Discovery of a Hepatitis C Virus NS5B Replicase Palm Site Allosteric

Article pubs.acs.org/jmc

Discovery of a Hepatitis C Virus NS5B Replicase Palm Site Allosteric Inhibitor (BMS-929075) Advanced to Phase 1 Clinical Studies Kap-Sun Yeung,*,† Brett R. Beno,† Kyle Parcella,† John A. Bender,† Katherine A. Grant-Young,† Andrew Nickel,† Prashantha Gunaga,‡ Prakash Anjanappa,‡ Rajesh Onkardas Bora,‡ Kumaravel Selvakumar,‡ Karen Rigat,† Ying-Kai Wang,† Mengping Liu,† Julie Lemm,† Kathy Mosure,† Steven Sheriff,§ Changhong Wan,§ Mark Witmer,§ Kevin Kish,§ Umesh Hanumegowda,† Xiaoliang Zhuo,† Yue-Zhong Shu,§ Dawn Parker,† Roy Haskell,† Alicia Ng,§ Qi Gao,# Elizabeth Colston,§ Joseph Raybon,§ Dennis M. Grasela,§ Kenneth Santone,† Min Gao,† Nicholas A. Meanwell,† Michael Sinz,† Matthew G. Soars,† Jay O. Knipe,† Susan B. Roberts,† and John F. Kadow† †

Bristol-Myers Squibb Research and Development, P.O. Box 5100, 5 Research Parkway, Wallingford, Connecticut 06492, United States ‡ Department of Discovery Chemistry, Biocon Bristol-Myers Squibb Research and Development Center, Biocon Park, Jigani Link Road, Bommasandra IV, Bangalore 560099, India § Bristol-Myers Squibb Research and Development, P.O. Box 4000, Princeton, New Jersey 08543, United States # Bristol-Myers Squibb Research and Development, 1 Squibb Drive, New Brunswick, New Jersey 08901, United States S Supporting Information *

ABSTRACT: The hepatitis C virus (HCV) NS5B replicase is a prime target for the development of direct-acting antiviral drugs for the treatment of chronic HCV infection. Inspired by the overlay of bound structures of three structurally distinct NS5B palm site allosteric inhibitors, the high-throughput screening hit anthranilic acid 4, the known benzofuran analogue 5, and the benzothiadiazine derivative 6, an optimization process utilizing the simple benzofuran template 7 as a starting point for a fragment growing approach was pursued. A delicate balance of molecular properties achieved via disciplined lipophilicity changes was essential to achieve both high affinity binding and a stringent targeted absorption, distribution, metabolism, and excretion profile. These efforts led to the discovery of BMS-929075 (37), which maintained ligand efficiency relative to early leads, demonstrated efficacy in a triple combination regimen in HCV replicon cells, and exhibited consistently high oral bioavailability and pharmacokinetic parameters across preclinical animal species. The human PK properties from the Phase I clinical studies of 37 were better than anticipated and suggest promising potential for QD administration.

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INTRODUCTION

Americas, Europe, and Asia, followed by genotypes 2 and 3, whereas genotypes 4 and 5 are more prevalent in Africa. The HCV genome encodes a single polyprotein of ∼3000 amino acids. Over the past 25 years, significant efforts have been invested in understanding the HCV replication cycle and the structures and functions of the viral proteins with the goal of identifying viable therapies for treating HCV infection.5 In particular, three of the HCV nonstructural proteins, the NS3/ 4A serine protease, the NS5A replication cofactor, and the NS5B replicase, have been extensively exploited as drug targets.6 This intensive research has culminated in the approval of several HCV-targeted direct-acting antiviral agents that are

Hepatitis C virus (HCV) infection is a major medical problem on a global scale. Between 130 and 150 million people are chronically infected with HCV worldwide with an estimated 2.7−3.9 million infections in the United States alone.1,2 Patients with HCV-related chronic liver diseases are at a very high risk of developing liver cirrhosis and progressing to hepatocellular carcinoma. Chronic HCV infection is therefore the leading cause of liver transplantation in the US and is responsible for approximately 700,000 deaths per year worldwide.1,2 HCV was identified in 1989.3 It is a single-stranded positivesense RNA virus belonging to the Hepacivirus genus within the Flaviviridae family. HCV has been classified into six major genotypes with multiple subtypes, and a seventh genotype was identified recently.4 Genotypes 1a and 1b predominate in the © 2017 American Chemical Society

Received: March 3, 2017 Published: April 21, 2017 4369

DOI: 10.1021/acs.jmedchem.7b00328 J. Med. Chem. 2017, 60, 4369−4385

Journal of Medicinal Chemistry

Article

Figure 1. (a) Structure of the HCV NS5B protein.10 The finger loops are colored cyan, and the fingers, palm, thumb, and C-terminal domains are depicted in green, blue, red, and white, respectively. The “primer grip” and active site GDD motif are shown in yellow and orange, respectively, and the location of the palm site is denoted with a magenta sphere. (b) Close-up view of the HCV NS5B active site and palm binding pocket highlighting key residues and structural features. α-Helices are labeled according to the nomenclature in ref 12. Image generated with The PyMol Molecular Graphics System (v. 1.8, Schrödinger, LLC).

adopted in this report.13−15 Several different structural classes of palm site inhibitors have been reported to be in various stages of clinical development.13−15 These include nesbuvir (HCV-796, 1)16 (Figure 2), which established clinical proof of

used in combination in the clinic to replace pegylatedinterferon/ribavirin-based therapy.7 These advanced all-oral treatment options offer a cure for chronic HCV infection with increased sustained virological response rates, shorter treatment durations, improved safety profiles, and expanded genotype coverage.8,9 The HCV NS5B replicase is an RNA-dependent RNA polymerase that performs the synthesis of HCV genomic RNA within the HCV replication complex machinery and is therefore an indispensable enzyme for HCV replication.5 From the initial positive-sense RNA template, a negative-sense RNA intermediate is synthesized, which in turn is used to generate progeny positive-sense RNA for further translation and packaging into new virions. Early on, three X-ray crystallographic structures of constructs of HCV genotype 1b (gt-1b) BK strain NS5B with the C-terminal membrane anchor region truncated were determined.10−12 The structures revealed that the catalytic domain adopts a right-hand configuration with fingers, palm, and thumb domains typical of polymerase enzymes (Figure 1a). The highly conserved active site that harbors the GDD aspartic acid motif, Gly317-Asp318-Asp319, is located within the palm domain. The pivotal role of the NS5B replicase in the HCV replication cycle and the absence of a mammalian counterpart together with the structural information that enables structure-based drug design have made NS5B an excellent anti-HCV drug target. The discovery and development of HCV NS5B replicase active site-directed nucleoside and nucleotide inhibitors and their prodrugs, as well as non-nucleoside inhibitors that bind in the three allosteric sites (thumb I, thumb II, and palm site), have been comprehensively reviewed.13−15 The palm binding pocket is near the active site and is surrounded by the Tyr448 β-hairpin loop from the thumb domain, the Ile363-Asn369 primer grip loop, and two α-helices (αI, Pro197-Ala210 and αP, Ser407-Tyr410) from the palm domain. The side chain of residue 316 on the β-sheet preceding the catalytic Gly317-Asp318-Asp319 loop extends into the binding site, and movement of the Arg200 side chain accommodates inhibitor binding (Figure 1b). The palm site is the largest of the three allosteric binding sites and is deeply recessed and hydrophobic in nature. The palm site has been divided into site I and site II in some publications. However, unlike thumb site I and site II, the two palm pockets are overlapping and contiguous; thus, a single palm site naming is

Figure 2. Structures of benzofuran NS5B palm site inhibitors 1−3.

concept in HCV-infected patients for benzofuran-based inhibitors that bind to the allosteric palm site. A boronic acid derivative 2 (GSK-5852)17 and a tetracyclic analogue 3 (MK8876)18 have also been reported recently.19

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MEDICINAL CHEMISTRY STRATEGY A high-throughput screening campaign conducted against HCV gt-1b NS5B enzyme identified an anthranilic acid hit compound 4 (Figure 3). An X-ray cocrystal structure of 4 in complex with the ΔC18 truncated gt-1b Con1 strain NS5B protein revealed that 4 bound in the palm site of the enzyme.20 Interestingly, the bound structure of 4 partially overlapped with that of benzofuran analogue 5,21 as observed in an NS5B/5 cocrystal structure, with its benzoic acid moiety projecting from the direction of the benzofuran C5 position of 5 (Figure 4a). Another NS5B complex structure with 6 in the benzothiadiazine series of NS5B inhibitors22 was determined and showed that the bound structure of 6 intersected with both of those of 4 and 5 with its thiadiazine ring placed in the location of the benzoic acid of 4 (Figure 4a). Overall, the three-dimensional structures of the protein molecules in the three complexes were very similar with pairwise RMSD values derived from protein structural alignments varying from 0.3 to 0.7 Å. The protein molecules were also structurally similar in the vicinity of the bound ligands with variations observed in a few flexible loops. The overlapping structures created an opportunity to merge the 4370



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replicon assay, as a template to explore the structure−activity relationship (SAR) at the benzofuran C5 position in the palm binding site. The C5 oxygen atom provided a convenient handle for library synthesis, and reagent selection was facilitated by iterative molecular modeling based on NS5B/inhibitor complex structures. Although the molecular weight (MW = 327) and lipophilicity (cLogP = 4.18) of 7 are somewhat outside of the boundaries of those associated with a good fragment,24 its ligand efficiency (LE) value of 0.27 (calculated from (1.37/HAC) × pIC50, IC50 determined from the gt-1b NS5B enzymatic assay and HAC = heavy atom count)25,26 was considered a suitable starting point for a fragment growing approach.

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RESULTS AND DISCUSSION An initial benzofuran C5 aryl library obtained from Suzuki cross-coupling between the C5-triflate derivative of compound 7 and commercially available aryl boronic acids or esters quickly established the importance of a carboxylate anion group for enhancing inhibitory activity against the NS5B enzyme, as summarized in Table 1. Both the meta-benzoic acid 8 and para-

Figure 3. Structures of NS5B palm site inhibitors 4−7 and HCV inhibitory potency toward gt-1b NS5B enzyme (IC50) and replicons (EC50) for 4 and 7 (the IC50 value for 4 was the average of n ≥ 2 independent experiments; values for 7 were from a single experiment performed in duplicate).

