CS195709B2 - Method of producing novel antibiotics based on polycyclic ethers - Google Patents

Description

The invention relates to novel polycyclic ether antibiotics having the character of acid, a class of compounds biologically characterized by their effect on the transfer of cations to mitochondria. This group of antibiotics includes:

monensin (J. Amer. Chem. Soc., 89, 5737, 1967), nigericin (Biochem. Biopliys. Res. Comm., 33, 2 2, 1968), grisorixin (J. Chem. Soc. Chem. Coinmun., 1421 (1970), dianemycin (J. Antibiotics, 22, 161, 1969), salinomycin (J. Antibotics, 27, 814, 1974), antibiotic X-537A (J. Chem. Soc. Chem. Commun., 967, 1972) , antibiotic X-206 (J. Chem. Soc. Chem. Commun., 927, 1971), antibiotic A204A (J. Amer. Chem. Soc., 95, 3399, 1973).

The polycyclic ether-based antibiotics mentioned above are active against gram-positive bacteria, fungi and protozoa. They develop significant activity against coccidia.

U.S. Pat. No. 3,839,557 discloses a method of improving the use of feed by ruminants and monogastric animals, fed fibrous matter of plant origin, by administering monensin, dianemycin, nigericin or other polycyclic ether antibiotics.

The invention relates to a novel polycyclic ether antibiotic known as compound 38 295 of the formula:

The antibiotic compound is produced by culturing Streptomyces hygroscopicus ATCC 31050 at 28-36 ° C in an aqueous nutrient medium containing an assimilable source of carbon, nitrogen, and inorganic salts under submerged aerobic conditions, and isolating the antibiotic either by separation from the fermentation liquid by extraction liquid with a water-insoluble organic solvent and chromatographically concentrated extract on a silica gel column or by evaporation of the fermentation liquid.

The microorganism used to prepare the antibiotic of the invention was isolated from a soil sample from Japan. It was grown in a number of nutrient media used to identify this genus and the result was that a substantially similar description to that of Streptomyces hygroscopicus was achieved.

The culture (Pfizer F. D. 23604) was compared in a number of characterizing features on various nutrient soils by Streptomyces hygroscopicus 482.48, obtained from the Dutch Public Culture Collection, Centraalbureau voor Schimmelcultures (CBS). Comparisons and decisions were made on the basis of the description of this species in Applied Microbiology, 4,243-250, 1956. The following compares two cultures:

Morphology of strings.

The chains of Pfizer F. D. 23604 in spirals of 6 to 10 turns or less on different media were similar to those described in Streptomyces hygroscopicus CBS shown in Figure 1 of the aforementioned work.

The spores in Pfizer FD 23604 were examined by electron microscope analysis and took the form exactly as in the strain CBS Streptomyces hygroscopicus, shown in Applied Microbiology, 18, 695, -696, where the spores are described as having rough surface pits. surrounded by dikes of wall materials, spore. When stained for microscopic purposes, both cultures appeared identical.

Color of colonies.

The aerial mycelium appeared gray in the line. However, the exact shielding is different for both cultures. Pfizer 23604 culture on sucrose. Czape.k agar exhibited growth forms thickened irregularly wavy, greyish-white, while S. hygroscopicus CBS culture was flat, thin and gray. The Pfizer F. D. 23604 culture on oatmeal agar after 4 weeks of incubation developed yellow airy mycelium that was previously gray. The Pfizer F. D. 23604 culture on the yeast extract and malt extract had yellow growth spots on the gray mycelium.

Melanin was not formed on either of the peptone-iron agar cultures.

Carbon utilization.

The method of Pridham and Gottlieb, J. Bact., 56, 107-114, 1948).

