CA1074307A - Antibiotic - Google Patents
AntibioticInfo
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- CA1074307A CA1074307A CA324,111A CA324111A CA1074307A CA 1074307 A CA1074307 A CA 1074307A CA 324111 A CA324111 A CA 324111A CA 1074307 A CA1074307 A CA 1074307A
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- dihydromocimycin
- mocimycin
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Abstract
ABSTRACT OF THE DISCLOSURE
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 growth-promoting 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.
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 growth-promoting 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 inventi~n relatc~ to a n~w ~nti~iotic ~esignated dihydromocimycin, a process for its production and compositions containing it. The antibiotic is especially useful against the pigs' disease called Treponema dysentery, Vibrio Doyle, etc.
The invention further relates to a process for using dihydromo-cimycin as a starting material for the production of mocimycin.
Dihydromocimycin has the following structural formula ~" f ~ocn3 o ~
OH
Dihydromocimycin is produced by fermenting Streptomyces ramocissimus, or suitable mutants thereof and is formed in addition to mocimycin. Streptomyces ramocissimus is a microorganism described in British Specification No. 1325200;
the microorganism is deposited with the culture collection of Centraal Bureau voor Schimmelcultures at Baarn, The Netherlands, where it obtained the number CBS 190.69 and is available to the public.
In British Specification No. 1325200 a process is described and claimed for the production of mocimycin (then called MYC ~003) by the above indicated microorganism. The structure of mocimycin is indicatea by Vos and Verwiel in Tetrahedron Letters 52 (1973) pp. 5173-5176. It has now been discovered that Streptomyces ramocissimus, or suitable mutants thereof, produce dihydromocimycin in addition to mocimycin.
Dihydromocimycin is a pale yellow solid substance, weakly acid and further characterized by the following physico-chemical properties:
i~7'~307 Solubilit~:
The solubility of the compound is good in chloroform, methyl isobutyl ketone, ethyl acetate, butyl acetate, acetone, dioxane, 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, cyclo-hexane and petroleum ether.
Optical rotation:
1~D = -85 (1~ methanolic solution).
Melting point:
The compound does not melt, but decomposition starts at 123 C.
Elementary analysis:
The following values were found:
Found:Calculated for C43H62N2O12 2 C: 61.8% 61.8%
H: 7.5% 8.0%
N: 3.4% 3.3%
O: 27.2% (by difference) 26.8 Ultra-violet spectrum:
The ultra-violet spectrum of dihydromocimycin in a 1:1 mixture of water and methanol at various pH values is shown in Figure 1 of the attached drawings. The concentration of the solutions measured was 18.3 ~g/ml. The ultra-violet spectrum is dependent on the pH. The following maxima were found (the molecular extinctions are indicated between brackets):
methanol - water: 233.5 nm ( = 63,000~; 267 nm (E = 23,000) 291 nm ( = 19,000) and 333 nm ( = 18,000); curve 1;
methanol - 0.5N NaOH: 235 nm ( = 62,000); 277 nm ( = 25,000) and 308 nm (~ = 29,000); curve 2;
iO74307 methanol - 0.5N HCl: 233.5 n~ (~ = 65,000); 268 nm (~ = 25,~00) and 338 nm (~ = 14,000);
curve 3.
Infr_-red spectrum:
The I.R. spectrum of dihydromocimycin in chloroform and in potassium bromide is shown in Figures 2 and 3, respectively. The following principal absorptions were found:
chloroform (v in cm 1): +3445 (sh); 3430; 2979; 2941; 2886;
1662-1653; 145~; 1100; 1080; 1040; 998; 944; 893; 870 and 840;
potassium bromide (v in cm ): +3aoo-3320; 2972; 2935; 2880;
1650; 1535; 145S; 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 Mc).
Thin layer chromatography-Thin layer chromatograms of dihydromocimycin were made on Kieselgel F 254 plates (Merck, ready-for-use plates), and after drying the spots were detected by fluorescence extinction, or by carbonization after spraying with a diethyi ether-sulphuric acid mixture. Investigations showed that the spot ascribed to dihydromocimycin, even 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-dimention~l chromatogram.
The following Rf-values were found for dihydromocimycin in various eluents (the Rf-value for the additional spot is indicated between brackets):
50:45:5 mixture of methyl isobutyl ketone, acetone and water:
0.44 (about 0.44);
70:2Q:lQ:0.5 mixture of ethyl acetate, methanol, water and 25%
ammonia:
iO7~307 0.29 (0.36);
65:40:9 mixture of benzene, 10~ ethanol and 33~ ammonia:
0.16 (0.22) 60:~2:10 mixture of chloroform, 96% ethanol and 25~ ammonia:
0.4.
The structural formula of dihydromocimycin is confirmed on the following grounds: The PMR spectra (220 Mc) of mocimycin (cf. Tetrahedron ~etters 52 (1973) pp. 5173-5176 for its structural formula) and dihydromocimycin showed that both compounds are similar, except that two doublets at ~ 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 dihydromocimycin. Those signals are caused by the protons of the 5th and 6th carbon atoms of the pyridone nucleus.
