Antitumor active compounds derived from marine fungi and their preparation methods

Abstract

This invention relates to antitumor active compounds derived from marine fungi and methods for their preparation. The antitumor active compounds have the structures shown in compounds 1 and 2.

Description

Technical Field

[0001] This invention belongs to the field of fungal active secondary metabolites, specifically relating to antitumor active compounds derived from marine fungi and their preparation methods. Background Technology

[0002] The endophytic fungus HSG11-9 of the mangrove sago palm (fruit) was isolated from the fruit of the mangrove sago palm in Hainan. The applicant previously isolated a series of antitumor active compounds from this fungus (Chinese Invention Patent Application No.: 202111133295.6). The applicant further studied other fractions of the fermentation product of this fungus and obtained two antitumor active compounds. Summary of the Invention

[0003] This invention provides an antitumor active compound derived from marine fungi or a pharmaceutically acceptable salt thereof, characterized in that the antitumor active compound has the structures shown in compounds 1 and 2:

[0004]

[0005] Another embodiment of the present invention provides a method for preparing compound 1 and / or 2, characterized by comprising the following steps:

[0006] (1) Prepare seed culture medium by inoculating the endophytic fungus HSG11-9 of Sorbus aegypti into the seed culture medium and culturing at 26°C for 3 days to obtain seed culture solution.

[0007] (2) Inoculate the seed culture medium obtained in step (1) into the fermentation medium and incubate at a constant temperature of 26℃ for 40-45 days to obtain the fermentation product;

[0008] (3) Separate the fermentation broth and cells from the fermentation product obtained in step (2). Extract the fermentation broth with an equal volume of ethyl acetate 2 to 4 times. Combine the extracts and concentrate under reduced pressure to obtain an extract. Separate compounds 1 and / or 2 by chromatography.

[0009] The chromatographic separation step (3) is as follows: The extract is subjected to reduced pressure silica gel column chromatography, using petroleum ether-ethyl acetate gradients of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100. Two column volumes are collected for each gradient. The fractions obtained from the 80:20 and 60:40 gradients are combined, concentrated, and then subjected to Sephadex LH-20 gel column chromatography with chloroform:methanol = 1:1 as the eluent. The mixture is then prepared by high performance liquid chromatography (HPLC) using an Agilent C18 column (9.4 × 250 mm, 7 μm), a flow rate of 2 mL / min, and a mobile phase of CH3OH:H2O = 35:65. Compounds 1 and 2 are finally obtained.

[0010] The proportions of the eluent or mobile phase are all volume ratios; the seed culture medium contains 1.5%–3.0% glucose, 0.1%–0.5% yeast extract, 0.1%–0.5% peptone, 0.11%–0.6% crude sea salt, and an appropriate amount of water; the fermentation culture medium contains 1.6%–3.5% glucose, 0.1%–0.5% yeast extract, 0.1%–0.5% peptone, 0.11%–0.6% crude sea salt, and an appropriate amount of water; all the above percentages are weight percentages; both the seed culture medium and the fermentation culture medium need to be sterilized at 120°C for 25–30 minutes.

[0011] Another embodiment of the present invention provides the use of the above-mentioned endophytic fungus HSG11-9 from the fruit of the *Sonneratia spp.* in the preparation of antitumor compounds 1 and / or 2. Preferably, it is targeted at HeLa tumor cells.

[0012] The preservation information of the endophytic fungus (HSG11-9) from *Sargentodoxa cuneata* described in this invention is as follows: Preservation Institution: China General Microbiological Culture Collection Center (CGMCC); Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Preservation Date: September 8, 2021; Accession Number: CGMCC No. 23226; Classification: *Aspergillus terreus*. (Chinese Invention Patent Application No.: 202111133295.6)

[0013] Another embodiment of the present invention provides the use of the above-described compounds 1, 2 or pharmaceutically acceptable salts thereof in the preparation of antitumor drugs.

[0014] The present invention provides an antitumor pharmaceutical composition, characterized in that compounds 1 and 2 or their pharmaceutically acceptable salts are used as active ingredients.

[0015] The antitumor drug composition provided by the present invention may also contain other antitumor drugs; it may also contain pharmaceutically acceptable excipients (preferably pharmaceutically acceptable carriers, diluents, or excipients). The dosage form of the above drug composition may be a solid dosage form, a semi-solid dosage form, or a liquid dosage form.

