Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Pleuromutilin (also now known as pleuromulin) is
an antibiotic produced by cultures of the basidiomycete
Pleurotus mutilis. The antibiotic has antibacterial
activity. A group of new derivatives of pleuromutilin
having antibacterial activity is described in German
Offenlegungschrift 2,248,237 (April 12, 1973). One of
these derivatives in particular, 14-deoxy-14-[(2-diethyl-
aminoethyl)mercaptoacetoxy]mutilin (known generically
as tiamutilin or tiamulin), as the hydrogen fumarate salt,
1~ has been found to be very active against microorganisms
such as Streptococci, Staphylococci and Mycoplasmas and
particularly useful in veterinary medicine. See
Antimicrobial Agents and Chemotherapy, May 1975, pages
507-516, 517-S21. This substance, however, causes
difficulties in manufacture, formulation and administration.
Research on the substance has now demonstrated that the
formation of the neutral fumarate salt provides a material
at least equally active on a weight basis but which has
markedly different physical properties which overcome the
more serious disadvantages of the hydrogen fumarate.
~ GC144a
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This invention relates to the new compound
14-deoxy-14-[(2-diethylaminoethyl)mercaptoacetoxy]mutilin
neutral fumarate and to compositions containing it.
[See Hodgin et al., Eur. J. Biochem. 47, 527-533 (1974?
for the structure and this system of nomenclature.~
This compound can also be named as the 14-(2-diethyl-
aminoethyl)thioacetate ester of mutilin, neutral fumaratesalt. By neutral fumarate salt is meant the salt containing
one moie of fumaric acid and two moles of 14-deoxy-14-
[(2-diethylaminoethyl)mercaptoacetoxy]mutilin, i.e.,
both acid groups of the fumaric acid are neutralized.
[For conveneince, 14-deoxy-14-[(2-diethylaminoethyl)-
mercaptoacetoxy]mutilin is referred to hereinafter for
brevity as mutilin derivative.] This is distinguished
from the hydrogen fumarate discussed in the two articles
in Antimicrobial Agents and Chemotherapy, supra, which
contains one mole of fumaric acid and one mole of mutilin
derivative.
The mutilin derivative is difficult to isolate
from the impurities and by products found in the reaction
mixture in which it is produced. Tiamutilin forms
few readily isolatable salts. One of these is the
salt with fumaric acid. It has been found, however,
that even the previously used hydrogen furamate requires
numerous steps for isolation and purification involving
formation of a non-aqueous solvent adduct and employment
of several solvents in large amounts which must then be
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disposed of.
By forming the neutral fumarate under conditions
as described below, a reproducible, readily crystallizable
salt is formed in one step.
The new neutral fumarate lS produced from the mutilin
derivative(produced as described in German Offenlegungsschrift
2,248,237) by adding one mole of fumaric acid to two moles
of mutilin derivative. The mutilin derivative is preferably
dissolved in a solvent in which the final product is insoluble,
e.g., isobutyl acetate (which is preferred), butanol, ethyl
acetate, methyl isobutyl ketone, xylene or the like. The
fumaric acid can be added as a solid but it is preferably
added as a concentrated solution in dimethylformamide.
By adding the fumaric acid to the solution of mutilin
derivative the neutral fumarate is formed in high yield in
the form of well developed prisms. When the order of
addition is reversed, i.e., the mutilin derivative is added
to the fumaric acid then the tendency is to form the hydrogen
fumarate salt. The crystalline neutral fumarate product is
isolated by filtration or centrifugation, washing with the
same solvent and drying.
According to the preferred method, the crude mutilin
derivative,as the free base, in the reaction mixture is
extracted into isobutyl acetate, a solution containing
fumaria acid in dimethylformamide is added to the isobutyl
acetate solution in a proportion of one mole of fumaric
acid to two moles of mutilin derivative at room temperature.
The precipitated crystalline product is then isolated by
filtering or centrifuging.
--3--
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1077056
.
The neutral fumarate is obtained in crystalline form
which has properties distinct from the hydrogen fumarate.
A comparison of properties of the neutral fumarate and
hydroyen fumarate is as follows:
Property Neutral llydrogen
fumarate fumarate
Molecular weight 1103.5 609.8
Melting point, C 137-138 147-1~9
Solubility in Water at 25 C, 380 70
g/ltr
pH of Saturated Solution 4.9 3.3
Bulk Density gm/cc 0.5 0.27
HygroscopiOity at 70% r.h. none none
and 25 C
Residual Solvents traces traces
Volatiles in Wet Cake, <10 >30
% w/w
Active Base in Salt,% w/w 89.5 ~1.0
Crystal Porm Stout Prisms Thin Plates
and needles
The new neutral fumarate of this invention overcomes
various disadvantages encountered with thc known hydrogen
fumarate in various areas. In manufacturing, the hydrogen
fumarate has a tendency to form polymorphs and solvates which
make it difficult to reliably reproduce acceptable crystals
on a bulk manufacturing scale. No solvates of the neutral
fumarate have been encountered in manufacturing so reproducible
results are consistently obtained.
