24-membered macrolide compound as well as preparation method and application thereof

Abstract

The invention discloses a 24-membered macrolide compound as well as a preparation method and application thereof. Three macrolide compounds [1-3] with glycosyl side chains are prepared from bacillus amyloliquefaciens SCSIO 41392, as shown in the formula (I), the compounds 1-3 are newly reported compounds, generation of pseudomonas aeruginosa virulence factors can be remarkably inhibited, and the compound 1-3 can be used for preparing the glycosyl side chains of the macrolide compounds [1-3] with the glycosyl side chains of the macrolide compounds [1-3] with the glycosyl side chains of the macrolide compounds [1-3] with the glycosyl side chains of the macrolide compounds [1-3] with the glycosyl side chains of the macrolide compounds [1-3] as shown in the formula (I). Wherein the compounds 1 and 2 can inhibit the generation of a PQS quorum sensing system of pseudomonas aeruginosa and a virulence factor'pyocyanine 'regulated by the PQS quorum sensing system, and the compound 3 can significantly inhibit the generation of a pseudomonas aeruginosa virulence factor'siderophore', so that the compounds 1-3 can be used for developing antibacterial drugs. According to the invention, a new application is provided for the polar region bacterium Bacillus amyloliquefaciens SCSIO 41392, and an alternative lead compound is provided for the development of a new antibacterial agent derived from polar region microorganisms.

Description

Technical Field

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

[0002] Currently, polar regions have experienced climate change, such as global warming and an increase in the duration of ice-free periods. Many studies have focused on polar microorganisms. Due to the extreme environment, polar marine microorganisms are considered to be a source of new antimicrobial compounds that have not yet been fully developed and can meet the needs of dealing with new multi-drug resistant pathogens. In the process of studying microorganisms from Arctic deep-sea sediments, we isolated a class of macrolide compounds with sugar side chains, which can significantly inhibit the formation of Pseudomonas aeruginosa virulence factors including pyocyanin and siderophore, further enriching the understanding of polar microbial resources, and also providing new candidate compound resources for the discovery of inhibitory drugs for the production of Pseudomonas aeruginosa virulence factors. Summary of the invention

[0003] The first object of the present invention is 24-membered macrolide compounds (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 separated and purified the liquid fermentation extract of Bacillus amyloliquefaciens SCSIO 41392 from Arctic deep-sea sediments, and obtained macrolide compounds (1-3) therefrom. Compounds 1-3 are all new compounds, and their specific structures are shown in formula (I). By evaluating the biofilm inhibition activity of macrolide compounds (1-3), it was found that compounds 1-3 have obvious inhibitory effects on Pseudomonas aeruginosa biofilm, quorum sensing system and siderophore and other pathogenic bacteria virulence regulation systems, and can be used as lead compounds for the development of antibacterial drugs.

[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 the 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 production of the PQS quorum sensing system, pyocyanin and / or siderophore of Pseudomonas aeruginosa.

[0010] The fourth object of the present invention is to provide Bacillus amyloliquefaciens SCSIO41392, whose deposit number is 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. preparing a fermentation product 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 volume ratio of 75%:25% to 80%:20%. Fraction fr6 was separated by semi-preparative HPLC at a flow rate of 3 mL / min. When acetonitrile and ultrapure water containing 0.6% formic acid in a volume ratio of 42:58 were used as the mobile phase, the component with a retention time of 35.5 min was compound 2 The component with a retention time of 13 min is fraction fr6-3, and the component with a retention time of 32.6 min is fraction fr6-6; fraction fr6-6 is separated by semi-preparative HPLC with a flow rate of 3 mL / min, and when acetonitrile and ultrapure water containing 0.8% formic acid in a volume ratio of 35:65 are used as the mobile phase, the component with a retention time of 33 min is compound 1; fraction fr6-3 is separated by semi-preparative HPLC, and when acetonitrile and ultrapure water containing 0.6% formic acid in a volume ratio of 39:61 are used as the mobile phase, the component with a retention time of 10 min is compound 3.

[0015] Preferably, the preparation method of the fermentation product is as follows: inoculating Bacillus amyloliquefaciens SCS IO 41392 into a culture medium, culturing at 28° C., 180 rpm to obtain a seed solution, inoculating the seed solution into the culture medium at an inoculation rate of 3% by volume, culturing at 28° C., 180 rpm to obtain a fermentation product of Bacillus amyloliquefaci ens SCS IO 41392, wherein each 1000 mL of the culture medium is prepared as follows: 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, and the preparation method is to add each component into solvent water, sterilize and set aside.

