CN106957216B - 一种抗真菌化合物制备方法和抗真菌用途 - Google Patents
一种抗真菌化合物制备方法和抗真菌用途 Download PDFInfo
- Publication number
- CN106957216B CN106957216B CN201610009266.1A CN201610009266A CN106957216B CN 106957216 B CN106957216 B CN 106957216B CN 201610009266 A CN201610009266 A CN 201610009266A CN 106957216 B CN106957216 B CN 106957216B
- Authority
- CN
- China
- Prior art keywords
- compound
- antifungal
- culture
- alpha
- preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 68
- 230000000843 anti-fungal effect Effects 0.000 title claims abstract description 24
- 229940121375 antifungal agent Drugs 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 241000222122 Candida albicans Species 0.000 claims abstract description 16
- 239000003814 drug Substances 0.000 claims abstract description 15
- 241000222173 Candida parapsilosis Species 0.000 claims abstract description 14
- 201000007336 Cryptococcosis Diseases 0.000 claims abstract description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 241000221204 Cryptococcus neoformans Species 0.000 claims abstract description 13
- 229940095731 candida albicans Drugs 0.000 claims abstract description 12
- 229940055022 candida parapsilosis Drugs 0.000 claims abstract description 12
- 238000000855 fermentation Methods 0.000 claims abstract description 12
- 230000004151 fermentation Effects 0.000 claims abstract description 12
- 239000011347 resin Substances 0.000 claims abstract description 11
- 229920005989 resin Polymers 0.000 claims abstract description 11
- 241000970324 Kitasatospora albolonga Species 0.000 claims abstract description 7
- 239000000287 crude extract Substances 0.000 claims abstract description 7
- 238000010828 elution Methods 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 7
- 239000000126 substance Substances 0.000 claims abstract description 6
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 239000000306 component Substances 0.000 claims description 12
- 238000004440 column chromatography Methods 0.000 claims description 9
- 208000024386 fungal infectious disease Diseases 0.000 claims description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- 239000012043 crude product Substances 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000001963 growth medium Substances 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 239000000945 filler Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 241001337994 Cryptococcus <scale insect> Species 0.000 claims description 4
- 241000233866 Fungi Species 0.000 claims description 4
- 241000187747 Streptomyces Species 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 229920005654 Sephadex Polymers 0.000 claims description 3
- 239000012507 Sephadex™ Substances 0.000 claims description 3
