DK141582B - Process for the preparation of lasalocid antibiotics or pharmaceutically tolerable salts thereof. - Google Patents

Description

(cf. (11) PUBLICATION 141582 DAMMARS (51) int. Cl.3 C 07 D 407/04 UAINIVIAK c 12 P 17/16 // c 12 r 1Λ65 (21) Note 1576/75 (22) Filing « the 1 * aPr * 1975 \ W® (23) Bill day 1 Apr 1975 \ £ y (44) The note presented and in accordance with the petition published on the aPr · s

DIRECTORATE OF

PATENT AND TRADEMARKET (3 °) Received 'desire' from it

Apr 2 1974, 457296, US

(71) P. HOFPMANN-LA ROCHE & CO. AKTIENGESELLSCHAFT, Grenzacherstrasee 124-184, Postfach, CH-4002 Basel, CH

(72) Inventor: John Westley, 111 Pollard Road, Mountain Lakes, New Jersey, US.

(74) The Plenipotentiary:

Plougmann & Vingtoft Patent Office. _____________ (64) Process for the preparation of lasalocid antibiotics or farms? * Ceutically tolerable salts thereof.

The present invention relates to a process for the preparation of novel lasalocide antibiotics or pharmaceutically tolerable salts thereof, characterized in that from a crude product obtained by submersing and aerobically culturing a microorganism of the species Streptomyces X-537 in an assimilable carbonic acid. drate and nitrogen source-containing nutrient solution isolate lasalocide B, C, D and E of the formula COOH f | 3 C / 5 R *, C 2 H 5

H0 V- / x "" C2H5 I

t fj I I V ^ O ^ H r1 H

Wherein one of the substituents R, R, R and R is ethyl and the others are methyl and a product obtained, if desired, is converted into a pharmaceutically tolerable salt thereof.

It has been found that antibiotics of formula I and their pharmaceutically tolerable salts have coccidiostatic and antibacterial effects.

Thus, they can be used as coccidiostatic and antibacterial agents.

The antibiotic-forming microorganism belongs to the genus Streptomyces and is isolated from a soil sample found in Hyde Park, Massachusetts. Freeze-dried specimens of laboratory labeled X-537 culture are 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 is commonly available through NRRL.

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

When culture is tested after those in Shirling, E.B. and D. Gottlieb, "Methods for Characterization of Streptomyces Species", Intern. J. Systematic Bacteriol. 16, 313 - 340, 1966 "(hereinafter referred to as" Shirling & Gottlieb, 1966 "), it shows a well-developed and branched mycelium. The spore chains exhibit pulled twists (retinaculum apertum according to that of Ralf Hutter: Systematic der Streptomycetes, page 10, Publ. S. Karger, 1967 given terminology.) Each trace chain carries an average of about 25 spores; the spore surface is rough.

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

Agar Medium 1) Colony Surface Back of Colony Soluble Color 2) Color 3) Pigment 4) Yeast-Painted 2dc (Light Brown-0c5r (Yellow-Yellow-Brown (ISP No. 2) Light-Gray)-Brown) 3 141582

Oatmeal e (light metal Co5a (whitish Light yellow-brown (ISP # 3) lily gray) yellow)

Inorganic e Coo4s (red-yellow-brown salty and starchy yellow-brown) see (ISP # 4)

Glucose-asparagus- 2dc Coo2m (golden Light yellow liggin (ISP # 5) yellow-brown)-brown 1) Media according to Shirling & Gottlieb, 1966 2) Tresner-Backus color scale 3) Prauser's (1964) color scale 4) No change in 0.05N HCl or NaOH.

When tested according to Shirling & Gottlieb, 1966, the culture utilizes carbohydrates as follows:

Arabinose 3+, cellulose +, fructose 3+, glucose 3+, inositol 4+, mannitol 4+, raffinose +, rhamnose 2+, sucrose + and xylose 3+.

When the culture is tested 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, observed the following physiological reactions:

Hydrolysis of: adenine +, casein +, hypoxanthine +, tyrosine +, potato starch +.

Formation of: urease, nitrate reductase -.

Utilization of: citrate +, lactate -, malate +, mucate -, oxalate -, succinate +.

Acid of: adonitol -, arabinose -, dulcitol -, erythritol -, galactose + glucose +, inositol +, lactose +, maltose +, mannitol +, mannose +, melibiosis +, α-methyl-D-glucoside +, raffinose -, rhamnose +, sorbitol -, trehalose +, xylose +.

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Growth at: 10 ° C +, 42 ° C +, 53 ° C

Growth in: lysozyme culture liquid, salicylate culture liquid, glycerol culture liquid +.

