from the tail vein;

[0548] Blood sample processing method: Blood samples for hematological tests were treated with EDTA-K 2 Anticoagulation, the whole blood sample is directly injected for testing, the sample is stored at 15-25℃, and the test is completed within 8 hours;

[0549] 3.4 Gross anatomical observation

[0550] Dissection time: Day 16; all surviving animals in each group;

[0551] Anesthesia and dissection method: The rats were anesthetized with 10% chloral hydrate (400 mg / kg) according to their body weight, and blood was collected from the abdominal aorta for euthanasia. The rats were subjected to gross dissection observation, and the thymus was removed and weighed to calculate the thymus coefficient (organ weight (mg) per 10 g body weight)

[0552] 4 Experimental results

[0553] 4.1 Body weight (Table 25 and Fig.10 )

[0554] Compared with the normal control group, the body weight of the model control group decreased significantly on D2 and D9 (P≤0.05), and the rest of the time was comparable to that of the normal control group. Compared with the model control group, the body weight of the P32-0 treatment group decreased significantly in the first week (P≤0.05, P≤0.01). There was no significant difference in body weight between the other treatment groups and the model control group, and there was a gradual increase trend.

[0555] The average body weight of the normal control group increased by 2.06% on D2 and increased by 21.86% on D9, while the average body weight of the model control group decreased by 4.39% on D2, increased by 2.40% on D3, increased by 6.64% on D5, and increased by 12.02% on D7. The average body weight of the P32-0 treatment group decreased by 9.62%, 7.84%, and 0.61% on D2, D3, and D5, respectively, and increased by 5.72% on D7.

[0556] The statistical data of animal weight are shown in Table 25. The weight growth trend of each group is shown in Fig.10 .

[0557] Table 25 Statistical summary of weight changes of rats in each group

[0558] Weight (average) D0 D1 D2 D3 D5 D7 D9 D11 D14 D16 Normal control group 278.53 282.81 288.64 299.02 315.88 331.95 344.63 356.39 372.33 389.04 Model control group 273.44 280.94 <![CDATA[268.62 * ]]> 287.70 299.59 314.71 <![CDATA[322.50 * ]]> 340.09 354.86 365.19 <![CDATA[ Plinabulin Treatment Group]]> 272.11 282.46 257.70 275.89 289.76 301.53 316.32 327.89 343.93 355.38 P32-0 treatment group 271.05 276.42 <![CDATA[249.82 # ]]> <![CDATA[254.75 # ]]> <![CDATA[274.74 # ]]> <![CDATA[292.22 # ]]> 307.70 319.79 337.23 351.23 P5-0 treatment group 270.60 273.48 266.76 278.64 293.09 306.88 323.03 335.59 348.32 363.77 P12-0 treatment group 273.94 278.73 270.58 282.00 300.39 315.43 331.93 336.22 357.24 371.28 P10-0 treatment group 281.50 287.67 278.49 292.81 309.88 321.74 336.56 347.53 365.02 377.87 P11-0 treatment group 272.35 281.18 271.75 286.77 300.48 315.73 332.82 341.45 358.99 370.65

[0559] * P ≤ 0.05, normal control group vs model control group; # P≤0.05, ## P≤0.01, model control group vs each treatment group.

[0560] 4.2 Food intake

[0561] During the experiment, the food intake of the model control group, Plinabulin treatment group, P5-0 treatment group, P12-0 treatment group, P10-0 treatment group, and P11-0 treatment group decreased on D1 and returned to normal on D2; the food intake of the P32-0 treatment group decreased on D1 and D2 and returned to normal on D6.

[0562] Table 26 Statistical data on animal food intake

[0563]

[0564] 4.3 Hematological indicators

[0565] 1) Neutrophil count (Neut, ×10 9 cells / L) results (Table 27 and Fig.11 ):

[0566] On the first day, the neutrophil count in the model control group was significantly higher than that in the normal control group (P≤0.001); from the 2nd to the 7th day, the neutrophil count in the model control group was significantly lower than that in the normal control group (P≤0.05, P≤0.01, P≤0.001); on the 12th day, the neutrophil count in the model control group was significantly higher than that in the normal control group (P≤0.05), and the overall trend was that the neutrophil count increased after modeling, then decreased, then increased, and then returned to normal levels.

[0567] Compared with the model control group, the neutrophil count in the Plinabulin treatment group was significantly increased on the 1st and 2nd days after administration (P≤0.05, P≤0.01), and was significantly decreased on the 7th day (P≤0.05); the neutrophil count in the P32-0 treatment group was significantly increased on the 2nd, 3rd, and 11th days after administration (P≤0.05, P≤0.01); the neutrophil count in the P12-0 treatment group and the P11-0 treatment group was significantly increased on the 2nd day after administration (P≤0.05).

[0568] The statistical data of animal neutrophils are shown in Table 27. The changes of neutrophils in each group are shown in Fig.11 .

[0569] Table 27 Statistical summary of neutrophil counts in each group of rats

[0570]

[0571] * 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.

