CN107903277B - Mould chlorins compound of a kind of N- methyl-Mei Da and its preparation method and application - Google Patents
Mould chlorins compound of a kind of N- methyl-Mei Da and its preparation method and application Download PDFInfo
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- -1 chlorins compound Chemical class 0.000 title claims abstract description 25
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 150000001875 compounds Chemical class 0.000 claims abstract description 27
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 18
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 18
- 239000000287 crude extract Substances 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 239000000945 filler Substances 0.000 claims description 7
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
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- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Inorganic materials [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- BBNQQADTFFCFGB-UHFFFAOYSA-N purpurin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC(O)=C3C(=O)C2=C1 BBNQQADTFFCFGB-UHFFFAOYSA-N 0.000 description 1
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- NLVFBUXFDBBNBW-PBSUHMDJSA-N tobramycin Chemical compound N[C@@H]1C[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N NLVFBUXFDBBNBW-PBSUHMDJSA-N 0.000 description 1
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- C07D493/04—Ortho-condensed systems
-
- 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
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses mould chlorins compounds of a kind of N- methyl-Mei Da and its preparation method and application.The mould chlorins compound of the N- methyl-Mei Da is named as 3 '-N- methyl-Mei Da mycins.The mould chlorins compound of N- methyl-Mei Da of the present invention is to isolate and purify to obtain from a kind of actinomyces, is identified as a kind of mould chlorins compound of N- methyl-Mei Da, is specifically named as 3 '-N- methyl-Mei Da mycins.The experiment proved that the compound has stronger inhibiting effect to Human Prostate Cancer Cells, IC50 value reaches 0.51255 μM.The compounds of this invention has a good application prospect in terms of the drug of preparation treating cancer.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of mould chlorins compound of N- methyl-Mei Da and its preparation
Methods and applications.
Background technique
Marine actinomycete and fungi are concerned because it generates structure novel, natural active product vdiverse in function.It is special
The marine eco-environment of different multiplicity imparts marine actinomycete and the complicated unique metabolic pathway of fungi, secondary metabolite
Structure type from terms of all show the feature and diversity different with fungi with terrestrial actinomyces, become
It was found that the important sources of natural active product and new drug lead compound.
Simultaneously in the secondary metabolite of streptomycete, the antibiotic of aromatic polyketones class is that a kind of structure function is more various
The compound of change.In the biosynthesis pathway of streptomycete, this kind of compound is polyketide synthase, ketone reduction in cell first
The aromatic polyketones carbon skeleton of compound is synthesized under the action of enzyme, aromatizing enzyme and cyclase etc., and aoxidized, glycosylated,
The modification such as methylation, forms the secondary metabolite of unique aromatic polyketones class.These diversified modification means make fragrance
The antibiotic of polyketone class has diversified structure and activity.
In the antibiotic of aromatic polyketones class, pyranone alpha naphthol quinones (pyranonaphthoquinones, BIQs) generation
Table one kind has the antibiotic of similar aromatic polyketones carbon skeleton, including beauty reaches mycin (Medermycin), kalafungin, puts
Line bacterium purpurine, granaticin etc..Its Sino-U.S. reaches mycin, is a kind of carbon tobramycin antibiotic with aromatic polyketones carbon skeleton, initially
Streptomyces sp.K73 is isolated from by Takano et al. in 1976, there is very strong antibacterial and antitumor activity,
Promise to be the lead compound of antibacterial anti-tumor drug.
It is shown for the activity research of beauty up to mycin, when such small molecule compound and serine/threonine protein kinase
After cysteine residues in enzyme (AKT/PKB) active site combine, it can irreversibly inhibit serine/threonine protein kinase
Enzyme loses substrate phosphorylation ability.But for some serine/threonines for lacking cysteine residues in active site
For protein kinase, beauty loses the inhibiting effect to it up to mould chlorins compound.Furthermore the compound of beauty up to mycin class also has
Inhibit mammal rapamycin target protein (mammalian target of rapamycin, mTOR) signal path in cell,
And inhibit the activity of capped mRNA translation initiation process.
