DE2908845A1 - PROCEDURES, COMPOSITIONS AND NEW CHEMICAL COMPOUNDS FOR USE IN THE BREEDING OF DOMESTIC RURAL REWING - Google Patents

Description

PATENT Attorney * _Λ 'J, HSfZ 1979

DR.RICHARD KNEiSSL / I ^

D-80D0 iVrjNCrZM 22
TeLCS9 / 235125

Folder 24 537

ICI case no. PH. 29958 / DT

IMPERIAL CIdEIuICAL INDUSTRIES LIMITED London / Great Britain

Method Zusanunensetzimgen and new chemical compounds for use in the breeding of domestic ruminants

909848 / Ö532

description

The invention relates to methods, compositions and novel chemical compounds for use in the Breeding of domesticated ruminants, such as cattle, sheep, goats and deer, in order to reduce the amount of ruminal fermentation to reduce methane formed and to increase the amount of propionic acid in the volume fluid and around thereby improving the growth rate and / or the utilization of the forage.

In ruminants, a considerable proportion of the total energy intake in the form of feed is lost as methane. This forms in the rumen during the fermentation of the feed. Another significant part of the total energy consumption is lost as fermentation heat. For example, in lambs, methane formation can consume around 10% of the total energy intake and the heat of fermentation around another 6%. The compositions and compounds according to the invention have the effect of increasing the amount of propionic acid in the rumen fluid. In particular, they have the effect of increasing the amount of propionic acid at the expense of methane and / or acetic acid. It is known that this is a desirable effect in the forage of ruminants, since propionic acid is a much more effective precursor to glucose, from which the animal derives its energy and growth, than is the case with acetic acid. On the other hand, that part of the feed given to the animal that is converted into methane is lost to the animal. The methane escapes in the form of flatulence. Thus, the modification of the rum material alternately by the compositions and compounds according to the invention is an extremely useful effect. It is expected an increase in the speed of growth and the utilization of the feed of ruminants.

The invention therefore relates to a method of growing

9O9848 / O5S2

from domesticated ruminants, which is carried out by giving orally to these animals a compound of the formula:

administered in which

1 ?

R and R each represent a hydrogen atom or an acetyl radical stand} and

either the carbon atoms 3, 3 ¹ and 9 'are all in the R configuration, X is an ethylene or trans-vinylene radical and Y is a radical Y of the formula:

Me

a-

II

wherein X is bonded to the CHp group and the ring carbon atom is bonded to the oxygen atom and wherein X is an ethylene or trans-vinylene radical and Z ^{+ is} a sodium, potassium, lithium, cesium or ammonium ion or an ion of the formula R ³ R ^ R ⁵ R ⁶ N ⁺ , where R ³ , R ⁴ , R ⁵ and R ^{6 are} each hydrogen or Cj C-alkyl; or

909848/05 $$

BATH ORIGINAL

the carbon atoms 3, 3 ¹ and 9 ^s are all in the S configuration, X is an ethylene radical and Y is a radical Y ^{2 of} the formulaϊ

-CH ₂ -X ² -CH ₂ -CH-CH (CH ₃ ) .O.CO.CH-CH (CH ₃ ) ₂ or

NH ₂
-CH2-X ² -CH ₂ -CH-CH (CH ₃ ) .0.CO.CH-CH (CH ₃ ) ₂ .Z ⁺

represents wherein the _CH2 group is attached to the ₂ group shown in formula I CH, and the CH group to the oxygen atom overall

is bonded and X stands for an ethylene or cis-vinylene radical? or

the carbon atoms 3, 3 ¹ and 9 ¹ are all in the S configuration, X stands for an ethylene radical and Y stands for a radical Y ^{3 of} the formula:

~ CH ₂ -X ² -CH ₂ -CH-CH (CH ₃ ) .OH. Z ⁺ is in which X and Z ^{+ have} the meanings given above.

Special Yerbindtingen of the formula I, which in the invention Methods that can be used are:

1. Aplasmomycin (3R, 3 ¹ S, 9 ¹ R, R ¹ = R ² = H, X - trans-vinylene _s Y = Y, where X is trans-vinylene and Z is sodium), Ss is a Metabo-Iiten, which was isolated during the fermentation of Streptomyces griseus strain SS-20 und.von Okami et al. in "The Journal of Antibiotics"! 1976, Volume XXIX, pages 1019-1025 and Kitahara, ibid, 1977, Volume XXXj, pages 714-719. This strain is deposited at the Fermentation Research Institute, Chlba, Japan under the reference number FERM-P No. 3448} '■ - ■. .

909848/0512

ICI 122,378, a metabolite that occurs in fermentation of the S. griseus strain, listed in the National Collection of Bacteria, Ministry of Agriculture, Fisheries and Food, Torry Research Station, 135 Abbey Road, Aberdeen AB9 8DG, Scotland, under reference number NCIB 11371, -This material is identical to aplasmomycin,

Boromycin (3S, 3'S, 9 ¹ S, R ¹ = R ² = H, X = ethylene, Y = Y ² , where X is cis-vinylene). This is a metabolite that was isolated during the fermentation of S. antibioticus (Waksman and Woodruff) strain ETH 28829 and was described by Hutter et al., Helvetica Chimica Acta, 1967, volume 50, pages 1533 to 1539, and Dunitz et al., ibid, 1971, vol. 54, pages 1709-1713. This strain is also deposited in the United States Department of Agriculture, Peoria, Illinois, USA under the reference number NRRL 3207.

