GPR35 receptor agonist or medicine and application thereof

Abstract

The invention relates to the technical field of medicines, and finds that a natural product amentoflavone can be used as a GPR35 (G-protein coupled receptor 35, GPR35) receptor agonist and application thereof, the compound is amentoflavone, has GPR35 receptor agonist activity, is a GPR35 receptor agonist, can be used for treating thromboembolic diseases, spring conjunctivitis, intracranial embolism, primary sclerosing cholangitis and myocardial infarction, and can be used for treating various diseases such as thromboembolic diseases, spring conjunctivitis, intracranial embolism, primary sclerosing cholangitis and myocardial infarction. Therefore, a novel GPR35 receptor stimulant seedling compound with a clear action target can be provided for the related diseases.

Description

Technical Field

[0001] This invention belongs to the field of GPR35 (G-protein coupled receptor 35) receptor agonists, and relates to the discovery of the target of biflavonoid compounds in Ginkgo biloba, specifically the discovery of the target of cephalotaxel biflavonoids. The biflavonoid compound cephalotaxel biflavonoids and its derivatives, as well as their corresponding pharmaceutically acceptable salt-forming compounds; the target is the GPR35 receptor; the applications include thromboembolic diseases, vernal conjunctivitis, intracranial embolism, primary sclerosing cholangitis, and myocardial infarction. Background Technology

[0002] Ginkgo biloba, belonging to the genus Ginkgo in the family Ginkgoaceae, is also known as maidenhair tree, maidenhair tree, duck's foot tree, duck's palm tree, and fan tree. It is one of the oldest relict tree species from the Mesozoic Era, often referred to as a "living fossil." Ginkgo biloba has a complex chemical composition, with flavonoids and terpene lactones being the most important active substances. Flavonoids can be further divided into monoflavonoids and biflavonoids, with biflavonoids being characteristic components of gymnosperms and widely distributed in Ginkgoaceae plants. Ginkgo biflavonoids are dimers composed of two monoflavonoid molecules linked together, exhibiting higher biological activity compared to monomers and showing promising application prospects.For example, compared with apigenin, ginkgo biloba flavonoids showed more obvious anti-hepatotoxic and renal protective effects; clinical pharmacological studies have shown that ginkgo biloba flavonoids have anti-inflammatory, antioxidant, antimicrobial, anticancer, neuroprotective, cardiovascular and hypoglycemic effects (Zhang Zhongpeng, Liu Xiufen. Overview of the development of ginkgo leaf extract. Journal of Traditional Chinese Medicine Research and Information, 2005, 7(2):38-40; Tu Qingbo, Sun Yun, Xu Ting et al. Research progress on pharmacological effects of ginkgo biloba flavonoids. Shandong Medical Journal, 2018, 58(19):112-114; JIA X, CHEN J, LIN H, et al. Disposition of flavonoids via enteric recycling: enzyme-transporter coupling affects metabolism of biochanin A and formononetin and excretion of their phase II conjugates. J Pharmacol Exp Ther, 2004, 310(3):1103-1113; Zhang Zunting, Gao Runli, Zhuang Sukai. Synthesis of biflavonoids and their interaction with DNA. Acta Pharmaceutica Sinica, 2009, 44(8):873-878; SAGRERA G, BERTUCCI A, VAZQUEZ A, et al. Synthesis and antifungal activities of natural and synthetic biflavonoids. Bioorg Med Chem, 2011, 19(10):3060-3073; JOYEUX M, LOBSTEINA, ANTONR, et al. Comparative antilipoperoxidant, antinecrotic and scavenging properties of terpenes and biflavones from Ginkgo and some flavonoids. Planta Med, 1995, 61(2):126-129; QIAO Y, LIU X, LI X, et al. Biflavonoids from Juniperus oblongainhibit organic anion transporter 3. Biochem Biophys Res Commun, 2019, 509(4):931-936).

