DE2621615A1 - DIHYDROMOCIMYCINE, ITS SALT WITH ACIDS, PROCESS FOR ITS MANUFACTURING AND FEED - Google Patents

Description

DIPL-CHEM. DR. VOLKER VOSSIUS PATENT ADVERTISER

uZ: L 689 Case: DUI-2042 GIST - BROCADES Ή.Υ. Delft, the Netherlands

MUNICH 86,,. , -. _; .,

PO BOX 86 07 67 I * h HPi 'MD

SIEBERTSTRASSE 4 PHONE: (O 89) 47 40 75

CABLE ADDRESS: BENZENE PATENT MÖNCHEN TELEX 5-29453 VOPAT D

"Dihydromocimycin, its salts with acids, method too its manufacture and feed "

Priority: May 16, 1975, Great Britain, Ur. 20926/1975

July 22nd, 1975, Great Britain, Fr. 30646/1975

The invention relates to the antibiotic dihydrotnocimycin formula

and its salts with acids, in particular the non-toxic, pharmacologically acceptable salts. The alkali metal, Ammonium and amine salts.

609848/0999

ORIGiMAL INSPECTED

2821615

The invention also relates to a method of production of dihydromocimycin, which is characterized in that the microorganism Streptomyces ramocissimus CBS 190.69 or a dihydromocimycin producing mutant of this strain in an assimilable Sources of carbon and nitrogen, as well as inorganic ones Culture medium containing compounds and the separates dihydro'mocimycin formed during cultivation. Of the The aforementioned microorganism strain is in GB-PS 1,325,200 described. According to this publication, this microorganism is cultivated to produce mocimycin (MYC 8003). The structure of mocimycin has been reported by Vos and Verwiel, Tetrahedron Letters, 52: 5173-5176 (1973).

It has now been found according to the invention that this strain of Streptomyces ramocissimus or corresponding mutants of this strain not only for the formation of mocimycin but also for the formation of Dihydromocimycin are capable.

Dihydromocimycin is a pale yellow solid that reacts slightly acidic and can be characterized by the following physico-chemical properties:
Solubility:

The compound is readily soluble in chloroform, methyl isobutyl ketone, Ethyl acetate, butyl acetate, acetone, dioxane, methanol, ethanol, tetrahydrofuran and weakly alkaline, aqueous solutions. It is moderately soluble in carbon tetrachloride and benzene and insoluble in diethyl ether, water, weakly acidic, aqueous solutions, cyclohexane and petroleum ether.

609848/0999

2621616

Optical rotation;
85 °

(1 percent solution in methanol).

Melting point:

The connection does not melt. Decomposition occurs from 123 ° C.

Elemental analysis:

C ₄₃ H ₆₂ N ₂ O ₁₂ .2H ₂ O: CH IT 0 (difference)

calc .: 61.8 8.0 3.3 26.8

found: 61.8 7.5 3.4 27.2

UV spectrum:

The UV spectrum of dihydromocimycin in a 1: 1 G-emiseh from water and methanol at different pH values results from Pig. 1. The concentration of the solutions is 18.3 ^ gZmI. The UV spectrum depends on the pH value. The following maxima can be determined (the molar extinction coefficient is given in round brackets):
Methanol - water: 233.5 nm (ε = 63,000); 267 nm (ε = 23,000);

291 nm (ε = 19,000) and 333 nm (e. = 18,000);

Curve 1;
Methanol - 0.5 η NaOH: 235 nm (£ = 62,000); 277 nm (ε = 25,000 ) and

308 nm (ε = 29,000.); Curve 2; Methanol - 0.5 η HCl: 233.5 nm (e = 65,000); 268 nm (ε. = 25,000) and

338 nm _λ (£ = H 000) .; Curve 3.

IR spectrum:

The IR spectrum of dihydromocimycin in chloroform and potassium

'609848/0999

bromide is shown in Figures 2 and 3, respectively. The following main absorption bands are found:

Chloroform (vin cm " ¹ ): + 3445 (sh); 3430; 2979? 29415 2886; 1662-1653 5 1458; 1100; 1080; 1040; 998; 944; 893; 870 and 840; potassium bromide (vin cm" ¹ ) : + 3400-3320; 2972; 29355 2880; 1650; 1535; 1455; 10995 1040s 990; 943; 860; 840 and 790.

PMR spectrum:

The PMR spectrum. of dihydromocimycin in deuterochloroform under Use τοη tetramethylsilane as an internal standard is in Fig. 4 (60 MHz) shown.

