A novel skeleton type cytochalasin and its application in the preparation of drugs with anti-tumor activity

Abstract

The present invention relates to the technical field of pharmaceutical chemistry, and specifically discloses a cytochalasin compound with a novel skeleton type of 5 / 5 / 13 tricyclic system isolated from endophytic fungi of Fritillaria hupehensis Boeremia exigua . The cell viability was detected by the MTT method. The experimental results showed that the compound had obvious inhibitory activity against MCF-7 (human breast cancer cells), and the IC 50 value was 11.81 mM.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and chemistry, and particularly relates to a cytochalasin compound derived from the endophytic fungus Boeremia exigua of Fritillaria hupehensis and its application in the preparation of a drug with anti-tumor activity. Background Art

[0002] Due to gene mutations, changes in living habits, and the instability of the external living environment, the risk of normal cells in the human body mutating into cancer cells has increased, and cancer has become one of the high-incidence diseases in humans. Apoptosis is the natural mechanism of cell death. Due to the overexpression of anti-apoptotic proteins and the low expression of pro-apoptotic proteins in cancer cells, multiple apoptosis pathways are inhibited, which leads to enhanced drug resistance of cancer cells, and it has become difficult for traditional anti-cancer drugs to combat the increasingly evolving cancer cells. In the process of treating cancer, multiple means are usually used to activate the apoptosis pathway of cancer cells to make them undergo natural apoptosis, so as to achieve the treatment purposes of controlling the deterioration of the condition and curing the disease. Currently, compounds derived from fungi show significant anti-cancer effects by activating different apoptosis pathways, becoming a new hope for combating drug-resistant cancer cells.

[0003] Fritillaria hupehensis is a perennial herb of the Liliaceae family, with the effects of resolving phlegm and relieving cough, and detoxifying and dissipating nodules. It is currently widely cultivated in Jianshi and Xuan'en in Hubei Province and is a commonly used traditional Chinese medicine for treating tracheitis and chronic bronchitis. Endophytic fungi live in healthy plant tissues and organs and are in a mutually beneficial symbiotic balance with plants. The biosynthesis pathways of endophytic fungi themselves are rich and diverse and ever-changing. They provide a series of novel compounds with complex structures that are difficult to obtain through artificial chemical synthesis pathways and are an important source of novel structurally active natural products and clinical drugs. Many pesticides, anti-cancer drugs, and antibacterial drugs are directly or indirectly derived from endophytic fungi. Based on the advantages of endophytic fungi such as rich resources, diverse species, short culture cycles, and high feasibility of genetic manipulation, and cytochalasin compounds having various biological activities such as anti-tumor, antibacterial, anti-inflammatory, and antioxidant. The applicant explores the anti-tumor activity of a cytochalasin with a novel skeleton isolated from the endophytic fungi of Fritillaria hupehensis. Summary of the Invention

[0004] Aiming at the deficiencies existing in the prior art, the purpose of the present invention is to provide a cytochalasin and its application in the preparation of a drug with anti-tumor activity.

[0005] A cytochalasin with anti-tumor activity has the following structural formula:

[0006]

[0007] Molecular formula: C 29 H 37 NO4

[0008] Named: boerelasin A

[0009] The application of the cytochalasin in the preparation of a drug that can inhibit the growth of MCF-7 (human breast cancer cells) also belongs to the protection scope of the present invention.

[0010] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0011] 1. The present invention discloses a cytochalasin with a novel skeleton type of 5 / 5 / 13 tricyclic system derived from the endophytic fungus B. exigua of Fritillaria hupehensis.

[0012] 2. The present invention provides a cytochalasin with anti-tumor activity, which can inhibit the proliferation of MCF-7 cells in a dose-dependent manner and has the potential use for preparing novel anti-tumor drugs.

[0013] 3. The cytochalasin whose use the present invention intends to protect can be obtained by extraction and purification from plant endophytic fungi, and has the advantages of short culture period, high operation feasibility, no chemical pollution, environmental friendliness, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 1H NMR spectrum (600 MHz, MeOD) of the compound prepared in Example 1 in the specific embodiment;

[0015] Figure 2 13C NMR spectrum (150 MHz, MeOD) and DEPT spectrum of the compound prepared in Example 1 in the specific embodiment;

[0016] Figure 3 HMQC spectrum of the compound prepared in Example 1 in the specific embodiment;

[0017] Figure 4 HMBC spectrum of the compound prepared in Example 1 in the specific embodiment;

[0018] Figure 5 1H- 1 H COSY spectrum of the compound prepared in Example 1 in the specific embodiment; 1 H COSY spectrum;

[0019] Figure 6 ROESY spectrum of the compound prepared in Example 1 in the specific embodiment;

