The astins, a family of natural antitumor cyclopeptides, from the roots of Aster tataricus, consist of a 16-membered ring system containing uncoded amino acid residues. The backbone conformation, with a cis-3,4-dichlorinated proline residue, plays an important role in antineoplastic activity. The acyclic astins, on the other hand, do not show antitumor activity, suggesting that the cyclic nature of astins may be a key role in their biological properties. Although the antineoplastic activity of natural astins has been screened in vitro and in vivo, the mechanism of action has never been investigated. With the aim at elucidating the influence of conformational flexibility on biological activity, we have designed and synthesized several astin analogues containing either Aib and the nonproteinogenic Abu and (S)beta3-hPhe residues, able to modify the peptide backbone structure, or the peptide bond surrogate -SO2-NH-. Tested for their antitumor effect, our astin-related cyclopeptides are able to inhibit the growth of tumor cell lines, while the acyclic astins are inefficacious. The present work reports on the structure-activity study of a selected synthetic cyclotetrapeptide corresponding to the sequence c[Thr-Aib-(S)beta3-hPhePsi(CH2-SO2-NH)-Abu], synthesized by classical methods and characterized conformationally by two-dimensional NMR and molecular dynamics analyses.