Substituted 4-quinolinone compound as well as preparation method and application thereof

Abstract

The invention is applicable to the technical field of biological medicines, and provides a substituted 4-quinolinone compound as well as a preparation method and application thereof. A substituted 4-quinolinone compound with a brand new structure is developed, an in-vitro anti-hepatic fibrosis activity test shows that most of the compounds have a remarkable inhibition effect on fibronectin (FN) expression in LX-2 cells (human hepatic stellate cell lines) stimulated by TGF-beta1 at the concentration of 10 [mu] M, the inhibition rate of part of the compounds exceeds 90%, and the inhibition rate of the compound I-3 reaches 98%. The compound can inhibit activation of hepatic stellate cells and excessive deposition of extracellular matrixes and improve liver tissue damage, so that the compound has important drug value and wide clinical application prospect in the field of anti-hepatic fibrosis, and a new choice can be provided for research and development of anti-hepatic fibrosis drugs. In addition, the preparation method has the advantages of easily available raw materials, simple synthesis, high product yield and easy purification, and lays a foundation for compound structure optimization and further drug research and development.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a substituted 4-quinolinone compound, a preparation method and an application thereof. Background Art

[0002] Liver fibrosis is a major global health problem, and the number of patients is huge and still growing. Data show that approximately 2 million people die each year from cirrhosis and hepatocellular carcinoma caused by liver fibrosis, accounting for 4% of the global death toll. Liver fibrosis is essentially the liver's repair response to various inflammations and tissue damage. It usually occurs after chronic liver cell damage, and its core mechanism is the activation of hepatic stellate cells (HSCs). Under inflammatory stimulation, hepatic stellate cells are activated and transformed into a pro-fibrotic cell phenotype, secreting large amounts of extracellular matrix (ECM) such as collagen and fibronectin. These matrices are excessively deposited in the liver, destroying the normal structure of the liver and ultimately leading to liver fibrosis. If not effectively treated, liver fibrosis will eventually develop into malignant diseases such as cirrhosis and even liver cancer.

[0003] The pathological mechanisms of liver fibrosis are extremely complex, involving the interplay of multiple cells, signaling pathways, and molecules. Currently, several drugs, including the FXR agonist obeticholic acid, the ASK1 inhibitor selonsertib, and the CCR2 / CCR5 antagonist cenicriviroc, have entered clinical trials. However, these drugs can only partially slow the progression of liver fibrosis, but cannot completely block or reverse fibrosis, and they are often associated with toxic side effects. Although pirfenidone (PFD), a broad-spectrum antifibrotic drug, has been approved for the treatment of idiopathic pulmonary fibrosis and is undergoing a Phase II clinical trial for liver fibrosis (NCT04099407), its severe hepatotoxicity limits its clinical application in the treatment of liver fibrosis. To date, no drugs directly targeting liver fibrosis have been approved for marketing. Therefore, the development of novel antifibrotic compounds with novel structures, improved efficacy, and reduced toxicity and side effects is urgent.

[0004] The 4-quinolinone skeleton, with its unique chemical structure and rich biological activities, holds a prominent position in drug development. 4-Quinolinone compounds have demonstrated a variety of activities, including antibacterial, antiviral, antimalarial, and antitumor activities. However, it is worth noting that there are currently no reports on the application of 4-Quinolinone compounds in the treatment of liver fibrosis. Based on this, the present invention proposes a substituted 4-Quinolinone compound, its preparation method, and its application, which is expected to open a new path for the development of drugs for the treatment of liver fibrosis. Summary of the Invention

[0005] The purpose of the present invention is to provide a substituted 4-quinolinone compound and a preparation method and application thereof, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A substituted 4-quinolinone compound, including an N-allyl-substituted 4-quinolinone compound (i.e., an N-allyl-4-quinolinone compound) and a 3-allyl-substituted 4-quinolinone compound (i.e., a 3-allyl-4-quinolinone compound). The structures of the N-allyl-substituted 4-quinolinone compound and the 3-allyl-substituted 4-quinolinone compound are shown in formula (I) and formula (II), respectively:

[0008]

[0009] in:

[0010] R1 is selected from -H, -F, -Cl, -Br, -I, -OH, -R, -OR, -CF3, -CN, -NO2, -OCF3, -CO2R, -CO2H, wherein R is an alkyl group having 1 to 6 carbon atoms. The group R1 may be one or more. When multiple R1 groups are present, R1 may be the same or different. Preferably, R1 is selected from -H, -OMe, -Me, -F, Cl, or -Br. R1 may be one or two. When two R1 groups are present, R1 may be the same or different.

