Natural product-based drug design method, pentacyclic triterpenoid, preparation method and application thereof

Abstract

This invention discloses a drug design method based on natural products, a pentacyclic triterpenoid compound, its preparation method, and its application. The design method includes the following steps: (1) obtaining a template molecule; (2) selecting natural products with structural similarity to the template within a threshold range and possessing the specific biological activity as a set of candidate reference molecules; (3) selecting one or more from the set of reference molecules, comparing them with the template molecule, and obtaining different active functional groups; and constructing the different active functional groups onto the molecular skeleton shared by the template molecule and the reference molecules. This invention successfully designed a cheap and readily available lead compound to replace triterpenoid molecules by using the bioavailability of plant triterpenoid natural products in the host plant and the structural similarity value with triptolide and crosolic acid compounds as screening conditions. Experiments have confirmed that it has significant anti-inflammatory and anti-metabolic syndrome physiological activities caused by chronic inflammation.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, relates to a drug design method based on natural products, pentacyclic triterpenoids, their preparation methods and applications. Background Technology

[0002] Despite the significant human and material resources invested in combinatorial chemistry by drug researchers worldwide, only sorafenib, approved by the FDA in 2005 for the treatment of renal cell carcinoma, has been discovered through this pathway to date. Natural products play an indispensable and irreplaceable role in new drug development. Compared to synthetic compounds, natural products possess more stereoisomer centers, more fused, bridged, or spirocyclic structures, diverse molecular structures, and a greater ease of binding to biological macromolecules. Their novel structures and significant biological activities determine their unparalleled advantages in participating in vital physiological processes.

[0003] However, many effective natural products have very low natural abundance. For example, the first-line chemotherapy drug paclitaxel is found in less than 0.01% of the bark of the yew tree, making its large-scale production extremely difficult. Metabolic syndrome is another example, a group of chronic diseases that seriously endanger human health and impose a huge economic burden on society. Studies have found that insulin resistance and type 2 diabetes caused by obesity are closely related to immune system dysfunction. Patients with this condition secrete large amounts of inflammatory factors in their adipose tissue: tumor necrosis factor-α (TNF-α). Tripterygium wilfordii has anti-inflammatory and anti-obesity properties; however, it is extremely toxic, with a low oral LD50. 50 At a dose of 20.5 mg / kg, 4 mg / kg resulted in a 40% lethality in mice, and 1 mg / kg showed severe toxicity to the brain, heart, and liver of mice. Furthermore, triptolide exhibits low bioavailability (0.1-0.3% dry weight) and low bioavailability (17.06%) in its host plant. Summary of the Invention

[0004] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a drug design method based on natural products. The aim is to modify the natural product to be improved by screening for reference molecules with similar or high abundance of natural products. This method also provides a pentacyclic triterpenoid compound, its preparation method, and its applications, thereby solving the technical problems of many currently effective natural products having low abundance, leading to environmental unfriendliness, high costs, or unfavorable efficacy or toxicity for drug development.

[0005] To achieve the above objectives, according to one aspect of the present invention, a drug design method based on natural products is provided, comprising the following steps:

[0006] (1) Obtain the molecular structure of natural products with specific biological activities that need to be improved;

[0007] (2) Using the molecular structure obtained in step (1) as the template molecule, select natural products with similarity to the template structure within the threshold range and having the specific biological activity as the candidate reference molecule set.

[0008] (3) Select one or more reference molecules from the set of reference molecules obtained in step (2), compare them with the template molecule, and obtain active functional groups with differences; construct the active functional groups with differences onto the molecular backbone shared by the template molecule and the reference molecule to obtain an improved molecule with specific biological activity.

[0009] Preferably, in the drug design method based on natural products, the threshold range in step (2) is within the range where the similarity score with the template molecule structure is above 0.2, and more preferably, the similarity score is between 0.2 and 0.9.

[0010] Preferably, in the natural product-based drug design method, the natural products in the candidate reference molecule set have a relative biomass in the corresponding host plant that exceeds a preset biomass threshold; the biomass threshold is preferably 1%.

[0011] Preferably, in the natural product-based drug design method, the selection of one or more reference molecules from the set of reference molecules obtained in step (2) specifically involves screening one or more reference molecules from the set of reference molecules obtained in step (2) that have the same target and / or pathway of action.

[0012] The specific steps for obtaining differentiated active functional groups are as follows: the active functional groups of the reference molecule are screened and determined through physiological models or molecular docking models, and compared with the template to obtain differentiated active functional groups.

[0013] According to another aspect of the present invention, a method for preparing compound molecules designed by the drug design method based on natural products is provided, characterized by comprising the following steps:

[0014] S1. Select compounds from the template molecule and the reference molecule that have a relatively high biomass content in the corresponding host plant species as raw materials; preferably, use the reference molecule as the raw material.

[0015] S2. Through chemical reaction, active functional groups with differences between the template molecule and the reference molecule are constructed onto the raw material to obtain the compound molecule designed by the drug design method.

[0016] Preferably, in the method for preparing the compound molecule designed by the drug design method based on natural products, the specific biological activity is anti-inflammatory activity, and the natural product to be improved with the specific biological activity is triptolide or corosolic acid; the reference molecule of the natural product is 18-β-glycyrrhetinic acid, and the preparation method includes the following steps:

[0017] S1. 18-β-glycyrrhetinic acid is selected as the raw material;

[0018] S2. Through chemical reaction, active functional groups with differences between the template molecule and the reference molecule are constructed onto the raw material to obtain the compound molecule designed by the drug design method.

[0019] Preferably, triptolide and 18-β-glycyrrhetinic acid are used to construct the 18-β-glycyrrhetinic acid molecule by substituting the active differential functional group (hydroxyl group on ring A) with conjugated enone structure; or corosolane and 18-β-glycyrrhetinic acid are used to construct the 18-β-glycyrrhetinic acid molecule by substituting the active differential functional group (α,β-hydroxyl group on ring A) with conjugated enone structure.

[0020] According to another aspect of the invention, a pentacyclic triterpenoid compound is provided, having a molecular skeleton shared by a template pentacyclic triterpenoid compound and a natural product having a structural similarity score of 0.2 or higher with respect to the template pentacyclic triterpenoid compound, and active functional groups differing between the template pentacyclic triterpenoid compound and the natural product; the template pentacyclic triterpenoid compound is tripterycin or corosolic acid; the natural product has anti-inflammatory activity, preferably having a relative biomass content of more than 1% in the corresponding host plant.

[0021] Preferably, the natural product of the pentacyclic triterpenoid compound is 18-β-glycyrrhetinic acid.

[0022] Preferably, the pentacyclic triterpenoid compound has an oleanane-type triterpenoid-30 carboxylic acid skeleton and a 3-carbonyl-1,2-enol structure on its A ring, preferably 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanane-30 carboxylic acid or 11-dicarbonyl-12-en-2α,3β-dihydroxy-oleanane.

[0023] According to another aspect of the invention, the pentacyclic triterpenoid compound and its pharmaceutically acceptable salts are used in the preparation of anti-chronic inflammatory drugs or drugs for the treatment of metabolic syndrome; preferably in the preparation of drugs for the treatment of surgery-free obesity or drugs for the treatment of type 2 diabetes.

[0024] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects:

[0025] The natural product-based drug design method provided by this invention, through screening based on molecular structural similarity and bioavailability, starts with low-cost, high-abundance reference molecules and combines structural design with chemical synthesis. This reduces the production cost of drugs using natural products as raw materials and effectively improves the drug-likeness of natural products, i.e., reducing toxicity or increasing bioactivity. Therefore, this method is of great significance for natural product-based drug development. This drug design method can be applied to the research of more structurally complex natural drugs.

[0026] Specifically, this invention uses the bioavailability of plant triterpenoid natural products in the host plant and the structural similarity to compounds such as triptolide and crosolic acid as screening criteria. By performing a core-structure conversion on the lead compound, a cheap and readily available lead compound was successfully designed to replace the triterpenoid molecule. The active structural fragment of the lead compound was fused into the cheap template molecule, and two triterpenoid derivatives with significant weight loss and anti-diabetic effects were obtained through molecular structure modification. This invention avoids drug research methods such as single-target drug design and direct derivation of lead compounds, and focuses on structural similarity conversion of natural product lead compounds with significant drug effects and complex structures that have not been well understood in terms of drug action mechanism.

[0027] The pentacyclic triterpenoid compounds provided by this invention have similar physiological effects to triptolide and corosolic acid. They are also easy to prepare in large quantities and are inexpensive natural product derivatives. Experiments have confirmed that they have significant anti-inflammatory and anti-metabolic syndrome physiological activities caused by chronic inflammation. Attached Figure Description

[0028] Figure 1 The flowchart of the design method based on natural products provided in Embodiment 1 of the present invention includes: a: simplified flowchart; b: two-dimensional correlation diagram of 3D molecular structure similarity and plant host biomass content; c: 3D molecular superposition diagram of triptolide and glycyrrhetinic acid.

