Preparation method and application of emodin derivative with hepatic fibrosis relieving effect

Abstract

The invention discloses a preparation method and application of an emodin derivative with an effect of relieving hepatic fibrosis, and belongs to the technical field of medicine synthesis. According to the technical scheme, the emodin derivative molecule has the structure, wherein R1 is a hydrogen atom or R is hydroxyl or X is a phenyl derivative or a benzyl derivative. The invention designs and synthesizes an emodin derivative with a novel structure, and the compound can regulate and control copper ions in a targeted manner so as to have an effect of relieving hepatic fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to a method for preparing and applying a rhein derivative that can alleviate liver fibrosis. Background Technology

[0002] Rhubarb is a commonly used herb in Traditional Chinese Medicine (TCM). There are 39 varieties and 2 variants in my country, and it is recorded in ancient texts such as the *Shennong Bencao Jing* and *Bencao Gangmu*. As a renowned medicinal herb, rhubarb is considered to possess the power to "expel and renew," and is hailed as one of the "Four Great Pillars" of clinical TCM, along with ginseng, rehmannia root, and aconite, for its potent effects. The main components of rhubarb include rhein, rheinic acid, chrysophanol, rhubarb polysaccharides, and sennosides. The pharmacological effects of rhubarb include: laxative effect, immunomodulatory effect, free radical scavenging effect, antibacterial, anti-inflammatory, and antiviral effects, diuretic effect, protective effect against cerebral ischemia-reperfusion injury, cardiovascular pharmacological effects, and antitumor effects. The main pharmacologically active components of rhubarb are anthraquinone compounds and their derivatives. Modern research has confirmed that sennosides and anthraquinone glycosides in rhubarb are the effective laxative components, free anthraquinones are the effective antibacterial and antitumor components, and free anthraquinones and anthraquinone glycosides are the effective lipid-lowering and antioxidant components. Anthraquinone derivatives such as rhein methyl ether, rhein, aloe-rhein, rhein, and rhein acid have anti-angiogenic effects. Rhein acid has antitumor activity and, due to its tetracycline-like structural features, exhibits bone affinity. As a compound with a known structure, rhein acid possesses various pharmacological activities, including anti-inflammatory, antitumor, antibacterial, antiviral, antioxidant, hypoglycemic, lipid-regulating, hepatoprotective, and anti-liver fibrosis effects. It has shown outstanding performance in treating osteoarthritis, diabetic nephropathy, and synergistic antitumor effects, making it a research hotspot.

[0003] Liver fibrosis refers to the pathological changes in the liver caused by long-term chronic stimulation of the liver by various damaging factors such as hepatitis viruses, alcohol, drugs and toxins, schistosomiasis, metabolic and genetic factors, cholestasis, and autoimmune liver disease. This leads to excessive proliferation and abnormal deposition of extracellular matrix (ECM) components in liver tissue, resulting in an imbalance between proliferation and metabolism, and causing abnormalities in liver structure and / or function. It is an inevitable stage in the progression of chronic liver disease to cirrhosis. According to statistics, there were 821 million people with liver fibrosis worldwide in 2022, mainly caused by viral hepatitis and chronic alcoholism. If liver fibrosis is not effectively controlled, it will gradually develop into irreversible cirrhosis, and may further evolve into liver cancer. These diseases pose a significant threat to human survival and health. Currently, treatment for liver fibrosis is limited to controlling the cause or traditional Chinese medicine; no chemical drugs have yet entered clinical application.

[0004] Copper death, a newly discovered programmed cell death mechanism, involves the accumulation of copper ions in cells and their binding to mitochondrial lipoylated proteins. This induces a series of reactions, including protein aggregation, inactivation of iron-sulfur cluster proteins, and oxidative stress imbalance, ultimately leading to apoptosis. Copper death is particularly important in liver diseases, with studies showing a close correlation between disordered copper metabolism in liver tissue and the development of chronic liver disease. Some literature indicates that during liver fibrosis, copper ions exacerbate the process by regulating fibroblast activation, inflammatory factor release, and redox homeostasis. Inducing or regulating copper death may provide a new target for alleviating liver fibrosis. Therefore, developing emodin and its derivatives with the ability to regulate copper death not only expands their application in the field of anti-liver fibrosis but also holds promise for providing candidate compounds for novel therapeutic strategies targeting the mechanism of copper death, possessing significant scientific value and application prospects. Summary of the Invention

[0005] The present invention discloses a method for preparing and applying a rhein derivative with an effect of alleviating liver fibrosis, characterized in that the molecular structure of the rhein derivative is as follows: Where R1 is a hydrogen atom or Wherein X is a phenyl derivative or a benzyl derivative; or the molecular structure of the said emodin derivative is: Where R is a hydroxyl group or Where X is a phenyl derivative or a benzyl derivative.

