IL23314A - Esters of the antibiotic pleuro-mutilin,their production and animal feed supplement containing same - Google Patents

Description

*8X€QBDI2g" Gesellachaitj mit t-eaotue i lster fiaituag o£ mmt TIROL, Austria .

O HEREBY DECLARE the nature of this invention and in what manner the same is to be performed to be particularly described and ascertained in and by the following statement: She present invention relates to novel antibiotiee derivatives useful in animal feed stuffs and to the production of the same. It also relates to ready- mixed animal feeds and feed supplements for preparing tiie feeds eaoh containing the antibiotic derivatives.

We have found that a antibiotic may be produced by the fermentation of fllitopilus passeckerianua(Pil. ) Sing. RRL 3100(which belongs to the family of Tricholomatacaea in the order Agaricales of the class Basidiomycetes) in a aqueous nutrient medium in the presence of sources of assimilable carbon and nitrogen. ' aoh o p!Pooooo io claimed in pplic ion ¾fo. 562Qg/67.

The antibiotic produced by the above process has an empirical molecular formula (molecular weight 378), is readily soluble in alcohol, ether, butyl acetate, acetone, methylisobutylketone, tetra- glycol, and chloroform, but difficulty soluble in water, petroleum ether and cyclohexane, melts at 167°<3. £ orystaHized from Eg© 0^QH( il)]# has an optical rotationζ«1 m t 3?° ( 0 in chloroform), and exhibits characteristic absorption maxima in the ultra-violet spectrum at 205 and 293 κ (in ether) or 209 and 290 πιμ (in 60 ethanol), respectively, and characteristic absorption bands in the infra-red spectrum(potassium bromide pressings) at 3400,3000, 1235 ani1095 and in a methylene chloride solution at 3570, 3610, 1730, 1740 and 1630 cm. !Ehe antibiotic was originall named BC 757 by us but recent investigations have shown that the antibiotic BC 757 is identical with the known antibiotic pleuromutilin which is described for example, in Proc. Nat. Acad. Sci. Vol. 37, (1951) , page 570 (Frederick Kavanagh et al) , and whose structural formula has been shown to be probably OH The present invention provides novel compounds which are esters of pleuromutilln(BC 757) or of its hydrogenation product. The hydrogenation product of pleuromutilin is the compound in which the vinyl group of pleuromutilin has been hydrogenated to an ethyl group. The esters are formed by reaction between one or both of the hydroxy groups on pleuromutilin or its hydrogenation product and an ester-forming group on the other reactant. Specifically the novel esters of the present invention are the reaction products of pleuromutilin or its hydrogenation product with carbamate ester forming derivatives of isocyanic acid, with acylating derivatives of aliphatic monocarboxylic acids having from 3 to 19 carbon atoms or of haloformic or monohaloacetic acids, with acylating derivatives of aliphatic dicarboxylic acids having from 3 to 14 carbon atoms, or with sulphonylating ^ivatives of sulphonic acids. Y/here the acid has an additional group capable of reacting with a base to form a salt, for example a free acid group or a saponifiable ester group, the first-formed eater may be reacted with a baee such as NaOH, sodium bicarbonate or a triallcylamine to Obtain a water-soluble salt of the ester. Such salts are also included in the present invention.

The esters of the hydrogenation product of pleuromutilin may be obtained by reaction of the hydrogenation product of pleuromutilin with ester-forming derivatives of the above mentioned acids.

Alternatively they may be prepared by hydrogenation of the esters after formation. When the esters are subsequently reacted with a base to form the salt , hydrogenation may be carried out before or after reaction with base. Hydrogenation of water-soluble esters of the invention may be carried out without destroying the water-solubility and activity of the esters.

The preferred classes of esters of the present invention are (a) carbamate esters which may be obtained by reacting pleuromutilin or its hydrogenation product with isocyanates of the general formula R1 - CH - Z - C00R2 I NCO wherein is hydrogen, alkyl having up to six carbon 2 atoms, carbalkoxy, carbalkoxyalkyl or aryl, R is alkyl having up to five carbon atoms and Z is a direct C - C bond or the grouping "^σΗ2^η~ w*1®1*® 11 n is an integer from 1 to 6, the esters thus obtained being optionally selectively hydrolysed in the presence of 2 base to saponify the ester grouping - COOR ; (b) carboxylate esters which may be obtained by reacting plueromutilin or its hydrogenation product with a haiide or ahnydride of an aliphatic mono-carboxylic acid of the general formula R3 - (R4)m - 000H 3 wherein R represents a halogen atom, particularly 4 chlorine or bromine, R represents a branched or unbranched alk lene radical, particularly methylene, and m represents an integer from 0 to 18, the esters thus obtained being optionally reacted with a dialkyl amine for example dimethylamine to replace the halogen atom by a dialkylamino group, which product in the form of its hydrochloride is a water-soluble derivative (c) carboxylate esters which may be obtained by reac¾bn of pleuromutilin or its hydrogenation product with a haiide or anhydride of a dicarboxylic acid of the general formula COOH from 1 to 12, the esters thus obtained being optionally reacted with a base to form a salt of the free acid group} (d) esters which may be obtained by reaction of pleuromutili or its reaction product with a sulphonlc acid of the formula HOOC - R5 - SOgOH wherein R is a branched or unbranched alkylene radical, particularly ethylene, or a phenylene radical, the esters thus obtained being optionally reacted with base to form a salt with the free acid group.

