Synthesis and Biological Characterization of Aryl Uracil Inhibitors of Hepatitis C Virus NS5B Polymerase: Discovery of ABT-072, a trans-Stilbene Analog with Good Oral Bioavailability.

ABT-072 is a non-nucleoside HCV NS5B polymerase inhibitor that was discovered as part of a program to identify new direct-acting antivirals (DAAs) for the treatment of HCV infection. This compound was identified during a medicinal chemistry effort to improve on an original lead, inhibitor 1, which we described in a previous publication. Replacement of the amide linkage in 1 with a trans-olefin resulted in improved compound permeability and solubility and provided much better pharmacokinetic properties in preclinical species. Replacement of the dihydrouracil in 1 with an N-linked uracil provided better potency in the genotype 1 replicon assay. Results from phase 1 clinical studies supported once-daily oral dosing with ABT-072 in HCV infected patients. A phase 2 clinical study that combined ABT-072 with the HCV protease inhibitor ABT-450 provided a sustained virologic response at 24 weeks after dosing (SVR24) in 10 of 11 patients who received treatment.

In vitro and in vivo antiviral activity and resistance profile of ombitasvir, an inhibitor of hepatitis C virus NS5A.

Ombitasvir (ABT-267) is a hepatitis C virus (HCV) NS5A inhibitor with picomolar potency, pan-genotypic activity, and 50% effective concentrations (EC50s) of 0.82 to 19.3 pM against HCV genotypes 1 to 5 and 366 pM against genotype 6a. Ombitasvir retained these levels of potency against a panel of 69 genotype 1 to 6 chimeric replicons containing the NS5A gene derived from HCV-infected patients, despite the existence of natural sequence diversity within NS5A. In vitro resistance selection identified variants that conferred resistance to ombitasvir in the HCV NS5A gene at amino acid positions 28, 30, 31, 58, and 93 in genotypes 1 to 6. Ombitasvir was evaluated in vivo in a 3-day monotherapy study in 12 HCV genotype 1-infected patients at 5, 25, 50, or 200 mg dosed once daily. All patients in the study were HCV genotype 1a infected and were without preexisting resistant variants at baseline as determined by clonal sequencing. Decreases in HCV RNA up to 3.1 log10 IU/ml were observed. Resistance-associated variants at position 28, 30, or 93 in NS5A were detected in patient samples 48 hours after the first dose. Clonal sequencing analysis indicated that wild-type virus was largely suppressed by ombitasvir during 3-day monotherapy, and at doses higher than 5 mg, resistant variant M28V was also suppressed. Ombitasvir was well tolerated at all doses, and there were no serious or severe adverse events. These data support clinical development of ombitasvir in combination with inhibitors targeting HCV NS3/4A protease (ABT-450 with ritonavir) and HCV NS5B polymerase (ABT-333, dasabuvir) for the treatment of chronic HCV genotype 1 infection. (Study M12-116 is registered at ClinicalTrials.gov under registration no. NCT01181427.).

In vitro activity and resistance profile of dasabuvir, a nonnucleoside hepatitis C virus polymerase inhibitor.

Dasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50 resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.

Discovery of ABT-267, a pan-genotypic inhibitor of HCV NS5A.

We describe here N-phenylpyrrolidine-based inhibitors of HCV NS5A with excellent potency, metabolic stability, and pharmacokinetics. Compounds with 2S,5S stereochemistry at the pyrrolidine ring provided improved genotype 1 (GT1) potency compared to the 2R,5R analogues. Furthermore, the attachment of substituents at the 4-position of the central N-phenyl group resulted in compounds with improved potency. Substitution with tert-butyl, as in compound 38 (ABT-267), provided compounds with low-picomolar EC50 values and superior pharmacokinetics. It was discovered that compound 38 was a pan-genotypic HCV inhibitor, with an EC50 range of 1.7-19.3 pM against GT1a, -1b, -2a, -2b, -3a, -4a, and -5a and 366 pM against GT6a. Compound 38 decreased HCV RNA up to 3.10 log10 IU/mL during 3-day monotherapy in treatment-naive HCV GT1-infected subjects and is currently in phase 3 clinical trials in combination with an NS3 protease inhibitor with ritonavir (r) (ABT-450/r) and an NS5B non-nucleoside polymerase inhibitor (ABT-333), with and without ribavirin.

High potency improvements to weak aryl uracil HCV polymerase inhibitor leads.

Described herein is the development of a potent non-nucleoside, small molecule inhibitor of genotype 1 HCV NS5B Polymerase. A 23 µM inhibitor that was active against HCV polymerase was further elaborated into a potent single-digit nanomolar inhibitor of HCV NS5B polymerase by additional manipulation of the R and R1 substituents. Subsequent modifications to improve physical properties were made in an attempt to achieve an acceptable pharmacokinetic profile.