In tumour cell, since the mutation of tumor cell gene causes a series of variation of mechanism, as PTEN is active
Missing, causes the level of the protein kinase AKT activated in tumour cell to be sharply increased.Beauty passes through mutual with protein kinase up to mycin
Effect combines, and prevents the combination of ATP and protein kinase under physiological status, therefore have the ability for inhibiting the enzymatic activity, influences
The normal processes of cell cycle.But research finds beauty up to the no specificity of mycin, while playing a role to tumour cell
The multiple fission of normal cell is influenced, it is too big as clinical development application toxicity, therefore can not patent medicine.Therefore it is based on such chemical combination
The mechanism of action of object continually develops research and obtains a series of structure and functions and beauty up to the similar compound of mycin.
Summary of the invention
The present invention has found that a kind of new N- methyl-Mei Da with anti-tumor activity is mould by carrying out research to actinomyces
Chlorins compound.
A kind of mould chlorins compound of N- methyl-Mei Da is named as 3 '-N- methyl-Mei Da mycins, structural formula such as Formulas I:
Invention further provides the mould chlorins compound of N- methyl-Mei Da answering in the drug of preparation treating cancer
With.
The cancer be melanoma, acute myeloid leukemia, colon cancer, non-small cell lung cancer, leukaemia, liver cancer,
Kidney, thyroid cancer, cutaneum carcinoma, cancer of pancreas, oophoroma, breast cancer, celiothelioma or Peripheral Nerve Sheath Tumours etc..Preferably,
The cancer is prostate cancer.
The present invention also provides the preparation methods of the mould chlorins compound of N- methyl-Mei Da, comprising 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 mould chlorins compound of the N- methyl-Mei Da,
Wherein, the actinomyces are Streptomyces cavourensis strain NRRL 2740, and purchase is certainly Chinese
Research for Industrial Microbial Germ preservation administrative center.
Fermentation is prepared by Gause I fluid nutrient medium and sea salt in 10mL: 1g ratio using culture medium.
Isolation and purification method is as follows in step (3):
(a) crude extract is extracted with ethyl acetate；
(b) after extract liquor concentration, using silica gel chromatography, and accounted for methylene chloride volume 0%~20% dichloro
Methane-methanol system gradient elution；
(c) it after merging comprising the eluted product of purpose compound, is purified using gel column chromatography, and with methylene chloride body
Product accounts for 50% methylene chloride-methanol system elution；
(d) after merging comprising the eluted product of purpose compound, using described in high performance preparative liquid chromatography purifying acquisition
The mould chlorins compound of N- methyl-Mei Da.
Extracting process in step (a) are as follows: extracted 3 times with the isometric ethyl acetate of crude extract.
The filler that silica gel chromatography uses in step (b) is silica gel.
The filler that gel column chromatography purifying uses in step (c) is hydroxypropyl sephadex.
The filler that high performance preparative liquid chromatography purifying uses in step (d) is octadecyl silane, and mobile phase is body
The methanol-water solution of product concentration 20%-100%, and the trifluoroacetic acid of volumetric concentration 0.5 ‰ is also added in mobile phase, with gradient
Elution.
The mould chlorins compound of N- methyl-Mei Da of the present invention is to isolate and purify to obtain from a kind of actinomyces, is identified as one
The kind mould chlorins compound of N- methyl-Mei Da, is specifically named as 3 '-N- methyl-Mei Da mycins.The experiment proved that the compound is to people
Prostate gland cancer cell has stronger inhibiting effect, IC50Value reaches 0.51255 μM.The compounds of this invention is in preparation treating cancer
Drug in terms of have a good application prospect.
Detailed description of the invention
Fig. 1 is the compounds of this invention1H-NMR map.
Fig. 2 is the compounds of this invention13C-NMR map.
Fig. 3 is the structure chart of the compounds of this invention.
