Polyketone compound as well as preparation method and application thereof

Abstract

The invention discloses a polyketone compound as well as a preparation method and application thereof, and belongs to the technical field of marine microorganisms. The preparation method of the polyketone compound comprises the following steps: firstly, obtaining a fermentation product containing the compound through microbial fermentation culture, and then separating and purifying the fermentation product by adopting methods such as VLC normal-phase column chromatography, C-18 ODS reversed-phase column chromatography and semi-preparative HPLC (High Performance Liquid Chromatography) to obtain the polyketone compound. The microorganism is Streptomyces coelicola A3 (2) / p15A-KOspiH3, and the preservation number of the microorganism is CCTCC (China Center for Type Culture Collection) NO: M 20241470, and the microorganism is Streptomyces coelicola A3 (2) / p15A-KOspiH3, and the preservation number of the microorganism is CCTCC NO: M 20241470. Experiments prove that the polyketone compound has the dual activities of resisting bacteria and reducing uric acid, the characteristics of the dual functions of resisting bacteria and resisting hyperuricemia are relatively rare in current medicines, and a novel treatment strategy for combined infection or gout complications can be developed.

Description

Technical Field

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

[0002] Although the discovery and clinical use of antibiotics have revolutionized the history of human health against microbial infections, the rapid development of bacterial resistance to several antibiotics has attracted worldwide attention. The World Health Organization (WHO) has reported antimicrobial drug resistance as one of the potential public health crises. In addition, the annual consumption and overuse of antibiotics have increased several-fold, leading to a significant increase in cases of antibiotic resistance. It is worth noting that in the past few decades, the number of antibiotics approved by the US Food and Drug Administration (FDA) is far less than the reported cases of antibiotic-resistant bacteria. Importantly, newly synthesized antibiotics have similar antibacterial mechanisms to existing antibiotics. There are also some other limitations in the discovery of antibiotics, such as complex chemical synthesis methods, poor bioavailability, and systemic toxicity, etc. Therefore, there is an urgent need for new antibacterial drugs with innovative antibacterial mechanisms to combat multi-drug resistant bacteria. Summary of the Invention

[0003] One of the purposes of the present invention is to provide a new polyketide compound and a preparation method of the compound, and another purpose is to provide a specific application of the compound to make up for the deficiencies of the prior art.

[0004] To achieve the above purposes, the specific technical solutions adopted by the present invention are as follows: A polyketide compound, the structural formula of the compound is shown in formula (I): (I).

[0005] The preparation method of the polyketide compound is to first obtain a fermented product containing the compound through microbial fermentation culture, and then separate and purify the fermented product by methods such as VLC normal-phase column chromatography, C-18 ODS reverse-phase column chromatography, and semi-preparative HPLC to obtain the polyketide compound.

[0006] Further, 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, Hubei 430072.

[0007] Use of the polyketide compound in the preparation of antibacterial drugs.

[0008] Use of the polyketide compound in the preparation of drugs for reducing uric acid.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention prepares a polyketide compound through microbial fermentation, and through experimental verification, this polyketide compound has broad-spectrum and highly efficient antibacterial activity: it not only has an obvious inhibitory effect on common Gram-positive bacteria (such as Bacillus cereus Bacillus cereus , Staphylococcus aureus Staphylococcus aureus , Bacillus subtilis Bacillus subtilis ), but also can effectively inhibit drug-resistant strains (such as methicillin-resistant Staphylococcus aureus MRSA and methicillin-resistant coagulase-negative Staphylococcus 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 avoid common drug-resistant pathways. In addition, this compound has dual activities of antibacterial and uric acid reduction: this characteristic of dual functions of antibacterial and anti-hyperuricemia is relatively rare in current drugs, which helps to develop new treatment strategies for combined infections or gout complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 It is a diagram showing the XOD inhibitory activity results of Compound I.

[0011] Figure 2 It is the nuclear magnetic resonance hydrogen spectrum of Compound I (DMSO- d 6 , 500 MHz).

[0012] Figure 3 It is the nuclear magnetic resonance carbon spectrum of Compound I (DMSO- d 6 , 125 MHz).

[0013] Figure 4 It is the HSQC spectrum of Compound I (DMSO- d 6 ).

[0014] Figure 5 It is the 1 H- 1 H COSY spectrum of Compound I (DMSO- d 6 ).

[0015] Figure 6 It is the HMBC spectrum of Compound I (DMSO- d6 ).

