CH513976A - 3-amino-4-hydroxy-7-2-o acyl-3-o-5-methyl-2-pyrrole - Google Patents

Abstract

Cpds. (I):- where Acyl=RCO, R being alkyl, aralkyl, phenyl, and alkyl- or halo- substd. phenyl. R is pref. CH3(II) Intermediates in antibiotic synthesis. Thus, reaction of (I) with a 2,4-dihalocarbonyl-3-methylpyrrole gives a product with activity against Gram-positive and -negative bacteria. The coumarin OH is not acylated, using, for example, acetic anhydride-pyridine. Allowing a mixture of 6.5 g (III), 40 ml acetic anhydride, and 20 ml pyridine to stand for 18 hr. gave the monoacetyl deriv. (IV), m 151-3 deg. decompn. (isopropanol). Hydrogenation of 5.6 g (IV) in 250 ml ethanol using Pd-C catalyst, filtering, evaporating, washing a soln. of the residue in ethyl acetate with 0.5N-HCl, and evaporating gave 5.3 g (I; acyl=CH3CO), m. 200 deg. decompn.

Description

  

  
 



  Process for the manufacture of antibiotics
The present invention relates to a process for the preparation of a novel mixture containing antibiotics. These antibiotics can be produced by cultivating microorganisms belonging to the genus Streptomyces.



   The organism which 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 the sake of simplicity, this organism is referred to as Streptomyces hazeliensis. A culture of this Streptomyces hazeliensis has been found in the Microorganism Collection of the United States Department of Agriculture, Northern Utilization Research and Development Division, Peoria, III. (USA) deposited under No. NRRL 2938.



   The antibiotics contained in the mixture obtainable according to the invention are referred to as sugordomycins.



  They can be represented by the following general formula:
EMI1.1
 where R1 and R2 represent hydrogen, 2-pyrroyl or 5-methyl-2pyrroyl, only one of the radicals Rr and R2 being hydrogen.



   Particularly interesting antibiotics which can be produced by the process according to the invention are given by the general formula
EMI1.2
  in which the substituents R1 and R2 have the meaning given above.



   The process according to the invention relates to the preparation of an anti-biotic mixture containing compounds of the formula I and is 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 formed antibiotic mixture from the fermentation solution wins.



   The salts which can be prepared according to the invention are obtained by reaction with suitable bases. Examples of such salts are the sodium salt, potassium salt, ammonium salt or other pharmaceutically suitable base addition salts.



   The process product can contain the particularly preferred compounds of the formulas I and 11: a) N, N'-bis [4-hydroxy-8-methyl-1-14-0-methy dimethyl-3-0 (5'-methyl- 2'-pyrrole) -α-L-] yxopyranosyloyl-2-oxo-2H-1-venzopyran-3-yl-3-methyl-pyrroldicarboxamide (R1 and R2 = 5-methyl-2-pyrroyl).



   The following antibiotics of the formula 11 which can be produced by the process according to the invention include: b) N-14-hydroxy-8-methyl-7- [4-0-methyl] -5,5 dimethyl-3-0 (2'-pyrroyl) -α-L] yxopyranosyloxyl-2-oxo-2H-1-benzopyran-3-yl] -N4- [4-hydroxy-8-methyl7- [4-0-methyl-5,5 -dimethyl-3-0 (5'-methyl-2'-pyrrole) α-L-lyxopyranosyloxyl-2-oxo-2H-1-benzopyran-3-yl] -3-methyl-2,4-pyrroldicarboxamide (R1 = 2-pyrroyl and R2 = 5-methyl-2-pyrroyl).



   c) Nê- (4-Hydroxy-8-methyl-7- [4-0-methyl-5,5-dimethyl-3-0 = (5'-methyl-2'-pyrroyl) -α-L-lyxopyranosyloxyl- 2-oxo-2H-1-benzopyran-3-yll-N4-14-hydroxy-8-methyl-7- [4-0-methyl-5,5-dimethyl-3-0 (2 'pyrrole) -α; -L-lyxopyranosyloxyl-2-oxo-2H-1-benzopyran-3-yll-3-methyl-2,4-pyrroldicarboxamide (R1 = 5-methyl-2-pyrroyl and R2 = 2-pyrroyl).



   d) N, N'-bis- [4-hydroxy-8-methyl-7- [4-0-methy dimethyl-3-0- (2'-pyrroyl) -α-L-lyxopyranosyloxyl-2-oxo- 2H-1-benzopyran-3-yll-3-methyl-2,4-pyrroledicarboxamide (R1 and R2 = 2-pyrroyl).



