Application of natural derivative in preparation of medicine for treating non-alcoholic fatty liver disease and / or hepatic fibrosis

Abstract

The invention relates to the technical field of biological medicines, and discloses an application of an inosine derivative in preparation of medicines for treating non-alcoholic fatty liver diseases and hepatic fibrosis, and the pharmacological activity of the inosine derivative subjected to structural modification and transformation in treatment of the non-alcoholic fatty liver diseases and / or hepatic fibrosis is detected. The pharmacological activity of the compound is tested in various animal disease models, and data for preventing and treating different types of non-alcoholic fatty liver diseases and / or hepatic fibrosis activity is provided, so that the compound is proved to have good activity, and the effect is obviously superior to that of a clinically common positive drug nintedanib; the effect is obviously superior to those of hypoxanthine derivatives A, B and C and other hypoxanthine derivatives in the prior art. The hypoxanthine derivative disclosed by the invention can provide a novel skeleton for screening a novel compound for preparing a medicine for treating the non-alcoholic fatty liver disease and / or hepatic fibrosis, and lays a theoretical foundation for developing a novel lead compound.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of a hypoxanthine derivative in preparation of a medicine for treating non-alcoholic fatty liver disease and / or liver fibrosis. BACKGROUND

[0002] Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease caused by factors other than alcohol and other clear liver damage, which is mainly characterized by excessive deposition of liver parenchymal cells. NAFLD includes a wide range of diseases, which can be divided into benign non-alcoholic fatty liver (NAFL) and more serious non-alcoholic steatohepatitis (NASH) according to the course of the disease. Influenced by metabolism, oxidative stress and heredity, its onset involves multiple organs and multiple mechanisms, and its development is mainly related to lipid accumulation, oxidative stress, endoplasmic reticulum stress and lipid toxicity[1].

[0003] Non-alcoholic steatohepatitis (NASH) is characterized by steatosis, hepatocyte ballooning, lobular inflammation and extensive fibrosis. In order to regenerate new cells, NASH progresses to cirrhosis, and liver cells are replaced by I collagen scar tissue produced by stellate cells[2]. Liver fibrosis is the activation of hepatic stellate cells, which is further transformed into myofibroblasts, which is characterized by excessive proliferation and abnormal deposition of a large amount of extracellular matrix, leading to destruction of liver structure and pseudolobule formation[3]. Non-alcoholic fatty liver disease can lead to liver fibrosis in the later stage.

[0004] In recent years, the incidence of NAFLD is increasing, and it is reported that one-fourth of the world's population is affected. NAFLD is extremely harmful to the human body, and if not intervened in time, it may further develop into liver cancer and eventually lead to death, but so far no drug for treating NAFLD has been officially approved. Therefore, it is of great importance to find a drug that can prevent and treat non-alcoholic fatty liver disease and / or liver fibrosis for the prevention and treatment of non-alcoholic fatty liver disease and / or liver fibrosis.

[0005] Currently, there are only two anti-fibrosis drugs used in clinical practice, namely nintedanib and pirfenidone, but the FDA has only approved their clinical indications for pulmonary fibrosis. The characteristics of pulmonary fibrosis are the proliferation of fibroblasts and the deposition of extracellular matrix (ECM), leading to the destruction of lung tissue structure. Fibroblasts synthesize and secrete proteins to form lung extracellular matrix, maintain the normal scaffold of lung epithelium and endothelium, participate in effective gas exchange and normal repair of damaged tissue. Many pro-fibrotic factors stimulate the proliferation of fibroblasts, induce the transformation of fibroblasts into myofibroblasts, and lead to increased ECM synthesis[4].

[0006] Nintedanib is approved for the treatment of idiopathic pulmonary fibrosis (IPF), non-small cell lung cancer (NSCLC), systemic sclerosis-associated interstitial lung disease (SSc-ILD), and progressive fibrosing interstitial lung disease (PF-ILD). Nintedanib is also being investigated for the treatment of endometrial fibrosis in phase II and III (NCT05635071), which is not yet approved. Pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis (IPF). Pirfenidone is also being investigated for the treatment of liver fibrosis in phase II (NCT02161952), kidney fibrosis in phase II (NCT04258397), myocardial fibrosis after acute myocardial infarction in phase II (NCT05531955), and multiple sclerosis in phase I and II (NCT03109288), which are not yet approved. Due to the different pathogenesis of fibrosis in different organs, nintedanib and pirfenidone are not effective for the treatment of non-alcoholic fatty liver disease and liver fibrosis.

[0007] Metformin can be used for the treatment of NASH, which can effectively control blood glucose, improve metabolism, reduce body weight, improve NASH, reduce steatosis and inflammation, but does not improve fibrosis.

[0008] Hypoxanthine (Hypoxanthine) is also known as "6-hydroxy purine", which is a naturally occurring purine compound, and is a synthetic precursor of purine nucleotides of nucleic acids. There is no report on hypoxanthine derivatives that can block the development of non-alcoholic fatty liver disease and / or liver fibrosis and reverse pathological damage. We have developed a series of hypoxanthine derivatives that can effectively improve non-alcoholic fatty liver disease and / or liver fibrosis. SUMMARY

[0009] The purpose of the present application is to provide a hypoxanthine derivative for the treatment of non-alcoholic fatty liver disease and / or liver fibrosis, in order to accelerate the development of new drugs for the treatment of non-alcoholic fatty liver disease and / or liver fibrosis. The technical effects of the preferred technical solution of the present application are described in detail below.

