Euphorbia lathyris type diterpene lactone compound as well as preparation method and application of euphorbia lathyris type diterpene lactone compound

Abstract

The invention provides a euphorbia lathyris diterpene lactone compound as well as a preparation method and application thereof, and belongs to the technical field of natural medicines. A structural formula of the euphorbia lathyris diterpene lactone compound is shown as a formula I: # imgabs0 #. According to the invention, the euphorbia lathyris diterpene lactone compound capable of inhibiting cardiac valve calcification is extracted from euphorbia fischeriana for the first time; the compound has great development value as a novel medicine for preventing or treating the calcified aortic valve disease, and the design thought of the compound also provides a new thought and approach for development of the novel medicine for preventing or treating the calcified aortic valve disease.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines, and particularly relates to a euphorbolane-type diterpenoid lactone compound, a preparation method thereof, and an application thereof. Background Art

[0002] Calcific aortic valve disease (CAVD) is a degenerative heart disease characterized by lipoprotein deposition, chronic inflammation, and progressive thickening and calcification of valve leaflets. Its core pathological manifestation is the sclerosis of the aortic valve structure and the loss of function. As one of the most common heart diseases, the incidence of CAVD reaches 6% in people over 65 years old and climbs to 4.6% in people over 75 years old, and it has become an important inducement for heart failure and sudden death in the elderly. At present, clinical treatment mainly relies on surgical aortic valve replacement (SAVR) or transcatheter aortic valve replacement (TAVR), but there is no effective drug to reverse or block the progression of early lesions. Research shows that aortic valve interstitial cells, as the core cell population maintaining valve homeostasis, their abnormal proliferation, osteogenic differentiation, and calcium and phosphorus metabolism disorders are the key links driving valve calcification, which provides potential targets for early drug intervention. However, existing chemically synthesized drugs are often limited by single targets or toxic side effects. Therefore, exploring multi-target and low-toxic active ingredients from natural products has become a research hotspot. In recent years, traditional Chinese medicines and their extracts have shown unique advantages by virtue of mechanisms such as regulating inflammation and inhibiting calcification, opening up a new direction for the development of new anti-CAVD drugs.

[0003] Euphorbia fischeriana Steud., a perennial herb of the Euphorbiaceae family, has thick and fleshy roots with white latex and is cylindrical. It is one of the source plants of the traditional Chinese medicine Stellera chamaejasme L. It has a pungent taste, is flat, and is poisonous. It belongs to the liver and spleen meridians and has the efficacy of dissipating binds and killing insects. It is widely used in Chinese patent medicines and clinical prescriptions. In traditional medicine, Stellera chamaejasme L. is used for treating phlegm, food, and insect accumulation, edema and abdominal distension, abdominal pain, mass accumulation, scrofula, scabies, etc. Modern research results show that traditional Chinese medicine Stellera chamaejasme L. has various pharmacological activities, such as anti-tuberculosis, anti-HIV virus, anti-tumor, antibacterial, anti-inflammatory, and insecticidal effects. Modern research shows that the chemical constituents of Euphorbia fischeriana Steud. mainly include phenolic acids and terpenoids. Among them, terpenoids are mainly diverse diterpenoids, which are also considered to be the main material basis for Euphorbia fischeriana Steud. to exert many pharmacological activities. Regarding a brand-new euphorbolane-type diterpenoid lactone compound involved in the present invention and its inhibitory activity against heart valve calcification, there has been no patent or literature report so far. Summary of the Invention

[0004] Based on the above content, the object of the present invention is to provide a euphorbolane-type diterpenoid lactone compound, a preparation method thereof, and an application thereof. The euphorbolane-type diterpenoid lactone compound has good inhibitory activity against heart valve calcification and can be used for preparing drugs for calcific aortic valve disease.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] One of the technical solutions of the present invention is a euphane-type diterpenoid lactone compound, and the structural formula is shown in Formula I:

[0007]

[0008] Another technical solution of the present invention is a preparation method of the above-mentioned euphane-type diterpenoid lactone compound, comprising the following steps:

[0009] Using the dried root of Euphorbia fischeriana as the raw material, an ethanol extract is obtained by ethanol percolation extraction;

[0010] The ethanol extract is extracted with ethyl acetate to obtain an ethyl acetate extract;

[0011] The ethyl acetate extract is eluted successively through silica gel column chromatography 1, MCI resin column chromatography, silica gel column chromatography 2, Sephadex LH-20 gel column chromatography, and ODS medium-pressure column chromatography, and the collected eluate is prepared by preparative ODS liquid chromatography to obtain the euphane-type diterpenoid lactone compound.

