DE1467941A1 - Process for the manufacture of antibiotics - Google Patents

Description

F. Hoffmann-La Roche A Co .. Public company . Basel, Switzerland)

Process for the manufacture of antibiotics

The present invention relates to a method for Manufacture of new antibiotics. These antibiotics can be produced by growing microorganisms that the Belong to genus Streptomyces.

The organism that was used for the new antibiotics that can be produced according to the invention is called Streptomyces hazeliensis var. Hazeliensis nov. sp. This organism was isolated from a soil sample found in Matane, Gaspe ", Canada. For convenience, this organism will be referred to as Streptomyces hazeliensis. A culture of this Streptomyces hazeliensis was stored in the United States Department of Agriculture's collection of microorganisms, Northern Utilization Research and Development Division, Peoria, 111. (USA) deposited under No. NRRL 2938.

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According to one embodiment of the process according to the invention, the new antibiotics are obtained by cultivating the organism Streptomyces hazeliensis submerged and aerobically in an aqueous culture liquid containing an organic C and N source and extracting the antibiotic mixture formed from the fermentation solution and, if necessary converted into a salt.

The antibiotics that can be produced according to the invention are referred to as sugordomycins. You can go through the following

general formula are shown;

wherein R ₁ and R _{2 are} hydrogen, 2-pyrroyl or 5-methyl-2-pyrroyl, only one of the radicals R and Rp being hydrogen.

Particularly interesting according to the process according to the invention Manufactured antibiotics are made by the general formula

in which the substituents R, and R _{2 have} the meaning given above,
characterized.

The salts which can be prepared according to the invention are

obtained by reacting with suitable bases. Examples for such salts are the sodium salt, potassium salt, ammonium salt or other pharmaceutically suitable base addition salts.

A particularly preferred process product of the formulas I and II is (III) N _f N'-bis {4-hydroxy-8-methyl-7 [4-0-methyl-5,5-dimethyl-3-O- (5 '-methyl-2'-pyrroylJ-cc-L-lyxopyra nosyloxy] - ^ - oxo- ^ Hl-benzopyran-S-yl} -3-methyl-2,4-pyrrole-

dicarboxamide.

This compound has the following structural formula

To the other antibiotics by the invention Processes that can be made include the following:

(IV)

2N £ 4-Hydroxy-8-methyl-7 [4-0-methyl-5,5 «dimethy1-3- _t 0 (2 · -pyrroyl) -aL-lyxopyranosyloxy] -2-oxo-2H-1-benzopyran- 3-y2j -4N ^l - / 4-hydroxy-8-methyl-7 [4-0-methyl-5,5-dimethyl-3-O (5'-methyl-2 ^l -pyrroyl) -aL-lyxopyranoeyloxy] - 2-oxo-2H-1-benzopyran-3-y33 -3-methyl-2,4-pyrroledicarboxamide.

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CBaO

2N {4-hydroxy-8-methyl-7 [4-0-methy 1-5,5-dimethy 1-3-0 = (5 '-methyl-2' -pyrroylJ-a-L-lyxopyranosyloxyJ ^ -oxo ^ H-l-benzopyran-3-yl} 4-N ' - {4-hydroxy-8-methyl-7 [4-0-methyl-5,5-dimethyl-3-0 (2'-pyrroyl) -aL-lyxopyranosyloxy] -2-oxo-2H-1-benzopyran-3 -yl} -3-methyl-2,4-pyrroledicarboxamide.

CH ₉ Q

co «:

(VI)

N, N ^! -bis {4-Hydroxy-8-methyl-7 [4-0-methyl-5,5-dimethyl-3-0 (2'-pyrroyl) -aL-lyxopyrano8yloxy] -2-oxo-2H-1-benzopyran- 3-yl} -3-methyl-2,4-pyrroldicarboxamide.

(vii;

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U67S4t

(5'-methyl-2 · pyrroyl) -aL-l3rxopyranosyloxy] -2-pyran-3-yl} 4-if ^f - {4-hydro3ty-8-methyl-7 [4-0-meti3yl-5,5 -dimethyl-α-II-lyxopyranosyloxy] -2-oxo-2H-1-benzopyran-3-yl} -3-iaethyl-2,4-pyrroldicarboxamld.

-7 [4-0-methyl-5,5-dimethyl

8-methyl-7 [4-O-iaethyl-5,

-methyl-2-pyrroyl) -

2 ^ -pyrroldicarboxamide.

2 "{4-Hydroxy-8-ae1 * yl-714-0-methyl-5,5-dime-thy 1-3-0" (2 ■ -pyrroyl) -aL-lyxopyrene © eyloiy] -2-oxo- 45 » -

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CH ₉

2N £ 4-Hydroxy-8-methyl-7 [4-0-methyl-5,5-dimethyl-aL-13Txopyranosyloxy] -2-oxo-2H-1 ~ benzopyran-3-yl} -41f ^l 14-hydroxy- 8-methyl-7 [4-0-methyl-5,5-dimethyl-3-O (2 '-pyrroyl) -aL-lyxopyranosyloxy] -2-oxo-2H-l-benÄ © pyraji-3-y3 | -3-methyl-2,4-pyrroldicarboxamide.

According to another AasftikrungBform asm inventive method the compounds of formulas III and VI are prepared in such a way that a compound of the formula my

in which R _{5 is} alkyl, aralkyl, phenyl, alkyl-substituted phenyl or halogen-substituted phenyl and R 1 is hydrogen or methyl , with a compound of the formula

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~ ⁷ ~ H67941

■ COX

XII XOC-J

in which X is either chlorine or bromine,

converts and, if desired, the product obtained into a salt convicted.

Streptomyces hazeliensis is an aerobic, air-sporulating representative of the order Actinomycetales and belongs to the genus Streptomyces (Bergey's Manual of Determinative Bacteriology, 7th edition, 1957). Although this organism is associated with Streptomyces griseoflavus strain no. 160, Streptomyces niveus BHRL 2446, Streptomyces spheroides MRL 2449 »Streptomyces Hr. 58383, Streptomyces spirogriseus NRRL 2590, and Streptomyces griseus ATGC 12,318, there are numerous differences between the above organisms and Streptomyces hazeliensis which allow it to be clearly identified that the latter is not identical to any of the earlier species. The differences in morphology and spore color are sufficiently clear to distinguish Streptomyces hazeliensis from all antibiotic-producing Streptomyces species, according to the classification of TG Pridham, C. ¥. Hesseltine and RG Benedict, Appl. Micorbiology (>: 52 - 79 (1958): "A guide for the classification of Streptomyces according to selected groups. ¹¹

Another crucial differentiation is the difference between the antibiotics made from Streptomyces hazeliensis and the novobiocin made from the above organisms. 909841/1692

