DE2450813A1 - ANTIBIOTICA - Google Patents

Description

Patent attorneys

Dr. ! ng. Walter Abte

Dr. Dieter F. Mo.rf 245.0813

Dr. Hans-A. Browns

8 Munich 86, Pisnzafiauefsir. 28

. 15 523Y

MERCK & CO., INC. Railway, New Jersey 07065, V.St.A.

Antibiotics

The invention relates to a new antibiotic which also growth-promoting activity on v / eist, from the fermentation liquid of Streptomyces lactamdurans by solvent extraction isolated from and referred to as FR-02A.

FR-02A is an antibiotic that is effective against both gram-positive and gram-negative bacteria, and consequently can be used to treat a wide variety of animal infections. In particular, FR-02A is effective against PPIO in chickens, pigs and cattle. It is also effective against mouse coccidiosis and against the most prevalent types of Chicken coccidiosis. The antibiotic also acts subcutaneously against air suction caused by M. gallisepticum In chickens and orally in mice against systemic infections caused by Bordetella bronchiseptica. Furthermore, FR-02A can be used as growth promoting agents in animals such as chickens, pigs and cattle.

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The invention relates to a new production process by fermentation and isolation of a valuable antibiotic Substance that is not yet known. In particular, the invention relates to the manufacture of the antibiotic FR-02A by fermenting Streptomyces lactamdurans under controlled conditions and then isolating the hitherto antibiotic FR-O2A not yet described.

The antibiotic FR-02A is controlled by cultivating the already known microorganism Streptomyces lactamdurans Conditions in a fermentation liquid and extracting the whole fermentation liquid with one with water obtained immiscible polar organic solvents. Fermentation can take place in media that contain suspended nutrients contain, or be carried out in predominantly clear media that are practically free of suspended nutrients are.

If the fermentation is carried out in media that are suspended Containing nutrients, the antibiotic is found in both the solids of the mycelium and the suspended nutrients as well as in the fermentation liquid. The antibiotic is isolated from the solids by removing the solids separates from the fermentation liquid by filtration, centrifugation or in some other way and the solid cake extracted with an organic solvent, preferably a polar organic solvent. The antibiotic, that remained in the liquid from which the solids were previously separated becomes from the liquid isolated by extraction with a water-immiscible polar organic solvent. It should be noted that by delayed harvesting, the total amount of solids in the fermentation liquid is reduced and less Amount of the antibiotic is found in the solids recovered from the liquid.

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For fermentation media that do not contain suspended nutrients, most of the FR-02A is found predominantly in the clear fermentation liquid. In these cases the entire liquid becomes immiscible with one with water polar organic solvent, such as chloroform, is extracted to recover the antibiotic. But you can too any solids, such as mycelium, from the fermentation liquor separate before extracting the latter with the water-immiscible polar organic solvent. Furthermore, in order to win all remaining FR-02A, that of the mycelium separated from the fermentation liquid with a suitable polar organic solvent extracted.

The preferred method for obtaining the antibiotic according to of the invention is the cultivation of the known microorganism Streptomyces lactamdurans under controlled conditions in a medium containing suspended solids or in a clear medium free of suspended solids and extracting all of the liquid with a water-immiscible polar organic solvent. The extraction is carried out by adjusting the pH of the liquid to an acidic range and adding the solvent to the liquid. After mixing, the solids are separated from the liquid. You let the liquid stand until the solvent is deposited Has. The solvent layer is peeled off, rinsed with water wash, dry with a suitable desiccant and evaporate in vacuo. The residue comes with a non-polar organic solvents, such as petroleum ether or hexane, washed and air dried. This is how you get the antibiotic FR-02A.

A preferred method for obtaining the antibiotic FR-02A is to cultivate the known microorganism Streptomyces lactamdurans under controlled conditions in a medium containing suspended nutrients. Including the solids the mycelium and the suspended nutrients are

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which is filtered off from the permentation fluid. ! aehdem the Filter cake has been blown as dry as possible, it is stirred into a polar organic solvent and filtered off again. After washing the filter cake with The combined solvent extracts and washing liquids are evaporated in a vacuum using more solvent, with an aqueous slurry remains. The pH is adjusted to an acidic range. The aqueous slurry is washed with a non-polar organic solvent such as petroleum ether, hexane and the like until the wash liquids are colorless. Then the aqueous slurry is extracted with a polar organic solvent. The solvent extract is dried, filtered and evaporated in a vacuum, leaving the antibiotic FR-Q2A as a residue remains behind. According to the method according to the invention, for example, the antibiotic FR-O2A can be used in about 40 to 50 percent preserved in pure form.

In the other method, the fermentation can be carried out in a medium which is practically free of suspended solids Nutrients is. The myeel is filtered off from the fermentation liquid and mixed with a polar organic solvent extracted. The extract is dried and evaporated to dryness in a vacuum. The residue is with a non-polar organic solvents such as petroleum ether, hexane and the like, shaken. The organic solvent is decanted after the residue has been allowed to settle by centrifugation. The residue is at the Air dried and the antibiotic FR-02A is obtained.

An even more preferred method of obtaining the antibiotic According to the invention, the cultivation of the microorganism Streptomyces laetamdurans is under controlled conditions in a fermentation medium and extraction of the medium after separating the solids, namely the mycelium or the mycelium and suspended nutrients. The pH of the fermentation liquid is adjusted to the acidic range

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and the solids, namely either just mycelium or Mycelium and suspended nutrients are separated. The fermentation liquid, which is free of pesticides, is mixed with a Water immiscible polar organic solvent mixed. After mixing, the layers are allowed to separate from each other. The organic layer is peeled off with dried using a suitable desiccant and evaporated to dryness in vacuo. The residue comes with a non-polar organic solvents, such as petroleum ether or hexane, washed and air-dried, the antibiotic FR-Q2A remains.

If the extractions are carried out in the procedures described above with a water-immiscible polar organic solvent can be carried out, solvents such as alkyl esters of lower alkanoic acids such as methyl formate, Ethyl formate, methyl acetate, ethyl acetate, n-butyl acetate, isobutyl acetate, Ethyl propionate, ketones such as cyclohexanone, or lower halogenated hydrocarbons such as chloroform, methylene chloride, Carbon tetrachloride, ethylene dichloride, 1-chloro-2,2-dimethylpropane, Use tetrachlorethylene or bromoform.