However, two triplets appeared in the spectrum of dihydromocimycin at ~ 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 pH 12 during 5 minutes at O~C. After reduction of the reaction mixture obtained with hydrogen catalyzed by PtO2 and after hydrolysis with concentrated hydrochloric acid, ~-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 difference:
a low concentration of mocimycin added to the feed of animals h~ing fattened, such as chickens or pigs, improves the growth and the feed-conversion markedly. Dihydromocimycin, on the contrary, does not show any improvement of the growth or feed-conversion of those animals, which is unexpected, since in vitro it shot~s activity against the same microor~anisms; in manlr cases it shows even a better activity than mocimycin. A comp~rison of the antimicrobial activities of the two antibiotics is indicated in the following table:
Agar dilution tests Organism tested Minimum inhibitory concentration (~g./ml.) in anaerobic culture mocimycindihydromocimycin _ _ Staph~lococcus aureus A 2000 >100 >100 Staphylococc~s 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 Listerla monocyt~genes A2131 6 6 Listeria monocytogenes A2132 6 6 Clostridium perfrinqens A738 >100 >100 Clostridium septicum A2152 10 10 Strëptococcus zooepidemicus R-Streptococcen A2148 6 3 Brucella suis (smooth) A2126 0.75 0.4 Pasteurella haemolytica A2136 3 1.5 Treponema spec. A2275 30 10 .
Liquid dilution tests Organism ~ested Minimum inhibitory concentra-tion (~g./ml.) mocimycin dihydromocimycln ._ --Bacillus subtilis ATCC 6633 100 50 Bacillus subtilis ATCC 6051 100 50 Bacillus subtilis 6346 D167 1.2 0.9 Bacillus subtilis 220 D17875 75 Bacillus subtilis T~ 10 100 50 Bacillus cereus D166 0.6 0.6 Bacillus cerëus 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 Wll1 2 0 9 Bacillus mesenterium D169100 50 Bacillus cereus var. mycoides 1.2 0.45 Streptococcus haemolyticus A266 0.45 0.45 Streptococcus haemolYticus A2182 0.25 0 12 Mycoplasma ~ rhi~ AZ730 1 0 3 Steptom~es virid~chromoyenes 2~5 0.9 _ lV'7430~7 S~reptomyces ramocissimus, under suitable conditions, -does produce 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 Streptomyces ramocissimus (CBS 190.69), or a dihydromocimycin-producingmutant thereof, in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances, and separating the dihydromocimycin 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 40C, preferably between 26 and 34C, and the pH between 5 and 9, preferably between 6.5 and 8.
It will be appreciated that the aforesaid process is similar to that for the production of mocimycin, and it has been found that dihydromocimycin is.formed under suitable conditions in addition to mocimycin in the fermentation of Streptomyces ramocissimus, or its mocimycin- (and dihydromocimycin-) producing mutants.
To obtain a greater yield of dihydromocimycin relative t~ that of mocimycin, 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 mocimycin. However, dihydromocimycin has been found to be produced more abundantly then mocimycin by better aeration of 1~4307 the culture liquid, which may be achievecl ~y techni~ues known per se, such as a higher aeration rate ~volume of air per volume of culture meaium per unit o~ time), a higher agitation rate of the cultu.~e medium in the fermenter. Suitable aeration rates of the culture medium, e.g. of about 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, e.g. 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 ~ihydxomocimycin 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 solution3 at a temperature ~etween about -12C to about +15 DC ~
preferably between -5C and +8C. 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 the 10 to about 15 minutes (corresponding to about 25 to about 40 74;~07 litres ~f ~a~eous anunonia) 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 by 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 less than 5% of mocimycin.
Highly purified dihydromocimycin can be obtained by passing the product obtained in the way just described over a SEPHADEX (Trade Mark) LH 20 column using the difference in adsorption of mocimycin and dihydromocimycin. The SEPHADEX is 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 ~e 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 F 254 plates, size 20x5 cm (Merck). The eluent is a 60:42:10 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).
In an early stage of investigation it W25 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 that time showed that mocimycin was active against this disease, but not more so than tylosin. For that reason no further investigations were carried out.
As indicated hereinbefore, dihydromocimycin was found to be more active than mocimycin against many microorganisms, and consequently an investigation with this substance was made against the microorganism causing Treponema dysentery. From the experiments it appeared that dihydromocimycin possesses a markedly higher activity against the microorganisms than tylosin and, in addition, was active against tylosin-resistant strains. Thus, dihydromocimycin may be regarded as being superior to tylosin (and also to mocimycin) in the treatment of Treponema dysentery.
According to another feature of the invention there are provided pig feedstuffs supplemented by a significant proportion of dihydromocimycin or a slat, e.g. sodium salt, thereof. The antibiotic or its alkali metal or ammonium or amine salt thereof may also be dispersed in, or mixed with, any suitable inert, physiologically innocuous carrier or diluent, which is orally administrable to a pig, non-reactive with the antibiotic and not harmful to the pig on oral administration. Effective amounts of dihydromocimycin incorporated in a pig feedstuff for the prevention or treatment of ~reponema dysentery are about 10 to about 200 ppm, preferably 20 to 40 ppm, of dihydromocimycin, based on the weight of the feed.
Dihydromocimycin obtained by the procedure hereinbefore described is a fine, easily dusting powder. This could lead to difficulties in the mi~ing procedure with the f~e~ 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 dihy~romocimycin 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 water-soluble salt thereof, suspended or dissolved in a usual injection liquid, for example, saline, propylene glycol, glycerol-water mixtures, etc.
Mocimycin shows advantageous growth-promoting properties when fed to lîve-stock including chickens and other fowl, but dihydromocimycin does not show the aforesaid properties. In addition, therefore, attempts have been made to convert dihydromocimycin into the, for this application, more useful mocimycin.
It has thus been found that dehydrogenation of dihydromocimycin is possible with a special dehydrogenating substance and procedure. Quinones, e.g. 2,3-dicyano-5,6-dichloro-1,4-quinone, p-chloranil and o-chloranil, are not satisfactory for the dehy~roqenation of dihydromocimycill as other products are formed. Halogenation with cupric bromide, bromine, iodine or N-bromosuccinimide, followed by dehydrohalogenation, did not give the desired result either, even in the presence of catalysts such as benzoyl peroxide and ~ azo-isobutyronitrile. Attempts to dehydrogenate dihydromocimycin catalytically involve too high temperatures which would lead to decomposition of dihydromocimycin.
