Use of Compounds as CYP2E1 Inhibitors

Abstract

The present invention discloses the use of compounds as CYP2E1 inhibitors, in which the compound represented by formula (I) or a salt thereof targets CYP2E1 as an inhibitor, binds to the target, and exerts an inhibitory effect on CYP2E1. The inhibitors are useful for the prevention and treatment of tumors such as liver cancer, glioma, ovarian cancer, lung cancer, bladder cancer, and gallbladder cancer. The inhibitors are also useful for the prevention and treatment of other inflammation-related diseases, such as liver injury, fatty liver, hepatitis, liver fibrosis, pulmonary fibrosis, rheumatic fever and rheumatoid arthritis, pyemia, Alzheimer's disease, ischemic stroke, Parkinson's disease, hyperlipidemia, atherosclerosis, coronary artery disease, and diabetes.

Description

[Technical field]

[0001] The present invention relates to the field of targets and drugs for preventing and treating inflammation-related diseases, and in particular to the use of compounds as CYP2E1 inhibitors. [Background technology]

[0002] It is now recognized that the occurrence of many diseases, such as tumors (liver cancer, cervical cancer, nasopharyngeal cancer, colorectal cancer, glioma, lung cancer, etc.) and non-tumor diseases (Alzheimer's disease, Parkinson's syndrome, stroke, arteriosclerosis, diabetes, liver fibrosis, pulmonary fibrosis, etc.), is associated with inflammation, and the above diseases associated with inflammation are collectively called inflammation-mediated diseases (IMDs). In general, it is believed that a long-term unregulated inflammatory microenvironment is associated with the occurrence of inflammation-related diseases. In 2005, the 125th year since the founding of "Science," it was reported that "inflammation is the primary cause of all chronic diseases."

[0003] The tumor microenvironment plays a crucial role in the development and progression of tumors, and research on the tumor microenvironment (TME) has developed rapidly over the past decade. In 2010, Professor Karin M of the University of California, USA, pointed out in "Cell" that immunity and inflammation in the tumor microenvironment are closely related to the development and progression of tumors. In recent years, groundbreaking progress has been made in the immune research of TME, and the immunotherapeutic drugs PD-1 and PD-L1 that target the tumor microenvironment have been clinically successful and have become broad-spectrum antitumor drugs. The 2018 Nobel Prize in Physiology or Medicine was awarded to James P. Allison, an American immunologist, and Tasuku Honjo, a Japanese immunologist, for their outstanding and original contributions in the field.

[0004] Chronic uncontrolled inflammation is an important feature of the tumor microenvironment. Take liver cancer as an example. Liver cancer usually develops from hepatitis and liver cirrhosis. In addition to hepatoma extracellular mesenchymal cells, hepatic stellate cells are also an important component of the general tumor microenvironment, and the activation of hepatic stellate cells can cause collagen deposition. Persistent liver injury caused by factors such as alcohol abuse, nonalcoholic steatohepatitis (NASH), and chronic hepatitis B virus (HBV) infection can lead to oxygen deficiency and the formation of chronic uncontrolled inflammation, which is an important feature of the liver cancer tumor microenvironment.

[0005] Tumor development and progression are closely related to the tumor microenvironment, and drugs targeting the tumor microenvironment have already become effective means of treating liver cancer, such as immune checkpoint inhibitors PD-1 and PDL-1 and angiogenesis inhibitor bevacizumab. Drugs targeting the tumor microenvironment generally have a broad spectrum of antitumor activity, for example, the antiangiogenesis drug bevacizumab is useful for the treatment of colorectal cancer, non-small cell lung cancer, renal cell carcinoma, ovarian cancer, cervical cancer, and liver cancer, while immune checkpoint inhibitors PD-1 and PDL-1 are useful for the treatment of melanoma, lung cancer, breast cancer, liver cancer, pancreatic cancer, gastrointestinal tumors, gynecological tumors, urological tumors, myeloma, and lymphoma.

[0006] The success of drugs targeting immunity and angiogenesis in the tumor microenvironment has provided a reference for successful research on tumors. Indeed, conventional anti-inflammatory drugs, such as nonsteroidal anti-inflammatory drugs such as COX-2 inhibitors, have exerted great effects in terms of antipyresis, analgesia, anti-inflammation, and anti-rheumatism, but no satisfactory therapeutic effect has been observed against inflammation-related tumors, and currently there are no antitumor drugs that target the inflammatory microenvironment of tumors. The reason for this may be related to the existence of new targets of inflammation that have not yet been discovered in the chronic uncontrolled inflammatory microenvironment.

[0007] Cytochrome P450 2E1 (CYP2E1) is a protein mainly present in the endoplasmic reticulum of hepatocytes, and the applicant has discovered that the content of cytochrome P450 2E1 is the highest in the liver cytochrome P450 (CYP450), accounting for about 24.8%. The main function of CYP2E1 is metabolism, which is involved in the biological transformation of drugs, procarcinogens and environmental toxins, and can metabolize and activate more than 85 kinds of exogenous substances, including carcinogens such as nitrosamines, benzene, and 13-butadiene, to produce hepatotoxic and carcinogenic substances, and CYP2E1 can metabolize and activate nitrosamines, toluene, chloroform, acetone, and tobacco-specific carcinogen NNK.

[0008] The metabolism and activation of CYP2E1 is closely related to inflammation-related diseases such as tumors. In the case of liver cancer, various factors such as hepatitis viruses, nitrosamines, aflatoxins, and alcohol are involved in the development of liver cancer. Nitrosamines (N-nitrosamines) are strong carcinogens, and generate carcinogens through metabolic activation by CYP2E1 in vivo, which further form adducts with DNA to induce liver cancer. In traditional Chinese foods and beverages, such as aged meat products, bacon, ham, and pickles, the nitrosamine content often exceeds the standard value, and epidemiological studies have proven that the nitrosamine content in foods and beverages is closely related to the development of liver cancer. Animal experiments have found that CYP2E1 gene knockout mice significantly inhibited mouse liver cancer caused by diethylnitrosamine. It was suggested that CYP2E1 may affect the development of liver cancer by affecting the metabolic activation of nitrosamines in vivo.

[0009] The proinflammatory effect of CYP2E1 is closely related to inflammation-related diseases such as tumors. CYP2E1 has obvious proinflammatory effects and has been involved in the development and progression of many inflammation-related diseases. CYP2E1 is associated with inflammation-related tumors such as hepatocellular carcinoma, glioma, ovarian cancer, lung cancer, nasopharyngeal cancer, bladder cancer and gallbladder cancer, and CYP2E1 is also involved in the development and progression of other inflammation-related diseases such as liver injury, nonalcoholic steatohepatitis (NASH), liver fibrosis, rheumatoid arthritis, pyemia, Alzheimer's disease, hyperlipidemia, diabetes, ischemic stroke and pulmonary fibrosis. CYP2E1 enhances the release of TNF-α in Kupffer cells, causing inflammatory necrosis of hepatocytes. In non-alcoholic steatohepatitis (NASH) model mice, inhibiting CYP2E1 activity can prevent and treat the disease through reducing TNF-α expression and restoring endothelial nitric oxide synthase (eNOS) activity, and in cyp2e1 gene knockout mice, it can significantly inhibit the inflammatory response induced by chronic alcohol exposure. The CYP2E1 inhibitor allyl sulfide can prevent and treat non-alcoholic steatohepatitis by inhibiting CYP2E1 and reducing the release of IL-1β and IL-12.

[0010] The proinflammatory action of CYP2E1 is related to the promotion of oxidative stress and lipid peroxidation. CYP2E1 promotes the production of reactive oxygen species (ROS), which induces oxidative stress and lipid peroxidation, causing inflammation, apoptosis, and liver fibrosis in hepatocytes. High expression of CYP2E1 in hepatocytes promotes the production of ROS. ROS activates the cell surface molecule Fas ligand, a member of the tumor necrosis factor family, causing a protease binding reaction and leading to cell degradation and apoptosis. Apoptotic hepatocytes promote the aggregation of inflammatory cells, inducing the production of various inflammatory factors such as TNF-α and IL-6, causing liver inflammation and steatohepatitis. CYP2E1 also affects arachidonic acid (AA) metabolism, promoting the invasion and metastasis of hepatoma cells. Research has found that CYP2E1 increases AA toxicity mainly through reactive oxygen species and lipid peroxidation products. CYP2E1 and AA increase intracellular Ca through ROS peroxidation. 2+It activates phospholipase A2 (PLA2) by inducing the release of AA, promoting the production of AA. In hepatoma cells, AA is converted to prostaglandin 2 (PGE2) by the action of cyclooxygenase 2 (COX-2), binds to EP receptors linked to G proteins on the cell membrane, activates the EGFR / Met signaling pathway, and induces the invasion and metastasis of hepatoma cells.

[0011] In recent years, the applicant has established a relatively large liver sample bank, including 127 cases of healthy livers and 102 cases of cirrhotic livers of liver cancer patients, and conducted systematic research on the physiology and pathology of CYP450. The individual difference of CYP2E1 is about 10 times higher, and the CYP2E1 metabolic activity of liver cancer patients is significantly higher by about 2.13 times, with a positive rate of about 44.6%. Moreover, CYP2E1 activity is significantly negatively correlated with the postoperative survival time of patients, with the survival times of CYP2E1 positive and negative patients being 238 days and 612 days, respectively, and the increase in CYP2E1 activity is an independent risk factor for the development and progression of liver cancer. It has been proved by using a rat primary liver cancer model that the congenital activity of CYP2E1 (before the model was created) and the development of liver cancer have a good causal relationship, that is, the higher the congenital activity of CYP2E1, the more likely it is to develop liver cancer. These results suggest that CYP2E1 may be a new target for the prevention and treatment of liver cancer, and that drugs targeting CYP2E1, a new target in the tumor inflammatory microenvironment, may have a broad-spectrum preventive and therapeutic effect against inflammation-related diseases.

[0012] In conclusion, CYP2E1 is involved in the occurrence and progression of many inflammation-related diseases through its metabolic activation and proinflammatory effects, so inhibiting CYP2E1 activity is important for the prevention and treatment of related diseases, and therefore, the study of CYP2E1 inhibitors is of great theoretical and practical significance. Currently, there are no CYP2E1 specific inhibitors in clinical use. Compounds and drugs with CYP2E1 inhibitory activity that have been reported include 4-methylpyrazole, disulfiram, diethyldithiocarbamate, isothiocyanate, orphenadrine, and clomethiazole. Many of them have poor selectivity and toxicity for CYP2E1 inhibition, so they are mainly used in basic research. Currently, there are no CYP2E1 specific inhibitors in clinical use. Therefore, it is necessary to screen and synthesize CYP2E1 inhibitors based on the role of CYP2E1 in the development and progression of many diseases, including liver disease. Summary of the Invention

[0013] One aspect of the present invention discloses the use of a compound represented by formula (I) or a salt thereof as an inhibitor that targets CYP2E1, binds to the target, and exerts an inhibitory effect on CYP2E1 as a CYP2E1 inhibitor.

[0014] According to a first aspect of the present application, there is provided use of a compound represented by formula (I) or a salt thereof as an inhibitor, which selects CYP2E1 as a target, binds to the target, and exerts an inhibitory effect on CYP2E1 as a CYP2E1 inhibitor. [ka] (Wherein, R1 is hydrogen, C1 to C 10 is any one selected from the group consisting of alkyl, alkylene oxide, R2 is hydrogen, substituted C1-C 10 is any one selected from the group consisting of alkyl I, a substituent represented by formula O-1, a substituent represented by formula O-2, and a substituent represented by formula O-3; [ka] [ka] [ka] R21 , R 22 , R 23 , R 24 are independently hydrogen, alkoxy, halogen, C1-C3 alkyl, C6-C 10 At least one selected from the group consisting of aryl, R 25 is any one selected from hydroxy and alkoxy; R3 is hydrogen, C1-C 10 Alkyl, substituted C1-C 10 At least one selected from the alkyl groups II.

[0015] Preferably, the alkylene oxide is selected from butylene oxide.

[0016] Preferably, R1 is any one selected from hydrogen, C1-C4 alkyl, and alkylene oxide.

[0017] Preferably, R3 is at least one selected from hydrogen, C1-C4 alkyl, and substituted C1-C4 alkyl II.

[0018] Suitably, the compounds of formula I are used as CYP2E1 inhibitors.

[0019] Preferably, the substituted C1-C 10 The substituent in the alkyl I is at least one selected from the group consisting of a substituted amino I, a substituent represented by the formula M-1, and a substituent represented by the formula M-2. [ka] [ka]

[0020] Preferably, the substituents in the substituted amino I are C6-C 10 and substituted C1 to C3 alkyl III.

[0021] Preferably, the substituents in the substituted C1-C3 alkyl III are C6-C 10 The aryl is selected from the group consisting of

[0022] Preferably, the substituted C1-C 10 The substituent in the alkyl II is at least one selected from the group consisting of a substituted amino II, a substituent represented by the formula M-3, and a substituent represented by the formula M-4. [ka] [ka]

[0023] Preferably, the substituents in the substituted amino group II are selected from substituted C1-C3 alkyl groups IV; The substituent in the substituted C1-C3 alkyl IV is at least one selected from pyridyl and halogen.

[0024] Suitably, the inhibitor is selected from the compounds of formula (I) or a pharma- ceutically acceptable salt thereof. [ka] (Wherein, R1 is hydrogen, C1 to C 10 At least one selected from the group consisting of alkyl, alkylene oxide, R2 is hydrogen, substituted C1-C 10 is at least one selected from the group consisting of alkyl, substituted carbonyl, and substituted imino; R3 is hydrogen, C1-C 10 Alkyl, substituted C1-C 10 At least one selected from the alkyl groups II.

[0025] Preferably, the substituted C1-C 10The substituent in the alkyl I is at least one selected from halogen, substituted amino I, a substituent represented by the formula M-1, and a substituent represented by the formula M-2. [ka] [ka]

[0026] The substituent in the substituted carbonyl is a substituted C1-C 10 is selected from the alkenyl The substituent in the substituted imino is at least one selected from hydroxy and C1-C3 alkenylkoxy.

[0027] Preferably, the substituents in the substituted amino I are C1-C 10 and at least one selected from the group consisting of aryl, substituted C1-C3 alkyl, and Preferably, the substituents in the substituted C1-C3 alkyl III are C1-C 10 The aryl is selected from the group consisting of:

[0028] Preferably, the substituted C1-C 10 The substituents in the alkenyl are C1-C 10 Aryl, substituted C1-C 10 At least one selected from the group consisting of aryl, Preferably, the substituted C1-C 10 The substituent in the aryl is at least one selected from C1 to C3 alkenylkoxy, halogen, and C1 to C3 alkyl.

[0029] Preferably, the substituted C1-C 10 The substituent in the alkyl II is at least one selected from the group consisting of a substituted amino II, a substituent represented by the formula M-3, and a substituent represented by the formula M-4. [ka] [ka]

[0030] Preferably, the substituents in the substituted amino group II are selected from substituted C1-C3 alkyl groups IV; The substituent in the substituted C1-C3 alkyl IV is at least one selected from pyridyl and halogen.

[0031] Preferably, at least one of the following compounds targets CYP2E1 as an inhibitor, binds to the target and exerts an inhibitory effect on CYP2E1. [ka]

[0032] Preferably, at least one of the following compounds targets CYP2E1 as an inhibitor, binds to the target and exerts an inhibitory effect on CYP2E1. [ka]

[0033] Preferably, at least one of the following compounds targets CYP2E1 as an inhibitor, binds to the target and exerts an inhibitory effect on CYP2E1. [ka]

[0034] Preferably, the compound represented by formula (I) is reacted with an acid to obtain an acid salt of the compound represented by formula (I), The acid is at least one selected from inorganic acids and organic acids.

[0035] Preferably, the inorganic acid is at least one selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; The organic acid is at least one selected from acetic acid, oxalic acid, butanedioic acid, tartaric acid, succinic acid, malic acid, lactic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, resin acid, maleic acid, fumaric acid, salicylic acid, and acetylsalicylic acid.

[0036] Preferably, the compound of formula (I) has the structural formula [ka] and the X-ray powder diffraction pattern of crystalline form A of the hydrochloride salt of compound of formula (I) includes three or more 2θ values ​​selected from the group consisting of 8.4±0.2°, 13.1±0.2°, 14.8±0.2°, 16.6±0.2°, 24.1±0.2°, 27.2±0.2°, 30.5±0.2°, 31.8±0.2°, 33.5±0.2°, 35.4±0.2°, and 35.7±0.2°; As shown in the DSC-TGA diagram of the crystalline form A of the hydrochloride salt of the compound represented by formula (I), there is an obvious endothermic peak between 70°C and 220°C, and thermal decomposition occurs between 80°C and 170°C.

[0037] Preferably, the compound of formula (I) has the structural formula [ka] and the X-ray powder diffraction of crystalline form B of the sulfate salt of the compound of formula (I) includes five or more 2θ values ​​selected from the group consisting of 10.1±0.2°, 15.1±0.2°, 16.0±0.2°, 16.7±0.2°, 19.2±0.2°, 19.9±0.2°, 23.4±0.2°, 24.0±0.2°, 25.8±0.2°, 26.5±0.2°, 28.9±0.2°, 30.3±0.2°, and 32.2±0.2°; As shown in the DSC-TGA diagram of crystalline form B of the sulfate salt of the compound represented by formula (I), there are at least one endothermic peak between 30°C and 85°C, 90°C and 160°C, and 215°C and 330°C, and thermal decomposition occurs between 150°C and 350°C.

[0038] Suitably, the process for preparing the compounds of formula (I) as inhibitors comprises at least one of the following methods: Method 1: Compound A, an aprotic solvent, and a raw material containing a Grignard reagent are reacted at -20 to 25°C for 0.5 to 3 hours to obtain CYP2E1 inhibitor A. The compound A is at least one selected from compounds having a structural formula represented by formula II, [ka] The CYP2E1 inhibitor A is at least one selected from compounds having a structural formula represented by formula III, [ka] Method 2: reacting a compound containing the structural formula represented by formula III with aqueous ammonia in the presence of an alkaline source I to obtain a CYP2E1 inhibitor B; The CYP2E1 inhibitor B is at least one selected from compounds having a structural formula represented by formula III-1, [ka] Method 3: A compound having a structural formula represented by formula III is reacted with an aromatic aldehyde compound in the presence of an alkaline source II at 25 to 100°C for 2 to 8 hours to obtain a CYP2E1 inhibitor C; The CYP2E1 inhibitor C is at least one selected from compounds having a structural formula represented by formula III-2, [ka] Method 4: Reacting a compound containing the structural formula represented by formula III, an amine compound, and a reducing agent in the presence of an acid source to obtain a CYP2E1 inhibitor D; The amine compound is at least one selected from aniline and benzylamine, The CYP2E1 inhibitor D is at least one selected from the compounds having the structural formula represented by formula III-3. [ka]

[0039] Preferably, the method 2 comprises at least the step of reacting a compound containing a structural formula represented by formula III, ethanol and aqueous ammonia in the presence of an alkaline source I to obtain a CYP2E1 inhibitor B.

