WO1993013094A1 - Archazolides, method of preparing them and agents containing them - Google Patents

Archazolides, method of preparing them and agents containing them Download PDF

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WO1993013094A1
WO1993013094A1 PCT/EP1992/002980 EP9202980W WO9313094A1 WO 1993013094 A1 WO1993013094 A1 WO 1993013094A1 EP 9202980 W EP9202980 W EP 9202980W WO 9313094 A1 WO9313094 A1 WO 9313094A1
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archazolide
methanol
archazolides
nych
nhch
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PCT/EP1992/002980
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German (de)
French (fr)
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Gerhard Höfle
Hans Reichenbach
Florenz Sasse
Heinrich Steinmetz
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GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF)
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • 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
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings

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  • the invention relates to archazolides of the following general formula:
  • NYCH 3 is NHCH 3 or
  • NYCH 3 is N (CH 3 ) 2
  • the invention relates to an archazolid of the empirical formula C 42 H 62 N 2 SO 7 with the following parameters:
  • the invention relates to archazolides which can be prepared by isomerizing the above-described archazolide by UV radiation and, if appropriate, separating the isomer mixture by chromatography in a manner known per se.
  • the invention relates to a process for the preparation of the archazolide of the empirical formula C 42 H 62 N 2 SO 7 with the parameters specified above, this process being characterized in that
  • This method according to the invention can be characterized in that - adsorbed on Amberlite XAD16,
  • Variant (b) receives, or
  • the invention further relates to an agent that consists of one or more archazolides according to the invention or comprises these in addition to customary auxiliaries, for example carriers and diluents.
  • the bacterium Archangium gephyra belongs to the order of the
  • the Ar 3548 production strain was isolated in January 198 at the Deutschen für Biotechnologische Anlagen mbh (GBF) from a soil sample from the Karawanken in Austria. The strain was deposited on November 13, 1991 at the German Collection of Microorganisms (DSM) under number DSM 6806. The characteristics of the production strain are consistent with the description of Archangium gephyra in Bergey's Manual of Systematic
  • the stock culture is carried out on agar plates, preferably on yeast agar (VY / 2 agar).
  • This medium contains 0.5% baker's yeast, 0.1% CaCl 2 x 2 H 2 O, 0.1 ⁇ g / l vitamin B 12 and 1.2% agar.
  • the pH is adjusted to 7.4.
  • the plates are incubated at 30 ° C. A.3. Morphological description
  • the vegetative cells are long, slender rods with tapered ends, approximately 0.8 x 6 - 10 ⁇ m. Due to the
  • the myxospores are short, clumsy, very thick and well rounded rods, often somewhat irregular in outline, and always strongly refractive. They measure approximately 1.4-1.8 x 1.8-2.2 ⁇ m.
  • the Ar 3548 strain produces a substance, namely archazolide, that inhibits the growth of human cancer cell lines and other animal cell cultures and the growth of fungi.
  • the inhibitor can be isolated both from the cells and from the culture supernatant.
  • a typical fermentation proceeds as follows: A fermentor with 350 1 working volume is filled with 300 1 culture medium (composition: 0.5%
  • Adsorber resin (Amberlite XAD-16) added. Inoculation is carried out with 10 l of a 2-day-old preculture which was produced in the same medium in a correspondingly smaller fermenter. It is fermented at 30 ° C. with a stirring speed of 150 rpm and aeration of 10% by volume per min. Initial foam formation is prevented by adding 50 ml of silicone anti-foam (e.g. Tegosipon). The pH increases during the fermentation. The increase is limited to 7.8 by adding 5% sulfuric acid. The fermentation is ended after 6 days.
  • silicone anti-foam e.g. Tegosipon
  • Archazolid very effectively inhibits the growth of human cancer cell lines and other animal cell cultures without immediately killing the cells. Under the influence of archazolide, adherently growing cells round off and become freely mobile. Archazolid inhibits the growth of certain fungi (see table).
  • HeLa (ATCC CCL 2) 1ng / ml
  • the resin is sieved from a 100 l fermentation batch of the Ar 3548 strain in the presence of 1 l adsorber resin (Amberlite XAD 16) and eluted with 5 l methanol over a period of 2.5 hours.
