In addition to their antibiotic potency, beta-lactams have recently been investigated as inhibitors of serine proteinase such as leukocyte elastase (LE), released by inflammatory cells. We describe the synthesis of a series of 4-alkylidene-beta-lactams, and investigate how substitutions on C-3, C-4, and N-1 of the beta-lactam ring affect the activity of human LE and gelatinases MMP-2 and MMP-9. LE activity was measured using a chromogenic substrate, while gelatin-zymography assay was used to evaluate gelatinase activity. We demonstrate that C-4 unsaturation on the beta-lactam ring determines the degree of biological activity, with a selectivity over LE by 3-[1-(tert-butyldimethylsilyloxy)-ethyl] derivatives (lowest IC(50) was 4 microM), and over gelatinase MMP-2 by C-3-unsubstituted 4-[1-ethoxycarbonyl]-ethylidene-beta-lactams (lowest IC(50) was 60 microM). (3S)-3-[(1R)-1-hydroxyethyl]-4-(1-ethoxycarbonyl)-ethylidene-azetidin-2-one inhibits gelatinase MMP-9. The compounds tested showed no cytotoxicity against NIH-3T3 murine fibroblasts. This is the first example of beta-lactams inhibiting metallo-proteinases instrumental in cancer invasion and angiogenesis. These molecules are good candidates for prototype drugs showing selective antibiotic, anti-inflammatory, and anti-invasion properties.