Application of 2H-pyrazolo [3, 4-d] pyrimidine derivative in preparation of medicine for preventing, relieving, treating and / or adjunctively treating non-alcoholic steatohepatitis

Abstract

The invention provides application of 2H-pyrazolo [3, 4-d] pyrimidine derivatives in preparation of drugs for prevention, alleviation, treatment and / or adjuvant therapy of non-alcoholic steatohepatitis, and belongs to the technical field of biological medicines. The 2H-pyrazolo [3, 4-d] pyrimidine derivative disclosed by the invention has a good effect on treating non-alcoholic steatohepatitis and has no adverse reaction. It is verified that the 2H-pyrazolo [3, 4-d] pyrimidine derivative can improve liver fat accumulation of two different mouse NASH models induced by high-fat, high-cholesterol and high-fructose diet and methionine and choline deficiency diet, inhibit liver inflammation and relieve liver fibrosis progression, and can be used for preparing the compound. The pharmaceutical composition has a good application prospect in the aspect of preventing, treating, relieving or improving the non-alcoholic steatohepatitis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the use of 2H-pyrazolo[3,4-d]pyrimidine derivatives in the preparation of drugs for preventing, alleviating, treating and / or assisting in the treatment of non-alcoholic fatty hepatitis. Background Art

[0002] Non-alcoholic steatohepatitis (NASH), also known as metabolic steatohepatitis, is a more serious type of non-alcoholic fatty liver disease (NAFLD) in the disease spectrum. It is a chronic liver disease characterized by liver cell degeneration and necrosis, accompanied by varying degrees of inflammatory cell infiltration or interstitial fibrosis. It can progress to cirrhosis and induce hepatocellular carcinoma. The FDA, EMA and NMPA have not approved any drug for NAFLD and NASH, and there is still a huge treatment gap for NASH.

[0003] Currently, there are no reports on the use of 2H-pyrazolo[3,4-d]pyrimidine derivatives in the treatment of NASH. Summary of the invention

[0004] The object of the present invention is to provide the use of 2H-pyrazolo[3,4-d]pyrimidine derivatives in the preparation of drugs for preventing, alleviating, treating and / or assisting in the treatment of non-alcoholic steatohepatitis.

[0005] The present invention provides the use of 2H-pyrazolo[3,4-d]pyrimidine derivatives in the preparation of drugs for preventing, alleviating, treating and / or assisting in the treatment of non-alcoholic steatohepatitis; the chemical structural formula of the 2H-pyrazolo[3,4-d]pyrimidine derivatives is shown in Formula I:

[0006]

[0007] Preferably, the drug is a unit dose preparation based on mice; the unit dose preparation is formulated in a dosage form for administration of 1 to 500 mg / kg of mouse body weight.

[0008] Preferably, the drug further comprises a pharmaceutically acceptable excipient of a 2H-pyrazolo[3,4-d]pyrimidine derivative; the excipient comprises one or more of a salt, an ester and a hydrate.

[0009] Preferably, the salt comprises an organic salt and / or an inorganic salt.

[0010] Preferably, the organic salt includes one or more of methanesulfonate, formate, acetate, trifluoroacetate, maleate, tartrate, succinate, fumarate, citrate, benzenesulfonate, p-toluenesulfonate, naphthalenesulfonate, lactate and benzoate; the inorganic salt includes one or more of hydrochloride, hydrobromide, sulfate and phosphate.

[0011] Preferably, the dosage form of the drug includes one or more of oral preparations, injections, implants, external preparations, sprays and inhalants.

[0012] Preferably, the oral preparation includes an oral solid preparation or; the oral solid preparation includes a tablet, a capsule, a pill, a granule or a powder; the oral liquid preparation includes a syrup, a tincture, an alcoholic preparation or a dew; the implant includes a suppository; the spray includes an aerosol; the external preparation includes a film.

[0013] Preferably, the 2H-pyrazolo[3,4-d]pyrimidine derivative is the only active ingredient of the drug.

[0014] Preferably, the non-alcoholic steatohepatitis includes non-alcoholic steatohepatitis induced by a high-fat, high-cholesterol, and high-fructose diet and / or non-alcoholic steatohepatitis induced by a methionine and choline deficient diet.