Particular examples of ester- orming derivatives which may react with pleuromutilin or its hydrogenation product in accordance with the present invention are ethyl alpha-lsocyanatopropionate, phosgene, diethyl alpha-isocyanatoglutarate, ethyl isocyanatoacetate, palmitic acid chloride, succinic anhydride, beta-sulphopropionic anhydride, m - catooxybenzene sulphochloride and chloracetyl chloride.

It has been found and confirmed by numerous feeding tests that esters of pleuromutilin are useful additives to animal feeds and that they afford advantage over the antibiotics used hitherto as feed supplements, despite the recent conclusion that they, in common with pleuromutilin itself, are not sufficiently valuable antibiotics to/e used in human therapy. It is an object of the present invention to provide ready-mixed animal feeds (and feed supplements for preparin such feeds) having a content of an antibiotic not applicable to human therapy.

The invention therefore provides ready mixed feeds containing an addition of antibiotically active substances an¾ feed supplements for preparing such ready mixed feeds containing the above-mentioned derivatives of the antibiotic pl^romutilin. Besides these antibiotically active agents the ready mixed feeds or feed supplements proposed by the invention may contain the usual additives incorporated in feed mixtures, such as protein concentrates, fish meals, dried mycelia, particular^ from the production of penicillin, dried blood and/or mineral substances and/or vitamins.

The difficultly water-soluble pleuromutilin is only slightly resorbed in the intestine. The antibiotic bacitracin, however, which is a substance that is not resorbed in the intestinal tract, is already being used in the animal feed-producing industry.

High resorption is therefore clearly not essential to the efficacy of an antibiotic in an animal feed.

A particular advantage of employing derivatives of the antibiotic pleuronutilin instead of other antibiotics is that pleuEomutilin is entirely stable both as a pure substance and in admixture with fish meals which often considerably attack antibiotic additives.

The storage keeping properties of both a feed supplement according to the invention containing the equivalent P.A.23514 File 131I6 2.1.69 concentrate with a content equivalent to 150 and of a ready mixed fattening feed with a content equivalent to 21Y of pleuromutilin per gramme were tested at 24 and 37°C. At intervals of 1 month samples were taken over a period of six months, the antibiotic being extracted and tested. A drop in the content of antibiotic was not found to have occurred.

For achieving an optimum rate of gain an an improved atilization of the feed by farm animals addition of the above-mentioned derivatives of the antibiotic pleuromutilin, in concentrations that are conventional in the case of antibiotics hitherto used is sufficient. In other words, ready mixed feeds according to the invention should contain quantities of plueormutilin derivatives equivalent to the following quantities of pleuromutilin per kg. of the mixture for poultry 2.5 to 50 mg. for pigs 10.0 to 50 mg. and for calves and beef cattle the feed supplements according to the invention should be fed in quantities to provide the animal with between 25 and 70 mg. of pleuromutilin per day.

The following examples illustrate animal feeds and feed supplements according to the invention.

EXAMPLE 1.

Ready mixed feed for fattening poultry.

A feed supplement was prepared from 100 mg. of P.A.23314 Pile 13116 2.1.69 mycelium from the production of penicillin. This was incorporated in the following feeds to provide a fattening feed containing 10 mg. of pleuromutilin succinate per kg»-590g6f wholemeal maize, 190 g. of course sjrybean meal, 90 g. of fish meal, 74.4 g. of wheat bran, 20 g. of phosphate of lime, 12.5 g. of carbonatedf lime, 5 g. of iodized cattle salt, 10 g. of shredded green lucerne, 0.5 g. of DL-methionine and 7.5 g. of a conventional mixture of vitamins and trace elements.

EXAMPLE 2 A ready mixed feed for fattening poultry was prepared analogously to Example 1 excepting that pleuromutilin (BC 757) palmitate was used instead of pleuromutilin succinate. The first named compound can be prepared as follows: 3g of palmitic acid chloride are heated in a benzanepyridine mixture (10:1) together with 4.1 g of BC 757 kept at 100°C for 30 minutes. The solvent is then completely removed under reduced pressure.