[0029] Figure 2 The structural formula of the template molecule triptolide provided in Example 1 is shown.

[0030] Figure 3 This is an evaluation of the anti-inflammatory bioactivity of triterpenoid candidate molecules;

[0031] Figure 4 The template molecule is the corosolic acid molecular structure provided in Example 2;

[0032] Figure 5 Example 3 is a molecular structure design based on the triptolide structural fragment and the glycyrrhetinic acid skeleton structure;

[0033] Figure 6 is a diagram of the structural identification results of GA-01 provided in Example 3, where a is the proton NMR spectrum and b is the carbon NMR spectrum.

[0034] Figure 7 shows the structural identification results of GA-02 provided in Example 3, where a is the proton NMR spectrum and b is the carbon NMR spectrum.

[0035] Figure 8 Example 4 is a molecular structure design based on the corosolic acid structural fragment and the glycyrrhetinic acid backbone structure;

[0036] Figure 9 The image shown is the structural identification result of GA-03 provided in Example 4, which is a hydrogen nuclear magnetic resonance spectrum.

[0037] Figure 10 This is a diagram illustrating the GA-02 inhibition verification effect provided in Example 5, where a: GA-02 inhibits LPS-induced ICAM-1 expression activity. 1 μg / ml LPS activated the inflammatory pathway in HMEC-1 cells, and the effects of triptolide and GA-02 on LPS-induced ICAM-1 expression were measured. b: The effect of GA-02 on NF-κB signaling. HEK293 cells were transfected with the NF-κB luciferase reporter plasmid, and the effects of triptolide and GA-02 on TNF-α-induced NF-κB activity were measured. c: The relative levels of IL-1β, TNF-α, IL-6, and MCP-1 mRNA were detected by quantitative real-time RT-PCR.

[0038] Figure 11 The effect of GA-02 provided in Example 6 on body weight and food intake in high-fat induced obese mice was investigated. DIO mice were intraperitoneally injected with GA-02 (4, 12, 20 mg / kg) and blank solvent for 14 days. (a) Daily body weight change of mice, (b) Percentage change in body weight of mice (%), (c) Mass reduction in body weight of mice in each group after 14 days of administration (g). The ac experiment was conducted in two independent groups, with 6 obese model mice in each cage.

[0039] Figure 12 Example 6 describes the effect of GA-02 on body fat content in high-fat induced obese mice, where a: changes in fat and lean body mass in obese model mice treated with low, medium, and high doses (4, 12, 20 mg / kg); b: changes in body fat in obese model mice treated with low, medium, and high doses (4, 12, 20 mg / kg); c: NMR imaging of body fat in obese model mice treated with low, medium, and high doses (4, 12, 20 mg / kg).

[0040] Figure 13The figures provided in Example 6 show the changes in body size, liver, epididymal fat, and fasting blood glucose in obese mice before and after GA-02 treatment. Figure a shows the changes in the back shape of obese mice treated with low, medium, and high doses (4, 12, 20 mg / kg); figure b shows the changes in the abdomen shape of obese mice treated with low, medium, and high doses (4, 12, 20 mg / kg); figure c shows the changes in the shape and mass of liver and epididymal fat in obese mice treated with low, medium, and high doses (4, 12, 20 mg / kg); and figure f shows the fasting blood glucose levels in obese mice treated with low, medium, and high doses (4, 12, 20 mg / kg).

[0041] Figure 14 Example 6 provides the experimental results of the average daily food intake of GA-02-treated high-fat induced obese mice in the first three days, where a: average daily food intake of GA-02-treated obese mice in the first three days; b: daily weight change of GA-02-treated obese mice in the first three days; c: percentage of weight loss of GA-02-treated obese mice in the first three days; d: average daily food intake of GA-02-treated obese mice in the first and second weeks.

[0042] Figure 15 These are anatomical diagrams of the main internal organs of obese mice induced by high-fat diet and treated with GA-02, provided in Example 6; where a: heart and attached fat of obese model mice; b: liver of obese model mice; c: kidney and perirenal attached fat of obese model mice; d: abdominal fat of obese model mice; e: heart, kidney and abdominal cavity of obese model mice 14 days after GA-02 administration.

[0043] Figure 16 The images shown in Example 6 are the results of liver function tests, including Oil Red O staining and H&E staining of liver sections in obese mice induced by high-fat diet and treated with GA-02 before and after treatment. In the images, a: Oil Red O staining and H&E staining of liver sections in obese mice after two weeks of treatment with blank solvent and high-dose GA-02 (20 mg / kg); b: Changes in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in obese mice before and after treatment (blank control group n=4; GA-02 group n=4).

[0044] Figure 17 The figures provided in Example 6 show the results of glucose tolerance and insulin sensitivity in obese mice before and after GA-02 treatment, where a: blood glucose change curves at different time points in the glucose tolerance test for mice in the solvent control group and GA-02 treatment group; b: AUC of blood glucose change curves in the glucose tolerance test for mice in the solvent control group and GA-02 treatment group; c: blood glucose change curves at different time points in the insulin sensitivity test for mice in the solvent control group and GA-02 treatment group; d: AUC of blood glucose change curves in the insulin sensitivity test for mice in the solvent control group and GA-02 treatment group.

[0045] Figure 18Example 6 describes the effects of GA-02 administration on body weight and food intake in non-obese mice. (ac) Lean mice weighing 22g were treated with a blank solvent and GA-02 for 14 days; (a) changes in body weight and (b) percentage changes in body weight, (c) average daily food intake; (n=6, blank solvent group; n=8, GA-02 treatment group); (df) Mice fed a standard diet for 14 weeks and weighing approximately 28g were treated with a blank solvent and GA-02 for 14 days; (d) changes in body weight and (e) percentage changes in body weight, (f) average daily food intake; (n=6, blank solvent group; n=8, GA-02 treatment group).

[0046] Figure 19 Example 6 provides a graph showing the effects of GA-02 on body weight and food intake in ob / ob and db / db mice, where a: the effect of solvent control and continuous intraperitoneal administration of GA-02 for 14 days on db / db body weight; b: the percentage decrease in db / db body weight after continuous intraperitoneal administration of GA-02 after 14 days in both solvent control and GA-02; c: the average daily food intake of db / db mice after continuous intraperitoneal administration of GA-02 after 14 days in both solvent control and GA-02; d: the effect of solvent control and continuous intraperitoneal administration of GA-02 on body weight in ob / ob mice; e: the percentage decrease in ob / ob body weight after continuous intraperitoneal administration of GA-02 after 14 days in both solvent control and GA-02; f: the average daily food intake of ob / ob mice after continuous intraperitoneal administration of GA-02 after 14 days in both solvent control and GA-02.

[0047] Figure 20 Example 6 shows the effect of GA-02 gavage administration on body weight and food intake in mice with different degrees of obesity. (a–c) are high-fat induced obese mice, (d–f) are slightly obese mice fed with conventional diet, and (g–i) are lean mice treated with blank solvent and GA-02 (40 mg / kg) by gavage for 14 days. a: body weight (g) and (b) percentage change in body weight (%) of obese mice, c: average daily food intake (g), (5 mice / group); d: body weight (g) and (e) percentage change in body weight (%) of slightly obese mice, f: average daily food intake (g), (6 mice / group); g: body weight (g) and (h) percentage change in body weight (%) of lean mice, i: average daily food intake (g), (6 mice / group).

[0048] Figure 21 The figure provided in Example 7 shows the effect of GA-03 on body weight and food intake in high-fat induced obese mice. DIO mice were intraperitoneally injected with GA-03 (2, 4, 8 mg / kg) and blank solvent for 21 days. (a) Daily body weight change of mice, (b) Percentage change in body weight of mice (%), (c) Mass reduction in body weight of mice in each group after 14 days of administration (g). The ac experiment was conducted in two independent groups, with 6 obese model mice in each cage.

[0049] Figure 22Example 7 shows the effect of GA-03 on body fat content in high-fat induced obese mice, where a: average daily food intake of GA-03-treated obese mice in the first three days; b: daily weight change of GA-03-treated obese mice in the first three days; c: percentage decrease in weight of GA-03-treated obese mice in the first three days; and d: average daily food intake of GA-03-treated obese mice in the first, second, and third weeks.

[0050] Figure 23 The figures provided in Example 7 show the changes in body size, liver, epididymal fat, and fasting blood glucose in obese mice before and after GA-03 treatment. Figure a shows the changes in the back shape of obese mice treated with low, medium, and high doses (2, 4, 8 mg / kg); figure b shows the changes in the abdomen shape of obese mice treated with low, medium, and high doses (2, 4, 8 mg / kg); figure c shows the changes in the shape and mass of liver, kidney, and epididymal fat in obese mice treated with low, medium, and high doses (2, 4, 8 mg / kg); and figure f shows the fasting blood glucose levels in obese mice treated with low, medium, and high doses (2, 4, 8 mg / kg).

