Synthesis of several new 9-anilin0, phenylhydrazino, and sulphonamido analogs of 2or 4-methoxy-6-nitroacridine derivatives is described. The prepared compounds were tested for their in v i m antiturnor activity against 60 human tumor cell lines by the National Cancer Institute (NCI) and showed a potential anticancer activity. Compounds 9-(phenylhydrazino)-2-methoxy-6-nitroac- ridine (8a) and 9-(4-chlorophenylhydrazino)-4-methoxy-6-nitroacridine (9b) exhibited a broad spectrum antitumor activity with full panel (MG-MID) median growth inhibition ( G h ) , of 16.1 and 10.9 pM and total growth inhibition (TGI) of 66.7 and 37.9 pM, respectively. Meanwhile, compounds 1Sa and 1Sb showed moderate selectivity toward leukemia cell lines. As a trial to explore the mode of action of their antitumor activity, the 6-nitroacridine analogs were evaluated for their inhibitory effect on major cell cycle control proteins cdc2 kinase and cdc25 phosphatase as possible molecular targets that may account for antimitotic potency. None of the tested compounds proved to exert their activity via this antimitotic mode of action.