Use of benzofuro[3,2-c]quinolinone compounds in preventing and treating metabolic dysfunction-related fatty hepatitis

Abstract

The application discloses application of a benzofuro[3,2-c]quinolinone compound in preventing and treating metabolic dysfunction related steatohepatitis. The compound P82, i.e. 3,8-dihydroxybenzofuro[3,2-c]quinolin-6(5H)-one, is prepared through synthesis and purification of an intermediate, and can obviously improve liver steatosis, inflammatory infiltration and fibrosis in a metabolic dysfunction related steatohepatitis model induced by HFHC, thereby effectively delaying the progress of metabolic dysfunction related steatohepatitis. Meanwhile, the compound can improve lipid accumulation of liver cells induced by oleic acid and palmitic acid, and is expected to be developed into a new drug for preventing and treating metabolic dysfunction related steatohepatitis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the synthesis and application of benzofurano[3,2-c]quinolinone compounds, particularly the application of these compounds in the preparation of drugs for the prevention and / or treatment of metabolic dysfunction-related steatohepatitis. Background Technology

[0002] Metabolic dysfunction-associated steatohepatitis (MASH) has become a major challenge in the global health field. MASH is the advanced stage of nonalcoholic fatty liver disease (NAFLD), which is a collective term for a series of diseases characterized by lipid accumulation and steatosis in hepatocytes.

[0003] Mastalgia pilaris (MASH) is a complex liver disease characterized by abnormal fat accumulation in the liver, accompanied by inflammation and hepatocellular damage, which can lead to liver fibrosis, liver failure, and even liver cancer in severe cases. However, treatment options for this disease remain limited. To date, only one thyroxine receptor β (THR-β) agonist, resmetirom, has been approved by the US FDA for the treatment of MASH, but its patient response rate is only 25%–30%, and its relatively high cost (US$40,000–50,000 per year) also limits its clinical application. Some technologies related to this drug have proposed methods for preparing intermediates or specific crystal forms of resmetirom (e.g., CN114907327A). Therefore, there is an urgent need to find more novel and effective candidate compounds to provide new avenues for the drug treatment of MASH.

[0004] Benzofuran is a common structural unit in natural products, and numerous reports have shown that its derivatives possess significant anti-inflammatory and antioxidant activities. For example, 2,3-dihydrobenzofuran derivatives can inhibit the expression of cyclooxygenase-2 and nitric oxide synthase-2, thereby suppressing lipopolysaccharide-stimulated inflammatory responses and further inhibiting the production of inflammatory mediators (International Journal of Molecular Sciences, 2023, 24(12): 10399.); 2-arylbenzofuran derivatives can significantly inhibit the production of interleukin-6 and NO, reduce the level of inflammatory factors in serum, and weaken the infiltration of inflammatory cells and excessive mucus secretion in lung tissue (Scientific Reports, 2019, 9(1): 862.); 5-amide benzofuran derivatives, as an inhibitor of the NLRP3 inflammasome, can enhance the release of IL-1β and significantly improve peritoneal inflammation in model mice (Bioorganic & Medicinal Chemistry Letters, 2021, 46: 128160.); In addition, benzofuran-2(3H)-one derivatives also have antioxidant activity, which can scavenge free radicals and thus reduce oxidative stress (Molecules, 2018, 23(4): 710.). However, these benzofuran compounds have not shown hepatoprotective activity, nor have there been any reports of their use in the treatment of MASH patients with inflammation and hepatocellular damage.

[0005] In addition, CN107021958A and CN107021957A proposed compounds for the prevention and / or treatment of non-alcoholic fatty liver disease mediated by FXR receptors, but their efficacy in preventing and treating MASH was not verified; CN116693456A proposed the use of aryl carboxylic acid compounds (specifically isoquinolinone compounds) as dual-target inhibitors of FABP4 / FABP5, specifically including the prevention and treatment efficacy against NAFLD (and MASH), but the experiments were limited to the cellular level, and their role in guiding clinical drug use was limited; the tetracyclic quinolinone alkaloid derivatives proposed in CN108623590A are mainly used as antiviral, antibacterial and antiparasitic drugs. Summary of the Invention

[0006] To address the scarcity of therapeutic drugs for metabolic dysfunction-associated steatohepatitis (MASH), this invention provides the application of benzofurano[3,2-c]quinolinone compounds in the prevention and treatment of MASH.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the use of a benzofurano[3,2-c]quinolinone compound in the preparation of a medicament for the prevention and / or treatment of metabolic dysfunction-related steatohepatitis is provided, wherein the benzofurano[3,2-c]quinolinone compound is any one of a compound with the structure shown below or a pharmaceutically acceptable salt thereof:

[0009]

[0010] Among them, R 1 For H, R 2 It is any one of H or C1~C3 alkyl groups (e.g., -CH3).

[0011] Preferably, the benzofurano[3,2-c]quinolinone compound improves hepatic steatosis.

[0012] Preferably, the benzofurano[3,2-c]quinolinone compound improves liver fibrosis.