Specific embodiment
Strain source
Streptomycete Streptomyces cavourensis strain NRRL 2740: purchase is from positioned at Beijing's southern exposure
The Chinese industrial Microbiological Culture Collection administrative center (CICC) in No. 6 building of No. 24 institutes in area winebibber's bridge Road orders network address:
http://www.china-cicc.org/。
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.
Embodiment 1
1, seed liquor
Streptomycete Streptomyces cavourensis strain NRRL 2740 is inoculated in containing 250mL Gao Shi
In the 500mL conical flask of No.1 fluid nutrient medium, with 180rpm shaken cultivation 3 days at 28 DEG C in shaking table, seed liquor is obtained.
2, it ferments
Above-mentioned seed liquor is inoculated into culture medium that (culture medium is 250mL Gao Shi in every bottle by the amount for being inoculated with 12mL with every bottle
25g sea salt is added in No.1 fluid nutrient medium, then through obtained by high pressure moist heat sterilization), it is shaken in shaking table at 28 DEG C with 180rpm
Fermentation is terminated after swinging culture culture 7 days.
3, 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, crude extract (oily medicinal extract) is concentrated under reduced pressure to obtain.
4, compound separates
(a) the isometric ethyl acetate of above-mentioned crude extract is extracted 3 times, the concentration of gained extract liquor；
(b) after extract liquor concentration, using silica gel chromatography, and accounted for methylene chloride volume 0%~20% dichloro
Methane-methanol system gradient elution, every 1/4 column volume are a fraction, and thin-layer chromatography (TLC) analysis, which merges, contains target chemical combination
The fraction of object；
(c) it after merging comprising the eluted product of purpose compound, is purified using gel column chromatography, and with methylene chloride body
Product accounts for 50% methylene chloride-methanol system elution, and every 1/4 column volume is a fraction, and thin-layer chromatography (TLC) analysis, which merges, to be contained
There is the fraction of target compound；
(d) after merging comprising the eluted product of purpose compound, (Agilent is purified using high performance preparative liquid chromatography
Pursuit C-18 (10 μm, 21.2 × 250mm) chromatographic column, filler is octadecyl silane, Detection wavelength 254nm), stream
It is dynamic to be mutually the methanol-water solution of volumetric concentration 20%-100%, and the trifluoro second of volumetric concentration 0.5 ‰ is also added in mobile phase
Acid collects the chromatographic peak where the mould chlorins compound of the N- methyl-Mei Da with gradient elution, and then recycling design obtains institute
The mould chlorins compound of N- methyl-Mei Da is stated, is in rufous crystalloid.
Embodiment 2
The compound that embodiment 1 is isolated and purified carries out Structural Identification.
The nuclear magnetic resonance data of the compound is as shown in table 1, nuclear magnetic resonance parameter 1H 500MHz, 13C 125.7MHz.
Table 1
In conjunction with mass spectrometric data (Fig. 1 and Fig. 2), show that the molecular formula of the compound is C23H25NO8([M+H]+
444.1659), structural formula is as shown in figure 3, identify that the Compound nomenclature is 3 '-N- methyl-Mei Da mycins.
Embodiment 3
Obtained compound is purified to the proliferation of Human carcinoma of prostate cell line PC3 cell using SBR method detection embodiment 1
Inhibiting effect.
The cell of logarithmic growth phase, is configured to 5 × 104A/mL is laid on 96 well culture plates, CO with 100 holes μ L/2Culture
It is cultivated 24 hours in case, the sample to be tested of various concentration is added after taking-up culture plate in every hole, each concentration sets 3 multiple holes,
After the completion of dosing, it is placed in CO2Culture plate is taken out after continuing culture in incubator 72 hours, discards culture solution, 100 μ are added in every hole
10% trichloroacetic acid (TCA) of L4 DEG C of refrigerator pre-cooling is fixed, stands after five minutes, then culture plate is moved to 4 DEG C of refrigerator overnights.