[0572] 4) White blood cell count (WBC, ×10 9 cells / L) results (Table 28 and Fig.12 ):

[0573] On days 2 to 7 and 11, the white blood cell count in the model control group was significantly lower than that in the normal control group (P≤0.05, P≤0.001). Overall, it increased after modeling, then decreased, and then slowly increased to the normal level.

[0574] Compared with the model control group, the leukocyte count in the Plinabulin treatment group increased significantly on the 1st and 2nd day after administration (P≤0.05, P≤0.01), and decreased significantly on the 7th day (P≤0.05); the leukocyte count in the P32-0 group increased significantly on the 2nd day after administration (P≤0.01); the leukocyte count in the P12-0 and P11-0 treatment groups increased significantly on the 2nd day after administration (P≤0.01);

[0575] The statistical data of animal leukocytes are shown in Table 28. The changes of leukocytes in each group are shown in Fig.12 .

[0576] Table 28 Summary of statistical data on white blood cell counts of rats in each group

[0577]

[0578] * 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.

[0579] 4.4 Thymus weight and thymus coefficient

[0580] 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 was significantly decreased (P≤0.05); the thymus coefficient of the P12-0 treatment group and the P10-0 treatment group was significantly decreased (P≤0.05).

[0581] The statistical data of animal thymus weight and thymus coefficient are shown in Table 29.

[0582] Table 29 Statistical data of animal thymus weight and thymus coefficient

[0583] average value Thymus weight (g) Thymus index (mg / 10g) Normal control group 0.6868 0.1760 Model control group 0.6409 0.1765 <![CDATA[ Plinabulin Treatment Group]]> 0.5863 0.1644 P32-0 treatment group 0.4670 0.1321 P5-0 treatment group <![CDATA[0.4514 # ]]> 0.1243 P12-0 treatment group <![CDATA[0.4561 # ]]> <![CDATA[0.1248 # ]]> P10-0 treatment group <![CDATA[0.4387 # ]]> <![CDATA[0.1158 # ]]> P11-0 treatment group 0.5170 0.1381

[0584] # P≤0.05, model control group vs each treatment group.

[0585] 4 Summary

[0586] Under the conditions of this experiment, after the model control group was modeled with 50 mg / kg cyclophosphamide intraperitoneally, there was no abnormality in general clinical observation, and there was no significant change in body weight and food intake. About one week after modeling, the neutrophil and white blood cell counts decreased significantly, indicating that the cyclophosphamide-induced neutropenia model was successfully established at this dose.

[0587] During the experiment, the neutrophil counts in the Plinabulin treatment group and the P32-0 treatment group generally increased significantly on the 2nd and 3rd days after administration, indicating that compound P32-0 has a significant preventive and therapeutic effect on neutropenia; compounds P12-0 and P11-0 also showed a certain white blood cell-raising effect at a dose of 12 mg / kg.

[0588] Example 25: Efficacy test of a series of compounds on cyclophosphamide-induced neutropenia in rats 4

[0589] 1 Experimental Materials and Methods

[0590] 1.1 Preparation of series of compounds

[0591] Use an appropriate amount of sterile 5% glucose injection to dissolve the compounds. The concentrations of P32-0, P38-0, P39-0 and P41-0 are 1.2 mg / mL; the concentration of P40-0 is 0.8 mg / mL; the concentration of P42 is 2.4 mg / mL. Shake gently to fully dissolve and mix, filter through a 0.22 μm filter for sterilization, and strictly avoid light during and after the preparation process.

[0592] 1.2 For other experimental materials and methods, please refer to Effect Example 22.

[0593] 2 Test methods and detection indicators

[0594] 2.1 Group design

[0595] The experiment set up 9 groups, each with 6 male rats, for a total of 54 rats; they were 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, and P42 treatment group; rats with uniform neutrophil levels before modeling were selected and randomly divided into groups.

[0596] Table 30 Experimental Animal Grouping

[0597]

[0598] 2.2 Dosage design

[0599] After the experiment was divided into groups, except for the normal control group, the animals in the other groups were intraperitoneally injected with cyclophosphamide for modeling. The modeling dose was 50 mg / kg and the administration volume was 10 mL / kg.

[0600] 30 minutes after the administration of the modeling drug, the normal and model control groups were injected with 5% glucose solution at 10 mL / kg through the tail vein.

[0601] The Plinabulin treatment group was given a single dose of 7.5 mg / kg via intravenous infusion over 20 minutes 30 minutes after model establishment.

[0602] The P32-0, P38-0, P39-0, P40-0 and P41-0 treatment groups were intravenously injected with the corresponding compounds at 12 mg / kg 30 minutes after modeling, and the P42 treatment group was intragastrically administered with 24 mg / kg 30 minutes after modeling, for a single dose.

[0603] Table 31 Dosage design table

[0604]

[0605] Note: Due to the poor solubility of P40-0, the administration volume was adjusted to 15 mL / kg.

