Discovery of Daclatasvir, a Pan-Genotypic Hepatitis C Virus NS5A

Drug Annotation pubs.acs.org/jmc

Discovery of Daclatasvir, a Pan-Genotypic Hepatitis C Virus NS5A Replication Complex Inhibitor with Potent Clinical Effect Makonen Belema* and Nicholas A. Meanwell* Department of Discovery Chemistry, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut 06492, United States

ABSTRACT: The discovery and development of the first-in-class hepatitis C virus (HCV) NS5A replication complex inhibitor daclatasvir (6) provides a compelling example of the power of phenotypic screening to identify leads engaging novel targets in mechanistically unique ways. HCV NS5A replication complex inhibitors are pan-genotypic in spectrum, and this mechanistic class provides the most potent HCV inhibitors in vitro that have been described to date. Clinical trials with 6 demonstrated a potent effect on reducing plasma viral load and, in combination with mechanistically orthogonal HCV inhibitors, established the ability to cure even the most difficult infections without the need for immune stimulation. In this Drug Annotation, we describe the discovery of the original high-throughput screening lead 7 and the chemical conundrum and challenges resolved in optimizing to 6 as a clinical candidate and finally we summarize the results of select clinical studies.

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INTRODUCTION Although contemporary drug discovery practices have evolved to focus heavily on the design and implementation of targetbased biochemical screens to discover and optimize lead molecules, phenotypic screening remains an important source of drug candidates, particularly those with novel modes of action.1−3 Indeed, it has been highlighted that the majority of drugs approved between 1999 and 2008 had their origins in phenotypic screens.1a This kind of screening approach offers the opportunity to identify molecules acting by mechanisms that are unanticipated, allowing interrogation of the function of a target that may not be accessible in the isolated protein, such as those that exist in complex with other proteins within the cellular environment and are thus difficult to recapitulate in a biochemical assay. Phenotypic screening for antiviral agents has been a useful approach to lead generation that juxtaposes the diversity inherent to a compound collection with the panoply of viral targets that are represented in either a partial or full virus replication cycle in cell culture.2,3 However, virus-encoded proteins often play multiple roles in virus reproduction in a fashion that is distinct and temporally coordinated. This is particularly true for the smaller RNA viruses like hepatitis C virus (HCV) or human immunodeficiency virus 1 (HIV-1) where phenotypic screening may be considered to offer an additional dimension in which the unique role of viral targets at different points of the replication cycle can readily and effectively be assessed. As an example, the HIV-1 integrase strand transfer inhibitors that bind to the active site of the enzyme interfere with the integration of the viral cDNA into the host cell chromosome.4 In contrast, allosteric inhibitors that © XXXX American Chemical Society

associate with the lens epithelium-derived growth factor (LEDG-F) binding site appear to act at multiple points in the replication cycle, with the antiviral activity in cell culture manifested most prominently postintegration and associated with effects on the maturation of virus particles.5 We have exploited phenotypic screening to identify mechanistically novel antiviral agents that include the influenza fusion inhibitor 1, the respiratory syncytial virus fusion inhibitor 2, the HIV-1 attachment inhibitor 3, the influenza nucleoprotein (NP) inhibitor 4, and the HCV entry inhibitor 5 (Chart 1).6−10 Elucidation of the mode of action of antiviral agents discovered in this fashion is facilitated by a process involving the generation of resistant virus, the careful mapping of mutations, and demonstration of the resistant phenotype when mutations are reverse-engineered into the viral genome. However, phenotypic screening for antiviral agents may identify inhibitors of host cell processes, and in order to confirm that the biochemical target is a viral protein, we have frequently augmented this approach by preparing and utilizing affinity probes to confirm target engagement. These probes have also proven to be important tool molecules that have not only furthered our understanding of the mode of action of lead molecules but also provided unique and important insights into drug−target interactions or illumined fundamental aspects of mechanism of action that have been important for the evolution of a program.6b,7d,e Received: March 3, 2014

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dx.doi.org/10.1021/jm500335h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Drug Annotation

Chart 1

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DISCUSSION At the initiation of our drug discovery efforts to identity and develop inhibitors of HCV, we adopted a strategy that anticipated the use of combination therapy to address the problem associated with the development of resistance that frequently occurs with therapeutic agents targeting an RNA virus. Viral RNA polymerases are associated with poor fidelity of replication due to a limited proofreading capability that results in the rapid emergence of resistant virus under the selective pressure of a drug, an evolutionary defensive posture for the virus. Combination antiviral therapy has its basis in teachings from the arena of HIV-1 drug development, and clinical trials with HCV inhibitors have not only confirmed the validity of concerns involving the emergence of resistance but have also led to a deeper understanding of viral kinetics and a fuller appreciation of the challenges associated with HCV drug development.11−13 Consequently, we conducted multiple discovery programs simultaneously with a focus initially directed toward the NS3 protease and NS5B RNA-dependent RNA polymerase, since biochemical assays for these targets could be reconstituted in vitro. However, the advent of cellbased systems that efficiently recapitulated key aspects of HCV replication using subgenomic constructs, originally reported by Bartenschlager in 1999 almost a decade after the identification of HCV as an RNA virus, allowed the implementation of phenotypic screens that sought mechanistically unique inhibitors of virus replication.14 The development of subgenomic replicons was an important advance in the HCV field since, in contrast to HIV-1, conditions under which the virus would replicate in cell culture system remained elusive until 2005.11,14 Although the first generation HCV replicon systems, which were based on a genotype-1b (GT-1b) virus, captured only the basic elements of virus replication, excluding viral entry, assembly, and egress, they nevertheless facilitated drug discovery by allowing an assessment of the effects of NS3 and NS5B inhibitors in a more physiologically relevant setting. Prior to the development of the GT-1b replicon system, the only cell culture assays that were available for the evaluation of HCV inhibitors were based on bovine viral diarrhea virus (BVDV), a pestivirus that is a member of the same Flaviviridae family as HCV and has been considered to be sufficiently close phylogenetically to function as a surrogate for screening purposes.15 However, our experience of using BVDV as a surrogate to identify HCV inhibitors had not proven to be