[0016] The invention discloses a method for preparing macrolide compounds from Bacillus amyloliquefaciens SCSIO 41392, which is derived from Arctic deep-sea sediments, and an application of the macrolide compounds in inhibiting the production of virulence factors of Pseudomonas aeruginosa. The 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, which can significantly inhibit the production of virulence factors of Pseudomonas aeruginosa. Compounds 1 and 2 can inhibit the production of the PQS quorum sensing system of Pseudomonas aeruginosa and the virulence factor "psyorin" regulated by it, and compound 3 can significantly inhibit the production of the virulence factor "iron carrier" of Pseudomonas aeruginosa. Therefore, compounds 1-3 can be used to develop antibacterial drugs. The invention provides a new application for the polar bacterium Bacillus amyloliquefaciens SCSIO 41392 and provides 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 Microbiological 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 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 from Arctic deep-sea sediments.

[0025] Each 1000 mL of the culture medium is prepared as follows: 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 into solvent water, sterilize and set aside. Bacillus amyloliquefaciens SCSIO 41392 is inoculated into the above culture medium, cultured at 28°C, 180 rpm, for 3 days to obtain seed liquid, and the seed liquid is inoculated into the culture medium at an inoculation rate of 3% by volume, and cultured at 28°C, 180 rpm, for 7 days to obtain a shaking culture fermentation product of Bacillus amyloliquefaciens SCSIO 41392.

[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 with a small amount of methanol, 25 g of diatomaceous earth was added and the sample was evenly mixed, air-dried, loaded into a 100 g capacity loading column, and a 120 g loading capacity Spherical C18 column (model SW-5222-120-SP) was selected for reverse phase medium pressure separation. The H2O-CH3OH system was used, and a gradient was set every 10% of the first 10% CH3OH according to the volume ratio, and a gradient was set every 5% of the last 90% CH3OH. Each gradient was separated by 5 column volumes of mobile phase. According to thin layer chromatography analysis, the fractions of similar polarity of the combined components were fr1 to 6. Fraction fr6 was obtained under a gradient system of methanol and water in a volume ratio of 75%:25%-80%:20%. Fraction fr6 was separated by semi-preparative high performance liquid chromatography, under the monitoring of wavelengths at 230nm and 260nm, with an ODS (5μm, 10*250mm) separation column as the stationary phase, a flow rate of 3mL / 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 35.5min was compound 2, the component with a retention time of 13min was fraction fr6-3, and the component with a retention time of 32.6min was fraction fr6-6; Fraction fr6-6 was separated by semi-preparative high performance liquid chromatography, under the monitoring of wavelengths at 230nm and 260nm, with Chole ster (5μm, 10*250mm) separation column was used as the stationary phase, the flow rate was 3mL / min, and acetonitrile and ultrapure water containing 0.8% formic acid in a volume ratio of 35:65 were used as the mobile phase, and the component with a retention time of 33min was compound 1; fraction fr6-3 was separated by semi-preparative high performance liquid chromatography, under monitoring at 230nm and 260nm wavelengths, with Cholester (5μm, 10*250mm) separation column as the stationary phase, the flow rate was 3mL / min, and acetonitrile and ultrapure water containing 0.6% formic acid in a volume ratio of 39:61 were used as the mobile phase, and the component with a retention time of 10min 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 spectrum m / z 677.3194 [MH] - The suggested molecular formula is 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 Hybridized methylene, 1 sp 3 Methyl, 4 oxygen-linked methines, 1 methoxyl group, 1 lactone carbonyl carbon, 1 sugar fragment and 1 succinic acid fragment. Based on the above NMR data and mass spectrometry data, it is speculated that the structure type 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 confirmed that CH2-8′ and CH2-9′ were connected. The key related information of HMBC (H2-8′ was related to C-7′, H2-6′ was related to C-7′, and H2-9′ was related to C-10′) further confirmed that the succinic acid fragment was linked to the C-6′ position. According to Scifinder search, compound 1 was a new structure of natural origin and was named amylomacrolactine A.

[0034] New natural product - compound 2 structure identification: light yellow oil, high-resolution mass spectrometry shows that compound 2 is consistent with compound 1 in molecular formula, 1D NMR data show that compound 2 is almost completely consistent with compound 1, but the double bond coupling constants at C-8 and C-9 and C-17 and C-18 are different, so it is speculated that the double bond configuration of compound 2 at C-8 and C-17 is opposite to that of 1. Further 2D NMR data COSY, HSQC, and HMBC spectra also confirmed the above inference. According to Scifinder search, compound 2 is a new structure of natural origin and is named amylomacrolactine B.

[0035] New natural product - compound 3 structure identification: light yellow oil, high-resolution mass spectrometry shows that compound 3 has one less CH2 than compound 1, and its molecular weight is 14 less than 1. 1D NMR data show that compound 3 is almost the same as compound 1, but there is no methoxy substitution at C-19. Further 2D NMR data COSY, HSQC, and HMBC spectra show that C-19 should be substituted by hydroxyl. Therefore, it is speculated that the substitution at C-19 of compound 3 is hydroxyl rather than methoxy. According to Scifinder search, compound 3 is a new structure of natural origin and is 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 the production of virulence factors of Pseudomonas aeruginosa by the macrolide compound 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 15.1mM (NH4)2SO4, 33.7mM Na2HPO4·2H2O, 22mM K2HPO4, 0.05mM NaCl, 1mM MgCl2·6H2O, 100μM CaCl2·2H2O, 10μM 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 can be used after the temperature drops to room temperature.

