CH630411A5 - Process for preparing dihydromocimycin - Google Patents

Description

The invention relates to a process for the preparation of the antibiotic dihydromocimycin of the formula

HO OH

S

and its non-toxic, pharmacologically acceptable salts. The alkali metal, ammonium and amine salts are preferred.

The process according to the invention is characterized in that the microorganism Streptomyces ramo-cissimus CBS 190.69 or a mutant of this strain which forms dihydromocimycin is cultivated under aerobic conditions in an aqueous medium which contains assimilable carbon and nitrogen sources and inorganic substances, and that which is formed during the cultivation Separates dihydromocimycin from the mixture of mocimycin and dihydromocimycin obtained. The aforementioned microorganism strain is described in GB-PS 1 325 200. According to this document, mocimycin (MYC 8003) is formed by cultivating this microorganism. The structure of mocimycin was described by Vos and Verwiel, Tetrahedron Letters, Vol. 52 (1973), S.

5173-5176. Streptomyces ramocissimus was deposited on February 19, 1969 with the Centraal Bureau voor Schimmelcultu-55 res in Baarn, Netherlands, under the number CBS 190.69.

It has now been found that this strain of Streptomyces ramocissimus or corresponding mutants of this strain are also capable of forming dihydromocimycin in addition to the formation of mocimycin.

Dihydromocimycin is a pale yellow solid that reacts weakly acidic and can be characterized by the following physicochemical properties:

65 Solubility:

The compound is readily soluble in chloroform, methyl isobutyl ketone, ethyl acetate, butyl acetate, acetone, dioxane, methanol, ethanol, tetrahydrofuran and weak

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.

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

Melting point:

The connection doesn't melt. Decomposition starts at 123 ° C.

Elemental analysis C43H62N2O12.2H2O:

calc .: C 61.8, H 8.0, N 3.3.0 26.8 (difference)

found: C 61.8, H 7.5, N 3.4.0 27.2

UV spectrum:

The UV spectrum of dihydromocimycin in a 1: 1 mixture of water and methanol at different pH values results from FIG. 1. The concentration in solutions is 18.3 ng / 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 (e = 63,000); 267 nm (e = 23,000); 291 nm (e = 19,000) and 333 nm (e = 18,000); Curve 1;

Methanol-0.5 n NaOH: 235 nm (e = 62,000); 277 nm (e = 25,000) and 308 nm (e = 29,000); Curve 2;

Methanol-0.5N HCl: 233.5 nm (e = 65,000); 268 nm (e = 25,000) and 338 nm (e = 14,000); Curve 3.

IR spectrum:

The IR spectrum of dihydromocimycin in chloroform and potassium bromide is shown in Figures 2 and 3, respectively. The following main absorption bands are found:

Chloroform (v in cm-1): ± 3445 (sh); 3430; 2979; 2941; 2886; 1662-1653; 1458; 1100; 1080; 1040; 998; 944; 893; 870 and 840; Potassium bromide (v in cm "1): ± 3400-3320; 2972; 2935; 2880; 1650; 1535; 1455; 1099; 1040; 990; 943; 860; 840 and 790.

PMR spectrum:

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

Thin layer chromatography:

A thin layer chromatogram of dihydromocimycin is recorded on silica gel F 254 plates (coated finished plates from Merck). After drying, the stains are detected by fluorescence, absorbance measurement or charring after spraying with a mixture of diethyl ether and sulfuric acid. Highly purified preparations of dihydromocimycin also result in a small additional stain in addition to a main stain. This is due to a tautomeric equilibrium, as can be shown by a two-dimensional chromatogram.

