A class of 24-membered macrolide compounds and their preparation method and application

Abstract

The present invention discloses a class of 24-membered macrolide compounds, their preparation methods, and applications. Three macrolide compounds [1-3] with glycosyl side chains were prepared from Bacillus amyloliquefaciens SCSIO 41392. As shown in formula (I), compounds 1-3 are newly reported compounds that can significantly inhibit the production of virulence factors of Pseudomonas aeruginosa. Compounds 1 and 2 can inhibit the PQS quorum sensing system of Pseudomonas aeruginosa and the production of the virulence factor "pyocyanin" regulated by it, while compound 3 can significantly inhibit the production of the virulence factor "siderophore" of Pseudomonas aeruginosa. Therefore, compounds 1-3 can be used to develop antibacterial drugs. The present invention provides new applications for the polar bacterium Bacillus amyloliquefaciens SCSIO 41392 and provides alternative lead compounds for the development of new antibacterial drugs derived from polar microorganisms.

Description

Technical Field

[0001] The present invention belongs to the field of marine natural product chemistry, and particularly relates to macrolide compounds, a preparation method thereof, and application thereof in the preparation of drugs for inhibiting the production of virulence factors of Pseudomonas aeruginosa. Background Art

[0002] Polar regions are already experiencing climate change, such as global warming and increased ice-free periods. Much research has focused on polar microorganisms. Due to their extreme environments, polar marine microorganisms are considered an underexplored source of novel antimicrobial compounds that could address emerging multidrug-resistant pathogens. During our study of microorganisms derived from Arctic deep-sea sediments, we isolated a class of macrolide compounds with sugar side chains that significantly inhibit the formation of Pseudomonas aeruginosa virulence factors, including pyocyanin and siderophores. This further enriches our understanding of polar microbial resources and provides a new candidate resource for the discovery of drugs that inhibit the production of Pseudomonas aeruginosa virulence factors. Summary of the Invention

[0003] The first object of the present invention is a 24-membered macrolide compound (1-3) with a glycosyl side chain.

[0004] The macrolide compound of the present invention has a structure as shown in any one of formula (I)

[0005]

[0006] The inventors isolated and purified a fermentation extract of Bacillus amyloliquefaciens SCSIO 41392, derived from Arctic deep-sea sediments, to obtain macrolide compounds (1-3). Compounds 1-3 are all novel compounds, with their specific structures shown in Formula (I). Evaluation of the biofilm inhibitory activity of the macrolide compounds (1-3) revealed that compounds 1-3 exhibited significant inhibitory effects on Pseudomonas aeruginosa biofilms, quorum sensing systems, and siderophore-mediated virulence regulation systems, suggesting their potential as lead compounds for antimicrobial drug development.

[0007] The second object of the present invention is to provide the use of Bacillus amyloliquefaciens SCSIO 41392 derived from Arctic deep-sea sediments in the preparation of the above-mentioned macrolide compounds.

[0008] The third object of the present invention is to provide the use of a macrolide compound (1-3) as shown in formula (I) in the preparation of a drug for inhibiting the production of virulence factors of Pseudomonas aeruginosa.

[0009] Preferably, the drug is a drug that inhibits the PQS quorum sensing system, pyocyanin and / or siderophore production of Pseudomonas aeruginosa.

[0010] The fourth object of the present invention is to provide Bacillus amyloliquefaciens SCSIO41392, which has a deposit number of GDMCC 65022.

[0011] The fifth object of the present invention is to provide a method for preparing the macrolide compound (1-3) as shown in formula (I), which is prepared and separated from the fermentation product of Bacillus amyloliquefaciens SCSIO 41392.

[0012] Preferably, the specific steps are as follows:

[0013] A. Preparation of fermentation products of Bacillus amyloliquefaciens SCSIO 41392;

[0014] B. The fermentation product was extracted with ethyl acetate, and the ethyl acetate extract was concentrated to obtain a crude extract. The crude extract was subjected to reverse phase medium pressure separation using a H2O-CH3OH system, eluted from 90%:10% to 0%:100% by volume, and fraction fr6 was obtained under a gradient system of methanol and water in a ratio of 75%:25% to 80%:20% by volume. Fraction fr6 was separated by semi-preparative high performance liquid chromatography at a flow rate of 3 mL / min. When the mobile phase was acetonitrile and ultrapure water containing 0.6% formic acid in a ratio of 42:58 by volume, the component with a retention time of 35.5 min was compound 2 The component with a retention time of 13 min was fraction fr6-3, and the component with a retention time of 32.6 min was fraction fr6-6; fraction fr6-6 was separated by semi-preparative high performance liquid chromatography with a flow rate of 3 mL / min, and when acetonitrile and ultrapure water containing 0.8% formic acid were used as the mobile phase in a volume ratio of 35:65, the component with a retention time of 33 min was compound 1; fraction fr6-3 was separated by semi-preparative high performance liquid chromatography, and when acetonitrile and ultrapure water containing 0.6% formic acid were used as the mobile phase in a volume ratio of 39:61, the component with a retention time of 10 min was compound 3.