Table 1. HCV Inhibitory Potency towards NS5B Enzyme (IC50) for Compounds 8−13 (Data Obtained from Single Experiment Performed in Duplicate)

Figure 4. (a) Overlay of the bound structures of anthranilic acid 4 (magenta), benzofuran 5 (orange), and benzothiadiazine 6 (green) in the palm site of the wild-type (WT) gt-1b NS5B protein. (b) X-ray structure of 5 bound to the gt-1b NS5B WT protein illustrating key interactions of 5 with palm site residues. Images generated with The PyMol Molecular Graphics System (v. 1.8, Schrödinger, LLC).

three chemotypes by growing a benzofuran inhibitor into available binding regions via substitution at the benzofuran C5 position. The primary objective of the project was to identify an NS5B replicase palm site inhibitor clinical candidate that could be utilized in combination with NS5A and NS3/4A inhibitors. Moreover, included in this goal was to achieve activity toward the pre-existing C316N mutant, which is present in approximately 40% of gt-1b HCV-infected patients. Benzofuran 1 was reported to exhibit 26-fold resistance in a BB7 gt1bC316N cell-based replicon assay compared to that of wildtype.23 This was considered to be a deficiency in the profile of 1, which would substantially limit its clinical application. As exhibited in the NS5B/5 complex cocrystal structure, the NH and carbonyl of the methyl amide from the inhibitor engage in two key hydrogen bonding interactions with the side chains of Ser365 in the primer grip loop and Arg200, respectively (Figure 4b), whereas the 4-fluorophenyl substituent forms tight van der Waals contacts with the surrounding hydrophobic residues in the binding site. These two moieties together serve to anchor the core benzofuran ring to the palm site. The initial chemistry strategy was therefore to utilize the structurally simpler C6-unsubstituted benzofuran analogue 7 (Figure 3), which showed weak but detectable activity in both the in vitro enzymatic assay and cell-based HCV

a

Average value of n ≥ 2 independent experiments.

analogue 9 were equally active with >20-fold improvements in IC50 values against gt-1b as compared to that of the starting benzofuran 7. However, ortho-analogue 11 was essentially inactive, reflecting the importance of both the hydrogen bonding distance and angle for interaction in the binding site.27 The desired position of the acid group for inhibitory activity was recapitulated in thiophene-2-carboxylic acid 10 in which the 5-membered thiophene ring with its longer C−S bond and smaller C−S−C bond angle served as a suitable isostere for the phenyl ring of 8 and 9.28 The loss in activity of ester 12 and benzyl alcohol 13 suggested that an oxygen atom 4371



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with strong hydrogen bond-accepting potential29 was necessary for potency against the enzyme. These SAR results were consistent with a strong hydrogen bond between the carboxylate of the inhibitor and backbone N−H (O−N distance 2.8 Å) of Tyr448, as observed in the cocrystal structure of the NS5B/8 complex that was obtained subsequently. The existence of a binding pocket adjacent to the carboxylate group, as revealed in the complex structure, provided an opportunity to further functionalize the acid moiety via amide coupling and potentially form additional interactions with the enzyme. Such modification could also convert these polar anionic compounds to neutral species that were anticipated to show enhanced cell permeability for increasing potency in the cell-based replicon assay, which was far from satisfactory in the initial acidic lead compounds 8 and 9 (gt-1b EC50 = 12 and 4.5 μM, respectively, and gt-1a EC50 > 20 μM). As shown in Table 2, a notable improvement in the activity against gt-1b and gt-1a replicon cells was obtained starting from

engaged in hydrogen bonding with the hydroxyl group of the side chain from Tyr452 and the backbone carbonyl of Ile447 via a water bridge (Figure 5a). The change in the amide conformation from a secondary to a tertiary amide could also affect the favorable interaction. In general, compounds from the meta-amide series provided better potency profiles than the corresponding para-amides, which were more than 10-fold less active as represented by 20, the para-isomer of 18. Thus, subsequent SAR studies were concentrated on the meta-amide series. The Me to Ph matched molecular pair transformation on the amide moiety improved the potency against the gt-1b NS5B enzyme by up to 9-fold (compare 16 and 21, 17 and 22, and 18 and 23). This phenomenon was attributed to a favorable πstacking interaction between the amide phenyl ring and the phenyl ring from the Phe551 side chain. However, the 1.4 unit LogP increase in lipophilicity30 associated with a Me → Ph transform in this molecular context might also play a role. Although the activity in the replicon cells, particularly in gt-1a, was not as sensitive to the introduction of an amide phenyl ring, when the gem-dimethyl group of 23 was cyclized into a cyclopropyl ring, single digit nanomolar potency against both the NS5B enzyme and gt-1b replicon cells was realized, as represented by 24. The cyclopropyl ring in 24 presumably fills the volume enclosed by Met414, Ile447, and Tyr452 more effectively, resulting in improved van der Waals contacts. Encouragingly, the potency against the gt-1a replicon cells was also driven below 50 nM in phenylcyclopropyl analogue 24, which became the new lead structure for further optimization. Compared to starting template 7, the ligand efficiency was improved (LE = 0.31 for 18 and 0.30 for 24) even though the heavy atom count was increased by 38% in 18 and as much as 58% in 24. During the course of the SAR studies, multiple inhibitor/ NS5B complex cocrystal structures were determined. In these structures, remarkable conformational flexibility of the NS5B Cterminal region and Tyr448 loop that comprise part of the palm site was observed. Because of this plasticity, the size and shape of the binding pocket vary significantly in response to different sizes and shapes of benzofuran inhibitor C5 substituents, as illustrated in Figure 5b. Thus, the NS5B palm site offers an interesting example of an inducible binding pocket.31 This conformational flexibility suggested that diverse amide structures could be accommodated, providing an opportunity for further optimization focused on potency enhancement and/ or modulation of physicochemical properties for an improved absorption, distribution, metabolism, and excretion (ADME) profile as noted below and to be disclosed in future publications. Subsequent optimization performed on inhibitor 24 was again guided by observations from the NS5B/inhibitor complex cocrystal structures. Ligand elaboration to fill a small hydrophobic cavity proximal to the 2′-position of the amide phenyl ring and formed by the side chains from Phe193, Cys316, Cys366, and Tyr448 appeared to be feasible (Figure 5c). Indeed, substitution at the 2′-position with small groups of increasing size (25−28, Table 3) not only maintained the single digit nanomolar potency against gt-1b in the replicon assay but also led to a stepwise increase in the activity against both gt-1a replicon and gt-1bC316N mutant replicon. The 2′-methyl analogue 28 emerged from these SAR studies with a more balanced inhibitory potency profile and exhibited a favorable cytotoxicity window (CC50 > 25 μM in the replicon Huh-7 host cell line). However, a major drawback to the in vitro profile of

Table 2. HCV Inhibitory Potency towards NS5B Enzyme (IC50) and Replicon (EC50) for Compounds 14−24 (Data Are Average Values of n ≥ 2 Independent Experiments)

a

Data from a single experiment performed in duplicate; ND = not determined.

methyl amide 15. This was attributed to a combination of increased inhibition of the NS5B enzyme and improved cell penetration, reflected by the ∼2-fold ratio between the gt-1b enzyme IC50 and replicon EC50 values. A key discovery from this amide library SAR exercise was that successive addition of a methyl group to the α-carbon of the alkyl amide series (from 15 to 18) as well as the benzyl amide analogues (from 21 to 23) enhanced the potency both toward the enzyme and in replicon cells. Pleasingly, potent activity in the nanomolar range in the replicon was achieved with these compounds (gt-1b EC50 = 18 nM for 18 and 10 nM for 23). N-Methylation of the secondary amide NH decreased the potency by 3-fold in the enzymatic assay, and this decline translated into the replicon assay as exemplified by 19, the N-methyl analogue of 18. This was subsequently rationalized by the fact that the amide NH 4372



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Figure 5. (a) X-ray crystal structure of 18 bound to gt-1b NS5B WT protein showing direct and water-bridged hydrogen bonds between the protein and ligand. Hydrogen bonds are indicated with blue dashes. (b) Overlay of cocrystal structures of NS5B complexed with 8 (green) and 18 (orange) highlighting the Y448 loop, C-terminal region, and F551, which adopt different conformations depending on the identity of the benzofuran C5 substituent. (c) The hydrophobic cavity proximal to the 2′-position of the inhibitor amide phenyl ring and formed by the side chains from F193, C316, C366, and Y448 as illustrated in the close-up view of that region in the NS5B/18 complex structure in (a). Images generated with The PyMol Molecular Graphics System (v. 1.8, Schrödinger, LLC).

for solving this problem. Incorporation of a nitrogen atom into the phenyl ring to increase polarity of the molecule would be desirable in light of the already high lipophilicity of the 2′methyl lead compound 28 (cLogP = 6.28).30 It was determined that the 2-pyridyl analogue 29 with a reduced cLogP value of 5.44 provided excellent stability in both HLM and RLM while maintaining the potency profile of 28 (Table 4) compared to the 3- and 4-pyridyl analogues 30 and 31, respectively. The microsomal stability of 29 in RLM (t1/2 = 61 min) together with its high membrane permeability (999 nm/s, pH 7.4) as measured in a parallel artificial membrane permeability assay (PAMPA) translated into good plasma exposure following oral administration of the compound to rats in a 6 h pharmacokinetic screen. A high concentration of 29 (15.7 μM) was also obtained in rat liver at the 6 h time point (Figure 6), indicating a favorable exposure level in the target organ could be achieved with this compound series. The clearance estimated from the screen was a modest 14 mL min−1 kg−1. These encouraging data established the direction for further optimization. During these SAR studies, it was noted that the 3- and 4pyridyl analogues 30 and 31 appeared to be almost instantly metabolized in liver microsomes and were significantly less stable than the 2-pyridyl lead 29 and the parent phenyl analogue 28, although both 30 and 31 are less lipophilic than 29 and 28 by approximately 0.4−1.5 calculated LogP units (Table 4). Quantum chemical calculations performed at the MP2/6-31+G** level on the N-(1-(pyridin-2-yl)cyclopropyl)-

