A gamma-lactam alkaloid compound with activity against methicillin-resistant staphylococcus aureus and preparation method and use thereof

Abstract

This invention discloses a γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus (MRSA), its preparation method, and its uses. The compound's structural formula is shown in Figure I. The preparation method includes the following steps: fermenting Aspergillus pyrolyticus (accession number CCTCC NO: M2014086) to obtain a γ-lactam alkaloid ferment; extracting the ferment with ethyl acetate to obtain a crude extract; removing the crude extract with hexane and dichloromethane to obtain a final extract; and purifying the final extract using normal-phase silica gel column chromatography, reversed-phase medium-pressure column chromatography, and reversed-phase semi-preparative high-performance liquid chromatography. The advantage is that this γ-lactam alkaloid compound exhibits activity against MRSA and its antibacterial mechanism has been elucidated, making it a potential drug against foodborne pathogens.

Description

Technical Field

[0001] This invention relates to a γ-lactam alkaloid compound, and more particularly to a γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus extracted from marine fungal fermentation products, its preparation method, and its uses. Background Technology

[0002] Staphylococcus aureus is a common foodborne pathogen, widely distributed in the natural environment. Under suitable conditions, Staphylococcus aureus can produce enterotoxins, causing food poisoning. With the emergence of methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus infections have become more severe. MRSA is a common, highly virulent bacterium in clinical practice, causing severe gastroenteritis, nausea, vomiting, diarrhea, and abdominal pain within 1 to 6 hours after ingesting contaminated food. MRSA refers to Staphylococcus aureus carrying the mecA gene and resistant to all penicillins, cephalosporins, and carbapenems.

[0003] γ-lactam alkaloids, an important class of secondary metabolites in natural products, have a unique 10-phenyl-substituted perhydroisoindole-1-one skeleton as their main structural backbone. Most existing marketed antibacterial drugs are β-lactam antibiotics. The discovery of this compound provides a novel natural product for the development of drugs against foodborne pathogens. The inventors discovered this new γ-lactam alkaloid natural product during a chemical investigation of the ethyl acetate extract of the marine fungus Aspergillus sp. NBU4698 fermented in culture medium. Currently, there are no reports on the chemical structure and anti-MRSA mechanism of this compound, and therefore, no related drugs are available on the market. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus, as well as its preparation method and uses.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0006] 1. A γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus, the structural formula of which is shown in (I):

[0007]

[0008] 2. The preparation method of the above-mentioned γ-lactam alkaloid compounds with activity against methicillin-resistant Staphylococcus aureus includes the following steps:

[0009] (1) Fermentation production

[0010] Aspergillus ustus with accession number CCTCC NO: M2014086 was streaked on a PDA solid medium plate and incubated upside down in an incubator at 28℃ for 3 days. After that, a single colony was picked and inoculated into PDB liquid medium and cultured on a shaker at 28℃ and 150 rpm for 3 days to obtain the seed liquid. Rice medium was then placed in Erlenmeyer flasks and autoclaved at 121℃ for 15 minutes. 15 mL of seed liquid was then inoculated into each flask of rice medium and cultured at 28℃ under static conditions for 30 days to obtain the fermentation product.

[0011] (2) Extraction of crude extract

[0012] Add an equal volume of ethyl acetate to the fermentation product obtained in step (1), and extract repeatedly until the extract is colorless. Then, evaporate the ethyl acetate extract under vacuum to obtain a crude extract.

[0013] (3) Degreasing and dehydration of crude extract

[0014] The crude extract obtained in step (2) is first dissolved in 95% methanol, and then hexane of the same volume as methanol is added for extraction to remove oil. After repeating the operation three times, the methanol solution is taken and vacuum evaporated to dryness; then it is dissolved in dichloromethane, and water of the same volume as dichloromethane is added for extraction to remove water. The dichloromethane solution is then vacuum evaporated to dryness to obtain the extract.