[0011] R2 is selected from C1-6 alkyl, C1-6 cycloalkyl, aromatic ring or aromatic heterocycle containing one or more substituents, and the substituents are selected from -H, -F, -Cl, -Br, -I, -OH, -CH2OH, -CH2OR, -R, -OR, -CF3, -CN, -NO2, -NHC(=O)R, -OCF3, -C(=O)OR, -C(=O)OH, -C(=O)N(R) n , wherein R is an alkyl group having 1 to 6 carbon atoms, n represents the number of substituents, and n is 0, 1, or 2. When n=2, the groups R may be the same or different. Preferably, R2 is selected from methyl, ethyl, isopropyl, n-butyl, or one of the following structures:

[0012]

[0013] R3 is selected from hydrogen, C 3-6 Cycloalkyl, aryl or heteroaryl containing one or more substituents. The substituents on the aryl or heteroaryl are selected from -H, -F, -Cl, -Br, -I, -OH, -CH2OH, -CH2OR, -R, -OR, -CF3, -CN, -NO2, -NHC(=O)R, -OCF3, -C(=O)OR, -C(=O)OH, -C(=O)N(R) n, wherein R is an alkyl group having 1 to 6 carbon atoms, n represents the number of substituents, and n is 0, 1, or 2. When n=2, the groups R may be the same or different. Preferably, R3 is selected from H, cyclohexyl, or one of the following structures:

[0014]

[0015] R 4 Selected from -C(=O)OR, -C(=O)OH, -C(=O)N(R) n Or -CN. The substituent R is selected from C 1-6 Alkyl, -Bn; n represents the number of substituents, and n is 0, 1 or 2.

[0016] The double bond geometric configurations of the nitrogen atom at position 1 of the 4-quinolinone ring in formula (I) and the allyl group at the carbon atom at position 3 of the 4-quinolinone ring in formula (II) can be E or Z configuration alone, or a mixture of the two.

[0017] A method for preparing substituted 4-quinolinone compounds according to the above-mentioned method, wherein the preparation of the compounds represented by formula (I) and formula (II) both uses a 4-quinolinone compound 1 and an allyl reagent 2 as common starting materials, and the difference lies in the difference in the reaction conditions (such as reaction solvent, temperature, catalyst, etc.).

[0018] The synthetic routes of the compounds represented by formula (I) and formula (II) are as follows:

[0019]

[0020] The preparation method of the compound represented by formula (I) comprises the following steps:

[0021] The 4-quinolinone compound 1 and the allylation agent 2 are mixed in an organic solvent according to the feed ratio, and then an organic or inorganic base catalyst is added, and the mixture is heated to react under stirring conditions to obtain the N-allyl substituted 4-quinolinone compound represented by formula (I).

[0022] The molar ratio of the 4-quinolinone compound 1 to the allylation agent 2 is 1:0.8 to 1:1.5, and the preferred molar ratio is 1:1.2.

[0023] The organic solvent is selected from tert-butanol, n-butanol, tetrahydrofuran, 1,4-dioxane, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, and acetonitrile, preferably acetonitrile or tert-butanol. The volume of the organic solvent used is 10 to 50 mL / mmol, preferably 10 mL / mmol, based on the amount of 4-quinolinone compound 1.

[0024] The catalyst is selected from DMAP, triethylamine, pyridine, potassium carbonate, sodium carbonate, potassium phosphate, potassium tert-butoxide, sodium tert-butoxide, preferably DMAP. The amount of the catalyst used is 0.01 to 0.5 equivalents, preferably 0.2 equivalents.