[0006] The present invention relates to a method for preparing and applying a rhein derivative with an effect of alleviating liver fibrosis, characterized in that:

[0007] (1) A certain amount of 1,3,8-trihydroxy-6-methylanthraquinone, potassium carbonate, and 3-bromopropyne were added to N,N-dimethylformamide. The mixture was heated to 100°C under nitrogen protection and reacted for a period of time. After stirring with brine, the mixture was extracted multiple times with dichloromethane. The organic phases were combined, concentrated under vacuum to remove the organic solvent, and then separated by silica gel column chromatography to obtain the final product.

[0008] (2) Put a certain amount Azide compounds, sodium ascorbate, and CuSO4 were added to a mixed solution of water, tert-butanol, and tetrahydrofuran. The mixture was heated to reflux and reacted for a period of time. After stirring, brine was added. The organic phase was dried over anhydrous sodium sulfate and extracted multiple times with dichloromethane. The organic phases were combined, concentrated under vacuum to remove the organic solvent, and then separated by silica gel column chromatography to obtain the target compound.

[0009] (3) Add rhein, propargylamine, HATU, and DIPEA to DMF, heat under nitrogen protection to 60-80℃ and stir for a period of time. Then pour into brine, extract the reaction system with dichloromethane multiple times, combine the organic phases, dry with anhydrous sodium sulfate, and concentrate under vacuum. After concentration, separate by silica gel column chromatography to obtain...

[0010] (4) Put a certain amount Azide compounds, sodium ascorbate, and CuSO4 were added to a mixed solution of water, tert-butanol, and tetrahydrofuran. The mixture was heated to reflux and reacted for a period of time. After stirring, brine was added. The organic phase was dried over anhydrous sodium sulfate and extracted multiple times with dichloromethane. The organic phases were combined, concentrated under vacuum to remove the organic solvent, and then separated by silica gel column chromatography to obtain the target compound.

[0011] The emodin derivatives described in this invention have the following technical advantages: (1) This invention designs and synthesizes novel derivatives with acidic groups based on the structure of emodin; (2) These derivatives with acidic groups can alleviate liver fibrosis; (3) These derivatives with acidic groups can interact with copper ions in liver fibrotic cells and thus alleviate liver fibrosis by regulating copper death. Attached Figure Description

[0012] Figure 1 This is the mass spectrum of compound 3i.

[0013] Figure 2 This is the mass spectrum of compound 6a. Detailed Implementation

[0014] The following examples further illustrate the above-described content of the present invention, but it should not be construed as limiting the scope of the subject matter of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

[0015] Example 1

[0016]

[0017] In a stirred reaction flask, 0.01 mol of 1,3,8-trihydroxy-6-methylanthraquinone and 0.02 mol of potassium carbonate were added to N,N-dimethylformamide, followed by 0.01 mol of 3-bromopropyne. The mixture was heated to 100 °C under nitrogen protection and reacted for 5 h. After washing with brine and extraction with dichloromethane, the organic phases were combined, concentrated under vacuum to remove the organic solvent, and separated by silica gel column chromatography to obtain compound 2 (1.62 g). 1H NMR (400MHz, CDCl3): 12.29 (s, 1H), 12.07 (s, 1H), 7.62 (s, 1H), 7.42 (d, J = 4.0Hz, 1H), 7.08 (s, 1 H), 6.79 (d, J = 4.0Hz, 1H), 4.83 (d, J = 4.0Hz, 2H), 2.61 (t, J1 = 4.0Hz, J2 = 4.0Hz, 1H), 2.45 (s, 3H).

[0018] Example 2

[0019]

[0020] Compound 2 (1 mmol), 4-fluorobenzyl azide (1.2 mmol), sodium ascorbate (2 mmol), and CuSO4 (1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. After the reaction was complete, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 3a (0.317 g).

[0021] Example 3

[0022]

[0023] Compound 2 (1 mmol), 2-trifluoromethoxyphenyl azide (1.2 mmol), sodium ascorbate (2 mmol), and CuSO4 (1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. After the reaction was complete, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 3b (0.277 g).

[0024] Example 4

[0025]

[0026] Compound 2 (1 mmol), 3-chlorobenzyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. After the reaction was complete, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 3c (0.152 g).

[0027] Example 5

[0028]

[0029] Compound 2 (1 mmol), 4-bromobenzyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. After the reaction was complete, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 3d (0.206 g).