[0016] Figure 7 It is the HRESIMS spectrum of Compound Ⅰ. Detailed implementation mode

[0017] The present invention will be further explained and illustrated below through specific embodiments in conjunction with the accompanying drawings.

[0018] Example 1 Fermentation production, separation and purification of Compound Ⅰ 1 Fermentation production Fermentation culture of the production strain: According to the conventional method of culturing microorganisms, take Streptomyces coelicolor Streptomyces coelicolor A3(2) / p15A-KO spi An appropriate amount of H3, first culture it on an MS solid medium containing 50 μ g / mL of Apra in a 28 °C incubator for 7 days.

[0019] Take the Streptomyces coelicolor Streptomyces coelicolor A3(2) / p15A-KO spi An appropriate amount of H3, inoculate it into a 500 mL conical flask containing 100 mL of the medium, and the composition of the medium (g / L): soluble starch 10 g, peptone 2 g, yeast extract 4 g, water 1 L, pH 7.2 - 7.4], and culture it in a 28 °C shaker (180 rmp) for 8 days to obtain the fermentation product.

[0020] Obtaining the extract Filter the fermentation broth with gauze to obtain the supernatant, extract it three times with an equal volume of ethyl acetate, combine all the ethyl acetate phases, and concentrate it under reduced pressure to obtain a crude extract, a total of 50 grams.

[0021] 3 Separation and purification of the compound The extract (50 g) was dissolved in 90% methanol and extracted with petroleum ether to remove the fat components. After the 90% methanol solution was evaporated to dryness, normal-phase column chromatography was carried out using dichloromethane (DCM)-methanol (CH3OH) as the elution system, and it was divided into 5 fractions, namely DCM:CH3OH = 80:1, DCM:CH3OH = 60:1, DCM:CH3OH = 40:1, DCM:CH3OH = 20:1, DCM:CH3OH = 1:1. Subsequently, the fraction of DCM:CH3OH = 40:1 was subjected to gradient elution on a C-18 ODS reversed-phase column with methanol-water as the mobile phase (methanol:water = 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%). The fraction of methanol:water = 60% was further separated by reversed-phase semi-preparative high-performance liquid chromatography (acetonitrile:water = 55:45) to obtain compound I (30 mg).

[0022] The chemical structure of compound I (the Arabic numerals in the structural formula are the carbon atom positions in the chemical structure) is as follows: 。

[0023] Compound I is a brown solid, with the molecular formula C 16 H 10 O4, as Figure 7 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 13C NMR nuclear magnetic attribution is shown in Table 1, Figure 2 、 Figure 3 。

[0024] Table 1 1 1H and 13 13C NMR data (500 and 125 MHz, in DMSO-d6) a 。

[0025] (a) The signal attribution in this table is based on the results of COSY, HSQC and HMBC spectra analysis (as Figure 4 、 Figure 5 、 Figure 6 shown). The hydrogen signals are represented by s (singlet), d (doublet), t (triplet) and q (quartet), m (multiplet).

[0026] (b) The numbers and codes in this column respectively represent the 1 H- 1 H nuclei that give coupling-related signals with the 1 H in the corresponding row in the 1 H COSY spectrum.

[0027] (c) The numbers and codes in this column respectively represent the 1 C nuclei that give coupling-related signals with the 13 H in the corresponding row in the HMBC spectrum.

[0028] Example 2 Determination of the Bacteriostatic Activity of Compound I 1 Experimental Samples Preparation of the sample solution to be tested: The test sample is the pure compound I separated and purified in Example 1 above. Weigh an appropriate amount of the sample precisely and prepare a stock solution of 25.6 mg / mL with DMSO.

[0029] 2 Preparation of Bacterial Solutions According to the guidelines of the Clinical and Laboratory Standards Institute (CLSI), the MIC values of the compound and the drug are measured by the microdilution broth dilution method. Inoculate the target bacteria on a Mueller-Hinton agar (MHA) plate at 37 °C. After culturing for 24 h, pick a single colony into MHB broth medium and shake it on a shaker for 4 - 6 h (220 rpm, 37 °C) until the bacterial content reaches approximately 1×10 8 CFU / mL. Dilute it 100 times with MHB broth medium to obtain a bacterial solution containing approximately 1×10 6 CFU / mL for standby. Test strains: 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), Methicillin-resistant coagulase-negative Staphylococcus (MRCNS).

[0030] 3 Activity Test The microdilution broth dilution method is adopted. Take a 96-well plate and add 198 μ μL of medium (except for the 11th column) in the first well of the first column. The following 8 rows are 100 μ μL of medium. The 10th, 11th, and 12th columns are for positive and negative controls.

