A cyclic peptide compound, its preparation method and application

Abstract

The present invention discloses a cyclic peptide compound, a preparation method and an application thereof. For the first time, a high-yield cyclic peptide compound (I) is isolated from the fermentation product of Fusarium oxysporum, and the yield is 10 mg / L. The cyclic peptide compound of the present invention is produced by microbial liquid fermentation, with simple operation process, low cost and no pollution to the environment. The cyclic peptide compound (I) of the present invention has anti-tumor activity, and its structure can be modified in the later stage to prepare its derivatives to greatly improve its activity, so as to prepare anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to an anti-drug resistant tumor lead compound, in particular to a cyclodextrin compound and a preparation method and application thereof. Background Art

[0002] Malignant tumors seriously threaten human health and have become the second leading cause of death in humans. The research on anti-tumor drugs remains a major area of ​​medical development. There are thousands of biological species in the marine environment, which are a valuable source of new functional proteins and peptides and have been applied to the treatment of many diseases, including cancer.

[0003] Plant endophytic fungi are considered to be one of the richest sources of natural products. They play a potentially important role in the development of new drugs. Due to the special living environment of coastal plants, their endophytic fungi may have different metabolic mechanisms from endophytic fungi from terrestrial and marine plants. Fusarium is one of the three major filamentous fungi. Polyketide synthase, non-ribosomal peptide synthetase and terpene synthase are commonly found in the fungus, which can produce novel and diverse secondary metabolites (alkaloids, peptides, amides, terpenes, quinones and pyrones, etc.). These secondary metabolites generally have potential pharmacological activities, such as antibacterial, anti-tumor, anti-malaria, antioxidant and enzyme inhibition. Summary of the invention

[0004] The purpose of the present invention is to provide a cyclodextrin compound and a preparation method and application thereof. The cyclodextrin compound is synthesized by a biological method, has no pollution to the environment, solves the problem of large-scale production of the compound, and has certain anti-tumor activity, which promotes the discovery of active drug lead compounds.

[0005] The technical solution adopted by the present invention is:

[0006] The present invention provides a cyclodepsipeptide compound represented by formula (I), which has the following chemical structure:

[0007]

[0008] The present invention provides a method for preparing a cyclodextrin compound represented by formula (I), wherein the method is carried out according to the following steps:

[0009] (1) Strain fermentation: Fusarium oxysporum CGMCC No. 17763 was inoculated into GPY medium, and fermented at room temperature (28° C.) in the dark for 10-30 days (preferably 20 days) to obtain a fermentation mixture; the GPY medium composition: 10 g / L glucose, 5 g / L peptone, 2 g / L yeast extract, 2 g / L L-tryptophan, 2 g / L sea salt, the solvent was water, and the pH value was 7.5;

[0010] (2) Compound extraction: Centrifuge the fermentation mixture prepared in step (1) (preferably centrifuge at 20 °C and 4000 rpm for 20 min), take the precipitate, add methanol a, and then perform ultrasonic-assisted extraction. The organic layer is concentrated under reduced pressure until no liquid flows out to obtain a crude extract paste; dissolve all the crude extract paste with methanol b and filter it through a 0.22 μm organic filter head. The filtrate is subjected to isocratic elution using an analytical HPLC column. The mobile phase is acetonitrile and water with a volume ratio of 70:30. Collect the effluent at the 16th minute, remove the solvent, and dry (preferably dry at 25 °C) to obtain the cyclic peptide compound shown in formula (I).

[0011] Preferably, before the fermentation of Fusarium oxysporum CGMCC No. 17763 in step (1), it is first activated and then subjected to seed expansion culture, and then the seed liquid is inoculated into the GPY medium at an inoculation amount of 50 mL / L; the activation and seed expansion culture are carried out according to the following steps: ① Activation culture: Inoculate Fusarium oxysporum CGMCC No. 17763 into the PDA slant medium and culture it in an incubator at 28 °C for 3 - 4 days to activate the strain; the final concentration composition of the PDA slant medium is: glucose 20 g / L, potato 200 g / L, agar 15 - 18 g / L, the solvent is distilled water, and the pH is natural; ② Seed culture: Pick one inoculation loop of the bacterial body from the activated colony in step ① and inoculate it into the PDB seed medium, and shake culture it at 200 rpm and 28 °C for 3 days to obtain the seed liquid; the final concentration composition of the PDB seed medium is: potato 200 g / L, glucose 20 g / L, the solvent is distilled water, and the pH is natural.

