An aurone compound and medical use thereof

Abstract

The application provides an aurone compound and a medical use thereof, and is an aurone compound and application thereof in preparation of prevention and treatment of myelosuppression or blood system diseases. The aurone compound provided by the application can significantly improve the myelosuppression state caused by ruxolitinib and radiotherapy, can restore the generation of red blood cells after irradiation and use of ruxolitinib, and has a significant effect on the recovery of white blood cells, especially neutrophils and lymphocytes, of mice after irradiation. The application provides a new potential drug for the treatment of myelosuppression or blood system diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of biomedicine and pharmacy, and relates to an aurone compound and a medical use thereof, and is an aurone compound and an application of the aurone compound in the preparation of prevention and treatment of myelosuppression or blood system diseases. BACKGROUND

[0002] Myelosuppression refers to the decrease in the activity of blood cell precursors in bone marrow, which can be caused by chemical factors, radiation factors, infection factors, blood system diseases, drug factors, etc. Myelosuppression is a common and serious side effect in cancer patients receiving radiotherapy. Irradiation can damage hematopoietic stem and progenitor cells in bone marrow, leading to a significant decrease in the production of red blood cells, white blood cells (especially neutrophils and lymphocytes), and platelets. This condition not only causes anemia, but also significantly reduces the immune function of patients, increases the risk of infection, bleeding, and tumor recurrence, and even threatens life in severe cases.

[0003] In addition to myelosuppression, there are many factors that can cause dysfunction of the hematopoietic system. Among them, drug-induced blood system diseases are common and relatively serious, including drug-induced anemia, drug-induced allergic purpura, drug-induced thrombocytopenia, drug-induced agranulocytosis, and drug-induced leukemia, etc. (Zhao Xianglan, Huang Zhumin, ed. Clinical Pharmacology. 2007: 149)

[0004] Ruxolitinib (CAS No. 941678-49-5) and other JAK2 inhibitors are widely used in the treatment of myeloproliferative diseases, but they are prone to cause adverse reactions such as drug-induced anemia during application. These drugs interfere with erythropoiesis by inhibiting the JAK2-STAT5 signaling pathway, leading to impaired function of bone marrow hematopoietic stem and progenitor cells, and then causing anemia.

[0005] The spleen, as a secondary hematopoietic organ, plays an important role in extramedullary hematopoiesis (EMH) when the function of bone marrow is impaired. When bone marrow hematopoiesis is impaired, organs such as the spleen can compensate for the generation of blood cells, alleviating the state of insufficient hematopoiesis. This process is particularly crucial in dealing with bone marrow damage and drug-induced hematopoietic suppression, and can restore blood cell production to some extent, maintaining the balance of hematopoiesis in the body.

[0006] Currently, the common treatment methods for bone marrow suppression or hematopoietic system dysfunction caused by radiotherapy, drugs and other factors include red blood cell transfusion, use of erythropoietin (EPO) and its derivatives, granulocyte colony-stimulating factor (G-CSF) and platelet transfusion. However, these methods have many limitations. For example, although red blood cell transfusion can temporarily relieve anemia symptoms, it needs frequent blood transfusion, which can easily cause transfusion syndrome and iron overload; G-CSF can promote the generation of neutrophils, but can cause side effects such as bone pain and splenomegaly, and its effect on restoring lymphocytes and immune function is limited; platelet transfusion also has similar risks and can increase the risk of blood transfusion-related infections. Therefore, there is an urgent need for an innovative therapeutic drug that can comprehensively restore red blood cell, white blood cell and platelet production, and at the same time enhance immune function. SUMMARY

[0007] The present application aims to provide an aurone compound, the chemical structure of which is shown as formula I:

[0008]

[0009] The present application provides a preparation method of the above-mentioned aurone compound of formula I, which is prepared by 6-hydroxy-2H-benzofuran-3-ketone and 2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxylic acid ethyl ester under the action of a base, preferably piperidine. The preparation method comprises the following steps: (1) equimolar amounts of 6-hydroxy-2H-benzofuran-3-ketone and 2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxylic acid ethyl ester are added to an organic solvent, preferably ethanol, and then 1.5-2.5 molar equivalents of piperidine, preferably 2.0 molar equivalents, are added; (2) reaction at room temperature for 10-22 hours; (3) the reaction liquid is filtered to obtain a solid, which is washed with an organic solvent and dried to obtain the aurone compound of formula I.

