IE43106B1 - Mocimycin derivatives - Google Patents

Abstract

A new antibiotic (now called dihydromocimycin) being a yellow, salt-forming weak acid and its non-toxic, pharmaceutically acceptable salts produced by the microorganism Streptomyces ramocissimus or suitable mutants thereof. The antibiotic has antibacterial properties and has been found to be effective against Treponema dysentery, one of the most common swine diseases. Dihydromocimycin may also be converted into mocimycin, which is an antibiotic which possesses interesting growthpromoting properties when added to animal feed, by a dehydrogenation agent. The dehydrogenation may be carried out with selenium dioxide, preferably in the presence of an organic solvent.

Description

THIS INVENTION relates to a new antibiotic designated dihydromocimycin/ a process for its production and compositions containing it. The antibiotic is especially useful against the pigs' disease called Treponema dysentery or Vibrio Doyle. The invention further relates to a process for using dihydromocimycin as a starting material for the production of the known antibiotic mOcimycin.

The present invention provides dihydromocimycin having the following structural formula:- its alkali metal/ ammonium and amine salts.

Dihydromocimycin is produced by fermenting Streptomvces ramocissimus/ or a suitable mutant thereof/ and ls formed in addition to moeimycin. Streptomvces ramocissimus is a microorganism described in Patent Specification No. 36056 the microorganism is deposited /ith the culture collection of Centraal Bureau voor •chimmelcultures at Baarn, The Netherlands, where it obtained :he number CBS 190.69 and is available to the public.

In Patent Specification No, 36056 a process is described and claimed for the production of mocimycin (then called MYC 8003) by the abovementioned microorganism. The structure of mocimycin is indicated by Vos and Verwiel in Tetrahedron Letters 52 (1973), pp. 5173-5176. It has now been discovered that Streptomvces ramocissimus, or a suitable mutant thereof, produces dihydromocimycin in addition to mocimycin.

Dihydromocimycin is a pale yellow solid substance, weakly acid and further characterized by the following physico-chemical properties: Solubility: The solubility of the compound is good in chloroform, methyl isobutyl ketone, ethyl acetate, butyl 15 acetate, acetone, dioxan, methanol, ethanol, tetrahydrofuran, and in weakly alkaline aqueous solutions. The solubility is moderate in carbon tetrachloride and benzene, and the compound is insoluble in diethyl ether, water, weakly acid aqueous solutions, cyclohexane and petroleum ether.

Optical rotation: [a]p° = -85° (1% methanolic solution).

Melting point: The compound does not melt, but decomposition starts at 123°C. - 3 )6 Elementary analysis s She following values were founds Found; Calculated for c43H62N2°12*2H2Os Cs 61.8% 61.8% Hs 7.5% 8.0% Ns 3,.4% 3.3% Os 27.2% (by difference) 26„8% Ultra-violet spectrums The ultra-violet spectrum of dihydromocimycin in a lsl mixture of water and methanol at various pH values is shown in Figure 1 of the accompanying drawings. The concentration of the solutions measured was 18.3 pg./ml. The ultra-violet spectrum is dependent on the pH. The following maxima were found (the molecular extinctions are indicated between brackets); methanol - waters 233.5 nm (8 = 63,000); 267 nm (£ = 23,000); 291 nm (E => 19,000) and 333 nm (8 = 18,000)s curve 1; methanol - 0.5N NaOHs 235 nm (£ = 62,000); 277 nm (£ = 25,000 and 308 nm (ε = 29,000)s curve 2; methanol - 0.5N HCl; 233.5 nm (ε = 65;000); 268 na (£ = 25,000) and 338 nm (£ = 14,000)s . curve 3. Infra-red spectrums The I.R. spectrum of dihydromocimycin in chloroform - 4 43106 and in potassium bromide is shown in Figures 2 and 3, respectively. The following principal absorptions were found: chloroform (Ό in cm4): + 3445 (sh); 3430; 2979; 2941; 2886; 1662-1653; 1458; 1100; 1080; 1040; 998; 944; 893; 870 and 840; potassium bromide (p in cm-4·): + 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 deutero-chloroform< using tetramethylsilane as the internal reference, is shown in Figure 4 (60 Me).

Thin layer chromatography: Thin layer chromatograms of dihydromocimycin were made on Kieselgel F 254 (Merck - a registered Trade Mark, ready-for-use plates), and after drying the spots were detected by fluorescence extinction, or by carbonization after spraying with a diethyl ether-sulphuric acid mixture. Investigations showed that the spot ascribed to dihydromocimycin, even with the purest preparations, was always accompanied by a small additional spot. The appearance of the two spots is due to a tautomeric equilibrium, as can be shown by a two-dimensional chromatogram.

The following Rf-values were found for dihydro- 5 6 mocimycin in various eluents (the Rf-value for the additional spot is indicated between brackets)s 50;45ί5 mixture of methyl isobutyi 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 335(, ammonia: 0.16 (0.22); 60;42sl0 mixture of chloroform, 96% ethanol and 25% ammonia: 0.4.