[0010] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0011] The first aspect of the present application relates to the use of a hypoxanthine derivative for the preparation of a medicament for the treatment of non-alcoholic fatty liver disease and / or liver fibrosis, wherein the hypoxanthine derivative has activity in treating non-alcoholic fatty liver disease and / or liver fibrosis, and the hypoxanthine derivative has one of the following structures:

[0012]

[0013] In which:

[0014] R1 can be any of O, N, C, S or = O;

[0015] R2, R3, and R4 can be chosen as H and C1-C, respectively. 18 Alkyl or halogen-substituted C1-C 18 Alkyl, trifluoromethyl, sulfonyl, sulfonamide, sulfinyl, amino acid, 2-[bis(neopentyloxy)methoxy]phosphonomethoxyethyl, C1-C 18 Fatty acid group, C3-C 12 Heterocyclic groups, C1-C 18 Fatty acids; or C1-C atoms in which R2, R3, and R4 are optionally substituted with oxygen, sulfur, or nitrogen atoms. 18 The alkyl or fatty acid group; when R3 or R4 is substituted, the double bond is attached to the unsubstituted N position, and when all are substituted, there is no double bond.

[0016] The second aspect of this invention relates to compounds having the following structure:

[0017]

[0018] in:

[0019] R1 can be any of O, N, C, S or = O;

[0020] R2, R3, and R4 can be chosen as H and C1-C, respectively. 18 Alkyl or halogen-substituted C1-C 18 Alkyl, trifluoromethyl, sulfonyl, sulfonamide, sulfinyl, amino acid, 2-[bis(neopentyloxy)methoxy]phosphonomethoxyethyl, C1-C 18 Fatty acid group, C3-C 12 Heterocyclic groups, C1-C 18 Fatty acids; or C1-C atoms in which R2, R3, and R4 are optionally substituted with oxygen, sulfur, or nitrogen atoms. 18 The alkyl or fatty acid group; when R3 or R4 is substituted, the double bond is attached to the unsubstituted N position, and when all are substituted, there is no double bond.

[0021] According to a preferred embodiment, the compound is selected from the group consisting of:

[0022]

[0023] A third aspect of the present invention relates to pharmaceutical compositions comprising the compounds of the present invention or pharmaceutically acceptable salts thereof.

[0024] According to a preferred embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients or auxiliary ingredients.

[0025] According to a preferred embodiment, the pharmaceutical composition is an oral formulation, an injectable formulation, or a nasal mucosal administration formulation.

[0026] The fourth aspect of the present invention relates to a method for treating non-alcoholic fatty liver disease and / or liver fibrosis by administering an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention, to an individual in need.

[0027] According to a preferred embodiment, the medication for treating non-alcoholic fatty liver disease and / or liver fibrosis includes medications that have the efficacy of preventing and treating non-alcoholic fatty liver disease and / or liver fibrosis and their complications.

[0028] According to a preferred embodiment, the drug for treating non-alcoholic fatty liver disease and / or liver fibrosis is a formulation prepared by adding pharmaceutically acceptable excipients or auxiliary ingredients, with hypoxanthine derivatives or their salts as the active ingredient.

[0029] According to a preferred embodiment, the formulation is an oral formulation, an injectable formulation, or a nasal mucosal administration formulation.

[0030] Terminology definition:

[0031] The compounds and derivatives provided by this invention can be named according to the IUPAC (International Union of Pure and Applied Chemistry) or CAS (Chemical Abstracts Service, Columbus, OH) nomenclature systems.

[0032] The term "alkyl" refers to a straight-chain or branched saturated hydrocarbon group. Examples of C1 to C3 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), and isopropyl (C3).

[0033] The term "pharmaceutically acceptable" means that a carrier, delivery substance, diluent, excipient, and / or the salt formed therefrom is generally chemically or physically compatible with other components constituting a drug dosage form and physiologically compatible with receptors.

[0034] The term "pharmaceutically acceptable salt" refers to acidic and / or basic salts formed by the compounds of this invention with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compounds. Alternatively, they can be obtained by mixing the aforementioned compounds with appropriate (e.g., equimolar) amounts of an acid or base. These salts may form precipitates in solution and be collected by filtration, or be recovered after solvent evaporation, or be prepared by freeze-drying after reaction in an aqueous medium. The salts described in this invention can be hydrochlorides, sulfates, citrates, benzenesulfonates, hydrobromides, hydrofluoric acids, phosphates, acetates, propionates, succinates, oxalates, malates, succinates, fumarates, maleates, tartrates, or trifluoroacetates of the compounds.

[0035] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

[0036] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with: (a) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin wax; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0037] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0038] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0039] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0040] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0041] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0042] Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays, and inhalers, such as nasal mucosal delivery formulations. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0043] The pharmaceutically acceptable excipients described in this invention refer to substances other than the active ingredient contained in the dosage form.

[0044] The pharmaceutically acceptable adjuvant component described in this invention possesses certain physiological activities. However, the addition of this component does not alter the dominant role of the aforementioned pharmaceutical composition in the disease treatment process; rather, it merely exerts an adjuvant effect. These adjuvant effects are simply the utilization of the known activity of the component, and are a commonly used adjuvant therapy method in the pharmaceutical field. If the aforementioned adjuvant component is used in combination with the pharmaceutical composition of this invention, it should still fall within the scope of protection of this invention.

[0045] Specifically, the application of the hypoxanthine derivatives provided by this invention in the preparation of drugs for non-alcoholic fatty liver disease and / or liver fibrosis can significantly improve non-alcoholic fatty liver disease and / or liver fibrosis.