The following Rr values for dihydromocimycin result in different solvents (the Rp value for the additional stains is given in parentheses):

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 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 following structural formula results for dihydromocimycin: The PMR-

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Spectra (220 MHz) of mocimycin (see Tetrahedron Letters, Vol. 52 (1973), pp. 5173-5176, where the structural formula of mocimycin is given) and dihydromocimycin show that the two compounds are very similar, with two doublets at ô = 5.9 and 7.3 ppm (tetramethylsilane is used as standard) occur in the spectrum of mocimycin and are not present in the spectrum of dihydromocimyin. These signals are caused by the protons of the fifth and sixth carbon atoms in the pyridone nucleus. In the spectrum of dihydromocimycin, however, two triplets occur at 8 = 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 ozonization of 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 PtCh as catalyst and after hydrolysis with concentrated hydrochloric acid, ß-alanine can be detected, which 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. However, an important difference between these two compounds 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 cause increased growth or improved feed conversion in these animals. This is surprising since both compounds are active against the same microorganisms in vitro. Dihydromocimycin is often even more effective than mocimycin. The following table shows a comparison of the antimicrobial effects of the two antibiotics:

Agar dilution tests

Microorganism examined Minimal

Inhibitory concentration (Hg / ml) in aerobic cultures

Mocimycin dihydromocimycin

Staphylococcus aureus A 2000

> 100

> 100

Staphylococcus aureus A 2001

> 100

> 100

Diplococcus pneumoniae L54

1.5

<0.75

Salmonella typhimurium R172

> 100

> 100

Escherichia coli U20

> 100

100

Listeria monocytogenes A2130

6

1.5

Listeria monocytogenes A2131

6

6

Listeria monocytogenes A2132

6

6 '

Clostridium perfringens A738

> 100

> 100

Clostridium septicum A2152

10th

10th

Streptococcus zooepidemicus A2144

6

6

R streptococci A2148

6

6

Brucella suis (smooth) A2126

0.75

0.4

Pasteurella haemolytica A2136

3rd

1.5

Treponema spec. A2275

30th

10th

3rd

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

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Liquid dilution tests

Microorganism examined Minimal

Inhibitory concentration (ag / ml)

Mocimycin dihydromocimycin

Bacillus subtilis ATCC 633

100

50

Bacillus subtilis ATCC 6051

100

50

Bacillus subtilis 6346 Dl67

1.2

0.9

Bacillus subtilis 220 Dl78

75

75

Bacillus subtilis TH 10

100

50

Bacillus cereus Dl 66

0.6

0.6

Bacillus cereus D261

0.9

0.6

Bacillus cereus D220

1.2

0.9

Bacillus cereus B569

2.5

1.2

Bacillus cereus TH 1

1.8

1.2

Bacillus thuringiensis W11

1.2

0.9

Bacillus mesenterium D169

100

50

Bacillus cereus var. Mycoides

1.2

0.45

Streptococcus haemolyticus A266

0.45

0.45

Streptococcus haemolyticus A2182

0.25

0.12

Mycoplasma hyorhinus A2230

1

0.3

Streptomyces viridochromogenes

2.5

0.9

As already mentioned, the strain Streptomyces ramocissimus CBS 190.69 or its corresponding mutant forms dihydromocimycin in addition to mocimycin. The microorganism can be grown in liquid nutrient media which contain conventional sources of carbon, nitrogen, phosphorus, calcium, iron, sulfur, magnesium, potassium, vitamins and trace elements. For example, media can be used that contain beet molasses, malt paste, peanut flour, lactose, potato starch, corn swell products and yeast extract. The growth temperature of the medium is generally 20 to 40 ° C, preferably 26 to 34 ° C and the pH 5 to 9, preferably 6.5 to 8.

The process according to the invention for producing dihydromocimycin is very similar to the process for producing mocimycin. It has been found that when Streptomyces ramocissimus or its corresponding mutants are grown under appropriate conditions, dihydromocimycin is also formed in addition to mocimycin.

It was surprisingly found that the yield of dihydromocimycin compared to the yield of mocimycin can be increased by increasing the oxygen pressure in the growth medium.

It can be assumed from the structure of dihydromocimycin that the yield of dihydromocimycin decreases compared to mocimycin due to a higher oxygen pressure in the growth medium. However, the above-mentioned effect occurs that dihydromocimycin compared to mocimycin results in better ventilation of the breeding liquid in larger amounts is formed. The ventilation can be carried out according to conventional procedures. For example, the aeration speed (volume part of air per volume part of growth medium per unit time) or a higher stirring speed of the growth medium in the fermenter is used. Suitable aeration speeds of the growth medium, the volume of which is, for example, 2 liters, 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 to the growth medium.

