Extraction method and application of a class of azasesterterpenoids

Abstract

The application discloses an extraction method and application of an azasesterterpene compound with anti-prostate cancer activity from marine fungi, and the azasesterterpene compound or a medicinal salt thereof has a structure as shown in formula I-3: wherein compound 1 is pyripyropene E, compound 2 is pyripyropene O, and compound 3 is 11-deacetyl PPPA. Molecular docking analysis shows that the compound has anti-prostate cancer activity, and further experiments find that the compound pyripyropene O (compound 2) has significant cytotoxic activity on prostate cancer cells, which provides an alternative compound for developing a new prostate cancer treatment and has important significance for developing Chinese marine drug resources.

Description

Technical field:

[0001] The present invention relates to the field of natural products, and in particular to an extraction method and application of an aza-sesquiterpenoid compound. Background technology:

[0002] Prostate cancer, a typical malignancy of the male reproductive system, ranks second in incidence among male malignancies worldwide. Although the exact cause of prostate cancer remains unclear, it is generally believed that its development is closely related to multiple factors, including genetics, environmental factors, and sex hormone levels. Most prostate cancers originate in the peripheral regions of the gland and often present in a multifocal distribution.

[0003] Given the high morbidity and mortality rates of prostate cancer, the development of effective treatments is particularly urgent. Marine ecosystems, with their unique environmental conditions, foster rich biodiversity, providing new perspectives and opportunities for the exploration of natural drug resources. The diversity and uniqueness of marine biological resources make them an important source for new drug development, especially for the discovery of lead compounds with potential for new drug development. Against this backdrop, marine microorganisms, as an important biological resource, have attracted particular attention for their anti-tumor activity. Screening and identifying natural products with anti-prostate cancer cell activity from marine microorganisms not only enriches the existing drug library but also has important scientific significance and clinical application value for the development of new drugs for the treatment of prostate cancer. Therefore, in-depth research on the active ingredients in marine microorganisms and the exploration of their anti-tumor mechanisms are of immeasurable importance for promoting the development of prostate cancer therapeutics. Summary of the invention:

[0004] The first object of the present invention is to provide an aza-sesquiterpenoid compound derived from marine fungi with anti-prostate cancer activity or a pharmaceutically acceptable salt thereof, the structure of which is shown in Formula I-3:

[0005]

[0006] Among them, compound 1 is pyripyropene E, compound 2 is pyripyropene O, and compound 3 is 11-deacetyl pyripyropene A (11-deacetyl PPPA).

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned azasesquiterpenoid compounds, which are prepared and isolated from the fermentation culture of the marine fungus Aspergillus fumigatus SCSIO 41220 (GDMCC No. 64921). The specific preparation method is as follows: 1) preparing the fermentation culture of the marine fungus Aspergillus fumigatus SCSIO 41220 GDMCC No. 64921; 2) soaking the fermentation culture with ethyl acetate, concentrating the extract to obtain an extract, and subjecting the extract to medium-pressure silica gel column chromatography. PE :V EtOAc =100:0~1:1 petroleum ether / ethyl acetate at 50mL·min -1 The first part was obtained by gradient elution with a flow rate of V PE :V EtOAc :V MeOH =20:20:1~0:0:100 petroleum ether / ethyl acetate / methanol at a flow rate of 50 mL·min -1 The second part was obtained by gradient elution at a flow rate of 100 nm. The two parts were combined to obtain 8 fractions after thin layer chromatography (TLC), namely Fr.1 to Fr.8. The sixth fraction Fr.6 was subjected to reverse phase medium pressure chromatography (ODS) and purified by V MeOH :V H2O = 5:95~100:0 methanol / water gradient elution to obtain 33 sub-fractions, namely Fr.6-1~Fr.6-33, among which the 10th sub-fraction Fr.6-10 was semi-preparative liquid phase with V MeOH :V H2O =64:36 methanol / water purification to obtain compound 2; wherein, the eighth fraction Fr.8 was subjected to reverse phase medium pressure chromatography (ODS) with V MeOH :V H2O = 5:95~100:0 methanol / water gradient elution to obtain 21 sub-fractions, namely Fr.8-1~Fr.8-21, among which the 9th sub-fraction Fr.8-9 was semi-preparative liquid phase with V MeOH :V H2O =63:37 methanol / water purification to obtain compound 3 (11-deacetylpyrimidine A) and pyrimidine A, the 18th sub-fraction Fr.8-18 was semi-preparative liquid phase with V MeOH :V H2O =70:30 methanol / water to obtain compound 1 (pyripyropene E).

