Note: Descriptions are shown in the official language in which they were submitted.
CA 02271847 1999-OS-11
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PROCESS FOR THE PREPARATION OF SALTS AND ESTERS
OF CLAW'LANIC ACID
s The present invention relates to a process for the pre-
paration of pharmaceutically acceptable alkali metal salts and
esters of clavulanic acid, especially potassium clavulanate.
Clavulanic acid and its alkali metal salts and esters are
!3-lactamase inhibitors, able to enhance the effectiveness of
~o penicillins and cephalosporins.
Clavulanic acid is normally prepared by the fermentation
of a microorganism which produces clavulanic acid as for
instance Streptomyces strains such as Streptomyces clavuliQer-
us. The resulting aqueous broth is normally subjected to
~s conventional purification and concentration processes, such
as disclosed in GB-1508977.
EP-A-26044 discloses the use of the tertiary butylamine
salt of clavulanic acid as an useful intermediate in the pre-
paration of clavulanic acid. Other amine salts of clavulanic
zo acid are disclosed in for example WO 93/25S57, WO 94/22873,
EP-A-0387178, EP-A-562583, WO 96/26944 and WO 96/20199. In the
present application also diamino ethers as disclosed in the
latter application are considered as amines. All these cited
references are incorporated in the present application by
zs reference.
The aim of this invention is to prepare clavulanic acid
and its pharmaceutically acceptable salts, such as potassium,
wherein the desired substance is obtained in a high yield and
of high purity.
3o A lot of process improvements were surprisingly found.
First of a11, whole broth extraction appears to be an advan-
tageous alternative for removing solids from the fermentation
broth. A further improvement relates to the application of an
amine which forms with clavulanic acid an amine clavulanate,
3s which sticks to the walls in the normally applied ethyl acetate
/acetone solution. The addition of a water miscible organic
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solvent like an alcohol, preferably absolute alcohol, avoids
the tendency to stick to the walls of the diamine crystals of
the crystallising vessel which results in a yield loss.
Furthermore, it appears to be possible to influence the crys-
s tal morphology of the end product by regulating the amount of
water added during the crystallisation stage.
According to the present invention a process for the
preparation of a pharmaceutically acceptable salt or ester of
clavulanic acid has been provided comprising one or more of
~o the following steps:
- fermentation of a clavulanic producing microorganism;
- acidifying the clavulanic acid containing broth;
- optional addition of a small amount of a water miscible
solvent;
~s - extraction of the acidified clavulanic acid containing
broth with a water immiscible organic solvent;
- concentration of the clavulanic acid containing water
immiscible organic solution by evaporation;
- purification of the concentrated clavulanic acid so-
zo lution by adsorption;
- filtration of the clavulanic acid solution;
- optional addition of one or more water miscible organic
solvent(s);
- addition of an amine, optionally together with a water
zs miscible solvent, to prepare a clavulanate salt of said
amine;
optional recrystallization of the clavulanate salt of
amine formed;
- conversion into purified clavulanic acid by acidifying
30 or into a pharmaceutically acceptable salt or ester
clavulanate by adding a source of the corresponding salt
or ester;
- separation of the pharmaceutically acceptable salt or
ester clavulanate from the solution.
35 This applies for instance to the application of amines
especially of N,N,N',N'-tetramethylethylenediamine, 1,3-
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bis(di-methylamino)-2-propanol, t-butylamine, t-octylamine,
benzhydrylamine and bis (2-(dimethyl-amino)ethyl)ether.
' In almost a11 prior art processes it is emphasized that
it should be necessary to remove any of the suspended solids
s prior to solvent extraction. Although filtration or
ultrafiltration does give favourable results, it turns out
that whole broth extraction does give a comparable end result
in terms of yield and purity of the end product . An important
advantage of leaving out the filtration step is of course a
~o larger process efficiency by leaving out a process step. The
possible difficulties of broth instability and emulsion forma-
tion can be met by adding a small (up to about 25% of the
total volume of the fermentation broth) amount of solvent such
as a water miscible ketone or alcohol and/or pH adjustment to
is denature proteins in the broth and/or addition of a suitable
demulsifier and therefore to improve the whole broth extrac-
tion performance.
