Indolone compound and application thereof in treating acute and chronic liver diseases and promoting tissue repair

Abstract

The invention discloses an indolone compound and application thereof in treatment of acute and chronic liver diseases and promotion of tissue repair, and belongs to the field of biological medicines, and the structural formula of the indolone compound is shown in the specification. The indolone compound provided by the invention is the first small molecule compound which is designed aiming at the structure of the MFN2 protein, takes the MFN2 protein as a target spot, resists liver injury and promotes liver repair, can effectively activate the biological activity of the MFN2 protein in a targeted manner after being specifically combined with the MFN2 protein, can be used as an agonist of the MFN2 protein, and has a good application prospect. Therefore, the compound can be used for preparing medicines for treating liver diseases, especially medicines for treating liver injury and metabolic related liver diseases. # imgabs0 # # imgabs1 # # imgabs2 #

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 is involved in regulating the morphology and function of mitochondria. MFN2 protein plays a key role in the mitochondrial fusion process, helping mitochondria to fuse with each other, maintaining the integrity of the mitochondrial network, and maintaining the health of cells. The main functions of MFN2 protein include: Mitochondrial fusion: MFN2 protein promotes fusion between mitochondria and maintains the stability and integrity of the mitochondrial network by interacting with other mitochondrial fusion proteins; Regulating mitochondrial morphology: MFN2 protein can affect the morphology and structure of mitochondria and regulate the size and shape of mitochondria; Regulating mitochondrial function: MFN2 protein is also closely related to the functional regulation of mitochondria, including cell energy metabolism, cell apoptosis, intracellular calcium ion balance, etc. Abnormal expression or mutation of MFN2 protein may be associated with the occurrence and development of a variety of diseases, such as neurodegenerative diseases, metabolic diseases, cardiovascular diseases, etc. Studies have shown that the loss or abnormal function of MFN2 protein may lead to pathological and physiological processes such as abnormal mitochondrial function, increased cell apoptosis, and disordered cell energy metabolism. Therefore, MFN2 protein is of great significance in the study of cell biology and disease mechanisms. The absence or dysfunction of MFN2 protein may be associated with metabolic diseases such as obesity, diabetes and non-alcoholic fatty liver disease. Currently, drugs developed for MFN2 protein targets are still in the preclinical research stage, and there are no reports of any compounds targeting MFN2 to prevent liver damage or promote liver repair. Summary of the invention

[0005] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention provides an indolinone compound, which can be used to treat acute and chronic liver diseases and promote tissue repair, solving the problem that there is no drug in the prior art that targets MFN2 to prevent liver damage and promote liver repair.

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

[0007] Technical solution: In order to achieve the above-mentioned purpose, the indolinone compound or a pharmaceutically acceptable salt thereof of the present invention is characterized in that the structural formula of the indolinone compound is as shown in any one of the structures of Formula I-III: .

[0008] The method for preparing the indole ketone compound of the present invention comprises the following steps: (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 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, add hydrochloric acid dropwise at room temperature, heat to react, and directly spin-dry the solvent 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 (I-11): 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-diazacyclohexyl-3-yl)indol-2-one (I-12): 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.

[0009] The 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: .

[0010] The invention discloses an indolinone compound represented by any one of the structures of Formula I-IV or a pharmaceutically acceptable salt thereof for use in the preparation of a drug capable of promoting cell mitochondrial fusion and improving mitochondrial biological activity and energy metabolism.

[0011] Furthermore, the compound represented by any one of the structures of Formulae I-IV or a pharmaceutically acceptable salt thereof specifically binds to the MFN2 protein, allosterically activates MFN2, promotes mitochondrial fusion, and improves liver cell viability.

[0012] Use of an indolinone compound represented by any one of the structures of Formulas I-IV of the present invention or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating acute and chronic liver diseases.

[0013] Among them, the acute and chronic liver diseases include drug-induced liver injury, septic liver injury, liver injury caused by liver resection or metabolic disorder fatty hepatitis.

[0014] The invention provides an application of an indolinone compound represented by any one of the structures of Formulas I-IV or a pharmaceutically acceptable salt thereof in the preparation of a drug for promoting liver tissue repair.

[0015] The tissue repair is the repair of liver tissue after it is damaged by drugs, infection sources or trauma.

[0016] The pharmaceutical composition for treating acute and chronic liver diseases and promoting tissue repair of the present invention comprises the indolinone compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, diluent, adjuvant or vehicle.

[0017] Among them, the pharmaceutical composition is preferably a capsule, powder, tablet, granule, pill, injection, syrup, oral solution, inhalant, ointment, suppository or patch.

[0018] The present invention designs and constructs an indolinone compound, which can specifically bind to the MFN2 protein, allosterically activate MFN2, promote mitochondrial fusion, and improve liver cell activity, thereby playing a role in treating acute and chronic liver diseases and promoting tissue repair.

[0019] The indolinone compounds designed and prepared by the present invention further verified that the compounds can bind to MFN2 protein through surface plasmon resonance (SPR) experiments. The effect of the compounds on the GTPase activity of MFN2 protein was further verified, and it was found that the compounds can increase the GTPase activity of MFN2 protein. In the in vitro biological activity evaluation experiment, the compounds can significantly increase the mitochondrial aspect ratio of mouse liver primary cells and promote mitochondrial fusion. In order to evaluate the in vivo biological activity of the compounds, APAP (acetaminophen)-induced drug-induced liver injury model, 70% liver resection-induced liver injury model, S. typhimurium-induced septic liver injury model, MCDHFD (choline-deficient-methionine-reduced high-fat diet)-induced metabolic fatty liver disease model and GAN (Gubra-Amylin NASH) diet-induced metabolic fatty liver disease were constructed. The compounds can significantly reduce liver cell death, alleviate liver damage, promote liver lipid metabolism and liver tissue repair and regeneration.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following advantages: 1. The indolinone compounds provided by the present invention are the first small molecule compounds designed based on the structure of MFN2 protein, which target MFN2 protein and can resist liver damage and promote liver repair. After the indolinone compounds specifically bind to the MFN2 protein, they can effectively target and activate the biological activity of the MFN2 protein, and can serve as agonists of the MFN2 protein, thereby being used to prepare drugs for treating liver diseases, especially for preparing drugs for treating liver damage and metabolic-related liver diseases.

[0021] 2. The present invention designs and prepares a small molecule compound with a completely new structure, which has the effect of promoting mitochondrial fusion of hepatocytes, improving hepatocyte vitality, and promoting hepatocyte proliferation, thereby repairing liver damage. It can be used to treat acute and chronic liver diseases and promote tissue repair.