4. Factor B, a compound with the molecular formula C ^ 2% 2 which is a cometabolite of aplasmomycin in the fermentation of S. griseus, NCIB 11371, and is a monoacetyl derivative thereof where the relative stereochemistry may be the same as in aplasmomycin or neither.

5. Factor C, a compound having the molecular ^{formula c} 44 ^H 64 ^BNa0- j6 »which is a cometabolite of aplasmomycin in the fermentation of S. griseus, NCIB 11371, and is a diacetyl derivative thereof, wherein the relative stereochemistry may be the same as in aplasmomycin or not.

In the method according to the invention, the compound of the formula is preferably administered orally to animals as a supplement to their normal diet

909848/0532

"fcers administered, i.e. in a mixture with the usual solid Putter, in food cubes or salt licks, dissolved or suspended in drinking water or, for young animals such as lambs or calves, dissolved or suspended in whole milk or skimmed milk. The compound of formula I is in the putter, the food cubes, the salt lick, the drinking water, the whole milk or the skimmed milk incorporated to such an extent that each animal is 0.01 mg / kg body weight to 30 mg / kg body weight per day, preferably 0.01 mg / kg to 10 mg / kg per day of the compound of formula I absorbs.

Alternatively, the compound of formula I can also be administered orally to animals be administered in the form of intraryminal pellets or pills with slow release of active ingredient, so that the animal has a similar Amount per day of the compound of formula I absorbed.

The animals can use a compound of the formula I during practically the entire growth period or only during part of the Growth period, for example during the first part and / or before slaughter. The by the invention The method of gaining weight allows meat animals to be brought to market or slaughter weight in a shorter time than usual or allows heavier animals to be obtained at the end of a normal growing season. The improved utilization of the feed achieved by the method according to the invention also enables animals to achieve a desired weight, where they need less feed than an untreated one, animal bred to the same weight. With optimal, growth-promoting amounts there is no sign of a poisonous effect determined on the basis of the compound of formula I.

According to the invention, a non-toxic, orally administrable composition is also proposed which comprises a compound of the formula I ₄ , wherein i

oil © carbon atoms 3 »3 * and 9 ⁵ are all in the R configuration b0 £ lnä, en _f X for an ethylene» or treas-finylearadical

909848IiSIf

and Y stands for a radical Y as defined above; or

the carbon atoms 3 »3 'and 9' are all in the S configuration are located, X stands for an ethylene radical and Y for a radical Y ^ as defined above, wherein X is an ethylene radical is · or

the carbon atoms 3 »3 'and 9 ¹ are all in the S configuration, X stands for an ethylene radical and Y stands for a radical Y ³ as defined above} together with a solid or liquid, non-toxic, orally administrable diluent or Contains carrier.

A suitable liquid diluent or carrier is, for example, drinking water, whole milk or skimmed milk.

A suitable solid, non-toxic, orally administrable diluent or carrier is, for example, a common nutritionally balanced ruminant feed, such as a typical cattle or sheep feed, consisting of cereal products such as barley flour, corn flour or wheat meal, and seed products such as peanut cake or cottonseed cake, or extracted cottonseed cake, together with smaller amounts of, for example, feather meal, seaweed meal, bone meal, chalk, algae, urea, molasses, vitamins and trace minerals. But it can also be an inert solid diluent or carrier with no nutritional value, such as kaolin, talc, calcium carbonate, fuller's marriage Earth, attapulgus clay, ground oyster shells, or ground limestone. After all, it can also be about strength or lactose act.

The compositions according to the invention may take the form of a supplementary feed for direct administration to animals, in which case the feed contains 5 to 3000 ppm of a compound of formula ί in admixture with a conventional ruminant feed. However, the compositions can also take the form of a

909348/0632

concentrated premix to be diluted with a conventional ruminant feed to produce a supplemented feed that is suitable for direct feeding to animals. Such a premix is 0.3 wt -.% To 50 wt .- ^ a compound of formula I in admixture with either a conventional nutritionally balanced ruminant feed, an inert solid diluent without nutritional value, such as. contain ground limestone, or starch or lactose.

The invention also relates to a method of production a solid composition according to the invention, which is characterized by, it is stated that a compound of formula I with a solid, non-toxic, orally administrable diluent or vehicle uniformly mixes.

The compound of formula I is preferably gradually diluted with a diluent or carrier in two or more stages in order to ensure uniform mixing.

of the formula I According to the invention, a process for the preparation of a compound /

1 2
proposed, in which R and R each represent a hydrogen atom or an acetyl radical, X represents a trans-vinylene radical

1 1

and Y is a radical Y where X is a trans-vinylene radical and Z ^{+ is} a sodium ion which is carried out by placing an aplasmomycin producing strain or mutants of Streptomyces griseus in an aqueous nutrient medium which are sources of assimilable carbon, assimilable Contains nitrogen and assimilable boron and a sodium salt, ^{cultivated under aerobic conditions at a temperature between 10 and 37 °} C., the culture is filtered and the product is isolated from the filtrate by conventional means.

In the method according to the invention, a suitable source of assimilable carbon is, for example, a polyhydric alcohol, such as glycerine, glucose, xylose, arabinose, rhamnose, fructose, galactose, mannitol or inositol. As a carbon source

909848/0532

-IQ-

glucose syrup is preferred, which is expediently incorporated into the medium in a ratio of 1 to 5% (w / v).

A suitable source of assimilable nitrogen is, for example, peptone, which is conveniently incorporated in an amount of 0.05 to 2% (w / v).

