Process for preparing cephalosporin derivatives

Details Process for preparing cephalosporin derivatives Process for preparing cephalosporin derivatives

2002-04-19

2003-11-04

Lilling, Herbert J. (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

C435S049000, C540S224000, C540S229000

Reexamination Certificate

active

06642020

ABSTRACT:

The invention relates to a process for the preparation of 3-cephalosporin C derivatives which are used in the preparation of &bgr;-lactam antibiotics. In particular, the invention relates to a process for the preparation of 3-thiolated derivatives of 3-acetoxy-methyl-7-amino-ceph-3-em-carboxylic acid.
BACKGROUND
All cephalosporins used in therapeutic applications are semi-synthetic and are produced by modifying the basic &bgr;-lactam structure in the material obtained from a fermentation broth of
Acremonium chrysogenum
or, after chemical transformation, from the products obtained from fermentation broths of
Penicillium chrysogenum
using different precursors.
Typically, cephalosporin C [3-acetoxymethyl-7-(D-5-amino-5-carboxy pentan amido)-ceph-3-em-4-carboxylic acid] is converted into 3-acetoxymethyl-7-amino-ceph-3-em-carboxylic acid, usually known as 7-amino cephalosporanic acid (7-ACA), by removing the lateral aminoadipic chain of the &bgr;-lactam ring. The 7-ACA is purified and crystallised, and is then used as starting material for subsequent modifications at the 7- and 3-position. 7-ACA is the base building block used in the synthesis of many important semi-synthetic cepahalosporin antibiotics that are of current interest in the biopharmaceutical industry.
3-thiomethyl Cephalosporin C derivatives have been disclosed in U.S. Pat. Nos. 3,278,531; 3,516,997; 3,647,788; GB 1,400,804; GB 1,565,053 and GB 1,566,515. A few examples of 3′-thiomethyl glutaryl 7-ACA derivatives are described in WO-A-9535020 and EP 0846695.
7-ACA is the most widely used intermediate for reaction with heterocyclic thiols as it can be obtained either chemically or enzymatically on an industrial scale. This has been disclosed in several patents including U.S. Pat. Nos. 3,502,665; 3,954,745; 3,516,997; 3,979,383; 4,115,645; 4,317,907; 5,387,679; JP 55,139,327; EP 0167651 and WO-A-9302085.
U.S. Pat. No. 5,387,679 describes the reaction of 7-amino cephalosporanic acid with 2-mercapto-5-methyl-1,3,4-thiadiazole (MMTD) in the presence of sodium bicarbonate in aqueous acetone, at pH 6-7. A yield of about 60-65% is achieved.
In U.S. Pat. No. 4,317,907, the yield of the above reaction in anhydrous medium was increased to about 86% when acetic acid or nitromethanol was used as a solvent, in the presence of boron trifluoride or boron trifluoride etherate. However the purity is low, 80% max, as it contains unreacted 7-ACA and degradation products. In WO-A-9302085 the use of dialkyl carbonate in the presence of a dialkyl carbonate trifluoride complex and an aliphatic acid increased the yield to a maximum of 89%. However, WO-A-9302085 shows the problems associated with using toxic and very expensive gas such as BF
3
and the handling of waste effluent which contains borides and fluoroborides.
The deacylation of cephalosporin C, i.e., the elimination of the 7′-lateral side chain, is usually carried out chemically, for example using nitrosyl chloride in formic acid in the presence of acetonitrile (U.S. Pat. No. 3,367,933). Another method of deacylation involves the protection of the carboxyl group of aminopenteanoic chain, reaction with phosphorus pentachloride at −55° C. and subsequent hydrolysis at low temperature with a mixture of water and methanol (BE 718,824).
These known methods must generally be carried out at low temperatures, and require the use of costly and toxic solvents or reagents, consequently they may have a serious environmental impact. In addition, because of the chemical instability of the &bgr;-lactam nucleus and the reactivity of the groups present in positions 3 and 7 of the ring, special reaction conditions must be applied, which makes the process on an industrial scale complex.
