CN112300243A

CN112300243A - Cyclopeptide compound and preparation method and application thereof

Info

Publication number: CN112300243A
Application number: CN202011032764.0A
Authority: CN
Inventors: 吴斌
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-09-27
Filing date: 2020-09-27
Publication date: 2021-02-02
Anticipated expiration: 2040-09-27
Also published as: CN112300243B

Abstract

The invention discloses a cyclopeptide compound and a preparation method and application thereof, belonging to the technical field of marine fungus active ingredient analysis. The invention extracts and separates two cyclic peptide compounds and molecules with novel structures from fermentation culture of marine fungiAre respectively C₂₀H₂₃N₃O₆、C₂₀H₂₃N₃O₅The preparation method comprises the following steps: inoculating activated Talaromyces fungi CX11 into culture solution for fermentation culture; separating to obtain mycelium and fermentation broth, leaching the mycelium with methanol, separating to obtain leaching solution, and extracting the leaching solution with ethyl acetate or chloroform; extracting the fermentation liquor by using ethyl acetate or chloroform, and then concentrating, separating and purifying the extract liquor to obtain the cyclic peptide compound. The cyclic peptide compound has good antitumor and antibacterial activities and has good development prospects in the field of pharmacy.

Description

Cyclopeptide compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of marine fungus active ingredient analysis, in particular to an oxygen-containing bridged cyclic peptide compound with a novel structure and a preparation method and application thereof.

Background

The marine microorganisms can tolerate extreme conditions unique to the sea, such as high salt, high pressure, low oxygen, low light, and the like, and the specificity of the living environment leads to the diversity of the marine microorganisms in species, genetic composition, and ecological functions. Marine fungi in marine microorganisms are abundant sources of active secondary metabolites, and 70-80% of the secondary metabolites of marine fungi have biological activity and comprise small molecule lactone compounds; a mycotoxin; novel substances having inhibitory activity on the central nervous system; 1-dodecanol, unsaturated hydrocarbon, acid, ester; lipopeptide antibiotics which can inhibit plant and human fungal viruses from acting on the cell wall of fungi to synthesize new targets.

The discovery of natural products with specific structure types by taking marine fungi as raw materials has important significance for the research and development of marine drugs. With the progress of the marine microbial resource acquisition technology, unprecedented opportunities are brought to the research of marine microbial source natural drug source compounds.

Patent document CN 108485987 a discloses a marine fungus WH4-2 with antibacterial activity against multiple pathogenic bacteria is separated from bottom sediment in the sea area near the wei hai, and identified as a basophilus by ITS full sequence analysis, the fungus has high-efficiency anti-vibrio parahaemolyticus activity, and the yield of an active substance, namely isopentenyl-containing dibenzoxepin compounds, is high, and is expected to be applied to the development of novel anti-vibrio parahaemolyticus drugs.

Few studies are currently conducted on the chemical composition of the Talaromyces fungus (Talaromyces). The Kunming plant institute of Chinese academy of sciences separated 5 alpha, 6 alpha-epoxy-24 (R) -methyl cholesteric-7, 22-diene-3 beta-alcohol, quinizarine, bryonic acid and m-methyl phenol from Taxus yunnanensis endophytic fungus Talaromyces sp.TIBF in 2011, and 5 alpha, 6 alpha-epoxy-24 (R) -methyl cholesteric-7, 22-diene-3 beta-alcohol separated from Cordyceps sinensis under activity tracking such as Bok (1999) has good antitumor activity (Lilianggang, Yanyan light, Zengying, Cheng, Pepper, Guangxi plant, Taxus plant endophytic fungus Talaromyces sp.TIBF chemical composition research, 2011,31(5), pp 699-701).

The prophase of the subject group separates and obtains Talaromyces sp.CX11 fungus CX11 from marine plants, and extracts a heteroterpene compound which has a novel structure and good antiviral activity to pseudorabies virus from the fungus CX11 fungus, see patent document CN 110452247A.