[0015] (4) Isolation and preparation of compounds

[0016] The extract obtained in step (3) was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel and subjected to normal-phase medium-pressure column chromatography. Gradient elution was performed using a 100:1 petroleum ether-ethyl acetate solution as the eluent, and the eluent was collected. The collected eluent was then subjected to reverse-phase medium-pressure column chromatography. Linear gradient elution was performed using a methanol-water solution with a methanol volume percentage of 45-100% as the eluent, and the eluent fractions were collected and arranged in descending order of polarity to obtain 6 components. The fourth component was purified by semi-preparative reversed-phase high-performance liquid chromatography using a mixed solution of acetonitrile-water with a volume ratio of 53:47 as the mobile phase to obtain a monomeric compound, the structure of which is shown in (I).

[0017]

[0018] Furthermore, the PDA solid culture medium preparation method described in step (1) is as follows: 6g of potato starch, 20g of glucose and 20g of agar are added to 1000mL of distilled water to prepare the medium.

[0019] Furthermore, the preparation method of the PDB liquid culture medium in step (1) is as follows: dissolve 6g of potato starch and 20g of glucose in 1000mL of water.

[0020] Furthermore, the rice culture medium preparation method described in step (1) is as follows: 90g of rice and 3g of sea salt are dissolved in 110mL of water.

[0021] Furthermore, in step (4), the methanol volume percentage in the reversed-phase medium-pressure column chromatography linear gradient elution ranges from 45% to 100%, and the elution time is 120 min.

[0022] Furthermore, the flow rate for the semi-preparative reversed-phase high-performance liquid chromatography compound separation preparation described in step (4) is 2.0 mL / min.

[0023] 3. The use of the above-mentioned γ-lactam alkaloid compounds in the preparation of inhibitors against methicillin-resistant Staphylococcus aureus.

[0024] Compared with existing technologies, the advantages of this invention are as follows: This invention discloses a γ-lactam alkaloid compound, its preparation method, and its uses. The method involves obtaining a fermentation product through the fermentation culture of isolated microorganisms, then extracting the fermentation product with ethyl acetate to obtain a crude extract. The crude extract is then subjected to hexane deoiling and dichloromethane dehydration to obtain a final extract. This final extract is then purified by medium-pressure normal-phase column chromatography, medium-pressure reversed-phase column chromatography, and semi-preparative high-performance liquid chromatography to obtain a monomeric compound. This compound exhibits activity against methicillin-resistant Staphylococcus aureus (MRSA) with low toxicity. Studies on its antibacterial mechanism have revealed that this compound causes irregular and wrinkled bacterial surfaces, thereby disrupting the structure of the bacterial cell wall and cell membrane. It can be used in drug development to inhibit diseases caused by methicillin-resistant Staphylococcus aureus.

[0025] The aforementioned Aspergillus ustus strain DJ003, with accession number CCTCC NO: M2014086, was deposited on March 14, 2014, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China. Attached Figure Description

[0026] Figure 1 The HR-ESI-MS spectra of the compounds of this invention are shown below.

[0027] Figure 2 The UV spectrum of the compound of this invention;

[0028] Figure 3 The nuclear magnetic resonance (IR) spectrum of the compound of this invention;

[0029] Figure 4 The proton NMR spectrum of the compound of this invention;

[0030] Figure 5 The carbon NMR spectrum of the compound of this invention;

[0031] Figure 6 The DEPT-135 NMR spectrum of the compound of this invention;

[0032] Figure 7 The COSY NMR spectrum of the compound of this invention;

[0033] Figure 8 The HSQC nuclear magnetic resonance spectrum of the compound of this invention;

[0034] Figure 9 The nuclear magnetic resonance HMBC spectrum of the compound of this invention;

[0035] Figure 10 The NMR NOESY spectrum of the compound of this invention;

[0036] Figure 11 This is a single-crystal diffraction pattern of the compound of the present invention;

[0037] Figure 12 This is a visual schematic diagram of the COSY and HMBC of the compounds of this invention;

[0038] Figure 13 This is a schematic diagram of the bactericidal curve of the compound of the present invention;

[0039] Figure 14 These are the results of cytotoxicity analysis of the compounds of this invention;

