Combinatorial and structure-based medicinal chemistry strategies were used together to advance a lead compound with an activity of K(i) = 58 microM via a potency enhancement of >70 000-fold to an analogue with an activity of K(i) = 0.8 nM against influenza neuraminidase (A/Tokyo/67). Lead optimization was initiated using molecular modeling and combinatorial chemistry. Protein crystal structures revealed that inconsistent structure-activity relationship (SAR) data resulted from different binding orientations of the inhibitor core five-membered rings from one series to another. Binding modes for a series of compounds showed up to a 180 degrees variation in orientation of the five-membered ring within the active site. Potent analogues were only achieved with chemical series that were observed to bind in the same orientation and yielded consistent SAR. In one series, consistent binding was obtained by an unprecedented occupation of a negatively charged binding pocket by a neutral methyl ester unit. The structural rationale for this novel SAR variation, based on protein crystallographic data, is given.

The structure-activity relationship (SAR) of a novel hydrophobic binding interaction within a subsite of the influenza neuraminidase (NA) active site was characterized and optimized for a series of trisubstituted pyrrolidine inhibitors modified at the 4-position. Previously, potent inhibitors have targeted this subsite with hydrophilic substituents such as amines and guanidines. Inhibitor-bound crystal structures revealed that hydrophobic substituents with sp(2) hybridization could achieve optimal interactions by virtue of a low-energy binding conformation and favorable pi-stacking interactions with the residue Glu119. From a lead methyl ester, investigation of five-membered heteroaromatic substituents at C-4 produced a 3-pyrazolyl analogue that improved activity by making a targeted hydrogen bond with Trp178. The SAR of substituted vinyl substituents at C-4 produced a Z-propenyl analogue with improved activity over the lead methyl ester. The C-1 ethyl ester prodrugs of the substituted C-4 vinyl analogues gave compounds with excellent oral bioavailability (F > 60%) when dosed in rat.

A new series of H3 receptor antagonists was discovered with nanomolar and subnanomolar affinities at human and rat H3 receptors. Starting from an earlier, more structurally limited series of benzofurans, the present series of compounds demonstrated increased structural variety and flexibility with greater in vitro potency. One compound in particular, [2-[2-(2-(R)-methylpyrrolidin-1-yl)ethyl]benzofuran-5-yl](5-nitropyridin-2-yl)amine (7h), gave the best binding potency (human K(i) of 0.05 nM, rat K(i) of 0.11 nM), which represented a 9-fold (in human) and an 11-fold (in rat) improvement over ABT-239 (compound 5), a compound previously reported to have excellent in vitro potency and in vivo efficacy. The synthesis, SAR of the H3 binding affinities, in vitro assay for phospholipidosis, and pharmacokinetic properties of the new compounds are described.