Extraction method and application of aza sesquiterpenoids

Abstract

The invention discloses an extraction method and application of an aza-sesquiterpenoid compound which is derived from marine fungi and has anti-prostatic cancer activity. The structure of the aza-sesquiterpenoid compound is shown as a formula I-3 or pharmaceutical salt of the aza-sesquiterpenoid compound: # imgabs0 #, wherein the compound 1 is pyripyropene E, the compound 2 is pyripyropene O, and the compound 3 is 11-deacetylated pyripyropene A (11-deacetylated PPPA). The invention further discloses a preparation method of the aza-sesquiterpenoid compound with the anti-prostatic cancer activity and application of the aza-sesquiterpenoid compound with the anti-prostatic cancer activity and application of the aza-sesquiterpenoid compound with the anti-prostatic cancer activity. Molecular docking analysis shows that the compound shows anti-prostatic cancer activity, further experiments find that the compound pyripyroene O (compound 2) has remarkable cytotoxic activity on prostatic cancer cells, an alternative compound is provided for developing new prostatic cancer treatment, and the compound 2 has important significance on development of marine drug resources in China.

Description

Technical field:

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

[0002] Prostate cancer is a typical representative of male reproductive system malignancies, and its incidence ranks second among male malignancies worldwide. Although the exact cause of prostate cancer is still unclear, it is generally believed that its occurrence may be closely related to multiple factors such as genetic factors, environmental factors, and sex hormone levels. Most prostate cancers originate from the peripheral area of ​​the gland and often present multifocal distribution.

[0003] Given the high incidence and mortality of prostate cancer, it is particularly urgent to develop efficient treatment methods. The marine ecosystem, due to its unique environmental conditions, has nurtured rich biodiversity, providing a new perspective and space for the exploration of natural drug resources. The diversity and uniqueness of marine biological resources make it an important source for new drug development, especially in the discovery of lead compounds with new drug development potential. In this context, marine microorganisms, as an important type of biological resource, are particularly attractive in the study of anti-tumor activity. Screening and identifying natural products with anti-prostate cancer cell activity from marine microorganisms can not only enrich the existing drug library, but also have 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 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 or a pharmaceutically acceptable salt thereof having anti-prostate cancer activity derived from marine fungi, 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 aza-sesquiterpenoid compounds, which are prepared and separated 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 SCSIO41220 GDMCC No. 64921; 2) soaking the fermentation culture with ethyl acetate for extraction, concentrating the extract to obtain an extract, subjecting the extract to medium-pressure silica gel column chromatography, and using V 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 50mL·min -1 The second part was obtained by gradient elution at a flow rate of 1.5 ℃ and 2.5 ℃. The two parts were combined by thin layer chromatography (TLC) to obtain 8 fractions, namely Fr.1 to Fr.8. The sixth fraction Fr.6 was purified by reverse phase medium pressure chromatography (ODS) with 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 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-deacetylpyranopyrine A) and pyranopyrine A. The 18th subfraction Fr.8-18 was semi-preparative liquid phase with V MeOH :V H2O =70:30 methanol / water to obtain compound 1 (pyriproxypine E).

[0008] The specific steps of preparing the fermentation culture of marine fungus Aspergillus fumigatus SCSIO 41220 GDMCC No.64921 are as follows: the marine fungus Aspergillus fumigatus SCSIO 41220 is inoculated into a 500mL triangular flask, each flask containing 200mL of seed culture medium, and cultured at 25°C and 180rpm for 72 hours to obtain a seed solution, the seed solution is inoculated into a 1000mL triangular flask container containing a rice culture medium, a total of 78 flasks are cultured at a temperature of 25°C and a static condition 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: 200g rice, 2g refined sea salt, 200mL distilled water.

[0010] The third object of the present invention is to protect the use of the aza-sesquiterpenoid compound or its pharmaceutically acceptable salt 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 objective of the present invention is to protect the use of marine fungus Aspergillus fumigatus SCSIO41220GDMCC No: 64921 in the preparation of the aza-sesquiterpenoid compounds or their pharmaceutically acceptable salts.

[0013] The invention discloses four heteroterpenoid compounds, pyripyropene E (compound 1), pyripyropene O (compound 2), pyripyropene A (compound 4) and 11-deacetyl PPPA (compound 3), prepared by the marine fungus Aspergillus fumigatus SCSIO 41220. Molecular docking analysis shows that the four compounds exhibit anti-prostate cancer activity, and further experiments show that the compound pyripyropene O (compound 2) has significant cytotoxic activity against prostate cancer cells, providing alternative compounds for the development of new prostate cancer treatments, and having important significance for the development of China's marine drug 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 City, 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 It is the molecular docking analysis of pyripyropene O (compound 2) and cyclin-dependent kinase 4 (PDB ID: 6P8E);