Aryl uracil inhibitors of hepatitis C virus NS5B polymerase: synthesis and characterization of analogs with a fused 5,6-bicyclic ring motif.

The synthesis and structure-activity relationships of a novel aryl uracil series which contains a fused 5,6-bicyclic ring unit for HCV NS5B inhibition is described. Several analogs display replicon cell culture potencies in the low nanomolar range along with excellent rat pharmacokinetic values.

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.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Exploratory study of oral combination antiviral therapy for hepatitis C.

BACKGROUND: There is a need for interferon-free treatment regimens for hepatitis C virus (HCV) infection. The goal of this study was to evaluate ABT-450, a potent HCV NS3 protease inhibitor, combined with low-dose ritonavir (ABT-450/r), in addition to ABT-333, a nonnucleoside NS5B polymerase inhibitor, and ribavirin, for the treatment of HCV infection. METHODS: We conducted a 12-week, phase 2a, open-label study involving patients who had HCV genotype 1 infection without cirrhosis. All patients received ABT-333 (400 mg twice daily) and ribavirin (1000 to 1200 mg per day) and one of two daily doses of ABT-450/r. Groups 1 and 2 included previously untreated patients; group 1 received 250 mg of ABT-450 and 100 mg of ritonavir, and group 2 received 150 mg and 100 mg, respectively. Group 3, which included patients who had had a null or partial response to previous therapy with peginterferon and ribavirin, received daily doses of 150 mg of ABT-450 and 100 mg of ritonavir. The primary end point was an undetectable level of HCV RNA from week 4 through week 12 (extended rapid virologic response). RESULTS: A total of 17 of the 19 patients in group 1 (89%) and 11 of the 14 in group 2 (79%) had an extended rapid virologic response; a sustained virologic response 12 weeks after the end of treatment was achieved in 95% and 93% of the patients, respectively. In group 3, 10 of 17 patients (59%) had an extended rapid virologic response, and 8 (47%) had a sustained virologic response 12 weeks after therapy; 6 patients had virologic breakthrough, and 3 had a relapse. Adverse events included abnormalities in liver-function tests, fatigue, nausea, headache, dizziness, insomnia, pruritus, rash, and vomiting. CONCLUSIONS: This preliminary study suggests that 12 weeks of therapy with a combination of a protease inhibitor, a nonnucleoside polymerase inhibitor, and ribavirin may be effective for treatment of HCV genotype 1 infection. (Funded by Abbott; ClinicalTrials.gov number, NCT01306617.).

Hepatitis C NS5B polymerase inhibitors: functional equivalents for the benzothiadiazine moiety.

A series of quinoline derivatives was synthesized as potential bioisosteric replacements for the benzothiadiazine moiety of earlier Hepatitis C NS5B polymerase inhibitors. Several of these compounds exhibited potent activity in enzymatic and replicon assays.

Synthesis and biological characterization of B-ring amino analogues of potent benzothiadiazine hepatitis C virus polymerase inhibitors.

Benzothiadiazine inhibitors of the HCV NS5B RNA-dependent RNA polymerase are an important class of non-nucleoside inhibitors that have received considerable attention in the search for novel HCV therapeutics. Research in our laboratories has identified a novel series of tetracyclic benzothiadiazine inhibitors of HCV polymerase bearing a benzylamino substituent on the B-ring. Compounds in this series exhibit low-nanomolar activities in both genotypes 1a and 1b polymerase inhibition assays and subgenomic replicon assays. Optimization of pharmacokinetic properties in rat led to compound 30, which has good oral bioavailability (F = 56%) and a favorable tissue distribution drug profile, with high liver to plasma ratios. Compound 30 is a potent inhibitor in replicon assays, with EC(50) values of 10 and 6 nM against genotypes 1a and 1b, respectively.

Inhibitors of hepatitis C virus polymerase: synthesis and biological characterization of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines.

The hepatitis C virus (HCV) NS5B polymerase is essential for viral replication and has been a prime target for drug discovery research. Our efforts directed toward the discovery of HCV polymerase inhibitors resulted in the identification of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines 2 and 3. The most active compound displayed activity in genotypes 1a and 1b polymerase and replicon cell culture inhibition assays at subnanomolar and low nanomolar concentrations, respectively. It also displayed an excellent pharmacokinetic profile in rats, with a plasma elimination half-life after intravenous dosing of 4.5 h, oral bioavailability of 77%, and a peak liver concentration of 21.8 microg/mL.

Development of a cell-based assay for high-throughput screening of inhibitors against HCV genotypes 1a and 1b in a single well.