Using the International Streptomyces Project (ISP) (Shirling & Gottlieb, 1966) methods and Nonomura (J. Nonomura: Key for Classification and Identification of 458 Species of Streptomycetes Included in ISP; J. Ferment. Technol. 52 : 78-92, 1974), it appears that the culture does not match some of the streptomycetes examined in these studies. The culture contains the L-isomer of diaminopimelic acid.

The formerly known antibiotic X-537A (lasalocid A) is known as 6- [7 (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-creatinic acid, ie a compound of the formula COOH CH3 Cp3 C2H5 PH3 h ° Jxy * I I I h / \ o / A A oh J OH 0%

Up / ^ “3 ^ CH3

The antibiotic X-537A (lasalocid A) is described in J. Am. Chem. Soc. Vol. 73, 1951, pages 5295-98, and this compound can be prepared by fermentative route by cultivation of Streptomyces X-537. The fermentative preparation of the subject homologue of the antibiotic lasalocid A does not differ significantly from the known methods. However, according to the invention, using other isolation methods, it has been found that the culture fluid contains novel homologues of Formula I which are distinguished from lasalocid A by having surprising advantages such as lower toxicity or more intense antibiotic action.

5 1A1582

The lasalocld homologs in question are compounds of the following formulas and nomenclature:

Lasalocid B

CO OH CHo §R3 pH3 II f X / 2Hr-A * C2H5 \ # / V 2 5 T i I ηΛ / α-Λοη η c OH 0 H5C2 CH3 6- [7 (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-hydroxy-3-ethylbenzoic acid.

Lasalocid C: COOH C2H5 CH, * 2H5 _ H0

II T T Λ0 / Λ / oH

6x [7 (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] -3 (R) -ethyl-4 (S) - hydroxy-5 (S) -methyl-6-oxononyl] 2,3-cresotic.

Lasalocid D: COOH CH, C_H5 C2H fH3 F ^ I Ϊ i / - \ ^ 2H5 / -WC2H5 / Vy oh o% H3C ch3 6- [7 (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] -5 (S) ethyl - 4 (S) -hydroxy-3 (R) -methyl-6-oxononyl] -2,3-cresotic.

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Lasalocid E:

COOH CH3 5H3 S2H5f * CH

H0 \ JvV2 VVCA

TT 1 'AMT

6 OH [% H-C3 CH3 6- [7 (R) - [5 (S) -ethyl 1-5- (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-oxononylJ-2,3-cresotic.

The aforementioned lasalocide homologues are prepared as mentioned by fermentation of Streptomyces X-537 under aerobic sub-ambient conditions, setting the pH of the fermentation solution to approximately neutral value, i.e. ca. 6.5 - 7.5. The nutrient liquid used contains a nitrogen source, e.g. yeast, a yeast product, cornmeal or bean flour, which soybean meal is preferred; salts such as potassium phosphate and calcium carbonate and trace elements; a source of carbohydrate such as sugar or molasses, preferably brown sugar; and vegetable or animal fat or oil, e.g. soybean oil or lard oil, as a carbon source and anti-foaming agent and to improve the yield of the desired final product. The fermentation is carried out at slightly elevated temperature, ie. between approx. 25 and 35 ° C, especially at approx. 28 ° C. After an incubation time of approx. For 4 to 6 days, the fermentation solution is filtered and the crude product containing the lasalocides is isolated by extraction.

After isolation of the crude product, several methods can be used to isolate and purify the lasalocide homologs. Examples thereof are solvent extraction methods, e.g. intermittent extraction or liquid-liquid extraction in countercurrent distribution methods or gel permeation chromatography in a non-aqueous system.

Due to the particular advantages of the isolation of the lasa-locid homologs, it is preferred that the crude product be isolated by filtration of the aqueous nutrient solution, extraction of the filtrate with a water-insoluble solvent and separation of final products from the solvent extract, especially the filtrate extracted with n. -butyl acetate and the extract is treated with petroleum ether to obtain a mother liquor enriched with the desired end products.

A particularly preferred method of isolating the lasalocides from the crude product preferred by the invention is that the crude product is subjected to countercurrent distribution and that countercurrent distribution is carried out by first using a solvent system consisting of heptane-ethyl acetate-methanol water. whereupon the fraction containing the desired products is subjected to a further countercurrent distribution with heptane-ethyl acetate-ethanol-water-glacial acetic acid as a solvent system. Due to the particularly good yields thereby obtained, a preferred variant according to the invention assumes 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 acetic acid in a volume ratio of approx. 10: 5: 9: 3: 1.

