Epoxy discodermolone compounds, methods of making and use thereof in the preparation of medicaments for the treatment of kidney disease

Abstract

The application discloses an epoxy discodermolide compound, a preparation method thereof and application of the compound in preparation of a drug for treating kidney diseases. It is found for the first time that the epoxy discodermolide compound with similar structure can significantly inhibit expression of a kidney injury molecule KIM-1 in damaged kidney cells, significantly improve a survival rate of HK-2 cells after H / R injury, and has potential for treating acute kidney injury and related kidney diseases. The epoxy discodermolide compound can be used as an active ingredient for preparing a drug for treating acute kidney injury and related kidney diseases, and promotes development and utilization of marine microbial drug resources.

Description

Technical Field

[0001] This invention belongs to the field of microbial and natural product application technology. More specifically, this invention relates to a class of marine fungal-derived epoxyphenanthrene compounds, their preparation methods, and their application in the preparation of drugs for treating kidney diseases such as acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function, manifested as a rapid increase in serum creatinine levels and decreased urine output. Common precipitating factors include sepsis, nephrotoxic drugs, major surgery, heart failure, cirrhosis, and pregnancy. Currently, the core treatment of AKI focuses on correcting reversible factors, maintaining homeostasis, and managing complications; renal replacement therapy is only used when conventional treatments are ineffective. AKI has a high probability of poor long-term prognosis, which can manifest as various kidney diseases such as chronic kidney disease and end-stage renal disease. At present, there are still no drugs that can effectively prevent or reverse AKI. Therefore, despite continuous advancements in supportive care, developing novel drug leads that can effectively prevent or reverse AKI and related kidney diseases remains a crucial clinical need.

[0003] Marine fungi, due to their unique ecological environment, have evolved distinctive biosynthetic pathways, producing a rich variety of bioactive natural products, making them a treasure trove of resources for drug development. Azafiron is a common natural product derived from fungi, and activity screening shows that most of it possesses antitumor, antibacterial, and anti-inflammatory biological activities. The oxygen on the pyran ring in the azafiron structure readily reacts with primary amines (such as proteins, amino acids, and nucleic acids), hence its nickname "azophile," which is also the main reason for its broad biological activity. However, this lack of selective affinity presents challenges for new drug development. Summary of the Invention

[0004] The first object of the present invention is to provide a class of epoxy pseudophenanthrene ketone compounds having the structure shown in the following general formula (I); General Formula (I)

[0005] Where R1 is H or OH or OCOCH3; R2 is H or OH; R3 is H or OH; R4 is H or OH or OCOCH3; and R5 is H or OH.

[0006] These compounds have similar structures, differing only in the substitution of hydroxyl or acetyl groups at R1-R5 of the branched chain. They belong to the azafirone (azophile) class of natural products, characterized by C-9 methylation and C-9, C-10 epoxidation of the branched chain. This unique structural segment affects the conjugated system between the pyran ring and the branched chain, influencing the non-unique affinity of the compounds.

[0007] These epoxy pseudophenanthrene ketone compounds are preferably compounds with the following structure, as shown in formula (II): Formula (II).

[0008] In formula (II), 14- epi -PPD, 13- epi Five compounds, -16OH-PPA, PPK, 14OH-PPC, and PPL, are novel compounds not previously reported. Four compounds, PPB-PPE, have been previously studied in terrestrial plant endophytic fungi. Pestalotiopsis oxyanthi It was discovered in (Phytochemistry 2022, 194:113045), but this is the first time it has been found in marine fungi or the first time it has been found in... Neopestalotiopsis It was found in fungi. This invention provides a method for identifying new compounds.

[0009] The second objective of this invention is to provide a method for preparing the aforementioned epoxy-p-phenanthroline compound, specifically comprising the following steps:

[0010] (a) Preparation of fungi Neopestalotiopsis Fermentation product of sp. SCSIO41422;

[0011] (b) The fermentation product obtained in step (a) was extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain an extract. The extract was eluted by a forward silica gel column gradient with a petroleum ether / ethyl acetate / methanol system as the eluent. The fraction was then purified by medium-pressure reverse silica gel column chromatography and semi-preparative HPLC to obtain the epoxy phenanthrene compound.

[0012] Preferably, the method for preparing the fermentation product is as follows: taking fungi Neopestalotiopsis Sp. SCSIO41422 was used for seed fermentation culture. The culture medium contained 15 g of malt extract, 24 g of crude sea salt, and 15 g of agar per liter. The culture was shaken to obtain seed fermentation broth. The seed fermentation broth was then inoculated into rice culture medium containing 200 g of rice, 250 mL of water, and 6 g of sea salt per liter. The culture was allowed to stand for fermentation to obtain fermented product.

