Tackling lipophilicity of peptide drugs: Replacement of the backbone n -methyl group of Cilengitide by N -oligoethylene glycol (N -OEG) chains

Cilengitide is an RGD-peptide of sequence cyclo[RGDfNMeV] that was was developed as a highly active and selective ligand for the α _vβ₃ and α_vβ₅ integrin receptors. We describe the synthesis of three analogues of this peptide in which the N-Me group has been replaced by N-oligoethylene glycol (N-OEG) chains of increasing size: namely N-OEG₂, N-OEG₁₁, and N-OEG₂₃, which are respectively composed of 2, 11, and 23 ethylene oxide monomer units. The different N-OEG cyclopeptides and the original peptide were compared with respect to lipophilicity and biological activity. The N-OEG₂ analogue was straightforward to synthesize in solid phase using an Fmoc-N-OEG₂ building block. The syntheses of the N-OEG ₁₁ and N-OEG₂₃ cyclopeptides are hampered by the increased steric hindrance of the N-substituent, and could only be achieved by segment coupling, which takes place with epimerization and thus requires extensive product purification. All the N-OEG analogues were found to be more hydrophobic than the parent peptide, and their hydrophobicity was systematically enhanced upon increasing the length of the OEG chain. The N-OEG₂ cyclopeptide displayed the same capacity as Cilengitide to inhibit the integrin-mediated adhesion of HUVEC endothelial, DAOY gliobastoma, and HT-29 colon cancer cells to their ligands vitronectin and fibrinogen. The N-OEG₁₁ and N-OEG ₂₃ analogues also inhibited cell adhesion to these immobilized ligands, but their IC₅₀ values dropped by 1 order of magnitude with respect to the parent peptide. These results indicate that replacement of the backbone N-Me group of Cilengitide by a short N-OEG chain provides a more lipophilic analogue with a similar biological activity. Upon increasing the size of the N-OEG chain, liophilicity is enhanced, but synthetic yields drop and the longer polymer chains may impede targeted binding.