However, no report has been found on the isolation of an anti-tumor oxacyclopeptide compound from fungi of the genus Talaromyces.

Disclosure of Invention

The invention aims to extract natural active substances with medicinal value from marine Talaromyces fungi (Talaromyces).

In order to achieve the above object, the present invention provides a cyclopeptide compound, the structural formula of which is shown in formula (i):

wherein R is OH or H.

The two compounds are cyclic peptide compounds with an oxygen-bridged cyclic depsipeptide structure separated from Talaromyces fungi CX 11. A cyclic peptide compound of R ═ OH is herein designated compound 1, of formula C₂₀H₂₃N₃O₆The cyclic peptide compound of R ═ H is denoted as compound 2, and the molecular formula is C₂₀H₂₃N₃O₅。

The invention also provides a preparation method of the cyclic peptide compound, which comprises the following steps:

(1) the preservation number is CCTCC NO: activating M2018338 Talaromyces purpurogenus CX11(Talaromyces purpurogenus. CX11), inoculating into culture solution, and statically culturing at 20-30 deg.C for 10-40 days;

the culture solution comprises the following raw materials in a volume of 1L: 1-5g of starch, 10-20g of bran, 3-15g of yeast extract and KH₂PO₄ 1-8g，MgSO₄·7H₂0.1-0.8g of O and the balance of water;

or, the culture solution comprises the following raw materials in a volume of 1L: 600g of potato 200-; the initial pH value of the culture solution is 6.0-7.0;

or, the culture solution comprises the following raw materials in a volume of 1L: 10-40g of cane sugar, 5-20g of corn flour and NaNO₃1-4g, 1-4g yeast extract and KH₂PO₄ 0.2-0.8g、MgSO₄·7H₂O 0.2-1g、KCl 0.2-1g、FeSO₄0.001-0.005g and the balance of water.

(2) After the fermentation culture is finished, mycelium and fermentation liquor are obtained through separation;

(3) adding the mycelium into methanol for leaching, separating to obtain a leaching liquor, concentrating the leaching liquor, suspending the leaching liquor with distilled water to obtain an aqueous suspension, extracting the aqueous suspension with ethyl acetate or chloroform, concentrating, separating and purifying the extract to obtain a cyclic peptide compound;

or extracting the fermentation liquor by using ethyl acetate or chloroform, concentrating the extract liquor, separating and purifying to obtain the cyclic peptide compound;

the separation and purification comprises the following steps: a. and (3) carrying out normal phase silica gel column chromatography separation on the extract liquor, and sequentially carrying out the following steps of: 1. 7:1, 6:1, 5:1, 4:1, 3: 1. carrying out gradient elution on the petroleum ether/ethyl acetate mixed solution and ethyl acetate in a ratio of 2:1, 1:3 and 1:9, and collecting fractions eluted from the petroleum ether/ethyl acetate mixed solution in a volume ratio of 4: 1; b. subjecting the collected fraction to reverse phase silica gel column chromatography, gradient eluting with methanol/water mixed solution at volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and collecting 6:4 fraction; c. separating the collected 6:4 fraction by using an SEC chromatographic column LH-20, using methanol as a mobile phase to obtain a target fraction, and then separating by using high performance liquid chromatography, wherein the mobile phase is a methanol/water mixed solution with a volume ratio of 55:45, the flow rate is 10mL/min, the peak of the retention time of 41 minutes is the cyclic peptide compound with R ═ OH, and the peak of the retention time of 46 minutes is the cyclic peptide compound with R ═ H.

In the step (1), Talaromyces sp.CX11 is subjected to fermentation culture on the Talaromyces fungus CX 11.

