Butenolide compound, and preparation method and application thereof

Abstract

The application discloses a butenolide compound from a marine fungus, a preparation method and application thereof, and has the advantages that the butenolide compound is a novel compound discovered from metabolic products of Aspergillus terreus, similar skeleton compounds have not been reported, and the application proves for the first time that the butenolide compound has better tumor cell proliferation inhibition ability on colon cancer cells compared with other butenolide compounds.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical compounds, and relates to a butenolide compound from marine fungi as well as a preparation method and application thereof. BACKGROUND

[0002] Drug therapy is an important part of clinical treatment of tumors, including traditional chemotherapeutic drugs, molecular targeted drugs and tumor immunological drugs. Marine natural products are an important way to discover active lead compounds. Since the first report of spongothymidine, a marine natural product with biological activity, in 1950, about 30,000 marine natural products have been discovered so far (accounting for 7.5% of all natural products), of which more than 9,000 have pharmacological activities, and currently 12 have entered clinical application. Extraction of chemical components from marine natural products has been an important means for natural medicinal chemistry to discover active compounds in the past few decades.

[0003] Curing tumors has always been a big problem in the medical field. Among them, colon cancer is a malignant disease that threatens human health and life, and its morbidity and mortality are very high. Colon cancer ranks third in male cancer morbidity rate after lung cancer and prostate cancer, and ranks second in female cancer morbidity rate after lung cancer and breast cancer. In China, the number of new cases of colon cancer each year is as high as 400,000, and the number of new cases of colon cancer each year worldwide is about 10 million. Colon cancer may have no symptoms in the early stage, and in the middle and late stages, it can manifest as abdominal distension, indigestion, and then changes in bowel habits, abdominal pain, or hematochezia. After tumor ulceration, hemophilia or toxin absorption, symptoms such as anemia, low fever, fatigue, weight loss or lower extremity edema often occur. Because the symptoms of early colon cancer are not obvious, the detection means for early colon cancer is not perfect, resulting in the result that colon cancer is found in the late stage. At present, for the treatment of colon cancer, some patients can be treated by surgery, but the risk of surgery is high, and the postoperative effect is not ideal.

[0004] Therefore, it is of urgent practical significance to develop new natural active ingredients for preventing and treating cancer, especially colon cancer. SUMMARY

[0005] The present application aims at at least one of the problems existing in the prior art to some extent, and therefore provides a butenolide compound from marine fungi as well as a preparation method and application thereof.

[0006] According to one aspect of the present application, a butenolide compound is provided, characterized in that the structure is shown in formula (I):

[0007]

[0008] According to still another aspect of the present application, there is provided a method for preparing a butenolide compound as shown in formula (I), comprising: culturing a marine fungus Aspergillus terrestris in a culture medium containing inorganic sulfur; extracting the culture medium with ethyl acetate to obtain an extract; and purifying the extract to obtain the butenolide compound as shown in formula (I).

[0009] Preferably, the culture medium is a seed culture medium of the marine fungus Aspergillus terrestris.

[0010] Preferably, purifying the extract to obtain the butenolide compound as shown in formula (I) comprises: refluxing the extract under reduced pressure to obtain a total extract; separating the total extract by silica gel column chromatography to obtain a separated extract; and eluting the separated extract with a petroleum ether-ethyl acetate system (20:1) and then repeatedly eluting the separated extract by MCI column chromatography, Sephadex LH-20 and ODS column chromatography to obtain the butenolide compound as shown in formula (I).

[0011] Preferably, the butenolide compound as shown in formula (I) is obtained by repeatedly eluting with an MCI column chromatography using a mobile phase of 85% methanol, a Sephadex LH-20 using a mobile phase of methanol and an ODS column chromatography using a mobile phase of 60% methanol.

[0012] According to still another aspect of the present application, there is provided a use of a butenolide compound as shown in formula (I) or a butenolide compound as shown in formula (I) obtained by the above method in the preparation of an anti-tumor drug.

[0013] Preferably, the tumor is colon cancer.

[0014] According to still another aspect of the present application, there is provided an anti-tumor drug, characterized in that the raw material component of the drug comprises a butenolide compound as shown in formula (I) or a butenolide compound as shown in formula (I) obtained by the above method or a salt thereof.

[0015] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0016] Preferably, the drug is an oral preparation or a non-oral preparation.