[0022] 3. The present invention proposes a new synthesis route for indole ketone compounds. The compounds of the present invention are cleverly designed, simple in structure, and made of cheap and readily available raw materials. The synthesis process is safe and environmentally friendly and is easy to mass produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Fitting curve for the binding affinity between compound AH-262 at different concentrations and MFN2 protein detected by SPR; Figure 2 The response value curve of the binding of compound AH-262 with different concentrations to MFN2 protein detected by SPR; Figure 3 This is the dose-effect curve of the compound AH-262 on the GTPase activity of MFN2 protein; Figure 4 The test results of the effect of compound AH-262 on the mitochondrial morphology of mouse liver primary cells, wherein the solvent control group is marked as the solvent control group, i.e., serum-free DMEM high-glucose medium containing 1‰ DMSO, and the AH-262 group is marked as the drug administration group, i.e., 25uM AH-262 prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO; Figure 5 The results are for the efficacy study of compound AH-262 in the acetaminophen-induced hepatocellular injury model and the cell survival rate results; Figure 6 The pharmacodynamic study of compound AH-262 in the APAP (acetaminophen)-induced drug-induced liver injury model, schematic diagram of mouse liver pathological tissue morphology observation (H&E×200, scale bar 100μm); Figure 7 The results of the activity test of serum aspartate aminotransferase and alanine aminotransferase in mice were used to study the efficacy of compound AH-262 in the APAP (acetaminophen)-induced drug-induced liver injury model. Figure 8 The pharmacodynamics study of compound AH-262 in a liver injury model induced by 70% hepatectomy, and the statistical results of mouse liver weight ratio; Fig. 9 The results of the activity test of serum aspartate aminotransferase and alanine aminotransferase in mice are shown in the pharmacodynamic study of compound AH-262 in a liver injury model induced by 70% hepatectomy. Fig.10 Schematic diagram of Ki67 immunohistochemical staining observation of mouse liver for the efficacy study of compound AH-262 in a liver injury model induced by 70% hepatectomy (IHC×200, scale bar 100μm); Fig.11 The pharmacodynamic study of compound AH-262 in a liver injury model induced by 70% hepatectomy shows the statistical results of the percentage of Ki67 immunohistochemical staining positive cells in mouse liver; Fig.12The pharmacodynamic study of compound AH-262 in the septic liver injury model induced by S. typhimurium shows the pathological tissue morphology of mouse liver (H&E×200, scale bar 100μm); Fig.13 The results of the activity test of serum aspartate aminotransferase and alanine aminotransferase in mice are shown in the pharmacodynamic study of compound AH-262 in the septic liver injury model induced by S. typhimurium. Fig.14 This is a study on the efficacy of compound AH-262 in the septic liver injury model induced by S. typhimurium, and the results of the bacterial load detection in mouse liver tissue; Fig.15 The results of the efficacy study of compound AH-262 in the septic liver injury model induced by S. typhimurium and the statistical results of bacterial load in mouse liver tissue; Fig.16 The pharmacological effect study of compound AH-262 in the metabolic fatty liver disease model induced by MCDHFD (choline-deficient-methionine-reduced high-fat diet), schematic diagram of mouse liver pathological tissue morphology observation (H&E×200, Masson×200, Oil Red O×200, scale bar is 100μm); Fig.17 The results of the activity test of serum aspartate aminotransferase and alanine aminotransferase in mice were used to study the efficacy of compound AH-262 in the metabolic fatty liver disease model induced by MCDHFD (choline-deficient-methionine-reduced high-fat diet); Fig.18 To study the efficacy of compound AH-262 in the MCDHFD (choline-deficient-methionine-reduced high-fat diet)-induced metabolic fatty liver disease model, the mRNA levels of inflammatory factors MCP-1, TNF-ɑ, IL-1β, and IL-6 in mouse liver; Fig.19 To study the efficacy of compound AH-262 in the MCDHFD (choline-deficient-methionine-reduced high-fat diet)-induced metabolic fatty liver disease model, the mRNA levels of mouse liver fibrosis factors ɑ-SMA, Col1ɑ1, and TGF-β; Fig. 20 The pharmacodynamic study of compound AH-262 in the GAN (Gubra-Amylin NASH) diet-induced metabolic fatty liver disease model, schematic diagram of mouse liver pathological tissue morphology observation (H&E×200, Masson×200, Oil Red O×200, scale bar is 100μm); Fig.21This is a study on the efficacy of compound AH-262 in the GAN (Gubra-Amylin NASH) diet-induced metabolic fatty liver disease model. The results of mouse body weight were recorded. Fig. 22 The results of the activity test of serum aspartate aminotransferase and alanine aminotransferase in mice are shown in the pharmacodynamic study of compound AH-262 in the metabolic fatty liver disease model induced by GAN (Gubra-Amylin NASH) diet. Fig.23 To study the efficacy of compound AH-262 in the GAN (Gubra-Amylin NASH) diet-induced metabolic fatty liver disease model, the mRNA levels of inflammatory factors MCP-1, TNF-ɑ, IL-1β, and IL-6 in mouse liver; Fig.24 The pharmacodynamic study of compound AH-262 in the GAN (Gubra-Amylin NASH) diet-induced metabolic fatty liver disease model, the mRNA levels of mouse liver fibrosis factors ɑ-SMA, Col1ɑ1, and TGF-β; Fig.25 Fitting curve for the binding affinity between compound Y-1 at different concentrations and MFN2 protein detected by SPR; Fig.26 Fitting curve for the binding affinity between compound Y-2 and MFN2 protein at different concentrations detected by SPR; Fig. 27 Fitting curve for the binding affinity between compound Y-3 at different concentrations and MFN2 protein detected by SPR; Fig.28 It is the response value curve of the binding of compound Y-1 with different concentrations to MFN2 protein detected by SPR; Fig.29 It is the response value curve of the binding of compound Y-2 with different concentrations to MFN2 protein detected by SPR; Fig.30 The response value curve of the binding of compound Y-3 with different concentrations to MFN2 protein detected by SPR; Fig.31 This is the dose-effect curve of compound Y-1 on the GTPase activity of MFN2 protein; Fig.32 This is the dose-effect curve of compound Y-2 on the GTPase activity of MFN2 protein; Fig.33 This is the dose-effect curve of compound Y-3 on the GTPase activity of MFN2 protein; Fig.34The test results of the effect of compound AH-262 on the mitochondrial morphology of mouse liver primary cells, wherein the solvent control is the solvent control group, i.e., serum-free DMEM high-glucose medium containing 1‰ DMSO, and the Y-1, Y-2 and Y-3 are the drug administration groups, i.e., 25uM Y-1, Y-2 and Y-3 prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO; Fig.35 The results are for the efficacy study of compounds Y-1, Y-2 and Y-3 in the acetaminophen-induced hepatocellular injury model and cell survival rate results. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments.