[0003] The GPR35 receptor is an orphan receptor containing 309 amino acids and belongs to the G protein-coupled receptor superfamily. GPR35 receptors are expressed in various tissues, including immune cells, the colon, pancreas, small intestine, and spleen, with higher expression levels in human colon cancer cells (HT-29). Current research indicates that the GPR35 receptor is associated with diseases such as asthma, heart failure, hypertension, ulcerative colitis, primary sclerosing cholangitis, coronary heart disease, metabolic syndrome, pain, and cancer. The discovery and confirmation of new ligands for the GPR35 receptor is of great significance for elucidating its pharmacological and biological functions, which will provide new directions for targeted therapy of the disease (O'DowdB F, Nguyen T, Marchese A, Cheng R, Lynch KR, Heng HHQ, Kolakeowski LF, George SR. Genomics 47(1998)310-313; Taniguchi Y, Tonai-Kachi H, Shijo K. FEBS Lett 580(2006)5003-5008; Wang J, Simonavicius N, Wu X, Swaminath G, Reagan J, Tian H, Ling LJ Biol Chem 281(2006)22021-22028; YYang, JY LLu, X Wu, S Summer, J Whoriskey, C Saris, JD Reagan.Pharmacology86(2010)1-5;HDeng,Y Fang.Pharmacology 89(2012)211-219;Huayun Deng,Ye Fang.Med Chem Commun3(2012)1270-1274;Abdalhameed MM,Zhao PW,Hurst DP,Reggio PH,Abood ME, CroattMP, Bioorganic & Medicinal Chemistry Letters 27 (2017) 612-615).

[0004] Currently, there are no reports on the effects of cephalotaxus diflavonoids, a type of biflavonoid compound, on diseases such as thromboembolic diseases, vernal conjunctivitis, intracranial embolism, primary sclerosing cholangitis, and myocardial infarction based on the GPR35 receptor (G-protein coupled receptor 35). Summary of the Invention

[0005] This invention relates to the discovery of the target of action of paclitaxel flavonoids and the application of such compounds. One objective is to provide that the target of paclitaxel flavonoids is the GPR35 receptor; the other objective is to provide the scope of clinical applications of such compounds.

[0006] The technical solution of this invention is as follows:

[0007] The GPR35 receptor agonist is the compound Taxodium flavonoids.

[0008] The chemical structure of the compound paclitaxel flavonoid is as follows:

[0009]

[0010] The present invention relates to the application of biflavonoid compounds in the preparation of drugs for the prevention and / or treatment of thromboembolic diseases, vernal conjunctivitis, intracranial embolism, primary sclerosing cholangitis, and myocardial infarction, wherein the biflavonoid compound is Taxodium biflavonoid.

[0011] The beneficial effects of this invention are:

[0012] In vitro cell experiments showed that the compound in this invention, Taxodium flavonoids, acts on the GPR35 receptor, which is a G protein-coupled receptor. The GPR35 receptor is associated with diseases such as thromboembolic diseases, vernal conjunctivitis, intracranial embolism, primary sclerosing cholangitis, and myocardial infarction. Based on the correlation between the target and the disease, the clinical application scope of such compounds can be broadened.

[0013] This invention discovers that the natural product paclitaxel flavonoids can serve as a GPR35 (G-protein coupled receptor 35, GPR35) receptor agonist and its application. Specifically, it identifies the target of flavonoid compounds, namely the GPR35 receptor. The GPR35 receptor agonist is paclitaxel flavonoids or its derivatives, and one or more of its corresponding pharmaceutically acceptable salts as active ingredients. In vitro cell experiments demonstrate that the compounds of this invention are GPR35 receptor agonists. Current research indicates that the GPR35 receptor is associated with diseases such as thromboembolic diseases, vernal conjunctivitis, intracranial embolism, primary sclerosing cholangitis, and myocardial infarction. GPR35 agonists can prevent and treat these diseases, thus providing novel and targeted GPR35 receptor agonist precursor compounds for the aforementioned related diseases. Attached Figure Description

[0014] Figure 1The maximum DMR response concentration curves of different concentrations of paclitaxel flavonoids (A) and menthol (B) on HT-29 cells within 60 min; and the maximum DMR response concentration curves of menthol on HT-29 cells within 60 min after 60 min pretreatment with different concentrations of paclitaxel flavonoids and menthol.

