Sulfur-containing prodrug compounds and medical uses thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a sulfur-containing prodrug compound and medical use thereof. 1‑6 alkyl, substituted C 1‑6 alkyl, C 6‑10 aryl, substituted C 6‑10 aryl; R2 is selected from H or C 1‑6 alkyl, substituted C 1‑6 alkyl; R3 is selected from H or C1-6 alkyl, substituted C 1‑6 alkyl; R4 is selected from H, COOH or COOR 5 ; wherein R 5 is selected from C 1‑6 alkyl, substituted C 1‑6 alkyl or benzyl; L is selected from or a chemical bond; wherein X is selected from H, halogen, C 1‑6 alkyl, CH3O, CN or NO2; X is single substitution or multiple substitution at any position; Y is selected from O, S, NH or NCH3; R6 is selected from H or C 1‑3 alkyl; R7 is selected from H or C 1‑3 alkyl; n is selected from 1, 2, 3 or 4.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a class of sulfur-containing prodrug compounds, and also to the pharmaceutical use of such compounds as antioxidants. Background Technology

[0002] Hydrogen sulfide (H2S) is an endogenous bioactive molecule involved in diverse biological processes and regulating the functions of multiple organs, including the liver, kidneys, gastrointestinal tract, gut microbiota, and immune system. The mechanisms by which H2S exerts its biological activity are complex. In recent years, increasing research has confirmed that H2S can participate in the formation of hydropersulfides (RSSH) in vivo, thereby directly intervening in oxidative stress or regulating post-translational modifications of proteins. Similar to H2S, RSSH is also widely present in various cells and tissues; however, compared to H2S, RSSH has stronger nucleophilicity and is an important intermediate in responding to intracellular electrophilic / oxidative stress. For example, elevated oxidative stress levels lead to irreversible oxidative modifications of cysteine ​​residues in proteins; however, hydropersulfides formed from cysteine ​​can resist these oxidative modifications and restore the activity of cysteine ​​residues. Furthermore, RSSH possesses excellent single-electron reducing power, directly quenching superoxide / oxygen free radicals in cells and tissues and downregulating stress levels. In conclusion, RSSH has become a highly promising antioxidant molecule.

[0003] Designing and developing prodrugs for RSSH (reactive skeletal enzymes) to directly deliver small RSSH molecules in vitro and in vivo is an important strategy for studying the biological activity of RSSH and developing therapeutically significant antioxidant molecules. Although several RSSH prodrugs have been reported, those released via in vivo enzymatic reactions are limited to esterases (Angew. Chem. Int. Ed. 2017, 56, 11749; Org. Lett. 2018, 20, 6364) and glucosidases (Chem. Commun. 2022, 58, 2987). Therefore, we still need to develop novel RSSH prodrugs to achieve different types of enzymatic release and obtain active molecules with ideal antioxidant activity and therapeutic effects on oxidative stress-related diseases. Therefore, based on NAD(P)H quinone dehydrogenase 1 (NQO1) highly expressed under oxidative stress, we designed and synthesized a series of sulfur-containing compounds and found that these compounds have outstanding antioxidant and cytoprotective effects. Summary of the Invention

[0004] In view of the problems existing in the prior art, the first objective of the present invention is to provide a novel sulfur-containing prodrug compound, which has outstanding antioxidant and cell protection effects.

[0005] A second object of the present invention is to provide the pharmaceutical use of the aforementioned compounds as antioxidants.

[0006] The technical solution adopted by this invention to solve its technical problem is:

[0007] In a first aspect, the present invention protects a sulfur-containing compound of formula (I), or a pharmaceutically acceptable salt, tautomer, meso compound, racemic compound, stereoisomer, metabolite, metabolic precursor, or solvate thereof:

[0008]

[0009] Among them, R 1 Selected from: C 1-6 Alkyl, substituted C 1-6 Alkyl, C 6-10 Aryl, substituted C 6-10 Aryl;

[0010] R 2 Selected from: H or C 1-6 Alkyl, substituted C 1-6 alkyl;

[0011] R 3 Selected from: H or C 1-6 Alkyl, substituted C 1-6 alkyl;

[0012] R 4 Selected from: H, COOH or COOR 5 ;

[0013] Among them, R 5 Selected from: C 1-6 Alkyl, substituted C 1-6 Alkyl or benzyl;

[0014] L is selected from: Or chemical bonds;

[0015] Wherein, X is selected from: H, halogen, C 1-6 Alkyl, CH3O, CN, or NO2; X can be monosubstituted or polysubstituted at any position;

[0016] Y is selected from: O, S, NH or NCH3;

[0017] R 6 Selected from: H or C 1-3 alkyl;

[0018] R 7 Selected from: H or C 1-3 alkyl;

[0019] n is selected from: 1, 2, 3 or 4.

[0020] In the specific implementation plan

[0021] R 1 Selected from: C 1-6 Alkyl, phenyl, or substituted phenyl groups;

[0022] R 2 Selected from: H or C 1-3 alkyl;

[0023] R 3 Selected from: H or C 1-3 alkyl;

[0024] R 4 Selected from: H, COOH or COOR 5 ;

[0025] Among them, R 5 Selected from: C 1-6 Alkyl or benzyl;

[0026] L is selected from: Or chemical bonds;

[0027] Wherein, X is selected from: H, halogen, C 1-6 Alkyl, CH3O, CN, or NO2; X can be monosubstituted or polysubstituted at any position;

[0028] Y is selected from: O, S, NH or NCH3;

[0029] R 6 Selected from: H or C 1-3 alkyl;

[0030] R 7 Selected from: H or C 1-3 alkyl;

[0031] n is selected from: 1 or 2.

[0032] In the specific implementation plan

[0033] Among them, R 1 Selected from: C 1-6 Alkyl, phenyl, or substituted phenyl groups;

[0034] R 2 Selected from: H or methyl;

[0035] R 3 Selected from: H or methyl;

[0036] R 4 Selected from: H, COOH or COOR 5 ;

[0037] Among them, R 5 Selected from: C1-6 alkyl or benzyl;

[0038] L is selected from: Or chemical bonds;

[0039] Wherein, X is selected from: H, F, Cl, Br, I, methyl, CH3O, CN or NO2; X can be monosubstituted or polysubstituted at any position;

[0040] Y is selected from: O, S, NH or NCH3;

[0041] R 6 Selected from: H or methyl;

[0042] R 7 Selected from: H or methyl;

[0043] n is selected from: 1 or 2.