[0025] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0026] ATPase / GTPase Assay kit was purchased from Abcam, ab272520.

[0027] EGTA / HBSS solution was purchased from ECOTOP, ED-8006-500ml.

[0028] Collagenase was purchased from Sigma, C1899.

[0029] APAP (acetaminophen) was purchased from Targetmol, T0065.

[0030] Silybin was purchased from Targetmol, T1660.

[0031] AST (aspartate aminotransferase) activity detection kit was purchased from Nanjing Jiancheng Bioengineering Institute, C010-2-1.

[0032] ALT (alanine aminotransferase) activity detection kit was purchased from Nanjing Jiancheng Bioengineering Institute, C009-2-1.

[0033] S. typhimurium was purchased from ATCC, strain number 14028.

[0034] MCDHFD (choline-deficient-methionine-reduced high-fat diet) was purchased from Research Diet, A06071309.

[0035] GAN (Gubra-Amylin NASH) was purchased from Research Diet, D09100310.

[0036] Example 1

[0037] The synthetic route of compound AH-262 of formula IV: Preparation of 1-ethylindole-2,3-dione (I-1): 2.9 g (20 mmol) 1H-indole-2,3-dione and 6.9 g (50 mmol) K2CO3 were dissolved in 30 mL DMF solution, 1.8 mL (24 mmol) ethyl bromide was added dropwise, and the mixture was stirred at 60 °C for 2 h. After the reaction was completed, 120 mL of water was added to quench the reaction and the mixture was extracted three times with 150 mL of ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by flash chromatography on a silica gel column (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).

[0038] Preparation of 1-ethylindolin-2-one (I-2): 3.3 g (18.6 mmol) of 1-ethylindole-2,3-dione was dissolved in 20 mL of 80% hydrazine hydrate solvent and stirred at 130 °C for 2 h. After the reaction, the reaction solution was cooled to room temperature and diluted with 20 mL of water. The aqueous phase was extracted three times with 40 mL of EA, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by flash chromatography on silica gel column (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).

[0039] Preparation of 3-(dimethylamino)methylidene-1-ethylindol-2-one (I-3): 2.7 g (16.8 mmol) of 1-ethylindolin-2-one was dissolved in 20 mL of ethanol solution, and 4.3 mL (25.2 mmol) of N,N-dimethylformamide diethyl acetal was added dropwise thereto. The mixture was stirred at room temperature for 3 h. After the reaction, the solvent was dried by spin drying, and diluted with 30 mL of water. The aqueous phase was extracted three times with 30 mL of EA, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by flash chromatography on a 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).

[0040] Preparation of 4-(3-((dimethylamino)methylene)-1-ethyl-2-oxoindoline-6-yl)-4-oxobutanoic acid (I-4): 3.5 g (16.2 mmol) of 3-(dimethylamino)methylidene-1-ethylindol-2-one, 1.9 g (19.4 mmol) of succinic anhydride, and 5.4 g (40.5 mmol) of anhydrous aluminum chloride were dissolved in 60 mL of 1,2-dichloroethane solution. The reaction solution was first stirred at 0 ° C for 1.5 h, and then stirred at 50 ° C for 1.5 h. After the reaction, the reaction solution was cooled to room temperature and poured into 60 mL of 17% hydrochloric acid solution to precipitate a large amount of white solid. The mixture was stirred for 30 min in an ice bath and vacuum filtered 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).

[0041] Preparation of 1-ethyl-3-(hydrazinomethylene)-6-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)indolin-2-one (I-5): 1.5 g (4.7 mmol) of 4-(3-((dimethylamino)methylene)-1-ethyl-2-oxoindolin-6-yl)-4-oxobutanoic acid was dissolved in 30 mL of isopropanol solution, heated to 90°C, 0.6 mL (9.4 mmol) of 80% hydrazine hydrate was added dropwise thereto, and stirred at 90°C for 3 h. After the reaction, the solvent was dried by spin drying, and diluted with 30 mL of water. The aqueous phase was extracted three times with 30 mL of DCM, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and purified by flash chromatography on silica gel column (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).

[0042] Preparation of 1-ethyl-3-((Z)-(2-((2E)-butan-2-ylidene)ethylamino)methylidene)-6-(6-oxydeoxy-4H,5H,6H,1H-1,2-diazacyclohexyl-3-yl)indol-2-one (AH-262): 0.8 g (4.7 mmol) of 1-ethyl-3-(hydrazinomethylene)-6-(6-oxo-1,4,5,6-tetrahydropyridazine-3-yl)indolin-2-one was dissolved in 10 mL of isopropanol solution, heated to 90°C, 0.48 mL (5.4 mmol) of 2-butanone and a drop of acetic acid were added dropwise, and stirred at 90°C for 3 h. After the reaction, the solvent was dried by spin drying, 10 mL of 70% methanol aqueous solution was added to dissolve, and the solution was separated and purified using a MorphlingTM WD-C18 preparative liquid chromatography column (chromatographic column specifications: MorphlingTM WD-C18, 10 μm, 30x250 mm), the mobile phase was 70% aqueous solution, the flow rate was 20 mL / min, and the component with a retention time of 27.5 min was collected. After separation, the collected solution was concentrated by rotary evaporator, diluted with 20 mL of water, and the aqueous phase was extracted three times with 20 mL of DCM. After the DCM was dried by rotary evaporator, it was vacuum dried to obtain 115 mg of compound AH-262 (yield 44%). 1H NMR (600MHz, 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).

[0043] Example 2 In vitro binding experiment of compound AH-262 and MFN2 protein For the compound AH-262 prepared by the present invention, the dissociation constant between the compound and the MFN2 protein was detected using a biomolecular interaction instrument Biacore T200 (General Electric Company). The specific detection process is as follows: 1. Experimental preparation: Prepare the Biacore T200 biomolecular interaction instrument and the matching buffer, chip (CM5 chip is used for amino coupling fixed protein), syringe, sample tube and other consumables. 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 determine the protein concentration and dilute it to 1 mg / ml with buffer for fixation; the small molecule compound AH-262 is also dissolved with buffer and prepared into a series of solutions with different concentrations (gradient concentrations from 1 nM to 10 μM) for subsequent binding experiments. 2. Chip pretreatment: Install the CM5 chip on the Biacore T200 instrument, and use activation reagents (N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)) to activate the carboxyl groups on the chip surface for 15 minutes to make the chip surface active for binding with proteins. 3. Protein fixation: Inject the diluted protein solution onto the chip surface at a flow rate of 5μL / min for 30 minutes. The protein reacts with the activated carboxyl groups through the amino groups and covalently binds to the chip surface. After fixation, use ethanolamine to block the unreacted active groups to terminate the fixation reaction. 4. Baseline stability: Inject buffer to balance the chip surface until a stable baseline is detected to ensure that the detection environment is stable and there is no interference signal. 5. Binding determination: Inject small molecule compound solutions of different concentrations in sequence, set the flow rate to 20μL / min, and the injection time is 5 minutes. Record the signal changes generated during the binding process of small molecules and proteins fixed on the chip, that is, the changes in the resonance unit (RU) value, reflecting the binding of the two. 6. Dissociation assay: After the binding reaction is completed, inject buffer to rinse the chip surface, monitor the change of RU value over time during the dissociation of small molecules 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, deduct the signal of the blank control (only buffer injection), correct the baseline drift, etc. By fitting the binding and dissociation curves, the binding rate constant (ka) and dissociation rate constant (kd) are obtained, and then the equilibrium dissociation constant (KD = kd / ka) is calculated. The smaller the KD value, the stronger the binding force between the small molecule compound and the protein.

