Use of a CYP2E1 inhibitor in the preparation of a drug for treating or preventing inflammation-related diseases

Abstract

The present application discloses the use of a CYP2E1 inhibitor in the preparation of a drug for treating or preventing an inflammation-related disease, wherein the inflammation-related disease is selected from lung cancer or pulmonary fibrosis; a compound or a salt thereof selected from formula (I) is used as an inhibitor to target CYP2E1 and bind thereto, thereby having a good preventive / therapeutic effect on lung cancer or pulmonary fibrosis.

Description

Technical Field

[0001] The present application relates to the use of a CYP2E1 inhibitor in the preparation of a drug for treating or preventing inflammation-related diseases, and belongs to the field of pharmaceutical technology. Background Art

[0002] It is now generally recognized that the occurrence of many diseases, such as lung cancer and pulmonary fibrosis, is related to inflammation, which is called inflammation-mediated disease (IMD). Cytochrome P450 2E1 (CYP2E1) is a protein mainly present in the endoplasmic reticulum of liver cells. The applicant found that its content in liver cytochrome P450 (CYP450) accounts for the highest proportion of about 24.8%. The metabolic activation of CYP2E1 is closely related to inflammation-related diseases such as tumors. Animal experiments have found that CYP2E1 gene knockout mice can significantly inhibit diethylnitrosamine-induced mouse liver cancer. This suggests that CYP2E1 may affect the occurrence of liver cancer by affecting the metabolic activation of nitrosamines in the body. CYP2E1 has obvious inflammatory effects and is involved in the occurrence and development of many inflammation-related diseases. CYP2E1 is associated with inflammation-related tumors such as liver cancer, glioma, ovarian cancer, lung cancer, nasopharyngeal carcinoma, bladder cancer, and gallbladder cancer. CYP2E1 is also implicated in the development and progression of liver diseases such as liver injury, non-alcoholic steatohepatitis (NASH), and liver fibrosis, as well as other inflammation-related diseases such as rheumatoid arthritis, sepsis, Alzheimer's disease, hyperlipidemia, diabetes, ischemic stroke, and pulmonary fibrosis. The proinflammatory 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 ligand, a cell surface molecule of the tumor necrosis factor family, triggering a protease cascade that leads to cell lysis and apoptosis. Apoptotic hepatocytes can promote the aggregation of inflammatory cells, induce the production of TNF-α, IL-6 and other inflammatory factors, and cause liver inflammation and fatty liver disease.

[0003] Currently, there are no specific CYP2E1 inhibitors for clinical use. The development of a new CYP2E1 inhibitor for use in the prevention of inflammatory-related diseases, especially the treatment or prevention of lung cancer and pulmonary fibrosis, is of great significance. Summary of the Invention

[0004] On the one hand, the present invention discloses the use of a CYP2E1 inhibitor in the preparation of a drug for treating or preventing an inflammation-related disease, wherein the inflammation-related disease is selected from lung cancer or pulmonary fibrosis. The compound represented by formula (I) or a salt thereof is selected as an inhibitor, which targets CYP2E1 and binds thereto, and has a good preventive / therapeutic effect on lung cancer or pulmonary fibrosis.

[0005] Use of a CYP2E1 inhibitor in the preparation of a medicament for treating or preventing an inflammation-related disease, wherein the inflammation-related disease is selected from lung cancer or pulmonary fibrosis;

[0006] The CYP2E1 inhibitor is selected from at least one compound represented by formula (I) or a salt thereof;

[0007]

[0008] wherein R1 is selected from hydrogen, C1-C 10 Any of alkyl and epoxyalkyl;

[0009] R2 is selected from hydrogen, substituted C1-C 10 Any one of the alkyl group I, the substituent represented by formula O-1, the substituent represented by formula O-2, and the substituent represented by formula O-3;

[0010]

[0011] 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 aromatic groups;

[0012] R 25 Any one selected from hydroxyl group and alkoxy group;

[0013] R3 is selected from hydrogen, C1-C 10 Alkyl, substituted C1-C 10 At least one of the alkyl groups II.

[0014] Optionally, the alkylene oxide group is selected from butylene oxide groups.

[0015] Optionally, R1 is selected from any one of hydrogen, C1-C4 alkyl, and alkylene oxide.

[0016] Optionally, R3 is selected from at least one of hydrogen, C1-C4 alkyl, and substituted C1-C4 alkyl II.

[0017] Alternatively, the compound represented by formula (I) reacts with an acid to obtain an acid salt of the compound represented by formula (I);

[0018] The acid is selected from at least one of an inorganic acid and an organic acid.

[0019] Optionally, the inorganic acid is selected from at least one of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid;

[0020] 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, resin acid, maleic acid, fumaric acid, salicylic acid, and acetylsalicylic acid.

[0021] Optionally, the compound represented by formula I is used as a CYP2E1 inhibitor.

[0022] Optionally, the substituted C1-C 10 The substituent in the alkyl group I is selected from at least one of a substituted amino group I, a substituent represented by formula M-1, and a substituent represented by formula M-2;

[0023]

[0024] Optionally, the substituent in the substituted amino group I is selected from C6-C 10 At least one of an aryl group, a substituted C1-C3 alkyl group III.

[0025] Preferably, the substituent in the substituted C1-C3 alkyl group III is selected from C6-C 10 of aromatic groups.

[0026] Optionally, the substituted C1-C 10 The substituent in the alkyl group II is selected from at least one of a substituted amino group II, a substituent represented by formula M-3, and a substituent represented by formula M-4;

[0027]

[0028] Optionally, the substituent in the substituted amino group II is selected from substituted C1-C3 alkyl groups IV;

[0029] The substituent in the substituted C1-C3 alkyl IV is selected from at least one of pyridyl and halogen.

[0030] Optionally, the inhibitor is selected from the compounds represented by formula (I) or pharmaceutically acceptable salts thereof, wherein:

[0031]

[0032] wherein R1 is selected from hydrogen, C1-C 10 At least one of an alkyl group or an epoxyalkyl group;

[0033] R2 is selected from hydrogen, substituted C1-C 10 At least one of an alkyl group I, a substituted carbonyl group, and a substituted imino group;

[0034] R3 is selected from hydrogen, C1-C 10 Alkyl, substituted C1-C 10 At least one of the alkyl groups II.

[0035] Optionally, the substituted C1-C 10 The substituent in the alkyl group I is selected from at least one of halogen, substituted amino group I, a substituent represented by formula M-1, and a substituent represented by formula M-2;

[0036]

[0037] The substituents in the substituted carbonyl group are selected from substituted C1-C 10 alkenyl;

[0038] The substituent in the substituted imino group is selected from at least one of a hydroxyl group and a C1-C3 alkoxy group.

[0039] Optionally, the substituent in the substituted amino group I is selected from C1-C 10 At least one of an aryl group, a substituted C1-C3 alkyl group III;

[0040] Preferably, the substituent in the substituted C1-C3 alkyl group III is selected from C1-C 10 of aromatic groups.

[0041] Optionally, the substituted C1-C 10 The substituents in the alkenyl group are selected from C1-C 10 Aryl, substituted C1-C 10 At least one of the aromatic groups;

[0042] Preferably, the substituted C1-C 10 The substituents in the aryl group are selected from at least one of a C1-C3 alkoxy group, a halogen group, and a C1-C3 alkyl group.

[0043] Optionally, the substituted C1-C 10 The substituent in the alkyl group II is selected from at least one of a substituted amino group II, a substituent represented by formula M-3, and a substituent represented by formula M-4;

[0044]

[0045] Optionally, the substituent in the substituted amino group II is selected from substituted C1-C3 alkyl groups IV;

[0046] The substituent in the substituted C1-C3 alkyl IV is selected from at least one of pyridyl and halogen.

[0047] Optionally, the CYP2E1 inhibitor is selected from at least one of the following compounds;

[0048]

[0049] Optionally, the CYP2E1 inhibitor is selected from at least one of the following compounds as an inhibitor that targets CYP2E1 and binds to it, thereby inhibiting CYP2E1;

[0050]

[0051] Preferably, the CYP2E1 inhibitor is selected from at least one of the following compounds as an inhibitor that targets CYP2E1 and binds to it, thereby inhibiting CYP2E1;

[0052]

[0053] Alternatively, the structural formula of the compound represented by formula (I) is

[0054] The X-ray powder diffraction pattern of Form A of the hydrochloride salt of the compound represented by formula (I) includes three or more 2θ values selected from the group consisting of 8.4±0.2°, 13.1±0.2°, 14.8±0.2°, 16.6±0.2°, 24.1±0.2°, 27.2±0.2°, 30.5±0.2°, 31.8±0.2°, 33.5±0.2°, 35.4±0.2°, and 35.7±0.2°;

[0055] The DSC-TGA chart of the crystalline form A of the hydrochloride salt of the compound represented by formula (I) shows that it has an obvious endothermic peak between 70°C and 220°C and thermally decomposes at 80°C to 170°C.

