Semi-cage-shaped sorbitol polyketone compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of natural medicines, in particular to a semi-cage-shaped sorbitol polyketone compound as well as a preparation method and application thereof. The method comprises the following steps: by taking a rice secondary metabolite of trichoderma reesei fungus as a raw material, soaking, extracting and concentrating to obtain an extract, suspending the extract in water to obtain a suspension, and extracting and concentrating to obtain an ethyl acetate part extract; the extract is taken and subjected to separation through a positive and negative phase silica gel column, MCI medium-pressure column chromatography, Sephadex LH-20 gel column chromatography and high performance liquid chromatography purification, and the semi-cage-shaped sorbitol polyketone compound is prepared. The compounds show significant potential in the aspect of being developed into innovative drugs for preventing and treating calcified aortic valve diseases, and the unique molecular structure of the compounds opens up an original design path for research and development of novel drugs for the diseases.

Description

Technical Field

[0001] This invention relates to the field of natural medicine technology, and in particular to a semi-cage-like sorbitol polyketide compound, its preparation method, and its application. Background Technology

[0002] Novel natural products have always been a vital source of molecular inspiration for drug development, expanding the chemical space of bioactive small molecules and revealing new paradigms in chemical and biosynthesis. Sorbitol compounds are typical examples of this type of molecule: their characteristic sorbitol side chains are linked to highly oxidized polyketide cores, forming unique hexakone-derived structures. Since their initial discovery, over 200 sorbitol compounds, primarily derived from Penicillium and Trichoderma, have been reported, exhibiting a wide range of biological activities, including antibacterial, antiviral, and cytoprotective effects. Their conjugated diene sorbitol group, as a chemically rich pharmacophore, can regulate molecular recognition, redox behavior, and protein interactions. However, the inherent structural complexity and low natural abundance of these compounds severely hinder systematic structure-activity relationship studies and their broader development and utilization as chemical resources.

[0003] Calcific aortic valve disease is a progressive disease driven by chronic inflammation, oxidative stress, and pathological tissue remodeling, with its pathogenesis primarily regulated by phenotype transformation of valvular interstitial cells. Conventional systemic therapies (including statins and angiotensin-converting enzyme inhibitors) have failed to effectively delay disease progression in large clinical trials, highlighting a pressing clinical need in this field. Therefore, therapeutic strategies that directly target early pathological events, particularly redox imbalances and pro-inflammatory signaling pathways, are receiving increasing attention. Summary of the Invention

[0004] Based on the above, the present invention provides a semi-cage-like sorbitol polyketide compound, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a semi-cage-like sorbitol polyketide compound, the structural formula of which is shown in compounds 1-8 below:

[0006]

[0007] .

[0008] The second technical solution of the present invention is a method for preparing the above-mentioned semi-cage-like sorbitol polyketide compound, comprising the following steps: (1) Using rice secondary metabolites of Trichoderma reesei as raw material, extract was obtained by soaking, extraction and concentration. The extract was suspended in water to obtain a suspension, and then extracted and concentrated to obtain a crude extract. (2) The crude extract was separated by silica gel column chromatography and eluted with petroleum ether-ethyl acetate at volume ratios of 5:1, 3:1, 2:1, 1:1, 1:2, and 1:5 to obtain six major fractions 1-6. (3) Fraction 3 was separated by medium-pressure preparative liquid chromatography with octadecylsilane-bonded silica gel and eluted with methanol-water gradient to obtain 25 subfractions 3.1–3.25; (4) Fraction 3.18 was separated by Sephadex LH-20 gel column chromatography to obtain 5 secondary fractions 3.18.1–3.18.5; (5) The fraction 3.18.4 was further purified by semi-preparative high performance liquid chromatography to obtain compound 1, compound 4 and compound 5; (6) Fraction 3.22 was separated by silica gel column chromatography to obtain 9 components 3.22.1–3.22.9; (7) Fraction 3.22.3 was separated by Sephadex LH-20 gel column chromatography to obtain 5 subfractions 3.22.3.1–3.22.3.5; (8) The fractions 3.22.3.4 were purified by semi-preparative high performance liquid chromatography to obtain compounds 2 and 3; (9) Fraction 3.22.4 was separated by preparative high performance liquid chromatography to obtain 5 subfractions 3.22.4.1–3.22.4.5; (10) Fraction 3.22.6 was separated by Sephadex LH-20 gel column chromatography to obtain 8 components (3.22.6.1–3.22.6.8). (11) Fraction 3.22.6.3 was separated by semi-preparative high performance liquid chromatography to obtain 5 components 3.22.6.3.1–3.22.6.3.5; (12) The fraction 3.22.6.3.1 was purified by semi-preparative high performance liquid chromatography to obtain compound 6 and compound 7; (13) Fraction 4 (32 g) was eluted by silica gel column chromatography gradient to obtain 6 fractions 4.1–4.6; (14) Fraction 4.1 was separated by preparative high performance liquid chromatography to obtain 5 subfractions 4.1.1–4.1.5; (15) Fraction 4.1.5 was purified by semi-preparative high performance liquid chromatography to obtain compound 8.

[0009] The third technical solution of this invention is the application of the above-mentioned semi-cage-like sorbitol polyketide compound in the preparation of a drug for preventing and treating calcified aortic valve disease.

