Application of compound as CYP2E1 inhibitor

Abstract

The invention discloses an application of a compound as a CYP2E1 inhibitor, which is characterized in that the compound or a salt thereof as an inhibitor takes CYP2E1 as a target spot and is combined with the CYP2E1, and the CYP2E1 inhibitor has an inhibition effect on CYP2E1. The inhibitor can be used for preventing and treating tumors such as liver cancer, glioma, ovarian cancer, bladder cancer and gallbladder cancer. Similarly, the inhibitor can also be used for preventing and treating other inflammation-related diseases such as liver injury, fatty liver, hepatitis, hepatic fibrosis, rheumatic and rheumatoid arthritis, sepsis, Alzheimer's disease, ischemic cerebral apoplexy, Parkinson's disease, hyperlipidemia, atherosclerosis, coronary heart disease, diabetes mellitus and the like.

Description

[0001] This invention is a divisional application of application number 202010906508.3 filed on September 1, 2020, entitled "Application of a compound as a CYP2E1 inhibitor". Technical Field

[0002] This invention relates to the field of targets and drugs for the prevention and treatment of inflammatory diseases, specifically to the application of a compound as a CYP2E1 inhibitor. Background Technology

[0003] It is now widely accepted that many diseases are related to inflammation, such as tumors (liver cancer, cervical cancer, nasopharyngeal carcinoma, colorectal cancer, glioma, etc.) and non-tumor diseases (Alzheimer's disease, Parkinson's syndrome, stroke, arteriosclerosis, diabetes, etc.). These inflammation-related diseases can be collectively referred to as inflammation-mediated diseases (IMD). It is generally believed that a long-term, uncontrollable inflammatory microenvironment is associated with the occurrence of IMD.

[0004] The tumor microenvironment (TME) plays a crucial role in tumorigenesis and development, and research on the TME has progressed rapidly over the past decade. In 2010, Professor Karin M of the University of California wrote in *Cell* that immunity and inflammation within the TME are closely related to tumorigenesis and development. Recent breakthroughs have been made in immunological research on the TME, with immunotherapies targeting the TME, such as PD-1 and PD-L1, successfully used clinically as broad-spectrum anti-tumor drugs. James P. Allison, an American immunologist, and Tasuku Honjo, a Japanese immunologist, were awarded the 2018 Nobel Prize in Physiology or Medicine for their outstanding original contributions to this field.

[0005] Chronic uncontrolled inflammation is a key characteristic of the tumor microenvironment. Taking liver cancer as an example, which typically originates from hepatitis and cirrhosis, liver cancer cells, in addition to other components of the tumor microenvironment, include hepatic stellate cells, which can lead to collagen deposition upon activation. Persistent liver damage caused by factors such as alcohol abuse, non-alcoholic steatohepatitis (NASH), and chronic hepatitis B virus (HBV) infection can result in hypoxia and the formation of chronic uncontrolled inflammation, which are also important characteristics of the liver cancer tumor microenvironment.

[0006] Tumor development and progression are closely related to the tumor microenvironment. Targeting the tumor microenvironment with drugs has become an effective treatment for liver cancer, such as immune checkpoint inhibitors PD-1 and PDL-1, and angiogenesis inhibitors like bevacizumab. These drugs generally have broad-spectrum anti-tumor effects. For example, the anti-angiogenic drug bevacizumab can be used to treat colorectal cancer, non-small cell lung cancer, renal cell carcinoma, ovarian cancer, cervical cancer, and liver cancer. Immune checkpoint inhibitors PD-1 and PDL-1 can be used to treat melanoma, lung cancer, breast cancer, liver cancer, pancreatic cancer, gastrointestinal tumors, gynecological tumors, urinary system tumors, myeloma, and lymphoma.

[0007] The success of drugs targeting the immune and angiogenesis mechanisms of the tumor microenvironment has provided valuable lessons for research in oncology and other fields. While traditional anti-inflammatory drugs, such as COX-2 inhibitors and other nonsteroidal anti-inflammatory drugs (NSAIDs), have played a significant role in antipyretics, analgesia, and anti-inflammatory and antirheumatic effects, they have not shown satisfactory efficacy against inflammation-related tumors. Currently, there are no anti-tumor drugs that target the inflammatory microenvironment of tumors. This may be due to the existence of undiscovered novel inflammatory targets within the chronic, uncontrolled inflammatory microenvironment.

[0008] Cytochrome P450 2E1 (CYP2E1) is a protein mainly found in the endoplasmic reticulum of hepatocytes. The applicant found that it constitutes the highest proportion of hepatocyte cytochrome P450 (CYP450), approximately 24.8%. The main function of CYP2E1 is metabolism, participating in the biotransformation of drugs, procarcinogens, and environmental toxins. For example, it can metabolize and activate more than 85 exogenous substances to generate hepatotoxic or carcinogenic substances, including carcinogens such as nitrosamines, benzene, and 1,3-butadiene. CYP2E1 is associated with nitrosamines, toluene, chloroform, acetone, and the tobacco-specific carcinogen NNK.

[0009] CYP2E1 metabolic activation is closely related to inflammatory diseases such as tumors. Taking liver cancer as an example, its occurrence is related to multiple factors, including hepatitis viruses, nitrosamines, aflatoxin, and alcohol. Nitrosamines (N-nitrosamines) are potent carcinogens that require CYP2E1 metabolic activation in the body to generate carcinogenic substances, which then form adducts with DNA, leading to liver cancer. Traditional Chinese diets often contain excessive levels of nitrosamines, such as processed meat products, cured meats, ham, and pickled vegetables. Epidemiological studies have shown a close correlation between the content of nitrosamines in food and the occurrence of liver cancer. Animal experiments have found that CYP2E1 gene knockout mice can significantly inhibit diethylnitrosamine-induced liver cancer in mice. This suggests that CYP2E1 may affect the occurrence of liver cancer by influencing the metabolic activation of nitrosamines in the body.

[0010] The inflammatory effects of CYP2E1 are closely related to inflammatory diseases such as tumors. CYP2E1 has significant inflammatory effects and participates in the development and progression of numerous inflammatory diseases. CYP2E1 is associated with inflammatory tumors such as liver cancer, glioma, nasopharyngeal carcinoma, bladder cancer, and gallbladder cancer. Simultaneously, CYP2E1 is also associated with liver injury, non-alcoholic steatohepatitis (NASH), liver fibrosis, and other liver diseases, as well as the development and progression of other inflammatory diseases such as rheumatoid arthritis, sepsis, Alzheimer's disease, hyperlipidemia, diabetes, and ischemic stroke. CYP2E1 can enhance the release of TNF-α from Kupffer cells, leading to inflammatory necrosis of hepatocytes. In a mouse model of non-alcoholic steatohepatitis (NASH), inhibiting CYP2E1 activity can prevent and treat NASH by reducing TNF-α expression and restoring endothelial nitric oxide synthase (eNOS) activity. Cyp2E1 knockout mice can significantly inhibit the inflammatory response induced by chronic alcohol exposure. The CYP2E1 inhibitor allyl sulfide can prevent and treat NASH by inhibiting CYP2E1 and reducing the release of IL-1β and IL-12.

[0011] The pro-inflammatory effects of CYP2E1 are related to the promotion of oxidative stress and lipid peroxidation. CYP2E1 promotes the production of reactive oxygen species (ROS), inducing oxidative stress and lipid peroxidation, leading to hepatocyte inflammation, apoptosis, and liver fibrosis. High expression of CYP2E1 in hepatocytes promotes ROS production. ROS can activate Fas ligands, cell surface molecules of the tumor necrosis factor family, generating protease-linked reactions that cause cell lysis and apoptosis. Apoptotic hepatocytes can promote the aggregation of inflammatory cells, inducing the production of various inflammatory factors such as TNF-α and IL-6, causing liver inflammation and fatty liver disease. CYP2E1 can also affect arachidonic acid (AA) metabolism, promoting the invasion and metastasis of liver cancer cells. Studies have found that CYP2E1 mainly increases AA toxicity through reactive oxygen species and lipid peroxidation products. CYP2E1 and AA, through ROS peroxidation, cause intracellular calcium... 2+ The release of phospholipase A2 (PLA2) activates phospholipase A2, promoting the production of amino acids (AA). In liver cancer cells, AA is converted into prostaglandin 2 (PGE2) by cyclooxygenase 2 (COX-2), which binds to the EP receptor coupled to the G protein on the cell membrane, activating the EGFR / Met signaling pathway and causing liver cancer cell invasion and metastasis.

[0012] In recent years, the applicant has established a large liver specimen bank including over 127 normal human livers and 102 cirrhotic livers from patients with hepatocellular carcinoma, and has conducted a relatively systematic study on the physiology and pathology of CYP450. The study found that there was an individual variation of more than 10-fold in CYP2E1 levels, with hepatocellular carcinoma patients showing a significantly increased CYP2E1 metabolic activity of approximately 2.13 times, a positive rate of approximately 44.6%. Furthermore, CYP2E1 activity was significantly negatively correlated with postoperative survival, with survival times of 238 days for CYP2E1-positive patients and 612 days for CYP2E1-negative patients. Increased CYP2E1 activity is an independent risk factor for the development and progression of hepatocellular carcinoma. Using a rat model of primary hepatocellular carcinoma, the study demonstrated a strong causal relationship between innate CYP2E1 activity (before modeling) and hepatocellular carcinoma development; that is, the higher the innate CYP2E1 activity, the more likely hepatocellular carcinoma will occur. This suggests that CYP2E1 may be a novel target for the prevention and treatment of hepatocellular carcinoma, and it is speculated that drugs targeting the novel CYP2E1 target in the tumor inflammatory microenvironment may have broad-spectrum preventive and therapeutic effects on inflammation-related diseases.

[0013] In summary, CYP2E1 participates in the occurrence and development of numerous inflammation-related diseases through metabolic activation and pro-inflammatory effects. Therefore, inhibiting CYP2E1 activity is of great significance for the prevention and treatment of these diseases. Thus, research on CYP2E1 inhibitors has significant theoretical and practical implications.

[0014] Currently, there are no specific CYP2E1 inhibitors for clinical use. Compounds or drugs reported to have CYP2E1 inhibitory activity, including 4-methylpyrazole, disulfiram, diethyldithiocarbamate, isothiocyanate, o-toluene-hyphenate, and chlorometrazazole, mostly exhibit poor selectivity for CYP2E1 inhibition and have high toxicity, thus primarily used in basic research. Therefore, given the role of CYP2E1 in the development and progression of numerous diseases such as liver diseases, there is an urgent need to screen and synthesize CYP2E1 inhibitors. Summary of the Invention

[0015] In one aspect, this invention discloses the application of a compound as a CYP2E1 inhibitor, which is selected from the compound shown in formula (I) or its salt as an inhibitor to target and bind to CYP2E1, thereby inhibiting CYP2E1.

[0016] According to a first aspect of this application, the use of a compound as a CYP2E1 inhibitor is provided.

[0017] Compounds selected from formula (I) or their salts act as inhibitors, targeting and binding to CYP2E1, thereby inhibiting CYP2E1; wherein,

[0018]

[0019] R1 is selected from hydrogen, C1-C 10 Any one of alkyl groups and epoxyalkyl groups;

[0020] R2 is selected from hydrogen, substituted C1-C 10 Any one of the alkyl group I, the substituent shown in formula O-1, the substituent shown in formula O-2, and the substituent shown in formula O-3;

[0021]

[0022] R 21 R 22 R 23 R 24 Independently selected from hydrogen, alkoxy, halogen, C1-C3 alkyl, C6-C 10 At least one of the aryl groups;

[0023] R 25 Selected from hydroxyl and alkoxy groups;

[0024] R3 is selected from hydrogen, C1-C 10 Alkyl, substituted C1-C 10 At least one of the alkyl II groups.

[0025] Preferably, the epoxy alkyl group is selected from epoxy butyl group.

[0026] Preferably, R1 is selected from hydrogen, C1-C4 alkyl groups, and epoxy alkyl groups.

[0027] Preferably, R3 is selected from at least one of hydrogen, C1-C4 alkyl, and substituted C1-C4 alkyl II.

[0028] Optionally, the compound shown in Formula I is used as a CYP2E1 inhibitor.

[0029] Optionally, the substituted C1-C 10 The substituent in the alkyl I is selected from at least one of the substituted amino I, the substituent shown in formula M-1, and the substituent shown in formula M-2;

[0030]

[0031] Optionally, the substituents in the substituted amino I are selected from C6-C6. 10 At least one of aryl and substituted C1-C3 alkyl III.

[0032] Preferably, the substituents in the substituted C1-C3 alkyl III are selected from C6-C6. 10 Aryl groups.