- 235000019764 Soybean Meal Nutrition 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- WGLUMOCWFMKWIL-UHFFFAOYSA-N dichloromethane;methanol Chemical compound OC.ClCCl WGLUMOCWFMKWIL-UHFFFAOYSA-N 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 239000004455 soybean meal Substances 0.000 claims description 3
- 239000008107 starch Substances 0.000 claims description 3
- 235000019698 starch Nutrition 0.000 claims description 3
- 235000013619 trace mineral Nutrition 0.000 claims description 3
- 239000011573 trace mineral Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims 3
- 239000012046 mixed solvent Substances 0.000 claims 1
- 238000005481 NMR spectroscopy Methods 0.000 abstract description 6
- 244000053095 fungal pathogen Species 0.000 abstract description 6
- 241000186361 Actinobacteria <class> Species 0.000 abstract description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 3
- 239000000741 silica gel Substances 0.000 abstract description 3
- 229910002027 silica gel Inorganic materials 0.000 abstract description 3
- 238000001179 sorption measurement Methods 0.000 abstract description 3
- FXRDDHAARFQORE-UHFFFAOYSA-N [2-(2-methylpropyl)cyclohexyl] acetate Chemical compound CC(C)CC1CCCCC1OC(C)=O FXRDDHAARFQORE-UHFFFAOYSA-N 0.000 abstract description 2
- 239000000178 monomer Substances 0.000 abstract description 2
- 238000000338 in vitro Methods 0.000 abstract 1
- 238000004949 mass spectrometry Methods 0.000 abstract 1
- 238000001228 spectrum Methods 0.000 description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 229910052799 carbon Inorganic materials 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 4
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 3
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 3
- 229960003942 amphotericin b Drugs 0.000 description 3
- 244000052616 bacterial pathogen Species 0.000 description 3
- 238000005100 correlation spectroscopy Methods 0.000 description 3
- 238000000990 heteronuclear single quantum coherence spectrum Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 241001446247 uncultured actinomycete Species 0.000 description 3
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 2
- 206010007134 Candida infections Diseases 0.000 description 2
- 206010017533 Fungal infection Diseases 0.000 description 2
- 208000031888 Mycoses Diseases 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- 201000003984 candidiasis Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001052 heteronuclear multiple bond coherence spectrum Methods 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 2
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- XMAYWYJOQHXEEK-OZXSUGGESA-N (2R,4S)-ketoconazole Chemical compound C1CN(C(=O)C)CCN1C(C=C1)=CC=C1OC[C@@H]1O[C@@](CN2C=NC=C2)(C=2C(=CC(Cl)=CC=2)Cl)OC1 XMAYWYJOQHXEEK-OZXSUGGESA-N 0.000 description 1