Cultures of Streptomyces hazeliensis which are grown on suitable culture media, such as tomato-soy agar at 28 ⁰ C for 2-10 days, form an aerial mycelium, dae usually is white and is colored gray to gray-brown in sporulation. A colorless to yellow exudate forms on various nutrient media. Sporophores on Bennett agar and on asparagine agar are mostly elongated to serpentine, sometimes ending in a wide hook or in a loop or in loose, open, characteristically wide (diameter 7 μ) spirals with 1 to 5 loops, the spirals being irregular in shape Shape and distance between the loops are formed. The spores are oval to cylindrical and vary in width from 1 to 1.2 μ, in length from 1.4 to 2.4 μ. Well-formed spirals could not be observed either on Czapek agar or on yeast-malt agar, but small hooks or primitive spirals were observed. Sometimes, starting from one point, up to 5 sporophores appear in a broom-like shape. There are no ball-like, dense or compact spirals, as they are present in the culture of Streptomyces spheroides. Although some sporophores appear in clusters, at their end they are not, as is characteristic of Streptomyces niveus, coiled like a corkscrew. On the contrary, the sporophores of Streptomyces hazeliensis are larger, wider and looser at their end and become narrower and denser at the point of attachment of the hyphae.

To Streptomyces hazeliensis with known Streptomyces, which in a classical way both on complex nitrogenous Culture media, as well as being grown on chemically defined culture media [Pridham and Gottlieb, J. Bact. £ 6,107 115 (1948)], these two types of nutrient media were used. For the methodology and the culture media, see

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ORIGINAL

Littraturetelle by Fridham and Gottlieb and also Gottlieb D., Appl. Microbiology £ s 55-65 (1961) - "A Manual of Method for Pure Culture Study of Bacteria" by the "Committee on Bacteriologio Technic of the Society of American Bacteriologists", 1946) and Wakgoan, "The Actinomyoetea * (Ghronica Botanica Co «1950).

To those used by Bennett and Ceapek all agar media 3treptomycea-hazeliensie-Stämrae agile grew at 22 °, 28 ° and 35 ⁰ C. At the higher temperatures was observed better sporulation. On the other hand, Streptomyces niveue and Streptomyces spheroideß grew well at the lower temperatures but not at all at 35 ° 0. Streptomyces No. 160 and Streptomyces sp. Ifr. Also 583β3 not grow at 35 ⁰ C

The growth of Streptomyces hazeliensis on different culture media is described below. The listed color tones match the color scale available in the Color Harmony Manual 4th Volume, Container Corporation of America, Chicago, Illinois, 1956

Sucrose nitrate aftar (Czapek sucrose); Growth good but no sporulation; The color of the air mycelium is cream (between shade 2 cc and chm gray, scale d). Color of the underside cream-brown, pigment cream (shade approx. 2).

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Glucose- »Ajcaragin-Aaar t Lively growth, slightly raised, smooth surface! color of the Luftmycels pale gray, with pale gray spores, color of the underside pale cream | no soluble pigment.

Qlt & oae-Nahr-Aaari Growth massive, flat, leathery, old strong hold in the agar »color of the aerial mycelium white, color of the underside dark cream; soluble pigment brownish in color (shade 4 ne). Streptomyces spheroides shows massive growth with no soluble figment, while Streptomyces nlveus grows well with the formation of soluble pigment. Streptomyces sp. No. 58383 shows vigorous growth but no soluble figment.

Gl uk ο ae - 31v cerlfl- »agar ;. Lively growth, very deep folds in the agarj surface, wrinkled, with cracks. Vegetative mycelium pale tan, with some rather large drops of pale yellow-colored sxudate. color of the air mycelium partly white to gray-brown (chm * near gray 5 de), approx. 25 # sporulated. Color of the underside coffee brown} soluble pigment pale brown-yellow (shade 3 ie) colored brown.

Amidex-Agar ι Waohetum moderate to good, flat, color of the air mycelium medium gray; color of the underside cream-brown to dark coffee-brown. soluble figment red-brown (shade 4 pe-pg)

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Tomato Paste Oatmeal Agar: Strong growth with good spore formation; a few drops of exudate. Color of the air mycelium gray to gray-brown (ehm. Near gray 5 fe). Color of the underside dark brownish olive with dirty olive-colored soluble pigment.

Skimmed milk agar: substrate growth good, doughy, smooth, without aerial mycelium. Strong production of soluble, dark brown pigment, colored pale brown at the thread ends (shade 5 ni and shade 3 ie).

Peptone iron agar: only a few capsules smooth, doughy, regularly furrowed cones with a bright white surface. The color of the air mycelium is dark cream, the surface is colored white. Soluble pigment of dark brown color (shade 4 pn).

Bennett's Agar: Growth vivid, color of the aerial mycelium gray with gray-brown (reddish) spores. Formation of pale yellow drops of exudate. Color of the underside yellow-brown; soluble pigment colored light yellow-brown (shade 3 pe). A second strain showed no exudate. A third strain formed a spreading yellow exudate that wetted the entire growth surface.

Potato pieces: Growth after two weeks: massive, blackening of the potatoes already after 4 days. On the other hand, Streptomyces spheroides and Streptomyces niveus initially showed massive growth, then slowly increasing good growth with gray

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lent white spore formation with brownish discoloration of the Potato. Streptomyces No. 160 showed a lively, badly wrinkled one Growth.

Carrot pieces; Very good substrate growth of white color, which turns smoke-gray to blue-gray when the spores are formed. Slightly clear yellow exudate. Streptomyces niveus showed massive growth with white aerial mycelium, while no growth was observed with Streptomyces spheroides.

Proteolytic activity

(a) Gelatin: Three tubes were inoculated with a spore suspension using a needle. Before the assessment, the tubes were kept at 3 ° C. for one hour and then brought back to room temperature. After 7 days: no liquefaction, small white colonies on the surface with dark brown, soluble pigment (shade 2 pi-pi) directly next to the colonies. After 14 days: approx. 25-6 liquefaction, fairly good growth on the surface, furthermore a few spherical colonies submerged in the liquefied phase; dark brown soluble pigment. After 21 days: 10076-owned liquefaction, color gelatin brown-yellow (hue 2 pe) while the color of the control tube with a shade von.l - i ca showed. In the literature it is stated that Streptomyces niveus and Streptomyces spheroides show good growth on gelatin but do not form any soluble pigment. The former species shows a partial

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Liquefaction, the latter a rapid liquefaction (occurring within 3-10 days). Streptomyces No. 160 liquefies quickly but does not form a soluble pigment. Streptomyces sp. No. 58383 grows on gelatin scanty and does not liquefy.

(b) milk; All five Streptomyces hazeliensis strains tested form faint, cream-colored surface growth membranesj no coagulation or peptonization within 2 days; pH (glass electrode) unchanged as control tube: 6.0 to 6.2.