  • the eluate is concentrated to the water phase and extracted 3 times with ethyl acetate in order to separate polar constituents.
  • the ethyl acetate extract is concentrated.
  • the remaining crude extract (12 g) is dissolved in 200 ml of methanol and fractionated in 2 portions over a Sephadex-LH20 column (62 mm x 63 mm) at a flow of 3.5 ml / min.
  • Eluent methanol Detection at 227 nm and by TLC chromatography.
  • the fractions containing archazolide (4.1 g) are in 10 0.41 g portions over a preparative Nucleosil C-18, 7 ⁇ m column (250 x 16 mm) with the mobile solvent methanol / water 85/15 and a flow of 15 ml / min (detection at 227 nm) chromatographed. After evaporation of the fractions containing Archazolid until

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Abstract

The invention concerns archazolides of general formula (I), as well as methods of preparing them and agents containing them.

Description

Archazolide, Herstellungsverfahren und Mittel  Archazolide, manufacturing process and agents
Die Erfindung betrifft Archazolide der folgenden allgemeinen Formel: The invention relates to archazolides of the following general formula:
Figure imgf000003_0001
mit den folgenden Bedeutungen:
Figure imgf000003_0001
with the following meanings:
(a) 7-OX und 15-OX sind OH  (a) 7-OX and 15-OX are OH
NYCH3 ist NHCH3 oder NYCH 3 is NHCH 3 or
(b) 7-OX und 15-OX sind OAcyl (C1 bis C6, Benzoyl) NYCH3 ist NHCH3 oder (b) 7-OX and 15-OX are OAcyl (C 1 to C 6 , benzoyl) NYCH 3 is NHCH 3 or
(c) 7-OX und 15-OX sind OCH3 (c) 7-OX and 15-OX are OCH 3
NYCH3 ist N(CH3)2 Gemäß einer speziellen Ausführungsform betrifft die Erfindung ein Archazolid der Summenformel C42 H62N2SO7 mit folgenden Parametern: NYCH 3 is N (CH 3 ) 2 According to a special embodiment, the invention relates to an archazolid of the empirical formula C 42 H 62 N 2 SO 7 with the following parameters:
UV (Methanol): lambda max (log epsilon) = 228 nm (4,6)  UV (methanol): lambda max (log epsilon) = 228 nm (4.6)
Massenspektrum (EI, 70 eV): m/z = 738 [M] H+ Mass spectrum (EI, 70 eV): m / z = 738 [M] H +
Dünnschichtchromatographie (DC-Alufolie 60F-254,  Thin layer chromatography (DC aluminum foil 60F-254,
Laufmittel Dichlormethan/Methanol 95:5): Rf = 0,56 Mobile phase dichloromethane / methanol 95: 5): R f = 0.56
Analytische HP-Flüssigchromatographie (Nucleosil C-18 10μm, 250x4 mm, Laufmittel Methanol/Wasser 8:2, 1,5 ml Fluß/min) : Rt = 12,0 min. Analytical HP liquid chromatography (Nucleosil C-18 10μm, 250x4 mm, mobile solvent methanol / water 8: 2, 1.5 ml flow / min): R t = 12.0 min.
Gemäß einer weiteren speziellen Ausführungsform betrifft die Erfindung Archazolide, die dadurch herstellbar sind, daß man das vorstehend beschriebene Archazolid durch UV-Bestrahlung isomerlsiert und gegebenenfalls das Isomerengemisch in an sich bekannter Weise chromatographisch auftrennt. According to a further special embodiment, the invention relates to archazolides which can be prepared by isomerizing the above-described archazolide by UV radiation and, if appropriate, separating the isomer mixture by chromatography in a manner known per se.
