DK143606B - PROCEDURE FOR THE PREPARATION OF DIHYDROMOCIMYCINE OR SALTS THEREOF - Google Patents

Description

(19) DENMARK

| J | (12) PUBLICATION MANUAL OR 143606 B

DIRECTORATE OF THE PATENT AND TRADEMARKET SYSTEM

(21) Application No. 2151/76 (51) intci.9 C 12 P 19/26 (22) Filing date 15 · May 1976 C 07 H 7/06 (24) Race day 15 · May 1976 (41) Aim. available Nov. 17 1976 (44) Presented 14. s ep. 1 981 (86) International application no. - (86) International filing day - (85) Continuation day - (62) Master application no. -

(30) Priority 16 May 1975, 20926/75 # GB 22 Jul. 1975, 50646/75, GB

(71) Applicant GIST-BR0CADES N.V., Delft, NL.

(72) Inventor Hendrik Marten Jongsma, NL: Hermanus Jacobus Koore = husband, NL: Jan Lambert van Os, NL: Cornells Vos, NL.

(74) Clerk of the International Patent Office.

(54) Process for the preparation of dlhydromoclmycin or its salts.

The invention relates to a process for the preparation of a novel antibiotic called dihydromocimycin or a non-toxic pharmaceutically acceptable salt thereof. This antibiotic is particularly valuable for the swine disease called Treponema dysentery, Vibrio Doyle, etc.

Dihydromocimycin has the following formula

CD

ISLAND

CO

ISLAND ISLAND

-ct *

Q

143606 2 / n ° o ho oh N oh

y <A. A / Y / V

S \ 0 æ% H

H OH

D ihydromocimycin is produced by fermenting Streptomyces ramocissimus and produced in addition to mocimycin. Streptomyces ramocissimus is a microorganism described in English Patent Specification No. 1325200. The microorganism is deposited in the culture collection at the Centraal Bureau voor Schimmelcultures in Baarn, the Netherlands, where it has been given the number CBS 190.69 and from which it is available to the public.

British Patent Specification No. 1325200 describes a process for the preparation of mocimycin (at the time called MYC 8003) from the above-mentioned microorganism. The structure of mocimycin is given by Vos and Verwiel in Tetrahedron Letters 52 (1973) pp. 5173-5176. It has now been found that Streptomyces ramocissimus produces dihydromocimycin in addition to mocimycin.

Dihydromocimycin is a pale yellow solid, weakly acidic, further characterized by the following physicochemical properties:

Solving equality.

The solubility of the compound is good in chloroform, methyl isobutyl ketone, ethyl acetate, butyl acetate, acetone, dioxane, methanol, ethanol, tetrahydrofuran and in slightly alkaline aqueous solutions. The solubility is moderate in carbon tetrachloride and benzene and the compound is insoluble in diethyl ether, water, slightly acidic aqueous solutions, cyclohexane and petroleum ether.

Optical rotation.

[α] p = -85 ° (1% methanolic solution).

SmeItepunkt:

The compound does not melt, but decomposition begins at 123 ° C.

Elemental Analysis: The following values were found:

Found: Calculated for 2 N 2 by difference) 26.8% 3 143606

Ultraviolet spectrum:

The ultraviolet spectrum of dihydromocimycin in a 1: 1 mixture of water and methanol at various pH values is depicted in FIG. 1 of the accompanying drawing. The concentration of the measured solutions was 18.3 µg / ml. The ultraviolet spectrum is dependent on the pH. The following maxima were measured (molecular extinction is indicated between the brackets): methanol - water: 233.5 nm (f = 63,000); 267 nm (f = 23,000); 291 nm (£ = 19,000) and 333 nm (£ = 18,000); curve 1; methanol - 0.5N NaOH: 235 nm (δ = 62,000); 277 nm (£ = 25,000) and 308 nm (fc = 29,000); curve 2; methanol - 0.5N HCl: 233.5 nm (δ = 65,000); 268 nm (£ = 25,000) and 338 nm (t-14,000); curve 3.

