FI56979B - FOERFARANDE FOER FRAMSTAELLNING AV NYA LASALOSIDANTIBIOTIKA - Google Patents

Description

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Patent raeddelat ^ T ^ (51) Kv.lk. * / Int.ci. * c 12 D 9/14 A 25 K 1/1? C O? D 407/0 * FINLAND I — FI N LAN D (21) Patanttihakamu * - PatantansSknlnf 750930 (22) HakamlipUvt - Anseknlngadag 02.0U.75 (23) AlkupUvt —GHtlghotadag 02.0U.75 (41) Become Public - Bllvlt offant 75

Patent and Registration Office .... .. . . . ,,,., _ ,, (44) Date of publication and date of publication. -

Patent and regulatory authorities' Anaöktn utlagd och utl.akrlftan publkarad 31.01.30

(32) (33) (31) Privilege claimed— Bagird prlorltet 02.0U. 7U

USA (US) U57296 (71) F. Hofftaann-La Roche & Co. Aktiengesellschaft, Basel, Switzerland-Switzerland (CH) '(72) John Westley, Mountain Lakes, NJ, USA (7 ^ +) Oy Kolster Ab (5U) Method for the preparation of new lasalocid antibiotics - Förfarande för framställning av nya lasalosidibiotika relates to a process for the preparation of new lasalocid antibiotics of the general formula

RU

CO H R R CpH l__ c H __k c H

"0" X χ 2 5 if R 1 OH 0 / "CH 3 wherein R 1, R 2, R 2 and R 2 represent methyl or ethyl, wherein only one of the substituents R 1 -R 2 represents ethyl, in isolated form, as well as for the preparation of pharmaceutically acceptable salts of these compounds.

f 2 56979

The antibiotic X-537A (lasalocid A) (j. Am. Chem. Soc. Vol. 73, 1951, pages 5295-98) is known, which can be prepared by fermentation. The fermentative preparation of lasalocid A homologues to be prepared according to the invention is not substantially different from the known preparation. However, according to the invention, using a different isolation method, new homologues of the formula I are obtained which, compared with the known lasalocid A, have advantageous properties, such as lower toxicity and stronger antibiotic activity.

It has been found that the antibiotics of the formula I and their pharmaceutically acceptable salts have coccidiostatic and antibacterial activity.

They are therefore useful as coccidiostats and antibacterial agents.

The novel antibiotics of the formula I and their pharmaceutically acceptable salts are prepared according to the invention by a process characterized in that a microorganism belonging to the species Streptomyces lasaliensis (Streptomyces lasaliensis X-537), preferably strain NRRL 3382, is cultured in contact carbohydrate and nitrogen sources and aerobically, until the antibiotic activity of the medium is appreciated, leaving nm nm of the prepared compounds of formula I isolated from the aqueous medium and, if desired, converted into a pharmaceutically acceptable salt thereof.

The microorganism producing the antibiotics of the invention belongs to the genus Streptomyces and was isolated from a soil sample found in Hyde Park, Massachusetts. Freeze-dried samples from a culture labeled X-537 were deposited under the NRRL 3382 microorganism collection of the Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois, USA. The culture is available to the public under the label NRRL 3382.

A typical strain of Streptomyces lasaliensis (X-537) is generally described below.

When examining the culture according to the instructions given by E.B.Shirling and D.Gottlieb in "Methods for Characterization of Streptomyces Species", Intern. J. Systematic Bacteriol. 16, 313-3 ^ 0, 1966, (hereinafter abbreviated as "Shirling & Gottlieb, 1966"), it appears to have a well-developed and branched mycelium. Spore chains have different threads (according to Retinaculm-Apertum terminology presented by Ralf Hiitter in Systematic der Streptomyceten, page 10, published by S. Karger, 1967) - Each spore chain has an average of about 25 spores, the surface of the spores is rough.

