CN111153908A

CN111153908A - Azophilic ketone alkaloid with anti-tumor activity, preparation method and application thereof

Info

Publication number: CN111153908A
Application number: CN202010078082.7A
Authority: CN
Inventors: 王伟毅; 覃江江; 邵宗泽; 杨静
Original assignee: China Ocean Mineral Resources R & D Association (china's Ocean Affairs Administration); Third Institute of Oceanography MNR
Current assignee: China Ocean Mineral Resources R & D Association (china's Ocean Affairs Administration); Third Institute of Oceanography MNR
Priority date: 2020-02-02
Filing date: 2020-02-02
Publication date: 2020-05-15
Anticipated expiration: 2040-02-02
Also published as: CN111153908B

Abstract

The invention discloses a diazotrophone alkaloid with anti-tumor activity, a preparation method and application thereof. The said azaphilone alkaloid has obvious cytotoxicity to gastric cancer cell line.

Description

Azophilic ketone alkaloid with anti-tumor activity, preparation method and application thereof

Technical Field

The invention relates to the field of compounds, in particular to a diazotrophone alkaloid with anti-tumor activity, a preparation method and application thereof.

Background

Malignant tumors have long been a serious threat to human health and life, being a creditable "first killer". According to the world health organization statistics, about 600 million people are killed by cancer every year around the world, and the estimated increase will be 1000 million people by 2020. However, there is still no good drug for clinical use to eliminate cancer. In the clinical use process of many antitumor drugs, the low selectivity of the antitumor drugs is gradually found, and the antitumor drugs have great lethality to normal cells while inhibiting tumor growth, so that strong toxic and side effects are caused. The weak can be manifested as vomiting, fatigue, anorexia, dizziness, and in severe cases, it can cause damage to the heart. Therefore, the search for novel antitumor drugs with novel structures and small toxic and side effects is urgent.

Due to the special living environment, the marine microorganisms form a unique genetic metabolic mechanism and a unique chemical defense mechanism in the evolution process of adapting to the environment, and are the main sources of the current natural products with new structures or new activities. The research of marine anticancer drugs has always played a leading role in the research of marine drugs. 1500 marine products are isolated annually in the United states, 1% of them have anticancer activity, and at least 10 marine anticancer drugs are currently in clinical or preclinical research. In recent years, the activity screening of marine microorganisms is enhanced, and efficient and low-toxicity anticancer drugs are searched, and the drugs are directly used in clinic or used as lead compounds for structural modification, and have become the development trend of marine anticancer drug research. In addition, marine microorganisms are easy to collect and culture, and have the characteristics of large-scale acquisition, environmental friendliness, low application cost and the like, and the development and application prospects of the marine microorganisms are internationally recognized.

Disclosure of Invention

The invention aims to provide a diazotrophone alkaloid with anti-tumor activity.

In order to achieve the aim, the invention provides a diazotrophone alkaloid with anti-tumor activity, which is characterized in that the structural formula is shown as a formula (A),

wherein n is 0 to 5; preferably, n is 0 to 2, more preferably, n is 0 or 1.

Further R₁Is composed of

Wherein 4 'is R configuration, and 5' is R configuration; or

Wherein 4 'is S configuration, 5' is R configuration; or

Or

The invention also provides a preparation method of the azone alkaloid with the anti-tumor activity, which is characterized by comprising the following steps:

preparation of strains: sterilizing at high temperature with seawater PDA culture medium, making into plate, and inoculating activated Chaetomium globosum as strain;

preparing a fermentation seed solution: filling a seawater PDA liquid culture medium into a conical flask, sterilizing at high temperature, inoculating the strain for culture, and taking the culture as a seed solution;

inoculation: preparing a fermentation bottle by adopting a solid fermentation mode, adding a rice solid fermentation culture medium subjected to high-temperature sterilization, inoculating the seed solution, and standing at room temperature for culture;