When the extraction processes described above are carried out with a polar organic solvent, can one solvents, such as lower alkyl esters of lower alkanecarboxylic acids, such as methyl formate, ethyl formate, methyl acetate, Ethyl acetate, n-butyl acetate, isobutyl acetate, ethyl propionate, Ketones, such as acetone, methyl ethyl ketone or cyclohexanone, or lower halogenated hydrocarbons, such as chloroform, methylene chloride, carbon tetrachloride, ethylene dichloride, 1-chloro-2,2-dimethylpropane, Use tetrachlorethylene or bromoform.

The microorganism not only produces PR-O2A, but how is known from the literature, Streptomyces lactamdurans NRRL 3802 produces the antibiotic cephamycin C (cf. "Antimicrobial Agents and Chemotherapy", September 1972, p

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122-131, Vol. 2, No. 3, "Cephamycins, a New Family of β-Lactam Antibiotics ", as well as BE-PS 764-160). Since FR-02A in with water immiscible organic solvents, while cephamycin C is highly soluble in water-immiscible organic solvents Solvent is as good as insoluble, these two substances can easily be separated from each other, and you get by extracting the fermentation liquor with a water-immiscible solvent of each of the two antibiotics in a form not contaminated by the other antibiotic.

The antibiotic FR-02A isolated from the fermentation liquid is further processed by chromatography on a molecular sieve and subsequent chromatography on a surface-active one Adsorbent cleaned. A molecular sieve suitable for this purpose is a cross-linked dextran, such as the molecular sieve "Sephadex LH-20" from Pharmacia Fine Chemicals Inc. FR-02A can be made from this molecular sieve with a lower Alkanol, such as methanol, can be eluted. A suitable surface-active adsorbent is a hydrophobic, nonionic, macroporous, crosslinked with divinylbenzene copolymer of styrene, which is available from Rohm and Haas under under the names "Amberlite XAD-1 to XAD-12" is brought. A preferred resin for purifying FR-02A is "XAD-2". Suitable for eluting the adsorbed FR-O2A Solvents are aqueous solutions of lower alkanols, e.g. aqueous solutions of methanol, ethanol, isopropanol, Butanol and the like. The preferred solvent for eluting FR-02A from the "XAD-2" resin is one 50 percent solution of isopropanol in water.

The microorganism that produces FR-02A is a well-known one Strain of Streptomyces lactamdurans, which is in the culture collection of Merck & Co., Ine ", Rahway, New Jersey, is designated MA-2908. It was isolated from a soil sample and collected from the Northern Utilization Culture Collection Research and Development Division, Agricultural Research

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Service, U.S. Department of Agriculture (formerly Northern Regional Research Laboratories), Peoria, Illinois 61604, for the farmer with no accessibility restrictions and is available to the public under culture number NRRI 3802.

Complete taxonomic and morphological studies over Streptomyces lactamdurans can be found in BE-PS 764 160. On the basis of taxonomic studies, Streptomyces lactamdurans was identified as a new Actonomycete. It was found, that it belongs to the genus Streptomyces and has many characteristics of the known species Streptomyces fradiae. Biochemically, the two agree almost completely; morphologically, however, there are important differences. For example, the color of the aerial mycelium of S. fradiae is sea shell pink, whereas S. lactamdurans is cream-colored. On> reason this difference and other characteristic features gave the microorganism the species name Streptomyces lactamdurans given.

Streptomyces lactamdurans is just one example of a microorganism strain which can be used to produce FR-02A; however, the invention is not to use limited by microorganisms that correspond to this special label. The invention also includes the Use of other microorganisms including the strains from actinomycetes that are either isolated from nature or obtained by mutation, e.g. microorganisms, that are obtained through natural selection, or microorganisms that are caused by mutation agents, such as X-ray irradiation, Ultraviolet radiation, N-mustard gas, and the like, and produce FR-O2A under suitable conditions.

FR-02A is inoculated in the aerobic fermentation of a suitable aqueous nutrient medium under controlled conditions produced with the organism Streptomyces lactamdurans. Aqueous media such as those used for the production of other antibiotics

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are also suitable for the production of the antibiotic FR-02A. Such media contained by the microorganism assimilable sources of carbon, nitrogen and inorganic salts. The choice of medium is not decisive and the fermentation can be carried out in media containing suspended nutrients or in media which are predominantly clear and practically free of suspended nutrients.

In general, carbohydrates such as sugars, e.g. dextrose, Glucose, arabinose, maltose, raffinose, xylose, mannitol and the like, and starches such as grain, e.g. oats, rye, corn starch, Cornmeal and the like, in the nutrient medium either alone or in combination as sources of assimilables Carbon can be used. The exact amount of the carbohydrate source or sources in the medium depends in part from the other components of the medium; in general, however, the amount of carbohydrate will vary between about 1 and 6 percent by weight of the medium. A single substance can be used as the carbon source, or several carbon sources can be used in the Combine medium with each other. As N sources in the fermentation process many protein compounds can be used. Suitable N sources are e.g. nutrient broth, yeast extract, yeast hydrolysates, Primary yeast, soybean meal, cottonseed meal, casein hydrolysates, corn steep liquor, soluble pulp components or tomato paste and the like. The N sources can be used either on their own or in combination with one another Amounts of about 0.2 to 6 percent by weight of the aqueous medium can be used.

The permeation media described in the examples are used only to explain the multitude of different possible media and are not to be interpreted restrictively.

The fermentation takes place at temperatures of about 20 to 37 ° C; fermentation is used to achieve the best results but preferably at temperatures from about 24 to

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32 ° C. The pH of the culture medium for cultivation the culture of Streptomyces lactamdurans and for the production of the antibiotic FR-02A is said to be in the range of about 6.0 "to 8.0.

On a small scale, the fermentation of the AntiMoticum is expediently carried out by adding a suitable nutrient medium inoculated with the culture producing the antibiotic and, after transferring it to the production medium, fermentation several times Days in the shaker at a constant temperature of about 28 ° C. At the end of the incubation period the mycelium and the suspended nutrients can be centrifuged off or filtered off and extracted with a solvent or the entire liquid can be extracted with chloroform or with liquids immiscible with water. But you can also extract the fermentation liquid after you have the solids, consisting of mycelium or from mycelium and suspended nutrients, separated from it.

