A new naphthoquinone heteroterpenoid compound and its preparation method and application

Abstract

The present invention belongs to the field of natural medicines, and specifically discloses a naphthoquinone heteroterpenoid compound with a novel chemical structure, a preparation method thereof, and an application thereof in the preparation of an antibacterial agent for animals. Streptomyces A new naphthoquinone heteroterpenoid compound was prepared from sp. HBERC-16614 and named spironapthomeroterpenoid. The compound spironapthomeroterpenoid of the present invention was confirmed by in vitro antibacterial tests to have a significant inhibitory effect on Staphylococcus aureus ( Staphylococcus aureus ) growth. It can be used to prepare veterinary antibacterial drugs. This invention provides alternative compounds for the development of new veterinary antibiotics and is of great significance to the development of my country's microbial drug resources.

Description

Technical Field

[0001] The present invention belongs to the field of natural medicine preparation, and particularly relates to a new naphthoquinone heteroterpenoid compound, a preparation method thereof, and application thereof in the preparation of veterinary antibacterial drugs. Background Art

[0002] Staphylococcus aureus, also known as "golden Staphylococcus," belongs to the genus Staphylococcus and is a representative of Gram-positive bacteria. It is a common foodborne pathogen that can be transmitted through the ingestion of contaminated milk and dairy products, and meat products (including cured meats), and has the characteristics of cross-infection between humans and animals. Streptococcus suis is an important zoonotic pathogen that can cause a variety of pathological conditions such as sepsis, meningitis, arthritis, and pneumonia. Currently, animals infected with these pathogens are mainly treated with antibiotics, but different pathogen strains have different drug sensitivities, and the misuse of antibiotics can also cause bacterial resistance. Therefore, the development of new antimicrobial drugs to control these pathogens has become an urgent problem that the pig industry needs to solve. Finding safe and effective antibiotics from natural products will be an important way to develop new drugs.

[0003] Naphthoquinone terpenes are a large class of natural products isolated from soil and marine Streptomyces. They include compounds such as napyradiomycins, naphterpins, marinones, merochlorins, and furaquinocins, which exhibit a wide range of biological activities, including antibacterial, antitumor, cytotoxic, and neuroprotective activities. Napyradiomycins represent a large family of this class of compounds, with over 50 compounds of related structures reported. These compounds exhibit diverse biological activities, particularly excellent antibacterial activity against Gram-positive bacteria. In particular, napyradiomycins A1 and B1, the first compounds reported in this family, not only exhibit excellent activity against Staphylococcus aureus (MIC < 0.78 μg / mL), but also exhibit strong antibacterial activity against Streptococcus suis, with MIC values ​​of 3.125 and 6.25 μg / mL, respectively. In addition, during our previous screening of veterinary antibiotics, a new C-16 oxidized napyradiomycin A1 derivative, napyradiomycin A4, was isolated from soil-derived Streptomyces kebangsaanensis WS-68302. This derivative showed significant antiviral activity against PRV (pseudorabies virus), with an IC 50 The value was 2.056 μM, which showed moderate activity against Erysipelothrix rhusiopathogenes. This is the first report of the activity of this family of compounds against zoonotic pathogens.

[0004] Naphterpins, naphthgeranines, and naphthablins belong to another family of naphthoquinone terpenoids. Naphterpin, naphterpins B, and C are derived from Streptomyces CL-190 and exhibit significant antioxidant activity. The more complex naphthoquinone terpenoid, naphthablin, was isolated from the fermentation broth of Streptomyces punctatus as an inhibitor of the Abl oncogene. Subsequently, naphthablins B and C were isolated from Streptomyces CP26-58 and exhibited weak activity against HeLa cells. Although these compounds exhibit a variety of biological activities, no antibacterial activity has been reported.

[0005] Microorganisms are a treasure trove of resources for discovering bioactive natural products. Secondary metabolites from actinomycetes provide a precursor to new drug discovery. Most antibiotics used in medicine, veterinary practice, and agriculture originate from Streptomyces. During an earlier screening for veterinary antibiotics, our research team discovered a strain of Streptomyces, identified as Streptomyces spp. and designated HBERC-16614. Its fermentation extract exhibited strong antibacterial activity against Staphylococcus aureus. Therefore, we isolated and purified a large amount of this fermentation extract and systematically investigated its chemical composition and biological activity. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a new naphthoquinone heteroterpenoid compound prepared from the fermentation product of Streptomyces sp. HBERC-16614, and a preparation method and application thereof.

