DK141999B - Process for the preparation of antibiotic substance designated 30,504 RP, its metal salts and its salts with nitrogenous bases. - Google Patents

Description

(11) SUBMISSION 6 ELSCRIPTION 141999 (¾) DENMARK (51) lnt 0, 3 c 12 p ^, 06 L-TMIM ΐνίΛΛΓΤίν C gy g jj / qq '(21) Application no. 5Ο65 / 77 (22) Filed on 15 · · Nov · 1977 (23) Race day 28 Feb 1975 (44) The application presented and the petition published on 4 'Suk · 1980 DIRECTORATE OF THE PATENT AND TRADEMARKET ^ Priority requested from the

Mar 1 1974, 7407066, FR

(71) RHONE-POULENC INDUSTRIES, 22, Avenue Montaigne, 75560 Paris Cedex 08, FR.

(72) Inventor: Jean Florent, 6, rue Raffaelli, Paris 16s, FR: Jean Runel, 6, rue de Toul, "Paris 12eme, FR: Denise Mancy, 7th Avenue Stinvil = le, Charenton (Val-de-Marne) ), FR.

(74) Plenipotentiary in the proceedings:

The engineering firm Budde, Schou & Co. (64) Process for the preparation of antibiotic substance designated 50.5 ° 4 RP, its metal salts and its salts with nitrogenous bases.

The present invention relates to a process for the preparation of a novel antibiotic substance, hereinafter referred to as the number 30,504 RP, its metal salts or its salts with nitrogenous bases.

30,504 RP is of particular interest as a result of its action against coccidia manifesting alongside its antibacterial and anti-malarial effects.

The method according to the invention is characterized in that a strain of Streptomyces gallinarius DS 25.881 (NRRL 5.785) is grown aerobically on a usually suitable medium and under conditions usual for this type of culture, after which 30.504 RP formed during cultivation is separated, optionally in the form of a salt, and purified.

141999 2 30,504 RP is characterized in that it has the following physical and chemical properties: appearance: white, microcrystalline powder, solubility; 30,504 RP is readily soluble in chlorinated solvents such as methylene chloride and chloroform, in alcohols such as methanol and in dimethylformamide, and it is relatively soluble in acetone and ethyl acetate, heavily soluble in hexane and virtually insoluble in water (the terms are according to Pharraacopée Francaise IX Part, page 13, (1972), percent composition: 30,504 RP contains carbon, hydrogen and oxygen, its percent composition is about: C 69.4%, H 9.0%, O 21.0%, neutralization equivalent: 30.504 RP is a substance with acidic properties whose neutralization equivalent (determined in dimethylformamide solution by titration with tetrabutylammonium hydroxide) is 830, m.p.: (determined on a Kofler bench): near 200 °, turning ability: (determined on a 1% solution in methanol ) the specific rotational power of 30.504 RP is about: [α] 2 ° = -790 [α] 206 = -188 ° [α] 5 = -378 ° ultraviolet spectrum: 30.504 RP has no characteristic absorption in the ultraviolet range. , infrared spectrum: (determined using potassium bromide tablets)

This spectrum is shown in the drawing, where outwardly the abscissa is indicated partly the wavelength in μ (upper scale) and partly the wavelength in cm ^ (lower scale) and outward the ordinate the optical density.

In the following Table I, the main absorption band of this product in the infrared range is indicated in wavelength (cm 2).

3 141999

Table I.

3440 P 1325 m 915 m 3030 ép 1315 ép. 880 f 2960 F 1295 ép. 865 tf 2938 F 1272 m 845 tf 2880 F 1225 f 825 f 2855 ép. 1200 tf 792 m 2540 tf 1172 m 775 f 2060 tf 1140 m 735 f 1945 tf 1115 ép. 700 f 1900 tf 1100 ép. 675 ép.

1785 ép. 1085 F 655 f 1705 F 1070 ép. 630 ép.

1632 m 1045 ép. 615 m 1622 ép. 1038 F 565 ep.