[0044] In the specific implementation plan

[0045] R 1 Selected from: methyl, ethyl, isopropyl, phenyl;

[0046] R 2 Selected from: H or methyl;

[0047] R 3 Selected from: H or methyl;

[0048] R 4 Selected from: H, COOH or COOR 5 ;

[0049] Among them, R 5 Selected from: methyl, ethyl, or isopropyl;

[0050] L is selected from: Or chemical bonds;

[0051] Wherein, X is selected from: H;

[0052] Y is selected from: O, S, NH or NCH3;

[0053] R 6 Selected from: H or methyl;

[0054] R 7 Selected from: H or methyl;

[0055] n is selected from: 1 or 2.

[0056] This invention also protects any of the compounds shown in the table below, or their pharmaceutically acceptable salts, tautomers, mesosomes, racemates, stereoisomers, metabolites, metabolic precursors, prodrugs, or solvates:

[0057]

[0058] Secondly, the present invention protects a pharmaceutical composition comprising the aforementioned compound or a pharmaceutically acceptable salt thereof, a tautomer, a meso compound, a racemic compound, a stereoisomer, a metabolite, a metabolic precursor, a prodrug, or a solvate, and a pharmaceutically acceptable carrier.

[0059] Thirdly, this invention protects the use of any of the compounds described above or their pharmaceutically acceptable salts, tautomers, mesosomes, racemates, stereoisomers, metabolites, metabolic precursors, prodrugs or solvates, or the pharmaceutical compositions described above in the preparation of antioxidants.

[0060] Fourthly, this invention protects the use of any of the compounds described above or their pharmaceutically acceptable salts, tautomers, mesosomes, racemates, stereoisomers, metabolites, metabolic precursors, prodrugs or solvates, or the pharmaceutical compositions described above in the preparation of a medicament.

[0061] Preferably, the drug is for the prevention and / or treatment of diseases related to abnormal oxidative stress, or the drug is for the prevention and / or treatment of one or more of the following: autoimmune diseases, inflammatory diseases, allergic diseases, metabolic diseases, infection-based diseases, fibrotic diseases, cardiovascular diseases, respiratory diseases, kidney diseases, dermatological diseases, liver diseases, gastrointestinal diseases, oral diseases, and hematopoietic diseases.

[0062] More preferably, the drug is used for the prevention and / or treatment of drug-induced liver injury, liver fibrosis, drug-induced kidney injury, renal fibrosis, acute and chronic lung injury, pulmonary fibrosis, ulcerative colitis, or gastrointestinal toxicity caused by chemotherapy.

[0063] The compounds, pharmaceutical compositions, antioxidants, and drugs of the present invention can be administered locally or systemically, for example, for enteral administration, such as rectal or oral administration, or for parenteral administration to mammals (especially humans). The compounds of the present invention can also be administered parenterally, for example, by inhalation, injection, or infusion, such as via intravenous, intra-arterial, intra-bone, intramuscular, intracerebral, extraventricular, intrasynovial, intrasternal, intrathecal, intralesional, intralesional, intracranial, intratumoral, intradermal, and subcutaneous injection or infusion.

[0064] The effective amount of the compound, pharmaceutical composition, or drug described in this invention depends on the species, weight, age, individual condition, individual pharmacokinetic parameters, the disease to be treated, and the route of administration of the mammal.

[0065] The effective amount of the compounds, pharmaceutical compositions, or drugs described in this invention can be easily determined by routine experiments. The most effective and convenient route of administration and the most appropriate formulation can also be determined by routine experiments.

[0066] The compounds, pharmaceutical compositions, antioxidants, or drugs of the present invention can be designed into different dosage forms as needed, such as tablets, pills, suppositories, or injections.

[0067] To form tablets, any excipient known and widely used in the art can be used. Examples include carriers such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, and silica; binders such as water, ethanol, propanol, common syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; disintegrants such as dry starch, sodium alginate, agar powder, and kelp powder, sodium bicarbonate, calcium carbonate, fatty acid esters of polyvinyl sorbitol, sodium lauryl sulfate, glyceryl monostearate, starch, and lactose; disintegration inhibitors such as white sugar, glyceryl tristearate, coconut oil, and hydrogenated oil; adsorption promoters such as quaternary ammonium base and sodium lauryl sulfate; wetting agents such as glycerin and starch; adsorbents such as starch, lactose, kaolin, bentonite, and colloidal silica; and lubricants such as pure talc, stearates, boric acid powder, and polyethylene glycol. It can also be made into sugar-coated tablets, gelatin-coated tablets, sausage-coated tablets, coated tablets, double-layered tablets and multilayered tablets by using common coating materials as needed.

[0068] To form the pills, any known and widely used excipients in the art may be used, such as carriers, like lactose, starch, coconut oil, hardened vegetable oil, kaolin, and talc; binders, like gum arabic, tragacanth, gelatin, and ethanol; and disintegrants, like agar and kelp powder.

[0069] To form the suppository, any excipient known and widely used in the art can be used, such as polyethylene glycol, coconut oil, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides.

[0070] To prepare injections, solutions or suspensions can be sterilized and made isotonic with blood. Any commonly used carrier in the art can be used in the preparation of injections, such as water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and fatty acid esters of polyvinyl sorbitol. In addition, common solvents, buffers, and analgesics can be added.

[0071] The diluent may be a conventional diluent in the art.

[0072] The drug may be in the form of oral administration or as a sterile aqueous solution for injection, and may be prepared in accordance with any method known in the art for preparing pharmaceutical compositions.

[0073] Beneficial effects

[0074] Compared with existing technologies, this invention has the following beneficial effects: these compounds can be degraded by the in vivo oxidoreductase NQO1, possess ideal antioxidant activity, and can effectively alleviate oxidative stress damage to cells. It is worth noting that none of the previously reported RSSH precursors have demonstrated the intracellular release of RSSH, while this invention has proven that the disclosed compounds can release RSSH intracellularly, laying the foundation for subsequent pharmacological studies and drug development. Attached Figure Description

[0075] Figure 1 This is the hydrogen spectrum of compound I-5.

[0076] Figure 2 This is the hydrogen spectrum of compound I-6.

[0077] Figure 3 Intracellular RSSH generation for compound I-6.

[0078] Figure 4 The compound was used to alleviate DSS-induced weight loss in mice. Detailed Implementation

[0079] The following embodiments illustrate the content of the present invention. In this invention, the embodiments described below are for better explanation and are not intended to limit the scope of the invention. Various changes and modifications can be made to the present invention without departing from its spirit and scope.

