CN109280034A - A kind of Benzoxazepin class compound and the preparation method and application thereof with bacteriostatic activity - Google Patents
A kind of Benzoxazepin class compound and the preparation method and application thereof with bacteriostatic activity Download PDFInfo
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- CN109280034A CN109280034A CN201810866115.7A CN201810866115A CN109280034A CN 109280034 A CN109280034 A CN 109280034A CN 201810866115 A CN201810866115 A CN 201810866115A CN 109280034 A CN109280034 A CN 109280034A
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Abstract
The invention belongs to native compound technical fields, and in particular to a kind of Benzoxazepin class compound and the preparation method and application thereof with bacteriostatic activity.Benzoxazepin class compound of the present invention is novel compound, it is by the fermented extraction gained of wart spore bacterium V.GIFhornensis FIM06-0025 that applicant voluntarily screens, after measured, the Benzoxazepin class compound has wide spectrum inhibitory activity to Gram-negative bacteria and gram-positive bacteria, especially there is stronger inhibitory activity to Gram-negative bacteria helicobacter pylori and Friedlander's bacillus and gram-positive bacteria staphylococcus aureus, can be used for preparing extensive pedigree antibiotic.
Description
Technical field
The invention belongs to native compound extractive technique fields, and in particular to a kind of benzo nitrogen oxygen with bacteriostatic activity
Miscellaneous Zhuo class compound and the preparation method and application thereof.
Background technique
Since nineteen twenty-nine finds medical antibiotics penicillin, discovery farm antibiotics blasticidin-S S in 1958, so far entirely
The world has found more than 4000 antibiotic, has played important work in terms of person poultry disease's prevention and control and agricultural pest prevention and treatment
With, pushed human civilization and science and technology progress.
But the use of a large amount of broad-spectrum antibiotics, the evolution of pathogenic bacteria is accelerated instead, also leads to pathogen especially
The drug resistance of bacterial pathogens enhances increasingly, so that the curative effect of existing drug declines.In addition, a large amount of uses of broad-spectrum antibiotic
Also the type of multi-drug resistant bacteria is caused also to increase, so that drug resistance develops to gram-positive bacteria from gram negative bacilli, by
Nosocomial infection develops to community infection.The emergence of drug-resistant bacteria and multidrug resistance bacterium and fast propagation, make bacterium infection
Prevent, treat and be faced with stern challenge with control, therefore, bacterial resistance problem is the publilc health of 21 century most serious
One of problem.
Enterobacteriaceae lactobacteriaceae and non-fermented of the existing germy resistance problems outstanding behaviours in Carbapenem-resistant class antibiotic
Sugared bacterium, wherein it is especially the most serious with the drug resistance of Klebsiella Pneumoniae, Acinetobacter bauamnnii, pseudomonas aeruginosa etc., it is clinical
Treatment is also the most difficult.Although many scholars have carried out a large amount of research work to this, in past 30 years, only dislike
(oxazolidinon-5-yl-methyl)-2-thiophene-carboxamides and two class resisting gram-positive bacteria medicine of cyclic lipopeptide go through to list, and the drug of anti-Gram-negative bacteria is then less,
Clinical application status still allows of no optimist.Face the continuous upgrading of bacterial drug resistance, antibacterials curative effect reduces and clinical
No medicine can with etc. stubborn problems, the work for researching and developing novel drug-resistance bacteria medicine it is extremely urgent.
It is the world that active compound is found from Secondary Metabolites of Microorganisms as lead compound Development of New Drugs
One of the effective way that pharmacy worker generally acknowledges, and microbial resources are even more that infinite source is provided for innovation drug research.
The rich and varied secondary metabolite of microorganism provides a large amount of extremely precious modes for chemicals new drug research and exploitation
Structure and prodrug small molecule are the important material bases of drug discovery source treatment and Continuous Innovation, to entire new medicine
The research of object has decisive significance.
Actinomyces are the main sources of microbial medicine, wherein based on streptomyces and some rare actinomycetes category.But
From after the seventies, the probability of discovery noval chemical compound is with regard to fewer and fewer from streptomycete, and the repetitive rate of noval chemical compound is also more next
It is higher.The few actinomyces (being commonly called as rare actinomycete) of these types for obtaining at present such as wart spore bacterium, Nocard's bacillus are increasingly closed
Note.It is living to improve acquisition target for the reactive compound for containing a large amount of structure novels in the secondary metabolite of rare actinomycete
The probability of property compound.
