CN106518643B - A kind of cyclopentenone compound and its preparation method and use - Google Patents
A kind of cyclopentenone compound and its preparation method and use Download PDFInfo
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- CN106518643B CN106518643B CN201610895077.9A CN201610895077A CN106518643B CN 106518643 B CN106518643 B CN 106518643B CN 201610895077 A CN201610895077 A CN 201610895077A CN 106518643 B CN106518643 B CN 106518643B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
- C07C49/587—Unsaturated compounds containing a keto groups being part of a ring
- C07C49/703—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups
- C07C49/707—Unsaturated compounds containing a keto groups being part of a ring containing hydroxy groups a keto group being part of a three- to five-membered ring
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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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
- C12P7/38—Cyclopentanone- or cyclopentadione-containing products
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Abstract
The invention discloses a kind of cyclopentene ketone compounds and its preparation method and application, feature is the structural formula of the cyclopentene ketone compounds as shown in I, preparation methods steps include obtaining the fermentation liquid for containing new cyclopentene ketone compounds by the aspergillus ustus fermented and cultured that deposit number is CCTCC No.M2014086, then fermentation liquid is extracted with ethyl acetate, obtain coarse extract, by the extract through depressurizing silica gel column chromatography, reverse phase medium pressure column chromatography, last half preparative high-performance liquid chromatographic of reverse phase isolates and purifies to obtain, the cyclopentene ketone compounds, which have, inhibits cell activity, antitumor action, advantage is compound I and various pharmaceutically acceptable carriers, excipient or supplementary product compatibility, it can be made into anti-tumor drug, treatment for tumour, compound I is alternatively arranged as inhibiting the low molecule biology of cell Proliferation to visit Needle is used for life science.
Description
Technical field
The present invention relates to a kind of cyclopentene ketone compounds, more particularly, to a kind of cyclopentene ketone compounds and its preparation
Method and such compound are preparing the application in tumor cell proliferation inhibitor or antitumor agent.Background technique
Cyclopentene ketone compounds usually have the compound of significant physiological activity, such as prostaglandin, pentenomycin
Deng.The present inventor studies and learns, marine fungiAspergillus ustusDJ003(is preserved in China typical culture collection
Center, deposit number are as follows: CCTCC No:M2014086) ethyl acetate extract of liquid fermentation has preferable tumour cell to increase
Inhibitory activity is grown, its active constituent is studied then.The chemical structure and tumour cell for having not yet to see the compound increase
The report of inhibitory activity is grown, therefore in the market also there is not yet drug related to this.
Summary of the invention
Have technical problem to be solved by the invention is to provide one kind and inhibits tumor cell proliferation and anti-tumor activity
Cyclopentene ketone compounds and its preparation method and application.
The technical scheme of the invention to solve the technical problem is: a kind of cyclopentene ketone compounds, the ring penta
The structural formula of ketene compounds is as shown in I:
(I).
The preparation method of above-mentioned cyclopentene ketone compounds, specifically comprises the following steps:
(1) fermenting and producing
By deposit number be M2014086 aspergillus ustus (Aspergillus ustusDJ003) scribing line is brought back to life, and is inoculated into PDA
In solid slope culture medium, after being cultivated 5 days in 28 DEG C of incubators；The bacterium colony that inclined-plane culture obtains all is inoculated into and is equipped with
In 300mL PDB culture medium, in 28 DEG C, stationary culture 45 days, fermentation liquid is obtained；
(2) acquisition of medicinal extract
After fermentation liquid is removed thallus with filtered through gauze, the isometric ethyl acetate of fermentation liquid is repeated into extraction 3 times, is closed
And gained extract liquor is extracted three times, extract liquor is concentrated under reduced pressure and removes ethyl acetate, obtains coarse extract；
(3) separation and purification of compound
After coarse extract methylene chloride and methanol mixed solvent dissolution, add 200-300 mesh silica gel mixed sample, using petroleum
Ether/ethyl acetate is eluted using volume ratio 5:1 gradient as eluant, eluent, carries out decompression silica gel column chromatography to coarse extract, collection is washed
De- component uses elution ratio for 10-100% methanol/water gradient elution by elution fraction by compression leg chromatographic isolation in reverse phase
120 minutes, flow velocity 20ml/min, the flow point that appearance time is 100 minutes is collected, finally the flow point is high through more than half preparation reverse phases
Effect liquid phase chromatogram isolates and purifies to obtain cyclopentene ketone compounds, and structure is shown in formula I:
(I).