Table 3. Inhibitory Potency towards HCV Replicon (EC50) for Compounds 24−28 (Data Are Average Values of n ≥ 2 Independent Experiments)

replicon EC50 (nM)

a

compd

R

gt-1b

gt-1a

gt-1bC316N

24 25 26 27 28

H F Cl OMe Me

3.6 3.6 2.5 1.8 1.3

36 16 6.2 7.9 2.1

NDa 150 63 32 35

ND = Not determined.

this series of potent inhibitors was their low metabolic stability in liver microsomes. For example, only 68 and 39% of lead compound 28 remained unmetabolized in human and rat liver microsomes (HLM and RLM), respectively, after a 10 min incubation in a high-throughput metabolism screen (Table 4). Met-ID studies on the earlier compound 23 identified the electron-rich gem-dimethyl phenyl moiety as the major metabolic soft spot, thus suggesting the introduction of a heteroatom to the phenyl ring to increase polarity and potentially block metabolic sites could be a viable approach

Table 4. Inhibitory Potency towards HCV Replicon (EC50) and In Vitro Profiling for Compounds 28−31 (Data Are Average Values of n ≥ 2 Independent Experiments)

replicon EC50 (nM)

a

H/R LMa

compd

N−Ar

gt-1b

gt-1a

gt-1bC316N

% remain

t1/2 (min)

cLogPb

28 29 30 31

phenyl 2-pyridyl 3-pyridyl 4-pyridyl

1.3 1.9 2.2 0.83

2.1 2.8 17 8.3

35 55 193 63

68/39 89/82 0.7/0.6 11/11

11/ND 108/61 ND ND

6.28 5.44 5.05 5.05

cpKab

CYP3A4 IC50 (μM)

CYP2C8 IC50 (μM)

3.95 4.77 5.01

2.1 4.1 0.063 0.073

1.5 1.0 0.35 0.026

H/R LM = human and rat liver microsomes. bCalculated values;30 ND = not determined. 4373



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as metabolically stable in dog and monkey liver microsomes (t1/2 = 14 and 20 min, respectively), possessed low micromolar inhibition of CYP3A4 and CYP2C8 enzymes, hERG inhibition in a Tl+ ion flux assay with an IC50 of 5.8 μM, and activity in a pregnane X receptor (PXR) transactivation assay with an EC50 of 1.6 μM. Controlling the lipophilicity of the molecule (cLogP for 29 = 5.44, shake flask LogDpH6.5 = 5.42) to avoid these offtarget liabilities while preserving the good qualities of 29 was desirable at this stage of the optimization process. However, this represented a challenge because the palm site is deeply recessed and highly hydrophobic, and a trend of decreasing potency particularly against gt-1a and the gt-1bC316N mutant was noted in molecules with reduced lipophilicity. A subtle balance of molecular properties was thus needed to achieve both potent antiviral activity and favorable ADME properties. It was anticipated that the conformational flexibility of the NS5B C-terminal region, in which the cyclopropyl amide portion of the molecule resides as described above, would allow productive structural modifications of the inhibitor that improved potency while permitting optimization of molecular properties. Thus, further introduction of heteroatoms into the amide region by surveying 5- and 6-membered heterocycles was pursued. One avenue that proved to be effective was the addition of a second nitrogen atom into the pyridine ring of 29, and walking the nitrogen atom around the ring to deliver the four isomeric diazine analogues 32−35 (Table 5). Among

Figure 6. Six hour rat pharmacokinetic screen of 2-pyridyl analogue 29 (IV, 2 mg/kg; PO, 6 mg/kg; 9:1 PEG400/EtOH; n = 2); PO exposure at the 6 h time point: plasma = 1.64 μM and liver = 15.7 μM. The open and filled circles and diamonds indicate IV data obtained from two individual rats; the open and filled squares and triangles indicate PO data obtained from two individual rats.

benzamide model system predict a 0.8 kcal/mol preference for a conformation in which the plane of the pyridine ring bisects the cyclopropyl C2−C3 bond (Figure 7).32 This is in contrast

Table 5. Inhibitory Potency towards HCV Replicon (EC50) and cLogP Values for Compounds 29 and 32−36 (Data Obtained from Single Experiment Performed in Duplicate)

Figure 7. Relative energies of aryl-cyclopropyl bond rotamers for model systems (left) and bisected and perpendicular conformers of the 2-pyridyl model (right) calculated at the MP2/-6-31+G** level of theory.32

to the corresponding phenyl model system, which prefers a perpendicular conformation.33 It is possible that shielding of the pyridyl nitrogen lone pair in 29 by the cyclopropyl methylene moiety is partially responsible for the reduced basicity of 29 relative to 30 and 31, where the pyridyl nitrogen atoms are more accessible. Diminished steric encumbrance and higher basicity together contribute to the greater susceptibility of the pyridine rings in 30 and 31 toward potential N-oxidation by microsomal CYP enzymes and potent inhibition of CYPs by 30 and 31 via coordination to the heme iron at the nanomolar level in an in vitro assay using recombinant CYPs (Table 4). These findings are in line with the general trend observed in the change in lipophilicity and CYP3A4 inhibition when a monosubstituted phenyl ring is replaced by the three pyridine regioisomers.34 Although the 2-pyridyl lead compound 29 exhibited promising replicon potency, microsomal stability in HLM and RLM, membrane permeability, and oral exposure, other in vitro parameters still required optimization. For example, 29 was not

Average value of n ≥ 2 independent experiments. values.30 a

b

Calculated

them, only the more lipophilic 2-pyrimidinyl analogue 32 (cLogP = 4.83, shake flask LogDpH6.5 = 4.76) maintained the activity profile of the 2-pyridyl parent compound 29. The replicon activity against gt-1a and the gt-1bC316N mutant deteriorated with increasing polarity in the other three isomers. Other analogues possessing 5-membered heterocyclic rings (e.g., imidazole, pyrazole, triazole, tetrazole, oxazole, isoxazole) were also inferior primarily due to unfavorable replicon potency against the gt-1bC316N mutant (EC50 values ranged from 100 nM to >1 μM) occasionally accompanied by an undesirable 4374



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Table 6. In Vitro Profiling Data and Pharmacokinetic Parameters in Rats (0 to 24 h) for 32a H/R/Mk/D LMb t1/2 (min)

PAMPA pH 7.4 (nm/s)

CYP3A4 IC50 (μM)

CYP2C8 IC50 (μM)

769

11.4

3.9

>120/86/51/46 IV

hERG flux IC50 (μM)

hERG patch clamp

PXR-TA EC50 (μM)

4.8

96% Inh @10 μM

>50

PO

CL (mL min−1 kg−1)

IV t1/2 (hr)

Vss (L/kg)

Cmax (μM)

Tmax (hr)

AUC (μM h)

C6 liver (μM)

C24 liver (μM)

C24 liver/plasma

%F

5.5

5.3

6.1

6.7

4.7

48.7

8.7

0.614

2.4

>100

a

IV, 2 mg/kg; PO, 6 mg/kg; 9:1 PEG400/EtOH; n = 3. bH/R/Mk/D LM denotes human, rat, monkey, and dog liver microsomes, respectively.

CYP inhibition profile (IC50 < 1 μM). The oxadiazole analogue 36, which interestingly has the same cLogP value as the 2pyrimidyl derivative 32, was the only heterocyclic analogue surveyed in this series that exhibited a potency profile comparable to 32. However, 36 lacked stability in rat and monkey liver microsomes (t1/2 = 10 and 25 min, respectively). New lead compound 32 not only maintained good stability in HLM and RLM and high membrane permeability compared to parent compound 29 but also improved upon the metabolic stability in dog and monkey liver microsomes, off-target activity against CYP enzymes, and PXR transactivation (Table 6). In a 24 h pharmacokinetic study in rats, compound 32 exhibited low clearance and a high oral bioavailability. Although the concentration of 32 in the liver at the 6 h time point (8.7 μM) following oral dosing was approximately half of that obtained with 29, distribution into the liver was still favored for the neutral molecule (C24 liver/plasma ratio = 2.4). These in vitro and in vivo properties, together with a modest 4-fold shift in potency in the presence of 40% human serum and favorable cytotoxicity (CC50 = 20 μM in Huh-7 cells) exhibited by 32, set the stage for fine-tuning toward a viable development candidate. At this point, improving inhibitory potency against the gt1bC316N mutant to an EC50 value of less than 50 nM without increasing lipophilicity, while minimizing hERG activity (Tl+ flux IC50 = 4.8 μM; whole cell patch clamp: 96% inhibition @ 10 μM for 32) in this class of neutral molecules was challenging. Improving the gt-1bC316N inhibitory potency and resolving the hERG off-target liability represented a dilemma because modulation of the two parameters involved opposing lipophilicity requirements. One approach that had been contemplated to increase gt-1bC316N inhibition was to engage the N316 residue via a direct interaction with the inhibitor. It was observed from crystal structures of NS5B WT/ inhibitor complexes that the C−H bond at the benzofuran 4position points directly toward the side chain of the C316 residue with approximately 3.0 Å separating the benzofuran C4−H and the C316 sulfur atom. Thus, introduction of a dipole at the benzofuran 4-position could potentially capture a dipole−dipole or a hydrogen bonding interaction with the N316 side chain. Installation of a fluorine atom that can fit into the binding space (C−F bond length = ∼1.35 Å) as a dipole probe at the 4-position of the benzofuran ring of 32 provided compound 37 (BMS-929075),35 which exhibited a 2.8-fold improvement in the activity against the gt-1bC316N mutant (EC50 = 18 nM, Table 7). Attempts to obtain a cocrystal structure of 37 bound to gt-1bC316N mutant protein to provide direct structural evidence for interaction between the 4F and N316 side chain were not successful. However, a model of a 4-F-benzofuran analogue bound to the gt-1bC316N mutant protein based on a cocrystal structure of a gt1bC316N/inhibitor complex (Supporting Information) that was subsequently determined revealed that the negatively

Table 7. Inhibitory Potency towards HCV Replicons (EC50) Representing Genotypes 1−6 for Compound 37 (Data Are Average Values of n ≥ 2 Independent Experiments)

replicon EC50 (nM)

a

1a

1b

1bC316N

2a

2b

3a

4a

5a

6a

9

4

18

481

115

22

3

2a

5

HCV NS5B enzyme IC50.

polarized fluorine atom at the 4-position of the benzofuran ring can form a favorable electrostatic interaction with the positively polarized carbonyl carbon in the primary amide group of the N316 side chain (Figure 8). Both the angle (82°) and distance

Figure 8. Computer model of a 4-fluorobenzofuran analogue in complex with N316 mutant enzyme based on a cocrystal structure of gt-1bC316N/inhibitor complex. Image generated with The PyMol Molecular Graphics System (v. 1.8, Schrödinger, LLC).