[0033] Optionally, the substituted C1-C 10 The substituent in the alkyl II is selected from at least one of the substituted amino II, the substituent shown in formula M-3, and the substituent shown in formula M-4;

[0034]

[0035] Optionally, the substituent in the substituted amino II is selected from substituted C1-C3 alkyl IV;

[0036] The substituents in the substituted C1-C3 alkyl IV are selected from at least one of pyridyl and halogen.

[0037] Optionally, the inhibitor is selected from compounds of formula (I) or their pharmaceutically acceptable salts, wherein,

[0038]

[0039] R1 is selected from hydrogen, C1-C 10 At least one of alkyl groups and epoxyalkyl groups;

[0040] R2 is selected from hydrogen, substituted C1-C 10 At least one of alkyl I, substituted carbonyl, and substituted imine;

[0041] R3 is selected from hydrogen, C1-C 10 Alkyl, substituted C1-C 10 At least one of the alkyl II groups.

[0042] Optionally, the substituted C1-C 10 The substituent in the alkyl I is selected from at least one of halogens, substituted amino I, substituents shown in formula M-1, and substituents shown in formula M-2;

[0043]

[0044] The substituents in the substituted carbonyl group are selected from substituted C1-C groups. 10 alkenyl groups;

[0045] The substituent in the substituted imine group is selected from at least one of hydroxyl and C1-C3 alkoxy groups.

[0046] Optionally, the substituents in the substituted amino I are selected from C1-C1. 10 At least one of aryl and substituted C1-C3 alkyl III;

[0047] Preferably, the substituents in the substituted C1-C3 alkyl III are selected from C1-C3. 10 Aryl groups.

[0048] Optionally, the substituted C1-C 10 The substituents in the alkenyl group are selected from C1-C1. 10 aryl, substituted C1-C 10 At least one of the aryl groups;

[0049] Preferably, the substituted C1-C 10 The substituents in the aryl group are selected from at least one of C1-C3 alkoxy groups, halogens, and C1-C3 alkyl groups.

[0050] Optionally, the substituted C1-C 10 The substituent in the alkyl II is selected from at least one of the substituted amino II, the substituent shown in formula M-3, and the substituent shown in formula M-4;

[0051]

[0052] Optionally, the substituent in the substituted amino II is selected from substituted C1-C3 alkyl IV;

[0053] The substituents in the substituted C1-C3 alkyl IV are selected from at least one of pyridyl and halogen.

[0054] Optionally, at least one of the following compounds can act as an inhibitor to target and bind to CYP2E1, thereby inhibiting CYP2E1.

[0055]

[0056] Optionally, at least one of the following compounds can act as an inhibitor to target and bind to CYP2E1, thereby inhibiting CYP2E1.

[0057]

[0058] Preferably, at least one of the following compounds acts as an inhibitor to target and bind to CYP2E1, thereby inhibiting CYP2E1.

[0059]

[0060] Optionally, the compound represented by formula (I) reacts with an acid to give an acidic salt of the compound represented by formula (I);

[0061] The acid is selected from at least one of inorganic acids and organic acids.

[0062] Optionally, the inorganic acid is selected from at least one of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid;

[0063] The organic acid is selected from at least one of acetic acid, oxalic acid, succinic acid, tartaric acid, succinic acid, malic acid, lactic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, resinic acid, maleic acid, fumaric acid, salicylic acid, and acetylsalicylic acid.

[0064] Optionally, the structural formula of the compound shown in formula (I) is: The X-ray powder diffraction pattern of crystal form A of the hydrochloride salt of the compound shown in formula (I) includes three or more 2θ values ​​selected from the following group: 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°, 35.7±0.2°;

[0065] The DSC-TGA plot of the hydrochloride salt of the compound shown in formula (I) shows that it has a distinct endothermic peak between 70 °C and 220 °C, and thermally decomposes between 80 °C and 170 °C.

[0066] Optionally, the structural formula of the compound shown in formula (I) is:

[0067] The X-ray powder diffraction of the sulfate form B of the compound shown in 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°.

[0068] The DSC-TGA plot of the sulfate form B of the compound shown in formula (I) shows that it has at least one endothermic peak between 30℃~85℃, 90℃~160℃, and 215℃~330℃, and thermally decomposes at 150℃~350℃.

[0069] Optionally, the method for preparing the compound shown in formula (I) as an inhibitor includes at least one of the following methods;

[0070] Method 1: React the raw materials containing compound A, an aprotic solvent and Grignard reagent at -20 to 25°C for 0.5 to 3 hours to obtain CYP2E1 inhibitor A;

[0071] Compound A is selected from at least one of compounds having the structural formula shown in Formula II:

[0072]

[0073] The CYP2E1 inhibitor A is selected from at least one compound having the structural formula shown in Formula III:

[0074]

[0075] Method 2: Reacting a compound containing the structure shown in Formula III with ammonia in the presence of base source I yields CYP2E1 inhibitor B;

[0076] The CYP2E1 inhibitor B is selected from at least one compound having the structural formula shown in Formula III-1:

[0077]

[0078] Method 3: Reacting a compound containing the structure shown in Formula III with an aromatic aldehyde compound in the presence of base source II at 25–100 °C for 2–8 h yields CYP2E1 inhibitor C.

[0079] The CYP2E1 inhibitor C is selected from at least one compound having the structural formula shown in Formula III-2:

[0080]

[0081] Method 4: Reacting a compound containing the structure shown in Formula III, an amine compound, and a reducing agent in the presence of an acid source yields CYP2E1 inhibitor D;

[0082] The amine compound is selected from at least one of aniline and benzylamine;

[0083] The CYP2E1 inhibitor D is selected from at least one compound having the structural formula shown in Formula III-3:

[0084]

[0085] Optionally, the second method includes at least the following steps: reacting a compound containing the structure shown in Formula III, ethanol, and ammonia in the presence of base source I to obtain CYP2E1 inhibitor B.

[0086] Optionally, in Method 1, the aprotic solvent is selected from at least one of tetrahydrofuran and diethyl ether; the Grignard reagent is selected from at least one of methyl magnesium bromide and methyl magnesium chloride.

[0087] In Method 2, the alkali source I is selected from at least one of potassium hydroxide, sodium hydroxide, sodium carbonate, pyridine, triethylamine, and N,N-diisopropylethylamine;

[0088] In method three, the alkali source II is selected from at least one of sodium hydroxide, potassium hydroxide, potassium tert-butoxide, sodium methoxide, and potassium fluoride;

[0089] The aromatic aldehydes are selected from at least one of p-methoxybenzaldehyde, chlorobenzaldehyde, m-methoxybenzaldehyde, p-chlorobenzaldehyde, chlorobenzaldehyde, m-chlorobenzaldehyde, p-phenylbenzaldehyde, p-isopropylbenzaldehyde, and 3,4-difluorobenzaldehyde.

[0090] In method four, the acid source is selected from at least one of formic acid, acetic acid, and hydrochloric acid;

[0091] The reducing agent is selected from at least one of sodium cyanoborohydride, sodium borohydride, and lithium aluminum hydride.

[0092] Optionally, in Method 1, the molar ratio of compound A to Grignard reagent is 1:1 to 1:3;

[0093] In the second method, the molar ratio of the compound with the structure shown in Formula III to ammonia is 1:1 to 1:6.

[0094] In Method 3, the molar ratio of the compound with the structure shown in Formula III to the aromatic aldehyde compound is 1:1 to 1:5;

[0095] In Method 4, the molar ratio of the compound with the structure shown in Formula III to the reducing agent is 1:1 to 1:5.

[0096] Optionally, compound A is obtained by the following method:

[0097] Compound A can be obtained by reacting a raw material containing compound A-1, a condensing agent, N,O-dimethylhydroxylamine hydrochloride, and an aprotic solvent at 20–60°C for 10–20 h in the presence of alkali source III.

[0098] The compound A-1 is selected from at least one of compounds having the structural formula shown in Formula II-1:

[0099]

[0100] Optionally, the condensing agent is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dilaurate carbonate, N,N-carbonyldiimidazole, dicyclohexylcarbonylimide, and N-(4-carboxyphenyl)maleimide;

[0101] The alkali source III is selected from at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, pyridine, triethylamine, and N,N-diisopropylethylamine.

[0102] Optionally, the molar ratio of compound A-1, condensing agent, and N,O-dimethylhydroxylamine hydrochloride is 1:1:1 to 1:5:5.

[0103] Optionally, obtaining compound A-1 includes at least the following steps:

[0104] The raw material containing compound A-2 is hydrolyzed in the presence of alkali source IV to obtain a mixture. Then, the pH of the mixture is adjusted to 2-3 using an acid source to obtain compound A-1.

[0105] The compound A-2 is selected from at least one of the compounds having the structural formula shown in Formula II-2:

[0106]

[0107] Optionally, the alkali source IV is selected from at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and potassium carbonate.

[0108] Optionally, the acid source is selected from concentrated hydrochloric acid;

[0109] The process of adjusting the pH of the mixture to 2-3 using an acid source is carried out at 10-50°C.

[0110] Optionally, the method for preparing the acid salt of the compound shown in formula (I) includes at least the following:

[0111] By reacting the material containing the compound shown in formula (I) and solvent A at -20 to 80°C for 0.5 to 10 hours, the acid salt of the compound shown in formula (I) can be obtained.

[0112] Preferably, the method for preparing the acid salt of the compound represented by formula (I) includes at least the following:

[0113] By reacting materials containing the compound shown in formula (I) and solvent A at -15 to 60°C for 1 to 4 hours, an acid salt of the compound shown in formula (I) can be obtained.

[0114] More preferably, the method for preparing the acid salt of the compound shown in formula (I) includes at least:

[0115] By reacting materials containing the compound shown in formula (I) and solvent A at -10 to 40°C for 1 to 4 hours, an acidic salt of the compound shown in formula (I) can be obtained.

[0116] The present invention also provides an acid salt of the CYP2E1 inhibitor SMI0, wherein the acid salt is obtained by reacting SMI0 with an organic acid or an inorganic acid.

[0117] Optionally, the organic acid is selected from acetic acid, oxalic acid, succinic acid, tartaric acid, succinic acid, malic acid, lactic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, resinic acid, maleic acid, fumaric acid, salicylic acid, or acetylsalicylic acid.

[0118] Optionally, the inorganic acid is selected from hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, or phosphoric acid.

[0119] This invention also provides a method for preparing the acid salt of the CYP2E1 inhibitor SMI0. The method includes the following steps: preparing solutions of SMI0 and acid in a soluble solvent, mixing the two systems according to a molar ratio of SMI0 to acid in the acidic solution of 1:0.5 to 3, reacting at a temperature of -20 to 80°C for 0.5 to 10 hours to generate the target product, and drying the solvent under reduced pressure to obtain the acid salt of SMI0.

[0120] Optionally, the organic solvent used in the reaction includes one or more of the following organic solvents in any proportion: ethers, alcohols, esters, nitriles, ketones, haloalkanes, alkanes, or aromatics. Preferably, the soluble solvent is methanol, acetonitrile, acetone, ethyl acetate, ethanol, or diethyl ether.

[0121] Optionally, the molar ratio of SMI0 to acid in the acidic solution in the reaction is 1:0.5 to 3, preferably 1:1 to 2.

[0122] Optionally, the reaction temperature varies with the reagent or solvent, but is typically between -20 and 80°C, preferably between -15 and 60°C, and more preferably between -10 and 40°C. The reaction time also varies with the reagent or temperature, typically between 0.5 and 10 hours, preferably between 1 and 4 hours.

[0123] This invention also provides a crystalline form A of the hydrochloride salt of the CYP2E1 inhibitor SMI0, wherein the X-ray powder diffraction of 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°. Simultaneously, the DSC-TGA plot shows a distinct endothermic peak between 70°C and 220°C, and thermal decomposition between 80°C and 170°C.

[0124] This invention also provides a crystalline form B of the sulfate of the CYP2E1 inhibitor SMI0, wherein the X-ray powder diffraction of 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°. Simultaneously, the DSC-TGA plot shows at least one endothermic peak between 30℃ and 85℃, 90℃ and 160℃, and 215℃ and 330℃, and thermal decomposition at 150℃ and 350℃.

[0125] Optionally, the CYP2E1 inhibitor is used as an active substance in drugs for the treatment of liver damage, fatty liver, hepatitis, and liver fibrosis.

[0126] Optionally, the CYP2E1 inhibitor is used in a kit for the prevention and treatment of liver diseases.

[0127] Optionally, the CYP2E1 inhibitor is used in the active ingredient of a medicament for treating or preventing inflammation-related tumors;

[0128] The inflammation-related tumors include at least one of liver cancer, glioma, ovarian cancer, bladder cancer, and gallbladder cancer.

[0129] Optionally, the CYP2E1 inhibitor is used in the active ingredient of a medicament for treating or preventing inflammation-related diseases; the inflammation-related diseases include at least one of liver injury, fatty liver, hepatitis, liver fibrosis, rheumatoid arthritis, sepsis, Alzheimer's disease, ischemic stroke, Parkinson's syndrome, hyperlipidemia, atherosclerosis, coronary heart disease, and diabetes.

