NO762923L - - Google Patents

Description

The present invention relates to a method for producing the compound S 11743/A' in the acid form with formula I

and its salts.

The peculiarity of the method according to the invention for producing the compound S 11743/A in the form of the acid and its salts is that an S 11743/A producing strain of the actinorayceten species streptomyces rautabills is grown in the presence of a nutrient medium.

The compound S 11743/ft in the form of its amorphous and acetone-containing, crystalline sodium salts and crystalline potassium salts, as described in the examples/has the following characteristic properties Na salts of S 11743/A

The elemental composition of the amorphous sodium salt gives the following

values for c4iHQ9°x2Na:

theoretical C 63.4% H 9.0% Na 2.9% 0 24.7% found C 63.5% H 9.1% Na 3.2% O 24.1%

The IB spectrum of the amorphous sodium salt in CH0Cl0 is shown in fig. 1.

Fig. 2 shows the cH-NMR spectrum (90 MHz) of the amorphous Na salt in CDCl3 with tetramethylsilane as internal standard.

The Na salts are well soluble in organic solvents such as toluene, benzene, dichloromethane, ethyl acetate, isopropyl acetate, ethanol and methanol.

In water, the salts are only slightly soluble.

In the table, the Rf values for the amorphous salt in thin-layer chromatograms on silica gel plates, Merck, Kieselgel 60, F 254, with a layer thickness of 0.25 mra are shown in comparison with two other ionophores in the form of their sodium salts.

Footnote f coloring agent (Flm«):

1) Toluene-acetone (4 s 1) + 1% triethylamine

2) Chlorophora-ethyl acetate (1:1)

3) Chlorophorar-methanol (95 s 5)

To make the spots visible, iodine or a solution of 0.2% Ce(S04)2 in 50% sulfuric acid is used and subsequent heating to 120 - 150°C.

K-salt of S 11743/A

The elemental composition of the crystalline potassium salt gives f51genda values for

<C>41<H>69°12<K:>

theoretical C 62.1% H 8.8% K 4.9% 0 24.2%

found C 61.9% H 8.9% K 4.8% O (not determinable)

The method according to the invention can be carried out according to known methods. A preferred culture of the S 11743/A producing strain was deposited at the United States Department of Agriculture (Northern Utilization Research and Development Division), Peoria, 111., USA and is available to the public under the designation NRRL 8088.

However, S 11743/A producing strains can also be used, which could be obtained from the starting strain of the actinomycete species streptomyces mutabilis by treatment with mutagenic substances or radiation or by selection.

The vessel is in sering of the strain NRRL 8088

The new strain of the actinomycete species Streptomyces mutabilis used according to the invention was isolated from soil collected in 1965 in the Central African Republic.

The new strain HHRL 8088 grows well on the complex and synthetic nutrient media. The air tmucel is lead- to silver-grey and the spore chains are mainly monopodial but many times also sympodially branched. The spore chains can be composed of irregular, open spirals with many times 2 wrong 3 windings, but generally fit the terminologlen "Retinaculum-Apertum". Especially on synthetic media, wavy spore chains are also formed. In an electron microscope, the spore surfaces appear smooth, the spore shape is oval, elliptical to cylindrical. The tracks 0.7 - 0.9 ^u wide and 1.33 - 1.5 ai long. Among the "grey series" of streptomycetes with "Spira-type" spore chains, smooth spore surfaces and lack of melanin formation, the strain WRRL 8088 could be identified by the key of H. Nonomura, J. Ferment. Technol. 52, 78-92

(1974) are not identified under the "gray series'*. By assignment to the "white-grey series" the identification as streptomyces mutabilis was fulfilled. Using the Tresner-Backus color disc system (1963) the strain NRRL 8088 fell unambiguously under the "gray series" and could then be determined with the help of Bergey's handbook, pages 749, 772 and 777 (8th edition) as streptomyces mutabilis Pridham et al. 1958 in Gauze et al. 1957,

The growth characteristics of strain NRRL 8088 on normal biological media and its carbon assimilation are listed in the following tables.

Growth characteristics

Assimilation of carbon compounds i

The new strain WRRL 8088 has the following physiological properties:

The new strain NRRL 8088 can be grown on different nutrient media with the usual nutrients, e.g. as described in the following example.

The cultivation of the strain NRRL 8088 can be done by means of aerobic surface cultivation or immersion cultivation.