[0040] Preferably, in the method 1, the aprotic solvent is at least one selected from tetrahydrofuran and ethyl ether, and the Grignard reagent is at least one selected from methylmagnesium bromide and methylmagnesium chloride; In the method 2, the alkali source I is at least one selected from potassium hydroxide, sodium hydroxide, sodium carbonate, pyridine, triethylamine, and N,N-diisopropylethylamine; In the method 3, the alkali source II is at least one selected from sodium hydroxide, potassium hydroxide, potassium t-butoxide, sodium methoxide, and potassium fluoride; The aromatic aldehyde compound is at least one selected from p-methoxybenzaldehyde, o-methoxybenzaldehyde, m-methoxybenzaldehyde, p-chlorobenzaldehyde, o-chlorobenzaldehyde, m-chlorobenzaldehyde, p-phenylbenzaldehyde, p-isopropylbenzaldehyde, and 3,4-difluorobenzaldehyde; In the method 4, the acid source is at least one selected from formic acid, acetic acid, and hydrochloric acid; The reducing agent is at least one selected from sodium cyanoborohydride, sodium borohydride, and lithium aluminum hydride.

[0041] Preferably, in the method 1, the molar ratio of the compound A to the Grignard reagent is 1:1 to 1:3; In the method 2, the molar ratio of the compound represented by the structural formula III to aqueous ammonia is 1:1 to 1:6; In the method 3, the molar ratio of the compound represented by the structural formula III to the aromatic aldehyde compound is 1:1 to 1:5; In the method 4, the molar ratio of the compound of the structural formula represented by formula III to the reducing agent is 1:1 to 1:5.

[0042] Preferably, compound A is The compound A can be obtained by reacting raw materials containing compound A-1, a condensing agent, N,O-dimethylhydroxylamine hydrochloride and an aprotic solvent in the presence of an alkali source III at 20 to 60° C. for 10 to 20 hours to obtain the compound A.

[0043] The compound A-1 is at least one selected from compounds having a structural formula represented by formula II-1. [ka] Preferably, the condensing agent is at least one selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dilauryl carbonate, N,N-carbonyldiimidazole, dicyclohexylcarbodiimide, and N-(4-carboxyphenyl)maleimide; The alkali source III is at least one selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, pyridine, triethylamine, and N,N-diisopropylethylamine.

[0044] Preferably, the molar ratio of compound A-1, condensing agent, and N,O-dimethylhydroxylamine hydrochloride is 1:1:1 to 1:5:5.

[0045] Preferably, the compound A-1 is obtained by The method includes at least a step of hydrolyzing a raw material containing compound A-2 in the presence of an alkali source IV to obtain a mixture, and then adjusting the pH of the mixture to 2 to 3 with an acid source to obtain compound A-1; The compound A-2 is at least one selected from compounds having a structural formula represented by formula II-2. [ka]

[0046] Preferably, the alkalinity source IV is at least one selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and potassium carbonate.

[0047] Preferably, the acid source is selected from concentrated hydrochloric acid; The pH of the mixture is adjusted to 2 to 3 using the acid source at 10 to 50°C.

[0048] Preferably, the process for preparing an acid salt of a compound of formula (I) comprises the steps of: The method includes at least a step of reacting a compound represented by formula (I) with a material containing a solvent A at −20 to 80° C. for 0.5 to 10 hours to obtain an acid salt of the compound represented by formula (I), Preferably, the method for preparing the acid salt of the compound represented by formula (I) comprises the steps of: The method includes at least a step of reacting a material containing a compound represented by formula (I) and a solvent A at −15 to 60° C. for 1 to 4 hours to obtain an acid salt of the compound represented by formula (I).

[0049] More preferably, the method for preparing the acid salt of the compound represented by formula (I) comprises the steps of: The method includes at least a step of reacting a material containing a compound represented by formula (I) and a solvent A at −10 to 40° C. for 1 to 4 hours to obtain an acid salt of the compound represented by formula (I).

[0050] The present invention also provides an acid salt of the CYP2E1 inhibitor SMI0 obtained by reacting SMI0 with an organic or inorganic acid.

[0051] Preferably, the organic acid is one selected from acetic acid, oxalic acid, butanedioic acid, tartaric acid, succinic acid, malic acid, lactic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, resin acid, maleic acid, fumaric acid, salicylic acid or acetylsalicylic acid.

[0052] Preferably, the inorganic acid is one selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid or phosphoric acid.

[0053] The present invention also provides a method for producing an acid salt of the CYP2E1 inhibitor SMI0, which includes the steps of preparing solution systems in a solvent that dissolves SMI0 and an acid, respectively, mixing the two systems so that the molar ratio of SMI0 to the acid in the acidic solution is 1:0.5-3, producing a target product at a reaction temperature of -20 to 80°C for a reaction time of 0.5 to 10 hours, and rotary evaporating the solvent under reduced pressure to obtain the acid salt of SMI0.

[0054] Preferably, the organic solvent used in the reaction comprises one or more of ethers, alcohols, esters, nitriles, ketones, halogenated alkanes, alkanes or aromatic hydrocarbon organic solvents mixed in any ratio.Preferably, the soluble solvent is methanol, acetonitrile, acetone, ethyl acetate, ethanol or ethyl ether.

[0055] Suitably, in the reaction, the molar ratio of SMI0 to the acid in the acidic solution is 1:0.5-3, preferably 1:1-2.

[0056] In the above reaction, the reaction temperature varies depending on the reagents, solvent, etc., but is usually −20 to 80° C., preferably −15 to 60° C., and more preferably −10 to 40° C. The reaction time also varies depending on the reagents and temperature, but is usually 0.5 to 10 hours, and preferably 1 to 4 hours.

[0057] The present invention also provides a crystalline form A of the hydrochloride salt of the CYP2E1 inhibitor SMI0, wherein the powder X-ray diffraction of the crystalline form A includes three or more 2θ values ​​selected from the group consisting of 8.4±0.2°, 13.1±0.2°, 14.8±0.2°, 16.6±0.2°, 24.1±0.2°, 27.2±0.2°, 30.5±0.2°, 31.8±0.2°, 33.5±0.2°, 35.4±0.2°, and 35.7±0.2°. In addition, as shown in the DSC-TGA diagram, there is an obvious endothermic peak between 70°C and 220°C, and thermal decomposition occurs between 80°C and 170°C.

[0058] The present invention also provides a crystalline form B of sulfate of CYP2E1 inhibitor SMI0, wherein the powder X-ray diffraction of said crystalline form B includes five or more 2θ values ​​selected from the group consisting of 10.1±0.2°, 15.1±0.2°, 16.0±0.2°, 16.7±0.2°, 19.2±0.2°, 19.9±0.2°, 23.4±0.2°, 24.0±0.2°, 25.8±0.2°, 26.5±0.2°, 28.9±0.2°, 30.3±0.2°, and 32.2±0.2°. In addition, as shown in the DSC-TGA diagram, there is at least one endothermic peak between 30°C to 85°C, 90°C to 160°C, and 215°C to 330°C, and thermal decomposition occurs between 150°C to 350°C.

[0059] Preferably, said CYP2E1 inhibitor is used as an active substance in drugs for treating liver damage, fatty liver, hepatitis and liver fibrosis.

[0060] Preferably, the CYP2E1 inhibitor is used in a kit for preventing or treating liver disease.

[0061] Preferably, the use of said CYP2E1 inhibitor in the active ingredient of a drug for treating or preventing inflammation-associated tumors, comprising: The inflammation-associated tumors include at least one of liver cancer, glioma, ovarian cancer, lung cancer, bladder cancer, and gallbladder cancer.

[0062] Preferably, the CYP2E1 inhibitor is used as an active ingredient in a drug for treating or preventing an inflammation-related disease, the inflammation-related disease including at least one of liver damage, fatty liver, hepatitis, liver fibrosis, pulmonary fibrosis, rheumatic fever and rheumatoid arthritis, pyemia, Alzheimer's disease, ischemic stroke, Parkinson's disease, hyperlipidemia, atherosclerosis, coronary artery disease, and diabetes.

[0063] In the present invention, preferred CYP2E1 inhibitors are shown in the table below. [Table 0]

[0064] Preferably, the present application uses the CYP2E1 inhibitor SMI0 [ka] The synthesis method of has the following synthesis route. [ka]

[0065] Here, the reaction conditions are as follows: a. Hydrolysis and acidification are performed under alkaline conditions. b React with N,O-dimethylhydroxylamine hydrochloride in the presence of an alkali and a condensing agent. c React with Grignard reagents in anhydrous aprotic solvents at low temperature. d Under alkaline conditions, when heated, it undergoes an aldol condensation reaction with aromatic aldehydes. e Reacts with ammonia under alkaline conditions. f Reacts with amines under weak acid conditions and in the presence of a reducing agent.

[0066] Preferably, in the condition a, the alkali may be one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, etc., the acid may be one of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, etc. or a mixture thereof, the solvent used may be a mixture of water and one of water-soluble solvents such as methanol, ethanol, propanol, etc., the reaction temperature is 10°C to 50°C, Preferably, in condition b, the alkali may be one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, pyridine, triethylamine, N,N-diisopropylethylamine, etc., the condensing agent may be one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dilauryl carbonate, N,N-carbonyldiimidazole, N-(4-carboxyphenyl)maleimide, etc., the reaction temperature is 20°C to 60°C, and the solvent used may be one of aprotic solvents such as tetrahydrofuran, ethyl ether, etc.

[0067] Preferably, under condition c, the reaction temperature is -20°C to 25°C, the solvent used is one of aprotic solvents such as tetrahydrofuran and ethyl ether, and the equivalent ratio of compound 3 to the Grignard reagent is 1:1 to 1:3.

[0068] Preferably, in condition d, the alkali may be one of sodium hydroxide, potassium hydroxide, potassium t-butoxide, sodium methoxide, potassium fluoride, etc., the reaction temperature is 25°C to 100°C, and the solvent is one of water-soluble solvents such as methanol, ethanol, propanol, etc.

[0069] Preferably, in condition e, the alkali may be one of potassium hydroxide, sodium hydroxide, sodium carbonate, pyridine, triethylamine, N,N-diisopropylethylamine, etc., and the solvent is a mixture of water and one of water-soluble solvents such as methanol, ethanol, etc.

[0070] Suitably, in condition f, the acid may be one of organic or inorganic acids such as formic acid, acetic acid, hydrochloric acid, or a mixture thereof, and the solvent is one of water-soluble solvents such as methanol, ethanol, propanol, or the like.

[0071] In this application, C1 to C 10 The limit on the carbon atoms in the above-mentioned "substituted alkyl" and "substituted aryl" is not the number of carbon atoms after unsubstitution, but the number of carbon atoms contained in the alkyl or aryl itself. For example, C1 to C 10 The substituted alkyl refers to an alkyl group having 1 to 10 carbon atoms in which at least one hydrogen atom is substituted with a substituent.

[0072] In the present application, "alkyl" refers to a group formed by removing any hydrogen atom from an alkane compound molecule, which may be a straight-chain alkane, a branched alkane, an alkane, or a branched alkane.

[0073] In the present application, "alkenyl" refers to a group formed by removing any hydrogen atom from an olefin compound molecule. The alkane compound may be a straight-chain alkane, a branched alkane, an alkane, or a branched alkane.

[0074] As used herein, "aryl" refers to a group formed by the loss of one hydrogen atom on an aromatic ring from an aromatic molecule, such as p-tolyl formed by toluene losing the hydrogen atom para to the methyl on the benzene ring.

[0075] In the present application, "furyl" refers to a group formed by losing any hydrogen atom from a furan compound molecule.

[0076] As used herein, "imino" refers to the divalent group remaining after removal of two hydrogen atoms from an ammonia molecule, with the structural formula HN=.

[0077] As used herein, "pyridyl" refers to a group formed by losing any hydrogen atom from a pyridine compound molecule.

[0078] In the present application, the term "alkylene oxide" refers to a group formed by losing any hydrogen atom from an epoxy compound molecule. [Brief description of the drawings]

