Compound for preventing and treating fatty degenerative liver disease related to metabolic dysfunction and application thereof

Abstract

The invention provides a compound for preventing and treating fatty degenerative liver diseases related to metabolic dysfunction and application thereof. The invention discloses Eupatulin (EUP) and a novel derivative thereof, and also discloses a novel application of EUP and the novel derivative thereof, and the EUP and the novel derivative thereof are used for preventing and / or treating metabolic disorder related fatty degenerative liver disease (MASLD) or MASLD related metabolic diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology; more specifically, the present invention relates to eupatilin or its derivatives, and their use in the preparation of a composition for preventing and treating metabolic dysfunction-associated steatohepatitis (MASLD) or MASLD-related metabolic diseases. Background Art

[0002] Metabolic Dysfunction Associated Fatty Liver Disease (MASLD), formerly known as Nonalcoholic Fatty Liver Disease (NAFLD), is a fatty liver disease closely related to systemic metabolic disorders. MASLD encompasses a wide spectrum of liver injury, progressing from simple steatosis (lipid accumulation) to metabolic dysfunction-associated steatohepatitis (MASH), and may further develop into liver fibrosis, cirrhosis, and even liver cancer. Compared with NAFLD, the definition of MASLD emphasizes the core role of metabolic factors more, and its associated diagnosis and treatment with metabolic diseases such as diabetes and hyperlipidemia contribute to the regulation of the overall metabolic homeostasis of the human body. Over the past 20 years, the global incidence of MASLD has increased sharply and currently affects more than 30% of the global population (LE M H, YEO Y H, LI X, et al. 2019 Global NAFLD Prevalence: A Systematic Review and Meta-analysis [J]. Clin Gastroenterol Hepatol, 2022, 20(12): 2809-17.e28).

[0003] The pathogenesis of MASLD is complex and driven by multiple factors, mainly including abnormal lipid metabolism (such as lipid accumulation and changes in fatty acid metabolic pathways), oxidative stress and inflammation (such as lipotoxicity and immune-mediated inflammation), genetic variation and environmental factors (such as unhealthy lifestyles), enterohepatic circulation, and gut microbiota dysbiosis, etc.; in addition, MASLD is closely related to multiple metabolic abnormalities, especially obesity, insulin resistance, impaired vascular function, and dyslipidemia. These metabolic factors together constitute the MASLD-related metabolic disease spectrum, including obesity, type 2 diabetes, hypertension, and dyslipidemia, etc. Among them, insulin resistance, abnormal lipid metabolism, and excessive liver lipid accumulation are the core mechanisms of MASLD pathogenesis, leading to intrahepatic fat accumulation, and further causing liver function damage, aggravated inflammatory response, and fibrosis.

[0004] The diagnosis of MASLD is mainly based on evidence of hepatic steatosis and metabolic dysfunction. So far, the FDA has only approved one THR-β receptor agonist drug, Rezdiffra, for the treatment of MASLD. However, in patients receiving this drug, up to 91% of individuals developed at least one adverse reaction, including elevated liver enzymes, hypothyroxinemia, arrhythmia, and gastrointestinal side effects. Among them, gastrointestinal symptoms (such as diarrhea and nausea) were the most common, and the median duration of diarrhea was 15 to 20 days (HARRISON S A, BEDOSSA P, GUY C D, et al. A Phase 3, Randomized, Controlled Trial of Resmetiromin NASH with Liver Fibrosis[J]. N Engl J Med, 2024, 390(6):497-509). Therefore, lifestyle interventions (such as weight loss, dietary improvement, and increased exercise, etc.) are still the only widely applicable treatment measures for MASLD at present. However, lifestyle interventions have inherent limitations such as a long onset time, poor patient compliance, and unclear efficacy.

[0005] Therefore, there is an urgent need to find a treatment drug for MASLD with good therapeutic effect, clear mechanism of action, and few side effects. Summary of the Invention

[0006] The purpose of the present invention is to provide a class of compounds for preventing and treating metabolic dysfunction-related steatohepatitis, as well as their applications.

[0007] In the first aspect of the present invention, there is provided the use of a compound having a tricyclic parent nucleus structure shown in formula (I) or a pharmaceutically acceptable salt thereof for preparing a composition for preventing and / or treating metabolic dysfunction-related steatohepatitis (MASLD) or MASLD-related metabolic diseases;

[0008]

[0009] Wherein:

[0010] X is selected from: O, S, Se (selenium), C═S (sulfur double bond);

[0011] Y is a linking group between ring B and ring C, selected from H, C1-C4 alkyl (such as C1, C2, C3 alkyl), halogen (such as chlorine (-Cl), fluorine (-F), bromine (-Br), iodine (-I)).

[0012] In one or more embodiments, the compound having the tricyclic parent nucleus structure shown in formula (I) belongs to flavonoids, has a typical flavonoid skeleton, and its parent nucleus structure is mainly composed of three rings (ring A, ring B, and ring C).

[0013] In one or more embodiments, the compound having the tricyclic parent nucleus structure shown in formula (I) includes the compound of formula (II);

[0014]

[0015] Wherein:

[0016] R0 or R1 is independently selected from: hydroxy (-OH), (-O-) glycosyl (the sugar such as glucose, lactose, galactose, arabinose), carboxyl (such as -COOH, or substituted carboxyl such as -COOCH3, -COOCH2CH3), C1-C4 alkoxy (such as methoxy (-OCH3), ethoxy (-OC2H5), propoxy (-OC3H7)), C1-C4 alkyl (such as methyl (-CH3), ethyl (-C2H5), propyl (-C3H7)), hydrogen (-H), halogen (such as chlorine (-Cl), fluorine (-F), bromine (-Br), iodine (-I)), nitro (-NO2), cyano (-CN), amino (-NH2), substituted amino (-NHRa, -NRaRb)), acyl (such as formyl (-CHO), acetyl (-COCH3)), trifluoromethyl (-CF3); wherein, Ra or Rb is independently selected from a hydrogen atom (-H) or an organic group (such as C1-C4 alkyl, aryl such as phenyl (-C6H5));

[0017] R2 or R3 is independently selected from: C1-C4 alkoxy (such as methoxy (-OCH3), ethoxy (-OC2H5), propoxy (-OC3H7)), halogen (such as chlorine (-Cl), fluorine (-F), bromine (-Br), iodine (-I)), hydrogen (-H), C1-C4 alkyl (such as methyl (-CH3), ethyl (-C2H5), propyl (-C3H7)), hydroxy (-OH), cyano (-CN), nitro (-NO2), amino (-NH2), substituted amino (-NHRa, -NRa2), acyl (such as formyl (-CHO), acetyl (-COCH3)), trifluoromethyl (-CF3);

[0018] R4 is independently selected from: C1-C4 alkoxy (such as methoxy (-OCH3), ethoxy (-OC2H5), propoxy (-OC3H7)), hydrogen (-H), halogen (such as chlorine (-Cl), fluorine (-F), bromine (-Br), iodine (-I)), C1-C4 alkyl (such as methyl (-CH3), ethyl (-C2H5), propyl (-C3H7)), hydroxy (-OH), cyano (-CN), nitro (-NO2), amino (-NH2), substituted amino (-NHRa, -NRa2), acyl (such as formyl (-CHO), acetyl (-COCH3)), trifluoromethyl (-CF3).

[0019] In one or more embodiments, each of Ra or Rb independently represents an organic group (such as an alkyl group like methyl (-CH3), ethyl (-C2H5), etc., or an aryl group like phenyl (-C6H5)), or a hydrogen atom. The Ra or Rb groups can be the same. For example, when both Ra and Rb are methyl, -NRaRb is dimethylamino (-N(CH3)2); the Ra or Rb groups can also be different. For instance, when Ra is methyl and Rb is ethyl, it forms methyl ethyl amino (-N(CH3)(C2H5)).

[0020] In one or more embodiments, the compounds having the tricyclic parent nucleus structure shown in formula (I) include compounds selected from the following group:

[0021] (1) Eupatilin (EUP);

[0022] (2) Eupatilin-7-O-glucoside (R0 or R1 is independently -O-glucoside);

[0023] (3) Eupatilin-7-O-acetyl derivative (R0 or R1 is independently acetyl (-COCH3));

[0024] (4) 4'-chloro-eupatilin (R2 is independently -Cl);

[0025] (5) Thioeupatilin (X in ring B is independently S, preferably forming a "thioflavone" structure);

[0026] (6) Having the tricyclic parent nucleus structure, where one hydroxyl group (-OH) is replaced by a methoxy group (-OCH3);

[0027] (7) Having the tricyclic parent nucleus structure, where one hydroxyl group (-OH) is replaced by a carbonyl group (-C=O);

[0028] (8) Having the tricyclic parent nucleus structure, where one hydroxyl group (-OH) is replaced by a vinyl group (-CH=CH2);

[0029] (9) Having the tricyclic parent nucleus structure, where two hydroxyl groups (-OH) are replaced by methoxy groups (-OCH3);

[0030] (10) Having the tricyclic parent nucleus structure, where two hydroxyl groups (-OH) are replaced by carbonyl groups (-C=O);

[0031] (11) Having the tricyclic parent nucleus structure, where one methoxy group (-OCH3) is replaced by a hydroxyl group (-OH);

[0032] (12) Having the tricyclic parent nucleus structure, wherein one methoxy group (-OCH3) is replaced by a carbonyl group (-C=O);

[0033] (13) Having the tricyclic parent nucleus structure, wherein one hydroxyl group (-OH) and one methoxy group (-OCH3) are replaced by a vinyl group (-CH=CH2);

[0034] (14) Having the tricyclic parent nucleus structure, wherein one hydroxyl group (-OH) and one carbonyl group (-C=O) are replaced by a vinyl group (-CH=CH2);

[0035] (15) Having the tricyclic parent nucleus structure, wherein one hydroxyl group (-OH) is replaced by a hydrogen atom (-H);

[0036] (16) Having the tricyclic parent nucleus structure, wherein one methoxy group (-OCH3) is replaced by a hydrogen atom (-H);

[0037] (17) Having the tricyclic parent nucleus structure, wherein one hydroxyl group (-OH) is replaced by a methyl group (-CH3).

