A polyketone alkaloid hybrid compound, and a preparation method and application thereof

Abstract

The present application relates to a kind of polyketide alkaloid hybrid compound and its preparation method and application, wherein, the polyketide alkaloid hybrid compound is selected from at least one of the compounds with the structural formula shown in formula I.The compound 1~6 structure includes an atypical angular anthracene ring polyketide ring and open-loop ternary nitrogen ring, both are novel compounds of skeleton.The polyketide alkaloid hybrid compound of the present application has the activity of inhibiting neuraminidase and influenza virus H1N1 and H3N2, and can be used for preparing anti-influenza virus drugs;Meanwhile, it also has the activity of resisting bacteria and actinomycetes, and can be used for preparing antibacterial agent, with good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a polyketide alkaloid hybrid compound, its preparation method, and its application. Background Technology

[0002] Natural products and their derivatives have long been a treasure trove of promising drug candidates and lead compounds. Despite numerous challenges in this field, such as long commercialization times, high costs, and high compound repetition rates, their contributions to anti-infection research have sparked intense interest among scientists. Recent bioinformatics studies have revealed the enormous potential of microorganisms in synthesizing secondary metabolites, particularly those from rare or untapped microbial sources. Notably, several types of compounds, including alkaloids, terpenes, polyketides, peptides, and polyethers, have been reported to date, all exhibiting strong activity. However, bioinformatics indicates that 71% of secondary metabolites remain unexplored.

[0003] Kibdosporagium is a typical genus of filamentous actinomycetes, widely distributed in various ecosystems including deserts, oceans, terrestrial mines, rhizosphere soils, plants, and lichens, and is believed to possess multifaceted ecological and biological functions. However, there are currently few reports on research into the secondary metabolites of this organism. Summary of the Invention

[0004] The technical problem to be solved by this invention is: This invention provides a polyketide alkaloid hybrid compound, its preparation method and application. This invention studies the active components produced by the fermentation of marine actinomycete Kibdelosporangium sp. XY-R10, and obtains six polyketide alkaloid hybrid compounds with different chemical structures by using various column chromatography and high performance liquid chromatography. These compounds can be used in the preparation of anti-influenza virus and antibacterial drugs.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a polyketone alkaloid hybrid compound, wherein the polyketone alkaloid hybrid compound is selected from at least one of compounds having the structural formula shown in Formula I;

[0007]

[0008] This invention proposes a polyketide alkaloid hybrid compound, wherein compounds 1-6 of Formula I comprise an atypical horn-anthracene polyketide ring and an open-ring three-membered nitrogen ring, all of which are novel skeletal structures. This compound exhibits inhibitory activity against neuraminidase and influenza viruses H1N1 and H3N2, and can be used to prepare anti-influenza virus drugs. Simultaneously, it also possesses antibacterial and actinomycete activities, and can be used to prepare antibacterial agents, demonstrating promising application prospects.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned polyketone alkaloid hybrid compound, comprising the following steps:

[0010] S1. Preparation of fermentation broth for marine actinomycete Kibdelosporangium sp. XY-R10;

[0011] S2. Extract the fermentation broth with ethyl acetate and concentrate it to obtain an ethyl acetate extract;

[0012] S3. The ethyl acetate extract was subjected to reversed-phase column chromatography for fractionation, and gradient elution was performed in a water-methanol system with a volume ratio of 9:1 to 0:10. The components were tracked and combined by thin-layer chromatography and high-performance liquid chromatography to obtain 10 crude components F1-F10.

[0013] S4. F5 obtained in a water-methanol system with a volume ratio of 5:5 was separated by silica gel column chromatography, followed by gradient elution with a chloroform-methanol system with a volume ratio of 0:1 to 1:0, and then purified by semi-preparative liquid chromatography to obtain the polyketide alkaloid hybrid compound.

[0014] This invention utilizes various column chromatography and liquid chromatography techniques to isolate polyketide alkaloid hybrid compounds with Formula I from the fermentation products of the actinomycete Kibdelosporangium. This preparation method has the advantages of stability, reliability, and high efficiency, and can lay a good theoretical foundation for the subsequent research on unknown active ingredients in the secondary metabolites of natural organisms.

[0015] Optionally, in step S1, the preparation method of the fermentation broth specifically includes the following steps: inoculating marine actinomycete Kibdelosporangium sp. XY-R10 into ISP2 agar medium and culturing it at 28°C for 7 days to obtain a plate containing the inoculum; then inoculating the inoculum on the plate into ISP2 agar medium and culturing it at 28°C with rotation at 200 rpm for 3 days to obtain a seed culture; then inoculating the seed culture at a rate of 5% into TXY medium and culturing it at 28°C with rotation at 225 rpm for 7 days to obtain the fermentation broth.

[0016] Optionally, the TXY culture medium comprises: 20g soluble starch, 20g glycerol, 20g Peptone, 20g artificial sea salt, 5g CaCO3, and 1L water.

