Synthesis method and application of a kidney fibrosis inhibitor derived from marine fungi

Abstract

The application discloses a synthesis method of a renal fibrosis inhibitor from marine fungi and application of the renal fibrosis inhibitor in preparation of an anti-renal fibrosis drug, wherein the renal fibrosis inhibitor from marine fungi is a ergosterol-pyrone hybrid steroid compound with a structure as shown in formula I, and the method comprises the following steps: in a non-proton organic solvent, taking ergosterol and a pyrone derivative as substrates, performing one-step non-enzymatic coupling reaction in the presence of an acid promoter, and after the reaction is completed, performing separation and purification to obtain the renal fibrosis inhibitor from marine fungi, wherein the synthesis route is extremely simple, the yield is high, a production bottleneck is broken, and the obtained striasteroid B has significant inhibitory renal fibrosis activity and drug-making potential: I, wherein R is -CH3 or -CH2OH.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for synthesizing a marine fungal-derived renal fibrosis inhibitor and its application in the preparation of anti-renal fibrosis drugs. Background Technology

[0002] Renal fibrosis caused by chronic kidney disease (CKD) is a major global medical challenge and the common pathological pathway leading to end-stage renal failure in various CKD diseases. Its main characteristics are epithelial-mesenchymal transition and excessive deposition of extracellular matrix, which irreversibly damages the structure and function of kidney tissue. However, currently, there are no effective drugs in clinical practice that can reverse or block this pathological process. Therefore, the search for lead compounds with novel structures and significant anti-fibrotic activity to develop safe and effective new targeted drugs is an urgent need in current innovative drug development.

[0003] Striasteroid A and Striasteroid B, natural products isolated from the marine fungus *Striaticonidium cinctum* SCSIO41432, possess novel ergosterol-pyranone fused polycyclic skeletons (*Organic Letters* 2025, 27, 3737-3741). However, the abundance of these bioactive natural products in fungal ferments is extremely low, and the extraction and isolation processes are cumbersome, making it difficult to meet the large-scale usage requirements for subsequent drug efficacy evaluation and new drug development. Therefore, efficient synthesis is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for synthesizing a marine fungal-derived renal fibrosis inhibitor and its application in the preparation of anti-renal fibrosis drugs.

[0005] This invention is achieved through the following technical solutions: A method for synthesizing a marine fungal-derived renal fibrosis inhibitor, wherein the marine fungal-derived renal fibrosis inhibitor is an ergosterol-pyranone hybrid steroid compound with the structure shown in Formula I, the method comprising the following steps: in an aprotic organic solvent, using ergosterol and pyranone derivatives as substrates, a one-step non-enzymatic coupling reaction is carried out in the presence of an acidic promoter; after the reaction is completed, the ergosterol-pyranone hybrid steroid compound is obtained by separation and purification. .

[0006] The aprotic organic solvent is selected from ethyl acetate (EA) or dichloromethane (DCM); ethyl acetate is most preferred. The acidic promoter is a Lewis acid or a protic acid, selected from boron trifluoride diethyl ether complex (BF3·Et2O), trifluoroacetic acid (TFA), citric acid, or oxalic acid, with boron trifluoride diethyl ether complex being most preferred.

[0007] Preferably, the pyranone derivative is 4-hydroxy-6-methyl-2H-pyran-2-one or 4-hydroxy-6-hydroxymethyl-2H-pyran-2-one. When the pyranone derivative is selected from 4-hydroxy-6-methyl-2H-pyran-2-one, the resulting ergosterol-pyranone hybrid steroid compound is striasteroid B; when the pyranone derivative is 4-hydroxy-6-hydroxymethyl-2H-pyran-2-one, the resulting ergosterol-pyranone hybrid steroid compound is striasteroid A. When the acid promoter is selected from boron trifluoride diethyl ether complex (BF3·Et2O), the specific equation is as follows: .

[0008] Preferably, the molar ratio of ergosterol, pyranone derivatives and boron trifluoride diethyl ether complex is 1:(1.5~3):(3~10).

[0009] More preferably, the molar ratio of ergosterol, pyranone derivatives and boron trifluoride diethyl ether complex is 1:2.5:5.

[0010] Preferably, the coupling reaction is carried out at room temperature (28°C) for 5 days.

[0011] The inventors discovered that striasteroid B, as an inhibitor of renal fibrosis, can significantly inhibit TGF-β1-induced epithelial-mesenchymal transition and extracellular matrix deposition, specifically by downregulating the expression of fibrosis marker proteins fibronectin (FN) and α-smooth muscle actin (α-SMA) in renal tubular epithelial cells. Therefore, this invention also protects the use of striasteroid B in the preparation of anti-renal fibrosis drugs. The drug comprises an effective amount of striasteroid B as the active ingredient and a pharmaceutically acceptable carrier.