[0013] Preferably, the benzofurano[3,2-c]quinolinone compound reduces inflammatory infiltration of the liver and ballooning degeneration of hepatocytes in the liver.

[0014] Preferably, the metabolic dysfunction-associated steatohepatitis is induced by a high-fat, high-cholesterol (HFHC) diet.

[0015] Preferably, the drug further includes any one or more combinations of pharmaceutically acceptable excipients.

[0016] Secondly, the use of the aforementioned benzofurano[3,2-c]quinolinone compounds in the preparation of medicaments for delaying the progression of non-alcoholic fatty liver disease (particularly metabolic dysfunction-related steatohepatitis).

[0017] Preferably, the drug further includes any one or more combinations of pharmaceutically acceptable excipients.

[0018] Thirdly, a drug for treating metabolic dysfunction-related steatohepatitis is provided, the drug comprising (e.g., containing a therapeutically effective amount) any one of a compound with the structure shown in the following formula or a pharmaceutically acceptable salt of the compound:

[0019]

[0020] Among them, R f It is any one of C1 to C3 alkyl groups (e.g., -CH3; that is, the drug may be a newly synthesized benzofurano[3,2-c]quinolinone compound or a pharmaceutically acceptable salt thereof as the active ingredient and used for the prevention and / or treatment of metabolic dysfunction-related steatohepatitis).

[0021] Preferably, the drug further includes any one or more combinations of pharmaceutically acceptable excipients.

[0022] Preferably, the drug can be formulated into dosage forms such as tablets, granules, capsules, pills, oral liquids, or sustained-release formulations.

[0023] Preferably, the pharmaceutically acceptable salt is selected from any one of the following: medicinal ammonium salts, alkali metal salts (e.g., sodium salts, potassium salts) and alkaline earth metal salts (e.g., magnesium salts, calcium salts), as well as salts of aminobutanetriol, diethanolamine, ethylenediamine, etc.

[0024] Fourthly, a method for preparing a benzofurano[3,2-c]quinolinone compound is provided, comprising the following steps:

[0025] Compounds with the following structures were prepared from 2-bromo-5-alkoxyaniline (e.g., 2-bromo-5-methoxyaniline) as starting materials and used as intermediates:

[0026]

[0027] Among them, R k R is any one of the C1 to C3 alkyl groups. f It is any one of the C1 to C3 alkyl groups;

[0028] The obtained intermediate was used to prepare benzofurano[3,2-c]quinolinone compounds with the following structure by a dealkylation reaction (e.g., demethylation reaction):

[0029]

[0030] Among them, R 1 For H, R 2 It is any one of H or C1~C3 alkyl groups (e.g., -CH3).

[0031] Preferably, the preparation method of the benzofurano[3,2-c]quinoline compound specifically includes the following steps:

[0032] In the first step, under nitrogen (N2) protection, 2-bromo-5-methoxyaniline, cuprous iodide (CuI), palladium dichloride bis(triphenylphosphine) chloride (Pd(PPh3)2Cl2), and triphenylphosphine (PPh3) are added. Then, triethylamine (Et3N) and toluene are added as a mixed reaction solvent, followed by trimethylsilylacetylene. The resulting system has a molar ratio of 2-bromo-5-methoxyaniline:trimethylsilylacetylene:cuprous iodide:palladium dichloride bis(triphenylphosphine) chloride:triphenylphosphine of 100:150:2:2:5. Then, under nitrogen protection... Under protection, the system was heated to 80-120℃ and subjected to the Sonogashira coupling reaction. After reacting for 16-30 hours, the system was filtered, and the filter cake was washed with ethyl acetate (or dichloromethane). The filtrates and eluents were combined, and all solvents (e.g., ethyl acetate, triethylamine, and toluene) were removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography with a 200-300 mesh filter, wherein the eluent was petroleum ether:ethyl acetate (volume ratio) = 60:1-40:1. Gradient elution yielded 5-methoxy-2-((trimethylsilyl)ethynyl)aniline.

[0033] The second step uses 5-methoxy-2-((trimethylsilyl)ethynyl)aniline as the first intermediate compound, anhydrous potassium carbonate (K2CO3), potassium tert-butoxide (t-BuOK), or sodium tert-butoxide (t-BuONa) to provide an alkaline environment, and anhydrous methanol (MeOH) as the reaction solvent. The molar ratio of 5-methoxy-2-((trimethylsilyl)ethynyl)aniline to anhydrous potassium carbonate (or potassium tert-butoxide or sodium tert-butoxide) in the resulting system is 1:(1.2~2). The system is then heated to 25°C. The detrimethylsilylation reaction was carried out at ~28℃. After 2-4 hours of reaction, the system was filtered, and the filter cake was washed with ethyl acetate (or dichloromethane). The filtrates and eluents were combined, and all solvents (e.g., ethyl acetate and methanol) were removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography with a 200-300 mesh, wherein the eluent was petroleum ether:ethyl acetate (volume ratio) = 50:1-30:1. Gradient elution yielded the second intermediate compound (specifically 2-ethynyl-5-methoxyaniline).