The separation of dihydromocimycin from the growth medium is partially similar to the separation of mocimycin. In the last stage, where the compounds are precipitated from an organic solvent, differences in the solubility of mocimycin and dihydromocimycin are used. Mocimycin precipitates first when gaseous ammonia is passed through the solution. 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 be followed by thin layer chromatography. After the Mocimycin precipitate has been separated off, for example by filtering off, from the solution, ammonia is passed in until the dihydromocimycin is practically completely precipitated. The dihydromocimycin can then also be separated off, for example by filtration. Ammonia is, for example, 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 ° C, initiated. By further introducing gaseous ammonia, the hydrogen ion concentration decreases to such an extent that dihydromocimycin becomes insoluble. For example, under the above conditions, it is introduced for 10 to 15 minutes (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. Examples include sodium methylate and triethylamine.

The dihydromocimycin separated from the growth medium in this way can be further cleaned of impurities in mocimycin by dissolving the precipitate in a strongly diluted ammoniacal solution of pH 9 and extracting this solution with a solvent such as chloroform or methylene chloride. Mocimycin is only slightly soluble in such solvents. If the extract obtained is poured into an apolar solvent such as petroleum ether, cyclohexane or pentane which is present in excess, a dihydromocimycin precipitate is formed which contains less than 5 percent mocimycin.

Highly purified dihydromocimycin is obtained by chromatography on three-dimensionally cross-linked dextran (for example Sephadex LH 20, using the adsorption differences of mocimycin and dihydromocimycin. The dextran is suspended in 100% methanol and carefully poured into the column. After the methanol has been displaced, chloroform is used to replace the methanol The above-mentioned dihydromocimycin precipitate is applied to the column. Chloroform is used as the eluent. This gives first dihydromocimycin and then mocimycin. Both compounds can be detected in the eluate since they absorb in the UV range at 350 nm. The pure compounds can be precipitated from the chloroform solution with an apolar solvent such as cyclohexane or pentane.

The identity of the compounds can be confirmed by thin layer chromatography. For this, silica gel F254 plates measuring 20 x 5 cm (Merck) are used. A 60:42:10 mixture of chloroform, ethanol and 25 percent ammonia is used as the eluent. The run time is 2 hours. Mocimycin shows an Rr value of 0.3 (main tautomer) and dihydromocimycin of 0.4 (main tautomer).

Based on previous studies, it was assumed that mocimycin, which has an antimicrobial spectrum of activity similar to that of tylosin (cf. the most common

4th

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

5 630411

Pigs diseases, is effective. Investigations of bromine, iodine or N-bromosuccinimide under the following genes, which were carried out in the context of the present invention, did not lead to the desired dehydrohalogenation, but it was found that the effect of Moci result, not even in the presence of catalyst.

mycin is not larger than that of tylosin. ren, such as benzoyl peroxide or a, a-azobisisobutyronitrile. To

From the table above it follows that dihydromocatalytic dehydrogenation of dihydromocimycin to cimycin are necessary to achieve high temperatures effective against a number of microorganisms, so that the risk of decomposition

is more than mocimycin. There have been studies into the effectiveness of dihydromocimycin. It has been found that the dehydration of dihydromocimycin against the causative agent of Treponema dysentery has only been found to mean that dihydromocimycin can be carried out satisfactorily against this selenium dioxide. Microorganism is much more effective than tylosin and therefore the invention also relates to the use of the herbal

dihydromocimycins also used against strains which are resistant to tylosin for the production of mocimy-

is effective. Thus, dihydromocimycin is a treatment for, by dehydration of dihydromocimycin with Selendi-von Treponema dysentery tylosin and thus also mocimycin oxide. This process can also be applied to mixtures of dihy-

think. Apply dromocimycin and mocimycin, for example

Pig feeds can contain an effective amount of 15 when working up mocimycin from fermentation liquids

Dihydromocimycin or a salt of this compound can be obtained.

