DE3527335A1 - Process for the microbial hydroxylation of forskolin and its derivatives by Mortierella strains - Google Patents
Process for the microbial hydroxylation of forskolin and its derivatives by Mortierella strainsInfo
- Publication number
- DE3527335A1 DE3527335A1 DE19853527335 DE3527335A DE3527335A1 DE 3527335 A1 DE3527335 A1 DE 3527335A1 DE 19853527335 DE19853527335 DE 19853527335 DE 3527335 A DE3527335 A DE 3527335A DE 3527335 A1 DE3527335 A1 DE 3527335A1
- Authority
- DE
- Germany
- Prior art keywords
- forskolin
- mortierella
- hydrogen
- ddf
- derivatives
- 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.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 16
- 241000235575 Mortierella Species 0.000 title claims abstract description 5
- 230000000813 microbial effect Effects 0.000 title claims abstract description 5
- 230000033444 hydroxylation Effects 0.000 title claims description 5
- 238000005805 hydroxylation reaction Methods 0.000 title claims description 5
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 title description 24
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 title description 14
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 title description 12
- 239000003814 drug Substances 0.000 claims abstract description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- 229910052739 hydrogen Inorganic materials 0.000 claims description 8
- 239000001257 hydrogen Substances 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- 230000003647 oxidation Effects 0.000 claims description 5
- 238000007254 oxidation reaction Methods 0.000 claims description 5
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 4
- 241000907999 Mortierella alpina Species 0.000 claims description 4
- 150000002431 hydrogen Chemical class 0.000 claims description 4
- 241000306282 Umbelopsis isabellina Species 0.000 claims description 3
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 claims description 3
- 230000004151 fermentation Effects 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000002425 crystallisation Methods 0.000 claims description 2
- 230000008025 crystallization Effects 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 244000005700 microbiome Species 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims description 2
- 150000003505 terpenes Chemical class 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 9
- 150000004160 forskolin derivatives Chemical class 0.000 abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 208000010412 Glaucoma Diseases 0.000 abstract description 3
- 206010019280 Heart failures Diseases 0.000 abstract description 3
- 206010020751 Hypersensitivity Diseases 0.000 abstract description 3
- 206010020772 Hypertension Diseases 0.000 abstract description 3
- 206010061218 Inflammation Diseases 0.000 abstract description 3
- 230000007815 allergy Effects 0.000 abstract description 3
- 230000004054 inflammatory process Effects 0.000 abstract description 3
- 208000014181 Bronchial disease Diseases 0.000 abstract description 2
- 208000035475 disorder Diseases 0.000 abstract description 2
- 210000000987 immune system Anatomy 0.000 abstract description 2
- 239000008177 pharmaceutical agent Substances 0.000 abstract description 2
- 230000009466 transformation Effects 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 230000036983 biotransformation Effects 0.000 description 5
- SUZLHDUTVMZSEV-WESICCPUSA-N (3R,4aR,5S,6S,6aS,10S,10aS,10bS)-3-ethenyl-6,10-dihydroxy-3,4a,7,7,10a-pentamethyl-1-oxododecahydro-1H-benzo[f]chromen-5-yl acetate Chemical compound O=C([C@@H]12)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C SUZLHDUTVMZSEV-WESICCPUSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- PXYAFNGUEZPJBI-OAUGPMCWSA-N [(3r,4ar,5s,6s,6as,10as,10bs)-3-ethenyl-6,10b-dihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-5,6,6a,8,9,10-hexahydro-2h-benzo[f]chromen-5-yl] acetate Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)CCCC1(C)C PXYAFNGUEZPJBI-OAUGPMCWSA-N 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- WDJJFDJQYGLZPA-CGPDBNODSA-N (3R,4aS,5S,6S,6aS,10S,10aS,10bR)-3-ethenyl-5,6,10-trihydroxy-3,4a,7,7,10a-pentamethyl-2,5,6,6a,8,9,10,10b-octahydrobenzo[f]chromen-1-one Chemical compound O1[C@@](C)(C=C)CC(=O)[C@@H]2[C@@]3(C)[C@@H](O)CCC(C)(C)[C@@H]3[C@H](O)[C@H](O)[C@]21C WDJJFDJQYGLZPA-CGPDBNODSA-N 0.000 description 3