[0017] Figure 3 It 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, rather than 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] The fungus Aspergillusfumigatus SCSIO 41220 was inoculated into a 500 mL triangular flask container, each containing 200 mL of seed culture medium (MB culture medium), cultured at 25°C, 180 rpm, for 72 hours to obtain a seed solution, and the seed solution was inoculated into a rice culture medium in a 1000 mL triangular flask container, and a total of 78 bottles were cultured at a temperature of 25°C and static conditions for 50 days to obtain a rice culture product of the fungus Aspergillusfumigatus SCSIO 41220. The seed culture medium is MB culture medium: 15 g of malt extract, 10 g of refined sea salt, 1000 mL of distilled water, and PH 7.4-7.8. Rice culture medium: 200 g of rice, 2 g of refined sea salt, and 200 mL of 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 crushed, 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, mixed with 100-200 mesh silica gel, 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 ~ 0:0:100, flow rate 50mL 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), of which 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) to obtain compound 3 (4.02 mg) and compound 4 pyriproxypyrimidine 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] II. 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) are consistent with the reported pyripyropene E data, and the high-resolution mass spectrometry (HR-ESIMS) gives a quasi-molecular ion peak of m / z 452.2440 ([M+H] + ), molecular formula is C 27 H 33 NO5, this data is consistent with the molecular weight of pyripyropene E being 451.2, and therefore it was determined to be pyripyropene E (Compound 1).

[0030] Compound 2: light brown oil, 1 H-NMR, 13 The C-NMR (Table 1) is consistent with the reported pyripyropene O data, and the low-resolution mass spectrometry (LR-ESIMS) gives a quasi-molecular ion peak of 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 (Table 2) was consistent with the reported 11-deacetyl PPPA data, and the low-resolution mass spectrometry (LR-ESIMS) gave a quasi-molecular ion peak of 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 the reported pyripyropene A data, and the low-resolution mass spectrometry (LR-ESIMS) gave a quasi-molecular ion peak of m / z 447.2 ([M+H] + ), which is consistent with the molecular weight of pyripyropene A of 288.1, so 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 Antiproliferative Activity of Compound 1, Compound 2, Compound 3 and Compound 4 against Prostate Cancer Cells

[0041] 1. Computer 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 interacted well 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. In silico molecular docking evaluation of compounds with 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, and the specific operation was performed according to the literature description (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 Up to 1×10 5 cells. The inoculated cells adhere to the wall in 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 24-72 hours to observe the effects of the compounds on cell growth. After the incubation, MTT solution is added to each well, which is reduced to purple formamide crystals under the action of mitochondrial dehydrogenases in living cells. After another 4 hours of incubation, 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 an enzyme reader, and 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), that is, the concentration required for the compound to inhibit cell growth by 50%, can be calculated. 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 ability of human prostate cancer cell PC-3 cells is shown in the following table. Figure 6 Show.

Claims

1. Azasesquiterpenoid compound derived from marine fungi or a pharmaceutically acceptable salt thereof, characterized in that: Its structure is shown in Formula I-3:

2. The method for preparing the aza-sesquiterpenoid compound according to claim 1, characterized in that: It is prepared and separated from the fermentation culture of marine fungus Aspergillus fumigatus SCSIO 41220GDMCC No.64921. The specific preparation method is as follows: 1) preparing the fermentation culture of marine fungus Aspergillus fumigatus SCSIO41220GDMCC No.64921; 2) soaking the fermentation culture with ethyl acetate to extract, and concentrating the extract to obtain an extract, and the extract is subjected to medium-pressure silica gel column chromatography, and V 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 obtained fractions were identified by thin layer chromatography 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 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 to obtain compound 2; wherein the eighth fraction Fr.8 was purified by 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 to obtain compound 1.

3. The preparation method according to claim 2, characterized in that: The specific steps of preparing the fermentation culture of marine fungus Aspergillus fumigatus SCSIO 41220 GDMCC No: 64921 are as follows: the marine fungus Aspergillus fumigatus SCSIO 41220 is inoculated into a 500mL triangular flask, each flask containing 200mL of seed culture medium, and cultured at 25°C and 180rpm for 72 hours to obtain a seed solution, the seed solution is inoculated into a 1000mL triangular flask container containing a rice culture medium, a total of 78 flasks are cultured at a temperature of 25°C and a static condition for 50 days to obtain a rice culture product of the fungus Aspergillus fumigatus SCSIO 41220.

4. The preparation method according to claim 3, 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.

5. Use of the aza-sesquiterpenoid compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-prostate cancer drug.

6. A medicine comprising an effective amount of the aza-sesquiterpenoid compound or a pharmaceutically acceptable salt thereof according to claim 1 as an active ingredient, and a pharmaceutically acceptable carrier.

7. Use of the marine fungus Aspergillus fumigatus SCSIO 41220 GDMCC No: 64921 in the preparation of the aza-sesquiterpenoid compound or its pharmaceutically acceptable salt as claimed in claim 1.

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