CN109280034B

CN109280034B - Benzoxazepine compound with antibacterial activity and preparation method and application thereof

Info

Publication number: CN109280034B
Application number: CN201810866115.7A
Authority: CN
Inventors: 陈名洪; 江红; 张文龙; 林如; 谢阳; 方东升; 连云阳
Original assignee: Fujian Shengwei Biotechnology Co ltd
Current assignee: FUJIAN SHENGWEI BIOTECHNOLOGY Co.,Ltd.
Priority date: 2018-08-01
Filing date: 2018-08-01
Publication date: 2021-06-15
Anticipated expiration: 2038-08-01
Also published as: CN109280034A

Abstract

The invention belongs to the technical field of natural compounds, and particularly relates to a benzoazepine compound with antibacterial activity, and a preparation method and application thereof. The benzoazepine compound is a new-structure compound, is obtained by fermenting and extracting verruca bacteria V.gifhornensis FIM06-0025 screened by the applicant, and has broad-spectrum inhibitory activity on gram-negative bacteria and gram-positive bacteria through determination, particularly has strong inhibitory activity on gram-negative bacteria helicobacter pylori, Klebsiella pneumoniae and gram-positive bacteria staphylococcus aureus, and can be used for preparing broad-spectrum antibacterial drugs.

Description

Benzoxazepine compound with antibacterial activity and preparation method and application thereof

Technical Field

The invention belongs to the technical field of natural compound extraction, and particularly relates to a benzoazepine compound with antibacterial activity, and a preparation method and application thereof.

Background

Since the discovery of medical antibiotic penicillin in 1929 and the discovery of agricultural antibiotic blasticidin S in 1958, more than 4000 antibiotics have been discovered all over the world so far, play an important role in the prevention and control of human and animal diseases and the prevention and control of agricultural harmful organisms, and promote the progress of human civilization and scientific technology.

However, the use of a large number of broad-spectrum antibiotics accelerates the evolution of pathogenic bacteria, and causes the drug resistance of pathogens, particularly bacterial pathogens, to be enhanced day by day, so that the curative effect of the existing drugs is reduced. In addition, the use of a large number of broad-spectrum antibiotics has led to an increase in the number of types of multidrug-resistant bacteria, resulting in the development of resistance from gram-negative bacteria to gram-positive bacteria, and from nosocomial infections to nosocomial infections. The continuous emergence and rapid spread of drug-resistant bacteria and multidrug-resistant bacteria have made the prevention, treatment and control of bacterial infections pose serious challenges, and thus the problem of bacterial resistance is one of the most serious public health problems in the 21 st century.

The drug resistance problem of the existing bacteria is highlighted in the carbapenem antibiotic resistant enterobacteriaceae and non-fermentation sugar bacteria, particularly the drug resistance of Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and the like is the most serious, and the clinical treatment is the most difficult. Although a great deal of research work is carried out by many scholars, only two classes of anti-gram-positive bacteria, namely oxazolidinone and cyclic lipopeptide, are approved to be on the market in the last 30 years, and the anti-gram-negative bacteria are fewer, so that the current clinical application is still not optimistic. In the face of the intractable problems of continuous upgrade of bacterial drug resistance, reduced curative effect of antibacterial drugs, clinical no drug availability and the like, the work of developing novel anti-drug-resistant bacteria drugs is urgent.

The search of active compounds from microbial secondary metabolites as lead compounds to create new drugs is one of the accepted effective ways of pharmaceutical workers in the world, and the microbial resources provide an infinite source for the research of innovative drugs. The abundant and diverse secondary metabolites of the microorganisms provide a large number of precious mode structures and drug precursor micromolecules for the research and development of new chemical drugs, are important material bases for the innovation of drug discovery sources and continuous innovation, and have decisive significance for the research of the whole innovative drugs.

Actinomycetes are the main sources of microbial drugs, and Streptomyces and some rare actinomycetes are the main sources. However, since the 70 s, the chances of finding new compounds from streptomyces became less and the repetition rate of new compounds became higher. These currently available actinomycetes of a small species (commonly called rare actinomycetes) such as verrucella, nocardia, and the like have been attracting increasing attention. A large number of active compounds with novel structures are contained in secondary metabolites of rare actinomycetes, so that the probability of obtaining target active compounds is improved.