- SBMKZIAQTQIEHN-NRADYMPXSA-N Coleonol C Natural products [H][C@@]12[C@H](OC(C)=O)[C@@H](O)[C@@]3(C)O[C@@](C)(CC(=O)[C@]3(O)[C@@]1(C)[C@@H](C)CCC2(C)C)C=C SBMKZIAQTQIEHN-NRADYMPXSA-N 0.000 description 3
- 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 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000131459 Plectranthus barbatus Species 0.000 description 3
- CLOQVZCSBYBUPB-VDCUXWMXSA-N [(3r,4ar,5s,6s,10s,10ar,10bs)-3-ethenyl-5,10,10b-trihydroxy-3,4a,7,7,10a-pentamethyl-1-oxo-5,6,6a,8,9,10-hexahydro-2h-benzo[f]chromen-6-yl] acetate Chemical compound O[C@H]([C@@]12C)CCC(C)(C)C1[C@H](OC(=O)C)[C@H](O)[C@]1(C)[C@]2(O)C(=O)C[C@](C)(C=C)O1 CLOQVZCSBYBUPB-VDCUXWMXSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- WPDITXOBNLYZHH-UHFFFAOYSA-N desacetylforskolin Natural products O1C(C)(C=C)CC(=O)C2(O)C3(C)C(O)CCC(C)(C)C3C(O)C(O)C21C WPDITXOBNLYZHH-UHFFFAOYSA-N 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- ZKZMDXUDDJYAIB-SUCLLAFCSA-N 1,9-dideoxyforskolin Natural products O=C([C@@H]12)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)CCCC1(C)C ZKZMDXUDDJYAIB-SUCLLAFCSA-N 0.000 description 2
- ZKZMDXUDDJYAIB-MLITWPTNSA-N 1,9-dideoxyforskolin Chemical compound O=C([C@@H]12)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)C1[C@]2(C)CCCC1(C)C ZKZMDXUDDJYAIB-MLITWPTNSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 235000021508 Coleus Nutrition 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- ZKZMDXUDDJYAIB-UHFFFAOYSA-N dideoxy-forskolin Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)CCCC2(C)C ZKZMDXUDDJYAIB-UHFFFAOYSA-N 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- PNQDGVYRLQBNOL-UHFFFAOYSA-N (1S,7S,9S,5R,8R)-8,9-dihydroxy-1,5,7,11,11-pentamethyl-6-oxa-vinyltricyclo[8.4.0.02,7]tetradecan-3-one Natural products O1C(C)(C=C)CC(=O)C2C3(C)CCCC(C)(C)C3C(O)C(O)C21C PNQDGVYRLQBNOL-UHFFFAOYSA-N 0.000 description 1
- PNQDGVYRLQBNOL-GPRZKFDUSA-N 1,9-Dideoxy-7-deacetylforskolin Natural products O1[C@@](C)(C=C)CC(=O)[C@@H]2[C@@]3(C)CCCC(C)(C)[C@@H]3[C@H](O)[C@H](O)[C@]21C PNQDGVYRLQBNOL-GPRZKFDUSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010006482 Bronchospasm Diseases 0.000 description 1
- 235000005320 Coleus barbatus Nutrition 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000007885 bronchoconstriction Effects 0.000 description 1
- 230000003491 cAMP production Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- WBKFWQBXFREOFH-UHFFFAOYSA-N dichloromethane;ethyl acetate Chemical compound ClCCl.CCOC(C)=O WBKFWQBXFREOFH-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 229930004069 diterpene Natural products 0.000 description 1
- 125000000567 diterpene group Chemical group 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- -1 labdane terpenoids Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 238000012746 preparative thin layer chromatography Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/92—Naphthopyrans; Hydrogenated naphthopyrans
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- 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
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Virology (AREA)
- Botany (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
Die vorliegende Erfindung betrifft Forskolin-Derivate, ein Verfahren zu deren Herstellung mittels mikrobieller Umwandlung durch Mortierella-Stämme und die Verwendung der Substanzen als pharmazeutische Mittel, insbesondere als Medikamente zur Behandlung von Glaukom, Herzinsuffizienz, Hypertonie, Entzündung, Allergien, Bronchialerkrankungen und Erkrankungen des Immunsystems.The present invention relates to forskolin derivatives, a process for their production by means of microbial Conversion by Mortierella strains and use the substances as pharmaceutical agents, in particular as Medicines to treat glaucoma, heart failure, Hypertension, inflammation, allergies, bronchial diseases and diseases of the immune system.