After 7 days, all stems form well-formed, firm, creamy-to-cream slightly brown-colored surface growth membranes, with a brown ring on the tube wall; Color of the soluble Pigments creamy yellow to slightly orange. Am trunk showed slight peptonization; pH range 5 »3 - 5.7 (control pH 5.7 - 6.1).

Growth of the surface after 14 days good without sporulation; Aerial mycelium leathery; Color pink- ^ cream to pale brown or purple: no coagulation but slight peptonization. Color of the non-peptonized phase: cream-orange (shade 4 ca-en); pH 5.2-5.7 (control pH 5.6).

Streptomyces Mr. 160 showed strong coagulation and strong peptonization of milk,

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Oil reductase activity

(a) Organic solution: After 14 days: good growth, thick, white skins; dark yellow colored, soluble pigment. Weak nitrite formation that increases after 21 days ; strong surface growth with beginning spore formation, color light gray.

(b) inorganic solution; Slight growth with no reduction within 14 days; good surface growth after 21 days, but no nitrite formation, nitrate still present. Smith et al., Streptonivicin, A New Antibiotic, Antib. and Chemother. 6, 135-142 (1956), found that Streptomyces niveus shows no nitrate reduction, neither in synthetic nor in natural nitrate solution; Streptomyces spheroides shows only a slight reduction of nitrate agar to nitrite in 4 days, while Streptomyces sp. No. 58383 a strong positive nitrite formation was observed.

Formation of hydrogen sulfide; After 21 hours, agar slant cultures showed very weak growth but strong H ₂ S development; after 14 days: colonies with a shiny surface with discoloration of the culture medium from dark blue to dark brown.

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Tyrosine agar: For all 5 strains: tyrosinase reaction positive. Slight growth after 14 days. The color of the air mycelium is white, the underside is white. No pigment formation after 7 days. Color of the pigment after 14 days: beige-gray, shade 3 ec-ge (control shade 1 - \ db).

Utilization of carbon: xylose, arabinose, rhamnose, glucose, fructose, sucrose, lactose, raffinose (+), inositol, cellobiose, trehalose, citrate, maleate and succinate are utilized. Inulin, maltose, mannitol, sorbitol, acetate, formate, oxalate and tartrate are not used.

It should be noted that Streptomyces niveus and Streptomyces spheroides contain inulin, maltose, mannitol and sorbitol utilize in contrast to Streptomyces hazeliensis, whereby, in contrast to the latter, they do not rely on citrate or succinate culture media. Streptomyces niveus utilizes formate, oxalate, tartrate and sodium citrate.

Utilization of nitrogen: L (+) arginine is utilized. Methionine, sarcosine, creatine, amino isobutyric acid, taurine and betaine are not utilized.

Antagonistic properties: Forms the sugurdomycin antibiotics, but does not form novobiocin even under the most varied of conditions. Streptomyces spheroides and Streptomyces niveus, on the other hand, both produce novobiocin but no sugordomycin antibiotic, even on nutrient media on which strepto-

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myces hazeliensis only forms sugordomycin antibiotics.

In summary, it can be said that Streptomyces hazeliensis differs morphologically from both Streptomyces niveus as well as from Streptomyces spheroides, in that the tips of the sporophores do not have corkscrew-like loops form, as is characteristic of Streptomyces niveus and in the absence of the dense, compact, ball-like spirals, as they are typical for Streptomyces spheroides.

While Streptomyces niveus and Streptomyces spheroides very soon tear the agar medium in a characteristic way and split, this phenomenon is not usually observed in Streptomyces hazeliensis. Streptomyces niveus a soluble pigment on various agar media while Streptomyces spheroides does not form any pigment. On the other hand, Streptomyces hazeliensis normally forms a pigment, namely stronger than Streptomyces niveus.

It has also been found that the composition of the culture media in which Streptomyces hazeliensis is grown significantly influences the following variables: (a) the amount of antibiotics formed, (b) the rate of formation the antibiotics and (c) the type of antibiotic mixture in the solution.

The content of fermented solutions in bottles or

Fermenting kettles are made by the usual "cup-plate ^H agar diffusion

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sion method, using Staphylococcus aureus as a Test organism, determined (D.C. Grove and W.A. Randall, Assay-Methods of Antibiotics. A Laboratory Manual, Medical Encyclopedia Inc., New York 1955, pages 7-16).

The amount of antibiotics dissolved in 1 ml of aqueous buffer, a zone of inhibition of 18 mm in diameter on the Test plate forms is arbitrarily referred to as a unit of activity. The colonies are 8 mm in diameter. That pure antibiotic III has an activity of 5500 units per mg.

As a preferred source of nitrogen for growing the Antibiotics are used green or yellow pea meal. Also with soybean flour, corn "Distillers Solubles" (dry grain residue and soluble part from corn burning), cottonseed flour, coconut protein flour, flaxseed flour and "SoIuferm" (Dissolvable dry residues and grains from the maize Fermentation.) Medium to good yields were achieved, but that usually below those obtained with peas. An addition of small amounts of ferrous sulfate, or bone ash dried residue from corn steep liquor to the peas did not increase the yield of the antibiotics; however, the Yield after adding calcium carbonate and dipotassium phosphate.

Adding a carbohydrate to natural nitrogen sources had several effects. Certain Carbohydrates impaired the formation of the antibiotics.

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Only then did other carbohydrates deliver a good yield Antibiotics, when the ratio between carbon and nitrogen has been put in a certain relation. A Addition to a yellow pea medium for $ 0.25 a sodium salt of organic acids, completely inhibited the formation of antibiotics. In this experiment, a Basic nutrient medium used, which is $ 3 from yellow pea meal, 0.1 $ calcium carbonate and 0.1 $ dipotassium phosphate.

The effect that was achieved by adding carbohydrates to the production of antibiotics is from the following The table in which the basic nutrient medium contained 2 $ yellow pea meal can be seen:

Table 1

Addition to the basic nutrient medium

$ 1 cornstarch (control) $ 1 mannitol 1 $ glycerin 2 $ glycerin 1 $ maltose 1 $ arabinose 1 $ rhamnose 1 $ lactose $ 1. Inositol

S. aureus units per ml Gesaj r'tl dissolution in 7 days

180

120

50

traces

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In the following experiments, which are compiled in Table 2 A basic culture medium was made of yellow pea meal with 0.1 $ calcium carbonate and 0.1 # dipotassium phosphate used.

Table 2

Addition to the basic nutrient medium

S. aureus units Maximum output per ml of total solution prey after

Days

l, 0 # corn starch (control) 590 7th 0.596 corn starch 310 6th 2.0 # cornstarch 400 7th I96 glucose 230 7th 29 & glucose 25th 7th l? o brown sugar IQO 7th 296 brown sugar 0 7th 1 # dextrin 360 7th 296 dextrin 290 VJl 196 xylose 0 7th

It was already noted above that an addition of 0, l # Calcium carbonate and 0.1? Ί dipotassium phoephate the yield of antibiotics elevated. When both calcium carbonate and dipotassium phosphate are absent, or when the concentration of one or both Salts are increased to $ 0.5, the yield of antibiotics is significantly worsened.