The structural formula of dihydromocimycin ie confirmed on the following grounds; The PMR spectra (220 Me) of mocimycin (ef. Tetrahedron Letters 52 (1973), pp. 5173-5176, for its structural formula) and dihydromocimycin Showed that both compounds are similar, except that two doublets at δ 5.9 and 7.3 ppm (tetramethylsilane was used as the reference), which occurred in the spectrum of mocimycin, did not occur in the spectrum of dihydromocimycina Those signals are caused hy the protons of the 5th and 6th carbon atoms of the pyridone nucleus. However, two triplets appeared in the spectrum of dihydromocimycin at δ 2.5 and 3.4 ppm.

This is an indication that the bond between the 5th and 6th carbon atoms in the pyridone nucleus of dihydromocimycin is saturated. This interpretation was confirmed by an ozonisation of an aqueous solution of dihydromocimycin at - 6 43106 pH 12 during 5 minutes at 0*C. After reduction of the reaction mixture obtained with hydrogen catalysed by J?tO2 and after hydrolysis with concentrated hydrochloric acid, β-alanine was detected, indicating that the bond between the 5th and 6th carbon atoms of the pyridone nucleus of dihydromocimycin is saturated.

Many properties of dihydromocimycin are similar to those of mocimycin. However, there is one important differences a low concentration of mocimycin added to the feed of animals being fattened, such as chickens or pigs, improves the growth and the feed-conversion markedly.

I Dihydromocimycin, on the contrary, does not produce any improvement in the growth or feed-conversion of those animals, which is unexpected, since in vitro it shows antibiotic activity against the same microorganisms; in many cases it showe even a better activity than mocimycin. A comparison of the antimicrobial activities of the two antibiotics is indicated in the following Table, ... 7 _ 6 TABLE I Agar dilution tests Organism tested Minimum inhibitory concentration (gg./ml.) in 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 coli U20 >100 100 Listeria monocytogenes A2130 6 1.5 Listeria monocytogenes A2131 6 6 Listeria monocytogenes A2132 6 6 Clostridium perfrinqens A738 >100 >100 Clostridium septicum A2152 10 10 Streptococcus zooepidemieus Α2Ϊ44 6 6 R-streptococcen A2148 6 3 Brucella suis (smooth) A2126 0.75 0.4 PasteUrella haemolytica A2136 3 1.5 Treponema spec. A2275 30 10 - 8 4310 liquid dilution teats Organism tested Minimum inhibitory concentration (pg./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 Ώ220 1.2 0.9 Bacillus cereus B569 2.5 1.2 Bacillus cereus TH 1 1.8 1.2 Bacillus thurinqiensis Wll 1.2 0.9 Bacillus mesenterium 0169 100 50 Bacillus cereus var. mvcoides 1.2 0.45 Streptococcus haemolyticus A266 0.45 0.45 Streptococcus haemolyticus A2182 0.25 0.12 Mycoplasma hyorhinus A2230 1 0.3 Streptomvces viridochromoaenes 2.5 0.9 Streptomvces ramocissimus, under suitable conditions, produces dihydromocimycin in addition to mocimycin and, therefore, according to a feature of the invention, dihydromocimycin is produced by the process which comprises aerobically cultivating the microorganism Streptomvces ramocissimus (CBS 190.69), or a dihydromocimycin producing mutant thereof, in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances, and separating the dihydromocimycin - 9 3106 formed during the cultivation. Fermentation of the microorganism may be carried out with the liquid media containing the usual carbon, nitrogen, phosphorus, calcium, iron, sulphur, magnesium, potassium, vitamin and trace-element sources, such as media containing beet molasses, malt paste, peanut flour, lactose, potato starch, corn steep and yeast extract. The temperature of the fermentation medium 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 appreciated that the aforesaid process is substantially similar to that described in . Patent Specification No. 36056. for the production of mociffiycin.

To obtain a greater yield of dihydromocimycin relative to that of moeimycin, it has unexpectedly been found that this is achieved by increasing the oxygen pressure in the culture medium.

From the structure of dihydromocimycin it would be expected that a higher oxygen pressure in the culture medium would decrease the production of dihydromocimycin with respect to that of moeimycin. However, dihydromocimycin has been found to be produced more abundantly than moeimycin by better aeration of the culture liquid, which may be achieved by techniques known per se such as a higher aeration rate (volume of air per volume of culture medium per unit of - 10 43106 time) and a higher agitation rate of the culture medium in the fermenter. Suitable aeration rates of the culture medium, for example of 2 litres, are 1 litre to 3 litres of air (preferably 1.5-2.5 litres) per minute. A further improvement of the yield of dihydromocimycin is obtained by adding low concentrations of certain metal ions, such as the ions of iron, cobalt and nickel, to the culture medium.

The separation of dihydromocimycin from the culture medium is partially similar to the separation of mocimycin.

