Nineteen new phenanthrene-based tylophorine analogues with various functional groups on the piperidine moiety were designed, synthesized, and evaluated for in vitro anticancer activity against four human tumor cell lines. Analogues 15 and 21 showed approximately 2-fold enhanced inhibitory activity as compared with our prior lead compound (PBT-1). Analogues 23 and 24 with S- and R-configured substituents, respectively, at the piperidine 3'-position exhibited comparable cytotoxicity to that of PBT-1. Furthermore, mechanistic studies to investigate the effects of the new compounds on Akt protein in lung cancer cells and the NF-kB signaling pathway suggested that the compounds may exert their inhibitory activity on tumor cells through inhibition of activation of both Akt and NF-kB signaling pathway.