Aurone compound and medical application thereof

Abstract

The invention provides an aurone compound and medical application thereof, and relates to an aurone compound and application thereof in preparation of drugs for preventing and treating myelosuppression or blood system diseases. The aurone compound provided by the invention can significantly improve the myelosuppression state caused by rusotinib and radiotherapy, can recover the generation of erythrocytes after mouse irradiation and use of rusotinib, and has a significant effect on the recovery of leukocytes, especially neutrophils and lymphocytes, of the irradiated mouse. The invention provides a new potential drug for treatment of myelosuppression or blood system diseases.

Description

Technical Field

[0001] The invention relates to the fields of biomedicine and pharmacy, and relates to an aurone compound and its medical use, and is an aurone compound and its use in the preparation and prevention of bone marrow suppression or blood system diseases. Background Art

[0002] Myelosuppression refers to the decrease in the activity of blood cell precursors in the 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 cells in the bone marrow, leading to a significant decrease in the production of red blood cells, white blood cells (especially neutrophils and lymphocytes) and platelets. This state not only causes anemia, but also significantly reduces the patient's immune function, increases the risk of infection, bleeding and tumor recurrence, and even threatens life in severe cases.

[0003] In addition to bone marrow suppression, there are many other factors that can lead to hematopoietic system dysfunction. Among them, drug-induced blood system diseases are relatively common and serious, including drug-induced anemia, drug-induced purpura, drug-induced thrombocytopenia, drug-induced agranulocytosis and drug-induced leukemia. (Zhao Xianglan, Huang Minzhu, ed. Clinical Pharmacology. 2007:149)

[0004] JAK2 inhibitors such as ruxolitinib (CAS No. 941678-49-5) are widely used to treat myeloproliferative diseases, but adverse reactions such as drug-induced anemia are prone to occur during their use. This type of drug inhibits the JAK2-STAT5 signaling pathway, interferes with erythropoiesis, causes damage to the function of bone marrow hematopoietic stem and progenitor cells, and then causes anemia.

[0005] As a secondary hematopoietic organ, the spleen can play an important role through extramedullary hematopoiesis (EMH) when bone marrow function is impaired. When bone marrow hematopoiesis is impaired, organs such as the spleen can compensatory produce blood cells to alleviate the state of insufficient hematopoiesis. This process is particularly critical in dealing with bone marrow damage and drug-induced hematopoietic inhibition, and can restore blood cell production to a certain extent and maintain the body's hematopoietic balance.

[0006] At present, common treatments for bone marrow suppression or hematopoietic system dysfunction caused by various factors such as radiotherapy and drugs include red blood cell transfusion, the 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 requires frequent transfusions and is prone to problems such as transfusion syndrome and iron overload; G-CSF can promote the production of neutrophils, but may 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 may increase the risk of transfusion-related infection. Therefore, there is an urgent need for an innovative therapeutic drug that can fully restore the production of red blood cells, white blood cells and platelets and enhance immune function at the same time. Summary of the invention

[0008] The object of the present invention is to provide an aurone compound, the chemical structure of which is shown in Formula I:

[0009] The present invention provides a method for preparing the orange ketone compound of the above formula I, that is, preparing the compound by reacting 6-hydroxy-2H-benzofuran-3-one and 2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxylic acid ethyl ester under the action of a base, wherein the base is preferably piperidine. The steps of the preparation method are as follows: (1) adding 6-hydroxy-2H-benzofuran-3-one and 2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxylic acid ethyl ester in equal molar equivalents to an organic solvent, wherein the organic solvent is preferably ethanol, and then adding 1.5 to 2.5 molar equivalents of piperidine, preferably 2.0 molar equivalents; (2) reacting at room temperature for 10 to 22 hours; (3) filtering the reaction solution to obtain a solid, washing with an organic solvent, and drying to obtain the orange ketone compound of formula I.