[0008] The fermentation culture of the marine fungus Aspergillus fumigatus SCSIO 41220 GDMCC No. 64921 is prepared as follows: the marine fungus Aspergillus fumigatus SCSIO 41220 is inoculated into 500-mL Erlenmeyer flasks, each containing 200 mL of seed culture medium, and cultured at 25° C. and 180 rpm for 72 hours to obtain a seed solution. The seed solution is then inoculated into 78 1000-mL Erlenmeyer flasks containing rice culture medium, and the culture is allowed to stand at 25° C. for 50 days to obtain a rice culture product of the fungus Aspergillus fumigatus SCSIO 41220.

[0009] The seed culture medium is MB culture medium: 15g malt extract, 10g refined sea salt, 1000mL distilled water, pH 7.4-7.8; the rice culture medium is: 200g rice, 2g refined sea salt, and 200mL distilled water.

[0010] The third object of the present invention is to protect the use of the aza-sesquiterpenoid compound or a pharmaceutically acceptable salt thereof in the preparation of anti-prostate cancer drugs.

[0011] The fourth object of the present invention is to protect a drug comprising an effective amount of the aza-sesquiterpenoid compound or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier.

[0012] The fifth object of the present invention is to protect the use of the marine fungus Aspergillus fumigatus SCSIO41220GDMCC No: 64921 in the preparation of the aza-sesquiterpenoid compound or its pharmaceutically acceptable salt.

[0013] The present invention discloses four heteroterpenoid compounds, pyripyropene E (Compound 1), pyripyropene O (Compound 2), pyripyropene A (Compound 4), and 11-deacetyl PPPA (Compound 3), produced from the marine fungus Aspergillus fumigatus SCSIO 41220. Molecular docking analysis revealed that these four compounds exhibited anti-prostate cancer activity. Further experiments revealed that pyripyropene O (Compound 2) exhibited significant cytotoxic activity against prostate cancer cells, providing potential candidates for the development of new prostate cancer treatments and significantly contributing to the development of China's marine pharmaceutical resources.

[0014] The marine fungus Aspergillus fumigatus SCSIO 41220 of the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on July 29, 2024, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, Deposit Number: GDMCC No: 64921. Description of the drawings:

[0015] Figure 1 It is the molecular docking analysis of pyripyropene E (compound 1) and cyclin-dependent kinase 4 (PDB ID: 6P8E);

[0016] Figure 2 is the molecular docking analysis of pyripyropene O (compound 2) and cyclin-dependent kinase 4 (PDB ID: 6P8E);

[0017] Figure 3 is the molecular docking analysis of pyripyropene A (compound 4) and cyclin-dependent kinase 4 (PDB ID: 6P8E);

[0018] Figure 4 is the molecular docking analysis of 11-deacetyl PPPA (compound 3) and cyclin-dependent kinase 4 (PDB ID: 6P8E);

[0019] Figure 5 is the effect of pyripyropene O (compound 2) on the viability of normal prostate cancer cells and prostate cancer cells;

[0020] Figure 6 The effect of pyripyropene O (compound 2) on the proliferation ability of PC-3 cells. Specific implementation method:

[0021] The following is a further description of the present invention, but not a limitation of the present invention.