[0056] Alternatively, the structural formula of the compound represented by formula (I) is

[0057]

[0058] The X-ray powder diffraction pattern of Form B of the sulfate salt of the compound represented by formula (I) includes 5 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°;

[0059] The DSC-TGA diagram of the crystalline form B of the sulfate salt of the compound represented by formula (I) shows that it has at least one endothermic peak between 30°C and 85°C, 90°C and 160°C, and 215°C and 330°C, and thermally decomposes at 150°C to 350°C.

[0060] Optionally, the method for preparing the compound represented by formula (I) as an inhibitor includes at least any one of the following methods;

[0061] Method 1: React a raw material containing compound A, an aprotic solvent, and a Grignard reagent at -20 to 25°C for 0.5 to 3 hours to obtain CYP2E1 inhibitor A;

[0062] The compound A is selected from at least one compound having the structural formula shown in Formula II:

[0063]

[0064] The CYP2E1 inhibitor A is selected from at least one compound having the structural formula shown in Formula III:

[0065]

[0066] Method 2: reacting a compound having the structure represented by formula III with ammonia in the presence of an alkaline source I to obtain a CYP2E1 inhibitor B;

[0067] The CYP2E1 inhibitor B is selected from at least one compound having the structural formula shown in Formula III-1:

[0068]

[0069] Method 3: reacting a compound having the structure represented by formula III with an aromatic aldehyde compound in the presence of an alkaline source II at 25-100° C. for 2-8 hours to obtain a CYP2E1 inhibitor C;

[0070] The CYP2E1 inhibitor C is selected from at least one compound having the structural formula shown in Formula III-2:

[0071]

[0072] Method 4: reacting a compound having the structure represented by Formula III, an amine compound and a reducing agent in the presence of an acid source to obtain a CYP2E1 inhibitor D;

[0073] The amine compound is selected from at least one of aniline and benzylamine;

[0074] The CYP2E1 inhibitor D is selected from at least one compound having the structural formula shown in Formula III-3:

[0075]

[0076] Optionally, the second method comprises at least the following steps: reacting a compound having the structural formula represented by Formula III, ethanol and aqueous ammonia in the presence of an alkaline source I to obtain the CYP2E1 inhibitor B.

[0077] Optionally, in the 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 methylmagnesium bromide and methylmagnesium chloride;

[0078] In the second method, the alkaline source I is selected from at least one of potassium hydroxide, sodium hydroxide, sodium carbonate, pyridine, triethylamine, and N,N-diisopropylethylamine;

[0079] In the method three, the alkaline source II is selected from at least one of sodium hydroxide, potassium hydroxide, potassium tert-butoxide, sodium methoxide, and potassium fluoride;

[0080] The aromatic aldehyde compound is selected from at least one of p-anisaldehyde, para-anisaldehyde, m-anisaldehyde, p-chlorobenzaldehyde, para-chlorobenzaldehyde, m-chlorobenzaldehyde, p-phenylbenzaldehyde, p-isopropylbenzaldehyde, and 3,4-difluorobenzaldehyde;

[0081] In the fourth method, the acid source is selected from at least one of formic acid, acetic acid, and hydrochloric acid;

[0082] The reducing agent is selected from at least one of sodium cyanoborohydride, sodium borohydride, and lithium aluminum hydride.

[0083] Optionally, in the method 1, the molar ratio of the compound A to the Grignard reagent is 1:1 to 1:3;

[0084] In the second method, the molar ratio of the compound of formula III to aqueous ammonia is 1:1 to 1:6;

[0085] In the third method, the molar ratio of the compound of formula III to the aromatic aldehyde compound is 1:1 to 1:5;

[0086] In the fourth method, the molar ratio of the compound of the structural formula represented by Formula III to the reducing agent is 1:1 to 1:5.

[0087] Optionally, the compound A is obtained by the following method:

[0088] The raw materials containing compound A-1, a condensing agent, N,O-dimethylhydroxylamine hydrochloride and an aprotic solvent are reacted in the presence of an alkali source III at 20-60° C. for 10-20 hours to obtain the compound A.

[0089] The compound A-1 is selected from at least one compound having the structural formula shown in formula II-1:

[0090]

[0091] Optionally, the condensing agent is selected from at least one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dilauryl carbonate, N,N-carbonyldiimidazole, dicyclohexylcarbodiimide and N-(4-carboxyphenyl)maleimide;

[0092] The alkaline 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.

[0093] Optionally, the molar ratio of compound A-1, the condensing agent, and N,O-dimethylhydroxylamine hydrochloride is 1:1:1 to 1:5:5.

[0094] Optionally, the obtaining of the compound A-1 comprises at least the following steps:

[0095] The raw material containing compound A-2 is hydrolyzed in the presence of an alkali source IV to obtain a mixture, and then an acid source is used to adjust the pH of the mixture to 2 to 3 to obtain the compound A-1; the compound A-2 is selected from at least one compound having the structural formula shown in formula II-2:

[0096]

[0097] Optionally, the alkaline source IV is selected from at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and potassium carbonate.

[0098] Optionally, the acid source is selected from concentrated hydrochloric acid;

[0099] The step of adjusting the pH of the mixture to 2-3 using an acid source is performed at 10-50°C.

[0100] Optionally, the preparation method of the acid salt of the compound represented by formula (I) comprises at least:

[0101] The material containing the compound represented by formula (I) and solvent A is reacted at -20 to 80° C. for 0.5 to 10 hours to obtain the acid salt of the compound represented by formula (I);

[0102] Preferably, the method for preparing the acid salt of the compound represented by formula (I) comprises at least:

[0103] The material containing the compound represented by formula (I) and solvent A is reacted at -15 to 60° C. for 1 to 4 hours to obtain the acid salt of the compound represented by formula (I).

[0104] Further preferably, the method for preparing the acid salt of the compound represented by formula (I) at least comprises:

[0105] The material containing the compound represented by formula (I) and solvent A is reacted at -10 to 40° C. for 1 to 4 hours to obtain the acid salt of the compound represented by formula (I).

[0106] 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.

[0107] Optionally, the organic acid is selected from one of acetic acid, oxalic acid, succinic acid, tartaric acid, succinic acid, malic acid, lactic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, resin acid, maleic acid, fumaric acid, salicylic acid or acetylsalicylic acid.

[0108] Optionally, the inorganic acid is selected from one of hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid or phosphoric acid.

[0109] The present invention also provides a method for preparing an acid salt of the CYP2E1 inhibitor SMI0, comprising the following steps: separately preparing a solution system of SMI0 and an acid in a soluble solvent, mixing the two systems at a molar ratio of SMI0 to the acid in the acidic solution of 1:0.5-3, reacting at a temperature of -20-80°C for 0.5-10 hours to generate a target product, and then drying the solvent under reduced pressure to obtain the SMI0 acid salt.

[0110] Optionally, the organic solvent used in the reaction includes a mixture of one or more of ethers, alcohols, esters, nitriles, ketones, halogenated alkanes, alkanes or aromatic hydrocarbons in any proportion. Preferably, the soluble solvent is methanol, acetonitrile, acetone, ethyl acetate, ethanol or diethyl ether.

[0111] Optionally, in the reaction, the molar ratio of SMIO to the acid in the acidic solution is 1:0.5-3, preferably 1:1-2.

[0112] Optionally, the reaction temperature varies with the reagents or solvents, but is generally -20 to 80° C., preferably -15 to 60° C., and more preferably -10 to 40° C. The reaction time also varies with the reagents or temperature, and is generally 0.5 to 10 hours, preferably 1 to 4 hours.

[0113] The present invention also provides a crystalline Form A of the hydrochloride salt of the CYP2E1 inhibitor SMI0, wherein the X-ray powder diffraction pattern of the crystalline Form A comprises 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°. Furthermore, a DSC-TGA pattern shows a distinct endothermic peak between 70°C and 220°C, and thermal decomposition between 80°C and 170°C.

[0114] The present invention also provides a crystalline Form B of the sulfate salt of the CYP2E1 inhibitor SMI0, wherein the X-ray powder diffraction pattern of the crystalline Form B comprises 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°. Furthermore, a DSC-TGA pattern shows at least one endothermic peak between 30°C and 85°C, 90°C and 160°C, and 215°C and 330°C, and thermal decomposition between 150°C and 350°C.

[0115] The preferred CYP2E1 inhibitors of the present invention are shown in the following table:

[0116]

[0117] Optionally, the CYP2E1 inhibitor SMI0 in this application SMI7

[0118] SMI16 and SMI20 The synthetic method has Figure 31 The synthetic route shown:

[0119] Among them, the reaction conditions are: a. hydrolysis under alkaline conditions and acidification;

[0120] b. reacting with N,O-dimethylhydroxylamine hydrochloride in the presence of a base and a condensing agent;

[0121] c. React with Grignard reagent in anhydrous aprotic solvent at low temperature;

[0122] d. Under alkaline conditions, heating can cause aldol condensation reaction with aromatic aldehydes;

[0123] e reacts with ammonia under alkaline conditions;

[0124] f Reacts with amines in weak acid conditions and with reducing agents.