The fermentation is carried out on a small scale in a sterilized flask through one-stage, two-stage, three-stage or four-stage vaccine development carried out. As a nutrient medium any suitable combination of C and N sources can be used for the vaccination step. The inoculation flask is kept in for 1 to 2 days shaken in a constant temperature chamber at about 28 ° C, and some of the culture obtained is used to inoculate either a second stage inoculation medium or the production medium used. When dealing with intermediate vaccination development flasks works, the inoculated material is developed in these essentially in the same way; i.e. a Part of the contents of the flask is used to inoculate the production medium used. The inoculated flasks are shaken for several days at constant temperature, and at the end of the incubation period, the antibiotic FR-02A, as already described, isolated.

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On a large scale, fermentation is preferably carried out in suitable Permenters carried out, which are equipped with a stirrer and devices for aerating the fermentation medium are. According to this method, the nutrient medium is prepared in the fermenter and heated to temperatures up to about Sterilized at 120 ° C. After cooling, the sterilized medium is inoculated with a previously grown inoculum from the cultivated culture and fermentation for several days, e.g., 2 to 4 days, with agitation and / or aeration of the nutrient medium and pause carried out at a temperature of about 28 ° C. The yield of FR-02A is generally 20 to 200 mg per liter Growing volume determined by bioanalysis.

Analytical method

The analyzes are carried out by the test disc and plate method using 9.5 mm filter paper discs. The analysis plates are made with Difco nutrient agar and 2.0 g of Difco yeast extract per liter in an amount of 10 ml per plate. An overnight cultivation of the test organism Vibrio percolans (American Type Culture Collection ATCC. 8461) in nutrient broth with a content of 0.2 <fo yeast extract is diluted in sterile saline solution to form a suspension with a permeability of 40 tfo at a wavelength of 660 μm. Before the plates are poured, this suspension is added to the medium in an amount of 20 ml per liter.

The test plates are kept at 4 ° C until use (maximum 5 days). After applying the antibiotic With the test discs saturated, the plates are incubated at 28 ° C. for 16 to 24 hours. The zones of inhibition are in mm in diameter read and used to determine the relative strength or, if compared to a purified reference standard, the strength in γ / ml used. Analysis of the FR-02A in fermentation liquids, Mycelium and the suspended nutrients separated from the fermentation fluids as well as in the Fermentation liquids free of nutrients are after

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extracting the FR-02A with a suitable solvent carried out. The analyzes of solutions that FR-02A in one Containing a concentration of 200 γ / ml show zones of inhibition of 16 mm when using 9.5 mm leaflets. If such Analysis is carried out quantitatively, 50 to 100 γ / ml antibiotic can be detected.

FR-02A is active against gram-negative and gram-positive bacteria, Coccidia and species of Mycoplasma. In vitro, FR-02A is effective against E-acervulina, Bordetella, and Streptococcus faecalis, Streptococcus faecum, Streptococcus agalactiae, Streptococcus pyogenes, Proteus vulgaris, M. hyorhinis, M. synoviae, M. arthritidis, M. galliseptieum and species of Pasteurella. There was also activity against Vibrio percolans (ATCC 8461), Salmonella gallinarum (MB 1287), Escherichia coli (MB 1418), Klebsiella pneumoniae (MB 1264), Pseudomonas stutzeri (MB 1231) and (MB 2765), Bacillus subtilis (MB 964) and (MB 797), Staphylococcus aureus (MB 108), (MB 210) and (MB 703) and against Pseudomonas aeruginosa (MB 3210).

PR-02A is both an antibiotic and a growth promoter Means can be used for animals.

When 3PR-O2A is used as an antibiotic, that is special Type of administration to animals not particularly decisive; all currently used or used to treat infected methods available to animals at risk of infection are satisfactory.

FR-02A can be used as an antibiotic, e.g. in the form of pharmaceutical Preparations, which are also an organic or inorganic, solid or liquid pharmaceutical Contain an extender that is suitable for enteral, parenteral or topical administration. Suitable extenders are substances that do not react with the antibiotic, e.g. water, gelatine, lactose, starches, stearyl alcohol,

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magnesium stearate, talc, vegetable oils, benzyl alcohol, resins, Propylene glycol, polyalkylene glycols, white petrolatum, cholesterol, or other "known medical excipients". The pharmaceutical preparations can, for example, as tablets, coated tablets, ointments, creams or capsules or in liquid form in the form of solutions, suspensions or emulsions. They can be sterilized and / or aids such as preservatives, Stabilizers, wetting or emulsifying agents, solubilizers, salts for regulating the osmotic pressure or Buffer, included.

If the antibiotic is to be presented in dry, solid unit dosage form, capsules, pills, or are used Tablets containing the desired amount of antibiotic. These dosage forms are made by taking the active ingredient intimately and evenly with suitable finely divided diluents, Fillers, disintegrating agents and / or binders such as starch, lactose, talc, magnesium stearate, Vegetable resins and the like. Such unit dose formulations may vary depending on factors such as the type of treatment being treated Host animal, the severity and type of infection and the weight of the host animal, in terms of their total weight and their FR-02A content vary within wide limits. The antibiotic can be used in amounts of about 5 to 100 mg per kg of body weight are administered.

The invention also encompasses the non-toxic, pharmaceutically acceptable salts of FR-02A, e.g., the alkali and Alkaline earth salts, such as the salts of sodium, potassium, ammonium and calcium, or salts with organic bases, such as triethylamine, N-ethylpiperidine and dibenzylethylenediamine.

In addition to being an antibiotic, the FR-02A can also be used as a feed additive to support the growth of animals such as chickens and sheep and cattle, speed up. Using FR-02A will reduce the time it takes for the Animals reach the weight required for sale.

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When PR-02A is used as a growth promoting agent for animals it can be given as part of the putter or in solution or suspension in drinking water.

When PR-02A is used as part of the putter, it is first formulated as a putter additive. In such putter accessories is PR-02A in relatively high concentrations contained in intimate distribution in an inert carrier or diluent. The putter attachment can be added directly to the Putter added or processed into a premix in a dilution or mixing stage. An inert carrier is one that does not react with the antibiotic and that can be safely administered to the animals. Preferably a carrier is used which is or can be a component of the animal's diet. Typical carriers or diluents, which are suitable for such mixtures are e.g. dried distillery grain residues, corn flour, citrus flour, permentation residues, ground oyster shells, welzen milling waste, soluble molasses, corn on the cob flour, edible bean mill feed, soy groats, crushed limestone and the like. The antibiotic is administered according to methods such as stirring, grinding or tumbling, intimately in the carrier dispersed. Mixtures containing the antibiotic in concentrations of about 5 to 50 percent by weight are suitable especially as putter additives.