Of rare actinomycetes, Verrucosispora (Verrucosispora) is known to belong to the order actinomycetes, Micromonosporaceae (Micromonosporaceae). Since the earliest isolation of wartwort from marsh sludge by rheoims et al in 1998, scientists continued to isolate this genus of strain from marine muds, mangrove, ascidians and sponges. It is reported that verrucaria is becoming an important bacterial source of microbial drugs due to its abundance in natural product resources. In 2004, verrucosispora maris AB 18-032 belonging to Micromonosporaceae was isolated from 289m deep sediments in the Japanese sea, and the strain can produce polyketide abysomicin C with MRSA and VRSA activity, and the compound has a new antibacterial effect target and has positive significance for searching novel high-efficiency antibiotics. Bull and Stach were found to contain at least 20 gene clusters synthesized from natural products by genomic analysis of verrucosispora maris AB 18-032, which together with the findings of Net et al demonstrated that Verrucaria verruculosaThe bacteria not only can synthesize antibacterial active substances, but also can metabolize and synthesize more natural compounds. As another example, verrucosispora sp.mg-37 isolated from sediments 250 m deep in the norwegian sea in 2008, synthesized anti-tumor fosfomes compounds (proximicins); in 2010, Dai et al isolated verrucosispora sediminis MS426 from sediments in the south sea at 3602 m depth^TAnd 8 cyclic-dipeptides (cyclo-dipeptides) and two nocardiam leisures (nocardamine-like) were isolated from the strain, some of which had antibacterial and antifungal activity; in 2010, Shirai et al isolated two new diterpenoid compounds (gifhornenolones A and B) with anti-prostate cancer from the strain verrucosispora gifhornensis YM 28-088. In addition, compounds with novel structures and excellent activity, such as antineoplastic compounds thiochromdriline C and Harrucomicin C, are also separated from the verruca. The antibacterial agent with a novel structure or a novel action mechanism is screened from the verruca acuminata, so that the method has practical feasibility for coping with multi-drug resistant bacteria.

Disclosure of Invention

Therefore, the invention aims to provide a benzoazepine compound and further discloses a preparation method and application thereof.

In order to solve the technical problems, the benzoazepine compound or the pharmaceutically acceptable salt thereof has a structure shown in the following formula (1):

the invention also discloses a method for preparing the benzoazepine compound, which comprises the steps of carrying out fermentation culture on Verrucosispora gifhornensis FIM06-0025 and extracting the benzoazepine compound from the obtained fermentation liquor;

the verrucaria Verrucosispora gifhornensis FIM06-0025 is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.15242 and the preservation date of 2018, 01 month and 18 days.

Specifically, the fermentation culture step comprises the step of inoculating the verrucaria V.gifhornensis FIM06-0025 into a fermentation culture medium for fermentation, and collecting fermentation liquor;

the fermentation medium contains per liter: soluble starch 20.0g, K₂HPO₄ 0.5g，KNO₃ 5.0g， MgSO₄·7H₂O 0.5g，NaCl 0.5g，FeSO₄·7H₂O 0.01g，CaCO₃1.0g, and the balance of seawater, and adjusting the pH value to 7.2-7.5.

Specifically, the step of extracting the benzoazepine compound specifically comprises the following steps:

(1) performing solid-liquid separation on the collected fermentation liquor to obtain mycelium and supernatant for later use;

(2) adding the separated mycelia into a mixed solvent of methanol and acetone for extraction, and concentrating the obtained extract to remove the solvent to obtain a first extract; mixing the obtained first extract with the separated supernatant, adding ethyl acetate for extraction, and concentrating to obtain a second extract;

(3) and (3) carrying out C18 reverse phase silica gel column chromatography on the obtained second extract, and respectively carrying out the following steps of: 100. 50: 100. 70: 100. 70: performing gradient elution by using a methanol-water solution of 90 as an eluent, and collecting the elution concentration of the eluent as 70: 100 to obtain a first component a-1;

(4) the obtained first component a-1 was subjected to silica gel column chromatography with chloroform-methanol solution as an eluent in a volume ratio of 9: 1-1: 1, performing gradient elution, collecting eluent in parts, detecting by TLC, and performing gradient elution according to the volume ratio of 10: 1, taking chloroform-methanol as a developing agent and iodine as a color developing agent, combining the same components, and collecting the component with Rf value of 0.55 by TLC detection to obtain a second component A-2;

(5) and carrying out Sephadex LH-20 column chromatography on the obtained second component A-2, and carrying out mass transfer on the second component A-2 according to a volume ratio of 1: 1 in methanol-acetonitrile as eluent, collecting elution fractions, detecting by TLC, and separating by using a solvent with a volume ratio of 10: 1, and collecting the component with Rf value of 0.61 to obtain the benzoazepine compound with the structure as shown in claim 1.

