HU182776B - Process for preparing new metabolytes - Google Patents

Abstract

The present invention relates to a process for the preparation of new antibiotics S-41062 / F-1, S41062 / F-6 and S-41062 / F-7. According to the method, a new fungal strain belonging to the Cryptosporiopsis genus was grown on a suitable medium. The novel compounds have a significant antibiotic effect, especially against Candida infections. -1-

Description

The present invention relates to a process for the preparation of compound 8 41062 / F-1, S 41062 / F-6 and S 41062 / F-7.

The process according to the invention is described in S 41062 / F-1. S 41062 / F-6 and / or S 41062 / F-7 are prepared as follows. and culturing a strain producing S 41062 / F-1 and / or S 41062 / F-6 and / or S 41062 / F-7, such as 1 strain of Cryptosporiopsis fungagenus, in the presence of medium.

S 41062 / F-1, S 41062 / F-6 and S 41062 / F-7 have approximately the following properties.

S 41062 / F-1

- white microcrystalline powder

- o.p. 235-240 ° 0 / gradual decomposition, especially above 265 ° C /

- / alpha7p0 = -6 ° / c = 0.5 in methanol /

- UV spectrum in methanol _of 193 nm, lambda ^, El% = 786 max cm χ / 222 / nm "= 140

279 nm '= 14

IR spectrum in KBr: Figure 1;

- 1 H-HMR spectrum (90 mHz in DMSO / tetramethylsilane as internal standard): Figure 4;

? -N-RI spectrum in Dn-methanol / 36 mg in 0.3 mL / tetramethylsilane as internal standard: 1. column A of Table 2;

- in situ desorption mass spectrum peaks: M / + li / ⁺ 1055 and

M / Na + / ⁺ 1071, corresponding to θ ο ^ ^ ^ δοΑδ Ιβ ^em P ^.mu.l -Kus formula, molecular weight 1048;

- analysis after drying at room temperature under high vacuum / θ ^ οΗβθΝβΟ ^ θ /:

57.5 Η 7.7 N 10.7 0 24.4% found:

C 55.7 H 7.7 N 10.1 0 24.7% σ 56.5 H 7.8 N 10.5 0.24.1%

- solubility: freely soluble in methanol, ethanol, pyridine, dimethylsulfoxide (DMSO); poorly soluble in water, chloroform, ethyl acetate, ether, benzene, hexane;

the compound is stable in aqueous methanol at pH 4-7. It decomposes under alkaline or strongly acidic conditions and loses its antifungal activity;

acidic hydrolysis / 6N HCl * 110 ° C, 24 hours / yields a decomposition product of a saturated fatty acid which shows a mass spectrum peak at M * 270 in methyl ester form.

- Amino acid analysis (according to Stein and Moore). Decomposition products having the same or similar retention times, e.g. ab aspartic acid, hydroxyproline, serine, and ammonia; and an unknown amino acid having a retention time between leucine and tyrosine.

S 41062 / F-1 is a neutral cyclic hexapeptide containing a branched, amide-linked, saturated fatty acid moiety.

Chemical assays (amino acid analysis, etherification) and spectral data (IFI, 15 C-NMR) suggest that the following 2182 766 amino acids are present: serine, 4-hydroxyproline, 4-methyl-3-hydroxy -proline, 3-hydroxy-homothyrosine and most likely

4,5-hihidroxi-ornithine; the sixth unknown amino acid unit contains an HpNCO group and is believed to contain a secondary hydroxyl group. S 41062 / F-1 can be represented by structural formula I wherein R is a branched C 16 fatty acid group.

Thin-layer chromatography (Table 1).

Antifungal activity: Table 5.

S 41062 / F-6

Based on the acid decomposition products, S 41062 / F-6 is a mixture of at least 3 components.

white microcrystalline powder;

- o.p. 240-244 ° C (decomposition gradually);

- [alpha] 7 [deg.] C = -9 [deg.] C. = 0.5 in methanol;

P

UV spectrum in methanol ^{lamb <y} max ^{193 E} ™ l5 _m / 222 /

278 nm nm = 861 = 340 = 15

IR spectrum KBr-bans Figure 1 a;

@ 1 H-NMR spectrum / 90 mHz in DMSO / tetramethyl azilane as internal standard: 1. Figure 5;