(2.8 Å) are favorable for a C−F···CO interaction and consistent with those values reported by Banner, Müller, and Diederich.36,37 It is conceivable that this atypical molecular interaction contributed to the enhancement, though modest, of the potency against the gt-1bC316N mutant. Although 37 is 98.2% bound in human serum, it exhibited only a modest 5.5-fold right shift in the replicon EC50 against gt-1a and gt-1b in the presence of 40% human serum. At the onset of the project, the targeted antiviral profile was for coverage of both WT gt-1 virus and the pre-existing gt1bC316N mutant. Further evaluation of 37 in hybrid replicons representing the NS5B replicase of other HCV genotypes that became available in the later stages of the project revealed that the compound retained high inhibitory potency against genotypes 3−6 but was less potent toward gt-2 (Table 7). 4375



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Figure 9. Ligand efficiency of HCV NS5B inhibitors 7, 18, and 37 with the % increase in HAC indicated.

methyl amide forms two strong hydrogen bonds, one as an acceptor with Arg200 and one as a donor to Ser365. The 4fluorine atom on the benzofuran ring is in close contact with the thiol of the Cys316 side chain in a hydrogen bonding-type interaction. The benzofuran ring and the meta-amide phenyl ring form favorable electrostatic interactions with Arg200 and the phenyl ring of Tyr448. The 2′-methyl group fills a hydrophobic hole. The cyclopropyl ring projects into a hydrophobic pocket, and the pyrimidine ring forms an edgeto-face interaction with the phenyl ring of the Phe551 side chain. Based on the higher-resolution X-ray structures of similar compounds bound to gt-1b NS5B WT protein, it is likely that the secondary meta-amide forms two water-bridged hydrogen bonds: one to Arg200 and one to the hydroxyl group of the Tyr452 side chain. The small molecule crystal structure of the unsolvated neat form of 37 exhibits two conformers per asymmetric unit (Figure 10b). The relative three-dimensional arrangements of the two conformers are largely the same, with subtle differences in the orientation of the C2-methyl amide and the 4-fluorophenyl ring, and are close to the NS5B-bound conformation of 37. The bound and unbound molecular conformations of 37 differ in the positioning of the pyrimidinylcyclopropyl moiety by a 180° rotation about the cyclopropane-amide C1−N bond. However, in both the bound and unbound forms, the pyrimidinylcyclopropyl group adopts a bisected conformation, which is predicted to be the only energy minimum for rotation about the cyclopropane−pyrimidine bond based on MP2/6-31+G** calculations on a model system (Figure 7).32 These observations on the solid state structures of 37 suggest that the entropic cost of binding of 37 to NS5B can be substantially reduced. The extended structure of 37 as displayed in both the bound and unbound states suggests the solvent accessible surface area of 37 can be maximized for binding. Here, maximization of enthalpic gain and accessible surface area combined with reduction of entropic cost as ligand size increases are beneficial design approaches for improving ligand efficiency in this fragment-growing optimization process. The incorporation of the 4-F atom on the benzofuran ring also served to mitigate hERG inhibition (Table 8) despite an increase in the lipophilicity of the neutral molecule (cLogP = 4.98, shake flask LogDpH6.5 = 5.09 for 37) compared to that of 4-H analogue 32. Compound 37 exhibited an IC50 of 24 μM in the hERG ion flux assay, which was consistent with an IC50 of 18 μM in a whole cell patch clamp electrophysiology assessment. This favorably compared to the 96% inhibition @ 10 μM observed with 32. The lipophilicity value of 37 is also above the upper limit (cLogP = 4, LogD = 3.3) estimated for a neutral molecule to ensure an hERG inhibition IC50 value of >10 μM.43 Further analysis of the benzofuran series suggested that the small structural change from benzofuran to 4-Fbenzofuran constituted a matched pair transformation for attenuation of hERG inhibition although accompanied by a

Combination studies of 37 with the approved NS5A inhibitor daclatasvir38 and the NS3/4A protease inhibitor asunaprevir39 in a colony elimination experiment40 revealed that the triple combination eradicated HCV gt-1b replicons more effectively than the dual combination of daclatasvir/asunaprevir or monotherapy with any of the single inhibitors at similar total concentrations of ≥10 times the replicon EC50 value, suggesting that 37 can be a very effective agent for combination therapy. In the optimization from simple template 7 to the initial meta-amide lead compound 18 and eventually to the clinical candidate 37, ligand efficiency was maintained if not improved despite a significant increase in HAC (38 and 67%, Figure 9). This is in contrast to the generally observed trend that LE often decreases as ligand size increases.41,42 The conservation of LE as HAC increased during this lead optimization process is because of the fact that each additional functional moiety growing out from template 7 was optimally deployed based on SAR analysis coupled with insights from molecular modeling. Each part of 37 engages the protein in the palm binding pocket as observed in the gt-1b NS5B/37 complex cocrystal structure (Figure 10a), maximizing the enthalpic gain in binding: The 4fluorophenyl ring makes tight van der Waals contacts. The

Figure 10. (a) gt-1b NS5B/37 complex cocrystal structure with key interacting residues highlighted (see text). The omit Fo-Fc electron density is contoured at 3 RMSD in magenta mesh, and hydrogen bonding, π-cation, and π-stacking interactions are denoted with blue, red, and cyan dashes, respectively. (b) Small molecule crystal structure of the unsolvated neat form of 37, showing the two conformers per asymmetric unit. Images generated with The PyMOL Molecular Graphics System (v. 1.8, Schrödinger, LLC). 4376



Article

Table 8. In Vitro Profiling Data for Compound 37 cLogPa

a

LogDpH6.5 (shake flask)

4.98 CYP3A4 IC50 (μM)

5.09 CYP2C8 IC50 (μM)

>13

6.7

H/R/Mk/D LMb t1/2 (min)

PAMPA pH 7.4 (nm/s)

Caco Pc A to B (nm/s)

>120/>120/>120/>120 972 hERG Flux IC50 hERG patch clamp PXR-TA EC50 (μM) IC50 (μM) (μM) 24

18

H/R/Mk/Db protein binding (% bound)

>100 Huh-7 CC50 (μM)

HepG2 CC50 (μM)

60

>12.5

>50

98.2/99.7/97.2/98.1 primary human hepatocytes CC50 (μM) >50

Calculated values.30 bH/R/Mk/D LM denotes human, rat, monkey, and dog liver microsomes, respectively.

0.2−0.3 unit increase in measured LogD or cLogP value.44 As shown in Figure 11, the 4-F atom reduces the negative

and PXR transactivation profile of 32 while improving upon microsomal stability. The molecule was equally stable in liver microsomes across all species (LM t1/2 > 120 min, Table 8) while maintaining good intrinsic membrane permeability as indicated by the high PAMPA value (972 nm/s, pH 7.4). In a multiconcentration bidirectional Caco-2 permeability assay, 37 displayed an A to B value of >100 nm/s with insignificant efflux. These properties translated into promising oral exposure in rats. The exposure of 37 in rat liver following oral dosing was increased by 2-fold at the 6 h time point (15.1 μM) compared to that of 32, an improvement that extended to the 24 h time point (1.12 vs 0.614 μM). The PO exposure in rat plasma as measured by Cmax and AUC were also higher for 37 (Table 9). The improved IV clearance (1.7 mL min−1 kg−1) exhibited by 37 likely contributed to its higher exposure. Thus, the introduction of a single fluorine atom to 32 yielding 37 imparted multiple beneficial effects including improved potency, metabolic stability, and oral exposure.47 Subsequent pharmacokinetic studies in dogs and monkeys revealed low IV clearance across species for 37 with generally good oral exposure and good to excellent bioavailability. Remarkably, the curves in both the dose-normalized IV and PO plasma concentration/time profiles of compound 37 in rats, dogs, and monkeys appeared to be superimposable.48 However, this consistent in vivo performance of 37 in the three different preclinical species came with a caveat. Although 37 has excellent permeability as mentioned above, because of its low crystalline aqueous solubility (100

a

IV, 2 mg/kg; PO, 6 mg/kg; 9:1 PEG400/EtOH, n = 3. bASD formulation with 10% (W/W) drug loading in 0.02:0.5:99.48 docusate sodium/ methocel/water. cThe liver/plasma ratio was obtained in separate in vivo studies; ND = not determined. 4377



Article

Scheme 1. Discovery Synthesis of 37a

Reagents and conditions: (a) Selectfluor (1.2 equiv), MeCN, rt, 24 h; (b) NaOH (1N, aq), 1:1 MeOH/THF, 90 °C, 24% 2 steps; (c) MeNH2·HCl, HOBT·H2O, EDC, i-Pr2NEt, DMF, rt, 93%; (d) PhN(OTf)2, Et3N, CH2Cl2, rt, 98%, (e) (PPh3)4Pd, Cs2CO3, 1:10 H2O/Dioxane, 95 °C, 58%; (f) TBTU, i-Pr2NEt, DMF, rt 79%.

a

mg and higher, the C24 concentration in the SAD study and the steady state C24 values from the MAD study were at or above 10-fold the EC50 value for the gt-1bC316N replicon. These results suggest that 37 has promising potential as a QD drug.