[0025] The reaction temperature is 60-150° C., preferably 80-110° C. The reaction time is 12-72 hours.

[0026] The preparation method of the compound represented by formula (II) comprises the following steps:

[0027] The 4-quinolinone compound 1 and the allylation agent 2 are mixed in an organic solvent according to the feed ratio, and then an organic base catalyst is added and heated to react under stirring conditions to prepare a 3-allyl substituted 4-quinolinone compound shown in formula (II).

[0028] The molar ratio of the 4-quinolinone compound 1 to the allylation agent 2 is 1:0.8 to 1:1.5, and the preferred molar ratio is 1:1.2.

[0029] The organic solvent is selected from tetrahydrofuran, 1,4-dioxane, chlorobenzene, ethyl acetate, chloroform, acetonitrile, and 1,2-dichloroethane, preferably 1,2-dichloroethane or chloroform. The volume of the organic solvent used is 10 to 50 mL / mmol based on the amount of the 4-quinolinone compound 1.

[0030] The catalyst is selected from DMAP, DABCO, triethylamine, pyridine, and diisopropylethylamine, preferably DABCO. The amount of the catalyst used is 0.01 to 0.5 equivalents, preferably 0.1 equivalents.

[0031] The reaction temperature is 60-150° C., preferably 80-100° C. The reaction time is 12-72 hours.

[0032] A use of the substituted 4-quinolinone compound described above in the preparation of a medicament for treating liver fibrosis. Preferably, the liver fibrosis comprises at least one of viral hepatitis, alcoholic steatohepatitis, non-alcoholic steatohepatitis, autoimmune hepatitis, hereditary liver disease, cirrhosis, liver cancer, and liver fibrosis.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention develops a novel substituted 4-quinolinone compound, which differs from existing anti-hepatic fibrosis drugs in structure. In vitro anti-hepatic fibrosis activity tests showed that most of these compounds significantly inhibited FN expression in TGF-β1-stimulated LX-2 cells at a concentration of 10 μM, with some compounds exhibiting inhibition rates exceeding 90%, including compound I-3, which achieved an inhibition rate of 98%. These compounds can inhibit hepatic stellate cell activation and excessive extracellular matrix deposition, improving liver tissue damage, demonstrating their significant pharmaceutical value and broad clinical application prospects in the field of anti-hepatic fibrosis, providing new options for the development of anti-hepatic fibrosis drugs. Furthermore, the preparation method for these compounds utilizes readily available raw materials, a simple synthesis process, high product yields, and ease of purification, laying the foundation for compound structure optimization and further drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The figure shows the inhibitory effect of compound I-3 on FN and COL1A1 proteins in LX-2 cells; wherein, A is a Western Blot protein band diagram, B is a quantitative analysis of the relative expression of FN protein, and C is a quantitative analysis of the relative expression of COL1A1 protein.

[0036] Figure 2 Pathological sections showing the effect of compound I-3 on alleviating CCl4-induced liver damage. DETAILED DESCRIPTION

[0037] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0038] The specific implementation of the present invention is described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art; the starting material 4-quinolinone compound can be purchased directly or prepared according to the method reported in the literature (Ulmer, Brunner, et al., Chem. Eur. J. 2016, 22, 3660-3664; Jones, Anderson, Buchwald. J Org Chem. 2007, 72, 7968-7973.); the starting material allyl reagent (Morita-Baylis-Hillman carbonate, referred to as MBH carbonate) is prepared according to the literature method (Yang, X Het al., Chem. Commun., 2019, 55, 9144-9147; Pautigny, C. et al., Adv. Synth. Catal. 2008, 350, 2525-2532.). Unless otherwise specified, other reagents and materials used in the following examples can be obtained from commercial sources.

