An antitumor active compound and a method for preparing the same

Abstract

The present application relates to a kind of antitumor active compound and its preparation method, and the antitumor has the structure shown in the following compound 1-3:

Description

[0001] Based on the examination opinion issued by the State Intellectual Property Office (Document No. 2022122702491480) regarding the lack of unity of invention, the applicant filed this divisional application for Chinese Patent Application No. 202111133295.6. The original application was filed on September 27, 2021, and the original invention was entitled "An Endophytic Fungus of Sangka Fruit and Its Application in the Preparation of Antitumor Active Compounds". Technical Field

[0002] This invention belongs to the field of fungal active secondary metabolites, specifically relating to an endophytic fungus of *Sargentodoxa cuneata* and its application in the preparation of antitumor active compounds. Background Technology

[0003] According to IUCN standards, among the 20 endangered mangrove species in my country, four are classified as critically endangered: *Avicennia marina*, *Avicennia hainanensis*, *Avicennia ovata*, and *Avicennia latifolia*. However, currently, there are very few reports on the chemical constituents of *Avicennia* species, resulting in a rich variety of compound structures, including novel skeletons such as dimeric α-alkylbutyrolactones, pentacyclic triterpenoids, sesquiterpenoids, 5,8-dioxanone ergosterols, and lignans. This invention provides an endophytic fungus (HSG11-9) from *Avicennia marina* fruit and its application in the preparation of antitumor active compounds. Summary of the Invention

[0004] This invention provides an endophytic fungus (HSG11-9) from the fruit of the mulberry tree, characterized by its preservation information: 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; Preservation number: CGMCC No. 23226; Classification: Aspergillus terreus.

[0005] Another embodiment of the present invention provides a method for simultaneously preparing compounds 1-3 using the endophytic fungus HSG11-9 of *Sarcasmum hainanense*, characterized by comprising the following steps:

[0006] (1) Prepare seed culture medium by inoculating the above-mentioned 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 1-3 by chromatography.

[0009] 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 70:30 and 50:50 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 = 30:70 to 40:60. Finally, compounds 1, 2, and 3 are obtained.

[0010]

[0011] 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.

[0012] Another embodiment of the present invention provides the use of the above-mentioned endophytic fungus HSG11-9 from the fruit of the mulberry tree in the preparation of antitumor compounds 1-3. Preferably, it is targeted at A549 tumor cells.

[0013] Another embodiment of the present invention provides the use of the above-described compounds 1-3 or pharmaceutically acceptable salts thereof in the preparation of antitumor drugs.

[0014] Another embodiment of the present invention provides the use of the above compounds 1-3 or pharmaceutically acceptable salts thereof in the preparation of antitumor drug lead compounds.

[0015] Another embodiment of the present invention provides the use of the above compounds 1-3 or pharmaceutically acceptable salts thereof in the preparation of antitumor drug candidates.

[0016] The present invention provides an antitumor pharmaceutical composition, characterized in that compounds 1-3 above or their pharmaceutically acceptable salts are used as active ingredients.

[0017] 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.

[0018] 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.

[0019] The endophytic fungus (HSG11-9) of the mangrove fruit described in this invention was isolated from the mangrove fruit of the Hainan mangrove, which was harvested by the inventor, Ms. Luo Youping, from the Hainan Dongzhaigang Wild Pineapple Island Mangrove Nature Reserve. Attached Figure Description

[0020] Figure 1 It is compound 1 1 H NMR spectrum;

[0021] Figure 2 It is compound 1 13 C NMR spectrum;

[0022] Figure 3 It is compound 2. 1 H NMR spectrum;

[0023] Figure 4 It is compound 2. 13 C NMR spectrum;

[0024] Figure 5 It is compound 3. 1 H NMR spectrum;

[0025] Figure 6 It is compound 3. 13 C NMR spectrum. Detailed Implementation

[0026] 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.

[0027] Example 1

[0028] (1) Culture of endophytic fungus HSG11-9 from Sinapis alba fruit

[0029] 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.

[0030] 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.

[0031] (2) Fermentation of endophytic fungi HSG11-9 in Sinapis alba fruit

[0032] 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.

[0033] 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.

[0034] (3) Preparation of extract

[0035] 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).