[0130] Preferred CYP2E1 inhibitors in this invention are shown in the table below:

[0131]

[0132] Optionally, the CYP2E1 inhibitor SMI0 in this application SMI7 SMI16 and SMI20 The synthetic method has the following synthetic route:

[0133]

[0134] Among them, the reaction conditions are: a) hydrolysis and acidification under alkaline conditions;

[0135] b reacts with N,O-dimethylhydroxylamine hydrochloride in the presence of a base and a condensing agent;

[0136] c reacts with Grignard reagents in anhydrous aprotic solvents under low temperature conditions;

[0137] d Under alkaline conditions, upon heating, it undergoes an aldol condensation reaction with aromatic aldehydes;

[0138] e reacts with ammonia under alkaline conditions;

[0139] f reacts with amines under weakly acidic conditions and with reducing agents.

[0140] Optionally, the base mentioned in condition a can be one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, etc., and the acid can be one or a mixture of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, etc., and the solvent used is a mixture of one of water-soluble solvents such as methanol, ethanol, propanol and water, and the reaction temperature is from 10 degrees to 50 degrees.

[0141] Optionally, the base mentioned in condition b can be one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, pyridine, triethylamine, N,N-diisopropylethylamine, etc., and the condensing agent can be one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dilaurate carbonate, N,N-carbonyldiimidazole, and N-(4-carboxyphenyl)maleimide, etc., the reaction temperature is from 20 degrees to 60 degrees, and the solvent used is one of aprotic solvents such as tetrahydrofuran and diethyl ether.

[0142] Optionally, the reaction temperature described in condition c ranges from -20 degrees to 25 degrees, the solvent used is one of the aprotic solvents such as tetrahydrofuran or diethyl ether, and the equivalence ratio of compound 3 to Grignard reagent is 1:1 to 1:3.

[0143] Optionally, the base mentioned in condition d can be one of sodium hydroxide, potassium hydroxide, potassium tert-butoxide, sodium methoxide, potassium fluoride, etc., the reaction temperature is from 25 degrees to 100 degrees, and the solvent is one of water-soluble solvents such as methanol, ethanol, propanol, etc.

[0144] Optionally, the base mentioned in condition e may be one of potassium hydroxide, sodium hydroxide, sodium carbonate, pyridine, triethylamine, N,N-diisopropylethylamine, etc., and the solvent may be a mixture of one of water-soluble solvents such as methanol and ethanol and water.

[0145] Optionally, the acid mentioned in condition f can be one or a mixture of organic or inorganic acids such as formic acid, acetic acid, and hydrochloric acid, and the solvent can be one of water-soluble solvents such as methanol, ethanol, and propanol.

[0146] In this application, C1-C 10This refers to the number of carbon atoms contained. The limitation on carbon atoms in "substituted alkyl" and "substituted aryl" refers to the number of carbon atoms inherent in the alkyl or aryl group itself, not the number of carbon atoms after substitution. For example, C1-C... 10 A substituted alkyl group refers to an alkyl group having 1 to 10 carbon atoms, in which at least one hydrogen atom is replaced by a substituent.

[0147] In this application, "alkyl" refers to a group formed by losing any one hydrogen atom from an alkane compound molecule. The alkane compound includes straight-chain alkanes, branched alkanes, cycloalkanes, and branched cycloalkanes.

[0148] In this application, "alkenyl" refers to a group formed by losing any one hydrogen atom from an alkene compound molecule. The alkane compounds include straight-chain alkanes, branched alkanes, cycloalkanes, and branched cycloalkanes.

[0149] In this application, "aryl" is a group formed by the loss of a hydrogen atom from the aromatic ring of an aromatic compound molecule; such as p-tolyl formed by the loss of a hydrogen atom at the para position of the methyl group on the benzene ring of toluene.

[0150] In this application, "furanyl" is a group formed by losing any one hydrogen atom from a furan compound molecule.

[0151] In this application, "imine group" is the divalent group remaining after removing two hydrogen atoms from an ammonia molecule, with the structural formula NH=.

[0152] In this application, "pyridyl" is a group formed by losing any one hydrogen atom from a pyridine compound molecule.

[0153] In this application, "epoxyalkyl" is a group formed by losing any one hydrogen atom from an epoxy compound molecule. Attached Figure Description

[0154] Figure 1 This is the 1H NMR spectrum of compound SMI0;

[0155] Figure 2 This is the 1H NMR spectrum of compound SMI7;

[0156] Figure 3 This is the 1H NMR spectrum of compound SMI16;

[0157] Figure 4 This is the 1H NMR spectrum of compound SMI20;

[0158] Figure 5 This is the X-ray powder diffraction (XRPD) pattern of SMI0 hydrochloride crystal form A;

[0159] Figure 6The differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) spectra of SMI0 hydrochloride crystal form A are shown.

[0160] Figure 7 This is the X-ray powder diffraction (XRPD) pattern of SMI0 sulfate crystal form B;

[0161] Figure 8 The differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) spectra of SMI0 sulfate crystal form B are shown.

[0162] Figure 9 The graph shows the double reciprocal plot of the inhibitory effect of compound SMI0 on human liver metabolism of chlorzoxazone.

[0163] Figure 10 It is a secondary plot based on the double reciprocal plot of the inhibitory effect of compound SMI0 on chlorzoxazone metabolism in human liver CYP2E1.

[0164] Figure 11 The graph shows the inhibitory effects of 21 small molecule compounds on the in vitro metabolic activity of CYP2E1.

[0165] Figure 12 The graph shows the inhibitory effect of compound SMI0 on the metabolism of diethylnitrosamine in rat CYP2E1 cells.

[0166] Figure 13 The graph shows the inhibitory effect of compound SMI0 on a rat model of liver injury induced by diethylnitrosamine.

[0167] Figure 14 The graph shows the inhibitory effect of compound SMI0 on a mouse model of hepatic steatosis and hepatitis induced by a high-fat diet. Among them, A is a representative image of the liver of each group of mice in the hepatitis model; B is the histopathological features of inflammation-related tissues; H&E is hematoxylin and eosin staining; Masson staining is Masson staining; Oil red staining is Oil Red O staining; LCA staining is common leukocyte antigen staining.

[0168] Figure 15 This is a quantitative graph of relevant parameters in a mouse model of hepatic steatosis and hepatitis induced by a high-fat diet, induced by compound SMI0; where A is the liver coefficient of each group of animals in the hepatitis model; B is the steatosis score of each group of animals; C is the Masson staining score of each group of animals; D is the percentage of Oil Red O staining area of ​​each group of animals; and E is the LCA score of each group of animals.

[0169] Figure 16The graph shows the inhibitory effect of compound SMI0 on a mouse model of liver fibrosis induced by a high-fat diet. A represents representative images of the livers of mice in each group of the liver fibrosis model; B represents the histopathological features related to inflammation and liver fibrosis; H&E represents hematoxylin and eosin staining; Masson represents Masson staining; and Oil red represents Oil Red O staining.

[0170] Figure 17 This is a quantitative graph of relevant parameters in a mouse model of liver fibrosis induced by a high-fat diet, where A represents the liver coefficient of each group of animals in the liver fibrosis model; B represents the steatosis score of each group of animals; C represents the Masson staining score of each group of animals; and D represents the percentage of Oil Red O staining area of ​​each group of animals.

[0171] Figure 18 The graph shows the inhibitory effect of compound SMI0 on a rat model of liver fibrosis induced by diethylnitrosamine. A represents representative images of the livers of rats in each group of the liver fibrosis model; B represents the histopathological features related to liver fibrosis; H&E represents hematoxylin and eosin staining; Masson represents Masson staining; α-SMA represents α-smooth muscle actin; and Collagen I represents type I collagen.

[0172] Figure 19 This is a quantitative graph of relevant parameters in a rat liver fibrosis model induced by the inhibition of diethylnitrosamine by compound SMI0; where A is the liver coefficient of each group of animals in the liver fibrosis model; B is the Ishark score of each group of animals; C is the Masson staining score of each group of animals; D is the α-SMA histochemical score of each group of animals; and E is the Collagen I histochemical score of each group of animals.

[0173] Figure 20 The graph shows the inhibitory effect of cyp2e1 gene knockout on a rat hepatocellular carcinoma model with orthotopic implantation of Walker256 cells in the liver.

[0174] Figure 21 The graph shows the inhibitory effect of compound SMI0 on a mouse liver cancer model in which H22 liver cancer cells are implanted in situ.

[0175] Figure 22 The graph shows the high expression level of CYP2E1 in the tissue adjacent to the tumor in glioma patients.

[0176] Figure 23 The graph shows the inhibitory effect of cyp2e1 gene knockout on a mouse glioma model of GL261 cells implanted in situ in the brain.

[0177] Figure 24The graph shows the inhibitory effect of compound SMI0 on a mouse glioma model in which GL261 cells are implanted in situ in the brain. Among them, A is a gross image of the brain of each group of mice; B is a representative image of HE staining of brain tissue of each group of mice; C is the tumor volume of each group of mice; and D is the in vitro inhibitory effect of SMI0 on GL261 cells.

[0178] Figure 25 The graph shows the inhibitory effect of compound SMI0 on a mouse ovarian cancer model in which ID-8 cells are implanted in situ in the ovary. In this graph, A shows the gross images of the ovaries and tumors of mice in each group; B shows the total tumor weight of mice in each group; and C shows the in vitro inhibitory effect of SMI0 on ID-8 cells.

[0179] Figure 26 The graph shows the inhibitory effect of compound SMI0 on mouse peritoneal xenografts of ovarian cancer ID-8 cells; where A represents the body weight of mice in each group, and B represents the ascites volume of mice in each group.

[0180] Figure 27 This is a gross image of the foot of a rat model of rheumatoid arthritis induced by complete Freund's adjuvant inhibition of compound SMI0.

[0181] Figure 28 This is a quantitative graph of relevant parameters in a rat model of rheumatoid arthritis induced by compound SMI0 with complete Freund's adjuvant; where A is the paw edema rate of rats in each group at different time points; B is the paw edema rate of rats at 24 h; C is the paw edema rate of rats at 36 h; and D is the paw edema rate of rats at 48 h.

[0182] Figure 29 The graph shows the dose-response relationship of compound SMI0 inhibiting a rat model of rheumatoid arthritis induced by complete Freund's adjuvant. Among them, A is the dose-response relationship of rat paw swelling rate at different time points; B is the 24-hour dose-response relationship; C is the 36-hour dose-response relationship; and D is the 48-hour dose-response relationship.

[0183] Figure 30 The graph shows the inhibitory effect of compound SMI0 on the changes in body temperature in a rat model of sepsis induced by lipopolysaccharide; where A represents the body temperature of rats in each group at different time points; B represents the body temperature of rats in each group at 4 h; C represents the body temperature of rats in each group at 5 h; and D represents the body temperature of rats in each group at 6 h.

[0184] Figure 31 The graph shows the inhibitory effect of compound SMI0 on the changes in body temperature in a mouse model of lipopolysaccharide-induced sepsis. In the graph, A represents the body temperature of mice in each group at different time points; B represents the body temperature of mice in each group at 6 h; C represents the body temperature of mice in each group at 12 h; and D represents the body temperature of mice in each group at 24 h.

[0185] Figure 32The graph shows the effect of compound SMI0 on improving renal function and renal function impairment in a mouse model of lipopolysaccharide-induced sepsis. In the graph, A represents the 24-hour serum urea nitrogen level of each group of mice; B represents the 24-hour serum creatinine level of each group of mice; C represents the 24-hour creatine kinase level of each group of mice; and D represents the 24-hour serum lactate dehydrogenase level of each group of mice.

[0186] Figure 33 The graph shows how compound SMI0 improves cognitive impairment in a rat model of Alzheimer's disease induced by streptozotocin; where A represents the latency of rats in each group at different training time points; B represents the time spent in the platform quadrant of rats in each group; and C represents the number of times rats in each group crossed the platform.

[0187] Figure 34 The graph shows the inhibitory effect of compound SMI0 on focal cerebral ischemia-reperfusion injury in rats.

[0188] Figure 35 The graph shows the effect of compound SMI0 on cerebral infarction and cerebral edema in rats with focal cerebral ischemia-reperfusion injury; where A represents the cerebral infarction rate in rats and B represents the cerebral edema rate in rats.

[0189] Figure 36 It is compound SMI0 that reduces ApoE caused by a high-fat diet. - / - Plotting blood lipid levels in a mouse model of hyperlipidemia;

[0190] Figure 37 It is compound SMI0 that inhibits ApoE caused by a high-fat diet. - / - A graph showing the inhibitory effect on the formation of atherosclerotic plaques in the aorta of mice; where A represents the total area of ​​plaques in each group; and B represents the percentage of Oil Red O area in the total plaque area in each group.