[0017] Optionally, in step S2, the concentration is reduced pressure concentration.

[0018] Optionally, in step S4, the separation and purification conditions for the semi-preparative liquid chromatography are as follows: the chromatographic column is C10 ... 18 The column was used with a detection wavelength of 254 nm, a flow rate of 3 mL / min, and a mobile phase of acetonitrile-water with a volume ratio of 27:73.

[0019] Thirdly, the present invention provides the application of the above-mentioned polyketone alkaloid hybrid compound or the polyketone alkaloid hybrid compound obtained by the above preparation method in the preparation of anti-influenza virus drugs.

[0020] Fourthly, the present invention provides the application of the above-mentioned polyketone alkaloid hybrid compound or the polyketone alkaloid hybrid compound obtained by the above preparation method in the preparation of antibacterial drugs.

[0021] The polyketide alkaloid hybrid compound obtained by this invention can be effectively used to prepare anti-influenza virus drugs and antibacterial drugs, expanding the medicinal and commercial value of the actinomycete Kibdelosporangium, and bringing significant economic and social benefits. Attached Figure Description

[0022] Figure 1 The ECD spectrum of the polyketide alkaloid hybrid compound prepared in Example 1 of this invention;

[0023] Figure 2 This is an X-ray single-crystal diffraction pattern of the polyketide alkaloid hybrid compound prepared in Example 1 of the present invention. Detailed Implementation

[0024] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the present invention can be understood more clearly and thoroughly, and that the scope of the present invention can be fully conveyed to those skilled in the art.

[0025] Example 1

[0026] A method for preparing a polyketide alkaloid hybrid compound includes the following steps:

[0027] S1. Prepare the fermentation broth of marine actinomycete Kibdelosporangium sp. XY-R10.

[0028] Specifically, the steps include: inoculating marine actinomycete Kibdelosporangium sp. XY-R10 into ISP2 agar medium and culturing it at 28°C for 7 days to obtain a plate containing the inoculum; then inoculating the inoculum on the plate into ISP2 agar medium and culturing it at 28°C with rotation at 200 rpm for 3 days to obtain a seed culture; then inoculating the seed culture at a rate of 5% into TXY medium and culturing it at 28°C with rotation at 225 rpm for 7 days to obtain the fermentation broth.

[0029] The TXY culture medium consists of: 20g soluble starch, 20g glycerol, 20g Peptone, 20g artificial sea salt, 5g CaCO3, and 1L water.

[0030] S2. Extract the fermentation broth three times with an equal volume of ethyl acetate, concentrate under reduced pressure to obtain an ethyl acetate extract, weighing 30g.

[0031] In this embodiment, the ethyl acetate extract is further analyzed by mass spectrometry (MS), and the secondary mass spectrometry (MS / MS) data is submitted to the global network mass spectrometry platform (https: / / gnps.ucsd.edu / ProteoSAFe / static / gnps-splash.jsp) for data visualization using Cytoscape 3.9.0.

[0032] S3. The ethyl acetate extract was subjected to reverse-phase column chromatography for fractionation, with gradient elution in a water-methanol system with a volume ratio of 9:1 to 0:10. The fractions were tracked and combined by thin-layer chromatography and high-performance liquid chromatography, the elution solvent was recovered, the fractions were evaporated to dryness and transferred with methanol to obtain 10 crude fractions F1-F10.

[0033] S4. Based on the above mass spectrometry analysis results, F5 obtained in a water-methanol system with a volume ratio of 5:5 was separated by silica gel column chromatography, followed by gradient elution in a chloroform-methanol system with a volume ratio of 0:1 to 1:0, and then purified by semi-preparative liquid chromatography to obtain the polyketide alkaloid hybrid compound.

[0034] The separation and purification conditions for the semi-preparative liquid chromatography are as follows: the chromatographic column is C10 ... 18 The column was used with a detection wavelength of 254 nm, a flow rate of 3 mL / min, and a mobile phase of acetonitrile-water with a volume ratio of 27:73.

[0035] The polyketide alkaloid hybrid compound prepared in Example 1 was subjected to nuclear magnetic resonance spectroscopy (Tables 1 and 2) and ECD spectroscopy (Tables 1 and 2). Figure 1 ) and X-ray single-crystal diffraction analysis ( Figure 2The final chemical structures of compounds 1–6 are shown in Formula I below:

[0036]

[0037] Table 1. 1H NMR spectra (δin ppm, J in Hz) of polyketide alkaloid hybrid compounds 1–6

[0038]

[0039] Table 2. Carbon spectra (δin ppm, J in Hz) of polyketide alkaloid hybrid compounds 1–6

[0040]

[0041] The polyketide alkaloid hybrid compound prepared in Example 1 was subjected to anti-influenza virus tests and antibacterial activity tests.