[0125] Optionally, the base described in condition a can be one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, etc., the acid can be one of hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, etc. or a mixture thereof, the solvent used is a mixture of one of a water-soluble solvent such as methanol, ethanol, propanol, etc. and water, and the reaction temperature is from 10 to 50 degrees;

[0126] Optionally, the base described in condition b can be one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, pyridine, triethylamine, N,N-diisopropylethylamine, etc., the condensing agent can be one of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dilauryl 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.

[0127] Optionally, the reaction temperature in condition c is from -20 degrees to 25 degrees, the solvent used is one of aprotic solvents such as tetrahydrofuran and diethyl ether, and the equivalent ratio of compound 3 to Grignard reagent is 1:1 to 1:3.

[0128] Optionally, the base described 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, and propanol.

[0129] Optionally, the base in condition e can be one of potassium hydroxide, sodium hydroxide, sodium carbonate, pyridine, triethylamine, N,N-diisopropylethylamine, etc., and the solvent is a mixture of one of water-soluble solvents such as methanol and ethanol and water.

[0130] Optionally, the acid described 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.

[0131] In this application, C1-C 10 The carbon number of the "substituted alkyl" and "substituted aryl" refers to the number of carbon atoms contained in the alkyl and aryl groups, not the number of carbon atoms after substitution. 10 The 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.

[0132] In this application, "alkyl" refers to a group formed by losing any hydrogen atom from an alkane compound molecule. The alkane compound includes straight-chain alkanes, branched-chain alkanes, cycloalkanes, and branched cycloalkanes.

[0133] In the present application, "alkenyl" is a group formed by losing any hydrogen atom from an alkene compound molecule. The alkane compound includes straight-chain alkanes, branched-chain alkanes, cycloalkanes, and branched cycloalkanes.

[0134] In the present application, "aryl" is a group formed by losing a hydrogen atom on the aromatic ring of an aromatic compound molecule; for example, toluene loses the hydrogen atom at the para position of the methyl group on the benzene ring to form p-tolyl.

[0135] In the present application, "furanyl" refers to a group formed by losing any hydrogen atom from a furan compound molecule.

[0136] In the present application, an "imino group" is a divalent group remaining after removing two hydrogen atoms from an ammonia molecule, and has the structural formula HN=.

[0137] In the present application, "pyridyl" refers to a group formed by losing any hydrogen atom from a pyridine compound molecule.

[0138] In the present application, "epoxyalkyl" refers to a group formed by losing any hydrogen atom from an epoxy compound molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1 is the H NMR spectrum of compound SMI0;

[0140] Figure 2 This is the H NMR spectrum of compound SMI7;

[0141] Figure 3 This is the H NMR spectrum of compound SMI16;

[0142] Figure 4 This is the H NMR spectrum of compound SMI20;

[0143] Figure 5 is the X-ray powder diffraction pattern (XRPD) of SMIO hydrochloride Form A;

[0144] Figure 6 is the differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) of SMIO hydrochloride Form A;

[0145] Figure 7 is the X-ray powder diffraction pattern (XRPD) of SMIO sulfate Form B;

[0146] Figure 8is the differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) of SMI0 sulfate salt Form B;

[0147] Figure 9 is a double reciprocal plot of the inhibitory effect of compound SMI0 on human liver metabolism of chlorzoxazone;

[0148] Figure 10 It is a secondary plot based on double reciprocal plotting of the inhibitory effect of compound SMI0 on the metabolism of chlorzoxazone by human liver CYP2E1;

[0149] Figure 11 It is a plot of the inhibitory effects of 21 small molecule compounds on the in vitro metabolic activity of CYP2E1;

[0150] Figure 12 The inhibitory effect of compound SMI0 on the metabolism of diethylnitrosamine by rat CYP2E1 is plotted.

[0151] Figure 13 The increased CYP2E1 content in adjacent lung tissues of lung cancer patients was plotted.

[0152] Figure 14 The figure shows the inhibitory effect of cyp2e1 gene knockout on the lung cancer model of mice orthotopically implanted with Lewis lung cancer cells; A, gross image of lung tumors in each mouse; B, weight of lung tumors in each mouse.

[0153] Figure 15 The graph shows the inhibitory effect of compound SMI0 on the lung cancer model of mice orthotopically implanted with Lewis lung cancer cells; wherein, A, representative images of tumor-bearing lung tissues of mice in each group; B, gross images of lung tumors of each mouse; C, weight of lung tumors of each mouse.

[0154] Figure 16 The graph shows the inhibitory effect of compound SMI0 on the lung metastasis model of colorectal cancer cell CT26 injected into the tail vein; wherein, A, the gross image of the tumor-bearing lung tissue of each mouse; B, the lung weight of each mouse.

[0155] Figure 17 The graph shows the inhibitory effect of compound SMI0 on the lung metastasis cancer model of melanoma cells B16-F10 injected into the tail vein; wherein, A, the general picture of the tumor-bearing lung tissue of each mouse; B, the lung weight of each mouse.

[0156] Figure 18 Figure 3. Changes in CYP2E1 activity in a mouse lung cancer model in which Lewis cells were orthotopically implanted in the lungs and their correlation with tumor severity. A: Changes in CYP2E1 activity in each group of mice. B: Correlation between CYP2E1 activity in each mouse and lung tumor weight.

[0157] Figure 19The compound SMI0 inhibits the inflammatory microenvironment of the adjacent lung tissue in a mouse lung cancer model in which Lewis lung cancer cells are orthotopically implanted in the lung; A, Western Blot graph of the expression of inflammatory factors and related signaling pathway proteins in the adjacent lung tissue of each group of mice; B, quantitative mapping of the expression of inflammatory factors and related signaling pathway proteins in the adjacent lung tissue of each group of mice.

[0158] Figure 20 The effects of compound SMI0 on apoptosis and autophagy-related proteins in cancer tissue of a mouse lung cancer model in which Lewis cells were orthotopically implanted in the lungs are plotted; A, Western Blot graph of apoptosis and autophagy-related protein expression in lung cancer tissue of each group of mice; B, quantitative plot of apoptosis and autophagy-related protein expression in lung cancer tissue of each group of mice.

[0159] Figure 21 The graph shows that compound SMI0 has no direct inhibitory effect on lung cancer cells; A, Lewis lung cancer cells; B, A549 lung cancer cells.

[0160] Figure 22 The graph shows the effect of compound SMI0 on inhibiting the proliferation of A549 lung cancer cells by inhibiting the M2 polarization of macrophages.

[0161] Figure 23 Figure 3 is a graph showing the inhibitory effect of cyp2e1 gene knockout on the lipopolysaccharide-induced mouse pulmonary fibrosis model; A, HE and Masson staining of lung tissues of mice in each group; B, percentage of collagen-positive area and histological score of lung tissues of mice in each group.

[0162] Figure 24 Figure 3 is a graph showing the inhibitory effect of compound SMI0 on lipopolysaccharide-induced lung injury in mice; A, HE staining of lung tissues of mice in each group; B, lung injury scores of mice in each group.

[0163] Figure 25 Figure 3 is a graph showing the inhibitory effect of compound SMI0 on the mouse pulmonary fibrosis model induced by lipopolysaccharide; A, HE and Masson staining of lung tissues of mice in each group; B, percentage of collagen-positive area and histological score of lung tissues of mice in each group.

[0164] Figure 26 The figure shows the changes in CYP2E1 activity in the mouse pulmonary fibrosis model induced by lipopolysaccharide and the correlation with the severity of pulmonary fibrosis; A, the changes in CYP2E1 activity in each group of mice; B, the correlation between CYP2E1 activity in each mouse and the lung index.

[0165] Figure 27Figure 3 is a graph showing the inhibitory effect of compound SMI0 on the myeloperoxidase (MPO) level in the lung tissue of mice with lipopolysaccharide-induced pulmonary fibrosis; A, MPO staining of lung tissue of mice in each group; B, percentage of MPO-positive area in lung tissue of mice in each group.

[0166] Figure 28 The graph shows the inhibitory effect of compound SMI0 on inflammatory factors in the lung tissue of mice with pulmonary fibrosis induced by lipopolysaccharide; A, relative expression levels of TNF-α in the lung tissue of mice in each group; B, relative expression levels of IL-1β in the lung tissue of mice in each group.

[0167] Figure 29 Figure 3. The inhibitory effect of compound SMI0 on oxidative stress in lung tissue and the expression of epithelial cell marker E-cadherin and apoptosis-related proteins in the mouse pulmonary fibrosis model induced by lipopolysaccharide; A, CAT level, an indicator of oxidative stress, in lung tissue of each group of mice; B, C Western Blot graphs showing the expression of epithelial cell marker E-cadherin and apoptosis-related proteins in lung tissue of each group of mice; D, E, F Quantitative graphs showing the expression of epithelial cell marker E-cadherin and apoptosis-related proteins in lung tissue of each group of mice.