[0016] In this invention, the term "pharmaceutically acceptable salt" refers to the addition salt of a nontoxic inorganic or organic acid and / or base, see "Salt selection for basic drugs", Int. J. Pharm. (1986), 33, 201–217. Attached Figure Description

[0017] Figure 1 This is a COSY and HMBC correlation diagram for compound 1;

[0018] Figure 2 It is compound 1 1 H NMR spectrum;

[0019] Figure 3 It is compound 1 13 C NMR spectrum;

[0020] Figure 4 This is the DEPT plot of compound 1;

[0021] Figure 5 This is the HH COSY diagram of compound 1;

[0022] Figure 6 This is the HSQC diagram of compound 1;

[0023] Figure 7 This is the HMBC diagram of compound 1;

[0024] Figure 8 This is the NOESY diagram of compound 1. Detailed Implementation

[0025] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed description. However, these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following.

[0026] Example 1

[0027] (1) Culture of endophytic fungus HSG11-9 from Sinapis alba fruit

[0028] Prepare seed culture medium: 20g glucose, 2g peptone, 2g yeast extract, 2.5g coarse sea salt, 1.0L water, evenly dispensed into two 1000mL Erlenmeyer flasks, and sterilized at 120℃ for 25 minutes.

[0029] The endophytic fungus strain HSG11-9 of Sorbus aegypti was inoculated into the prepared seed culture medium and cultured at 26℃ for 3 days to obtain the seed culture solution.

[0030] (2) Fermentation of endophytic fungi HSG11-9 in Sinapis alba fruit

[0031] Prepare the fermentation medium: 1.1 kg glucose, 100 g peptone, 100 g yeast extract, 125 g sea salt, and 50 L water. Distribute the mixture evenly into 100 1000 mL Erlenmeyer flasks and inactivate at 120 °C for 25–30 minutes.

[0032] Take an appropriate amount of the seed culture solution (10 mL / bottle) obtained in step (1) and inoculate it into an Erlenmeyer flask containing fermentation medium. Incubate at a constant temperature of 26℃ for 45 days to obtain the fermentation product.

[0033] (3) Preparation of extract

[0034] Separate the fermentation broth and cells from the fermentation product obtained in step (2). Extract the fermentation broth three times with an equal volume of ethyl acetate. Combine the extracts and concentrate under reduced pressure to obtain an extract (26.3g).

[0035] (4) Extraction and separation of compounds 1 and 2

[0036] The extract obtained in step (3) was subjected to reduced pressure silica gel column chromatography, using petroleum ether-ethyl acetate gradients of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100. Two column volumes were collected for each gradient. The fractions obtained from the 80:20 and 60:40 gradients were combined, concentrated, and then subjected to Sephadex LH-20 gel column chromatography with chloroform:methanol = 1:1 as the eluent. The extract was then prepared by high performance liquid chromatography (HPLC) using an Agilent C18 column (9.4 × 250 mm, 7 μm), a flow rate of 2 mL / min, and a mobile phase of CH3OH:H2O = 35:65. Compounds 1 (18.3 mg) and 2 (15.2 mg) were finally obtained.

[0037]

[0038] Compound 1: White powder, absorbs under 254 nm UV light, high-resolution mass spectrometry (HR-ESI-MS) (m / z 347.2501, [M+H) + Calculated as C 22 H 35 O3 + (347.2581) indicates the molecular formula is C 22 H 34 O3 has an unsaturation degree of 6. 1 The 1H NMR spectrum (table) shows that this compound has 6 methylene (δ) groups. H 2.82, 1H; 2.32, 1H; 2.10, 2H; 2.05, 2H; 1.95, 2H; 1.42, 4H;), 6 methines, 5 methyl groups (δ H 1.37,3H; 0.88,3H). 13 C10 NMR and DEPT spectra showed that the compound had a total of 22 carbon signals, including 5 quaternary carbons (δ¹⁸ C⁻¹). C 139.45, 135.39, 134.26, 71.28, 64.33), 6 methylene carbons (δ C 43.53, 40.06, 39.72, 31.15, 26.05, 22.67), 5 methyl carbons (δ C(29.34, 29.26, 19.12, 16.40, 16.16). Literature review revealed that the NMR data of compound 1 is essentially consistent with that of compound yanuthone M, with the main difference being that the carbonyl group at C-18 is replaced by an epoxy group. Therefore, the structure of compound 1 is identified as 2,4,6-trimethyl-12-(14,15,13,18-diepoxy-16-methyl-16-cyclohexenyl)-6,10-dodecadie n-2-ol.