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Antifoam agents are commonly used to prevent the To prevent foaming, which occurs with large fermentation batches. It has been found that quite a number of vegetable oils from e.g. saffron, cottonseed, sesame, coconut, soybeans or animal oils such as lard or oleic acid, etc. can be used without reducing the yield of the antibiotics. Silicone-based antifoam agents also decreased the yield of antibiotics does not.

The following table compares the yields of the antibiotics obtained on the same culture media Streptomyces hazeliensis, Streptomyces niveus and Streptomyces spheroides were formed. It should be noted that, unlike Streptomyces hazeliensis, the distinct, forms inhibition zones with sharp edges, in Streptomyces niveus and Streptomyces spheroides only fuzzy and less pronounced Inhibition zones occur. There is therefore a clear difference in the formation of these inhibition zones between the sugordomycins and the novobiocin.

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Table 3

co
O
co
ex" Nutrient components in % Staphylococcus aureus "Cup-plate" units / ml Strep,
hazeliensis Strep,
niveus Strep,
spheroides 6 days Strep,
hazeliensis Strep,
niveus Strep,
spheroides 1692 Pea meal: 2, corn starch: 1
CaCO-: 0.1, K ₂ HPO ₄ : 0.1 4 days 69 50 47 320 53 5 Corn steeple: 4, glucose: 5
KH2PO .: 0.05, NaNO ,: 0.3,
'MgSO ₄ ⁴ . 7H ₂ O: 0.1 ^D 0 0 0 0 traces 9 Soy flour: 2, brown sugar: 2
Dried residue from corn swell
water: 0.25
K ₂ HPO ₄ : 0.1 traces 0 0 13th 0 1
0! R
* I.
Ϋ
I. Dried "Distillers Solubles"
(Soludri): 2
Glucose: 2, K ₂ HPO ₄ : 0.1, CaCO: 0.1 0 54 37 0 69 5 Pharmamedia: 2.5, glucose: 2.5 0 48 10 0 3 traces "Fermamine" ² type III: 2, strength: 1
K ₂ HPO ₄ : 0.1, MgSO ₄ : 0.05 0 8th 19th 0 6th 6th

^ ■ Pharmamedia is a 56% protein and 24% carbohydrate material made from cottonseed meal, (Traders Oil Mill Col., Fort Worth, Texas)
"Fermamine" is an enzymatic protein hydrolyzate (Sheffield Chemical Co., Norwich, NYj

U67941

As can be seen, a "preferred method of production is." of a mixture of sugordomycins by cultivating Streptomyces hazeliensis as indicated above, characterized in that that pea meal is used as both a carbon and nitrogen source. There is an advantageous way of working in that the cultivation is carried out in the presence of approx. 0.5 - 3%, in particular approx. 0.1 $ dipotassium phosphate and approx. 0.1 3 $, especially about 1-2 $ of a carbohydrate e.g. one Starch or any of the sugars mentioned on page 15, lines 6-8. The breeding becomes advantageous carried out for about 3-8 days and at a temperature of about 20 - 35 ° C with an air volume of 15 - 300 liters / minute aerated per 200 liters of nutrient solution. The antibiotic geraisch can be isolated from the fermentation solution in several ways:

His method is to give preference to the fermentation solution with phosphoric acid, to a pH of 27, in particular pH 4 - 4.5 adjusts, although other mineral acids e.g. hydrochloric acid and sulfuric acid can also be used. If necessary, a filter aid, e.g. diatomaceous earth, is added. The acidified solution is filtered, the filter cake obtained is in an organic solvent, for example in a lower one Alkanol such as methanol, ethanol, butanol, isobutanol etc. or in acetone, methyl isobutyl ketone, butylacetab oclor in a mixture of these solvents, e.g. acetone / methyl chloride Methanol / benzene etc., especially digested in butanol md then filtered or centrifuged. The clear filtrate

ORIGINAL INSPECTED

" ²³ " 1A679A1

or the extract is adjusted to a pH of approx. 6-7 with alkali, for example with a 50 # sodium hydroxide solution. The solvent is removed, eg, the concentrate formed by distillation under reduced pressure at a temperature below 50 ⁰ C. of about 1/40 of the original volume with a non-polar solvent, for example with petroleum ether (boiling range 60-90 ° C), benzene, Hexane, pentane, diethyl ether etc. are added, the crude antibiotic mixture precipitating as the sodium salt or as a mixture of the sodium salt with the free form, which can easily be separated off by sedimentation, filtration or centrifugation.

Another way of working is to isolate the raw antibiotic from the fermentation solution in such a way that the mycelium is filtered off, the filtrate is adjusted to a pH of approx. 1-7 with a mineral acid and then the acidified filtrate with a water-immiscible solvent such as butanol, butyl acetate, ethyl acetate, ether, chloroform, MethyIisobuty! Ketone etc., shaken out.

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The organic phase is separated from the aqueous layer. The raw, antibiotic Geraisch can either Evaporation of the solvent obtained or on an ion exchanger, such as Zeo-Karb H, Dowex-50 W, Amberlite IRC-50, Amberlite IR-4, Amberlite IR-100, Ionac A-300, Dowex-1-Chloride,

• Dowex-1-Base, Dowex-1-Pormat etc. or on activated charcoal adsorbed and then isolated by elution with a solvent such as methanol. The mycelium is filtered off and with an organic solvent such as a lower alkanol, e.g., methanol, ethanol, butanol, isobutanol Etc.; or extracted with acetone, butyl acetate, acetomethylene chloride, methyl isobutyl ketone, methanol benzene, etc. That after

raw antibiotic left after evaporation of the solvent The mixture can then be combined with the antibiotic mixture obtained from the piltrate.

One can purify the raw sodium salt of the antibiotic mixture or the raw acidic antibiotic by using if the antibiotic mixture is dispersed in acetone, a sufficient amount of mineral acid, e.g. hydrochloric acid, is added to an acidic pH, e.g. of about 2-6, preferably about pH 4, the insoluble material filtered off and the filter cake washes out with a small amount of acetone. The piltrate is combined with the washing solution and reduced under reduced pressure Pressure evaporated. The concentrate, reduced to about 1/6 of the original volume, is mixed with water and benzene

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offset. The solid suspension that forms here in both liquid phases consists of the free acidic antibiotic Mixture which is filtered off and digested in warm methanol. The solid that separates out on cooling Shares are separated. They consist of the purified, free, acidic, antibiotic mixture.