[0606] 2.3 Administration

[0607] Administration route: tail vein injection; tail vein infusion; oral administration; single administration 30 minutes after the modeling drug is administered; administration speed: tail vein infusion is performed slowly and completed within 20 minutes;

[0608] Note: The day of drug administration is defined as Day 1 (D1).

[0609] 2.4 Detection indicators

[0610] After administration, general condition observations were performed once a day, including but not limited to the injection site, food and water intake, slow movement, skin ulceration, lymphadenopathy, death and other abnormal symptoms of the rats.

[0611] 2.4.1 Weigh before administration, and 2, 3, 7, 11, 14, and 16 days after administration;

[0612] 2.4.2 Food intake measurement time: 1-2, 2-3, 6-7, 13-14 after administration;

[0613] Hematological tests were performed once a day before and after administration. On the 15th day after administration, all rats were dissected, and the thymus of each animal was taken, weighed, and its organ coefficient (weight of the organ per 10 g body weight (mg)) was calculated.

[0614] 2.4.3 Hematological tests

[0615] Detection time: before administration, and on D2, D3, D5, D7, D8, D9, D11, D12, D14, and D16 after administration; about 0.1 mL of blood was collected from the tail vein;

[0616] Blood sample processing method: Blood samples for hematological tests were treated with EDTA-K2 Anticoagulation, the whole blood sample is directly injected for testing, the sample is stored at 15-25℃, and the test is completed within 8 hours;

[0617] 2.4.4 Gross anatomical observation

[0618] Dissection time: Day 16; all surviving animals in each group;

[0619] Anesthesia and dissection method: The rats were anesthetized with 10% chloral hydrate (400 mg / kg) according to body weight, and blood was collected from the abdominal aorta for euthanasia. The rats were subjected to gross dissection observation, and the thymus was removed and weighed to calculate the thymus coefficient (organ weight (mg) per 10 g body weight).

[0620] 3 Experimental results

[0621] 3.1 Body weight (Table 32 and Fig.13 )

[0622] Compared with the model control group, the body weight of the P32-0 treatment group decreased significantly in the first week (P<0.05), and the body weight of the P38-0 treatment group decreased significantly on D2 and D3 (P<0.05). There was no significant difference in body weight between the other treatment groups and the model control group, and the body weight showed a gradual increase trend.

[0623] The statistical data of animal weight are shown in Table 32. The weight growth trend of each group is shown in Fig.13 .

[0624] Table 32 Statistical summary of weight changes of rats in each group

[0625] Group D0 D1 D2 D3 D7 D11 D14 D16 Normal control group 254.54 259.97 270.62 273.88 297.47 315.77 327.67 337.59 Model control group 259.32 260.11 261.52 270.70 291.46 313.08 322.24 334.92 <![CDATA[ Plinabulin Treatment Group]]> 254.55 258.70 247.69 256.29 282.62 300.87 311.62 321.19 P32-0 treatment group 257.42 262.58 249.82 244.47 264.85 292.02 302.08 313.30 P38-0 treatment group 254.96 258.38 240.87 249.24 283.07 303.25 317.24 330.19 P39-0 treatment group 254.73 257.13 254.45 265.51 286.83 313.94 321.16 330.39 P40-0 treatment group 254.42 257.65 253.58 263.63 278.07 288.74 302.56 311.78 P41-0 treatment group 257.03 260.83 254.07 262.57 289.52 304.94 322.02 331.54 P42 treatment group 256.62 262.29 260.65 268.01 295.04 311.43 326.88 333.37

[0626] # P<0.05, model control group vs each treatment group.

[0627] 3.2 Food intake

[0628] During the experiment, the food intake of the model control group, P39-0 treatment group, P40-0 treatment group, P41-0 treatment group, and P42 treatment group decreased on D1 and returned to normal on D2; the food intake of the Plinabulin treatment group, P32-0 treatment group, and P38-0 treatment group decreased on D1 and D2 and returned to normal on D6.

[0629] Table 33 Statistical summary of changes in food intake of rats in each group

[0630]

[0631] 3.3 Hematological indicators

[0632] 1) Neutrophil count (Neut, ×10 9cells / L) results (Table 34 and Fig.14 ):

[0633] On days 3 to 8, the neutrophil count in the model control group was significantly lower than that in the normal control group (P<0.01, P<0.001); on days 14 and 16, the neutrophil count in the model control group was significantly higher than that in the normal control group (P<0.05). The overall trend was that the neutrophil count increased after modeling, then decreased, then increased, and then dropped to near the normal level.

[0634] Compared with the model control group, the neutrophil count in the Plinabulin treatment group was significantly increased on the 2nd day after administration (P<0.05), and was significantly decreased on the 8th day (P<0.01); the neutrophil count in the P32-0 treatment group was significantly increased on the 2nd and 7-14th days after administration (P<0.05, P<0.01, P<0.001); the neutrophil count in the P38-0 treatment group and the P41-0 treatment group was significantly increased on the 2nd day after administration (P<0.05, P<0.01); the neutrophil count in the P40-0 treatment group was significantly increased on days 7-14.