[0020] Figure 7 Four possible configurations of the compound prepared in Example 1 inferred according to Figures 1-6 speculation;

[0021] Figure 8 Comparison of the measured carbon spectrum and the calculated carbon spectrum of the compound prepared in Example 1 in the specific implementation mode;

[0022] Figure 9 Comparison of the measured CD spectrum and the calculated CD spectrum of the compound prepared in Example 1 in the specific implementation mode;

[0023] Figure 10 Effect of the compound prepared in Example 1 in the specific implementation mode on the viability of MCF-7 cells. Specific implementation mode

[0024] To make the invention purpose and the invention content of the present application presented more clearly, the applicant will clearly and completely describe the technical solution of the present invention in combination with specific embodiments below.

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels. Example 1:

[0027] The endophytic fungus Boeremia exigua was isolated from the roots of fresh Fritillaria thunbergii plants collected from Enshi, Hubei, China. It was identified as Boeremia exigua by ITS sequencing. This strain was preserved in the Microbial Strain Bank of the School of Pharmacy, South-Central University for Nationalities (already publicly available, see: Lv Xiao, Ye Ke, Ma Xujun, etc. Chemical Constituents and Anti-Inflammatory Activities of Endophytic Fungus Boeremia exigua from Fritillaria thunbergii [J]. Journal of South-Central University for Nationalities (Natural Science Edition), 2022, 41(2): 174-179). The endophytic fungus Boeremia exigua of Fritillaria thunbergii was amplified and cultured by solid rice fermentation: The test tube storing the strain was taken out of the refrigerator and placed at room temperature in a sterile environment for 1 hour. Then, a 5-mm-diameter fungal block was inoculated onto a PDA medium plate under aseptic conditions. After growing for about 8 days under normal temperature and light-shielded conditions, a 5-mm-diameter fungal block was picked from the PDA medium plate and inoculated onto the rice medium, a total of 340 bottles. The culture conditions were: dark culture at 25 °C for 30 d. The ratio of the rice medium was: 50 g of rice per bottle, 50 mL of distilled water per bottle, placed in a 500-mL culture bottle, and sterilized at 120 °C for 20 minutes.

[0028] Isolation and purification process of the compound cytochalasin described in the claims and the invention content of the specification: Mash the solid rice medium (36 kg in total) covered with mycelia, soak it with 25 L of organic solvent (dichloromethane - methanol = 1:1, v / v) for 2 days, centrifuge, concentrate the extract under reduced pressure, and repeat the extraction operation 3 times; Combine the extracts concentrated 3 times, concentrate and evaporate the solvent to dryness under reduced pressure, add a small amount of water to dissolve, and then extract with ethyl acetate 3 times repeatedly (the amount of ethyl acetate used for each extraction is 4 L). After combining the ethyl acetate parts, concentrate under reduced pressure to obtain 660 g of crude extract paste. Chromatograph the paste on a normal-phase silica gel column with 80 - 100 mesh, elute with a gradient of dichloromethane - methanol (dichloromethane: methanol = 100:0, 50:1, 20:1, 10:1, 5:1, v / v), and detect and develop color with thin-layer chromatography (the developing agent is dichloromethane: methanol = 10:1, v / v) to roughly divide into five components A - E.

[0029] Component D (the component eluted when the volume ratio of dichloromethane / methanol is 10:1, 56.8 g) was passed through a high-performance medium-pressure liquid preparative chromatography (Biotage SP1, reverse-phase packing material: RP-18, 20 - 45 μm, Fuji Silysia Chemical Ltd., Japan) with a methanol-water system (methanol / water = 50:50, 60:40, 70:30, 80:20, 90:10, v / v). The elution flow rate was 20 mL / min, and the elution time for each of the five ratios was 60 min. Thin-layer chromatography (developing agent: dichloromethane:methanol = 10:1, v / v) was used for detection and color development. The same or similar components were combined to obtain six sub-components, which were sequentially labeled as D1 - D6 according to increasing polarity. Component D3 (27.3 g), which was eluted when the volume ratio of methanol-water was 60:40, was subjected to Sephadex LH-20 gel column chromatography (Pharmacia Fine Chemical Co., Ltd., Sweden) and eluted with methanol (the amount of methanol used was 10 column volumes). Then, thin-layer chromatography (developing agent: dichloromethane:methanol = 10:1, v / v) was used for detection and color development. The same or similar components were combined to obtain 8 sub-components, which were sequentially labeled as D3-1 to D3-8 according to increasing polarity. The component D3-4 (2.6 g) eluted with the 4th column volume was further subjected to normal-phase silica gel column chromatography with a petroleum ether-acetone system (petroleum ether:acetone = 10:1 - 1:1, v / v) for gradient elution. Thin-layer chromatography (developing agent: petroleum ether:acetone = 4:1, v / v; thin-layer chromatography silica gel plate, Qingdao Marine Chemical Factory) was used for detection and color development to obtain 5 components, which were sequentially numbered as D3-4-1 to D3-4-5 in the order of elution. Among them, component D3-4-3 (13 mg, eluted when petroleum ether:acetone = 4:1) was further subjected to high-performance liquid preparative chromatography (Agilent 1260; chromatographic column: Agilent Zorbax SB-C18, specification: 9.4 mm × 150 mm, 5 μm; acetonitrile-water = 60:40 - 75:25, v / v; flow rate: 4 mL / min) for gradient elution for 35 min to prepare 2.5 mg of the compound of the present invention (retention time: 18 min).