[0039] Example 1: Preparation of Compound I-3;

[0040]

[0041] 2-Phenylquinolin-4(1H)-one 1-1 (44 mg, 0.2 mmol) and MBH carbonate 2-3 (78 mg, 0.24 mmol, 1.2 eq) were dissolved in t-BuOH (tert-butanol, 2 mL), and DMAP (0.2 equiv) was added. The reaction mixture was then heated to 80°C with magnetic stirring and maintained at this temperature for 30 h. After completion of the reaction, the reaction mixture was cooled to room temperature, then water (20 mL) was added and extracted three times with 20 mL of ethyl acetate. The organic layers were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The solution was concentrated under reduced pressure to obtain the crude product. Finally, the crude product was separated and purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1, v / v) to obtain the target compound I-3 (E / Z = 2:1), 65 mg, in a 76% yield.

[0042] Referring to the preparation method of compound I-3 above, N-allyl-4-quinolinone compounds I-1, I-2, I-4 to I-16 were prepared. The specific structures of I-1 to I-16 are as follows:

[0043]

[0044] The characterization data of N-allyl-4-quinolinone compounds I-1 to I-16 are shown in Table 1:

[0045] Table 1. Characterization data of N-allyl-4-quinolinone compounds I-1 to I-16

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] Example 2: Preparation of Compound II-1;

[0053]

[0054] 2-Phenylquinolin-4(1H)-one 1-1 (44 mg, 0.2 mmol) and MBH carbonate 2-1 (88 mg, 0.3 mmol, 1.5 eq) were dissolved in 1,2-dichloroethane (DCE, 2 mL). DABCO (0.02 mmol, 0.1 equiv) was added and the mixture was reacted at 100°C for 30 h. The reaction was monitored by TLC (PE:EA = 1:1) for completion. After completion, the cooled mixture was poured into water (20 mL) and extracted with ethyl acetate (20 mL x 3). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:ethyl acetate = 10:1, v / v) to obtain the target compound II-1 (E / Z = 3:1) in 86% yield.

[0055] Referring to the preparation method of compound II-1 above, 3-allyl-4-quinolinone compounds II-2 to II-20 were prepared. The specific structures are as follows:

[0056]

[0057] The characterization data of 3-allyl-4-quinolinone compounds II-1 to II-20 are shown in Table 2:

[0058] Table 2. Characterization data of 3-allyl-4-quinolinone compounds II-1 to II-20

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] Example 3: Preparation of Compound II-21;

[0065]

[0066] Compound II-7 (43 mg, 0.1 mmol) was dissolved in dichloromethane (CH2Cl2, 2 mL) and stirred at -20°C. A 1 mol / L BBr3 solution in dichloromethane (250 μL, 2.5 equiv) was slowly added dropwise. The mixture was stirred for 30 min and then returned to room temperature. The reaction was monitored for completion by TLC (PE:EA = 1:1). The reaction time was 18 h. After completion, saturated sodium bicarbonate (20 mL) was poured into the mixture and extracted with ethyl acetate (20 mL x 3). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:ethyl acetate = 10:1) to obtain the target compound II-21 as a yellow solid in 80% yield, mp: 77-79°C. 1 H NMR(400MHz,DMSO)δ11.45(s,1H),9.88(s,1H),8.03(s,1H),7.59–7.56(m ,2H),7.36–7.20(m,10H),6.88(d,J=8.5Hz,2H),3.71(s,2H),3.60(s,3H). 13 C NMR (101MHz, DMSO) δ176.2,168.3,158.5,148.9,139.4,135.9,135.4,132.6,131.2 ,130.5,129.4,128.2,127.9,125.5,125.0,123.5,122.6,118.1,115.1,51.5,25.8.

[0067] Example 4: Preparation of Compound II-22;

[0068]

[0069] Compound II-17 (97 mg, 0.2 mmol) was dissolved in methanol / water (2:1 v / v, 3 mL). 1 mol / L aqueous NaOH (3 mL) was added and the mixture was reacted at 80°C. TLC confirmed the reaction was complete (DCM:MeOH = 50:1) for 6 h. After completion, the mixture was concentrated under reduced pressure. Water (10 mL) was poured into the residue, and 10% aqueous hydrochloric acid was slowly added dropwise until no more precipitate formed. The resulting precipitate was collected by filtration and washed thoroughly with plenty of water to obtain the target compound II-22 in a 96% yield.

