A series of 5,6-heteroaromatically annulated pyridine-2,4-diamines have been synthesized and their in vitro cytotoxic activities evaluated against six human cancer cell lines. Benzo[g] annulated pyrido[2,3-b]indolediamines 7a-b and 8 showed relatively high cytotoxic activity as well as most of the diamines with pyrrolo[2,3-b]pyridine 17, thieno[2,3-b]pyridine and furo[2,3-b]pyridine 26-28, 1,8-naphthyridine 32 and 34 and benzo[h]quinoline 37 skeletons. Surprisingly, pyrido[2,3-b]indolediamines 13 and 14 without benzo[g] annulation were inactive. None of the new compounds were as potent as ellipticine, the reference compound.