CN102070588A - Alpha-pyrone compounds, and preparation method and application thereof - Google Patents

Alpha-pyrone compounds, and preparation method and application thereof Download PDF

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CN102070588A
CN102070588A CN2011100293278A CN201110029327A CN102070588A CN 102070588 A CN102070588 A CN 102070588A CN 2011100293278 A CN2011100293278 A CN 2011100293278A CN 201110029327 A CN201110029327 A CN 201110029327A CN 102070588 A CN102070588 A CN 102070588A
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compound
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candida albicans
methanol
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朱伟明
付鹏
王乂
刘培培
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Ocean University of China
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Abstract

The invention relates to alpha-pyrone compounds, and a preparation method and application thereof. The alpha-pyrone compounds with novel structure are prepared by nocardiopsis dassonvillei HR10-5 extracted from sea samples. Tests prove that the compounds can be used as a candida albicans inhibitor.

Description

Alpha-pyranone compound and its production and use
Technical field:
The present invention relates to prepare method (the culture presevation numbering: CCTCC M 2010369 of alpha-pyranone compound with nocardia sp darsonville bacterium HR10-5 (Nocardiopsis dassonvillei HR10-5), preservation date: on December 28th, 2010, preservation place: Chinese Wuhan, Wuhan University.); The invention still further relates to the purposes of this compounds in preparation Candida albicans inhibitor.
Background technology:
Candida albicans is subordinate to Saccharomycetales, Saccharomycetaceae, mycocandida, is present in skin surface, small intestine, cavity and the moistening position of human body widely.It is primary pathogenic bacteria (Edwards JE Jr.Management of severe candidal infections:integrationand review of current guidelines for treatment and prevention.Curr.Clin.Top.Infect.Dis.2001, the 21:135-147 of fungi infestation; Klepser ME, Lewis RE, Pfaller MA.Therapy of Candida infections:susceptibility testing, resistance, andtherapeutic options.Ann.Pharmacother.1998,32:1353-1361; Reyes G., Ghannoum MA.Antifungalsusceptibility testing ofyeasts:uses and limitations.Drug Resist.Updates 2000,3:14-19).Along with extensively carrying out of immunosuppressant therapy in the widespread usage of Broad spectrum antibiotics, the organ transplantation and increasing of immune deficiency patient such as acquired immune deficiency syndrome (AIDS), the candida albicans infection rate was increasing sharply in recent years, the deep monilial infection has become major reason (the White TC of patient with severe symptoms's death, Marr KA.Clinical, cellular, and molecular factors that contribute to antifungal drug resistance.Clin.Microbiol.Rev.1998,11:382-402; LawD, Moore CB, Wardle HM, Ganguli LA, Keaney MG, Denning DW.High prevalence of antifungal resistance inCandida spp.from patients with AIDS.JAntimicrob chemother 1994,34 (5): 659-668).Medicine at candida albicans infection mainly contains (Kowalsky SF, Dixon DM.Fluconazole:a new antifungal agent.Clin Pharm.1991,10:179-94 such as KETOKONAZOL, clotrimazole and fluconazole at present; Palacin C, Tarrago C, Agut J, Guglietta, A.In vitro activity of sertaconazole, fluconazole, ketoconazole, fenticonazole, clotrimazole and itraconazole against pathogenic vaginal yeast isolates.Methods Find Exp Clin Pharmacol2001,23:61-64).But because the abuse of medicine, Candida albicans has produced resistance and resistance to these medicines, becomes a great problem of clinical treatment, seeks novel structure, acts on that the anti-candida albicans infection medicine is extremely urgent efficiently.