[0010] The fourth technical solution of the present invention is a drug for preventing and treating calcified aortic valve disease, the effective ingredient of which includes the above-mentioned semi-cage-like sorbitol polyketide compound.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This invention involves the isolation and purification of an ethanol extract of rice secondary metabolites from *Trichoderma reesei* fungus, yielding a series of novel compounds. Using various spectroscopic analyses and other methods, their structures were determined to be semi-cage-like sorbitol polyketides. Evaluation of the lipopolysaccharide (LPS)-induced anti-inflammatory activity in RAW 264.7 macrophages revealed that compounds 2 and 3 exhibited potent anti-inflammatory activity at a concentration of 100 μM. Compound 3, in particular, demonstrated a potent inhibitory effect on aortic valve calcification by restoring GPX4 expression and reducing ferroptosis, proving its potential as a lead compound for the development of novel drugs for the prevention or treatment of calcified aortic valve disease. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 In the image, A represents two possible structures proposed for compound 1 based on spectroscopic analysis; B represents compound 1 (left) and 1... (Right) Key ROESY related signals in the 3D geometric optimization model; C represents candidate structures 1 and 1 The DP4+ probability analysis results; D represents candidate structures 1 and 1 Comparison of NMR calculation data.

[0014] Figure 2 In the diagram, A represents the four possible stereoisomers of compound 1; B represents the experimental and calculation results for 1a and 1b. 13 Linear correlation of 1 / 2C NMR chemical shift and DP4+ analysis; C shows the comparison of experimental and calculated ECD spectra of compounds 1, 2, and 9; D shows the experimental and calculated spectra of modified compounds 1–11. 13 Linear correlation analysis of C NMR chemical shifts.

[0015] Figure 3In the table, A represents the detection of nitrite concentration in LPS-stimulated cells by compounds 1, 2, 3, 8, 10, and 11; BD represents the analysis of ALP and RUNX2 protein expression in hVICs by the 11 compounds; EF represents the alizarin red staining of hVICs by the 11 compounds; GI represents the analysis of ALP and RUNX2 protein expression in hVICs by different concentrations of compound 3 (RSC); JK represents the alizarin red staining of hVICs by different concentrations of RSC; LM represents the staining and quantification of ALP activity under three conditions; and N represents the determination of the cytotoxic IC50 value of RSC.

[0016] Figure 4 A shows an RNA-seq differential gene heatmap, displaying the expression and pathways of C1 and C3 modules; B shows log2FC validation of GPX4 expression; CD shows Western blot analysis of ALP, RUNX2, and GPX4; EF shows C11 BODIPY assay for lipid peroxidation levels; G shows FerroOrange assay for intracellular iron levels. Figure 4 The compounds verified by AG are all compounds 3).

[0017] Figure 5 AC represents the ultrasound detection and quantification of peak jet velocity and mean pressure gradient of the aortic valve in each group of mice 6 weeks post-surgery; DF represents the Von Kossa and Masson staining of aortic valve tissue in each group; GH represents the GPX4 immunofluorescence staining analysis of aortic valve tissue in each group. Figure 5 All of the compounds verified by AF were compound 3). Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The first aspect of this invention provides a semi-cage-like sorbitol polyketide compound with the structural formulas shown in Compounds 1-11 below:

[0024] A second aspect of the present invention provides a method for preparing the above-mentioned semi-cage-like sorbitol polyketide compound, comprising the following steps: (1) Using rice secondary metabolites of Trichoderma reesei as raw material, the extract was obtained by soaking, extraction and concentration. The extract was suspended in water to obtain a suspension, and then extracted and concentrated to obtain a crude extract. (2) The crude extract was separated by silica gel column chromatography and eluted with petroleum ether-ethyl acetate at volume ratios of 5:1, 3:1, 2:1, 1:1, 1:2, and 1:5 to obtain six main fractions A to F; (3) Fraction 3 (78.3 g) was separated by medium-pressure preparative liquid chromatography with octadecylsilane-bonded silica gel and eluted with a methanol-water gradient (20%→100%) to obtain 25 subfractions (3.1–3.25). (4) Fraction 3.18 was separated by Sephadex LH-20 gel column chromatography (methanol elution) to obtain 5 secondary fractions (3.18.1–3.18.5). (5) The fraction 3.18.4 was further purified by semi-preparative high performance liquid chromatography (methanol-water, 62:38, v / v) to give compounds 1, 4 and 5; (6) Fraction 3.22 was separated by silica gel column chromatography (petroleum ether-ethyl acetate system, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 0:1, v / v) to obtain 9 components (3.22.1–3.22.9). (7) Fraction 3.22.3 was separated by Sephadex LH-20 gel column chromatography (methanol elution) to obtain 5 secondary fractions (3.22.3.1–3.22.3.5). (8) The fractions 3.22.3.4 were purified by semi-preparative high performance liquid chromatography (methanol-water, 60:40, v / v) to give compounds 2 and 3; (9) The fractions 3.22.4 were separated by preparative high performance liquid chromatography (methanol-water, 67:33, v / v) to obtain 5 components (3.22.4.1–3.22.4.5). (10) The fraction 3.22.4.2 was purified by semi-preparative high performance liquid chromatography (acetonitrile-water, 52:48, v / v) to give compound 9; (11) The fractions 3.22.4.3 were purified by semi-preparative high performance liquid chromatography (acetonitrile-water, 51:49, v / v) to give compounds 10 and 11; (12) Fraction 3.22.6 was subjected to Sephadex LH-20 gel column chromatography (methanol elution) to obtain 8 fractions (3.22.6.1–3.22.6.8); (13) The fractions 3.22.6.3 were separated by semi-preparative high performance liquid chromatography (acetonitrile-water, 62:38, v / v) to obtain 5 components (3.22.6.3.1–3.22.6.3.5). (14) Fraction 3.22.6.3.1 was purified by semi-preparative high performance liquid chromatography (methanol-water, 50:50, v / v) to give compounds 6 and 7; (15) Fraction 4 (32 g) was subjected to silica gel column chromatography (petroleum ether-ethyl acetate, 10:1→5:1→1:1) gradient elution to obtain 6 fractions (4.1–4.6). (16) 4.1 The fractions were separated by preparative high performance liquid chromatography (methanol-water, 65:35, v / v) to obtain 5 subfractions (4.1.1–4.1.5). (17) Fraction 4.1.5 was purified by semi-preparative high performance liquid chromatography (acetonitrile-water, 53:47, v / v) to give compound 8.

