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The development of a robust palladium-catalysed urea N-arylation protocol to install various ureas at the 3-position of the 2(1H)-pyrazinone scaffold is described. The method involves Pd(OAc)2 in combination with bidentate ligands, xantphos [4,5-bis(diphenylphosphino)-9,9-dimethylxanthene] in particular, and resulted in good to excellent coupling yields of aliphatic, aromatic, and sterically hindered ureas. Furthermore, the C-3 chlorine was shown to be selectively displaced in the presence of aryl halide ureas, and this finding was supported by density functional theory (DFT) calculations. This allows further diversification of the scaffold for the production of compound libraries. Overall, the protocol facilitates further exploitation of pyrazinones as beta-sheet-inducing scaffolds in the development of sophisticated peptidomimetics/protease inhibitors. This is exemplified here by the synthesis of a new pyrazinone-based hepatitis C virus (HCV) NS3 protease inhibitor.

Herein, novel hepatitis C virus NS3/4A protease inhibitors based on a P2 pyrimidinyloxyphenylglycine in combination with various regioisomers of an aryl acyl sulfonamide functionality in P1 are presented. The P1′ 4-(trifluoromethyl)phenyl side chain was shown to be particularly beneficial in terms of inhibitory potency. Several inhibitors with Ki-values in the nanomolar range were developed and included identification of promising P3-truncated inhibitors spanning from P2–P1′. Of several different P2 capping groups that were evaluated, a preference for the sterically congested Boc group was revealed. The inhibitors were found to retain inhibitory potencies for A156T, D168V, and R155K variants of the protease. Furthermore, in vitro pharmacokinetic profiling showed several beneficial effects on metabolic stability as well as on apparent intestinal permeability from both P3 truncation and the use of the P1′ 4-(trifluoromethyl)phenyl side chain.

Herein we describe the design, synthesis, inhibitory potency, and pharmacokinetic properties of a novel class of achiral peptidomimetic HCV NS3 protease inhibitors. The compounds are based on a dipeptidomimetic pyrazinone glycine P3P2 building block in combination with an aromatic acyl sulfonamide in the P1P1′ position. Structure–activity relationship data and molecular modeling support occupancy of the S2 pocket from elongated R6 substituents on the 2(1H)-pyrazinone core and several inhibitors with improved inhibitory potency down to Ki = 0.11 μM were identified. A major goal with the design was to produce inhibitors structurally dissimilar to the di- and tripeptide-based HCV protease inhibitors in advanced stages of development for which cross-resistance might be an issue. Therefore, the retained and improved inhibitory potency against the drug-resistant variants A156T, D168V, and R155K further strengthen the potential of this class of inhibitors. A number of the inhibitors were tested in in vitro preclinical profiling assays to evaluate their apparent pharmacokinetic properties. The various R6 substituents were found to have a major influence on solubility, metabolic stability, and cell permeability.

BACKGROUND: HCV infections are a serious threat to public health. An important drug target is the NS3 protease, for which several inhibitors are in clinical trials. Because of the high mutation rate of the virus, resistance against any HCV-specific drug is likely to become a substantial problem. Structure-activity data for the major resistant variants are therefore needed to guide future designs of protease inhibitors. METHODS: The inhibitory potency of tripeptide NS3 protease inhibitors, with either a P2 proline or phenylglycine, in combination with different P3 and P1-P1' groups, was assessed in enzyme activity assays using the full-length NS3 protein with known resistance-conferring substitutions A156T or D168V. The results obtained from these variants were compared with the inhibition of the wild-type enzyme. Molecular modelling was used to rationalize the biochemical results. RESULTS: Inhibitors combining the P2 proline and P1 (1R,2S)-1-amino-2-vinylcyclopropyl-carboxylic acid (vinylACCA) lost much of their potency on the resistant variants. Exchange of the P2 proline for phenylglycine yielded inhibitors that were equipotent on the wild-type and on the A156T and D168V variants. The same result was obtained from the combination of either the P2 residue with a norvaline or an aromatic scaffold in the P1 position. CONCLUSIONS: The combination of a substituted P2 proline and P1 vinylACCA appears to be the main problem behind the observed resistance. Molecular modelling suggests an enforced change in binding conformation for the P2 proline-based inhibitors, whereas the phenylglycine-based inhibitors retained their wild-type binding conformation in the substituted forms of the enzyme.

Inhibition of the hepatitis C virus (HCV) NS3 protease has emerged as an attractive approach to defeat the global hepatitis C epidemic. In this work, we present the synthesis and biochemical evaluation of HCV NS3 protease inhibitors comprising a non-natural aromatic P-1 moiety. A series of inhibitors with aminobenzoyl sulfonamides displaying submicromolar potencies in the full-length NS3 protease assay was prepared through a microwave-irradiated, palladium-catalyzed, amidocarbonylation protocol.