[0012] The beneficial effects of this invention are as follows: 1) The synthetic route is extremely simple, with high yield, breaking through the bottleneck of mass production: This invention breaks through the limitations of traditional complex steroid compounds that rely on lengthy total synthesis or inefficient natural extraction. For the first time, it achieves the construction of an extremely complex ergosterol-pyranone fused polycyclic skeleton in just one reaction step. The method is simple to operate, and under optimal conditions (EA / BF3·Et2O), the substrate conversion rate reaches as high as 92%, the separation yield reaches 71%, and the purity reaches 98%. It successfully realizes the gram-scale preparation of striasteroid B, providing sufficient material support for subsequent drug development evaluation.

[0013] 2) Mild reaction conditions, green and efficient: The preparation method of this invention does not require expensive metal catalysts or harsh anhydrous and oxygen-free extreme conditions. It can be carried out efficiently at room temperature and in conventional organic solvents with very few by-products, which greatly reduces production costs and environmental pollution, and has a very high prospect for industrial application.

[0014] 3) Striasteroid B exhibits significant inhibitory activity against renal fibrosis and has drug potential: The striasteroid B prepared in this invention showed no significant toxicity to HK-2 cells at a concentration of 100 μM; it significantly inhibited the expression of TGF-β1-induced fibrosis markers (FN and α-SMA) in a dose-dependent manner within the concentration range of 10–100 μM. Pharmacokinetic studies in animals showed that striasteroid B exhibited slow elimination in mice, with an oral bioavailability of approximately 9.59%, demonstrating good drug potential. Attached Figure Description

[0015] Figure 1 This is an evaluation of the anti-renal fibrosis activity of striasteroid B in a TGF-β1-induced HK-2 cell model; Among them, A indicates that striasteroid B has no significant toxicity to HK-2 cells at a concentration of 100 µM; B indicates that striasteroid B inhibits TGF-β1-induced HK-2 cell fibrosis in a concentration-dependent manner and significantly reduces the expression intensity of fibrosis markers fibronectin (FN) and α-smooth muscle actin (α-SMA); C indicates that striasteroid B inhibits TGF-β1-induced α-SMA overexpression in a concentration-dependent manner; and D indicates that striasteroid B inhibits TGF-β1-induced FN overexpression in a concentration-dependent manner. Detailed Implementation

[0016] The following is a further description of the invention, but not a limitation thereof.

[0017] Example 1: Gram-scale preparation of the ergosterol-pyranone hybrid steroid compound striasteroid B ; 1) The substrate ergosterol (compound 3, 1.5 g, 1.0 equivalent) and 4-hydroxy-6-methyl-2H-pyran-2-one (compound 5, 2.5 equivalent) were placed in a dry reaction flask, and an appropriate amount of ethyl acetate (EA) was added as a solvent to dissolve them completely. The mixture was stirred at room temperature (28 °C) until homogeneous. 2) Add BF3·Et2O (5.0 equivalents) as an acidic promoter to the above mixed solution. After the addition is complete, stir the mixture continuously at 28 °C for 5 days, and monitor the reaction progress by HPLC until the substrate conversion reaches 92%. 3) After the reaction is complete, saturated sodium bicarbonate aqueous solution is slowly added to the reaction solution to quench the reaction. The mixture is transferred to a separatory funnel and extracted multiple times with ethyl acetate. The organic phases are combined and washed successively with water and saturated brine. After drying with anhydrous sodium sulfate, the mixture is filtered and concentrated under reduced pressure to remove the solvent, and the crude product of the reaction is obtained. 4) The crude product was separated and purified by column chromatography or high performance liquid chromatography. The eluent containing the target product was collected and concentrated under reduced pressure. Finally, it was dried under vacuum to obtain the purified target product striasteroid B (1.065 g) with a purity of 98% and a separation yield of 71%.

[0018] Example 2: Preparation of the ergosterol-pyranone hybrid steroid compound striasteroid A ; ; 1) The starting material 4-methoxy-6-methyl-2H-pyran-2-one (compound 6) and selenium dioxide (SeO2) were placed in a sealed glass tube, and dichloromethane (DCM) was added as a solvent. The oxidation reaction was carried out under heating conditions of 140 °C. After the reaction was completed, the product 4-methoxy-6-(hydroxymethyl)-2H-pyran-2-one (compound 7) was obtained by cooling, extraction, vacuum concentration and column chromatography purification. 2) The obtained compound 7 was placed in a dry reaction flask, dissolved in dichloromethane, and then trimethyliodosilane (TMSI) was added. The demethylation reaction was carried out under reflux conditions. After the substrate was completely consumed, a quencher was added to stop the reaction. The mixture was extracted, washed, dried, concentrated and separated by silica gel column chromatography to obtain the key pyranone precursor 4-hydroxy-6-(hydroxymethyl)-2H-pyran-2-one (compound 4). 3) The substrate ergosterol (compound 3, 0.012 mg, 1.0 equivalent) and the compound 4 (2.5 equivalent) prepared above were placed in a dry reaction flask, and an appropriate amount of ethyl acetate was added as a solvent to dissolve it completely. The mixture was stirred evenly at room temperature (28 °C), and BF3·Et2O (5.0 equivalent) was slowly added dropwise as an acidic promoter. The non-enzymatic coupling reaction was carried out by continuous stirring. 4) Monitor the coupling reaction until it is complete by thin-layer chromatography or high-performance liquid chromatography. Quench the reaction by adding saturated sodium bicarbonate aqueous solution. Extract the mixture multiple times with ethyl acetate. Combine the organic phases and wash them successively with water and saturated brine. Dry with anhydrous sodium sulfate, filter, concentrate under reduced pressure to obtain crude product. Finally, separate and purify the crude product by silica gel column chromatography. Collect the target component and dry it under vacuum to obtain the purified final target product striasteroid A (0.004 g), with a separation yield of 25%.