[0191] Figure 38 The graph shows the effect of compound SMI0 on reducing blood glucose levels in rats with diabetes induced by a high-fat diet and streptozotocin. A represents fasting blood glucose in each group of rats; B represents blood glucose at different time points in each group of rats during the glucose tolerance test; and C represents the area under the curve in each group of rats during the glucose tolerance test.

[0192] Figure 39 The graph shows the high expression level of CYP2E1 in adjacent tissues of bladder cancer.

[0193] Figure 40 The graph shows the high expression level of CYP2E1 in the tissue adjacent to gallbladder cancer. Detailed Implementation

[0194] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0195] The high-resolution mass spectrometer used for the synthesis of CYP2E1 inhibitors in Examples 2, 3, 4 and 5 of this application is a Q-Tof micro type from Waters Corporation, USA.

[0196] In Examples 2, 3, 4 and 5 of this application, the CYP2E1 inhibitors were synthesized using a nuclear magnetic resonance spectrometer, Bruker DPX-400, Germany.

[0197] In Examples 10, 11 and 12 of this application, the inhibitory effect of CYP2E1 in vivo and in vitro was determined using a high-performance liquid chromatograph (HPLC) from Agilent Technologies, model Agilent 1260.

[0198] The mouse glioma model in Example 14 of this application was prepared using a stereotaxic instrument, model ZR-09, manufactured by Shanghai Puxin Instrument Technology Co., Ltd.

[0199] Example 1: Preparation of human liver microsomes

[0200] Liver specimens were thawed and weighed using differential centrifugation. A liver homogenate was prepared by adding 50 mM Tris-HCl (pH = 7.0) buffer (containing 150 mM KCl and 2 mM EDTA) at a ratio of 1:4 (w / v) and homogenizing with a glass homogenizer. The homogenate was centrifuged at 9000 × g for 20 min at 4°C, and the supernatant was centrifuged at 100000 × g for 60 min at 4°C. The precipitate was resuspended in 4 mL of 0.15 M Tris-HCl (pH = 7.6) and centrifuged again at 100000 g for 60 min at 4°C. The precipitate was then resuspended in 0.25 M sucrose at a ratio of 1:2 (w / v) to prepare 2 mL of microsomal suspension per gram of liver tissue. The suspension was aliquoted and stored overnight in liquid nitrogen, then transferred to -80°C for long-term storage the following day. All operations were performed on ice. The microsomal protein content (mg / mL) was determined using the Bradford method.

[0201] This method was used for liver microsomes in both patients with liver injury and healthy individuals.

[0202] Example 2: Synthesis of SMI0

[0203] 4-Methylthiazol-5-carboxylate (1 mol) and NaOH (1.6 mol) were reacted overnight at room temperature in a mixture of ethanol and water. The reaction was monitored by TLC (using pure ethyl acetate). After the reaction was complete, the ethanol was evaporated to dryness under reduced pressure, the pH was adjusted to 2-3 with concentrated sulfuric acid, and the solid was obtained by filtration, washing, and drying. 4-Methylthiazol-5-carboxylic acid (1 mol) and DDC (1 mol) were stirred in anhydrous tetrahydrofuran at room temperature for 2-3 hours. Then, dimethylhydroxylamine hydrochloride (1.2 mol) was added, followed by the dropwise addition of triethylamine (1.5 mol). The mixture was stirred overnight at room temperature and monitored by TLC (PE:EA = 3:1). After the reaction was complete, the tetrahydrofuran was evaporated to dryness under reduced pressure, extracted three times with ethyl acetate, washed twice with saturated sodium bicarbonate aqueous solution, dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness under reduced pressure. The product (1 mol) was dissolved in anhydrous tetrahydrofuran under nitrogen protection and pre-cooled in a cold trap at -10 to 15 °C. Grignard reagent CH3MgCl (1.5 mol) was added dropwise, and the reaction was monitored by TLC (PE:EA = 3:1). After the reaction was complete, it was quenched with saturated NH4Cl, extracted with ethyl acetate, dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness under reduced pressure. The crude product was purified by vacuum distillation. The NMR data of the product are as follows (e.g., Figure 1 As shown):

[0204] 1 H NMR (400MHz, CDCl3) δ8.79(s,1H),2.80(s,3H),2.60(s,3H).

[0205] Example 3: Synthesis of compound SMI7

[0206] 3 mmol of hydroxylamine hydrochloride was placed in a round-bottom flask, 3 mL of ethanol was added, and the mixture was stirred at 25°C for 10 min. 3 mL of 1 M NaOH solution was added, followed by 3 mmol of SMI. The mixture was refluxed in an oil bath at 80°C. After the reaction was complete as monitored by TLC, the reaction solution was neutralized with 10% dilute hydrochloric acid, extracted with water and ethyl acetate, and the organic phases were combined. The mixture was dried over anhydrous magnesium sulfate, and the magnesium sulfate was removed by filtration. The filtrate was concentrated under vacuum and separated by silica gel column chromatography with petroleum ether and ethyl acetate in a ratio of 1:2 to obtain compound SMI7. The NMR data of the product are as follows (e.g., Figure 2 As shown): 1 H NMR (400MHz, DMSO) δ11.50(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).

[0207] Example 4: Synthesis of compound SMI16

[0208] 3,4-Dichlorobenzaldehyde (1 mmol) was placed in a round-bottom flask, 2 mL of anhydrous ethanol was added, and the mixture was stirred at 50 °C to dissolve. 30 μL of 3M KOH solution was added, followed by SMI0 (1 mmol). The mixture was stirred at 50 °C until complete. After TLC monitoring, the reaction solution was neutralized with 10% dilute hydrochloric acid, extracted with water and ethyl acetate, and the organic phases were combined. The solution was dried over anhydrous magnesium sulfate, and magnesium sulfate was removed by filtration. The filtrate was concentrated under vacuum, and separated by silica gel column chromatography with petroleum ether:ethyl acetate in a 2:1 ratio to obtain compound SMI16. The NMR detection data of the product are as follows (e.g., Figure 3 As shown): 1 H NMR (400MHz, CDCl3) δ8.84(s,1H),7.68(d,J=11.2Hz,2H),7.51(d,J=8.3Hz,1H),7.44(d,J=8.2Hz,1H),7.19(d,J=15.5Hz,1H),2.86(s,3H).

[0209] Example 5: Synthesis of compound SMI20

[0210] Aniline (2 mmol) and SMI20 (2 mmol) were placed in a round-bottom flask, and 3 mL of anhydrous ethanol was added. The mixture was stirred at 25°C for 10 min. Then, BH3CNNa (2 mmol) and acetic acid (1 mmol) were added, and the mixture was stirred at 25°C. After the reaction was completed by TLC monitoring, the reaction solution was neutralized with 10% dilute hydrochloric acid, extracted with water and ethyl acetate, and the organic phases were combined. The mixture was dried over anhydrous magnesium sulfate, and the magnesium sulfate was removed by filtration. The filtrate was concentrated under vacuum and separated by silica gel column chromatography with petroleum ether:acetone = 4:1 as the eluent to obtain compound SMI20. The NMR detection data of the product are as follows (e.g.) Figure 4 As shown): 1 H NMR (400MHz, CDCl3) δ8.55(s,1H),7.13(t,J=7.7Hz,2H),6.71(t,J=7.2Hz,1H),6.51(d ,J=7.9Hz,2H),4.74(q,J=6.2Hz,1H),4.00(s,1H),2.50(s,3H),1.56(d,J=6.6Hz,3H).

[0211] Example 6: Synthesis of SMI0 hydrochloride

[0212] 1 g (7.08 mmol) of SMI0 was dissolved in 5 mL of ethanol in a round-bottom flask. 11 mL of 1 mol / L hydrochloric acid-ethanol solution was slowly added dropwise under stirring at room temperature for 1 hour. The solution was concentrated under reduced pressure, cooled to crystallize, filtered, washed with 0.5 mL of cold anhydrous ethanol, and dried to give 1.06 g of a white solid, yield 85%, purity 99.8% (HPLC), melting range 160–162 °C. The X-ray powder diffraction (XRPD) pattern of SMI0 hydrochloride crystal form A is shown below. Figure 5 As shown, the differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) spectra are as follows: Figure 6 As shown.

[0213] Example 7 Synthesis of SMI0 sulfate

[0214] 1 g (7.08 mmol) of SMI0 was placed in a round-bottom flask and dissolved in 5 mL of ethanol. 8 mL of 1 mol / L sulfuric acid-ethanol solution was slowly added dropwise under stirring at room temperature, and the reaction was carried out for 1 hour. The solvent was evaporated to dryness, and the solution was recrystallized from a small amount of methanol, yielding 0.85 g of a yellow solid. The yield was 70%, and the purity was 99.5% (HPLC). The X-ray powder diffraction (XRPD) pattern of SMI0 sulfate crystal form A is shown below. Figure 7 As shown, the differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) spectra are as follows: Figure 8 As shown.

[0215] Example 8: Determination of the inhibitory effect on CYP2E1 metabolic activity

[0216] Chlorzoxazone was used as the probe substrate to detect the inhibitory effect (IC50) on the CYP2E1 metabolic activity of mixed liver microsomes in normal individuals. 50 Half-maximal inhibitory concentration; K i Suppression constant; IC 50 The smaller K is i The smaller the value, the higher the inhibitory strength, to determine the inhibitory effect of the test inhibitor on human liver microsome CYP2E1.

[0217] Inhibition of CYP2E1 activity IC 50 The determination

[0218] The total volume of the incubation system was 100 μL, and it included the substrate, inhibitors of different concentrations, liver microsomal protein, and phosphate buffer. The reaction was carried out in a 37°C water bath. After a 5-minute pre-incubation, the reduced coenzyme was added to initiate the reaction, and the reaction was terminated by placing the container on ice for 30 minutes. In specific experiments, the concentration of the inhibitor can be selected as needed.

[0219] In this embodiment, the incubation system includes 62.5 μM chlorzoxazone, 100 mM (pH=7.4) phosphate buffer, 0.3 mg / mL liver microsomal protein, and 1 mM NADPH.

[0220] In other preferred embodiments, the incubation system may include 7.8–1000 μM chlorzoxazone as a substrate. The concentration of liver microsomal protein may be 0.1–0.5 mg / mL. 50 mM–100 mM phosphate buffer or 50 mM–100 mM Tris-HCl buffer may be used as needed.

[0221] An NADPH regeneration system can also be used. Preferably, the incubation system comprises 62.5 μM chlorzoxazone, 100 mM phosphate buffer (pH 7.4), 0.3 mg / mL liver microsomal protein, and 1 mM NADPH.

[0222] In this embodiment, the reaction was terminated with 1 mL of ethyl acetate. In other preferred embodiments, the reaction can also be terminated with 1 mL of methyl tert-butyl ether, 1 mL of diethyl ether, and 100 μL of methanol.

[0223] Inhibition of CYP2E1 activity K i The determination

[0224] Once it was determined that the inhibitor had a good inhibitory effect on CYP2E1, in vitro metabolic incubation inhibition experiments were conducted using different concentrations of substrate and inhibitor to calculate the inhibition constant K of the inhibitor on CYP2E1 metabolism of chlorzoxazone. i .

[0225] The total volume of the incubation system was 100 μL, and it included substrate, inhibitors of different concentrations, liver microsomal protein, and phosphate buffer. The reaction was carried out in a 37°C water bath. After a 5-minute pre-incubation, the reduced coenzyme was added to initiate the reaction, and the reaction was terminated by incubating on ice for a certain period of time.

[0226] In this embodiment, the incubation system included chlorzoxazone at concentrations of 15.6, 31.25, 62.5, 125, and 250 μM, respectively; the IC50 values ​​were determined above. 50 Based on this, different concentration gradients of the inhibitor were determined in a ratio of 1 / 4 to 4:100 mM (pH=7.4) phosphate buffer, 0.3 mg / mL liver microsomal protein, and 1 mM NADPH.

[0227] In other preferred embodiments, the incubation system may include 7.8–1000 μM chlorzoxazone as a substrate, more preferably, it may include 15.6–250 μM (15.6, 31.25, 62.5, 125, and 250 μM) chlorzoxazone. The concentration of liver microsomal protein may be 0.1–0.5 mg / mL. 50 mM–100 mM phosphate buffer or 50 mM–100 mM Tris-HCl buffer may be used as needed.

[0228] Alternatively, one of the 1mM NADPH or NADPH regeneration systems can be used. The NADPH regeneration system contains 1.3mM NADP+, 3.3mM glucose-6-phosphate, 0.4U / mL glucose dehydrogenase, and 3.3mM magnesium chloride.

[0229] In this embodiment, the reaction was terminated with 1 mL of ethyl acetate. In other preferred embodiments, the reaction can also be terminated with 1 mL of methyl tert-butyl ether, 1 mL of diethyl ether, and 100 μL of methanol.

