HCV NS3 Protease Research Articles

Discovery of Danoprevir (ITMN-191/R7227), a Highly Selective and Potent Inhibitor of Hepatitis C Virus (HCV) NS3/4A Protease

HCV serine protease NS3 represents an attractive drug target because it is not only essential for viral replication but also implicated in the viral evasion of the host immune response pathway through direct cleavage of key proteins in the human innate immune system. Through structure-based drug design and optimization, macrocyclic peptidomimetic molecules bearing both a lipophilic P2 isoindoline carbamate and a P1/P1' acylsulfonamide/acylsulfamide carboxylic acid bioisostere were prepared that possessed subnanomolar potency against the NS3 protease in a subgenomic replicon-based cellular assay (Huh-7). Danoprevir (compound 49) was selected as the clinical development candidate for its favorable potency profile across multiple HCV genotypes and key mutant strains and for its good in vitro ADME profiles and in vivo target tissue (liver) exposures across multiple animal species. X-ray crystallographic studies elucidated several key features in the binding of danoprevir to HCV NS3 protease and proved invaluable to our iterative structure-based design strategy.

Achiral Pyrazinone-Based Inhibitors of the Hepatitis C Virus NS3 Protease and Drug-Resistant Variants with Elongated Substituents Directed Toward the S2 Pocket

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 R(6) 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 R(6) substituents were found to have a major influence on solubility, metabolic stability, and cell permeability.

847 ACH-2684 DEMONSTRATES POTENT VIRAL SUPPRESSION IN GENOTYPE 1 HEPATITIS C PATIENTS WITH AND WITHOUT CIRRHOSIS: SAFETY, PHARMACOKINETIC, AND VIRAL KINETIC ANALYSIS

Background: ACH-2684 is a second-generation HCV NS3 protease inhibitor in Phase Ib development for the treatment of chronic HCV infection. ACH-2684 demonstrates potent activity in vitro against genotype-1 (GT1) HCV and, as compared to other protease inhibitors, has improved activity against multiple viral variants. Presented here are phase I study results in cirrhotic and non-cirrhotic patients with chronic HCV GT1 infection. Methods: ACH-2684 or placebo was administered to non-cirrhotic (n = 30) and cirrhotic (n = 8) patients with chronic HCV GT1 infection. Pharmacokinetic, (PK) viral load and safety assessments were collected on Days 1 through 35. Serial blood samples were collected on Day 1 and Day 3, and plasma non-compartmental pharmacokinetic parameters were determined. Drug efficiency was estimated by applying a single population viral dynamics model (Neumann et al, Science 1998, 282,103). Results: ACH-2684 was safe and well tolerated in non-cirrhotic and cirrhotic patients. There were no serious adverse events, and no discontinuations for safety concerns. Mean maximum reductions in plasma HCV RNA were 3.36, 3.73 and 4.16 log10 (IU/ml) at doses of 100 mg daily, 400 mg daily and 400 mg twice daily, respectively. In cirrhotic patients administered 400 mg daily, mean maximum reduction was 3.67 log10 (IU/ml). The systemic exposures were generally greater than doseproportional for the dose range studied in non-cirrhotics, and approximately 2-fold higher in cirrhotic versus non-cirrhotic patients. The drug efficiency of the 400 mg daily dose in non-cirrhotic and cirrhotic subjects was similar at 0.9993 ± 0.0005 and 0.9991 ± 0.0007, respectively. Conclusions: ACH-2684 was safe and well-tolerated, and produced more than a 3 log10 (IU/ml) mean maximum reduction in plasma HCV RNA levels in all groups. A 400 mg dose of ACH-2684 yielded approximately a 2-fold higher exposure in cirrhotics than non-cirrhotics, but was still safe and well-tolerated, and was equally effective in reducing HCV RNA levels. Viral kinetics parameters were robust and demonstrated high drug efficiency. These data provide evidence that ACH-2684 is a safe and well-tolerated protease inhibitor with potent anti-viral activity in both cirrhotic and non-cirrhotic patients with chronic GT-1 HCV infection, making it a promising candidate for future treatment of chronic HCV infection.