The inventor has found that a strain can produce the nocardia sp darsonville bacterium HR10-5 (Nocardiopsis dassonvilleiHR10-5) of anti-candida albicans active substance from the marine actinomycete of Huanghe delta earth sample separation.From its liquid fermentate, separation and purification its activeconstituents, and be accredited as the alpha-pyranone compound of novel structure through spectrum and chemical means.Active testing shows, this type of alpha-pyranone compound has stronger Candida albicans and suppresses active, minimum inhibition concentration MIC is 0.05～0.11 μ M, can be used as the Candida albicans inhibitor and be used for the treatment of diseases that causes by candida albicans infection, also can be used as the low molecular biosciences probe that suppresses Candida albicans and be used for life science.
Summary of the invention:
What the present invention aimed to provide a class formation novelty has an active alpha-pyranone compound of anti-candida albicans, and structure is suc as formula (Fig. 1) shown in the I.
Figure BSA00000428048100011
R 1=-CH 3, C 2～C 4Alkyl
R 2=-H ,-OR (R=H, C 1～C 4Alkyl)
R 3, R 4, R 5, R 6=-H ,-CH 3, C 2～C 4Alkyl
Fig. 1. the chemical structure of formula I
The present invention also aims to provide the preparation method and the purposes in preparation Candida albicans inhibitor thereof of a compounds.
The production bacterium of alpha-pyranone compound involved in the present invention is nocardia sp darsonville bacterium HR10-5 (Nocardiopsis dassonvilleiHR10-5).
Preferred The compounds of this invention is described formula I compound, the wherein R of compound 1 1, R 3For-CH 3, R 2, R 4And R 5Be hydrogen, R 6For-CH 2CH 3The R of compound 2 1, R 3, R 6For-CH 3, R 2, R 4, R 5Be hydrogen; The R of compound 3 1, R 3For-CH 3, (configuration S-), R 6For-CH 2CH 3, R 4, R 5Be hydrogen; Its structural formula is seen Fig. 2.
Figure BSA00000428048100021
Fig. 2. the chemical structure of compound 1-3
Most preferred is that liquid fermentate by the nocardia sp darsonville bacterium HR10-5 (Nocardiopsis dassonvilleiHR10-5) of marine source is through silica gel column chromatography in the foregoing invention, with sherwood oil, sherwood oil-methylene dichloride, methylene chloride-methanol is that solvent carries out gradient elution, the eluate of sherwood oil-methylene dichloride (v/v 1: 1), again through Sephadex LH-20 gel filtration chromatography (1: 2 wash-out of methyl alcohol-methylene dichloride), obtained component 1-2 through preparation HPLC separation and purification, obtains compound 1 and compound 2 with methanol-water (v/v 80: 20) wash-out again; The component of methylene chloride-methanol (v/v 100: 1) wash-out is through Sephadex LH-20 column chromatography (1: 2 wash-out of methyl alcohol-methylene dichloride), and obtained component 3-2 through preparation HPLC separation and purification, obtains compound 3 with methanol-water (v/v 65: 35) wash-out again.
The present invention adopts paper disk method to test the bacteriostatic activity of formula I compound to Candida albicans (Candida albicans).Experiment confirm, the growth of this compounds dialogue look oidiomycetic has had strong inhibitory effects, and the minimum inhibition concentration of compound 1, compound 2 and compound 3 is respectively 0.10,0.11 and 0.05 μ M.Therefore formula I compound of the present invention can be used as the inhibitor of Candida albicans.
Formula I compound and various medicine acceptable carrier, vehicle or supplementary product compatibility can be made into the anti-candida albicans medicine, are used for the treatment of candida albicans infection.
Formula I compound also can be used as the low molecular biosciences probe that suppresses Candida albicans and is used for life science, and when using as probe, formula I compound dissolves in chloroform, the methyl alcohol, also dissolves in the aqueous solution of dimethyl sulfoxide (DMSO) to be applied.