Thin layer chromatography:

A thin layer chromatogram of dihydromocimycin is carried out with silica gel F 254 plates (coated prefabricated plates from Merck). After drying, the stains will appear by fluorescence, absorbance measurement or charring Spraying with a mixture of diethyl ether and sulfuric acid proven. Even highly purified preparations of dihydromocimycin produce a small additional stain in addition to a main stain Stains. This is due to a tautomeric equilibrium, as can be shown by a two-dimensional chromatogram:

The following R _f values result for dihydromocimycin in different solvents (the R _f value for the additional stain is given in round brackets):

609848/0999

50: 45-5 mixture of methyl isobutyl ketone, acetone and water:

0.44 (about 0.44);

70: 20: 10: 0.5 mixture of ethyl acetate, methanol and water and 25 percent ammonia solution: 0.29 (0.36); 65 J 40 ^ mixture of benzene, 100 percent ethanol and 33 percent Ammonia solution: 0.16 (0.22);

60:42:10 mixture of chloroform, 96 percent ethanol and 25 percent ammonia solution: 0.4.

The structural formula given at the beginning results for dihydromocimycin on the basis of the following facts: The PMR spectra (220 MHz) of mocimycin Tygl7 Tetrahedron letters, Vol. 52 (1973), pp. 5173-5176, where the structural formula of mocimycin is given) and dihydromocimycin show that both compounds are very similar, with two doublets at δ = 5.9 and 7.3 ppm (tetramethylsilane is used as standard) appearing in the spectrum of mocimycin and not being present in the spectrum of dihydromocimycin. These signals are caused by the protons of the fifth and sixth carbon atoms in the pyridone nucleus. In the spectrum of dihydromocimycin, on the other hand, there are two triplets at 5 = 2.5 and 3.4 ppm. This is an indication that the bond between the fifth and sixth carbon atoms in the pyridone nucleus of dihydromocimycin is saturated. This interpretation can be confirmed by ozonizing an aqueous solution of dihydromocimycin at ⁰ ° C. and a pH of 12 for 5 minutes. After reduction of the reaction mixture obtained with hydrogen using PtO ₂ as a catalyst and after hydrolysis with concentrated hydrochloric acid, ß-alanine can be obtained

609848/0999

demonstrate what suggests that the bond between the fifth and sixth carbon atom of the pyridone nucleus of dihydromocimycin is saturated.

Dihydromocimycin is similar to mocimycin in many properties. An important difference between these two compounds, however, is that mocimycin can be used as a feed additive in small amounts Quantities in animals such as chickens and pigs result in weight gain and an increase in size growth and feed conversion causes. In contrast, dihydromocimycin contributes to no increased growth or improved feed conversion in these animals. This is surprising as both connections are effective in vitro against the same microorganisms. Dihydromocimycin is often even more effective than mocimycin. A comparison of the antimicrobial effects of the two antibiotics results from the following table:

609848/0999

Agar dilution tests

examined microorganism ' -Minimum home concentration. '.
(ug / ml) for aerobic cultures' - 6th
6th
0.75 Dihydromoc imycin - ■ .Mocimycin 3 > 100 6th
3rd
0.4 Staphylococcus aureus A 2000 > 100 30th > 100 1.5 'Staphylococcus aureus A 2001 > 100 <0 _? 75 10 Diplococcus pr.eurnoniae L54 1.5 MOO 'SaJjnonellQ tvoiiiniuriurn R172 > 100 100 Escherichia coli U20 > 100 . 1.5 Listeria rnonocvtocsnes A213O 6th 6th "Listeria monocvtocienes A2131 '6 6th Listeria monocvtoaenes Ä2132 6th > 100 Clostridium perfrincsns A738 > 100 10 Clostridium se-Dticuin A2152 10 Streptococcus zcoepideniicus A2144
R-streptococci A214S
Brucella suis (smooth) A2126 Pasteurella haemolytica A2136 Treponema spec. A2275

Liquid dilution tests

Investigated microorganism Minimal. Inhibitory Concentration- - ■
Oig / ml) ...... Dihydromo c imyc in • 0.45 Bacillus subtilis ATCC 6633 .Mocimycin 50 0.12
0.3 Bacillus subtilis ATCC 6051 100 50 Baci3.1us subtilis 6346 D167 100 0.9 Bacil-lus subtilis 220 D178 1.2 75 Bacillus subtilis TH 10 75 50 Bacillus cereus D166 100 0.6 Bacillus cereus D261 0.6 0.6 Bacillus cersus D22O 0.9 0.9 Bacillus cereus B569 1.2 1.2 Bacillus cereus TK 1 2.5 Ψ. Bacillus thurinqiensis WlI 1.8 0.9 Bacillus mesenterium D169 1.2 50 Bacillus cereus var. Raycoides 100 0.45 Streptococcus haeniolvticus 1.2 A266
Streptococcus haemolvticus A21S2
Mycoplasma livorhinus A2230
Strotrtomyces viridochiconuqep.es 0.45 0.25
1
2.5

609848/0999

As already mentioned, the Streptomyces ramocissimus CBS 190.69 strain or its corresponding mutants produce dihydromocimycin in addition to mocimycin. The microorganism can be cultivated in liquid carrier media containing the usual sources of carbon, nitrogen, phosphorus, calcium, iron, sulfur, magnesium, potassium, vitamins and trace elements. For example, media containing beet molasses, malt paste, peanut flour, lactose, potato starch, soaked corn products, and yeast extract can be used. The growth temperature of the medium should, preferably 26 to 34 ° C and the pH ™ ert 5 to 9 »to 8 are 20 to 4-0 ⁰ C preferably 6.5.