[0040] Figure 15 The following are indicators of the antibacterial mechanism of the compounds of this invention: A represents the scanning electron microscopy (SEM) analysis of MRSA ATCC43300 treated with compound I; B represents the PI staining flow cytometry analysis of MRSA ATCC43300 treated with compound I for 30 or 120 minutes, where red line represents no treatment as a negative control, blue line represents treatment with 1×MIC compound for 30 minutes, orange line represents treatment with 2×MIC compound for 30 minutes, pink line represents treatment with 2×MIC compound for 120 minutes, and purple line represents treatment with 4×MIC compound for 120 minutes; C represents the interaction between compound I and MRSA ATCC43300 genomic DNA, where M represents DNA marker and CK represents untreated DNA. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0042] Example 1: The structural formula of a γ-lactam alkaloid compound with activity against methoxysporin-resistant Staphylococcus aureus is shown in (I):

[0043]

[0044] Example 2: A method for preparing γ-lactam alkaloid compounds as shown in structural formula (I) of Example 1, specifically including the following steps:

[0045] (1) Fermentation production

[0046] Aspergillus ustus with accession number CCTCC NO: M2014086 was streaked onto a plate of PDA solid medium (prepared by adding 6g potato starch, 20g glucose, and 20g agar to 1000mL distilled water). After incubating upside down in a 28℃ incubator for 3 days, a single colony was picked and inoculated into PDB liquid medium (prepared by dissolving 6g potato starch and 20g glucose in 1000mL water). The culture was then incubated on a shaker at 28℃ and 150rpm / min for 3 days to obtain the seed culture. Rice culture medium (90g rice, 3g sea salt, and 110mL water) was added to a 1L Erlenmeyer flask and autoclaved at 121℃ for 15 minutes. Finally, 15mL of the seed culture was inoculated into each flask of rice culture medium and cultured at 28℃ for 30 days to obtain the fermentation product.

[0047] (3) Extraction of crude extract

[0048] Add an equal volume of ethyl acetate to the fermentation product obtained in step (1), and extract repeatedly until the extract is colorless. Then, evaporate the ethyl acetate extract under vacuum to obtain a crude extract.

[0049] (3) Degreasing and dehydration of crude extract

[0050] The crude extract obtained in step (2) is first dissolved in 95% methanol, and then hexane of the same volume as methanol is added for extraction to remove oil. After repeating the operation three times, the methanol solution is taken and vacuum evaporated to dryness; then it is dissolved in dichloromethane, and water of the same volume as dichloromethane is added for extraction to remove water. The dichloromethane solution is then vacuum evaporated to dryness to obtain the extract.

[0051] (4) Isolation and preparation of compounds

[0052] The extract obtained in step (3) was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel and subjected to normal-phase medium-pressure column chromatography. Gradient elution was performed using a 100:1 petroleum ether-ethyl acetate solution as the eluent, and the eluent was collected. The collected eluent was then subjected to reverse-phase medium-pressure column chromatography. Linear gradient elution was performed using a methanol-water solution with a methanol volume percentage of 45-100% as the eluent for 120 min. The eluent fractions were collected, arranged in descending order of polarity, and combined to obtain 6 fractions. The 4th fraction was purified by semi-preparative reversed-phase high-performance liquid chromatography using a 53:47 acetonitrile-water mixture as the mobile phase at a flow rate of 2.0 mL / min to obtain a monomeric compound, the structure of which is shown in (I).

[0053]

[0054] Structural identification of γ-lactam alkaloids prepared by the method in Example 3 and Example 2.