[0013] Preferably, step (b) is as follows: the fermentation product obtained in step (a) is extracted with ethyl acetate, the solvent is recovered under reduced pressure to obtain an extract, and the extract is eluted by a positive silica gel column gradient using a petroleum ether / ethyl acetate / methanol system as the eluent. The volume fractions are: petroleum ether / ethyl acetate / methanol = 100% / 0 / 0→90% / 10% / 0→80% / 20% / 0→70% / 30% / 0→50% / 50% / 0→40% / 60% / 0→30% / 70% / 0→20% / 80% / 0→18% / 82% / 0→1 4 / 86% / 0→10% / 90% / 0→0 / 100% / 0%→0 / 99% / 1%→0 / 95% / 5%→0 / 80% / 20%→0 / 70% / 30% gradient elution, collecting the following fractions: petroleum ether / ethyl acetate / methanol = 70% / 30% / 0 (Fr.4), petroleum ether / ethyl acetate / methanol = 50% / 50% / 0 (Fr.5), petroleum ether / ethyl acetate / methanol = 30% / 70% / 0 (Fr.7), and petroleum ether / ethyl acetate / methanol = 20% / 80% / 0 (Fr.8).

[0014] Fr.4 was subjected to reversed silica gel column chromatography with a methanol / water gradient elution. The volume fractions were: methanol / water = 10% / 90% → 24% / 76% → 31% / 69% → 38% / 62% → 45% / 55% → 52% / 48% → 59% / 41% → 66% / 34% → 73% / 27% → 94% / 6% → 100% / 0. The methanol / water = 24% / 76% elution fraction Fr.4-2 was then purified by semi-preparative high-performance liquid chromatography (HPLC) at ChromCore 120 C18, 10 × 250 mm, 5 µm, 40% MeCN / H2O eluent, 2.5 mL / min, yielding compound PPK t. R 17 min;

[0015] Fr.5 was subjected to reversed-direction silica gel column chromatography with methanol / water as the eluent. The elution gradient was methanol / water = 10% / 90% → 17% / 83% → 24% / 76% → 31% / 69% → 38% / 62% → 45% / 55% → 52% / 48% → 59% / 41% → 66% / 34% → 73% / 27% → 87% / 13% → 94% / 6% → 100% / 0. The methanol / water elution fraction Fr.5−6 was separated by semi-preparative high-performance liquid chromatography (HPLC) at ChromCore 120 C18, 10 × 250 mm, 5 µm; eluent 56% MeOH / H2O, 2.5 mL / min, yielding compound 14-epi-PPD. R20 min; eluent 40% MeCN / H2O, 2.5 mL / min 13-epi-16OH-PPA t R 12 min; eluent 40% MeCN / H2O, 2.5 mL / min PPC t R 13 min; eluent 56% MeOH / H2O, 2.5 mL / min; compound PPD t R 16 min; The methanol / water = 66% / 34% elution fraction Fr.5−9 was further purified by semi-preparative high-performance liquid chromatography (HPLC) at ChromCore 120 C18, 10 × 250 mm, 5 µm, with 65% MeOH / H2O as eluent at 2.5 mL / min to obtain compound PPB. R 21 min;

[0016] Fr.7 was subjected to reversed-phase silica gel column chromatography with a methanol / water gradient elution. The volume fractions were: methanol / water = 10% / 90% → 24% / 76% → 31% / 69% → 38% / 62% → 45% / 55% → 52% / 48% → 59% / 41% → 66% / 34% → 73% / 27% → 94% / 6% → 100% / 0. The methanol / water elution fraction Fr.7-3 was then subjected to semi-preparative high-performance liquid chromatography (HPLC) at ChromCore 120 C18, 10 × 250 mm, 5 µm, with 40% CH3CN / H2O as eluent at 2.5 mL / min, from which compound PPLt was isolated. R 21 min;

[0017] Fr.8 was further purified by semi-preparative high-performance liquid chromatography (HPLC) using ChromCore 120 C18, 10 × 250 mm, 5 µm, with 25% CH3CN / H2O as eluent at 2.5 mL / min to give compound 14OH-PPC. R 9 min; eluent 35% CH3CN / H2O, 2.0 mL / min PPE t R 8 min.

[0018] A third object of the present invention is to provide the use of a class of epoxy-p-phenanthrene compounds, such as those represented by general formula (I), in the preparation of medicaments for treating kidney disease.

[0019] The drug comprises an effective dose of an epoxyphenanthrene compound of general formula (I) or a pharmaceutical salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

[0020] The kidney disease mentioned is acute kidney injury.

[0021] A fourth object of the present invention is to provide a medicament for treating kidney disease, comprising an effective dose of an epoxyphenanthrone compound as shown in general formula (I) or a pharmaceutical salt thereof.

