DE2022452A1 - Process for the manufacture of a new antibiotic - Google Patents

Description

Sandoz AG

Basel Case 100-3072

Process for the preparation of a new antibiotic 4 |

The present invention relates to the neje antibiotic ll ^ Desaeetoxy-wortmannin (Formula I, see ^ ormelblatt), Process for the preparation of ll-deacetoxy-wortmannin and pharmaceutical preparations thereof.

According to the invention one arrives at 11-deacetoxy-wortmannin, by either

a) Δ ° ^ '-8,9-dihydro-ll-desacetoxy-wortmannin (formula II} isomerized or

10. b) a new strain of the fungal species Penicillium funicu » losum Thorn or the mushroom species Aspergillus janus Raper and Thorn grows on or in a nutrient medium and thereon 11-deacetoxy-wortmannin from the fermentation broth in a manner known per se, e.g. by extraction or Adsorption, isolates and purifies.

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After a) the isomerization is carried out in an anhydrous organic Base carried out. Basically all are organic Bases can be used, but organic nitrogen bases such as pyridine or quinoline are preferred. Since the inventive Isomerization proceeds faster at elevated temperature, it is, for example, at the boiling point the organic base carried out.

One embodiment of the process consists in dissolving £ i ^ -8,9-dihydro-ll ~ .desaceto: ^ - wortmannin in pyridine and refluxing it under nitrogen. The resulting 11-deacetoxy-wortmannin is purified after evaporation by recrystallization in a manner known per se.

The δ ° ^ '-8,9-dihydro-lldesacetoxy-wortmannin used as the starting product can be produced by reacting wortmannin (formula III) with zinc dust in an organic Solvent in the presence of an acid but in the absence of water, for example at the boiling point of the organic solvent. The starting compound will after filtering off the zinc dust and evaporating the reaction mixture purified by recrystallization and / or chromatography in a manner known per se.

According to b) one arrives at 11-deacetoxy-wortmannin by as starting material either a new strain of the PiIz-

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species Penicillium funiculosum Thorn or a new strain the mushroom species Aspergillus Janus Raper and Thorn are used.

The new strain S 3196 from Penicillium used according to the invention funiculosum Thorn was isolated from a soil sample found in Clarksburg, Maryland (USA) and one sample of which at the United States Department of Agriculture (Northern Utilization Research and Development Division), Peoria, 111., USA, deposited under number NRRL 3563. The new tribe the fungus species Peniciilium funiculosum Thorn grows up a malt yeast extract agar at 18-27 ° and forms a dense green conidia lawn with bright yellow conidia carriers. The back of the colony shows a greenish yellow Discoloration of the substrate.

The strain corresponds in its morphology and physiology to the description of C. Thorn, USDept.Agr.Bur.Anim.Ind.Bul. 118 , p. 69, fig. 27 (1910) and is described and illustrated in detail in the manual KB Raper and Ch. Thorn, "A Manual of the Penicillia", The Williams and Wilkins Co. 1949, Baltimore, on pages 616 to 620.

The new strain S 8033 / P of Aspergillus janus used according to the invention was derived from one in the Central African Republic found soil sample isolated and a sample of it at the United States Department of Agriculture (Northern Utilization Research and Development Division), Peoria, 111.,.

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USA, deposited under number NRRL j? 8O7 and is there accessible.

The new strain of the mushroom species Aspergillus Janus is growing on a glucose-yeast extract-malt extract-peptone agar 18 to 53 ° C, but is preferably grown at 18 to 27 °. The fungus grows on this medium with a loose, spreading mycelium. The nutrient substrate and the back of the Colonies are discolored by a yellowish pigment. In its morphology and physiology the new one corresponds Strain of Aspergillus janus described in: K.B. Raper and D.J. Penneil, "The Genus Aspergillus", The Williams & Wilkins Comp., Baltimore, 1965, pages 476-480.

Strains can also be used for the method according to the invention as they are e.g. by selection or mutation under the action of ultraviolet or X-rays or by applying other measures, such as treating laboratory cultures with suitable chemicals, from the new strains of the fungal species Penicillium funiculosum Thorn and the fungal species Aspergillus janus Raper and Thorn can be won.

