CS196262B2 - Method of producing antibiotics - Google Patents

Abstract

1479063 Antibiotics PFIZER Inc 16 April 1975 [16 April 1974 17 Jan 1975] 15748/75 Heading C2A A synergistic antibiotic mixture comprises compounds 35, 763, 36, 926, 37, 277 and 37, 932 and miscellaneous lesser antibiotic components produced by cultivating Actinoplanes auranticolor ATCC 31011 under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable source of carbon and nitrogen until substantial antibiotic activity is obtained and separating the antibiotic mixture therefrom. Antibiotic compound 37, 277 is in crystalline form and has an optical rotation of [α] D <SP>25‹</SP>= + 11‹ at a concentration of 1% in ethanol, absorption maxima in ethanol in the ultra-violet region at 225, 274, 282, 303 and 355 mÁ with E<SP>1%</SP> 1cm values of 309À3, 36.67, 45.01, 70 and 20, an average composition by weight of 60.91% C, 5.98% H, 10.45% N and 22.66% O, when dissolved in chloroform exhibits the absorption characteristics indicated in Fig. 1 (not shown), on heating decomposes between 145‹ and 150‹ C., is insoluble in diethyl ether, Lexane, treptane and water, slightly soluble in acetone and leuzene, and readily soluble in methanol ethanol, chloroform and methylene chloride. Antibiotic compound 36, 926 has an average composition by weight of 57.89% C, 6.78% H, 8.04% N and 27.29% O, a molecular formula C 26 H 35 N 3 0 7 , an optical rotation of [α] D <SP>25‹</SP>=-130‹ at a concentration of 1% in ethanol, absorption maximum in ethanol in the ultra-violet region of the spectrum at 214 mÁ. with E<SP>1%</SP> 1cm of 723À8, 8 when spectrum at 214 mÁ with E<SP>1%</SP> 1cm of 723À8, when pelleted on KBr exhibits the absorption characteristics indicated in Fig. 2 (not shown), and on heating, decomposition commences at 100‹ C., is soluble in ethanol, methanol, chloroform and methylene chloride and insoluble in diethyl ether, hexane and heptane. Antibiotic compound 35, 763 has the molecular formula C 26 H 27 N 3 O 7 , an optical rotation of [α] D <SP>25‹</SP>=-114‹ at a concentration of 1% in ethanol, absorption maximum in ethanol in the ultra violet region at 218 mÁ with E<SP>1%</SP> 1cm of 668.9, when pelleted in KBr exhibits the absorption characteristic indicated in Fig. 3 (not shown), is soluble in methanol, ethanol, chloroform and methylene chloride, insouble in diethyl ether, heptane and hexane, on heating decomposition commences at 100‹ C., has a M.Wt of 503 and an average composition by weight of 61À29% C, 6À73% H, 8À83% N and 23À15% O. Antibiotic compound 37, 932 is in crystalline form and has an optical rotation of [α] D <SP>25‹</SP> = + 5.0‹ at a concentration of 0.25% in chloroform, absorption maxima in ethanol in the ultra-violet region of the spectrum at 226, 276, 283, 305 and 355 mÁ with E<SP>1%</SP> 1cm values of 304.4, 36.8, 43.49, 70.25 and 20.07, an average composition by weight of 59.41% C, 6À01% H, 10À66% N and 23.92% O when pelleted in KBr exhibits the absorption characteristics indicated on Fig. 4 (not shown) and is soluble in methanol, ethanol, chloroform and methylene chloride, is insoluble in diethyl ether, hexane, heptane and water and on heating decomposition commences at 185‹ C. The antibiotic mixture or one or more of the antibiotics may be admixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition. The antibiotic mixture or one or more of the antibiotics may be incorporated in an animal feed composition.

Description

In literature dealing with. The synergism is extensively described: The Journal of Antibiotics 25, No. 6, 371 (1972), J. Chem. Soc. 19C 1653 (1966); Bull. Soc. Chim. Belg. 68, 716 (1959); J. Amer. Chem. Soc. 82, 4414 (1960); Tetrahedron 'Letters, 2687 (1971); J. Antibiotics, Ser. A 14, 14 (1961); Nature 187, 598 (1960), J. Chem. Soc. 2286 (1960); Antimicrobial Agents and Chemotherapy 360-365 (1964); Tetrahedron Letters 4231-4238 (1966); Organic Mass

Spectrometers, 6, 151-166 (1972); Tetrahedron Letters 369-372 (1966) and J. Chem. Soc. 19C, 1669-1766 (1966).

The novel antibiotic synergistic compositions prepared according to the present invention are coupled to other synergistic compositions already described: micamycline, pristinamycin, ostreogrycin, streptogramin, P.A. 114, vernamycin, and vlglniamycin.