[0022] Example 1: Preparation and structural identification of pyripyropene E (Compound 1), pyripyropene O (Compound 2), pyripyropene A (Compound 4) and 11-deacetyl PPPA (Compound 3)

[0023] 1. Preparation

[0024] 1. Microbial culture conditions:

[0025] Aspergillus fumigatus SCSIO 41220 was inoculated into 500 mL Erlenmeyer flasks, each containing 200 mL of seed culture medium (MB medium), and cultured at 25°C, 180 rpm, for 72 hours to produce a seed solution. The seed solution was then inoculated into 78 1000 mL Erlenmeyer flasks containing rice culture medium. The culture was then allowed to stand at 25°C for 50 days to obtain a rice culture product of Aspergillus fumigatus SCSIO 41220. The seed culture medium consisted of MB medium (15 g malt extract, 10 g refined sea salt, 1000 mL distilled water, pH 7.4-7.8). The rice culture medium consisted of 200 g rice, 2 g refined sea salt, and 200 mL distilled water.

[0026] 2. Extraction and separation:

[0027] After fermentation, the rice fermentation product was soaked in twice the volume of ethyl acetate for 2 days, then the rice was mashed, ultrasonicated for 15 minutes, extracted with ethyl acetate three times, and concentrated under reduced pressure to obtain a crude extract (163 g) of ethyl acetate phase. The crude extract was separated by medium pressure chromatography using 100-200 mesh silica gel as the sample, and the mobile phase was petroleum ether:ethyl acetate (V:V = 100:0-1:1, flow rate 50 mL min -1 ) gradient elution to obtain the first part, and then petroleum ether: ethyl acetate: methanol (V:V:V = 20:20:1 to 0:0:100, flow rate 50 mL min -1 ) gradient elution to obtain the second part. The two parts were identified by thin layer chromatography (TLC) and combined to obtain 8 fractions, namely Fr.1 to Fr.8. The sixth fraction Fr.6 was purified by reverse phase medium pressure chromatography (ODS) (V MeOH :V H2O =5:95~100:0) gradient elution to obtain 33 sub-fractions, namely Fr.6-1~Fr.6-33, among which the 10th sub-fraction Fr.6-10 was subjected to semi-preparative liquid phase (V MeOH :V H2O =64:36) to obtain compound 2 (9.32 mg), wherein the eighth fraction Fr.8 was purified by reverse phase medium pressure chromatography (ODS) (V MeOH :V H2O =5:95~100:0) gradient elution to obtain 21 sub-fractions, namely Fr.8-1~Fr.8-21, among which the 9th sub-fraction Fr.8-9 was subjected to semi-preparative liquid phase (V MeOH :V H2O =63:37) were purified to obtain compound 3 (4.02 mg) and compound 4 pyripridine A (7.56 mg). The 18th subfraction Fr.8-18 was purified by semi-preparative liquid phase (V MeOH :VH2O =70:30) to obtain compound 1 (12.98 mg).

[0028] 2. Structural Identification of Compounds 1, 2, 3, and 4

[0029] Compound 1: light brown oil, 1 H-NMR, 13 The C-NMR data (Table 1) were consistent with those of the reported pyripyropene E. High-resolution mass spectrometry (HR-ESIMS) gave a quasi-molecular ion peak at m / z 452.2440 ([M+H] + ), molecular formula is C 27 H 33 NO5, these data are consistent with the molecular weight of pyripyropene E of 451.2, and thus it was identified as pyripyropene E (Compound 1).

[0030] Compound 2: light brown oil, 1 H-NMR, 13 The C-NMR data (Table 1) were consistent with those of the reported pyripyropene O, and the low-resolution mass spectrometry (LR-ESIMS) gave a quasi-molecular ion peak at m / z 447.2 ([M+H] + ), which is consistent with the molecular weight of pyripyropene O being 288.1, and thus it was determined to be pyripyropene O.

[0031] Compound 3: light brown oil, 1 H-NMR, 13 The C-NMR data (Table 2) were consistent with the reported 11-deacetyl PPPA data, and the low-resolution mass spectrometry (LR-ESIMS) gave a quasi-molecular ion peak at m / z 247.1 ([M+H] + ), which is consistent with the molecular weight of 11-deacetyl PPPA being 288.1, and thus it was determined to be 11-deacetyl PPPA.