   e) Nê- [4-Hydroxy-8-methyl-7- [4-0-methyl-5,5-dimethyl-3-0 (5'-methyl-2'-pyrrole) -α-L-lyxopyranosyloxyl -2-oxo-2H-1-benzopyran-3-yll-N4-14hydroxy-8-methyl-7-14-0-methyl-5,5-dimethyl-α-L] yxopyranosyloxyl] -2-oxo-2H- 1-benzopyran-3-yll-3-methyl 2,4-pyrroldicarboxamide (R1 = 5-methyl-2-pyrroyl and R2 = hydrogen).



   f) Nê- (4-Hydroxy-8-methyl-7- [4-0-methyll-5,5-dimethyl-α-L-lyxopyranosyloxyl] -2-oxo-2H-1-benzopyran-3-yll- N4- [4-hydroxy8-methyl-7-14-0-methyl-5,5-dimethyl-3-0 (5'-methyl-2'-pyrroyl) α-L-lyxopyranosyloxyl-2-oxo-2H- 1-benzopyran-3-yll-3-methyl 2,4-pyrroledicarboxamide (R1 = hydrogen and R2 = 5-methyl-2-pyrroyl).



   g) Nê-14-Hydroxy-8-methyl-7- [4-0-methyl-5,5-dimethyl-3 (2'-pyrroyl) -α-L-lyxopyranosyloxyl-2-oxo-2H-1- benzopyran 3-yll N4- [4-hydroxy-8-methyl-7- [4-0-methyl-5,5-dimethyl-α-L-lyxo-pyranosyloxyl-2-oxo-2H-2H-1-benzopyran -3-yll-3-methyl-2,4-pyrroldicarboxamide (R1 = 2-pyrroyl and R2 = hydrogen).



   h) Nê- [4-Hydroxy-8-methyl-7- [4-0-methyl-5,5-dimethyl-α-L-lyxoparansyloxyl-2-oxo-2H-1-benzopyran-3-yll-N4 -14-hydroxy 8-methyl-7-14-0-methyl-5,5-dimethyl-3-0- (2'-pyrroyl) -α-L-lyxo-pyranosyloxy-2-oxo-2H-1- benzopyran-3-yll-3-methyl-2,4-pyrrole-dicarboxamide (R1 = hydrogen and R2 = 2-pyrroyl).



   Streptomyces hazeliensis is an aerobic, air-sporulating representative of the order Actinòmycetales 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. l60, Streptomyces niveus NRRL 2446, Streptomyces sphaeroides NRRL 2449, Streptomyces No.



     58383, Streptomyces spirogriseus NRRL 2590, and Streptomyces griseus ATCC 12 318, there are numerous differences between the above organisms and Streptomyces hazeliensis which allow it to be clearly stated 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, as classified by T. G. Pridham, C. W. Hesseltine and R. G Benedict, Appl.

  Microbiology 6: 52-79 (1958): A guide for the classification of Streptomyces according to selected groups.
Another crucial differentiation is the difference between the antibiotics made from Streptomyces hazeliensis and the novobiocin made from the above organisms.



   Cultures of Streptomyces hazeliensis grown on suitable culture media, such as tomato-soy,.



  Agar at 28 C for 2-10 days, form aerial mycelium, which is usually white and turns gray to gray-brown when sporulated. A colorless to yellow exudate forms on various nutrient media. Sporophores on Bennett agar and on asparagine agar are mostly elongated to serpentine, sometimes in a wide hook or in a loop or in loose, open, characteristically wide (diameter 7, u) spirals ending with 1 to 5 loops, the spirals being irregular are designed in the shape and distance between the loops. The spores are oval to cylindrical and vary in width from 1 to 1.2 y, in length from 1.4 to 2.4, u.



  Well-formed spirals could not be observed on either Czapek agar or yeast-malt agar, but small hooks or primitive spirals could. Occasionally, starting from one point, up to 5 sporophores appear in a broom-like shape. They are not ball-like, dense or compact spirals like those found in the culture of Streptomyces sphaeroides. 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 ends and become narrower and denser at the point of attachment of the hyphae.



   To Streptomyces hazeliensis with known Streptomyces, which are cultivated in the classical way both on complex nitrogenous nutrient media as well as on chemically defined nutrient media [Pridham and Gottlieb, J. Bact. 56, 107-115 (1948) 1, these two types of nutrient media were used. For the methodology and the culture media, see the above-described literature by Pridham and Gottlieb and also Gottlieb D., Appl.