[0080] Example 1: Synthesis of Compound I-1

[0081]

[0082] Synthesis of 6-hydroxy-4,4,5,7,8-pentamethylbenzopyran-2-one (10):

[0083] Trimethylhydroquinone (4.86 g, 32 mmol) and 3-methyl-2-enoic acid (3.68 g, 36.8 mmol) were placed in a dry double-necked flask (100 mL), Ar was used for displacement, and methanesulfonic acid (50 mL) was added. The mixture was refluxed in an oil bath at 85 °C with stirring for 3 h. The reaction was monitored by TLC until complete. The mixture was quenched with ice water (100 g), extracted with ethyl acetate (5 × 50 mL), washed with saturated sodium bicarbonate, and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was hot-washed in an oil bath at 85 °C for 4 h with a solvent of PE:EA = 2:1, and filtered to give 10 (6.3 g, 84%) of brown solid. 1 H NMR (300MHz, DMSO-d6) δ7.98(s,1H),2.57(s,2H),2.27(s,3H),2.10(s,3H),2.09(s,3H),1.35(s,6H).

[0084] Synthesis of 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)butyric acid (6):

[0085] Compound 10 (6.27 g, 26.8 mmol) was placed in a dry round-bottom flask (200 mL), and a solution of acetonitrile:water = 5:1 (75 mL total) was added. The mixture was cooled to 0 °C, and NBS (5 g, 28.4 mmol) was added at this temperature. The mixture was stirred at room temperature for 1 h, and the reaction was monitored by TLC until completion. After the reaction was complete, the mixture was extracted with ethyl acetate (5 × 75 mL), and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was slurried with diethyl ether to give a yellow solid 6 (3.72 g, 56%). 1 H NMR(300MHz,Chloroform-d)δ3.04(s,2H),2.16(s,3H),1.97(s,3H),1.95(s,3H),1.45(s,6H).

[0086] Synthesis of 4-(((tert-butyldimethylsilyl)oxy)methyl)phenol (11):

[0087] p-Hydroxybenzyl alcohol (744 mg, 6 mmol) and imidazole (490 mg, 7.2 mmol) were placed in a 50 mL double-necked flask, DMF (10 mL) was added, and the mixture was cooled to 0 °C. Then, TBSCl (1.1 g, 7.2 mmol) was added dropwise over 10 min, and the reaction was carried out under these conditions for 2 h. The reaction was monitored by TLC until complete, quenched with ice water (100 g), and extracted with ethyl acetate (5 × 20 mL). The organic phases were combined, washed with 10% LiCl solution (20 mL), saturated sodium bicarbonate solution (20 mL), and saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 20 / 1) to give a golden yellow oil 11 (1.2 g, 84%). 1 H NMR (300MHz, Methanol-d4) δ7.15 (d, J = 8.1 Hz, 2H), 6.75 (d, J = 8.6 Hz, 2H), 4.64 (s, 2H), 0.94 (s, 9H), 0.10 (s, 6H).

[0088] Synthesis of 4-((tert-butyldimethylsiloxy)methyl)phenyl-3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohex-1,4-dienyl)butyrate (12):

[0089] Compound 11 (476 mg, 2 mmol), compound 6 (600 mg, 2.4 mmol), and DMAP (36 mg, 0.3 mmol) were placed in a double-necked flask (50 mL), and anhydrous dichloromethane (20 mL) was added. Then, EDCI (768 mg, 4 mmol) was added in portions. The resulting mixture was stirred overnight at room temperature. The reaction was monitored by TLC until completion. After the reaction was complete, the mixture was extracted with dichloromethane (2 × 20 mL), and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was subjected to column chromatography (petroleum ether / ethyl acetate = 19 / 1) to give a pure yellow oil (838 mg, 89.1%). 1 H NMR(300MHz,DMSO-d6)δ7.30(d,J=8.3Hz,2H),6.96(d,J=8.5Hz,2H),4.68(s,2H ),3.16(s,2H),2.12(s,3H),1.87(s,6H),1.47(s,6H),0.89(s,9H),0.07(s,6H).

[0090] Synthesis of 4-hydroxymethylphenyl-3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dienyl)butyrate (2a):

[0091] Compound 12 (838 mg, 1.8 mmol) was placed in a dry round-bottom flask (50 mL), and triethylamine trifluoride (1.2 g, 7.1 mmol) was added, followed by anhydrous THF (18 mL). The resulting solution was stirred for 6 h. The reaction was monitored by TLC until completion. After the reaction was complete, the mixture was extracted with ethyl acetate (5 × 20 mL), and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was subjected to column chromatography (petroleum ether / ethyl acetate = 9 / 1) to give a pure yellow oil (370 mg, 59%). 1 H NMR (300MHz, DMSO-d6) δ7.31(d,J=8.4Hz,2H),6.94(d,J=8.4Hz,2H),5.21(s,1H),4.47(s,2H),3.16(s,2H),2.11(s,3H),1.87(s,6H),1.47(s,6H).

[0092] Synthesis of 4-(bromomethyl)phenyl-3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dienyl)butyrate (3a):

[0093] 2a (1.4 g, 4 mmol) and CBr4 (2.6 mg, 8 mmol) were placed in a dry double-necked flask (100 mL), and anhydrous dichloromethane (15 mL) was added. PPh3 (2.1 mg, 8 mmol) dissolved in anhydrous dichloromethane (15 mL) was then added dropwise under ice bath conditions. The reaction was monitored by TLC until completion. After completion, the mixture was extracted with DCM (3 × 40 mL), and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was subjected to column chromatography (petroleum ether / ethyl acetate = 10 / 1) to give a pure yellow solid (1.3 g, 80.3%). 1 H NMR (300MHz, DMSO-d6) δ7.45(d,J=8.4Hz,2H),7.01(d,J=8.1Hz,2H),4.76(s,2H),3.18(s,2H),2.12(s,3H),1.87(s,6H),1.47(s,6H).

[0094] Synthesis of 4-(mercaptomethyl)phenyl-3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dienyl)butyrate (4a):

[0095] Compound 3a (356 mg, 1 mmol), thiourea (312 mg, 4 mmol), and THF (5 mL) were added to a 20 mL sealed tube. The mixture was stirred at 85 °C for 5 h, concentrated under reduced pressure, and the crude product was directly used in the next step. The crude product was dissolved in degassed CHCl3 (4 mL) and placed in a sealed tube. Na2S2O3 (380 mg, 2 mmol) was dissolved in water (2 mL) and added to the reaction system all at once. The mixture was stirred vigorously at 85 °C for 5 h. The reaction was monitored by TLC until complete, extracted with DCM (3 × 10 mL), and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was directly used in the next step to avoid dimerization during column chromatography.