Examples of typical putter additives, the PR-02A in dispersion contained in a solid support are:

(A) PR-02A 5

Wheat milling waste 95

(B) PR-02A 50

Corn distillery grain residues .... 50

These and similar putter additives are made by evenly mixing the antibiotic with the carrier. .

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The putter additive can be added directly to the putter or by further diluting or mixing with an orally presentable Carriers can be processed into a premix. Mixtures containing the antibiotic in concentrations of 0.03 to 5 percent by weight are particularly suitable as premixes. These premixes are made by adding the Mix the antibiotic evenly with an orally administrable carrier.

Such additives or premixes are used in the putter Amounts added so that the finished putter holds the PR-02A in the concentration desired to accelerate growth contains. PR-02A is given pure chickens in a concentration of 50 to 300 g per ton of putter to achieve the desired Achieve acceleration of growth. In the palle of pigs, including pigs infected with M. hyorhinis, PR-02A can be administered in the putter at similar concentrations.

In the above description of the invention, reference has primarily been made to solid mixtures in which the PR-O2A in the putter additive, called the premix or is present in the finished putter in a mixture with an edible carrier. This is the preferred method of administration by PR-02A. Another method is to dissolve or suspend the PR-02A in the animals' drinking water. The amount that can be suspended in the water without the risk of excessive settling is limited. One can therefore also add emulsifiers or surfactants.

The putter additives and finished animal feed mixes that PR-02A may also contain vitamins, other antibiotics and growth-promoting agents or other nutrients.

PR-02A is against poultry PPLO in amounts ranging from 5 to 100 mg / kg effective. A preferred area for a single

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dose is 35 "to 45 mg / kg. For the sake of simplicity there is the preferred method of administering the antibiotic to treat PPLO in the FR-O2A with the animal feed to mix. For the control of PPLO, a preferred one is Range 0.0055 to 0.02 percent by weight of the lutter.

For the treatment of aerial sacculitis in chickens, the ED ^ value is 40 to 100 mg / kg. Accordingly, the dose can vary FR-02A vary in the range of 10 to 150 mg / kg.

A solution or suspension for subcutaneous injection for the treatment of aerial sacculitis in chickens may be as follows getting produced:

Ampoule with subcutaneous solution or suspension containing 20 mg FR-02A

FR-02A 20 mg

Diluent: sterile water for those

Injection 2 cm.

The preferred method for treating coccidiosis is in that the FR-02A is presented in the feed at a concentration of about 0.05 to 2 percent by weight of the feed.

The dose to be used depends, of course, largely on the condition and weight of the host animal, and for the Oral administration is preferred for the treatment of PPLO and coccidiosis. For use as a growth promoting agent the FR-02A is preferably given mixed with the feed.

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example 1

Production of the antibiotic FR-02A by shake flask fermentation from Streptomyces lactamdurans

A freeze-dried culture of Streptomyces lactamdurans MA-2908 is used as inoculum.

The freeze-dried culture is in one with three baffles 250 ml Erlenmeyer flask provided with the 40 ml first-stage inoculation medium contains:

First stage inoculation medium

Primary yeast in distilled water 1 fo adjust the pH to 7.0 with NaOH

The first-stage inoculation flask as well as the subsequent second-stage flask and the production flask are in one with 220 rpm working shaker incubated at 28 ° C.

After 2 days in the first-stage medium, 1 ml of the first-stage Inoculation material for inoculating 40 ml of the two-stage inoculation medium in a one provided with three deflection organs, 250 ml Erlenmeyer flask used.

Second stage inoculation medium

"Ardamine YEP" (99F) '11 °

in distilled water;
pH to 7.0 with NaOH
set"

The second stage inoculation flask is incubated for one day, after which ten 250 ml Erlenmeyer flasks not provided with deflectors, each containing 40 ml of production medium, each with 1 ml inoculated from this culture.

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Production mediuro

Primary yeast 1 #

Soluble slurry ingredients $ 3

Glycine $ 0.05

! -Phenylalanine 0.3 #

Corn starch 2.0 fo

Dimethylformamide 1.0 V0I.-9S

"Mobil par-S" as foam ν relief 0.25 Vl $

medium in distilled water;

pH adjusted to 7.0 with UaOH.

A sodium thio sulfate solution is prepared by dissolving 12.5 g of Na ₂ S _{2 O, .5H 2} O in 100 ml of distilled water. This solution is filter-sterilized, after which 1 ml of the solution is added to each of the 10 production flasks.

The 10 flasks are incubated for 4 days at 28 ° C. and then harvested.

Isolation of FR-02A

The pH of 400 ml of the fermentation liquid thus obtained is adjusted to 5 'with hydrochloric acid, whereupon 2 parts by volume of chloroform are added to the liquid. After thorough The liquid is mixed through a filter plug filtered. Water is added to accelerate the filtration. The filtrate is allowed to stand until the chloroform layer has separated. The chloroform layer is peeled off, washed twice with 500 ml of water each time, dried over anhydrous magnesium sulfate and evaporated to dryness in a vacuum. The solid residue is mixed with 500 ml of petroleum ether, filtered off, washed with 50 ml of petroleum ether and turned on air dried. 144 mg of FR-02A are obtained. The FR-02A is converted into the ammonium salt by adding a solution of the FR-02A in 50 ml of water, which with ammonium hydroxide a pH of 10 has been adjusted, is freeze-dried. After freeze drying, the product weighs 140 mg.

Since FR-02A accumulates together with the mycelium and the suspended nutrients, the mycelium and the suspended nutrients can be

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substances first of all from the fermentation liquid sbfiltrioren and the FR-O2A from the filter cake with a polar, with Extract water-miscible organic solvents such as acetone. This method of isolating FR-O2A is as follows carried out:

From 400 ml of the total fermentation liquid obtained in the fermentation described above, the mycelium and the suspended nutrients filtered off. The filter cake is stirred with 40 ml of acetone for 30 minutes and filtered again. The cake is then washed with 15 ml of acetone. The combined acetone extracts and washing liquids are im Evaporated in vacuo at 30 ° C to drive off the acetone. The residue is taken up in 15 ml of water. The pH will be adjusted to 4.0 with hydrochloric acid.

An equal volume of hexane is added and the mixture is stirred for 10 minutes. After settling, the hexane is decanted and discarded. Man extracted twice more with the same volume of hexane, the last extract being colorless.