Of particular note, the method for producing formula I compound of the present invention through organism of fermentation can adopt other any microorganism that can produce this compounds, all can be used as and produces bacterium and be used for preparation I compound as long as can produce the microorganism of this compounds.
Enumerated the example of the preferred compound that utilizes nocardia sp darsonville bacterium HR10-5 (Nocardiopsis dassonvillei HR10-5) preparation formula I of the present invention in the embodiments of the invention, but the new compound of the present invention's preparation is not confined to the preparation method and the application example of this compound in the present embodiment.
This actinomycetes strain HR10-5 separates to obtain from the ooze that picks up from the Dongying Huanghe delta, Shandong, is accredited as the nocardia sp darsonville bacterium through polyphase sort research, is decided to be Nocardiopsis dassonvillei HR10-5; And by China's typical culture collection center preservation (numbering: CCTCC M 2010369, date: on December 28th, 2010, place: Chinese Wuhan, Wuhan University).
This nocardia sp darsonville bacterium HR10-5 (Nocardiopsis dassonvillei HR10-5) bacterial strain has following morphological feature: aerial hyphae is longer, and suitable branch is arranged, and white mycelium or faint yellow produces water-soluble faint yellow pigment; Get inserted sheet and observe its microscopic morphology and show that aerial hyphae is fractured into spore, spore is long and narrow, smooth.
This nocardia sp darsonville bacterium HR10-5 (Nocardiopsis dassonvillei HR10-5) bacterial strain has following chemotaxonomy feature: the amino acid of cell walls contains meso-DAP (meso diaminopimelic acid) in constituting; Atypism sugar.
This nocardia sp darsonville bacterium HR10-5 (Nocardiopsis dassonvillei HR10-5) bacterial strain has following molecular biological characteristics: its 16S rRNA gene order information is:
Figure BSA00000428048100022
Figure BSA00000428048100031
Embodiment:
The fermentative production and the separation and purification of [embodiment 1] compound 1～3
1 fermentative production
Produce the fermentation culture of bacterium: by the ordinary method of culturing micro-organisms, it is an amount of to get nocardia sp darsonville bacterium HR10-5 (Nocardiopsisdassonvillei HR10-5), is inoculated on the Gause I slant medium, cultivates 5 days in 28 ℃ of incubators.It is an amount of to get 5 days nocardia sp darsonville bacterium HR10-5 of slant culture, is inoculated into 150mL nutrient solution [substratum composition (%): glucose 2%, extractum carnis 0.3%, yeast extract 1%, Zulkovsky starch 1%, peptone 1%, K is housed 2HPO 40.05%, MgSO 40.05%, CaCO 30.2%, artificial seawater element 3.3%, pH 7.0] the 500mL triangular flask in (400 bottles), be loaded on 28 ℃, the 180 rev/mins shaking tables, carry out 8 days by a definite date production fermentation, obtain fermented liquid.Fermented liquid equal-volume ethyl acetate extraction three times, combined ethyl acetate extraction liquid concentrating under reduced pressure gets crude extract 30 grams.
2 separation and purifications
Behind medicinal extract (30 gram) dissolve with methanol, add 60 gram 200-300 order silica gel Hs (Qingdao Haiyang Chemical Industry Group Corp.'s product) and mix sample, after the removal of solvent under reduced pressure, use silica gel column chromatography, with sherwood oil, sherwood oil-methylene dichloride, methylene chloride-methanol is that solvent carries out gradient elution, the eluate of sherwood oil-methylene dichloride (v/v 1: 1), again through Sephadex LH-20 gel filtration chromatography (1: 2 wash-out of methyl alcohol-methylene dichloride), obtained component 1-2 is again through preparation HPLC separation and purification, obtain compound 1 (310mg, t with methanol-water (v/v 80: 20) wash-out R8.6min) and compound 2 (52mg, t R7.2min); The component of methylene chloride-methanol (v/v 100: 1) wash-out is through Sephadex LH-20 column chromatography (1: 2 wash-out of methyl alcohol-methylene dichloride), obtained component 3-2 is again through preparation HPLC separation and purification, obtain compound 3 (12.5mg, t with methanol-water (v/v 65: 35) wash-out R7.8min).