[0042] (2) Fluorescence reporter system detection

[0043] 0.5 mg of compound 1 and 2 were each dissolved in 50 μL of DMSO to form a concentration of 10 mg mL -1 The PQS quorum sensing fluorescent reporter strain Pseudomonas aeruginosa PAO1 / pUCP22::pqsA-gfp strain (J.Med.Chem.2017,60,215-227) cultured overnight was washed twice with PBS and the bacterial solution OD 600 Adjust the density to 0.01 and dilute it again in 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, add each group into the 96-well plate at a volume of 150 μL / well, and then incubate the 96-well plate in a 37°C incubator for 24 hours. After 24 hours, use a fluorescent microplate reader to measure the OD of each well. 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 / OD of each group 600 Value, the result is Figure 2 As shown, compared with the Blank group and the DMSO group, the fluorescence intensity values ​​of the 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 3.5 mL ABTGC medium containing DMSO, and the DO of wild-type Pseudomonas aeruginosa PAO1 cultured overnight and the double knockout strain ΔlasIΔrhlI with both Las quorum sensing system and rhl quorum sensing system knocked out were added. 600 Adjust to 0.01, incubate at 37℃ for 24h, and record the OD of each group of bacterial solution 600 , and centrifuged at 10000rpm for 10min, 3mL of supernatant was added to 3mL of chloroform and vortexed for 10min, the mixture was allowed to stand for 20min and then centrifuged at 10000rpm for 10min, 3mL of blue layer was added to 0.2M hydrochloric acid (1.5mL) to extract pyocyanin, the upper pink layer containing pyocyanin was transferred to a 96-well microtiter plate, and the absorbance at 520nm 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 knockout of the quorum sensing system gene resulted in almost zero pyocyanin production. 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 PQ S quorum sensing system of Pseudomonas aeruginosa, and the difference was 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, and each group was repeated 4 times. After the 96-well plate was cultured in a 37°C incubator for 22 hours, the OD of each group was detected using an enzyme reader. 600 The iron carrier pyoverdine was produced (excitation wavelength: 398nm, emission wavelength: 460nm). 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 separated and prepared by the present invention provide new ideas for the development and research of new anti-Pseudomonas aeruginosa drugs in clinical practice.

[0049] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A macrolide compound, the structure of which is shown in any one of 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.

3. The use according to claim 2, characterized in that: The drug is a drug that inhibits the PQS quorum sensing system, pyocyanin and / or iron carrier production of Pseudomonas aeruginosa.

4. Bacillus amyloliquefaciens SCSIO 41392, whose deposit number is GDMCC No: 65022.

5. A method for preparing the macrolide compound according to claim 1, characterized in that: The invention is prepared and separated from the fermentation product of Bacillus amyloliquefaciens SCSIO 41392 as claimed in claim 4.

6. The preparation method according to claim 5, characterized in that: The specific steps are as follows: A. preparing a fermentation product 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 HPLC at a flow rate of 3 mL / min. When acetonitrile and ultrapure water containing 0.6% formic acid in a volume ratio of 42:58 were used as the mobile phase, the component with a retention time of 35.5 min was compound 2 The component with a retention time of 13 min is fraction fr6-3, and the component with a retention time of 32.6 min is fraction fr6-6; fraction fr6-6 is separated by semi-preparative HPLC with a flow rate of 3 mL / min, and when acetonitrile and ultrapure water containing 0.8% formic acid in a volume ratio of 35:65 are used as the mobile phase, the component with a retention time of 33 min is compound 1; fraction fr6-3 is separated by semi-preparative HPLC, and when acetonitrile and ultrapure water containing 0.6% formic acid in a volume ratio of 39:61 are used as the mobile phase, the component with a retention time of 10 min is compound 3.

7. The preparation method according to claim 6, characterized in that: The preparation method of the fermentation product is as follows: Bacillus amyloliquefaciens SCSIO 41392 is inoculated into a culture medium, and cultured at 28°C and 180rpm to obtain a seed solution, and the seed solution is inoculated into the culture medium at an inoculation amount of 3% by volume, and cultured at 28°C and 180rpm to obtain a fermentation product of Bacillus amyloliquefaciens SCSIO 41392. Each 1000mL culture medium is prepared as follows: 10g of malt extract, 4g of yeast extract, 0.2% of calcium carbonate, 0.4% of glucose, and 3.2% of sea salt. The preparation method is as follows: each component is added into solvent water, and sterilized for standby use.

8. Use of Bacillus amyloliquefaciens SCSIO 41392 according to claim 4 in the preparation of the macrolide compound according to claim 1.

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