DE3248280A1 - Salbomycin and process for its preparation - Google Patents

Abstract

Salbomycin, process for its preparation and compositions containing salbomycin.

Description

Salbomycin and process for its preparation

The invention relates to a completely new, biologically active substance, which is called salbomycin in the following and has interesting properties relating to it their use in human and veterinary medicine.

The invention also relates to a process for the production of salbomycin, as well as preparations and pharmaceuticals containing salbomycin - including the cultivation of salbomycin-producing microorganisms and the processing of the resulting cultures.

On the one hand, salbomycin shows a remarkable antibiotic effectiveness, for example against trichomonads, on the other hand it is capable of a cytostatic To have an effect. These properties depend on the specific molecular Structure of salbomycin together that enable this antibiotic to be special To develop activities on biological membranes, for example. So is suitable Salbomycin for use in all those areas where malfunction due to the above mentioned properties of salbomycin can be remedied.

The salbomycin is represented by the structural formula shown in FIG is marked. The configuration of the asymmetrically substituted carbon atoms Elier is described by the letters R and S (for the meaning of R and S compare e.g. L.F. Fieser and M. Fieber "Textbook of Organic Chemistry", published published by Verlag Chemie, Weinheim, 2nd improved edition, 1972, pp. 90 f.).

The present invention thus also relates to salbomycin a process for the preparation of salbomycin, which is characterized7 that a Salbomycin-producing Streptomyces in a fermentation medium in the submerged process cultivated t and the salbomycin from the Mycelium and the culture filtrate extracts and cleanses.

According to the invention for the production of Salbomyclns of Streptomyces albus ATCC 21 838 (DSM 2566) used. But it can also be its variants and Mutants are used, even other Streptomycetes from Streptomyces albus, but are able to produce salbomycin. The taxonomic Properties of Streptomyces albus (ATCC 21 838 - DSM 2566) are from Miyazaki et al.

in J. Antibiotics 27, pp. 815-816 (1974).

To obtain the salbomycin, Streptomyces albus ATCC 21 838 (DSM 2566) in an aqueous nutrient medium, preferably under submerged aerobic conditions Conditions grown until a sufficient concentration of the salbomycin is obtained. On the one hand, the nutrient medium contains carbon sources, such as carbohydrates, on the other hand, nitrogen quarries, including any suitable nitrogen compound, such as protein-containing sterals. Preferred carbon donating compounds are glucose, sucrose, glycerine, malt extract, starch, oils, fats and the like.

Preferred nitrogen-supplying substances are, for example, corn steep liquor, Yeast extracts, soy flour, fish meal, skimmed milk powder, digested casein or meat extract.

So-called "synthetic" nutrient solutions can also be used, which, however, often result in lower yields. It can still be useful be, trace elements such as zinc, magnesium, iron, cobalt or manganese in the fermentation medium admit.

The fermentation that leads to the formation of salbomycin can take place in can be carried out in a known manner in a wide temperature range, for example at temperatures between 10 and 400C, preferably between approximately 25 and 350C. The pH value of the medium is also kept at values that support the growth of the microorganisms are beneficial, for example with values between 4.0 and 8.0, preferably between 5.0 and 7.0. Depending on the nutrient medium, such as its composition and concentration and the fermentation conditions such as ventilation rate, temperature or pH, salbomycin is usually formed in the culture solution after about 2 to 10 days.

The salbomycin can be found both in the mycelium and in the culture filtrate the fermentation cells are located. The majority of the salbomycin is generally in Find mycelium.

Therefore, the mycelium is advantageously removed from the aqueous phase, For example, separated by filtration or centrifugation and the salbomycin from the respective phases with suitable solvents such as methanol, methylene chloride, Acetone, butanol or ethyl acetate or extracted with mixtures of these solvents. A large number of methods can be used to purify the salbomycin, such as Solvent extraction, partition chromatography, Graig distribution, crystallization, Chromatography on various carriers, such as aluminum oxide, molecular sieves, Silica gel or adsorbent resins can be used.

A preferred enrichment method for the recovery of the salbomycin consists in removing the antibiotic cell from the mycelium, preferably with acetone or methanol or from the aqueous phase with a suitable one, with water not miscible organic solvents such as butanol, ethyl acetate, chloroform or extracted methylene chloride.

The extract is then dried using known methods, such as adding of the usual desiccants, and it is concentrated in a vacuum, whereby the crude product is obtained. The crude product can then by one or more of the above methods, preferably enriched to the pure substance by repeated chromatography on silica gel will. The last step of the isolation is advantageously the crystallization, for example from methanol, tetrahydrofuran or benzene.

The crystal mass is then by methods known per se, for example separated by filtration and dried.

The pure salbomycin is a colorless, crystalline solid, its Elemental analysis of the presence of carbon, hydrogen and oxygen, as well the absence of nitrogen, phosphorus, sulfur and halogens in the molecule results. That Ultraviolet light is absorbed by salbomycin in methanol with #max = 252 nm, (E1 cm1% = 570).

The melting point is 189-1900C, the specific rotation [α] D = -52 <c = 0.5 in chloroform). Salbomycin is in chloroform, methylene chloride, Acetone and aliphatic alcohols are readily soluble, in water and petroleum ether it is Poor solubility. The biological properties of salbomycin are in view to its use as a therapeutic, growth promoter or preservative Interesting.

The following are an example of the microbiological spectrum of activity Called values.