[0044] like Figure 1 , Figure 2As shown, the binding force fitting curves of compound AH-262 with different concentrations and MFN2 protein were detected by SPR. The results confirmed that compound AH-262 could bind to MFN2 protein with a dissociation constant of 4.753E-8M.

[0045] Example 3

[0046] Effect of compound AH-262 on the GTPase activity of MFN2 protein The ATPase / GTPase Assay kit (Abcam, ab272520) was used to detect the effect of compound AH-262 on the GTPase activity of MFN2 protein according to the instructions.

[0047] result Figure 3 As shown, the results confirmed that compound AH-262 can significantly increase the GTPase activity of MFN2 protein, with an EC50 value of 23.33uM. The stronger the GTPase activity of MFN2 protein, the stronger its activity in mediating mitochondrial fusion, further proving that compound AH-262 can bind to MFN2 protein.

[0048] Example 4

[0049] Effects of compound AH-262 on mitochondrial morphology of mouse primary liver cells Extraction of primary mouse liver cells: Preheat EGTA / HBSS and collagenase solution at 40℃ for 30min. Wash the pump hose with 75% ethanol, then insert the hose into the EGTA / HBSS solution to remove bubbles. 6-week-old male C57BL / 6J mice were intraperitoneally injected with 2.5% tribromoethanol at a dose of 10ml / kg until fully anesthetized. Cut the abdominal hair and disinfect with 75% ethanol. Fix, open the abdominal cavity, put the intestines to the right, and expose the portal vein and inferior vena cava. Use cotton swabs and cotton to separate the inferior vena cava, bury the suture needle, carefully support the blood vessel with the forceps handle, and slowly insert the indwelling needle into the blood vessel at the same time, about 1.5cm to 2.5cm, and then tie it with the pre-buried thread. At the same time, it was found that the liver blood vessels flowed back into the indwelling needle. Open the chest cavity, ligate the aorta with an artery clamp, cut the portal vein, and perfuse, adjusting the flow rate to 7ml / min, 40ml / mouse EGTA / HBSS solution. When it is less than 5 minutes, add 40ml collagenase solution, adjust the flow rate to 10ml / min, and digest for about 2 minutes. Press a few more times with tweezers to fully digest the cells. Put the collagenase and digested liver into a dish, remove the gallbladder, add an appropriate amount of EGTA / HBSS solution (15mL / liver), quickly wash once and tear it apart. Pass a digested liver through a 100-mesh sieve (15ml per sieve), centrifuge at 4℃50g for 5min. Discard the supernatant, take 5mL PM solution to resuspend and blow evenly, add 5mL Percoll, centrifuge at 4℃120g for 5min, aspirate the upper cell suspension, resuspend with 2mL PM solution, centrifuge at 4℃50g for 5min, discard the supernatant, resuspend with 2mL PM solution, count and plate.

[0050] The isolated primary cells were incubated at 37°C with 5% CO 2 Drugs were administered after 36 hours of incubation. The solvent control group was serum-free DMEM high-glucose medium containing 1‰ DMSO, and the drug administration group was 25uM AH-262 prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO. After administration, the cells were placed at 37°C and 5% CO 2 Incubate in the chamber for 30 minutes. After administration, stain with Mitotraker deepred and Hoechst live cell dyes. After staining, observe and photograph under a confocal microscope.

[0051] The results of the test on the effect of compound AH-262 on the mitochondrial morphology of mouse liver primary cells are as follows Figure 4As shown, the solvent control group is marked as the solvent control group, i.e., a serum-free DMEM high-glucose medium containing 1‰ DMSO, and the AH-262 group is marked as the drug group, i.e., 25uM AH-262 configured in a serum-free DMEM high-glucose medium containing 1‰ DMSO. The results show that the mitochondrial aspect ratio of the drug group increased significantly, and the degree of mitochondrial fusion was higher. The compounds of the present invention can increase the degree of mitochondrial fusion of cells, maintain the morphology and structural integrity of mitochondria, promote mitochondrial function by optimizing energy metabolism, etc., participate in intracellular material transport and signal transduction, and ensure the stability of mitochondrial DNA and achieve its complementation and recombination.

[0052] Example 5 Protective effect of compound AH-262 on APAP (acetaminophen)-induced injury in mouse primary liver cells The mouse liver primary cell extraction procedure was the same as in Example 4. The isolated primary cells were incubated at 37°C, 5% CO 2 Drugs were administered after 36 hours of incubation. The solvent control group was serum-free DMEM high-glucose medium containing 1‰ DMSO, the drug administration group was 5uM AH-262 prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO, and the positive control group was 5uM silybin prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO. Pre-administration was performed for 24 hours. After the pre-administration, the culture medium was replaced with 10mM acetaminophen solution (prepared in serum-free DMEM high-glucose medium). After 24 hours, the cell viability was detected using the CCK8 method.

[0053] The results of the pharmacodynamic study on the protective effect of compound AH-262 on APAP (acetaminophen)-induced injury in mouse primary liver cells are as follows: Figure 5 As shown, the solvent control group is marked with solvent control, i.e., serum-free DMEM high-glucose medium containing 1‰ DMSO; the drug group is marked with AH-262, i.e., 5uAH-262 prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO; the Silibinin group is marked with Silybinin (positive control) group. The results showed that the cell viability of the drug group was significantly increased compared with the solvent control group, and the effect was equivalent to that of the positive control group, indicating that compound AH-262 has a certain protective effect on APAP (acetaminophen)-induced mouse primary liver cell damage.