One-Pot, Two-Step, Microwave-Assisted Palladium-Catalyzed Conversion of Aryl Alcohols to Aryl Fluorides via Aryl Nonaflates

A convenient procedure for converting aryl alcohols to aryl fluorides via aryl nonafluorobutylsulfonates (ArONf) is presented. Moderate to good one-pot, two-step yields were achieved by this nonaflation and microwave-assisted, palladium-catalyzed fluorination sequence. The reductive elimination step was investigated by DFT calculations to compare fluorination with chlorination, proving a larger thermodynamic driving force for the aryl fluoride product. Finally, a key aryl fluoride intermediate for the synthesis of a potent HCV NS3 protease inhibitor was smoothly prepared with the novel protocol.

Synthesis and optimization of a novel series of HCV NS3 protease inhibitors: 4-Arylproline analogs

In this report we describe the synthesis and evaluation of diverse 4-arylproline analogs as HCV NS3 protease inhibitors. Introduction of this novel P2 moiety opened up new SAR and, in combination with a synthetic approach providing a versatile handle, allowed for efficient exploitation of this novel series of NS3 protease inhibitors. Multiple structural modifications of the aryl group at the 4-proline, guided by structural analysis, led to the identification of analogs which were very potent in both enzymatic and cell based assays. The impact of this systematic SAR on different drug properties is reported.

A phase 2a trial of 12-week interferon-free therapy with two direct-acting antivirals (ABT-450/r, ABT-072) and ribavirin in IL28B C/C patients with chronic hepatitis C genotype 1

ABT-450 (combined with low-dose ritonavir, ABT-450/r) is a potent HCV NS3 protease inhibitor, and ABT-072 is a non-nucleoside NS5B polymerase inhibitor. The goal of this study was to evaluate the safety, tolerability, and efficacy of the peginterferon-free combination of ABT-450/r and ABT-072 with ribavirin in treatment-naïve patients with IL28B CC genotype, infected with HCV genotype 1. This was a phase 2a, multicenter, open-label, single-arm study in 11 treatment-naïve, non-cirrhotic HCV GT1-infected patients with IL28B rs12979860 genotype CC. Patients received ABT-450/r 150/100 mg once daily and ABT-072 400 mg once daily with weight-based ribavirin 1000-1200 mg/day dosed twice daily for 12 weeks. Eight (73%) patients were male, 9 (82%) were Caucasian (including 3 who self-identified as Hispanic); mean baseline HCV RNA was 6.9 log₁₀ IU/ml (range 6.5-7.3 log₁₀ IU/ml). All 11 patients completed 12 weeks of treatment and maintained HCV RNA <25 IU/ml from weeks 4 through 12 of treatment. Ten patients (91%) achieved sustained virologic response 24 weeks post-treatment, with a second patient relapsing 36 weeks post-treatment. There were no deaths, serious or severe adverse events, or premature discontinuations. Adverse events were mostly mild and the most frequent were headache, fatigue, nausea, and dry skin. A 12-week regimen of ABT-450/r and ABT-072 with ribavirin was well tolerated with 9/11 patients achieving sustained virologic response through 36 weeks of post-treatment observation. These findings suggest that peginterferon-free regimens may have the potential to cure a high proportion of HCV genotype 1-infected patients.