[0012] In the present invention, using the dried root of Euphorbia fischeriana as the raw material, an ethanol extract is obtained by ethanol percolation extraction, specifically: percolating and extracting the dried root of Euphorbia fischeriana with 95% ethanol for 120 h at a flow rate of 0.5 L / h, and the extract is rotary evaporated and concentrated to obtain an extract-like ethanol extract.

[0013] In the present invention, the ethanol extract is extracted with ethyl acetate to obtain an ethyl acetate extract, specifically: suspending the extract-like ethanol extract in water to obtain a suspension, and then adding ethyl acetate equal in volume to the suspension for extraction, and concentrating the extraction solution to obtain an extract, which is the ethyl acetate extract.

[0014] In the present invention, the elution method through silica gel column chromatography 1 is gradient elution; the eluents during elution are mixed solutions of petroleum ether and ethyl acetate with volume ratios of 10:1, 3:1, 2:1, 1:1, 1:2, and 1:10 respectively; the eluate collected by elution through silica gel column chromatography 1 is the eluate obtained by elution with a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:1.

[0015] In the present invention, the elution method through MCI resin column chromatography is gradient elution; the eluents during elution are mixed solutions of methanol and water with volume ratios of 20:80, 40:60, 60:40, 80:20, and 90:10 respectively; the eluate collected by elution through MCI resin column chromatography is the eluate obtained by elution with a mixed solution of methanol and water with a volume ratio of 80:20.

[0016] In the present invention, the elution method through silica gel column chromatography 2 is gradient elution; the eluents during elution are mixed solutions of petroleum ether, acetone and methanol with volume ratios of 10:1:1, 8:1:1, 5:1:1, 3:1:1, and 1:1:1 respectively; the eluate collected by eluting through silica gel column chromatography 2 is the eluate eluted with a mixed solution of petroleum ether, acetone and methanol with a volume ratio of 3:1:1.

[0017] In the present invention, the elution method through Sephadex LH-20 gel column chromatography is isocratic elution; the eluent during elution is a mixed solution of dichloromethane and methanol with a volume ratio of 1:1; the elution rate during elution is 1 mL / min, the elution time is 10 h, the eluate is collected every 2 h, and the eluate eluted in the 4th time period is collected.

[0018] In the present invention, the elution method through ODS medium pressure column chromatography is gradient elution; the eluents during elution are mixed solutions of methanol and water with volume ratios from 60:40 to 90:10; during elution, the volume ratio of methanol to water increases from 60:40 to 90:10 in a gradient manner, with an increase rate of 2 ratios per hour, the flow rate is 25 mL / min, the eluate is collected every 1 h, the total elution time is 15 h, and 15 eluates, namely Fr.1 to Fr.15, are obtained; the eluate Fr.10 is used to prepare the euphorbolane-type diterpenoid lactone compounds through preparative ODS liquid chromatography.

[0019] In the present invention, during the preparation through preparative ODS liquid chromatography, the mobile phase is a mixed solution of acetonitrile and water; the volume ratio of acetonitrile to water is 60:40.

[0020] Technical solution three of the present invention: an application of the above-mentioned euphorbolane-type diterpenoid lactone compounds in the preparation of a drug for preventing or treating calcific aortic valve disease.

[0021] Technical solution four of the present invention: a drug for preventing or treating calcific aortic valve disease, the raw materials of which include the euphorbolane-type diterpenoid lactone compounds described in claim 1 and pharmaceutically acceptable excipients.

[0022] In the present invention, the dosage form of the drug for preventing or treating calcific aortic valve disease is tablets, capsules, granules, oral liquids, infusion preparations, dripping pills or pellets.

[0023] The present invention discloses the following technical effects:

[0024] The present invention has for the first time achieved the extraction of euphane - type diterpene lactone compounds with the ability to inhibit cardiac valve calcification from Euphorbia fischeriana. These compounds have great development value as novel drugs for the preparation of preventive or therapeutic agents for calcific aortic valve disease. The design concept of such compounds also provides new ideas and approaches for the development of novel drugs for the prevention or treatment of calcific aortic valve disease. Brief Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0026] Figure 1 1H - NMR spectrum of the euphane - type diterpene lactone compound in Example 1 1 1H - NMR spectrum.

[0027] Figure 2 13C - NMR spectrum of the euphane - type diterpene lactone compound in Example 1 13 13C - NMR spectrum.