for example, the sodium salt. The antibiotic or the dehydration of dihydromocimycin with selenium dioxide, the alkali metal, ammonium or amine salt can also be carried out at normal ambient temperature.

a corresponding inert, physiologically acceptable. However, the reaction is preferably carried out at elevated temperatures

Carriers or diluents which are administered orally to pigs, for example about 65 to about 110 ° C. and preferably

can be followed, do not react with the antibiotic wise at 80 to 95 ° C. The reaction time can and is safe for pigs when administered orally, from about 10 hours to about 20 minutes at a temperature of 1 ° C

be dispersed or mixed. Effective amounts range from about 65 to about 110 ° C. Dihydromocimycin, which for the prophylaxis or therapy of Tre-, the reaction time is about 3 hours to about 1 hour in the ponema dysentery, is added to the feed, is in the preferred temperature range. At normal temperatures about 10 to about 200 ppm, preferably 20 to 40 ppm, the reaction time is about 1 week.

on the feed weight. The reaction is preferably carried out in a solvent

The dihydromo obtained in the manner described above was carried out. Examples of corresponding solvents are cimycin, a fine, slightly dusting powder. Since this is taking hexamethylphosphoric triamide (HMPT), dimethyl sulfoxide

Difficulty mixing with the Fut-30 (DMSO), tert-butanol, tert-amyl alcohol, sec-butanol, hexy-

ter, a premix with a lenglycol, n-butanol, isopropanol, ethylene glycol monomethyl

or several components of the pig feed. ether, dimethylformamide, water, phenylmethylcarbinol and This premix contains, for example, 9 to 99 times the propanol and mixtures of two or more of this solution desired amount of dihydromocimycin. Premixes can be used. HMPT, DMSO and tert-butanol are preferred. for example with corn flour, potato flour or soy flour 35 HMPT is particularly preferred.

getting produced. Investigations have shown that dihy- Preferably in the dehydrogenation, the selenium dioxide in dromocimycin is also in the pelletization, i. H. Granulate stoichiometric amounts or in excess, based on under high pressure and high temperatures with use dihydromocimycin.

formation of water vapor, proves to be stable. Because the isolation of both products from the fermentation liquid

The premix can be a preventative measure of the feed, while the separation of dihydro-

treatment or for treatment of mild Treponema dysentemocimycin and mocimycin requires more effort

of pigs. This method, which converts dihy-

When pigs are so strongly affected by Treponema dysentery from dromocimycin to mocimycin, a great advantage with oils is that they have lost their appetite or are healthy. It is also possible to use a mixture of both compounds as the starting material for the animals to be pushed away from the feeding site, whereby the

Dihydromocimycin preferably administered with drinking water- Mocimycin is essentially unaffected. Thus, preferably together with a taste-improving mixture, the dihydromocimycin follows together with a detergent. For this purpose, dihydromocimycin is contained in water-soluble mocimycin, directly for the dehydration of dihydro

cher form, for example as a potassium, sodium or amine salt mocimycin used without prior isolation of the individual compounds. 50 can be used.

Pigs very severely affected by dysentery can use. The examples illustrate the invention.

Injections of dihydromocimycin or an equivalent water-soluble salt are treated. The active ingredient example 1

is suspended or dissolved in a customary injection liquid, for example production of dihydromocimycin, a saline solution, propylene glycol or mixtures. 55 Streptomyces ramocissimus CBS 190.69 is suspended in 2000 liters from glycerol and water. a nutrient medium containing 20 g malt paste, 10 g yeast ex-

Mocimycin has beneficial growth-promoting tracts and 5 g of solids obtained from corn steep liquor when fed to livestock, including contains, at a pH of around 7 with stirring and aeration.