Table 1. compound 1～3 1H and 13C NMR data (600and 150MHz in CDCl 3)
Figure BSA00000428048100032
Compound 1, faint yellow oily, HRESIMS:193.1237[M+H] +IR (KBr) v Max2973,2937,2879,1708,1638,1577,1459,1379,1273,1122,1092,1025,960cm -1 1H reaches 13C NMR data see Table 1.
Compound 2, faint yellow oily, HRESIMS:179.1073[M+H] +IR (KBr) v Max2928,1706,1641,1579,1456,1377,1345,1278,1122,1090,994cm -1 1H reaches 13C NMR data see Table 1.
Compound 3, faint yellow oily,
Figure BSA00000428048100033
HRESIMS:209.1170[M+H] +IR (KBr) v Max3426,2968,2928,2868,1686,1645,1559,1449,1252,1121,1062,1024cm -1 1H reaches 13C NMR data see Table 1.
The active test of [embodiment 2] anti-candida albicans
1 laboratory sample and experimental technique
The preparation of sample and positive control drug solns: specimen is the pure product compound 1,2,3 of separation and purification in the foregoing description 1, and the pure product of positive control drug KETOKONAZOL (Tokyo changes into).Accurately take by weighing an amount of sample, be mixed with the solution of desired concn with methyl alcohol by doubling dilution, promptly 50 μ g/mL, 25 μ g/mL, 12.5 μ g/mL, 6.25 μ g/mL, 3.125 μ g/mL, 1.5625 μ g/mL, 0.78125 μ g/mL, 0.390625 μ g/mL, 0.1953125 μ g/mL are for active testing.
The pathogenic bacterium Candida albicans is cultivated: the Candida albicans of getting frozen preservation is an amount of, is inoculated into activation (substratum consists of: yeast extract paste 1%, glucose 2%, peptone 2%, agar 2%, pH=7.0) on the inclined-plane of YPD substratum.It is an amount of to get 3 days Candida albicans of slant culture, be inoculated in the 500mL Erlenmeyer flask that 150mL nutrient solution (substratum consists of: yeast extract paste 1%, glucose 2%, peptone 2%, pH=7.0) is housed, shaking table was cultivated 24 hours under 28 ℃, the condition of 180rps/min, obtained the seed culture fluid of Candida albicans.With spreading rod this seed culture fluid is spread upon on the flat board of YPD cultivation bacterium, for active testing uniformly.
The bacteriostatic activity experiment of paper disk method determination experiment sample: with the sample of above-mentioned each concentration for preparing, drawing 10 μ L dropping is on the sterilization filter paper of 6mm at diameter.The filter paper that aseptic technique will be added with sample is attached on the above-mentioned flat board that has bacterium for preparing.Cultivating 12 hours for 28 ℃, observe the antibacterial circle diameter size, is the minimum inhibition concentration (MIC) of this sample with peak concentration that can't see inhibition zone.
2 experimental results
1,2,3 pairs of Candida albicans of compound have strong inhibition activity, and its MIC is respectively 0.10,0.11,0.05 μ M, and the MIC of positive control drug KETOKONAZOL is 0.02 μ M.
3 conclusions
1,2,3 pairs of oidiomycetic growths of white of compound have strong restraining effect.Therefore, formula I compound of the present invention can be used as or is used to prepare the Candida albicans inhibitor, and is used for the treatment of the disease that candida albicans infection causes, and also can be used as the low molecular biosciences probe that suppresses Candida albicans and is used for life science.
Figure ISA00000428048300021
Figure ISA00000428048300011