[0046] The hypoxanthin derivatives provided by this invention have at least the following beneficial technical effects:

[0047] This invention relates to the application of hypoxanthine derivatives in the preparation of drugs for treating non-alcoholic fatty liver disease (NAFLD) and / or liver fibrosis. The pharmacological activity of the modified hypoxanthine derivatives in treating NAFLD and / or liver fibrosis was tested in various animal disease models. Data on the activity in preventing and treating different types of NAFLD and / or liver fibrosis were provided, confirming that they all exhibit good activity, with effects significantly superior to the clinically commonly used drug nintedanib, and also significantly superior to hypoxanthine derivatives A, B, and C, and other hypoxanthine derivatives in the prior art. This invention provides a novel framework for screening new compounds for the preparation of drugs for treating NAFLD and / or liver fibrosis, laying a theoretical foundation for the development of novel lead compounds. Brief description of the attached figures

[0048] Figure 1 HE images of livers in different experimental groups in a mouse model of non-alcoholic fatty liver disease induced by a high-fat diet.

[0049] Figure 2 HE images of livers from different experimental groups in a CCl4-induced liver fibrosis mouse model.

[0050] Figure 3 Masson plots of livers in different experimental groups in a CCl4-induced liver fibrosis mouse model.

[0051] Figure 4 HE images of livers in mouse models of non-alcoholic fatty liver disease and liver fibrosis induced by a high-fat diet and CCl4.

[0052] Figure 5 Masson plots of livers in mouse models of non-alcoholic fatty liver disease and liver fibrosis induced by a high-fat diet and CCl4. Detailed Implementation

[0053] To make the objectives, advantages, and technical solutions of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementations obtained by those skilled in the art without creative effort, including extended studies on the treatment of non-alcoholic fatty liver disease and / or liver fibrosis using hypoxanthine derivatives of this invention, are within the scope of protection of this invention.

[0054] Through research on the medicinal chemistry and chemical processes of natural products, numerous plant endogenous compounds and derivatives have been developed. Modification and alteration of the structures of natural products have yielded many derivatives with excellent pharmacological and chemical activities. This invention provides the application of hypoxanthine derivatives in the preparation of drugs for treating non-alcoholic fatty liver disease (NAFLD) and / or liver fibrosis. The pharmacological activity of the modified hypoxanthine derivatives against NAFLD and / or liver fibrosis was tested in various animal disease models. Data on the activity in preventing and treating different types of NAFLD and / or liver fibrosis are provided, confirming that they all exhibit good activity, with effects significantly superior to the clinically commonly used drug nintedanib, and also significantly superior to hypoxanthine derivatives A, B, and C, and other hypoxanthine derivatives in the prior art. This invention provides a novel framework for screening new compounds for the preparation of drugs against NAFLD and / or liver fibrosis, laying a theoretical foundation for the development of novel lead compounds.

[0055] The structures of hypoxanthine derivatives A (control 1), B (control 2), and C (control 3) are shown below:

[0056]

[0057] Hypoxanthin derivative A (control 1) was commercially available. Compounds B (control 2) and C (control 3) were prepared according to the method used to prepare compounds 3 and 5, respectively.

[0058] The application of the hypoxanthin derivatives provided by the present invention in the preparation of drugs for treating non-alcoholic fatty liver disease and / or liver fibrosis is described in detail below with reference to Examples 1 to 4.

[0059] Example 1: Preparation method of compounds 1 to 18

[0060] According to a preferred embodiment, compounds 1 to 18 are prepared by alkylation of hypoxanthin.

[0061] This embodiment provides preparation methods for 18 compounds, and the structures of all obtained compounds were determined by nuclear magnetic resonance spectroscopy and mass spectrometry.

[0062] Preparation of Compound 1 and Compound 5

[0063] The synthetic routes for compounds 1 and 5 are shown below:

[0064]

[0065] Reaction: (1) At 0℃, 3.45g NaH (4.5eq) was added to a 250mL four-necked flask. After evacuating the gas 3 times, 40mL (8V) of anhydrous THF was slowly added under N2 atmosphere; (2) 12.3mL of isopropanol (4.5eq) was slowly added dropwise to the system and the reaction was allowed to proceed for 30min; (3) Subsequently, 5g of compound 1 (1.0eq) was slowly added dropwise to a 100mL (20V) isopropanol mixture to the reaction system; (4) The temperature was raised to 80℃ and the system was allowed to react for 10h.

[0066] Post-processing: (1) After the reaction was complete, water was added to quench the reaction and acetic acid was added to neutralize the pH to 8-10; (2) Ethyl acetate was extracted 5 times and the organic phases were combined; (3) The organic phase was dried with anhydrous sodium sulfate, concentrated and then separated by rapid silica gel column chromatography to obtain a pale yellow solid; (4) TLC monitoring: developing solvent: dichloromethane / methanol = 10:1 Rf(compound 1) = 0.4.

[0067] The relevant spectral data for compounds 1 and 5 are as follows:

[0068] Compound 1: 1H NMR (400MHz, DMSO-d6) δ 13.33 (s, 1H), 8.44 (s, 1H), 8.32 (s, 1H), 5.55 (hept, J = 6.1Hz, 1H), 1.37 (d, J = 6.2Hz, 6H). HRMS (ESI-TOF) calculated for C8H10N4OH+[M+H+]: 179.09; found 179.10.

[0069] Compound 5: 11H NMR (400MHz, DMSO-d6) δ 13.37 (s, 1H), 8.47 (s, 1H), 8.35 (s, 1H), 5.57 (hept, J = 6.2Hz, 1H), 1.39 (d, J = 6.2Hz, 6H). HRMS (ESI-TOF) [M+H+]: 179.09; found 179.00.

