Discovery of BMS-961955, an allosteric inhibitor of the hepatitis C virus NS5B polymerase.

The synthesis, structure-activity relationship (SAR) data, and further optimization of the metabolic stability and pharmacokinetic (PK) properties for a previously disclosed class of cyclopropyl-fused indolobenzazepine HCV NS5B polymerase inhibitors are described. These efforts led to the discovery of BMS-961955 as a viable contingency backup to beclabuvir which was recently approved in Japan for the treatment of HCV as part of a three drug, single pill combination marketed as XimencyTM.

Discovery and preclinical evaluation of potent, orally bioavailable, metabolically stable cyclopropylindolobenzazepine acylsulfonamides as thumb site 1 inhibitors of the hepatitis c virus NS5B RNA-dependent, RNA polymerase.

Herein, we describe the synthesis, antiviral structure-activity relationships (SAR), metabolic stability, and pharmacokinetic (PK) properties for a series of cyclopropylindolobenzazepine acylsulfonamide HCV NS5B polymerase inhibitors. Optimization of SAR, metabolic stability and PK led to the identification of compound 19 which was advanced into pre-IND enabling toxicology studies.

Resensitizing daclatasvir-resistant hepatitis C variants by allosteric modulation of NS5A.

It is estimated that more than 170 million people are infected with hepatitis C virus (HCV) worldwide. Clinical trials have demonstrated that, for the first time in human history, the potential exists to eradicate a chronic viral disease using combination therapies that contain only direct-acting antiviral agents. HCV non-structural protein 5A (NS5A) is a multifunctional protein required for several stages of the virus replication cycle. NS5A replication complex inhibitors, exemplified by daclatasvir (DCV; also known as BMS-790052 and Daklinza), belong to the most potent class of direct-acting anti-HCV agents described so far, with in vitro activity in the picomolar (pM) to low nanomolar (nM) range. The potency observed in vitro has translated into clinical efficacy, with HCV RNA declining by ~3-4 log10 in infected patients after administration of single oral doses of DCV. Understanding the exceptional potency of DCV was a key objective of this study. Here we show that although DCV and an NS5A inhibitor analogue (Syn-395) are inactive against certain NS5A resistance variants, combinations of the pair enhance DCV potency by >1,000-fold, restoring activity to the pM range. This synergistic effect was validated in vivo using an HCV-infected chimaeric mouse model. The cooperative interaction of a pair of compounds suggests that NS5A protein molecules communicate with each other: one inhibitor binds to resistant NS5A, causing a conformational change that is transmitted to adjacent NS5As, resensitizing resistant NS5A so that the second inhibitor can act to restore inhibition. This unprecedented synergistic anti-HCV activity also enhances the resistance barrier of DCV, providing additional options for HCV combination therapy and new insight into the role of NS5A in the HCV replication cycle.

Potency and resistance analysis of hepatitis C virus NS5B polymerase inhibitor BMS-791325 on all major genotypes.

BMS-791325 is a hepatitis C virus (HCV) inhibitor binding to the thumb domain of the NS5B RNA-dependent RNA polymerase. BMS-791325 is well characterized in genotype 1 (GT1) and exhibits good inhibitory activity (50% effective concentration [EC50], <10 nM) against hybrid replicons containing patient NS5B sequences from GT3a, -4a, and -5a while potency against GT2 is significantly reduced (J. A. Lemm et al., Antimicrob. Agents Chemother. 58:3485-3495, 2014, doi:http://dx.doi.org/10.1128/AAC.02495-13). BMS-791325 potency against GT6a hybrid replicons is more variable, with two of three hybrid clones having EC50s similar to that for GT1 while a third patient clone was ∼ 10 times less susceptible to BMS-791325. To characterize the resistance profile of BMS-791325 beyond GT1, curing studies were performed across GT1a and -3a to -6a and demonstrated that GT1a has the highest resistance barrier versus BMS-791325 while GT6a has the lowest. Selection of GT3 to -6 NS5B chimeric replicon cells at different concentrations of BMS-791325 revealed substitutions in the thumb domain of NS5B at residues 494 and 495 that conferred different levels of resistance to BMS-791325 but remained susceptible to NS5A or NS3 protease inhibitors. In addition, we demonstrate that the reduced potency of BMS-791325 against one GT6a patient is due to an A494 polymorphism present in ∼ 21% of sequences in the European HCV database. The results from this report suggest that BMS-791325 is a candidate for combination treatment of HCV GT3 to -6 chronic infections, and the resistance profiles identified will provide useful information for future clinical development.

