A polyketone compound and its preparation method and application

Abstract

The present invention discloses a polyketide compound, a preparation method and an application thereof, and belongs to the field of marine microbial technology. The preparation method of the polyketide compound is to first obtain a fermentation product containing the compound by microbial fermentation culture, and then separate and purify the fermentation product by VLC normal phase column chromatography, C-18 ODS reverse phase column chromatography and semi-preparative HPLC to obtain the polyketide compound. The microorganism is: Streptomyces coelicolor A3(2) / p15A-KOspiH3 Streptomyces coelicolor A3(2) / p15A-KOspiH3, with a preservation number of CCTCC NO:M 20241470. After experimental verification, the polyketide compound has dual activity of antibacterial and anti-hyperuricemia: this dual function of antibacterial and anti-hyperuricemia is relatively rare in current drugs, and is helpful to develop new treatment strategies for combined infections or gout complications.

Description

Technical Field

[0001] The present invention relates to the technical field of marine microorganisms, and in particular to a polyketide compound and a preparation method and application thereof. Background Art

[0002] Although the discovery and clinical use of antibiotics have revolutionized human health against microbial infections, the rapid development of bacterial resistance to several antibiotics has garnered worldwide attention. The World Health Organization (WHO) has designated antimicrobial resistance as a potential public health crisis. Furthermore, annual antibiotic consumption and overuse have increased severalfold, leading to a significant increase in cases of antibiotic resistance. Notably, over the past few decades, the number of antibiotics approved by the US Food and Drug Administration (FDA) has far exceeded the number of reported cases of antibiotic-resistant bacteria. Importantly, newly synthesized antibiotics possess antimicrobial mechanisms similar to those of existing antibiotics. Antibiotic discovery also has several other limitations, such as complex chemical synthesis methods, poor bioavailability, and systemic toxicity. Therefore, new antimicrobial agents with innovative antimicrobial mechanisms are urgently needed to combat multidrug-resistant bacteria. Summary of the Invention

[0003] One of the purposes of the present invention is to provide a new polyketone compound and a method for preparing the compound. Another purpose is to provide a specific application of the compound to make up for the deficiencies of the prior art.

[0004] In order to achieve the above object, the specific technical solution adopted by the present invention is:

[0005] A polyketone compound, the structural formula of the compound is shown in formula (I):

[0006] (I).

[0007] The preparation method of the polyketide compound comprises the following steps: firstly obtaining a fermentation product containing the compound by microbial fermentation culture; then separating and purifying the fermentation product by VLC normal phase column chromatography, C-18 ODS reverse phase column chromatography and semi-preparative HPLC to obtain the polyketide compound.

[0008] Furthermore, the microorganism is specifically: Streptomyces coelicolor A3(2) / p15A-KOspiH3, deposit number: CCTCC NO: M 20241470, deposit date: July 3, 2024, deposit unit: China Center for Type Culture Collection, deposit address: School of Life Sciences, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430072.

[0009] Application of the polyketone compound in the preparation of antibacterial drugs.

[0010] Application of the polyketide compound in the preparation of uric acid-lowering drugs.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The present invention prepares polyketide compounds by microbial fermentation, and the polyketide compounds have been verified by experiments to have broad-spectrum and high-efficiency antibacterial activity: not only against common Gram-positive bacteria (such as Bacillus cereus Bacillus cereus , Staphylococcus aureus Staphylococcus aureus , Bacillus subtilis Bacillus subtilis ) has a significant inhibitory effect and can also effectively inhibit drug-resistant strains (such as methicillin-resistant Staphylococcus aureus MRSA and methicillin-resistant coagulase-negative Staphylococci MRCNS). In the context of the increasingly serious problem of drug resistance, it has obvious advantages. Compared with existing antibiotics, this polyketide compound may act through different mechanisms and has the potential to circumvent common resistance pathways. In addition, this compound has dual antibacterial and anti-hyperuricemia activities: this dual antibacterial and anti-hyperuricemia function is relatively rare in current drugs, which will help develop new treatment strategies for co-infections or gout complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the XOD inhibitory activity result of compound Ⅰ.

[0014] Figure 2 This is the H NMR spectrum of compound Ⅰ (DMSO- d 6 , 500 MHz).

[0015] Figure 3 is the C NMR spectrum of compound Ⅰ (DMSO- d 6 , 125 MHz).