[0015] Preferably, the prevention and / or treatment of non-alcoholic fatty liver disease includes any one of the following: 1) inhibiting liver fat accumulation; 2) inhibiting fatty vacuolar degeneration of liver tissue; 3) inhibiting hepatocyte ballooning lesions; 4) inhibiting liver inflammation; 5) improving liver damage; 6) alleviating liver fibrosis and / or slowing down the progression of liver tissue fibrosis; 7) maintaining body weight; 8) reducing serum alanine aminotransferase and / or aspartate aminotransferase activity; 9) reducing the content of triglycerides and / or cholesterol in liver tissue; 10) reducing liver collagen deposition.

[0016] The present invention provides the use of 2H-pyrazolo[3,4-d]pyrimidine derivatives in the preparation of drugs for preventing, alleviating, treating and / or assisting in the treatment of non-alcoholic fatty liver disease. 2H-pyrazolo[3,4-d]pyrimidine derivatives have good efficacy in treating non-alcoholic fatty liver disease and have no adverse reactions. It has been verified that 2H-pyrazolo[3,4-d]pyrimidine derivatives can improve liver fat accumulation, inhibit liver inflammation and alleviate liver fibrosis progression in two different mouse NASH models induced by a high-fat, high-sugar, high-cholesterol diet and a methionine choline-deficient diet, and have good application prospects in preventing, treating, alleviating or improving non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 This is the weight change curve of each group of mice in the high-fat model;

[0019] Figure 2 The following are the graphs of the activities of alanine aminotransferase and aspartate aminotransferase in the serum of each group of mice in the high-fat model, as well as the contents of triglycerides and total cholesterol in liver tissue (*, p<0.05, i.e., statistically significant differences);

[0020] Figure 3 This is the staining result of liver tissue pathology sections of mice in each group of high-fat model;

[0021] Figure 4 This is the body weight change curve of mice in each group of MCD model;

[0022] Figure 5 The graphs are the activity of alanine aminotransferase and aspartate aminotransferase in the serum of mice in each group of the MCD model, and the content of triglyceride and total cholesterol in liver tissue (*, p<0.05, i.e., statistically significant difference);

[0023] Figure 6 These are the results of staining of liver tissue pathological sections of mice in each group of the MCD model. DETAILED DESCRIPTION

[0024] The present invention provides the use of 2H-pyrazolo[3,4-d]pyrimidine derivatives in the preparation of drugs for preventing, alleviating, treating and / or assisting in the treatment of non-alcoholic steatohepatitis; the chemical structural formula of the 2H-pyrazolo[3,4-d]pyrimidine derivatives is shown in Formula I:

[0025]

[0026] In the specific implementation of the present invention, the preparation method of the 2H-pyrazolo[3,4-d]pyrimidine derivative refers to the preparation method of compound 9 disclosed in patent CN 112574216A (see Example 9 for details).

[0027] In patent CN 112574216A, the chemical structure of compound 9 is shown in formula II.

[0028]

[0029] In the specific implementation of the present invention, the drug is a unit dose preparation based on mice; the unit dose preparation is formulated into a dosage form for administration of 1 to 500 mg / kg mouse body weight, preferably, the unit dose preparation is formulated into a dosage form for administration of 6.25 to 25 mg / kg mouse body weight.

[0030] In the specific implementation process of the present invention, the drug also includes a pharmaceutically acceptable excipient of a 2H-pyrazolo[3,4-d]pyrimidine derivative; the excipient includes one or more of a salt, an ester and a hydrate; the salt includes an organic salt and / or an inorganic salt; the organic salt includes one or more of methanesulfonate, formate, acetate, trifluoroacetate, maleate, tartrate, succinate, fumarate, citrate, benzenesulfonate, p-toluenesulfonate, naphthalenesulfonate, lactate and benzoate; the inorganic salt includes one or more of a hydrochloride, a hydrobromide, a sulfate and a phosphate.

[0031] In the specific implementation of the present invention, the dosage form of the drug includes one or more of oral preparations, injections, implants, external preparations, sprays and inhalants.

[0032] In the specific implementation process of the present invention, the oral preparation includes oral solid preparation or; the oral solid preparation includes tablets, capsules, pills, granules or powders; the oral liquid preparation includes syrup, tincture, wine or dew; the implant includes suppository; the spray includes aerosol; the external preparation includes film.

[0033] In the specific implementation of the present invention, the 2H-pyrazolo[3,4-d]pyrimidine derivative is the only active ingredient of the drug.

[0034] In the specific implementation of the present invention, the non-alcoholic fatty liver disease includes non-alcoholic fatty liver disease induced by a high-fat, high-sugar, and high-cholesterol diet and / or non-alcoholic fatty liver disease induced by a methionine choline-deficient diet.