[0010] Another object of the present invention is to provide the use of the aurone compound described in 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 further 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] Another object of the present invention is to provide an application of aurone compounds of formula I in the preparation of drugs for preventing or treating blood system diseases, wherein the blood system diseases are preferably blood system diseases caused by drugs. Among the blood system diseases induced by drug mediation, the mediated drugs are preferably selected from one or more of chemotherapeutic drugs, targeted anti-tumor drugs, antibacterial drugs, immunosuppressants, antipsychotic drugs, and antithyroid drugs, and the blood system diseases induced by drug mediation are preferably one or more of drug-induced anemia, drug-induced thrombocytopenia, and drug-induced agranulocytosis, and the immunosuppressant is preferably a JAK2 inhibitor, and more preferably ruxolitinib.

[0012] The present invention also provides an application of a pharmaceutical composition in the preparation of a method for preventing and treating bone marrow suppression or blood system diseases, wherein the pharmaceutical composition is composed of the aurone compound according to claim 1 and a pharmaceutically acceptable excipient, and contains an excipient, and the pharmaceutical dosage form composed of the pharmaceutical composition and the excipient is preferably one or more of a capsule, a tablet, an oral suspension, an injection, and a lyophilized powder injection.

[0013] The chemical structure of the orange ketone compound HT5002 provided by the present invention is significantly different from the existing drugs for preventing and treating bone marrow suppression or blood system diseases, and has a significant anti-bone marrow suppression effect in a mouse model. In the bone marrow suppression mouse model induced by irradiation, HT5002 can significantly increase the number of red blood cells, white blood cells (including neutrophils and lymphocytes) and platelets in peripheral blood. In addition, HT5002 shows strong efficacy in the treatment of drug-induced anemia model induced by ruxolitinib, which can promote the proliferation of bone marrow hematopoietic stem and progenitor cells and significantly improve the anemia symptoms caused by JAK2 inhibitors.

[0007] At present, there is still a lack of effective drugs in clinical practice that can simultaneously activate bone marrow hematopoiesis and spleen stress hematopoiesis to fully restore the production of red blood cells, white blood cells and platelets. The aurone compound (structure shown in Formula I, numbered HT5002) provided by the present invention provides new possibilities for the development of such drugs. It can not only restore the hematopoietic function of the bone marrow, but also alleviate the bone marrow suppression caused by radiation and drugs through the spleen stress hematopoietic pathway, and has important clinical application prospects.

[0014] Compared with existing therapeutic drugs (such as amifostine, erythropoietin and red blood cell transfusion), HT5002 can not only fully restore the production of multiple blood cells, but also effectively alleviate anemia caused by bone marrow suppression through the mechanism of spleen stress hematopoiesis. Its mechanism of action is significantly different from that of the existing technology. The technical solution of the present invention is expected to provide a drug with a new mechanism of action for the prevention and treatment of bone marrow suppression or blood system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1The figure shows the therapeutic effect of HT5002 on the irradiation-induced bone marrow suppression mouse model. 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 showed that in the irradiation-induced bone marrow suppression model, HT5002 significantly improved the generation of these cells and showed a better effect than the positive drug amifostine.

[0016] Figure 2 The effect of HT5002 in the semi-in vitro colony proliferation experiment is shown. The figure shows that HT5002 promotes the colony formation number and colony size of spleen hematopoietic stem and progenitor cells, and also has a tendency to promote 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.

[0017] Figure 3 The figure shows the therapeutic effect of HT5002 on the drug-induced anemia mouse model induced by ruxolitinib. The figure shows that HT5002 significantly up-regulated the hemoglobin (Hb), red blood cell count (RBC) and hematocrit (HCT) of ruxolitinib-induced anemia mice, and significantly promoted the expression of bone marrow hematopoietic stem and progenitor cells (Lin - cKit + Sca1 + ) proliferation, indicating its promoting effect on bone marrow hematopoietic function. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The materials, reagents, instruments and methods used in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer, and can be obtained through commercial channels without special instructions. GraphPad Prism 8.0.1 software was used for statistical analysis, and the data were expressed as X±SD. The t-tests test was used to analyze the differences between the two groups, and One-way ANOVA was used for variance analysis of multiple groups of data. *P<0.05, **P<0.01, ***P<0.001.

