2015 Philip S. Portoghese Medicinal Chemistry Lectureship. Curing

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2015 Philip S. Portoghese Medicinal Chemistry Lectureship. Curing Hepatitis C Virus Infection with Direct-Acting Antiviral Agents: The Arc of a Medicinal Chemistry Triumph† Nicholas A. Meanwell* Department of Discovery Chemistry, Bristol-Myers Squibb Research & Development, Wallingford, Connecticut 06492, United States ABSTRACT: The development of direct-acting antiviral agents that can cure a chronic hepatitis C virus (HCV) infection after 8−12 weeks of daily, well-tolerated therapy has revolutionized the treatment of this insidious disease. In this article, three of Bristol-Myers Squibb’s HCV programs are summarized, each of which produced a clinical candidate: the NS3 protease inhibitor asunaprevir (64), marketed as Sunvepra, the NS5A replication complex inhibitor daclatasvir (117), marketed as Daklinza, and the allosteric NS5B polymerase inhibitor beclabuvir (142), which is in late stage clinical studies. A clinical study with 64 and 117 established for the first time that a chronic HCV infection could be cured by treatment with direct-acting antiviral agents alone in the absence of interferon. The development of small molecule HCV therapeutics, designed by medicinal chemists, has been hailed as “the arc of a medical triumph” but may equally well be described as “the arc of a medicinal chemistry triumph”.

1. INTRODUCTION Over the past century, small molecule therapeutic agents have contributed significantly to the reduction in mortality and morbidity that has translated into a positive effect on human health and longevity.1,2 Of particular note are those individuals who are infected with human immunodeficiency virus 1 (HIV-1) and hepatitis B virus (HBV), pathogens that establish chronic infections with the former incurable based on available drugs and the current understanding of latency, while curing the latter remains a significant challenge.3−5 For HIV-1 infection, contemporary combinations of antiretroviral therapeutics have transformed the prospects of those infected from the fate of certain death in the 1980s and early 1990s to that of a manageable, chronic infection compatible with a near normal life span when therapy is initiated before damage is inflicted on the immune system, although the long-term effect of living with HIV-1 is not without its morbidities as we are learning more about the manifestations of the infection.6 For HBV, although viral replication can effectively be controlled by nucleos(t)ide-based inhibitors, persistent protein production from the covalently closed circular viral DNA appears to prevent a sterilizing cure.7 Having established how to effectively control HIV-1 and HBV replication in vivo, attention is now being directed toward developing approaches to cure these chronic infections, a significant challenge, particularly for HIV-1 infection where a copy of the viral DNA is integrated into the host cell chromosome.8,9 A third problematic chronic viral infection that was recognized in the early 1970s as non-A, non-B hepatitis is the result of a virus that remained anonymous until 1989 when Michael Houghton, Daniel Bradley, and colleagues isolated a cDNA © XXXX American Chemical Society

clone of what was designated as hepatitis C virus (HCV) after amplifying viral RNA isolated from an infected patient using the polymerase chain reaction.10−12 The virus was classified as a member of the flavivirus family, and the information encoded in the RNA genome was quickly deciphered into three structural (E1, E2, and capsid) and seven nonstructural (NS) proteins that included the p7 ion channel protein (Figure 1).13,14 Within the HCV NS proteins it was determined that the virus encoded for two proteases (the NS2 metalloprotease and the NS3 serine protease), while the viral RNA-dependent RNA polymerase (RdRp) was associated with the NS5B protein.15−17 It was subsequently demonstrated that the NS4A protein was a cofactor for the NS3 protease and that the NS3 protein comprised the protease in the 181-residue amino terminus while the carboxy terminus was associated with helicase activity.18,19 The 447residue protein encoded by NS5A, although critical for viral replication in vivo, was viewed as enigmatic in nature, with no known enzymatic activity and little information about its function that could be gleaned from an analysis of its amino acid sequence.20 Although these studies provided a very basic understanding of the virus, the lack of cell culture systems that supported viral replication significantly hampered progress toward understanding the virus lifecycle in more detail and its inevitably complex modulation of the host cell environment.21 While this problem would take almost a decade to solve, early drug discovery efforts by necessity focused on the NS3 protease and NS5B RdRp, since these were considered the most tractable Received: June 18, 2016

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DOI: 10.1021/acs.jmedchem.6b00915 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Genetic organization of HCV that comprises 5′- and 3′-untranslated regions and encodes for structural and nonstructural proteins with the sites of cleavage of the polypeptide depicted.

targets based on a basic understanding of their function, role in viral replication, and the ability to biochemically recapitulate enzymatic activity in vitro.22,23 Importantly, since HCV replication occurs in the cytosolic compartment and the replicative intermediates do not involve a reverse transcription step to afford a viral DNA, HCV is a curable infection, unlike HIV-1 and HBV, with chronicity a function of effective control of the host immune response by the virus.24 The complexity of HCV was ultimately revealed when it was determined to be comprised of seven major genotypes and 67 subtypes that demonstrated differential worldwide distribution, with genotype-1 (GT-1) the most prevalent in the United States, Japan, and Europe.25 HCV is most commonly transmitted by percutaneous exposure to infected blood and establishes a chronic infection in the majority of subjects exposed to the virus, exerting a slowly progressing burden on the liver over a period of 20−30 years. Over this extended period, the virus inflicts severe damage to the liver, causing cirrhosis, hepatocellular carcinoma, and end stage liver disease, while the early stages of infection are largely asymptomatic such that most are unaware of their infection.26 The discovery and characterization of HCV coupled with its prevalence, worldwide distribution, and associated morbidity and mortality stimulated a significant effort within the pharmaceutical industry to identify and develop specific, direct-acting inhibitors that would have the potential to be effective therapeutic agents to treat the disease. At Bristol-Myers Squibb, our initial drug discovery efforts were focused on developing and implementing HCV NS3 protease inhibition assays for use in high throughput screening (HTS) campaigns. In addition, in parallel we probed for inhibitors of bovine viral diarrhea virus (BVDV) using a cell-based screen in the anticipation that since this was the closest phylogenetic relative of HCV, leads discovered in this fashion might provide a productive avenue into the identification of HCV inhibitors. However, analogous to the experiences of others, our HTS campaigns failed to identify structural matter that could be optimized into potent inhibitors of NS3 protease. In contrast, cell-based BVDV screening identified the substituted benzimidazole-2-one derivative 1 as an inhibitor of replication, EC50 ≈ 2.2 μM, for which resistance mapped to a single Glu291Gly mutation in the viral polymerase. However, the inhibitory activity observed with 1 could not be detected in a BVDV polymerase biochemical assay, only in a membrane-based assay, severely compromising this strategic approach as an entree into HCV inhibitors which was conducted several years before the advent of cell-based HCV replication systems.27

