CN104212852B - A kind of phenol piperazine class antitumor antibiotics and preparation method thereof - Google Patents
A kind of phenol piperazine class antitumor antibiotics and preparation method thereof Download PDFInfo
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- CN104212852B CN104212852B CN201410409077.4A CN201410409077A CN104212852B CN 104212852 B CN104212852 B CN 104212852B CN 201410409077 A CN201410409077 A CN 201410409077A CN 104212852 B CN104212852 B CN 104212852B
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Abstract
The present invention is a kind of new phenol piperazine class antitumor antibiotics, is characterized in the streptomyces strain for CCTCC No.M2013727 by fermented and cultured preservation registration number(Streptomycessp.)YIM R4, then its tunning is isolated and purified, molecular formula is obtained for C25H16N4O4, molecular weight is a kind of 436 new antitumor antibiotics, be the experiment proved that, antitumor antibiotics of the invention has inhibitory action to human gastric cancer and human cervical carcinoma's tumour cell.
Description
Technical field
The present invention relates to a kind of new phenol piperazine class antitumor antibiotics and preparation method thereof, belong to biological technical field.
Background technology
The new disease continuously emerged and the enhancing of pathogen Antibiotic Resistance force people to accelerate grinding for new drug
Hair, the natural source new drug lead compound that high-efficiency low-toxicity is found from microorganism is one of focus of Recent study.Due to mesh
The existing longer history of the preceding research to normal soil environmental microorganism, therefrom finds that the difficulty of novel structure reactive compound is more next
Bigger, in order to find antibiotics lead compound, people turn to sight the microorganism for inhabiting special habitats.
The endophyte in plant is widely present in due to common evolutionary for a long time with its host plant, possesses special generation
Thank to approach, the probability for producing active noval chemical compound is larger, is the new microbe resource that a class has preferable potentiality to be exploited.Yunnan
Xishuangbanna possesses abundant resources of medicinal plant, wherein containing substantial amounts of endophyte of plant, carries out and therefrom finds activity newly
The research of lead compound, it is significant to be that the follow-up new drug development with independent intellectual property right lays the foundation.
The content of the invention
It is an object of the invention to provide a kind of novel anti-tumor antibiotic for being different from known phenol piperazine class antibiotic structure, it is right
Human gastric cancer and human cervical carcinoma's tumour cell have inhibitory action.
It is a further object to provide a kind of preparation method of new phenol piperazine class antitumor antibiotics.
Technical scheme is as follows:
A kind of new phenol piperazine class antitumor antibiotics, the producing bacterial strain of this antibiotic has been deposited in China typical culture collection
Center;Address:Chinese Wuhan;Preservation date:On December 29th, 2013;Preservation numbering of registering on the books is CCTCC No.
M2013727 ;By cultivating one plant of streptomycete bacterial strain being isolated from In Xishuangbanna of Yunnan medicinal plant tissue
(Streptomyces Sp.) YIM R4, then its tunning is isolated and purified, obtain this antitumor antibiotics;This is anti-
Anti-neoplastic antibiotic is that molecular formula is C25H16N4O4, molecular weight is 436 compound, and the compound has following structure:
(I)。
The preparation method of the phenol piperazine class antitumor antibiotics of the present invention, comprises the following steps:
(l) strain liquid seed culture:1. the preparation of liquid seed culture medium:Take 2.0 ~ 6.0g of yeast extract;Portugal
2.0 ~ 6.0g of grape sugar;3.0 ~ 7.0g of malt extract;2.5 ~ 4.5mg of B B-complex;1000ml, pH 7.2 are added water to,
120 °C of sterilizing 30min;2. ferment:By bacterial strain YIM R4 be inoculated into sterilizing after culture medium in, under 28 °C in rotating speed be 190 ~
Shake flask fermentation 2 days on 250 revs/min of shaking table.
(2) strain fermentation culture:1. the preparation of fermentation medium:Take 20.0 ~ 30.0g of starch;Beef extract 1.0 ~
5.0g ;0.5 ~ 1.5g of glucose;3.0 ~ 7.0g of yeast extract;1.0 ~ 5.0g of peptone;CaCO32.0~6.0g ;Add water to
7.0,120 °C of 30 min of sterilizing of 1000ml, pH;2. ferment:By the bacterial strain YIM R4 seeds obtained in the above method (l)
Liquid is added in the fermentation medium after sterilizing, plus 10 % that strain amount is fermentation medium weight, under 28 °C in rotating speed be 190 ~
Shake flask fermentation 6 ~ 8 days on 250 revs/min of shaking table.