1 H-NMR spectrum in D-methanol / 44 mg in 0.3 mL / tetramethylsilane as internal standard: 1. column B of Table 2;

- analysis after drying at room temperature under high vacuum:

t Kill á7 *

G 56.3 * H 7.7 N 10.9 o 26.0%

C 56.8 II 7.9 N 10.7 o 24.8%

- solubility: easily soluble in methanol, ethanol, pyridine, dimethylsulfoxide (DL1SO); poorly soluble in water, chloroform, ethyl acetate, ether, benzene and hexane;

the compound is stable in aqueous methanol at pH 4-7. It decomposes under alkaline or strongly acidic conditions with a decrease in antifungal activity;

acidic hydrolysis / 6N IIC1 _? 110 ° G, 24 h / decomposition product, yields a saturated fatty acid which exhibits a mass spectrum peak at M + 270 in the methyl ester form;

amino acid analysis (Stein and Moore method): Degradation products having the same or similar retention times e.g. aspartic acid, hydroxyproline, azeroline and ammonia, and an unknown amino acid having a retention time between leucine and tyrosine; thin-layer chromogram: Table 1;

antifungal activity: Table 5.

41062 / F-7

- White powder;

- 0 _x p. From 2 ° C to 5 ° C (shrinks with gradual decomposition);

- [alpha] 7 = -6 [deg.] C. (c = 0.5 in methanol);

- UV spectrum in methanol lambda "" _v max 194 nm, '/ 222 / nm 278 nm

1 'cm = 890 = 559 «15

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IR spectrum in KBr: 1. Figure 3;

J-II-IILR spectrum (90 ml / DMZ / tetramethylsilane as internal standard): Figure 6;

Spectrum in DMSO-methanol / 38 mg in 0.25 ml / tetramethylsilane as% 1 standard: 1. Column C of Table 2;

- analysis after drying at room temperature under high vacuum: found:

C 54.5 H 7.5 H 9.9 0 25.0%

C 55.1 H 7.5 H 10.2%

A sample was dissolved in ethyl acetate / isopropanol, extracted with very dilute hydrochloric acid, evaporated and precipitated from methanol with acetone. The analysis then gives the following results:

found:

G 57.4 H 8.0 N 11.0 0 24.6%

C 56.0 H 7.9 N 10.3 0 24.8%

- Solubility: freely soluble in methanol, ethanol, pyridine, dimethylsulfoxide (DMSO); poorly soluble in water, chloroform; ethyl acetate, ether, benzene, hexane;

- acid hydrolysis / 6N 1101, 110 ° C, 24 hours / decomposition product provided a saturated fatty acid which can be considered as dinethyl ryristic acid based on gas chromatographic retention times and mass spectroscopic peaks.

- Amino acid analysis: Degradation products are obtained which have the same or similar retention times eg. aspartic acid, hydroxyproline / allo-hydroxyproline, serine and ammonia; and an additional amino acid having a retention time between leucine and tlrozine.

TLC: Table 1.

Antifungal Activity: Table 5.

Table 1

Thin Layer Food Chromatography on Silica Gel Plates / Merck; 0.25 mm film thickness / provides the following Rf results:

Eluent methylene chloride id methanol

41062 / F1 0.25 S 41062 / F6 0.20 341062 / F7 0.40 Echinocandin B / Reference substance / 0.28 methylene chloride: methanol: water 71: 25: 4 0.32 0.31 0.42 0, 35 appropriate reagents

The substance may be used in the presence of iodine vapor or mannose spray.

Suitable sprayable reagents may be prepared, e.g. by dissolving 0 mg of ferric chloride in 1 ml of ethyl acetate and adding 100 ml of nitrate-free concentrated sulfuric acid.

A completely solvent-free thin film is sprayed with an reagent and heated to a white glow temperature until strongly brown spots appear.