[0030] Structure identification of the compound: The compound cytochalasin prepared in Example 1 was dissolved in 0.5 mL of deuterated methanol and transferred to a nuclear magnetic resonance tube with a 200 μL pipette. The hydrogen spectrum, carbon spectrum, and two-dimensional spectrum were detected on a nuclear magnetic resonance spectrometer (Bruker Avance III 600 MHz, Germany) as shown in Figures 1-6 ). Based on the comprehensive physical and chemical data, the structure of the compound was solved and named boerelasin A.

[0031] Nuclear magnetic resonance data of the obtained cytochalasin:

[0032]

[0033]

[0034] The absolute configuration of the compound was determined by quantum chemistry. Among them, C-3, C-4, C-5, C-16 and C-20 are the carbons on the skeleton, so their configurations should be consistent with the basic skeleton of cytochalasin, that is, 3S4R5S16R20R. The remaining C-8 and C-9 have four possible configurations (1a-8R9S, 2a-8R9R, 3a-8S9S, 4a-8S9R, as Figure 7 shown). We calculated the NMR at the mPW1PW91-SCRF / 6-31+G(d,p) / / M06-2X / def2SVP (PCM solvent model) theoretical level, and the results showed that the calculated values of 3a-3S4R5S8S9S16R20R were in the best agreement with the experimental values (R 2 = 0.9986)( Figure 8 ). Therefore, we can determine that the absolute configuration of boerelasin A is 3S4R5S8S9S16R20R. In addition, we also calculated the ECD spectra of the four configurations, and the results showed that the ECD spectra of the 8S9S configuration were in better agreement with the measured values ( Figure 9 ), which was consistent with the results of the carbon spectrum calculation.

[0035] Other physicochemical data:

[0036] Appearance: light yellow amorphous powder. According to high-resolution mass spectrometry HRESIMS ([M+H] + , m / z 464.2797), according to the above test results, the structural formula of the compound obtained in this example was confirmed as:

[0037]

[0038] Molecular formula: C 29 H 37 NO4

[0039] Example 2: MTT assay for cell viability

[0040] Principle: The MTT method, namely the MTT colorimetric method, is a method for testing cell viability that is simple to operate, highly sensitive, cost-effective, and has intuitive results. MTT is a tetramethyl thiazolyl tetrazolium salt, with the full name of 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide, and its property is yellow powder. Succinate dehydrogenase in the mitochondria of living cells has the function of reducing exogenous MTT, which can reduce MTT to insoluble blue (or bluish-purple) crystal formazan, while dead cells do not have this function. DMSO (dimethyl sulfoxide) can dissolve the formazan deposited in the cells, and the absorbance value is measured by an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 570 nm, so as to measure the content of formazan. Generally, since the amount of formazan generated is proportional to the number of living cells, the relative number of living cells can be calculated according to the optical density OD value (setting the living cells in the blank control group as 100%)

[0041] Experimental materials and instruments:

[0042] Cell line: Human breast cancer cell line (MCF-7) (Kunming Institute of Botany, Chinese Academy of Sciences); DMEM medium and sterile PBS (pH = 7.4, 0.01 M) (Hyclone), fetal bovine serum (Zhejiang Tianhang Biotechnology Co., Ltd.), MTT (Sigma), DMSO (Biosharp); 96-well cell plates, cell scrapers, cell culture flasks (Corning);

[0043] Multifunctional ELISA reader (TECAN, Germany), carbon dioxide incubator (Thermo), inverted microscope (Nikon), autoclave (Shanghai Boxun Medical Biotechnology Co., Ltd.), ice maker (Panasonic), electronic balance (Sartorius).

