A class of chain polyene vicinal diol compounds and their preparation method and use

Abstract

The present invention provides a class of chain polyene vicinal diol compounds and their preparation methods and uses. The inventors obtained compounds Aspertrienediols A and B by separating and purifying the liquid fermentation extract of Antarctic Aspergillus sp.SCSIO 05702. Structural analysis showed that compounds 1 and 2 were both new compounds, and their specific structures were shown in formula (I). Through the evaluation of the anti-inflammatory activity of compounds 1 and 2, it was found that the new compound Aspertrienediol B (2) had a significant inhibitory effect on nitric oxide produced by mouse macrophage cell line RAW264.7 induced by lipopolysaccharide (LPS), and simultaneously inhibited the mRNA expression of pro-inflammatory factors such as MCP-1, IL-6 and TNF-α. At the same concentration, it had no cytotoxic activity on RAW264.7 cells, and could be used as a lead compound for the development of anti-inflammatory drugs.

Description

Technical Field

[0001] The invention belongs to the field of marine natural product chemistry, and particularly relates to chain polyene vicinal diol compounds, a preparation method thereof, and application thereof in the preparation of anti-inflammatory drugs. Background Art

[0002] Microorganisms from special marine ecological environments, especially those from extreme environmental stresses such as the polar regions, deep sea, and hydrothermal regions, have evolved special metabolic pathways that can produce metabolites with significant pharmacological activity, and are an important source for new drug research and development. At present, obtaining active secondary metabolites with novel skeletons from microorganisms from extreme environments such as the polar regions has gradually become one of the important directions of natural medicine research. The unique environment and ecological pressures faced by polar microorganisms are the main driving factors for the production of secondary metabolites and the emergence of various bioactive compounds. During the three years from 2018 to 2020, 201 natural products from polar regions were reported, and 94 new compounds were discovered, which also had rich and diverse biological activities. In the process of studying secondary metabolites derived from Antarctic fungi, we isolated a class of novel chain polyene vicinal diol compounds, which have a significant inhibitory effect on nitric oxide production in the mouse macrophage cell line RAW264.7 induced by lipopolysaccharide (LPS), and no obvious cytotoxicity was found at effective doses. At the same time, they inhibited the mRNA expression of pro-inflammatory factors such as MCP-1, IL-6 and TNF-α. This not only provides ideal candidate compounds for screening novel, highly effective and low-toxic anti-inflammatory drugs, but also has important significance for the development and utilization of microbial resources in special environments such as the polar regions. Summary of the Invention

[0003] The first object of the present invention is the chain polyene vicinal diol compounds Aspertrienediols A and B (1 and 2).

[0004] The chain polyene vicinal diol compound of the present invention has a structure as shown in formula (I):

[0005]

[0006] The third object of the present invention is to provide the use of the chain polyene vicinal diol compound represented by formula (I) in the preparation of anti-inflammatory drugs.

[0007] Preferably, the chain polyene vicinal diol compound is compound 2.

[0008] The third object of the present invention is to provide Aspergillus sp. SCSIO 05702, deposited with CGMCC No. 10279.

[0009] The fourth object of the present invention is to provide the use of the above-mentioned Antarctic Aspergillus sp. SCSIO 05702 in the preparation of the above-mentioned chain polyene vicinal diol compounds.

[0010] A fifth object of the present invention is to provide a method for preparing a chain polyene vicinal diol compound, comprising the following steps:

[0011] a) Preparation of a fermentation culture of Aspergillus sp. SCSIO 05702.

[0012] b) separating the fermentation broth and mycelium from the fermentation culture using a large Büchner funnel, extracting the fermentation broth with ethyl acetate, combining the ethyl acetate extracts, and concentrating to obtain extract A; first extracting the mycelium with acetone, combining the extracts, extracting the remaining water mixture after recovering the acetone with ethyl acetate, concentrating the ethyl acetate extracts to obtain extract B, combining extracts A and B to obtain a crude extract; subjecting the crude extract to medium-pressure normal-phase silica gel column chromatography with a dichloromethane / methanol gradient elution from 100:0 to 0:100 to obtain five fractions (fr1-fr5) in this gradient elution sequence, collecting fraction fr1 eluted with a dichloromethane / methanol volume ratio of 100:0; and subjecting the fraction to Sephadex chromatography. LH-20 chromatography purification was performed with dichloromethane / methanol volume ratio of 1:1 as mobile phase to obtain four fractions (fr1-1-fr1-4); fraction fr1-3 was detected at a wavelength of 220 nm and a flow rate of 3 mL / min with acetonitrile:water (25:75, v / v) for isocratic elution and semi-preparative high performance liquid phase separation. HPLC (YMC-pack ODS-A, 10×250 mm, 5 μm) was used to obtain compound 1 (4.6 mg, retention time t R 10.7 min) and compound 2 (4.8 mg, retention time t R 16.1min).