[0019] Example 1: Synthesis of HT5002

[0020] To a mixture of 6-hydroxy-2H-benzofuran-3-one (0.90 g, 6.0 mmol) and ethyl 2,4-dimethyl-5-formyl-1H-pyrrole-3-carboxylate (1.17 g, 6.0 mmol) in anhydrous EtOH (18 ml), piperidine (1.02 g, 12.0 mmol) was added. The mixture was stirred at room temperature for 16 hours. After sufficient reaction, a large amount of precipitate was generated. The solid was retained by suction filtration, washed with a small amount of anhydrous EtOH, and dried to obtain an orange-yellow solid.

[0021] Yield: 42.6%; ESI-MS: m / z=328 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ11.03(s,1H),7.57(d,J=8.4Hz,1H),6.82(d,J=2.0Hz,1H),6.7 1–6.65(m,2H),4.19(q,J=7.1Hz,2H),2.57(s,3H),2.33(s,3H),1.29(t,J=7.1Hz,3H).

[0022] Example 2: Therapeutic effect of HT5002 on irradiation-induced myelosuppression mouse model

[0023] Test materials: C57BL / 6 female mice (8 weeks old), purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. HT5002 was synthesized by the method of Example 1; the positive drug amifostine was purchased from Sigma-Aldrich.

[0024] Experimental methods: Mice were randomly divided into a control group, an irradiation group, and a treatment group. The control group was given a blank solvent, and the irradiation group was given a half-lethal dose of 4Gy whole-body radiation to establish a bone marrow suppression model. The treatment group was intraperitoneally injected with 100mg / kg of HT5002 or positive drug amifostine 30 minutes before irradiation. On the 12th day, blood samples were collected from the eyeballs of the mice, and blood routine indicators were detected using a fully automatic blood analyzer.

[0025] 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 ( Figure 1 A). In addition, HT5002 can also significantly increase red blood cell count (RBC), hemoglobin (Hb), hematocrit (HCT) and platelet count (PLT), and the effect is comparable to that of the positive control amifostine ( Figure 1 B), indicating that HT5002 has a significant alleviating effect on irradiation-induced bone marrow suppression and anemia.

[0026] Example 3: Experiment on the promotion of colony formation of spleen hematopoietic stem and progenitor cells and bone marrow hematopoietic stem and progenitor cells by HT5002

[0027] Experimental materials: C57BL / 6 female mice (8 weeks old) were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.; hematopoietic stem and progenitor cell sorting reagents were purchased from BioLegend; colony culture medium was purchased from STEMCELL.

[0028] Experimental method: Mice were irradiated with 4 Gy whole body radiation and then sacrificed 24 hours later. Bone marrow and spleen were collected and single cell suspension was prepared. TM Mouse Hematopoietic Progenitor Cell Isolation Kit was used to isolate hematopoietic stem and progenitor cells from spleen and bone marrow. The isolated cells were inoculated into MethoCult TM M3434 semi-solid medium was used for colony formation and proliferation experiments. In the experiment, colony-forming units (Colony-Forming Unit-Erythroid, CFU-E) were counted on the 3rd day, and burst-forming units (BFU-E) were counted on the 7th day, and the number of colonies was recorded. The counts at these two time points respectively reflect the proliferation of erythroid precursors at different stages in the hematopoietic process. CFU-E mainly characterizes the proliferation ability of mature erythroid precursors, while BFU-E represents the proliferation and differentiation potential of early erythroid progenitor cells.

[0029] Experimental results: According to Figure 2 The results showed that HT5002 significantly promoted the formation of erythroid burst-forming unit-E (BFU-E) colonies in spleen hematopoietic stem and progenitor cells, and also promoted the formation of erythroid colony-forming unit-E (CFU-E). Figure 2 A, C). In addition, HT5002 showed a certain promotion trend on the colony formation of bone marrow hematopoietic stem and progenitor cells ( Figure 2 B, C). These results indicate that HT5002 has a dual role in promoting hematopoiesis, which can simultaneously enhance the proliferation of erythroid precursors in the spleen stress hematopoietic pathway and the proliferation of bone marrow hematopoietic stem and progenitor cells, and has a broad potential for hematopoietic support.