BACKGROUND OF THE INVENTION The present invention relates to a process for the production of a mixture of antibiotics produced by submerged aerobic propagation of Actinoplanes auranticolor ATCC 31011 in an aqueous medium, as well as to the isolation of individual antibiotics therefrom. These mixtures containing macrocyclic lactones and depsipeptides can be separated and recovered from the fermentation broth by solvent extraction, countercurrent partitioning, column chromatography, or a combination thereof. The individual antibiotic components have significant antibiotic activity. The crude antibiotic mixture or the combination of the pure macrocyclic lactone and the pure depsipeptide obtained from the crude mixture exhibit considerable synergistic antibiotic activity. Crude antibiotic mixture, pure individual antibiotic components, and mixtures of pure macrocyclic lactones and depsipeptides are potent growth promoters of chickens and pigs and a drug for combating pig dysentery.

A method for producing these antibiotics according to the present invention is to cultivate Actinoplanes auranticolor ATCC 31011 under submerged aerobic conditions in an aqueous nutrient medium containing an assimilable source of carbon and nitrogen, preferably at a temperature of 28 ° to 36 ° C and preferably at a pH of about 7, and upon completion of the culture, the antibiotic mixture is isolated from the culture filtrate or mycelium, for example by solvent extraction with chloroform, ethyl acetate or methyl isobutyl ketone, followed by removal of the extraction agent to obtain a solid residue of the antibiotic mixture.

If necessary, further processing thereof, for example by countercurrent distribution and / or chromatography, is isolated individually

antibiotics, namely macrocyclic lactone. compounds 36 926 and 35 763, and depsipeptide compounds 37 277 and 37 932, respectively.

The microorganism useful for preparing the antibiotics of the invention was isolated from a soil sample in Egypt. It grew on potato agar with carrots and was found to belong to the class of sporangia-forming actinomycetes, as is the case of Actinoplanes. Therefore, it was grown in numerous environments used to study this genus. Culture suspensions were prepared by grinding culture pieces derived from sloping agar in small tubes containing about 0.2 ml of sterile distilled water each time, rinsing the contents and combining the contents with another amount of sterile water to a volume of about 5 ml for culture. These suspensions are used to grow cultures in different media in test tubes, in slanted cultures, or in petri dishes. The incubation temperature was 28 ° C unless otherwise indicated. Subtraction of results - some tests were performed after 22 days, but most results were recorded after 14 days. The colors of cultures are by Maerz and Paul, Dictionary of Colors, 2nd edition 1950, as well as personal descriptive names. This new culture (Pfizer FD 24090) was deposited in the American Type Culture Collection (ATCC) in Rockville, Maryland on March 11, 1974, and was designated Actinoplanes auranticolor ATCC 31011. Permanent storage and easy access to culture will be ensured if: Until a decision on the application of the invention applies to the availability of the culture, rules 14 and 35 of USC 112. All restrictions on the availability of the culture to the public will be irrevocably canceled after the grant of the patent.

The identification of the environment used to characterize the culture and the references to its composition are as follows:

1. · 2% · · aqueous agar

- 2. · Potato · carrot agar Μ. P. Lechevalier, J. Lab. and Clin. Copper. 71, 934-944 (1968). Only 30 grams of potatoes and 2.5 grams of carrots are used, but 20 grams of agar.

3. Gzapek's sucrose agar. Waksman, S.A .; · The Actinomycetes 2, 328 (1961) Medium No. 1, p., 328..

- 4. · · Glucose-asparagine agar. Waksman, see previous point, · Medium, No. 2, p. 328.

5. Agar · yeast and malt extract, Accinotics · Ann. 1956 · (1957), pp. 947-953.

6. Hickey and Tresner agar. J. Bact. 64, 891-892 (1952).

7. Potato-glucose agar. Peel, finely chop and boil 100 g potatoes with 500 · ml water, filter through the fabric, add 10 · g glucose, 20 g agar and make up to 1 liter with water.

8. Starch agar. J. Bact. 73, 15-27 (1957) .. ......

9. Gelatine J. Bact. 73, 15-27 (1957).

10. Tyrosine agar. J. Bact. 69, 147-150 (1955).

11. Difco ferric agar peptone.

12. Collected milk Difco.

13. Dextrose-nitrate culture medium. Waksman S.A., The Actinomycetes, 2, 328 (1961). Medium # 1 with 3.0 g glucose instead of sucrose and without agar.