human dose, which would surpass by 10-fold the gt-1bC316N EC50 at C24 in the liver, was 79 mg QD. Although, except for the topological polar surface area, the molecular properties of 37 (LogDpH6.5 = 5.09, cLogP = 4.98; MW = 538; tPSA = 86; Hbond donors = 2, N/O count = 7; no. of rotatable bonds = 6; no. of aromatic rings = 5) are at the upper limits of those considered for orally bioavailable drugs,50 compound 37 exhibited consistently high oral bioavailability in preclinical animal species that projected a similar pharmacokinetic performance in humans. The dissolution-limited absorption was adequately addressed by utilizing an ASD formulation with stable high drug loading that enabled flexibility for dose escalation. Compound 37 was evaluated in an in-house safety screening panel of 43 off-targets that included receptors, ion channels, and enzymes, and no significant response was observed. In a single-dose oral toxicokinetic/tolerability study in rats and dogs, compound 37 was well tolerated at doses ≤100 mg/kg. In a 2week toxicity study in rats, there were no significant findings at doses ≤100 mg/kg/day. At 100 mg/kg/day on day 14, the mean plasma AUC values of compound 37 in rats were up to 231-fold higher than the projected human efficacious AUC value. In the one month IND toxicology studies in rats and dogs, the exposures at NOAELs were 7- to 55-fold higher, respectively, than the human AUC at the predicted efficacious dose. The promising preclinical pharmacokinetic results and favorable toxicological profile supported the advancement of 37 into Phase I clinical studies. In a randomized, double-blind, placebo-controlled Phase I clinical evaluation using the ASD formulation with 30% drug loading, compound 37 was safe and well-tolerated in normal healthy volunteers in a single ascending dose (SAD) study at doses up to 400 mg as well as in a multiple ascending dose (MAD) study at doses up to 400 mg/day for 14 days. The human pharmacokinetic parameters were better than projected with a dose proportional increase in plasma exposure and a longer human t1/2.48 The Cmax/C24 exhibited a characteristic small ratio in both the SAD and MAD studies. At doses of 25

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CONCLUSIONS Insights from the superposition of bound structures of the three structurally different NS5B palm site allosteric inhibitors, anthranilic acid 4, benzofuran 5, and benzothiadiazine 6, inspired an optimization process originating with the simple benzofuran template 7. Elaboration of the C5 position in 7 through a fragment growing approach ultimately resulted in the identification of clinical candidate 37. The recessed and hydrophobic nature of the palm site binding pocket necessitated a careful modulation of lipophilicity to achieve a balance of high inhibitory potency and favorable ADME properties. The conformational flexibility of the Tyr448 loop and C-terminal region residues that form part of the binding pocket and adapt to the inhibitor structure was exploited for productive structural modifications. Noteworthy SAR observations include the multiple advantageous effects conferred by a single fluorine atom strategically deployed in the molecule, and the impact of electrostatic potential modulation on hERG channel affinity of a lipophilic neutral molecule. The pyrimidinylcyclopropyl moiety emerged from the different pyridine and diazine regioisomers as a metabolically stable group across species. Compound 37 maintained the ligand efficiency of 7 even though the HAC was increased significantly by ∼70%. This was realized by maximizing binding interactions and minimizing ligand reorganization. Compound 37 expressed potent HCV inhibition toward all genotypes with the exception of gt-2. The activity against gt-2 of this and related series was subsequently improved during a second generation optimization process.51 Triple combination of 37 with NS5A and NS3/ 4A inhibitors demonstrated efficacy against HCV gt-1b replicon cells. Compound 37 exhibited consistent high oral bioavailability and promising pharmacokinetic parameters across the three preclinical animal species, which predicted a similar 4378



Article

7.32, 1H,), 7.23 (apparent d, J = 7.10, 2H), 7.17 (t, J = 7.17, 1H), 2.85 (appeared as d, J = 4.58, 3H), 1.30 (appeared as d, 4H). LC/MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 4 mL/min. Column: Xterra MS 7 μm, C18, 3.0 × 50 mm. (ES+) m/z = 523.44 (M + H)+; Rt = 1.803 min. 5-(2-Chloro-5-(1-phenylcyclopropylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran-3-carboxamide (26). 1H NMR (500 MHz, CD3OD) δ 9.36 (broad s, 1H), 7.98−7.94 (overlapping m, 3H), 7.85 (dd, J = 8.39, 2.29, 1H), 7.75 (d, J = 1.83, 1H), 7.66 (d, J = 8.55, 1H), 7.63 (d, J = 8.00, 1H), 7.48 (dd, J = 8.55, 1.83, 1H), 7.29−7.25 (overlapping m, 6H), 7.17 (m, 1H), 2.95 (s, 3H), 1.36−1.34 (appeared as d, 4H). LC/MS method: solvent A = 10% MeOH− 90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 4 mL/min. Column: Xterra MS 7 μm, C18, 3.0 × 50 mm. (ES+) m/z = 539.37, 541.38 (M + H)+; Rt = 1.868 min. Analytical HPLC method: solvent A = 5% MeCN−95% H2O−0.1% TFA, solvent B = 95% MeCN−5% H2O−0.1% TFA, start % B = 50, final % B = 100, gradient time = 15 min, stop time = 18 min, flow rate = 1 mL/min. Column: Sunfire C18, 3.5 μm, 4.6 × 150 mm; Rt = 10.84 min. Column: Xbridge Phenyl, 3.5 μm, 4.6 × 150 mm; Rt = 8.37 min. 2-(4-Fluorophenyl)-5-(2-methoxy-5-(1phenylcyclopropylcarbamoyl)phenyl)-N-methylbenzofuran-3-carboxamide (27). 1H NMR (400 MHz, CD3OD) δ 8.05−7.94 (m, 4H), 7.85 (d, J = 1.51, 1H), 7.67−7.62 (m, 1H), 7.62−7.56 (m, 1H), 7.38− 7.28 (m, 6H), 7.26−7.18 (m, 2H), 3.94 (s, 3H), 3.01 (s, 3H), 1.40 (dd, J = 7.03, 2.01, 4H). LC/MS method: solvent A = 10% MeCN− 90% H2O−0.1% TFA, solvent B = 90% MeCN−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 3 min, stop time = 4 min, flow rate = 4 mL/min. Column: Waters Sunfire 5 μm C18, 4.6 × 50 mm. (ES+) m/z = 535.16 (M + H)+; Rt = 2.373 min. Analytical HPLC method: solvent A = 5% MeCN−95% H2O−0.1% TFA, solvent B = 95% MeCN−5% H2O−0.1% TFA, start % B = 10, final % B = 100, gradient time = 10 min, stop time = 20 min, flow rate = 1 mL/min. Column: Sunfire C18, 3.5 μm, 4.6 × 150 mm; Rt = 10.31 min. Column: Xbridge Phenyl, 3.5 μm, 4.6 × 150 mm; Rt = 12.23 min. 2-(4-Fluorophenyl)-N-methyl-5-(2-methyl-5-(1phenylcyclopropylcarbamoyl)phenyl)benzofuran-3-carboxamide (28). 1H NMR (500 MHz, CD3OD) δ 7.97 (dd, J = 8.85, 5.49, 2H), 7.79 (m, 2H), 7.65−7.64 (d overlapping s, 1H), 7.64 (s, 1H), 7.42 (m, 1H), 7.37 (dd, J = 8.24, 1.83, 1H), 7.29−7.26 (overlapping m, 6H), 7.16 (m, 1H), 2.95 (s, 3H), 2.34 (s, 3H), 1.36−1.33 (appeared as d, 4H). LC/MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 4 mL/min. Column: Xterra MS 7 μm, C18, 3.0 × 50 mm. (ES+) m/z = 519.40 (M + H)+; Rt = 1.832 min. Analytical HPLC method: solvent A = 5% MeCN−95% H2O−0.1% TFA, solvent B = 95% MeCN−5% H2O−0.1% TFA, start % B = 50, final % B = 100, gradient time = 15 min, stop time = 18 min, flow rate = 1 mL/min. Column: Sunfire C18, 3.5 μm, 4.6 × 150 mm; Rt = 9.93 min. Column: Xbridge Phenyl, 3.5 μm, 4.6 × 150 mm; Rt = 7.64 min. 2-(4-Fluorophenyl)-N-methyl-5-(2-methyl-5-(1-(pyridin-2-yl)cyclopropylcarbamoyl)phenyl)benzofuran-3-carboxamide (29). 1H NMR (500 MHz, CD3OD) δ 8.61 (d, J = 5.80, 1H), 8.44 (t, J = 7.32, 1H), 7.97 (dd, J = 8.85, 5.19, 2H), 7.87 (s, 1H), 7.85 (d, J = 6.71, 1H), 7.83−7.81 (m, 2H), 7.67 (d, J = 8.24, 1H), 7.64 (d, J = 0.92, 1H), 7.47 (d, J = 8.55, 1H), 7.38 (dd, J = 8.39, 1.68, 1H), 7.29 (t, J = 8.85, 2H), 2.95 (s, 3H), 2.36 (s, 3H), 1.82 (m, 2H), 1.75 (m, 2H). LC/MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 4 mL/min. Column: Xterra MS 7 μm, C18, 3.0 × 50 mm. (ES+) m/z = 520.41 (M + H)+; Rt = 1.513 min. Analytical HPLC method: solvent A = 5% MeCN−95% H2O−0.1% TFA, solvent B = 95% MeCN−5% H2O− 0.1% TFA, start % B = 10, final % B = 100, gradient time = 15 min, stop time = 18 min, flow rate = 1 mL/min. Column: Sunfire C18, 3.5

pharmacokinetic performance in humans. The dissolutionlimited absorption of this BCS class II compound was resolved by utilizing a stable amorphous solid dispersion formulation and, together with the flexibility of high drug loading, allowed dose escalation. The human PK properties in normal healthy volunteers from both the SAD and MAD Phase I clinical studies were better than projected, suggesting that 37 has promising potential as a QD drug.