The hydrogen fumarate which is obtained in the form
of fine crystals shows a tendency to disperse into the atmosphere
and cause the handlers to sneeze, thus providing a hazard
to the manufacturing workcrs. Thc neutral fumaratc is
1077056 GC144a
characterized by large crystal size and high bulk density
and does not display this tendency. The large prisms in
which the neutral fumarate forms are easily obtained in
pure form during manufacture and are easy to handle.
The amount of volatiles retained in the wet cake after
production of the salt/as seen from the table, is greater
than 30% in the case of the hydrogen fumarate compared with
less than 10% (usually about 8%) for the neutral fumarate.
This makes it easier to work up the neutral fumarate,
requiring less washing of the cake to remove impurities.
From the point of view of formulation, the high
bulk density of the neutral fumarate simplifies formulation
work, especially in tablets and capsules. The product is
useful in preparing injectable compositions, particularly
for larger animals like pigs and cattle, and also to
produce concentrates for dilution with drinking water in
automatic proportioning devices like "Auto-Medic" used
in administering medicaments to poultry.
The higher pH of the neutral fumarate in solution is an
advantage from the point of view of irritation at the site of
injection since it is known that such irritation occurs more fre-
quently as the pH decreases. In addition, the local tolerance on
injection is improved when the neutral fumarate is used.
The neutral fumarate is at least as active as the
hydrogen fumarate in vitro and in vivo on a weight basis.
The product is thus useful as an antibacterial agent,
particularly in veterinary medicine, for the treatment of a
variety of infections particularly against pathogenic gram
positive bacteria such as Streptococci and Staphylococci
as well as Mycoplasma and Treponema. Some gram positive
1077056 GC144a
organisms such as Shigella, Klebsiella and Escherichia coli
are also responsive to this substance.
The neutral fumarate of 14-deoxy-14-[(2-diethylamino-
ethyl)mercaptoacetoxy]mutilin is useful for the treatment of
swine dysentery (due to Treponema _yodysenteriae), enzootic
pneumonia of pigs, atrophic rhinitis in pigs, mycoplasmosis,
chronic respiratory disease in poultry (CRD), air sac disease,
infectious synovitis, fowl cholera, bovine pneumonia,
respiratory infections of horses, sheep, dogs, cats, etc.
The substance can be administered orally in drinking
water or in feed compositions or parenterally in physiologically
acceptable vehicle or carrier such as water, which is the
preferred carrier, aqueous ethanol solutions, naturally
occurring vegetable oil or physiologically acceptable modified
naturally occurring oils or synthetic oils. Effective doses
lie in the range of 1 to 50 mg/kg, preferably about 5 to 20 mg/kg.
Compositions containing the 14-deoxy-14-[(2-diethylaminoethyl~-
mercaptoacetoxy]mutilin neutral fumarate, dependiny on the
intended use and mode of administration as discussed below,
can contain abouto-oo2 to 20~ by weight of active substance.
The preferred mode of administration is by intramuscular
injection. The vehicle can be sterile water for
injection, aqueous ethanol solutions, naturally occurring
vegetable oil solutions, e.g., corn oil, sesame oil,
coconut oil, cottonseed oil and the like, or synthetic oil
solutions, e.g., ethyl oleate, fractionated coconut oil, etc.
The drug is administered utilizing sufficient
material in about 5 to 10 ml. of vehicle, providing conccntra-
tions of about-5 to 20% (weight/vol.). The dosage is in
general administered 1 to 2 times daily for 5 to 10 days.
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Other substances can be included in the formulations as
required to produce a stable composition of pharmaceutical
elegance. For example, the composition can include
preservatives like phenol, chlorobutanol, methyl paraben,
propylaraben or benzyl alcohol, antioxidants like sllT
(butylated hydroxytoluene), sH~ (butylated hydroxyanisole),
buffering agents, etc. as required by conventional pharmaceutical
practice in formulating drug compositions, especially for
veterinary use.
The neutral fumarate of this invention can also be
administered by other methods. For example, it can be
administered to poultry for the treatment of Mycoplasma
infections in the drinking water. The active material is
dissolved in the water for the poultry at a concentration of
about 0.002 to 2~, preferably about 0.005 to 0.05% (wt/vol).
Treatment is usually continued for about 7 to 10 days.