Claims (6)

1. formula I compound, wherein R 1=-CH 3, C 2～C 4Alkyl, R 2=-H ,-OR (R=H, C 1～C 4Alkyl), R 3, R 4, R 5, R 6=-H ,-CH 3, C 2～C 4Alkyl:
Figure FSA00000428048000011
2. the described formula I compound of claim 1, the wherein R of compound 1 1, R 3For-CH 3, R 2, R 4And R 5Be hydrogen, R 6For-CH 2CH 3The R of compound 2 1, R 3, R 6For-CH 3, R 2, R 4, R 5Be hydrogen; The R of compound 3 1, R 3For-CH 3, R 2For-OH (configuration S-), R 6For-CH 2CH 3, R 4, R 5Be hydrogen:
Figure FSA00000428048000012
3. the preparation method of the described formula I compound of claim 2, it is characterized in that fermentation culture nocardia sp darsonville bacterium HR10-5 (Nocardiopsisdassonvillei HR10-5), deposit number: CCTCC M 2010369, preservation date: on December 28th, 2010, preservation place: Chinese Wuhan, Wuhan University.HR10-5 obtains the fermented product that contains above-mentioned formula I compound by fermentation culture nocardia sp darsonville bacterium, and separation and purification goes out alpha-pyranone compound 1, compound 2 and compound 3 from fermented product then.
4. the described preparation method of claim 3, wherein with described fermented product through silica gel column chromatography, with sherwood oil, sherwood oil-methylene dichloride, methylene chloride-methanol is that solvent carries out gradient elution, the eluate of sherwood oil-methylene dichloride (v/v 1: 1), again through Sephadex LH-20 gel filtration chromatography (1: 2 wash-out of methyl alcohol-methylene dichloride), obtained component 1-2 through preparation HPLC separation and purification, obtains compound 1 and compound 2 with methanol-water (v/v 80: 20) wash-out again; The component of methylene chloride-methanol (v/v 100: 1) wash-out is through Sephadex LH-20 column chromatography (1: 2 wash-out of methyl alcohol-methylene dichloride), and obtained component 3-2 through preparation HPLC separation and purification, obtains compound 3 with methanol-water (v/v 65: 35) wash-out again.
5. the purposes of the described formula I compound of claim 1 in preparation Candida albicans inhibitor.
6. the purposes of the described formula I compound of claim 1 in preparation anti-candida albicans medicine.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019022294A1 (en) * 2017-07-25 2019-01-31 한국해양과학기술원 Novel compound produced by means of marine microorganism and composition for improving skin wrinkles, enhancing elasticity and skin whitening comprising same compound as active ingredient
CN114773305A (en) * 2022-05-16 2022-07-22 山东瑞捷新材料有限公司 Preparation method and application of 2-ring aropyranone pH fluorescence ratio probe
CN115677640A (en) * 2022-10-17 2023-02-03 浙江省海洋开发研究院 Pyran 2 ketone compound and preparation method and application thereof

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019022294A1 (en) * 2017-07-25 2019-01-31 한국해양과학기술원 Novel compound produced by means of marine microorganism and composition for improving skin wrinkles, enhancing elasticity and skin whitening comprising same compound as active ingredient
KR20190011853A (en) * 2017-07-25 2019-02-08 한국해양과학기술원 New compounds produced by a marine microorganism and composition for wrinkle improvement, elasticity improvement and whitening effect of skin
KR101971977B1 (en) * 2017-07-25 2019-04-25 한국해양과학기술원 New compounds produced by a marine microorganism and composition for wrinkle improvement, elasticity improvement and whitening effect of skin
US11234919B2 (en) 2017-07-25 2022-02-01 Korean Institute of Ocean Science & Technology Compound produced by means of marine microorganism and composition for improving skin wrinkles, enhancing elasticity and skin whitening comprising same compound as active ingredient
CN114773305A (en) * 2022-05-16 2022-07-22 山东瑞捷新材料有限公司 Preparation method and application of 2-ring aropyranone pH fluorescence ratio probe
CN114773305B (en) * 2022-05-16 2023-07-14 山东瑞捷新材料有限公司 Preparation method and application of 2-cycloarone pH fluorescence ratio probe
CN115677640A (en) * 2022-10-17 2023-02-03 浙江省海洋开发研究院 Pyran 2 ketone compound and preparation method and application thereof

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