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CHEMISTRY The discovery synthesis of 37 to provide sufficient quantities of materials at the required purity for all biological activity assays, in vitro profiling, initial PK studies in rat, dog, and monkey, evaluations in different formulations, and an initial 4-day minitox study in rat is shown in Scheme 1. The Suzuki crosscoupling of triflate 42 with boronic ester 43 to provide penultimate acid 44, followed by amide coupling with 1(pyrimidin-2-yl)cyclopropan-1-amine 45 to deliver 37, is representative of the general synthetic approach for the synthesis of the analogues described in this report. Coupling intermediates 4352 and 4553 were prepared in large scale by following literature procedures. The key intermediate triflate 42 was derived from 5-hydroxybenzofuran 38, synthesized according to ref 21. Fluorination of 38 with Selectfluor provided a crude mixture of desired 4-F intermediate 39 and the 6-F isomer in ∼3:1 ratio. A pure sample of 39 was obtained after purification to confirm its identity by NMR studies. The crude mixture of the fluorinated product was subjected to ester hydrolysis and then purification to provide desired compound 40 in 24% overall yield for the two steps. Methyl amide formation at C3 followed by installation of the triflate at C5 converted 40 smoothly to triflate 42 in almost quantitative yield. After consecutive coupling of 42 with boronic ester 43 and then amine 45 and recrystallization of the crude product from methanol/water mixture, 37 was obtained in 79% yield.

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EXPERIMENTAL SECTION

Biological methods and the preparation of analogues presented in Tables 1 and 2 can be found in ref 35. The 1-(diazin-2-yl)cyclopropan1-amines for analogues 33−35 were prepared as described in ref 53. General. All reagents were purchased from commercial suppliers and used without purification unless otherwise stated. All anhydrous reactions were performed under a nitrogen atmosphere using anhydrous solvents from commercial sources. Reaction progress was monitored by analytical TLC or LC/MS. 1H NMR spectra were recorded on a Bruker AvanceIII 500 MHz spectrometer or a Bruker Ultrashield 400 MHz spectrometer. All spectra were determined in the deuterated solvent indicated, and residual protic solvent was used as an internal standard for chemical shift assignments. Proton coupling constants were reported in hertz (Hz) and chemical shifts δ in ppm. Multiplicity patterns were designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; dd, doublet of doublets; dt, doublet of triplets; dq, doublet of quartets. All compounds were determined to be ≥95% purity as detemined by liquid chromatography methodologies. LC/MS were performed using a Shimadzu-VP instrument with UV detection at 220 nm and Waters Micromass. Analytical HPLC were performed using a Shimadzu-VP instrument with UV detection at 220 and 254 nm. HPLC conditions are as indicated for each compound. Rt denotes retention time in minutes. 5-(2-Fluoro-5-(1-phenylcyclopropylcarbamoyl)phenyl)-2-(4-fluorophenyl)-N-methylbenzofuran-3-carboxamide (25). 1H NMR (500 MHz, DMSO-d6) δ 9.33 (s, 1H), 8.51 (q, J = 4.58, 1H), 8.15 (dd, J = 7.48, 2.29, 1H), 8.00 (dd, J = 9.00, 5.34, 2H), 7.97 (m, 1H), 7.83 (s, 1H), 7.82 (d, J = 8.60, 1H), 7.64 (d, J = 8.55, 1H), 7.46 (dd, J = 10.22, 8.70, 1H), 7.41 (t, J = 9.00, 2H), 7.29 (t, J = 7.63, 1H), 7.28 (d, J = 4379



Article

μm, 4.6 × 150 mm; Rt = 8.90 min. Column: Xbridge Phenyl, 3.5 μm, 4.6 × 150 mm; Rt = 9.43 min. 2-(4-Fluorophenyl)-N-methyl-5-(2-methyl-5-(1-(pyridin-3-yl)cyclopropylcarbamoyl)phenyl)benzofuran-3-carboxamide (30). 1H NMR (500 MHz, CD3OD) δ 8.76 (d, J = 2.44, 1H), 8.71 (dd, J = 5.80, 0.92, 1H), 8.50 (ddd, J = 8.32, 2.21, 1.37, 1H), 8.04 (dd, J = 8.39, 5.65, 1H), 7.96 (dd, J = 9.00, 5.34, 2H), 7.83 (d, J = 1.53, 1H), 7.82 (m overlapped with d, 1H), 7.66 (d, J = 8.24, 1H), 7.64 (d, J = 1.83, 1H), 7.45 (d, J = 8.55, 1H), 7.28 (t, J = 8.70, 2H), 2.95 (s, 3H), 2.35 (s, 3H), 1.60 (dt, J = 4.20, 2.59, 4H). LC/MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O− 0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 5 mL/min. Column: Phenomenex-Luna, 3.0 × 50 mm, S10. (ES+) m/z = 520.08 (M + H)+; Rt = 1.482 min. 2-(4-Fluorophenyl)-N-methyl-5-(2-methyl-5-(1-(pyridin-4-yl)cyclopropylcarbamoyl)phenyl)benzofuran-3-carboxamide (31). 1H NMR (500 MHz, CD3OD) δ 8.68 (d, J = 7.32, 1H), 7.97 (dd, J = 8.85, 5.19, 2H), 7.87 (d, J = 2.14, 1H), 7.86 (m overlapped with d, 1H), 7.81 (d, J = 7.02, 1H), 7.67 (d, J = 8.55, 1H), 7.65 (d, J = 1.22, 1H), 7.48 (d, J = 8.55, 1H), 7.39 (dd, J = 8.39, 1.68, 1H), 7.28 (t, J = 8.85, 2H), 2.95 (s, 3H), 2.37 (s, 3H), 1.83 (appeared as d, 4H). LC/MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 5 mL/min. Column: Phenomenex-Luna, 3.0 × 50 mm, S10. (ES+) m/z = 520.15 (M + H)+; Rt = 1.435 min. Analytical HPLC method: solvent A = 5% MeCN−95% H2O−0.1% TFA, solvent B = 95% MeCN−5% H2O− 0.1% TFA, start % B = 50, final % B = 100, gradient time = 15 min, stop time = 18 min, flow rate = 1 mL/min. Column: Sunfire C18, 3.5 μm, 4.6 × 150 mm; Rt = 8.03 min. Column: Xbridge Phenyl, 3.5 μm, 4.6 × 150 mm (start % B = 10, final % B = 100); Rt = 14.09 min. 2-(4-Fluorophenyl)-N-methyl-5-(2-methyl-5-(1-(pyrimidin-2-yl)cyclopropylcarbamoyl) phenyl) benzofuran-3-carboxamide (32). 1 H NMR (400 MHz, DMSO-d6): δ 9.20 (s, 1H), 8.68−8.67 (d, J = 4.8, 2H), 8.50−8.47 (m, 1H), 8.02−7.98 (m, 2H), 7.87−7.86 (d, J = 4.4, 2H), 7.78−7.76 (d, J = 8.4, 1H), 7.59 (s, 1H), 7.45−7.39 (m, 4H), 7.29−7.26 (t, J = 4.8, 1H), 2.83−2.82 (d, J = 4.8, 3H), 2.31 (s, 3H), 1.61−1.59 (m, 2H), 1.36−1.33 (m, 2H). LC/MS method: solvent A = 2% MeCN−98% H2O−10 mM NH4CHO2, solvent B = 98% MeCN− 2% H2O−10 mM NH4CHO2, time (min)/% B = 0/0, 1.5/100, 3.2/ 100, 3.6/0, flow rate = 1 mL/min. Column: Ascentis Express C18 5 × 2.1 mm, 2.7 μm. (ES+) m/z = 521.2 (M + H)+; Rt = 1.883 min. Analytical HPLC method: solvent A = 5% MeCN−95% H2O−0.05% TFA, solvent B = 95% MeCN−5% H2O−0.05% TFA, time (min)/% B = 0/10, 25/100, 30/100, flow rate = 1 mL/min. Column: Sunfire C18, 3.5 μm, 4.6 × 150 mm; Rt = 17.12 min; Xbridge Phenyl, 3.5 μm, 4.6 × 150 mm; Rt = 15.73 min. 2-(4-Fluorophenyl)-N-methyl-5-(2-methyl-5-(1-(pyrazin-2-yl)cyclopropylcarbamoyl)phenyl)benzofuran-3-carboxamide (33). 1H NMR (400 MHz, CD3OD): δ 8.59 (s, 1H), 8.52 (t, 1 H), 8.37 (d, J = 2.4, 1H), 8.00−7.95 (m, 2H), 7.87−7.84 (m, 2H), 7.67 (d, J = 7.2 Hz, 1H), 7.66 (s, 1H), 7.47 (d, J = 8.4, 1H), 7.40 (dd, J = 8.4, 2.0, 1H), 7.31−7.25 (m, 2H), 2.95 (s, 3H), 2.37 (s, 3H), 1.74−1.71 (m, 2H), 1.46−1.43 (m, 2H). 19F NMR (376.51 MHz, CD3OD): δ −113.35. LC/MS method: solvent A = 2% MeCN−98% H2O−10 mM NH4CHO2, solvent B = 98% MeCN−2% H2O−10 mM NH4CHO2, time (min)/% B = 0/0, 1.5/100, 3.2/100, flow rate = 1 mL/min. Column: Ascentis Express C18, 5 × 2.1 mm, 2.7 μm. (ES+) m/z = 521.2 (M + H)+; Rt = 1.902 min. Analytical HPLC method: buffer, 0.05% TFA in water, pH 2.5; mobile phase A, 95:5 buffer/MeCN; mobile phase B, 95:5 MeCN/buffer, time (min)/% B = 0/10, 12/100, 15/100, flow rate = 1 mL/min. Column: Sunfire C18, 3.5 μm, 4.6 × 150 mm; Rt = 10.81 min; Xbridge Phenyl, 3.5 μm, 4.6 × 150 mm; Rt = 9.90 min. 2-(4-Fluorophenyl)-N-methyl-5-(2-methyl-5-(1-(pyrimidin-4-yl)cyclopropyl carbamoyl)phenyl)benzofuran-3-carboxamide (34). 1H NMR (400 MHz, CD3OD): δ 9.03 (d, J = 1.0, 1 H), 8.61 (d, J = 5.8, 1 H), 8.04−7.94 (m, 2 H), 7.91−7.83 (m, 2 H), 7.72−7.64 (m, 2 H), 7.57−7.45 (m, 2 H), 7.43−7.37 (m, 1 H), 7.34−7.24 (m, 2 H), 2.96 (s, 3 H), 2.37 (s, 3 H), 1.90−1.81 (m, 2 H), 1.57−1.49 (m, 2 H). 19F