The raw antibiotic mixture can also be purified by making the benzylamine salt. For this the antibiotic mixture is in an organic solvent, e.g. in n-butanol, n-butyl acetate, ether, ethyl acetate etc., dissolved and with an excess of benzylamine treated. The benzylamine salt of the antibiotic mixture precipitates, with numerous impurities contained in the antibiotic mixture were present, remain in the organic solvent. The precipitated benzylamine salt is filtered off. The free antibiotics can be obtained from the benzylamine salt by treating with an aqueous mineral acid, e.g., aqueous hydrochloric acid. The free antibiotic mixture can be removed from the aqueous phase with the aid an organic solvent which is immiscible or only slightly miscible with water, e.g. with n-butanol, n-butyl acetate, ether, Ethyl acetate, etc., are extracted. The mixture remaining after evaporation of the solvent becomes e.g. a warm mixture of methanol / benzene reprecipitated.

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Other amines experimentally used in cleaning the antibiotic mixture used have not turned out to be proven. Only the benzylamine salt was found to be completely satisfactory. To other bases that are not found to be suitable proven to include calcium hydroxide, procaine, ethylenediamine, diethanolamine, monoethanolamine, N, N'-dibenzylethylenediamine, Dimethylethanolamine, hydroxyethylethyldiamine, diisobutylamine and PurfuryIamin.

The raw antibiotic mixture can also be purified by dividing the raw mixture between an organic one Solvent, e.g. butyl acetate, ether or mixtures of chloroform, isopropanol etc. and an aqueous one Buffer solution with a pH of approx. 6.8-11, distributed. The antibiotic mixture can pass through from the aqueous buffer solution Acidification, e.g. with a mineral acid such as hydrochloric acid, sulfuric acid, etc. up to a pH of approx. 2-5 can be isolated. the resulting suspension can with an organic, with water not miscible or only slightly miscible, solvents, e.g. with n-butanol, n-butyl acetate, ether, ethyl acetate, etc., extracted will. The cleaned, acidic, antibiotic mixture is then, e.g. by evaporating the organic solvent, isolated.

The arcibiotic purified by one of the above methods Mixture can then with the help of a counter

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Power distribution system, e.g. according to Craig, can be separated into its components. The organic one used here solvent is immiscible or only slightly miscible with water, e.g. n-butanol, n-butyl acetate, ether, ethyl acetate, Chloroform isopropanol, etc. The aqueous phase is on a pH of approx. 6-9, in particular approx. 8-8.9 »is set. The buffers usually used for this purpose can be used as buffers Phosphates and borates can be used.

The compounds of the formula XI, which are used as starting compounds used in one embodiment of the process according to the invention for the production of sugordomyeins can be obtained in the following ways:

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XIII

XIY

XY

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In this reaction scheme, R_ and R have the above given meaning.

The process shown in the reaction scheme above can e.g. be carried out as follows:

a) 4-Benzyloxy-7a- (2,3-0-carbony 1) -noviosyloxy-8-methyl-coumarin (XIII) is with a 2-methyl-pyrrole-Grignard compound, e.g. with a 2-methyl-pyrrole-magnesium halide, especially with the bromide. The reaction product obtained is in an aqueous, acidic medium, for example in an aqueous Mineral acid solution, such as dilute hydrochloric acid, decomposed.

b) The 4-benzyloxy-7a-3-0- (5 ^l- methyl-2 ^l pyrroyl) -noviosyloxy-8-methyl-coumarin formed is treated with hydrogen in the presence of a hydrogenation catalyst, for example palladium carbon, etc.

c) The 4-hydroxy-7a-3-0- [5'-methyl-2 ^l pyrroyl] -novioxyloxy-8-methyl-coumarin formed is diazotized! Treated aniline.

d) The 3-benzene-diazo-4-hydroxy-7a-3-0- [5'-methyl-2'-pyrroyl] -novioByloxy-8-methyl-coumarin obtained is treated with an acylating agent containing, for example, the group R, -C-, where R, has the above meaning, e.g. with acetic anhydride in

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Pyridine implemented. Other suitable acylating agents also include the acyl halides, e.g., acetyl chloride, etc.

e) The 3-benzene-diazo-4-hydroxy-7a-2-0-acyl-3-0- [5'-methyl-2'-pyrroyl] -noviosyloxy-8-methyl-coumarin formed in this way is then treated with hydrogen in the presence of a hydrogenation catalyst, e.g. palladium carbon, to 3-amino-4-hydroxy-7oc-2-0-acyl-3-0- [5'-methyl-2'-pyrroyl] -noviosyloxy-8-methyl-coumarin reduced.

The reaction of the compounds of the formula XI with compounds of the formula XII is preferably carried out in such a way that 3-amino-4-hydroxy-7a-2-0 ^ 5'-methyl-2-pyrroyl] - _Q novosyloxymethyl -coumarin, in which acyl represents the above R, -6 group, in which R has the meaning given above, with a 2,4-dihalocarbonyl-3-methyl-pyrrole, in the halogen chlorine or bromine means, converts to a compound of the formula III.

The antibiotics obtainable according to the invention are characterized by being effective against Staphylococcus aureus are highly active. In addition, the antibiotics are against a large class of gram-positive and gram-negative bacteria, e.g. against Aerobacter aerogenes, Mycobacterium ph IhL, Proteus vulgaris, Sarcina lutea PCI-IOOl, etc. effective.

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The antibiotics available according to the invention are therefore suitable for combating bacterial infections such as those caused by Staphylococcus aureus. Alternatively, the purified antibiotic mixture can also be used as a Therapeuticura.

The products of the process can be used as medicaments, e.g. in the form of pharmaceutical preparations, which they or their salts in a mixture with a pharmaceutical suitable for enteral or parenteral administration, organic or inorganic inert carrier material, such as water, gelatin, lactose, starch, magnesium stearate, Contains talc, vegetable oils, gum, polyalkylene glycols, petroleum jelly, etc. The pharmaceutical preparations can be in solid form, e.g. as tablets, coated tablets, suppositories, capsules, or in liquid form, e.g. as solutions, suspensions or emulsions. If necessary, they are sterilized and / or contain auxiliaries, such as Preservatives, stabilizers, wetting agents or emulsifiers, Salts to change the osmotic pressure or buffers. They can also contain other therapeutically valuable substances.

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example 1

Streptomyces hazeliensis is stored in spore form either on a tomato and oatmeal agar in Medicinal bottles or in freeze-dried form, with the spores suspended in 10 - 6 bovine serum or in 10% skimmed milk are. In a 6-liter shake bottle with 3 liters of water Nutrient solution, consisting of:

1% pea meal,

1% corn starch,

0.196 calcium carbonate,

0.1% dipotassium phosphate, and

0.15c lard

is added to an aqueous spore suspension and incubated at 28 ⁰ C for 3 to 5 days on a shaker at 200 rpm until a brisk growth occurs. The incubation is carried out in a vessel that is suitable for aeration of the nutrient solution, for example in the above-mentioned shaker bottle or in a gas washing bottle. The inoculation solution obtained in this way is used to inoculate 250 liters of aqueous nutrient solution in a 400-liter fermentation kettle. The nutrient solution consists of:

3% pea meal,

0.1% calcium carbonate,

0.1% dipotassium phosphate,

0.03% silicone paste (Dow-Corning silicone A paste), and

90984 1/1692 _0> £ 1 pork lard.