The Talaromyces sp.CX11 fungus CX11 is a fungus, and can be used for fermentation culture by using a conventional PDA liquid culture medium or a wort culture medium. In order to increase the yield and provide sufficient nutrients for the growth and metabolism of the microorganism, the culture solution preferably comprises the following raw materials in a volume of 1L: 3g of starch, 14g of bran, 6g of yeast extract and KH₂PO₄5g，MgSO₄·7H₂0.4g of O and the balance of water;

or, the culture solution comprises the following raw materials in a volume of 1L: 400g of potato, 6g of peptone, 2g of yeast extract, 10g of glucose and the balance of water;

or, the culture solution comprises the following raw materials in a volume of 1L: 25g of cane sugar, 10g of corn flour and NaNO₃2g, 2g of yeast extract and KH₂PO₄ 0.5g，MgSO₄·7H₂O 0.5g，KCl 0.5g， FeSO₄0.001g, and the balance being water.

The static culture mode is not to carry out shake flask culture. Preferably, the temperature of the fermentation culture is 22-26 ℃. More preferably, the culture is carried out at 25 ℃ for 20 days.

And (2) separating to obtain mycelium and fermentation liquor, wherein the cyclic peptide compound can be obtained by extraction and separation from the mycelium and the fermentation liquor.

Wherein, when the mycelium is used for obtaining the cyclopeptide compound, the mycelium is soaked in methanol for 7-14 days, the mycelium is fully broken, and intracellular substances are effectively dissolved out.

When the cyclic peptide compound is obtained by using the fermentation liquor, the fermentation liquor and diatomite are stirred and then extracted by adopting ethyl acetate reflux.

The separation and purification are as follows: separating the extractive solution with normal phase silica gel column chromatography, and separating the obtained fraction with reverse phase silica gel column chromatography and high performance liquid chromatography. The cyclic peptide compound with higher purity can be obtained by multi-step separation and purification.

The research of the invention shows that the cyclopeptide compound obtained by separating from the fermentation culture of Talaromyces sp.CX11 (Talaromyces sp. 11) by the method has better anti-tumor activity and antibacterial activity, so the invention also provides the application of the cyclopeptide compound in preparing anti-tumor drugs or antibacterial drugs. In particular, the tumor is cervical cancer; the pathogenic bacteria are staphylococcus aureus, especially methicillin-resistant staphylococcus aureus.

The medicine is prepared by taking the cyclopeptide compound as a main active ingredient and adding pharmaceutically acceptable auxiliary materials, and can be prepared into preparations according to preparation methods recorded in pharmaceutics. The preparation can be injection, infusion solution, powder for injection, granule, tablet, granule, powder, oral liquid, sugar coated tablet, film coated tablet, enteric coated tablet, buccal preparation, granule, pill, unguent, pill, spray, dripping pill, disintegrant, orally disintegrating tablet, pellet, etc.

The invention has the following beneficial effects:

(1) the invention utilizes the polarity difference of the cyclic peptide to extract and separate the cyclic peptide compound with a novel structure from the fermentation culture of the marine fungi, and the method has simple and convenient operation, high extraction yield and high product purity and is suitable for large-scale production.

(2) In vitro anti-tumor tests show that the cyclopeptide compound provided by the invention has better anti-tumor activity and aims at tumor cells Hela and IC of the compound 1₅₀Value of 21.2. mu.M, IC of Compound 2₅₀The value is 32.8 mu M, can be used for preparing anti-tumor drugs and has good development prospect.

(3) In-vitro antibacterial tests show that the cyclopeptide compound provided by the invention has better in-vitro antibacterial activity, aiming at methicillin-resistant staphylococcus aureus, the MIC value of the compound 1 is 4 mu M, the MIC value of the compound 2 is 4 mu M, the cyclopeptide compound can be used for preparing anti-infective drugs, and the development prospect is good.

Drawings

FIG. 1 shows the structural formulas of two cyclic peptide compounds of the present invention.

FIG. 2 shows Compound 1 of the present invention¹H NMR spectra (in CD)₃OD)。

FIG. 3 is a drawing showing a scheme of Compound 1 of the present invention¹³C NMR spectra (in CD)₃OD)。

FIG. 4 is a drawing showing Compound 2 of the present invention¹H NMR spectra (in CD)₃OD)。

FIG. 5 shows Compound 2 of the present invention¹³C NMR spectra (in CD)₃OD)。

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.