[0051] Figure 24 The images shown in Example 7 are the results of liver function tests, including Oil Red O staining and H&E staining of liver sections in obese mice induced by high-fat diet and treated with GA-03 before and after treatment. In the images, a: Oil Red O staining and H&E staining of liver sections in obese mice after three weeks of treatment with blank solvent and high-dose GA-03 (8 mg / kg); b: Changes in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in obese mice before and after treatment (blank control group n=5; GA-03 group n=5).

[0052] Figure 25 The figures provided in Example 7 show the results of glucose tolerance and insulin sensitivity in obese mice before and after GA-03 treatment, where a: blood glucose change curves at different time points in the glucose tolerance test for mice in the solvent control group and the GA-03 treatment group; b: AUC of blood glucose change curves in the glucose tolerance test for mice in the solvent control group and the GA-03 treatment group; c: blood glucose change curves at different time points in the insulin sensitivity test for mice in the solvent control group and the GA-03 treatment group; d: AUC of blood glucose change curves in the insulin sensitivity test for mice in the solvent control group and the GA-03 treatment group.

[0053] Figure 26 The effects of GA-03 administration provided in Example 7 on body weight and food intake in lean and mildly obese mice were investigated. Lean mice were fed 20g and 30g of normal diet and intraperitoneally injected with GA-03 (2, 4, 8 mg / kg) and a blank solvent for 21 days. (a) Changes in daily body weight and (b) average food intake of 20g lean mice, and (c) changes in daily body weight and (d) average food intake of 30g lean mice were also shown. The experiment was conducted in two independent groups, with 6 mice per cage.

[0054] Figure 27Example 7 shows the results of glucose tolerance and insulin sensitivity in lean mice before and after GA-03 treatment. Lean mice were intraperitoneally injected with GA-03 (2, 4, 8 mg / kg) and a blank solvent for 21 days before glucose tolerance and insulin tolerance tests were performed. Figure a shows the blood glucose change curves at different time points in the glucose tolerance test for mice in the solvent control group and the GA-03 treatment group; figure b shows the AUC of the blood glucose change curves in the glucose tolerance test for mice in the solvent control group and the GA-03 treatment group; figure c shows the blood glucose change curves at different time points in the insulin sensitivity test for mice in the solvent control group and the GA-03 treatment group; and figure d shows the AUC of the blood glucose change curves in the insulin sensitivity test for mice in the solvent control group and the GA-03 treatment group. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0056] The present invention provides a drug design method based on natural products, comprising the following steps:

[0057] (1) Select natural products with specific biological activities to be improved and obtain their molecular structures;

[0058] (2) Using the molecular structure obtained in step (1) as the template molecule, select natural products with similarity to the template molecule within the threshold range and having the specific biological activity as the candidate reference molecule set.

[0059] The threshold range is a range where the structural similarity score with the template molecule is above 0.2, preferably below 0.9.

[0060] Preferably, the natural products in the candidate reference molecular set have a relative biomass in the corresponding host plant that exceeds a preset biomass threshold; the biomass threshold is preferably 1%.

[0061] (3) Select one or more reference molecules from the set of reference molecules obtained in step (2), compare them with the template molecule, and obtain active functional groups with differences; construct the active functional groups with differences onto the molecular backbone shared by the template molecule and the reference molecule to obtain an improved molecule with specific biological activity.

[0062] The selection of one or more reference molecules from the set of reference molecules obtained in step (2) specifically involves: selecting one or more reference molecules from the set of reference molecules obtained in step (2) that have the same target and / or action pathway;

[0063] The specific steps for obtaining differentiated active functional groups are as follows: the active functional groups of the reference molecule are screened and determined through physiological models or molecular docking models, and compared with the template to obtain differentiated active functional groups.

[0064] The compound molecules designed by the drug design method can be prepared according to the following method:

[0065] S1. Select compounds with relatively high bioavailability in the corresponding host plant species from the template molecule and reference molecule as raw materials; usually, the template molecule has excellent biological activity, but it may be limited by its bioavailability and therefore has poor drug-like properties, while the reference molecule is obtained through molecular screening, so its natural source cost is controllable, so the compound of the reference molecule is selected as the raw material.

[0066] S2. Through chemical reaction, active functional groups with differences between the template molecule and the reference molecule are constructed onto the raw material to obtain the compound molecule designed by the drug design method.

[0067] This invention targets metabolic syndrome caused by chronic inflammation, such as obesity. According to the method provided by this invention, compounds with anti-inflammatory and therapeutic effects on metabolic syndrome, including obesity, were designed and screened.

[0068] The present invention provides a pentacyclic triterpenoid compound, a molecular skeleton shared by a template pentacyclic triterpenoid compound and a natural product having a structural similarity score of 0.2 or higher with the template pentacyclic triterpenoid compound, and active functional groups that differ between the template pentacyclic triterpenoid compound and the natural product; the template pentacyclic triterpenoid compound is tripterycin or corosolic acid; the natural product has anti-inflammatory activity, preferably with a relative biomass content of more than 1% in the corresponding host plant.

[0069] More preferably, the reference molecule of the natural product is 18-β-glycyrrhetinic acid;

[0070] The pentacyclic triterpenoid compound has an oleanane-type triterpenoid-30 carboxylic acid skeleton and a 3-carbonyl-1,2-enol structure on its A ring.

[0071] The pentacyclic triterpenoid compound is 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanolane-30 carboxylic acid or 11-dicarbonyl-12-ene-2α,3β-dihydroxy-oleanolane.

[0072] The pentacyclic triterpenoid compounds provided by this invention are prepared according to the following method:

[0073] S1. 18-β-glycyrrhetinic acid is selected as the raw material;

[0074] S2. Through a chemical reaction, the active functional groups that differ between the template molecule and the reference molecule are constructed onto the raw material to obtain the compound molecule designed by the drug design method; preferably, the active differential functional groups of triptolide and 18-β-glycyrrhetinic acid are: hydroxyl substitution on ring A and conjugated enone structure, which are constructed onto the 18-β-glycyrrhetinic acid molecule; or the active differential functional groups of corosolane and 18-β-glycyrrhetinic acid are: α and β hydroxyl substitution on ring A and conjugated enone structure, which are constructed onto the 18-β-glycyrrhetinic acid molecule.

[0075] By incorporating the active differential functional groups of triptolide and 18-β-glycyrrhetinic acid—hydroxyl substitution on ring A and a conjugated enone structure—into the 18-β-glycyrrhetinic acid molecule, the following steps were taken:

[0076] S2-1, Carbonyl groups are constructed onto 18-β-glycyrrhetinic acid via the Jones oxidation reaction, preferably yielding 3,11-dicarbonyl-12-en-oleanolane-30 carboxylic acid;

[0077] S2-2, The 2-enol structure is constructed onto 3,11-dicarbonyl-12-en-oleanane-30 carboxylic acid by oxygen oxidation mediated by tert-butanol / potassium tert-butoxide, preferably obtaining 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanane-30 carboxylic acid.

[0078] The active differential functional groups of corosolic acid and 18-β-glycyrrhetinic acid—α- and β-hydroxy substitutions on the A ring, and the conjugated enone structure—were constructed onto the 18-β-glycyrrhetinic acid molecule, specifically:

[0079] S2-1, Carbonyl groups are constructed onto 18-β-glycyrrhetinic acid via the Jones oxidation reaction, preferably yielding 3,11-dicarbonyl-12-en-oleanolane-30 carboxylic acid;

[0080] S2-2, The 2-enol structure was constructed onto 3,11-dicarbonyl-12-en-oleanane-30 carboxylic acid by oxygen oxidation mediated by tert-butanol / potassium tert-butoxide, to obtain 3,11-dicarbonyl-1,12-dien-2-hydroxy-oleanane-30 carboxylic acid.

[0081] S2-3, 2-α-3β-dihydroxy is constructed onto 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanol-30 carboxylic acid by Jones oxidation, potassium tert-butoxide / tert-butanol / oxygen oxidation, and sodium borohydride reduction, to obtain 11-dicarbonyl-12-ene-2α,3β-dihydroxy-oleanol.

[0082] The present invention provides pentacyclic triterpenoid compounds or pharmaceutically acceptable salts thereof (such as NH4) + Na + K + or Mg 2+ Salt, as demonstrated in experiments, has the ability to inhibit the expression of ICAM-1 in human microvascular endothelial cells, thus exhibiting a good inhibitory effect on chronic inflammation. It also shows significant therapeutic effects on metabolic syndrome, including obesity caused by overnutrition and type 2 diabetes.

[0083] The following is an example:

[0084] Example 1 applies the natural product-based design method provided by this invention, such as... Figure 1 The design of the anti-inflammatory active molecule shown includes the following steps:

[0085] (1) Tripterygium wilfordii was selected as the natural product molecule to be improved, i.e., the template molecule;

[0086] Tripterygium wilfordii is an active pentacyclic triterpenoid natural product isolated from the rhizome of Tripterygium wilfordii, a plant in the Celastraceae family. Its structure is as follows: Figure 2 As shown. Although triptolide possesses potent anti-inflammatory, anti-tumor, and obesity-treating activities, some undesirable pharmacological properties and its low abundance severely hinder its drug development. Tripterygium wilfordii is not a drug molecule with excellent medicinal properties, but it is a promising lead molecule for drug research.