In the absence of screening leads, we designed and synthesized prospective libraries of potential NS3 inhibitors that took advantage of known peptidic and nonpeptidic mechanismbased serine protease inhibitor chemotypes. This approach was also unsuccessful in identifying lead HCV NS3 inhibitors, although the saccharin derivative 2 emerged from this work as a potent, mechanism-based inhibitor of human mast cell tryptase that is notable for its lack of a basic moiety capable of being recognized by the S1 subsite of this enzyme.28,29 A more productive approach was the result of a collaboration with Axys Pharmaceuticals that sought to identify inhibitors of NS3 by building upon their original observation that serine protease inhibitors based on a bis-benzimidazolemethane motif create a binding site for Zn 2+ when appropriately juxtaposed with the catalytic histidine and serine side chains (His57 and Ser139 in HCV NS3 protease).30 The recruitment of Zn2+ ion to a serine protease/inhibitor complex with appropriately deployed functionality leads to enhanced potency by stabilization of the complex. A screening campaign using this technology identified 3 as a lead inhibitor of NS3/NS4A activity that exhibited a Ki of 200 nM in the presence of Zn2+ ion.31a In the presence of EDTA, which sequesters the Zn2+, the Ki increased to 167 μM providing a measure of the stabilizing effect conferred by metal coordination. However, despite this seemingly promising start, further probing of this inhibitor class failed to identify suitable replacements for the 2-amino-3-phosphonopropanoic acid moiety that might facilitate membrane permeability.31b Rather, polyacidic compounds exemplified by the poly-2-amino-3-phosphonopropanoic acid derivative 4, which displayed Ki values of 27 nM and 1 μM in the presence and absence of Zn2+ ion, respectively, emerged as the only more potent inhibitors. However, these compounds did not offer a clear line of sight to more druglike molecules and, as a consequence, this approach had to be abandoned.

2. DISCOVERY OF THE HCV NS3 PROTEASE INHIBITOR ASUNAPREVIR An extensive series of biochemical studies conducted at both academic and industrial laboratories led to a deeper understanding of the function and activity of the HCV NS3/4A protease that illuminated fundamental aspects associated with substrate specificity, the choreography of viral protein cleavage, B



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and its role in viral homeostasis.23,32 The solid state structure of the NS3/4A complex was solved by two groups simultaneously, revealing the full extent of the challenge that this protein presented as an antiviral drug target, with its extended, shallow, and featureless substrate binding groove that lacked the kind of invaginations more commonly associated with mammalian serine proteases.33 While this architecture provided an explanation for the failure to identify lead inhibitors from screening campaigns, an important discovery to emerge from the biochemical studies was that the HCV NS3/4A protease was subject to product inhibition.34 This observation led to the characterization of the hexapeptide 5 as a potent inhibitor of the NS3 enzyme with a Ki value of 600 nM.34 The extended, polyacidic nature of this peptidic inhibitor, which echoes elements of the discoveries that we had made with the bisbenzimidazolemethane derivatives 3 and 4 described above, provided a significant challenge for optimization into druglike molecules. Structure−activity relationship (SAR) studies were unable to identify noncharged replacements for all of the carboxylic acid Cα side chains with something more amenable to optimization into cell permeable inhibitors. Some progress was made when it was demonstrated that the P2 glutamate could be replaced by a cyclohexylmethyl glycine while a CF2H moiety was identified as an effective isostere of the P1 cysteine thiol, the value of which is exemplified by comparing the activities if the homologous series of inhibitors 6−8.35,36 Further refinement identified the keto acid 9 as a smaller, slow-binding, mechanism-based inhibitor of HCV NS3 protease activity that relied upon the electrophilicity of the keto carbonyl to react at its si-face with the catalytic hydroxyl of Ser139, forming a covalently bound, tetrahedral intermediate that was observed in a cocrystal structure. While the X-ray structure confirmed the isosteric relationship between the CF2H and SH moieties, 9 displayed only modest inhibitory potency in biochemical assays with a Ki of 17 μM and a Ki* of 27 nM.37 Nevertheless, the α-keto acid moiety was incorporated into telaprevir (10) and boceprevir (11), the first NS3 protease inhibitors to be approved by the United States (U.S.) Food and Drug Administration (FDA) in 2011 for use as add-on therapy to pegylated interferon-α (PEG-IFN-α) and ribavirin (12), the dual drug combination that by that time had been developed as the standard of care (SOC) for treating HCV infection.38,39 An important breakthrough in HCV NS3 inhibitor design occurred with the description of ciluprevir (13, BILN-2061), a macrocyclic tripeptidic acid derivative that was profiled as a potent enzyme inhibitor in vitro and that demonstrated rapid antiviral activity following administration to HCV-infected subjects.40,41 Oral dosing of a solution of 13 (200 mg) in

PEG 400 and ethanol twice daily for 2 days to eight HCV GT-1-infected subjects produced a 2−3 log10 IU mL−1 decline in viremia in all patients, with most subjects achieving undetectable levels of HCV RNA by 48 h following the first dose.40a The design of 13 was the result of an extensive medicinal chemistry campaign that originated with the hexapeptide 14, a poorly potent enzyme inhibitor, IC50 = 150 μM, that was optimized by a careful and systematic examination of the effect of structural modifications introduced at the each of the P1, P2, P3, and P4 residues.40,41 The tetrapeptide derivative 15, IC50 = 3.5 μM, incorporated a cyclopropyl glycine at P1 that was associated with a 3-fold increase in potency when compared to a norvaline-containing progenitor. However, it was the large, lipophilic naphthyl moiety attached to C-4 of the P2 proline residue that was the critical SAR breakthrough, providing a 380-fold enhancement in potency when introduced into 14.41 This discovery, combined with additional optimization guided by X-ray and NMR structure analyses, allowed truncation to a tripeptide-based inhibitor that was further preorganized C



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warhead to interact covalently with the catalytic serine hydroxyl.46 The methyl acylsulfonamide 18 was the first representation of the concept to be prepared, a compound that exhibited potency in both the biochemical and cell-based assays that was comparable to the progenitor carboxylic acid 17 (Table 1).45 The next phase of the structure−activity survey focused on the systematic optimization of the methyl substituent of the acylsulfonamide element of 18, studies that were guided by computer models of the drug−inhibitor complex. The result of this survey was a very clear inflection point with the cyclopropyl derivative 21 which expressed potent inhibitory activity in both in vitro assays, with an EC50 value in the GT-1b replicon of 4 nM, a value that met our target criteria of 50 >100 >150 41 >100 23

purely prospective library, and while iminothiazolidinone derivatives were well represented in the literature, they are typically C-5-benzylidine derivatives that are readily prepared by a Knoevenagel condensation between the C-5 unsubstituted parent and an aldehyde.63 The unique C-5-phenyl substituent in 65 would turn out to be of significance for the interesting discoveries that lay ahead as this chemotype was adopted and explored in more depth. The in vitro antiviral properties of 65 are summarized in Table 9 and met the targeted selectivity criteria with respect to BVDV inhibition, with antiviral specificity confirmed by profiling in respiratory syncytial virus (RSV), human rhinovirus (HRV), and HIV-1 cell culture assays where the compound was essentially inactive.62,64 The absence of inhibitory effects in HCV NS3 protease and polymerase biochemical assays suggested that 65 was acting via a novel mode of action in the replicon, a hypothesis confirmed by the generation of resistant replicons which mapped sensitivity to mutations in the amino terminus domain (domain I) of the HCV NS5A phosphoprotein. More specifically, the resistant phenotype was mapped to a Tyr93His mutation or a dual Leu31Val/Gln54Leu combination in NS5A, both of which conferred at least 10-fold reduced inhibitory sensitivity.62,64 At the time that 65 was discovered, the function of NS5A and its role in virus replication was poorly understood, although it had been defined as essential for replication in vivo and there were several lines of evidence suggesting that NS5A was a mediator of the response of infection to the antiviral effects of M



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Figure 7. Structure and replicon EC50 values for 65 and a select set of analogues that define fundamental aspects of the SARs of the screening lead.

