Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound having a 1-thia-5-aza-bicyclo
Reexamination Certificate
1998-06-05
2003-01-07
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound having a 1-thia-5-aza-bicyclo
C435S043000, C435S044000, C435S045000, C435S046000, C435S047000, C435S048000, C435S049000, C435S050000, C435S147000
Reexamination Certificate
active
06503727
ABSTRACT:
The invention relates to a process for the preparation of an antibiotic wherein a &bgr;-lactam core is acylated and the antibiotic is recovered from the reaction mixture and subsequently the remaining mother liquor is worked up.
In the preparation of antibiotics involving the acylation of a &bgr;-lactam core with an acylation agent, for instance a derivative of a D-phenyl glycine, the recovery of the antibiotic and the working up of the reaction mixture are difficult in general. Thus WO-A-93/12250 for instance describes that the acylation reaction never runs to completion and the ultimate purification of the final product is hindered because the acid/base properties and solubilities of some other components that are present (in particular 7-ADCA and phenyl glycine in the case of cephalexin preparation as described in U.S. Pat. No. 4,003,896) differ little from those of the final product. As a result, coprecipitation occurs, so that impure antibiotic, i.c. cephalexin, is obtained. In WO-A-93/12250 and U.S. Pat. No. 4,003,896 the use of a complexing agent such as naphthol is proposed. However, this entails the drawback that an additional substance alien to the process has to be added.
The objective of the invention is to provide a simple, general and widely applicable process, enabling the antibiotic to be recovered pure, without using such organic compounds that are foreign to the process and without large losses of costly (starting) material, i.c. &bgr;-lactam core.
This is achieved according to the invention in that in the working-up step the mother liquor—which still contains a relatively high concentration of antibiotic—is subjected to a hydrolysis reaction and subsequently the &bgr;-lactam core is at least partially re-used for instance in an acylation reaction.
The acylation reaction can be effected either chemically or enzymatically. In the case of a chemical acylation and subsequent working up, racemisation will generally also result in the formation of a very small amount of acylation product with the wrong side chain enantiomer. If the acylation product in the mother liquor is recycled, strong accumulation of this side product may occur. The process according to the invention, with the acylation product and the acylation agent (side chain) being hydrolyzed again, offers the additional advantage of circumventing this problem.
The process according to the invention is preferably carried out in combination with an enzymatic acylation reaction. In the state-of-the-art processes a large excess of acylation agent has to be used in an enzymatic acylation reaction in order to ensure a high yield of antibiotic relative to &bgr;-lactam core. This entails the drawback that either large losses of acylation agent occur as a result of hydrolysis or that a large quantity of the reaction mixture (that which remains after recovery of the antibiotic in the solid state) has to be worked up.
The applicant now has found a process wherein the use of a large excess of acylation agent during the acylation reaction is obviated and wherein nevertheless the &bgr;-lactam core losses are limited, by subjecting the mother liquor to a hydrolysis reaction in which antibiotic still present in the mother liquor is decomposed into its initial compounds and the acylation agent is hydrolized as well, with the ability to subsequently recover or recycle the &bgr;-lactam core. The fact is that it has appeared that the process according to the invention enables a simple process to be realized, with no need for full conversion of the &bgr;-lactam core, while nevertheless only minor losses of &bgr;-lactam core occur.
Surprisingly, it has been found that the solubility of the &bgr;-lactam core is influenced by the presence of remaining acylation agent and antibiotic in the reaction mixture, in the sense that the solubility of the &bgr;-lactam core is unexpectedly high at relatively high concentrations of acylation agent and antibiotic. As a result, it was found to be possible to recover virtually quantitatively the &bgr;-lactam core in the solid state by hydrolizing the mother liquor obtained after isolation of the antibiotic, which results in hydrolysis of the antibiotic and the acylation agent. Moreover, it was found that the &bgr;-lactam core, in the solid state, was recovered in a purer form than in a process in which hydrolysis of th)e mother liquor had not been effected. Especially the content of &agr;-substituted acetic acid in the &bgr;-lactam core appeared to have been strongly decreased, which in turn had a surprisingly large effect on the acylation reaction speed. In particular, the content of phenylacetic acid in 7-ADCA and the content of phenyl or phenyloxy acetic acid in 6-APA appeared to be strongly decreased. In addition it appeared that the &bgr;-lactam core crystals contained little adhering moisture, which means a little amount of impurities. An additional advantage appears in that, because of their low solubility, the &bgr;-lactam core crystals could also be washed without large losses. This in turn, means a lower build-up of impurities, possibly accumulating in the antibiotic, in recycling.
The &bgr;-lactam core obtained after the hydrolysis reaction constitutes a novel composition that has particular advantages in the enzymatic preparation of &bgr;-lactam antibiotics. Applicant has found that the free phenyl or phenoxy acetic acid content in the &bgr;-lactam core recovered after hydrolysis, is significantly lower than that of the &bgr;-lactam core starting material, in particular it has appeared in relation to the 7-ADCA &bgr;-lactam core that where the free phenylacetic acid content of the 7-ADCA starting material was 120 ppm, the free phenylacetic acid content of the 7-ADCA obtained after hydrolysis was 69 ppm; for 6-APA these figures were 30 ppm and <15 ppm, respectively (ppm calculated with respect to the amount of &bgr;-lactam core). The invention, therefore, also relates to 7-ADCA with less than 100, in particular less than 80, preferably less than 70 ppm phenylacetic acid, and to 6-APA with less 20, preferably less than 15 ppm &agr;-aryl- or aryloxy acetic acid, particularly &agr;-phenyl- or &agr;-phenyoxyacetic acid.
Another advantage of the process according to the invention is that as a rule the—generally valuable—hydrolized acylation agent can effectively be recovered.
Optionally, the &bgr;-lactam core can be recycled in solution to the acylation reaction after the hydrolysis reaction. Preferably, the &bgr;-lactam core is recovered, however, for instance by lowering the pH and isolating the &bgr;-lactam core precipitated in the solid state. The hydrolized acylation agent can be recovered at various places in the process as a whole, for instance after the hydrolysis reaction, after the recovery of the &bgr;-lactam core or after the condensation reaction.
The hydrolysis is preferably carried out in the presence of a suitable enzyme. Suitable enzymes for the enzymatic hydrolysis reaction are for instance the enzymes that are used in the preparation of &bgr;-lactam cores and in the enzymatic acylation reactions, for instance amidases or acylases, in particular penicillin amidases or acylases. Such enzymes are described for instance by J. G. Shewale et al. in Process Biochemistry, August 1989, pp. 146-154, and in Process Biochemistry International, June 1990, pp. 97-103. Examples of suitable enzymes are enzymes derived from Acetobacter, in particular
Acetobacter pasteurianum,
Aeromones, Alcaligenes, in particular
Alcaligenes faecalis,
Aphanocladium, Bacillus sp., in particular
Bacillus megaterium,
Cephalosporium, Escherichia, in particular
Escherichia coli,
Flavobacterium, Kluyvera, Mycoplana, Protaminobacter, Pseudomonas en Xanthomonas, in particular
Xanthomonas citril.
Preferably an immobilized enzyme is used, since the enzyme can be easily isolated and re-used then. A suitable immobilization technology is described for instance in EP-A-222462. Another suitable technology consists in immobilizing the Penicillin G acylase on a carrier which contains a gelating agent, f
Boesten Wilhelmus H. J.
Dekkers Rocus M.
Van Den Tweel Wilhelmus J. J.
Gist-Brocades B.V.
Marx Irene
Pillsbury & Winthrop LLP
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