(3) fermentation liquor treatment:1. zymotic fluid is through 2500 ~ 3500 revs/min of centrifugations, and centrifugation time is 15 ~ 25 minutes;
2. with isometric ethyl acetate extractive fermentation supernatant 3 ~ 5 times, ethyl acetate is reclaimed, mycelium acetone soak and ultrasound is broken
It is broken, acetone is reclaimed, merges two extracting section things, obtains the sepia paste at active position;3. silica gel column layer is carried out
Analysis, with chloroform:Methanol 40:1 to 1:1 gradient elution, collects the component containing the compound, and recycling design obtains thick
A kind of antitumor antibiotics of product;4. a kind of antitumor antibiotics of crude product is subjected to methanol gel and chloroform successively:Methanol
30 :1 silica gel column chromatography, recycling design obtains a kind of antitumor antibiotics compared with sterling;5. it is a kind of antitumor antibiotics is purer
Product carry out methanol gel column chromatography, and recycling design obtains a kind of antitumor antibiotics sterling.
It is preferably that yeast extract is 4.0g in the preparation of liquid seed culture medium;Glucose is 4.0g;Malt extract is
5.0g ;B B-complex is 3.5mg.
It is preferably that starch is 24.0g in the preparation of fermentation medium;Beef extract is 3.0g;Glucose is 1.0g;Ferment
Female cream is 5.0g;Peptone is 3.0g;CaCO3For 4.0g.
Shaking table revolution is advisable for 220 revs/min during shake flask fermentation, and fermentation time was advisable with 7 days.
When zymotic fluid is centrifuged, centrifuge speed is advisable with 3000 revs/min, and described centrifugation time is to be advisable for 20 minutes,
The extraction times of described ethyl acetate are advisable with 4 times.
A kind of antitumor antibiotics sterling is through analytic type HPLC detections, chromatographic column(5 μm of 250 × 4.6mm, Luna,
Phenomenex, C18);Methanol:Water 7:3 isocratic elutions, Detection wavelength 254nm, flow velocity 1.0ml/min, it was demonstrated that one kind is anti-swollen
Knurl antibiotic sterling purity is higher than 98%.
Antitumor antibiotics of the present invention is through conventional mtt assay it is demonstrated experimentally that it has inhibitory action to human tumor cell line, and it resists
Tumor promotion such as table(1)It is shown.
Table(1)The anti tumor activity in vitro of antitumor antibiotics of the present invention
Tumour cell | IC50(ug/ml) |
Human gastric carcinoma cell line BGC-823 | 14.9 |
Human cervical carcinoma cell lines Hela | 28.8 |
Brief description of the drawings
Fig. 1 is the chemical structural formula of antibiotic of the present invention;
Fig. 2 is the infrared absorption spectroscopy of antibiotic of the present invention;
Fig. 3 composes (DMSO- for the hydrogen of antibiotic of the present inventiond 6) ;
Fig. 4 for antibiotic of the present invention carbon compose (13C-NMR、DEPT) ;
Fig. 5 composes for the HMBC of antibiotic of the present invention;
Fig. 6 is the hsqc spectrum of antibiotic of the present invention;
Fig. 7 composes for the H-H COSY of antibiotic of the present invention;
Fig. 8 composes for the NOESY of antibiotic of the present invention;
Fig. 9 is the mass spectrum of antibiotic of the present invention.
Embodiment
With reference to specific embodiment, the invention will be further described:
Embodiment 1
The preparation of compound (I)
(l) strain liquid seed culture:1. the preparation of liquid seed culture medium:Take yeast extract 4.0g;Glucose
4.0g ;Malt extract 5.0g;B B-complex 3.5mg;Add water to 1000ml, 7.2,120 °C of sterilizing 30min of pH;2. ferment
:Bacterial strain YIM R4 are inoculated into the culture medium after sterilizing, in shake flask fermentation 2 on the shaking table that rotating speed is 220 revs/min under 28 °C
My god.
(2) strain fermentation culture:1. the preparation of fermentation medium:Take starch 24.0g;The g of beef extract 3.0;Grape
Sugared 1.0g;Yeast extract 5.0g;Peptone 3.0g;CaCO34.0g ;Add water to 1000ml, 7.0,120 °C of sterilizings 30 of pH
min ;2. ferment:The bacterial strain YIM R4 seed liquors obtained in the above method (l) are added in the fermentation medium after sterilizing,
Plus 10 % that strain amount is fermentation medium weight, under 28 °C in rotating speed be shake flask fermentation on 220 revs/min of shaking table 7 days.
(3) fermentation liquor treatment:1. zymotic fluid is through 3000 revs/min of centrifugations, and centrifugation time is 20 minutes;2. with grade body
Product ethyl acetate is extracted 4 times, reclaims ethyl acetate, mycelium acetone soak and ultrasonication, reclaims acetone, merges two parts
Extract, obtains the sepia paste at active position;3. silica gel column chromatography is carried out, with chloroform:Methanol 40:1
To 1:1 gradient elution, collects the component containing the compound, and recycling design obtains a kind of antitumor antibiotics of crude product;4. will
A kind of antitumor antibiotics of crude product carries out methanol gel and chloroform successively:Methanol 30:1 silica gel column chromatography, recycling design,
A kind of antitumor antibiotics is obtained compared with sterling;5. a kind of antitumor antibiotics is subjected to methanol gel column chromatography compared with sterling, reclaimed
Solvent, obtains a kind of antitumor antibiotics sterling.