Scheme 2. Chemical Degradation Pathways Associated with the Thiazolidinone Derivative 77

peak in the HPLC trace, and where the bioactivity assay identified active material, the UV trace was comparable to baseline, suggestive of small amounts of potently active material. At this point, a preparative experiment was conducted that required the development of practically important enrichment procedures prior to HPLC analysis in order to isolate the active components from the replicon medium.71 This process comprised extracting the complex medium matrix with CH3CN and centrifuging the organic phase which was then frozen at −20 °C to promote separation of the aqueous phase. The unfrozen CH3CN layer was recovered and further refined using a CHCl3−MeOH−H2O partitioning step and then concentrated and the residue fractionated by HPLC, guided by antiviral assessment in both wild-type and Tyr93His resistant GT-1b HCV replicons. This process resulted in the identification of two active components that were isolated in sufficient quantity to allow their detailed characterization by 1 H and 13C NMR spectroscopy. These fractions, which were

since neither 81 nor 82 demonstrated significant antiviral activity in the GT-1b replicon. In an effort to understand the degradation pathway more completely, 77 was incubated in replicon medium at a concentration of 5 μM for 48 h before being extracted with CH3CN and subjected to a HPLC biogram analysis. Biogram methodology, which was developed to analyze natural product extracts, combines semipreparative HPLC separation techniques, automated compound handling and distribution, high throughput biological screening, and informatics tools to develop a graphical report in which bioactivity is overlaid with HPLC retention time.72 The initial analysis surveyed 80 HPLC fractions that were collected and concentrated to allow evaluation of the residue in the GT-1b replicon. The results of this experiment were that HCV inhibitory was found to be associated with fractions eluting from the reversed phase HPLC column after both parent 77 and the known degradation products 81 and 82. However, the active fractions were not associated with a distinct and readily detectable UV N



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designated as peak 4 and peak 6 in the HPLC trace, exhibited potent antiviral activity in the GT-1b replicon with EC50 values of 43 and 0.6 nM, respectively. The 1H NMR spectral data acquired for the compound designated as peak 4 indicated preservation of all of the major structural elements of 77 with the exception of the C-5 methine proton which was absent while the 13C NMR resonance for the C-5 carbon atom was deshielded relative to 77, shifted from δ 51.4 to δ 70.2. Both of the peak 4 and peak 6 compounds were found to be high molecular weight species with the mass determined to be 1139.3379 Da (MH+) by high resolution analysis, a value that is just 2 mass units less than double that of 77. Moreover, the peak 4 and peak 6 components were determined to be structurally related when heating of a sample of the peak 6 material in CD3CN at 55 °C in order to homogenize the amide resonances in the 1H NMR spectrum resulted in its conversion to the peak 4 component. These data and collective observations led to the suggestion that the two components were symmetrical dimers represented generically by 83 that were derived from 77 by dimerization across C-5 of the thiazolidinone ring, with the peak 4 component the thermodynamically more stable diastereomer.71 The mechanistic interpretation of these observations is summarized in Scheme 2 and envisages hydrogen atom abstraction from C-5 of the thiazolidinone ring as the activating step, a process presumed to be mediated by molecular oxygen which in the ground state is a diradical species. The C-5 radical 84 that is generated by this process is stabilized in a captodative fashion by the phenyl ring, sulfur atom, and carbonyl substituents.73 The combination of 84 with molecular oxygen would produce the hydroperoxide 85 that may be subject to reduction in (by) DMSO to afford the corresponding C-5 alcohol which would be anticipated to ringopen to the ketoamide 86 and reclose to give the more stable thiohydantoin 81. In aqueous media, the α-ketoamide 86 would be susceptible to hydrolytic degradation to provide the thiourea 82 and, presumably, the α-ketoacid 87, which was not isolated. The stability of the radical species 84 presumably allows adequate time for radical combination in assay media to give the dimeric species 83, possibly facilitated by aggregation of the parent compound 77 under aqueous conditions.74 Evidence that the C-5 hydrogen atoms of 77 and 79 are capable of participating in radical-based chemistry was obtained by heating these compounds with Mn(OAc)3 and Cu(OAc)2 in AcOH at 80 °C, reaction conditions that have been shown to promote the oxidation of radicals to cations that can then be trapped by the solvent.75 In this particular case, the acetates represented by 88 were produced which upon methanolysis afforded the substituted thiohydantoin derivative 81 and its proline homologue (structure not shown).70b The potency associated with the two putative diastereomeric dimers represented by 83 and their sensitivity to the Tyr93His mutation in HCV NS5A suggested that these compounds retained the antiviral phenotype of the screening hit 65 and its analogues 77 and 79, a profile that encouraged continued interest in the chemotype. On the basis of the observations that the antiviral activity of 65 was highly sensitive to small structural changes to the alanine moiety while variation of the thiazolidinone ring N-substituents was associated with more nebulous SARs, it was hypothesized that the NS5A-inhibiting pharmacophore in 83 was defined by the embedded bibenzyl element and that the remainder of the thiazolidinone ring and its substituents were superfluous for antiviral activity.70b This scenario envisioned the thiazolidinone ring acting as a scaffolding

element to convene a symmetrical molecule in the cell culture assay, a pharmacophore concept that was readily and straightforwardly tested by preparing 89 which incorporates the proline cap moiety. This compound exhibited an EC50 value of 30 nM in the GT-1b replicon, but its synthetic precursor, the conformationally less mobile stilbene 90, was a considerably more interesting and compelling inhibitor based on its picomolar antiviral potency, EC50 = 0.086 nM in the GT-1b replicon, an advantage of more than 300-fold over 89.70b,71 The antiviral activity of 90 remained sensitive to the NS5A Tyr93His mutation with the EC50 value shifting to 4 μM in this replicon, and the compound was inactive in the BVDV counterscreen. Collectively, these data indicated that we had finessed an important NS5A-inhibiting pharmacophore expressed in the dimeric species 83 that was cryptically expressed by 79 in cell culture. At this juncture of the project, the dimeric species represented by 89 and 90 became the primary focus of further study. While the high level of antiviral potency associated with stilbene 90 was attractive, this compound presented three problems that were considered to be of significance in further optimization. From the perspective of chemical structure, the stilbene olefin was subject to configurational instability via cis−trans isomerism, observed in studies with analogues of 90, while the embedded aniline moiety was of concern because of the potential for release in vivo by protease- or esterasemediated cleavage of the proline amide bond.76,77 Anilines are potentially mutagenic and genotoxic after metabolic activation, arousing considerable anxiety around this potential safety issue.77 While it was anticipated that these issues could be addressed by a series of structure−activity studies, the discovery of 89 and 90 coincided with the development of a GT-1a replicon where both compounds were determined to be essentially inactive, with EC50 values of >10 μM. This was considered to be a significant problem, since a compound with selectivity for HCV GT-1b would be of limited value as a therapeutic agent based on demographic analyses that revealed the worldwide prevalence and importance of GT-1a infections.78 Nevertheless, against the backdrop of the unique mechanism of the chemotype, a broad-based approach to structural exploration was implemented that focused on further defining the NS5Ainhibiting pharmacophore while, where possible, integrating O



