Indolinone compounds and their applications in the treatment of acute and chronic liver diseases and the promotion of tissue repair

Abstract

The present invention discloses an indolone compound and its application in the treatment of acute and chronic liver diseases and the promotion of tissue repair, belonging to the field of biomedicine. The structural formula of the indolone compound in the present invention is shown as follows. The indolone compound provided by the present invention is the first small molecule compound targeting the MFN2 protein for anti-liver injury and promoting liver repair designed based on the structure of the MFN2 protein. After the compound of the present invention specifically binds to the MFN2 protein, it can effectively target and activate the biological activity of the MFN2 protein, and can be used as an agonist of the MFN2 protein, thereby being used for the preparation of drugs for treating liver diseases, especially for the preparation of drugs for treating liver injury and metabolic-related liver diseases.

Description

Technical Field

[0001] The invention belongs to biomedicine, and particularly relates to an indolinone compound and its application in treating acute and chronic liver diseases and promoting tissue repair. Background Art

[0002] Common liver diseases include: metabolic fatty liver disease, hepatitis (including viral hepatitis, drug-induced hepatitis, autoimmune hepatitis, etc.), acute and chronic liver damage, liver fibrosis, liver cancer, etc. Acute liver damage is a serious liver damage disease caused by multiple factors. The clinical characteristics of acute liver damage are a sudden increase in transaminases (as a marker of liver damage) in patients without chronic liver disease, accompanied by impaired liver function, namely jaundice and coagulation dysfunction.

[0003] Mitochondrial dynamics is a discipline that studies the structure, function, and movement characteristics of mitochondria in cells. Mitochondria are the energy centers in cells, mainly responsible for energy production and regulation in cells. Mitochondrial dynamics studies have found that the morphology and function of mitochondria are closely related to the metabolic state of cells. Changes in mitochondrial morphology and increases or decreases in the number of mitochondria may affect metabolic activities in mitochondria, thereby affecting energy production and metabolic regulation of cells. For example, mitochondrial dysfunction can lead to insufficient energy supply to cells, which in turn affects the metabolic activities of cells and even causes cell apoptosis.

[0004] Mitofusin 2 (MFN2) protein is an important mitochondrial fusion protein that participates in regulating the morphology and function of mitochondria. MFN2 protein plays a key role in the process of mitochondrial fusion, helping mitochondria fuse with each other, maintaining the integrity of the mitochondrial network, and maintaining the healthy state of cells. The main functions of MFN2 protein include: Mitochondrial fusion: MFN2 protein promotes the fusion between mitochondria by interacting with other mitochondrial fusion proteins, maintaining the stability and integrity of the mitochondrial network; Regulating mitochondrial morphology: MFN2 protein can affect the morphology and structure of mitochondria, regulating the size and shape of mitochondria; Regulating mitochondrial function: MFN2 protein is also closely related to the regulation of mitochondrial function, including cellular energy metabolism, apoptosis, intracellular calcium ion balance, etc. Abnormal expression or mutation of MFN2 protein may be related to the occurrence and development of various diseases, such as neurodegenerative diseases, metabolic diseases, cardiovascular diseases, etc. Studies have shown that the deletion or dysfunction of MFN2 protein may lead to pathophysiological processes such as abnormal mitochondrial function, increased apoptosis, and disordered cellular energy metabolism. Therefore, MFN2 protein has important significance in the research of cell biology and disease mechanisms. The deletion or dysfunction of MFN2 protein may be associated with metabolic diseases such as obesity, diabetes, and non-alcoholic fatty liver disease. Currently, the drugs developed targeting the MFN2 protein target are still in the preclinical research stage, and there is no report on any compound targeting MFN2 for anti-hepatic injury and promoting liver repair. Summary of the Invention

[0005] Object of the Invention: To solve the problems existing in the prior art, the present invention provides an indolone compound, which can be used for treating acute and chronic liver diseases and promoting tissue repair, and solves the problem that there is no drug targeting MFN2 for anti-hepatic injury and promoting liver repair in the prior art.

[0006] The present invention also provides a preparation method and application of the compound.

[0007] Technical Solution: To achieve the above object, the indolone compound or a pharmaceutically acceptable salt thereof according to the present invention is characterized in that the indolone compound has a structural formula shown in any one of Formula I-III:

[0008] 。

[0009] The preparation method of the indolone compound according to the present invention comprises the following steps:

[0010] (1) Synthesis of 6-bromo-1-ethylindole-2,3-dione:

[0011] Dissolve 6-bromoindigo red and anhydrous potassium carbonate in an organic solvent, add bromoethane dropwise, stir overnight at room temperature. After the reaction is completed, dilute with water, extract, wash, dry, and purify to obtain the compound 6-bromo-1-ethylindole-2,3-dione;

[0012] (2)Synthesis of 6-bromo-1-ethyl-2,3-dihydro-1H-indol-2-one:

[0013] Dissolve 6-bromo-1-ethylindole-2,3-dione in a hydrazine hydrate solution, heat for reaction. After the reaction is completed, cool the reaction solution to room temperature, quench with water, then extract, wash, dry, and purify to obtain the compound 6-bromo-1-ethyl-2,3-dihydro-1H-indol-2-one;

[0014] (3)Synthesis of 1-ethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-2,3-dihydro-1H-indol-2-one:

[0015] Dissolve 6-bromo-1-ethyl-2,3-dihydro-1H-indol-2-one, bis(pinacolato)diboron, potassium acetate, and DPPF dichloropalladium in an organic solvent, add water under inert gas protection, heat for reaction. After the reaction is completed, evaporate the solvent, dissolve in water and filter through diatomaceous earth, extract the filtrate, wash, dry, and purify to obtain the compound 1-ethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-2,3-dihydro-1H-indol-2-one;

[0016] (4)Synthesis of 1-ethyl-6-(6-methoxy-1,2-diazacyclohex-3-yl)-2,3-dihydro-1H-indol-2-one:

[0017] Dissolve 1-ethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-2,3-dihydro-1H-indol-2-one, 3-bromo-6-methoxypyridazine, cesium fluoride, and tetrakis(triphenylphosphine)palladium in an organic solvent, add water under inert gas protection, heat and react overnight. After the reaction is completed, evaporate the solvent, dissolve in water and filter through diatomaceous earth, extract, wash, dry, and purify to obtain the compound 1-ethyl-6-(6-methoxy-1,2-diazacyclohex-3-yl)-2,3-dihydro-1H-indol-2-one;

[0018] (5)Synthesis of 1-ethyl-6-(6-oxo-1H-1,2-diazacyclohex-3-yl)-2,3-dihydro-1H-indol-2-one:

[0019] 1 - ethyl - 6 - (6 - methoxy - 1,2 - diazacyclohept - 3 - yl) - 2,3 - dihydro - 1H - indol - 2 - one was dissolved in an organic solvent, hydrochloric acid was added dropwise at room temperature, and the mixture was heated for reaction. After the reaction was completed, the solvent was directly rotary evaporated without extraction, and the compound 1 - ethyl - 6 - (6 - oxo - 1H - 1,2 - diazacyclohept - 3 - yl) - 2,3 - dihydro - 1H - indol - 2 - one was obtained by purification;

[0020] (6)Synthesis of 3 - [(dimethylamino)methylene] - 1 - ethyl - 6 - (6 - oxo - 1H - 1,2 - diazacyclohept - 3 - yl)indol - 2 - one (I - 11):

[0021] 1 - ethyl - 6 - (6 - oxo - 1H - 1,2 - diazacyclohept - 3 - yl) - 2,3 - dihydro - 1H - indol - 2 - one was dissolved in an organic solvent, N,N - dimethylformamide diethyl acetal was added dropwise at room temperature, and the mixture was stirred at room temperature overnight. After the reaction was completed, the solvent was rotary evaporated and diluted with water, then extracted and purified to obtain the compound 3 - [(dimethylamino)methylene] - 1 - ethyl - 6 - (6 - oxo - 1H - 1,2 - diazacyclohept - 3 - yl)indol - 2 - one;

[0022] (7)Synthesis of 3 - (ethylazanylmethylene) - 1 - ethyl - 6 - (6 - oxo - 1H - 1,2 - diazacyclohept - 3 - yl)indol - 2 - one (I - 12):

[0023] 3 - [(dimethylamino)methylene] - 1 - ethyl - 6 - (6 - oxo - 1H - 1,2 - diazacyclohept - 3 - yl)indol - 2 - one was dissolved in an organic solvent, hydrazine hydrate was added dropwise at room temperature, and the mixture was heated for reaction. After the reaction was completed, it was rotary evaporated and slurried to obtain the compound 3 - (ethylazanylmethylene) - 1 - ethyl - 6 - (6 - oxo - 1H - 1,2 - diazacyclohept - 3 - yl)indol - 2 - one;

[0024] (8)Synthesis of 3 - [{2 - [butan - 2 - ylidene]ethylazanyl}methylene] - 1 - ethyl - 6 - (6 - oxo - 1H - 1,2 - diazacyclohept - 3 - yl)indol - 2 - one:

[0025] 3 - (ethylazanylmethylene) - 1 - ethyl - 6 - (6 - oxo - 1H - 1,2 - diazacyclohept - 3 - yl)indol - 2 - one was dissolved in an organic solvent, 2 - butanone and acetic acid were added dropwise at room temperature, and the mixture was heated for reaction. After the reaction was completed, the solvent was rotary evaporated, diluted with water, extracted, washed, dried, and purified to obtain the compound of formula I, 3 - [{2 - [butan - 2 - ylidene]ethylazanyl}methylene] - 1 - ethyl - 6 - (6 - oxo - 1H - 1,2 - diazacyclohept - 3 - yl)indol - 2 - one;

[0026] Synthesis of 1-ethyl-6-(6-oxo-1H-1,2-diazocine-3-yl)-3-[[2-(3,4,5,6-tetrahydro-2H-pyran-4-ylidene)ethylamino]methylene]indol-2-one:

[0027] Dissolve 3-(ethylaminomethylene)-1-ethyl-6-(6-oxo-1H-1,2-diazocine-3-yl)indol-2-one in an organic solvent, add tetrahydropyranone and acetic acid dropwise at room temperature, heat the reaction, evaporate the solvent after the reaction is completed, dilute with water, extract, wash, dry, and purify to obtain the compound of formula II, 1-ethyl-6-(6-oxo-1H-1,2-diazocine-3-yl)-3-[[2-(3,4,5,6-tetrahydro-2H-pyran-4-ylidene)ethylamino]methylene]indol-2-one;

[0028] Synthesis of 1-ethyl-6-(6-oxo-1H-1,2-diazocine-3-yl)-3-[[2-(tetrahydrothiopyran-4-ylidene)ethylamino]methylene]indol-2-one:

[0029] Dissolve 3-(ethylaminomethylene)-1-ethyl-6-(6-oxo-1H-1,2-diazocine-3-yl)indol-2-one and tetrahydrothiopyran-4-one in an organic solvent, add acetic acid dropwise at room temperature, heat the reaction, evaporate the solvent after the reaction is completed, dilute with water, extract, wash, dry, and purify to obtain the compound of formula III, 1-ethyl-6-(6-oxo-1H-1,2-diazocine-3-yl)-3-[[2-(tetrahydrothiopyran-4-ylidene)ethylamino]methylene]indol-2-one.