To overcome the drawbacks of the chemical route to 7-ACA, alternative enzymatic cleavage of cephalosporin C has been described. Direct one-step removal of the lateral 7-aminoadipic side-chain of cephalosporin C is possible by using specific cephalosporin acylases (FR 2,241,557; U.S. Pat. No. 4,774,179; EP 283,248; WO-A-9512680; WO-A-9616174). These processes, however, are often not reproducible and are characterised by low yields and lengthy reaction times as described in U.S. Pat. No. 5,296,358. No industrial application of this technology (single-step conversion of cephalosporin C to 7-ACA) has been reported at this time (Parmar et al, Crit. Rev. Biotechnol. 18, 1, 1998).
On the other hand, processes that transform the cephalosporin C into 7-ACA by means of two enzymatic steps are important from an industrial point of view. The first stage consists of using a D-amino acid oxidase (E.C. 1.4.3.3, hereinbelow indicated as DAAO) from different sources (
Trigonopsis variabilis
, GB 1,272,769;
Rhodotorula gracilis
, EP 0,517,200; or
Fusarium solari
M-0718, EP 0,364,275). DAAO oxidises the lateral D-5-amido-carboxypentanoyl chain of cephalosporin C in the presence of molecular oxygen, to produce 7&bgr;-(5-carboxy-5-oxopent-amido)-ceph-3-em-carboxylic acid (or &agr;-ketoadipyl-7-aminocephalo-sporanic acid, hereinbelow indicated as &agr;-ketoadipyl-7-ACA) and hydrogen peroxide, which chemically decarboxylate the &agr;-ketoadipyl-7-ACA to 7&bgr;-(4-carboxy butanamido)-ceph-3-em-4-carboxylic acid (or glutaryl-7-aminocephalosporanic acid, hereinbelow indicated as GL-7-ACA).
In a second stage, a specific acylase for GL-7-ACA, glutaryl-7-ACA acylase (E.C. 3.5.1.3), is used, for example that of a Pseudomonas type microorganism (U.S. Pat. No. 3,960,662, EP 0496993) over expressed in
E. coli
, which deacylates the GL-7-ACA into 7-ACA.
This two-step enzymatic process for obtaining 7-ACA has been used on an industrial scale (Conlon et al. Biotechnol. Bioeng. 46, 510, 1995).
An environmental-friendly alternative to produce 3′-heterocyclic thiomethyl cephalosporanic acid derivatives was disclosed in EP 0846695. Chemical nucleophilic displacement of the 3-position of glutaryl-7-ACA in aqueous medium is followed by an enzymatic transformation of the 3′glutaryl-7-ACA-derivative by using the enzyme glutaryl-7-ACA acylase. The quantity of the derivative is about 65% with no environmental impact. This procedure can be defined as an enzymatic-chemical-enzymatic (ECE) process, since the isolated GL-7-ACA comes from a bioconversion of solubilised cephalosporin C, then GL-7-ACA is reacted with the heterocyclic thiols and finally the 3-heterocyclic thio-derivative is enzymated with GL-7-ACA acylase. The problem with this method is the need to isolate GL-7-ACA, which given its high water solubility, is technically difficult and expensive, as described in WO-A-9535020.
An additional problem is that the enzyme is only reusable for a few cycles. In fact no more than three cycles are described with 5-mercaptotriazol and MMTD, where the residual amount of MMTD after crystallisation is 2.8 mg/ml. The other two thiols used as examples are only used in one cycle due to the high amounts of the thiols remaining in solution. This is the case with 5-mercapto-1-methyltetrazole (MMTZ) or 2,5-dihydro-3-mercapto-2-methyl-5,6-dioxo-1,2,4-triazine (TTZ), which are very soluble in water and not removable by decreasing the pH. This poisoning effect of the thiol has also been described for MMTD on Penicillin G Amidase from
E. coli
CFC-04017 (Won et al, App. Biochem. Biotech. 69, 1, 1998) during the enzymatic synthesis of cefazolin. To overcome this inhibitory effect the molar ratio of MMTD/7-ACA is decreased to 1.2:1 in order to extend the lifetime of the enzyme. This low molar ratio reduces the yield of the 3-thio derivative and avoids the use of the other published process on the chemo-enzymatic synthesis of 3-modified cephalosporins, in which an enzymatic-enzymatic-chemical process (EEC) is proposed, using D-amino acid oxidase—glutaryl-7-ACA acylase and a chemical reaction with heterocyclic thiols (Jistiz et al., J. Org. Chem. 62, 9099, 1997).
There is therefore a need for an improved process for preparing 3-cephalosporin C derivatives.
STATEMENTS OF INVENTION
According to the invention there is provided an enzymatic

Affiliated with

Also associated with

Rating

Process for preparing cephalosporin derivatives does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Process for preparing cephalosporin derivatives, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Process for preparing cephalosporin derivatives will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3181312