[0230] Determination of the selectivity of CYP2E1 inhibitors

[0231] Meanwhile, using normal human mixed liver microsomes as the research subject, probe drugs of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were selected respectively to determine the in vitro inhibitory effect of the test inhibitors on the metabolic probe drugs of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in normal human mixed liver microsomes, and to evaluate the selectivity of the test inhibitors on the inhibitory effect of CYP2E1.

[0232] The CYP1A2 probe is one of 6.25–800 μM phenacetin or 27.5–12520 μM caffeine;

[0233] The CYP2A6 probe is one of 0.156–20 μM coumarin or 12.5–2000 μM nicotine;

[0234] The CYP2B6 probe is one of 7.8–500 μM bupropion or 0.25–30 mM cyclophosphamide;

[0235] The CYP2C8 probe is one of 2.5–80 μM paclitaxel or 0.125–128 μM amodiaquine;

[0236] The CYP2C9 probe is one of 31.25–2000 μM tolbutamide or 0.1–200 μM diclofenac.

[0237] The CYP2C19 probe is one of 3.9–500 μM omeprazole or 1.95–1000 μM mephenytoin;

[0238] The CYP2D6 probe is one of 0.625–96 μM dextromethorphan or 0.0195–80 μM propafenone;

[0239] The CYP3A4 probe is one of 0.39–50 μM midazolam or 1.98–1000 μM testosterone;

[0240] The total volume of the incubation system was 100 μL, and it included substrate, inhibitors of different concentrations, liver microsomal protein, phosphate buffer, and a 37°C water bath. After a 5-minute pre-incubation, the reduced coenzyme was added to initiate the reaction, and the reaction was terminated by incubating on ice for a certain period.

[0241] In this embodiment, the incubation system includes at least one of the following: 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, and 1.56 μM midazolam; 100 mM phosphate buffer (pH = 7.4); 0.3 mg / mL liver microsomal protein; and 1 mM NADPH.

[0242] In other preferred embodiments, the substrate may include 7.8–1000 μM chlorzoxazone. The concentration of liver microsomal protein may be 0.1–0.5 mg / mL. The buffer may be selected from 50 mM phosphate buffer, 100 mM phosphate buffer, 50 mM Tris-HCl buffer, and 100 mM Tris-HCl buffer. Alternatively, a 1 mM NADPH or NADPH regeneration system may be used.

[0243] In this embodiment, the reaction was terminated with 1 mL of ethyl acetate. In other preferred embodiments, the reaction can also be terminated with 1 mL of methyl tert-butyl ether, 1 mL of diethyl ether, and 100 μL of methanol.

[0244] Example 9: In vitro screening results of CYP2E1 inhibitors

[0245] SMI0 (2E1 suppression IC) 50 1.64 μM, for 2A6 suppression IC 50 The concentration was 76.20 μM, and it had no significant inhibitory effect on other CYP enzymes.

[0246]

[0247] Now for reference Figure 9 All reactions were performed using a 100 μL incubation system. Chlorzoxazone concentrations were 15.6, 31.2, 62.5, 125, and 250 μM, and SMI0 series concentrations were 0, 0.4638, 0.9276, 1.855, and 3.710 μM. All results were derived from the average of three independent experiments. Figure 9 This is a double reciprocal plot showing the inhibitory effect of compound SMI0 on human liver metabolism of chlorzoxazone. It indicates that compound SMI0 is a mixed inhibitor of CYP2E1.

[0248] Now for reference Figure 10 , Figure 10 This is a quadratic plot based on a double reciprocal plot, which investigates the inhibitory effect of compound SMI0 on chlorzoxazone metabolism in human liver CYP2E1. Linear regression was performed on a series of SMI0 concentrations (0, 0.4638, 0.9276, 1.855, 3.710 μM) with different inhibition curve slopes. The absolute value of the intersection of the line and the horizontal axis is K. i It is 0.8970 μM.

[0249] Referring to the specific research methods in Example 8, the inhibitory effects of 21 small molecule compounds (Table 1) on CYP2E1 were tested in vitro. The results showed that SMI1 and SMI8 had significant inhibitory effects on CYP2E1, with an IC50 value for CYP2E1 inhibition of [missing value]. 50 The concentrations were 7.99 and 17.03 μM, respectively. SMI10 showed a slight inhibitory effect on CYP2E1, with an IC50 concentration of 7.99 μM and 17.03 μM, respectively. 50 The concentration was 114.5 μM. Compared to SMI0, SMI1, SMI8, and SMI10, other small molecules showed weaker inhibitory effects on CYP2E1 (e.g., Figure 11 (As shown).

[0250] Furthermore, selective studies have shown that SMI1 inhibits the IC50 of CYP2A6. 50 The IC50 value for inhibiting CYP2C9 is 55.63 μM. 50 The concentration was 1.86 μM. The structures of the 21 small molecules are shown in Table 1.

[0251] Table 1

[0252]

[0253]

[0254]

[0255]

[0256] Example 10: Results of in vivo inhibitory effect of SMI0 compound on CYP2E1

[0257] Experimental Methods: A self-controlled crossover experimental design was used to study the inhibitory effect of SMI0 compound on CYP2E1 in rats. In the first round of the experiment, 30 SD rats were intraperitoneally injected with diethylnitrosamine (DEN) at 50 mg / kg during the first week. One week later, in the second round, each rat was first administered a low, medium, or high dose of SMI0 compound (6 mg / kg, 30 mg / kg, or 150 mg / kg) via gavage, followed by a second intraperitoneal injection of DEN at 50 mg / kg (10 rats for each low, medium, and high dose). Blood samples were collected at 2, 7, 15, and 30 mins and at 1, 2, 4, 6, 9, 12, 24, 36, 48, and 60 h. Plasma DEN concentrations were measured at these different time points to determine the toxicokinetic parameters of DEN. The detection method for plasma DEN was performed using high-performance liquid chromatography (HPLC). 10 μL of plasma was added to 10 μL of perchloric acid, vortexed for 3 min, centrifuged at 12000 rpm for 10 min, and 10 μL of the supernatant was injected. The mobile phase was methanol:water = 50:50, and the detection wavelength was 240 nm.

[0258] Experimental Results: The effects of low, medium, and high doses of SMI0 compound on CYP2E1 activity in rats were analyzed, using DEN toxicokinetics as a representative of CYP2E1 activity. The results showed that compared with the DEN-only model group, low, medium, and high doses of SMI0 decreased the DEN clearance rate (CL) by (63.98±7.78)%, (79.63±7.29)%, and (85.42±3.74)%, respectively, with inhibition rates of 62.43%, 80.42%, and 86.77%, respectively (e.g., ). Figure 12 As shown in Table 2, * P<0.05, *** P < 0.001 (vs. model group). Similarly, low, medium, and high doses of SMI0 can shorten the half-life of DEN. 1 / 2 The curve lengths were extended by (133.60±116.80)%, (619.97±363.57)%, and (868.70±241.26)%, respectively, with the area under the curve (AUC) increasing accordingly. 0-t The area under the curve (AUC) increased by (196.82±73.63)%, (407.70±184.30)%, and (529.67±153.72)%, respectively. 0-∞ The inhibitory effects were increased by (189.99±67.55)%, (455.90±219.26)%, and (626.56±187.15)%, respectively. Furthermore, the medium-dose SMI0 compound showed significantly better inhibitory effects than the low-dose compound, and the high-dose SMI0 compound showed significantly better inhibitory effects than both the low-dose and medium-dose compounds. #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 different doses of SMI0 compound can significantly inhibit the metabolic activity of CYP2E1 on DEN in rats, and this inhibition has a good dose-dependent relationship.

[0259] The above results indicate that SMI0 has a significant inhibitory effect on CYP2E1 activity in rats.

[0260] Table 2. Toxicokinetic parameters of SMI0 compound in inhibiting the in vivo metabolism of diethylnitrosamine by CYP2E1.

[0261]

[0262] Note: Mean in Table 2 represents the mean; SD represents the standard deviation.

[0263] C max Peak drug concentration (PSC): The highest blood drug concentration observed after drug administration. This parameter is an important indicator reflecting the rate and extent of drug absorption in the body.

[0264] T max Time to peak concentration (TBC): The time required to reach the peak drug concentration after administration. This parameter reflects the rate at which the drug enters the body; a faster absorption rate results in a shorter TBC.

[0265] V d Apparent volume of distribution (ADI) is the ratio of the amount of drug in the body to its blood concentration when the drug reaches dynamic equilibrium in the body, typically expressed in liters (L). This parameter reflects the extent of drug distribution in the body; a higher value indicates wider distribution. ADI is numerically derived from the ratio of clearance rate to terminal elimination rate.

[0266] CL: Clearance rate, the apparent volume of distribution of a drug cleared from the body per unit time, typically expressed in L / h. This parameter is an important indicator reflecting the body's response to drugs and is closely related to physiological factors. Clearance rate varies depending on dose and AUC. (0-∞) The ratio is worth getting.

[0267] AUC: Area Under the Chance Curve, the area enclosed by the blood drug concentration curve on the time axis. This parameter is an important indicator for evaluating the extent of drug absorption and reflects the drug's exposure characteristics in the body. Since blood drug concentration can only be observed up to a specific time point t in pharmacokinetic studies, AUC is expressed in two ways: AUC (0-t) and AUC (0-∞) The former is obtained using the trapezoidal area method, and the latter is calculated using the formula: AUC (0-∞) =AUC(0-t) +Terminal concentration / Terminal elimination rate.

[0268] Example 11: SMI0 compound inhibits diethylnitrosamine-induced liver injury in rats

[0269] Experimental Methods: A liver injury model in SD rats was established using intermittent intraperitoneal injection of diethylnitrosamine (DEN). The model group received intraperitoneal injections of DEN 50 mg / kg twice weekly for the first 4 weeks, and once weekly from weeks 5 to 8. The SMI0 intervention group was divided into low-dose and high-dose DEN-concurrent groups and a high-dose continuous administration group. The low-dose and high-dose DEN-concurrent groups received SMI0 compound 30 mg / kg and 150 mg / kg via gavage 5 minutes before each DEN administration, respectively. The high-dose continuous administration group received SMI0 compound 150 mg / kg via gavage daily from week 1 until the model ended in week 8. At the end of the model, blood was collected from the orbital sinus, and plasma liver function indicators were measured using an automated biochemical analyzer. [Model group, n = 10 rats; Low-dose SMI0 compound group, n = 21 rats; (150 mg / kg)] a SMI0 compound accompanied by high-dose DEN, n=28 rats; (150mg / kg) b [High-dose continuous administration of SMI0 compound, n=24 rats]

[0270] Experimental Results: Compared with the model group, the SMI0 intervention group showed significantly increased albumin (ALB2) and cholinesterase (CHE2), and significantly decreased alkaline phosphatase (ALP2S), alanine aminotransferase (ALTL), direct bilirubin (BILD2), total bilirubin (BILT3), and gamma-glutamyl transferase (GGTI2). The low-dose SMI0 group showed significantly increased TP2, while the high-dose SMI0 concomitant medication group and the high-dose continuous SMI0 group showed significantly decreased GGTI2 (e.g., ...). Figure 13 As shown, * P<0.05, ** P<0.01, *** P < 0.001 vs. model group; where 150 mg / kg a For the high-dose concomitant medication group of SMI0, 150 mg / kg b (This refers to the SMI0 high-dose continuous dosing group). Compared with the SMI0 low-dose group, the SMI0 high-dose concomitant dosing group showed significantly decreased ALTL, BILD2, BILT3, and GGTI2, and significantly increased TP2; the SMI0 high-dose continuous dosing group showed significantly increased ALB2, and significantly decreased ALP2S, ALTL, ASTL, BILD2, CHE2, BILT3, and GGTI2. ## P<0.01, ###P<0.001 vs SMI0 low-dose group). Compared with the SMI0 high-dose concomitant medication group, the SMI0 high-dose continuous medication group showed significantly increased ALB2 and significantly decreased ALP2S, ALTL, ASTL, BILD2, CHE2, BILT3, and GGTI2. $ P<0.05, $$$ P<0.001 (vs SMI0 high-dose concomitant drug group). This suggests that SMI0 administration can significantly improve diethylnitrosamine-induced liver injury in rats.

[0271] The above results indicate that SMI0 has a significant preventive and therapeutic effect on diethylnitrosamine-induced liver injury in rats. This suggests that SMI0 compounds may be used for the clinical prevention and treatment of liver injury.

[0272] Example 12: SMI0 inhibits fatty liver and hepatitis induced by a high-fat diet in mice.

