CN107353295B - A kind of mould chlorins compound of lattice Féraud and its preparation method and application - Google Patents
A kind of mould chlorins compound of lattice Féraud and its preparation method and application Download PDFInfo
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- CN107353295B CN107353295B CN201710556313.9A CN201710556313A CN107353295B CN 107353295 B CN107353295 B CN 107353295B CN 201710556313 A CN201710556313 A CN 201710556313A CN 107353295 B CN107353295 B CN 107353295B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/08—Bridged systems
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
Abstract
The invention discloses a kind of mould chlorins compounds of lattice Féraud and its preparation method and application.The compound is isolated and purified from the streptomycete (Streptomyces sp.) that deposit number is CICC No.11030 and is obtained, shown in structural formula such as formula (I), through experimental tests find the compound to Human Prostate Cancer Cells (PC3) activity with higher, its activity is more taller than cis-platinum, has a good application prospect.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of mould chlorins compound of lattice Féraud and preparation method thereof and
Using.
Background technique
Malignant tumour is a kind of common disease and frequently-occurring disease for seriously threatening human health, in China or even worldwide
Column second, is only second to cardiovascular and cerebrovascular disease in interior death toll.Therefore novel, efficient, less toxic antineoplastic is searched out
Object is a great problem of the world today.
New anti-tumor drug is found from natural resources becomes the trend of research, and wherein microorganism is due to special existence
Environment, it is possible to produce the unique active secondary metabolites of structure novel, mechanism of action.Therefore activity is found from microorganism
Ingredient prevents and treats tumour, increasingly attracts people's attention.
Actinomyces are as a kind of important microorganism, to generate miscellaneous bioactive substance extensively by weight
Depending on.Currently, actinomyces are still the major microorganisms source of antibiotic, the antibiosis of clinical use, which is known as 70%, to be generated by actinomyces
's.Up to more than 4000 kinds of antibiotic generated by actinomyces for having now found that and isolating, wherein had 50 Multiple Classes of Antibiotics
It is widely used, such as streptomysin, erythromycin, Polyoxin, aureomycin, kanamycins, chloramphenicol and gentamicin are used
In a variety of diseases of clinical treatment people.Raising with pathogenic microorganism to antibiotic resistance, finding has novel mechanism of action
Antibiotic seem very necessary.
Actinomyces are gained the name because mycelia is radial, since Cohn in 1877 from the neck of ox swollen disease discovery actinomyces with
Afterwards, until initial stage 1940's, the inappreciable microorganism of one kind that actinomyces are not still known, at that time only
Not bery specific four or five categories of meaning are reported, more well-known is there are pathogenic actinomyces to humans and animals
(Actinomycetes), Nocardia (Nocardia) and be considered as bacterium mycobacteria (Mycobacterium).
With going deep into for understanding, it is identified it is prokaryotes to the actinomyces sixties, it is extremely close with the relationship of bacterium, it might even be possible to wrap
It includes in the bacterium of broad sense.Currently, in " primary Jie Shi systematic bacteriology handbook " Volume Four, actinomyces in Prokaryota,
Firmicutes, Actinomycetes, Actinomycetal (Actinomycetales).
Actinomycetal and its relevant microorganism are that group the most diversified occurs for form in gram-positive bacteria.Unwrapping wire
Bacterium is the gram-positive bacterium of a kind of height (G+C) mol%.It has now been found that tens of thousands of kinds of microbe-derived biological active matters
It is as synthesized by actinomyces there are about 70% in matter.
Streptomyces is a maximum category in actinomyces, because they can generate abundant and have certain bioactivity
The secondary metabolites of effect and receive significant attention.
Summary of the invention
The present invention provides a kind of lattice Féraud mycin (gephyromycin) class compound by carrying out research to actinomyces
And its preparation method and application.
A kind of compound, structural formula such as Formulas I:
Invention further provides application of the compound in the drug of preparation treating cancer.
The cancer is prostate cancer.
The present invention also provides the preparation methods of the compound, and the preparation method comprises the following steps:
(1) fermented and cultured actinomyces obtain tunning；
(2) tunning filtering removal mycelium obtains crude extract；
(3) crude extract, which isolates and purifies, obtains the compound,
Wherein, the actinomyces deposit number is CICC No.11030.Streptomycete (Streptomyces sp.), deposit number
For CICC No.11030, purchase is located at the Chinese industrial microbial bacteria in No. 6 building of the institute of Jiuxianqiao, Chaoyang District, Beijing City Road 24 certainly
Kind preservation administrative center (CICC).