Only one dehydrogenation agent, selenium dioxide, has been found to be useful for the dehydrogenation of dihydromocimycin.
Therefore the present invention further relates to a 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 about 65 to about 110C, preferably from 80 to 95~. The reaction time is from about 10 hours to about 20 minutes in the temperature range of about 65 to about 110C, and is preferably from about 3 hours to about 1 hour in the preferred temperature ran~e. At ambient temperatures the reaction takes about a week.
The reaction is preferably carried out in a solvent me2ium. Suitable solvents are, for example, hexamethylphosphor-triamide (HMPT), dime~hyl sulphoxide (DMSO), t-butanol, t-amyl alcohol, sec-butanol, hexylene glycol, n-butanol, isopropanol, methylcellosolve, dimethylformamide, water, phenylmethylcarbinol and propanol, and mixtures o 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 and mocimycin is much more difficult, the discovery that dihydromocimycin may be converted by chemical means into - for the growth promotion valuable properties -mocimycin, even in a mixture of both compounds, leaving the latter compound substantially unaffected by the process, is very important. Therefore, isolati~on of the compounds is not necessary for the dehydrogenation of dihydromocimycin in mixtures containing dihydromocimycin and mocimycin.
The invention is illustrated by the following examples.
Pre~_ration of dihydromocimycin The microorganism Strep~ ces ramocissimus (CBS 190.69) was fermented in 2,000 litres of a medium containing 20 g of malt paste, 10 g of yeast extract and 5 g of corn steep solids per litre at a pH of about 7 with agitation and aeration. After fermentation the culture medium was mixed with about 2% of dicalite (an expanded perlite, an aluminium silicate containing potassium, sodium and trace elements) as filter aid, and the mixture was filtered. The filtrate was acidified with 8N
sulphuric acid to pH 6.0 and extracted twice with 1/5th of its volume of methyl isobutyl ketone (MIBK) and emulsions formed were broken with *Hyflo Supercel filter aid (a diatomaceous earth). The organic liquids were mixed and concentrated to about 1 litre by evaporation under reduced pressure and evaporation with a rotary evaporator. From the concentrate formed a crude product was obtained by adding it to 5 times its own volume of petroleun-l ether (b.p. 40 to 60 C) and the precipitate formed -~Trade Mark was filtered off with a c31ass 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 150 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.
The sodium salt of dihydromocimycin was obtained by dissolving dihydromocimycin in water with addition of 0.lN
sodium hydroxide to pH 9 until a saturated solution was obtained.
The solution was filtered and evaporated azeotropically with addition of butanol tin vacuo at about 45C), 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 i~ a similar manner.
Preparation of dihydromocimycin in higher ~ields To obtain a higher yield of dihydromocimycin, a lyophilised culture of Streptomyces ramocissimus (CBS 190.~9), 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 comn~sition: 2Q g of malt paste, 10 g of ye2st extr2ct and 5 g of corn steep solids per litre of tap water, p~l 7Ø
After incubation on a rotating sha~ing device (300 rpm, stroke 2.5 cm) at 30C for three days, the culture obtained was used for inoculating small fermenters containing 2000 ml of the above-mentioned medium to which 20 mg of CoC12.6H20 per litre was added. This growth phase, also effected at 30C, 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 timP 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 follows: 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 1/5th 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/lOth 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 was formed which was filtered off. The filtrate was treated once more with gaseous ammonia for 10 to 15 minutes. The precipitate then o~tained was dissolved in dilute ammonia (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.
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 2: 100 ppm of tylosin were added to the feed;
group 3: 25 ppm of dihydromocimycin were added to the feed;
group 4: 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 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 1~74307 living animals. The consistency of the faeces is indicated as follows: -+ means thin liquid; + means thicl; liquid; - means normal.
The results of the immuno-fluorescence technique are indicated quantitatively by indicatinq the number of Treponemas in the visual field: 5 means very crowded; 4 means many; 3 means about 10 Treponemas; 2 means 1 or 2 Treponemas, 1 means more visual fields necessary to find one Treponema and - means negative.
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 + + ~ + + + + + + _ + + + ~ ~ + +
after 2 weeks + + + + + + + + + + + + + + + + ~ +
after 3 weeks + + + + ~ + + + + + _ _ + + _ _ + _ _ .
Immunofluorescence after 1 week 4 4 4 4 5 4 4 3 4 - S 4 5 5 ~ 5 - 5 3 3 after 2 weeks 4 4 4 - 4 4 4 2 2 - 4 4 3 1 2 - ~ 4 after 3 weeks 2 2 2 2 ~ 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 immunofluorescence observations it appeared that the animals treated with dihydromocimycin were cured markedly faster than 30 the animals treated with tylosin. When the administered amounts of antibiotics were also taken into account, it can be concluded . .
that dihydromocimycin is at least 4 times as active as tylosin.
D ~_omocimycin against Treponema dysentery Pigs, from a farmwhere 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 1: 66 pigs were treated with feed containing 100 ppm of tylosin;
group 2: 34 pigs were treated with feed containing 25 ppm of dihydromocimycin;
group 3: 40 pigs were treated with feed con-taining 50 ppm of dihydromocimycin;
group 4: 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 in group 1 were cured only after 3 to 4 days. The animals of groups 2 and 3 were cured in periods lying between those of groups 1 and 4.
The animals treated with dihydromocimycin appeared to be much more liverly than before the treatment. The animals were not averse to feed containing dihydromocimycin and the general conclusion was that a dosage of 25 ppm of dihydromocimycin was better than a dosage of 100 ppm of tylosin in the curing of '~reponema dysentery.