In order to avoid a delay in the production of S 11743/A, the vegetative form is preferably used instead of the spore form of the microorganism for the inoculation of the production medium. Similarly, a vegetative inoculum of the microorganism is first prepared by inoculating a small amount of a culture medium with a spore suspension of the strain NRRL 8088.

The mycelial part of the fermentation broth is possibly digested and the compound is recovered by extraction and/or adsorption methods in a manner known per se. However, you can also centrifuge the mycelium from the culture broth, extract the culture filtrate and the mycelium separately after digesting and further process the extracts individually or separately Compound S.11743/A. is preferably isolated in the form of the salts, preferably alkali metal, alkaline earth metal or ammonium salts being mentioned.

The compound S 11743/A is suitable as a medicine.

The compound S 11743/A shows an inhibitory effect against microorganisms such as Gram positive bacteria and fungi. In the following table, the minimum inhibitory concentrations (MlC values) against some microorganisms are indicated. The MIC values were determined in a known manner using dilution series. The determination was made for the bacteria by incubation 1 Brain Heart Infusion broth at pH 7.4 and 37°C, for the fungi in malt extract medium (2%) at pH 5.2 to 5.4 and 27°C for 48 to 72 hours*

Furthermore, the compound S 11743/A excels in the form of the acid and the salts

itself in the pharmacological testing by strengthening heart muscle power and increasing the coronary flow and can therefore also find use as a medicine. The compound S 11743/A and their physiologically tolerable salts are indicated for use in acute heart failure, for shock therapy as well as for the treatment of coronary diseases.

As a medicine, S 11743/A or its physiologically tolerable salts can be administered alone or in a suitable preparation form with pharmacologically indifferent excipients.

The compound S 11743/A in the form of the acid or its salts can be used for human medical purposes as a medicine alone, and then in pure form or in corresponding preparations for intravenous, enteral or parenteral administration.

In the continued description, all temperature indications are in °C.

The following examples illustrate the implementation of the method according to the invention.

EXAMPLE 1: Cultivation of the metabolite S 11743/A in a shaking culture

a) Agar initial culture

It used agar culture of strain NRRL 8088 as starting material

is obtained by inoculating a culture medium with the following composition:

with a spore suspension of the originally isolated strain NRRL 8088 prepared in a manner known per se.

The medium is adjusted for the sterilization with NaOH to pH 8.3 to 8.6. After the sterilization (20 minutes at 120°C), the pH has adjusted to 6.9 to 7.3.

b) Sporesusgens^ori

A well sporulating agar stock culture of strain WRRL 8088

5 ral of sterile, 0.9% sodium bicarbonate solution is added, which gives a thick spore suspension.

c) Preculture

1 ml of this spore suspension is used to inoculate a 500 ml

Erlenmeyer flask, containing 100 ml of pre-culture medium:

The pH value is 6.5 to 6.8. The pre-culture medium is sterilized in an autoclave at 120°C for 20 minutes, whereby the pH value is 6.7 to 7.2.

d) Main culture

The inoculated pre-culture is incubated aerobically for 3 days at 27°C

on a rotary shaker (200 rpm) and then used directly for inoculation of a main culture, whereby 5 ml of the pre-culture in a 500 ral Erlenmeyer flask is inoculated with 100 ml of f Saying medium:

The pH is adjusted after the sterilization (20 minutes at 120°C) with NaOH to 6.8 to 7.2. The thus inoculated main culture is incubated for 16 days at 27°C on a rotary shaking machine (200 rpm).

EXAMPLE 2: Cultivation of the compound S 11743/A in a fermentation culture

a) Preculture

A culture of the originally isolated strain 13RRL 8088 is obtained

by cultivation for 10 days at 27°C on a medium III. The spores and mycelium in this culture are taken up in physiological saline solution.

With this suspension, 1 liter of nutrient solution containing the following medium is inoculated:

b) Main culture

This pre-culture is incubated for 3 days at 27°C on a rotary

shaking machine (180 rpm). 10 liters of this nutrient solution containing medium II is inoculated with the pre-culture and incubated in a glass fermentation vessel under stirring (200 rpm) and aeration (1 liter of air/min./liter of nutrient solution) for 5 days at 27°C.