[0022] This invention isolates an epoxide-containing compound called *Neopestalotiopsis* sp. SCSIO41422 from the fermentation product of this marine fungus. Content screening for high renal protective activity showed that the epoxide-containing compound significantly inhibited the expression of the renal injury marker KIM-1 and significantly increased the survival rate of HK-2 cells after H / R injury. By reducing renal tubular damage, it exerts therapeutic effects on acute kidney injury and related kidney diseases. Therefore, the epoxide-containing compound of this invention can be used to treat kidney diseases such as acute kidney injury, while also promoting the development and utilization of marine microbial drug resources. Attached Figure Description

[0023] Figure 1 The chemical structure of the epoxy-p-phenanthrene ketone compound;

[0024] Figure 2 Two-dimensional NMR related information diagrams for COSY, HMBC and NOESY of epoxy pseudophenanthrene ketone compounds;

[0025] Figure 3 To assess the renal protective activity of epoxide-based phenanthrene compounds. Detailed Implementation

[0026] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the embodiments described in this specification are merely illustrative of the invention and are not intended to limit the invention.

[0027] Example 1: Isolation and identification of marine-derived Neopsalotiopsis sp. SCSIO41422

[0028] The sponge-associated fungus *Neopestalotiopsis* sp. SCSIO41422 was isolated from sponge samples collected in October 2020 from Weizhou Island, Guangxi, China. This strain was preserved on MH broth (MB) agar slants (15 g malt extract, 24 g artificial sea salt, 18 g agar) at 4°C and deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), Guangdong, China, Institute of Microbiology, Guangdong Academy of Sciences, on August 4, 2022, with accession number GDMCC No: 62689. It is disclosed in patent CN202510362339.4, "A small molecule compound derived from marine fungi and its application in the preparation of drugs for treating renal fibrosis."

[0029] Example 2 Fungi Neopestalotiopsis Isolation and purification of epoxide-derived phenanthrene compounds from sp. SCSIO41422

[0030] (a) Take fungi Neopestalotiopsis Sp. SCSIO41422 was used for seed fermentation culture (the culture medium consisted of 15 g malt extract, 24 g crude sea salt, 15 g agar, 950 mL distilled water, pH 7.2–7.4, diluted to a final volume of 1000 mL, and sterilized). 200 mL of the culture medium was placed in a 500 mL Erlenmeyer flask and cultured at 25 °C with shaking at 180 rpm. After 3 days of culture, the seed fermentation broth was obtained. 10 mL of the seed fermentation broth was transferred to a sterilized Erlenmeyer flask containing rice culture medium (200 g rice, 250 mL water, 6 g sea salt / flask, totaling 100 flasks) and allowed to ferment statically at 25 °C to obtain the fermentation product.

[0031] (b) The fermentation product obtained in step (a) was extracted three times with ethyl acetate, and the solvent was recovered under reduced pressure to obtain the extract. The extract (120.0 g) was dissolved in a small amount of ethyl acetate and then eluted by a gradient on a normal silica gel column. The eluent was a petroleum ether / ethyl acetate / methanol system (by volume fraction, petroleum ether / ethyl acetate / methanol = 100% / 0 / 0→90% / 10% / 0→80% / 20% / 0→70% / 30% / 0→50% / 50% / 0→40% / 60% / 0→30% / 70% / 0→20% / 80% / 0→18% / 82% / 0→14 / 86% / 0→10% / 90% / 0→0 / 100% / 0%→0 / 99% / 1%→0 / 95% / 5%→0 / 80% / 20%→0 / 70% / 30%), yielding 16 fractions Fr.1–Fr.16. Fr.4 (petroleum ether / ethyl acetate / methanol = 70% / 30% / 0 elution fraction) was subjected to reverse silica gel column chromatography with a methanol / water gradient elution (methanol / water = 10% / 90%→24% / 76%→31% / 69%→38% / 62%→45% / 55%→52% / 48%→59% / 41%→66% / 34%→73% / 27%→94% / 6%→100% / 0 by volume fraction), separating into 11 subfractions (Fr.4−1~4−13). Fr.4−2 (methanol / water = 24% / 76%) was further purified by semi-preparative high performance liquid chromatography (ChromCore 120 C18, 10 × 250 mm, 5 µm, eluent 40% MeCN / H2O) to give compound PPK (1.6 mg, t R 17 min, 40% MeCN / H2O, 2.5 mL / min, 210 nm). Fr.5 (petroleum ether / ethyl acetate / methanol = 50% / 50% / 0 elution fraction) was subjected to reverse silica gel column chromatography, eluted with a methanol / water gradient (methanol / water = 10% / 90%→17% / 83%→24% / 76%→31% / 69%→38% / 62%→45% / 55%→52% / 48%→59% / 41%→66% / 34%→73% / 27%→87% / 13%→94% / 6%→100% / 0), and separated into 13 subfractions (Fr.5−1~5−13). Fr.5−6 (methanol / water = 45% / 55% elution fraction) was separated by semi-preparative high performance liquid chromatography (ChromCore 120 C18, 10 × 250 mm, 5 µm) to give compound 14- epi -PPD (6.0 mg, t R20 min, eluent 56% MeOH / H2O, 2.5 mL / min, 210 nm), 13- epi -16OH-PPA (4.1 mg, t R 12 min, eluent 40% MeCN / H2O, 2.5 mL / min, 210 nm), PPC (8.8 mg, t R 13 min, eluent 40% MeCN / H2O, 2.5 mL / min, 210 nm) and PPD (13.3 mg, t R 16 min, eluent 56% MeOH / H2O, 2.5 mL / min, 210 nm; Fr.5−9 (methanol / water = 66% / 34%) was further purified by semi-preparative high performance liquid chromatography (ChromCore 120 C18, 10 × 250 mm, 5 µm, eluent 65% MeOH / H2O) to obtain compound PPB (4.1 mg, t R 21 min, 65 % MeOH / H2O, 2.5 mL / min, 210 nm). Fr.7 (petroleum ether / ethyl acetate / methanol = 30% / 70% / 0) was eluted with a methanol / water gradient (methanol / water = 10% / 90%→24% / 76%→31% / 69%→38% / 62%→45% / 55%→52% / 48%→59% / 41%→66% / 34%→73% / 27%→94% / 6%→100% / 0 by volume fraction) and subjected to reversed-phase silica gel column chromatography, yielding 10 fractions (Fr.7−1~7−10). Fr.7-3 (methanol / water = 31% / 69%) was separated by semi-preparative high-performance liquid chromatography (ChromCore 120 C18, 10 × 250 mm, 5 µm, eluent 40% CH3CN / H2O) to obtain compound PPL (2.1 mg). t R 21 min, 40% CH3CN / H2O, 2.5 mL / min, 210 nm). Fr.8 (petroleum ether / ethyl acetate / methanol = 20% / 80% / 0 elution fraction) was further purified by semi-preparative high performance liquid chromatography (ChromCore 120 C18, 10 × 250 mm, 5 µm) to give compound 14OH-PPC (4.4 mg, t R9 min, 25 % CH3CN / H2O, 2.5 mL / min, 210 nm) and PPE (5.0 mg, t R 8min, 35% CH3CN / H2O, 2.0 mL / min, 210 nm).