Infrared spectrum: The IR spectrum of dihydromocimycin in chloroform and in potassium bromide is depicted in Figures 2 and 3. The following main absorptions were found: chloroform (0 in cm-1): - 3445 (sh); 3430; 2979; 2941; 2886; 1662-1653; 1458; 1100; 1080; 1040; 998; 944; 893; 870 and 840; potassium bromide (0 in cm'1): ΐ 3400-3320; 2972; 2935; 2880; 1650; 1535; 1455; 1099; 1040; 990; 943; 860; 840 and 790.

PMR spectrum: The PMR spectrum of dihydromocimycin dissolved in deuterochloroform, using tetramethylsilane as internal reference ^ is depicted in FIG. 4 (60 Mc).

Thin layer chr oraa t ograf in:

Thin-layer chromatograms of dihydromocimycin were performed on silica gel F 254 plates (Merck, plates that are equally applicable) and after drying the spots were revealed by fluorescence extinction or by carbonization after spraying with a diethyl ether sulfuric acid mixture. The studies revealed that even with the purest preparations, the stain corresponding to dihydromocimycin was always accompanied by a small additional stain. The appearance of two spots is due to a tautomeric equilibrium, which can be shown by a two-dimensional chromatogram.

The following R ^ values were found for dihydromocimycin in various eluents (the R ^ values for the extra stain are given in the 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% ammonia: 0.29 (0.36); 65: 40: 9 mixture of benzene, 100% ethanol and 33% ammonia: 0.16 (0.22); 60:42:10 mixture of chloroform, 96% ethanol and 25% ammonia: 0.4.

143606 4

The structural formula for dihydromocimycin was confirmed from the following evidence: The PMR spectrum (220 Mc) of mocimycin (cf. Tetrahedron Letters 52 (1973) pages 5173-5176 on its structural formula) and dihydromocimycin showed that both compounds were similar except two doublets at S 5.9 and 7.3 ppm (tetramethylsilane were used as reference) found in the spectrum of mocimycin but not found in the spectrum of dihydromocimycin. These signals are caused by the protons at the 5th and 6th carbon atoms belonging to the pyridone nucleus. However, two triplets in the spectrum of dihydromocimycin were found at S 2.5 and 3.4 ppm. This suggests that the bond between the 5th and 6th carbon atoms of the dihydromocimycin pyridone nucleus is saturated. This interpretation was confirmed by ozonization of an aqueous solution of dihydromocimycin at pH 12 over 5 minutes at 0 ° C. After reduction of the reaction mixture with hydrogen catalyzed by PtCl 2 and after hydrolysis with concentrated hydrochloric acid, β-alanine was detected, indicating that the bond between the 5th and 6th carbon atom of the pyridone core of dihydromocimycin is saturated.

Many properties of dihydromocimycin are similar to those found for mocimycin. However, there is one important difference: a low concentration of mocimycin added to the feed for animals to be fed such as chickens or pigs, increase growth and food conversion remarkably. Dihydromocimycin, on the other hand, shows no such improvement in growth or food conversion in these animals, which is unexpected as in vitro experiments have shown activity against the same microorganisms. In many cases, it even shows better activity than mocimycin.

A comparison of the antimicrobial effects of the two antibiotics is shown in the following table:

Agarfortyndingsprøver.

Organism tested Minimum inhibitory concentration _ (/ g / ml) anaerobic culture_ 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 coll U20> 100 100

Listeria monocytogenes A2130 6 1.5

Listeria monocytogenes Δ2131 6 6

Listeria monocytogenes A2132 6 6

Clostridium perfrincens A738> 100> 100

Clostridium septicum A2152 10 10 5 143606

Table (continued) _

Organism tested Minimum inhibitory concentration (/ tg / ml) anaerobic culture____ mocimycin dihydromocimycin

Streptococcus zooepidemicus A2144 6 6 R-Streptococcus A2148 6 3

Brucella suis (smooth) A2126 0.75 0.4

Pasteure11a haemolytica A2136 3 1.5

Treponema spec. A2275 30 10 Liquid Dilution Samples. _

Organism tested Minimum inhibitory concentration (/ tg / ml) __ mocimycin dihydromocimycin