The Pfizer strain FD 23604 used glucose, L-arabinose, dextrin, D-fructose, D (+ j-galactose, glycerin, inosit, inulin, lactose,: maltose, D-mannitol, raffinose, rhamnose, sodium acetate, D-sorbitol, These results are consistent with those reported in S. hygroscopicus strains in Applied Microbiology, 15, 637-639, 1967 and in S. hygroscopicus CBS strain in Industrial Microbiology, 10 , 183-221, except that inositol was not used in the CBS strain.

Positive results in terms of hydrogen sulfide production were obtained in both cultures on iron peptone agar (sloping agar) with lead acetate strips.

In both cultures, the same results were obtained with respect to milk, no coagulation, complete hydrolysis or approximately complete in 12 days, coloring, soluble pigment.

Gelatine

The cultures were similar in growth, coloration and moderate degree of liquefaction.

Special characteristics.

The S. hygroscopicus CBS strain developed within 3 weeks on. Glucose-asparagine agar, oat-agar and agar containing black yeast and malt extracts of the black hygroscopic area, whereas Pfizer F, D. 23604 showed black spots only on yeast and malt extract agar. Further incubation for 1 week and storage on Petri cultures at 4 to 10 ° C for 4 weeks increased the blackness in Pfizer FD 23604 cultures. Pfizer FD 23604 strain on oat agar on Czapek sucrose agar and yeast extract agar, and malt produced an odor similar to that of wild onion or garlic with a certain earthy odor, whereas S. hygroscopicus CBS produced only an earthy odor, and there is no description of the wild onion odor in this strain. Pfizer F. D. 23604 S. hygroscopicus produces compound 38,295, which is a novel polycyclic ether antibiotic, whereas S. hygroscopicus CBS does not produce this antibiotic and no reports of the production of this antibiotic have been reported with other strains.

S. hygroscopicus. .

Pfizer F, D. 23604 was deposited with the American Type Culture Collectlon (Rockville, Md.) And was designated as ATCC 31050 in this collection.

The cultivation of Streptomyces hygroscopicus ATCC 31050 is preferably carried out in an aqueous broth at 28 to 36 ° C in a submerged aerobic culture with stirring. The nutrient medium useful for this purpose includes a source of assimilable carbon such as sugar, starches and glycerin, further comprising an organic nitrogen source such as casein or enzymatic degradation product of casein, soy flour, cotton cake, peanut flour, wheat gluten, soybean cakes, meat and fish meal. Growth-promoting sources such as soluble granular components and yeast extract as well as salts such as sodium chloride, calcium carbonate and trace elements such as iron, magnesium, zinc, cobalt and manganese can also be used with advantageous results. If excessive foaming occurs during fermentation, antifoam additives such as vegetable oils or silicones can be used which are added to the fermentation environment. The breeding of the culture medium in the submerged growth tanks is preferably carried out at a rate of about half to two volumes of fresh air per volume of liquid culture medium per minute. Stirring can be carried out using stirrers customary in the fermenting industry. However, it is necessary to maintain aseptic conditions during transport and growth.

The vaccine for the preparation of the antibiotic can be obtained by using partial cultures. Growth of the organism can be accomplished by inoculation of the skin into bottles or inoculation tanks, or alternatively, inoculation tanks can be inoculated with shake-bottle cultures. Shake flask culture generally reaches its maximum in 3 to 5 days, whereas the vaccine in submersible inoculation tanks is usually achieved in the most favorable period of 2 to 3 days. Significant antibiotic activity is achieved at the final, fermentation stage in approximately 3 to 5 days. The amount of antibiotic varies between 50 and 500 mg / liter.

The method of producing the antibiotic is preferably carried out by carrying out biological testing of the culture during fermentation using sensitive strains of Staphylococcus aureus or Bacillus subtilis. A standard plate test method is used, where it is used as a measure for the antibiotic activity of the inhibition zone that surrounds a filter paper soaked with culture medium.