[0168] Figure 30 Figure 3 is a graph showing the inhibitory effect of compound SMI0 on the expression of TGF-β1 and α-SMA in lung tissue of a mouse pulmonary fibrosis model induced by lipopolysaccharide; A, C are staining images of TGF-β1 and α-SMA in lung tissue of mice in each group; B, D are the percentages of positive areas for TGF-β1 and α-SMA staining in each group.

[0169] Figure 31 It is the synthetic route of SMI0, SMI7, SMI16 and SMI20. DETAILED DESCRIPTION

[0170] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0171] The high-resolution mass spectrometer used in the synthesis of the CYP2E1 inhibitors in Examples 2, 3, 4 and 5 of the present application was a Q-Tof micro mass spectrometer from Waters, USA.

[0172] The synthesis of the CYP2E1 inhibitors in Examples 2, 3, 4 and 5 of the present application was performed using a nuclear magnetic resonance spectrometer (DPX-400, Bruker, Germany).

[0173] Example 10 of the present application uses a high performance liquid chromatograph, Agilent Technologies, model Agilent 1260.

[0174] Example 1 Preparation of human liver microsomes

[0175] Liver specimens were thawed and weighed using differential centrifugation. 50 mM Tris-HCl (pH 7.0) (containing 150 mM KCl and 2 mM EDTA) was added at a 1:4 (w / v) ratio and the liver was homogenized using a glass homogenizer. The mixture was centrifuged at 9000 × g for 20 min at 4°C. The supernatant was centrifuged at 100,000 × g for 60 min at 4°C. The resulting pellet was resuspended in 4 mL of 0.15 M Tris-HCl (pH 7.6) and centrifuged again at 100,000 × g for 60 min at 4°C. The pellet was then resuspended in 0.25 M sucrose at a 1:2 (w / v) ratio to prepare 2 mL of microsomal suspension per gram of liver tissue. Aliquots were stored in liquid nitrogen overnight and then transferred to -80°C the next day for long-term storage. All procedures were performed in an ice bath. Microsomal protein content (mg / mL) was determined using the Bradford method.

[0176] This method was used for liver microsomes from patients with liver damage and normal subjects.

[0177] Example 2 Synthesis of SMI0

[0178] Ethyl 4-methylthiazole-5-carboxylate (1 mol) and NaOH (1.6 mol), using a mixture of ethanol and water as the solvent, were reacted at room temperature overnight and monitored by TLC (pure ethyl acetate). After completion, the ethanol was evaporated to dryness under reduced pressure, the pH was adjusted to 2-3 with concentrated sulfuric acid, and the solid was filtered, washed, and dried. 4-Methylthiazole-5-carboxylic acid (1 mol) and DDC (1 mol) were stirred in anhydrous tetrahydrofuran at room temperature. After activation for 2-3 hours, dimethylhydroxylamine hydrochloride (1.2 mol) was added, followed by the dropwise addition of triethylamine (1.5 mol), and the mixture was stirred at room temperature overnight. The reaction was monitored by TLC (PE:EA = 3:1). After completion, the tetrahydrofuran was evaporated to dryness under reduced pressure, extracted three times with ethyl acetate, washed with saturated sodium bicarbonate solution, dried over anhydrous magnesium sulfate, filtered, and evaporated to dryness under reduced pressure. The product (1 mol) was dissolved in anhydrous tetrahydrofuran, protected by nitrogen, and pre-cooled in a cold trap to control the temperature at -10 to 15 ° C. Grignard reagent CH3MgCl (1.5 mol) was added dropwise. The reaction was detected 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 detection data of the product are as follows (such as Figure 1 shown):

[0179] 1 H NMR (400MHz, CDCl3) δ8.79(s,1H),2.80(s,3H),2.60(s,3H).

[0180] Example 3 Synthesis of Compound SMI7

[0181] Hydroxylamine hydrochloride (3 mmol) was placed in a round-bottom flask, 3 mL of ethanol was added, and the mixture was stirred at room temperature at 25°C for 10 min. 3 mL of 1 M NaOH solution was added, followed by SMI0 (3 mmol). The mixture was refluxed in an oil bath at 80°C. After the reaction was complete, the reaction solution was neutralized with 10% dilute hydrochloric acid, extracted with water and ethyl acetate, and the organic phases were combined and dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration, and the filtrate was concentrated under vacuum and separated by silica gel column chromatography with a ratio of petroleum ether: ethyl acetate = 1:2 as the eluent to obtain compound SMI7. The NMR data of the product are as follows (e.g. Figure 2 shown):

[0182] 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).

[0183] Example 4 Synthesis of Compound SMI16

[0184] 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 for dissolution. 30 μL of 3M KOH solution was added, followed by SMI0 (1 mmol), and the mixture was stirred at 50°C. After the reaction was completed 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 and dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration, and the filtrate was concentrated under vacuum and separated by silica gel column chromatography with a ratio of petroleum ether:ethyl acetate = 2:1 as the eluent to obtain compound SMI16. The NMR detection data of the product are as follows (e.g. Figure 3 shown):

[0185] 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).

[0186] Example 5 Synthesis of Compound SMI20

[0187] Aniline (2 mmol) and SMI0 (2 mmol) were placed in a round-bottom flask, 3 mL of anhydrous ethanol was added, and the mixture was stirred at room temperature at 25°C for 10 min. BH3CNNa (2 mmol) and acetic acid (1 mmol) were then added, and the mixture was stirred at room temperature 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 and dried over anhydrous magnesium sulfate. The magnesium sulfate was removed by filtration, and the filtrate was concentrated under vacuum and separated by silica gel column chromatography with a ratio of 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 shown):

[0188] 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).

[0189] Example 6 Synthesis of SMI0 Hydrochloride

[0190] SMI0 (1 g, 7.08 mmol) was placed in a round-bottom flask and dissolved in 5 mL of ethanol. 11 mL of a 1 mol / L hydrochloric acid-ethanol solution was slowly added dropwise with stirring at room temperature and allowed to react for 1 hour. The solution was concentrated under reduced pressure, cooled, and crystallized. The solution was filtered, washed with 0.5 mL of cold anhydrous ethanol, and dried to obtain 1.06 g of a white solid with an 85% yield and a purity of 99.8% (HPLC). The melting range was 160-162°C. The X-ray powder diffraction pattern (XRPD) of the hydrochloride form A of compound SMI0 is shown below. Figure 5 As shown, differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) Figure 6 shown.

[0191] Example 7 Synthesis of SMI0 Sulfate

[0192] SMI0 (1 g, 7.08 mmol) was placed in a round-bottom flask and dissolved in 5 mL of ethanol. 8 mL of a 1 mol / L sulfuric acid-ethanol solution was slowly added dropwise with stirring at room temperature and allowed to react for 1 hour. The solvent was evaporated to dryness, and the mixture was recrystallized from a small amount of methanol to precipitate 0.85 g of a yellow solid with a yield of 70% and a purity of 99.5% (HPLC). The X-ray powder diffraction pattern (XRPD) of the SMI0 sulfate salt, Form B, is shown below. Figure 7 As shown, differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) Figure 8 shown.

[0193] Example 8 Determination of Inhibition of CYP2E1 Metabolic Activity

[0194] Chlorzoxazone was used as the probe substrate to detect the inhibitory effect of the inhibitor on the metabolic activity of CYP2E1 in normal human mixed liver microsomes (IC 50 , half inhibitory concentration; K i , inhibition constant; IC 50 The smaller the K i The smaller the value, the higher the inhibitory strength of the inhibitor) was used to determine the inhibitory effect of the tested inhibitor on human liver microsomal CYP2E1.

[0195] Inhibits CYP2E1 action IC 50 Determination

[0196] The total incubation volume is 100 μL, and the incubation system includes substrate, varying concentrations of inhibitor, liver microsomal protein, and phosphate buffer. Reactions are incubated at 37°C in a water bath. After a 5-minute preincubation, the reduced coenzyme is added to initiate the reaction, and the reaction is terminated after 30 minutes in an ice bath. The inhibitor concentration can be adjusted based on specific experimental needs.

[0197] 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.

[0198] In other preferred embodiments, the incubation system may include 7.8 to 1000 μM chlorzoxazone as a substrate. The concentration of liver microsomal protein may be 0.1 to 0.5 mg / mL. 50 mM to 100 mM phosphate buffer or 50 mM to 100 mM Tris-HCl buffer may be used as needed.

[0199] An NADPH regeneration system can also be used. Preferably, 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.

[0200] In this embodiment, 1 mL of ethyl acetate was used to terminate the reaction. In other preferred embodiments, 1 mL of methyl tert-butyl ether, 1 mL of ethyl ether, and 100 μL of methanol may also be used to terminate the reaction.