- 238000005084 2D-nuclear magnetic resonance Methods 0.000 description 1
- 241000283095 Elephas maximus Species 0.000 description 1
- 241000406668 Loxodonta cyclotis Species 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- -1 amphotericin B) Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 238000007876 drug discovery Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 1
- 238000002114 high-resolution electrospray ionisation mass spectrometry Methods 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 229960004125 ketoconazole Drugs 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000013213 metal-organic polyhedra Substances 0.000 description 1
- 238000012011 method of payment Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 150000004291 polyenes Chemical class 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C35/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring
- C07C35/22—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring polycyclic, at least one hydroxy group bound to a condensed ring system
- C07C35/23—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring polycyclic, at least one hydroxy group bound to a condensed ring system with hydroxy on a condensed ring system having two rings
- C07C35/36—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring polycyclic, at least one hydroxy group bound to a condensed ring system with hydroxy on a condensed ring system having two rings the condensed ring system being a (4.4.0) system, e.g. naphols
-
- 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
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明涉及一种抗真菌化合物制备方法和抗真菌用途。本发明公开了一种以放线菌Streptomyces albolongus为来源分离制备的具有下述结构式I的化合物,化学名为(1β,4β,4aβ,8aα)‑4,8a‑dimethyloctahydronap hthalene‑1,4a(2H)‑diol,分子式为C12H22O2。本发明还提供了该化合物的制备方法和应用。本发明所述方法包括:菌株S.albolongus经发酵培养后,离心获得培养液,培养液经过大孔吸附树脂柱吸附,95%乙醇洗脱部分减压浓缩获得粗提物;粗提物经正相硅胶,葡聚糖凝胶LH‑20和反相硅胶柱色谱获得结构I所示的单体化合物,采用核磁共振波谱和质谱鉴定其结构。体外抗真菌活性研究表明,本发明提供的化合物I对多种致病真菌,包括白色念珠菌、近平滑念珠菌和新型隐球菌具有明显的抑制作用,有望开发成为新的抗真菌药物。
Description
技术领域
本发明涉及具有抗真菌活性的化合物,具体的说,涉及一种从放线菌Streptomyces albolongus发酵液中提取得到的抗真菌活性的化合物及其在治疗真菌感染疾病中的用途,属于医药技术领域。
背景技术
近年来随着免疫功能低下而被真菌感染的人群数量不断增加,尤其是深部真菌感染发病率的大幅上升,抗真菌药物研究成为热点领域。目前抗真菌药物主要为咪唑类(如酮康唑)和多烯类(如两性霉素B),它们对人体均有严重的毒副作用。同时,临床上耐药病原菌的不断出现,使发现新型高效低毒抗真菌药物成为当务之急。
念珠菌(Candida)又称假丝酵母菌,为条件致病性真菌。在机体免疫功能低下时,可引起皮肤黏膜念珠菌病、阴道念珠菌病等。另外,在深部真菌感染病人中,念珠菌已成为第四位最常见医院内血液感染的致病菌,死亡率高达40%。对人类致病的念珠菌中白色念珠菌(Candida albicans)和近平滑念珠菌(Candida parapsilosis)临床分离率较高。
新型隐球菌(Cryptococcus neoformans)属于隐球菌属,是一种临床重要条件致病真菌,可以引起致死性新型隐球菌性脑膜炎。近年来新型隐球菌感染发生率呈明显上升趋势,突出表现在艾滋病人群中,己成为艾滋病患者死亡的首要原因。
放线菌是药物发现的重要资源,临床治疗和农业生产中应用的抗生素大约其中75%来源于放线菌。本发明实验研究从来自大象(Elephas maximus)粪便中分离得到的放线菌Streptomyces albolongus发酵液中分离得到一种抗真菌活性化合物。同时,抗真菌实验显示,该化合物对条件致病真菌白色念珠菌、近平滑念珠菌和新型隐球菌显示了较好的抗真菌活性。
发明内容