The aqueous slurry is then an equal volume Chloroform extracted. It is then extracted again with an equal volume and the second extract is combined with the first. The aqueous phase is discarded.

The chloroform extract is dried over anhydrous sodium sulfate dried, filtered and evaporated in vacuo to give 106 mg FR-02A.

By chromatography of the fermentation process described above recovered material on "Sephadex LH-20" the molecular weight of the FR-02A is determined to be about 1,000. The material obtained from this treatment is chromatographed again, this time on "Amberlite XAD-2", to get an analytically pure sample.

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Chromatography on "Sephadex LH-20" gel

0.5 ml of a methanol solution containing 106 mg of the FR-02A obtained according to Example 1 are applied to a 75 ml layer (1.25 cm diameter and 56 cm high) "Sephadex IH-20" gel applied in methanol (Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey). The column is developed with methanol. The eluate stream is measured using a differential refractometer (Meeco) monitored, and the diagram obtained in this way shows 3 maxima. Fractions are in 1 to 10 fold Dilute with 12.7 mm paper discs on agar diffusion Test plates inoculated with Vibrio percolans analyzed. Zones of inhibition are observed that correspond to the established correspond to the third mass maximum (Kj, = 0.3). The dem Fractions corresponding to the third mass maximum are combined with one another and evaporated. 43 mg of FR-02A are obtained.

If the product according to the above-described biological Agar diffusion method is analyzed with Vibrio percolans, it gives a zone of inhibition of 16 mm at one concentration of 200 γ / ml. The ultraviolet absorption in a phosphate buffer solution with a pH of 7 * 0 gives the following values:

^E 1 cm - ²¹⁶

1 4

E "= 464 ι cm

If the product obtained according to Example 1 in water at a pH value of 9 (which is adjusted by adding dilute sodium hydroxide solution) is dissolved, the insoluble material is filtered off and the filtrate is freeze-dried, the purification with "Sephadex IH-2O" gel can be omitted.

Chromatography on Amberlite XAD-2 ^U resin

The 43 mg of product obtained during purification with "LH-20". are further by chromatography on a 150 ml layer (1.25 cm diameter and 112 cm high) "Amberlite XAD-2" resin (Rohm & Haas Co., Philadelphia, Pa.) In 50 percent iso-

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propanol purified in water. The output is on a Acidified to pH 2 to convert it to the free acid before adding it to the column. The column will monitored with the aid of a differential refractometer (Meeco), and the diagram obtained shows 2 mass maxima. By Agar diffusion with 6.4 mm discs using Biological analyzes carried out by Vibrio percolans show that the mass maxima centered at K-p3.5 to 4-, O is the antibiotic contain. The fractions that correspond to these K-ß values, are combined with one another and evaporated to dryness in vacuo, so 16.4 mg of the antibiotic are obtained FR-02A, - PR-02A is obtained as a pale yellow solid material, which is resistant to normal handling and is purely analytical.

Example 2

Production of the antibiotic FR-02A by shake flask fermentation from Streptomyces lactamdurans

A freeze-dried culture of Streptomyces lactamdurans MA-29O8 is used as the inoculation material «

The freeze-dried culture is in one with three baffles 250 ml Erlenmeyer flask provided with the 40 ml of first-stage inoculation medium contains the following composition:

First stage inoculation medium

Primary yeast in distilled water 1 tfo ■ with NaOH to a pH value
from 7 _? 0 set

The first-stage inoculation vial and the subsequent second-stage The flask and production flask are incubated in a shaker operating at 220 rpm at 28 ° C.

After 2 days of incubation in the first-stage medium, 1 ml of the first-stage inoculum is used to inoculate 40 ml of the second-

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stepped inoculation medium in a three-sided enkcp? ganen, 250 ml Erlenmeyer flask used.

Second stage inoculation medium

"Ärdamine YEP" (99B ¹ ). Λ fo

in distilled water;
with NaOH to pH
set from 7.0.

The second-stage inoculation flask is incubated for one day, after which ten 250 ml Erlenmeyer flasks not provided with deflectors, each containing 40 ml of production medium, each with 1 ml inoculated in every culture. The production medium has the following composition:

Production medium

Corn steep water 2.8 wt .- ^

Cerelose 5.6 wt. -Fo

"ProfIo" 2.8 wt .- # ·

Glycerine 1.4 vol. -4>

Dimethylformamide 1.4 ToI .- $ in tap water, adjusted to pH 7.3 with NaOH.

One drop of anti-foaming agent ("P-2000") is added to each flask prior to autoclaving.

A sodium thiosulfate solution is prepared by dissolving 6.25 g of Na ₂ SpO- * 5H ₂ O in 100 ml of distilled water. This solution is filter-sterilized, whereupon 0.5 ml of this sodium thiosulfate solution is added to each of the production flasks after the autoclave treatment.

The ten production flasks (400 ml in total) are incubated at 28 ° C. for 5 days and then harvested.

The contents of the ten flasks are combined, the pH is adjusted to 5.5 with hydrochloric acid, whereupon 500 ml of chloroform added and the mixture stirred for 1/2 hour at room temperature. The mixture is then centrifuged to separate the phases to separate, and the aqueous phase is discarded. the

15 523Y

The chloroform layer is dried over anhydrous magnesium sulfate and the solvent evaporated in vacuo. The residue is shaken with 30 ml of hexane. After centrifugation the hexane is decanted from the residue. The residue is mixed with 30 ml of water, previously with ammonium hydroxide have been adjusted to a pH of 10, a somewhat cloudy solution being obtained. For the biological Analysis, the pH value is then reduced to 8.3 with hydrochloric acid and the solution with water from a pH value of 8.3 for the analysis diluted after the disc test. Biological analysis shows that the yield of the fermentation flask 327 γ FR-02A per ml of permentation liquid or in total 130 mg.

Example 3

Production of the antibiotic PR-02A by shake flask fermentation from Streptomyces lactamdurans

A freeze-dried culture of Streptomyces lactamdurans MLA-2908 is used as inoculum.

The freeze-dried culture is in one with three baffles 250 ml Erlenmeyer flask provided with the 40 ml of first-stage inoculation medium contains the following composition:

First stage inoculation medium

Primary yeast in distilled water $ 1>

with NaOH to pH
set from 7.0.

The first-stage inoculation flask and the subsequent production flask are incubated in a shaking machine operating at 220 rpm at 28 ° C.