The pharmaceutically tolerable salts of the present lasalocid homologs may be prepared in the usual manner. These salts are prepared from the free acid form of the antibiotic or a derivative thereof by methods known per se, e.g. by washing the free acid in solution with a suitable base or salt. Examples of such pharmaceutically tolerable basic salts are alkali metal bases, e.g. sodium hydroxide, potassium hydroxide or lithium hydroxide; alkaline earth metal bases such as calcium hydroxide and potassium hydroxide; and ammonia. Alkali metal or alkaline earth metal salts which can be used to prepare the pharmaceutically tolerable salts of this invention are e.g. carbonates, hydrogen carbonates and sulfates.

The relative antibiotic effect of the lasalocid homologs B, C, D and E in in vitro experiments against the microorganism Bacillus TA is found by comparing a pure sodium salt of lasalocid A with the four homologs using the agar diffusion method on hollow plates. The calculated numerical values for the five components are based on any set activity of 100 for the lasalocid A base and are shown in the table below: 1 Al 582

Compound In vitro activity against ______ Bacillus TA_

Lasalocid A 100

Lasaloeid B 90

Lasalocid C 180

Lasalocid D 160

Lasalocid E 170

In terms of toxicity, the lasalocides A, C, D and E lasalocid A produced by the method according to the invention are superior, as can be seen from the acute toxicity data (experiments performed on mice, values after 24 hours):

Product LD50 mg / kg orally for mice

Lasalocid B + C

(in a 1: 1 ratio) 210 - 30

Lasalocid D + E

(in a 1: 1 ratio) 195 - 32

Lasalocid A 146

It is apparent from the values given in the tables that the novel substances have surprising advantages over lasalocid A.

Coccidiostatic compositions containing as an active ingredient homologous to the antibiotic Lasalocid A or a pharmaceutically tolerable salt thereof can be prepared by mixing the active ingredient with an inert carrier. The inert carrier may be a feed or substitute material. By the term "inert carrier" is meant a material which does not act as an antiparasitic agent, e.g. as coccidiostats and which are harmless to the animals to be treated.

The active ingredient suppresses coccidiosis in utility poultry, especially turkeys and chickens, either by prophylaxis or, in the presence of disease, its healing after oral administration as a constituent of the feed. Furthermore, the treated animals retain their weight, or even increase their weight compared to control animals. Thus, the agents in question not only combat coccidiosis but also act as growth promoters.

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The concentration of the active substance in feed can be varied according to individual needs. Eg. For example, a feed premix or a complete feed may be enriched with a sufficient amount of active ingredient such that it constitutes approx. 0.006 - approx. 0.03% by weight of daily feed quantity. Preferably, approx. 0.015 -0.03% by weight. In general, approx. 0.015% by weight of the active substance to effectively suppress or combat coccidiosis. Larger amounts than 0.015% are effective, but generally show no benefits over 0.015% and in some cases can adversely affect growth, feed conversion and mortality.

Although amounts greater than 0.03 wt.% Are effective in controlling coccidiosis, these amounts for economic reasons are the preferred upper limit, ie. the cost per unit of action is lowest in this area. Below 0.006% by weight, the active substances are not effective in the control of coccidiosis. A lower limit of 0.015% is preferred as this concentration ensures optimum effect.

The particularly preferred concentration, i.e. Therefore, 0.015% by weight of daily feed intake is particularly preferred as it produces maximum effect at minimal dosage.

For the treatment or prophylaxis of coccidiosis, the active substances can first be brought in the form of a concentrate, a feed additive mixture or a feed additive. From these, whole feed or feed additive can be prepared by dilution. Examples of carriers are: commercial poultry feed, milled cereals, by-products of the milling industry, plant protein concentrates (eg soy and peanuts), by-products of fermentations, salt, limestone and inorganic compounds or mixtures thereof. Liquid dispersions can be prepared using water or vegetable oils, preferably with the incorporation of a surfactant or emulsifier, e.g. ethylene diamine tetraacetic acid, and solvent. As inert carriers, other carriers or substitutes may also be used if they are inert to the active substance and are non-toxic to the animals to which they are to be administered.