Preclinical characterization of BMS-791325, an allosteric inhibitor of hepatitis C Virus NS5B polymerase.

BMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC50) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC50s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC50, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC50s of >4 µM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC50. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposures in vivo in several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥ 10-fold above the inhibitor EC50s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.

Discovery and development of hepatitis C virus NS5A replication complex inhibitors.

Lead inhibitors that target the function of the hepatitis C virus (HCV) nonstructural 5A (NS5A) protein have been identified by phenotypic screening campaigns using HCV subgenomic replicons. The demonstration of antiviral activity in HCV-infected subjects by the HCV NS5A replication complex inhibitor (RCI) daclatasvir (1) spawned considerable interest in this mechanistic approach. In this Perspective, we summarize the medicinal chemistry studies that led to the discovery of 1 and other chemotypes for which resistance maps to the NS5A protein and provide synopses of the profiles of many of the compounds currently in clinical trials. We also summarize what is currently known about the NS5A protein and the studies using NS5A RCIs and labeled analogues that are helping to illuminate aspects of both protein function and inhibitor interaction. We conclude with a synopsis of the results of notable clinical trials with HCV NS5A RCIs.

Identification of a novel series of potent HCV NS5B Site I inhibitors.

Efforts investigating spatially comparative alternates of the ethylene-bridged piperazine in BMS-791325 that would offer a maintained or improved virologic and pharmacokinetic profile have been multifaceted. One foray involved the utilization of various octahydropyrrolo[3,4-c]pyrrole propellanes. Many of the propellane analogs described in this work exhibited better than targeted potency (less than 20 nM). Additionally, improved exposure in rats was achieved through the employment of two newly invented and now readily accessible carbon bridged propellanes as compared to their heteroatom bridged analogs.

Hepatitis C virus NS5A replication complex inhibitors: the discovery of daclatasvir.

The biphenyl derivatives 2 and 3 are prototypes of a novel class of NS5A replication complex inhibitors that demonstrate high inhibitory potency toward a panel of clinically relevant HCV strains encompassing genotypes 1-6. However, these compounds exhibit poor systemic exposure in rat pharmacokinetic studies after oral dosing. The structure-activity relationship investigations that improved the exposure properties of the parent bis-phenylimidazole chemotype, culminating in the identification of the highly potent NS5A replication complex inhibitor daclatasvir (33) are described. An element critical to success was the realization that the arylglycine cap of 2 could be replaced with an alkylglycine derivative and still maintain the high inhibitory potency of the series if accompanied with a stereoinversion, a finding that enabled a rapid optimization of exposure properties. Compound 33 had EC50 values of 50 and 9 pM toward genotype-1a and -1b replicons, respectively, and oral bioavailabilities of 38-108% in preclinical species. Compound 33 provided clinical proof-of-concept for the NS5A replication complex inhibitor class, and regulatory approval to market it with the NS3/4A protease inhibitor asunaprevir for the treatment of HCV genotype-1b infection has recently been sought in Japan.

Hepatitis C virus NS5A replication complex inhibitors. Part 6: Discovery of a novel and highly potent biarylimidazole chemotype with inhibitory activity toward genotypes 1a and 1b replicons.

A medicinal chemistry campaign that was conducted to address a potential genotoxic liability associated with an aniline-derived scaffold in a series of HCV NS5A inhibitors with dual GT-1a/-1b inhibitory activity is described. Anilides 3b and 3c were used as vehicles to explore structural modifications that retained antiviral potency while removing the potential for metabolism-based unmasking of the embedded aniline. This effort resulted in the discovery of a highly potent biarylimidazole chemotype that established a potency benchmark in replicon assays, particularly toward HCV GT-1a, a strain with significant clinical importance. Securing potent GT-1a activity in a chemotype class lacking overt structural liabilities was a critical milestone in the effort to realize the full clinical potential of targeting the HCV NS5A protein.