[0016] Figure 4 is the HSQC spectrum of compound Ⅰ (DMSO- d 6 ).

[0017] Figure 5 For compound Ⅰ 1 H- 1 H COSY spectrum (DMSO- d 6 ).

[0018] Figure 6is the HMBC spectrum of compound Ⅰ (DMSO- d 6 ).

[0019] Figure 7 This is the HRESIMS pattern of compound Ⅰ. DETAILED DESCRIPTION

[0020] The present invention is further explained and illustrated below through specific embodiments in conjunction with the accompanying drawings.

[0021] Example 1 Fermentation production and separation and purification of compound I

[0022] 1 Fermentation production

[0023] Fermentation culture of production bacteria: According to the conventional method of culturing microorganisms, Streptomyces coelicolor Streptomyces coelicolor A3(2) / p15A-KO spi H3 appropriate amount, first containing antibiotic Apra 50 μ The cells were cultured on MS solid medium at 500 μg / mL in a 28°C incubator for 7 days.

[0024] Take Streptomyces coelicolor cultured on the slant for 7 days Streptomyces coelicolor A3(2) / p15A-KO spi An appropriate amount of H3 was inoculated into 100 mL of culture medium, the composition of which (g / L) was as follows: 10 g soluble starch, 2 g peptone, and yeast extract. 4 g, 1 L of water, pH 7.2-7.4] in a 500 mL Erlenmeyer flask and cultured at 28 °C in a shaking incubator (180 rpm) for 8 days to obtain the fermentation product.

[0025] Obtaining the extract

[0026] The fermentation liquid was filtered through gauze to obtain the supernatant, which was extracted three times with equal amounts of ethyl acetate. All ethyl acetate phases were combined and concentrated under reduced pressure to obtain a crude extract, totaling 50 g.

[0027] 3. Separation and purification of compounds

[0028] The extract (50 g) was dissolved in 90% methanol and extracted with petroleum ether to remove the oily components. The 90% methanol-soluble fraction was evaporated to dryness and then purified by normal phase column chromatography using a dichloromethane (DCM)-methanol (CH3OH) eluent. The fraction separated into five fractions: DCM:CH3OH = 80:1, DCM:CH3OH = 60:1, DCM:CH3OH = 40:1, DCM:CH3OH = 20:1, and DCM:CH3OH = 1:1. The fraction with DCM:CH3OH = 40:1 was then eluted on a C-18 ODS reverse-phase column with a methanol-water mobile phase (methanol:water = 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%). The fraction with 60% methanol:water was further purified by reverse-phase semi-preparative HPLC (acetonitrile:water = 55:45) to obtain compound I (30 mg).

[0029] The chemical structure of compound I (the Arabic numerals in the structural formula represent the positions of carbon atoms in the chemical structure) is:

[0030] .

[0031] Compound I is a brown solid with the molecular formula C 16 H 10 O4, such as Figure 7 As shown, HR-ESI-MS m / z : 265.0505 [M-H] - , (calculated value: 265.0506); IR (KBr) ν max 3442, 2922, 1682, 1436,1213 cm -1 ; 1 Hand 13 C NMR nuclear magnetic assignments are shown in Table 1. Figure 2 、 Figure 3 .

[0032] Table 1 Compound Ⅰ 1 H and 13 C NMR data (500 and 125 MHz, in DMSO-d6) a

[0033] .

[0034] (a) The signal assignments in this table are based on the analysis results of COSY, HSQC and HMBC spectra (e.g. Figure 4 、 Figure 5 、 Figure 6The hydrogen signals are represented by s (singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).

[0035] (b) The numbers and codes in this column represent 1 H- 1 The H COSY spectrum and the corresponding row 1 H gives the coupled correlation signal 1 H core.

[0036] (c) The numbers and symbols in this column represent the 1 H gives the coupled correlation signal 13 C core.

[0037] Example 2 Determination of antibacterial activity of compound I

[0038] 1 Experimental samples

[0039] Preparation of the test sample solution: The test sample is the pure compound I isolated and purified in Example 1. Accurately weigh an appropriate amount of sample and prepare it into a 25.6 mg / mL stock solution with DMSO.