[0007] One object of the present invention is to provide a new naphthoquinone heteroterpenoid compound, spironapthomeroterpenoid.

[0008] Another object of the present invention is a method for preparing the naphthoquinone heteroterpenoid compound spironapthomeroterpenoid.

[0009] Another object of the present invention is to provide the use of a new naphthoquinone heteroterpenoid compound in the preparation of animal antibacterial drugs, especially to provide the use of a new naphthoquinone heteroterpenoid compound spironapthomeroterpenoid in the preparation of antibacterial drugs targeting Staphylococcus aureus.

[0010] In order to achieve the above object, the technical solution of the present invention is:

[0011] When the applicant was applying microbial resources to screen livestock antibiotics, he discovered a strain of Streptomyces, which was identified as Streptomyces sp. and numbered HBERC-16614. Its fermentation extract has strong antibacterial activity against Staphylococcus aureus. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on June 13, 2024, address: Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M 20241209 and a classification name: Streptomyces sp. HBERC-16614.

[0012] Furthermore, the applicant separated and extracted the fermentation product of the strain and obtained a new naphthoquinone heteroterpenoid compound, chemically named spironapthomeroterpenoid, whose chemical structure is as follows:

[0013]

[0014] The protection scope of the present invention includes:

[0015] The preparation method of the above-mentioned compound spironapthomeroterpenoid comprises the following steps: freeze-drying the fermentation culture of Streptomyces sp. HBERC-16614, extracting with ethyl acetate to obtain a crude ethyl acetate extract; and subjecting the crude ethyl acetate extract to column chromatography separation and purification to obtain the monomer compound spironapthomeroterpenoid.

[0016] The above-mentioned preparation method, preferably, the preparation method of the fermentation culture comprises: inoculating the activated Streptomyces sp. HBERC-16614 strain into a fermentation medium; the fermentation medium contains, per liter, the following: mannitol 5-15g, glucose 5-15g, malt extract powder 5-15g, yeast extract powder 3-8g, calcium carbonate 1-5g, corn steep liquor 0.5-1.5g, pH 7.0, and the solvent is water.

[0017] In the above-mentioned preparation method, preferably, the step of subjecting the crude ethyl acetate extract to column chromatography for purification comprises: ① subjecting the crude ethyl acetate extract to silica gel column chromatography, first using a petroleum ether-ethyl acetate mixed solvent for gradient elution in sequence, and then using an ethyl acetate-methanol mixed solvent for gradient elution, and collecting the petroleum ether-ethyl acetate eluate; ② taking the petroleum ether-ethyl acetate eluate with a volume ratio of 12:1 and separating it by preparative HPLC, using acetonitrile-water as the mobile phase, and using a gradient elution with 40-100% acetonitrile-water by volume fraction, adding acetic acid to the mobile phase, collecting the components containing naphthoquinone heteroterpenoid compounds, and concentrating to obtain the monomer compound spironapthomeroterpenoid.

[0018] The protection scope of the present invention also includes:

[0019] A combination containing the compound spironapthomeroterpenoid.

[0020] The invention relates to the use of the compound spironapthomeroterpenoid as the sole active ingredient or one of the active ingredients in the preparation of a medicine for treating or preventing Staphylococcus aureus infection.

[0021] The invention relates to an application of the compound spironapthomeroterpenoid as the sole active ingredient or one of the active ingredients in the preparation of a Staphylococcus aureus antibacterial agent.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a novel naphthoquinone heteroterpenoid compound, spironapthomeroterpenoid, which exhibits significant antibacterial activity, particularly against Staphylococcus aureus, with an MIC of 3.125 μg / mL, which is stronger than current antibiotics. Spironapthomeroterpenoid has significant antibacterial activity and can serve as a lead compound for the development of antibacterial drugs, thus significantly contributing to the development of microbial drug resources in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a compound of spironapthomeroterpenoid 1 H-NMR spectrum.

