NO762267L - - Google Patents

Description

"Procedure for the preparation of a new organic compound."

The present invention relates to the preparation of the compound S 31794/F-1.

The distinctive feature of the method in Haibold's invention is that an S 31794/F-l producing strain of the mushroom species Acrophialophora limonispora nov. spee Dreyfuss + Muller is grown in the presence of a nutrient medium.

The compound S 31794/F-l shows the following properties:

A white amorphous powder which can be crystallized as indicated in example 3. Unless otherwise indicated, the following numerical indications refer to the amorphous as well as the crystalline form.

Melting point 178 - 18o8c (cleavage) (amorphous)

Melting point 181 - 183°C (decomposition) (crystalline)

[o^° = - 24° (c = 0.5 in methanol)

[a^<0>= + 37° (c =0.5 in pyridine) (crystalline)

In the UV spectrum in methanol (fig. 1) the absorption maxima appear at ^max 194nm'?<]*>= 807'- Xmax225nm (shoulder),

Eicm<=>132<")l maxx275™'EJcm= 12'8'jl max 284nra 'atalte)'

Elom=10'5-

IR spectrum in KBr (amorphous) (Fig. 2).

IR spectrum in Nujol (crystalline) (Fig. 3).

''"H-NMR spectrum (100 MHz in DMSO) with tetramethyl silane as internal standard (Fig. 4).

<13>C-NMR spectrum in D^-methanol (190 mg in 1.5 ml of D^-methanol)

with tetramethylsilane as internal standard, see Table 1 (crystalline).

For the analysis, the amorphous substance was dried at 20°C in high vacuum. The elemental analysis gave the following limit values: C 53.9% H 7.5% N 10.1% 0, 27.3%

For the analysis, the crystalline substance was dried in high vacuum for 2 hours at 100°C. The elemental analysis gave the following limit values: Found C 55.5 - 56.5% H 7.5 - 7.7% N 10.5 - 10.8%

O 25.5 -226.0%.

The compound is easily soluble in methanol, ethanol, pyridine, dimethyl sulphoxide, sparingly soluble in water, chloroform, ethyl acetate, ether, benzene, hexane.

The compound is stable in aqueous methanol at pH 3 - 7.5

but in the alkaline or strongly acidic pH range, cleavage begins during the loss of the antifungal activity.

During acid hydrolysis (6N HC1/110°C), myristic acid was found as a breakdown product, which was identified in the form of the methyl ester.

In thin-layer chromatograms on silica gel plates merck, 0.25 mm layer thickness, S 31794/F-l shows the following Rf values in the indicated fluids:

(comparison width for uracil in parentheses)

The spots can be made visible with iodine or with the help of

a suitable spraying reagent.

The following spraying reagents have proven suitable:

The plate, which has been completely freed of solvent, is pre-sprayed with a mixture of 0.5 glacial acetic acid in ethanol acetone c(>1 : 1). It is then set for approx. 7 minutes in a chlorine atmosphere and excess Cl'2 is then blown away in a powerful. air flow. Then spray with a solution of 1 part 0.05 molar KJ solution in water + 4 parts of a 9.5% benzidine solution in 20% acetic acid. The antibiotic S 31794/F-l produces a blue spot during this development.

With ninhydrin, S 31794/F-l gives no color.

The procedure can be carried out according to known methods for fermentation extraction. A preferred culture of the S 31794/F-1 producing strain was deposited at the United States Department of Agriculture (Northern Utilization Research and Development Division), Peoria, 111., USA and is there available to the public under the designation NRRL 8095.

One can also use S 31794/F-l- producing strains, which can be extracted from the starting strain, of the fungal species of limonispora nov. spee. Dreyfuss + Muller by treatment with mutagenic substances or radiation or by selection.

Characteristics of the strain NRRL 8095

The new strain of the mushroom species Acrophialophora limonispora nov. spee. Dreyfuss + Muller used in the invention was isolated

from a in British Columbia, Canada collected soil sample.

The new strain NRRL 8095 was, due to its morphological characteristics, assigned to the hyphomycete genus Acrophialophora Edward, but could not be reconciled with the three hitherto described species of this genus (Samson and Tariq Mahmood, 1970: The Genus Acrophialophora Acta bot. Neerl . 19 (6) 804 - '808) so that the new strain NRRL 8095 is hereinafter designated as a new species, Acrophialophora limonispora nov.

spee.

The light to deep brown conodiophores of A. limonispora

NRRL 8095 is mostly 700 to 1800 yu long and 4 to 8 yu thick, septate; straight or slightly boyde, in the lower or middle part unbranched or a few times forked and may be sculptured with warts along the entire length. At the tips of the conidiophores, a symmetrical or asymmetrical, most often compact, penicillium-like branched system develops, with most frequently on the latter cells of the main conidiophore axis growing out one- to few-cell def1 ~ > primary side branches singly alternating, but most often opposite in pairs or grown in clusters of 3 (rarely 4).

