A thioether compound and its preparation method and use
The sulfide-based compounds isolated from marine actinomycetes have solved the problem that the prior art is difficult to inhibit MRSA, and the effective inhibition of the bacteria is achieved, which has important drug development value.
Patent Information
- Application Number
- CN202310293587.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-24
- Publication Date
- 2025-05-09
- Estimated Expiration
- 2043-03-24
AI Technical Summary
The prior art is difficult to effectively inhibit the spread of methicillin-resistant Staphylococcus aureus (MRSA), resulting in the bacteria becoming the main drug-resistant pathogen in healthcare and community settings.
A new sulfide compound was isolated from the ethyl acetate extract fermented by marine actinomyces diastaticus in rice medium, which was prepared by fermentation, extract extraction and medium pressure column chromatography.
The sulfide compound showed significant anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, could effectively inhibit the growth of the bacteria, and had potential drug development applications.
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Abstract
Description
Technical Field
[0001] The invention relates to a thioether compound, in particular to a thioether compound extracted from marine actinomycetes and a preparation method and application thereof. Background Art
[0002] Methicillin-resistant Staphylococcus aureus (MRSA) has been around since the 1960s and has spread globally. Within a decade, many local and hospital outbreaks of MRSA occurred, and it has become a major cause of bacterial infections in health care and community settings. Since the beginning of the 21st century, the landscape of hospital-based MDR pathogens in clinical practice has changed dramatically. MRSA has been shown to be one of the most persistent resistant pathogens in health care and community settings, and it remains a significant cause of death worldwide (especially as a pathogen of endocarditis and bacteremia). In the face of this situation, the discovery of new antibiotics has become particularly important.
[0003] Organic thioether compounds have been widely used in the fields of medicine, biology, agriculture, optical materials, etc. Thioether derivatives are an important synthetic strategy in drug discovery. In recent years, due to their extensive agricultural activities, such as antibacterial, insecticidal, acaricidal, nematicidal, antiviral, herbicidal and plant growth regulating activities, they have become increasingly important in the field of plant protection. In recent years, thioethers and their analogs have been a field of great interest. The inventors discovered a new natural product of thioethers in a chemical investigation of the ethyl acetate extract of marine actinomycete Streptomyces diastaticus fermented in rice culture medium. At present, there is no report on the chemical structure of the compound and its anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, so there is no drug related to this on the market. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide a thioether compound having an inhibitory effect against methicillin-resistant Staphylococcus aureus (MRSA) and a preparation method and use thereof.
[0005] The technical solution adopted by the present invention to solve the above technical problems is:
[0006] 1. A thioether compound, the structural formula of which is shown in (I);
[0007]
[0008] 2. A method for preparing a thioether compound, comprising the following steps:
[0009] (1) Fermentation production
[0010] The marine actinomycetes (Steptomyces sp.4-7) with the accession number of CCTCC NO: M2020953 were streaked on a plate of Gao's solid medium No. 1, and after inverted culture in an incubator at 28° C. for 7 days, a single colony was picked and inoculated into Gao's liquid medium No. 1, and cultured on a shaker at a temperature of 28° C. and a speed of 180 rpm / min. After culturing for 3 days, the seed liquid was collected, and then the seed liquid was inoculated into a rice solid medium at an inoculum amount of 10% by volume, and cultured at a temperature of 28° C. for 30 days to obtain a fermentation product;
[0011] (2) Extraction
[0012] Add an equal amount of ethyl acetate to the fermented product obtained in step (1), extract repeatedly for 3 times, and then evaporate the ethyl acetate extract to dryness under reduced pressure to obtain a crude extract;
[0013] (3) Isolation and preparation of compounds
[0014] The crude extract obtained in step (2) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and gradient elution is performed using a petroleum ether-ethyl acetate solution in a volume ratio of 1:1 as an eluent, and the eluent is collected; the collected eluent is subjected to reverse phase medium pressure column chromatography linear gradient elution, using methanol-water in a volume percentage of 25-100% as an eluent, and the eluted fractions are collected, arranged in descending order according to the polarity of the fractions, and 14 components are obtained by merging; the 10th component obtained is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a solution of acetonitrile and water in a volume ratio of 40:60 as a mobile phase to obtain a compound, whose structure is shown in (I)
[0015]
[0016] Furthermore, the Gao's No. 1 solid culture medium described in step (1) is prepared as follows: 20g of soluble starch, 1g of KNO3, 0.5g of K2HPO4, 0.5g of MgSO4·7H2O, 0.5g of NaCl, 0.01g of FeSO4·7H2O and 20g of agar are added to 1000mL of distilled water, and the pH is adjusted to 7.4-7.6; the Gao's No. 1 liquid culture medium is prepared as follows: 20g of soluble starch, 1g of KNO3, 0.5g of K2HPO4, 0.5g of MgSO4·7H2O, 0.5g of NaCl and 0.01g of FeSO4·7H2O are added to 1000mL of distilled water, and the pH is adjusted to 7.4-7.6; the rice solid culture medium is prepared as follows: 80g of rice, 120mL of water and 3.6g of sea salt are mixed and sterilized.