Test organisms Minimum inhibitory concentration [mcg / ml] Staph. aureus SG 511 1.56 staph. aureus 285 1.56 Staph. aureus 503 1.56 Steptoc. pyogenes 308 A 1.56 Streptoc. pyogenes 77 A 1.56 Streptoc. faecium D 3.13 pseudom. aeruginosa ATCC 9027 > 100.0 E. coli 055> 100.0 Trichophyton mentagrophytes (100/25)> 125 Candida albicans (200/175) 125 Aspergillus niger (500/284)> 125 Trichomonas vaginalis (carneri) 1.95 Entamoeba histolytica 15.6 Eimeria tenella in vivo efficacy of leukemia cells the mouse, L-1210 0.2 Salbomycin - quite generally - unfolds one Influence on biological membranes. This effect is e.g. via the action potential or to determine other measurands. Salbomycin also has a beneficial effect for immune stimulation in the so-called macrophage test detectable and measurable, as well like cytostatic activity. The influence of biological membranes is generally required in human and veterinary medicine. Salbomycin is everywhere of great value where its distinctive ability to work with biological membranes to be able to change the right direction, can be used.

The invention also relates to preparations, in particular medicinal preparations, for example for topical or parenteral application, which by a content are characterized by salbomycin. The salbomycin can also be used in combination with other active ingredients are used.

Preparations that contain salbomycin as an active ingredient can be made by combining salbomycin with one or more pharmacologically acceptable, known carriers or diluents and mixed in a for brings the desired application suitable preparation form.

The following exemplary embodiments serve for further explanation of the invention, but do not limit it to it.

Example 1 The vaccine cultivation takes place - as in the microbiological Production usual - from a freeze-dried permanent form of the organism Streptomyces albus ATCC 21 838 / DSM 2566 via single colony passage and inclined tubes. The for The fermentation necessary for mass production of spores is also carried out on solid Nutrient testicles carried out in Roux bottles.

Agay medium for plate, slant tube and Roux bottle: glycerine 2.6 % Yeast extract 0.2% table salt 0.1% agar-agar 2.0 90 pH value 7.0 sterilization at 1200C 20 min.

Incubation at 30 ° C for 9 days.

With the spore removal from the Roux bottle in 1 00 ml sterile Water is inoculated as a vegetative fermentation precursor in a Fernbach bottle (Working volume 1.2 1).

Precursor medium: glucose 4.0% soy flour 1.0% dry yeast 1.0% calcium carbonate 0.2% pH value 6.7 sterilization 1200C 20 min incubation at 28 ° C for 2 days on a Shaking machine with 150 rpm and 5 cm amplitude 2. Pre-fermentation: After 48 hours of preculture (see above), 200 l of pre-fermentation medium with 1.2 l of vaccine were added inoculated from the Fernbach bottle.

Medium: soy flour 1.0% dry yeast 1.0% calcium carbonate 0.2 g glucose 4.0% pH value 6.3 sterilization 120 ° C 30 min.

Incubation at 33 ° C with stirring at a peripheral speed 5 m / sec. and ventilation at 0.6 vvm for 2 days.

3. Main fermentation: After 48 hours of pre-fermentation (see above) 2400 1 main fermentation medium inoculated with 35 -l culture broth from the pre-fermentation.

Medium: soy oil 10.0% glucose 0.5 90 soy flour 0.5% wheat germ 0.5 % Ammonium sulfate 0.3% table salt 0.1% calcium carbonate 0.5% potassium chloride 0.5% di-potassium hydrogen phosphate 0.1% pH 7.5 sterilization at 120 ° C for 20 min incubation at 330 ° C with stirring with a peripheral speed of 5 m / sec. and loading ventilation of 0.76 vvm 9 days.

The pH falls to 5.5 within about 48 hours and is dried with sodium chloride (30 gig) controlled to 5.5 - 6.0.

Yield: approx. 1,600 kg of culture solution containing salbomycin.

Example 2 1 80 l of culture solution, obtained according to Example 1, were through Centrifugation in cell mass and aqueous supernatant separated and the cell mass with 60 l of acetone, the aqueous supernatant extracted with 75 l of n-butanol. The clear organic ones Phases were combined and concentrated in vacuo to 6 l. The anhydrous, still butanol-containing 60 l of petroleum ether were added to the suspension. The resulting precipitate was centrifuged off and washed out with methanol. The methanol phase contained the salbomycin as a crude product, which was dried in vacuo. 25 g of this raw material were dissolved in 50 ml of chloroform-methanol (mixing ratio 95: 5) and on a Column applied that contained 500 g of silica gel in chloroform. The chromatography column had the dimensions 5.5 x 45 cm. After the order, the column contents were initially with 2 1 chloroform, then washed with 2 1 chloroform-methanol (95: 5) and then eluted with 2 l of chloroform-methanol (90:10).

The eluate was collected in fractions, which was towards the end of the elution occurring salbomycin-containing fractions combined and reduced in vacuo to 1/30 of the Confined in volume.

The salbomycin crystallized in this concentrate. It was cut off recrystallized in methanol and dried. 1 g of salbomycin was obtained.

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Claims (5)

Claims: 1. Salbomycin of the formula shown in Fig. 1. 2. Process for the production of salbomycin, characterized in that that a salbomycin-producing Streptomyces in a fermentation medium im Cultivated the submerged method and the salbomycin from the mycelium and the culture filtrate extracts and cleanses. 3. The method according to claim 2, characterized in that as Streptomyces a Streptomyces albus is used. 4. The method according to claim 3, characterized in that as Streptomyces albus of Streptomyces albus ATCC 21 838 (DSM 2566) is used. 5. Preparations, characterized by a content of salbomycin.