HCV NS5A replication complex inhibitors. Part 4. Optimization for genotype 1a replicon inhibitory activity.

A series of symmetrical E-stilbene prolinamides that originated from the library-synthesized lead 3 was studied with respect to HCV genotype 1a (G-1a) and genotype 1b (G-1b) replicon inhibition and selectivity against BVDV and cytotoxicity. SAR emerging from an examination of the prolinamide cap region revealed 11 to be a selective HCV NS5A inhibitor exhibiting submicromolar potency against both G-1a and G-1b replicons. Additional structural refinements resulted in the identification of 30 as a potent, dual G-1a/1b HCV NS5A inhibitor.

Characterizations of HCV NS5A replication complex inhibitors.

The hepatitis C virus NS5A protein is an established and clinically validated target for antiviral intervention by small molecules. Characterizations are presented of compounds identified as potent inhibitors of HCV replication to provide insight into structural elements that interact with the NS5A protein. UV-activated cross linking and affinity isolation was performed with one series to probe the physical interaction between the inhibitors and the NS5A protein expressed in HCV replicon cells. Resistance mapping with the second series was used to determine the functional impact of specific inhibitor subdomains on the interaction with NS5A. The data provide evidence for a direct high-affinity interaction between these inhibitors and the NS5A protein, with the interaction dependent on inhibitor stereochemistry. The functional data supports a model of inhibition that implicates inhibitor binding by covalently combining distinct pharmacophores across an NS5A dimer interface to achieve maximal inhibition of HCV replication.

HCV NS5A replication complex inhibitors. Part 5: discovery of potent and pan-genotypic glycinamide cap derivatives.

The isoquinolinamide series of HCV NS5A inhibitors exemplified by compounds 2b and 2c provided the first dual genotype-1a/1b (GT-1a/1b) inhibitor class that demonstrated a significant improvement in potency toward GT-1a replicons compared to that of the initial program lead, stilbene 2a. Structure-activity relationship (SAR) studies that uncovered an alternate phenylglycine-based cap series that exhibit further improvements in virology profile, along with some insights into the pharmacophoric elements associated with the GT-1a potency, are described.

HCV NS5A replication complex inhibitors. Part 3: discovery of potent analogs with distinct core topologies.

In a recent disclosure, we described the discovery of dimeric, prolinamide-based NS5A replication complex inhibitors exhibiting excellent potency towards an HCV genotype 1b replicon. That disclosure dealt with the SAR exploration of the peripheral region of our lead chemotype, and herein is described the SAR uncovered from a complementary effort that focused on the central core region. From this effort, the contribution of the core region to the overall topology of the pharmacophore, primarily vector orientation and planarity, was determined, with a set of analogs exhibiting <10 nM EC(50) in a genotype 1b replicon assay.

Combinations of lambda interferon with direct-acting antiviral agents are highly efficient in suppressing hepatitis C virus replication.

The clinical efficacy of a pegylated form of human lambda 1 interferon (IFN-λ1; also referred to herein as lambda) has been demonstrated in patients chronically infected with hepatitis C virus (HCV) representing genotypes 1 through 4. In these proof-of-concept studies, lambda showed an improved safety profile compared to the pegylated form of alpha interferon (referred to herein as alfa). In the study described in this report, an assessment of the in vitro antiviral activity of type III IFNs toward different HCV replicons revealed that the unpegylated recombinant form of IFN-λ1 (rIFN-λ1) exerted the most robust effect, while rIFN-λ3 exhibited greater activity than rIFN-λ2. More importantly, cross-resistance to rIFN-λ1 was not observed in replicon cell lines known to have reduced susceptibility to investigational direct-acting antiviral (DAA) agents targeting the essential HCV nonstructural protein NS3, NS5A, or NS5B. When combined with either rIFN-α, the NS3 protease inhibitor (NS3 PI) asunaprevir (ASV), the NS5A replication complex inhibitor (NS5A RCI) daclatasvir (DCV), or the NS5B polymerase site I inhibitor (NS5B I) BMS-791325, rIFN-λ1 displayed a mixture of additive and synergistic effects. In three-drug combination studies, inclusion of lambda with ASV and DCV also yielded additive to synergistic effects. In line with these observations, it was demonstrated that a regimen that used a combination of rIFN-λ1 with one or two DAAs was superior to an IFN-free regimen in clearing HCV RNA in genotype 1a cell lines representing wild-type and NS3 protease inhibitor-resistant sequences. Overall, these data support further clinical development of lambda as part of alternative combination treatments with DAAs for patients chronically infected with HCV.