Forskolin und seine natürlich vorkommenden Analoge stellen eine Gruppe von Labdan-Terpenoiden dar, die aus der Pflanze Coleus forskolii (Briqu.) isoliert werden. Die Strukturformel von Forskolin und seinen natürlich vorkommenden, isolierten Analogen werden in der Formel I dargestellt. Forskolin and its naturally occurring analogues are a group of Labdan terpenoids that are isolated from the Coleus forskolii (Briqu.) Plant. The structural formula of Forskolin and its naturally occurring, isolated analogs are shown in Formula I.
Für Forskolin ist R1, R2 = OH, R′ = Ac, sowie R3 bis R7 und R″ = HFor forskolin, R 1 , R 2 = OH, R ′ = Ac, and R 3 to R 7 and R ″ = H
Bis heute sind folgende natürlich vorkommenden Forskolin-
Analoge nachgewiesen worden:
1,9-Didesoxy-forskolin: R′ = Ac, sowie R1 bis R7 und R″ = H
9-Desoxy-forskolin: R1 = OH, R′ = Ac, sowie R2 bis R7 und
R″ = H
1,9-Didesoxy-7-deacetyl-forskolin: R′, R″ und R1 bis R7 = H
1-Desoxy-forskolin: R2 = OH, R′ = Ac, sowie R′ und R1, R3 bis R7 = H
7-Deacetyl-forskolin: R1, R2 = OH, R′, R″, sowie R3 bis R7 = H
6-Acetyl-7-deacetyl-forskolin: R1 = OH, R″ = Ac, sowie R′ und
R2 bis R7 = H
(Tetrahedron Lett. 19, 1669 (1977); J. Med. Chem. 26, 486
(1983)).To date, the following naturally occurring forskolin analogs have been detected:
1,9-dideoxy-forskolin: R ′ = Ac, and R 1 to R 7 and R ″ = H
9-deoxy-forskolin: R 1 = OH, R ′ = Ac, and R 2 to R 7 and R ″ = H
1,9-dideoxy-7-deacetyl-forskolin: R ′, R ″ and R 1 to R 7 = H
1-Deoxy-forskolin: R 2 = OH, R '= Ac, and R' and R 1 , R 3 to R 7 = H
7-deacetyl-forskolin: R 1 , R 2 = OH, R ′, R ″, and R 3 to R 7 = H
6-acetyl-7-deacetyl-forskolin: R 1 = OH, R ″ = Ac, and R ′ and R 2 to R 7 = H
(Tetrahedron Lett. 19, 1669 (1977); J. Med. Chem. 26, 486 (1983)).
Forskolin ist eines der wichtigen Diterpene der Pflanze Coleus forskolii. Diese Substanz und seine Derivate können verwendet werden als wertvolle Arzneimittel bei der Behandlung verschiedener Erkrankungen wie Glaukom, Hypertonie, Herzinsuffizienz, Entzündung, Allergien, Bronchokonstriktion und ganz allgemein von Erkrankungen, die durch Störungen der cAMP-Produktion und -Metabolismus hervorgerufen werden (Indisches Patent Nr. 1 43 875 und entsprechendes DE-OS 25 57 784, Indisches Patent Nr. 1 45 926, Indisches Patent Nr. 1 47 630 und entsprechende DE-OS 26 40 275; Med.Res. Revs. 3, 201-19 (1983)).Forskolin is one of the plant's important diterpenes Coleus forskolii. This substance and its derivatives can are used as valuable medicines in treatment various diseases such as glaucoma, hypertension, Heart failure, inflammation, allergies, bronchoconstriction and more generally of diseases caused by disorders of the cAMP production and metabolism (Indian Patent No. 1 43 875 and corresponding DE-OS 25 57 784, Indian Patent No. 1 45 926, Indian Patent No. 1 47 630 and corresponding DE-OS 26 40 275; Medical Res. Revs. 3, 201-19 (1983)).
