Discovery of a Potent Boronic Acid Derived Inhibitor of the HCV RNA

Subscriber access provided by FLORIDA STATE UNIV

Article

Discovery of a Potent Boronic Acid Derived Inhibitor of the HCV RNA-Dependent RNA Polymerase Andrew Maynard, Renae M. Crosby, Byron Ellis, Robert Hamatake, Zhi Hong, Brian A. Johns, Kirsten M. Kahler, Cecilia Koble, Anna L. Leivers, Martin R. Leivers, Amanda Mathis, Andrew J. Peat, Jeffrey J. Pouliot, Christopher D. Roberts, Vicente Samano, Rachel M. Schmidt, Gary K. Smith, Andrew Spaltenstein, Eugene L. Stewart, Pia Thommes, Elizabeth M. Turner, Christian Voitenleitner, Jill T. Walker, Greg Waitt, Jason Weatherhead, Kurt L Weaver, Shawn Williams, Lois Wright, Zhiping Z. Xiong, David Haigh, and J. Brad Shotwell J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400317w • Publication Date (Web): 14 May 2013 Downloaded from http://pubs.acs.org on May 17, 2013

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 43

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Discovery of a Potent Boronic Acid Derived Inhibitor of the HCV RNA-Dependent RNA Polymerase Andrew Maynard2, Renae M. Crosby1, Byron Ellis2, Robert Hamatake1, Zhi Hong1, Brian Johns1, Kirsten M. Kahler2, Cecilia Koble1, Anna Leivers1, Martin R. Leivers1, Amanda Mathis4, Andrew J. Peat1, Jeffrey J. Pouliot1, Christopher D. Roberts1, Vicente Samano1, Rachel M. Schmidt2, Gary K. Smith2, Andrew Spaltenstein1, Eugene L. Stewart2, Pia Thommes3, Elizabeth M. Turner1, Christian Voitenleitner5, Jill T. Walker1, Greg Waitt2, Jason Weatherhead1, Kurt Weaver2, Shawn Williams2, Lois Wright2, Zhiping Z. Xiong1, David Haigh3, and J. Brad Shotwell1,* 1

GlaxoSmithKline, Infectious Diseases Medicines Discovery Unit, 5 Moore Drive, Research

Triangle Park, NC 27709-3398 2

GlaxoSmithKline, Platform Technology and Science, 5 Moore Drive, Research Triangle Park,

NC 27709-3398 3

GlaxoSmithKline, Infectious Diseases Centre for Excellence for Drug Discovery, Gunnels

Wood Road, Stevenage, Hertfordshire, UK SG1 1NY 4

Current Address: Salix Pharmaceuticals Inc., 8510 Colonnade Center Drive, Raleigh, NC

27615

ACS Paragon Plus Environment

Page 2 of 43

Page 3 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


5

Current Address: Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404

KEYWORDS: HCV RNA-Dependent RNA Polymerase, boron, NS5B ABSTRACT: A boronic acid moiety was found to be a critical pharmacophore for enhanced in vitro potency against wild type hepatitis C replicons and known clinical polymorphic and resistant HCV mutant replicons. The synthesis, optimization, and structure-activity relationships associated with inhibition of HCV replication in a sub-genomic replication system for a series of non-nucleoside boron-containing HCV RNA-Dependent RNA Polymerase (NS5B) inhibitors are described. A summary of the discovery of 3 (GSK5852), a molecule which entered clinical trials in subjects infected with HCV in 2011, is included. Introduction. Of all new chemical entities approved by the US FDA only bortezomib incorporates the element boron.1 Despite the opportunity for a tremendous increase in compositional diversity and novelty2 via the incorporation of boronic acids or related boron heterocycles into drug candidates, boron remains poorly represented in the patent literature and in the clinic. This lack of representation has perhaps at its roots the perception within the medicinal chemistry community that boron functions as an intrinsically (chemically or metabolically) unstable Lewis acid. Recently a growing awareness has emerged that this view is likely a false one.3,4,5 Reports describing the characterization of novel boron-containing substructures6 in the context of both pre-clinical7,8,9 and clinical10,11 efforts are becoming more common. Boronic acids have been well-characterized as pharmacophores for the reversible covalent inhibition of hydroxyl proteases,12 and recently we13 and others14 have disclosed several series of potent inhibitors of the HCV protease which incorporate a boronic acid warhead at the P4 and P1



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

sites respectively. We became interested in a unique opportunity to profile boronic acid isosteres within the context of HCV RNA-dependent RNA polymerase (RdRp) inhibitors. The NS5B protein consists of 591 amino acids and is responsible for synthesizing new viral RNA. We envisioned 115 (Figure 1), a prototypical NS5B Palm II inhibitor, could serve as a starting scaffold for leveraging boron-based chemistry. We recognized that the orientation of the Palm II pocket allowed for the positioning of an inhibitor with a boronic acid (or other polar moiety) near the catalytic site and/or RNA/NTP channel. Here, a boronic acid could also interact directly with the incoming NTP or the growing RNA chain itself. 16 Boronic acids are known to couple to the ribose units of nucleotides, 17 and modeling suggested the benzofuran scaffold of 1 could position such a group within proximity of a growing viral RNA chain. There was also an opportunity to specifically interact with key nucleophilic residues (for example S365) flanking the Palm II allosteric site by a direct reversible covalent bond, which could enhance the desirable slow binding kinetics reported for 1.18 Consistent with the high sequence conservation of the Palm II site, 1 exhibits similar potency across wild-type genotypes 1-6 (as assessed by HCV replicons). This is in contrast to inhibitors that bind to NS5B at other allosteric sites. Despite initially promising clinical results, progression of 1 was ultimately discontinued due to safety findings.19 Additionally, a significant shift in potency against genotype (GT) 1b 316N, a key polymorph present in 40% of GT1b patients,20 potentially limited efficacy in a large number of patients. Additionally, the emergence of highly resistant GT1a virus bearing a C316Y mutation during clinical trials in patients treated with 1 was observed. Generally, the design of allosteric inhibitors of NS5B has been complicated by (i) a lack of sequence homology within most known allosteric binding sites of the diverse HCV genotypes 1-621, (ii) intra-genotype polymorphism within the allosteric sites in


Page 4 of 43

Page 5 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


significant portions of the HCV patient population (including 316N in Palm II)22, and (iii) the propensity for rapid emergence of drug-resistant mutations as a result of the low genetic barrier to resistance associated with previously reported allosteric inhibitors and the error-prone replication of HCV.23,24 It was therefore of interest to investigate whether introduction of a boronic acid pharmacophore might provide unique advantages for targeting a subset of NS5B polymorphs and resistant mutants. As multiple ligand-bound crystal structures were available for this protein, structure-based design of boron-based Palm II inhibitors offered a direct approach to explore robust HCV inhibition by addressing known mutational deficiencies (GT 1a316Y, GT 1b316N, GT 1b316Y) of 1. Herein we report the discovery, synthesis, and structure activity relationships of potent, pan-genotype boronic acid HCV polymerase inhibitors where the boronic acid functionality was found to be a key pharmacophore for replicon potency. We include the first crystallographic data for clinically relevant GT1a and GT1b NS5Bs (316Y), an efficient diastereoselective synthesis of novel chiral oxaborole 2, evidence of slow binding kinetics to isolated NS5B protein, and the first disclosure of boronic acid 3 (GSK5852, Figure 1), an aryl boronic acid which was progressed to proof of concept studies in human HCV subjects in 2011.25 Palm II site structural biology, modeling, and initial design strategy. Of the four well established NS5B allosteric inhibitor sites, the Palm II non-nucleoside inhibitor (NNI) site is the most proximal to the catalytic site of the polymerase.21 Crystallographic studies carried out with 1 demonstrated that the Palm II site is strategically positioned adjacent to the conserved catalytic GDD motif at the junction formed between the incoming nucleotide triphosphate (NTP) substrate and RNA template channels.18 This key RNA junction serves as the active site for RNA priming, initiation, and elongation. The Palm II inhibitor pocket consists of a highly conserved