1535 tf 1012 m 525 m 1458 F 980 F 495 m 1405 ép. 958 F 465 tf 1380 F 935 m F = heavy m = medium f = weak tf = very weak ép. = shoulder

Chromatographic migration: 30.504 RP can be characterized by ascending thin layer chromatography on silica gel using two systems of solvent mixtures: ethyl acetate / cyclohexane / water / butanol (50/50/25/5 volume ratio):

Rf = 0.45, methylene chloride / methanol (94/6 by volume): Rf = 0.55. Bacteriostatic effect in vitro: 30,504 RP has a bacteriostatic effect that is particularly exerted on certain gram-positive bacteria.

The bacteriostatic effect of 30,504 RP against a certain number of organisms is determined by one of the dilution methods commonly used for this purpose. For each organism, the smallest concentration of the active substance is determined, which under defined conditions prevents the visible development of the organism in a suitable medium. The results of the various determinations are summarized in the following Table II, where the minimum bacteriostatic 3 concentrations are given in micrograms per gram. cm of the nutrient medium:

Table II

Minimal bacteriostatic

Bacterial organism concentrations investigated _ (in pg / cm3) _

Staphylococcus aureus, strain 209 P - ATCC 6538 P 0.2

Staphylococcus aureus, strain Smith 0.5

Sarcina lutea - ATCC 9341 0.9

Streptococcus faecalis - ATCC 8043 0.1

Streptococcus pyogenes hemolyticus, strain Dig (Insitut Pasteur) 0.2

Diplococcus pneumoniae, strain To (Institut Pasteur) 0.05

Neisseria catarrhalis (A 152, Institute Pasteur) 6

Bacillus subtilis - ATCC 6633 0.2

Bacillus cereus - ATCC 6630 0.1

Mycobacterium species - ATCC 607 6

Escherichia coli - ATCC 9637> 150

Shigella dysenteriae, Shiga L (Institute Pasteur)> 150

Salmonella paratyphi A (strain Lacasse, Institut

Pasteur)> 150

Salmonella schottmuelleri (paratyphi B) - (strain Fougenc, Institut Pasteur)> 150

Proteus vulgaris> 150

Pseudomonas aeruginosa> 150

toxicity:

For chickens, DL ^g is 85 mg / kg by a single oral administration.

Effect against coccidia:

The effect against coccidia is determined on chickens that have been particularly infected with Eimeria tenella and Eimeria acervulina.

The effect of RP 30,504 incorporated in the chickens' feed against coccidia is shown by non-toxic concentrations of 0.005-0.04% by weight of the product in the feed mixture.

5 141999

Impact against malaria; 30,504 RP also has an effect against malaria in experimentally induced plasmodium infections in mice and chickens.

The organism producing the antibiotic substance 30,504 RP is a strain of Streptomyces isolated from a soil sample taken in India and this organism is designated by the number DS 25881. A sample of this organism is deposited in the Northern Regional Research Laboratory of the U.S., Department of Agriculture in Peoria, 111.

(USA), where it is registered under the designation NRRL 5785.

This organism, which has properties that do not allow it to be identified with a species already described, must be considered a novel species and has been named Streptomyces gallinarius, DS 25881.

This strain has been isolated using the usual method of suspending a small amount of soil in distilled, sterile water, diluting the suspension to various concentrations, and dispensing a small volume of each dilution on the surface into petri dishes containing a nutritional agar. After incubation for a few days at 26 ° C, whereby the microorganisms develop, the colonies desired to be isolated for further study are removed and seeded on nutrient agar to obtain larger cultures.

Streptomyces gallinarius, DS 25,881 forms cylindrical to oval spores measuring 1.0-1.2 μ / 0.8-1.0 μ. Its sporophores are arranged in bunches, and the spore-forming filaments, which are soft and curved, are often wound up at the ends, either assuming a slightly bent shape or forming a helical winding, sometimes several. Due to the mode of spore formation, this strain belongs to the Retinaculum-Apertum group according to Pridham's classification.

Streptomyces gallinarius DS 25,881 develops well at 26 ° C and at 37 ° C, but not at 50 ° C. It has a spore-forming gray mycelium. The color of the vegetative mycelium generally varies, depending on the culture medium, from yellow or greyish-yellow to brown-yellow or yellow-brown. Apart from a special production of soluble brownish rosaviolet pigment on an agar according to Hickey and Tresner, the organism on all culture media where it has been observed yields a production of soluble pigment which is generally of low intensity and which goes from brownish yellow or brownish-gray to yellowish-brown.