[0017] The application provides a butenolide compound from a marine fungus, a preparation method and application thereof. BRIEF DESCRIPTION OF DRAWINGS

[0018] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0019] Figure 1 A chemical structure of 2-thiomethylbutenolide is shown

[0020] Figure 2 A mass spectrum analysis diagram of 2-thiomethylbutenolide is shown

[0021] Figure 3 A hydrogen spectrum analysis diagram of 2-thiomethylbutenolide is shown

[0022] Figure 4 A carbon spectrum analysis diagram of 2-thiomethylbutenolide is shown

[0023] Figure 5 A DEPT135 analysis diagram of 2-thiomethylbutenolide is shown

[0024] Figure 6 A HSQC analysis diagram of 2-thiomethylbutenolide is shown

[0025] Figure 7 A HMBC analysis diagram of 2-thiomethylbutenolide is shown 1 H- 1 H COSY analysis diagram of 2-thiomethylbutenolide is shown

[0026] Figure 8 A HMBC analysis diagram of 2-thiomethylbutenolide is shown

[0027] Figure 9 Effects of 2-thiomethylbutenolide on growth of colon cancer cells are shown

[0028] Figure 10 Effects of 2-thiomethylbutenolide on HCT116 clone formation of colon cancer cells are shown

[0029] Figure 11 Effects of 2-thiomethylbutenolide on HCT116 death process of colon cancer cells are shown

[0030] Figure 12The effect of 2-thiomethyl butenolide on apoptosis-related proteins of colon cancer cells HCT116 is shown. DETAILED DESCRIPTION

[0031] The following examples are provided to enable those skilled in the art to more clearly understand the present application. It should be noted that the following examples do not limit the scope of protection required by the present application, but are merely illustrative examples. The raw materials, reagents or devices mentioned in the following examples, if not specifically stated, can be obtained commercially or by known existing methods.

[0032] Example 1: Isolation and purification of compound 2-thiomethyl butenolide

[0033] (1) Seed liquid preparation

[0034] The glycerol tube of the fungus Aspergillus terreus ZSD520-1 isolated from the internal tissue of corals in the Xisha Archipelago of China or Aspergillus terreus M7 isolated from a sponge sample in the South China Sea, which was stored in a ultra-low temperature refrigerator at -80°C, was inoculated on a PDA plate and incubated at 28°C for 3 days. After obvious mycelium grew on the PDA plate (fungal medium: 5.0 g potato infusion powder; 20.0 g glucose; 20.0 g agar; sea water crystal 33.0 g; H2O 1 L; adjust PH to 6.0), an appropriate amount of sterile water was added, and the mycelium was washed off with a sterile inoculation loop. The spore suspension was inoculated into 200 ml PDB liquid medium (fungal medium: 10.0 g potato infusion powder; 20.0 g glucose; sea water crystal 33.0 g; H2O 1 L; adjust PH to 6.0) at 28°C, 200 rpm for 3 days. Among them, Aspergillus terreus ZSD520-1 was preserved in Sun Yat-sen University on November 6, 2022, with the preservation number ZSD520202211-1; Aspergillus terreus M7 was preserved in China Center for Type Culture Collection, Wuhan University, on June 7, 2021, with the preservation number CTCTCCNO: M 2021679; in the BLAST database, the conserved ITS rDNA of Aspergillus terreus ZSD520-1 (GenBank KF 231662) matched 100% with Aspergillus terreus.

[0035] (2) Strain fermentation

[0036] A 100L large-scale liquid medium was prepared by additionally adding inorganic sulfur reagent (sodium sulfate) to the fungal medium. The above seed liquid was added at a mass ratio of 1:1000, and the culture solution was collected after 30 days of culture. The culture solution was extracted with ethyl acetate for 3-5 times, the extract was combined, and the total extract was obtained by reduced pressure reflux.

[0037] The total extract was separated preliminarily by silica gel column chromatography with petroleum ether-ethyl acetate system (20:1) as eluent, and then repeatedly by MCI column chromatography (85% methanol), Sephadex LH-20 (methanol) and ODS (60% methanol). Finally, 2-thiomethylbutenolide was purified by preparative liquid chromatography (mobile phase: 70% methanol, column: C18 packing, peak elution time: 35 min).

