A new type of sulfur-containing silybin derivatives, and preparation method and application thereof

Abstract

The application discloses a novel sulfur-containing silybin derivative, a preparation method and application thereof. The novel sulfur-containing silybin derivative is shown as formula (I), wherein C2-C3 is a carbon-carbon single bond or a carbon-carbon double bond; R1 is H, aliphatic acyl, aromatic acyl, sulfonyl, carbamoyl, thiocarbamoyl or R4; R2 and R3 are H or alkyl-substituted amine methylene. The silybin derivative and a medicinal salt thereof can be used for preparing a liver protection drug. The sulfur-containing silybin derivative has good antioxidant activity and free radical scavenging activity. A representative compound 2 can effectively improve liver function, reduce glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase levels, improve liver oxidative stress level, and relieve acute liver injury and chronic liver injury.

Description

Technical Field

[0001] The present invention relates to a novel sulfur-containing silybin derivative, a preparation method thereof, and applications thereof in liver protection and the like. Background Art

[0002] The liver is the largest digestive gland in the human digestive system. When the liver is continuously stimulated by viruses, alcohol, drugs, etc., it may cause liver cell damage, which can then develop into liver disease. According to statistics, approximately 2 million people die from liver disease each year. Among them, non-alcoholic fatty liver disease (NAFLD) is currently the most common chronic liver disease in the world. It is estimated that the number of people suffering from NAFLD has reached 25% of the global population and will continue to increase in the next 10 years. NAFLD can be divided into early primary fatty liver, steatohepatitis, liver fibrosis, and finally cirrhosis and liver cancer according to the degree of liver tissue lesions. Non-alcoholic steatohepatitis (NASH) is a key transition stage in the development of NAFLD to hepatocellular carcinoma. Liver pathology at this stage can still be reversed. How to control the degree of liver fibrosis and reverse liver damage at this stage is particularly important.

[0003] Silymarin is a polyphenolic flavonoid lignin compound with antioxidant activity extracted and isolated from the seeds of the Asteraceae plant, Silybum marianum. Silybin, the primary hepatoprotective active ingredient in Silybum marianum, has been used to treat various liver diseases, protecting the liver from oxidative stress, inflammation, steatosis, fibrosis, and liver cancer. However, silybin's poor solubility results in a low oral bioavailability (less than 1%), significantly limiting its clinical application. Summary of the Invention

[0004] One of the purposes of the present invention is to provide a new class of sulfur-containing silybin derivatives, the structure of which is shown in formula (I):

[0005]

[0006] Among them, (1) there is a carbon-carbon single bond -CH-CH- or a carbon-carbon double bond -C=C- between C2 and C3.

[0007] (2) R1 is H, fatty acyl, aromatic acyl, sulfonyl, carbamoyl, thiocarbamoyl or R4,

[0008] R4 is shown below:

[0009]

[0010] (3) R2 and R3 are H or alkyl-substituted amine methylene.

[0011] Preferably, the novel sulfur-containing silybin derivatives include but are not limited to compounds represented by Formulas 1 to 15:

[0012]

[0013] The second object of the present application is to provide a preparation method of the novel S-containing silybin derivatives.

[0014]

[0015]

[0016] (1) Preparation of compounds 1 and 4

[0017] Under the protection of nitrogen, triphenylphosphine is dissolved in anhydrous tetrahydrofuran, and diisopropyl azodicarboxylate is slowly added dropwise under ice-bath stirring. After the addition is completed, white precipitates are observed. Then, silybin or 2,3-dehydrosilybin dissolved in anhydrous tetrahydrofuran is added to the reaction solution, and finally thioacetic acid is added. The reaction is continued at room temperature for 30 minutes. After the reaction is completed, ethyl acetate is used for extraction, and the organic phases are combined and washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent is rotary evaporated and purified by silica gel column chromatography to obtain compounds 1 and 4. The molar mass ratio of silybin or 2,3-dehydrosilybin, triphenylphosphine, diisopropyl azodicarboxylate, and thioacetic acid is 1:1-6:1-6:1-6, and the amount of the solvent tetrahydrofuran used is 1-50 milliliters per 1 gram of silybin.

[0018] (2) Preparation of compounds 2 and 5

[0019] Compound 1 or compound 4 is dissolved in 1M HCl / MeOH solution, and stirred at room temperature for 3-4 hours. After the reaction is completed, ethyl acetate is used for extraction, and the organic phases are combined and washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent is rotary evaporated and purified by silica gel column chromatography to obtain compounds 2 and 5. The amount of 1M HCl / MeOH solution used is 1-50 milliliters per 1 gram of compound 1 or 4.

[0020] (3) Preparation of compounds 3 and 6

[0021] Compound 2 or compound 5 is subjected to condensation reaction under the action of an oxidant iodine for about 10-20 minutes. After the reaction is completed, saturated sodium thiosulfate solution is used for quenching, ethyl acetate is used for extraction, and the organic phases are combined and washed with saturated brine, dried with anhydrous sodium sulfate, and the solvent is rotary evaporated to obtain compounds 3 and 6. The molar mass ratio of compound 2 or 5 to iodine is 1:0.1-3, and the solvent used is dimethyl sulfoxide, with 1-50 milliliters of solvent used per 500 milligrams of compound 2 or 5.

[0022] (4) Preparation of compounds 7 to 15

[0023] Compound 1 or 2 was dissolved in methanol, and 37% formaldehyde aqueous solution and the corresponding secondary amine were added sequentially. The reaction was carried out at room temperature for 12 to 30 hours. After the reaction, the mixture was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin drying. The mixture was then separated and purified by silica gel column chromatography to obtain compounds 7 to 15. The molar mass ratio of compound 1 or 2, 37% formaldehyde aqueous solution, and the corresponding secondary amine was 1:0.5-4:0.5-4. The amount of methanol solvent used was 1 to 25 ml per 250 mg of compound 1 or 2.

[0024] The third object of the present invention is to provide the novel sulfur-containing silybin derivatives and pharmaceutically acceptable salts thereof for use in preparing hepatoprotective drugs.

[0025] The novel sulfur-containing silybin derivatives provided by the present invention exhibit excellent antioxidant and free radical scavenging activities. Representative compound 2 can effectively improve liver function, reduce alanine aminotransferase and aspartate aminotransferase levels, alleviate liver oxidative stress, and alleviate acute and chronic liver damage. Furthermore, rat studies have shown that compound 2 has an oral bioavailability 28 times that of silybin. It can be used to prevent and treat acute and chronic liver damage caused by various causes, including nonalcoholic steatohepatitis, alcoholic steatohepatitis, drug-induced liver injury, liver fibrosis, and cirrhosis.