[0055] Compound I of this invention is a white solid. Figure 1 HR-ESI-MS of the cation yielded a quasi-molecular ion peak at m / z 464.2424 [M+H]. + Its molecular formula was determined to be C 28 H 34 O5 (calculated molecular weight 464.2437) indicates an unsaturation degree of 13. This compound... 1 H and 13 C NMR spectra, two-dimensional data, and single crystal data are available in [reference]. Figure 4-12 And Table 1: Figure 6-10 The main two-dimensional correlation of the compounds of this invention; Figure 4 of 1 The HNMR spectrum showed four methyl signals (CH3-11; CH3-12; CH3-22; CH3-23), two methylene signals (CH2-10; CH2-15), and one monosubstituted phenyl signal (δ). H 7 . 14, 7.19, 7.26), 2 pairs of olefin hydrogen signals (CH-13 / 14; CH-19 / 20), the 13-ene and 19-ene groups have trans configurations, and their chemical shifts have large coupling constants (J). H -13 / H-14 = 15.2 Hz and J H -19 / H-20=15.5Hz), 2 oxygen-containing sps 3 Hybridized carbon signal (δ C 88.7, C-9; δ C 80.4, C-18), two sps 2Quaternary carbon signal (δ C 140.5, C-6; δ C 137.5, C-1′), and a conjugated carbonyl signal (δ C 213.1, C-17) and two ester or amide carbonyl signals (δ C 166.2, C-21; δ C 171.5, C-1); by Figure 5 and Figure 6 It is known that this compound contains 28 carbons, among which, according to 13 The C and DEPT 135 spectra indicate the presence of a total of 7 quaternary carbons (C-1, C-6, C-9, C-17, C-18, C-21, C-1′), 2 methylene groups (C-10, C-15), and 15 methine groups (C-3, C-4, C-5, C-7, C-8, C-13, C-14, C-16, C-19, C-20, C-2′ / 3′, C-5′ / 6′, C-4′) in the compound, along with... Figure 4 of 1 Four methyl carbon signals (C-11, C-12, C-22, C-23) were detected by high-field ¹H NMR signal analysis; Figure 7 The COSY spectrum shows that H-19 and H-20, H-2′ / H-3′ / H-4′ / H-5′ / H-6′, H-3 / H-4 / H-5 / H2-10 / H3-11, and H-7 / H-8 / H-13 / H-14 / H2-15 / H-16 / H3-22 exhibit COSY correlations. Furthermore, combined with... Figure 8 The HSQC spectrum reveals the chemical shifts of protons and atoms, indicating that I has a 10-phenyl-substituted perhydroisoindole-1-one skeleton. For example... Figure 9 As shown, the HMBC correlations from H3-11 to C-4 and C-6, and from H3-12 to C-5 and C-7, indicate that C-5 and C-6 in I are connected to methyl groups (CH3-11, CH3-12). The HMBC correlations of CH3-22 with C-15 and C-17 confirm that CH3-22 is connected to C-16, and the HMBC correlations of CH3-23 with C-18 and C-19 confirm that CH3-23 is connected to C-18. Furthermore, the key COSY correlations of H-7 / H-8 / H-13 / H-14 / H2-15 / H-16 / H3-22 show that the olefinic carbon (C-7) in I is connected to C-8, and H-3 / H-4 / H-5 / H2-10 / H3-11 confirm that the methylene carbon (C-10) in I is connected to C-3, thus determining the planar structure of I and ultimately identifying the compound as a new compound. Figure 10 The NOESY spectrum lacks key NOESY correlations, through Figure 11The X-ray single-crystal diffraction pattern determined the absolute configuration of I.

[0056] Table 1. Compound I 1 H and 13 C10 NMR data (DMSO-d6)

[0057]

[0058]

[0059] Note 1: s - singlet, d - doublet, t - triplet, dd - quartet, m - multiplet.

[0060] Note 2: 1 H was obtained by 600MHz NMR; 13 C was obtained by 150MHz NMR.

[0061] Figure 11 This is a single-crystal diffraction pattern of the compound of this invention. Figure 12 The diagrams provided are intuitive examples of the COSY and HMBC of the compounds of this invention. Figure 12 This provides all the information from the aforementioned nuclear magnetic resonance spectrum.

[0062] Example 4, determination of the activity of γ-lactam alkaloid compounds against methicillin-resistant Staphylococcus aureus in Example 1.

[0063] 1. Determination of Minimum Inhibitory Concentration (MIC)

[0064] Experimental materials: methicillin-resistant Staphylococcus aureus ATCC43300 (MRSAATCC43300), MH medium, vancomycin, sterile water, dimethyl sulfoxide (DMSO).