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

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Agar väliaine1 'colonies colony Soluble back surface color 2) side of the color pigment k)

Yeast Malt 2dc 0c5r Yellowish brown (ISP No. 2) (light brown (yellowish brown) solid gray)

Oatmeal e (ISP No. 3) (bright metal- Co5a Slightly gray) (rather yellowish-yellow)

Inorganic e Goo4s Yellowish salts and starches (reddish brown yellowish brown) (ISP No. U)

Glucose- 2dc Coo2m. Bright yellow asparagine (i§ ^ e ^ Pke 1 "1 θ r * ivan (ISP No. 5) brown) brown ^ Media according to Shirling & Gottlieb, 1966 2)" 'Tresner-Backus color scale ^ Prauser' in (1964) color scale 'No change in 0.09 n Helium or NaOH - When a culture is examined according to "Shirling & Gottlieb, 1966", it utilizes carbohydrates in the following order arabinose 3 + * cellulose +, fructose 3+, glucose 3+ »Inositol 4+» raffinose +, rhamnose 2+, sucrose +, xylose 3 + ·

In studying the culture in H.E · Gordon *, "The Taxonomy of Soil Bacteria" in Ecology of Soil Bacteria, T.B.G * Gray & D.Parkinson,

According to Liverpool University Press, Liverpool, 1967f, the following physiological reactions are observed!

Hydrolysis of the following substances: adenine +, casein +, hypoxanthine +, tyrosil +, potato starch +.

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Generation of the following substances: urease, nitiate reductase

Utilization of: citrate +, lactate, malate +, lapse, oxalate, succinate +.

Acids from the following substances: adonltol, arabinose, dulcitol, erythritol, galactose +, glucose +, inositol +, lactose +, maltose +, mannitol +, mannose +, melibiose +, α-methyl-D-glycoside +, raffinose -, rhamnose +, sorbitol +, trehalose +, xylose +.

Growth at the following temperatures: 10 ° C +, 42 ° C +, 53 ° C -.

Growth in the following substances: lysozyme broth, salicylate broth, Gly-serol broth +.

Using methods consistent with the International Streptomyces Project * in (iSP) (Shirling & Gottlieb, 1966) and Nonomura (j. Nono mura: Key for Classification and Identification of 436 Species of Streptomycetes included in ISP | J. Ferment Teohnol. 52: 76-92, 1974) »it appears that the culture does not correspond to any of the Streptomycetes species blocked in these studies. The culture contains the L-isomer of diaminopropylic acid.

To date, the antibiotic labeled antibiotic X 537A (Lasaleside A) has been considered by laboratory analysis to be 6-E (7) (H) - (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) -dimethyl-6- oxononyl-2,3-creothioic acid, i.e. as a compound of formula 9 ° 2H CH3 CH3 C2H5 p H (A xv JL X / \ Co He r— \

Ai «T" 3 ch3

According to the present invention, lasalocid A homologues and pharmaceutically acceptable salts thereof are obtained. These lasalocid homologues have the following formulas and chemical names:

Lasalocid B: 5 56979 f * "f1 f", c *? 4 VV ^ vSr ^ X> 2V ^ * e> "·

AJ) J. J

ch3 6- [7 (R) - [5 (s) -ethyl-5- (5 (R) -ethyltetrahydro-5-hydro-6 (S) -methyl-2H-pyran-2 (R) -yl) - Tetrahydro-3 (s) -methyl-2 (S) -furyl] -4 (S) -hydroxy-3 (R), 5 (s) -diaethyl-6-oceononyl-2-hydroxy-3-ethylhenoic acid.

Lasaloeidi C: 9 ° 2h c2h5 rH c H 9¾

BiCAJ f »I

* ch3 6- [7 (R) - [5 (S) -ethyl-5- (5 (R) -ethyltetrahydro-3-hydro-6 (S) -methyl-2H-pyran-2 (R) -yl) -tetrahydro-3 (s) -methyl-2 (S) -furyl] -3 (R) -ethyl-4 (S) -hydroxy-3 (S) -ethyl-6-oceononyl] -2,3-creothic acid.