Anemia Management in Patients with Chronic Viral Hepatitis C

To review the literature regarding current strategies for the management of anemia associated with treatment for chronic viral hepatitis C (HCV) in adults. The MEDLINE/PubMed, EMBASE, and Cochrane databases were searched (January 1980-October 2012) for articles in English using the search terms anemia, ribavirin, dose reduction, erythropoietin stimulating agents, hepatitis C, HIV, liver transplant, telaprevir, and boceprevir. All relevant original studies, meta-analyses, systematic reviews, guidelines, and review articles were assessed for inclusion. References from pertinent articles were examined for additional content not found during the initial search. Standard of care for patients infected with HCV genotype 1 now requires a triple therapy regimen including an HCV NS3 protease inhibitor. These regimens lead to significantly higher rates of anemia compared to prior dual therapy regimens. Development of an optimal management strategy should begin with risk stratification. Ribavirin dose reductions have been recommended in the package inserts for the pegylated interferon products and studies have demonstrated the need for maintenance of 80% of the initial ribavirin dose to achieve optimal sustained virologic response (SVR) with dual therapy. The use of erythropoietin-stimulating agents has been shown to be effective for anemia caused by peginterferon and ribavirin without compromising SVR rates. Limited data have been published regarding the management of anemia with triple therapy; however, efficacy studies for boceprevir and telaprevir have used ribavirin dose reduction and erythropoietin-stimulating agents to successfully manage anemia. Anemia is a common adverse event associated with the use of ribavirin, and, more recently, the new HCV protease inhibitors. Ribavirin dose reduction should continue to be used as an initial anemia management strategy, with the use of erythropoietin alfa 40,000 units once weekly reserved for patients whose hemoglobin does not adequately respond to initial management strategies.

Concise asymmetric synthesis of a (1R,2S)-1-amino-2-vinylcyclopropanecarboxylic acid-derived sulfonamide and ethyl ester

The development and demonstration of short, robust and chromatography-free sequences for the preparation of a (1R,2S)-1-amino-2-vinylcyclopropane-carboxylic acid-derived sulfonamide and ethyl ester in ≥99% ee are described. Both compounds are common building blocks in multiple preparations of potent HCV NS3 protease inhibitors. The robustness of the asymmetric cyclopropanation of (E)-N-benzylideneglycine ethyl ester under phase transfer catalysis conditions is significantly improved based on a detailed mechanistic investigation that included an analysis of the catalyst decomposition pathway, a postulated model for the stereo-selectivity that was guided by calculations and rigorous quality control of the starting materials and reagents. Wet milling has been demonstrated to dramatically accelerate this phase transfer reaction. A bench stable benzylidene-protected primary 1-amino-2-vinylcyclopropane amide intermediate was isolated and its reliable enantiomeric enrichment was achieved by a controlled crystallization process. A chemical resolution procedure was identified using di-p-toluoyl-(D)-tartaric acid to access (1R,2S)-1-amino-2-vinyl-cyclopropanecarboxylic ester in high ee.

Rapid and Sharp Decline in HCV upon Monotherapy with Ns3 Protease Inhibitor, Ach-1625

ACH-1625 is a linear peptidomimetic inhibitor that non-covalently binds to HCV NS3 protease with high potency and specificity. Short-term monotherapy of HCV genotype-1 infection with ACH-1625 was found to be safe and resulted in ≥3.3 log(10) IU/ml mean viral load reduction. These viral load decay data were analysed to compare HCV dynamics with prior reports and estimate the antiviral efficiency of ACH-1625. Drug efficiency was estimated by analysing the viral decay following initiation of up to 5 days of monotherapy with ACH-1625 in 36 chronically infected HCV genotype-1 patients. During this monotherapy study, ACH-1625 was administered either twice-a-day for 4.5 days or once daily for 5 days at 5 different dose levels in 36 patients. A sharp viral decay during the first 48 h following the initiation of ACH-1625 treatment afforded high drug efficiency estimates (≥0.9934). In addition, an increase in the estimated drug efficiency was observed with increasing ACH-1625 dose. The observed anti-HCV response was fairly uniform in this proof-of-concept study across the population of 36 patients. Estimates of the treatment-independent viral kinetics parameters were consistent with prior reports and the estimated drug efficiency of ACH-1625 monotherapy was very high (≥0.9934) in fasted and fed states.

Synthesis and antiviral activity of novel HCV NS3 protease inhibitors with P4 capping groups

We have synthesized and evaluated a series of novel HCV NS3 protease inhibitors with various P4 capping groups, which include urea, carbamate, methoxy-carboxamide, cyclic carbamate and amide, pyruvic amide, oxamate, oxalamide and cyanoguanidine. Most of these compounds are remarkably potent, exhibiting single-digit to sub-nanomolar activity in the enzyme assay and cell-based replicon assay. Selected compounds were also evaluated in the protease-inhibitor-resistant mutant transient replicon assay, and they were found to show quite different potency profiles against a panel of HCV protease-inhibitor-resistant mutants.

