Application of Dipheylarazine A in preparation of antithrombotic drugs
By developing an antithrombotic drug using Diphenylalazine A, the shortcomings of existing cardiovascular disease treatments in angiogenesis and thrombus inhibition have been addressed, achieving safe and effective angiogenesis and antithrombotic effects.
Patent Information
- Application Number
- CN202511522717.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-23
- Publication Date
- 2025-11-21
AI Technical Summary
Existing cardiovascular disease treatments are insufficient in promoting angiogenesis and inhibiting thrombus formation, especially lacking effective means to inhibit platelet activation and aggregation.
Using Diphenylalazine A as the active ingredient, an antithrombotic drug was developed to promote angiogenesis and inhibit thrombus formation. Its safety and efficacy were verified using a zebrafish model.
Diphenylalazine A exhibits no developmental toxicity to zebrafish within safe concentration ranges. It can effectively reverse vascular damage, promote angiogenesis, and significantly inhibit thrombus formation, thus possessing broad market application prospects.
Smart Images
Figure CN120983437A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical technology, specifically the application of Diphenylalazine A in the preparation of antithrombotic drugs. Background Technology
[0002] Cardiovascular disease (CVD) is a major disease that seriously affects the health of the Chinese population. Current evidence suggests that its occurrence is closely related to insufficient angiogenesis. Angiogenesis refers to the process by which differentiated vascular endothelial cells form new capillaries at the site of existing blood vessels. Imbalance in the regulation of this process can lead to cardiovascular disease. Currently, my country's population is aging rapidly, and the incidence of cardiovascular disease is rising accordingly. Drugs that promote angiogenesis hold promise for increasing the length and number of blood vessels and improving vascular function, thereby alleviating cardiovascular disease. For example, "Diagnosis and Treatment of Lower Limb Ischemic Diseases" points out that the goal of treating lower limb ischemic diseases is to improve limb ischemia and salvage the limbs. The treatment mechanism is vascular recanalization or increased collateral circulation and vascular regeneration. Currently, the main drugs used clinically are various drugs that promote vasodilation and collateral circulation formation, such as prostacyclin drugs like acetaminophen and styrazides. "Vascular Diseases" points out that in gene therapy for lower limb arteriosclerosis obliterans, vascular endothelial growth factor (VEGF) induces angiogenesis, which can promote the establishment of collateral circulation, increase the ankle-brachial ratio, reduce rest pain, and promote the healing of ischemic ulcers. "912 Questions for Attending Physicians in Vascular Surgery" states that the main principles of drug treatment for arteriosclerosis obliterans include reducing blood viscosity, inhibiting thrombin activity, inhibiting platelet aggregation, relieving vasospasm, promoting collateral formation, and improving microcirculation and blood flow. Therefore, the development of drugs that promote angiogenesis is of crucial significance for the prevention and treatment of cardiovascular diseases. In recent years, thrombotic diseases have become one of the most concerning chronic diseases worldwide. Platelet hyperactivation plays a crucial role in the development and progression of thrombotic diseases. Activated platelets promote adhesion and aggregation, forming platelet-rich thrombi that obstruct arteries, impairing arterial blood flow and thus triggering thrombotic disorders. Therefore, inhibiting platelet activation and aggregation is a primary means of preventing and treating thrombosis.
[0003] Natural product active ingredients generally refer to compounds with unique biological functions extracted from animals, plants, and microorganisms. They can serve as a source for drug development, and approximately 60% of approved drugs are natural products or their derivatives. Compound Diphenylalazine A is a piperazine dione compound isolated from the fungus that parasitizes Cordyceps. Cordyceps -Colonizing Fungus Epicoccum nigrumIn recent years, researchers have also studied other fungi, such as... Epicoccum nigrum The compound (Cytotoxic Thiodiketopiperazine Derivatives from the Deep Sea-Derived Fungus) was isolated from SD-388. Epicoccum nigrum Zebrafish (SD-388) are small in size, have a high reproductive capacity, develop quickly, are easy to observe, and exhibit high homology with humans, which gives them unique advantages in drug development. This invention aims to use zebrafish as a disease model to explore the application of Diphenylalazine A in cardiovascular diseases. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides the application of Diphenylalazine A in the preparation of antithrombotic drugs.
[0005] The technical solution of this invention is as follows: The application of Diphenylalazine A in the preparation of antithrombotic drugs, wherein the chemical structural formula of Diphenylalazine A is shown in Formula I:
[0006] Formula I.