[0025] In a preferred embodiment of the present invention, in step (1), ethanol with a volume fraction of 95% is used for soaking and extraction, and the soaking and extraction is performed three times, with each soaking and extraction lasting for 24 hours.

[0026] In a preferred embodiment of the present invention, in step (1), ethyl acetate is used for extraction; the volume ratio of ethyl acetate to the suspension is 1:1.

[0027] A third aspect of the present invention provides the use of the above-mentioned semi-cage-like sorbitol polyketide compound in the preparation of a medicament for the prevention and treatment of calcified aortic valve disease.

[0028] The fourth aspect of the present invention provides a medicament for preventing and treating calcific aortic valve disease, the active ingredient of which includes the above-mentioned semi-cage-like sorbitol polyketide compound.

[0029] In a preferred embodiment of the present invention, the medicament for preventing and treating calcified aortic valve disease further includes pharmaceutically acceptable excipients.

[0030] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0031] The method for preparing rice secondary metabolites from Trichoderma reesei used in this embodiment of the invention comprises the following steps: The fungus *Trichoderma reesei* was initially cultured for 5 days on potato dextrose agar (PDA) plates at 28°C, with each plate containing 10 ml of standard PDA (0.0461 g / ml), for a total of 100 plates. Under aseptic conditions, the inoculum was inoculated onto 40 kg of rice culture medium and statically fermented at room temperature for 30 days. The inoculum ratio was 1:40 (m / m), and the rice culture medium consisted of a mixture of rice and water at a mass-to-volume ratio of 1 g:2 ml.

[0032] The *Trichoderma reesei* fungus in this invention was isolated from a plant sample of *Gastrodia elata* Blume, collected in September 2021 from Yingshan County, Hubei Province, China. The sequence data of this strain has been submitted to the Japanese DNA Database (DDBJ), the European Molecular Biology Laboratory Nucleic Acid Database (EMBL), and the National Center for Biotechnology Information (GenBank), with accession number KJ767092.1. This strain has been published in the literature “Trichodenoids A and B, Two Skeletally Unprecedented Polyketides from *Trichoderma reesei* with Cardioprotective Effects against H-Induced Injury”.

[0033] In this embodiment of the invention, when preparative or semi-preparative high-performance liquid chromatography (HPLC) is used for separation or purification, preparative HPLC is performed on a Ruihe Instruments instrument (China), equipped with a UV detector and a YMC C18 column (5 μm, 250 × 21.2 mm, Welch Materials, Inc.). Semi-preparative HPLC analysis is performed using an Agilent 1220 HPLC system equipped with a reversed-phase (RP) C18 column (5 μm, 10 × 250 mm, Welch Materials, Inc.), with multiple detection wavelengths (210 nm, 230 nm, 254 nm, 280 nm, 365 nm) and a column temperature of 30 °C. In the embodiments of this invention, column chromatography separation was performed using normal phase chromatography (CC) with silica gel (200-300 mesh, Qingdao Ocean Chemical Co., Ltd., Qingdao, China), octadecylsilane-bonded silica gel (ODS, 50 μm, YMC Corporation, Tokyo, Japan) and Sephadex LH-20 (40-70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden).

[0034] The type I collagenase used in the embodiments of this invention is from (Biosharp, Beijing Lanjieke Technology Co., Ltd.).

[0035] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1 Using 40 kg of rice secondary metabolites from *Trichoderma reesei* fungus as raw material, the extract was obtained by soaking in 95% ethanol three times, each time for 24 h. The concentrate was then suspended in water to obtain a suspension, which was extracted three times with ethyl acetate (1:1, v / v), and concentrated to obtain a crude extract (200 g). The crude extract was separated by silica gel column chromatography using a petroleum ether (PE)-ethyl acetate (EA) gradient system (5:1, 3:1, 2:1, 1:1, 1:2, 1:5, v / v, eluting 45 L at each ratio, collecting once every 45 L), yielding six major fractions (1–6).

[0037] Fraction 3 was separated by medium-pressure preparative liquid chromatography (C18 ODS, methanol-water 25%→100%, 25 h, 25 mL / min, collected every 1 h) to obtain twenty-five subfractions (3.1–3.25). Fraction 3.18 was separated by Sephadex LH-20 gel column chromatography (methanol elution, 1 mL / min, elution time 10 h, collected every 2 h) to obtain five secondary fractions (3.18.1–3.18.5); Fraction 3.18.4 was purified by semi-preparative high-performance liquid chromatography (methanol-water, 62:38, v / v, 2 mL / min) to obtain compounds 1 (8.8 mg, retention time 17.6 min), 4 (1.5 mg, retention time 27.3 min), and 5 (2.7 mg, retention time 32.6 min).