Lasaloeidi D:? ° 2h t3 Tl hcl λ JL X * / \ c2He / - \ r

JL (J * UH O ^^ OH

oh o ο- ^ ς h3C CHj 6- [7 (R} -5'S) -ethyl-5- (5 (R) -it3Tr11-hydro-5-hydrol (1-6 (s) -methyl) -2H-pyran-2 (R) -yl) -tetrahydro-3 (s) -methyl-2 (s) -furyl] -5 (s) -ethyl-4 (s) -hydroxy-3 (R) -methyl -6-oxonoyl] -2,3-creothlinic acid.

Lasaloeidi Ei 56979 COgH CHj CHj C2H5 ys V "° 2h5

T jj H H <^ 0 OH

H3C oh ° 'ch3 6 - [(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-N- (S) -hydroxy-3 (R), 5 (S) -dimethyl-6-oxononyl--2,3-crotinic acid .

According to the invention, lasalocid homologues are prepared by fermentation of Streptomyces lasaliensis ~ X-537 under aerobic conditions in submerged conditions, whereby the pH of the fermentation broth is adjusted to approximately neutral, i. approximately between 6.5 and 7.5. The medium used contains a nitrogen source such as e.g. yeast, yeast product, corn flour or bean flour, with soybean meal being preferred, salts such as potassium phosphate, calcium carbonate and trace elements, a carbohydrate source such as sugar or molasses, preferably brown, brown vegetable or animal fats or oils, such as, for example, soybean oil or frying oil, and substances to prevent foaming and to improve the yield of the desired final product. The fermentation is carried out at a slightly elevated temperature, i.e. between about 25-35 ° C, especially at about 28 ° C. After an incubation period of about k-6 days, the fermentation broth is filtered and the antibiotics are recovered by extraction.

After fermentation, many methods can be used to isolate and purify the lasalocid homologs of the invention. Examples of these are solvent extraction methods, such as, for example, batch extraction or liquid-liquid extraction in a countercurrent partitioning method, or also gel permeation chromatography in an anhydrous system.

Pharmaceutically acceptable salts of lasalocid homologues prepared according to the invention may be prepared according to conventional methods. These salts are prepared from the free acid form of the antibiotic or its derivatives according to methods known per se, for example by washing the free acids in solution with a suitable base or a suitable salt. Examples of such pharmaceutically acceptable basic salt-forming agents are alkali metal bases such as sodium hydroxide, potassium hydroxide or lithium hydroxide, alkaline earth metal bases such as calcium hydroxide and ammonium hydroxide. Alkali metal or alkaline earth metal salts that can be used to prepare pharmaceutically acceptable salts include, for example, carbonates, bicarbonates, and sulfates.

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The relative antibiotic potency of lasalocid homologues B, C, D and E against BacillusTA used as a microorganism in vitro was determined by comparing the potency of the pure lasalocid A sodium salt with four homologues using the perforated agar diffusion method. The calculated numerical values for the five components are based on an arbitrarily selected activity number of 100 for lasalocid A base and are shown in the following table:

Compound Calculated values for in vitro activity

Against Bacillus TA

Lasalocid A 100

Lasalocid B 90 ^ Lasalocid C 10

Lasalocid D 160

Lasalocid E 170.

“The toxicity of lasalocid homologues B, C, D and E is lower than that of the known lasalocid A, as shown in the following table. The experiments were performed in mice, and the LD 2 value indicates acute toxicity (2 h hour value).

Compound LD50 q mg / kg p.o. the mouse

Lasalocid B + C 210-30 (ratio 1: 1)

Lasalocid D + E 195 “32 (ratio 1: 1)

Lasalocid A 1L6.

The lasalocid homologues B, C, D and E prepared according to the invention thus have a more advantageous effect and lower toxicity compared to the known lasalocid A.

Example 1 «, Preparation of lasalocid homologues B, C, II and E.