[0028] Figure 3 Effect of the euphane - type diterpene lactone compound of the present invention on the calcification of VIC cells; wherein, A: X - ray single - crystal diffraction structure of the euphane - type diterpene lactone compound of the present invention; B: Effect of the euphane - type diterpene lactone compound of the present invention on the expression levels of calcification - and fibrosis - related molecules Runx2 and Fn1 in a VICs cell model intervened with calcified medium; C: IC 50 value of the euphane - type diterpene lactone compound of the present invention in human primary VICs cells is 200.5 μM; D - F: Immunohistochemical staining results; G - K: Western blot results; L - M: Effect of the euphane - type diterpene lactone compound of the present invention on the ALP protein activity in VICs cells; N - O: Effect of the euphane - type diterpene lactone compound of the present invention on calcium salt deposition in primary VICs cells.

[0029] Figure 4Binding of the ingenane diterpenoid lactone compound of the present invention to Fn1 protein; wherein, A: Schematic diagram of DARTS experiment; B-C: Silver staining and mass spectrometry results of DARTS experiment; D: Schematic diagram of the structure of Fn1 protein; E-F: Verification of the samples in DARTS by Western blotting experiment; G-H: Results of DARTS experiment with human recombinant Fn1 protein; I: Molecular docking results of the ingenane diterpenoid lactone compound of the present invention and Fn1 protein; J-K: Results of CETSA experiment with lysate of human primary VICs cells; L-M: Results of TSA experiment with human recombinant Fn1 protein; N-O: SPR analysis results.

[0030] Figure 5 Effect of the ingenane diterpenoid lactone compound of the present invention on valvular calcification in the Apoe - / - mouse high-fat feeding model; wherein, A: Animal experiment design; B-D: Effect of the ingenane diterpenoid lactone compound of the present invention on the peak velocity and transvalvular pressure difference at the aortic valve orifice of the heart of Apoe - / - mice; E-H: Results of Von Kossa staining, alizarin red staining and Masson staining of the aortic valve tissue of mice; I: Analysis result of the thickness of aortic valve leaflets; J-K: Effect of long-term injection of the ingenane diterpenoid lactone compound of the present invention on the HAP deposition in the aortic valve tissue of mice; L-N: Results of alizarin red staining and Von Kossa staining in the in vitro culture experiment of human valve tissue. Detailed implementation manners

[0031] The various exemplary implementation manners of the present invention will be described in detail below. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although only preferred methods and materials are described in this invention, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0034] Without departing from the scope or spirit of this invention, various improvements and variations can be made to the specific embodiments of the description of this invention, which will be obvious to those skilled in the art. Other embodiments obtained from the description of this invention will be obvious to those skilled in the art. The description and examples of this invention are merely exemplary.

[0035] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0036] The technical solutions described in this invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been made public.

[0037] To better understand this invention, the content of this invention will be further clarified below in conjunction with examples, but the content of this invention is not limited to the following examples only.

[0038] Example 1

[0039]

[0040] The preparation method of the diterpene lactone compounds of the euphorbol type in Formula I is as follows:

[0041] (1) Using the dried root (1 kg) of Euphorbia fischeriana as the raw material, percolating and extracting with 95% ethanol for 120 h at a flow rate of 0.5 L / h, concentrating by rotary evaporation to obtain an ethanol extract in the form of an extract, suspending the ethanol extract in water to obtain a suspension, adding ethyl acetate for extraction with a volume equal to that of the suspension, and concentrating the extraction solution to obtain an extract;

[0042] (2) Subjecting the ethyl acetate extract to gradient elution by silica gel column chromatography. Specifically, the eluent is an eluent with a volume ratio of petroleum ether - ethyl acetate of 10:1, 3:1, 2:1, 1:1, 1:2, 1:10. Six eluates are collected by gradient elution with the eluent in segments;

[0043] (3) The eluate obtained by eluting with the eluent with a petroleum ether - ethyl acetate volume ratio of 1:1 in step (2) is subjected to MCI resin column chromatography and gradient eluted with a methanol - aqueous solution. Specifically, the eluents are eluents with a methanol - water volume ratio of 20:80, 40:60, 60:40, 80:20, and 90:10. Five eluates are collected by gradient elution with the eluents in segments;

[0044] (4) The eluate eluted with the eluent with a methanol - water volume ratio of 80:20 in step (3) is subjected to silica gel column chromatography and gradient eluted with a petroleum ether - acetone - methanol solvent. Specifically, the eluents are eluents with a petroleum ether - acetone - methanol volume ratio of 10:1:1, 8:1:1, 5:1:1, 3:1:1, and 1:1:1. Five eluates are collected by gradient elution with the eluents in segments;