[0038] Fraction 3.22 was eluted by silica gel column chromatography with a petroleum ether (PE)-ethyl acetate (EA) gradient system (20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 0:1, v / v, eluting 1 L at each ratio, collecting every 1 L) to obtain nine fractions (3.22.1–3.22.9). Fraction 3.22.3 was separated by Sephadex LH-20 gel column chromatography (methanol elution, 1 mL / min, elution time 10 h, collecting every 2 h) to obtain five subfractions (3.22.3.1–3.22.3.5); Fraction 3.22.3.4 was purified by semi-preparative high-performance liquid chromatography (methanol-water, 60:40, v / v, 2 mL / min) to obtain compounds 2 (8.2 mg, retention time 47.2 min) and 3 (7.8 mg, retention time 53.2 min). 3.22.4 Preparative high-performance liquid chromatography (methanol-water, 67:33, v / v, 8 mL / min) yielded five subfractions (3.22.4.1–3.22.4.5); 3.22.4.2 Semi-preparative high-performance liquid chromatography (acetonitrile-water, 52:48) yielded compound 9 (1.8 mg, retention time 35.6 min); 3.22.4.3 Semi-preparative high-performance liquid chromatography (acetonitrile-water, 51:49, v / v) yielded 10 (13.9 mg, retention time 66.3 min) and 11 (2.5 mg, retention time 68.6 min). 3.22.6 Separation by Sephadex LH-20 gel column (methanol elution, 1 mL / min, elution time 16 h, collection every 2 h) yielded 8 subfractions (3.22.6.1–3.22.6.8); 3.22.6.3 Separation by semi-preparative high performance liquid chromatography (acetonitrile-water, 62:38, v / v, 2 mL / min) yielded five fractions (3.22.6.3.1–3.22.6.3.5); 3.22.6.3.1 Purification by semi-preparative high performance liquid chromatography (methanol-water, 50:50, v / v, 2 mL / min) yielded compounds 6 (1.8 mg, retention time 53.9 min) and 7 (2.1 mg, retention time 70.1 min).

[0039] Fraction 4 was subjected to gradient elution by silica gel column chromatography (petroleum ether-ethyl acetate, 10:1→5:1→1:1, eluting 4 L at each ratio, collecting every 2 L) to obtain six fractions (4.1–4.6). Fraction 4.1 was prepared by preparative high-performance liquid chromatography (methanol-water, 65:35, v / v, 8 mL / min) to obtain five subfractions (4.1.1–4.1.5); Fraction 4.1.5 was purified by semi-preparative high-performance liquid chromatography (acetonitrile-water, 53:47, v / v, 2 mL / min) to obtain compound 8 (13.2 mg, retention time 69.9 min).

[0040] Example 2: Structural identification of compounds 1–11 Data analysis was performed on compounds 1–11 using NMR, mass spectrometry, optical rotation, infrared spectroscopy, ultraviolet spectroscopy, and circular dichroism spectroscopy. NMR quantum calculations were also performed on compounds 1–11. DP4+ calculations and error parameter analysis were conducted on compounds 1, 6, and 7 (e.g., Figure 2 ECD data calculations were performed on compounds 1, 2, and 9 (as shown). Figure 2 As shown), the structures of compounds 1–11 are determined as follows: .

[0041] Compound 1 (Rebisorbicillin A): Colorless gel; [ α 25 D +64 (c 0.1, methanol); UV (methanol) l max (log) e ): 207, 289 nm; IR (potassium bromide) n max 3364, 1759, 1648 cm -1 HRESIMS m / z 521.1717[M + Na] + (C 27 H 30 O9Na (calculated value 521.1788); 1 H and 13 The C NMR data are shown in Table 1.

[0042] Compound 2 (Rebisorbicillin B): White amorphous powder; α 25 D +101 (c 0.1, methanol); UV (methanol) l max (log) e ): 202, 297 nm; IR (potassium bromide) nmax : 3363, 1754, 1630 cm -1 HRESIMS m / z 517.1831 [M + Na] + (C 28 H 30 O8Na (calculated value 517.1838); 1 H and 13 The C NMR data are shown in Table 2.

[0043] Compound 3 (Rebisorbicillin C): White amorphous powder; α 25 D +75 (c 0.1, methanol); UV (methanol) l max (log) e ): 203, 296 nm; IR (potassium bromide) n max : 3363, 1754, 1630 cm -1 HRESIMS m / z 517.1891 [M + Na] + (C 28 H 30 O8Na (calculated value 517.1838); 1 H and 13 The C NMR data are shown in Table 2.

[0044] Compound 4 (Rebisorbicillin D): Yellow oily substance; α 25 D +53 (c 0.1, methanol); UV (methanol) l max (log) e ): 204, 287 nm; IR (potassium bromide) n max 3396, 1760, 1646 cm -1 HRESIMS m / z 553.2031[M + Na] + (C 28 H 34 O 10 Na (calculated value 553.2050); 1 H and 13 The C NMR data are shown in Table 3.

[0045] Compound 5 (Rebisorbicillin E): Yellow oily substance; α 25 D +55 (c 0.1, methanol); UV (methanol) l max (log) e ): 207, 290 nm; IR (potassium bromide) n max 3431, 1760, 1680 cm -1 HRESIMS m / z 553.2032[M + Na] + (C 28 H 34 O 10 Na (calculated value 553.2050); 1 H and 13 The C NMR data are shown in Table 3.

[0046] Compound 6 (Rebisorbicillin F): A colorless oily substance; α 25 D +64 (c 0.1, methanol); UV (methanol) l max (log) e ): 209, 289 nm; IR (potassium bromide) n max 3444, 1759, 1716 cm -1 HRESIMS m / z 552.1951[M + Na + H] + (C 28 H 33 O 10 Na (calculated value 552.1966); 1 H and 13 The C NMR data are shown in Table 4.

[0047] Compound 7 (Rebisorbicillin G): A colorless oily substance; α 25 D +68 (c 0.1, methanol); UV (methanol) l max (log) e ): 202, 290 nm; IR (potassium bromide) n max : 3433, 1761, 1714 cm -1 HRESIMS m / z 524.1618[M + Na + H]+ (C 26 H 29 O 10 Na (calculated value 524.1653); 1 H and 13 The C NMR data are shown in Table 4.

