Application of benzofuro [3, 2-c] quinolinone compound in prevention and treatment of metabolic dysfunction related steatohepatitis

Abstract

The invention discloses an application of a benzofuro [3, 2-c] quinolinone compound in prevention and treatment of metabolic dysfunction related steatohepatitis. For example, a compound P82, namely 3, 8-dihydroxybenzofuro [3, 2-c] quinoline-6 (5H)-ketone, which is prepared through synthesis and purification of an intermediate, can obviously improve liver fatty degeneration, inflammatory infiltration and fibrosis in an HFHC-induced metabolic dysfunction-related steatohepatitis model, so that the progress of metabolic dysfunction-related steatohepatitis is effectively delayed; meanwhile, liver cell lipid accumulation induced by oleic acid and palmitic acid can be improved, so that the compound is expected to be developed into a new medicine for preventing and treating metabolic dysfunction related steatohepatitis.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to the synthesis and application of benzofurano[3,2-c]quinolinone compounds, and in particular to the application of such compounds in the preparation of drugs for preventing and / or treating metabolic dysfunction-related fatty liver inflammation. Background Art

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

[0003] MASH is a complex liver disease characterized by abnormal accumulation of fat in the liver, accompanied by inflammation and liver cell damage, which may lead to liver fibrosis, liver failure and even liver cancer in severe cases. However, the choice of therapeutic drugs for this disease is still limited. So far, only one thyroxine receptor β (THR-β) agonist drug, Resmetirom, has been approved by the US FDA for the treatment of MASH, but the patient response rate of this drug is only 25% to 30%. At the same time, the relatively expensive price (annual treatment cost of 40,000 to 50,000 US dollars) also limits its clinical application. In some technologies related to this drug, methods for preparing intermediates or specific crystal forms of Resmetirom have been proposed (for example, CN114907327A). Therefore, there is an urgent need to find more candidate compounds with novel structures and definite efficacy, so as to provide new approaches for the drug treatment of MASH.

[0004] Benzofuran structure is a common structural unit of natural products. Many reports show that its derivatives have significant anti-inflammatory and antioxidant activities. For example, 2,3-dihydrobenzofuran derivatives can inhibit the inflammatory response stimulated by lipopolysaccharide by inhibiting the expression of cyclooxygenase-2 and nitric oxide synthase 2, and further inhibit 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-amidebenzofuran derivatives, as NLRP3 inflammasome inhibitors, 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 and can scavenge free radicals, thereby reducing oxidative stress (Molecules, 2018, 23(4): 710.). However, these benzofuran compounds did not show hepatocellular protective activity, and there are no reports on their use in the treatment of MASH accompanied by inflammation and hepatocellular damage in clinical practice.

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

[0006] In view of the shortage of drugs for treating metabolic dysfunction-associated steatohepatitis (MASH), the present invention provides the use of benzofurano[3,2-c]quinolinone compounds in preventing and treating metabolic dysfunction-associated steatohepatitis.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, a benzofurano[3,2-c]quinolinone compound is provided for use in the preparation of a medicament for preventing and / or treating metabolic dysfunction-related fatty hepatitis, wherein the benzofurano[3,2-c]quinolinone compound is a compound having a structure shown in the following formula or any one of the pharmaceutically acceptable salts of the compound:

[0008] Among them, R 1 For H, R 2 It is any one of H or C1~C3 alkyl (e.g. -CH3).

[0009] Preferably, the benzofurano[3,2-c]quinolinone compound improves hepatic steatosis.

[0010] Preferably, the benzofurano[3,2-c]quinolinone compound improves liver fibrosis.

[0011] Preferably, the benzofurano[3,2-c]quinolinone compound reduces liver inflammatory infiltration and hepatocellular ballooning in the liver.

[0012] Preferably, the metabolic dysfunction-associated steatohepatitis is induced by a high-fat, high-cholesterol diet (HFHC).

[0013] Preferably, the drug further comprises any one or more combinations of pharmaceutically acceptable excipients.

[0014] In a second aspect, there is provided a use of the above-mentioned benzofurano[3,2-c]quinolinone compound in the preparation of a drug for delaying the progression of non-alcoholic fatty liver disease (especially metabolic dysfunction-related steatohepatitis).

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

[0016] In a third aspect, a drug for treating metabolic dysfunction-related fatty liver disease is provided, the drug comprising (for example, containing a therapeutically effective amount of) a compound having a structure shown in the following formula or any one of the pharmaceutically acceptable salts of the compound:

[0017] Among them, R f is any one of the C1~C3 alkyl groups (e.g. -CH3; that is, the drug can use the newly synthesized benzofurano[3,2-c]quinolinone compound or a pharmaceutically acceptable salt thereof as an active ingredient and is used to prevent and / or treat metabolic dysfunction-related fatty liver disease).