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Table 2 13 'XJ-HMR spectrum / instrument: Bruker HX-90-E; registration 22.63 muz-nj sweeping width: 6000 Hz /

S 41062 / P-1 41062 / P-6 . S 41062 / P- ' delta / ppm / delta / ppm / delta / ppm / 176.9 176.9 176.1 176.0 176.0 174.0 174.0 174.0 173.5 173.5 173.7 172.8 172.7 172.6 171.8 171.8 172.0 169.0 169.0 169.1 157.0 157.0 156.9 131.4 131.4 131.4 129.7 129.7 129.7 116.2 116.1 116.1 76.2 81.5 ' '73, 9 74.3 76.2 71.2 71.2 74.3 70.6 70.6 71.2 69.7 69.9 70.5 63.6 69.5 69.9 62.3 63.6 63.6 57.4 62.4 62.3 56.1 57.3 57.3 55.4 56.0 56.1 50.8 55.4 55.4 49.8 '51.8 53.0 48.9 50.8 51.8 48.0 49.9 50.8 47.1 48.9 49.9 45.8 48.0 48.9 40.7 47.1 48.0 39.0 45.8 47.1 38.0 40.6 46.1 36.7 39.0 45.8 34.5 38.0 40.7 32.8 36.7 .59,0 31.1 32.8 38.0 30.7 31.1 36.7 30.5 30.6 35.2 30.4 30.3 32.8 30.2 27.9 31.1 27.9 26.9 30.5 26.6 20.7 30.2 20.7 20.1 27.9 20.2 11.2 26.9 11.6 20.7 20.1 11.6 11.2 1.1

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The process of the invention can be carried out by known methods. A sub-culture of the S 41062 / F-1, S 41062 / F-6 and / or S 41062 / F-7 producing strain was deposited on January 2, 1980 at the American Type Culture Collectlon / ATCC, Rockville, Maryland. USA / and it is available to the public under accession number ATCC 20594. The culture is also available from Sandoz Ltd, Basel, Switzerland.

An additional sub-culture was deposited on June 16, 1980, with the US Department of Agriculture (Northern Utilization Research and Development Division, Peoria, Illinois, USA) and is available to the public under accession number NRRL 12192.

However, production strains S 41062 / F-1, S 41062 / F-6 and / or S 41062 / F-7, which may be derived from the parent strain ATCC 20594 or NRRL 12192, may also be used, e.g. irradiation, treatment with conventional mutagenic substances or selection.

Characteristics of ATCC 20594 / and NRRL 12192 /

This strain was isolated from the root of a plant near Soyhieres (Switzerland); the strain forms dense open conidia / acervuli in pure culture. Based on this characteristic, the strain deposited under ATCC 20594 and NRRL 12192 is clearly classified as Melanconiaceae. Based on its microscopic characteristics and B.C. Applying Sutton Identification Key / Alnsvíorth, Sparrow and Sussmannj The Fungi, vol.

IV.a., Academic Press 1973, Chapter 11, p. ^555/9 fungus considered as belonging to the genus Cryptosporiopsis BUB and Kab. With the exception of some indeterminate features, this classification is in good agreement with JA von Arx / Review of fungi classified as Gloeosporium, Bibliotheka Mycologica, vol. 24., J. Cramer, 1970, p. 5 ·, θ · »27-29 · / · Considering the fact that no fruit bodies were observed on the original substrate and that no fungi perfecti condition was observed so far, and the fact that the systemically relevant culture descriptions are very rare in the literature / JW Groves, 1939 * Some Pezicula species and their conidial phases, Can. J. Research 17, 125-145; J · W. Groves, 1939. Some Pezicula species turning on the sinus βίο, Mycologia 52, 112-125; JW · Groves, 1941: Pezicula cafnea and Pezicula subcarnea, Mycologia 53, 510-522; JW Groves,

1947: Pezicula Morthieri rhamnuson, Mycologia 59, 528-353 /, currently no more accurate classification can be made.

The Cryptosporlopais ATCC 20594 strain S 41062-F-1, S 41062 / F-6 and / or S 41062 / F-7 has the following physiological and morphological characteristics. Good growth is observed on agar medium, preferably at lower temperatures. The minimum and maximum growth temperatures are 0 ° C and 10 ° C, respectively. 31-33 ° C, while optimum growth is 15-24 ° C.

Table 5 shows the growth characteristics as a function of the incubation temperature and the medium. Colon diameters (average 6 colonies / mm) after incubation for 14 days in punctured inoculated Petri dishes; these diameters serve as a measure of growth rate.