[0054] Example 6 Construction of APAP (acetaminophen)-induced drug-induced liver injury model and study of the efficacy of compound AH-262 APAP (acetaminophen)-induced drug-induced liver injury model: 12 C57BL / 6J mice were purchased (Shanghai Slake Co., Ltd.). After one week of normal feeding, they were randomly divided into two groups according to body weight, with 6 mice in each group: model group (administered with normal saline for 3 days and then injected with acetaminophen (300 mg / kg) intraperitoneally) and drug group (administered with small molecule compound AH-262 (10 mg / kg) for 3 days and then injected with acetaminophen (300 mg / kg) intraperitoneally). 24 hours after intraperitoneal injection of acetaminophen, mice were anesthetized by intraperitoneal injection of tribromoethanol, and the peritoneal cavity was opened after orbital blood collection to remove the liver for further analysis. After removing the liver, the residual blood was washed with 4°C precooled normal saline, the water was absorbed on filter paper, and it was immediately fixed in 4% paraformaldehyde precooled at 4°C. After fixation for 24 hours, the mice were embedded, sliced, and stained with H&E. The mouse blood was taken out and allowed to stand at room temperature for 30 minutes, then centrifuged at 6000g for 15 minutes. The supernatant (i.e., serum) was taken and tested using AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity detection kits.

[0055] Pharmacodynamic study of compound AH-262 in APAP (acetaminophen)-induced drug-induced liver injury model, schematic diagram of mouse liver pathological tissue morphology observation (H&E×200) Figure 6 As shown, the results showed that the mice in the solvent control group showed obvious liver damage and necrosis 24 hours after APAP (acetaminophen) injection, while the mice in the compound AH-262 administration group did not show obvious liver damage and necrosis, indicating that compound AH-262 has a certain protective effect on APAP (acetaminophen)-induced drug-induced liver injury.

[0056] The results of the mouse serum AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity test are shown in the figure. Figure 7 As shown, the results showed that the serum AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activities of mice in the solvent control group were significantly increased 24 hours after APAP (acetaminophen) injection, indicating that a large number of liver cells died and ruptured, while the serum AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activities 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 APAP (acetaminophen)-induced drug-induced liver injury.

[0057] Example 7 Construction of a 70% hepatectomy-induced liver injury model and study of the efficacy of compound AH-262 Liver injury model caused by 70% hepatectomy: 60 C57BL / 6J mice (Shanghai Slake Co., Ltd.) were purchased and fed normally for one week. They were randomly divided into two groups according to their body weight, with 30 mice in each group: model group (70% hepatectomy was performed 3 days after intragastric administration of normal saline) and drug group (70% hepatectomy was performed 3 days after intragastric administration of small molecule compound AH-262 (10 mg / kg). 2 / 3 hepatectomy: After intraperitoneal injection of tribromoethanol to anesthetize the mice, the abdomen was opened at the midline, and 2 / 3 of the mouse liver lobe (left lateral lobe and middle lobe) was removed by ligating the corresponding branch vessels, retaining the right upper, right lower and caudate lobes, and the abdomen was closed for observation. 48 hours after hepatectomy, the mice were anesthetized by intraperitoneal injection of tribromoethanol, and the abdominal cavity was opened to remove the liver after orbital blood collection for further analysis. The liver was removed and weighed immediately, and then the residual blood was washed with 4°C pre-cooled physiological saline, the water was absorbed on filter paper, and it was immediately placed in 4% paraformaldehyde pre-cooled at 4°C for fixation. After fixation for 24 hours, it was embedded, sliced, and subjected to H&E staining and immunohistochemical staining. The mouse blood was removed and allowed to stand at room temperature for 30 minutes, then centrifuged at 6000g for 15 minutes, and the supernatant (i.e. serum) was taken and tested using AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity detection kits.

[0058] The efficacy of compound AH-262 in a liver injury model induced by 70% hepatectomy was studied. The liver weight / body weight ratio of mice 48 hours after 70% hepatectomy was as follows: Figure 8 As shown in the figure, 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. The results of the detection of serum AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activities in mice are shown in the figure. Fig. 9 As shown in the figure, the results showed that the serum AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activities of mice in the solvent control group increased significantly 48 hours after 70% hepatectomy, indicating that a large number of liver cells died and ruptured, while the serum AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activities 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 damage induced by 70% hepatectomy. Schematic diagram of Ki67 immunohistochemical staining observation of mouse liver (IHC×200) Fig.10 The results show that compound AH-262 can significantly increase the proliferation of hepatocytes after 70% hepatectomy and promote liver tissue regeneration. The statistical results of Ki67 immunohistochemical staining positive cells in mouse liver are shown in Fig.11 As shown, the results showed that compound AH-262 could significantly increase hepatocyte proliferation after 70% hepatectomy and promote liver tissue regeneration.

[0059] Example 8 Construction of a septic liver injury model induced by S. typhimurium and study of the efficacy of compound AH-262 Septic liver injury model induced by S. typhimurium: Twelve C57BL / 6J mice were purchased (Shanghai Slake Co., Ltd.). After one week of normal feeding, they were randomly divided into two groups according to their body weight, with 6 mice in each group: model group (administered normal saline by gavage for 3 days and then injected with S. typhimurium (2×10 6 FU / 20g)) and the drug-treated group (administered intragastrically with the small molecule compound AH-262 (10 mg / kg) 3 days later with intraperitoneal injection of S. typhimurium (2×10 6 CFU / 20g). 24 hours after the tail vein injection of S.typhimurium, the mice were anesthetized by intraperitoneal injection of tribromoethanol. After orbital blood collection, the abdominal cavity was opened to remove the liver for further analysis. After orbital blood collection, the abdominal cavity was opened to remove the liver, 1 mg of fresh liver tissue was accurately weighed, 1 ml of normal saline was added, and homogenized. The homogenate was diluted 10 times and spread on the LB plate. After incubation in a 37°C incubator for 24 hours, it was taken out, photographed and the number of colonies was counted. The bacterial load of mouse liver tissue = Log (10000×plate colony number). The remaining liver was washed with 4°C pre-cooled normal saline to remove residual blood, the water was blotted on filter paper, and it was immediately placed in 4°C pre-cooled 4% paraformaldehyde for fixation. After fixation for 24 hours, it was embedded, sliced, and H&E stained. The mouse blood was taken out and allowed to stand at room temperature for 30 minutes, then centrifuged at 6000g for 15 minutes, and the supernatant (i.e. serum) was taken and tested using AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity detection kits.