[0065] Experimental procedure: Methicillin-resistant Staphylococcus aureus (MRSA) ATCC43300 was transferred and activated overnight. The activated bacteria were incubated to the logarithmic phase and then diluted to 5 log₂. 10 Compound I was diluted with DMSO to final concentrations of 128, 64, 32, 16, and 8 μg / mL and added to 96-well plates. Vancomycin was diluted with sterile water to a final concentration of 2×MIC and added to 96-well plates to form a positive control group. Diluted bacterial cultures were added to the experimental wells, and the plates were incubated at 37°C for 24 hours to observe bacterial growth. The determination of the minimum inhibitory concentration (MIC) showed that compound I had a MIC of 64 μg / mL against MRSAATCC43300.

[0066] 2. Sterilization curve

[0067] Experimental materials: MRSAATCC43300, MH medium, vancomycin, sterile water, dimethyl sulfoxide (DMSO), PBS phosphate buffer, agar powder.

[0068] Experimental Procedure: MRSAATCC43300 was transferred and activated overnight, then transferred to the logarithmic growth phase. MH solid medium was poured into bacterial culture dishes and allowed to cool and solidify. The bacteria were diluted to a final concentration of 5 log₂. 10 CFU / ml, add 5ml of MH medium to an Erlenmeyer flask, add bacterial suspension, and add compounds at concentrations of 1×MIC, 2×MIC, and 4×MIC according to calculations. For the blank control group, add DMSO at the same volume as the highest drug concentration. For the antibiotic control group, add vancomycin at 2×MIC according to calculations. After mixing, dilute tenfold to the appropriate concentration. Repeat this dilution process to obtain the appropriate concentration, plate the mixture, and perform three parallel experiments. Repeat the procedure at 0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h after treatment. Count and plot after 24h.

[0069] The results are as follows Figure 13 As shown, by Figure 13 It can be seen that for the MRSAATCC43300 control group, the colony count (log) 10 The cfu / mL concentration increased rapidly over 2 to 4 hours and maintained this upward trend over 12 hours, eventually reaching 7.0 log₂O₅. 10 cfu / mL. At a concentration of 1×MIC, the bacterial count increased linearly, reaching 7.4 log [unclear value]. 10 cfu / mL. Within 2–12 h, the bacterial count continued to decrease at a 4×MIC concentration, falling below 1.0 log. 10 At a concentration of 2×MIC, the cfu / mL level slightly increased within 6-8 hours, with the colony count approaching 2.9 log. 10 The concentration of cfu / mL eventually stabilized at 2.5 log10 cfu / mL. Therefore, 2×MIC and 4×MIC exhibit certain antibacterial effects.

[0070] 2. Cytotoxicity

[0071] Experimental materials: RAW264.7 macrophages, DMEM medium, PBS phosphate buffer, dimethyl sulfoxide (DMSO), CCK-8 solution.

[0072] Experimental Procedure: RAW264.7 macrophages were cultured in DMEM medium at 37°C, 5% CO2, and saturated humidity. When the cells reached the logarithmic growth phase, they were resuspended in DMEM medium containing 10% fetal bovine serum and cultured at 2.5 × 10⁻⁶ cells / mL. 5Cells were seeded at a density of 100 μL per well in 96-well plates and incubated in a CO2 incubator for 24 h. After incubation, the DMEM medium was discarded, and the cells were washed twice with PBS buffer. Then, 100 μL of compounds at concentrations of 128, 64, 32, 16, 8, and 4 μg / mL were added to each well according to a concentration gradient. A blank control was prepared by adding only culture medium. Cells were incubated for another 24 h. After incubation, 10 μL / well of CCK-8 solution was added to each well of the 96-well plate, and the plates were incubated at 37°C for 4 h. The absorbance of each well was measured at 460 nm using a microplate reader. Each sample was tested in triplicate.

[0073] The results are as follows Figure 14 As shown, by Figure 14 It is known that when the drug concentration is 128 μg / mL, it exhibits some toxicity, with a survival rate of approximately 65%. The toxicity decreases as the drug concentration decreases. Therefore, Compound I is safe for use in food processing and storage environments, with minimal impact on the host.

[0074] 3. Observe the morphology of biofilms using scanning electron microscopy.