[0039]

[0040]

[0041] Compound 2: White solid. 1 H NMR(CD3OD,400MHz):7.27(1H,ddm,J=15.2,10.1Hz,H-9),6.73(1H,d,J=15.0Hz,H-8),4.14( 1H,d,J=7.0Hz,H-4),6.31(2H,m,H-11),3.80(1H,dd,J=11.0,2.5Hz,H-6),3.55(1H,dd,J=11 .0,5.5Hz,H-6),3.51(1H,ddd,J=7,5.5,2.5Hz,H-5),6.25(1H,m,H-10),2.21(2H,m,H-12),1 .29(12H,m,H-14,15,16,17,18,19),1.45(2H,q,J=7.5Hz,H-13),0.89(3H,t,J=7Hz,H-20),. 13 C NMR (CD3OD, 100MHz): δ C 198.74 (C-7), 175.31 (C-2), 145.50 (C-9), 124.22 (C-8), 88.08 (C-3), 79.05 (C-4), 148.62 (C-11), 62.09 (C-6), 60.34 (C-5), 130.49 (C-10), 34.22 (C-12), 33.04 (C-18), 30.66, 30.55, 30.43, 30.31 (C-14, 15, 16, 17), 29.84 (C-13), 23.72 (C-19), 14.43 (C-20). Literature review showed that the NMR data of compound 2 was basically consistent with the known NMR data of compound Pramanicin A; therefore, the structure of compound 2 was identified as Pramanicin A.

[0042] Example 2

[0043] (1) Culture of endophytic fungus HSG11-9 from Sinapis alba fruit

[0044] Prepare seed culture medium (10.0L): 1.5% glucose (by weight, the same below), 0.5% yeast extract, 0.1% peptone, 0.11% crude sea salt, and the remainder water; dispense evenly into 16 1000mL Erlenmeyer flasks and incinerate at 120℃ for 25–30 minutes.

[0045] The endophytic fungus HSG11-9 of Sorbus aegypti was inoculated into the prepared seed culture medium and cultured at 26℃ for 3 days to obtain the seed culture solution.

[0046] (2) Fermentation of endophytic fungi HSG11-9 in Sinapis alba fruit

[0047] Prepare fermentation medium (100L): 1.6% glucose (by weight, the same below), 0.5% yeast extract, 0.1% peptone, 0.11% crude sea salt, and the remainder water; dispense evenly into 200 1000mL Erlenmeyer flasks and sterilize at 120℃ for 30 minutes.

[0048] Take an appropriate amount of the seed culture solution obtained in step (1) and inoculate it into an Erlenmeyer flask containing fermentation medium. Incubate at 26°C for 40 days to obtain the fermentation product.

[0049] (3) Preparation of extract

[0050] Separate the fermentation broth and cells from the fermentation product obtained in step (2). Extract the fermentation broth four times with an equal volume of ethyl acetate. Combine the extracts and concentrate under reduced pressure to obtain an extract.

[0051] (4) Extraction and separation of compounds 1 and 2

[0052] The extract obtained in step (3) was subjected to vacuum silica gel column chromatography, using petroleum ether-ethyl acetate gradients of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100. Two column volumes were collected for each gradient. The fractions obtained from the 80:20 and 60:40 gradients were combined, concentrated, and then subjected to Sephadex LH-20 gel column chromatography with chloroform:methanol = 1:1 as the eluent. The mixture was then prepared by high performance liquid chromatography (HPLC) using an Agilent C18 column (9.4 × 250 mm, 7 μm), a flow rate of 2 mL / min, and a mobile phase of CH3OH:H2O = 35:65. Compounds 1 and 2 were finally obtained.

[0053] Example 3

[0054] (1) Culture of endophytic fungus HSG11-9 from Sinapis alba fruit

[0055] Prepare seed culture medium (1.0L): 3.0% glucose (by weight, the same below), 0.1% yeast extract, 0.5% peptone, 0.6% crude sea salt, and the remainder water; dispense evenly into three 500mL Erlenmeyer flasks and incinerate at 120℃ for 25–30 minutes.

[0056] The endophytic fungus strain HSG11-9 of Sorbus aegypti was inoculated into the prepared seed culture medium and cultured at 28℃ for 3 days to obtain the seed culture solution.

[0057] (2) Fermentation of endophytic fungi HSG11-9 in Sinapis alba fruit

[0058] Prepare fermentation medium (10L): 3.5% glucose (by weight, the same below), 0.1% yeast extract, 0.5% peptone, 0.6% crude sea salt, and the remainder water; dispense evenly into 20 1000mL Erlenmeyer flasks and sterilize at 120℃ for 25 minutes.

[0059] Take an appropriate amount of the seed culture solution obtained in step (1) and inoculate it into an Erlenmeyer flask containing fermentation medium. Incubate at a constant temperature of 26℃ for 42 days to obtain the fermentation product.