[0032] Compound 4: light brown oil, 1 H-NMR, 13 The C-NMR data (Table 1) were consistent with those of the reported pyripyropene A. The low-resolution mass spectrometry (LR-ESIMS) gave a quasi-molecular ion peak at m / z 447.2 ([M+H] + ), which is consistent with the molecular weight of pyripyropene A of 288.1, and thus it was determined to be pyripyropene A.

[0033] Table 1. Compound 1-2 1 H NMR and 13 C NMR data

[0034]

[0035]

[0036]

[0037] Table 2. Compound 3 and Compound 4 1 H NMR and 13 C NMR data

[0038]

[0039]

[0040] Example 2: Determination of the Anti-Prostate Cancer Cell Proliferation Activity of Compound 1, Compound 2, Compound 3 and Compound 4

[0041] 1. Computational molecular docking of compounds 1, 2, 3, and 4

[0042] AutoDockVina software was used for molecular docking analysis to screen multiple aza-sesquiterpenoid natural products. The specific operation was carried out according to the literature description (Dai, Y.; Li, K.; She, J.; Zeng, Y.; Wang, H.; Liao, S.; Lin, X.; Yang, B.; Wang, J.; Tao, H.; Dai, H.; Zhou, X.; Liu, Y. Lipopeptide Epimers and a Phthalide Glycerol Ether with AChE Inhibitory Activities from the Marine-Derived Fungus Cochliobolus Lunatus SCSIO41401. Marine drugs 2020, 18.). Compounds 1-4 were docked with the prostate cancer CCDN1 gene target (PDB ID: 6P8E) and their interactions were evaluated. The molecular interactions between the ligand binding domain and the compounds were studied and analyzed. The results are shown in Table 3 and Figure 1-Figure 4 It can be seen that the screened compounds 1-4 all produced good interactions with the target protein, indicating that these compounds may have significant prostate cancer cell inhibitory activity, among which compound 2 has a lower docking binding energy.

[0043] Table 3. Computational molecular docking evaluation of compounds with the prostate cancer CCDN1 gene target (PDB ID: 6P8E)

[0044]

[0045] II. Determination of the inhibitory activity of compounds 1, 2, 3 and 4 against prostate cancer cells

[0046] The MTT method was used to further evaluate the anti-prostate cancer cell activity of the compound. The specific operation was carried out according to the description in the literature (Cai, J.; Wang, X.; Gan, X.; Zhou, Q.; Luo, X.; Yang, B.; Liu, Y.; Ratnasekera, D.; Zhou, X. New Chlorinated Metabolites and Antiproliferative Polyketone from the Mangrove Sediments-Derived Fungus Mollisia sp. SCSIO41409. Mar. Drugs 2023, 21, 32.). First, prostate cancer cells need to be cultured in a suitable culture medium until they reach a density of 80%-90%. Subsequently, cell separation agents such as trypsin are used to separate the cells from the culture dish and prepare a single cell suspension. The suspension is counted using a cell counting plate or an automatic counter to determine the cell concentration. Next, the cell suspension is diluted to an appropriate concentration and inoculated into a 96-well plate, with 1×10 4 to 1×10 5 cells. The seeded cells adhere to the wall of the incubator and begin to grow. After the cells adhere, different concentrations of compound solutions are added to the 96-well plate to evaluate their effects on cell activity. The incubation process usually lasts for 24-72 hours to observe the effect of the compound on cell growth. After the incubation is completed, MTT solution is added to each well. The solution is reduced to purple formamide crystals under the action of mitochondrial dehydrogenase in living cells. After another 4 hours of incubation, to ensure that the MTT is fully reacted. After the reaction is completed, acidic isopropanol or other solvents are added to dissolve the formamide crystals. The absorbance of each well is measured at a specific wavelength using a microplate reader. The change in absorbance reflects the cell activity. Through data analysis, the effect of the compound on the growth of prostate cancer cells can be evaluated and the half-maximal inhibitory concentration (IC50) can be calculated, that is, the concentration required for the compound to inhibit cell growth by 50%. Finally, based on the experimental results, the anti-prostate cancer cell activity of the compound is evaluated and compared with known drugs or standard compounds to determine its potential anti-tumor activity.

