Tormentic acid compound as CB2 receptor agonist and medicine and application of tormentic acid compound as CB2 receptor agonist and medicine

Abstract

The invention relates to the technical field of medicines, discovers that a natural product tormentic acid can be used as a CB2 receptor agonist and application thereof, and particularly relates to discovery of a tormentic acid action target spot, the target spot is a CB2 receptor, the CB2 receptor agonist is tormentic acid or a derivative thereof, and the derivative of the tormentic acid or the derivative of the tormentic acid is a derivative of the tormentic acid or the derivative of the tormentic acid or the derivative of the tormentic acid. And one or more than two of the corresponding pharmaceutically acceptable salts thereof are active ingredients. In vitro cell experiments show that the compound is a CB2 receptor agonist. The current research shows that the CB2 receptor is related to pain, inflammatory diseases, neurodegenerative diseases, cancers and autoimmune diseases such as multiple sclerosis and asthma, and the CB2 receptor agonist can prevent and treat pain, inflammatory diseases, neurodegenerative diseases, cancers and autoimmune diseases such as multiple sclerosis and asthma. Therefore, a novel CB2 receptor agonist Miao compound with a clear action target can be provided for the related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of CB2 (Cannabinoid receptor 2, CB2) receptor agonists, and relates to the discovery of the action target of triterpenoid compounds in Perilla leaves, and specifically to the discovery of the action target of potentilla acid in triterpenoid compounds. The triterpenoid compound potentilla acid and its derivatives, and the corresponding pharmaceutically acceptable salt compounds; the target is CB2 receptor; the application is autoimmune diseases, multiple sclerosis and asthma, etc. Background Art

[0002] Perilla frutescens (L.) Britt. is a plant of the genus Perilla in the family Lamiaceae. It is an annual, upright herb and a traditional medicinal and edible plant with a unique aroma. Its leaves, stems and fruits can all be used as medicine. [1] , is widely cultivated in my country and Southeast Asia, from Indonesia in the south to Japan in the east [2] Perilla was first recorded in Er Ya and first used as medicine in Ming Yi Bie Lu. It has the effects of dispelling cold, promoting qi and harmonizing the stomach. It can be used for colds, coughs, nausea, vomiting during pregnancy, fish and crab poisoning, etc. [3] . Modern pharmacological studies have shown that perilla also has pharmacological effects such as antioxidant, anti-inflammatory, antibacterial, anti-allergic and anti-tumor. However, most of the current research focuses on the cellular level, and there is little research on the target of pure compounds (Yu Shuling, Zhang Dongting. Edible and medicinal uses of perilla and its principles. Anhui Agricultural Sciences, 2006, 34(13): 3047-3048; Flora of China Editorial Committee, Chinese Academy of Sciences. Flora of China (Volume 61). Beijing: Science Press, 1978; Pharmacopoeia of the People's Republic of China: Volume 1 [S]. 2020: 354).

[0003] CB2 (Cannabinoid receptor 2, CB2) is encoded by the gene CNR. Human CB2 contains 360 amino acids and has a global sequence homology of 44% and a transmembrane sequence homology of 68% with CB1R. Unlike CB1, CB2 is widely distributed in peripheral tissues. For example, in the immune system, CB2 can regulate physiological functions such as immune function, cell migration and cytokine release. Activation of CB2 is related to neural defense function and ensures the maintenance of bone mass and relief of inflammatory symptoms. Studies have found that CB2 agonists can delay certain neurodegenerative diseases, such as Huntington's disease and Alzheimer's disease. Although CB2 agonists have made important progress in the role of various diseases, there are currently no drugs that selectively act on CB2 receptors on the market, and only a few synthetic CB2 ligands have entered clinical trials. In addition, some candidate drugs have also encountered setbacks in clinical trials. For example, the CB2 agonist S-777469 has good safety but did not show ideal therapeutic effects, and the research was eventually abandoned. Therefore, discovering more CB2 receptor selective ligands with high activity, high selectivity and excellent drugability is of great significance for clarifying its pharmacological and biological functions, which will provide a new direction for the targeted treatment of diseases (Hu, J., Feng, Z., Ma, S., Zhang, Y., Tong, Q., Alqarni, MH, Gou, X., Xie, X.-Q. Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to DrugDiscovery.J.Chem.Inf.Model.2016,56,1152-1163;Li,X.,Hua,T.,Vemuri,K.,Ho,J.-H.,Wu,Y.,Wu,L.,Popov,P.,Benchama,O.,Zvonok,N.,Locke,Ka, Qu,L.,Han,GW,Iyer,MR,Cinar,R.,Coffey,NJ,Wang,J.,Wu,M.,Katritch,V.,Zhao,S.,Kunos,G.,Bohn,LM,Makriyannis,A.,Stevens,RC,Liu,Z.-J.Crystal Structure of the HumanCannabinoid Receptor CB2.Cell 2019, 176, 459-467; Xing, C., Zhuang, Y., Xu, T.-H., Feng, Z., Zhou, X. E., Chen, M., Wang, L.,Meng,X.,Xue,Y.,Wang,J.,Liu,H.,McGuire,TF,Zhao,G.,Melcher,K.,Zhang,C.,Xu,HE,Xie,X.-Q.Cryo-EM Structure of the HumanCannabinoid Receptor CB2-Gi Signaling Complex.Cell 2020,180,645-654;Hua,T.,Li,X.,Wu,L.,Iliopoulos-Tsoutsouvas,C.,Wang,Y.,Wu,M.,Shen,L.,Johnston,CA,Nikas,SP,Song,F.,Song,X., Yuan,S.,Sun,Q.,Wu,Y.,Jiang,S.,Grim,TW,Benchama,O.,Stahl,EL,Zvonok,N.,Zhao,S.,Bohn,LM,Makriyannis,A.,Liu,Z.-J.Activationand Signaling Mechanism Revealed by Cannabinoid Receptor-Gi ComplexStructures.Cell 2020,180,655-665; Pacher,P.,Bátkai,S.,Kunos,G.The Endocannabinoid System as an Emerging Target of Pharmacotherapy. Pharmacol. Rev. 2006, 58, 389-462). .