[0018] Preferably, the drug further comprises any one or more combinations of pharmaceutically acceptable excipients.

[0019] Preferably, the drug can be prepared into dosage forms such as tablets, granules, capsules, pills, oral liquids, or sustained-release preparations.

[0020] Preferably, the pharmaceutically acceptable salt is selected from pharmaceutically acceptable ammonium salts, alkali metal salts (such as sodium salts, potassium salts) and alkaline earth metal salts (such as magnesium salts, calcium salts) and other base addition salts, and any one of tromethamine, diethanolamine, ethylenediamine salts and the like.

[0021] In a fourth aspect, a method for preparing a benzofurano[3,2-c]quinolinone compound is provided, comprising the following steps: A compound having the following structure is prepared using 2-bromo-5-alkoxyaniline (e.g. 2-bromo-5-methoxyaniline) as a starting material and as an intermediate:

[0022] Among them, R k is any one of the C1~C3 alkyl groups, R f Any one of C1~C3 alkyl groups; The obtained intermediate is subjected to a dealkylation reaction (eg, a demethylation reaction) to prepare a benzofurano[3,2-c]quinolinone compound having the following structure:

[0023] Among them, R 1 For H, R 2 It is any one of H or C1~C3 alkyl (e.g. -CH3).

[0024] Preferably, the method for preparing the benzofurano[3,2-c]quinoline compound specifically comprises the following steps: In the first step, under the protection of nitrogen (N2), 2-bromo-5-methoxyaniline, cuprous iodide (CuI), bis(triphenylphosphine palladium dichloride) (Pd(PPh3)2Cl2) and triphenylphosphine (PPh3) are first added, followed by the addition of triethylamine (Et3N) and toluene as a mixed reaction solvent, and then trimethylsilyl acetylene is added. The molar ratio of 2-bromo-5-methoxyaniline: trimethylsilyl acetylene: cuprous iodide: bis(triphenylphosphine palladium dichloride): triphenylphosphine in the obtained system is 100:150:2:2:5; then, under the protection of nitrogen, the reaction mixture is stirred for 1 h. The system is heated to 80-120° C. under protection and a Sonogashira coupling reaction is carried out. After the reaction for 16-30 hours, the system is filtered, and the filter cake is rinsed with ethyl acetate (or dichloromethane). The filtrate and the rinsing liquid are combined, and all solvents (such as ethyl acetate, triethylamine and toluene) are removed by vacuum distillation to obtain a crude product; the crude product is purified by 200-300 mesh silica gel column chromatography, wherein the eluent is petroleum ether:ethyl acetate (volume ratio) = 60:1-40:1, and 5-methoxy-2-((trimethylsilyl)ethynyl)aniline is obtained by gradient elution.

[0025] In the second step, 5-methoxy-2-((trimethylsilyl)ethynyl)aniline is used as the first intermediate compound, anhydrous potassium carbonate (K2CO3), potassium tert-butoxide (t-BuOK) or sodium tert-butoxide (t-BuONa) provides an alkaline environment, and anhydrous methanol (MeOH) is used as a reaction solvent. The molar ratio of 5-methoxy-2-((trimethylsilyl)ethynyl)aniline: anhydrous potassium carbonate (or potassium tert-butoxide or sodium tert-butoxide) in the obtained system is 1: (1.2~2); then the system is heated at 25 The detrimethylsilyl reaction is carried out at 400 °C to 28 °C. After 2 to 4 hours of reaction, the system is filtered, the filter cake is rinsed with ethyl acetate (or dichloromethane), the filtrate and the rinsing liquid are combined, and all solvents (such as ethyl acetate and methanol) are removed by vacuum distillation to obtain a crude product; the crude product is purified by 200-300 mesh silica gel column chromatography, wherein the eluent is petroleum ether:ethyl acetate (volume ratio) = 50:1-30:1, and the second intermediate compound (specifically 2-ethynyl-5-methoxyaniline) is obtained by gradient elution.

[0026] In the third step, under nitrogen protection, 2-ethynyl-5-methoxyaniline, cuprous iodide and bistriphenylphosphine palladium dichloride are first added, followed by the addition of triethylamine as a reaction solvent, and then 1-bromo-2-iodo-4-methoxybenzene is added. The molar ratio of 2-ethynyl-5-methoxyaniline: 1-bromo-2-iodo-4-methoxybenzene: cuprous iodide: bistriphenylphosphine palladium dichloride in the obtained system is 50:60:2:1; then the system is subjected to Sonogas reaction at 25-28°C under nitrogen protection. Hira coupling reaction, after reacting for 5 to 10 hours, the system is filtered, the filter cake is rinsed with ethyl acetate (or dichloromethane), the filtrate and the rinsing liquid are combined, and all solvents (such as ethyl acetate and triethylamine) are removed by reduced pressure distillation to obtain a crude product; the crude product is purified by 200-300 mesh silica gel column chromatography, wherein the eluent is petroleum ether:ethyl acetate (volume ratio) = 40:1-25:1, and 2-((2-bromo-5-methoxy)ethylalkynyl)-5-methoxyaniline is obtained by gradient elution.