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

hydrophobic cleft formed between the serine-rich S365CSS loop and P197-S210 helix of the palm. Typically, this pocket is induced upon binding of the ligand, whereby the sidechains of conserved R200 and S365 rotate to accommodate inhibitors.18 A model of HCV RdRp with a growing RNA strand was constructed using the homologous polio RdRp:RNA crystal structure. Boronic acid containing derivatives of 1 were then added and the location of the boronic acid was optimized in silico. Optimization was guided by potential interactions with the incoming NTP or growing RNA strand. Figure 2 depicts the binding mode of 3 in the Palm II site. While the majority of inhibitor 3 contacts are located in the palm domain (gray), there are also contacts with the β-hairpin flap (yellow), specifically Y448 (See Figure 5 and 7). Y448 is conserved across HCV genotypes. The β-flap has been implicated in the regulation of polymerase initiation and the binding of template RNA. 26 More recently, Y448 has been reported to orient GTP for polymerase de novo initiation.27 We superimposed NS5B with experimental structures of related RdRps, some including the presence of co-crystallized viral RNA, to assess variation of the Palm II site in the presence or absence of RNA. Based on these structural comparisons, we found either an aryl boronic acid (as depicted for 3 in Figure 2) or an extension of the hydroxyl ethyl sidechain present in 1 with a methylene boronic acid moiety (i.e. 5, Scheme 1), would position the polar boronic acid moiety close to the putative incoming NTP.27,28 Figure 2b depicts the location of the aryl boronic acid of 3 at the center of the NTP/RNA channel. This view is magnified in Figure 2c in the context of incoming NTP (a GTP dinucleotide primer) by superposition with a HCV GT1a construct.29 Similarly, Figure 2d depicts the location of 3 relative to the viral RNA primer-template duplex, based on NS5B superposition with poliovirus RdRp in “active” complex with an extended RNA primer-template duplex.30 The orientation of the nucleotides in the GT1a NS5B:GTP structure31 is in register


Page 6 of 43

Page 7 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with the base-pairing of the polio RdRp:RNA structure. Thus, homology modeling suggested that 3 and related analogs could disrupt polymerase initiation, as well as priming of the +0 nucleotide position. Therefore, locating a boronic acid at this position suggested potential inhibition of NS5B through multiple mechanisms, including restriction of the β-flap to an inactivated state and/or direct interference of RNA processing. We anticipated this strategy could offer novel opportunities to resolve many of the deficiencies associated with 1 (and generally other NS5B NNIs), particularly with respect to achieving potency across polymorphic NS5B variants.32,33 Chemistry and Structure-Activity Results and Discussion. Initially this hypothesis was tested by directly modifying the sulfonamide of 1 by extension of the hydroxyethyl moiety or introduction of an aryl linker group to access the NTP/RNA channel. Alkylation of 1 directly with commercially available 2-(bromomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane and potassium carbonate in DMF followed by acidic deprotection of the pinacol ester in the presence of polymer-supported benzeneboronic acid to sequester residual pinacol afforded boronic acid 5 (Scheme 1).34,35 Similarly, direct alkylation of sulfonamide 6 with either 3- or 4(bromomethyl)phenyl)boronic acids afforded 7 and 8, respectively.34,35 At first, boronic acids 5, 7, and 8 were profiled alongside 1 in three cell-based HCV replication systems (replicon GT1a, GT1b, and GT1b 316N; after 2 day incubations) as well as in a GT1b biochemical polymerase assay (Table 1). Boronic acids 5, 7, and 8 demonstrated improved potency in the HCV replicon systems relative to 1 (Table 1). In addition to modest (2-5 fold) improvements against GT1a and GT1b wild-type replicons, 5 showed a roughly 40-fold improvement in potency relative to 1 against a GT1b replicon containing key clinical polymorph 316N (Table 1). The aryl boronic acid 7 also demonstrated improvements in in vitro potency against this polymorph. The



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

differences in biochemical potency with isolated NS5B were not as apparent as IC50s in this assay were within error for all analogs profiled and likely approached the limit of detection in our enzyme assay (Table 1, see Supporting Information for protocol, NS5B concentration = 30 nM). To test whether the boronic acid was unique relative to other polar functionality with respect to 1b316N replicon potency, we profiled a number of direct boronic acid replacements for 7 including a phosphate (9), carboxylic acid (10), sulfonic acid (11), phosphonate ester (12) and phenyl ring (13). The corresponding dimethyl phosphate, methyl amide, sulfone, nitrile, methyl ester, phenol, sulfonamide, and hydroxyamide (see Supporting Information Table SI-3) were also profiled. Consistent with our hypothesis, 5 showed inhibition of GT1b C316N with a shift relative to WT 1b replicon potency much less than other analogs. While polar anionic functionality in general afforded potent biochemical inhibitors (i.e. 9-11), as anticipated these inhibitors suffered from poor permeability. This was in contrast to the potency and good permeability seen with boronic acids 5, 7, and 8. Notably, even those analogs which had good permeability (1, 12, 13) demonstrated a characteristic shift between GT1b and GT1b C316N replicon potency (≥ 5 fold) whereas linear boronic acid 5 was equipotent when profiled in both replicons (See Supplementary Table SI-7 for chromatographic log D, artificial membrane permeability, and MDCK permeability data for 1, 3, 5, 7-13). We considered a shift in potency against GT1b 316N for 1 a risk to be recapitulated in human clinical trials and as such we considered it an important property requiring optimization. Incorporation of a boronic acid functionality elsewhere on the benzofuran core, specifically replacement of the pendant methyl amide with boron functionality anticipated to engage conserved S365 directly, did not result in improved antiviral properties (see Supporting Information Table SI-4). As such, additional efforts focused on targeting the RNA/NTP tunnel with boron-containing groups.


Page 8 of 43

Page 9 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Both boronic acids 5 and 7 exhibited poor in vivo DMPK in rat. Linear boronic acid 5 was readily oxidized to 1 in vivo in rat (Cl = 25 ml/min/kg) while 7 demonstrated 10:1 diastereoselectivity over two steps. Direct reduction to alcohol 25 followed by Mitsunobu esterification with 6 and subsequent hydrogenation afforded 2. The absolute configuration assigned earlier by VCD was consistent with the anticipated diastereoselectivity from the aldol reaction and analysis using the earlier separation method indicated 2 was obtained in >95% ee. Notably, the present approach is complementary to recent reports by Molander wherein ephedrine-based auxilliaries are implemented to install boron moieties (via alkylation with bromo-methyl dioxaborolanes) in the diastereoselective aldol step.37 Due to the propensity for β-hydroxy amides to undergo elimination under related alkylation conditions, the synthesis of 2 was best accomplished through


Page 11 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the strategy illustrated in Scheme 3. Indeed it was found that a β-boryl group was compatible with the aldol chemistry and not prone to elimination. Access to gram quantities of 2 allowed for its broader virologic characterization and higher species DMPK profiling. Benzofuran 1 has shown significantly reduced potency against a number of other mutations near the palm site and a marked shift in its ability to inhibit replicon carrying the 316N polymorph. Specifically, other mutations at residues C316 and S365 have been reported as particularly problematic, giving rise to EC50 shifts of >100 fold in most cases. Oxaborole 2 shows an improvement in its ability to inhibit the replication of these mutant GT1b replicons relative to 1 (Table 3), but showed a 100 fold loss in potency for a GT1a 316Y mutant replicon (EC50 = 830 nM) relative to wild type replicon (EC50 = 7.6 nM, Table 2). The poor activity against GT1a 316Y mutant virus was of particular concern, as the mutation was observed in individuals treated with 1 monotherapy indicating that patient viruses containing GT1a 316Y are viable resistant mutants. Additionally, further in vitro fitness increases of nearly tenfold have been reported with secondary fitness mutations in combination with 316Y in replicons.18, 20, 32 To minimize the potential GT1a 316Y liability for 2, we assessed the activity of other compounds in the series to ascertain their profiles against GT1a 316Y. Initially we focused on boronic acid 7 for further structure-activity studies because it was more potent than 2 against GT1a 316Y (Table 3). Because this potency was improved (7-fold) relative to 2, in addition to representing a smaller fold-shift relative to the GT1b 316N potency, we synthesized a set of functionalized aryl boronic acids in an attempt to further enhance potency. To access bicyclic boron containing motifs, 26 was subjected to sequential carbonylation, Sandmeyer, and radical bromination conditions which afforded 27. Direct alkylation of 6 with 27 followed by borylation