EXAMPLE S
Dehydro~enat-ion of dihydromoc mycin A solution was made of 1 g of dihydromocimycin in 15 ml of HMPT (technical grade, dried over a molecular sieve 3A) and an amount of 139 mg (1.25 mmoles) of selenium dioxide was added.
The mixture was heated on a steam bath for 100 minutes and an additional amount of 139 mg of selenium dioxide was added after 60 minutes. The selenium formed after cooling was separated by filtration through a G4 glass filter and the precipitate was washed with a small amount of methanol. The filtrate was poured into 350 ml of distilled water and the precipitate formed was filtered off and washed with distilled water. The filtrate was stored.
The precipitate was dissolved in methanol and diluted with MIBK. The solution was evaporated at 40C under reduced pressure until methanol and water were removed. A precipitate was formed which did not contain mocimycin as indicated 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 6~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.
Dehydrogenation of dih~dromocimycin This experiment was carried out in duplicate. 2.4 g of ~ composition containing 22.8~ of mocimycin and 35.5~ of dihydromocimycin was dissolved in 50 ml of HMPT. ~n amount of 350 mg of selenium dioxide was added and the mixture was kept at 105C for 45 minutes with stirring. According to a thin layer chromatographic test dihydromocimycin was not present anymore.
The mixture was cooled to room temperature and filtered through a glass filter G 4, removing the blac~ selenium formed and a small amount of unreacted selenium dioxide.
The precipitate was washed with MIBK and to the combined filtrate and MIBK washings 350 ml of water, 10 g of sodium chloride and 5 ml of 4 N hydrogen chloride were added.
This mixture was extracted 3 times with 100 ml of MIBK each.
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 20C until 25 ml were left. The residue was added dropwise to 500 ml of stirred petroleum ether (40 - 60C) and the precipitate formed was filtered off, washed with petroleum ether (40 - 60C) and dried in a vacuum drying oven at room temperature during the night.
In the first experiment the following yield was obtained: 1.64 g of a product containing 41.2% of mocimycin and C 2~ of dihydromocimycin ~as determined by high pressure liquid chromatography).
In the second experiment the yield was 1.79 g of a product containing 41.6~ o~ mocimycin and ~ 1% of dihydromocimycin.
Dehydrogenation of dihydromocimycin, influence of solvents In this example several solvents were tested to find out which one is preferred for the dehydrogenation of dihydromoci-mycin. In all experiments 50 mg of a technical preparation containing dihydromocimycin and mocimycin were reacted with 20 mg (0.18 m~ole) of selenium dioxide in 3 ml of solvent at a temperature of 80C. At 2, 4 and 7 hours after the start 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: ~ecomposition or no reaction was obtained in propargyl alcohol, diacetone alcohol, nitromethane, propylene carbonate, acetonitrile, pyridine, sulpholane, benzylalcohol, mesityl oxide, ethylene carbonate, N-methyl-2-pyrrolidone, N,N-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 NaHCO3/Na2CO3 of pH 9.1, n-propanol, phenylmethylcarbinol, water, and dimethylformamide.
A somewhat better conversion was obtained in methylcellosolve, 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 W2S obtained in HMPT.
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 ~he 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:
The invention further relates to a process for using dihydromo-cimycin as a starting material for the production of mocimycin.
Dihydromocimycin has the following structural formula ~" f ~ocn3 o ~
OH
Dihydromocimycin is produced by fermenting Streptomyces ramocissimus, or suitable mutants thereof and is formed in addition to mocimycin. Streptomyces ramocissimus is a microorganism described in British Specification No. 1325200;
the microorganism is deposited with the culture collection of Centraal Bureau voor Schimmelcultures at Baarn, The Netherlands, where it obtained the number CBS 190.69 and is available to the public.
In British Specification No. 1325200 a process is described and claimed for the production of mocimycin (then called MYC ~003) by the above indicated microorganism. The structure of mocimycin is indicatea by Vos and Verwiel in Tetrahedron Letters 52 (1973) pp. 5173-5176. It has now been discovered that Streptomyces ramocissimus, or suitable mutants thereof, produce dihydromocimycin in addition to mocimycin.
Dihydromocimycin is a pale yellow solid substance, weakly acid and further characterized by the following physico-chemical properties:
i~7'~307 Solubilit~:
The solubility of the compound is good in chloroform, methyl isobutyl ketone, ethyl acetate, butyl acetate, acetone, dioxane, 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, cyclo-hexane and petroleum ether.
Optical rotation:
1~D = -85 (1~ methanolic solution).
Melting point:
The compound does not melt, but decomposition starts at 123 C.
Elementary analysis:
The following values were found:
Found:Calculated for C43H62N2O12 2 C: 61.8% 61.8%
H: 7.5% 8.0%
N: 3.4% 3.3%
O: 27.2% (by difference) 26.8 Ultra-violet spectrum:
The ultra-violet spectrum of dihydromocimycin in a 1:1 mixture of water and methanol at various pH values is shown in Figure 1 of the attached drawings. The concentration of the solutions measured was 18.3 ~g/ml. The ultra-violet spectrum is dependent on the pH. The following maxima were found (the molecular extinctions are indicated between brackets):
methanol - water: 233.5 nm ( = 63,000~; 267 nm (E = 23,000) 291 nm ( = 19,000) and 333 nm ( = 18,000); curve 1;
methanol - 0.5N NaOH: 235 nm ( = 62,000); 277 nm ( = 25,000) and 308 nm (~ = 29,000); curve 2;
iO74307 methanol - 0.5N HCl: 233.5 n~ (~ = 65,000); 268 nm (~ = 25,~00) and 338 nm (~ = 14,000);
curve 3.