The nutrient medium can also contain small amounts of other elements, such as phosphorus, in the form of, for example, potassium dihydrogen orthophosphate or diammonium hydrogen phosphate; Magnesium, in the form of, for example, magnesium sulfate or magnesium carbonate; Sulfur, in the form of, for example, a sulfate salt; Potassium, in the form of, for example, potassium chloride, potassium carbonate or Potassium dihydrogen orthophosphate; and trace amounts of salts of such elements as copper, zinc, iron, manganese and molybdenum, contain.

A suitable temperature for fermentation is 28 ° C. The product can be isolated from the culture filtrate by conventional means, for example by extracting the filtrate with a water-immiscible organic solvent without adjustment of the pH value, subsequent evaporation of the solvent and chromatography of the residue.

According to the invention, the Streptomyces griseus, the in the National Collection of Industrial Bacteria under the designation NCIB 11371, and variants and mutants suggested of it. This organism is suitable for carrying out the method according to the invention and has the following Description:

909848/0532

ORIGINAL INSPECTED

(The media used are determined according to the recipes for the "International Streptomyces Project (ISP)" produced and are from Shirling E.B. & Gottlieb, D (1966) in "Methods for characterization of Streptomyces species" in International Journal of Systematic Bacteriology, £ 1 (3) »313 to 340 described).

Conditions for Incubation

Temperature 30 ⁰ C

Growth in the dark.
ISP2 yeast extract / malt extract

4 days : 3 to 4 mm. Good raised, wrinkled, glassy colonies. Dark fawn. Lapel - brown-gray. 10 days ; 10 mm. Submerged growth at the margin with raised, wrinkled watery-fawn-gray colonies. Reverse - unchanged. No color in the agar »
ISP3 oatmeal agar

4 days ; 1 to 2 mm. Thin, watery colonies, sometimes with a torn depression in the center. Revers very pale brown-gray.

10 days ; 4 to 6 mm. Waxy-yeast-like, smooth, gray-brown colonies. Reverse - unchanged. Very weak brown color transferred to the agar «ISP4 A n organic salts / starch agar

4 days ; 1 to 2 mm. Good, fairly moist, very compact colonies. Pale brown. Lapel - pale fawn / gray. 10 days : Slightly more growth. "Now waxy, somewhat raised, wrinkled colonies with uneven edges. Pale fawn. Reverse - unchanged. No color transferred to the agar.

ISP5 glycerine / asparagine agar

4 days : 1 to 2 mm. Sublime colonies. Colonies wrinkled and the agar lowered at the edge. Glassy fawn brown. Lapel - pale brown-gray,

909848 / OS32

- 2C -

10 days : 3 to 5 mm. Waxy, raised, and wrinkled colonies. Yellowish fawn. Reverse - unchanged. ISP7 tyrosine agar

4 days ; 2 to 3 mm. Good raised, wrinkled, glassy, fawn colonies. Lapel - gray-brown. Medium turned pale brown.

10 days : 10 mm. Waxy, wrinkled, yellowish-dark brown-gray colonies with some powdery gray areas. Reverse - dark-gray-brown, but discoloration of the agar can just be seen.
ISP9 carbon exploitation extra

Encore

4 days

Days

classification

water

glucose

Arabinose

Cellulose fructose

Inositol mannitol

Raffinose rhamnose sucrose xylose

up to 2 mm. Submers, very thin, "Rausehr thin, 'chig', submerged "smoky"

1 to 2 mm. Light, quite gelatinous, fawn-gray

something better than water

indefinable

1 mm. Dense, raised, glassy colonies, fawn-gray. Fawn-gray lapel

like arabinose

2 mm. Dense, raised, glassy fawn colonies with torn ones Centers. Lapel fawn

like arabinose like water

like water

1 to 2 mm. Very thin, short, velvety, fawn brown

up to 3 mm. Dense, raised, v / axelike, fawn-gray. Reverse - unchanged

like water

like water

4 to 5 mm. Dense, raised, wakeful, • wrinkled, torn, fawn gray

something better than water

2 to 3 mm. Dense, raised, waxy, fawn-brown. Lapel - fawn brown.

like water
like V / ater
like water

2 to 3 mm. Slightly raised, velvety, light fawn- gray, possibly spore-forming SG9848 / 8S32

Partial air tolerance

On tryptone / yeast nutrient broth (ISP1) .. Lowest temperature tested 19 ° C Grows pretty well. Best around 28 ° C, does not tolerate temperatures above 37 ° C.
morphology

It is a weakly spore-forming strain, if however, spores are seen, then they are in short chains and are smooth-walled and barrel-shaped.

According to the invention, a new compound of the
Proposed formula I, in which R and R each represent a hydrogen atom or an acetyl radical and;

(a) the carbon atoms 3, 3 'and 9 ¹ are all in the R configuration, Y stands for a radical Y ¹ , one of the symbols X and X stands for an ethylene radical and the other stands for a trans-vinylene radical or X and X both represent ethylene radicals and Z " ¹ " has the meaning given above; or

(b) the carbon atoms 3, 3 'and 9 ^! are all in the R configuration, X is a trans-vinylene radical and Y is a radical Y, where X ^{1 is} a trans-vinylene radical and Z ^{+ is} a potassium, lithium, cesium or ammonium ion or an ion of the formula R ⁵ RTl ⁵ RN ⁺ according to the above
Definition stands; or

(c) the carbon atoms 3, 3 'and 9' are all in the S configuration are, X is an ethylene radical and Y is a radical Y ^, wherein X is

909848/0532

Is ethylene radical; or

(d) 'the carbon atoms 3, 3 ¹ and 9 ^f are all in the S configuration, X stands for an ethylene radical and Y stands for a radical Ί ⁵ , wherein X ^{2 is} as above
has given meaning and A ⁺ for a potassium, lithium, cesium or ammonium ion or an ion of
Formula R ⁵ RR ⁵ R Ii ^{+ as} defined above
stands.