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

under standard conditions afforded 28 (Scheme 4). The boronic acid moiety proved stable to a variety of conditions, including direct reduction with superhydride to give 4 (29-33 were prepared in an analogous manner).34,35 Clinical candidate 3 and related ortho substituted boronic acids were prepared in a similar manner to 7 via direct alkylation with the corresponding benzyl bromide containing either the boronic acid moiety (for 3,7, and 8) or an aryl bromide precursor which was subsequently converted to the boronic acid (for 34 and 35).34,35 The structure-activity trends for the oxaborole analogs (Table 4) were consistent with those observed for 7 and 8. Specifically, analogs which have a para substituted aryl boronic acid relative to the connection to the core (4 and 29) proved superior to analogs with meta-disposed boronic acids (30 and 31) when profiled against the 1b316N polymorph. This observation is consistent with the predicted positioning of the boronic acid within the NTP/RNA tunnel. Other arrangements of the boronic acid about the aryl ring including chiral 32 and 6-membered ring 33 showed promising potencies across wild type replicons. Additionally, ortho-substitution adjacent to the boronic acid was well tolerated, with fluoro-analog 3 having higher potency to electron-donating methoxy 34 or the methyl analog 35 against the GT1b 316N replicon. Installation of fluorine ortho to the boron moiety of 29 afforded 4, but did not result in a boost in GT1b 316N potency relative to 29 as was observed for the 7, 3 pair. As observed in other series, direct inhibition of isolated NS5B protein was relatively invariant to compound, while the cell-based replicon assays proved to be more discriminating. Although 3, 4, 32, and 33 showed similar activities against GT1b (≤ 6-fold) and the GT1b 316N polymorph (Table 4), substantial differences in potencies were observed against other HCV mutant replicons (Table 3). For example a ≥ 50-fold difference in in vitro GT1a 316Y potency was observed between the compounds, with only 3 potently inhibiting this mutant (EC50 < 10 nM).


Page 12 of 43

Page 13 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Although 3 was a potent inhibitor, its mechanism of action was not well understood. Potency in the replicon system had been systematically optimized, but a relationship between the cell-based and biochemical potency as measured for the series had not been established (especially for 1 vs. 3). Additionally, although in silico modeling has been utilized by others,18 no crystallographic data for GT1a or GT1b NS5B possessing a 316Y mutation had been previously reported. Unfortunately, the models offered little explanation for the exceptional potency of 3 relative to 2 or 4 against the GT1a 316Y mutant.18 To address these concerns, we prioritized obtaining both biochemical kinetic data and crystallographic structural data so as to better understand the unique profile of 3 and to guide future compound design.

Measurement of koff for 3 and 1. The enzyme IC50 data in Tables 1, 2, and 4 were determined after a pre-incubation of enzyme with inhibitor for 15 minutes. Suggestive of slow-binding kinetics, increased preincubation time to 45 or 90 minutes results in increased potency of 3 (data not shown). Accordingly, the primary driver for compound potency throughout the optimization phase of this series focused on the cell-based replicon system (2-day compound incubation). Off-rates for 3 and 1 were measured to further explore the slow-binding kinetics with isolated GT1b 316N protein. The enzyme was incubated with either 3 or 1 to pre-form an enzyme:inhibitor complex. The solution was then rapidly diluted 20-fold into an excess of the other inhibitor (that is, the 3:NS5B complex was diluted into excess 1, and vice versa) to allow the bound inhibitor to dissociate and to prevent rebinding. The rate of dissociation of the inhibitor from the enzyme was determined by rapid size exclusion filtration of the NS5B:inhibitor complexes. This procedure was followed by measurement of the concentration



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of each bound inhibitor by mass spectrometry. The results of these experiments are shown in Figure 3. Our method measures both 3 and 1 simultaneously, so that 3 (top panel) dissociation is measured both as a decrease in bound 3 and an equal increase in bound 1. Similarly, dissociation of 1 (bottom panel) is measured both as a decrease in bound 1 and as an increase in bound 3. As shown in Figure 3, the dissociation of 3 is qualitatively much slower than dissociation of 1 from isolated GT1b 316N NS5B protein. These data are consistent with similar experiments carried out using 3H-3 (not shown). Dissociation rate constants were calculated from these data. The dissociation of 1 was best fit to a single exponential that gives a calculated t1/2 of 1.7 hours, and is in good agreement with previous reports that state the 1:NS5B complex requires two hours to reach equilibrium.18 We attempted to fit the dissociation of 3 to a single exponential, however, the data are better fit to a mechanism in which 21% of the compound dissociates with a “fast” off-rate, t1/2 value of 4.3 hrs, and 79% dissociates with a much slower off-rate, t1/2 value of 69.3 hours. The determination of two dissociation rates can be indicative of a structural reorganization of the enzyme inhibitor complex. Overall, the data show that the majority of 3 dissociates from NS5B approximately 40-fold slower than does 1. The slower dissociation rate is consistent with an increased GT1b 316N replicon potency for 3 (1.9 nM) relative to 1 (100 nM). Compound 3 inhibits NS5B Initiation. Due to the partial overlap in compound binding sites, it has been suggested that allosteric NS5B Palm II binders should behave mechanistically similar to NS5B Palm site I binders and inhibit the initiation step of the polymerase cycle. This concept was tested directly for 3 by measuring inhibition of a biochemical assay that used purified NS5B to catalyze de novo initiation. An RNA template of sequence 5’-A8CG-3’ was used to drive production of a pCpG dimer product in a non-primer driven reaction. NS5B was capable of


Page 14 of 43

Page 15 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


adding GTP to a radiolabeled CTP nucleoside in uninhibited reactions (Figure 4, Lane 1). Addition of increasing amounts of 3 inhibited this reaction (Figure 4, Lanes 3-7) until no product was visible at high concentrations of inhibitor. This de novo initiation assay demonstrates 3 is capable of blocking initiation and 3 is the first Palm II NS5B inhibitor for which experimental evidence has been published supporting this mechanism of action. NS5B:Inhibitor Crystallographic Studies. HCV NS5B proteins from GT1b 316N, GT1a, GT1b 316Y, and GT1a 316Y were cloned, expressed, purified, and found to be suitable for crystallography. All proteins were crystallized and then co-incubated with 1, 3, 4 and 32. Figure 5 shows crystal structures of GT1a, GT1a 316Y, and GT1b 316N NS5B proteins with 3. In all structures the orientation of the inhibitor core is essentially identical. The pendant fluoro phenyl ring occupies the hydrophobic recess of the Palm II cleft and the benzofuran core forms a cationπ interaction with R200. R200 is conserved across all HCV genotypes and plays a critical role in the binding mode of 3 in all NS5B constructs. Relative to the apo crystal structure, R200 pivots to accommodate and interact with 3. Additionally, R200 anchors a network of key hydrogen bond interactions to the sulfonamide and carboxamide groups of the inhibitor, the backbone carbonyl of G192, and theY448 hydroxyl. S365 also consistently hydrogen bonds with the carboxamide of 3 in the pocket. Towards the solvent/RNA front, the benzyl boronic acid group of 3 forms favorable interactions with M414 and Y448. The boronic acid also has interactions with bound water molecules. In the GT1a 316Y mutant (Figure 5b) F193 rotates to accommodate the C316Y mutation, as 316Y is oriented towards the solvent front in proximity to the boronic acid of 3. In Figure 6a, the boronic acid of 3 is observed to form a through-water hydrogen bond with 316Y in the 1b316Y mutant, while compound 4 and 32 are able to make direct hydrogen bonds with 316Y (Figure 6b and Figure 6c). In contrast, the hydroxyl ethyl