[0026] The present invention uses a semi-synthetic method to prepare silybin C 23 The antioxidant active group sulfhydryl was cleverly introduced, and then its thioether dimer and a series of silybin C were synthesized. 23 A series of novel sulfur-containing silybin derivatives were designed and synthesized by combining thioester compounds at the C6 and C8 positions with hydrophilic tertiary amines to improve solubility. Compared to silybin, these novel sulfur-containing silybin derivatives exhibit better antioxidant activity and solubility, and are therefore valuable for the development and utilization of hepatoprotective drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The proliferation inhibitory activity of silybin and its sulfur-containing derivatives 1-5 on HSC-T6 cells;

[0028] Figure 2 Effects of compound 2 on body weight growth rate (A) and liver index (B) in rats with acute liver injury induced by CCl4;

[0029] Figure 3 Effects of compound 2 on the pathological morphology of liver tissue in rats with acute liver injury (A) and the oil droplet area stained with Oil Red O (B);

[0030] Figure 4 Effects of compound 2 on serum liver injury indicators AST (A), ALT (B), and LDH (C) in rats with acute liver injury;

[0031] Figure 5 Effect of compound 2 on SOD (A) and MDA (B) in acute liver injury rats

[0032] Figure 6 Effect of compound 2 on body weight gain rate (A) and liver index (B) in CCl4 and high-fat diet induced chronic liver injury mice

[0033] Figure 7 Effect of compound 2 on liver morphology (A), liver histopathology (B), oil red O staining oil droplet area (C) and Masson staining fibrosis area (D) in chronic liver injury mice

[0034] Figure 8 Effect of compound 2 on ALT (A) and AST (B) in chronic liver injury mice

[0035] Figure 9 Effect of compound 2 on TG (A), CHO (B), LDL (C) and HDL (D) in chronic liver injury mice

[0036] Figure 10 Effect of compound 2 on SOD (A), MDA (B) and GSH-Px (C) in chronic liver injury mice

[0037] Figure 11 Pharmacokinetic curve of shugan jingui and compound 2 in rats. DETAILED DESCRIPTION

[0038] Example 1

[0039] Preparation of compound 1

[0040]

[0041] Under nitrogen, triphenylphosphine (3.144 g, 12 mmol, 3 eq) was dissolved in 30 ml of anhydrous tetrahydrofuran. Diisopropyl azodicarboxylate (DIAD, 2.360 ml, 12 mmol, 3 eq) was slowly added dropwise at 0°C. After the addition was complete, a milky white precipitate was observed. Silybin (1.928 g, 4 mmol, 1 eq) and thioacetic acid (0.857 ml, 12 mmol, 3 eq) dissolved in 10 ml of anhydrous tetrahydrofuran were added to the reaction solution, which was then warmed to room temperature. The reaction solution was stirred at room temperature for 30 minutes and the reaction progress was monitored by TLC. After the reaction, calcium bromide (4.797 g, 24 mmol, 6 eq) was added to the reaction solution and the mixture was stirred at room temperature for 4 h. A white precipitate was observed and the triphenylphosphine oxide-calcium bromide complex was removed by filtration. The filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by silica gel column chromatography with an eluent of PE / DCM / EA = 4 / 2 / 1 (v / v / v) to obtain compound 1 (1.5316 g, 71% yield) as a light yellow solid. 1 H NMR (500MHz, DMSO-d6) δ11.89(s,1H),10.85(s,1H),9.23(s,1H),7.11(dd,J=4.0,2.0Hz,1H),7.03(d,J=7.6Hz, 2H),6.97(dd,J=8.3,1.8Hz,1H),6.88(dd,J=8.2,1.9Hz,1H),6.83(d,J=8.1Hz,1H),5.92(d,J=2.1Hz,1H),5.88 (t,J=2.4Hz,1H),5.82(d,J=6.2Hz,1H),5.09(d,J=11.3Hz,1H),4.83(d,J=7.8Hz,1H),4.61(dt,J=11.7,6.0Hz, 1H), 4.38 (m, J = 4.3Hz, 1H), 3.79 (s, 3H), 3.08 (dd, J = 14.1, 3.5Hz, 1H), 2.95 (dd, J = 14.1, 7.6Hz, 1H), 2.33 (s, 3H). 13 C NMR (126MHz, DMSO-d6) δ198.2,194.7,170.8,167.3,163.8,162.9,148.2,147.8,143.7,143.4,131.1,127.1,121.9,121.3,117.1,1 16.8,115.9,112.3,101.0,96.5,95.5,82.9,78.4,76.1,71.9,60.2,56.2,30.9,30.1,21.2,14.6.HRMS(ESI)m / z[M+H]+calcd.For[C 27 H25 O 10 S]+,541.1163; found 541.1165.

[0042] Example 2

[0043] Preparation of compound 2

[0044]

[0045] Compound 1 (2.16 g, 4 mmol, 1 eq) was dissolved in 1 mol / L hydrochloric acid methanol solution (20 mL, 5 eq). The reaction mixture was reacted at 40°C for 4 h. The reaction progress was monitored by TLC. After the reaction was completed, water was added to quench the mixture, and the mixture was extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography with an eluent of PE / EA = 2 / 1 (v / v) to obtain compound 2 (1.43 g, 72% yield) as a light yellow solid. 1 H NMR(500MHz,DMSO-d6)δ11.90(s,1H),10.88(s,1H),9.24(s,1H),7.11(dd,J=3.9,1.9Hz,1H),7.06 –7.02(m,2H),7.01(dd,J=8.3,1.7Hz,1H),6.89(d,J=8.0Hz,1H),6.82(d,J=8.1Hz,1H),5.92(d,J= 2.1Hz,1H),5.88(t,J=2.6Hz,1H),5.84(d,J=6.2Hz,1H),5.09(d,J=11.3Hz,1H),4.91(d,J=7.6Hz, 1H),4.62(dt,J=11.4,6.6Hz,1H),4.36–4.30(m,1H),3.79(s,3H),2.65(m,1H),2.56–2.47(m,1H). 13 C NMR (151MHz, DMSO-d6) δ198.3,167.3,163.8,162.9,148.3,147.7,143.7,130.9,127.4,122.0,121.8,121.1,117.2,117.1,11 6.9,116.8,115.9,112.1,100.9,96.5,95.5,82.9,77.9,77.4,71.9,60.2,56.2,25.4,14.6.HRMS(ESI)m / z[M+H]+calcd.For[C 25 H 23 O9S]+,499.1057; found 499.1052.

[0046] Example 3

[0047] Preparation of compound 3

[0048]

[0049] Compound 2 (50 mg, 0.1 mmol, 1 eq) was dissolved in 1 ml of DMSO, and iodine (12.5 mg, 0.05 mmol, 0.5 eq) was added. The mixture was stirred at room temperature for 30 min, and the reaction progress was monitored by TLC. After completion, the reaction was quenched by the addition of saturated sodium thiosulfate solution. The mixture was extracted with EA (3 × 10 mL). The organic phases were combined, washed three times with saturated NaCl solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the title compound 3 (47.8 mg, 96% yield) as a white solid. 1 H NMR (500MHz, DMSO-d6) δ11.89(s,1H),10.86(s,1H),9.22(s,1H),7.11(d,J=6.0Hz,1H),7.02(dd,J=14 .0,6.4Hz,2H),6.92–6.86(m,1H),6.82(d,J=11.0Hz,2H),5.93(d,J=2.0Hz,1H),5.88(dt,J=3.2,1.9H z,1H),5.84(dd,J=6.3,2.7Hz,1H),5.10(d,J=11.5Hz,1H),4.86–4.82(m,1H),4.63(dd,J=14.5,10.4H z,1H),4.45(dt,J=7.7,3.4Hz,1H),3.76(s,3H),2.93(dd,J=17.7,6.4Hz,1H),2.80(d,J=13.8Hz,1H). 13 C NMR (126MHz, DMSO-d6) δ198.2,167.3,163.8,162.9,148.3,147.7,143.6,143.3,131.0,127.2,122.0,121.8,121.2,121. 1,117.2,117.1,116.8,115.9,112.1,100.9,96.5,95.5,82.9,78.0,76.0,71.9,56.1.HRMS(ESI)m / z[M+H]+calcd.For[C 50 H 43 O 18 S2]+,995.1885; found 995.1885.