For analysis of the antibiotic produced in the fermentation broth and for the composition of the raw and purified material extracted from the fermentation broths, a thin layer chromatography using silica gel is a useful tool. Spray with a 3% w / v solution of vanillin, in ethanolic sulfuric acid solution (98.5: -1.5%, v / v), thin-layer chromatograph after developing with ethyl acetate, then heat to 60-80 ° C after couple of minutes. The antibiotic is observed as a bright pink-red spot with a white background.

The antibiotic compound 38295 can be separated and recovered from the clarified fermentation broth by extraction with an organic solvent such as chloroform, ethyl acetate or methyl isobutyl ketone. The major portion of the antibiotic is contained in a separate mycelium and is preferably extracted therefrom by forming a suspension of mycelium in a water-soluble solvent such as methanol.

A preferred method of separating and recovering antibiotic compound 38,295 is as follows:

Aggregate unfiltered fermentation kapaliha extracted twice using about 1/5 to ^1/2 by volume of methylisobutyl ketone. The solvent extract is concentrated to an oily residue which is then suspended in a 1: 1 mixture of chloroform and hexane and loaded onto a silica gel column (preferably silica gel PF254, both from E. Merck, Darmstadt) is loaded onto silica gel 60. . The silica gel column was progressively developed with hexane, 1: 1 chloroform: hexane and chloroform. The major antibiotic fraction was eluted with ethyl acetate. The eluate was concentrated in vacuo, taken up in acetone and stirred for about 30 to 60 minutes with activated charcoal (Darco G 60). The charcoal was collected by filtration, the solution was concentrated to a small volume in vacuo, a small amount of water was added and the pH adjusted to 8.0-8.5 with sodium hydroxide. The crystalline material was collected by filtration, washed with water and dried.

The antibiotic isolated in this step is a mixed sodium and potassium salt of compound 38,295 formed by the action of both sodium and potassium ions found in the fermentation liquid and obtained from the added sodium hydroxide. Thin layer chromatograms may show the presence of trace amounts of at least two other multi-polar antibiotics which, like compound 38,295, exhibit characteristic pink-red spots when sprayed with vanillin in an ethanolic sulfuric acid solution.

Compound 38295 can be obtained as a pure unit by column chromatography on silica gel PF254 using hexane. The mixed sodium potassium salts were added as a solution in ethyl acetate or chloroform and the column was developed at a pressure of 5.5 kp / cm ² using hexane containing increasing proportions of ethyl acetate. Progress in separation was monitored by thin layer chromatography. Appropriate fractions were combined, evaporated in vacuo and the antibiotic crystallized in a mixture of acetone and water.

The free acid of compound 38295 can be obtained from the mixed sodium potassium salt by adjusting the pH in ethyl acetate or chloroform to 4.5 with dilute phosphoric acid. The organic layer was dried over anhydrous sodium sulfate, evaporated in vacuo and the residue crystallized from chloroform / hexane.

The sodium salt of compound 38295 may be obtained •

by adjusting the solution of ethyl acetate or chloroform of the free acid to pH 8.5 with 1 N sodium hydroxide solution. The material crystallized from acetone and water. The crystalline potassium salt is similarly obtained using potassium hydroxide instead of sodium hydroxide.

Crystalline silver salt can be prepared by adding an aqueous solution of silver nitrate to an ethanolic solution of the mixed sodium potassium salt of compound 38295. Other readily obtainable compounds are the salts of compound 38295 copper, zinc, ammonium, calcium, magnesium, and lithium. Due to the final use for the prevention and treatment of poultry concdiosis, the entire fermentation liquid containing compound 38295 can be dried, preferably by freeze-drying, and added to the poultry feed in an amount corresponding to the desired level of antiblottic activity.

Compound 38295 is characterized by a growth inhibitory effect on a number of microorganisms, as shown in Table I below. Test organisms are inoculated into a series of test vessels containing nutrient medium and different concentrations of compound 38295 to determine the minimum antibiotic concentration in mcg / ml that inhibits growth. organism within 24 hours.

