DE918162C - Process for the production of antibiotic active ingredients - Google Patents

Description

Method of Obtaining Antibiotic Active Invention concerns the extraction of new antibiotic active ingredients from Streptomyces collinus n. sp. This strain is in the »Archive for Microbiol. a, Vol. 17, pp. 28o to 282. The new antibiotics are valuable because of their bacteriostatic effectiveness.

It has been found that by cultivating Streptomyces collinus in suitable nutrient media and by extracting the antibiotic substances, preferably from the separated mycelium, two active ingredients are obtained, which are distinguished by Have adsorption methods isolated. It is rubromycin and one second antibiotic agent called collinomycin.

Rubromycin has the following properties: It crystallizes from methanol or ethyl acetate in thin bar-shaped crystals that look pale purple under the microscope, Macroscopically, on the other hand, look red and decompose from 2z5 ° (Berl block). Rubromycin is moderately soluble in acetone and chloroform, slightly soluble in ether and low molecular weight Alcohols and practically insoluble in water, petroleum ether and in sodium hydrogen carbonate solution. From aqueous alkali and concentrated sulfuric acid it turns reddish-violet Color added (absorption maxima: in 2N sodium hydroxide solution 584 mm, in concentrated Sulfuric acid 518 to 52o my.) When shaking his red methylene chloride solution With aqueous sodium hydrogen carbonate, a red-violet color appears at the phase interface Precipitation of the sodium salt of the antibiotic. The red solution of the antibiotic in water-containing dioxane, sodium hyposulfite (not sodium bisulfite) discolored and red again when standing in the air. Rubromycin gives in pyridine-methanol with titanium trichloride a blue-green solution that turns red-violet after a short time. The red solution in acetic anhydride changes color when pyroboroacetate is added blue-violet and after brief boiling shows two absorption maxima at 581 and 515 mu-rubromycin does not contain nitrogen and thus differs characteristically from rhodomycin (B. 84, 700). A preparation dried in a high vacuum at 154 'gave the following analytical figures: C 6o, ßo, H 4.26, 0 3391 (sum 98.47).

With acetic anhydride-pyridine, kubromycin is converted into benzene-petroleum ether Acetate crystallizing in yellow needles with a melting point of 193 to 195 ° (uncorr.) (Found C 59.45. H 4.21, CH, C0 17.4). The acetate can be extracted from its yellow benzene solution Do not shake out with sodium hydrogen carbonate or sodium carbonate.

When boiling a solution of rubromycin in methylene chloride with dilute Hydrochloric acid forms a red compound in almost quantitative yield, which consists of Acetone in red, bar-shaped crystals from Sehmp. 265 to 268 ° (uncorr.) Crystallized. It inhibits the growth of staph. aureus in a dilution of more than 1:15 ooo ooo, d. H. in the same order of magnitude as rubromycin. The connection differs from rubromycin in that its ethereal solution has absorption maxima at 547 and 532 n111, also shows by their behavior in adsorption and distribution. Used to produce a mixture of rubromycin and its conversion product from chloroform Calcium sulphate is adsorbed, two are formed as the chromatogram develops clearly separated zones, of which the upper one contains the rubromycin. When distributing in the system dioxane - water - gasoline (volume ratio 12: 2 : 3) Rubromycin goes predominantly into the hydrophilic phase, the conversion product on the other hand, is distributed evenly over both phases. Also by paper chromatography the antibiotic and the conversion product can be separated. Rubromycin crystallizes from chloroform-methanol in orange prisms from melting point 28o to 282 ° (uncorr.) and cannot be distilled without decomposition in a high vacuum.

Collinomycin is sparingly soluble in chloroform, acetone and dioxane, difficult soluble in ether and low molecular weight alcohols, practically insoluble in petroleum ether, Water and sodium hydrogen carbonate solution. When shaking its yellow ethereal solution With 2N sodium carbonate, the 1sodium salt of the antibiotic precipitates at the phase interface as a violet precipitate, while the aqueous phase is only slightly violet colors. The new compound with red-violet (absorption maxima: 575, 535 mA, of concentrated sulfuric acid with a carmine red color (absorption maxima: 525, 48o mA). If titanium trichloride is added to collinomycin in pyridine-methanol, so the solution first turns olive green and soon afterwards red. The orange (in UV light with the same color fluorescent) solution in acetic anhydride is based on the addition of pyroboroacetate carmine red and after a short boiling shows absorption maxima at 608, 555 and 515 mY. The yellow-red dioxane solution becomes pale yellow due to sodium hyposulfite; when standing at the Air returns to its original color. Like rubromycin, it contains collinomycin no nitrogen, but in contrast to this shows a considerable methoxyl content. A preparation dried in a high vacuum at 15q. ° gave the following analytical figures: C. 59.41, H 4.o1, 0 34.83, C H30 16.2.

With acetic anhydride-pyridine, collinomycin is converted into benzene-petroleum ether Acetate crystallizing in yellow needles with a melting point of 228 to 230 ° (Gef. C 61.18, H 4.17, CH3C0 2z, 4), which do not shake out from benzene with 2N sodium carbonate leaves.