According to the invention, a process for the preparation of new compounds of the formula I is also proposed, which thereby
is carried out that one

(1) for the preparation of the new compounds defined under (a) above, a corresponding compound of the formula I, where X and X each represent a trans-vinylene radical stand, selectively or non-selectively hydrogenated over a platinum or palladium catalyst; or

(2) to produce the new ones defined under Cb) above

or ICI 122378
Compounds an aplasmomycinj producing strain of

S. griseus as defined above in the presence of a potassium, lithium, cesium or ammonium or
R ^ R R- ⁵ RN ⁺ salt fermented in an aqueous nutrient medium; or

(3) for the preparation of the new compounds defined under (c) above, a corresponding compound of the formula I, where X is an ethylene radical and Y is a radical Y ^, where X is a cis-vinylene radical, hydrogenated over a platinum or palladium catalyst; or

(4) for the preparation of the new compounds defined under (d) above, a compound of the formula I in which X

909848/0532

represents an ethylene radical and Y represents a radical Y ² , with potassium, lithium or cesium hydroxide or potassium, lithium or cesium carbonate.

It should, of course, be appreciated that a compound of formula I wherein Z ^{+ has} one meaning can be converted to a similar compound wherein Z ⁺ has another meaning using conventional techniques such as the use of ion exchange resins.

Aplasmomycin and factors B and C also show anticoccicidal activity. This activity is demonstrated by its effect in preventing coccicidal growth in cultures of ulcerated kidney cells inoculated with sporocites according to the standard test described in Journal of Parasitology, 58 . 664 to 668 (1972). In this test, aplasmomycin shows an activity against Eimeria tenella at a concentration of 0.037 ppm, but shows a toxic effect against the host cells only at a concentration of 9 ppm. Similarly, a mixture of factors B and C shows activity against E. tenella at a concentration of 0.012 ppm, but only shows toxic effects on host cells at a concentration of 1 ppm.

The invention is illustrated in more detail by the following examples. example 1

Streptomyces griseus NCIB 11371 has been considered as slant culture to 45 ml dextrin-agar for 7 days at 30 ⁰ C. The slant was scraped into 100 ml of sterile water and the resulting suspension was used to inoculate three 500 ml flasks, each of which contained 50 ml of the following medium!

Söjatoöimen flour - 1 ₉ 5% (w / v) -

Bifco Bacto Casitone (trademarks) 0.196 "

909848/0632

ORIGINAL

Sodium Nitrate 0.3? £ (W / V)

Glucose Syrup 2, Q ^ ^fl

Calcium carbonate 0.25% ^M.

desalinated water to 100

which is pre-sterilized in an autoclave at 1 atm. 1/2 hour with the pH being approximately 7.0.

The three flasks obtained in this way were ^{shaken on a rotary shaker at 27 °} C. for 75 hours. The contents of the three flasks were then combined and used to inoculate a stainless steel fermenter containing 30 liters of sterile medium of the same composition as described above. The contents of the fermenter were ^{stirred at 27 °} C. for 88 hours, using a turbine with 4 flat blades which rotated at 350 rpm. It was aerated at a rate of 7.5 l / min. The resulting broth was filtered through diatomaceous earth and the filter bed was washed with 3 liters of water. The filtrate and washes, which totaled 28 liters, were combined and extracted at their natural pH with 1x91 and 1x71 ethyl acetate. The ethyl acetate extracts were combined, dried with sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to give 29 g of an oily residue.

The desired product can be obtained from the above concentrate by one of two methods described below

(A) The oily residue (29 g) became in the smallest volume of a mixed solvent of the following composition dissolved: 98 parts of chloroform, 1 part of methanol, 1 part Formic acid. The solution obtained was applied to the top of a 90 cm χ 5 1/2 cm measuring silica gel column, which made up as a slurry with the same solvent mixture. The column was filled with the same solution

909848 / Θ532

medium mixture eluted and 500 ml fractions were collected after the solvent front exited the column. Fractions 4 and 5 were combined and the solvent was evaporated to give 3.5 g of a gum which was further determined by preparative thin layer chromatography on silica (Merck Kieselgel 60 P250 - Viarenzeichen - 2mm thick) using 30? A ( 60 was purified as eluant mixture to 80 ⁰ C) V / V) ethyl acetate in petroleum ether (bp. The band with Rp = 0.25 (approximate) was scraped from the plate and extracted with ethyl acetate and the solvent was evaporated to give 140 mg of a white crystalline solid which was crystallized from aqueous methanol and dried under vacuum for 5 h. This gave ICI 122.378 without a defined melting point, Ca] Jp = + 210 ° (C = 0.5 in chloroform). Analysis: found C = 60.1, H = 7.7, Na «2.8. calculated for ^C 40 ^H 60 ^BNa0 14 ^{i C = 60} » ² ' ^{H = 7} ' ⁵ » ^Έα ~ ² > 9%. Mass spectrum: M ⁺ = 798.396, calculated for C ^ Hg ₀ BIJaO ₁ ^ = 798.396. R _p = 0.74 (thin layer chromatography on Merck 0.25 mm thick plates 60F-254, developed with 10 ^ ^ i / V) methanol in chloroform, visualized as a brown spot after spraying with 3N sulfuric acid and heating to 100 ⁰ C or as a pink spot after spraying with a solution of carminic acid in 3N sulfuric acid and heating at 70 ⁰ C% rährend 5 min).