Streptomyces X-537 (Streptomyces lasaliensis) strain NRRL 3382 is incubated in shake flasks immersed in an air-conditioned nutrient solution. The pH of the fermentation broth is adjusted to 6.5 ~ 7 → 5 by adding aqueous potassium hydroxide solution, then the nutrient solution is sterilized. Fermentation is performed in a tank using a 5-10% inoculum comprising a 3-day-old submerged culture taken from aerated flasks. The nutrient medium contains 2% soybean meal, 2% brown sugar, 0.1% dipotassium phosphate and 0.5% corn steep water. The fermentation is carried out at 28 ° C under a positive atmospheric pressure, in which case 150-300 liters of air per minute are blown into the solution per 150-300 liters of solution. The fermentation is stopped after k-6 days, the fermentation broth is filtered and the antibiotic is recovered by extraction. The extraction is performed as follows:

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204 liters of fermentation broth 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, the aqueous layer separated and discarded, a solution of n-butyl acetate containing an antibiotic concentration corresponding to 30 million Bacillus E units is 20-22 mm in diameter), concentrated to 3 liters under reduced pressure, washed with 10% aqueous sodium carbonate solution and dried over anhydrous sodium sulfate.

The solution is concentrated to 300 ml and 350 ml of light petroleum ... o (boiling range 50-60 ° C) are added to give 41 g of a solid corresponding to 25 million Bacillus E units in the experiment, which is separated. This solid is then extracted in a 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 filtered. After recrystallization, an enriched mother liquor is obtained for lasalocid homologues B, C, D and E.

Example 2

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

A portion (corresponding to 22 g of dry matter) of the mother liquor obtained according to Example 1 is chromatographed in a countercurrent separator equipped with 200 small tubes (80 ml each). The sample is dissolved in 160 ml of the phase mixture (heptane / ethyl acetate / methanol / water, 27: 18: 18: 2) and the solution is placed in the first two small tubes. After 380 partitions, the following fractions are obtained, the solids obtained being identified after evaporation as follows: I. mixture of lasalocid homologues B, C, D and E

II. lasalocid A

III. isolaloside A. This product is an isomer of lasalocid A with a structure different from lasalocid A - E.

Fraction I is dissolved in 20 ml of the heptane / ethyl acetate / ethanol / water / glacial acetic acid phase mixture (10: 5: 9: 3: 1) of the solvent system and chromatographed on a 500 small tube countercurrent separator. After 2 800 distributions, approximately 200 mg of lasalocid B, Cf D and E with the following melting points are separated in each case:

Lasalocid B - 85-87 ° C

Lasalocid C - 97 ~ 100 ° C

Lasalocid D - 102-104 ° C

Lasalocid E - 90 ° C

Example 3

Sodium salt of lasalocid E.

About 100 mg of lasalocid E is dissolved in methylene chloride and treated with saturated aqueous sodium carbonate solution. The methylene chloride phase is concentrated with 9,56979 hexane. This gives 104 mg of crystalline sodium salt of lasalocid E (melting point 182-182.5 ° C).

Use of lasalocid additives as a feed additive

As a first feed, medicated poultry feed for chickens for fattening is prepared by mixing 0.015 wt.% And 0.006 wt.% Of lasalocid B and C (50/50) and lasalocid D and E (50 / 50) poultry feed consisting of:

Ingredients Maize flour ground per unit weight 510 kg / tonne stabilized fat or vegetable oil 27 kg / tonne soybean meal (low fiber; 50% protein) 200 kg / tonne corn gluten meal 23 kg / tonne fishmeal, treated with antioxidant 1 kg / tonne protein 60% protein soluble parts ("fish solubles"), based on dry weight 5 kg / tonne of meat and bone waste, 50% protein 64 kg / tonne of dried, soluble residues from the corn bulb 23 kg / tonne of lucerne meal, 17% protein, 200,000 A / kg 14 kg / tonne manganese sulphate, feed grade 0.35 kg / tonne zinc carbonate or oxide 0.11 kg / tonne riboflavin 3 g vitamin B12 6 mg calcium pantothenate 5 g niacin 30 g stabilized vitamin A, US Pharmacopoeia units 6,000,000 2) vitamin D_, international . 650,000 chicken units. . . 3. . ..... 3) vitamin E acetate, kansamval. units 5,000 vitamin K, (menadione sodium bisulphite) 2 g DL-methionine a or its hydroxy analogue 0.1 + 5 kg / tonne antioxidant (ethoxyquin or butylated hydroxytoluene) 0.11 kg / tonne 1 Vitamin A unit = 0.0006 mg β-carotene (m.p. 184 ° C) 2) 1 Vitamin D unit = 0.000025 mg of 7-dehydrocholesterol (dimethyldihydrocalciferol) 3) .....

1 Vitamin E unit = 1.0 mg d, l-et-tocopherol acetate 10 56979 10 chickens raised in the laboratory were infected with Eimeria tenella in each experiment. 10 chickens were used as weight control and 10 chickens as infected control animals. The antibiotic was administered U8 hours before infection. 1 g of antibiotic was mixed in a mechanical mixer with the required amount of chicken feed to achieve the desired dosage.

The infection comprised approximately 200,000 Eimeria tenella oocysts administered orally by pipette. The experiment lasted 11 days, after which the surviving animals were necropsied and visually examined for colonic lesions. The number of surviving animals and the number of colonic injuries were recorded. The result is expressed as the "average degree of infection". An average infection rate of less than 1.5 is considered to be remarkably effective at that dose.

As will be seen below, the antibiotic mixture is effective in treating coccidiosis.

Dosage in feed, Mean Mortality weight # Infection rate #

Uninfected, untreated comparison 0.0 0

Infected, untreated comparison not at all 2.6-2.8 10-20

Lasalocid B /,, 0.015 1.0 0

Lasalocid C.) 0.006 1.2 0

Lasalocid D (..., 0.015 0.0 0

Lasalocid E_J 0.006 1.2 0

In the next experiment, 0.01% by weight of lasalocid D was mixed with the basic feed described above. The effect of the antibiotic against Eimeria tenella was examined in the same manner as above. The following results show that the antibiotic has an effect against coccidiosis.

Dosage in feed, Mean Mortality weight # Infection rate #

Uninfected, untreated - comparison none 0.0 0

Infected, untreated comparison none 3.0 20

Lasalocid D 0.01 0.5 0

Claims (6)

A process for the preparation of novel lasaloside antibiotics of the general formula COgH Rg R3 C2H5> H0 OH O CH3 wherein R, Rg, R 2 and R denotes ethyl, in isolated form, together with pharmaceutically acceptable salts of these compounds, characterized by the cultivation of a species of Streptomyces lasaliensis (Streptomyces lasaliensis X-537) belonging to the microorganism, preferably strain NRRL 33θ2, in an assimilable carbohydrate containing nitrogenase under submersic and aerobic conditions until the medium exhibits significant antibiotic activity, after which the compounds of formula I produced are isolated from the aqueous nutrient medium and, if desired, transferred to the pharmaceutically acceptable site thereof. Process according to claim 1, characterized in that the cultivation of Streptomyces X-537 is carried out at a pH of about 6.5-7.5 and at a temperature of about 25_35 ° C. 3. A method according to claim 1 or 2, characterized in that the cultivation is carried out in the presence of a vegetable or animal fat or an oil. Process according to any of claims 1-3, characterized in that the final product is recovered so as to filter the aqueous nutrient medium, extract the filtrate with a water-insoluble solvent and separate the final product from the solvent extract. 5. A process according to claim U, characterized in that the filtrate is extracted with n-butyl acetate and treated with the extract with petroleum ether to obtain a mother liquor enriched with respect to the final product. Process according to claim U or 5, characterized in that the separation of the final product from the solvent extract is carried out, so that the latter is subjected to a countercurrent distribution.