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Instead of this nutrient solution, 250 l of a nutrient solution of the first-mentioned composition can also be used. The fermentation kettle is kept at a temperature of 28 C for 3 to 4 days. The air volume is 225 liters / minute. During this time the viscosity increases significantly, while the pH falls to about 6.6 to 5.9. The inoculum thus obtained is transferred to a 35'OOO-liter fermentor with 23 ¹ 1 OOO sterilized nutrient solution. The nutrient solution consists of:

3 % ground pea meal $ 0.1 calcium carbonate, $ 0.1 dipotassium phosphate and

0.03-0.05 # Dow-Corning Silicone A suspended in

$ 0.1 soybean oil.

The solution is left to ^{ferment at 28 °} C. for 5-8 days. The fermentation process is controlled with the help of the in vitro texts. The air volume is set to 700 liters / minute during the first 24 hours, then increased to 2800 liters / minute during the following 24-72 hours and to 4000 liters / minute during the further 72-168 hours. The pH of the fermentation solution is then adjusted to 3.9 by adding a syrupy, 85% phosphoric acid. After adding 750 kg of diatomaceous earth, the acidified solution is filtered. The filter cake is slurried in 7,500 liters of n-butanol and stirred vigorously. The slurry is allowed to stand overnight without stirring. The clear butanol extract obtained by centrifugation is then

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closing with 1482 ml of 50 $ NaOH solution, neutralized to a pH value of about 7, and carefully concentrated under reduced pressure at a temperature below 5O ⁰ C to a volume of 200. 1

This concentrate is added with vigorous stirring in 1000 1 of petroleum ether (boiling range 60-9O ⁰ G). The precipitating crude sodium salt of the antibiotic mixture presumably consists of a mixture of the acidic and partially neutral form of the various components of the antibiotic mixture. The crude sodium salt is isolated by centrifugation and then dried.

Example 2 Purification of the raw sodium eel ze3

a) By partitioning between a solvent and a buffer

50 g of the crude sodium salt obtained above are suspended in 1 liter of methanol and stirred for 30 minutes. Dan insoluble Material is filtered off. The filtrate is concentrated under reduced pressure to a thick syrup. This is suspended in 25 ml of methanol and 625 ml of 0.1 η hydrochloric acid. The suspension is extracted 3 times with 1.5 liters of ether each time.

90984 1/1692

shakes. The combined ether extracts (4.24 liters) are mixed twice with 1.1 liters of A M phosphate buffer pH 6.2, then twice extracted with 1.1 liters of ^ M phosphate buffer pH 7.0. Little activity is lost with this treatment. Afterward the ethereal solution is added twice with 1.1 liters of .g M phosphate buffer each time pH 7.8 extracted, whereupon the antibiotic mixture migrates into the aqueous phase. The suspension is on a pH adjusted to 7.0 and extracted with n-butanol, whereby the precipitate goes into solution. The n-butanol is evaporated off under reduced pressure. The residue is in r ^ M phosphate buffer pH 6.5 suspended. The insoluble, purified antibiotic mixture is filtered off and dried.

b) By making the benzylamine salts

100 g of the crude sodium salt are dissolved in 10 liters of r = · M disodium phosphate buffer pH 9.5 dissolved. The insoluble fractions are filtered off in the presence of a piltering aid. The dark colored filtrate is adjusted to a pH of 2.5 with concentrated hydrochloric acid. The suspension obtained is then extracted with the same amount of butyl acetate, the precipitated antibiotic going into solution. the Butyl acetate solution is reduced to volume under reduced pressure concentrated from 1800 ml. After separating a small amount of insoluble material, the filtrate becomes with a solution 100 ml of benzylamine in 100 ml of butyl acetate are added. The benzylamine salt which settles in the form of a fine precipitate the antibiotics are filtered off, washed with a little butyl acetate and then with anhydrous ether.

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20 g of this benzylamine salt of antibiotics is in 140 ml Dissolved methanol and, after adding 190 ml of water, adjusted to a pH of 2.5 with the aid of hydrochloric acid. To the To lower the viscosity of the suspension, another 300 ml Water added. The mixture is then extracted twice with 400 ml of butyl acetate each time. The combined butyl acetate extracts are concentrated to approximately 800 ml. The crude acid which precipitates out at this concentration is filtered off and mixed with washed in cold methanol. This product can be reprecipitated by dissolving it in a mixture of hot methanol and benzene. The antibiotics, which are deposited in finely divided form, melt after drying at 22'8 - 230 C.

c) By extraction with acetone

50 g of the crude sodium salt are suspended in 600 ml of acetone and, after addition of 30 ml of 10% aqueous hydrochloric acid, stirred for 12 hours. The insoluble components are filtered off and washed with a little acetone. The combined filtrate and washing acetone are evaporated under reduced pressure to about 100 ml. 500 ml of water and 200 ml of benzene are added to this concentrate, resulting in a suspension of solid material in 2 liquid phases. The precipitate of this suspension consists of the crude, acidic, antibiotic mixture. It is dispersed in 100 ml of methanol at 40-5O ⁰ C, then cooled to room temperature and left to stand in the cold for 12 hours. The solid that separates out consists of

the purified antibiotic mixture.

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Example 5

Separation of the purified antiseptic mixture into its Components

5 g of the purified antibiotic mixture obtained by the purification process with acetone are placed in a 200-tube Craig countercurrent distribution apparatus between an upper aqueous phase of γ- K ₂ HPO. Buffer (pH 8.2) and a lower organic phase consisting of equal parts of isopropyl alcohol and chloroform distributed. After equilibrium has been established between the buffer and the organic phase, partitioning is carried out in the usual manner through the 200 tubes, each containing 40 ml of each phase. The dispenser provides a mixture of antibiotics III, IV, V and VI in tubes 1-140 and a mixture of antibiotics VII, VIII, IX and X in tubes 141-200.

This procedure is then repeated in 200 additional tubes with a pH 8.9 buffer.

In the upper phase of the repeated procedure (total of 400 tubes) the antibiotic III is in the tubes 120 - 152. The antibiotics IV and V are in the tubes 161 - 170. The antibiotics V and VI are in the Tubes 185-195. It has not been established with certainty whether tubes 161-170 contain the antibiotic of the formula IV or V is present and which antibiotic is in the

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Tube 185-195 is present. Contain tubes 153-160 either a mixture of antibiotics III and IV or III and V and the tubes 171-184 contain a mixture of the Antibiotics IV and V.