[0048] Compound 8 (Rebisorbicillin H): White amorphous powder; α 25 D +21 (c 0.1, methanol); UV (methanol) l max (log) e ): 218, 289 nm; IR (potassium bromide) n max 3435, 1746, 1671 cm -1 HRESIMS m / z 579.2204 [M + Na] + (C 30 H 36 O 10 Na (calculated value 579.2206); 1 H and 13 The C NMR data are shown in Table 5.

[0049] Compound 9 (Rebisorbicillin I): Colorless gel; [ α 25 D +30 (c 0.1, methanol); UV (methanol) l max (log) e ): 206, 283 nm; IR (potassium bromide) n max 3392, 1746, 1647 cm -1 HRESIMS m / z 496.2095[M + Na + H] + (C 26 H 33 O8Na (calculated value 496.2073); 1 H and 13 The C NMR data are shown in Table 5.

[0050] Compound 10 (Rebisorbicillin J): A colorless oily substance; α 25 D +38 (c 0.1, methanol); UV (methanol) lmax (log) e ): 227, 284 nm; IR (potassium bromide) n max 3393, 1747, 1723 cm -1 HRESIMS m / z 527.2259 [M + Na] + (C 27 H 36 O9Na (calculated value 527.2257); 1 H and 13 The C NMR data are shown in Table 6.

[0051] Compound 11 (Rebisorbicillin K): Colorless gel; [ α 25 D +37 (c 0.1, methanol); UV (methanol) l max (log) e ): 204, 284 nm; IR (potassium bromide) n max 3444, 1745, 1631 cm -1 HRESIMS m / z 527.2250[M + Na] + (C 27 H 36 O9Na (calculated value 527.2257); 1 H and 13 The C NMR data are shown in Table 6.

[0052] Table 1. Compound 1 1 H NMR and 13 C NMR data ( d in ppm, J (in Hz)

[0053] Table 2. Compounds 2 and 3 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data ( d in ppm, J (in Hz)

[0054] Table 3. Compounds 4 and 5 1 H NMR (600 MHz) and 13C NMR (150 MHz) data ( d in ppm, J (in Hz)

[0055] Table 4. Compounds 6 and 7 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data ( d in ppm, J (in Hz)

[0056] Table 5. Compounds 8 and 9 1 H NMR and 13 C NMR data ( d in ppm, J (in Hz)

[0057] Table 6. Compounds 10 and 11 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data ( d in ppm, J (inHz)

[0058] Figure 1 In the image, A represents two possible structures proposed for compound 1 based on spectroscopic analysis; B represents compound 1 (left) and 1... (Right) Key ROESY related signals in the 3D geometric optimization model; C represents candidate structures 1 and 1 The DP4+ probability analysis results; D represents candidate structures 1 and 1 Comparison of NMR calculation data.

[0059] Figure 2 In the diagram, A represents the four possible stereoisomers of compound 1; B represents the experimental and calculation results for 1a and 1b. 13 Linear correlation of 1 / 2C NMR chemical shift and DP4+ analysis; C shows the comparison of experimental and calculated ECD spectra of compounds 1, 2, and 9; D shows the experimental and calculated spectra of modified compounds 1–11. 13 Linear correlation analysis of C NMR chemical shifts.

[0060] The structural formulas of compounds 1-11 are shown below:

[0061] Example 3 The pharmacological activity was tested as follows: Human Samples and Ethics: Aortic valve samples used for primary cell extraction were collected from patients with dilated cardiomyopathy during heart transplantation surgery. Echocardiographic evaluation confirmed the absence of aortic valve calcification in these patients. Informed consent was obtained from all participants prior to inclusion in the study. This study protocol was approved by the Ethics Committee of Fuwai Hospital, Central China, and this study was conducted in accordance with the principles of the Declaration of Helsinki.

[0062] Isolation and Culture of Human Heart Valve Interstitial Cells: Human aortic valve interstitial cells (hVICs) were isolated from non-calcified valve leaflets. After washing three times with phosphate-buffered saline (PBS), aortic valve tissue was digested with 1 mg / mL type I collagenase at 37 °C for 12 hours. The cell suspension was then centrifuged at 1000 rpm for 10 minutes. The isolated valvular interstitial cells (VICs) were cultured in high-glucose Duchenne modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37 °C in a humidified incubator containing 5% carbon dioxide. Cells from passages 3 to 5 were used for all subsequent experiments.

[0063] In vitro osteogenic differentiation: Osteogenic medium (OM) was prepared by adding 2% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 10 mM calcium to Duchenne modified Eagle medium (DMEM). β The cells were prepared from glycerophosphate, 0.1 μM dexamethasone, and 50 μg / mL ascorbic acid. Valvular interstitial cells (VICs) were seeded at an appropriate density in each well of a 6-well or 12-well plate. When the cells reached confluence, the growth medium was replaced with osteogenic medium to induce osteogenic differentiation.