The inventive method for the preparation of dihydromocimycin is very similar to the process used to make mocimycin. It was found that when Streptomyces ramocissimus or its corresponding mutants under appropriate conditions in addition to mocimycin at the same time Dihydromocimycin is formed.

It was' surprisingly found that the yield of dihydromocimycin compared to the yield of mocimycin by increasing the oxygen pressure in the culture medium leaves.

From the structure of dihydromocimycin it can be assumed that the higher oxygen pressure in the culture medium Yield of dihydromocimycin compared to mocimycin decreases.

609848/0999 ^C0PY

However, there is the aforementioned effect that dihydromocimycin compared to mocimycin due to better ventilation the culture liquid is formed in larger quantities. The ventilation can be carried out by methods which are customary per se will. For example, the ventilation speed (part by volume of air per part by volume of cultivation medium per unit of time) or a higher stirring speed of the culture medium in the fermenter is used. Appropriate ventilation speeds of the culture medium, the volume of which is 2 liters, for example, are 1 to 3 liters of air, preferably 1.5 to 2.5 liters of air per minute. A further improvement in the yield of dihydromocimycin can be achieved by adding certain Metal ions in low concentrations, for example by adding iron, cobalt or nickel ions for breeding purposes. achieve medium.

The separation of dihydromocimycin from the culture medium is in part similar to the separation of mocimycin. In the In the last stage, v / o the compounds are precipitated from an organic solvent, differences in solubility are used from mocimycin and dihydromocimycin. Mocimycin fails when gaseous ammonia is passed through the solution first. The separation can thus be carried out by introducing ammonia until the mocimycin is practically completely precipitated. The dihydromocimycin remains practically completely in solution. It can can be followed by thin layer chromatography. After severing

609848/0999. copy

For example, by filtering off the mocimycin precipitate from the solution, further ammonia is passed in until the dihydromocimycin has practically completely precipitated. The dihydromocimycin can then also be separated off, for example by filtration. Ammonia is used for about 1 to 4 minutes at a rate of 150 liters per liter of solution per hour (i.e. about 2.5 to about 10 liters of gaseous ammonia per liter of solution) at a temperature of about -12 to about + 15 ⁰ C, preferably -5 to +8 ⁰ C, initiated. If gaseous ammonia is introduced further, the hydrogen ion concentration decreases to such an extent that dihydromocimycin becomes insoluble. For example, under the aforementioned conditions, a period of 10 to 15 minutes is passed (corresponding to about 25 to about 40 liters of gaseous ammonia). In addition to ammonia, all alkaline compounds can generally be used to separate mocimycin and dihydromocimycin. Sodium methylate and triethylamine may be mentioned as examples.

The dihydromocimycin separated from the culture medium in this way Mocimycin impurities can be further purified by keeping the precipitate in a strong dilute ammoniacal solution of pH 9 and this solution with a solvent such as chloroform or methylene chloride, extracted. Mocimycin is only sparingly soluble in such solvents. The extract obtained is poured into an apolar solvent present in excess, such as petroleum ether, cyclohexane or pentane, forms a dihydromoci

609848/0999

mycine precipitate that contains less than 5 percent mocimycin.

Highly purified dihydromocimycin is obtained by chromatography in three-dimensional networks! Dextran (for example Sephadex LH 20, using the adsorption differences between mocimycin and dihydromocimycin. This is where the dextran suspended in 100 percent methanol and carefully poured into the column. Compartment displacement of the methanol by chloroform the aforementioned dihydromocimycin precipitate is applied to the column. Chloroform is used as the eluent. This first gives dihydromocimycin and then mocimycin. Both compounds can be detected in the eluate because they absorb in the W range at 350 nm. The pure ones Compounds can be extracted from the chloroform solution by precipitation with an apolar solvent such as cyclohexane or pentane, be won.

The identity of the compounds can be determined by thin layer chromatography confirm. To do this, .Kieselgel 3? 254 plates of the Dimensions 20 χ 5 cm (Merck) used. Serves as a solvent a 60:42:10 mixture of chloroform, ethanol and 25 percent Ammonia. The running time is 2 hours. Mocimycin shows an R n value of 0.3 (major tautomeric) and dihydromocimycin of 0.4 (main tautomeric).

Based on previous research, it was believed that mocimycin,. which has a similar spectrum of antimicrobial activity as TyIosin (cf. Merck Index, - 8th edition, p. 1089), against

, 609848/0999

Treponema hyodysenteriae, the causative agent τοη Treponema dysentery or Vibrio Doyle, one of the most common diseases in pigs, is effective. Through studies carried out in the context of the present Invention were carried out, however, it was found that the effect of mocimycin is no greater than that of tylosin.