14. Organic nitrate culture medium. J. Bact., 73, 15-27 (1957).

15. ATCC Environment 172. ATCC Catalog, 10th Edition, p. 235 (1972).

16. Utilization of carbon, J. Bact., 56, 107-114 (1948).

The description of this new culture is as follows:

Aqueous agar - weak growth, thin, flat near 9D2 (very pale pink), no aerial mycelium; 9D2 colorless substrate mycelium, no soluble pigment.

Czapek's sucrose · agar - moderate to good growth, flat, · near · 9G6 (pale orange), no · aerial mycelium, · substrate mycelium near · 9G6, no · soluble pigment.

Glucose-asparagine agar - growth mild, raised, rugged, close to 9L9 (light orange), no aerial mycelium, no soluble pigment.

Yeast and malt extract agar - no growth. ’

Hickey and Tresner agar - growth mild to · good, · slightly raised · and rough, close to 9F9 (dull · orange), faint whitish efflorescence on surface, substrate · mycelium · close to 918, light brownish, · soluble pigment.

Potato-glucose agar - · growth mild, raised, rough, near · 9L9 (light orange) ·, no air mycelium, substrate mycelium close to 9L9, · no soluble pigment. . .

Tyrosine agar - growth weak to mild, flat, close to 13A10 (dull reddish orange), no airborne, mycelium, · substrate mycelium close to 10D11, brown soluble pigment. :

Gelatin - moderate growth, close to 9K12 (reddish orange), traces of whitish efflorescence, substrate mycelium close to 9K12, no soluble pigment.

Starch agar · - mild growth · to good, raised, close to 9K10 (orange), · light whitish efflorescence, substrate mycelium · close to 9K10, · pale yellow soluble pigment.

The starch was poorly hydrolyzed, the liquefaction of the gelatin was strong, the nitrates were not reduced to nitrites in any of the nitrate media, even after 22 days. (Growth was very poor in dextrose nitrate medium but good in organic nitrate medium). hydrogen sulphide was produced in a small amount, there was none in peptone ferric agar. soluble · pigment, milk does not precipitate., and. . nehydroiy.žovala · not even after. expiration of 22 · days, weekly rosin was not digested, growth in ATCC environment 172 occurred at temperature · 28. to ‘3.7. . ° C. at "" "temperature. No growth occurred at 45 ° C. Arabinose, fructose, glucose, mannitol were used. Rhinosis, rhamnosis ,. sucrose, inositol was not used. · There is no odor in any environment. · '-.

Sporangia. arise. · only. on. ·. potato agar with carrots. The palisade layer is formed. · Dimensions are: · 5.5 to · 11 · x 4.5 · · to .8 microns (width) and 9 to · · 12 microns. · (Height). They are very numerous, irregular in shape and release spores as they progressively. softening. Sporangia · from potato · agar .s. carrot uvolňuíí · · · p ^on day incubation in a few hours of spores at ASI. 21 “C; . if the pieces of culture are immersed in. a small amount of a solution of 1 g of glucose, and · T · ml of Tween 80 · in one liter of water (a modification of the solution used by M. L. Higgins, J. Bact., 94, 495-498, Spores are, in irregularly shaped strings, in spores, but when released they are almost spherical and 1.6 microns wide to wide: elliptical, 1.6 to .2.2 x 1.1 to 1.6 microns, almost all are movable ...

. An attempt to identify led to a comparison of this culture with A. auranticolor ATCC 15300. auranticolor ATCC 31011 and A. · auranticolor ATCC 15330 have essentially similar appearance in morphological, color and soluble pigment · on · Bennett agar, on nutrient · agar, on yeast extract agar, · on glucose-asparagine agar, glycerol- asparagine agar, calcium malate agar, and tyrosine agar. Neither culture reduces nitrates to nitrites; both produce weakly hydrogen sulfide and do not have the ability to generate melanin on peptone ferric agar; both hydrolyzed Starch. Auranticolor ATCC 15330 does not cause any change in the milk collection tubes,. whereas a new culture. causes clarification in three of the six used milk tubes and forms a yellow-cream soluble pigment after 21 days.

A. auranticolor aTcC 31011 utilizes glucose, arabinose, fructose, mannitol, raffinose, rhomnose, sucrose and xylose. A. auranticolor ATCC 15330 uses all these sugars except raffinose. Sporangia and spores of these two cultures · · have. . similar to A. auranticolor 15330 spores, rather a rod shape. ,

Most importantly, A. auranticolor ATCC 15330 has no antibiotic effect under fermentation conditions in which A. auranticolor ATCC 31011 forms a mixture of antibiotics according to the invention. .