[0034] In the third step, under nitrogen protection, 2-ethynyl-5-methoxyaniline, cuprous iodide, and palladium dichloride bis(triphenylphosphine) were first added, followed by triethylamine as a reaction solvent, and then 1-bromo-2-iodo-4-methoxybenzene. The resulting system had a molar ratio of 2-ethynyl-5-methoxyaniline:1-bromo-2-iodo-4-methoxybenzene:cuprous iodide:palladium dichloride bis(triphenylphosphine)) of 50:60:2:1. The system was then subjected to Sonogas reaction under nitrogen protection at 25–28 °C. The hira coupling reaction was carried out for 5-10 hours, and the system was filtered. The filter cake was washed with ethyl acetate (or dichloromethane). The filtrates and eluents were combined and all solvents (e.g., ethyl acetate and triethylamine) were removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography with a 200-300 mesh screen, wherein the eluent was petroleum ether:ethyl acetate (volume ratio) = 40:1-25:1. Gradient elution yielded 2-((2-bromo-5-methoxy)ethylynyl)-5-methoxyaniline.

[0035] In the fourth step, 2-((2-bromo-5-methoxy)ethylynyl)-5-methoxyaniline was used as the third intermediate compound, copper acetate (Cu(OAc)2) as a catalyst, silver carbonate (Ag2CO3) as an additive (oxidizing agent), cesium carbonate (Cs2CO3) as an oxidant and to provide an alkaline environment, and dimethyl sulfoxide (DMSO) as a reaction solvent. The resulting system had a molar ratio of 2-((2-bromo-5-methoxy)ethylynyl)-5-methoxyaniline:copper acetate:silver carbonate:cesium carbonate of 10:1:10:40. The system was then heated to 120-180℃. The reaction proceeded with an oxidative ring-closure reaction. After 24-48 hours, the system was subjected to vacuum distillation and ethyl acetate / water extraction to remove the solvent (specifically DMSO). The resulting ethyl acetate phase was washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to vacuum distillation to remove the solvent (specifically ethyl acetate) to obtain the crude product. The crude product was purified by silica gel column chromatography with a 200-300 mesh screen, using dichloromethane:methanol (volume ratio) of 80:1-50:1 as the eluent. Gradient elution yielded 3,8-dimethoxybenzofurano[3,2-c]quinoline-6(5H)-one, i.e., compound P80 (structure shown below):

[0036]

[0037] In the fifth step, under nitrogen protection, compound P80 was first added, followed by dichloromethane (CH2Cl2) as the reaction solvent. Then, a dichloromethane solution of boron tribromide (BBr3) was added at 0-5℃, resulting in a molar ratio of compound P80 to boron tribromide of 1:(3-6). The system was then heated to 25-28℃ under nitrogen protection for demethylation. After 12-24 hours of reaction, water was added at 0-5℃ to precipitate a solid. The solid was allowed to stand, and then separated by filtration to obtain the crude product. The crude product was purified by dry chromatography using a 200-300 mesh silica gel column with dichloromethane:methanol (volume ratio) of 50:1-30:1 as the eluent. Gradient elution yielded 3-hydroxy-8-methoxybenzofurano[3,2-c]quinoline-6(5H)-one, compound P81 (structure shown below).

[0038]

[0039] In the sixth step, under nitrogen protection, compound P81 was first added, followed by dichloromethane as the reaction solvent. Then, a dichloromethane solution of boron tribromide was added at 0-5°C, resulting in a molar ratio of compound P81 to boron tribromide of 1:(3-6). The system was then heated to 25-28°C under nitrogen protection for demethylation. After 12-24 hours of reaction, water was added at 0-5°C to precipitate a solid. The solid was allowed to stand and then separated by filtration to obtain the crude product. The crude product was purified by dry chromatography using a 200-300 mesh silica gel column with dichloromethane:methanol (volume ratio) of 35:1-25:1 as the eluent. Gradient elution yielded 3,8-dihydroxybenzofurano[3,2-c]quinoline-6(5H)-one, compound P82 (structure shown below).

[0040]

[0041] Fifthly, a method for preparing a pharmaceutical intermediate is provided, comprising the following steps:

[0042] 2-Bromo-5-alkoxyaniline (e.g., 2-bromo-5-methoxyaniline) is subjected to a Sonogashira coupling reaction with trimethylsilylacetylene to give a first intermediate compound (i.e., 5-alkoxy-2-((trimethylsilylacetyl)ethynyl)aniline), wherein the alkyl group contained in the alkoxy group is any one of C1-C3 alkyl groups (e.g., -CH3); the first intermediate compound is subjected to a detrimethylsilylation reaction under basic conditions to give a second intermediate compound (i.e., 2-ethynyl-5-alkoxyaniline); the second intermediate compound is then reacted with 1 -Bromo-2-iodo-4-alkoxybenzene (e.g., 1-bromo-2-iodo-4-methoxybenzene) undergoes a Sonogashira coupling reaction to give a third intermediate (i.e., 2-((2-bromo-5-alkoxy)ethylynyl)-5-alkoxyaniline), wherein the alkyl group in the alkoxy group is any one of C1-C3 alkyl groups (e.g., -CH3); the third intermediate is then subjected to an oxidative cyclization reaction with the addition of a silver salt (e.g., silver carbonate) or silver oxide and under basic conditions to give a compound with the following structure, i.e., the intermediate:

[0043]

[0044] Among them, R k R is any one of the C1 to C3 alkyl groups. f It can be any one of C1 to C3 alkyl groups; for example, the intermediate obtained has the following structure:

[0045] .