From the table above it can be seen that dihydromocimycin is more effective than a number of microorganisms Mocimycin. In studies of the effectiveness against the causative agent of treponemal dysentery, it was found that that dihydromocimycin is much more effective than tylosin against this microorganism and also against strains that resistant to tylosin is effective. Thus, for the treatment of treponemal dysentery, dihydromocimycin is tylosin and therefore also superior to mocimycin.

The invention thus also relates to feedstuffs for pigs which contain an effective amount of dihydromocimycin or a salt this compound, for example the sodium salt. The antibiotic or its alkali metal, ammonium or Amine salt can also be in a corresponding inert, physiologically harmless carrier or diluent, which can be administered orally to pigs, do not react with the antibiotic and are safe for pigs when administered orally are dispersed or mixed. Effective amounts of dihydromocimycin used for prophylaxis or therapy

609848/0999

of treponemal dysentery are added to the feed at about 10 to about 200 ppm, preferably 20 to 40 ppm, based on the weight of the feed.

The dihydromocimycin obtained in the above manner is a fine, lightly dusting powder. As this can lead to difficulties when mixing with the feed can lead, a premix is preferably prepared with one or more components of the pig feed. This Premix contains, for example, 9 to 99 times the desired amount of dihydromocimycin. Premixes can for example made with corn flour, potato flour or soy flour will. Investigations have shown that dihydromocimycin also dissolves when pelletized, i.e. granulated under high pressure and high temperature using water vapor, proves stable.

The premix can be added to the feed as a preventive measure or for the treatment of mild treponemal dysentery in pigs can be added.

Vv ^r hen pigs are so heavily infested by Treponema dysentery that they have lost their appetite or be pushed away from healthy animals from the feeding site, Dihydromocimycin is preferably administered in the drinking water, preferably together with a taste-improving agent. For this purpose, dihydromocimycin is used in water-soluble form, for example as

609848/0999

-H-

Potassium, sodium or amine salt used.

Pigs that are severely affected by dysentery can receive injections of dihydromocimycin or a water-soluble equivalent Salt to be treated. The active ingredient is in a conventional injection liquid, for example a Saline solution, propylene glycol or mixtures of glycerine and water, suspended or dissolved.

Mocimycin has beneficial growth-promoting properties when fed to livestock, including chickens and other poultry. Dihydromocimycin exhibits these properties not on. Attempts have therefore been made to convert the dihydromocimycin into mocimycin.

It was found according to the invention that dihydromocimycin if a certain dehydrating agent is used, can be dehydrated under certain conditions. Ghinones, such as 2,3-dicyano-5,6-dichloro-1,4-quinone, p-chloranil and o-chloranil are not suitable for the dehydration of dihydromocimycin because when used, other products are formed. Also halogenation with copper (II) bromide, bromine, iodine or H-bromosuccinimide with subsequent dehydrohalogenation did not lead to the desired result, not even in the presence VGn catalysts, such as benzoyl peroxide or Qr, <X-azobisisobutyronitrile. Too high temperatures are necessary for the catalytic dehydrogenation of dihydromocimycin, so that the danger there is decomposition of dihydromocimycin. It was fixed-

609848/0999

posed that the dehydration of dihydromocimycin only can be carried out with selenium dioxide in a satisfactory manner.

The invention thus also relates to a method for the dehydrogenation of dihydromocimycin to mocimycin, which is characterized is that dihydromocimycin is reacted with selenium dioxide. This procedure can also be applied to mixtures of dihydromocimycin and Use mocimycin, which is obtained, for example, from the processing of mocimycin from fermentation liquids.

The dehydrogenation of dihydromocimycin with selenium dioxide can be carried out at normal ambient temperature. Preferably, however, the reaction is carried out at elevated temperatures, for example from about 65 to about 110.degree. C. and preferably at from 80 to 95.degree. 'The reaction time may be at a temperature range of about 65 to about 110 ⁰ C for about 10 hours to about 20 minutes. The reaction time is preferably about 3 hours to about 1 hour in the preferred temperature range. At normal temperatures, the reaction time is about 1 week.

The reaction is preferably carried out in a solvent. Examples of suitable solvents are hexamethylphosphoric triamide (HMPT), dimethyl sulfoxide _. (DMSO), tert-butanoi, tert-amyl alcohol, sec-butanol, hexylene glycol, n-buta-

Isopropanol,
nol / ethylene glycol monomethyl ether, dimethylformamide, water, phenylmethylcarbinol and propanol and mixtures of two or more of these solvents. HMPT, DMSO and tert-butanol are preferred. HMPT is particularly preferred.

'609848/0999

In the dehydrogenation, the selenium dioxide is preferably in stoichiometric amounts or in excess, based on dihydromocimycin, before.

Since the isolation of both products from the fermentation liquid is a simple matter Procedure is while the separation of dihydromocimycin and mocimycin requires more effort, this process involves the conversion of dihydromocimycin to mocimycin allowed to have a great advantage with it. It is also possible to use a mixture of both compounds as the starting material, the mocimycin is essentially unaffected. Thus, G-emic mixtures containing dihydromocimycin can be put together with mocimycin, used directly for the dehydration of dihydromocimycin without prior isolation of the individual compounds v / er den-.