Strain basketball fungus CX11(Talaromyces purpurogenus. cx11) was isolated from large marine plants in the coast of zhejiang and was deposited in the chinese type culture collection at 6 and 4 months in 2018 at the deposit address: wuhan, Wuhan university, China; the preservation number is as follows: CCTCC NO: m2018338.

EXAMPLE 1 fermentation culture of Talaromyces fungi

Preparing activated Talaromyces fungus into spore suspension, inoculating into culture solution, and performing static fermentation culture at 25 deg.C for 20 days.

Wherein, the formula of the culture solution is as follows: 3g of starch, 14g of bran, 6g of yeast extract and KH₂PO₄5g， MgSO₄·7H₂O0.4 g and water 1000 mL.

Example 2 fermentation culture of Talaromyces fungi

Preparing activated Talaromyces fungus into spore suspension, inoculating into culture solution, and performing static fermentation culture at 24 deg.C for 20 days.

Wherein, the formula of the culture solution is as follows: 400g of potato, 6g of peptone, 2g of yeast extract, 10g of glucose and 1000mL of water; the initial pH of the broth was 6.5.

EXAMPLE 3 fermentation culture of Talaromyces fungi

Wherein, the formula of the culture solution is as follows: 25g of cane sugar, 10g of corn flour and NaNO₃2g, 2g of yeast extract and KH₂PO₄ 0.5g，MgSO₄·7H₂O 0.5g，KCl 0.5g，FeSO₄0.001g of water, 1000 mL.

EXAMPLE 4 preparation of Cyclic peptide Compounds

After fermentation culture of Talaromyces fungi, taking 5L of fermentation culture solution, centrifuging, and taking precipitate to obtain mycelium; soaking mycelia in methanol for 1 week, concentrating, suspending with 1L distilled water, mixing the water suspensions, extracting with 6L ethyl acetate, and concentrating the ethyl acetate extractive solution to obtain extract 10 g; mixing with silica gel (100 mesh, 100g), separating by normal phase silica gel column chromatography (200 mesh, 300 mesh, 100 g; silica gel column size L50 mm,

) Gradient elution is sequentially carried out on petroleum ether/ethyl acetate mixed solution and ethyl acetate in the volume ratio of 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:3 and 1:9, and each time is 300 mL; detecting fractions by TLC; the 4:1 fraction was collected and recrystallized from methanol to obtain crude crystals a of mainly Compound 1. The mother liquor is continuously recrystallized to obtain crude crystals b mainly of the compound 2. The crude crystals a and b were recrystallized from methanol to obtain pure compound 1 and pure compound 2, respectively.

EXAMPLE 5 preparation of Cyclic peptide Compounds

After fermentation culture of Talaromyces fungi, taking 5L of fermentation culture solution, centrifuging, and taking supernatant to obtain fermentation solution; concentrating the fermentation liquid, mixing with 10g of diatomite, refluxing with 1L of ethyl acetate, performing normal phase silica gel column chromatography separation (200 meshes, 300 meshes, 1 kg; silica gel column size L50 mm,

) Gradient elution is sequentially carried out on petroleum ether/ethyl acetate mixed liquor and ethyl acetate in the volume ratio of 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:3 and 1:9, and 4:1 fractions are collected; subjecting the collected fraction to reverse phase silica gel column chromatography, gradient eluting with methanol/water mixture at volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and collecting 6:4 fraction; reverse phase chromatography 6:4 separating with SEC chromatography LH-20 (methanol as solvent, separating to obtain 7 fractions), collecting the 3 rd fraction, separating with high performance liquid chromatography, wherein the mobile phase is methanol/water mixture with volume ratio of 55:45, flow rate is 10mL/min, and retention time is 41 minThe peak of (1) is a cyclic peptide compound of R ═ OH, and the peak of retention time for 46 minutes is a cyclic peptide compound of R ═ H.