[0038] Example 2: Structural identification of compound 2-thiomethylbutenolide

[0039] Optical rotation (OR) was recorded on a rudolph AUTOPOL IV polarimeter, ultraviolet (UV) spectra were recorded on a UH5300 UV-visible double-beam spectrophotometer, infrared spectra were recorded on an IR Tracer-100 Fourier transform infrared spectrophotometer, 1D and 2D spectra were recorded on a Bruker Avance III 600 MHz spectrometer with TMS as internal standard. CD spectra were recorded using a Chirascan-Plus CD spectrometer (Applied Photophysics), and HR-ESI-MS was recorded using an ultra-high performance liquid chromatography (HPLC) system and a Q-Exactive HF mass spectrometer. Silica gel (200-300 mesh and 500-800 mesh, Qingdao Marine Chemical Inc., Qingdao, China), RP-18 gel (20-45 μm, Fuji Silysia Chemical Ltd, Kasugai, Japan) and Sephadex LH-20 (Amersham Biosciences, Upsala, Sweden) were used for column chromatography. Medium pressure liquid chromatography was performed on a Biotage Isolera one system using an RP-18 column. Preparative high performance liquid chromatography was performed using an Agilent 1260 liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) with a Zorbax SB-C18 column (5 μm, 9.4 x 150 mm) and a Daicel chiral column (AS-H, 5 μm, 4.6 x 250 mm) and a diode array detector. Thin layer chromatography (Qingdao Marine Chemical Co., Ltd., China) was used to monitor fractions, and compounds were observed after spraying silica gel plates with 10% sulfuric acid solution and heating.

[0040] The chemical structure of compound 2-thiomethylbutenolide is shown in Figure 1 .

[0041] Mass spectral analysis of compound 2-thiomethylbutenolide is shown in Figure 2 .

[0042] NMR analysis of compound 2-thiomethylbutenolide is shown inFigures 3-8 and shown in Table 1.

[0043] Table 1. Activity of 2-thiomethylbutenolide 1 H (600 MHz) and 13 C (150 MHz) NMR analysis of 2-thiomethylbutenolide

[0044]

[0045]

[0046] Example 3: Analysis of the ability of compound 2-thiomethylbutenolide to inhibit tumor cell proliferation

[0047] The effect of 2-thiomethylbutenolide on cell proliferation was evaluated using sulforhodamine B staining for a variety of tissue-derived tumor cells, such as colon cancer cells HCT116 and Caco2. Cells in the logarithmic growth phase were seeded in 96-well plates (5000 cells / well, 100 μl / well). After overnight attachment growth, 100 μl / well (concentration gradient experiment) of fresh medium containing the drug was added, 2 wells per dose, and incubated at 37°C, 5% CO2for 48 hours. 50 ml of cold 50% (w / v) trichloroacetic acid (TCA) was added to each well and fixed at 4°C for 1 hour. The fixing solution was discarded and the wells were rinsed with distilled water at low flow rate for 5 times and dried naturally in air. 100 μl of 0.4% (w / v) SRB solution was added to each well and stained at room temperature for 10 minutes. The supernatant was discarded and rinsed with 1% acetic acid for 5 times to remove the non-specifically bound dye and dried naturally in air. 200 μl of 10 mM Tris solution (pH 10.5) was added to each well. The OD value was measured at 515 nm wavelength after shaking for 5 minutes. The fitted curve of drug concentration versus relative cell number was plotted and the 50% inhibition concentration (IC50) was calculated. The results showed that 2-thiomethylbutenolide had inhibitory activity for both colon cancer cells. Figure 9 , Table 2).

[0048] Table 2. Activity of 2-thiomethylbutenolide to inhibit the growth of colon cancer cells (IC50, μM)

[0049]

[0050] As a control, the butenolide compound of structure (I) and the butenolide compound of structure (II) were tested as the drug / concentration variable factor by the method of this example. The results showed that the butenolide compound of structure (I) 2-thiomethylbutenolide had stronger inhibitory effect on the proliferation of tumor cells (colon cancer cells HCT116 and Caco2). This was related to the different mechanisms of regulating apoptosis. The butenolide compound of structure (II) regulated apoptosis by inducing p53 protein, while the butenolide compound of structure (I) was not found to induce p53 protein to regulate apoptosis in the study of this patent.

[0051]

[0052] Example 4: Analysis of the ability of compound 2-thiomethylbutenolide to inhibit tumor cell clonogenicity

[0053] Colon cancer cells HCT116 were seeded in a 12-well plate at 1000 cells / well, and after 24 hours, different concentrations of 2-thiomethylbutenolide were added, and cultured for 14 days. The culture medium was discarded, and washed once with PBS, and 2 ml of 0.2% crystal violet solution (PBS containing 3.7% paraformaldehyde) was added. Incubation was performed at room temperature for 20 minutes, and washed with PBS for 3 times. The PBS was discarded, and dried, and the colonies were counted. The colony formation inhibition rate was calculated by the following formula: Figure 10 It can be seen that 2-thiomethylbutenolide can inhibit the clonogenicity of tumor cells.