In known rare actinomycete, wart spore bacterium (Verrucosispora) belongs to Actinomycetal Micromonosporaceae
(Micromonosporaceae).From Rheims in 1998 etc. after isolated wart spore bacterium earliest in the mud of marsh, science
Family the category bacterial strain is separated to from ocean mud sample, mangrove, ascidian and sponge one by one.It is reported that wart spore bacterium due to
Contain natural products resource extremely abundant, just becomes an important bacterium source of microbial medicine.Such as 2004, from Japan
The verrucosispora maris AB 18-032 for belonging to Micromonosporaceae, the bacterium are isolated in the deposit of extra large 289m depth
Strain can generate the active polyketides deep-sea element (Abyssomicin C) of anti-MRSA, VRSA, which has new
Bacteriostasis target spot has positive effect to new and effective antibiotic is found.Bull and Stach by pair
The genome analysis of verrucosispora maris AB 18-032 finds that it has included at least 20 natural products synthesis
Gene cluster, this demonstrated together with the research conclusion of Nett etc. wart spore bacterium not only can with the active material of synthetic antimicrobial, and
The more native compounds of synthesis can also be metabolized.For another example, it 2008, is separated to from the deposit of the 250 meters of depths in the Norwegian Sea
Verrucosispora sp.MG-37, Fuan class compound (proximicins) of the bacterial strain synthesizing antitumor；2010,
Dai etc. isolates verrucosispora sediminis MS426 from the deposit of the 3602 meters of depths in the South SeaT, and from the bacterium
Strain is separated to 8 Cyclic dipeptides (cyclo-dipetides) and two kinds of Nocardamin thunder scholar objects (nocardamine-like),
In some with antibacterium and fungi activity；2010, Shirai etc. was from bacterial strain verrucosispora gifhornensis
Be isolated to have in YM28-088 two of anti-prostate cancer new diterpene-kind compounds (gifhornennolones A and
B).In addition, being also separated to the knot such as antitumoral compounds thiochondrilline C and Harrucomicin C from wart spore bacterium
The compound of structure novelty, good activity.The antibacterials of structure novel or novel mechanism are screened from wart spore bacterium to cope with
Multidrug resistant bacteria has realistic feasibility.
Summary of the invention
For this purpose, technical problem to be solved by the present invention lies in a kind of Benzoxazepin class compound is provided, one is gone forward side by side
Walk open preparation method and application.
In order to solve the above technical problems, a kind of Benzoxazepin class compound of the present invention or its pharmaceutically may be used
The salt of receiving, the Benzoxazepin class compound have the structure as shown in following formula (1):
The invention also discloses a kind of methods for preparing the Benzoxazepin class compound, including with wart spore bacterium
Verrucosispora gifhornensis FIM06-0025 carries out the step of fermented and cultured, and from gained fermentation liquid
The step of extracting the Benzoxazepin class compound；
The wart spore bacterium Verrucosispora gifhornensis FIM06-0025 is preserved in Chinese microorganism strain
Preservation administration committee common micro-organisms center, deposit number are CGMCC No.15242, and the deposit date is 01 month 2018 18
Day.
Specifically, the fermented and cultured step includes meeting the wart spore bacterium V.gifhornensis FIM06-0025
The step of kind is fermented into fermentation medium, and collect fermentation liquid；
Every liter of the fermentation medium contains: soluble starch 20.0g, K2HPO40.5g, KNO35.0g, MgSO4·
7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, CaCO31.0g, surplus are seawater, adjust pH7.2-7.5.
Specifically, the step of extraction Benzoxazepin class compound, specifically includes:
(1) fermentation liquid of collection is separated by solid-liquid separation, obtains mycelium and supernatant, it is spare；
(2) mixed solvent of the mycelium addition methanol and acetone that take separation extracts, and gained extracting solution is concentrated
Solvent is removed, the first medicinal extract is obtained；The first medicinal extract of gained is merged with the supernatant that separates, and be added ethyl acetate into
Row extraction, it is concentrated to spray the second medicinal extract；
(3) by the second medicinal extract of gained through C18 reversed-phase silica gel column chromatography, and respectively with volume ratio 30:100,50:100,70:
100, the methanol-water solution of 70:90 is that eluant, eluent carries out gradient elution, and the wash-out concentration for collecting eluant, eluent is 70:100's
Component obtains the first component A-1；
(4) by the first component of gained A-1 through silica gel column chromatography, and using chloroform-methanol as eluant, eluent, from volume ratio
9:1-1:1 carries out gradient elution, and fraction collection eluent is detected through TLC, is expansion with the chloroform-methanol of volume ratio 10:1
Agent, iodine are color developing agent, merge same composition, and collect the component that TLC detection Rf value is 0.55, obtain the second component A-2；
(5) gained the second component A-2 is chromatographed through Sephadex LH-20 column, and with the methanol-acetonitrile of volume ratio 1:1
Solution is eluant, eluent, collects elution fraction and detects through TLC, using the chloroform-methanol of volume ratio 10:1 as solvent, and collects Rf
It is worth the component for being 0.61 to get the Benzoxazepin class compound of the structure as shown in claim 1.