During the crystallisation stage of the amine clavulanate
it has been found advantageous to add a small amount of an
zo alcohol like absolute ethanol to the mixture comprising clavu
lanic acid and the extracting solvent, preferably ethylacetate
and the crystallising solvent, preferably acetone. In a pre-
ferred embodiment, the alcohol is added before or simulta-
neously with the amine. The application of this three compo-
25 nent mixture of solvent diminishes the exhibition of the
crystals to stick to the walls resulting in a loss of yield
and it also leads surprisingly to an enhancement of the purity
and general quality characteristics. A11 ways of addition of
the amine to the mixture comprising clavulanic acid result
so then in favourable crystallization results, for instance
besides addition of the amine to the mixture comprising clavu-
lanic acid both simultaneous addition of the amine and the
clavulanic acid mixture to a reaction vessel and the addition
of clavulanic acid mixture to the amine. Furthermore,
35 recrystallisation can optionally be applied by treating the
aqueous solution of amine clavulanate, either neat or partly
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diluted with solvent, with charcoal and then sterile filtering
the resulting solution prior to sterile crystallisation.
During the formation of the potassium clavulanate it has
been found advantageous to form the same by slurrying the
s amine salt in a solvent, preferably a water miscible ketone
or alcohol and then adding a potassium source, preferably
potassium ethyl hexanoate in a solvent, also preferably a
water miscible ketone or alcohol. Use of a slurry system
reduces significantly the amount of solvent required which
~o reduces costs and increases the yield of the endproduct.
Furthermore, it has been found advantageous to add a small
amount of water to the (slurry) solvent. Surprisingly it has
been found that by doing this it is possible to influence the
crystal morphology of the final product. Thus it is possible
~s to obtain crystals which can be washed more readily and which
have improved filtration and drying characteristics. In this
way it is possible to form either rosette or needle formed
crystals, preferably a cluster of needles formed crystals.
Furthermore, especially in case as amine the bis(2-(di
zo methylamino)ethyl)ether is applied the residual amine in the
endproduct is extremely low, lower than 0.05% w/w, preferably
lower than 0.02% w/w appears to be possible, viz. ten times
lower than the level of tertiary butylamine permitted by the
British Pharmacopoeia. A low amine content in the amine
2s clavulanate intermediate is desirable since it results in a
more stable endproduct. Therefore, it is really advantageous
to apply potassium clavulanate, prepared in this way, in a
pharmaceutical composition comprising potassium clavulanate
and amoxycillin trihydrate.
3o The following examples are given for illustration purposes
only.
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Examples
Example 1
Whole broth extraction of a clavulanic acid containing broth
Broth resulting from a fermentation with Streptomyces
clavuliQerus, (3.5 1, containing 13.3 g of clavulanic acid)
was cooled to 2 °C. This broth was added to 10.5 1 ethylaceta-
1o to at 1-3 °C while stirring. After addition of the broth, the
pH of the mixture was adjusted to pH=1.6 with the aid of 3 M
sulphuric acid. The mixture was rested to settle for 5 min-
utes, and the layers were separated. 9.25 1 of ethylacetate
layer was collected, containing 7.5 g of clavulanic acid.
' Example 2
Evaporation of clavulanic acid containing ethyl acetate sol-
ution
The ethylacetate solution (9.25 1) from the preceding
example was evaporated in vacuo at 30-35 °C, using a natural
circulation evaporator. A concentrated solution (0.88 1) was
obtained, containing 7.5 g of clavulanic acid. This solution
z5 was concentrated using a rotatory thin film evaporator (va-
cuum, 30-35 °C). A concentrate (0.11 1) was obtained, contai-
ning 7.4 g of clavulanic acid.
3o Example 3
. Preparation of crystalline bis (2-(dimethylamino)ethyl)ether
diclavulanate
Into a one litre three-necked roundbottom flask fitted
35 with a thermometer, two dropping funnels and a stirrer was
placed ethanol (50 ml). The temperature was brought to 10° C
and under stirring an impure solution of clavulanic acid
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(400 ml; 32.9 g clavuianic acid/litre) in ethyl acetate and
a solution of bis(2-(dimethylamino)ethyl)ether (13.5 g) in
ethanol (230 ml) were added simultaneously. The temperature
during the addition was maintained between 10-15° C. After the
addition (15-20 minutes) , the mixture was stirred for one hour
at 5° C. The crystals were filtered, washed twice with ethyl
acetate 40 ml) and dried under vacuum at room temperature. 16
g of bis(2-(dimethylamino)ethyl)ether diclavulanate was
obtained.