[0038] In one or more embodiments, the MASLD or MASLD-related metabolic diseases include:

[0039] (a) Liver diseases related to systemic metabolic disorders, preferably including: simple steatosis (lipid deposition), metabolic dysfunction-associated steatohepatitis (MASH), MASLD-related liver fibrosis, MASLD-related cirrhosis, MASLD-related hepatocellular carcinoma;

[0040] (b) Diseases related to metabolic factors including obesity, insulin resistance, vascular dysfunction, or dyslipidemia, preferably including: overweight, obesity, hyperglycemia, hypertension, dyslipidemia.

[0041] In one or more embodiments, the diseases related to metabolic factors include, but are not limited to, the following diseases:

[0042] a) Overweight or obesity: BMI ≥ 25 kg / m 2 (for Asians ≥ 23 kg / m 2 ) or male waist circumference > 94 cm, female waist circumference > 80 cm;

[0043] b) Hyperglycemia: fasting blood glucose ≥ 100 mg / dL or 2-hour postprandial blood glucose ≥ 140 mg / dL or glycated hemoglobin (HbA1c) ≥ 5.7%;

[0044] c) Hypertension: blood pressure ≥ 140 / 90 mmHg;

[0045] d) Dyslipidemia: Metabolic diseases such as triglyceride (TG) ≥ 150 mg / dL, male plasma high-density lipoprotein cholesterol (HDL) < 40 mg / dL, and female plasma high-density lipoprotein cholesterol (HDL) < 50 mg / dL.

[0046] In one or more embodiments, the preventive effect refers to the early administration of drugs to high-risk populations of MASLD or MASLD-related metabolic diseases without a clear diagnosis of MASLD.

[0047] In one or more embodiments, the therapeutic effects include: alleviating hepatic steatosis, reducing hepatic lipid deposition, reducing fat mass, reducing body weight, reducing liver weight, reducing the liver weight / body weight ratio, improving blood lipid indices, improving liver function indices, reducing blood glucose, and alleviating the inflammatory response in MASLD patients.

[0048] In one or more embodiments, the high-risk populations of MASLD or MASLD-related metabolic diseases mainly include populations related to metabolic factors (such as obesity, type 2 diabetes, dyslipidemia, hypertension, etc.), populations related to lifestyle (such as sedentary, poor eating habits, etc.), populations related to genetic factors (family history of metabolic diseases), and patients with other co-existing metabolic diseases, etc.

[0049] In one or more embodiments, the composition is also used for: reducing serum liver function indices such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT); optimizing blood lipid profiles such as high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and total cholesterol (TC); reducing the levels of inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the liver and serum; increasing the activity of superoxide dismutase (SOD) and reducing the content of malondialdehyde (MDA); or increasing antioxidant substances (such as glutathione peroxidase (GSH), etc.).

[0050] In another aspect of the present invention, there is provided a compound having the structure shown in formula (II) or a pharmaceutically acceptable salt thereof;

[0051]

[0052] Wherein:

[0053] X is selected from: O, S, Se (selenium), C═S (sulfur bond);

[0054] Y is a linking group between ring B and ring C, and is selected from H, C1-C4 alkyl (such as C1, C2, C3 alkyl), halogen (such as chlorine (Cl), fluorine (F), bromine (Br), iodine (I));

[0055] R0 or R1 is independently selected from: hydroxy (-OH), (-O-) glycosyl (said sugar such as glucose, lactose, galactose, arabinose), carboxyl (such as -COOH, or substituted carboxyl such as -COOCH3, -COOCH2CH3), C1-C4 alkoxy (such as methoxy (-OCH3), ethoxy (-OC2H5), propoxy (-OC3H7)), C1-C4 alkyl (such as methyl (-CH3), ethyl (-C2H5), propyl (-C3H7)), hydrogen (-H), halogen (such as chlorine (-Cl), fluorine (-F), bromine (-Br), iodine (-I)), nitro (-NO2), cyano (-CN), amino (-NH2), substituted amino (-NHRa, -NRaRb), acyl (such as formyl (-CHO), acetyl (-COCH3)), trifluoromethyl (-CF3); wherein, Ra or Rb is independently selected from a hydrogen atom (-H) or an organic group (such as C1-C4 alkyl, aryl such as phenyl (-C6H5)).

[0056] R2 or R3 is independently selected from: C1-C4 alkoxy (such as methoxy (-OCH3), ethoxy (-OC2H5), propoxy (-OC3H7)), halogen (such as chlorine (-Cl), fluorine (-F), bromine (-Br), iodine (-I)), hydrogen (H), C1-C4 alkyl (such as methyl (-CH3), ethyl (-C2H5), propyl (-C3H7)), hydroxy (-OH), cyano (-CN), nitro (-NO2), amino (-NH2), substituted amino (-NHRa, -NRa2), acyl (such as formyl (-CHO), acetyl (-COCH3)), trifluoromethyl (-CF3);

[0057] R4 is independently selected from: C1-C4 alkoxy (such as methoxy (-OCH3), ethoxy (-OC2H5), propoxy (-OC3H7)), hydrogen (H), halogen (such as chlorine (Cl), fluorine (F), bromine (Br), iodine (I)), C1-C4 alkyl (such as methyl (-CH3), ethyl (-C2H5), propyl (-C3H7)), hydroxy (-OH), cyano (-CN), nitro (-NO2), amino (-NH2), substituted amino (-NHRa, -NRa2), acyl (such as formyl (-CHO), acetyl (-COCH3)), trifluoromethyl (-CF3);

[0058] And, the compound of the structure shown in formula (II) does not include eupatilin.

[0059] In another aspect of the present invention, there is provided a composition comprising the compound of the structure shown in formula (II) described above or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

[0060] In one or more embodiments, the composition is a pharmaceutical composition.

[0061] In one or more embodiments, eupatilin is not included in the composition.

[0062] In one or more embodiments, the compound of the structure shown in formula (II) or a pharmaceutically acceptable salt thereof is in an effective amount in the pharmaceutical composition; preferably, the effective amount is, by weight content, for example (but not limited to), 0.001 - 50%, 0.03 - 30%, 0.05 - 10%, 0.01 - 5%, 0.03 - 3%, 0.05 - 1%, 20 - 30%, 40 - 50%.

[0063] In one or more embodiments, the effective amount of the compound of the structure shown in formula (II) or a pharmaceutically acceptable salt thereof in the pharmaceutical composition is 0 - 800 μM or 1 - 800 μM; for example (but not limited to) 2, 5, 10, 20, 50, 100, 200, 300, 500, 600 or 800 μM.

[0064] In one or more embodiments, the composition is a preparation for internal use.

[0065] In one or more embodiments, the dosage forms of the pharmaceutical composition include: capsules, pills, tablets, granules, oral preparations (such as oral liquids), powders, powders for external use, sustained-release agents, controlled-release agents, injections, infusion solutions (such as injection solutions), suspensions.

[0066] In one or more embodiments, the pharmaceutical composition includes: pharmaceutical adjuvants, excipients, carriers, pharmaceutical compositions, bioactive agent compositions, health product compositions or food compositions, etc.

[0067] In one or more embodiments, the pharmaceutical composition includes a pharmaceutically or physiologically acceptable excipient or diluent.

[0068] In another aspect of the present invention, there is provided an application of the aforementioned defined compound (including its salt) or composition in the preparation of a kit for preventing and / or treating metabolic dysfunction-associated steatohepatitis (MASLD) or MASLD-related metabolic diseases.

[0069] In another aspect of the present invention, there is provided a kit, including: the compound of the structure shown in formula (II) as described in any one of the foregoing or a pharmaceutically acceptable salt thereof.

[0070] In another aspect of the present invention, there is provided a kit, including the composition.

[0071] In another aspect of the present invention, there is provided a method for preventing and / or treating metabolic dysfunction-associated steatohepatitis (MASLD) or MASLD-related metabolic diseases, comprising: administering to a subject in need of treatment an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof; preferably, administering to a subject in need of treatment an effective amount of a compound of formula (II) or a pharmaceutically acceptable salt thereof.

[0072] Other aspects of the present invention will be apparent to those skilled in the art from the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 : Effects of EUP on body weight, liver, epididymal and subcutaneous fat weights, and liver index in mice;

[0074] (A) Photographs of mice and body weight curves of four experimental groups: normal negative control group (CON), high-fat-induced MASLD model group (HFD), MASLD model combined with low-concentration EUP group (HFD+EUP-L), and MASLD model combined with high-concentration EUP group (HFD+EUP-H);

[0075] (B) Liver weight and liver / body weight ratio of four groups of mice at 16 weeks;

[0076] (C) Photographs of epididymal and subcutaneous fat and fat / body weight ratio of four groups of mice at 16 weeks.