Organism

Table I

Compound 38295 (mixed sodium potassium salt)

Compound 38295 (free acid)

Stapylococcus aureus 01A005 01A052 01A109 01A110 01A400

Streptomyces faecalls Streptomyces pyogenes Bacillus subtilis Lactobacillus casei var casei Lactobacillus catenaforme Clostridlum innocum Clostridlum blfermentans Treponema hyodysenteriae

0.78 0.39 0.78 3.12 0.78 0.78 0.39 1.56 0.39 1.56 0.20 1.56 <0.10 <0.10 <0.10 <0.10 <0.10 <0.10 1.56 - 0.78 - 0.20 - 0.78 - 0.39 -

Compound 38295 and its salts have excellent activity against coccidiosis in poultry. When added to the diet of chickens in an amount of 15 to 250 ppm, these compounds are effective to combat infections caused by individual microorganisms Eimeria tenella, E. acervulina, E. maxima and others, as well as against mixed infections caused by these microorganisms.

Data on the efficacy of Compound 38292 and its salts against infectious coccidiosis in chickens are obtained as follows. Groups of 3-5 day old SPF chickens (cocks) are fed a mixed diet containing compound 38295 or one of its salts uniformly dispersed therein. After feeding in this manner for about 24 hours of each chicken, it is vaccinated orally by individual Eimeria species to be tested. Other chickens fed without antibiotics are similarly infected and serve as infected control animals. Uninfected and non-treated chickens form normal control animals. Treatment results are evaluated after 5 days for Eimeria acervulina and after 6 days for all other Eimeria species.

Table II shows the results obtained using the mixed sodium potassium salt of compound 38,295.

^! Table II

Infecting organism sp. Dose (ppm) Average grade. infection* Degree of damage (ratio) Eimeria tenella 250 0.0. 0.0 0.0 125 0.0 Mi / ·· .. ' ¹ 60 0.7 0.21 30 1.7 0.54 15 Dec 2.7 0.86 Eimeria acérvulina 250 0.0 0.0 125 0.2 0.1 60 0.0 0.0 45 0.4 0.2 30 1,2 0.6 15 Dec 2,0 1.0 Eimeria nfefcatrix 250 0.0 0.0 125 0.2 0.13 60 0.2 0.13 Eimeria maxima 250 0.0 0.0 125 0.0 0.0 60 0.4 0.25 45 0.6 0.33 30 0.8 0.44 15 Dec 1,8 0.66 Eimeria brunettl 250 0.4 0.22 125 0.4 0.22 60 0.8 0.44 45 ‘ 1,2 0.66 30 1.6 0.88 15 Dec 1.6 0.88 infection of mixtures of microorganisms 250 0.0 0.0 ** 0.0 0.0 (Coccivac D, Sterwine Labo- 125 0,2 0,4 0.08 0.18 ratoťies, Opelika, Alabama) 60 0,2 0,0 0,08 0,0 50 1,0 0,6 0.38 0.21 40 1,2 1,0 0.46 0.35 V ' 30 2,6 2,0 0.85 0.71

Note:

* For the measurement of anticoctidiosis activity, the scale of damage rate from 0 to 4 for E. tenella (JE Lynch, Am. J. Vet. Res. 22, 324-326, (1961) and 0 to 3 for other species (J. Johnson and WH Reid.Exp. Parasit., 28, 30-36, 1970) The constant ratio is determined by dividing the degree of injury in each treatment group by the degree of injury of infected control animals.

** The first number refers to viscera damage and the second number to vision damage.

Similar results can be obtained using free acid, sodium salt, potassium salt, or a dried fermentation broth containing compound 38295 in batches of food at the desired level of antibiotic activity.

Properties of compound. 38295 underlining growth is explained by tests conducted on reared domestic animals.