Bacillus subtilis ATCC 6633 100 50

Bacillus subtilis ATCC 6051 100 50

Bacillus subtilis 6346 D167 1.2 0.9

Bacillus subtilis 220 D178 75 75

Bacillus subtilis TH 10 100 50

Bacillus cereus D166 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 ΤΗ 1 1.8 1.2

Bacillus thuringiensis Wil 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 where A2230 is 0.3

Streptomyces viridochromogenes 2.5 0.9

As mentioned, Streptomyces ramocissimus produces dihydromocimycin under suitable conditions in addition to mocimycin. The process of the invention for the preparation of dihydromocimycin is accordingly characterized by aerobically culturing a strain of Streptomyces ramocissimus in an aqueous nutrient substrate containing assimilable sources of carbon, nitrogen and inorganic compounds and separating the dihydromocimycin formed by animals. , optionally in the form of a salt. The fermentation of the microorganism can be carried out in a liquid substrate containing the usual carbon, nitrogen, phosphorus, calcium, iron, sulfur, magnesium, potassium, vitamin and trace element sources, e.g. substrates containing beet pulp, malt paste, peanuts1, lactose, potato starch, cornstarch and yeast extract. The ferment substrate temperature should be between 20 and 40 ° C, preferably between 26 and 34 ° C and the pH between 5 and 9, preferably between 6.5 and 8.

It will be seen that the aforementioned process is similar to that of mocimycin.

In order to obtain a higher yield of dihydromocimycin over the yield of mocimycin, it has unexpectedly been found that this can be achieved by increasing the oxygen pressure in the culture substrate.

From the formula of the dihydromocimycin, it could be expected that a higher oxygen pressure in the culture substrate would decrease the production of dihydromocimycin relative to the production of mocimycin. However, it has been found that dihydromocimycin is produced more abundantly than mocimycin by better aeration of the culture substrate, which can be achieved by measures well known to those skilled in the art, e.g. at a higher aeration rate (volume of air per volume of culture substrate per unit time), or a higher rate of stirring of the culture substrate in fermenters. According to the invention, preferably, aeration rates are used for the culture substrate of 1-3 liters of air, and preferably 1.5 to 2.5 liters of air, per liter. 2 liters of culture medium per minute. A further improvement in the yield of dihydromocimycin is achieved according to the invention by providing the presence of e.g. low concentration iron, cobalt or nickel ions in the culture substrate.

The separation of dihydromocimycin from the culture substrate is partially equal to the separation hf mocimycin. In the final step, where the precipitation of the compounds occurs from an organic solvent, the difference in solubility of mocimycin and dihydromocimycin is taken advantage of. According to the invention, the separation-1 · advantage is advantageously carried out by fractional precipitation from an organic solution by means of an alkaline compound, preferably gaseous ammonia. By passing gaseous ammonia through the solution, mocimycin is precipitated first and the separation can be carried out by passing ammonia through the solution until substantially all of the mocimycin has precipitated and substantially all of the dihydromocimycin is left in the solution. This can be controlled by e.g. thin layer chromatography tests. After discarding, e.g. by filtration, the mocimycin bottom fluid from the solution is continued to feed ammonia until substantially all of the dihydromocimycin is precipitated so that it can be separated, e.g. by filtration. The ammonia is conducted e.g. through the solution at a rate of approx. 150 liters per liter. liters of solution per liter. hour in approx. 1 to approx. 4 minutes (e.g., about 2.5 to 10 liters of gaseous ammonia per liter of solution) at a temperature between about -12 ° C to approx. + 15 ° C, preferably between -5 ° C and + 8 ° C. By continuing the addition of gaseous ammonia to the solution, the hydrogen ion concentration is sufficiently increased to render the dihydromocimycin insoluble, when the ammonia is allowed at the above rate over the course of 10 to about 30 minutes. 15 minutes (corresponding to about 25 to about 40 liters of gaseous ammonia) under the same conditions. In addition to ammonia, usually all alkaline compounds can be used for the separation of mocimycin and dihydromocimycin, e.g. sodium methoxide and triethylamine.