[0017] Furthermore, the elution gradient volume ratios of the petroleum ether-ethyl acetate solution in step (3) are 1:0, 9:1, 6:1, 4:1, 7:3, 3:2, 1:1, 2:3 and 0:1, respectively.
[0018] Furthermore, in the reversed-phase MPLC gradient elution in step (3), the volume percentage of acetonitrile ranges from 25 to 100%, and the elution time is 150 min.
[0019] Furthermore, the flow rate of the compound separation preparation by semi-preparative reverse-phase HPLC described in step (3) is 2.0 mL / min.
[0020] 3. Use of the above thioether compounds in the preparation of methicillin-resistant Staphylococcus aureus (MRSA) inhibitors.
[0021] Compared with the prior art, the advantages of the present invention are as follows: the present invention provides a thioether compound and a preparation method and use thereof, obtains a fermentation product by microbial fermentation culture, then extracts the fermentation product by soaking it in ethyl acetate to obtain a crude extract, and then separates and purifies the crude extract by medium-pressure normal-phase silica gel column chromatography, medium-pressure reversed-phase column chromatography, and reversed-phase semi-preparative high-performance liquid chromatography, obtains the compound, has significant anti-methicillin-resistant Staphylococcus aureus (MRSA) activity, and can be used for drug development for inhibiting related diseases caused by methicillin-resistant Staphylococcus aureus (MRSA).
[0022] The above-mentioned marine actinomycetes (Steptomyces sp.4-7) is the NBU2966 strain, with the preservation number of CCTCCNO: M2020953. It was deposited in the China Center for Type Culture Collection on December 21, 2020, and the preservation address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of the compound of the present invention;
[0024] Figure 2 is the carbon nuclear magnetic resonance spectrum of the compound of the present invention;
[0025] Figure 3 is the nuclear magnetic resonance DEPT-135 spectrum of the compound of the present invention;
[0026] Figure 4 is the COSY nuclear magnetic resonance spectrum of the compound of the present invention;
[0027] Figure 5 is the nuclear magnetic resonance HSQC spectrum of the compound of the present invention;
[0028] Figure 6 is the nuclear magnetic resonance HMBC spectrum of the compound of the present invention;
[0029] Figure 7 It is the ECD absolute configuration fitting curve diagram of the compound of the present invention. DETAILED DESCRIPTION
[0030] The present invention is further described in detail below with reference to the embodiments.