[0079] [Figure 1] 1 is a hydrogen nuclear magnetic resonance spectrum of compound SMI0. [Diagram 2] 1 is a hydrogen nuclear magnetic resonance spectrum of the compound SMI7. [Diagram 3] 1 is a hydrogen nuclear magnetic resonance spectrum of compound SMI16. [Figure 4] 1 is a hydrogen nuclear magnetic resonance spectrum of compound SMI20. [Diagram 5] 1 is a powder X-ray diffraction pattern (XRPD) of crystalline form A of SMI0 hydrochloride. [Figure 6] 1 is a differential scanning calorimetry-thermogravimetry analysis pattern (DSC-TGA) of crystalline form A of SMI0 hydrochloride. [Figure 7] 1 is a powder X-ray diffraction pattern (XRPD) of crystalline form B of SMI0 sulfate. [Figure 8] 1 is a differential scanning calorimetry-thermogravimetry analysis pattern (DSC-TGA) of crystalline form B of SMI0 sulfate. [Figure 9] 1 is a double reciprocal plot of the inhibitory effect of compound SMI0 on chlorzoxazone metabolism in human liver. [Figure 10] 1 is a quadratic plot based on a double reciprocal plot of the inhibitory effect of compound SMI0 on the metabolism of chlorzoxazone in human hepatic CYP2E1. [Figure 11] 1 is a plot of the inhibitory effects of 21 small molecule compounds on the in vitro metabolic activity of CYP2E1. [Figure 12] 1 is a plot of the inhibitory effect of compound SMI0 on the in vivo metabolism of diethylnitrosamine in rat CYP2E1. [Figure 13] 1 is a plot of the inhibitory effect of compound SMI0 on a diethylnitrosamine-induced rat liver injury model. [Figure 14]Plot of the inhibitory effect of compound SMI0 on high-fat diet-induced hepatic steatosis and hepatitis in mice. A: Representative images of the liver of mice in each group of hepatitis models. B: Pathological findings of inflammation-related tissues. H&E: Hematoxylin-eosin staining. Masson: Masson staining. Oil red: Oil red O staining. LCA: Leukocyte common antigen staining. [Figure 15] Plots quantifying the inhibition parameters of compound SMI0 on high-fat diet-induced hepatic steatosis and hepatitis model in mice. A: Hepatic index for each group of animals in the hepatitis model; B: Steatosis score for each group of animals; C: Masson staining score for each group of animals; D: Oil Red O stained area percentage for each group of animals; E: LCA score for each group of animals. [Figure 16] Plots of the inhibitory effect of compound SMI0 on a high-fat diet-induced mouse liver fibrosis model. A: Representative images of the livers of mice in each group of the liver fibrosis model. B: Pathological findings of tissues related to inflammation and liver fibrosis. H&E: Hematoxylin-eosin staining, Masson: Masson staining, Oil red: Oil red O staining. [Figure 17] FIG. 1 is a plot quantifying the parameters for the inhibition of compound SMI0 against a high-fat diet-induced mouse liver fibrosis model, where A is the hepatic index for each group of animals in the liver fibrosis model, B is the steatosis score for each group of animals, C is the Masson staining score for each group of animals, and D is the Oil Red O stained area percentage for each group of animals. [Figure 18] This is a plot of the inhibitory effect of compound SMI0 on diethylnitrosamine-induced rat liver fibrosis model. A is a representative image of the rat liver in each group of the liver fibrosis model, B is the pathological findings of tissues related to liver fibrosis, H&E is hematoxylin-eosin staining, Masson is Masson staining, α-SMA is α-smooth muscle actin, and Collagen I is type I collagen. [Figure 19]FIG. 1 is a plot quantifying the inhibition parameters of compound SMI0 in a diethylnitrosamine-induced rat liver fibrosis model, where A is the liver index for each group of animals in the liver fibrosis model, B is the Ishark score for each group of animals, C is the Masson staining score for each group of animals, D is the α-SMA histochemical score for each group of animals, and E is the Collagen I histochemical score for each group of animals. [Figure 20] 13 is a plot of the inhibitory effect of cyp2e1 gene knockout on lipopolysaccharide-induced mouse pulmonary fibrosis model; A is the HE and Masson staining patterns of lung tissue from mice in each group, and B is the collagen-positive area percentage and histological score of lung tissue from mice in each group. [Figure 21] 1 is a plot of the inhibitory effect of compound SMI0 on lipopolysaccharide-induced lung injury in mice, in which A is the HE staining image of the lung tissue of mice in each group, and B is the lung injury score of mice in each group. [Figure 22] 1 is a plot of the inhibitory effect of compound SMI0 on a lipopolysaccharide-induced mouse pulmonary fibrosis model; A is the HE and Masson staining patterns of lung tissue from mice in each group, and B is the collagen-positive area percentage and histological score of lung tissue from mice in each group. [Figure 23] 13 shows plots of the change in CYP2E1 activity in a lipopolysaccharide-induced mouse pulmonary fibrosis model and the correlation with the severity of pulmonary fibrosis. A shows the change in CYP2E1 activity in mice of each group, and B shows the correlation between CYP2E1 activity in each mouse and the pulmonary index. [Figure 24] FIG. 1 is a plot of the inhibitory effect of compound SMI0 on myeloperoxidase (MPO) levels in lung tissue from a lipopolysaccharide-induced mouse lung fibrosis model, in which A is the MPO staining pattern of lung tissue from each group of mice, and B is the percentage of MPO staining-positive area in lung tissue from each group of mice. [Diagram 25] 1 is a plot of the inhibitory effect of compound SMI0 on inflammatory factors in lung tissue of a mouse pulmonary fibrosis model induced by lipopolysaccharide, where A is the relative expression level of TNF-α in the lung tissue of mice in each group, and B is the relative expression level of IL-1β in the lung tissue of mice in each group. [Figure 26]FIG. 1 shows plots of the inhibitory effect of compound SMI0 on oxidative stress in lung tissue of a mouse pulmonary fibrosis model induced by lipopolysaccharide and on the expression of epithelial cell marker E-cadherin and apoptosis-related proteins. A shows the oxidative stress index CAT level in the lung tissue of mice in each group. B and C show Western blot plots of the expression of epithelial cell marker E-cadherin and apoptosis-related proteins in the lung tissue of mice in each group. D, E, and F are plots quantifying the expression of epithelial cell marker E-cadherin and apoptosis-related proteins in the lung tissue of mice in each group. [Figure 27] This is a plot of the inhibitory effect of compound SMI0 on the expression of TGF-β1 and α-SMA in lung tissue of a lipopolysaccharide-induced mouse lung fibrosis model. A and C are the TGF-β1 and α-SMA staining patterns in lung tissue of mice from each group, and B and D are the percentage of TGF-β1 and α-SMA staining-positive area in each group. [Figure 28] 1 is a plot of high expression levels of CYP2E1 in peritumoral tissues of lung cancer. [Figure 29] 1 is a plot of the inhibitory effect of cyp2e1 gene knockout on the lung orthotopic transplantation of lung cancer Lewis cells in a mouse model of lung cancer, where A is a schematic diagram of the lung tumor in each mouse, and B is the weight of the lung tumor in each mouse. [Diagram 30] FIG. 1 is a plot of the inhibitory effect of compound SMI0 on a Lewis cell lung orthotopic transplanted mouse lung cancer model; A: representative images of tumor-bearing lung tissue from each group of mice; B: schematic diagram of the lung tumors from each mouse; C: lung tumor weight from each mouse. [Diagram 31] 1 is a plot of the inhibitory effect of compound SMI0 on a lung metastasis cancer model induced by tail vein injection of colorectal cancer cells CT26, in which A is a schematic diagram of the tumor-containing lung tissue of each mouse, and B is the weight of the lung of each mouse. [Diagram 32] 1 is a plot of the inhibitory effect of compound SMI0 on a lung metastasis cancer model induced by tail vein injection of B16-F10 melanoma cells, in which A is a schematic diagram of the tumor-containing lung tissue of each mouse, and B is the weight of the lung of each mouse. [Diagram 33]This is a plot of the correlation between changes in CYP2E1 activity and tumor severity in a mouse lung cancer model orthotopically transplanted with Lewis cells. The left graph shows the changes in CYP2E1 activity in each group of mice, and the right graph shows the correlation between CYP2E1 activity in each mouse and lung tumor weight. [Diagram 34] FIG. 1 shows plots of the inhibition of the inflammatory microenvironment of the peritumoral lung tissue of a Lewis cell lung orthotopic tumor model by compound SMI0. FIG. 1 shows Western blot plots of the expression of inflammatory factors and related signal pathway proteins in the peritumoral lung tissue of mice in each group. FIG. 1 shows plots of the quantification of the expression of inflammatory factors and related signal pathway proteins in the peritumoral lung tissue of mice in each group. [Diagram 35] FIG. 1 shows plots of the effect of compound SMI0 on apoptosis- and autophagy-related proteins in cancer tissues of a Lewis cell lung orthotopic tumor model. A is a Western blot plot of the expression of apoptosis- and autophagy-related proteins in lung cancer tissues of mice in each group. B is a plot quantifying the expression of apoptosis- and autophagy-related proteins in lung cancer tissues of mice in each group. [Diagram 36] Plots showing the absence of direct inhibitory activity of compound SMI0 on lung cancer cells, A being Lewis lung cancer cells and B being A549 lung cancer cells. [Figure 37] 1 is a plot of the inhibitory effect of compound SMI0 on A549 lung cancer cell proliferation by inhibiting M2 polarization of macrophages. [Figure 38] 1 is a plot of elevated CYP2E1 in peritumor liver tissue of liver cancer patients. [Figure 39] This is a plot of the inhibitory effect of cyp2e1 gene knockout on a liver cancer model using rats orthotopically transplanted with Walker256 cells. A is a representative image of the liver containing a tumor from each group of rats, B is an image of the liver tumor from each group of rats, and C is the total weight and maximum tumor diameter of the liver tumor from each rat. [Diagram 40]This is a plot of the inhibitory effect of compound SMI0 on a mouse liver cancer model orthotopic transplantation of liver cancer cell line H22 cells into the liver and lung. A is a representative image of the liver containing the tumor in each group of mice, B is an image of the liver tumor in each group of mice, and C is the total weight and maximum tumor diameter of the liver tumor in each mouse. [Diagram 41] FIG. 1 shows plots of the inhibition of the inflammatory microenvironment of peritumoral liver tissue in the H22 liver-lung orthotopic transplant mouse tumor model by compound SMI0. FIG. 1 shows Western blot plots of the expression of inflammatory factors and related signaling pathway proteins in peritumoral liver tissue of mice in each group. FIG. 1 shows plots of the quantification of the expression of inflammatory factors and related signaling pathway proteins in peritumoral liver tissue of mice in each group. [Diagram 42] FIG. 1 shows plots of the effect of compound SMI0 on cancer tissue apoptosis and related signaling pathway proteins in the peritumoral liver tissue of the H22 liver-lung orthotopic transplant mouse tumor model; A is a Western blot plot of apoptosis and related signaling pathway protein expression in the liver cancer tissue of mice in each group; B is a plot quantifying apoptosis and related signaling pathway protein expression in the lung cancer tissue of mice in each group. [Diagram 43] Plots showing the case where compound SMI0 has no direct inhibitory effect on hepatoma cells, A being HepG2 hepatoma cells, and B being H22 hepatoma cells. [Diagram 44] These are plots showing the anti-liver cancer effect of compound SMI0 by influencing macrophages. A, B, and C are plots quantifying the effect of SMI0 on apoptosis, migration, and proliferation of HepG2 liver cancer cells co-cultured with macrophages. D and E are plots quantifying the effect of SMI0 on apoptosis and migration of HepG2 liver cancer cells co-cultured with macrophages. [Diagram 45] Plots of high expression levels of CYP2E1 in peritumoral tissues of glioma patients, showing immunohistochemistry results in A and qPCR results in B. [Figure 46] 13 is a plot of the inhibitory effect of cyp2e1 gene knockout on glioma GL261 cell brain orthotopically implanted mouse glioma model. [Figure 47]FIG. 1 shows plots of the inhibitory effect of compound SMI0 on glioma GL261 cell brain orthotopically implanted mouse glioma model. A: Schematic diagram of mouse brain tissue from each group; B: Representative images of HE staining of mouse brain tissue from each group; C: Tumor volume of mouse brain tissue from each group; D: In vitro inhibitory effect of SMI0 on GL261 cells. [Figure 48] Plots showing the change in CYP2E1 activity in a mouse model of orthotopic glioma tumor transplantation and its correlation with tumor severity. The left graph shows the change in CYP2E1 activity in each group of mice, and the right graph shows the correlation between CYP2E1 activity in each mouse and lung tumor weight. [Figure 49] Plots showing that compound SMI0 has no direct effect on GL261 (left panel) or U251 (right panel) glioma cells. [Figure 50] Plots of compound SMI0 inhibiting GL261 or U251 glioma via primary microglial cells or derived microglial cells HMC-3, where A and C are inhibiting tumor cell proliferation, and B and D are promoting tumor cell apoptosis. [Figure 51] 1A and B are plots of compound SMI0 inhibiting GL261 glioma via primary astrocytes, A inhibiting tumor cell proliferation, and B promoting tumor cell apoptosis. [Figure 52] 13 is a plot of the inhibition of compound SMI0 on primary microglial cell-mediated M2 polarization. [Diagram 53] 1 is a plot of compound SMI0 inhibition on cholesterol metabolism in stellate cells. [Figure 54] FIG. 1 is a plot of the inhibitory effect of compound SMI0 on ovarian cancer ID-8 cell ovarian orthotopic transplant mouse ovarian cancer model, where A is the total tumor weight of each group of mice, B is the ascites weight of each group of mice, and C is the in vitro inhibitory effect of SMI0 on ID-8 cells. [Figure 55] 1 is a plot of the inhibitory effect of compound SMI0 on ovarian cancer ID-8 cell tumors implanted in the abdominal cavity of mice, the left graph showing the total tumor weight in each group of mice, and the right graph showing the ascites weight in each group of mice. [Figure 56] 13 is a plot of the inhibitory effect of cyp2e1 gene knockout on a collagen-induced rat rheumatoid arthritis model, where A is a schematic representative image of the paws of each group, and B is a graph of the hind paw score, hind paw thickness and hind paw volume of rats in each group. [Figure 57] 1 is a plot of the inhibitory effect of compound SMI0 on a collagen-induced rat rheumatoid arthritis model, in which A is the paw swelling index of each group of rats, and B is the total score, paw thickness and paw volume of each group of rats. [Figure 58] 1 is a plot of the dose-effect relationship of the inhibition of rheumatoid arthritis by compound SMI0, where A is the recovery rate of paw swelling in rats in each dose SMI0 group, and B is the correlation between SMI0 dose and the recovery rate of paw swelling in rats on days 2, 8, and 20. [Figure 59] FIG. 1 is a schematic diagram of a rat paw in a complete Freund's adjuvant model of rheumatoid arthritis inhibited by compound SMI0. [Figure 60] FIG. 1 shows plots quantifying the inhibitory parameters of compound SMI0 in a rat rheumatoid arthritis model in complete Freund's adjuvant. A shows the paw swelling ratio of rats in each group at each time point, B shows the paw swelling ratio of rats at 24 h, C shows the paw swelling ratio of rats at 36 h, and D shows the paw swelling ratio of rats at 48 h. [Figure 61] 1 shows plots of dose-effect relationship of compound SMI0 for inhibition of rheumatoid arthritis in a rat complete Freund's adjuvant model. A shows the dose-effect relationship of rat paw swelling rate at each time point, B shows the dose-effect relationship at 24 h, C shows the dose-effect relationship at 36 h, and D shows the dose-effect relationship at 48 h. [Figure 62] 1 is a plot of the correlation between changes in CYP2E1 activity in a rheumatoid arthritis rat model and the severity of paw swelling, where A is a graph showing the correlation between CYP2E1 activity in rheumatoid arthritis model rats and B is a graph showing the correlation between CYP2E1 activity in each rat and the severity of paw swelling. [Figure 63]1 is a plot of the inhibitory effect of compound SMI0 on body temperature changes in a lipopolysaccharide-induced pyotoxemia model in rats, where A is the body temperature of rats in each group at each time, B is the body temperature of rats in each group at 4 hours, C is the body temperature of rats in each group at 5 hours, and D is the body temperature of rats in each group at 6 hours. [Figure 64] This is a plot of the inhibitory effect of compound SMI0 on body temperature changes in a lipopolysaccharide-induced mouse pyotoxemia model, where A is the body temperature of each group of mice at each time point, B is the body temperature of each group of mice at 6 hours, C is the body temperature of each group of mice at 12 hours, and D is the body temperature of each group of mice at 24 hours. [Figure 65] This is a plot of the improving effect of compound SMI0 on the damage of renal function and cardiac function in a lipopolysaccharide-induced pyotoxemia model in mice. A: 24-h serum urea nitrogen level of each group of mice, B: 24-h serum creatinine level of each group of mice, C: 24-h creatine kinase level of each group of mice, D: 24-h serum lactate dehydrogenase level of each group of mice. [Figure 66] FIG. 1 is a plot of the improvement of cognitive impairment in a rat Alzheimer's disease model induced by streptozocin using compound SMI0. A is the latency of each group of rats at each training time point, B is the time spent in the platform quadrant of each group of rats, and C is the number of times each group of rats crosses the platform. [Figure 67] 1 is a plot of the inhibitory effect of compound SMI0 on focal cerebral ischemia-reperfusion injury in rats. [Figure 68] 1 is a plot of the inhibition of cerebral infarction and cerebral edema caused by focal cerebral ischemia-reperfusion injury in rats by compound SMI0, where A is the cerebral infarction rate in rats and B is the cerebral edema rate in rats. [Figure 69] 1 is a plot of the reduction in blood lipid levels by compound SMI0 in a high-fat diet-induced ApoE− / − mouse hyperlipidemia model. [Figure 70] FIG. 1 is a plot of the inhibitory effect of compound SMI0 on high-fat diet-induced atherosclerotic aortic plaque formation in ApoE− / − mice, where A is the total plaque area for each group of animals, and B is the percentage of Oillet O area to the total plaque area for each group of animals. [Figure 71]FIG. 1 is a plot of the reduction in blood glucose levels in high-fat diet and streptozotocin-induced diabetic rats by compound SMI0, where A is the fasting blood glucose of each group of rats, B is the blood glucose level of each group of rats at each time point in a glucose tolerance test, and C is the area under the curve of each group of rats in a glucose tolerance test. [Figure 72] 1 is a plot of high expression levels of CYP2E1 in peritumoral tissue of bladder cancer. [Figure 73] 1 is a plot of high expression levels of CYP2E1 in peritumoral tissue of gallbladder cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] Hereinafter, the present application will be described in detail with reference to examples, but the present application is not limited to these examples.

[0081] High-resolution mass spectrometer used in the synthesis of CYP2E1 inhibitors in Examples 2, 3, 4, and 5 of the present application: Q-Tof micro model, manufactured by Waters Corporation, USA. Nuclear magnetic resonance apparatus used for synthesizing CYP2E1 inhibitors in Examples 2, 3, 4, and 5 of the present application: DPX-400 model, manufactured by Bruker, Germany. High performance liquid chromatograph used to measure the in vivo and in vitro inhibitory effects of CYP2E1 in Examples 8, 9, and 10 of the present application: Agilent 1260 model, manufactured by Agilent Technologies Co. Ltd. In Example 17 of the present application, a stereotaxic apparatus was used to prepare the mouse glioma model: Shanghai Puxinyi Instrument Technology Co., Ltd., ZR-09 model.

[0082] Example 1 Preparation of human liver microsomes Using differential centrifugation, liver samples were removed, thawed, and weighed. 50 mM Tris-HCl (pH = 7.0) (containing 150 mM KCl and 2 mM EDTA) buffer was added at a ratio of 1:4 (W / V) and crushed in a glass homogenizer to prepare liver homogenates. The mixture was centrifuged at 9000 × g for 20 min at 4 °C, and the supernatant was centrifuged at 100,000 × g for 60 min at 4 °C. The resulting precipitate was resuspended in 4 mL 0.15 M Tris-HCl (pH = 7.6), centrifuged at 100,000 × g for 60 min at 4 °C, and the precipitate was added to a 0.25 M sucrose heavy suspension at a ratio of 1:2 (W / V). Finally, 2 mL of microsomal suspension per 1 g of liver tissue was prepared. After aliquoting, the suspension was first stored in liquid nitrogen overnight and then transferred to -80 °C the next day for long-term storage. All of the above operations were performed in an ice bath. Microsomal protein content (mg / mL) was measured using the Bradford method. This method was used for liver microsomes from both patients with liver injury and healthy subjects.

[0083] Example 2 Synthesis of SMI0 4-Methylthiazole-5-carboxylate ethyl (1 mol) and NaOH (1.6 mol) were used as a solvent, and the mixture was reacted at room temperature overnight. The reaction was detected by TLC (pure ethyl acetate). After the reaction was completed, the ethanol was evaporated under reduced pressure, the pH was adjusted to 2-3 with concentrated sulfuric acid, and the solid was obtained by suction filtration, washed, and dried. 4-Methylthiazole-5-carboxylate (1 mol) and DDC (1 mol) were stirred at room temperature in anhydrous tetrahydrofuran, activated for 2-3 h, and then dimethylhydroxylamine hydrochloride (1.2 mol) was added, triethylamine (1.5 mol) was added dropwise, and the mixture was stirred at room temperature overnight. The reaction was detected by TLC (PE:EA=3:1). After the reaction was completed, the tetrahydrofuran was evaporated under reduced pressure, and ethyl acetate was added and extracted three times, washed twice with saturated aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulfate, suction filtered, and evaporated under reduced pressure. The product (1 mol) was dissolved in anhydrous tetrahydrofuran, protected with nitrogen gas, precooled in a cold trap, and the temperature was controlled to -10 to 15°C. Grignard reagent CH3MgCl (1.5 mol) was added dropwise, and the reaction was detected by TLC (PE:EA = 3:1). After the reaction was completed, the reaction was quenched with saturated NH4Cl, extracted with ethyl acetate, dried with anhydrous magnesium sulfate, suction filtered, and evaporated under reduced pressure. The crude product was distilled under reduced pressure to obtain a pure product. The nuclear magnetic detection data of the product is as follows (see Figure 1). 1 H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H), 2.80 (s, 3H), 2.60 (s, 3H).

[0084] Example 3 Synthesis of compound SMI7 Hydroxylamine hydrochloride (3mmol) was taken into a round-bottom flask, 3mL of ethanol was added, and the mixture was stirred at room temperature of 25℃ for 10min, 3mL of 1M NaOH solution was added, and SMI0 (3mmol) was added, and the mixture was refluxed at 80℃ in an oil bath, and monitored by TLC. After the reaction was completed, the reaction solution was neutralized with 10% dilute hydrochloric acid, extracted with water and ethyl acetate, the organic phase was combined, dried with anhydrous magnesium sulfate, and filtered by suction to remove magnesium sulfate. The filtrate was concentrated in vacuum, and the compound SMI7 was obtained by silica gel column chromatography (eluent and its ratio is petroleum ether: ethyl acetate = 1:2). The nuclear magnetic detection data of the product is as follows (see Figure 2). 1 H NMR (400 MHz, DMSO) δ 11.50 (s, 2 / 3H), 11.48 (s, 1 / 3H), 9.09 (s, 1 / 3H), 8.95 (s, 2 / 3H), 2.57 (s, 2 / 3H), 2.53 (s, 1 / 3H), 2.28 (s, 1 / 3H), 2.26 (s, 2 / 3H).

[0085] Example 4 Synthesis of Compound SMI16 3,4-Dichlorobenzaldehyde (1mmol) was taken in a round-bottom flask, 2mL of anhydrous ethanol was added, and the mixture was stirred at 50℃ to dissolve. 30μL of 3M KOH solution was added, and SMI0 (1mmol) was added, and the mixture was stirred at 50℃ to monitor the reaction by TLC. After the reaction was completed, the reaction solution was neutralized with 10% dilute hydrochloric acid, extracted with water and ethyl acetate, the organic phase was combined, dried with anhydrous magnesium sulfate, and filtered by suction to remove magnesium sulfate. The filtrate was concentrated in vacuum, and the product was separated by silica gel column chromatography (the eluent and its ratio are petroleum ether: ethyl acetate = 2:1) to obtain compound SMI16. The nuclear magnetic detection data of the product is as follows (see Figure 3): 1H NMR (400 MHz, CDCl3) δ 8.84 (s, 1H), 7.68 (d, J = 11.2 Hz, 2H), 7.51 (d, J = 8.3 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.19 (d, J = 15.5 Hz, 1H), 2.86 (s, 3H).

[0086] Example 5 Synthesis of Compound SMI20 Aniline (2mmol) and SMI0 (2mmol) were taken into a round-bottom flask, 3mL of anhydrous ethanol was added, and the mixture was stirred at room temperature of 25℃ for 10min. BH3CNNa (2mmol) and acetic acid (1mmol) were added, and the mixture was stirred at room temperature of 25℃. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was neutralized with 10% dilute hydrochloric acid, extracted with water and ethyl acetate, the organic phase was combined, dried with anhydrous magnesium sulfate, and filtered by suction to remove magnesium sulfate. The filtrate was concentrated in vacuum and separated by silica gel column chromatography (the eluent and its ratio are petroleum ether:acetone=4:1) to obtain compound SMI20. The nuclear magnetic detection data of the product is as follows (see Figure 4). 1 H NMR (400 MHz, CDCl3) δ 8.55 (s, 1H), 7.13 (t, J = 7.7 Hz, 2H), 6.71 (t, J = 7.2 Hz, 1H), 6.51 (d, J = 7.9 Hz, 2H), 4.74 (q, J = 6.2 Hz, 1H), 4.00 (s, 1H), 2.50 (s, 3H), 1.56 (d, J = 6.6 Hz, 3H).

[0087] Example 6 Synthesis of SMI0 hydrochloride SMI0 (1 g, 7.08 mmol) was placed in a round-bottom flask, dissolved by adding 5 mL of ethanol, and reacted for 1 hour by slowly dropping 11 mL of 1 mol / L hydrochloric acid-ethanol solution while stirring at room temperature. The mixture was concentrated under reduced pressure, cooled, crystallized, filtered, washed with 0.5 mL of cold anhydrous ethanol, and dried to obtain 1.06 g of a white solid (yield 85%, purity 99.8% (HPLC), melting range 160-162 ° C). The powder X-ray diffraction pattern (XRPD) of the crystalline form A of compound SMI0 hydrochloride is shown in Figure 5, and the differential scanning calorimetry-thermogravimetry pattern (DSC-TGA) is shown in Figure 6.

[0088] Example 7 Synthesis of SMI0 sulfate SMI0 (1 g, 7.08 mmol) was placed in a round-bottom flask, dissolved by adding 5 mL of ethanol, and reacted for 1 hour by slowly dropping 8 mL of 1 mol / L sulfuric acid-ethanol solution while stirring at room temperature. The solvent was removed by evaporation, and recrystallized with a small amount of methanol to precipitate 0.85 g of a yellow solid (yield 70%, purity 99.5% (HPLC)). The powder X-ray diffraction pattern (XRPD) of the crystalline form B of compound SMI0 sulfate is shown in Figure 7, and the differential scanning calorimetry-thermogravimetry pattern (DSC-TGA) is shown in Figure 8.