~o
Example 4
Purification of clavulanic acid solution by treatment with
charcoal
The concentrated ethyl acetate solution of example 2
(7.4 g of clavulanic acid in 110 ml) was subjected to a char-
coal treatment . The solution was cooled to 5 °C, and charcoal
(16.5 g, Norit SX Ultra) was added. The mixture was stirred
zo during two hours at 5-10 °C. Filter aid (Dicalite, 5 g) was
added, and the mixture was filtered. The solid cake was washed
with ethyl acetate until the volume of the collected filtrate
was 185 ml.
Example 5
Crystallization of bis(2-(dimethylamino)ethyl)ether
diclavulanate from a concentrated clavulanic acid solution in
ethyl acetate
The following experiment was carried out in nitrogen
atmosphere. Absolute ethanol (450 ml) was added to a solution
of clavulanic acid in ethyl acetate (900 ml, concentration
g of clavulanic acid/litre). The solution was stirred,
35 cooled to 10 °C, and bis(2-(dimethylamino)ethyl)ether
(35.l ml, 0.18 moles) was added. The mixture was cooled to
3 °C, and stirred during one hour. The crystal suspension was
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filtered, and the cake was washed with 200 ml of acetone, and
again with 200 ml of acetone. The wet cake was dried in vacuum
at room temperature. 45 g of bis (2- (dimethylamino) ethyl) ether
diclavulanate was obtained.
Example 6
Crystallization of potassium clavulanate starting from bis(2-
(dimethylama.no)ethyl)ether diclavulanate
~o
The following experiment was carried out in nitrogen
atmosphere. Bis(2-(dimethylamino)ethyl)ether diclavulanate
(10 g) was suspended in a mixture of acetone (300 ml) and
water (6 ml). The mixture was stirred, cooled to 12 °C, and
~s a solution of potassium 2-ethylhexanoate in acetone (125 ml
of a 0.34 M solution) was added in the course of 10 minutes.
The mixture was cooled to 5 °C, and stirred during one hour.
The crystal suspension was filtered and the cake was washed
with three cake volumes acetone. The wet cake was dried in
zo vacuum at 20 °C. 8.22 g of potassium clavulanate was obtained.
Example 7
Purification of bis(2-(dimethylamino)ethyl)ether diclavulanate
zs by recrystallization
The following experiment was carried out in nitrogen
atmosphere. Bis(2-(dimethylamino)ethyl)ether diclavulanate
(10 g) was dissolved in a mixture of absolute ethanol (45 ml)
3o and water (5 ml) at 20 °C. The clear solution was added to
acetone (250 ml) in the course of 20 minutes while stirring
the mixture at 20 °C. The mixture was cooled to 5 °C, and
_ stirred during 30 minutes. The crystal suspension was fil
tered, and the cake was washed with 2 cake volumes of acetone .
35 The crystals were dried in vacuum at 20 °C. 9.44 g of bis(2
(dimethylamino)ethyl)ether diclavulanate was obtained.
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_ g _
Example 8
Crystallization of potassium clavulanate starting from bis(2-
(dimethylamino)ethyl)ether diclavulanate with intermediate
charcoal treatment
The following experiment was carried out in nitrogen
atmosphere.Bis(2-(dimethylamino)ethyl)ether diclavulanate(10
g) was dissolved in a mixture of absolute ethanol (45 ml) and
water (5 ml) at 20 °C. The pH of the mixture was adjusted to
~o pH=6.5 with the aid of 2-ethylhexanoic acid. Charcoal (1 g)
was added, and the mixture was stirred during 30 minutes.
Filter aid was added (Dicalite, 0.3 g), and the mixture was
filtered. The cake was washed with absolute ethanol (5 ml),
and acetone (10 ml). The combined filtrates were .added to
~s acetone (250 ml) at 10 °C. A crystal suspension was obtained.
A solution of potassium 2-ethylhexanoate in acetone (125 ml
of a 0.34 M solution) was added in the course of 10 minutes.
The mixture was cooled to 5 °C, and stirred during one hour.
The crystal suspension was filtered and the cake was washed
zo with three cake volumes of acetone. The wet cake was dried in
vacuum at 20 °C. 7.56 g of potassium clavulanate clusters of
needles was obtained.