Gemäß einer weiteren Ausführungsform betrifft die Erfindung ein Verfahren zur Herstellung des Archazolids der Summenformel C42H62N2SO7 mit den vorstehend angegebenen Parametern, wobei dieses Verfahren dadurch gekennzeichnet ist, daß man According to a further embodiment, the invention relates to a process for the preparation of the archazolide of the empirical formula C 42 H 62 N 2 SO 7 with the parameters specified above, this process being characterized in that
- Archangium gephyra DSM 6806 in einem Kohlenstoff- und Stick- stoffquellen sowie Mineralsalze enthaltenden Medium kultiviert und das Archazolid produzieren läßt,  - Archangium gephyra DSM 6806 cultivated in a medium containing carbon and nitrogen sources as well as mineral salts and having the archazolide produced,
- das gebildete Archazolid an einem Adsorberharz adsorbiert, adsorbs the archazolide formed on an adsorber resin,
- das Adsorberharz vom Kulturmedium abtrennt, separates the adsorber resin from the culture medium,
- danach das Archazolid vom Adsorberharz mit einem Elutionsmittel freisetzt und gefriertrocknet.  - Then the archazolide is released from the adsorber resin with an eluent and freeze-dried.
Dieses erfindungsgemäße Verfahren kann dadurch gekennzeichnet sein, daß man - an Amberlite XAD16 adsorbiert, This method according to the invention can be characterized in that - adsorbed on Amberlite XAD16,
- mit Methanol eluiert,  - eluted with methanol,
- das Eluat bis zur Wasserphase einengt,  - the eluate is concentrated to the water phase,
- mit Ethylacetat extrahiert,  - extracted with ethyl acetate,
- den Ethylacetat-Extrakt einengt,  - concentrates the ethyl acetate extract,
- in Methanol aufnimmt,  - takes up in methanol,
- an einer Sephadex-Säule (LH20; Laufmittel: Methanol) fraktioniert,  - fractionated on a Sephadex column (LH20; mobile solvent: methanol),
- an einer Umkehrphase-Säule (Nucleosil C-18; Laufmittel:  - on a reverse phase column (Nucleosil C-18; mobile solvent:
Methanol/Wasser) chromatographiert und  Methanol / water) and chromatographed
- einengt und gefriertrocknet.  - concentrated and freeze-dried.
Archazolide der vorstehend angegebenen allgemeinen Formel lassen sich dadurch herstellen, daß man Archazolides of the general formula given above can be prepared by
(b') bei einem Archazolid gemäß Anspruch 2 oder 3 7-OH und 15-OH acyliert und Archazolide gemäß der allgemeinen Formel (b ') in an archazolide according to claim 2 or 3, 7-OH and 15-OH acylated and archazolides according to the general formula
Variante (b) erhält, oder Variant (b) receives, or
(b') bei einem Archazolid gemäß Anspruch 2 oder 3 7-OH, 15-OH sowie NHCH3 zu 7-OCH3, 15-OCH3 und N(CH3)2 methyliert und(b ') methylated in an archazolide according to claim 2 or 3 7-OH, 15-OH and NHCH 3 to 7-OCH 3 , 15-OCH 3 and N (CH 3 ) 2 and
Archazolide gemäß der allgemeinen Formel Variante (c) erhält. Archazolide according to the general formula variant (c) is obtained.
Ferner betrifft die Erfindung ein Mittel, daß aus einem oder aus mehreren erfindungsgemäßen Archazoliden besteht oder diese neben üblichen Hilfsmitteln, beispielsweise Trägern und Verdünnungsmittel, umfaßt. The invention further relates to an agent that consists of one or more archazolides according to the invention or comprises these in addition to customary auxiliaries, for example carriers and diluents.
Nachstehend wird die Erfindung durch ein Beispiel näher erläutert. Beispiel (Herstellung von Archazolid A) The invention is explained in more detail below by an example. Example (production of Archazolid A)
Figure imgf000006_0001
Figure imgf000006_0001
A.1. Produktionsstamm A.1. Production master
Das Bakterium Archangium gephyra gehört zur Ordnung der  The bacterium Archangium gephyra belongs to the order of the
Myxococcales, Unterordnung Cystobacterineae, Familie Myxococcales, subordination Cystobacterineae, family
Archangiaceae. Der Produktionsstamm Ar 3548 wurde im Januar 198 an der Gesellschaft für Biotechnologische Forschung mbh (GBF) aus einer Bodenprobe aus den Karawanken in Österreich isoliert. Der Stamm wurde am 13.11.91 bei der Deutschen Sammlung von Mikroorganismen (DSM) unter Nummer DSM 6806 hinterlegt. Die Merkmale des Produktionsstamms stimmen mit der Beschreibung von Archangium gephyra in Bergey's Manual of Systematic Archangiaceae. The Ar 3548 production strain was isolated in January 198 at the Gesellschaft für Biotechnologische Forschung mbh (GBF) from a soil sample from the Karawanken in Austria. The strain was deposited on November 13, 1991 at the German Collection of Microorganisms (DSM) under number DSM 6806. The characteristics of the production strain are consistent with the description of Archangium gephyra in Bergey's Manual of Systematic
Bacteriology., vol. 3 (1989), Seite 2148, überein. Bacteriology., Vol. 3 (1989), page 2148.