[0032] Example 3: Structural Identification of Epoxy-Pseudophenanthrene Compounds

[0033] The epoxy-p-phenanthrene ketone compounds PPB−PPE and 14-PPE isolated in Example 2 epi -PPD, 13- epi The chemical structures of -16OH-PPA, PPK, 14OH-PPC, and PPL are as follows: Figure 1 As shown. Among them, 14- epi -PPD, 13- epi Five compounds, -16OH-PPA, PPK, 14OH-PPC, and PPL, are novel compounds not previously reported. Four compounds, PPB-PPE, have been previously studied in terrestrial plant endophytic fungi. Pestalotiopsis oxyanthi It was discovered in (Phytochemistry 2022, 194:113045), but this is the first time it has been found in marine fungi or the first time it has been found in... Neopestalotiopsis It was found in fungi.

[0034] The nuclear magnetic properties of the four compounds PPB-PPE are as follows:

[0035] PPB. Yellow solid; 13 C NMR (125 MHz, MeOD) d C 201.5 (C-6), 163.0 (C-3), 147.7 (C-4a), 146.6 (C-1), 137.5 (C-12), 132.1 (C-11), 122.8 (C-8a), 106.6(C-5), 106.1 (C-4), 77.9 (C-7), 73.4 (C-8), 66.4 (C-10), 61.0 (C-9), 59.2 (C-17), 34.8 (C-13), 31.3 (C-14), 21.4 (C-16), 19.1 (C-19), 13.5 (C-18), 12.5(C-15); 1 H NMR (500 MHz, MeOD) d H 7.48 (t, J= 1.6 Hz, 1H, H-1), 6.46 (s, 1H, H-4), 5.41 (d, J = 1.2 Hz, 1H, H-5), 5.31 (d, J = 10.1 Hz, 1H, H-12), 4.55 (d, J =2.0 Hz, 1H, H-8), 4.24 (s, 2H, H-17), 3.74 (s, 1H, H-10), 2.58 (m, 1H, H-13),1.46 (s, 3H, H-18), 1.42 (m, 1H, H2-14), 1.28 (m, 1H, H2-14), 1.17 (s, 3H, H-19), 1.01 (d, J = 6.6 Hz, 3H, H-16), 0.89 (t, J = 7.4 Hz, 3H, H-15)。

[0036] PPC. yellow solid; 13 C NMR (125 MHz, MeOD) d C 201.5 (C-6), 162.9 (C-3),147.5 (C-4a), 146.5 (C-1), 134.8 (C-12), 130.2 (C-11), 122.9 (C-8a), 106.7(C-5), 105.9 (C-4), 77.9 (C-7), 74.5 (C-13), 73.4 (C-8), 68.4 (C-10), 60.6(C-9), 37.1 (C-14), 28.9 (C-16), 19.1 (C-19), 14.9 (C-17), 13.1 (C-18), 8.8(C-15); 1 H NMR (500 MHz, MeOD) d H 7.50 (t, J = 1.6 Hz, 1H, H-1), 6.45 (s, 1H, H-4), 5.48 (m, 1H, H-12), 5.43 (d, J = 1.3 Hz, 1H, H-5), 4.56 (d, J = 2.0 Hz, 1H,H-8), 3.53 (s, 1H, H-10), 1.94 (d,J = 1.5 Hz, 3H, H-17), 1.66 (q, J = 7.5 Hz,2H, H-14), 1.49 (s, 3H, H-18), 1.35 (s, 3H, H-16), 1.19 (s, 3H, H-19), 0.95(t, J = 7.5 Hz, 3H, H-15)。