Concentrates for liquid proportioning devices containing
2 ounces per gallon for mixing with 128 gallons water provide
a 0.016% solution.
The medicament can also be administered by incorporating
the substances in animal feed compositions, e.g., swine
starter rations, grower and finisher rations, starter and
finisher rations for broiler chickens and turkeys, layer
rations, etc. Such compositions are generally prepared as
concentrated feed premixes (containing about 50 to 500 gm.,
preferably 50 to 250 gm. of medicament per pound of premix
material) which are then incorporated in the bulk animal eed
material resulting in a concentration of about 50 to 500 (jm.
of neutral fumarate per ton of feed compositioJl.
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1077056 GC144a
The following examples are illustrative of thc
invention.
~xample 1
Pre aration of 14-Deox -1~-[(2-diethvlaminoethyl)mercapto-
P Y,
acetoxy]mutilin Neutral Fumarate From Pleuromutilin Tosyla~c
Pleuromutilin tosylate~(53.3 grams) and ~-diethylamino-
ethanethiol hydrochloride (18.7 grams) are stirred with
acetone (160 ml.) in an ice bath. After cooling to +5 C,
a methanolic solution of sodium methylate (50 ml. of a
25~ solution) is added in one portion. The temperature
rises rapidly to about 30 C. The mixture is again cooled
to 20 C. and isobutyl acetate (250 ml.) and water (250 ml.)
are added. The batch is equilibrated and the layers are
allowed to separate. The aqueous layer is discarded. The
organic layer is stirred with water (100 ml.) and sufficient
dilute HCl to lower the pH to 6.5. The aqueous phase,
containing disulfide impurities, is again discarded. The
organic phase is then extracted with 1 N E~Cl (100 ml.),
followed by water (25 ml.). The combined aqueous layers are
made basic with lN NaOH (105 ml.) to a pH above 8 in the
presence of fresh isobutyl acetate (150 ml.). The mixture is
equilibrated, the phases are allowed to separate and the
aqueous layer is discarded. The organic layer is dried over
MgSO4 and is polish filtered. The filtrate contains the
desired mutilin derivative as the free base. Titration
indicates a yield of about 92-94 mole~, based upon the
pleuromutilin tosylate input.
The equivalent amount of fumaric acid (5.3 grams) is
dissolved in dimethylformamide (17.5 ml.). About 10~ of
this solution is added with stirring to the solution of
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the free base from above. Crystallizatioll is allowed to
proceed for about lO minutes, in the presence of some secds
of the desired product. Subsequently, the remainder of the
fumaric acid solution is added and the crystallizat~on is
completed by stirring for two hours at room temperature.
The crystals are collected on a filter and are
washed with fresh isobutyl acetate (about 50 Ml.). Drying
furnishes pure 14-deoxy-14[(2-diethylaminoethyl)mercapto-
acetoxy]mutilin neutral fumarate (37 cJrams, correspondiny
to 67 mole %, based upon the pleuromutilin tosylate input).
Most of the mutilin derivative remaining in the mother .
liquors (about 22 mole %, based upon the pleuromutilin tosylate
input) is recovered by washing the liquid with aqueous sodium
hydroxide to remove fumaric acid, followed by extraction into
aqueous hydrochloric acid at a pH below 2. This acidic
extract is then returned into a subsequent batch for a
substantial overall yield increase.
Example 2
A solution of the free base of the mutilin derivative
in isobutyl acetate is prepared as described in Example l.
The solution is heated to about 60-70 C. and the equivalent
amount of solid fumaric acid (5.3 grams) is added in one
portion with efficient agitation. The fumaric acid dissolves
gradually and the characteristic prismatic crystals of the
desired product separate. The mixture is allowed to cool
to room temperature and the crystallization is completed
by stirring for two more hours. The crystals are collected
on a filter and are washed with fresh isobutyl ace~ate (75 ml.).
Drying furnishes pure 14-deoxy-14-[(2-dietllylaminoethyl)mercai)to-
acetoxy]mutilin neutral fumarate (4G.3 yrams or 84 mole %,
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based upon the pleuromutilin tosylate charged).
Example 3
14-Deoxy-14-[(2-diethylaminoethyl)mercaptoacetoxy]-
mutilin is produced as described in Example 1 and the mixture
is worked up to the aqueous acidic extract. For the
subsequent back-extraction, ethyl acetate (200 ml.) is
substituted for the isobutyl acetate. The extract is dried
and filtered as described in ~xample 1. Titration indicates
the presence of about 92 mole % of the free base of the
mutilin derivative in the filtrate.