The formula of the PDB culture medium is as follows: 200 g of potato, glucose 20 g and seawater 1L.
The volume ratio of methylene chloride and methanol is 1:1 in the methylene chloride and methanol mixed solvent.
The eluent of the half preparation reversed-phase high performance liquid chromatography is that 1:1 is mixed methanol by volume with water.
The application of above-mentioned cyclopentene ketone compounds, the cyclopentene ketone compounds are preparing tumor cell proliferation suppression
Purposes in terms of preparation or anti-tumor drug.
Compared with the prior art, the advantages of the present invention are as follows: a kind of cyclopentene ketone compounds of the present invention and its preparation side
Method and purposes, the present invention obtain the fermentation liquid for containing new cyclopentene ketone compounds by microbial fermentation culture, then will hair
Zymotic fluid is extracted with ethyl acetate, and obtains coarse extract, which is depressurized silica gel column chromatography, reverse phase medium pressure column chromatography, last anti-
Half preparative high-performance liquid chromatographic of phase isolates and purifies to obtain, which, which has, inhibits cell activity, antitumor work
With.Compound I and various pharmaceutically acceptable carriers, excipient or supplementary product compatibility, can be made into anti-tumor drug, for tumour
Treatment.Compound I is alternatively arranged as inhibiting the low molecule bioprobe of cell Proliferation for life science, as probe application
When, compound I is dissolved in methanol, water or aqueous methanol, is also dissolved in the aqueous solution of dimethyl sulfoxide and is applied.
Above-mentioned aspergillus ustus (Aspergillus ustus), which is DJ003 bacterial strain, and deposit number is CCTCC No.
M2014086, China typical culture collection center was preserved on 03 14th, 2014, and preservation address is the China Wuhan military
Chinese university.
Specific embodiment
Present invention is further described in detail with reference to embodiments.
Embodiment 1
A kind of cyclopentene ketone structural formula of compound is as shown in I:
(I).
Embodiment 2
The preparation method of cyclopentene ketone compounds, specifically comprises the following steps: as shown in I formula
(1) fermenting and producing
By deposit number be M2014086 aspergillus ustus (Aspergillus ustusDJ003) scribing line is brought back to life, and is inoculated into PDA
In solid slope culture medium, after being cultivated 5 days in 28 DEG C of incubators；The bacterium colony that inclined-plane culture obtains all is inoculated into and is equipped with
In 300mL PDB culture medium (200 g of potato, 20 g of glucose, seawater 1L), in 28 DEG C, stationary culture 45 days, sent out
Zymotic fluid；
(2) acquisition of medicinal extract
After fermentation liquid is removed thallus with filtered through gauze, the isometric ethyl acetate of fermentation liquid is repeated into extraction 3 times, is closed
And gained extract liquor is extracted three times, extract liquor is concentrated under reduced pressure and removes ethyl acetate, obtains coarse extract；
(3) separation and purification of compound
After coarse extract methylene chloride and methanol (volume ratio of methylene chloride and methanol is 1:1) mixed solvent dissolution,
Add 200-300 mesh silica gel mixed sample, petrol ether/ethyl acetate is used to be eluted using volume ratio 5:1 gradient as eluant, eluent, to thick leaching
Cream carries out decompression silica gel column chromatography, collects elution fraction, by elution fraction by compression leg chromatographic isolation in reverse phase, using elution ratio
Example is 10-100% methanol/water gradient elution 120 minutes, flow velocity 20ml/min, collects the flow point that appearance time is 100 minutes,
Finally the flow point isolates and purifies to obtain cyclopentene ketone compounds, structure such as Formulas I through more than half preparation reversed-phase high performance liquid chromatography
It is shown:
(I).