[0030] Use of the indolone compound represented by any one of the structures of formula I-IV or a pharmaceutically acceptable salt thereof in the preparation of an agonist for targetedly activating the biological activity of MFN2 protein:

[0031] .

[0032] Use of the indolone compound represented by any one of the structures of formula I-IV or a pharmaceutically acceptable salt thereof in the preparation of a drug capable of promoting cell mitochondrial fusion and enhancing mitochondrial bioactivity and energy metabolism.

[0033] Furthermore, the compound represented by any one of the structures of formula I-IV or a pharmaceutically acceptable salt thereof specifically binds to the MFN2 protein, allosterically activates MFN2, promotes mitochondrial fusion, and enhances hepatocyte viability.

[0034] Use of the indolone compound represented by any one of the structures of formula I-IV or a pharmaceutically acceptable salt thereof in the preparation of a drug for the treatment of acute and chronic liver diseases.

[0035] Among them, the acute and chronic liver diseases include drug-induced liver injury, septic liver injury, liver injury caused by hepatectomy, or metabolic disorder non-alcoholic steatohepatitis.

[0036] Use of an indolinone compound represented by any one of the structures of Formulas I-IV or a pharmaceutically acceptable salt thereof according to the present invention in the preparation of a drug for promoting liver tissue repair.

[0037] Among them, the tissue repair is the repair of liver tissue after being damaged by drugs, infectious agents or trauma.

[0038] The pharmaceutical composition for treating acute and chronic liver diseases and promoting tissue repair according to the present invention comprises the indolinone compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant or vehicle.

[0039] Among them, the pharmaceutical composition is preferably a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhalant, ointment, suppository or patch.

[0040] The present invention designs and constructs an indolinone compound, which can specifically bind to the MFN2 protein, allosterically activate MFN2, promote mitochondrial fusion, improve hepatocyte viability, and thus play a role in treating acute and chronic liver diseases and promoting tissue repair.

[0041] The indolinone compound designed and prepared by the present invention is further verified by surface plasmon resonance (SPR) experiments that the compound can bind to the MFN2 protein. The effect of the compound on the GTPase activity of the MFN2 protein is further verified, and it is found that the compound can increase the GTPase activity of the MFN2 protein. In the in vitro biological activity evaluation experiment, the compound can significantly increase the mitochondrial aspect ratio of primary mouse hepatocytes and promote mitochondrial fusion. In order to evaluate the in vivo biological activity of the compound, a drug-induced liver injury model induced by APAP (paracetamol), a liver injury model caused by 70% hepatectomy, a septic liver injury model induced by S. typhimurium, a metabolic fatty liver disease model induced by MCDHFD (choline-deficient and methionine-reduced high-fat diet), and a metabolic fatty liver disease induced by GAN (Gubra-Amylin NASH) diet are constructed. The compound can significantly reduce hepatocyte death, alleviate liver injury, promote liver lipid metabolism and liver tissue repair and regeneration.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0043] 1. The indole ketone compounds provided by the present invention are the first small molecule compounds targeting the MFN2 protein for anti - liver injury and promoting liver repair designed based on the structure of the MFN2 protein. After specifically binding to the MFN2 protein, the indole ketone compounds can effectively target and activate the biological activity of the MFN2 protein, and can serve as agonists of the MFN2 protein, thereby being used for preparing drugs for treating liver diseases, especially drugs for treating liver injury and metabolic - related liver diseases.

[0044] 2. The small molecule compounds with a brand - new structure designed and prepared by the present invention have the function of promoting mitochondrial fusion of hepatic parenchymal cells, improving hepatocyte viability, and promoting hepatocyte proliferation, thereby repairing liver injury, and can be used for treating acute and chronic liver diseases and promoting tissue repair.

[0045] 3. The present invention proposes a new synthetic route for indole ketone compounds. The compounds of the present invention are ingeniously designed, have a simple structure, the raw materials are cheap and easily available, and the synthesis process is safe, environmentally friendly, and easy for large - scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the fitting curve for detecting the binding force between compound AH - 262 and MFN2 protein at different concentrations by SPR;

[0047] Figure 2 It is the response value curve for detecting the binding of compound AH - 262 and MFN2 protein at different concentrations by SPR;

[0048] Figure 3 It is the dose - effect curve for the influence of compound AH - 262 on the GTPase activity of MFN2 protein;

[0049] Figure 4 It is the detection result of the influence of compound AH - 262 on the mitochondrial morphology of primary mouse hepatocytes. Among them, the one marked with solvent control is the solvent control group, that is, serum - free DMEM high - glucose medium containing 1‰ DMSO, and the one marked with AH - 262 is the administration group, that is, 25 μM AH - 262 prepared with serum - free DMEM high - glucose medium containing 1‰ DMSO;

[0050] Figure 5 It is the pharmacodynamic study of compound AH - 262 in the hepatocyte injury model induced by acetaminophen, and the cell survival rate result;

[0051] Figure 6 It is the pharmacodynamic study of compound AH - 262 in the drug - induced liver injury model induced by APAP (acetaminophen), and the schematic diagram of the morphological observation of mouse liver pathological tissues (H&E×200, the scale is 100 μm);

[0052] Figure 7Pharmacodynamic study of compound AH-262 in an APAP (acetaminophen)-induced drug-induced liver injury model, detection results of the activities of aspartate aminotransferase and alanine aminotransferase in mouse serum;

[0053] Figure 8 Pharmacodynamic study of compound AH-262 in a liver injury model induced by 70% hepatectomy, statistical results of mouse liver weight ratio;

[0054] Figure 9 Pharmacodynamic study of compound AH-262 in a liver injury model induced by 70% hepatectomy, detection results of the activities of aspartate aminotransferase and alanine aminotransferase in mouse serum;

[0055] Figure 10 Pharmacodynamic study of compound AH-262 in a liver injury model induced by 70% hepatectomy, schematic diagram of mouse liver Ki67 immunohistochemical staining observation (IHC×200, scale bar is 100μm);

[0056] Figure 11 Pharmacodynamic study of compound AH-262 in a liver injury model induced by 70% hepatectomy, statistical results of the percentage of Ki67 immunohistochemical staining positive cells in mouse liver;

[0057] Figure 12 Pharmacodynamic study of compound AH-262 in a septic liver injury model induced by S. typhimurium, schematic diagram of mouse liver pathological tissue morphology observation (H&E×200, scale bar is 100μm);

[0058] Figure 13 Pharmacodynamic study of compound AH-262 in a septic liver injury model induced by S. typhimurium, detection results of the activities of aspartate aminotransferase and alanine aminotransferase in mouse serum;

[0059] Figure 14 Pharmacodynamic study of compound AH-262 in a septic liver injury model induced by S. typhimurium, detection result diagram of bacterial load in mouse liver tissue;

[0060] Figure 15 Pharmacodynamic study of compound AH-262 in a septic liver injury model induced by S. typhimurium, statistical results of bacterial load in mouse liver tissue;

[0061] Figure 16Pharmacodynamic study of compound AH-262 in a metabolic fatty liver disease model induced by MCDHFD (choline-deficient and methionine-reduced high-fat diet). Schematic diagram of the morphological observation of mouse liver pathological tissues (H&E×200, Masson×200, Oil Red O×200, scale bar = 100μm);

[0062] Figure 17 Pharmacodynamic study of compound AH-262 in a metabolic fatty liver disease model induced by MCDHFD (choline-deficient and methionine-reduced high-fat diet). Detection results of the activities of serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase in mice;

[0063] Figure 18 Pharmacodynamic study of compound AH-262 in a metabolic fatty liver disease model induced by MCDHFD (choline-deficient and methionine-reduced high-fat diet). mRNA levels of liver inflammatory factors MCP-1, TNF-α, IL-1β, and IL-6 in mice;

[0064] Figure 19 Pharmacodynamic study of compound AH-262 in a metabolic fatty liver disease model induced by MCDHFD (choline-deficient and methionine-reduced high-fat diet). mRNA levels of liver fibrosis factors α-SMA, Col1α1, and TGF-β in mice;

[0065] Figure 20 Pharmacodynamic study of compound AH-262 in a metabolic fatty liver disease model induced by GAN (Gubra-Amylin NASH) diet. Schematic diagram of the morphological observation of mouse liver pathological tissues (H&E×200, Masson×200, Oil Red O×200, scale bar = 100μm);

[0066] Figure 21 Pharmacodynamic study of compound AH-262 in a metabolic fatty liver disease model induced by GAN (Gubra-Amylin NASH) diet. Recorded results of mouse body weight;