[0030] Example 6

[0031]

[0032] Compound 2 (1 mmol), 2-chlorophenyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. After the reaction was complete, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain the solid product 3e (0.172 g).

[0033] Example 7

[0034]

[0035] Compound 2 (1 mmol), 3-chlorophenyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. After the reaction was complete, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 3f (0.249 g).

[0036] Example 8

[0037]

[0038] Compound 2 (1 mmol), 2-fluorophenyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min until completely dissolved. The mixture was then heated under reflux with stirring. After the reaction was complete, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. The mixture was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to the mixture, and after thorough stirring, it was allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain 3 g (0.183 g) of solid product.

[0039] Example 9

[0040]

[0041] Compound 2 (1 mmol), 2-bromophenyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min until completely dissolved. The mixture was then heated under reflux with stirring. After the reaction was complete, the mixture was extracted multiple times with dichloromethane, and the lower organic phase was collected. The mixture was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to the mixture, and after thorough stirring, it was allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain a solid product 3 h (0.307 g).

[0042] Example 10

[0043]

[0044] Compound 2 (1 mmol), 2-trifluoromethylbenzyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water:tert-butanol:THF = 1:1:1 (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min until completely dissolved. The mixture was then heated to reflux with stirring. After the reaction was complete as detected by TLC, the mixture was extracted three times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration. Dichloromethane was removed by vacuum distillation at 35 °C to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 3i (0.225 g). 1 H NMR (400MHz, CDCl3): 12.30 (s, 1H), 12.09 (s, 1H), 7.74 (d, J = 8.0Hz, 1H), 7.63 (s, 1H), 7.58-7.46 (m, 3 H),7.42(d,J=4.0Hz,1H),7.09(s,1H),6.80(d,J=4.0Hz,1H),5.78(s,2H),5.31(s,2H),2.45(s,3H).

[0045] Example 11

[0046]

[0047] Compound 2 (1 mmol), 4-trifluoromethylbenzyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min until completely dissolved. The mixture was then heated to reflux with stirring. After the reaction was complete as detected by TLC, the mixture was extracted three times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration. Dichloromethane was removed by vacuum distillation at 35 °C to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 3d (0.294 g). 1 H NMR(400MHz, CDCl3):12.23(s,1H),12.01(s,1H),7.60-7.55(m,4H),7.34-7.32( m, 3H), 7.02 (s, 1H), 6.72 (d, J = 4.0Hz, 1H), 5.56 (s, 2H), 5.25 (s, 2H), 2.38 (s, 3H).

[0048] Example 12

[0049]

[0050] Compound 2 (1 mmol), 2-trifluoromethylphenyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added to the flask. The mixture was stirred at room temperature for 10 min until completely dissolved. The mixture was then heated to reflux with stirring. After the reaction was complete as detected by TLC, the mixture was extracted three times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration. Dichloromethane was removed by vacuum distillation at 35 °C to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 3k (0.197 g).

[0051] Example 13

[0052]

[0053] In a reaction flask, rhein (1 mmol), 3-ethynylpropamine (1.2 mmol), HATU (1 mmol), and DIPEA (2 mmol) were added to DMF. The mixture was heated to 60-80°C under nitrogen protection and stirred for a period of time. The mixture was then poured into brine and the reaction system was extracted with dichloromethane multiple times. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under vacuum. After concentration, compound 5 (0.208 g) was obtained by silica gel column chromatography.

[0054] Example 14

[0055]

[0056] Compound 5 (1 mmol), benzyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. After the reaction was complete as detected by TLC, the mixture was extracted three times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added to this phase, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 6a (0.355 g).

[0057] Example 15

[0058]

[0059] Compound 5 (1 mmol), 2-fluorobenzyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. After the reaction was complete as detected by TLC, the mixture was extracted three times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 6b (0.227 g).

[0060] Example 16

[0061]

[0062] Compound 5 (1 mmol), 3-fluorobenzyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. After the reaction was complete as detected by TLC, the mixture was extracted three times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 6c (0.188 g).

[0063] Example 17

[0064]

[0065] Compound 5 (1 mmol), 4-fluorobenzyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. The mixture was extracted three times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 6d (0.303 g).

[0066] Example 18

[0067]

[0068] Compound 5 (1 mmol), 3-chlorophenyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. The mixture was extracted three times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 6e (0.261 g).