[0037] PPD. yellow solid; 13 C NMR (175 MHz, MeOD) d C 201.6 (C-6), 162.9 (C-3),147.6 (C-4a), 146.5 (C-1), 132.0 (C-12), 129.1 (C-11), 122.9 (C-8a), 106.7(C-5), 105.9 (C-4), 77.9 (C-7), 73.4 (C-8), 72.6 (C-14), 67.9 (C-10), 60.6(C-9), 41.1 (C-13), 21.5 (C-15), 19.1 (C-19), 17.2 (C-16), 14.4(C-17), 13.3(C-18); 1 H NMR (700 MHz, MeOD) d H 7.47 (t, J = 1.6 Hz, 1H, H-1), 6.43 (s, 1H, H-4), 5.40 (d, J = 1.3 Hz, 1H, H-5), 5.3 (m, 1H, H-12), 4.54 (d, J = 2.0 Hz, 1H, H-8), 3.53 (m, 1H, H-14), 3.53 (s, 1H, H-10), 2.47 (dt, J = 10.1, 6.8 Hz, 1H, H-13), 1.74 (d, J = 0.8 Hz, 3H, H-17), 1.45 (s, 3H, H-18), 1.17 (s, 3H, H-19),1.12 (d, J = 6.3 Hz, 3H, H-15), 1.03 (d, J= 6.7 Hz, 3H, H-16).

[0038] PPE. Yellow solid; 1 H NMR (500 MHz, MeOD) d H 7.46 (t, J = 1.5 Hz, 1H, H-1), 6.41 (s, 1H, H-4), 5.39 (d, J = 1.2 Hz, 1H, H-5), 5.23 (dt, J = 9.9, 1.5 Hz,1H, H-12), 4.53 (d, J = 2.0 Hz, 1H, H-8), 3.55 (m 1H, H-15), 3.53 (s, 1H, H-10), 3.48 (m 1H, H-15), 2.68 (dt, J = 15.5, 8.5 Hz, 1H, H-13), 1.72 (d, J = 1.0Hz, 3H, H-17), 1.60 (m, 1H, H-14), 1.48 (m, 1H, H-14), 1.43 (s, 3H, H-18), 1.16 (s, 3H, H-18), 1.00 (d, J = 6.7 Hz, 3H, H-16).

[0039] Structural analysis was performed on five new compounds, and important two-dimensional NMR related information for COSY, HMBC, and NOESY is shown in the figure below. Figure 2 As shown, the physicochemical properties data are as follows:

[0040] 14- epi -PPD. Yellow oil; UV (MeOH) l max (log e ): 238 (2.59), 288 (2.34), 350 (3.11) nm; ECD (0.3 mg / mL, MeOH) l max (Δ e ): 212 (+13.58), 228 (−0.30), 255(+11.70), 313 (−5.01), 333 (−3.00), 370 (−6.73); IR (film) nmax 3410, 2976,2877, 1672, 1653, 1553, 1253, 1167, 831 cm -1 ; –84.02 ( c 0.1, MeOH);HRESIMS m / z 363.1803 [M+H] + (calcd for C 20 H 27 O6 + , 363.1802)。

[0041] 13- epi -16OH-PPA. yellow solid; UV (MeOH) l max (log e ): 236 (2.64), 254(2.75), 287 (2.34), 351 (3.20) nm; ECD (0.3 mg / mL, MeOH) l max (Δ e ): 209 (+16.03), 229 (−1.21), 255 (+14.03), 317 (−6.20), 345 (−3.41), 369 (−7.92); IR(film) n max 3379, 2961, 2872, 1614, 1551, 1167, 1125, 833 cm -1 ; –72.71( c 0.1, MeOH); HRESIMS m / z 363.1805 [M+H] + (calcd for C 20 H 27 O6 + , 363.1802)。

[0042] PPK. yellow solid; UV (MeOH) l max (log e ): 236 (2.77), 251 (2.81), 285(2.38), 350 (3.13) nm; ECD (0.3 mg / mL, MeOH) lmax (Δ e ): 210 (+20.98), 230 (−0.26), 254 (+11.85), 311 (−4.84), 343 (−2.37), 370 (−6.14); IR (film) n max 2967, 2876, 1740, 1626, 1558, 1231, 1167, 831 cm -1 ; –66.33 ( c 0.1, MeOH);HRESIMS m / z 405.1910 [M+H] + (calcd for C 22 H 29 O7 + , 405.1908)。