A.2. Stammkultur A.2. Root culture
Die Stammkultur erfolgt auf Agarplatten, bevorzugt auf Hefe-Agar (VY/2-Agar). Dieses Medium enthält 0,5 % Bäckerhefe, 0.1 % CaCl2 x 2 H2O, 0,1 μg/l Vitamin B12 und 1,2 % Agar. Der pH-Wert wird auf 7,4 eingestellt. Die Platten werden bei 30 ºC bebrütet. A.3. Morphologische Beschreibung The stock culture is carried out on agar plates, preferably on yeast agar (VY / 2 agar). This medium contains 0.5% baker's yeast, 0.1% CaCl 2 x 2 H 2 O, 0.1 μg / l vitamin B 12 and 1.2% agar. The pH is adjusted to 7.4. The plates are incubated at 30 ° C. A.3. Morphological description
Die vegetativen Zellen sind lange, schlanke Stäbchen mit verjüngten Enden, etwa 0,8 x 6 - 10 μm. Bedingt durch die  The vegetative cells are long, slender rods with tapered ends, approximately 0.8 x 6 - 10 μm. Due to the
Gleitbewegung der Bakterien, breiten sich die Kolonien rasch über die Kulturplatte aus. Die Schwarmkolonie auf Hefeagar ist dünn, filmartig, rötlich braun. Wie an dem um die Kolonien entstehenden Klärhof zu erkennen, werden die Hefezellen im Sliding movement of the bacteria, the colonies spread quickly across the culture plate. The swarm colony on Hefeagar is thin, film-like, reddish brown. As can be seen from the clarification center that is emerging around the colonies, the yeast cells in the
Medium abgebaut. Medium dismantled.
Auf Ausstrichen von lebenden Escherichia-coli-Bakterien auf Was- seragar (1,5 % Agar, 0,1 % CaCl22H2O, pH 7,2, autoklaviert), aber auch auf VY/2-Agar bildet der Organismus hellorange bis blaß bräunliche Fruchtkörper. Bei diesen handelt es sich um Rippen und wulstige Massen, die oft kammartig hochgezogen oder sogar koralloid verzweigt sind und aus erhärtetem Schleim und Myxosporen bestehen. Die Myxosporen sind kurze plumpe, sehr dicke und gut gerundete Stäbchen, oft etwas unregelmäßig im Umriß, und immer stark lichtbrechend. Sie messen etwa 1,4-1,8 x 1 ,8-2,2 μm. The streak of living Escherichia coli bacteria on water agar (1.5% agar, 0.1% CaCl 2 2H 2 O, pH 7.2, autoclaved), but also on VY / 2 agar, the organism forms light orange to pale brown fruiting bodies. These are ribs and bulky masses that are often raised like a comb or even branched coralloid and consist of hardened mucus and myxospores. The myxospores are short, clumsy, very thick and well rounded rods, often somewhat irregular in outline, and always strongly refractive. They measure approximately 1.4-1.8 x 1.8-2.2 μm.
A.4. Leistungen A.4. Services
Der Stamm Ar 3548 produziert eine Substanz, nämlich Archazolid, die das Wachstum von humanen Krebszellinien und anderer tierischer Zellkulturen und das Wachstum von Pilzen hemmt. Der Hemmstoff kann sowohl aus den Zellen wie auch aus dem Kulturüberstand isoliert werden.  The Ar 3548 strain produces a substance, namely archazolide, that inhibits the growth of human cancer cell lines and other animal cell cultures and the growth of fungi. The inhibitor can be isolated both from the cells and from the culture supernatant.