(B) The oily residue (60 g) from the ethyl acetate extract obtained from a similar 80-1 fermentation was dissolved in 400 ml of methanol, and the solution was washed with 800 ml of petroleum ether (b.p. 60 to 80 ° C) shaken. The bottom phase (methanol layer) was separated and concentrated under reduced pressure, dissolved in the minimum volume of a mixture of 35 parts of ethyl acetate and 65 parts of petroleum ether (b.p. 60 to 80 ⁰ C) and on the top of a silica gel column with 24 g of a gum, (51 x 4 1/2 cm; 450 g), which had been made up as a slurry with the same mixture of solvents, was applied. 25 ml fractions were collected after the solvent front was removed from the

909848/8532

- 2ό -

Column had exited. Fractions 25 to 70 were combined, and the solvent was evaporated to obtain 3.3 g of a viscous gum. This rubber got wider purified by preparative thin layer chromatography on silica (as described above under A), with 300 mg ICI 122,378 which was identical to the material obtained in Method A above.

Example 2

The property of ICI 122,378, the formation of methane in the room of ruminants and to inhibit the formation of eropionate (Ac / Pr) and butyrate (Ac / Bu) at the expense of the acetate in the formed to increase volatile fatty acids (VFA) without simultaneously affecting the entire digestive process affect is demonstrated as follows:

Rumen fluid is regularly and routinely obtained from two bulls collected, which are fed with the same hay plus concentrate feed. Sampling is carried out as far as possible standardized, and the fluid from the two animals is pooled on a 50/50 basis. Large particulate solids are removed by filtering the pooled fluid through four layers of muslin cloth. The filtrate is then in a ratio of 1 volume of filtrate to 3 volumes an artificial rumen fluid (prepared as described by G.L. Bales et al. in Journal of Diary Science, 1976, vol. 59, p.1850, described, but omitting the acetic acid), and the pH is adjusted with saturated aqueous sodium carbonate solution set to 6.9 to 7.0. 50 ml samples of this mixture are transferred to 100 ml conical flasks containing 0.5 g dry milled hay and each flask is used to: to test a test compound at a specific concentration.

The test compound is applied to the conical flask as a solution in etha-

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nol added, the flask is flushed with carbon dioxide gas, stoppered with a suba seal and 15 to 16 st incubated at 39 ⁰ G. After 1 hour, a pierced needle is inserted through the Suba occluder to release the gas pressure and the needle is withdrawn 30 minutes before the incubation is complete. The fermentation is then stopped by placing the flask in ice. After cooling for 15 minutes, the gas above the liquid is analyzed for methane by gas chromatography. The contents of the flask are then filtered through a previously dried and weighed sintered glass funnel, the funnel is oven-dried and the difference in weight is used to determine the digested hay.

Control flasks that do not contain hay are made in a similar fashion treated to determine the non-cellulosic residue. Three samples of the filtrate are analyzed for VFA by gas chromatography and by comparison with that before incubation certain VFA content, the net VFA (acetate, Propionate and butyrate) formed during incubation, certainly.

The following results were obtained (Monensin, a known growth promoter that acts on the rumen, is called positive comparison included, together with a negative comparison in which no test compound is used) ι

% Methane 'Ac / Pr Ae / Bu

all in one

gas

Comparison . 8.37 1.90 3.92

ICI 122 »378i 3 ^ t g / ml

1 µg / ml

0.5 jig / ml

0.3 jx g / ml

0.1 / l

809848 / ISSf

2.58 1.34 3.52 3.81 1.26 4.26 4.87 1.29. 4.56 5.72 1.36 4.75 7.87 1.63 4.22

1.54 2908845 4.53 1.51 3.39 5.62 1.61 3.21 6.75 1.65 4.03 7.53 1.80 4.11 8.35 4.22

Monensin:
3 η g / ml
1 ^ ig / ml
0.5 »g / ml
0.3 ^ g / ml
0.1 ^ g / ml

Example 3

The fermentation described in Example 1 was repeated, the resulting culture broth was filtered through diatomaceous earth, and the filter bed was washed with 3 liters of water. The filtrate and washes were combined and added their natural pH 1 χ with 9 1 and 1 χ with 7 1 ethyl acetate extracted. The ethyl acetate extracts were combined with sodium sulfate dried and filtered, and the filtrate was concentrated under reduced pressure to give 8.2 g of an oily Residue were obtained. The wet mycelium was in methanol homogenized, and after filtration, the wet methanol solution was concentrated under reduced pressure to give a predominantly aqueous solution was obtained. This solution was extracted twice with 1/3 volume of ethyl acetate and the ethyl acetate extracts were combined, dried with sodium sulfate and filtered. The resulting filtrate was reduced under reduced pressure Pressure concentrated to give 1.5 g of a gum which was then combined with the ethyl acetate extract from the cooling filtrate so that a total extract weight of 9 »7 g was obtained.