[0035] In the specific implementation process of the present invention, the prevention and / or treatment of non-alcoholic fatty liver disease includes any one of the following: 1) inhibiting liver fat accumulation; 2) inhibiting fatty vacuolar degeneration of liver tissue; 3) inhibiting ballooning lesions of liver cells; 4) inhibiting liver inflammation; 5) improving liver damage; 6) alleviating liver fibrosis and / or slowing down the progression of liver tissue fibrosis; 7) maintaining body weight; 8) reducing serum alanine aminotransferase and / or aspartate aminotransferase enzyme activity; 9) reducing the content of triglycerides and / or cholesterol in liver tissue; 10) reducing liver collagen deposition.

[0036] In the specific implementation process of the present invention, 2H-pyrazolo[3,4-d]pyrimidine derivatives can alleviate the weight gain induced by a high-fat, high-sugar, high-cholesterol diet, reduce the activity of serum alanine aminotransferase and aspartate aminotransferase, reduce the content of triglycerides and cholesterol in liver tissue, improve liver damage and fat accumulation, and 2H-pyrazolo[3,4-d]pyrimidine derivatives can alleviate fatty degeneration and inflammatory cell aggregation, reduce liver collagen deposition, and thus alleviate liver fibrosis of NASH mice induced by a high-fat diet to a certain extent. 2H-pyrazolo[3,4-d]pyrimidine derivatives can also improve liver fat accumulation induced by a high-fat diet.

[0037] In the specific implementation process of the present invention, 2H-pyrazolo[3,4-d]pyrimidine derivatives can significantly alleviate the weight loss induced by the methionine choline deficiency diet (MCD) and improve health status. 2H-pyrazolo[3,4-d]pyrimidine derivatives can also reduce the activity of serum alanine aminotransferase and aspartate aminotransferase and reduce the content of triglycerides and cholesterol in liver tissue, thereby improving liver damage and fat accumulation. 2H-pyrazolo[3,4-d]pyrimidine derivatives can also alleviate fatty degeneration and inflammatory cell aggregation, as well as alleviate liver collagen accumulation induced by the MCD diet, thereby alleviating liver fibrosis of NASH mice induced by the MCD diet to a certain extent.

[0038] In order to further illustrate the present invention, the use of the 2H-pyrazolo[3,4-d]pyrimidine derivatives provided by the present invention in the preparation of drugs for preventing, alleviating, treating and / or assisting in the treatment of non-alcoholic fatty hepatitis is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0039] The preparation method of the 2H-pyrazolo[3,4-d]pyrimidine derivative used in the following examples refers to the preparation method of compound 9 disclosed in patent CN112574216A (see Example 9 for details). In patent CN 112574216A, the chemical structural formula of compound 9 is shown in Formula II. In the following examples, the 2H-pyrazolo[3,4-d]pyrimidine derivative is referred to as compound 1.

[0040] Example 1

[0041] Effect of compound 1 on the NASH model of mice induced by a high-fat, high-sugar, and high-cholesterol diet

[0042] 1. Preparation of NASH animal model

[0043] C57BL / 6J, (8-10 weeks old) wild-type mice were fed a high-fat, high-sugar, high-cholesterol diet (composed of the following components in mass percentage: 78.8% basal feed + 10% lard + 10% egg yolk powder + 1% cholesterol + 0.2% sodium cholate) and high-sugar water (23.1 g fructose and 18.9 g glucose dissolved in 1 L water) for 16 weeks.

[0044] 2. Grouping

[0045] Control group: intragastrically injected with the same volume of normal saline;

[0046] Model group: The mice were given a high-fat, high-sugar, high-cholesterol diet and high-sugar water as described above to establish the model, starting from the first day until the end of the experiment, for a total of 16 weeks;

[0047] Positive drug group: starting from the 5th week, mice were given 100 mg / kg silymarin positive drug once a day by oral gavage for 12 consecutive weeks;

[0048] Compound 1 low-dose group: starting from the 5th week, mice were given 6.25 mg / kg of compound 1 twice a day by oral gavage for 12 consecutive weeks;

[0049] Compound 1 high-dose group: starting from the 5th week, mice were given 25 mg / kg of Compound 1 twice a day by oral gavage for 12 consecutive weeks.

[0050] The body weight of mice was recorded once a week, and the body weight, liver weight, and liver appearance of mice were recorded at the end of the 16th week; serum and liver tissue biochemical indices were subsequently tested, and pathological sections were stained to detect the improvement in lipid accumulation, inflammation, and fibrosis.