Cultivation of A. auranticolor ATCC. Preferably, it is carried out in aqueous nutrient media at a temperature. 28 to 36 ° C under submerged aerobic conditions and with shaking. · Media environments that:. can be used for this purpose comprising an assimilable carbon source such as sugars, starch and molasses, an organic nitrogen source such as casein, enzymatically hydrolyzed casein, flour. from soya beans. Cottonseed flour,. seed flour · groundnuts and wheat gluten. They can also be used, with favorable results, as · sources of · distillery growth substances · · stillage, · · fish meal, · yeast. extracts as well as salts such as sodium chloride and calcium carbonate and trace minerals. such as iron, · magnesium, zinc, · cobalt, manganese. If excessive foaming occurs during fermentation, antifoam agents may be added to the fermentation medium. vegetable oils or silicones. in tanks, for submerged. the growth is preferably maintained at a ratio of about% to 2 volumes of air per volume of culture medium per minute. Shaking may be carried out using shakers commonly used in the fermentation industry. However, aseptic conditions must be maintained during the transfer of the organism and throughout its growth.

The inoculum for preparing the antibiotic composition can be obtained using an increase. from inclined cultures in an environment such as ATCC environment 172 to which reference has been made above. The increments can be used either to inoculate shake bottles or seed tanks, or the seed tanks can be seeded from shake bottles. The maximum growth in shake bottles is. reached about · within four days, while inoculum in · submerged vaccination tanks ·. most preferably within 2 to 3 days. A substantial antibiotic effect is achieved in the final fermentation stage after a period of 20 to 30 hours. -.

Antibiotic formation is conveniently monitored during fermentation. biological tests of the broth using a sensitive strain of Sta-phylococcus aureus. Standard plate testing is used in which the inhibition zone surrounding the disc of the filter paper saturated with the broth is used as a measure of antibiotic activity. When the fermentation broth reaches the desired level of antibiotic activity, the products are either isolated from the whole broth or from the filtrate from the broth. nutrient media. In the latter case, the mycelium is removed by filtration or centrifugation. These devices can be used such as pressure filters, centrifuges, etc. ·

For analyzing antibiotic mixtures produced in fermentation media and for analyzing the composition of raw and purified materials extracted from. The fermentation broths are utilized by thin layer chromatography using silica gel. The differentiation of the components of the antibiotic composition is primarily dependent on the antibiotic activity of the system. Antibiotic activity too low - it does not reveal. minority. of antibiotic components too high antibiotic efficacy leads to veiling and thus to a low resolution result.

The developing system for thin layer chromatography is chloroform-ethanol (9: 1). Thin layer chromatograms can be observed after ultraviolet light at 254 ημ and 366 μM after development. Biological detection of antibiotic components can be accomplished by coating a thin layer of nutrient agar with a sensitive strain. Staphylococcus aureus or other susceptible organism.

'The primary components in the antibiotic mixtures produced by A.' auranticolor ATCC 31011 are various macrocyclic lactones and - depsipeptide antibiotic components. The composition of these antibiotic compositions varies from fermentation to fermentation and is a function of time, pH, environmental composition, etc. Under the conditions given in the Examples, the most important antibiotic components in the antibiotic compositions are: 37,277 (depsipeptide) and 36,926 ( macrocyclic lactone), while less important antibiotic components are compounds 37,932 (depsipeptide) and 35,763 - (macrocyclic lactone).

Macrocyclic lactones are compounds containing a ring with - more than 8 carbon atoms and also a lactone moiety, depsipeptides - are cyclic polypeptides with a lactone moiety.

The components of the antibiotic mixtures can be separated and - recovered from the fermented broth by various methods, such as solvent extraction, Craig countercurrent extraction, column chromatography or a combination of these methods. Various organic solvents, such as chloroform, ethyl acetate and methyl isobutyl ketone, are useful for the extraction of antibiotics from the broth. The solvent extraction is preferably carried out by extracting the nutrient broth twice at pH 7, with a volume of solvent equal to approximately Vs to% of the broth volume, from which the antibiotic mixture is to be recovered. Depending on the volume of the nutrient medium, extraction devices such as separating funnels, mixing tanks and mechanical extraction devices such as centrifuges can be used for extraction purposes.

A preferred method of separating and recovering the components of the antibiotic compositions is as follows:

the whole or clarified broth is adjusted to pH 7 and extracted twice with Vs-2

(B)

V2 volume of methylisobutylketin. The extract is concentrated in vacuo and the concentrate is defatted with heptane or petroleum ether. The defatted concentrate is then evaporated to dryness under vacuum. The solid residue was subjected to Craig countercurrent separation (6 levels) using toluene, 5 parts: ethanol, 2 parts: - aqueous phosphate buffer pH 4.5, 3 parts. After separating the layers into the upper and lower phases in the counter separation process, the phases are monitored by thin-layer chromatography. The separated fractions were evaporated in vacuo.