[0015] Preferably, the fermentation product is prepared by inoculating Bacillus amyloliquefaciens SCS 10 41392 into a culture medium, culturing at 28°C and 180 rpm to obtain a seed solution, inoculating the seed solution into the culture medium at an inoculum rate of 3% by volume, and culturing at 28°C and 180 rpm to obtain a fermentation product of Bacillus amyloliquefaciens SCS 10 41392. The culture medium is prepared as follows per 1000 mL: 10 g of malt extract, 4 g of yeast extract, 0.2% of calcium carbonate, 0.4% of glucose, and 3.2% of sea salt. The preparation method is to add each component to solvent water and sterilize it for later use.

[0016] The present invention discloses a method for preparing macrolide compounds using Bacillus amyloliquefaciens SCSIO 41392, a bacterium derived from Arctic deep-sea sediments, and their use in inhibiting the production of virulence factors of Pseudomonas aeruginosa. The present invention prepares three macrolide compounds [1-3] with glycosyl side chains from Bacillus amyloliquefaciens SCSIO 41392 (as shown in formula (I)). Compounds 1-3 are newly reported compounds that can significantly inhibit the production of virulence factors of Pseudomonas aeruginosa. Compounds 1 and 2 can inhibit the PQS quorum sensing system of Pseudomonas aeruginosa and the production of the virulence factor "psyocyanin" regulated by it, while compound 3 can significantly inhibit the production of the virulence factor "siderophore" of Pseudomonas aeruginosa. Therefore, compounds 1-3 can be used to develop antibacterial drugs. The present invention provides a new application for the polar bacterium Bacillus amyloliquefaciens SCSIO 41392 and an alternative lead compound for developing new antibacterial drugs derived from polar microorganisms.

[0017] The Arctic deep-sea sediment-derived Bacillus amyloliquefaciens SCSIO 41392 of the present invention was deposited on August 20, 2024 in the Guangdong Provincial Microbial Culture Collection Center (GDMCC), 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070, and its deposit number is GDMCC 65022. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Main COSY and HMBC information of macrolide compounds 1-3;

[0019] Figure 2 : Inhibitory effects of macrolide compounds 1 and 2 on the PQS quorum sensing system of Pseudomonas aeruginosa;

[0020] Figure 3 : Inhibitory effects of macrolide compounds 1 and 2 on pyocyanin production by Pseudomonas aeruginosa;

[0021] Figure 4 : Inhibitory effect of macrolide compound 3 on the production of virulence factor siderophore of Pseudomonas aeruginosa. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1: Preparation and structural identification of macrolide compounds

[0024] 1. Prepare a fermentation culture of Bacillus amyloliquefaciens SCSIO 41392 derived from Arctic deep-sea sediments.

[0025] The culture medium (per 1000 mL) is prepared as follows: 10 g malt extract, 4 g yeast extract, 0.2% calcium carbonate, 0.4% glucose, and 3.2% sea salt. The preparation method is to add each component to a water solvent and sterilize it for later use. Bacillus amyloliquefaciens SCSIO 41392 is inoculated into the above-mentioned culture medium and cultured at 28°C, 180 rpm, for 3 days to produce a seed solution. This seed solution is then inoculated into the culture medium at a volume percentage of 3% and cultured at 28°C, 180 rpm, for 7 days to produce a fermentation product of Bacillus amyloliquefaciens SCSIO 41392 in a shaker culture.