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Table 3

Column diameter, mm, at next incubation temperature, ° C broth TODAY ° 2 0-1 + + 4-5 15 3 12 - 13 20 7 14-15 26 20 18 - 19 53 35 21 - 22 33 59 24 - 25 25 38 26.5-27.5 10 16 30-32 + + 32-35 Θ Θ

MA: 2% maize extract + 0.4% yeast extract + 2% agar in 1000 ml deionized water; sterilization for 20 minutes at 120θ0-οη

0 ₂ : Czapek Agar, 1. B. Raper, DI Eennelli Aapergillus does not. EM'lioms und Wllkins Company, 1965 ·, ρ · 36 · +: gjenge growth

Θ: no growth

In young cultures, the vigorous cotton wool - cotton wool, white-to-gray aerial mycelium gradually develops into light gray-azure blue or light brown levels during further incubation. From below, the colonies appear to be ocher or hazel and at ca. Cultures incubated at 27 ° C secrete dark brown pigment in the agar.

In liquid MA medium (i.e., MA medium containing no agar), the pH of which can be adjusted with hydrochloric acid or sodium hydroxide, the ATCC 20594 grows quite well at 2 pH η with shaking / 180 rpm and 21-22 ° No increase was observed at pH 9.3 and above pH 9.3. Good growth was observed in the pH range of 2.3-8.0.

Lower temperatures are also preferred for sporulation of the ATCC 20594 Cryptosporiopsis strain.

Today, after 7-10 days of incubation at 18 ° C, many micro- and macro-conidia have developed. These form cream-colored light brown disc-shaped conidia / acervuli / with a diameter of 50-200 / u or max. They form conidium aggregates of size 3 mm and accumulate mucus-like on the surface of the conidium.

Conidiogenic cells present in the upper conidia margin were classified as fialides based on dish microscopic observations. The two micro-conidia-forming fialides are often located directly behind each other, while the bottoms of the end-form fialides are often swollen and have a size of 6-10 x-3-5 U. There is an opening on the side of the lower fialides. Fialide-forming macroconidia are often much longer (8-26 x 3-5 / u /) and generally do not lie behind each other.

It's single-celled. hyaline microconidiae are small and cylindrical with a slightly bungular shape, while the wider end is round and the narrower is caonucous. Colorless to light amber single-cell macroconidia, on the other hand, are large and usually allantoid. One end is spherical, while the other has a 7182,778 rue projecting base.

Culturing conditions can significantly influence the formation and size of micro- and macroconion unions, as summarized in Table 4 below.

Table 4

Formation and size of macro- and micro-conidia as a function of temperature and medium after 21 days of incubation + some conidium ++ many conidium +++ very conidium - no conodium present

In each case, 10 statistically selected conidia are weighed microscopically. The range as well as the average length and width of the conidium 10 are given in u.

% malt extract Czapek agar

Macro - Micro- Macro- Microconidium, / u conidium, / u

Range 4-5 ° C average 25.5 37.5 ^ 6.4 to 9.0 30,8x7,3 12-13 ° 0 +++ ++ ++ - province 27.5 to 35.7 4.8 to 6.5 14.5 to 21.0 X X X. 6.4 to 8.3 1.1-1.3 7.3 to 9.0 average 31,2x7,0 5,6x1,2 18,5x8,1 14-15 ° 0 ++ + ++ 4 province 26.0 to 34.2 4.8-5.7 11.3 to 21.0 3.2 to 5.7 X X X X 6.6 to 8.8 1.1-1.3 6.5 to 9.0 1.7-2.4 average 30,0x7,8 5,2x1,2 15,3x7,9, 18-19 ° C province average 28.3 to 35.0 6.5 to 9.0 31,6x7,6 + 4.0-6.0 X 1.1-1.3 4,9x1,2 21-22 ° C province ++ 21.0 to 32.4 X 6.4 to 9.0 +++ 4.0 to 5.7 X 1.1-1.3 average 26,7x7,1 4,8x1,2 24-25 ° 0 province average + 26.0 to 30.0 6.6 to 9.0 27,8x7,9 4.1-5.8 1.1- i, 3 4,8x1,2 26.5 to 27.5 ° C - - - -

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The matured macroconidia are filled with drop-smeared material, which is rich in Sudan III dye and deep in Sudan Black B. Microconidia cannot be stained.

Germination of macroconidia is optimal at 24 ° C on MA medium in the narrow pH range of 3.1-3 * 8 / 70-80% germination /. Ph

Above 5.5, the germination rate is less than 5%. The germination rate of microconidia is less than 1% under all conditions tested.