A Novel Peptidomimetic Compounds as HCV-NS3 Protease Inhibitors: Spectroscopic Analysis

The presented molecular modeling is utilized to innovate new peptidomimetic compounds. These proposed compounds are designed to act as HCV NS3 protease antiviral. The suggested antivirals are divided into two groups. The first group has hexapeptide (Glu-Asp-Val-Val-Cys-Cys) binding to cellulose monomer at positions 2, 3 or 6 while the second group has hexapeptide (Glu-Asp-Val-Val-Cys-Cys) binding to cellulose dimer at positions 2, 3, 6, 2', 3'or 6'. Semi- empirical PM3 quantum mechanical method is first utilized for optimization, then to calculate the vibrational spectra of these novel compounds. It is found that higher dipole moment (11.907 Debye) corresponds to the hexapeptide (Glu-Asp- Val-Val-Cys-Cys) binding to cellulose dimer at position 2' compound. Accordingly, calculation is repeated at HF/3- 21g**and B3LYP/3-21g** for such compound for verification.

1200 ANTIVIRAL ACTIVITY OF EP-NI266, A POTENT NUCLEOTIDE HCV POLYMERASE INHIBITOR

1198 HCV NS3/4A PROTEASE RESISTANCE-ASSOCIATED VARIANTS (RAVS) IDENTIFIED IN GENOTYPE 1A PATIENTS EXHIBIT DIFFERENCES IN PHENOTYPIC RESISTANCE TO BOCEPREVIR AND TELAPREVIR IN GENOTYPE 1A REPLICON CELLS S. Black, P. McMonagle, R. Chase, S. Curry, R.J.O. Barnard, D.J. Hazuda, A.Y. Howe, J.A. Howe, R.A. Ogert. Merck & Co., Inc., Kenilworth, NJ, USA E-mail: robert.ogert@merck.com Background: The HCV NS3 protease inhibitors boceprevir (BOC) and telaprevir (TPV) in combination with pegylated interferon alpha-2 plus ribavirin (PR) have been approved for treatment of HCV genotype 1 (GT 1) infection. The major RAVs identified in nonsustained virologic response (SVR) GT 1a infected patients were similar for BOC and TPV. These RAVs were analyzed for phenotypic resistance to BOC and TPV using an in-vitro GT 1a replicon assay. Methods: RAVs were detected by population sequencing in samples from SPRINT-2 (treatment naive) and RESPOND-2 (prior nonresponder/relapser) patients. RAVs were engineered into a GT 1a replicon derived from the H77 1a strain. Replicon cell lines were treated with BOC or TPV for 3 days, and HCVRNA was quantified by Taqman real time PCR analysis. Results: For patients treated with BOC, RAVs were detected in 53% of non-SVR patients with NS3 sequences available. In BOC treated, non-SVR HCV GT 1a infected patients; the most common RAVs were V36M, T54S and R155K. T54A and A156S/T were also identified in HCV GT 1a non-SVR treated patients but were observed more frequently in HCV GT 1b. A similar pattern of HCV GT 1a RAVs was identified in TPV treated patients (Bartels DJ, et al. Hepatology. 2011;54(S1):1328). These RAVs were analyzed for resistance to BOC and TPV in a GT 1a replicon assay. T54A and T54S produced similar fold-shifts in EC50 with BOC and TPV. However, V36M, R155K and A156S each produced a larger fold-shift in resistance to TPV than BOC. The fold shift in EC50 for each RAV was 2to 6-fold for BOC and 5to 16-fold for TPV. Also, the V36M + R155K double variant resulted in a >20-fold increase in EC50 for TPV but <10-fold for BOC.