[0087] (2) Using the molecular structure of triptolide as a template molecule, natural triterpenoid molecules with similar skeletal structures were screened from natural product databases. Simultaneously, the bio-content of these triterpenoids in host plants was reviewed from original research literature on related natural products, and their molecular structural formulas, CAS numbers, physicochemical properties, plant sources, and original literature were recorded. The obtained information was then compiled into a triterpenoid natural product database.

[0088] The 3D molecular structures of each natural product in the constructed triterpenoid natural product database were exported one by one from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ). These structures were then compared with the 3D molecular structure of triptolide using structure alignment software (Sybyl X2.0) to obtain a structural similarity score. A two-dimensional correlation diagram was constructed by combining the relative bio-content of the obtained natural products in their respective host plants with the structural similarity score of triptolide. A subset of triterpenoid compounds with a structural similarity score higher than 0.2 and a bio-content higher than 1% was selected. A comprehensive analysis of each natural product in this subset was then performed to select a candidate compound subset, including: oleanolic acid, corosolic acid, hawthorn acid, betulinic acid, ursolic acid, desmethylzelamylaldehyde, 18-α-glycyrrhetinic acid, 18-β-glycyrrhetinic acid, and asiatic acid.

[0089] (3) By simulating the inflammatory physiological model of lymphocyte infiltration into damaged tissue through blood vessels mediated by the expression of cell adhesion molecule (ICAM-1) in human microvascular endothelial cells (HMEC-1), candidate molecules with the strongest inhibitory activity of ICAM-1 were screened. The specific steps are as follows:

[0090] Pharmacological experimental methods and results of pentacyclic triterpenoid natural products inhibiting the expression of ICAM-1 in human microvascular endothelial cells:

[0091] 1) HMEC-1 cell culture was as follows: HMEC-1 cells were cultured in MCDB131 medium (purchased from Sigma-Aldrich, USA), supplemented with 10% fetal bovine serum (FBS, purchased from Zhejiang Tianhang Biotechnology Co., Ltd.), penicillin (100 U / ml and streptomycin, 1 μg / ml hydrocortisone (Sangon Biotech (Shanghai) Co., Ltd.), and human recombinant growth factor (hEGF, Sangon Biotech (Shanghai) Co., Ltd.), and cultured at 37℃, 5% CO2, and 100% humidity.

[0092] 2) HMEC-1 cells were seeded into 24-well plates and cultured in a cell culture incubator at 37°C, 5% CO2, and 100% humidity. When the cells reached 80% confluence, different concentrations of drugs were added for 3 hours (1 μM triptolide was used as a positive control). After treatment, 1 μg / ml LPS was added for 12 hours to activate the NF-κB signaling pathway. After activation, the supernatant was discarded, and the cells were washed three times with PBS. 100 μL of 0.25% trypsin (containing 0.5 mM EDTA) was added and the cells were digested in a 37°C incubator for 3 minutes. After most of the cells detached, 150 μL of MCDB131 complete medium was added to stop the digestion. The cells were shaken for 3 minutes and then transferred to V-shaped 96-well plates (Corning). The cells were centrifuged at 4°C for 3 minutes and 2000 rpm to pellet the cells. The supernatant was discarded, and 100 μL of pH 7.4 buffer A (PBS + 0.5% BSA + 1 mM EDTA) was added to each well. Wash once with MgCl2, centrifuge at 3000 rpm for 3 min at 4℃, remove buffer A, add 100 μL of PBS solution containing 5% bovine serum albumin to each well, and block by shaking at 120 rpm for 30 min at room temperature. After blocking, centrifuge at 2000 rpm for 3 min at 4℃, discard the supernatant, add 20 μL of buffer A containing 5 μg / ml primary anti-ICAM-1 antibody LB-2, and incubate by shaking at 120 rpm for 1 h at room temperature. Use buffer A without anti-ICAM-1 antibody LB-2 as a negative control. The expression level of ICAM-1 protein in HMEC-1 cells was indirectly detected by detecting the anti-ICAM-1 fluorescence value by flow cytometry. Triple-dose wells were set for each concentration, with PBS as a blank control and 1 μM triptolide as a positive control to analyze the inhibitory effect of different concentrations of drugs on ICAM-1 expression levels.

[0093] like Figure 3 As shown, the results indicate that 18-β-glycyrrhetinic acid is the natural product with the best inhibitory activity against ICAM-1, the highest bioavailability, and the lowest cost among candidate triterpenoid natural products, but its anti-inflammatory bioactivity is still somewhat inferior to that of triptolide.

[0094] The structures of the screened natural products 18-β-glycyrrhetinic acid and tripterygium oleanol were compared and analyzed. Based on the differences in molecular structure, a modification scheme for the active functional group was made, and the target compound 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanol-30 carboxylic acid (GA-02) was designed.

[0095] The GA-02 synthesis route and actual synthesis method are described in Example 3. Its anti-inflammatory and anti-metabolic syndrome activities were evaluated using the HMEC-1 cell screening model, as described in Examples 5 and 6.

[0096] Example 2

[0097] The steps are the same as in Example 1. The template molecule is corosolic acid, and the reference molecule is 18-β-glycyrrhetinic acid with a biological content of more than 1%. The similarity score between the two is 0.4.

[0098] Corosolic acid is a triterpenoid compound naturally found in Lagerstroemia indica (Figure 4), a plant in the Rosaceae family. It exists in the plant in free form or as a saponin. In the plant, it often coexists with its isomer, ursolic acid (2α-hydroxyoleanolic acid), which has similar structures and chemical properties, making isolation difficult. In vitro and in vivo experiments show that corosolic acid can promote glucose transport, thereby enhancing cellular glucose absorption and utilization, thus achieving its hypoglycemic effect. Its stimulatory effect on glucose transport is similar to that of insulin; therefore, corosolic acid is also known as plant insulin. Animal experiments show that corosolic acid has a significant hypoglycemic effect in both normal rats and hereditary diabetic mice. Its stimulatory effect on glucose transport is similar to that of insulin; therefore, corosolic acid is also known as plant insulin.

[0099] Structural comparison analysis was performed on 18-β-glycyrrhetinic acid and corosolic acid. Based on the structural differences between the two, a molecular modification scheme was designed, and the target compound 11-dicarbonyl-12-ene-2α,3β-dihydroxy-oleanolane (GA-03) was designed.

[0100] The GA-03 synthesis route and actual synthesis method are described in Examples 3 and 4; the bioactivity of the target molecule and corosolic acid was measured at the cellular level, and its similar pharmacological effects to corosolic acid were verified using obese and type 2 diabetic mouse models, as described in Examples 5 and 7.

[0101] Example 3 Synthesis and Identification of 3,11-Dicarbonyl-1,12-diene-2-hydroxy-oleanolane-30 carboxylic acid (GA-02)

[0102] Structural modification schemes and synthetic routes using 18-β-glycyrrhetinic acid as template molecules are as follows: Figure 5 As shown. The 3-ene-3-hydroxy-2-one structural module of triptolide was transferred to the A ring of glycyrrhetinic acid, while keeping the other structures of 18-β-glycyrrhetinic acid unchanged; the preparation method of the designed compound is as follows: 18-β-glycyrrhetinic acid was oxidized with John's reagent to prepare 3,11-dicarbonyl-12-ene-oleanolane-30 carboxylic acid (2); compound (2) was oxidized in tert-butanol solvent by potassium tert-butoxide / oxygen to prepare 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanolane-30 carboxylic acid (3); compound (3) was recrystallized in methanol / dichloromethane mixed solvent to obtain a crystalline pure product. The specific implementation scheme is as follows:

[0103] Synthesis of 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanolane-30 carboxylic acid

[0104] S2-1. Carbonyl groups are constructed onto 18-β-glycyrrhetinic acid via the Jones oxidation reaction to obtain 3,11-dicarbonyl-12-en-oleanolane-30 carboxylic acid.

[0105] Option 1: Glycyrrhetinic acid (GA, 23.5g) Add 50 mmol of chromium trioxide to a 500 mL round-bottom flask, add 300 mL of acetone and 50 mL of dichloromethane, and stir at 30 °C for 1 hour until completely dissolved. Add 20 mL of freshly prepared John's reagent (2.62 g of chromium trioxide dissolved in a small amount of water, then slowly added to 2.3 mL of concentrated sulfuric acid, and then diluted with water to 10 mL). Monitor the reaction of the starting material by TLC (petroleum ether: ethyl acetate: acetic acid = 2:1:0.01) until complete. Filter to remove insoluble matter, remove most of the acetone under reduced pressure, add 200 mL of distilled water to the residue, and extract with 250 mL of ethyl acetate three times. Wash the organic phase with 100 mL of saturated brine twice, dry with anhydrous sodium sulfate for 24 hours, and then recover the solvent under reduced pressure. Dissolve the remaining crude product in 300 mL of ethanol at 60 °C. After the solution is clear, filter while hot. Crystallize the filtrate at room temperature for 48 hours, filter and collect the colorless crystals, and dry at 60 °C for 24 hours to obtain 12.87 g of pure product, yield 55%, MP > 300 °C.