[0046] The beneficial effects of this invention are reflected in:

[0047] This invention targets benzofurano[3,2-c]quinolinone compounds (such as compounds P81 and P82 mentioned above), and for the first time demonstrates their role in improving lipid accumulation in hepatocytes through cellular level experiments. In vivo experiments were also conducted to screen candidate drug molecules (such as compound P81 mentioned above). Experimental results show that these benzofurano[3,2-c]quinolinone compounds have the potential to be developed into novel anti-MASH drugs. Their significant effects in reducing hepatic steatosis, inflammation, and fibrosis levels demonstrate their ability to alleviate MASH-related symptoms and thus slow the progression of non-alcoholic fatty liver disease (especially MASH).

[0048] Furthermore, this invention, through intervention experiments on multiple benzofurano[3,2-c]quinolinone compounds induced by chemical induction (specifically palmitic acid and oleic acid), discovered the structure-activity relationship of these compounds in improving lipid accumulation in liver cells (specifically involving normal human liver cells and human liver cancer cells) (see the substituent differences or changes in compounds P80, P81, and P82 based on the "benzofurano[3,2-c]quinolinone" core), providing a basis for screening drug molecules for the prevention and / or treatment of MASH.

[0049] Furthermore, this invention experimentally determined that compound P82 can significantly improve hepatic steatosis, reduce hepatocyte ballooning degeneration and inflammatory infiltration in the liver, and improve liver fibrosis in HFHC-induced MASH animal models. At the same time, it reduces the levels of total cholesterol (TC), triglycerides (TG), and blood lipids in the liver, as well as the levels of liver damage indicators such as aspartate aminotransferase (ALT) and alanine aminotransferase (AST). These results suggest that compound P82 can play a better role in the prevention and / or treatment of MASH.

[0050] The benzofurano[3,2-c]quinolinone compound of this invention, as a drug molecule, can be prepared using similar compounds (such as compound P80) as intermediates. Moreover, the synthesis and purification process is simple and can better meet the needs of large-scale and intensive production in the pharmaceutical industry.

[0051] Furthermore, by controlling reaction conditions (including appropriate reaction time, avoiding excessively high reaction temperature, and the proportion of reaction reagents), this invention effectively solves the problem that the target compound (e.g., P81 or P82) is generated to varying degrees during the preparation of the target compound using an intermediate (e.g., compound P80), making it difficult to separate and purify, thus ensuring a high yield of the target compound.

[0052] Furthermore, the method for preparing the intermediate (e.g., compound P80) proposed in this invention can use the cheaper 2-bromo-5-methoxyaniline instead of 2-iodo-5-methoxyaniline as a starting material, thereby reducing the synthesis cost. Attached Figure Description

[0053] Figure 1 The results of compounds P80, P81, and P82 reducing OA+PA-induced lipid accumulation in hepatocytes; including: (A) Oil Red O staining and Bodipyridine (BRP) fluorescence staining of human HepG2 liver cancer cells; (B) Oil Red O staining and Bodipyridine (BRP) fluorescence staining of normal human L02 liver cells; p <0.01 Vs. Blank control group,** p<0.01 Vs. Model group.

[0054] Figure 2 Results of compound P82 in alleviating HFHC-induced symptoms in MASH mice; including: (A) grouping and treatment; (B) liver weight; (C) liver weight / body ratio; (D) HE staining (H&E) and NAS score statistics; (E) Oil Red O staining and Oil Red O-positive area statistics; (F) Masson staining and Masson positive area statistics; (G) serum TG content; (H) serum TC content; (I) AST activity; (J) ALT activity;** p <0.01 Vs. Model group.

[0055] Figure 3 HE staining results of the effects of compound P82 on mouse organs in toxicity evaluation.

[0056] Figure 4-1 This is the synthetic route for benzofurano[3,2-c]quinolinone compound P80 (i.e., compound P80).

[0057] Figure 4-2 The 1H NMR spectrum of benzofurano[3,2-c]quinolinone compound P80 is shown.

[0058] Figure 5-1 This is the synthetic route for benzofurano[3,2-c]quinolinone compound P81 (i.e., compound P81).

[0059] Figure 5-2 The 1H NMR spectrum of benzofurano[3,2-c]quinolinone compound P81 is shown.

[0060] Figure 6-1 This is the synthetic route for benzofurano[3,2-c]quinolinone compound P82 (i.e., compound P82).