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structural modifications that were designed to address the inherent chemical liabilities described above. The oral delivery of the symmetrical chemotype was also of some concern given the relatively high molecular weight (640), cLogP (6.2) and rotatable bond count (10), but this was allayed to some extent by studies with the cyclopropylmethyl amide 91, a potent HCV GT-1b inhibitor, which displayed 64% oral bioavailability in the rat. However, it was the GT-1a inhibition that would ultimately prove to be the most challenging problem to solve and the endeavor that ultimately succeeded in resolving this issue required considerable experimentation and persistence.

modification (Figure 8). The amide element of the α-keto amide 97 was designed to preserve the H-bond accepting function of the isoquinoline ring N atom of prototype compound 95 while introducing the potential for both conformational relaxation and, by manipulation of the keto moiety, the incorporation of substituents that explored new vectors.84 The addition of mandelic acid caps to the core pyrrolidine rings afforded the diastereomeric alcohols 98 and 99 that exhibited discordant antiviral activity, with the (R)-isomer 98 190-fold more potent than the (S)-analogue 99 toward the GT-1b replicon, although the GT-1a replicon was far less discriminatory (Figure 8). More interestingly, the antiviral activities associated with the methyl carbinols 100 and 101 were similarly discordant but potency was resolved in a complementary fashion with respect to the absolute configuration since the (S)-isomer 101 was more potent than the (R)-isomer 100, particularly toward the GT-1a replicon where the values were titrated.84 While this survey of the cap elements was being conducted, structural changes to the core had continued to be explored in parallel, which facilitated the hybridization of several of the key discoveries and concepts with SAR developments that emerged from modifications to the cap elements.84 Replacement of the

While a wide range of derivatives and analogues of 90 that explored structural variation of the proline, phenylacetic acid, and olefin moieties were prepared and profiled for antiviral activity, none provided a clear seam of coherent and tractable SARs with respect to GT-1a inhibition.79,80 Representative examples of molecules with measurable GT-1a inhibition are provided by the oxazole 92, which was unique within a series of olefin replacements, while the substituted proline analogue 93 and the ortho-substituted benzoic acid 94 were the only compounds within a series of stilbene derivatives to demonstrate detectable GT-1a inhibition.81,82 The critical breakthrough came with the synthesis and evaluation of the isoquinoline-1-carboxamide 95 that, although demonstrating only modest antiviral activity in replicons, proved to be a bona fide lead inhibitor of GT-1a HCV replication.83 For example, the 3,5-disubstituted homologue 96 was 20-fold more potent in the GT-1a replicon, although this was accompanied by a more modest increase in GT-1b inhibitory activity. While additional studies that explored the substitution pattern of the isoquinoline heterocycle in more detail expanded the SARs associated with this moiety, it was a more radical structural surgery in which the isoquinoline ring was deconstructed to give the α-keto amide 97 that proved to be the most informative P



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Figure 8. Antiviral activity of the series of stilbene-based proline amide HCV NS5A RCIs 97−101.

potential explanation for the weaker GT-1a inhibition observed with both 108 and 109 when compared to their anilide analogues. In order to restore the topological relationship between the cap elements and the core, additional structural adjustments were given consideration. Recognition that the linearity associated with the 1,2-diphenyl alkyne core of 103 and 105 could be reproduced by a biphenyl system led to this scaffolding element being explored, but its deployment required a compensatory structural adjustment based on the appreciation that this modification would shorten the spatial relationship between the two cap elements. This topology problem was addressed by dismantling the fused ring system of the heterocycle in a process of deannelation, which identified an imidazole−phenyl−phenyl−imidazole motif as a potential core on which to deploy the cap elements, a design process delineated in Figure 9.

olefin moiety of the stilbene 90 with an alkyne was designed to address the cis−trans isomerism problem, and although the antiviral profile of 102 was poorly balanced with potent GT-1b but low GT-1a inhibition, substitution of the phenylacetic acid cap was restorative (Table 10). The (R)-dimethylamino Table 10. SARs Associated with 1,2-Diphenylethyne-Based HCV NS5A RCIs 102−107

derivative 103 demonstrated potent and more balanced antiviral properties, while the (S)-isomer 104 was considerably less impressive as an inhibitor of GT-1a replication. The (R)-NHCO2CH3 analogue 105 performed similarly to 103, leading to the suggestion that a H-bond donor in the cap element was an important pharmacophoric element, particularly for GT-1a inhibition, and fulfilled in 103 by the protonated form of the dimethylamine functionality. This hypothesis was supported by the lesser antiviral activity associated with the morpholine derivative 106 compared to what is presumed to be the (R)-Nmethylpiperazine 107 where the potency differences were attributed to their likely divergent protonation states at physiological pH.84 The potent antiviral activity associated with 103 and 105 provided a structural basis from which to explore functional alternatives to the anilide moiety that were designed to abrogate the potential for the in vivo release of an aniline derivative.85 Both the NH and carbonyl oxygen atom of the anilide moiety were considered to be of importance for antiviral activity based on the extant SARs, and it was recognized that both of these structural elements could be preserved in a benzimidazole heterocycle. However, this motif alters the topology of the vectors between the core and the terminal cap elements, providing a

Figure 9. Structural evolution of the 1,2-diphenyl alkyne core of NS5A inhibitors to that of an imidazole−4,4′-biphenyl−imidazole motif.