[0064] Western blot analysis: Cells were lysed on ice for 10 minutes in RIPA lysis buffer (New Cell & Molecular Biotechnology Co., Ltd., Product No.: WB3100) containing a mixture of protease and phosphatase inhibitors (New Cell & Molecular Biotechnology Co., Ltd., Product No.: P002) to extract cellular proteins. The lysates were centrifuged at 12,000 rpm for 15 minutes at 4 °C. The supernatant was aliquoted into new microcentrifuge tubes. After denaturation, proteins were separated by electrophoresis on SDS-polyacrylamide gels (Anshunda Biotechnology Co., Ltd., Product No.: ET15420LGel) with a 4-20% gradient, and then electroporated onto polyvinylidene fluoride (PVDF) membranes (Millipore, Product No.: 03010040001). The membranes were blocked with TBST containing 5% skim milk at room temperature for 1 hour, and then incubated overnight at 4 °C with primary antibody. After washing with TBST, the membrane was incubated with species-matched horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour. Protein expression was detected using a Bio-Rad imaging system and chemiluminescent substrate (Thermo Fisher Scientific, product number: 32209), and the intensity of the labeled bands was quantified using ImageJ software (National Institutes of Health). Primary antibody information is as follows: anti-RUNX2 antibody (1:1000, Cell Signaling Technologies, product number: 8486), anti-alkaline phosphatase (ALP) antibody (1:1000, R&D Systems, product number: MAB29092), anti-glutathione peroxidase 4 (GPX4) antibody (1:1000, Abogen, product number: T56959), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:10000, Abogen, product number: AC001).

[0065] Alizarin Red Staining and Alkaline Phosphatase (ALP) Activity Assay: Calcium deposition in human valvular interstitial cells (hVICs) was assessed using alizarin red staining. Cells were washed twice with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature. After fixation, cells were washed twice with double-distilled water (ddH2O). Cells were stained with 2% alizarin red S solution (pH 4.2; ServiceBio, product number G1038) for 30 minutes at room temperature. Cells were rinsed thoroughly with double-distilled water to remove unbound dye. Cell images were observed and captured using an Olympus microscope. The calcification area was quantified using ImageJ software. Alkaline phosphatase (ALP) activity was detected using the BCIP / NBT alkaline phosphatase colorimetric kit (Beyotime Biotech, product number C3206) according to the manufacturer's protocol.

[0066] Cell viability assay: Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Absin, product number abs50003, Shanghai, China) according to the manufacturer's protocol. Valvular interstitial cells (VICs) were counted at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells / well in 96-well plates and cultured in serum-free medium for 24 hours. Cells were then treated with escalating concentrations of the compound for 72 hours. After treatment, cells were incubated with CCK-8 reagent (1:10 diluted in fresh medium) at 37 °C for 2 hours. Absorbance was measured at 450 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific). The half-maximal inhibitory concentration (IC50) of the compound was calculated using nonlinear regression analysis. 50 )value.

[0067] Lipid peroxidation assay: Cellular lipid peroxidation was assessed using a lipid peroxidation assay kit (BODIPY 581 / 591 C11) (Beyotime Biotechnology, product number S0043S) according to the manufacturer's instructions. Cells were incubated with 2 μM BODIPY 581 / 591 C11 working solution (prepared with PBS) at 37 °C in the dark for 30 minutes, following the manufacturer's protocol. Cells were then washed twice with PBS to remove unbound probes. Fluorescence images were acquired using a Leica TCS SP8 laser scanning confocal microscope (Leica microsystem). Both oxidized (green fluorescence; excitation / emission wavelengths approximately 488 / 510 nm) and reduced (red fluorescence; excitation / emission wavelengths 581 / 591 nm) forms were detected simultaneously. The level of lipid peroxidation was quantified using ImageJ software by calculating the ratio of green (oxidized) to red (reduced) fluorescence intensity.

[0068] RNA Extraction and Library Construction: Total RNA was extracted using TRIzol reagent (Invitrogen, a Thermo Fisher Scientific company) according to the manufacturer's instructions. RNA purity and concentration were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). RNA integrity was assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, California, USA). RNA-seq libraries were prepared using the VAHTS Universal V5 RNA-seq Library Preparation Kit (Nanjing Novizan Biotechnology Co., Ltd., China) according to the manufacturer's instructions. RNA sequencing and subsequent bioinformatics analysis were performed by Shanghai Ouyi Biomedical Technology Co., Ltd.

[0069] RNA sequencing and differentially expressed gene analysis: The library was sequenced using the Illumina NovaSeq 6000 platform, generating 150 bp paired-end reads. The raw FASTQ reads were processed using FASTP software to remove low-quality reads, resulting in clean reads for subsequent data analysis. HISAT2 was used for reference genome alignment, and gene expression levels were calculated as fragments per kilobase transcript (FPKM). HTSeq-count was used to obtain read counts for each gene. Principal component analysis (PCA) was performed on the gene counts using R (v 3.2.0) to assess biological repeats. Differential expression analysis was performed using DESeq2, defining genes with a q-value < 0.05 and a fold change > 2 or < 0.5 as differentially expressed genes (DEGs). Hierarchical cluster analysis was performed on differentially expressed genes in R (v 3.2.0) to visualize expression patterns across different groups and samples. The ggradar package in R was used to generate radar plots to visualize the expression changes of the top 30 upregulated or downregulated genes. Subsequently, the hypergeometric distribution algorithm was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses on differentially expressed genes to identify significantly enriched functional terms. Bar plots, chord plots, or enrichment analysis loop diagrams were created using R (v 3.2.0) to visualize the significantly enriched functional terms.

[0070] Animal Studies: To investigate the effect of RSC on aortic valve calcification, this invention employed a previously established mouse model of aortic valve endothelial injury. Briefly, 8-week-old male C57BL / 6J mice were anesthetized with ketamine / toluidine (100 / 10 mg / kg). A 10 mm cervical incision was made to expose the right carotid artery. A 0.36 mm angioplasty guidewire (Abbott Laboratories) was inserted, passing through the aortic arch, across the aortic valve, and into the left ventricle. Mild endothelial injury was induced by gently maneuvering the guidewire back and forth 30 times at the valve orifice under echocardiographic guidance. The guidewire was removed, the arterial incision was sutured with 10-0 sutures, and the skin was sutured with 6-0 sutures. Mice were allowed to recover on oxygen at a 37 °C heating pad until they were able to move (≥30 minutes), during which time buprenorphine (0.1 mg / kg, every 12 hours for 48 hours) was administered subcutaneously. A sham control group underwent the same procedure but without penetrating the valve. Eight weeks post-surgery, prior to euthanasia via cervical dislocation, terminal echocardiography and hemodynamic assessment were performed under anesthesia. Aortic valves were harvested and immediately frozen or fixed with 4% paraformaldehyde for histological examination.