Ein Verfahren zur Isolierung des Hauptanteils von Forskolin aus Coleus forskohlii ist bereits beschrieben (s. Indische Patente Nr. 1 43 875 und 1 45 926, DE-OS 25 57 784). Bei diesem Verfahren werden außerdem Forskolin-Analoge gewonnen. Diese Analoge können verwendet werden als Zwischenprodukte zur Herstellung von Forskolin. Es sind bereits Methoden beschrieben worden, durch die die Analoge mit gleicher Oxidationsstufe wie Forskolin, z. B. 7-Deacetyl-Forskolin und 6-Acetyl-7-deacetylforskolin in Forskolin umgewandelt werden können (Indisches Patent 1 47 007, J.Chem. Perk. Trans. 1, 767 (1982)). A process to isolate the bulk of forskolin from Coleus forskohlii has already been described (see Indian Patents No. 1 43 875 and 1 45 926, DE-OS 25 57 784). At This process also provides forskolin analogs. These analogs can be used as intermediates for the production of forskolin. They are methods through which the analogs with the same Oxidation level such as forskolin, e.g. B. 7-deacetyl forskolin and 6-acetyl-7-deacetylforskolin converted to forskolin (Indian Patent 1 47 007, J. Chem. Perk. Trans. 1, 767 (1982)).
Es ist bereits ein Verfahren der mikrobiellen Umwandlung beschrieben worden, durch das die weniger hydroxylierten Forskolin-Analoge in Forskolin, 7-Deacetyl-Forskolin oder 6-Acetyl-7-deacetylforskolin, oder in solche Derivate umgewandelt werden, die sich durch bekannte chemische Methoden leicht in Forskolin umwandeln lassen (Deutsche Patentanmeldung P 35 02 685.5).It is already a process of microbial conversion through which the less hydroxylated Forskolin analogs in Forskolin, 7-Deacetyl-Forskolin or 6-acetyl-7-deacetylforskolin, or converted into such derivatives that are characterized by known chemical Easily convert methods into forskolin (German Patent application P 35 02 685.5).
Gegenstand der vorliegenden Erfindung ist nun ein Verfahren zur Hydroxylierung bzw. Oxidation von Labdan-Terpenoiden der Formel I worin R1 und R3 bis R7 Wasserstoff, R2 Wasserstoff oder Hydroxy, R′ die Acetylgruppe oder Wasserstoff und R′ Wasserstoff bedeuten, durch Mortierella-Stämme, insbesondere Mortierella alpina ATCC 8979, Mortierella alpina Peyron CBS 52872 und Mortierella isabellina ATCC 16007.The present invention now relates to a process for the hydroxylation or oxidation of labdane terpenoids of the formula I. wherein R 1 and R 3 to R 7 are hydrogen, R 2 is hydrogen or hydroxy, R 'is the acetyl group or hydrogen and R' is hydrogen, by Mortierella strains, in particular Mortierella alpina ATCC 8979, Mortierella alpina Peyron CBS 52872 and Mortierella isabellina ATCC 16007 .
Das Verfahren ist dadurch gekennzeichnet, daß man eine Verbindung der Formel I, worin R1 bis R7, R′ und R″ die oben genannten Bedeutungen haben, in einem üblichen Nährmedium definierter Zusammensetzung, das einen der genannten Mikroorganismen-Stämme enthält, fermentiert. Die Fermentation erfolgt mehrere Tage, bevorzugt 5 Tage lang bei einer Temperatur von 25-30°C, bevorzugt bei 28°C unter Schütteln.The process is characterized in that a compound of the formula I, in which R 1 to R 7 , R ′ and R ″ have the meanings given above, is fermented in a customary nutrient medium of defined composition which contains one of the said microorganism strains. The fermentation takes place for several days, preferably for 5 days at a temperature of 25-30 ° C, preferably at 28 ° C with shaking.
Das Nährmedium enthält Kohlenstoff- und Stickstoffquellen, gegebenenfalls auch anorganische Nährsalze sowie Spurenelemente.The nutrient medium contains carbon and nitrogen sources, optionally also inorganic nutrient salts and trace elements.