Further, the volume ratio of the methanol to the acetone is 1: 1, the volume ratio of the mixed solvent of the methanol and the acetone to the mycelium is 1.5-2.0: 1.

in the step (2), the extraction step is ultrasonic extraction.

The invention also discloses application of the benzoazepine compound or pharmaceutically acceptable salt thereof in preparing antibacterial drugs.

Specifically, the antibacterial drugs comprise anti-helicobacter pylori, pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, escherichia coli, staphylococcus aureus, candida albicans or enterococcus faecium drugs.

The invention also discloses an antibacterial drug which takes the benzoazepine compound or the pharmaceutically acceptable salt thereof as an active ingredient.

The invention also discloses a verruca vulgaris strain which is classified and named as Verrucosispora gifhornensis and has been preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.15242, and the preservation date is 2018, 01 month and 18 days.

The invention also discloses the use of the verruca vulgaris strain for preparing the benzoazepine compound according to claim 1 by fermentation.

The benzoazepine compound is a new-structure compound, is obtained by fermenting and extracting verruca v.gifhornensis FIM06-0025 screened by the applicant, and has broad-spectrum inhibitory activity on gram-negative bacteria and gram-positive bacteria through determination, particularly has strong inhibitory activity on gram-negative bacteria helicobacter pylori, klebsiella pneumoniae and gram-positive bacteria staphylococcus aureus, and can be used for preparing broad-spectrum antibacterial drugs.

The benzoazepine compound is obtained by fermenting and extracting verrucaria V.gifhornnensis FIM06-0025, and the preparation method is simple and easy to implement.

Drawings

In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with the accompanying drawings, which,

FIG. 1 shows the single colony morphology of the strain FIM06-0025 of the present invention in nutrient agar medium;

FIG. 2 is a scanning electron micrograph of the strain FIM06-0025 of the present invention;

FIG. 3 is an HRESI-TOF-MS spectrum of Compound FW251 of the present invention;

FIG. 4 is an IR spectrum of compound FW251 of the present invention;

FIG. 5 shows the preparation of compound FW251 according to the invention¹H-NMR spectrum;

FIG. 6 shows the preparation of compound FW251 according to the invention¹H-¹H COSY spectrum;

FIG. 7 is an HMBC spectrum of compound FW251 of the present invention;

FIG. 8 shows the preparation of compound FW251 according to the invention¹³C-NMR spectrum;

FIG. 9 is an HSQC spectrum of compound FW251 of the present invention;

FIG. 10 is a DEPT spectrum of compound FW251 of the present invention;

FIG. 11 shows the chemical structure and essential structure of the compound FW251 of the present invention¹H-¹H COSY and HMBC correlation spectra.

Detailed Description

Example 1

A strain is selected from sponge of east China sea of Fujian province, named FIM06-0025, and the taxonomic characteristics of the strain are as follows through detection.

1.1 physicochemical Properties of the Strain

The single colony morphology of strain FIM06-0025 in nutrient agar medium is shown in FIG. 1. From the scanning electron microscope image (FIG. 2) of the strain FIM06-0025, the intrabasal mycelia developed well with septate branches and a diameter of 0.4 μm, and a single spore grew on a short spore stem, and the spore had a handle and a wart-like projection on the surface.

The selected strain FIM06-0025 was inoculated with an inoculating loop under aseptic conditions onto agar slant medium of ISP1-ISP7 as follows, cultured at 32 ℃ for 2-5d, and the culture characteristics of the FIM06-0025 strain in different media were observed and recorded (see Table 1 below).

The nutrient agar culture medium comprises the following components (g/L): 3.0 parts of beef extract, 10.0 parts of peptone, 2.0 parts of yeast extract, 5.0 parts of sodium chloride, 12.0 parts of agar and 1000mL of distilled water, and the pH value is adjusted to 7.2-7.5 before sterilization.