The new ATCO 20594 strain can be grown at appropriate temperatures in various conventional media under aerobic surface or submerged conditions.

After sufficient amounts of the new compounds S 41062 / F-1, S 41062 / F-6 and / or 341062 / F-7 have been formed in the culture, e.g. based on activity against Candida species, myelium can be isolated from the fermentation broth and extracted by conventional means.

A preferred separation method consists of homogenizing the mycelium with methanol and separating the liquid phase and concentrating. Extraction is then performed with a water immiscible organic solvent, e.g. ethyl acetate and then the solvent was removed in vacuo. After washing with hexane, the solution is evaporated to dryness in vacuo.

S 41062 / F-lj S 41062 / E-6 and / or S 41062 / P-7 can be isolated and purified by conventional means, e.g. using chromatographic techniques or countercurrent separation.

The invention also relates to the production of fermentation broths obtained by culturing strains of the Oryptosporiopsis genus S 41062 / P-1, S 41062 / P-6 and / or S 41062 / F-7.

S 41062 / F-1, S 41062 / F-6 and S 41062 / F-7 show antibiotic activity. They have growth inhibitory effects on certain microorganisms, e.g. yeasts, but show no significant activity against Gram-positive and Gram-negative bacteria.

The broad spectrum activity against a wide variety of yeasts and fungi is particularly evident in various strains of human pathogen Candida.

Table 5 shows the minimum inhibitory concentration (MIC) of S 41062 / F1, S 41062 / P-6 and S 41062 / P-7 against various microorganisms, as determined by a serial dilution assay in malt extract medium / incubation temperature 27 °. C, 48-72 hours.

Table 5

S 41062

F-l F-6 F-7

Micro-organism MIC, / G / ml Candida albicans 0.1 0.5 0.5 Candida albicane II 12 0.1 0.5 0.3 Candida albicans 5897 0.1 0.5 0.3 Candida albicane 439 0.1 0.5 0.1 Candida albicane 422 0.1 0.5 0.5 Candida krusei 1 100 100

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Candida tropicalls Candida tropicalls CK 4 Aspergillus niger Mucor pusillus

n .: larger than Trichophyton quinckeanum Trichophyton mentagrophytes M i strong powder um cania Epidermophyton floceoaum Blaatomycea brasiliensis

10 n.100 100 naóo n.100 0.3 n.100 n.100 n.100 n.100 n.100 n.100 n.100 1 30 30 n. 10 10 10 30 30 10

This activity was observed in vivo, in a genitalia model infection test. In one such test, mice and rats pretreated with estradiol benzoate were challenged intravaginally and then administered orally or parenterally / mouse / ill for 5 consecutive days. intravaginally (rat) with test substance. The success of the treatment is determined by the presence or absence of a fungus in the vagina or uterus.

At topical application, antimycotic activity is observed at concentrations of 2% and above.

Systemic activity is observed with in vivo subcutaneous administration, with approximately 5 to 50 mg / kg body weight in the dosage range.

S 41062 / F-1, S 41062 / F-6 and S 41062 / F-7 are therefore considered useful antibiotics.

For such use, the dosage will, of course, depend on the compound employed, the mode of administration and the disease to be treated, but generally satisfactory results will be obtained. if 5-kb. A daily dose of 50 mg / kg body weight is preferably administered in divided doses 2 to 4 times daily or in sustained release form. In the case of larger mammals, the total daily dose is ca. 300 - approx. 3000 mg, e.g. Up to 500 mg. Unit dosage forms suitable for oral administration may have a dosage of approx. 75 mg - approx. max. 1500 mg, e.g. They contain 250 mg of the compound, using a solid or liquid pharmaceutically acceptable carrier or diluent.

Therefore, the present invention also provides methods of treating infectious diseases caused by yeasts and fungi by the use of S 41062 / F-1, S 41062 / F-6 and / or S 41062 / F-7. The present invention includes within its scope these compounds as medicaments, e.g. as an antibiotic; and in the therapeutic treatment of the human or animal body.

The compounds are preferably administered orally or parenterally. conveniently, the usual pharmaceutically acceptable diluents and carriers, and, if desired, other components, are admixed.

The preparation of these compositions is also an object of the present invention. The invention is illustrated by the following examples. All temperatures are given in ° 0.