In cultures of the organism incubated at 26 ° C, it has the following biochemical properties: 6 U1999 production, of melanin: negative hydrogen sulphide production: negative tyrosinase: negative gelatin melting: positive utilization of cellulose: positive nitrite formation from nitrates : strongly positive, both in nitrate-containing broth and in synthetic media starch hydrolysis: positive culture on skimmed milk: peptonization without coagule ring acidity increases quite easily.

The cultural characteristics of Streptomyces gallinarius DS 25.881 are summarized in the following Table III. It is the characteristics of cultures that have reached a good stage of development, ie. 2-3 weeks at 26 ° C, unless otherwise stated. These properties are observed on nutrient agar and the broth usually used to determine the morphological properties of Streptomyces strains, with sloped agar cultures used on agar media. A certain number of the culture media used are prepared according to the recipes set forth in The Actinomycetes, S.A. Waksman, pp. 193-197, Chronica Botanica Company, Waltham, Mass., USA, 1950, and in this case, they are indicated by the letter W followed by the number they have in this work. The references or compositions of the other media are as follows: - Ref. A - "Hickey and Tresner's Agar" - T.G. Pridham et al. - Antibiotics Annual, 1956-1957, pp. 950 - Ref. B - "Bennett's Agar" - S.A. Waksman - The Actinomycetes, Volume 2, p. 331 - No. 30 - The Williams and Wilkins Company, Baltimore, 1961 - Ref. C - Recipe W-23, added 2% agar - Ref. D - "Yeast Extract Agar". T. G. Pridham et al. - Antibiotics Annual, 1956-1957, pp. 950 - Ref. E - "Tomato Paste Oatmeal Agar" - T.G. Pridham et al. - Antibiotics Annual, 1956-1957, pp. 950 - Ref. F - "Melanin formation medium" - S.A. Waksman - The Actinomycetes, Volume 2, p. 333, No. 42 - The Williams and Wilkins Company, Baltimore, 1961 - Ref. G - W.E. Grundy et al. - Antibiotics and Chem. 2, 401, 1952 - Ref. H - "Inorganic Salts - Starch Agar" - T.G. Pridham et al. Antibiotics Annual 1956-1957, p. 951 - Ref. I - corresponds to recipe W-1, where 3% sucrose has been replaced with 1.5% glucose - Ref. J - corresponds to recipe W-1, where 3% sucrose is replaced by 1.5% glycerol - Ref. K - corresponds to recipe W-18, where 3% sucrose is replaced by 1.5% glucose - Ref. L - corresponds to recipe W-18 where sucrose is omitted and replaced with small filter paper tapes partially submerged in the liquid -Ref. M - "Manual of Methods for Pure Culture Study of Bacteria" -Society of American Bacteriologists - Geneva, N.Y. H ^ q - 18 - Ref. N - "Plain gelatin" - prepared as specified in "Manual of Methods for Pure Culture Study of Bacteria" - Society of American Bacteriologists - Geneva, N.Y. 11 ^ -18 - Ref. P - Skimmed milk powder (commodity) - reconstituted - Ref. Q - Medium designated for examination of production of H2S by H.D. Tresner and F. Danga - Journal of Bacteriology, 76, 239-244, 1958.

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Streptomyces gallinarius DS 25881 has a set of properties that do not exactly coincide with the properties of some of the strains already described, and it must therefore be considered a new species.

By considering the species described in "The Actinomycetes" (Volume 2, S.A. Waksman, The Williams and Wilkins Company, Baltimore, 1961) as well as in Bergey's "Manual of Determinative Bacteriology" (7th edition, The Williams and Wilkins Company , Baltimore, 1957), is the species with which it can logically be compared to Streptomyces hygroscopicus. Like this, S. gallinarius DS 25,881 thus develops a vegetative mycelium which is generally yellow or greyish yellow to brownish-yellow or yellow-brown, and generally has a gray, spore-forming air mycelium. It does not form soluble melanin pigment on organic media, does not produce HjS and slowly melts gelatin on which it does not form soluble pigment. In all its culture media, both synthetic and organic, where it does not form soluble pigment or where it forms only soluble pigment, which is generally of low intensity, it is brownish-yellow or brownish-yellow to yellow-brown. In addition to these properties, Streptomyces gallinarius DS 25,881 has the property that it can in a particular way produce a soluble, brownish rosaviolet pigment on one of the media in which it has been grown (Hickey and Tresner agar), which H.D. Tresner and E.J. Backus (Applied Microbiology 4, 243-250, 1956) has been recognized as a property of numerous strains belonging to the species Streptomyces hygroscopicus and has been observed on various media depending on the strains. However, it differs from this species by two of the three essential characteristics by which it is defined. In part, the shape of its sporophores does not correspond to the sporophores of the species Streptomyces hygroscopicus, and secondly, no shiny black areas are formed in its spore-forming air mycelium in any case, which is characteristic of the species Streptomyces hygroscopicus.