  Microbiology 9: 55-65 (196I); A Manual of Methods for Pure Culture Study of Bacteria from the Committee on Bacteriologic Technic of the Society of American Bacteriologists, 1948; and Waksman, The Actinomycetes (Chronica Botanica Co. 1950).



   On the agar media used by Bennett and Czapek, all Streptomyces hazeliensis strains grew vigorously at 22, 28 and 35 C. Better sporulation was observed at the higher temperatures. On the other hand, Streptomyces niveus and Streptomyces sphaeroides grew well at the lower temperatures but not at all at 35 "C. Streptomyces No. 160 and Streptomyces sp. No. 58 383 also do not grow at 35" C.



   The growth of Streptomyces hazeliensis on different culture media is described below. The colors listed correspond to the color scale available in the Color Harmony Manual 4th Edition, Container Corporation of America, Chicago, lllinois, 1958:
Sucrose nitrate agar (Czapek sucroseµ. Growth good but no sporulation; color of the air mycelium cream (between color shade 2 cc and cm gray, scale d).



  Color of the underside cream-brown, pigment cream (shade approx. 2).



   Glucose-Asparagine-Agar: Growth vivid, slightly raised, smooth surface; Color of the aerial mycelium pale gray, with pale gray spores. Color of the underside pale cream; no soluble pigment. One stem shows tiny clear drops of exudate at the filament end.



   Glucose nutrient agar: Growth moderate, flat, leathery, with strong folds in the agar. Color of the air mycelium white, color of the underside dark cream; soluble pigment brownish in color (shade 4 ne). Streptomyces sphaeroides shows moderate growth with no soluble pigment, while Streptomyces niveus grows well with the formation of soluble pigment. Streptomyces sp.



  No. 58,383 shows vigorous growth but no soluble pigment.



   Glucose-Glycerin-Agar: Lively growth, very deep wrinkles in the agar; Surface wrinkled, with cracks.



  Vegetative mycelium pale tan, with some rather large drops of pale yellow exudate.



  Color of the air mycelium partly white to gray-brown (chm.



  near gray 5 dc), approx. 25% sporulated. Color of the underside coffee brown; soluble pigment slightly brown-yellow (shade 3 ie) to brown in color.



   Amidex agar: growth moderate to good, flat, color of the aerial mycelium medium gray; Color of the underside cream-brown to dark coffee-brown. Soluble pigment red-brown (shade 4 pe-pg).



   Tomato Paste Oatmeal Agar: Strong growth with good spore formation; a few drops of exudate. Color of the air mycelium gray to gray-brown (chm. Near gray 5 fe).



  Color of the underside dark brownish olive with dirty olive-colored soluble pigment.



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



   Peptone Iron Agar: Only a few colonies; 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 pc). 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: moderate. Blackening of the potatoes already after 4 days.



  On the other hand, Streptomyces sphaeroides and Streptomyces niveus showed initially moderate growth, then slowly increasing good growth with greyish white spore formation and brownish discoloration of the potatoes. Streptomyces No.



  160 showed vigorous, severely wrinkled growth.



   Carrot pieces: Very good substrate growth of white color, which becomes smoky gray to blue-gray when the spores are formed. Slightly clear yellow exudate. Streptomyces niveus showed moderate growth with white aerial mycelium, whereas no growth was observed with Streptomyces sphaeroides.



  Proteolytic activity a) Gelatin: three tubes were inoculated with a spore suspension using a needle. Before the assessment, the tubes were kept at 3 OC for one hour and then returned 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. 25/0 liquefaction, fairly good growth on the surface, furthermore a few spherical colonies submerged in the liquefied phase; dark brown soluble pigment. After 21 days: 10000 liquefaction, color of the gelatin brown-yellow (shade 2 pc) while the color of the control tubes showed a shade of 1-17 approx.

  In the literature it is stated that Streptomyces niveus and Streptomyces sphaeroides show good growth on gelatin but do not form any soluble pigment. The former shows partial liquefaction, the latter rapid liquefaction (occurring within 3-10 days). Streptomyces No. 160 liquefies quickly but does not form a soluble pigment. Streptomyces sp. No. 58383 grows poorly on gelatine and does not liquefy.



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



   After 7 days, all trunks form well-formed, firm, cream to light brown-colored surface growth membranes with a brown ring on the tube wall; Color of the soluble pigment is creamy yellow to slightly orange.