Example 1

Cultivation of ATCO 20594 strain in glass fermenter a / Starting agar culture

Spore and mycelium suspension required for inoculation10

Z-10182.776 was prepared from the slant agar culture of ATCC 20594 obtained by incubation at 21 ° C for 21 days on agar medium of the following composition:

malt extract / syrup / 20 g yeast clone 4 g agar 20 g deionized water per liter b / Pre-culture

The spores obtained from the culture obtained in (a) and the mycelium are suspended in physiological saline and used to inoculate a 1 liter volume of the following medium in a 2 L Erlenmeyer flask;

malt extract 20 g yeast extract 4 g deionized water per liter

Incubation is done on a shaker / 180 rpm for 7 days at 21 °.

c / Production culture

The pre-culture obtained under (b) is used to inoculate 10 liters of the following medium, weighed into a 14-liter glass fermenter:

naloutahus can / liquid / 20 g élesztőklvonat 4 g citric acid 12 g NaOH / IN / 112 ml HC1 / IN / 44 ml deionized water 1 l

Prior to sterilization, the pH was adjusted to 1N HCl and / or Using NaOII

Adjust to 5.8-4.1.

Incubation is carried out for 5 days at 21 ° using agitation / 150 rpm / aeration / 1 liter / min / liter medium.

Example 2

Culturing ATCC 20594 in a Steel Fermenter a / Starting Agar Culture

The culture was carried out for 21 days at 21 ° using 400 ml Eoux flasks containing 150 ml of agar medium according to Example 1a /.

b / Pre-culture

To prepare the suspension for inoculation, 50 ml of physiological saline is added and the spore and mycelium suspension is obtained using a sterile scraper.

250 ml of the thus prepared inoculum are used to inoculate 50 liters of pre-culture medium having the same composition as in Example 1b / 75 L in a steel fermenter.

The incubation was carried out for 5 days at 21 °; aeration rate 0.5 liter / min / liter medium at 0.5 bar, agitation speed 150 rpm.

c / Interculture

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100 liters of the above preculture was inoculated with 500 liters of medium containing the following composition in a 750-liter steel fermenter:

malt extract / syrup / 50g yeast extract 10g FeS0n.7H _p 0 16.68 mg ZnS0 ^., 7H ₂ 0 6.88 mg deionized water 1 liter

Culture conditions: 4 days at 21 °, 100 rpm, bar and 0.5 liter / min / liter aeration! sebes0, / ei / Main Breeding

Intermediate culture was inoculated with 3,000 liters of lc / exemplary production medium in a 4,500 liter aoal fermenter. The culture was carried out for 5 days at 21 ^° C-0.5 ° C at a pressure of 0.5 bar and aeration rate of 1.0 L / min / L.

3 · Example

Isolation of S 41062 / F-1, S 41062 / F-6 and S 41062 / F-7

The pH of 300 liters of fermentation broth prepared in Example 2 is adjusted to 6-7 with dilute sulfuric acid or sodium hydroxide and the mycelium precipitate is separated from the filtrate of the culture using a tfestphalia separator.

The wet mycelium is homogenized in the Dispax reactor with 10 times the amount of methanol and the liquid phase is separated. This procedure was repeated twice with 90% aqueous methanol. The filtrates were combined, de-methanol removed in vacuo, and extracted three times with ethyl acetate and once with 4: 1 ethyl acetate / isopropanol. The extracts were freed of solvent in vacuo and the residue was dissolved in ten volumes of 90% aqueous methanol. After washing with hexane, the polar phase is again evaporated to dryness in vacuo.

This residue was dissolved in 1.5 liters of 7: 20: 2: 2 methylene chloride: methanol: water and partitioned by chromatography on 20 kg of silica gel using Aleick, 0.05-0.2 mm / ml, using the latter solvent mixture as eluent. Fractions of 13 liters were obtained.

Fractions 8 and 89, showing activity against Candida alblcans, containing primarily S 41062 / F1 and S 41062 / F-7 are combined and 1.5 kg of Sephadex LH 20 / methanol are added. ml / fraction / chromatography, with the two metabolites being the same as in Figures 42-62. fraction significantly enriched.

The concentrated residue of these fractions was partitioned into 4 portions and each fraction was chromatographed on 1.2 kg silica gel (Merck H, 10 cm diameter columns) using methylene chloride: methanol (4: 1); Collect 100 ml fractions.