The examples illustrate the invention.

Example 1 Production of Dihydromocimycin

Streptomyces ramocissimus CBS 190.69 is made in 2000 liters of one Culture medium containing 20 g of malt paste and 10 g of yeast extract per liter and contains 5 g of pesticides extracted from corn steep liquor, grown at a pH of about 7 with stirring and aeration. After cultivation, the medium becomes about 2 percent Dicalite (expanded perlite, aluminum silicate with a content of potassium, sodium and trace elements) as a filter aid offset. The mixture is then filtered. The filtrate will

609848/0999

COPY

acidified to pH 6.0 with 8 η sulfuric acid and extracted twice with a fifth of its volume with methyl isobutyl ketone (MIBK). The emulsions formed are broken with Hyflo Supercel filter aid (a diatomaceous earth product). The organic liquids are mixed and concentrated under reduced pressure in a rotary evaporator to a volume of about 1 liter optionally ⁰ to 60 g. the precipitated crude product is filtered off with a glass filter (G 3). the residue is washed with fresh petroleum ether and dried. this gives a yellow-colored powder which contains MOCIMYCINE and Dihydromocimycin.

Purified dihydromocimycin with a mocimycin content of at most 5 percent is obtained in the following manner: Gaseous ammonia is introduced into the concentrate for 1 minute at a temperature of 2 ° C. at a rate of 150 liters / liter of concentrate / hour. This creates a precipitate that contains mocimycin. The filtrate obtained after filtering off the precipitate is treated with gaseous ammonia for a further 10 to 15 minutes. The precipitate that has now formed is filtered off and dissolved in dilute ammonia solution with a pH of 9.0. The resulting solution is extracted with an equal volume of methylene chloride. The extract is poured into 3 to 5 times the volume of cyclohexane. The precipitate obtained is filtered off, dried and pulverized.

COPY 609848/0999

2621 © 15

Dissolves for the preparation of the sodium salt of dihydromocimycin one dihydromocimycin in water and mixed with 0.1 η sodium hydroxide solution to pH 9j whereby a saturated solution is formed. The solution is filtered off and, with the addition of Butanol evaporated under reduced pressure at about 45 G under azeotropic conditions. The butanol residue is in a small amount of anhydrous butanol added. Petroleum ether is then added dropwise to the solution with stirring until the salt has completely precipitated. The precipitate is filtered off, washed and dried. The E sodium salt is obtained of dihydromocimycin. Other salts of dihydromocimycin can also be prepared in an analogous manner.

Example 2

Production of dihydromocimycin in higher yields In order to achieve higher yields of dihydromocimycin, a lyophilized culture of Streptomyces ramocissimus GBS 190.69 or a well-sporulated agar culture of this microorganism is inoculated into a 500 ml Erlenmeyer flask containing 100 ml of sterilized medium of the following composition contains: 20 g malt paste, 10 g yeast extract and 5 g solids from maize source liquid per liter of tap water (pH value 7 _} 0).

After 3 days of incubation at 3O ⁰ G on a rotary shaker'vorrichtung (300 U / min, stroke 2.5 cm), the culture obtained in small fermenters, 2000 ml of the above medium plus 20 mg _CoCl? .6Hp0 contained per liter, inoculated. In order to promote the formation of dihydromocimycin, this

609848/0999

Growth phase ^{carried out at 3O 0} C with very good ventilation. For this purpose, at least 2 liters of sterilized air per minute are introduced at a stirring speed of 1000 rpm. The formation of dihydromocimycin begins about 12 hours after the start of cultivation and reaches its maximum after about 120 hours. Larger scale cultivation is possible by using a culture grown in the small fermenter for 48 hours as an inoculum for large fermenters.

The fermentation liquid is converted into dihydromocimycin in the following way won: Bach added 2 percent diatomaceous earth as a pilgrim aid the culture is filtered off. The filtrate is acidified to pH 5 to 6 with 8 η sulfuric acid and twice with one fifth of its volume extracted with MIBK. One if necessary The emulsion formed is broken by filtering the mixture after adding diatomaceous earth. The organic phases are collected and concentrated under reduced pressure to about one tenth of the original volume. Then will Gaseous ammonia was introduced into the piltrate for 1 minute at a rate of 150 liters / liter concentrate / hour. The precipitate formed is filtered off. The PiItrat is treated with gaseous ammonia for a further 10 to 15 minutes. The resulting precipitate is dissolved in dilute ammonia solution with a pH of 9.0. By extracting the ' Ammonia solution with an equal volume of methylene chloride becomes purified dihydromocimycin (containing mocimycin of a maximum of 5 percent). The extract is poured into 3 to 5 times the volume of cyclohexane. The precipitation will

609848/0999

- 20 filtered off, dried and powdered.