[0096] Synthesis of N-acetyl-S-(4-(3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dienyl)butyryl)oxy)benzyl)thio)-L-cysteine ​​(I-1):

[0097] Compound 4a (372 mg, 1 mmol) and compound 5a (200 mg, 0.91 mmol) were placed in a two-necked flask, Ar was used for displacement, methanol (5 mL) was added, and the mixture was stirred overnight at room temperature. The reaction was monitored by TLC until completion. After the reaction was complete, the solvent was removed by direct vacuum distillation, and the crude product was subjected to column chromatography (dichloromethane / methanol = 100 / 1) to give a yellow solid I-1 (126 mg, yield 23.6%). 1H NMR (300MHz, DMSO-d6) δ8.33(d,J=7.8Hz,1H),7.33(d,J=7.9Hz,2H),6.96(d,J=7.8Hz,2H),4.56-4.40(m,1 H),3.96(s,2H),3.17(s,2H),3.08-2.93(m,1H),2.90-2.72(m,1H),2.11(s,3H),1.86(s,9H),1.46(s,6H).

[0098] Example 2 Synthesis of Compound I-2

[0099]

[0100] Synthesis of N-(4-(hydroxymethyl)phenyl)-3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)butyramide (2b):

[0101] Compound 6 (3.5 g, 14 mmol) was placed in a double-necked flask, Ar was substituted, THF (50 mL) and N-methylmorpholine (1.5 g, 15.2 mmol) were added, and the resulting solution was cooled to -20 °C. Isobutyl chlorocarbonate (2.1 g, 15.2 mmol) was then added dropwise. After reacting for 1 h under these conditions, 4-aminopropanol (2.6 g, 21 mmol) dissolved in THF (10 mL) was added, and the mixture was reacted overnight at room temperature. The reaction was monitored by TLC until complete, quenched with ice water (100 g), extracted with ethyl acetate (5 × 50 mL), and the organic phases were combined, washed with saturated NaHCO3 (2 × 50 mL) and brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 5 / 1) to give a brown solid, which was further purified by slurrying with diethyl ether to give a yellow powder 2b (3.1 g, 62%). 1 H NMR (300MHz, Chloroform-d) δ7.40(d,J=8.2Hz,2H),7.29(d,J=5.2Hz,2H),4.64(s,2H),3.02(s,2H),2.17(s,3H),1.96(s,6H),1.68(s,3H),1.51(s,6H).

[0102] Synthesis of N-acetyl-S-(4-(3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dienyl)butamido)benzyl)thio)-L-cysteine ​​(I-2):

[0103]

[0104] Following the method in Example 1, 2a in the example was replaced with 2b to prepare a yellow oily liquid I-2 (102 mg, 19%). 1 H NMR (300MHz, DMSO-d6) δ9.97 (s, 1H), 8.30 (d, J = 8.0Hz, 1H), 7.44 (d, J = 8.2Hz, 2H), 7.22 (d,J=8.2Hz,2H),4.53-4.41(m,1H),3.91(s,2H),3.05-2.97(m,1H),2.95(s,2H),2.85 -2.76(m,1H),2.05(s,3H),1.90(s,6H),1.86(s,3H),1.39(s,6H). 13 C NMR (75MHz, DMSO) δ190.8,187.3,172.5,170.8,169.9,154.6,144.0,138.5,137.1,136.6, 132.3,130.1,119.6,51.7,49.3,42.0,38.1,28.6,22.8,14.2,13.1,12.2.HRMS(ESI)forC 26 H 32 N₂O₆S₂Na[M+Na] + calcd 555.1594,found555.1598.

[0105] Example 3 Synthesis of Compound I-3

[0106] Synthesis of N-(4-(hydroxymethyl)phenyl)-N,3-dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)butyramide (2c):

[0107]

[0108] The yellow solid 2c (802 mg, 82.4%) was prepared according to the preparation steps of 2b above. 1 H NMR(300MHz,Chloroform-d)δ7.46(d,J=7.8Hz,2H),7.21(d,J=6.6Hz,2H),4.78(s, 2H),3.17(s,3H),2.76(s,2H),2.12(s,3H),2.02(s,3H),1.98(s,3H),1.32(s,6H).

[0109] Synthesis of N-acetyl-S-(4-(N,3-dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dienyl)butamido)benzyl)thio)-L-cysteine ​​(I-3):

[0110]

[0111] Following the method in Example 1, 2a in the example was replaced with 2c to prepare a yellow oily liquid I-3 (120 mg, 22%). 1 H NMR (300MHz, DMSO-d6) δ12.84(s,1H),8.26(d,J=8.0Hz,1H),7.41(d,J=7.7Hz,2H),7.20(d,J=7.8Hz,2H),4.52-4.38(m,1H),4.00( s,2H),3.03(s,3H),2.86(d,J=11.6Hz,1H),2.75(d,J=9.7Hz,1H),2.66(s,2H),1.99(s,3H),1.88(s,6H),1.84(s,3H),1.21(s,6H). 13 C NMR (75MHz, DMSO) δ190.8,187.3,172.5,171.4,169.9,155.4,143.8,143.2,137.5,137.2,13 5.6,131.1,127.8,51.6,47.3,41.7,38.2,37.1,28.5,22.9,14.2,13.1,12.2.HRMS(ESI)for C 27 H 34 N₂O₆S₂Na[M+Na] + calcd 569.1750, found 569.1741.

[0112] Example 4 Synthesis of Compound I-4

[0113]

[0114] Synthesis of 2-(pyridyl-2-disulfonyl)ethane-1-amine (5e):

[0115] Mercaptoethylamine (1 g, 8.8 mmol) was placed in a dry, double-necked flask (50 mL), and anhydrous methanol (20 mL) was added. Ar gas was bubbled in for 30 min, and dithiodipyridine (2.3 g, 10.6 mmol) was added. The reaction was allowed to proceed overnight at room temperature. The reaction was monitored by TLC until complete. The solvent was removed by direct vacuum distillation, and the residue was slurried with diethyl ether to give solid compound 5e (1.4 g, 61.3%).