The young heifers of Angus-Aberdeen cattle are arbitrarily divided into pens - 5 animals per pens - and are supplied to the dog by compound 38295, for example in drinking water, etc. 11 to 33 ppm. Compound 38295 may be in various forms as the free acid, sodium salt, potassium salt, mixed sodium potassium salt, or as a dried fermentation liquid produced by Streptomyces hygroscopicus ATGC 31050.

Feed mixtures with or without Compound 38295 are administered in any amount to test animals. The feed composition contains 12.5% protein, 75:25 concentrate in bulk cattle feed in adjusted doses containing corn, soybean meal, alfalfa meal, calcium phosphate, molasses, vitamins and trace elements. Compound 38295 is included in the premix together with ground corn with minerals and Vitamins.

Basic ration (12.5% protein - 75:25, concentrate: ration by volume of feed for cattle)

ground corn 55.6 soy flour, 48% protein 6.3 alfalfa flour, 17% protein 25.0 dried molasses * 10.0 soya oil 2.0 calcium phosphate (24% calcium, 18.5% phosphate 0.40 (trace mineral salts ** 0,50 mixture of vitamins and minerals *** 0,20

Note:

* Cane molasses on a carrier consisting of ground maize, not less than 3% raw protein, not more than 15% fiber, not less 42% inert sugar, not more than 6% water.

** Provides this trace element rate in micros: manganese 10, iron 6.25, copper 1.25, iodine 0.025, cobalt 0.86, zinc 45.

*** Produces the following levels of vitamins per 454 g of ration: vitamin A 1497 M.J .; vitamin

D 200 M.J .; vitamin E 25 M.J. Pre-mix < Folders: Quantity (1816 g) Vitamin A Supplemental (30,000 M.J./g) vitamin D3 supplementary (200,000 M.J, / g] vitamin supplement (0.27000 M.J./g) Zinc oxide (79.4% zinc) cobalt carbonate (45% cobalt) soya pulp or finely ground maize Compound 38295 100 g 2,00 g 182 g 38,0 g 1,70 g 1485 g 40.0 g

454 g of premix per tonne of the complete batch produces a final level of 11 ppm of 38295.

Table III explains the results of the 28-day trial obtained in three repeated groups of 5 calves each, with an approximate initial weight of calves of 115 kg for each calf.

Table III (mixed potassium sodium salts 38295)

Effect (ppm) Average Average Feed / Increment Daily Increment Daily Feed Ration

kg index kg index ratio index control 0.56 100.0 6.32 100.0 1.92 100.0 Compound 38295 (11) 0.64 110.6 5.68 95.1 1.65 117.0 compound 38295 (33) 0.67 116.9 5.58 87.3 1.43 136.0

Substantially the same results can be obtained using the free acid, sodium salt, potassium salt or dried fermentation liquid with compound 38295.

Comparable results may be obtained 1 in other ruminants such as sheep or in monogastric animals such as horses, pigs and rabbits.

The active compound 38295 may be administered together with an animal feed or beverage, or it may be used from a premix for mixing with an animal feed or beverage. The liquid antibiotic concentrate in the premix for addition to drinking water to increase animal feed efficiency can be prepared at the desired concentrations by dissolving in a suitable solvent such as ethanol or propylene glycol compound 38295.

Liquid premix concentrate

The liquid antibiotic premix concentrate suitable for addition to drinking water for animals can be prepared at the desired concentrations by dissolving in an appropriate solvent such as ethanol or propylene glycol of antibiotic 38295.