[0047] The results showed that compound 2 had significant inhibitory activity against a variety of prostate cancer cells. Figure 5 Show.

[0048] The effect of pyripyropene O (compound 2) on the proliferation of human prostate cancer cell PC-3 cells is shown in the following table. Figure 6 Show.

Claims

1. A method for preparing an aza-sesquiterpenoid compound, characterized in that: The product is prepared and isolated from the fermentation culture of the marine fungus Aspergillus fumigatus SCSIO 41220GDMCC No. 64921. The specific preparation method is as follows: 1) preparing the fermentation culture of the marine fungus Aspergillus fumigatus SCSIO 41220GDMCC No. 64921; 2) soaking the fermentation culture with ethyl acetate, concentrating the extract to obtain an extract, and subjecting the extract to medium-pressure silica gel column chromatography. PE: V EtOAc =100:0~1:1 petroleum ether / ethyl acetate gradient elution to obtain the first part, and then V PE :V EtOAc :V MeOH =20:20:1~0:0:100 petroleum ether / ethyl acetate / methanol gradient elution to obtain the second part, the two parts of the flow obtained by thin layer chromatography identification and combined to obtain 8 fractions, namely Fr.1~Fr.8, of which the sixth fraction Fr.6 was subjected to reverse phase medium pressure chromatography, V MeOH :V H2O = 5:95~100:0 methanol / water gradient elution to obtain 33 sub-fractions, namely Fr.6-1~Fr.6-33, among which the 10th sub-fraction Fr.6-10 was semi-preparative liquid phase with V MeOH :V H2O =64:36 methanol / water purification to obtain compound 2; wherein, the eighth fraction Fr.8 was subjected to reverse phase medium pressure chromatography using V MeOH :V H2O = 5:95~100:0 methanol / water gradient elution to obtain 21 sub-fractions, namely Fr.8-1~Fr.8-21, among which the 9th sub-fraction Fr.8-9 was semi-preparative liquid phase with V MeOH :V H2O =63:37 methanol / water purification to obtain compound 3 and pyriproxypyrimidine A, the 18th subfraction Fr.8-18 was semi-preparative liquid phase with V MeOH :V H2O =70:30 methanol / water purification to obtain compound 1; the structure of compound 1-3 is as follows:

2. The preparation method according to claim 1, characterized in that The fermentation culture of the marine fungus Aspergillus fumigatus SCSIO 41220 (GDMCC No. 64921) is prepared as follows: the marine fungus Aspergillus fumigatus SCSIO 41220 is inoculated into 500-mL Erlenmeyer flasks, each containing 200 mL of seed culture medium, and cultured at 25° C. and 180 rpm for 72 hours to obtain a seed solution. The seed solution is then inoculated into 78 1000-mL Erlenmeyer flasks containing rice culture medium, and the culture is allowed to stand at 25° C. for 50 days to obtain a rice culture product of the fungus Aspergillus fumigatus SCSIO 41220.

3. The preparation method according to claim 2, characterized in that The seed culture medium is MB culture medium: 15g malt extract, 10g refined sea salt, 1000mL distilled water, pH 7.4-7.8; the rice culture medium: 200g rice, 2g refined sea salt, 200mL distilled water.

4. Use of the aza-sesquiterpenoid compound of Formula 2 or Formula 3 or a pharmaceutically acceptable salt thereof in the preparation of an anti-prostate cancer drug, 5. Use of the marine fungus Aspergillus fumigatus SCSIO 41220GDMCC No: 64921 in the preparation of azasesquiterpenoid compounds or pharmaceutically acceptable salts thereof according to any one of formulas 1-3:

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