[0070] Preparation of compounds 2, 4 and 6

[0071] The synthetic routes for compounds 2, 4, and 6 are shown below:

[0072]

[0073] The relevant spectral data for compounds 2, 4, and 6 are as follows:

[0074] Compound 2: 1found 179.30.

[0075] Compound 4: 1 H NMR(400MHz,Chloroform-d)δ8.58(s,1H),8.07(s,1H),5.63(hept,J=6.2Hz,1H),4.90(hept,J =6.7Hz,1H),1.59(d,J=6.8Hz,6H),1.44(d,J=6.2Hz,6H).HRMS(ESI-TOF)[M+H+]:221.27; found 221.20.

[0076] Compound 6: 1 found 179.30.

[0077] Preparation of compound 3

[0078] The synthetic route for compound 3 is shown below:

[0079]

[0080] The relevant spectral data for compound 3 are as follows:

[0081] Compound 3: 1 H NMR (400MHz, DMSO-d6) δ8.43 (s, 1H), 8.19 (s, 1H), 5.08 (hept, J = 6.9 Hz, 1H), 4.71 (hept, J = 6. 8Hz,1H),1.52(d,J=6.8Hz,6H),1.41(d,J=6.9Hz,6H).HRMS(ESI-TOF)[M+H+]:221.27; found 221.30.

[0082] Preparation of compounds 7 to 18

[0083] Compounds 7 through 18 were prepared using the same method as compound 3 described above.

[0084] The relevant spectral data of compounds 7 to 18 are as follows:

[0085] Compound 7: 1 found 193.10.

[0086] Compound 8: 1 H NMR(500MHz,Chloroform-d)δ8.55(s,1H),7.95(d,J=0.9Hz,1H),4.85(heptd,J=5.2,0.5Hz,1H) ,4.62(q,J=6.5Hz,2H),1.57(s,3H),1.50(t,J=6.5Hz,3H).HRMS(ESI-TOF)[M+H+]:207.12; found 207.10.

[0087] Compound 9: 1 H NMR (500MHz, Chloroform-d) δ8.51 (s, 1H), 7.57 (d, J = 0.3Hz, 1H), 4.57 (heptd, J = 5.0, 0.7Hz, 1H), 4.61 (t, J = 5. 4Hz,2H),1.90(qt,J=7.3,5.9Hz,2H),1.63(s,6H),1.15(t,J=7.4Hz,3H).HRMS(ESI-TOF)[M+H+]:221.13; found 221.10.

[0088] Compound 10: 1 H NMR(500MHz,Chloroform-d)δ8.37(s,1H),5.33–5.27(m,3H),4.74–4.64(m,2H),1.25 (d,J=6.2Hz,6H),1.17(dd,J=6.7,5.2Hz,12H).HRMS(ESI-TOF)[M+H+]:265.20; found 265.20.

[0089] Compound 11: 1H NMR (500MHz, Chloroform-d) δ7.99–7.88 (m, 2H), 4.83 (pd, J=5.6, 0.8Hz, 1H), 2.32 (s, 3H), 1.53 (s, 6H). HRMS (ESI-TOF) [M+H+]: 221.10; found 221.10.

[0090] Compound 12: 1 H NMR(500MHz,Chloroform-d)δ7.99–7.90(m,2H),4.85(heptd,J=5.2,0.5Hz,1H),2.54( q,J=7.7Hz,2H),1.55(s,3H),1.19–1.11(m,3H).HRMS(ESI-TOF)[M+H+]:235.10; found 235.10.

[0091] Compound 13: 1 H NMR(500MHz,Chloroform-d)δ8.12(s,1H),7.84(s,1H),4.62(s,1H),3.60(s,3H),1.52(s,6H).HRMS(ESI-TOF)[M+H+]:193.11; found193.11.

[0092] Compound 14: 1 H NMR(500MHz,Chloroform-d)δ8.14(s,1H),7.83(s,1H),4.62(s,1H),4.11(s,2H),1.52(s,6H),1.20(s,3H).HRMS(ESI-TOF)[M+H+]:207.12; found 207.12.

[0093] Compound 15: 1 H NMR (500MHz, Chloroform-d) δ7.84 (d, J = 12.2Hz, 2H), 4.62 (s, 1H), 4.42 (s, 1H), 1.52 (s, 6H), 1.46 (s, 6H). HRMS (ESI-TOF) [M+H+]: 221.14; found 221.14.

[0094] Compound 16: 1H NMR(500MHz,Chloroform-d)δ8.11(s,1H),7.85(s,1H),4.23(s,1H),3.93(s,3H),1.45(s,6H).HRMS(ESI-TOF)[M+H+]:193.11; found193.11.

[0095] Compound 17: 1 H NMR(500MHz,Chloroform-d)δ7.94(s,1H),7.85(s,1H),4.37(s,1H),4.24(s,2H),1.51(s,3H),1.43(s,6H).HRMS(ESI-TOF)[M+H+]:207.12; found 207.12.

[0096] Compound 18: 1 H found 221.14.