After 2 days of incubation in the first-stage inoculation medium, ten 250 ml Erlenmeyer flasks not provided with deflectors, each containing 40 ml production medium, which

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practically free of suspended nutrients, each with ': ml of the first-stage inoculation medium. The production medium has the following composition:

Production medium

Uährbrühe (Mfco) 0.8 fo

Yeast Extract (Mfco) 0.2 <fo

Dextrose 1.0 fo in distilled water.

One drop of anti-foaming agent ("P-2000") is added to each flask prior to autoclaving.

The ten production flasks are then incubated at 28 ° C for 5 days and harvested.

Isolation of FR-02A

The contents of the ten production flasks (400 ml in total) are combined, the pH is adjusted to 5.5 with hydrochloric acid and filtered the fermentation liquor to remove the mycelium. The clear filtrate is mixed with 500 ml of chloroform and stirred for 1/2 hour. Then the mixture is centrifuged, to separate the phases and the aqueous phase is discarded. The chloroform layer is dried over anhydrous magnesium sulfate dried and evaporated to dryness in vacuo. The residue is washed with 30 ml of hexane, whereupon by Air dry the antibiotic FR-02A.

Example 4

Production of the antibiotic FR-O2A by shake flask fermentation from Streptomyces lactamdurans

A freeze-dried culture of Streptomyces serves as the inoculation material lactamdurans MA-2908,

The freeze-dried culture is placed in one with three steering organs 250 ml Erlenmeyer flask provided with the 40 ml of first stage inoculation medium of the following composition contains:

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First stage in the cultivation medium primary yeast in distilled water 1 $

with NaOH to pH
set by 7f0.

The first-stage inoculation flask and the subsequent production flask are incubated in a shaker operating at 220 rpm at 28 ° C.

After 2 days of incubation in the first-stage inoculation medium, ten 250 ml Erlenmeyer flasks not provided with deflectors, each containing 40 ml production medium, which is practically free of suspended nutrients, with 1 ml each of the first-stage inoculation medium inoculated »The production medium has the following composition;

Production medium

Nutrient broth (Difco) 0.8 #

Yeast Extract (Difco) $ 0.2

Dextrose 1.0 # in distilled water.

One drop of anti-foaming agent ("P-2000") is added to each flask prior to autoclaving.

The ten production flasks are then incubated at 28 ° C. for 5 days and harvested.

Isolation of FR-02A

The contents of the ten production flasks (a total of 4-00 ml) will combined, the pH adjusted to 5.5 with hydrochloric acid and the mixture filtered to recover the mycelium. The mycelium is extracted with 500 ml of chloroform. The chloroform extract is dried over anhydrous magnesium sulfate and in vacuo evaporated to dryness. The residue is washed with 30 ml of hexane and obtained by drying in air the antibiotic! FR-02A.

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15 523Y
Example 5

Production of the antibiotic FR-02A by shake flask fermentation τοη Streptomyces lactamdurans

A freeze-dried culture of Streptomyces serves as the inoculation material lactamdurans MA-2908.

The freeze-dried culture is in one with three baffles 250 ml Erlenmeyer flask provided with the 40 ml of first stage inoculation medium of the following composition contains:

First stage inoculation medium

Primary yeast in distilled v / ater 1 fo

with liaOH to pH
set from 7.0.

The first-stage inoculation flask and the subsequent production flasks are incubated in a shaking machine operating at 220 rpm at 28 ° C.

After 2 days of incubation in the first-stage inoculation medium ten 250 ml Erlenmeyer flasks not provided with deflectors, each containing 40 ml of production medium, which is practically free of suspended nutrients, with 1 ml each of the first-stage inoculation medium. The production medium has the following composition:

Production medium

Nutrient broth (Difco) $ 0.8>

Yeast Extract (Difco) $ 0.2

Dextrose 1.0 ψ in distilled water.

One drop of anti-foaming agent ("P-2000") is added to each flask prior to autoclaving.

The ten production flasks are then incubated at 28 ° C for 5 days and harvested.

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Isolation of FR-02A

The contents of the ten production flasks (400 ml in total) are combined and the pH is adjusted to 5.5 with hydrochloric acid. After adding 500 ml of chloroform, the mixture is stirred for 1/2 hour at room temperature. Then the mixture is centrifuged, to separate the phases and the aqueous phase is discarded. The chloroform layer becomes over anhydrous Magnesium sulfate dried and evaporated to dryness in a vacuum. The residue is washed with 30 ml of hexane and air dried. This is how you get the antibiotic FR-02A.

Example 6

Production of the antibiotic FR-02A by shake flask fermentation from Streptomyces lactamdurans

A freeze-dried culture of Streptomyces lactamdurans MA-2908 is used as inoculum.

The freeze-dried culture is in one with three baffles 250 ml Erlenmeyer flask provided with the 40 ml of first-stage inoculation medium contains the following composition:

First-stage inoculation medium Primary yeast in distilled water 1 fo with NaOH to a pH value
set from 7.0.

The first-stage inoculation flask as well as the subsequent second-stage flask and the production flask are in one at 220 rpm working shaker incubated at 28 ° C.

After 2 days of incubation in the first-stage medium, 1 ml of the first-stage inoculum is used to inoculate 40 ml of the second-stage inoculation medium is used in a 250 ml Erlenmeyer flask equipped with three deflectors.

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Second stage vaccination medium

"Ardamine YEP" (99F) in distilled water. 1 fo

adjusted to pH 7.0 with NaOH.

The second-stage inoculation flask is incubated for one day, whereupon ten 250 ml Erlenraeyer's flasks not provided with deflectors, each containing 40 ml of production medium, each with 1 ml inoculated in every culture. The production medium has the following composition:

Production medium

Corn steep water 2.8 wt .- ^

Cerelose 5.6 wt. - $>

"Proflo" 2.8 wt / α

Glycerin $ 1.4 vol

Dimethylformamide 1.4 vol .-? £

in tap water with FaOH

adjusted to a pH of 7.3.

One drop of anti-foaming agent ("P-2000") is added to each flask prior to autoclaving.

A sodium thiosulfate solution is prepared by dissolving 6.25 g of Na ₂ S ₂ O., 5H ₂ O in 100 ml of distilled water. This solution is filter-sterilized, whereupon 0.5 ml of this sodium thiosulfate solution is added to each of the production bottles after autoclaving. . '

The ten production flasks (400 ml in total) are incubated for 5 days at 28 ° C. and then harvested.

