Cephamycins

Early work on the reactivity of the acetoxy grouping in (179) showed that displacement reactions occurred fairly readily with certain heterocyclic tertiary bases (139), while hydrolysis to deacetylcephalosporin C was readily effected using an acetyl esterase (140). The structure elucidation of this first cephalosporin derivative in 1961 has been followed by the discovery of a number of other natural metabolites produced by fungi and various actinomycete species. The various structures are listed in Table 6. Aspects of the history, chemistry and biology of the group are covered in the extensive review edited by Flynn (6). 

Cleavage of the amide bond of the aminoadipyl side chain of cephalosporin C affords the 7-aminocephalosporanic acid (7-ACA) nucleus (180). This was first achieved in small yield by direct acid hydrolysis (151). The inability to cleave the side chain enzymatically as in the penicillins (6) resulted in much effort to find an efficient chemical method to provide (180), since this is the source of many clinically important semi-synthetic cephalosporins. Many of these compounds have advantages over the penicillins in terms of acid stability and resistance to P-lactamases. 

The first effective method of side chain cleavage made use of the reaction of (179) with nitrosyl chloride (152). Loss of nitrogen from the intermediate diazo compound (181) gives imine (182) which on hydrolysis provides 7-ACA (180) and the lactone (183). This provided a route to substantial quantities of (180) thus allowing the preparation of many side chain derivatives. Later a more efficient procedure made use of phosphorus pentachloride followed by cleavage of an imino ether (153). The utility of other phosphorus halogen compounds for cleavage of the amide bond has also been described (154). 

A large number of nuclear analogues of the cephalosporin ring system have been synthesised and comprehensively reviewed (46, 155, 156). Approaches to the natural products themselves have been limited. The Roussel (157) and Squibb (158) groups have utilised suitably protected intermediates of type (184) to make the amino acid (185). Cyclisation as in Sheehan s penicillin synthesis followed by deprotection afforded the amino lactone (186). 

The only complete synthesis of cephalosporin C provides a classic example of the synthetic art of the late R. B. Woodward, and formed the subject of his Nobel Prize winning lecture of 1965. The synthesis, published in 1966, starts from L(+)-cysteine (189) 164, 165). Protection of the nitrogen, sulphur and acid functional groups provided the cyclic intermediate (191) ideally suited for the introduction of the amino function which was to become the nitrogen atom of the crucial -lactam intermediate (195). This was achieved in a completely stereocontrolled manner by a novel substitution method. Thermal introduction of the hydrazo-substituent to form (192) was followed by oxidation and conversion to the tran -hydroxy ester (193). Inversion by displacement of the mesylate with azide and subsequent reduction gave the cw-amino ester (194) which afforded the (i-lactam (195) on cyclisation. 

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Occurrence, Fermentation, and Biosynthesis. Although a large number of Streptomjces species have been shown to produce carbapenems, only S. cattkja (2) and S. penemfaciens (11) have been reported to give thienamycin (2). Generally the antibiotics occur as a mixture of analogues or isomers and are often co-produced with penicillin N and cephamycin C. Yields are low compared to other P-lactams produced by streptomycetes, and titres are of the order of 1—20 p-g sohdusmL despite, in many cases, a great deal of effort on the optimization of the media and fermentation conditions. The rather poor stabiUty of the compounds also contributes to a low recovery in the isolation procedures. The fermentation and isolation processes for thienamycin and the olivanic acids has been reviewed in some detail (12). 

Fig. 1. Biosynthesis of cephalosporins and cephamycins. a, Cephalosporium acremonium, b, Penicillium chjsogenum, c, Streptomjces clavuligerus-, d, Streptomjces lipmanir, e, Streptomjces wadajamensis, REX is a ring expansion en2yme (deacetoxycephalosporin C synthethase).

At present all of the cephalosporins ate manufactured from one of four P-lactams, cephalosporin C (2), penicillin V [87-08-17, penicillin G [113-98-4] and cephamycin C (8), which ate all produced in commercial quantities by fermentation (87). The manufacturing process consists of three steps fermentation, isolation, and chemical modification. 