The ISP1 culture medium comprises the following components (g/L): tryptone 0.5, yeast extract 0.3, agar 2, distilled water 1L, adjusting pH to 7.2-7.5 before sterilization.

The ISP2 culture medium comprises the following components (g/L): 0.4% of yeast extract, 1% of malt extract, 0.4% of glucose, 2% of agar and 1L of distilled water, and adjusting the pH value to 7.3 before sterilization.

The ISP3 culture medium comprises the following components (g/L): oat flour 2, agar 2, trace elements 1mL, agar 2 and distilled water 1L, and adjusting the pH value to 7.4 before sterilization.

The ISP4 culture medium comprises the following components (g/L): soluble starch 10.0, K₂HPO₄ 1.0，MgSO₄ 1.0，NaCl 1.0，(NH₄)₂SO₄ 2.0，CaCO₃2.0, 1mL of trace elements, 2 parts of agar and 1L of distilled water, and adjusting the pH value to 7.2 before sterilization.

The ISP5 culture medium comprises the following components (g/L): L-Aspartame 1.0, K₂HPO₄1.0, 10.0 glycerol, 20.0 agar, 1mL microelement, 2 agar and 1L distilled water, and adjusting pH to 7.2-7.4 before sterilization.

The ISP6 culture medium comprises the following components (g/L): 36.0 parts of peptone yeast extract iron agar, 1.0 part of yeast extract, 2 parts of agar and 1L of distilled water, and adjusting the pH value to 7.2-7.4 before sterilization.

The ISP7 culture medium comprises the following components (g/L): glycerol 1.5, L-tyrosine 0.05, L-aspartyl 0.1, K₂HPO₄0.05，MgSO₄0.05, NaCl 0.05, 1mL/L of trace elements, 1.5 of agar and 1L of distilled water, and the pH value is adjusted to 7.2-7.4 before sterilization.

The trace element solution comprises the following components (g/L): FeSO₄·7H₂O 0.1，MnCl·4H₂O 0.1， ZnSO₄·7H₂O0.1, distilled water 100 mL.

TABLE 1 cultivation characteristics of FIM06-0025 in different media

Culture medium	Growth conditions	Color of base filament
			ISP1	+	Moderate Orange Yellow
ISP2	+++	Deep Brown
			ISP3	+++	Moderate Orange
ISP4	+++	Moderate Orange
			ISP5	+	Deep Brown
ISP6	+++	Deep Orange Yellow
			ISP7	+-++	Brownish Black

Note: , + + + + indicates good growth; + indicates general growth; + represents a growth difference; -means no growth; color and color values were referenced to the ISCC-NBC standard.

The strength of the utilization of such carbon source by the microorganism is characterized by the growth rate of the microorganism under the condition of a single carbon source. ISP9 basic medium is selected for testing.

The carbon source was examined as 21 carbon sources including melibiose, raffinose, D-mannose, L-xylose, inositol, saligenin, L-rhamnose, dulcitol, sorbitol, dithiothreitol, D-galactose, dulcitol, esculin, erythritol, cellobiose, adonitol, melezitose, trehalose, L-arabinose, D-glucose and chitin. 0.8g/L of each carbon source was added and 3 replicates of each treatment were run with media without carbon source as a control.

Under aseptic conditions, the strain FIM06-0025 was inoculated with a inoculating loop onto ISP9 minimal medium petri dish plates containing different carbon sources as described above, cultured upside down at 32 ℃, the results were recorded at different culture times and compared with a blank, and the results of carbon source utilization were analyzed (as shown in Table 2 below).

The ISP9 basic medium comprises the following components (g/L): k₂HPO₄ 5.65，KH₂PO₄ 2.38， (NH₄)₂SO₄2.64，MgSO₄·7H₂O1.0, agar 12.0, carbon source 1.0, trace elements 1mL, distilled water 1000mL, adjusting pH to 7.5.

The trace element preparation comprises the following components (g/L): CuSO₄·5H₂O 0.64g，FeSO₄·7H₂O 0.11g，ZnSO₄·7H₂O 0.15g，MnCl₂·4H₂O0.79 g, distilled water 100 mL.

TABLE 2 carbon source utilization of strain FIM06-0025

Note: + indicates that it is fully available; ± means partially available; -means completely unusable.