[0050] Example 4

[0051] Preparation of compound 4

[0052]

[0053] Following the procedure for synthesis of compound 1, from the starting material dehydro- silychristin (0.960 g, 2 mmol), 312 mg of compound 4 was obtained as a yellow solid in 28% yield. 1 H NMR (500 MHz, DMSO-d6) δ 12.41 (s, 1H), 10.84 (s, 1H), 9.63 (s, 1H), 9.26 (s, 1H), 7.77 (d, J = 9.3 Hz, 2H), 7.11 (d, J = 8.4 Hz, 1H), 7.06 (d, J = 1.9 Hz, 1H), 6.90 (dd, J = 8.1, 1.9 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 6.46 (d, J = 2.1 Hz, 1H), 6.20 (d, J = 2.1 Hz, 1H), 4.89 (d, J = 7.7 Hz, 1H), 4.48 (td, J = 7.7, 3.4 Hz, 1H), 3.80 (s, 3H), 3.09 (dd, J = 14.1, 3.5 Hz, 1H), 2.97 (dd, J = 14.2, 7.7 Hz, 1H), 2.34 (s, 3H). 13 C NMR (126 MHz, DMSO-d6) δ 194.7, 176.5, 164.6, 161.2, 156.7, 148.3, 147.9, 146.0, 144.7, 143.8, 136.9, 126.9, 124.7, 121.9, 121.4, 117.3, 116.7, 115.9, 112.3, 103.6, 98.8, 94.1, 78.4, 76.4, 56.2, 30.9, 30.0. HRMS (ESI) m / z [M+H]+ calcd. For [C27H23O10S]+, 539.1006; found 539.1010.

[0054] Example 5

[0055] Preparation of compound 5

[0056]

[0057] Following the procedure for synthesis of compound 2, from the starting material compound 4 (0.992 g, 2 mmol), 280 mg of compound 5 was obtained as a yellow solid in 28% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.41 (s, 1H), 10.84 (s, 1H), 9.62 (s, 1H), 9.24 (s, 1H), 7.78 (d, J = 9.4 Hz, 2H), 7.16 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 2.0 Hz, 1H), 6.92 (dd, J = 8.1, 2.0 Hz, 1H), 6.83 (d, J = 8.1 Hz, 1H), 6.47 (d, J = 2.0 Hz, 1H), 6.20 (d, J = 2.0 Hz, 1H), 4.96 (d, J = 7.6 Hz, 1H), 4.44 (dt, J = 9.2, 5.9 Hz, 1H), 3.80 (s, 3H), 2.71 - 2.64 (m, 1H), 2.57 (q, J = 7.4 Hz, 1H). 13 C NMR (126 MHz, DMSO-d6) δ 176.5, 164.6, 161.2, 156.7, 148.3, 147.8, 146.1, 145.0, 143.8, 136.9, 127.2, 124.6, 121.8, 121.1, 117.4, 116.7, 115.9, 112.1, 103.6, 98.7, 94.1, 77.9, 77.8, 60.2, 56.2, 25.4, 21.2, 14.6. HRMS (ESI) m / z [M+H]+ calcd. For [C25H21O9S]+, 497.0901; found 497.0903.

[0058] Example 6

[0059] Preparation of compound 6

[0060]

[0061] The compound 6 was prepared according to the synthetic method of reference compound 3, from the starting material compound 4 (50 mg, 0.1 mmol), to give 44 mg of compound 6 as a yellow solid in 88% yield. 1H NMR(500MHz,DMSO-d6)δ12.29(s,1H),10.72(s,1H),9.40(s,1H),9.27(s,1H),7.75–7 .72(m,1H),7.58(d,J=2.1Hz,1H),7.04(d,J=8.1Hz,2H),6.88(d,J=7.5Hz,1H),6.84(d ,J=8.1Hz,1H),6.33(d,J=2.0Hz,1H),6.13(d,J=2.0Hz,1H),4.86(d,J=7.6Hz,1H),4. 53(t,J=6.1Hz,1H),3.81(s,3H),2.92(dd,J=14.5,8.6Hz,1H),2.72(d,J=13.3Hz,1H). 13 C NMR (126MHz, DMSO-d6) δ176.2,164.4,161.1,156.5,148.3,147.8,145.7,144.2,143.5,136.8,127.0,124.8,121.8,121.2,117.1,116.5,1 16.0,112.1,103.4,98.6,93.9,77.9,75.7,56.2,22.6,14.4.HRMS(ESI)m / z[M+H]+calcd.For[C50H39O18S2]+,991.1572; found991.1570.

[0062] Example 7

[0063] Preparation of compound 7

[0064]

[0065] Compound 1 (108 mg, 0.2 mmol, 1 eq) was dissolved in 6 mL of methanol. 37% formaldehyde aqueous solution (7.5 μL, 0.1 mmol, 0.5 eq) and 40% dimethylamine aqueous solution (12.7 μL, 0.1 mmol, 0.5 eq) were added to the reaction solution in sequence. The mixture was stirred at room temperature for 18 h. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column chromatography with an eluent of DCM / MeOH = 30 / 1-10 / 1 (v / v) to obtain 47 mg of compound 7 as a white solid in a yield of 39%. 1HNMR (500 MHz, CDC13) δ 7.15 (dd, J = 5.2, 2.1 Hz, 1H), 7.08 - 7.05 (m, 1H), 7.02 (dd, J = 8.2, 2.1 Hz, 1H), 6.96 (d, J = 8.7 Hz, 1H), 6.92 - 6.88 (m, 2H), 6.04 (d, J = 5.6 Hz, 1H), 4.95 (d, J = 11.3 Hz, 1H), 4.74 (d, J = 7.5 Hz, 1H), 4.49 (dd, J = 11.9, 6.1 Hz, 1H), 4.26 - 4.20 (m, 1H), 4.12 (q, J = 7.1 Hz, 1H), 3.93 (s, 3H), 3.91 (s, 2H), 3.23 (dd, J = 14.0, 3.7 Hz, 1H), 2.86 (dd, J = 14.0, 7.2 Hz, 1H), 2.56 (s, 6H), 2.33 (s, 3H). 13 C NMR (126 MHz, CDC13) δ 195.0, 194.8, 171.9, 162.7, 160.8, 147.2, 146.8, 143.8, 129.9, 127.7, 121.3, 121.2, 117.5, 117.4, 116.6, 114.8, 109.7, 100.8, 99.0, 97.2, 82.9, 78.7, 76.1, 72.3, 56.19, 56.18, 54.5, 44.0, 30.62, 29.8.

[0066] Example 8

[0067] Preparation of compound 8

[0068]

[0069] Referring to the synthesis method of compound 7, 44 mg of compound 8 was obtained as a white solid from the starting material compound 2 (100 mg, 0.2 mmol) in the form of a white solid, and the yield was 40%. 1H NMR (500 MHz, DMSO-d6) δ 7.08 (t, J = 2.3 Hz, 1H), 7.04 (t, J = 1.5 Hz, 1H), 7.01 - 6.99 (m, 2H), 6.89 (dd, J = 7.9, 1.9 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H), 5.54 (d, J = 2.6 Hz, 1H), 4.96 (dd, J = 10.8, 1.5 Hz, 1H), 4.90 (d, J = 7.6 Hz, 1H), 4.46 (dd, J = 10.9, 8.2 Hz, 1H), 4.32 (qd, J = 6.4, 3.3 Hz, 1H), 3.83 (s, 2H), 3.79 (s, 3H), 2.65 (dd, J = 14.5, 3.5 Hz, 1H), 2.57 - 2.52 (m, 1H), 2.51 (s, 6H). 13 C NMR (126 MHz, DMSO-d6) δ 194.9, 175.2, 162.2, 161.9, 148.3, 147.8, 143.6, 143.5, 131., 127.4, 121.6, 121.1, 117.0, 116.8, 115.9, 112.2, 100.2, 97.5, 97.1, 82.6, 77.9, 77.4, 71.7, 56.2, 53.2, 43.0, 25.5.