Further, the volume ratio of the methanol and acetone is 1:1, the dosage of the mixed solvent of the methanol and acetone
It is 1.5-2.0:1 with the mycelial volume ratio.
In the step (2), the extraction step is ultrasonic extraction.
It is anti-in preparation that the invention also discloses the Benzoxazepin class compounds or its pharmaceutically acceptable salt
Purposes in bacterium drug.
Specifically, the antibacterials include anti-helicobacter pylori, pseudomonas aeruginosa, Acinetobacter bauamnnii, pneumonia gram
The primary bacterium of thunder, Escherichia coli, staphylococcus aureus, Candida albicans or enterococcus faecium drug.
The invention also discloses a kind of antibacterials, the drug with the Benzoxazepin class compound or its
Pharmaceutically acceptable salt is active constituent.
Specifically, the antibacterials include anti-helicobacter pylori, pseudomonas aeruginosa, Acinetobacter bauamnnii, pneumonia gram
The primary bacterium of thunder, Escherichia coli, staphylococcus aureus, Candida albicans or enterococcus faecium drug.
The invention also discloses one plant of wart spore bacterial strain, classification naming is Verrucosispora gifhornensis,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.15242, is protected
Hiding the date is on 01 18th, 2018.
The invention also discloses the wart spore bacterial strains to prepare Benzoxazepin as described in claim 1 for fermenting
The purposes of class compound.
Benzoxazepin class compound of the present invention is novel compound, the wart voluntarily screened by applicant
Obtained by the fermented extraction of spore bacterium V.gifhornensis FIM06-0025, after measured, the Benzoxazepin class compound pair
Gram-negative bacteria and gram-positive bacteria have wide spectrum inhibitory activity, especially to Gram-negative bacteria helicobacter pylori and lung
Scorching Klebsiella and gram-positive bacteria staphylococcus aureus have stronger inhibitory activity, can be used for preparing wide spectrum
Antibacterials.
Benzoxazepin class compound of the present invention is fermented by wart spore bacterium V.gifhornensis FIM06-0025
Gained is extracted, preparation method is simple.
Detailed description of the invention
In order to make the content of the present invention more clearly understood, it below according to specific embodiments of the present invention and ties
Attached drawing is closed, the present invention is described in further detail, wherein
Fig. 1 is single colonie form of the bacterial strain FIM06-0025 of the present invention in nutrient agar；
Fig. 2 is the scanning electron microscope (SEM) photograph of bacterial strain FIM06-0025 of the present invention；
The HRESI-TOF-MS that Fig. 3 is the compounds of this invention FW251 is composed；
The IR that Fig. 4 is the compounds of this invention FW251 is composed；
Fig. 5 is the compounds of this invention FW251's1H-NMR spectrum；
Fig. 6 is the compounds of this invention FW251's1H-1H COSY spectrum；
The HMBC that Fig. 7 is the compounds of this invention FW251 is composed；
Fig. 8 is the compounds of this invention FW251's13C-NMR spectrum；
Fig. 9 is the hsqc spectrum of the compounds of this invention FW251；
The DEPT that Figure 10 is the compounds of this invention FW251 is composed；
Figure 11 is the chemical structure of the compounds of this invention FW251 and main1H-1H COSY and HMBC Correlated Spectroscopy.
Specific embodiment
Embodiment 1
One plant of bacterial strain is filtered out from the sponge of the Fujian East Sea, is named as FIM06-0025, through detecting, taxology feature
It is as follows.
1.1, the physicochemical property of bacterial strain
Single colonie form of the bacterial strain FIM06-0025 in nutrient agar is as shown in Figure 1.From bacterial strain FIM06-
0025 electron-microscope scanning figure (Fig. 2) as it can be seen that substrate mycelium physically well develops, branch have every, 0.4 μm of diameter, single spore it is raw
In on short sporophore, spore has handle, and there is verruca on surface.
