Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2000-10-31
2003-05-06
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S253500
Reexamination Certificate
active
06558931
ABSTRACT:
A subject of the present invention is a new process for the preparation of Fexofenadine.
Fexofenadine is a medicament which has a significant antihistaminic activity and which is free from side effects. (Efficacy and safety of Fexofenadine hydrochloride for treatment of seasonal allergic rhinitis, Bernstein D. I. et al, Ann. Allergy-Asthma-Immunol. (1997) 79(5) 443-448).
This compound is the principal metabolite of Terfenadine, itself an antihistaminic agent (D. Mc Tavish, K L Goa and M. Ferill, “terfenadine: an updated review of its pharmacological properties and therapeutic efficiency, Drug 39, 552-574 (1989)).
Fexofenadine is currently prepared by chemical route, in numerous stages with a yield of less than 10% (U.S. Pat. No. 5,578,610 and 5,581,011).
The Applicant therefore proposes to find another synthesis route for Fexofenadine. The choice is then concentrated on a bioconversion method using one of the following very specific two types of microorganisms: either the filamentous fungi of the
Absidia Corymbifera
genus and in particular
Absidia Corymbifera
LCP 63-1800, or a Streptomyces and in particular
Streptomyces platensis
NRRL 2364. The specificity of the bioconversion with these microorganisms was unexpected: among the numerous strains studied during a screening, these are the only ones which allow Fexofenadine to be obtained with a good yield and little or no by-products.
A subject of the invention is a process for the preparation of the compounds of formula (I):
characterized in that a bioconversion of the compound or compounds of formula (II) is carried out:
with either a microorganism culture of the
Absidia corymbifera
genus or a microorganism culture of the Streptomyes genus, at a pH comprised between 5.0 and 8.0, in order to obtain the expected compound or compounds of formula (I), which if appropriate are isolated, purified and/or salified, the compounds of formulae (I) or (II) being able to be in the two possible enantiomeric forms, isolated or in mixtures.
The asterisk indicates the position of the asymmetrical carbon.
A particular subject of the invention is a preparation process as described above in which the
Absidia corymbifera
is
Absidia corymbifera
LCP 63-1800 or in which the Streptomyces is
Streptomyces platensis
NRRL 2364.
Fexofenadine is a racemic mixture of the enantiomers of formula (I). A more particular subject of the process is therefore the process as described previously in which a bioconversion of Terfenadine, corresponding to a racemic mixture of the two enantiomers of formula (II), is carried out in order to obtain Fexofenadine, corresponding to a racemic mixture of the enantiomers of formula (I).
Absidia corymbifera
and in particular
Absidia corymbifera
LCP 63-1800 are available from the Laboratoire de Cryptogamie du Museum d'Histoire Naturelle de Paris [The Cryptogam Laboratory at the Paris Natural History Museum].
Among the Streptomyces which can be used in the process, which is a subject of this Application, the following Streptomyces can be mentioned:
Streptomyces albus
Streptomyces ambofaciens
ATCC 15154
Streptomyces antibioticus
ATCC 31771
Streptomyces aureofaciens
ATCC 10762
Streptomyces djakartensis
Streptomyces erythraeus
Streptomyces felleus
DSM 40130
Streptomyces fradiae
W3554
Streptomyces griseus
NRRL B150
Streptomyces JSP-2 (FH2126)
Streptomyces lividans
JT46/pCS2
Streptomyces narbonensis
FH 2102
Streptomyces olivaceus
ATCC 3335
Streptomyces platensis
ATCC 13865
Streptomyces platensis
NRRL 2364
Streptomyces rimosus
2234
Streptomyces venezuelae
NRRL B-2446.