Fragment-Based Development of HCV Protease Inhibitors for the Treatment of Hepatitis C

A novel computational technology based on fragmentation of the chemical compounds has been used for the fast and efficient prediction of activities of prospective protease inhibitors of the hepatitis C virus. This study spans over a discovery cycle from the theoretical prediction of new HCV NS3 protease inhibitors to the first cytotoxicity experimental tests of the best candidates. The measured cytotoxicity of the compounds indicated that at least two candidates would be suitable further development of drugs.

Discovery of GS-9451: An acid inhibitor of the hepatitis C virus NS3/4A protease

The discovery of GS-9451 is reported. Modification of the P3 cap and P2 quinoline with a series of solubilizing groups led to the identification of potent HCV NS3 protease inhibitors with greatly improved pharmacokinetic properties in rats, dogs and monkeys.

Single- and Multiple-Ascending-Dose Studies of the NS3 Protease Inhibitor Asunaprevir in Subjects with or without Chronic Hepatitis C

Hepatitis C virus (HCV) protease inhibitors combined with pegylated alfa interferon-ribavirin have demonstrated improved efficacy compared with pegylated alfa interferon-ribavirin alone for the treatment of chronic hepatitis C. Asunaprevir (BMS-650032), a novel HCV NS3 protease inhibitor in clinical development, was evaluated for safety, antiviral activity, and resistance in four double-blind, placebo-controlled, sequential-panel, single- and multiple-ascending-dose (SAD and MAD) studies in healthy subjects or subjects with chronic HCV genotype 1 infection. In SAD studies, subjects (healthy or with chronic HCV infection) were randomized to receive asunaprevir in dose groups of 10 to 1,200 mg or a placebo. In MAD studies, healthy subjects were randomized to receive asunaprevir in dose groups of 10 to 600 mg twice daily or a placebo for 14 days; subjects with HCV infection received asunaprevir in dose groups of 200 to 600 mg twice daily, or a placebo, for 3 days. Across all four studies, headache and diarrhea were the most frequent adverse events in asunaprevir recipients. Asunaprevir at doses of 200 to 600 mg resulted in rapid HCV RNA decreases from the baseline; maximal mean changes in HCV RNA over time were 2.7 and 3.5 log(10) IU/ml in the SAD and MAD studies, respectively. No enrichment of signature asunaprevir-resistant viral variants was detected. In conclusion, the novel NS3 protease inhibitor asunaprevir, when administered at single or multiple doses of 200 to 600 mg twice daily, is generally well tolerated, achieving rapid and substantial decreases in HCV RNA levels in subjects chronically infected with genotype 1 HCV.

New Antiviral Agents and New Treatments on the Horizon for the Management of Viral Hepatitis

Transfusion dependant patients are at risk of acquiring transfusiontransmitted viral infections including Hepatitis B virus (HBV) and Hepatitis C (HCV). These infections can lead to cirrhosis and hepatic cancer. Standard treatment, although with improved therapeutic results still exhibits resistance and relapses. New antiviral agents have been developed to further improve results and reduce adverse events. For hepatitis B, along with pegylated interferon α-2α, other drugs that have been approved include lamivudine, adefovir, entecavir, telbivudine and tenofovir, while emtricitabine and clevudine are awaiting FDA approval. Possible combination drug therapy may improve efficacy without engendering resistance. For hepatitis C, standard therapy has been the combination of Peg-IFN/Ribavarin. Genotype 1 of the virus, which is widespread in the USA and Europe, can be resistant to treatment especially with high viral load. Directly acting antiviral agents (DAAs), are being developed. These are: (i) HCV NS3 protease inhibitors, such as telaprevir and boceprevir, which are currently approved by the FDA. Several other compounds are in phase I-II development; (ii) NS5B polymerase inhibitors, which target HCV replication. These include mericitabine (a nucleoside analogue inhibitor) currently in phase III trials, and nonnucleiside inhibitors; (iii) New intreferons such as pgylated interferonl λ, which are also on trial. Triple therapy using pegylated IFNa/ Ribavarin along with telaprevir or boceprevir are also under trial.