[0010] Another object of the present application is to provide the use of the aurone compound of formula I in the preparation of a drug for preventing or treating bone marrow suppression, wherein the pathogenic factors of the bone marrow suppression are preferably one or more of chemical factors, radiation factors, infection factors and drug factors, more preferably bone marrow suppression caused by radiation factors, including bone marrow suppression caused by whole body or local radiotherapy, further preferably bone marrow suppression caused by whole body radiotherapy, and more preferably one or more of anemia, granulocytopenia and thrombocytopenia caused by bone marrow suppression caused by whole body radiotherapy, and the irradiation intensity of the whole body radiotherapy is preferably 4Gy.

[0011] Still another object of the present application is to provide the use of the aurone compound of Formula I in the preparation of a medicament for preventing or treating a hematological disease, preferably a drug-induced hematological disease. The drug-induced hematological disease is preferably one or more of drug-induced anemia, drug-induced thrombocytopenia, and drug-induced agranulocytosis, the drug is preferably one or more of a chemotherapeutic drug, a targeted anti-tumor drug, an antibacterial drug, an immunosuppressant, and an antipsychotic drug, and the immunosuppressant is preferably a JAK2 inhibitor, further preferably ruxolitinib.

[0012] The present application also provides the use of a pharmaceutical composition in the preparation of a medicament for preventing or treating myelosuppression or a hematological disease, wherein the pharmaceutical composition comprises the aurone compound of claim 1 and a pharmaceutically acceptable excipient, and the pharmaceutical composition is in the form of one or more of a capsule, a tablet, an oral suspension, an injection, and a lyophilized powder.

[0013] The aurone compound HT5002 provided by the present application has a chemical structure significantly different from that of the existing drugs for preventing or treating myelosuppression or a hematological disease, and has a significant anti-myelosuppression effect in a mouse in vivo model. In a mouse model of radiation-induced myelosuppression, HT5002 can significantly increase the number of red blood cells, white blood cells (including neutrophils and lymphocytes), and platelets in the peripheral blood. In addition, HT5002 shows strong efficacy in treating a ruxolitinib-induced drug-induced anemia model, can promote the proliferation of bone marrow hematopoietic stem and progenitor cells, and significantly improve anemia symptoms caused by JAK2 inhibitors.

[0014] Currently, there is still a lack of effective drugs in clinical practice that can simultaneously activate bone marrow hematopoiesis and spleen stress hematopoiesis to comprehensively restore the production of red blood cells, white blood cells, and platelets. The aurone compound (structure shown in Formula I, numbered as HT5002) provided by the present application provides a new possibility for the development of such drugs, which not only restores the hematopoietic function of the bone marrow, but also relieves radiation- and drug-induced myelosuppression through the spleen stress hematopoiesis pathway, and has important clinical application prospects.

[0015] Compared with existing treatment drugs (such as amifostine, erythropoietin, and red blood cell transfusion), HT5002 can not only comprehensively restore the production of multiple blood cells, but also effectively relieve anemia caused by myelosuppression through the mechanism of spleen stress hematopoiesis, and the mechanism of action is significantly different from that of the prior art. The technical solution of the present application is expected to provide a drug with a new mechanism of action for preventing or treating myelosuppression or a hematological disease. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1The therapeutic effect of HT5002 on the irradiation-induced myelosuppression mouse model is shown. The figure shows the effect of HT5002 on the number of white blood cells (WBC), neutrophils (NEUT), lymphocytes (LYMPH), red blood cells (RBC), hemoglobin (Hb), hematocrit (HCT), and platelets (PLT) in peripheral blood. The results show that HT5002 significantly improves the generation of these cells in the irradiation-induced myelosuppression model, and exhibits more excellent effect than the positive drug amifostine.

[0017] Figure 2 The effect of HT5002 in the semi-in vitro colony proliferation experiment is shown. The figure shows the promoting effect of HT5002 on the number of colony formation and colony size of spleen hematopoietic stem and progenitor cells, and also has a promoting trend on the formation of bone marrow hematopoietic stem and progenitor cell colonies, indicating that HT5002 can play a protective role through the spleen stress hematopoietic pathway and bone marrow hematopoiesis.