A.5. Produktion des Archazolids A.5. Production of the Archazolid
Die Substanz wird während der logarithmischen bis hin zur stationären Wachstumsphase produziert. Eine typische Fermentation verläuft wie folgt: Ein Fermentor mit 350 1 Arbeitsvolumen wir mit 300 1 Kulturmedium gefüllt (Zusammensetzung: 0,5 %  The substance is produced during the logarithmic to the stationary growth phase. A typical fermentation proceeds as follows: A fermentor with 350 1 working volume is filled with 300 1 culture medium (composition: 0.5%
Einzel lerprotein aus Methylomonas clarae (Probion; Hoechst); 0,5 % Stärke; 0,2 % Glucose; 0,1 % Hefeextrakt; 0,1 % MgSO4 x 7 H2O; 0,1 % CaCl2 x 2 H2O; 0,1 μg/l Vitamin B12). Der pH-Wert wird mit NaOH auf 7,4 eingestellt. Zur Bindung des ins Medium Single learning protein from Methylomonas clarae (Probion; Hoechst); 0.5% starch; 0.2% glucose; 0.1% yeast extract; 0.1% MgSO 4 x 7 H 2 O; 0.1% CaCl 2 x 2 H 2 O; 0.1 μg / l vitamin B 12 ). The pH is adjusted to 7.4 with NaOH. For binding the in the medium
freigesetzten Hemmstoffes wird dem Medium 1 % (V/V) eines  released inhibitor is 1% (V / V) of the medium
Adsorberharzes (Amberlite XAD-16) zugesetzt. Beimpft wird mit 10 1 einer 2 Tage alten Vorkultur, die im gleichen Medium in einem entsprechend kleineren Fermentor erzeugt wurde. Fermentiert wird bei 30 ºC mit einer Rührgeschwindigkeit von 150 Upm und einer Belüftung von 10 Vol. % pro min. Anfängliche SchaumbiIdung wird durch Zugabe von 50 ml Silikon-Antischäum (z.B Tegosipon) verhindert. Der pH-Wert steigt im Laufe der Fermentation an. Der Anstieg wird durch Zugabe von 5 %i ger Schwefelsäure auf 7,8 begrenzt. Die Fermentation wird nach 6 Tagen beendet.  Adsorber resin (Amberlite XAD-16) added. Inoculation is carried out with 10 l of a 2-day-old preculture which was produced in the same medium in a correspondingly smaller fermenter. It is fermented at 30 ° C. with a stirring speed of 150 rpm and aeration of 10% by volume per min. Initial foam formation is prevented by adding 50 ml of silicone anti-foam (e.g. Tegosipon). The pH increases during the fermentation. The increase is limited to 7.8 by adding 5% sulfuric acid. The fermentation is ended after 6 days.
B. Wirkung B. Effect
Archazolid hemmt sehr effektiv das Wachstum von humanen Krebszellinien und anderen tierischen Zellkulturen, ohne die Zel l en unmittelbar abzutöten. Adhärent wachsende Zellen runden sich unter dem Einfluß von Archazolid ab und werden frei beweglich. Archazolid hemmt das Wachstum von bestimmten Pilzen (vgl. Tabelle).  Archazolid very effectively inhibits the growth of human cancer cell lines and other animal cell cultures without immediately killing the cells. Under the influence of archazolide, adherently growing cells round off and become freely mobile. Archazolid inhibits the growth of certain fungi (see table).