Example 3

Effect of dihydromocimycin against treponemal dysentery 20 3-month-old pigs are infected with the causative agent of treponemal dysentery by giving them a feed with a homogeneous mixture of intestinal contents and intestinal mucosa from 2 animals suffering from this disease. The infected pigs are divided into 4 groups of 5 animals each. For 1 week, the animals receive a pulpy feed diluted with water in a ratio of 1: 1. The total amount of feed per animal and day is 1.2 kg and is given in 2 portions.

! After 5 days, the first symptoms of the disease appear in the form of thin droppings. The disease can be confirmed with a microbiological feces investigation. After 1 week the Animals treated as follows:

Group 1: No addition of antibiotics to the feed; Group 2: addition of 100 ppm tylosin to the feed; Group 3: addition of 25 ppm dihydromocimycin to the feed; Group 4s addition of 50 ppm dihydromocimycin to the feed.

The feed, optionally enriched with antibiotics, is fed for 1 week. Then normal food is again given without the addition of antibiotics. To determine whether the animals have recovered from the infection, body weight is used determined and the feces macroscopically for its texture and microscopically according to a special immuno-

609848/0999

fluorescence method investigated.

During the examination, 1 animal from groups 1, 3 and 4 is perished. The test results are compiled in the following table. The weight data are average weights of the animals living at the respective point in time. The quality of the manure is assessed as follows:
+ Thin; + thick; - normal.

During the immunofluorescence study, the number of treponemas indicated in the field of vision as follows:

very close

numerous

about 10 treponemes

1 or 2 treponemes

several fields of view are necessary to find 1 treponema negative.

609848/0999

2621815

2. Woehe (1) .Group 1 Group 2 24.4 + + + + - 4434- Group 3 + x group 4th 3 -Weeks Weight in kg 22.2 + + + + ^ -t- 4 4 2 2- + Weeks 24.6 21.4 +, + +, +, i 3 - - 2 - 24.2 23> 4 just before
the / infection 1 23.7 23.2 .Imm.un.ofluore s cence 21 j3 21.6 after _2 week 20.9 Excrement quality 4 4 4 4 5 20.2 5 χ 21.2 after: * 3 Weeks 19.8 + + + + + 4 4 4-4 24.0 1 23.0 -after Weeks + + + + + 2 2 2 25? 1 1 + ± + + χ + + + + + + + + after 2 Where before ± ± ± + + X + + ·. after .3 Weeks - -f + - after Weeks 5 4 5 5-53 3 _ after 4 4 3 2 - χ 4 1 after _ _ _ - . after

(1): start of antibiotic treatment χ: an animal dies

From the quality of the faeces and from the immunofluorescence examination it turns out that those treated with dihydromocimycin Animals recover much faster than those treated with tylosin Animals. If the amount of antibiotic administered is also taken into account, it can be assumed that Dihydromocimycin is at least 4 times as effective as tylosin »

Example 4

Effect of dihydromocinrycin against treponemal dysentery Pigs are treated under veterinary supervision in a breeding facility where the pigs suffered from severe diarrhea. The pigs are divided into 4 groups and treated for 4 days in the following way:
Group 1: 66 pigs received putters with 100 ppm tylosin; Group 2: 34 pigs received feed with 25 ppm dihydromocimycin;

609848/0999

Group 3 'x 40 pigs received! Feed with 50 ppm dihydromocimycin;

Group 4: 40 pigs are fed a diet with 100 ppm dihydromocimycin.

The faeces of all animals before the start of treatment is thin or very thin. Faecal samples turn out to be Treponema-positive and Salmonella-negative. Occasionally, worm eggs are found.

1 day after the start of treatment, the faeces of the pigs are from Group 4 normal. The animals of group 1 are only cured after 3 "to 4 days. In the animals of groups 2 and 3, the Treatment success after a period between the corresponding Group 1 and Group 4 times. The animals treated with dihydromocimycin make a livelier one Impression than before the treatment. The animals show no aversion to putters containing dihydromocimycin. Overall it can be stated that for the treatment of treponemal dysentery a dose of 25 ppm dihydromocimycin is more beneficial than a dose of 100 ppm tylosin.

Example 5 Dehydration of Dihydromocimycin

A solution of 1 g of dihydromocimycin in 15 ml of HMPT (technical Product dried over a molecular sieve 3 A) is with 139 mg

(1.25 mmol), added selenium dioxide. The mixture is 100 minutes warmed on a steam bath and added another 139 mg selenium dioxide after 60 minutes. After cooling down

- 609848/0999

precipitated selenium is filtered off through a G4 glass filter. The precipitate is washed with a small amount of methanol. The filtrate is poured into 350 ml of distilled water. The precipitate is filtered off and washed with distilled water. The piltrate is kept.

The precipitate is dissolved in methanol and diluted with MIBK. The solution is ^{evaporated at 40 0} G under reduced pressure to remove methanol and water. Thin-layer chromatography shows that the precipitate does not contain any mocimycin and only consists of polar impurities. The precipitate is filtered off and washed with MIBK. The Piltrat is treated dropwise with an excess of petroleum ether of boiling range 40 to 60 ⁰ C. The precipitate formed is filtered off, washed with petroleum ether and dried to give 500 mg of product.