[0089] Example 8 Measurement of inhibitory effect on CYP2E1 metabolic activity Chlorzoxazone was used as a probe substrate to measure the inhibitory effect of the target inhibitor on the metabolic activity of CYP2E1 in mixed liver microsomes from healthy subjects (IC 50 : half inhibitory concentration, K i : Inhibition constant, IC 50 The smaller is, the more i The inhibitory effect of the inhibitor on human hepatic microsomal CYP2E1 was confirmed. CYP2E1 inhibitor IC 50 Decision The total volume of the incubation system was 100 μL, and the incubation system contained substrates, inhibitors at different concentrations, liver microsomal protein, and phosphate buffer, and the reaction was carried out in a water bath at 37 °C. After 5 min, reduced coenzymes were added to start the reaction, and after 30 min, the reaction was terminated in an ice bath. In specific experiments, the concentration of the inhibitor can be selected according to needs. In this example, the incubation system contains 62.5 μM chlorzoxazone, 100 mM (pH=7.4) phosphate buffer, 0.3 mg / mL liver microsomal protein, and 1 mM NADPH. In another preferred embodiment, the incubation system may contain 7.8 to 1000 μM chlorzoxazone as a substrate. The concentration of liver microsomal protein may be 0.1 to 0.5 mg / mL. If necessary, phosphate buffer may be used at 50 mM to 100 mM, and Tris-HCL buffer may be used at 50 mM to 100 mM. An NADPH regenerating system can also be used. Preferably, the incubation system contains 62.5 μM chlorzoxazone, 100 mM (pH=7.4) phosphate buffer, 0.3 mg / mL liver microsomal protein, and 1 mM NADPH. In this example, the reaction is quenched with 1 mL of ethyl acetate. In other preferred examples, the reaction may be quenched with 1 mL of methyl tert-butyl ether, 1 mL of ethyl ether, or 100 μL of methanol. K for CYP2E1 inhibition i Decision To determine whether an inhibitor has a good inhibitory effect on CYP2E1, select different concentrations of substrate and different concentrations of inhibitor and perform an in vitro incubation inhibition test to determine the inhibition constant K of the inhibitor against chlorzoxazone metabolism by CYP2E1. i was calculated. The total volume of the incubation system for the incubation reaction was 100 μL, and the incubation system contained substrate, inhibitors at different concentrations, liver microsomal protein, and phosphate buffer, and the reaction was carried out in a water bath at 37°C. After 5 minutes of incubation, reduced coenzymes were added to start the reaction, and after a certain period of incubation, the reaction was terminated in an ice bath. In this example, the incubation system contained 15.6, 31.25, 62.5, 125, and 250 μM chlorzoxazone, 100 mM (pH = 7.4) phosphate buffer, 0.3 mg / mL liver microsomal protein, and 1 mM NADPH, respectively. 50 Based on this, different concentration gradients of inhibitors were determined in the ratio of 1 / 4 to 4 times. In another preferred embodiment, the incubation system may contain 7.8 to 1000 μM chlorzoxazone as a substrate, more preferably 15.6 to 250 μM (15.6, 31.25, 62.5, 125, and 250 μM) chlorzoxazone. The concentration of liver microsomal protein may be 0.1 to 0.5 mg / mL. If necessary, phosphate buffer may be used at 50 mM to 100 mM, and Tris-HCL buffer may be used at 50 mM to 100 mM. Either 1 mM NADPH or an NADPH regenerating system can be used. The NADPH regenerating system contains 1.3 mM NADP+, 3.3 mM 6-phosphate glucose, 0.4 U / mL glucose dehydrogenase, 3.3 mM magnesium chloride. In this example, the reaction is quenched with 1 mL of ethyl acetate. In other preferred examples, the reaction may be quenched with 1 mL of methyl tert-butyl ether, 1 mL of ethyl ether, or 100 μL of methanol. Determining the selectivity of CYP2E1 inhibitors In addition, using healthy human mixed liver microsomes as the study subject, probe drugs for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were selected, and the inhibitory effect of the inhibitors to be measured on the metabolism of the probe drugs by healthy human mixed liver microsomes CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 was measured to evaluate the selectivity of the inhibitory effect of the inhibitors to be measured against CYP2E1. The CYP1A2 probe was either 6.25-800 μM phenacetin or 27.5-12520 μM caffeine. The CYP2A6 probe was either 0.156-20 μM coumarin or 12.5-2000 μM nicotine. The CYP2B6 probe is either 7.8 to 500 μM bupropion or 0.25 to 30 mM cyclophosphamide. The CYP2C8 probe is either 2.5-80 μM paclitaxel or 0.125-128 μM amodiaquine. The CYP2C9 probe was either 31.25-2000 μM tolbutamide or 0.1-200 μM diclofenac. The CYP2C19 probe was either 3.9-500 μM omeprazole or 1.95-1000 μM mephenytoin. The CYP2D6 probe was either 0.625-96 μM dextromethorphan or 0.0195-80 μM propafenone. The CYP3A4 probe was either 0.39-50 μM midazolam or 1.98-1000 μM testosterone. The total volume of the incubation system was 100 μL, and the incubation system contained substrate, inhibitors at different concentrations, liver microsomal protein, and phosphate buffer, and the reaction was carried out in a water bath at 37°C. After preincubation for 5 min, reduced coenzymes were added to start the reaction, and after incubation for a certain period of time, the reaction was terminated in an ice bath. In this example, the incubation system contains at least one of 62.5 μM chlorzoxazone, 2.5 μM coumarin, 62.5 μM bupropion, 10 μM paclitaxel, 250 μM tolbutamide, 62.5 μM omeprazole, 20 μM dextromethorphan, 62.5 μM chlorzoxazone, 1.56 μM midazolam, 100 mM (pH=7.4) phosphate buffer, 0.3 mg / mL liver microsomal protein, and 1 mM NADPH. In another preferred embodiment, the substrate may include 7.8-1000 μM chlorzoxazone as a substrate. The concentration of liver microsomal protein may be 0.1-0.5 mg / mL. The buffer may be one selected from 50 mM phosphate buffer, 100 mM phosphate buffer, 50 mM Tris-HCL buffer, and 100 mM Tris-HCL buffer. Either 1 mM NADPH or an NADPH regenerating system may be used. In this example, the reaction is quenched with 1 mL of ethyl acetate. In other preferred examples, the reaction may be quenched with 1 mL of methyl tert-butyl ether, 1 mL of ethyl ether, or 100 μL of methanol.

[0090] Example 9 Results of in vitro screening test of CYP2E1 inhibitors SMI0 (inhibitory IC against 2E1 50 : 1.64 μM, inhibitory IC against 2A6 50 :76.20 μM, and no significant inhibitory effect on other CYP enzymes was observed. [ka] Now, referring to Figure 9, all reactions used 100μL incubation system, chlorzoxazone concentrations were 15.6, 31.2, 62.5, 125, 250μM, and series concentrations of SMI0 were 0, 0.4638, 0.9276, 1.855, 3.710μM. All experiments were obtained from the average value of three independent experiments. Figure 9 is a double reciprocal plot of the inhibitory effect of SMI0 compound on chlorzoxazone metabolism by human liver. It was shown that SMI0 compound is a mixed inhibitor of CYP2E1. Referring now to Figure 10, Figure 10 is a quadratic plot based on a double reciprocal plot of the inhibitory effect of the SMI0 compound on the metabolism of chlorzoxazone by human hepatic CYP2E1. A series of concentrations of SMI0 (0, 0.4638, 0.9276, 1.855, 3.710 μM) were linearly regressioned with different inhibition curve slopes, and the absolute value K i was 0.8970 μM. In addition, referring to the specific research method of Example 8, the inhibitory effect of 21 small molecule compounds (Table 1) on CYP2E1 was tested in vitro. The results showed that SMI1 and SMI8 showed significant inhibitory effect on CYP2E1, and the inhibitory IC 50 The IC values ​​were 7.99 and 17.03 μM, respectively. SMI10 had a slight inhibitory effect on CYP2E1, with IC 50 The inhibitory effect of other small molecules on CYP2E1 was weaker for SMI0, SMI1, SMI8, and SMI10 (see FIG. 11). Additionally, selectivity studies have demonstrated that SMI1 has an IC 50 55.63 μM, IC for CYP2C9 inhibition 50 The structures of these 21 small molecules are shown in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0091] Example 10 Test results of in vivo inhibitory effect of SMI0 compound on CYP2E1 Experimental method: The study on the inhibitory effect of SMI0 compound on CYP2E1 in vivo in rats was carried out using a self-controlled crossover experimental design. In the first experiment, diethylnitrosamine (DEN) was injected intraperitoneally at 50 mg / kg (a total of 30 SD mice) alone in the first week. After a one-week interval, the second experiment was carried out, that is, each rat was given SMI0 compound by intragastric force at low, medium or high doses (6 mg / kg, 30 mg / kg or 150 mg / kg), and 5 min later, DEN was injected intraperitoneally at 50 mg / kg (10 rats for each low, medium and high dose). Blood sampling times were 2, 7, 15, 30 min and 1, 2, 4, 6, 9, 12, 24, 36, 48, 60 h. The plasma DEN concentration at each of the above blood sampling times was measured, and the toxicokinetic parameters of DEN were obtained. High performance liquid chromatography was used to detect plasma DEN. 100μL of plasma was mixed with 10μL of perchloric acid, vortex mixed for 3 minutes, centrifuged at 12000rpm for 10 minutes, and 10μL of the supernatant was injected. The mobile phase was methanol:water = 50:50, and the detection wavelength was 240nm. Experimental results: CYP2E1 activity was expressed by the toxicokinetic parameters of DEN, and the effects of low, medium, and high doses of SMI0 compound on CYP2E1 activity in rats were analyzed. The results showed that compared with the model group of DEN alone, low, medium, and high doses of SMI0 reduced the DEN clearance rate CL by (63.98±7.78), (79.63±7.29), and (85.42±3.74), respectively, and the inhibition rates were 62.43%, 80.42%, and 86.77%, respectively (Figure 12, Table 2). * P < 0.05, *** P<0.001 vs. model group). Similarly, the low, medium, and high doses of SMI0 significantly reduced the half-life of DEN 1 / 2 were extended by (133.60±116.80)%, (619.97±363.57)%, and (868.70±241.26)%, respectively, and the area under the curve AUC 0-t were increased by (196.82±73.63)%, (407.70±184.30)%, and (529.67±153.72)%, respectively, and the area under the curve AUC 0-∞were increased by (189.99±67.55)%, (455.90±219.26)%, and (626.56±187.15)%, respectively. In addition, the inhibitory effect of the medium dose of SMI0 compound was significantly superior to that of the low dose, and the inhibitory effect of the high dose of SMI0 compound was significantly superior to that of the low and medium doses ( # P < 0.05, ## P < 0.01, ### P<0.001 vs SMI0 low dose group; $ P < 0.05, $$$ P<0.001 vs. SMI0 medium dose group). This suggests that each dose of the SMI0 compound can significantly inhibit the metabolic activity of rat CYP2E1 against DEN, and that there is an excellent dose-dependent relationship. These results demonstrated that SMI0 has a significant inhibitory effect on CYP2E1 activity in rats. [Table 2] Note: In Table 2, Mean stands for the average value and SD stands for the standard deviation. C max Peak concentration: The highest blood concentration that occurs after administration. This parameter is an important indicator that reflects the rate and extent of drug absorption in vivo. T max Peak time: The time required to reach a peak concentration after administration. This parameter reflects the rate at which a drug enters the body; the faster the absorption rate, the shorter the time to reach the peak. V d Apparent volume of distribution is the proportional constant between the amount of drug in the body and the blood concentration when the drug reaches dynamic equilibrium in the body, and is generally expressed in units of L. This parameter reflects how widely the drug is distributed in the body, with higher values ​​indicating more widespread distribution. The apparent volume of distribution is calculated numerically from the ratio of the clearance rate to the terminal elimination rate. CL: Clearance rate, which is the apparent distribution volume of a drug removed from the body per unit time, and is generally expressed in L / h. This parameter is an important parameter that reflects the drug treatment characteristics of the body and is closely related to physiological factors. The clearance rate is a function of dose and AUC (0-∞) It is obtained from the ratio of AUC: Area under the drug blood concentration-time curve, the area enclosed by the blood concentration curve against the time axis. This parameter is an important index for evaluating the degree of drug absorption and reflects the exposure characteristics of the drug in vivo. In pharmacokinetic studies, blood concentrations can only be observed up to a certain time point t, so AUC is expressed as AUC (0-t) and AUC (0-∞) The former is obtained by the trapezoidal area method, and the latter is calculated by the AUC (0-∞) =AUC (0-t) + Final concentration / final rate of elimination.

[0092] Example 11 Inhibition of diethylnitrosamine-induced liver injury in rats by SMI0 compounds Experimental method: SD rat liver injury model was created by intermittent intraperitoneal injection of diethylnitrosamine (DEN). The model group was intraperitoneally injected with DEN 50 mg / kg twice a week for the first 4 weeks, and once a week for the 5th to 8th weeks. The SMI0 treatment group was divided into a low-dose DEN combination group, a high-dose DEN combination group, and a high-dose continuous administration group. Among them, the low-dose DEN combination group and the high-dose DEN combination group were forced to intragastrically administer 30 mg / kg and 150 mg / kg of SMI0 compound 5 min before each DEN administration, respectively, and the high-dose continuous administration group was forced to intragastrically administer 150 mg / kg of SMI0 compound every day from the 1st week, and the model was completed at the 8th week. When the model was completed, blood was collected from the orbit and rat plasma liver function index was measured using a fully automated biochemical analyzer [model group, n = 10 rats; SMI0 compound low-dose group, n = 21 rats (150 mg / kg)]. a ; High dose group of SMI0 compound combined with DEN, n=28 rats (150mg / kg) b ; SMI0 compound high-dose continuous administration group, n = 24 rats]. Experimental results: Compared with the model group, in the SMI0 treatment group, albumin (ALB2), cholinesterase (CHE2) were significantly increased, and alkaline phosphatase (ALP2S), glutamine transaminase (ALTL), direct bilirubin (BILD2), total bilirubin (BILT3) and glutamyl transpeptidase (GGTI2) were significantly decreased. In the SMI0 low-dose group, TP2 was significantly increased, and in the SMI0 high-dose combined administration group and the SMI0 high-dose continuous administration group, GGTI2 was significantly decreased (as shown in Figure 13A to Figure 13I). * P < 0.05, ** P < 0.01, *** P<0.001 vs. model group; 150mg / kg a SMI0 high dose combination group, 150mg / kg b In the SMI0 high-dose continuous administration group, ALTL, BILD2, BILT3, and GGTI2 were significantly decreased and TP2 was significantly increased in the SMI0 high-dose combination administration group compared to the SMI0 low-dose group, and in the SMI0 high-dose continuous administration group, ALB2 was significantly increased and ALP2S, ALTL, ASTL, BILD2, CHE2, BILT3, and GGTI2 were significantly decreased ( ## P < 0.01, ## P<0.001 vs. SMI0 low dose group). Compared with the SMI0 high dose combined administration group, ALB2 was significantly increased, and ALP2S, ALTL, ASTL, BILD2, CHE2, BILT3 and GGTI2 were significantly decreased in the SMI0 high dose continuous administration group ( $ P < 0.05, $$$ P<0.001 vs. high-dose SMI0 co-administration group), suggesting that administration of SMI0 could significantly improve diethylnitrosamine-induced liver injury in rats. These results demonstrated that SMI0 has obvious preventive and therapeutic effects on diethylnitrosamine-induced liver injury in rats, suggesting that SMI0 compound may be useful in the clinical prevention and treatment of liver injury.

[0093] Example 12 Inhibition of SMI0 on high-fat diet-induced steatosis and hepatitis in mice Experimental method: Healthy male C57 / 6J mice were fed a high-fructose, high-lipid, and high-cholesterol diet to create a mouse steatosis and hepatitis model, and the control group was fed a low-lipid, low-sugar control diet. The SMI0 treatment group was divided into a low-dose treatment group and a high-dose treatment group, i.e., 30 mg / kg and 150 mg / kg of SMI0 compound were administered intragastrically every day at the start of model creation, respectively, and the model was completed at 22 weeks. At the end of the experiment at 22 weeks, blood was collected from the orbit, and the mice were killed after recording their weight. Liver weight and appearance (liver color, texture, presence or absence of nodules, etc.) were recorded. After HE staining of some liver samples, the steatosis scores of mice in each group were observed and recorded, and the liver fibrosis status of mice was evaluated by Masson staining, and the Oil Red O stained area percentage and LCA stained inflammation were scored (model group, n = 8 mice; SMI0 compound low dose group, n = 10 rats; SMI0 compound high dose group, n = 10 rats). The pathological results of HE staining of liver tissue are quantitatively scored according to the following criteria to determine the severity of fatty degeneration. (1) Fatty degeneration area less than 5%: 0 points (2) Fat change area: 5%-20% 1 point (3) Fat change area >20%~35% 2 points (4) Fat change area >35-50% 3 points (5) Fat change area >50%~65% 4 points (6) Fatty degeneration area >65%~80% 5 points (7) Steatosis area >80% 6 points The pathological results of Masson staining of liver tissue are quantitatively scored according to the following criteria to determine the severity of fibrosis. (1) No fibrosis (2) Slight fibrous hyperplasia is observed around the central vein and in the sinusoids (1 point) (3) Moderate fibrous hyperplasia is observed around the central vein and in the sinusoids. 2 points (4) Clear fibrous hyperplasia is observed in the portal vein or around the portal vein. 3 points (5) Clear fibrous hyperplasia is observed in the sinusoids, portal vein, or around the portal vein. 4 points (6) Fiber bridging occurred: 5 points (7) Liver cirrhosis: 6 points Experimental results: Compared with the control group, in the model group, the liver index, degree of steatosis (steatosis score and percentage of the area stained with Oil Red O), and level of liver inflammation (number of LCA-positive cell stained foci) were all significantly increased, although to different degrees (for example, as shown in Figures 14A to 14B and Figures 15A to 15E, * P < 0.05, ** P < 0.01, *** P<0.001 vs. control group; all scales in Fig. 14 are 100 μm). Compared with the model group, the liver index and degree of steatosis (steatosis score and percentage of Oil Red O stained area) were significantly decreased in the SMI0 low dose treatment group ( # P < 0.05, ## P < 0.01, ### P<0.001 vs. model group). In the high-dose SMI0 treatment group, the degree of steatosis (steatosis score and percentage of Oil Red O stained area) and the level of liver inflammation (number of LCA-positive cell stained foci) were all significantly reduced. This suggests that the SMI0 compound significantly reduces high-fat diet-induced liver steatosis and inflammation. These results demonstrated that SMI0 has a clear preventive and therapeutic effect on high-fat diet-induced hepatic steatosis and hepatitis in mice, suggesting that the SMI0 compound may be useful for the clinical prevention and treatment of fatty liver and hepatitis.