The Hepatitis C (HCV) replicon system is a useful tool for the high-volume screening of inhibitors of HCV replication. In this report, a cell-based assay has been described, which monitors the inhibition of HCV genotypes 1a and 1b as well as cytotoxicity, from a single well of a 96-well plate. A mixture of two stable replicon cell lines was used: one containing a 1a-H77 replicon expressing a firefly luciferase reporter, and the other one containing a 1b-N replicon with a secreted alkaline phosphatase reporter, thus allowing us to monitor replication of two HCV genotypes in the same well. Cytotoxicity was measured using the Resazurin cytotoxicity assay. The assay was validated with known HCV inhibitors and showed that the antiviral activity and cytotoxicity of compounds were reproducibly measured under screening conditions. It was also showed that the assay's signal-to-noise ratio and Z' coefficient were suitable for high-throughput screening. A panel of HCV inhibitors showed a good correlation between EC(50) and TD(50) values for 1a and 1b replicon activity and cytotoxicity measured using either a single replicon format or mixed replicon format. Thus, the use of this mixed replicon format provides an economical method for simultaneous measurement of compound activity against two HCV genotypes as well as cytotoxicity, thereby reducing cost of reagents and labor as well as improving throughput.

Methods to measure the intracellular concentration of unlabeled compounds within cultured cells using liquid chromatography/tandem mass spectrometry.

A high-throughput and sensitive liquid chromatography/tandem mass spectrometry assay was established to detect total unlabeled hepatitis C virus inhibitor concentrations in replicon cells. The intracellular concentrations determined by this assay correlated well with concentrations obtained using radiolabeled compound. Some compounds accumulated inside the cells, with concentrations up to 300-fold higher than the input concentration. Confocal microscopic evaluation of two fluorescent-tagged inhibitors confirmed high accumulation inside the cells, sequestered inside vesicles within the cytoplasm. Incubation of cells with compound at 4 degrees C revealed that nonspecific binding to the outside of the cell membrane and to the cell culture plate occurred for some compounds. Therefore, the total concentration of compound extracted at 37 degrees C was reduced by the amount that was nonspecifically bound at 4 degrees C to yield the amount of compound inside the cells. A modification of the protocol was used for compounds with low intracellular concentrations in which cells were harvested with trypsin-EDTA prior to extraction. This eliminated the nonspecific binding to the cell culture plate and decreased the overall background of the assay. This assay was used to understand differences in cellular potency between compounds and the effects of serum proteins on the metabolic stability of compounds during incubation with cells.

Des-A-ring benzothiadiazines: inhibitors of HCV genotype 1 NS5B RNA-dependent RNA polymerase.

In our program to discover non-nucleoside, small molecule inhibitors of genotype 1 HCV polymerase, we investigated a series of promising analogs based on a benzothiadiazine screening hit that contains an ABCD ring system. After demonstrating that a methylsulfonylamino D-ring substituent increased the enzyme potency into the low nanomolar range, we explored a minimum core required for activity by truncating to a three-ring system. Described herein are the syntheses and structure-activity relationship of a set of inhibitors lacking the A-ring of an ABCD ring system. We observed that small aromatic rings and alkenyl groups appended to the 5-position of the B-ring were optimal, resulting in inhibitors with low nanomolar potencies.

The emerging field of HCV drug resistance.

BACKGROUND: With 170 million people infected worldwide and an inadequate current standard of care, hepatitis C virus (HCV) infection represents a major unmet medical need. Multiple companies are working on the discovery and development of specific HCV antiviral drugs, including inhibitors of HCV polymerase, protease and NS5A. Because of the error-prone nature of viral RNA replication, resistance mutants will develop that could present a potentially significant challenge to developing antiviral treatment regimens. OBJECTIVE: Here, we review the major drug classes currently in preclinical and clinical development and the resistance mutations specific for each class that have been identified from cell culture and/or in vivo studies. METHODS: We have analyzed currently available scientific literature to create a comprehensive review of the current state of the art in the field of HCV resistance to specific antiviral agents, in vitro and in vivo. RESULTS/CONCLUSION: Most specific HCV inhibitors described in the literature can select resistant viral variants in cell culture and in the clinic. Interplay of a mutant's fitness and its level of resistance will determine its clinical importance. Combinations of non-cross-resistant classes of drugs will be key to successful antiviral therapy. The number of drugs in a combination as well as the optimal duration of antiviral treatment, are important issues that need to be addressed in future studies.

Synthesis and SAR of novel 1,1-dialkyl-2(1H)-naphthalenones as potent HCV polymerase inhibitors.

A series of gem-dialkyl naphthalenone derivatives with varied alkyl substitutions were synthesized and evaluated according to their structure-activity relationship. This investigation led to the discovery of potent inhibitors of the hepatitis C virus at low nanomolar concentrations in both enzymatic and cell-based HCV genotype 1a assays.