[0069] Example 19

[0070]

[0071] Compound 5 (1 mmol), 4-bromobenzyl azide (1.2 mmol), sodium ascorbate (0.2 mmol), and CuSO4 (0.1 mmol) were added to a round-bottom flask equipped with a stir bar. Then, 150 mL of a mixed solvent of water, tert-butanol, and THF (v / v / v) was added. The mixture was stirred at room temperature for 10 min, then heated to reflux with stirring. The mixture was extracted three times with dichloromethane, and the lower organic phase was collected. This was then back-extracted three times with saturated brine, and the lower organic phase was collected. Anhydrous sodium sulfate was added, and the mixture was stirred thoroughly and allowed to stand for 30 min. The anhydrous sodium sulfate was then removed by filtration, and dichloromethane was removed by vacuum distillation to obtain the crude product. The crude product was then purified by column chromatography to obtain solid product 6f (0.113 g).

[0072] Example 20

[0073] CCK-8 assay for cell proliferation activity: LX-2 cells in logarithmic growth phase were digested and counted, and then analyzed at a concentration of 1×10⁻⁶. 4 LX-2 cells were seeded per well in a 96-well plate, with 100 μL of ethanol (to a final concentration of 100 mM) added to each well to activate the cells. Experimental groups, negative controls (cells + solvent), and blank controls (cell-free medium) were set up. The plates were incubated at 37°C and 5% CO2 for 24 hours. After LX-2 cells adhered, the original medium was discarded. The experimental groups were then treated with a drug-containing medium (100 μL / well, containing gradient concentrations of rhein, rhein positive control, emodin, and emodin derivatives). The control group underwent medium replacement. The plates were incubated for another 48 hours. The medium was then discarded, and 100 μL of DMEM solution containing CCK-8 (10 μL CCK-8 + 90 μL DMEM) was added to each well. The mixture was shaken thoroughly and incubated at 37°C in the dark for 1–4 hours (until the negative control OD). 450(≈1.0), absorbance at 450 nm was measured using an ELISA reader. If air bubbles were found in the wells, they needed to be punctured with a needle. The cell inhibition rate was calculated using the formula. The 11 emodin derivatives (3a-3k) designed in this study were validated. Most drugs were effective at a concentration of 100 μM, inhibiting LX-2 cell activity within a certain range. The inhibition rate after 48 hours ranged from 20% to 70.9%, with compound 3i showing the best effect at 70.9%. Furthermore, when 3i was applied at the same concentration to mouse hepatocytes AML-12, the inhibition rate was measured after 48 hours. Compound 3i had the least effect on AML-12 within the normal range, with an inhibition rate of 15.42%. We simultaneously examined the effects of synthetic rhein analogues (4, 6a-6f) on LX-2 cells and measured the CCK-8 results after 48 hours. We found that rhein (4) significantly inhibited alcohol-induced LX-2 cell growth, with inhibition rates of 75.17% and 52.35% against LX-2 and AML-12 cells, respectively, at a concentration of 100 μM. Compounds 3i and 4 were selected for further investigation.

[0074] Example 21

[0075] After digesting and counting LX-2 cells in the logarithmic growth phase, they were stored at a high density of 5 × 10⁻⁶ cells. 5 Cells were seeded per well in a 6-well plate, with 2 mL of complete culture medium added to each well. The plate was incubated at 37°C with 5% CO2 until the cell density reached 90-95% confluence. Using a sterile pipette tip (200 μL), a straight scratch was gently made perpendicular to the bottom of the plate with constant pressure. The cells were then gently washed three times with PBS to remove detached cells. The medium was replaced with 100 mM low-concentration FBS (3%) containing alcohol. Two groups were established: 50 mM rhein 4 and 50 mM emodin 3. The drugs were added, and the scratch images were immediately photographed under an inverted microscope at 0 hours, with the locations marked. The plates were then returned to the incubator for further culture. The plates were removed at preset time points of 12, 24, and 48 hours, and photographed again at the same marked locations. The scratch width was measured using ImageJ software, and the migration rate (%) was calculated as [(0-hour width - T-hour width) / 0-hour width] × 100. Each group had 3 replicates. The results are as follows: The migration rates of LX-2 cells treated with rhein 4 at 24 h and 48 h were 21% and 59%, respectively; the migration rates of LX-2 cells treated with emodin compound 3i at 24 h and 48 h were 28% and 62%, respectively, lower than the migration rates of LX-2 cells in the alcohol group under the same conditions (30% and 70%). The compounds rhein 4 and emodin 3i synthesized in this study can inhibit alcohol-induced migration of LX-2 cells.