Aseptically, the agar for bacterium FIM06-0025 being inoculated into following ISP1-ISP7 with oese is oblique
On the culture medium of face, culture 2-5d is carried out in 32 DEG C, observes and records the cultural characteristic of FIM06-0025 bacterial strain in different medium
(see the table below shown in 1).
The nutrient agar training culture medium includes following component (g/L): beef extract 3.0, peptone 10.0, yeast extract
2.0, sodium chloride 5.0, agar 12.0, distilled water 1000mL, it is 7.2-7.5 that pH value is adjusted before sterilizing.
ISP1 culture medium includes following component (g/L): tryptone 0.5, yeast extract 0.3, agar 2, distilled water 1L, before sterilizing
Adjust pH7.2-7.5.
ISP2 culture medium includes following component (g/L): yeast extract 0.4, malt extract 1, glucose 0.4, agar 2, distilled water
1L adjusts pH7.3 before sterilizing.
ISP3 culture medium includes following component (g/L): oatmeal 2, agar 2, microelement 1mL, agar 2, distilled water
1L adjusts pH7.4 before sterilizing.
ISP4 culture medium includes following component (g/L): soluble starch 10.0, K2HPO41.0, MgSO41.0, NaCl
1.0, (NH4)2SO42.0, CaCO32.0, microelement 1mL, agar 2, distilled water 1L adjust pH 7.2 before sterilizing.
ISP5 culture medium includes following component (g/L): L- asparagine 1.0, K2HPO41.0, glycerol 10.0, agar 20.0,
Microelement 1mL, agar 2, distilled water 1L adjust pH 7.2-7.4 before sterilizing.
ISP6 culture medium includes following component (g/L): peptone yeast extract iron agar 36.0, yeast extract 1.0, agar 2,
Distilled water 1L adjusts pH 7.2-7.4 before sterilizing.
ISP7 culture medium includes following component (g/L): glycerol 1.5, l-tyrosine 0.05, L- asparagine 0.1, K2HPO4
0.05, MgSO40.05, NaCl 0.05, microelement 1mL/L, agar 1.5, distilled water 1L adjust pH 7.2-7.4 before sterilizing.
Trace element solution includes following component (g/L): FeSO4·7H2O 0.1, MnCl4H2O 0.1, ZnSO4·
7H2O 0.1, distilled water 100mL.
The cultural characteristic of 1 FIM06-0025 of table in different medium
| Culture medium | Upgrowth situation | Base silk color |
| ISP1 | + | Moderate Orange Yellow |
| ISP2 | +++ | Deep Brown |
| ISP3 | +++ | Moderate Orange |
| ISP4 | +++ | Moderate Orange |
| ISP5 | + | Deep Brown |
| ISP6 | +++ | Deep Orange Yellow |
| ISP7 | +-++ | Brownish Black |
Note: +++ indicate well-grown；++ indicate that growth is general；+ indicate that growth is poor；Expression is not grown；Color and color value
Referring to ISCC-NBC standard.
Microorganism is characterized to the utilization intensity of this carbon source with growth rate of microorganism under the conditions of single carbon source.
ISP9 basal medium is selected to be tested.
Investigation carbon source is 21 kinds of carbon sources, including melibiose, raffinose, D-MANNOSE, L- xylose, inositol, salicin, L-
Rhamnose, dulcitol, sorbierite, dithiothreitol (DTT), D- galactolipin, sweet and pure, aesculin, erythrite, cellobiose, adonose
Alcohol, melezitose, trehalose, L-arabinose, D-Glucose and chitin.Every kind of carbon source adds 0.8g/L, and carbon source is not added
Culture medium is as control, 3 repetitions of each processing.
Aseptically, bacterial strain FIM06-0025 is inoculated into the above-mentioned ISP9 base containing different carbon source with oese
It on basal culture medium culture dish plate, is cultivated in 32 DEG C of inversions, different incubation times record result and compare with blank, and analyze
Result (2) as shown in the table of utilization of carbon source.
ISP9 basal medium includes following component (g/L): K2HPO45.65 KH2PO42.38, (NH4)2SO4
2.64 MgSO4·7H2O 1.0, agar 12.0, carbon source 1.0, microelement 1mL, distilled water 1000mL adjust pH7.5.
It includes following component (g/L): CuSO that microelement, which is prepared,4·5H2O 0.64g, FeSO4·7H2O 0.11g,
ZnSO4·7H2O 0.15g, MnCl2·4H2O 0.79g, distilled water 100mL.
2 bacterial strain FIM06-0025 utilization of carbon source situation of table
Note :+indicate to be fully utilized；± indicate partially to utilize；Expression cannot utilize completely.