[0045] (5) The eluate eluted with the eluent with a petroleum ether - acetone - methanol volume ratio of 3:1:1 in step (4) is subjected to Sephadex LH - 20 gel column chromatography and isocratically eluted with a dichloromethane - methanol solvent. Specifically, the elution rate of the eluent is 1 mL / min, the elution time is 10 h, and it is collected once every 2 h. Five eluates are collected by elution with the eluent in segments;

[0046] (6) The eluate eluted with the eluent in the 4th time period in step (5) is subjected to ODS medium - pressure column chromatography and gradient eluted with a methanol - water solvent. Specifically, the eluents are eluents with a methanol and water volume ratio that rises from 60:40 to 90:10 with a rising rate of 2 ratios per hour, the flow rate is 25 mL / min, and it is collected once every 1 h. The total elution time is 15 h. Fifteen eluates Fr.1 - Fr.15 are collected by gradient elution with the eluent in segments;

[0047] (7) The Fr.10 part in step (6) is prepared by preparative DS liquid chromatography with an acetonitrile - aqueous solution with a volume ratio of 60:40 as the mobile phase to obtain the compound of formula I.

[0048] Example 2

[0049] The compound of formula I prepared in Example 1 is subjected to structure identification.

[0050] The identification results are as follows:

[0051] The compound shown in formula I is a colorless block crystal (methanol), highly soluble in methanol and dichloromethane. The 10% sulfuric acid - ethanol solution shows a purplish - red color. Nuclear magnetic resonance, mass spectrometry, optical rotation, infrared spectroscopy, ultraviolet spectroscopy, X - ray single - crystal diffraction and other data tests are carried out on the compound shown in formula (I) to determine that the compound of formula I is the novel euphorbolane - type diterpenoid lactone compound of the present invention.

[0052] Physical and chemical data of compound Ⅰ (ELA): colorless block crystals; melting point: 187–188 °C; −136.5 (methanol, c 0.1); UV (methanol) λ max (log ε): 204 (4.34) nm, 234 (4.17) nm, 276 (3.99) nm; IR (KBr) ν max : 3576, 2943, 2923, 2859, 1708, 1643, 1613, 1453, 1325, 1315, 1297, 1280, 1268, 1223, 1146, 1117, 1071, 1058, 1027, 998, 985, 902, 863, 715 cm –1 ; (+)-HRESIMSm / z 445.2337 [M + Na] + (C 27 H 34 O4Na + The calculated value is m / z 445.2355); 1 H and 13 13C NMR data are shown in Table 1.

[0053] X-ray single crystal diffraction data of compound Ⅰ (ELA) ( Figure 3 in A, CDCC No. 2412796): C 27 H 34 O4, M = 422.54 g / mol, α = 90°, β = 90°, γ = 90°, T = 293(2) K, space group C2221, Z = 8, μ(Cu Kα) = 0.624 mm -1 , 14136 reflections measured, 4641 independent reflections (R int = 0.0261). The final R1 values were 0.0317 (I > 2σ(I)). The final wR(F 2 ) values were 0.0823 (I > 2σ(I)). The final R1 values were 0.0322 (all data). The final wR(F 2 ) values were 0.0827 (all data). The goodness of fit on F 2 was 1.041. Flack parameter was 0.07(6).

[0054] Table 1 of the compounds of Formula I 1 1H-NMR (600 MHz, CDCl3) and 13 13C-NMR (150 MHz, CDCl3) data

[0055]

[0056]

[0057] Pharmacological Activity of Example 3

[0058] Test Method

[0059] Animal model: Male C57BL / 6J and apolipoprotein E knockout (Apoe - / - ) mice (weighing 20 - 25 g, 6 weeks old) were purchased from Wuhan Shulaibao Biotechnology Co., Ltd. The wild-type control group (C57BL / 6J, n = 4) was fed a standard maintenance diet, and the Apoe - / - group (n = 6) was fed a Western diet for 24 weeks. After completion of transthoracic echocardiography and hemodynamic evaluation, the mice were euthanized, and the aortic valves were collected for histopathological examination and spatial transcriptome sequencing. In the animal experiment verification of Compound I, the Apoe - / - group was fed a Western diet for 24 weeks, and Compound I (20 mg / kg) was intraperitoneally injected once a week during this period.