[0076] Example 22

[0077] LX-2 cells in logarithmic growth phase were seeded into six-well plates and cultured. The alcohol group and the drug intervention group were labeled separately. After 24 hours of cell adhesion, the medium was replaced with 100 mM alcohol-containing medium. The drug group was supplemented with 50 mM of rhein 4, and the alcohol group was supplemented with the same dose of PBS. Culture was continued for another 48 hours. Total cell protein was extracted according to Example 24. According to the Elabscience Cell Copper Ion Kit instructions, under acidic conditions, copper ions dissociate from ceruloplasmin and react with a complexing agent to produce a purple complex. Within a certain range, the absorbance is directly proportional to the concentration. A chromogenic solution was added to the extracted cell protein. Simultaneously, according to the copper ion standard (provided in the kit), sample wells were added along with the chromogenic solution. After incubation at 37°C in the dark for 5 minutes, the OD value was measured at 580 nm using a microplate reader. Based on the standard curve, the copper ion concentration in the sample was calculated. The results of three repeated experiments showed that the compound rhein 4 designed in this study could reduce the copper ion concentration in LX-2 cells activated by alcohol. After intervention with this drug, the copper ion concentration was 0.52 μmol / gprot, which was lower than 0.60 μmol / gprot in the alcohol group. The copper ion concentration in the drug group was 86.67% of that in the alcohol group. No significant changes were observed in the compound rhein 3i.

[0078] Example 23

[0079] After digesting and counting LX-2 cells in the logarithmic growth phase, the cells were counted at a ratio of 2 × 10⁻⁶. 4Cells were seeded per well on 24-well cell culture plates and incubated in a 37°C incubator containing 5% CO2. After 24 hours of culture and firm cell attachment, alcohol and drug intervention groups were established. The drug intervention groups were treated with 50mM rhein 4 and 50mM emodin 3, respectively, while the alcohol group was treated with the same dose of PBS. Cells were incubated for 48 hours. The culture medium was discarded, and the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes, followed by three washes with PBS. 0.2% Triton X-100 (200 μL) was added to each well for permeabilization for 10 minutes, followed by three washes with PBS. 200 μL of 5% BSA was added to each well for blocking for 30 minutes. Diluted primary antibodies (1:50, antibodies α-SMA and SLC31A1) were added to cover the cells, and the cells were incubated overnight at 4°C. The overnight primary antibody was discarded, and the cells were washed three times with PBS. Fluorescently labeled secondary antibody was added in the dark, and the cells were incubated at room temperature in the dark for 1 hour, followed by three washes with PBS. A DAPI-containing anti-fluorescence quenching mounting medium was added in the dark, and the cells were covered with coverslips. Images were observed and acquired under a laser confocal microscope. Results showed that positive results indicated the target protein was present in specific regions of the cell, with specific fluorescent signals in the cytoplasm (488-labeled α-SMA appeared green, Alexa Fluor 594-labeled SLC31A1 appeared red), and the cell nucleus showed blue fluorescence after DAPI staining. Protein expression was analyzed by fluorescence intensity and distribution patterns. The results were as follows: Compared with the alcohol group (100%), the relative fluorescence intensity of α-SMA, an indicator of hepatic stellate cell activation, was reduced in the drug intervention group by 48% and 66%, respectively. This indicates that the synthesized compounds rhein 4 and rhein 3i can effectively reduce the activation state of alcohol-stimulated LX-2 cells. Simultaneously, we found that compound rhein 4 could reduce the expression of SLC31A1 protein in alcohol-activated LX-2 cells (57%). SLC31A1 acts as a "gatekeeper" of cellular copper homeostasis, actively transporting extracellular reduced copper ions into cells through a trimer channel structure to maintain life activities and influence disease progression. Copper ions are a common cause of fibrosis in various organs. Therefore, we speculate that the compound rhein 4 synthesized in this study may improve alcoholic liver fibrosis by reducing the concentration of copper ions in hepatic stellate cells to a certain extent.

[0080] Example 24

[0081] LX-2 cells in the logarithmic growth phase were seeded into six-well plates and cultured. The alcohol group and the drug intervention group were labeled separately. After the cells adhered for 24 hours, the medium was replaced with 100 mM alcohol-containing medium. The drug group was supplemented with 50 mM of the compound emodin 3i, and the alcohol group was supplemented with the same dose of PBS. The cells were cultured for another 48 hours.

[0082] Extraction of total cellular protein: Discard the culture medium in the well plate; wash adherent cells 2-3 times with pre-chilled PBS, carefully discarding the PBS; digest with trypsin by pipetting, collect the digestion solution in an EP tube, centrifuge and discard the supernatant; prepare cell lysis buffer containing phosphatase inhibitor (add 5 μL of phosphatase inhibitor to 1 mL of cell lysis buffer); add 40-100 μL of pre-chilled cell lysis buffer (depending on the number of cells) to the cell pellet, gently shake, and then lyse on ice for 30 min, vortexing every 5 min; centrifuge at 12000 rpm for 1 min in a pre-chilled 4°C centrifuge. -1 Centrifuge for 20 min, collect the supernatant into a new EP tube for later use; aspirate the supernatant and use the BCA protein concentration kit to quantify cellular proteins.

