Formation of fluorescent pyrazine derivatives via a novel degradation pathway of the carbacephalosporin loracarbef

Abstract

Recently an unknown degradation product of loracarbef (structure 1) was observed at low levels (--0.1%) in samples of the bulk drug substance that had been stored at 30°C for 36 months. Identification of this unknown was desired because of the potential for this unknown to form in the product during the shelf-life of the drug [1]. Isolation of such a minor impurity from limited quantities of sample is difficult and, therefore, more severe conditions were evaluated for generating higher levels of this degradation product. LC analysis with photodiode array detection indicated that this degradation product was present at higher levels in a sample of loracarbef stressed at 85°C for 8.5 months. Therefore, the degradation product (structure 4) and two other closely eluting degradation products (structures 5 and 6) were isolated concurrently from the 85°C stressed sample by preparative LC. Visualization of all three of these products under long wavelength UV indicated that they were fluorescent. MS and NMR spectroscopic characterization of these three products indicated the structures (structures 4-6) were derivatives of the highly fluorescent 2-hydroxy3-phenylpyrazine (structure 3). It is well established that [3-1actam antibiotics containing the phenyl glycine side chain will degrade under certain conditions to 2-hydroxy3-phenyl pyrazine (structure 3) [2-4]. Degradation studies of cefaclor (structure 2) [5] and other phenyl glycine-containing 13-1actams [4, 6] led to a proposed pathway for the formation of this pyrazine derivative (Scheme 1). The pathway to these 2-hydroxy-3-phenyl pyrazine derivatives involves hydrolysis of carbon-6 to reveal the masked aldehyde, and subsequent cyclization and aromatization leading to the pyrazine structure. In the case of the loracarbef, the sulphur at position 5 is replaced with a methylene, effectively blocking the possibility of hydrolysis leading to an aldehyde at position 6. Thus it was predicted that carbacepahlosporins such as loracarbef would not degrade to 2-hydroxy-3phenyl pyrazine derivatives. In agreement with this prediction, no pyrazine derivatives were detected in a previous study of the aqueous degradation of loracarbef [7]. The discovery that substituted pyrazines were formed during solid-state degradation of Ioracarbef indicates the existence of a novel degradation pathway to pyrazines, distinct from the established cephalosporin degradation pathway. This report describes the isolation, characterization and proposed mechanism of formation for