NMR (376.51 MHz, CD3OD): δ −113.31. LC/MS method: solvent A = 10% MeCN−90% H2O−20 mM NH4OAc, solvent B = 90% MeCN−10% H2O−20 mM NH4OAc, time (min)/% B = 0/0, 2/100, 2.5/100, 3/0, flow rate = 2.5 mL/min. Column: Purospher STAR RP18 (4 × 55) mm, 3 μm. (ES+) m/z = 521.2 (M + H)+; Rt = 1.828 min. Analytical HPLC method: buffer, 0.05% TFA in water, pH 2.5; mobile phase A: 95:5 buffer/MeCN; mobile phase B: 95:5 MeCN/buffer, time (min)/% B = 0/10, 25/100, 30/100, flow rate = 1 mL/min. Column: Sunfire C18, 3.5 μm, 4.6 × 150 mm; Rt = 16.43 min; Xbridge Phenyl, 3.5 μm, 4.6 × 150 mm; Rt = 15.27 min. 2-(4-Fluorophenyl)-N-methyl-5-(2-methyl-5-(1-(pyridazin-3-yl)cyclo propylcarbamoyl)phenyl)benzofuran-3-carboxamide (35). 1H NMR (400 MHz, CDCl3): δ 9.17 (s, 1 H), 7.92−7.96 (m, 3 H), 7.77− 7.69 (m, 4 H), 7.65 (s, 1 H), 7.56 (d, J = 8.4, 1 H), 7.38 (d, J = 8.0, 1 H), 7.30 (dd, J = 8.4, 1.6, 1H), 7.23−7.19 (m, 2 H), 5.94 (bs, 1 H), 3.0 (s, 3 H), 2.33 (s, 3 H), 1.89−1.86 (m, 2 H), 1.60−1.57 (m, 2 H). 19F NMR (376.47 MHz, CDCl3): δ −109.29. LC/MS method: solvent A = 2% MeCN−98% H2O−10 mM NH4CHO2, solvent B = 98% MeCN−2% H2O−10 mM NH4CHO2, time (min)/% B = 0/0, 1.5/ 100, 3.2/100, flow rate = 1 mL/min. Column: Ascentis Express C18 5 × 2.1 mm, 2.7 μm. (ES+) m/z = 521.0 (M + H)+; Rt = 1.858 min. Analytical HPLC method: buffer, 0.05% TFA in water, pH 2.5; mobile phase A: 95:5 buffer/MeCN; mobile phase B: 95:5 MeCN/buffer, time (min)/% B = 10/10, 12/100, 15/100, flow rate = 1 mL/min. Column: Sunfire C18, 3.5 μm, 4.6 × 150 mm; Rt = 9.77 min; Xbridge Phenyl, 3.5 μm, 4.6 × 150 mm; Rt = 9.33 min. 2-(4-Fluorophenyl)-N-methyl-5-(2-methyl-5-(1-(3-methyl-1,2,4oxadiazol-5-yl)cyclopropylcarbamoyl)phenyl)benzofuran-3-carboxamide (36). A mixture of methyl 1-(3-(2-(4-fluorophenyl)-3(methylcarbamoyl)benzofuran-5-yl)-4-methylbenzamido)cyclopropanecarboxylate (25.3 mg, 0.051 mmol), N′-hydroxyacetimidamide (11.23 mg, 0.152 mmol), and K2CO3 (41.9 mg, 0.303 mmol) in toluene (1 mL) under N2 in a reusable sealed tube was stirred at 150 °C for 2 h. The mixture was evaporated, diluted with MeOH, and purified by Shimadzu-VP preparative reverse-phase HPLC using the separation method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 50, final % B = 100, gradient time = 10 min, stop time = 12 min, flow rate = 25 mL/min. Column: Waters-Sunfire 19 × 100 mm S5. Fraction collection: 7.54−8.08 min (UV detection at 220 nm) to give the product as a white solid (19.9 mg, 75%). 1H NMR (500 MHz, CD3OD) δ 7.98 (m, J = 9.00, 5.34, 2H), 7.82 (d, J = 2.14, 1H), 7.81 (m, 1H), 7.66 (d, J = 8.55, 1H), 7.65 (d, J = 1.50, 1H), 7.44 (d, J = 8.55, 1H), 7.38 (dd, J = 8.24, 1.83, 1H), 7.28 (m, J = 8.85, 2H), 2.95 (s, 3H), 2.36 (s, 3H), 2.31 (s, 3H), 1.75 (m, 2H), 1.58 (m, 2H). LC/ MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 5 mL/min. Column: Phenomenex-Luna, 3.0 × 50 mm, S10. (ES+) m/z = 525.12 (M + H)+; Rt = 1.700 min. 4-Fluoro-2-(4-fluorophenyl)-N-methyl-5-(2-methyl-5-(1-(pyrimidin-2-yl)cyclopropylcarbamoyl)phenyl)benzofuran-3-carboxamide (37). To a mixture of 3-(4-fluoro-2-(4-fluorophenyl)-3(methylcarbamoyl)benzofuran-5-yl)-4-methylbenzoic acid (44) (1.87 g, 4.44 mmol), 1-(pyrimidin-2-yl)cyclopropanamine·2HCl (45) (1.108 g, 5.33 mmol), and 2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium tetrafluoroborate (2.85 g, 8.88 mmol) in DMF (40 mL) at rt under N2 was added N,N-diisopropylethylamine (3.10 mL, 17.75 mmol) dropwise. The mixture was stirred at rt for 25 h. The mixture was then added with H2O (210 mL) slowly, and the light pinkish-white solid was filtered, washed with H2O (4 × 55 mL), and dried. The solid was further washed with CH2Cl2 (3 × 21 mL) (during the first wash, the solid first appeared to dissolve in CH2Cl2 and then reappeared as solid), and dried to give a white solid (2.03 g). The solid was dissolved in MeOH (200 mL) and filtered through a sintered glass funnel (with 2 × 10 mL washings with MeOH). H2O was added to the solution while being rigorously swirled (with 2.5−5 mL each time (total 170 mL)) until it turned cloudy. The round-bottom flask was covered with a wiper tissue paper and left standing at rt for 25 h. The white crystalline solids were filtered and washed with a mixture of 4380



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Phenomenex-Luna, 3.0 × 50 mm, S10. (ES+) m/z = 319.14 (M + H)+; Rt = 1.798 min. 4-Fluoro-2-(4-fluorophenyl)-5-hydroxybenzofuran-3-carboxylic Acid (40). To a mixture of ethyl 2-(4-fluorophenyl)-5-hydroxybenzofuran-3-carboxylate (38) (3 g, 9.99 mmol) in acetonitrile (200 mL) at rt under N 2 was added 1-(chloromethyl)-4-fluoro-1,4diazoniabicyclo[2.2.2]octane tetrafluoroborate (4.25 g, 11.99 mmol) portion-wise. The mixture was stirred at rt for 24 h. The mixture was then filtered to remove the white insoluble portion, and the filtrate was evaporated. The residue was dissolved in CH2Cl2 (30 mL) and purified by Biotage Horizon chromatography (0−70% EtOAc/hexane, 3 × 160 g silica gel columns, 18 mm tube−27 mL × 48 tubes) to give an orange solid (1.89 g). To a mixure of the solid obtained from above (1.89 g, 5.94 mmol) in a mixture of MeOH (35 mL)/THF (35 mL) at rt under N2 was added sodium hydroxide (17.81 mL, 17.81 mmol, 1 N aqueous). The mixture was stirred at 90 °C for 6 h. The mixture was cooled to rt, acidified with 1 N HCl (20 mL), and then evaporated to dryness. To the solid residue was added H2O (30 mL); the light brown solid was filtered, washed with H2O (4 × 10 mL), and dried. To the solid was added MeOH (30 mL), which was purified by Shimadzu-VP preparative reverse-phase HPLC using the separation method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH− 10% H2O−0.1% TFA, start % B = 40, final % B = 100, gradient time = 10 min, stop time = 12 min, flow rate = 25 mL/min. Column: Sunfire Prep C18 19 × 100 5um. Fraction collection: 6.80−7.40 min (UV detection at 220 nm). Evaporation of the combined desired fractions gave desired C4−F product 40 as an off-white solid (690 mg, 24% 2 steps). 1H NMR (500 MHz, CD3OD) δ 7.98 (m, 2H), 7.25 (t overlapping with dd, 2H), 7.24 (dd, 1H), 7.02 (t, J = 8.39, 1H). LC/ MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 5 mL/min. Column: Phenomenex-Luna, 3.0 × 50 mm, S10. (ES+) m/z = 291.01 (M + H)+; Rt = 1.478 min. 4-Fluoro-2-(4-fluorophenyl)-5-hydroxy-N-methylbenzofuran-3carboxamide (41). To a mixture of 4-fluoro-2-(4-fluorophenyl)-5hydroxybenzofuran-3-carboxylic acid (40) (546.5 mg, 1.883 mmol), methylamine·HCl (191 mg, 2.82 mmol), HOBT hydrate (490 mg, 3.20 mmol), and EDC hydrochloride (650 mg, 3.39 mmol) in DMF (20 mL) at rt under N2 was added N,N-diisopropylethylamine (1.644 mL, 9.42 mmol). The mixture was stirred at rt for 19.5 h. The mixture was concentrated to approximately half of the volume, and then 1 N HCl (30 mL) was added, and the solution was further diluted with H2O (120 mL). The off-white solid was filtered, washed with H2O (3 × 30 mL), and dried (528.5 mg, 93%). 1H NMR (500 MHz, CD3OD) δ 7.89 (dd, J = 8.09, 5.34, 2H), 7.25 (t overlapping with dd, 2H), 7.23 (dd, 1H), 6.99 (t, J = 8.55, 1H), 2.96 (s, 3H). LC/MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH− 10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 5 mL/min. Column: PhenomenexLuna, 3.0 × 50 mm, S10. (ES+) m/z = 304.06 (M + H)+; Rt = 1.262 min. 4-Fluoro-2-(4-fluorophenyl)-3-(methylcarbamoyl)benzofuran-5yl trifluoromethanesulfonate (42). To a mixture of 4-fluoro-2-(4fluorophenyl)-5-hydroxy-N-methylbenzofuran-3-carboxamide (41) (1 g, 3.30 mmol) in CH2Cl2 (30 mL) at rt under N2 was added triethylamine (0.914 mL, 6.59 mmol) dropwise. The white suspension was cooled to 0 °C using an ice−water bath, and then 1,1,1-trifluoroN-phenyl-N-(trifluoromethylsulfonyl)methanesulfonamide (1.767 g, 4.95 mmol) was added portion-wise. The mixture, which slowly turned to a clear light yellow solution, was stirred at rt for 6 h. The mixture was evaporated to dryness, and H2O (30 mL) was added. The white powder was filtered, washed with H2O (4 × 15 mL), and dried (1.4121 g, 98%). 1H NMR (500 MHz, CD3OD) δ 7.95 (m, 2H), 7.59 (dd, J = 9.00, 1.00, 1H), 7.50 (dd, J = 9.00, 7.50, 1H), 7.30 (t, J = 8.55, 2H), 2.99 (s, 3H). LC/MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3