Infr_-red spectrum:
The I.R. spectrum of dihydromocimycin in chloroform and in potassium bromide is shown in Figures 2 and 3, respectively. The following principal absorptions were found:
chloroform (v in cm 1): +3445 (sh); 3430; 2979; 2941; 2886;
1662-1653; 145~; 1100; 1080; 1040; 998; 944; 893; 870 and 840;
potassium bromide (v in cm ): +3aoo-3320; 2972; 2935; 2880;
1650; 1535; 145S; 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 Mc).
Thin layer chromatography-Thin layer chromatograms of dihydromocimycin were made on Kieselgel F 254 plates (Merck, ready-for-use plates), and after drying the spots were detected by fluorescence extinction, or by carbonization after spraying with a diethyi ether-sulphuric acid mixture. Investigations showed that the spot ascribed to dihydromocimycin, even 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-dimention~l chromatogram.
The following Rf-values were found for dihydromocimycin in various eluents (the Rf-value for the additional spot is indicated between brackets):
50:45:5 mixture of methyl isobutyl ketone, acetone and water:
0.44 (about 0.44);
70:2Q:lQ:0.5 mixture of ethyl acetate, methanol, water and 25%
ammonia:
iO7~307 0.29 (0.36);
65:40:9 mixture of benzene, 10~ ethanol and 33~ ammonia:
0.16 (0.22) 60:~2:10 mixture of chloroform, 96% ethanol and 25~ ammonia:
0.4.
The structural formula of dihydromocimycin is confirmed on the following grounds: The PMR spectra (220 Mc) of mocimycin (cf. Tetrahedron ~etters 52 (1973) pp. 5173-5176 for its structural formula) and dihydromocimycin showed that both compounds are similar, except that two doublets at ~ 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 dihydromocimycin. Those signals are caused by the protons of the 5th and 6th carbon atoms of the pyridone nucleus.
However, two triplets appeared in the spectrum of dihydromocimycin at ~ 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 pH 12 during 5 minutes at O~C. After reduction of the reaction mixture obtained with hydrogen catalyzed by PtO2 and after hydrolysis with concentrated hydrochloric acid, ~-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 difference:
a low concentration of mocimycin added to the feed of animals h~ing fattened, such as chickens or pigs, improves the growth and the feed-conversion markedly. Dihydromocimycin, on the contrary, does not show any improvement of the growth or feed-conversion of those animals, which is unexpected, since in vitro it shot~s activity against the same microor~anisms; in manlr cases it shows even a better activity than mocimycin. A comp~rison of the antimicrobial activities of the two antibiotics is indicated in the following table:
Agar dilution tests Organism tested Minimum inhibitory concentration (~g./ml.) in anaerobic culture mocimycindihydromocimycin _ _ Staph~lococcus aureus A 2000 >100 >100 Staphylococc~s 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 Listerla monocyt~genes A2131 6 6 Listeria monocytogenes A2132 6 6 Clostridium perfrinqens A738 >100 >100 Clostridium septicum A2152 10 10 Strëptococcus zooepidemicus R-Streptococcen A2148 6 3 Brucella suis (smooth) A2126 0.75 0.4 Pasteurella haemolytica A2136 3 1.5 Treponema spec. A2275 30 10 .
Liquid dilution tests Organism ~ested Minimum inhibitory concentra-tion (~g./ml.) mocimycin dihydromocimycln ._ --Bacillus subtilis ATCC 6633 100 50 Bacillus subtilis ATCC 6051 100 50 Bacillus subtilis 6346 D167 1.2 0.9 Bacillus subtilis 220 D17875 75 Bacillus subtilis T~ 10 100 50 Bacillus cereus D166 0.6 0.6 Bacillus cerëus 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 Wll1 2 0 9 Bacillus mesenterium D169100 50 Bacillus cereus var. mycoides 1.2 0.45 Streptococcus haemolyticus A266 0.45 0.45 Streptococcus haemolYticus A2182 0.25 0 12 Mycoplasma ~ rhi~ AZ730 1 0 3 Steptom~es virid~chromoyenes 2~5 0.9 _ lV'7430~7 S~reptomyces ramocissimus, under suitable conditions, -does produce 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 Streptomyces ramocissimus (CBS 190.69), or a dihydromocimycin-producingmutant thereof, in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic substances, and separating the dihydromocimycin 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 40C, preferably between 26 and 34C, and the pH between 5 and 9, preferably between 6.5 and 8.
It will be appreciated that the aforesaid process is similar to that for the production of mocimycin, and it has been found that dihydromocimycin is.formed under suitable conditions in addition to mocimycin in the fermentation of Streptomyces ramocissimus, or its mocimycin- (and dihydromocimycin-) producing mutants.
To obtain a greater yield of dihydromocimycin relative t~ that of mocimycin, 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 mocimycin. However, dihydromocimycin has been found to be produced more abundantly then mocimycin by better aeration of 1~4307 the culture liquid, which may be achievecl ~y techni~ues known per se, such as a higher aeration rate ~volume of air per volume of culture meaium per unit o~ time), a higher agitation rate of the cultu.~e medium in the fermenter. Suitable aeration rates of the culture medium, e.g. of about 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, e.g. 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 ~ihydxomocimycin 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 solution3 at a temperature ~etween about -12C to about +15 DC ~
preferably between -5C and +8C. 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 the 10 to about 15 minutes (corresponding to about 25 to about 40 74;~07 litres ~f ~a~eous anunonia) 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 by 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 less than 5% of mocimycin.
Highly purified dihydromocimycin can be obtained by passing the product obtained in the way just described over a SEPHADEX (Trade Mark) LH 20 column using the difference in adsorption of mocimycin and dihydromocimycin. The SEPHADEX is 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 ~e 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 F 254 plates, size 20x5 cm (Merck). The eluent is a 60:42:10 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).