Typical poultry feed materials which can be mixed with the active ingredient contain e.g. cereal products such as maize, wheat, oats, barley, milk cereals, oatmeal and after-meal (eg wheat aftermeal); stabilized fats; plant proteins such as soybean meal, corn gluten meal and peanuts1; vegetable proteins such as fish meal, water-soluble parts of fish (fish solubles) and meat waste; sources of UGF ("unidentified growth factor") and other vitamin B carriers such as dry milk products, dried brewer's yeast, dried soluble residues from the distillation industry, dried residues from fermentations and lucerne green flour; and various special additives such as riboflavin, vitamin B 2 / calcium pantothenate, niacin, choline, vitamin K, vitamin E, stabilized vitamin A, vitamin (activated animal sterols), calcium and phosphorus supplements such as dicalcium phosphate, steam-treated bone meal, defluorinated phosphate and limestone, iodized salt , manganese sulphate, zinc carbonate, methionine or hydroxy analogs thereof, antioxidants and other antibiotically active feed additives.

The process according to the invention is further illustrated by the following example:

Example.

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

Streptomyces x-537 is grown in the aerated submersible nutrient solution in shake bottles. The pH of the gas ring solution is adjusted to 6.5 - 7.5 by the addition of aqueous potassium hydroxide solution; then the nutrient solution is sterilized. A tank fermentation is performed using an S - 10% inoculum consisting of a 3 day old submers culture from the aerated flasks. The nutrient medium contains 2% bean flour, 2% brown sugar, 0.1% dipotassium phosphate and 0.5% corn cast water. The fermentation is carried out at 28 ° C under positive air pressure, with air volumes of 150 - 300 liters / minute per day being injected. 150-300 liters of solution. The fermentation is stopped after 4-6 days, the fermentation solution is filtered and the antibiotic is isolated by extraction. The extraction is carried out as follows: Filter 204 liters of fermentation solution. The wet filter cake is poured into 100 liters of n-butyl acetate and the mixture is stirred overnight at room temperature. The mixture is then filtered and the aqueous phase is separated and discarded, the n-butyl acetate solution containing an anti-biotic concentration corresponding to 30 million Bacillus E units, evaporated to 3 liters under reduced pressure, washed with 10% aqueous sodium carbonate solution and dried over anhydrous sodium sulfate.

After further evaporation to 300 ml and dilution with 350 ral petroleum ether (boiling range 50 - 60 ° C), 41 g of solid, corresponding to 25 million Bacillus E units, is separated. This solid is then extracted in Soxhlet® apparatus with 4 liters of petroleum ether (boiling range 50 - 60 ° C) for 40 hours. The extract is evaporated to dryness under reduced pressure and the crystalline residue is dispersed in petroleum ether and the filters. After repeated crystallisations, one enriched mother liquor with the lasalocide homologues B, C, D and E. -

Isolation of the lasalocid homologs B, C, D and E.

A portion (corresponding to 22 g of solid) of the obtained mother liquor is chromatographed in a counterflow distribution apparatus equipped with 200 small tubes (each with a volume of 80 ml). The sample is dissolved in 160 ml of the mixed phases (heptane-ethyl acetate-methanol - 27: 18: 18: 2 water) and the solution is poured onto the first two small tubes. After 380 distributions, the following fractions are obtained, the solids obtained after evaporation being identified as follows: A. Mixture of the lasalocid homologs B, C, D and E, B. Lasalocid A, C. Isolasalocide A. This product is an isomer of Lasalocid A with a structure different from the lasalocides A - E.

Fraction A is dissolved in 20 ml of the mixed phases of the solvent system heptane-ethyl acetate-ethanol-water glacial acetic acid (10: 5: 9: 3;) and chromatographed in a counterflow distribution apparatus equipped with 500 small tubes. After 2800 distributions, approx. 200 mg of each of Lasalocid B, C, D and E with the following melting points:

Claims (2)

12 141582 Lasalocid B - 85 - 87 ° C Lasalocid C - 97 - 100 ° C Lasalocid D - 102 - 104 ° C Lasalocid E - 90 ° C. The sodium salt of Lasalocid E. Approx. Dissolve 100 mg of Lasalocid E in methylene chloride and treat with saturated aqueous sodium carbonate solution. The methylene chloride phase is evaporated with hexane. 104 mg of crystalline sodium salt of Lasalocid E is obtained (m.p. 182. -182.5 ° C). Claims .. A process for the preparation of lasalocid antibiotics or pharmaceutically tolerable salts thereof, characterized in that from a crude product obtained by submersing and aerobically culturing a microorganism of the species Streptomyces X-537 in an assimilable carbohydrate and nitrogen source nutrient solution. lasalocid B, C, D and E of formula R1 B.2 = 2R5 R3 p °° H Ξ Ξ II \ o '_ / Oli ·. Wherein one of the substituents R, R, R and R is ethyl and the others are methyl and a product obtained, if desired, is converted into a pharmaceutically tolerable salt thereof . Process according to Claim 1, 2, characterized in that the raw product is subjected to countercurrent distribution 3.