[0061] Figure 6-2 The 1H NMR spectrum of benzofurano[3,2-c]quinolinone compound P82 is shown. Detailed Implementation

[0062] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0063] The benzofurano[3,2-c]quinolinone compound synthesized in this invention has the general structural formula shown in Formula 1:

[0064]

[0065] For the benzofurano[3,2-c]quinolinone compounds (e.g., compound P80: R 1 = R 2 = -CH3; Compound P81: R 1 = H, R 2 = -CH3; Compound P82: R 1 = R 2 = H), to intervene and evaluate metabolic dysfunction-associated steatohepatitis (MASH) at the cellular and in vivo levels; the results showed that these compounds have certain anti-MASH activity. Details are as follows.

[0066] (I) Preparation of benzofurano[3,2-c]quinolinone compound P80

[0067] The compound P80 has the following structural formula and is named 3,8-dimethoxybenzofurano[3,2-c]quinoline-6(5H)-one:

[0068]

[0069] The synthetic route of compound P80 is as follows: Figure 4-1 As shown, the specific preparation steps are as follows:

[0070] (1) Under nitrogen protection, 10.10 g (50 mmol) of 2-bromo-5-methoxyaniline, 190 mg (1 mmol) of cuprous iodide, 700 mg (1 mmol) of bis(triphenylphosphine)palladium dichloride, and 650 mg (2.5 mmol) of triphenylphosphine were added sequentially to a round-bottom flask. The flask was then purged with nitrogen three times. Under nitrogen protection, 50 mL of triethylamine and 200 mL of toluene were added to the flask. Finally, 7.4 g (75 mmol) of trimethylsilylacetylene was added dropwise. After the addition was complete, the system was heated to 100 °C under nitrogen protection. The Sonogashira coupling reaction was carried out at 100 °C for 24 hours. After the reaction, the system was filtered through a Buchner funnel. The filter cake was washed with ethyl acetate. The filtrate and eluent were combined and distilled under reduced pressure (40 °C, -0.09 ~ The solvent was removed at -0.1 MPa, and the crude product was purified by silica gel column chromatography with a 200-300 mesh. The eluent was petroleum ether:ethyl acetate = 60:1-40:1. Gradient elution was performed to give 7.1 g of 5-methoxy-2-((trimethylsilyl)ethynyl)aniline, with a yield of 65%.

[0071] (2) 4.39 g (20 mmol) of 5-methoxy-2-((trimethylsilyl)ethynyl)aniline, 5.53 g (40 mmol) of anhydrous potassium carbonate, and 80 mL of anhydrous methanol were added sequentially to a round-bottom flask. After the addition was complete, the system underwent a detrimethylsilylation reaction at room temperature with stirring for 2 hours. After the reaction was completed, the system was filtered through a Buchner funnel, and the filter cake was washed with ethyl acetate. The filtrate and the eluent were combined and the solvent was removed by vacuum distillation (45℃, -0.09 ~ -0.1 MPa). The crude product was purified by silica gel column chromatography with a 200~300 mesh. The eluent was petroleum ether:ethyl acetate = 50:1~30:1. Gradient elution was performed to obtain 2.92 g of 2-ethynyl-5-methoxyaniline, with a yield of 99%.

[0072] (3) Under nitrogen protection, 1.47 g (10 mmol) of 2-ethynyl-5-methoxyaniline, 76 mg (0.4 mmol) of cuprous iodide, and 140 mg (0.2 mmol) of bis(triphenylphosphine)palladium dichloride were added sequentially to a round-bottom flask. The flask was then purged with nitrogen three times. Under nitrogen protection, 80 mL of triethylamine was added to the flask. Finally, 3.76 g (12 mmol) of 1-bromo-2-iodo-4-methoxybenzene was slowly added dropwise. After the addition was complete, the system continued to undergo the Sonogashira coupling reaction under nitrogen protection (the reaction is better at room temperature). The reaction lasted for 5 hours. After the reaction was complete, the system was filtered through a Buchner funnel. The filter cake was washed with ethyl acetate. The filtrate and the eluent were combined and distilled under reduced pressure (40 °C, -0.09 ~ The solvent was removed at -0.1 MPa, and the crude product was purified by silica gel column chromatography with a 200-300 mesh. The eluent was petroleum ether:ethyl acetate = 40:1-25:1. Gradient elution was performed to give 2.16 g of 2-((2-bromo-5-methoxy)ethylynyl)-5-methoxyaniline, with a yield of 65%.

[0073] (4) 1.66 g (5 mmol) of 2-((2-bromo-5-methoxy)ethylynyl)-5-methoxyaniline, 91 mg (0.5 mmol) of copper acetate, 1.38 g (5 mmol) of silver carbonate, and 6.52 g (20 mmol) of cesium carbonate were added sequentially to a round-bottom flask, followed by 75 mL of dimethyl sulfoxide. The system was then heated to 130 °C in an oil bath, where an oxidative ring-closure reaction occurred. The reaction lasted for 48 hours. After the reaction was completed, most of the dimethyl sulfoxide solvent was removed by vacuum distillation (60~80 °C, -0.09 MPa). 200 mL of water was added to the remaining system, and the mixture was extracted with ethyl acetate (200 mL × 3 times) to remove the remaining small amount of dimethyl sulfoxide. The ethyl acetate phases were combined, washed with 100 mL of saturated brine, and the ethyl acetate layer was separated and dried over anhydrous sodium sulfate. The mixture was then vacuum distilled (40 °C, -0.09 ~ The solvent was removed at -0.1 MPa, and the crude product was purified by silica gel column chromatography with a mesh size of 200-300. The eluent was dichloromethane:methanol = 80:1-50:1. Gradient elution was performed to obtain 0.59 g of the target compound (i.e., compound P80), with a yield of 40%.