  One strain 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 weak peptonization. Color of the non-peptonized phase: cream-orange (shade 4 ca-en); pH 5.2-5.7 (control pH 5.6).



   Streptomyces No. 160 showed severe coagulation and severe peptonization of milk.

 

  Nitrate 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 in either synthetic or natural nitrate solution; Streptomyces sphaeroides shows only a weak reduction of nitrate agar to nitrite in 4 days, while Streptomyces sp. No. 58383 a strongly positive nitrite formation was observed.



   Formation of hydrogen sulfide: after 21 hours, agar slant cultures showed very weak growth but strong development of H2S; after 14 days: colonies with a shiny surface with discoloration of the culture medium from dark blue to dark brown.



   Tyrosine agar: For all 5 strains: tyrosinase reaction positive. Slight growth after 14 days. 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 - '/ 2 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 sphaeroides utilize inulin, maltose, mannitol and sorbitol in contrast to Streptomyces hazeliensis, whereby they, in contrast to the latter, did not grow on citrate or succinate culture media in parallel experiments.



  Streptomyces niveus utilizes formate, oxalate, tartrate and sodium citrate.



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



   Production of antibiotics: By breeding Streptomyces hazeliensis, sugordomycins are obtained, but no novobiocin, even under the most varied of conditions. Streptomyces sphaeroides and Strepiomyces niveus, on the other hand, both produce novobiocin but not a sugordomycin antibiotic, even on culture media on which Streptomyces hazeliensis only produces sugordomycin antibiotics.



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



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



   It has also been found that the composition of the culture media in which Streptomyces hazeliensis is grown has a decisive influence on the following variables: (a) the amount of antibiotics formed, (b) the rate at which the antibiotics are formed and (c) the type of antibiotic Mixture in the solution.



   The content of fermented solutions in bottles or fermentation kettles is determined by the usual cup-plate agar diffusion method using Staphylococcus aureus as the test organism (DC Grove and WA 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 forms a zone of inhibition 18 mm in diameter on the test plate is arbitrarily referred to as a unit of activity. The colonies are 8 mm in diameter. The pure antibiotic III has an activity of 5500 units per mg.



   Green or yellow pea meal is used as the preferred nitrogen source for the cultivation of antibiotics. Medium to good yields were also achieved with soybean meal, corn distillers solubles (dry grain residue and soluble fraction from corn distillation), cottonseed meal, coconut protein flour, flaxseed meal and Soluferm (soluble dry residues and grains from corn fermentation), but these were usually below those obtained with peas . Adding small amounts of ferrous sulfate, bone ash, or dried corn steep liquor residue to the peas did not increase the yield of the antibiotics; however, the yield increased after the addition of calcium carbonate and dipotassium phosphate.



   Adding a carbohydrate to natural nitrogen sources had several effects. Certain carbohydrates interfered with the production of antibiotics. Other carbohydrates only gave a good yield of antibiotics if the ratio between carbon and nitrogen was set in a certain relation. An addition to a yellow pea medium in the amount of 0.25% of a sodium salt of organic acids completely inhibited the formation of antibiotics. In this experiment, a basic nutrient medium was used which consisted of 3% yellow pea meal, 0.1% calcium carbonate and 0.1% dipotassium phosphate.



   The effect that was achieved by adding carbohydrates in the production of antibiotics can be seen from the table below, in which the basic nutrient medium contained 2% yellow pea meal:
Table 1 Addition to the basic culture medium S. aureus units per ml
Total solution in 7 days 1 / o corn starch (control) 180 1 0/0 mannitol 120 1% glycerin 50 2% glycerin traces 1/0 maltose 55 1% arabinose 4 1/0 rhamnose 12 1/0 lactose 18 1 0/0 inositol 3
In the following experiments, which are compiled in Table 2, a basic medium of yellow pea meal with 0.1% calcium carbonate and 0.1% dipotassium phosphate was used.

 

   Table 2 Addition to S aureus units Maximum basic culture medium per ml of total solution Yield after days
1.0 0/0 corn starch 590 7 (control) 0.5% corn starch 310 6 2.0 0/0 corn starch 400 7 1 0/0 glucose 230 7 2% 0/0 glucose 25 7 1 0/0 brown sugar 100 7 2 0/0 brown sugar 0 7 Addition to S.aureus units Maximum basic nutrient medium per ml of total solution Cut out after days
1 0/0 dextrin 360 7
2 0/0 dextrin 290 5 i o / O xylose 0 7
It has already been noted above

   that an addition of
0.1 0/0 calcium carbonate and 0.1 0/0 dipotassium phosphate the
Increased yield of antibiotics; If both calcium carbonate and dipotassium phosphate are absent, or if the concentration of one or both salts are increased to 0.5%, the yield of antibiotics is significantly impaired.