[0019] Example 3: Evaluation of the anti-renal fibrosis activity of Striasteroid B 1) Human renal tubular epithelial cells were cultured in DMEM / F12 (Gibco) medium containing 10% fetal bovine serum (FBS) (ExCell, FSP500). The cytotoxicity of striasteroid B was detected by the CCK-8 assay. The results showed that it had no significant toxicity to HK-2 cells at a concentration as high as 100 µM. Figure 1 A); 2) Establish a TGF-β1-induced HK-2 cell model to simulate epithelial-mesenchymal transition and extracellular matrix deposition. After the cells were starved of serum for 24 hours, TGF-β1 was added for stimulation, and at the same time, different concentrations (10 µM, 50 µM, 100 µM) of striasteroid B were added for 48 hours. 3) Collect cells and extract total protein, and detect the expression levels of fibrosis markers FN and α-SMA by Western blot; Experimental results showed that striasteroid B could significantly downregulate α-SMA in a concentration-dependent manner. Figure 1 B, Figure 1 C) and FN ( Figure 1 B, Figure 1 The expression of D) was inhibited at a concentration of 10 µM, which was comparable to that of the clinical antifibrotic drug pirfenidone (PFD), while at concentrations of 50 µM and 100 µM, its anti-renal fibrosis activity was superior to that of pirfenidone.

[0020] Example 4: In vivo pharmacokinetic evaluation of Striasteroid B 1) The experimental mice were randomly divided into groups and administered purified striasteroid B via intravenous injection (IV) and gavage (IG), respectively. The intravenous injection dose was 10 mg / kg and the gavage dose was 25 mg / kg. 2) Blood samples were collected from mice at different time points after drug administration, plasma was separated, and the concentration of striasteroid B in plasma was quantitatively determined using a validated high performance liquid chromatography-tandem mass spectrometry method. 3) Based on the measured plasma concentration-time data, calculate the pharmacokinetic parameters of striasteroid B, including maximum plasma concentration, area under the curve, and clearance rate. Experimental results showed that striasteroid B exhibited slow elimination regardless of whether it was administered via IV or IG. The oral bioavailability of IG administration was calculated to be 9.59 ± 2.01%. This pharmacokinetic characteristic provides drug-like support for its subsequent new drug development and in vivo pharmacodynamic studies.

Claims

1. A method for synthesizing a marine fungal-derived renal fibrosis inhibitor, wherein the marine fungal-derived renal fibrosis inhibitor is an ergosterol-pyranone heterosteroid compound with the structure shown in Formula I, characterized in that... The method includes the following steps: in an aprotic organic solvent, using ergosterol and pyranone derivatives as substrates, a one-step non-enzymatic coupling reaction is carried out in the presence of an acidic promoter. After the reaction is completed, the mixture is separated and purified to obtain an ergosterol-pyranone hybrid steroid compound. 。 2. The synthesis method according to claim 1, characterized in that, The aprotic organic solvent is selected from ethyl acetate or dichloromethane.

3. The synthesis method according to claim 1, characterized in that, The acid promoter is a Lewis acid or a protic acid.

4. The synthesis method according to claim 1, characterized in that, The acidic accelerator is selected from one of boron trifluoride ether complex, trifluoroacetic acid, citric acid, or oxalic acid.

5. The synthesis method according to claim 1, characterized in that, The pyranone derivative is 4-hydroxy-6-methyl-2H-pyran-2-one or 4-hydroxy-6-hydroxymethyl-2H-pyran-2-one.

6. The synthesis method according to claim 4, characterized in that, When the acidic accelerator is selected from boron trifluoride ether complex, the molar ratio of ergosterol, pyranone derivatives to boron trifluoride ether complex is 1:(1.5~3):(3~10).

7. The synthesis method according to claim 6, characterized in that, The molar ratio of ergosterol, pyranone derivatives and boron trifluoride diethyl ether complex is 1:2.5:

5.

8. The synthesis method according to claim 1, characterized in that, The coupling reaction was carried out at a temperature of 28°C for 5 days.

9. The use of striasteroid B prepared by the synthetic method according to claim 1 in the preparation of anti-renal fibrosis drugs.

10. The application according to claim 9, characterized in that, The drug contains an effective amount of striasteroid B as the active ingredient and a pharmaceutically acceptable carrier.

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