HCV NS5A replication complex inhibitors. Part 2: investigation of stilbene prolinamides.

In a previous disclosure,(1) we reported the dimerization of an iminothiazolidinone to form 1, a contributor to the observed inhibition of HCV genotype 1b replicon activity. The dimer was isolated via bioassay-guided fractionation experiments and shown to be a potent inhibitor of genotype 1b HCV replication for which resistance mapped to the NS5A protein. The elements responsible for governing HCV inhibitory activity were successfully captured in the structurally simplified stilbene prolinamide 2. We describe herein the early SAR and profiling associated with stilbene prolinamides that culminated in the identification of analogs with PK properties sufficient to warrant continued commitment to this chemotype. These studies represent the key initial steps toward the discovery of daclatasvir (BMS-790052), a compound that has demonstrated clinical proof-of-concept for inhibiting the NS5A replication complex in the treatment of HCV infection.

Effect on hepatitis C virus replication of combinations of direct-acting antivirals, including NS5A inhibitor daclatasvir.

Three hepatitis C virus (HCV) inhibitors, asunaprevir (ASV; BMS-650032), daclatasvir (DCV; BMS-790052), and BMS-791325, each targeting a different nonstructural protein of the virus (NS3, NS5A, and NS5B, respectively), have independently demonstrated encouraging preclinical profiles and are currently undergoing clinical evaluation. Since drug-resistant variants have rapidly developed in response to monotherapy with almost all direct-acting antiviral agents (DAAs) for HCV, the need for combination therapies to effectively eradicate the virus from infected patients is clear. These studies demonstrated the additive-synergistic effects on replicon inhibition and clearance of combining NS3 protease or NS5B RNA polymerase inhibitors with the first-in-class, NS5A replication complex inhibitor daclatasvir (DCV) and reveal new resistance pathways for combinations of two small-molecule inhibitors that differ from those that develop during monotherapy. The results suggest that under a specific selective pressure, a balance must be reached in the fitness costs of substitutions in one target gene when substitutions are also present in another target gene. Further synergies and additional novel resistance substitutions were observed during triple-combination treatment relative to dual-drug therapy, indicating that, in combination, HCV inhibitors can exert cross-target influences on resistance development. Enhanced synergies in replicon inhibition and a reduced frequency of resistance together lend strong support to the utility of combinations of DAAs for the treatment of HCV, and the identification of altered resistance profiles during combination treatment provides useful information for monitoring resistance in the clinic.

Synthesis and SAR studies of novel heteroaryl fused tetracyclic indole-diamide compounds: potent allosteric inhibitors of the hepatitis C virus NS5B polymerase.

Presented here are initial structure-activity relationship (SAR) studies on a series of novel heteroaryl fused tetracyclic indole-based inhibitors of the hepatitis C viral polymerase, NS5B. The introduction of alternative heterocyclic moieties into the indolo-fused inhibitor class significantly expands the reported SAR and resulted in the identification of pyridino analogs, typified by compounds 44 and 45 that displayed excellent potency against the NS5B polymerase of both HCV 1a and HCV 1b genotypes.

The effects of NS5A inhibitors on NS5A phosphorylation, polyprotein processing and localization.