[0635] The statistical data of animal neutrophils are shown in Table 34. The changes of neutrophils in each group are shown in Fig.14 .

[0636] Table 34 Statistical summary of neutrophil counts in each group of rats

[0637] Group D0 D2 D3 D5 D7 D8 D9 D11 D12 D14 D16 Normal control group 0.81 0.95 0.94 1.13 1.07 1.10 1.12 1.09 1.43 0.92 0.55 Model control group 0.83 0.60 <![CDATA[0.41 ** ]]> <![CDATA[0.14 *** ]]> <![CDATA[0.35 ** ]]> <![CDATA[0.41 * ]]> 1.81 1.66 1.78 1.51 0.96 <![CDATA[ Plinabulin Treatment Group]]> 0.80 <![CDATA[1.60 # ]]> 0.40 <![CDATA[0.08 *** ]]> 0.19 0.23 0.81 1.24 1.78 1.13 1.16 P32-0 treatment group 0.82 <![CDATA[3.23 ### ]]> 0.51 <![CDATA[0.11 *** ]]> 0.88 2.20 2.75 <![CDATA[3.52 ## ]]> <![CDATA[3.09 # ]]> 2.39 1.00 P38-0 treatment group 0.80 <![CDATA[2.54 ## ]]> 0.44 0.08 0.28 0.37 1.30 1.15 1.89 1.19 0.52 P39-0 treatment group 0.85 0.92 0.38 0.07 0.31 0.44 1.33 1.76 2.03 1.69 1.17 P40-0 treatment group 0.80 0.59 0.23 0.07 0.50 0.80 1.61 3.29 3.56 3.03 0.85 P41-0 treatment group 0.80 <![CDATA[2.09 # ]]> 0.37 0.06 0.23 0.49 1.12 1.97 1.99 1.97 0.89 P42 treatment group 0.80 0.21 0.16 0.11 0.18 0.36 1.10 1.16 1.18 1.22 0.49

[0638] * 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.

[0639] 5) White blood cell count (WBC, ×10 9 cells / L) results (Table 35 and Fig.15 ):

[0640] On days 2 to 8 and 11, the white blood cell count in the model control group was significantly lower than that in the normal control group (P < 0.05, P < 0.001), and overall it decreased after modeling, and then slowly increased to normal levels.

[0641] Compared with the model control group, the white blood cell count in the P32-0 treatment group increased significantly on the 2nd and 11-14th days after administration (P<0.05); the white blood cell count in the P38-0 treatment group and the P41-0 treatment group increased significantly on the 2nd day after administration (P<0.05, P<0.01); the white blood cell count in the P40-0 treatment group increased significantly on the 11th-14th day after administration.

[0642] The statistical data of animal leukocytes are shown in Table 35. The changes of leukocytes in each group are shown in Fig.15 .

[0643] Table 35 Animal leukocyte statistics (×10 9 cells / L)

[0644] Group D0 D2 D3 D5 D7 D8 D9 D11 D12 D14 D16 Normal control group 10.28 9.57 10.45 10.92 11.32 10.77 11.83 12.69 12.86 10.34 9.46 Model control group 10.25 4.70 3.27 1.65 4.37 4.90 8.11 8.92 11.09 11.16 8.09 <![CDATA[ Plinabuli nTreatment Group]]> 9.42 6.38 2.79 1.62 3.35 3.67 5.42 8.08 9.07 7.72 8.06 P32-0 treatment group 9.50 7.50 3.13 1.26 5.48 7.66 9.96 13.02 11.10 10.50 6.79 P38-0 treatment group 9.70 7.16 2.40 1.30 3.66 3.73 6.11 6.98 8.83 7.73 6.05 P39-0 treatment group 10.55 5.54 3.33 1.53 4.70 4.75 8.06 9.82 9.94 9.97 7.40 P40-0 treatment group 9.22 3.46 2.08 1.27 3.41 4.11 6.66 10.99 13.48 11.17 5.64 P41-0 treatment group 9.82 7.22 3.29 1.47 4.62 5.29 7.37 9.75 9.96 10.31 6.44 P42 treatment group 9.37 3.34 2.67 1.70 3.13 4.07 6.64 8.13 8.94 8.67 6.19

[0645] * 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.

[0646] 3.4 Thymus weight and thymus coefficient

[0647] Compared with the normal control group, the thymus weight and thymus coefficient of the model control group decreased significantly (P<0.05). There were no significant differences among the other groups.

[0648] The statistical data of animal thymus weight and thymus coefficient are shown in Appendix 36.

[0649] Table 36 Statistical data of animal thymus weight and thymus coefficient

[0650] Group Thymus weight (g) Thymus index (mg / 10g) Normal control group 0.6071 0.1802 Model control group 0.4305 0.1275 <![CDATA[ Plinabulin Treatment Group]]> 0.4622 0.1443 P32-0 treatment group 0.4921 0.1564 P38-0 treatment group 0.4702 0.1426 P39-0 treatment group 0.4965 0.1497 P40-0 treatment group 0.3557 0.1133 P41-0 treatment group 0.4756 0.1435 P42 treatment group 0.4761 0.1426

[0651] 4. Summary

[0652] Under the conditions of this experiment, after the model control group was modeled with 50 mg / kg cyclophosphamide intraperitoneally, there was no abnormality in general clinical observation, and there was no significant change in body weight and food intake. One week after modeling, neutrophils and white blood cells decreased significantly, indicating that the cyclophosphamide-induced neutropenia model was successfully established at this dose.

