br Hepatitis C virus HCV infection is a
Hepatitis C virus (HCV) infection is a major cause of chronic liver disease that can lead to cirrhosis and hepatocellular carcinoma. It is estimated that nearly 200 million individuals worldwide are currently infected with HCV and it is the leading cause of liver transplants. The current standard course of therapy, pegylated interferon-α in combination with ribavarin, suffers from low response rates and severe side effects. Thus, a specific and broadly effective therapeutic agent for the treatment of HCV remains a critical unmet medical need. HCV is a member of the Flaviviridae family of enveloped, single-stranded, positive-sense RNA viruses. The 9.6-kilobase genome of HCV encodes a single polyprotein that is post-translationally processed into at least 10 structural and nonstructural (NS) proteins by a combination of both host and viral proteases. The NS3 protease has been shown to be essential for viral replication and the first antiviral proof of principle studies in man with BILN2061 demonstrated that the NS3 protease is a relevant clinical target. We reported previously the discovery of figure 1 containing a 7-methoxy-2-phenyl-4-oxo-quinoline on the P2 proline (). We have demonstrated that the aryl group on the 2-position of the quinoline contributed substantially to the potency of the inhibitor. Effectively, H NMR studies demonstrated that the aryl group is positioned above the catalytic triad pair (Asp81 and His57) in the bound conformation, partially shielding the active site from solvent. In our continuing efforts to discover novel NS3 protease inhibitors, we envisioned employing an azide () as a useful handle to evaluate substituted triazoles at the C4-position of proline (). This would provide a diverse class of inhibitors that could mimic the effect of the aryl-substituted quinoline and allow us to map the large S2 surface binding site on the enzyme. Introduction of the azide substituent (, ) was straightforward via displacement of the corresponding brosylate () with inversion of stereochemistry to yield the desired azide. The unsubstituted triazole was then prepared by thermal cycloaddition of with prop-2-yn-1-oic acid, followed by hydrolysis of the P1-ester with concomitant decarboxylation to afford triazole that constituted the initial point of diversity for SAR. In order to gain insight on the binding conformation of these series of inhibitors and to guide the SAR, compound was docked and minimized into the active site of the NS3 protease ()., The docking was based on previously reported structures of related analogues in complex with the NS3 protease. In this model, the P1, P2, and P3 moieties of , including the C-terminal acid, were docked to invoke the known intermolecular hydrogen bond network with the NS3 protease enzyme. Importantly, the model indicated that the triazole substituent adopts a pseudo axial conformation on the P2 proline ring which induces a large, flat lipophilic surface in the protein defined by the side chains of Asp168 and Arg155. Although the exact orientation of the triazole ring could not be unambiguously established from this docking protocol, it was speculated that C4-substitution would provide a basis for further exploration of this binding area. To evaluate this hypothesis, we employed a synthetic strategy that provided both the C4- and C5-arylated inhibitors (). Thermal cycloaddition of azide () with phenylacetylene yielded a 1:1 mixture of the two regioisomers, which were separated and hydrolyzed for independent testing. As predicted, the C4-phenyltriazole (, ) was indeed the most potent of the two isomers and provided an 84-fold improvement in intrinsic potency over the unsubstituted triazole (). Although the C5-arylated inhibitor () also led to an improvement in potency (4-fold) over , we focused all further efforts on C4-substituted analogues. In an effort to further understand the binding mode of these inhibitors, transfer nOe NMR experiments were performed on in the presence of the NS3 protease (). This study was consistent with the model which was obtained and confirmed that the P2 substituent occupies a pseudo axial conformation on the proline ring in both the free and bound states. Further, the C5–H of the triazole ring of has specific nOe’s with the δ-hydrogens of the proline ring indicating the ring is pointing toward the cyclopentyl carbamate in the bound state. This conformation is likely due to the increased interactions of π–π stacking of the phenyl group with Arg155.