In an early stage of investigation it W25 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 that time showed that mocimycin was active against this disease, but not more so than tylosin. For that reason no further investigations were carried out.
As indicated hereinbefore, dihydromocimycin was found to be more active than mocimycin against many microorganisms, and consequently an investigation with this substance was made against the microorganism causing Treponema dysentery. From the experiments it appeared that dihydromocimycin possesses a markedly higher activity against the microorganisms than tylosin and, in addition, was active against tylosin-resistant strains. Thus, dihydromocimycin may be regarded as being superior to tylosin (and also to mocimycin) in the treatment of Treponema dysentery.
According to another feature of the invention there are provided pig feedstuffs supplemented by a significant proportion of dihydromocimycin or a slat, e.g. sodium salt, thereof. The antibiotic or its alkali metal or ammonium or amine salt thereof may also be dispersed in, or mixed with, any suitable inert, physiologically innocuous carrier or diluent, which is orally administrable to a pig, non-reactive with the antibiotic and not harmful to the pig on oral administration. Effective amounts of dihydromocimycin incorporated in a pig feedstuff for the prevention or treatment of ~reponema dysentery are about 10 to about 200 ppm, preferably 20 to 40 ppm, of dihydromocimycin, based on the weight of the feed.
Dihydromocimycin obtained by the procedure hereinbefore described is a fine, easily dusting powder. This could lead to difficulties in the mi~ing procedure with the f~e~ 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 dihy~romocimycin 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 water-soluble salt thereof, suspended or dissolved in a usual injection liquid, for example, saline, propylene glycol, glycerol-water mixtures, etc.
Mocimycin shows advantageous growth-promoting properties when fed to lîve-stock including chickens and other fowl, but dihydromocimycin does not show the aforesaid properties. In addition, therefore, attempts have been made to convert dihydromocimycin into the, for this application, more useful mocimycin.
It has thus been found that dehydrogenation of dihydromocimycin is possible with a special dehydrogenating substance and procedure. Quinones, e.g. 2,3-dicyano-5,6-dichloro-1,4-quinone, p-chloranil and o-chloranil, are not satisfactory for the dehy~roqenation of dihydromocimycill as other products are formed. Halogenation with cupric bromide, bromine, iodine or N-bromosuccinimide, followed by dehydrohalogenation, did not give the desired result either, even in the presence of catalysts such as benzoyl peroxide and ~ azo-isobutyronitrile. Attempts to dehydrogenate dihydromocimycin catalytically involve too high temperatures which would lead to decomposition of dihydromocimycin.
Only one dehydrogenation agent, selenium dioxide, has been found to be useful for the dehydrogenation of dihydromocimycin.
Therefore the present invention further relates to a 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 about 65 to about 110C, preferably from 80 to 95~. The reaction time is from about 10 hours to about 20 minutes in the temperature range of about 65 to about 110C, and is preferably from about 3 hours to about 1 hour in the preferred temperature ran~e. At ambient temperatures the reaction takes about a week.
The reaction is preferably carried out in a solvent me2ium. Suitable solvents are, for example, hexamethylphosphor-triamide (HMPT), dime~hyl sulphoxide (DMSO), t-butanol, t-amyl alcohol, sec-butanol, hexylene glycol, n-butanol, isopropanol, methylcellosolve, dimethylformamide, water, phenylmethylcarbinol and propanol, and mixtures o 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 and mocimycin is much more difficult, the discovery that dihydromocimycin may be converted by chemical means into - for the growth promotion valuable properties -mocimycin, even in a mixture of both compounds, leaving the latter compound substantially unaffected by the process, is very important. Therefore, isolati~on of the compounds is not necessary for the dehydrogenation of dihydromocimycin in mixtures containing dihydromocimycin and mocimycin.
The invention is illustrated by the following examples.
Pre~_ration of dihydromocimycin The microorganism Strep~ ces ramocissimus (CBS 190.69) was fermented in 2,000 litres of a medium containing 20 g of malt paste, 10 g of yeast extract and 5 g of corn steep solids per litre at a pH of about 7 with agitation and aeration. After fermentation the culture medium was mixed with about 2% of dicalite (an expanded perlite, an aluminium silicate containing potassium, sodium and trace elements) as filter aid, and the mixture was filtered. The filtrate was acidified with 8N
sulphuric acid to pH 6.0 and extracted twice with 1/5th of its volume of methyl isobutyl ketone (MIBK) and emulsions formed were broken with *Hyflo Supercel filter aid (a diatomaceous earth). The organic liquids were mixed and concentrated to about 1 litre by evaporation under reduced pressure and evaporation with a rotary evaporator. From the concentrate formed a crude product was obtained by adding it to 5 times its own volume of petroleun-l ether (b.p. 40 to 60 C) and the precipitate formed -~Trade Mark was filtered off with a c31ass 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 150 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.
The sodium salt of dihydromocimycin was obtained by dissolving dihydromocimycin in water with addition of 0.lN
sodium hydroxide to pH 9 until a saturated solution was obtained.
The solution was filtered and evaporated azeotropically with addition of butanol tin vacuo at about 45C), 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 i~ a similar manner.