Fractions residues were examined by thin-layer dietary chromatography for purity and pure fractions were collected individually. Pure S 41062 / F-1 is found at ca. In fractions 75-85 and pure S 41062 / F-7 in fractions 40-65.

The concentrated residues containing S 41062 / F-1 are dissolved in methanol, combined, treated with activated carbon and the solutions are filtered through a pad of talc. It's almost colorless

The -12182.776 filtrate was concentrated in a slight vacuum and slowly stirred with acetone. This affords S 41062 / F-1 as a highly hygroscopic compound in the form of a highly hygroscopic compound with a white and solvent state. Mp 235-240 ° / decomposes gradually /.

Chromatographic fractions which showed only thin layer chromatography on S 41062 / F-7 were combined and filtered through a talc layer as a methanol solution. concentrated in vacuo and slowly mixed with acetone. As an amorphous precipitate, S 41062 / F-7 is obtained. From 205 (shrinks with gradual decomposition).

Fractions 10 and 11 from the first chromatogram, containing mainly S 41062 / F-6, were combined and partitioned on 500 g silica gel (Merck, 0.04-0.06 mm / 4: 1 methylene chloride: methanol); Fractions of 80 ml are collected.

Fractions active against Candida albicans were evaporated to dryness in vacuo and the purity of the residues was determined by thin layer chromatography.

The fractions containing almost exclusively S41062 / -F-6 are combined, dissolved in methanol, filtered through a clear talc layer and, after concentration in vacuo, slowly mixed with acetone. S 41062 / F-6 precipitates as a white microcrystalline compound, m.p. 240-244 ° / gradual decay.

Claims (4)

Patent claims A process according to formula I, wherein R is a C 16 branched fatty acid group S 41062 / F-1, which has the following characteristics in the form of a white microcrystalline powder: / 1 / Ζ57ρθ = -6 ° / o = 0.5 in methanol /; / 11 / analysis results: C 55.7 H 7.7 N 10.1 0 24.7%; IR spectrum in KBr is shown in Figure 1; (iv) 1 H-NMR spectrum (90 mHz in DMSO) with tetramethylsilane as internal standard d Figure 4; / v / UV spectrum in methanol: lambda 193 nm e} ⁷ '= 786 max 1 cm / 222 / nm = 140 279 nm '= 14; / vi / antibiotic activity is shown in Table 5; / vii / has a molecular weight of 1048 - and / or a related compound S 41062 / F-6 - which has the following approximate properties as a white microcrystalline powder: / 1 / Z 2 ° D ⁼ 9 ° (0-0.5 in methanol); / 11 / analysis results: 0 56.3 H 7.7 N 10.9 0 26.0%; IR spectrum in KBr is shown in Figure 2; / iv / 1H NMR spectrum (90 mHz in DMSO / tetramethylsilane as internal standard) is shown in Figure 5; / v / UV spectrum in methanol: -13182.776 lambda. , 1% = 861 / vi / box ^{195 nm} Bon / 222 / nm '= 340 278 nm '= 15; its antibiotic activity is shown in Table 5 and / or the related compound S 41062 / F-7, which has the following approximate characteristics as a white powder /? analysis results: C 54.5 H 7.5 N 9.9 0 25.0%? / 111 / IR spectrum in KBr shown in Figure 3? / iv / the spectrum of Ih-HHR / 90 ml / d in DMSO / tetramethylsilane as an internal standard in Figure 6? / v / UV spectrum to methanol #: ^{lanbda nax 194 nm B} 1 is “/ 222 / nm” 278 nm / µ / antibiotic activity, characterized in that a strain producing S 41062 / F1 and / or S 41062 / F-6 and / or S 41062 / F-7 is aerobically or immersed in a medium containing carbon and nitrogen. cultivated. in / 1 / / 11 / 890 339 15; Table 5 - pre2. The method of claim 1, wherein the producing strain is a Cryptosporiopsis strain. 3. The method of claim 2, wherein the Cryptosporiopsis strain is ATCC 20594 or NRRL 12192. . A process for the preparation of a pharmaceutical composition comprising two active compounds S-41062 / F-1 and / or S-41062 / F-6 and / or 41062 / F-7, characterized in that the active ingredient produced by the process according to any of claims 1-3 · prepared with conventional carriers, diluents, fillers and / or other excipients.