NO143669B - PROCEDURE FOR THE PREPARATION OF LASALOCID ANTIBIOTICS - Google Patents

Description

The present invention relates to a method for the production of new antibiotics of the general formula

where R 1 , R 2 , and R 4 are methyl or ethyl, with only one of the substituents R 2 —- R 4 being ethyl,

in isolated form as well as pharmaceutically acceptable salts of these compounds.

It was found that the antibiotics of formula I and their pharmaceutically acceptable salts in addition to coccidiostatic and anti-bacterial action. They are thus usable as coccidiostatic and antibacterial agents.

The new antibiotics of formula I and their pharmaceutically acceptable salts are produced according to the invention by a method which is characterized by submerging and aerobically cultivating a microorganism belonging to the species Streptomyces lasaliensis (Streptomyces X-537), preferably the strain NRRL 3382, in a nutrient medium containing assimilable carbohydrate and nitrogen sources, after which the obtained compounds of formula I are isolated from the aqueous culture medium and, if desired, a compound obtained is transferred to a pharmaceutically acceptable salt thereof.

The microorganism that produces the antibiotics belongs to the genus Streptomyces and was isolated from a soil sample found in Hyde Park, Massachusetts. Freeze-dried samples of the culture designated with the laboratory designation X-537 were deposited in the microorganism collection of the United States Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois, USA under No. NRRL 3382. This culture has been made available to the public under the NRRL .

Below follows a general description of a typical strain of Streptomyces X-537.

When the culture is tested according to the regulations in Shirling, E.B. and

D. Gottlieb, "Methods for Characterization of Streptomyces Species", Intern. J. Systematic Bacteriol. 16, 313-340, 1966

(in the following brief termed "Shirling & Gottlieb, 1966" it exhibits a well-developed and branched mycelium. The spore chains exhibit windings pulled apart (Retinaculum-Apertum according to the terminology of Ralf Huttér: Systematic der Strepto-myceten, page 10, PubL.S . Karger, 1967).Each spore chain carries an average of about 25 spores 5 the spore surface is rough.

The morphology of the colonies on different agar media is as follows:

When the culture is tested according to Shirling & Gottlieb, 1966, it utilizes the following carbohydrates: Arabinose 3+, cellulose i, fructose 3+, glucose -3+, inositol 4+, mannitol 4+, raffinose rhamnose 2+, sucrose +, xylose 3+ .

When the culture according to R.E. Gordon "The Taxonomy of Soil Bacteria" in Ecology of Soil Bacteria, publisher T.R.G. Gray & D. Parkinson, Liverpool University Press, Liverpool, 1967 prove, the following physiological reactions were observed: Hydrolysis of: Adenine +, casein hypoxanthine +, tyrosine +,

potato starch -.

Production of: Urease -, nitrate reductase -.

Utilization of: Citrate +, lactate -, malate +, mucate -,

oxalate -, succinate +.

Acid from: Adonitol -, arabinose -, dulcitol -, erythritol -, galactose +, glucose + , inositol +, lactose +, maltose +, mannitol +, mannose +, melibiose +, a-methyl-D-glucoside +, raffinose -, rhamnose +, sorbitol -, trehalose +, xylose +.

Growth at: 10°C +, 4 2°C +, 53°C -.

Growth in: Lysozyme soup -, salicylate soup -, glycerol soup +.

By applying the methods according to the International Streptomyces Project (ISP) (Shirling & Gottlieb, 1966) and the key by Nonomura (J. Nonomura: Key for Classification and Identifica-tion of 458 Species of Streptomycetes included in ISP; J. Fer-ment. Technol. 52: 78-92, 1974) it appears that the culture does not correspond to any Streptomycetes examined in these studies. The culture contains the L-isomer of diaminopimelic acid.

The antibiotic hitherto characterized as antibiotic X-537A (Lasalocid A) was characterized in the laboratory analysis as 6-\ 1<C>R)-[5(S)-ethyl-5-(5(R)-ethyltetrahydro-5-hydroxy- 6(S)-methyl-2H-pyran-2(R)-yl)-tetrahydro-3(S)-methyl-2(S)-furyl]-4(S)-hydroxy-3(R), 5(S)-dimethyl-6-oxononyl} - 2,3-cresotinic acid, i.e.

a compound of the formula

The present invention provides homologues of the antibiotic lasalocid A and their pharmaceutically acceptable salts. The lasalocid homologues are compounds of the following formulas and nomenclature:

Lasalocid B:

6-(7(R)-15(S)-ethyl-5-(5(R)-ethyltetrahydro-5-hydroxy-6(S)-methyl-2H-pyran-2(R)-yl)-tetrahydro -3(S)-methyl-2(S)-furyl-4(S)-hydroxy-3(R),5(S)-dimethyl-6-oxononyl}-2-hydroxy-3-ethylbenzoic acid.

