DE2621615B2 - DIHYDROMOCIMYCINE, ITS SALT, METHOD FOR ITS MANUFACTURING AND ITS USE AS AN ADDITIVE TO FEED - Google Patents

Description

The invention relates to the subject matter characterized in the claims.

From the DT-OS 2140 674 is a method for Production of mocimycin known, which is characterized in that one microorganisms of the Strain Streptomyces ramocissimus (CBS 190 69) or obtained therefrom by customary methods, the Antibiotic-producing mutants or variants in an aqueous, usable carbon and nitrogen source and culture medium containing inorganic salts and the resulting antibiotic isolated from the fermentation mash by extraction with organic solvents at pH values of about 5 to 8 and, if desired, further cleaning. The structure of mocimycin was derived from V ο s and V e r w i e 1, Tetrahedron Letters, 52: 5173 to 5176 (1973).

It was found according to the invention that microorganisms of the strain Streptomyces ramocissimus (CBS 190 69) or by customary methods obtained therefrom, the antibiotic-forming mutants or Variants when growing in an aqueous, utilizable carbon and nitrogen sources and Nutrient medium containing inorganic salts not only for the formation of mocimycin but also for the formation of Dihydromocimycin are capable.

Dihydromocimycin is a weak acid and forms salts, e.g. B. alkali metal, ammonium and amine salts. It has the following physical and 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.

ίο Optical rotation:

[α] = -85 ° (1 percent solution in methanol).

Melting point:

The connection does not melt. From 123 ° C occurs - a ¹⁵ decomposition.

Elemental analysis:

C ₄₃ H ₆₂ N ₂ O, ■ 2 H ₂ O
Ben:

• »° C 61.8, H 8.0, N 3.3, O (difference) 26.8;
gel'.:
C 61.8, H 7.5, N 3.4, O (difference) 27.2.

UV spectrum:

The UV spectrum of dihydromocimycin in a 1: 1 mixture of water and methanol at various pH values is shown in FIG. 1 reproduced. The concentration of the solutions is 18 ^ g / ml. 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 (ε = 18,000);

Curve 1;
Methanol -0, Sn-NaOH:

235 nm (ε = 62,000); 277 nm (ε = 25,000) and

308 nm (ε = 29,000); Curve 2;
Methanol - 0.5 n-HCl:

233.5 nm (ε = 65,000); 268 nm (ε = 25,000) and

338 nm (f = 14,000); Curve 3.

1R spectrum:

The IR spectrum of dihydromocimycin in chloroform and as a potassium bromide pellet is shown in FIG. 2 or 3 reproduced. The most important absorption maxima in chloroform are ± 3445 (sh), 3430, 2979, 2941,

26th

2161

»886, 1662-1653, 1458, 1100, 1080, 1040, 998, 944, 893, } 7O and 840 cm '. They are in the potassium bromide pellet \ absorption maxima at ± 3400-3320,2972,2935,2880, 1650, 1535, 1455, 1099, 1040, 990, 943, 86Ü, 840 and 790 cm- '. s

PMR spectrum:

The PMR spectrum of dihydromocimycin in deuterochloroform using tetramethylsilane as an internal standard is shown in Fig. 4 (60MHz) ι ο reproduced.

Thin layer chromatography:

The thin-layer chromatogram of dihydromocimycin is recorded on silica gel F254 plates. After drying, the stains are determined by fluorescence, absorbance measurement or charring detected after spraying with a mixture of diethyl ether and sulfuric acid. Also highly purified Preparations of dihydromocimycin give, in addition to a main spot, a small additional spot. this is due to a tautomeric equilibrium, such as by a two-dimensional chromatogram can be shown.

The following R ₍ values for dihydromocimycin in various solvents are obtained (the R, value for the additional stain is given in round brackets):

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, water and 25 percent ammonia solution: 0.29 (0.36);

