In an attempt to further optimize interactions

In an attempt to further optimize interactions in the lipophilic P4 pocket, we next prepared compounds – () which possessed the preferred stereochemistry at P4, but with R-groups of varying size and shape. In addition to providing important structural information for maximizing potency, these compounds were engineered with the goal of improving PK. We reasoned that the terminal methyl ether oxygen of – might form an intramolecular hydrogen-bond with the urea moiety, effectively presenting the end of the molecule as a pseudo 5-membered ring, and thereby masking a portion of the urea polarity and potentially improving permeability., Compound proved to be the most active in this series, with anti-NS3 protease activity in the GT 1a and GT 3a biochemical assays ∼3-fold more potent than that observed for compound . SAR trends in this series were sensitive to steric bulk, with anti-NS3 protease activities generally deteriorating as R became either smaller (compounds –) or larger (compounds –) than the -butyl moiety of compound . With respect to PK properties, a 6-fold increase in plasma AUC was observed for compound compared to urea while liver concentrations were found to be similar for these analogues. Interestingly, this PK improvement was coincidental with a ∼50-fold increase in aqueous solubility of when compared to . Given these data, we speculated that the terminal P4 ether moiety in may be driving solubility by providing a more polar terminus compared to . This enhanced solubility may also explain the increase in 1400W dihydrochloride mg for compared to . However, the potential for the terminal methyl ether in to facilitate the desolvation of the P4 urea through the formation of an intramolecular hydrogen-bond that obscures the polarity of the urea should also be considered as a source of improved absorption of . Although the PK result for marked progress within the urea series, the exposure observed for this compound was inferior to that of carbamate and, hence, attention was focused on replacement of the P4 methyl ether end cap in an effort to further modulate anti-NS3 protease activity and improve PK. The P4 cap functionality explored in this part of the survey included esters, amides and acidic groups, as exemplified by compounds – in . It should be noted that the P4 -butyl group present in was a fixed structural element in this series since the SAR suggested optimal complementarity between this lipophilic substituent and the complementary pocket of the enzyme. In the event, esters and and amides – were found to exert similar anti-NS3 protease activity in the biochemical assay although they did not provide a significant improvement in potency compared to . This observation suggested that the immediate P4 caps were not interfacing with the enzyme surface but were instead solvent-exposed. The PK profile of compounds and provided no significant improvement over the simpler congener . Interestingly, compound , which bears a carboxylic acid as a P4 cap, demonstrated a significant improvement in potency in the biochemical assays across NS3 proteases representing GTs 1–3. Most notable was the IC value toward the GT 1a enzyme which, at 50 pM, was 6-fold more potent than compound . Docking studies suggested that the enhanced potency of could be attributed to hydrogen-bonding interactions between the terminal carboxylate of and both Arg-123 as well as the backbone NH of Cys-159 (). While this level of activity in the biochemical assay was significant, the PK profile of was less than optimal and hence this compound was not progressed. As noted above, a model of , bound to the NS3/4a protease enzyme complex suggested that the P4 urea moiety could potentially bind to the carbonyl moiety of Ala-157 in a bidentate fashion (), while P4 carbamates such as interface with the enzyme through a single H-bond interaction., Given the potency of carbamate , we sought to examine the idea that acceptable inhibition of the NS3 protease may be achievable with substrates possessing a single H-bond donor projected distal rather than proximal to the P3 moiety. To this end, both -methylated urea derivatives as well as reverse carbamates were prepared in effort to explore this concept and the results are summarized in . Synthetic schema for the preparations of these compounds can be found in the .