[0060] Schematic diagram of the pharmacological efficacy of compound AH-262 in the septic liver injury model induced by S. typhimurium. Schematic diagram of the pathological tissue morphology of mouse liver (H&E×200) Fig.12 As shown in the results, the mice in the solvent control group showed obvious inflammatory infiltration and tissue necrosis in the liver 24 hours after the tail vein injection of S. typhimurium, while the mice in the compound AH-262 administration group did not show obvious damage and necrosis in the liver, indicating that compound AH-262 has a certain protective effect on septic liver injury induced by S. typhimurium. The activity test results of mouse serum aspartate aminotransferase and alanine aminotransferase are shown in Fig.13 The results show that compound AH-262 can significantly reduce the increase of liver transaminase caused by S. typhimurium infection and has a certain protective effect on the liver. Fig.14As shown, the results show that compound AH-262 can significantly enhance the bacterial clearance rate in the liver. The statistical results of bacterial load in mouse liver tissue are as follows: Fig.15 As shown, the results showed that compound AH-262 could significantly enhance the bacterial clearance rate in the liver.

[0061] Example 9 Construction of MCDHFD (choline-deficient-methionine-reduced high-fat diet)-induced metabolic fatty liver disease model and efficacy study of compound AH-262 MCDHFD (choline-deficient-methionine-reduced high-fat diet)-induced metabolic fatty liver disease model: 12 C57BL / 6J mice (Shanghai Slake Co., Ltd.) were purchased and fed normally for one week. They were randomly divided into two groups according to body weight, with 6 mice in each group: model group (administered MCDHFD diet and gavaged with normal saline every day) and drug group (administered MCDHFD diet and gavaged with small molecule compound AH-262 (10 mg / kg) every day). After 6 weeks, mice were anesthetized by intraperitoneal injection of tribromoethanol, and the peritoneal cavity was opened to remove the liver after orbital blood collection. The residual blood was washed with 4°C precooled normal saline, and the water was blotted on filter paper. The liver was immediately fixed in 4% paraformaldehyde precooled at 4°C. After fixation for 24 hours, the mice were embedded, sliced, and stained with H&E, Oil Red O, and Masson. 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. RNA was then reverse transcribed using the HiScript III RT SuperMix forqPCR (+gDNA wiper) kit. The 2-ΔΔCT semi-quantitative analysis method was used to determine the relative differences in the expression of target genes in different samples, including MCP-1, TNF-ɑ, IL-1β, IL-6, ɑ-SMA, Col-1ɑ1, and TGF-β. The mouse blood was taken out and allowed to stand at room temperature for 30 minutes, then centrifuged at 6000g for 15 minutes, and the supernatant (i.e., serum) was taken and tested using AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity detection kits.

[0062] Pharmacological efficacy study of compound AH-262 in the MCDHFD (choline-deficient-methionine-reduced high-fat diet)-induced metabolic fatty liver disease model, schematic diagram of mouse liver pathological tissue morphology observation (H&E×200, Masson×200, Oil Red O×200) Fig.16The results showed that after 6 weeks of feeding MCDHFD, the mice in the solvent control group had obvious lipid accumulation, inflammatory infiltration and fibrosis in the liver, while the lipid accumulation, inflammatory infiltration and fibrosis in the liver of the mice in the AH-262 administration group were significantly reduced, indicating that the compound AH-262 can promote liver lipid metabolism and reduce liver damage caused by lipid accumulation. The activity test results of serum aspartate aminotransferase and alanine aminotransferase in mice are shown in Figure 2. Fig.17 As shown in the figure, the compound AH-262 can significantly reduce the increase of liver transaminase caused by MCDHFD (choline-deficient-methionine-reduced high-fat diet), and has a certain protective effect on the liver. The mRNA levels of inflammatory factors MCP-1, TNF-ɑ, IL-1β, and IL-6 in mouse liver are shown in the figure. Fig.18 The results showed that the level of liver inflammation in mice in the AH-262-treated group was significantly lower than that in the solvent control group. The mRNA levels of fibrosis factors ɑ-SMA, Col1ɑ1, and TGF-β in the mice liver were shown in Figure 2. Fig.19 As shown, the results showed that the liver fibrosis level of mice in the compound AH-262 administration group was significantly lower than that in the solvent control group.

[0063] Example 10 Construction of GAN (Gubra-Amylin NASH) diet-induced metabolic fatty liver disease model and pharmacodynamic study of compound AH-262 GAN (Gubra-Amylin NASH) diet-induced metabolic fatty liver disease model: 12 C57BL / 6J mice (Shanghai Slake Co., Ltd.) were purchased and fed normally for one week. They were randomly divided into two groups according to body weight, with 6 mice in each group: model group (administered GAN diet and gavaged with normal saline every day after 10 weeks) and drug group (administered GAN diet and gavaged with small molecule compound AH-262 (10 mg / kg) every day after 10 weeks). After 22 weeks of modeling, mice were anesthetized by intraperitoneal injection of tribromoethanol. After orbital blood collection, the abdominal cavity was opened to remove the liver, and the residual blood was washed with 4°C pre-cooled normal saline. The water was absorbed on filter paper and immediately placed in 4% paraformaldehyde pre-cooled at 4°C for fixation. After fixation for 24 hours, the mice were embedded, sliced, and stained with H&E, Oil Red O, and Masson. 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. RNA was then reverse transcribed using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit. The 2-ΔΔCT semi-quantitative analysis method was used to determine the relative differences in the expression of target genes in different samples, including MCP-1, TNF-ɑ, IL-1β, IL-6, ɑ-SMA, Col-1ɑ1, and TGF-β.

[0064] The mouse blood was taken out and allowed to stand at room temperature for 30 minutes, then centrifuged at 6000g for 15 minutes. The supernatant (i.e., serum) was taken and tested using AST (aspartate aminotransferase) and ALT (alanine aminotransferase) activity detection kits.

[0065] Pharmacological study of compound AH-262 in the metabolic fatty liver disease model induced by GAN (Gubra-Amylin NASH) diet, schematic diagram of mouse liver pathological tissue morphology observation (H&E×200, Masson×200, Oil Red O×200) Fig. 20 As shown in the results, the mice in the solvent control group had obvious lipid accumulation, inflammatory infiltration and fibrosis in the liver after being fed with GAN (Gubra-Amylin NASH) feed for 21 weeks, while the lipid accumulation, inflammatory infiltration and fibrosis in the liver of the mice in the AH-262 administration group were significantly reduced, indicating that the compound AH-262 can promote liver lipid metabolism and reduce liver damage caused by lipid accumulation. The results of mouse weight recording are shown in the figure. Fig.21 The results show that compound AH-262 can increase lipid metabolism and reduce the body weight of model mice. The results of the activity test of serum aspartate aminotransferase and alanine aminotransferase in mice are shown in Fig. 22As shown in the figure, the results showed that compound AH-262 could significantly reduce the increase of liver transaminase caused by GAN (Gubra-Amylin NASH) diet, and had a certain protective effect on the liver. The mRNA levels of inflammatory factors MCP-1, TNF-ɑ, IL-1β, and IL-6 in mouse liver were shown in the figure. Fig.23 The results showed that the level of liver inflammation in mice in the AH-262-treated group was significantly lower than that in the solvent control group. The mRNA levels of fibrosis factors ɑ-SMA, Col1ɑ1, and TGF-β in the mice liver were shown in Figure 2. Fig.24 As shown, the results showed that the liver fibrosis level of mice in the compound AH-262 administration group was significantly lower than that in the solvent control group.