[0106] Option 2: Add glycyrrhetinic acid (GA, 23.5g 50mmol) to a 500mL round-bottom flask, add 200mL of dichloromethane and 200mL of acetone mixture, and stir at 30℃ for 1 hour until completely dissolved. Add 30mL of freshly prepared John's reagent (dissolve 13.1g of chromium trioxide in a small amount of water, then slowly add 11.5mL of concentrated sulfuric acid, and then dilute with water to 50mL). Monitor the reaction of the starting materials by TLC (petroleum ether: ethyl acetate: acetic acid = 2:1:0.01) until complete, then filter to remove the precipitate. The insoluble matter was removed under reduced pressure to remove most of the solvent. The residue was added to 200 mL of distilled water and extracted with ethyl acetate (250 mL × 3). The organic phase was washed with saturated brine (100 mL × 2). After drying with anhydrous sodium sulfate for 24 hours, the solvent was recovered under reduced pressure. The remaining crude product was dissolved in 300 mL of ethanol at 60 °C. After the solution became clear, it was filtered while hot. The filtrate was crystallized at room temperature for 48 hours. The colorless crystals were collected by filtration and dried at 60 °C for 24 hours to obtain 17.5 g of pure product, yield 75%, MP > 300 °C.

[0107] The product's 1H NMR spectrum is as follows Figure 6a As shown, the proton and carbon NMR spectra are as follows: Figure 6b As shown.

[0108] S2-2, The 2-enol structure was constructed onto 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanol-30 carboxylic acid via an oxygen oxidation reaction mediated by tert-butanol / potassium tert-butoxide, to obtain 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanol-30 carboxylic acid.

[0109] Option 1:

[0110] Potassium tert-butoxide (22.44 g, 20 mmol) was dissolved in 250 mL of tert-butanol and stirred at 40 °C for 30 minutes. GA-01 (4.68 g, 10 mmol) was added to the reaction solution in one go, and the mixture was stirred rapidly until completely dissolved. The reaction was stirred at 40 °C for 3 hours. The reaction was detected by TLC (petroleum ether: ethyl acetate: acetic acid = 3:1:0.01) to indicate the end of the reaction. The pH was adjusted to 3-4 with 4 mol / L sodium hydroxide solution, and most of the tert-butanol was removed under reduced pressure. 200 mL of distilled water was added, and the mixture was extracted with 250 mL of ethyl acetate three times. The organic phase was washed twice with 200 mL of saturated brine and dried over anhydrous sodium sulfate for 24 hours. The solvent was recovered under reduced pressure to obtain the crude product. The crude product was subjected to column chromatography with a petroleum ether: ethyl acetate: acetic acid ratio of 4:1:0.01. The pure fraction was collected, and the solvent was recovered to obtain 2.65 g of pure white powder, with a yield of 55%.

[0111] Option 2: GA-01 (4.68 g, 10 mmol) was dissolved in 250 mL of tert-butanol and stirred at 40 °C for 30 minutes. Potassium tert-butoxide (22.44 g, 20 mmol) was added to the reaction solution in one go, and the mixture was stirred rapidly until completely dissolved. The reaction was carried out at 45 °C for 3 hours. The reaction was detected by TLC (petroleum ether: ethyl acetate: acetic acid = 3:1:0.01) to indicate the end of the reaction. The pH was adjusted to 3-4 with 4N sodium hydroxide solution, and most of the tert-butanol was removed under reduced pressure. 200 mL of distilled water was added, and the mixture was extracted with 250 mL of ethyl acetate three times. The organic phase was washed twice with 150 mL of saturated brine and dried over anhydrous sodium sulfate for 24 hours. The solvent was recovered under reduced pressure to obtain the crude product. The crude product was then subjected to column chromatography with a petroleum ether: ethyl acetate: acetic acid = 4:1:0.01. The pure fraction was collected and the solvent was recovered to obtain 2.16 g of pure white powder GA-02, with a yield of 45%.

[0112] Option 3: Dissolve potassium tert-butoxide (33.66 g, 30 mmol) in 250 mL of tert-butanol and stir at 40 °C for 30 minutes. Add GA-01 (4.68 g, 10 mmol) to the reaction solution all at once and stir rapidly until completely dissolved. Introduce air into the reaction solution using a delivery tube and stir at 45 °C for 3 hours. Detect the reaction by TLC (petroleum ether: ethyl acetate: acetic acid = 3:1:0.01) to indicate completion. Adjust the pH to 3-4 with 4 mol / L sodium hydroxide solution and reduce the pressure. Most of the tert-butanol was removed, and the product was extracted with 200 mL of distilled water and 250 mL of ethyl acetate three times. The organic phase was washed twice with 200 mL of saturated brine and dried over anhydrous sodium sulfate for 24 hours. The solvent was recovered under reduced pressure to obtain a white solid powder. The crude product was dissolved in a mixed solvent of 300 mL methanol and 50 mL dichloromethane and heated to 60 °C until completely dissolved. The solution was filtered while hot, and the filtrate was recovered and recrystallized at room temperature for 48 hours. The resulting colorless crystals were collected and dried at 60 °C to obtain 3.13 g of pure GA-02 product (65% yield).

[0113] Scheme 4: Potassium tert-butoxide (33.66 g, 30 mmol) was dissolved in 250 mL of tert-butanol and stirred at 40 °C for 30 minutes. GA-01 (4.68 g, 10 mmol) was added to the reaction solution all at once and stirred rapidly until completely dissolved. Air was introduced into the reaction solution using a delivery tube, and the reaction was stirred at 45 °C for 3 hours. The reaction was detected by TLC (petroleum ether: ethyl acetate: acetic acid = 3:1:0.01) to indicate the end of the reaction. The pH was adjusted to 3-4 with 4 mol / L sodium hydroxide solution, and most of the tert-butanol was removed under reduced pressure. 200 mL of distilled water was added, and the mixture was extracted with 250 mL of ethyl acetate three times. The organic phase was washed twice with 200 mL of saturated brine and dried over anhydrous sodium sulfate for 24 hours. The solvent was recovered under reduced pressure to obtain a white solid powder. The product was placed in 300 mL of methanol, and 50 mL of ammonia was added. The mixture was heated at 50 °C until completely dissolved. The product was filtered while hot, and the filtrate was recovered and recrystallized at room temperature for 48 hours. The obtained colorless crystals were collected and dried at 60 °C to obtain 2.16 g of pure GA-02 product (yield 45%). The product's 1H NMR spectrum is as follows Figure 7a As shown, the proton and carbon NMR spectra are as follows: Figure 7b As shown.

[0114] Example 4 Synthesis and Identification of 11-Dicarbonyl-12-ene-2α,3β-dihydroxy-oleanane (GA-03)

[0115] For modification schemes and synthetic routes using corosolic acid as the template molecule and 18-β-glycyrrhetinic acid as the reference molecule, such as... Figure 8As shown: The α- and β-hydroxyl groups on the A ring of corosolic acid were substituted, and the conjugated enone structure was transferred to the A ring of glycyrrhetinic acid, while keeping the other structures of 18-β-glycyrrhetinic acid unchanged. The preparation method of the designed compound is as follows: 18-β-glycyrrhetinic acid was oxidized with John's reagent to prepare 3,11-dicarbonyl-12-en-oleanolane-30 carboxylic acid (GA-01); compound GA-01 was oxidized in tert-butanol solvent by potassium tert-butoxide / oxygen to prepare 3,11-dicarbonyl-1,12-dien-2-hydroxy-oleanolane-30 carboxylic acid (GA-02); compound GA-02 was recrystallized in a methanol / dichloromethane mixed solvent to obtain a crystalline pure product; GA-02 was reduced in tetrahydrofuran solvent by sodium borohydride to prepare GA-03. The specific implementation scheme for the preparation of GA-02 is the same as in Example 3. The steps for synthesizing GA-03 using GA-02 as a raw material are as follows:

[0116] Option 1

[0117] Compound GA-02 (4.82 g, 10 mmol) was added to a 500 mL round-bottom flask, dissolved in 100 mL of tetrahydrofuran, and heated in a 42 °C water bath. Sodium borohydride (378 mg, 100 mmol) was added in batches, and the mixture was stirred with a magnetic rotor. During the reaction, the reaction was monitored by TLC (petroleum ether:acetone:acetic acid = 3:1:0.01 or petroleum ether:acetone:acetic acid = 5:2:0.01) to ensure complete reaction. After the reaction was complete, 50 mL of freshly prepared 2 mol / L dilute hydrochloric acid was added to neutralize the reaction solution and adjust the pH to 3-4. Extract with 250 mL of ethyl acetate (3 times). Wash the extract with 500 mL of distilled water 1-2 times. After separation, wash the aqueous phase with 250 mL of saturated brine (3 times). Mix the organic phases and dry with anhydrous sodium sulfate for 24 hours. Then, recover the solvent under reduced pressure. Dissolve the crude product in 300 mL of ethanol at 60 °C. After the solution is clear, filter while hot. Crystallize the filtrate at room temperature for 48 hours. Collect the colorless crystals by filtration and dry at 60 °C for 24 hours to obtain 3.1 g of pure product, yield 64.6%, MP > 300 °C.

