Stereoisomeric 2,5-diazabicyclo[2.2.2]octanes 14 and 15 were prepared in a chiral-pool synthesis starting from (S)- or (R)-aspartate. The key step in the synthesis was a Dieckmann-analogous cyclization of (dioxopiperazinyl)acetates 8, which involved trapping of the intermediate hemiketal anion with Me3SiCl. The σ1 affinity was tested using membrane preparations from animal (guinea pig) and human origin. The binding of bicyclic compounds was analyzed by molecular dynamics simulations based on a 3D homology model of the σ1 receptor. The good correlation between Ki values observed in the σ1 assays and calculated free binding energy, coupled with the identification of four crucial ligand/receptor interactions, allowed the formulation of structure-affinity relationships. In an in vitro antitumor assay with seven human tumor cell lines, the bicyclic compounds inhibited selectively the growth of the cell line A427, which is due to induction of apoptosis. In this assay, the compounds behave like the known σ1 receptor antagonist haloperidol.