[0027] In the fourth step, 2-((2-bromo-5-methoxy)ethylalkynyl)-5-methoxyaniline is used as the third intermediate compound, copper acetate (Cu(OAc)2) is used as a catalyst, silver carbonate (Ag2CO3) is used as an additive (oxidation aid), cesium carbonate (Cs2CO3) is used as an oxidant and provides an alkaline environment, and dimethyl sulfoxide (DMSO) is used as a reaction solvent. The molar ratio of 2-((2-bromo-5-methoxy)ethylalkynyl)-5-methoxyaniline: copper acetate: silver carbonate: cesium carbonate in the obtained system is 10:1:10:40; then the system is heated to 120~180°C The oxidative ring-closing reaction is carried out. After reacting for 24 to 48 hours, the system is successively subjected to reduced pressure distillation and ethyl acetate / water extraction to remove the solvent (specifically DMSO). The obtained ethyl acetate phase is successively washed with saturated brine, dried over anhydrous sodium sulfate, and distilled under reduced pressure to remove the solvent (specifically ethyl acetate) to obtain a crude product. The crude product is purified by 200 to 300 mesh silica gel column chromatography, wherein the eluent is dichloromethane:methanol (volume ratio) = 80:1 to 50:1, and 3,8-dimethoxybenzofurano[3,2-c]quinoline-6(5H)-one is obtained by gradient elution, i.e., compound P80 (the structure is shown in the following formula):

[0028] Step 5: Under nitrogen protection, compound P80 is first added, followed by dichloromethane (CH2Cl2) as a reaction solvent, and then a dichloromethane solution of boron tribromide (BBr3) is added at 0-5°C, wherein the molar ratio of compound P80 to boron tribromide in the obtained system is 1:(3-6); then, the system is heated to 25-28°C under nitrogen protection and a demethylation reaction is performed. After reacting for 12-24 hours, water is added to the system at 0-5°C to precipitate a solid, and the solid is allowed to stand, and the precipitated solid in the system is separated by filtration to obtain a crude product; the crude product is purified by dry-method loading on a 200-300 mesh silica gel column, wherein the eluent is dichloromethane:methanol (volume ratio) = 50:1-30:1, and 3-hydroxy-8-methoxybenzofurano[3,2-c]quinoline-6(5H)-one is obtained by gradient elution, i.e., compound P81 (the structure is shown in the following formula):

[0029] Step 6: Under nitrogen protection, compound P81 is first added, followed by dichloromethane as a reaction solvent, and then a dichloromethane solution of boron tribromide is added at 0-5°C, wherein the molar ratio of compound P81 to boron tribromide in the obtained system is 1:(3-6); then, the system is heated to 25-28°C under nitrogen protection and a demethylation reaction is performed. After reacting for 12-24 hours, water is added to the system at 0-5°C to precipitate a solid, and the solid is allowed to stand, and the precipitated solid in the system is separated by filtration to obtain a crude product; the crude product is purified by dry-method loading on a 200-300 mesh silica gel column, wherein the eluent is dichloromethane:methanol (volume ratio) = 35:1-25:1, and 3,8-dihydroxybenzofurano[3,2-c]quinoline-6(5H)-one is obtained by gradient elution, i.e., compound P82 (the structure is shown in the following formula):

[0030] A fifth aspect provides a method for preparing a pharmaceutical intermediate, comprising the following steps: 2-bromo-5-alkoxyaniline (e.g., 2-bromo-5-methoxyaniline) is subjected to a Sonogashira coupling reaction with trimethylsilyl acetylene to obtain a first intermediate compound (i.e., 5-alkoxy-2-((trimethylsilyl)ethynyl)aniline), wherein the alkyl group contained in the alkoxy group is any one of the C1-C3 alkyl groups (e.g., -CH3); the first intermediate compound is subjected to a detrimethylsilylation reaction under alkaline conditions to obtain a second intermediate compound (i.e., 2-ethynyl-5-alkoxyaniline); the second intermediate compound is reacted with 1 -bromo-2-iodo-4-alkoxybenzene (e.g., 1-bromo-2-iodo-4-methoxybenzene) is subjected to a Sonogashira coupling reaction to obtain a third intermediate compound (i.e., 2-((2-bromo-5-alkoxy)ethylalkynyl)-5-alkoxyaniline), wherein the alkyl group contained in the alkoxy group is any one of the C1 to C3 alkyl groups (e.g., -CH3); the third intermediate compound is subjected to an oxidative ring-closing reaction under alkaline conditions with the addition of a silver salt (e.g., silver carbonate) or silver oxide to obtain a compound with the following structure, i.e., the intermediate:

[0031] Among them, R k is any one of the C1~C3 alkyl groups, R f is any one of the C1~C3 alkyl groups; for example, the structure of the obtained intermediate is shown in the following formula: .