In the lower phase, the tubes contain the same antibiotic Distribution as in the upper phase.

The antibiotics are poured together from the solvent phase isolated from tubes of the same content. The antibiotic III is e.g. by pouring the tubes together 120-152 and obtained by evaporating the collected solvents to dryness. The buffer phase of the above Groups of tubes are acidified to pH 2.5. The antibiotic is in butanol or in a mixture of Chloroforin / isopropyl alcohol (1: l) extracted. The solvent extract is evaporated to dryness, the antibiotic separating out as a residue.

The antibiotic III obtained by the countercurrent distribution described above is then passed through a further 200 tubes. 3 isomers are obtained: namely in tubes 90-100, in tube 120 and in tube 140. This indicates the presence of various closely related isomers, the absolute tint of which is currently unknown. All of these isomers are bi > ■ ·,] ': iv, dh. they have high activity against Staphylococcus us.

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■ ψ U679A1

The mixture of antibiotics VII, VII, IX and X isolated above is obtained with the aid of the countercurrent distribution according to Craig with 200 tubes separated into a mixture of antibiotics VII and VIII and into a mixture of antibiotics IX and X. That used Solvent system consists of the same parts of the same organic phase as used above, i.e. an equal volume mixture of isopropyl alcohol and chloroform, while the aqueous phase consists of a ^ M dipotassium phosphate buffer pH 7.0. After the equilibrium between the solvent and the buffer has been established, the The above antibiotic mixture VII - X was poured into the apparatus and passed through the 200 tubes. The mixture of antibiotics VII and VIII is in the tube 110 and the mixture of antibiotics IX and X in the tube 170.

Example 4

5.3 g of 3-amino-4-hydroxy-7a-2 ~ 0-acetyl-3-0- [5'-methyl-2'-pyrroyl] -noviosyloxy-8-methyl-coumarin are in 50 ml abs. Pyridine dissolved with a solution of 700 mg of 2,4-dichlorocarbonyl-3-methyl-pyrrole in 20 ml abs. Tetrahydrofuran was added dropwise with stirring and cooling. The reaction mixture is 12 Held at room temperature for hours. The dark colored solution is then diluted with ethyl acetate, one after the other washed with 1 η sulfuric acid and water, dried over sodium sulfate and concentrated under reduced pressure. The one left behind brown foam (516 g) is dissolved in approx. 80 ml of liquid ammonia. The ammonia is slowly evaporated. Of the

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I -T

Residue turns yellow in water and in a small amount of alcohol. The solution is washed with ethyl acetate. The precipitate which separates out after acidification with dilute phosphoric acid is extracted with ethyl acetate. The solution is washed with water, dried over sodium sulfate and evaporated to dryness. The residue, a brown foam, is crystallized from a mixture of methanol / benzene; Melting point 234 235 ⁰ C. After recrystallization from acetone! Tril / acetone, the antibiotic N, N! -Bis {4-hydroxy-8-methyl-7 [4-0-methy 1-5,5 -dime thy 1- 3-0- (5 * -me thy 1-2'-pyrroyl) -α-L-lyxopyranosyloxy] -2-oxo-2H-1-benzopyran-3-yl} -3-methyl-2,4-pyrroldicarboxamide ( III), at 238 - 239 ⁰ C (decomposition).

A umkristallieierte again from a mixture of Aceteasigeeter / dimethylformamide / water sample melts at 258 ⁰ C with decomposition.

The antibiotic given above has a decomposition point which is difficult to determine precisely because it is strongly influenced by the rate of temperature rise and the melting point apparatus.

The 3-amino-4-hydroxy-7ct-2-0-acetyl-3-0 [5'-methyl-2'-propyl] -noviosyloxy-8-methylcoumarin used as the starting product can be made as follows:

In a solution of methylmagnesium iodide (prepared from 1.25 g of magnesium and 3 ml of abs ml methyl iodide in j 50th ether) is added dropwise

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8 g of 2-methylpyrrole in 30 ml of abs. Aether too. The solution is heated under reflux conditions for ΙΟ minutes and then poured into a solution of 4.8 g of 4-benzyloxy- [2,3-0-carbonyl-a-noviosyloxy] -8-methyl-coumarin (VIII) in 2OG ml of abs. Benzene was added dropwise, with a precipitate separating out immediately. The mixture is stirred for 30 minutes and then diluted hydrochloric acid is carefully added. After addition of ethyl acetate, the organic phase is washed successively with water, dilute potassium bicarbonate solution and again with water, dried over sodium sulfate and evaporated under reduced pressure. The remaining 4-benzyloxy-7 <x-3-0- [5'-methyl-2'-pyrroyl] -novi-> osyloxy-8-methyl-coumarin is crystallized from benzene. The compound melts after re-crystallisation from acetonitrile at 131-132 ⁰ C with decomposition.

4 g of 4-benzyloxy-7a-3-0- [5'-methyl-2'-pyrroyl] -novioeyloxy-8-methyl-coumarin are dissolved in 200 ml of ethyl acetate and hydrogenated after adding 2 g of palladium-carbon. Ee are 170 ml Absorbed oxygen (the theoretical amount is 150 ml). The solution is filtered and evaporated to dryness. One Sample of the remaining yellowish compound (3.2 g) is crystallized from acetonitrile for analysis; Melting point 173 175 ° C (Decomposition).

1.6 g of aniline are dissolved in 120 ml of water and 3.2 ml of concentrated hydrochloric acid and at 5 ° with a solution of 1.1 g of sodium nitrite in 10 ml of water are diazotized. 10 minutes after the addition of the nitrite is complete, a solution of 6.4 g

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Sodium acetate in 30 ml of water was added. After 5 minutes, a solution of 3.2 g of 4-hydroxy-7oc-3-0- [5'-methyl-2'-pyrroyl] -noviosyloxy-8-methyl-coumarin in 40 ml of alcohol is added dropwise with stirring at 5 ° registered. A yellow precipitate forms. The reaction mixture is stirred for a further 3 hours at 5 °. The precipitate is then extracted with ethyl acetate, washed successively with water, 1 η hydrochloric acid and again with water, dried over sodium sulfate and evaporated under reduced pressure. The remaining 3-benzene-diazo-4-hydroxy-7a-3-0- [5'-methyl-2 ^! -pyrroyl] -noviosyloxy-8-methyl-coumarin melts after recrystallization from benzene at 174-176 ⁰ C.

