Steroidal compounds with anti-lipotoxicity and antioxidant activity, their preparation methods and applications

Abstract

This invention discloses a steroidal compound with anti-lipotoxicity and antioxidant activity, its preparation method, and its applications. The steroidal compound is β-sitosterol-3-β-O-glucopyranoside-6′-O-palmitate, with the structural formula shown below. The steroidal compound BSBD of this invention exhibits significant anti-lipotoxicity and antioxidant activity in a HepG2 cell model induced by the combined effects of free fatty acids and hydrogen peroxide, showing great promise for application in the preparation of drugs for the prevention or treatment of non-alcoholic steatohepatitis.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a steroid compound with anti-lipotoxicity and antioxidant activity, its preparation method, and its application. Background Technology

[0002] Nonalcoholic steatohepatitis (NASH) is a complex global health problem, and currently, there are no NASH-specific drugs approved by the U.S. Food and Drug Administration (FDA). For the past decade or so, researchers worldwide have been dedicated to developing multi-pathway, multi-target NASH treatments. Steroid compounds, as a class of natural products with a cyclopentane-polyhydrophenanthrene core skeleton, are widely distributed in the animal, plant, and microbial kingdoms, playing an irreplaceable role in key physiological processes such as membrane structure maintenance, signal transduction regulation, and metabolic homeostasis. Their structural diversity and broad bioactivity spectrum make them an important lead molecular library for modern drug development, especially in the fields of endocrine regulation, anti-inflammatory immunity, and the treatment of metabolic diseases, where they have significant clinical value. Summary of the Invention

[0003] This invention provides a steroidal compound with anti-lipotoxicity and antioxidant activity, its preparation method, and its applications. The steroidal compound of this invention belongs to the acylated sterol glucosides and can be used to prepare drugs for the prevention or treatment of non-alcoholic steatohepatitis.

[0004] The steroidal compound with anti-lipotoxicity and antioxidant activity described in this invention is β-sitosteryl-3-ObD-glucopyranoside-6′-O-palmitate (BSBD), with the following structural formula: .

[0005] The method for synthesizing the above-mentioned steroidal compounds with anti-lipotoxicity and antioxidant activity includes the following steps:

[0006] (1) Synthesis of β-sitosterol-β-D-glucoside: β-sitosterol and acetyl bromide glucose were dissolved in anhydrous diethyl ether, and then silver oxide and anhydrous sodium sulfate were added. The reaction was carried out at room temperature in the dark with shaking. A small amount of dilute nitric acid was added to cause the silver oxide to condense. The silver salt was removed by filtration. The filtrate was washed with water and dried with anhydrous sodium sulfate. The solution was concentrated under reduced pressure. The residue was recrystallized with ethanol to remove the water-soluble byproducts derived from acetyl bromide glucose. The product was dissolved in ethanol at 50~60℃. An excess of 10% KOH aqueous solution was added to remove the acetyl group. After cooling, water and diethyl ether were added. The flocculent layer formed at the interface was collected and purified by precipitation with a mixed solvent of pyridine and 95% ethanol to obtain β-sitosterol-β-D-glucoside.

[0007] (2) 2,4,6-Trimethylpyridine (sym-Collidine)-mediated 6′-regioselective palmitoylation: β-sitosterol-β-D-glucoside was dissolved in anhydrous pyridine with stirring. 2,4,6-Trimethylpyridine and anhydrous N,N-dimethylformamide (DMF) were added. The mixture was stirred at room temperature and cooled to 0°C in an ice bath. Anhydrous DMF solution of palmitoyl chloride was slowly added dropwise. The mixture was stirred and mixed evenly at 0°C and then brought to room temperature. The reaction was continued with stirring. TLC monitoring was performed. After the starting material spot basically disappeared, methanol was added to quench the reaction. After stirring, the mixture was concentrated under reduced pressure to remove most of the solvent. The residue was dissolved in ethyl acetate and washed successively with 1 M HCl, saturated NaHCO3, water, and saturated brine. The organic phase was dried with anhydrous Na2SO4 and concentrated under reduced pressure. The mixture was then purified by silica gel column chromatography to obtain β-sitosterol-3-β-O-glucopyranoside-6′-O-palmitate.