[0032] The beneficial effects of the present invention are embodied in: The present invention aims at benzofurano[3,2-c]quinolinone compounds (such as the above-mentioned compounds P81 and P82), and for the first time confirms their role in improving lipid accumulation in liver cells through cell-level experiments, and screens candidate drug molecules (such as the above-mentioned compound P81) in combination with in vivo experiments. The experimental results show that this type of benzofurano[3,2-c]quinolinone compound has the potential to be developed as a new anti-MASH drug, and through its significant role in reducing liver fatty degeneration, inflammation and fibrosis levels, it is proved that it can alleviate MASH-related symptoms and thus delay the progression of non-alcoholic fatty liver disease (especially MASH).

[0033] Furthermore, the present invention conducted intervention experiments on hepatocyte fatty degeneration caused by chemical induction (specifically palmitic acid and oleic acid induction) on multiple benzofurano[3,2-c]quinolinone compounds, and discovered the structure-activity relationship of this class of compounds related to improving lipid accumulation in liver cells (specifically involving normal human liver cells and human liver cancer cells) (specifically, see the differences and similarities or changes in the substituents of compounds P80, P81, and P82 based on the "benzofurano[3,2-c]quinolinone" parent nucleus), which provides a basis for screening drug molecules for the prevention and / or treatment of MASH.

[0034] Furthermore, the present invention experimentally determined that compound P82 can significantly improve liver fatty degeneration in HFHC-induced MASH animal models, reduce hepatocyte ballooning and inflammatory infiltration in the liver, and improve liver fibrosis, while reducing liver total cholesterol (TC), triglycerides (TG) and blood lipid levels, and reducing liver damage indicators aspartate aminotransferase (ALT) and alanine aminotransferase (AST) levels. These results suggest that compound P82 can play a better application effect in the prevention and / or treatment of MASH.

[0035] The benzofurano[3,2-c]quinolinone compound of the present invention can be used as a drug molecule and can be prepared using similar compounds (such as compound P80) as intermediates. The synthesis and purification process is simple and can better meet the needs of large-scale and intensive production in the pharmaceutical industry.

[0036] Furthermore, the present invention solves the problem that in the process of preparing the target compound from the intermediate (such as compound P80), the target compound (such as P81 or P82) is generated to varying degrees as the reaction proceeds and is difficult to separate and purify by controlling the reaction conditions (including appropriate reaction time, avoiding excessively high reaction temperature, and the ratio of the reaction reagent feed amounts), thereby ensuring a higher yield of the target compound.

[0037] Furthermore, the preparation method of the intermediate (such as compound P80) proposed in the present invention can use cheaper 2-bromo-5-methoxyaniline instead of 2-iodo-5-methoxyaniline as the starting material, thereby reducing the synthesis cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The results of compounds P80, P81, and P82 reducing OA+PA-induced lipid accumulation in hepatocytes; (A) Oil Red O staining and Bodipy fluorescence staining of human hepatoma cells HepG2; (B) Oil Red O staining and Bodipy fluorescence staining of human normal hepatocytes L02; ## p <0.01 Vs. blank control group, ** p<0.01 Vs. Model group.

[0039] Figure 2 The results of compound P82 alleviating the symptoms of HFHC-induced 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.

[0040] Figure 3 HE staining results of the effects of compound P82 on mouse organs in toxicity evaluation.

[0041] Figure 4-1 The synthetic route of benzofurano[3,2-c]quinolinone compound P80 (i.e., compound P80) is shown.

[0042] Figure 4-2 This is the hydrogen nuclear magnetic resonance spectrum of benzofurano[3,2-c]quinolinone compound P80.

[0043] Figure 5-1 The synthetic route of benzofurano[3,2-c]quinolinone compound P81 (i.e., compound P81) is shown.

[0044] Figure 5-2 This is the hydrogen nuclear magnetic resonance spectrum of the benzofurano[3,2-c]quinolinone compound P81.

[0045] Figure 6-1 The synthetic route of benzofurano[3,2-c]quinolinone compound P82 (i.e., compound P82) is shown.

[0046] Figure 6-2 This is the hydrogen nuclear magnetic resonance spectrum of the benzofurano[3,2-c]quinolinone compound P82. DETAILED DESCRIPTION

[0047] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. The embodiments are only used to explain the present invention but not to limit the protection scope of the present invention.