[0116] Synthesis of (2-(pyridyl-2-disulfonyl)ethyl)acetamide (5d):

[0117] Compound 5e (1.4 g, 5.4 mmol) was placed in a dry double-necked flask (50 mL), and dichloromethane (22 mL) was added. Acetyl chloride (0.7 mL, 10.8 mmol) and triethylamine (2.3 mL, 16.2 mmol) were added dropwise at 0 °C. The reaction was carried out at 0 °C for 30 min, and then transferred to room temperature for 6 h. The reaction was monitored by TLC until complete. The mixture was extracted with DCM (3 × 20 mL), and the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The crude product was subjected to column chromatography (dichloromethane / methanol = 30 / 1) to give a yellow oily liquid 16 (1 g, 83%).

[0118] Synthesis of N-(4-(((2-acetamidoethyl)dithioalkyl)methyl)phenyl)-N,3-dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dienyl)butyramide (I-4):

[0119] Compound 5e (70 mg, 0.25 mmol) and compound 4c (99 mg, 0.26 mmol) were placed in a dry double-necked flask (10 mL), tetrahydrofuran (3 mL) was added, Ar was bubbled for 30 min, and the reaction was carried out overnight at room temperature. The reaction was monitored by TLC until complete. 1N HCl (10 mL) and dichloromethane (3 × 10 mL) were added. The organic phases were combined, washed with saturated sodium bicarbonate and saturated brine, and dried over anhydrous sodium sulfate. The crude product was subjected to column chromatography (dichloromethane / methanol = 50 / 1) to give a yellow oily liquid I-4 (113 mg, 87%). 1 H NMR(300MHz,Chloroform-d)δ7.39(d,J=8.3Hz,2H),7.15(d,J=7.9Hz,2H),5.81(s,1H),3.92(d,J=3.9Hz,2H),3 .57-3.29(m,2H),3.13(s,3H),2.71(d,J=8.6Hz,2H),2.62-2.33(m,2H),2.18-1.54(m,12H),1.50-1.04(m,6H); 13C NMR (126MHz, CDCl3) δ191.30,187.68,172.00,170.20,154.65,143.52,143.41,137.90,137.04,136.45 ,130.77,127.66,47.83,42.79,38.09,37.42,37.13,28.58,23.26,14.12,12.74,12.13.HRMS(ESI)for C26H34N2O4S2[M+H] + calcd502.19600, found 503.20260.

[0120] Example 5: Synthesis of Compound I-5

[0121]

[0122] Following the method in Example 1, 4a in Example 1 was replaced with 4c (432 mg, 1 mmol), and 5a was replaced with 5b, to obtain a yellow oily liquid I-5 (73 mg, 12%). 1 H NMR (500MHz, CDCl3) δ7.38(d,J=7.7Hz,2H),7.17(d,J=8.0Hz,2H),6.32-6.18(m,1H),4.79(d,J=9.0Hz,1H),3.99(s,2H),3.78 (s,3H),3.16(s,3H),2.76(s,2H),2.12(s,3H),2.08(s,3H),2.02(s,3H),1.98(s,3H),1.44(s,3H),1.40(s,3H),1.32(s,6H). 13 C NMR (126MHz, CDCl3) δ191.2,187.7,172.0,170.7,169.8,154.7,143.6,143.4,137.8,136.7,136.3,130.7 ,127.7,58.8,52.5,52.3,47.7,44.7,38.1,37.1,28.5,26.1,24.8,23.3,14.1,12.7,12.1.HRMS(ESI)for C 30 H 40 N₂O₆S₂Na[M+Na] + calcd 611.2220, found 611.2212.

[0123] Example 6 Synthesis of Compound I-6

[0124]

[0125] Synthesis of tert-butyl(2-(N,3-dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)butamido)ethyl)(methyl)carbamate (7):

[0126] Compound 6 (498 mg, 2 mmol), methyl (2-(methylamino)ethyl)carbamate tert-butyl ester (404 mg, 2 mmol), and DMAP (163 mg, 1.34 mmol) were placed in a round-bottom flask, and dichloromethane (20 mL) was added. Then, EDCI (499 mg, 2.6 mmol) was added in portions, and the mixture was reacted at room temperature for 3 h. The reaction was monitored by TLC until complete, and the mixture was extracted with DCM (3 × 20 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the crude product was purified by column chromatography (dichloromethane / methanol = 50 / 1 to 10 / 1) to give a yellow oily liquid 7 (724 mg, 86%).

[0127] Synthesis of N,3-dimethyl-N-(2-(methylamino)ethyl)-3-(2,4,5-trimethyl-3,6-dioxocyclohex-1,4-dien-1-yl)butyramide trifluoroacetate (8):

[0128] Compound 7 (419 mg, 1 mmol) was placed in a round-bottom flask, and dichloromethane (24 mL) was added. Trifluoroacetate (8 mL) was added dropwise, and the reaction mixture was allowed to react at room temperature for 2 h. The reaction was monitored by TLC until complete, and the solution was concentrated by rotary evaporation to obtain a yellow solution. Then, NaHCO3 solution (50 mL) was added, and the resulting mixture was extracted with DCM (3 × 15 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (dichloromethane / methanol = 50 / 1 to 10 / 1) to give a white solid 8 (275 mg, 63%).

[0129] Synthesis of (R)-2-acetamido-3-((2-(N,3-dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)butamido)ethyl)(methyl)carbamoyl)dithio)-3-methylbutyrate (I-6):

[0130] N-acetyl-penicillin methyl ester (246 mg, 1.2 mmol) was placed in a double-necked flask (25 mL), and anhydrous dichloromethane (5 mL) was added. After Ar replacement, chlorocarbonyl sulfinyl chloride (0.11 mL, 1.26 mmol) dissolved in anhydrous dichloromethane (2 mL) was added dropwise at 0 °C. The resulting solution was stirred for 1 h and concentrated by rotary evaporation. The residue (crude product 9) was used directly in the next step without purification.