The compounds of this invention as obtained above, with following physicochemical properties:
1. character:Yellow powder.
2. molecular weight:Molecular weight is 436.
3. molecular formula:Molecular formula is C25H16N4O4。
4. elementary analysis:C 68.79%, H 3.70%, N 12.84%, O 14.67%.
5. the characteristic group in infrared absorption spectroscopy:(KBr) IR (3427,1708,1627,1579cm-1Wave number) card
There are phenolic hydroxyl group, carboxylic carbonyl and phenyl ring in bright molecular structure.
6. integrate1H spectrums,13C spectrums, HMBC, HSQC, H-H COSY spectrums, NOESY spectrums and mass spectrum (ESI-MS) [M+Na]+ =
459 analyze datas, it is determined that the molecular weight of the compounds of this invention is 436, and molecular formula is C25H16N4O4, its structure such as institute of accompanying drawing 1
Show.
Embodiment 2
The compound prepared in embodiment 1(I)Inhibitory activity to tumour cell is determined using conventional mtt assay.
It is demonstrated experimentally that compound(I)There is inhibitory action to human tumor cell line.
To the IC of Human gastric carcinoma cell line BGC-82350(ug/ml)For 14.9.
To human cervical carcinoma cell lines Hela IC50(ug/ml)For 28.8.
Above tumor cell line is preserved by Northeastern University.
Embodiment 3
The preparation of compound (I)
(l) strain liquid seed culture:1. the preparation of liquid seed culture medium:Take yeast extract 2.0g;Glucose
2.0g ;Malt extract 3.0g;B B-complex 2.5mg;Add water to 1000ml, 7.2,120 °C of sterilizing 30min of pH;2. ferment
:Bacterial strain YIM R4 are inoculated into the culture medium after sterilizing, in shake flask fermentation 2 on the shaking table that rotating speed is 190 revs/min under 28 °C
My god.
(2) strain fermentation culture:1. the preparation of fermentation medium:Take starch 20.0g;Beef extract 1.0g;Glucose
0.5g ;Yeast extract 3.0g;Peptone 1.0g;CaCO32.0g ;Add water to 1000ml, 7.0,120 °C of 30 min of sterilizing of pH
;2. ferment:The bacterial strain YIM R4 seed liquors obtained in the above method (l) are added in the fermentation medium after sterilizing, plus bacterium
Plant under 10 % measured as fermentation medium weight, 28 °C in shake flask fermentation 6 days on the shaking table that rotating speed is 190 revs/min.
(3) fermentation liquor treatment:1. zymotic fluid is through 2500 revs/min of centrifugations, and centrifugation time is 15 minutes;2. with grade body
Product ethyl acetate is extracted 3 times, reclaims ethyl acetate, mycelium acetone soak and ultrasonication, reclaims acetone, merges two parts
Extract, obtains the sepia paste at active position;3. silica gel column chromatography is carried out, with chloroform:Methanol 40:1
To 1:1 gradient elution, collects the component containing the compound, and recycling design obtains a kind of antitumor antibiotics of crude product;4. will
A kind of antitumor antibiotics of crude product carries out methanol gel and chloroform successively:Methanol 30:1 silica gel column chromatography, recycling design,
A kind of antitumor antibiotics is obtained compared with sterling;5. a kind of antitumor antibiotics is subjected to methanol gel column chromatography compared with sterling, reclaimed
Solvent, obtains a kind of antitumor antibiotics sterling.
Embodiment 4
The preparation of compound (I)
(l) strain liquid seed culture:1. the preparation of liquid seed culture medium:Take yeast extract 6.0g;Glucose
6.0g ;Malt extract 7.0g;B B-complex 4.5mg;Add water to 1000ml, 7.2,120 °C of sterilizing 30min of pH;2. ferment
:Bacterial strain YIM R4 are inoculated into the culture medium after sterilizing, in shake flask fermentation 2 on the shaking table that rotating speed is 250 revs/min under 28 °C
My god.
(2) strain fermentation culture:1. the preparation of fermentation medium:Take starch 30.0g;Beef extract 5.0g;Glucose
1.5g ;Yeast extract 7.0g;Peptone 5.0g;CaCO36.0g ;Add water to 1000ml, 7.0,120 °C of 30 min of sterilizing of pH
;2. ferment:The bacterial strain YIM R4 seed liquors obtained in the above method (l) are added in the fermentation medium after sterilizing, plus bacterium
Plant under 10 % measured as fermentation medium weight, 28 °C in shake flask fermentation 8 days on the shaking table that rotating speed is 250 revs/min.