The solids containing the depsipeptides were dissolved in chloroform, decolorized with activated carbon and evaporated to dryness under vacuum. The residue obtained by evaporation of chloroform is dissolved in - acetone. The solid precipitated by the addition of heptane was dissolved in a small amount of chloroform and applied to a column of buffered silica gel (pH 6) in chloroform: n-propanol (9S9: 1% v / v). Column -. is developed in the same solvent mixture under a pressure of 0.55 MPa. The individual fractions collected are chromatographed on a thin layer. The fractions containing the separated depsipeptides were combined, evaporated in vacuo and crystallized from acetone-heptane.

The 'from - countercurrent extraction fractions containing macrocyclic' - - lactones are pooled, -. evaporate in vacuo and the residue is taken up in ethyl acetate. The solution was stirred with silica gel, filtered and the solvent was removed in vacuo. The residue was again ^- taken - ethyl acetate - and precipitated by addition of hexanes. The solids are dissolved in a small amount of chloroform and chromatographed under a pressure of 0.55 MPa. On a column of buffered silica gel (pH 6) in ethyl acetate. The digestion mixture is composed of ethyl acetate - tetrahydrofuran - hexane (80: 20: 20) and is saturated with aqueous phosphate buffer (pH-value 6). Separate fractions were monitored by thin layer chromatography. The fractions containing the separated macrocyclic lactones were combined and evaporated in vacuo. Individual - fractions are divided by Craig's counter-current. partitioning and / or column chromatography using different developing systems. Careful identification in each purification operation ensures that the individual macrocyclic lactones are sufficiently isolated so that the liquid fractions can be evaporated to give pure compounds.

Actinoplanes aurWticolor ATCC 310100 produces at least four depsipeptides and at least four macrocyclic lactones. However, the primary components are depsipeptides: compounds 37 277 (significant) and 37 932 (minor), and macrocyclic lactones: compounds 36 926 (significant) and 35 763 (minor).

Crude antibiotic mixtures directly obtained from the broth and purified individual components have broad antibacterial spectra. As . The microorganisms which stop reproducing in the presence of these antibiotics are Salmonella typhosa. Shigella dysenteriae,

Escherichia coli, Klebsiella pneumoniae, Sta196262 phylococcus aureus, Streptococcus pyogenes, Streptococcus fecalis, Diplococcus pneumoniae, Bacillus subtilis, Corynebacterium diphteriae, Clostridium septicum ^ Brucella abortus, Lactus sacurus acid.

Antibiotics prepared according to the present invention, either as a raw mixture or in the form of purified individual components or mixtures thereof, can be used in combating various infections in humans and animals. In general, for these antibiotics, administration at daily oral doses of 0.5 to 1 g or parenteral doses of from 100 to 500 mg, depending on the type and severity of the infection and the weight of the organism being treated, is most suitable.

The compounds of the invention may be, alone or in combination with pharmaceutically usable vehicles, and may be. administered in single or multiple doses.

Tablets for oral administration contain various fillers such as sodium citrate, calcium carbonate and calcium phosphate, and various disintegrants such as starch, alginic acid and complex silicates may be added together with a binder such as polyvinylpyrrolidone, sucrose, gelatin and arabic gum. In addition, glidants such as magnesium stearate, sodium lauryl sulfonic acid and talc for tabletting purposes are often used. Solid form preparations of a similar type can be used as fillers in soft and hard gelatin capsules, suitable materials are lactose, as well as high molecular weight polyethylene glycols. Aqueous suspensions and / or elixirs are suitable for oral administration, the essential component of which may be combined with various sweetening or flavoring agents as well as diluents such as water, ethanol, propylene glycol, glycerol and mixtures thereof.

Solutions of these antibiotics in sesame and peanut oil or aqueous propylene glycol can be used for parenteral administration.

Interestingly, the individual antibiotics of the present invention exhibit an antimicrobial effect which is largely bacteriostatic in nature. However, crude antibiotic mixtures or mixtures of purified depsipeptides and purified macrocyclic lactones exhibit a synergistic effect which is predominantly bactericidal in nature.