[0201] Inhibits CYP2E1 action K i Determination

[0202] When it is determined that the inhibitor has a good inhibitory effect on CYP2E1, different concentrations of substrate and inhibitor are selected to conduct in vitro metabolic incubation inhibition test to calculate the inhibition constant K of the inhibitor on CYP2E1 metabolism of chlorzoxazonei .

[0203] The total volume of the incubation reaction system was 100 μL, and the incubation system included substrate, inhibitors at different concentrations, liver microsomal protein, and phosphate buffer. The reaction was incubated in a 37°C water bath. After a 5-min preincubation, the reduced coenzyme was added to initiate the reaction. After a certain incubation time, the reaction was terminated by ice bath.

[0204] In this embodiment, the incubation system includes 15.6, 31.25, 62.5, 125, and 250 μM chlorzoxazone, respectively; the IC 50 Based on the above, different concentration gradients of the inhibitor were determined according to a ratio of 1 / 4 to 4 times; 100 mM (pH = 7.4) phosphate buffer, 0.3 mg / mL liver microsomal protein, 1 mM NADPH.

[0205] In other preferred embodiments, the incubation system can include 7.8 to 1000 μM chlorzoxazone as a substrate, more preferably 15.6 to 250 μM (15.6, 31.25, 62.5, 125, and 250 μM) chlorzoxazone. The concentration of liver microsomal protein can be 0.1 to 0.5 mg / mL. 50 mM to 100 mM phosphate buffer or 50 mM to 100 mM Tris-HCl buffer can be used as needed.

[0206] Alternatively, 1 mM NADPH or an NADPH regeneration system containing 1.3 mM NADP+, 3.3 mM glucose-6-phosphate, 0.4 U / mL glucose dehydrogenase, and 3.3 mM magnesium chloride may be used.

[0207] In this embodiment, 1 mL of ethyl acetate was used to terminate the reaction. In other preferred embodiments, 1 mL of methyl tert-butyl ether, 1 mL of ethyl ether, and 100 μL of methanol may also be used to terminate the reaction.

[0208] Determination of CYP2E1 inhibitor selectivity

[0209] At the same time, normal human mixed liver microsomes were used as research objects, and probe drugs of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were selected respectively to determine the in vitro inhibitory effects of the test inhibitors on the probe drugs metabolized by 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 for CYP2E1 inhibition.

[0210] The CYP1A2 probe was either 6.25–800 μM phenacetin or 27.5–12520 μM caffeine;

[0211] The CYP2A6 probe was either 0.156–20 μM coumarin or 12.5–2000 μM nicotine;

[0212] The CYP2B6 probe was either 7.8–500 μM bupropion or 0.25–30 mM cyclophosphamide;

[0213] The CYP2C8 probe was either 2.5–80 μM paclitaxel or 0.125–128 μM amodiaquine;

[0214] The CYP2C9 probe was either 31.25–2000 μM tolbutamide or 0.1–200 μM diclofenac;

[0215] The CYP2C19 probe was either 3.9–500 μM omeprazole or 1.95–1000 μM mephenytoin;

[0216] The CYP2D6 probe was either 0.625–96 μM dextromethorphan or 0.0195–80 μM propafenone;

[0217] The CYP3A4 probe was either 0.39–50 μM midazolam or 1.98–1000 μM testosterone;

[0218] The total volume of the incubation system was 100 μL, and the system included substrate, inhibitors at varying concentrations, liver microsomal proteins, and phosphate buffer. The reaction was incubated at 37°C in a water bath. After a 5-minute preincubation, the reduced coenzyme was added to initiate the reaction. After a certain incubation period, the reaction was terminated by placing the enzyme in an ice bath.

[0219] In this embodiment, the incubation system includes at least one of 62.5 μM chlorzoxazone, 2.5 μM coumarin, 62.5 μM bupropion, 10 μM paclitaxel, 250 μM tolbutamide, 62.5 μM omeprazole, 20 μM dextromethorphan, 62.5 μM chlorzoxazone, 1.56 μM midazolam, 100 mM (pH = 7.4) phosphate buffer, 0.3 mg / mL liver microsomal protein, and 1 mM NADPH.

[0220] In other preferred embodiments, the substrate can include 7.8 to 1000 μM chlorzoxazone. The concentration of liver microsomal protein can be 0.1 to 0.5 mg / mL. The buffer can be selected from 50 mM phosphate buffer, 100 mM phosphate buffer, 50 mM Tris-HCl buffer, and 100 mM Tris-HCl buffer. 1 mM NADPH or an NADPH regeneration system can also be used.

[0221] In this embodiment, 1 mL of ethyl acetate was used to terminate the reaction. In other preferred embodiments, 1 mL of methyl tert-butyl ether, 1 mL of ethyl ether, and 100 μL of methanol may also be used to terminate the reaction.

[0222] Example 9 In vitro screening test results of CYP2E1 inhibitors

[0223] SMI0 (2E1 inhibitory IC 50 :1.64μM, IC for 2A6 inhibition 50 was 76.20 μM, and had no significant inhibitory effect on other CYP enzymes).

[0224]

[0225] Now refer to Figure 9 All reactions were incubated in 100 μL volumes with chlorzoxazone concentrations of 15.6, 31.2, 62.5, 125, and 250 μM and SMI0 concentrations of 0, 0.4638, 0.9276, 1.855, and 3.710 μM. All experiments are the mean of three independent experiments. Figure 9 The figure shows the inhibitory effect of SMI0 on the human liver metabolism of chlorzoxazone, indicating that SMI0 is a mixed inhibitor of CYP2E1.

[0226] Now refer to Figure 10 , Figure 10 This is a quadratic plot based on double reciprocal plotting of the inhibitory effect of SMI0 compounds on the metabolism of chlorzoxazone by human liver CYP2E1. Linear regression was performed on the series of SMI0 concentrations (0, 0.4638, 0.9276, 1.855, 3.710 μM) using the slopes of different inhibition curves. The absolute value of the intersection of the straight line and the horizontal axis is the K i It is 0.8970μM.

[0227] In addition, referring to the specific research method in Example 8, 21 small molecule compounds (Table 1) were tested for their inhibitory effects on CYP2E1 in vitro. The results showed that SMI1 and SMI8 had significant inhibitory effects on CYP2E1, and the IC values for CYP2E1 inhibition were 50SMI10 has a slight inhibitory effect on CYP2E1, with IC 50 Compared with SMI0, SMI1, SMI8 and SMI10, other small molecules have weaker inhibitory effects on CYP2E1 (such as Figure 11 shown).

[0228] In addition, selective studies have shown that SMI1 inhibits CYP2A6 with an IC 50 The IC for inhibiting CYP2C9 is 55.63 μM 50 The structures of the 21 small molecules are shown in Table 1.

[0229] Table 1

[0230]

[0231]

[0232]

[0233]

[0234] Example 10 Results of the Inhibitory Effect of SMI0 Compound on CYP2E1 in Vivo

[0235] Experimental Methods: The inhibitory effect of SMI0 compounds on CYP2E1 in rats was investigated using a self-controlled crossover design. In the first round of experiments, diethylnitrosamine (DEN) was administered intraperitoneally at a dose of 50 mg / kg (30 SD rats) for the first week. One week later, a second round of experiments was conducted in which each rat was gavaged with a low, medium, or high dose of SMI0 compound (6 mg / kg, 30 mg / kg, or 150 mg / kg). Five minutes later, DEN was administered intraperitoneally at a dose of 50 mg / kg (10 rats each). Blood samples were collected at 2, 7, 15, and 30 minutes, and at 1, 2, 4, 6, 9, 12, 24, 36, 48, and 60 hours. Plasma DEN concentrations were measured at these different time points to determine DEN toxicokinetic parameters. Plasma DEN was detected using high-performance liquid chromatography. 10 μL of perchloric acid was added to 100 μL of plasma, vortexed for 3 minutes, centrifuged at 12,000 rpm for 10 minutes, and 10 μL of the supernatant was injected. The mobile phase consisted of 50:50 methanol:water, and the detection wavelength was 240 nm.

[0236] Experimental results: The toxicokinetic parameters of DEN were used to represent CYP2E1 activity, and the effects of low, medium and high doses of SMI0 compounds on CYP2E1 activity in rats were analyzed. The results showed that compared with the model group treated with DEN alone, low, medium and high doses of SMI0 could reduce the DEN clearance rate CL by (63.98±7.78)%, (79.63±7.29)% and (85.42±3.74)%, respectively, and the inhibition rates were 62.43%, 80.42% and 86.77%, respectively (e.g. Figure 12 , as shown in Table 2, * P<0.05, *** P<0.001vs model group). Similarly, low, medium and high doses of SMI0 could shorten the half-life of DEN to t 1 / 2 The area under the curve (AUC) was prolonged by (133.60±116.80)%, (619.97±363.57)%, and (868.70±241.26)%, respectively. 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 effect of the medium dose of SMI0 compound was significantly better than that of the low dose, and the inhibitory effect of the high dose of SMI0 compound was significantly better than that of the low dose and medium dose ( # P<0.05, ## P<0.01, ### P<0.001vsSMI0 low-dose group; $ P<0.05, $$$ P<0.001 vs SMI0 medium-dose group). This suggests that different doses of SMI0 compounds can significantly inhibit the metabolic activity of rat CYP2E1 on DEN, and have a good dose-dependent relationship.