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multi-functional protein that is expressed in basally phosphorylated (p56) and in hyperphosphorylated (p58) forms. NS5A phosphorylation has been implicated in regulating multiple aspects of HCV replication. We recently reported the identification of a class of compounds that potently inhibit HCV RNA replication by targeting NS5A. Although the precise mechanism of inhibition of these compounds is not well understood, one activity that has been described is their ability to block expression of the hyperphosphorylated form of NS5A. Here, we report that an NS5A inhibitor impaired hyperphosphorylation without affecting basal phosphorylation at the C-terminal region of NS5A. This inhibitor activity did not require NS5A domains II and III and was distinct from that of a cellular kinase inhibitor that also blocked NS5A hyperphosphorylation, results that are consistent with an inhibitor-binding site within the N-terminal region of NS5A. In addition, we observed that an NS5A inhibitor promoted the accumulation of an HCV polyprotein intermediate, suggesting that inhibitor binding to NS5A may occur prior to the completion of polyprotein processing. Finally, we observed that NS5A p56 and p58 separated into different membrane fractions during discontinuous sucrose gradient centrifugation, consistent with these NS5A phosphoforms performing distinct replication functions. The p58 localization pattern was disrupted by an NS5A inhibitor. Collectively, our results suggest that NS5A inhibitors probably impact several aspects of HCV expression and regulation. These findings may help to explain the exceptional potency of this class of HCV replication complex inhibitors.

Discovery of potent hepatitis C virus NS5A inhibitors with dimeric structures.

The exceptional in vitro potency of the hepatitis C virus (HCV) NS5A inhibitor BMS-790052 has translated into an in vivo effect in proof-of-concept clinical trials. Although the 50% effective concentration (EC(50)) of the initial lead, the thiazolidinone BMS-824, was ~10 nM in the replicon assay, it underwent transformation to other inhibitory species after incubation in cell culture medium. The biological profile of BMS-824, including the EC(50), the drug concentration required to reduce cell growth by 50% (CC(50)), and the resistance profile, however, remained unchanged, triggering an investigation to identify the biologically active species. High-performance liquid chromatography (HPLC) biogram fractionation of a sample of BMS-824 incubated in medium revealed that the most active fractions could readily be separated from the parental compound and retained the biological profile of BMS-824. From mass spectral and nuclear magnetic resonance data, the active species was determined to be a dimer of BMS-824 derived from an intermolecular radical-mediated reaction of the parent compound. Based upon an analysis of the structural elements of the dimer deemed necessary for anti-HCV activity, the stilbene derivative BMS-346 was synthesized. This compound exhibited excellent anti-HCV activity and showed a resistance profile similar to that of BMS-824, with changes in compound sensitivity mapped to the N terminus of NS5A. The N terminus of NS5A has been crystallized as a dimer, complementing the symmetry of BMS-346 and allowing a potential mode of inhibition of NS5A to be discussed. Identification of the stable, active pharmacophore associated with these NS5A inhibitors provided the foundation for the design of more potent inhibitors with broad genotype inhibition. This culminated in the identification of BMS-790052, a compound that preserves the symmetry discovered with BMS-346.

Inhibitors of HCV NS5A: From Iminothiazolidinones to Symmetrical Stilbenes.

The iminothiazolidinone BMS-858 (2) was identified as a specific inhibitor of HCV replication in a genotype 1b replicon assay via a high-throughput screening campaign. A more potent analogue, BMS-824 (18), was used in resistance mapping studies, which revealed that inhibitory activity was related to disrupting the function of the HCV nonstructural protein 5A. Despite the development of coherent and interpretable SAR, it was subsequently discovered that in DMSO 18 underwent an oxidation and structural rearrangement to afford the thiohydantoin 47, a compound with reduced HCV inhibitory activity. However, HPLC bioassay fractionation studies performed after incubation of 18 in assay media led to the identification of fractions containing a dimeric species 48 that exhibited potent antiviral activity. Excision of the key elements hypothesized to be responsible for antiviral activity based on SAR observations reduced 48 to a simplified, symmetrical, pharmacophore realized most effectively with the stilbene 55, a compound that demonstrated potent inhibition of HCV in a genotype 1b replicon with an EC50 = 86 pM.