1.2 identification of 16S rRNA of Strain

5mL of FIM06-0025 bacterial liquid cultured to logarithmic phase is taken, centrifugation is carried out for 15min at 8000r/min, supernatant is poured off, and thalli are collected.

Extracting thallus genome DNA by adopting a CTAB/NaCl method, and specifically comprising the following steps: adding 1.35mL of TE solution (pH 8.0) into the thallus, suspending, adding 0.3mL of 10% Sodium Dodecyl Sulfate (SDS), 150 μ L of lysozyme, 100mg/mL of lysozyme, 150 μ L of protease K and 100mg/mL of protease K, uniformly mixing, carrying out water bath at 60 ℃ for 1h, adding 0.25mL of NaCl, 5mol/L of NaCl and 0.2mL of LCTAB/NaCl solution, carrying out water bath at 65 ℃ for 10min, extracting for 3 times by using equal volumes of phenol-chloroform-isoprene solvent and chloroform-isoprene solvent respectively, carrying out centrifugal treatment at 10000r/min for 10min, sucking supernatant, adding 0.6-fold volume of isopropanol, placing in an environment at-20 ℃ for precipitating DNA, dissolving the dissolved DNA in 50 μ L of TE, and storing at-20 ℃ for later use. The integrity of the extracted genomic DNA was checked by electrophoresis on a 1% agarose gel using the following universal primers for the 16S rRNA gene:

27F (5'-AGAGTTTGATCCTGGCTCAG-3') and

1492R (5'-GGTTACCTTGTTACGACTT-3') PCR amplified the extracted DNA.

The amplified DNA product is sent to Shanghai bioengineering Co., Ltd for sequencing, and the 16S rRNA sequence is shown as SEQ ID NO. 1. BLAST software was used to compare and analyze the 16S rRNA sequences recorded in the NCBI GenBank database and BLAST searches for homologous sequences. By comparison, strain FIM06-0025 and Verrucosispora gifhornensis DSM 44337^TSimilarity of 16S rRNA Gene sequences99 percent, and the strain FIM06-0025 is determined to be verruca verrucosa Verrucosissispora according to the results of physiological and biochemical and molecular identification of the strain.

The strain is classified and named as Verrucosispora gifhornensis, which is preserved in China general microbiological culture Collection center (CGMCC), and the address is as follows: no. 3 of Xilu No.1 of Beijing, Yangyang, with the preservation number of CGMCC No.15242 and the preservation date of 2018, 01 month and 18 days.

EXAMPLE 2 fermentation of the Strain

Inoculating a platinum loop preserved strain Verrucosispora gifhornensis FIM06-0025 Gao's aspartyl agar slant culture into a seed culture medium, and performing shake cultivation for 223d at 26 ℃ and 240rpm to obtain a seed culture solution of V.gifhornensis FIM 06-0025.

Inoculating the obtained seed culture solution of the strain FIM06-0025 into the prepared fermentation culture medium in an inoculation amount of 5-10% by volume fraction, and performing shake fermentation culture at 26 ℃ and 240rpm for 4-5 days to obtain the fermentation culture solution of the wart cell strain V.gifhornnensis FIM 06-0025.

The asparagkis-agar slant culture medium comprises the following components (g/L): 20.0g of soluble starch, 0.5g of aspartyl, 16.5g of sea salt, 0.5g of NaCl and KNO₃ 1.0g，K₂HPO₄ 0.5g，MgSO₄·7H₂O 0.5g，CaCO₃1.0g, agar 12.0g, 1000mL of seawater, and natural pH value; the preparation method comprises mixing the above materials according to their content, and sterilizing at 121 deg.C for 30 min.

The seed culture medium comprises the following components (g/L): 15.0g of soluble starch, 5.0g of yeast powder, 5.0g of peptone, 5.0g of glucose and K₂HPO₄ 0.5g，MgSO₄·7H₂O 0.5g，NaCl 0.5g， (NH₄)₂SO₄ 0.5g，CaCO₃1.0g and 1L of seawater, and is prepared by mixing the above components uniformly according to their contents, adjusting pH to 7.5 before sterilization, and sterilizing at 121 deg.C for 30 min.

