Influence of amino acid size at the P3 position of N-Cbz-tripeptide Michael acceptors targeting falcipain-2 and rhodesain for the treatment of malaria and human african trypanosomiasis.

In recent decades, several structure-activity relationship (SAR) studies provided potent inhibitors of the cysteine proteases falcipain-2 (FP-2) and rhodesain (RD) from Plasmodium falciparum and Trypanosoma brucei rhodesiense, respectively. Whilst the roles of the warhead and residues targeting the P1 and P2 pockets of the proteases were extensively investigated, the roles of the amino acids occupying the S3 pocket were not widely assessed. Herein we report the synthesis and biological evaluation of a set of novel Michael acceptors bearing amino acids of increasing size at the P3 site (1a-g/2a-g, SPR20-SPR33) against FP-2, RD, P. falciparum, and T. brucei. Overall, the Michael acceptors bearing small amino acids at the P3 site exhibited the most potent inhibitory properties towards FP-2. In contrast, analogues with bulky residues at the P3 position were very potent rhodesain inhibitors. In cell based assays, single-digit micromolar EC50 values against the two protozoa were observed. These findings can be a starting point for the development of peptide-based FP-2 and RD inhibitors.