[0070] Example 9

[0071] Preparation of compound 9

[0072]

[0073] To compound 1 (108 mg, 0.2 mmol, 1 eq) dissolved in 6 mL of methanol, 37% formaldehyde aqueous solution (7.5 μL, 0.1 mmol, 0.5 eq) and diethylamine (10.3 μL, 0.1 mmol, 0.5 eq) were added successively, and stirred at room temperature for 24 h. The reaction progress was monitored by TLC. After the reaction was completed, the crude product was concentrated under reduced pressure. The crude product was purified by flash silica gel column chromatography with DCM / MeOH = 30 / 1-10 / 1 (v / v) as eluent to give 50 mg of compound 9 as a white solid with a yield of 40%. 1H NMR(500MHz,DMSO-d6)δ7.07(t,J=2.6Hz,1H),7.05–6.97(m,3H),6.94(dd,J=8.5,1.6Hz,1H),6.87(dd ,J=8.1,2.0Hz,1H),6.82(d,J=8.0Hz,1H),5.53(d,J=2.5Hz,1H),4.96(d,J=10.9Hz,1H),4.82(d,J=7.7 Hz,1H),4.46(dd,J=10.9,7.6Hz,1H),4.37(tdd,J=7.9,5.5,3.6Hz,2H),3.89(s,2H),3.78(s,3H),3.0 7(dd,J=14.0,3.6Hz,1H),2.97–2.89(m,1H),2.83(q,J=7.2Hz,4H),2.32(s,3H),1.13(t,J=7.2Hz,6H). 13 C NMR (126MHz, DMSO-d6) δ194.7,194.3,174.8,161.6,161.1,147.8,147.3,143.2,142.8,131.1,126.7,120.8,1 16.6,116.5,116.3,115.4,111.8,99.2,97.2,96.6,82.1,77.9,75.6,71.2,55.7,47.8,45.8,30.4,29.6,9.7.

[0074] Example 10

[0075] Preparation of compound 10

[0076]

[0077] Referring to the synthesis method of compound 9, 51 mg of white solid compound 10 was obtained from the starting material compound 2 (100 mg, 0.2 mmol) with a yield of 44%. 1H NMR (500 MHz, DMSO-d6) δ 7.08 (t, J = 2.3 Hz, 1H), 7.10 - 6.96 (m, 3H), 6.89 (d, J = 8.2 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 5.53 (d, J = 2.9 Hz, 1H), 4.96 (d, J = 10.8 Hz, 1H), 4.90 (d, J = 7.6 Hz, 1H), 4.46 (dd, J = 10.9, 8.1 Hz, 1H), 4.32 (tp, J = 8.7, 5.4, 4.3 Hz, 1H), 3.89 (s, 2H), 3.79 (s, 3H), 2.84 (q, J = 7.3 Hz, 4H), 2.65 (dd, J = 14.4, 3.5 Hz, 1H), 2.57 - 2.52 (m, 1H), 1.14 (t, J = 7.2 Hz, 6H). 13 C NMR (126 MHz, DMSO-d6) δ 195.2, 175.3, 162.1, 161.6, 148.3, 147.8, 143.7, 143.5, 131.5, 127.4, 121.6, 121.1, 117.0, 116.8, 115.9, 112.2, 99.8, 97.8, 97.1, 82.6, 77.9, 77.4, 71.7, 56.2, 48.3, 46.3, 42.3, 25.5, 12.6, 10.2.

[0078] Example 11

[0079] Preparation of compound 11

[0080]

[0081] Referring to the synthesis method of compound 9, 54 mg of compound 11 in the form of a white solid was obtained from starting material compound 1 (108 mg, 0.2 mmol), pyrrolidine (8.2 μL, 0.1 mmol) at a yield of 43%. 1H NMR(500MHz,DMSO-d6)δ7.07(t,J=2.6Hz,1H),7.03–6.99(m,2H),6.94(dd,J=8.3,1.7Hz,1H), 6.87(dd,J=8.1,1.9Hz,1H),6.82(d,J=8.1Hz,1H),5.48(t,J=2.4Hz,1H),4.96–4.90(m,1H),4 .82(d,J=7.7Hz,1H),4.42(m,1H),4.37(m,1H),3.94(s,2H),3.79(s,3H),3.07(dd,J=14.1,3. 6Hz,1H),2.98(d,J=6.6Hz,4H),2.93(dd,J=14.1,7.7Hz,1H),2.32(s,3H),1.90–1.83(m,4H). 13 C NMR (126MHz, DMSO-d6) δ194.3,193.7,175.6,161.6,161.5,147.8,147.4,143.1,142.7,131.3,126.7,121.4,12 0.8,116.6,116.3,115.4,111.8,100.1,96.9,96.5,82.0,77.9,75.6,71.1,55.7,52.6,49.1,30.4,29.6,22.9.

[0082] Example 12

[0083] Preparation of compound 12

[0084]

[0085] Referring to the synthesis method of compound 9, 47 mg of white solid compound 12 was obtained from raw material compound 2 (100 mg, 0.2 mmol) and pyrrolidine (8.2 μL, 0.1 mmol) with a yield of 40%. 1H NMR (500 MHz, DMSO-d6) δ 7.07 (t, J = 2.4 Hz, 1H), 7.04 - 6.97 (m, 3H), 6.89 (d, J = 8.1 Hz, 1H), 6.82 (d, J = 8.1 Hz, 1H), 5.48 (d, J = 2.6 Hz, 1H), 4.95 - 4.88 (m, 2H), 4.42 (dd, J = 10.8, 8.4 Hz, 1H), 4.31 (qd, J = 6.5, 3.3 Hz, 1H), 3.94 (s, 2H), 3.78 (s, 3H), 2.99 (d, J = 6.4 Hz, 4H), 2.65 (dd, J = 14.4, 3.5 Hz, 1H), 2.57 - 2.52 (m, 1H), 1.89 - 1.84 (m, 4H). 13 C NMR (126 MHz, DMSO-d6) δ 194.2, 176.2, 162.1, 162.0, 148.3, 147.8, 143.6, 143.5, 131.6, 127.4, 121.8, 121.1, 117.0, 116.8, 115.9, 112.2, 100.7, 97.5, 96.9, 82.5, 77.9, 77.4, 71.6, 56.2, 55.4, 53.1, 49.6, 25.5, 23.4.

[0086] Example 13

[0087] Preparation of compound 13

[0088]

[0089] Referring to the synthesis method of compound 9, compound 13 was obtained as a white solid from starting material compound 1 (108 mg, 0.2 mmol), piperidine (9.9 μL, 0.1 mmol) in 53 mg, yield 42%. 1H NMR(500MHz,DMSO-d6)δ7.07(t,J=2.5Hz,1H),7.04–6.99(m,2H),6.95(dd,J=8.3,1.6Hz,1H),6.87(dd, J=8.1,2.0Hz,1H),6.82(d,J=8.1Hz,1H),4.99(d,J=11.0Hz,1H),4.82(d,J=7.7Hz,1H),4.49(dd,J=11. 0,7.1Hz,1H),4.37(tdd,J=8.0,5.0,3.5Hz,1H),3.79(s,5H),3.07(dd,J=14.1,3.6Hz,1H),2.94(dd,J= 14.1,7.6Hz,1H),2.71(t,J=5.4Hz,4H),2.32(s,3H),1.59(t,J=5.8Hz,4H),1.47(q,J=7.6,6.5Hz,2H). 13 C NMR (126MHz, DMSO-d6) δ196.0,194.7,173.2,162.0,161.5,148.3,147.9,143.7,143.3,131.5,127.1,121.7,121. 3,117.0,116.8,115.9,112.3,100.0,98.5,96.7,82.7,78.4,76.1,71.8,56.2,52.7,52.6,30.8,30.1,24.8,23.1.

