CN111499649A

CN111499649A - Benzodifuranone compound with anti-tumor activity, preparation method and application thereof

Info

Publication number: CN111499649A
Application number: CN202010439985.3A
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: 2020-05-22
Filing date: 2020-05-22
Publication date: 2020-08-07
Anticipated expiration: 2040-05-22
Also published as: CN111499649B

Abstract

The invention discloses a benzodifuranone compound with antitumor activity, a preparation method and application thereof. The benzodifuranone compound has obvious cytotoxicity on a gastric cancer cell line.

Description

Benzodifuranone compound with anti-tumor activity, preparation method and application thereof

Technical Field

The invention relates to the field of compounds, in particular to a benzodifuranone compound with antitumor activity, a preparation method and application thereof.

Background

Cancer is a general term for a large group of malignant tumors. Cancer cells are characterized by unlimited and endless proliferation, so that nutrients in a patient body are greatly consumed; the cancer cells release various toxins, so that a series of symptoms are produced by a human body; cancer cells can also be transferred to various parts of the whole body to grow and reproduce, which causes emaciation, weakness, anemia, inappetence, fever, serious organ function impairment and the like.

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.

Chemical drug therapy is an important clinical means for treating cancer, and the development of antitumor drugs focuses on the research of targeting drugs. In the process of tumor development, a plurality of cancer promoting transcription factors play an important role in regulating the gene expression of cancer cells, and provide important molecular targets for developing novel cancer treatment drugs. Wherein the signal transduction and transcription activator 3 (signaltranducers and activators of transcription 3(STAT3)) is one of STAT family members and can regulate gene transcription initiated by a tyrosine kinase activated membrane receptor. Aberrant activation of STAT3 protein may lead to abnormal proliferation and apoptotic dysfunction of cells and promote transformation of normal cells into malignant cells. Therefore, the development of STAT 3-targeted inhibitors is becoming a hot spot for the development of cancer treatment drugs by international bio-corporation.

The antitumor mechanisms of STAT3 inhibitors mainly include: (1) inhibits the formation of STAT 3-STAT 3 dimer; (2) inhibition of STAT3 phosphorylation; (3) inhibits binding of STAT3 to DNA within the nucleus. The STAT3 inhibitor comprises natural products, peptides, peptidomimetics and small molecule inhibitors; the natural product STAT3 inhibitor has a complex structure and is less researched; peptides and peptidomimetics have good activity, but the development of clinical drugs is difficult due to low bioavailability in vivo; therefore, the small molecule inhibitor with the advantages of good activity, ideal physicochemical property and pharmacokinetic property is a hotspot of STAT3 inhibitor research.

Disclosure of Invention

The invention aims to provide a benzodifuranone compound AtrA with anti-tumor activity.

In order to achieve the purpose, the invention provides a benzodifuranone compound Aspeterreurone A with antitumor activity, which is characterized in that the structural formula is shown as a formula (A),

the invention also provides a preparation method of the benzodifuranone compound Aspeterreurone A with the antitumor 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 Aspergillus fungus as strain at normal temperature;

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; the rice solid fermentation medium comprises 70g of rice, 0.3g of peptone, 0.1g of sodium glutamate and 100ml of seawater;

and (3) extraction: standing and culturing at room temperature for 20-30 days, adding ethyl acetate solution, soaking the fermentation product, dissolving the fermentation product in the ethyl acetate solution, pouring out the upper layer of ethyl acetate 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 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: 5-70: 30 to obtain 9 components which are respectively named as components F1-F9; the component F3 is subjected to gel column chromatography to obtain 7 sub-components which are respectively named as sub-components SF3-1 to SF 3-7; subfractions SF3-7 were separated by preparative high performance liquid chromatography to give the compound benzodifuranone compound AtrA.

Further, the Aspergillus fungus is Aspergillus terreus CC-S06-18;

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

the rice solid fermentation medium comprises 70g of rice, 0.3g of peptone, 0.1g of sodium glutamate and 100ml of seawater; preferably seawater from the beach of white city, mansion city.

Further, the extraction is that after standing and culturing for 25 days at room temperature, ethyl acetate solution 500m L/bottle is added to soak the fermentation product, the fermentation product is dissolved in the ethyl acetate solution, after soaking, the upper layer ethyl acetate 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, so that ethyl acetate extract is obtained;

optionally, in the separation step, petroleum ether-ethyl acetate is used as an eluent, and gradient elution is performed from 95:5 to 70:30 in a volume ratio of 95:5, 92:8, 90:10, 88:12, 85:15, 83:17, 80:20, 75:25 and 70:30 in sequence.