The residue is twice recrystallized from benzene. .2 g of pleuromutilin palmitate are thus obtained.

The empirical formula was ascribed to this compound.

Calculated C « 73.97 B» 10.47 f* Found C « 73.3$ He 10.2 The following examples illustrate the production of water-soluble derivatives of the antibiotic pleuromutilin which ma be used in the read mixed feeds or in P.A.23314 Pile 13H6 2.1.69 Example 3 The reaction product of pleuromutilin with the ethyl ester of a-isocyanatopropionic acid. 1.0 g (0.007 mols) of the ethyl ester of a-isocyanato-propionic acid was refluxed with 2.6 g. of pleuromutilin(0.00"2> mole) in 25 ml of xylene for 5 hours, at the end of which 2.5 ml of pyridine were added and heating was continued for another 25 minutes. The solvent was driven off in a vacuum, the residue taken up in alcohol and the resultant reaction product treated with equivalent quantities of alcoholic NaOH at room temperature. The ethyl ester was thus saponified. After removal of the alcohol at reduced pressure the sodium salt precipitated in the form of a white amorphous powder. This was filtered off on a suction filter, washed with ether and dried. the 1.6 g of sodium salt of ,α-carboxy-ethylcarbamate of pleuromutilin was produced wftihh was readily soluble in water. The empirical formula was ascribed to the compound.

Calculated N« 2.72 ≠ Pound NB 2.701> Equivalent weight of the free acid Calculated 493 Pound 488 ■The-"reac tJir product υΓ plyuruiuuliliii "with C tr di tlayX.,-^ P.A.251 File 13116 2.1.69 Example 4 The reaction product of pleuromutilin with the diethyl ester of a-isocyanatoglutaric acid.

In a manner analogous to that described in Example 3 pleuromutilin was reacted with the diethyl ester of a-isocyanatoglutaric acid and the carbamate ester so formed was treated with alcoholic NaOH to saponify both ethyl ester groups. The di-sodium salt of the 1,3 dicarboxy-propyl-carbamate of plmaromutilin was obtained which very readily dissolved in water.

The empirical formula ascribed to this substance.

Calculated N* 2.35$ Pound N» 2.30 Example 5 The reaction product of pleuromutilin with the ethyl ester of isocyanatoacetic acid.

The reaction was performed in the same way as in Examples 3 an 4, both hydroxy groups of pleuromutilin being esterified by the ethyl isocyanatoacetate. The compound thus obtained(the di-sodium salt of the bis-( carboxymeth lcarbamate) of pleuromutilin) was readily soluble in water and was ascribed the empirical formula Calculated MM-.49^ Pound N«4.42# P.A.23314 Pile 13116 2.1.69 Example 6 The reaction product of pleuromutilin with β-Bulphopropionic anhydride. 1 g. of β-sulphopro ionic anhydride and 2.8 g. of pleuromutilin were dissolved in 50 ml. of dioxan. After reaction, the solution was evaporated in a vacuum until dry and the residue taken up in 50 ml of dioxan containing 3.7 ml of a 2N-solution of NaOH. After again concentrating at reduced pressure the sodium salt was precipitated in the form of a white finely crystalline powder. The novel compound, the sodium salt of pleuromutilin β-sulphopropionate dissolves well in water.

Summary formula of the compound: C^^H^^O^SiTa Calculated C= 55.95 # H» 6.95 Pound 0= 55.7 He 6.8$ Example 7 The reaction product of pleuromutilin with m-carboxybenzene sulphochloride. 2 g of m-carboxybenzene sulphochloride were reacted with 3*2 g. of pleuromutilin in pyridine.

The solvent was then driven off in a vacuum and the residue taken up in ether. The organic phase was extracted with water and then shaken out with a solution of NaHC0„ ~ 3 in order to convert the free acid to the sodium salt.

After the aqueous phase had been submitted to freeze-drying 3*1 g of Na pleuromutilin m-sulphobenzoate were obtained.

Calculated C= 59.6 H» 6.4% Found C= 59.2% He 6*2% Example 8 g. of chloroacetyl chloride (CHgClCOCl) were reacted with8.5 g. of pleuromutilin in a mixture of pyridine and dioxan. The mixture was introduced into water with cooling, the precipitated reaction product (the bis~(chloroacetate) of pleuromutilin) dissolved in ethyl acetate, extracted with water, the organic phase de-watered and concentrated in vacuo until dry. 2.5 g. of an absolutely dry dimethylamine in 50 ml. of methyl cyanide were added to the residue at -10°C.