and (3) extraction: standing at room temperature for 25 days, adding methanol to soak the fermentation product, dissolving the fermentation product in methanol solution, soaking, pouring out the upper layer methanol solution, extracting for 1-4 times per bottle, filtering the obtained extractive solution with filter paper, and concentrating under reduced pressure to solid state to obtain methanol extract; dispersing the methanol extract with distilled water, performing liquid-liquid extraction with ethyl acetate, collecting ethyl acetate part, and concentrating under reduced pressure to dry to obtain ethyl acetate extract;

separation: subjecting the ethyl acetate extract to silica gel column chromatography, and performing gradient elution by using petroleum ether-ethyl acetate as an eluent according to a volume ratio of 95: 5-80: 20 to obtain 4 components which are named as Fr1, Fr2, Fr3 and Fr4 respectively; the components Fr2 to Fr4 are combined, gradient elution is carried out by MCI macroporous resin column chromatography and methanol-water as an eluent according to the volume ratio of 50: 50-100: 0, and then 4 sub-components are obtained and named as SF1, SF2, SF3 and SF4 respectively; wherein the subfraction SF1 is subjected to silica gel column chromatography, reversed phase preparative high performance liquid chromatography and gel column chromatography to obtain compound 2 and compound 3;

separating the second subfraction SF2 by gel column chromatography to obtain two subfractions, which are named as SF2a and SF2b respectively; wherein subfraction SF2a is subjected to reverse phase preparative high performance liquid chromatography to provide Compound 1; subfraction SF2b is subjected to reverse phase preparative high performance liquid chromatography to give compound 4;

gradient elution of the third subfraction SF3 through reversed phase C18 column to obtain two subfractions SF3a and SF3 b; wherein subfraction SF3b is separated by reversed phase preparative high performance liquid chromatography to give compound 5 and compound 7;

separating the fourth subfraction SF4 by reverse phase preparative high performance liquid chromatography and gel column chromatography to obtain compound 6 and compound 8; the compounds 1-8 are the azone alkaloid with anti-tumor activity.

Further, the Chaetomium globosum is Chaetomium globosum MP 4-S01-7.

Optionally, the seawater PDA culture medium comprises 200g of potato, 20g of glucose, 15-20g of agar and 1L of seawater;

the rice solid fermentation medium comprises 70g of rice, 2g of D-glucose, 1g of maltose, 1g of mannitol, 0.3g of peptone, 0.1g of corn flour, 0.1g of sodium glutamate and 100ml of seawater, and the pH is adjusted to 6.5.

Further, in the step of inoculating: 70g of rice, 2g of D-glucose, 1g of maltose, 1g of mannitol, 0.3g of peptone, 0.1g of corn flour, 0.1g of sodium glutamate and 100ml of seawater are added into a 1L triangular conical flask, and the pH is adjusted to 6.5.

Further, the extraction is that after standing and culturing for 25 days at room temperature, 500mL of methanol solution is added into each bottle to soak the fermentation product, the fermentation product is dissolved in the methanol solution, after soaking, the upper layer of methanol solution is poured out, each bottle is extracted for 3 times, the obtained extracting solution is filtered by filter paper, and the filtering solution is concentrated to a solid state under reduced pressure to obtain methanol extract; dispersing the methanol extract with 1L of distilled water, performing liquid-liquid extraction with ethyl acetate, collecting ethyl acetate part, and concentrating under reduced pressure to dry to obtain ethyl acetate extract;

optionally, in the separation step, petroleum ether-ethyl acetate is used as an eluent, and gradient elution is performed from 95:5 to 80:20 in a volume ratio of 95:5, 90:10, 85:15 and 80:20 in sequence.

Further, in the separation step, silica gel column chromatography is eluted with petroleum ether and acetone, wherein the ratio of petroleum ether: the volume ratio of acetone is 9: 1;

optionally, SephadexLH-20 is used as a filler and methanol is used as an elution solvent in a gel column chromatography method;

optionally, the reverse phase C18 column gradient elution is from 50% methanol to 100% methanol;

reversed phase preparative high performance liquid chromatography is eluted with acetonitrile and flow rate of 8 ml/min; wherein 75% acetonitrile is used for the separation of the subfraction SF 1; 88% acetonitrile was used for the separation of subfraction SF2a and subfraction SF3 b; subfraction SF4 and subfraction SF2b were separated using 70% acetonitrile.