The crude dihydromocimycin thus excreted from the culture substrate is further purified from mocimycin. . by dissolving the precipitate in a strong dilute ammonia solution (pH 9) and extracting this solution with a solvent such as chloroform or methylene chloride. The mocimycin is poorly soluble in such a solvent1, and by pouring the extract thus obtained into an excess of an apolar solvent (e.g., petroleum ether, cyclohexane or pentane), a precipitate of dihydromocimycin containing less than 57 mocimycin is formed.

Very pure dihydromocimycin can be obtained by passing the product thus obtained over a 'SEPHADEX "(trademark) LH 20 column, using the difference in adsorption of mocimycin and dihydromocimycin." SEPHADEX "is suspended in 100¾ methanol and carefully poured onto the column. replacing the methanol with chloroform, the aforementioned dihydromocimycin precipitate is transferred to the column. Chloroform is used as an eluent. The pure compounds can be recovered from the chlorine of the worm by precipitation with an apolar solvent such as cyclohexane or pentane.

Confirmation of the identity of the compounds can be obtained by thin layer chromatography.

«I II

fi. Plates of the product negotiated under the designation Kiselgel F 254, size 20x5 cm (Merck), are used. The eluent is a mixture 60:42:10 of chloroform, ethanol and 25% ammonia. The elution time is 2 hours. Mocimycin shows a value of 0.3 (main tautomer) and dihydromocimycin shows an R 2 value of 0.4 (main tautomer).

At an early stage of the studies, it was believed that mocimycin had a similar antimicrobial spectrum as tylosin (Merck Index, 8th edition, page 1089) and that it would be effective against Treponema hyodysenteriae causing Treponema dysentery or Vibrio Doyle, one of the most common swine diseases. Studies performed at that time showed that mocimycin was effective in this disease, but no more than tylosin. For this reason, no further studies were conducted.

143606 8

As indicated above, dihydromocimycin was found to be more active than moci-mycin to many microorganisms, and consequently studies with this compound were conducted against the microorganism causing Treponema dysentery. The tests showed that dihydromocimycin had a significantly higher effect on the microorganisms than tylosin and further that it was effective on tylosin resistant strains. Thus, dihydromocimycin must be considered better than tylosin (and also than mocimycin) in the treatment of Treponema dysentery.

Dihydromocimycin. may be used in that a certain portion of dihydromocimycin or a salt, e.g. the sodium salt, thereof. The anti-biotic or its alkali metal or ammonium or amine salt may also be dispersed in or mixed with any suitable, inert, physiologically susceptible carrier or diluent which can be administered orally and which does not react with the antibiotic and is not harmful to the pig by oral administration. Effective amounts of dihydromocimycin incorporated in swine feed to prevent or treat Treponema dysentery are approx. 10 to approx. 200 ppm, preferably 20 to 40 ppm dihydromocimycin, based on the weight of the feed.

Dihydromocimycin produced by the present process is a fine, light dusting powder. This could lead to difficulties in the mixing procedure with the feed and, therefore, a premix containing the dihydromocimycin in e.g. a 9- to 99-double amount. Suitable components for making a premix are e.g. corn flour, potato flour and soy flour. Studies have shown that dihydromocimycin has good stability to pelleting (high temperature granulation at high temperature using steam).

The premix can be added to the feed as a prophylactic or as a treatment for pigs that are only slightly attacked by Treponema dysentery.

If the pigs are so severely affected by Treponema dysentery that there is loss of appetite, or the diseased pigs are pushed away from the feeding trough by the healthy pigs, the dihydromocimycin is preferably administered in the drinking water, preferably in the presence of a taste-correcting agent. For this purpose, dihydromocimycin is used in water-soluble form, e.g. in the form of a salt such as a potassium, sodium or amine salt.

Pigs severely affected by dysentery may be treated by injection with dihydromocimycin or a water-soluble salt thereof suspended or dissolved in conventional injections, e.g. saline solution, propylene glycol, glycerol, water mixtures, etc.

g 143606

The process is further explained in the following examples.

Example 1

Preparation of dihydromocimycin.