[0008] Furthermore, in step (1), the mass ratio of β-sitosterol, acetylglucose bromide, silver oxide and anhydrous sodium sulfate is 18:18:28:30.

[0009] Furthermore, in step (1), the reaction time is 8-10 h with shaking at room temperature in the dark.

[0010] Furthermore, in step (2), the ratio of β-sitosterol-β-D-glucoside, 2,4,6-trimethylpyridine and palmitoyl chloride is 1g:0.63mL:0.71g.

[0011] Furthermore, in step (2), the developing solvent used for TLC monitoring is dichloromethane / methanol = 10:1, V:V.

[0012] Furthermore, in step (2), the eluent used for silica gel column chromatography purification is: dichloromethane / methanol = 98:2~95:5, V:V.

[0013] The present invention also provides the use of the above-mentioned steroidal compounds in the preparation of drugs for the prevention or treatment of non-alcoholic steatohepatitis.

[0014] The steroid compound BSBD of this invention showed that in the viability test of HepG2 cells induced by the combined effects of free fatty acids (FFA) and hydrogen peroxide (H2O2), BSBD effectively improved the cell viability of the model HepG2 cells. Particularly in the lipid accumulation experiment, the results showed that BSBD reduced lipid accumulation in HepG2 cells in a significant dose-dependent manner, while significantly reducing the levels of malondialdehyde (MDA) and reactive oxygen species (ROS) in the model HepG2 cells, and increasing the inhibition rate of superoxide dismutase (SOD) in the model HepG2 cells. This indicates that BSBD effectively improves lipid metabolism and antioxidant capacity of hepatocytes. Therefore, the steroid compound BSBD of this invention can be used to prepare drugs for the treatment of non-alcoholic steatohepatitis (NAH), providing an active compound or lead compound for the development of specific drugs for the prevention or treatment of NHA, and also providing a valuable drug-derived molecule for the medical industry. Attached Figure Description

[0015] Figure 1 This study aimed to protect HepG2 cells from FFA-H2O2-induced lipotoxicity and oxidative stress induced by the steroid compound BSBD. The results included: (a) a schematic diagram of BSBD structure; (b) cell compatibility results; (c) the effect of BSBD on lipid accumulation in HepG2 cells; (d) oil red staining microscopy images of HepG2 cells (L: 5 μM, M: 10 μM, H: 20 μM); (e) laser confocal microscopy images of ROS content in each group; (f) quantitative analysis results of ROS detected by a fluorescence microplate reader in each group; (g) SOD activity in each group; and (h) MDA content in each group. Data are presented as mean ± standard deviation (x ± s, n = 4 / 6). A comparison was made between the control and model. **** P<0.0001, compared with the model group, the drug treatment group, #### P<0.0001, ### P<0. L / M / H: Low, medium, and high concentrations of compound BSBD were used in treatment groups.

[0016] Figure 2 For the steroid compound BSBD 13 C-NMR spectrum.

[0017] Figure 3 For the steroid compound BSBD 1 H-NMR spectrum. Detailed Implementation

[0018] The present invention will now be described in further detail with reference to specific embodiments and accompanying drawings.

[0019] Example 1

[0020] The synthesis of the steroid compound BSBD includes the following steps:

[0021] (1) Synthesis of β-sitosterol-β-D-glucoside. In a 1 L dry three-necked flask, β-sitosterol (18.0 g) and acetylglucose (18.0 g) were added and dissolved in 300 mL of anhydrous diethyl ether. Freshly precipitated dry silver oxide (28.0 g) and anhydrous sodium sulfate (30.0 g) were added. The mixture was shaken at room temperature in the dark for 8 hours. A small amount of dilute nitric acid was added to coagulate the silver oxide, and the silver salt was removed by filtration. The filtrate was washed twice with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure.

[0022] The residue was recrystallized from ethanol to remove water-soluble byproducts derived from acetylglucose bromide. The product was dissolved in ethanol at 60°C, and an excess of 10% KOH aqueous solution was added. After digestion for several minutes, the acetyl groups were removed. After cooling, water and diethyl ether were added, and the flocculent layer (crude glucoside) formed at the interface was collected. The mixture was purified by precipitation with a mixed solvent of pyridine and 95% ethanol to give 1 g of β-sitosterol-β-D-glucoside.