The chemical compounds I colorless oil, molecular formula C7H9O3Cl, anion HRESIMSm/z: 175.0162,177.0149
[M – H]–,1H and13C-NMR data are shown in Table 1.
1 chemical compounds I of table1H and13C NMR data (500 and 125MHz, in DMSO-d 6)a
Note: a) indicate this table signals assignment be based on DEPT,1H-1H COSY, HSQC and HMBC spectrum analysis result.Hydrogen signal
Multiplicity uses s(singlet respectively), d(doublet), t(triplet) and m(multiplet) table；B) indicate in this column number and
Code name respectively represents1H-1Coupling coherent signal is provided in H COSY spectrum with the 1H in corresponding line1H core；C) it indicates in this column
Number and code name respectively represent HMBC compose in and in corresponding line1H provides coupling coherent signal13C core.
Embodiment 3
The test (cell inhibitory effect active testing) of anti tumor activity in vitro
(1) laboratory sample
The preparation of sample solution: test sample is the chemical compounds I sterling isolated and purified in above-described embodiment 1, accurate
Appropriate amount of sample is weighed, the solution of required concentration is configured to methanol, for surveying activity.
The squamous subculture of cell line and cell uses Non-small cell lung carcinoma A549 cell and human hepatoma HepG2 cell,
Use 10%BCS(OEG cell growth hormone) 1640 culture mediums, 37 DEG C in be passed through 0.5% carbon dioxide incubator relay
It is commissioned to train feeding.
(2) experimental method
Cell inhibitory effect activity test method: the people of tetramethyl azo azoles salt (MTT) method logarithmic growth phase is non-small thin
Born of the same parents' lung cancer A549 cell and human hepatoma HepG2 cell, after cell is digested with pancreatin, adjusting density is 2~3 × 104A/
Hole makes an addition in 96 orifice plates, and 195 microlitres of every hole is placed in 37 DEG C, 0.5%CO2It cultivates 24 hours, is then added not in incubator
With concentration samples solution into 96 orifice plates, each concentration of sample is respectively provided with three holes in 96 orifice plate of same, using methanol as
Negative right, 37 DEG C, 0.5%CO2It is cultivated 48 hours in incubator；96 orifice plates are taken out, 20 microlitres of MTT are added in every hole, and (tetramethyl is even
Nitrogen azoles salt) (5 mg/ml of concentration), continue culture 4 hours；Culture solution is abandoned, 150 microlitres of DMSO (dimethyl sulfoxide) are added in every hole,
The light absorption that each hole is measured at 37 DEG C of oscillations 6 min, 492 nm, takes three hole mean OD values by IR(%)=(OD negative control-OD
Sample)/OD negative control × 100% formula calculates the cell proliferation inhibition rate (IR%) under each concentration.
(3) experimental result
In mtt assay test, the chemical compounds I of various concentration is to Non-small cell lung carcinoma A549 cell and human liver cancer
The Proliferation Ability result of HepG2 cell is shown in Table 2 respectively.
Inhibiting rate (%) of the chemical compounds I of 2 various concentration of table to cancer cell multiplication
Chemical compounds I has apparent Cytostatic to tumor cell effect as seen from the above table, can be used as inhibition of cell proliferation
Or antitumor agent is used for antitumor research.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range.