[0030] Example 4: Therapeutic effect of HT5002 on the drug-induced anemia mouse model induced by ruxolitinib

[0031] Test materials: C57BL / 6 female mice (8 weeks old), purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. HT5002 was synthesized by the method of Example 1; Ruxolitinib was purchased from Wuhan Costan Biotechnology Co., Ltd.

[0032] Experimental methods: Mice were randomly divided into a control group, a ruxolitinib modeling group, and a treatment group. The control group was given a blank solvent, and the modeling group mice were intraperitoneally injected with ruxolitinib (200 mg / kg, bid) twice a day for 20 consecutive days to establish a drug-induced anemia model. The treatment group mice were also intraperitoneally injected with 50 mg / kg of HT5002 once a day. On the 21st day, blood samples were collected from the mice's eyes, and blood routine indicators were tested using an automatic blood analyzer; bone marrow was obtained from the femur and tibia of the mice, and single cell suspensions were prepared by flushing the bone marrow cavity with PBS. Subsequently, the cells were resuspended in flow cytometry buffer, and 3% BSA was added and incubated at 4°C for 30 minutes to block nonspecific binding. Next, specific fluorescent antibodies are 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 the bone marrow), c-Kit (CD117) antibody and Sca-1 antibody to mark hematopoietic stem and progenitor cells. After mixing the antibodies with the cells, incubate them at 4°C in the dark for 30 minutes. After staining, wash the cells twice with cold PBS and resuspend them in flow cytometry buffer. After staining, use a BD flow cytometer for data acquisition, and exclude Lin + c-Kit + and Sca-1 + Cell gating to identify Lin-c-Kit + Sca-1 + The proportion and number of hematopoietic stem and progenitor cells (LKS) were analyzed by flow cytometry analysis software (such as FlowJo) to evaluate the changes in the proportion of LKS cells in each experimental group.

[0033] Experimental results: HT5002 significantly increased the red blood cell count (RBC), hemoglobin (Hb) level and hematocrit (HCT) of ruxolitinib (Ru)-induced anemia 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 and progenitor cells in vivo, revealing its potential mechanism in improving hematopoietic function ( Figure 3 B). The above data show that HT5002 can effectively alleviate drug-induced anemia caused by ruxolitinib and has potential clinical application value.

Claims

1. An aurone compound, characterized in that: The chemical structure of the aurone compound is shown in Formula I:

2. Use of the aurone compound according to claim 1 in the preparation of a drug for preventing or treating myelosuppression, wherein the pathogenic factors of myelosuppression are selected from one or more of chemical factors, radiation factors, infection factors, and drug factors.

3. Use of the aurone compound according to claim 1 in the preparation of a drug for preventing or treating a blood system disease, wherein the blood system disease refers to a blood system disease induced by drug mediation.

4. The use according to claim 2, characterized in that: The bone marrow suppression refers to bone marrow suppression caused by radiation factors, including bone marrow suppression caused by systemic or local radiotherapy.

5. The use according to claim 3, characterized in that: The drug-mediated blood system disease may include one or more of chemotherapy drugs, targeted anti-tumor drugs, antibacterial drugs, immunosuppressants, antipsychotic drugs, and antithyroid drugs. The drug-mediated blood system disease may include one or more of drug-induced anemia, drug-induced thrombocytopenia, and drug-induced granulocytosis.

6. The use according to claim 5, wherein the immunosuppressant is the JAK2 inhibitor ruxolitinib.

7. Use of a pharmaceutical composition for the preparation of a drug for preventing and treating bone marrow suppression or blood system diseases, characterized in that: The pharmaceutical composition is composed of the aurone compound according to claim 1 and pharmaceutically acceptable excipients, and contains excipients. The pharmaceutical dosage form of the pharmaceutical composition and excipients is one or more of capsules, tablets, oral suspensions, injections, and lyophilized powder injections.

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