[0044] Experimental steps:

[0045] Passage culture MCF-7 cells in DMEM medium containing 10% fetal bovine serum in a constant temperature incubator (5% CO2, 37°C). Take MCF-7 cells in the logarithmic growth phase with good growth state, prepare a cell suspension with DMEM medium containing 10% fetal bovine serum, and adjust the concentration to 1×10 5cells / mL were inoculated into a 96-well plate, 100 μL per well. To avoid edge effects, cells were seeded only in the middle 6×10 wells, and 100 μL of sterile PBS was added to the outer wells around the cell wells. The cells were cultured for 8 h at 5% CO2 and 37 °C. Compounds obtained in Example 1 were prepared at six concentration gradients with DMEM medium without fetal bovine serum, namely 40, 30, 20, 10, 5, and 1 μM. After the cells adhered, the DMEM medium in the wells was discarded. Different concentrations of the compounds were added to the drug groups, 100 μL per well, 100 μL of sterile PBS was added to each well in the blank group, and cisplatin (prepared in the same way as the compounds obtained in Example 1) was added to the positive control group. Then the 96-well plate was placed in a constant temperature incubator and cultured for another 12 h. Then 100 μL of MTT solution with a concentration of 0.5 mg / mL was added to each cell well. After culturing for 4 h at 5% CO2 and 37 °C, the culture was terminated, the supernatant was discarded, 100 μL of DMSO was added to each cell well, and the plate was placed on a shaker and shaken well to fully dissolve the formazan deposited in the cells. The absorbance value of each well was measured at a wavelength of 572 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The OD value was recorded and the survival rate of each test compound was calculated (survival rate % = drug group / blank group × 100%), and then the IC 50 value was calculated.

[0046] Experimental results:

[0047] The results showed that compound boerelasin A had obvious inhibitory activity against MCF-7 (human breast cancer cells). As the concentration of the compound increased, the inhibitory effect also increased accordingly, and there was statistical significance. Therefore, it was considered that the inhibitory effect of this compound on the growth of MCF-7 cells was dose-dependent, and its IC 50 value was 11.81 μM, which was better than that of the positive control drug cisplatin (21.69 μM).

Claims

1. A cytochalasin compound, whose structural formula is:

2. The cytochalasin compound according to claim 1, characterized in that, The structural formula of the said compound is as follows:

3. A method for preparing the compound according to claim 1 or 2, characterized in that, It includes the following steps: 1) Soak and extract the solid fermentation product of the endophytic fungus B. exigua in Fritillaria hupehensis Hsiao et K. C. Hsia with an organic mixed solvent, concentrate the extract to dryness, dissolve it in water again, and extract with ethyl acetate; 2) Perform silica gel column separation on the ethyl acetate extract to obtain five components A - E; 3) Perform medium - pressure liquid chromatography preparative separation on component D to obtain six sub - components D1 - D6; 4) Component D3 is separated by gel column chromatography to obtain eight sub - components D3 - 1 - D3 - 8; 5) Perform silica gel column separation on component D3 - 4 to obtain five sub - components D3 - 4 - 1 - D3 - 4 - 5; 6) Purify component D3 - 4 - 3 by high - performance liquid chromatography preparative separation to obtain the said compound; The organic mixed solvent in step 1) is a dichloromethane - methanol mixed solvent with a volume ratio of 1:1; The conditions for silica gel column separation in step 2) include: using dichloromethane - methanol as the eluent, eluting successively in the volume ratios of 100:0, 50:1, 20:1, 10:1, 5:1, detecting by TLC, to obtain five eluted components, where the developing agent is dichloromethane:methanol = 10:1, v / v; The conditions for medium - pressure liquid chromatography preparative separation in step 3) include: an RP - 18 chromatographic column, using methanol - water as the eluent, eluting successively in the volume ratios of 50:50, 60:40, 70:30, 80:20, 90:10, with a flow rate of 20 mL / min, and the elution time for each ratio being 60 min, detecting by TLC, to obtain six eluted sub - components, where the developing agent is dichloromethane:methanol = 10:1, v / v; For the gel column chromatography separation in step 4), using methanol as the eluent, detecting by TLC, to obtain eight eluted sub - components, where the developing agent is dichloromethane:methanol = 10:1, v / v; The conditions for silica gel column separation in step 5) include: using petroleum ether - acetone as the eluent, eluting with a gradient of volume ratios from 10:1 to 1:1, detecting by TLC, to obtain 5 eluted components, where the developing agent is petroleum ether:acetone = 4:1, v / v; The conditions for high - performance liquid chromatography preparative separation in step 6) include: a C18 chromatographic column, using acetonitrile - water as the eluent, performing gradient elution for 35 min with the initial volume ratio of acetonitrile and water being 60:40 and the final volume ratio being 75:25, and a flow rate of 4 mL / min.

4. Use of the compound according to claim 1 or 2 in the preparation of an anti-breast cancer drug.

5. Use of the compound according to claim 1 or 2 in the preparation of a drug for inhibiting the growth of human breast cancer cell MCF-7.

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