CN101585809B - Novel compound with antibacterial and antitumor activities - Google Patents

Novel compound with antibacterial and antitumor activities Download PDF

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CN101585809B
CN101585809B CN 200910009796 CN200910009796A CN101585809B CN 101585809 B CN101585809 B CN 101585809B CN 200910009796 CN200910009796 CN 200910009796 CN 200910009796 A CN200910009796 A CN 200910009796A CN 101585809 B CN101585809 B CN 101585809B
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鲍时翔
吴晓鹏
黄惠琴
朱军
方哲
孙前光
郑业辉
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Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
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Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
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Abstract

The invention provides a novel compound with antibacterial and antitumor activities, discloses a novel lactam compound and a preparation method and application thereof, in particular relates to a novel lactam compound prepared by microbial fermentation and application thereof, and belongs to the field of biotechnology. The chemical name of the lactam compound is (6S, 8aS, 9S, 11S, 12aR)-6-hydroxy-9,10-dimethyldecahydrobenzo[d]aza-decm-pentaene-2,4,12(3H)-triketone. The novel lactam compound has the advantages that experiments show that the compound has obvious inhibiting effect on the growthof fusarium oxysopoyum f.sp.cubence race 4 (FOC4), human hepatocarcinoma cell SMMC-7721 and mouse sarcoma cell s180, and has lower toxicity; therefore, the compound can be used as a raw material of anti-fungal pesticide or an anticancer medicament and has better application prospect; moreover, the preparation method for the compound is a biofermentation method, has simple steps and lower cost andis propitious to industrialized popularization and implementation.