[0653] During the experiment, the neutrophil counts in the Plinabulin treatment group, P32-0 treatment group, P38-0 treatment group and P41-0 treatment group increased significantly on the 2nd day after administration, and the neutrophil counts in the P40-0 treatment group increased significantly on the 7th to 14th days after administration, indicating that these compounds have a certain degree of preventive and therapeutic effects on neutropenia.

[0654] Example 26: Pharmacodynamics experiment of compound P32-0 combined with G-CSF in the treatment of cyclophosphamide-induced neutropenia in rats

[0655] 1. Materials and Methods

[0656] 1.1 Plinabulin preparation

[0657] Solvent: 1,2-propylene glycol and HS-15 (80 mg Plinabulin: 12 mL 1,2-propylene glycol, 8 mL HS-15)

[0658] Preparation method: Each preparation shall be made according to the actual required amount and the prescription ratio. The specific preparation process is as follows:

[0659] 1) Under strict light-proof conditions, take the prescribed amount of 1,2-propylene glycol, add Plinabulin to dissolve at 60°C, and keep warm and stir until the API is completely dissolved, and keep warm and stir for 0.25-0.5h;

[0660] 2) Continue to add half of the HS-15 while stirring and keep warm, stir for 10 minutes, and then add half of the HS-15 again;

[0661] 3) Under heat preservation conditions, the drug solution was sterilized by filtering through a 0.22 μm microporous filter and then prepared into a concentrated solution with a concentration of 4 mg / mL;

[0662] 4) Take an appropriate amount of concentrated solution and dilute it to 0.375 mg / mL with 5% glucose injection;

[0663] Preparation frequency: The concentrated solution is prepared once a week, stored in a dry place at room temperature away from light, and transported to the animal room in a light-proof condition; the injection diluent needs to be prepared on the day of administration, stored in a dry place at room temperature away from light, and transported to the animal room in a light-proof condition, and used immediately after preparation; the injection diluent should be administered within 6 hours after preparation.

[0664] 1.2 Preparation of PEG-rhG-CSF injection

[0665] Manufacturer: Jiangsu Hengrui Medicine Co., Ltd.; Batch number: 210219CG / 210208CG;

[0666] Properties: Colorless, clear liquid; Specification: 0.6mL: 6mg, prefilled injection, sealed with chlorobutyl rubber piston, 1 tube / box; Store at 2-8℃ away from light.

[0667] Preparation method: Under sterile conditions, dilute the mother solution (0.6mL: 6mg = 10mg / mL) of the prefilled injection solution with 0.9% sodium chloride injection to the required concentration of 0.6mg / mL; store in a dry place at 2-8℃ away from light, and transport to the animal room under light-proof conditions;

[0668] 1.3P32-0 Configuration

[0669] Use an appropriate amount of sterile 5% glucose injection solution to dissolve 0.6 and 1.2 mg / mL P32-0, shake gently to fully dissolve and mix, filter and sterilize the sample for tail vein injection, and strictly protect from light during and after the preparation process.

[0670] 2.4 Cyclophosphamide preparation

[0671] Dissolve cyclophosphamide in an appropriate amount of sterile 0.9% sodium chloride injection, shake gently to fully dissolve and mix to 5 mg / mL. Store in a dry place at room temperature away from light.

[0672] 2.5 Experimental animals

[0673] SPF grade SD male rats weighed about 210-230g when purchased and about 280-290g when grouped.

[0674] 2 Experimental design and drug administration method

[0675] 2.1 Group design

[0676] Group design: There were 9 groups in the experiment, including normal control group, model control group, Plinabulin treatment group, G-CSF (sulfopegfilgrastim) 0.25mg / kg group, G-CSF (sulfopegfilgrastim) 0.5mg / kg group, P32-06mg / kg group, P32-012mg / kg group, P32-06mg / kg + G-CSF (sulfopegfilgrastim) 0.25mg / kg group, P32-012mg / kg + G-CSF (sulfopegfilgrastim) 0.5mg / kg group; each group had 6 male rats, a total of 54 rats;

[0677] Grouping method: Rats with uniform neutrophil levels before modeling were randomly divided into groups.

[0678] Table 37. Experimental animal grouping table

[0679]

[0680]

[0681] 2.2 Dosage design

[0682] After the experiment was divided into groups, except for the normal control group, the animals in the other groups were intraperitoneally injected with cyclophosphamide for modeling. The modeling dose was 50 mg / kg and the administration volume was 10 mL / kg.

[0683] 30 minutes after the administration of the modeling drug, the normal control group and the model control group were injected with 5% glucose solution at 10 mL / kg through the tail vein.