The invention also protects the application of the azophilic alkaloid and the azophilic alkaloid prepared by the preparation method in preparing anti-tumor drugs.

Further, the anti-tumor drug is a drug for preventing and treating gastric cancer.

The invention also provides a pharmaceutical preparation, which is characterized by comprising the azophilic alkaloid.

Further, the preparation is pills, granules, tablets, capsules or injections.

The compound has obvious cytotoxicity to two gastric cancer cell lines MGC803 and AGS. Can be used for preparing medicine for preventing gastric cancer.

Drawings

FIG. 1 shows compounds 1 to 4 in^aThe measurement is carried out at the frequency of 600MHz,^btabulated nuclear magnetic resonance data measured at 850 MHz.

FIG. 2 shows compounds 5 to 8 in^aThe measurement is carried out at the frequency of 600MHz,^btabulated nuclear magnetic resonance data measured at 850 MHz.

FIG. 3 is a table showing the results of the effects of compounds 1-8 on human gastric cancer cell activity.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1: fermentation of strain ChaetomiumMP4-S01-7

(1) Preparation of strains: sterilizing at high temperature with seawater PDA culture medium, making into plate, and inoculating activated Chaetomium globosum MP4-S01-7 at normal temperature as strain; the components of the seawater PDA culture medium are 200g of potatoes, 20g of glucose, 15-20g of agar and 1L of seawater;

the deposit information of Chaetomium globosum MP4-S01-7 is as follows:

and (4) strain preservation: chaetomium globosum MP4-S01-7,

the preservation unit: the China Center for Type Culture Collection (CCTCC) is,

and (4) storage address: the university of Wuhan and Wuhan in China,

the preservation number is: the CCTCC No. M2019509,

the preservation date is as follows: 7/3/2019.

(2) Preparing a fermentation seed solution: respectively filling seawater PDA liquid culture medium into a plurality of conical flasks, sterilizing at high temperature, inoculating the strain, and culturing to obtain seed solution;

(3) inoculation: preparing a fermentation bottle by adopting a solid fermentation mode, adding a rice solid fermentation culture medium, sterilizing at high temperature, inoculating the seed solution, and standing and culturing at room temperature; the rice solid fermentation medium comprises 70g of rice, 2g of D-glucose, 1g of maltose, 1g of mannitol, 0.3g of peptone, 0.1g of corn flour, 0.1g of sodium glutamate and 100ml of seawater (pH is adjusted to 6.5) in each 1L of triangular pyramid bottle.

Example 2: extraction and separation of azaphilone alkaloids

(1) And (3) extraction: standing and culturing at room temperature for 25 days, adding 500mL of methanol solution per bottle, soaking the fermentation product, dissolving the fermentation product in the methanol solution, soaking overnight, pouring out the upper layer of methanol solution to obtain extractive solution, extracting for 3 times per bottle, filtering the extractive solution with filter paper, and concentrating under reduced pressure to solid state to obtain methanol extract; dispersing the methanol extract with 1L distilled water, performing liquid-liquid extraction with ethyl acetate, collecting ethyl acetate part, and concentrating under reduced pressure to dry to obtain ethyl acetate extract.