This concept was reduced to practice in the context of the phenylglycine derivatives 110 and 111, both of which expressed potent and balanced antiviral activity in the GT-1a and GT-1b replicons.85 However, these compounds could not be progressed further because of an absence of plasma and liver exposure following oral administration to rats. The approach adopted to address this deficiency focused on assessing the effect of reducing the molecular weight of the molecule by Q



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abridging the aromatic rings of the phenylglycine moieties to simpler and more sp3 carbon atom-rich isopropyl substituents. Implementation of this concept led to the synthesis of the bisD-valine derivative 112, but this compound suffered from a dramatic 44 000-fold decline in potency toward the GT-1a virus, although GT-1b inhibition was affected to a much lesser extent (29-fold). Further probing of this SAR observation revealed that manipulation of just a single phenylglycine to smaller structural elements resulted in significant declines in GT-1a inhibition, with values ranging from 400 to 11 000-fold.85 Although this result was somewhat perplexing and markedly disappointing, the two representative compounds 113 and 114 were nevertheless advanced into PK studies in an effort to substantiate the validity of the proposed strategy. Both compounds exhibited poor oral bioavailability in the rat but were somewhat improved in the mouse and considerably better in the dog, where the fraction of the dose reaching the systemic circulation was 60% for 113 and 45% for 114. Despite the poor oral bioavailability of 114 in rats (6.8%), an oral dose of 5 mg/kg delivered liver levels of 116 nM measured at 24 h postdose, with 26-fold lower levels in plasma at this time point. Collectively, these data were viewed from the positive perspective of providing support for the strategy of reducing molecular weight in order to improve oral exposure. With these results in hand, further attention was focused on modifying the cap moieties where the effort was directed toward identifying compounds that combined high antiviral potency with diminutive C-α substituents. While considerable experimentation was devoted to the exploration of analogues and homologues of 113 and 114, it was the synthesis and evaluation of the unsymmetrical derivative 115, in which one of the cap elements of 114 has been exchanged for an N-methoxycarbonyl-L-alanine moiety, that provided the seminal and unanticipated insight. Compound 115 demonstrated subnamomolar EC50 values in the HCV genotype replicons, and potent antiviral activity was preserved in the fully symmetrical homologue 116 that incorporated the natural amino acid at each cap. Homologation of L-Ala to L-Val afforded 117 (BMS-790052), the molecule that would eventually be identified as daclatasvir, which is 5- to 10-fold more potent than the alanine progenitor.86 The SARs surrounding the amino acid nitrogen and C-α substituents were quickly and systematically explored, guided, in part, by established insights, and the most promising inhibitors were profiled in PK studies. This initiative identified 117 and the unsymmetrical compound 118 as the leading contenders for nomination to advance into IND-enabling toxicology studies, and the antiviral properties and PK profiles of these two compounds are compiled in Tables 11 and 12, respectively. The antiviral data reveal that both compounds exhibit potent activity toward all of the genotype replicons and hybrid replicons tested with comparable EC50 values, with the exception of GT-3a virus where 118 is an order of magnitude more potent than 117. The PK profiles show some differences, with the plasma exposure (AUC and concentration at 24 h) of 118 lower than that for 117, although the liver levels of both compounds in the rat were high. The unsymmetrical 118 exhibited a more hepatotropic profile than 117 based on the ratio of the 24 h drug levels, while the oral bioavailability of 117 was higher across the species than 118. In an effort to further differentiate the compounds, both were assessed in a 4-day toxicological study conducted in mice where both were well tolerated, but the analysis of plasma, liver, and heart levels revealed a 2-fold

Table 11. Antiviral and Preclinical PK Profile of 117 Antiviral Activity EC50 (pM)

replicon

GT-1a 50 GT-1b 9 GT-2a (JFH) 71 GT-2a (JFH)a 103 GT-3aa 146 GT-4aa 12 GT-5aa 33 Pharmacokinetic Profile after Oral Dosingb species (dose, mpk) parameter

rat (5)

dog (2.3)

cynomolgus monkey (2.8)

AUC (μM·h) plasma concn (nM) at 24 h liver concn (nM) at 24 h F (%)

4.8 18 103 50

11 26 ND 108

1.93 6.5 ND 38

a

Data from hybrid replicons. bND = not determined.

accumulation of 118 at all doses over the 4 days of study while the tissue levels of 117 were similar on days 1 and 4, an observation that led to the selection of 117 for advancement. While addressing the GT-1a inhibition had proven to be a significant challenge, the solution that was ultimately devised encompassed all of the genotypes that were available for testing in replicons or hybrid replicons at the time of compound nomination. As 117 was advancing toward clinical studies, an infectious GT-2a virus was developed that was found to be sensitive to the compound with an EC50 value of 28 pM, potency comparable to that recorded in the GT-2a hybrid replicon.87 This result provided additional confidence in the potential of a R



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cell lysates before the mixture was passed over a streptavidin column, separated by SDS−polyacrylamide gel electrophoresis and then probed with an antibody specific for HCV NS5A. However, this protocol failed to isolate significant amounts of viral protein and the experiment was redesigned such that 119 was incubated in the GT-1b replicon for 18 h before the cells were lysed, passed over the streptavidin column, separated by SDS−polyacrylamide gel electrophoresis, and then probed with antibodies. This experiment pulled down the HCV NS5A protein but neither NS3 nor NS5B could be detected by antibody probes, while the inactive D-proline-based diastereomer failed to pull down any viral proteins. More recent studies have demonstrated the binding of 117 and structurally related inhibitors to the NS5A protein using complementary experimental techniques.88

Table 12. Antiviral and Preclinical PK Profile of 118 Antiviral Activity replicon

EC50 (pM)

GT-1a 36 GT-1b 12 GT-2a (JFH) 20 GT-3aa 8 GT-4aa 14 GT-5aa 21 Pharmacokinetic Profile after Oral Dosingb species (dose, mpk)

AUC (μM·h) plasma concn (nM) at 24 h liver concn (nM) at 24 h F (%)

rat (5)

dog (3.5)

cynomolgus monkey (3.0)

0.165 BD 178 3.6

1.2 9.0 ND 66

0.497 4.2 ND 20.9

a

Data from hybrid replicons. bBD = below the level of detection. ND = not determined.

An X-ray crystal structure of a portion of the amino terminus (domain 1) of HCV NS5A was published 3 years after the discovery of the stilbene inhibitor chemotype and revealed a dimeric species in which the geometry of the protein interface created a U-shaped surface rich in basic amino acids, suggested to be an RNA binding domain.89 This solid state structure complemented the palindromic topology that we had discovered with 83, 89, and 90 and which is retained in 117, and the location of resistance mutations suggested a binding site located between the NS5A protein and the membrane, with the compounds spanning the dimer interface to engage both protein monomers.89 Subsequent X-ray structures of NS5A have confirmed the preferred shape of the protein but identified alternate dimer interfaces that anticipate an oligomeric species. This concept provides a potential explanation for calculations that suggest that at the EC50 concentration in replicons, the molecular ratio of NS5A to 117 in cells is approximately 47 000 which translates to 1 molecule of 117 for 23 500 dimers of NS5A.90−92 In addition, the RNA binding properties of NS5A have been confirmed and the dimeric association of the protein has been shown to be important for viral replication in replicons.88,93,94 Phase 1 clinical trials with 117 began with a randomized, double-blind SAD study conducted in NHVs which revealed dose-related increases in plasma exposure of the drug which was administered over the dose range of 1−200 mg as an oral solution.62 The drug was well tolerated, with concentrations in plasma measured at 24 h postdose and beyond above the protein binding-adjusted EC90 value determined in the less sensitive GT-1a replicon at all doses tested, reflecting a long plasma half-life, data that collectively predicted the potential for once daily (q.d.) dosing. On the basis of these results, doses of 1, 10, and 100 mg were selected for proof-of-concept studies which were conducted in HCV GT-1-infected subjects. Plasma exposure values of the drug in these individuals were comparable to those in the NHVs given the same dose, with concentrations of 117 at 24 h determined to be more than 10-fold above the GT-1a protein binding-adjusted EC90 value.