Benzoyl chloride-pyridine converts the antibiotic into one made from acetone-ether Benzoate crystallizing in lemon-yellow needles with a melting point of 226 '(Gef. C 66.43, H 3.94, C, H "C0 38.8).

By prolonged heating in acetone containing sulfuric acid, collinomycin is eliminated into a compound of m.p. which crystallizes from acetone in orange needles. 273 to 275 ° above, which differs from the starting material in that its Ether solution shows absorption bands at 529 and 515 nu and when shaken with sodium hydrogen carbonate solution a red-violet, almost water-insoluble sodium salt of the conversion product ruled out. If the methanol solution of the conversion product is mixed with methanolic Lead acetate, a dark brown precipitate falls out, while collinomycin is used this reaction only shows a color change from red-yellow to brown-violet.

The following table shows the effectiveness of rubromycin against various microorganisms in the dilution test (inhibition values after 80 hours at 37 'in Hiß's solution): Bact. coli about ..... i: 1o ooo B. typhi. . . . . . . . . . . i: io ooo to i: 2o ooo staphylococci ..... i: ioo ooo to i: zoo ooo enterococci ....... i: ioo ooo streptococci ....... i: 8oo ooo Co. diphth .......... i: 80o ooo B. subtilis. . . . . . . . . i: 80o, ooo The following values were found in serum-free nutrient solution: St. aureus approx. . . . . . . . . . . . . . . i: 2o ooo ooo B. subtilis. . . . . . . . . . . . . . . . . . . . 1: 25,000,000 Collinomycin inhibits Staph. aureus at a dilution of 1: 2,000,000.

To produce the antibiotics, one can proceed as follows, for example: The inoculation material from St. collinus is grown on glycerine-glycocolla agar slants and inoculated onto culture flasks after 8 to 10 days. The culture solution has the following composition: 2% glycerine, 0.25 O / o glycocolla, o, i% NaC1, o, i% K2HP04, o, oi% FeS04, 010.0 / 0 Mg S 04, 0.50 /, Yeast cooking juice, traces of CaC03. After inoculation, 1.5 l each of this culture solution are sterilized in flasks for 20 minutes at 2 atmospheres and incubated for 25 to 27 days at 27 °.

After 8 to 10 days of incubation, a thin layer of mycelium covers the surface of the culture solution, and dye formation begins. To achieve greater mycelial yields the pistons will be at this age shaken vigorously once. To about 25 days the growth is finished, the culture solution then has a deep dark brown color to brown-red color, the mycelium is gray-white on the surface and on the underside colored almost black and forms a strong cover. The culture solution only contains small amounts of antibiotic substances, so that the work-up little is worthwhile.

The mycelium contains most of the antibiotic substance. For work-up and separation, the mycelium filtered off from the culture solution becomes weak squeezed out, frozen out to burst the cell walls and after thawing by squeezing and vacuum drying at 3o to 35 ° freed from adhering water. Then it will be fine grind. Let by extraction with petroleum ether, ether or carbon disulfide remove lipoid-like accompanying substances. For the extraction of the raw antibiotic the dry mycelium is treated with acidic acetone (z 180 ° / qiges acetone + 120 ccm 2 n-H2S 04) largely extracted in the cold. Other suitable extractants are Acetone also chloroform, methylene chloride, ethyl acetate, tetrahydrofuran and dioxane.

The acidic acetone extract is used for further processing (with N a 0 H) brought to pH 6.0, in vacuo at 35 ° to a quarter of its original Reduced in volume and shaken vigorously with water and ether. In the hydrophilic Phase there are red and brown ineffective accompanying dyes, in the ether more Fat in addition to colorings and small amounts of antibiotics. The bulk of the antibiotic substance precipitates at the phase boundary layer. This precipitation is centrifuged off, washed thoroughly with water and dried. By exhaustive Extraction with methylene chloride (acetone, chloroform, methanol are also suitable) the antibiotics can be obtained from it.

The dyes are made from the deep red methylene chloride extract Saturated sodium hydrogen carbonate solution largely shaken out, whereby a violet precipitate forms at the boundary between the two phases, which is predominantly consists of dye salt. The methylene chloride phase is carefully neutralized, washed with water and then evaporated to dryness. The deep red residue leaves from chloroform to precipitated gypsum, activated by heating, by chromatographic Separate adsorption.

The chromatogram eluate of the two lower chromatograms collected in fractions After narrowing to dryness, zones deliver almost pure rubromycin or collinomycin. They are obtained purely by recrystallization, e.g. B. from chloroform, methanol or ethyl acetate.

Claims (2)

PATENT CLAIMS: e.g. Process for the production of antibiotic active substances, characterized in that Streptomyces collinus is in a suitable nutrient medium and from the resulting culture, preferably from the separated mycelium, the antibiotic active ingredients are obtained by extraction with organic solvents. 2. The method according to claim r, characterized in that after separation of the antibiotic agents separate them from one another by adsorption methods and so isolated rubromycin and collinomycin.