Synthesis of multimeric integrin ligands for the selective tumor-targeted delivery of cytotoxic drugs

Conjugation of cytotoxic agents to targeting carriers (e.g. antibodies or small molecules) capable of selectively binding to tumor-specific antigens, is emerging as a successful strategy to overcome the lack of selectivity typical of traditional chemotherapeutics. αVβ3 integrin has been widely investigated as receptor for tumor-targeting strategies, as it is over-expressed on the blood vessels of several human cancers[1]. Extracellular matrix proteins are recognized by this receptor through the Arg-Gly-Asp (RGD) sequence, which has been incorporated in numerous peptide- and peptidomimetic ligands[2]. Among them, our research group developed the new integrin ligand DKP3-RGD[3], in which the RGD sequence is constrained into a cycle by a bifunctional 2,5-diketopiperazine (DKP) scaffold. Our research group has recently developed the RGD-PTX conjugate 1, containing a dipeptide linker that can be selectively cleaved by the proteases present inside the target cell [4]. Compound 1 displayed a fairly effective integrin-targeting, inhibiting cell proliferation with increased selectivity towards αVβ3-expressing cells compared to the free PTX. In order to increase the binding affinity towards the cells overexpressing integrin αVβ3, the dimeric conjugate 2 has been synthesized. The ability of compound 2 to compete with biotinylated vitronectin for the binding to the purified αVβ3 integrin has been tested in vitro. As expected, the dimeric compound 2 showed higher binding affinity than the monomeric analog 1 (IC50 = 4 nM vs. 13 nM). Remarkably, the IC50 obtained for the dimeric compound 2 is identical to the one reported for the free ligand DKP3-RGD.