Synthesis and Biological Evaluation of RGD and isoDGR–Monomethyl Auristatin Conjugates Targeting Integrin αVβ3

This work reports the synthesis of a series of small-molecule–drug conjugates containing the α_Vβ₃-integrin ligand cycloDKP-RGD] or cycloDKP-isoDGR], a lysosomally cleavable Val-Ala (VA) linker or an “uncleavable” version devoid of this sequence, and monomethyl auristatin E (MMAE) or F (MMAF) as the cytotoxic agent. The conjugates were obtained via a straightforward synthetic scheme taking advantage of a copper-catalyzed azide–alkyne cycloaddition as the key step. The conjugates were tested for their binding affinity for the isolated α_vβ₃ receptor and were shown to retain nanomolar IC₅₀ values, in the same range as those of the free ligands. The cytotoxic activity of the conjugates was evaluated in cell viability assays with α_vβ₃ integrin overexpressing human glioblastoma (U87) and human melanoma (M21) cells. The conjugates possess markedly lower cytotoxic activity than the free drugs, which is consistent with inefficient integrin-mediated internalization. In almost all cases the conjugates featuring isoDGR as integrin ligand exhibited higher potency than their RGD counterparts. In particular, the cycloDKP-isoDGR]-VA-MMAE conjugate has low nanomolar IC₅₀ values in cell viability assays with both cancer cell lines tested (U87: 11.50±0.13 nm ; M21: 6.94±0.09 nm ) and is therefore a promising candidate for in vivo experiments.