Testing of major macrocyclic lactone, compound 36 926 (A) and major depsipeptides, compound 37 277 [B] individually and in a mixture by dilution method in tubes against the following microorganisms showed a minimum inhibitory concentration in micrograms / ml / MIC:

MicroorganismA

Štaph. aureus 0051,56

Staph. aureus 4003,12

Strept. faecalis100

Neisséria sicca0,39

Treponemahyodysenteriae—

Shard, pyogenes 0.39

Bacteriodes fragilis 35 61425

Clostridium inocuum6,25

Lactobacillus cassi var. casei3,12

В A + В Crude mixture 25 0,10> <0.10 12.5 0,78> <0.10 12.5 1.56 0.20 50 0.10 <0.10 - - 0.19 12.5 0.10 <0.10 50 0.73 0.78 > 100 0.20 0.39 6.25 0.20 0.39

Comparable results were obtained when testing minor macrocyclic lactone, compound 35 763 (A ') and minor depsipeptides, compound 37 932 (B'), individually or at appropriate concentrations, ie A'sA, B '=== B or ( A '+ B'j = (A' + B ') = ξ [A + B') = (A Η-B).

The maximum synergistic efficacy of the mixtures of pure macrocyclic lactones and depsipeptides is about 1 to 2: 1. Approximately such a ratio of components occurs in fermented broths. AND.

auranticolor ATCC 31011 and in crude antibiotic mixtures isolated therefrom. This finding contradicts other synergistic mixtures described, where the synergistic factors appear in fermented soils and raw mixtures at suboptimal ratios.

In vivo data on the protective effect achieved with oral and subcutaneous administration in mice experimentally infected with Staphylococcus aureus is shown in the following table.

TABLE

PD 50 values (mg / kg)

S. aureus 01A005

orally subcutaneously Compound 36 926 200 200 Compound 37 277 200 200 compound 36 926 + compound 37 277 (1: 1) 210 60 crude antibiotic mixture 150 to 200 72 to 120

6'29 2

Example 1

The sterile aqueous medium has the following composition and preparation. se:

grams / liter glucose10,0 starch solution20,0 yeast extract5,0 enzymatic casein hydrolyzate 5,0 · calcium carbonate · 1,0 pH - 7,0

Actinoplanes auranticolor ATCC 31011 slant agar cells grown on ATCC medium 172 are transferred to a series of 300 ml Erlenmeyer flasks each containing 50 ml of this medium and agitated on a rotary shaker for 3-4 days at 28-30 ° C. . An aliquot of 5 ml of grown inoculum is transferred to 300 ml Erlenmeyer flasks each containing 100 ml of the sterile medium described above. After stirring for 3 to 4 days at 28 to 30 ° C. Transfer 5 to 10% by volume of the grown inoculum into a 4 liter fermentation tank containing two liters of sterile medium as follows:.

grams / liter yeast extract, 2.0 glucose10.0

Corn steep · liquor 1ml enzymatic casein hydrolyzate 5,0 cobalt chloride0,002 pH - 7,0

The fermentation is carried out for 20 to 30 hours at 30 ° C with stirring at 1700 rpm and drying with about one volume of air per volume of culture medium per minute.

The whole broth is adjusted, if necessary, to a pH of 7 and extracted twice with Us up to ¥ 2 of its volume with methyl isobutyl ketone. The extract was concentrated under reduced pressure and degreased by extraction with petroleum ether. The efficacy in the broth, extract and subsequent fractions is monitored by thin layer chromatography on silica gel in a chloroform-ethanol (9: 1) system and observed under ultraviolet light at 254 and 366 ταμ.

The defatted concentrate was evaporated to dryness under reduced pressure. The solid was subjected to Craig countercurrent separation on a 6-story apparatus using toluene, 5 parts ethanol, 2 parts: aqueous phosphate buffer, pH 4.5, 3 parts. The separated layers provide the upper and lower phases of the countercurrent distribution system. After separation, the layers are monitored by thin layer chromatography.

The depsipeptide, compound 37 277, is concentrated in the upper layer, preferably on the 0 th floor. The second depsipeptide, compound 37 932, is obtained from the top layer on floors 1, 2, 3. Macrocyclic lactone, compound 35 763 is preferably found in the lower layers of trays 0 and 1. Compound 36 926 'is present at the highest concentration in the lower stages of the trays. 2, 3, 4 and 5.

The higher phase on tray 0, which contains compound 37,277, was evaporated to dryness under reduced pressure, dissolved in chloroform and stirred for about 30 minutes with activated carbon. The solution was filtered and evaporated under reduced pressure. The residue was dissolved in acetone and the solid precipitated by the addition of heptane. The precipitate was dissolved in a small amount of chloroform and chromatographed on a silica gel column buffered to pH 6 with chloroform: n-propanol (99: 1% v / v). The column develops in the same solvent system under a pressure of 0.55 MPa. The individual fractions were monitored by thin layer chromatography. Fractions containing the separated compound 37 277 were combined, evaporated under reduced pressure, and the compound crystallized from acetone-heptane.

Compound 37 277 does not have a defined melting point. Decomposition occurs at a temperature of 140-150 ° C. This compound is insoluble in diethyl ether, hexane, heptane and benzene and readily soluble in methanol, ethanol, chloroform and methylene chloride.