[0060] Cell and tissue culture: The healthy human aortic valve tissue used as the heart transplantation donor in the present invention was obtained from patients with dilated cardiomyopathy. The isolation method of primary human valvular interstitial cells (hVICs) referred to the previous literature (PMID: 37441596). The tissue blocks were placed in DMEM medium containing 10% heat-inactivated fetal bovine serum (FBS, Thermo Fisher Scientific, USA) and 1% penicillin / streptomycin (HyClone) for culture, and the fourth-generation cells were used for experiments. The isolation method of mouse valvular interstitial cells (mVICs) referred to the technical system established in PMID: 34028451. Tissue culture protocol: Under sterile conditions, the healthy human aortic valve was rinsed with PBS and then cut into tissue blocks of 5 mm 2 in size; the tissue blocks were placed in the medium with different treatment conditions and continuously cultured for 6 weeks; after the culture was completed, the human aortic valve tissue was fixed in 4% paraformaldehyde for subsequent experiments.

[0061] Preparation of key reagents: Osteogenic induction medium (OM): containing 10 mM β-glycerophosphate, 100 nM dexamethasone, 50 μg / ml L-ascorbic acid, 2% FBS and 1% penicillin / streptomycin; The polypeptide pUR4 (sequence SEQ ID NO.1: GSKDQSPLAGESGETEYITEVYGNQQNPVDIDKKLPNETGFSGNMVETEDTKLN) was custom synthesized by Wuhan Tianda Biotechnology Co., Ltd. (Wuhan, China), and the working concentration was 40 μM; The concentration of compound I used in tissue culture experiments was 20 μM.

[0062] Spatial transcriptome sequencing: Obtain aortic root tissue samples from anesthetized mice, and perform sectioning after cryopreservation. The tissue sections were fixed on slides and stained with hematoxylin-eosin (H&E) routinely. To construct a barcode-labeled library, the aortic valve tissue samples were permeabilized, and their original spatial localization information was analyzed by cell clustering. The spatial transcriptome sequencing was completed by OE Biotech Co., Ltd. (China).

[0063] Compound screening: Primary human valve interstitial cells (hVICs) were seeded in 96-well plates. In the presence of osteogenic medium (OM), individual compounds were added to the wells, and the medium was changed every 3 days. After 21 days of culture, alizarin red staining was performed and imaging was carried out using the Leica MICA system. For high-content screening, Fn1 immunofluorescence staining was performed 7 days after cell culture. The cell nuclei were counterstained with DAPI. Then, the stained cells were imaged and analyzed using the Opera Phenix high-content screening system (Revvity).

[0064] Western blotting: The protein immunoblotting experiment was performed according to the standard procedure. The specific steps included: extracting the total proteins from cell and tissue samples, separating them by polyacrylamide-sodium dodecyl sulfate gel electrophoresis and then transferring them to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked in TBST buffer (Tris-HCl buffer containing 0.05% Tween-20) containing 5% skim milk powder, and then incubated with the primary antibody at 4 °C overnight. After incubation with the secondary antibody, protein expression was detected using a Bio-Rad imaging system combined with chemiluminescent substrate. Key antibody information: RUNX2 (cat. no. 8486, 1:1000, Cell Signaling Technology, USA); alkaline phosphatase ALP (cat. no. MAB29092, 1:1000, R&D Systems, USA); type I collagen COL1A1 (cat. no. ab138492, 1:1000, Abcam, UK); fibronectin Fn1 (cat. no. ab268020, 1:1000, Abcam, UK); GAPDH (cat. no. AC001, 1:10000, Abclonal, China).

[0065] IHC staining: In this experiment, primary cells were inoculated into confocal culture dishes for culturing. After the cells were fixed with 4% paraformaldehyde, they were permeabilized with PBS solution containing 0.25% Triton X-100. Nonspecific binding sites were blocked with 5% bovine serum albumin (BSA) at room temperature for 30 minutes, and then incubated with specific primary antibody overnight at 4°C. After washing with PBS, HRP-labeled secondary antibody was incubated at room temperature for 30 - 60 minutes. A brown precipitate was generated by the chromogenic reaction of diaminobenzidine (DAB), the cell nuclei were counterstained with hematoxylin, and microscopic imaging analysis was performed after mounting the slides.