本发明人从放线菌Streptomyces albolongus发酵液中分离得到一种化合物I,命名为(1β,4β,4aβ,8aα)-4,8a-dimethyloctahydronaphthalene-1,4a(2H)-diol,并对此进行了深入地研究,证明该化合物可以用于预防和治疗真菌感染性疾病,从而完成了本发明。
本发明的一个目的在于提供一种如结构I所示抗真菌活性化合物I。
本发明的另一目的在于提供菌株Streptomyces albolongus制备化合物I的方法。
本发明的再一目的在于提供所述的化合物I在制备治疗或预防真菌感染性疾病药物中的应用。
本发明的又一目的在于提供所述的化合物I在制备治疗或预防念珠菌属和隐球菌属真菌感染性疾病药物中的应用。
本发明的又一目的在于提供所述的化合物I在制备治疗或预防条件致病真菌白色念珠菌(C.albicans)、近平滑念珠菌(C.parapsilosis)和新型隐球菌(Cryptococcusneoformans)感染药物中的应用。
为达上述目的,本发明提供了一种化合物I的制备方法,所述方法包括:
(1)Streptomyces albolongus的种子培养:
种子培养基:酵母膏2~10g,葡萄糖2~10g,麦芽膏2~10g,1~5mL复合维生素,1~5mL微量元素,蒸馏水1L。培养条件:pH 6.5~8.0,培养温度25~32摄氏度,培养时间24~72小时。
(2)Streptomyces albolongus的发酵培养:
发酵培养基:大豆粉10g/L,蛋白胨2g/L,葡萄糖20g/L,可溶性淀粉5g/L,酵母膏2g/L,氯化钠4g/L,磷酸氢二钾0.5g/L,硫酸镁0.5g/L,碳酸钙2g/L。发酵培养条件:pH 6.5~8.0,培养温度25~32摄氏度,培养时间96~192小时。
(3)获得粗提物:将步骤(2)获得的发酵液离心,上清液经过大孔树脂柱层析,先用水洗脱除去培养基成分,再用体积为大孔树脂填料体积10-30倍的体积浓度为75-95%的乙醇水溶液洗脱,收集乙醇溶液并进行浓缩,得到粗提物。
(4)单体化合物分离、纯化:上述步骤(3)获得的膏状物,采用正相硅胶柱色谱二氯甲烷-甲醇(50:1~1:1)梯度洗脱,通过薄层检测合并含有化合物I的组分,该组分经过进一步Sephadex LH-20葡聚糖凝胶柱色谱甲醇洗脱进行纯化,薄层检测合并含有化合物I的组分得到化合物I粗品,最后化合物I粗品经ODS反相柱层析,甲醇-水(70:30)洗脱,得到化合物I。
根据本发明所述的方法所述的大孔树脂可以为本领域常规的大孔树脂,譬如D101,XAD16等树脂。
(5)结构鉴定:本发明综合应用高分辨质谱(HRMS)、核磁共振氢谱(1H NMR)、核磁共振碳谱(13C NMR)和二维核磁共振波谱(包括COSY、NOESY、HSQC和HMBC谱)确定了单体化合物结构。
另一方面,本发明还提供了所述方法制备的化合物I。
再一方面,本发明还提供了所述的化合物I在制备治疗或预防真菌感染性疾病药物中的应用。
又一方面,本发明还提供所述的化合物I在制备治疗或预防条件致病真菌白色念珠菌(Candida albicans)、近平滑念珠菌(Candida parapsilosis)和新型隐球菌(Cryptococcus neoformans)感染药物中的应用。
综上所述,本发明提供了一种抗真菌化合物及其制备方法和应用。
附图说明
图1化合物I结构。
图2化合物I的高分辨质谱。
图3化合物I核磁共振氢谱图1H NMR(600MHz,DMSO-d6)。
图4化合物I核磁共振碳谱图13C NMR(150MHz,DMSO-d6)。
图5化合物I的COSY谱图。
图6化合物I的NOESY谱图。
图7化合物I的HSQC谱图。
图8化合物I的HMBC谱图。
具体实施方式
以下通过具体实施例详细说明本发明的实施过程和产生的有益效果,旨在帮助阅读者更好地理解本发明的实质和特点,不作为对本案可实施范围的限定。
实施例1
化合物I的制备
1.1 Streptomyces albolongus的种子培养。
种子培养基:酵母膏0.4g,葡萄糖0.4g,麦芽膏0.5g,0.1mL复合维生素,1.0mL微量元素,蒸馏水100mL。培养条件:pH 7.2,培养温度28摄氏度,培养时间48小时。
1.2 Streptomyces albolongus的发酵培养。
发酵培养基:大豆粉10g/L,蛋白胨2g/L,葡萄糖20g/L,可溶性淀粉5g/L,酵母膏2g/L,氯化钠4g/L,磷酸氢二钾0.5g/L,硫酸镁0.5g/L,碳酸钙2g/L,蒸馏水70L。培养条件:pH 7.8,培养温度28摄氏度,培养时间168小时。
1.3提取。
将步骤(2)获得的发酵液离心,上清液经过XAD-16大孔吸附树脂(大孔吸附树脂体积为35L)柱层析,先用12倍填料体积的20%乙醇洗脱除去培养基成分,再用体积为大孔树脂填料体积12倍的体积浓度为95%的乙醇水溶液洗脱,收集乙醇溶液并进行减压浓缩,得到粗提物。
1.4分离纯化
上述步骤(3)获得的膏状物,甲醇溶解后与100~200目硅胶拌样,采用200~300目硅胶干法装柱,干法上样。分别采用二氯甲烷甲醇(50:1,20:1,10:1,4:1)梯度洗脱,通过薄层检测合并相同组分,得到13个组分(Fr.1~13)。组分Fr.3含有化合物I,该组分经过进一步Sephadex LH-20葡聚糖凝胶柱色谱甲醇洗脱进行纯化,薄层检测合并成6个组分(Fr.3.1~Fr.3.6)。组分Fr.3.1主要含有化合物I,减压蒸干后得到化合物I粗品,最后化合物I粗品经ODS反相柱层析,甲醇-水(70:30)洗脱,得到化合物I。
1.5结构鉴定
化合物I,为白色固体,ESIMS m/z 221[M+Na]+,419[2M+Na]+;HRESIMS m/z221.1513[M+Na]+(calcd for C12H22O2Na,221.1518),提示该化合物分子量为198,分子式为C12H22O2。化合物I碳谱显示12个碳信号,包括2个季碳信号,2个次甲基信号,6个亚甲基信号和2个甲基信号。氢谱中在化学位移δ0.70(3H,d,6.0Hz)和0.89(3H,s)处给出2个甲基质子信号,在化学位移δ3.27(1H,m)处给出含氧次甲基质子信号,在化学位移δ5.38(1H,d,4.5Hz)和4.75(1H,s)处给出2个活泼氢质子信号,在氢谱中其余的质子信号属于6个亚甲基质子信号。结合上述信息推测化合物I的不饱和度为2,分子中含有2个羟基基团。通过HSQC谱归属了化合物碳和相应氢的信号(见表1),结合化合物的COSY、HMBC和NOESY谱确定了化合物的结构如下:
化学名为:(1β,4β,4aβ,8aα)-4,8a-dimethyloctahydronaphthalene-1,4a(2H)-diol.