Mean analysis of compound 37 277: 60.91 percent carbon, 5.98 in hydrogen, 10.45% nitrogen, 22.66% oxygen (by difference).

Compound 37 277 is optically active and has an optical rotation of [α] 25 ° - + 11 ° (c = 1.0, EtOH). The ultraviolet absorption spectra were taken in ethanol and showed peaks at 255, 274, 282, 303, 355 μα with E 'values of 309.3, 36.67, 45.01, 70 and 20.

The infrared spectrum of Compound 37 277 is shown in Figure 1. The chloroform solution exhibits characteristic infrared absorption at the following wavelengths in microns: 3.05, 3.40, 5.77, 5.93, 6.07, 6.62 , 6.82 and 7.67.

The countercurrent fraction contains 35,763- and 36,926, evaporated under reduced pressure, the residue taken up in ethyl acetate and stirred with silica gel. The filtered solution was evaporated under reduced pressure, the residue was taken up in ethyl acetate and solids were precipitated by addition of hexane. The precipitated solids were dissolved in a small amount of chloroform and chromatographed on a silica gel column in ethyl acetate buffered to pH 6.0. The column is developed with ethyl acetate: tetrahydrofuran: hexane (80:20:20) saturated with aqueous phosphate buffer (pH 6.0) at 0.55 MPa. The first 17 fractions, each of 20 ml, contained a yellow oil which was discarded. Fractions 26 to 40 contain predominantly compound 36 926. Fractions 41 to 50 are a mixture of compounds 36 926 and 35 763. 'Fractions' 26 to 40 are combined, concentrated under reduced pressure

Re-chromatograph on silica gel, collecting 20 ml fractions. The first 15 fractions contained a yellow oil which was discarded. Fractions 16 to 30 mainly contain compound 36 926, Fraction 31. to 40 contain a mixture of compounds 36,926 and 35,763, fractions 16 to 30 sec. evaporate under reduced pressure, dissolve the residue in a small amount of chloroform and apply to a column of buffered silica gel (pH 6.0) in chloroform. The column was developed with chloroform: ethanol (95.5: 4.5% v / v) at a pressure of 0.9 MPa and 160 fractions of 6 ml were collected. Fractions 60-90 'contained only compound 36 926. The fractions were combined and evaporated under reduced pressure. The residue was dissolved in a minimal amount of ethanol and precipitated with ether to give the amorphous pure compound 36 926.

Compound 36,926 is soluble in methanol, ethanol, chloroform, and methylene chloride. It is insoluble in diethyl ether, hexane and heptane.

Compound 36'926 does not have a defined melting point. Decomposition begins around 100 ° C. Average analysis of compound:

carbon 57.89% hydrogen 6.78% nitrogen 8.04% oxygen (difference) 27.29%

The molecular weight determined from high resolution mass spectra is 501 and the sum formula C35H36N5O7.

Compound 36 926 is optically active, and has a rotation of [α] D 25 = -130 ° (c = 1.0, EtOH). Its ultraviolet absorption spectra in ethanol show a maximum of 214 ταμ with E 1 * 723.8 values. ,

The infrared spectrum of compound 36,926, Figure 2, is attached. The spectra obtained by the KBr technique exhibited characteristic infrared absorption at the following wavelengths in microns: 2.95, 3.40, 5.75, 5.98, 6.23, 6.58, 6.87, 7.45 , 8.25, 8.38, 8.80, 9.08, 10.15, 10.35, 11.10 and 13.30.

This compound may be similar. or indistinguishable from. Antibiotic A2315B described in. Fourteenth Interscience Conference on Antimicrobial Agents and Chemotherapy, 11-13, September 1974.

Compound 36 763 is isolated and purified in a very similar manner to compound 36 926. The pure compound is soluble in methanol, ethanol, chloroform, and methylene chloride. It is insoluble in dimethyl ether, hexane and

heptane. It has not been defined temperature thaw. Decomposition begins around temperature Mp 100 ° C. Prů- specific analysis of the compound is: carbon 61.29 % hydrogen 6.73 % nitrogen 8.83 % oxygen (from difference) 23.15 %

and is 503 and the sum formula C26H37N3O7.

Compound 35 763 is optically active and has a rotation of [α] _D -25 ° = -114 ° (c ~ 1.0, EtOH). Its ultraviolet absorption spectrum in ethanol shows a maximum at 218 τημ with an E * of 668.9.