[0684] The Plinabulin treatment group was given a single dose of Plinabulin at 7.5 mg / kg via intravenous infusion over 20 minutes 30 minutes after model establishment.

[0685] The G-CSF 0.25 mg / kg group and the G-CSF 0.5 mg / kg group were given a single subcutaneous injection of PEG-rhG-CSF at a low dose of 0.25 mg / kg and a high dose of 0.5 mg / kg, respectively, 24 hours after model establishment.

[0686] The P32-06mg / kg group and the P32-012mg / kg group were given a single intravenous injection at 30 minutes after modeling, with a low dose of 6mg / kg and a high dose of 12mg / kg, respectively.

[0687] The P32-06mg / kg+G-CSF0.25mg / kg group was intravenously injected with P32-0 at 6mg / kg 30min after modeling, and PEG-rhG-CSF was subcutaneously injected once at 0.25mg / kg 24h after modeling.

[0688] In the P32-012mg / kg+G-CSF0.5mg / kg group, P32-0 was intravenously injected at 12mg / kg 30min after modeling, and PEG-rhG-CSF was subcutaneously injected once at 0.5mg / kg 24h after modeling.

[0689] Table 38. Dosage design table

[0690]

[0691] 3. Experimental results

[0692] 3.1 Weight

[0693] There was no difference in body weight between the model control group and the normal control group. Compared with the model control group, the body weight of the P32-012mg / kg group and the P32-012mg / kg+G-CSF0.5mg / kg group was significantly reduced on D3 and D5 (P≤0.05, P≤0.01). Compared with the G-CSF0.5mg / kg group, the body weight of the P32-012mg / kg+G-CSF0.5mg / kg group was significantly reduced on D3 (P≤0.01). There was no significant difference in body weight between the rats in the other groups and the model control group, and there was an increasing trend.

[0694] The statistical data of animal weight are shown in Table 39. The weight growth trend of each group is shown in Fig.16 .

[0695] Table 39 Statistical data of animal weight (g)

[0696]

[0697]

[0698] 3.2 Food intake

[0699] During the experiment, the food intake of the model control group and each drug-treated group decreased on D1, and the higher the dose of P32-0, the lower the food intake. Except for the P32-012mg / kg group and the P32-012mg / kg+G-CSF0.5mg / kg group on D2, the other groups basically resumed normal food intake. The P32-012mg / kg group and the P32-012mg / kg+G-CSF0.5mg / kg group resumed normal food intake on D6.

[0700] Table 40. Food intake of each group of animals (g / day)

[0701]

[0702] 3.3 Hematological indicators

[0703] 1) Neutrophil count (Neut, ×10 9 cells / L) results (Table 41 and Fig.17 )

[0704] On the 2nd to 8th day, the neutrophil count in the model control group was significantly lower than that in the normal control group (P≤0.01, P≤0.001); on the 9th to 11th day, the neutrophil count in the model control group was significantly higher than that in the normal control group (P≤0.05, P≤0.01). Overall, the neutrophil count first decreased, then increased, and then returned to normal levels after modeling.

[0705] As shown in Table 41 and Fig.17 As shown in A, compared with the model control group, the neutrophil count in the Plinabulin treatment group increased significantly on the 2nd day after administration (P≤0.05), the neutrophil count in the G-CSF0.25mg / kg group increased significantly on the 2nd to 7th and 11th days after administration (P≤0.05, P≤0.01), the neutrophil count in the P32-06mg / kg group increased significantly on the 2nd day after administration (P≤0.01), and the neutrophil count in the P32-06mg / kg+G-CSF0.25mg / kg group increased significantly on the 2nd to 9th days after administration (P≤0.05, P≤0.01). Compared with the P32-06mg / kg group, the neutrophil count in the P32-06mg / kg+G-CSF0.25mg / kg group increased significantly on the 3rd to 8th days after administration (P≤0.05, P≤0.01).

[0706] like Fig.17 As shown in B, compared with the model control group, the neutrophil count in the Plinabulin treatment group was significantly increased on the 2nd day after administration (P≤0.05), the neutrophil count in the G-CSF0.5mg / kg group was significantly increased on the 2nd to 8th and 12th days after administration (P≤0.05, P≤0.01, P≤0.001), the neutrophil count in the P32-012mg / kg group was significantly increased on the 2nd and 10th to 12th days after administration (P≤0.05, P≤0.01), and the neutrophil count in the P32-012mg / kg+G-CSF0.5mg / kg group was significantly increased on the 2nd to 9th and 11th to 12th days after administration (P≤0.05, P≤0.01, P≤0.001). Compared with the G-CSF0.5mg / kg group, the neutrophil count in the P32-012mg / kg+G-CSF0.5mg / kg group increased significantly on days 9 to 10 after administration (P≤0.05). Compared with the P32-012mg / kg group, the neutrophil count in the P32-012mg / kg+G-CSF0.5mg / kg group increased significantly on days 3 to 8 after administration (P≤0.05, P≤0.01).