Further, in the separation step, Sephadex L H-20 is used as a filler in a gel column chromatography, and methanol is used as an elution solvent;

optionally, the high performance liquid chromatography is a gradient elution from 20% acetonitrile to 70% acetonitrile for 20 minutes at a flow rate of 10 ml/min.

Further, the benzodifuranone compound Aspeterreurone A is used for preparing antitumor drugs; preferably, 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 benzodifuranone compound Aspeterreurone A.

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

The invention determines marine-derived benzodifuranone (Aspertrereurone A, short for AttA) as a novel STAT3 inhibitor. Meanwhile, the benzodifuranone compound AtrA is found to have cytotoxic effect on gastric cancer cell lines and can induce cell cycle arrest and apoptosis. Further studies have shown that the benzodifuranones, AtrA, inhibit STAT3 phosphorylation and induce cleavage of PARP proteins. Overall, the benzodifuranone compound AtrA represents a novel class of STAT3 inhibitors with potential for development as a drug for the treatment of human cancer, particularly gastric cancer.

Drawings

FIG. 1 is a graph showing the effect of compound AtrA on cell cycle progression in human gastric cancer cells.

FIG. 2 is a graph showing the effect of compound AtrA on apoptosis of human gastric cancer cells.

FIG. 3 is a graph showing the statistical results of the compound AtrA inducing apoptosis in human gastric cancer cells.

FIG. 4 is a graph showing the effect of compound AtrA on phosphorylation of STAT 3.

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: extraction and separation of benzodifuranones

Preparing strains, namely using a seawater PDA culture medium, sterilizing at high temperature, preparing a flat plate, placing the flat plate at a normal temperature, and inoculating activated Aspergillus terreus CC-S06-18 as strains, wherein the seawater PDA culture medium comprises 200g of potatoes, 20g of glucose, 15-20g of agar and 1L of seawater;

the deposited information of Aspergillus terreus CC-S06-18 is as follows:

and (4) strain preservation: aspergillus terreus CC-S06-18,

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. M2019793,

the preservation date is as follows: 10 and 10 months and 10 days in 2019.

Preparing fermented seed liquid by respectively filling seawater PDA liquid culture medium (potato 200g, glucose 20g, 1L seawater) into multiple conical flasks, sterilizing at high temperature, inoculating the above strain, and culturing to obtain seed liquid;

inoculating, namely 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 culturing, wherein the rice solid fermentation culture medium comprises 70g of rice, 0.3g of peptone, 0.1g of sodium glutamate and 100ml of seawater (collected from Baicheng beach of Xiamen city) in each 1L triangular pyramid bottle;

and (3) extraction, namely after standing and culturing for 25 days at room temperature, adding an ethyl acetate solution of 500m L/bottle to soak the fermentation product, dissolving the fermentation product in the ethyl acetate solution, pouring out the upper ethyl acetate solution after soaking, extracting for 3 times in each bottle, filtering the obtained extracting solution by using filter paper, and concentrating under reduced pressure to a solid state to obtain an ethyl acetate extract.

Separating, namely extracting an extract from ethyl acetate, performing silica gel column chromatography, performing gradient elution by using petroleum ether-ethyl acetate as an eluent according to the volume ratio of 95:5, 92:8, 90:10, 88:12, 85:15, 83:17, 80:20, 75:25 and 70:30, and eluting 1L in each ratio to obtain 9 components (components F1 to F9), performing gel column chromatography (Sephadex L H-20 is used as a filler and methanol is used as an elution solvent) on the component F3 to obtain 7 subfractions (subfractions SF3-1 to SF3-7), and performing high performance liquid chromatography (gradient elution is performed for 20 minutes from 20% acetonitrile to 70% acetonitrile and the flow rate is 10 ml/minute) on the subfraction SF3-7 to obtain the compound (1.8mg and the retention time is 16.5 minutes).

Example 2: structural analysis of benzodifuranones

The structure of the benzodifuranones is determined based on mass spectrometry and nuclear magnetic resonance data analysis.

A random dark red powder; the nuclear magnetic data are shown in the table (measured at 600 MHz); molecular formula C₂₀H₁₈O₄High resolution mass spectrum HRESIMS (positive) M/z 345.1106[ M + Na ]]⁺(the theoretical formula is C)₂₀H₁₈O₄Na, theoretical molecular weight 345.1103).

The structure is as follows:

the nuclear magnetic data are shown in table 1.