The microorganism Streptomyces ramocissimus (CBS 190.69) was fermented in 2000 liters of a substrate containing 20 g of malt paste, 10 g of yeast extract and 5 g of solids from corn molding liquid per day. liter at a pH of approx. 7 with stirring and aeration. After fermentation, the growth substrate was mixed with ca. 27. dicalite (an expanded perlite, an aluminum silicate containing potassium, sodium and trace elements) as filter aid, after which the mixture was filtered. The filtrate was acidified with 8 N sulfuric acid to pH 6.0, then extracted twice with methyl isobutyl ketone (MIBK) in an amount of 1/5 of its volume. The emulsions formed were broken with Hyflo Supercel filter aid (a diatomaceous earth). The organic liquids were mixed and concentrated to ca. 1 liter by evaporation under reduced pressure, the evaporation being done with a rotary evaporator. From the obtained concentrate, a crude product was obtained by adding 5 times its volume of petroleum ether (bp 40-60 ° C) and the resulting precipitate was filtered off on a glass filter (G 3). The precipitate was washed with fresh petroleum ether and dried to give a yellow colored powder containing mocimycin and dihydromocimycin.

A purified form of dihydromocimycin containing less than 57 mocimycin was obtained as follows: Gaseous ammonia passed through the concentrate at a rate of 150 liters per minute. per liter of concentrate per hour for 1 minute at a temperature of 2 ° C. A precipitate containing mocimycin was formed. The precipitate was filtered off and the filtrate was again treated with gaseous ammonia for 10-15 minutes. The precipitate now formed was filtered off and dissolved in dilute ammonia (pH 9.0). This solution was extracted with an equal volume of methylene chloride and the extract was retained in the 3-5 twice volume of cyclohexane. The resulting precipitate was filtered off, dried and pulverized.

The sodium salt of dihydromocimycin was obtained by dissolving dihydromocimycin in water while adding 0.1 N sodium hydroxide to pH 9 until a saturated solution was obtained. The solution was filtered and azeotropically evaporated with the addition of butanol (in vacuo at about 45 ° C) and the butanolic residue was collected in a small amount of anhydrous butanol. Petroleum ether is added dropwise with stirring to the solution until all the salt has precipitated. The precipitate was filtered off, washed and dried to give the sodium salt of dihydromocimycin. Other salts of dihydromocimycin were prepared in a similar manner.

10 143606

Example 2

Preparation of higher yield dihydromocimycin.

To obtain higher yields of dihydromocimycin, a lyophilized culture of Streptomyces ramocissimus (CBS 190.69) or a well-sporulated agar culture of this organism was used to seed the contents of a 50 ml erlenmeyer flask containing 100 ml of a sterilized substrate of the following composition: 20 g malt paste, 10 g yeast extract and 5 g solids from corn dusting fluid per liter of tap water, pH 7.0.

After incubation on a rotary shaker (300 rpm, 2.5 cm fluctuation) at 30 ° C for 3 days, a culture was used to seed small fermenters containing 2000 ml of the above-mentioned substrate to which 20 mg ΟοΟ ^ .β ^ Ο pr. liter. This part of the growth, also carried out at 30 ° C, occurred under very good aeration conditions to stimulate the production of dihydromocimycin. To this end, more than 2 liters of sterile air was passed through the growth substrate per well. The wafer substrate was stirred at a rate of up to 1000 rpm. The production of dihydromocimycin began after a fermentation time of approx. 12 hours and was maximum after approx. 120 hours. Large-scale fermentation was possible, using 48-hour-old growth substrates obtained in small fermenters as inoculants for large fermenters.

The dihydromocimycin thus produced was recovered from the growth medium as follows: After adding 2% diatomaceous earth as a filter aid, the culture was filtered and the filtrate was acidified with 8 N sulfuric acid to a pH 5 to 6. Thereafter, extracted twice with MIBK using 1 / 5 of the filtrate volume. If an emulsion was formed, the emulsion was broken by filtration of the mixture after the addition of some diatomaceous earth. The organic layer was collected and concentrated in vacuo to ca. 1/10 of the original volume of the growth medium. Gaseous ammonia was passed through the concentrate at a rate of 150 liters per minute. per liter of concentrate per hour for 1 minute. The formed precipitate is filtered off. The filtrate was again treated with gaseous ammonia for 10-15 minutes. The precipitate thus obtained was dissolved in dilute ammonia (pH 9.0) and then purified dihydromocimycin (containing no more than 5% mocimycin) was obtained by extracting the solution in ammonia with equal volume of methylene chloride. The extract was poured into 3-5 times its volume of cyclohexane, after which the precipitate formed was filtered, dried and pulverized.