65:40: 9 mixture of 100 percent benzene 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 (cf. 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 appearing at O = 5.9 and 7.3 ppm (tetramethylsilane is used as the standard) in the spectrum of mocimycin and absent 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 dihydromociriiycin, on the other hand, there are two triplets at ό = 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 carried for 5 minutes ozonation an aqueous solution of Dihydromocimycin at 0 ⁰ C and f> o confirm a pH of 12th After reduction of the reaction mixture obtained with hydrogen using PtCh as a catalyst and after hydrolysis with concentrated hydrochloric acid, /} - alanine can be detected, which suggests that the bond between the fifth and sixth carbon atom of the pyridone chain of dihydromocimycin is dihydromocimycin similar in many properties to mocimycin. An important difference between these two compounds, however, is that mocimycin as a feed additive in small amounts in animals such as chickens and pigs causes weight gain and an increase in size growth and feed conversion. In contrast, dihydromocimycin does not lead to increased growth or improved feed conversion in these animals. This is surprising since both compounds are active in vitro against the same microorganisms. In many cases, dihydromocimycin is even more effective than mocimycin. The MIC values of the two antibiotics against various germs are summarized in Table I (agar dilution test) and Table II (tube dilution test).

Table 1

20th Test germ MIC ₁ : 'g / rr il nl Mocimycin Dihydro Dihydro mocimy- mocimy cin cin Staphylococcus aureus A 2000 > 100 > 100 Staphylococcus aureus A 2001 > 100 > 100 Diplococcus pneumoniae L 54 1.5 <0.75 Salmonella typhimurium Rl 72 > 100 > 100 Escherichia coli U 20 > 100 100 3 ° Listeria monocytogenes A 2130 6th 1.5 Listeria monocytogenes A 2131 6th 6th Listeria monocytogenes A 2132 6th 6th Clostridium perfringens A 738 > 100 > 100 Clostridium septicum A 2152 10 10 35 Streptococcus zooepidemicus 6th 6th A 2144 R-streptococci A 2148 6th 3 Brucella suis (smooth) A 2126 0.75 0.4 Pasteurella haemolytica A 2136 3 1.5 40 Treponema spec. A 2275 30th 10 Table II Test germ MIC, μξ / χ] 45 Mocimycin

Bacillus subtilis ATCC 6633 100 50

Bacillus subtilis ATCC 6051 100 50
Bacillus subtilis 6346 Dl 67 1.2 0.9

Bacillus subtilis 220 Dl 78 75 75

Bacillus subtilis TH 10 100 50
Bacillus ceraus Dl 66 0.6 0.6

Bacillus cereus D 261 0.9 0.6

Bacillus cereus D 220 1.2 0.9

Bacillus cereus B 569 2.5 1.2

Bacillus cereus TH 1 1.8 1.2

Bacillus thuringiensis W 11 1.2 0.9

Bacillus mesenterium D 169 100 50
Bacillus cereus var. Rnycoides 1.2 0.45

Streptococcus haemolytieus 0.45 0.45

A 266

Streptococcus haemolyticus 0.25 0.12

A 2182

Mycoplasma hyorhinus A 2230 1 0.3

Streptotnyces viridochromogenes 2.5 0.9

The microorganisms can be cultivated in liquid nutrient media which contain 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, corn steep liquor solids, and yeast extract can be used. The cultivation temperature is 20 to 40 ⁰ C, preferably 26 to 34 ° C, and the pH 5 to 9, preferably from 6.5 to eighth

Surprisingly, the yield of dihydromocimycin can be compared to the yield of Increase mocimycin by increasing the oxygen partial pressure in the nutrient medium, from the structure of Dihydromocimycin can be assumed to be due to a higher oxygen partial pressure in the nutrient medium the yield of dihydromocimycin decreases compared to mocimycin. However, the aforementioned occurs Effect on dihydromocimycin compared to mocimycin through better aeration of the nutrient medium is formed in larger quantities. The ventilation can be carried out in a manner known per se. For example, the ventilation speed (part by volume of air per part by volume of nutrient medium per Time unit) or the agitation speed of the nutrient medium in the fermenter is increased. Appropriate ventilation speeds of the nutrient medium, the volume of which is 2 liters, for example, are 1 to 3 liters Air, preferably 1.5 to 2.5 liters of air, per minute. Another improvement in the yield of dihydromocimycin can be by adding certain metal salts in low concentrations, for example by adding iron, cobalt or nickel salts to the nutrient medium.