表1化合物I的1H NMR(600MHz)和13C NMR(150MHz)数据(DMSO-d6)
实施例2:抗真菌活性实验
化合物I溶于DMSO中,倍比稀释配置成9个不同浓度(22.0,11.0,5.5,2.75,1.38,0.69,0.36,0.18,0.09mg/mL)。病原菌条件致病真菌白色念珠菌(Candida albicans ATCCMYA-2876)、近平滑念珠菌(Candida parapsilosis ATCC 22019)和新型隐球菌(Cryptococcus neoformans ATCC 208821)分别接种在RPMI-1640培养基(10.4g L-1RPMI-1640,2g L-1NaHCO3,34.53g L-1MOPs,pH 7.0)制成菌悬液,白色念珠菌(C.albicans)和近平滑念珠菌(C.parapsilosis)菌悬液浓度为2×103cfu/mL,新生隐球菌(C.neoformans)菌悬液浓度为5×104cfu/mL。样品采用RPMI-1640培养基稀释10倍,10μL的样品和100μL的菌悬液加到96孔板中,等体积的DMSO溶液作阴性对照,两性霉素B作阳性对照。结果表明化合物I对白色念珠菌和近平滑念珠菌显示较强的抑制活性,MIC值分别为12.5和3.3μg/mL,对新型隐球菌显示较弱的抑菌活性,MIC值为200μg/mL。阳性对照药两性霉素B对三株菌的MIC值分别为1.0,2.0和2.0μg/mL。
Claims (5)
2.一种制备权利要求1所述的具有抗真菌活性化合物I的方法,其特征在于包括以下步骤:
(1)菌株Streptomyces albolongus的种子培养:种子培养基:酵母膏2~10g,葡萄糖2~10g,麦芽膏2~10g,1~5mL复合维生素,1~5mL微量元素,蒸馏水1L;
培养条件:pH 6.5~8.0,培养温度25~32摄氏度,培养时间24~72小时;
(2)菌株Streptomyces albolongus的发酵培养:发酵培养基:大豆粉10g/L,蛋白胨2g/L,葡萄糖20g/L,可溶性淀粉5g/L,酵母膏2g/L,氯化钠4g/L,磷酸氢二钾0.5g/L,硫酸镁0.5g/L,碳酸钙2g/L;
培养条件:pH 6.5~8.0,培养温度25~32摄氏度,培养时间96~192小时;
(3)获得粗提物:将步骤(2)获得的发酵液离心,上清液经过大孔树脂柱层析,大孔树脂用量和发酵液按体积比0.5~1:1,先用体积为大孔树脂填料体积10-20倍体积的0~20%乙醇洗脱除去培养基成分,再用体积为大孔树脂填料体积10-20倍体积的浓度为75-95%的乙醇水溶液洗脱,收集乙醇溶液并进行浓缩,得到粗提物;
(4)分离纯化:上述步骤(3)获得的膏状物,经正相硅胶柱色谱,采用体积比50:1至体积比1:1的二氯甲烷和甲醇混合溶剂进行梯度洗脱,通过薄层检测合并含有化合物I的组分,该组分经过进一步Sephadex LH-20葡聚糖凝胶柱色谱甲醇洗脱进行纯化,薄层检测合并含有化合物I的组分得到化合物I粗品,最后化合物I粗品经ODS反相柱层析,体积浓度70%的甲醇水溶液洗脱,得到化合物I。
3.如权利要求1所述的抗真菌活性化合物I在制备治疗或预防真菌感染性疾病药物中的用途。
4.如权利要求3所述的具有抗真菌活性化合物I在制备治疗或预防真菌感染性疾病药物中的用途,其中特征在于,所述的真菌为念珠菌属和隐球菌属真菌。
5.如权利要求4所述的具有抗真菌活性化合物I在制备治疗或预防真菌感染性疾病药物中的用途,其中特征在于,所述的真菌为白色念珠菌(Candida albicans)、近平滑念珠菌(Candida parapsilosis)和新型隐球菌(Cryptococcus neoformans)。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610009266.1A CN106957216B (zh) | 2016-01-08 | 2016-01-08 | 一种抗真菌化合物制备方法和抗真菌用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610009266.1A CN106957216B (zh) | 2016-01-08 | 2016-01-08 | 一种抗真菌化合物制备方法和抗真菌用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN106957216A CN106957216A (zh) | 2017-07-18 |
CN106957216B true CN106957216B (zh) | 2021-04-02 |
Family
ID=59480424
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610009266.1A Expired - Fee Related CN106957216B (zh) | 2016-01-08 | 2016-01-08 | 一种抗真菌化合物制备方法和抗真菌用途 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN106957216B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113402369B (zh) * | 2021-05-28 | 2022-07-08 | 南方医科大学 | 具有抗流感病毒活性的倍半萜类化合物及应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103784427A (zh) * | 2014-03-03 | 2014-05-14 | 中国科学院昆明植物研究所 | 含桉烷型倍半萜的药物组合物及其在制药中的应用 |