[0071] The activity results are as follows: Compounds inhibit osteogenic differentiation of human primary valvular interstitial cells: Sorbic acid compounds possess anti-inflammatory and antioxidant activities. In preliminary screening using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, compounds 2 and 3 showed potent anti-inflammatory activity at a concentration of 100 μM. Figure 3 (A). Given that the pathogenesis of calcific aortic valve disease (CAVD) involves a cascade of valvular oxidative stress and inflammation that drives pathological mineralization, and that human valvular interstitial cells (hVICs) play a key role through osteogenic differentiation, this invention evaluated the effects of these compounds on the calcification pathway. hVICs were treated in osteogenic medium (OM) with 11 compounds (100 μM). Western blot analysis showed that compounds 3, 6, and 9 significantly reduced alkaline phosphatase (ALP) expression, an early marker of osteogenic differentiation, compared to the OM group. Figure 3 (Middle BC). Notably, only compound 3 significantly downregulated RUNX2 expression, and RUNX2 is a major transcription factor regulating osteogenic activity. Figure 3 (D). After 21 days of culture in OM, alizarin red staining—a method for quantifying calcium deposition—showed that compounds 3 and 9 significantly reduced calcification compared to the OM group. Figure 3 These results collectively demonstrate that compound 3 of the present invention effectively alleviates osteogenic differentiation and subsequent mineralization of hVICs. Compound 3 (RSC) was selected as the lead compound due to its superior anti-calcification efficacy and good purification yield among 11 candidate compounds. To determine the minimum effective concentration (MEC), human aortic valve interstitial cells (hVICs) in osteogenic medium (OM) were treated with RSC (0–100 μM). Western blotting showed that at concentrations ≥50 μM, RSC downregulated the expression of RUNX2 and alkaline phosphatase (ALP). Figure 3 After 21 days of osteogenic medium culture, compared with the osteogenic medium group, RSC (≥50 μM) significantly reduced the alizarin red positive area (G-I). Figure 3 (JK). After culturing in osteogenic medium for 7 days, the alkaline phosphatase activity of hVICs treated with RSC (50 μM) was significantly inhibited. Figure 3 (Middle LM). Cell viability assays were used to determine the half-maximal inhibitory concentration (IC50) of RSCs. 50 The value was 282.2 μM. Figure 3 (N).

[0072] Compound RSC alleviates human valvular interstitial cell calcification by regulating ferroptosis: To investigate the mechanism by which RSC alleviates human valvular interstitial cell (hVIC) calcification, RNA sequencing was performed on three experimental groups: control group (CTR), osteogenic culture medium group (OM), and OM + 50 μM RSC group (…). Figure 4 (A). Wien analysis was performed on differentially expressed genes (DEGs; FDR < 0.05, |log2FC| > 1). The results showed that, compared with the OM group, RSC reversed 99.7% of OM-induced upregulated genes (3 overlaps out of 940) and 98.3% of downregulated genes (16 overlaps out of 909) (Fisher exact test). p <0.0001), a protective transcriptome distinct from OM-induced calcification was established. Weighted gene co-expression network analysis (WGCNA) identified four treatment-related modules ( Figure 4 (A). Pathway enrichment analysis (FDR < 0.05) showed that ferroptosis (NES = 2.4), TGF-β signaling pathway (NES = 2.1), cytoskeleton remodeling (NES = 1.8), and p53 pathway (NES = 1.9) were significantly regulated processes. Figure 4 (A). Meanwhile, FerroOrange staining showed a significant increase in iron accumulation in the ossification model (OM) group compared to the control group. p <0.01), while RSC treatment significantly reduced iron levels (0.16 times compared to the OM group). p <0.01). These results suggest that RSC may inhibit ferroptosis by restoring glutathione peroxidase 4 (GPX4) function and chelating free iron ions, thereby alleviating osteogenic differentiation and calcification of valvular interstitial cells. Ferroptosis and lipid peroxidation in hVIC drive key pathological processes in the progression of aortic stenosis (CAVD), including inflammation, osteogenic differentiation, and calcification. RNA sequencing showed that OM downregulated GPX4 expression (log2FC = -2.1, FDR <0.001), while RSC treatment restored GPX4 levels (log2FC = +1.8, FDR <0.01 compared to the OM group); Figure 4 (B) Western blot analysis confirmed that, compared with the CTR group, OM significantly increased the expression of alkaline phosphatase (ALP) (1.8-fold) and RUNX2 (1.7-fold), while decreasing the expression of GPX4 (0.8-fold). p Expression of <0.01). Combined RSC treatment reversed these effects: compared to the OM group, ALP (0.7-fold), RUNX2 (0.6-fold), and GPX4 (1.6-fold) were all significantly reduced. p <0.05; Figure 4(CD). BODIPY C11 staining quantified lipid peroxidation levels by calculating the ratio of green (BODIPY 510, oxidized state) to red (BODIPY 591, reduced state) fluorescence intensity. Compared to the OM group, treatment with compound RSC significantly reduced this ratio ( p The result was <0.01, demonstrating its efficacy in reducing oxidative stress.