Wirkungspektrum Spectrum of activity
Humane Krebszell inien IC50 Human cancer cell in IC 50
HeLa (ATCC CCL 2) 1 ng/ml HeLa (ATCC CCL 2) 1ng / ml
K-562 (ATCC CCL 243) 1 ng/ml  K-562 (ATCC CCL 243) 1ng / ml
Tierische Zeil inien  Animal Zeil inien
Maus, L 929 (ATCC CCL 1) 1 ng/ml Mouse, L 929 (ATCC CCL 1) 1ng / ml
Ente (ATCC CCL 141) 0,1 ng/ml  Duck (ATCC CCL 141) 0.1ng / ml
Hefen Hemmhof im Agardiffusionstest [mm] Yeast Inhibition zone in the agar diffusion test [mm]
(20 μg pro Testblättchen mit 5 mm Durchmesser) (20 μg per test sheet with 5 mm diameter)
Metschnikowia pulcherrima 10 Metschnikowia pulcherrima 10
Pichia membranaefaciens 10  Pichia membranaefaciens 10
Torulopsis glabrata 9  Torulopsis glabrata 9
Lipomyces lipofer 9  Lipomyces lipofer 9
Nematospora coryli 9  Nematospora coryli 9
Saccharomyces cerevisiae 9  Saccharomyces cerevisiae 9
Hansenula anomala 7  Hansenula anomala 7
Andere Pilze  Other mushrooms
Ustilago zeae 12  Ustilago zeae 12
Aspergillus niger 11  Aspergillus niger 11
Mucor hiemalis 10  Mucor hiemalis 10
Pythium debaryanum 10  Pythium debaryanum 10
Sclerotinia sclerotiorum 10  Sclerotinia sclerotiorum 10
Botrytis cinerea 8  Botrytis cinerea 8
C. Isolierung von Archazolid C. Isolation of Archazolid
Aus einem 100 1 Fermentationsansatz des Stammes Ar 3548 in Gegenwart von 1 1 Adsorberharz (Amberlite XAD 16) wird das Harz abgesiebt und mit 5 1 Methanol in einem Zeitraum von 2,5 Stunden eluiert. Das Eluat engt man bis zur Wasserphase ein und extrahiert 3 mal mit Ethylacetat, um polare Bestandteile abzutrennen. Der Ethylacetatextrakt wird eingeengt. Der verbleibende Rohextrakt (12 g) wird in 200 ml Methanol gelöst und in 2 Portionen über eine Sephadex-LH20-Säule (62 mm x 63 mm) bei einem Fluß von 3,5 ml/min fraktioniert. Laufmittel Methanol; Detektion bei 227 nm und durch DC-Chromatographie. Die Archazolid enthaltenden Fraktionen (4,1 g) werden in 10 0,41 g Portionen über eine präparative Nucleosil C-18, 7 μm-Säule (250 x 16 mm) mit dem Laufmittel Methanol/Wasser 85/15 und einem Fluß von 15 ml/min (Detektion bei 227 nm) chromatographiert. Nach Eindampfen der vereinigten Archazolid enthaltenen Fraktionen bis zur  The resin is sieved from a 100 l fermentation batch of the Ar 3548 strain in the presence of 1 l adsorber resin (Amberlite XAD 16) and eluted with 5 l methanol over a period of 2.5 hours. The eluate is concentrated to the water phase and extracted 3 times with ethyl acetate in order to separate polar constituents. The ethyl acetate extract is concentrated. The remaining crude extract (12 g) is dissolved in 200 ml of methanol and fractionated in 2 portions over a Sephadex-LH20 column (62 mm x 63 mm) at a flow of 3.5 ml / min. Eluent methanol; Detection at 227 nm and by TLC chromatography. The fractions containing archazolide (4.1 g) are in 10 0.41 g portions over a preparative Nucleosil C-18, 7 μm column (250 x 16 mm) with the mobile solvent methanol / water 85/15 and a flow of 15 ml / min (detection at 227 nm) chromatographed. After evaporation of the fractions containing Archazolid until
Wasserphase wird gefriergetrocknet. Man erhält 100 mg Archazolid als amorphe Substanz. Water phase is freeze-dried. 100 mg of archazolide are obtained as an amorphous substance.