The stored Piltr t is extracted with MIBK. The extract is added dropwise to petroleum ether. A further 100 mg of product of the same quality are obtained. The total yield is 650 mg. Thin-layer chromatography can be used to determine that the end product consists of mocimycin, which contains only traces of dihydromocimycin.

Example 6 Dehydration of Dihydromocimyein

This experiment is carried out in a double approach. 2.4 g of a mass containing $ 22.8> mocimycin and

60984 8/09 99

35 »5 percent dihydromocimycin is dissolved in 50 ml of HMPT. 350 mg of selenium dioxide are then added. The mixture is stirred at 105 ° C. for 45 minutes. Through a thin layer chromatographic Examination shows that dihydromocimycin is no longer present. The G-emic becomes on Cooled to room temperature and to remove black selenium formed and a small amount of unreacted Selenium dioxide filtered through a G4 glass filter.

The precipitate is washed with MIBK. Mitrat and MIBK washing liquid are combined and mixed with 350 ml of water, 10 g of sodium chloride and 5 ml of 4 η hydrochloric acid. The mixture is extracted 3 times with 100 ml of MIBK each time. The combined MIBK extracts are washed with 100 ml v / ater. The water phase is removed and the MIBK phase is dried over anhydrous magnesium sulfate. The magnesium sulfate is washed with a small amount of MIBK. The MIBK layer was then concentrated ml under reduced pressure at about 2O ⁰ G to a volume of 25th The residue is treated dropwise with stirring with petroleum ether of boiling range 40 to 6O ⁰ C. The precipitate formed is filtered off, washed with petroleum ether of boiling range 40 to 6O ⁰ · G, and dried overnight at room temperature under reduced pressure.

The yield in the first batch is 1.64 g of product with a content of 41 »2 percent, mocimycin and < 2 percent dihydromocimycin (determined by high pressure liquid chromatography).

609848/0999

In the second batch, the yield is 1.79 g of product containing 41 »6 percent mocimycin and £ 1 percent dihydromocimycin.

Example?

Influence of Solvents in the Dehydrogenation of Dihydromocimycin The influence of various solvents on the dehydrogenation of dihydromocimycin is investigated. In each case, 50 mg of a technical product containing Dihydromocimycin and MOCIMYCINE with 20 mg (0.18 mmol) of selenium dioxide reacted in 3 ml of solvent at a temperature of 80 ⁰ C. Samples are taken 2, 4 and 7 hours after the start of the experiment and examined by thin layer chromatography on silica gel 60 discs (Merck) using a 50: 45: 5 mixture of MIBK, acetone and water as the mobile phase. The verification is carried out by spraying on a mixture of sulfuric acid and diethyl ether. This allows the course of the reaction to be followed qualitatively.

Decomposition occurs when the following solvents are used or no conversion: propargyl alcohol, diacetone alcohol, nitromethane, propylene carbonate, acetonitrile, pyridine, suIfolane, Benzyl alcohol, mesityl oxide, ethylene carbonate, H-methyl-2-pyrrolidone, IT, U-Dimet hy! Acetamid, dioxane, MIBK, Buty lace tat, amyl acetate, IT-Me thy! Acetamide, anisole, diethylene glycol methyl ether and 1,2-dichloroethane. A low degree of conversion is obtained with Use of tetramethylurea, 0.1 M UaHGO ^ / iTapCO ^ buffer of pH 9j1j n-propanol, phenylmethylcarbinol, water and Dimethylformamide. Reached a slightly better degree of implementation

609848/0999

when using τοη ethylene glycol monomethyl ether, isopropanol, n-butanol, hexylene glycol, sec-butanol and tert-amyl alcohol. A moderate conversion is achieved in tert-butanol and dimethyl sulfoxide. The best results will be when using received from HMPT.

Examples

Dehydration of Dihydromocimycin in Solvent Mixtures The suitability of solvent mixtures with HMPT as a constituent is being investigated. In each case 2.4 g of the starting material from Example 7 are used. After the conversion of all of the dihydromocimycin determined by thin-layer chromatography, the reaction mixture is worked up as follows: After cooling, the selenium formed is filtered through a D4 glass filter and washed with a small amount of methanol. The filtrate is poured into excess water and acidified to pH 3. It is then extracted 3 times with MIBK in an amount of a quarter of the volume each time. A tar-like intermediate layer is extracted with MIBK by first dissolving it again in methanol and then diluting with MIBK and water. The combined MIBK extracts are washed 3 times with water in an amount of one third of the volume each time. The organic phase is concentrated at about 40 ° C. under reduced pressure. In all cases the precipitate consists essentially of decomposition products, as can be shown by thin-layer chromatography. The precipitate is filtered off and