1.2 bacterial strain 16S rRNA identification
Above-mentioned culture is taken to carry out centrifugation 15min in 8000r/min to the FIM06-0025 bacterium solution 5mL of logarithmic growth phase,
Incline supernatant, collects thallus.
Phage gene group DNA is extracted using CTAB/NaCl method, the specific steps are as follows: 1.35mL TE is added into thallus
Solution (pH 8.0) suspends and is added the lauryl sodium sulfate (SDS) and 150 μ L, 100mg/ml lysozyme of 0.3mL 10%
With 150 μ L, 100mg/mL Proteinase K, mix, 60 DEG C of progresss water-bath 1h, and plus 0.25mL, 5mol/L NaCl and
0.2mLCTAB/NaCl solution, it is different in 65 DEG C of water-bath 10min, then with isometric phenol chloroform-isoamyl alcoholic solvent and chloroform-
Amylalcohol solvent respectively extracts 3 times, in 10000r/min centrifugal treating 10min, Aspirate supernatant, and the different of 0.6 times of volume is added
Propyl alcohol is placed in -20 DEG C of environment and precipitates DNA, and the DNA of dissolution is dissolved in 50 μ L TE, is saved backup in -20 DEG C.It utilizes
The genomic DNA integrality of 1% agarose gel electrophoresis detection extracting, and using the universal primer of following 16S rRNA gene:
27F (5'-AGAGTTTGATCCTGGCTCAG-3') and
1492R (5'-GGTTACCTTGTTACGACTT-3') carries out PCR amplification to the DNA of extraction.
Supreme marine growth Engineering Co., Ltd is sent to be sequenced the DNA product after amplification, 16S rRNA sequence such as SEQ
Shown in ID NO.1.The 16S rRNA sequence included with the GenBank database of BLAST software and NCBI is compared,
Blast searches for homologous sequence.Through comparing, bacterial strain FIM06-0025 and wart born of the same parents bacterium Verrucosispora gifhornensis
DSM 44337TThe similitude of 16S rRNA gene order be 99%, the Physiology and biochemistry and Molecular Identification knot of comprehensive bacterial strain
Fruit determines that bacterial strain FIM06-0025 is wart born of the same parents bacterium Verrucosispora.
The strain classification is named as Verrucosispora gifhornensis, is preserved in Chinese microorganism strain
Preservation administration committee common micro-organisms center (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number
For CGMCC No.15242, the deposit date is on 01 18th, 2018.
The fermentation of 2 bacterial strain of embodiment
The bacterial strain Verrucosispora gifhornensis FIM06-0025 Gao Shi asparagine fine jade for taking a platinum loop to save
Rouge slant culture accesses in seed culture medium, and shaking table culture 223d is carried out under the conditions of 26 DEG C, 240rpm, is obtained
The seed culture fluid of V.gifhornensis FIM06-0025.
The seed culture fluid of obtained bacterial strain FIM06-0025 is inoculated into preparation with the inoculum concentration of volume fraction 5-10%
Fermentation medium in, under the conditions of 26 DEG C, 240rpm carry out shaker fermentation culture 4-5d, obtain wart born of the same parents bacterium
The fermentation culture of V.gifhornensis FIM06-0025.
The Gao Shi asparagine agar slant culture-medium includes following component (g/L): soluble starch 20.0g, asparagine
0.5g, sea salt 16.5g, NaCl 0.5g, KNO31.0g, K2HPO40.5g, MgSO4·7H2O 0.5g, CaCO31.0g, agar
12.0g, seawater 1000mL, natural ph；Preparation method is to be uniformly mixed each component by its content, is gone out in 121 DEG C of high temperature
Bacterium 30min, it is spare.
The seed culture medium includes following component (g/L): soluble starch 15.0g, yeast powder 5.0g, peptone
5.0g, glucose 5.0g, K2HPO40.5g, MgSO4·7H2O 0.5g, NaCl 0.5g, (NH4)2SO40.5g, CaCO3
1.0g, 1L seawater are prepared, and preparation method is to be uniformly mixed each component by its content, adjust pH7.5,121 DEG C of high temperature before sterilizing
Sterilize 30min, spare.
The fermentation medium includes following component (g/L): soluble starch 20.0g, K2HPO40.5g, KNO3
5.0g, MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, CaCO31.0 g, seawater 1000mL, preparation
Method is to be uniformly mixed each component by its content, pH 7.2-7.5, spare in 121 DEG C of high-temperature sterilization 30min.