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

sidechain of 1 is oriented away from 316Y, though a water is observed to contact 316Y (Figure 6d). R200 is also flipped in the interaction with 1 compared its orientation with 3, 4, and 32. Contrary to our original hypothesis, the tremendous potency enhancements associated with the boronic acid functionality of 3 do not appear to be due to the formation of a covalent bond with any nearby residue. Additionally, we have seen no crystallographic evidence of hydration or a charged boronate. From the standpoint of inactivating the β-hairpin flap which regulates polymerization, the binding of 3 completes the formation of a hydrophobic pocket (comprised of F193, P197, M414, F551 and 3) in which Y448 is buried (Figure 7). Y448 is located at the tip of the β-flap and is conserved across all HCV genotypes. The benzyl ring of 3 provides a hydrophobic buttress against Y448, while the Y448 hydroxyl forms an intermolecular hydrogen bond with the sulfonamide of 3 and/or an intra-NS5B hydrogen bond with R200 in the back of this pocket. This is consistent with the SAR of 13, which has reasonable replicon potency (Table 1, GT1a EC50 = 58 nM, GT1b EC50 = 18 nM) and features only a simple unsubstituted benzyl ring. Removal of the benzyl ring substantially degrades replicon potency (EC50 > 500 nM). Restricting Y448 in this manner likely traps the β-flap in an inactivated state, inhibiting polymerase initiation, RNA binding, and progression to RNA elongation. All attempts to co-crystallize RNA in the presence of inhibitors were unsuccessful. Recently, a HCV NS5B GT2a construct, with the β-flap deleted, enabled resolution of NS5B in complex with viral primer-template RNA.27 Applying NS5B homology, superposition of these RNAbound structures with inhibitor co-crystal structures reported here suggests 3 can also interact with the +0 nucleotide of the primer strand to disrupt polymerization, in addition to inactivating


Page 16 of 43

Page 17 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the β-flap/Y448. Intriguingly, superposition yields the ribose of the +0 nucleotide oriented towards the boronic acid of 3, such that the 2’ hydroxyl is within 3Å of the boron (Figure SI-12, Supplementary Information), suggestive of a transient covalent interaction with the ribose or potential steric interference. Figure 8 depicts the superposition of the HCV NS5B β-flap along the axis of the double stranded RNA product resulting from polymerization, emphasizing the inactivated conformation of the regulatory β-flap (yellow). Taken together, comparison of crystallographic data suggests that 3 inhibits polymerization primarily through two mechanisms: (i) stabilization of the β-flap in a closed, inactivated state to inhibit template binding and primer initiation, including restricting Y448 to participate in de novo initiation (consistent with the initiation studies in Figure 4), and/or (ii) disruption of the RNA processing channel either through direct steric contacts or transient adduct formation between the +0 ribose and the boronic acid of 3. Notably, the introduction of boronic acid moieties uniquely enhance the robustness of replicon inhibition. Structural data reveals direct and through-water hydrogen bond contacts with the boronic acid and key mutants, (particularly 316Y). Additional interactions, potentially including interaction with viral RNA, or altered NS5B:inhibitor interactions in the presence of RNA, await further characterization. Conclusions. Despite a growing number of reports highlighting applications of boronic acids within the context of inhibitor design, few studies describe the optimization of boron-containing molecules with a balance of properties ultimately suitable for clinical investigations in man. We have discovered a series of potent inhibitors of the NS5B polymerase wherein a boronic acid moiety has been demonstrated to be a critical pharmacophore. Potency against key resistance mutants was optimized to provide clinical candidate 3. Clinical candidate 3 shows slow binding



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

kinetics with isolated GT1b 316N protein, has been characterized with a dissociation half-life of > 40 hours (Figure 3), and inhibits the initiation step of the polymerase RNA replication cycle (Figure 4). We have reported elsewhere a full virologic (including several resistant replicons specific to 3)33 and pharmacokinetic25 characterization of 3 in mouse, rat, dog, monkey, and man. Notably, 3 showed a statistically significant reduction in serum HCV ribonucleic acid (RNA) following a single dose of 420 mg (-1.33 log10 copies/mL) compared with placebo (-0.09 log10 copies/mL) at 24 h post-dose in subjects infected with hepatitis C virus but is not being further pursued in clinical trials.25 Acknowledgements. We thank Dean Phelps for the VCD analysis for compound 2. All authors are current or former employees of GlaxoSmithKline. Experimental Section HCV Stable and Transient Replicon assays and NS5B polymerase assays. Experimental protocols and method for IC50 determination can be found in the Supporting Information. NS5B Initiation Assay. The initiation assay utilized genotype 1b NS5B (strain BK) containing the mutations L47Q, F101Y, and K114R, a C-terminal ∆21 truncation, and a C-terminal hexahistidine tag to drive de novo initiation from an oligo template of the sequence 5’AAAAAAAAGC-3’ (Integrated DNA Technologies, Coralville IA). Reactions contained 2.7 µM NS5B, 1 mM GTP, 100 µM CTP, 10 µCi [α33P]-CTP, and 23 µM RNA template in a buffer of 50 mM HEPES pH 7.3, 10 mM magnesium chloride, and 5 mM dithiothreitol, and were begun either by addition of enzyme or by addition of RNA template. After a 30 minute incubation at 30oC, reactions were terminated by addition of 90% formamide and freezing on dry ice.


Page 18 of 43

Page 19 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Samples were run on 25% polyacrylamide 7M urea TBE gels and quantitated with a Typhoon system. Compound Purities & Identity. All compounds underwent an immediate processing quality control protocol to confirm identity and determine relative purity immediately before processing in the replicon or NS5B polymerase assays. Analysis is by UPLC-MS with UV diode array detection to determine purity and MS was used to confirm molecular weight. A Waters Acquity UPLC system comprising Binary Solvent manager, Sample Manager, PDA Detector, Waters ZQ or SQD mass spectrometer, Waters Acquity Evaporative Light Scattering Detector or Polymer Laboratories Evaporative Light Scattering Detector were employed. Mobile phases: acetonitrile + 0.1% formic acid; water + 0.1% formic acid. Wash solutions: strong wash 100% acetonitrile + 0.1% formic acid; weak wash 50:50 acetonitrile:water + 0.1% formic acid. All individual lots of compounds tested ≥ 95% pure (unless otherwise noted) according to this protocol (see Supporting Information Table SI-2 for purity ranges). Absolute Stereochemical Assignments by VCD. Absolute stereochemical assignments for 2 and 17 were initially made by comparing an ab initio VCD analysis (see Supporting Information Section VI) with spectra obtained from enantiomerically pure (> 95% ee) samples of 2 and 17 obtained by resolution of 16 using supercritical fluid chromatography (25% MeOH/CO2, 140 bar, 10 mL/min, ChiralPak IC 10X250 mm, 254 nM). The estimated confidence limit of this analysis (see Supporting Information Figure SI-11) was 94% and afforded absolute configurational assignments consistent with the synthesis of 2 using the diastereoselective route described in Scheme 3.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 43

Diastereoselective synthesis of 2. (4R,5S)-3-acryloyl-4-methyl-5-phenyl-1,3-oxazolidin-2-one (23). A solution of 2-propenoic acid (12.59 mL, 183 mmol) and triethylamine (49.2 mL, 353 mmol) in tetrahydrofuran (900 mL) maintained at -20°C was treated dropwise with acryloyl chloride (13.8 mL, 169 mmol). A thick white precipitate formed and the mixture was maintained with stirring at -20°C for 1 hour. Lithium chloride (7.78 g, 183 mmol) was added (as a solid) in one portion followed by (4R,5S)-4-methyl-5-phenyl-1,3-oxazolidin-2-one (25g, 141 mmol) as a solution in tetrahydrofuran (150 mL). The mixture was warmed to room temperature, maintained with stirring for four hours, and concentrated under reduced pressure. The solids were dissolved in dichloromethane and water and the organic layer was washed with brine, dried over sodium sulfate, filtered, stripped onto celite, and purified by column chromatography to afford (4R,5S)-3-acryloyl-4-methyl-5-phenyl-1,3-oxazolidin-2-one (23) (25.26 g, 109 mmol, 77 % yield) as a clear oil. 1H NMR (400 MHz, CDCl3) δ ppm 7.36 - 7.60 (m, 4 H) 7.29 - 7.36 (m, 2 H) 6.58 (dd, J=17.00, 1.76 Hz, 1 H) 5.93 (dd, J=10.36, 1.76 Hz, 1 H) 5.72 (d, J=7.23 Hz, 1 H) 4.73 - 4.93 (m, 1 H) 0.95 (d, J=6.64 Hz, 3 H).