Als Kohlenstoffquelle eignen sich z. B. Glucose, Stärke, Dextrin und Glycerin. Geeignete Stickstoffquellen sind z. B. Sojamehl, Hefeextrakt, Fleischextrakt, Malzextrakt, Maisquellwasser, Pepton und Casein. Als anorganische Nährsalze kommen z. B. Natriumchlorid, Magnesiumsulfat oder Calciumcarbonat, als Spurenelemente z. B. Eisen, Magnesium, Kupfer, Zink und Kobalt in Form geeigneter Salze in Frage.As a carbon source z. B. glucose, starch, Dextrin and glycerin. Suitable nitrogen sources are e.g. B. soy flour, yeast extract, meat extract, malt extract, Corn steep liquor, peptone and casein. As inorganic Nutrient salts come e.g. B. sodium chloride, magnesium sulfate or calcium carbonate, as trace elements e.g. B. iron, Magnesium, copper, zinc and cobalt in the form of suitable salts in question.
Nach Abschluß der Umsetzung wird das Pilzmycel abfiltriert und das resultierende Kulturfiltrat mit organischen Lösungsmitteln wie Dichlormethan, Chloroform oder Ethylacetat extrahiert. Der Extrakt wird zweckmäßig über Trocknungsmitteln wie Na2SO4 getrocknet und im Vakuum konzentriert. Der Rückstand wird dann durch Trennungsverfahren wie beispielsweise Chromatographie und durch Kristallisation gereinigt.After completion of the reaction, the fungal mycelium is filtered off and the resulting culture filtrate is extracted with organic solvents such as dichloromethane, chloroform or ethyl acetate. The extract is expediently dried over drying agents such as Na 2 SO 4 and concentrated in vacuo. The residue is then purified by separation methods such as chromatography and by crystallization.
Die hydroxylierung erfolgt bevorzugt in 1, 2, 3 und/oder 12-Stellung, die Oxidation zur Ketogruppe in 3-Stellung.The hydroxylation is preferably carried out in 1, 2, 3 and / or 12-position, oxidation to the keto group in 3-position.
a) Stammhaltung und Anzuchta) Maintaining and breeding
Sporensuspensionen der obengenannten Stämme werden auf
Schrägröhrchen mit folgendem Nährmedium beimpft und 14
Tage bei 30°C bebrütet:
Stammhaltungsmedium:
Haferflocken 40 g/l
Agar 20 g/l
pH 7,2Spore suspensions of the above-mentioned strains are inoculated on inclined tubes with the following nutrient medium and incubated for 14 days at 30 ° C:
Maintaining medium:
Oatmeal 40 g / l
Agar 20 g / l
pH 7.2
Zur Anzucht in Flüssigkulturen werden von dem Stamm Sporensuspensionen
durch Abschwemmen der Schrägröhrchen mit
steriler NaCl 0,8% gewonnen und diese auf einen Titer
von 2 × 107 Sporen/ml verdünnt. 1 ml dieser Suspension
dienen als Inokulum von 50 ml Hauptkultur (in 100 ml Erlenmeyer-Kolben)
folgender Zusammensetzung:
Glucose 10 g/l
Pepton 10 g/l
Hefeextrakt 2 g/l
Cornsteep fl. 2 ml
pH 5,5
Nach 24 Stunden bei 28°C und einer Schüttelfrequenz von
190 Upm erzielt man ein dichtes, mycelartiges Wachstum.For cultivation in liquid cultures, spore suspensions are obtained from the strain by washing off the oblique tubes with sterile NaCl 0.8% and diluting them to a titer of 2 × 10 7 spores / ml. 1 ml of this suspension serve as an inoculum of 50 ml of the main culture (in 100 ml Erlenmeyer flasks) of the following composition:
Glucose 10 g / l
Peptone 10 g / l
Yeast extract 2 g / l
Cornsteep fl. 2 ml
pH 5.5
After 24 hours at 28 ° C and a shaking frequency of 190 rpm, dense, mycelium-like growth is achieved.
b) Biotransformationb) Biotransformation
Zur Biotransformation werden die 50 ml Hauptkultur in einen sterilen 300 ml-Erlenmeyer-Kolben überführt und 16 mg Substrat (in Methanol gelöst) zugesetzt. Anschließend wird die Suspension bei 28°C und 240 Upm 5 Tage geschüttelt. Der Fortgang der Biotransformation wird mittels DC (HPTLC- Platten, Methylenchlorid-Ethylacetat 3 : 1; Anis- aldehyd-Schwefelsäure als Nachweisreagenz) verfolgt. Nach Abschluß der Umsetzung wird das Pilzmycel abfiltriert, das resultierende Kulturfiltrat mit der gleichen Menge Dichlormethan extrahiert und der Extrakt im Vakuum eingedampft.For biotransformation, the 50 ml main culture is put into one sterile 300 ml Erlenmeyer flask transferred and 16 mg Substrate (dissolved in methanol) added. Subsequently the suspension is shaken at 28 ° C. and 240 rpm for 5 days. The progress of the biotransformation is monitored using DC (HPTLC Plates, methylene chloride-ethyl acetate 3: 1; Anise- aldehyde-sulfuric acid as detection reagent). To At the end of the implementation, the fungal mycelium is filtered off, the resulting culture filtrate with the same amount Dichloromethane extracted and the extract evaporated in vacuo.