• 609848/0999

washed with MIBK. The combined MIBK concentrates are added slowly with stirring to an excess of petroleum ether from ¹ Siedebereic 10 to 6O ⁰ C. The precipitate formed is filtered off, washed with petroleum ether and dried. The results are compiled in the following table:

609848/0999

Amount of
Solution
middle leactions
fcemperatu
(° G) - Entire
r reaction
Time
(Hours.) SeO ^ addition
in g;
Time after
Reaction
beginning .usage
'Weight
arocent) Moles of SeO ₂
per mole
mixture HMPT 25 ml
H ₂ O 35 ml 90 2 0.5 at the beginning
+0.5 after
55 minutes 42 3: 1 HMPT 15 ml
0.1 m
NaHCO ^ -
Ka ₉ CO ^
Puffei pH
9.1 25 ml 90 2 3/4 0.1 at the beginning
0.1 after 20
Minutes
0.1 after 85
Minutes
0.1 after 130
Minutes 50 1.2: 1 t-BuOH 20
ml
BMPT 10 ml 84 3 0.1 at the beginning
0.1 after 25
Minutes
0.1 after 45
Minutes
0.1 after 75
Minutes
0.1 after 120
Minutes 42 1.5: 1 t-AmOH
100 ml
HMPT 50 ml 96 3 0.35 at the beginning
0.35 after 70
Minutes
0.35 after 110
Minutes 71 3.2: 1 t-AmOH
150 ml 96 3 0.35 at the beginning
0.35 after 75
. Minutes
0.35 after 150
Minutes 55 3.2: 1 t-BuQH
100 ml *
HMPT 50 ml 80 2 1.0 at the beginning
1.0 after 35
Minutes 42 6: 1 HJiEPT 150
ml 95 end of
Reaction
time after
1 H. 0.4 at the beginning 79 1.2: 1

HMPT = hexamethylphosphoric triamide t-BuOH = tert-butanol t-AmOH = tert-amyl alcohol

609848/0999

The table shows that the highest yield (79 percent) is obtained when using pure HMPT as a solvent. A high yield is obtained even if it is used a mixture of HKPT and tertiary amy alcohol.

609848/0999

Claims (1)

Claims 1 ♦ / Dihydromocimycin of the formula HO OH and its salts. .2. Alkali metal, ammonium or amine salts of dihydromocimyein according to claim 1. 3. A method for the preparation of JDihydromocimycin according to claim !, characterized in that one Streptomyces ramocissimus CBS 190.69 or a dihydromocimycin forming mutant of this strain in an "aqueous Uährmedium, that contains assimilable sources of carbon and nitrogen and inorganic substances, breeds and that in breeding separates formed dihydromocimycin. 4. The method according to claim 3 »characterized in that that one can start breeding at a ventilation speed from 1 to 3 liters of air per 2 liters of nutrient medium and per minute. 609848/0999 5. The method according to claim 3, characterized in that the cultivation is carried out at a ventilation rate of 1.5 to 2.5 liters of air per 2 liters of nutrient medium and per minute. 6. The method according to claim 3, characterized in that that the cultivation in the presence of low concentrations of iron, cobalt or Uickelionen performs. 7. The method according to claim 3 _5, characterized in that the mocimycin is separated off with the aid of alkaline compounds such as ammonia, llatriummethylat or triethylamine. 8.Feed for pigs effective against treponemal dysentery, characterized in that they contain at least one compound according to claim 1. 9. Feed according to claim 8, characterized in that that there is about 10 to about 200 ppm of a compound according to claim 1, based on the weight of the feed, contains. 10. Feed according to claim 8, characterized in that it contains 20 to 40 ppm of a compound according to claim 1, based on the weight of the feed. 609848/0999 11. Medicinal preparation for the treatment of Treponema diseases in pigs, characterized by a content on a connection according to claim 1. 12. Process for the dehydration of dihydromocimycin too Mocimycin, characterized in that dihydromocimycin is reacted with selenium dioxide. 13. The method according to claim 12, characterized in that that a mixture of mocimycin and dihydromocimycin is used as the starting material. 14. The method according to claim 12, characterized in that that the reaction is carried out at elevated temperatures. 15. The method according to claim 12, characterized in that one carries out the reaction at temperatures from about 65 to about 110 G ^0. " 16. The method according to claim 12, dadur. ch marked, characterized in that one carries out the reaction at temperatures of 80 to 95 ⁰ C. 17. The method according to claim 12, characterized in that that the reaction in Ilexamethylphosphortriamid, Dimethyl sulfoxide, tert-butanol, tert-amyl alcohol, sec-butanol, hexylene glycol, n-butanol, isopropanol, ethylene 609848/0939 Glykolmoiiometliylätlier, dimethylformamide, water, phenylmethylcarbenol, propanol or mixtures of two or more of these
Solvent carries out. 18. The method according to claim 12, characterized in that that the reaction in hexamethylphosphoric triamide, Dimethyl sulfoxide or tert-butanol carries out. 19 «Method according to claim 12, characterized in that that the reaction in hexamethylphosphoric triamide performs as a solvent. 609848/0999