[0013] The fermentation culture of Aspergillus sp. SCSIO 05702 is prepared by the following method: activated Aspergillus sp. SCSIO 05702 is inoculated into a seed culture medium, cultured at 24° C. and 180 rpm for 3 days to obtain a seed solution, the seed solution is inoculated into a fermentation culture medium at an inoculum rate of 5% by volume, and statically cultured at 24° C. for 70 days to obtain a fermentation culture. The formula of the seed culture medium and the fermentation culture medium is as follows: 10 g of soluble starch and 1 g of peptone per liter of culture medium liquid.

[0014] The present inventors isolated and purified the liquid fermentation extract of Aspergillus sp. SCSIO 05702 to obtain compounds Aspertrienediols A and B (1 and 2). Structural analysis revealed that compounds 1 and 2 are both new compounds, with their specific structures shown in formula (I). Evaluation of the anti-inflammatory activity of compounds 1 and 2 revealed that the new compound Aspertrienediol B (2) significantly inhibited lipopolysaccharide (LPS)-induced nitric oxide production in the mouse macrophage cell line RAW264.7, while also inhibiting the mRNA expression of pro-inflammatory factors such as MCP-1, IL-6, and TNF-α. At the same concentration, it showed no cytotoxic activity against RAW264.7 cells, suggesting that it could be used as a lead compound for the development of anti-inflammatory drugs.

[0015] The Aspergillus sp. SCSIO 05702 of the present invention was deposited in the China General Microbiological Culture Collection (CGMCC) on January 6, 2015, with the address: Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, and the deposit number is: CGMCC No. 10279. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 :Chain-like polyene vicinal diol compounds 1 and 2 are mainly 1 H- 1 H COSY, HMBC information;

[0017] Figure 2 :ECD calculated spectra of chain polyene vicinal diol compounds 1 and 2

[0018] Figure 3 : Anti-inflammatory activity of chain polyene vicinal diol compound 2 (from left to right in each column in b are Ctrl, LPS, LPS + compound 2) DETAILED DESCRIPTION

[0019] The present invention is further explained below with reference to the following examples, but the examples do not limit the present invention in any form.

[0020] Example 1: Preparation and structural identification of compounds 1 and 2

[0021] 1. Prepare a fermentation culture of Aspergillus sp. SCSIO 05702.

[0022] Each 1000 mL of culture medium (seed medium and fermentation medium) was prepared as follows: 10 g of soluble starch and 1 g of peptone were dissolved in an appropriate amount of water, the volume was made up to 1000 mL, and the mixture was sterilized at 121°C for 20 minutes before use. Antarctic Aspergillus sp. SCSIO 05702 was inoculated into the above culture medium and cultured at 24°C, 180 rpm, for 3 days to produce a seed solution. This seed solution was then inoculated into the fermentation medium at a volume percentage of 5% and incubated at 24°C for 70 days to produce a static culture fermentation product of Antarctic Aspergillus sp. SCSIO 05702.

[0023] 2. Separation and purification of compounds 1 and 2

[0024] The fermentation culture was separated from the mycelium using a large Buchner funnel, the fermentation broth was extracted with ethyl acetate, the ethyl acetate extracts were combined, and the extracts were concentrated to obtain extract A; the mycelium was first extracted with acetone, the extracts were combined, the remaining water mixture after the extract recovered the acetone was extracted with ethyl acetate, the ethyl acetate extract was concentrated to obtain extract B, and extracts A and B were combined to obtain a crude extract; the crude extract was subjected to medium-pressure normal phase silica gel column chromatography, and dichloromethane / methanol was gradient eluted from 100:0 to 0:100. The gradient elution sequence obtained 5 components (fr1-fr5), and the fraction fr1 eluted with a dichloromethane / methanol volume ratio of 100:0 was collected; the fraction was subjected to Sephadex gel chromatography. LH-20 chromatography purification was performed with dichloromethane / methanol in a volume ratio of 1:1 as the mobile phase to obtain four fractions (fr1-1-fr1-4); fraction fr1-3 (TLC thin layer chromatography Rf value was 0.7, the developing solvent was dichloromethane / methanol = 10 / 1, volume ratio) was detected at a wavelength of 220 nm, a flow rate of 3 mL / min, and acetonitrile: water (25:75, v / v) was used for isocratic elution for semi-preparative high performance liquid separation, HPLC (YMC-pack ODS-A, 10×250 mm, 5 μm), to obtain compound 1 (4.6 mg, retention time t R 10.7 min) and compound 2 (4.8 mg, retention time t R 16.1min).

[0025] Table 1. 500MHz 1 H and 125MHz 13 C NMR spectral data of1 and2 in CD3OD

[0026]

[0027] 3. Structural identification of compounds 1 and 2

[0028] The obtained chain polyene vicinal diol compounds 1 and 2 were subjected to nuclear magnetic resonance (NMR), mass spectrometry (MS), circular dichroism (CD) and other data tests to determine the chemical structures of the compounds.