[0025] Figure 2 It is a compound of spironapthomeroterpenoid 13 C-NMR spectrum.

[0026] Figure 3 This is the HSQC spectrum of the compound spironapthomeroterpenoid.

[0027] Figure 4 It is a compound of spironapthomeroterpenoid 1 H- 1 HCOSY spectrum.

[0028] Figure 5 This is the HMBC spectrum of the compound spironapthomeroterpenoid.

[0029] Figure 6This is the NOSEY spectrum of the compound spironapthomeroterpenoid.

[0030] Figure 7 This is the HREIMS spectrum of the compound spironapthomeroterpenoid.

[0031] Figure 8 This is the ultraviolet absorption spectrum of the compound spironapthomeroterpenoid. DETAILED DESCRIPTION

[0032] The following examples are used to illustrate the present invention but are not intended to limit the scope of the invention. The experimental methods in the following examples where specific conditions are not specified are generally based on conventional experimental conditions.

[0033] Example 1:

[0034] Obtaining Streptomyces sp. HBERC-16614:

[0035] When the applicant was applying microbial resources to screen livestock antibiotics, he discovered a strain of Streptomyces, which was identified as Streptomyces sp. and numbered HBERC-16614. Its fermentation extract has strong antibacterial activity against Staphylococcus aureus. The strain was deposited in the China Center for Type Culture Collection (CCTCC) on June 13, 2024, address: Wuhan University, Wuhan, China, with a deposit number of CCTCC NO: M 20241209, and a classification name: Streptomyces sp. HBERC-16614.

[0036] Example 2:

[0037] Fermentation method of Streptomyces HBERC-16614

[0038] The strain of Streptomyces HBERC-16614 was inoculated into a 500 mL conical flask containing 100 mL of seed culture medium (1% mannitol, 1% glucose, 1% malt extract, 0.5% yeast extract, 0.25% calcium carbonate, 0.1% corn steep liquor, pH 7.0) and fermented for 96 hours.

[0039] Under sterile conditions, 10% of the seed culture was transferred to a 500-mL conical flask containing 100 mL of fermentation medium (containing per liter: 5-15 g mannitol, 5-15 g glucose, 5-15 g malt extract powder, 3-8 g yeast extract powder, 1-5 g calcium carbonate, 0.5-1.5 g corn steep liquor, pH 7.0, solvent: water), and cultured on a shaker at 28° C. and 150 rpm for 120 h to obtain a fermentation broth of strain HBERC-16614.

[0040] Determination of antibacterial activity of fermentation broth

[0041] The purified bacteria were streaked onto LB or TSA solid medium and cultured at 37°C for 24 h. A single colony was picked and inoculated into LB or TSB liquid medium and cultured at 37°C and 200 r / min on a shaker for 12 h. The bacterial suspension was inoculated into LB or TSB liquid medium at a ratio of 1:1000 and cultured at 37°C and 200 r / min on a shaker until the OD 600 nm About 0.5 (bacterial count is about 1×10 8 CFU / mL), and then dilute the bacterial suspension 1:100 for later use. Centrifuge the fermentation broth at 12000rpm for 15min, take the supernatant, and dilute it in series with LB or TSB liquid culture medium. Add 50μL of fermentation broth of different concentrations to each well of a 96-well culture plate, add 50μL of diluted bacterial solution, and repeat each fermentation broth sample 3 times for each strain of bacteria. At the same time, set up a negative control (add 50μL culture medium) and a positive control (50μL of 50μg / mL penicillin). Place it in a 37℃ incubator and incubate for 16-20h, and read the OD 600nm Results. Visually observe the microdilution wells for the presence of no bacterial growth (or OD 600nm The results were similar to those of the positive control wells and showed antibacterial activity.

[0042] Antibacterial activity results of the fermentation broth of Streptomyces HBERC-16614

[0043] The fermentation broth of Streptomyces sp. HBERC-16614 obtained in Example 2 was diluted 2, 4, 8, and 16 times, respectively. The antibacterial activity of the fermentation broth dilutions against two Gram-positive bacteria, Staphylococcus aureus and Streptococcus suis, was tested according to the antibacterial activity test method in Example 2. The results are shown in Table 1. The fermentation broth had a strong antibacterial effect against both bacteria when diluted 2, 4, and 8 times, respectively. In particular, when diluted 2 times, the OD values ​​of Staphylococcus aureus and Streptococcus suis were significantly higher. 600nm The values ​​were 0.033 and 0.042 respectively, and the antibacterial activity was better than that of the positive control penicillin.