Preparation of dihydromocimycin in higher ~ields To obtain a higher yield of dihydromocimycin, a lyophilised culture of Streptomyces ramocissimus (CBS 190.~9), 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 comn~sition: 2Q g of malt paste, 10 g of ye2st extr2ct and 5 g of corn steep solids per litre of tap water, p~l 7Ø
After incubation on a rotating sha~ing device (300 rpm, stroke 2.5 cm) at 30C for three days, the culture obtained was used for inoculating small fermenters containing 2000 ml of the above-mentioned medium to which 20 mg of CoC12.6H20 per litre was added. This growth phase, also effected at 30C, 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 timP 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 follows: 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 1/5th 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/lOth 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 was formed which was filtered off. The filtrate was treated once more with gaseous ammonia for 10 to 15 minutes. The precipitate then o~tained was dissolved in dilute ammonia (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.
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 2: 100 ppm of tylosin were added to the feed;
group 3: 25 ppm of dihydromocimycin were added to the feed;
group 4: 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 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 1~74307 living animals. The consistency of the faeces is indicated as follows: -+ means thin liquid; + means thicl; liquid; - means normal.
The results of the immuno-fluorescence technique are indicated quantitatively by indicatinq the number of Treponemas in the visual field: 5 means very crowded; 4 means many; 3 means about 10 Treponemas; 2 means 1 or 2 Treponemas, 1 means more visual fields necessary to find one Treponema and - means negative.
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 + + ~ + + + + + + _ + + + ~ ~ + +
after 2 weeks + + + + + + + + + + + + + + + + ~ +
after 3 weeks + + + + ~ + + + + + _ _ + + _ _ + _ _ .
Immunofluorescence after 1 week 4 4 4 4 5 4 4 3 4 - S 4 5 5 ~ 5 - 5 3 3 after 2 weeks 4 4 4 - 4 4 4 2 2 - 4 4 3 1 2 - ~ 4 after 3 weeks 2 2 2 2 ~ 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 immunofluorescence observations it appeared that the animals treated with dihydromocimycin were cured markedly faster than 30 the animals treated with tylosin. When the administered amounts of antibiotics were also taken into account, it can be concluded . .
that dihydromocimycin is at least 4 times as active as tylosin.
D ~_omocimycin against Treponema dysentery Pigs, from a farmwhere 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 1: 66 pigs were treated with feed containing 100 ppm of tylosin;
group 2: 34 pigs were treated with feed containing 25 ppm of dihydromocimycin;
group 3: 40 pigs were treated with feed con-taining 50 ppm of dihydromocimycin;
group 4: 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 in group 1 were cured only after 3 to 4 days. The animals of groups 2 and 3 were cured in periods lying between those of groups 1 and 4.
The animals treated with dihydromocimycin appeared to be much more liverly than before the treatment. The animals were not averse to feed containing dihydromocimycin and the general conclusion was that a dosage of 25 ppm of dihydromocimycin was better than a dosage of 100 ppm of tylosin in the curing of '~reponema dysentery.
EXAMPLE S
Dehydro~enat-ion of dihydromoc mycin A solution was made of 1 g of dihydromocimycin in 15 ml of HMPT (technical grade, dried over a molecular sieve 3A) and an amount of 139 mg (1.25 mmoles) of selenium dioxide was added.
The mixture was heated on a steam bath for 100 minutes and an additional amount of 139 mg of selenium dioxide was added after 60 minutes. The selenium formed after cooling was separated by filtration through a G4 glass filter and the precipitate was washed with a small amount of methanol. The filtrate was poured into 350 ml of distilled water and the precipitate formed was filtered off and washed with distilled water. The filtrate was stored.
The precipitate was dissolved in methanol and diluted with MIBK. The solution was evaporated at 40C under reduced pressure until methanol and water were removed. A precipitate was formed which did not contain mocimycin as indicated 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 6~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.
Dehydrogenation of dih~dromocimycin This experiment was carried out in duplicate. 2.4 g of ~ composition containing 22.8~ of mocimycin and 35.5~ of dihydromocimycin was dissolved in 50 ml of HMPT. ~n amount of 350 mg of selenium dioxide was added and the mixture was kept at 105C for 45 minutes with stirring. According to a thin layer chromatographic test dihydromocimycin was not present anymore.
The mixture was cooled to room temperature and filtered through a glass filter G 4, removing the blac~ selenium formed and a small amount of unreacted selenium dioxide.
The precipitate was washed with MIBK and to the combined filtrate and MIBK washings 350 ml of water, 10 g of sodium chloride and 5 ml of 4 N hydrogen chloride were added.
This mixture was extracted 3 times with 100 ml of MIBK each.
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 20C until 25 ml were left. The residue was added dropwise to 500 ml of stirred petroleum ether (40 - 60C) and the precipitate formed was filtered off, washed with petroleum ether (40 - 60C) and dried in a vacuum drying oven at room temperature during the night.
In the first experiment the following yield was obtained: 1.64 g of a product containing 41.2% of mocimycin and C 2~ of dihydromocimycin ~as determined by high pressure liquid chromatography).
In the second experiment the yield was 1.79 g of a product containing 41.6~ o~ mocimycin and ~ 1% of dihydromocimycin.
Dehydrogenation of dihydromocimycin, influence of solvents In this example several solvents were tested to find out which one is preferred for the dehydrogenation of dihydromoci-mycin. In all experiments 50 mg of a technical preparation containing dihydromocimycin and mocimycin were reacted with 20 mg (0.18 m~ole) of selenium dioxide in 3 ml of solvent at a temperature of 80C. At 2, 4 and 7 hours after the start 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: ~ecomposition or no reaction was obtained in propargyl alcohol, diacetone alcohol, nitromethane, propylene carbonate, acetonitrile, pyridine, sulpholane, benzylalcohol, mesityl oxide, ethylene carbonate, N-methyl-2-pyrrolidone, N,N-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 NaHCO3/Na2CO3 of pH 9.1, n-propanol, phenylmethylcarbinol, water, and dimethylformamide.
A somewhat better conversion was obtained in methylcellosolve, 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 W2S obtained in HMPT.