[0075] Experimental materials: MRSAATCC43300, MH medium, dimethyl sulfoxide (DMSO), PBS buffer, anhydrous ethanol, glutaraldehyde solution.

[0076] Experimental procedure: MRSAATCC43300 was activated overnight, then transferred and cultured to mid-log phase. The bacteria were diluted with PBS to a concentration of 1×10⁻⁶. 8 CFU / mL bacterial suspension was added 1 mL / tube to centrifuge tubes. Compound I was added to the bacterial suspension to a final concentration of 4×MIC. Untreated bacterial suspension was used as a negative control. The mixture was incubated at 37°C for 2 h. After incubation, the mixture was centrifuged at 5000 rpm for 5 min at room temperature. The supernatant was discarded, and the bacterial cells were washed twice with PBS buffer. 1 mL of 2.5% glutaraldehyde solution was added to the bacterial pellet, and the cells were gently resuspended by pipetting. The cells were then fixed for 15 min. After glutaraldehyde fixation, the cells were washed twice more with PBS buffer. Then, a gradient dehydration was performed using different concentrations of ethanol solution (20%-50%-70%-85%-95%×2-100%×2), 5 min each time. The sample was then dropped onto a silicon wafer and dried overnight at room temperature. Finally, a metal film was formed on the sample surface using ion sputtering, and the morphology of the gold-palladium sputtered bacteria was observed using an S4800 scanning electron microscope.

[0077] The results are as follows Figure 15As shown in Figure A, scanning electron microscopy results indicate that the bacterial surface in the blank control group was smooth and intact. However, after treatment with compound I at a concentration of 4×MIC, the bacterial surface developed obvious irregular wrinkles, the cell membranes adhered to each other to form filaments, and multiple pores of varying sizes were formed, leading to cytoplasmic leakage and cell death. This suggests that compound I may have the ability to disrupt bacterial cell walls and cell membranes.

[0078] 4. The effect of compound I on bacterial cell membranes

[0079] Experimental materials: MRSAATCC43300, MH medium, dimethyl sulfoxide (DMSO), PBS phosphate buffer, propidium iodide (PI) solution.

[0080] Experimental procedure: Incubate MRSAATCC43300 overnight, then inoculate and culture to the logarithmic growth phase. Dilute the bacteria to 1×10⁻⁶. 8 CFU / mL was added to 1 mL of the solution in a centrifuge tube, followed by the addition of compound I to achieve final concentrations of 1×, 2×, and 4× MIC, respectively. The tubes were then incubated at 37°C for 30 min and 120 min, respectively. After incubation, the tubes were centrifuged at 5000 rpm for 5 min, and then washed twice with PBS buffer. Finally, PI solution was added to a final concentration of 50 μg / mL, and the tubes were incubated at room temperature for 15 min. The untreated group served as a negative control. Fluorescence was finally detected by flow cytometry.

[0081] The results are as follows Figure 15 As shown in B, by Figure 15 As shown in Figure B, the fluorescence intensity of MRSA ATCC43300 without compound I was 1.78%, indicating that the bacterial cell membrane was intact before the application of compound I. With increasing drug concentration, the fluorescence intensity increased from 13.8% (1×MIC) to 16.9% (2×MIC), but decreased at a concentration of 4×MIC. This may be due to the difficulty in capturing bacterial fragments under high drug concentrations, resulting in lower values. After 120 minutes of treatment with compound I, the fluorescence intensity decreased (1×MIC: 2.09%, 2×MIC: 2.37%, 4×MIC: 5.48%). This may be because prolonged high-concentration treatment led to complete bacterial lysis, preventing the bacteria from maintaining their morphology, thus some PI-stained cells were not captured, resulting in decreased fluorescence intensity. Therefore, compound I has a strong effect on disrupting the cell wall and cell membrane.

[0082] 5. The effect of compound I on bacterial DNA

[0083] Experimental materials: MRSA ATCC43300, bacterial genome extraction kit, DMSO.