[0077] Figure 2 : H&E staining and Oil Red O staining diagrams of the liver tissue of mice in four experimental groups, and H&E staining diagram of subcutaneous fat.

[0078] Figure 3 : Effects of EUP on inflammatory factors in four experimental groups of mice;

[0079] (A) Serum tumor necrosis factor-α level in mice;

[0080] (B) Serum interleukin-6 level in mice;

[0081] (C) Liver tumor necrosis factor-α in mice;

[0082] (D) Liver interleukin-6 level in mice.

[0083] Figure 4 : Effects of EUP on the oxidative stress levels of mice in four experimental groups;

[0084] (A) Serum superoxide dismutase (Serum SOD) level in mice;

[0085] (B) Serum glutathione peroxidase (Serum GSH-PX) level in mice;

[0086] (C) Serum malondialdehyde (Serum MDA) level in mice;

[0087] (D) Liver superoxide dismutase (Liver SOD) level in mice;

[0088] (E) Liver glutathione peroxidase (Liver GSH-PX) level in mice;

[0089] (F) Liver malondialdehyde (Liver MDA) level in mice.

[0090] Figure 5 : Effects of EUP on serum biochemical indexes and liver function indexes of mice in four experimental groups;

[0091] (A) Serum total cholesterol (Serum TC) level in mice;

[0092] (B) Serum triglyceride (Serum TG) level in mice;

[0093] (C) Serum alanine aminotransferase (Serum ALT) level in mice;

[0094] (D) Serum aspartate aminotransferase (Serum AST) level in mice;

[0095] (E) Serum high-density lipoprotein cholesterol (Serum HDL-C) level in mice;

[0096] (F) Serum low-density lipoprotein cholesterol (Serum LDL-C) level in mice;

[0097] (G) Liver total cholesterol (Liver TC) level in mice;

[0098] (H) Liver triglyceride (Liver TG) level in mice;

[0099] (I) Liver high-density lipoprotein cholesterol (Liver HDL-C) level in mice;

[0100] (J) Liver low-density lipoprotein cholesterol (Liver LDL-C) level in mice.

[0101] Figure 6 : EUP on the serum glucose (Serum Glu) levels of mice in four experimental groups.

[0102] Figure 7 : Metabolic cages were used to detect the effect of EUP on energy metabolism in mice in two experimental groups (HFD: high-fat-induced mouse MASLD model group; HFD+EUP: EUP-treated MASLD model group);

[0103] (A) Curves of O2 consumption levels and average daily levels in mice;

[0104] (B) Curves of CO2 production levels and average daily levels in mice;

[0105] (C) Curves of energy consumption levels and average daily levels in mice;

[0106] (D) Curves of respiratory exchange ratio (RER) levels and average daily levels in mice.

[0107] Figure 8 : The effect of EUP on the proliferation of AML12 cells in mouse hepatocyte line;

[0108] (A) The effect of EUP treatment for 24 hours on the proliferation of AML12 cells;

[0109] (B) The effect of EUP treatment for 48 hours on the proliferation of AML12 cells.

[0110] Figure 9 : The Oil Red O staining results of EUP on AML12 cells in four experimental groups;

[0111] Four experimental groups were set up: Control: negative control group; PO: positive control group induced by oleic acid and palmitic acid; PO+Eup40μM: experimental group induced by PO with 40μM concentration intervention; PO+Eup 80μM: experimental group induced by PO with 80μM concentration intervention.

[0112] Figure 10 : The effect of EUP on the TC and TG levels in AML12 cell line in four experimental groups;

[0113] (A) Total cholesterol (TC) content in AML12 cell line in four experimental groups;

[0114] (B) Triglyceride (TG) content in AML12 cell line in four experimental groups.

[0115] Figure 11 : The Nile Red staining results of EUP on human liver organoids induced by oleic acid and palmitic acid (PO).

[0116] Figure 12 : Effects of EUP and four derivatives on the body weight and liver of mice.

[0117] Figure 13 : H&E staining and Oil Red O staining images of the liver tissue of mice in the experimental groups of EUP and four derivatives.

[0118] Figure 14 : Indicators of ALT, TG, TC, and blood glucose in the sera of mice in the experimental groups of EUP and four derivatives.

[0119] Figure 15 , High-performance liquid chromatography analysis results of eupatilin-7-O-glucoside (derivative 1).

[0120] Figure 16 , High-performance liquid chromatography analysis results of eupatilin-7-O-acetyl (derivative 2).

[0121] Figure 17 , 4 ’-chloro-eupatilin (derivative 3) high-performance liquid chromatography analysis results.

[0122] Figure 18 , High-performance liquid chromatography analysis results of thioeupatilin (derivative 4). Detailed implementation manners

[0123] The present invention discloses eupatilin (EUP) and its novel derivatives, and also discloses new uses of EUP and its novel derivatives for preventing and / or treating metabolic dysfunction-associated steatohepatitis (MASLD) or MASLD-related metabolic diseases.

[0124] Terms

[0125] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art to which the present invention pertains.

[0126] As used herein, the "compounds of the present invention", "EUP or its derivatives", "compounds having the EUP parent nucleus structure", and "compounds having the tricyclic parent nucleus structure shown in formula (I)" can be used interchangeably.

[0127] As used herein, "C1-C4 alkyl" refers to a straight-chain or branched-chain alkyl group having 1-4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, or similar groups.

[0128] As used herein, "C1-C4 alkoxy" refers to a straight-chain or branched alkoxy group having 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy or similar groups.

[0129] As used herein, "alkoxy" refers to -O-(alkyl). Representative examples include methoxy, ethoxy, propoxy, etc.

[0130] As used herein, "halogen" includes fluorine, chlorine, bromine or iodine.

[0131] As used herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on a group are replaced by substituents selected from (but not limited to) the following group: C1-C3 alkyl, halogen, hydroxy, carboxyl (-COOH), C2-C4 acyl, C2-C4 ester, amino, phenyl; said phenyl includes unsubstituted phenyl or substituted phenyl having 1 to 3 substituents, and the substituents are for example selected from (but not limited to): halogen, C1-C3 alkyl, C1-C3 haloalkyl, hydroxy, C1-C3 alkoxy.

[0132] As used herein, the term "comprising" means that various components can be applied together in the mixtures or compositions of the present invention. Thus, the terms "consisting essentially of" and "consisting of" are included in the term "comprising".

[0133] As used herein, "consisting essentially of" means that in the said composition, in addition to containing the necessary components or necessary ingredients, it may also contain a small amount of secondary components and / or impurities that do not affect the active ingredients. For example, it may contain sweeteners or flavoring agents to improve the taste, antioxidants to prevent oxidation, pigments to adjust the color, and other commonly used additives in the art.

[0134] As used herein, "consisting essentially of", "consisting essentially of" or "consisting of" are usually used in compositions to indicate the key components that play an active role in the composition. For example, the composition "consisting essentially of the compound of formula (I)" or "consisting of the compound of formula (I)" means that in the said composition, the main active component is the compound of formula (I) or only the compound of formula (I).

[0135] As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, having a reasonable benefit / risk ratio.

[0136] As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically or food-acceptable solvent, suspending agent or excipient for delivering the compounds, isomers, solvates, precursors of the tricyclic parent nucleus structure of the present invention, or their pharmaceutically acceptable salts to animals or humans. The carrier can be liquid or solid.

[0137] As used herein, "isomers" include: geometric isomers, enantiomers, diastereoisomers (such as cis-trans isomers, conformational isomers).

[0138] As used herein, "solvate" refers to a compound carrying solvent molecules. For example, the solvate may be a hydrate.

[0139] As used herein, the terms "unit dosage" and "unit amount" refer to the form in which cysteine or its analogs of the present invention are prepared into the amount required for a single dose for convenient administration. For example, the daily dosage is in one package / container, or the daily amount is split into several separate doses and placed in independent packages / containers.

[0140] EUP or its derivatives

[0141] To solve the problems existing in the treatment of MASLD, the present invention provides a compound (EUP or its derivatives) with a tricyclic parent nucleus structure represented by formula (I), which is applied to the prevention and / or treatment of MASLD and its related metabolic diseases.

[0142] As a preferred embodiment of the present invention, the EUP derivatives of the present invention include compounds of formula (II).

[0143] As a preferred embodiment of the present invention, the compound of formula (I) is EUP. EUP is a lipophilic flavonoid compound extracted from Artemisia argyi, with a molecular weight of 344.32 and a molecular formula of C 18 H 16 O7. Studies have shown that EUP has a variety of pharmacological effects, including anti-inflammatory, anti-tumor, anti-ulcer, anti-allergic and neuroprotective effects. Its mechanism of action is complex and diverse. For example, it exerts anti-inflammatory effects by activating the PPARα signaling pathway and inhibiting the activities of inflammation-related transcription factors such as NF-κB; at the same time, EUP is also involved in regulating the cell cycle and inducing the expression of apoptosis-related proteins. However, there is no publicly reported evidence showing that EUP has preventive or therapeutic effects on MASLD and its related metabolic diseases.

