Hepatitis C virus (HCV) infection is the major cause of chronic liver disease, leading to cirrhosis and hepatocellular carcinoma, which affects more than 170 million people worldwide. Currently the only therapeutic regimens are subcutaneous interferon-alpha or polyethylene glycol (PEG)-interferon-alpha alone or in combination with oral ribavirin. Although combination therapy is reasonably successful with the majority of genotypes, its efficacy against the predominant genotype (genotype 1) is moderate at best, with only about 40% of the patients showing sustained virological response. Herein, the SAR leading to the discovery of 70 (SCH 503034), a novel, potent, selective, orally bioavailable NS3 protease inhibitor that has been advanced to clinical trials in human beings for the treatment of hepatitis C viral infections is described. X-ray structure of inhibitor 70 complexed with the NS3 protease and biological data are also discussed.

Introduction of various modified prolines at P(2) and optimization of the P(1) side chain led to the discovery of SCH6 (24, Table 2), a potent ketoamide inhibitor of the HCV NS3 serine protease. In addition to excellent enzyme potency (K(i)*= 3.8 nM), 24 was also found to be a potent inhibitor of HCV subgenomic RNA replication with IC(50) and IC(90) of 40 and 100 nM, respectively. Recently, antiviral activity of 24 was demonstrated with inhibition of the full-length genotype 2a HCV genome. In addition, 24 was found to restore the responsiveness of the interferon regulatory factor 3 (IRF-3) in cells containing HCV RNA replicons.

The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were determined for a series of ketoamide inhibitors in complex with the protease. The changes in the inhibitor potency were correlated with changes in the buried surface area upon binding the inhibitor to the active site. The largest contribution to the binding energy arises from the hydrophobic interactions of the P1 and P2 groups as they bind to the S1 and S2 pockets [the numbering of the subsites is as defined in Berger, A.; Schechter, I. Philos. Trans. R. Soc. London, Ser. B 1970, 257, 249-264]. This correlation of the changes in potency with increased buried surface area contributed directly to the design of a potent tripeptide inhibitor of the HCV NS3/4A protease that is currently in clinical trials.

Boceprevir (SCH 503034), 1, a novel HCV NS3 serine protease inhibitor discovered in our laboratories, is currently undergoing phase III clinical trials. Detailed investigations toward a second generation protease inhibitor culminated in the discovery of narlaprevir (SCH 900518), 37, with improved potency (∼10-fold over 1), pharmacokinetic profile and physicochemical characteristics, currently in phase II human trials. Exploration of synthetic sequence for preparation of 37 resulted in a route that required no silica gel purification for the entire synthesis.KEYWORDS Hepatitis C virus NS3 serine protease inhibitor, R-ketoamide, narlaprevir, SCH 900518 H epatitis C virus (HCV) infection is a global health crisis leading to liver cirrhosis, hepatocellular carcinoma and liver failure in humans. 1 An estimated 3% of the human population worldwide is chronically infected with HCV. 2 Currently the only available treatment regimens are subcutaneous R-interferon or long-acting pegylated-interferon, alone or in combination with oral antiviral agent ribavirin. 3 The approved therapy is still far from ideal for the hard to treat genotype-1 patients 4 and is frequently accompanied by adverse side effects. There is an unmet medical need to discover new, more effective and tolerable regimens for the treatment of HCV infection. Advances in the understanding of molecular pathways of HCV replication have resulted in several small molecule direct-acting antivirals entering clinical trials in the past few years.Since identification of this virus, the NS3 serine protease contained within the N-terminal region of the NS3 protein has been studied extensively. 5 This chymotrypsin-like serine protease plays a pivotal role in viral replication and, therefore, is an attractive target for HCV antiviral therapeutics. 6,7 Intense efforts were focused in the past decade to discover novel small molecule agents that inhibit NS3 serine protease. 8 Proof of concept studies in humans with BILN 2061, a noncovalent P1-P3 macrocyclic inhibitor, validated this hypothesis. 9 Since then, several NS3 protease inhibitors have progressed to human clinical trials. Currently the most advanced among those are boceprevir (SCH 503034), 1, 10,11 and telaprevir (VX950), 12,13 from the slow-binding reversible R-ketoamide class, in phase III human evaluation. Inhibitors in phase II studies, from the structurally distinct noncovalent macrocyclic class, include 14 15 and MK-7009 (P2-P4 macrocycle). 16 Other NS3 protease inhibitors currently in clinical evaluation (structure not yet disclosed) include BI-201335, ABT-450, PHX-1766, ACH-1625 and VX-813. 8 Inhibitor 1 exhibited K i * = 14 nM in the enzyme binding assay, 17 EC 90 = 350 nM in the cell-based replicon assay, 18 and acceptable pharmacokinetic profile in rats and dogs (Figure 1). In our efforts to discover a second generation HCV protease inhibitor, we focused mainly on improving the in vitro potency and preclinical pharmacokinetic profile of the inhibitor, specifically exposure in monkeys. Further...

Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K i * ) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC 90 ) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5؋ EC 90 of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Despite recent advances, the eradication of chronic HCV infection remains challenging. Approximately 50% of patients infected with the most prevalent form of the virus (genotype 1) fail to respond to treatment with the current standard of care of pegylated alpha interferon in combination with ribavirin (4, 13). Thus, there is a great unmet medical need for the development of new agents with activity against HCV to improve the sustained virological response (SVR). The HCV genome is translated as a single polyprotein precursor which is processed by cellular and viral proteases into structural proteins (the C, E1, and E2 proteins), followed by nonstructural (NS) proteins (the p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B proteins). The NS3 protein is multifunctional, with the N-terminal region (amino acids 1 to 181) encoding a serine protease and the remaining polypeptide encoding a helicase. The NS3 protease noncovalently binds to the cofactor NS4A, which contributes to the formation and stability of the active site and helps to anchor the NS3/NS4A complex to the membrane. The NS3 protease is responsible for the processing of the HCV polyp...