[0083] Clean the glass plate and prepare the separating gel according to the molecular weight. After sealing the gel, let it stand until it solidifies. Prepare the stacking gel and add it to the remaining space in the glass plate. Immediately insert the comb and wait for the gel to solidify before removing the comb. Prepare the cooked protein in advance and place it on ice to prevent protein degradation. Use a pipette to add the sample vertically, ensuring the pipette tip does not touch the gel to prevent protein from floating out. Set the initial voltage to 80V. When electrophoresis reaches the boundary between the two gels, increase the voltage to 100V. Stop electrophoresis when the molecular weight of the target protein has separated. Prepare the electrotransfer tank in advance. Pour a small amount of 1× transfer buffer into the tray and filter paper. Cut the PVDF membrane according to the molecular weight range for electrophoresis and activate it with methanol beforehand. Cut the gel with a gel plate, remove air bubbles, add some transfer buffer to moisten it, and then cover it using the sandwich method. Place the transfer tank in an ice-water mixture. Typically, constant current transfer is performed at 200mA. Determine the transfer time according to the molecular weight. Pay attention to cooling during the transfer process. After transfer, the membrane was incubated in 5% skim milk at room temperature for 2 hours. After milk powder blocking, the PVDF membrane was washed three times with 1xPBST for 5 minutes each time, and then placed in primary antibody at 4°C overnight. The next day, after removing the primary antibody from the refrigerator, the membrane was washed three times with 1xPBST for 5 minutes each time, and then placed in secondary antibody at room temperature for 1 hour. After incubation, the membrane was washed with 1xPBST. The chromogenic solution A and B from ECL chemiluminescence solution were mixed at a 1:1 ratio and immersed on the membrane. Finally, the images were photographed and saved using an exposure machine, and the gray values ​​of the bands were analyzed using ImageJ software. The results showed that under the action of 50 mM emodin 3i, the expression of stellate cell activation marker proteins such as TGF-β1, α-SMA, and Col1 in LX-2 cells decreased to 83.12%, 90.53%, and 79.06% of those in the alcohol group, respectively. We hypothesize that the compound emodin 3i synthesized in this study improved the activation state of alcohol-activated LX-2 cells and, to some extent, delayed the progression of alcoholic liver fibrosis.

[0084] Example 25

[0085] LX-2 cells in logarithmic growth phase were seeded into six-well plates and cultured, labeled as alcohol group and drug intervention group, respectively. After 24 h of cell adhesion, the medium was replaced with 100 mM alcohol-containing medium. The drug group was supplemented with 50 mM of compound rhein 4, and the alcohol group was supplemented with the same dose of PBS. Cells were cultured for another 48 h. Following Example 24, cellular proteins were extracted for Western blotting experiments. The results showed that the designed compound rhein 4 could inhibit the activation state of alcohol-activated LX-2 cells. Activation markers such as TGF-β1, α-SMA, Col1, and PDGF were all downregulated, with expression levels of 63.64%, 74.74%, 58.35%, and 75.14% in the alcohol group, respectively. We further examined changes in copper ion metabolism-related proteins in the samples. The results showed that compound rhein 4 also reduced the expression of SCL31A1 and LOX in LX-2 cells, with levels of 75.69% and 60.82% in the alcohol group, respectively. LOX is a 50 kDa glycoprotein that enables collagen and elastin to form covalent bonds with the help of copper ions. LOX activity is regulated by copper ions; copper overload enhances LOX activity and can promote pathological processes such as fibrosis. These results further indicate that the synthesized compound rhein-4 can interfere with copper ion metabolism in alcohol-activated LX-2 cells and may influence liver fibrosis through the regulation of the copper-LOX axis.

[0086] Example 26

[0087] LX-2 cells in logarithmic growth phase were fed at a rate of 2 × 10⁻⁶. 4 Cells were seeded per well in 24-well plates and cultured at 37°C and 5% CO2 for 24 hours until 70% confluence. The medium was replaced with 100 mM alcohol-containing medium. The drug group received 50 mM of compound rhein 4, while the alcohol group received the same dose of PBS. Cultures were continued for 48 hours. The medium was discarded, and 10 μM DCFH-DA working solution diluted with serum-free medium was added. The cells were incubated at 37°C in the dark for 30 minutes. The probe solution was discarded, and the cells were gently washed three times with pre-warmed PBS to remove any probes that had not entered the cells. Green fluorescence was immediately observed under a fluorescence microscope. Blank wells (cell-free + probe), unstimulated controls (cells + probe without irritant), probe background wells (cells + probe-free medium), and positive controls were simultaneously set up. The results showed that compound rhein 4 improved the oxidative stress effect of alcohol on ROS-induced cells in LX-2 cells to a certain extent, reducing the relative fluorescence intensity of ROS by 68.48% of that in the alcohol group.