[0004] At present, there are no reports on the effect of potentilla acid in triterpenoid compounds on CB2 receptor (Cannabinoid receptor 2, CB2). Summary of the invention

[0005] The present invention relates to the discovery of the action target of potentianic acid in triterpenoid compounds and the application of such compounds. One purpose is to provide the action target of the compound potentianic acid as CB2 receptor; the second purpose is to provide the clinical application range of such compounds.

[0006] The technical solution of the present invention is:

[0007] The CB2 receptor agonist is the compound potentilla acid.

[0008] The chemical structure of Potentilla acid is as follows:

[0009]

[0010] The triterpenoid compound of the present invention is used in the preparation of drugs for preventing and / or treating autoimmune diseases, multiple sclerosis, asthma and the like, wherein the triterpenoid compound is potentilla acid.

[0011] Beneficial effects of the present invention:

[0012] In vitro cell experiments show that the compound of the present invention is cinnamic acid that acts on the CB2 receptor, which is a G protein-coupled receptor. The CB2 receptor is associated with pain, inflammatory diseases, neurodegenerative diseases, cancer, autoimmune diseases such as multiple sclerosis and asthma. Based on the correlation between the target and the disease, the clinical application scope of this type of compound can be broadened.

[0013] The present invention has found that the natural product potentilla acid can be used as a CB2 (Cannabinoid receptor 2, CB2) receptor agonist and its application, specifically the discovery of the target of triterpenoid compounds, the target is CB2 receptor, the CB2 receptor agonist is potentilla acid or its derivatives, and one or more of their corresponding pharmaceutically acceptable salts as active ingredients. In vitro cell experiments show that the compound of the present invention is a CB2 receptor agonist. Current studies have shown that CB2 receptors are related to diseases such as pain, inflammatory diseases, neurodegenerative diseases, cancer, and autoimmune diseases such as multiple sclerosis and asthma. CB2 receptor agonists can prevent and treat pain, inflammatory diseases, neurodegenerative diseases, cancer, and autoimmune diseases such as multiple sclerosis and asthma. Based on this, a novel CB2 receptor agonist lead compound with a clear target can be provided for the above-mentioned related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The negative control group (HBSS buffer containing 0.1% DMSO and 20 mM HEPES), CP 55940 (1.25 μM), potentilla acid (100 μM), and the maximum DMR response (%) of potentilla acid_CP 55940 on CHO-CB2 cells within 60 min;

[0015] Figure 2 The maximum DMR response dose-effect curves of different concentrations of potentilla acid on CHO-CB2 cells within 60 minutes of stimulation and desensitization;

[0016] Figure 3 The maximum DMR response dose-effect curve of CP 55940 at different concentrations on CHO-CB2 cells within 60 min of stimulation and desensitization. DETAILED DESCRIPTION

[0017] The present invention is further described with reference to examples. The examples are only intended to illustrate the present invention, but not to limit the present invention.

[0018] Potentilla acid (brand: Chengdu Pusi; item number: PS1766) was purchased from Chengdu Pusi Biotechnology Co., Ltd. CHO cells (item number: GNHa7) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; the stable cell line CHO-CB2 cells with high expression of human CB2 receptors was constructed according to the literature (Zhou, H., Peng, X., Hou, T., Zhao, N., Qiu, M., Zhang, X., Liang, X., 2020a. Identification of novel phytocannabinoids from Ganoderma by label-free dynamic mass redistribution assay. J. Ethnopharmacol. 246, 112218). CP 55940 (brand: Yuanye Bio; item number: T52304) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. F12 culture medium (brand: Gibco; item number: C11765500BT) was purchased from Yingwei Jieji (Shanghai) Trading Co., Ltd. Dimethyl sulfoxide (DMSO, catalog number: A503039-0250). 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 (Buffer) preparation volume ratio: HEPES: HBSS: ultrapure water = 1:5:44, used for compound dilution and preparation during the experiment. The detection platform is Corning third generation The imager detects the wavelength shift caused by the dynamic mass resetting (DMR) of the cell.