[0031] Example 1
[0032] The structural formula of a thioether compound is shown in (I):
[0033]
[0034] Example 2
[0035] The method for preparing the sulfide compound shown in structural formula (I) in Example 1 specifically comprises the following steps:
[0036] (1) Fermentation production
[0037] The marine actinomycetes with the accession number of CCTCC NO: M2020953 were streaked on a plate of Gao's solid culture medium No. 1, and after inverted culture in an incubator at 28°C for 7 days, a single colony was picked and inoculated into Gao's liquid culture medium No. 1, and cultured on a shaker at a temperature of 28°C and a speed of 180rpm / min. After culture for 3 days, the seed liquid was collected, and then the seed liquid was inoculated into a rice solid culture medium at an inoculum volume ratio of 10%, and cultured at a temperature of 28°C for 30 days to obtain a fermentation product. The preparation method of Gao's solid culture medium No. 1 is as follows: 20g of soluble starch, 1g of KNO3, 0.5g of K2HPO4, 0.5g of MgSO4·7H2O, 0.5g of NaCl, and 0.5g of FeSO4·7H2O. 0.01g and 20g agar, 1000mL distilled water, adjust pH = 7.4-7.6; Gao's No. 1 liquid culture medium is prepared as follows: 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl and 0.01g FeSO4·7H2O, 1000mL distilled water, adjust pH = 7.4-7.6; The preparation method of rice solid culture medium is as follows: 80g rice, 120mL water, 3.6g sea salt are mixed and sterilized;
[0038] (2) Extraction
[0039] The fermented product obtained in step (1) is added with ethyl acetate in an amount equal to that of the fermented product, and the mixture is repeatedly soaked for three times, and then the ethyl acetate extract is evaporated under reduced pressure to dryness to obtain a crude extract;
[0040] (3) Isolation and preparation of compounds
[0041] The crude extract obtained in step (2) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and gradient elution is performed using a petroleum ether-ethyl acetate solution in a volume ratio of 1:1 as an eluent, and the eluent is collected; the collected eluent is subjected to reverse phase medium pressure column chromatography linear gradient elution, using methanol-water with a volume percentage of 25-100% as an eluent, and the elution time is 150 minutes, and the eluted fractions are collected, arranged in descending order according to the polarity of the fractions, and 14 components are obtained by merging; the obtained 10th component is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a solution of acetonitrile and water in a volume ratio of 40:60 as a mobile phase to obtain a compound, whose structure is shown in (I).
[0042]
[0043] Example 3
[0044] Compound I of the present invention is a black oil, and high-resolution mass spectrometry (HR-ESI-MS) in positive ion mode gives its quasi-molecular ion peak m / z 324.0809 [M+H] + (calcd for C 17 H 14 N3O2S, 324.0807), combined 13 C NMR confirmed that its molecular formula is C 17 H 13 N3O2S, the compound 1 H and 13 C NMR and 2D data are shown in Figure 1-7 and Table 1. 1 H and 13 C NMR combined with HSQC spectra showed 10 methine carbon δ C 62.7 (C-2), 127.3 (C-6), 117.2 (C-7), 133.3 (C-8), 114.5 (C-9), 116.3 (C-3′), 127.5 (C-6′), 119.5 (C-7′), 131.4 (C-8′), 116.8 (C-9′), 2 carbonyl carbons δ C 163.4 (C-4), 165.0 (C-4′), no methyl or methylene groups. H 7.29, δ H 8.47 and δ H The NH proton at 11.08 lacks HSQC correlation. Figure 4 As shown, the COSY spectrum shows that H-1(δ H 7.29) and H-2(δ H 5.87) related, H-3(δH 8.47) and H-2(δ H 5.87), and the HMBC data show that H-1 (δ H 7.62) is related to C-5, H-3 (δ H 8.47) is associated with C-5, which indicates the connection between NH. Figure 6 As shown, HMBC data showed that H-3′(δ H 7.52) is related to C-2′ and C-4′, H-2(δ H 5.87) is associated with C-2′ and C-10, thus establishing a C-2–S–C-2′ connection. 1 H- 1 The H COSY spectra were coupled to each other, revealing the correlation of C-6, C-7, C-8, C-9 and the correlation of C-6′, C-7′, C-8′, C-9′. Therefore, the planar structure of the compound was determined. Figure 7 As shown, the absolute configuration was determined by comparison with the calculated ECD spectrum, and the compound was concluded to have a 2S structure. Finally, the structure of the compound was identified as (S)-2-((4-oxo-1,4-dihydroquinolin-2-yl)thio)-2,3-dihydroquinazolin-4(1H)-one (2-(4-oxo-1,4-dihydroquinolin-2-yl)thio)-2,3-dihydroquinazolin-4(1H)-one), and the compound was found to be a new compound by SciFinder query.
[0045] Table 1. NMR data of compound Ⅰ (CDCl3-d6)
[0046]
[0047]
[0048] Note 1: s—singlet, d—doublet, t—triplet.
[0049] Note 2: 1 H was obtained at 600 MHz NMR; 13 C was obtained at 150 MHz NMR.