The differences in solubility of these compounds in an alkaline medium are used to separate dihydromocimycin from mocimycin. When gaseous ammonia is introduced into a solution obtained by extracting a culture liquid acidified to a pH of 5 to 6 with methyl isobutyl octone, the mocimycin initially precipitates practically completely. The dihydromocymycin remains practically completely in solution. After the mocimycin precipitate has been separated off, for example by filtration, 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 (ie about 2.5 to about 10 liters of gaseous ammonia per liter of solution) at a temperature of about -12 to about + 15 ° C, preferably -5 to +8 ⁰ C, initiated. Further introduction of gaseous ammonia decreases the hydrogen ion concentration to such an extent that dihydromocimycin becomes insoluble. For example, under the aforementioned conditions, a period of 10 to 15 minutes is introduced (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 off in this way can carry impurities on mocimycin be cleaned by putting the precipitate in a

ίο

strongly diluted ammoniacal solution of pH 9 dissolves and this solution with a Solvent such as chloroform or methylene chloride extracted. In such solvents there is mocimycin only slightly soluble. The extract obtained is poured into a non-polar one that is present in excess Solvents, such as petroleum ether, cyclohexane or pentane, form a dihydromocimycin precipitate, which contains less than 5% mocimycin.

Highly purified dihydromocimycin is obtained by chromatography on three-dimensionally cross-linked Dextran, using the adsorption differences between mocimycin and dihydromocimycin. Included the dextran is suspended in 100% methanol and carefully poured into the column. To Displacement of the methanol by chloroform, the dihydromoemycin precipitate is applied to the column. Chloroform is used as the eluent. This initially gives dihydromocimycin and then mocimycin. Both compounds can be detected in the eluate because they are in the UV range absorb at 350 nm. The pure compounds can be extracted from the chloroform solution by precipitation with a non-polar solvent such as cyclohexane or pentane can be obtained.

The identity of the compounds can be determined by thin layer chromatography confirm. For this purpose, silica gel F 254 plates measuring 20 × 5 cm are used used. A 60:42:10 mixture is used as the mobile phase from chloroform, ethanol and 25 percent ammonia. The running time is 2 hours. Mocimycin shows an Rr value of 0.3 (main tautomer) and dihydromocimycin of 0.4 (main tautomer).

Based on previous research, it was believed that mocimycin, which is a similar antimicrobial Spectrum of activity like tylosin (see Merck Index, 8th edition, p. 1089), against Treponema hyodysentcriae, the causative agent of Treponema-Dysentric or Vibrio Doyle, one of the most common diseases Pigs, is effective. Studies have shown, however, that the effect of mocimycin is no greater is than that of tylosin.

Tables 1 and II show that dihydromocimycin against a number of Problcmkcimctii is more effective than mocimycin. When investigating the effectiveness against the pathogen of Treponcma dysentery, it has been found that dihydromocimycin is active against this microorganism is much more effective than tylosin and is also effective against tylosin-resistant strains Somii is dihydromocimycin for the treatment of treponemal dysentery tylosin and therefore also mocimycin think.

The invention thus also relates to the use of dihydromocimycin or its salts, for example the sodium salt, as a feed additive for pigs. The antibiotic or its alkali metal ammonium or amine salt can also be dispersed or mixed in a corresponding inert, physiologically harmless carrier or diluent which can be administered to pigs ora without reacting to the antibiotic and is harmless for pigs when administered orally of dihydromocimycin, which are added to the feed for prophylaxis or therapy of treponemal dysentery, are around 10 bi! about 200 ppm, preferably 20 to 40 ppm. based on the feed weight.

(P

The dihydromocimycin obtained according to the invention is a fine, easily dusting powder. Since this is under Circumstances that can lead to difficulties in mixing with the feed, preferably one Premix made with one or more components of the pig feed. This premix contains for example 9 to 99 times the desired amount of dihydromocimycin. Premix can for example, be made with corn flour, potato flour or soy flour. Have investigations show that dihydromocimycin also dissolves when pelletized; H. Granulating under high pressure and high temperatures using steam, proves stable.

When pigs are so affected by treponemal dysentery that they have lost their appetite or are pushed away from the feeding site by healthy animals, dihydromocimycin is preferably administered with the Drinking water administered, preferably together with a taste-improving agent. This will be Dihydromocimycin used in water-soluble form, for example as the potassium, sodium or amine salt.

Very dysentery pigs can be given injections of dihydromocimycin or a appropriate water-soluble 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.