[0144] EUP is a flavonoid compound extracted from Artemisia argyi, with a CAS number of 22368-21-4, a chemical formula of C 18 H 16 O7, a molecular weight of 344.32, and its parent nucleus has a typical flavonoid skeleton, including three rings (ring A, ring B and ring C).

[0145] The present invention also includes hybrids formed by covalently linking EUP and its derivative monomers with natural products (such as coumarins or terpenoid compounds).

[0146] As a preferred embodiment of the present invention, in addition to EUP, the compound having the tricyclic parent nucleus structure of formula (I) further includes compounds selected from the following group:

[0147] Eupatilin-7-O-glucoside (R0 or R1 is independently -O-glucoside); Molecular formula: C 24 H 26 O 13 ; Molecular weight: 524.48;

[0148] Eupatilin-7-O-acetyl derivative (R0 or R1 is independently acetyl (-COCH3)); Molecular formula: C 20 H 18 O8; Molecular weight: 387.36

[0149] 4'-Chloro-eupatilin (R2 is independently -Cl); Molecular formula: C 17 H 13 ClO6; Molecular weight: 348.74;

[0150] Sulpho-eupatilin (X in the B ring is independently S, preferably forming a "sulphoflavone" structure); Molecular formula: C 18 H 16 O6S; Molecular weight: 360.38;

[0151] EUP parent nucleus structure, in which one hydroxyl group (-OH) is replaced by a methoxy group (-OCH3); Molecular formula: C 19 H 18 O7; Molecular weight: 362.34;

[0152] EUP parent nucleus structure, in which one hydroxyl group (-OH) is replaced by a carbonyl group (-C=O); Molecular formula: C 18 H 14 O8; Molecular weight: 358.30;

[0153] EUP parent nucleus structure, in which one hydroxyl group (-OH) is replaced by a vinyl group (-CH=CH2); Molecular formula: C 20 H 18 O7; Molecular weight: 370.35;

[0154] EUP parent nucleus structure, in which two hydroxyl groups (-OH) are replaced by methoxy groups (-OCH3); Molecular formula: C 20 H 20 O7; Molecular weight: 372.37;

[0155] EUP parent nucleus structure, in which two hydroxyl groups (-OH) are replaced by carbonyl groups (-C=O); Molecular formula: C 18 H 12O9; Molecular weight: 374.28;

[0156] The EUP parent nucleus structure, in which one methoxy group (-OCH3) is replaced by a hydroxyl group (-OH); Molecular formula: C 17 H 14 O7; Molecular weight: 330.29;

[0157] The EUP parent nucleus structure, in which one methoxy group (-OCH3) is replaced by a carbonyl group (-C=O); Molecular formula: C 17 H 12 O8; Molecular weight: 340.28;

[0158] The EUP parent nucleus structure, in which one hydroxyl group (-OH) and one methoxy group (-OCH3) are replaced by a vinyl group (-CH=CH2); Molecular formula: C 21 H 20 O7; Molecular weight: 384.38;

[0159] The EUP parent nucleus structure, in which one hydroxyl group (-OH) and one carbonyl group (-C=O) are replaced by a vinyl group (-CH=CH2); Molecular formula: C 20 H 18 O8; Molecular weight: 386.36;

[0160] The EUP parent nucleus structure, in which one hydroxyl group (-OH) is replaced by a hydrogen atom (-H) (removing the hydroxyl group); Molecular formula: C 18 H 14 O6; Molecular weight: 326.30;

[0161] The EUP parent nucleus structure, in which one methoxy group (-OCH3) is replaced by a hydrogen atom (-H) (removing the methoxy group); Molecular formula: C 17 H 12 O6; Molecular weight: 312.27;

[0162] The EUP parent nucleus structure, in which one hydroxyl group (-OH) is replaced by a methyl group (-CH3); Molecular formula: C 19 H 16 O6; Molecular weight: 340.33.

[0163] The therapeutic effects of the above compounds mainly include: reducing serum liver function indicators such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT); improving lipid profiles such as high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), and total cholesterol (TC); reducing hepatic lipid deposition; lowering blood glucose; reducing inflammatory factors such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the liver and serum; and regulating the activities of hepatic antioxidants such as superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH).

[0164] The present invention also includes isomers, solvates, precursors of the compounds with the tricyclic parent nucleus structure, or pharmaceutically acceptable salts thereof, provided that they also have the same or substantially the same functions as the compounds with the tricyclic parent nucleus structure. The "pharmaceutically acceptable salts" can be salts formed by reacting with inorganic acids, organic acids, alkali metals, or alkaline earth metals, etc. These salts include (but are not limited to): (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, carbonic acid, nitric acid, phosphoric acid; (2) salts formed with the following organic acids, such as hydrobromic acid, citric acid, tartaric acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, or p-toluenesulfonic acid. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium, or magnesium), in the form of esters, carbamates, or other conventional "prodrugs".

[0165] The compounds have one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereoisomers, mixtures of diastereoisomers, cis or trans isomers.

[0166] The "precursor of the compound" refers to a compound that, when administered by an appropriate method, undergoes metabolism or a chemical reaction in the patient's body and is converted into a compound of structural formula (I), or a salt or solution composed of a compound of chemical structural formula (I).

[0167] Those skilled in the art should understand that after knowing the structure of the compounds of the present invention, the compounds of the present invention can be obtained by methods well-known in the art or the methods disclosed in the examples of the present invention, using known raw materials, such as chemical synthesis or extraction from organisms (such as animals or plants). These methods are all included in the present invention.

[0168] Application

[0169] Based on the above new findings of the present inventors, the present invention provides the use of the compound of the tricyclic parent nucleus structure or its isomers, solvates, precursors or pharmaceutically acceptable salts thereof for preparing a composition or a kit for preventing and / or treating metabolic dysfunction-associated steatohepatitis (MASLD) or MASLD-related metabolic diseases.

[0170] The present invention reveals for the first time that eupatilin and its various derivatives (including eupatilin-7-O-glucoside, eupatilin-7-O-acetyl derivative, 4'-chloro-eupatilin and thioeupatilin) can significantly improve the related symptoms of MASLD, which are specifically manifested in the following aspects: significantly improving serum liver function indexes, such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT); optimizing the blood lipid profile, such as high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglyceride (TG) and total cholesterol (TC); reducing liver lipid deposition; lowering blood glucose (GLU); reducing inflammatory factors in the liver and serum, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and regulating the redox level of the liver, such as activating superoxide dismutase (SOD) and glutathione peroxidase (GSH) and reducing the level of malondialdehyde (MDA).

[0171] In the examples of the present invention, it is confirmed for the first time at three levels of cells, organoids and animal models that EUP has a therapeutic effect on MASLD and its related metabolic syndrome, providing an experimental basis for the subsequent development of therapies for preventing and treating MASLD and its related metabolic syndrome with EUP as the core component. At the same time, the inventors also confirmed through designing experimental groups with different doses that a lower dose of EUP has a good therapeutic effect.

[0172] According to the above, the beneficial effects of the present invention mainly include the following:

[0173] 1) Reducing liver lipid deposition: During the progression of MASLD, liver lipid deposition is a pathological state of excessive fat accumulation in hepatocytes. In the initial stage, it usually presents as simple fatty liver. As the disease progresses, it may evolve into steatohepatitis, accompanied by hepatocyte inflammation and necrosis, and then lead to liver fibrosis, and finally may progress to cirrhosis. The results show that EUP or its derivatives can effectively reduce the deposition of liver lipids, reduce the degree of steatosis and liver fibrosis, thus having an improving effect on the pathological process of MASLD.

[0174] 2) Regulating blood lipid metabolism: MASLD is often accompanied by abnormal blood lipid metabolism. EUP or its derivatives can reduce the serum blood lipid levels, such as TC, LDL-C and TG levels.

[0175] 3) Blood glucose reduction: Hyperglycemia is a common accompanying symptom of MASLD. In the state of hyperglycemia, insufficient insulin secretion or insulin resistance will exacerbate hepatic fat deposition and inflammatory responses, thereby promoting the occurrence and development of MASLD. The results show that EUP or its derivatives can significantly reduce blood glucose levels.

[0176] 4) Anti-inflammatory effect: In the pathological process of MASLD, the inflammatory response is a key link. The activation of the inflammatory response leads to liver tissue damage and fibrosis, thus accelerating the progression of the disease. The results show that EUP or its derivatives can effectively inhibit the occurrence and development of the inflammatory response by inhibiting the release of inflammatory factors (such as IL-6 and TNF-α).

[0177] 5) Alleviating oxidative stress: In the development process of MASLD, oxidative stress plays an important role. Oxidative stress can lead to the excessive production of oxygen free radicals in hepatocytes, triggering membrane lipid peroxidation and other damages, thereby exacerbating liver inflammation and injury. The results show that EUP can effectively improve the oxidative state of hepatocytes and alleviate the damage of oxidative stress to the liver by increasing the activities of antioxidant enzymes (such as SOD and GSH) and reducing the level of the oxidative stress marker MDA.

[0178] 6) Improving the energy metabolism level: MASLD is often accompanied by metabolic dysfunction. EUP can significantly increase CO2 production and O2 consumption, thereby improving the energy metabolism efficiency.