[0054] Example 5: Analysis of the ability of compound 2-thiomethylbutenolide to induce tumor cell apoptosis

[0055] HCT116 in the logarithmic growth phase was seeded in a 96-well plate at 7500 cells per well. After adherent growth culture for 24 hours, compound 2-thiomethylbutenolide and Annexin V and Syto green dyes were added. After 24 hours of treatment, the cells were imaged using ImageXpress Pico. The imaging results showed that after treatment with 2-thiomethylbutenolide, the phosphatidylserine on the inner side of the outer cell membrane lipid bilayer was stained red, which was a clear feature of early apoptosis, indicating that 2-thiomethylbutenolide promoted tumor cell apoptosis. Figure 11 Western blot analysis of apoptosis-related proteins also showed that the related proteins increased with the increase of drug concentration. Figure 12

[0056] ​The above merely provides the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the scope of the present application.

Claims

1. A butenolide compound, characterized by, The structure is shown as formula (I): 。 2. A method for producing the butenolide compound according to claim 1, characterized by, Comprising: Culturing marine fungus Aspergillus terrestris in a culture medium containing inorganic sulfur; Extracting the culture medium with ethyl acetate to obtain an extract; Separating and purifying the extract to obtain butenolide compounds shown as formula (I).

3. The method of claim 2, wherein, Comprising: The culture medium is a seed culture medium of the marine fungus Aspergillus terrestris.

4. The method of claim 2, wherein, Separating and purifying the extract to obtain butenolide compounds shown as formula (I), comprising: Refluxing the extract under reduced pressure to obtain total extract; Separating the total extract by silica gel column chromatography to obtain a separation liquid; After eluting the separation liquid with petroleum ether-ethyl acetate system 20:1, repeatedly eluting by MCI column chromatography-Sephadex LH-20-ODS column chromatography to obtain butenolide compounds shown as formula (I).

5. The method of claim 4, wherein, Eluting by MCI column chromatography with 85% methanol as the mobile phase, Sephadex LH-20 with methanol as the mobile phase, and ODS with 60% methanol as the mobile phase to obtain butenolide compounds shown as formula (I).

6. Use of the butenolide compounds of claim 1 or the butenolide compounds prepared by the method of any one of claims 2 to 5 in the preparation of an antitumor drug.

7. Use according to claim 6, characterized in that, The tumor is colon cancer.

8. An antitumor agent, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The raw material component of the drug contains the butenolide compounds of claim 1 or the butenolide compounds prepared by the method of any one of claims 2 to 5 or a salt thereof.

9. The medicament according to claim 8, characterized in that, The drug further contains a pharmaceutically acceptable excipient.

10. The medicament according to claim 9, characterized in that, The drug is an oral preparation or a non-oral preparation. The structure is shown as formula (I): Comprising: Culturing marine fungus Aspergillus terrestris in a culture medium containing inorganic sulfur; Extracting the culture medium with ethyl acetate to obtain an extract; Separating and purifying the extract to obtain butenolide compounds shown as formula (I). Comprising: The culture medium is a seed culture medium of the marine fungus Aspergillus terrestris. Separating and purifying the extract to obtain butenolide compounds shown as formula (I), comprising: Refluxing the extract under reduced pressure to obtain total extract; Separating the total extract by silica gel column chromatography to obtain a separation liquid; After eluting the separation liquid with petroleum ether-ethyl acetate system 20:1, repeatedly eluting by MCI column chromatography-Sephadex LH-20-ODS column chromatography to obtain butenolide compounds shown as formula (I). Eluting by MCI column chromatography with 85% methanol as the mobile phase, Sephadex LH-20 with methanol as the mobile phase, and ODS with 60% methanol as the mobile phase to obtain butenolide compounds shown as formula (I).

6. Use of the butenolide compounds of claim 1 or the butenolide compounds prepared by the method of any one of claims 2 to 5 in the preparation of an antitumor drug. The tumor is colon cancer. The raw material component of the drug contains the butenolide compounds of claim 1 or the butenolide compounds prepared by the method of any one of claims 2 to 5 or a salt thereof. The drug further contains a pharmaceutically acceptable excipient. The drug is an oral preparation or a non-oral preparation.

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