[0067] Figure 22 Pharmacodynamic study of compound AH-262 in a metabolic fatty liver disease model induced by GAN (Gubra-Amylin NASH) diet. Detection results of the activities of serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase in mice;

[0068] Figure 23 Pharmacodynamic study of compound AH-262 in a metabolic fatty liver disease model induced by GAN (Gubra-Amylin NASH) diet. mRNA levels of liver inflammatory factors MCP-1, TNF-α, IL-1β, and IL-6 in mice;

[0069] Figure 24 Pharmacodynamic study of compound AH-262 in a GAN (Gubra-Amylin NASH) diet-induced metabolic fatty liver disease model, levels of mouse liver fibrosis factors ɑ-SMA, Col1ɑ1, and TGF-β mRNA;

[0070] Figure 25 To fit the binding curve of compound Y-1 at different concentrations to MFN2 protein by SPR;

[0071] Figure 26 To fit the binding curve of compound Y-2 at different concentrations to MFN2 protein by SPR;

[0072] Figure 27 To fit the binding curve of compound Y-3 at different concentrations to MFN2 protein by SPR;

[0073] Figure 28 To obtain the response value curve of the binding of compound Y-1 at different concentrations to MFN2 protein by SPR;

[0074] Figure 29 To obtain the response value curve of the binding of compound Y-2 at different concentrations to MFN2 protein by SPR;

[0075] Figure 30 To obtain the response value curve of the binding of compound Y-3 at different concentrations to MFN2 protein by SPR;

[0076] Figure 31 To obtain the dose-effect curve of the effect of compound Y-1 on the GTPase activity of MFN2 protein;

[0077] Figure 32 To obtain the dose-effect curve of the effect of compound Y-2 on the GTPase activity of MFN2 protein;

[0078] Figure 33 To obtain the dose-effect curve of the effect of compound Y-3 on the GTPase activity of MFN2 protein;

[0079] Figure 34 To obtain the detection results of the effect of compound AH-262 on the mitochondrial morphology of primary mouse hepatocytes. Among them, the one labeled with solvent control is the solvent control group, namely serum-free DMEM high-glucose medium containing 1‰ DMSO, and the ones labeled with Y-1, Y-2, and Y-3 are the drug administration groups, namely 25 uM Y-1, Y-2, and Y-3 prepared with serum-free DMEM high-glucose medium containing 1‰ DMSO;

[0080] Figure 35Pharmacodynamic study of compounds Y-1, Y-2 and Y-3 in a paracetamol-induced hepatocyte injury model, cell survival rate results. Detailed implementation mode

[0081] The present invention will be further described below in conjunction with specific embodiments.

[0082] The experimental methods described in the examples are all conventional methods unless otherwise specified; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0083] The ATPase / GTPase Assay kit was purchased from Abcam, ab272520.

[0084] The EGTA / HBSS solution was purchased from ECOTOP, ED-8006-500ml.

[0085] Collagenase was purchased from Sigma, C1899.

[0086] APAP (paracetamol) was purchased from Targetmol, T0065.

[0087] Silibinin was purchased from Targetmol, T1660.

[0088] The AST (aspartate aminotransferase) activity detection kit was purchased from Nanjing Jiancheng Bioengineering Institute, C010-2-1.

[0089] The ALT (alanine aminotransferase) activity detection kit was purchased from Nanjing Jiancheng Bioengineering Institute, C009-2-1.

[0090] S. typhimurium was purchased from ATCC, strain number 14028.

[0091] MCDHFD (choline-deficient and methionine-reduced high-fat diet) was purchased from Research Diet, A06071309.

[0092] GAN (Gubra-Amylin NASH) was purchased from Research Diet, D09100310.

[0093] Example 1

[0094] Synthesis route of compound AH-262 of formula IV:

[0095]

[0096] Preparation of 1-ethylindole-2,3-dione (I-1):

[0097] Dissolve 2.9 g (20 mmol) of 1H-indole-2,3-dione and 6.9 g (50 mmol) of K2CO3 in 30 mL of DMF solution. Dropwise add 1.8 mL (24 mmol) of bromoethane to it and stir at 60 °C for 2 h. After the reaction is completed, quench with 120 mL of water and extract three times with 150 mL of ethyl acetate. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, and purify by flash column chromatography on silica gel (PE:EA = 5:1) to obtain 3.3 g of compound I-1 (yield 95%). 1H NMR (300 MHz, DMSO-d6) δ 7.66 (td, J = 7.7, 1.4 Hz, 1H), 7.54 (ddd, J = 7.4, 1.4, 0.6 Hz, 1H), 7.19 (dt, J = 8.0, 0.8 Hz, 1H), 7.12 (td, J = 7.5, 0.8 Hz, 1H), 3.70 (q, J = 7.2 Hz, 2H), 1.18 (t, J = 7.2 Hz, 3H).

[0098] Preparation of 1-ethylindolin-2-one (I-2):

[0099] Dissolve 3.3 g (18.6 mmol) of 1-ethylindole-2,3-dione in 20 mL of 80% by mass hydrazine hydrate solvent and stir at 130 °C for 2 h. After the reaction is completed, cool the reaction solution to room temperature, dilute with 20 mL of water, and extract the aqueous phase three times with 40 mL of EA. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, and purify by flash column chromatography on silica gel (PE:EA = 7:1) to obtain 2.7 g of compound I-2 (yield 90%). 1H NMR (300 MHz, DMSO-d6) δ 7.29 - 7.20 (m, 2H), 7.04 - 6.96 (m, 2H), 3.68 (q, J = 7.2 Hz, 2H), 3.53 (d, J = 1.1 Hz, 2H), 1.13 (t, J = 7.2 Hz, 3H).

[0100] Preparation of 3-(dimethylamino)methylene-1-ethylindolin-2-one (I-3):

[0101] Dissolve 2.7 g (16.8 mmol) of 1-ethylindoline-2-one in 20 mL of ethanol solution. Dropwise add 4.3 mL (25.2 mmol) of N,N-dimethylformamide diethyl acetal thereto and stir at room temperature for 3 h. After the reaction is completed, rotary evaporate the solvent. After diluting with 30 mL of water, extract the aqueous phase with 30 mL of EA three times. Wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, and purify by flash column chromatography on silica gel column (PE:EA = 1:1) to obtain 3.5 g of compound I-3 (yield 95%). 1H NMR (300 MHz, CDCl3) δ 7.63 (s, 1H), 7.44 (dd, J = 7.8, 1.2 Hz, 1H), 6.99 - 6.84 (m, 3H), 3.89 (p, J = 7.2 Hz, 4H), 3.35 (s, 6H), 1.46 - 1.23 (m, 3H).

[0102] Preparation of 4-(3-((dimethylamino)methylene)-1-ethyl-2-oxoindolin-6-yl)-4-oxobutyric acid (I-4):

[0103] Dissolve 3.5 g (16.2 mmol) of 3-(dimethylamino)methylene-1-ethylindol-2-one, 1.9 g (19.4 mmol) of succinic anhydride, and 5.4 g (40.5 mmol) of anhydrous aluminum chloride in 60 mL of 1,2-dichloroethane solution. Stir the reaction solution at 0 °C for 1.5 h first, and then at 50 °C for 1.5 h. After the reaction is completed, cool the reaction solution to room temperature and pour it into 60 mL of 17% hydrochloric acid solution by mass fraction to precipitate a large amount of white solid. Stir in an ice bath for 30 min, and vacuum filter using a Buchner funnel to obtain 1.5 g of compound I-4 (yield 30%). 1H NMR (300 MHz, DMSO-d6) δ 10.76 (s, 1H), 8.06 (s, 1H), 7.82 (d, J = 1.8 Hz, 1H), 7.63 - 7.37 (m, 2H), 6.97 (t, J = 7.7 Hz, 1H), 5.24 (s, 1H), 3.79 (dt, J = 13.8, 7.0 Hz, 2H), 2.96 (q, J = 8.5 Hz, 2H), 2.48 - 2.37 (m, 2H), 1.18 - 1.08 (m, 3H).

[0104] Preparation of 1-ethyl-3-(hydrazonomethylene)-6-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)indolin-2-one (I-5):

[0105] Dissolve 1.5 g (4.7 mmol) of 4-(3-((dimethylamino)methylene)-1-ethyl-2-oxoindolin-6-yl)-4-oxobutyric acid in 30 mL of isopropanol solution, heat to 90 °C, add dropwise 0.6 mL (9.4 mmol) of 80% hydrazine hydrate, stir at 90 °C for 3 h. After the reaction is completed, evaporate the solvent under reduced pressure, dilute with 30 mL of water, extract the aqueous phase with 30 mL of DCM three times, wash the organic phase with saturated brine, dry over anhydrous sodium sulfate, and purify by flash column chromatography on silica gel (DCM:MeOH = 20:1) to obtain 0.8 g of compound I-5 (yield 56%). 1H NMR (300 MHz, DMSO-d6) δ 12.10 (s, 1H), 7.74 (d, J = 49.7 Hz, 1H), 7.57 (ddd, J = 7.1, 5.4, 1.6 Hz, 1H), 7.52 - 7.41 (m, 2H), 3.82 (p, J = 7.2 Hz, 2H), 3.72 (s, 2H), 3.35 (d, J = 11.0 Hz, 6H), 2.60 - 2.53 (m, 2H), 1.14 (td, J = 7.1, 2.1 Hz, 3H).

