Abstract: To discover an efficient approach for the conversion of antibacterial fluoroquinolones into an antitumor activity, a fused heterocycle ring core, thiazolo［3, 2-b］［1, 2, 4］triazol-5-one was used as an isostere and further modified with an arylidene group. Then, 12 novel C-3 fused heterocyclic unsaturated ketones, 1- ethyl-6-fluoro-7-(4-methyl-piperazin-1-yl)-3-［6-arylidene-thiazolo［3, 2-b］［1, 2, 4］triazol-5(6H)-one-3-yl］- quinolon-4(1H)-ones( 6a - 6l ), were designed and synthesized from pefloxacin( 1 ). The structures were characterized by elemental analysis and spectral data, and the in vitro antitumor activity of the title compounds against SMMC-7721, Capan-1 and HL60 cell lines was evaluated. The preliminary pharmacological results demonstrated that the title compounds exhibited more significantly antiproliferative activity than the parent 1 . The compounds with fluorophenyl or o-methoxyphenyl showed comparable activity to the comparasion doxorubicin. Thus, it appears to be an alternative route for a fused heterocyclic unsaturated ketone as an isostere of the C-3 carboxylic group to improve the antitumor activity.