[0088] Example 27

[0089] Thirty sexually mature male clean-grade C57BL / 6N mice (5-6 weeks old; weighing 20-23g) were used. The mice were housed in an SPF (Special Purpose Facility) animal facility at a temperature of 20-22°C and a relative humidity of 50%-60% (control room: 25±2°C, 45-60% humidity; 12-hour light-dark cycle). They had free access to food and water. Before the experiment, the mice were acclimatized to the environment by being fed rodent food for one week. The mice were randomly divided into three groups of 10 each.

[0090] Group A (Normal Group): The normal control group was fed with control liquid diet (Lieber-DeCarli, TP4028AC) for 8 weeks, and a saline negative control mouse model group was established.

[0091] Group B (alcoholic fibrosis model group): A mouse model of progressive alcoholic liver fibrosis was established by feeding the mice with 5% alcohol liquid diet (Lieber-Decarli, TP4028A) for 8 weeks, combined with 31.5% ethanol by gavage (twice a week, 5g / kg).

[0092] Group C (alcohol + rhein group) was fed with 5% alcohol liquid feed (Lieber-Decarli, TP4028A) for 8 weeks, combined with 31.5% ethanol by gavage (twice a week, 5g / kg). Half an hour after gavage, rhein solution (50mg / kg) was administered by gavage again.

[0093] Throughout the modeling process, the mice were observed daily for their activity, fur luster, activity level, diet, and excretion. They were weighed once a day to closely monitor changes in body weight. The experimental animals were euthanized at the end of the experiment.

[0094] Example 28

[0095] Prepare tissue lysis buffer (add 5 μl of phosphatase inhibitor to 1 ml of cell lysis buffer). Label and weigh mouse liver tissue, and add it to a homogenizer at a ratio of 10 ml of tissue lysis buffer per 1 g of liver tissue. Maintain the temperature at -30°C throughout the process to prevent protein degradation due to temperature rise. After each 5-second low-speed homogenization, cool the homogenizer for 10 seconds until the tissue is completely fragmented. Centrifuge at 12000 rpm·min⁻¹ at 4°C for 10 min. Aliquot the supernatant into EP tubes and store immediately at -80°C for later use. Quantify the proteins in each group using the BCA protein concentration kit.

[0096] Example 29

[0097] Mice were injected with alcohol until deeply intoxicated and then fixed to a dissecting board. The eyeballs were gently pressed around the eyes with sterile forceps to make them protrude. The eyeballs were then quickly removed with ophthalmic curved forceps, and the blood flowed naturally into a 1.5 mL EP tube (tilted at a 45° angle to increase blood flow). The blood was allowed to stand for 30-60 minutes until it coagulated. After coagulation, the blood was centrifuged at 4°C and 3000×g for 15 minutes. The upper layer of pale yellow, transparent serum was aspirated with a pipette, avoiding contact with the middle layer of lipids or the bottom layer of cell debris. The serum was then aliquoted into sterile EP tubes and stored at -80°C.

[0098] Example 30

[0099] According to the Elabscience Cell Copper Ion Kit instructions, under acidic conditions, copper ions dissociate from ceruloplasmin and react with a complexing agent to form a purple complex. Within a certain range, absorbance is directly proportional to concentration. Chromogenic solution was added to extracted mouse liver tissue protein and mouse serum. Simultaneously, according to the copper ion standard (provided in the kit), chromogenic solution was added to sample wells, and the mixture was incubated at 37°C in the dark for 5 min. OD values ​​were then measured at 580 nm using a microplate reader. The copper ion concentration of each group of samples was calculated based on the standard curve. The experiment was repeated three times with different samples. Results indicated that rhein reduced the serum copper ion concentration in mice with alcoholic liver fibrosis. The average serum copper ion concentration in the alcohol group was 18.68 μmol / L, while the average serum copper ion concentration in mice fed with compound rhein 4 was 15.04 μmol / L.