The examples illustrate the invention.

example 1

Microorganisms of the Slrepiomyccs ramocissimus strain (CBS 190 69) are in 2000 liters of a nutrient medium that 20 ι per liter; Barley malt paste, Contains 10 g corn extract and 5 g corn steep liquor, at a pH wide of about 7 with stirring and Ventilation gc / .üchtct. After cultivation, the cooling liquid with about 2% expanded perlite is used as Filter aid added and filtered. The filtrate is unacidified with 8 N-Schwcfclsäurc to a pH value of b.0 and extracted twice with one fifth of its volume of methyl isobutyl octone (MIBK). The resulting Emulsions are broken with diatomaceous earth as a filter aid. The organic phases will combined and under reduced pressure in a rotary evaporator to a volume of about 1 liter constricted. The concentrate is slowly added to 5 liters of petroleum ether (boiling range 4ü to W) "C). The The resulting precipitate is filtered off with a glass filter frit (G 3). The filter residue is with washed and dried with fresh Petroiather. A yellow colored powder is obtained which contains mocimycin and Contains dihydromocimycin.

Purified dihydromocimycin with a mocimycin content of at most 5 percent is obtained as follows: Gaseous ammonia is passed into the concentrate for 1 minute at a temperature of 2 "C at a rate of 150 liters / liter of concentrate / hour. The filter 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 of pH 9.0 The extract is poured into 3 to 5 times the volume of cyclohexane

.n>

The precipitate obtained is filtered off, dried and pulverized.

To prepare the sodium salt of dihydromocimycin, dihydromocimycin is dissolved in water and 0.1 η sodium hydroxide solution is added to pH 9, a saturated solution being formed. The solution is filtered off and, with the addition of butanol, ^{evaporated under reduced pressure at about 45 °} C. under azeotropic conditions. The butanol residue is taken up in a small amount of anhydrous butanol. Petroate is then added dropwise to the solution with stirring until the salt has completely precipitated. The precipitate is filtered off, washed and dried. The sodium salt of dihydromocimycin is obtained. The ammonium salt and amine salts of dihydromocimycin can also be prepared in an analogous manner.

Example 2

A lyophilized culture of microorganisms of the strain Streptomyces ramocissimus (CBS 190 69) or a well-sporulating agar culture of these microorganisms is placed in a 500 ml Erlenmeyer flask inoculated, which contains 100 ml of a sterilized nutrient medium of the following composition: 20 g of barley malt paste, 10 g yeast extract and 5 g corn steep liquor solids per liter of tap water (pH 7.0).

After 3 days incubation at 3O ⁰ C to a Drchschüttelmaschine (300 U / min, stroke 2.5 cm) was inoculated the culture obtained in small fermenters, the 2000 ml of the nutrient medium and an additional 20 mg CoCl: · fc H: O per liter . To improve the formation of dihydromocimycin, this growth phase is ^{carried out at 30 D} 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. Cultivation on a larger scale is possible by using a culture grown for 48 hours in the small fermenters as inoculum for large fermenters.

The Kultuiflüssiykeil becomes Dihydromocimycin aiii Obtained in the following way: After adding 2 ° / c Diatomeenirdc as a filter aid, the cooling liquid is filtered off. The filtrate is acidified with 8N acidified to a pH value of Γ »to b and twice extracted with a fifth of its volume of MIBK.

Any emulsion that may have formed is broken up with diatomaceous earth as a filter aid. The organi see phases are united and under reduced pressure to about one tenth of the original! Confined in volume. Gaseous ammonia with a gel is then poured into the filtrate for a minute speed of 150 liters / liter of concentrate / hour initiated. Film the resulting precipitate. The filtrate is mi for an additional 10 to 15 minutes treated with gaseous ammonia. The resulting precipitate is in dilute ammonia solution of pH 9.0 dissolved. By extracting the ammonia oil solution with an equal volume of methyl chloride in purified dihydromocimycin (with a content ai Mocimycin of 5% or less). The Iraq is poured into 3 to 5 times the volume of cyclohexane The resulting precipitate is filtered off, gel-dried and pulverized.

709 627/6

example

10

Effect of dihydromocimycin against treponemal dysentria

20 pigs at the age of 3 months are infected with the causative agent of treponemal dysentria by gives them a feed that has a homogeneous mixture of intestinal contents and intestinal mucosa of 2 on this Is mixed with animals suffering from disease. The infected pigs are divided into 4 groups of 5 each Animals. For 1 week, the animals received a paste-like substance diluted with water in a ratio of 1: 1 Lining. 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 feces. The disease can be confirmed by a microbiological examination of the faeces. After 1 week, the animals are treated as follows:
Group 1:

No antibiotics added to the feed;
Group 2:

Addition of 100 ppm tylosin to feed;
Group 3:

Addition of 25 ppm dihydromocimycin to feed; Group 4:

Addition of 50 ppm dihydromocimycin to the feed. The optionally enriched with antibiotic Feed is fed for 1 week. Then normal feed is returned to without the addition of antibiotics given. To determine whether the animals have recovered from the infection, body weight is used found and the feces macroscopically on his

ίο texture and microscopic according to a special Investigated immunofluorescence methods.