[0015] Figure 2 The maximum DMR response concentration curves of paclitaxel flavonoids (A) (5 μM) and menthol (B) (150 nM) in HT-29 cells within 60 min after 10 min of pretreatment with different concentrations of ML145 were obtained. Detailed Implementation

[0016] The present invention will now be further illustrated with examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0017] Taxodium biflavonoids (brand: Yuanye Biotechnology; catalog number: B21229) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. HT-29 cells (catalog number: THu103) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; Zaprinast (catalog number: Z0878) was purchased from SIGMA. ML145 cells (brand: Tocris; catalog number: 4172) were purchased from Shanghai Unimicron Biotechnology Co., Ltd. McCoy's 5A culture medium (brand: Biological Industries; catalog number: 01-075-1ACS) was purchased from Nanjing Fomax Biotechnology Co., Ltd. Dimethyl sulfoxide (DMSO, catalog number: A503039-0250) was also purchased. HBSS (brand: Gibco; catalog number: 14065056; specification: (10×) 500mL) and HEPES (brand: Gibco; catalog number: 15630080; specification: (100×) 500mL) buffer solutions were purchased from Invitrogen (Shanghai) Trading Co., Ltd. Each compound was dissolved in DMSO to prepare a 100mM stock solution. The buffer solution was prepared at a volume ratio of HEPES:HBSS:ultrapure water = 1:5:44, and was used for compound dilution during the experiment. The detection platform was Corning Gorilla Glass 3. The imager detects the wavelength shift caused by dynamic mass resetting (DMR) of cells.

[0018] Example 1: Evaluation of GPR35 receptor agonist activity of Taxodium spicata flavonoids

[0019] HT-29 cells in the logarithmic growth phase were seeded into... In the 384-well biosensor microplate, each well was seeded with 40 μL of cell suspension (McCoy's 5A medium containing cells), and the number of cells seeded per well was 3.0 × 10⁶.4 Then, place the 384-well plate in a cell culture incubator (air with a volume concentration of 5% CO2, 37°C) and culture for 22-24 hours. When the cell confluence reaches about 95%, conduct the experiment.

[0020] First, 10 μL of different final concentrations of paclitaxel flavonoids (12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM, 0.78 μM, 0.39 μM, 0.20 μM, 0.10 μM, 0.05 μM, 0.02 μM) and menthol (1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.63 nM, 7.81 nM, 3...) were added respectively. 0.91 nM, 1.95 nM, 0.98 nM, 0.49 nM, 0.24 nM, and 0.12 nM solutions were added to different wells seeded with HT-29 cells, and their DMR response signals were monitored in real time for 1 hour. Then, a final concentration of 150 nM of the GPR35 receptor agonist, phenobarbital, prepared with buffer (containing 0.1% DMSO, volume concentration), was added to different wells seeded with HT-29 cells, and the results were detected on an EPIC instrument for 1 hour. The results are as follows: Figure 1 As shown in Figure A, paclitaxel flavonoids produced a concentration-dependent DMR response signal in HT-29 cells, and also exhibited a desensitizing effect on the DMR response signal of the GPR35 receptor agonist menthol in HT-29 cells in a concentration-dependent manner. This indicates that paclitaxel flavonoids possess GPR35 receptor agonist activity, and its agonist and desensitizing effect on EC... 50 Value and IC 50 The values ​​were 0.85±0.09 μM and 0.78±0.10 μM, respectively. Positive molecules ( Figure 1 B) The stimulant and desensitizing effects of phenobarbital on EC. 50 Value and IC 50 The values ​​are 22.89±0.01nM and 15.53±0.01nM, respectively.