D. Charakterisierung von Archazolid D. Characterization of Archazolid
Farblose amorphe Substanz UV (Methanol): λ max(lge) = 228 nm (4,6) Colorless amorphous substance UV (methanol): λ max (lge) = 228 nm (4.6)
El-MS (70 eV): m/z - 738 [M]H+ El-MS (70 eV): m / z - 738 [M] H +
Hochauflösung High resolution
ber.: 738,4278 für C42H62N2SO7 calc .: 738.4278 for C 42 H 62 N 2 SO 7
gef.: 738,4239  found: 738.4239
1H-NMR s. Tab. 1 1H NMR s. Tab. 1
13C-NMR s. Tab. 2 1 3 C-NMR see. Tab. 2
DC: (DC-Alufolie 60 F-254, Merck; Laufmittel Dichlormethan / Methanol 95:5) Rf = 0,56 TLC: (TLC aluminum foil 60 F-254, Merck; mobile solvent dichloromethane / methanol 95: 5) R f = 0.56
Detektion: UV-Absorption oder Ansprühen mit Vanillin  Detection: UV absorption or spraying with vanillin
/Schwefelsäure-Reagenz und Erhitzen auf 120 ºC (violetter / Sulfuric acid reagent and heating to 120 ºC (purple
Fleck). Spot).
Analytische HPCL: Laufmittel Methanol/Wasser 8:2, Säule: Nucleosil C-18 10 μm (250 x 4 mm), Fluß: 1,5 ml/min, Detektion: 227 nm; Retentionszeit Rt = 12,0 min. Analytical HPCL: eluent methanol / water 8: 2, column: Nucleosil C-18 10 μm (250 x 4 mm), flow: 1.5 ml / min, detection: 227 nm; Retention time R t = 12.0 min.
1H- und 13C- NMR Daten von Archazolid In CD3OD 1 H and 13 C NMR data from Archazolid In CD 3 OD
Figure imgf000011_0001
Figure imgf000011_0001
*) vertauschbar  *) interchangeable

Claims

P A T E N T A N S P R Ü C H E PATENT CLAIMS
1. Archazolide der folgenden allgemeinen Formel: 1. Archazolides of the following general formula:
Figure imgf000012_0001
Figure imgf000012_0001
mit den folgenden Bedeutungen: with the following meanings:
(a) 7-OX und 15-OX sind OH  (a) 7-OX and 15-OX are OH
NYCH3 ist NHCH3 oder NYCH 3 is NHCH 3 or
(b) 7-OX und 15-OX sind OAcyl (C1 bis C6, Benzoyl) NYCH3 ist NHCH3 oder (b) 7-OX and 15-OX are OAcyl (C 1 to C 6 , benzoyl) NYCH 3 is NHCH 3 or
(c) 7-OX und 15-OX sind OCH3 (c) 7-OX and 15-OX are OCH 3
NYCH3 ist N(CH3)2 NYCH 3 is N (CH 3 ) 2
2. Archazolid der Summenformel C42H62N2SO7 mit folgenden Parametern: 2. Archazolid of the empirical formula C 42 H 62 N 2 SO 7 with the following parameters:
UV (Methanol): lambda max (log epsilon) = 228 nm (4,6) UV (methanol): lambda max (log epsilon) = 228 nm (4.6)
Massenspektrum (EI, 70 eV): m/z = 738 [M] H+ Mass spectrum (EI, 70 eV): m / z = 738 [M] H +
Dünnschichtchromatographie (DC-Alufolie 60F-254, Thin layer chromatography (DC aluminum foil 60F-254,
Laufmittel Dichlormethan/Methanol 95:5): Rf = 0,56 Mobile phase dichloromethane / methanol 95: 5): R f = 0.56
Analytische HP-Flüssigchromatographie (Nucleosil C-18 10μm, 250x4 mm, Laufmittel Methanol /Wasser 8:2, 1,5 ml Fluß/min) : Rt = 12,0 min Analytical HP liquid chromatography (Nucleosil C-18 10μm, 250x4 mm, mobile solvent methanol / water 8: 2, 1.5 ml flow / min): R t = 12.0 min
3. Archazolide, dadurch herstellbar, daß man das Archazolid gemäß Anspruch 2 durch UV-Bestrahlung isomerisiert und gegebenenfalls das Isomerengemisch in an sich bekannter Weise chromatographisch auftrennt. 3. Archazolides, which can be prepared by isomerizing the archazolide according to claim 2 by UV radiation and optionally separating the isomer mixture by chromatography in a manner known per se.