[0043] 14OH-PPC. yellow oily; UV (MeOH) l max (log e ) 236 (5.52), 254 (5.76),286 (4.75), 351 (6.85) nm; ECD (0.4 mg / mL, MeOH) l max (Δ e ) 211 (+15.73), 229 (–1.89), 255 (+15.78) , 371 (–8.99) nm; IR (film) n max 3371, 2978, 2935, 2358,1606, 1456, 1166, 1020, 833 cm −1 ; –105 ( c 0.1, MeOH); HRESIMS m / z 379.1754 [M+H] + (calculated for C 20 H 27 O7 + , 379.1751)。

[0044] PPL. dark yellow oily substance; UV (MeOH) lmax (log e ) 209 (6.45), 241(5.65), 251 (5.70), 290 (4.93) nm; ECD (0.4 mg / mL, MeOH) l max (Δ e ) 209 (+10.56), 228 (–1.32), 253 (+8.38) , 308 (–4.99) nm; IR (film) n max 3442, 2972,2358, 1730, 1612, 1373, 1026, 831 cm −1 ; –45.5 ( c 0.1, MeOH); HRESIMS m / z 405.1911 [M+H] + (calculated for C 22 H 29 O7 + , 405.1908).

[0045] 1 H and 13 The C10 NMR data are shown in Table 1-2.

[0046] Table 1 Compound 14- epi -PPD, 13- epi -16OH-PPA, PPK, 14OH-PPC and PPL 1 H NMR [ d ,mult, ( J [in Hz)] data

[0047] a: 1 H (500 MHz) and 13 C (125 MHz) NMR data (CD3OD).

[0048] b: 1 H (700 MHz) and 13 C (175 MHz) NMR data (CD3OD).

[0049] Table 2 Compound 14- epi -PPD, 13- epi-16OH-PPA, PPK, 14OH-PPC and PPL 13 C NMR (ppm, type) data

[0050] a: 1 H (500 MHz) and 13 C (125 MHz) NMR data (CD3OD).

[0051] b: 1 H (700 MHz) and 13 C (175 MHz) NMR data (CD3OD).

[0052] Example 4: Screening for the renal protective activity of epoxyphenanthrene compounds

[0053] To efficiently screen potential therapeutic drugs with renal protective and renal injury-alleviating effects, and to address the current situation of drug scarcity and limited efficacy in clinical practice, this invention specifically constructs a scientific and efficient drug screening system, providing a reliable experimental basis and technical support for the development of renal protective drugs. Specifically, this invention uses HK-2 cells as an experimental cell model, employing H / R treatment to induce HK-2 cell damage, simulating the core pathological process of renal tubular epithelial cell damage caused by ischemia and hypoxia in clinical acute kidney injury. Simultaneously, KIM-1 is selected as a core detection indicator. This indicator is a highly sensitive and specific biomarker for renal tubular injury, and its expression level is positively correlated with the degree of renal tubular injury. Under normal physiological conditions, HK-2 cells show low expression, while in the H / R-induced injury model, it is significantly upregulated. By detecting the expression level of KIM-1, the protective and inhibitory effects of drugs on renal tubular injury can be accurately determined, providing a reliable evaluation standard for drug activity screening.

[0054] The specific implementation of the H / R-induced damage model and cytotoxicity detection is as follows.

[0055] a) H / R-induced damage model: HK-2 cells were seeded in well plates with appropriate compatibility. When the cell density reached 60%, the test drug or blank solvent was added and the cells were treated for 12 h. Then, the medium was replaced with sugar-free medium and the cells were placed in an anaerobic incubator for 12 h of hypoxia. The cells were then replaced with complete medium and reoxygenated for 2 h to construct a hypoxia / reoxygenation cell model. Subsequent experiments such as cell viability and damage index detection were then carried out.

[0056] b) Cytotoxicity assay: Logarithmic growth phase HK-2 cells were seeded in 96-well plates. After culturing at 37 ℃ and 5% CO2 for 12–24 hours, the supernatant was discarded. Complete culture medium containing the corresponding treatment was added to each group (control group treated with 0.1% DMSO; treatment groups treated with different drugs containing 0.1% DMSO at a concentration of 10 μM; 6 replicates per group). After culturing for another 24 hours, CCK-8 reagent and DMEM / F12 were mixed at a 1:10 ratio to prepare the working solution, and incubated in an incubator for 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader.

[0057] Based on the cell models and detection indicators described above, this invention evaluated the cytotoxicity of all compounds.

[0058] The results showed that no significant decrease in cell viability or morphological abnormalities were observed when the compound concentration reached 10 μM. Figure 3 (A) Subsequent experiments all used 10 μM as a uniform dosage concentration to ensure that the observed phenotypic changes were due to the pharmacological activity of the compound rather than cytotoxic interference.