successful, an observation that led to the use of this virus as a counter screen designed to act as a stringent arbiter of selectivity for HCV that would help to eliminate modulators of host cell function and rapidly triage lead inhibitors.15e,16 Thus, the screen implemented to discover the iminothiazolidinone chemotype that proved to be a cryptic precursor to daclatasvir (6, Chart 2) assessed inhibition of GT-1b HCV and BVDV Chart 2

replicons constructed in the same Huh-7 host cell background.16 These cell lines were deployed in the same screening well in a fashion that took advantage of orthogonal reporters for each virus, thus allowing the selection of leads with high specificity for HCV. Alamar Blue staining was used to assess host cell viability at the end of the incubation period, and the cells were subsequently lysed to calibrate the inhibition of HCV replication using a FRET-based reporter assay that monitored cleavage of a substrate by the NS3/4A protease while the BVDV replicon utilized a luciferase read-out. BMS-858 (7) emerged from this screening campaign as a selective HCV inhibitor that targeted the NS5A protein (vide infra), and although over one million compounds were ultimately screened, it was the only chemotype identified with this antiviral mechanism. This kind of hit rate has precedent with the other high throughput screening leads described above B



Drug Annotation

resistant to 8 retained sensitivity to inhibitors of NS3 protease.18,19 At the time of this discovery, our understanding of the function of the HCV NS5A protein in the virus replication cycle was rudimentary, with no known enzymatic activity.20 As a consequence, biochemical characterization of NS5A activity in a fashion similar to that developed for the NS3 protease and NS5B RNA-dependent RNA polymerase enzymes was precluded.20 Studies with NS5A inhibitors in HCV replicons, replicating virus preparations and clinical trials, have since illuminated several aspects of NS5A function, and this protein has been suggested to be a master regulator of virus replication.20b However, although NS5A has been shown to coordinate both HCV replication and assembly while modulating host cell responses that contribute to creating an environment conducive to virus replication and propagation, the current level of understanding of NS5A function remains elementary at best.20g As a screening lead, 7 suffered from several potential shortcomings including a high molecular weight (MW = 587), a cLogP of >5, an extensive polar surface area of 139 Å2, and a stereocenter at C-5 that was prone to epimerization during routine handling. However, its unique mode of action was attractive and initial structure−activity studies focused on assessing the effect of changes to the peripheral elements bound to the core scaffold of 7 (the amino acid moiety and the Nphenyl and the N-furanylmethyl substituents), a synopsis of which is captured in Figure 2 where all compounds were assessed as mixtures containing both isomers at the C-5 center of the thiazolidinone core.18,21 Modifications to the phenyl and furanylmethyl elements modulated replicon inhibitory potency over a broad dynamic range, with more polar moieties associated with higher antiviral activity. However, the structure−activity relationships (SARs) for the amino acid were much more precise, with inversion of the alanine stereoconfiguration reducing antiviral activity by more than 100-fold. Moreover, substitution of alanine revealed that glycine was 10-fold inferior, proline improved antiviral potency modestly, while all other amino acids afforded analogues that were poorly active in the replicon. A key finding in this phase of the program was that changing the carboxybenzyl moiety to a phenylacetamide cap markedly increased potency in both the

which were typically either singletons or members of a series with only a limited number of structural homologues. These results highlight the importance of past drug discovery campaigns and the practices associated with library enrichment that shape the complexion of corporate compound collections.17 Compound 7 was profiled as a modestly potent GT-1b HCV inhibitor with an EC50 between 0.57 and 1.0 μM, representing over 50-fold selectivity toward both BVDV (EC50 > 50 μM) and host cell toxicity (CC50 > 50 μM).18 This iminothiazolidinone chemotype originated as part of an opportunistic library of molecules designed and prepared for general screening campaigns in an era when that was common practice. Although iminothiazolidinones are well represented in the literature, they are frequently obtained by Knoevenagel condensation of C-5unsubstituted compounds with aldehydes or derivatives thereof that affords C-5 vinylidene derivatives, as depicted in Figure 1.

Figure 1. Structure of the parent iminothiazolidinone chemotype and conversion to C-5-unsaturated analogues.

However, although benzylidene homologues of 7 prepared from aromatic aldehydes in this fashion inhibited HCV replication with single digit micromolar EC50 values, they were insensitive to replicons expressing mutations that conferred resistance to 7, suggesting an alternative mode of action. Ultimately, the C-5 phenyl substituent of 7 and the attendant chemical properties turned out to be of considerable importance to the discovery of 6. Resistance selection and mapping studies with 7 and the more potent analogue 8 (EC50 = 0.005 μM) implicated NS5A as the target with a Y93H substitution in domain I of the protein conferring resistance that was >5-fold to 7 and >625-fold to 8.18,19 Additional resistance selection and mapping experiments identified a combination of L31V and Q54L as consensus mutations that conferred 354-fold reduced sensitivity to 8, but replicons

Figure 2. Synopsis of structure−activity relationships associated with 7 as an inhibitor of replication of an HCV GT-1b replicon. C



Drug Annotation

Figure 3. Decomposition of 8 in DMSO.