(2) Separation: extracting an extract by using ethyl acetate, performing silica gel column chromatography, and performing gradient elution by using petroleum ether-ethyl acetate as an eluent according to a volume ratio of 95: 5-80: 20 to obtain 4 components (Fr1, Fr2, Fr3 and Fr4) (detecting by using TLC, combining the same components, and performing the same procedures in the following steps); combining the components Fr2 to Fr4, performing MCI macroporous resin column chromatography, taking methanol-water as an eluent, and performing gradient elution from a volume ratio of 50: 50-100: 0 to obtain 4 sub-components (SF1, SF2, SF3 and SF 4). Fraction SF1 was subjected to silica gel column chromatography (petroleum ether/acetone 9/1), reverse phase preparative high performance liquid chromatography (eluting with 75% acetonitrile and 8 ml/min) and gel column chromatography (sephadex lh-20 as a packing and methanol as an eluting solvent) to give compound 3 and compound 2; the component SF2 is separated by gel column chromatography (SephadexLH-20 as filler and methanol as elution solvent) to obtain two sub-components (SF2a and SF2 b). Fraction SF2a was prepared by reverse phase preparative high performance liquid chromatography eluting with 88% acetonitrile and a flow rate of 8 ml/min to give compound 1; fraction SF2b was prepared by reverse phase preparative high performance liquid chromatography eluting with 70% acetonitrile and a flow rate of 8 ml/min to give compound 4. Fraction SF3 was gradient eluted through reverse phase C18 column chromatography (50% methanol to 100% methanol) to give two subfractions SF3a and SF3b, where fraction SF3b was separated by gel column chromatography (sephadex lh-20 as packing, methanol as eluting solvent) and reverse phase preparative high performance liquid chromatography (elution with 88% acetonitrile and 8 ml/min flow rate) to give compound 5 and compound 7. Fraction SF4 was separated by reverse phase preparative high performance liquid chromatography (eluting with 70% methanol and 8 ml/min) and gel column chromatography (SephadexLH-20 as packing, methanol as eluting solvent) to give compound 6 and compound 8.

Example 3: structural analysis of Azotone alkaloids

The structure of the azaphilone alkaloids is determined based on their mass spectrometry, nuclear magnetic resonance data analysis.

Compound 1

A random dark red powder; nuclear magnetic data are shown in FIG. 1(^aMeasured at 600 MHz.^bMeasured at 850 MHz); molecular formula C₃₃H₄₂ClNO₅High resolution mass spectrum HRESIMS (positive) M/z 568.2835[ M + H ]]⁺(calcd forC₃₃H₄₃ ³⁵ClNO₅,568.2830). The structure is as follows:

compound 2

A random dark red powder; the nuclear magnetic data are shown in figure 1; molecular formula C₃₃H₄₂ClNO₅High resolution mass spectrum HRESIMS (positive) M/z 568.2851[ M + H ]]⁺(calcd for C₃₃H₄₃ ³⁵ClNO₅,568.2830). The structure is as follows:

compound 3

Irregular deep red colorPowder; the nuclear magnetic data are shown in figure 1; molecular formula C₂₈H₃₄ClNO₅High resolution mass spectrum HRESIMS (positive) M/z 500.2199[ M + H ]]⁺(calcd for C₂₈H₃₅ ³⁵ClNO₅,500.2204). The structure is as follows:

compound 4

A random dark red powder; the nuclear magnetic data are shown in figure 1; molecular formula C₂₈H₃₄ClNO₅High resolution mass spectrum HRESIMS (positive) M/z 500.2197[ M + H ]]⁺(calcd for C₂₈H₃₅ ³⁵ClNO₅,500.2204). The structure is as follows:

compound 5

A random orange powder; the nuclear magnetic data are shown in FIG. 2; molecular formula C₃₃H₄₀ClNO₄High resolution mass spectrum HRESIMS (positive) M/z 550.2711[ M + H ]]⁺(calcd for C₃₃H₄₁ ³⁵ClNO₄,550.2724). The structure is as follows:

compound 6

A random orange powder; the nuclear magnetic data are shown in FIG. 2; molecular formula C₂₈H₃₂ClNO₄High resolution mass spectrum HRESIMS (positive) M/z 482.2102[ M + H ]]⁺(calcd for C₂₈H₃₃ ³⁵ClNO₄,482.2098). The structure is as follows:

compound 7

A random dark red powder; nuclear magnetic numberAs seen in FIG. 2; molecular formula C₃₁H₃₈ClNO₄High resolution mass spectrum HRESIMS (positive) M/z 524.2597[ M + H ]]⁺(calcd for C₃₁H₃₉ ³⁵ClNO₄,524.2856). The structure is as follows:

compound 8

A random dark red powder; the nuclear magnetic data are shown in FIG. 2; molecular formula C₂₆H₃₀ClNO₄High resolution mass spectrum HRESIMS (positive) M/z 456.1935[ M + H ]]⁺(calcd for C₂₆H₃₁ ³⁵ClNO₄,456.1942). The structure is as follows:

example 4: antitumor activity of a diazotrophone alkaloid

(1) Culturing of tumor cell lines: human gastric cancer cell lines MGC803 and AGS were both at 37 ℃ and 5% CO₂Cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin under humid conditions.