Chickens and other poultry. Dihydromocimycin has grown. After cultivation, the medium with about these properties will not. An attempt was therefore made to use 60 2 percent dicalite (expanded perlite, aluminum silicate with

Convert dihydromocimycin to mocimycin. containing potassium, sodium and trace elements) as fil-

According to the invention, it was found that dihydrogenation aid is shifting. The mixture is then filtered. The fil

mocimycin when using a certain dehydration step is acidified to pH 6.0 with 8N sulfuric acid by means of dehydration under certain conditions. and twice with a fifth of its volume with methyl isobu

Quinones such as 2,3-dicyano-5,6-dichloro-1,4-quinone, p-chloroanil 65 tylketone (MIBK) extracted. The emulsions formed and o-chloranil are not suitable for the dehydration of Dihy- with Hyflo Supercel filter aid (a diatomaceous earth product)

dromocimycin, as other products are broken when used. The organic liquids will be formed mixed. Halogenation with copper (II) bromide and under reduced pressure in a rotary evaporator

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6

concentrated to a volume of about 1 liter. This concentrate is added to five times the volume of petroleum ether with a boiling range of 40 to 60 ° C. The precipitated crude product is filtered off with a glass filter (G 3). The residue is washed with fresh petroleum ether and dried. A yellow-colored powder is obtained which contains mocimycin and dihydromocimycin.

Purified dihydromocimycin with a Mocimycinge content of at most 5 percent is obtained in the following way: Gaseous ammonia is introduced into the concentrate for 1 minute at a temperature of 2 ° C. at a rate of 150 liters / liter 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 now formed is filtered off and dissolved in dilute ammonia solution with a pH of 9.0. The solution obtained is extracted with the same 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.

To prepare the sodium salt of dihydromocimycin, dihydromocimycin is dissolved in water and 0.1 N sodium hydroxide solution is added to pH 9, a saturated solution being formed. The solution is filtered off and evaporated with the addition of butanol under reduced pressure at about 45 ° C. under azeotropic conditions. The butanol residue is taken up in a small amount of anhydrous butanol. Petroleum ether is then added dropwise to the solution while stirring until the salt has completely precipitated. The precipitate is filtered off, washed and dried. The sodium salt of dihydromocimycin is obtained. 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 CBS 190.69 or a well spurred agar culture of this microorganism is inoculated into a 500 ml Erlenmeyer flask containing 100 ml sterilized medium of the following composition: 20 g malt paste , 10 g yeast extract and 5 g solids from corn steep liquor per liter of tap water (pH 7.0).

After 3 days of incubation at 30 ° C. on a rotary shaker (300 rpm, stroke 2.5 cm), the culture obtained is inoculated into small fermenters containing 2000 ml of the aforementioned medium and an additional 20 mg of CoCU-6H2O per liter . In order to promote the formation of dihydromocimycin, this growth phase is carried out at 30 ° C with very good ventilation. For this purpose, at least 2 liters of sterilized air are introduced per minute at a stirring speed of 1000 rpm. The formation of dihydromocimycin begins about 12 hours after the start of breeding and reaches the maximum after about 120 hours. Growing on a larger scale is possible by using a culture grown in the small fermenters for 48 hours as an inoculum for large fermenters.

Dihydromocimycin is obtained from the fermentation liquid in the following way: After adding 2 percent diatomaceous earth as a filter aid, the culture is filtered off. The filtrate is acidified to pH 5 to 6 with 8N sulfuric acid and extracted twice with one fifth of its volume with MIBK. Any emulsion formed is broken by filtering the mixture after adding diatomaceous earth. The organic phases are collected and under reduced pressure to about one tenth of the original

Volume constricted. Gaseous ammonia is then passed into the filtrate for 1 minute at a rate of 150 liters / liter concentrate / hour. The precipitate formed is filtered off. The filtrate is treated with gaseous ammonia for a further 10 to 15 minutes. The precipitate obtained is dissolved in dilute ammonia solution with a pH of 9.0. By extracting the ammonia solution with an equal volume of methylene chloride, purified dihydromocimycin (with a maximum mocimycin content of 5 percent) is obtained. The extract is poured into 3 to 5 times the volume of cyclohexane. The precipitate is filtered off, dried and pulverized.

Effect of dihydromocimycin against Treponema dysentery

20 3-month-old pigs are infected with the pathogen of Treponema dysentery by giving them a feed mixed 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. The animals receive a slurry-like feed diluted with water in a ratio of 1: 1 for 1 week. 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 faecal examination. After 1 week, the animals are treated as follows:

Group 1: no antibiotic added to the feed;

Group 2: addition of 100 ppm tylosin to the feed;

Group 3: addition of 25 ppm dihydromocimycin to the feed;

Group 4: addition of 50 ppm dihydromocimycin to the feed.