[0021] Example 2: Verification of the antagonistic effect of GPR35 receptor agonist activity of Taxodium styracifolium biflavonoids

[0022] HT-29 cells in the logarithmic growth phase were seeded into... In the 384-well biosensor microplate, each well was seeded with 40 μL of cell suspension (McCoy's 5A medium containing cells), and the number of cells seeded per well was 3.0 × 10⁶. 4 Then, place the 384-well plate in a cell culture incubator (air with a volume concentration of 5% CO2, 37°C) and culture for 22-24 hours. When the cell confluence reaches about 95%, conduct the experiment.

[0023] DMR antagonism assay: HT-29 cells were pretreated with ML145 at different final concentrations (20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.313 μM, 0.156 μM, 0.078 μM, 0.039 μM, 0.020 μM, 0.010 μM, 0.005 μM, 0.002 μM, containing 0.1% DMSO (volume ratio)) for 10 min. Then, cephalosporin (final concentration 5 μM, calculated based on the final volume per well) and menthol (final concentration 150 nM, calculated based on the final volume per well) were added, and the cells were monitored on an EPIC instrument for 1 h. Results are as follows: Figure 2 As shown, the DMR response signal of paclitaxel flavonoids in HT-29 cells can be antagonized by the GPR35 receptor antagonist ML145 in a concentration-dependent manner, with an antagonistic IC50 value of [missing value]. 50 The value was 0.04 ± 0.01 μM, indicating that cypermethrin is a GPR35 receptor agonist. The IC50 of ML145 antagonism against menthol was... 50 The value is 0.44±0.14μM.

[0024] Pharmacological DMR experiments have shown that the GPR35 receptor is the target of the compound cephalotaxel. Current research indicates that the GPR35 receptor is associated with diseases such as thromboembolic diseases, vernal conjunctivitis, intracranial embolism, primary sclerosing cholangitis, and myocardial infarction. Based on the correlation between the target and diseases, the clinical application scope of this compound can be broadened.

Claims

1. The application of Taxus chinensis flavonoids as a GPR35 receptor agonist or in the preparation of drugs, characterized in that: The GPR35 receptor agonist or the prepared drug contains one or more of the following as active ingredients: a paclitaxel flavonoid compound, a derivative of a paclitaxel flavonoid compound, or a pharmaceutically acceptable salt corresponding to a paclitaxel flavonoid compound.

2. The application according to claim 1, characterized in that: The structural formula of the compound of Taxus chinensis biflavonoids is as follows:

3. The application according to claim 1, characterized in that: The prepared drug is one or more of the following: drugs for treating and / or preventing diseases including thromboembolic diseases, vernal conjunctivitis, intracranial embolism, primary sclerosing cholangitis, and myocardial infarction.

4. The application according to any one of claims 1-3, characterized in that: The GPR35 receptor agonist or drug also includes a pharmaceutically acceptable carrier and / or excipient.

5. A drug or GPR35 receptor agonist, characterized in that: The active ingredient is one or more of the following: a compound of Taxodium biflavonoids, or a derivative thereof, and a pharmaceutically acceptable salt thereof.

6. The drug or GPR35 receptor agonist according to claim 5, characterized in that: The chemical structure of cyperus rotundus flavonoids is shown below:

7. The drug according to claim 5 or 6, characterized in that: The drug or GPR35 receptor agonist also includes a pharmaceutically acceptable carrier and / or excipient.

8. The drug or GPR35 receptor agonist according to claim 5 or 6, characterized in that: The drug is for the treatment and / or prevention of one or more of the following diseases: thromboembolic diseases, vernal conjunctivitis, intracranial embolism, primary sclerosing cholangitis, and myocardial infarction.

Images