(4R,5S)-3-((S)-3-(benzyloxy)-2-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)methyl)propanoyl)-4-methyl-5-phenyloxazolidin-2-one (24). A suspension of sodium tertbutoxide (0.419 g, 4.36 mmol), DPEPhos (0.78 g, 1.45 mmol), and copper(I) chloride (0.14 g, 1.45 mmol) in THF (50 mL) was stirred at room temperature, cooled to 0°C, and treated dropwise with bis(pinacolato)diboron (12.91 g, 50.9 mmol) in THF (50 mL). Stirring was maintained for 45 minutes and then (4R,5S)-3-acryloyl-4-methyl-5-phenyl-1,3-oxazolidin-2-one (23) (11.20 g, 48.4 mmol, in THF, 50 mL) was added dropwise. The solution was warmed to room temperature and maintained with stirring for 3 hours. The mixture was passed through


Page 21 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


glass filter paper, diluted with celite, concentrated, and purified by column chromatography to afford intermediate (4R,5S)-4-methyl-5-phenyl-3-[3-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2yl)propanoyl]-1,3-oxazolidin-2-one (8.13 g, 22.63 mmol, 47% yield) as a white solid. 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.32 - 7.47 (m, 5 H) 5.78 (d, J=7.41 Hz, 1 H) 4.77 (t, J=6.93 Hz, 1 H) 2.86 - 3.16 (m, 2 H) 1.16 - 1.31 (m, 12 H) 0.95 (q, J=6.50 Hz, 2 H) 0.83 (d, J=6.63 Hz, 3 H). A solution of (4R,5S)-4-methyl-5-phenyl-3-[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)propanoyl]-1,3-oxazolidin-2-one (5.0 g, 13.92 mmol) in DCM (69.6 ml) was cooled to 0°C and then treated by the addition of TiCl4 (1M in DCM, 16.70 ml, 16.70 mmol). The yellow reaction was stirred for 10 minutes, then DIEA (3.04 ml, 17.40 mmol) was added to the reaction. The reddish reaction was treated with chloromethyl phenylmethyl ether (2.51 ml, 18.09 mmol). The mixture was slowly allowed to warm to room temperature with stirring for 2 hours. The reaction was then diluted with additional dichloromethane, saturated aqueous NH4Cl, and water. The layers were separated and the combined organic layers were washed with saturated NaHCO3 and brine. The combined organic layers were dried over Na2SO4, filtered, and concentrated to afford

(4R,5S)-3-((S)-3-(benzyloxy)-2-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)methyl)propanoyl)-4-methyl-5-phenyloxazolidin-2-one as a crude residue (quant. yield, used without further purification).

(R)-3-(benzyloxy)-2-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methyl)propan-1-ol (25).

A

solution of (4S,5R)-4-methyl-5-phenyl-3-{(2R)-3-[(phenylmethyl)oxy]-2-[(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)methyl]propanoyl}-1,3-oxazolidin-2-one (24) (3.46g, 8.30 mmol) in THF (41.5 ml) and MeOH (0.40 ml, 9.96 mmol) was placed under a nitrogen atmosphere, cooled to 0°C, and treated with LiBH4 (2M solution in THF, 4.98 ml, 9.96 mmol). After stirring at room



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 43

temperature overnight, the reaction was quenched by the addition of MeOH and saturated NH4Cl and extracted with ethyl acetate. The combined organics were washed with water, dried over Na2SO4, filtered, and concentrated. The crude residue was purified by column chromatography to afford (R)-3-(benzyloxy)-2-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methyl)propan-1-ol (25) as a clear oil (1.03g, 41% yield). 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.16 - 7.39 (m, 5 H) 4.48 (s, 2 H) 4.09 (q, J=7.23 Hz, 1 H) 3.51 - 3.62 (m, 1 H) 3.41 - 3.51 (m, 2 H) 3.33 3.41 (m, 1 H) 2.02 - 2.11 (m, 1 H) 1.20 (d, J=2.93 Hz, 12 H) 0.75 (dd, J=7.23, 2.93 Hz, 2 H).

5-cyclopropyl-2-(4-fluorophenyl)-6-[{[(4R)-2-hydroxy-1,2-oxaborolan-4 yl]methyl}(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide (2). A solution of 5cyclopropyl-2-(4-fluorophenyl)-N-methyl-6-[(methylsulfonyl)amino]-1-benzofuran-3carboxamide (175 mg, 0.435 mmol), (2R)-3-[(phenylmethyl)oxy]-2-[(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)methyl]-1-propanol (233 mg, 0.761 mmol), and triphenylphosphine (257 mg, 0.98 mmol) in tetrahydrofuran (5 mL) was maintained at 0°C with stirring and treated dropwise with DIAD (0.169 mL, 0.870 mmol). The mixture was warmed to room temperature, maintained with stirring for 16 hours, diluted with celite, concentrated, and the residue was absorbed on celite. The product was purified by column chromatography (20 to 50% EtOAc/hexanes) to afford

5-cyclopropyl-2-(4-fluorophenyl)-N-methyl-6-((methylsulfonyl){(2R)-3-

[(phenylmethyl)oxy]-2-[(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methyl]propyl}amino)-1benzofuran-3-carboxamide (235 mg, 0.340 mmol, 78 % yield) as a white foam with NMR spectroscopic properties identical to 10. A suspension of 5-cyclopropyl-2-(4-fluorophenyl)-Nmethyl-6-((methylsulfonyl){(2R)-3-[(phenylmethyl)oxy]-2-[(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)methyl]propyl}amino)-1-benzofuran-3-carboxamide (0.600 g, 0.869 mmol)


Page 23 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and 10% Pd/C (Degussa Type (wet). (300mg, 0.869 mmol) in tetrahydrofuran (10 mL) was maintained under 40 psi hydrogen gas with rapid stirring for 16 hours. The mixture was filtered through celite, concentrated under reduced pressure, dissolved in tetrahydrofuran (10 mL) and aqueous 5.0N HCl (1.216 mL, 6.08 mmol), and polymer supported benzene boronic acid (2.6 mmol/g) (1.67 g, 4.34 mmol) was added. The mixture was maintained with stirring at room temperature for 2 hours, filtered through celite, concentrated, and partitioned between dichloromethane and HCl (pH < 3). The organic layer was dried over sodium sulfate, filtered, taken to a residue under reduced pressure, and purified by column chromatography to afford 5cyclopropyl-2-(4-fluorophenyl)-6-[{[(4R)-2-hydroxy-1,2-oxaborolan-4yl]methyl}(methylsulfonyl)amino]-N-methyl-1-benzofuran-3-carboxamide (2) (297 mg, 0.594 mmol, 68.3 % yield) as a white foam with > 95% purity and > 95% ee% as determined by chiral chromatography (see supporting information). The residue was dissolved in acetonitrile/water, frozen, and stored on a lyophilizer for 16 hours to give a free-flowing white solid.