Example 6 structural identification of Cyclic peptide Compounds

Performing purity identification on the prepared compound by HPLC, performing structure identification on a sample with the purity of more than 98% by mass spectrometry and nuclear magnetic resonance technology, measuring the nuclear magnetic resonance by using a Bruker AVANCE DRX-600 NMR secctrometer, and using TMS as an internal standard; high resolution mass spectra fticrrms were measured using a Bruker Apex Spectrometer; ESI-MS electrospray ionization mass spectrometry with Bruker Esquire 3000^plusSpectrometer assay.

From the results of one-dimensional NMR analysis (see Table 1) and two-dimensional NMR analysis of the compound, it was found that the compound 1 had the formula C₂₀H₂₃N₃O₆The molecular formula of the compound 2 is C₂₀H₂₃N₃O₅The structure is shown in figure 1:

TABLE 1 NMR data for cyclopeptide compounds

Example 7 analysis of antitumor Activity of Cyclic peptide Compound

Hela cells were cultured in RP-MI 1640 medium containing 10% calf serum, 100IU/mL penicillin and 100 g/mL streptomycin, 1 change every 3 days, and 1 passage every 5 days. The cells were all placed at 37 ℃. Taking logarithmic growth phase cells, diluting to 5 × 10 with RPMI 1640 medium⁴Single cell suspension/mL, seeded in 96-well cell culture plates, 3 wells per concentration, 180. mu.L per well. After incubation in incubator for 12h, 20 μ L of test solution with different concentrations was added to each well of drug group, and blank control group (equal volume of RPMI 1640 medium was used to replace the drug to be tested) and positive control group (5-FU) were set in parallel for 48h of co-culture. mu.L of 1mg/mL MTT solution was added to each well, after further incubation for 4h, the supernatant was aspirated off, and 150. mu.L of dimethyl sulfoxide (DMSO) was added to each well to dissolve the MTT-reduced product sufficiently. Measuring optical density (D) of each drug group and blank group at 492nm wavelength on an enzyme labeling instrument, and calculating to obtain half inhibition of drug on tumor cells according to formulaConcentration (IC)₅₀) And the drug effect is preliminarily evaluated.

IR (%) - (1-mean D value of dosing group/mean D value of control group) × 100%.

As a result of the experiment, IC of Compound 1 was found₅₀Value of 21.2. mu.M, IC of Compound 2₅₀IC of positive control 5-FU with a value of 32.8. mu.M₅₀The value is 22.9 mu M, which shows that the compound has better anti-tumor effect.

EXAMPLE 8 analysis of the antibacterial Activity of Cyclic peptide Compounds

The broth dilution method is adopted for the bacteriostatic activity test. Staphylococcus aureus [ CMCC (B)26003] was selected for the test, using tetracycline as a positive control.

The cultivation of bacteria is performed in a sterile operating table in a sterile room. Adding 2mL of culture solution into a 15mL sterile centrifuge tube, adding 20 mu L of bacterial solution into the culture solution, uniformly mixing, and placing into a constant-temperature incubator at 37 ℃ for respective culture for 20-24 h. Adding 100 mu L of culture solution into each well of a sterile 96-well plate, adding 2 mu L of test sample, adding an appropriate amount of bacteria into the culture solution, diluting the bacteria, adding 100 mu L of diluted bacteria solution into each well, establishing a blank control group, measuring the absorbance value of the 96-well plate of the sample and bacteria solution mixture at 650nm by using a microplate reader, placing the 96-well plate into a constant-temperature incubator at 37 ℃ for culturing for 20-24h, and measuring the absorbance value again.

The MIC value for Compound 1 was calculated to be 4. mu.M, the MIC value for Compound 2 was calculated to be 4. mu.M, and the MIC value for the positive control tetracycline was calculated to be 4. mu.M.

EXAMPLE 9 preparation of cyclopeptide Compound-containing dripping pill preparation

0.5g of cyclopeptide compound and 10.5g of polyethylene glycol-6000 are uniformly mixed, heated and melted, the mixture is moved to drop pills for drip irrigation, the medicine is dropped into liquid paraffin with the temperature of 6-8 ℃, oil is removed, and 300 drop pills are prepared.