Fermentation. The commercial P-lactam antibiotics which act as starting material for all of the cephalosporins ate produced by submerged fermentation. The organisms used for the commercial production of the penicillins and cephalosporins ate mutants of PenicU/in chTysogenum and Cephalosporium acremonium respectively (3,153,154). Both ate tme fungi (eucaryotes). In contrast, the cephamycins ate produced by certain species of procaryotic Streptomyces including Streptomyces clavuligerus and Streptomyces lipmanii (21,103). 

Isolation. Isolation procedures rely primarily on solubiHty, adsorption, and ionic characteristics of the P-lactam antibiotic to separate it from the large number of other components present in the fermentation mixture. The penicillins ate monobasic catboxyHc acids which lend themselves to solvent extraction techniques (154). Pencillin V, because of its improved acid stabiHty over other penicillins, can be precipitated dkecdy from broth filtrates by addition of dilute sulfuric acid (154,156). The separation process for cephalosporin C is more complex because the amphoteric nature of cephalosporin C precludes dkect extraction into organic solvents. This antibiotic is isolated through the use of a combination of ion-exchange and precipitation procedures (157). The use of neutral, macroporous resins such as XAD-2 or XAD-4, allows for a more rapid elimination of impurities in the initial steps of the isolation (158). The isolation procedure for cephamycin C also involves a series of ion exchange treatments (103). 

In organisms which produce cephalosporin and cephamycins, the configuration of the O -aminoadipyl side chain of (30) is D, while penicillin producers yield the l isomer. The exact point at which the configuration is inverted is unknown. Subsequent steps in cephalosporin biosynthesis are believed to involve ring expansion to deacetoxycephalosporin C (31), which may proceed by a mechanism analogous to the chemical pathway (see Section 5.10.4.2), followed by hydroxylation and acetylation at C-3 to produce cephalosporin C (32). 

Cefmetazole (78) is a cephamycin-inspired cephalosporin differing from the mainstream compounds in having an aliphatic amide moiety attached to C-7. Its antibacterial spectrum is similar to the second generation agent cefoxitin. The synthesis starts with 7-aminocephalosporan-... 

Figure 6.10 Biosynthetic pathways from isopenicillin N to penicillin G and cephalosporin C. Some strains have the ability to convert deacetylcephalosporin C into cephamycin C.

The biosynthetic route to cephalosporin C is identical to that of the penicillins as far as isopeniciUin N (section 3.4.3). The further route to cephalosporin C is shown on p. 160. Note the branch into a third series of /3-lactam drugs, the cephamycins (see Chapter 5). 

While screening for p-lactam antibiotics stable to p-lactamases, a strain of Streptomyces lactamdurans was found to contain several such agents which have a 6-a-methoxy group whose electronic and steric properties protect the antibiotic from enzymatic attack. Cephamycin C (29a), one of these substances, is not of commercial value, but side chain exchange has led to much more potent materials. Of the various ways of effecting this transformation, one of the more direct is to react cephamycin C with nitrous acid so that the aliphatic diazo product (29b) decomposes by secondary amide participation giving cyclic iminoether 30. The imino ether moiety solvolyzes more readily than the p-lactam to produce 7-aminocephamycinic... 

There has also been extensive activity towards the replacement of the entire chemical route to 7-ADCA (Scheme 1.14) with a biocatalytic one. This is somewhat more complex than the above example, as the penicillin fermentation product requires ring expansion as well as side-chain hydrolysis in order to arrive at the desired nucleus. The penicillin nucleus can be converted to the cephalosporin nucleus using expandase enzymes, a process that occurs naturally during the biosynthesis of cephalosporin C by Acremonium chryso-genum and cephamycin C by Streptomyces clavuligems from isopenicUhn N (6-APA containing a 6-L-a-aminoadipoyl side chain). ... 

The ribosome-associated ppGpp synthetase (RelA) is required for antibiotic production under the conditions of nitrogen limitation in S. coelicolor A3 and for cephamycin C production in S. clavuligerus. Deletion of the relA in S. coelicolor A3 results in the loss of production of the antibiotics actinorhodin (Act) and undecylprodigiosin (Red) and... 

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