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  • Biological evaluations of the compounds were done

    2021-09-27

    Biological evaluations of the compounds were done both using HCV protease and replicon assays. Results are shown in . The protease inhibitory IC’s were determined using a FRET assay with HCV NS3/4A 1a protease domain. The replicon EC’s were determined using a replicon luciferase cell-based assay. The initially designed compound proved equipotent than danoprevir (ITMN-191) with enzymatic activity IC of 0.4nM. While was potent in the genotype 1b assay (EC 3.7nM), the P4 structural change turned out to be detrimental to compound’s potency against genotype 1a replicon activity (EC 159nM). Encouraged by this result, we set out to further explore the linker SAR. Changing the urea linker to its corresponding carbamate (compound ), the potency dropped off in both enzymatic or replicon assays. Compound is a regioisomer of compound , its potency was even worse. However, homologation of the urea linker (in compound ) restored enzyme activity and, compared to lead compound , improved replicon EC by 4 fold for genotype 1b, although similar for genotype 1a. The amide linker in compound provided similar potency or slightly inferior to compound . Next, we explored substitutions on benzoxaborole ring system by improving lipophilicity, reducing polar surface area (PSA) of the ring while maintaining the oxaborole functionality, aimed to improve replicon potency for genotype 1a. We kept the urea linker for this exploration because it showed best potency and ease of synthesis. Compound is a fluoro derivative of compound with excellent enzyme potency and improvement in both genotype 1a and 1b replicon activity by 2 to 3-fold. The compound with both methyl and fluoro substitutions on benzoxaborole ring improved further cellular potency in genotype 1a. Preliminary SAR in this series strongly suggests that it should be possible to further optimize cellular potency by further modifications in the benzoxaborole ring. We decided to explore the impact of P2∗ groups on the anti-HCV potency of P4-benzoxoborole substituted macrocyclic compounds. We picked representative isoquinoline and quinolone groups from other ZJ 43 of HCV protease inhibitors, namely BMS-791325 and TMC-435. The in vitro profiles of these compounds are summarized in . Interestingly, compounds and with isoquinoline P2∗ have similar enzyme and replicon potency profile to isoindoline P2∗. The more bulky, elaborated quinoline P2∗-containing compounds and showed better replicon potency, although enzymatic potency fell off. We believe that some of the differences between the enzyme and replicon potency may originate from factors that differentiate both assays, such as different transcellular transport. Selected compounds were evaluated in rats for their pharmacokinetic parameters, blood samples from both jugular and portal vein were drawn and drug concentrations were measured. The results are shown in . The oral absorption was calculated from the portal vein drug concentrations and oral bioavailability was calculated from jugular vein drug concentrations as compared to the drug concentrations after IV administration. ITMN-191 exhibits calculated 17.4% absorption and 20% oral bioavailability in rats. However, benzoxaborole-substituted macrocylic inhibitors , and displayed minimal to undetectable level of absorption and oral bioavailability. We noticed good water solubility of these inhibitors compared to ITMN191 when the PK samples were made, however, their permeability and absorption are almost certainly limited by their high molecular weight and high polar surface area (PSA). In summary, we have designed and synthesized a series of P4-benzoxaborole-substituted macrocyclic HCV protease inhibitors. We suggest that the benzoxaborole moiety can be a useful moiety towards developing compounds retaining potency against resistant enzymes. These compounds exhibited potent inhibitory activity against HCV NS3/4 protease. Their cellular replicon potencies were impacted by substitutions on the benzoxaborole ring system and P2∗ groups, but even a limited exploration with compounds and suggest that further potency optimization should be possible. These compounds had high polar surface area (PSA), which may initially limit their oral absorption and bioavailability. However, our results with a related series suggest that relatively simple structural changes can bring these molecules back into more drug-like parameters.