[0066] Embodiment 11 Preparation methods of compounds Y-1 (Formula I), Y-2 (Formula II) and Y-3 (Formula III) having in vitro activity and efficacy equivalent to compound AH-262:

[0067] Step 1: Synthesis of 6-bromo-1-ethylindole-2,3-dione (I-6): Dissolve 4521 mg (20 mmol) of 6-bromoindigo carmine and 8293 mg of anhydrous potassium carbonate (60 mmol) in 30 mL of DMF solution, add 1792 μL (24 mmol) of ethyl bromide, and stir overnight at room temperature. After the reaction is completed, dilute with 120 mL of water, extract with 150 mL of EA three times, wash with saturated brine, dry with anhydrous sodium sulfate, and flash chromatography on a silica gel column (PE:EA=7:1) to obtain 4421 mg (17.4 mmol) of a red solid (yield 87%).

[0068]

[0069] Step 2: Synthesis of 6-bromo-1-ethyl-2,3-dihydro-1H-indol-2-one (I-7): 4421 mg (17.4 mmol) of 6-bromo-1-ethylindole-2,3-dione was dissolved in 30 mL of 80% hydrazine hydrate solution and reacted at 130 ° C for 3 h. After the reaction, the reaction solution was cooled to room temperature, 30 mL of water was added to quench the reaction, and then extracted three times with 60 mL of EA, washed with saturated brine, dried over anhydrous sodium sulfate, and flash chromatography on a silica gel column (PE: EA = 7: 1) to obtain 3635 mg (15.1 mmol) of a white greenish solid (yield 87%).

[0070]

[0071] 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): 1781mg (7.42mmol) 6-bromo-1-ethyl-2,3-dihydro-1H-indol-2-one, 2262mg (8.9mmol) biboric acid pinacol ester, 2185mg potassium acetate (22.6mmol), 543mg (0.75mmol) DPPF palladium dichloride were dissolved in 30mL 1,4-dioxane solution, 1mL water was added under nitrogen protection, and the reaction was carried out at 80°C for 3h. After the reaction, the solvent was dried by spin drying, 30mL water was added to dissolve, and then filtered through diatomaceous earth, the filtrate was extracted three times with 30mL EA, washed with saturated brine, dried over anhydrous sodium sulfate, and flash chromatography silica gel column chromatography (PE: EA = 20:1) was performed to obtain 2046mg white solid (7.12mmol) (yield 96%).

[0072] Step 4: Synthesis of 1-ethyl-6-(6-methoxy-1,2-diazacyclohexan-3-yl)-2,3-dihydro-1H-indol-2-one (I-9): 2046mg (7.12mmol) 1-ethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-indol-2-one, 1683mg (8.9mmol) 3-bromo-6-methoxypyridazine, 3382mg cesium fluoride (22.3mmol), 858mg (0.742mmol) tetrakistriphenylphosphine palladium were dissolved in 15mL 1,4-dioxane solution, 5mL water was added under nitrogen protection, and the reaction was carried out at 90°C overnight. After the reaction, the solvent was dried by spin drying, 30mL water was added to dissolve, and the insoluble Pd was removed by diatomaceous earth filtration, and then extracted three times with 30mL EA, washed with saturated brine, dried over anhydrous sodium sulfate, and flash chromatography silica gel column chromatography (PE:EA=1:1) was performed to obtain 457mg (1.7mmol) (yield 24%).

[0073] Step 5: Synthesis of 1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexyl-3-yl)-2,3-dihydro-1H-indol-2-one (I-10): 457 mg (1.7 mmol) of 1-ethyl-6-(6-methoxy-1,2-diazacyclohexane-3-yl)-2,3-dihydro-1H-indol-2-one was dissolved in 8 mL of 1,4-dioxane and 2N HCl (v / v, 1 / 1), and stirred at 80°C for 4 h. After the reaction was completed, the solvent was directly dried without extraction, and flash chromatography on a silica gel column (DCM:MeOH=50:1) gave 238 mg (0.93 mmol) of a light red solid (yield 55%).

[0074] Step 6: Synthesis of 3-[(dimethylamino)methylidene]-1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexyl-3-yl)indol-2-one (I-11): 238mg (0.93mmol) 1-ethyl-6-(6-oxy-1H-1,2-diazacyclohexyl-3-yl)-2,3-dihydro-1H-indol-2-one was dissolved in 5mL ethanol solution, 190μL (1.116mmol) N,N-dimethylformamide diethyl acetal was added dropwise at room temperature, and the reaction was stirred overnight at room temperature. After the reaction, the ethanol was dried and diluted with 20mL water, and the aqueous phase was extracted three times with 20mL DCM, and flash chromatography on silica gel column (DCM: MeOH = 30:1) was performed to obtain 260mg (0.84mmol) of yellow-black solid (yield 90%).

[0075] Step 7: Synthesis of 3-(ethylidene)-1-ethyl-6-(6-oxydene-1H-1,2-diazacyclohexyl-3-yl)indol-2-one (I-12): 260 mg (0.84 mmol) of 3-[(dimethylamino)methylidene]-1-ethyl-6-(6-oxyidene-1H-1,2-diazacyclohexane-3-yl)indol-2-one was dissolved in 5 mL of isopropanol solution, and 68 μL (1 mmol) of 80% hydrazine hydrate was added dropwise at room temperature, and the mixture was reacted at 80°C for 1 h. After the reaction was completed, the mixture was evaporated to leave a small amount of isopropanol, and then slurried with methyl tert-butyl ether to obtain 230 mg (0.78 mmol) of brown-black solid (yield 92%).

[0076] Step 8: Synthesis of 3-[{2-[butan-2-ylidene]ethylamino}methylidene]-1-ethyl-6-(6-oxoylidene-1H-1,2-diazacyclohexan-3-yl)indol-2-one (Y-1): 60 mg (0.2 mmol) of 3-(ethylaminomethylidene)-1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexyl-3-yl)indol-2-one was dissolved in 2 mL of isopropanol solution, 22 μL (0.24 mmol) of 2-butanone and one drop of acetic acid were added dropwise at room temperature, and the mixture was reacted at 80°C for 1 h. After the reaction, the solvent was dried by spin drying, diluted with 10 mL of water, extracted three times with 10 mL of EA, washed with saturated brine, dried over anhydrous sodium sulfate, and flash chromatographed on a silica gel column (DCM: MeOH = 30:1) to obtain 56 mg (0.16 mmol) of a bright yellow solid (yield 80%).