A replicon-based shuttle vector system for assessing the phenotype of HCV NS5B polymerase genes isolated from patient populations.

Hepatitis C virus (HCV) replicon-based shuttle vectors that permit phenotypes of NS5B polymerase genes from a large number of patient isolates to be rapidly assessed when transiently expressed in cultured cells were designed. When used to test responses to an inhibitor of HCV RNA-dependent RNA polymerase, IC(50) values for inhibition covered a several hundred-fold range among 47 patient samples tested. This observation highlights the variability that can be found by testing isolates derived from HCV-infected subjects. Partial suppression with a polymerase inhibitor of the most sensitive species permitted detection of minor quasispecies that were 7-200-fold more resistant than the bulk population in approximately half of the samples. Sequence analysis showed a wide range of amino acid changes not detected by conventional selection methods using laboratory-derived strains. This approach provides a means to assess variation in antiviral efficacy, and to predict possible responses in a clinical setting.

In vitro selection and characterization of human immunodeficiency virus type 2 with decreased susceptibility to lopinavir.

Lopinavir (LPV)-ritonavir has demonstrated durable antiviral activity in human immunodeficiency virus type 1 (HIV-1)-infected antiretroviral-naïve and protease inhibitor (PI)-experienced patients. However, information on LPV activity against HIV-2 and the patterns of mutations in HIV-2 in response to selection by LPV is limited. The activity of LPV against three strains of HIV-2 was assessed and compared to activity against a reference HIV-1 strain. LPV demonstrated activity similar to that observed against HIV-1 in two HIV-2 strains (HIV-2(MS) and HIV-2(CBL-23)) tested. On the other hand, approximately 10-fold-reduced susceptibility was observed with the third HIV-2 strain, HIV-2(CDC310319). Passage of HIV-2(MS) with increasing concentrations of LPV selected mutations V47A and D17N in the HIV-2 protease gene. The introduction of both 17N and 47A either individually or together into HIV-2(ROD) molecular infectious clones showed that the single V47A substitution in HIV-2 resulted in a substantial reduction in susceptibility to LPV. In contrast, this mutant retained wild-type susceptibility to other PIs and appeared to be hypersusceptible to atazanavir and saquinavir.

Identification and characterization of mutations conferring resistance to an HCV RNA-dependent RNA polymerase inhibitor in vitro.

Compound A-837093, a non-nucleoside HCV RNA-dependent RNA polymerase inhibitor, displayed nanomolar potencies against HCV genotypes 1a and 1b replicons. It also exhibited an excellent metabolic profile and achieved high plasma and liver concentrations in animals. In order to characterize the development of resistance to this anti-HCV agent, HCV subgenomic 1b strain N replicon cells were cultured in the presence of A-837093 with G418. Mutations S368A, Y448H, G554D, Y555C, and D559G in the NS5B polymerase gene were identified that led to substantial decreases in the susceptibilities of 1b genotype replicons to the inhibitor A-837093. However, the resistant mutants remained susceptible to HCV protease inhibitor BILN-2061 and alpha interferon as well as to a different class of non-nucleoside HCV polymerase inhibitor. In addition, each single resistant mutation identified significantly reduced the replication capacity of mutant compared to wild-type replicon. These findings provide a strategic guide for the future development of non-nucleoside inhibitors of HCV NS5B polymerase.

Identification of host genes involved in hepatitis C virus replication by small interfering RNA technology.

UNLABELLED: Hepatitis C virus (HCV) replication is highly dependent on host cell factors. Identification of these host factors not only facilitates understanding of the biology of HCV infection but also enables the discovery of novel targets for anti-HCV therapy. To identify host genes important for HCV RNA replication, we screened a library of small interfering RNA (siRNA) that targets approximately 4,000 human genes in Huh7-derived EN5-3 cells harboring an HCV subgenomic replicon with the nonstructural region NS3-NS5B from the 1b-N strain. Nine cellular genes that potentially regulate HCV replication were identified in this screen. Silencing of these genes resulted in inhibition of HCV replication by more than 60% and exhibited minimal toxicity. Knockdown of host gene expression by these siRNAs was confirmed at the RNA level and, in some instances, at the protein level. The level of siRNA silencing of these host genes correlated well with inhibition of HCV. These genes included those that encoded a G-protein coupled receptor (TBXA2R), a membrane protein (LTbeta), an adapter protein (TRAF2), 2 transcription factors (RelA and NFkappaB2), 2 protein kinases (MKK7 and SNARK), and 2 closely related transporter proteins (SLC12A4 and SLC12A5). Of interest, some of these genes are members of the tumor necrosis factor/lymphotoxin signaling pathway. CONCLUSION: Findings of this study may provide important information for understanding HCV replication. In addition, these cellular genes may constitute a novel set of targets for HCV antiviral therapy.