[0048] The benzofurano[3,2-c]quinolinone compound synthesized in the present invention has a general structural formula as shown in Formula 1:

[0049] The benzofurano[3,2-c]quinolinone compound (for example, 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 fatty liver disease (MASH) at the cellular and in vivo levels; the results showed that this type of compound has certain anti-MASH activity. The specific description is as follows.

[0050] (I) Preparation of benzofurano[3,2-c]quinolinone compound P80 The structural formula of the compound P80 is as follows, and it is named as 3,8-dimethoxybenzofurano[3,2-c]quinolin-6(5H)-one:

[0051] The synthetic route of compound P80 is as follows Figure 4-1 As shown, the specific preparation steps are as follows: (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 to a round-bottom flask in sequence. The flask was then evacuated and replaced with nitrogen three times. Under nitrogen protection, 50 mL of triethylamine and 200 mL of toluene were added to the round-bottom flask. Finally, 7.4 g (75 mmol) of trimethylsilyl acetylene was added dropwise to the round-bottom flask. After the addition was completed, the system was heated to 100°C under nitrogen protection. The Sonogashira coupling reaction was carried out at 100°C under nitrogen protection for 24 hours. After the reaction was completed, the system was filtered with a Buchner funnel, the filter cake was rinsed with ethyl acetate, the filtrate and the rinse were combined, and distilled under reduced pressure (40°C, -0.09 ~ -0.1MPa) to remove the solvent, and the obtained crude product was purified by 200-300 mesh silica gel column chromatography, with petroleum ether:ethyl acetate = 60:1-40:1 as the eluent, and gradient elution was performed to obtain 7.1 g of 5-methoxy-2-((trimethylsilyl)ethynyl)aniline, with a yield of 65%.

[0052] (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 to a round-bottom flask in sequence. After the addition was completed, the system was subjected to a detrimethylsilylation reaction at room temperature under stirring for 2 hours. After the reaction was completed, the system was filtered using a Buchner funnel, and the filter cake was rinsed with ethyl acetate. The filtrate and the eluent were combined and the solvent was removed by vacuum distillation (45°C, -0.09 to -0.1 MPa). The crude product was purified by 200-300 mesh silica gel column chromatography with an eluent of petroleum ether:ethyl acetate = 50:1 to 30:1, and gradient elution was performed to obtain 2.92 g of 2-ethynyl-5-methoxyaniline with a yield of 99%.

[0053] (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 to the round-bottom flask in sequence. The flask was then evacuated and replaced with nitrogen three times. Under nitrogen protection, 80 mL of triethylamine was added to the round-bottom flask. Finally, 3.76 g (12 mmol) of 1-bromo-2-iodo-4-methoxybenzene was slowly added dropwise to the round-bottom flask. After the addition was completed, the system continued to undergo Sonogashira coupling reaction under nitrogen protection (the reaction was better at room temperature). The reaction lasted for 5 hours. After the reaction was completed, the system was filtered with a Buchner funnel, the filter cake was rinsed with ethyl acetate, the filtrate and the rinse were combined, and distilled under reduced pressure (40°C, -0.09 ~ -0.1MPa) to remove the solvent, and the obtained crude product was purified by 200-300 mesh silica gel column chromatography with petroleum ether:ethyl acetate = 40:1-25:1 as the eluent, and gradient elution was performed to obtain 2.16 g of 2-((2-bromo-5-methoxy)ethylalkynyl)-5-methoxyaniline with a yield of 65%.

[0054] (4) 1.66 g (5 mmol) of 2-((2-bromo-5-methoxy)ethylalkynyl)-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 to a round-bottom flask in sequence, and 75 mL of dimethyl sulfoxide was added. The system was then heated to 130°C in an oil bath, and an oxidative ring-closure reaction occurred at 130°C for 48 hours. After the reaction, most of the dimethyl sulfoxide solvent was removed by vacuum distillation (60-80°C, -0.09 MPa), and 200 mL of water was added to the remaining system. The system was extracted with ethyl acetate (200 mL × 3 times) to remove the remaining small amount of dimethyl sulfoxide. The ethyl acetate phases were combined and washed with 100 mL of saturated brine. The ethyl acetate layer was separated and dried over anhydrous sodium sulfate, and vacuum distilled (40°C, -0.09 ~ -0.1MPa) to remove the solvent, and the obtained crude product was purified by 200-300 mesh silica gel column chromatography with dichloromethane:methanol=80:1-50:1 as the eluent, and gradient elution was performed to obtain 0.59 g of the target compound (i.e., the compound P80) with a yield of 40%.

[0055] The identification results of the obtained target compound are: 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 Figure 4-2 ).