[0074] The identification results of the target compound obtained are as follows: 1 H NMR (400 MHz, DMSO) δ 11.87 (s, 1H),7.96 (d, J = 8.8 Hz, 1H), 7.72 (d, J = 9.0 Hz, 1H), 7.51 (d, J = 2.6 Hz, 1H), 7.04 (dd, J = 9.0, 2.6 Hz, 1H), 7.02 (d, J = 2.4 Hz, 1H), 6.98 (dd, J = 8.8, 2.4 Hz, 1H), 3.86 (s, 6H). The data are consistent with the structure of the target compound (see [link to relevant documentation]). Figure 4-2 ).

[0075] (II) Preparation of benzofurano[3,2-c]quinolinone compound P81

[0076] The compound P81 has the following structural formula and is named 3-hydroxy-8-methoxybenzofurano[3,2-c]quinoline-6(5H)-one:

[0077]

[0078] Compound P81 was obtained by a one-step demethylation reaction based on compound P80 synthesized in (I) above. The synthetic route of compound P81 is as follows: Figure 5-1 As shown, the specific preparation steps are as follows:

[0079] Under nitrogen protection, 590 mg (2 mmol) of compound P80 was placed in a round-bottom flask. The flask was evacuated three times with nitrogen. Then, under nitrogen protection, 25 mL of dry dichloromethane was added to the flask. The flask was then moved from room temperature to an ice-water bath and cooled to 0°C. Under nitrogen protection and an ice-water bath, 5 mL of a 2M dichloromethane solution of boron tribromide (containing 10 mmol of boron tribromide) was slowly added dropwise. After the addition was complete, the system was brought to room temperature, and a demethylation reaction occurred under nitrogen protection. After 24 hours, the system was placed in an ice-water bath at 0°C. 20 mL of water was slowly added to the system to quench the reaction and cause the solid to precipitate rapidly. After the system was allowed to stand at room temperature overnight, it was filtered through a Buchner funnel. The crude solid obtained by filtration was loaded onto a dry sample and purified by silica gel column chromatography with a 200-300 mesh screen. The eluent was dichloromethane:methanol = 50:1-30:1, and gradient elution was performed to obtain 478 mg of the target compound (i.e., compound P81), with a yield of 85%.

[0080] The identification results of the target compound obtained are as follows: 1 H NMR (400 MHz, DMSO) δ 11.77 (s, 1H),9.54 (s, 1H), 7.94 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.40 (s,1H), 7.01 (s, 1H), 6.96 (d, J = 8.8 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 3.85(s, 3H). 13 C NMR (101 MHz, DMSO) δ 161.33 (s), 159.46 (s), 158.78 (s), 154.61 (s), 148.50 (s), 140.25 (s), 124.85 (s), 122.59 (s), 113.85 (s), 111.92 (s), 111.34 (s), 107.80 (s), 105.80 (s), 104.85 (s), 99.15 (s), 55.49 (s). The data are consistent with the structure of the target compound (see [link to relevant data]). Figure 5-2 ).

[0081] (III) Preparation of benzofurano[3,2-c]quinolinone compound P82

[0082] The compound P82 has the following structural formula and is named 3,8-dihydroxybenzofurano[3,2-c]quinoline-6(5H)-one:

[0083]

[0084] Compound P82 was obtained by a one-step demethylation reaction based on compound P81 synthesized in (II) above. The synthetic route of compound P82 is as follows: Figure 6-1 As shown, the specific preparation steps are as follows:

[0085] Under nitrogen protection, 281 mg (1 mmol) of compound P81 was placed in a round-bottom flask. The flask was purged with nitrogen three times. Then, under nitrogen protection, 13 mL of dry dichloromethane was added to the flask. The flask was then moved from room temperature to an ice-water bath and cooled to 0°C. Under nitrogen protection and an ice-water bath, 2.5 mL of a 2M dichloromethane solution of boron tribromide (containing 5 mmol of boron tribromide) was slowly added dropwise. After the addition was complete, the system was brought to room temperature and a demethylation reaction occurred under nitrogen protection. After 24 hours, the system was placed in an ice-water bath at 0°C. 10 mL of water was slowly added to the system to quench the reaction and cause the solid to precipitate rapidly. After the system was allowed to stand at room temperature overnight, it was filtered through a Buchner funnel. The crude solid obtained by filtration was loaded onto a dry sample and purified by silica gel column chromatography with a 200-300 mesh screen. The eluent was dichloromethane:methanol = 35:1-25:1, and gradient elution was performed to obtain 232 mg of the target compound (i.e., compound P82), with a yield of 87%.