[0179] The inventor of the present invention also analyzed the effective dose of eupatilin in the treatment of MASLD. The present invention first confirms that eupatilin and its derivatives have the effects of improving liver function, reducing lipid deposition, regulating blood glucose, inhibiting inflammatory responses, alleviating oxidative stress and improving the energy metabolism level on MASLD. Based on this component, new drugs or new therapies for preventing and / or treating MASLD can be developed.

[0180] Composition

[0181] The present invention also provides a composition containing an effective amount of the compound with the tricyclic parent nucleus structure, or its isomer, solvate, precursor, or their pharmaceutically acceptable salts.

[0182] In a preferred manner, the composition contains an effective dose of EUP and pharmaceutically acceptable pharmaceutical adjuvants, excipients, carriers, pharmaceutical compositions, bioactive preparation compositions, health product compositions or food compositions, etc.

[0183] In the present invention, in the said pharmaceutical composition, the content of the compound with a tricyclic parent nucleus structure or its isomer, solvate or precursor, or their pharmaceutically acceptable salts is an effective amount. For example, it may contain the compound with a tricyclic parent nucleus structure or its pharmaceutically acceptable salts in a weight ratio of 0.001 - 50%. Preferably, the said pharmaceutical composition contains the compound with a tricyclic parent nucleus structure or its pharmaceutically acceptable salts in a weight ratio of 0.01 - 20%.

[0184] In a preferred embodiment, the effective dose is, for example, 0 - 800 μM or 1 - 800 μM.

[0185] The dosage forms of the pharmaceutical composition of the present invention can be diverse, as long as they can effectively deliver the active ingredient to the mammalian body. For example, it can be selected from: powder, powder for oral use, tablet, pill, capsule, sustained-release agent, controlled-release agent, injection, infusion, suspension. Those skilled in the art can select a convenient dosage form according to the type of disease to be treated by the compounds of the present invention.

[0186] From the perspective of easy preparation and storage, the preferred pharmaceutical composition is a solid composition, especially tablets and solid-filled or liquid-filled capsules. From the perspective of easy administration, the preferred pharmaceutical composition is an oral preparation. The compounds or their pharmaceutical compositions of the present invention can also be stored in a sterilized device suitable for injection or infusion.

[0187] The effective administration dose of the compound with a tricyclic parent nucleus structure as the active ingredient may vary depending on the mode of administration and the severity of the disease to be treated. However, generally, when the compound of the present invention is administered at a dose of about 0.01 - 100 mg / kg of animal body weight per day, satisfactory effects can be obtained, preferably administered in 1 - 3 divided doses per day, or in a sustained-release form. This dosage regimen can be adjusted to provide the best therapeutic response. For example, depending on the urgency of the treatment condition, several divided doses can be administered per day, or the dose can be proportionally reduced.

[0188] The said pharmaceutical composition can be placed in a kit. The kit may also include an instruction manual for explaining the usage method of the said pharmaceutical composition.

[0189] The present invention will be elaborated in detail through several specific embodiments. These examples are only for illustration purposes to deeply analyze the present invention and do not impose any form of restriction on its application scope or core content. The purpose of providing these examples is to deepen the understanding of the present invention rather than establishing a sole or optimal mode that must be followed. Therefore, the protection scope of the present invention is extensive and not limited to the specific forms described. Any result inspired by the idea of the present invention or integrating the core technology of the present invention with the prior art for innovation, as long as it is essentially the same as or highly similar to the present invention, falls within the protection scope of the present invention.

[0190] Example 1: EUP treatment of a mouse model of MASLD induced by a high-fat diet

[0191] 1. Experimental method

[0192] (1) MASLD mice induced by a high-fat diet and drug intervention

[0193] Eupatilin (the parent compound), abbreviated as EUP, was purchased from MCE (product number HY-N0783).

[0194] Twenty 5- to 6-week-old male C57 mice of the same batch were selected. After 1 week of adaptive feeding, they were randomly divided into a normal negative control group (CON) with 5 mice, a high-fat-induced MASLD model group (HFD) with 5 mice, a low-dose EUP treatment group (HFD+EUP-L), and a high-dose EUP treatment group (HFD+EUP-H), with 5 mice in each group. The CON group was fed with a basal diet, and the HFD group and the EUP groups were fed with a high-fat and high-sugar diet. After 8 weeks of feeding, one mouse was sacrificed from each group, and liver tissues were collected for H&E staining to detect liver pathological changes, and blood samples were collected to detect liver function indicators. If the liver H&E staining showed obvious fat vacuoles and the liver function indicators (such as AST and ALT) were significantly elevated, it was determined that the MASLD model was successfully established.

[0195] After the model was successfully established, corresponding treatments were given. Among them, the normal negative control group (CON) and the model group (HFD) were given 2 mL of normal saline by gavage every day, and the two dose groups of HFD+EUP-L and HFD+EUP-H were given EUP at 20 and 40 mg / kg / d by gavage every day respectively. When treating, the corresponding drugs were dissolved in 2 mL of normal saline and given by gavage. The treatment time was 8 weeks, and the body weight was recorded every week.

[0196] (2) Collection of serum, liver, and epididymal subcutaneous fat samples

[0197] After 16 weeks of feeding, the mice were anesthetized and sacrificed, and serum and liver samples were collected. Specifically, after fasting all the mice for 12 hours, the mice were anesthetized by intraperitoneal injection, blood was taken from the orbital cavity, left standing at room temperature for 2 hours, then centrifuged at 4000 rpm for 10 minutes at 4°C, and the serum samples were collected and stored at -80°C. The liver and epididymal fat were quickly removed and weighed. A small piece of liver tissue was taken from the same part of the right lobe of the liver, rinsed with normal saline at 4°C, blotted dry with filter paper, fixed with 4% paraformaldehyde solution, and the remaining liver tissue was stored at -80°C for subsequent index detection.

[0198] The pathological morphological characteristics, lipid deposition and liver injury of the livers of mice in the normal negative control group, model group and EUP intervention group were detected by H&E staining and Oil Red O staining.

[0199] Detection of liver lipid content and oxidative stress:

[0200] Detection of triglyceride (TC) in the liver: After thawing the liver tissue stored at -80°C on ice, about 20 mg was weighed accurately, added to 9 times the volume of absolute ethanol, homogenized under low temperature conditions, and then the tissue homogenate was centrifuged at 5000 rpm / min for 10 min. The supernatant was taken and determined with a triglyceride kit;

[0201] Detection of superoxide dismutase (SOD) in the liver: After thawing the liver tissue stored at -80°C on ice, about 20 mg was weighed accurately, added to 9 times the volume of normal saline, homogenized under low temperature conditions, and then the tissue homogenate was centrifuged at 3000 rpm / min for 10 min. The supernatant was taken and determined with a superoxide dismutase kit;

[0202] Detection of malondialdehyde (MDA) in the liver: 0.3 mL of reagent was pipetted into a 1.5 mL centrifuge tube, and then 0.1 mL of the sample was added and mixed well. Incubate in a water bath at 95°C for 30 min (cover tightly to prevent water loss), cool in an ice bath, centrifuge at 10000 rpm / min at 25°C for 10 min. Pipette 200 μL of the supernatant into a 96-well plate and detect according to the kit instructions.

[0203] Detection of serological indicators: The contents of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TG), triglyceride (TC), low density lipoprotein (LDL-C) and high density lipoprotein (HDL-C) were detected by a biochemical analyzer.

[0204] Metabolic cage energy metabolism detection: At the end of the 8th week of drug intervention, 3 mice were randomly selected from the model group and the high-dose EUP group and placed in metabolic cages for a one-week observation of energy metabolism. By monitoring indicators such as oxygen consumption, carbon dioxide production, energy consumption, and respiratory exchange rate of the mice, the energy metabolism status of each group of mice was evaluated to analyze the effect of EUP on the energy metabolism of MASLD model mice.

[0205] 2. Experimental results

[0206] (1) In terms of the phenotypes of body weight and liver weight, compared with the negative control group, the body weight, liver weight, epididymal subcutaneous fat weight, and liver index of the mice in the MASLD model group were significantly increased. However, interventions with high-dose and low-dose EUP both significantly reduced the body weight, liver weight, epididymal subcutaneous fat weight, and liver-to-body ratio index of the mice in the MASLD model group (*P﹤0.05, **P﹤0.01, ***P﹤0.005)( Figure 1 ).

[0207] (2) In the pathological analysis of the liver and subcutaneous fat, the results of H&E staining and Oil Red O staining showed that significant fatty degeneration and liver damage occurred in the hepatocytes of the mice in the MASLD model group. However, after interventions with high-dose and low-dose EUP, the fatty degeneration and liver damage of the hepatocytes of the mice were significantly improved( Figure 2 ).

[0208] (3) Compared with the negative control group, the levels of IL-6 and TNF-α in the liver and serum of the mice in the MASLD model group were increased. However, interventions with high-dose and low-dose EUP both significantly inhibited the release of these inflammatory factors (*P﹤0.05, **P﹤0.01)( Figure 3 ).