In the last step, wherein the precipitation of the compounds is from an organic solvent, use is made of a difference in solubilities of mocimycin and dihydromocimycin. Mocimycin is precipitated first after passage of gaseous ammonia through the solution, and the separation may be carried out by introducing ammonia through the solution until substantially all mocimycin is precipitated, and substantially all dihydromocimycin is left in the solution. This may be controlled, for example, by thin layer chromatographic tests. After separating, e.g. filtering off, the mocimycin precipitate from the solution, the passage of ammonia is continued until substantially all dihydromocimycin is precipitated so that it can be separated, e.g. filtered off.

The ammonia is, for example, passed through the solution at a speed of about 150 litres per litre of solution per hour during about 1 to about 4 minutes (i.e. about 2.5 to about 10 litres of gaseous ammonia per litre of solution) at a temperature between about -12®C. to about +15°G., preferably between -5*0. and +8®C. Upon continued passage of gaseous ammonia through the solution, the hydrogen ion concentration decreases sufficiently to make dihydromocimycin insoluble when the ammonia is passed with the above indicated speed during a period of 10 to about 15 minutes (corresponding to ahout 25 to about.40 litres of gaseous ammonia) under the same circumstances. In addition to ammonia, generally all alkaline compounds may be used for the separation of mocimycin and dihydromocimycin, e.g. sodium methoxide and triethylamine.

The crude dihydromocimycin thus separated from the culture medium is further purified from mocimycin by dissolving the precipitate in a highly diluted ammoniacal solution (pH 9) and extracting this solution with a solvent such as chloroform or methylene chloride. Mocimycin is poorly soluble in such a solvent, and hy pouring the extract obtained into an excess of an apolar solvent (e.g. petroleum ether, cyclohexane or pentane) a precipitate is formed of dihydromocimycin containing lees than 5% of mocimycin.

Highly purified dihydromocimycin can be obtained by passing the product obtained in the way just described over a SBPHADEX (Trade Mark) Iii 20 column using the difference - 12 43106 in adsorption of mocimycin and dihydromocimycin. The SEPHADEX xs suspended in 100% methanol and poured carefully into the column. After displacement of the methanol with chloroform, the above-mentioned dihydromocimycin precipitate is brought into the column. The eluent used is chloroform. After some time dihydromocimycin is obtained first, followed by mocimycin. Both compounds can be detected in the eluate since they show absorption in the ultra-violet spectrum at, 350 nm. The pure compounds may be recovered from the chloroform 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. Use is made of Kieselgel P 254 plates, size 20 x 5 cm. (Merck). The eluent is a 60s42;10 mixture of chloroform, ethanol and 25% ammonia, respectively. Elution time 2 hours. Mocimycin shows an Rf value of 0.3 (main tautomer) and dihydromocimycin shows an Rf value of 0.4 (main, tautomer).

Salts of dihydromocimycin, e.g. alkali metal salts, can be prepared by methods known per se.

In an early stage of investigation it was presumed that mocimycin, having a similar anti-microbial spectrum to that of tylosin (Merck Index, 8th Ed. Page 1089), might be effective against Treponema hyodysenteriae causing Treponema dysentery or Vibrio Doyle, one of the most common swine diseases. Experiments at the time wed that moeimycin was active against this disease, but not e so than tylosin. For that reason no further sstigations were carried out.

As indicated hereinbefore, dihydromocimycin has been id to be more active than moeimycin against many microorganisms consequently an investigation with this substance was made Lnst the microorganism causing Treponema dysentery. From the sriments it appeared that dihydromocimycin possesses a markedly ler activity against the microorganism than tylosin and, in .tion, was active against tylosin-resistant strains. Thus, ’dromocimycin may be regarded as being superior to isin (and also to moeimycin) in the treatment of Treponema ntery.

According to another feature of the invention there are ided pig feedstuffs useful against Treponema dysentery lemented by an effective amount of at least one compound cted from dihydromocimycin and non-toxic pharmaceutically ptable salts, e.g. sodium salt, thereof. The antibiotic, or lkali metal or ammonium or amine salt thereof, may also be ersed in, or mixed with, any suitable inert, physiologically suous carrier or diluent, which is orally administrable to a non-reactive with the antibiotic and not harmful to the pig ral administration. Effective amounts of dihydromocimycin or ι-toxic pharmaceutically acceptable salt thereof incorporated pig feedstuff for the prevention or treatment of Treponema itery are suitably 10 to 200 ppm, preferably 20 to 40 of dihydromocimycin or such a salt thereof, based on the - 14 43106 weight of the feed.

Dihydromocimycin obtained by the procedure hereinbefore described is a fine, easily dusting powder.

This could lead to difficulties in the mixing procedure with the feed and, therefore, a premix is preferably made with one or more of the components of the pig feed containing, for example, a 9 to 99-fold amount of the dihydromocimycin. Suitable components for preparing the premix are, for example, corn flour, potato flour and soya flour.