   Antifoam agents are usually used to prevent the foaming that occurs in large fermentation batches. It has been found that a number of vegetable oils from z. B. saffron, cottonseed, sesame, coconut, soybean or animal oils such as lard or oleic acid, etc. can be used without reducing the yield of antibiotics.



  Even silicone-based antifoams did not reduce the yield of antibiotics.



   The following table compares the yields of the antibiotics produced by Streptomyces hazeliensis, Streptomyces niveus and Streptomyces sphaeroides on the same culture media. It must also be noted that, in contrast to Streptomyces hazeliensis, which forms clear, sharply bordered zones of inhibition, in Streptomyces niveus and Streptomyces sphaeroides only blurred and poorly defined zones of inhibition occur. There is therefore a clear difference in the formation of these inhibition zones between the sugordomycins and the novobiocin.



   Table 3 Nutrient medium components in% StaphSlococcus aureus Cup-plate units / ml
4 days 6 days
Strep. Strep. Strep. Strep. Strep. Strep.



   hazeliensis niveus sphaeroides hazeliensis niveus sphaeroides pea meal: 2, corn starch: 1 69 50 47 320 53 5 CaCO3: 0.1, K2HPO4: 0.1 corn steep liquor: 4, glucose: 5 0 0 0 0 traces 9 KH2PO4: 0.05, NaNO3 : 0.3, MgSO4 7H2O: 0.1 Soya flour: 2, brown sugar: 2 traces 0 0 13 0 0 Dried residue from corn steep liquor: 0.25 K2HPO4: 0.1 Dried distillers solubles 0 54 37 0 69 5 (Soludri) : 2 Glucose: 2, K2HPO4: 0.1, CaCO3: 0.1 Pharmamedia ': 2.5, Glucose: 2.5 0 48 10 0 3 traces of Fermamine 2 type III: 2, strength: 1 0 8 19 0 6 6 K2HPO4: 0.1, MgSO4:

   0.05 1 Pharmamedia is a 56 0/0 protein and 24 0/0 carbohydrate material from cottonseed meal (Traders Oil Mill Col., Fort Worth, Texas) 2 Fermamine is an enzymatic protein hydrolyzate (Sheffield Chemical Co., Norwich, N.Y.)
As can be seen, a preferred method of producing a mixture of sugordomycins by growing Streptomyces hazeliensis as indicated above is characterized by using pea meal as both a carbon and nitrogen source. An advantageous way of working is 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%, in particular approx. 1-2%, of one Carbohydrates e.g.

  B. a starch or any sugar mentioned above with utilization of carbon. The cultivation is advantageously carried out for about 3-8 days and aerated at a temperature of about 20-35 "C with an amount of air of 15-300 liters / minute per 200 liters of nutrient solution. The antibiotic mixture can be isolated from the fermentation solution in various ways will:
One method is to adjust the fermentation solution, preferably with phosphoric acid, to a pH of 2-7, in particular to pH 4-4.5, although other mineral acids, e.g. B. hydrochloric acid and sulfuric acid can also be used. If necessary, a filter aid z. B. diatomaceous earth, mixed in. The acidified solution is filtered, the filter cake obtained is dissolved in an organic solvent, e.g.

  B. in a lower alkanol such as methanol, ethanol, butanol, isobutanol, etc. or in acetone, methyl isobutyl ketone, butyl acetate or in a mixture of these solvents z. B. acetone / methylene chloride, methanol / benzene, etc., especially digested in butanol and then filtered or centrifuged. The clear filtrate or extract is treated with alkali z. B. with a 500 / above sodium hydroxyl solution to a pH of about 6-7. The solvent is removed, e.g. B. by distillation under reduced pressure at a temperature below 50 "C. The concentrate formed, approximately 40 of the original volume, is mixed with a non-polar solvent, for example with petroleum ether (boiling range 60-90" C), benzene, hexane, pentane , Diethyl ether etc.



  added, the raw antibiotic mixture as sodium salt or as a mixture of the sodium salt with the free
Form precipitated by sedimentation, filtration or
Centrifugation can be easily separated in a simple manner.



   According to another procedure, the raw antibiotic mixture is isolated from the fermentation solution in such a way that the mycelium is filtered off, the filtrate with a
Adjust the mineral acid to a pH of approx. 1-7 and then shake the acidified filtrate with a water-immiscible solvent such as butanol, butyl acetate, ethyl acetate, ether, chloroform, methyl isobutyl ketone, etc.