Example 1

Prepare a sterile aqueous medium containing the following composition:

g / 1

glucose 10.0 soluble starch 20.0 yeast extract 5.0 enzyme product degradation of casein 5.0 meat meal 5.0 calcium carbonate 1.0

pH - 7.0

Cells of Streptomyces hygroscopicus ATCC 31050 are transferred to a series of 300 ml flasks each containing 50 ml of said sterile broth and shaken on a rotary shaker for 3-4 days at 28-38 ° C. 5 ml aliquots of the propagated vaccine are transferred aseptically to 300 ml large bottles containing 100 ml each of the sterile broth described above. After shaking for 3 to 4 days at 28 to 30 ° C, the multiplied inoculum is transferred to a 4 liter fermenter, each containing 2 liters of the following sterile broth:

g / 1

glucose 10.0 cornstarch 10.0 soy flour 10.0 soluble components from cereals 5.0 sodium chloride 5.0 calcium carbonate 1.0

pH - 6.6

The fermentation is carried out for 90 to 120 hours at 28 to 36 ° C with stirring at 1700 rpm. and with ventilation so that about 1 volume of air per 1 volume of culture medium is supplied per minute. Large fermentation vessels containing from 300 L to 37,000 L of broth can be inoculated with about 2% of this multiplied vaccine. Fermentation is not complete until antibiotic activity is at least 50 mg / l (90 to 120 hours).

100 liters of complete unfiltered fermentation liquid is extracted 2X with 5% by volume of methyl isobutyl ketone. The separated extract in solvent was concentrated in vacuo on an oily residue (769 g) which was dispersed on silica gel by adding to a solution in 2 liters of chloroform per kg of silica gel PF254, after which the solvent was suspended in 2 L of chloroform and hexane (1: 1) and poured onto a bed of 300 g silica gel onto which a layer of 300 g silica gel PF254 was placed. The silica gel was then sequentially washed with 5.6 L of hexane, 5.6 L of chloroform with hexane (1: 1), and

5.6 chloroform. The antibiotic was eluted with 3.8 L of ethyl acetate. The ethyl acetate is evaporated in vacuo and the residue (38 g is dissolved in 400 ml of acetone. The acetone solution is stirred at room temperature for about 40 minutes with about 40 g of activated charcoal (Darco G 60). and diluted with 100 mL of water, Compound 38295 was separated as a mixed sodium potassium salt and adjusted to pH 8.5 by addition of 1 N sodium hydroxide solution. The column is developed at a pressure of 5.6 kp / cm ² with hexane using increasing amounts of ethyl acetate.The sequence of separation in sequence 14 is thin layer chromatography. water.

The pure crystalline mixed sodium potassium salt of compound 38295 has a melting point of 200 ° C and an optical rotation of [α] _D ²⁵ = + 36 ° C at a concentration of 1% in methanol. After drying under vacuum overnight at 70 ° C with phosphorus pentoxide, the resulting analysis for carbon was 60.58% and hydrogen was 8.56%.

The compound is soluble in methanol, ethanol, acetone, chloroform, methylene chloride, diethyl ether, ethyl acetate, and methyl isobutyl ketone. It is partially soluble in hexane and insoluble in water.

The mixed sodium potassium salt of compound 38295 has no characteristic absorption lines in the ultraviolet spectrum.

Figure 1 shows the infrared spectrum of the mixed sodium potassium salt. The sodium-potassium salt shows characteristic absorption lines in the infrared region at the following wavelengths expressed in microns, 92, 3.40, 6.27, 6.85, 7.22, 8.40, 9.00, 9.15, 9, 25, 9.48, 9.98, 10.45, 10.68 and 11.45.

Example 1 a d 2

The method of Example 1 was repeated using the clarified fermentation liquid instead of the complete unfiltered fermentation liquid.

Example 1 a d 3

The mycelium separated from the clarified fermentation liquid of Example 2 is suspended several times in methanol, the methanol extract is concentrated in vacuo and the residue is treated as in Example 1.