[0237] The above results showed that SMI0 had a significant inhibitory effect on CYP2E1 activity in rats.

[0238] Table 2 Toxicokinetic parameters of SMI0 compounds inhibiting the metabolism of diethylnitrosamine by CYP2E1

[0239]

[0240] Note: Mean in Table 2 stands for mean; SD stands for standard deviation;

[0241] C max Peak drug concentration: The highest blood drug concentration after drug administration. This parameter is an important indicator of the rate and extent of drug absorption in the body.

[0242] T max : Time to peak concentration: The time it takes to reach peak drug concentration after administration. This parameter reflects how quickly the drug enters the body; faster absorption means a shorter time to peak concentration.

[0243] V d Apparent volume of distribution: The constant proportional to the drug's amount in the body and its plasma concentration when the drug reaches dynamic equilibrium in the body, generally expressed in L. This parameter reflects the extent of the drug's distribution in the body, with higher values indicating wider distribution. The apparent volume of distribution is numerically derived from the ratio of clearance to terminal elimination rate.

[0244] CL: Clearance, the apparent distribution volume of the drug cleared from the body per unit time, generally expressed in L / h. This parameter is an important parameter that reflects the body's characteristics of drug disposal and is closely related to physiological factors. Clearance is based on the dose and AUC. (0-∞) The ratio is worth getting.

[0245] AUC: Area under the drug-time curve, the area enclosed by the blood drug concentration curve on the time axis. This parameter is an important indicator for evaluating the degree of drug absorption and reflects the exposure characteristics of the drug in the body. Since the blood drug concentration in pharmacokinetic studies can only be observed up to a certain time point t, AUC has two ways of expression: AUC (0-t) and AUC (0-∞) The former is obtained according to the trapezoidal area method, and the latter is calculated as: AUC (0-∞) =AUC (0-t) +End point concentration / end point elimination rate.

[0246] Example 11: SMI0 inhibits the proliferation of mouse lung cancer

[0247] (1) Changes in CYP2E1 in clinical lung cancer patients

[0248] Experimental methods: 30 normal subjects were used as controls, and the changes in CYP2E1 expression in paracancerous tissues of 30 clinical lung cancer patients were studied to compare the changes in CYP2E1 content in paracancerous tissues of lung cancer patients.

[0249] Experimental results: Immunohistochemistry results showed that the expression of CYP2E1 in the adjacent tissues of lung cancer patients was significantly higher than that in normal lung tissues (such as Figure 13 ***P < 0.001 vs normal lung tissue group).

[0250] (2) Orthotopic lung tumor transplantation in cyp2e1 knockout mice

[0251] Experimental method: Lewis cells of lung cancer cell line were used to prepare female C57 mice lung cancer orthotopic transplant tumor model, with the cell number of 2*10 4C57BL / 6N mice were divided into control, model, and cyp2e1 knockout groups. The model was established after 18 days (control group, n=8; model group, n=8; knockout group, n=8). At the end of the experiment, the mice were weighed, blood was collected from the orbitals, and the mice were sacrificed. Lung and tumor tissues were collected. After weighing, lung specimens were stained with HE to observe lung lesions and tumor development in each group.

[0252] Experimental results: Compared with the model group, the weight of lung cancer in cyp2e1 knockout mice was significantly reduced (such as Figure 14 A to 14B), the tumor proliferation inhibition rate can reach up to 56.9%, indicating that cyp2e1 knockout can significantly inhibit the occurrence and development of Lewis cell lung cancer orthotopic transplanted tumor model.

[0253] (3) SMI0 intervention of orthotopic lung transplanted tumors in mice

[0254] Experimental method: Lewis cells of lung cancer cell line were used to establish orthotopic transplanted tumor model in female C57 mice, with the cell number of 2*10 4 . C57BL / 6N mice were divided into control, model, positive drug, and SMI0 intervention groups. The model was terminated after 18 days [control group, n=20; model group, n=20; positive drug group (20 mg / kg), n=15; SMI0 (3.3 mg / kg), n=15; SMI0 (10 mg / kg), n=20; SMI0 (30 mg / kg), n=20)]. At the end of the experiment, the body weight of each group of mice was recorded, and the mice were sacrificed after orbital blood sampling. Lung tissue and tumor tissue were collected. After weighing, lung weight and appearance (lung color, texture, etc.) were recorded. All tumors were accumulated and arranged together to represent the tumor occurrence of one animal. The cumulative weight of lung tumors in each animal was measured. Some lung specimens were stained with HE to observe the lung lesions and tumor occurrence in each group of mice.

[0255] Experimental results: Compared with the model group, the proliferation of lung tumors in mice in the SMI0 intervention groups at different doses was significantly inhibited. That is, SMI0 intervention can significantly inhibit the growth of lung tumors in mice, with the highest tumor proliferation inhibition rate reaching 68.3% (e.g. Figure 15 As shown in A to 15C, *P < 0.05 vs. model group, where Figure 15 Figure 15A is a representative image of tumor-bearing lung tissue in each group of mice, Figure 15B is the lung tumor tissue of each mouse, and Figure 15C is the tumor weight of each group of animals. These results indicate that SMIO can significantly inhibit the development and progression of Lewis lung cancer cell orthotopic transplanted tumors in mice.

[0256] (4) SMI0 intervention in mouse CT26 cell lung metastasis

[0257] Experimental method: The lung metastasis model of female Balb / C mice was established by tail vein injection of colorectal cancer cells CT26, with a cell number of 3*10 5 Mice were divided into control, model, and SMI0 intervention groups, and the model was terminated after 14 days (control group, n=5; model group, n=5; SMI0 (30 mg / kg), n=5). At the end of the experiment, the body weight of each group of mice was recorded. Blood was collected from the orbital cavity and then sacrificed. Lung tissue was collected and weighed. Lung tissue was fixed in Burkholderia stain for 24 hours, and the number of lung nodules in each group was recorded.

[0258] Experimental results: Compared with the model group, the lung weight of mice in the SMI0 intervention group at different doses was significantly reduced, the number of nodules was significantly reduced, and the tumor proliferation inhibition rate was as high as 66.7% (such as Figure 16 As shown in A to 16B, *P < 0.05 vs. model group, where Figure 16 A is a representative picture of the tumor-bearing lung tissue of each group of mice. Figure 16 (B) Lung weight and number of lung nodules of each mouse. This indicates that SMI0 can significantly inhibit the occurrence and development of the lung metastasis model induced by tail vein injection of CT26 cells.

[0259] (5) SMI0 intervention in B16-F10 cell lung metastasis in mice

[0260] Experimental method: Melanoma B16-F10 cells were injected into the tail vein to establish a lung metastasis model in female C57BL / 6J mice. The cell number was 8*10 5 Mice were divided into control, model, and SMI0 intervention groups, and the model was terminated after 14 days (control group, n=5; model group, n=5; SMI0 (30 mg / kg), n=4). At the end of the experiment, the body weight of each group of mice was recorded. Blood was collected from the orbital cavity and then sacrificed. Lung tissue was collected and weighed. Lung tissue was fixed in Burkholderia stain for 24 hours, and the number of lung nodules in each group was recorded.

[0261] Experimental results: Compared with the model group, the lung weight of mice in the SMI0 intervention group at different doses was significantly reduced, the number of nodules was significantly reduced, and the tumor proliferation inhibition rate was as high as 58.3% (as shown in Figures 17A and 17B, *P<0.05 vs the model group, where Figure 17 A is a representative picture of the tumor-bearing lung tissue of each group of mice. Figure 17 B is the lung weight of each mouse. This indicates that SMI0 can significantly inhibit the occurrence and development of lung metastasis in the B16-F10 cell tail vein injection mouse model.

[0262] These results indicate that the CYP2E1 inhibitor SMI0 has a significant preventive and therapeutic effect on the development and progression of Lewis cell orthotopic lung xenografts and lung metastases induced by tail vein injection of CT26 and B16-F10 cells, suggesting that SMI0 compounds may be used for the prevention and treatment of lung cancer.