[0118] Option 2

[0119] Compound GA-02 (4.82 g, 10 mmol) was added to a 500 mL round-bottom flask and dissolved in 100 mL of anhydrous methanol at room temperature. Sodium borohydride (756 mg, 200 mmol) was added in batches and stirred with a magnetic rotor. During the reaction, the reaction was monitored by TLC (petroleum ether: acetone: acetic acid = 5:2:0.01) until complete. After the reaction was complete, some methanol was evaporated, and the pH was adjusted to 3-4 by adding 50 mL of 2 mol / L dilute hydrochloric acid. The mixture was extracted with 250 mL of ethyl acetate three times. The extract was washed 1-2 times with 500 mL of distilled water, and the aqueous phase was washed with 250 mL of saturated brine three times. The ethyl acetate phase was mixed and dried with anhydrous sodium sulfate for 24 hours. The solvent was recovered under reduced pressure. The residual crude product was dissolved in 300 mL of ethanol at 60 °C. After the solution was clear, it was filtered while hot. The filtrate was crystallized at room temperature for 48 hours. The colorless crystals were collected by filtration and dried at 60 °C for 24 hours to obtain 3.5 g of pure product, yield 71.5%, MP > 300 °C. The product's 1H NMR spectrum is as follows Figure 9 As shown.

[0120] Example 5 Anti-inflammatory activity evaluation

[0121] HMEC-1 cells were cultured as follows: HMEC-1 cells were cultured in MCDB131 (purchased from Sigma) medium supplemented with 10% fetal bovine serum (FBS, purchased from Zhejiang Tianhang Biotechnology Co., Ltd.), penicillin (100 U / ml and streptomycin, 1 μg / ml hydrocortisone (purchased from Sangon Biotech) and human recombinant growth factor (hEGF, purchased from Sangon Biotech), and cultured at 37℃, 5% CO2 and 100% humidity.

[0122] HMEC-1 cells were seeded into 24-well plates and cultured in a cell culture incubator at 37°C, 5% CO2, and 100% humidity. When the cells reached 80% confluence, different concentrations of drugs were added for 3 hours (1 μM triptolide was used as a positive control). After treatment, 1 μg / ml LPS was added for 12 hours to activate the NF-κB signaling pathway. After activation, the supernatant was discarded, and the cells were washed three times with PBS. 100 μL of 0.25% trypsin (containing 0.5 mM EDTA) was added and the cells were digested in a 37°C incubator for 3 minutes. After most cells detached, 150 μL of MCDB131 complete medium was added to stop the digestion. The cells were shaken for 3 minutes and then transferred to V-shaped 96-well plates (Corning). The cells were centrifuged at 4°C for 3 minutes and 2000 rpm to pellet the cells. The supernatant was discarded, and 100 μL of pH 7.4 buffer A (PBS + 0.5% BSA + 1 mM EDTA) was added to each well. Wash once with MgCl2, centrifuge at 3000 rpm for 3 min at 4℃, remove buffer A, add 100 μL of PBS solution containing 5% bovine serum albumin to each well, and block by shaking at 120 rpm for 30 min at room temperature. After blocking, centrifuge at 2000 rpm for 3 min at 4℃, discard the supernatant, add 20 μL of buffer A containing 5 μg / mL primary anti-ICAM-1 antibody LB-2, and incubate by shaking at 120 rpm for 1 h at room temperature. Use buffer A without anti-ICAM-1 antibody LB-2 as a negative control. The expression level of ICAM-1 protein in HMEC-1 cells was indirectly detected by detecting the anti-ICAM-1 fluorescence value by flow cytometry. Triple-dose wells were set for each concentration, with PBS as a blank control and 1 μM triptolide as a positive control to analyze the inhibitory effect of different concentrations of drugs on ICAM-1 expression levels. Results are as follows. Figure 10 As shown.

[0123] Combined with the ICAM-1 cell expression and quantitative detection model, 1 μM triptolide significantly inhibited LPS-activated ICAM-1 expression, with an inhibitory effect comparable to that of the control group; GA-02 showed concentration-dependent ICAM-1 inhibitory activity, and 10 μM GA-02 showed ICAM-1 inhibitory activity comparable to that of 1 μM triptolide.

[0124] ICAM-1 is a downstream effector molecule regulated by the NF-κB signaling pathway, and NF-κB plays an important regulatory role in ICAM-1 gene expression. Tripterygium wilfordii can inhibit LPS-induced activation of the IKK / NF-κB signaling pathway. To verify that GA-02 also functions through the IKK / NF-κB signaling pathway, we constructed an NF-κB luciferase reporter gene system to verify the inhibitory activity of triptolide and GA-02 on the luciferase reporter gene. Experimental results showed that 1 μg / ml LPS activated the NF-κB pathway, significantly enhancing the fluorescence signal compared to the control group. 1 μM triptolide suppressed 70% of the fluorescence signal, and GA-02 showed concentration-dependent inhibition of fluorescence intensity. The activity of 10 μM triptolide was comparable to that of 1 μM triptolide. Simultaneously, both triptolide and GA-02 downregulated the transcription levels of LPS-activated inflammatory factors IL-1β, TNF-α, IL-6, and MCP-1 mRNA.

[0125] These experimental results indicate that triptolide and GA-02 exert similar anti-inflammatory activities through their similar molecular structures. Although the anti-inflammatory activity of GA-02 is 10 times lower than that of triptolide, its activity is significantly enhanced compared to 18β-glycyrrhetinic acid, demonstrating the effectiveness of the molecular design scheme. Using the same method, GA-03 showed anti-inflammatory activity that was essentially equivalent to GA-02 and superior to 18β-glycyrrhetinic acid.

[0126] Example 6: Experimental Verification of the Anti-metabolic Syndrome Effect of 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanolane-30 carboxylic acid (GA-02) Prepared in Example 2.

[0127] The experimental method for verifying the anti-metabolic syndrome effect is as follows:

[0128] 1) Establishment of an obese mouse model of metabolic syndrome and drug treatment experiments

[0129] The rearing method was as follows: 6-week-old SPF-grade C57BL / 6 mice (purchased from Hubei Provincial Center for Disease Control and Prevention) were housed in an SPF animal room at 22℃, at a rate of 4 mice / cage, on a 60% calorie high-fat diet (purchased from Bio-medicine) for 14 weeks. Mice had free access to food, and bedding was changed every two days. Body weight and blood glucose were monitored weekly. Drug administration began when blood glucose exceeded 11 mmol / L and body weight exceeded 42 g. The drug was dissolved in DMSO and administered intraperitoneally at doses of 4, 12, and 20 mg / kg for 14 consecutive days, with an injection volume of 25 μL per day. A solvent (25 μL DMSO) and a positive control (triptothion) were also included. Food intake and weight changes of mice in each group were measured daily. Blood routine and blood biochemical parameters were monitored on day 0 and day 14. Glucose tolerance (GTT) and insulin sensitivity (ITT) tests were performed, and body fat parameters of mice were measured on day 0 and day 14. Mice in the blank group and GA-02 treatment group were pathologically dissected to observe changes in organs before and after drug treatment, and samples were taken to prepare pathological sections.

[0130] 2) Establishment of a lean mouse model and drug treatment experiments

[0131] The feeding and experimental methods were as follows: Six-week-old SPF male C57BL / 6 mice (purchased from Hubei Provincial Center for Disease Control and Prevention) were housed in an SPF animal room at 22℃ (4 mice / cage) for two weeks on standard mouse feed (purchased from [source missing]). Mice had free access to food, and bedding was changed every two days. Body weight and blood glucose were measured weekly. Once the mice reached a weight of 22g, drug administration began. The drug was dissolved in DMSO and administered via intraperitoneal injection at a volume of 25uL per day. A solvent and positive drug control were also provided. Food intake and body weight were monitored daily for each group of mice. Complete blood count and blood biochemical parameters were monitored on days 0 and 14, and glucose tolerance test (GTT) and insulin sensitivity test (ITT) were performed simultaneously.