[0018] Figure 3 The therapeutic effect of HT5002 on the ruxolitinib-induced drug-induced anemia mouse model is shown. The figure shows that HT5002 significantly up-regulates hemoglobin (Hb), red blood cell count (RBC), and hematocrit (HCT) in ruxolitinib-induced anemia mice, and can significantly promote the proliferation of bone marrow hematopoietic stem and progenitor cells (Lin - cKit + Sca1 + ) in ruxolitinib-induced anemia mice, indicating its promoting effect on bone marrow hematopoiesis. DETAILED DESCRIPTION

[0019] The present application will be further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present application and are not used to limit the scope of the present application. The materials, reagents, instruments and methods used in the examples are generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer, and are commercially available unless otherwise specified. Statistical analysis was performed using GraphPad Prism 8.0.1 software, and data were expressed as X ± SD. t-tests were used to analyze the differences between two groups, and One-way ANOVA was used for variance analysis of multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001.

[0020] Example 1: Synthesis of HT5002

[0021]

[0022] To a mixture of 6-hydroxy-2H-benzofuran-3-one (0.90 g, 6.0 mmol) and 2,4-dimethyl-5-formyl-lH-pyrrole-3-carboxylic acid ethyl ester (1.17 g, 6.0 mmol) in anhydrous EtOH (18 ml) was added piperidine (1.02 g, 12.0 mmol). The mixture was stirred at room temperature for 16 hours. After the reaction was complete, a large amount of precipitate was formed. The solid was retained by suction filtration and washed with a small amount of anhydrous EtOH, dried to give an orange-yellow solid.

[0023] Yield: 42.6%; ESI-MS: m / z = 328 [M+H] + ; 1 H NMR (400 MHz, DMSO-d6) δ 11.03 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 2.0 Hz, 1H), 6.71 - 6.65 (m, 2H), 4.19 (q, J = 7.1 Hz, 2H), 2.57 (s, 3H), 2.33 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H).

[0024] Example 2: Therapeutic effect of HT5002 on irradiation-induced myelosuppression mouse model

[0025] Test materials: C57BL / 6 female mice (8 weeks old) were purchased from Zhejiang Vantoll Life Experimental Animal Technology Co., Ltd. HT5002 was synthesized by the method of Example 1; the positive drug amifostine was purchased from Sigma-Aldrich.

[0026] Experimental method: The mice were randomly divided into control group, irradiation group and treatment group. The control group was given blank solvent, the irradiation group was given a sublethal dose of 4 Gy whole body radiation to establish a myelosuppression model, and the treatment group was injected intraperitoneally with 100 mg / kg of HT5002 or the positive drug amifostine 30 minutes before irradiation. On the 12th day, the eye blood samples of the mice were collected, and the blood routine indexes were detected using a full-automatic blood analyzer.

[0027] Experimental results: HT5002 significantly increased the number of white blood cells (WBC), neutrophils (NEUT) and lymphocytes (LYMPH) in mice after irradiation (IR), and the effect was better than that of the positive control amifostine (A). In addition, HT5002 also significantly increased red blood cell count (RBC), hemoglobin (Hb), hematocrit (HCT) and platelet count (PLT), and the effect was comparable to that of the positive control amifostine (B), indicating that HT5002 had a significant alleviating effect on irradiation-induced myelosuppression and anemia. Figure 1 Figure 1

[0028] ​​Example 3: HT5002 promotes colony formation of splenic and bone marrow hematopoietic progenitor cells

[0029] Test materials: C57BL / 6 female mice (8 weeks old) were purchased from Zhejiang Vantoll Life Experimental Animal Technology Co., Ltd. Hematopoietic progenitor cell sorting reagents were purchased from BioLegend. Colony culture medium was purchased from STEMCELL.

[0030] Experimental method: Mice were sacrificed 24 hours after receiving 4Gy whole-body radiation, and their bone marrow and spleen were collected to prepare single-cell suspensions. MojoSort TM Mouse Hematopoietic Progenitor Cell Isolation Kit was used to sort hematopoietic progenitor cells in the spleen and bone marrow, respectively. The sorted cells were seeded at a density of 150,000 cells / mL in MethoCult TM M3434 semi-solid medium for colony formation and proliferation experiments. In the experiment, red cell colony-forming units (Colony-Forming Unit-Erythroid, CFU-E) were counted on day 3, and red cell burst-forming units (Burst-Forming Unit-Erythroid, BFU-E) were counted on day 7, and the number of colonies was recorded. The counts at these two time points reflect the proliferation of different stages of red blood cell precursors in the hematopoietic process. CFU-E mainly represents the proliferation ability of mature red blood cell precursors, while BFU-E represents the proliferation and differentiation potential of early red cell progenitor cells.