The fermentation medium comprises the following components (g/L): soluble starch 20.0g, K₂HPO₄ 0.5g， KNO₃5.0g，MgSO₄·7H₂O 0.5g，NaCl 0.5g，FeSO₄·7H₂O 0.01g，CaCO₃1.0g and 1000mL of seawater, and is prepared by mixing the above components uniformly according to their contents, sterilizing at 121 deg.C for 30min with pH of 7.2-7.5, and keeping.

HPLC detection shows that the fermentation titer of the target compound benzoazepine compound in the obtained fermentation liquor is 0.064 mg/L.

Example 3 extraction of the Compound

50L of the fermentation culture solution of the verruca plana V.gifhornensis FIM06-0025 prepared in the example 2 is centrifuged at 4500rpm to obtain mycelium and supernatant respectively for later use;

adding 1.5-2.0 times volume of methanol-acetone mixed solvent (1: 1, v: v) into the separated mycelium, mixing, performing ultrasonic extraction for 2 times, controlling ultrasonic temperature below 40 deg.C, and performing ultrasonic treatment for 30 min; collecting extractive solution, and concentrating under reduced pressure at temperature below 40 deg.C to remove methanol-acetone solvent to obtain first extract (210 g); mixing the obtained first extract with the separated supernatant, adding 1.5-2.0 times volume of ethyl acetate into the obtained mixed solution, extracting for 2 times, and concentrating under reduced pressure at below 40 deg.C to remove ethyl acetate to obtain second extract (169 g);

and (3) carrying out chromatography on the obtained second extract by using a C18 reverse phase silica gel column, wherein the volume ratio is 30: 100. 50: 100. 70: 100 and 70: 90 methanol-water solution is used as eluent for gradient elution, and the concentration of the combined methanol-water eluent is 70: 100 to give a first fraction a-1(110 mg);

mixing the obtained first component A-1 with 100-mesh 200-mesh silica gel, filling the mixture into a column by a dry method, and then, taking chloroform-methanol as eluent to obtain the first component A-1, wherein the volume ratio of the first component A-1 to the second component A-1 is 9: 1-1: 1, performing gradient elution, collecting each part of eluent respectively, detecting by TLC, and performing gradient elution according to the volume ratio of 10: 1, taking chloroform-methanol as a developing agent and iodine as a color developing agent, combining the same components to respectively obtain five components, namely, the components with Rf values of 0.27, 0.39, 0.55, 0.67 and 0.75, and collecting the component (30mg) with Rf value of 0.55 to obtain a second component A-2;

and (3) carrying out Sephadex LH-20 column chromatography on the obtained second component A-2, wherein the volume ratio is 1: 1, taking methanol-acetonitrile solvent as eluent, detecting elution fractions by TLC, and performing detection on the elution fractions by using a solvent with a volume ratio of 10: 1 as developing agent, and collecting the component (3.2mg) with Rf value of 0.61, namely the required monomer compound, which is marked as compound FW 251.

Example 4 structural characterization

And performing data tests such as mass spectrum, ultraviolet spectrum, infrared spectrum, nuclear magnetic resonance and the like on the compound FW251 to determine the structure of the compound.

Compound FW251 is a white powder, which

UV(MeOH),λ max:241nm，301nm；HR-TOF-MS(m/z 208.1032[M+H]⁺(Calcd for 208.0974) (see FIG. 3), it can be seen that its molecular formula is C₁₁H₁₃NO₃The unsaturation was calculated to be 6.

The infrared absorption spectrum (IR) as shown in FIG. 4 shows that the spectrum was measured at 3075, 1614, 1493, and 1582cm^-1The absorption at (B) indicates that the aromatic benzene ring group is contained, 1644cm^-1The strong absorption peak at (a) indicates the presence of the amide.

As shown in fig. 5¹H NMR spectrum showed 1 methyl proton [ delta ]_H1.11(3H)]Signal, group 1 methylene proton [ delta ]_H(4.44,4.32)]Signal, 2 methine protons [ delta ]_H4.32、δ_H3.76]Signal, 1 amino hydrogen proton [ delta ] of 9-NH_H4.8]And 1 10-OH benzene hydroxyl proton [ delta ]_H12.3]Signal and 4 phenyl protons [ delta ]_H7.43(1H,J＝7.8Hz)、δ_H7.59(1H,J＝7.8Hz)、δ_H6.92(1H,J＝7.5Hz)、 δ_H6.96(1H,J＝8.3Hz)]A signal. From the coupling constants of 4 phenyl proton signals, it can be seen that the compound contains 1 ortho-substituted benzene ring,¹H-¹the H COSY and HMBC spectra further confirmed the above-described junction theory (see fig. 6, 7).