[0050] Example 4
[0051] Activity and application of sulfide compounds described in Example 1
[0052] (1) Experimental samples
[0053] Preparation of the sample solution: The test sample is the pure compound I separated and purified in Example 1 above. Accurately weigh an appropriate amount of the sample and use DMSO to prepare a solution of the required concentration for testing the antibacterial activity. The indicator bacteria used in this experiment is methicillin-resistant Staphylococcus aureus.
[0054] (2) Experimental methods
[0055] 96-well plate antibacterial test: Pick a single colony of methicillin-resistant Staphylococcus aureus (MRSA) and inoculate it into broth MH liquid culture medium, add 4μL of 12.8mg / mL sample and 96μLMH culture medium solution to each well. Through gradient dilution, the final concentrations were 128μg / mL, 64μg / mL, 32μg / mL, 16μg / mL, 8μg / mL, 4μg / mL, 2μg / mL, 1μg / mL, and 0.5μg / mL. DMSO was used as a negative control, and vancomycin at the same concentration was used as a positive control. After culturing at 28°C for 2 days, the growth of methicillin-resistant Staphylococcus aureus (MRSA) was observed, and the entire experiment was repeated 3 times.
[0056] (3) Experimental results
[0057] In the 96-well plate antibacterial test, the MIC of compound Ⅰ against methicillin-resistant Staphylococcus aureus (MRSA) was determined to be 8 μg / mL.
[0058] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.
Claims
1. A thioether compound, characterized in that The structural formula of the thioether compound is shown in (I); 2. A method for preparing a sulfide compound according to claim 1, characterized in that The steps include: (1) Fermentation production The marine actinomycetes with the accession number of CCTCC NO: M2020953 were streaked on a plate of Gao's solid culture medium No. 1, and after inverted culture in an incubator at 28°C for 7 days, a single colony was picked and inoculated into Gao's liquid culture medium No. 1, and cultured on a shaker at a temperature of 28°C and a speed of 180rpm / min. After culture for 3 days, the seed liquid was collected, and then the seed liquid was inoculated into a rice solid culture medium at an inoculum volume ratio of 10%, and cultured at a temperature of 28°C for 30 days to obtain a fermentation product, wherein the Gao's solid culture medium No. 1 was prepared as follows: 20g of soluble starch, 1g of KNO3, 0.5g of K2HPO4, 0.5g of MgSO4·7H2O, 0.5g of NaCl, 0.5g of FeSO4·7H2O 0.01g and 20g agar, 1000mL distilled water, adjust pH=7.4-7.6; the preparation method of Gao's No. 1 liquid culture medium is as follows: 20g soluble starch, 1g KNO3, 0.5g K2HPO4, 0.5g MgSO4·7H2O, 0.5g NaCl and 0.01g FeSO4·7H2O, 1000mL distilled water, adjust pH=7.4-7.6; the preparation method of the rice solid culture medium is as follows: 80g rice, 120mL water, 3.6g sea salt are mixed and sterilized; (2) Extraction Add an equal amount of ethyl acetate to the fermented product obtained in step (1), extract repeatedly for 3 times, and then evaporate the ethyl acetate extract under reduced pressure to obtain a crude extract; (3) Isolation and preparation of compounds The crude extract obtained in step (2) is first dissolved in a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then 200-300 mesh silica gel is added to mix the sample, and normal phase medium pressure column chromatography is performed, and gradient elution is performed using a petroleum ether-ethyl acetate solution in a volume ratio of 1:1 as an eluent, and the eluent is collected; the collected eluent is subjected to reverse phase medium pressure column chromatography linear gradient elution, using methanol-water with a volume percentage of 25-100% as an eluent, and the elution time is 150min, and the eluted fractions are collected, arranged in descending order according to the polarity of the fractions, and 14 components are obtained by merging; the 10th component obtained is separated and purified by semi-preparative reverse phase high performance liquid chromatography using a solution of acetonitrile and water in a volume ratio of 40:60 as a mobile phase to obtain a compound, whose structure is shown in (I), The flow rate of the compound separation and preparation by semi-preparative reverse-phase high performance liquid chromatography is 2.0 mL / min.
3. Use of the thioether compound according to claim 1 in preparing a methicillin-resistant Staphylococcus aureus inhibitor.