[0023] (2) 2,4,6-Trimethylpyridine-mediated regioselective palmitoylation at the 6′ position. In a dry 100 mL three-necked flask, β-sitosterol-β-D-glucoside (1.00 g) and 25 mL of anhydrous pyridine were added and stirred to dissolve. 2,4,6-Trimethylpyridine (0.63 mL) and 10 mL of anhydrous DMF were added, and the mixture was stirred at room temperature for 10 min. The mixture was cooled to 0 °C in an ice bath, and a solution of palmitoyl chloride (0.71 g) in anhydrous DMF (5 mL) was slowly added dropwise. The mixture was stirred at 0 °C for 30 min, then brought to room temperature and stirred for another 2 h. TLC monitoring (developing solvent: dichloromethane / methanol = 10:1) was performed. After the starting material spot had essentially disappeared, 5 mL of methanol was added to quench the reaction, and the mixture was stirred for 10 min. The mixture was concentrated under reduced pressure to remove most of the solvent, and the residue was dissolved in ethyl acetate (50 mL). The sample was washed successively with 1 M HCl (30 mL), saturated NaHCO3 (30 mL), water (30 mL), and saturated brine (30 mL). The organic phase was dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography (eluent: dichloromethane / methanol gradient 98:2 to 95:5) to give 0.9 g of the steroid compound BSBD as a white solid.

[0024] Example 2

[0025] The steroid compound BSBD alleviated lipotoxic damage in HepG2 cells by dose-dependently inhibiting lipid accumulation and regulating oxidative stress.

[0026] The in vitro therapeutic effect of the steroid compound BSBD on non-alcoholic steatohepatitis (NASH) was investigated primarily by constructing in vitro hepatocyte or liver tissue models to simulate the pathological features of NASH, such as lipid deposition, inflammation, oxidative stress, and hepatocyte damage, thereby evaluating the intervention effects of candidate drugs on these pathological processes. The HepG2 hepatocyte cell line was used to construct an in vitro model through combined induction of lipotoxicity and oxidative stress. After drug intervention, the regulatory effects of candidate drugs on lipid metabolism and oxidative stress were assessed by evaluating intracellular lipid accumulation, reactive oxygen species (ROS) and malondialdehyde (MDA) levels, and superoxide dismutase (SOD) activity.

[0027] The specific experimental method is as follows:

[0028] 1. Seeding cells: Prepare a single-cell suspension using culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum, and seed 8,000 to 15,000 cells per well into a 96-well plate, with a volume of 100 μl per well. The cells should be seeded and cultured 12 to 24 hours in advance.

[0029] 2. In vitro model establishment: The human hepatocellular carcinoma cell line HepG2 was cultured in DMEM medium supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 IU penicillin, and 100 mg / ml streptomycin. Cells were placed in a humidified CO2 (5%) incubator at 37°C. When the cell density reached 90%, cells were seeded at 8000 cells / well in 96-well plates. After 12 h of cell adhesion, the cells were divided into groups of six replicates, each treated with a gradient concentration of H2O2 (10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 80 μM, and 160 μM) and 0.2 mM FFA (palmitic acid:oleic acid ratio = 1:2) for 12 h of induction. Cell viability was assessed using a CCK-8 assay kit, and absorbance was measured at 450 nm after 2 h to verify the successful establishment of the model.

[0030] 2. Addition of BSBD solution: Dissolve BSBD in DMSO and adjust the final concentration to 10 μM. Seed cells at 8000 cells / well in 96-well plates. After 12 h of cell adhesion, add the model drug and BSBD for another 12 h. Cell viability is then assessed using a CCK-8 assay kit.

[0031] 3. Oil Red O staining analysis: The steroid compound BSBD was dissolved in DMSO, and high, medium, and low dose groups were set up with concentrations of 20 μM, 10 μM, and 5 μM, respectively. Cells were cultured at 3 × 10⁶ cells / year. 5Cells were seeded per well in 6-well plates, with 3 parallel wells per group. After 12 h of cell adhesion, the model drug and the test compound were added and treated for another 12 h. Oil Red O staining was performed on the treated cells using an Oil Red O staining kit (Nanjing Jiancheng Biotechnology Institute, China). Staining was performed according to the kit instructions, and lipid droplet accumulation was observed under a microscope for qualitative analysis. Then, 500 μL of isopropanol was added to each well of the 6-well plate, and the plate was shaken for 15 min to elute the stain. 100 μL of the eluent was added to a 96-well plate, and the absorbance at 510 nm was measured using a microplate reader for quantitative analysis.