As shown in fig. 8-9¹³C-NMR and DEPT135 (DMSO-d)₆125MHz) spectrum showed that the compound contained 11 carbon signals, including 1 methylene1 methyl group, 6 methine groups and 3 quaternary carbons.¹H-¹H COSY (see FIG. 6) shows that 11-CH₃(δ_H1.11)、10-H(δ_H3.76)、7-H(δ_H4.32) and 8-H (. delta.))_H4.44,4.32) can yield the following structural fragment: CH (CH)₃-C₁₀-C₇-C₈In the HMBC spectra shown in FIG. 7, 8-H and 9-C (. delta.) (delta.)_C164.9) related known fragment CH of amide carbon₃-C₁₀-C₇-C₈-NH-CO-, then, 2-H (. delta.) -_H7.59) coupling to the amide carbon of 9-C it is possible to deduce the fragment CH₃-C₁₀-C₇-C₈-NH-CO-is attached to the aromatic group through the amide carbon 9-C. Combining the above analyses, and combining the HSQC of fig. 10 and the HMBC spectrum shown in fig. 7, the overall carbon signal and hydrogen signal assignments for compound FW251 were obtained, as shown in table 3 below. Finally, the chemical structure of the compound FW251 is determined according to the degree of unsaturation and the molecular weight (as shown in FIG. 11) as shown in the following formula (1), namely the benzo-nitrogen-oxygen hetero compound with a new structure. Thus, the chemical structure and main content of the compound FW251 were determined¹H-¹The correlation spectra of H COSY and HMBC are shown in FIG. 11.

TABLE 3 1H NMR (in Chloroform-d4,400MHz) and 13C-NMR (in Chloroform-d4,100 MHz) data for Compound FW251

Example 5 antimicrobial Activity test

Compound FW251 was tested for Minimal Inhibitory Concentration (MIC) using a broth dilution method with cefotaxime sodium as a positive control.

Test bacteria include gram-negative bacteria: helicobacter pylori (h.pylori) ATCC 43504, klebsiella pneumoniae (k.pneumoniae) ATCC 4352, escherichia coli (e.coli) ATCC 25922, pseudomonas aeruginosa (p.aeruginosa) ATCC 27853, acinetobacter baumannii (a.baumannii) ATCC 17978, and gram-positive bacteria: staphylococcus aureus (s. aureus) ATCC 25923, candida albicans (c. albicans) ATCC 90028, and enterococcus faecium (e.faecalis) ATCC 35667 (see table 4 below).

The specific operation steps are as follows:

(1) MH broth culture medium preparation: weighing MH broth culture medium 21.0g, adding into 1L distilled water, heating and boiling to dissolve completely, subpackaging in test tubes, and autoclaving at 121 deg.C for 15 min;

(2) early culture of test bacteria: under aseptic condition, test bacteria are inoculated into 100mL MH broth culture medium and cultured in an incubator at 35 ℃ for 12h for later use;

(3) preparation of a stock solution: weighing a proper amount of a sample to be detected and a positive control substance respectively, dissolving the positive control substance by using 1mL of sterile water, dissolving the sample to be detected by using 1mL of methanol, wherein the initial concentration of each stock solution is more than 1000 mug/mL;

(4) preparation of test bacterium solution: the cultured test bacteria are diluted with MH broth to 0.5 McLee unit turbidity standard at a ratio of 1:20 under aseptic condition to obtain a bacterial solution with a concentration of about 5 × 10⁶CFU/mL for standby;

(5) dilution of stock and inoculation of test bacteria: and (3) performing sterile operation, namely taking one sterile 96-well plate, adding 100 mu L of MH broth into each hole except the first hole, adding 40 mu L of stock solution of the sample to be detected into the 1 st hole, uniformly mixing, sucking 100 mu L of the stock solution to the 2 nd hole, uniformly mixing, sucking 100 mu L of the stock solution to the 3 rd hole, uniformly mixing, diluting to the 3 rd reciprocal hole in a sequential multiple ratio manner, and sucking 100 mu L of the total amount from the 3 rd reciprocal hole and discarding. The penultimate 2 wells are growth controls without drug and the last 1 well is an ungaccinated control. Then, 10. mu.L of the prepared inoculum of the test bacteria was added to each well to give a final concentration of about 5X 10 bacteria per well⁵CFU/mL；

