Abstract: To explore a new strategy for the transformation of antibacterial activity of fluoroquinolone into antitumor activity, twelve new title compounds, 1-ethyl-6-fluoro-7-(4-methyl-pipreazin-1-yl)-quinolin-4-one-3-carboxylicacid(5-arylidene-2-thioxo-1, 3-thiozolidin-2, 4-dione-3-yl)amides( 6a - 6l ), were designed and synthesized with an amide group and a rhodanine unsaturated ketone moiety as an isostere and modified group, respectively, from pefloxacin( 1 ). Their structures were characterized by elemental analysis and spectral data. The in vitro antitumor activity of the title compounds against Hep-3B, Capan-1 and L1210 cell lines exhibited more significant potency than pefloxacin. The compounds with aromatic heterocyclic or flurophenyl displayed comparable activity to the comparasion doxorubicin. Thus, rhodanine unsaturated ketone hybrided amide group as an isostere of the C-3 carboxylic acid group appears to be an alternative route for further design of antitumor fluoroquinolone.