   The organic phase is separated from the aqueous layer. The raw, antibiotic mixture can either be obtained by evaporating the solvent or on an ion exchanger, such as. B. 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-Format etc. or adsorbed on activated carbon and then isolated by elution with a solvent such as methanol. The mycelium is filtered off and washed with an organic solvent such as a lower alkanol, e.g. B. methanol, ethanol, butanol, isobutanol, etc .; or with acetone,
Butyl acetate, acetomethylene chloride, methyl isobutyl ketone,
Methanolbenzene, etc. extracted.

  The crude antibiotic mixture remaining after evaporation of the solvent can subsequently be combined with the antibiotic mixture obtained from the filtrate.



   One can use the raw sodium salt of the antibiotic
Mixture or the crude acidic antibiotic thereby purify by dispersing the antibiotic mixture in acetone, a sufficient amount of mineral acid, e.g. B.



   Hydrochloric acid, added to bring about an acidic pH, e.g. B. from approx. 2-6, preferably approx. PH 4, the insoluble material is filtered off and the filter cake is washed out with a small amount of acetone. The filtrate is combined with the washing solution and evaporated under reduced pressure. That narrowed to about 1/6 of the original volume
Water and benzene are added to the concentrate. The solid suspension formed here in both liquid phases consists of the free acidic antibiotic mixture, which is filtered off and digested in warm methanol. The solid fractions that separate out on cooling are separated off. They consist of the purified, free, acidic, antibiotic mixture.



   The raw antibiotic mixture can also be purified by making the benzylamine salt.



  For this purpose, the antibiotic mixture is in an organic solvent, e.g. B. in n-butanol, n-butyl acetate, ether, ethyl acetate, etc., dissolved and treated with an excess of benzylamine. The benzylamine salt of the antibiotic mixture precipitates, with numerous impurities that were present in the antibiotic mixture remaining in the organic solvent.



  The precipitated benzylamine salt is filtered off. The free antibiotics can be prepared from the benzylamine salt by treatment with an aqueous mineral acid, e.g. B. aqueous hydrochloric acid can be obtained. The free antibiotic mixture can be extracted from the aqueous phase with the aid of an organic solvent that is immiscible or only slightly miscible with water, e.g. B. with n-butanol, n-butyl acetate, ether, ethyl acetate etc. can be extracted. The mixture remaining after evaporation of the solvent is z. B. reprecipitated from a warm mixture of methanol / benzene.



   Other amines that have been used experimentally in the purification of the antibiotic mixture have not proven themselves. Only the benzylamine salt was found to be completely satisfactory. Other bases which have not been found to be suitable include calcium hydroxide, procaine, ethylenediamine, diethanolamine, monoethanolamine, N, N'-dibenzylethylenediamine, dimethylethanolamine, hydroxyethylethylenediamine, diisobutylamine and furfurylamine.



   The raw antibiotic mixture can also be purified by separating the raw mixture between an organic solvent, e.g. B. butyl acetate, ether or mixtures of chloroform, isopropanol etc. and an aqueous buffer solution with a pH of about 6.8-11 distributed. The antibiotic mixture can be made from the aqueous buffer solution by acidification, e.g. B. be isolated with a mineral acid such as hydrochloric acid, sulfuric acid, etc. up to a pH of about 2-5. The resulting suspension can be mixed with an organic solvent that is immiscible or only slightly miscible with water, e.g. B. with n-butanol, n-butyl acetate, ether, ethyl acetate etc. can be extracted. The purified, acidic, antibiotic mixture is then, for. B. by evaporating the organic solvent isolated.



   The purified by one of the above methods antibiotic mixture can then with the help of a countercurrent distribution system, e.g. B. after Craig, be separated into its components. The organic solvent used here is immiscible or only slightly miscible with water, such as. B. n-butanol, n-butyl acetate, ether, ethyl acetate, chloroform isopropanol, etc. The aqueous phase is adjusted to a pH of approx. 6-9, in particular approx. 8-8.9. The phosphates and borates customarily used for this purpose can be used as buffers.



   The antibiotics contained in the mixture obtainable according to the invention are characterized in that they are highly active against Staphylococcus aureus. In addition, the antibiotics are against a large class of gram-positive and gram-negative bacteria, e.g. B. against Aerobacter aerogenes, Mycobacterium phlei, Proteus vulgaris, Sarcina lutea PCI-1001, etc. effective.