[0097] Example 2: Activity of compounds 1-18 obtained in Example 1 against non-alcoholic fatty liver disease

[0098] Experimental Methods: A high-fat diet-induced non-alcoholic fatty liver disease mouse model was established: SPF-grade C57BL / 6 male mice (weighing approximately 22-25g) were acclimatized for one week and then randomly divided into several groups according to body weight, including a blank control group, a model group, a compound 1-18 intervention model group, and control groups 1-4 drug intervention model groups (compound AC intervention and positive control metformin group), with 15 mice in each group. Blank control group: fed with normal feed. Model group, compound 1-18 group, control groups 1-3 (hypoxanthin derivative AC) and control group 4 (positive control metformin) were fed a diet consisting of 78.8% basal feed + 10% lard + 10% egg yolk powder + 1% cholesterol + 0.2% sodium cholate. Mice in each group were administered medication via gavage. The blank control group and model group received the same volume of physiological saline as the drug groups, while the model group and drug intervention group received an appropriate volume of 60 mg / kg / day of drug compound 1-18, control drugs 1-3 (compounds A, B, and C, respectively), and the positive control metformin (control 4), administered twice daily. The administration lasted 12 weeks, with mouse weight measured weekly. Mice were fasted for 12 hours before sampling. They were anesthetized with tribromoethanol solution (300 mg / kg), and blood was collected from the right atrium after opening the thoracic cavity. The blood was centrifuged to separate the supernatant serum, and biochemical indicators (ALT, AST, TC, TG) were measured to evaluate liver function. Liver tissue was collected for HE analysis. Data were analyzed using SPSS 19.0 statistical software, expressed as mean ± standard deviation, with P < 0.05 considered statistically significant. Results are shown below.

[0099] From the liver HE images of each group ( Figure 1 It can be seen that the hepatic steatosis in the compound 1-18 intervention group was significantly reduced compared with the model group, and the infiltration of inflammatory cells in the liver was significantly reduced in the compound 1-18 drug intervention group. This indicates that the hypoxanthine derivative 1-18 has a significant effect on non-alcoholic fatty liver disease and is superior to the control group 1-3 (hypoxanthine derivative AC) and control group 4 metformin.

[0100] Table 1 shows that compared with the normal feed group (i.e., the blank control group), the body weight of mice in the model group was significantly increased (P<0.001), while the body weight increase in the compound 1-18 intervention group was lower than that in the model group (P<0.05), and also lower than that in the control group 1-3 and the positive control group 4 (metformin). These results indicate that the intervention of compound 1-18 can significantly control body weight and significantly improve non-alcoholic fatty liver disease.

[0101] Table 1. Changes in body weight of mice in each group (body weight at sample collection / body weight at drug intervention)

[0102] Group Body weight gain index (%) Group Body weight gain index (%) Blank control group 14±2 Compound 7 23±2 Model group 45±3 Compound 8 25±3 Control group 1 43±4 Compound 9 20±3 Control group 2 43±5 Compound 10 19±2 Control group 3 47±4 Compound 11 19±1 Control group 4 31±2 Compound 12 23±2 Compound 1 19±2 Compound 13 19±3 Compound 2 18±2 Compound 14 18±2 Compound 3 16±2 Compound 15 16±3 Compound 4 21±2 Compound 16 24±1 Compound 5 19±3 Compound 17 20±2 Compound 6 22±3 Compound 18 22±2

[0103] As shown in Table 2, compound 1-18 prepared in Example 1 significantly inhibited serum ALT, AST, and TG levels in a mouse model of non-alcoholic fatty liver disease induced by a high-fat diet (P < 0.05), and its improvement effect was significantly greater than that of control groups 1-3 and the positive control group 4 (metformin group). These results indicate that compound 1-18 significantly improves non-alcoholic fatty liver disease, and is significantly superior to its analogues and positive control drugs, achieving unexpected results.

[0104] Table 2. Serum marker test results for each group

[0105]

[0106]

[0107] Example 3: Anti-liver fibrosis activity of compounds 1-18 obtained in Example 1

[0108] Experimental methods: The high-fat diet-induced NAFLD model can better reflect the liver histopathology of human NAFLD, but it is difficult to induce liver fibrosis. CCL4 causes hepatocyte degeneration and necrosis by damaging the plasma membrane, initiating lipid peroxidation, and destroying the membrane structure of hepatocytes, thereby inducing hepatotoxicity, fibrosis, lipid peroxidation, and cell membrane damage [5].

[0109] A CCl4-induced liver fibrosis mouse model was established: SPF-grade C57BL / 6 mice (approximately 22–25 g) were acclimatized for one week and then randomly divided into several groups according to body weight, including a blank control group, a model group, a compound 1-18 intervention model group, and control group 1-4 drug intervention model group (compound AC intervention and positive control group 4 nintedanib group), with 15 mice in each group. The blank control group was intraperitoneally injected with olive oil (2 mL / kg), while the model group, drug intervention group, and control group were intraperitoneally injected with 10% CCl4 (diluted with olive oil), twice a week. Mice in each group were administered medications via gavage. Mice in the blank control group and model group received the same volume of physiological saline as the drug groups, while the model group and drug intervention group received an appropriate volume of 60 mg / kg / day of compound 1-18, control drugs 1-3 (compounds A, B, and C, respectively), and positive control 4 nintedanib, twice daily. The mice were weighed weekly for 6 weeks. Mice were fasted for 12 hours before sampling. They were anesthetized with tribromoethanol solution (300 mg / kg), the thoracic cavity was opened, and blood was collected from the right atrium. The blood was centrifuged to separate the supernatant serum, and biochemical indicators such as ALT, AST, and TG were detected to evaluate liver function. Liver tissue was taken for HE and MASSON staining, and the liver activity score (NAS) was used for histological scoring. The results are shown below.

