Synthesis of cyclic RGD peptidomimetics, containing a bifunctional diketopiperazine scaffold, as new integrin ligands

Integrins are glycoprotein heterodimers that control diverse cell functions such as growth, differentiation, proliferation and migration, therefore contributing to important physiological processes such as hemostasis and angiogenesis. A great amount of work has been devoted to the study and use of short peptide sequences of the endogenous ligands, that are known to be essential regulators of integrin activation or inactivation. In particular, the RGD sequence, a common motif of several endogenous integrin ligands, has been widely utilized for the construction of biologically active peptides and peptidomimetics. Conformationally rigid spacers have also been used to induce the correct spatial presentation of the RGD pharmacophoric groups. In the frame of a program aimed at the rational design of peptidomimetics with defined secondary structure, we envisioned the synthesis, conformational analysis and investigation of the biological activity of cyclic RGD-peptidomimetics, containing our recently reported bifunctional diketopiperazine scaffold DKP-1 or its trans-analogue DKP-2. The RGD-derivatives were prepared by solution phase peptide synthesis using a Boc protection strategy and t-Bu/Mtr side chain protection, giving peptidomimetics 3 and 4 in moderate to good yields. Conformational studies were performed in solution by 1H-NMR spectroscopy and computational methods (restrained molecular dynamics and docking studies), revealing that derivative 4 is better pre-organized for the interaction with the alphavbeta3 integrin receptor. The cyclic RGD peptides were then examined in vitro for their abilities to compete with biotinylated vitronectin for binding to the purified alphavbeta3 receptor, obtaining an excellent value of IC50 = 3.2 nM for derivative 4. Further studies aimed at the synthesis of modified cyclic RGD-peptidomimetics and screening with different biological targets are currently underway.