The oily residue (30 g), which had been obtained from three such 30-1 fermentations as described above, was dissolved in the smallest volume of petroleum ether (b.p. 60 to 80 ⁰ C), and the solution was applied to the top of a column made of neutral aluminum oxide (45 cm χ 4 1/4 cm, Woelm, 500 g), which had been made up with the same solvent, abandoned. The column was first filled with 2 liters of petroleum ether (b.p. 60 to

909348/6552

ORIGINAL INSPECTED

80 ° C) and then eluted with 2 1 of a mixture of W% (v / v) ethyl acetate in petroleum ether (b.p. 60 to 80 ⁰ C), whereby substantial amounts of oily components came to light, which were discarded. The elution was then changed to 30! & (V / v) ethyl acetate in petroleum ether (b.p. 60 to 80 ⁰ C), and 25 ml fractions of the eluent were collected on an automated fraction collector. Factors B and C were eluted as a mixture in tubes 60 to 80, the contents of which were pooled and concentrated to give a syrup of 730 mg. This syrup also contained small amounts of aplasmomycin (factor A), however the bulk of this compound was located in tubes 81-120. The Rp of factors A, B and C in thin layer chromatography on 0.25 mm thick Merck silica gel plates 60F-254, which were developed with 5Ο5έ (V / Y) ethyl acetate in petroleum ether (bp 60 to 80 ° C)., Was approximately as follows:

A. (Aplasmomycin) ^R F Pactor B. 0.36 factor C. 0.25 factor 0.21

The factors were visualized as brown spots after spraying with 3N sulfuric acid and heating to 100 ⁰ C.

The syrup (60 mg), which mainly contained factors B and C, was further purified by preparative thin layer chromatography on silica (Merck "Kieselgel 60 F250" - trademark - 2 mm thick), using 50% (V / V) ethyl acetate in petroleum ether ( Kp 60 to 80 ⁰ C) was used as the elution mixture. The R " 0.25 (approximate) band was scraped from the plate and extracted with ethyl acetate and the solvent was evaporated to give Factor B as 20 mg of a white crystalline solid which was crystallized from aqueous methanol and dried under vacuum , Mp 260 to 262 ⁰ Cj IR 3360, 1735,

909848 / S5S2

ORIGINAL INSPECTED

-1

1710, 1285, 1080 cm "'(Nujol mull). Analysis: found C = 58.3, H =. 7.4; calculated for C ₄₂ H ₅₂ BNaO ₁₅ -H ₂ OC = 58.7, H = 7, 4Jf.Mass spectrum -M ⁺ = 840.403, calculated for C ₄₂ H ₅₂ BNaO ₁₅ * 840.408, The MR spectrum (in CDCl,) shows a predominant peak at h2.1, probably due to an acetyl group.

The band with Rp = 0.2 (approximate) was also scraped off the plate and extracted with ethyl acetate, whereupon the solvent was evaporated. Factor C was obtained as 6 mg of white crystalline solid. Mp> 320 ° C, IR 1735, 1715, 1285, 1265 cm " ¹ (NuJoI muli). Mass spectrum - M ⁺ = 882.418, calculated for C ₄₄ H ₅₄ BNaO ₁₅ = 822.419.

Example 4

The test procedure described in Example 2 was repeated, comparing aplasmomycin with factors B and C. There were the following results are obtained, expressed as percentages of the values obtained with untreated comparative samples became;

3 ppm % Inhibition Ac / Pr Ac / Bu 3 ppm of methane Aplasmomyc in 3 ppm 69 -40 -49 Factor B 1 ppm 76 -45 -18 Factor C 1 ppm 44 -41 +22 Aplasmomycin 1 ppm 57 -41 -36 Factor B 63 -48 +21 Factor C 15th -33 +18 Example 5

The short-term effects of ICI 122,378 and factor B on the room metabolism of sheep were demonstrated as follows:

909848/8532

The rtimenf ermentation scheme of a group of sheep was stabilized by feeding them on a standard diet
dried grass (2 500 g per day) were fed for a few months. 6 test animals were used for each inclusion value of each test compound. Groups of animals were also given monensin, a known modifier for the metabolism of rumen, as a positive control. 12 untreated animals were used as negative controls. Samples of the spatial fluid were collected through a gastric tube from each animal 6 hours after treatment. The gastric fluid was analyzed for acetate, propionate and butyrate by gas chromatography. This gave the following results:

8098A8 / §5S2

Treating

Dose MoI- ^ Rumen VFA

(mg / kg) acetate propionate butyrate

Day 4 0 64.5 24.0 10.2, Comparison. 0.5
0.25 59.8
60 ·, 5 27.8
28.5 11.3
9.9 Monensin 0.5
0.25 63.4
63.7 25.5
25.1 9.9
9.8 ICI 122,378 0.5
0.25 65.0
65.1 22.4
22.3 11.3
11.3 Factor B Day 7 0 65.6 22.9 10.7 comparison 0.5
0.25 60.2
61.3 26.2
26.1 12.6
11.4 Monensin 0.5
0.25 62.6
63.8 26.6
24.8 9.2
9.9 ICI 122,378 0.5
0.25 63.5
63.5 25.9
22.1 8.9
'11 .7 Factor B

909848/9532

Claims (16)