[0051] 3. Record of mouse weight changes

[0052] The weight of mice was recorded every week for 16 weeks. The weight of mice was counted and the weight changes of mice were observed for 16 weeks.

[0053] 4. Detection of serum alanine aminotransferase and aspartate aminotransferase activity

[0054] On the last day of the experiment, blood was collected from the mice by orbital vein, and the blood was placed in a 4°C refrigerator overnight. The next day, the blood was centrifuged at 3500g and 4°C for 10 minutes, and the supernatant was placed on ice for testing. The activity of alanine aminotransferase and aspartate aminotransferase in serum was detected using the ALT Activity Detection Kit (BC1555-100T / 48S, Solebao) and the AST Activity Detection Kit (BC1565-100T / 48S, Solebao). The standard (sodium pyruvate) was diluted to 2μmol / mL with distilled water, and the standard and reagent one were mixed to obtain a concentration gradient standard tube. 5μl of the sample to be tested and 25μl of reagent one were added to the 96-well plate, mixed, and reacted at 37°C for 30min. Then 25μl of reagent two was added, mixed, and reacted at 37°C for 20min. Finally, 240μl of reagent three was added, mixed, and placed at room temperature for 10min, and the absorbance of each tube was measured at a wavelength of 505nm. The test tube and control tube are added with 5μl of the sample to be tested when adding reagent 1 and reagent 2. (Note: Reagent 1, reagent 2 and reagent 3 in this paragraph are all reagents in the kit.) The standard curve is made with the concentration of each standard solution as the x-axis and ΔA (A standard tube-A blank tube) as the y-axis to obtain the equation y=kx+b. Substitute (A test tube-A control tube) into the equation to find the x value. Then calculate the activity of alanine aminotransferase and aspartate aminotransferase in unit volume of serum by volume.

[0055] 5. Detection of triglyceride content in liver tissue

[0056] Weigh about 0.1g of mouse liver tissue, add 1mL of reagent 1 (n-heptane and isopropanol mixed in a volume ratio of 1:1) and homogenize in an ice bath. Centrifuge at 8000g, 4℃ for 10min, take the supernatant and place it on ice for testing. Use the triglyceride content detection kit (BC0625-100T / 96S, Solebao) to detect the triglyceride content in liver tissue. Prepare several EP tubes, first add 120μl of the sample or standard to be tested, and 375μl of reagent 1 and 75μl of reagent 2. After adding reagent 1, mix thoroughly, then add reagent 2, shake vigorously for 30s, let stand for 3-5min, and then shake vigorously for 30s, repeat this 3 times, let stand for a certain period of time at room temperature, and then take 30μl of the upper solution and place it in a new EP tube. Then add 100μl of reagent 3 and 30μl of reagent 4, mix thoroughly, bath at 65℃ for 3min, and cool. Then add 100μl of reagent 5 and 100μl of reagent 6, mix thoroughly, place in a 65℃ water bath for 15min, and cool. (Note: Reagent 1, reagent 2, reagent 3, reagent 4, reagent 5, and reagent 6 in this paragraph are all reagents in the kit.) After cooling, draw 200μl into a 96-well plate to measure the absorbance at 420nm, and record it as A blank, A standard, and A test. Finally, calculate the content of triglyceride per unit mass of liver tissue according to the sample mass.

[0057] 6. Detection of total cholesterol content in liver tissue

[0058] Weigh about 0.1g of mouse liver tissue, add 1mL of extract (isopropanol) and homogenize in an ice bath. Centrifuge at 10000g, 4℃ for 10min, take the supernatant and place on ice for testing. Use the total cholesterol content detection kit (BC1985-100T / 96S, Solebao) to detect the total cholesterol content in liver tissue. Dilute the 50μmol / mL cholesterol standard solution with isopropanol to obtain 2.5, 2, 1.25, 0.625, 0.3125, 0.15625μmol / mL standard solutions for use. Add 20μl of the sample to be tested, the standard or blank control, and 180μl of the working solution to a 96-well plate, mix well, and let stand at 37℃ for 15min. After the reaction is completed, measure the absorbance A at 500nm, and record them as A determination, A standard and A blank, respectively. ΔA determination = A determination - A blank, ΔA standard = A standard - A blank. According to the concentration of the standard tube (x, μmol / mL) and the absorbance ΔA standard (y, ΔA standard), a standard curve is established. According to the standard curve, the ΔA determination (y, ΔA determination) is substituted into the formula to calculate the sample concentration (x, μmol / mL). Finally, the total cholesterol content per unit mass of liver tissue is calculated according to the sample mass.