[0070] Example 5: Preparation of Compound II-23;

[0071]

[0072] Compound II-22 (47 mg, 0.1 mmol) was dissolved in dichloromethane (2 mL) and stirred at 0°C. EDCI (0.15 mmol, 1.5 equiv) and DMAP (0.015 mmol, 0.15 equiv) were added, followed by benzylamine (0.12 mmol, 1.2 equiv). The mixture was stirred for 30 min and then returned to room temperature. The reaction was monitored for completion by TLC (DCM:MeOH = 50:1). The reaction time was 6 h. After completion of the reaction, saturated sodium bicarbonate (20 mL) was poured into the mixture and extracted with ethyl acetate (20 mL x 3). The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol = 100:1) to obtain the target compound II-23 (E / Z = 5:1) in 70% yield. (E)-II-23: 1 H NMR (400MHz, DMSO) δ11.50(s,1H),8.67(s,1H),7.40(s,1H),7.30–7.19(m,7H),7.09–7.02(m, 2H),7.02–6.95(m,5H),6.85–6.78(m,2H),4.22(s,2H),3.84(s,3H),3.82(s,3H),3.76(s,5H). 13 C NMR (101MHz, DMSO) δ175.7,168.3,159.7,152.9,148.6,146.6,139.4,137.2,136.0,135.0,131.6,129.9,129.2,12 7.9,127.8,127.1,126.8,126.6,126.3,117.7,115.0,113.4,104.2,99.0,55.7,55.5,55.1,42.4,24.2; (Z)-Ⅱ-23: 1 HNMR(400MHz, CDCl3)δ9.08(s,1H),7.61(s,1H),7.51(s,1H),7.41(d,J=8.0Hz,2H),7.34–7.27(m,1H),7.25–7.18(m,3H),7.18–7.11 (m,6H),7.08–7.01(m,2H),6.89–6.80(m,3H),6.21(s,1H),4.31(d,J=6.0Hz,2H),3.94(s,3H),3.85(s,3H),3.76(s,3H),3.52(s,2H).

[0073] Example 6: Cell-based test of the anti-hepatic fibrosis activity of compounds I-1 to I-16 and II-1 to II-23;

[0074] Using pirfenidone (500uM, PDF) as a positive control, immunofluorescence was used to evaluate the anti-fibrotic activity of the target compounds (I-1 to I-16 and II-1 to II-23) at a concentration of 10uM by monitoring fibronectin (FN) expression in LX-2 cells (a human hepatic stellate cell line) stimulated with transforming growth factor-β1 (TGF-β1). The inhibition rate of the test compound on FN production was calculated by analyzing fluorescence intensity and cell confluence. The specific experimental method is as follows:

[0075] LX-2 cells in the logarithmic growth phase were seeded in 96-well plates at an appropriate density. After the cells were cultured for 24 hours, they were replaced with 2% fetal bovine serum medium for starvation treatment for 12 hours, and the test compound (prepared by Examples 1 to 6) was added at a concentration of 10uM, and 10ng / mL of TGF-β1 was added at the same time to induce hepatic stellate cell activation and FN expression. A blank group (untreated), a TGF-β1 stimulation group (only TGF-β1 was added, without compound), a positive drug group (TGF-β1+PDF) and a test compound treatment group (TGF-β1+test compound) were set up. After 48 hours of culture, the PBS cells were fixed, permeabilized, blocked, incubated with FN primary antibody at 4°C overnight, and incubated with FITC-conjugated secondary antibody for 1 hour. FITC green immunofluorescence images were taken using a live cell workstation, and the cell confluence was taken under bright field at the same time. The inhibition rate of FN production by each test substance was calculated by analyzing the fluorescence intensity and cell confluence. The specific calculation formula is as follows:

[0076]

[0077] The cell activity test results are shown in Table 3:

[0078] Table 3. Activity test results of compounds

[0079] Compound (10uM) Inhibition rate (%) Compound Inhibition rate (%) I-1 88 Ⅱ-5 94 I-2 79 Ⅱ-6 72 I-3 98 Ⅱ-7 95 I-4 90 Ⅱ-8 63 I-5 52 Ⅱ-9 84 I-6 75 Ⅱ-10 67 I-7 75 Ⅱ-11 75 I-8 49 Ⅱ-12 0 I-9 81 Ⅱ-13 51 I-10 96 Ⅱ-14 0 I-11 65 Ⅱ-15 49 I-12 82 Ⅱ-16 0 I-13 61 Ⅱ-17 0 I-14 59 Ⅱ-18 56 I-15 72 Ⅱ-19 56 I-16 60 Ⅱ-20 62 Ⅱ-1 89 Ⅱ-21 27 Ⅱ-2 76 Ⅱ-22 41 Ⅱ-3 63 Ⅱ-23 58 Ⅱ-4 88 PDF(500uM) 89

[0080] As shown in Table 3, most compounds significantly inhibited FN expression in TGF-β1-stimulated LX-2 cells at a concentration of 10 uM. Compound I-3 exhibited the best inhibitory activity, with an inhibition rate of 98% at a concentration of 10 uM on FN production in TGF-β1-stimulated LX-2 cells.

[0081] Example 7: Western blot detection;

[0082] LX-2 cells were plated at 1×10 5Cells were plated in 6-well plates at a density of 100 μg / mL and cultured in DMEM (Gibco) containing 10% fetal bovine serum at 37°C and 5% CO₂ for 24 hours. The culture medium in the 6-well plates was then removed and replaced with DMEM (Gibco) containing 2% fetal bovine serum, followed by starvation for 12 hours. TGF-β1 (5 ng / mL) was added to induce a cell response, and compound I-3 was added at varying concentrations (10 μM, 20 μM, and 50 μM). A control group (no TGF-β1 induction) and a TGF-β1-induced group (only TGF-β1 induction) were also set up and cultured under the same conditions for another 24 hours.

[0083] Protein was extracted by adding 200 μL of RIPA cell lysis buffer (containing 10% protease inhibitors) to each well of a 6-well plate, and protein concentration was determined using the BCA assay. Samples were loaded at 15 μg / 15 μL per well, and the cells were electrophoresed, transferred to the membrane, blocked with 5% skim milk, and incubated overnight with the corresponding primary antibody. The membrane was then washed and incubated with an HRP-conjugated secondary antibody for 1 hour. Ultrasensitive ECL luminescent solution (Thermo Fisher) was applied to the membrane, and the desired protein bands were visualized using a Bio-rad chemiluminescence imaging system.

[0084] from Figure 1 As can be seen in Figures AC, under the induction of TGF-β1, the expression levels of liver fibrosis-related proteins FN and COL1A1 were significantly increased. Compound I-3 can reverse the TGF-β1-induced upregulation of FN and COL1A1 expression in a concentration-dependent manner, suggesting that compound I-3 has potential inhibitory activity against liver fibrosis.

[0085] Example 9: In vivo pharmacodynamic evaluation in animals;

[0086] Eight-week-old male C57BL / 6J mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were selected and housed in an SPF environment in the animal center. A 12-h circadian rhythm was maintained, and the bedding was changed every 3 days. The mice were free to eat and drink. All animal experiments were approved by the Experimental Animal Ethics Committee of the School of Pharmacy of Jilin University (approval number: 20250010). After one week of adaptive feeding, the mice were randomly divided into five groups according to their body weight (normal control group, model group, and three treatment groups, with 8 mice in each group). Carbon tetrachloride (CCl4) and olive oil were mixed in a volume ratio of 1:9 to prepare a 10% CCl4 solution. The mice were weighed, and the volume of CCl4 solution required for each mouse was calculated based on a dose of 1 mL / kg. The normal control group (Control) was intraperitoneally injected with olive oil, and each mouse was intraperitoneally injected 3 times a week for 6 consecutive weeks; the drugs were administered at the same time as the modeling, and the mice in the treatment group were orally gavaged. Compound I-3 was administered at 5 mg / kg, 10 mg / kg, and 20 mg / kg (corresponding to the groups Model + I-3 (5 mg / kg), Model + I-3 (10 mg / kg), and Model + I-3 (20 mg / kg)) every other day. The model group (CCl4Model) received an equal volume of saline containing DMSO. After modeling, blood was collected from the orbital venous plexus of the mice, and serum was separated. The mice were sacrificed, and liver tissue was isolated from the same portion of the largest lobe. The tissue was embedded in paraffin, sectioned, and then stained with H&E and Masson staining for analysis.