[0132] 3) Establish a mildly obese mouse model

[0133] The feeding and experimental methods were as follows: Six-week-old SPF male C57BL / 6 mice (purchased from Hubei Provincial Center for Disease Control and Prevention) were housed at a rate of 4 mice / cage in an SPF animal room at 22°C. They were fed standard mouse feed (purchased from [source not specified]) for 14 weeks with free access to food. Bedding was changed every two days, and body weight and blood glucose were measured weekly. Once the mice reached a weight of 30g, drug administration began. The drug was dissolved in DMSO solvent and administered via intraperitoneal injection at a volume of 25uL per day. A solvent and positive drug control were also included. Food intake and body weight were monitored daily for each group of mice. Complete blood count and blood biochemical parameters were monitored on days 0 and 14.

[0134] 4) Treatment experiments in ob / ob and db / db model mice

[0135] Eight-week-old male ob / ob and db / db mice were acclimatized to their environment under normal conditions for one week. They were then divided into two groups (two cages per group, five mice per cage) based on body weight and blood glucose levels. The experimental group mice were acclimatized to the solvent for 3 days (25 μL DMSO / day) before receiving an intraperitoneal injection of the drug (20 mg / kg; 25 μL per injection). The control group received the same volume of solvent. Drug administration began at 18:00 daily and continued for 14 days. Mouse body weight and food intake were recorded daily. The experimental results are as follows: Figure 11 As shown.

[0136] Modeling was achieved by feeding male C57BL / 6 mice a high-fat diet for 14 weeks. The results were as follows: Figure 12 As shown, mice weighing more than 42g and with blood glucose levels higher than 11mmol / L were considered obese model mice. The model mice were grouped according to blood glucose and body weight. Each group of model mice was given three doses of GA-02 (low, medium, and high, 4, 12, and 20 mg / kg, 25 μL / day) via intraperitoneal injection for 14 consecutive days. The blank control group was given intraperitoneal injection of DMSO (25 μL / day). Food intake and body weight were recorded daily. Two weeks after administration, the body weight of mice in the solvent control group did not fluctuate significantly. The body weight of mice in the low-dose group decreased from 42.50±1.71g to 38.46±0.95g, a decrease of 9.88±2.37%; the body weight of mice in the medium-dose group decreased from 41.41±1.17g to 35.45±1.15g, a decrease of 13.15±2.73%; and the body weight of mice in the high-dose group decreased from 42.14±1.37g to 30.07±1.23g, a decrease of 26.42±0.73%. The body weight of mice in each group showed a significant dose-dependent reduction.

[0137] During the 14-day drug administration period, the average daily food intake of mice in the solvent control group was 2.31±0.18 g in the first week, while the average daily food intake of the low, medium, and high dose groups was 1.83±0.28 g, 1.68±0.35 g, and 0.85±0.23 g, respectively, representing decreases of 20.8%, 27.3%, and 63.2% compared to the solvent control group. In contrast, the average daily food intake of mice in the solvent control group was 2.47±0.13 g in the first week, while the average daily food intake of the low, medium, and high dose groups was 1.55±0.12 g, 1.35±0.18 g, and 0.70±0.16 g, respectively, representing decreases of 37.2%, 45.3%, and 68.8% compared to the solvent control group. These results indicate that the body weight and average daily food intake of obese mice showed a drug concentration-dependent decreasing trend, with the high-dose group exhibiting the most significant appetite suppression and weight reduction.

[0138] Magnetic resonance imaging analysis of body fat showed that the lean body mass of mice treated with low, medium and high doses of drugs did not change significantly, but the fat content was significantly reduced compared with the obese model mice. In vivo fat imaging also showed that the body fat in the drug-treated group was significantly reduced compared with the model mice. By comparing the reduced body weight and the reduced fat weight, it was found that the reduction in body weight was mostly due to the reduction in adipose tissue mass. It is speculated that the reduction in body weight in mice is due to the reduction in food intake, which leads to the reduction of body weight by burning fat.

[0139] Dissection of mice revealed the following results: Figure 13 As shown, the mice treated with GA-02 were thinner than the control group. The large amount of fat accumulated in the abdomen of the obese mice was significantly reduced after two weeks of treatment with GA-02. Dissection of organs revealed that the obese mice had a large amount of fat in the heart, perirenal area, epididymis and subcutaneous tissue. The liver of the obese mice showed grayish-white fatty liver symptoms with visible lipid droplets. After treatment with GA-02, the liver color turned dark red. Moreover, the liver and epididymal fat weight of the mice in the low, medium and high dose groups decreased in a dose-dependent manner compared with the control group, and the fasting blood glucose level of the mice also dropped to normal levels.

[0140] Daily food intake in a high-fat-induced obese mouse model during the first three days, the first week, and the second week is shown in the experimental results. Figure 14 As shown, the average daily food intake in the high-dose treatment group was 2.0±0.18g on day 0. On the first day of treatment, the average daily food intake decreased to 0.60±0.02g, and the body weight decreased from 42.09±0.42g to 40.98±0.57g. On the second day, the average daily food intake decreased to 0.47±0.16g, and the body weight decreased to 39.94±0.62g. On the third day, the average daily food intake decreased to 0.39±0.11g, and the body weight decreased to 39.23±0.71g. The trend of body weight reduction in the high-dose group was the same as that in the paired quantitative feeding group, indirectly indicating that the decrease in body weight was due to the reduction in food intake.

[0141] Images showing changes in the internal organs of obese mice before and after treatment with GA-02, such as... Figure 15 As shown.

[0142] Dissection of obese model mice fed a high-fat diet revealed significant fat accumulation in major organs, with substantial fat deposits around the heart, visible lipid droplets in the liver, and obvious fatty liver symptoms. More seriously, a thick layer of white fat tightly encased the kidney tissue around the kidneys, and two large lumps of epididymal fat in the abdomen (Fig. 15a-d). After two weeks of treatment with GA-02, the accumulated fat in the visceral tissues of the mice completely disappeared, and it was difficult to find any visible fat (Fig. 15e-h). In the high-dose treatment group, the fat around the heart and kidneys completely disappeared, and no obvious visible fat was found in the subcutaneous tissue or viscera. No obvious lipid droplet accumulation was observed in the liver, and no fatty liver symptoms were observed, indicating that the previous fatty liver was completely relieved.

[0143] The results of liver sections stained with oil red O and H&E are as follows: Figure 16 As shown in Figure 16a, a large number of lipid droplets were visible in the liver of the blank control group, while the lipid droplets in the liver of the high-dose GA-02 treatment group completely disappeared. Liver function indicators such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly lower than those in the solvent control group (Figures 16b-c), returning to normal levels. Fasting blood glucose levels in the solvent treatment group were higher than 10 mmol / L, but fasting blood glucose levels in all groups were significantly lower than those in the solvent control group and returned to normal levels. These results indicate that the symptoms of fatty liver induced by a high-fat diet were effectively alleviated.

[0144] Results of glucose tolerance (GTT) and insulin sensitivity (ITT) tests are as follows Figure 17 As shown: Obesity caused by diet is often accompanied by symptoms of diabetes such as hyperglycemia and insulin resistance. We tested the glucose tolerance and insulin sensitivity of mice in the high-dose treatment group. The results showed that the mice treated with GA-02 had significantly improved glucose processing ability and insulin sensitivity, and fasting blood glucose levels were significantly reduced to normal levels, indicating that the symptoms of diabetes caused by obesity were well recovered. Figure 17 ).

[0145] Changes in body weight and food intake in lean and slightly obese mice after drug administration are as follows: Figure 18 As shown: If the decrease in body weight and food intake in obese mice is caused by toxic reactions, then the same phenomenon will also occur in the normal mouse group. We injected normal lean mice (weighing approximately 22g) intraperitoneally with blank solvent and high-dose GA-02. During the two-week treatment period, the food intake of the lean mice did not change significantly compared with the solvent control group, and their body weight did not decrease but instead showed an increasing trend. Figure 18 a) In the mildly obese group (weight approximately 28g), the solvent had no significant effect on food intake and body weight, while the high-dose GA-02 administration group showed significantly lower food intake and body weight compared to the solvent control group. Figure 18These experimental results indicate that the appetite-suppressing and weight-reducing effects of GA-02 are not due to toxicity, but are positively correlated with the degree of obesity in mice.

[0146] Changes in body weight and food intake in db / db and ob / ob mice after drug administration are as follows: Figure 19 As shown: Leptin receptor-deficient db / db mice were treated with blank solvent and GA-02 via intraperitoneal injection for 14 days. The food intake and body weight of the mice in the solvent group did not change significantly. The food intake of the mice in the GA-02 group did not change significantly compared with the solvent control group, and the body weight did not decrease but increased by 15% (Fig. 19a-c). This indicates that GA-02 has no effect on suppressing appetite and reducing weight in db / db mice. Similarly, leptin-deficient ob / ob mice were intraperitoneally injected with blank solvent and GA-02 for 14 days. Compared with the blank solvent group, the GA-02 treatment group showed a decrease in food intake, but this was not significant. The mice gained 10% in the first five days, but then returned to their initial experimental weight and remained stable. Overall, the weight did not decrease significantly (Figure 19d-f). The results also indicate that GA-02 did not significantly inhibit appetite or reduce weight in ob / ob mice. The experimental results indirectly suggest that the appetite inhibition and weight reduction effects of GA-02 in obese mice are related to the leptin pathway, and that GA-02 has a positive regulatory effect on leptin.