Description

A kind of have a new compound antibiotic, anti-tumor activity
Technical field
The present invention relates to a kind of novel lactam analog compound and preparation method thereof and application, more specifically say so and utilize a kind of novel lactam analog compound of Production by Microorganism Fermentation and application thereof, belong to biological technical field.
Background technology
Nineteen twenty-eight Fleming finds penicillium mould, and Waksman found Streptomycin sulphate in 1945, obtained the Nobel prize in succession, thereby has promoted microbiotic research in global rise.20th century 50 and the golden period that is the microbiotic research and development sixties.This section period, from actinomycetes, found a large amount of microbiotic, and put into production, cured the lots of clinical transmissible disease.Begin to occur some antitumor, antiviral and other non-antibiotic materials simultaneously.These active substances have 70%～80%, are produced by actinomycetes, have shown that actinomycetes produce survivor's immense value as medicine.
About 22500 kinds of biologically active substances of from mikrobe, finding at present have nearly 53% to be that actinomycetes produce, and wherein streptomyces just produces 8366 kinds, account for all antibioticly 51%, and any microorganism belonging to genus can't be compared; Soil is the important place of actinomycetes perch, and it is development new pharmaceutical and the requisite element task of novel pesticide in the present world wide that the actinomycetes that from soil, obtain to have high-efficiency antimicrobial and anti-tumor activity produce microbiotic.
This seminar is through studying the pedotheque that picks up from parrot mountain range, Hainan Island virgin forest; Find that bacterial strain Streptomycesnoursei Da07210 can produce a kind of novel lactam analog compound; This compound is to No. 4 microspecies of banana sickle-like bacteria Cuba specialized form (Fusarium oxysporum f.sp.cubense race 4; FOC4), the growth of human liver cancer cell SMMC-7721 and murine sarcoma cell S180 has the obvious suppression effect; And toxicity is lower, can be used for antimycotic agricultural chemicals or anti-cancer medicament raw material, has application promise in clinical practice.
Summary of the invention
First purpose of the present invention provides a kind of novel lactam analog compound---210-A with No. 4 microspecies of anti-banana sickle-like bacteria Cuba's specialized form, anti-tumor activity, and its chemistry is by name: (6S, 8aS; 9S, 11S, 12aR)-6-hydroxyl-9; 10-dimethyl-decahydro benzo [d] azepine pentaene-2 between the last of the ten Heavenly stems; 4,12 (3H)-triketones, chemical structural formula is following:
Figure G2009100097966D00011
(6S，8aS，9R，11S，12aR)-6-hydroxy-9，11-dimcthyldccahydrobcnzo[d]azccinc-2，4，12(3H)-trione
Second purpose of the present invention provides the preparation method that bacterial strain Streptomyces noursei Da07210 prepares new compound 210-A.
The 3rd purpose of the present invention provides the purposes of 210-A in the medicine of antimycotic agricultural chemicals of preparation and inhibition growth of tumour cell.
For realizing above-mentioned purpose, the present invention adopts following technical scheme:
The bacterial strain of above-mentioned lactam analog compound is produced in one strain, separates from Hainan Island parrot mountain range virgin forest soil, called after Da07210.This bacterial strain is kept at China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCCNo.2561, and on June 27 2008 preservation day, the name of suggestion is called streptomyces noursei Streptomyces noursei Da07210.
A kind of method for preparing above-mentioned lactam analog compound may further comprise the steps:
(1) seed culture of Streptomyces noursei Da07210:
Seed culture medium: glucose 5-15g, Zulkovsky starch 2-20g, soyflour 5-15g, yeast extract paste 0-20g, potassium primary phosphate 0-1g, sodium-chlor 0-20g, zero(ppm) water 1000ml;
Culture condition is: pH6.5～7.5,25～32 ℃ of culture temperature, incubation time 24～72 hours;
(2) fermentation culture of Streptomyces noursei Da07210:
Fermention medium: glucose 0-20g, glycerine 0-20g, soybean cake powder 5-20g, ammonium sulfate 0-10g, potassium hydrogenphosphate 0-1g, sodium-chlor 0-20g, zero(ppm) water 1000ml.
Fermentation condition is: pH 6.5～7.5,25～32 ℃ of leavening temperatures, fermentation time 96～168 hours, inoculum size 5%～10%;
(3) separate general extractive:
The fermented liquid that step (2) obtains is centrifugal, get supernatant, through chloroform or ethyl acetate extraction, concentrating under reduced pressure gets brown paste substance;
(4) purified extract:
With the paste that dissolve with methanol above-mentioned steps (3) obtains, utilize normal phase silica gel column chromatography (appearance on the dry method) to carry out the initial gross separation purifying; Eluent is a hexanaphthene: acetone, increase the amount of acetone gradually, and use methanol-eluted fractions at last, 500ml is a stream part, decompression and solvent recovery obtains corresponding component; Inspect and merge same composition through thin-layer chromatography; Find to have the active ingredient of anti-FOC4 through biological activity assay; This component is carried out purifying with the preparation thin-layer chromatography, and developping agent is a hexanaphthene: chloroform: acetone=2: 2: 1 obtains reactive site; This reactive site is carried out purifying with Sephadex LH-20, and methanol-eluted fractions obtains novel lactam analog compound.
Advantage of the present invention is: novel lactam analog compound provided by the invention; Growth to banana sickle-like bacteria Cuba No. 4 microspecies of specialized form (FOC4), human liver cancer cell SMMC-7721 and murine sarcoma cell S180 has the obvious suppression effect; And toxicity is lower; Can be used for antimycotic biological pesticide or anti-cancer medicament raw material, have application promise in clinical practice; The method for preparing compound provided by the invention is a biological fermentation process, and step is simple, cost is lower, help Industry Promotion implements.