[0026] 2. Separation and purification of macrolide compounds (1-3)

[0027] The fermentation product was extracted with ethyl acetate, and the ethyl acetate extracts were combined and concentrated to obtain a crude extract. 43.3 g of the crude extract was dissolved in a small amount of methanol, mixed evenly with 25 g of diatomaceous earth, air-dried, and loaded onto a 100 g column. Reverse-phase medium-pressure separation was performed on a 120 g Spherical C18 column (model SW-5222-120-SP). Separation was accomplished using a H2O-CH3OH system with a 90%:10% to 0%:100% (volume ratio) mobile phase gradient, with a 10% increment of CH3OH followed by a 5% increment of CH3OH over 5 column volumes. Thin-layer chromatography analysis revealed that fractions of similar polarity were pooled and designated as fractions fr1-6. Fraction fr6 was obtained using a methanol:water gradient system with a volume ratio of 75%:25% to 80%:20%. Fraction fr6 was separated by semi-preparative high performance liquid chromatography, with an ODS (5 μm, 10*250 mm) separation column as the stationary phase, a flow rate of 3 mL / min, and a mobile phase of recovered acetonitrile and ultrapure water containing 0.6% formic acid in a volume ratio of 42:58. The component with a retention time of 35.5 min was compound 2, the component with a retention time of 13 min was fraction fr6-3, and the component with a retention time of 32.6 min was fraction fr6-6; Fraction fr6-6 was separated by semi-preparative high performance liquid chromatography, with a Cholesterol column as the stationary phase, a flow rate of 3 mL / min, and a mobile phase of recovered acetonitrile and ultrapure water containing 0.6% formic acid in a volume ratio of 42:58. The component with a retention time of 35.5 min was compound 2, the component with a retention time of 13 min was fraction fr6-3, and the component with a retention time of 32.6 min was fraction fr6-6. The separation column was used as the stationary phase at a flow rate of 3 mL / min, and acetonitrile and ultrapure water containing 0.8% formic acid in a volume ratio of 35:65 were used as the mobile phase. The component with a retention time of 33 min was compound 1. Fraction fr6-3 was separated by semi-preparative high performance liquid chromatography, and under monitoring at wavelengths of 230 nm and 260 nm, a Cholester (5 μm, 10*250 mm) separation column was used as the stationary phase at a flow rate of 3 mL / min. When acetonitrile and ultrapure water containing 0.6% formic acid in a volume ratio of 39:61 were used as the mobile phase, the component with a retention time of 10 min was compound 3.

[0028] Table 1.700MHz 1 H and 175MHz 13 C NMR spectral data of 1-3in CD3OD

[0029]

[0030]

[0031] 3. Structural identification of compounds 1-3

[0032] The obtained macrolide compounds 1-3 were subjected to nuclear magnetic resonance (NMR), mass spectrometry (MS) and other data tests to determine the chemical structures of the compounds.

[0033] New natural product - compound 1 structure identification: light yellow oil, high resolution mass spectrometry m / z 677.3194 [MH] - The molecular formula is suggested to be C 35 H 50 O 13 Contains 11 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 the 17 carbon atom signals include: 12 sp 2 Hybridized double bond methine, 6 sp 3 Hybrid methylene, 1 sp 3 Methyl, 4 oxymethyl groups, 1 methoxy group, 1 lactone carbonyl carbon, 1 sugar fragment and 1 succinic acid fragment. The above NMR data and mass spectrometry data suggest that the structure of compound 1 should be a macrolide compound with a sugar side chain. Figure 1 As shown, combined with the two-dimensional NMR spectrum 1 H- 1 H COSY analysis confirmed the linkage of CH2-8′ and CH2-9′. Key HMBC information (H2-8′ linked to C-7′, H2-6′ linked to C-7′, and H2-9′ linked to C-10′) further confirmed the succinate fragment's attachment to the C-6′ position. A Scifinder search revealed compound 1 to be a novel structure of natural origin and named amylomacrolactine A.

[0034] Structural identification of the new natural product, compound 2: A pale yellow oil was identified. High-resolution mass spectrometry (HRMS) showed that the molecular formula of compound 2 was consistent with that of compound 1. 1D NMR data revealed that compound 2 was nearly identical to compound 1, but with differences in the double bond coupling constants at C-8 and C-9, and C-17 and C-18. This suggests that the double bond configurations at C-8 and C-17 are opposite to those of compound 1. Further 2D NMR data, including COSY, HSQC, and HMBC spectra, confirmed this hypothesis. A Scifinder search confirmed that compound 2 is a novel structure of natural origin and named amylomacrolactine B.

[0035] Structure identification of the new natural product, compound 3: A pale yellow oil. High-resolution mass spectrometry revealed that compound 3 had one less CH2 group in its molecular formula than compound 1 and a molecular weight 14 less than compound 1. 1D NMR data showed that compound 3 was nearly identical to compound 1, but lacked a methoxy group at C-19. Further 2D NMR data, including COSY, HSQC, and HMBC spectra, suggested that the C-19 position should be substituted by a hydroxyl group. Therefore, the C-19 substitution in compound 3 is presumed to be a hydroxyl group, not a methoxy group. A Scifinder search confirmed that compound 3 is a novel structure of natural origin and named amylomacrolactine C.