[0090] Example 14

[0091] Preparation of compound 14

[0092]

[0093] Referring to the synthesis method of compound 9, 42 mg of white solid compound 14 was obtained from the starting material compound 2 (100 mg, 0.2 mmol) and piperidine (9.9 μL, 0.1 mmol) with a yield of 35%. 1H NMR(500MHz,DMSO-d6)δ7.12(dd,J=4.1,1.8Hz,1H),7.06–6.99(m,3H),6.89(dt,J=8.1,1.7Hz,1H), 6.83(d,J=8.0Hz,1H),6.13(d,J=4.3Hz,1H),5.75(s,1H),5.16(d,J=11.4Hz,1H),4.91(d,J=7.6Hz,1 H),4.67(dd,J=11.3,7.1Hz,1H),4.33(ddt,J=11.3,8.9,4.4Hz,1H),4.09(s,2H),3.79(d,J=1.4Hz,3 H),3.34(m,2H),2.94(m,2H),2.66(ddd,J=14.0,8.2,3.4Hz,1H),2.55(q,J=7.4Hz,1H),1.69(m,6H). 13 C NMR (126MHz, DMSO-d6) δ198.8,198.7,166.6,163.4,163.3,148.3,147.8,143.8,143.7,130.6,127.4,122.0,12 1.1,117.1,117.0,116.0,112.2,100.6,97.8,95.2,83.2,77.9,77.5,71.8,56.2,55.4,52.8,25.5,22.7,21.5.

[0094] Example 15

[0095] Preparation of compound 15

[0096]

[0097] Compound 1 (108 mg, 0.2 mmol, 1 eq) was dissolved in 8 mL of methanol. 37% aqueous formaldehyde solution (60 μL, 0.8 mmol, 4 eq) and morpholine (71 μL, 0.8 mmol, 4 eq) were added to the reaction solution in sequence. The mixture was stirred at room temperature for 8 h. The reaction progress was monitored by TLC. After the reaction, the mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column chromatography with DCM / MeOH = 30 / 1-10 / 1 (v / v) as the eluent to afford 121 mg of compound 15 as a white solid in a yield of 82%. 1H NMR (500 MHz, CDC13) δ 7.09 - 7.05 (m, 1H), 7.01 - 6.94 (m, 2H), 6.90 (d, J = 8.2 Hz, 1H), 6.87 - 6.82 (m, 2H), 4.85 (d, J = 11.8 Hz, 1H), 4.69 (dd, J = 10.0, 7.8 Hz, 1H), 4.37 (dd, J = 11.8, 1.4 Hz, 1H), 4.20 (m, 1H), 3.88 (d, J = 2.2 Hz, 3H), 3.75 - 3.57 (m, 12H), 3.18 (dt, J = 14.1, 3.2 Hz, 1H), 2.79 (m, 1H), 2.52 (d, J = 35.6 Hz, 8H), 2.27 (s, 3H). 13 C NMR (126 MHz, CDC13) δ 195.3, 194.6, 168.4, 161.1, 159.9, 147.2, 146.8, 143.8, 129.7, 127.4, 121.3, 120.8, 117.3, 116.4, 116.1, 114.8, 109.7, 102.4, 101.3, 99.2, 82.9, 78.7, 76.0, 72.4, 66.7, 56.1, 53.0, 52.8, 51.4, 30.5, 30.4.

[0098] Example 16

[0099] In vitro antioxidant activity experiment

[0100] 16.1 DPPH free radical scavenging experiment

[0101] Prepare 0.2 mM DPPH solution and sample solutions with different concentrations (6.25, 12.5, 25, 50, 100, 200 mM) respectively with methanol solution. The determination is carried out in a 96-well plate, the test group (A1) is 100 μl DPPH solution and 100 μl sample solution with different concentrations in each well, mixed uniformly, three duplicate wells for each concentration; the negative control group (A2) is 100 μl sample solution with different concentrations and equal volume of methanol solution in each well; the blank control group (A0) is 100 μl DPPH solution and equal volume of methanol solution in each well. The mixture is reacted for 30 minutes at room temperature in the dark, and then the absorbance of each well is determined at 517 nm by an enzyme marker. The DPPH free radical scavenging rate is calculated: 16.2 Superoxide anion radical scavenging experiment

[0102] Tris-HCl buffer solution (pH 8.2) was added to a 96-well plate, and incubated at 25°C for 20 minutes, 80 μL of sample solution of different concentrations was added to each well, and incubated at 25°C for 20 minutes, 20 μL of 3 mM pyrogallol solution was added, mixed well, and reacted at 25°C for 5 minutes, then 20 μL of 10 mM hydrochloric acid solution was immediately added to terminate the reaction. Then the absorbance of each well was measured by a microplate reader at 320 nm, three replicate wells were taken for each concentration, and the average value was taken. The blank control was replaced by the same volume of methanol instead of the sample solution. The superoxide anion radical scavenging rate was calculated:

[0103] 16.3 Iron reduction capacity test experiment

[0104] 250 μL of sodium phosphate buffer solution (0.2 mol / L, pH 6.6) and 250 μL of 1% potassium ferricyanide solution were mixed in a 1.5 mL centrifuge tube, 250 μL of sample solution of different concentrations was added, mixed well, heated in a 50°C water bath for 20 minutes, then 250 μL of 10% trichloroacetic acid (w / v) was added. Then the centrifuge tube was centrifuged in a centrifuge at 1000 rpm for 10 minutes. After centrifugation, 500 μL of the upper layer solution was mixed with 500 μL of deionized water and 100 μL of 0.1% FeCl3, and the absorbance was measured at 700 nm. The assay was performed in a 96-well plate, and the sample was added to the 96-well plate according to the solution volume of 200 μL per well, and the absorbance of each well was measured by a microplate reader at 700 nm, three replicate wells were taken for each concentration, and the average value was taken. The blank control was replaced by the same volume of methanol instead of the sample solution. The iron reduction capacity was calculated:

[0105] 16.4 Lipid peroxidation inhibition rate determination

[0106] (1) Preparation of yolk suspension: Fresh eggs were removed from the egg white, and the yolk was mixed with an equal volume of phosphate buffer solution (0.1 mol / L, pH 7.4) to prepare a 1:1 suspension. The mixture was magnetically stirred at 37°C for 10 minutes, then diluted with PBS buffer to 1:25 yolk suspension, and stored in the refrigerator for standby.

[0107] (2) Color development and determination: 40 μl of the above-mentioned egg yolk suspension, 10 μl of sample solution of different concentrations, and 40 μl of FeSO4 solution (25 mMol / L) were added to a centrifuge tube in sequence. The volume of the solution was made up to 400 μl with PBS buffer, and the centrifuge tube was then incubated at 37°C for 15 minutes. After removal, 100 μl each of 20% trichloroacetic acid solution and 0.8% thiobarbituric acid solution were added to the centrifuge tube, and the mixture was heated in a water bath at 100°C for 15 minutes. After cooling to room temperature, the centrifuge tube was centrifuged at 1000 rpm for 10 minutes, the supernatant was collected, and its absorbance was measured at 532 nm. The determination was performed in a 96-well plate, and the supernatant was added to each well of the 96-well plate at a volume of 200 μl. The absorbance of each well was measured at 532 nm using a microplate reader. Three replicates were used for each concentration, and the average value was taken. A blank control was performed by replacing the sample solution with an equal volume of methanol. Calculation of lipid peroxidation inhibition rate:

[0108] 16.5 Experimental Results

[0109] The in vitro antioxidant activities of compounds 1–6 are shown in Table 1. Compared to silibinin, most of the new sulfur-containing silibinin derivatives exhibited excellent in vitro antioxidant activity, particularly compounds 2 and 5, which exhibited higher activity than all other tested compounds in DPPH and superoxide anion radical scavenging assays. Oxidative stress plays a significant role in the development of various liver diseases, and the potent antioxidant activity of these sulfur-containing derivatives may therefore make them excellent candidates for hepatoprotection.