[0209] (4) Compared with the negative control group, the activities of the antioxidant enzymes SOD and GSH-PX in the liver of the mice in the MASLD model group were significantly decreased, while the level of the oxidative stress marker MDA was significantly increased. High-dose EUP intervention could significantly increase the activity of SOD and at the same time decrease the level of MDA (*P﹤0.05, **P﹤0.01, ***P﹤0.005). It was shown that EUP had the effect of improving the oxidative stress state of the liver. However, the changes in the levels of oxidative stress markers in the sera of the mice in the four experimental groups were not obvious, suggesting that the antioxidant effect of EUP might be mainly concentrated in the liver tissue( Figure 4 ).

[0210] (5) Compared with the negative control group, the levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in the serum and liver tissues of mice in the MASLD model group were significantly increased. However, both high-dose and low-dose EUP interventions could significantly reverse the accumulation of these lipids in the serum and liver (*P﹤0.05, **P﹤0.01, ***P﹤0.005), indicating that EUP has a significant improvement effect on MASLD-related lipid metabolism disorders and liver function damage( Figure 5 ).

[0211] (6) Compared with the negative control group, the glucose level in the serum of mice in the MASLD model group was significantly increased, and both high-dose and low-dose EUP interventions could significantly reduce the blood glucose level in the MASLD group (*P﹤0.05, **P﹤0.01, ***P﹤0.005)( Figure 6 ).

[0212] (7) In terms of energy metabolism, mice treated with EUP showed a significantly improved energy metabolism state in the metabolic cage experiment. Specifically, the oxygen consumption and carbon dioxide production increased, and at the same time, the energy metabolism and respiratory exchange rate (RER) were significantly increased (**P﹤0.01, ***P﹤0.005). These results indicate that EUP can effectively improve the energy metabolism of mice( Figure 7 ).

[0213] Example 2: Effects of EUP on the activity and lipid deposition of mouse hepatocytes AML12

[0214] 1. Experimental method

[0215] (1) Detection by CCK8 assay

[0216] Culture, drug treatment, and CCK8 detection of AML12 cells: AML12 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) in an incubator at 37 °C and 5% CO2.

[0217] Preparation of the standard curve: In a 96-well plate, 1×10 4 , 2×10 4 , 4×10 4 , 8×10 4 , 1.6×10 5 , 3.2×10 5 cells were seeded in each well to form a cell density gradient. After overnight culture, 10 μL of CCK8 reagent was added to each well, incubated for 1 - 4 hours, and the absorbance value (OD value) at 450 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader to plot the standard curve of cell number vs. OD value.

[0218] Drug treatment of AML12 cells: Seed AML12 cells into a 96-well plate at a density of 1×10 4 / well and incubate overnight. Prepare complete medium containing different concentrations of EUP (0, 10, 20, 40, 60, 80, 160 μM). Add the drug-containing medium to the 96-well plate and incubate for 48 hours. Remove the old medium, add 100 μL of fresh complete medium to each well, then add 10 μL of CCK8 reagent, and continue to incubate for 1 - 4 hours. Measure the absorbance at 450 nm using a microplate reader, and calculate the cell survival rate / % = [(Aexperimental group - Ablank group) / (Acontrol group - Ablank group)] × 100%. Through the above steps, the effect of EUP on the survival rate of AML12 cells can be evaluated, and its safe concentration range can be determined.

[0219] (2) Oil Red O staining

[0220] Modeling: Adjust AML-12 to a cell suspension with a density of 1×10 5 / mL, seed 1 mL / well into a 6-well plate, and culture overnight in a constant temperature incubator at 37°C and 5% carbon dioxide. When the cell confluence in the culture plate reaches 50% - 60%, discard the old medium, add DMEM complete medium containing 500 μM PO (oleic acid and palmitic acid, 1:1 ratio), and culture in a constant temperature incubator at 37°C and 5% carbon dioxide for 24 hours to construct a steatotic cell model.

[0221] After determining the EUP administration concentration, next analyze the effect of EUP on the PO-induced AML-12 cell NAFLD model. Divide the cultured cells into four groups: ① CON group: cell control group, given medium; ② PO group: PO model group, given 500 μM PO (oleic acid and palmitic acid, 1:1 ratio); ③ PO + EUP group: given 500 μM PO and 40, 80 μM EUP. After 24 hours of drug treatment, detect the corresponding lipid metabolism indicators and perform Oil Red O staining of the cells to observe, and evaluate the improvement effect of EUP in the in vitro model.

[0222] Staining procedure: Remove the cell culture medium, wash twice with PBS, add an appropriate amount of Oil Red O fixative and fix for 30 min; prepare Oil Red O staining solution; prepare 60% isopropanol, 6 ml of isopropanol + 4 ml of ddH2O; discard the fixative, wash twice with ddH20; add 60% isopropanol and soak for 20 s; discard the 60% isopropanol and then add the newly prepared Oil Red O staining solution, stain for 20 min; discard the staining solution, rinse with 60% isopropanol for 20 s until the stroma is clear. Wash with water 2 - 5 times until there is no excess staining solution; add Mayer hematoxylin staining solution and counterstain the nucleus for 1 - 2 min. Discard the staining solution and then wash 5 times with water; immerse in Oil Red O buffer for 1 min and discard; add distilled water to cover the cells and observe and photograph under the microscope.

[0223] (3) Detection of TC and TG levels

[0224] Detection of TC and TG levels in PO-induced AML12 cells: After successful modeling, the AML12 cells were detected according to the kit instructions.

[0225] 2. Experimental results

[0226] (1) EUP had no effect on the viability of AML12 cells at concentrations up to 80 μM for 24 h and 48 h ( Figure 8 ).

[0227] (2) Observation of lipid accumulation in hepatocytes by Oil Red O staining: The results of Oil Red O staining showed that compared with the control group, after 24 h of PO treatment, a large amount of red lipid deposition could be caused in AML12 cells; EUP treatment could significantly reduce the accumulation of lipid droplets in cells ( Figure 9 ).

[0228] (3) EUP significantly reduced the levels of TC and TG in PO-treated AML12 cells and reduced the lipid deposition in cells ( Figure 10 ).

[0229] Example 3: Effect of EUP on lipid deposition in human liver organoids

[0230] 1. Experimental method

[0231] (1) Culture of human liver organoids

[0232] 1) To isolate human hepatocytes, the fresh material should be processed as soon as possible after liver recovery. Wash the liver tissue with 0.9% (wt / vol) NaCl solution and observe under the microscope. Obvious white dendritic structures can be seen in the liver parenchyma. Remove them to avoid the growth of liver duct organoids.

[0233] 2) Aspirate the excess liquid, in human liver tissue (0.5 - 1 cm 3) Add 5 mL of freshly prepared type IV collagenase digestion solution for digestion (2 - 5 min, incubation at 37 °C). During this period, use forceps to cut into pieces of 2 - 5 mm and further pipette to mix evenly. Transfer to a 50 mL centrifuge tube and add 35 mL of adDMEM / F12 washing solution to terminate digestion, then centrifuge at low speed (300 - 400 g, 5 min, 4 °C).

[0234] 3) Wash and filter multiple times with ice-precooled adDMEM / F12 medium, and centrifuge at low speed (300 - 400 g, 5 min, 4 °C). At the same time, it is recommended to remove red blood cells.

[0235] 4) Resuspend the cell pellet with organoid matrix gel (Amoolo, A21122 - 0010) and aliquot into each well. Add 50 μL of matrix gel / hepatocyte mixture to the center of each well in a 24-well plate to form a dome-shaped embedding structure.

[0236] 5) Place the culture plate in the cell culture incubator and incubate for 15 minutes to allow the organoid matrix gel to polymerize.

[0237] 6) Add an appropriate volume of initial liver organoid medium, approximately 500 μL per well.

[0238] 7) Place the culture plate containing liver organoid cultures back into the cell culture incubator to promote organoid growth.

[0239] 8) After three days, aspirate the medium from each well and add fresh liver organoid expansion medium every other day; approximately 500 μL per well.

[0240] 9) Passage the organoids or start differentiation after 7 - 10 days.

[0241] (2) Differentiation of liver organoids

[0242] 1) After 7 - 10 days of expansion, aspirate the liver organoid expansion medium, wash once with PBS, and then add the same volume of liver organoid differentiation medium. Approximately 500 μL per well. Then incubate for 11 - 13 days.

[0243] 2) Aspirate the medium from each well every other day and then add fresh liver organoid differentiation medium.

[0244] (3) Nile red staining

[0245] After the differentiation of liver organoids, Nile red staining was performed. First, it was pre-incubated with 500 μM PO (oleic acid and palmitic acid, 1:1 ratio) for 3 days to cause fatty degeneration; then, in the presence of 500 μM PO, the organoids were treated with 40 and 80 μM concentrations of EUP for 7 days; all the organoids were collected for lipid staining. The organoids were collected in pre-cooled Advanced DMEM and washed once with pre-cooled Advanced DMEM. The precipitated organoids were fixed in 4% formaldehyde at room temperature (RT) for 30 - 60 minutes; the organoids were washed twice with PBS and incubated with Nile red (0.5 μg / ml) and DAPI (1 μg / ml) at RT for 20 minutes; the organoids were washed twice with PBS, and 100 μl of PBS was transferred to a 96-well black light-shielding plate for imaging analysis.

[0246] 2. Experimental results

[0247] Observation of lipid deposition in liver organoids by Nile red staining: The results showed that compared with the control group, there was a large amount of red lipid deposition in the liver organoid cells of the PO treatment group; the EUP intervention group could significantly reduce the accumulation of lipid droplets in the organoids ( Figure 11 ).