During the investigation, 1 animal in each of groups 1, 3 and 4 dies. The test results are in Table 111 summarized. The weights are

is the average weight of the animals living at the time. The quality of the manure is assessed as follows:
+ Thin; ± thick; - normal.
In the immunofluorescence examination, the

: o Number of treponemas in the field of vision stated as follows:
5: very dense,
4: numerous,
3: about lOTreponemas,

; s 2: 1 or 2 treponemes,

1: several fields of view are necessary to 1

To find treponema
-: negative.

Table III Group i I. in kg + H- H- 4th 4th S. Group 2 H- H- H- Group 1 H- H- H- group 4th H- + H weight H H- H- 4th - 4th H- H- ± ± ± -1- X + H 24.6 ± + X 2 2 X 24.4 + ± ± 24.2 - ± ± 23.4 + - Just before Immunofluorescence the infection 23.7 4 4 22.2 4th 3 4th 21.3 4th 5 5 21.6 S. 3: After 1 week ¹ ) 20.9 4 4 21.4 4th 2 2 20.2 4th 3 1 21.2 χ 4 1 After 2 weeks 19.8 2 2 23.2 - - 2 24.0 - - 1 23.0 _ After 3 weeks •1- . ₍ .. X + -1- After 1 week H- -I- ± H- H- After 2 weeks ± ± - - - After 3 weeks 4th - 5th X S - After 1 week 4th - 4th After 2 weeks 3 - - After 3 weeks KolbschalTenhcit + + HI- H- ±

'): Start treatment with antibiotics.
x; An animal dies.

The quality of the faeces and the immunofluorescence examination show that the animals treated with dihydromocimycin are much faster get well than animals treated with tylosin. One also takes into account the amount of administered Antibiotic, it can be assumed that dihydromocimycin is at least 4 times as effective how tylosin is.

Example 4

Effect of dihydromocimycin against Treponemal Dysentria

Under veterinary supervision pigs are It a breeding establishment, In which the pigs at heavy Suffered from diarrhea, treated. The pigs are in <

Divided into groups and treated for 4 days in the following way:

Group 1:

66 pigs received feed with 100 ppm tylosin;

Group 2:>

34 pigs are fed a diet containing 25 ppm dihydromocimycin;

Group 3:

40 pigs are fed with 50 ppm dihydromocimycin; to

Group 4:

40 pigs are fed with 100 ppm dihydromocimycin.
The faeces of all animals before the start of treatment is

thin or very thin. Fecal 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 in group 4 are normal. The animals from group 1 are only healed after 3 to 4 days. In the animals of groups 2 and 3, the treatment success occurs after one Time that lies between the corresponding times of Group 1 and Group 4. The ones with dihydromocimycin treated animals make a livelier impression than before the treatment. The animals do not show any Aversion to feed containing dihydromocimycin. Overall it can be stated that For the treatment of Treponena Dysentria, a dose of 25 ppm dihydromocimycin is cheaper than one Dose of 100 ppm tylosin.

For this purpose 3 sheets of drawings

Claims (4)

Palent claims: 1. Dihydromocimycin of the formula and its salts. 2. A process for the preparation of dihydromocitnycin according to claim 1, characterized in that that one microorganisms of the strain Streptomyces ramocissimus (CBS 190 69) or after customary methods obtained therefrom, the antibiotic forming mutants or variants in one aqueous nutrient medium containing usable carbon and nitrogen cues and inorganic salts cultivates in a manner known per se, the culture broth is filtered off, the filtrate on one acidified pH 5 to 6, the acidified filtrate extracted with methyl isobutyl ketone, the Treated the extract with a base, the precipitated mocimycin and the mother liquor worked up on dihydromocimycin and, if desired, converted into a salt. 3. The method according to claim 2, characterized in that that the extract with the help of ammonia, sodium methylate or triethylamine as Base treated. 4. Use of the dihydromocimycin according to claim 1 as a feed additive.