The new fungal strains of the fungal species Penicillium funiculosum Thorn and the mushroom species Aspergillus janus Raper and Thorn can be grown on a variety of nutrient media that use the usual nutrients

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contain substances, breed. So such tribes use the nutrients commonly used for carbon-heterotrophic organisms, e.g. glucose, starch, dextrin, Lactose, cane sugar, etc. as a carbon source, organic and inorganic, nitrogen-containing "compounds, such as peptone, Yeast or meat extracts, ammonium sulfate, ammonium nitrate, amino acids, etc. as a nitrogen source, as well as the usual ones. Mineral salts and trace elements.

11-Desacetoxy-wortmannin can be produced in the way that a liquid nutrient medium can be mixed directly with a spore suspension or with a preculture of a new strain of Penicillium produced using a spore suspension funiculosum Thorn or inoculated by Aspergillus janus Raper and Thorn. In the case of the strain of Penicillium funiculosum Thorn is the culture 5 to 10 days at 27 °, in the case of the Tribe of Aspergillus janus Raper and Thom / 5 to 4 days J incubated at 18 °. Cultivation can be carried out under aerobic conditions in a surface culture or submerged with shaking or in fermenters with aeration by air or oxygen take place with stirring. Once a maximum amount of antibiotic has been produced, v / lrd filtered and the antibiotic by extractive or adsorptive working methods obtained from the filtrate in a manner known per se.

A method that has been found to be preferable

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is the extraction of the filtrate with ethyl acetate, but other organic solvents, such as e.g. Benzene, chloroform, butyl acetate, methylene chloride or butanol can be used. Then · the extracts from Freed solvent, e.g. by distillation, and the residue to isolate the desired antibiotic chromatographic route on adsorbing agents such as clay, silica gel, magnesium silicate and the like or cleaned by means of countercurrent distribution.

11-Desacetoxy-Wortniannin is a valuable remedy.

In particular, "it has a very high fungistatic Effect on various pathogens causing fungal infections of human pathogenic or plant parasitic fungi. In vitro 11-Desacetoxy-Wortmannin is particularly effective against Histoplasma capsulatum, Candida krusei, Trichophyton tonsurans, Epidermophyton floccosum and Microsporum canis.

11-Desacetoxy-wortmannin also shows an anti-edema and anti-inflammatory activity, as shown in the carrageenan edema test, the carrageenan granuloma bag test and the CMC granuloma bag test is evident from the waking rat.

The dose to be administered naturally varies; depending on the Type of administration and the condition to be treated. in the in general, however, satisfactory results are obtained with a dose of 1 to 15 mg / kg body weight »This dose

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BADORONAL

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If necessary, it can be administered in 2 to 5 portions or as a sustained release form. For larger mammals, the daily dose is around 1 to 50 mg. For oral administration, a suitable form of administration contains 1 to 50 mg of the active substance, mixed with a liquid or solid carrier. For topical application, powders or sprays or ointments or tinctures that contain 0.1 to 2 # of the active substance can be used.

'■■■■: "' .: ■ - - - ■ '.'. ■■ i In the following examples, which explain the implementation of the process but are not intended to limit the scope of the invention in any way, all temperatures are given in degrees Celsius The melting or decomposition points are determined on the Kofler block.

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BADORFQINAL

100-5072

CH _x O-CH ₀

CH 0-CH ₂

II

OAc

CH ^ O-CH

III

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Example 1:

5 g of Δ '-8,9-dihydro-II-desacetoxy-wortmannin are in Dissolved 200 ml of pyridine and refluxed βθ hours under nitrogen. The reaction mixture is then evaporated and the residue was recrystallized twice from methanol. Pure 11-deacetoxy-wortmannin is obtained with a melting point. 178 - 180 °.

The zrj ^ -8,9-dihydro-lldesacetoxy-wortmannin used as starting material can be prepared as follows:

10 g of Wortmannin are dissolved in 1 liter of ethanol and after Add 10 g of zinc dust and 10 ml of glacial acetic acid and refluxed for 15 minutes. After cooling, the reaction mixture becomes filtered and evaporated. The residue is taken up in methylene chloride, filtered and evaporated again.

Subsequent recrystallization from methanol gives crude δ "^ '-8,9-dihydro-ll-desacetoxy-wortmannin. This is given to (J Chromatographed 26o g of silica gel. Chloroform / methanol (49: 1) is used as the eluent, with fractions of 500 ml. be caught. Fractions 3 to 8 are put together, the eluent is evaporated and the residue is recrystallized from methylene chloride with the addition of methanol. 202-206 °.