[0007] The application of Diphenylalazine A in the preparation of drugs for treating cardiovascular diseases, wherein the cardiovascular disease is lower limb ischemic disease and arteriosclerosis obliterans.
[0008] Preferably, the arteriosclerosis obliterans is lower extremity arteriosclerosis obliterans.
[0009] An antithrombotic drug composition, with Diphenylalazine A as the active ingredient.
[0010] A pharmaceutical composition for treating cardiovascular diseases, wherein Diphenylalazine A is the active ingredient, and the cardiovascular disease is one of lower extremity ischemic disease and arteriosclerosis obliterans.
[0011] Preferably, the arteriosclerosis obliterans is lower extremity arteriosclerosis obliterans.
[0012] Preferably, the pharmaceutical composition contains one or more pharmaceutically acceptable carriers and / or excipients.
[0013] More preferably, the excipient is one or more of the following: a sustained-release agent, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, and a lubricant.
[0014] Beneficial effects: This invention has found that Diphenylalazine A can reverse PTK787-induced vascular damage and arachidonic acid-induced thrombosis in zebrafish, exhibiting angiogenesis-promoting and antithrombotic activities. It can provide candidates for the development of drugs that promote angiogenesis to treat cardiovascular diseases and antithrombotic drugs, and has broad market application prospects. Attached Figure Description
[0015] Figure 1 The test results are for the safety evaluation of Diphenylalazine A. Figure 2 Images of intersegmental vessels in zebrafish after treatment with Diphenylalazine A; Figure 3 The results show the statistical results of intersegmental vessel length in zebrafish treated with Diphenylalazine A. Figure 4 Images were acquired of the heart region of zebrafish after treatment with Diphenylalazine A. Figure 5 The statistical results show the staining area of zebrafish hearts after treatment with Diphenylalazine A. Detailed Implementation
[0016] The following description is based on specific embodiments: The preparation method of Diphenylalazine A was based on the preparation of compound 5a in the literature "Synthesis of Monodehydro-Diketopiperazines Enabled by Cp*Rh(III)-Catalyzed Amine-Directed N−HFunctionalization" (Jenna C. Molas, Emily M. Poag, and Jonathan A. Ellman).
[0017] The zebrafish embryo culture water consisted of the following components: CaCl2 0.4 mM, NaCl 5 mM, MgSO4 0.16 mM, KCl 0.17 mM, and deionized water.
[0018] Example 1: Safety evaluation of Diphenylalazine A 1. Zebrafish sample pretreatment: Healthy, sexually mature AB-type zebrafish were placed in a mating tank at a 1:1 female-to-male ratio, with a partition between them. The tank was kept in darkness. The partition was removed the next day before the lights were turned on, and light stimulation was applied to induce ovulation. The ovulation time was controlled within half an hour to minimize the difference in development time between embryos. Half an hour after ovulation, the adult fish were removed, and the fertilized eggs were collected, disinfected, and cleaned. The fertilized eggs were then transferred to clean zebrafish embryo culture water, with 0.2 ppm methylene blue added. The tank was cultured at 28°C under a 14-hour light / 10-hour dark cycle for 12 hours.
[0019] 2. Treatment with Diphenylalazine A: After 12 hours of light-controlled culture, the zebrafish embryos described in step 1 were placed in 12-well plates and randomly divided into a blank control group, a low-dose Diphenylalazine A treatment group, a medium-dose Diphenylalazine A treatment group, and a high-dose Diphenylalazine A treatment group, with 3 parallel wells in each group and 10 embryos per well. The embryo culture water in the plates was removed, and 2 mL of embryo culture water was added to the blank control group. 2 mL of embryo culture water containing 5 μM, 10 μM, and 20 μM Diphenylalazine A was added to the low-dose, medium-dose, and high-dose Diphenylalazine A treatment groups, respectively. The plates were then cultured at 28°C for another 72 hours.
[0020] 3. Safety evaluation test of Diphenylalazine A: The zebrafish in each group described in step 2 were anesthetized by being placed in a 0.02% tricaine solution. Images of the zebrafish at 24 hpf, 48 hpf, and 72 hpf were acquired under a microscope, and the hatching rate and mortality rate were statistically analyzed. The results are as follows: Figure 1 As shown in the figure, "0" represents the blank control group, "5" represents the low-dose Diphenylalazine A treatment group, "10" represents the medium-dose Diphenylalazine A treatment group, and "20" represents the high-dose Diphenylalazine A treatment group.