[0036] The structural formulas of compounds 1, 2, and 3 are shown below:

[0037]

[0038] Example 2: Experiment on the inhibition of virulence factor production of Pseudomonas aeruginosa by macrolide compounds amylomacrolactines AC (1-3)

[0039] 1. Fluorescence reporter screening experiment of PQS system

[0040] (1) ABTGC culture medium preparation

[0041] The following components were prepared in a glass bottle according to 15.1 mM (NH4)2SO4, 33.7 mM Na2HPO4·2H2O, 22 mM K2HPO4, 0.05 mM NaCl, 1 mM MgCl2·6H2O, 100 μM CaCl2·2H2O, 10 μM FeCl3·6H2O, 20% fructose (w / v), and 20% casein hydrolysate (w / v). The mixture was mixed and sterilized in a high-pressure steam autoclave at 121°C for 20 min. After sterilization, the mixture was cooled to room temperature before use.

[0042] (2) Fluorescence reporter system detection

[0043] 0.5 mg of compound 1 and 2 were dissolved in 50 μL of DMSO to form a concentration of 10 mg mL -1 Then, the overnight cultured PQS quorum sensing fluorescent reporter strain Pseudomonas aeruginosa PAO1 / pUCP22::pqsA-gfp strain (J.Med.Chem.2017,60,215-227) was washed twice with PBS and the bacterial solution OD 600 Adjust the density to 0.01 and dilute it into ABTGC culture medium. There are 4 groups in the experiment, namely compound 1, compound 2, DMSO group and blank control group. Take 4 1.5mL EP tubes and add 500μL OD600 Compounds 1 and 2 were then added in turn at a final concentration of 50 μg mL -1 Take a 96-well plate and add each group at a volume of 150 μL / well to the 96-well plate. Then, incubate the 96-well plate in a 37°C incubator for 24 hours. After 24 hours, measure the OD value of each well with a fluorescence microplate reader. 600 The activity of the final reporter system was measured by measuring the OD value of the bacteria. 600 The values ​​were standardized, that is, the GFP value of each group / OD 600 Value, the result is Figure 2 As shown in the figure, compared with the Blank group and DMSO group, the fluorescence intensity values ​​of compound 1 and compound 2 groups were significantly reduced, indicating that compounds 1 and 2 can effectively inhibit the PQS quorum sensing system of Pseudomonas aeruginosa PAO1.

[0044] 2. Pyocyanin formation inhibition experiment

[0045] After adding compounds 1 and 2 (50 μg mL -1 ) or DMSO in 3.5 mL ABTGC medium, and the DO of overnight cultured wild-type Pseudomonas aeruginosa PAO1 and the double knockout strain ΔlasIΔrhlI that knocked out the Las quorum sensing system and rhl quorum sensing system was 4.37 mmol / l. 600 Adjust to 0.01, incubate at 37℃ for 24h, and record the OD of each group of bacterial solution. 600 , and centrifuged at 10000 rpm for 10 min, 3 mL of the supernatant was added to 3 mL of chloroform and vortexed for 10 min, the mixture was allowed to stand for 20 min and then centrifuged at 10000 rpm for 10 min, 3 mL of the blue layer was taken and 0.2 M hydrochloric acid (1.5 mL) was added to extract pyocyanin, the upper pink layer containing pyocyanin was transferred to a 96-well microtiter plate, and the absorbance at 520 nm was read. The OD 520 The data were divided by the final OD 600 value, normalize the data, and finally calculate the OD 520 / OD 600 Multiply by the molar extinction coefficient 17.072 ([OD 520 / OD 60 ]×17.072) is the obtained pyocyanin concentration (μg mL -1 )The result is as follows Figure 3 As shown, the ΔlasIΔrhlI group is the control group, in which the pyocyanin production is almost zero due to the knockout of the quorum sensing system gene. Figure 3As shown, compared with the Blank group and DMSO group, compound 1 and compound 2 groups significantly inhibited the production of virulence factor pyocyanin regulated by the PQS quorum sensing system of Pseudomonas aeruginosa, and the differences were statistically significant.