As soon as the mixture had reached room temperature the solvent was removed in vacuum, the residue in the orm of the hydrochloride dissolved in vxater, extracted with ether and then lyophilised from this solvent. The product (8.2 g. of pleuromutilin bis(dimethylaminoacetate) is readily water-soluble in the form of the hydrochloride. The empirical formula was ascribed to the free base.

Calculated C= 65.65 H=8.85% N=5.H% Found G= 6 .1 H=8.65% N=4.95%

Claims (21)

P.A.23314 File 13116 2.1.69 WHAT IS CLAIMED IS:

1. A process for producing esters of the antibiotic pleuromutilin or of its hydrogenation product, which comprises reacting pleuromutilin or its hydrogenation product with an ester-forming derivative of an acid selected from isocyanic acid, an aliphatic monocarboxylio acid having from 3 to 19 carbon atoms, a haloformic or monohaloacetic acid, an aliphatic dicarboxylic acid having from 3 to 14 carbon atoms,, or a sulphonic acid.,

2. A process according to Claim 1, wherein the acid contains an additional group capable of reacting with a base to form a salt,, followed :by reacting the first-formed ester with a base to form the salt.

3. A process according to Claim 2, for producing an ester of the hydrogenation product of the antibiotic pleuromutilin wherein pleuromutilin is reacted with the ester-forming derivative of an acid and the resulting product is hydrogeaated before or after treatment with the base to form the salt.

4. A process according to any of Claims 1 to 3 for producing a carbamate ester of pleuromutilin or its hydrogenation product, wherein the ester-forming derivative of isocyanic acid is an isocyanate of the general formula R1 - OH - Z - C00R2 t NCO in which R^" signifies hydrogen, an alkyl radical having from 1 to 6 carbon atoms, a carbalkoxy radical, a 2 carbalkoxyalkyl radical, or an aryl radical, R signifies P·A«. File 1. 2.1.69 Z signifies a carbon-carbon bond or - (Clig) -» beinS an integer from 1 to 6, and the first formed ester is optionally selectively reacted with a base to saponify 2 the -CQOR ester group.

5. A process according to Claim 1 for producing a carboxylate ester of pleuromutilin or its hydrogenation product, wherein the ester-forming derivative of an acid is a halide or anhydride of an acid of the general formula R5 - (R4)m- C00H 3 4 in which R signifies a halogen atom, R signifies a branched or unbranched alkylene radical, and m is an integer between 0 and 18.

6. A process according to Claim 5» followed by reacting the ester, thus produced with dimethylamine, whereby the halogen atom is replaced by a dimethylamine group.

7. process according to any of Claims 1 to 3 for producing an ester of pleuromutilin or its hydrogenation product, wherein the ester forming derivative of an acid is a halide or an anhydride of a sulphonic acid of the general formula HOOC - R5 - S02 - OH 5 in which R is an ethylene radical or a phenylene radical.

8. A process for producing esters of the antibiotic according to Claim 1 pleuromutilin or of its hydrogenation product, substantially P.A. 23314 Pile 131I6 2.1.69

9. Esters of the antibiotic pleuromutilin or of its hydrogenation product, whenever produced by the process claimed in any one of Claims 1 to 8.

10. The succinate of the antibiotic pleuromutilin or its hydrogenation product.

11. » The mono-palmitate of the antibiotic pleuromutilin or its hydrogenation product.

12. The a-carboxyethylcarbamate of the antibiotic pleuromutilin or its hydrogenation product.

13. The lt3-dicarboxypropylcarbamate of the antibiotic pleuromutilin or its hydrogenation product.

14. The β-sulphopropionate of the antibiotic pleuromutilin or its hydrogenation product.

15. The m-sulphobenatoate of the antibiotic pleuromutilin or its hydrogenation product.

16. The bis-(c loroacetate) of the antibiotic pleuromutilin or its hydrogenation product.

17. The bis-(dimethylaminoacetate) of the antibiotic pleuromutilin or its hydrogenation product.

18. The bis-(carboxymeth lcarbamate) of the antibiotic pleuromutilin or its hydrogenation product.

19. An animal feed or feed supplement containing one or more esters of pleuromutilin or its hydrogenation product*claimed in any of claim 9 to 18.

20. An animal feed or feed supplement containing one or more esters of the antibiotic plemomutilin or its hydrogenation product produced in accordance with the process claimed in any one of Claims 1 to 8. Ρ.Α.23314 Pile 13116 2.1.69

21. An animal feed or feed supplement containing one or more esters of the antibjotic piearomutilin or its hydrogenation product as claimed in any one of Claims 9 to 18. 22, An animal feed or feed supplement according to Claimsl9,20 or 21 substantially as herein described. Attorneys for Applicants.