11 143606

Anvendelsesekserapler:

Example A

Dihydromocimycin for Treponema dysentery.

Twenty-three-month-old pigs were infected with the microorganism that causes Treponema dysentery. This was done by feeding them with feed mixed with a homogeneous mixture of intestinal contents and intestinal mucosa from two animals suffering from this disease. The infected pigs were divided into 4 groups of 5 animals each and the animals were fed the feed in the form of a slurry diluted 1: 1 with water for one week. The total amount of feed per day. animals were 1.2 kg per day given in 2 servings.

After 5 days, the first symptoms of the disease in the form of thin faeces were observed and confirmed by microbiological examination of faeces. After one week, the animals were treated as follows:

Group 1: no antibiotics for the feed; group 2: 100 ppm tylosin added to the feed; group 3: 25 ppm dihydromocimycin added to the feed; group 4: 50 ppm dihydromocimycin added to the feed.

The antibiotic-enriched feed was given for one week. After this week, feed without antibiotics was given again. Whether the animals overcame the infection was examined by weight of the animals and by examining faecal samples macroscopically for their consistency and microscopically by special immunofluorescence technique.

During the trial, 1 animal died for each of groups 1, 3 and 4.

The results are shown in the following table. Here, the weights indicated are the mean values of the still living animals at the specified time. The consistency of .'faeces is given as follows: + means thin fluid; - means viscous; and - means normal.

The results of the immunofluorescence study are quantitatively indicated by indicating the number of Treponemas in the field of view: 5 means crowded; 4 means many; 3 means approx. 10 Treponemas; 2 means 1 or 2 Treponemas, 1 means more than one field of view to find 1 Treponema and - means negative.

12 143806

Group 1 Group 2 Group 3 Group 4 Weight in kg

Just before infection 24.6 24.4 24.2 23.4

After 1 week (1) 23.7 22.2 21.3 21.6

After 2 weeks 20.9 21.4 20.2 21.2

After 3 weeks 19.8 23.2 24.0 23.0

Faeces consistency

After 1 week + + + + + + + + + - + + + + ώ + + + + +

After 2 weeks +++++++++++++++++++ ++++++++++++++

After 3 weeks_ + - + ώ - + - - - - - - - + _

Immune or lu or e s cenc

After 1 week 44445 4434 - 54554 5-533

After 2 weeks 444-4 4422- 4431 2 - in 4 1

After 3 weeks 2 2 2 2 4 3 - · 2 - 1 - - - (l) The time for starting antibiotic administration; ώ means an animal dead.

From the consistency of faeces as well as from the immunofluorescence studies, it was found that the animals treated with dihydromocimycin were cured significantly faster than the animals treated with tolysin. When the administered amount of antibiotics was also taken into account, it can be concluded that dihydromocimycin is at least 4 times as active as tylosin.

Example B

Dihydromocimycin against Treponema dysentery.

From a farm that had for some time had problems with pig diarrhea, pigs were treated under the supervision of the local veterinarian and under the supervision of the inspector of the Health Service Station. The pigs are divided into groups and treated for 4 days as follows:

Group 1: 66 pigs were treated with feed containing 100 ppm tolysin; group 2: 34 pigs were treated with feed containing 25 ppm dihydromocimycin; group 3: 40 pigs were treated with feed containing 50 ppm dihydromocimycin; group 4: 40 pigs were treated with feed containing 100 ppm dihydromoci mycine.

before treatment, all animals gave thin or very thin faeces. Specimens of faeces were examined and found to be Treponema positive and Salmonella negative. Some worm eggs were found.

The day after the start of treatment, faeces from the pigs were found