first-in-class molecule that targeted a protein with no known enzymatic activity and imprecisely and poorly defined functions in the viral lifecycle. That the chemotype represented by 117 bound to the NS5A protein was established in studies with the biotinylated probe 119 which was synthesized and evaluated in replicons along with the analogue in which both prolines were of the unnatural D-configuration. The chemical probe 119 inhibited replication of the GT-1b replicon with an EC50 value of 33 nM, antiviral activity that was sensitive to the Tyr93His mutation, EC50 > 10 μM, while the analogue with the unnatural D-proline moieties was inactive, EC50 > 10 μM. The initial pulldown experiments that were conducted with 119 followed a design in which the probe was incubated with GT-1b replicon S



Perspective

Figure 10. Mean maximal reductions in viral load following oral administration of 117 to HCV-infected subjects.

through the lens of physicochemical properties. This compound was active in a NS5B enzyme biochemical assay, IC50 = 120 nM, and was of considerably lower molecular weight with a reduced burden of sp2 carbon atom count. Conformational constraint was explored by linking the indole N-substituent to the ortho position of the 2-phenyl ring, a concept evaluated initially with the simple alkyl chain incorporated in 123, which resulted in a modest enhancement of antiviral potency. Both activity and physicochemical properties were further improved by the introduction of a polar amide moiety into the bridging element which afforded 124 in the initial iteration, a functionality that simultaneously provided an opportunity to explore new vectors of structural modification. The in vitro criteria set for compound progression for this series were identical to those established for NS3 and NS5A inhibitors with target EC50 values of 50 μM), and higher solubility (34 μg/mL as amorphous material). The final steps toward the identification of beclabuvir (142, BMS-791325) explored the introduction of a bridging element in the piperazine ring which identified differences in potency and, most notably, PK properties based on its relative location with respect to the core indole heterocycle. Installation of an ethylene bridge distal to the indole core, as represented by the racemic mixture 140, was associated with lower antiviral potency compared to its racemic isomer 141. The PK profiles of 140 and 141 in the rat differed more markedly, with iv clearance of the former measured as 20.6 mg mL−1 kg−1 while liver levels were 0.7 μM 4 h after a 10 mg/kg dose. In contrast, the iv clearance for 141 was much lower at 7.2 mg mL−1 kg−1 and a 10 mg/kg oral dose delivered liver levels at 4 h of 55.4 μM. Resolution of 141 identified 142 as the eutomer that displayed EC50 values of 3 and 6 nM for GT-1a and GT-1b inhibition, respectively, while the distomer displayed EC50 values of 208 and 200 nM toward GT-1a and GT-1b replicons, respectively. The in vitro profiling data for 142 that are compiled in Table 13 reveal a compound with potent inhibitory activity toward HCV NS5B enzymes and replicons derived from GT-1, GT-3, GT-4, GT-5, and GT-6 while representative GT-2 enzymes and replicons are less sensitive.101,104−106 Mechanistic studies indicated that 142 is a time-dependent, noncompetitive inhibitor of the polymerase that interferes with initiation of replication, with resistance mapping to a Pro495Leu mutation located in the thumb domain and a Leu30Ser change located in the Δ1 loop of the fingers domain.106,107 A two-step binding mechanism was determined from biochemical and biophysical experiments, with the Pro495Leu mutation modulating the kinetics of the second binding step such that the dissociation rate of the compound is faster than for wild type enzyme.

Table 13. In Vitro Profiling Data for 142 HCV NS5B enzyme assay

IC50 (nM)

GT-1a GT-1b GT-2a JFH-1

3.3 4.2 165

GT-2b GT-3a GT-4a

164 1.8 19.9

GT-5a GT-6a

4.8 61.6

assay

result

HCV GT-1a replicon HCV GT-1b replicon HCV GT-2a JFH-1 replicon 40% human serum effect BVDV replicon poliovirus, rhinovirus, coronavirus, coxsackie virus, HIV-1, influenza virus cytotoxicity (7 cell lines) mammalian polymerases α, β, γ

EC50 = 3 nM EC50 = 6 nM EC50 = 87 nM 14 μM EC50 > 14 μM

EC50 = 14−30 μM IC50 > 25 μM

In contrast, 142 bound several-fold faster to the Leu30Ser resistant enzyme, but the forward and reverse processes to form the final complex were found to be equivalent.107 Additional preclinical profiling established the aqueous solubility of 142 to be >1.6 mg/mL at pH = 2.2 and pH = 7.8, but this was reduced considerably at pH 6.5 (∼0.02 mg/mL), reflecting the amphoteric nature of the molecule which has pKa values of 4.6 and 6.8. Protein binding was found to be 98.8% for human and 97.8−98.7% for the other species tested, and while permeability across an artificial membrane was high, efflux was observed in Caco-2 cells, an indication that 142 was a P-gp substrate although it was not an inhibitor. Demethylation of the sulfamide moiety of 142 was observed in LM, and the compound was determined to be a substrate of CYP 3A4, forming 143 (BMS-794712), a compound with antiviral activity comparable to 142 but which exhibited poor oral bioavailability in preclinical species thereby precluding consideration of this metabolite as a potential clinical candidate.108 In phase 1 clinical trials, single doses of 100, 300, 600, and 900 mg of 142 were administered to IFN-α-naive and -experienced, HCV GT-1-infected subjects following a double blind, placebo-controlled study design that was conducted after a standard SAD evaluation in NHVs to assess PK and establish tolerability.109 Plasma concentrations of 143 increased in a dose-related fashion and were maximal between 2 and 4 h postdose, with plasma levels at 24 h exceeding the protein-binding-adjusted EC90 value in the GT-1 replicon. V