[0056] (II) Preparation of benzofurano[3,2-c]quinolinone compound P81 The structural formula of the compound P81 is as follows, and it is named as 3-hydroxy-8-methoxybenzofurano[3,2-c]quinolin-6(5H)-one:

[0057] The compound P81 is prepared by a one-step demethylation reaction based on the compound P80 synthesized in (I) above. The synthetic route of the compound P81 is as follows: Figure 5-1As shown, the specific preparation steps are as follows: Under nitrogen protection, 590 mg (2 mmol) of the compound P80 was placed in a round-bottom flask, and the vacuum was replaced with nitrogen three times. 25 mL of dry dichloromethane was added to the round-bottom flask under nitrogen protection. Then, the round-bottom flask was moved from room temperature to an ice-water bath and cooled to 0°C. 5 mL of a 2M dichloromethane solution of boron tribromide (containing 10 mmol of boron tribromide) was slowly added dropwise to the round-bottom flask under nitrogen protection and ice-water bath. After the addition was completed, the system was warmed to room temperature and a demethylation reaction was carried out under nitrogen protection. The reaction After 24 hours, the system was placed in an ice-water bath at 0°C, 20 mL of water was slowly added dropwise to the system to quench the reaction, and solids were quickly precipitated from the system. After the system was allowed to stand at room temperature overnight, it was filtered using a Buchner funnel. The solid crude product obtained by filtration was dry-loaded and purified by 200-300 mesh silica gel column chromatography. The eluent was dichloromethane:methanol = 50:1-30:1, and gradient elution was performed to obtain 478 mg of the target compound (i.e., the compound P81) with a yield of 85%.

[0058] The identification results of the obtained target compound are: 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) δ161.33 (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 were consistent with the structure of the target compound (see Figure 5-2 ).

[0059] (III) Preparation of benzofurano[3,2-c]quinolinone compound P82 The structural formula of the compound P82 is as follows, and it is named as 3,8-dihydroxybenzofurano[3,2-c]quinolin-6(5H)-one:

[0060] Based on the compound P81 synthesized in (II) above, the compound P82 is prepared by a one-step demethylation reaction. The synthetic route of the compound P82 is as follows: Figure 6-1 As shown, the specific preparation steps are as follows: Under nitrogen protection, 281 mg (1 mmol) of the compound P81 was placed in a round-bottom flask, and the vacuum was replaced with nitrogen three times. 13 mL of dry dichloromethane was added to the round-bottom flask under nitrogen protection. Then, the round-bottom flask was moved from room temperature to an ice-water bath and cooled to 0°C. 2.5 mL of a 2M dichloromethane solution of boron tribromide (containing 5 mmol of boron tribromide) was slowly added dropwise to the round-bottom flask under nitrogen protection and ice-water bath. After the addition was completed, the system was warmed to room temperature and a demethylation reaction was carried out under nitrogen protection. The reaction After 24 hours, the system was placed in an ice-water bath at 0°C, 10 mL of water was slowly added dropwise to the system to quench the reaction, and solids were quickly precipitated from the system. After the system was allowed to stand at room temperature overnight, it was filtered using a Buchner funnel. The solid crude product obtained by filtration was dry-loaded and purified by 200-300 mesh silica gel column chromatography. The eluent was dichloromethane: methanol = 35:1-25:1, and gradient elution was performed to obtain 232 mg of the target compound (i.e., the compound P82) with a yield of 87%.

[0061] The identification results of the obtained target compound are: 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 Figure 6-2 ).

[0062] (IV) Effects of benzofurano[3,2-c]quinolinone compounds P80, P81 and P82 on lipid accumulation in hepatocytes induced by oleic acid (OA) and palmitic acid (PA) Normal human liver cells L02 and human liver cancer cells HepG2 were cultured in DMEM medium (i.e., normal culture medium) containing 10% fetal bovine serum (BSA) and 1% double antibody (penicillin-streptomycin mixture) at 37°C and 5% CO2. When the cell confluence reached about 95%, 2×10 cells were added to each well. 5cell / mL were plated in a 6-well plate and randomly divided into 6 groups, namely, blank control group, model group, compound P80 group, compound P81 group, compound P82 group, and positive drug Resmetirom group. Resmetirom was from TargetMol Chemicals Inc., with product number: 158082, referred to as Resm. After the cells adhered to the wall, 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]quinolinone compound P80 and the above concentration of PO (i.e. PO+ P80 40 μM), 40 μM benzofurano[3,2-c]quinolinone compound P81 and the above concentration of PO (i.e. PO+ P81 40 μM), 40 μM benzofurano[3,2-c]quinolinone compound P82 and the above concentration of PO (i.e. PO+ P82 40 μM), and 10 μM positive drug Resmetirom and the above concentration of PO (i.e. PO+ Resm 10 μM). After replacing the culture medium according to the above groups, the cells were cultured for 24 h, and the cultured cells were stained with Oil Red O and Bodipy fluorescence staining respectively.

