Tryprostatin A 1 and B 2 are indole alkaloid-based fungal products that act in the G2/M phase of the cell cycle. Tryprostatin A and B as well as their two enantiomers and four diastereomers have been synthesized via a common strategy. As a measure of cytotoxicity, these eight stereoisomers were assayed for their growth inhibitory properties in human breast, prostate, and lung cancer cell lines. The ability of the tryprostatins and the tryprostatin stereoisomers to induce topoisomerase II-mediated DNA relaxation or to inhibit tubulin polymerization was also examined. Although none of the stereoisomers were significantly active in topoisomerase II- or tubulin-based assays, ds2-try B 11 was found to exhibit a cytotoxicity profile more potent than etoposide 3 in the human cancer cell lines examined. In addition, ds2-try B 11 is comprised of an L-tryptophan derivative coupled to a D-proline moiety, the latter stereochemistry of which may enhance the activity of 11 and potential analogues in vivo.