[0059] 7. Staining of liver tissue pathology sections

[0060] Mouse liver tissue was fixed with paraformaldehyde, routinely dehydrated, embedded in paraffin, sliced, stained with H&E and Sirius red, and sealed with neutral gum. H&E and Sirius red staining image data were collected using an upright microscope. Oil red staining used frozen sections. Fresh mouse liver tissue of appropriate size was taken, fixed with embedding agent, placed in a -80℃ refrigerator overnight, sliced, and stained with oil red before staining with an upright microscope.

[0061] 8. Experimental results

[0062] The experimental results are as follows Figure 1 to Figure 3 As shown by Figure 1 It can be seen that after feeding a high-fat, high-sugar, and high-cholesterol diet for 16 weeks, the high-fat model group had a greater weight gain than the control group, and high-dose compound 1 could significantly alleviate the weight gain of mice induced by a high-fat diet. Figure 2 It can be seen that compound 1 significantly reduced the serum alanine aminotransferase and aspartate aminotransferase activities of NASH mice induced by a high-fat diet, and reduced the content of triglycerides and cholesterol in liver tissue, indicating that compound 1 can improve liver damage and fat accumulation, and p<0.05, that is, there is a statistically significant difference. Figure 3It can be seen that H&E staining observations showed that the liver tissue of the control group was tightly arranged, without fat cavitation and degeneration, and without inflammation. The liver tissue of the high-fat model group showed a large amount of fat cavitation and deformation of liver tissue, balloon-like deformation of liver cells, and inflammatory cells gathered around the portal vein. Fatty degeneration and inflammatory cell aggregation were significantly alleviated after the administration of compound 1. Sirius red staining observations showed that compared with the control group, the collagen deposition in the liver tissue of the high-fat model group increased, and the liver collagen deposition decreased after the administration of compound 1, indicating that compound 1 can alleviate the liver fibrosis of NASH mice induced by high-fat diet to a certain extent. Oil red staining observations showed that compared with the control group, there was a large amount of fat accumulation in the liver tissue of the high-fat model group, and high-dose compound 1 can significantly improve the fat accumulation in the liver of mice induced by high-fat diet.

[0063] Example 2

[0064] Effects of compound 1 on the NASH model of mice induced by methionine and choline deficient diet

[0065] 1. Preparation of NASH animal model

[0066] C57BL / 6J (8-10 weeks old) wild-type mice were fed a methionine choline-deficient diet (MCD) (MCD feed was purchased from Beijing Ke'ao Xieli Feed Co., Ltd.) for 8 weeks.

[0067] 2. Grouping

[0068] Control group: intragastrically injected with the same volume of normal saline;

[0069] Model group: The mice were given a methionine and choline deficient diet as described above, starting from the first day until the end of the experiment, which lasted for 8 weeks;

[0070] Positive drug group: starting from the 5th week, mice were given 100 mg / kg of silymarin positive drug once a day by oral gavage for 4 consecutive weeks;

[0071] Compound 1 low-dose group: starting from the 5th week, mice were given 6.25 mg / kg of compound 1 twice a day by oral gavage for 4 consecutive weeks;

[0072] Compound 1 high-dose group: starting from the 5th week, mice were given 25 mg / kg of Compound 1 twice a day by oral gavage for 4 consecutive weeks.

[0073] The body weight of mice was recorded once a week, and the body weight, liver weight, and liver appearance of mice were recorded at the end of the 8th week; serum and liver tissue biochemical indices were subsequently tested, and pathological sections were stained to detect the improvement in lipid accumulation, inflammation, and fibrosis.

[0074] 3. Record of mouse weight changes

[0075] The weight of mice was recorded every week for 8 weeks. The weight of mice was counted and the weight changes of mice were observed for 8 weeks.

[0076] 4. The specific methods for detecting the activities of alanine aminotransferase and aspartate aminotransferase in serum, detecting the contents of triglycerides and total cholesterol in liver tissue, and staining of liver tissue pathological sections are similar to those in Example 1, as described above.