[0248] Example 4: New derivatives of eupatilin and their synthesis schemes

[0249] The inventors first synthesized four derivatives of eupatilin:

[0250] 1. Derivative 1

[0251] Derivative 1 is eupatilin-7-O-glucoside, and the structural formula and synthesis steps are as follows:

[0252]

[0253] Raw material preparation:

[0254] Eupatilin (the parent nucleus compound).

[0255] Activated glucose donors (including glucose bromide or glucose trifluoroacetate), and in this experiment, it was glucose bromide.

[0256] Glycosylation catalyst boron trifluoride diethyl ether (BF3·Et2O) and deprotection reaction catalyst phosphorus oxychloride (POCl3).

[0257] Reaction steps:

[0258] 1) Protect other hydroxyl groups:

[0259] Due to the presence of multiple hydroxyl groups (including 5-OH and 4'-OH) on the eupatilin nucleus, it is necessary to protect the non-target hydroxyl groups first to avoid side reactions. Specifically, trimethylchlorosilane (TMS-Cl) is used to protect 5-OH and 4'-OH:

[0260] Eupatilin + TMS-Cl → Protected Eupatilin.

[0261] 2) Glycosylation reaction:

[0262] Under anhydrous conditions, the protected eupatilin is reacted with activated glucose bromide under the action of the catalyst BF3·Et2O:

[0263] Protected Eupatilin + Glucose-Bromo + BF3·Et2O → Eupatilin-7-O-Glucoside

[0264] The reaction temperature is controlled at 0 °C to room temperature, and the time is 6 - 12 hours.

[0265] 3) Deprotection:

[0266] Phosphorus oxychloride (POCl3) is used to remove the protecting group to obtain the target product:

[0267] Protected Eupatilin-7-O-Glucoside → Eupatilin-7-O-Glucoside (Final Product).

[0268] 4) Product purification:

[0269] The target compound is separated and purified by column chromatography (silica gel column) or high performance liquid chromatography (HPLC). In this experiment, silica gel column chromatography is used.

[0270] The results of high performance liquid chromatography analysis are as Figure 15 .

[0271] 2. Derivative 2

[0272] Derivative 2 is a eupatilin-7-O-acetyl derivative, and its structural formula and synthesis steps are as follows:

[0273]

[0274] Required raw materials:

[0275] Eupatilin (the parent nucleus compound).

[0276] Acetylation reagent: acetic anhydride (Ac2O) or acetyl chloride (CH3COCl). Ac2O was used in this experiment.

[0277] Catalyst: organic base (such as pyridine) or Lewis acid (such as DMAP, 4-dimethylaminopyridine). DMAP was used in this experiment.

[0278] Solvent: anhydrous dichloromethane (CH2Cl2) or acetonitrile (CH3CN). Anhydrous dichloromethane (CH2Cl2) was used in this experiment.

[0279] Reaction steps:

[0280] 1) Protect other hydroxyl groups:

[0281] Eupatilin contains multiple hydroxyl groups (including 5-OH and 4'-OH). To avoid side reactions, it is necessary to protect the non-target hydroxyl groups first.

[0282] Common protecting groups are silyl (TMS, trimethylsilyl) or benzyl (Bn, benzyl). Trimethylchlorosilane (TMS-Cl) was used to protect 5-OH and 4'-OH in this experiment:

[0283] Eupatilin + TMS-Cl → Protected Eupatilin.

[0284] 2) Acetylation reaction:

[0285] Dissolve the protected eupatilin in anhydrous dichloromethane (CH2Cl2).

[0286] Slowly add acetic anhydride and a small amount of DMAP as a catalyst.

[0287] Control the reaction temperature at 0 °C to room temperature and stir for 4 - 6 hours to complete the acetylation at the 7-OH position:

[0288] Protected Eupatilin + Ac2O → 7-O-Acetylated Eupatilin

[0289] 3) Deprotection:

[0290] After the reaction is completed, use an acid (trifluoroacetic acid (TFA)) or a reducing agent (Pd / C-catalyzed hydrogenation) (TFA was used in this experiment) to remove the protecting group to obtain the target product:

[0291] Protected 7-O-Acetylated Eupatilin → Eupatilin-7-O-Acetate

[0292] 4) Product purification:

[0293] The solvent was removed by vacuum distillation or rotary evaporation (vacuum distillation was used in this experiment).

[0294] The eupatilin-7-O-acetyl derivative was purified by silica gel column chromatography (the eluent was ethyl acetate / n-hexane).

[0295] The results of high performance liquid chromatography analysis were as Figure 16 .

[0296] 3. Derivative 3

[0297] Derivative 3 is 4'-chloro-eupatilin, and its structural formula and synthesis steps are as follows:

[0298]

[0299] Raw materials required:

[0300] Eupatilin (the parent compound).

[0301] Chlorination reagent: In the site-specific demethoxylation reaction, the 4'-methoxy group can be activated by aluminum chloride (AlCl3) or phosphorus pentachloride (PCl5), and AlCl3 was used in this experiment; in the site-specific chlorination reaction, the chlorination reagent can be phosphorus oxychloride (POCl3) or thionyl chloride (SOCl2), and POCl3 was used in this experiment.

[0302] Solvent: Dichloromethane (CH2Cl2) or toluene, and dichloromethane (CH2Cl2) was used in this experiment.

[0303] Reaction steps:

[0304] 1) Selective demethoxylation:

[0305] In the molecule containing a methoxy group, the methoxy group was activated by a Lewis acid (AlCl3) or an acidic solvent (trichloroacetic acid) (AlCl3 was used in this experiment) to remove the methyl group, generating the intermediate 4'-hydroxy-eupatilin.

[0306] Reaction conditions: The solvent can be dichloromethane or toluene (dichloromethane was used in this experiment), the reaction temperature was controlled at room temperature to 50 °C, and the time was 6 to 12 hours.

[0307] Eupatilin + AlCl3 → 4'-hydroxy-eupatilin

[0308] 2) Chlorination reaction:

[0309] The obtained 4'-hydroxy-eupatilin was dissolved in anhydrous dichloromethane, and the chlorination reagent POCl3 was slowly added for the chlorination reaction, and the hydroxyl group was replaced by a chlorine atom to generate the target product 4'-chloro-eupatilin.

[0310] Reaction conditions: anhydrous environment, control the temperature for 0 - 6 hours.

[0311] 4’-Hydroxy eupatilin + POCl3 → 4’-Chloro - eupatilin

[0312] 3) Product purification:

[0313] Remove the solvent by distillation under reduced pressure. The obtained crude product is separated by silica gel column chromatography (eluent: n - hexane / ethyl acetate) to obtain pure 4’-Chloro - eupatilin.

[0314] The results of high - performance liquid chromatography analysis are as Figure 17 .

[0315] 4. Derivative 4

[0316] Derivative 4 is thio - eupatilin, and its structural formula and synthesis steps are as follows:

[0317]

[0318] Raw material preparation:

[0319] Eupatilin (the parent compound).

[0320] Sulfurizing reagent (such as phosphorus sulfide (P4S 10 ) or hydrogen sulfide (H2S)), in this experiment, P4S 10 .

[0321] Solvent (such as pyridine or dimethylformamide (DMF)), in this experiment, it is pyridine.

[0322] Reaction steps:

[0323] 1) Sulfurization reaction:

[0324] Dissolve eupatilin in pyridine and add a sulfurizing reagent (such as P4S 10 ).

[0325] The reaction is usually heated at 80℃ - 100℃ for 48 hours to replace the oxygen atom in the C - ring with a sulfur atom:

[0326] Eupatilin + P4S 10 → Thio - Eupatilin

[0327] 2) Neutralization treatment:

[0328] Cool the reaction mixture to room temperature and neutralize it with 5% HCl to precipitate the crude product.

[0329] 3) Product purification:

[0330] Separate and purify eupatilin using column chromatography (silica gel column, eluent: ethyl acetate / n-hexane).

[0331] The results of high performance liquid chromatography analysis are as Figure 18 .

[0332] Example 5: Treatment of high-fat diet-induced MASLD model in mice with four newly synthesized EUP derivatives

[0333] 1. Experimental method

[0334] (1) High-fat diet-induced MASLD mice and drug intervention

[0335] Select 30 male C57 mice of the same batch, 5 - 6 weeks old, and feed them with a high-fat and high-sugar diet to establish a fatty liver model. After 8 weeks of feeding, randomly select three mice for sacrifice, collect liver samples for HE staining to detect liver pathology, and collect blood samples to detect liver function. If obvious fat vacuoles are observed in the liver HE staining and the liver function indicators ALT, TG, and TC are significantly increased, the establishment of the MASLD model is considered successful. After confirming the successful establishment of the model, randomly divide the mice into a fatty liver model (HFD) group, a high-dose EUP group, an eupatilin-7-O-glucoside group, an eupatilin-7-O-acetyl group, a 4'-chloro-eupatilin group, and an eupatilin group, a total of 6 groups, with 5 mice in each group. Among them, the HFD group is treated with 2 mL of normal saline by gavage, and the high-dose EUP group and the four new derivative groups are treated with 40 mg / kg / d of the corresponding drugs by gavage. When treating, disperse the corresponding drugs in 2 mL of normal saline for gavage. The treatment time is 8 weeks, and the body weight is recorded weekly.