[0046] 3. Siderophore Formation Inhibition Experiment

[0047] The ability of the strain to form pyoverdine virulence factors was tested using the minimal culture medium ABTGC with or without the addition of dronedarone hydrochloride. The wild-type PAO1 cultured overnight was washed twice with PBS, the OD600 was adjusted to 1, and then the bacteria were diluted to the corresponding ABTGC with or without compound No. 3 at a ratio of 1:100 in a 96-well plate for culture. The experiment was divided into three groups, namely the blank group, the compound group, and the DMSO solvent group, with four replicates in each group. After the 96-well plate was cultured in a 37°C incubator for 22 hours, the OD of each group was detected using a microplate reader. 600 The iron carrier Pyoverdine was produced (excitation wavelength: 398 nm, emission wavelength: 460 nm). The final Pyoverdine production between the groups was expressed as Pyoverdine amount / OD 600 To calculate, the result is Figure 4 shown.

[0048] Depend on Figure 2-4 It can be seen that the addition of compounds can inhibit the PQS quorum sensing system of Pseudomonas aeruginosa and the production of virulence factors pyocyanin and siderophore; combined with the previous results, it can be concluded that compounds 1 and 2 provided by the present invention can significantly inhibit the production of virulence factor pyocyanin regulated by the PQS quorum sensing system of Pseudomonas aeruginosa. Compound 3 can significantly inhibit the production of virulence factor siderophore of Pseudomonas aeruginosa. The virulence factors of Pseudomonas aeruginosa, including pyocyanin and siderophore, play an important role in Pseudomonas aeruginosa invading the host immune system and regulating bacterial resistance. The three macrolide compounds isolated and prepared by the present invention provide new ideas for the development and research of new anti-Pseudomonas aeruginosa drugs in clinical practice.

[0049] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. Macrolide compounds, the structure of which is shown in any one of formula (I) Formula (I).

2. Use of the macrolide compound according to claim 1 in the preparation of a drug for inhibiting the production of virulence factors of Pseudomonas aeruginosa, wherein compounds 1 and 2 inhibit the production of the virulence factor pyocyanin regulated by the PQS quorum sensing system of Pseudomonas aeruginosa, and compound 3 inhibits the production of siderophores of Pseudomonas aeruginosa.

3. Bacillus amyloliquefaciens SCSIO 41392, deposited under GDMCC No: 65022.

4. A method for preparing the macrolide compound according to claim 1, characterized in that: It is prepared and isolated from the fermentation product of Bacillus amyloliquefaciens SCSIO 41392 according to claim 3; The specific steps are as follows: A. Preparation of fermentation products of Bacillus amyloliquefaciens SCSIO 41392; B. The fermentation product was extracted with ethyl acetate, and the ethyl acetate extract was concentrated to obtain a crude extract. The crude extract was subjected to reverse phase medium pressure separation using a H2O-CH3OH system, eluted from 90%:10% to 0%:100% by volume, and fraction fr6 was obtained under a gradient system of methanol and water in a volume ratio of 75%:25% to 80%:20%. Fraction fr6 was separated by semi-preparative high performance liquid chromatography at a flow rate of 3 mL / min. When the mobile phase was acetonitrile and ultrapure water containing 0.6% formic acid in a volume ratio of 42:58, the component with a retention time of 35.5 min was compound 2. The component with a retention time of 13 min was fraction fr6-3, and the component with a retention time of 32.6 min was fraction fr6-6; fraction fr6-6 was separated by semi-preparative high performance liquid chromatography with a flow rate of 3 mL / min. When acetonitrile and ultrapure water containing 0.8% formic acid were used as the mobile phase in a volume ratio of 35:65, the component with a retention time of 33 min was compound 1; fraction fr6-3 was separated by semi-preparative high performance liquid chromatography. When acetonitrile and ultrapure water containing 0.6% formic acid were used as the mobile phase in a volume ratio of 39:61, the component with a retention time of 10 min was compound 3.

5. The preparation method according to claim 4, characterized in that The fermentation product is prepared by inoculating Bacillus amyloliquefaciens SCSIO 41392 into a culture medium, culturing at 28°C and 180 rpm to obtain a seed solution, inoculating the seed solution into the culture medium at an inoculum rate of 3% by volume, and culturing at 28°C and 180 rpm to obtain a fermentation product of Bacillus amyloliquefaciens SCSIO 41392. The culture medium is prepared as follows per 1000 mL: 10 g of malt extract, 4 g of yeast extract, 0.2% of calcium carbonate, 0.4% of glucose, and 3.2% of sea salt. The preparation method is to add each component to solvent water and sterilize it for later use.

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