Lasalocid C:

6-{7(R)-1.5(S)-ethyl-5-(5(R)-ethyltetrahydro-5-hydroxy-6(S)-methyl-2H-pyran-2(R)-yl)-tetrahydro- 3(S)-methyl-2(S)-furylJ-3(R)-ethyl-4(S)-hydroxy-5(S)-methyl-6-oxononyl} - 2,3-cresotinic acid.

Lasalocid D:

6-(7(R)-15(S)-ethyl-5-(5(R)-ethyltetrahydro-5-hydroxy-6(S)-methyl-2H-pyran-2(R)-yl)-tetrahydro -3(S)-methyl-2(S)-furylJ-5(S)-ethyl-4(S)-hydroxy-3(R)-methyl-6-oxononyl} - 2,3-cresotinic acid .

Lasalocid E:

6-{7(R)-[5<S)-ethyl-5-(5(R)-ethyltetrahydro-5-hydroxy-6(S)-methyl-2H-pyran-2(R)-yl)-tetrahydro -3(S)-ethyl-2(S)-furyl]-4(S)-hydroxy-3(R),5(S)-dimethyl-6-oxononyl}-2,3-cresotinic acid.

The lasalocid homologues are produced according to the invention by fermentation of Streptomyces lasaliensis (Streptomyces X-537) under aerobic, submerged conditions, the pH value for the yeast solution being set approximately neutral, i.e. about. 6.5 to 7.5.

The nutrient base used contains a nitrogen source, such as yeast, a yeast product, corn flour or bean flour and soybean flour are preferred: salts such as potassium phosphate, calcium carbonate and trace elements; a carbohydrate source, such as sugar or flour, preferably brown sugar, and a vegetable or animal fat or oil, such as e.g. soybean oil or lard oil, as carbon source and agent for foam control and for improving the yield of the desired end product. The fermentation is carried out at a slightly elevated temperature, i.e. between approx. 25° and 35°C, especially at approx. 28°C. After an incubation time of approx. After 4 to 6 days, the yeast solution is filtered and the antibiotics are recovered by extraction.

After complete fermentation, many methods can be used for the isolation and purification of the lasalocid homologues according to the invention. Examples of these are the solvent extraction procedure, which e.g. intermittent extraction or liquid-liquid extraction in a countercurrent partitioning process or also gel permeation chromatography in a non-aqueous system.

The pharmaceutically acceptable salts for the Lasalocid homologues produced according to the invention can be produced by traditional methods. These salts are prepared from the free acid form of the antibiotic or its derivatives according to methods known per se, e.g. by washing the free acid in solution with a suitable base acc. a suitable salt. Examples of such pharmaceutically acceptable basic substances suitable for salt formation are alkali metal bases, such as e.g. sodium hydroxide, potassium hydroxide, and lithium hydroxide; alkaline earth metal bases such as calcium hydroxide and potassium hydroxide and ammonium hydroxide. Alkali metal or alkaline earth metal salts, which can be used for the preparation of the pharmaceutically acceptable salts according to the invention, are e.g. carbonates, bicarbonates and sulphates.

The relative antibiotic activity of the lasalocid homologues

B, C, D and E in the in vitro test against the microorganism 3acyl-lus TA was shown by comparison of a pure sodium salt of lasalocid A with the four homologues using the agar diffusion method on well plates. The calculated numerical value for the five components is based on an arbitrarily set activity of 100 for the lasalocid A base and appears in the table below:

With regard to toxicity, the lasalocids B, C, D and E obtained through the present invention are better than the known lasalocid A, as can be seen from the following data for acute toxicity in mice (24-hour value):

The lasalocids B, C, D and E which are obtained through the present invention consequently show advantages with regard to effect and/or toxicity compared to previously known products which could not be derived from the state of the art. They therefore constitute a surprising and valuable addition to the range of medicines.

The coccidiostatic preparations contain as active ingredient homologues of the antibiotic lasalocid A or a pharmaceutically acceptable salt thereof and are prepared by mixing the active ingredient with an inert carrier. The inert

carrier can consist of lining or additive materials. The term "inert carrier" means a material that does not work

as an antiparasitic agent, e.g. as a coccidiostat, and. which is harmless to the animals to be treated.

The active ingredient suppresses coccidiosis in productive poultry, especially turkeys and chickens, either by preventing or, in the case of already present disease, curing it after oral administration as a component of the sheep. Moreover, the animals to be treated maintain their weight or even increase their weight compared to the control animals. The agents according to the invention thus do not control bare coccidiosis, but they also act as growth promoters.

