In continuation of our studies directed towards metabolically stable, 3-acyloxymethyl cephem derivatives, we report the synthesis, antibacterial activity and biological properties of 2a-f, a series of 3-acryloxymethyl cephalosporins. Cefotaxime 1a, a pioneering third-generation cephalosporin, possesses high in vitro activity against a wide range of gram-positive and gram-negative bacterial pathogens. The mechanism of action of such 3-acetoxymethyl derivatives has been demonstrated to comprise acylative attack by the β-lactam moiety on the penicillin-binding proteins bound to the cytoplasmic membrane of the bacterial cell-wall, with subsequent expulsion of the acetoxy nucleofuge. 2-6 In vivo, however, the molecule is vulnerable to esterase hydrolysis of the acetate linkage; the resulting 3-hydroxymethyl metabolite is considerably attenuated in activity. 7 In a previous report, we described an extension of our investigations 8 of cephalosporins containing novel 3-substituents to establish a strategy for circumventing such esterase deactivation. This provided an activating lactonyl substituent at the 3-position which could recyclise in the event of hydrolytic cleavage; 9 the series retained the in vitro antibacterial potency of cefotaxime. We now describe an alternative approach, based on the reduced reactivity of α,β-unsaturated esters 2 towards nucleophilic attack by water at the enoate carbonyl group, in comparison with their saturated counterparts. 10 This is a consequence of their electronic structure and led us to predict that the conjugated series would be less compromised by esterase hydrolysis. Furthermore, we anticipated that the provision of double bond substituents would also inhibit this cleavage sterically. The hydroxymethyl cephem 3 was obtained by known methods from 7-aminocephalosporanic acid. 11 Attempted reactions of this compound with activated acrylates (acid chloride, anhydride, mixed methanesulphonic anhydride) gave mixtures of Δ²- and Δ³-cephems, though in good yield (>70%). However, under Mitsunobu conditions (Scheme 1) almost instantaneous reaction between 3 and various unsaturated acids occurred to give the required Δ³-cephems 4a-f as the major products; little of the Δ²-cephem isomers (cf. 11, Scheme 2) was observed. Transamidation of the 7-side chain substituent using oximinoacetic acids 8 and 99 then allowed elaboration to the free acids and their sodium salts 2a-f. In an alternative strategy, benzhydryl 6-phenylacetamido-3-chloromethyl-cep-3-em-4-carboxylate (10, known as 'G Cl H') 12 was investigated as a starting material for the preparation of these acrylate derivatives, by simple nucleophilic displacement of the halogen atom by acrylate salts in dimethyl formamide as solvent. Sodium acrylate gave a complex mixture of products, largely polymeric. Higher acrylates gave some of the desired products but contaminated with inseparable Δ²-cephems 11. Oxidation of these mixtures with 3-chloroperbenzoic acid to restore the Δ³-double bond via the Δ³-cephem sulphoxide 12 (cf. Scheme 2) was unsatisfactory; the Δ²- and Δ³-esters oxidised at sulphur at considerably different rates, leading irreversibly to partial over-oxidation to the sulphones. However, the use of phase-transfer conditions for the acrylate-halogen displacement conveniently provided the Δ²-cephem 11 as the sole product (Scheme 2). This obviated the foregoing complication, permitting efficient access to the required Δ³-cephems via an oxidation/reduction cycle. The methacrylate 2c and the regioisomeric dimethacrylates 2a and 2e were found to exhibit interesting in vitro biological activities. They showed excellent activity against gram +ve and gram -ve bacteria (except for Pseudomonads) (Table), including β-lactamase producers. They also exhibited useful activity against Staphylococci and Streptococci possessing target site-mediated resistance. Our predictions for stability of the enoate linkage proved to be correct; for example, dimethyl acrylate 2a retained aqueous stability and was completely stable to a rat liver homogenate preparation in which 1a and 1b had half-lives of the order of 0.25h or less, thus demonstrating its insusceptibility to esterases (and other degradative liver enzymes) (Figs. 1 & 2). Compounds 2 (Table) lack pseudomonal activity necessary for parenteral usage. Nonetheless the good broad-spectrum antibacterial profiles would be suitable for an oral agent. 15 Sodium salts 2c and 2e showed good blood levels and long serum half-lives in mice, by the subcutaneous route, but almost no oral absorption. To enhance the oral absorption properties of these compounds their respective in vivo hydrolysable esters 7c and 7e (R³ = pivaloyloxymethyl or acetoxyethyl) were prepared. These were derived either from the salts 2c,e by phase-transfer alkylation (causing no Δ²-cephem formation) or by the modified synthesis from 'G Cl H' as shown in full in Scheme 2. This also allowed an improved preparation of the salt forms. Administration of the esters to mice by the oral route greatly enhanced the oral bioavailability of the parent salts 7c, 7e to ca. 25% of that produced on subcutaneous dosage.