Streptomyces gallinarius DS 25,881 can also be compared to Streptomyces aureofaciens, which also does not form melanin, generally produces a yellow to orange-yellow or banana-vegetative mycelium, and which has a gray, spore-forming air mycelium. In addition, Streptomyces aureofaciens does not form soluble pigment on numerous culture media, and, when this is the case, it forms soluble pigments with a yellow to brownish color which is generally rather weak. In some cases, the vegetative mycelium of Streptomyces aureofaciens reddish-brown is stained purple, but in this case it turns out to be a pigment which does not diffuse into the medium, 14 141999 and, as a result, cannot match the soluble, brownish, rosaviolet pigment that can be produced by Streptomyces gallinarius DS 25,881.

In addition, S. aureafaciens does not melt gelatin on which it forms a cream-colored, vegetative mycelium and neither coagulates nor peptonizes skimmed milk, whereas S. gallinarius DS 25,881, on the other hand, melts gelatin on which it forms a yellow-brown vegetative mycelium. and regular peptonization of skim milk. It is thus apparent from this that S. gallinarius DS 25,881 also differs from this latter species.

The ability of S. gallinarius DS 25.881 to utilize various carbon or nitrogen sources has been determined according to the principle of the method of Pridham and Gottlieb (J. of Bact. 56, 1Q7-114, 1948). The degree of development has been observed on the base medium stated by the authors, with glucose being replaced with different carbon sources and ammonium sulfate being replaced with different nitrogen sources there. The results obtained are given in the following table: 15 141999

Exploitation Examined Exploitation Carbon Sources ^ _ Nitrogen Sources __ D-Ribose Negative NaNO ^ Positive D-Xylose Positive NaN02 Positive L-Arabinose Positive (NH4) 2S04 Positive L-Rhamnose Positive (NH4) 2HPC> 4 Positive D-Glucose Positive Urea Positive D- Galactose positive L-asparagine positive D-Fructose positive glucosamine positive D-Mannose positive glycocol positive L-Sorbose negative sarcosine negative

Lactose positive DL-alanine positive

Maltose positive DL-Valine positive

Sucrose positive DL-aspartic acid positive

Trehalose positive L-glutamic acid positive

Cellobiose positive L-arginine positive

Raffinose negative L-Lysine positive

Dextrin positive DL serine positive

Inulin negative DL-threonine positive

Starch positive DL-methionine positive

Glycogen positive taurine negative

Glycerol positive DL-phenylalanine positive

Erythritol negative L-tyrosine positive

Adonitol negative DL-proline positive

Dulcitol negative L-hydroxyproline positive D-Mannitol positive L-tryptophan positive D-Sorbitol negative betaine negative

Inositol positive

Salicin positive 16 141999

The cultivation of Streptoxnyces gallinarius DS 25881 can be carried out by any aerobic cultivation method on the surface or most recently. but the latter method is preferred for convenience reasons. For this purpose, various types of apparatus commonly used in the fermentation industry are used.

In particular, the following workflow can be used to carry out the operations:

Streptomyces gallinarius DS 25.881 (stem culture) agar culture in rv charcoal culture seed culture for fermentation in production culture for fermentation.

The fermentation medium should substantially contain an assimilable carbon source and an assimilable nitrogen source, inorganic elements, especially chlorides, and; possibly growth factors, since all these substances can be added 'in? in the form of well-defined products or complex mixtures, such as biological products of different origins.

Carbohydrates such as glucose, saucbarose, maltose, dextrins, starch or other carbon and hydrogen-containing substances such as sugar alcohols (glycerol) or certain organic acids, e.g. lactic or citric acid.