Pate ntansprüche / Ι. , Method of breeding domesticated ruminants, thereby '^, / marked that you can orally to these animals a compound of the formula: administered in which 1 2
R and R each represent a hydrogen atom or an acetyl radical; and either the carbon atoms 3, 3 'and 9 ¹ are all in the R configuration, X is an ethylene or trans-vinylene radical and Y is a radical Y of the formula: -X ^J Me -H II .Z " "in which X _{is bonded to the CH 2} group and the ring carbon atom is bonded to the oxygen atom and in which X is an ethylene or trans-vinylene radical and Z ^{+ is} a sodium, potassium, lithium, cesium or ammonium ion 909848/0532 .Or an ion of the formula R ³ R ⁴ R ⁵ R ⁶ Nt where R ⁵ , R ⁴ , R ⁵ and R ^{6 are} each hydrogen or C 1-6 alkyl; or the carbon atoms of 3, 3 ^{1 1} and 9 are all located in the S-configuration, X represents an ethylene radical and Y is a radical of the formula ι: ₃
-CH ₂ -X ² -CH ₂ -iH-CH (CH ₃ ) .O.CO.CH-CH (CH,) ₂ or NH ₂
-CH ₂ -X ² -CH ₂ -CH-CH (CH ₃ ) .0.CO.CH-CH (CH ₃ ) ₂ .Z ⁺ represents wherein the _CH2 group is attached to the ₂ group shown in formula I CH, and the CH group to the oxygen atom is bonded and X is an ethylene or cis-vinylene radical standsj or the carbon atoms 3, 3 'and 9 ¹ are all in the S configuration, X stands for an ethylene radical and Y for a radical Y * of the formula: -CH ₂ -X ² -CH ₂ -CH-CH (CH ₃ ) .0H. Z ⁺ 2 + stands in which X and Z have the meanings given above. 2. The method according to claim 1, characterized in that that the compound of formula I is aplasmomycin. 3 · The method of claim 1, characterized in that the compound of formula I is a factor B, a compound of the molecular formula C ^ ₂ Hg ₂ BNaO _15> which is a co-metabolite \ ^r on Aplasmomycin in the fermentation of Streptomyces griseus NCIB 11371, and is a monoacetyl derivative thereof, wherein the relative stereochemistry may or may not be the same as in aplasmomycin. 909848/8532 4. The method according to claim 1, characterized in that the compound of formula I or boromycin Factor C is a compound of the molecular formula C ^ Hg ^ BNaO ^, -, which is a cometabolite of aplasmomycin in fermentation from Streptomyces griseus NCIB 11371, and a diacetyl derivative of which is where the relative stereochemistry may or may not be the same as in aplasmomycin. 5. The method according to claim 1, characterized in that that the compound of formula I on animals in a mixture with a usually solid feed, in feed cubes or salt licks, dissolved or suspended in drinking water or, for young animals, dissolved or suspended in whole milk or skimmed milk, or in the form of intraruminal pellets or slow release pills. 6. The method according to claim 5, characterized in that that each treated animal between 0 * 01 mg / kg body weight / day and 30 mg / kg body weight / day of a compound of formula I receives. 7. Non-toxic, orally administrable composition, thereby characterized in that they contain a compound of formula I wherein the carbon atoms 3, 3 ¹ and 9 ^s see all in the R configuration, X stands for an ethylene or trans-vinylene radical and Y stands for a radical Y as defined above or stents the carbon atoms 3, 3 * and 9 * are all in the S configuration, X stands for an ethylene radical and Y stands for a radical Y ² as defined above, in which X ^{2 is} . The ethylene radical is | or the carbon atoms 3, 3 ¹ and 9 * are all in the S configuration, X stands for an ethylene radical and Y stands for a radical Y ^ as defined above} 909848/6512 together with a solid or liquid, non-toxic, orally administrable diluent or carrier. 8. Composition according to claim 7 »characterized in that the solid or liquid, non-toxic, orally administrable diluents or carriers made from drinking water, whole milk, skimmed milk, a conventional one nutritionally balanced ruminant feed, an inert solid diluent or carrier with no nutritional value, Starch or lactose. 9. Composition according to claim 8, characterized in that it is in the form of a supplemented putter for direct feeding to animals, which contains 5 to 3000 ppm of a compound of formula I. 10. Composition according to claim 8, characterized in that it is in the form of a concentrated premix, which contains 0.3 to 50% of a compound of the formula I, for dilution with a conventional ruminant feed to produce a supplemented feed that is suitable for direct feeding to animals. 11. Process for the preparation of a solid composition according to Claim 7, characterized in that a compound of formula I with a solid, non-toxic, orally administrable diluent or carrier mixes uniformly. 12. A process for the preparation of a compound of formula I, wherein 1 2
R and R each represent a hydrogen atom or an acetyl radical, X represents a trans-vinylene radical and Y represents 1 1 is a radical Y, wherein X is a trans-vinylene radical and Z ^{+ is} a sodium ion, characterized in that an aplasmomycin-producing strain or mutants of Streptomyces griseus in an aqueous nutrient medium which 909848/0532 Sources of assimilable carbon, assimilable nitrogen and assimilable boron and a sodium salt contains, cultivated under aerobic conditions at a temperature between 10 and 37 ° C, the culture is filtered and the Product isolated from the piltrate by conventional means. 13. The method according to claim 12, characterized in that that the aplasmomycin producing strain or mutants of S. griseus is that; the at the National Collection of Industrial Bacteria, Ministry of Agriculture, Fisheries and Food, Torry Research Station, Abbey Road, Aberdeen, AB9 8DG, Scotland and is described as follows: (The media used are prepared according to the recipes for the "International Streptomyces Project (ISP)" and are from Shirling EB & Gottlieb, D (1966) in "Methods for characterization of Streptomyces species" in the International Journal of Systematic Bacteriology, 16, ( 3 ) _f 313 to 340 described). Conditions for Incubation Temperature 30 ⁰ C Growth in the dark.