The preparation method, fermentation are rice medium using culture medium, and the rice medium presses following weight group
Divide and be made: rice 40g, 25% sea salt water 60mL.
The method isolated and purified in step (3) is as follows:
(a) crude extract is extracted with ethyl acetate；
(b) it after extract liquor concentration, is purified using reversed column chromatography, and eluted with methanol-water solution；
(c) after merging comprising the eluted product of purpose compound, using described in reversed-phase high performance liquid chromatography separation acquisition
Compound.
Extracting process in step (a) are as follows: extracted 3 times with the isometric ethyl acetate of crude extract.
Elution process is the methanol-water solution gradient elution for the use of volume ratio being 10: 90~100: 0 in step (b).
The filler that reversed-phase high performance liquid chromatography separation uses in step (c) is octadecyl silane, and mobile phase is body
Product collects the chromatographic peak of 21-24min than being 42: 58 acetonitrile-water systems with 10mL/min isocratic elution.
The compounds of this invention is to isolate and purify to obtain from a kind of actinomyces, is identified as a kind of lattice Féraud mycin
(gephyromycin) class compound, through experimental tests find that the compound is with higher to Human Prostate Cancer Cells (PC3)
Activity, activity is more taller than cis-platinum, has a good application prospect.
Detailed description of the invention
Fig. 1 is the structural formula that the present invention has active compound for anti tumor.
Fig. 2 is that there is the present invention structure of active compound for anti tumor to mark figure, wherein arrow indicates that carbon-hydrogen is related, adds
Key is learned in roughening indicates that hydrogen-hydrogen is related.
Fig. 3 is present invention compound with anti-tumor activity1H NMR spectra.
Fig. 4 is present invention compound with anti-tumor activity13C NMR spectra.
Fig. 5 is the HSQC map of present invention compound with anti-tumor activity.
Fig. 6 is the HMBC map of present invention compound with anti-tumor activity.
Fig. 7 is present invention compound with anti-tumor activity1H-1H COSY map.
Specific embodiment
Strain source
Streptomycete (Streptomyces sp.), deposit number are CICC No.11030, and purchase is from positioned at Chaoyang District, Beijing City
The Chinese industrial Microbiological Culture Collection administrative center (CICC) in No. 6 building of No. 24 institutes in winebibber's bridge Road.
Culture medium
Gause I fluid nutrient medium: in terms of fermentation medium 1L, soluble starch 20g, KNO31g, K2HPO40.5g,
MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g adds water to 1L, adjusts pH 7.2.
Gause I solid medium: in terms of fermentation medium 1L, soluble starch 20g, KNO31g, K2HPO40.5g,
MgSO4·7H2O 0.5g, NaCl 0.5g, FeSO4·7H2O 0.01g, 12g agar adds water to 1L, adjusts pH 7.2.
Rice medium: rice and sea salt water press following mass volume ratio: rice 40g, 25% sea salt water 60mL, i.e., greatly
Rice: 25% sea salt water=40g: 60mL.
Embodiment 1
1, it activates
Inclined-plane is saved from original or glycerol tube takes fungi appropriate, is inoculated on plating medium, is stood in 28 DEG C of incubators,
Activation culture 5 days.
2, seed liquor
Oneself activated above-mentioned streptomycete is inoculated in Gause I solid medium, streptomycete is carried out in culture dish
Culture 5-7 days is inverted in culture at room temperature.Then picking single colonie is inoculated in the 500mL taper containing 250mL fluid nutrient medium
In bottle, every bottle of fluid nutrient medium of Gause I containing 250mL obtains seed with 180rpm shaken cultivation 5 days at 28 DEG C in shaking table
Liquid.
3, it ferments
Above-mentioned seed liquor is inoculated into 300 bottles of rice fermentation culture mediums (by following components by weight percent by the amount for being inoculated with 8mL with every bottle
It is made: rice 40g, 25% sea salt water 60mL) in, 28 DEG C terminate fermentation after stationary culture 30 days.