The equivalent amount of fumaric acid is added as a
solid to the stirred filtrate at reflux temperature. The
fumaric acid dissolves quickly and the neutral fumarate salt
separates in the form of prismatic crystals. The mixture is
cooled to room temperature and crystallization is completed
by stirring for two additional hours. Filtration, washing
with fresh ethyl acetate (50 ml.) and dryiny furnishes pure
14-deoxy-14-[(2-diethylaminoethyl)mercaptoacetoxy]mutilin
neutral fumarate (42.4 grams or 77 mole %, based upon the
pleuromutilin tosylate input).
Example 4
A 10% multiple dose solution is prepared by dissolviny
1 kg. of 14-deoxy-14-[(2-diethylaminoethyl)mercaptoacetoxy]-
mutilin neutral fumarate in 10 liters of a 50% solution ofaqueous ethanol at room temperature. The solution is
bottled in 100 ml. multi-dose bottles. This solution is
administered intramuscularly to pigs infected with swine
dysentery in 1 to 10 ml. dosaqes (10 mg/kg) once daily
for five days.
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10770S6 GC144a
Examplc 5
1 kg. of 14-deoxy-14[(2-diethylaminoethyl)mercaptoacetoxy]-
mutilin neutral fumarate is mixed with 5 liters of ethyl oleate
under sterile conditions until thoroughly suspended. S mg. of
a mixture of methyl paraben and propyl paraben (10:1) are added.
The 20% oil suspension is bottled in 100 ml. multiple dose bottles
for intramuscular injection.
Example 6
500 gm. of 14-deoxy-14-[(2-diethylaminoethyl)mercapto-
acetoxy]mutilin neutral fumarate are suspended in 10 liters
of fractionated coconut oil (Neobee 20) and 100 mg. of
benzyl alcohol are added under sterile condltions. The
5~ suspension is subdivided into sterile vials each containing
5 ml. for intramuscular injection.
~ mple 7
An aqueous concentrate for use in a liquid proportioning
device like "Auto-Medic" is prepared by dissolying 6 k~. oE
14-deoxy-14-[(2-diethylaminoethyl)mercaptoacetoxy]mu~ilin
neutral fumarate in 100 gal. of water. 3.78 kg. of benzyl
alcohol are added. The solution is bottled in 1 gal.
bottles. The contents of one bottle are placed in a liquid
proportioning device adjusted to mix with 128 gal. of water
to provide a .0125% concentration drinking water solution for
administration to broiler chickens infected with CRD.
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l'xarnple 8
.
A feed premix is prepared by thoroucJhly admixiny 10 kg.
of 14-deoxy-14-[(2-diethylaminoethyl)mercaptoacetoxy]mutilin
neutral fumarate with 100 lb. of soybean meal. The mixture
is subdivided into packages each containing 100 gm. o~
active ingredient per lb. of premix. Two lbs. of premix
is added to one ton of feed to provide a medicated feed
containing 200 gm. per ton.
Example 9
A swine starter ration is admixed consistiny of the
following ingredients
Lbs
Corn meal 1315
Wheat midds 100
Soybean 405
Menhaden meal 40
Dried Whey 50
Calcium phosphate 30
Salt 10
Molasses 50
2000
To the swine starter ration is added 100 gm of 14-
deoxy-14-[(2-diethylaminoethyl)mercaptoacetoxy~mutilin
neutral fumarate and the mixture is thoroughly blended.
The feed composition contains 100 gm. of activ,e medicament
per ton of feed. The medicated feed composition is fed to
5 to 8 week old pigs infected with Treponema hvodysenteriae
for 14 days. ^
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.. . .
1~77056 GC144a
E:xample 10
A swine starter ration is prepared containing the
following ingredients:
Lbs
Corn meal 1311
Wheat midds 100
Soybean 405
Menhaden meal 40
Dried Whey 50
Calcium phosphate 30
Salt 10
Molasses 50
Mineral Mix 2
Vitamin Mix 2
2000
The mineral mix is made up in the followiny proportions:
Gm
Copper 5 4
Iron 68.0
Manganese 18.2
Zinc 45.4
Iodine 0.2
The vitamin mix is made up in the following
proportions:
Vitamin A 3000 IU
Vitamin D 1000 IU
Vitamin E 20 IU
Vitamin B12 20 1i(3 ;
Riboflavin 3 ~lg
~clntotl~en.ic (~ O ~ r
~o770s6 GC144a
Niacin 20 mg
Choline . 200 mg
The feed composition including vitamin and mineral
mixes is thoroughly admixed with 200 gms. of 14-deoxy-14-
[(2-diethylaminoethyl)mercaptoacetoxy]mutilin neutral
fumarate providing a medicated feed composition containing
200 gm. of active ingredient per tOIl of feed. Pigs infected
with Treponema hyodysenteriae are permitted to feed ad libitum
on this feed composition for 14 days.