[0263] Example 12: CYP2E1 activity in mice after SMI0 inhibition is correlated with tumor severity in a lung carcinoma in situ model

[0264] Experimental Methods: Mouse liver microsomes were prepared using the calcium precipitation method, and microsomal protein concentration was determined using the Braford method. The incubation system was prepared using 2× PBS buffer, chlorzoxazone solution, and a final concentration of 0.5 mg / mL. The incubation was performed at 37°C for 5 minutes. The reaction was initiated by the addition of NADPH, incubated at 37°C for 30 minutes, and terminated on ice. 6-Hydroxychlorzoxazone was extracted with ethyl acetate, vortexed, centrifuged, and the upper organic phase was aspirated and dried under nitrogen. The peak area of the chlorzoxazone metabolite, 6-hydroxychlorzoxazone, was determined by high-performance liquid chromatography (HPLC) with a methanol:water ratio of 56:44 and a detection wavelength of 287 nm. Substitute the standard curve for the concentration of the metabolite, 6-hydroxychlorzoxazone, into the reaction curve to calculate ΔC. The reaction rate of chlorzoxazone to 6-hydroxychlorzoxazone was calculated as V (pmol / min / mg) = (ΔC*1000) / (B*T), where B is the microsomal protein concentration (mg / mL) and T is the incubation time (min). This was used to assess CYP2E1 enzyme activity. Correlation analysis was performed between CYP2E1 activity and lung tumor weight in the Lewis lung carcinoma in situ model.

[0265] Experimental results: Compared with the sham operation group, the CYP2E1 activity of mice in the model group was significantly increased (P<0.05); compared with the model group, the CYP2E1 activity of mice in the SMI0 intervention group was significantly decreased (P<0.05), indicating that SMI0 can effectively inhibit the increase of CYP2E1 enzyme activity in the liver tissue of Lewis lung carcinoma in situ model mice, and the CYP2E1 activity of mice was significantly positively correlated with the tumor weight of Lewis lung carcinoma in situ model mice (r=0.70, P<0.01) (e.g. Figure 18 The results indicate that CYP2E1 activity is positively correlated with the severity of Lewis lung carcinoma in situ, and the anti-lung cancer effect of SMI0 may be related to the inhibition of CYP2E1 activity.

[0266] Example 13: SMI0 inhibits the inflammatory microenvironment of orthotopic transplanted tumors in mice lungs

[0267] Experimental methods: Western-blot method was used to detect the expressions of pro-inflammatory cytokines TGF-β, IL-10, IL-4, and inflammation-related signaling pathway proteins (IL-6 / p-STAT3, p-ERK1 / 2 / ERK1 / 2, and epithelial-mesenchymal transition-related proteins MMP-2 and MMP-9) in the adjacent lung tissues of mice in each group. At the same time, the expressions of anti-apoptosis-related proteins caspase3 and Bcl-2, and autophagy-related protein p53 in tumor tissues were detected.

[0268] Experimental results: Compared with the sham operation group, the expression of proinflammatory cytokines (TGF-β, IL-10), inflammation-related signaling pathways (IL-6 / p-STAT3, p-ERK1 / 2 / ERK1 / 2) and matrix metalloproteinases (MMP-2, MMP-9) in the adjacent lung tissues of mice in the model group were significantly increased (P<0.05); further, compared with the model group, the expression of the above proteins in the SMI0 intervention group was significantly decreased (P<0.05) (e.g. Figure 19 Similarly, compared with the model group, the expression of anti-apoptotic proteins caspase3 and Bcl-2 in tumor tissues of the SMI0 intervention group (30 mg / kg) was significantly decreased (P<0.05), and the expression of autophagy-related protein p53 was significantly increased (P<0.01) (as shown in Figure 19B). Figure 20 (A-20B) These results suggest that SMI0 can significantly inhibit inflammation in adjacent lung tissues of Lewis lung carcinoma in situ mice, the activation of related signaling pathways, the expression of matrix metalloproteinases, and enhance p53-mediated autophagy in tumor tissues.

[0269] Example 14: SMI0 inhibits M2 polarization of macrophages in the peritumoral microenvironment

[0270] (1) SMI0 has no direct inhibitory effect on lung cancer cells

[0271] Experimental method: Lewis lung cancer cells and A549 lung cancer cells in the logarithmic growth phase were selected and 1*10 5 Cells were seeded at a concentration of 100 μL / mL in a 96-well plate. 100 μL of basal medium containing varying concentrations of SMI0 (0, 0.16, 0.8, 4, 20, and 100 μmol / L) was added to each well. After 24 hours, 10 μL of CCK8 reagent was added to each well and cultured for another 2 hours. The absorbance (OD) of each well at 450 nm was measured using a microplate reader. This was repeated three times and the average value was calculated. Proliferation activity was calculated based on the OD value.

[0272] Experimental results: SMI0 at the tested concentration (up to 100 μmol / L) had no significant inhibitory effect on the proliferation of Lewis lung cancer cells and A549 lung cancer cells (e.g. Figure 21 (as shown in A to 21B).

[0273] (2) SMI0 inhibits lung cancer cell proliferation by inhibiting M2 polarization of macrophages

[0274] Experimental method: 100 μmol / L PMA was used to induce human monocytic leukemia cells THP-1 into M0 macrophages, and then 20 ng / ml IL-4 and IL-13 were used to induce M0 macrophages into M2 macrophages. The supernatant was extracted and co-cultured with A549 lung cancer cells to simulate the M2 macrophage microenvironment of lung cancer. The SMI0 intervention group was treated with SMI0 (50 μmol / L) while adding interleukin induction, and the supernatant of M0 macrophages, M2 macrophages, and SMI0 intervention group cells was extracted to prepare conditioned medium. Lewis lung cancer cells in the logarithmic growth phase were selected and cultured at 1*10 5 Cells were seeded at a concentration of 100 μL / mL in a 96-well plate. 100 μL of different conditioned media was added to each well for 24 hours. 10 μL of CCK8 reagent was then added to each well. After incubation for another 2 hours, the absorbance (OD) of each well was measured at 450 nm using a microplate reader. This was repeated three times and the average value was calculated. Proliferation activity was calculated based on the OD value.

[0275] Experimental results: Compared with the A549 lung cancer cell single culture group, the proliferation activity of A549 cells in the M2 macrophage co-culture group was significantly enhanced (P<0.01); compared with the M2 macrophage co-culture group, the proliferation activity of A549 cells in the SMI0 intervention group (50μmol / L) was significantly weakened (P<0.05) (e.g. Figure 22 These results suggest that the inhibition of Lewis lung orthotopic xenograft tumors by the CYP2E1 inhibitor SMI0 may be related to the suppression of the inflammatory microenvironment surrounding lung cancer, especially the M2 polarization of macrophages in the surrounding microenvironment.

[0276] Example 15: SMI0 inhibits the development of pulmonary fibrosis in mice

[0277] (1) Pulmonary fibrosis in cyp2e1 knockout mice

[0278] Experimental Methods: A pulmonary fibrosis model was established in C57BL / 6N mice using intratracheal instillation of 8 mg / kg lipopolysaccharide. The C57BL / 6N mice were divided into control, model, and cyp2e1 knockout groups. The model was terminated after 28 days (control group, n=8; model group, n=8; knockout group, n=8). At the end of the experiment, the mice were weighed, and orbital blood was collected before being sacrificed. Lung tissue was then collected and weighed. The left lung was fixed in 4% formalin and stained with HE and Masson's staining. Lung tissue was graded for fibrosis and histological scoring to reflect the severity of pulmonary fibrosis.

[0279] Experimental results: Compared with the control group, the lung index of the model group mice was significantly increased, pathological examination showed thickening of alveolar septa, shrinkage or even disappearance of alveolar cavities, obvious inflammatory cell infiltration in the lung parenchyma, and significantly increased fibrosis and histological scores. Compared with the model group, the lung index of cyp2e1 knockout mice was significantly decreased, pathological examination showed no obvious inflammatory cell infiltration, and fibrosis and histological scores were significantly decreased (such as Figure 23 A to 23B). This suggests that cyp2e1 knockout can significantly inhibit the occurrence of lipopolysaccharide-induced pulmonary fibrosis.

[0280] (2) SMI0 intervention in mouse lung injury

[0281] Experimental Methods: A pulmonary fibrosis model was established in C57BL / 6N mice using intratracheal instillation of 8 mg / kg lipopolysaccharide. The SMI0 intervention group was divided into low- and high-dose groups, each receiving daily oral administration of 30 mg / kg and 90 mg / kg of the SMI0 compound starting three days prior to modeling and continuing until the model was established 6 hours later (control group, n=8; model group, n=8; high-dose SMI0 compound group, n=8). At the end of the experiment, mice were weighed, blood was collected from the orbital cavity, and the mice were sacrificed. Lung tissue was then collected and weighed, and the left lung was fixed in 4% formalin for HE staining.

[0282] Experimental results: Compared with the control group, the lung index of mice in the model group was significantly increased (P<0.001); compared with the model group, the lung index of mice in the low-dose and high-dose SMI0 groups was significantly decreased (P<0.01). This suggests that SMI0 can significantly reduce the increase in the lung index of mice with acute lung injury induced by lipopolysaccharide. Similarly, compared with the control group, the lung tissue structure of mice in the model group was significantly damaged, mainly manifested as alveolar edema, alveolar wall thickening, alveolar cavity shrinkage and a large number of inflammatory cell infiltration, and the lung injury score was significantly increased; compared with the model group, the SMI0 medication group showed reduced pulmonary edema, narrowing of alveolar septa, reduced inflammatory cell infiltration, and lung tissue results tending to normal, and the lung injury score was significantly reduced (such as Figure 24 A to 24B). These results suggest that SMI 0 can alleviate LPS-induced acute lung injury in mice.