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 ~he 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:
- 2~ -After coolin~ the reaction mixture was filtered through a G~ glass filtcr 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 1/4th 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 40C. In all cases a precipitate was formed essentially consisting of decomposition products (according to TLC). The precipitate was filtered and washed with MIBK and the combined MIBK
concentrates were added slowly, with stirring, to an excess of petroleum ether (b.p. 40 to 60C). The precipitate formed was filtered, washed with petroleum ether and dried. The results of the experiments are shown in the following table:
1~74307 ~mount of Reactlon O~erall SeO - Yield Moles solvent temperature reaction add2tion (% by SeO
(C) time in grams weight) added (hrs) and time per of mole of addition mixture start of reaction .
HMPT 25 ml 90 2 0.5 start 42 3~1 H20 35 ml + 0.5 . aifner 55 HMPT 15 ml 90 2 3/4 0.1 start 50 1 2~1 0.1 M 0.1 after . .
NaHCO3- 20 min Na CO3 0.1 after bu~fer pH 85 min 9.1 25 ml 0.1 after 130 min t-BuOH 84 3 0.1 start 421.5:1 20 ml 0.1 after HMPT 10 ml 0451mafter 0 1 after 0 1 after . _ ._ t-AmOH 96 3 0.35 start 71 3.2:1100 ml 0.35 after HMPT S0 ml 70 min liO min . . .
t-AmOH 96 3 0.35 start 55 3 2~1150 ml 0 35 after . .
150 min t-BuOH 80 2 1.0 start 42 6:1 100 ml 1.0 after HMPT 50 ml 35 min _ HMPT 95reaction 0.4 start 791.2:1 150 ml after 1 hr The following abbrevations are used:
H.~IPT = 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 H~PT and t-amyl alcohol.
concentrates were added slowly, with stirring, to an excess of petroleum ether (b.p. 40 to 60C). The precipitate formed was filtered, washed with petroleum ether and dried. The results of the experiments are shown in the following table:
1~74307 ~mount of Reactlon O~erall SeO - Yield Moles solvent temperature reaction add2tion (% by SeO
(C) time in grams weight) added (hrs) and time per of mole of addition mixture start of reaction .
HMPT 25 ml 90 2 0.5 start 42 3~1 H20 35 ml + 0.5 . aifner 55 HMPT 15 ml 90 2 3/4 0.1 start 50 1 2~1 0.1 M 0.1 after . .
NaHCO3- 20 min Na CO3 0.1 after bu~fer pH 85 min 9.1 25 ml 0.1 after 130 min t-BuOH 84 3 0.1 start 421.5:1 20 ml 0.1 after HMPT 10 ml 0451mafter 0 1 after 0 1 after . _ ._ t-AmOH 96 3 0.35 start 71 3.2:1100 ml 0.35 after HMPT S0 ml 70 min liO min . . .
t-AmOH 96 3 0.35 start 55 3 2~1150 ml 0 35 after . .
150 min t-BuOH 80 2 1.0 start 42 6:1 100 ml 1.0 after HMPT 50 ml 35 min _ HMPT 95reaction 0.4 start 791.2:1 150 ml after 1 hr The following abbrevations are used:
H.~IPT = 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 H~PT and t-amyl alcohol.
Claims (11)
1. A process for the dehydrogenation of dihydromocimycin having the structural formula:
into mocimycin which comprises the step of reacting dihydromocimycin with selenium oxide.
into mocimycin which comprises the step of reacting dihydromocimycin with selenium oxide.
2. The process of claim 1 wherein a mixture of mocimycin and dihydromocimycin is used as the starting material.
3. The process of claim 1 wherein the reaction is carried out at an elevated temperature.
4. The process of claim 1 wherein the reaction is carried out at a temperature between about 65° and about 110°C.
5. The process of claim 1 wherein the reaction is carried out at a temperature between 80° and 95°C.
6. The process of claim 1 wherein the reaction is carried out in a solvent selected from the group consisting of hexamethyl-phosphortriamide, dimethyl sulphoxide, t-butanol, t-amyl alcohol, sec-butanol, hexylene glycol, n-butanol, iso-propanol, methyl-sellosolve, dimethylformamide, water, phenylmethylcarbinol, propanol and mixtures of two or more of those solvents.
7. The process of claim 1 wherein the reaction is carried out in a solvent selected from the group consisting of hexamethylphosphortriamide, dimethylsulphoxide and t-butanol.
8. The process of claim 1 wherein the reaction is carried out in hexamethylphosphortriamide as a solvent.
9. Mocimycin whenever prepared according to the process of claim 1, 2 or 3.
10. Mocimycin whenever prepared according to the process of claim 4, 5 or 6.
11. Mocimycin whenever prepared according to the process of claim 7 or 8.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA324,111A CA1074307A (en) | 1975-05-16 | 1979-03-26 | Antibiotic |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB20926/75A GB1545474A (en) | 1975-05-16 | 1975-05-16 | Mocimycin derivatives |
GB3064675 | 1975-07-22 | ||
CA252,534A CA1070630A (en) | 1975-05-16 | 1976-05-14 | Dihydromocimycin from streptomyces ramocissimus |
CA324,111A CA1074307A (en) | 1975-05-16 | 1979-03-26 | Antibiotic |
Publications (1)
Publication Number | Publication Date |
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CA1074307A true CA1074307A (en) | 1980-03-25 |
Family
ID=27425913
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CA324,111A Expired CA1074307A (en) | 1975-05-16 | 1979-03-26 | Antibiotic |
Country Status (1)
Country | Link |
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CA (1) | CA1074307A (en) |
-
1979
- 1979-03-26 CA CA324,111A patent/CA1074307A/en not_active Expired
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