Experiments have shown that dihydromocimycin has a good stability against pelletising (granulating under high pressure at high temperatures, using steam).

The premix can be added to the feed as a prophylactic means or as a means for the treatment of pigs only lightly attacked with Treponema dysentery.

When pigs are attacked so heavily with Treponema dysentery that their appetite is lost, or that the sick pigs are pushed away from the feeding trough by healthy pigs, dihydromocimycin is preferably administered through the drinking water, preferably in the presence of a flavouring corrigent. For that purpose dihydromocimycin is used in a water-soluble form, such as a salt, e.g. a potassium, sodium or amine salt.

Pigs badly attacked with dysentery may be treated by injection of dihydromocimycin or a non-toxic pharmaceutically acceptable salt thereof, suspended or dissolved in a suitable injection liquid, for example, saline, propylene glycol and glycerol-water mixtures. - 15 6 The present invention includes within its scope a method ; protecting pigs from, or treating pigs infected with, Treponema 'sentery which comprises administering to the pigs an effective lount of at least one compound selected from dihydromocimycin d non-toxic pharmaceutically acceptable salts thereof.

Mocimycin shows advantageous growth-promoting properties en fed to live-stock including chickens and other fowl, but hydromocimycin does not show the aforesaid properties. In dition, therefore, attempts have been made to convert hydromocimycin into the, for this application, more eful mocimycin.

It has thus been found that dehydrogenation of hydromocimycin is possible with a special dehydrogenatihg bstance and procedure. Quinones, e.g. 2,3-dicyano-5,6chloro-1,4-quinone, p-chloranil and ρ-chloranil, are not tisfactory for the dehydrogenation of dihydromocimycin as her products are formed. Halogenation with cupric bromide, omine, iodine or N-bromosuccinimide, followed by dehydrologenation, did not give the desired result either, even the presence of catalysts such as benzoyl peroxide and a, ceo-bis-isobutyronitrile. Attempts to dehydrogenate dihydromomycin catalytically involve too high temperatures which would ad to decomposition of dihydromocimycin. Only one hydrogenation agent, selenium dioxide, has been found to be aful for the dehydrogenation of dihydromocimyciil.

Therefore, the present invention further relates to process for the dehydrogenation of dihydromocimycin into mocimycin, which comprises reacting dihydromocimycin with selenium dioxide. This process may advantageously be applied to mixtures of dihydromocimycin and mocimycin as obtained, for example, by the recovery of mocimycin from fermentation liquids in which it is formed.

The dehydrogenation of dihydromocimycin with selenium dioxide may be carried out at ambient temperature, but is preferably carried out at elevated temperatures, e.g. from 65° to 110°C., preferably from 80°C to 95°C.

The reaction time is from about 10 hours to about 20 minutes in the temperature range of about 65°C. to about 11O°C., and is preferably from about 3 hours to about 1 hour in the preferred temperature range. At ambient temperatures the reaction takes about a week.

The reaction is preferably carried out in a solvent (preferably an organic solvent) medium. Suitable solvents are, for example, hexamethylphosphortriamide (HMPT), dimethyl sulphoxide (DMSO), t-butanol, t-amyl alcohol, sec-butanol, hexylene glycol, n-butanol, isopropanol, 2-methoxyethanol, dimethylformamide, water, phenylmethylcarbinol and propanol, and mixtures of two or more of those solvents. Preferred solvents are HMPT, DMSO and t-butanol.

The most preferred solvent is HMPT.

Based on dihydromocimycin, a stoichiometric amount or an excess of selenium dioxide is preferred to carry out the dehydrogenation reaction.

Since isolation of both products together from the fermentation liquid is a simple procedure, whereas the separation, of dihydromocimycin from mocimycin is much more difficult, the discovery that dihydromocimycin may be converted by chemical means into - for the growth promotion, zaluable properties - mocimycin, even in a mixture of both Compounds, leaving the latter compound substantially inaffected by the process, is very important. Therefore, isolation of the compounds is not necessary for the lehydrogenation of dihydromocimycin in mixtures containing lihydromocimycin and mocimycin.

The invention is illustrated by the following xamples.

EXAMPLE 1. reparation of dihydromocimycin.

The microorganism Streptomyces ramocissimus CBS 190,69) was fermented in 2,000 litres of medium ontaining 20 g. of malt .paste, 10 g, of yeast extract nd 5 g. of corn steep solids per litre at a pH of about with agitation and aeration. After fermentation the ilture medium was mixed with about 2% of Dicalite - a sgistered Trade Mark (an expanded perlite, an aluminium .licate containing potassium, sodium and trace elements) as .Iter aid, and the mixture was filtered. The filtrate was - 18 43106 acidified with 8N sulphuric acid to pH 6.0 and extracted twice with l/5th of its volume of methyl isobutyi ketone (hereinafter abbreviated to MIBK) and emulsions formed were broken with Hyflo Supercel filter aid (a diatomaceous earth). Hyflo and Supercel are registered Trade Marks. The organic liquids were mixed and concentrated to about 1 litre by evaporation under reduced pressure and evaporation with a rotary evaporator. From the so-formed concentrate a crude product was obtained by adding it to 5 times its own volume of petroleum ether (b.p. 4o°C. to 60°C.), and the precipitate formed was filtered off with a glass filter (G 3). The precipitate was washed with fresh petroleum ether and dried to obtain a yellow coloured powder containing mocimycin and dihydromocimycin.