4, it slightly mentions
Every bottle of solid fermentation object EA (ethyl acetate) about 200mL soaked overnight, three layers of filtered through gauze remove mycelium, receive
Collect filtrate, be concentrated under reduced pressure crude extract (oily medicinal extract) amount to 40g (300 bottles in total).
5, compound separates
The isometric ethyl acetate of above-mentioned crude extract is extracted 3 times, the concentration of gained extract liquor.By gained ethyl acetate portion
Reversed-phase column chromatography purifying is carried out, the methanol-water solution gradient elution for being 10: 90 to 100: 0 with volume ratio uses thin-layer chromatography point
The fraction containing noval chemical compound is analysed, is merged.
Gained fraction separates (Agilent Pursuit C-18 (10 μm, 21.2 × 250mm) with reversed-phase high performance liquid chromatography
Chromatographic column, Detection wavelength 254nm), the mobile phase used is that volume ratio is that 42: 58 acetonitrile-water systems are washed so that 10mL/min is isocratic
It is de-, the chromatographic peak of 21-24min is collected, for recycling design to get the compounds of this invention, appearance is colourless bulk on Rotary Evaporators
Crystal.
Embodiment 2
Purifying gained compound is identified.This is speculated as C according to high resolution mass spectrum HR-ESI-MS19H20O8([M+Na]+
399.1052).Infrared spectra adsorption are as follows: 3389cm-1(hydroxyl), 1753cm-1, 1635cm-1(carbonyl).Nuclear magnetic resonance data and
Signals assignment as shown in table 1 (1H NMR 400MHz,13C NMR 100MHz, solvent methanol-d4).It is marked such as Fig. 2 institute
Show.
Table 1
Present invention compound with anti-tumor activity1H NMR (nuclear magnetic resonance spectroscopy) figure is as shown in figure 3, the present invention
Compound with anti-tumor activity13C NMR (carbon-13 nmr spectra) figure is as shown in figure 4, the present invention has anti-tumor activity
Compound HSQC (heteronuclear list quantum relation) map as shown in figure 5, present invention compound with anti-tumor activity
HMBC (the hydrocarbon relationship of multikey) map is as shown in fig. 6, the present invention has the compound of antibacterial activity1H-1H COSY (hydrogen-hydrogen phase
Closing spectrum) map in conjunction with above-mentioned qualification result as shown in fig. 7, analyze, and the compound structure is as shown in Figure 1, be lattice Féraud mycin
(Gephyromycin) a kind of analog is named as lattice Féraud mycin B (Gephyromycin B).
Embodiment 3
The anti-tumor activity of compound detects, and is detected using mtt assay, detection method is as follows:
1, inoculating cell: by Human Prostate Cancer Cells (PC3) with containing 10% fetal calf serum culture solution (DMEM or
RMPI1640 it) is made into individual cells suspension, with 10000, every hole cell inoculation to 96 orifice plates, every pore volume 100 μ L is adherent thin
Born of the same parents shift to an earlier date 12 hours inoculated and cultureds.
2, be added compound of formula I and positive control drug cisplatin solution (40 μM of primary dcreening operations of fixed concentration, it is thin to tumour in the concentration
Compound of the intracellular growth inhibition near 50% sets 5 concentration and enters gradient secondary screening), every 200 μ L of hole final volume, every kind of processing is
If 3 multiple holes.
3, develop the color: after 37 degrees Celsius are cultivated 48 hours, every hole adds 20 μ L of MTT solution.Continue to be incubated for 4 hours, terminate culture,
It inhales and abandons culture supernatant in hole, every hole adds the SDS solution (10%) of 200 μ L, is incubated overnight (37 DEG C of temperature), keeps crystal abundant
Melt.
4, colorimetric: selection 595m wavelength, enzyme-linked immunosorbent assay instrument (Bio-Rad Imark) read each hole absorbance value, note
Record is as a result, using concentration as abscissa, and cell survival rate is that ordinate draws cell growth curve, using two-point method (Reed and
Muench method) calculate compound IC50Value.The results are shown in Table 2, and Gephyromycin B has significant inhibition prostate cancer
The effect of cell Proliferation.