[0066] ALP activity, alizarin red and Von Kossa staining: ALP activity staining: BCIP / NBT alkaline phosphatase color development kit (Beyotime) was used for staining, and the operation strictly followed the instructions of the kit. This kit forms an insoluble dark blue precipitate through the reaction of the hydrolysis product of BCIP with NBT, specifically labeling the alkaline phosphatase activity region. Alizarin red staining: After the cells were rinsed with PBS, they were fixed with 4% paraformaldehyde at room temperature for 10 minutes; rinsed with distilled water 3 times to remove residual fixative; stained with alizarin red solution (Servicebio) to detect calcium nodule deposition. Von Kossa staining: The slides were immersed in 5% silver nitrate solution (Sigma) for 30 minutes; exposed to direct sunlight for 1 hour to promote the reduction of silver ions to black metallic silver particles; treated with 5% sodium thiosulfate (Sigma) to remove unreacted silver salts. Bright-field images of the stained samples were collected with a Zeiss microscope (Axio Imager Z2), and quantitative analysis was performed using ZEN software.

[0067] DARTS: Preparation of cell lysate: hVICs cells were collected, and after lysis, the protein concentration was adjusted to 2 mg / mL; compound I (100 μM and 200 μM) was added to the experimental groups respectively, and an equal volume of DMSO was added to the control group; incubated at room temperature for 2 hours to induce the formation of drug-target protein complexes. Proteolysis treatment: Pronase was added at a mass ratio of 1:4000; digested at room temperature for 10 minutes (accurately controlling the proteolysis time to retain stable complexes). Reaction termination and sample treatment: The reaction was terminated by adding 0.5 M EDTA (pH 8.0) (final concentration of EDTA 50 mM); the enzyme activity was terminated by rapid cooling on ice; 5× SDS loading buffer was added, and the proteins were denatured by boiling at 100°C for 10 minutes. Electrophoresis and staining analysis: Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); stained with Pierce TM Silver staining reagent (product number 24600, Thermo Fisher) was used for staining. Mass spectrometry identification: The differential protein bands were excised (compared with the DMSO control group); the drug-binding targets were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0068] CETSA (Cell Thermal Shift Assay) and TSA (Thermal Shift Assay) were used to collect lysates of human primary valvular interstitial cells (hVICs). In the experimental group, 200 μM of Compound I was added, and in the control group, an equal volume of DMSO was added. Incubation was carried out at room temperature for 2 hours to induce the formation of drug - target protein complexes. Gradient heating was performed using a PCR instrument (51°C → 72°C, linear heating for 10 minutes), and then the samples were equilibrated at room temperature for 5 minutes to promote protein conformational rearrangement. 5× SDS loading buffer was added, and the reaction was terminated by boiling at 100°C for 10 minutes. Proteins were separated by SDS - polyacrylamide gel electrophoresis (SDS - PAGE), and after transfer to the membrane, Western blot analysis was performed using the Fn1 (1:200, CST) antibody. Purified human fibronectin Fn1 (R&D Systems, catalog number 4305 - FNB - 200) was taken. In the experimental group, 200 μM of Compound I was added, and in the control group, DMSO was added. Temperature - programmed treatment was carried out (57°C → 72°C, gradient heating for 15 minutes; after heating, the samples were immediately placed on ice to terminate the reaction), and the thermal stability of the proteins was analyzed by SDS - PAGE and immunoblotting, and the target protein was detected using the anti - Fn1 antibody (1:200, CST 26836).

[0069] SPR (Surface Plasmon Resonance Analysis): At 25°C, the equilibrium dissociation constant (K d ) of Compound I with human Fn1 protein was determined using surface plasmon resonance (SPR) on a Biacore 1K SPR system (Cytiva). The Fn1 protein (R&D, 4305 - FNB - 200) was immobilized on a Series S CM5 sensor chip (Cytiva) through amine coupling chemistry, achieving a fixed level of 10,000 - 12,000 resonance units (RU). In the running buffer solution, Compound I was serially diluted to a concentration range of 80 μM to 0.4 nM and injected into the chip in ascending order of concentration. Each cycle included injecting 200 μL of the sample at the specified concentration, with a contact time of 120 seconds and then a dissociation time of 180 seconds. Data were analyzed using the Biacore Insight Evaluation Software (Version 3.0.12), and the binding affinity was calculated using the steady - state 1:1 binding model.

[0070] Molecular docking: Molecular docking simulations were performed using AutoDock Vina (version 1.2.3) (PMID: 19499576, PMID: 34278794) to study the binding mode of Compound I with the Fn1 protein. The structure of the Fn1 protein (PDB ID: 3M7P) was obtained from the RCSB PDB website (http: / / www.rcsb.org / ).