Chart 3

exhibited EC50 values of 43 and 0.6 nM, respectively, in the replicon, and their antiviral activity was sensitive to the NS5A Y93H mutant. A detailed analysis of the 1H and 13C NMR spectra combined with MS spectroscopy revealed that the compounds were derived from 8 via a process of dimerization and that the connectivity for peak 4 was at C-5, leading to their identification as isomers of 9. Interestingly, an attempt to simplify the NMR spectrum of peak 6 by warming a CD3CN solution to 55 °C, in order to homogenize the amide rotamer resonances, led to its conversion to peak 4, suggesting the latter to be the thermodynamically more stable isomer. However, the relative isomeric composition of the two dimeric compounds has not been definitively established and it is not clear if the conversion of peak 6 to peak 4 represents interconversion of atropisomers across a rotational barrier or dimer dissociation into monomeric species that recombine in a fashion that affords the alternative, more stable isomer. Dimerization of 8 to afford 9 is envisaged to arise by way of coupling of radicals generated at C-5 of 8 by hydrogen atom abstraction, presumably mediated by oxygen that is a diradical in its ground state, with the phenyl, carbonyl, and sulfur substituents providing sufficient captodative stabilization of the radical to allow dimeric combination to occur in the assay medium.19,21 The discovery of biologically active dimeric derivatives of 8 propelled the program in a markedly different direction that addressed the challenge associated with the high molecular weight (MW = 1139) and structural complexity of 9.21 On the basis of the premise that the precise SARs associated with the amino acid moiety identified a key pharmacophoric element, it was hypothesized that the chemical reactivity of the thiazolidinone heterocycle was functioning to convene an embedded symmetrical dimeric species and that the two thiazolidinone heterocycles of the dimer 9 could be dispensed

alanine (8) and proline series, affording compounds with single digit nanomolar EC50 values in the GT-1b replicon.21 As the examination of the iminothiazolidinone chemotype continued, we became aware of a chemical instability issue associated with 8 which was observed to partially decompose on standing for several days as a dimethyl sulfoxide (DMSO) solution. Initial studies identified a degradation pathway involving oxidation at C-5 leading to a thiohydantoin derivative along with small amounts of a thiourea and ketoacid products arising from hydrolytic decomposition of the putative acylated thiourea intermediate (Figure 3). Follow-up studies demonstrated that 8 was also unstable in replicon medium, although surprisingly the observed HCV inhibitory activity was preserved regardless of the extent of degradation of the compound. An HPLC analysis of material extracted from the replicon medium revealed no significant additional peaks by UV detection, suggesting that the antiviral activity maybe associated with minor components of the mixture. In elucidating the origin of biological activity, the HCV replicon played a critical role by providing a highly sensitive assay with which to detect antiviral components that were not readily apparent from the initial HPLC trace because of their low concentration. After developing an extraction process that captured the biologically active fraction, an HPLC biogram analysis was used to guide identification of the active components that showed reduced potency toward the NS5A Y93H mutation. Incubating 8 in replicon media on a preparative scale led to the identification of three additional products by HPLC in addition to those characterized in the earlier decomposition study discussed above. The major additional components, two structurally related active species, were isolated in sufficient quantity to allow detailed characterization.19,21 These compounds, designated as peak 4 and peak 6 from the biogram analysis, D



Drug Annotation

Chart 4

It quickly became apparent from initial SAR studies surrounding 10a that the peripheral structural elements and their topological disposition were primarily responsible for antiviral potency while the stilbene element was determined to be largely playing the role of a scaffold on which to display the pharmacophore. Moreover, pharmacokinetic (PK) profiling of analogues of 10a established the potential of this chemotype to deliver compounds that combined GT-1b replicon inhibitory activity with reasonable bioavailability following oral dosing to rats, although these properties required some compromise, as exemplified by the data associated with 11 (Chart 4).25,26 Extensive variation of the phenylacetamide moiety of 10 and optimization of compounds demonstrating modestly potent GT-1a inhibition led to the discovery of 12a as the first compound with balanced inhibition of both the GT-1a and GT1b viral subtypes.27 Gratifyingly, this compound also displayed potent antiviral activity toward replicons and hybrid replicons representing HCV genotypes 2a, 3a, and 5a with EC50 values ranging from 2.2 to 14 nM, data that further heightened our interest in the NS5A mechanistic approach.27 Simplification of the isoquinolinamide moiety to the phenylglyoxamide 13, envisioned as a deannelated analogue of the heterocycle, compromised GT-1a inhibitory potency by 20-fold, but this compound displayed an antiviral profile comparable to that of 12b, the progenitor of 12a, further identifying salient elements of the embedded pharmacophore.28 Attention was then focused on modifying the benzylic carbonyl moiety of 13 with a variety of substituents introduced in order to probe interactions with the NS5A protein. Examination of a series of mandelamidebased caps revealed that while potency toward both GT-1a and GT-1b replicons could be improved, there was considerable subtlety associated with stereochemical preferences.28 For example, the (R)-mandelamide 14a was more potent than its diastereomer 14b, whereas this scenario was reversed with the α-methylated homologues 15 where the (S)-capped analogue 15b is the more potent diastereomer. In this series, enhanced GT-1a inhibitory activity was accompanied by a significant boost in potency toward the GT-1b replicon, with the single digit picomolar EC50 associated with 15b being one of the earliest demonstrations of the high level of inhibitory potency that could be achieved by disrupting the function(s) of HCV NS5A.

