Several 9-chloro-11H-indeno[1,2-c]quinolin-11-one derivatives have been designed which is replacing side chains with different groups containing oxygen, nitrogen or sulfur atoms. Substitution of C-6 on the starting structure, 6,9-dichloro-11H-indeno[1,2-c]quinolin-11-one, using apposite nucleophilic group with a suitable base or acid could be obtained 28 novel tetracyclic azafluorenone derivatives. The cytotoxic activity of these analogues was examined in cancer cell lines by MTT assay and compounds 4, 5, 13, and 26 were selected to evaluate in topoisomerase I drug screening assay, respectively. At the same time, 17 compounds were selected for NCI-60 anticancer drug screen to prevent the narrower concept of an in vitro screening model. Its worth to find that 9-chloro-6-(piperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (12) showed greater cytotoxicity than another azafluorenone derivatives with an average GI50 of 10.498μM over 60 cell lines. We also found that another analogue, 9-chloro-6-(2-methylpiperazin-1-yl)-11H-indeno[1,2-c]quinolin-11-one (13), exhibited preferential growth inhibition effect toward cancer cell lines and showed a significant inhibitory effect on topoisomerase I. © 2016.