Perspective

following several days of dosing as monotherapy and were followed by combination studies with PEG-IFN-α and 12. The NS3 protease inhibitor 13 was the first HCV DAA to demonstrate a clinical effect on viral load following oral dosing, results that were reported in 2002 (Figure 12). NM-283 (144), administered as its C-3′ valine ester prodrug valopicitabine (145), and nesbuvir (HCV-796, 146) established proof-of-concept for active site nucleoside analogue and allosteric NS5B RdRp inhibitors in 2004 and 2006, respectively, while daclatasvir (117) was the pioneering NS5A inhibitor, with efficacy in HCV-infected subjects disclosed in 2008 (Figure 12). While 13 was abandoned without progressing further because of preclinical cardiotoxicity, 145 and 146 were advanced into phase 2 clinical trials where they were evaluated in conjunction with PEG IFN-α and 12.111 However, while both compounds augmented the effects of PEG IFN-α and 12 on viral load, both were abandoned because of toxicity issues (gastrointestinal toxicity for 145 and hepatotoxicity for 146) that were observed several weeks into a phase 2 combination study. Despite their shortcomings, these compounds provided important insights into the development of HCV DAAs and helped to define a path to approval for the first small molecules 10 and 11 which were licensed in the spring of 2011 as add-on therapy to PEG IFN-α and 12.112 The addition of either of these drugs to a backbone of PEG IFN-α and 12 improved SVR response rates significantly, but these regimens still required 24−48 weeks of therapy; both DAAs were administered on a t.i.d. schedule, and both were associated with additional side effects that added to the considerable burden imposed by the SOC backbone.113−115 Ultimately, the marketing of both 10 and 11 in the U.S. was abandoned by their sponsors late in 2014 due to the combination of lower efficacy in a real world setting than had been observed in clinical trials and a poor side effect profile (10 acquired a black box warning for problems associated with

The exposure profile of the metabolite 143 was similar to 142, but the plasma AUC was ∼22% of that of the parent drug. In general, 142 was well tolerated in these studies with no serious adverse events or discontinuations noted and plasma viral load declined by 1.30−2.78 log10 IU/mL, with the higher doses associated with improved efficacy (Figure 11). These results were sufficient to encourage further clinical study of the compound (vide infra).

5. CLINICAL TRIALS WITH DIRECT-ACTING HCV INHIBITORS AND THE DEVELOPMENT OF HIGHLY EFFECTIVE AND CURATIVE COMBINATION THERAPIES The initial clinical trials with direct-acting HCV inhibitors typically established efficacy after a single dose of drug or W



Perspective

Figure 11. Mean maximal reductions in viral load following oral administration of 142 to HCV-infected subjects.

Figure 12. Historical timelines for key events in the development of HCV DAAs.

severe rash), a decision that reflected a response to the rapidly changing availability of HCV DAA therapeutic agents with improved antiviral and side effect profiles.116 What, however, was not abundantly clear at the time that these early clinical trials were conducted was whether PEG IFN-α and/or 12 would remain as an important component of SOC and whether shorter durations of therapy would be effective at producing a SVR.117 The anticipation of the need for combinations of DAAs for effective HCV therapy was confirmed with early clinical trials, notably in studies with 10 where resistance emerged within 2 weeks of starting treatment.118 Despite its markedly increased potency and efficacy, resistance also proved to be problematic with 117 when viral rebound was observed in a 14-day monotherapy study, with GT-1a virus evolving resistance more

rapidly than GT-1b virus.110,119 This is a function of the underlying viral genetic constitution where only a single base pair change is typically required for GT-1a virus to encode for an alternative amino acid in contrast to the two base pair changes that are necessary for GT-1b virus.120 This result clearly indicated a need for combination therapy, and two phase 3 clinical X



Perspective

potential of an interferon-free DAA regimen.124 In contrast, the study of the combination of 64 and 117 was specifically designed to evaluate the full potential of the two drugs in a cohort of 21 GT-1-infected null responders, a difficult-to-treat patient population who had failed to respond to therapy with PEG IFN-α and 12.123 Most of the patients (19 of 21, 90%) selected for the study of the combination of 64 and 117 were determined to have an unfavorable IL28B CT or TT genotype that had been established as being predictive of a lack of response to PEG IFN-α rather than the CC genotype that predicts sensitvity.125 The 21 patients were randomized into two groups, designated A and B, with A receiving 64 (600 mg q.d.) and 117 (60 mg b.i.d.) while group B received the two DAAs administered on the same dosing regimen in combination with PEG IFN-α and 12, with both arms scheduled to examine the outcome of 24 weeks of combination therapy. The cohort receiving the quadruple therapeutic regimen achieved 100% SVR12, and 90% achieved SVR24, with one patient experiencing detectable but not quantifiable HCV RNA at week 24 postdosing, although this subject was determined to have undetectable viral RNA 35 days later (Figure 13a).123 In the dual DAA arm, 36% (4 of 11) of the subjects achieved

trials of 117 with PEG IFN-α and 12 were embarked upon where it was demonstrated that the therapeutic regimen could be shortened to 24 weeks of treatment with equivalent or improved efficacy as measured by SVR24 and without the burden of additional side effects.121 In a similar clinical protocol conducted in treatment-naive GT-1 and GT-4 infected subjects in which 64 (200 mg b.i.d.) was combined with PEG IFN-α and 12, 24 weeks of therapy were associated with SVR24 rates of 64% and 89% compared to 44% and 43% in the placebo groups, respectively.122 However, compounds 64 and 117 had entered clinical trials in a contemporaneous fashion with safety and efficacy data obtained in parallel, a circumstance that presented a unique opportunity to conduct a small phase 2 clinical trial of the dual combination therapy.123 This study was not the first clinical trial of a combination of DAAs, since the NS3 inhibitor danoprevir (147) and the nucleoside-based NS5B inhibitor mericitabine (148), a bis-valine ester prodrug, had been administered to noncirrhotic subjects chronically infected with HCV GT-1.124 However, this clinical study relied upon a protocol of 13 days of dosing with the dual DAA combination followed by a day of drug washout and then 46 weeks of therapy with PEG IFN-α and 12 and was thus not designed to assess the full

Figure 13. SVR12 and SVR24 rates following 24 weeks of therapy with drug regimens containing 64 and 117. (a) Percentage of patients achieving undetectable HCV RNA in response to the combination of 64, 117, PEG IFN-α, and 12 administered to GT-1-infected subjects for 24 weeks. (b) Percentage of patients achieving undetectable HCV RNA in response to the dual combination of the HCV DAAs 64 and 117 administered to GT-1-infected subjects for 24 weeks. Y