with. This concept was readily and rapidly explored in the context of the more potent proline derivative and led to the synthesis of stilbene 10a along with its saturated bibenzyl analogue 10b. While 10b exhibited an EC50 value of 30 nM in the GT-1b replicon, 10a was considerably more potent with an EC50 of 86 pM, a result attributed to the conformational constraint afforded by the olefin moiety.19,21 Moreover, 10a exhibited >10 000-fold reduction in inhibitory potency toward the NS5A Y93H resistant mutant, indicative of a similar mode of inhibition as the iminothiazolidinone progenitor 8. This dimeric pharmacophore model was corroborated several years later with the determination of the solid state structures of domain I of initially GT-1b and subsequently GT-1a NS5A as a dimeric species that complemented the symmetry associated with the structures of 9 and 10 (Chart 3).22 While the discovery of stilbene 10a represented a significant simplification of the chemotype and an advance for the program, the potential of this chemotype as a vehicle for drug discovery was compromised by two liabilities inherent to the structure. The potential for the olefin to undergo cis/trans isomerization was viewed as problematic, while the anilide moieties were of greater concern because of the risk for the release of a potentially toxic aniline moiety in vivo should the molecule be recognized by amidases or be subject to hydrolytic decomposition in the gut.23 An additional and more substantial complication that emerged shortly after the discovery of 10a was associated with the development of a replicon system that allowed evaluation of inhibitory activity toward the GT-1a subtype. In this assay, the EC50 value for 10a was determined to be >10 μM, a result presenting a more significant issue that needed to be resolved if we were to advance an inhibitor with a broader antiviral profile. The clinical relevance of the GT-1a subtype as a therapeutic target is underscored by demographic analyses that reveal it to be the HCV variant with the highest prevalence in North America, while there is significant additional presence in South America, Europe, and Australia.24 Moreover, GT-1a infections have proven to be a difficult subtype to treat clinically. Although enhancing GT-1a inhibitory activity was the major focus of the next phase of compound optimization, solving this problem turned out to be a considerable challenge. E



Drug Annotation

derivatives.26 Additional examination of the cap revealed that phenylglycinamide derivatives imparted enhanced potency toward GT-1a and/or GT-1b replicons, with a marked preference for the (R)-stereoconfiguration at the benzylic center (compare 16b with 16c in Table 1).28 While both a basic dimethylamine (16b) and a carbamate (16e) substituent at the benzylic carbon were associated with good antiviral potency, the less basic morpholine substituent in 16d was more than an order of magnitude weaker than 16b toward the GT-1a replicon. It is hypothesized that the more basic analogues such as 16b are protonated at physiological pH, allowing them to engage the GT-1a NS5A protein through a H-bond interaction with a proximal acceptor in the binding pocket, an interaction that can be mimicked by the carbamate NH of 16e. However, this type of H-bonding interaction with the GT-1b NS5A protein appears to be less important for maintaining antiviral activity, since all of the modifications in 16b−e are associated with potent GT-1b inhibition with EC50 values of less than 4.6 nM. Having firmly established the importance of the cap region for securing potent GT-1a inhibitory activity, the final stages of the campaign to identify a clinical candidate were directed toward addressing the embedded anilide moiety. The SARs that had emerged suggested that both the NH and the carbonyl moiety of the anilide were of importance for antiviral activity

Although replacement of the alkene moiety of 10a with an alkyne, exemplified by 16a (Table 1), was accompanied by Table 1. Antiviral Activity Associated with Alkyne Derivatives 16a−e

some loss of GT-1b antiviral activity, this compound was selected as the prototype for further study because it obviated the cis−trans isomerization liability associated with stilbene Chart 5

F



Drug Annotation

Scheme 1. Synthesis of 6

evaluation. Compound 6, one of 10 possible stereoisomers, derives its chirality from the two natural amino acids L-proline and L-valine, facilitating its preparation in an efficient and convergent manner by two routes, the more efficient of which is outlined in Scheme 1.31,32 Alkylation of the N-protected proline 25 with the 1,1′-([1,1′-biphenyl]-4,4′-diyl)bis(2-bromoethanone) 24 afforded the bis-ketoester 26 which was converted to the bis-imidazole 27 in a one pot, multistep transformation by exposing the compound to NH4OAc in hot toluene or xylenes.31,32 Deprotection of 27 under acidic conditions gave the salt of amine 28 which was coupled with the L-valine derivative 29 to afford 6. Compound 6 exhibited potent inhibition in all of the wildtype replicons and hybrid replicons representing the range of genotypes and subtypes that were available at the time of its discovery. These data are summarized in Table 2 where activity

and the modifications that were probed preserved these structural elements. The benzimidazoles 17 and 18 (Chart 5) fulfilled these structural criteria and were potent inhibitors of GT-1b HCV, but the reduction in GT-1a potency for both of these compounds necessitated further optimization which focused on adjusting the topology of the molecule based on findings from earlier studies with GT-1b-biased inhibitors.26,29 After considerable experimentation, deannelation of the benzimidazole of 17 combined with excision of the alkyne moiety to accommodate the changes in topology and core length afforded 19a as a compound with potent antiviral activity toward both GT-1a and GT-1b that established a remarkable new benchmark for potency not only for NS5A inhibition but for HCV inhibitors in general.29 Consistent with the SAR for 16b and 16e, the dimethylamine analogue 19b exhibited a similar level of antiviral potency with balanced GT1a and GT-1b inhibition. However, both 19a and 19b demonstrated poor absorption in rat PK studies, attributed to their high molecular weight (807 and 747 Da, respectively) and structural composition. These results catalyzed an examination of truncation of the phenyl ring within the context of carbamate 19a as an approach to address these shortcomings. However, the initial product of this exercise, the D-valine derivative 20, which retained the cap benzylic stereoconfiguration of 19a, exhibited a large (>40 000-fold) erosion of GT-1a inhibitory potency.29 Alternative approaches that were based on abridging a single cap moiety, exemplified by the tetrahydrofuran 21, also incurred a significant reduction in GT-1a antiviral activity, but the measurable exposure observed for this compound following oral dosing across species supported the strategy of reducing molecular weight and aromatic content in order to improve oral bioavailability. The final breakthrough emerged from an unanticipated SAR finding that the optimal antiviral activity for the alkyl- and arylglycine cap series resided in different absolute stereoconfigurations. The initial productive step in this direction occurred when the tetrahydrofuran moiety of 21 was replaced with L-alanine which afforded 22, a compound with significantly enhanced GT-1a inhibitory properties. As a followup, the symmetrical L-alanine analogue 23 was synthesized and found to be a potent inhibitor of both GT-1a and GT-1b subtypes with subnanomolar EC50 values. A rapid optimization effort centered around 23 ultimately yielded the bis-L-valine derivative 6, in which the stereoconfiguration of the caps is inverted compared with that of 20.30−32 This C-2 symmetric compound 6 exhibited antiviral, absorption, distribution, metabolism, and excretion (ADME) and in vitro and in vivo toxicological profiles that supported its selection for clinical