A purified form of dihydromocimycin containing not more than 5% of mocimycin was obtained as follows: Gaseous ammonia was passed through the concentrate at a rate of I5o litres per litre of concentrate per hour for 1 minute at a temperature of 2°C. A precipitate was formed which contained mocimycin. The precipitate was filtered off and the filtrate was treated once more with gaseous ammonia for 10 to 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 poured out into 3 to 5 times its own volume of cyclohexane. The precipitate obtained was filtered off, dried and powdered. - 19 The sodium salt of dihydromocimycin was obtained by dissolving dihydromocimycin in water with addition of 0.1N sodium hydroxide to pH 9 until a saturated solution was obtained. The solution was filtered and evaporated azeotropieally with addition of butanol (in vacuo at about 45°C.), and the butanolic residue was collected in a small amount Of anhydrous butanol. Petroleum ether was added dropwise to the stirred solution until all the salt was 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.

EXAMPLE 2 Preparation of dihydromocimycin in higher yields.

To obtain a higher yield of dihydromocimycin, a lyophilised culture of Streptomvces ramocissimus (CBS 190.69), or a well sporulated agar culture of the said microorganism, was used for inoculating the contents of a 500 ml. erlenmeyer flask containing 100 ml. of a sterilised medium of the following compositions 20 g. of malt paste, g. of yeast extract and 5 g. of com steep solids per litre of tap water, pH 7.0.

After incubation on a rotating shaking device (300 rpm, stroke 2.5 cm) at 30°C. for three days, the culture obtained was used for inoculating small fermenters containing 2000 ml. of the above-mentioned medium to which mg. of ΟοΟ^-δΗ^Ο per litre were added. This growth phase, also effected at 30°C., was carried out under conditions of very good aeration in order to stimulate production of dihydromocimycin. For that purpose, more than 2 litres of sterile air were blown through the culture medium per minute, and the culture medium was stirred at a speed up to 1000 rpm. The production of dihydromocimycin started after a fermentation time of about 12 hours and was maximal after about 120 hours. Fermentation on a larger scale was possible by using a 48 hours old culture medium obtained in small fermenters as inoculum for large fermenters.

The dihydromocimycin so produced was recovered from the culture medium as followss After addition of 2% of diatomaceous earth as a filter aid, the culture was filtered and the filtrate was acidified with 8N sulphuric acid to a pH of 5 to 6 and was extracted twice with l/5th of its volume of MIBK. If an emulsion formed, it was broken by filtration of the mixture after addition of some diatomaceous earth.

The organic layers were collected and concentrated in vacuo to about l/10th of the original culture volume. Gaseous ammonia was passed through the concentrate at a rate of 150 litres per litre of concentrate per hour for 1 minute. A precipitate formed which was filtered off. The filtrate was treated once more with gaseous ammonia for 10 to 15 minutes. The precipitate then obtained was dissolved in dilute ammonia · 21 » (pH 9.0) and purified dihydromocimycin (containing not more than 5% of mocimycin) was obtained by extracting the Solution in ammonia with an equal volume of methylene chloride. The extract was poured into 3 to 5 times its own volume of cyclohexane and the precipitate obtained was filtered off, dried and powdered.

EXAMPLE 3 Dihydromocimycin against Treponema dysentery.

Twenty pigs, three months old, were infected with the microorganism causing Treponema dysentery by administering to them feed mixed with a homogeneous mixture of contents of the intestines and intestinal mucous membranes of two animals suffering from the disease. The infected pigs were divided up into four groups of five animals each and the animals were fed with the feed as a slurry diluted 1:1 with water for 1 week. The total amount of feed per animal was 1.2 kg. each day and was given in two portions.

After 5 days the first symptoms of the disease were observed from the thin faeces and confirmed by a microbiological investigation of the faeces. After a week the animals were treated as follows: group 1: no antibiotic was added to the feed; group 2s 100 ppm of tylosin were added to the feed; group 3s 25 ppm of dihydromocimycin were added to the feed; group 4s 50 ppm of dihydromocimycin were added to the feed.

The antibiotic-enriched feed was administered for a week. After that week feed without any antibiotic was given again. Recovery from the infection was observed from the weights of the animals and from inspection of samples of the faeces, macroscopically from their consistencies, and microscopically by means of a specific immuno-fluorescence technique.

During the test one animal of each of groups 1, 3 and 4 died.

The results are shown in the following Table, wherein the weights indicated are the averages at that time of the still living animals. The consistency of the faeces is indicated as follows; + means thin liquid; + means thick liquid; - means normal.