[0036] (4) Extraction and separation of compounds 1-3

[0037] 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 70:30 and 50:50 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 = 30:70 to 40:60. The final results yielded compounds 1 (22 mg), 2 (11 mg), and 3 (16 mg).

[0038]

[0039] Compound 1, white powder; ESI-MS m / z: 470; 1H NMR (600MHz, DMSO): 7.79 (1H, d, J = 8.9 Hz, H-1), 7.02 (1H, d, J = 8.9 Hz, H-2), 7.02 (1H, d, J = 8.9 Hz, H-4), 7.79 (1H, d, J = 8.9 Hz, H-5), 6.56 (1H,s,H-8), 2.38(1H,d,J=16.2Hz,H-12α), 2.72(1H,d,J=16.6Hz,H-12β),1.28(1H,m,H-15α),2.47(1H,ddd,J=4.3,13.7,15.2Hz,H-15 β),1.70(1H,m,H-16α),1.91(1H,ddd,J=4.3,13.7,15.0Hz,H-16β),1.28(1H,m,H-19α),2.23(1H,m,H-19β),1.70(2H,m,H-20),3.53(1H ,dd,J=2.1,8.9Hz,H-21),1.12(3H,s,CH3-23),1.05(3H,s,CH3-24),1.47(3H,s,CH3-25),1.23(3H,s,CH3-26),3.85(3H,s,-OCH3-27). 13 C NMR (150MHz, DMSO) δ126.86(C-1), 114.48(C-2), 161.10(C-3), 114.48(C-4), 126.86(C-5), 123.67(C -6),156.99(C-7),97.25(C-8),163.05(C-9),97.25(C-10),161.1(C-11),28.73(C-12),75.97(C-13 The NMR data of compound 1 are 81.57 (C-14), 28.73 (C-15), 25.27 (C-16), 81.61 (C-17), 42.69 (C-18), 24.66 (C-19), 24.34 (C-20), 75.97 (C-21), 41.03 (C-22), 23.81 (C-23), 20.77 (C-24), 24.34 (C-25), 21.23 (C-26), and 55.40 (-OCH3). Literature review shows that the NMR data of compound 1 is basically consistent with the NMR data of the known compound Arisugacin M; therefore, the structure of compound 1 is identified as Arisugacin M.

[0040] Compound 2, white powder; ESI-MS m / z: 526; 1H NMR(600MHz,CD3CO CD3):5.65(1H,d,J=10.3Hz,H-2), 6.31(1H,d,J=10.3Hz,H-3), 1.97-1.99(2H,m,H-5 α,H-5β), 1.79-1.81(1H,m,H-6α), 2.32-2.36(1H,m,H-6β), 6.61(1H,s,H-8), 3.50(1H ,d,J=17.6Hz,H-12α),2.78(1H,d,J=17.5Hz,H-12β),1.22(3H,s,4α-CH3),1.09(3H, s,4β-CH3),1.45(3H,s,6α-CH3),1.43(3H,s,12β-CH3),3.86(9H,s,3',4',5'-OCH3). 13 C NMR (150MHz, CD3COCD3) δ202.69 (C-1), 123.71 (C-2), 153.53 (C-3), 42.73 (C-4), 79.84 (C-4a), 25.69 (C-5), 27 .39(C-6),79.84(C-6a),163.58(C-7a),98.09(C-8),158.06(C-9),164.69(C-11),98.32(C-11a),27.39(C-12) The NMR spectra of compound 2 are: 76.08 (C-12a), 56.38 (C-12b), 23.59 (4α-CH3), 25.69 (4β-CH3), 23.51 (6a-CH3), 21.99 (12b-CH3), 127.29 (C-1'), 103.23 (C-2', C-6'), 154.11 (C-3', C-5'), 140.65 (C-4'), 56.33 (-OCH3-3', -OCH3-5'), 60.40 (-OCH3-4'). Literature review shows that the NMR data of compound 2 is essentially consistent with the known NMR data of compound territrem B; therefore, the structure of compound 2 is identified as territrem B.