[0086] The identification results of the target compound obtained are as follows: 1 H NMR (400 MHz, DMSO) δ 11.66 (s, 1H), 10.33 (s, 1H), 9.48 (s, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.38 (s, 1H), 6.89 (s, 1H), 6.82 (dd, J = 18.7, 8.9 Hz, 2H), the data are consistent with the structure of the target compound (see [reference]). Figure 6-2 ).

[0087] (iv) Effects of benzofurano[3,2-c]quinolinone compounds P80, P81 and P82 on oleic acid (OA) and palmitic acid (PA)-induced lipid accumulation in hepatocytes

[0088] Human normal hepatocytes L02 and human hepatocellular carcinoma cells HepG2 were cultured in DMEM medium (i.e., normal medium) containing 10% fetal bovine serum (BSA) and 1% penicillin-streptomycin mixture at 37°C and 5% CO2. After the cells reached approximately 95% confluence, they were seeded at 2 × 10⁶ cells per well. 5 Cells / mL were seeded in 6-well plates and randomly divided into 6 groups: control group, model group, compound P80 group, compound P81 group, compound P82 group, and positive control drug Resmetirom group. Resmetirom was from TargetMol Chemicals Inc., catalog number: 158082, abbreviated as Resm. After the cells adhered, the blank control group was given only normal culture medium containing DMSO, the model group was given normal culture medium containing 1 mM OA and 0.5 mM PA (i.e., OA+PA, represented by PO) and DMSO (solvent), and the drug groups were given normal culture medium containing 40 μM benzofurano[3,2-c]quinolone compound P80 and the above concentration of PO (i.e., PO+P80 40 μM), 40 μM benzofurano[3,2-c]quinolone compound P81 and the above concentration of PO (i.e., PO+P81 40 μM), 40 μM benzofurano[3,2-c]quinolone compound P82 and the above concentration of PO (i.e., PO+P82 40 μM), and 10 μM positive control drug Resmetirom and the above concentration of PO (i.e., PO+Resm 10 μM). After changing the culture medium according to the above groups, continue culturing the cells for 24 h, and then perform Oil Red O staining and Bodipy fluorescence staining on the cultured cells.

[0089] The Oil Red O staining procedure is as follows: Discard the cell culture medium, wash twice with PBS, add Oil Red O fixative, let stand for 20 min, remove the fixative, wash twice with distilled water, rinse with 60% isopropanol for 20 s, and air dry. Add freshly prepared Oil Red O staining solution (the staining solution must be filtered through a 0.45 μm filter before use; add 500 μL of Oil Red O staining solution to each well), immerse for 10 min, remove the staining solution, and wash 3-5 times with PBS until no excess staining solution remains. Immerse the stained cells in PBS and observe and photograph them under a microscope.

[0090] The Bodipy fluorescence staining procedure is as follows: Discard the cell culture medium, wash twice with PBS, add 4% paraformaldehyde, fix at room temperature for 15 min, wash twice with distilled water, add freshly prepared Bodipy 493 / 503 staining solution, immerse for 10 min, remove the staining solution, wash 3-5 times with PBS, add an appropriate volume of stabilizer, incubate at room temperature in the dark for 10 min, wash twice with PBS. Immerse the stained cells in PBS and observe and photograph them under a fluorescence microscope.

[0091] The results are as follows Figure 1 As shown: Compared with the model group, benzofurano[3,2-c]quinolone compounds P81 and P82 significantly reduced lipid accumulation in human normal hepatocytes L02 and human hepatocellular carcinoma cells HepG2 at a dose of 40 μM. Furthermore, benzofurano[3,2-c]quinolone compound P82 was significantly better than benzofurano[3,2-c]quinolone compound P81 in improving lipid accumulation. Benzofurano[3,2-c]quinolone compound P80 was almost ineffective (although P80 reduced lipid accumulation in the corresponding hepatocytes compared with the model group, the difference was not significant).

[0092] (v) Verify the pharmacological effects and toxicity of benzofurano[3,2-c]quinolinone compound P82.