The results of the immuno-fluorescence technique are indicated quantitatively by indicating the nuittber of Treponemas in the visual field; 5 means very crowded; 4 means many; 3 means about 10 Treponemas; 2 means 1 or 2 Treponemas, means more visual fields necessary to find one Treponema and - means negative. 6 TABLE II Group 1 Group 2 Group 3 Group 4 Weights 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 + + + + + + + + + + + + + + + + ώ + + after 3 weeks + + + + is + + + + + — — + + --+ _ — - Immuno-fluorescence after 1 week 4 4 4 4 5 4 4 3 4 - 5 4 5 5 4 5-533 after 2 weeks 4 4 4 -4 4 4 2 2 - 4 4 3 1 2-441 after 3 weeks 2 2 2 2 έ 3--2- - --1 (1) at which time the administration of the antibiotics is started;.. & means ; an animal died.

From the faeces consistency as well as from itomuno-fluorescence observations it appeared that the animals treated with dihydromocimycin were cured markedly faster than the animals treated with tyIosin» When the administered amount» of antibiotics were also taken into account, it can be concluded that dihydromocimycin is at least 4 times as active as tylosin.

EXAMPLE 4 Dihydromocimycin against.Treponema dysentery.

Pigs, from a farm where problems with pigs scour had existed for some time, were treated under the supervision of the local veterinary surgeon and the inspector of the Health Service Station, The pigs were divided into groups and treated for 4 days in the following manner: group ls 66 pigs were treated with feed containing 100 ppm of tylosin; group 2s 34 pigs were treated with feed containing 25 ppm of dihydromocimycin; group 3s 40 pigs were treated with feed containing 50 ppm of dihydromocimycin; group 4s 40 pigs were treated with feed containing 100 ppm of dihydromocimycin.

Before the treatment all animals lost thin or very thin faeces. Samples thereof were investigated and found to be Treponema-positive, and Salmonella-negative. Sometimes worm eggs were found.

One day after the start of the treatment the faeces of the pigs of group 4 were normal. The animals of group 1 were cured only after 3 to 4 days. The animals of ips 2 and 3 were cured in periods lying between those (roups 1 and 4. The animals treated with dihydromocimycin iared to be much more lively than before the treatment, animals were not averse to feed containing dihydromocimycin the general conclusion was that a dosage of 25 ppm of dromocimycin was better than a dosage of 100 ppm of tylosin he curing of Treponema dysentery.

EXAMPLE 5 droqenation of dihydromocimycin.

A solution was made of 1 g. of dihydromocimycin in L. of HMPT (technical grade, dried over a molecular 3 3A) and an amount of 139 mg. (1.25 mmoles) of selenium Lde was added. The mixture was heated on a steam bath LOO minutes and an additional amount of 139 mg. of iium dioxide was added after 60 minutes. The selenium :d after cooling was separated by filtration through a .ass filter and the precipitate was washed with a small it of methanol. The filtrate was poured into 350 ml..of lied water and the precipitate formed was filtered off ashed with distilled water. The filtrate was stored.

The precipitate was dissolved in methanol and subsequently ed with MIBK. The Solution was evaporated at 40°C. reduced pressure until methanol and water were removed, cipitate was formed which did not contain mocimycin as ated by a thin-layer chromatographed (TLC) test and it consisted of polar impurities only. The precipitate was filtered off and washed with MIBK. The filtrate was added dropwise to an excess of petroleum ether (b.p. 40° to 60°C) and the precipitate formed was filtered off, washed with petroleum ether and dried to obtain 500 mg. of product.

The stored filtrate was extracted with MIBK, and the extract was added dropwise to petroleum ether to obtain another 100 mg. of product of the same quality so that the total yield was 650 g. A thin layer chromatographic test showed that the final product contained mocimycin.with only a trace of dihydromocimycin.

EXAMPLE 6 Dehydrogenation of dihydromocimycin.

This experiment was carried out in duplicate. 2.4 g. of a composition containing 22.8% of mocimycin and 35.5% of dihydromocimycin was dissolved in 50 ml. of HMPT.

An amount of 350 mg. of selenium dioxide was added and the mixture was kept at 105°C. for 45 minutes with stirring. According to a thin layer chromatographic test dihydromocimycin was no longer present. The mixture was cooled to room temperature and filtered through a g4 glass filter, removing the black selenium formed, and. a small amount of unreacted selenium dioxide.

The precipitate was washed with MIBK and to the 6 combined filtrate and MIBK washings 350 ml. of water, 10 g. of sodium chloride and 5 ml. of hydrogen chloride were added. This mixture was extracted 3 times with 100 ml. of MIBK each time. The combined MIBK extracts were washed with 100 ml of water and the water layer was removed. The MIBK phase was dried over anhydrous magnesium sulphate which eventually was washed with a little MIBK. The MIBK phase was then evaporated in vacuo at a temperature of about 20°C until 25 ml. were left. The residue was added dropwise to 500 ml. of stirred petroleum ether (4o°-6O°C.) and the precipitate formed was filtered off, washed with petroleum ether (40°50°C.) and dried in a vacuum drying oven at room temperature overnight.