There is an urgent need for more efficient therapies for people infected with hepatitis C virus (HCV). HCV NS3 protease inhibitors have shown proof-of-concept in clinical trials, which make the virally encoded NS3 protease an attractive drug target. Product-based NS3 protease inhibitors comprising a P1 C-terminal carboxylic acid have shown to be effective and we were interested in finding alternatives to this crucial carboxylic acid group. Thus, a series of diverse P1 functional groups with different acidity and with possibilities to form a similar, or an even more powerful, hydrogen bond network as compared to the carboxylic acid were synthesized and incorporated into potential inhibitors of the NS3 protease. Biochemical evaluation of the inhibitors was performed in both enzyme and cell-based assays. Several non-acidic C-terminal groups, such as amides and hydrazides, were evaluated but failed to produce inhibitors more potent than the corresponding carboxylic acid inhibitor. The tetrazole moiety, although of similar acidity to a carboxylic acid, provided an inhibitor with mediocre potencies in both assays. However, the acyl cyanamide and the acyl sulfinamide groups rendered compounds with low nanomolar inhibitory potencies and were more potent than the corresponding carboxylic acid inhibitor in the enzymatic assay. Additionally, results from a pH-study suggest that the P1 C-terminal of the inhibitors comprising a carboxylic acid, an acyl sulfonamide or an acyl cyanamide group binds in a similar mode in the active site of the NS3 protease.

The inhibition mechanism of electrophilic peptide-based protease inhibitors of full-length hepatitis C virus (HCV) NS3 has been investigated by determining the Ki-values for a series of compounds differing in the electrophilicity and acidity of the C-terminal residue at pH-values above and below the pKa of the catalytic histidine (6.85) and at two different ionic strengths. Electrophilic compounds with a pentafluoroethyl ketone group showed stronger inhibition at pH 8 than pH 6, as expected for a mechanism requiring an unprotonated catalytic histidine. However, the difference was only significant at high ionic strength. In contrast, electrophilic compounds with an acidic C-terminal group or a cyclic P1 residue showed a lower inhibitory effect at pH 8 than at pH 6, inconsistent with a mechanism-based inhibition. Moreover, all electrophilic compounds had an unexpectedly strong inhibition at pH 6, when mechanism-based inhibition is unlikely. The results suggest that for some of the electrophilic compounds the reactive group may not be properly positioned in the active site and that binding of these inhibitors is a result of non-covalent interactions. The nature of these interactions is discussed.

Background: The NS3 protease of hepatitis C virus (HCV) is a prime target for anti-HCV drugs but resistance towards inhibitors of the enzyme is likely to emerge because of mutations in the viral genome that modify the structure of the protein. Enzyme inhibition data supporting this is limited to studies with few compounds and analysis performed with truncated NS3. Experimental: The potential of HCV acquiring resistance towards NS3 protease inhibitors and the structural features associated with resistance has been explored with a series of inhibitors and by using full-length NS3 protease/helicase variants with amino acid substitutions (A156T, D168V and R155Q) in the protease domain. Results: The A156T and D168V substitutions did not influence the kinetic properties of the protease, whereas the R155Q substitution reduced the catalytic efficiency 20 times, as compared with the wild type. Inhibition studies revealed that these substitutions primarily affected the potency of compounds which effectively inhibit the wild-type enzyme, and had little effect on weak or moderate inhibitors. As a consequence, all compounds had similar inhibitory potencies to the substituted enzyme variants. An exception was VX-950, which inhibited the D168V enzyme more efficiently than the wild type. For this inhibitor, the present data correlated better with replicon data than data from assays with truncated enzyme. Conclusions: These results have provided a structural basis for designing inhibitors that may be less susceptible to resistance by three known mutations, and suggest that the present variants of full-length NS3 constitute effective models for resistance profiling of NS3 protease inhibitors.

A method for solid-phase peptide synthesis in the N- to C-direction that delivers good coupling yields and a low degree of epimerization is reported. The optimized method involves the coupling, without preactivation, of the resin-bound C-terminal amino acid with excess amounts of amino acid tri-tert-butoxysilyl (Sil) esters, using HATU as coupling reagent and 2,4,6-trimethylpyridine (TMP, collidine) as a base. For the amino acids investigated, the degree of epimerization was typically 5%, except for Ser(t-Bu) which was more easily epimerized (ca. 20%). Five tripeptides (AA(1)-AA(2)-AA(3)) with different properties were used as representative model peptides in the development of the synthetic method: Asp-Leu-Glu, Leu-Ala-Phe, Glu-Asp-Val, Asp-Ser-Ile, and Asp-D-Glu-Leu. The study used different combinations of HATU and TBTU as activating agents, N, N-diisopropylethylamine (DIEA) and TMP as bases, DMF and dichloromethane as solvents, and cupric chloride as an epimerization suppressant. The epimerization of AA(2) in the coupling of AA(3) was further reduced in the presence of cupric chloride. However, the use of this reagent also resulted in a decrease in loading onto the resin and significant cleavage between AA(1) and AA(2). Experiments indicated that the observed suppressing effect of cupric chloride on epimerization in the present system merely seemed to be a result of a base-induced cleavage of the oxazolone system, the key intermediate in the epimerization process. Consequently, the cleavages were most pronounced in slow couplings. An improved synthesis of fully characterized amino acid tri-tert-butoxysilyl (Sil) ester hydrochloride building blocks is presented. The amino acid Sil esters were found to be stable as hydrochlorides but not as free bases. Although only a few peptides have been used in this study, we believe that the facile procedure devised herein should provide an attractive alternative for the solid-phase synthesis of short (six residues or less) C