[0012] Preferably, the addition amount of methanol a in step (2) is 100 mL / L based on the total volume of the fermentation broth; the addition amount of methanol b is 10 mL / mg based on the weight of the crude extract paste; both methanol a and methanol b are methanol, and they are named to distinguish the different amounts used in different steps, and the letters themselves have no meaning; preferably, ultrasonic extraction is performed 3 times, and the ultrasonic conditions for each time are: ultrasonic at 30 - 50 kHz for 15 - 20 min (preferably 40 kHz and 20 min).

[0013] Preferably, the HPLC conditions in step (2) are: liquid phase instrument: UV - VIS, detector: Shimadzu SPD - 16; HPLC pump: Shimadzu LC - 16P; chromatographic conditions: analytical column, C 18 Column 4.6×250 mm, flow rate 1.0 mL / min, column temperature 40 °C, detection wavelength 209 nm.

[0014] The Fusarium oxysporum ZZP-R1 of the present invention is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The deposit date is May 29, 2019, and the deposit number is CGMCC No. 17763, which has been disclosed in the patent application CN110563740A.

[0015] The present invention also provides the use of a cyclopeptide compound shown in formula (I) in the preparation of a drug for inhibiting the activity of tumor cells, and the tumor cells include the human breast cancer drug-resistant cell line MCF-7 / ADR.

[0016] The present invention also provides the use of a cyclopeptide compound shown in formula (I) in the preparation of an anti-tumor drug.

[0017] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0018] (1) The present invention first isolates a high-yield cyclopeptide compound (I) from the fermentation product of Fusarium oxysporum, and the yield is 10 mg / L.

[0019] (2) The cyclopeptide compound of the present invention is produced by microbial liquid fermentation, and the operation process is simple, the cost is low, and there is no pollution to the environment.

[0020] (3) The cyclopeptide compound (I) of the present invention has anti-tumor activity, and its structure can be modified in the later stage to prepare its derivatives to greatly improve its activity, so as to prepare anti-tumor drugs. Description of the Drawings

[0021] Figure 1 It is the positive ion ESI-MS spectrum of compound (I).

[0022] Figure 2 For compound (I) 1 1H NMR spectrum (CDCl3-d1, 600 MHz).

[0023] Figure 3 For compound (I) 13 13C NMR spectrum (CDCl3-d1, 100 MHz). Detailed Embodiments

[0024] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0025] The room temperature described in the present invention is 25 - 30 °C.

[0026] Example 1: Fermentation Culture of Fusarium oxysporum CGMCC No. 17763

[0027] (1) Activation culture: Fusarium oxysporum CGMCC No. 17763 was inoculated into a PDA slant medium and cultured in an incubator at 28 °C for 3 - 4 days to obtain activated strains; the final concentration composition of the PDA slant medium was: glucose 20 g / L, potato 200 g / L, agar 18 g / L, the solvent was distilled water, and the pH was natural.

[0028] (2) Seed culture: A loopful of bacteria was picked from the activated colony in step (1) and inoculated into a PDB seed medium, and cultured on a shaker at 200 rpm and 28 °C for 3 days to obtain a seed solution; the final concentration composition of the PDB seed medium was: glucose 20 g / L, potato 200 g / L, the solvent was distilled water, and the pH was natural.

[0029] (3) Fermentation culture: The seed solution obtained in step (2) was inoculated into 40 L of GPY medium at an inoculation amount of 50 mL / L, and fermented statically in the dark at room temperature for 20 days to obtain a fermentation mixture. The composition of the GPY medium was: glucose 10 g / L, peptone 5 g / L, yeast extract 2 g / L, L-tryptophan 2 g / L, sea salt 2 g / L, the solvent was water, and the pH was 7.5.

[0030] Example 2: Extraction, separation and identification of cyclopeptide compounds (I)

[0031] 1. Extraction and separation of compound (I)

[0032] All the fermentation mixtures prepared in Example 2 were centrifuged at 20 °C and 4000 rpm for 20 min, the mycelium was filtered out, the precipitate was taken and 4 L of methanol was added (the addition amount of methanol was 100 mL / L based on the total volume of the fermentation mixture), and ultrasonic extraction was carried out three times at 40 kHz, 20 min each time. The organic layers were combined and concentrated under reduced pressure until no liquid flowed out to obtain a crude extract paste; all the crude extract pastes were dissolved in 10 mL of methanol (the addition amount of methanol was 10 mL / mg based on the weight of the crude extract paste) and filtered through a 0.22 μm organic filter head. The filtrate was subjected to isocratic elution using an analytical chromatographic column of high performance liquid chromatography, with acetonitrile: water at a volume ratio of 70:30 as the mobile phase. The effluent at the 16th minute was collected, the solvent was removed by rotary evaporation, and dried at 25 °C to obtain 80 mg of the cyclopeptide compound shown in formula (I).