[0273] Experimental Methods: Healthy male C57 / 6J mice were fed a high-fructose, high-fat, and high-cholesterol diet to establish a mouse model of hepatic steatosis and hepatitis. The control group was fed a low-fat, low-sugar diet. The SMI0 intervention group was divided into low-dose and high-dose intervention groups, receiving 30 mg / kg and 150 mg / kg of SMI0 compound daily by gavage at the beginning of the model, respectively, until the model ended at 22 weeks. At the end of the 22-week experiment, blood was collected from the orbital sinus, and the mice were sacrificed after their weight was recorded. Liver weight and appearance (liver color, texture, presence of nodules, etc.) were recorded. Hematoxylin and eosin (HE) staining was performed on some liver specimens, and the steatosis score of each group was observed and recorded. Masson staining was used to evaluate the liver fibrosis status, and the percentage of Oil Red O staining area and LCA staining inflammation score were also recorded. (Model group, n = 8 mice; low-dose SMI0 compound group, n = 10 rats; high-dose SMI0 compound group, n = 10 rats).

[0274] The pathological results of HE staining of liver tissue were quantitatively scored according to the following criteria to determine the severity of steatosis.

[0275]

[0276] The pathological results of Masson staining of liver tissue were quantitatively scored according to the following criteria to determine the severity of fibrosis.

[0277]

[0278] Experimental results: Compared with the control group, the model group showed significant increases in liver coefficient, degree of steatosis (Steatosis score and percentage of Oil Red O staining area), and level of liver inflammation (number of LCA-positive cell foci). Figure 14 A- Figure 14 B Figure 15 A- Figure 15 E, * P<0.05, ** P<0.01, *** P<0.001 vs. control group; Figure 14 (Scale is 100 μm); compared with the model group, the liver coefficient and degree of steatosis (steatosis score and percentage of Oil Red O staining area) were significantly reduced in the SMI0 low-dose intervention group. # P<0.05, ## P<0.01, ### (P<0.001 vs. model group) The high-dose SMI0 intervention group showed significantly reduced levels of steatosis (Steatosis score and percentage of Oil Red O staining area) and liver inflammation (number of LCA-positive cell foci). This suggests that SMI0 compounds can significantly reduce high-fat diet-induced hepatic steatosis and inflammation.

[0279] The above results indicate that SMI0 has a significant preventive and therapeutic effect on fatty liver and hepatitis induced by a high-fat diet in mice. This suggests that SMI0 compounds may be used clinically for the prevention and treatment of fatty liver and hepatitis.

[0280] Example 13: SMI0 inhibits liver fibrosis induced by a high-fat diet in mice.

[0281] Experimental Methods: Male C57 / 6J mice were fed a high-fructose, high-fat, and high-cholesterol diet to establish a liver steatosis and hepatitis model. The control group was fed a low-fat, low-sugar diet. The SMI0 intervention group was divided into low-dose and high-dose intervention groups, which were administered SMI0 compound 30 mg / kg and 150 mg / kg daily by gavage at the beginning of the model until the end of 26 weeks. At the end of the 26-week experiment, blood was collected from the orbital sinus, and the mice were sacrificed after their weight was recorded. Liver weight and appearance (liver color, texture, presence of nodules, etc.) were recorded. Some liver specimens were stained with HE and the steatosis score of each group of mice was observed and recorded. Masson staining was used to evaluate the liver fibrosis status of the mice, and the percentage of Oil Red O staining area was also scored. (Model group, n = 10 mice; low-dose SMI0 compound group, n = 10 rats; high-dose SMI0 compound group, n = 10 rats).

[0282] Experimental results: Compared with the control group, the model group showed significant increases in liver coefficient, indicators reflecting the degree of steatosis (steatodeosis score and percentage of Oil Red O staining area), and Masson staining score reflecting the degree of fibrosis (e.g., Figure 16 A- Figure 16 B Figure 17 A- Figure 17D, *P<0.05, **P<0.01 vs control group; Figure 16 In B, HE and Masson staining scales were 100 μm, and Oil Red staining scales were 50 μm; compared with the model group, the degree of fatty degeneration (fatty degeneration score and percentage of Oil Red O stained area) and the degree of fibrosis (Masson staining score) were significantly different in the low- and high-dose SMI0 intervention groups. # P<0.05, ## P<0.01, ### P < 0.001 (vs. model group). This suggests that the SMI0 compound can significantly reduce the level of liver fibrosis induced by a high-fat diet in mice.

[0283] The above results indicate that SMI0 has a significant preventive and therapeutic effect on liver fibrosis induced by a high-fat diet in mice. This suggests that SMI0 compounds may be used for the clinical prevention and treatment of liver fibrosis.

[0284] Example 14: SMI0 compound inhibits diethylnitrosamine-induced liver fibrosis in rats.

[0285] Experimental Methods: A liver injury model in SD rats was established using intermittent intraperitoneal injection of diethylnitrosamine (DEN). The model group received intraperitoneal injections of DEN 50 mg / kg twice weekly for the first 4 weeks, and once weekly from weeks 5 to 12. The SMI0 intervention group was divided into low-dose and high-dose DEN-concurrent groups and a high-dose continuous administration group. The low-dose and high-dose DEN-concurrent groups received SMI0 compound 30 mg / kg and 150 mg / kg via gavage 5 minutes before each DEN administration, respectively. The high-dose continuous administration group received SMI0 compound 150 mg / kg via gavage daily from week 1 until the end of the 12-week model. At the end of the model, orbital blood samples were collected, and plasma liver function indicators were measured using an automated biochemical analyzer (model group, n = 10 rats; low-dose SMI0 compound group, n = 21 rats; high-dose SMI0 compound with DEN-concurrent group, n = 28 rats; high-dose SMI0 compound continuous administration group, n = 24 rats).

[0286] Experimental results: Compared with the model group, the SMI0 intervention group showed varying degrees of reduction in liver coefficient, Ishark score for liver fibrosis, percentage of Masson staining area for liver fibrosis, α-SMA, and Collagen I (e.g., Figure 18 A- Figure 18 B Figure 19 A- Figure 19 As shown in E, *P<0.05, **P<0.01, ***P<0.001 vs. model group; Figure 18B scale is 100μm), and the high-dose (150mg / kgb) continuous SMI0 treatment group was significantly better than the high-dose diethylnitrosamine (150mg / kgb) and low-dose SMI0 groups. ## P<0.01, ### P<0.001 vs SMI0 low-dose group; &P<0.05, &&& P<0.001 vs. high-dose group with SMI0).

[0287] The above results indicate that SMI0 has a significant preventive and therapeutic effect on diethylnitrosamine-induced liver fibrosis in rats. This suggests that SMI0 compounds may be used for the clinical prevention and treatment of liver fibrosis.

[0288] Example 15: SMI0 inhibits the development and progression of liver cancer in mice.

[0289] (1) Cyp2e1 knockout rat xenografts

[0290] Experimental methods: A liver cancer xenograft model was established in SD rats by orthotopic implantation of Walker256 breast cancer sarcoma cell line into the liver. The cell concentration was 4*102 6 SD rats were divided into a model group and a cyp2e1 gene knockout group. The modeling period ended after 21 days (model group, n = 9 rats; gene knockout group, n = 8 rats). At the end of the experiment, blood was collected from the orbital sinus, and the rats were sacrificed after their weight was recorded. Liver weight and appearance (liver color, texture, etc.) were recorded. After HE staining of some liver specimens, the occurrence of liver lesions and tumors in each group of rats was observed.

[0291] Experimental results: Compared with the model group, the cyp2e1 gene knockout group showed significantly inhibited liver tumor growth in rats, with a tumor proliferation inhibition rate of up to 82.0% (e.g., Figure 20 (As shown in the image). This suggests that cyp2e1 gene knockout can significantly inhibit the occurrence of a rat hepatocellular carcinoma xenograft model by orthotopic implantation of Walker256 cells into the liver.

[0292] (2) SMI0 intervention in mouse xenografts

[0293] Experimental methods: A male BALB / c mouse hepatocellular carcinoma xenograft model was established by orthotopic seeding of H22 hepatocellular carcinoma cells into the liver. The cell concentration was 1.5*102 5The SMI0 intervention group was divided into low-, medium-, and high-dose groups. Starting two days before model establishment, mice were administered SMI0 compound at doses of 3.3 mg / kg, 10 mg / kg, and 30 mg / kg daily via gavage until the model was established (model group, n = 19 mice; low-dose SMI0 group, n = 19 mice; medium-dose SMI0 group, n = 18 mice; high-dose SMI0 group, n = 29 mice). At the end of the experiment, blood was collected from the orbital rim, and mice were sacrificed after their weight was recorded. Liver weight and appearance (liver color, texture, etc.) were recorded. Hematoxylin and eosin (HE) staining was performed on some liver specimens, and liver lesions and tumor occurrence were observed in each group of mice.

[0294] Experimental results: Compared with the model group, the incidence of H22 tumors in the liver of mice in the SMI0 intervention group was significantly reduced, from 100% in the model group to 78.9%, 72.2%, and 68.4% in the low, medium, and high dose SMI0 groups, respectively (e.g., ...). Figure 21 As shown in Table 3 (A~21C), *P<0.05, **P<0.01 (vs. model group). Figure 20 Images of liver tumor tissue from various mice were provided. If multiple tumors formed in the liver of a single mouse, all tumors were accumulated and arranged together to represent the tumor development in that animal. The cumulative weight of liver tumors in each animal was measured. The results showed that tumors formed in the livers of all mice in the model group. Compared with the model group, tumor proliferation was significantly inhibited in the SMI0 intervention group. That is, SMI0 intervention can significantly inhibit the growth of liver tumors in mice, with a tumor proliferation inhibition rate of up to 71.8% (e.g., ...). Figure 21 As shown in Table 3, from A to 21C. * P<0.05, ** P<0.01 (vs. model group). This suggests that SMI0 treatment can significantly inhibit the occurrence and development of H22 cell orthotopic liver transplantation in mouse hepatocellular carcinoma xenograft models.

[0295] In addition, in vitro inhibition experiments ( Figure 21 D) showed that SMI0, when applied to H22 cells at concentrations ranging from 0 to 128 μM for 48 h, had no significant inhibitory effect on H22 cells.

[0296] The above results indicate that SMI0 has a significant preventive and therapeutic effect on the occurrence and development of hepatocellular carcinoma in a mouse model of orthotopic implantation of H22 cells in the liver. This suggests that the SMI0 compound may be used for the clinical prevention and treatment of hepatocellular carcinoma.

[0297] Table 3. Quantification of relevant parameters in the SMI0 compound-induced inhibition of H22 hepatocellular carcinoma cell line in a mouse model of orthotopic liver implantation.

[0298]

[0299] *P<0.05,**P<0.01 vs. model group.

[0300] Example 16: SMI0 inhibits the proliferation of mouse gliomas.

[0301] (1) Changes in CYP2E1 in clinical glioma patients

[0302] Experimental methods: Using 46 normal individuals as controls, the study investigated the changes in CYP2E1 expression in adjacent tissues of 32 clinical glioma patients, comparing the changes in CYP2E1 content in adjacent tissues of glioma patients.

[0303] Experimental results: Immunohistochemical results showed that CYP2E1 expression in the peritumoral tissue of glioma patients was significantly higher than that in normal brain tissue (e.g., ...). Figure 22 As shown, ***P<0.001 vs normal brain tissue group).

[0304] (2) Cyp2e1 knockout mouse xenografts

[0305] Experimental methods: A male C57 / 6 mouse glioma model was established by orthotopic implantation of GL261 glioma cells into the brain. The cell concentration was 1*102 6 Mice were divided into a model group and a cyp2e1 knockout group. The model was established in approximately 21 days (model group, n = 10 mice; cyp2e1 knockout group, n = 6 mice). At the end of the experiment, blood was collected from the orbital rim, the mice were recorded, and then sacrificed for brain extraction. The brain tissue specimens were stained with hematoxylin and eosin (HE) to observe the occurrence of brain lesions and tumors in each group of mice.

[0306] Experimental results: Compared with the model group, cyp2e1 gene knockout significantly inhibited the growth of mouse glioma tumors. (e.g.) Figure 23 As shown in the figure, *P<0.05 (vs. wild-type model group). This suggests that cyp2e1 gene knockout can significantly inhibit the occurrence of GL261 cell orthotopic brain implantation in mouse glioma models.

[0307] (3) SMI0 intervention in mouse xenografts

[0308] Experimental methods: A male C57 / 6 mouse glioma model was established by orthotopic implantation of GL261 glioma cells into the brain. The cell concentration was 1*102 6The SMI0 intervention group was divided into low-, medium-, and high-dose groups. Starting two days before modeling, mice were administered SMI0 compound at doses of 3.3 mg / kg, 10 mg / kg, and 30 mg / kg daily via gavage, respectively. The positive control group was administered temozolomide at 50 mg / kg daily via gavage from days 3 to 7 and from days 10 to 14 post-surgery. The model was terminated after 21 days. (Sham-operated group: n = 9 mice; Model group: n = 10 mice; Temozolomide group: n = 11 mice; Low-dose SMI0 compound group: n = 13 mice; Medium-dose SMI0 compound group: n = 13 mice; High-dose SMI0 compound group: n = 13 mice). At the end of the experiment, blood was collected from the orbital rim, and mice were sacrificed after their weight was recorded. Brain tissue samples were stained with hematoxylin and eosin (HE) to observe brain lesions and tumor development in each group.