Table 2. Potency of 6 in Replicons and Hybrid Repliconsa replicon genotype

EC50 (nM)

GT-1a H77 GT-1b Con1 GT-2a JFH-1 GT-2a J6 GT-3a GT-3a Y-H GT-3a Y-H* GT-4a GT-5a GT-6

0.050 0.009 0.071 7.5 0.146 364 1451 0.012 0.033 0.054

a Excluding the first three entries, the remaining data are for hybrid replicons in either GT-2a JFH-1 or GT-1b Con1 backbone: GT-2a J6 (JFH-1); GT-3a or GT-3a Y-H (Con1); GT-3a Y-H* (JFH-1); GT-4a (Con1); GT-5a (JFH-1); GT-6a (JFH-1).

toward some of the resistant mutants is also included. Just prior to the compound entering clinical trials, we established a GT-2a JFH replicating virus assay where potent inhibition, EC50 = 28 pM, was also observed.14,30,31,33 This result correlated well with GT-2a JFH replicon inhibition and provided an additional level of confidence for an untested mechanism of HCV inhibition as the compound advanced into clinical evaluation. Liver exposure of HCV inhibiting drugs is considered to be an important factor in treating a disease that replicates primarily in hepatocytes and has been the subject of considerable study in an effort to understand the relationship between the PK and pharmacodynamic properties of direct-acting HCV inhibitors.34 G



Drug Annotation

In the preclinical setting, we routinely evaluated liver exposure as part of rat PK studies with HCV inhibitors as a means of demonstrating target organ exposure. The PK properties of 6 in preclinical species are compiled in Table 3 and reveal good

Chart 6

Table 3. Pharmacokinetic Properties of 6 in the Rat, Dog, and Cynomolgus Monkey species (no. of animals)

dose (mg/kg)

plasma AUC (μM·h)

plasma concn at 24 h (nM)

F (%)

rat (3) dog (2) cynomolgus monkey (3)

5.0 2.3 2.8

4.8 11 1.93

18 26 6.5

50 108 38

failed to pull down any viral proteins. However, by incubating GT-1b replicons with 29 for 18 h before cell lysis and then passing the lysate over streptavidin agarose beads, the HCV NS5A protein was selectively pulled down. This result indicated that in order for the probe to bind to the viral protein, it needed to be present at the time of replicon complex formation. Notably, the inactive (R,R) isomer 30 failed to pull down the HCV NS5A protein under the same experimental conditions, thus establishing a direct correlation between antiviral activity and target engagement, recently described as one of the four pillars of in vitro profiling underlying successful drug candidates.41 The biotin probe 29 has proven to be a particularly useful tool to further illuminate aspects of NS5A inhibitor−protein interaction. Most interestingly, when a similar experiment was conducted with replicons that are insensitive to the inhibitory activity of 29, including a GT-1b replicon harboring a Y93H resistant mutation or a GT-1a replicon, the HCV NS5A protein was pulled down in each case. This unanticipated finding suggests that the lack of sensitivity to HCV NS5A inhibitors may not rely upon exclusion of the inhibitor from binding but rather that the viral protein accommodates the inhibitor in a fashion that restores the function of the viral protein.40 Consistent with this notion, many of the resistance mutations involve substitutions with amino acids that have smaller or more flexible side chain moieties that may allow the NS5A protein to articulate its function in the presence of the inhibitor. These observations indicate that binding of a molecule to the NS5A protein is a necessary but not sufficient condition for the expression of an antiviral effect. This hypothesis anticipates that the poor activity toward HCV GT-1a replicons associated with prototypical GT1b inhibitors maybe a reflection of subtle differences in the function of the NS5A proteins that allow GT-1b to be more susceptible to inhibition by structurally diverse molecules while GT-1a NS5A has a more stringent demand for dimeric species that display specific pharmacophoric features.