The concentration of the active substance in contraceptives can be varied according to individual needs. E.g. a feed pramix or a complete feed can be enriched with sufficient active substance to contain approx. 0.006 to approx. 0.03% by weight of the daily feed intake. Preferably, approx. 0.015 to 0.03 wt% is used. In general, approx. 0.015% by weight of active substance is sufficient to suppress or successfully combat the cocci. Larger amounts than 0.015% are indeed effective, but they generally show no advantages over 0.015% and in some cases can have an adverse effect on growth, and mortality.

Only amounts above 0.03% by weight are effective in combating coccidiosis. is this amount for economic reasons the preferred upper limit, i.e. the costs per unit of action is the lowest within this range. Below 0.006% by weight, the active substances for combating coccidiosis are not effective. A lower limit of 0.015% is preferred, because this concentration provides optimal activity. The particularly preferred concentration, i.e. about. 0.015% by weight of the daily feed consumption is therefore particularly preferred, as it gives the maximum effect at a minimal dosage.

For the treatment or prevention of coccidiosis, the active substance can first be brought into the form of a concentrate, a sheep additive "pramix" or a feed additive. Based on this, full-fledged forage or additive forage can be produced by dilution. Examples of carriers are: commercial poultry feed, ground cereals, by-products from the molasses industry, plant protein concentrates (soya, peanut, etc), by-products from fermentations, salt, limestone, inorganic compounds, etc. or mixtures of these. Liquid dispersions can be prepared from water or vegetable oils, preferably with the co-use of a surface-active agent or an emulsifier, etc., such as ethylenediaminetetraacetic acid, etc., and solubilizers. Other carriers or additive materials can also be used as inert carriers, if they are inert to the active substance and are non-toxic to the animals to which they are to be administered.

Typical poultry meats that can be mixed with the active ingredient contain e.g. cereal products, such as corn, wheat, oats, barley, "milo", oat flour, "Nachmel" (e.g. "tfeizennachmehl") etc.; stabilized fats; plate proteins, such as soy flour, corn flour, peanut flour, etc.; animal proteins such as fishmeal, water-soluble portions of fish ("fish solubles"), meat waste, etc.; sources for UGF ("unidentified growth factor") and other B-vitamin carriers such as dry milk products, dried brewer's oats, dried, soluble residues from the distillation industry, soluble residues from fermentations etc, alfalfa flour and various special additives such as "Riboflavin", vitamin B-^? calcium pantothenate, niacin, choline, vitamin K, vitamin E, stabilized vitamin A, vitamin D (activated animal sterols), calcium and phosphorus supplements such as dicalcium phosphate, steam-treated bone meal, defluorinated phosphate, limestone, etc., iodized salt, manganese sulphate, zinc carbonate, methionine or its hydroxy analogues, antioxidants as well as further antibiotically effective additives.

EXAMPLE 1

Preparation of the lasalocid homologues B, C, D and E.

The Streptomyces organism NRRL 3 3 82 is grown in aerated submersible nutrient solution in shaking flasks. The pH value of the fermentation solution is adjusted to 6.5 to 7.5 by adding aqueous potassium hydroxide solution, and then the nutrient solution is sterilized. A container fermentation is carried out, with a 5-10% inoculum consisting of a 3-day-old submerged culture from the aerated bottles being used. The nutrient medium contains 2% soybean meal, 2% brown sugar, 0.1% dipotassium phosphate and 0.5% corn spring water. The fermentation is carried out at 28°C under positive air pressure, with air quantities of 150-300 liters per minute per 150-300 liters of solution are blown in. Fermentation is stopped after 4 to 6 days, the yeast solution is filtered and the antibiotic is recovered by extraction. The extraction is carried out as follows: 204 liters of fermentation solution are filtered. The wet filter cake is slurried in 100 liters of n-butyl acetate and the mixture is stirred overnight at room temperature. The mixture is then filtered and the aqueous layer is separated and discarded, the n-butyl acetate solution, which contains an antibiotic concentration corresponding to 30 million Bacillus E units, is concentrated under reduced pressure to 3 liters, washed with 10% aqueous sodium carbonate solution and dried over anhydrous sodium sulfate.

After further concentration to 300 ml and dilution with 350 ml of petroleum ether (boiling point 50-60°C), 41 g of solid substance is secreted, which in the test corresponds to 25 million Bacillus E units. This solid substance is then extracted for 40 hours in a Soxhlet apparatus with 4 liters of petroleum ether (boiling point 50-60°C). The extract is evaporated under reduced pressure to dryness and the crystalline residue is dispersed in petroleum ether and filtered.

After repeated crystallizations, a mother liquor enriched in Lasalocid homologues B, C, D and E is obtained.