EXAMPLE 3: Isolation of the compound S 11743/A (the JJa salt)

100 liters of culture broth, prepared in ex. 1 or 2, centrifuged

whereby approximately* 90 liters of growing felt rat are obtained and approx. 6 layers of mycelium. The culture filtrate is adjusted to pH 9 with 1 M sodium hydroxide solution and filtered clear using the filter aid "Celite 545'<*>. The filtrate is extracted 3 times at 20°C with 90 liters of ethyl acetate each time and the extracts are washed with 15 liters of water. After evaporation of the organic phase in vacuum at 20 - 40°C

is achieved approx. 16 g crude extract.

The mycelium is first extracted with 12 liters of methanol, then twice

with each time 12 liters of methane&L water (9 s 1) in each time for 1 hour at 20 - 25°C, whereby it is simultaneously homogenized using an ultra-turraks apparatus. The cell material is then filtered off through "Celite 545". Add 5 liters of water to the filtrate, evaporate it in a vacuum at 20 - 40°C to a volume of approx. 3 liters and it is adjusted to pH 9 with 1 N sodium hydroxide solution. It is extracted three times with 3 liters of ethyl acetate each time, the extracts are washed with

1.5 liters of water and the organic phase is evaporated in a vacuum at 20 - 40°C whereby 9.4 g of extract is obtained.

The two extracts are combined, taken up in 500 ml of water and extracted three times. with each time 250 ral toluene. The toluene phase is washed once with 100 ml of water, dried over sodium sulphate and evaporated in vacuo at 40°C, whereby 11 g of a yellow, oily residue is obtained. The residue is chromatographed on the 50-fold amount of silica gel 60, Merck, grain size 0.06 - 0.2 mm. Toluene-acetone (98 j 2) + 1% triethylamine is used for preparation of the column and for application of the substance. The elution with toluene-acetone (98:2) + 1% triethylamine and with toluene-acetone (9:1) + 1% triethylamine gives mainly inactive byproducts. S 11743/A is eluted in enriched form with toluene-acetone (4:1) and toluene-acetone (1 s I) with the addition of, in each case, 1% triethylamine. The fractions are evaporated in a vacuum at 20 - 40°C and the remainder is tested by thin-layer chromatography and biologically against staphylococcus aureus for the content of S 11743/A. The active fractions are combined and chromatographed again on the 40-fold amount of silica gel, using chloroform with an increasing methanol content for the elution. Chloroform methanol (98 : 2) elutes S 11743/A in highly enriched form. After evaporation of the active fractions in a vacuum at 20 - 40 C, the light yellow residue is dissolved in chloroform, shaken once with 1 N sodium hydroxide, the organic phase is washed with water, evaporated in a vacuum and the residue is crystallized from the 1.5-fold amount of acetone, whereby S 11743/A-Na salt is obtained in the form of colourless, acetone-containing crystals with a melting point of 159 - 163°C. To remove the crystalline acetone, heat in a vacuum several times with benzene and then with methanol.*The amorphous, pure sodium salt of S 11743/A is dried in a high vacuum at 60°C and then melts at 166 - 169 C.

EXAMPLE 4: Potassium salt of S 11743/A

280 rag S 11743/A Na salt 15sed in 40 ml chloroform and shaken twice. times with each time 40 ral of 2 N hydrochloric acid and wash with 40 ml of water. The organic phase with the free acid of S 11743/A is then added

once extracted twice with each time 40 ml of 2 N KOH and washed once with 40 ml of water. The chloroform phase is dried with fine potassium carbonate and evaporated after filtration under vacuum. The residue is dissolved in 2 ml of methanol and filtered through talc. After 4 hours, the crystallisate is filtered off and recrystallized from 2 ml of boiling methanol. After standing overnight, it is filtered off and the residue is dried under high vacuum for 1 hour at 40°C, whereby colorless crystals with a melting point of 158 - 160°C are obtained.

Claims (3)

1. Process for the preparation of the compound S 11743/A in the form of the acid of formula I and its salts, characterized in that a S 11743/A producing new strain of the actinoray ceten species streptomyces mutabilis is cultivated on or in a nutrient medium and then the compound S 11743/A is isolated from the nutrient medium in the form of the acid and its salts and purified. 2. Method as stated in claim 1, characterized in that the strain NRRL 8088 is used as a new strain of the acinoray ceten species streptomyces mutabilis. 3. Method as stated in claim 1, characterized by using a strain obtained from the strain NRRL 8088 by treatment with mutagenic substances or radiation or by selection.