[0063] The Oil Red O staining process is as follows: discard the cell culture medium, wash twice with PBS, add Oil Red O fixative, let stand for 20 minutes, remove the fixative and wash twice with distilled water, rinse with 60% isopropanol for 20 seconds and dry, add the newly prepared Oil Red O staining solution (the staining solution needs to be filtered with a 0.45μm filter membrane before use, and add 500μL Oil Red O staining solution to each well), remove the staining solution after immersion for 10 minutes, and wash with PBS 3~5 times until there is no excess staining solution left. Soak the stained cells in PBS and observe and take pictures under a microscope.

[0064] The process of Bodipy fluorescence staining is as follows: discard the cell culture medium, wash twice with PBS, add 4% paraformaldehyde, fix at room temperature for 15 minutes, wash twice with distilled water, add freshly prepared Bodipy493 / 503 staining solution, remove the staining solution after immersion for 10 minutes, wash 3-5 times with PBS, add an appropriate volume of stabilizer, incubate at room temperature in the dark for 10 minutes, and wash twice with PBS. Soak the stained cells in PBS and observe and photograph them under a fluorescence microscope.

[0065] The results are as follows Figure 1As shown: Compared with the model group, benzofurano[3,2-c]quinolinone compounds P81 and P82 at a dose of 40 μM can significantly reduce lipid accumulation in human normal liver cells L02 and human liver cancer cells HepG2 cells, and the effect of benzofurano[3,2-c]quinolinone compound P82 in improving lipid accumulation is significantly better than that of benzofurano[3,2-c]quinolinone compound P81; benzofurano[3,2-c]quinolinone compound P80 is almost ineffective (compared with the model group, although P80 reduced lipid accumulation in the corresponding liver cells, there was no significant difference).

[0066] (V) Verification of the pharmacological effects and toxicity of benzofurano[3,2-c]quinolinone compound P82 Male C57BL / 6 mice were randomly divided into a normal group, a model group, a low-dose administration (oral gavage, 25 mg / kg) of the benzofurano[3,2-c]quinolinone compound P82 group, a high-dose administration (oral gavage, 50 mg / kg) of the benzofurano[3,2-c]quinolinone compound P82 group, and a positive drug Resmetirom administration (oral gavage, 15 mg / kg; Resmetirom was from TargetMolChemicals Inc., catalog number: 158082, referred to as Resm) group, with 6 animals in each group. At the beginning of the experimental process, the normal group was given a normal diet (NC), and the model group and each treatment group were given a high-fat and high-cholesterol diet (HFHC). After feeding for 8 weeks (8W), the three treatment groups were gavaged with 25 mg / kg of benzofurano[3,2-c]quinolinone compound P82 (i.e. HFHC+P82 25 mg / kg), 50 mg / kg of benzofurano[3,2-c]quinolinone compound P82 (i.e. HFHC+P82 50 mg / kg) and 15 mg / kg of Resmetirom (i.e. HFHC+Resm 15 mg / kg) every day and continued to be given a high-fat and high-cholesterol diet for 8 consecutive weeks (i.e. the experimental process totaled 16 weeks, as shown in the figure). Figure 2 As shown in A); during the administration period, the model group was given an equal volume of saline (and continued to be given a high-fat, high-cholesterol diet), and the normal group was given an equal volume of saline (and continued to be given a normal diet). After the experimental process, the mice were fasted but not watered for 1 day, blood was collected from the orbital venous plexus, and serum was separated (3500rpm, 20min, 4℃) and stored in a -80℃ refrigerator; the mice were anesthetized and killed, the liver was quickly removed, and the residual blood was cleaned with saline. The right lobe of the liver of all mice was fixed in 4% paraformaldehyde solution and OTC embedded for liver tissue pathology. The remaining liver tissue was quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator for use.

[0067] The heart, liver, spleen, lung and kidney of the animals that received single drug administration for 8 weeks were also collected for toxicity evaluation.

[0068] The pharmacological indicators are tested as follows: (1) Animal liver weight and liver-to-body weight ratio; (2) Biochemical indicators: ALT, AST activity and TG, TC content; (3) Liver tissue pathology: HE staining, Oil Red O staining, MASH score (hepatic steatosis, intralobular inflammation, hepatocyte ballooning, comprehensive score, i.e. NAS score); (4) Fibrosis detection: Masson staining.