[0087] The results are as follows Figure 2 As shown, H&E staining showed that in the liver tissue of mice in the normal control group (Control), the hepatocytes were uniform and orderly, the hepatic lobule structure was clear, and there was no inflammatory cell infiltration or fibrous tissue proliferation. In the model group (CCl4Model), hepatocytes were degenerated and necrotic, the fibrous connective tissue in the tissue proliferated, and the hepatic lobule structure was disordered. In the treatment group (Model+I-3), as the dose of the compound increased (5→10→20 mg / kg), the range of hepatocyte necrosis gradually narrowed, and the cell arrangement became more regular; the fibrous connective tissue proliferation also decreased, and the hepatic lobule structure gradually recovered. The liver tissue morphology of the Model+I-3 (20 mg / kg) group was close to that of the normal control group, which directly reflected the dose-dependent improvement of compound I-3 on liver tissue morphology.

[0088] In Masson staining, collagen fibers appear blue and liver parenchyma appears red. The figure shows that in the normal control group (Control), only a small amount of blue collagen fibers are found around normal structures such as blood vessels and bile ducts. The liver parenchyma area (red) is clear and uniform, and there is no abnormal deposition of ECM. In the model group (CCl4Model), due to the progression of liver fibrosis, a large amount of blue collagen fibers are deposited in the liver tissue, forming extensive fibrous septa that divide the liver parenchyma, and the degree of ECM deposition is significantly increased. In the treatment group (Model+I-3), as the dosage increases (5→10→20 mg / kg), the area of ​​blue collagen fibers gradually decreases, the fibrous septa become thinner and broken; the degree of ECM deposition continues to decrease, the improvement in the 10 mg / kg group is more obvious, and the 20 mg / kg group is close to the normal level, verifying that compound I-3 can dose-dependently inhibit CCl4-induced excessive ECM deposition.

[0089] In summary, the results of H&E staining and Masson staining jointly confirmed that compound I-3 can improve CCl4-induced liver fibrosis damage in mice in a dose-dependent manner by protecting hepatocyte morphology, inhibiting fibrous tissue proliferation and abnormal ECM deposition.

[0090] The above are only preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention. These should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A substituted 4-quinolinone compound, characterized in that: The compounds include N-allyl-substituted 4-quinolinone compounds and 3-allyl-substituted 4-quinolinone compounds, whose structures are shown in formula (I) and formula (II), respectively: in: R1 is selected from -H, -F, -Cl, -Br, -I, -OH, -R, -OR, -CF3, -CN, -NO2, -OCF3, -CO2R, -CO2H, wherein R is an alkyl group having 1 to 6 carbon atoms; R2 is selected from C1-6 alkyl, C1-6 cycloalkyl, aromatic ring or aromatic heterocycle containing one or more substituents, and the substituents are selected from -H, -F, -Cl, -Br, -I, -OH, -CH2OH, -CH2OR, -R, -OR, -CF3, -CN, -NO2, -NHC(=O)R, -OCF3, -C(=O)OR, -C(=O)OH, -C(=O)N(R) n , wherein R is an alkyl group having 1 to 6 carbon atoms, n represents the number of substituents, and n is 0, 1 or 2; R3 is selected from hydrogen, C 3-6 Cycloalkyl, aryl or heteroaryl containing one or more substituents; wherein the substituents on the aryl or heteroaryl are selected from -H, -F, -Cl, -Br, -I, -OH, -CH2OH, -CH2OR, -R, -OR, -CF3, -CN, -NO2, -NHC(=O)R, -OCF3, -C(=O)OR, -C(=O)OH, -C(=O)N(R) n , wherein R is an alkyl group having 1 to 6 carbon atoms, n represents the number of substituents, and n is 0, 1 or 2; R 4 Selected from -C(=O)OR, -C(=O)OH, -C(=O)N(R) n or -CN; wherein R is an alkyl group or a benzyl group having 1 to 6 carbon atoms; n represents the number of substituents, and n is 0, 1 or 2.