[0131] Compound 8 (419 mg, 1 mmol) and triethylamine (0.3 mL, 2.1 mmol) were placed in a two-necked flask (25 mL), and dichloromethane (5 mL) was added. The mixture was cooled to 0 °C, and compound 9 was dissolved in dichloromethane. Compound 9 was added dropwise at 0 °C, and the reaction was continued for 3 h under the same conditions. The reaction was monitored by TLC until complete. The mixture was quenched with ice water (10 mL) and extracted with DCM (3 × 10 mL). The organic phases were combined, washed with saturated saline solution, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by column chromatography (dichloromethane:methanol = 50 / 1 to 10 / 1) to give a yellow solid I-6 (325 mg, 56%). 1 H NMR(500MHz,Chloroform-d)δ4.58-4.47(m,1H),3.72(s,3H),3.60-3.42(m,4H),3.06(s,2H),3.01(s,3H),2.99-2.93(m,2 H),2.12(d,J=4.0Hz,3H),2.07-2.03(m,3H),1.95-1.89(m,6H),1.45(s,3H),1.41(s,6H),1.33(s,3H),1.28-1.22(m,2H); 13 C NMR (126MHz, CDCl3) δ191.48,187.94,172.84,170.74,167.65,154.67,143.32,138.46,136.90,59.51,53.77,52.93,52 .45,48.38,47.90,45.21,37.77,36.17,29.99,29.01,28.96,26.95,26.28,23.24,14.55,12.98,12.42.HRMS(ESI)forC 27 H 41 N3O7S2[M+H] + calcd584.2459,found 584.2461.

[0132] Example 7 Synthesis of Compound I-7

[0133] Synthesis of (R)-2-benzylamidinyl-3-((2-(N,3-dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)butamidinyl)ethyl)(methyl)carbamoyl)dithio)-3-methylbutyrate (I-7):

[0134]

[0135] Referring to the method of Example 6, the raw material (R)-2-acetamido-3-mercapto-3-methylbutyrate methyl ester in Example 6 was replaced with (R)-2-benzylamino-3-mercapto-3-methylbutyric acid to prepare Example 7. HRMS(ESI) for C 32 H 44 N3O7S2[M+H] + calcd646.2615, found 646.2618.

[0136] Example 8 Synthesis of Compounds I-8

[0137] Synthesis of (R)-3-(((2-(N,3-dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)butamido)ethyl)(methyl)carbamoyl)dithio)-2-isobutamido-3-methylbutyrate isopropyl ester (I-8):

[0138]

[0139] Referring to the method of Example 6, the raw material (R)-2-acetamido-3-mercapto-3-methylbutyrate methyl ester in Example 6 was replaced with (R)-2-isobutyramide-3-mercapto-3-methylbutyrate isopropyl ester to prepare Example 8. HRMS(ESI) for C 31 H 50 N3O7S2[M+H] + calcd 640.3085, found 640.3088.

[0140] Example 9: Synthesis of Compounds I-9

[0141] Synthesis of (R)-2-acetamido-3-((3-(N,3-dimethyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dien-1-yl)butamido)propyl)(methyl)carbamoyl)dithio)-3-methylbutyrate isopropyl ester (I-9):

[0142]

[0143] Referring to the method of Example 6, the raw material methyl (2-(methylamino)ethyl)carbamate tert-butyl ester was replaced with methyl (3-(methylamino)propyl)carbamate tert-butyl ester; and (R)-2-acetamido-3-mercapto-3-methylbutyrate methyl ester was replaced with (R)-2-isobutyrylamino-3-mercapto-3-methylbutyrate methyl ester, to obtain compound I-10. HRMS(ESI) for C 30 H 48 N3O7S2[M+H] +calcd626.2928,found 626.2931.

[0144] Example 10 Synthesis of Compound I-10

[0145]

[0146] Synthesis of 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dienyl)butyritin S-triphenyl ester (21):

[0147] Compound 6 (196 mg, 0.8 mmol) and EDCI (226 mg, 1.2 mmol) were placed in a round-bottom flask, DCM (7 mL) was added, the mixture was cooled to 0 °C, TrtSH (216 mg, 0.8 mmol) and DMAP (10 mg, 0.1 mmol) were added, and the mixture was transferred to room temperature and reacted for 24 h. The reaction was monitored by TLC until complete. The mixture was extracted with dichloromethane (3 × 20 mL), the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 3 / 1) to give a white solid 21 (266 mg, 67%).

[0148] Synthesis of 3-methyl-3-(2,4,5-trimethyl-3,6-dioxane-1,4-dienyl)butyritin (22):

[0149] Compound 21 (254 mg, 0.5 mmol) was placed in a double-necked flask, DCM (6 mL) was added, Ar was used for displacement, then triisopropylsilane (158 mg, 1 mmol) and trifluoroacetic acid (113 mg, 1 mmol) were added, and the reaction was carried out at room temperature for 30 min. After the reaction was completed by TLC monitoring, the solvent was removed by rotary evaporation, and the crude product was purified by column chromatography (petroleum ether / ethyl acetate = 2 / 1) to give colorless liquid 22.

[0150] Synthesis of (R)-2-isobutyramide-3-methyl-3-(pyridyl-2-dithioalkyl)butyrate methyl ester (24):

[0151] Compound 23 (233 mg, 1 mmol) and dithiopyridine (264 mg, 1.2 mmol) were placed in a double-necked flask, purge with Ar for 30 min, and reacted overnight at room temperature. The reaction was monitored by TLC until complete. The solvent was removed by direct rotary evaporation, and the crude product was purified by column chromatography (dichloromethane / methanol = 20 / 1) to give a pale yellow solid (266 mg, 78%).

[0152] Synthesis of (R)-2-acetamido-3-methyl-3-((3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexyl-1,4-dienyl)butyryl)dithioalkyl)isopropyl butyrate (I-10):

[0153] Compound 24 (171 mg, 0.5 mmol) and compound 22 (133 mg, 0.5 mmol) were placed in a double-necked flask, DCM (8 mL) was added, and Ar was bubbled over the atmosphere for 30 min. The reaction was allowed to proceed overnight at room temperature. The reaction was monitored by TLC until complete. The solvent was removed by rotary evaporation, and the crude product was purified by column chromatography (dichloromethane / methanol = 20 / 1) to give I-10. HRMS (ESI) for C 30 H 48 N3O7S2[M+H] + calcd626.2928,found 626.2931.

[0154] Example 11 In vitro antioxidant activity assay

[0155] (1) Cell culture: Complete culture medium preparation: DMEM / F-12 (1:1) medium with 10% FBS and 1% penicillin and streptomycin added; HK-2 cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences, cultured in complete culture medium, seeded in T25 vials, and cultured in a 5% CO2 incubator (37℃). The medium was changed every other day. When the cells grew to about 80%, they were digested and passaged with 0.1% trypsin for 3 min each time, and centrifuged at 1000 rpm for 5 min.

[0156] (2) Preparation of H2O2 solution: The molar absorptivity of H2O2 at a wavelength of 240 nm is ε = 43.6 M. -1 cm -1 The solution was diluted 1000 times, and the absorbance A was measured to be 0.384. The concentration was determined according to the Lambert-Beer law, and then diluted with ultrapure water as needed.