[0029] Structural identification of new compound 1: light yellow oil, high-resolution mass spectrometry m / z 177.0890 [M+Na] + (calcdfor177.0886) suggests its molecular formula is C9H 14 O2 contains 3 degrees of unsaturation, 1 H and 13 C NMR data are shown in Table 1. 13 C NMR combined with DEPT-135 spectrum suggests that its 9 carbon atom signals include: 1 methyl, 2 sp 3 Hybridized oxymethylenyl, 6 sp 2 Hybridized double bond methine. 2D NMR spectrum 1 H- 1 H COSY( Figure 1 ) confirmed an independent self-selective coupling system of CH2-1 / CH-2 / CH-3 / CH-4 / CH-5 / CH-6 / CH-7 / CH-8 / CH3-9. Key related information of HMBC ( Figure 1 ) (H-5 correlates with C-3 and C-7; H-6 correlates with C-4 and C-8), further confirming the displacement of the linked oxygen-linked vicinal diol fragment at the 5- and 6-positions of the long-chain alkane. BC correlation signals between CH3-9 and C-7 and C-8 confirmed the terminal methyl group at position 9, directly connected to the olefinic carbon C-8. Further ECD quantum chemical calculations confirmed the configuration of compound 1 to be 5S, 6R. A Scifinder search revealed compound 1 to be a novel structure, designated Aspertrienediol A.

[0030] Structural identification of new compound 2: A pale yellow oil was identified. High-resolution mass spectrometry data revealed that compound 2 had an identical molecular formula to compound 1, and 1D NMR data were also nearly identical. Given the presence of only two chiral carbon atoms in the vicinal diol group, compounds 2 and 1 were presumed to be diastereomers. Further 2D NMR data, including COSY, HSQC, and HMBC spectra, confirmed this diastereomerism. ECD quantum chemical calculations confirmed the 5S, 6S configuration of compound 2. A Scifinder search confirmed compound 2 to be a novel structure and named Aspertrienediol B.

[0031] Example 2: Experimental data on the anti-inflammatory activity of chain polyene vicinal diol compounds 1 and 2

[0032] The Griess method was used to detect the effects of chain polyene vicinal diol compounds on NO in RAW264.7 cells. The ELISA kit was used to detect the effects of pentanone thiophene compounds on the levels of proinflammatory factors in RAW264.7 cells. RT-PCR was used to detect the gene expression levels of factors related to the polarization state of macrophages.

[0033] Experimental results ( Figure 3 ) showed that the chain polyene vicinal diol compound Aspertrienediol B (2) can effectively inhibit the trend of LPS-induced proinflammatory cytokine levels (MCP-1, IL-6 and TNF-α), and can also effectively inhibit the mRNA expression of proinflammatory factors such as MCP-1, IL-6 and TNF-α. This is consistent with the inhibitory effect of the chain polyene vicinal diol compound Aspertrienediol B (2) on NO release. At the same concentration, it has no cytotoxic activity against RAW264.7 cells and can be used as a candidate lead compound for the development of anti-inflammatory drugs.

Claims

1. Chain polyene vicinal diol compounds, whose structure is as shown in formula (I) 2. Use of the chain polyene vicinal diol compound according to claim 1 in the preparation of anti-inflammatory drugs.

3. Use of Antarctic Aspergillus sp. SCSIO 05702 in the preparation of chain polyene vicinal diol compounds, wherein the Aspergillus sp. SCSIO 05702 has a deposit number of CGMCC No. 10279; The chain polyene vicinal diol compound is:

4. A method for preparing a chain polyene vicinal diol compound, characterized in that: The following steps are involved: a) preparing a fermentation culture of Aspergillus sp. SCSIO 05702; b) separating the fermentation broth and mycelium from the fermentation culture using a large Buchner funnel, extracting the fermentation broth with ethyl acetate, combining the ethyl acetate extracts, and concentrating to obtain extract A; extracting the mycelium with acetone, combining the extracts, extracting the remaining water mixture after recovering the acetone with ethyl acetate, concentrating the ethyl acetate extracts to obtain extract B, combining extracts A and B to obtain a crude extract; subjecting the crude extract to medium-pressure normal-phase silica gel column chromatography, gradient elution with dichloromethane / methanol from 100:0 to 0:100, and obtaining five fractions in the gradient elution sequence, collecting the fraction fr1 eluted with a dichloromethane / methanol volume ratio of 100:0; and subjecting the fraction to Sephadex chromatography. The product was purified by LH-20 chromatography using a mobile phase of dichloromethane / methanol (volume ratio: 1:1) to obtain four fractions, fr1-1 to fr1-4. Fraction fr1-3 was detected at a wavelength of 220 nm and eluted isocratically with acetonitrile:water (25:75, v / v) at a flow rate of 3 mL / min. Semi-preparative high-performance liquid separation was performed by HPLC (YMC-pack ODS-A, 10×250 mm, 5 μm) to obtain compounds 1 and 2. The chain polyene vicinal diol compound is:

5. The method according to claim 4, characterized in that The fermentation culture of Aspergillus sp. SCSIO 05702 is prepared by the following method: activated Aspergillus sp. SCSIO 05702 is inoculated into a seed culture medium, cultured at 24°C and 180 rpm for 3 days to obtain a seed solution, the seed solution is inoculated into a fermentation culture medium at an inoculum rate of 5% by volume, and statically cultured at 24°C for 70 days to obtain a fermentation culture. The seed culture medium and fermentation culture medium have a formula of 10 g of soluble starch and 1 g of peptone per liter of culture medium liquid.

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