TABLE 1 nuclear magnetic data Table

Position	δ_C,Type	δ_H(J in Hz)
			2	88.0,CH	4.48,qd(6.5,4.5)
2-CH₃	21.0,CH₃	1.34,d(6.5)
			3	45.0,CH	3.18,qd(6.9,4.5)
3-CH₃	19.4,CH₃	1.31,d(6.9)
			4	108.6,C
4-CH₃	11.0,CH₃	2.18,s
			5	146.3,C
6	112.6,C
			7	135.3,C
8	142.9,C
			9	133.5,C
1'	166.7,C
			2'	123.5,C
3'	142.0,CH	8.23,s
			4'	136.6,C
5'	132.4,CH	8.13,dd(8.2,1.9)
			6'	129.1,CH	7.46,m
7'	131.1,CH	7.45,m

Example 3: functional test of benzodifuranone Compound AtrA

1. AtrA cytotoxic assay

The CCK8 assay was used to determine the effect of compound AtrA on the viability of the gastric cancer cell lines HGC27, MGC803, BGC823, AGS and the normal gastric epithelial cell line GES-1 cells.

Culture of human gastric cancer cell lines:

human gastric cancer cell lines MGC803, BGC823, AGS, HGC27 and human normal gastric epithelial cell line GES-1 were obtained from the institute of cell research, national academy of sciences (Shanghai, China). These cells are commercially available or can be purchased from ATCC. All cell lines were cultured in RPMI1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ with 5% CO₂Cultured in an incubator.

Cell viability assay experiment: the gastric cancer cell lines MGC803, BGC823, AGS and HGC27 and the normal gastric epithelial cell line GES-1 were cultured overnight in 96-well plates and then treated with different concentrations of AtrA or paclitaxel. After 72 hours, a CCK8 assay was performed to examine the effect of AtrA on gastric cancer cell activity and absorbance was measured at 450 nm. IC calculation Using Graphpad Prism5 software₅₀。

The results show that the compound has obvious cytotoxic effect on the cancer cell lines and is concentration-dependent, and the IC of the gastric cancer cell lines HGC27, MGC803, BGC823 and AGS₅₀The values were 3.4, 7.0, 6.2, 8.2. mu.M, respectively. GES-1 cell line on Compound AtrA (IC)₅₀29 μ M) was much less sensitive than gastric cancer cell lines, indicating that the compound was selectively cytotoxic to cancer cell lines. However, paclitaxel as a positive control showed strong cytotoxicity, IC, against all cell lines₅₀Values were 8.4, 3.8, 1.3, 53, 7.9nM, respectively.

2. Effect of AtrA on cell cycle distribution

Cell cycle analysis using flow cytometry was used to evaluate the effect of compound AtrA on cell cycle distribution.

MGC803 cells were plated in 6-well plates (1 × 10)⁵Cells/well) were left overnight and then treated with 0, 2, 5, 10, 20 μ M of AtrA for 24 hours, the cells were harvested, washed with pre-cooled PBS and fixed with 95% alcohol overnight at 4 ℃, the fixed cells were incubated with RNase and staining reagent in the dark at 37 ℃ for 30 minutes and measured using a flow cytometer, data analysis was performed using ModFit L T software.

The results are shown in FIG. 1, with the number of cells on the ordinate. 0 in FIG. 1 indicates the effect of AtrA at a concentration of 0. mu.M on the cell cycle progression of human gastric cancer cells; 2 represents the influence of the AtrA concentration of 2. mu.M on the cell cycle progression of human gastric cancer cells, 5 represents the influence of the AtrA concentration of 5. mu.M on the cell cycle progression of human gastric cancer cells, 10 represents the influence of the AtrA concentration of 10. mu.M on the cell cycle progression of human gastric cancer cells, and 20 represents the influence of the AtrA concentration of 20. mu.M on the cell cycle progression of human gastric cancer cells.

As can be seen from FIG. 1, AtrA resulted in a significant increase in MGC803 cells arrested in the G2/M phase at a concentration of 5. mu.M (P <0.05), and a significant decrease in cells in the G2/M phase when the concentration was increased to 20. mu.M (P < 0.05).

Apoptosis assay:

the effect of compound AtrA on MGC803 apoptosis was determined using flow cytometry analysis after Annexin V-FITC/PI staining.