It is detected through HPLC, in gained fermentation liquid, the fermentation titer of target compound Benzoxazepin class compound is
0.064mg/L。
The extraction of 3 compound of embodiment
The fermentation culture 50L of wart born of the same parents bacterium V.gifhornensis FIM06-0025 prepared by above-described embodiment 2,
It is centrifuged under the conditions of 4500rpm, respectively obtains mycelium and supernatant, it is spare；
Isolated mycelium is taken, the methanol-acetone mixed solvent (1:1, v:v) that 1.5-2.0 times of volume is added is mixed
It is even, it carries out ultrasonic extraction 2 times, control ultrasonic temperature is lower than 40 DEG C, ultrasonic time 30min；It collects extracting solution and is being lower than 40 DEG C of items
It is concentrated under reduced pressure under part to remove methanol-acetone solvent, obtains the first medicinal extract (210g)；By the first medicinal extract of gained with it is above-mentioned
Isolated supernatant merges, and the ethyl acetate that 1.5-2.0 times of volume is added in gained amalgamation liquid is carried out extraction 2 times, then
It is concentrated under reduced pressure under the conditions of lower than 40 DEG C to remove ethyl acetate, obtains the second medicinal extract (169g)；
It takes the second medicinal extract of gained to be chromatographed through C18 reverse phase silica gel column, is respectively 30:100,50:100,70 with product ratio:
The methanol-water solution of 100 and 70:90 is that eluant, eluent carries out gradient elution, and merging methanol-water eluant strength is 70:100
Elution fraction, obtain the first component A-1 (110mg)；
It is elution with chloroform-methanol after dry column-packing by gained the first component A-1 100-200 mesh silica gel mixed sample
Liquid carries out gradient elution from volume ratio 9:1-1:1, collects each section eluent respectively, detect through TLC, with volume ratio 10:1's
Chloroform-methanol is solvent, using iodine as color developing agent, merges same composition, respectively obtains five kinds of components, i.e. Rf value is respectively
0.27,0.39,0.55,0.67 and 0.75 component collects the component (30mg) that Rf value is 0.55, obtains the second component A-2；
It is chromatographed by gained the second component A-2, then through Sephadex LH-20 column, it is molten with the methanol-acetonitrile of volume ratio 1:1
Agent is eluant, eluent, and elution fraction is detected through TLC, and using the chloroform-methanol of volume ratio 10:1 as solvent, collecting Rf value is 0.61
Component (3.2mg), as needed for monomeric compound, be denoted as compound FW251.
4 Structural Identification of embodiment
The data tests such as mass spectrum, ultraviolet spectra, infrared spectroscopy and nuclear magnetic resonance are carried out to compound FW251, so that it is determined that
The structure of compound.
Compound FW251 is white powder,UV (MeOH), λ max:241nm,
301nm；HR-TOF-MS(m/z 208.1032[M+H]+(Calcd for 208.0974) (see Fig. 3), it is seen that its molecular formula is
C11H13NO3, calculating its degree of unsaturation is 6.
Infrared absorption spectrum (IR) display as shown in Figure 4, in 3075,1614,1493 and 1582cm-1Place has absorption to say
It is bright to contain fragrant benzene radicals, 1644cm-1The strong absworption peak at place illustrates the presence for having amide.
As shown in Figure 51H NMR spectra shows there is 1 methyl proton [δH1.11 (3H)] signal, 1 group of methene proton
[δH(4.44,4.32)] signal, 2 methine protons [δH4.32、δH3.76] signal, the amino Hydrogen Proton [δ of 1 9-NHH4.8] and 1 10-OH benzene hydroxyl proton [δH12.3] signal and 4 phenyl protons [δH7.43 (1H, J=7.8Hz), δH7.59 (1H, J=7.8Hz), δH6.92 (1H, J=7.5Hz), δH6.96 (1H, J=8.3Hz)] signal.From 4 phenyl protons
The coupling constant of signal can be seen that the phenyl ring containing 1 ortho position substitution in compound,1H-1H COSY and HMBC wave spectrum are into one
Step demonstrates above-mentioned conclusion (such as Fig. 6, Fig. 7).