[0073] Compound RSC reduces aortic valve calcification in vivo: To evaluate the therapeutic effect of RSC in vivo, this invention employed an established wire injury-induced aortic valve calcification model. Male C57BL / 6J mice were randomly assigned to: a sham-operated group (n = 8), an injury group (n = 8), and an injury + RSC group (20 mg / kg intraperitoneally every 72 hours, n = 8). Eight weeks post-operation, transthoracic echocardiography (Vevo 2100) was performed by unsuspecting researchers. The results showed that the injury led to an increase in peak flow velocity (maximum flow velocity). V max The mean amplitude was 1631.8±155.6 mm / s, while that in the sham surgery group was 813.9±102.3 mm / s; p <0.0001) and increased mean pressure gradient (mean pressure gradient PGmean: 10.7±2.2 mmHg, compared to 2.7±0.7 mmHg in the sham surgery group; p<0.0001). RSC significantly decreased V max (1346.6±229.2 mm / s, compared with the injury group, p <0.05) and PGmean (7.4±2.4 mmHg, compared with the injury group, p <0.05; Figure 5 (Central AC). Histomorphometry showed that RSC reduced calcification (von Cossa stain: 0.75-fold compared to the lesion group). p <0.01) and fibrosis (Type I collagen: 0.83 times compared with the damage group, p <0.01; Figure 5 (D-F). Immunofluorescence quantitative analysis showed that, compared with the sham-operated group, the expression of glutathione peroxidase 4 (GPX4) in the injury group was reduced (0.47-fold). p <0.001), while RSC partially restored its expression (1.59-fold compared to the damaged group). p <0.01; Figure 5 (G-H). These data suggest that RSCs alleviate aortic valve calcification in vivo through GPX4-mediated ferroptosis regulation.

[0074] In summary, the semi-cage-like sorbitol polyketide compounds prepared in this invention exhibit good inhibitory activity against cardiac valve calcification and can be used to prepare drugs for calcified aortic valve disease. These compounds have significant development value as novel drugs for the prevention or treatment of calcified aortic valve disease, and the design concept of these compounds also provides new ideas and approaches for the development of novel drugs for the prevention or treatment of calcified aortic valve disease.

[0075] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A semi-cage-like sorbitol polyketide compound, characterized in that, The structural formulas are shown in compounds 1-8 below: 。 2. A method for preparing the semi-cage-like sorbitol polyketide compound according to claim 1, characterized in that, Includes the following steps: (1) Using rice secondary metabolites of Trichoderma reesei as raw material, extract was obtained by soaking, extraction and concentration. The extract was suspended in water to obtain a suspension, and then extracted and concentrated to obtain a crude extract. (2) The crude extract was separated by silica gel column chromatography and eluted with petroleum ether-ethyl acetate at volume ratios of 5:1, 3:1, 2:1, 1:1, 1:2, and 1:5 to obtain six major fractions 1-6. (3) Fraction 3 was separated by medium-pressure preparative liquid chromatography with octadecylsilane-bonded silica gel and eluted with methanol-water gradient to obtain 25 subfractions 3.1–3.25; (4) Fraction 3.18 was separated by Sephadex LH-20 gel column chromatography to obtain 5 secondary fractions 3.18.1–3.18.5; (5) The fraction 3.18.4 was further purified by semi-preparative high performance liquid chromatography to obtain compound 1, compound 4 and compound 5; (6) Fraction 3.22 was separated by silica gel column chromatography to obtain 9 components 3.22.1–3.22.9; (7) Fraction 3.22.3 was separated by Sephadex LH-20 gel column chromatography to obtain 5 subfractions 3.22.3.1–3.22.3.5; (8) The fractions 3.22.3.4 were purified by semi-preparative high performance liquid chromatography to obtain compounds 2 and 3; (9) Fraction 3.22.4 was separated by preparative high performance liquid chromatography to obtain 5 subfractions 3.22.4.1–3.22.4.5; (10) Fraction 3.22.6 was separated by Sephadex LH-20 gel column chromatography to obtain 8 components ( 3.22.6.1–3.22.6.8）； (11) Fraction 3.22.6.3 was separated by semi-preparative high performance liquid chromatography to obtain 5 components 3.22.6.3.1–3.22.6.3.5; (12) The fraction 3.22.6.3.1 was purified by semi-preparative high performance liquid chromatography to obtain compound 6 and compound 7; (13) Fraction 4 (32 g) was eluted by silica gel column chromatography gradient to obtain 6 fractions 4.1–4.6; (14) Fraction 4.1 was separated by preparative high performance liquid chromatography to obtain 5 subfractions 4.1.1–4.1.5; (15) Fraction 4.1.5 was purified by semi-preparative high performance liquid chromatography to obtain compound 8.

3. The preparation method according to claim 2, characterized in that, In step (1), 95% ethanol was used for soaking and extraction. The soaking and extraction were carried out three times, and the soaking and extraction time was 24 h each time.

4. The preparation method according to claim 2, characterized in that, In step (1), ethyl acetate is used for extraction; the volume ratio of ethyl acetate to the suspension is 1:

1.

5. The use of the semi-cage-like sorbitol polyketide compound as described in claim 1 in the preparation of a medicament for the prevention and treatment of calcified aortic valve disease.

6. The application according to claim 5, characterized in that, It also includes pharmaceutically acceptable excipients.

7. A drug for preventing and treating calcified aortic valve disease, characterized in that, The active ingredient includes the semi-cage-like sorbitol polyketide compound as described in claim 1.

8. The medicament for preventing and treating calcified aortic valve disease according to claim 7, characterized in that, It also includes pharmaceutically acceptable excipients.

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