Table 2
| Compound | IC50(μM) |
| Cis-platinum | 17.0 |
| Gephyromycin B | 13.32 |
Claims (7)
1. a kind of compound, structural formula such as Formulas I:
2. application of the compound as described in claim 1 in the drug of preparation treating cancer.
3. application as claimed in claim 2, which is characterized in that the cancer is prostate cancer.
4. the preparation method of compound as described in claim 1, which is characterized in that the preparation method comprises the following steps:
(1) fermented and cultured actinomyces obtain tunning；
(2) tunning filtering removal mycelium obtains crude extract；
(3) crude extract, which isolates and purifies, obtains the compound,
Wherein, the actinomyces deposit number is CICC No.11030,
The method isolated and purified in step (3) is as follows:
(a) crude extract is extracted with ethyl acetate；
(b) it after extract liquor concentration, is purified using reversed column chromatography, and eluted with methanol-water solution；
(c) after merging comprising the eluted product of purpose compound, the chemical combination is obtained using reversed-phase high performance liquid chromatography separation
Object,
Wherein, the filler that reversed-phase high performance liquid chromatography separation uses in step (c) is octadecyl silane, and mobile phase is body
Product collects the chromatographic peak of 21-24min than being 42: 58 acetonitrile-water systems with 10mL/min isocratic elution.
5. preparation method as claimed in claim 4, which is characterized in that fermentation is rice medium using culture medium, described big
Rice culture medium is made by following components: the sea salt water 60mL that mass concentration is 25% being added in every 40g rice.
6. preparation method as claimed in claim 4, which is characterized in that extracting process in step (a) are as follows: with the bodies such as crude extract
Long-pending ethyl acetate extracts 3 times.
7. preparation method as claimed in claim 4, which is characterized in that it is 10 that elution process, which is using volume ratio, in step (b):
90~100: 0 methanol-water solution gradient elution.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4945108A (en) * | 1988-04-26 | 1990-07-31 | Hoechst Aktiengesellschaft | Angucyclinones from streptomycetes, a process for the preparation thereof, and the use thereof |
| DE102004004906A1 (en) * | 2004-01-30 | 2005-09-01 | Johannes-Gutenberg-Universität Mainz | New oxygen-bridged angucyclinone derivatives, useful for treating bacterial infections and dementia, are modulators of intracellular calcium levels |
| CN1923825A (en) * | 2006-09-14 | 2007-03-07 | 中国医学科学院医药生物技术研究所 | Novel antibiotic Chemomycin A and preparation method thereof |
| CN101054403A (en) * | 2007-06-21 | 2007-10-17 | 中国医学科学院医药生物技术研究所 | Novel antibiotic Chemomycin A, B, C, D and preparation method thereof |
| CN103215281A (en) * | 2013-04-03 | 2013-07-24 | 中国科学院南海海洋研究所 | Biosynthetic gene cluster of grincamycin and P-1894B and application thereof |
-
2017
- 2017-07-10 CN CN201710556313.9A patent/CN107353295B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4945108A (en) * | 1988-04-26 | 1990-07-31 | Hoechst Aktiengesellschaft | Angucyclinones from streptomycetes, a process for the preparation thereof, and the use thereof |
| DE102004004906A1 (en) * | 2004-01-30 | 2005-09-01 | Johannes-Gutenberg-Universität Mainz | New oxygen-bridged angucyclinone derivatives, useful for treating bacterial infections and dementia, are modulators of intracellular calcium levels |
| CN1923825A (en) * | 2006-09-14 | 2007-03-07 | 中国医学科学院医药生物技术研究所 | Novel antibiotic Chemomycin A and preparation method thereof |
| CN101054403A (en) * | 2007-06-21 | 2007-10-17 | 中国医学科学院医药生物技术研究所 | Novel antibiotic Chemomycin A, B, C, D and preparation method thereof |
| CN103215281A (en) * | 2013-04-03 | 2013-07-24 | 中国科学院南海海洋研究所 | Biosynthetic gene cluster of grincamycin and P-1894B and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| "Angucycline/Angucyclinone类抗生素的研究进展";汪月等，;《国外医药抗生素分册》;20071231;第28卷(第3期);第97-102页 |
| "Gephyromycin, the first bridged angucyclinone, from Streptomyces griseus strain NTK 14";Gerhard Bringmann等，;《Phytochemistry》;20050507;第66卷;第1366-1373页 |
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