1

H NMR

(CDCl3) δ: 7.78-7.95 (m, 2H), 7.47 (d, J = 7.6 Hz, 1H), 7.29 (d, J = 2.1 Hz, 1H), 7.20 (m, 2H), 5.75 (br. s.,1H), 4.32 (br. s., 1H), 4.05 - 4.21 (m, 1H), 3.54 - 4.00 (m, 3H), 2.91 - 3.14 (m, 6H), 2.17 2.68 (m, 2H), 1.03 - 1.25 (m, 3H), 0.66 - 1.02 (m, 3H). LCMS (m/z, ES+) = 501 (M+1).

Author Information Corresponding Author: For JBS: phone, (919) 483-3918; email, [email protected] Abbreviations used HCV, hepatitis C virus; NTP, nucleotide triphophate; NS5B, nonstructural protein 5B; GT, genotype; NNI, non-nucleoside inhibitor; HCV RdRp, Hepatitis C Virus RNA-Dependent



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

RNA Polymerase; MDCK, Madin-Darby Canine Kidney epithelial cells; SFC, supercritical fluid chromatography; VCD, vibrational circular dichroism spectroscopy

Associated Content. Supplementary information including references to chemical syntheses, purities of final compounds, potency and structure-activity relationships for additional analogs, protocols for the NS5B polymerase and replicon assays, in vivo and in vitro DMPK protocols and data, NMR spectra and chiral HPLC traces, and a full description of the VCD analysis can be accessed free of charge via the internet at http://pubs.acs.org.

References 1. Center for Drug Evaluation and Research, Food and Drug Administration, US Department of Health and Human Services. 2011 Novel New Drugs. (Accessed January 2012). http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugInnovation/ucm285554.htm

2. Bosdet, M. J. D.; Piers, W. E. B-N as a C-C Substitute in Aromatic Systems. Can. J. Chem. 2009, 87, 8-29.

3. Nielsen, F. H. Boron in Human and Animal Nutrition. Plant Soil 1997, 193, 199-208.

4. Hunter, P. Not Boring at All. EMBO Rep. 2009, 10, 125-128.

5. Ciani, L.; Ristori, S. Boron as a Platform for New Drug Design. Expert Opin. Drug Discov. 2012, 7, 1017-1027.


Page 24 of 43

Page 25 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6. Zhdankin, V. V.; Persichini, P. J.; Zhang, L.; Fix, S.; Kiprof, P. Synthesis and Structure of Benzoboroxoles: Novel Organoboron Heterocycles. Tetrahedron Lett. 1999, 40, 6705-6708.

7. Rock, F. L.; Mao, W.; Yaremchuk, A.; Tukalo, M.; Crépin, T.; Zhou, H.; Zhang, Y.-K.; Hernandez, V.; Akama, T.; Baker, S. J.; Plattner, J. J.; Shapiro, L.; Martinis, S. A.; Benkovic, S. J.; Cusack, S.; Alley, M. R. K. An Antifungal Agent Inhibits an Aminoacyl-tRNA Synthetase by Trapping tRNA in the Editing Site. Science 2007, 316, 1759-1761.

8. Tomsho, J. W.; Arnab, P.; Hall, D. G.; Benkovic, S. J. Ring Structure and Aromatic Substituent Effects on the pKa of the Benzoxaborole Pharmacophore. ACS Med. Chem. Lett. 2012, 3, 48-52.

9. Baker, S. J.; Tomsho, J. W.; Benkovic, S. J. Boron-containing Inhibitors of Synthetases. Chem. Soc. Rev. 2011, 40, 4279-4285.

10. Baker, S. J.; Zhang, Y.-K.; Akama, T.; Lau, A.; Zhou, H.; Hernandez, V.; Mao, W.; Alley, M. R. K.; Sanders, V.; Plattner, J. J. Discovery of a New Boron-containing Antifungal Agent, 5Fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), for the Potential Treatment of Onychomycosis. J. Med. Chem. 2006, 49, 4447-4450.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

11. Sutcliffe, J. A. Antibiotics in Development Targeting Protein Synthesis. Ann. N. Y. Acad. Sci. 2011, 1241, 122-152.

12. Smoum, R.; Rubinstein, A.; Dembitsky, V. M.; Srebnik, M. Boron Containing Compounds as Protease Inhibitors. Chem. Rev. 2012, 112, 4156-4220.

13. Li, X.; Zhang, S.; Zhang, Y.-K.; Liu, Y.; Ding, C. Z.; Zhou, Y.; Plattner, J. J.; Baker, S. J.; Bu, W.; Liu, L.; Kazmierski, W. M.; Duan, M.; Grimes, R. M.; Wright, L. L.; Smith, G. K.; Jarvest, R. L.; Ji, J.-J.; Cooper, J. P.; Tallant, M. D.; Crosby, R. M.; Creech, K.; Ni, Z.-J.; Zou, W.; Wright, J. Synthesis and SAR of Acyclic HCV NS3 Protease Inhibitors with Novel P4benzoxaborole Moieties. Bioorg. & Med. Chem. Lett. 2011, 21, 2048-2054.

14. Priestley, E. S.; De Lucca, I.; Ghavimi, B.; Erickson-Viitanen, S.; Decicco, C. P. P1 Phenethyl Peptide Boronic Acid Inhibitors of HCV NS3 Protease. Bioorg. & Med. Chem. Lett. 2002, 12, 3199-3202.

15. Kneteman, N. M.; Howe, A. Y. M.; Gao, T.; Lewis, J.; Pevear, D.; Lund, G.; Douglas, D.; Mercer, D. F.; Tyrrell, D. L. J.; Immermann, F.; Chaudhary, I.; Speth, J.; Villano, S. A.; O’Connell, J.; Collett, M. HCV796: A Selective Nonstructural Protein 5B Polymerase Inhibitor with Potent Anti-hepatitis C Virus Activity In Vitro, in Mice with Chimeric Human Livers, and in Humans Infected with Hepatitis C Virus. Hepatology. 2009, 49, 745-752.


Page 26 of 43

Page 27 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


16. O’Farrell, D.; Trowbridge, R.; Rowlands, D.; Jager, J. Substrate Complexes of Hepatitis C Virus RNA Polymerase (HC-J4): Structural Evidence for Nucleotide Import and De-novo Initiation. J. Mol. Biol. 2003, 326, 1025-1035.

17. Luvino, D.; Baraguey, C.; Smietana, M.; Vasseur, J.. Borononucleotides: Synthesis, and Formation of a New Reversible Boronate Internucleosidic Linkage. Chem. Comm. 2008, 23522354.

18. Hang, J. Q.; Yang, Y., Harris, S. F.; Leveque, V.; Whittington, H. J.; Rajyaguru, S.; Aoleong, G.; McCown, M. F.; Wong, A.; Giannetti, A. M.; Le Pogam, S.; Talamas, F.; Cammack, N.; Najera, I.; Klumpp, K.. Slow Binding Inhibition and Mechanism of Resistance of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus. J. Biol. Chem. 2009, 284(23), 1551715529.

19. Feldstein, A.; Kleiner, D.; Kravetz, D.; Buck, M.. Severe Hepatocellular Injury With Apoptosis Induced by a Hepatitis C Polymerase Inhibitor. J. Clin. Gastroenterol. 2009, 43(4), 374-381.

20. McCown, M. F.; Rajyaguru, S.; Kular, S.; Cammack, N.; Najera, I. GT-1A or GT-1b Subtype-Specific Resistance Profiles for Hepatitis C Virus Inhibitors Telaprevir and HCV-796. Antimicrob. Agents & Chem. 2009, 53(5), 2129-2132.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

21. Beaulieu, P. Non-nucleoside Inhibitors of the HCV NS5B Polymerase: Progress in the Discovery and Development of Novel Agents for the Treatment of HCV Infections. Curr. Opin. Invest. Drugs. 2007, 8(8), 614-634.