Perspective

moment in the annals of HCV therapy” because the study demonstrated for the first time that a chronic HCV infection could be cured in the absence of interferon therapy.127 This result set the stage for what would ultimately become an extensive series of clinical trials conducted by multiple pharmaceutical houses examining combinations of DAAs acting by orthogonal mechanisms. These studies defined curative regimens and durations for a broad range of HCV-infected populations, including those with and without liver cirrhosis, those co-infected with HIV-1, those infected with genotypes beyond GT-1, and individuals experiencing reinfection after a liver transplant.128 An expansion of the study of the dual combination of 64 and 117 confirmed the results observed with the sentinel cohort in GT-1b-infected subjects and set the stage for development of these two drugs in Japan where this viral subtype is predominant.129,130 The Pharmaceuticals and Medical Devices Agency (PMDA) granted approval for marketing the combination of 64 and 117 in Japan as Daklinza and Sunvepra, respectively, on July 4, 2014, the first HCV DAA combination to be approved worldwide.131 The two-drug combination was studied in two phase 3 clinical trials, one conducted in Japan and the second conducted worldwide, that demonstrated efficacy in a broad range of patient populations, including those naive and nonresponsive to PEG IFNα and 12 therapy and those either intolerant of or ineligible for this regimen, with a subset of cirrhotic patients included in each trial.130 When administered at doses of 100 mg b.i.d. of 64 and 60 mg q.d. of 117 for 24 weeks, the SVR12 rates in the Japanese study were 81% in nonresponders and 87% in intolerant/ineligible subjects while in the global study SVR12 rates in these patient populations were 90% and 82%, respectively. In these trials, it was determined that a Tyr93His polymorphism in the NS5A gene was predictive of the clinical outcome, with the probability of achieving SVR declining from 95% to 45% if this mutation preexisted at baseline. In order to broaden the scope of therapy to include the more difficult to treat GT-1a virus, a fixed dose combination of 64 (200 mg), 117 (30 mg), and 142 (75 mg) was developed to be administered clinically as a b.i.d. regimen to treat GT-1 HCV infection in treatment-naive and treatment-experienced subjects that encompasses those with or without liver cirrhosis.132 In these studies, SVR12 rates following 12 weeks of therapy were 87−93% dependent upon patient background and the inclusion or exclusion of 12 as part of the therapeutic regimen, with cure rates in GT-1a-infected subjects somewhat lower than for GT-1b.132,133 Regimens that include 117 have been used to successfully treat recurrent HCV infection following liver transplantation, with the first example of an SVR reported for the drug in combination with PEG-IFN-α and 12 therapy for 24 weeks.134 However, it was a study of 117 in combination with the nucleoside-based HCV NS5B inhibitor sofosbuvir (149), both administered orally daily for 24 weeks, that provided the first example of achieving a SVR in the post-liver-transplant setting using an interferon-free regimen.134b The patient, who was infected with GT-1b HCV and had plasma RNA levels of 12 × 106 IU/mL, had been refractory to therapy with the combination of PEG-IFN-α and 12, but the dual combination of 117 and 149 resulted in viral RNA being undetectable in plasma 9 months after completing therapy. Combinations of 117 with the NS3 protease inhibitors 64 or simeprevir (150), the latter in conjunction with 12, have also been found to cure infection following liver transplantation.134

SVR12 and all four continued to be free of detectable virus when assessed at 24 weeks after the final dose of the two drugs (Figure 13b). This cohort comprised nine subjects infected with GT-1a virus and two infected with GT-1b of which both GT-1b- and two of the GT-1a-infected subjects were cured of infection. The nine treatment failures were determined to have developed resistance mutations in both the NS3 and NS5A genes, with mutations in the latter including Gln30Arg, Leu31Met or Leu31Val, and Tyr93Cys or Tyr93Asn. Resistance variants in the NS3 protease gene included Arg155Lys and Glu168Ala/ Glu/Thr/Val/Tyr, and the single patient in group A who suffered viral relapse in the 12 weeks after drug dosing ended was determined to have the NS3 protease resistance variant Arg155Lys preexisting at baseline and present at the time of viral relapse, while the NS5A resistance variant Gln30Glu was observed only at relapse.123a The potential for the emergence of resistance in response to HCV DAA therapy has been elegantly explained by a series of studies that have analyzed viral replication rates and half-life in vivo in conjunction with estimates of the rate at which errors are incorporated by the viral polymerase.126 It has been estimated that in an HCV-infected subject, 1012 virions are produced each day, a replication rate that is 10- to 100-fold higher than that determined for HIV-1, with polymerase error rates estimated at 0.1 per genome synthesized (Table 14). This analysis predicts that while 91% of Table 14. Estimated HCV Replication Rates and Predicted Rates of the Production of Mutated Viruses HIV1 and HCV Replication Rates: Virions Produced per Day HIV-1 1010 to 1011 HCV 1012 Daily Production of HCV Mutant Viruses Based on an Error Rate of 0.1 per RNA Synthesized no. mutations

%

virions/day

0 1 2 3

91 8.7 0.42 0.013

910 × 109 87 × 109 4.2 × 109 0.13 × 109

virions will be faithfully reproduced, 9% of the virions produced each day will harbor at least one mutation, with the breakdown anticipating that 87 billion particles will be assembled carrying a single mutation, while 4.2 billion viruses will incorporate two mutations and 130 million virions will be produced with three base pair changes. Since the HCV genomic RNA comprises just over 10 000 bases, the consequence of this analysis is that all single and dual mutations pre-exist and DAA monotherapy will simply and rapidly select for resistant virus.

Despite the modest clinical success with the dual combination study with 64 and 117, this result was hailed as “a watershed Z



Perspective

the treatment of GT-1b infection. The treatment of GT-1a infection requires the addition of 12 for 12 weeks of therapy in the absence of cirrhosis which extends to 24 weeks for those with this clinical background. A synopsis of the clinical efficacy of Viekira Pak is compiled in Table 16, with cure rates of Table 16. Synopsis of Clinical Results with Viekira Pak, a Fixed Dose Combination of 152, 153, and 154 Co-Packaged with 155, Administered with or without 12 Depending on the Clinical Background of the Patient Population The combination of 117 and 149 has been shown to be an effective therapy for the treatment of naive and experienced subjects infected with GT-1 HCV and those co-infected with HIV-1.135,136 On July 24, 2015, the FDA licensed 117 for the treatment of HCV GT-3 infection when used in combination with 149 based on results from the ALLY-3 phase 3 clinical trial, with expansion to include GT-1 infections announced on February 9, 2016. In a phase II clinical trial conducted in GT-1b-infected subjects who were naive to therapy or who had experienced a null response, the combination of 117 and 150 with and without 12 for 12 or 24 weeks was associated with SVR12 rates ranging from 69.6% to 95%.137 The first fixed dose combination of DAAs to be licensed by the U.S. FDA was a regimen containing 149 (400 mg) and the NS5A RCI ledipasvir (151, 90 mg) that was approved on October 10, 2014, based on a series of phase 3 clinical trials conducted in HCV-infected subjects with a range of clinical backgrounds.138 This combination, which is well-tolerated and offers the convenience of once daily dosing, is marketed as Harvoni and is associated with cure rates in excess of 90% in the majority of patient populations, as summarized in Table 15.138,139

patient population treatment-naive

phase

GT, status

3

GT-1a, noncirrhotic GT-1b, noncirrhotic GT-1b, noncirrhotic GT-1a, cirrhotic GT-1a, cirrhotic GT-1b, cirrhotic

3 treatment-experienced

3

treatment-naive and -experienced

3

therapeutic regimen and duration

SVR12 (%)

12 weeks with 12

96

12 weeks

100

12 weeks

100

12 weeks with 12

89

24 weeks with 12

95

12 weeks with 12

99

Table 15. Synopsis of the Clinical Results Observed with Harvoni, a Fixed Dose Combination of 149 and 151, in GT-1 Infected Patients patient population

phase

status

naive

3

naive naive experienced experienced HIV co-infected

3 3 3 3 2

noncirrhotic

2015 Philip S. Portoghese Medicinal Chemistry Lectureship. Curing

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