Below in conjunction with embodiment the present invention is described further, embodiment of the present invention is not limited to this, and is every according to content disclosed by the invention or principle, any this area of enforcement be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 is the HR-ESI-MS collection of illustrative plates of compound 210-A.
Fig. 2 is the ESI-MS collection of illustrative plates of compound 210-A.
Fig. 3 is compound 210-A's 1The H-NMR collection of illustrative plates.
Fig. 4 is compound 210-A's 13The C-NMR collection of illustrative plates.
Fig. 5 is the DEPT135 ° of collection of illustrative plates of compound 210-A.
Fig. 6 is the HMBC collection of illustrative plates of compound 210-A.
Fig. 7 is the HSQC collection of illustrative plates of compound 210-A.
Fig. 8 is compound 210-A's 1H- 1H COSY collection of illustrative plates.
Fig. 9 is the NOESY collection of illustrative plates of compound 210-A.
Embodiment
Embodiment 1: the preparation of new compound 210-A
The seed culture of one .Streptomyces noursei Da07210:
Seed culture medium: glucose 10g, Zulkovsky starch 10g, soyflour 10g, yeast extract paste 10g, potassium primary phosphate 0.5g, sodium-chlor 10g, zero(ppm) water 1000ml.
Culture condition is: pH7.2,28 ℃ of culture temperature, incubation time 48 hours.
The fermentation culture of two .Streptomyces noursei Da07210:
Fermention medium: glucose 10g, glycerine 10g, soybean cake powder 15g, ammonium sulfate 5g, potassium primary phosphate 0.5g, sodium-chlor 10g, zero(ppm) water 1000ml.
Fermentation condition is: inoculum size 5%～10%, pH 7.0,28 ℃ of leavening temperatures, fermentation time 144 hours.
Three. separate general extractive:
With the centrifugal 10min of above-mentioned fermented liquid 8000r/min, get supernatant, through ethyl acetate extraction three times, concentrating under reduced pressure gets brown paste substance.
Four. purified extract:
With the mixing solutions of an amount of methyl alcohol and chloroform dissolving extract, supernatant joined in an amount of 100-200 order silica gel mixes appearance, treat that solvent volatilizees fully after, appearance on the dry method; Earlier carry out wash-out, increase the ratio of acetone then gradually,, carry out wash-out with methyl alcohol at last up to carrying out wash-out with pure acetone with hexanaphthene.The volume of each stream part is 500ml, and decompression and solvent recovery obtains 30 components; Inspect with thin-layer chromatography, developping agent is a hexanaphthene: acetone=3: 2, merge identical component, and obtain 22 components; With the activity of the anti-FOC4 of scraps of paper detection method each component of detection, find that component 17 has tangible activity; Component 17 usefulness preparation thin-layer chromatographys are carried out purifying, and eluent is a hexanaphthene: chloroform: acetone=2: 2: 1, be a blackening under the uv lamp 254nm, and blackening is scraped, chloroform soaks, and reclaims chloroform and obtains reactive site 210-17; This reactive site is carried out purifying with Sephadex LH-20, and methanol-eluted fractions obtains novel lactam analog compound 210-17-2.
Five. structure is identified
New compound is a colorless oil, is dissolved in chloroform, acetone, methyl alcohol; On thin-layer chromatography, spray improvement bismuth potassium iodide shows orange red with this chemical combination object point, prompts for to contain nitrogen compound; ESI-MS m/z 282 [M+H] +, 304 [M+Na] +, 585 [2M+Na] +HR-ESI-MS m/z 282.1691 [M+H] +(calcd for C 15H 24NO 4, 282.1705), 304.1527 [M+Na] +(calcd for C 15H 23NO 4Na, 304.1524) the prompting molecular weight is 281, and molecular formula is C 15H 23NO 4[α] D 23-14.55 (c0.0187, CHCL 3), UV λ MaxNm:214, IR bands (KBr, V MaxCm -1): 3433,2927,1693,1631,1381,1264; According to 1H-NMR, 13C-NMR and the DEPT135 ° of degree of unsaturation of releasing this compound is 5; Contain 1 ketone carbonyl, 2 carbonyls that link to each other with nitrogen, 2 methyl, 5 methylene radical, 5 methynes, 1 hydroxyl and 2 rings; Because 2 carbonylation displacements that link to each other with amino are very approaching, so their residing chemical environment basically identicals.According to 1H-NMR data (concrete data are seen table 1); This structure contains the reactive hydrogen on two methyl that link to each other with methyne respectively, methyne that links to each other with oxygen and the amino; Be respectively δ 0.95 (d, J=6.4Hz), 1.20 (d, J=7.2Hz), 4.2 (dd; J=8.4Hz is 2.6Hz) with 8.7 (S); Again according to HSQC, HMBC, 1H- 1HCOSY and NOESY finally confirm new compound be (6S, 8aS, 9S, 11S, 12aR)-6-hydroxyl-9,10-dimethyl-decahydro benzo [d] azepine pentaene-2,4 between the last of the ten Heavenly stems, 12 (3H)-triketones;
English name: (6S, 8aS, 9S, 11S, 12aR)-and 6-hydroxy-9,10-dimethyldecahydrobenzo [d] azecine-2,4,12 (3H)-trione
Structure is following:
Figure G2009100097966D00041
Compound 210-A's 1H with 13The C nuclear magnetic resonance data is seen table 1.
Table 1 compound 210-A's 1H NMR (400MHz) and 13C NMR (100MHz) data (CDCL 3)
Figure G2009100097966D00051
Embodiment 2: anti-banana sickle-like bacteria Cuba's No. 4 microspecies of specialized form (FOC4) activity test
This compound is dissolved with amount of methanol, be configured to the solution that concentration is 0.87mg/ml, 0.087mg/ml, 0.0087mg/ml, 0.00087mg/ml.Adopt disk diffusion method, application of sample amount 10 μ L measure antibacterial circle diameter behind the cultivation 24h, and antibacterial circle diameter is respectively 15mm, 11mm, 10mm, 9mm.
Embodiment 3: anti-tumor activity test
This compound is dissolved with a spot of DMSO, and add cell culture medium with 10,50,100,500,1000 times of its dilutions, the solution that detects different concns through mtt assay is to human liver cancer cell SMMC-7721, murine sarcoma cell S 180Inhibition active.Cell strain is the biochemical and cell research institute available from Chinese Academy of Sciences Shanghai all.
This new compound is to human liver cancer cell SMMC-7721, murine sarcoma cell S 180Very strong restraining effect is all arranged, IC 50Be respectively: 0.77 μ g/ml and 0.74 μ g/ml.