[0106] Preparation of 1-ethyl-3-((Z)-(2-((2E)-but-2-en-1-ylidene)ethylamino)methylene)-6-(6-oxo-4H,5H,6H,1H-1,2-diazepin-3-yl)indol-2-one (AH-262):

[0107] Dissolve 0.8 g (4.7 mmol) of 1-ethyl-3-(hydrazonomethylene)-6-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)indolin-2-one in 10 mL of isopropanol solution, heat to 90 °C, add dropwise 0.48 mL (5.4 mmol) of 2-butanone and a drop of acetic acid, stir at 90 °C for 3 h. After the reaction is completed, evaporate the solvent under reduced pressure, dissolve in 10 mL of 70% aqueous methanol solution, and separate and purify the solution using a MorphlingTM WD-C18 preparative liquid chromatography column (column specifications: MorphlingTM WD-C18, 10 μm, 30x250 mm). The mobile phase is 70% aqueous solution, the flow rate is 20 mL / min, and the fraction with a retention time of 27.5 min is collected. After separation, the collected solution is concentrated by a rotary evaporator, diluted with 20 mL of water, the aqueous phase is extracted with 20 mL of DCM three times, the DCM is evaporated by a rotary evaporator, and dried under vacuum to obtain 115 mg of compound AH-262 (yield 44%) 11H NMR (600 MHz, CDCl3) δ 11.61 (d, J = 10.3 Hz, 1H), 8.66 (s, 1H), 8.06 (d, J = 9.7 Hz, 1H), 7.39 (d, J = 1.4 Hz, 1H), 7.32 - 7.28 (m, 2H), 3.93 (q, J = 7.2 Hz, 2H), 3.02 (t, J = 8.1 Hz, 2H), 2.62 (t, J = 8.2 Hz, 2H), 2.37 (q, J = 7.4 Hz, 2H), 2.02 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H), 1.15 (t, J = 7.5 Hz, 3H).

[0108] Example 2

[0109] In vitro binding experiment of compound AH-262 to MFN2 protein

[0110] For the compound AH-262 prepared by the present invention, a biomolecular interaction analyzer Biacore T200 (General Electric Company) was used to detect the dissociation constant of the compound and MFN2 protein. The specific detection process is as follows: 1. Experimental preparation: Prepare the Biacore T200 biomolecular interaction analyzer and supporting consumables such as buffers, chips (CM5 chips for amino coupling and immobilizing proteins), syringes, and sample tubes. Prepare the small molecule compound AH-262, MFN2 protein, and HBS-EP+ buffer (containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) surfactant P20, pH 7.4). Accurately measure the protein concentration and dilute it to 1 mg / ml with the buffer for immobilization; the small molecule compound AH-262 is also dissolved with the buffer and formulated into a series of solutions with different concentrations (gradient concentrations from 1 nM - 10 μM) for subsequent binding experiments. 2. Chip pretreatment: Install the CM5 chip on the Biacore T200 instrument and activate the carboxyl groups on the chip surface with the activation reagents (N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)) for 15 minutes to make the chip surface have the activity to bind proteins. 3. Protein immobilization: Inject the diluted protein solution onto the chip surface at a flow rate of 5 μL / min for 30 minutes. The protein reacts with the activated carboxyl groups through amino groups and covalently binds to the chip surface. After immobilization, use ethanolamine to block the unreacted active groups to terminate the immobilization reaction. 4. Baseline stabilization: Inject the buffer to balance the chip surface until a stable baseline is detected to ensure a stable detection environment and no interference signals. 5. Binding assay: Inject the small molecule compound solutions with different concentrations in sequence, set the flow rate to 20 μL / min, and the injection time to 5 minutes. Record the signal changes generated during the binding of the small molecule to the protein immobilized on the chip, that is, the change in the resonance unit (RU) value, which reflects the binding situation between the two. 6. Dissociation assay: After the binding reaction is completed, inject the buffer to wash the chip surface and monitor the change in the RU value over time during the dissociation of the small molecule from the protein, and record the dissociation curve for 5 minutes. 7. Data processing: Use the software supporting the Biacore T200 instrument to process the collected binding and dissociation data, subtract the signal of the blank control (only buffer injection), and correct the baseline drift, etc. By fitting the binding and dissociation curves, obtain the association rate constant (ka), dissociation rate constant (kd), and further calculate the equilibrium dissociation constant (KD = kd / ka). The smaller the KD value, the stronger the binding force between the small molecule compound and the protein.

[0111] As Figure 1 , Figure 2As shown, the binding force fitting curve of compound AH-262 with different concentrations to MFN2 protein was detected by SPR. The results confirmed that compound AH-262 could bind to MFN2 protein, and its dissociation constant was 4.753E-8M.

[0112] Example 3

[0113] Effect of compound AH-262 on the GTPase activity of MFN2 protein

[0114] Using the ATPase / GTPase Assay kit (Abcam, ab272520), the effect of compound AH-262 on the GTPase activity of MFN2 protein was detected according to the instructions.

[0115] Results Figure 3 As shown, the results confirmed that compound AH-262 could significantly increase the GTPase activity of MFN2 protein, and the EC50 value was 23.33 uM. The stronger the GTPase activity of MFN2 protein, the stronger its activity in mediating mitochondrial fusion, further proving that compound AH-262 could bind to MFN2 protein.

[0116] Example 4

[0117] Effect of compound AH-262 on the mitochondrial morphology of primary mouse hepatocytes

[0118] Isolation of primary mouse liver cells: Preheat EGTA / HBSS and collagenase solution at 40 °C for 30 min. Wash the pump hose with 75% ethanol, then insert the hose into the EGTA / HBSS solution to remove air bubbles. Intraperitoneally inject 6-week-old male C57BL / 6J mice with 2.5% tribromoethanol at a dose of 10 ml / kg until fully anesthetized. Shave the abdominal hair and disinfect with 75% ethanol. Fix the mouse, open the abdominal cavity, move the intestines to the right side to expose the hepatic portal vein and inferior vena cava. Separate the inferior vena cava with a cotton swab and cotton, insert a suture needle, carefully support the blood vessel with the forceps handle, and slowly insert an indwelling needle into the blood vessel at the same time, about 1.5 cm to 2.5 cm, then tie it with the pre-buried thread. At the same time, it is found that the liver blood vessels flow back into the indwelling needle. Open the thoracic cavity, ligate the aortic cavity with an arterial clamp, cut open the hepatic portal vein, perfuse, adjust the flow rate to 7 ml / min, and perfuse 40 ml / animal of EGTA / HBSS solution. Add 40 ml of collagenase solution within 5 min, adjust the flow rate to 10 ml / min, and digest for about 2 min. Press a few more times with forceps to fully digest the cells. Put the collagenase and the digested liver into a dish, remove the gallbladder, add an appropriate amount of EGTA / HBSS solution (15 mL / animal), quickly wash once and disperse. Pass a digested liver through a 100-mesh sieve (15 ml / animal), centrifuge at 50 g for 5 min at 4 °C. Discard the supernatant, resuspend and pipette evenly with 5 mL of PM solution, add 5 mL of Percoll, centrifuge at 120 g for 5 min at 4 °C, aspirate the upper cell suspension, resuspend with 2 mL of PM solution, centrifuge at 50 g for 5 min at 4 °C, discard the supernatant, resuspend with 2 mL of PM solution, and count and plate.

[0119] The isolated primary cells were cultured in an incubator at 37 °C and 5% CO2 for 36 hours and then given drugs. The solvent control group was serum-free DMEM high-glucose medium containing 1‰ DMSO, and the drug administration group was 25 μM AH-262 prepared with serum-free DMEM high-glucose medium containing 1‰ DMSO. After administration, they were placed in an incubator at 37 °C and 5% CO2 for 30 minutes. After the administration was completed, Mitotraker deepred and Hoechst live cell dyes were used for staining. After staining, they were placed under a confocal microscope for observation and photography.

[0120] The detection results of the effect of compound AH-262 on the mitochondrial morphology of primary mouse liver cells are as Figure 4As shown, the one labeled with solvent control is the solvent control group, namely the serum-free DMEM high-glucose medium containing 1‰ DMSO, and the one labeled with AH-262 is the administration group, namely 25 μM AH-262 prepared with the serum-free DMEM high-glucose medium containing 1‰ DMSO. The results show that the mitochondrial length-width ratio in the administration group is significantly increased and the degree of mitochondrial fusion is higher. The compound of the present invention can improve the degree of mitochondrial fusion in cells, maintain the integrity of mitochondrial morphology and structure, promote mitochondrial function by optimizing energy metabolism, participate in intracellular material transport and signal transduction, and ensure the stability of mitochondrial DNA and achieve its complementation and recombination.

[0121] Example 5

[0122] Protective effect of compound AH-262 on APAP (acetaminophen)-induced damage of primary mouse hepatocytes

[0123] The steps for extracting primary mouse hepatocytes are the same as those in Example 4. The isolated primary cells are cultured in an incubator at 37°C and 5% CO2 for 36 hours and then given drugs. The solvent control group is the serum-free DMEM high-glucose medium containing 1‰ DMSO, the administration group is 5 μM AH-262 prepared with the serum-free DMEM high-glucose medium containing 1‰ DMSO, and the positive control group is 5 μM silybin prepared with the serum-free DMEM high-glucose medium containing 1‰ DMSO. After pre-administering the drugs for 24 hours, after the pre-administration ends, the culture medium is replaced with a 10 mM acetaminophen solution (prepared with the serum-free DMEM high-glucose medium), and the cell viability is detected by the CCK8 method after 24 hours.

[0124] The results of the pharmacodynamic study on the protective effect of compound AH-262 on APAP (acetaminophen)-induced damage of primary mouse hepatocytes are as Figure 5 shown. Among them, the one labeled with solvent control is the solvent control group, namely the serum-free DMEM high-glucose medium containing 1‰ DMSO, the one labeled with AH-262 is the administration group, namely 5 μM AH-262 prepared with the serum-free DMEM high-glucose medium containing 1‰ DMSO, and the one labeled with Silibinin is the silybin (positive control) group. The results show that the cell viability in the administration group is significantly increased compared with the solvent control group, and the effect is equivalent to that of the positive control group, indicating that compound AH-262 has a certain protective effect on APAP (acetaminophen)-induced damage of primary mouse hepatocytes.