[0094] Example 13 Inhibition of SMI0 in the development and progression of liver fibrosis (1) Inhibition of SMI0 in high-fat diet-induced liver fibrosis in mice Experimental method: Male C57 / 6J mice were fed a high-fructose, high-lipid, and high-cholesterol diet to create a hepatic steatosis and hepatitis model, and the control group was fed a low-lipid and low-sugar control diet. The SMI0 treatment group was divided into a low-dose treatment group and a high-dose treatment group, i.e., 30 mg / kg and 150 mg / kg of SMI0 compound were administered intragastrically every day at the start of model creation, respectively, and the model was completed at 26 weeks. At the end of the experiment at 26 weeks, blood was collected from the orbit and the mice were killed after recording their weight. Liver weight and appearance (liver color, texture, presence or absence of nodules, etc.) were recorded. After HE staining of some liver specimens, the steatosis scores of the mice in each group were observed and recorded, and the liver fibrosis status of the mice was evaluated by Masson staining, and the oil red O stained area percentage was also scored (model group, n = 10 mice; SMI0 compound low-dose group, n = 10 rats; SMI0 compound high-dose group, n = 10 rats). Experimental results: Compared with the control group, in the model group, the liver index, the indicators reflecting the degree of steatosis (steatosis score and percentage of the area stained with Oil Red O), and the indicator reflecting the degree of fibrosis, the Masson staining score, all increased significantly, although to different degrees (for example, as shown in Figures 16A to 16B and Figures 17A to 17D). * P < 0.05, ** P<0.01 vs. control group; scales for HE and Masson staining in Fig. 16B are 100 μm, scale for Oil red staining is 50 μm). Compared with the model group, the degree of steatosis (steatosis score and Oil red O stained area percentage) and the degree of fibrosis (Masson staining score) were significantly decreased in the low-dose and high-dose SMI0 treatment groups ( # P < 0.05, ## P < 0.01, ### P<0.001 vs. model group). This suggests that the SMI0 compound significantly reduces the level of liver fibrosis induced by a high-fat diet in mice. These results demonstrate that SMI0 has a clear preventive and therapeutic effect against high-fat diet-induced liver fibrosis in mice, suggesting that the SMI0 compound may be useful for the clinical prevention and treatment of liver fibrosis. (2) Inhibition of SMI0 in diethylnitrosamine-induced liver fibrosis in rats Experimental method: SD rat liver injury model was created by intermittent intraperitoneal injection of diethylnitrosamine (DEN). In the model group, DEN 50mg / kg was intraperitoneally injected twice a week for the first 4 weeks, and once a week for the 5th to 12th weeks. The SMI0 treatment group was divided into a low-dose DEN combination group, a high-dose DEN combination group, and a high-dose continuous administration group. Among them, the low-dose DEN combination group and the high-dose DEN combination group were forced to intragastrically administer 30mg / kg and 150mg / kg SMI0 compound, respectively, 5min before each DEN administration, and the high-dose continuous administration group was forced to intragastrically administer 150mg / kg SMI0 compound every day from the 1st week, and the model was completed at the 12th week. When the model was completed, blood was collected from the orbit and rat plasma liver function index was measured using a fully automated biochemical analyzer (model group, n = 10 rats; SMI0 compound low dose group, n = 21 rats; DEN combined SMI0 compound high dose group, n = 28 rats; SMI0 compound high dose continuous administration group, n = 24 rats). Experimental results: Compared with the model group, in the SMI0 treatment group, the liver index, liver fibrosis Ishark score, liver fibrosis Masson staining area percentage, α-SMA, and Collagen I were all decreased, although to different degrees (as shown in Figures 18A to 18B and Figures 19A to 19E). * P < 0.05, ** P < 0.01, *** P<0.001 vs. model group; all scales in Fig. 18B are 100 μm), SMI0 high dose (150 mg / kg b The continuous administration group was a high-dose group (150 mg / kg a ) and was significantly more effective than the SMI0 low dose group ( ## P < 0.01, ### P<0.001 vs SMI0 low dose group; & P < 0.05, &&& P<0.001 vs. high-dose combination SMI0 group). These results demonstrated that SMI0 has obvious preventive and therapeutic effects on diethylnitrosamine-induced hepatic fibrosis in rats, suggesting that the SMI0 compound may be useful for the clinical prevention and treatment of hepatic fibrosis.

[0095] Example 14 Inhibition of SMI0 on the development and progression of pulmonary fibrosis in mice (1) Pulmonary fibrosis in cyp2e1 gene knockout mice Experimental method: A C57BL / 6N mouse pulmonary fibrosis model was created by tracheal instillation of 8 mg / kg lipopolysaccharide. C57BL / 6N mice were divided into a control group, a model group, and a cyp2e1 gene knockout group, and the model was completed after 28 days (control group, n=8; model group, n=8; gene knockout group, n=8). At the end of the experiment, the weight of each group of mice was recorded, and the mice were killed after blood was taken from the orbit, and lung tissue was collected. After weighing, the whole left lung was fixed in 4% formalin, and HE and Masson staining were performed, and fibrosis grading of the lung tissue was performed, and fibrosis and histological scoring were performed to reflect the severity of pulmonary fibrosis. Experimental results: Compared with the control group, the model group mice had significantly increased lung index, pathologically showed thickening of alveolar intervals, shrinkage and disappearance of alveolar spaces, and obvious inflammatory cell infiltration in the lung parenchyma, and the fibrosis score and histological score were all significantly increased. Compared with the model group, the cyp2e1 gene knockout mice had significantly decreased lung index, no pathologically obvious inflammatory cell infiltration, and significantly decreased fibrosis score and histological score (see Figures 20A-20B). This suggests that cyp2e1 gene knockout can significantly inhibit the development of pulmonary fibrosis induced by lipopolysaccharide. (2) SMI0 treatment for mouse lung injury Experimental method: A C57BL / 6N mouse pulmonary fibrosis model was created by intratracheal instillation of 8 mg / kg lipopolysaccharide. The SMI0 treatment group was divided into a low-dose group and a high-dose group, and SMI0 compound was administered at 30 mg / kg and 90 mg / kg daily from 3 days before the model was created, and the model was completed 6 h later (control group, n=8; model group, n=8; SMI0 compound high-dose group, n=8). At the end of the experiment, the weight of each group of mice was recorded, and the mice were killed after blood was collected from the orbit, and lung tissue was collected. After weighing, the entire left lung was fixed in 4% formalin and stained with HE. Experimental results: Compared with the control group, the lung index of the mice in the model group was significantly increased (P<0.001). Compared with the model group, the lung index of the mice in the low-dose and high-dose SMI0 groups was significantly decreased (P<0.01). This suggested that SMI0 could significantly reduce the increase in the lung index of mice with acute lung injury induced by lipopolysaccharide. Similarly, compared with the control group, the mice in the model group had obvious destruction of lung tissue structure, mainly showing alveolar edema, thickening of alveolar walls, shrinkage of alveolar spaces and large amounts of inflammatory cell infiltration, and the lung injury score was significantly increased. Compared with the model group, all SMI0-treated groups showed reduced pulmonary edema, narrowing of alveolar intervals, reduced inflammatory cell infiltration, normalization of lung tissue results and a significant reduction in lung injury score (see Figures 21A-21B). This suggested that SMI0 could reduce acute lung injury induced by lipopolysaccharide in mice. (3) SMI0 treatment for pulmonary fibrosis in mice Experimental method: A C57BL / 6N mouse pulmonary fibrosis model was created by intratracheal instillation of 8 mg / kg lipopolysaccharide. The SMI0 treatment group was divided into a low-dose group and a high-dose group, and SMI0 compound was administered at 30 mg / kg and 90 mg / kg daily from 3 days before the model was created, and the model was completed on the 28th (control group, n=8; model group, n=8; SMI0 compound high-dose group, n=8). At the end of the experiment, the weight of each mouse was recorded, and the mice were killed after blood was collected from the orbit, and lung tissue was collected. After weighing, the whole left lung was fixed in 4% formalin, and HE and Masson staining were performed to grade the fibrosis of the lung tissue, and fibrosis and histological scoring were performed to reflect the severity of pulmonary fibrosis. Experimental results: Compared with the control group, the lung index of the mice in the model group was significantly increased, and pathological findings included thickening of the alveolar space, shrinkage and disappearance of the alveolar space, and obvious inflammatory cell infiltration in the lung parenchyma. Compared with the model group, the lung index of the mice in the SMI0 treatment group was significantly decreased, and no obvious inflammatory cell infiltration was observed pathologically (see Figures 22A-22B). This suggests that administration of SMI0 can significantly inhibit the development of pulmonary fibrosis induced by lipopolysaccharide. These results demonstrate that the CYP2E1 inhibitor SMI0 has a clear preventive and therapeutic effect on the development and progression of pulmonary fibrosis induced by tracheal instillation of lipopolysaccharide, suggesting that SMI0 is useful for the clinical prevention and treatment of pulmonary fibrosis. (4) Correlation between CYP2E1 activity and the severity of pulmonary fibrosis in mice after SMI0 inhibition Experimental method: Mouse liver microsomes were prepared using calcium precipitation method, and microsomal protein concentration was measured using Braford method. An incubation system was prepared using 2x PBS buffer, chlorzoxazone solution, and liver microsomes with a final concentration of 0.5mg / mL, and pre-incubated at 37℃ for 5min. The reaction was started by adding NADPH, incubated at 37℃ for 30min, and stopped by placing on ice. 6-hydroxychlorzoxazone was extracted with ethyl acetate, vortex mixed, centrifuged, and the upper organic phase was aspirated and dried with nitrogen gas. The peak area of ​​6-hydroxychlorzoxazone, a chlorzoxazone metabolite, was detected using high performance liquid chromatography with methanol:water=56:44 and detection wavelength of 287nm. The concentration ΔC of the metabolite 6-hydroxychlorzoxazone was calculated by substituting it into the standard curve, and the reaction rate of conversion of chlorzoxazone to 6-hydroxychlorzoxazone was calculated by V (pmol / min / mg) = (ΔC * 1000) / (B * T), where B is the microsomal protein concentration (mg / mL) and T is the incubation time (min), to evaluate the level of CYP2E1 enzyme activity. The correlation between mouse CYP2E1 activity and the severity of mouse pulmonary fibrosis was analyzed. Experimental results: Compared with the control group, the chlorzoxazone metabolic reaction rate V was significantly increased in the model group mice (P<0.01). Compared with the model group, the chlorzoxazone metabolic reaction rate V was significantly decreased in the SMI0 high dose group (P<0.05), suggesting that SMI0 can effectively inhibit the increase in CYP2E1 enzyme activity in pulmonary fibrosis model mice induced by lipopolysaccharide. Analysis of the correlation between the chlorzoxazone reaction rate V and the lung index of pulmonary fibrosis mice showed a correlation coefficient of r=0.81, P<0.01 (see Figures 23A-23B). This suggests that CYP2E1 enzyme activity has a good correlation with the severity of pulmonary fibrosis, and the anti-fibrotic effect of SMI0 may be linked to the inhibition of CYP2E1 enzyme activity. (5) Inhibition of SMI0 on the inflammatory microenvironment of mouse lung fibrosis (i) Inhibition of SMI0 on inflammatory responses in lung-injured mice Experimental Method: The neutrophil levels in the lung tissue of mice were evaluated using immunohistochemical staining, and the percentage of immunostained positive areas was quantified and evaluated using Image-Pro Plus software. RNA was extracted from the lung tissue of mice using a kit method, and the expression levels of TNF-α and IL-1β in the lung tissue of each mouse were measured using RT-PCR. Experimental results: Compared with the control group, the percentage of MPO immunostaining positive area in lung tissue of the model group mice was significantly increased (P<0.001) (see Figures 24A-24B), and the expression of TNF-α and IL-1β in lung tissue was significantly increased (P<0.01) (see Figures 25A-25B). Compared with the model group, the percentage of MPO immunostaining positive area in lung tissue of the SMI0 high dose group was significantly decreased (P<0.05), and the expression of TNF-α and IL-1β in lung tissue was significantly decreased (P<0.05). It was suggested that SMI0 could reduce neutrophil infiltration in lung tissue of mice with acute lung injury induced by lipopolysaccharide and inhibit the release of pro-inflammatory cytokines. (ii) Inhibition of SMI0 on the inflammatory microenvironment in pulmonary fibrosis mice Experimental Method: The ammonium molybdate method and the microplate method were used to process the mouse lung tissue homogenates, respectively, to detect the changes in the oxidative stress indicators CAT and GSH in the lung tissue of mice in each group. The expression levels of TGF-β1 and mesenchymal cell marker α-SMA in the mouse lung tissue were evaluated using immunohistochemical staining, and the percentage of positive areas was statistically calculated using Image-Pro Plus software. The expression levels of epithelial cell marker E-cadherin, apoptosis-related protein Bax, and anti-apoptosis-related protein Bcl-2 were detected using Western Blot method. Experimental results: Compared with the control group, in the model group, the levels of oxidative stress indicator CAT (P<0.01) and epithelial cell marker E-cadherin were significantly decreased (P<0.05), the percentage of α-SMA and TGF-β1 immunostaining positive areas was significantly increased (P<0.01), the expression level of the apoptotic factor Bax was significantly increased (P<0.01), and the expression level of the anti-apoptotic factor Bcl-2 was significantly decreased (P<0.05). Compared with the model group, in the SMI0-treated mice, the oxidative stress index CAT (P<0.01) and the epithelial cell marker E-cadherin levels were significantly increased (P<0.05), the percentage of α-SMA and TGF-β1 immunostaining positive areas was significantly decreased (P<0.01), the Bax level was significantly decreased (P<0.01), and the Bcl-2 level was significantly increased (P<0.001) (see Figures 26-27). This suggests that SMI0 reduces lipopolysaccharide-induced pulmonary fibrosis in mice, which may lead to improved antioxidant capacity, anti-apoptotic effects, and reduced levels of fibrosis-inducing factor TGF-β1 and reduced epithelial-mesenchymal cell transformation in the lung tissue of pulmonary fibrosis mice.