It is known to a person skilled in the art that the extreme simplicity of bacterial cells, without a distinct nucleus, allows them to be classified as prokaryotes. This is the case for Streptomyces, filamentous bacteria, which are aerobes and gram-positive. On the other hand, the other microorganisms are called eukaryotes suchas, for example, the filamentous fungi and quite particularly
Absidia corymbifera
. Their cells are differentiated in particular from the prokaryotes by the presence of a nucleus and numerous cytoplasmic organelles.
The process described above, which is a subject of the present Application, offers numerous advantages. On the one hand, it avoids the chemical route which requires numerous synthesis stages accompanied by necessary isolation processes at each of these reaction stages. In the case of an industrial use, this route can prove to be expensive and polluting.
In the case of the process which is a subject of this Application, a single operation is necessary and the optional purification stage essentially only has the purpose of eliminating a by-product which can form during the bioconversion, namely triolphosphate which corresponds to the alcohol non yet oxidized to acid, which is esterified in the form of a phosphate (formula (IIIb)) described below.
On the other hand this process lends itself to industrial use. By operating with a concentration of starting product of 0.5 g/l, the yield exceeds 70% relative to the starting product.
Among the other advantages of the use of the microbiological route against the chemical route, the non polluting aspect of this technique can be emphasized, all the operations taking place in aqueous media.
The purification is carried out according to the methods known to a person skilled in the art. It can be purification by crystallization, by chromatography or by ion exchange resin.
The salification reactions can be carried out under the usual conditions. For example the operation can be carried out in the presence of ethanolic soda. A sodium salt can also be used such as sodium or potassium carbonate or acid carbonate. The salts obtained can be the salts of alkali or alkaline-earth metals or optionally substituted ammonium.
The implementation of the oxidation is carried out according to the methods which are currently used for the microbiological oxidation of organic molecules using cultures of filamentous fungi (Holland HL, Organic synthesis with oxidative enzymes. VCH publisher, Inc, New York 1992; Lacroix I, Biton J and Azerad R, Microbial biotransformation of a synthetic immunomodulating agent HR325, Bioorg. Med. Chem. (1997) 7, 1369-1380; Sebek O K, Fungal transformations as a useful method for the organic synthesis of organic compounds, Mycologia 75(2) 383-394, 1983; Azerad, R. Microbial models for drug metabolism,
Advances in Biochemical Engineering and Biotechnology
, Vol. 63, page 169, 1999.
Thus, firstly, the most favourable fermentation conditions are determined by analytical route, in particular by thin layer chromatography or HPLC, in prior tests, such as for example the choice of the nutrient medium, the solvent of the appropriate substrate, the concentration of substrate, the technical conditions such as temperature, aeration, pH, and the optimum periods for the culture, addition of the substrate and contact of the substrate with the microorganism.
In a first phase, the culture is carried out from an inoculum (spores or mycelium) of
Absidia corymbifera
LCP 63-1800 or a bacteria of the Streptomyces genus, in particular
Streptomyces platensis
NRRL 2364, in a liquid nutrient medium at an initial pH of 5 to 7 and at a temperature of 20 to 30° C., preferably from 26 to 28° C. Aeration is ensured by rotary agitation of the culture receptacles (150 to 250 rpm) or, in the fermentation vessel, by the introduction of air at a flow rate of approximately 1 l/min and per litre of broth culture medium.
After a time varying from 24 to 72 hours, preferably 60 to 65 hours, the terfenadine is added at a concentration of 0.1 to 1 g/litre, preferably 0.4 to 0.6 g/litre, in solution in an organic solvent which is miscible with water, such as ethanol, acetone, tetrahydrofuran, dimethylformamide or dimethylsulphoxide (5 to 20 ml/litre of culture), preferably ethanol (10 ml/litre). The incubation is continued under the same conditions as the culture. The pH is optionally readjusted and maintained at a value of 5.0 to 8.0. The conversion of the substrate is advantageous
Azerad Robert
Biton Jacques
Lacroix Isabelle
Aventis Pharma S.A.
Lilling Herbert J.
Muserlian Lucas and Mercanti
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