[0157] (3) H2O2 modeling: H2O2 was diluted to the target concentration with DMEM / F-12 (1:1) medium, with a volume ratio of 1‰. The final concentrations were 0 (blank control group), 200, 400, 500, 800, 1000, and 2000 μM. HK-2 cells were seeded in 96-well plates at a density of 5000 cells / well. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. The medium in the 96-well plates was replaced with medium containing H2O2, 100 μL per well, and the plates were incubated for 24 hours. The medium was discarded, and the cells were washed three times with PBS. DMEM / F-12 (1:1) medium containing 10% CCK8 was added, and the plates were incubated at 37°C in a shaker for 1 hour until the absorbance of the blank control group was around 1.0. The absorbance was measured using a microplate reader (λ = 450 nm). Cell viability was calculated.

[0158] (4) Cisplatin-based cell modeling: Cisplatin was dissolved in DMSO and then diluted with DMEM / F-12 (1:1) medium at a volume ratio of 1‰, resulting in final concentrations of 2, 5, 10, 20, 40, and 80 μM. HK-2 cells were seeded in 96-well plates at a density of 5000 cells / well. The plates were incubated at 37°C with 5% CO2 for 24 hours. The medium in the 96-well plates was then replaced with cisplatin-containing medium, 100 μl per well, and incubated for 24 hours. The medium was discarded, and the cells were washed three times with PBS. DMEM / F-12 (1:1) medium containing 10% cck8 was added, and the plates were incubated at 37°C with a shaker for 1 hour until the absorbance of the blank control group was approximately 1.0. The absorbance was measured using a microplate reader (λ = 450 nm). Cell viability was calculated.

[0159] (5) Cell viability: The compound and the control drug N-acetylcysteine ​​(NAC) were dissolved in DMSO and then diluted with DMEM / F-12 (1:1) medium at a volume ratio of 1‰, resulting in final compound concentrations of 25, 50, 100, 200, and 400 μM. HK-2 cells were seeded in 96-well plates at a density of 5000 cells / well and incubated for 24 h. Replace the culture medium in the 96-well plate with drug-containing medium, adding 50 μl to each well. Incubate for 30 minutes, then add medium containing H2O2 (initial concentration 1000 mM, final concentration 500 μM) or medium containing cisplatin (initial concentration 80 μM, final concentration 40 μM). Incubate for 24 hours, then discard the medium. Wash cells three times with PBS, add DMEM medium containing 10% CCK8, and incubate at 37°C with a shaker for 1 hour until the absorbance of the blank control group is around 1.0. Measure the absorbance using a microplate reader (λ = 450 nm). Calculate cell viability.

[0160] (6) The experimental results are shown in Table 1: (n=6)

[0161] Table 1 Evaluation of the antioxidant activity of compounds

[0162]

[0163] The compounds exhibit varying degrees of antioxidant activity, with superior activity compared to NAC; compounds I-5 and I-6, in particular, demonstrate significantly enhanced antioxidant activity. This indicates that the compounds disclosed in this invention can be applied to kidney damage caused by oxidative stress or chemotherapy drugs (such as cisplatin), and provides new insights for related drug development and treatment.

[0164] Example 12 Validation of intracellular release of RSSH from the compound

[0165] Experimental Principle: The experiment was conducted in accordance with the literature (Chem. Eur. J. 2022, 28, e202200540 and Nitric Oxide 2021, 116, 47-64). One molecule of prodrug compound can release one molecule of RSSH and the metabolic byproduct NMI into the cell. RSSH can be captured by the trapping agent β-(4-hydroxyphenyl)ethyliodoacetamide (HPE-IAM). This invention investigates the intracellular release of the aforementioned drug by LC-MS / MS.

[0166]

[0167] Experimental methods:

[0168] HK-2 cell line was seeded in 12-well cell culture plates (2×10⁶ cells / well). 5 (Cells / well). Compound I-6 (0, 50, 100, and 200 μM) was added to each well containing serum-free DMEM / F-12 medium. After incubation at 37°C for 30 min, the drug-containing medium was removed, cells were washed three times with PBS, and lysed in cold methanol solution of HPE-IAM (1 mM). The mixture was vortexed for 5 min to obtain more complete analyte extraction and protein precipitation, and then centrifuged at 18,000 g for 5 min. Aliquots (50 μL) of the supernatant were transferred to new tubes, diluted with acetonitrile solution containing warfarin (250 μL), and finally evaporated with a nitrogen stream. The resulting dried sample was reconstituted with 100 μL of acetonitrile-water (1:1, v / v) and centrifuged again at 30,000 g before LC-MS / MS analysis. The analytical instruments and conditions were the same as those in the literature above. HPE-IAM was purchased from COMBI-BLOCKS, USA. Protein content was determined using the BCA colorimetric protein assay kit.

[0169] The mass spectrometry conditions and parameters are shown in Table 2 below:

[0170] Table 2 Mass Spectrometry Parameters

[0171]

[0172] (5) Experimental Results

[0173] The results of intracellular release of compound I-6 are shown in the appendix. Figure 3 The results showed that compound I-6 could release RSSH intracellularly in a dose-dependent manner. Furthermore, NMI, a metabolic byproduct associated with the release of RSSH, was detected intracellularly. Other compounds of this invention also showed similar results. These experimental results clearly demonstrate that the compounds disclosed in this invention possess the ideal property of intracellular RSSH release, which will provide a novel solution for studying the many biological properties of RSSH and related drug development.

[0174] Example 13: In vivo efficacy evaluation of compound I-6

[0175] Experimental principle: This invention uses sulfasalazine (SASP) as a positive control to investigate whether compound I-6 can alleviate acute ulcerative colitis induced by sodium dextran sulfate (DSS) in mice.

[0176] Experimental animals: C57BL / 6Slac mice (Shanghai Bikai Keyi Biotechnology Co., Ltd., weighing 20g), 6-8 weeks old, n=8.

[0177] Experimental protocol: ① Modeling and drug administration (drug dosage is 100mg / kg, solvent is 0.5% CMC-Na, once daily, administered by gavage; except for the blank group, animals in each group have free access to 2.5% DSS solution): Modeling was carried out from day 0, and drug administration was performed simultaneously with modeling for 7 days; ② DSS was stopped and drinking water was replaced on day 8, while drug administration continued; ③ Animals were sacrificed on day 11 and samples were collected.