As Figure 8-913C-NMR and DEPT135 (DMSO-d6, 125MHz) and map shows that compound contains 11
Carbon signal, including 1 methylene, 1 methyl, 6 methines and 3 quaternary carbons.1H-1Display in H COSY (such as Fig. 6),
11-CH3(δH1.11)、10-H(δH3.76)、7-H(δHAnd 8-H (δ 4.32)H4.44,4.32 coupled relation) can be obtained following
Structure fragment: CH3-C10-C7-C8, shown in HMBC spectrum as shown in Figure 7,8-H and 9-C (δC164.9) phase of amide carbon
Segment CH known to closing3-C10-C7-C8- NH-CO-, moreover, 2-H (δH7.59) coupled relation with the amide carbon of 9-C can be released
Segment CH3-C10-C7-C8- NH-CO- is connect with aromatic group by amide carbon 9-C.In summary it analyzes, and combines Figure 10's
HSQC and HMBC spectrogram shown in Fig. 7 obtain the whole carbon signals and hydrogen signal ownership of compound FW251, see the table below shown in 3.
It is last to be determined that the chemical structure (such as Figure 11) such as following formula (1) of compound FW251 is shown according to degree of unsaturation and molecular weight,
The as Benzoxazepin class compound of new construction.Thus determine the chemical structure of compound FW251 and main1H-1H
COSY and HMBC Correlated Spectroscopy such as Figure 11.
The 1H NMR (in Chloroform-d4,400MHz) and 13C-NMR (in of 3 compound FW251 of table
Chloroform-d4,100 MHz) data
The test of 5 antibacterial activity of embodiment
Using micro broth dilution method, using Cefotaxime Sodium as positive reference substance, the minimum suppression of test compound FW251
Bacteria concentration (minimum inhibitory concentration, MIC).
Test bacterium bag includes 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 and Acinetobacter bauamnnii (A.baumanniiin) ATCC 17978 and gram sun
Property bacterium: staphylococcus aureus (S.aureus) ATCC 25923, Candida albicans (C.albicans) ATCC 90028 and dung
Enterococcus (E.faecium) ATCC 35667 (see the table below shown in 4).
Specific steps are as follows:
(1) MH broth bouillon is prepared: being weighed MH broth bouillon 21.0g, is added in 1L distilled water, heating is boiled
It to being completely dissolved, is sub-packed in test tube, 121 DEG C of high pressure sterilization 15min are spare；
(2) test culture bacterium early period: under aseptic condition, test bacterium is inoculated into 100mL MH broth bouillon, is placed in
35 DEG C of incubator culture 12h, it is spare；
(3) storage liquid preparation: appropriate sample to be tested and positive reference substance, positive reference substance 1mL sterile water are weighed respectively
Dissolution, sample to be tested 1mL methanol dissolve, and the initial concentration for respectively storing liquid should be greater than 1000 μ g/mL；
(4) it tests the preparation of bacterium solution: under aseptic condition, above-mentioned cultured test bacterium being corrected to 0.5 with MH meat soup
It is diluted after maxwell unit turbidity standard in the ratio of 1:20, bacterial concentration is about 5 × 10 at this time6CFU/mL, it is spare；
(5) diluting stock solutions and inoculation test bacterium: sterile working takes sterile 96 orifice plate one, except the first hole is added
Outside 160 μ L MH meat soups, 100 μ L of MH meat soup is added in remaining every hole, and 40 μ L of sample to be tested stoste is added in the 1st hole and mixes, so
After draw 100 μ L to the 2nd hole, mixes after mixing drawing 100 μ L to the 3rd hole, so continuous doubling dilution to the 3rd hole of inverse,
And 100 μ L are always drawn from the 3rd hole reciprocal and are discarded.2nd hole reciprocal is the growth control of not drug containing, and last 1 hole is nonvaccinated
Control.Then the above-mentioned test bacterium 10 μ L of inoculum prepared are added in every hole, the bacterial concentration for keeping every hole final is about
It is 5 × 105CFU/mL；
(6) it is incubated for: 96 orifice plates for being inoculated with test bacterium being covered into lid, sets in 35 DEG C of biochemical cultivation cases and cultivates 16-20h；
(7) MIC terminal interpretation: with the naked eye can completely inhibit bacterial growth seen in 96 orifice plates is the sample to this
The minimum inhibitory concentration of kind bacterium, record result see the table below 4.
4 bacteriostatic test measurement result of table
| Test bacterium | FW251(μg/mL) | Cefotaxime Sodium (μ g/mL) |
| Helicobacter pylori | 150 | 28 |
| Friedlander's bacillus | 210 | 30 |
| Acinetobacter bauamnnii | 180 | 200 |
| Escherichia coli | 210 | 0.039 |
| Pseudomonas aeruginosa | 210 | 100 |
| Candida albicans | 220 | 180 |
| Staphylococcus aureus | 180 | 6 |
| Enterococcus faecium | 190 | 128 |
The results are shown in Table 4, and compound FW251 there is wide spectrum to inhibit to live Gram-negative bacteria and gram-positive bacteria
Property (MIC value be 3.4-200 μ g/mL), it is especially blue to Gram-negative bacteria helicobacter pylori and Friedlander's bacillus and leather
Family name's positive bacterium S. aureus has stronger inhibitory activity.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.
