SYNTHESIS AND BIOLOGICAL EVALUATION OF POTENT INTEGRIN LIGANDS CONTAINING A DIKETOPIPERAZINE SCAFFOLD, AND OF THEIR DUAL-ACTION CONJUGATES
Creators: name: MINGOZZI, MICHELE
Publisher: name: Universit� degli Studi di Milano
Year: 2014

description: 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 ?v?3 and ?v?5 integrin receptors. All of the ligands displayed low nanomolar affinity for ?V?3 integrin and nanomolar affinity for ?V?5 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 [C?(Asp)-C?(Arg) average distance ca. 9 �]. 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 ?v?3 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 ?v?3 integrin, establishing the electrostatic clamp as well as additional key-interactions. 
Since ?v 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 (?v?3 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 synergistic anticancer effects.
In addition, a new class of dual-action conjugates aimed at blocking the natural ?cross-talk? between integrins and vascular endothelial growth factor receptors (VEGFR) was developed. Starting from two selective ligands (MA peptide and c[DKP-f3-RGD]), two covalently-linked dual-action conjugates (MA-RGD and Ac-MA-RGD) were synthesized, aiming at inhibiting both ?v?3 and VEGFR1 receptors. The dual-action conjugates were prepared via a final CuAAC ?click? reaction (in solution phase and on solid phase, respectively) between the alkyne-bearing VEGFR ligand (prepared via microwave-assisted SPPS) and the azido-functionalized PEG8-RGD integrin ligand. The biological activities of both the two compounds were evaluated in vitro with binding assays on the isolated receptors ?V?3/?V?5: the affinity for the integrin ?V?3 was maintained. The anti-angiogenic activity of these compounds and their precursors was tested in vitro with morphogenesis assays on Matrigel (HUVEC); similar results were obtained, underlining a very complex angiogenesis regulation system. ?V?3/VEGFR dual-action conjugates can be considered a promising tool to further elucidate the cross-talk regulation mechanism in the examined cell biology.