[0178] Experimental groups: (A) Blank group: daily gavage with solvent, free access to purified water; (B) Model group: daily gavage with solvent, free access to 2.5% DSS solution; (C) SASP group: daily gavage with SASP, free access to 2.5% DSS solution; (D) Compound I-6 group: daily gavage with compound I-6, free access to 2.5% DSS solution.

[0179] Efficacy indicators:

[0180] ①DAI (Disease Activity Index) score, see Table 3:

[0181] Table 3 Scoring Criteria

[0182]

[0183] The fecal occult blood test uses the o-toluidine-glacial acetic acid method. The principle of this method is that heme in hemoglobin has peroxidase-like activity, which can catalyze hydrogen peroxide to release nascent oxygen, oxidizing o-toluidine to o-toluidine, which turns blue.

[0184] The experimental results are shown in Table 4:

[0185] Table 4. DAI score on the tenth day after modeling.

[0186]

[0187] Experimental conclusion: Compound I-6 reduced the degree of bloody stool and loose stool in mice caused by DSS, and its efficacy was better than that of the positive control SASP.

[0188] ② Weight changes

[0189] Experimental method: During the 10 days of drug administration, the weight of mice was measured and recorded daily, and the weight change of mice on the last day of drug administration and the first day of drug administration was calculated.

[0190] The experimental results are attached. Figure 4 :

[0191] Experimental conclusion: Compound I-6 can significantly inhibit DSS-induced weight loss in mice, and its remission effect is significantly better than that of the positive control drug SASP.

[0192] ③ Colon length

[0193] Experimental method: On day 11, the mice were sacrificed and their abdominal cavities were dissected along the midline. The cecal pouch and the lower end of the colon were removed. The length of the colon was measured and recorded using a ruler.

[0194] The experimental results are shown in Table 5:

[0195] Table 5. Colon Length Measurement

[0196]

[0197] Experimental conclusion: The colon length of mice in the compound I-6 group was significantly longer than that in the model group, and significantly better than that of the positive drug SASP. This indicates that compound I-6 can better alleviate the colon shortening and damage caused by DSS in mice.

[0198] ④ Content of myeloperoxidase (MPO) in colon tissue

[0199] Experimental methods: Mouse colon tissue was taken, and 30 mg of intestinal segment was cut into an EP tube. 300 μL of physiological saline was added to homogenize the tissue to prepare a colon homogenate with a concentration of 10 w / v%. After centrifugation at 5000g for 5 min, 100 μL of the tissue supernatant was collected into an EP tube. Myeloperoxidase activity in the tissue was measured by colorimetric method (myeloperoxidase test kit was obtained from Nanjing Jiancheng Bioengineering Institute).

[0200] The experimental results are shown in Table 6:

[0201] Table 6 MPO activity in colon tissue

[0202]

[0203] Experimental conclusions: MPO is a key enzyme in the production of hypochlorous acid, which is a significant cause of oxidative stress. A DSS-induced mouse ulcerative colitis model leads to increased MPO activity in the mouse colon. Unexpectedly, compound I-6 significantly reduced MPO activity in the mouse colon, to a greater extent than SASP and the control group. These results suggest that compound I-6 possesses ideal antioxidant activity and can inhibit MPO-induced colonic damage. Other compounds in this invention also showed similar results.

[0204] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that those skilled in the art can make improvements or modifications based on the above description, and such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof: ， Formula (I), in, R 1 Selected from: C 1-6 Alkyl, C 6-10 Aryl; R 2 Selected from: H or C 1-6 alkyl; R 3 Selected from: H or C 1-6 alkyl; R 4 Selected from: H, COOH or COOR 5 ; Among them, R 5 Selected from: C 1-6 alkyl; L is selected from: , , Or chemical bonds; Wherein, X is selected from: H, halogen, C 1-6 Alkyl, CH3O, CN, or NO2; X can be monosubstituted or polysubstituted at any position; Y is selected from: O, S, NH or NCH3; R 6 Selected from: H or C 1-3 alkyl; R 7 Selected from: H or C 1-3 alkyl; n is selected from: 1, 2, 3 or 4.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof: in, R 1 Selected from: C 1-6 Alkyl, phenyl; R 2 Selected from: H or C 1-3 alkyl; R 3 Selected from: H or C 1-3 alkyl; R 4 Selected from: H, COOH or COOR 5 ; Among them, R 5 Selected from: C 1-6 alkyl; L is selected from: , , Or chemical bonds; Wherein, X is selected from: H, halogen, C 1-6 Alkyl, CH3O, CN, or NO2; X can be monosubstituted or polysubstituted at any position; Y is selected from: O, S, NH or NCH3; R 6 Selected from: H or C 1-3 alkyl; R 7 Selected from: H or C 1-3 alkyl; n is selected from: 1 or 2.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, in, R 1 Selected from: C 1-6 Alkyl, phenyl; R 2 Selected from: H or methyl; R 3 Selected from: H or methyl; R 4 Selected from: H, COOH or COOR 5 ; Among them, R 5 Selected from: C1-6 alkyl groups; L is selected from: , , Or chemical bonds; Wherein, X is selected from: H, F, Cl, Br, I, methyl, CH3O, CN or NO2; X can be monosubstituted or polysubstituted at any position; Y is selected from: O, S, NH or NCH3; R 6 Selected from: H or methyl; R 7 Selected from: H or methyl; n is selected from: 1 or 2.

4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that... R 1 Selected from: methyl, ethyl, isopropyl, phenyl; R 2 Selected from: H or methyl; R 3 Selected from: H or methyl; R 4 Selected from: H, COOH or COOR 5 ; Among them, R 5 Selected from: methyl, ethyl, or isopropyl; L is selected from: , , Or chemical bonds; Wherein, X is selected from: H; Y is selected from: O, S, NH or NCH3; R 6 Selected from: H or methyl; R 7 Selected from: H or methyl; n is selected from: 1 or 2.

5. Any of the compounds shown in the table below, or a pharmaceutically acceptable salt thereof: 。 6. A pharmaceutical composition, characterized in that, The composition contains the compound of any one of claims 1-5 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

7. The use of any compound of claims 1-5 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 6 in the preparation of an antioxidant.

8. The use of any compound of claims 1-5 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 6 in the preparation of a medicament; The drug is used for the prevention and / or treatment of diseases related to abnormal oxidative stress.

9. The application according to claim 7 or 8, characterized in that, The antioxidant or drug dosage form is a tablet, pill, suppository, or injection.

10. The application according to claim 7 or 8, characterized in that, The antioxidants or drugs mentioned herein are administered intravenously or parenterally.

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