[0707] The statistical data of animal neutrophils are shown in Appendix 41. The changes of neutrophils in each group are shown in Fig.17 .

[0708] Table 41 Animal neutrophil statistics (×10 9 cells / L)

[0709]

[0710] 6) White blood cell count (WBC, ×10 9 cells / L) results (Table 42 and Fig.18 )

[0711] From the 2nd to the 8th day, the white blood cell count in the model control group was significantly lower than that in the normal control group (P≤0.05, P≤0.01), and the overall trend was that the count first decreased during modeling and then slowly increased to the normal level.

[0712] like Fig.18As shown in A, compared with the model control group, the white blood cell count of the G-CSF0.25mg / kg group increased significantly on days 2-3 and 7-8 after administration (P≤0.05, P≤0.01), the white blood cell count of the P32-06mg / kg group decreased significantly on days 3, 5 and 14 after administration (P≤0.05, P≤0.01), and the white blood cell count of the P32-06mg / kg+G-CSF0.25mg / kg group increased significantly on days 2-3 and 7-9 after administration (P≤0.05, P≤0.01). Compared with the G-CSF0.25mg / kg group, the white blood cell count of the P32-06mg / kg+G-CSF0.25mg / kg group increased significantly on days 7-9 after administration (P≤0.05). Compared with the P32-06 mg / kg group, the white blood cell count in the P32-06 mg / kg+G-CSF 0.25 mg / kg group increased significantly from 3 to 9 days after administration (P≤0.05, P≤0.01, P≤0.001).

[0713] like Fig.18 As shown in B, compared with the model control group, the white blood cell count in the G-CSF0.5mg / kg group increased significantly on days 2 to 8 after administration (P≤0.05, P≤0.01, P≤0.001), the white blood cell count in the P32-012mg / kg group decreased significantly on days 3 and 5 after administration (P≤0.05, P≤0.01), and the white blood cell count in the P32-012mg / kg+G-CSF0.5mg / kg group increased significantly on days 7 to 12 after administration (P≤0.05, P≤0.001). Compared with the G-CSF0.5mg / kg group, the white blood cell count in the P32-012mg / kg+G-CSF0.5mg / kg group increased significantly on days 8 to 12 after administration (P≤0.05, P≤0.01), and the white blood cell count in the 3rd and 5th days decreased significantly (P≤0.01, P≤0.001). Compared with the P32-012 mg / kg group, the white blood cell count in the P32-012 mg / kg+G-CSF0.5 mg / kg group increased significantly from 5 to 12 days after administration (P≤0.05, P≤0.001).

[0714] The statistical data of animal leukocytes are shown in Table 42; the changes of leukocytes in each group are shown in Fig.18 .

[0715] Table 42 Animal leukocyte statistics (×10 9 cells / L)

[0716]

[0717] 3.4 Thymus weight and thymus coefficient

[0718] Compared with the model control group, the thymus weight and thymus index of the Plinabulin treatment group, G-CSF0.25mg / kg group, G-CSF0.5mg / kg group, and P32-012mg / kg+G-CSF0.5mg / kg group were significantly reduced (P≤0.05, P≤0.01, P≤0.001). Compared with the G-CSF 0.25mg / kg group, the thymus weight and thymus index of the P32-06mg / kg+G-CSF0.25mg / kg group were significantly increased (P≤0.05). Compared with the Plinabulin treatment group, the thymus weight and thymus index of the P32-06mg / kg group were significantly increased (P≤0.05). There was no significant difference in thymus weight and thymus index among the other groups.

[0719] The statistical data of animal thymus weight and thymus coefficient are shown in Table 43.

[0720] Table 43 Statistical data of animal thymus weight and thymus coefficient

[0721]

[0722] 5. Summary

[0723] Under the conditions of this experiment, after the model control group was modeled with 50 mg / kg cyclophosphamide intraperitoneally, there were no abnormalities in general clinical observations, and there were no significant changes in body weight and food intake. About one week after modeling, the neutrophil and white blood cell counts decreased significantly, indicating that the cyclophosphamide-induced neutropenia model was successfully established at this dose.

[0724] During the experiment, the neutrophil count in the low-dose and high-dose G-CSF groups, the low-dose and high-dose P32-0 groups, and the combination group of the two was significantly increased after administration, indicating that G-CSF, P32-0, and the combination group of G-CSF and P32-0 have a certain degree of preventive and therapeutic effect on neutropenia.