[0110] From the liver HE images of each group ( Figure 2 It can be seen that the liver fibrosis process of compound 1-18 intervention was significantly reduced compared with the model group, and the liver inflammatory cell infiltration of compound 1-18 drug intervention group was significantly reduced, indicating that hypoxanthine derivative 1-18 has a significant anti-liver fibrosis effect, and is superior to control group 1-3 (hypoxanthine derivative AC) and control group 4 nintedanib.

[0111] Table 3 shows the liver fibrosis scoring table for each group of mice. Compared with the model group, compounds 1-18 showed significant anti-liver fibrosis effects (P<0.05), and were significantly better than control group 1-3 (i.e., hypoxanthine derivative AC) and positive control group 4 (nintedanib) (P<0.05).

[0112] Table 3. Liver fibrosis scoring table for each group of mice

[0113] Group NAFLD score Group NAFLD score Blank control group 0.41±0.2 Compound 7 1.1±0.3 Model group 4.35±0.7 Compound 8 1.2±0.3 Control group 1 4.2±0.4 Compound 9 1.3±0.4 Control group 2 4.2±0.5 Compound 10 1.4±0.2 Control group 3 4.4±0.4 Compound 11 1.2±0.2 Control group 4 3.8±0.4 Compound 12 0.7±0.3 Compound 1 1.1±0.3 Compound 13 2.4±0.4 Compound 2 1.7±0.3 Compound 14 2.8±0.3 Compound 3 0.6±0.1 Compound 15 1.5±0.2 Compound 4 1.5±0.3 Compound 16 1.7±0.2 Compound 5 2.1±0.1 Compound 17 1.8±0.3 Compound 6 1.4±0.3 Compound 18 1.7±0.3

[0114] Based on Masson staining to detect collagen deposition in a liver fibrosis model Figure 3 The study also quantified liver fibrosis (collagen volume fraction, Table 4). The results showed that hypoxanthin derivative compound 1-18 significantly reduced carbon tetrachloride-induced liver fibrosis, and its effect was significantly better than that of the positive control drug nintedanib (control group 4), and better than that of hypoxanthin derivative compound 1-3 (control group 1-3).

[0115] Table 4. Comparison of semi-quantitative analysis results of Masson staining collagen volume fraction in liver tissue of mice in each group.

[0116] Group Collagen volume fraction (%) Group Collagen volume fraction (%) Blank control group 0.7±0.09 Compound 7 4.3±0.4 Model group 20.5±2.7 Compound 8 2.8±0.3 Control group 1 19.8±1.8 Compound 9 3.8±0.4 Control group 2 21.5±2.5 Compound 10 2.7±0.4 Control group 3 18.5±1.5 Compound 11 4.2±0.5 Control group 4 17.8±11.4 Compound 12 4.8±0.6 Compound 1 2.5±0.3 Compound 13 1.5±0.5 Compound 2 3.2±0.2 Compound 14 1.8±0.5 Compound 3 1.7±0.2 Compound 15 2.5±0.3 Compound 4 4.4±0.4 Compound 16 2.7±0.2 Compound 5 5.5±0.4 Compound 17 3.1±0.6 Compound 6 3.6±0.2 Compound 18 2.0±0.4

[0117] As shown in Table 5, compound 1 obtained in Example 1 significantly inhibited liver index, serum ALT, AST, and tissue hydroxyproline (HyP) and TGF-β in a mouse model of CCl4-induced liver fibrosis. Table 5 clearly shows that the CCl4-induced liver fibrosis mouse model was significantly improved under the action of compounds 1-18, indicating that the compounds obtained in Example 1 have a significant anti-liver fibrosis effect, and the improvement effect is significantly better than that of control drugs 1-3 and the positive control nintedanib.

[0118] Table 5. Serum ALT, AST, and tissue hydroxyproline (HyP) and TGF-β levels in each group

[0119]

[0120]

[0121] Example 4: Compound 1-18 obtained in Example 1 exhibits anti-nonalcoholic fatty liver disease and liver fibrosis activity.

[0122] Experimental methods: A mouse model of non-alcoholic fatty liver disease and liver fibrosis induced by high-fat diet and CCl4 was established [6]: SPF-grade C57BL / 6 mice (weighing about 22-25g) were acclimatized for 1 week, and SPF-grade male C57BL / 6 mice (weighing about 22-25g) were acclimatized for 1 week and then randomly divided into several groups according to body weight, including blank control group, model group, low concentration group of compound 1, medium concentration group of compound 1, and high concentration group of compound 1, with 15 mice in each group. Blank control group: fed with ordinary feed, model group and drug intervention group: fed with high-fat feed (78.8% basic feed + 10% lard + 10% egg yolk powder + 1% cholesterol + 0.2% sodium cholate); Blank control group: injected with olive oil (2mL / kg) intraperitoneally, model group and drug intervention group: injected with 10% CCl4 (diluted with olive oil) intraperitoneally twice a week. Starting from week 5, mice in each group underwent intraperitoneal gavage intervention. The blank control group and model group received the same volume of physiological saline as the drug group based on body weight. The model group and drug intervention group received an appropriate volume of 60 mg / kg / day of drug compound 1-18, control drugs 1-3 (compounds A, B, and C, respectively), and the positive control nintedanib, twice daily for 4 weeks. Mouse weight was measured weekly. Mice were fasted for 12 hours before sampling. Mice were anesthetized with tribromoethanol solution (300 mg / kg), the thoracic cavity was opened, and blood was collected from the right atrium. The blood was centrifuged to separate the supernatant serum, and biochemical indicators (ALT, AST, TG) were measured to evaluate liver function. Liver tissue was collected for HE and MASSON staining, and scored according to the NAFLD Activity Score (NAS) histological scoring criteria. The results are shown below.