[0031] Add 2 μThe compound with the initial concentration was added to the first well. After mixing evenly, according to the serial dilution method, 100 μ μL of the liquid was pipetted from the first well and added to the second well, mixed evenly, and then 100 μ μL was added to the third well, and so on. After the tenth well was mixed evenly, 100 μ μL of the solution was discarded. Then 100 μ μL of the spare bacterial suspension (1×10 6 CFU / mL) was added to the first to the tenth wells in sequence and mixed evenly. Finally, the concentrations of the compound or drug in the first to the tenth wells were 128, 64, 32, 16, 8, 4, 2, 1 μ μg / mL. The final concentrations of the positive drugs were: 128, 64, 2, 16, 8, 4, 2, 0.5, 0.25, 0.125, 0.0625, 0.0312, 0.0156, 0.0078 μ μg / mL).

[0032] Each compound or drug was repeated in three groups, and ciprofloxacin, nystatin, vancomycin, and imipenem were used as positive drugs at the same time. The 96-well plate was placed in an incubator at 37 °C for 18 h, and the results were observed. The concentration corresponding to the clear well visible to the naked eye was the MIC value.

[0033] 4. Experimental results In the antibacterial activity test, the inhibitory activity results of compound I against seven pathogenic bacteria are shown in Table 2.

[0034] Table 2 Antibacterial activity results of compound I (MIC values, μ μg / mL) .

[0035] Example 3 Determination of the uric acid-lowering activity of compound I 1 Determination of the inhibitory activity of the compound against xanthine oxidase (XOD) The final concentration gradient of compound I was set as 10, 5, 2.5, 1.25, 0.625, 0.3125 μ mM. Using DMSO as the solvent control, different concentrations of compound I were co-incubated with XOD at 37 °C for 15 min, and then the substrate xanthine solution was added. Immediately, it was placed in an enzyme-labeled instrument, and the absorbance of the detection system at 295 nm was measured at 37 °C. It was measured once every 1 min for 10 consecutive minutes. According to XOD activity (%) = △OD of the compound group 295 nm ÷△OD of the control group 295 nm ×100 for calculation, and IC 50 fitting was performed using Graphpad prism 5 software.

[0036] Compound Ⅰ has good inhibitory activity against XOD, and the IC 50 is 0.6 μ M, as shown in Figure 1 .

[0037] The above experiments verified that the results of the activity screening of Compound Ⅰ showed that Compound Ⅰ had obvious inhibitory effects 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 could be used as an antibacterial lead compound for the treatment of bacterial infections. In addition, Compound Ⅰ had good inhibitory activity against XOD, indicating its uric acid-lowering activity and application.

[0038] Compound Ⅰ is a small molecule compound with a relatively simple structure and good pharmacokinetic potential (such as high oral bioavailability and strong tissue penetration). Compared with macromolecules or complex natural products, small molecules are easier to synthesize and optimize in structure, which helps to quickly establish the structure-activity relationship and develop derivatives. Small molecules can often directly act on intracellular targets by penetrating cell membranes, and have the possibility of developing new antibacterial mechanisms, providing a new direction for solving the serious problem of homogeneous antibacterial drug targets.

[0039] The specific embodiments described above further detailed the purpose, technical solutions, and beneficial effects of the present disclosure. It should be understood that the above are only specific embodiments of the present disclosure and are not used to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present disclosure should be included within the protection scope of the present disclosure.

Claims

1. A polyketide compound, characterized in that, The structural formula of the polyketide compound is shown in Formula (I): （I）。 2. The method for preparing a polyketide compound according to claim 1, wherein Firstly, a fermented product is obtained through microbial fermentation culture, and then the fermented product is successively separated and purified by VLC normal-phase column chromatography, C-18 ODS reversed-phase column chromatography, and semi-preparative HPLC methods to finally obtain the polyketide compound.

3. The preparation method according to claim 2, characterized in that, The microorganism is: Streptomyces coelicolor A3(2) / p15A-KOspiH3, deposit number: CCTCC NO: M 20241470, deposit date: July 3, 2024, depository: China Center for Type Culture Collection, depository address: School of Life Sciences, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan, Hubei 430072.

4. Use of the polyketide compound according to claim 1 in the preparation of antibacterial drugs.

5. Use of the polyketide compound according to claim 1 in the preparation of drugs for reducing uric acid.

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