[0110] Table 1 Antioxidant activity of silybin and compounds 1-6

[0111]

[0112] Example 17 Cytotoxicity Experiment

[0113] (1) Plating: Take HSC-T6 cells and perform the same cell passage procedure. After centrifugation, aspirate the supernatant and add 2 ml of fresh culture medium. Pipette and mix evenly. Pipette 20 μl of the cell suspension into a 1.5 ml centrifuge tube containing 180 μl of PBS buffer. Mix well and count the cells using a counting plate. Dilute to a cell concentration of 5 × 104 cells / ml and add 100 μl per well to a 96-well plate, i.e., 5 × 103 cells per well. Incubate in a cell culture incubator for 12 hours.

[0114] (2) Dosing: Prepare a 200 mmol / L stock solution of the compound using cell-grade DMSO and dilute it with culture medium to 20, 40, 80, and 120 μmol / L sample solutions. After observing cell attachment under a microscope, remove the old culture medium from the 96-well plate with a dispenser and add 100 μl of different sample solutions. Set up three replicate wells for each sample. Set up a blank group consisting of three wells containing only 100 μl of culture medium and a control group consisting of three wells without sample solution. Then, place the 96-well plate in a cell culture incubator and incubate for 24 hours.

[0115] (3) Assay: After incubating the 96-well plate in a cell culture incubator for 24 hours, add 20 μl of 5 mg / ml MTT solution to each well in a dark-protected environment and continue incubating in the cell culture incubator for another 4 hours. Then, remove the 96-well plate, aspirate the old culture medium, add 100 μl of dimethyl sulfoxide solution to each well, shake until the formazan is completely dissolved, and measure the absorbance of each well at 570 nm using a microplate reader.

[0116] (4) Experimental results: Figure 1 Silybin and its sulfur-containing derivatives, compounds 1-3, shown in (A), had no inhibitory effect on HSC-T6 cell proliferation at concentrations of 20, 40, and 80 μM. At 120 μM, compound 1 exhibited similar cell viability to silybin, with both near-100% viability. Furthermore, compounds 2 and 3 exhibited similar proliferation inhibition curves, with cell viability rates of 80.1±1.2% and 82.6±1.7%, respectively, at 120 μM, indicating that compounds 1-3 exhibited little toxicity to HSC-T6 cells at concentrations of 20, 40, 80, and 120 μM.

[0117] Example 18

[0118] Experimental study on acute liver injury induced by CCl4 in rats

[0119] 18.1 Experimental Methods

[0120] Twenty-four male SD rats were randomly divided into four groups: a blank control group, a model group, a silibinin group, and a thiosilibinin derivative group, with six rats in each group. After three days of acclimatization, the blank control group and the liver injury model group rats were gavaged with 0.3% sodium carboxymethylcellulose at a dose of 3 ml / kg. The experimental group rats were gavaged with a drug suspension (3 ml of 0.3% sodium carboxymethylcellulose suspension per 100 mg of drug) at a dose of 100 mg / kg. This was done once daily for seven consecutive days. Two hours after dosing on the seventh day, the blank control rats were intraperitoneally injected with corn oil (2 ml / kg), while the remaining rats were intraperitoneally injected with corn oil containing 50% CCl4 to induce acute liver injury. The rats were then deprived of food but not water. After 16 hours, they were weighed. After anesthesia with 5% chloral hydrate, 1 ml of blood was collected from the heart, placed in an EDTA-soaked EP tube and placed in an ice bath. The blood was centrifuged at 4°C, 8000 rpm / min for 10 min using a low-temperature centrifuge, and the upper serum was collected and frozen at -80°C.

[0121] The rats were then killed with 10% chloral hydrate, dissected, and liver tissue removed. The livers were washed with physiological saline, dried with filter paper, weighed, and photographed. After the rat livers were weighed, the left lobe of the rat liver was removed and immersed in 10% formalin tissue fixative for HE staining and Oil Red O staining. Another 100 mg portion of the right lobe of the rat liver was cut and mixed with physiological saline at a ratio of 1:9, placed in a grinding tube, and ground evenly in a grinder. The mixture was then centrifuged at 3500 rpm for 15 minutes, and the upper serum was aspirated and frozen at -80°C for detection of SOD activity and MDA content. The oil droplet area was counted, and the data were processed and plotted. All data are presented as the mean ± SD of six independent samples.

[0122] 18.2 Compound 2 Reduces Liver Index in Acute Liver Injury

[0123] The weight growth rate of SD rats during the experiment was as follows Figure 2 As shown in (A), during the administration period, the body weight of rats in the normal group, model group, silibinin group, and compound 2 group increased steadily, and the growth rate was similar, indicating that compound 2 had no obvious toxic side effects when administered orally at a dose of 100 mg / kg per day. Figure 2 (B) The results showed that compared with the normal group, the liver-to-body ratio of the model group rats increased significantly, indicating that the liver of the model group was damaged and swollen. Compared with the model group, the liver-to-body ratio of the rats treated with compound 2 was reduced, indicating that compound 2 can prevent liver enlargement caused by CCl4-induced acute liver injury and has a protective effect on the liver.

[0124] 18.3 Compound 2 improves liver tissue pathology in acute liver injury

[0125] Rat liver and pathologically stained sections Figure 3(A) As shown. Compared with the normal group, the liver of the model group rats was pale and rough in appearance, while the liver of the rats after administration of compound 2 was smooth and rosy, indicating that compound 2 can improve the appearance and morphology of the liver. The results of H&E staining showed that the liver tissue structure of the normal group rats was clear, the liver lobules were intact, the liver cell structure and morphology were normal, and there was no inflammation. The liver cells in the model group were obviously damaged, the liver tissue structure was disordered, the liver cells were swollen and necrotic, and there was inflammatory cell infiltration. The silybin group and the compound 2 group showed significant improvement, and the fatty degeneration and inflammatory cell infiltration were significantly reduced, especially for compound 2, the improvement was more obvious. In addition, the statistical results of Oil Red O staining and oil droplet area are shown as follows Figure 3 (B) Compared with the normal group, the model group showed severe fat accumulation. Compound 2 could reduce fat deposition caused by acute liver injury and improve hepatic fatty degeneration, and its effect was better than silybin.

[0126] 18.4 Compound 2 improves serum biochemical parameters in acute liver injury

[0127] Liver damage indicators such as Figure 4 Table 2. The results of plasma biochemical tests showed that compared with the normal group, the three liver injury indicators in the model group were significantly increased, indicating the occurrence of acute injury induced by CCl4, while the serum AST( Figure 4 (A))、ALT( Figure 4 (B)) and LDH( Figure 4 (C)) contents were significantly decreased, indicating that compound 2 has a good preventive effect on CCl4-induced acute injury and its effect is better than silybin.

[0128] Table 2 Effects of compound 2 on liver function indicators in rats

[0129] Group ALT(U / L) AST(U / L) LDH (U / L) Normal group 134.6±29.6 57.4±10.3 210.0±81.0 Model Group 817.7±30.9 439.4±50.0 1153.1±197.8 Silybin group 658.7±60.6 196.5±70.7 392.9±128.5 Compound Group 2 524.7±133.2 144.6±62.2 300.5±140.1

[0130] 18.5 Compound 2 improves oxidative stress indicators in acute liver injury

[0131] The malondialdehyde (MDA) content and superoxide dismutase (SOD) activity in rat liver tissue homogenate were analyzed. Figure 5 As shown in Table 3, the MDA content in the model group liver was approximately five times higher than that in the normal group, indicating abnormal lipid metabolism, increased lipid peroxidation, and oxidative stress in the rat liver. SOD activity was slightly decreased, suggesting insufficient antioxidant capacity in the rats. Both Compound 2 and silibinin reduced MDA content and restored SOD activity in the rats, with Compound 2 demonstrating a more significant improvement in oxidative stress than silibinin.