[0042] 1. Anti-influenza virus test

[0043] (I) Detection of Neuraminidase (NA) Inhibitory Activity

[0044] Test method: Mix the NA-containing solution with the sample of the test compound, incubate for 30 min, then add MU-NANA, and detect the fluorescence intensity on a microplate reader. Each compound was measured three times. The change in fluorescence intensity represents the strength of the crude extract activity. The fluorescence intensity change in the experimental group is denoted as I, and the fluorescence intensity change in the blank group is denoted as E. The percentage of enzyme activity inhibition is then calculated as: percentage of enzyme activity inhibition (%) = 1 - I / E.

[0045] Experimental results show that the IC50 of compounds 1–6 in Formula I is [missing information]. 50 The values ​​were 7.25, 15, 15, 7.25, 30, and 30 μg / mL, respectively, indicating that the polyketide alkaloid hybrid compounds shown in Formula I all have certain neuraminidase inhibitory activity.

[0046] (II) Virus Inhibition Test

[0047] Test Method: The antiviral activity of the compound was evaluated by observing the cytopathic effect induced by the virus after treatment. After MDCK cells grew into a monolayer, the culture medium was removed, and the cells were washed twice with Hanks' solution. Then, diluted virus solution was added to achieve a multiplicity of infection (MOI) of approximately 0.01. After incubation at 37°C for 2 hours, the infection solution was removed, and a compound-containing solution prepared with trypsin treated with 1 μg / mL TPCK and 0.3% BSA was added. After culturing for 72 hours, CCK-8 was added to detect cell viability. The absorbance at 450 nm was measured using a microplate reader, and the half-maximal effective concentration (IC50) was calculated. 50 .

[0048] Experimental results show that compound 6 has an IC50 value for H1N1 and H3N2. 50 The values ​​were 2 and 8 μg / mL, respectively, indicating that the compound with this structure has good inhibitory activity against the virus.

[0049] 2. Antibacterial activity test

[0050] Test method: 50 μL of the compound sample solution dissolved in LB broth was mixed with 50 μL of working solution from *Cordyceps militaris* in the logarithmic growth phase, and then continuously diluted using the two-fold dilution method (final concentration series: 0.12–250 μg / mL). After incubation at 37°C for 12 h, the turbidity of the culture medium in each well was observed visually, and the final concentration corresponding to the previous well where the culture medium became turbid was determined as the MIC value of the compound.

[0051] The experimental results showed that the MIC values ​​of compounds 1, 2, and 5 were 128, 64, and 32 μg / mL, respectively, indicating that the above compounds exhibited certain anti-Cyclocarya paliurus activity.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a polyketide alkaloid hybrid compound, characterized in that, The polyketone alkaloid hybrid compound is selected from at least one of compounds having the structural formula shown in Formula I; Formula I; The preparation method of the polyketide alkaloid hybrid compound includes the following steps: S1. Preparation of marine actinomycetes Kibdelosporangium Fermentation broth of sp.XY-R10; S2. Extract the fermentation broth with ethyl acetate and concentrate it to obtain an ethyl acetate extract; S3. The ethyl acetate extract was subjected to reversed-phase column chromatography for fractionation, and gradient elution was performed in a water-methanol system with a volume ratio of 9:1 to 0:

10. The components were tracked and combined by thin-layer chromatography and high-performance liquid chromatography to obtain 10 crude components F1-F10. S4. F5 obtained in a water-methanol system with a volume ratio of 5:5 was separated by silica gel column chromatography, followed by gradient elution with a chloroform-methanol system with a volume ratio of 0:1 to 1:0, and then purified by semi-preparative liquid chromatography to obtain the polyketide alkaloid hybrid compound.

2. The method for preparing the polyketide alkaloid hybrid compound as described in claim 1, characterized in that, In step S1, the preparation method of the fermentation broth specifically includes the following steps: [The text abruptly shifts to a seemingly unrelated topic about marine actinomycetes.] Kibdelosporangium sp.XY-R10 was inoculated into ISP2 agar medium and cultured at 28°C for 7 days to obtain a plate containing the inoculum. The inoculum on the plate was then inoculated into ISP2 agar medium and cultured at 28°C with a rotation speed of 200 rpm for 3 days to obtain the seed culture. The seed culture was then inoculated into TXY medium at a rate of 5% and cultured at 28°C with a rotation speed of 225 rpm for 7 days to obtain the fermentation broth. The TXY culture medium consists of: 20g soluble starch, 20g glycerol, 20g Peptone, 20g artificial sea salt, 5g CaCO3, and 1L water.

3. The method for preparing the polyketide alkaloid hybrid compound as described in claim 1, characterized in that, In step S2, the concentration is reduced pressure concentration.

4. The method for preparing the polyketide alkaloid hybrid compound as described in claim 1, characterized in that, In step S4, the separation and purification conditions for the semi-preparative liquid chromatography are as follows: the chromatographic column is C10 ... 18 The column was used with a detection wavelength of 254 nm, a flow rate of 3 mL / min, and a mobile phase of acetonitrile-water with a volume ratio of 27:73.

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