ISP2 ilefe extract / malt extract 4 days ; 3 to 4 mm. Good raised, wrinkled, glassy colonies. Dark fawn. Lapel - brown-gray. 10 days ; 10 mm. Submerged growth at the margin with raised, wrinkled, watery-fawn-gray colonies. Reverse - unchanged. No color in the agar.
ISP3 oat melil agar 4 days : 1 to 2 mm. Thin, watery colonies, sometimes with a torn depression in the center. Revers very pale brown-gray. ► 10 days : 4 to 6 mm. Machs-like-yeast-like, smooth, gray-beige-brown colonies. Reverse - unchanged. Very weak- 909848/0532 transferred brown color to the agar. ISP4 Inorganic Salts / Starch Agar 4 days : 1 to 2 mm. Good, fairly moist, very compact colonies. Pale brown. Lapel - pale fawn / gray. 10 days : some more growth. Now waxy, somewhat raised, wrinkled colonies with uneven edges. Pale fawn. Reverse - unchanged. No color transferred to the agar. ISP5 glycerine / asparagine agar 4 days : 1 to 2 mm. Sublime colonies. Colonies wrinkled and the agar lowered at the edge. Glassy fawn brown. Lapel - pale brown-gray. 10 days : 3 to 5 mm. Waxy, raised, and wrinkled colonies. Yellowish fawn. Reverse - unchanged. ISP7 tyrosine agar 4 days : 2 to 3 mm. Good raised, wrinkled, glassy, fawn colonies. Lapel - gray-brown. Medium turned pale brown. 10 days : 10 mm. Waxy, wrinkled, yellowish-dark brown-gray colonies with some powdery gray areas. Reverse - dark-gray-brown, but discoloration of the agar can just be seen.
ISP9 carbon utilization agar Addition 4 days 10 days grading Water 1 to 2 mm. Submersed, very thin, "Out very thin, 'chig", submersed "smoky" Glucose 1 to 2 mm. Light, 2 to 3 mm. Dense, quite gelatinous, waxy, fawn, fawn- ++ gray gray. Lapel - un changes Arabinose slightly better than water like water Cellulose as indeterminable as water 909848 / Θ532 Fructose 1 mm. Dense, raised, glassy colonies, fawn-gray. Fawn-gray lapel Inositol such as arabinose Mannitol 2 mm. Dense, raised, glassy fawn colonies with torn ones Centers. Lapel fawn Raffinose like arabinose rhamnose like water Sucrose like water Xylose 1 to 2 mm. Very thin, short, velvety, fawn brown. 4 to 5 mm. Dense, raised, vigilant, wrinkled, torn, fawn-gray slightly better than + water 2 to 3 mm. Dense, raised, waxy, fawn-brown. Lapel - fawn brown. like water like water - like water - 2 to 3 mm. Somewhat raised, velvety, ++ light fawn-gray, possibly spore forming Tempera tolerance On tryptone / kefe nutrient broth (ISP1). Lowest temperature tested 19 ° C. Grows pretty well. Best at around 28 ° C _f does not tolerate temperatures above 37 ° C. morphology It is a weakly spore-forming strain, but if spores are seen then they are in short chains and are smooth-walled and barrel-shaped. 14. Streptomyces griseus strain HCIB 11371 as in claim 13 described. 1 ? 15. A compound of the formula I in which R and R each represent a hydrogen atom or an acetyl radical and: (a) the carbon atoms 3, 3 ^f and 9 ¹ are all in the R configuration, Y stands for a radical Y, one of the symbols X and X ^ stands for an ethylene radical and the other for a trans-vinylene radical 9848/0532 1
or X and X both represent ethylene radicals and Z ^{+ has} the meaning given above; or (b) the carbon atoms 3, 3 'and 9 ^f are all in the R configuration, X for a trans-vinylene is radical and Y is a radical Y in which 1 + X is a trans-vinylene radical and Z is a potassium, lithium, cesium or ammonium ion or an ion of the formula rVr ⁵ RT according to the definition of claim 1 or (c) carbon atoms 3, 3 ¹ and 9 'are all in the S configuration, X is an ethylene radical and Y is a Y ² radical, where X ^{2 is} an ethylene radical; or (d) the carbon atoms 3, 3 ¹ and 9 'are all in the S configuration, X stands for an ethylene radical and Y stands for a radical Y *, where X ^{2 has} the meaning given above and A stands for a potassium, Lithium, cesium or ammonium ion or an ion of the formula rWrT according to the definition of claim 1. 16. A process for the preparation of a compound of the formula I as defined in claim 15, characterized in that one ' (1) for the preparation of the new compounds defined under (a) above, a corresponding compound of the formula I, in which X and X each represent a trans-vinylene radical, via a platinum or palladium catalyst selectively or non-selectively hydrogenated; or (2) for the preparation of the new compounds defined under (b) above, an aplasmomycin-producing strain of 909848/0532 S. grlseus as defined above in the presence of a potassium, lithium, cesium or ammonium or
R-% R ⁵ R IJ ^ salt fermented in an aqueous nutrient medium; or (3) to produce the new ones defined under (c) above Compounds a corresponding compound of the formula I in which X is an ethylene radical and Y is a Radical Y ^, where X is a cis-vinylene radical, hydrogenated over a platinum or palladium catalyst; or (4) for the preparation of the new compounds defined under (d) above, a compound of the formula I in which X
represents an ethylene radical and Y represents a radical Y ² , with potassium, lithium or cesium hydroxide or potassium, lithium or potassium carbonate. 909848/8532