The infrared spectrum of compound 35 '763, Figure 3, is attached. The spectrum obtained by the KBr technique exhibits characteristic infrared absorption at the following wavelengths in microns: 2.95, 3.38, 5.73, 6.00, 6.23, 6.60, 6.88, 7 , 23, 8.25, 8.38, 8.83, 10.20.

Compound 35,763 may be similar or indistinguishable from antibiotic A-2315 described in the Dutch patent application. No. 7,310,613.

The countercurrent fraction containing compound 37932 was evaporated to dryness under reduced pressure, the residue triturated with petroleum ether and chromatographed on a column. silica gel as described above. Fractions of appropriate composition are combined and recrystallized from acetone: heptane to give a solid. to give the compound. 37 932..

Compound 37,932 is soluble in methanol, ethanol, chloroform, and methylene chloride. It is insoluble in diethyl ether, hexane, heptane and water. The compound has no defined melting point. The decomposition begins at about 185 ° C. Elemental analysis gave the following values:

carbon 55.41 ¹⁰ / o hydrogen 6.01% nitrogen. 10.6% oxygen (by difference) 22.2%

Compound 37,932 is optically active with · an optical rotation [a] _D 50 ² = + 5.0 ° (c = 0.25, CHC). In its ultraviolet spectrum, taken in ethanol, peaks at 226, 276, 283, 305 and 335 ταμ occur with E values. . 304,4,36,8,43,49,70,25, 20,07.

Infrared. the spectrum of compound 37 932, Figure 4, is attached. The spectrum obtained by the KBr technique shows characteristic absorption in the infrared region. at the following wavelengths in microns: 2.96, 3.05, 3.38, 5.68, 5.73, 5.93, 5.98, 6.13, 6.50, 6.58, 6, 88, 7.40, 7.65, 8.08, 8.45, 8.60, 9.03, 9.45, 9.70, 9.98, 10.53, 11.0, 11.23, 11.60, 12.33, 13.25.

Example 2

Method . according to Example 1 can be repeated with comparable results using a fermentation broth of the following composition:

glucose8,0 tryptose .3,0 enzymatic casein hydrolyzate 1,0 glutamic acid1,0 soya flour0,5 sodium chloride8,3

Molecular weight was determined from mass spectra · high resolution196262 grams / litt1S

K2HPO4. ·. · 2,4

7KH2PO4 - η · '· .fl. Manganese chloride 0.02

Example. 3.

.Method. according to Example 1, it can be a repeat, with a comparable result using a fermentation environment of the following composition:. grams / liter glucose / soy flour. 10.0 Corn steep water (Gorn steep liquor) 1 ml

Example 1a and Procedure according to Example. 1 may be repeated. with comparable results using a fermentation broth of the following composition:

molasses. 10.0 an enzymatic casein hydrolyzate 1.0 yeast extract 2,0 water from steeping corn Corn steep liquor 1 ml casein 3,0 ··· Example.5 - -

The procedure of Example 1 is repeated: The methyl isobutyl ketone extract of the final fermentation broth is evaporated to dryness under reduced pressure and the residue is triturated with petroleum ether. The loose material is ground and the antibiotic components are quantitatively determined by high pressure liquid chromatography. Representative total crude fractions. The antibiotic mixtures from the individual fermentations were (as a percentage) determined as follows:

Share # 35 763 . 36 926 37 932 37 277 Ostatní sl. 1 8.5 32.0 8.7 19.3 1.3. 2 3.1 39.5 7.8 15.9 1,2 3 6.0 38.6 10.7 25.5 2.4. 4 9.4 47.8 0.9 26.9 0,5 - 5 . - 8,4 . 31,9: 2.2 20.3 . 1.4.

OBJECT OF THE INVENTION

Claims (4)

A process for the production of antibiotics. comprising macrocyclic lactones and. depsipeptides, characterized in that it is cultivated, the microorganism Actinoplanes. auranticolor ATCC 31011 under submerged aerobic conditions in an aqueous medium containing an assimilable source of carbon and nitrogen, preferably at a temperature of 28 to 36 ° C, and preferably at a pH of about 7, and after cultivation the antibiotic mixture is isolated from the culture filtrate or mycelium for example by solvent extraction with chloroform, ethyl acetate. or methyl isobutyl ketone, whereupon removal of the extraction agent yields a solid residue of the antibiotic mixture. Method according to claim 1, characterized in that the individual antibiotics, namely the macrocyclic lactone compounds 36 926 and 35 763, and the depsipeptide compound 37 277, are isolated by countercurrent distribution and / or chromatography. 3. The method of claim 1, wherein the ahtibiotic depsipeptide compound 37,932 is isolated by countercurrent distribution and / or chromatography. 4 sheets of drawings Severography, n. P., Plant 7, Most Giant. i 37,277 3β, & 26