[0125] Example 6

[0126] Construction of an APAP (acetaminophen)-induced drug-induced liver injury model and pharmacodynamic study of compound AH-262

[0127] APAP (paracetamol)-induced drug-induced liver injury model: Twelve C57BL / 6J mice (Shanghai SLAC Laboratory Animal Co., Ltd.) were purchased. After normal feeding for one week, they were randomly and evenly divided into 2 groups of 6 mice each according to body weight: the model group (intragastric administration of normal saline for 3 days followed by intraperitoneal injection of paracetamol (300 mg / kg)) and the administration group (intragastric administration of the small molecule compound AH-262 (10 mg / kg) for 3 days followed by intraperitoneal injection of paracetamol (300 mg / kg)). Twenty-four hours after intraperitoneal injection of paracetamol, the mice were anesthetized by intraperitoneal injection of tribromoethanol. After blood collection from the orbital cavity, the abdominal cavity was opened to remove the liver for further analysis. After removing the liver, the residual blood was washed away with precooled normal saline at 4°C, blotted dry on filter paper, and immediately fixed in precooled 4% paraformaldehyde at 4°C. After 24 hours of fixation, it was embedded, sectioned, and stained with H&E. The blood of the removed mice was allowed to stand at room temperature for 30 minutes and then centrifuged at 6000 g for 15 minutes. The supernatant (i.e., serum) was taken and detected using the AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity detection kits.

[0128] Pharmacodynamic study of compound AH-262 in an APAP (paracetamol)-induced drug-induced liver injury model. Schematic diagram of the morphological observation of mouse liver pathological tissues (H&E×200) is as Figure 6 shown. The results showed that obvious liver injury and necrosis occurred in the mice of the solvent control group 24 hours after APAP (paracetamol) injection, while no obvious liver injury and necrosis occurred in the mice of the compound AH-262 administration group, indicating that compound AH-262 has a certain protective effect on APAP (paracetamol)-induced drug-induced liver injury.

[0129] Schematic diagram of the detection results of the activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in mouse serum is as Figure 7 shown. The results showed that the activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in the serum of the mice in the solvent control group increased significantly 24 hours after APAP (paracetamol) injection, indicating a large number of hepatocyte deaths and ruptures. While the activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in the serum of the mice in the compound AH-262 administration group were significantly lower than those in the solvent control group, indicating that compound AH-262 has a certain protective effect on APAP (paracetamol)-induced drug-induced liver injury.

[0130] Example 7

[0131] Construction of a liver injury model induced by 70% hepatectomy and pharmacodynamic study of compound AH-262

[0132] Liver injury model induced by 70% hepatectomy: Sixty C57BL / 6J mice (Shanghai SLAC Laboratory Animal Co., Ltd.) were purchased. After being normally fed for one week, they were randomly and evenly divided into two groups of 30 mice each according to body weight: the model group (subjected to 70% hepatectomy 3 days after intragastric administration of normal saline) and the drug administration group (subjected to 70% hepatectomy 3 days after intragastric administration of the small molecule compound AH-262 (10 mg / kg)). 2 / 3 hepatectomy: After anesthetizing the mice by intraperitoneal injection of tribromoethanol, an abdominal incision was made along the midline. Two-thirds of the liver lobes (left lateral lobe and middle lobe) of the mice were resected by ligating the corresponding branch blood vessels, and the upper right, lower right, and caudate lobes were retained. The abdomen was closed and observed. Forty-eight hours after hepatectomy, the mice were anesthetized by intraperitoneal injection of tribromoethanol. Blood was collected from the orbital sinus, and then the abdomen was opened to remove the liver for further analysis. The removed liver was immediately weighed, and then the residual blood was washed away with precooled (4°C) normal saline, blotted dry on filter paper, and immediately fixed in precooled (4°C) 4% paraformaldehyde for 24 hours, followed by embedding, sectioning, and performing H&E staining and immunohistochemical staining. The blood taken from the mice was allowed to stand at room temperature for 30 minutes and then centrifuged at 6000 g for 15 minutes. The supernatant (i.e., serum) was taken and detected using AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity detection kits.

[0133] Pharmacodynamic study of compound AH-262 in a liver injury model induced by 70% hepatectomy. The results of the liver weight / body weight ratio of mice 48 hours after 70% hepatectomy are as Figure 8 shown. The results show that compound AH-262 can significantly increase the liver weight ratio of mice, indicating that compound AH-262 can promote liver regeneration and liver tissue repair. Schematic diagram of the detection results of the activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in the serum of mice is as Figure 9 shown. The results show that the activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in the serum of mice in the solvent control group were significantly increased 48 hours after 70% hepatectomy, indicating a large number of hepatocyte deaths and ruptures. However, the activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in the serum of mice in the compound AH-262 administration group were significantly lower than those in the solvent control group, indicating that compound AH-262 has a certain protective effect on liver injury induced by 70% hepatectomy. Schematic diagram of the observation of Ki67 immunohistochemical staining of mouse liver (IHC×200) is as Figure 10 shown. The results show that compound AH-262 can significantly increase the proliferation of hepatocytes after 70% hepatectomy and promote liver tissue regeneration. Statistical results of the percentage of Ki67 immunohistochemical staining positive cells in mouse liver are as Figure 11 shown. The results show that compound AH-262 can significantly increase the proliferation of hepatocytes after 70% hepatectomy and promote liver tissue regeneration.

[0134] Example 8

[0135] Construction of a murine model of Salmonella typhimurium-induced septic liver injury and pharmacological evaluation of compound AH-262

[0136] Salmonella typhimurium-induced septic liver injury model: Twelve C57BL / 6J mice (Shanghai SLAC Laboratory Animal Co., Ltd.) were purchased. After one week of normal feeding, the mice were randomly divided into two groups of six mice each according to body weight: a model group (intragastric administration of normal saline for 3 days followed by intravenous injection of Salmonella typhimurium (2×10 6 CFU / 20 g)) and a treatment group (intragastric administration of small molecule compound AH-262 (10 mg / kg) for 3 days followed by intraperitoneal injection of Salmonella typhimurium (2×10 6 CFU / 20 g)). Twenty-four hours after intravenous injection of Salmonella typhimurium, the mice were anesthetized by intraperitoneal injection of tribromoethanol. After blood collection from the orbital sinus, the abdomen was opened to remove the liver for further analysis. After blood collection from the orbital sinus, the abdomen was opened to remove the liver. 1 mg of fresh liver tissue was accurately weighed and added to 1 ml of normal saline for homogenization. The homogenate was diluted 10-fold and spread on an LB plate, and then incubated in an incubator at 37 °C for 24 hours. After removal, the plate was photographed and the number of colonies was counted. The bacterial load in the mouse liver tissue = Log(10000×number of colonies on the plate). The remaining liver was washed with ice-cold normal saline at 4 °C to remove residual blood, blotted dry on filter paper, and immediately fixed in 4% paraformaldehyde pre-cooled at 4 °C for 24 hours, then embedded, sectioned, and stained with H&E. The blood samples taken from the mice were allowed to stand at room temperature for 30 minutes and then centrifuged at 6000 g for 15 minutes. The supernatant (serum) was taken and detected using AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity assay kits.

[0137] Pharmacological evaluation of compound AH-262 in a murine model of Salmonella typhimurium-induced septic liver injury. Schematic diagram of the morphological observation of mouse liver pathological tissues (H&E×200). The results are as Figure 12 shown. The results showed that obvious inflammatory infiltration and tissue necrosis occurred in the livers of the mice in the solvent control group 24 hours after intravenous injection of Salmonella typhimurium, while no obvious damage and necrosis were observed in the livers of the mice in the compound AH-262 treatment group, indicating that compound AH-262 has a certain protective effect on Salmonella typhimurium-induced septic liver injury. The results of the determination of the activities of serum aspartate aminotransferase and alanine aminotransferase in the mice are as Figure 13 shown. The results showed that compound AH-262 could significantly reduce the increase in liver transaminases caused by Salmonella typhimurium infection, indicating a certain protective effect on the liver. The results of the determination of the bacterial load in the mouse liver tissue are shown in Figure 14As shown, the results show that compound AH-262 can significantly enhance the bacterial clearance rate of the liver. Statistical results of bacterial load in mouse liver tissue are as Figure 15 shown, the results show that compound AH-262 can significantly enhance the bacterial clearance rate of the liver.

[0138] Example 9

[0139] Construction of a metabolic fatty liver disease model induced by MCDHFD (choline-deficient and methionine-reduced high-fat diet) and pharmacological efficacy study of compound AH-262

[0140] Metabolic fatty liver disease model induced by MCDHFD (choline-deficient and methionine-reduced high-fat diet): Twelve C57BL / 6J mice (Shanghai SLAC Laboratory Animal Co., Ltd.) were purchased. After normal feeding for one week, they were randomly and evenly divided into 2 groups of 6 mice each according to body weight: the model group (fed with MCDHFD diet and gavaged with normal saline daily) and the administration group (fed with MCDHFD diet and gavaged with the small molecule compound AH-262 (10 mg / kg) daily). After 6 weeks, the mice were anesthetized by intraperitoneal injection of tribromoethanol. After taking blood from the orbital cavity, the abdominal cavity was opened to take out the liver. The residual blood was washed away with precooled normal saline at 4°C, blotted dry on filter paper, and immediately fixed in precooled 4% paraformaldehyde at 4°C. After fixation for 24 hours, it was embedded, sectioned, and subjected to H&E staining, Oil Red O staining, and Masson staining. A small part of the liver was reserved for RNA extraction and PCR analysis. RNA quantification and reverse transcription: RNA was quantified using NanoDrop, and its purity was evaluated using A260 / A280. Subsequently, the RNA was reverse transcribed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit. The relative differences in the expression of target genes in different samples were determined by the 2-ΔΔCT semi-quantitative analysis method. The target genes included MCP-1, TNF-α, IL-1β, IL-6, α-SMA, Col-1α1, and TGF-β. The blood taken from the mice was allowed to stand at room temperature for 30 minutes and then centrifuged at 6000 g for 15 minutes. The supernatant (i.e., serum) was taken and detected using the AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity detection kits.