Certain animal or vegetable oils, such as bacon or soybean oil, may advantageously replace these various carbon and hydrogen sources or be used in conjunction with them.

There are an enormous number of suitable sources of assimilable nitrogen. They can be very simple chemical substances, such as inorganic or organic ammonium salts, urea and certain amino acids. Nitrogen can also be supplied with complex substances containing primarily nitrogen in protein form, e.g. casein, lactalbumin, gluten and their hydrolysates, soybean meal, peanut flour, fishmeal, meat extract, yeast extract, distillers' solubles and maize cast water.

Among the added inorganic substances, some may have a buffering or neutralizing effect, such as alkali metal or alkaline earth metal phosphates or calcium or magnesium carbonates. Others provide the necessary ionic equilibrium for the development of Streptomyces gallinarius DS 25,881 and for the production of 30,504 RP, such as chlorides and sulphates of alkali metals and alkaline earth metals. Finally, certain substances act more specifically as activators of the metabolic reactions in Streptomyces gallinarius DS 25,881, and these are salts of zinc, cobalt, iron, copper and manganese.

The growth factors are substances of vitamin nature such as riboflavin, folic acid and pantothenic acid.

The pH value of the fermentant at the beginning of the culture should be between 5.8 and 7.8, preferably between 6.2 and 7.4. The optimum fermentation temperature is between 25 and 30 ° C, but satisfactory production is obtained at temperatures between 23 and 33 ° C. The aeration of the fermentation medium can be varied within fairly wide limits. However, it has been found that aerations of 0.3-3 liters of air per liters of medium and minute are particularly well suited. The maximum yield of RP 30,504 is obtained after 2-8 days of cultivation, this time being essentially dependent on the medium used.

It is apparent from the foregoing that the general conditions for cultivation of Streptomyces gallinarius DS 25,881 for the production of 30,504 RP can be varied over a wide range and adapted to any particular circumstance.

30.504 RP can be isolated from fermentation mixtures as follows:

The mixture is filtered at a pH in the acidic range, generally between 3 and 6 and preferably near 5, in the presence of a filtration aid.

The amount of active substance retained in the filter cake is extracted by a suitable organic solvent, e.g. a lower alcohol such as methanol, or a chlorinated solvent such as methylene chloride. The crude product can be isolated from the above-mentioned organic solutions by crystallization after concentration of these solutions under reduced pressure, optionally adding a poor solvent or a non-solvent and leaving it cold. . .

30,504 RP can be purified by commonly used methods such as recrystallization, chromatography on various adsorbents or countercurrent distribution.

The invention is illustrated by the following example.

Example.

(a) Fermentation: In a fermentation tank of 170 liters:

Peptone: 1200 g Yeast extract: 600 g

Glucose monohydrate: 1200 g

Partially hydrolyzed starch: 2400 g

Tap water up to 110 liters.

3 adjust the pH to 7.3 by adding 50 cm of 10 N sodium hydroxide solution. The medium is sterilized by purging with steam at a temperature of 122 ° C for 40 minutes. After cooling, the volume of the medium is 120 liters due to the condensation of steam during sterilization and the pH is 6.90.

200 cm 2 of a shake flask culture is seeded by Streptomyces gallinarius DS 25,881. The culture is developed over 21 hours with stirring and aeration with sterile air. It is then suitable for grafting the production culture.

The production culture is prepared in a fermentation tank of 800 liters in which the following substances are placed:

Distillers' solubles: 12.5 kg

Sucrose: 12.5 kg

Sodium chloride: 2.5 kg

Magnesium sulfate heptahydrate: 1.0 kg

A solution of 20 g of cobalt chloride hexanyrate per 0.5 liters of tap water up to 460 liters.

141999 19

The pH of the medium is adjusted to 7.5 by the addition of 720 cc of 10 N sodium hydroxide solution, and it is then sterilized by purging with steam at a temperature of 122 ° C for 40 minutes.

After cooling, the volume of the medium is 500 liters due to the condensation of steam during sterilization and the pH of the medium is 6.40. Inoculate with 50 liters of the above-described seed culture prepared in the 170 liter fermentation vessel. The culture is developed over 116 hours at 27 ° C with agitation with a turbine rotating at 205 rpm and aeration with 20 m sterile air per minute. hour. The lowering of the pH at the beginning of the cultivation is limited to 6.5 by the automatic addition of 5 N sodium hydroxide solution (total quantity used: 0.5 liter).