[0132] Table 3 Effects of A1 on oxidative stress indices in rats

[0133] Group MDA (nmol / mgprot) SOD(U / mgprot) Normal group 1.3±0.3 1733.9±218.5 Model Group 6.6±0.5 1327.8±95.3 Silybin group 4.4±0.4 1513.0±159.8 Compound Group 2 3.3±1.1 1793.7±122.8

[0134] Example 19

[0135] Experimental study on chronic liver injury in mice induced by CCl4 and high-fat diet

[0136] 19.1 Experimental Methods

[0137] Thirty-two C57BL / 6 mice (6 weeks, 20-25g) were randomly divided into four groups (8 mice each)—a normal group, a model group, a silibinin group, and two compound groups. After acclimating in a constant-temperature housing for three days, the animals were fasted for 24 hours on the morning of the third day. On the morning of the fourth day, the normal group received a standard diet, while the remaining groups received a high-fat diet. During this period, the mice were allowed to eat freely. The normal group received a weekly intraperitoneal injection of corn oil at a dose of 0.01ml / g, while the remaining groups received a weekly intraperitoneal injection of 2% CCl₄ at the same dose to induce liver damage. The model was maintained for five weeks, and the mice's body weights were recorded daily.

[0138] Five weeks after modeling, mice in the normal and model groups were gavaged with 0.3% (m) sodium carboxymethylcellulose at a dose of 5 ml / kg, while mice in the other groups were gavaged with the corresponding drug suspension at a dose of 150 mg / kg (drug suspension: 150 mg of drug suspended in 5 ml of 0.3% CMC-Na solution and ground evenly in a mortar). Dosing was continued once daily for four weeks. The normal group continued to eat a normal diet, while the other groups continued to receive a high-fat diet and intraperitoneal injection of CCl4 to maintain liver damage.

[0139] Four weeks after dosing, following the last dose, mice were fasted with or without water. After 16 hours of overnight dosing, they were weighed. Immediately after death by carbon dioxide asphyxiation, approximately 1 ml of intercardial blood was collected. The blood was placed in an EDTA-soaked EP tube and placed in an ice bath. The blood was then centrifuged at 8000 rpm / min for 10 minutes in a low-temperature centrifuge. The supernatant serum was aspirated and stored frozen at -80°C.

[0140] Mice were dissected and liver tissue was removed. The livers were washed with physiological saline, blotted dry with filter paper, weighed, and photographed. The left lateral lobe of the mouse liver was immersed in 10% formalin for preparation of sections stained with hematoxylin and eosin, Oil Red O, and Masson stains. Approximately 100 mg of the right lateral lobe of the mouse liver was excised and frozen in a centrifuge tube for assaying SOD activity, MDA content, and GSH-Px activity.

[0141] The oil droplet area and fibrosis area were statistically analyzed, and the data were processed and plotted. All data were shown as the mean ± SD of eight independent samples.

[0142] 19.2 Compound 2 Improves Liver Index in Chronic Liver Injury

[0143] The weight growth rate of mice Figure 6(A) Compared with the normal group, CCl4 has a more and more significant inhibitory effect on the weight gain rate of the model group mice. Compound 2 and silibinin can reduce the toxicity of CCl4 to a certain extent. Figure 6 (B) The results showed that compared with the normal group, the liver-to-body ratio index of the model group increased significantly, while the liver-to-body ratio index of the treatment group was significantly improved compared with the model group, indicating that compound 2 can alleviate the enlargement of liver volume and fat deposition caused by long-term injection of CCl4, and the effect is comparable to that of silybin.

[0144] 19.3 Compound 2 improves liver tissue pathology in chronic liver injury

[0145] The appearance of the liver of mice in each group was as follows Figure 7 (A), Pathologically stained sections Figure 7 (B) Compared with the normal group, the livers of the mice in the model group were pale and rough. H&E staining showed significant fatty degeneration, inflammatory cell infiltration, and ballooning degeneration of hepatocytes, indicating severe damage and inflammation in the liver tissue. Oil red O staining showed a large amount of fat accumulation, indicating abnormal lipid metabolism in the mouse liver. Masson staining showed that a large amount of collagen fibers were produced in the mouse liver tissue, indicating the occurrence of liver fibrosis. Compared with the model group, the livers of mice treated with silybin and compound 2 were smoother and more rosy. Pathological section results showed that fatty degeneration was significantly improved in the compound 2 group, inflammatory cell infiltration was reduced, and fat deposition and collagen fibers were significantly reduced. The statistical results of fat droplet and collagen fiber area are shown in Figure 2. Figure 7 (C) and Figure 7 (D) The above results indicate that compound 2 can improve the pathological morphology of chronic liver injury induced by CCl4 plus high-fat diet in mice, and its effect is better than silybin.

[0146] 19.4 Compound 2 Improves Liver Function Indices in Chronic Liver Injury

[0147] like Figure 8 As shown in Table 4, compared with the normal group, the ALT ( Figure 8 (A))、AST( Figure 8 (B)) content increased significantly, indicating the occurrence of liver damage. Silibinin and compound 2 treatment could reduce the level of transaminase in plasma, and the effect of compound 2 was better than that of silibinin.

[0148] Table 4 Effects of compound 2 on liver function indicators in mice

[0149]

[0150]

[0151] 19.5 Compound 2 improves blood lipid levels in chronic liver injury

[0152] like Figure 9 As shown in Table 5, compared with the normal group, the triglyceride (TG), cholesterol (CHO), and low-density lipoprotein (LDL) levels in the model group were increased, and the high-density lipoprotein (HDL) content was decreased, indicating that under this model, the blood lipids of mice increased significantly and there were problems with liver fat metabolism. Compound 2 had an improving effect on the above indicators, and the effect was more obvious than that of silybin.

[0153] Table 5 Effects of compound 2 on blood lipid levels in mice

[0154] Group TG mmol / L CHO mmol / L LDL mmol / L HDL mmol / L Normal group 0.72±0.08 2.27±0.15 0.20±0.03 1.47±0.12 Model Group 1.33±0.10 3.15±0.23 0.47±0.06 0.93±0.22 Silybin group 1.01±0.13 2.54±0.10 0.39±0.03 1.21±0.12 Compound Group 2 0.86±0.08 2.39±0.14 0.30±0.02 1.43±0.12

[0155] 19.6 Compound 2 improves oxidative stress indicators in chronic liver injury

[0156] like Figure 10 As shown in Table 6, compared with the normal control group, the model group mice had excessive liver MDA production and significantly decreased SOD and glutathione peroxidase (GSH-Px) activity, indicating abnormal liver lipid metabolism and severe oxidative stress. Treatment with silibinin and compound 2 significantly reduced MDA levels, and both SOD and GSH-Px activities returned to normal levels.

[0157] Table 6 Effects of compound 2 on oxidative stress indicators in mice

[0158] Group MDA (nmol / mgprot) SOD(U / mgprot) GSH-PX(U / mgprot) Normal group 0.38±0.05 758.02±79.11 237.52±23.76 Model Group 0.94±0.13 482.27±53.11 104.35±14.54 Silybin group 0.57±0.12 746.26±97.74 203.55±68.45 Compound Group 2 0.48±0.10 740.52±80.28 250.45±32.70

[0159] Example 20

[0160] Pharmacokinetic properties of compound 2 in rats

[0161] 20.1 Solution Preparation

[0162] (1) Preparation of Silybin and Compound 2 Standard Solutions: Accurately weigh 10.0 mg of Silybin reference substance into a 10 mL volumetric flask and dilute with methanol to a 1 mg / mL stock solution. Dilute the stock solution with methanol proportionally to prepare Silybin standard solutions with concentrations of 200, 500, 1000, 2000, 4000, 10000, 20000, and 100000 ng / mL. Take 10 μL of each standard solution and dilute it 20-fold with blank plasma to obtain plasma samples with blood drug concentrations of 10, 25, 50, 100, 200, 500, 1000, and 5000 ng / mL, respectively. Prepare plasma samples with compound 2 concentrations of 50, 100, 250, 500, 1000, 2000, 5000, and 10000 ng / mL, respectively, in the same manner. Store in a refrigerator at 4°C until use.