[0021] Depend on Figure 1The results showed that, compared with the blank control group, zebrafish treated with Diphenylalazine A (low, medium, and high doses) did not hatch at 24 hpf, with a mortality rate of 0% and no deformities. The zebrafish treated with Diphenylalazine A (low, medium, and high doses) all had a 100% hatching rate at 48 hpf and 72 hpf, with a mortality rate of 0% and no deformities. These results indicate that Diphenylalazine A at concentrations of 5 μM, 10 μM, and 20 μM have no developmental toxicity to zebrafish embryos and are all within the safe concentration range.
[0022] Example 2: Angiogenic activity of Diphenylalazine A PTK787 is a tyrosine kinase inhibitor that specifically inhibits the expression of vascular endothelial growth factor (VEGF) receptors, thereby inducing intersegmental vascular injury in zebrafish.
[0023] 1. Zebrafish sample pretreatment and streptomycin E treatment: The zebrafish sample pretreatment method was the same as in Example 1. After culturing under controlled light for 24 hours, the zebrafish embryos were demembranes removed with a 1 mg / mL streptomycin E solution, and the zebrafish embryos were rinsed with embryo culture water to remove residual streptomycin E.
[0024] 2. Treatment with Diphenylalazine A: The zebrafish embryos described in step 1 were placed in a 12-well plate and randomly divided into a blank control group, a PTK787 modeling group, a ferulic acid treatment group, a low-dose Diphenylalazine A treatment group, a medium-dose Diphenylalazine A treatment group, and a high-dose Diphenylalazine A treatment group, with 3 parallel wells in each group and 10 strips in each well. Remove the embryo culture water from the well plates. Add 2 mL of embryo culture water to the blank control group; add 2 mL of embryo culture water containing 0.3 μg / mL PTK787 to the PTK787 modeling group; add 2 mL of embryo culture water containing 0.3 μg / mL PTK787 and 20 μM ferulic acid to the ferulic acid treatment group; add 2 mL of embryo culture water containing 0.3 μg / mL PTK787 and 5 μM, 10 μM, and 20 μM Diphenylalazine A to the low-dose, medium-dose, and high-dose Diphenylalazine A treatment groups, respectively. Incubate the zebrafish in each group at 28℃ for 24 h.
[0025] 3. Statistics on the length of intersegmental blood vessels in zebrafish: The zebrafish in each group described in step 2 were anesthetized by being placed in a 0.02% tricaine solution, and images of the intersegmental vessels were acquired under a microscope. Based on the acquired images, the length of the intersegmental vessels in each group of zebrafish was calculated using Image-Pro Plus 6.0 software, and the data were statistically analyzed using GraphPad Prism 8.0.2 software.
[0026] Images of intersegmental vessels in zebrafish and statistical results of intersegmental vessel length are shown below. Figures 2-3 As shown in the figure, "0" represents the blank control group, "PTK787" represents the PTK787 model group, "FA" represents the ferulic acid treatment group, "5" represents the low-dose Diphenylalazine A treatment group, "10" represents the medium-dose Diphenylalazine A treatment group, and "20" represents the high-dose Diphenylalazine A treatment group.
[0027] Depend on Figures 2-3 The results showed that, compared with the blank control group, the PTK787 model group exhibited significant damage to the intersegmental vessels of zebrafish (##P<0.01), indicating successful modeling. Compared with the PTK787 model group, the intersegmental vessel length of zebrafish treated with ferulic acid, low-dose Diphenylalazine A, medium-dose Diphenylalazine A, and high-dose Diphenylalazine A was significantly increased (**P<0.01). Notably, compared with ferulic acid, an existing cardiovascular disease treatment drug, 10μM and 20μM Diphenylalazine A concentrations showed a more significant effect on improving the intersegmental vessel length of zebrafish and exhibited stronger activity in treating vascular damage.
[0028] The above results indicate that Diphenylalazine A can effectively reverse PTK787-induced intersegmental vascular damage in zebrafish and has angiogenesis-promoting activity.
[0029] Example 3: Antithrombotic effect of Diphenylalazine A Arachidonic acid can induce platelet aggregation and thrombus formation, which in turn leads to a decrease in cardiac return in zebrafish, specifically manifested as a reduction in the area of erythrocyte aggregation in the cardiac chamber and a decrease in staining intensity.