(3) fermentation liquor treatment:1. zymotic fluid is through 3500 revs/min of centrifugations, and centrifugation time is 25 minutes;2. with grade body
Product ethyl acetate is extracted 5 times, reclaims ethyl acetate, mycelium acetone soak and ultrasonication, reclaims acetone, merges two parts
Extract, obtains the sepia paste at active position;3. silica gel column chromatography is carried out, with chloroform:Methanol 40:1
To 1:1 gradient elution, collects the component containing the compound, and recycling design obtains a kind of antitumor antibiotics of crude product;4. will
A kind of antitumor antibiotics of crude product carries out methanol gel and chloroform successively:Methanol 30:1 silica gel column chromatography, recycling design,
A kind of antitumor antibiotics is obtained compared with sterling;5. a kind of antitumor antibiotics is subjected to methanol gel column chromatography compared with sterling, reclaimed
Solvent, obtains a kind of antitumor antibiotics sterling.
Claims (4)
1. a kind of preparation method of new phenol piperazine class antitumor antibiotics, it is characterized in that this method comprises the following steps:
(1) strain liquid seed culture:
1. the preparation of liquid seed culture medium:Take 2.0 ~ 6.0g of yeast extract, it is 2.0 ~ 6.0g of glucose, 3.0 ~ 7.0g of malt extract, multiple
Close the mg of vitamin 2.5 ~ 4.5, add water to 1000ml, 7.2,120 °C of sterilizing 30min of pH;2. ferment:Bacterial strain YIM R4 are connect
Plant into the culture medium after sterilizing, in shake flask fermentation 2 days on the shaking table that rotating speed is 190 ~ 250 revs/min under 28 °C;
(2) strain fermentation culture:
1. the preparation of fermentation medium:Take 20.0 ~ 30.0g of starch, 1.0 ~ 5.0g of beef extract, 0.5 ~ 1.5g of glucose, yeast extract
3.0 ~ 7.0g, 1.0 ~ 5.0g of peptone, CaCO32.0 ~ 6.0g, add water to 1000ml, 7.0,120 °C of sterilizing 30min of pH;②
Fermentation:The bacterial strain YIM R4 seed liquors obtained in the above method (l) are added in the fermentation medium after sterilizing, plus strain amount
For 10 % of fermentation medium weight, under 28 °C in rotating speed be shake flask fermentation on 190 ~ 250 revs/min of shaking table 6 ~ 8 days;
(3) fermentation liquor treatment:
1. zymotic fluid is through 2500 ~ 3500 revs/min of centrifugations, and centrifugation time is 15 ~ 25 minutes;2. extracted with isometric ethyl acetate
Take fermented supernatant fluid 3 ~ 5 times, reclaim ethyl acetate, mycelium acetone soak and ultrasonication, reclaim acetone, merge two parts
Extract, obtains the sepia paste at active position;3. silica gel column chromatography is carried out, with chloroform:Methanol 40:1
To 1:1 gradient elution, collects the component containing the compound, and recycling design obtains a kind of antitumor antibiotics of crude product;4. will
A kind of antitumor antibiotics of crude product carries out methanol gel and chloroform successively:Methanol 30:1 silica gel column chromatography, recycling design,
A kind of antitumor antibiotics is obtained compared with sterling;5. a kind of antitumor antibiotics is subjected to methanol gel column chromatography compared with sterling, reclaimed
Solvent, obtains a kind of antitumor antibiotics sterling, and the structure of the antitumor antibiotics is:
。
2. the preparation method of a kind of new phenol piperazine class antitumor antibiotics according to claim 1, it is characterized in that the step
(1) yeast extract in is that 4.0g, glucose are that 4.0g, malt extract are that 5.0g, B B-complex are 3.5mg.
3. the preparation method of a kind of new phenol piperazine class antitumor antibiotics according to claim 1, it is characterized in that the step
(2) starch in is that 24.0g, beef extract are that 3.0g, glucose are that 1.0g, yeast extract are that 5.0g, peptone are 3.0g, CaCO3
For 4.0g.
4. the preparation method of a kind of new phenol piperazine class antitumor antibiotics according to claim 1, it is characterized in that described shakes
Bed revolution is 220 revs/min, and the fermentation time is 7 days.
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CN201410195152.1 | 2014-05-09 | ||
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二株放线菌抗肿瘤活性次生代谢产物的初步研究;戎贺;《http://www.docin.com/p-873854865.html》;20140726;第3页第1.2.2-1.2.3节,第13页第1.3节,第14页第1.4节,第15页全文及图2-2,第16页全文,第17页图2-3,第25页最后一段,第29页第2段 * |
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