[0141] Pharmacological efficacy study of compound AH-262 in a metabolic fatty liver disease model induced by MCDHFD (choline-deficient and methionine-reduced high-fat diet). Schematic diagrams of morphological observation of mouse liver pathological tissues (H&E×200, Masson×200, Oil Red O×200) are as Figure 16As shown, the results showed that after 6 weeks of feeding the MCDHFD diet, the mice in the solvent control group had obvious lipid accumulation, inflammatory infiltration and fibrosis in the liver, while the degrees of liver lipid accumulation, inflammatory infiltration and fibrosis in the mice in the compound AH-262 administration group were significantly reduced, indicating that compound AH-262 can promote liver lipid metabolism and reduce liver damage caused by lipid accumulation. The results of the detection of the activities of serum glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase in mice are as Figure 17 shown. The results showed that compound AH-262 can significantly reduce the increase in liver transaminases caused by MCDHFD (choline-deficient and methionine-reduced high-fat diet), and has a certain protective effect on the liver. The mRNA levels of liver inflammatory factors MCP-1, TNF-α, IL-1β, and IL-6 in mice are as Figure 18 shown. The results showed that the liver inflammation level in the mice in the compound AH-262 administration group was significantly lower than that in the solvent control group. The mRNA levels of liver fibrosis factors α-SMA, Col1α1, and TGF-β in mice are as Figure 19 shown. The results showed that the liver fibrosis level in the mice in the compound AH-262 administration group was significantly lower than that in the solvent control group.

[0142] Example 10

[0143] Construction of a metabolic fatty liver disease model induced by GAN (Gubra-Amylin NASH) diet and pharmacological effect study of compound AH-262

[0144] GAN (Gubra-Amylin NASH) diet-induced metabolic fatty liver disease model: Twelve C57BL / 6J mice (Shanghai SLAC Laboratory Animal Co., Ltd.) were purchased. After being normally fed for one week, they were randomly and evenly divided into two groups of 6 mice each according to body weight: the model group (fed with GAN diet and gavaged with normal saline daily after 10 weeks) and the administration group (fed with GAN diet and gavaged with the small molecule compound AH-262 (10 mg / kg) daily after 10 weeks). After 22 weeks of modeling, the mice were anesthetized by intraperitoneal injection of tribromoethanol. After taking blood from the orbital cavity, the abdomen was opened to take out the liver. The residual blood was washed away with precooled normal saline at 4°C, and the water was blotted dry on filter paper. Then it was immediately fixed in precooled 4% paraformaldehyde at 4°C. After 24 hours of fixation, it was embedded, sectioned, and subjected to H&E staining, Oil Red O staining, and Masson staining. A small part of the liver was reserved for RNA extraction and PCR analysis. RNA quantification and reverse transcription: RNA was quantified using NanoDrop, and its purity was evaluated using A260 / A280. Subsequently, the RNA was reverse-transcribed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit. The relative differences in the expression of target genes in different samples were determined by the 2-ΔΔCT semi-quantitative analysis method. The target genes included MCP-1, TNF-α, IL-1β, IL-6, α-SMA, Col-1α1, and TGF-β.

[0145] The blood taken from the mice was allowed to stand at room temperature for 30 minutes and then centrifuged at 6000g for 15 minutes. The supernatant (i.e., serum) was taken and detected using the AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity detection kits.

[0146] Pharmacodynamic study of compound AH-262 in the GAN (Gubra-Amylin NASH) diet-induced metabolic fatty liver disease model. Schematic diagrams of the morphological observation of mouse liver pathological tissues (H&E×200, Masson×200, Oil Red O×200) are as Figure 20 shown. The results showed that after 21 weeks of feeding with the GAN (Gubra-Amylin NASH) diet, the livers of the mice in the solvent control group showed obvious lipid accumulation, inflammatory infiltration, and fibrosis. However, the degrees of lipid accumulation, inflammatory infiltration, and fibrosis in the livers of the mice in the compound AH-262 administration group were significantly reduced, indicating that compound AH-262 can promote liver lipid metabolism and reduce liver damage caused by lipid accumulation. The results of the mouse body weight records are as Figure 21 shown. The results showed that compound AH-262 can increase lipid metabolism and reduce the body weight of the model mice. The results of the detection of the activities of serum aspartate aminotransferase and alanine aminotransferase in the mice are as Figure 22As shown, the results show that compound AH-262 can significantly reduce the elevation of liver transaminases caused by the GAN (Gubra-Amylin NASH) diet, and has a certain protective effect on the liver. The mRNA levels of the liver inflammatory factors MCP-1, TNF-α, IL-1β, and IL-6 in mice are as Figure 23 shown. The results show that the liver inflammation level of the mice in the compound AH-262 administration group is significantly lower than that of the solvent control group. The mRNA levels of the liver fibrosis factors α-SMA, Col1α1, and TGF-β in mice are as Figure 24 shown. The results show that the liver fibrosis level of the mice in the compound AH-262 administration group is significantly lower than that of the solvent control group.

[0147] Example 11

[0148] Preparation method of compound AH-262 and compounds Y-1 (Formula I), Y-2 (Formula II), and Y-3 (Formula III) with equivalent in vitro activity and pharmacodynamic effect:

[0149]

[0150] Step 1: Synthesis of 6-bromo-1-ethylindole-2,3-dione (I-6):

[0151] Dissolve 4521 mg (20 mmol) of 6-bromoindigo red and 8293 mg of anhydrous potassium carbonate (60 mmol) in 30 mL of DMF solution, add dropwise 1792 μL (24 mmol) of bromoethane, and stir overnight at room temperature. After the reaction is completed, dilute with 120 mL of water, extract three times with 150 mL of EA, wash with saturated brine, dry over anhydrous sodium sulfate, and obtain 4421 mg (17.4 mmol) of red solid by flash column chromatography on silica gel (PE:EA = 7:1) (yield 87%).

[0152]

[0153] Step 2: Synthesis of 6-bromo-1-ethyl-2,3-dihydro-1H-indol-2-one (I-7):

[0154] Dissolve 4421 mg (17.4 mmol) of 6-bromo-1-ethylindole-2,3-dione in 30 mL of 80% hydrazine hydrate solution, and react at 130 °C for 3 h. After the reaction is completed, cool the reaction solution to room temperature, quench the reaction with 30 mL of water, extract three times with 60 mL of EA, wash with saturated brine, dry over anhydrous sodium sulfate, and obtain 3635 mg (15.1 mmol) of white-green solid by flash column chromatography on silica gel (PE:EA = 7:1), (yield 87%).

[0155]

[0156] Step 3: Synthesis of 1-ethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-indol-2-one (I-8):

[0157] Dissolve 1781 mg (7.42 mmol) of 6-bromo-1-ethyl-2,3-dihydro-1H-indol-2-one, 2262 mg (8.9 mmol) of bis(pinacolato)diboron, 2185 mg of potassium acetate (22.6 mmol), and 543 mg (0.75 mmol) of palladium(II) dichloride bis(diphenylphosphinoferrocene) in 30 mL of 1,4-dioxane solution. Under nitrogen protection, add 1 mL of water and react at 80 °C for 3 h. After the reaction is completed, rotary evaporate the solvent. Dissolve the residue in 30 mL of water, filter through diatomaceous earth, extract the filtrate with 30 mL of EA three times, wash with saturated brine, dry over anhydrous sodium sulfate, and purify by flash column chromatography on silica gel (PE:EA = 20:1) to obtain 2046 mg of a white solid (7.12 mmol) (yield 96%).

[0158]

[0159] Step 4: Synthesis of 1-ethyl-6-(6-methoxypyridazin-3-yl)-2,3-dihydro-1H-indol-2-one (I-9):

[0160] Dissolve 2046 mg (7.12 mmol) of 1-ethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-indol-2-one, 1683 mg (8.9 mmol) of 3-bromo-6-methoxypyridazine, 3382 mg of cesium fluoride (22.3 mmol), and 858 mg (0.742 mmol) of tetrakis(triphenylphosphine)palladium(0) in 15 mL of 1,4-dioxane solution. Under nitrogen protection, add 5 mL of water and react at 90 °C overnight. After the reaction is completed, rotary evaporate the solvent. Dissolve the residue in 30 mL of water, filter through diatomaceous earth to remove insoluble Pd, then extract with 30 mL of EA three times, wash with saturated brine, dry over anhydrous sodium sulfate, and purify by flash column chromatography on silica gel (PE:EA = 1:1) to obtain 457 mg (1.7 mmol) (yield 24%).

[0161]

[0162] Step 5: Synthesis of 1-ethyl-6-(6-oxo-1H-pyridazin-3-yl)-2,3-dihydro-1H-indol-2-one (I-10):

[0163] Dissolve 457 mg (1.7 mmol) of 1-ethyl-6-(6-methoxy-1,2-diazepan-3-yl)-2,3-dihydro-1H-indol-2-one in 8 mL of 1,4-dioxane and 2N HCl (v / v, 1 / 1), stir at 80 °C for 4 h. After the reaction is completed, directly evaporate the solvent without extraction. Obtain 238 mg (0.93 mmol) of a light red solid (yield 55%) by silica gel column chromatography of flash chromatography (DCM:MeOH = 50:1).