[0309] Experimental results: Compared with the model group, SMI0 intervention significantly inhibited the growth of mouse glioma tumors, with the high-dose SMI0 group showing the best inhibitory effect, achieving a tumor proliferation inhibition rate of up to 97.6% (e.g., Figure 24 A- Figure 24 As shown in Figure C, *P<0.05 vs. model group, **P<0.01 vs. model group). This suggests that SMI0 treatment can significantly inhibit the occurrence and development of GL261 cell orthotopic implantation in mouse glioma models.

[0310] In addition, in vitro inhibition experiments ( Figure 24 D) showed that SMI0, when applied to GL261 cells at concentrations ranging from 0 to 128 μM for 48 h, had no significant inhibitory effect on GL261 cells.

[0311] These results indicate that cyp2e1 gene knockout can significantly inhibit the occurrence and development of gliomas in mice, and the CYP2E1 inhibitor SMI0 has a significant preventive and therapeutic effect on the occurrence and development of gliomas in mice with GL261 cells implanted in situ in the brain. This suggests that the SMI0 compound may be used for the clinical prevention and treatment of gliomas.

[0312] Example 17: SMI0 inhibits the development and progression of ovarian cancer in mice.

[0313] (1) Ovarian cancer orthotopic xenograft

[0314] Experimental methods: A female C57 / 6 mouse orthotopic xenograft model of ovarian cancer was established using the mouse-derived ID-8 ovarian cancer cell line at a cell concentration of 1 x 10⁻⁶ cells. 6Mice were divided into a model group and an SMI0 intervention group. The intervention group received SMI0 compound 30 mg / kg daily via gavage starting three days before model establishment, continuing until the model ended after 60 days. (Model group: n = 6 mice; SMI0 compound 30 mg / kg: n = 7 mice). At the end of the experiment, blood was collected from the orbital rim, and mice were sacrificed after their weight was recorded. Ovaries and tumor tissues were collected. Ovarian tissue specimens were stained with hematoxylin and eosin (HE) to observe ovarian tumor tissue and tumor development in each group.

[0315] Experimental results: Compared with the model group, SMI0 intervention significantly inhibited the growth of ovarian cancer in mice, with a tumor proliferation inhibition rate as high as 72.51% (e.g., Figure 25 A- Figure 25 As shown in B, *P = 0.014 vs. the model group). This suggests that SMI0 treatment can significantly inhibit the occurrence and development of the ID-8 ovarian cancer orthotopic xenograft model.

[0316] In addition, in vitro inhibition experiments ( Figure 25 C) showed that SMI0, when applied to ID-8 cells at concentrations ranging from 0 to 320 μM for 48 hours, had no significant inhibitory effect on ID-8 cells.

[0317] (2) Ovarian cancer peritoneal xenograft

[0318] Experimental methods: A female C57 / 6 mouse ovarian cancer peritoneal xenograft model was established using the mouse-derived ID8 ovarian cancer cell line at a cell concentration of 2.5 x 10⁻⁶ cells. 6 Mice were divided into a model group and an SMI0 intervention group. The intervention group received SMI0 compound 30 mg / kg via gavage daily for three days prior to model establishment until the model ended on day 30. (Model group: n = 7 mice; SMI0 compound 30 mg / kg: n = 7 mice). At the end of the experiment, blood was collected from the orbital rim, and mice were sacrificed after their weight was recorded. Ovarian tissue was collected. The ovarian tissue specimens were stained with hematoxylin and eosin (HE) to observe ovarian lesions and tumor occurrence in each group of mice.

[0319] Experimental results: Compared with the model group, the mice in the SMI0 compound 30 mg / kg intervention group showed significantly reduced body weight and ascites volume, indicating that SMI0 compound treatment can significantly inhibit ascites formation and tumor progression in mouse ovarian cancer peritoneal xenografts, with an inhibition rate of 52.82% on ascites volume. Figure 26 A- Figure 26 As shown in Figure B, *P<0.05 (vs. model group). This suggests that SMI0 treatment can significantly inhibit the occurrence and development of the ID-8 cell ovarian cancer peritoneal xenograft model.

[0320] The above results indicate that the CYP2E1 inhibitor SMI0 has a significant preventive and therapeutic effect on the occurrence and development of ID-8 ovarian cancer orthotopic xenografts and peritoneal xenografts. This suggests that the SMI0 compound may be used for the prevention and treatment of ovarian cancer.

[0321] Example 18: SMI0 inhibits complete Freund's adjuvant-induced rheumatoid arthritis in rats.

[0322] Experimental Methods: A rheumatoid arthritis model was established in SD rats using complete Freund's adjuvant (CFA, 0.1 mL / rat). The SMI0 intervention group was divided into ultra-low dose, low dose, medium dose, and high dose groups, administered SMI0 compound by gavage daily for two days prior to model establishment (1 mg / kg, 6 mg / kg, 30 mg / kg, and 150 mg / kg, respectively). The positive control group received celecoxib by gavage (5 mg / kg). Ten animals were in each group. Rats were administered CFA according to the above groupings and dosages one day before CFA administration, followed by daily administration until the model ended on day 10. CFA was administered to the right hind paw of each rat, and the volume of the right hind paw was measured daily using a rat paw edema meter (every 12 hours for the first three days). The paw edema rate was calculated based on the change in right hind paw volume relative to before CFA administration at each time point, and the differences in paw edema rates among the groups were analyzed.

[0323] Experimental results: Compared with the control group, the model group rats showed significant paw swelling (e.g., Figure 27 (As shown). Compared with the model group, the SMI0 intervention group significantly alleviated rat paw edema induced by complete Freund's adjuvant (as shown). Figure 27 , Figure 28 A- Figure 28 As shown in D, Figure 28 B shows the paw edema rate of rats at 24 hours, 26C shows the paw edema rate of rats at 36 hours, and 28D shows the paw edema rate of rats 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 SMI0 compound achieved paw edema relief rates of 20.8%, 34.2%, 44.1%, and 52.5% at 36 hours (the time point when paw edema in the model group reached its maximum), respectively. Furthermore, the SMI0 dose showed a good dose-response relationship with the paw edema relief rate in rats (P<0.05). Figure 29 A- Figure 29 As shown in D, Figure 29 B represents the 24-hour dose-response relationship, 29C represents the 36-hour dose-response relationship, and 29D represents the 48-hour dose-response relationship. This suggests that SMI0 can significantly alleviate paw swelling in a rat model of rheumatoid arthritis induced by complete Freund's adjuvant.

[0324] The above results indicate that SMI0 has a significant preventive and therapeutic effect on rheumatoid arthritis induced by complete Freund's adjuvant in rats. This suggests that SMI0 compounds may be used clinically for the prevention and treatment of rheumatic and rheumatoid arthritis.

[0325] Example 19: SMI0 inhibits lipopolysaccharide-induced sepsis in rats and mice.

[0326] Experimental methods:

[0327] Rats: A sepsis model in SD rats was established by a single intraperitoneal injection of lipopolysaccharide (LPS, 5 mg / kg). Thirty minutes before LPS administration, rats in the model group were given physiological saline (0.5 mL / kg, ig), rats in the positive control group were given celecoxib (5 mg / kg, ig), and rats in the CYP2E1 inhibition group were given SMI0 compound (150 mg / kg, ig). Body temperature was measured and recorded hourly after LPS administration until 9 hours (control group, n = 8 rats; model group, n = 10 rats; celecoxib group, n = 10 rats; SMI0 compound group, n = 10 rats).

[0328] Mice: A mouse sepsis model was established by a single intraperitoneal injection of lipopolysaccharide (LPS, 15 mg / kg). Thirty minutes before LPS administration, mice in the model group were given physiological saline (0.5 mL / kg, ig), mice in the positive control group were given celecoxib (5 mg / kg, ig), and mice in the CYP2E1 inhibition group were given SMI0 compound (90 mg / kg, ig). Body temperature was measured and recorded at 6 h, 12 h, and 24 h after LPS administration, for a total of 10 measurements. Animals were sacrificed at 24 h, and blood samples were collected to measure renal and cardiac function indicators (control group, n = 8 mice; model group, n = 8 mice; celecoxib group, n = 8 mice; SMI0 compound group, n = 10 mice).

[0329] Experimental results:

[0330] Rats: Compared with the control group, the body temperature of rats in the model group was significantly elevated at 4h, 5h, and 6h; compared with the model group, the SMI0 compound treatment group and the positive control group could avoid the elevation of body temperature in the LPS-induced rat sepsis model. Figure 30 A- Figure 30 As shown in D, ** 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 intervention can significantly inhibit LPS-induced hyperthermia in rats and maintain normal body temperature.

[0331] Mice: Compared with the control group, the body temperature of mice in the model group was significantly lower at 6h, 12h, and 24h; compared with the model group, mice in the SMI0 compound treatment group avoided the decrease in body temperature induced by LPS in the mouse sepsis model. Compared with the control group, the body temperature of mice in the model group was significantly lower at 6h, 12h, and 24h compared with before LPS administration. Figure 31 A- Figure 31 As shown in D, ** 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. Meanwhile, compared with the control group, the model group mice showed significantly increased levels of renal function indicators (blood urea nitrogen and creatinine) and cardiac function indicators (creatine kinase and lactate dehydrogenase); compared with the model group, the SMI0 compound treatment group mice showed significantly decreased levels of renal and cardiac function-related indicators. Figure 32 A- Figure 32 As shown in D, ** P<0.01, *** P<0.001 vs. control group; &&& P<0.001 vs. model group; # P<0.01, ## P<0.001 (vs. celecoxib group). This suggests that SMI0 intervention can significantly inhibit LPS-induced hypothermia in mice and maintain normal body temperature; it also improves renal and cardiac function in an LPS-induced mouse sepsis model.

[0332] These results indicate that SMI0 can prevent body temperature changes in lipopolysaccharide (LPS)-induced sepsis models in rats and mice. It is speculated that the SMI0 compound can be used for the prevention and treatment of clinical sepsis.

[0333] Example 20: SMI0 improves cognitive impairment in streptozotocin (STZ)-induced Alzheimer's disease in rats.

[0334] Experimental Methods: An Alzheimer's disease (AD) model was established in male SD rats by bilateral ventricular injection of streptozotocin (STZ). STZ was injected on day 1 (3 mg / kg) and on day 3 (1.5 mg / kg). A stereotaxic instrument was used to inject STZ into the bilateral ventricles (0.9 mm posterior to the anterior fontanelle, 1.5 mm to the left and right of the sagittal suture, and 3.8 mm below the skull). The sham-operated group received the same volume of control solution under the same procedure. The low-dose and high-dose prevention groups were administered SMI0 compound at 10 mg / kg and 30 mg / kg by gavage, respectively, starting on day 11 after model establishment, for 21 consecutive days. On day 14 (day 25 of the model), spatial navigation training was conducted using the Morris water maze for 6 consecutive days. Spatial exploration experiments were performed 24 hours after the completion of spatial navigation training (15 rats in each group).

[0335] Experimental Results: During days 1-6 of the Morris water maze navigation experiment, the latency of rats in the model group was significantly prolonged compared to the sham-operated group; during days 2-6, the latency of rats in the high-dose SMI0 group was significantly shortened each day compared to the model group (Table 4 is a quantitative table of relevant parameters of SMI0 compound improving cognitive impairment in streptozotocin-induced rat Alzheimer's disease model, as shown in Table 4 and...). Figure 33 As shown in A, * P<0.05, ** P<0.01, *** P<0.001 vs sham surgery group # P < 0.05 (vs. model group). In the space exploration experiment on day 7, compared with the sham-operated group, the rats in the model group had significantly shorter time spent in the quadrant where the original platform was located and fewer times they crossed the platform. ** P<0.01, *** P<0.001 (vs. sham-operated group); compared with the model group, rats in the high-dose SMI0 group (30 mg / kg) had significantly increased time spent in the quadrant where the original platform was located and the number of times they crossed the platform (as shown in Table 4 and...). Figure 33 B- Figure 33 As shown in C, # P<0.05 (vs. model group); compared with the low-dose SMI0 group, the high-dose SMI0 group showed a significant increase in the number of times rats crossed the platform and the time spent in the quadrant where the original platform was located. & (P<0.05 vs. low-dose group). Regarding overall learning and memory function, compared to the model group, the high-dose SMI0 group significantly improved ICV-STZ-induced cognitive impairment, with an improvement rate of 46.7%; the low-dose SMI0 group showed an improvement trend, but no significant statistical difference, with an improvement rate of 32.6%. This suggests that SMI0 can significantly improve cognitive impairment induced by streptozotocin injection into the lateral ventricle.