[0096] Example 15 Inhibition of SMI0 on mouse lung cancer growth (1) Changes in CYP2E1 in clinical lung cancer patients Experimental method: Lung tissues from 30 healthy subjects were used as controls, and changes in CYP2E1 expression in the peritumoral tissues of 30 clinical lung cancer patients were examined, and changes in CYP2E1 content in the peritumoral tissues of lung cancer patients were compared. Experimental results: Immunohistochemistry results showed that the expression of CYP2E1 in the peritumoral tissues of lung cancer patients was significantly higher than that in the normal lung tissues (as shown in Figure 28, ***P<0.001 vs. normal lung tissue group). (2) Changes in lung orthotopic tumors in cyp2e1 knockout mice Experimental method: A lung cancer orthotopic tumor model was created in female C57 mice using Lewis cells, and the cell dose was 2*10 4 C57BL / 6N mice were divided into a control group, a model group, and a cyp2e1 gene knockout group, and the model was completed on the 18th day (control group, n=8; model group, n=8; gene knockout group, n=8). At the end of the experiment, the weight of each group of mice was recorded, and they were killed after blood was taken from the orbit, and lung tissues and tumor tissues were collected. After weighing, some lung samples were stained with HE, and the development of lung lesions and tumors in the mice of each group was observed. Experimental results: Compared with the model group, the lung tumor weight was significantly reduced in the cyp2e1 gene knockout mice (see Figures 29A-29B). This suggests that cyp2e1 gene knockout can significantly inhibit the development and progression of the Lewis cell lung cancer orthotopic transplantation tumor model. (3) SMI0 treatment of mouse Lewis cell lung orthotopic tumors Experimental method: Using the lung cancer cell line Lewis cells, a lung orthotopic tumor model was established in female C57 mice. The cell dose was 2*10 4C57BL / 6N mice were divided into control, model, positive drug and SMI0 treatment groups, and the model was completed on the 18th day [control, n=20; model, n=20; positive drug group (20mg / kg), n=15; SMI0 (3.3mg / kg), n=15; SMI0 (10mg / kg), n=20; SMI0 (30mg / kg), n=20)]. At the end of the experiment, the weight of each group of mice was recorded, and they were killed after blood was taken from the orbit, and lung tissue and tumor tissue were collected. After weighing, the weight and appearance of the lungs (lung color, texture, etc.) were recorded. The cumulative weight of all tumors was lined up to represent the tumor development status of one animal, and the cumulative weight of the tumor in the lungs of each animal was measured, and some lung samples were stained with HE, and then the lesions and tumor development in the lungs of the mice in each group were observed. Experimental results: Compared with the model group, the growth of lung tumors was significantly inhibited in the mice treated with SMI0, that is, SMI0 treatment could significantly inhibit the growth of lung tumors in mice, and the tumor growth inhibition rate reached 68.3% (as shown in Figures 30A-30C). * P<0.05 vs. model group, where Figure 30A is a representative image of the lung tissue containing tumors in each group of mice, Figure 30B is the lung tumor tissue in each mouse, and Figure 30C is the tumor weight in each group of animals.) This suggests that administration of SMI0 can significantly inhibit the development and progression of the Lewis lung cancer cell mouse lung orthotopic transplantation tumor model. (4) SMI0 treatment of mouse CT26 cell lung metastases Experimental method: A lung metastasis tumor model was created in female Balb / C mice by tail vein injection of colorectal cancer cells CT26. The cell dose was 3*10 5 The mice were divided into a control group, a model group, and an SMI0 treatment group, and the model was completed on the 14th day (control group, n=5; model group, n=5; SMI0 (30 mg / kg), n=5). At the end of the experiment, the weight of the mice in each group was recorded, and they were killed after blood was taken from the orbit, and the lung tissue was collected, weighed, and recorded. The lung tissue was fixed in Buzaglo´s staining solution for 24 hours, and the number of lung nodules in the mice in each group was recorded. Experimental results: Compared with the model group, the lung weight and nodule number in the SMI0-treated mice were significantly decreased, and the tumor growth inhibition rate reached a maximum of 66.7% (as shown in Figures 31A-31B). * P<0.05 vs. model group, where Fig. 31A shows representative images of lung tissue containing tumors in each group of mice, and Fig. 31B shows lung weight and lung nodule count for each mouse.) It was suggested that administration of SMI0 could significantly inhibit the development and progression of lung metastasis cancer model induced by tail vein injection of CT26 cells. (5) SMI0 treatment of mouse B16-F10 cell lung metastasis tumors Experimental method: A lung metastasis tumor model was created in female C57BL / 6J mice by tail vein injection of melanoma cells B16-F10. The cell dose was 8*10 5 The mice were divided into a control group, a model group, and an SMI0 treatment group, and the model was completed on the 14th day (control group, n=5; model group, n=5; SMI0 (30 mg / kg), n=4). At the end of the experiment, the weight of the mice in each group was recorded, and they were killed after blood was taken from the orbit, and the lung tissue was collected, weighed, and recorded. The lung tissue was fixed in Buzaglo´s staining solution for 24 hours, and the number of lung nodules in the mice in each group was recorded. Experimental results: Compared with the model group, the lung weight and nodule number in the SMI0-treated mice were significantly decreased, and the tumor growth inhibition rate reached 58.3% (as shown in Figures 32A-32B). * P<0.05 vs. model group, where FIG. 32A is a representative image of the lung tissue containing the tumor in each group of mice, and FIG. 32B is the lung weight of each mouse.) It was suggested that administration of SMI0 could significantly inhibit the development and progression of lung metastasis tumor model induced by tail vein injection of B16-F10 cells in mice. These results indicate that the CYP2E1 inhibitor SMI0 has a clear preventive and therapeutic effect on the development and progression of lung metastases induced by orthotopic transplantation of Lewis cells and tail vein injection of CT26 and B16-F10. The SMI0 compound is suggested to be useful for the prevention and treatment of lung cancer. (6) Correlation between mouse CYP2E1 activity after SMI0 inhibition and tumor severity in a lung orthotopic cancer model Experimental method: Mouse liver microsomes were prepared using calcium precipitation method, and microsomal protein concentration was measured using Braford method. An incubation system was prepared using 2x PBS buffer, chlorzoxazone solution, and liver microsomes with a final concentration of 0.5mg / mL, and pre-incubated at 37℃ for 5min. The reaction was started by adding NADPH, incubated at 37℃ for 30min, and stopped by placing on ice. 6-hydroxychlorzoxazone was extracted with ethyl acetate, vortex mixed, centrifuged, and the upper organic phase was aspirated and dried with nitrogen gas. The peak area of ​​6-hydroxychlorzoxazone, a chlorzoxazone metabolite, was detected using high performance liquid chromatography at methanol:water=56:44 and detection wavelength of 287nm. The concentration ΔC of the metabolite 6-hydroxychlorzoxazone was calculated by substituting it into the standard curve, and the conversion reaction rate of chlorzoxazone to 6-hydroxychlorzoxazone was calculated by V (pmol / min / mg) = (ΔC*1000) / (B*T), where B is the microsomal protein concentration (mg / mL) and T is the incubation time (min), to evaluate the level of CYP2E1 enzyme activity. The correlation between mouse CYP2E1 activity and lung tumor weight in Lewis lung orthotopic cancer model mice was analyzed. Experimental results: Compared with the sham operation group, the CYP2E1 activity in the model group mice was significantly increased (P<0.05), and compared with the model group, the CYP2E1 activity in the SMI0 treatment group mice was significantly decreased (P<0.05), which shows that SMI0 can effectively inhibit the increase in CYP2E1 enzyme activity in the liver tissue of Lewis lung orthotopic cancer model mice, and the mouse CYP2E1 activity showed a significant positive correlation with the tumor weight of Lewis lung orthotopic cancer model mice (r=0.70, P<0.01) (see Figure 33). This suggests that the high CYP2E1 enzyme activity is positively correlated with the severity of Lewis lung orthotopic cancer, and the anti-lung cancer effect of SMI0 may be linked to the inhibition of CYP2E1 enzyme activity. (7) SMI0 inhibits the inflammatory microenvironment of orthotopically transplanted tumors in mouse lungs Experimental method: Western blot was used to detect the expression of pro-inflammatory cytokines TGF-β, IL-10, IL-4, and inflammation-related signal pathway proteins (IL-6 / p-STAT3, p-ERK1 / 2 / ERK1 / 2, and epithelial-mesenchymal transition-related proteins MMP-2 and MMP-9) in the lung tissue surrounding the tumor of each group of mice. The expression of anti-apoptosis-related proteins caspase3, Bcl-2, and autophagy-related protein p53 in the tumor tissue was also detected. Experimental results: Compared with the sham-operated group, the expression of proteins such as proinflammatory cytokines (TGF-β, IL-10), inflammation-related signal pathways (IL-6 / p-STAT3, p-ERK1 / 2 / ERK1 / 2) and matrix metalloproteinases (MMP-2, MMP-9) in the lung tissue surrounding the tumor in the model group mice was significantly increased (P<0.05). Furthermore, compared with the model group, the expression of all the above proteins was significantly decreased (P<0.05) in the SMI0 treatment group (see Figures 34A-34B). Similarly, compared with the model group, the expression of anti-apoptotic proteins caspase3 and Bcl-2 in the tumor tissue in the SMI0 treatment group (30 mg / kg) was significantly decreased (P<0.05), and the expression of autophagy-related protein p53 was significantly increased (P<0.01) (see Figures 35A-35B). These results suggest that SMI0 can significantly inhibit inflammation and related signal pathway activation in the peritumor lung tissue of mouse Lewis lung orthotopically transplanted tumors, as well as matrix metalloproteinase expression, and enhance p53-mediated autophagy in tumor tissue. (8) SMI0 inhibits M2 polarization of macrophages in the tumor microenvironment (i) SMI0 does not have a direct inhibitory effect on lung cancer cells Experimental method: Lewis lung cancer cells and A549 lung cancer cells in logarithmic growth phase were cultured at 1*10 5The cells were inoculated into a 96-well plate at a concentration of 100 μL / mL, and 100 μL of basal medium containing different concentrations of SMI0 (0, 0.16, 0.8, 4, 20, 100 μmol / L) was added to each well. After 24 hours, 10 μL of CCK8 reagent was added to each well and cultured for 2 hours. The absorbance OD value at 450 nm wavelength of each well was detected using a microplate reader. This was repeated three times and the average value was taken. The proliferation activity was calculated based on the OD value. Experimental results: SMI0 at the tested concentrations (up to 100 μmol / L) had no obvious inhibitory effect on the proliferation of either Lewis lung cancer cells or A549 lung cancer cells (see Figures 36A-36B). (ii) SMI0 inhibits lung cancer cell proliferation by inhibiting M2 polarization of macrophages Experimental method: Human mononuclear leukemia cells THP-1 were induced to M0 macrophages using 100 μmol / L PMA, and then induced to M2 macrophages using 20 ng / ml IL-4 and IL-13. The supernatant and A549 lung cancer cells were extracted to construct a co-culture system to simulate the microenvironment of M2 macrophages in lung cancer. In the SMI0 treatment group, interleukin was added for induction and SMI0 (50 μmol / L) treatment was administered, and the cell supernatants of M0 macrophages, M2 macrophages, and SMI0 treatment groups were extracted to prepare conditioned medium. Logarithmic growth phase Lewis lung cancer cells were cultured at 1*10 5 The cells were inoculated into a 96-well plate at a concentration of 100 μL / mL, and 100 μL of different types of conditioned medium was added to each well and incubated for 24 hours. After that, 10 μL of CCK8 reagent was added to each well and incubated for 2 hours. The absorbance OD value of each well at a wavelength of 450 nm was then detected using a microplate reader. This was repeated three times and the average value was taken. The proliferation activity was calculated based on the OD value. Experimental results: Compared with the A549 lung cancer cell monoculture group, the proliferation activity of A549 cells was significantly improved in the M2 macrophage coculture group (P<0.01), and compared with the M2 macrophage coculture group, the proliferation activity of A549 cells was significantly decreased in the SMI0 treatment group (50 μmol / L) (P<0.05) (shown in Figure 37). This suggests that inhibition of Lewis lung orthotopic transplant tumors by the CYP2E1 inhibitor SMI0 may lead to inhibition of the peritumoral inflammatory microenvironment of lung cancer, especially the M2 polarization of macrophages in the peritumoral microenvironment.

[0097] Example 16 Inhibition of SMI0 on the development and progression of mouse hepatocellular carcinoma (1) Changes in CYP2E1 in clinical liver cancer patients Experimental method: Using liver tissues from 35 healthy subjects as controls, changes in CYP2E1 expression in the peritumoral tissues of 35 clinical liver cancer patients were examined, and changes in CYP2E1 content in the peritumoral tissues of liver cancer patients were compared. Experimental Results: Immunohistochemistry results revealed that the expression of CYP2E1 in the peritumoral tissues of liver cancer patients was significantly higher than that in normal liver tissues (as shown in Figure 38). *** P<0.0001 vs normal liver tissue group) (2) Orthotopic transplantation of liver tumors in cyp2e1 knockout rats Experimental method: A breast cancer sarcoma cell line, Walker 256 cells, was orthotopically transplanted into the liver of SD rats to create an orthotopic tumor model. The cell concentration was 4*10 6 SD rats were divided into a model group and a cyp2e1 gene knockout group, and the models were completed on the 21st day (model group, n = 9 mice; gene knockout group, n = 8 mice). At the end of the experiment, blood was collected from the orbit and the rats were killed after recording their weight. The weight and appearance of the liver (liver color, texture, etc.) were recorded. After HE staining of some liver samples, the rats in each group were observed for the development of liver lesions and tumors. Experimental results: Compared with the model group, the growth of liver tumors was significantly inhibited in the cyp2e1 gene knockout group of rats, with the tumor growth inhibition rate reaching 82.0% (see Figure 39). This suggests that cyp2e1 gene knockout can significantly inhibit the development of liver tumors in the Walker256 cell liver orthotopic transplantation rat liver model. (3) Treatment of orthotopic tumors transplanted into mouse liver with SMI0 Experimental method: A hepatoma tumor model was created in male BALB / c mice by orthotopic transplantation of hepatoma cell line H22 cells into the liver. The cell concentration was 1.5*10 5 The SMI0 treatment group was divided into a low-dose group, a medium-dose group, and a high-dose group, and each group was administered 3.3 mg / kg, 10 mg / kg, and 30 mg / kg of SMI0 compound every day from 2 days before the model was created, and the model was completed on the 24th day (model group, n = 19 mice; SMI0 compound low-dose group, n = 19 mice; SMI0 compound medium-dose group, n = 18 mice; SMI0 compound high-dose group, n = 29 mice). At the end of the experiment, blood was collected from the orbit and the mice were killed after recording their weight. The weight and appearance of the liver (liver color, texture, etc.) were recorded. After HE staining was performed on some of the liver specimens, the development of liver lesions and tumors in the mice of each group was observed. Experimental results: Compared with the model group, the incidence of liver H22 tumors in mice treated with SMI0 was significantly reduced, from 100% in the model group to 78.9%, 72.2%, and 68.4% in the low, medium, and high dose groups of SMI0 (as shown in Figures 40A-40C). * P < 0.05, ** P<0.01 vs. model group). Figure 40 shows the liver tumor tissue images of each mouse. When multiple tumors were formed in the liver of one mouse, all the tumors were arranged in a pile to show the tumor development status of one animal. The cumulative weight of the liver tumors of each animal was measured, and it was found that tumors were formed in the liver of each mouse in the model group, and tumor growth was significantly inhibited in the SMI0 treatment group compared to the model group. In other words, SMI0 treatment could significantly inhibit the growth of mouse liver tumors, with the tumor growth inhibition rate reaching 71.8%. This suggests that administration of SMI0 can significantly inhibit the development and progression of H22 cell orthotopic transplanted mouse liver cancer tumor models. (4) SMI0 inhibits the inflammatory microenvironment of orthotopically transplanted tumors in mouse liver Experimental method: The oxidative stress indicators MDA and T-AOC levels in the tumor-surrounding liver tissue of mice were measured using a kit method, the expression of chemokine CXCL-12 in the tumor-surrounding liver tissue of mice was measured using RT-PCR, and the expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β), inflammation and related signaling pathway proteins (ARRB1, Src, ITGAV, p-STAT3 / STAT3, p-ERK / ERK, p-P38MAPK / P38MAPK) in the tumor-surrounding lung tissue of mice in each group was measured using Western-blot. In addition, the expression of apoptosis-related proteins and signaling pathways casPase3, cleaved casPase3, p-ERK / ERK, p-p38MAPK / p38MAPK in the tumor tissue was measured using Western-blot. Experimental results: Compared with the sham-operated group, the expression of oxidative stress indicator MDA, chemokine CXCL-12, proinflammatory cytokines (TNF-α, IL-6, IL-1β), inflammation and related signal pathway proteins (ARRB1, Src, ITGAV, p-STAT3 / STAT3, p-ERK / ERK, p-p38MAPK / p38MAPK) in the tumor surrounding liver tissue of the model group mice was significantly increased (P<0.05 or P<0.01), and the expression of total antioxidant capacity indicator T-AOC was significantly decreased (P<0.01). Furthermore, compared with the model group, the expression of total antioxidant capacity indicator T-AOC in the SMI0 treatment group was significantly increased (P<0.01), and the expression of the other above proteins was significantly decreased (P<0.05 or P<0.01) (see Figures 41A-41B). Similarly, compared with the model group, in the SMI0 treatment group (30 mg / kg), the apoptosis-related proteins cleaved caspase3 / caspase3 in the tumor tissue were significantly increased (P<0.01), and p-ERK / ERK, p-p38MAPK / p38MAPK were significantly decreased (P<0.05 or P<0.01) (see Figures 42A-42B). This suggests that SMI0 significantly inhibits oxidative stress, inflammation and activation of related signal pathways in the tumor-surrounding liver tissue of mouse H22 liver orthotopic transplant tumors, promoting tumor apoptosis. (5) SMI0 exerts anti-liver cancer effect by affecting macrophages (i) SMI0 does not have a direct inhibitory effect on hepatoma cells Experimental method: Logarithmic growth phase HepG2 hepatoma cells and H22 hepatoma cells were cultured at 1 x 10 5 The cells were seeded into a 96-well plate at a concentration of 100 cells / mL, and 100 μL of basal medium containing different concentrations of SMI0 (0, 0.16, 0.8, 4, 20, 100 μmol / L) was added to each well. After 24 h, HepG2 proliferation activity was measured using CCK8, HepG2 migration ability was measured using a cell scratch experiment, and HepG2 apoptosis ability was measured using a flow cytometry method. Experimental results: SMI0 at the tested concentrations (up to 100 μmol / L) had no obvious inhibitory effect on the proliferation, migration and apoptosis of HepG2 and H22 hepatoma cells (see Figures 43A-43B). It was suggested that the CYP2E1 inhibitor SMI0 had no direct inhibitory effect on hepatoma cells. (ii) SMI0 exerts anti-hepatocellular carcinoma activity by affecting macrophages Experimental Method: Supernatant of mouse-derived macrophages RAW264.7 and HepG2 hepatoma cells were extracted, and a co-culture system was established to simulate the macrophage inflammatory microenvironment of hepatoma. The SMI0-treated group was induced with interleukin and treated with different concentrations of SMI0 (0, 0.16, 0.8, 4, 20, 100 μmol / L), and the cell supernatant of the SMI0-treated group was extracted to produce conditioned medium. HepG2 hepatoma cells in the logarithmic growth phase were selected, and HepG2 proliferation activity was measured using CCK8, HepG2 migration ability was measured using cell scratch experiments, and HepG2 apoptosis ability was measured using flow cytometry. Experimental results: Compared with the HepG2 hepatoma cell single culture group, the HepG2 cell proliferation activity (P<0.05) and migration ability were significantly increased (P<0.001), and the apoptosis ability was significantly decreased (P<0.001) in the RAW264.7 macrophage co-culture group. Compared with the macrophage co-culture group, the HepG2 hepatoma cell proliferation activity (P<0.001) and migration ability were significantly decreased (P<0.001), and the apoptosis ability was significantly increased (P<0.001) in the SMI0 treatment group (especially 4μmol / L) (see Figure 44). This suggests that the inhibition of hepatoma by the CYP2E1 inhibitor SMI0 may inhibit the proliferation and migration of HepG2 hepatoma cells and promote the apoptosis of HepG2 hepatoma cells by affecting the macrophages in the hepatoma tumor microenvironment. These results demonstrate that SMI0 has a clear preventive and therapeutic effect on the development and progression of hepatocellular carcinoma in mice orthotopically transplanted with H22 cells. SMI0 compound is suggested to be useful for clinical prevention and treatment of hepatocellular carcinoma.