[0164]

[0165] Step 6: Synthesis of 3-[(dimethylamino)methylidene]-1-ethyl-6-(6-oxo-1H-1,2-diazepan-3-yl)indol-2-one (I-11):

[0166] Dissolve 238 mg (0.93 mmol) of 1-ethyl-6-(6-oxo-1H-1,2-diazepan-3-yl)-2,3-dihydro-1H-indol-2-one in 5 mL of ethanol solution, add 190 μL (1.116 mmol) of N,N-dimethylformamide diethyl acetal dropwise at room temperature, and stir the reaction overnight at room temperature. After the reaction is completed, evaporate the ethanol and dilute with 20 mL of water. Extract the aqueous phase three times with 20 mL of DCM. Obtain 260 mg (0.84 mmol) of a yellowish-black solid (yield 90%) by silica gel column chromatography of flash chromatography (DCM:MeOH = 30:1).

[0167]

[0168] Step 7: Synthesis of 3-(ethylazanylmethylidene)-1-ethyl-6-(6-oxo-1H-1,2-diazepan-3-yl)indol-2-one (I-12):

[0169] Dissolve 260 mg (0.84 mmol) of 3-[(dimethylamino)methylidene]-1-ethyl-6-(6-oxo-1H-1,2-diazepan-3-yl)indol-2-one in 5 mL of isopropanol solution, add 68 μL (1 mmol) of 80% hydrazine hydrate dropwise at room temperature, and react at 80 °C for 1 h. After the reaction is completed, evaporate to dryness until a small amount of isopropanol remains and then triturate with methyl tert-butyl ether to obtain 230 mg (0.78 mmol) of a brownish-black solid (yield 92%).

[0170]

[0171] Step 8: Synthesis of 3-({2-[but-2-enylidene]ethylamino}methylene)-1-ethyl-6-(6-oxo-1H-1,2-diazepin-3-yl)indol-2-one (Y-1):

[0172] Dissolve 60 mg (0.2 mmol) of 3-(ethylaminomethylene)-1-ethyl-6-(6-oxo-1H-1,2-diazepin-3-yl)indol-2-one in 2 mL of isopropanol solution. At room temperature, add 22 μL (0.24 mmol) of 2-butanone and a drop of acetic acid dropwise, and react at 80 °C for 1 h. After the reaction, evaporate the solvent to dryness, dilute with 10 mL of water, extract three times with 10 mL of EA, wash with saturated brine, dry over anhydrous sodium sulfate, and perform silica gel column chromatography by flash chromatography (DCM:MeOH = 30:1) to obtain 56 mg (0.16 mmol) of a bright yellow solid (yield 80%).

[0173] 1 H NMR (300 MHz, DMSO) δ 13.08 (d, J = 2.1 Hz, 1H), 11.68 (dd, J =48.0, 10.5 Hz, 1H), 8.52 (dd, J = 10.4, 8.0 Hz, 1H), 8.11 (d, J = 9.9 Hz,1H), 7.64 (d, J = 7.9 Hz, 1H), 7.53 - 7.45 (m, 2H), 6.97 (dd, J = 9.9, 1.9 Hz,1H), 3.88 (p, J = 7.3 Hz, 2H), 2.35 (q, J = 7.4 Hz, 2H), 1.99 (d, J = 16.0Hz, 3H), 1.20 (t, J = 7.0 Hz, 3H), 1.15 - 1.04 (m, 3H).

[0174]

[0175] Step 9: Synthesis of 1-ethyl-6-(6-oxo-1H-1,2-diazepin-3-yl)-3-[[2-(3,4,5,6-tetrahydro-2H-pyran-4-ylidene)ethylamino]methylene]indol-2-one (Y-2):

[0176] Dissolve 60 mg (0.2 mmol) of 3-(ethylaminomethylene)-1-ethyl-6-(6-oxo-1H-1,2-diazepan-3-yl)indol-2-one in 2 mL of isopropanol solution. At room temperature, add dropwise 22 μL (0.24 mmol) of tetrahydropyran-4-one and a drop of acetic acid, and react at 80 °C for 1 h. After the reaction is completed, evaporate the solvent under reduced pressure, dilute with 10 mL of water and extract three times with 10 mL of EA. Wash with saturated brine, dry over anhydrous sodium sulfate, and obtain 65 mg (0.17 mmol) of a bright yellow solid (yield 86%) by silica gel column chromatography by flash chromatography (DCM:MeOH = 30:1).

[0177] 1 H NMR (300 MHz, DMSO) δ 13.09 (d, J = 1.9 Hz, 1H), 11.83 (d, J =10.4 Hz, 1H), 8.53 (d, J = 10.2 Hz, 1H), 8.10 (d, J = 10.0 Hz, 1H), 7.62 (d,J = 7.9 Hz, 1H), 7.49 (dd, J = 9.4, 1.6 Hz, 2H), 6.97 (dd, J = 10.0, 1.8 Hz,1H), 3.88 (q, J = 7.0 Hz, 2H), 3.78 (tt, J = 5.8, 3.1 Hz, 4H), 2.53 (s, 2H),2.44 (t, J = 5.6 Hz, 2H), 1.19 (t, J = 7.2 Hz, 3H).

[0178]

[0179] Step 10: Synthesis of 1-ethyl-6-(6-oxo-1H-1,2-diazepan-3-yl)-3-[[2-(tetrahydrothiopyran-4-ylidene)ethylamino]methylene]indol-2-one (Y-3):

[0180] Dissolve 60 mg (0.2 mmol) of 3-(ethylaminomethylene)-1-ethyl-6-(6-oxo-1H-1,2-diazepan-3-yl)indol-2-one and 28 mg (0.24 mmol) of tetrahydrothiopyran-4-one in 2 mL of isopropanol solution. At room temperature, add a drop of acetic acid, and react at 80 °C for 1 h. After the reaction is completed, evaporate the solvent under reduced pressure, dilute with 10 mL of water and extract three times with 10 mL of EA. Wash with saturated brine, dry over anhydrous sodium sulfate, and obtain 72 mg (0.18 mmol) of a bright yellow solid (yield 90%) by silica gel column chromatography by flash chromatography (DCM:MeOH = 30:1).

[0181] 1 1H NMR (400 MHz, DMSO) δ 13.12 - 13.06 (m, 1H), 11.83 (d, J = 10.3Hz, 1H), 8.52 (d, J = 10.1 Hz, 1H), 8.10 (d, J = 10.0 Hz, 1H), 7.62 (d, J =7.9 Hz, 1H), 7.53 - 7.46 (m, 2H), 6.98 (dd, J = 9.8, 1.8 Hz, 1H), 3.88 (q, J= 7.1 Hz, 2H), 2.85 (td, J = 8.2, 4.6 Hz, 4H), 2.72 (t, J = 5.8 Hz, 2H), 2.65(dd, J = 7.5, 4.5 Hz, 2H), 1.20 (t, J = 7.2 Hz, 3H).

[0182] Example 12

[0183] In vitro binding experiments of compounds Y-1, Y-2 and Y-3 to MFN2 protein

[0184] For compounds Y-1, Y-2 and Y-3 prepared in the present invention, the dissociation constants of these compounds with MFN2 protein were detected by a biomolecular interaction analyzer Biacore T200 (General Electric Company), and the specific detection process was as described in Example 2.

[0185] As Figures 25 - 30 shown, the binding force fitting curves of compounds Y-1, Y-2 and Y-3 with different concentrations to MFN2 protein were detected by SPR. The results confirmed that compounds Y-1, Y-2 and Y-3 could bind to MFN2 protein, and their dissociation constants were 2.548E-8 M, 1.949E-8 M and 1.918E-8 M, respectively.

[0186] Example 13

[0187] Effects of compounds Y-1, Y-2 and Y-3 on the GTPase activity of MFN2 protein

[0188] Using an ATPase / GTPase Assay kit (Abcam, ab272520), the effects of compounds Y-1, Y-2 and Y-3 on the GTPase activity of MFN2 protein were detected according to the instructions.

[0189] Results Figures 31 - 33As shown, the results confirmed that compounds Y-1, Y-2, and Y-3 could significantly increase the GTPase activity of MFN2 protein, with EC50 values of 24.30 uM, 23.49 uM, and 23.87 uM, respectively.

[0190] Example 14

[0191] Effects of Compounds Y-1, Y-2, and Y-3 on the Mitochondrial Morphology of Primary Mouse Hepatocytes

[0192] The specific experimental steps for extracting primary mouse hepatocytes were the same as those in Example 4. The isolated primary cells were cultured in an incubator at 37°C and 5% CO2 for 36 hours and then given drugs. The solvent control group was serum-free DMEM high-glucose medium containing 1‰ DMSO, and the drug administration groups were 25 uM Y-1, Y-2, and Y-3 prepared with serum-free DMEM high-glucose medium containing 1‰ DMSO. After administration, they were placed in an incubator at 37°C and 5% CO2 for 30 minutes. After the administration ended, Mitotraker deep red and Hoechst live cell dyes were used for staining, and after staining, they were placed under a confocal microscope for observation and photography.

[0193] The detection results of the effects of compounds Y-1, Y-2, and Y-3 on the mitochondrial morphology of primary mouse hepatocytes are as Figure 34 shown. Among them, the one labeled with solvent control is the solvent control group, that is, serum-free DMEM high-glucose medium containing 1‰ DMSO, and the ones labeled with Y-1, Y-2, and Y-3 are the drug administration groups, that is, 25 uM Y-1, Y-2, and Y-3 prepared with serum-free DMEM high-glucose medium containing 1‰ DMSO. The results showed that the mitochondrial length-width ratio in the drug administration groups increased significantly, and the degree of mitochondrial fusion was higher.