[0147] Changes in body weight and food intake in high-fat induced obese mice and lean mice after gavage administration are as follows: Figure 20 As shown: The experimental results indicate that intraperitoneal injection of GA-02 has no effect on the food intake and weight of lean mice, but has a significant effect on suppressing appetite and reducing weight in slightly obese mice, and a strong effect on suppressing appetite and reducing weight in obese mice. So, will the same phenomenon occur if the administration method is changed? Lean mice weighing about 25g, slightly obese mice weighing about 30g, and obese mice weighing about 45g were administered by gavage with blank solvent and high dose of GA-02 (40mg / kg), respectively.

[0148] Experimental results showed that GA-02 administered by gavage still had a strong appetite-suppressing and weight-reducing effect on obese mice, with a 21% reduction in body weight and a 50% reduction in average daily food intake after two weeks of administration; the mildly obese group experienced a 16% reduction in body weight and a 30% reduction in average daily food intake; GA-02 had no significant effect on the body weight and food intake of lean mice. These experimental results further demonstrate that the appetite-suppressing and weight-reducing effects of GA-02 are positively correlated with the degree of obesity in mice. Figure 20 ).

[0149] Example 7: Experimental Verification of the Anti-metabolic Syndrome Effect of 11-dicarbonyl-12-ene-2α,3β-dihydroxyoleanane (GA-03) Prepared in Example 3

[0150] 1) Establish an obese mouse model of metabolic syndrome

[0151] The feeding method was as follows: 6-week-old SPF-grade C57BL / 6 mice (purchased from Hubei Provincial Center for Disease Control and Prevention) were housed in an SPF animal room at 22℃, at a rate of 4 mice / cage, on a 60% calorie high-fat diet (purchased from Bio-medicine) for 14 weeks. Mice had free access to food, and their bedding was changed every two days. Weight and blood glucose were monitored weekly. Once blood glucose exceeded 11 mmol / L and weight exceeded 42 g, drug administration began. The drug was dissolved in DMSO and administered intraperitoneally at doses of 2, 4, and 8 mg / kg for 21 consecutive days, with an injection volume of 25 μL per day. A 25 μL DMSO solvent was prepared simultaneously. Food intake and weight changes were measured daily for each group of mice. Complete blood count and biochemical parameters were monitored on days 0 and 14. Glucose tolerance test (GTT) and insulin sensitivity test (ITT) were also performed. Body fat parameters were measured on days 0 and 14. Mice in the control group and GA-02 treatment group underwent pathological autopsy to observe organ changes before and after drug treatment, and samples were taken for pathological section preparation. Experimental results are as follows: Figure 21 As shown.

[0152] Modeling was achieved by feeding male C57BL / 6 mice a high-fat diet for 14 weeks, such as... Figure 22 As shown, mice weighing more than 42g and with blood glucose levels higher than 11mmol / L were considered obese model mice. The model mice were grouped according to blood glucose and body weight. Each group of model mice was given three doses of GA-03 (low, medium, and high, 2, 4, and 8 mg / kg, 25μL / day) via intraperitoneal injection for 14 consecutive days. The blank control group was given intraperitoneal injection of DMSO (25μL / day). Food intake and body weight were recorded daily. Three weeks after administration, the body weight of mice in the solvent control group did not fluctuate significantly. The body weight of mice in the low-dose group decreased from 47.11±1.32g to 37.49±1.28g, a decrease of 20.40±1.89%; the body weight of mice in the medium-dose group decreased from 46.94±1.41g to 33.99±2.30g, a decrease of 27.63±3.39%; and the body weight of mice in the high-dose group decreased from 47.49±0.97g to 31.45±1.81g, a decrease of 33.80±2.95%. The body weight of mice in each group showed a significant dose-dependent reduction.

[0153] Daily food intake in a high-fat-induced obese mouse model during the first three days, the first week, and the second week is shown in the experimental results. Figure 23As shown, in the high-dose treatment group, the average daily food intake on day 0 was 3.14±0.40g. On the first day of treatment, the average daily food intake decreased to 0.65±0.02g, and the body weight decreased from 47.49±0.97g to 46.18±0.95g. On the second day, the average daily food intake decreased to 0.57±0.01g, and the body weight decreased to 44.74±0.76g. On the third day, the average daily food intake decreased to 0.66±0.06g, and the body weight decreased to 43.13±0.65g. The trend of body weight reduction in the high-dose group was the same as that in the paired quantitative feeding group, indirectly indicating that the decrease in body weight was due to the reduction in food intake.

[0154] Dissection of mice revealed that the mice treated with GA-03 were thinner than the control group. The large amount of fat accumulated in the abdomen of obese mice was significantly reduced after two weeks of treatment with GA-03 (Fig. 23a-b). Organ dissection revealed that obese mice had a large amount of fat in the heart, perirenal area, epididymis, and subcutaneous tissue. The liver of the obese mice showed grayish-white fatty liver symptoms with visible lipid droplets. After treatment with GA-03, the liver color turned dark red (Fig. 23c). Moreover, the fat weight of the liver and epididymis of mice in the low, medium, and high dose groups decreased in a dose-dependent manner compared with the control group (Fig. 23d-e), and the fasting blood glucose level of the mice also dropped to normal levels (Fig. 23f).

[0155] Oil Red O staining and H&E staining results of liver sections showed that a large number of lipid droplets were visible in the liver of the blank control group, while the lipid droplets in the liver of the high-dose GA-03 treatment group completely disappeared. Figure 24 a) The liver function indicators AST and ALT were significantly lower than those in the solvent control group (Figure 24b-c), indicating that the symptoms of fatty liver caused by a high-fat diet were well relieved.

[0156] Results of glucose tolerance (GTT) and insulin sensitivity (ITT) tests are as follows Figure 25 As shown, obesity caused by overnutrition is often accompanied by symptoms of diabetes such as hyperglycemia and insulin resistance. We tested the glucose tolerance and insulin sensitivity of mice in the high-dose treatment group. The experimental results showed that the mice's ability to process blood glucose and insulin sensitivity were significantly improved after treatment with low, medium and high doses of GA-03, and the fasting blood glucose level was significantly reduced to the normal level, indicating that the symptoms of diabetes caused by obesity were well relieved (Figure 25).

[0157] 2) Establish a lean mouse model

[0158] The feeding and experimental methods were as follows: Six-week-old SPF male C57BL / 6 mice (purchased from Hubei Provincial Center for Disease Control and Prevention) were housed at a rate of 4 mice / cage in an SPF animal room at 22°C. They were fed standard mouse feed (purchased from [source not specified]) for two weeks with free access to food. Bedding was changed every two days, and body weight and blood glucose were measured weekly. Once the mice reached a weight of 22g, drug administration began. The drug was dissolved in DMSO solvent and administered via intraperitoneal injection at a volume of 25uL per day. A solvent and positive drug control were also included. Food intake and body weight were monitored daily in each group. Complete blood count and blood biochemical parameters were monitored on days 0 and 21, and glucose tolerance test (GTT) and insulin sensitivity test (ITT) were performed. Results are as follows: Figure 26 As shown

[0159] 3) Establish a mildly obese mouse model

[0160] The feeding and experimental methods were as follows: Six-week-old SPF male C57BL / 6 mice (purchased from Hubei Provincial Center for Disease Control and Prevention) were housed at a rate of 4 mice / cage in an SPF animal room at 22°C. They were fed standard mouse feed (purchased from [source not specified]) for 14 weeks with free access to food. Bedding was changed every two days, and body weight and blood glucose were measured weekly. Once the mice reached a weight of 30g, drug administration began. The drug was dissolved in DMSO solvent and administered via intraperitoneal injection at a volume of 25uL per day. A solvent and positive drug control were also included. Food intake and body weight were monitored daily for each group of mice. Complete blood count and blood biochemical parameters were monitored on days 0 and 21. Results are as follows: Figure 27 As shown.

[0161] Experimental results showed that intraperitoneal injection of GA-03 had no effect on food intake and weight in lean mice, but had a significant effect on appetite suppression and weight reduction in slightly obese mice, and a strong effect on appetite suppression and weight reduction in obese mice.

[0162] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of a pentacyclic triterpenoid and pharmaceutically acceptable salts thereof, characterized in that, The pentacyclic triterpenoid compound is 3,11-dicarbonyl-1,12-diene-2-hydroxy-oleanolane-30 carboxylic acid or 11-dicarbonyl-12-ene-2α,3β-dihydroxy-oleanolane, and is used to prepare drugs for the treatment of surgery-deficient obesity or drugs for the treatment of type 2 diabetes.

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