[0030] 1. Zebrafish sample pretreatment: The pretreatment method for zebrafish samples is the same as in Example 1.
[0031] 2. Treatment with Diphenylalazine A: After culturing zebrafish embryos under controlled light for 72 hours as described in step 1, they were placed in 12-well plates and randomly divided into a blank control group, an arachidonic acid model group, an aspirin treatment group, a low-dose Diphenylalazine A treatment group, a medium-dose Diphenylalazine A treatment group, and a high-dose Diphenylalazine A treatment group, with 3 parallel wells per group and 10 lines per well. The embryo culture water in the well plate was removed. 2 mL of embryo culture water was added to the blank control group and the arachidonic acid model group; 2 mL of embryo culture water containing 22.5 μg / mL aspirin was added to the aspirin treatment group; and 2 mL of embryo culture water containing 5 μM, 10 μM, and 20 μM Diphenylalazine A was added to the low-dose, medium-dose, and high-dose Diphenylalazine A treatment groups, respectively. After the zebrafish in each group were cultured in a constant temperature incubator at 28℃ for 6 hours, except for the blank control group, the remaining groups were added with arachidonic acid at a final concentration of 80 μM and cultured for another 1.5 hours.
[0032] 3. Statistical analysis of erythrocyte staining area in zebrafish hearts: Remove the culture water from each group of zebrafish embryos described in step 2, add o-anisidine staining solution, and stain for 10 minutes in the dark. Then wash three times with embryo culture water, fix with 4% paraformaldehyde, and fix the zebrafish juveniles on their sides on a glass slide coated with methylcellulose. Ten juveniles were randomly selected from each group, and images of the zebrafish heart were acquired under a microscope. Based on the acquired images, the stained area of erythrocytes in the heart was measured using Image-Pro Plus 6.0 software, and the data were statistically analyzed using GraphPad Prism 8.0.2 software.
[0033] Images of the zebrafish heart and statistical results of the stained area of erythrocytes in the heart are shown below. Figures 4-5 As shown in the figure, "0" represents the blank control group, "AA" represents the arachidonic acid model group, "ASP" represents the aspirin treatment group, "5" represents the low-dose Diphenylalazine A treatment group, "10" represents the medium-dose Diphenylalazine A treatment group, and "20" represents the high-dose Diphenylalazine A treatment group.
[0034] Depend on Figures 4-5It was found that, compared with the blank control group, the erythrocyte staining area of zebrafish hearts was significantly reduced in the arachidonic acid model group (##P<0.01), indicating that thrombosis occurred in the zebrafish circulatory system and the model was successful; compared with the arachidonic acid model group, the erythrocyte staining area of zebrafish in the aspirin treatment group, the medium-dose Diphenylalazine A treatment group, and the high-dose Diphenylalazine A treatment group were all significantly increased (**P<0.01).
[0035] The above results indicate that 10 μM and 20 μM Diphenylalazine A can effectively reverse arachidonic acid-induced thrombosis in zebrafish and has significant antithrombotic activity.
Claims
1. Application of Diphenylalazine A in the preparation of antithrombotic drugs, wherein the chemical structural formula of Diphenylalazine A is shown in Formula I: Formula I.
2. The application of Diphenylalazine A in the preparation of drugs for treating cardiovascular diseases, wherein the cardiovascular disease is one of lower limb ischemic diseases and arteriosclerosis obliterans.
3. The application as described in claim 2, characterized in that, The arteriosclerosis obliterans mentioned refers to arteriosclerosis obliterans of the lower extremities.
4. An antithrombotic drug composition, characterized in that, Diphenylalazine A is the active ingredient.
5. A pharmaceutical composition for treating cardiovascular diseases, characterized in that, With Diphenylalazine A as the active ingredient, the cardiovascular disease mentioned is one of the following: lower limb ischemic disease and arteriosclerosis obliterans.
6. The pharmaceutical composition according to claim 5, characterized in that, The arteriosclerosis obliterans mentioned refers to arteriosclerosis obliterans of the lower extremities.
7. The pharmaceutical composition according to any one of claims 4 to 6, characterized in that, The pharmaceutical composition contains one or more pharmaceutically acceptable carriers and / or excipients.
8. The pharmaceutical composition according to claim 7, characterized in that, The excipient is one or more of the following: sustained-release agent, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, and lubricant.