[0071] Fabrication and culture of human aortic valve microtissues: To generate aligned three-dimensional human valve tissue bundles, 14×12 mm 2 polydimethylsiloxane (PDMS, SYLGARD 184, Dow Corning) molds and 12-mm-long Velcro frames were used. A hydrogel solution composed of 20 μL fibrinogen (10 mg / ml), 10 μL Matrigel, and 20 μL collagen I was mixed with 5×10 5 human primary valve interstitial cells in 48 mL of culture medium to obtain a total of 100 μL of cell / gel solution. After adding 2.4 μL of thrombin, the cell / gel solution was placed in a PDMS mold containing a Velcro frame and polymerized at 37 °C for 1 hour. The obtained human valve tissue engineering microtissues were cultured in a valve tissue medium (10% FBS / DMEM + 1 mM sodium pyruvate (Gibco)), 2 mM glutamine (Gibco), 0.1 mM non-essential amino acids (Gibco), 50 μg / mL ascorbic acid (Sigma), 0.45 mM monothioglycerol (Sigma), 1 mg / mL 6-aminocaproic acid) on a shaker for 21 days. 10 μM ROCK inhibitor Y27632 was added on the first day; the medium was changed after 24 h to remove Y27632. Then the tissues were cultured in the valve tissue medium, and the medium was changed once every 3 days. The cells were cultured dynamically on a shaker and then transferred to a calcification induction medium.

[0072] Staining of mouse aortic valve tissue: The aortic root was microdissected and fixed in 4% paraformaldehyde (PFA) at 4 °C overnight. The tissues were washed 3 times in 1×PBS and stained overnight at 37 °C in freshly prepared OsteoSense TM 680EX (Revvity, NEV10020EX, 100 pmol / L PBS) solution. The cell nuclei were counterstained with Hoechst 33258 (Servicebio, G1011) at 37 °C for 1 hour. After staining, z-stack images of the entire aortic root volume were captured using an Olympus FV3000 confocal system. The positive area of Osteo Sense TM 680EX signal in each tissue was quantified using Image J software.

[0073] Activity results

[0074] The IC 50 value of compound I prepared in Example 1 in human primary VICs cells was 200.5 μM, and the cytotoxic effect was not obvious ([ Figure 3 in C). The results of immunohistochemical staining showed that in the VICs cell model intervened by OM, compound I (10 μM) could reduce the expression of Fn1 and Collagen I ([Figure 3 In Figures D - F). Western blot results showed that in the VICs cell model intervened by OM, compound I (10 μM) could reduce the expression of Fn1, Collagen I, Runx2 and ALP ( Figure 3 In Figures B, G - K). In the experiment of OM intervention for 7 days, compound I (10 μM) could reduce the ALP protein activity in human primary VICs cells ( Figure 3 In Figures L, M). In the experiment of long - term OM intervention for 21 days, alizarin red staining results showed that compound I (10 μM) could reduce the calcium salt deposition in human primary VICs cells ( Figure 3 In Figures N, O).

[0075] Confirmation of the interaction between compound I and Fn1 protein: The silver staining and mass spectrometry results of DARTS experiment showed that with the increase of compound I concentration, the number of polypeptide fragments of Fn1 identified in the gel strip increased ( Figure 4 In Figures A - D). Western blotting experiment was used to verify the sample concentration in DARTS, and it was found that the gray value of the Fn1 band increased with the increase of compound I concentration ( Figure 4 In Figures E - F). DARTS experiment was carried out using human recombinant Fn1 protein, and similar results were obtained by Western blotting detection ( Figure 4 In Figures G - H). Molecular docking results suggested that the docking binding energy between compound I and Fn1 protein was - 8.1 kcal / mol, and the possible binding sites of compound I and Fn1 were Leu - 323, Thr - 325, Gln - 330, Tyr - 372, Val - 406, Gln - 409, Ala - 465, Phe - 531 ( Figure 4 In Figure I). CETSA experiment was carried out using human primary VICs cell lysate, and the results showed that compound I could improve the thermal stability of Fn1 protein ( Figure 4 In Figures J - K). TSA experiment was carried out using human recombinant FN1 protein, and the results showed that compound I could improve the thermal stability of Fn1 protein ( Figure 4 In Figures L - M). SPR detection results showed that the dissociation constant K of compound I and human recombinant Fn1 protein ( d value was 9.19 pM ( Figure 4 In Figures N - O).