[0069] The pharmacological experiments using the HFHC-induced MASH model showed that compared with the model group, the administration of the benzofurano[3,2-c]quinolinone compound P82 could significantly reduce the liver weight of mice induced by HFHC ( Figure 2 B) liver-to-body weight ratio ( Figure 2 C); Compared with the model group, the low- and high-dose administration groups of the benzofurano[3,2-c]quinolinone compound P82 could significantly reduce the ballooning degeneration and inflammatory infiltration of hepatocytes in the liver of mice induced by HFHC ( Figure 2 D) Improves liver steatosis and lipid deposition in mice induced by HFHC and reduces lipid accumulation in liver cells ( Figure 2 E), and promoted the recovery of liver lobule structure in mice after HFHC induction, significantly reduced collagen deposition, and improved liver fibrosis in a dose-dependent manner ( Figure 2 F); Compared with the model group, the low- and high-dose administration groups of the benzofurano[3,2-c]quinolinone compound P82 could significantly reduce the activity values ​​of ALT and AST in the serum of mice induced by HFHC ( Figure 2 J and Figure 2 I, and reduced TC and TG levels in the liver and serum of mice after HFHC induction ( Figure 2 H and Figure 2 G).

[0070] In addition, the HE staining results in the toxicity evaluation showed that compared with the normal group (specifically, healthy mice that were not given the drug), there was no significant change in the pathological staining of the heart, liver, spleen, lung and kidney tissues of healthy mice after 8 weeks of drug administration (taking the benzofurano[3,2-c]quinolinone compound P82 as an example) ( Figure 3 ).

[0071] In summary, the present invention has confirmed through in vitro and in vivo experiments that benzofurano[3,2-c]quinolinone compounds represented by compound P82 have significant effects on alleviating MASH-related symptoms in model mice (including significantly reducing liver fatty degeneration, hepatocyte ballooning and inflammatory infiltration, and improving liver fibrosis), and are expected to be developed into new drugs for the prevention and treatment of MASH, with good application prospects.

Claims

1. Use of a benzofurano[3,2-c]quinolinone compound in the preparation of a medicament for preventing and / or treating metabolic dysfunction-related fatty hepatitis, characterized in that: The benzofurano[3,2-c]quinolinone compound is a compound having a structure shown in the following formula or any one of the pharmaceutically acceptable salts of the compound: Among them, R 1 For H, R 2 It is H or a C1~C3 alkyl group.

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

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

4. The use according to claim 1, characterized in that: The benzofuro[3,2-c]quinolinone compounds reduce liver inflammatory infiltration and hepatocellular ballooning in the liver.

5. The use according to claim 1, characterized in that: The metabolic dysfunction-associated steatohepatitis is diet-induced.

6. Use of a benzofurano[3,2-c]quinolinone compound in the preparation of a drug for delaying the progression of non-alcoholic fatty liver disease, characterized in that: The benzofurano[3,2-c]quinolinone compound is a compound having a structure shown in the following formula or any one of the pharmaceutically acceptable salts of the compound: Among them, R 1 For H, R 2 It is H or a C1~C3 alkyl group.

7. A drug for treating fatty liver disease associated with metabolic dysfunction, characterized in that: The drug includes any one of a compound having a structure as shown in the following formula or a pharmaceutically acceptable salt of the compound: Among them, R f It is a C1~C3 alkyl group.

8. The drug for treating metabolic dysfunction-related fatty liver disease according to claim 7, characterized in that: The medicine also includes excipients.

9. A method for preparing a benzofurano[3,2-c]quinolinone compound, characterized in that: The following steps are involved: 1) Prepare a compound having the following structure as an intermediate: Among them, R k is a C1~C3 alkyl group, R f is a C1~C3 alkyl group; 2) The intermediate prepared in step 1 is subjected to a dealkylation reaction to prepare a benzofurano[3,2-c]quinolinone compound having the following structure: Among them, R 1 For H, R 2 It is H or a C1~C3 alkyl group.

10. A method for preparing a pharmaceutical intermediate, characterized in that: The following steps are involved: 2-bromo-5-alkoxyaniline and trimethylsilyl acetylene are subjected to a Sonogashira coupling reaction to obtain a first intermediate compound, wherein the alkyl group contained in the alkoxy group of the 2-bromo-5-alkoxyaniline is a C1-C3 alkyl group; the first intermediate compound is subjected to a detrimethylsilylation reaction under alkaline conditions to obtain a second intermediate compound; the second intermediate compound is subjected to a Sonogashira coupling reaction with 1-bromo-2-iodo-4-alkoxybenzene to obtain a third intermediate compound, wherein the alkyl group contained in the alkoxy group of the 1-bromo-2-iodo-4-alkoxybenzene is a C1-C3 alkyl group; the third intermediate compound is subjected to an oxidative ring-closing reaction under alkaline conditions with the addition of silver salt or silver oxide to obtain a compound with the following structure, i.e., the drug intermediate: Among them, R k is a C1~C3 alkyl group, R f It is a C1~C3 alkyl group.

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