CN101585809A

CN101585809A - Novel compound with antibacterial and antitumor activities

Info

Publication number: CN101585809A
Application number: CNA2009100097966A
Authority: CN
Inventors: 鲍时翔; 吴晓鹏; 黄惠琴; 朱军; 方哲; 孙前光; 郑业辉
Original assignee: Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Current assignee: Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Priority date: 2009-01-22
Filing date: 2009-01-22
Publication date: 2009-11-25
Anticipated expiration: 2029-01-22
Also published as: CN101585809B

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 growth of 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 and is 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 penicillin, 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 sixties are the golden periods of microbiotic research and development.This section period, from actinomycetes, found a large amount of microbiotic, and put into production, cured a large amount of clinical transmissible diseases.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 finding from microorganism 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 to develop new pharmaceutical and the requisite element task of novel pesticide in the present world wide that the actinomycetes that obtain to have high-efficiency antimicrobial and anti-tumor activity from soil produce microbiotic.

This seminar is by 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, have 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, 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 as follows:

(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 achieving the above object, the present invention is by the following technical solutions:

The bacterial strain of above-mentioned lactam analog compound is produced in one strain, separates from parrot mountain range, Hainan Island 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, distilled 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, dipotassium hydrogen phosphate 0-1g, sodium-chlor 0-20g, distilled 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 liquor, 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 (sample 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 by thin-layer chromatography; Find to have the active ingredient of anti-FOC4 by 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.

The invention will be further described below in conjunction with embodiment, and embodiments of the present invention are not limited to this, 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, distilled 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, distilled 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 liquor, 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 liquor joined in an amount of 100-200 order silica gel mixes sample, treat that solvent volatilizees fully after, sample 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; Detect the activity of the anti-FOC4 of each component with scraps of paper detection method, 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 ultraviolet 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 ₂₄N O ₄, 282.1705), 304.1527[M+Na] ⁺(calcd for C ₁₅H ₂₃N O ₄Na, 304.1524) the prompting molecular weight is 281, and molecular formula is C ₁₅H ₂₃N O 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 see 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 determine 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 structures are as follows:

Compound 210-A's ¹H and ¹³The C nuclear magnetic resonance data sees Table 1.

Table 1 compound 210-A's ¹H NMR (400MHz) and ¹³C NMR (100MHz) data (CDCL ₃)

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, detect the solution of different concns to human liver cancer cell SMMC-7721, murine sarcoma cell S by mtt assay with 10,50,100,500,1000 times of its dilutions ₁₈₀The inhibition activity.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

1. a new lactam analog compound is characterized in that outward appearance is colorless oil, and molecular formula is C ₁₅H ₂₄NO ₄, chemical being called (6S, 8aS, 9S, 11S, 12aR)-and 6-hydroxyl-9,10-dimethyl decahydro benzo [d] azepine pentaene-2,4 between the last of the ten Heavenly stems, 12 (3H)-triketones, its chemical structural formula is as follows:

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, distilled water 1000ml;

Culture condition is: pH 6.5～7.5,25～32 ℃ 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%, dipotassium hydrogen phosphate 0.05%.

(3) separate general extractive:

The fermented liquid that step (2) obtains is centrifugal, get supernatant liquor, 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 (sample 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 by thin-layer chromatography; Find to have the active ingredient of anti-banana sickle-like bacteria Cuba's No. 4 microspecies of specialized form (FOC4) by 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 SephadexLH-20, methanol-eluted fractions obtains novel lactam analog compound.

3. the purposes of the described new compound of claim 1 in fungal farm chemicals such as preparation anti-banana sickle-like bacteria etc.

4. the described new compound of claim 1 suppresses purposes in the medicine of growth of tumour cell in preparation.