Tackling Lipophilicity of Peptide Drugs: Replacement of the Backbone N-Methyl Group of Cilengitide by N-Oligoethylene Glycol (N-OEG) Chains

Abstract

Cilengitide is an RGD-peptide of sequence cyclo[RGDfNMeV] that was was developed as a highly active and selective ligand for the αvβ3 and αvβ5 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 We describe the synthesis of three analogues of this peptide in which the N-Me group has been replaced by N-oligoethylene glycol (N-OEG) chains of increasing size: namely N-OEG2, N-OEG11, and N-OEG23, which are respectively composed of 2, 11, and 23 ethylene oxide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-OEG11 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-OEG23 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.

Review backbone N‐modified peptides: How to meet the challenge of secondary amine acylation

“…Similar findings were reported for cilengitide. For this N ‐methylated cyclopeptide, replacement of its backbone N ‐Me group by a short N ‐oligoethylene glycol ( N ‐OEG) chain provided a more lipophilic analog with a similar biological activity, and lipophilicity was enhanced upon increasing the length of the OEG chain . In another study, they synthesized an N ‐(4‐azidobutylated) analog of cilengitide, and conjugated it with a polyethylene glycol (PEG) chain through several chemical transformations.…”

“…In several reports on the acylation of N ‐substituted peptides in solid phase, bis(trichloromethyl)carbonate (BTC) was found to be the most efficient activating reagent . This activating reagent, which is considered to convert amino acids into their corresponding acid chlorides, was introduced by Gilon et al They demonstrated that a wide range of Fmoc‐amino acids can be efficiently coupled to various functionalized N‐ alkyl peptides using BTC (Table , entry v), and reported the use of BTC in the SPPS of several N ‐backbone cyclic peptides .…”

An alternative approach to create N-substituted cyclic dipeptides

“…26 More recently, Albericio and coworkers have reported the synthesis of N-oligoethylene glycol (N-OEG) substituted cyclic peptides: sansalvamide A with 3 repeat units of N-OEG 27 and cilengitide with different repeat units of N-OEG such as 2, 11 and 23. 28 The synthesis of cilengitide with N-OEG 2 and sansalvamide A with N-OEG 3 by solid-phase peptide synthesis in multiple steps was reported; however, peptides with longer N-OEG substitutions were very complicated to synthesize and were reported to require extensive purification steps. 28 In the current work, we are introducing anionic ring-opening polymerization (AROP) as a novel technique to create peptides with N-polyether derivatives in a one-step reaction without sophisticated purification steps.…”

“…28 The synthesis of cilengitide with N-OEG 2 and sansalvamide A with N-OEG 3 by solid-phase peptide synthesis in multiple steps was reported; however, peptides with longer N-OEG substitutions were very complicated to synthesize and were reported to require extensive purification steps. 28 In the current work, we are introducing anionic ring-opening polymerization (AROP) as a novel technique to create peptides with N-polyether derivatives in a one-step reaction without sophisticated purification steps. AROP is an effective method which is frequently used to synthesize various functional polymers readily applicable in the biomedical field.…”

In silico study of peptide inhibitors against BACE 1

“…Even though high biodegradability, low bioavailability and low cellular uptake may be some of the limitations of peptide drugs, they can benefit from novel synthetic strategies for enhancing their drug-like properties (Pandey et al 2009;Vlieghe et al 2010). Currently, modifications that a prospective peptide drug candidate can undergo to enhance their efficacy include peptide stapling (Verdine and Hilinski 2012), backbone replacement (Fernández-Llamazares et al 2014), non-natural amino acid incorporation, pseudo peptide bonds, cyclization (Gentilucci et al 2010), conjugation (Gauthier and Klok 2008;Ahrens et al 2012) and so on.…”