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 strains

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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
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forskolin
mortierella
hydrogen
ddf
derivatives
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Werner Dr Aretz
Dirk Dr Boettger
Klaus Dr Sauber
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Hoechst AG
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    • 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
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    • 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
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    • C12N1/145—Fungal isolates
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    • 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

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Abstract

The present invention relates to forskolin derivatives, a process for their preparation by means of microbial transformation by Mortierella strains and the use of these substances as pharmaceutical agents, in particular as medicaments for the treatment of glaucoma, cardiac insufficiency, hypertension, inflammation, allergies, bronchial disorders and disorders of the immune system.

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.

Allgemeine Verfahrensbeschreibung:General procedure description:

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

Beispiel 1example 1

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%.

Beispiel 2Example 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:

1,9-Didesoxy-3-keto-forskolin1,9-dideoxy-3-keto-forskolin

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)

1. Verfahren zur mikrobiellen Hydroxylierung oder 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, dadurch gekennzeichnet, daß man auf eine der genannten Verbindungen der Formel I als Substrat in einem üblichen Nährmedium einen Mikroorganismus- Stamm vom Genus Mortierella einwirken läßt und die gebildeten Verbindungen nach Beendigung der Fermentation aus der Kulturbrühe durch Extraktion mit einem organischen Lösungsmittel und anschließende Chromatographie und/oder Kristallisation isoliert.1. Process for the microbial hydroxylation or oxidation of Labdan 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, characterized in that a microorganism is used as a substrate in one of the compounds of the formula I mentioned in a conventional nutrient medium - strain of the genus Mortierella can act and, after the fermentation has ended, the compounds formed are isolated from the culture broth by extraction with an organic solvent and subsequent chromatography and / or crystallization. 2. Verfahren gemäß Anspruch 1, dadurch gekennzeichnet, daß man den Stamm Mortierella alpina ATCC 8979, Mortierella alpina Peyron CBS 52 872 oder Mortierella isabellina ATCC 16007 verwendet.2. The method according to claim 1, characterized in that the Mortierella alpina strain ATCC 8979, Mortierella alpina Peyron CBS 52 872 or Mortierella isabellina ATCC 16007 used. 3. Verfahren gemäß Anspruch 2, dadurch gekennzeichnet, daß man die Fermentation 5 Tage bei 25-30°C unter Schütteln durchführt und das Kulturfiltrat mit Dichlormethan extrahiert.3. The method according to claim 2, characterized in that the fermentation for 5 days at 25-30 ° C below Shakes and the culture filtrate with dichloromethane extracted. 4. Verfahren gemäß Anspruch 3, dadurch gekennzeichnet, daß man bei 28°C fermentiert.4. The method according to claim 3, characterized in that you ferment at 28 ° C. 5. Verfahren gemäß den Ansprüchen 1-4, dadurch gekennzeichnet, daß die Hydroxylierung in 1-, 2-, 3- und/oder 12-Stellung und die Oxidation in 3-Stellung erfolgt.5. The method according to claims 1-4, characterized in that the hydroxylation in 1-, 2-, 3- and / or  12 position and the oxidation in 3 position. 6. 1,9-Di-desoxy-3-keto-forskolin.6. 1,9-di-deoxy-3-keto-forskolin. 7. 1,9-Di-desoxy-12-hydroxy-forskolin.7. 1,9-di-deoxy-12-hydroxy-forskolin. 8. Arzneimittel, enthaltend eine Verbindung gemäß Anspruch 6 oder 7.8. Medicament containing a compound according to claim 6 or 7.
DE19853527335 1985-07-31 1985-07-31 Process for the microbial hydroxylation of forskolin and its derivatives by Mortierella strains Withdrawn DE3527335A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007046113A2 (en) * 2005-10-18 2007-04-26 Panacea Biotec Ltd. Novel pharmaceutical composition comprising alkaloid and process thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007046113A2 (en) * 2005-10-18 2007-04-26 Panacea Biotec Ltd. Novel pharmaceutical composition comprising alkaloid and process thereof
WO2007046113A3 (en) * 2005-10-18 2007-07-26 Panacea Biotec Ltd Novel pharmaceutical composition comprising alkaloid and process thereof

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