[0098] Example 17 Inhibition of SMI0 on mouse glioma growth (1) Changes in CYP2E1 in clinical glioma patients Experimental method: The brain tissues of 46 healthy subjects were used as controls, and the changes in CYP2E1 expression in the peritumoral tissues of 32 clinical glioma patients were examined, and the changes in CYP2E1 protein content in the peritumoral tissues of glioma patients were compared. The normal brain tissues of 6 subjects were used as controls, and the CYP2E1 mRNA levels in the peritumoral brains of 12 glioma patients were examined, and the changes in the peritumoral brain CYP2E1 mRNA levels were compared. Experimental results: Immunohistochemistry results showed that the expression of CYP2E1 in peritumoral tissues of glioma patients was significantly higher than that in normal brain tissues (as shown in Figure 45A). *** P<0.001 vs. normal brain tissue group). qPCR results showed that the expression of CYP2E1 mRNA levels in the peritumoral tissues of glioma patients was significantly higher than that in the normal brain tissues (as shown in Figure 45B). *** P<0.001 vs normal brain tissue group). (2) Orthotopic transplantation of tumors into the brain of cyp2e1 knockout mice Experimental method: A female C57 / BL6 rat brain orthotopic glioma model was created using mouse brain glioma cell line GL261 cells. Model creation method: A hole was made in the skull at a location 0.15 mm posterior and 2 mm to the right of the bregma, and 5 μL of a GL261 single cell suspension (total 1 × 10 cells) was injected with a microsyringe. 6 The mice were then aspirated with 1000 cells (cells), then the needle was slowly inserted vertically along the needle hole to a depth of 4 mm, then retracted 1 mm, and this step was repeated 10 times, after which the tumor-bearing cells were slowly injected at a rate of 1 μl / min. The experiment was divided into a model group and a cyp2e1 gene knockout group, and the model was completed on about the 21st day (model group, n = 10 mice; cyp2e1 gene knockout group, n = 6 mice). After recording the weight of the mice at the end of the experiment, blood was taken from the orbit, and the mice were killed and their brains were removed. HE staining was performed on the brain tissue samples to observe the tumor formation status and tumor size of the mice in each group. Experimental results: Compared with the model group, cyp2e1 gene knockout could significantly inhibit the growth of mouse glioma tumors (as shown in Figure 46). * P<0.05 vs. wild-type model group), suggesting that cyp2e1 gene knockout could significantly inhibit the development of GL261 cell brain orthotopically transplanted mouse glioma model. (3) Treatment of SMI0 on orthotopically transplanted tumors in mouse brain Experimental method: A glioma model was established in female C57 / BL6 mice by orthotopic transplantation of glioma cell line GL261 cells into the brain. The cell concentration was 1*10 6Experimental groups: sham operation group (n = 9), model group (n = 10), temozolomide (50 mg / kg, ig, n = 11), SMI0 low dose group (3.3 mg / kg, ig, n = 13), SMI0 medium dose group (10 mg / kg, ig, n = 13), SMI0 high dose group (30 mg / kg, ig, n = 13). In the SMI0 treatment group, the SMI0 compound was administered intragastrically every day from 3 days before the model was created, and in the positive drug group, it was administered for 3 to 7 days and 10 to 14 days after the operation, and the model was completed on about the 21st day. At the end of the experiment, blood was collected from the orbit, and the weight of the mice was recorded, and then they were killed and their brains were collected. After HE staining of the brain tissue samples, the development of brain tissue lesions and tumors in the mice of each group was observed. Experimental results: Compared with the model group, SMI0 treatment could significantly inhibit the growth of mouse glioma tumors, among which the inhibitory effect of the high dose group of SMI0 (30 mg / kg) was the best, with the tumor growth inhibition rate reaching up to 97.6% (as shown in Figure 47A-C). * P<0.05 vs. model group; ** P<0.01 vs. model group). These results suggest that SMI0 can significantly inhibit the development and progression of GL261 cell-brain orthotopically transplanted mouse glioma models. Furthermore, in vitro inhibition experiments (FIG. 47D) demonstrated that SMI0 acted on GL261 cells continuously for 48 h within the concentration range of 0 to 128 μM, and none of the SMI0 exerted any obvious inhibitory effect on GL261. These results indicate that CYP2E1 gene knockout can significantly inhibit the development and progression of mouse gliomas, and that the CYP2E1 inhibitor SMI0 has clear preventive and therapeutic effects on the development and progression of gliomas in mice orthotopically implanted with GL261 cells. These results suggest that the SMI0 compound may be useful for the clinical prevention and treatment of gliomas. (4) Correlation between CYP2E1 activity after SMI0 inhibition and tumor severity in a mouse glioma model Experimental method: Mouse liver microsomes were prepared using calcium precipitation method, and microsomal protein concentration was measured using Braford method. An incubation system was prepared using 2x PBS buffer, chlorzoxazone solution, and liver microsomes with a final concentration of 0.5mg / mL, and pre-incubated at 37℃ for 5min. The reaction was started by adding NADPH, incubated at 37℃ for 30min, and stopped by placing on ice. 6-hydroxychlorzoxazone was extracted with ethyl acetate, vortex mixed, centrifuged, and the upper organic phase was aspirated and dried with nitrogen gas. The peak area of ​​6-hydroxychlorzoxazone, a chlorzoxazone metabolite, was measured using high performance liquid chromatography with methanol:water=56:44 and detection wavelength of 287nm. The concentration ΔC of the metabolite 6-hydroxychlorzoxazone was calculated by substituting it into the standard curve, and the conversion reaction rate of chlorzoxazone to 6-hydroxychlorzoxazone was calculated by V (pmol / min / mg) = (ΔC * 1000) / (B * T), where B is the microsomal protein concentration (mg / mL) and T is the incubation time (min), to evaluate the level of CYP2E1 enzyme activity. The correlation between mouse CYP2E1 activity and tumor size in glioma model mice was analyzed. Experimental results: Compared with the sham operation group, the CYP2E1 activity in the model group mice was significantly increased (P<0.05), and compared with the model group, the CYP2E1 activity in the SMI0 treatment group mice was significantly decreased (P<0.001), which shows that SMI0 can effectively inhibit the increase in CYP2E1 enzyme activity in liver tissue of glioma orthotopic tumor model mice, and the mouse CYP2E1 activity showed a significant positive correlation with the tumor weight of glioma orthotopic model (r=0.5995, P=0.001) (see Figure 48). This suggests that the high CYP2E1 enzyme activity is related to glioma, and the anti-glioma effect of SMI0 may be related to the inhibition of CYP2E1 enzyme activity. (5) SMI0 inhibits the growth of GL261 glioma through the peritumoral microenvironment (i) SMI0 does not have a direct inhibitory effect on glioma cells Experimental method: 5*10 GL261 mouse glioma cells and U251 human glioma cells in the logarithmic growth phase 5 The cells were inoculated into a 96-well plate at a concentration of 100 μL / mL, and 100 μL of the cell suspension was added to each well. After 12 hours, the medium was discarded, and 200 μL of basal medium containing different concentrations of SMI0 (0, 0.16, 0.8, 4, 20, 100 μmol / L) was added to each well. After 24 hours, 10 μL of CCK8 reagent was added to each well and cultured for 2 hours. The absorbance OD value of each well at a wavelength of 450 nm was then measured using a microplate reader. This was repeated three times and the average value was taken. The proliferation activity was calculated based on the OD value. Experimental results: SMI0 at the tested concentrations (up to 128 μmol / L or 200 μmol / L) had no obvious inhibitory effect on the proliferation of either GL261 or U251 glioma cells (see FIG. 49). (ii) Glioma inhibition of SMI0 through peritumoral microglial cells and astrocytes Experimental method: Ten newborn C57BL / 6 wild mice aged 1-2 days were used. - / - Six mice were prepared, primary microglial cells and astrocytes were extracted, inoculated into 6-well plates, and the supernatants were collected after 24 h and co-cultured with glioma cells. Experimental groups were divided into: control group, M2 model group, high, medium, and low concentration SMI0 (50 μmol / L, 12.5 μmol / L, 3.1 μmol / L) groups, and Cyp2e1 gene knockout group. After 24 h, the supernatants were discarded, and in the M2 model group, high, medium, and low concentration SMI0 groups, 20 ng / ml of IL-4 and IL-13 were added to induce microglial cells into the M2 type, and in the treatment group, different concentrations of SMI0 (50 μmol / L, 12.5 μmol / L, 3.1 μmol / L) were added simultaneously for treatment. Glioma cell proliferation was measured using CCK8, and the apoptosis ratio was measured using Annexin V-FITC-PI apoptosis staining kit. Experimental results: Compared with the GL261 / U251 glioma cell single culture group, the proliferation activity of GL261 / U251 cells was significantly enhanced and apoptosis was significantly inhibited in the M2 microglial cell co-culture group; compared with the M2 microglial cell co-culture group, the proliferation of GL261 / U251 cells was significantly inhibited in the SMI0 treatment group (50μmol / L) (P<0.001) and promoted the apoptosis of GL261 cells (see Figure 50). This suggests that the inhibition of orthotopic transplantation of GL261 and U251 by the CYP2E1 inhibitor SMI0 may be related to microglial cells. Compared with the GL261 glioma cells cultured alone, the proliferation activity of GL261 cells was significantly enhanced (P<0.01) and apoptosis was significantly inhibited (P<0.01) in the M2 astrocyte co-culture group. Compared with the M2 astrocyte co-culture group, the proliferation activity of GL261 cells was significantly decreased (P<0.05) in the SMI0 treatment group (50 μmol / L) (P<0.01, as shown in Figure 51). This suggests that the inhibition of GL261 orthotopic tumor growth by the CYP2E1 inhibitor SMI0 may be related to astrocytes. (iii) SMI0-induced inhibition of glioma growth is associated with polarization of microglial cells into M2 and inhibition of astrocytic cholesterol metabolism. Experimental Method: The stellate cells from the co-culture group were extracted, RNA was extracted and reverse transcribed into cDNA, and the changes in inflammatory factors and cholesterol metabolism-related genes were detected using qPCR. Experimental results: Compared with the GL261 glioma cell monoculture group, the levels of IL-4, IL-10 and IL-13 in M2 type microglial cells were significantly higher, and the level of the related gene PPAR-α involved in lipid metabolism was significantly lower. Compared with the M2 type microglial cell coculture group, the levels of IL-4, IL-10 and IL-13 in the SMI0 treatment group (50 μmol / L) were significantly lower, and the level of the related gene PPAR-α involved in lipid metabolism was significantly higher (see Figures 52 and 53). This suggests that the inhibition of GL261 orthotopic transplantation tumors by the CYP2E1 inhibitor SMI0 may be related to the polarization of microglial cells to M2 and the inhibition of astrocyte cholesterol metabolism.

[0099] Example 18 Inhibition of SMI0 on the development and progression of mouse ovarian cancer (1) Orthotopic transplantation of ovarian cancer tumors Experimental method: Using mouse-derived ovarian cancer ID-8 cell line, an ovarian cancer orthotopic transplantation tumor model was created in female C57 / 6 mice. The cell concentration was 1 × 10 6 The mice were divided into a model group and an SMI0 treatment group, and the treatment group was forced to intragastrically administer 30 mg / kg of SMI0 compound every day from 2 days before the model was created, and the model was completed on the 60th day (model group, n = 15; SMI0 compound 30 mg / kg, n = 12). At the end of the experiment, blood was collected from the orbit, and the mice were killed after recording their weight, and the ovaries and tumor tissues were collected. After HE staining of the ovarian tissue samples, the ovarian tumor tissues and tumor development of the mice in each group were observed. Experimental results: Compared with the model group, SMI0 treatment could significantly inhibit the growth of ovarian cancer in mice, with the tumor growth inhibition rate reaching up to 63.14% and the inhibition rate of ascites weight reaching 76.40% (see Figure 54A). This suggests that the administration of SMI0 can significantly inhibit the development and progression of the ID-8 ovarian cancer orthotopic tumor model. Furthermore, in vitro inhibition experiments (FIG. 54B) demonstrated that SMI0 acted on ID-8 cells for 48 h within a concentration range of 0 to 320 μM, but had no clear inhibitory effect on ID-8. (2) Ovarian cancer peritoneal transplant tumor Experimental method: Using mouse-derived ID8 cell line of ovarian cancer, a tumor model of ovarian cancer was created in female C57 / 6 mice by abdominal transplantation at a cell concentration of 2.5 × 10 6 The mice were divided into a model group and an SMI0 treatment group. The treatment group was forced to intragastrically administer 30 mg / kg of SMI0 compound every day from 3 days before model creation, and the model was completed on the 30th day (model group, n = 10 mice; SMI0 compound 30 mg / kg, n = 11 mice). At the end of the experiment, blood was collected from the orbit and the mice were killed after recording their weights, and ovarian tissue was collected. After HE staining of the ovarian tissue samples, the ovarian tissue lesions and tumor development of the mice in each group were observed. Experimental results: Compared with the model group, the mice treated with 30mg / kg of SMI0 compound had a significant decrease in body weight and ascites volume, which means that the administration of SMI0 compound can significantly inhibit ascites formation and tumor progression of mouse ovarian cancer tumors implanted in the peritoneal cavity, among which the inhibition rate of omentum tumor growth reached a maximum of 58.70%, and the inhibition rate of ascites weight reached 83.78% (see Figure 55). This suggests that the administration of SMI0 can significantly inhibit the development and progression of ID-8 cell ovarian cancer tumor models implanted in the peritoneal cavity. These results demonstrated that the CYP2E1 inhibitor SMI0 has a clear preventive and therapeutic effect on the development and progression of orthotopic and peritoneal transplanted ID-8 ovarian cancer tumors, suggesting that the SMI0 compound may be useful for the prevention and treatment of ovarian cancer.

[0100] Example 19 Inhibition of SMI0 against rheumatoid arthritis in rats (1) Collagen inhibits cyp2e1 gene knockout in rat rheumatoid arthritis Experimental method: A rheumatoid arthritis model was created in SD rats using collagen emulsion prepared with collagen and incomplete Freund's adjuvant in a volume ratio of 1:1. The rats were divided into a model group and a cyp2e1 gene knockout group. The first immunization was performed by subcutaneously injecting 0.25 mL of collagen emulsion into the tail of each rat, and the second immunization was performed in the same manner as the first immunization one week later. If the rat arthritis score was 4 points or more (0 points: no erythroderma; 1 point: slight swelling of the little toe joint; 2 points: swelling of the toe joint and sole; 3 points: swelling of the toenail below the ankle joint; 4 points: swelling of the entire paw including the ankle joint; a total of 16 points for 4 paws) within one week of the second immunization, the model was considered to have been created successfully. After the rat model was successfully created, the thickness of the left and right hind paws and the left and right forepaws of the rats was measured daily using a vernier caliper. The volume of the left and right hind paws of the rats was measured using a rat paw swelling meter (measured once every 12 hours for the previous 3 days), and the paw swelling rate of each rat was calculated based on the change in the rat's hind paw volume at each time point compared to before model creation, and the difference in paw swelling rate between rats in each group was analyzed. After the model was successfully established, blood was collected from the orbit on the 7th day, and the rats were killed on the 20th day, blood was collected, and serum was separated. Tissues such as ankle joints, toe joints, spleen, and liver were collected. The tissues were decalcified and stained with hematoxylin-iosin (HE), and joint lesions, synovial hyperplasia, and connective tissue hyperplasia were observed under a microscope. Experimental results: Compared with the model group, the success rate of model creation in the cyp2e1 gene knockout group rats was 40%, significantly lower than the model group (90%), and the degree of joint swelling in the gene knockout group rats was also significantly lower than that in the model group (P<0.05) (see Figure 56). This suggests that cyp2e1 gene knockout can significantly reduce the incidence of collagen-induced rat rheumatoid arthritis and improve the degree of swelling. (2) Collagen inhibits SMI0 in rat rheumatoid arthritis Experimental method: A collagen emulsion was used to create a rheumatoid arthritis model in SD rats. 50 rats that had been successfully modeled were randomly divided into five groups: a model group (saline, 0.5 mL / kg, ig), a positive control group (celecoxib, 5 mg / kg, ig), and a low-dose group (18.75 mg / kg, ig), a medium-dose group (37.5 mg / kg, ig), and a high-dose group (75 mg / kg, ig). Administration began on the first day that the rat models were successfully created, and was administered once a day thereafter. Then, the thickness of the left and right hind paws and the left and right forepaws of the rats were measured every day using a vernier caliper, and the volume of the left and right hind paws of the rats was measured using a rat paw swelling meter (measured once every 12 hours for the previous three days). The paw swelling rate of each rat was calculated based on the change in the volume of the rat's hind paws at each time point compared to before model creation, and the difference in the paw swelling rate between rats in each group was analyzed. After the model was successful, blood was taken from the orbit on the 7th day, and on the 20th day, the rats were killed, blood was collected, and serum was separated. Tissues such as ankle joints, toe joints, spleen, and liver were collected. The tissues were decalcified and stained with hematoxylin-iosin (HE), and the joint lesions, synovial hyperplasia, and connective tissue hyperplasia were observed under a microscope. Experimental results: Compared with the model group, on the 21st day, the paw swelling degree was all significantly reduced in the SMI0 75mg / kg, 37.5mg / kg, and 18.75mg / kg groups, among which, the SMI0 (75mg / kg) group showed the best effect of improving rat paw swelling, which was similar to the swelling change in the case of 5mg / kg celecoxib (see Figure 57). In addition, the correlation analysis between the SMI0 dose and the paw swelling relief rate showed that the anti-rheumatoid arthritis effect of SMI0 had a significant dose-effect relationship, and from the 2nd to the 8th day, the SMI0 dose and the paw swelling relief rate showed a moderate correlation (P<0.01), and from the 8th to the 20th day, the SMI0 dose and the paw swelling relief rate all showed a strong correlation (P<0.001) (see Figure 58). These results suggest that SMI0 can significantly alleviate paw swelling induced by collagen in a rat rheumatoid arthritis model, and that this effect is dose-dependent. (3) Inhibition of SMI0 in rheumatoid arthritis rats by complete Freund's adjuvant Experimental method: A rheumatoid arthritis model was created in SD rats using complete Freund's adjuvant (CFA, 0.1mL / rat). The SMI0 treatment groups were divided into ultra-low dose group, low dose group, medium dose group and high dose group, and the SMI0 compound was administered intragastrically at 1mg / kg, 6mg / kg, 30mg / kg and 150mg / kg every day from 2 days before the model was created. The positive control group was administered intragastrically at 5mg / kg celecoxib, with 10 animals in each group. The rats were administered the above grouping and dose one day before CFA administration, and then once a day until the model was completed on the 10th day. Each rat was administered CFA into the plantar region of the right hind paw, and the volume of the right hind paw of each rat was measured every day using a rat paw swelling meter (measured every 12 h for the previous 3 days). The paw swelling rate of each rat was calculated based on the change in the volume of the right hind paw of each rat at each time point compared to before CFA administration, and the difference in paw swelling rate between rats in each group was analyzed. Experimental results: Compared with the control group, rats in the model group had obvious swelling of the paws (see FIG. 59). Compared with the model group, the SMI0-treated group significantly alleviated the swelling of rat paws caused by complete Freund's adjuvant (shown in FIG. 59 and FIG. 60-FIG. 60, where FIG. 60B is a graph of the swelling rate of rat paws at 24 hours, 60C is a graph of the swelling rate of rat paws at 36 hours, and 60D is a graph of the swelling rate of rat paws at 48 hours) ( *** P<0.001 vs. model group). At doses of 1 mg / kg, 6 mg / kg, 30 mg / kg, and 150 mg / kg, the alleviation rates of rat paw swelling at 36 h (when the degree of paw swelling in the model group reached its maximum) of the SMI0 compound were 20.8%, 34.2%, 44.1%, and 52.5%, respectively. The dose of SMI0 and the rat paw alleviation rate showed a good dose-effect relationship (P<0.05) (shown in Figures 61A to 61D, where Figure 61B is the dose-effect relationship at 24 h, Figure 61C is the dose-effect relationship at 36 h, and Figure 61D is the dose-effect relationship at 48 h). This suggests that SMI0 can significantly alleviate paw swelling in a rat rheumatoid arthritis model induced by complete Freund's adjuvant. (4) Correlation between CYP2E1 activity after SMI0 inhibition and severity of rheumatoid arthritis in rats Experimental method: Liver microsomes from collagen-induced rheumatoid arthritis model rats were prepared using calcium precipitation method, and microsomal protein concentration was measured using Braford method. An incubation system was prepared using 2xPBS buffer, chlorzoxazone solution, and liver microsomes with a final concentration of 0.5mg / mL, and pre-incubated at 37℃ for 5min. The reaction was started by adding NADPH, incubated at 37℃ for 30min, and stopped by placing on ice. 6-hydroxychlorzoxazone was extracted with ethyl acetate, vortex mixed, centrifuged, and the upper organic phase was aspirated and dried with nitrogen gas. The peak area of ​​6-hydroxychlorzoxazone, a chlorzoxazone metabolite, was detected using high performance liquid chromatography at methanol:water=56:44 and detection wavelength of 287nm. The concentration ΔC of the metabolite 6-hydroxychlorzoxazone was calculated by substituting it into the standard curve, and the conversion reaction rate of chlorzoxazone to 6-hydroxychlorzoxazone was calculated by V (pmol / min / mg) = (ΔC * 1000) / (B * T), where B is the microsomal protein concentration (mg / mL) and T is the incubation time (min), to evaluate the level of CYP2E1 enzyme activity. The correlation between rat CYP2E1 activity and the severity of rat rheumatoid arthritis was analyzed. Experimental results: Compared with the control group, CYP2E1 activity was significantly increased in the model group rats (P<0.05, Figure 62A), and CYP2E1 metabolic activity was significantly correlated with arthritis score, paw swelling thickness and volume (P<0.05, Figure 62B). This suggests that the high CYP2E1 enzyme activity is positively correlated with the severity of rheumatoid arthritis in rats, and the anti-rheumatoid arthritis effect of SMI0 may be related to the inhibition of CYP2E1 enzyme activity. These results demonstrated that SMI0 has a clear preventive and therapeutic effect against rheumatoid arthritis induced by collagen and complete Freund's adjuvant in rats, suggesting that the SMI0 compound may be effective in the clinical prevention and treatment of rheumatic fever and rheumatoid arthritis.

[0101] Example 20 SMI0 inhibition of lipopolysaccharide-induced pyemia in rats and mice Testing Method: Rats: A SD rat pyometra model was created by a single intraperitoneal injection of lipopolysaccharide (LPS, 5 mg / kg). 30 min before LPS administration, rats in the model group were administered saline (0.5 mL / kg, ig), rats in the positive control group were administered celecoxib (5 mg / kg, ig), and rats in the CYP2E1 inhibition group were administered SMI0 compound (150 mg / kg, ig). After LPS administration, rat body temperature was measured and recorded every hour for up to 9 h (control group, n = 8 rats; model group, n = 10 mice; celecoxib group, n = 10 rats; SMI0 compound group, n = 10 rats). Mice: A mouse pyometra model was established by a single intraperitoneal injection of lipopolysaccharide (LPS, 15 mg / kg). 30 min before LPS administration, mice in the model group were administered saline (0.5 mL / kg, ig), mice in the positive control group were administered celecoxib (5 mg / kg, ig), and mice in the CYP2E1 inhibition group were administered SMI0 compound (90 mg / kg, ig). The body temperature of the mice was measured and recorded at 6 h, 12 h, and 24 h after LPS administration, and was measured a total of 10 times during the entire experimental process. At 24 h, the animals were killed, blood samples were taken, and related indexes of renal and cardiac function were measured (control group, n = 8 mice; model group, n = 8 mice; celecoxib group, n = 8 mice; SMI0 compound group, n = 10 mice). Test Results: Rats: Compared with the control group, the body temperature of the rats in the model group was significantly increased at 4 h, 5 h, and 6 h. Compared with the model group, the SMI0 compound administration group and the positive group were able to avoid the increase in body temperature in the rat sepsis model induced by LPS (as shown in Figures 63A to 63D). ** P < 0.01, *** P<0.001 vs. control group; # P < 0.05, ## P < 0.01, ### P<0.001 vs. model group). This suggests that SMI0 treatment significantly inhibited the LPS-induced rise in body temperature in rats and could maintain the normal body temperature in rats. Mice: Compared with the control group, the body temperature of the mice in the model group was significantly decreased at 6 h, 12 h, and 24 h, and compared with the model group, the mice in the SMI0 compound administration group were able to avoid the decrease in body temperature in the mouse sepsis model induced by LPS. Compared with the control group, the body temperature of the mice in the model group was significantly decreased at 6 h, 12 h, and 24 h compared to before LPS administration (as shown in Figures 64A to 64D). ** P < 0.01, *** P<0.001 vs. control group; & P < 0.05, &&& P<0.001 vs. model group; ## P < 0.01, ### P<0.001 vs. celecoxib group). In addition, compared with the control group, the levels of urea nitrogen and creatinine, which are indicators of renal function, and creatine kinase and lactate dehydrogenase, which are indicators of cardiac function, were significantly increased in the model group mice, and the levels of indicators related to renal function and cardiac function were significantly decreased in the SMI0 compound-administered group mice compared with the model group (as shown in Figures 65A to 65D). ** P < 0.01, *** P<0.001 vs. control group; &&& P<0.001 vs. model group; # P < 0.01, ## P<0.001 vs. celecoxib group). These results suggest that SMI0 treatment significantly inhibited LPS-induced hypothermia in mice, maintained normal body temperature in mice, and improved renal and cardiac dysfunction in an LPS-induced mouse sepsis model. These results suggest that SMI0 can prevent the body temperature change induced by lipopolysaccharide (LPS) in rat and mouse pyemia models, and that SMI0 compound may be useful for the clinical prevention and treatment of pyemia.