   The antibiotics contained in the mixture available according to the invention are therefore suitable for bacterial infections, such as those found, for B. triggered by Staphylococcus aureus to fight. Alternatively, the purified antibiotic mixture can also be used as a therapeutic agent.



   The products of the process can be used as medicaments in the form of pharmaceutical preparations which contain them or their salts in a mixture with a pharmaceutical, organic or inorganic inert carrier material suitable for enteral or parenteral administration. The pharmaceutical preparations can be in solid form or in liquid form. They may contain auxiliaries such as preservatives, stabilizers, wetting agents or emulsifiers, salts for changing the osmotic pressure or buffers. They can also contain other therapeutically valuable substances.

 

   Example A. Preparation of the antibiotic mixture.



   Streptomyces hazeliensis is stored in spore form, either on a tomato-oatmeal agar in medicinal bottles or in freeze-dried form, the spores being suspended in 10% bovine serum or in 10% skimmed milk. In a 6 liter shaker bottle with 3 liters of aqueous nutrient solution, consisting of:
1 0/0 pea meal,
1 0/0 corn starch,
0.1 0/0 calcium carbonate,
0.1 0/0 dipotassium phosphate, and
0.1% of lard is added to an aqueous spore suspension and kept at 28 ° C. for 3 to 5 days on a shaker at 200
Incubated revolutions per minute until vigorous growth occurs.

  The incubation is carried out in a vessel that is suitable for aeration of the nutrient solution, e.g. B. in the above-mentioned shake bottle or in a gas washing bottle. With the inoculation solution obtained in this way, 250 liters of aqueous nutrient solution are inoculated in a 400-liter fermentation kettle. The nutrient solution consists of:
3 / O pea meal,
0.1% calcium carbonate,
0.1% dipotassium phosphate,
0.03% silicone paste (Dow-Corning silicone A paste), and
0.1 0/0 lard.



   Instead of this nutrient solution, 250 liters 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 amount of air is 225 liters / minute. During this time, the viscosity increases significantly, while the pH value increases to about 6.6 to
5.9 drops The inoculum obtained in this way is sterilized in a 35,000 liter fermentation tank of 23,000 liters
Transfer nutrient solution. The nutrient solution consists of:
3 0/0 ground pea meal
0.1% calcium carbonate,
0.1% dipotassium phosphate and 0.03-0.05% Dow-Corning silicone A, suspended in
0.1 / o soybean oil.



   The solution is left to ferment for 5-8 days at 28 "C. The course of fermentation is controlled with the aid of the in vitro texts. The air volume is adjusted to 700 liters / minute during the first 24 hours, then during the following 24-72 hours Hours to 2800 liters / minute and during the further 72-168 hours to 4000 liters / minute. The pH of the fermentation solution is then adjusted to 3.9 by adding a syrupy, 850 / above phosphoric acid. After adding 750 kg Diatomaceous earth, the acidified solution is filtered, the filter cake is slurried in 7500 liters of n-butanol and vigorously stirred. The slurry is left to stand overnight without stirring.

  The clear butanol extract obtained by centrifugation is then neutralized with 1482 ml of 500 / above NaOH solution up to a pH of approx. 7 and gently concentrated to a volume of 200 liters under reduced pressure at a temperature below 50 ° C.



   This concentrate is introduced into 1000 liters of petroleum ether (boiling range 60-90 "C) with vigorous stirring. The precipitating crude sodium salt of the antibiotic mixture probably consists of a mixture of the acidic and partially neutral form of the various components of the antibiotic mixture Centrifugation isolated and then dried.



   B. Purification of the Crude Sodium Salt 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.



  The insoluble material is filtered off. The filtrate is concentrated under reduced pressure to a thick syrup. This is dissolved in 25 ml of methanol and 625 ml of 0.1 n
Hydrochloric acid suspended. The suspension is used 3 times each
Shaken out 1.5 liters of ether. The combined ether extracts (4.24 liters) are extracted twice with 1.1 liters of 15 M phosphate buffer pH 6.2, then twice with 1.1 liters of 15 M phosphate buffer pH 7.0 each time. Little activity is lost with this treatment. The ethereal solution is then extracted twice with 1 liter of 1 M phosphate buffer pH 7.8 each time, whereupon the antibiotic mixture migrates into the aqueous phase. The suspension is adjusted to a pH of 7.0 and extracted with n-butanol, the precipitate going into solution.

