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  • In the course of studies leading to the discovery of

    2021-10-16

    In the course of studies leading to the discovery of , modifications at the P3-P4 interface of these tripeptide-based inhibitors were examined with the goal of improving potency while maintaining a PK profile similar to . In this report, we summarize those studies which led to the identification of a series of regioisomeric -methylated ureas and P4 reverse carbamates that exhibited potent inhibition against the NS3 protease while relying upon an alternative H-bond donor topology to that in and . In addition, we summarize efforts to optimize the antiviral activity in this urea series while also exploring structural modifications of this functionality designed to optimize the PK properties. Replacement of the -butyl carbamate endcap in with a -butyl urea afforded (), a Digoxigenin-11-UTP with inhibitory potencies comparable to against enzyme representing HCV NS3 GT 1a and against a HCV GT 1b replicon (). Urea also demonstrated a 3-fold improvement in potency against NS3 protease enzymes representing GT 2b and GT 3a relative to carbamate . Computational modeling of bound to a NS3/4A protease complex suggested that the urea motif of ligand could participate in a bidentate hydrogen-bonding interaction with the backbone carbonyl of Ala-157, as depicted in . Additionally, demonstrated a 10-fold improvement in aqueous solubility relative to its carbamate counterpart . Based on its promising antiviral activity, the pharmacokinetic (PK) profile of in the rat was examined. This compound was administrated intraduodenal dosing and exposure levels in the plasma and liver were measured over a 4 h period. The concentration of in both plasma and liver was found to be significantly lower than the levels observed after dosing under the same protocol (). Despite the inferior PK profile of compared to , the exploration of P4 urea caps remained of interest given the enhanced potency toward NS3 protease enzymes representing GT 1a, GT 2b and GT 3a. A general synthesis of P3-P4 ureas was devised and is depicted in . -Boc deprotection of with TFA in CHCl followed by conversion to the hydrochloride salt provided amine as a powder isolable by simple filtration. Treatment of with ’-disuccinimidyl carbonate afforded a mixture of the succinimidyl carbonate and isocyanate . The crude mixture was treated directly with excess amine to provide the desired ureas – in good overall yield. A series of P4-P5 amides was prepared hydrolysis of the -butyl ester in followed by subsequent HATU-mediated coupling of the resultant acid with various amines to give the series of amides –. All compounds were tested for enzyme inhibitory activity toward a homogeneous full-length HCV GT 1a NS3/4A protease complex with the more active molecules additionally tested against NS3/4A complexes representing GT 2b and 3a proteases., Activity was assessed using a fluorescence resonance energy transfer (FRET) assay and the 50% inhibitory concentration (IC) values were determined as previously described., The concentration required for half maximal inhibition of virus replication in a GT 1b (Con1) replicon was determined and the data reported as EC values. Construction of the replicon cell line has been described previously. Aqueous solubility was determined in phosphate buffer solution at pH = 6.5. These data are compiled in , . In the course of preparing a series of P3-P4 urea analogs, it was discovered that urea , which contains -valine methyl ester as the P4 endcap (), offered an ∼8-fold potency advantage over the corresponding -valine methyl ester . This observed stereochemical preference at P4 was recapitulated with the diastereomeric pair of ureas / and / where the -isomer showed a several-fold increase in potency relative to the -isomer. Computational modeling suggested that the enhancement in activity observed for compounds in the -stereochemical series was due to a beneficial hydrophobic surface interaction between enzyme and inhibitor at the lipophilic P4 pocket of the protease, as previously reported.,