[0123] From the liver HE images of each group ( Figure 4 It can be seen that the liver fibrosis process of compound 1-18 intervention was significantly reduced compared with the model group, and liver fibrosis and inflammatory cell infiltration were significantly reduced in the compound 1-18 drug intervention group, indicating that hypoxanthine derivative 1-18 has significant anti-liver fibrosis and anti-inflammatory effects, and is superior to the control group 1-3 (hypoxanthine derivative AC) and the control drug nintedanib.

[0124] Table 6 shows the liver tissue fibrosis scoring table for each group of mice. Compared with the model group, compounds 1-18 showed significant anti-liver fibrosis effects (P<0.05), and were significantly better than the control group 1-3, i.e., hypoxanthine derivative AC and the positive control nintedanib group (P<0.05).

[0125] Table 6. Liver fibrosis scoring table for each group of mice

[0126]

[0127]

[0128] Based on Masson staining to detect collagen deposition in a liver fibrosis model Figure 5 The study also quantified liver disease severity (collagen volume fraction, Table 7). The results showed that hypoxanthine derivative compound 1-18 significantly reduced non-alcoholic fatty liver disease and liver fibrosis induced by high-fat diet and carbon tetrachloride, and its effect was significantly better than the positive control drug nintedanib (compound 4) and better than hypoxanthine derivative compound 1-3 (control group 1-3).

[0129] Table 7. Comparison of semi-quantitative analysis results of Masson staining collagen volume fraction in liver tissue of mice in each group.

[0130] Group Collagen volume fraction (%) Group Collagen volume fraction (%) Blank control group 0.8±0.07 Compound 7 8.5±1.3 Model group 28.3±3.1 Compound 8 9.5±3.1 Control group 1 28.1±1.7 Compound 9 11.4±2.1 Control group 2 27.7±2.9 Compound 10 9.2±1.2 Control group 3 27.5±3.5 Compound 11 8.1±1.4 Control group 4 21.8±3.0 Compound 12 10.5±2.3 Compound 1 10.5±2.3 Compound 13 11.0±0.9 Compound 2 8.7±1.2 Compound 14 10.3±1.9 Compound 3 5.5±1.2 Compound 15 8.6±2.2 Compound 4 8.9±1.2 Compound 16 10.5±2.5 Compound 5 10.5±0.5 Compound 17 8.9±1.8 Compound 6 11.2±1.3 Compound 18 7.9±2.1

[0131] As can be seen from the data in Table 8, compounds 1-18 prepared in Example 1 have significant inhibitory effects on liver index, serum ALT, AST, and tissue hydroxyproline (HyP), TGF-β, TC (total cholesterol) and other indicators in a non-alcoholic fatty liver disease model induced by a high-fat diet and CCl4. This indicates that the compounds prepared in Example 1 have significant anti-non-alcoholic fatty liver disease and liver fibrosis effects, and the improvement effect is significant.

[0132] Table 8. Serum ALT, AST, and tissue hydroxyproline (HyP), TGF-β, and TC levels in each group

[0133] Group ALT (mmol / L) AST (mmol / L) Hyp (μg / g) TGF-β TC (mmol / L) Blank control group 13±2 16±3 134±14 1.1±0.3 2.1±0.2 Model group 75±8 121±13 421±51 4.9±0.5 6.3±0.5 Control group 1 72±9 130±16 428±40 4.9±0.5 5.7±0.4 Control group 2 75±8 128±14 421±47 4.8±0.5 5.9±0.3 Control group 3 72±4 119±20 431±51 4.7±0.5 5.8±0.5 Control group 4 58±7 105±5 385±21 3.7±0.2 6.0±0.3 Compound 1 24±2 28±9 201±35 2.1±0.3 2.8±0.4 Compound 2 21±6 31±5 167±24 1.9±0.4 3.0±0.3 Compound 3 17±3 22±5 164±17 1.5±0.4 2.5±0.5 Compound 4 27±5 51±6 204±23 1.9±0.2 3.2±0.3 Compound 5 21±5 41±5 241±25 2.1±0.4 2.9±0.4 Compound 6 31±6 46±5 218±35 2.2±0.3 3.5±0.5 Compound 7 26±4 51±5 196±17 2.1±0.4 2.4±0.3 Compound 8 31±4 35±6 196±24 1.9±0.3 3.1±0.4 Compound 9 19±6 41±5 204±27 1.8±0.1 2.6±0.5 Compound 10 21±6 54±8 212±27 1.9±0.2 2.7±0.3 Compound 11 19±5 28±5 191±17 1.7±0.3 3.4±0.4 Compound 12 29±8 35±6 216±18 2.3±0.3 2.3±0.5 Compound 13 22±3 41±5 251±27 2.5±0.4 3.3±0.3 Compound 14 24±6 26±9 167±20 2.2±0.3 2.5±0.4 Compound 15 30±7 50±6 235±22 1.8±0.2 3.0±0.5 Compound 16 23±9 39±5 215±18 2.0±0.5 2.8±0.3 Compound 17 32±7 40±7 190±26 1.6±0.4 3.2±0.5 Compound 18 21±2 33±9 223±19 2.5±0.3 3.1±0.4

[0134] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

[0135] References

[0136] 1. Guo X, Yin

[0137] 2. Kleiner, DE & Makhlouf, HR, 2016. Histology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis in Adults and Children. Clin Liver Dis, 20, 293-312.