[0044] Table 1 Antibacterial activity results of the fermentation broth of Streptomyces HBERC-16614

[0045]

[0046] Example 3:

[0047] Preparation method of compound spironapthomeroterpenoid

[0048] 1. The fermentation culture prepared in Example 2 was lyophilized. The lyophilized powder was moistened with a small amount of methanol / water and extracted with ethyl acetate three times. The extracts were combined and concentrated to dryness under reduced pressure to obtain 12.5 g of a crude ethyl acetate extract.

[0049] 2. Column chromatography purification of the crude ethyl acetate extract: The crude ethyl acetate extract was separated by silica gel column chromatography, first using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 20:1, 15:1, 12:1, 10:1, 8:1, 3:1, 1:1, and 100% ethyl acetate in a gradient elution, followed by elution with an ethyl acetate-methanol mixed solvent with a volume ratio of 20:1 and 10:1, respectively. The 12:1 volume ratio petroleum ether-ethyl acetate eluate was collected and concentrated to obtain 30.86 mg of a crude fraction. The crude fraction of the petroleum ether-ethyl acetate eluate was separated by preparative HPLC using an acetonitrile-water mobile phase with a gradient elution of 40-100% acetonitrile-water by volume. 0.2% acetic acid was added to the mobile phase. The fraction containing naphthoquinone heteroterpenoid compounds was collected and concentrated to obtain the monomer compound spironapthomeroterpenoid.

[0050] 3. Structure confirmation: The chemical structure of the compound spironapthomeroterpenoid was determined through comprehensive analysis of multiple modern spectral techniques such as UV spectroscopy, IR spectroscopy, 1D and 2D-NMR spectroscopy, and HR-ESIMS.

[0051] Spironapthomeroterpenoid: light brown amorphous solid, [α] D 24 =-16.8(c=0.22MeOH); IR(KBr)υ max 3393, 2951, 1707, 1641, 1614, 1456, 1377, 1248, 1161, 1020, 871, 746, 699cm -1 ; UV(MeOH)λ max(logε)249(3.23),303(2.87),350(2.90)nm; ESI-MS m / z425.25 / 427.27 / 429.20[M–H]-; HR-ESI-MS m / z 425.0931[M–H] - (calcd for C 21 H 23 Cl2O5,425.0928); 1 H and 13 The C NMR data are shown in Table 1.

[0052] Table 1 NMR data of compound spironapthomeroterpenoid (CD3OD)

[0053]

[0054] According to the comprehensive analysis of the above infrared, ultraviolet, mass spectrometry, one-dimensional and two-dimensional nuclear magnetic spectrum data ( Figures 1-8 ), the compound structure was deduced as follows and named spironapthomeroterpenoid:

[0055]

[0056] Example 4:

[0057] Antibacterial activity of the compound spironapthomeroterpenoid

[0058] 1. Biological activity assay

[0059] The minimum inhibitory concentration (MIC) of the drug was determined using the broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI 2012). Purified bacteria were streaked onto LB or TSA solid medium and incubated at 37°C for 24 h. A single colony was picked and inoculated into LB or TSB liquid medium and incubated at 37°C at 200 rpm for 12 h. The bacterial suspension was inoculated into LB or TSB liquid medium at a ratio of 1:1,000 and incubated at 37°C at 200 rpm until the OD reached 0. 600 nm is about 0.5 (the bacterial count is about 1×10 8CFU / mL), and then dilute the bacterial suspension 1:100 for later use. In a 96-well culture plate, add 50 μL of serially diluted drugs of different concentrations to each well, and add 50 μL of diluted bacterial solution. Repeat 3 times for each drug and each strain of bacteria. Set up negative and positive controls. Select cefpirome and streptomycin as control drugs and incubate in a 37°C incubator for 16-20 hours. Read the results. Visually observe the microdilution wells and the highest drug dilution concentration with no bacterial growth as the minimum inhibitory concentration of the drug for this type of bacteria. Repeat the operation 3 times in parallel, and take the average of the 3 results as the MIC of this drug for this bacteria.