(6) And (3) incubation: covering the 96-well plate inoculated with the test bacteria with a cover, and culturing in a biochemical incubator at 35 ℃ for 16-20 h;

(7) MIC endpoint interpretation: the lowest inhibitory concentration seen by the naked eye in a 96-well plate that completely inhibited bacterial growth was the lowest inhibitory concentration of the sample against that species of bacteria, and the results are reported in table 4 below.

TABLE 4 results of the bacteriostatic test

Test bacterium	FW251(μg/mL)	Cefotaxime sodium (mu g/mL)
			Helicobacter pylori	150	28
Klebsiella pneumoniae	210	30
			Acinetobacter baumannii	180	200
Escherichia coli	210	0.039
			Pseudomonas aeruginosa	210	100
Candida albicans	220	180
			Staphylococcus aureus	180	6
Enterococcus faecium	190	128

As shown in Table 4, the compound FW251 has broad-spectrum inhibitory activity (MIC value of 3.4-200 μ g/mL) against gram-negative bacteria and gram-positive bacteria, and particularly has strong inhibitory activity against gram-negative bacteria, helicobacter pylori, Klebsiella pneumoniae and gram-positive bacteria, Staphylococcus aureus.

It should be understood that the above-described embodiments are merely examples for clarity of description and are not intended to limit the scope of the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This list is neither intended to be exhaustive nor exhaustive. And obvious variations or modifications therefrom are within the scope of the invention.

Claims

1. A method for preparing a benzoazepine compound, characterized in that the benzoazepine compound has a structure represented by the following formula (1):

the method comprises the steps of carrying out fermentation culture on Verrucosispora gifhornensis FIM06-0025 and extracting the benzoxaazepine compound from the obtained fermentation liquor;

the Verrucaria verrucosa Gifhhornensis FIM06-0025 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.15242 and the preservation date of 2018, 01 month and 18 days;

the fermentation culture step comprises the steps of inoculating the Verrucosispora gifhhornnensis FIM06-0025 into a fermentation culture medium, performing shake fermentation culture at 26 ℃ and 240rpm for 4-5 days, and collecting fermentation liquor;

the fermentation medium contains per liter: soluble starch 20.0g, K₂HPO₄ 0.5g，KNO₃ 5.0g，MgSO₄·7H₂O 0.5g，NaCl 0.5g，FeSO₄·7H₂O 0.01g，CaCO₃1.0g, the balance being seawater, adjusting the pH value to 7.2-7.5;

the step of extracting the benzoazepine compound specifically comprises the following steps:

(4) the obtained first component a-1 was subjected to silica gel column chromatography with chloroform-methanol solution as an eluent in a volume ratio of 9: 1-1: 1, performing gradient elution, collecting eluates respectively, detecting by TLC, and performing gradient elution according to a volume ratio of 10: 1, taking chloroform-methanol as a developing agent and iodine as a color developing agent, combining the same components, and collecting the component with Rf value of 0.55 by TLC detection to obtain a second component A-2;

(5) and carrying out Sephadex LH-20 column chromatography on the obtained second component A-2, and carrying out mass transfer on the second component A-2 according to a volume ratio of 1: 1 in methanol-acetonitrile as eluent, collecting elution fractions, detecting by TLC, and separating by using a solvent with a volume ratio of 10: 1, taking chloroform-methanol as a developing agent, and collecting components with Rf value of 0.61 to obtain the benzoazepine compound with the structure shown in the formula (1).

2. The method for preparing benzoazepine compounds according to claim 1, wherein in step (2), said extraction step is ultrasonic extraction.

3. Use of a strain of verruca vulgaris for the fermentative preparation of a benzoazepine compound, characterized in that said benzoazepine compound has the structure shown in formula (1) below:

the verruca vulgaris strain is classified and named Verrucosispora gifhornensis FIM06-0025, is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, has the preservation number of CGMCC No.15242 and the preservation date of 2018, 01 month and 18 days.