22. Herlihy, K. J.; Graham, J. P.; Kumpf, R.; Patick, A. K.; Duggal, R.; Shi, S. T. Development of Intergenotypic Chimeric Replicons To Determine the Broad-Spectrum Antiviral Activities of Hepatitis C Virus Polymerase Inhibitors. Antimicrob. Agents & Chem. 2008, 52(10), 35233531.

23. Kim, A. Y.; Timm, J. Resistance Mechanisms in HCV: From Evolution to Intervention. Expert Rev. Anti Infect. Ther. 2008, 6(4), 463-478.

24. Sarrazin, C.; Zeuzem, S. Resistance to Direct Antiviral Agents in Patients with Hepatitis C Virus Infection. Gastroenterology 2010, 138, 447-462.

25. Wilfret, D.; Walker, J.; Voitenleitner, C.; Baptiste-Brown, S.; Lovern, M.; Kim, J.; Adkison, K.; Mathis, A.; Moss, L.; Yu, L.; Gan, J.; Spaltenstein, A. A Randomized, Double Blind, Dose Escalation, Fusion, First Time in Human Study to Assess the Safety, Tolerability, Pharmacokinetics, and Antiviral Activity of Single Doses of GSK5852 in Chronically Infected Hepatitis C Subjects.


Page 28 of 43

Page 29 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


26. Shim, J.-H.; Larson, G.; Wu, J.- Z.; and Hong, Z. Selection of 3’-Template Bases and Initiating Nucleotides by Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase. J. Virology 2002, 76(14), 7030-7039.

27. Mosley, R. T.; Edwards, T. E.; Murakami, E.; Lam, A. M.; Grice, R. L.; Du, J.; Sofia, M. J.; Furman, P. A.; Otto, M. J. Structure of Hepatitis C Virus Polymerase in Complex with PrimerTemplate RNA. J. Virology 2012, 86(12), 6503-6511.

28. Reich, S.; Golbik, R. P.; Geissler, R.; Lilie, H.; Behrens, S.-E. Mechanism of Activity and Inhibition of the Hepatitis C Virus RNA-dependent RNA Polymerase. J. Biol. Chem. 2010, 285(18), 13685-13693.

29. Butcher, S. J.; Grimes, J. M.; Makeyev, E. V.; Bamford, D. H.; and Stuart, D. I. A Mechanism for Initiating RNA-dependent RNA Polymerization. Nature 2001, 410, 235-240.

30. Gong, P.; and Peersen O. B. Structural Basis for the Active Site Closure by the Poliovirus RNA-dependent RNA Polymerase, Proc. Natl. Acad. Sci. USA 2010, 107, 22505-22510.

31. Harrus, D.; Ahmed-El-Sayed, N.; Simister, S.; Miller, M; Triconnet, M.; Hagedorn,C.; Mahias, K.; Rey, F.; Astier-Gin, T.; and Bressanelli, S. Further Insights into the Roles of GTP and the C Terminus of the HCV Polymerase in Initiation of RNA Synthesis, J. Biol. Chem.,2010, 285, 32906-32918



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

32. Nyanguile, O.; Devogelaere, B.; Vijgen, L.; Van den Broeck, W.; Pauwels, F.; Cummings, M. D.; De Bondt, H. L.; Vos, A. M.; Berke, J. M.; Lenz, O.; Vandercruyssen, G.; Vermeiren, K.; Mostmans, W.; Dehertogh, P.; Delouvroy, F.; Vendeville, S.; VanDyck, K.; Dockx, K.; Cleiren, E.; Raboisson, P.; Simmen, K. A.; Fanning, G. C. 1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus. J. Virology. 2010, 84(6), 2923-2934.

33. Voitenleitner, C.; Crosby, R.; Wang, A.; Walker, J.; Pouliot, J.; Woldu, E.; Van Horn, S.; Horton, J.; Creech, K.; Carballo, L.; You, S.; Remlinger, K.; Vamathevan, J.; Shotwell, J. B.; Spaltenstein, A.; Hong, Z.; Hamatake, R. Preclinical Characterization of GSK5852, a Novel HCV Polymerase Inhibitor.

34. Johns, B. A.; Shotwell, J. B., Haigh, D. Therapeutic Compounds. WO 2011/103063 A1. 2011.

35. Johns, B. A.; Shotwell, J. B., Haigh, D. Compounds. WO 2012/067663 A1. 2012.

36. Molander, G. A.; Jean-Gerard, L.; Scope of the Suzuki-Miyaura Cross-Coupling Reaction of Potassium Trifluoroboratoketohomoenolates. J. Org. Chem. 2009, 74, 1297-1303.


Page 30 of 43

Page 31 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


37. Molander, G. A.; Shin, I.; Jean-Gerard, L. Palladium-Catalyzed Suzuki-Miyaura Cross Coupling Reactions of Enantiomerically Enriched Potassium B-Trifluoroboratoamides with Various Aryl- and Hetaryl Chlorides. Org. Lett. 2010, 12(9), 4384-4387.

Figures/Schemes/Tables

Figure 1. Chemical structures for 1-4



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Syntheses of Linear and Aryl Boronates 5, 7, and 8

Conditions (i) 2-(bromomethyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, K2CO3, reflux, 24 hr., 38% Yield. (ii) [4-(bromomethyl)phenyl]boronic acid, KOtBu, THF, RT, 2 days, 49% Yield. (iii) [3-(bromomethyl)phenyl]boronic acid, KOtBu, THF, RT, 2 days, 20% Yield.


Page 32 of 43

Page 33 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. NS5B and Stable Replicon Data for 1, 5, 7-13*

Replicon EC50 (nM) Cmpd

1

5

IC50 (nM)

WT 1a

WT 1b

1b 316N

NS5B POL

27 (347)

18 (460)

170 (236)

40 (49)

6.7 (4)

3.2 (6)

3.9 (2)

40 (7)

2.7 (6)

2.6 (7)

13 (5)

32 (4)

8.8 (4)

6.9 (5)

120 (3)

30 (3)

2200 (3)

760 (6)

>5000 (4)

20 (5)

290 (3)

150 (3)

3200 (1)

25 (1)

>5000 (2)

1800 (2)

>5000 (2)

16 (1)

36 (2)

20 (2)

480 (2)

100 (3)

58 (3)

18 (4)

440 (4)

50 (3)

7

8

9

10

11

12

13 *number of independent measurements in parentheses, see SI for 95% confidence intervals & toxicity data in HUH-7 cells. All compounds ≥ 95% purity unless otherwise indicated.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 2. Racemic Synthesis of (±) 16

Conditions. (i) 6, K2CO3, DMF, RT, 2 days, (ii) Rh(CO)Cl(PPh3)2, HBPin, RT, 3 hr., 75% Yield, 2 steps; (iii) 10% Pd/C, H2, (iv) HCl, polymersupported benzene boronic acid, THF:H2O, 24 hours, Quant. Yield, 2 steps; (v) SFC resolution


Page 34 of 43

Page 35 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 2. Stable HCV Replicon Data for 2, 16-22*


(±) 16

(±) 18

19**

(±) 20

(±) 21

(±) 22

IC50 (nM)

WT 1a

WT 1b

1b (316N)

NS5B POL

2.9 (4)

2.8 (5)

7.6 (3)

50 (5)

6.1 (4)

3.1 (8)

5.6 (4)

50 (3)

9.6 (2)

1.9 (6)

13 (4)

50 (1)

8.8 (2)

6.5 (6)

52 (4)

100 (1)

9.7 (2)

6.4 (4)

56 (2)

63 (2)

9.1 (2)

3.7 (4)

38 (4)

80 (3)

7.6 (278)

4.3 (294)

13 (133)

63 (10)

5.3 (6)

2.9 (10)

11 (4)

50 (7)

(R)-2**

(S)-17** *number of independent measurements in parentheses, see SI for 95% confidence intervals & toxicity data in HUH-7 cells. All compounds ≥ 95% purity unless otherwise indicated. **19: Conglomerate EC50s for 2 lots with measured purities of 91 and 93%. 2: Conglomerate of EC50s for 14 individual lots, 13/14 lots > 95% purity, one lot measured 89% purity. 17: Conglomerate of EC50s for 3 individual lots with measured purities of 86, 93, and 98%.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 3. Diastereoselective Synthesis of (R)-2

Conditions. (i) CuI, DPEPhos, NaOtBu, (BPin)2; (ii) TiCl4, DIPEA, BOMCl, > 10:1 DE, 47% Yield, 2 steps; (iii) LiBH4, MeOH, 70% Yield; (iv) DIAD, PPh3, 6; (v) 10% Pd/C, H2; (vi) HCl, polymer supported benzene-boronic acid, THF:H2O, 49% Yield, 3 steps.