[0060] (3) Preparation of extract

[0061] Separate the fermentation broth and cells from the fermentation product obtained in step (2). Extract the fermentation broth twice with an equal volume of ethyl acetate. Combine the extracts and concentrate under reduced pressure to obtain an extract.

[0062] (4) Extraction and separation of compounds 1 and 2

[0063] The extract obtained in step (3) was subjected to vacuum silica gel column chromatography, using petroleum ether-ethyl acetate gradients of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100. Two column volumes were collected for each gradient. The fractions obtained from the 80:20 and 60:40 gradients were combined, concentrated, and then subjected to Sephadex LH-20 gel column chromatography with chloroform:methanol = 1:1 as the eluent. The mixture was then prepared by high performance liquid chromatography (HPLC) using an Agilent C18 column (9.4 × 250 mm, 7 μm), a flow rate of 2 mL / min, and a mobile phase of CH3OH:H2O = 35:65. Compounds 1 and 2 were finally obtained.

[0064] Example 4 Cytotoxic Activity Test

[0065] The cytotoxic activity of compound 1 against the HeLa tumor cell line was tested using the MTT assay. Tumor cells in the exponential growth phase were detached from the wall by adding 0.02% Trypsin-EDTA, and a single-cell suspension was prepared using RPMI 1640 medium containing 10% fetal bovine serum. After counting and adjusting the cell number, the cells were seeded into 96-well plates and incubated at 37°C for 24 h. The test compound was prepared in fractions of 2.00, 5.00, 10.00, and 20.00 μg / mL, with three replicates per group. The test samples were dissolved in DMSO, diluted with RPMI 1640, and added to 96-well plates. The plates were incubated at 37°C for 72 h. MTT was dissolved in serum-free RPMI 1640, with 50 μL added to each well. Incubate at 37℃ in a CO2 incubator for 4 hours. Remove the incubator, aspirate the supernatant, add 150 μL of DMSO to each well to dissolve the generated formazan, and measure the absorbance at 630 nm using a microplate reader to calculate the corresponding inhibition percentage and IC50. 50 value.

[0066] Compound 1 has an in vitro inhibitory effect on HeLa tumor cells. n=3)

[0067]

Claims

1. An antitumor active compound derived from marine fungi or a pharmaceutically acceptable salt thereof, characterized in that... The antitumor active compound has the structure shown in compound 1: 。 2. A method for preparing compound 1 according to claim 1, characterized in that... Includes the following steps: (1) Prepare seed culture medium, inoculate the endophytic fungus HSG11-9 of Sorbus aegyptium into the seed culture medium, and culture at 26℃ for 3 days to obtain seed culture solution; (2) Inoculate the seed culture medium obtained in step (1) into the fermentation medium and incubate at a constant temperature of 26℃ for 40-45 days to obtain the fermentation product; (3) Separate the fermentation broth and cell from the fermentation product obtained in step (2). Extract the fermentation broth with an equal volume of ethyl acetate 2 to 4 times. Combine the extracts and concentrate under reduced pressure to obtain the extract. Compound 1 was obtained by chromatographic separation; The chromatographic separation steps described in step (3) are as follows: The extract is subjected to reduced pressure silica gel column chromatography, using petroleum ether-ethyl acetate gradients of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:

100. Two column volumes are collected for each gradient. The fractions obtained from the 80:20 and 60:40 gradients are combined, concentrated, and then subjected to Sephadex LH-20 gel column chromatography with chloroform:methanol = 1:1 as the eluent. The mixture is then prepared by high performance liquid chromatography (HPLC) using an Agilent C18 column (9.4 × 250 mm, 7 μm), a flow rate of 2 mL / min, and a mobile phase of CH3OH:H2O = 35:65, ultimately yielding compound 1.

3. The use of the endophytic fungus HSG11-9 from the fruit of the mulberry tree in the preparation of compound 1 according to claim 1.

4. The use of compound 1 of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an antitumor drug, wherein the tumor is selected from HeLa tumor cells.

5. An antitumor drug composition, characterized in that... The active ingredient is compound 1 as described in claim 1 or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 5, characterized in that... It also includes other anti-tumor drugs.

7. The pharmaceutical composition according to any one of claims 5-6, characterized in that... It also contains pharmaceutically acceptable excipients.

8. The pharmaceutical composition of claim 7, characterized in that... The pharmaceutically acceptable excipients are selected from pharmaceutically acceptable carriers.

9. The pharmaceutical composition of claim 7, characterized in that... The dosage form of the pharmaceutical composition is selected from solid dosage forms, semi-solid dosage forms, or liquid dosage forms.

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