[0033] Conditions of high performance liquid chromatography:

[0034] Liquid phase instrument: UV-VIS, detector: Shimadzu SPD-16; high performance liquid chromatography pump: Shimadzu LC-16P; chromatographic conditions: analytical chromatographic column, C 18 Chromatographic column 4.6×250 mm, flow rate 1.0 mL / min, column temperature 40 °C, detection wavelength 209 nm, mobile phase was acetonitrile and water at a volume ratio of 70:30.

[0035] 2. Structure Identification of Compound (I)

[0036] (1) White solid, soluble in chloroform and methanol.

[0037] (2) ESI-MS spectrum was detected using a mass spectrometer (LCQ Fleet, Thermo), and the results are shown in Figure 1 ; 1H NMR spectrum was detected using a nuclear magnetic resonance spectrometer (ADVANCE III, Bruker) 1 and the results are shown in Figure 2 ; 13C NMR spectrum was detected using a nuclear magnetic resonance spectrometer (ADVANCE III, Bruker) 13 and the results are shown in Figure 3.

[0038] The mass spectrometry data was: ESI-MS m / z 623.3747 [M+Na] + , and combined with the nuclear magnetic resonance spectrum, the molecular formula of compound (I) was determined to be C 33 H 52 N4O6; the attribution of the nuclear magnetic data is shown in Table 1.

[0039] Table 1: 1 1H-NMR and 13 13C-NMR Nuclear Magnetic Data and Attribution (in CDCl3)

[0040]

[0041]

[0042] In summary, by comparison with the literature (N-Methylsansalvamide, a cytotoxic cyclic depsipeptide from a marine fungus of the genus Fusarium. Phytochemistry 2000, 55, 223-226.), the structural formula of compound (I) was determined to be:

[0043]

[0044] Example 3: Detection of the Antitumor Activity of Cyclic Depsipeptide Compound (I)

[0045] In this experiment, the sulforhodamine B (SRB) colorimetric method was used to conduct an in vitro growth inhibition experiment of tumor cells on the isolated monomeric compound.

[0046] 1. Tumor Cells

[0047] The human breast cancer drug-resistant cell line MCF-7 / ADR was obtained from the Cell Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences.

[0048] Tumor cells in the logarithmic growth phase were selected. After digestion with trypsin, the cell concentration was adjusted to 2×10 4 cells / mL with RPMI 1640 medium containing 10% fetal bovine serum to obtain a cell suspension of tumor cells.

[0049] 2. Drugs and Reagents

[0050] 0.4% SRB solution: Weigh 0.8 g of SRB and dissolve it in 200 mL of 1% acetic acid aqueous solution by volume, and store it at room temperature.

[0051] 50% TCA solution: Weigh 50 g of trichloroacetic acid (TCA), add water to make up the volume to 100 mL, and store it at 4°C.

[0052] 10 mM Tris-base solution: Weigh 0.6057 g of tris(hydroxymethyl)aminomethane (Tris-base), add water to make up the volume to 500 mL, pH 10.5, and store it at 4°C.

[0053] Sample solution: The cyclic peptide compound (I) prepared in Example 2 was formulated into a 100 μg / mL sample solution with dimethyl sulfoxide (DMSO).

[0054] Doxorubicin solution: Doxorubicin was formulated into a 100 μg / mL sample solution with dimethyl sulfoxide (DMSO).

[0055] 3. Tumor Cell Activity Experiment

[0056] The cell suspension of the tumor cells in Table 2 was inoculated into a 96-well culture plate at 190 μL per well and cultured at 37°C and 5% CO2 for 24 h. The culture wells were divided into drug wells, control wells, and blank wells.