[0336] (2) Collect serum and liver samples

[0337] At the end of 16 weeks of feeding, anesthetize and sacrifice the mice and collect serum and liver samples. Specifically, after fasting all mice for 12 hours, anesthetize the mice by intraperitoneal injection, take blood from the orbital cavity, let it stand at room temperature for 2 hours, then centrifuge at 4000 rpm for 10 minutes at 4 °C, collect the serum samples and store them at -80 °C. Quickly take out the liver and epididymal fat and weigh them. Take a small piece of liver tissue from the same part of the right lobe of the liver, rinse it with 4 °C normal saline, dry it with filter paper, fix it with 4% paraformaldehyde solution, and store the remaining liver tissue at -80 °C for subsequent index detection.

[0338] Detect the pathological morphological characteristics, lipid deposition, and liver injury of the livers of mice in each group by hematoxylin-eosin staining and oil red O staining. Serum index detection: Use a biochemical analyzer to detect the contents of serum ALT, TG, TC, and GLU.

[0339] 2. Experimental results

[0340] (1) The body weight of the MASLD model mice increased significantly, and high-dose EUP and the four new derivatives could all significantly reduce the body weight gain caused by HFD ( Figure 12 ), among which the body weights of the eupatilin-7-O-glucoside group and the eupatilin-7-O-acetyl group were close to that of the high-dose EUP group, while the body weights of the 4'-chloro-eupatilin group and the thioeupatilin group decreased more significantly than that of the high-dose EUP group (*P﹤0.05, **P﹤0.01, ***P﹤0.005), suggesting that 4'-chloro-eupatilin and thioeupatilin have better body weight control effects than EUP ( Figure 12 ).

[0341] (2) The results of hematoxylin-eosin staining and oil red O staining and their quantitative analysis showed that obvious fat vacuole structures existed in the livers of the mice in the HFD group, and lipid deposition in hepatocytes was significant. The lipid deposition in the livers of the high-dose EUP group and the four new derivative groups was significantly reduced, among which the improvement of lipid deposition in the 4'-chloro-eupatilin group and the thioeupatilin group was more obvious than that in the EUP group (*P﹤0.05, **P﹤0.01, ***P﹤0.005), suggesting that these two derivatives have better liver fat-reducing effects than EUP ( Figure 13 ).

[0342] (3) High-fat diet led to a significant increase in the levels of serum ALT, TG, TC, and GLU, while EUP and its 4 new derivatives significantly reversed the accumulation of lipids in serum and liver and the increase in blood glucose. Among them, the improvement of ALT, blood lipid, and blood glucose indexes in the 4'-chloro-eupatilin group and the thioeupatilin group was the most obvious (*P﹤0.05, **P﹤0.01, ***P﹤0.005), suggesting that these two derivatives have better effects on reducing liver damage and regulating blood lipid and blood glucose than EUP ( Figure 14 ).

[0343] The preferred embodiments disclosed in the present invention aim to elaborate its core idea, do not cover all details in detail, nor limit its only application form. Based on the content of this specification, extensive modifications and adjustments can be made. The selection and detailed description of the embodiments are intended to clearly show the principle of the present invention and its practical applications, facilitating understanding and application by technicians. The above embodiments are only examples, specific and detailed, but not a limitation on the scope of patent protection. Those skilled in the art can make various innovations and optimizations without departing from the core concept of the present invention, which all fall within the protection scope of the present invention. The protection scope of the present invention shall be subject to what is described in the claims.

Claims

1. Use of a compound having a tricyclic parent nucleus structure represented by formula (I) or a pharmaceutically acceptable salt thereof for preparing a composition for preventing and / or treating metabolic dysfunction-associated steatohepatitis or MASLD-related metabolic diseases; Wherein: X is selected from: O, S, Se, C═S; Y is a linking group between ring B and ring C and is selected from H, C1-C4 alkyl, halogen.

2. The application according to claim 1, characterized in that The compound having a tricyclic parent nucleus structure represented by formula (I) includes the compound of formula (II); Wherein: R0 or R1 is independently selected from: hydroxy, (-O-) glycosyl, carboxyl, C1-C4 alkoxy, C1-C4 alkyl, hydrogen, halogen, nitro, cyano, amino, substituted amino, acyl, trifluoromethyl; wherein, Ra or Rb are each independently selected from a hydrogen atom or an organic group; R2 or R3 is independently selected from: C1-C4 alkoxy, halogen, hydrogen, C1-C4 alkyl, propyl, hydroxy, cyano, nitro, amino, substituted amino, acyl, trifluoromethyl; R4 is independently selected from: C1-C4 alkoxy, hydrogen, halogen, C1-C4 alkyl, hydroxy, cyano, nitro, amino, substituted amino, acyl, trifluoromethyl.

3. The application according to claim 1, characterized in that, The compound having a tricyclic parent nucleus structure represented by formula (I) includes compounds selected from the following group: (1) Eupatilin; (2) Eupatilin-7-O-glucoside; (3) Eupatilin-7-O-acetyl derivative; (4) 4'-Chloro-eupatilin; (5) Thioeupatilin; (6) Having the tricyclic parent nucleus structure, wherein one hydroxy group is replaced by a methoxy group; (7) Having the tricyclic parent nucleus structure, wherein one hydroxy group is replaced by a carbonyl group; (8) Having the tricyclic parent nucleus structure, wherein one hydroxy group is replaced by a vinyl group; (9) Having the tricyclic parent nucleus structure, wherein two hydroxy groups are replaced by methoxy groups; (10) Having the tricyclic parent nucleus structure, wherein two hydroxy groups are replaced by carbonyl groups; (11) Having the tricyclic parent nucleus structure, wherein one methoxy group is replaced by a hydroxy group; (12) Having the tricyclic parent nucleus structure, wherein one methoxy group is replaced by a carbonyl group; (13) Having the tricyclic parent nucleus structure, wherein one hydroxy group and one methoxy group are replaced by vinyl groups; (14) Having the tricyclic parent nucleus structure, wherein one hydroxy group and one carbonyl group are replaced by vinyl groups; (15) Having the tricyclic parent nucleus structure, wherein one hydroxy group is replaced by a hydrogen atom; (16) Having the tricyclic parent nucleus structure, wherein one methoxy group is replaced by a hydrogen atom; (17) Having the tricyclic parent nucleus structure, wherein one hydroxy group is replaced by a methyl group.

4. The application according to any one of claims 1 to 3, characterized in that The MASLD or MASLD-related metabolic diseases include: (a) Liver diseases related to systemic metabolic disorders, preferably including: simple steatosis, metabolic dysfunction-associated steatohepatitis, MASLD-related liver fibrosis, MASLD-related cirrhosis, MASLD-related hepatocellular carcinoma; (b) Diseases related to metabolic factors including obesity, insulin resistance, vascular dysfunction or dyslipidemia, preferably including: overweight, obesity, hyperglycemia, hypertension, dyslipidemia.

5. The application according to any one of claims 1 to 3, characterized in that, The described composition is also used for: reducing serum liver function indicators such as aspartate aminotransferase and alanine aminotransferase; optimizing lipid profiles such as high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, and total cholesterol; reducing the levels of inflammatory factors such as interleukin-6 and tumor necrosis factor-α in the liver and serum; enhancing superoxide dismutase activity and reducing malondialdehyde content; or increasing antioxidant substances.

6. A compound having the structure shown in formula (II) or a pharmaceutically acceptable salt thereof; Wherein: X is selected from: O, S, Se, C═S; Y is a linking group between ring B and ring C and is selected from H, C1-C4 alkyl, halogen; R0 or R1 is independently selected from: hydroxy, (-O-) glycosyl, carboxyl, C1-C4 alkoxy, C1-C4 alkyl, hydrogen, halogen, nitro, cyano, amino, substituted amino, acyl, trifluoromethyl; wherein, Ra or Rb is independently selected from a hydrogen atom or an organic group; R2 or R3 is independently selected from: C1-C4 alkoxy, halogen, hydrogen, C1-C4 alkyl, hydroxy, cyano, nitro, amino, substituted amino, acyl, trifluoromethyl; R4 is independently selected from: C1-C4 alkoxy, hydrogen, halogen, C1-C4 alkyl, hydroxy, cyano, nitro, amino, substituted amino, acyl, trifluoromethyl; And, the compound having the structure shown in formula (II) does not include eupatilin.

7. The compound having the structure shown in formula (II) or a pharmaceutically acceptable salt thereof according to claim 6, characterized in that, The compound having the structure shown in formula (II) includes the compound defined in claim 3.

8. A composition comprising: the compound having the structure shown in formula (II) according to claim 6 or 7 or a pharmaceutically acceptable salt thereof; and, a pharmaceutically acceptable carrier.

9. A medicine box or kit, comprising: The compound having the structure shown in formula (II) according to claim 6 or 7 or a pharmaceutically acceptable salt thereof; or The composition according to claim 8.

10. A method for preventing and / or treating metabolic dysfunction-associated steatohepatitis or MASLD-related metabolic diseases, comprising: Administering an effective amount of the compound shown in formula (I) or a pharmaceutically acceptable salt thereof to a subject in need of treatment; Preferably, administering an effective amount of the compound shown in formula (II) or a pharmaceutically acceptable salt thereof to a subject in need of treatment.

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