[0032] 4. SOD and MDA content determination: The steroid compound BSBD was dissolved in DMSO and the final concentration was adjusted to 10 μM. Cells were cultured at a concentration of 3 × 10⁻⁶ cells / day. 5 Cells were seeded per well in 6-well plates, with 4 parallel wells per group. After 12 h of cell adhesion, the model drug and the test compound were added and treated for another 12 h. The superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were calculated according to the kit instructions (Nanjing Jiancheng Biotechnology Institute, China).

[0033] 5. Reactive Oxygen Species (ROS) Detection: The steroid compound BSBD was dissolved in DMSO and the final concentration was adjusted to 10 μM. Cells were cultured at a rate of 3 × 10⁻⁶ cells / day. 5 Cells were seeded at 10,000 cells / well in 6-well plates, with 4 parallel wells per group. After 12 h of cell adhesion, the model drug and the test compound were added and treated for another 24 h. Intracellular ROS levels were assessed using a ROS kit (Nanjing Jiancheng Biotechnology Institute, China) by observing changes in the DCFH-DA fluorescent probe. Qualitative analysis was performed under a microscope, and images were obtained. The steroid compound BSBD was dissolved in DMSO and adjusted to a final concentration of 10 μM. Cells were seeded at 10,000 cells / well in 96-well plates with black walls and transparent bottoms, with 6 parallel wells per group. After 12 h of cell adhesion, the model drug and the steroid compound BSBD were added and treated for another 12 h. Following the ROS kit instructions, the absorbance was measured using a fluorescence microplate reader 30 min later for quantitative analysis. Excitation wavelength: 488 nm; emission wavelength: 525 nm.

[0034] 6. Positive control compound: Rutin was set as the positive control compound in each experiment.

[0035] Table 1. Experimental results of the protective effect of BSBD against FFA-H2O2-induced lipotoxicity and oxidative stress in HepG2 cells.

[0036] Experimental group Cell vitality lipid accumulation ROS Level SOD Vitality MDA content Comparison Normal (100%) Extremely low Extremely low 18 U / mg Low lipid peroxidation level (2.6 nmol / mg) Model Significantly decreased by 75% Significantly increased (OD value increased to 1.4) Significantly increased It decreased to approximately 10.5 U / mg It rose to approximately 5.2 nmol / mg Rutin Significantly recovered to 95% Significantly reduced Significantly reduced Restored to approximately 18 U / mg It decreased to approximately 3.2 nmol / mg BSBD 5μM Partial recovery of 85% The OD value dropped to 1.15. Reduced strength Restored to approximately 14 U / mg It decreased to approximately 3.8 nmol / mg BSBD 10μM 90% recovered relatively well The OD value dropped to 1.02. Significantly weakened Recovery to approximately 16-17 U / mg It decreased to approximately 3.8 nmol / mg BSBD 20μM Nearly 100% The OD value dropped to 0.9 The ROS clearance rate reached 48.6%. Full recovery to 18-19 U / mg The concentration decreased significantly to 2.8 nmol / mg, a reduction of 44.8%.

[0037] Experimental results are as follows Figure 1 As shown in Table 1. Regarding cell viability ( Figure 1 b): Compound BSBD significantly reversed the decline in cell viability in the model group (P < 0.001), and its protective effect was comparable to that of rutin (P > 0.05), suggesting that it has good cell compatibility and basic protective ability. Regarding lipid accumulation ( Figure 1 c, d): Oil Red O staining and microscopic imaging consistently showed that the compound BSBD significantly reduced intracellular lipid droplet deposition in a concentration-dependent manner. Specifically, the high-concentration group (H) showed a reduction of approximately 45% in lipid droplet content compared to the model group, with effects approaching those of the rutin group, confirming its effective intervention in lipid metabolism disorders. Regarding oxidative stress markers ( Figure 1 eh): ROS fluorescence intensity significantly decreased ( Figure 1 e), quantitative analysis showed that its clearance rate reached 48.6 ± 3.2% (P < 0.001 vs. Model); SOD enzyme activity recovered from 10.3 ± 0.8 U / mg protein in the model group to 17.9 ± 1.1 U / mg protein (P < 0.01); MDA content decreased from 5.8 ± 0.3 nmol / mg protein in the model group to 3.2 ± 0.2 nmol / mg protein (P < 0.001), a decrease of 44.8%.