[0725] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof having anti-tumor efficacy, characterized in that: Its structural formula is shown in formula (I): Where: n=0-3; m=0-3; x=0-5; R 1 is a mono- or poly-substituted 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 methylthienyl; L is C1-C8 heteroalkylene, 2 Substituted C1-C8 heteroalkylene, C1-C8 alkylene or one or more R 2 Substituted C1-C8 alkylene; R 2 is C1-C8 alkyl or C3-C 10 Cycloalkyl; R 3 , R 4 , R 6 and R 7 is hydrogen, deuterium, C1-C8 alkyl, or one or more R 3-1 Substituted C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl, C1-C8 alkoxy, C3-C 10 Cycloalkyl, C6-C 10 Aryl or 3-10 membered heterocycloalkyl; wherein R 3-1 OR 3-1-1 , where R 3-1-1 is C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl or substituted by one or more C6-C 10 Aryl-substituted C1-C8 alkyl, C1-C8 alkenyl or C1-C8 alkynyl; Z is O, S, SO, SO2 or N(R 5 ); R 5 is hydrogen, deuterium, C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl, and is replaced by one or more R 5-1 Substituted C6-C 10 Aryl, benzyl, 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 is halogen; R 5-2 is hydrogen, C1-C8 alkoxy, benzyloxy, or 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 is C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl or C6-C 10 Aryl; wherein R 5-3-1 and R 5 -3-2 is hydrogen, C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl or C3-C 10 Cycloalkyl; R 5-4 is C1-C8 alkyl or C6-C 10 Aryl.

2. The 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof having anti-tumor efficacy according to claim 1, characterized in that: The heteroatoms in the C1-C8 heteroalkylene of L are selected from one or more of N, O, S and Se, and the number of heteroatoms is 1-4; 3 , R 4 , R 6 and R 7 The heteroatoms in the 3-10 membered heterocycloalkyl are selected from one or more of N, O and S, and the number of heteroatoms is 1-5.

3. The 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof having anti-tumor efficacy according to claim 2, characterized in that: In the structural formula: n=0-1; m=0-1; x=0-2; R 1 is a mono- or poly-substituted group selected from hydrogen, deuterium, vinyl, ethynyl, methyl, trifluoromethoxy, methoxy, halogen, cyano or benzoyl; L is R 2 is methyl, ethyl, isopropyl, tert-butyl or cyclopropyl; R 3 , R 4 , R 6 and R 7 is hydrogen, deuterium, methyl or ethyl, 3-1 substituted methyl, cyclohexyl, or pyridyl; wherein R 3-1 OR 3-1-1 , where R 3-1-1 is methyl, ethyl, n-propyl or n-butyl; Z is O, S, SO, SO2 or N(R 5 ); R 5 is hydrogen, deuterium, methyl, ethyl, n-propyl, isopropyl, n-butyl or 2-methylpropyl, C1-C8 alkenyl, C1-C8 alkynyl, and is replaced by one or more R 5-1 Substituted phenyl, benzyl, 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 is halogen; R 5-2 is hydrogen, C1-C8 alkoxy, benzyloxy, or 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 is C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl or C6-C 10 Aryl; wherein R 5-3-1 and R 5-3-2 is hydrogen, C1-C8 alkyl, C1-C8 alkenyl, C1-C8 alkynyl or C3-C6 cycloalkyl; R 5-4 is C1-C8 alkyl or C6-C 10 Aryl.

4. The 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof having anti-tumor efficacy according to claim 2, characterized in that: The specific structural formulas are:

5. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the 2,5-diketopiperazine compound with anti-tumor efficacy as claimed in claim 1 or its stereoisomer, tautomer, racemate, pharmaceutically acceptable salt, and pharmaceutically acceptable carriers and pharmaceutical excipients.

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

7. Use of the 2,5-diketopiperazine compound with anti-tumor efficacy or a pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 5 as a microtubule protein inhibitor for inhibiting microtubule protein.

8. Use of the 2,5-diketopiperazine compound with anti-tumor efficacy or a pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 5 in the preparation of a drug for treating tumors.

9. Use of the 2,5-diketopiperazine compound with anti-tumor efficacy or a pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 5 in combination with a chemotherapeutic agent in the preparation of a drug for treating tumors.

10. The use according to claim 9, characterized in that: The chemotherapeutic agent is docetaxel.

11. Use of the 2,5-diketopiperazine compound having anti-tumor efficacy or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for treating and / or preventing neutropenia.

12. The use according to claim 11, characterized in that: The neutropenia results from the administration of chemotherapy or from the administration of radiation therapy during the treatment of cancer.

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

14. The use according to claim 12 or 13, characterized in that: The chemotherapy is the administration of a single chemotherapeutic agent or the administration of a chemotherapeutic combination.

15. The use 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, vinblastine, doxorubicin and cyclophosphamide, or a combination of docetaxel and cyclophosphamide.

16. The use according to claim 11, characterized in that: The 2,5-diketopiperazine compound with anti-tumor efficacy or a pharmaceutically acceptable salt thereof is administered orally or by injection.

17. The use according to claim 16, characterized in that The 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof is administered alone during a chemotherapy cycle; or the 2,5-diketopiperazine compound or a pharmaceutically acceptable salt thereof is administered after administering the chemotherapeutic agent or chemotherapeutic composition.

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

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

20. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the 2,5-diketopiperazine compound with anti-tumor efficacy or a pharmaceutically acceptable salt thereof according to claim 1 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 2,5-diketopiperazine compound with anti-tumor efficacy or a pharmaceutically acceptable salt thereof.

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

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