[0102] Example 21: Improvement of SMI0 in cognitive dysfunction caused by Alzheimer's disease in rats by streptozotocin (STZ) Experimental method: Male SD rats were injected with streptozotocin (STZ) into both ventricles to create an Alzheimer's disease (AD) model, with STZ (3 mg / kg) injected on day 1 and STZ (1.5 mg / kg) injected on day 3. STZ was injected into both ventricles (0.9 mm posterior to bregma, 1.5 mm left and right of the sagittal suture, 3.8 mm from the skull below the brain surface) using a stereotaxic apparatus. The sham-operated group was injected with the same volume of control solvent using the same procedure. The low-dose and high-dose preventive treatment groups were forced to intragastrically administer 10 mg / kg and 30 mg / kg of the SMI0 compound, respectively, from the 11th day after model creation, for 21 consecutive days. On the 14th day of continuous administration (i.e., the 25th day after model creation), they underwent spatial orientation navigation training using the Morris water maze for 6 consecutive days, and 24 hours after the end of the spatial orientation navigation training, a spatial exploration experiment (15 rats per group) was conducted. Experimental results: On days 1 to 6 of the Morris water maze stereotaxic navigation experiment, the latency period was significantly extended in the model group rats compared to the sham operation group, and on days 2 to 6, the latency period was significantly shortened in the SMI0 high dose group rats compared to the model group (Table 3 is a table quantifying the relevant parameters of the SMI0 compound that improves cognitive dysfunction in a streptozotocin-induced rat Alzheimer's disease model. As shown in Table 3 and Figure 66A, * P < 0.05, ** P < 0.01, *** P<0.001 vs sham surgery group, # P<0.05 vs. model group). In the spatial exploration experiment on the 7th day, the time spent in the quadrant where the original platform was located and the number of times crossing the platform were significantly shorter in the model group compared with the sham-operated group ( ** P < 0.01, *** P<0.001 vs. sham-operated group). Compared with the model group, the time spent in the quadrant where the original platform was located and the number of times crossing the platform were significantly increased in the SMI0 high-dose group (30 mg / kg) rats (as shown in Table 3 and Figures 66B to 66C). # P<0.05 vs. model group). Compared with the low dose SMI0 group, the number of platform crossings and the time spent in the original platform quadrant were significantly increased in the high dose SMI0 group (& P<0.05 vs low-dose group). In terms of acquiring overall learning and memory function, compared with the model group, the SMI0 high-dose group could significantly improve the cognitive impairment induced by ICV-STZ, with the improvement rate reaching 46.7%, while the SMI0 low-dose group showed a tendency to improvement, with no significant statistical difference, with the improvement rate being 32.6%. This suggests that SMI0 can significantly improve the cognitive impairment induced by streptozocin injection into the lateral ventricle. [Table 3] These results demonstrate that SMI0 at 30mg / kg significantly improves cognitive impairment in a rat model of Alzheimer's disease induced by streptozocin. SMI0 compounds may be useful for the clinical prevention and treatment of Alzheimer's disease.

[0103] Example 22 Inhibition of SMI0 against focal cerebral ischemia-reperfusion injury in rats Experimental method: A rat middle cerebral artery occlusion-reperfusion (MCAO) model was prepared using the modified Zea-longa plugging technique. Rats were anesthetized intraperitoneally with 10% chloral hydrate and fixed on a thermostatic rat board in a supine position. The body temperature of the rat was maintained between 36.5℃ and 37.5℃. After disinfection with iodine bolts, the skin was incised in the middle of the neck, the left common carotid artery was exposed, the common carotid artery, external carotid artery, and internal carotid artery were separated, and the pterygopalatine artery was ligated. An incision was made on the external carotid artery side, and a nylon thread plug was inserted through the incision. Using the branching opening as a landmark, the thread plug was carefully and slowly pushed in when inserted about 17 mm. When resistance was felt at about 18±0.5 mm, indicating that the tip of the nylon thread had reached the anterior cerebral artery, the incision of the external carotid artery was closed and the skin was sutured. After 2 h of ischemia, rats were anesthetized with inhaled sevoflurane, and the nylon thread was slowly withdrawn, and the bulbous end of the nylon thread was retracted to the bifurcation of the common carotid artery, thus achieving reperfusion. Ischemia was 2 h, and reperfusion was 24 h. SMI0 compound (150 mg / kg) was administered intragastrically forcibly 10 min before reperfusion in the SMI0 treatment group, and an equal volume of control solvent was injected in the sham operation group and model group. Neurological behavior was scored 2.5 h and 6 h after surgery, and after 24 h of reperfusion, the rats' heads were cut off and the brains were removed and stained with triphenyltetrazolium chloride (TTC) to observe the changes in cerebral infarction volume and cerebral edema (10 rats per group). Experimental results: Compared with the model group, the cerebral infarction volume and cerebral edema volume were significantly reduced in the SMI0 treatment group (as shown in Figure 67, Figure 68A to Figure 68B). * P<0.05 vs. model group), cerebral infarction volume was reduced from 51.03% in the model group to 32.62%, and cerebral edema volume was reduced from 19.25% in the model group to 12.63%. The inhibition rates of cerebral infarction and cerebral edema volume by administration of SMI0 (150 mg / kg) were 36.06% and 34.39%, respectively. These results suggest that SMI0 significantly reduces cerebral infarction volume and cerebral edema volume in rats with medium artery occlusion focal cerebral ischemia-reperfusion injury. These results suggest that SMI0 can significantly improve cerebral infarction and cerebral edema in a rat middle cerebral artery occlusion-reperfusion model, and that the SMI0 compound may be useful for the clinical prevention and treatment of ischemic stroke.

[0104] Example 23 SMI0 reduces blood lipid levels in ApoE- / - mice with high-fat diet-induced hyperlipidemia Experimental method: ApoE- / - mice were used as the study subjects, and ApoE- / - mouse hyperlipidemia models were created by feeding them with a high-lipid, high-cholesterol diet. The SMI0 treatment group was divided into a low-dose treatment group and a high-dose treatment group, and 30 mg / kg and 150 mg / kg of SMI0 compound were administered daily during the model creation period, respectively, and the models were completed in the 16th week. At the end of the experiment, blood was collected from the orbit and the levels of correlation indicators of blood lipids in the serum of each mouse (8 mice per group) were measured using a fully automated biochemical analyzer. Experimental results: Compared with the control group, the total cholesterol and low-density lipoprotein levels of the mice in the model group were all significantly increased (Table 4, as shown in Figure 69). * P<0.05 vs. control group). Compared with the model group, total cholesterol and low-density lipoprotein were significantly decreased, and high-density lipoprotein was significantly increased in the high-dose SMI0 treatment group ( @ P<0.05 vs. model group). Compared with the model group, low-dose SMI0 treatment showed a certain improving effect on high blood lipid levels induced by high-fat diet, but the results were not statistically significant. In addition, administration of SMI0 can significantly inhibit the formation of aortic atherosclerotic plaques in ApoE- / - mice. For example, the results of HE staining showed that, compared with the model group, the high-dose SMI0 group (150 mg / kg) significantly reduced the total area of ​​aortic root lesions in mice (as shown in Figure 70). ** P<0.01 vs. model group), and the results of Oil Red O staining also showed that the high dose group of SMI0 (150 mg / kg) significantly reduced the total area of ​​aortic root lesions (as shown in FIG. 70). * P<0.05 vs. model group). These results suggest that administration of 150mg / kg SMI0 significantly improved the hyperlipidemia level (reducing total cholesterol and low-density lipoprotein and increasing high-density lipoprotein) induced by high-fat diet in mice, and inhibited the formation of aortic atherosclerotic plaque in ApoE- / - mice. [Table 4] These results demonstrate that SMI0 can significantly reduce the increase in blood lipid levels and the formation of atherosclerotic plaques in mice fed a high-fat diet. SMI0 compounds may be useful in the clinical prevention and treatment of hyperlipidemia, atherosclerosis, and coronary artery sclerosis.

[0105] Example 24 SMI0 reduces blood glucose levels in diabetic rats induced by high-fat diet and streptozotocin Experimental method: A rat diabetic model was created by intraperitoneally injecting streptozotocin (STZ, 40mg / kg) into a high-fat diet. The fasting blood glucose (FBG) level was ≥ 16.7mmol / L after 2 weeks. The model rats were divided equally into groups according to blood glucose levels. Among them, the rats in the model group were given saline, and the low-dose and high-dose SMI0 treatment groups were given 30mg / kg and 150mg / kg of SMI0 compound daily, respectively. The rat FBG was measured at scheduled intervals every week and recorded in 10 rats in each group until 6 weeks after administration. Experimental results: Compared to the model group, the fasting blood glucose levels of rats in the low-dose and high-dose SMI0 treatment groups were significantly lower in both the fifth and sixth weeks ( * P<0.05 vs. model group) (see FIG. 71A). The glucose tolerance test results showed that the low-dose and high-dose SMI0 treatment groups significantly improved the glucose tolerance of rats (see FIG. 71B-FIG. 71C). It was suggested that SMI0 treatment significantly improved the hyperglycemia level of rats induced by high-fat diet and streptozotocin. These results suggest that SMI0 can significantly lower blood glucose levels in diabetic rats induced by high-fat diet and streptozotocin, and that the SMI0 compound may be useful for the prevention and treatment of clinical diabetes.

[0106] Example 25 Increased expression of CYP2E1 in peritumoral cancer tissue of bladder cancer Experimental methods: Thirty healthy bladder tissues were used as controls, and changes in CYP2E1 expression in the peritumor tissues of 30 clinical bladder cancer patients were studied, and the changes in CYP2E1 content in the peritumor tissues of bladder cancer patients were compared. Experimental results: Immunohistochemistry showed that the expression of CYP2E1 in bladder cancer peritumor tissues was significantly higher than that in normal bladder tissues (P<0.05), as shown in Figure 72. Due to the significant increase in CYP2E1 expression in lung cancer, liver cancer and glioma, and the action of CYP2E1 in inflammation-related tumors, it was also suggested that SMI0 compound could significantly inhibit the development and progression of lung cancer, liver cancer and glioma, and the increased expression of CYP2E1 in bladder cancer peritumor tissues was related to the development of bladder cancer. SMI0 compound has preventive and therapeutic effects on the development and progression of bladder cancer, and SMI0 compound can be applied in the prevention and treatment of clinical bladder cancer.

[0107] Example 26 Increased expression of CYP2E1 in peritumoral tissue of gallbladder cancer Experimental Methods: We investigated the expression of CYP2E1 in gallbladder tissues from 31 healthy subjects and in the peritumor tissues of 33 clinical patients with gallbladder cancer. Experimental results: Immunohistochemistry showed that the expression of CYP2E1 in peritumoral tissues of gallbladder cancer was significantly higher than that in normal gallbladder tissues (P<0.05), as shown in Figure 73. Due to the significant increase in the expression of CYP2E1 in lung cancer, hepatic cancer and glioma, and the action of CYP2E1 in inflammation-related tumors, it was also suggested that SMI0 compound could significantly inhibit the occurrence and progression of lung cancer, hepatic cancer and glioma, and the increase in the expression of CYP2E1 in peritumoral tissues of gallbladder cancer is related to the occurrence of gallbladder cancer. SMI0 compound has preventive and therapeutic effects on the occurrence and progression of gallbladder cancer, and SMI0 compound can be applied in the prevention and treatment of clinical gallbladder cancer.

[0108] The above are merely some examples of the present specification, and are not intended to limit the present application. Although the present application is disclosed above as a better embodiment, it is not intended to limit the present application. If a technical person familiar with this field makes some changes or modifications using the technical contents disclosed above within the scope of the technical solution of the present application, these are equivalent to equivalent embodiments, and all of them belong to the scope of the technical solution.

Claims

1. The compound represented by formula (I) or a salt thereof is contained as an inhibitor, and the compound or salt thereof binds to CYP2E1 as a target and exerts an inhibitory effect on CYP2E1; 【Chemical 29】 (In the formula, R １ is hydrogen, C １ ~C １０ is any one selected from the alkyl of R ２ is any one selected from hydrogen, a substituent represented by formula O-2, and a substituent represented by formula O-3, 【Chemistry 31】 【Chemistry 32】 R ２５ is an alkoxy; R ３ is C １ ~C １０ Alkyl, substituted C １ ~C １０ At least one selected from the alkyl groups II The substituted C １ ~C １０ The substituent in the alkyl group II is a substituent represented by the formula M-3. 【Chemistry 35】 The CYP2E1 inhibitor is characterized in that the inhibitor contains at least one of the compounds represented by the following formula (38). 【Chemical Formula 38】

2. A method for producing a CYP2E1 inhibitor by reacting a compound represented by the following formula (Chemical Formula 39) with an acid and using an acid salt of the compound represented by the formula (Chemical Formula 39), The acid is at least one selected from hydrochloric acid and sulfuric acid, 【Chemical Formula 39】 The powder X-ray diffraction pattern of crystalline form A of the hydrochloride salt of the compound represented by [Chemical Formula 39] contains three or more 2θ values ​​selected from the group consisting of 8.4±0.2°, 13.1±0.2°, 14.8±0.2°, 16.6±0.2°, 24.1±0.2°, 27.2±0.2°, 30.5±0.2°, 31.8±0.2°, 33.5±0.2°, 35.4±0.2°, and 35.7±0.2°; As shown in the DSC-TGA diagram of crystalline form A of the hydrochloride of the compound represented by [Chemical formula 39], there is a clear endothermic peak between 70°C and 220°C, and thermal decomposition occurs between 80°C and 170°C. The powder X-ray diffraction of crystalline form B of the sulfate of the compound represented by [Chemical formula 39] includes five or more 2θ values ​​selected from the group consisting of 10.1±0.2°, 15.1±0.2°, 16.0±0.2°, 16.7±0.2°, 19.2±0.2°, 19.9±0.2°, 23.4±0.2°, 24.0±0.2°, 25.8±0.2°, 26.5±0.2°, 28.9±0.2°, 30.3±0.2°, and 32.2±0.2°. The method for producing a CYP2E1 inhibitor according to claim 1, characterized in that, as shown in the DSC-TGA diagram of crystalline form B of the sulfate salt of the compound represented by [Chemical Formula 39], there is at least one endothermic peak between 30°C to 85°C, 90°C to 160°C, and 215°C to 330°C, and thermal decomposition occurs at 150°C to 350°C.

3. The method for producing the compound represented by [Chemical Formula 39] as an inhibitor according to claim 2, characterized in that it comprises the following method: Method 1: Compound A, an aprotic solvent, and a raw material containing a Grignard reagent are reacted at −20 to 25° C. for 0.5 to 3 hours to obtain CYP2E1 inhibitor A; The compound A is a compound having a structural formula represented by Formula II, 【Chemistry 41】 The CYP2E1 inhibitor A is a compound having a structural formula represented by Formula III: 【Chemistry 42】 (In Formula II and Formula III, R 1 is any one selected from hydrogen and C 1 -C 10 alkyl; R 3 is at least one selected from C 1 -C 10 alkyl and substituted C 1 -C 10 alkyl II; The substituent in the substituted C 1 -C 10 alkyl II is a substituent represented by the formula M-3.

4. In the method 1, the aprotic solvent is at least one selected from tetrahydrofuran and ethyl ether, and the Grignard reagent is at least one selected from methylmagnesium bromide and methylmagnesium chloride; The method for producing a CYP2E1 inhibitor according to claim 3, characterized in that in the method 1, the molar ratio of compound A to the Grignard reagent is 1:1 to 1:

3.

5. The compound A is The compound A-1 is obtained by reacting raw materials including a compound A-1, a condensing agent, N,O-dimethylhydroxylamine hydrochloride, and an aprotic solvent in the presence of an alkali source III at 20 to 60° C. for 10 to 20 hours, The compound A-1 is a compound having a structural formula represented by formula II-1, 【Chemistry 46】 the condensing agent is at least one selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dilauryl carbonate, N,N-carbonyldiimidazole, dicyclohexylcarbodiimide, and N-(4-carboxyphenyl)maleimide, the alkali source III is at least one selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, pyridine, triethylamine, and N,N-diisopropylethylamine, the molar ratio of compound A-1, the condensing agent, and N,O-dimethylhydroxylamine hydrochloride is 1:1:1 to 1:5:5; To obtain the compound A-1, The method includes at least a step of hydrolyzing a raw material containing compound A-2 in the presence of an alkali source IV to obtain a mixture, and then adjusting the pH of the mixture to 2-3 with an acid source to obtain compound A-1; The compound A-2 is a compound having a structural formula represented by formula II-2, 【Chemistry 47】 the alkali source IV is at least one selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and potassium carbonate; the acid source is selected from concentrated hydrochloric acid; adjusting the pH of the mixture to 2-3 with the acid source is carried out at 10-50° C.; The method for producing an acid salt of the compound represented by formula (I) comprises the steps of: The method includes at least a step of reacting a material containing a compound represented by formula (I) and a solvent A at −20 to 80° C. for 0.5 to 10 hours to obtain an acid salt of the compound represented by formula (I), The method for producing a CYP2E1 inhibitor according to claim 3, characterized in that the solvent A is at least one selected from the group consisting of ether compounds, alcohol compounds, ester compounds, nitrile compounds, ketone compounds, halogenated alkanes, alkanes, and aromatic hydrocarbons.

6. The CYP2E1 inhibitor for treating or preventing an inflammation-associated tumor or an inflammation-associated disease, The inflammation-associated tumor includes at least one of liver cancer, glioma, ovarian cancer, lung cancer, bladder cancer, and gallbladder cancer; The CYP2E1 inhibitor of claim 1, characterized in that the inflammation-related disease includes at least one of liver injury, fatty liver, hepatitis, liver fibrosis, pulmonary fibrosis, rheumatic fever and rheumatoid arthritis, pyemia, Alzheimer's disease, ischemic stroke, Parkinson's disease, hyperlipidemia, atherosclerosis, coronary artery disease, and diabetes.

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