[0194] Example 15

[0195] Protective Effects of Compounds Y-1, Y-2, and Y-3 on APAP (Acetaminophen)-Induced Damage of Primary Mouse Hepatocytes

[0196] The steps for primary mouse liver extraction were the same as those in Example 4. The isolated primary cells were cultured in an incubator at 37°C and 5% CO2 for 36 hours and then given drugs. The solvent control group was serum-free high-glucose DMEM medium containing 1‰ DMSO, and the drug administration groups were 5 μM Y-1, Y-2, and Y-3 prepared with serum-free high-glucose DMEM medium containing 1‰ DMSO. The positive control group was 5 μM silybin prepared with serum-free high-glucose DMEM medium containing 1‰ DMSO. After pre-administering the drugs for 24 hours, at the end of the pre-administration, the medium was replaced with a 10 mM acetaminophen solution (prepared with serum-free high-glucose DMEM medium). After 24 hours, the CCK8 method was used to detect cell viability. The specific experimental method for detecting cell viability by the CCK8 method was as follows: First, prepare the CCK8 working solution by diluting the CCK8 stock solution 10-fold with serum-free high-glucose DMEM medium, and add 100 μl to each well of the 96-well plate after drug administration. After culturing in an incubator at 37°C and 5% CO2 for 2 hours, the absorbance values at wavelengths of 450 nm and 630 nm were read using an enzyme-linked immunosorbent assay (ELISA) reader. Finally, subtract the absorbance value at 630 nm from the absorbance value at 450 nm to obtain the actual absorbance value of the well. Taking the absorbance value of the control group as a reference, calculate the cell viability of each experimental group according to the formula "Cell viability (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%". Among them, the blank group was the wells that only added the medium and the CCK8 reagent without inoculating cells.

[0197] The results of the pharmacodynamic study on the protective effects of compounds Y-1, Y-2, and Y-3 against APAP (acetaminophen)-induced damage to primary mouse liver cells are as Figure 35 shown. Among them, the one marked with solvent control was the solvent control group, that is, serum-free high-glucose DMEM medium containing 1‰ DMSO. The ones marked with Y-1, Y-2, and Y-3 were the drug administration groups, that is, 5 μM Y-1, Y-2, and Y-3 prepared with serum-free high-glucose DMEM medium containing 1‰ DMSO. The one marked with Silibinin was the silybin (positive control) group. The results showed that the cell viability of the drug administration groups was significantly increased compared with the solvent control group, and the effect was comparable to that of the positive control group, indicating that compounds Y-1, Y-2, and Y-3 have a certain protective effect against APAP (acetaminophen)-induced damage to primary mouse liver cells.

Claims

1. An indolinone compound or a pharmaceutically acceptable salt thereof, characterized in that: The structural formula of the indole ketone compound is shown in any one of the structures of Formula I-III: 。 2. A method for preparing the indole ketone compound according to claim 1, characterized in that: The steps include: (1) Synthesis of 6-bromo-1-ethylindole-2,3-dione: Dissolve 6-bromoisatin and anhydrous potassium carbonate in an organic solvent, add ethyl bromide dropwise, stir overnight at room temperature, dilute with water after the reaction, extract, wash, dry, and purify to obtain the compound 6-bromo-1-ethylindole-2,3-dione; (2) Synthesis of 6-bromo-1-ethyl-2,3-dihydro-1H-indol-2-one: Dissolve 6-bromo-1-ethylindole-2,3-dione in a hydrazine hydrate solution, heat to react, cool the reaction solution to room temperature after the reaction is completed, add water to quench, extract, wash, dry, and purify to obtain a compound 6-bromo-1-ethyl-2,3-dihydro-1H-indole-2-one; (3) Synthesis of 1-ethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-indol-2-one: Dissolve 6-bromo-1-ethyl-2,3-dihydro-1H-indol-2-one, bipyraclostrobin, potassium acetate, and DPPF palladium dichloride in an organic solvent, add water under the protection of an inert gas, and heat to react. After the reaction is completed, spin-dry the solvent, add water to dissolve it, and filter it through diatomaceous earth. The filtrate is extracted, washed, dried, and purified to obtain a compound 1-ethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-indol-2-one; (4) Synthesis of 1-ethyl-6-(6-methoxy-1,2-diazacyclohexyl-3-yl)-2,3-dihydro-1H-indol-2-one: Dissolve 1-ethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-indol-2-one, 3-bromo-6-methoxypyridazine, cesium fluoride, and tetrakistriphenylphosphine palladium in an organic solvent, add water under the protection of an inert gas, and react overnight under heating. After the reaction, spin-dry the solvent, add water to dissolve it, filter it through diatomaceous earth, extract, wash, dry, and purify it to obtain the compound 1-ethyl-6-(6-methoxy-1,2-diazacyclohexyl-3-yl)-2,3-dihydro-1H-indol-2-one; (5) Synthesis of 1-ethyl-6-(6-oxydeoxy-1H-1,2-diazacyclohexyl-3-yl)-2,3-dihydro-1H-indol-2-one: Dissolve 1-ethyl-6-(6-methoxy-1,2-diazacyclohexyl-3-yl)-2,3-dihydro-1H-indol-2-one in an organic solvent, heat to react, add hydrochloric acid dropwise at room temperature, and spin-dry the solvent directly without extraction after the reaction, and purify to obtain the compound 1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexyl-3-yl)-2,3-dihydro-1H-indol-2-one; (6) Synthesis of 3-[(dimethylamino)methylidene]-1-ethyl-6-(6-oxydene-1H-1,2-diazacyclohexane-3-yl)indol-2-one: Dissolve 1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexane-3-yl)-2,3-dihydro-1H-indol-2-one in an organic solvent, add N,N-dimethylformamide diethyl acetal dropwise at room temperature, stir and react overnight at room temperature, spin-dry after the reaction, dilute with water, extract, and purify to obtain the compound 3-[(dimethylamino)methylidene]-1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexane-3-yl)indol-2-one; (7) Synthesis of 3-(ethylaminomethylidene)-1-ethyl-6-(6-oxydene-1H-1,2-diazacyclohexane-3-yl)indol-2-one: Dissolve 3-[(dimethylamino)methylidene]-1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexyl-3-yl)indol-2-one in an organic solvent, add hydrazine hydrate dropwise at room temperature, heat to react, and after the reaction is completed, perform rotary evaporation and slurrying to obtain the compound 3-(ethylidene)-1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexyl-3-yl)indol-2-one; (8) Synthesis of 3-[{2-[butan-2-ylidene]ethylamino}methylidene]-1-ethyl-6-(6-oxydeoxy-1H-1,2-diazacyclohexyl-3-yl)indol-2-one: 3-(Ethylaminomethylidene)-1-ethyl-6-(6-oxygenylidene-1H-1,2-diazacyclohexane-3-yl)indol-2-one is dissolved in an organic solvent, 2-butanone and acetic acid are added dropwise at room temperature, and the reaction is heated. After the reaction is completed, the solvent is spin-dried, diluted with water, extracted, washed, dried, and purified to obtain a compound of formula I, 3-[{2-[butan-2-ylidene]ethylamino}methylidene]-1-ethyl-6-(6-oxygenylidene-1H-1,2-diazacyclohexane-3-yl)indol-2-one; (9) Synthesis of 1-ethyl-6-(6-oxydeoxy-1H-1,2-diazacyclohexyl-3-yl)-3-[[2-(3,4,5,6-tetrahydro-2H-pyran-4-ylidene)ethylamino]methylidene]indol-2-one: Tetrahydropyrone and acetic acid are added dropwise to an organic solvent of 3-(ethylidene)-1-ethyl-6-(6-oxydeoxy-1H-1,2-diazacyclohexyl-3-yl)indol-2-one at room temperature, and the mixture is heated for reaction. After the reaction is completed, the solvent is dried by spin drying, and the mixture is diluted with water, extracted, washed, dried, and purified to obtain a compound of formula II, 1-ethyl-6-(6-oxydeoxy-1H-1,2-diazacyclohexyl-3-yl)-3-[[2-(3,4,5,6-tetrahydro-2H-pyran-4-ylidene)ethylidene]methylidene]indol-2-one; (10) Synthesis of 1-ethyl-6-(6-oxydeoxy-1H-1,2-diazacyclohexyl-3-yl)-3-[[2-(thiacyclohexyl-4-ylidene)ethylamino]methylidene]indol-2-one: 3-(Ethylaminomethylidene)-1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexane-3-yl)indole-2-one and tetrahydrothiopyran-4-one are dissolved in an organic solvent, acetic acid is added dropwise at room temperature, and the reaction is heated. After the reaction is completed, the solvent is spin-dried and diluted with water, extracted, washed, dried, and purified to obtain a compound of formula III, 1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexane-3-yl)-3-[[2-(thiacyclohexane-4-ylidene)ethylamino]methylidene]indole-2-one.

3. Use of an indolinone compound represented by any one of the structures of Formula I-IV or a pharmaceutically acceptable salt thereof in the preparation of an agonist for targeted activation of the biological activity of MFN2 protein: 。 4. Use of an indolinone compound represented by any one of the structures of Formulae I-IV or a pharmaceutically acceptable salt thereof in the preparation of a drug capable of promoting cell mitochondrial fusion and improving mitochondrial biological activity and energy metabolism: 。 5. The use according to claim 3 or 4, characterized in that: The compound represented by any one of the structures of Formulas I-IV or a pharmaceutically acceptable salt thereof specifically binds to the MFN2 protein, allosterically activates MFN2, promotes mitochondrial fusion, and improves liver cell activity.

6. Use of an indolinone compound represented by any one of the structures of Formula I-IV or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating acute or chronic liver disease: 。 7. The use according to claim 6, characterized in that: The acute and chronic liver diseases include drug-induced liver injury, septic liver injury, liver injury caused by liver resection or metabolic disorder fatty liver hepatitis.

8. Use of an indolinone compound represented by any one of the structures of Formula I-IV or a pharmaceutically acceptable salt thereof in the preparation of a drug for promoting liver tissue repair; 。 9. The use according to claim 8, characterized in that: The tissue repair is the repair of liver tissue after it is damaged by drugs, infection sources or trauma.

10. A pharmaceutical composition for treating acute and chronic liver diseases and promoting tissue repair, characterized in that: The invention comprises the indolinone compound or a pharmaceutically acceptable salt thereof as claimed in claim 1, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant or vehicle.

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