The feed, possibly enriched with antibiotics, is fed for 1 week. Then normal food is given again without the addition of antibiotics. To determine whether the animals have recovered from the infection, the body weight is determined and the faeces are examined macroscopically for their nature and microscopically using a special immunofluorescence method.

During the investigation, 1 animal from groups 1, 3 and 4 is received. The test results are summarized in the following table. The weights are average weights of the animals living at the time. The quality of the faeces is assessed as follows: + thin liquid; ± viscous; - normal.

In the immunofluorescence examination, the number of Trepo nema in the visual field is given as follows: 5: very dense 4: numerous

3: about 10 treponemes 2: 1 or 2 treponemes

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

Group 1 Group 2 Group 3 Group 4 Weight in kg immediately before infection 24.5

24.4

24.2

23.4

to

1 week (1) 23.7

22.2

21.3

21.6

2 weeks 20.9

21.4

20.2

21.2

3 weeks 19.8

23.2

24.0

23.0

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

7

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Group 1 Group 2

Group 3

Group 4

Weight in kg

Feces

1 week

+++++ ++++ -

+ + + + X

+ + + + +

2 weeks

+++++ +++ ± +

± ± ± +

+ + x + +

3 weeks

+ + + + X ± + ± fine

- - + +

- - + -

Immunofluorescence

1 week

44445 4434-

5455 X

5-533

2 weeks

444-4 4422-

443 1

2 - x 4 1

3 weeks

2222 x 3 2 -

1

io

(1): Start of antibiotic treatment x: an animal dies

15

The nature of the faeces and the immunofluorescence examination show that the animals treated with dihydromocimycin recover much faster than animals treated with tylosin. If one also takes into account the amount of the antibiotic administered, it can be assumed that dihydromocimycin is at least 4 times as effective as tylosin.

Effect of dihydromocimycin against Treponema dysentery 25

Under veterinary supervision, pigs are treated in a breeding facility where the pigs suffered from severe diarrhea. The pigs are divided into 4 groups and treated for 4 days as follows:

Group 1:66 pigs receive feed with 100 ppm Tylo- 30

sin;

Group 2: 34 pigs are fed 25 ppm dihydromocimycin;

Group 3: 40 pigs are fed 50 ppm dihydromocimycin; 35

Group 4: 40 pigs received feed with 100 ppm dihydromocimycin.

The faeces of all animals before the start of treatment are thin or very thin. Faecal samples are found to be treponema positive and salmonella negative. Worm eggs are occasionally found.

One day after the start of treatment, group 4 pigs' faeces are normal. The animals in group 1 are only healed after 3 to 4 days. In the animals of groups 2 and 3, treatment success occurs after a time that lies between the 45 corresponding times of group 1 and group 4. The animals treated with dihydromocimycin make a more vivid impression than before the treatment. The animals showed no aversion to food containing dihydromocimycin. Overall, it can be stated that for the treatment of 50 Treponema dysentery, a dose of 25 ppm dihydromocimycin is cheaper than a dose of 100 ppm tylosin.

Example 3

Dehydration of dihydromocimycin 55

A solution of 1 g dihydromocimycin in 15 ml HMPT (technical product dried over a 3 A molecular sieve) is mixed with 139 mg (1.25 mmol) selenium dioxide. The mixture is heated on a steam bath for 100 minutes and a further 139 mg of selenium dioxide are added after 60 minutes. The selenium which has precipitated 6o after cooling 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 filtrate is saved.

The precipitate is dissolved in methanol and diluted with M1BK. The solution is reduced at 40 ° C

Evaporated pressure to remove methanol and water. It can be determined by thin layer chromatography that the precipitate contains no mocimycin and only consists of polar impurities. The precipitate is filtered off and washed with M1BK. Excess petroleum ether with a boiling range of 40 to 60 ° C. is added dropwise to the filtrate. The precipitate formed is filtered off, washed with petroleum ether and dried to 500 mg of product.