4. Verfahren zur Herstellung des Archazolid gemäß Anspruch 2, dadurch gekennzeichnet, daß man 4. A process for the preparation of the archazolide according to claim 2, characterized in that
- Archangium gephyra DSM 6806 in einem Kohlenstoff- und Stickstoffquellen sowie Mineralsalze enthaltenden Medium kultiviert und das Archazolid produzieren läßt,  - Archangium gephyra DSM 6806 is cultivated in a medium containing carbon and nitrogen sources and mineral salts and has the archazolide produced,
- das gebildete Archazolid an einem Adsorberharz adsorbiert, adsorbs the archazolide formed on an adsorber resin,
- das Adsorberharz vom Kulturmedium abtrennt, separates the adsorber resin from the culture medium,
- danach das Archazolid vom Adsorberharz mit einem Elutionsmittel freisetzt und gefriertrocknet.  - Then the archazolide is released from the adsorber resin with an eluent and freeze-dried.
5. Verfahren nach Anspruch 4, dadurch gekennzeichnet, daß man5. The method according to claim 4, characterized in that one
- an Amberlite XAD16 adsorbiert, - adsorbed on Amberlite XAD16,
- mit Methanol eluiert,  - eluted with methanol,
- das Eluat bis zur Wasserphase einengt,  - the eluate is concentrated to the water phase,
- mit Ethylacetat extrahiert,  - extracted with ethyl acetate,
- den Ethylacetat-Extrakt einengt,  - concentrates the ethyl acetate extract,
- in Methanol aufnimmt, - an einer Sephadex-Säule (LH20; Laufmittel: Methanol) fraktioniert, - takes up in methanol, - fractionated on a Sephadex column (LH20; eluent: methanol),
- an einer Umkehrphase-Säule (Nucleosil C-18; Laufmittel: Methanol/Wasser) chromatographiert und  - Chromatographed on a reverse phase column (Nucleosil C-18; eluent: methanol / water) and
- einengt und gefriertrocknet  - concentrated and freeze-dried
6. Verfahren zur Herstellung von Archazoliden gemäß Anspruch 1, dadurch gekennzeichnet, daß man 6. A process for the preparation of archazolides according to claim 1, characterized in that
(b') bei einem Archazolid gemäß Anspruch 2 oder 3 7-OH und 15- acyliert und Archazolide gemäß Anspruch 1 (b) erhält oder (b ') in an archazolide according to claim 2 or 3, 7-OH and 15-acylated and archazolides according to claim 1 (b) is obtained or
(b') bei einem Archazolid gemäß Anspruch 2 oder 3 7-OH, 15-OH sowie NHCH3 zu 7-OCH3, 15-OCH3 und N(CH3 )2 methyliert und Archazolide gemäß Anspruch 1 (c) erhält. (b ') methylated in an archazolide according to claim 2 or 3 7-OH, 15-OH and NHCH 3 to 7-OCH 3 , 15-OCH 3 and N (CH 3 ) 2 and archazolides according to claim 1 (c).
7. Mittel, dadurch gekennzeichnet, daß es aus einem oder aus mehreren Archazoliden gemäß einem der Ansprüche 1 bis 3 besteht oder diese neben üblichen Hilfsmitteln, insbesondere Trägern und Verdünnungsmitteln, umfaßt. 7. Agent, characterized in that it consists of one or more archazolides according to any one of claims 1 to 3 or these in addition to conventional auxiliaries, in particular carriers and diluents.
PCT/EP1992/002980 1991-12-24 1992-12-22 Archazolides, method of preparing them and agents containing them WO1993013094A1 (en)

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WO1998013375A1 (en) * 1996-09-23 1998-04-02 GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) Compounds with antimycotic and cytostatic effect, preparation method, agent containing these compounds and dsm 11 092

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998013375A1 (en) * 1996-09-23 1998-04-02 GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) Compounds with antimycotic and cytostatic effect, preparation method, agent containing these compounds and dsm 11 092
DE19638870B4 (en) * 1996-09-23 2009-05-14 Helmholtz-Zentrum für Infektionsforschung GmbH Tubulysins, methods for their production and agents containing them

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