[0100] Example 31

[0101] Take thawed mouse serum samples and preheat the samples and matrix buffer (containing alanine / aspartic acid and α-ketoglutarate) in a 37°C water bath for 5 minutes according to the kit instructions. Add the substrate working solution to start the reaction (ALT detection tubes are precisely incubated at 37°C for 30 minutes, AST tubes for 60 minutes). Immediately add 2,4-dinitrophenylhydrazine stop solution and mix well to terminate the reaction. Let it stand at room temperature for 20 minutes. Finally, add 0.4M NaOH colorimetric solution, mix well, and incubate at 37°C for 15 minutes. Detect the absorbance at 510 nm using a microplate reader and calculate the enzyme activity according to the formula in the instructions: ALT (U / L) = [(OD of test tube - OD of control tube) / OD of standard tube] × standard concentration × sample dilution factor × K value (AST is the same method). Simultaneously set up standard tubes (pyruvate standard), blank tubes (distilled water instead of serum), and sample control tubes (add stop solution first, then serum). The results showed that, compared with the alcohol-treated group, liver damage indicators such as ALT and AST were reduced in the rhein-4 compound group, with serum ALT and AST at 51.48% and 31.10% of those in the alcohol-treated group, respectively. This indicates that the designed compound rhein-4 can improve hepatocellular damage caused by alcoholic liver fibrosis in C57 / BL6N mice.

[0102] Example 32

[0103] Liver tissue proteins from each group of mice were collected and Western blotting was performed according to Example 24. The results showed that the expression of indicators of stellate cell activation in the liver tissue of mice in the rhein 4 group was reduced, with the expression levels of TGF-β1, α-SMA, and COL1 being 63.00%, 80.07%, and 78.86% of those in the alcohol group, respectively. The expression levels of copper ion metabolism indicators SLC31A1 and LOX were 81.90% and 73.34% of those in the alcohol group, respectively. The designed compound rhein 4 may alleviate the progression of liver fibrosis by reducing copper ion accumulation in alcoholic liver fibrosis.

[0104] The above embodiments describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are only illustrative of the principles of the present invention. Various changes and modifications can be made to the present invention without departing from the scope of the principles of the present invention, and all such changes and modifications fall within the protection scope of the present invention.

Claims

1. A method for preparing and applying a rhein derivative with the effect of alleviating liver fibrosis, characterized in that... The structure of this emodin derivative is as follows: Where R1 is a hydrogen atom or Where R is a hydroxyl group or Where X is a phenyl derivative or a benzyl derivative.

2. The method for preparing a rhein derivative with the effect of alleviating liver fibrosis according to claim 1, characterized in that... The specific process is as follows: A certain amount of 1,3,8-trihydroxy-6-methylanthraquinone, potassium carbonate, and 3-bromopropyne are added to N,N-dimethylformamide. The mixture is heated to 100°C under nitrogen protection and reacted for a period of time. After stirring with brine, the mixture is extracted multiple times with dichloromethane. The organic phases are combined, concentrated under vacuum to remove the organic solvent, and then separated by silica gel column chromatography to obtain the final product.

3. The method for preparing a rhein derivative with the effect of alleviating liver fibrosis according to claim 1, characterized in that... The specific process is as follows: Take a certain amount of The target compound was obtained by adding azide compounds, sodium ascorbate, and CuSO4 to a mixed solution of water, tert-butanol, and tetrahydrofuran, heating to reflux, reacting for a period of time, adding brine and stirring, extracting multiple times with dichloromethane, combining the organic phases, drying the organic phase with anhydrous sodium sulfate, concentrating under vacuum to remove the organic solvent, and then separating by silica gel column chromatography.

4. The method for preparing a rhein derivative with the effect of alleviating liver fibrosis according to claim 1, characterized in that... The specific process is as follows: Rhein, propargylamine, HATU, and DIPEA are added to DMF, heated to 60-80℃ under nitrogen protection, and stirred for a period of time. The mixture is then poured into brine, and the reaction system is extracted multiple times with dichloromethane. The organic phases are combined, dried over anhydrous sodium sulfate, and then concentrated under vacuum. After concentration, the product is separated by silica gel column chromatography.

5. The method for preparing a rhein derivative with the effect of alleviating liver fibrosis according to claim 1, characterized in that... The specific process is as follows: Take a certain amount of The target compound was obtained by adding azide compounds, sodium ascorbate, and CuSO4 to a mixed solution of water, tert-butanol, and tetrahydrofuran, heating to reflux, reacting for a period of time, adding brine and stirring, extracting multiple times with dichloromethane, combining the organic phases, drying the organic phase with anhydrous sodium sulfate, concentrating under vacuum to remove the organic solvent, and then separating by silica gel column chromatography.

6. The application of the emodin derivative as described in claim 1 to alleviate liver fibrosis at the cellular level.

7. The application of the emodin derivative as described in claim 1, which can target and regulate copper ions to alleviate liver fibrosis in mice.

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