[0163] (2) Preparation of internal standard solution: chlorzoxazone and carbamazepine were used as internal standards for silybin and compound 2, respectively. 25.0 mg of chlorzoxazone and carbamazepine reference substances were accurately weighed and placed in a 25 mL volumetric flask. They were dissolved with acetonitrile and diluted to the mark to prepare an internal standard stock solution with a concentration of 1 mg / mL. 10 μL of the internal standard stock solution was placed in a 25 mL volumetric flask, diluted with acetonitrile and diluted to the mark to prepare an internal standard solution with a concentration of 1000 ng / mL. The solution was then refrigerated at 4°C for use.

[0164] 20.2 Plasma Sample Processing Methods

[0165] Take 100 μL of plasma sample and place it in a 1 mL centrifuge tube. Then add 100 μL of acetonitrile and 100 μL of internal standard solution. Vortex and shake for 1 min. Then, rotate at 13000 r·min- 1 After centrifugation for 5 min, 150 μL of the supernatant was collected for analysis.

[0166] 20.3 Chromatographic Conditions

[0167] (1) Chromatographic column: YMC-Pack ODS-AQ 250*4.6mm LDS-5μm, 12nm

[0168] (2) The mobile phase was acetonitrile (A): 0.1% trifluoroacetic acid in water (B); the flow rate was 1 mL min- 1 Injection volume: 10 μL; Column temperature: 25°C. Silybin isocratic elution: 30% (v / v) A; Gradient elution program for compound 2 is shown in Table 7.

[0169] Table 7 Gradient elution program of compound 2

[0170] Time (min) <![CDATA[流量(mL·min- 1 )]]> A(％) B(％) 0.00 1.00 40.0 60.0 5.00 1.00 50.0 50.0 12.00 1.00 60.0 40.0 14.00 1.00 40.0 60.0

[0171] 20.4 Experimental Methods

[0172] Twelve male SD rats were randomly divided into two groups: a silibinin group and a compound 2 group, with 6 rats in each group. After three days of acclimatization, the animals were gavage-administered at a dose of 150 mg / kg. Approximately 300 μL of blood was collected from the retroorbital venous plexus at 0.33 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h after administration for the silibinin group, and at 0.1 h, 0.33 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, and 12 h after administration for the compound 2 group. The blood was placed in an EDTA-soaked EP tube and placed in an ice bath. The blood was centrifuged at 8000 rpm min-1 for 30 min. 1 Centrifuge for 10 minutes, aspirate the supernatant serum, and freeze at -20°C. Process as described in 19.2 for analysis. Fit the mean plasma drug concentration data using a non-compartmental model to calculate the pharmacokinetic parameters of each component.

[0173] 20.5 Experimental Results

[0174] The plasma concentration-time curves of silibinin and compound 2 are shown in Figure 2. Figure 11 The pharmacokinetic parameters are shown in Table 8. The results of the in vivo pharmacokinetic experiment in rats showed that the Cmax and AUC of compound 2 in rat plasma were 0-8 h were 21 times and 28 times that of silybin, respectively, indicating that the absorption of compound 2 in rats was significantly improved compared with silybin. Using the formula (relative bioavailability = AUC 0-8 h derivative / AUC 0-8 The relative bioavailability of compound 2 was calculated by using the original drug as the base (h prototype drug * 100%). The relative bioavailability of compound 2 was 28.13%. The results showed that the oral bioavailability of compound 2 was significantly improved compared with silybin.

[0175] Table 8 Pharmacokinetic parameters of silybin and compound 2 in rats

[0176] Pharmacokinetic parameters Silybin Compound 2 AUC 0-8 h,ng / mL*h]]> 390.55±72.61 10971.66±1593.28 AUC 0-∞ , ng / mL*h 494.23±129.46 11599.60±1758.88 <![CDATA[MRT 0-∞ ,h]]> 5.22±2.20 3.51±0.25 t 1 / 2 ,h]]> 2.45±0.03 1.55±0.13 Cmax,ng / mL 194.34±23.06 4081.01±855.28 Vz / F,L 446.72±241.20 6.91±1.37 CLz / F,L / h 74.79±19.35 3.08±0.49

Claims

1. A class of sulfur-containing silybin derivatives, characterized in that: The sulfur-containing silybin derivatives are compounds shown in 1 to 15:

2. The method for preparing the sulfur-containing silybin derivative according to claim 1, wherein: The method comprises the following steps: (1) Preparation of Compounds 1 and 4 Under the protection of nitrogen, triphenylphosphine is dissolved in anhydrous tetrahydrofuran, and diisopropyl azodicarboxylate is slowly added dropwise under stirring in an ice bath. After the addition is completed, a white precipitate is observed to be generated; then, silybin or 2,3-dehydrosilybin dissolved in anhydrous tetrahydrofuran is added to the reaction solution, and finally thioacetic acid is added, and the temperature is restored to room temperature and the reaction is continued for 30 minutes; after the reaction is completed, extraction is performed with ethyl acetate, the organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent is spin-dried and separated and purified by silica gel column chromatography to obtain compound 1 or 4; the molar ratio of the silybin or 2,3-dehydrosilybin, triphenylphosphine, diisopropyl azodicarboxylate, and thioacetic acid is 1:1-6:1-6:1-6, and the amount of the tetrahydrofuran solvent used is 1-50 ml per 1 gram of silybin; (2) Preparation of Compounds 2 and 5 Compound 1 or 4 was dissolved in 1M HCl / MeOH solution and stirred at room temperature for 3-4 hours. After the reaction, the mixture was extracted with ethyl acetate, and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was dried by spin drying. The mixture was separated and purified by silica gel column chromatography to obtain compound 2 or 5. The amount of 1M HCl / MeOH solution used was 1-50 ml per 1 gram of compound 1 or 4. (3) Preparation of compounds 3 and 6 Compound 2 or compound 5 is subjected to a condensation reaction under the action of an oxidizing agent, iodine, for 10 to 20 minutes. After the reaction is completed, the mixture is quenched with a saturated sodium thiosulfate solution, extracted with ethyl acetate, the organic phases are combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent is spin-dried to obtain compound 3 or 6; the molar ratio of the compound 2 or 5 to the iodine element is 1:0.1 to 3, and the solvent used is dimethyl sulfoxide, with 1 to 50 ml of dimethyl sulfoxide used for every 500 mg of compound 2 or 5; (4) Preparation of compounds 7 to 15 Compound 1 or compound 2 was dissolved in methanol, and 37% formaldehyde aqueous solution and the corresponding secondary amine were added sequentially. The reaction was carried out at room temperature for 12 to 30 hours. After the reaction, the mixture was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was spin-dried and purified by silica gel column chromatography to obtain compounds 7 to 15; the molar ratio of the compound 1 or 2, 37% formaldehyde aqueous solution, and the corresponding secondary amine was 1:0.5-4:0.5-4, and the amount of methanol solvent used was 1 to 25 ml of methanol per 250 mg of compound 1 or 2; R had the same position as that of compound 7-15.

3. Use of the sulfur-containing silybin derivative or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of a liver protective drug for preventing and treating acute and chronic liver damage caused by various causes.

4. Use of the sulfur-containing silybin derivative or a pharmaceutically acceptable salt thereof as claimed in claim 3 in the preparation of a liver protection drug, wherein the liver damage is non-alcoholic steatohepatitis, alcoholic steatohepatitis, drug-induced liver injury, liver fibrosis and cirrhosis.

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