[0283] (2) SMI0 intervention in mouse pulmonary fibrosis

[0284] Experimental Methods: A pulmonary fibrosis model was established in C57BL / 6N mice using intratracheal instillation of 8 mg / kg lipopolysaccharide. The SMI0 intervention group was divided into low- and high-dose groups, each receiving daily oral administration of 30 mg / kg and 90 mg / kg of the SMI0 compound starting three days prior to model establishment and continuing until the 28th day of model establishment (control group, n=8; model group, n=8; high-dose SMI0 compound group, n=8). At the end of the experiment, mice were weighed, blood was collected from the orbital cavity, and lung tissue was harvested. After weighing, the left lung was uniformly fixed in 4% formalin and stained with HE and Masson's staining. Lung tissue was graded for fibrosis and histological scoring to reflect the severity of pulmonary fibrosis.

[0285] Experimental results: Compared with the control group, the lung index of the model group mice was significantly increased, pathologically, the alveolar septum was thickened, the alveolar cavity was reduced or even disappeared, and there was obvious inflammatory cell infiltration in the lung parenchyma. Compared with the model group, the lung index of the SMI0 intervention group mice was significantly reduced, and there was no obvious inflammatory cell infiltration in the pathological examination (such as Figure 25 A to 25B). This suggests that SMI0 can significantly inhibit the occurrence of pulmonary fibrosis induced by lipopolysaccharide.

[0286] These results indicate that the CYP2E1 inhibitor SMI0 has a significant preventive and therapeutic effect on the development and progression of pulmonary fibrosis induced by lipopolysaccharide intratracheal instillation, suggesting that SMI0 compounds may be used in the prevention and treatment of clinical pulmonary fibrosis.

[0287] Example 16: CYP2E1 activity in mice after SMI0 inhibition is correlated with the severity of pulmonary fibrosis

[0288] Experimental Methods: Mouse liver microsomes were prepared using the calcium precipitation method, and microsomal protein concentration was determined using the Braford method. The incubation system was prepared using 2× PBS buffer, chlorzoxazone solution, and a final concentration of 0.5 mg / mL. The incubation was performed at 37°C for 5 minutes. The reaction was initiated by the addition of NADPH, incubated at 37°C for 30 minutes, and terminated on ice. 6-Hydroxychlorzoxazone was extracted with ethyl acetate, vortexed, centrifuged, and the upper organic phase was aspirated and dried under nitrogen. The peak area of the chlorzoxazone metabolite, 6-hydroxychlorzoxazone, was determined by high-performance liquid chromatography (HPLC) with a methanol:water ratio of 56:44 and a detection wavelength of 287 nm. Substitute the standard curve for the concentration of the metabolite, 6-hydroxychlorzoxazone, into the reaction curve to calculate ΔC. The reaction rate of chlorzoxazone to 6-hydroxychlorzoxazone was calculated as V (pmol / min / mg) = (ΔC*1000) / (B*T), where B is the microsomal protein concentration (mg / mL) and T is the incubation time (min). This was used to assess CYP2E1 enzyme activity. Correlation analysis was performed between CYP2E1 activity and the severity of pulmonary fibrosis in mice.

[0289] Experimental results: Compared with the control group, the reaction rate V of chlorzoxazone metabolism in the model group mice was significantly increased (P<0.01); compared with the model group, the reaction rate V of chlorzoxazone metabolism in the high-dose SMI0 group was significantly decreased (P<0.05), indicating that SMI0 can effectively inhibit the increase of CYP2E1 enzyme activity in pulmonary fibrosis model mice induced by lipopolysaccharide. Correlation analysis between the reaction rate V of chlorzoxazone and the lung index of pulmonary fibrosis mice showed that the correlation coefficient between the two was r=0.81, P<0.01 (such as Figure 26 (A-26B). This suggests that CYP2E1 enzyme activity is well correlated with the severity of pulmonary fibrosis, and that the anti-fibrotic effect of SMI0 may be related to the inhibition of CYP2E1 enzyme activity.

[0290] Example 17: SMI0 inhibits the inflammatory microenvironment of pulmonary fibrosis in mice

[0291] (1) SMI0 inhibits the inflammatory response in mice with lung injury

[0292] Experimental Methods: Immunohistochemical staining was used to assess neutrophil counts in mouse lung tissue, and the percentage of immunostaining-positive areas was quantified using Image-Pro Plus software. RNA was extracted from mouse lung tissue using a kit, and RT-PCR was used to determine the expression levels of TNF-α and IL-1β in each mouse lung tissue.

[0293] Experimental results: Compared with the control group, the percentage of MPO immunostaining positive areas in the lung tissue of mice in the model group was significantly increased (P<0.001) (e.g. Figure 27 A to 27B), the expression of TNF-α and IL-1β in lung tissues was significantly increased (P < 0.01) (as shown in Figure 28 (A-28B); compared with the model group, the percentage of MPO-positive areas in the high-dose SMI0 group was significantly reduced (P < 0.05), and the expression of TNF-α and IL-1β in lung tissue was significantly decreased (P < 0.05). This suggests that SMI0 can alleviate neutrophil infiltration in the lung tissue of mice with LPS-induced acute lung injury and inhibit the release of proinflammatory cytokines.

[0294] (2) SMI0 inhibits the inflammatory microenvironment of mice with pulmonary fibrosis

[0295] Experimental Methods: Mouse lung tissue homogenates were processed using the ammonium molybdate method and the microplate method, respectively. Changes in oxidative stress markers CAT and GSH in the lung tissue of each group of mice were detected. Immunohistochemical staining was used to assess the expression levels of TGF-β1 and the mesenchymal cell marker α-SMA in the lung tissue of mice, and the percentage of positive areas was calculated using Image-Pro Plus software. Western blot was used to detect the expression levels of the epithelial cell marker E-cadherin, the apoptosis-related protein Bax, and the anti-apoptosis-related protein Bcl-2.

[0296] Experimental results: Compared with the control group, the levels of oxidative stress indicator CAT (P<0.01) and epithelial cell marker E-cadherin in the model group were significantly decreased (P<0.05), and the percentage of α-SMA and TGF-β1 immunostaining positive areas were significantly increased (P<0.01). At the same time, the expression level of apoptosis factor Bax was significantly increased (P<0.01), and the expression level of anti-apoptotic factor Bcl-2 was significantly decreased (P<0.05). Compared with the model group, the levels of oxidative stress indicator CAT (P<0.01) and epithelial cell marker E-cadherin in the SMI0 intervention group were significantly increased (P<0.05), and the percentage of α-SMA and TGF-β1 immunostaining positive areas were significantly decreased (P<0.01), the Bax level was significantly decreased (P<0.01), and the Bcl-2 level was significantly increased (P<0.001) (as shown in Figure 5). Figures 29 and 30 The results suggest that SMI0 alleviates LPS-induced pulmonary fibrosis in mice, which may be related to improving antioxidant capacity, anti-apoptosis, reducing the level of fibrosis-inducing factor TGF-β1 in the lung tissue of mice with pulmonary fibrosis, and reducing epithelial-mesenchymal transition.

[0297] The above descriptions are merely a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application discloses the preferred embodiments as above, they are not intended to limit the present application. Any technical personnel familiar with the present profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. Use of a CYP2E1 inhibitor in the preparation of a drug for treating or preventing inflammation-related diseases, characterized in that: The inflammation-related disease is selected from lung cancer or pulmonary fibrosis; The CYP2E1 inhibitor is selected from at least one compound represented by formula (I) or a salt thereof; 2. The use according to claim 1, characterized in that The compound represented by formula (I) reacts with an acid to obtain an acid salt of the compound represented by formula (I); The acid is selected from at least one of an inorganic acid and an organic acid.

3. The use 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, resin acid, maleic acid, fumaric acid, salicylic acid, and acetylsalicylic acid.

4. The use according to claim 1, characterized in that The X-ray powder diffraction pattern of Form A of the hydrochloride salt of the compound represented by formula (I) includes 3 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°; The DSC-TGA diagram of the crystalline form A of the hydrochloride salt of the compound represented by formula (I) shows that it has a significant endothermic peak between 70°C and 220°C and thermally decomposes at 80°C to 170°C.

5. The use according to claim 1, characterized in that The X-ray powder diffraction pattern of Form B of the sulfate salt of the compound represented by formula (I) comprises 5 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 diagram of the crystalline form B of the sulfate salt of the compound represented by formula (I) shows that it has at least one endothermic peak between 30°C and 85°C, 90°C and 160°C, and 215°C and 330°C, and thermally decomposes at 150°C to 350°C.

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