Isolation of PR-02A

The contents of the ten flasks are combined, the pH is adjusted to 5.5 with hydrochloric acid, and the mycelium as well as the suspended ones Nutrients are filtered off from the permentation fluid. The filtrate is mixed with 500 ml of chloroform and Stirred for 1/2 hour. The mixture is centrifuged to the

- 27 -.

509818/1246

15 523Y A.

Separate phases, whereupon the aqueous phase is discarded. The chloroform layer is dried over anhydrous magnesium sulfate dried and the solvent removed in vacuo. The residue is washed with 30 ml of hexane. By air drying the antibiotic FR-02A is obtained.

Physical Properties

The elemental analysis of the FR-02A provides the following result:

C = 60.98 ? O,
H = 7.60 %, Ή = 2.60 fo.

The empirical formula is CVqHq ₀ QgN ₂ O ₂₁ . This is in agreement with the molecular weight of 1168 as determined by mass spectrometry.

In the form of its aromonium salt, the FR-02A is in alcohol, and in Soluble in chloroform »At pH values of 7.0 or more, it is moderately soluble in water. The ultraviolet spectrum of the ammonium salt in water shows the following characteristics:

² ^ ^ ^E ] L ~ 320 528 nm; _B ] *, - 180

For FR-02A, 6 = 19 χ 10 ⁵

The nuclear magnetic resonance spectrum of the antibiotic FR-02A is recorded at 100 MHz in CDCl., As a solvent with tetramethylsilane (TMS) as the internal standard. Representative features of the spectrum are a doublet at 1.21, 1.31 and 4.63 τ and a singlet at 4.87 ν ₀

The infrared absorption spectrum of the antibiotic FR-02A in Nujol is shown in the drawing. FR-02A shows one characteristic absorption in the infrared of the spectrum at the following wavelengths, expressed in cnf:

50 9 8 18 / 12A6 - 28 -

15 523Y ^ *

Broad band "at 3400, strong bands at. 1640, 1460, 1380, 1080 and 1020, protruding bands at 1550, 1505, 1240, 1195, 940,

860, 720, 620.

FR-02A was determined according to various analytical methods with the fol investigated the following results:

1. "IH-20" column in methyl alcohol K ₀ = 0.302

(abandoned as NH « ⁺ salts)

2. "XAD-2" column; 50 percent

Isopropanol in water (given up as the free acid);

Leaving D.V. = 3.5 to 4.0

3. Thin layer chromatography

Silica gel eluted with a mixture of 80 parts of chloroform, 20 parts of methyl alcohol and 1 part of concentrated NH ₄ OH R _f = 0.344

4. Paper chromatography with a

Mixture of 70 parts of isopropanol and 30 parts of phosphate buffer (pH = 6.0; 0.01 molar) R _f = 0.9.

The FR-02A is a practically pure material as it is used in the Thin layer chromatography only provides a single spot and when monitoring the analysis in the "LH-20" column and gives a single Gaussian profile in the "XAD-2" column with the refractometer.

To further identify the 1R-02A in vitro, its Activity data using a profile of the antibiotic Spectrum determined. To carry out this test, a drop of about 0.015 ml of the antibiotic is used on the surface of inoculated complex agar plates

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"Bracht. The 3? R-O2A is in 10 percent solution
Methanol, which by itself does not create an inhibition zone. The results are shown in Table I as zones of inhibition in millimeters.

Table I.

Antibiotic spectrum profiles of FR.-02A
in the agar diffusion test

Diameter of
Inhibition zones, mm

Microorganism, MB

Bacillus sp. 633 Proteus vulgaris 1012 Pseudomonas aeruginosa 979 Serratia marcescens 252 Staphylococcus aureus 108 Bacillus subtilis 964 Sarcina lutea 1101 Staphylococcus aureus 698 * Streptococcus faecalis 753 Alcaiigenes faecalis 1018as vusicella bronchio percolansia coli 1287 aerogenes 835 Erwinia atroseptica 1159 Corynebact. pseudodipth. S. aureus 3032
S. aureus 2756
Strep, faecium 2820 Proteus vulgaris 838 E. eoli 60
S. aureus (res. Erythromycin) 1909

8th 2H 3 2 8th : -02A 0 1 5 23 0 1 mg / ml OH 9 0 20th 6th 1 OH 8th 6th 1 1 1 3 3 1 1 19th 21 2 1 1 28 1 27 1 1 1 1 1 1 1 1 1 1 1 • 1

H = cloudy zone,

* resistant to streptomycin, streptothricin,
Neomycin and viomycin.

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-37

When examined on mice, the Ar.tibiotiLouE FR-02A in vivo low toxicity and a "broad spectrum of antibacterial activity. An example of the results each for a gram-negative and a gram-positive bacterium are summarized in Table II.

Tabel

II

in vivo test results with FR-02A * Dar
reaching therapy on mice toxic infection IP
IP ^Dar ~ EI
reaching Toxicity** > 5.0
5.0 Microorganism IP 1,
IP 0, Ver
wear Proteus vulgaris
Strep, pyogenes 5.0
1.25 ^} 50 , 73
, 247

* The values in the table above are in mg per test animal expressed. Both the infection and the administration of the therapeutic doses of FR-02A are intraperitoneally. PR-O2A is used once at the time of infection and then given again after 6 hours.

** The toxicity studies with FR-02A on uninfected Mice show that two doses of 2.5 mg each per test animal, given at a time interval of 6 hours are tolerated by the animals, higher doses, namely 5 to 10 mg per test animal, but are toxic.

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Claims (30)