For those of ordinary skill in the art, other various forms of changes can also be made on the basis of the above description
Change or changes.There is no necessity and possibility to exhaust all the enbodiments.And obvious change extended from this
Change or changes still within the protection scope of the invention.
Claims (10)
1. a kind of Benzoxazepin class compound or its pharmaceutically acceptable salt, which is characterized in that the benzo nitrogen oxa-
Tall and erect class compound has the structure as shown in following formula (1):
2. a kind of method for preparing Benzoxazepin class compound described in claim 1, which is characterized in that including with wart spore
Bacterium Verrucosispora gifhornensis FIM06-0025 carries out the step of fermented and cultured, and from gained fermentation liquid
The step of extracting the Benzoxazepin class compound；
The wart spore bacterium Verrucosispora gifhornensis FIM06-0025 is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, deposit number are CGMCC No.15242, and the deposit date is on 01 18th, 2018.
3. the method according to claim 2 for preparing the Benzoxazepin class compound, which is characterized in that the hair
Ferment incubation step includes that the wart spore bacterium V.gifhornensis FIM06-0025 is inoculated into fermentation medium to send out
The step of ferment, and collect fermentation liquid；
Every liter of the fermentation medium contains: soluble starch 20.0g, K2HPO40.5g, KNO35.0g, MgSO4·7H2O
0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, CaCO31.0g, surplus are seawater, adjust pH7.2-7.5.
4. the method according to claim 2 or 3 for preparing the Benzoxazepin class compound, which is characterized in that institute
The step of extracting Benzoxazepin class compound is stated to specifically include:
(1) fermentation liquid of collection is separated by solid-liquid separation, obtains mycelium and supernatant, it is spare；
(2) mixed solvent of the mycelium addition methanol and acetone that take separation extracts, and gained extracting solution is concentrated and is removed
Solvent obtains the first medicinal extract；The first medicinal extract of gained is merged with the supernatant separated, and ethyl acetate is added and is extracted
It takes, it is concentrated to spray the second medicinal extract；
(3) by the second medicinal extract of gained through C18 reversed-phase silica gel column chromatography, and respectively with volume ratio 30:100,50:100,70:100,
The methanol-water solution of 70:90 is that eluant, eluent carries out gradient elution, and the wash-out concentration for collecting eluant, eluent is the component of 70:100,
Obtain the first component A-1；
(4) by the first component of gained A-1 through silica gel column chromatography, and using chloroform-methanol as eluant, eluent, from volume ratio 9:1-1:
1 carries out gradient elution, and fraction collection eluent is detected through TLC, and using the chloroform-methanol of volume ratio 10:1 as solvent, iodine is aobvious
Toner merges same composition, and collects the component that TLC detection Rf value is 0.55, obtains the second component A-2；
(5) gained the second component A-2 is chromatographed through Sephadex LH-20 column, and is with the methanol-acetonitrile solution of volume ratio 1:1
Eluant, eluent is collected elution fraction and is detected through TLC, and using the chloroform-methanol of volume ratio 10:1 as solvent, and collecting Rf value is 0.61
Component to get the structure as shown in claim 1 Benzoxazepin class compound.
5. according to the described in any item methods for preparing the Benzoxazepin class compound of claim 2-4, feature exists
In in the step (2), the extraction step is ultrasonic extraction.
6. Benzoxazepin class compound described in claim 1 or its pharmaceutically acceptable salt are in preparation antibacterials
Purposes.
7. purposes according to claim 6, which is characterized in that the antibacterials include anti-helicobacter pylori, verdigris vacation
Monad, Acinetobacter bauamnnii, Klebsiella Pneumoniae, Escherichia coli, staphylococcus aureus, Candida albicans or enterococcus faecium
Drug.
8. a kind of antibacterials, which is characterized in that the drug with Benzoxazepin class compound described in claim 1 or
Its pharmaceutically acceptable salt is active constituent.
9. one plant of wart spore bacterial strain, classification naming is wart spore bacterium Verrucosispora gifhornensis FIM06-0025,
It has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.15242,
The deposit date is on 01 18th, 2018.
10. wart spore bacterial strain as claimed in claim 9 prepares Benzoxazepin class compound as described in claim 1 for fermenting
Purposes.
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