c) Transformationsprodukte (Strukturen siehe Tabelle 1)
I 1,9-Didesoxyforskolin (1) → 1α-HO-DDF (5) = 9-Desoxyforskolin)
(DDF)
→ 2α-HO DDF-(8)
→ 2β-HO-DDF (6)
→ 3β-HO-DDF (7)
→ 3-Keto-DDF (4)
→ 12-HO-DDF (14)
(→ 4-Hydroxymethyl-DDF)
II 1-Desoxyforskolin (3) → Forskolin (2)
III 7-dAc-DDF → 7-dAc-3β-HO-DDF
c) Transformation products (structures see Table 1)
I 1,9-dideoxyforskolin ( 1 ) → 1α-HO-DDF ( 5 ) = 9-deoxyforskolin)
(DDF)
→ 2α-HO DDF- ( 8 )
→ 2β-HO-DDF ( 6 )
→ 3β-HO-DDF ( 7 )
→ 3-keto-DDF ( 4 )
→ 12-HO-DDF ( 14 )
(→ 4-hydroxymethyl-DDF)
II 1-deoxyforskolin ( 3 ) → forskolin ( 2 )
III 7-dAc-DDF → 7-dAc-3β-HO-DDF
Eine Umlagerung der Acetylgruppe von C-7 nach C-6 kann für alle Forskolinanaloge durch Anheben der pH-Wertes über 6,5 erreicht werden.A rearrangement of the acetyl group from C-7 to C-6 can for all forskolin analogs by raising the pH above 6.5 can be achieved.
Umsetzungsbeispiele mit Mortierella isabellina ATCC 16007Implementation examples with Mortierella isabellina ATCC 16007
I Biotransformation von DDF Durchführung gemäß 1 b) 120 Std. Die Bildung von 3-Keto-DDF kann durch Konstanthalten der Glucosekonzentration bei 2% unterdrückt werden.I Biotransformation of DDF according to 1 b) 120 hours The formation of 3-keto-DDF can be suppressed by keeping the glucose concentration constant at 2%.
Eine nach Beispiel 1 durchgeführte Biotransformation von DDF ergab nach Extraktion 150 mg Substanz. Über eine Vorsäule von geringem Volumen wurde an 15 g Kieselgel (Merck, 15-40 µm; Labomatic-Säule) mit dem System Petrolether/Ethylacetat = 2/1 bei einem Fluß von 1,5 ml/min chromatographiert. Neben der eingesetzten Ausgangsverbindung wurden 6,6 mg M1, 5,2 mg M2, 16 mg 3-β-Hydroxy-DDF, 2 mg M4 und einige kleinere Substanzmengen erhalten (M5, M1.2). A biotransformation of DDF carried out according to Example 1 gave 150 mg of substance after extraction. Chromatography was carried out over a small volume precolumn on 15 g of silica gel (Merck, 15-40 μm; Labomatic column) using the petroleum ether / ethyl acetate = 2/1 system at a flow of 1.5 ml / min. In addition to the starting compound used, 6.6 mg M 1 , 5.2 mg M 2 , 16 mg 3-β-hydroxy-DDF, 2 mg M 4 and some smaller amounts of substance were obtained (M 5 , M 1.2 ).
Eine Nachreinigung über präparative DC liefert 1,5 mg reines M1, 2 mg M2, 1,5 mg M4, 1,5 mg M5 und 1 mg M1.2.Subsequent cleaning via preparative TLC provides 1.5 mg of pure M 1 , 2 mg M 2 , 1.5 mg M 4 , 1.5 mg M 5 and 1 mg M 1.2 .