  The n-butanol is evaporated off under reduced pressure. The residue is suspended in tts M phosphate buffer pH 6.5. 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 / M disodium phosphate buffer pH 9.5. The insoluble fractions are filtered off in the presence of a filter 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 concentrated under reduced pressure to a volume of 1800 ml. After separating a small amount of insoluble material, the filtrate is treated with a solution of 100 ml of benzylamine in 100 ml of butyl acetate. The benzylamine salt of the antibiotics which settles in the form of a fine precipitate is filtered off, washed with a little butyl acetate and then with anhydrous ether.



   20 g of this benzylamine salt of antibiotics is used in
140 ml of methanol dissolved and after adding 190 ml
Water with the help of hydrochloric acid to a pH of
2.5 set. In order to lower the viscosity of the suspension, a further 300 ml of water are added. The
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 at this concentration is filtered off and washed with cold methanol. This product can be reprecipitated by dissolving it in a mixture of hot methanol and benzene. The antibiotics that are deposited in finely divided form melt after drying at 228-230 "C.



   c) By extraction with acetone
50 g of the crude sodium salt are suspended in 600 ml of acetone and, after the addition of 30 ml of a 100% 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 40-50 ° 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.



  C. Detection of active ingredient components in the antibiotic mixture
5 g of the purified antibiotic mixture obtained through the cleaning process with acetone are distributed in a 200-tube Craig countercurrent distribution apparatus between an upper aqueous phase of IsK2HPO4 buffer (pH 8.2) and a lower organic phase consisting of equal parts of isopropyl alcohol and chloroform. 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, VII, 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 (total of 400 tubes) there is antibiotic III in tubes 120-152. Antibiotics IV and V are in tubes 161-170. Antibiotics V and VI are in tubes 185-195. It has not been established with certainty whether the antibiotic of Formula IV or V is present in tubes 161-170 and which antibiotic is present in tubes 185-195. The tubes 153-160 contain either a mixture of antibiotics III and IV or III and V and the tubes 171-184 contain a mixture of antibiotics IV and V.



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



   The antibiotics are isolated from the solvent phase by pouring tubes with the same content together. The antibiotic III is z. B. obtained by pouring the tubes 120-152 together and evaporating the collected solvents to dryness.



  The buffer phase of the groups of tubes indicated above is acidified to a pH of 2.5.



  The antibiotic is extracted in butanol or in a mixture of chloroform / isopropyl alcohol (1: 1). The solvent extract is evaporated to dryness, the antibiotic separating out as a residue.



   The antibiotic III obtained through the countercurrent distribution described above is then passed through a further 200 tubes. 3 isomers are obtained: vial 90-100, vial 120 and vial 140. This indicates the presence of several closely related isomers, the absolute structure of which is not currently known. All of these isomers are bioactive; H. they have high activity against Staphylococcus aureus. The mixture of antibiotics VII, VIII, IX and X isolated above is separated into a mixture of antibiotics VII and VIII and a mixture of antibiotics IX and X using the Craig countercurrent distribution with 200 tubes.

  The solvent system used consists of equal parts of the same organic phase as used above; H. an equal volume mixture of isopropyl alcohol and chloroform, while the aqueous phase consists of a 1.5 M dipotassium phosphate buffer pH 7.0. After the equilibrium between the solvent and the buffer has been established, the above antibiotic mixture VII-X is poured into the apparatus and passed through the 200 tubes. The mixture of antibiotics VII and VIII is in tube 110 and the mixture of antibiotics IX and X is in tube 170.

 

Claims (1)

PATENT CLAIM Process for the preparation of a compound of the formula EMI8.1 wherein Rl and R2 are hydrogen, 2-pyrroyl or 5-methyl2-pyrroyl, where only one of the radicals Rl and R2 can be hydrogen, containing antibiotic mixture, characterized in that the organism Streptomyces hazeliensis in an aqueous culture liquid that has a Contains organic C and N sources, cultivated submerged and aerobically and the antibiotic mixture formed is obtained from the fermentation solution. SUBCLAIMS 1. The method according to claim, characterized in that the antibiotic mixture formed is converted into a salt. 2. The method according to claim, characterized in that pea meal is used as the C and N source. 3. The method according to claim, characterized in that the antibiotic mixture is obtained by extracting the clarified solution and the mycelium with the aid of a solvent. 4. The method according to claim, characterized in that inorganic salts are added to the culture liquid and the organism is cultivated for about 2 to 6 days until the medium has become strongly antimicrobial, after which the antibiotic mixture formed is obtained from the fermentation solution.