EXAMPLE 10 preparation of cyclopeptide Compound-containing lyophilized powder for injection

Taking 0.5g of cyclopeptide compound, 4.5g of glucose, 0.9g of sodium thiosulfate and 1000ml of distilled water, uniformly mixing the components, freeze-drying, and subpackaging 400 to obtain the pharmaceutical composition.

Claims

1. A cyclopeptide compound having the structural formula (I):

wherein R is OH or H.

2. The method for preparing a cyclic peptide compound according to claim 1, comprising the steps of:

the culture solution comprises the following raw materials in a volume of 1L: 1-5g of starch, 10-20g of bran, 3-15g of yeast extract and KH₂PO₄1-8g，MgSO₄·7H₂0.1-0.8g of O and the balance of water;

or, the culture solution comprises the following raw materials in a volume of 1L: 10-40g of cane sugar, 5-20g of corn flour and NaNO₃1-4g, 1-4g yeast extract and KH₂PO₄ 0.2-0.8g、MgSO₄·7H₂O 0.2-1g、KCl 0.2-1g、FeSO₄0.001-0.005g and the balance of water;

(3) adding the mycelium into methanol for leaching, separating to obtain a leaching liquor, concentrating the leaching liquor, suspending the leaching liquor with distilled water to obtain an aqueous suspension, extracting the aqueous suspension with ethyl acetate, chloroform, dichloromethane or diethyl ether, concentrating the extract, separating and purifying to obtain a cyclic peptide compound;

or extracting the fermentation liquor by using ethyl acetate, chloroform, dichloromethane or diethyl ether, concentrating the extract, separating and purifying to obtain the cyclic peptide compound;

the separation and purification comprises the following steps: a. carrying out normal phase silica gel column chromatography separation on the extract, carrying out gradient elution on petroleum ether/ethyl acetate mixed solution and ethyl acetate in volume ratios of 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:3 and 1:9 in sequence, and collecting fraction eluted from the petroleum ether/ethyl acetate mixed solution in volume ratio of 4: 1; b. subjecting the collected fraction to reverse phase silica gel column chromatography, gradient eluting with methanol/water mixture at volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and collecting 6:4 fraction; c. the 6:4 fraction collected by reverse phase chromatography was separated by using an SEC column to obtain a target fraction, which was then separated by high performance liquid chromatography using a methanol/water mixture at a volume ratio of 55:45 as a mobile phase at a flow rate of 10mL/min, and a cyclic peptide compound having a peak of R ═ OH at a retention time of 41 minutes and a cyclic peptide compound having a peak of R ═ H at a retention time of 46 minutes.

3. The method according to claim 2, wherein the culture solution comprises the following raw materials in a volume of 1L: 3g of starch, 14g of bran, 6g of yeast extract and KH₂PO₄ 5g，MgSO₄·7H₂0.4g of O and the balance of water.

4. The method according to claim 2, wherein the culture solution comprises the following raw materials in a volume of 1L: 400g of potato, 6g of peptone, 2g of yeast extract, 10g of glucose and the balance of water.

5. The method according to claim 2, wherein the culture solution comprises the following raw materials in a volume of 1L: 25g of cane sugar, 10g of corn flour and NaNO₃2g, 2g of yeast extract and KH₂PO₄ 0.5g，MgSO₄·7H₂O 0.5g，KCl 0.5g，FeSO₄0.001g, and the balance being water.

6. The method according to claim 2, wherein the fermentation culture in the step (1) is carried out at 25 ℃ for 20 days.

7. The use of the cyclic peptide compound of claim 1 for the preparation of an anti-tumor medicament.

8. The use of claim 7, wherein the neoplasm is cervical cancer.

9. Use of a cyclic peptide compound according to claim 1 for the preparation of an antibacterial medicament.

10. The use of claim 9, wherein the pathogenic bacteria is staphylococcus aureus.