Novel, potent and orally bioavailable indolizidinone-derived inhibitors of the hepatitis C virus NS3 protease

A novel, potent, and orally bioavailable class of product-like inhibitors of the HCV NS3 protease was discovered by constraining the P2-P3 amide bond and the P3 hydrocarbon substituent to the protease-bound conformation. This preorganization was accomplished by incorporation of the P2-P3 amide into a six-membered ring attached to the P2-proline 5-position. Isothermal calorimetric characterization of the role of hydrocarbon substitution of this six-membered ring, upon binding the HCV NS3 protease, was found to be exclusively entropic in nature. The synthesis, preliminary SAR and pharmacokinetic profiling of this compact, indolizidinone-derived scaffold are described.

Preparation of HCV NS3 and NS5B Proteins to Support Small‐Molecule Drug Discovery

Production of high-quality, well-characterized recombinant proteins facilitates screening of compound libraries. The protocols detailed in this unit are used to purify three recombinant enzymes that are widely used in HCV research: the HCV NS3 protease domain, the helicase domain as an NS3+NS4A complex, and the NS5B RNA-dependent RNA polymerase. The active enzymes are purified to homogeneity by two-column chromatography to support a screening program for HCV inhibitors.

Development of a Chimeric Replicon System for Phenotypic Analysis of Ns3 Protease Sequences from HCV Clinical Isolates

To support clinical development of HCV non-structural protein (NS) 3 protease inhibitors (PIs), phenotypic monitoring of patient isolates is a prerequisite for understanding the emergence of resistance. HCV isolates typically fail to replicate in cell culture, necessitating the use of alternative phenotyping methods. An NS3 protease chimeric replicon system was developed to monitor the phenotype of clinical isolates. The transfer of NS3 protease domain sequences from HCV-infected patients to the background of genotype (Gt) 1a-H77c, 1b-Con1 and 2a-JFH-1 lab strain replicons adapted to high-level cell culture replication was investigated. NS3 protease sequences derived from HCV Gt 1a or Gt 1b infected patients were transferred into Gt 1a and 1b replicons, respectively. Replication was detected for 20% of Gt 1a and 75% of Gt 1b sequences. Incorporation of known cell culture adaptive change NS3-E176G improved replication of Gt 1b but not of Gt 1a sequences. Transfer of Gt 1a clinical sequences into the Gt 1b background enhanced replication and allowed phenotypic analysis of all sequences. A correlation was observed between clinical isolate sequence polymorphisms and reduced susceptibility to NS3 PI. In mixed populations containing known NS3 PI resistance changes NS3-R155K or D168E/V, sensitivity of resistance detection was ≥ 10%. An HCV replicon capable of supporting phenotypic characterization of patient-derived HCV NS3 protease sequences was developed. Pre-existence of amino acid changes associated with NS3 PI resistance highlights the need for combination therapies in the treatment of HCV.

P2–P1′ macrocyclization of P2 phenylglycine based HCV NS3 protease inhibitors using ring-closing metathesis

Macrocyclization is a commonly used strategy to preorganize HCV NS3 protease inhibitors in their bioactive conformation. Moreover, macrocyclization generally leads to greater stability and improved pharmacokinetic properties. In HCV NS3 protease inhibitors, it has been shown to be beneficial to include a vinylated phenylglycine in the P2 position in combination with alkenylic P1′ substituents. A series of 14-, 15- and 16-membered macrocyclic HCV NS3 protease inhibitors with the linker connecting the P2 phenylglycine and the alkenylic P1′ were synthesized by ring-closing metathesis, using both microwave and conventional heating. Besides formation of the expected macrocycles in cis and trans configuration as major products, both ring-contracted and double-bond migrated isomers were obtained, in particular during formation of the smaller rings (14- and 15-membered rings). All inhibitors had Ki-values in the nanomolar range, but only one inhibitor type was improved by rigidification. The loss in inhibitory effect can be attributed to a disruption of the beneficial π–π interaction between the P2 fragment and H57, which proved to be especially deleterious for the d-phenylglycine epimers.