[0019] Example 1: Discovery that the triterpenoid compound Potentillaic acid acts on CB2 receptors

[0020] CHO-CB2 cells in the logarithmic growth phase were inoculated into In the 384-well biosensor microplate, the volume of the cell suspension (F12 culture medium containing cells) inoculated in each well is 40 μL, and the number of cells inoculated in each well is 1.5×10 4 Then, the 384-well plate was placed in a cell culture incubator (5% CO2 volume concentration in air, 37°C) for 22-24 hours. When the cell confluence reached about 95%, the experiment was performed.

[0021] First, 10 μL of a 100 μM final concentration of cinquefoil acid stock solution and a negative control group (0.1% DMSO (volume ratio) in 20 mM HEPES in HBSS buffer) were added to different wells inoculated with CHO-CB2 cells and detected on the EPIC instrument for 1 hour. Then, CP 55940 (1.25 μM, calculated based on the final volume of each well) was added and the detection was continued on the EPIC instrument for 1 hour. The results are shown in FIG. Figure 1 As shown, potentilla acid produced a strong DMR response signal on CHO-CB2 cells, and it could desensitize the DMR response signal of CB2 receptor agonist CP 55940, indicating that it may have CB2 receptor agonist activity.

[0022] Example 2: Verification of the effect of the triterpenoid compound Potentilla acid on CB2 receptors

[0023] CHO-CB2 cells in the logarithmic growth phase were inoculated into In the 384-well biosensor microplate, the volume of the cell suspension (F12 culture medium containing cells) inoculated in each well is 40 μL, and the number of cells inoculated in each well is 1.5×10 4 Then, the 384-well plate was placed in a cell culture incubator (5% CO2 volume concentration in air, 37°C) for 22-24 hours. When the cell confluence reached about 95%, the experiment was performed.

[0024] DMR agonist detection: Potentilla acid at different final concentrations (100 μM, 33.33 μM, 11.11 μM, 3.70 μM, 1.23 μM, 0.41 μM, 0.14 μM, in buffer containing 0.1% DMSO (volume ratio)) and CP 55940 at different concentrations (5 μM, 1.67 μM, 0.56 μM, 0.19 μM, 0.06 μM, 0.02 μM, 0.01 μM, in buffer containing 0.1% DMSO (volume ratio)) were added to CHO-CB2 cells and incubated on the EPIC instrument for 1 hour. Then, CB2 receptor agonist CP 55940 (1.25 μM, calculated according to the final volume of each well) was added and monitored on the EPIC instrument for 1 hour. The results are shown in the following table. Figure 2 Potentilla acid produced a dose-dependent DMR response signal on CHO-CB2 cells and desensitized the DMR response signal of the CB2 receptor agonist CP 55940 in a dose-dependent manner, indicating that it may have CB2 receptor agonist activity. 50 Value and desensitization IC 50 The values ​​were 33.30±4.41μM and 34.62±2.48μM, respectively. This indicates that cinquefoil acid has CB2 receptor agonist activity and is a CB2 receptor antagonist. In addition, the EC 50Value and desensitization IC 50 The values ​​were 1.05 ± 0.04 μM and 0.84 ± 0.08 μM, respectively. Figure 3 shown.

[0025] Pharmacological DMR experiments have shown that the CB2 receptor is the target of the compound for potentilla acid. Current studies have shown that the CB2 receptor is associated with pain, inflammatory diseases, neurodegenerative diseases, cancer, autoimmune diseases such as multiple sclerosis and asthma. Based on the correlation between the target and the disease, the clinical application range of this compound can be broadened.

Claims

1. A use of a potentilla acid compound as a CB2 receptor agonist or for preparing a medicament, characterized in that: The CB2 receptor agonist contains the following potentilla acid, or a derivative of potentilla acid, and one or more of the pharmaceutically acceptable salts of potentilla acid as active ingredients, and the compound structure is as follows:

2. The use according to claim 1, characterized in that: The potentilla acid compound described in claim 1 is used as an active ingredient in the preparation of therapeutic drugs for the following diseases, specifically including: one or more of autoimmune diseases, multiple sclerosis, asthma and the like.

3. The CB2 receptor agonist according to claim 1 or 2, characterized in that: The CB2 receptor agonist or drug also includes a pharmaceutically acceptable carrier and / or excipient.

4. A drug or CB2 receptor agonist, characterized in that: The active ingredients are one or more of the following compounds, derivatives of the compounds, and pharmaceutically acceptable salts of the compounds; The chemical structure is shown below:

5. The drug or CB2 receptor agonist according to claim 4, characterized in that: The medicine also includes a pharmaceutically acceptable carrier and / or excipient.

6. The drug or CB2 receptor agonist according to claim 4 or 5, characterized in that: The medicine is a medicine for treating and / or preventing the following diseases, which include one or more of autoimmune diseases, multiple sclerosis, asthma and the like.

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