[0076] In Apoe - / - mouse high - fat feeding model ( Figure 5 In Figure A), long - term intraperitoneal injection of compound I (20 mg / kg, once a week) could reduce Apoe - / - the peak flow velocity and trans - valve pressure difference at the aortic valve orifice of Apoe mice heart ( Figure 5 In Figures B - D), and could reduce Apoe- / - Leaf thickness of the aortic valve of mice ( Figure 5 I). The whole mouse aortic valve tissue was stained with OsteoSens 680EX dye to detect hydroxyapatite (HAP) in the mouse aortic valve tissue. The results showed that long-term injection of Compound I could reduce HAP deposition in the mouse aortic valve tissue ( Figure 5 J-K). In the in vitro culture experiment of human valve tissue, the alizarin red and Von Kossa staining results showed that Compound I could reduce calcium salt deposition in the calcified culture of human valve tissue ( Figure 5 L-N).

[0077] In summary, the euphorbol diterpenoid lactone compounds prepared by the present invention have good inhibitory activity against cardiac valve calcification and can be used for preparing drugs for calcific aortic valve disease. As a novel drug for preventing or treating calcific aortic valve disease, such compounds have great development value, and the design concept of such compounds also provides new ideas and approaches for the development of novel drugs for preventing or treating calcific aortic valve disease.

[0078] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A euphane-type diterpenoid lactone compound, characterized in that, The structural formula is as shown in Formula I:

2. A method for preparing the euphane - type diterpene lactone compound according to claim 1, characterized in that, It includes the following steps: Using the dry root of Euphorbia fischeriana as the raw material, an ethanol extract is obtained by ethanol percolation extraction; The ethanol extract is extracted with ethyl acetate to obtain an ethyl acetate extract; The ethyl acetate extract is eluted successively through silica gel column chromatography 1, MCI resin column chromatography, silica gel column chromatography 2, Sephadex LH-20 gel column chromatography, and ODS medium-pressure column chromatography, and the collected eluate is prepared by preparative ODS liquid chromatography to obtain the euphorbolane-type diterpene lactone compounds.

3. The preparation method according to claim 2, characterized in that, The elution method through silica gel column chromatography 1 is gradient elution; the eluents during elution are mixed solutions of petroleum ether and ethyl acetate with volume ratios of 10:1, 3:1, 2:1, 1:1, 1:2, and 1:10 respectively; the eluate collected by elution through silica gel column chromatography 1 is the eluate eluted with a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 1:

1.

4. The preparation method according to claim 2, wherein, The elution method through MCI resin column chromatography is gradient elution; the eluents during elution are mixed solutions of methanol and water with volume ratios of 20:80, 40:60, 60:40, 80:20, and 90:10 respectively; the eluate collected by elution through MCI resin column chromatography is the eluate eluted with a mixed solution of methanol and water with a volume ratio of 80:

20.

5. The preparation method according to claim 2, characterized in that, The elution method through silica gel column chromatography 2 is gradient elution; the eluents during elution are mixed solutions of petroleum ether, acetone, and methanol with volume ratios of 10:1:1, 8:1:1, 5:1:1, 3:1:1, and 1:1:1 respectively; the eluate collected by elution through silica gel column chromatography 2 is the eluate eluted with a mixed solution of petroleum ether, acetone, and methanol with a volume ratio of 3:1:

1.

6. The preparation method according to claim 2, characterized in that, The elution method through Sephadex LH-20 gel column chromatography is isocratic elution; the eluent during elution is a mixed solution of dichloromethane and methanol with a volume ratio of 1:1; the elution speed during elution is 1 mL / min, the elution time is 10 h, and the eluate is collected every 2 h, and the eluate eluted by the eluate in the 4th time period is collected.

7. The preparation method according to claim 2, characterized in that, The elution method through ODS medium-pressure column chromatography is gradient elution; the eluents during elution are mixed solutions of methanol and water with volume ratios of 60:40 to 90:10; during elution, the volume ratio of methanol to water increases from 60:40 to 90:10 in a gradient manner, the rising speed is 2 ratios per hour, the flow rate is 25 mL / min, the eluate is collected every 1 h, the total elution time is 15 h, and 15 eluates, namely Fr.1 to Fr.15, are obtained; the eluate Fr.10 is prepared by preparative ODS liquid chromatography to obtain the euphorbolane-type diterpene lactone compounds.

8. The preparation method according to claim 2, wherein During the preparation by preparative ODS liquid chromatography, the mobile phase is a mixed solution of acetonitrile and water.

9. Use of the euphorbolane-type diterpene lactone compound according to claim 1 in the preparation of a drug for preventing or treating calcific aortic valve disease.

10. A drug for preventing or treating calcific aortic valve disease, characterized in that, The raw materials include the euphorbolane-type diterpene lactone compound according to claim 1 and pharmaceutically acceptable excipients.

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