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BATH ORIGINAL

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Example 2t

150 liters of a nutrient solution are put into a fermenter per liter

20 g cerelose

2g malt extract

2 g yeast extract

2 g of KH ₂ PO ₄

Contains 2 g of MgSO ^ 7H ₂ O and 'demineralized water, inoculated with a spore suspension of the strain NRRL 3363 of Penicillium funiculosum Thorn and with aeration (150 L air / min.) And with stirring (100 rpm) 112 Incubated for hours at 27 °. The culture broth is filtered and the piltrate is extracted twice with 100 liters of ethyl acetate each time. The organic phase is washed once with 60 liters of water and concentrated to 5 liters in vacuo. The concentrate is dried over anhydrous magnesium sulfate and evaporated. The residue is partitioned three times between petroleum ether and methanol-water (9: 1). The methanol phase is concentrated and then extracted three times with ethyl acetate. The ethyl acetate solutions are washed once with water, dried over anhydrous magnesium sulfate and evaporated. The extract is chromatographed on a 50-fold amount of silica gel. With chloroform-methanol (99: 1) becomes 11-deacetoxy-wortmannin

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eluted, which melts after two recrystallization from methylene chloride-ether at 178-180 ^0.

example J:

In a fermenter 30 liters of a nutrient solution, which per Liter has the composition given in Example 2, with 3 liters of a preculture as described below of the strain NRRL 3807 of Aspergillus janus and under Ventilation (6-24 L air / min.) And stirring (150 rpm) Incubated for 90 hours at 18 °. The culture broth is filtered and the filtrate is extracted twice with 20 liters of ethyl acetate each time. The organic phase is washed once with 12 liters of water and concentrated in vacuo to 1 liter. The concentrate is over dried anhydrous magnesium sulfate and evaporated. The residue is washed three times between petroleum ether and methanol-water (9: 1) distributed. The methanol phase is concentrated and then

then extracted three times with ethyl acetate. The ethyl acetate solutions are washed once with water, dried over anhydrous magnesium sulfate and evaporated. The extract will chromatographed on 50 times the amount of silica gel. With ; Chloroform-methanol (99: 1) becomes 11-deacetoxy-wortmannin , eluted, which melts after two recrystallization from methylene chloride ether at 178-180 °.

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The preculture used is obtained as follows:

5 liters of the medium specified in Example 2 with additional 2 g of peptone per liter of solution are inoculated with a spore suspension of the strain NRRL 3807 and inoculated for 7 days at 27 ° Shaking incubated. ^

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Claims (8)

2,222,52 -13 - 100-3072 Patent claims: "_ 1. 11-Desacetoxy-wortmannin (Formula I, see Pormelblatt). .-- '"-—- 2. Process for the preparation of 11-deacetoxy-wortmannin .. -'- - ■ '■ " (Formula J, see Pormelblatt), characterized in that one either a) δ "V '-8,9-dihydro-II-desacetoxy-wortmannin (formula II) ^ r isomerized ™ or b) a new strain of the fungal species Penicillium funiculosum Thorn or the fungal species Aspergillus janus Raper and Thorn grows on or in a nutrient medium and on it 11-Desacetoxy-wortmannin is isolated from the nutrient solution and purified by methods known per se. 3. The method according to claim 2, characterized in that g the strain NRRL 3363 of the mushroom species is used as the new strain Penicillium funiculosum Thorn used. 4. The method according to claim 2, characterized in that the strain NRRL 3807 of the mushroom species is used as the new strain Aspergillus janus Raper and Thorn used. 5. The method according to claim 2, characterized in that the isomerization in an anhydrous organic base 009851/2272 - 14 - 100-5072 performs. 6. 'A process according to Ansprueh 5> characterized in that one uses efne ~ cigährsche nitrogen base. 7. The method according to claim 6, characterized by the use of pyridine. 8. The method according to claims 5 to 7> characterized in that the isomerization is carried out at an elevated temperature, for example the boiling point of the organic base. 9 «Medicines containing ll-D & sacetoxy-v / ortmannin. SANDOZ AG. 009851/2272