[0059] This invention constructs a high-content screening platform ( Figure 3 (B) A damage model was constructed by hypoxia / reoxygenation treatment of HK-2. The drug underwent a series of steps: fixation with 4% paraformaldehyde at room temperature, permeabilization with 0.2% Triton X-100, blocking with 5% BSA at room temperature, overnight incubation with primary antibody at 4°C, incubation with FITC-labeled secondary antibody at room temperature, hoechst nucleoside staining at room temperature, and finally, fluorescence images were acquired using the ImageXpress Micro confocal imaging system to construct a high-content screening platform. This platform enables simultaneous detection of multiple compounds and concentrations, significantly improving the efficiency and throughput of drug screening, effectively shortening the drug screening cycle, and ensuring the stability and repeatability of screening results. It overcomes the shortcomings of traditional screening methods, such as low efficiency, long processing time, and difficulty in large-scale screening.

[0060] This invention selects sponge-associated epiphytic fungi of the genus *Polytrichum*. Neopestalotiopsis sp. SCSIO41422 metabolites PPB, PPC, PPD, 14- epi- PPD and PPK were used to conduct inhibitory activity screening experiments on H / R-induced upregulation of KIM-1 expression in HK-2 cells using the high-content screening platform constructed above. The experimental conditions were strictly controlled during the experiment. HK-2 cells were seeded in wells with appropriate compatibility. When the cell density reached 60%, a blank control group, a model control group, and different compound treatment groups were set up (the control group was treated with 0.1% DMSO; the treatment groups were treated with different drugs containing 0.1% DMSO at a concentration of 10 μM; each group had 6 replicates). After 12 h of treatment, the medium was replaced with sugar-free medium and the cells were placed in an anaerobic incubator for 12 h of hypoxia, followed by reoxygenation in complete medium for 2 h to construct the HK-2 cell hypoxia / reoxygenation model. The cells were then subjected to a series of steps: fixation with 4% paraformaldehyde at room temperature, permeabilization with 0.2% Triton X-100, blocking with 5% BSA at room temperature, overnight incubation with primary antibody at 4 °C, incubation with FITC-labeled secondary antibody at room temperature, Hoechst staining at room temperature, and finally, fluorescence images were acquired using an ImageXpress Micro confocal imaging system. The expression level of KIM-1 in each group was determined using a standardized detection procedure. Statistical analysis showed that PPB, PPC, PPD, and 14- epi -PPD and PPK can significantly inhibit H / R-induced abnormal upregulation of KIM-1 in HK-2 cells ( Figure 3 C−D), possessing anti-acute kidney injury efficacy. Based on high-content screening results, this invention determined the cell viability of metabolites with anti-acute kidney injury efficacy. After statistical analysis, PPB, PPC, PPD, 14- epi Both PPD and PPK can significantly improve the survival rate of HK-2 cells after H / R injury. Figure 3 E).

[0061] In summary, this invention, by constructing a high-content screening system based on the KIM-1 phenotype and using an H / R-induced HK-2 cell damage model as a vector, successfully screened marine *Neoplasmosis* fungi. Neopestalotiopsis Activity screening of epoxy-based pseudophenanthrene compounds derived from sp. SCSIO41422 revealed structurally similar compounds such as PPB, PPC, PPD, and 14- epi Both PPD and PPK can significantly inhibit the expression of KIM-1 and significantly improve the survival rate of HK-2 cells after H / R injury. They have the activity of reducing renal tubular damage and protecting the kidneys. They can be used as lead compounds for the treatment of kidney diseases such as acute kidney injury, laying a solid experimental foundation for the structural optimization, efficacy verification and mechanism research of subsequent related kidney disease treatment drugs.

Claims

1. Epoxy-phenanthrene ketone compounds as shown in general formula (I); General Formula (I) in, R1 is H, OH, or OCOCH3; R2 is H or OH; R3 is H or OH; R4 is H, OH, or OCOCH3; R5 is H or OH.

2. The epoxy pseudophenanthrene ketone compound according to claim 1, characterized in that, As shown in equation (II): Formula (II).

3. A method for preparing the epoxy-p-phenanthroline compound of claim 2, characterized in that, Includes the following steps: (a) Preparation of fungi Neopestalotiopsis Fermentation product of sp. SCSIO41422; (b) The fermentation product obtained in step (a) was extracted with ethyl acetate, and the solvent was recovered under reduced pressure to obtain an extract. The extract was eluted by a forward silica gel column gradient with a petroleum ether / ethyl acetate / methanol system as the eluent. The fraction was then purified by medium-pressure reverse silica gel column chromatography and semi-preparative HPLC to obtain the epoxy phenanthrene compound.

4. The preparation method according to claim 3, characterized in that, The method for preparing the fermentation product is as follows: take fungi Neopestalotiopsis Sp. SCSIO41422 was used for seed fermentation culture. The culture medium contained 15g of malt extract, 24g of crude sea salt, and 15g of agar per liter. The culture was shaken to obtain seed fermentation broth. The seed fermentation broth was then inoculated into rice culture medium containing 200g of rice, 250mL of water, and 6g of sea salt per liter. The culture was allowed to stand for fermentation to obtain fermented product.