[0040] 2. Bacterial solution preparation

[0041] The MIC values of compounds and drugs were measured by the microbroth dilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. The target bacteria were inoculated on Mueller-Hinton agar (MHA) plates at 37°C. After 24 h of incubation, single colonies were picked and transferred to MHB broth medium. The plates were shaken on a shaker for 4-6 h (220 rpm, 37°C) until the bacterial count reached approximately 1 × 10 8 CFU / mL. Dilute 100-fold with MHB broth to obtain a bacterial count of approximately 1×10 6 CFU / mL of bacterial solution, set aside. Test strain: Acinetobacter baumannii ( A. baumannii ), Bacillus cereus ( B. cereus ), Pseudomonas aeruginosa ( P. aeruginosa ), Staphylococcus aureus ( S. aureus ), Bacillus subtilis ( B. subtilis )、Candida albicans ( C. albicans ), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-resistant coagulase-negative Staphylococci (MRCNS).

[0042] 3 Activity Test

[0043] Use the micro-broth dilution method. Take a 96-well plate, add 198 μL medium (except column 11), the following 8 rows are 100 μ L medium, columns 10, 11, and 12 for positive and negative controls.

[0044] 2 μ Add the compound with the initial concentration of L to the first well, mix well, and then use the serial dilution method to draw 100 μL of the compound from the first well with a pipette. μ L of liquid was added to the second well, mixed, and then 100 μ L is added to the third well, and so on, after mixing well in the 10th well, 100 μ L solution was discarded. Then 100 μ L of spare bacterial suspension (1 × 10 6 CFU / mL) were added to wells 1 to 10 and mixed. The final concentrations of the compound or drug in wells 1 to 10 were 128, 64, 32, 16, 8, 4, 2, and 1. μ g / mL. Final concentration of positive drug: 128, 64, 2, 16, 8, 4, 2, 0.5, 0.25, 0.125, 0.0625, 0.0312, 0.0156, 0.0078 μ g / mL).

[0045] Repeat three times for each compound or drug, using ciprofloxacin, nystatin, vancomycin, and imipenem as positive agents. Place the 96-well plate in a 37°C incubator for 18 hours and observe the results. The concentration corresponding to the wells with visible clarity is the MIC value.

[0046] 4. Experimental Results

[0047] In the antibacterial activity test, the inhibitory activity results of compound I against seven pathogenic bacteria are shown in Table 2.

[0048] Table 2 Antibacterial activity results of compound I (MIC values, μ g / mL)

[0049] .

[0050] Example 3 Determination of uric acid-lowering activity of compound I

[0051] 1. Xanthine oxidase (XOD) inhibitory activity assay of compounds

[0052] The final concentration gradient of compound I was set to 10, 5, 2.5, 1.25, 0.625, 0.3125 μM, using DMSO as solvent control, incubate different concentrations of compound I with XOD at 37°C for 15 min, then add substrate xanthine solution, immediately place in a microplate reader, and detect the absorbance of the system at 295 nm at 37°C, every 1 min for 10 min. According to XOD activity (%) = compound group △OD 295 nm ÷ control group△OD 295 nm ×100 was used for calculation, and IC was calculated using Graphpad Prism 5 software. 50 Fitting.

[0053] Compound Ⅰ has good inhibitory activity against XOD, IC 50 0.6 μ M, see Figure 1 .

[0054] The above experiments verified that the activity screening results of compound I showed that compound I had an effect on Bacillus cereus ( B. cereus ), Staphylococcus aureus ( S. aureus ), Bacillus subtilis ( B. subtilis ), methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-resistant coagulase-negative Staphylococci (MRCNS), and may be used as an antibacterial lead compound for the treatment of bacterial infections. Furthermore, compound I exhibits strong inhibitory activity against XOD, suggesting its potential for lowering uric acid and its potential application.

[0055] Compound I is a small molecule with a relatively simple structure and promising pharmacokinetic potential (e.g., high oral bioavailability and strong tissue penetration). Compared to large molecules or complex natural products, small molecules are easier to synthesize and optimize, facilitating rapid establishment of structure-activity relationships and the development of derivatives. Small molecules can often penetrate cell membranes to directly act on intracellular targets, potentially opening the door to novel antibacterial mechanisms and potentially addressing the current problem of severe target homogeneity in antibacterial drugs.

[0056] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects disclosed in the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polyketone compound, characterized in that The structural formula of the polyketone compound is shown in formula (I): （I）。 2. Use of the polyketide compound according to claim 1 in the preparation of antibacterial drugs.

3. Use of the polyketide compound according to claim 1 in the preparation of uric acid-lowering drugs.

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