2. The substituted 4-quinolinone compound according to claim 1, characterized in that The double bond geometric configurations of the nitrogen atom at position 1 of the 4-quinolinone ring in formula (I) and the allyl group at the carbon atom at position 3 of the 4-quinolinone ring in formula (II) are E configuration, Z configuration or a mixture of E / Z configurations.

3. A method for preparing a substituted 4-quinolinone compound according to claim 1 or 2, characterized in that: Among the substituted 4-quinolinone compounds, the synthetic routes of the N-allyl substituted 4-quinolinone compound represented by formula (I) and the 3-allyl substituted 4-quinolinone compound represented by formula (II) are as follows: The preparation method of the N-allyl substituted 4-quinolinone compound comprises the following steps: A 4-quinolinone compound 1 and an allylation agent 2 are mixed in an organic solvent, and then an organic or inorganic base catalyst is added, and the mixture is heated under stirring to react to obtain an N-allyl-substituted 4-quinolinone compound represented by formula (I); The preparation method of the 3-allyl substituted 4-quinolinone compound comprises the following steps: A 4-quinolinone compound 1 and an allylation agent 2 are mixed in an organic solvent, and then an organic base catalyst is added and heated to react under stirring to prepare a 3-allyl-substituted 4-quinolinone compound represented by formula (II).

4. The preparation method according to claim 3, characterized in that In the preparation method of the N-allyl substituted 4-quinolinone compound: The molar ratio of the 4-quinolinone compound 1 to the allylation agent 2 is 1:0.8 to 1:1.5; The organic solvent is selected from tert-butanol, n-butanol, tetrahydrofuran, 1,4-dioxane, ethyl acetate, N,N-dimethylformamide, dimethyl sulfoxide, and acetonitrile; The volume of the organic solvent is 10 to 50 mL / mmol based on the amount of the 4-quinolinone compound 1; The catalyst is selected from DMAP, triethylamine, pyridine, potassium carbonate, sodium carbonate, potassium phosphate, potassium tert-butoxide, sodium tert-butoxide; The amount of the catalyst is 0.01 to 0.5 equivalents; The reaction temperature is 60-150°C; The reaction time is 12 to 72 hours.

5. The preparation method according to claim 3, characterized in that In the preparation method of the 3-allyl substituted 4-quinolinone compound: The molar ratio of the 4-quinolinone compound 1 to the allylation agent 2 is 1:0.8 to 1:1.5; The organic solvent is selected from tetrahydrofuran, 1,4-dioxane, chlorobenzene, ethyl acetate, chloroform, acetonitrile, and 1,2-dichloroethane; The volume of the organic solvent is 10 to 50 mL / mmol based on the amount of the 4-quinolinone compound 1; The catalyst is selected from DMAP, DABCO, triethylamine, pyridine, and diisopropylethylamine; The amount of the catalyst is 0.01 to 0.5 equivalents; The reaction temperature is 60-150°C; The reaction time is 12 to 72 hours.

6. Use of the substituted 4-quinolinone compound according to claim 1 or 2 in the preparation of a medicament for treating liver fibrosis.

7. A pharmaceutical composition for treating liver fibrosis, characterized in that: The invention comprises the substituted 4-quinolinone compound according to claim 1 or 2 as an active ingredient, and pharmaceutically acceptable excipients.

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