[0031] Experimental results: According to the results of Figure 2 HT5002 significantly promoted the formation of red cell burst-forming units (BFU-E) colonies in splenic hematopoietic progenitor cells, and also promoted the formation of red cell colony-forming units (CFU-E) in splenic hematopoietic progenitor cells Figure 2 A, C). In addition, HT5002 showed a certain promotion trend for bone marrow hematopoietic progenitor cell colony formation Figure 2 B, C). These results indicate that HT5002 has a dual role in promoting hematopoietic function, which can simultaneously enhance the proliferation of red blood cell precursors in the splenic stress hematopoietic pathway and the proliferation of bone marrow hematopoietic progenitor cells, and has a broad hematopoietic support potential.

[0032] Example 4: Therapeutic effect of HT5002 on a mouse model of drug-induced anemia induced by Ruxolitinib

[0033] Test materials: C57BL / 6 female mice (8 weeks old) were purchased from Zhejiang Vantoll Life Experimental Animal Technology Co., Ltd. HT5002 was synthesized by the method of Example 1; Ruxolitinib was purchased from Wuhan Costar Biological Technology Co., Ltd.

[0034] Experimental method: Mice were randomly divided into control group, ruxolitinib modeling group and treatment group. The control group was given blank solvent, the modeling group of mice was given ruxolitinib (200 mg / kg, bid) intraperitoneally twice a day for 20 days to establish a drug-induced anemia model, and the treatment group of mice was given HT5002 50 mg / kg intraperitoneally once a day. On day 21, the eyeball blood samples of the mice were collected, and the blood routine indexes were detected by using an automatic blood analyzer; and the bone marrow was obtained from the femur and tibia of the mice, and a single cell suspension was prepared by flushing the bone marrow cavity with PBS. Then, the cells were resuspended in flow cytometry buffer and 3% BSA was added, and incubated at 4°C for 30 minutes to block non-specific binding. Next, specific fluorescent antibodies were added for staining, including Lineage (Lin) antibody mixture (such as anti-CD3, CD4, CD8, B220, Gr-1, Ter119, etc., used to exclude mature cells in bone marrow), c-Kit (CD117) antibody and Sca-1 antibody, to label hematopoietic stem / progenitor cells. After mixing the antibodies with the cells, incubate at 4°C for 30 minutes in the dark. After staining, wash the cells with cold PBS twice and resuspend in flow cytometry buffer. After staining is complete, use a BD flow cytometer to collect data, determine the proportion and number of Lin + c-Kit + and Sca-1 + cells by gating Lin + Sca-1 + (LKS) hematopoietic stem / progenitor cells, and finally use flow cytometry analysis software (such as FlowJo) to analyze the data and evaluate the changes in the proportion of LKS cells in each experimental group.

[0035] Experimental results: HT5002 significantly increased the red blood cell count (RBC), hemoglobin (Hb) level and hematocrit (HCT) of ruxolitinib (Ru)-induced anemic mice, indicating that it has significant efficacy in alleviating drug-induced anemia symptoms Figure 3 A). In addition, HT5002 significantly promoted the proliferation of bone marrow hematopoietic stem / progenitor cells in vivo, revealing its potential mechanism for improving hematopoietic function Figure 3 B). The above data show that HT5002 can effectively alleviate ruxolitinib-induced drug-induced anemia and has potential clinical application value.

Claims

1. An aurone compound, characterized by, The chemical structure of the aurone compound is shown in formula I: 。 2. Use of the aurone compound according to claim 1 for the manufacture of a medicament for preventing or treating myelosuppression, characterized in that, The myelosuppression refers to radiation-induced myelosuppression, including whole body or local radiotherapy-induced myelosuppression.

3. Use of the aurone compound according to claim 1 for the manufacture of a medicament for the prevention or treatment of hematological diseases, characterized in that, The hematological disease refers to drug-mediated hematological disease, which refers to drug-induced anemia, and the drug is JAK2 inhibitor ruxolitinib.

4. Use of a pharmaceutical composition for the preparation of a medicament for the prevention or treatment of radiation-induced myelosuppression or drug-induced anemia caused by ruxolitinib, characterized in that, The pharmaceutical composition consists of the aurone compound of claim 1 and pharmaceutically acceptable excipients, and contains excipients, and the pharmaceutical composition and excipients are in the form of one or more of capsules, tablets, oral suspensions, injection solutions, and freeze-dried powder injections.

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