[0093] Male C57BL / 6 mice were randomly divided into a normal group, a model group, a low-dose administration (gavage, 25 mg / kg) group of benzofurano[3,2-c]quinolinone compound P82, a high-dose administration (gavage, 50 mg / kg) group of benzofurano[3,2-c]quinolinone compound P82, and a positive control group of Resmetirom administration (gavage, 15 mg / kg; Resmetirom is from TargetMolChemicals Inc., catalog number: 158082, abbreviated as Resm), with 6 animals in each group. The experimental procedure began with the control group receiving a normal diet (NC), while the model group and all drug-treated groups received a high-fat, high-cholesterol diet (HFHC). After 8 weeks of feeding, the three drug-treated groups were administered 25 mg / kg of benzofurano[3,2-c]quinolone compound P82 (i.e., HFHC+P82 25 mg / kg), 50 mg / kg of benzofurano[3,2-c]quinolone compound P82 (i.e., HFHC+P82 50 mg / kg), and 15 mg / kg of Resmetirom (i.e., HFHC+Resm 15 mg / kg) daily by gavage, respectively, while continuing to receive the high-fat, high-cholesterol diet. This administration continued for 8 weeks (i.e., the experimental procedure lasted a total of 16 weeks, as follows). Figure 2 (As shown in A); During the drug administration period, the model group was given an equal volume of physiological saline (and continuously given a high-fat, high-cholesterol diet), while the normal group was given an equal volume of physiological saline (and continuously given a normal diet). After the experimental procedure, the mice were fasted but allowed to drink water for 1 day. Blood was collected from the orbital venous plexus, and the serum was separated (3500 rpm, 20 min, 4℃) and stored at -80℃. The mice were euthanized after anesthesia, and the liver was quickly removed. The residual blood was washed away with physiological saline. The right lobe of the liver of all mice was fixed in place and embedded in 4% paraformaldehyde solution and OTC for liver tissue pathological examination. The remaining liver tissue was flash-frozen in liquid nitrogen and stored at -80℃ for later use.

[0094] Hearts, livers, spleens, lungs, and kidneys of animals that were administered the drug alone for 8 weeks were used for toxicity evaluation.

[0095] The pharmacological indicators were tested as follows:

[0096] (1) Animal liver weight and liver-to-body weight ratio; (2) Biochemical indicators: ALT and AST activity and TG and TC content; (3) Liver histopathology: HE staining, Oil Red O staining, MASH score (hepatic steatosis, intralobular inflammation, hepatocyte ballooning degeneration, comprehensive score, i.e. NAS score); (4) Fibrosis detection: Masson staining.

[0097] The above pharmacological experimental results using the HFHC-induced MASH model show that, compared with the model group, administration of the benzofurano[3,2-c]quinolone compound P82 significantly reduced the liver weight of mice after HFHC induction. Figure 2 B) Liver-to-body weight ratio ( Figure 2 C); Compared with the model group, both low- and high-dose administration groups of benzofurano[3,2-c]quinolinone compound P82 significantly reduced hepatocyte ballooning degeneration and inflammatory infiltration in the liver of mice after HFHC induction. Figure 2 D) improves hepatic steatosis and lipid deposition in mice induced by HFHC and reduces lipid accumulation in hepatocytes. Figure 2 E), and significantly improved liver lobule structure recovery, reduced collagen deposition, and improved liver fibrosis in mice after HFHC induction, with dose-dependent reduction (E). Figure 2 F); Compared with the model group, both low- and high-dose administration of the benzofurano[3,2-c]quinolinone compound P82 significantly reduced the activity levels of ALT and AST in the serum of mice after HFHC induction. Figure 2 J and Figure 2 I), and reduced the levels of TC and TG in the liver and serum of mice after HFHC induction ( Figure 2 H and Figure 2 G).

[0098] In addition, HE staining results in the toxicity evaluation showed that, compared with the normal group (specifically, untreated healthy mice), the histopathological staining of heart, liver, spleen, lungs, and kidney tissues in healthy mice showed no significant changes after 8 weeks of administration (taking benzofurano[3,2-c]quinolinone compound P82 as an example). Figure 3 ).

[0099] In summary, this invention demonstrates through in vitro and in vivo experiments that benzofurano[3,2-c]quinolinone compounds, represented by compound P82, have a significant effect on alleviating MASH-related symptoms in model mice (including significantly reducing hepatic steatosis, hepatocyte ballooning degeneration, and inflammatory infiltration, while also improving liver fibrosis), and are expected to be developed into new drugs for the prevention and treatment of MASH, showing promising application prospects.

Claims

1. The use of benzofurano[3,2-c]quinolinone compounds in the preparation of medicaments for the prevention and / or treatment of metabolic dysfunction-related steatohepatitis, characterized in that: The benzofurano[3,2-c]quinolinone compound is any one of the compounds with the structure shown below or a pharmaceutically acceptable salt of such compound: Among them, R 1 For H, R 2 It can be H or -CH3.

2. The application according to claim 1, characterized in that: The benzofurano[3,2-c]quinolinone compound improves hepatic steatosis.

3. The application according to claim 1, characterized in that: The benzofurano[3,2-c]quinolinone compound improves liver fibrosis.

4. The application according to claim 1, characterized in that: The benzofurano[3,2-c]quinolinone compound reduces inflammatory infiltration in the liver and ballooning degeneration of hepatocytes in the liver.

5. The application according to claim 1, characterized in that: The metabolic dysfunction-related steatohepatitis mentioned above is induced by a high-fat, high-cholesterol diet.

6. The use of benzofurano[3,2-c]quinolinone compounds in the preparation of drugs for delaying the progression of non-alcoholic fatty liver disease, characterized in that: The benzofurano[3,2-c]quinolinone compound is any one of the compounds with the structure shown below or a pharmaceutically acceptable salt of such compound: Among them, R 1 For H, R 2 It can be H or -CH3.

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