bioavailability in all three species. Liver levels in the rat measured at 24 h following a 5 mg/kg dose of 6 were 103 nM, 5-fold higher than the plasma concentration and substantially higher than the replicon EC50 values for all genotypes tested at this time and which surpassed the protein binding-adjusted EC90 of 383 pM determined for the less sensitive GT-1a replicon.31 The first publication of an X-ray crystal structure of a portion of domain I of the NS5A protein appeared in the May 19, 2005, edition of Nature, revealing a dimeric species for which protein association created a groove lined with basic amino acids postulated as binding site for RNA.22a We viewed the structure with considerable interest and the C-2 symmetry of the complex corroborated the palindromic chemotype that we had discovered and allowed for the generation of drug−target interaction hypotheses. Subsequent studies have confirmed the suggestion that NS5A is an RNA binding protein, and virus replication has been shown to correlate with dimerization of the protein.35,36 A second X-ray structure of a slightly different sequence of domain I revealed a similarly shaped protein but with a very different dimer interface that placed the putative RNA binding domain on the outwardly facing surface.22b The two structures and the more-recently determined GT-1a NS5A domain I structure22c are viewed as anticipating an oligomeric association of NS5A that may provide potential mechanistic insights into the high inhibitory potency observed with 6, which is exceptional when compared, for example, to NS3 protease inhibitors that are assumed to depend on a stoichiometric association with the target.37 It has been estimated that the ratio of NS5A molecules to 6 at half maximal inhibitory concentrations in a replicon is in excess of 1000 and could be as high as 10 000.31,37−39 This has led to the suggestion that the association of 6 with an oligomeric form of NS5A effects a distortion that disrupts the function of the entire complex, by a local effect or one of an allosteric nature that is propagated through the entire complex. While resistance mapping implicated the NS5A protein as the target for the evolved chemotype classes leading to 6, evidence of target engagement was sought using the biotinylated probe 29 that incorporates the natural (S,S)configuration at both proline stereocenters, with the unnatural (R,R) isomer 30 used as a control (Chart 6).31,40 The (S,S) isomer 29 exhibited antiviral activity in a GT-1b replicon, EC50 = 33 nM, with sensitivity to the NS5A Y93H mutant, EC50 >10 μM, while the (R,R) isomer 30 was inactive, EC50 >10 μM, data that established a profile analogous to 6 and related compounds in the series. In the initial experiment, cells harboring GT-1b replicons were lysed and exposed to 29 and the mixture passed over streptavidin agarose beads, but this experimental design

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CLINICAL TRIALS WITH DACLATASVIR HCV replicates via only RNA-based intermediates with no incorporation of the viral genome into the host cell chromosome, as occurs with HIV-1. As a consequence, HCV is a curable infection with cure defined as being free of detectable virus in plasma 24 weeks after completion of therapy, referred to as a sustained virological response-24 (SVR24), although more recent studies suggest that SVR12 has predictive value. In the decade following the discovery and characterization of HCV, therapeutic treatment evolved to the use of a combination of pegylated interferon-α (PEG-IFN-α) administered as weekly subcutaneous injections in conjunction with twice-daily oral doses of the nucleoside analogue ribavirin (RBV) which was approved in 2001.42,43 However, this therapy H



Drug Annotation

Chart 7

an oral solution.31 Compound 6 was safe and well-tolerated at all doses with plasma exposure increasing proportionally with dose and drug levels at 24 h exceeding the protein bindingadjusted EC90 value measured in the least sensitive GT-1a replicon by over 10-fold at doses of 10 mg and above. The second phase of the study recruited subjects chronically infected with HCV GT-1 who were naive to therapy or who had not responded to PEG-IFN-α/RBV treatment or who were intolerant of this therapy. In this proof-of-concept trial, doses of 1, 10, and 100 mg of 6 were administered to subjects infected with GT-1a HCV with the exception of two subjects in the 10 mg dose cohort and three in the 100 mg group who were infected with GT-1b virus. Mean plasma viral load declined rapidly by 1.8 log10 IU/mL measured at 24 h following the single milligram dose of 6, while the higher doses produced a greater effect on viral RNA reduction, with a mean decline of 3.2 log10 at 24 h after the 10 mg dose while the 100 mg dose produced a maximal decline of 3.6 log10.31 The 100 mg dose cohort was composed of GT-1b-infected patients, where the antiviral effect was prolonged throughout the postdosing monitoring period of 144 h, with HCV RNA below the limit of quantification (25 IU/mL) in one subject. Collectively, these data confirmed that the high in vitro potency of 6 translated into a potent clinical effect while the duration of the suppression of viremia was related to the prolonged PK profile of the compound. However, most remarkably, the reduction in viral load that was observed following oral dosing of 6 was considerably faster than that associated with HCV inhibitors acting by other mechanisms. This profile has since been attributed to an initial and immediate effect of 6 on virion production that is followed by inhibition of viral replication, observations that have led to a recalibration of the half-life of HCV in vivo from 2.7 h that was based on the viral kinetics in response to PEG-IFN-α therapy to ∼45 min.45 A multiple ascending dose (MAD) study in which 6 was administered for 14 days to GT-1-infected subjects resulted in viral rebound due to the emergence of resistant virus that was faster in GT-1a-infected subjects than those infected with GT-

has significant shortcomings with the most prominent being the poor SVR observed in GT-1-infected subjects, which is typically only 40−50% following 48 weeks of therapy. In addition, neither of these agents exhibit specific antiviral activity toward HCV and both are associated with a significant incidence of side effects that, in the case of PEG-IFN-α, can be of sufficient severity to result in discontinuation of therapy.43,44 The molecular characterization of HCV provided a biochemical basis for the identification of direct-acting antiviral agents (DAAs) that has allowed the development of specifically targeted compounds with improved efficacy and tolerability profiles.42 The approval of the HCV NS3/4A protease inhibitors telaprevir (31) and boceprevir (32) in 2011, the NS3/4A protease inhibitor simeprevir (33), and the nucleoside analogue sofosbuvir (34) (Chart 7) in 2013 as add-on therapy to PEG-IFN-α/RBV or RBV represents important progress in that direction. However, there are many more DAAs in development with attractive profiles that act by a range of mechanisms and that, when used in combination, offer the potential to cure even the most difficult GT-1 HCV infections after just 12 weeks of therapy in the absence of the immune stimulation provided by PEG-IFN-α. Recent clinical studies with DAA combinations have assessed the effects of viral strain, baseline viral load, prior treatment experience with PEG-IFNα/RBV, host polymorphisms in key immune response genes, liver histological status, and HIV-1 co-infection, with the results establishing efficacy regardless of the background circumstances. Since entering clinical trials in 2007, 6 has played a critical role in several of these developments, validating NS5A as an attractive and important clinically relevant target while contributing to the shaping of the clinical landscape, which has evolved rapidly toward all DAA therapeutic regimens with pangenotype inhibition. The clinical program with 6 was initiated with a single ascending dose (SAD) study conducted in normal healthy volunteers under a double-blind, placebo-controlled protocol that assessed the plasma exposure and tolerability of the compound after escalating doses of 1−200 mg administered as I