[0138] 3. Li Qingqing, Yang Hongsheng, Li Jindou, Fang Meng, Liu Yuwei. Research progress on the pathogenesis of liver fibrosis and the anti-liver fibrosis effect of traditional Chinese medicine [J]. Journal of Jianghan University (Natural Science Edition), 2023, 51(03):75-81.

[0139] 4. Geng Y, Li L, Yan J, Liu K, Yang A, Zhang L, Shen Y, Gao H, Wu X, Noth I, Huang Y, Liu J, Fan

[0140] 5. Fang Chunqiu, Zhang Wenjun, Zhang Jingzhou, et al. Research status on the construction and application of common animal models of liver injury [J]. Chinese Journal of Clinical Pharmacology, 2022, 38(03):276-280.

[0141] 6. Tsuchida T, Lee YA, Fujiwara N, Ybanez M, Allen B, Martins S, Fiel MI, Goossens N, Chou HI, Hoshida Y, Friedman SL. A simplediet-and chemical-inducedmurine NASH model with rapid progression of steatohepatitis, fibrosis and liver cancer. J Hepatol. 2018Aug; 69(2):385-395.

Claims

1. The use of a hypoxanthine derivative in the preparation of a drug for treating non-alcoholic fatty liver disease and / or liver fibrosis, characterized in that, The hypoxanthin derivative has therapeutic activity for non-alcoholic fatty liver disease and / or liver fibrosis, and the hypoxanthin derivative has one of the following structures: in: R1 can be any of O, N, C, S or = O; R2, R3, and R4 can be chosen as H and C1-C, respectively. 18 Alkyl or halogen-substituted C1-C 18 Alkyl, trifluoromethyl, sulfonyl, sulfonamide, sulfinyl, amino acid, 2-[bis(neopentyloxy)methoxy]phosphonomethoxyethyl, C1-C 18 Fatty acid group, C3-C 12 Heterocyclic groups, C1-C 18 Fatty acids; or C1-C atoms in which R2, R3, and R4 are optionally substituted with oxygen, sulfur, or nitrogen atoms. 18 The alkyl or fatty acid group; when R3 or R4 is substituted, the double bond is attached to the unsubstituted N position, and when all are substituted, there is no double bond.

2. The use of the hypoxanthine derivative according to claim 1 in the preparation of drugs for treating non-alcoholic fatty liver disease and / or liver fibrosis, characterized in that, The hypoxanthine derivatives mentioned are one or more of the following compounds:

3. The use of the hypoxanthine derivative according to claim 1 in the preparation of drugs for treating non-alcoholic fatty liver disease and / or liver fibrosis, characterized in that, The medications mentioned for treating non-alcoholic fatty liver disease and / or liver fibrosis include those that have the efficacy of preventing and treating non-alcoholic fatty liver disease and / or liver fibrosis and their complications.

4. The use of the hypoxanthine derivative according to claim 1 in the preparation of a drug for treating non-alcoholic fatty liver disease and / or liver fibrosis, characterized in that, The aforementioned drug for treating non-alcoholic fatty liver disease and / or liver fibrosis is a formulation prepared with hypoxanthine derivatives or their salts as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients added.

5. The use of the hypoxanthine derivative according to claim 4 in the preparation of a drug for treating non-alcoholic fatty liver disease and / or liver fibrosis, characterized in that, The preparation is an oral preparation, an injectable preparation, or a nasal mucosal delivery preparation.

6. The use of the hypoxanthine derivative according to claim 1 in the preparation of a drug for treating non-alcoholic fatty liver disease and / or liver fibrosis, characterized in that, The non-alcoholic fatty liver disease includes one or more of idiopathic non-alcoholic fatty liver disease and secondary non-alcoholic fatty liver disease, and the liver fibrosis includes one or more of idiopathic liver fibrosis and secondary liver fibrosis.

7. Compounds having the following structure: in: R1 can be any of O, N, C, S or = O; R2, R3, and R4 can be chosen as H and C1-C, respectively. 18 Alkyl or halogen-substituted C1-C 18 Alkyl, trifluoromethyl, sulfonyl, sulfonamide, sulfinyl, amino acid, 2-[bis(neopentyloxy)methoxy]phosphonomethoxyethyl, C1-C 18 Fatty acid group, C3-C 12 Heterocyclic groups, C1-C 18 Fatty acids; or C1-C atoms in which R2, R3, and R4 are optionally substituted with oxygen, sulfur, or nitrogen atoms. 18 The alkyl or fatty acid group; when R3 or R4 is substituted, the double bond is attached to the unsubstituted N position, and when all are substituted, there is no double bond.

8. The compound according to claim 7, selected from the group consisting of:

9. A pharmaceutical composition comprising the compound of claim 7 or 8 or a pharmaceutically acceptable salt thereof.

10. The pharmaceutical composition according to claim 9, further comprising a pharmaceutically acceptable excipient or auxiliary ingredient.

11. The pharmaceutical composition according to claim 9 or 10, wherein it is an oral formulation, an injectable formulation, or a nasal mucosal administration formulation.

12. A method for treating non-alcoholic fatty liver disease and / or liver fibrosis, characterized in that, Administer to an individual in need an effective amount of the compound of claim 7 or 8 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claims 9-11.

13. The method according to claim 12, characterized in that, The non-alcoholic fatty liver disease includes one or more of idiopathic non-alcoholic fatty liver disease and secondary non-alcoholic fatty liver disease, and the liver fibrosis includes one or more of idiopathic liver fibrosis and secondary liver fibrosis.

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