Isolation of the lasalocid homologues B, C, D and E

A portion (corresponding to 22 g of solids) of the mother liquor extracted according to example 1 is chromatographed in a countercurrent distribution apparatus equipped with 200 small tubes (each with an absorption capacity of 80 ml). The sample is dissolved in 160 ml of the mixed phases (heptane-ethyl acetate-methanol-water, 27:18:18:2) and the solution is placed in front of the first two small stirrups. After 380 distributions, the following fractions are obtained, the recovered solids after evaporation being identified as follows:

A. Mixture of lasalocid homologues B, C, D and E

B. Lasalocid A

C. Isolasalocid A. This product is an isomer of lasalocid A with a different structure from the lasalocids A-E.

Fraction A is dissolved in 20 ml of the mixed phases of the solvent system heptane-ethyl acetate-ethanol-water-glacial vinegar (10:5:9:3:1) and is chromatographed on a countercurrent distribution apparatus equipped with 500 small stirrups. After 2,800 distributions, each approx. 200 mg lasalocid B, C, D and E with the following melting points:

Lasalocid B - 85-87°C

Lasalocid C - 97-100°C

Lasalocid D - 102-104°C

Lasalocid E - 90°C.

EXAMPLE 2

Sodium salt of lasalocid E

About. 100 mg lasalocid E is dissolved in methylene chloride and treated with saturated aqueous sodium carbonate solution. The methylene chloride phase is concentrated with hexane. 104 mg of crystalline sodium salt of lasalocid E is obtained (melting point 182-182.5°C).

Addition in livestock

This addition makes it possible to use the active substance in an animal feed. A medicated poultry feed intended as a starter feed for broilers is prepared by mixing 0.015% by weight and 0.006% by weight of a mixture of lasalocid B and C (50/50) and lasalocid D and E (50/50) in a poultry feed.

The following experiment illustrates the effect of the active substance: Laboratory chickens infected with Eimeria tenella Experimental methodology: 10 chickens are used per dose. 10 chickens are used as weight control and 10 chickens as infected controls. The antibiotic is added 48 hours before the infection. 1 g of the antibiotic is mixed in a mechanical mixing device with the appropriate amount of chicken manure to reach the desired dosage. The infection consists of approx. 200,000 The oocyst, administered orally using a pipette. The experiment lasts 11 days, after which the surviving animals are subjected to autopsy and examined for gross caecal damage. The number of surviving animals and the number of caecal injuries are determined. The results are expressed as "average infection rate". With an average degree of infection of less than 1.5, the required dose is considered significantly effective.

As can be seen from the following, the antibiotic mixture is effective in the treatment of coccidiosis.

Additive in animal feed.

This addition makes it possible to use a single active substance as a coccidiostat in an animal feed. A medicated poultry feed intended as a starter feed for broilers is produced. About. 0.01% by weight of lasalocid D is mixed into the poultry feed.

The test methodology described above is used for the investigation

of the effect against Eimeria tenella. As can be seen from the following, the antibiotic works against coccidiosis.

Claims (6)

1. Procedure for the production of antibiotics with the general formula where R 1 , R 2 , R 3 and R 3 are methyl or ethyl, as only one of the substituents R^-R^ is ethyl, in isolated form, and pharmaceutically acceptable salts of these compounds, characterized by submerging and aerobically cultivating a microorganism belonging to the species Streptomyces lasaliensis (Streptomyces X-537), preferably the strain NRRL 3382, in a nutrient medium containing assimilable carbohydrate and nitrogen sources, after which the obtained compounds of formula I are isolated from the aqueous culture medium and, if desired, a compound obtained is transferred to a pharmaceutically acceptable salt thereof. 2. Method according to claim 1, characterized in that the end products are recovered by filtering the aqueous nutrient base, extracting the filtrate with a water-insoluble solvent and separating the end products from the solvent extract. 3. Process according to claim 2, characterized in that the filtrate is extracted with n-butyl acetate and the extract is treated with petroleum ether to obtain a mother liquor enriched in the desired end products. 4. Method according to claim 2 or 3, characterized in that the final products are separated from the solvent extract in such a way that the latter is subjected to countercurrent distribution. 5. Method according to claim 4, characterized in that the countercurrent distribution is carried out in such a way that one first uses a solvent system consisting of heptane-ethyl acetate-methanol-water and then subjects the fraction containing the desired products to a second countercurrent distribution with heptane- ethyl acetate-ethanol-water-glacial vinegar as solvent system. 6. Method according to claim 5, characterized in that the solvent system for the first countercurrent distribution consists of heptane-ethyl acetate-methanol-water in a volume ratio of approx. 27:18:18:2 and that the solvent for the second countercurrent distribution consists of heptane-ethyl acetate-ethanol-water-glacial vinegar in a volume ratio of approx. 10:5:9:3:1.