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

Example 4

Dehydration of dihydromocimycin

This attempt is carried out in a double approach. 2.4 g of a mass containing 22.8% mocimycin and 35.5 percent dihydromocimycin are dissolved in 50 ml of HMPT. 350 mg of selenium dioxide are then added. The mixture is stirred at 105 ° C for 45 minutes. A thin-layer chromatographic examination shows that dihydromocimycin is no longer present. The mixture is cooled to room temperature and filtered through a G4 glass filter to remove black selenium formed and a small amount of unreacted selenium dioxide.

The precipitate is washed with MIBK. The filtrate and MIBK washing liquid are combined and 350 ml of water, 10 g of sodium chloride and 5 ml of 4N hydrochloric acid are added. The mixture is extracted 3 times with 100 ml MIBK each. The combined MIBK extracts are washed with 100 ml of water. 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 phase is then concentrated under reduced pressure at about 20 ° C to a volume of 25 ml. Petroleum ether with a boiling range of 40 to 60 ° C. is added dropwise to the residue with stirring. The precipitate formed is filtered off, washed with petroleum ether from the boiling range 40 to 60 ° C and dried overnight at room temperature under reduced pressure.

The yield in the first batch is 1.64 g of product containing 41.2 percent mocimycin and ^ 2 percent dihydromocimycin (determined by high pressure liquid chromatography). In the second approach, the yield is 1.79 g of product containing 41.6 percent mocimycin and 1 percent dihydromocimycin.

Example 5

Influence of solvents on dehydration of dihydromocimycin

The influence of various solvents on the dehydration of dihydromocimycin is examined. In each case 50 m of a technical product containing dihydromocimycin and mocimycin are reacted with 20 mg (0.18 mmol) of selenium dioxide in 3 ml of solvent at a temperature of 80 ° C. Samples are taken 2.4 and 7 hours after the start of the test and analyzed by thin layer chromatography on silica gel 60 disks (Merck) using a 50: 45: 5 mixture of MIBK, acetone and water as the eluent. The detection 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.

If the following solvents are used, decomposition or no reaction results: propargyl alcohol, diaceto alcohol, nitromethane, propylene carbonate, acetonitrile, pyridine,

630411

Sulfolane, benzyl alcohol, mesityl oxide, ethylene carbonate, N-methyl-2-pyrrolidone, N, N-dimethylacetamide, dioxane, MIBK, butyl acetate, amyl acetate, N-methylacetamide, anisole, diethyleneglycol methyl ether and 1,2-dichloroethane. A low degree of conversion is obtained when using tetramethylurea, 0.1 m NaHCCh / Na2CC) 3 buffer with a pH of 9.1, n-propa-nol, phenylmethylcarbinol, water and dimethylformamide. A somewhat better degree of conversion is achieved when using ethylene glycol monomethyl ether, isopropanol, n-butanol, hexylene glycol, sec-butanol and tert-amyl alcohol. Moderate conversion is achieved in tert-butanol and dimethyl sulfoxide. The best results are obtained when using HMPT.

Example 6

Dehydrogenation of dihydromocimycin in solvent mixtures

The suitability of solvent mixtures with HMPT as a component is being investigated. 2.4 g of the starting material from Example 7 are used in each case. After the reaction of the entire dihydromocimycin, as determined by thin layer chromatography, the reaction mixture is worked up as follows: After cooling, to remove selenium formed, it is filtered through a D4 glass filter and washed with a small amount of methanol. The filtrate is poured into an excess of water and acidified to pH 3. Then extracted three times with MIBK in an amount of a quarter of the volume. A tar-like intermediate layer is extracted with MIBK by first again dissolving 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. The organic phase is concentrated at about 40 ° C. under reduced pressure. In all cases, the precipitate essentially consists of decomposition products, as can be shown by thin layer chromatography. The precipitate is filtered off and washed with MIBK. The combined MIBK concentrates are slowly added with stirring to an excess of petroleum ether with a boiling range of 40 to 60 ° C. The precipitate formed is filtered off, washed with petroleum ether and dried. The results are summarized in the following table:

Amount of reaction temperature- total

Solvent for reaction time

(° C) (hrs)

SeCh addition in g;

Time after the start of the reaction

Yield mole SeCh per mole

(Weight percent) mixture

HMPT 25 ml 90

H2O 35 ml

HMPT 15 ml 90

0.1 m

NaHCCb-NazCCh buffer pH 9.1 25 ml t-BuOH 20 ml 84 HMPT 10 ml t-AmOH 96

100 ml

HMPT 50 ml t-AmOH 96

150 ml t-BuOH 80

100 ml

HMPT 50 ml HMPT 150 ml 95

2nd

23/4

End of reaction time after 1 hour

0.5 at the beginning 42

+ 0.5 after 55 minutes

0.1 in the beginning 50

0.1 after 20 minutes

0.1 after 85 minutes

0.1 after 130 minutes

0.1 at the beginning 42

0.1 after 25 minutes

0.1 after 45 minutes

0.1 after 75 minutes

0.1 after 120 minutes

0.35 at the beginning 71

0.35 after 70 minutes

0.35 after 110 minutes

0.35 at the beginning 55

0.35 after 75 minutes

0.35 after 150 minutes

1.0 at the beginning 42

1.0 after 35 minutes

0.4 at the beginning 79

3: 1 1.2: 1

1.5: 1

3.2: 1 3.2: 1 6: 1 1.2: 1

HMPT = hexamethylphosphoric triamide The table shows that the highest yield (79

t-BuOH = tert-butanol 55 percent) when pure HMPT is used as the solvent t-AmOH = tert-amyl alcohol. A high yield is also obtained when using a mixture of HMPT and tert-amyl alcohol.

G

2 sheets of drawings

Claims (12)

630411 2. The method according to claim 1, characterized in that one carries out the cultivation at an aeration speed of 1 to 3 liters of air per 2 liters of nutrient medium and per minute. 2nd PATENT CLAIMS 1. Process for the preparation of dihydromocimycin of the formula HO OH and its non-toxic, pharmaceutically acceptable salts, characterized in that Streptomyces ramocissi-mus CBS 190.69 is grown under aerobic conditions in an aqueous nutrient medium containing assimilable carbon and nitrogen sources and inorganic substances, and the dihydromocimycin formed during the cultivation separated from the mixture of mocimycin and dihydromocimycin obtained. 3. The method according to claim 1, characterized in that one carries out the cultivation at an aeration speed of 1.5 to 2.5 liters of air per 2 liters of nutrient medium and per minute. 4. The method according to claim 1, characterized in that one carries out the cultivation in the presence of low concentrations of iron, cobalt or nickel ions in the nutrient medium. 5. The method according to claim 1, characterized in that the mocimycin is initially separated from the mixture of mocimycin and dihydromocimycin obtained with the aid of alkaline compounds, such as ammonia, sodium methylate or triethylamine, leaving behind dihydromocimycin. 6. Use of the dihydromocimycin obtained by the process according to claim 1 for the preparation of Mocimycin by dehydration, characterized in that i5 one reacts dihydromocimycin with selenium dioxide. 7. Use according to claim 6, characterized in that one carries out the reaction at elevated temperatures. 8. Use according to claim 6, characterized in that the reaction at temperatures of 65 to 110 ° C. carries out. 9. Use according to claim 6, characterized in that one carries out the reaction at temperatures of 80 to 95 ° C. 25th 10. Use according to claim 6, characterized in that the reaction in hexamethylphosphoric triamide, dimethyl sulfoxide, tert-butanol, tert-amyl alcohol, sec-butanol, hexylene glycol, n-butanol, isopropanol, ethylene glycol monomethyl ether, dimethylformamide, water, Phenylmethylcarbi-3o noi, propanol or mixtures of two or more of these solvents. 11. Use according to claim 6, characterized in that one carries out the reaction in hexamethylphosphoric triamide, dimethyl sulfoxide or tert-butanol. 35 12. Use according to claim 6, characterized in that one carries out the reaction in hexamethylphosphoric triamide as a solvent.