Drug Annotation

1b virus.46 Sequencing of the virus in those experiencing breakthrough revealed mutations that correlated well with those observed in vitro using the replicon system. The observed pattern of viral breakthrough can be explained by GT-1a HCV typically requiring only a single base pair change in order to code for an amino acid, while GT-1b requires two base pair changes.47 These results indicate that although 6 is highly potent, it has a low genetic barrier to resistance, a profile that is somewhat analogous to that of the HIV-1 non-nucleoside reverse transcriptase inhibitors which are successful drugs when used as part of combination therapy. The clinical profile that emerged for 6 from these studies anticipated it being used in combination with PEG-IFN-α/RBV or mechanistically orthogonal HCV inhibitors.48 The first of those options was explored in a small phase IIa clinical trial conducted in treatment-naive GT-1 patients in which oncedaily doses of 3, 10, or 60 mg of 6 were added to a 48-week regimen of PEG-IFN-α/RBV.49 The results of this study, which established the long-term safety profile of 6, afforded an SVR24 rate of 83% for those administered the two higher doses of drug compared with 25% for the PEG-IFN-α/RBV control arm.49 A more impactful study was the first clinical trial to explore longterm administration of a DAA-only combination which was an open label study in which 6 was administered in conjunction with the NS3 protease inhibitor asunaprevir (35) (Chart 8).50a,51,52 This study selected what is considered to be one of

emerged in both the NS3 and NS5A proteins. This trial established for the first time that a combination of small molecule HCV inhibitors could cure viral infection in the absence of PEG-IFN-α/RBV therapy and established the potential of this drug combination for the treatment of GT1b-infected subjects. This population is prevalent in Japan to the extent of ∼75% of infections, and phase II clinical studies with the two drugs provided results that were supportive of expansion into a phase III registrational trial.53,54 These studies, which evaluated the combination of 6 (60 mg q.d.) and 35 (200 mg b.i.d.) administered to Japanese patients infected with GT1b HCV for 24 weeks, have been completed.53c,54 SVR24 rates ranged from 87.4% for PEG-IFN-α-intolerant or ineligible subjects to 80.5% for nonresponders to PEG-IFN-α/RBV, data that formed the basis of a filing in October, 2013 seeking marketing approval in Japan. On February 24, 2014, BristolMyers Squibb announced that the combination of 6 and 35 had been accorded Breakthrough Therapy Designation by the U.S. Food and Drug Administration (FDA) for the treatment of GT-1b HCV infection. On April 8th, 2014, Bristol-Myers Squibb announced the filing of NDAs with the US FDA seeking approval to market daclatasvir and asunaprevir for use in combination for the treatment of HCV GT-1b infection with the daclatasvir NDA also requesting approval for its use of in combination with other agents for the treatment of multiple HCV genotypes. In order to address the emergence of resistance in GT-1infected subjects when treated with 6 and 35, clinical trials evaluating a triple combination that supplements therapy with the non-nucleoside NS5B polymerase inhibitor BMS-791325 (36, Chart 8) (75 or 150 mg b.i.d.) have begun to be explored.55,56 The phase IIa study examined this triple combination at doses of 60 mg q.d. of 6 and 200 mg b.i.d. of 35 with 75 or 150 mg b.i.d. of 36 in a treatment-naive patient population composed of GT-1a and GT-1b infections in a ∼3:1 ratio. The drugs were administered for 12 or 24 weeks to 66 subjects with 92% achieving SVR12 based on a modified intention to treat analysis, with similar virologic response rates observed between the 12 and 24 weeks dosing arms. These results supported expansion of this study to include additional GT-1-infected subjects, including those naive to therapy, null responders to PEG-IFN-α/RBV and those with liver cirrhosis, and GT-4 infected patients. This combination is currently being evaluated in phase III clinical trials and was granted Breakthrough Therapy Designation by the FDA in March 2013.54 A combination of 6 and the nucleoside phosphoramidate polymerase inhibitor sofosbuvir 34, with and without RBV, has been examined in treatment-naive GT-1-, GT-2-, and GT-3infected patients and in treatment-experienced GT-1-infected subjects who failed a triple therapy regimen combining either of the protease inhibitors telaprevir or boceprevir with PEG-IFNα/RBV.57,58 Treatment durations of either 12 weeks (GT-1) or 24 weeks (GT-1, GT-2, and GT-3) were explored, and >90% of patients achieved an SVR12. Interestingly, the absence or inclusion of RBV in the therapeutic regimen had no significant impact on the cure rate, although including this drug did increase the incidence of adverse events.57 Market authorization for 6 in the European Union (EU) for the treatment of chronic genotypes 1, 2, 3, and 4 HCV infections in subjects with compensated liver disease in combination with other agents was sought in late 2013 with the compound granted accelerated regulatory review by the