Claims (3)
1. a kind of preparation method of cyclopentene ketone compounds, which comprises the following steps:
(1) fermenting and producing
The aspergillus ustus (Aspergillus ustus) that deposit number is M2014086 is crossed and is brought back to life, the training of PDA solid slope is inoculated into
It supports on base, after being cultivated 5 days in 28 DEG C of incubators；The bacterium colony that inclined-plane culture obtains all is inoculated into and is trained equipped with 300mL PDB
It supports in base, in 28 DEG C, stationary culture 45 days, obtains fermentation liquid；
(2) acquisition of medicinal extract
After fermentation liquid is removed thallus with filtered through gauze, the isometric ethyl acetate of fermentation liquid is repeated into extraction 3 times, merges three
Extract liquor is concentrated under reduced pressure and removes ethyl acetate by extract liquor obtained by secondary extraction, obtains coarse extract；
(3) separation and purification of compound
After coarse extract methylene chloride and methanol mixed solvent dissolution, add 200-300 mesh silica gel mixed sample, using petroleum ether/second
Acetoacetic ester is eluted using volume ratio 5:1 gradient as eluant, eluent, is carried out decompression silica gel column chromatography to coarse extract, is collected elution group
Point, by elution fraction by compression leg chromatographic isolation in reverse phase, use elution ratio for 10-100% methanol/water gradient elution 120
Minute, flow velocity 20ml/min collects the flow point that appearance time is 100 minutes, and finally the flow point prepares reversed phase high efficiency through more than half
Liquid chromatogram isolates and purifies to obtain cyclopentene ketone compounds, and structure is shown in formula I:
Wherein the eluent of the half preparation reversed-phase high performance liquid chromatography is that methanol is pressed with water
Volume ratio 1:1 is mixed.
2. a kind of preparation method of cyclopentene ketone compounds according to claim 1, it is characterised in that the PDB training
The formula for supporting base is as follows: potato 200g, glucose 20g and seawater 1L.
3. a kind of preparation method of cyclopentene ketone compounds according to claim 1, it is characterised in that the dichloro
The volume ratio of methylene chloride and methanol is 1:1 in methane and methanol mixed solvent.
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| CN107141335B (en) * | 2017-04-12 | 2020-10-20 | 宁波大学 | Cyclopeptide compound and preparation method and application thereof |
| CN108179114B (en) * | 2017-11-27 | 2021-08-20 | 南京晓庄学院 | Strain producing anti-anaerobic bacteria compound and fermentation method, anti-anaerobic bacteria compound extraction and preparation method and use method |
| CN111253222A (en) * | 2020-03-02 | 2020-06-09 | 福建省中科生物股份有限公司 | Phenolic compound ZKYY-037 and preparation method and application thereof |
| CN116874362B (en) * | 2023-06-27 | 2024-08-16 | 东海实验室 | A class of cyclopentenone compounds and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1230165A (en) * | 1996-09-27 | 1999-09-29 | 宝酒造株式会社 | Cyclopentenones, process for preparing same, and use thereof |
| CN1247528A (en) * | 1997-03-11 | 2000-03-15 | 宝酒造株式会社 | Cyclopentenone derivatives |
| CN1409715A (en) * | 1999-12-16 | 2003-04-09 | 查特豪斯治疗有限公司 | Cyclopenteneone derivatives |
| CN101613264A (en) * | 2008-06-27 | 2009-12-30 | 中国科学院广州生物医药与健康研究院 | Cycloenone compounds and their application in the preparation of antitumor drugs |
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- 2016-10-14 CN CN201610895077.9A patent/CN106518643B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1230165A (en) * | 1996-09-27 | 1999-09-29 | 宝酒造株式会社 | Cyclopentenones, process for preparing same, and use thereof |
| CN1247528A (en) * | 1997-03-11 | 2000-03-15 | 宝酒造株式会社 | Cyclopentenone derivatives |
| CN1409715A (en) * | 1999-12-16 | 2003-04-09 | 查特豪斯治疗有限公司 | Cyclopenteneone derivatives |
| CN101613264A (en) * | 2008-06-27 | 2009-12-30 | 中国科学院广州生物医药与健康研究院 | Cycloenone compounds and their application in the preparation of antitumor drugs |
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