[0057] Add 10 μL of sample solution to the drug wells to make the final drug concentration in the culture wells 5 μg / mL; add 10 μL of doxorubicin solution to the control wells to make the final drug concentration in the control wells 5 μg / mL; add 10 μL of 10% fetal bovine serum RPMI 1640 medium containing an equal volume of solvent (DMSO) to the blank wells. Incubate the culture plate at 37°C, 5% CO2 for 3 days, discard the culture medium, gently add 100 μL of 4°C pre-cooled 50% TCA solution to each well, let it stand for 5 minutes, and then move to 4°C for 1 hour to fix the cells. Pour out the fixative, wash with distilled water 5 times to remove TCA, and air dry for 1 hour. Add 80 μL of 0.4% SRB solution to each well and stain at room temperature for 30 minutes. Discard the staining solution, wash with 1% acetic acid aqueous solution 5 times to fully remove unbound SRB, and air dry for 1 hour. 150 μL of 10 mM Trisbase solution was added to each well for dissolution, and the mixture was shaken on a mini shaker (Kylin-Bell Lab instruments) for 5 min. A sample was taken from each well and the OD value at 570 nm was measured using an M5 microplate reader. The tumor cell growth inhibition rate was calculated according to the formula. The results are shown in Table 2.

[0058] Tumor cell growth inhibition rate (%) = (OD 对照 -OD 药物 ) / (OD 对照 -OD 空白 )×100%.

[0059] The results showed that the IC value of compound I against MCF-7 / ADR human breast cancer resistant cell line was 50 The value is 12.56 μM.

[0060] Table 2: Results of in vitro tumor cell growth inhibition experiment of compound I

[0061]

[0062] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that are not conceived through creative work should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope defined in the claims.

Claims

1. A method for preparing a cyclic peptide compound, characterized in that, The method is carried out according to the following steps: (1) Strain fermentation: Fusarium oxysporum CGMCC No. 17763 is inoculated into GPY medium and fermented statically in the dark at room temperature for 10 - 30 days to obtain a fermentation mixture; the composition of the GPY medium: glucose 10 g / L, peptone 5 g / L, yeast extract 2 g / L, L-tryptophan 2 g / L, sea salt 2 g / L, the solvent is water, pH 7.5; (2) Compound extraction: The fermentation mixture prepared in step (1) is centrifuged, the precipitate is added with methanol a and then subjected to ultrasonic-assisted extraction, the organic layer is concentrated under reduced pressure until no liquid flows out to obtain a crude extract paste; all the crude extract paste is dissolved with methanol b and filtered through a 0.22 μm organic filter head, the filtrate is subjected to isocratic elution using a high-performance liquid chromatography analytical column, the mobile phase is acetonitrile and water with a volume ratio of 70:30, the effluent at the 16th minute is collected, the solvent is removed and dried to obtain the cyclic peptide compound shown in formula (I); 2. The preparation method of the cyclic peptide compound according to claim 1, wherein Before the fermentation of Fusarium oxysporum CGMCC No. 17763 in step (1), it is first activated and then subjected to seed expansion culture, and then the seed liquid is inoculated into GPY medium at an inoculation amount of 50 mL / L; the activation and seed expansion culture are carried out according to the following steps: ① Activation culture: Fusarium oxysporum CGMCC No. 17763 is inoculated into PDA slant medium and cultured in an incubator at 28 °C for 3 - 4 days to activate the strain; the final concentration composition of the PDA slant medium: glucose 20 g / L, potato 200 g / L, agar 15 - 18 g / L, the solvent is distilled water, pH natural; ② Seed culture: One inoculation loop of the thallus is picked from the activated colony in step ① and inoculated into PDB seed medium, and cultured on a shaker at 200 rpm and 28 °C for 3 days to obtain a seed liquid; The final concentration composition of the PDB seed medium: potato 200 g / L, glucose 20 g / L, the solvent is distilled water, pH natural.

3. The preparation method of the cyclic peptide compound according to claim 1, wherein, The ultrasonic conditions in step (2) are: ultrasonic at 30 - 50 kHz for 15 - 20 min.

4. The preparation method of the cyclic peptide compound according to claim 1, characterized in that, The high-performance liquid chromatography conditions in step (2) are as follows: liquid phase instrument: UV-VIS, detector: Shimadzu SPD-16; high-performance liquid phase infusion pump: Shimadzu LC-16P; chromatographic conditions: analytical chromatographic column, C 18 chromatographic column 4.6×250 mm, flow rate 1.0 mL / min, column temperature 40 °C, detection wavelength 209 nm.

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