Die Rf-Werte auf Kieselgel-Methylenchlorid/Ethylacetat (2 / 1)
sind:
M1 (0.77)
M1.2 (0.64)
M2 (0.47)
3β-OH-DDF (0.43)
M4 (0.3)
M5 (0.21)
Die Substanzen wurden einer 1H-NMR-Analyse unterzogen.The Rf values on silica gel methylene chloride / ethyl acetate (2/1) are:
M 1 (0.77)
M 1.2 (0.64)
M 2 (0.47)
3β-OH-DDF (0.43)
M 4 (0.3)
M 5 (0.21)
The substances were subjected to 1 H-NMR analysis.
Durch authentische Vergleichssubstanzen konnten folgende
Verbindungen zugeordnet werden:
M1.2 = 1α-HO-DDF L 772584
M2 = 2β-HO-DDF L 847598
M3.1 = 2α-HO-DDF L 847597The following compounds could be assigned using authentic comparison substances:
M 1.2 = 1α-HO-DDF L 772584
M 2 = 2β-HO-DDF L 847598
M 3.1 = 2α-HO-DDF L 847597
M1 wurde anhand des 1H-NMR- und Massenspektrums charakterisiert. Bei der Substanz handelt es sich um: M 1 was characterized based on the 1 H-NMR and mass spectrum. The substance is:
1H-NMR (270 MHz, CDCl3, TMS) δ = 5.95 (dd, J = 18 Hz, J′ = 12 Hz; 14-H), 5.28 (d, J = 18 Hz; 15 t-H), 5.09 (d, J = 6 Hz; 7-H), 5.06 (d, J = 12 Hz; 15 c-H), 4.28 (m; 6α-H), 2.95-2.70 (m; COCH 2 und s; 9 α-H), 2.66 (AB, J = 18 Hz; 12-H), 2.29 (m; 5α-H), 2.22 (s; CH 3CO), 1.90-0.82 (m; 17 H darin: 1.62, 1.58, 1.41, 1.23 und 1.17, 5s; 5 CH 3). 1 H-NMR (270 MHz, CDCl 3 , TMS) δ = 5.95 (dd, J = 18 Hz, J ′ = 12 Hz; 14-H), 5.28 (d, J = 18 Hz; 15 tH), 5.09 ( d, J = 6 Hz; 7-H), 5.06 (d, J = 12 Hz; 15 cH), 4.28 (m; 6α-H), 2.95-2.70 (m; COC H 2 and s; 9 α-H ), 2.66 (AB, J = 18 Hz; 12-H), 2.29 (m; 5α-H), 2.22 (s; C H 3 CO), 1.90-0.82 (m; 17 H therein: 1.62, 1.58, 1.41 , 1.23 and 1.17, 5s; 5 C H 3 ).
MS m/z = 392 (M⁺), 377 (M⁺-CH3), 359 (M⁺-CH3-H2O), 332 (M⁺-AcOH), 317 (M⁺-CH3-AcOH), 249,95. MS m / z = 392 (M⁺), 377 (M⁺-CH 3 ), 359 (M⁺-CH 3 -H 2 O), 332 (M⁺-AcOH), 317 (M⁺-CH 3 -AcOH ), 249.95.
Claims (8)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19853527335 DE3527335A1 (en) | 1985-07-31 | 1985-07-31 | Process for the microbial hydroxylation of forskolin and its derivatives by Mortierella strains |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19853527335 DE3527335A1 (en) | 1985-07-31 | 1985-07-31 | Process for the microbial hydroxylation of forskolin and its derivatives by Mortierella strains |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| DE3527335A1 true DE3527335A1 (en) | 1987-02-05 |
Family
ID=6277207
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE19853527335 Withdrawn DE3527335A1 (en) | 1985-07-31 | 1985-07-31 | Process for the microbial hydroxylation of forskolin and its derivatives by Mortierella strains |
Country Status (1)
| Country | Link |
|---|---|
| DE (1) | DE3527335A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007046113A3 (en) * | 2005-10-18 | 2007-07-26 | Panacea Biotec Ltd | Novel pharmaceutical composition comprising alkaloid and process thereof |
-
1985
- 1985-07-31 DE DE19853527335 patent/DE3527335A1/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007046113A3 (en) * | 2005-10-18 | 2007-07-26 | Panacea Biotec Ltd | Novel pharmaceutical composition comprising alkaloid and process thereof |
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