Merck & Co., Inc. 15 523Y claims 1. Process for the preparation of the antibiotic FR-02A, characterized in that the microorganism Streptomyces lactamdurans cultivates under aerobic conditions in a fermentation liquid that produces a assimilable sources of carbohydrate, nitrogen and inorganic salts existing medium contains until itself has formed a substantial amount of FR-02A, whereupon the antibiotic FR-02Ä. from the entire fermentation liquid with a polar one immiscible with water organic solvent extracted. 2. The method according to claim 1, characterized in that one is practically with one of suspended nutrients free fermentation medium works. 3. The method according to claim 1, characterized in that one with a suspended nutrients containing fermentation medium is working. 4. The method according to claim 2, characterized in that before extracting the antibiotic FR-02A from the fermentation liquid with a water-immiscible one polar organic solvent separates the mycelium from the fermentation liquid. 5. The method according to claim 2, characterized in that the mycelium is separated from the fermentation liquid and the antibiotic FR-O2A extracted therefrom with a polar organic solvent. 509818/1246 - s2 - 523Y ♦ & · 6. The method according to claim 3> characterized in that before extracting the antibiotic FR-02A from the Fermentation liquid with a water-immiscible polar organic solvent the suspended Separates substances from the fermentation liquid. 7. Process for the preparation of the antibiotic FR-02A, characterized in that the microorganism Streptomyces lactamdurans grows under aerobic conditions in a fermentation liquid that is suspended Medium containing nutrients from assimilable sources of carbohydrate, nitrogen and inorganic Contains salts until a substantial amount of FR-O2A is formed has, whereupon the solids are separated from the fermentation liquid and the antibiotic FR-02A from extracted the solids with a polar organic solvent. 8. The method according to claim 1, characterized in that as a water-immiscible polar organic solvent, a lower alkyl ester of a lower one Alkanearboxylic acid, a ketone or a lower halogenated hydrocarbon used. 9 »The method according to claim 7, characterized in that the polar organic solvent is a lower alkyl ester of a lower alkanecarboxylic acid, a ketone or a lower halogenated hydrocarbon is used. 10. The method according to claim 8, characterized in that as water-immiscible polar organic Solvents methyl formate, ethyl formate, methyl acetate, Ethyl acetate, n-butyl acetate, isobutyl acetate, ethyl propionate, Cyclohexanone, chloroform, methylene chloride, Carbon tetrachloride, ethylene dichloride, 1-chloro-2,2-dimethylpropane, tetrachlorethylene and / or bromoform are used ^{applies #} 5G 9818/1246 - 33 - 523Y -3V. . 11. The method according to claim 9> characterized in that the polar organic solvent is methyl formate, Ethyl formate, methyl acetate, ethyl acetate, n-butyl acetate, Isobutyl acetate, ethyl propionate, acetone, methyl ethyl ketone, Cyclohexanone, chloroform, methylene chloride, carbon tetrachloride, ethylene dichloride, 1-chloro-2,2-dimethylpropane, Tetrachlorethylene and / or bromoform are used. 12. The method according to claim 1, characterized in that the antibiotic FR-02A is obtained in practically pure form. 13. The method according to claim 7, characterized in that the antibiotic FR-O2A is obtained in practically pure form. 14. The method according to claim 1, characterized in that the antibiotic FR-02A is applied by chromatography a molecular sieve and subsequent chromatography on a surface-active adsorbent in practical pure form wins. 15. The method according to claim 7 »characterized in that the antibiotic FR-02A is applied by chromatography a molecular sieve and subsequent chromatography on a surface-active adsorbent in practical pure form wins. 16. The method according to claim 1, characterized in that an aqueous medium is used, which is about 1 up to 6 percent by weight carbohydrates and about 0.2 to 6 percent by weight contains usable nitrogen. 17. The method according to claim 7, characterized in that an aqueous nutrient medium is used which contains about 1 to 6 percent by weight of carbohydrates and about 0.2 to 6 percent by weight contains usable nitrogen. 509818/1246 - 34 - 523Y ί.5 18. The method according to claim 1, characterized in that the fermentation at temperatures in the range of about 26 ■
leads* wa 26 to 30 ° C for about 2 to 5 days 19. The method according to claim 7, characterized in that the fermentation is carried out at temperatures in the range from about 26 to 30 ° C. for about 2 to 5 days. 20. The method according to claim 1, characterized in that the pH of the aqueous nutrient medium is adjusted to a range from about 6.0 to 8.0. "21. The method according to claim 7, characterized in that the pH of the aqueous carrier medium is adjusted to a range from about 6.0 to 8.0. 22. The method according to claim 1, characterized in that the microorganism is Streptomyces lactamdurans NERL 3802 used. 23. The method according to claim 7, characterized in that the microorganism is Streptomyces lactamdurans . NEIL 3802 used. - 24 »Antibiotic FR-02A in practically pure form or in form of pharmaceutically safe, non-toxic salts, characterized in that it is a pale yellow solid Substance with the following properties is: a) Soluble in lower alkyl esters of lower alkanecarboxylic acids, Ketones and chloroform, slightly soluble in water and insoluble in hexane; b) ammonium salt soluble in alcohol, chloroform and sparingly soluble in water with pH values of 7.0 or more, c) Composition $ 60.98 carbon, 7.60 fo water 5098 18/1246 - 35 - 523Y "^ ' substance, 2.60 fo nitrogen and 28.82 fo oxygen; d) molecular weight about 1168; e) empirical formula ^C 5g ^H go_.Q6 ^N 2 ^O 21 ' f) Ultraviolet absorption in phosphate buffer solution of pH 7.0 ⁵²⁷ ™ '> AL - ^{21β 2} 32 nm; ε] ^ = 464; g) Ultraviolet absorption of the ammonium salt in water: max. 233 nm; e] * _n = 320 max. ^{328 nffi} 5 ^E l cm ' ^18O 5 h) R ^ = 0.344 in thin layer chromatography on a Kieselsäuregelplatte under development with a solvent system of 80 i> chloroform, methanol and 1 20 ^ io concentrated ammonium hydroxide; i) IL. = 0.9 at the paper chromatography development in the solvent system of 70 $> isopropanol and 30 fo phosphate buffer solution (0.01 molar), pH 6.0; j) characteristic infrared absorption spectrum according to Illustration; k) nuclear magnetic resonance spectrum with a doublet at 1.21, 1.31 and 4.63 T and a singlet at 4.87 r. 25. Therapeutic agent, characterized in that it a therapeutically effective amount of FR-02A or a pharmaceutical one harmless salt thereof as well as a non-toxic, pharmaceutically harmless diluent contains. 26. Growth-promoting agent for animals, characterized in that it contains a growth-promoting amount of FR-02A or a pharmaceutical - 36 - 503818/1246 523Y -fr " The pharmaceutically acceptable salt of the same forms an inert one Includes carrier. 27. Composition according to claim 26, characterized in that it is the FR-O2A. contains in amounts of 0.005 to 50 percent by weight. 28. Agent according to claim 26, characterized in that it contains the FR-02A in amounts of 5 to 50 percent by weight, 29. Composition according to claim 26, characterized in that it is in the form of a premix and the FR-02Ä in quantities contains from 0.03 to 5 percent by weight. 30. Agent according to claim 26, characterized in that it is in the form of the finished animal feed and the FR-02A. contains in amounts of 0.005 to 0.03 percent by weight. - 37 - 509818/1246 Blank page