In the first experiment 1.64 g. of a product containing 41.2% of mocimycin and ¢2% of dihydromocimycin '.as determined by high pressure liquid chromatography) was ibtained.

In the second experiment the yield was 1.79 g of ι product containing 4l.6% of mocimycin and ¢1% of ίihydromocimycin.

EXAMPLE 7 ehydroqenation of dihydromocimycin, influence of solvents.

In this Example several solvents were tested to ind out which one is preferred for the dehydrogenation of ihydromocimycin.. In all experiments 50 mg. of a technical - 23 4310 preparation containing dihydromocimycin and moeimycin were reacted with 20 mg. (0.18 mmole) of selenium dioxide in 3 ml. of solvent at a temperature of 80°C. At 2, 4 and 7 hours after the start of the reaction a sample was taken for a thin-layer chromatographic test (if necessary after sample preparation) on a Kieselgel 60 disc (Merck) using a 50:45:5 mixture of MIBK, acetone and water as the eluent. Detection was carried out by carbonization after spraying with a sulphuric acid-diethyl ether mixture. A qualitative picture of the reaction was obtained in this manner.

The results are as follows: Decomposition-or no reaction was obtained in propargyl alcohol, diacetone alcohol nitromethane, propylene carbonate, acetonitrile, pyridine, sulpholane, benzyl alcohol, mesityl oxide, ethylene carbonate N-methyl-2-pyrrolidone, Ν,Ν-dimethylacetamide, dioxan, MIBK, butyl acetate, amyl acetate, N-methylacetamide, anisole, diglyme and 1,2-dichloroethane. A poor conversion was obtained in tetramethylurea, a 0.1 M buffer of NaHCO^/Na^CO^ of pH 9.1, n-propanol, phenylmethylcarbinol, water, and dimethylformamide. A somewhat better conversion was obtained in 2-methoxyethanol. isopropanol, n-butanol, hexylene glycol, sec-butanol and t-amyl alcohol. A moderate conversion was obtained in t-butanol, and dimethyl sulphoxide The best result was obtained in HMPT.

EXAMPLE 8 Dehydrogenation of dihydromocimycin in mixtures of solvents.

In this Example mixtures of solvents with HMPT as one of the components were tested to find out the preferred mixture for carrying out the dehydrogenation of dihydromocimycin.

In each of the experiments 2.4 g. of the preparation as used as the starting material in Example 7 were used.

After the reaction (i.e. after all dihydromocimycin was converted according to a TLC test), the reaction mixtures were recovered as follows: After cooling, the reaction mixture was filtered through a G4 glass filter to remove the selenium formed and the filter was washed with a small amount of methanol. The filtrate was poured into an excess of water and was acidified to pH 3 and then extracted three times with l/4th of its volume of MIBK. A tarry interlayer which was formed was extracted with MIBK again by dissolving it in methanol first and diluting it with MIBK and water thereafter. The combined MIBK extracts were washed three times with l/3rd of its volume of water , and the organic layer was concentrated in vacuo at about 40°C. In all cases a precipitate was formed sssentially consisting of decomposition products (according ;o TLC). The precipitate was filtered off and washed with MIBK md the combined MIBK concentrates were added slowly, with - 30 43106 stirring, to an excess of petroleum ether (b.p. 40° to 60°C.). The precipitate formed was filtered, washed with petroleum ether and dried. The results of the experiments are shown in the following Table. 6 α •Η §. c 4 ο 4 ο 81* S(tw Φ 8 ’η β Β εό η H ·· CO H • I CM • H H - ·· ΙΛ H ·« CM • CO H •t cM » CO H «· © H ·· N ι H <*» ?& CM 8 - cm «Ρ in m CM «Φ- © r< time of addition after start of reaction m « · - O d + e P in p in ti P P wo. p . in M4 .* ti o » .»c -S-S’g eeo o in co m co h ρ ρ ρ p Ρ Φ Φ Φ ti P +) P p m m m W ti ti ti Η Η Η H • · · 4 oooo • • · k c e ti c-h Se^o in in in cm CM Γ r-j fl P k Μ M M 0) 0 0 CD nj 4j +1 y +j +1 iy ih IW ih n ni nJ id io rjrj rlHrj · · · « o o o o o • • ti H g eo Ο H Ν H ρ ρ p P 0) Φ ti P P Ρ Μ M4 ti ti ti in in tn CO CO CO • · · ο o © • • β 4’g eo in in > H Ρ Ρ P Ρ Φ Φ ti Ρ P Ρ Μ M-l W ti ti in in in co co co « « « ο ο o w β H 6 m co P p Ρ Φ ti P -P Mm w ti , o o • · Η H -P p ti -P w t o , g ' i»C' CM co CM CO CO co CM c ; ο Ό •rl φ M A &M 3 m,§ φρρ , fi ti tiK ♦ O > 0 : © ; © © © 4 . co 10 . © © © • O CO in © ii in m ci ro S o 2 N . SB ' ri in i h co co p in SOO Φ cm y u • · o o CM H § 3Eh • « oo o m H g +> s • J © in H I fl « « o o o m H § 3ft m a fl s i o in K i The follovzing abbrevations are used: HMPT = hexamethylphosphortriamide t-BuOH = t-butanol t-AmOH = t-amyl alcohol The Table shows that the highest yield (79%) is obtained when pure HMPT is used as the solvent. A high yield is also obtained in a mixture of HMPT and t-amyl alcohol.