Chart 8

the more difficult to treat patient populations: those who had been treated with PEG-IFN-α/RBV for at least 12 weeks but failed to experience a decline in viral load of ≥2 log10 IU/mL. In this phase IIa trial, 6 (60 mg q.d.) and 35 (600 mg b.i.d.) were administered to 11 GT-1-infected subjects for 24 weeks with the comparator arm comprising 10 subjects who received the same dosing regimen of 6 and 35 in conjunction with PEGIFN-α/RBV therapy.50 In the dual drug combination arm, nine of the subjects were infected with GT-1a virus with the remaining two infected with GT-1b virus and both GT-1binfected subjects and two of the GT-1a-infected individuals achieved both SVR12 and SVR24. One GT-1a-infected patient was undetectable at the end of therapy but subsequently relapsed. All of the patients receiving quadruple therapy achieved SVR12, and 90% of this cohort had undetectable levels of virus 48 weeks after the cessation of therapy with the one remaining subject having detectable viral RNA in plasma at this point but who was determined to be undetectable 13 days later. All of the patients in the dual combination arm that experienced virological breakthrough had PEG-IFN-α/RBV added to their therapy, and although all responded initially with a reduction in viral load, this was not durable. Failure due to viral breakthrough was associated with resistance mutations that J



Drug Annotation

Notes

European Medicines Agency on January 8, 2014. This follows an EU recommendation on November 12, 2013, for the compassionate use of 6 in combination with 34 to treat chronic HCV infection in adults at a high risk of liver decompensation or death within 1 year if left untreated.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The discovery and development of daclatasvir represents the collective effort of a large number of individuals, the majority of whom are represented as authors of the articles cited in the reference section. From the discovery perspective, we acknowledge Min Gao for his leadership of the virology team, Lawrence B. Snyder and Lawrence G. Hamann for their chemistry leadership roles, and Carl P. Decicco for his consistent support of the program.

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CONCLUSION The discovery of 6 was the result of an extensive medicinal chemistry optimization campaign by a highly collaborative team that had to navigate through several significant challenges ranging from understanding the chemical instability associated with the lead to introducing potent GT-1a activity and optimizing for oral bioavailability. Although the high throughput screening lead 7 exhibited reasonable antiviral potency in a cell-based assay and excellent selectivity for HCV, the molecular properties of this lead molecule may be viewed as less than attractive as a starting point for a program. The high cLogP of >5 and a molecular weight of 586 represent less than optimal starting points for medicinal chemists who have typically increased both cLogP and molecular weight when optimizing leads.59 The characterization of a dimeric species with a molecular weight of 1139 as a contributor to the observed antiviral activity, although intriguing, heightened concerns, but the redefinition of the pharmacophore to that of stilbene 10 markedly simplified 9. At this point in the program, elucidating the structural elements that conferred GT1a inhibition and addressing concerns about the olefin and embedded aniline were the key problems to be solved. The ultimate solution embodied by 6 required extensive structural manipulation and refinement, along with an appreciation of SAR differences associated with the cap stereochemistry, to redefine and optimize molecular topology as structural changes were introduced that were designed to address targeted parameters. Resolving these problems required considerable effort and persistence, rewarded by the initial clinical results in which a profound and rapid effect on viral load was observed following dosing of a single milligram of 6 to HCV-infected patients. The discovery of 6 is a testament to the power of phenotypic screening to uncover unanticipated mechanisms and is particularly effective for those that cannot be readily assessed using biochemical assays as is the case for HCV NS5A. HCV is the leading cause of liver transplant in the United States, and in 2008 it surpassed HIV-1 infection as the leading viral cause of death.60 Unlike HIV-1 infection, HCV replication occurs without a DNA intermediate and is curable with small molecule DAAs. At the time of writing, we stand on the threshold of being able to eradicate a disease that in the absence of therapeutic intervention was predicted to be the underlying cause of deaths well in excess of 20 000 per year between 2022 and 2048 in the United States, a statistic projected to amount to over 1 million total deaths by 2060.61 Daclatasvir has been evaluated in more than 5500 patients and has had a meaningful and lasting impact on HCV therapy, and it is anticipated that NS5A inhibitors will play a key role in the eradication of HCV, a virus that is responsible for a significant amount of human suffering.20g

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ABBREVIATIONS USED ADME, absorption, distribution, metabolism, and excretion; AUC, area under the curve; b.i.d., bis in die (twice a day); BVDV, bovine viral diarrhea virus; CC50, concentration at which cytotoxicity is half-maximal; cDNA, complementary deoxyribonucleic acid; DAA, direct-acting antiviral agent; DMSO, dimethyl sulfoxide; EC50, concentration at which inhibition is half-maximal; EU, European Union; FDA, Food and Drug Administration; HCV, hepatitis C virus; HIV-1, human immunodeficiency virus 1; GT, genotype 1b; LEDG-F, lens epithelium-derived growth factor; MAD, multiple ascending dose; NS, nonstructural; PEG-IFN-α, pegylated interferon α; PK, pharmacokinetic; q.d., quaque die (once per day); RBV, ribavirin; RNA, ribonucleic acid; SAD, single ascending dose; SAR, structure−activity relationship; SVR, sustained virological response.

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AUTHOR INFORMATION

Corresponding Authors

*M.B.: phone, 203-677-6928; e-mail, makonen.belema@bms. com. *N.A.M.: phone, 203-677-6679; e-mail, nicholas.meanwell@ bms.com. K



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Drug Annotation

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Drug Annotation

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Discovery of Daclatasvir, a Pan-Genotypic Hepatitis C Virus NS5A

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