[0038] In summary, BSBD not only significantly inhibits lipid accumulation in hepatocytes in a dose-dependent manner, but also simultaneously enhances antioxidant defense capabilities, effectively improving the dual damage caused by FFA-H2O2-induced lipotoxicity and oxidative stress. Its multi-target synergistic mechanism (inhibiting lipid deposition, scavenging free radicals, and activating SOD) makes it a promising lead compound for the development of therapeutics for non-alcoholic steatohepatitis.

Claims

1. A steroidal compound with anti-lipotoxicity and antioxidant activity, characterized in that, It is β-sitosterol-3-β-O-glucopyranoside-6′-O-palmitate, with the following structural formula: .

2. The method for synthesizing steroidal compounds according to claim 1, characterized in that, Includes the following steps: (1) Synthesis of β-sitosterol-β-D-glucoside: β-sitosterol and acetyl bromide glucose were dissolved in anhydrous diethyl ether, and then silver oxide and anhydrous sodium sulfate were added. The reaction was carried out at room temperature in the dark with shaking. A small amount of dilute nitric acid was added to cause the silver oxide to condense. The silver salt was removed by filtration. The filtrate was washed with water and dried with anhydrous sodium sulfate. The solution was concentrated under reduced pressure. The residue was recrystallized with ethanol to remove the water-soluble byproducts derived from acetyl bromide glucose. The product was dissolved in ethanol at 50~60℃. An excess of 10% KOH aqueous solution was added to remove the acetyl group. After cooling, water and diethyl ether were added. The flocculent layer formed at the interface was collected and purified by precipitation with a mixed solvent of pyridine and 95% ethanol to obtain β-sitosterol-β-D-glucoside. (2) 2,4,6-Trimethylpyridine-mediated 6′-position regioselective palmitoylation: β-sitosterol-β-D-glucoside was dissolved in anhydrous pyridine, 2,4,6-trimethylpyridine and anhydrous DMF were added, stirred at room temperature, cooled to 0°C in an ice bath, and anhydrous DMF solution of palmitoyl chloride was slowly added dropwise. After stirring and mixing evenly at 0°C, the mixture was raised to room temperature and the reaction was continued with stirring. TLC monitoring showed that after the starting material spot basically disappeared, methanol was added to quench the reaction. After stirring, the mixture was concentrated under reduced pressure to remove most of the solvent. The residue was dissolved in ethyl acetate and washed successively with 1 M HCl, saturated NaHCO3, water and saturated brine. The organic phase was dried with anhydrous Na2SO4, concentrated under reduced pressure and purified by silica gel column chromatography to obtain β-sitosterol-3-β-O-glucopyranoside-6′-O-palmitate.

3. The synthesis method according to claim 2, characterized in that, In step (1), the mass ratio of β-sitosterol, acetylglucose bromide, silver oxide and anhydrous sodium sulfate is 18:18:28:

30.

4. The synthesis method according to claim 2, characterized in that, In step (1), the reaction time is 8-10 h with shaking at room temperature in the dark.

5. The synthesis method according to claim 2, characterized in that, In step (2), the ratio of β-sitosterol-β-D-glucoside, 2,4,6-trimethylpyridine and palmitoyl chloride is 1g:0.63mL:0.71g.

6. The synthesis method according to claim 2, characterized in that, In step (2), the developing solvent used for TLC monitoring is dichloromethane / methanol = 10:1, V:V.

7. The synthesis method according to claim 2, characterized in that, In step (2), the eluent used for silica gel column chromatography purification is: dichloromethane / methanol = 98:2~95:5, V:V.

8. The use of the steroidal compound according to claim 1 in the preparation of a drug for the prevention or treatment of non-alcoholic steatohepatitis.

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