Example 4

The fermentation procedure of Example 1 is repeated using a fermentation broth of the following composition:

g / 1

glucose 20.0 cornstarch 10.0 soya muoka 10.0 soluble components of grain 5.0 ferric sulphate 0.2 Manganese chloride 0.2 calcium carbonate 1.0 sodium chloride 5.0 oleic acid methyl ester 2,0 soybean oil 2,0

pH 6.6-6.7

At the end of the fermentation cycle, the total of unfiltered fermentation solids is dried to dryness by spray drying.

lf

Example 5 of

The mixed sodium potassium salt of compound 38295 was dissolved in ethyl acetate and adjusted to pH 4.5 with dilute phosphoric acid. Layer,. The solvent is dried over anhydrous sodium sulphate, evaporated in vacuo and the residue crystallized from chloroform / hexane to give a crystalline salt having a melting point of 135-138 ° C and an optical rotation [α] _D ²⁵ = + 38 °. At 1% concentration in methanol. After vacuum drying overnight at 70 ° C using phosphorus pentoxide, the mixture contains an average of 61.65 wt. % carbon, 8.94 wt. % hydrogen and

29.41 wt. % oxygen; (differential). The free acid of compound 38295 has no ultraviolet light characteristics.

The infrared spectrum of the free acid of compound 38295 is shown in Figure 2. The compound exhibits characteristic infrared absorption at the following wavelengths in microns: 2.92, 3.43, 5.47, 6.85, 7.25, 7.35, 8.28, 8.60, 8.80, 9.00, 9.15, 9.80,

10.10, 10.45, 10.68, 10.98 and 11.13,

Analysis of the silver crystal of compound 38295 by X rays yields a structural formula

Example 6

The sodium salt of compound 38295 is obtained by adding 1 N sodium hydroxide solution to the free acid solution of Example 5, having a pH of 8.5 in ethyl acetate. The material crystallized from a mixture of acetone and water has a melting point of 197-198 ° C and an optical rotation [α] d ²⁵ = + 38 ⁰ at a 1% concentration in methanol. After drying in vacuo overnight at 70 ° C with phosphorus pentoxide, the sodium salt was analyzed to contain 60.28% carbon to 8.76% hydrogen.

Figure 3 shows the infrared spectrum of sodium 38295. The compound exhibits characteristic infrared absorption at the following wavelengths in microns: 2.94, 3.40, 6.25, 6.85, 7.20, 7.45 , 8.38, 9.00, 9.27, 9.48, 10.00,

10.45, 10.65, 10.90, 11.13 and 11.47.

Example 7

The potassium salt of compound 38295 was prepared as in Example 6 using potassium hydroxide instead of sodium hydroxide. A substance crystallized from acetone and water has a melting point of 196-199 ° C and an optical rotation of [α] ²⁵ = +38 ° at 1.02 ° / concentration in methanol. After drying overnight under vacuum at 60 ° C with phosphorus pentoxide, the compound contains 60.58% carbon and 8.70% hydrogen.

Figure 4 shows the infrared spectrum of potassium spli of compound 38295. The compound exhibits characteristic infrared absorption at the following wavelengths in microns: 2.94, 3.42, 6.27, 6.84, 7.20, 8.75 , 9.00, 9.14, 9.30, 9.45, 10.45, 10.92, 11.45 and 11.47.

Example 8

The silver salt of compound 38295 was prepared by adding an aqueous solution of silver nitrate to the ethanolic solution of the mixed sodium potassium salt of Example 1. The solid which separated was crystallized from methanol. It has a melting point of 198-200 ° C and an optical rotation of [α] _D ²⁵ - + 27 ° at a 0.82% concentration in methanol. The average composition of the vacuum dried overnight at 70 ° C with phosphorus pentoxide is 55.95% carbon and 7.89% hydrogen. The molecular weight, crystalline spli of silver and compound 38295 determined by X-rays is 1060 + 4 atomic mass units.

Claims (1)

Method for producing new antibiotics based polycyclic ethers of formula wherein the microorganism Streptomyces hygroscopicus ATCC 31050 is cultivated at 28 to 36 ^Q C in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions, and the antibiotic was isolated either by separating from the fermentation liquid by extraction of the fermentation liquid with a water-immiscible organic solvent and chromatography of the concentrated extract on a silica gel column, or by evaporation of the fermentation liquid.