SYNTHESIS AND BIOLOGICAL EVALUATION OF POTENT INTEGRIN LIGANDS CONTAINING A DIKETOPIPERAZINE SCAFFOLD, AND OF THEIR DUAL-ACTION CONJUGATES

A library of six new bifunctional 2,5-diketopiperazine scaffolds (DKP-4-7, DKP-f4, DKP-f6) was prepared, in addition to the previously reported scaffolds DKP-1-3 and DKP-f2-f3. All the DKP scaffolds feature one carboxylic acid functionality and one protected amino group (DKP-4-7) or two protected amino groups (DKP-f4, DKP-f6). Moreover, the DKP scaffolds differ from each other for the substitution at the intracyclic nitrogen atoms (N-1, N-4), as they are either mono (DKP-4, DKP-6, DKP-f4, DKP-f6) or bis-benzylated (DKP-5, DKP-7). A different synthetic strategy, with respect to the one already reported, was devised: the key step was the formation of azido-dipeptide derivatives. Dibenzylated diketopiperazines DKP-5, and DKP-7 were easily accessed by benzylation of mono-substituted advanced intermediates. The six new scaffolds were used as templates for the synthesis of cyclic integrin ligands containing the RGD-recognition sequences. Ligands c[DKP-4-7-RGD] were tested for their ability to inhibit biotinylated vitronectin binding to the isolated αvβ3 and αvβ5 integrin receptors. All of the ligands displayed low nanomolar affinity for αVβ3 integrin and nanomolar affinity for αVβ5 integrin. The conformational preferences of ligands c[DKP-4-7-RGD] were investigated by NMR spectroscopy in water, detecting H-bonds and long-range NOE contacts. In addition, three-dimensional structures satisfying long-range NOE contacts were generated by restrained MC/SD simulations. These high affinity ligands display well-defined preferred conformations featuring intramolecular hydrogen-bonded turn motifs and an extended arrangement of the RGD sequence [Cβ(Asp)-Cβ(Arg) average distance ca. 9 Å]. Docking studies were performed: the highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex. Besides the well-known RGD motif, it was proposed that also the isoAsp-Gly-Arg (isoDGR) tripeptide is involved in integrin recognition. In fact, the isoDGR sequence can mimic RGD and interact with RGD binding site of integrins in an inverted orientation, maintaining all the typical electrostatic-clamp interactions of the RGD motif . Hence, two constrained peptides (c[DKP-2-isoDGR] and c[DKP-3-isoDGR]) containing the isoDGR motif and DKP-2 / DKP-3 diketopiperazine scaffolds were prepared, combining solid-phase and solution-phase synthesis. Very promising results were obtained for compounds c[DKP-2-isoDGR] and c[DKP-3-isoDGR], which displayed the ability to inhibit biotinylated vitronectin binding to the αvβ3 receptor, with IC50 values in the low nanomolar range. According to conformational and docking studies, the recognition sequence of compound c[DKP-2-3-isoDGR] can fit into the RGD-binding pocket of αvβ3 integrin, establishing the electrostatic clamp as well as additional key-interactions. Since αv integrins, which can be internalized by cells, are involved in tumor angiogenesis and are overexpressed on the surface of many cancer cells, integrin ligands can be usefully employed as tumor-homing peptidomimetics for site-directed delivery of cytotoxic drugs and other payloads (dual-action conjugates). This approach was applied to a new class of RGD/SMAC-mimetic dual-action conjugates. Aiming at enhancing and targeting the pro-apoptotic effect of this kind of molecules, two dual-action conjugates based on the RGD recognition motif and the SMAC mimetic unit were synthesized. The affinity of each subunit for its molecular target (αvβ3 receptor and BIR domain) was confirmed, and the moderate cytotoxicity of their monomeric precursors was maintained. In addition, preliminary results indicate that the combination of a tumor targeting cyclic RGD unit with a pro-apoptotic SMAC mimetic unit may lead to