These side branches in turn bear laterally and at the top more

secondary twigs which may analogously be tertiary branched.

At the top of the secondary or tertiary twigs sit two

to six bottle-shaped, 7 - 11 x 2.5 - 4 yu large phialides flaring at a tip. Particularly in older cultures, brush-like structures with greatly extended branching elements can be observed.

The hyaline conidia formed basipetally in chains that break easily are lemon-shaped when viewed from the side with small apiculi that appear darker, 3.2 - 4.2 x 3.0 - 3.5 yu in size and on the surface smooth to slightly warty sculpted.

The strain NRRL 8095 has a very broad growth optimum between 20°C and 27°C, as it forms approximately 35 to 40 mm large colonies on 2% malt extract agar in the course of seven days. The lower growth limit is at 4°C, the upper between 33°C and 34°C.

Colonies on 2% malt extract agar are first whitish-green and covered with porous, white aerial mycelium. With increasing age, the substrate mycelium is colored dark brown. Over the course of 5 - 10 days, the initially formed air mycelium is overlaid more and more strongly by the conidiophores formed in a thick mat. The conidial mats first appear golden yellow to yellowish green, later light yellow/strong to gray brown.

The new strain NRRL 8095 can be grown in or on suitably composed nutrient substrates in surface or submerged culture.

For the extraction of the new compound S 31794/F-1, submerged cultures are preferably used, as suitable nutrient substrates,

as described in examples 1 and 2, inoculated with a conidia or mycelial suspension of the new strain NRRL 8095 and inoculated at a pH value of 3 to 8, preferably 5 - 7.0 and a temperature of 15 - 30°C , preferably 18 - 27°C, with agitation shaking and/or aeration for 48 to 360 hours, preferred

120 to 288 hours.

The mycelial portion of the cultivation buUi ong is eventually swallowed up and

S 31794/F-l is recovered by extraction and/or adsorption methods in a manner known per se. However, one can also first centrifuge the mycelium from the culture broth, separately extract the culture filtrate and the mycelium after digestion and further process the extracts individually or collectively.

The composition of the culture broths used, which are obtained by cultivation of an S 31794/F-1-producing strain, appears in more detail in the following.

S 31794/F-l has interesting pharmacodynamic properties and can be used as medicine.

S 31794/F-l exhibits an antimicrobial effect, particularly against yeast and fungi. Against the usual representatives of gram-positive and gram-negative bacteria, no effect could be ascertained by means of known methods, but on the other hand, the effect against various human pathogenic !'cCandida strains is particularly pronounced. In a serial dilution experiment, which was carried out with a malt extract medium (2% malt extract), pH 5.2 0 5.4)

at an incubation temperature of 27°C and an incubation time of

48 to 72 hours, the following minimal inhibitory concentrations (MIC) were detected for S 31794/F-1.

The effect can also be demonstrated in vivo with the method of experimental sepsis infection in mice with candida albicans. The compound can therefore be used as an antimycotic. Daily dose is ■ 2 to 14 mg/kg s.c. respectively 100 to 300 mg/kg p.o. The daily dose for large mammals is 20 to 150 mg s.c. respectively

1 to 3 g p.o.

As medicine, S 31794/F-1 can be administered alone or in suitable preparation forms together with inorganic or organic, pharmacologically active excipients. For example, S 31794/F-1 is used as a component of an ointment, as the concentration

in the ointment can make up approx. 5 to 50 mg of active substance per gram of ointment.

The following examples shall illustrate the embodiment of the method according to the invention.

EXAMPLE 1 Pursuit_of_NRRL 8095

10 liters of a nutrient solution, which per literally demineralized

water contains 20 g glucose, 5 g casein peptone, 3 g NåNO^/

lg K 2 HPO 4 , 0/5 g KCl, 0.5 g MgSO 4 • 7H 2 O and 10 ml FeSO 4 . 7H20 was inoculated with 1 liter of a pre-culture of the strain NRRL 8095 and incubated in a 10 liter fermentation vessel under stirring (150 revolutions per minute) and aeration (1 liter of air/min./liter of nutrient solution) for 4 days at 18° C.

The pre-culture used as starting material is obtained in the following way: The spore and mycelial suspension used for inoculation is prepared from a culture of the strain NRRL 8095, which was grown for 10 days at 27°C on agar medium, which per liter of demineralized water contains 20 g malt extract, 20 g agar and 4 g yeast extract.

The spores and mycelium of this culture are taken up in a physiological saline solution. With this suspension, 1 liter of a nutrient solution is inoculated, which per liter of demineralized water contains 20 g of malt extract and 4 g of yeast extract, and is incubated in a 2 liter Erlenmeyer flask on a rotary shaker (.180 revolutions per minute) for 2 days at 27 oC. This cultivation broth serves

as seed material for the 10 liter glass fermentation vessel.