5. The preparation method according to claim 3 or 4, characterized in that, Step (b) is as follows: the fermentation product obtained in step (a) is extracted with ethyl acetate, the solvent is recovered under reduced pressure to obtain an extract, and the extract is eluted by a positive silica gel column gradient using a petroleum ether / ethyl acetate / methanol system as the eluent. The volume fractions are: petroleum ether / ethyl acetate / methanol = 100% / 0 / 0→90% / 10% / 0→80% / 20% / 0→70% / 30% / 0→50% / 50% / 0→40% / 60% / 0→30% / 70% / 0→20% / 80% / 0→18% / 82% / 0→14 / Elution was performed using a gradient of 86% / 0→10% / 90% / 0→0 / 100% / 0%→0 / 99% / 1%→0 / 95% / 5%→0 / 80% / 20%→0 / 70% / 30%, collecting the following fractions: petroleum ether / ethyl acetate / methanol = 70% / 30% / 0 (Fr.4), petroleum ether / ethyl acetate / methanol = 50% / 50% / 0 (Fr.5), petroleum ether / ethyl acetate / methanol = 30% / 70% / 0 (Fr.7), and petroleum ether / ethyl acetate / methanol = 20% / 80% / 0 (Fr.8). Fr.4 was subjected to reversed silica gel column chromatography with a methanol / water gradient elution. The volume fractions were: methanol / water = 10% / 90% → 24% / 76% → 31% / 69% → 38% / 62% → 45% / 55% → 52% / 48% → 59% / 41% → 66% / 34% → 73% / 27% → 94% / 6% → 100% / 0. The methanol / water = 24% / 76% elution fraction Fr.4-2 was then purified by semi-preparative high-performance liquid chromatography (HPLC) at ChromCore 120 C18, 10 × 250 mm, 5 µm, 40% MeCN / H2O eluent, 2.5 mL / min, yielding compound PPK t. R 17 min; Fr.5 was separated by reversed-direction silica gel column chromatography with methanol / water as the eluent. The elution gradient was methanol / water = 10% / 90% → 17% / 83% → 24% / 76% → 31% / 69% → 38% / 62% → 45% / 55% → 52% / 48% → 59% / 41% → 66% / 34% → 73% / 27% → 87% / 13% → 94% / 6% → 100% / 0. The methanol / water elution fraction Fr.5−6 was separated by semi-preparative high-performance liquid chromatography (HPLC) at ChromCore 120 C18, 10 × 250 mm, 5 µm; eluent 56% MeOH / H2O, 2.5 mL / min, yielding compound 14-epi-PPD. R 20 min; eluent 40% MeCN / H2O, 2.5 mL / min 13-epi-16OH-PPA t R 12 min; eluent 40% MeCN / H2O, 2.5 mL / min PPC t R 13 min; eluent 56% MeOH / H2O, 2.5 mL / min; compound PPD t R 16 min; The methanol / water = 66% / 34% elution fraction Fr.5−9 was further purified by semi-preparative high-performance liquid chromatography (HPLC) at ChromCore 120 C18, 10 × 250 mm, 5 µm, with 65% MeOH / H2O as eluent at 2.5 mL / min to obtain compound PPB. R 21 min; Fr.7 was subjected to reversed-phase silica gel column chromatography with a methanol / water gradient elution. The volume fractions were: methanol / water = 10% / 90% → 24% / 76% → 31% / 69% → 38% / 62% → 45% / 55% → 52% / 48% → 59% / 41% → 66% / 34% → 73% / 27% → 94% / 6% → 100% / 0. The methanol / water fraction Fr.7-3 was then subjected to semi-preparative high-performance liquid chromatography (HPLC) at ChromCore 120 C18, 10 × 250 mm, 5 µm, with 40% CH3CN / H2O as eluent at 2.5 mL / min, from which compound PPLt was isolated. R 21 min; Fr.8 was further purified by semi-preparative high-performance liquid chromatography (HPLC) using ChromCore 120 C18, 10 × 250 mm, 5 µm, with 25% CH3CN / H2O as eluent at 2.5 mL / min to give compound 14OH-PPC. R 9 min; eluent 35% CH3CN / H2O, 2.0 mL / min PPE t R 8 min.

6. The use of the epoxy-p-phenanthrene compound of claim 1 or its pharmaceutical salt in the preparation of a nephropathy treatment drug.

7. The application according to claim 6, characterized in that, The epoxy pseudophenanthrone compound is the epoxy pseudophenanthrone compound according to claim 2.

8. The application according to claim 6 or 7, characterized in that, The drug comprises an effective dose of an epoxyphenanthrene compound or a pharmaceutical salt thereof as the active ingredient, and a pharmaceutically acceptable carrier.

9. The application according to claim 6, characterized in that, The kidney disease mentioned is acute kidney injury.

10. A drug for treating kidney disease, characterized in that, The effective dose of the epoxy phenanthrene compound of claim 1 as represented by general formula (I) or a pharmaceutical salt thereof.

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