The present invention also includes within 10 its scope a method of combatting bacteria in warm-blooded animals other than humans which comprises administering to warm-blooded animals other than humans suffering from a bacterial infection ai effective amount of dihydromocimycin or a non-toxic pharmaceutically acceptable salt thereof.

Claims (25)

1. WE CLAIM;

1. Dihydromocimycin having the following structural formulas- and its non-toxic pharmaceutically acceptable salts.

2. A salt of dihydromocimycin according to claim 1 which is an alkali metal, ammonium or amine salt.

3. A process for the production of dihydromocimycin which comprises aerobically cultivating the microorganism Streptomyces ramocissimus (CBS 190.69), or a dihydromocimycin-producing mutant thereof, in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic substances,and separating the dihydromocimycin formed during the cultivation.

4. A process according to claim 3 in which the culture medium is aerated at. a rate of from 1 to 3 litres of air per 2 1 itres of culture medium per minute.

5. A process according to claim 3 in which the culture medium is aerated at a rate of from 1.5 to 2.5 litres of air per 2 litres ofculture medium per minute.

6. A process according to claim 3« 4 or 5 in which a low concentration of iron, cobalt or nickel ions is present in the culture medium.

7. A process according to any one of claims 3 to 6 in which the separation of dihydromocimycin from mocimycin (also produced during the aerobic cultivation of Streptomvces ramocissimus) is carried out by addition of an alkaline compound selected from ammonia, sodium methoxide and triethylamine to a solution of the two antibiotics in an organic solvent until mocimycin is precipitated, separating the mocimycin, and continuing the addition of the alkaline compound until the dihydromocimycin is precipitated, and separating the dihydromocimycin.

8. A process for the production of dihydromocimycin substantially as hereinbefore described with especial reference to Example 1 or 2.

9. Pig feedstuffs useful against Treponema dysentery supplemented by an effective amount of at least one compound as claimed in claim 1.

10. A composition according to claim 9 which contains 10 to 200 ppm of a compound as claimed in claim 1, based on the weight of the pig feedstuff.

11. A composition according to claim 9 which contains 20 to 40 ppm of a compound as claimed in claim 1, based on the weight of the pig feedstuff.

12. A method of protecting pigs from, or treating pigs with, Treponema dysentery which comprises administering to the pigs an effective amount of at least one compound as 3108 claimed in claim 1.

13. A method according to claim 12 in which the compound as claimed in claim 1 is contained in a pig feedstuff.

14. A method according to claim 12 in which a salt of dihydromocimycin is administered to the pigs as a solution in drinking water.

15. A method according to claim 12 in which the compound as claimed in claim 1 suspended or dissolved in a suitable injection liquid selected from saline, propylene glycol and glycerol-water mixtures is administered to the pigs by injection.

16. A process for the preparation of moeimycin which comprises the dehydrogenation of dihydromocimycin by reacting dihydromocimycin with selenium dioxide.

17. A process;, according to claim 16 in which a mixture of moeimycin and dihydromocimycin is used as the starting material.

18. A process according to claim 16 in which the reaction is carried out at a temperature between 65°C. and 110°C.

19. A process according to claim 16, 17 or 18 in which the reaction between the dihydromocimycin and selenium dioxide is carried out at a temperature between 30°C. and 95°C.

20. A process according to any one of claims 16 36 ^3106 to 19 in which the reaction is carried out in a solvent selected from hexamethylphosphortriamide, dimethyl sulphoxide» t-butanol» t-amyl alcohol, sec-butanol, hexylene glycol, n-butanol, isopropanol, 2-methoxyethanol, 5 dimethylformamide, water, phenylmethylcarbinol and propanol, or a mixture of two or more of such solvents.

21. A process according to claim 20 in which the reaction is carried out in hexamethylphosphortriamide.

22. A process according to claim 20 in which the 10 reaction is carried out in dimethylsulphoxide or t-butanol.

23. A method for the preparation of mocimycin substantially as hereinbefore described with especial reference to Example 5, 6, 7 or 8.

24. Mocimycin when prepared by the process 15 claimed in any one of claims 16 to 23.

25. A method of combatting bacteria which comprises administering to warm-blooded animals other than humans suffering from a bacterial infection an effective amount of at least one compound as claimed in claim 1.