Note: Descriptions are shown in the official language in which they were submitted.
1074307
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:
-- 1 --
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
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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
A2144 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
.
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
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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
-- 8 --
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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
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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
-- 10 --
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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.
1074307
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.
EXAMPLE 1
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
- 12 -
1074307
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.
EXAMPLE 2
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
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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
- 14 -
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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 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
- 16 --
. .
that dihydromocimycin is at least 4 times as active as tylosin.
EXAMPLE 4
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.
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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.
EXAMPLE 6
Dehydrogenation of dih~dromocimycin
This experiment was carried out in duplicate. 2.4 g
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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.
EXAMPLE 7
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
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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.
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 ~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:
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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:
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~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
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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.
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