The inhibition of urokinase-PA (u-PA) can block a series of pathological events, such as adhesion, migration and growth of malignant cells at the metastatic site. To discover new inhibitors the SS, RR, RS and SR of 3-(4-aminobutyl)-6-(1H-indole-3-ylmethyl)piperazine-2,5-dione (AIPZ) were designed, synthesized and evaluated. HPLC with a chiral column shows that their purity approaches 100%. The docking of these analogs toward the active site of u-PA resulted in high scores (118–134). The FT-MS spectra indicate that in aqueous solution the SS, RR, RS and SR of AIPZ exist as dimer, tetramer, trimer and trimer, respectively. The ROESY 2D NMR spectra give dimer, tetramer, trimer and trimer distinct conformations. The transmission electron microscopy and scanning electron microscopy show that the SS, RR, RS and SR can form distinct nano-species. The conversion of the plasminogen towards plasmin induced by UK is configuration-dependent, blocked by the SS, RR, RS and SR. The invasion and migration of HCCLM3 cells are configuration-dependent, inhibited by the SS, RR, RS and SR. The RR was selected as a representative for in vivo evaluation. At 5 μmol kg−1 of oral dose, RR not only blocked the metastasis of Lewis lung carcinoma cells towards the lung, but also slowed the growth of the primary solid tumors initiated either by S180 ascite tumor cells or by Lewis lung carcinoma cells, and it also prevented the mice from developing xylene-induced ear edema.