We have previously reported on

We have previously reported on the effects of replacing the isoquinoline P2∗ moiety with a naphthalene ring and modifying the connectivity for this element to the P2 proline ring, as demonstrated by compounds – (). Therein, it was hypothesized that the reduced potency observed with – when compared to and was caused by a conformational bias between the naphthalene and proline rings which resulted in a suboptimal overlay of the P2∗ region with the NS3 protease. Macrocyclization is an established strategy to enhance biological activity by limiting conformational ensembles and thereby favoring active conformers., , This has been an effective strategy to gain inhibitory activity against NS3 protease., Against this backdrop, tethering of the P2∗ naphthalene ring in to P4 was proposed as a potential approach to constrain the aromatic moiety in an active conformation. Models of the binding mode of acyclic 680C91 , 6-carbon tether macrocyclic naphthalene , and superimposed with on the HCV NS3 protease provided support for these hypotheses (). Hence, the initial goal of this work was to synthesize P2-P4 macrocycles and determine if potency could be secured in this series. To this end, compound was prepared which served as a synthetic precursor to these macrocycles. Removal of the Boc group on P4 provided the connection point for various tethers that were then cyclized through a ring closing metathesis (RCM) to P2∗. Compound exhibited modest potency that was comparable to in the GT 1b replicon (). A 6-carbon tether appeared to provide a favorable fit for this macrocyclic series based on modeling; however, tethers of four to eight carbon atoms were examined (compounds –, ). P2-P4 macrocycles with tether lengths of five () and six ( and ) carbon atoms were significantly more active than analogues incorporating tethers of four, seven, or eight carbons (, , and ). Compound , with the 6-carbon tether, proved to be the most active of this series with sub-nM replicon activity against GT 1a and 1b, respectively. Notably and importantly, was over 100-fold more active than the comparable acyclic analogue . Similarly, compound demonstrated a 140-fold potency increase versus the GT 1a (H77) replicon and a 250-fold increase against the GT 1b (Con1) replicon potency when compared to the analogous acyclic compound . These results suggested that conformational pre-organization P2-P4 macrocyclization was an effective tool to drive potency in this series. Since the six-carbon tether in demonstrated optimal activity within the series, this motif served as the basis for further analogue design that was focused on improving ADME properties. Metabolic stability data revealed a low to moderate half-life for in human liver microsomes (HLM), with oxidation along the macrocyclic tether considered as a potential site of metabolic modification. To address this issue, two approaches were taken. The first approach involved the introduction of polarity into the methylene tether and to this end, a series of analogues were prepared in which either an oxygen (, ) or nitrogen atom (–) was introduced (). As shown in , positioning an oxygen atom in the tether, as in , did not have a significant effect on microsomal stability but did result in an erosion of potency. Replacing the cyclopropyl vinyl group at P1 with a -cyclopropyl moiety had previously been reported as a structural modification in the tripeptide acylsulfonamide series that enhanced metabolic stability. In this P2-P4 macrocycle chemotype, this modification also led to enhanced metabolic stability, as exemplified by compound , accompanied by only a small reduction in potency. Compound , when compared to , also demonstrated a significant improvement in metabolic stability with incorporation of the P1 -cyclopropyl moeity. Based on these observations, evaluation of P2-P4 macrocycles containing nitrogen in the tether was conducted using the P1 -cyclopropyl moiety to afford –. Interestingly, potency tracked inversely with basicity. For example, the cyclopropylamine-based macrocycle was approximately 10-fold more potent than the corresponding isopropylamine homologue . Consistent with this observation, the carbamate analogue exhibited antiviral potency comparable to while also offering enhanced stability in the human liver microsomal assay.