Fixer is outwelled, every hole is washed with deionized 5 times, and drying is air-dried.70 μ L SRB solution are added in every hole, are placed at room temperature for
20 minutes, supernatant is removed, is washed 5 times, is air-dried with 1% acetic acid.In conjunction with 100 hole μ L/ 10mmol/L Tris alkali of SRB
Liquid (pH=10.5) oscillation dissolution.It is placed in microplate reader and measures each hole light absorption, measurement wavelength is 515nm.
Drug cell proliferation inhibiting rate: inhibiting rate=[1- (OD515 dosing holes/OD515 pairs is calculated according to each hole OD value
According to hole)] × 100%, according to each concentration inhibiting rate calculation of half inhibitory concentration IC50.The results are shown in Table 2, the compounds of this invention
To the IC of Human carcinoma of prostate cell line PC3 cell50Value is 0.51255 μM, illustrates there is stronger inhibiting effect.
Table 2
Claims (7)
1. a kind of preparation method of the mould chlorins compound of N- methyl-Mei Da, which is characterized in that the N- methyl-Mei Da mycin class
Compound is named as 3 '-N- methyl-Mei Da mycins, structural formula such as Formulas I:
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 mould chlorins compound of the N- methyl-Mei Da,
Wherein, the actinomyces are Streptomyces cavourensis strain NRRL 2740, are bought from Chinese industrial
Microbiological Culture Collection administrative center.
2. preparation method as described in claim 1, which is characterized in that fermentation is using culture medium by Gause I fluid nutrient medium
It is prepared with sea salt in 10mL: 1g ratio.
3. preparation method as described in claim 1, which is characterized in that isolation and purification method is as follows in step (3):
(a) crude extract is extracted with ethyl acetate；
(b) after extract liquor concentration, using silica gel chromatography, and accounted for methylene chloride volume 0%~20% methylene chloride-
Methanol system gradient elution；
(c) it after merging comprising the eluted product of purpose compound, is purified using gel column chromatography, and accounted for methylene chloride volume
50% methylene chloride-methanol system elution；
(d) after merging comprising the eluted product of purpose compound, the N- first is obtained using high performance preparative liquid chromatography purifying
The mould chlorins compound of Ji-Mei Da.
4. preparation method as claimed in claim 3, 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.
5. preparation method as claimed in claim 3, which is characterized in that the filler that silica gel chromatography uses in step (b)
For silica gel.
6. preparation method as claimed in claim 3, which is characterized in that gel column chromatography purifies the filler used in step (c)
For hydroxypropyl sephadex.
7. preparation method as claimed in claim 3, which is characterized in that high performance preparative liquid chromatography purifying uses in step (d)
Filler be octadecyl silane, mobile phase be volumetric concentration 20%-100% methanol-water solution, and in mobile phase also
The trifluoroacetic acid of volumetric concentration 0.5 ‰ is added, with gradient elution.
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| CN102452916A (en) * | 2010-10-25 | 2012-05-16 | 南京大学 | New aromatic polyketones, extraction method and application thereof |
| CN102844023A (en) * | 2010-03-08 | 2012-12-26 | 索隆-基特林癌症研究协会 | Cdc7 kinase inhibitors and uses thereof |
| CN105200072A (en) * | 2015-10-08 | 2015-12-30 | 中国科学院南海海洋研究所 | Biosynthetic gene cluster of romatic-polyketide atypical fluostatins and applications of biosynthetic gene cluster |
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| CN102844023A (en) * | 2010-03-08 | 2012-12-26 | 索隆-基特林癌症研究协会 | Cdc7 kinase inhibitors and uses thereof |
| CN102452916A (en) * | 2010-10-25 | 2012-05-16 | 南京大学 | New aromatic polyketones, extraction method and application thereof |
| CN105200072A (en) * | 2015-10-08 | 2015-12-30 | 中国科学院南海海洋研究所 | Biosynthetic gene cluster of romatic-polyketide atypical fluostatins and applications of biosynthetic gene cluster |
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