EXAMPLE 2 ?Y£]5ing_of_NRRL 8095_

For extraction of S 31794/F-l, a steel fermentation vessel is inoculated with

50 liters of medium, consisting of 200 g sucrose, 10 g soy egg white, 10 g yeast extract, 10 g malt extract, 2 g KE^PO^

2 g Mg SO^ • 7 H^ O to one liter of demineralized water, with 10

liters of secondary pre-culture. The fermentation took place for 12 days at 24°C

in a pH range of 5 to 7, an air supply of 0.8 to 1.0 liters of air/min./liter of medium and an increasing speed of 150 - 400 revolutions per minute.

The pre-culture used as starting material is produced in the following way:

The incubation of the strain NRRL 8095 in slanted agar tubes for the extraction of conidia took place over the course of 19 days at 27°C on a medium composed as follows: 100 g sorbitol (hexavalent alcohol, hexite), 10 g tryptone (peptone from casein , digested with thyrypsin), 250 mg KH2P04, 250 mg Mg S047H2°/250 mg Ca (N03)2• 4 H20, 125 mg K Cl, 16 mg Fe S047 H20, 7 mg Zn S047 H20, 15 g Agar to one liter demineralized water. To prepare a primary pre-culture, 1 liter of pre-culture medium, consisting of 20 g of malt extract and 4 g of yeast extract to 1 liter of demineralised water, is inoculated into a 2 liter Erlenmeyer flask with 8-9 10 9 conidia in 10 ml of 0.9% NaCl solution and incubated for 4 days at 24°C on a rotary shaker with 180 revolutions per minute.

To prepare the secondary pre-culture, 2 liters are incubated

of a primary pre-culture in a glass fermentation vessel with 10 liters of pre-culture medium and incubated for 4 days at 24°, 150 revolutions per minute and an air supply of 0.5 litres/litre medium/min.

EXAMPLE 3 ^solering_av_S 31794/F-l

90 liters of animal broth obtained according to examples 1 and 2 are added to 90 liters of a mixture of ethyl acetate and isopropanol (4:1) and homogenized at room temperature for 30 minutes. with an ultra-fast rudder device (ultraturrax). The organic phase is now separated with a separator and the extract is evaporated in vacuum at a maximum of 40°C. The extract residue is chromatographed on a tenfold amount of silica gel (Merck, 0.06 - 0.2 mm). For this, the crude extract is created on the 1-2 double amount of silica gel (Merck. 0.2 - 0.5 mm), adding the solution of the residue in as little methanol or methanol-water mixture as possible to the silica gel, and the solvent is then removed again under vacuum. The silica gel thus impregnated is suspended in chloroform-methanol (95:5) and the thick slurry is applied to the chromatography column. The elution begins with chloroform-methanol (95:5) and is then continued by doubling the methanol proportion to 40% in the individual case. The chromatogram fraction with antifungal activity against Candida albicans and metabolites in the thin layer chromatogram area of

the area of S 31794/F-l is united.

The fractions thus obtained are chromatographed on the 100-fold amount of sephadex LH-20 with methanol and the fractions are combined again corresponding to the antifungal activity and due to the thin-layer chromatographic result. The now highly enriched antibiotic is chromatographed again on the 100-fold amount, silica gel (Merck: 0.05 - 0.2 mm), whereby chloroform-methanol-water (71 : 25 : 4) is used for elution. The fractional residues are examined thin-layer chromatographically for the content of antibiotic S 31794/F-1 and the pure portions are combined. The substance is dissolved in a little methanol and poured by adding ether, whereby S 31794-1 is obtained in the form of a white, amorphous powder. After drying in high vacuum at 25 - 30°C, amoroft S 31794/F-l melts at 178 - 18o8c, (decomposition). The amorphous substance is crystallized from the 10-fold amount of the mixture ethyl acetate (methanol-water (80 : 12 ; 8) whereby thread-shaped crystals of the antibiotic S 31794/F-l are obtained. Melting point 181 - 183°C (cleavage) after drying in high vacuum for 20 °C.

Claims (4)

1. Process for the production of S 31794/F-l, characterized by the atenS 31794/F-l producing strain of the mushroom species acrophialophora limonispora nov. spee. Dreyfuss + Muller is grown on or in a nutrient medium and then S 31794/F-1 is isolated from the growth broth and purified in per se known ways. 2. Method as stated in claim 1, characterized in that as a new strain of the mushroom species Acrophialophora limonispora nov. spee. Dreyfuss + Muller used the strain NRRL 8095. 3. Method as stated in claim 2, characterized in that a strain is used which is extracted from the starting strain of the mushroom species Acrophialophora limonispora nov. spee. Dreyfuss + Muller by treatment, with mutagenic substances éLLec rays or by selection. 4. Cultivation broth, characterized in that it has been obtained by cultivating an S 31794/F-l producing strain of the mushroom species acrophialophora limonispora nov. spee. Dreyfuss + Muller.