After completion of the culture, the pH of the culture is 7.85 and the volume is 470 liters.

b) Extraction: 1000 liters of the fermentation mixture prepared under the above conditions is stirred for 1/2 hour after acidification to a pH of 5 by the addition of 7 liters of 6 N hydrochloric acid.

After mixing with 35 kg of filter aid, the fermentation mixture is filtered on a filter press. The filter cake is washed on the filter with 200 liters of water, the pH of which is adjusted to 5 by the addition of 6 N hydrochloric acid.

The filtrate and washing liquid are removed.

The filter cake is comminuted in 700 liters of methanol. The resulting mixture is adjusted to a pH of 7 by the addition of 470 cm 6 of sodium hydroxide solution and stirred for 1/2 hour.

The mixture is filtered on a filter press and the filter cake is washed on the filter with 100 liters of methanol.

The filtrate and wash liquid are combined and concentrated under reduced pressure (5-10 mm Hg) to give 60 liters of aqueous concentrate.

This concentrate is adjusted to a pH of 3 by adding 3 of 580 cm 6 of hydrochloric acid and stirring for 10 minutes with 40 liters of methylene chloride. The two stages are separated. The aqueous phase is then extracted successively with 30 liters and 20 liters of methylene chloride.

The methylene chloride extracts are combined and stirred with 9 liters of 3 water, acidifying to a pH of 3 in the aqueous phase (20 cm 6 N hydrochloric acid). The organic phase is separated and washed again with 18 liters of water, adding sodium hydroxide solution to a pH of 9.5 in the aqueous phase (20 cm 6 N sodium hydroxide solution).

20 141999

The methylene chloride phase is concentrated under reduced pressure (5-10 mm Hg) to a volume of approx. 1 liter.

The methylene chloride concentrate thus prepared is slowly poured into 10 liters of hexane. The solution is filtered. Thereby, 7.5 g of an inactive, insoluble substance is separated. The filtrate is concentrated to a volume of approx. 1 liter. The concentrate, to which is added 1 liter of ethyl acetate, is stirred in the presence of 1 liter of water. A pH of 3 is adjusted by the addition of 6 N hydrochloric acid.

The organic phase is separated and concentrated to a volume of 1 liter of »30,504 RP slowly crystallizes by recommending the solution at + 4 ° C. The resulting crystals are separated by filtration and washed with 50 cm 2 of hexane at 4 ° C and then dried at 35 ° C under reduced pressure (5-10 mm Hg). In this way, 76 g of 30,504 RP is obtained as crude product in acid form.

c) Purification: 3 76 g of the above product is dissolved in 1520 Jcm of acetone. The solution is stirred for 1/2 hour in the presence of activated charcoal.

3

The suspension is filtered and the filter cake washed with 100 cm of acetone.

3

The filtrate and washing liquid are combined. Then 850 cm 3 of vydid is added with slow stirring at 0 ° C and the mixture is allowed to stand at 4 ° C.

3

The crystals obtained are separated by filtration, washed with 100 cm of a mixture of acetone and water in a volume ratio of 1: 3 and dried under reduced pressure (5-10 mm Hg) at 35 ° C. 68.5 g of 30.504 RP are thus obtained.

d) Recrystallization: 68 g of the product thus obtained is dissolved in 2.8 liters of hexane. The solution is stirred for 7 hours, during which the product slowly crystallizes.

The suspension is left for 15 hours at + 4 ° C, after which the crystals are separated by filtration, washed with 100 cm of hexane and dried under reduced pressure (5-10 mm Hg) for 12 hours at 35 ° C. In this way, a first fraction of 54 g of 30,504 RP is obtained in acid form.

Another fraction can be prepared by concentrating the mother liquor under reduced pressure (5-10 mm Hg) to a volume of 0.4 liters and curing for 12 hours at + 4 ° C. The crystals formed 3 are separated by filtration, washed with 20 hexane and dried under reduced pressure (5-10 mm Hg) for 12 hours at 35 ° C. In this way, a second fraction of 10 g of 30,504 RP is obtained in acid form.