[0060] 2. Antibacterial activity results

[0061] The results showed that the compound spironapthomeroterpenoid obtained in Example 3 had a certain antibacterial effect on the tested S. aureus ATCC 25923 and S. suis SC19, but had no activity against the Gram-negative bacteria Escherichia coli.

[0062] Table 2 Antibacterial activity of compound spironapthomeroterpenoid (MIC, μg / mL)

[0063]

[0064] 3. This experiment shows that the compound spironapthomeroterpenoid has significant antibacterial activity against Staphylococcus aureus, with an MIC value of 3.125μg / mL, which is significantly higher than that of positive drugs. It is expected to be used as a compound or lead compound to develop new anti-Staphylococcus aureus therapeutic drugs.

[0065] The above description is only a preferred 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 naphthoquinone heteroterpenoid compound, characterized in that: The chemical name is spironapthomeroterpenoid, and the chemical structure is as follows: 。 2. A method for preparing the naphthoquinone heteroterpenoid compound according to claim 1, characterized in that The naphthoquinone heteroterpenoid compound is obtained from Streptomyces Streptomyces sp. HBERC-16614 fermentation culture, the specific steps are as follows: A. Preparation of Streptomyces Streptomyces Fermentation culture of sp. HBERC-16614; B. Lyophilize the fermentation culture, moisten the lyophilized powder with a small amount of methanol / water, and extract the lyophilized powder three times with ethyl acetate. Combine the extracts and concentrate the extracts to dryness under reduced pressure to obtain a crude ethyl acetate extract. C. The crude ethyl acetate extract is separated and purified by column chromatography to obtain the monomer compound spironapthomeroterpenoid.

3. The method for preparing naphthoquinone heteroterpenoid compounds according to claim 2, wherein The step A is as follows: Streptomyces Streptomyces sp. HBERC-16614 fermentation culture is Streptomyces sp. Streptomyces sp. HBERC-16614 was inoculated into a fermentation medium and fermented. The fermentation medium contained, per liter, 5-15 g of mannitol, 5-15 g of glucose, 5-15 g of malt extract powder, 3-8 g of yeast extract powder, 1-5 g of calcium carbonate, 0.5-1.5 g of corn steep liquor, pH 7.0, and the solvent was water.

4. The method for preparing naphthoquinone heteroterpenoid compounds according to claim 2, wherein: The step C is specifically as follows: ① The crude ethyl acetate extract was separated by silica gel column chromatography. First, a petroleum ether-ethyl acetate mixed solvent was used for gradient elution at a volume ratio of 20:1, 15:1, 12:1, 10:1, 8:1, 3:1, 1:1, and 100% ethyl acetate, and then an ethyl acetate-methanol mixed solvent was used for elution at a volume ratio of 20:1 and 10:

1. The petroleum ether-ethyl acetate eluate with a volume ratio of 12:1 was collected. ② The 12:1 petroleum ether-ethyl acetate eluate was concentrated to a paste and separated by preparative HPLC with acetonitrile-water as the mobile phase. A gradient elution of 40-100% acetonitrile-water was performed with 0.2% acetic acid added to the mobile phase. The fractions containing naphthoquinone heteroterpenoid compounds were collected and concentrated to obtain the monomer compound spironapthomeroterpenoid.

5. Use of the naphthoquinone heteroterpenoid compound according to claim 1 in the preparation of veterinary antibacterial drugs.

6. Use of the naphthoquinone heteroterpenoid compound according to claim 1 in the preparation of veterinary antibacterial drugs targeting Staphylococcus aureus and Streptococcus suis.

7. Use of the naphthoquinone heteroterpenoid compound according to claim 5 or 6 in the preparation of veterinary antibacterial drugs, characterized in that: The antibacterial target is Staphylococcus aureus ATCC 25923, or Streptococcus suis SC19, or Escherichia coli ATCC 25922.

8. The method for preparing naphthoquinone heteroterpenoid compounds according to claim 2, characterized in that: The Streptomyces Streptomyces sp. HBERC-16614 strain deposit number is CCTCC NO: M 20241209.

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