MGC803 cells were seeded in 6-well plates (1 × 10)⁵Cells/well) overnight and treated with AtrA (0, 2, 5, 10, 20 μ M) for 48 hours. Cells were then harvested and washed with pre-cooled PBS. And adding a binding buffer solution and a staining reagent in the FITC Annexin V apoptosis detection kit I to resuspend the cells. Sample analysis was performed using a flow cytometer. The results are shown in FIGS. 2-3. The ordinate of FIG. 2 is the number of cells. 0 indicates the effect of AtrA on MGC803 apoptosis at a concentration of 0 μ M; 2 indicates the effect of AtrA on MGC803 apoptosis at a concentration of 2. mu.M, 5 indicates the effect of AtrA on MGC803 apoptosis at a concentration of 5. mu.M, 10 indicates the effect of AtrA on MGC803 apoptosis at a concentration of 10. mu.M, 20 indicates the effect of AtrA on MGC803 apoptosis at a concentration of 20. mu.MThe effect of apoptosis.

As can be seen in FIGS. 2 and 3, the apoptosis rate of MGC803 cells increased significantly from 2.6% to 9.9% (P <0.001) after 48 hours of treatment with 20. mu.M AtrA.

3. AtrA inhibits STAT3 phosphorylation

Western Blot experiment:

MGC803 cells were seeded in 6 cm petri dishes (3 × 10)⁵Cells/well) overnight, treated with AtrA (0, 2, 5, 10, 20 μ M) for 24 hours, lysed with NP40 buffer containing serine protease and acetylcholinesterase inhibitor, protein quantitated and separated by SDS-PAGE gel, protein bands transferred to PVDF blots, blocked with 5% milk, incubated with primary antibodies β -actin antibody (13E5), PARP antibody (9542), STAT3 antibody (D3Z2G) and pStat3 antibody (Tyr705) (D3a7), respectively, overnight at 4 ℃, membranes washed three times with TBST and incubated with secondary anti-rabbit antibody (#7074) for 2 hours at room temperature, antibody-protein complexes detected using EC L luminescent reagent.

The expression levels of total STAT3 and phosphorylated STAT3(pSTAT3) in MGC803 cells after AtrA treatment were determined using the Western Blot experiment. AtrA significantly reduced the expression level of pSTAT3 starting at a dosing concentration of 2. mu.M. However, the expression of total STAT3 was reduced only at elevated concentrations up to 20 μ M, indicating that AtrA specifically inhibits STAT3 phosphorylation. And it can cause cleavage of PARP protein in a concentration-dependent manner, consistent with the results of apoptosis (fig. 4).

In the current study, the present invention identified a marine-derived benzodifuranone (known as AspeterreuroneA) as a novel STAT3 inhibitor. At the same time, the discovery shows that the AtrA has cytotoxic effect on gastric cancer cell lines and can induce cell cycle arrest and apoptosis. Further studies have shown that AtrA inhibits STAT3 phosphorylation and induces PARP cleavage. Overall, AtrA represents a novel class of STAT3 inhibitors with potential for development as drugs for the treatment of human cancer (particularly gastric cancer).

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 benzodifuranone compound Aspeterreurone A with anti-tumor activity is characterized in that the structural formula is shown as a formula (A),

2. a method for preparing the benzodifuranone compound Aspeterreurone a having antitumor activity according to claim 1, comprising the steps of:

3. The process for preparing the benzodifuranone type compound Asperterreurone A having antitumor activity according to claim 2, wherein the Aspergillus is Aspergillus terreus CC-S06-18;

4. The method for preparing the benzodifuranone compound Asperterreurone A with the antitumor activity according to claim 2, wherein the extraction is that after the mixture is subjected to static culture at room temperature for 25 days, an ethyl acetate solution 500m L/bottle is added to soak a fermentation product, the fermentation product is dissolved in the ethyl acetate solution, after the soaking, the upper ethyl acetate solution is poured out, each bottle is extracted for 3 times, the obtained extracting solution is filtered by using filter paper, and the obtained extracting solution is concentrated to a solid state under reduced pressure to obtain an ethyl acetate extract;

5. The method for preparing the benzodifuranone compound Aspeterreurone A having anti-tumor activity according to claim 2, wherein in the separation step, Sephadex L H-20 is used as a filler, and methanol is used as an elution solvent in gel column chromatography;

6. Use of the benzodifuranone compound Aspeterreurone A of claim 1 and Aspeterreurone A prepared by any of the preparation methods of claims 2-5 in the preparation of antitumor drugs; preferably, the anti-tumor drug is a drug for preventing and treating gastric cancer.

7. A pharmaceutical preparation comprising the benzodifuranone compound aspeterrene a according to claim 1.

8. The pharmaceutical formulation of claim 7, wherein the formulation is a pill, granule, tablet, capsule or injection.