[0041] Compound 3, white powder; ESI-MS m / z: 424; 1H NMR (600MHz, CD3CO) CD3):7.63(2H,d,J=5.8Hz,H-2',H-6'), 6.97(2H,d,J=5.8Hz,H-3',H-5') , 6.54(1H,dd,J=8.1,2.1Hz,H-6”), 6.58(1H,d,J=8.1,2.1Hz,H-5”), 6.49 (1H,d,J=2.1Hz,H-2”), 5.08(1H,m,H-8”), 3.74(3H,s,H-7),3.45(2H,d,J =13.7Hz,H-5),3.10(2H,m,H-5),1.62(3H,s,H-10'),1.56(3H,s,H-11'). 13 C NMR (150MHz, CD3COCD3) δ170.39(C-6), 168.47(C-1), 158.36(C-4'), 154.22(C-4”), 138.56(C-2),13 2.06(C-9”),131.83(C-2”),129.69(C-2’,C-6’),129.09(C-3”),127.98(C-3),127.45(C-6”),124.4 1 (C-1”), 122.86 (C-8”), 122.28 (C-1’), 116.19 (C-3’, C-5’), 114.59 (C-5”), 85.66 (C-4), 53.34 (C-7), 38.80 (C-5), 28.12 (C-7”), 25.49 (C-10”), 17.37 (C-11”). Literature review showed that the NMR data of compound 3 was basically consistent with the NMR data of the known compound butyrolactone I; therefore, the structure of compound 3 was identified as butyrolactone I.

[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 1-3

[0052] 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 70:30 and 50:50 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 = 30:70 to 40:60. The final results yielded compounds 1 (31 mg), 2 (22 mg), and 3 (29 mg).

[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 1-3

[0063] 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 70:30 and 50:50 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 = 30:70 to 40:60, ultimately yielding compounds 1-3.

[0064] Example 4 Cytotoxic Activity Test

[0065] The cytotoxic activity of compounds 1-3 against the A549 tumor cell line was tested using the MTT assay, with adriamycin as a positive control. Tumor cells in the exponential growth phase were collected, and adherent cells were detached by adding 0.02% Trypsin-EDTA. Single-cell suspensions were prepared using RPMI 1640 medium containing 10% fetal bovine serum. After cell count and adjustment, cells were seeded into 96-well plates and incubated at 37°C for 24 h. The test compounds were aliquoted at concentrations 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. The cells were incubated at 37℃ in a CO2 incubator for 4 hours. After incubation, the supernatant was aspirated, and 150 μL of DMSO was added to each well to dissolve the generated formazan. The absorbance was measured at 630 nm using a microplate reader, and the corresponding inhibition percentage was calculated. The results showed that compounds 1-3 exhibited certain cytotoxic activity against A549 tumor cells within the tested concentration range. Particularly at a concentration of 20.00 μg / mL, the inhibition rates of compounds 1-3 against A549 tumor cells were 73.70%, 74.50%, and 90.30%, respectively. Compound 3 showed the strongest cytotoxic activity against A549 tumor cells.

Claims

1. A method for simultaneously preparing compounds 1-3 using the endophytic fungus HSG11-9 from the fruit of the mulberry tree, characterized in that... Includes the following steps: (1) Prepare seed culture medium, inoculate the endophytic fungus HSG11-9 of the fruit of the sea mulberry 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. Compounds 1-3 were obtained by chromatographic separation; Preservation information of the endophytic fungus HSG11-9 from the fruit of the *Sonneratia natans*: 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*. The structures of compounds 1-3 are as follows: 。 2. The method according to claim 1, characterized in that... 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 70:30 and 50:50 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 = 30:70 to 40:

60. Finally, compounds 1, 2, and 3 are obtained.

3. The use of compounds 1-2 prepared by the method according to any one of claims 1-2 or pharmaceutically acceptable salts thereof in the preparation of antitumor drugs, wherein the tumor is selected from tumor cell line A549.

4. An antitumor drug composition, characterized in that... Compounds 1-2 prepared by the method of claim 1, or pharmaceutically acceptable salts thereof, are used as active ingredients.

5. The antitumor drug composition according to claim 4, characterized in that... It also contains pharmaceutically acceptable excipients.

6. The antitumor pharmaceutical composition according to any one of claims 4-5, characterized in that... The dosage form of the pharmaceutical composition is a solid dosage form, a semi-solid dosage form, or a liquid dosage form.

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