(2) Cytotoxic effects of compounds 1-8 on human gastric cancer cell lines: the effect of compounds 1-8 on gastric cancer cell viability was determined using the cell counting kit 8(CCK8) assay. Taxol (paclitaxel) was used as a positive drug. MGC803 and AGS cells were seeded in 96-well plates (2-3X 10, respectively)³Cells/well) overnight, treated with different concentrations (0.1-10 μ M) of compound 1-8 for 72 hours, incubated with CCK8 solution at room temperature for 3 hours, absorbance measured at 450nm, and IC calculated₅₀The value is obtained. The results are shown in FIG. 3.

As can be seen in FIG. 3, the compound was significantly cytotoxic to both cell lines, except for the IC of compound 8 for AGS₅₀In addition to values greater than 10, IC's of other compounds on both cells₅₀All values are less than 10. mu.M. Wherein the IC of the

compounds

1,2,5₅₀The values are all less than 1 μ M, and the most potent antitumor activity is shown.Furthermore, compound 2 exhibited similar inhibitory effects on both MGC803 and AGS cell lines, whereas the cytotoxic effects of

compounds

1 and 5 on AGS were more pronounced.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims

1. A diazotrophone alkaloid with anti-tumor activity is characterized in that the structural formula is shown as a formula (A),

wherein n is 0 to 5; preferably, n is 0 to 2, more preferably, n is 0 or 1.

2. The azaphilone alkaloid having anti-tumor activity of claim 1, wherein R is₁Is composed of

Wherein 4 'is R configuration, and 5' is R configuration; or

Wherein 4 'is S configuration, 5' is R configuration; or

3. A process for the preparation of the azone alkaloid with antitumor activity according to claim 1 or 2, characterized in that it comprises the following steps:

separating the fourth subfraction SF4 by reverse phase preparative high performance liquid chromatography and gel column chromatography to obtain compound 6 and compound 8;

the compounds 1-8 are the azone alkaloid with anti-tumor activity.

4. The method for preparing azophilic alkaloid with anti-tumor activity as claimed in claim 3, wherein Chaetomium globosum is Chaetomium globosum MP 4-S01-7;

5. The method for preparing a diazotrophic alkaloid having antitumor activity according to claim 3, characterized in that, in the inoculation step: 70g of rice, 2g of D-glucose, 1g of maltose, 1g of mannitol, 0.3g of peptone, 0.1g of corn flour, 0.1g of sodium glutamate and 100ml of seawater are added into a 1L triangular conical flask, and the pH is adjusted to 6.5.

6. The method for preparing a diazotrophone alkaloid with antitumor activity according to claim 3, wherein the extraction is that after standing and culturing for 25 days at room temperature, 500 mL/bottle of methanol solution is added to soak the fermentation product, the fermentation product is dissolved in the methanol solution, after soaking, the upper layer of methanol solution is poured out, each bottle is extracted for 3 times, the obtained extracting solution is filtered by filter paper, and the obtained extracting solution is concentrated to a solid state under reduced pressure to obtain a methanol extract; dispersing the methanol extract with 1L of distilled water, performing liquid-liquid extraction with ethyl acetate, collecting ethyl acetate part, and concentrating under reduced pressure to dry to obtain ethyl acetate extract;

7. The method for producing an antitumor agent-containing azaphilone alkaloid according to claim 3, wherein the separation step comprises silica gel column chromatography eluting with petroleum ether and acetone, wherein the ratio of petroleum ether: the volume ratio of acetone is 9: 1;

8. Use of the azone alkaloid of claim 1 or 2 and the azone alkaloid prepared by the preparation method of any one of claims 3 to 7 in the preparation of antitumor drugs; preferably, the anti-tumor drug is a drug for preventing and treating gastric cancer.

9. A pharmaceutical formulation comprising the azophilic alkaloid of claim 1.

10. The pharmaceutical formulation of claim 9, wherein the formulation is a pill, granule, tablet, capsule or injection.