[0336] Table 4. Quantification of parameters related to the improvement of cognitive impairment in streptozotocin (STZ)-induced Alzheimer's disease in rats by SMI0 compounds.

[0337]

[0338] These results indicate that SMI0 30 mg / kg significantly improved cognitive impairment in a streptozotocin-induced rat model of Alzheimer's disease. It is speculated that the SMI0 compound may be used for the clinical prevention and treatment of Alzheimer's disease.

[0339] Example 21: SMI0 inhibits focal cerebral ischemia-reperfusion injury in rats.

[0340] Experimental Methods: A rat model of focal cerebral ischemia-reperfusion injury (MCAO) caused by middle carotid artery occlusion was established using a modified Zea-longa suture method. Rats were anesthetized intraperitoneally with 10% chloral hydrate and fixed in a supine position on a temperature-controlled rat board to maintain their body temperature between 36.5℃ and 37.5℃. After disinfection with povidone-iodine, a midline incision was made in the neck to expose the left common carotid artery. The common carotid, external carotid, and internal carotid arteries were separated, and the pterygopalatine artery was ligated. A small incision was made on the side of the external carotid artery, and a nylon suture was inserted through the incision. Using the bifurcation as a marker, the suture was carefully and slowly pushed in when it was about 17 mm in depth. Resistance was felt when it was about 18 ± 0.5 mm in depth, indicating that the tip of the nylon suture had reached the anterior cerebral artery. The incision in the external carotid artery was then ligated, and the skin was sutured. Two hours after ischemia, the rats were anesthetized with inhaled sevoflurane, and the nylon suture was gently and slowly withdrawn until the bulbous end of the nylon suture returned to the bifurcation of the common carotid artery, thus achieving reperfusion. After 2 hours of ischemia and 24 hours of reperfusion, the SMI0 intervention group received SMI0 compound (150 mg / kg) by gavage 10 minutes before reperfusion, while the sham-operated group and model group received an equal volume of control solvent. Neurological and behavioral scores were assessed at 2.5 hours and 6 hours post-operation. At 24 hours post-reperfusion, the rats were decapitated and their brains were removed for triphenyltetrazolium chloride (TTC) staining to observe changes in cerebral infarction volume and cerebral edema (10 rats in each group).

[0341] Experimental results: Compared with the model group, the SMI0 intervention group showed a significant reduction in cerebral infarction volume and cerebral edema volume (e.g., Figure 34 , Figure 35 A- Figure 35 As shown in Figure B (*P<0.05 vs. model group), the infarct volume decreased from 51.03% to 32.62% in the model group, and the cerebral edema volume decreased from 19.25% to 12.63%. SMI0 (150 mg / kg) showed an inhibition rate of 36.06% and 34.39% on infarct and edema volumes, respectively. This suggests that SMI0 can significantly reduce the infarct and edema volumes in rats with focal cerebral ischemia-reperfusion injury caused by middle artery occlusion.

[0342] The above results indicate that SMI0 can significantly improve cerebral infarction and cerebral edema in a rat model of focal cerebral ischemia-reperfusion injury, suggesting that SMI0 compounds may be used for the prevention and treatment of ischemic stroke in clinical practice.

[0343] Example 22: SMI0 reduces lipid levels in ApoE- / - mice with high-fat diet-induced hyperlipidemia.

[0344] Experimental Methods: ApoE- / - mice were used as the research subjects to establish a hyperlipidemia model by feeding them a high-fat, high-cholesterol diet. The SMI0 intervention group was divided into low-dose and high-dose intervention groups, which were administered SMI0 compound at 30 mg / kg and 150 mg / kg daily, respectively, during the modeling period until the model was established at 16 weeks. At the end of the experiment, orbital blood samples were collected, and the serum levels of lipid-related indicators in each mouse (8 mice in each group) were measured using a fully automated biochemical analyzer.

[0345] Experimental results: Compared with the control group, the levels of total cholesterol and low-density lipoprotein in the model group mice were significantly increased (as shown in Table 5). Figure 36 As shown, * P < 0.05 (vs. control group). Compared with the model group, the SMI0 high-dose intervention group showed significantly lower total cholesterol and low-density lipoprotein (LDL), and significantly higher high-density lipoprotein (HDL). @ P<0.05 (vs. model group); compared with the model group, low-dose SMI0 intervention showed a certain improvement effect on high-fat diet-induced hyperlipidemia, but the effect did not reach statistical significance. Meanwhile, SMI0 administration significantly inhibited the formation of atherosclerotic plaques in the aorta of ApoE- / - mice. HE staining results showed that, compared with the model group, the high-dose SMI0 group (150 mg / kg) significantly reduced the total lesion area in the aortic root of mice (e.g., P<0.05 vs. model group). Figure 37 As shown, ** P<0.01 (vs. model group), Oil Red O staining results also showed that the high-dose SMI0 group (150 mg / kg) significantly reduced the total area of ​​aortic root lesions (e.g., P<0.01 vs. model group). Figure 37 As shown, * P<0.05 (vs. model group). This suggests that SMI0 150mg / kg significantly improved high-fat diet-induced hyperlipidemia in mice (reducing total cholesterol and LDL cholesterol, and increasing HDL cholesterol), while inhibiting the formation of atherosclerotic plaques in the aorta of ApoE- / - mice.

[0346] Table 5. Quantification of lipid-related parameters in a mouse model of hyperlipidemia induced by a high-fat diet using SMI0 compounds.

[0347]

[0348] These results indicate that SMI0 can significantly reduce the increase in blood lipid levels and atherosclerotic plaque formation in mice induced by a high-fat diet. It is speculated that the SMI0 compound may be used in the clinical prevention and treatment of hyperlipidemia, atherosclerosis, and coronary heart disease.

[0349] Example 23: SMI0 reduces blood glucose levels in rats with diabetes induced by a high-fat diet and streptozotocin.

[0350] Experimental Methods: A rat model of diabetes was established using a high-fat diet combined with a single intraperitoneal injection of streptozotocin (STZ, 40 mg / kg). Two weeks after modeling, a fasting blood glucose (FBG) level ≥16.7 mmol / L was used as the criterion for successful modeling. Rats with successful modeling were randomly divided into groups based on their blood glucose levels. The model group received saline, while the low-dose and high-dose SMI0 intervention groups received 30 mg / kg and 150 mg / kg of SMI0 compound daily, respectively. FBG was measured and recorded weekly until the sixth week after administration, with 10 rats in each group.

[0351] Experimental results: Compared with the model group, the fasting blood glucose levels of rats in the low- and high-dose SMI0 intervention groups were significantly lower in weeks 5 and 6. * P<0.05 vs model group) Figure 38 As shown in Figure A). The glucose tolerance test results showed that both low- and high-dose SMI0 intervention groups significantly improved glucose tolerance in rats. Figure 38 B- Figure 38 (As shown in C). This suggests that SMI0 intervention can significantly improve hyperglycemia levels in rats induced by a high-fat diet and streptozotocin.

[0352] The above results indicate that SMI0 can significantly reduce blood glucose levels in diabetic rats induced by a high-fat diet and streptozotocin, suggesting that SMI0 compounds may be used for the prevention and treatment of clinical diabetes.

[0353] Example 24: Increased expression of CYP2E1 in adjacent tissues of bladder cancer

[0354] Experimental methods: Using 30 healthy individuals as controls, the changes in CYP2E1 expression in the adjacent tissues of 30 clinical bladder cancer patients were studied, and the changes in CYP2E1 content in the adjacent tissues of bladder cancer patients were compared.

[0355] Experimental results: Immunohistochemical results showed that the expression of CYP2E1 in adjacent tissues of bladder cancer was significantly higher than that in normal bladder tissue (P<0.05). Figure 39As shown. Based on the significantly increased expression of CYP2E1 in liver cancer and glioma, the role of CYP2E1 in inflammation-related tumors, and the fact that SMI0 compounds can significantly inhibit the occurrence and development of liver cancer and glioma, it is speculated that the increased CYP2E1 expression in adjacent tissues of bladder cancer is related to the occurrence of bladder cancer, and that SMI0 compounds may have a preventive and therapeutic effect on the occurrence and development of bladder cancer. SMI0 compounds can be used for the clinical prevention and treatment of bladder cancer.

[0356] Example 25: Increased expression of CYP2E1 in adjacent tissues of gallbladder cancer.

[0357] Experimental methods: Using 31 healthy controls, the changes in CYP2E1 expression in adjacent tissues of 33 patients with gallbladder cancer were studied.

[0358] Experimental results: Immunohistochemical results showed that CYP2E1 expression in adjacent tissues of gallbladder cancer was significantly higher than that in normal gallbladder tissue (P<0.05). Figure 40 As shown. Based on the significantly increased expression of CYP2E1 in liver cancer and glioma, the role of CYP2E1 in inflammation-related tumors, and the fact that SMI0 compounds can significantly inhibit the occurrence and development of liver cancer and glioma, it is speculated that the increased CYP2E1 expression in adjacent tissues of gallbladder cancer is related to the occurrence of gallbladder cancer, and that SMI0 compounds may have a preventive and therapeutic effect on the occurrence and development of gallbladder cancer. SMI0 compounds can be used for the clinical prevention and treatment of gallbladder cancer.

[0359] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. The use of a compound or a salt thereof as a CYP2E1 inhibitor in the preparation of a medicament for treating or preventing inflammation-related tumors, characterized in that, Compounds selected from formula (I) or their salts are used as inhibitors to target and bind to CYP2E1, thereby inhibiting CYP2E1. The inflammation-related tumor is selected from one of the following: liver cancer, glioma, ovarian cancer, bladder cancer, and gallbladder cancer.

2. The application according to claim 1, characterized in that, The compound represented by formula (I) reacts with an acid to give the corresponding acid salt of the compound represented by formula (I); The acid is selected from at least one of inorganic acids and organic acids.

3. The application according to claim 2, characterized in that, The inorganic acid is selected from at least one of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; The organic acid is selected from at least one of acetic acid, oxalic acid, succinic acid, tartaric acid, succinic acid, malic acid, lactic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, resinic acid, maleic acid, fumaric acid, salicylic acid, and acetylsalicylic acid.

4. The application according to claim 2, characterized in that, The X-ray powder diffraction pattern of crystal form A of the hydrochloride salt of the compound shown in formula (I) includes three or more 2θ values ​​selected from the following group: 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°, 35.7±0.2°; The DSC-TGA plot of the hydrochloride salt of the compound shown in formula (I) shows that it has a distinct endothermic peak between 70 °C and 220 °C, and thermally decomposes between 80 °C and 170 °C.

5. The application according to claim 2, characterized in that, The X-ray powder diffraction of the sulfate form B of the compound shown in 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°. The DSC-TGA plot of the sulfate form B of the compound shown in formula (I) shows that it has at least one endothermic peak between 30℃~85℃, 90℃~160℃, and 215℃~330℃, and thermally decomposes at 150℃~350℃.

6. The application according to claim 1, characterized in that, The synthetic route for the compound shown in formula (I) is as follows: Among them, the reaction conditions are: a) hydrolysis and acidification under alkaline conditions; b reacts with N,O-dimethylhydroxylamine hydrochloride in the presence of a base and a condensing agent; c reacts with Grignard reagents in anhydrous aprotic solvents under low-temperature conditions.

7. The application according to claim 6, characterized in that, The base mentioned in condition a can be one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, etc., and the acid can be one or a mixture of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, etc. The solvent used is a mixture of one of water-soluble solvents such as methanol, ethanol, propanol and water, and the reaction temperature is from 10 degrees to 50 degrees.

8. The application according to claim 6, characterized in that, The base mentioned in condition b can be one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, pyridine, triethylamine, N,N-diisopropylethylamine, etc., and the condensing agent can be one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dilaurate carbonate, N,N-carbonyldiimidazole, and N-(4-carboxyphenyl)maleimide, etc., the reaction temperature is from 20 degrees to 60 degrees, and the solvent used is one of aprotic solvents such as tetrahydrofuran and diethyl ether.

9. The application according to claim 6, characterized in that, The reaction temperature described in condition c ranges from -20 degrees to 25 degrees, and the solvent used is one of the aprotic solvents such as tetrahydrofuran and diethyl ether. The equivalence ratio of compound 3 to Grignard reagent is 1:1 to 1:

3. Preferably, the Grignard reagent is selected from at least one of methyl magnesium bromide and methyl magnesium chloride.

10. The application according to claim 2, characterized in that, The method for preparing the acid salt of the compound shown in formula (I) includes at least the following: By reacting the material containing the compound shown in formula (I) and solvent A at -20 to 80°C for 0.5 to 10 hours, the corresponding acid salt of the compound shown in formula (I) can be obtained. Preferably, solvent A is selected from at least one of ether compounds, alcohol compounds, ester compounds, nitrile compounds, ketone compounds, haloalkanes, alkanes, and aromatics.

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