Scheme 4. Synthesis of 4

Conditions. (i) Pd(OAc)2, DPPF, Et3N, DMSO/MeOH, 80°C, 1.5 MPa CO, 75% Yield; (ii) MeCN, HBr, NaNO2, H2O, CuBr, 70°C, 46% Yield; (iii) benzoyl peroxide, NBS, CCl4, reflux, 48 hr., Quant. Yield; (iv) KI, K2CO3, DMF, RT, 6, 8 hr., 56% Yield, (v) bis(pinacolato)diboron, KOAc, PdDPPFCl2, 100°C, 16 hr., 71% Yield; (vi) LiBH4, THF, -5°C to RT, 18% Yield


Page 36 of 43

Page 37 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 4. Stable HCV Replicon Data for 3, 4, and 29-35*


IC50 (nM)

WT 1a

WT 1b

1b (316N)

NS5B POL

9.5 (6)

2.7 (8)

16 (3)

40 (3)

7.4 (2)

4.9 (3)

64 (3)

32 (4)

17 (2)

4.9 (3)

110 (3)

32 (3)

7.4 (2)

3.5 (6)

12 (4)

32 (2)

7.0 (2)

4.2 (4)

9.0 (2)

40 (1)

12 (2)

3.6 (4)

50 (2)

100 (1)

4.8 (2)

2.2 (4)

5.4 (2)

80 (2)

3.0 (82)

1.7 (110)

1.9 (26)

50 (49)

5.8 (184)

3.2 (197)

12 (129)

32 (6)

29

30

31

(±) 32

33

34

35

3**

4 *number of independent measurements in parentheses, see SI for 95% confidence intervals & toxicity data in HUH-7 cells.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

All compounds ≥ 95% purity unless otherwise indicated. ** Conglomerate of EC50s for 15 individual lots, 14/15 lots > 95% purity, one lot measured 94% purity.

Table 3. Transient Replicon EC50 (nM) for 1-4, 7, and 32-33* 1b Mutants C316Y 420 (18) 11 (6) 1.4 (8) 16 (6)

C316F 2500 (9) 37 (2) 3.8 (4) 110 (2)

S365T 1300 (12) 6.5 (6) 4.2 (8) 84 (6)

7

--

--

--

32

--

--

--

33

--

--

--

Cmpd 1 2 3 4

1a Mutant 316Y 1900 (40) 830 (5) 3.2 (8) 190 (4) 120 (2) 790 (2) 350 (2)

*number of independent measurements in parentheses, see SI for 95% confidence intervals & toxicity data in HUH-7 cells.


Page 38 of 43

Page 39 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3: Compound 3 exhibits slow binding kinetics to isolated NS5B enzyme. Measurement of inhibitor dissociation from GT1b 316N. 10uM NS5B∆21 1b 316N was saturated with either 12uM 3 (top) or 1 (bottom); then the samples were diluted 20-fold into 10 uM 1 (top) or 3 (bottom) respectively. To monitor kinetics, the NS5B-bound compounds were resolved from free compounds and analyzed for both 3 and 1 by LC-MS. Solid squares represent 3, and solid circles represent 1 bound to NS5B protein at times up to 75 hrs. IC50s for 1 and 3 vs. NS5B∆21 1b 316N as measured with a 15 minute pre-incubation are 160 nM (n = 40) and 130 nM (n = 36) respectively.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Compound 3 Inhibits NS5B initiation. HCV NS5B was incubated with RNA template and substrates in the presence of increasing concentrations of 3. Migration of CTP substrate and pCpG reaction product are noted. “X” designates a control reaction run in the absence of NS5B.

Figure 7. Hydrophobic pocket of Y448 (sticks, yellow carbons). Binding of 3 (green carbons) completes the formation of a hydrophobic pocket, in tandem with F193, F551 and M414, which encircles Y448. The hydroxyl group of Y448 also makes hydrogen bond contacts with R200 and the sulfonamide of 3 in the back of the pocket. Locking Y448 in this pose inactivates the β-flap (yellow ribbon) and restricts Y448 from participating in polymerase initiation.


Page 40 of 43

Page 41 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Structural biology of the NS5B Palm II binding site. The NS5B “fingers”, “thumb” and “palm” domains are colorcoded blue, red and gray, respectively. The β-hairpin “flap”, which regulates RNA-template binding and polymerization, is colorcoded yellow. a) Right-handed orientation of NS5B, looking down the NTP entrance. 3 occupies a strategic hydrophobic cleft in the palm, flanking conserved catalytic residues D318-D319 (sticks), with the boronic acid positioned at the “gateway” of RNA polymerization. b) Same view as (a) with solvent-accessible surface, emphasizing the central position of the boronic acid group within the NTP/RNA channel. c) Close-up of the NTP/RNA entrance with transparent surface, depicting the juxtaposition of 3 relative to NTP processing (here GTP, PDB: 1HI0).29 Conserved R386 and R394 support interactions with the incoming GTP triphosphate tail. d) The position of 3 from the viral RNA perspective (PDB: 3OL6).30 The figure has been rotated relative to (a) to view the RNA channel from the interior of the NS5B palm. The (+) RNA template is green and the resulting primer (-) RNA strand is red. Incoming NTP nucleotides (PDB: 3XI3)32 are colored magenta/blue with catalytic Mg2+ cofactors as orange spheres. In addition to stabilizing an inactive (“closed”) state of NS5B, the boronic head-group of 3 is poised to disrupt productive placement of incoming nucleotides at the site of polymerase initiation and propagation.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Crystal Structures of 3 with wild type and mutant NS5B constructs. Structures have been deposited in the RCSB. RCSB ID and PDB ID#s are listed in Table SI-12 in the Supporting Information (a) 3:WT1a, (b) 3:1a 316Y (c) 3:1b 316N.

Figure 6. Crystal Structures of 1b316Y. Structures have been deposited in the RCSB. RCSB ID and PDB ID#s are listed in Table SI-12 in the Supporting Information (a) 3:1b316Y, (b) 4:1b316Y, (c) 32:1b316Y, and (d) 1:1b316Y


Page 42 of 43

Page 43 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. The NS5B Palm II binding site, relative to the viral RNA entrance and exit channels, oriented along the dsRNA axis. HCV and polio viral RNA co-crystal structures were aligned according to HCV NS5B (PDB: 4E7A)27 and polio RdRp (PDB: 3OL6)30 protein homology alignment with the experimental GT1b NS5B:3 co-crystal structure of this study. The polio RNA primer (+) and template (-) strands, color-coded red and green respectively, are in register with the HCV RNA strands, color-coded dark teal. The boronic acid group of 3 is located at the initiation of the primer strand, able to make Van der Waals contact with the ribose of the incoming (+0) nucleotide (also see Figure SI-12). The β-hairpin “flap” in the NS5B:3 co-crystal, color-coded yellow, exists in an inactivated state which occludes the RNA-channel along the viral dsRNA axis.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TABLE OF CONTENTS GRAPHIC


Page 44 of 43

Discovery of a Potent Boronic Acid Derived Inhibitor of the HCV RNA

Recommend Documents