[0077] 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).

[0078] Step 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 (Y-2): 60 mg (0.2 mmol) of 3-(ethylaminomethylidene)-1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexyl-3-yl)indol-2-one was dissolved in 2 mL of isopropanol solution, 22 μL (0.24 mmol) of tetrahydropyrone and one drop of acetic acid were added dropwise at room temperature, and the reaction was carried out at 80°C for 1 h. After the reaction, the solvent was dried by spin drying, diluted with 10 mL of water, extracted three times with 10 mL of EA, washed with saturated brine, dried over anhydrous sodium sulfate, and flash chromatographed on a silica gel column (DCM: MeOH = 30:1) to obtain 65 mg (0.17 mmol) of a bright yellow solid (yield 86%).

[0079] 1H 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).

[0080] Step 10: Synthesis of 1-ethyl-6-(6-oxydeoxy-1H-1,2-diazacyclohexyl-3-yl)-3-[[2-(thiacyclohexane-4-ylidene)ethylamino]methylidene]indol-2-one (Y-3): 60 mg (0.2 mmol) of 3-(ethylaniline)-1-ethyl-6-(6-oxyylidene-1H-1,2-diazacyclohexyl-3-yl)indol-2-one and 28 mg (0.24 mmol) of tetrahydrothiopyran-4-one were dissolved in 2 mL of isopropanol solution, and a drop of acetic acid was added at room temperature. The reaction was carried out at 80°C for 1 h. After the reaction, the solvent was dried by spin drying, diluted with 10 mL of water, extracted with 10 mL of EA three times, washed with saturated brine, dried over anhydrous sodium sulfate, and flash chromatographed on a silica gel column (DCM: MeOH = 30:1) to obtain 72 mg (0.18 mmol) of a bright yellow solid (yield 90%).

[0081] 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).

[0082] Example 12 In vitro binding assay of compounds Y-1, Y-2 and Y-3 with MFN2 protein For the compounds Y-1, Y-2 and Y-3 prepared in the present invention, the dissociation constants between the compounds and the MFN2 protein were detected using a biomolecular interaction instrument Biacore T200 (General Electric Company). The specific detection process is as described in Example 2.

[0083] like Figure 25-30 As shown, the binding force fitting curves of compounds Y-1, Y-2 and Y-3 at different concentrations with 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-8M, 1.949E-8M and 1.918E-8M, respectively.

[0084] Example 13

[0085] Effects of compounds Y-1, Y-2 and Y-3 on the GTPase activity of MFN2 protein The ATPase / GTPase Assay kit (Abcam, ab272520) was used to detect the effects of compounds Y-1, Y-2 and Y-3 on the GTPase activity of MFN2 protein according to the instructions.

[0086] result Figure 31-33 As 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 24.30uM, 23.49 uM and 23.87 uM, respectively.

[0087] Embodiment 14

[0088] Effects of compounds Y-1, Y-2 and Y-3 on mitochondrial morphology of mouse primary liver cells The specific experimental steps for extracting mouse liver primary cells were the same as those in Example 4. The isolated primary cells were incubated at 37°C and 5% CO 2 Drugs were administered after 36 hours of incubation. The solvent control group was treated with serum-free DMEM high-glucose medium containing 1‰ DMSO, and the drug administration group was treated with 25uM Y-1, Y-2, and Y-3 prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO. After administration, the cells were placed at 37°C and 5% CO 2 Incubate in the chamber for 30 minutes. After administration, stain with Mitotraker deep red and Hoechst live cell dyes. After staining, observe and photograph under a confocal microscope.

[0089] The results of the test on the effects of compounds Y-1, Y-2 and Y-3 on the mitochondrial morphology of mouse liver primary cells are shown in Fig.34 As shown, the solvent control group is marked with solvent control, i.e., serum-free DMEM high-glucose medium containing 1‰ DMSO, and the groups marked with Y-1, Y-2 and Y-3 are drug-treated groups, i.e., 25uM Y-1, Y-2 and Y-3 prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO. The results showed that the mitochondrial aspect ratio of the drug-treated group was significantly increased, and the degree of mitochondrial fusion was higher.

[0090] Embodiment 15 Protective effects of compounds Y-1, Y-2 and Y-3 on APAP (acetaminophen)-induced injury in mouse primary hepatocytes The mouse liver primary cell extraction procedure was the same as in Example 4. The isolated primary cells were incubated at 37°C, 5% CO 2 After 36 hours of incubation, the drug was administered. The solvent control group was serum-free DMEM high-glucose medium containing 1‰ DMSO, the drug administration group was 5uM Y-1, Y-2 and Y-3 prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO, and the positive control group was 5uM silybin prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO. After 24 hours of pre-administration, the culture medium was replaced with 10mM acetaminophen solution (prepared in serum-free DMEM high-glucose medium), and the cell viability was detected by CCK8 method after 24 hours. The specific experimental method for detecting cell viability by CCK8 method is as follows: First, prepare CCK8 working solution, dilute CCK8 stock solution 10 times with serum-free DMEM high-glucose medium, add it to the 96-well plate after administration, 100ul per well, 37℃, 5% CO 2After 2 hours of incubation, the absorbance values ​​at wavelengths of 450nm and 630nm were read on a microplate reader. Finally, the absorbance value at 450nm was subtracted from the absorbance value at 630nm to obtain the actual absorbance value of the well. The absorbance value of the control group was used as a reference, and the cell viability of each experimental group was calculated according to the formula "Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%". Among them, the blank group is a well in which only culture medium and CCK8 reagent are added, and no cells are inoculated.

[0091] The results of the pharmacodynamic study on the protective effects of compounds Y-1, Y-2 and Y-3 on APAP (acetaminophen)-induced injury in mouse primary liver cells are shown in Fig.35 As shown, the solvent control group is marked with solvent control, i.e., serum-free DMEM high-glucose medium containing 1‰ DMSO; the groups marked with Y-1, Y-2 and Y-3 are drug-treated groups, i.e., 5uM Y-1, Y-2 and Y-3 prepared in serum-free DMEM high-glucose medium containing 1‰ DMSO; the group marked with Silibinin is Silybin (positive control) group. The results showed that the cell viability of the drug-treated group was significantly increased compared with the solvent control group, and the effect was equivalent to that of the positive control group, indicating that compounds Y-1, Y-2 and Y-3 have a certain protective effect on APAP (acetaminophen)-induced mouse primary liver cell damage.

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-diazacyclohexane-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 (I-11): 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 (I-12): 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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