6.5 g of 3-benzene-diazo-4-hydroxy-7a-3-0- [5 · -methyl-2'-pyrroyl] -noviosyloxy-8-methyl-coumarin are dissolved in 40 ml of acetic anhydride and 20 ml of pyridine and stand for 18 hours at room temperature calmly. The excess of acetic anhydride is then decomposed with water. The solution is extracted with ethyl acetate. The extract is made with 1 η hydrochloric acid and with water washed, dried over sodium sulfate and evaporated under reduced pressure. The remaining 3-benzene-diazo-4-hydroxy-7oc-2-0-acety 1-3-0- [5'-methyl-2'-pyrroyl] -noviosyloxy-8-methyl-coumarin After recrystallization from isopropanol, it melts at 151-153 ° C (decomposition).

5.6 g of 3-benzene-diazo-4-hydroxy-7a-2-0-acetyl-3-0- [5'-methyl-2'-pyrroyl] -noviosyloxy-8-methyl-coumarin are in 250 ml abs. Dissolved alcohol and hydrogenated after adding palladium-carbon.

-43- U67941

410 ml of hydrogen are taken up (the theoretical amount is 400 ml). The solution is filtered and used Dry evaporated. The residue is dissolved in ethyl acetate. The solution is washed with 0.5 η hydrochloric acid and with water, dried over sodium sulfate and evaporated under reduced pressure. 5.3 g of 3-amino-4-hydroxy-7a-2-0-acetyl-3-O- [5'-methyl-2'-pyrroyl-noviosyloxy-S-methyl-coumarin are obtained obtained as a yellow-brown foam.

Example 5

Manufacture of tablets with the following composition:

N, N ¹ -bisU-Hydroiy-8-methyl-7 [4-0-methyl-5,5-dimethyl-3-O- (5'-methyl-2'-pyrroyl) -aL-lyxopyranoeyloxy] -2- oxo-2H-1-benzopyran-3-yl} -3-methyl-2,4-pyrroldicarboxamide 100 mg

Dicalcium phosphate 60 mg

Corn starch 157 mg

Magnesium stearate 3 mg

320 mg

The active ingredient is mixed with the dicalcium phosphate, the corn starch and the magnesium stearate and passed through a grinding machine guided. The mixture is then briquetted on a tabletting machine, the resulting Briquettes are crushed again and passed through a sieve with a mesh size of approximately 1.19 mm. From the obtained homogeneous Mixture, tablets of 320 mg weight are pressed.

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Example 6

Manufacture of ampoules:

The active ingredient and the sterile water used for injection are ampouled separately. The active ingredient ampoule contains • 500 mg N, N'-bis {4-hydroxy-8-methyl-7 [4-0-methyl-5,5-dimethy1-5-0- (5'-methy1-2'- pyrroyl) -oc-L-lyxopyrano8yloxy] -2-oxo-2H-1-benzopyran-3-yl} -3-methyl-2,4-pyrroldicar "boxamide. The water ampoule contains 5 ml of water. Both ampoules are sealed for 2 hours Sterilized at 125 ^° C. The active ingredient is dissolved in the water immediately before the injection.

Example 7

Production of capsules with the following composition:

N, N'-bis {4-Hydroxy-8-methyl-7 [4-0-methyl-5,5-dimethyl-3-O- (5'-methyl-2'-pyrroyl) -aL-lyxopyranosyloxy] - 2-oxo-2H-1-benzopyran-3-ylJ 3-methyl-2,4-pyrroledicarboxamide

Cornstarch talc

The active ingredient is mixed homogeneously with the corn starch and passed through a grinding machine. After adding the Talks, the mixture is thoroughly homogenized. The filling) takes place in gelatin capsules of size 1.

9098 /, 1/1692

100 mg 200 mg 10 mg 310 mg

Claims (10)

Claims 1. A method for obtaining an antibiotic mixture, characterized in that the organism Streptomyces hazeliensis is cultivated submerged and aerobically in an aqueous culture liquid which contains an organic C and N source • and the antibiotic mixture formed wins from the fermentation solution and optionally converted into a salt. 2. The method according to claim 1, characterized in that pea meal is used as the C and N source. 3. The method according to claims 1-2, characterized marked ) draws that you can extract the antibiotic mixture by extracting it the clarified solution and the mycelium with the help of a solvent wins. 4. Process according to claims 1-3 »characterized in that the culture liquid is additionally inorganic > Add salts and cultivate the organism for approx. 2-6 days until the medium has become strongly antimicrobial, after which the antibiotic mixture formed is obtained from the fermentation solution and separates it into its antibiotic components. 9 O 9 8 A 1/16 9 2 U67941 5. The method according to claim 4, characterized in that one uses an antibiotic of the formula isolated by countercurrent distribution over multiple solvent systems from an organic phase that is only limited is miscible with water and an aqueous phase that is adjusted to a pH range of approx. 6-9 exist. 6. Process for the preparation of an antibiotic, characterized in that a compound of the formula in which R, alkyl, aralkyl, phenyl, alkyl-substituted Phenyl or halogen-substituted aa phenyl, and R. is either hydrogen or methyl, 9 0 9 8 A 1 /! B 9 2 -47- U67941 with a compound of the formula CHa JOX I! I XC in which X is either chlorine or bromine, converts and, if desired, the product obtained into a Salt transferred. 7. The method according to claim 6, characterized in that that R. represents the methyl radical. 8. The method according to claim 7, characterized in that the compound of formula XI required as starting material, in the R. means the methyl radical, in such a way that man (a) 4-Benzyloxy-7a- (2,3-0-carbonyl) -noviosyloxy-8-methylcoumarin with a 2-methyl-uzmyl-magnesium halide, the formed reaction product decomposed with an aqueous acid, the formed (b) 4-Benzyloxy-7a-3-0- [5'-methyl-2'-Bn "royl] -noviosyloxy-8-raethylcoumarin treated with hydrogen in the presence of a hydrogenation catalyst, the formed 90984 1/169? (c) 4-Hydroxy-7a-3-0- [5'-methyl-2'-pyrroyl] -noviosyloxy-methylcoumarin treated with diazotized aniline, the formed " (d) 3-Benzene-diazo-4-hydroxy-7a-3-0- [5'-methyl-2 · -pyrroyl] - noviosyloxy-8-methylcoumarin with an acylating agent implements and the educated (e) 3-Benzene-diazo-4-hydroxy-7a-2-0-acyl-3-0- [4'-methyl-2 · -
pyrroyl] noviosyloxy-8-methylcoumarin with hydrogen in
Reduced in the presence of a hydrogenation catalyst to 3-amino-4-hydroxy-7a-2-0-acyl-3-0- [5'-methyl-2'-pyrroyl] -noviosyloxy-8-methylcoumarin. 9. The method according to claim 7, characterized in that the acyl group is acetyl. 9098 4 1/1692 -.49 - U67941 10. Use of Streptomyces hazeliensis for the production of antibiotics of the general formula )H wherein R, and Rp represent hydrogen, 2-pyrroyl or 5-methyl-2-pyrroyl, where only one of the radicals R, and R ₂ can mean hydrogen, in the usual biological way and obtaining the antibiotics by extraction from the mycelium. 809841/1692