-
2016
- 2016-01-08 CN CN201610009266.1A patent/CN106957216B/zh not_active Expired - Fee Related
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103784427A (zh) * | 2014-03-03 | 2014-05-14 | 中国科学院昆明植物研究所 | 含桉烷型倍半萜的药物组合物及其在制药中的应用 |
Non-Patent Citations (5)
Title |
---|
Sesquiterpene lactones from North African Artemisia species;J. ALBERTO MARCO;《Phytochemistry》;19941231;第37卷(第2期);477-485 * |
The cultured mycelium of fungus Sarcodon produced a new cassane diterpenoid;Hisao SHIBATA;《Journal of the Faculty of Agriculture SHINSHU UNIVERSITY》;20021231;第38卷(第1-2期);55-58 * |
The First Atisane Diterpenoids from a Liverwort Polyols from Lepidolaena clavigera;Nigel B. Perry;《organic letters》;20011231;第3卷(第26期);4243-4245 * |
The new cassane-type diterpenes from Caesalpinia minax;Guo-Xu Ma;《Chem. Pharm. Bull.》;20121231;第60卷(第6期);759-763 * |
Total synthesis of (±)-(1β,4β,4aβ,8aα)-4,8a-dimethyl-octahydro-naphthal ene-1,4a(2H)-diol;Qingyin Liu;《ORGANIC CHEMISTRY FRONTIERS》;20121231;1-5 * |
Also Published As
Publication number | Publication date |
---|---|
CN106957216A (zh) | 2017-07-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112592350B (zh) | 聚酮类化合物lithocarpin E-G及其制备方法和应用 | |
CN111321095B (zh) | 具有α-葡萄糖苷酶抑制作用的丁烯酸内酯二聚体及其应用 | |
CN113637010B (zh) | 氢化氧杂蒽酮衍生物及其制备方法和应用 | |
CN112830949B (zh) | 海洋曲霉菌产生的抗真菌化合物及其制备方法 | |
CN106957216B (zh) | 一种抗真菌化合物制备方法和抗真菌用途 | |
KR100729437B1 (ko) | 항진균 활성이 우수한 히어리 잎 추출물과 이로부터 분리된텔리마그란딘 ⅰ | |
CN110218200B (zh) | 一种红树内生真菌中环缩肽化合物及其制备方法与应用 | |
CN115851454A (zh) | 嗜氮酮类化合物及其制备方法和在制备神经保护药物中的应用 | |
CN105399721B (zh) | 新化合物及其制备方法和在制备抗菌抗肿瘤药物中的应用 | |
CN115536645A (zh) | 化合物Phomolide B及其制备方法和在抗菌药物中的应用 | |
CN110655557A (zh) | 一种纽莫康定b0丝氨酸类似物的分离纯化方法 | |
CN111808088B (zh) | 化合物tersaphilone B和E及其制备方法和在制备抗肿瘤药物中的应用 | |
CN110229131B (zh) | 角果木内生真菌来源的苯并吡喃酮衍生物及其制备方法与应用 | |
CN110698441B (zh) | 一类2-甲基-4-(1-丙三醇)-呋喃类化合物及其制备方法和应用 | |
CN110330549B (zh) | 环肽emericellamide G及其制备方法和在制备酶抑制剂中的应用 | |
CN114149445A (zh) | 一种氧杂蒽酮类化合物的制备方法及其在抗耐药细菌方面的应用 | |
CN108441427B (zh) | 一种节菱孢属真菌及其生产的吡啶酮生物碱类化合物 | |
CN114989190B (zh) | 一类大环内酯类化合物kongjuemycins及其制备方法和应用 | |
CN111892574A (zh) | 一类非典型角环素类化合物及其制备方法和应用 | |
CN113651677B (zh) | 化合物eutyscoparins G及其制备方法和在制备抗菌药物中的应用 | |
CN109575040B (zh) | 一种具有抗菌活性的化合物及其制备方法 | |
CN111909856B (zh) | 一种开环芳香丁烯酸内酯的制备方法及其抗菌应用 | |
CN110627702B (zh) | 聚酮化合物及其制备方法和用途 | |
CN113603666B (zh) | 化合物eutyscoparol H和L及其制备方法和在制备抗菌药物中的应用 | |
CN114349762B (zh) | 一类骨架新颖的6/6/6/6/5/5环生物碱类化合物及其在制备抗菌药物中的应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210402 |