Claims (4)

1. a lactam analog compound is characterized in that outward appearance is colorless oil, and its chemical structural formula is following:
Figure 2009100097966100001DEST_PATH_IMAGE001
。
2. method for preparing the described lactam analog compound of claim 1 is characterized in that may further comprise the steps:
(1) seed culture of Streptomyces noursei Da07210:
Seed culture medium: glucose 5～15g, Zulkovsky starch 2～10g, soyflour 5～15g, potassium primary phosphate 0～1g, zero(ppm) water 1000ml;
Culture condition is: pH6.5～7.5,25～32 ℃ of culture temperature, incubation time 24～72 hours;
(2) fermentation culture of Streptomyces noursei Da07210:
Fermention medium: glucose 1.0%, glycerine 1.0%, soybean cake powder 1.5%, ammonium sulfate 0.5%, potassium hydrogenphosphate 0.05%;
Fermentation condition is: pH 6.5～7.5,25～32 ℃ of leavening temperatures, fermentation time 96～168 hours, inoculum size 5%～10%;
(3) separate general extractive:
The fermented liquid that step (2) obtains is centrifugal, get supernatant, through chloroform or ethyl acetate extraction, concentrating under reduced pressure gets brown medicinal extract;
(4) purified extract:
With the paste that dissolve with methanol above-mentioned steps (3) obtains, utilize normal phase silica gel column chromatography to carry out the initial gross separation purifying; Eluent is a hexanaphthene: acetone, increase the amount of acetone gradually, and use methanol-eluted fractions at last, 500ml is a stream part, decompression and solvent recovery obtains corresponding component; Inspect and merge same composition through thin-layer chromatography; Find to have the active ingredient of No. 4 microspecies of anti-banana sickle-like bacteria Cuba's specialized form through biological activity assay, this component is carried out purifying with the preparation thin-layer chromatography, developping agent is a hexanaphthene: chloroform: acetone=2: 2: 1 obtains reactive site; Then this reactive site is carried out purifying with Sephadex LH-20, methanol-eluted fractions obtains lactam analog compound.
3. the described compound of claim 1 is in the purposes of preparation in the anti-banana sickle-like bacteria agricultural chemicals.
4. the described compound of claim 1 suppresses the purposes in the medicine of growth of tumour cell in preparation.
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CN109942592B (en) * 2019-05-09 2020-07-14 江西中医药大学 Novel protopine type alkaloid and preparation method and application thereof
CN114540213B (en) * 2021-11-11 2024-03-19 中国热带农业科学院海口实验站 Actinomycetes with antibacterial activity and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
吴瑞阁等.链霉菌S024所产生的抗菌活性物质的研究.《安徽农业科学》.2007,第35卷(第34期),10963-10964. *
朱宏建等.一株链霉菌次生代谢产物抗菌活性的初步研究.《中国生物防治》.2008,第24卷(第2期),189-191. *
赵刚等.一株链霉菌发酵液的抗菌谱及稳定性.《农药》.2006,第45卷(第8期),525-526,528. *
鲍时翔.菌株Da07210产生的抗FOC4新结构化合物.《http://www.itbb.org.cn/con_view14.asp?id=149》.2008,第1页. *

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