[0077] 5. Experimental results

[0078] The experimental results are as follows Figures 4 to 6 As shown by Figure 4 It can be seen that after feeding the methionine choline-deficient diet for 8 weeks, the body weight of the MCD model group decreased significantly compared with the control group, and high-dose compound 1 can significantly alleviate the weight loss of mice induced by the MCD diet, indicating that compound 1 can improve the health of mice after treatment. Figure 5 It can be seen that compound 1 significantly reduced the serum alanine aminotransferase and aspartate aminotransferase activities of NASH mice induced by MCD diet, and reduced the content of triglycerides and cholesterol in liver tissue, indicating that compound 1 can improve liver damage and fat accumulation, and p<0.05, that is, there is a statistically significant difference. Figure 6 It can be seen that H&E staining observations showed that a large number of liver tissue fat vacuolation and deformation, balloon-like deformation of liver cells, and inflammatory cells gathered around the portal vein in the MCD model group. Fatty degeneration and inflammatory cell aggregation were significantly alleviated after the administration of compound 1, and the effect was obvious in the high-dose group. Sirius red staining observations showed that compared with the control group, the collagen deposition in the liver tissue of the MCD model group increased, and high-dose compound 1 could alleviate the liver collagen accumulation induced by the MCD diet, indicating that compound 1 could alleviate the liver fibrosis of NASH mice induced by the MCD diet to a certain extent.

[0079] In summary, compound 1 has good efficacy on non-alcoholic fatty liver disease and has no adverse reactions. Compound 1 can inhibit fatty vacuolar degeneration, hepatocyte ballooning lesions, fat accumulation and inflammation in the liver tissue of NASH mice induced by a high-fat, high-sugar, high-cholesterol diet and MCD diet, and slow down the progression of liver fibrosis in NASH mice induced by a high-fat, high-sugar, high-cholesterol diet and MCD diet. It has good application prospects in preventing, treating, alleviating or improving non-alcoholic fatty liver disease.

[0080] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of 2H-pyrazolo[3,4-d]pyrimidine derivatives in the preparation of drugs for preventing, alleviating, treating and / or assisting in the treatment of non-alcoholic steatohepatitis; the chemical structural formula of the 2H-pyrazolo[3,4-d]pyrimidine derivatives is shown in Formula I:

2. The use according to claim 1, characterized in that: The drug is a unit dose preparation for mice; the unit dose preparation is formulated into a dosage form for administration of 1 to 500 mg / kg of mouse body weight.

3. The use according to claim 1, characterized in that: The drug further comprises a pharmaceutically acceptable auxiliary material of a 2H-pyrazolo[3,4-d]pyrimidine derivative; the auxiliary material comprises one or more of a salt, an ester and a hydrate.

4. The use according to claim 3, characterized in that: The salt includes organic salt and / or inorganic salt.

5. The use according to claim 4, characterized in that: The organic salt includes one or more of methanesulfonate, formates, acetates, trifluoroacetates, maleates, tartrates, succinates, fumarates, citrates, benzenesulfonates, p-toluenesulfonates, naphthalenesulfonates, lactates and benzoates; the inorganic salt includes one or more of hydrochlorides, hydrobromides, sulfates and phosphates.

6. The use according to claim 1, characterized in that: The dosage form of the drug includes one or more of oral preparations, injections, implants, external preparations, sprays and inhalants.

7. The use according to claim 6, characterized in that: The oral preparations include oral solid preparations or; the oral solid preparations include tablets, capsules, pills, granules or powders; the oral liquid preparations include syrups, tinctures, wine or dew; the implants include suppositories; the sprays include aerosols; the external preparations include films.

8. The use according to claim 1, characterized in that: The 2H-pyrazolo[3,4-d]pyrimidine derivative is the only active ingredient of the drug.

9. The use according to claim 1, characterized in that: The non-alcoholic steatohepatitis includes non-alcoholic steatohepatitis induced by a high-fat, high-cholesterol, and high-fructose diet and / or non-alcoholic steatohepatitis induced by a methionine and choline deficient diet.

10. The use according to claim 1, characterized in that: The prevention and / or treatment of non-alcoholic fatty liver disease includes any one of the following: 1) inhibiting liver fat accumulation; 2) inhibiting fatty vacuolar degeneration of liver tissue; 3) inhibiting hepatocyte ballooning lesions; 4) inhibiting liver inflammation; 5) improving liver damage; 6) alleviating liver fibrosis and / or slowing down the progression of liver tissue fibrosis; 7) maintaining body weight; 8) reducing serum alanine aminotransferase and / or aspartate aminotransferase activity; 9) reducing the content of triglycerides and / or cholesterol in liver tissue; 10) reducing liver collagen deposition.

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