[0084] Experimental Procedure: MRSAATCC43300 culture was cultured to the logarithmic growth phase, and bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit. Then, compound I diluted to different concentrations (8–128 μg / mL) was mixed with bacterial DNA at a 1:1 volume ratio, and incubated at room temperature for 10 min. The migration of bacterial genomic DNA was then analyzed by 1% agarose gel electrophoresis. DNA not treated with compound I served as a negative control. After electrophoresis, the results were observed using a gel imaging system.

[0085] The results are as follows Figure 15 As shown in C, by Figure 15 As can be seen from C, the blocking effect on the bacterial genome is very small. Therefore, compound I has little effect on changing the structure of the bacterial genome and is even more difficult to bind to DNA.

[0086] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus, characterized in that... The structural formula of the compound is shown in (I); 2. A method for preparing the γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus as described in claim 1, characterized in that... Includes the following steps: (1) Fermentation production Aspergillus ustus with accession number CCTCC NO: M2014086 was streaked on a PDA solid medium plate and incubated upside down in an incubator at 28℃ for 3 days. After that, a single colony was picked and inoculated into PDB liquid medium and cultured on a shaker at 28℃ and 150 rpm for 3 days to obtain the seed liquid. Rice medium was then placed in Erlenmeyer flasks and autoclaved at 121℃ for 15 minutes. 15 mL of seed liquid was then inoculated into each flask of rice medium and cultured at 28℃ under static conditions for 30 days to obtain the fermentation product. (2) Extraction of crude extract Add an equal volume of ethyl acetate to the fermentation product obtained in step (1), and extract repeatedly until the extract is colorless. Then, evaporate the ethyl acetate extract under vacuum to obtain a crude extract. (3) Degreasing and dehydration of crude extract The crude extract obtained in step (2) is first dissolved in 95% methanol, and then hexane of the same volume as methanol is added for extraction to remove oil. After repeating the operation three times, the methanol solution is taken and vacuum evaporated to dryness; then it is dissolved in dichloromethane, and water of the same volume as dichloromethane is added for extraction to remove water. The dichloromethane solution is then vacuum evaporated to dryness to obtain the extract. (4) Isolation and preparation of compounds The extract obtained in step (3) was first dissolved in a 1:1 mixture of dichloromethane and methanol, then mixed with 200-300 mesh silica gel and subjected to normal-phase medium-pressure column chromatography. Gradient elution was performed using a 100:1 petroleum ether-ethyl acetate solution as the eluent, and the eluent was collected. The collected eluent was then subjected to reverse-phase medium-pressure column chromatography. Linear gradient elution was performed using a methanol-water solution with a methanol volume percentage of 45-100% as the eluent, and the eluent fractions were collected and arranged in descending order of polarity to obtain 6 components. The fourth component was purified by semi-preparative reversed-phase high-performance liquid chromatography using a mixed solution of acetonitrile-water with a volume ratio of 53:47 as the mobile phase to obtain a monomeric compound, the structure of which is shown in (I).

3. The method for preparing a γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus according to claim 2, characterized in that... The method for preparing the PDA solid culture medium in step (1) is as follows: 6g of potato starch, 20g of glucose and 20g of agar are added to 1000mL of distilled water to prepare the medium.

4. The method for preparing a γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus according to claim 2, characterized in that... The preparation method of PDB liquid culture medium in step (1) is as follows: dissolve 6g of potato starch and 20g of glucose in 1000mL of water.

5. The method for preparing a γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus according to claim 2, characterized in that... The rice culture medium prepared in step (1) is as follows: 90g rice, 3g sea salt, and 110mL water.

6. The method for preparing a γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus according to claim 2, characterized in that: In step (4), the methanol volume percentage in the reversed-phase medium-pressure column chromatography linear gradient elution ranges from 45% to 100%, and the elution time is 120 min.

7. The method for preparing a γ-lactam alkaloid compound with activity against methicillin-resistant Staphylococcus aureus according to claim 2, characterized in that: The flow rate for the semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC) preparation of compounds in step (4) is 2.0 mL / min.

8. Use of the γ-lactam alkaloid compound of claim 1, which has activity against methicillin-resistant Staphylococcus aureus (MRSA), in the preparation of an inhibitor against MRSA.

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