A new class of nonpeptidic inhibitors of the malarial aspartic protease plasmepsin II (PMII) with up to single-digit micromolar activities (IC50 values) was developed by structure-based de novo design. The active-site matrix used in the design was based on an X-ray crystal structure of PMII, onto which the major conformational changes seen in the structure of renin upon complexation of 4-arylpiperidines-including the unlocking of a new hydrophobic (flap) pocket - were modeled. The sequence identity of 35% between mature renin and PMII had prompted us to hypothesize that an induced-fit adaptation around the active site as observed in renin might also be effective in PMII. The new inhibitors contain a central 11-azatricyclo[6.2.1.0(2.7)]undeca-2(7),3,5-triene core, which, in protonated form, undergoes ionic H-bonding with the two catalytic Asp residues at the active site of PMII (Figs. 1 and 2). This tricyclic scaffold is readily prepared by a Diels-Alder reaction between an activated pyrrole and a benzyne species generated in situ (Scheme 1). Two substituents with naphthyl or 1,3-benzothiazole moieties are attached to the central core (Schemes 1-4) for accommodation in the hydrophobic flap and S1/S3 (or S2', depending on the optical antipode of the inhibitor) pockets at the active site of the enzyme. The most-potent inhibitors (+/-)-19a -19c (IC50 3-5 mum) and (+/-)-23b (2 mum) ( Table) bear an additional Cl-atom on the 1,3-benzothiazole moiety to fully fill the rear of the flap pocket. Optimization of the linker between the tricyclic scaffold and the 1,3-benzothiazole moiety, based on detailed conformational analysis (Figs. 3 and 4), led to a further small increase in inhibitory strength. The new compounds were also tested against other aspartic proteases. They were found to be quite selective against renin, while the selectivity against cathepsin D and E, two other human aspartic proteases, is rather poor (Table). The detailed SARs established in this investigation provide a valuable basis for the design of the next generations of more-potent and -selective PMII inhibitors with potential application in a new antimalarial therapy.