4.5:3.5 MeOH/H2O (2 × 40 mL) and dried under high vacuum overnight for four days (1.9036 g, 79%). HRMS (ES+) m/z observed 539.1873 [M + H]+; calculated for C31H25F2N4O3, 539.1889. 1H NMR (500 MHz, CD3OD) δ 8.65 (d, J = 5.04, 2 H), 7.96 (dd, J = 9.14, 5.36, 2 H), 7.91 (dd, J = 8.04, 2.05, 1 H), 7.85 (d, J = 1.89, 1 H), 7.54 (d, J = 8.51, 1 H), 7.46 (d, J = 7.88, 1 H), 7.33 (dd, J = 8.35, 7.09, 1 H), 7.30 (t, J = 8.83, 2 H), 7.23 (t, J = 4.89, 1 H), 2.96 (s, 3 H), 2.29 (s, 3 H), 1.77 (m, 2 H), 1.46 (m, 2 H); 13 C NMR (126 MHz, CD3OD) δ 171.48, 171.23, 167.11, 165.13 (d, J = 249.80), 158.38, 156.30 (d, J = 9.09), 154.14, 153.34 (d, J = 249.80), 142.48, 137.09, 133.40, 131.30, 130.92, 130.33, 130.26, 129.42 (d, J = 2.73), 128.42, 126.83 (d, J = 3.63), 124.47 (d, J = 15.44), 119.66, 118.01 (d, J = 20.90), 117.20 (d, J = 22.70), 112.11, 108.80 (d, J = 4.54), 38.24, 27.04, 20.57, 20.37; 19F NMR (470.45 MHz, CD3OD) δ −112.73, −123.11. Analytical HPLC was performed using a Waters Acquity HPLC with Waters PDA UV−vis detection and Waters SQ MS (ESCI probe). Flow = 0.35 mL/min. Gradient: hold 10% B 0−1 min, 10−98% B 1−32 min, hold 98% B 32−35 min, 98−10% B 35− 35.5 min, hold 10% B 35.5−40 min. UV detection: UV@295 nm (220 nm was also checked: no additional impurities were observed at 220 nm). Column: Waters Acquity BEH C18, 1.7 μm, 150 × 2.1 mm ID (at 35 °C). Mobile phase A: water with 0.05% TFA; mobile phase B: acetonitrile with 0.05% TFA; 99.6% purity. (ES+) m/z = 539.33 (M + H)+; Rt = 16.2 min. Column: Waters Acquity BEH C18, 1.7 μm, 150 × 2.1 mm ID (at 35 °C). Mobile phase A: 30 mM ammonium bicarbonate in water, pH 9.5; mobile phase B: acetonitrile; 99.6% purity; Rt = 16.3 min. Ethyl 4-Fluoro-2-(4-fluorophenyl)-5-hydroxybenzofuran-3-carboxylate (39). To a mixture of ethyl 2-(4-fluorophenyl)-5-hydroxybenzofuran-3-carboxylate (38) (500 mg, 1.665 mmol) in acetonitrile (10 mL) at rt under N2 was added 1-(chloromethyl)-4-fluoro-1,4diazoniabicyclo[2.2.2]octane tetrafluoroborate (708 mg, 1.998 mmol). The mixture was stirred at rt for 20 h and then evaporated. The residue was added with H2O (10 mL). The aqueous layer was decanted, and the residue was further washed with H2O (2 × 5 mL). The mixture was dissolved in MeOH (∼10 mL), and the insoluble portion was filtered. The filtrate was purified by Shimadzu-VP preparative reverse-phase HPLC using the separation method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH− 10% H2O−0.1% TFA, start % B = 60, final % B = 100, gradient time = 10 min, stop time = 12 min, flow rate = 25 mL/min. Column: WatersSunfire 19 × 100 mm S5. Fraction collection: 6.44−7.24 min (UV detection at 220 nm). The desired fractions were combined and evaporated to give a yellow solid. The yellow solid was further purified by Biotage Horizon flash chromatography (0−70% EtOAc/hexane, 3 × 80 g silica gel columns) to give 39 as a light yellow solid (108.9 mg). 1 H NMR (500 MHz, CD3OD) δ 7.95 (m, 2H), 7.26 (t overlapping with dd, 2H), 7.25 (dd, 1H), 7.03 (t, J = 8.39, 1H), 4.39 (q, J = 7.17, 2H), 1.36 (t, J = 7.17, 3H). 19F NMR (470.45 MHz, CD3OD) δ −112.36, −142.29 (the 19F chemical shift was referenced to CFCl3 at 0.0 ppm). The position of the F atom at C4 was confirmed by 1H−1H through bond correlation between H6 and H7, 1H−13C HMBC, and F−C4 coupling in 13C NMR (125.75 MHz, CD3OD) (δ 144.8 ppm, d, J = 247, C4). LC/MS method: solvent A = 10% MeOH−90% H2O− 0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 5 mL/min. Column: Phenomenex-Luna, 3.0 × 50 mm, S10. (ES +) m/z = 319.14 (M + H)+; Rt = 1.718 min. The minor fractions collected at approximately 7.69−8.20 min were confirmed by 1H−1H through bond correlation, 1H−13C HMBC, and F−C6 coupling in 13C NMR (125.75 MHz, CD3OD) (δ 152.5 ppm, d, J = 242 Hz, C6) to be the isomer of the F atom at C6 (∼3:1 C4/ C6 based on preparative HPLC % area of the UV trace). 1H NMR (500 MHz, CD3OD) δ 8.04 (dd, J = 8.55, 5.49, 2H), 7.59 (d, J = 8.85, 1H), 7.38 (d, J = 10.07, 1H), 7.25 (t, J = 8.70, 2H), 4.40 (q, J = 7.17, 2H), 1.41 (t, J = 7.17, 3H). 19F NMR (470.45 MHz, CD3OD) δ −112.29, −138.52. LC/MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 5 mL/min. Column: 4381



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min, flow rate = 5 mL/min. Column: Phenomenex-Luna, 3.0 × 50 mm, S10. (ES+) m/z = 436.04 (M + H)+; Rt = 1.678 min. 3-(4-Fluoro-2-(4-fluorophenyl)-3-(methylcarbamoyl)benzofuran5-yl)-4-methylbenzoic Acid (44). A mixture of 4-fluoro-2-(4fluorophenyl)-3-(methylcarbamoyl)benzofuran-5-yl trifluoromethanesulfonate (42) (1.563 g, 3.59 mmol), 4-methyl-3-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)benzoic acid (43) (1.129 g, 4.31 mmol), (Ph3P)4Pd (0.415 g, 0.359 mmol), and cesium carbonate (1.755 g, 5.39 mmol) in a mixture of water (5 mL)/dioxane (25 mL) under N2 was stirred at 90 °C for 6 h. The mixture was left standing at rt for 17 h. To the mixture was added 1,4-dioxane (50 mL), which was filtered through a short plug of Celite (with 4 × 10 mL washings with dioxane). The filtrate was concentrated to approximately half of the volume until precipitates started to form. The mixture was cooled in an ice−water bath, and 1 N HCl (70 mL) was added slowly, upon which yellow solids deposited on the wall of the flask. To the mixture was added H2O (150 mL), which was left to stand at rt for 45 min. The aqueous layer was decanted, and the yellow residue was washed with H2O (3 × 20 mL) and dried. To the residue was added MeOH (10 mL), which was filtered, and the solid was washed with MeOH (2 × 5 mL) to give an off-white solid of the product (620 mg). The MeOH filtrate was evaporated, and to the obtained residue was added Et2O (15 mL). The solids were filtered, washed with 1:5 MeOH/Et2O (∼5 mL) followed by Et2O (5 mL), and dried to give a further amount (260 mg) of the product as a white solid (880 mg total, 58%). 1H NMR (500 MHz, CD3OD) δ 7.99−7.95 (m, 3H), 7.89 (s, 1H), 7.54 (d, J = 8.55, 1H), 7.45 (d, J = 7.93, 1H), 7.31−7.27 (m, 3H), 2.96 (s, 3H), 2.29 (s, 3H). LC/MS method: solvent A = 10% MeOH−90% H2O−0.1% TFA, solvent B = 90% MeOH−10% H2O−0.1% TFA, start % B = 0, final % B = 100, gradient time = 2 min, stop time = 3 min, flow rate = 5 mL/min. Column: Phenomenex-Luna, 3.0 × 50 mm, S10. (ES+) m/z = 422.19 (M + H)+; Rt = 1.653 min. In Vivo Rat Pharmacokinetic Studies. All animal studies were performed under the approval of the Bristol-Myers Squibb Animal Care and Use Committee and in accordance with the American Association for Accreditation of Laboratory Animal Care (AAALAC). Male Sprague−Dawley rats (300−350 g) with dual indwelling cannulae implanted in the jugular or intraportal veins were used in the pharmacokinetic studies. After dosing, serial blood samples (0.3 mL) were obtained from the appropriate cannula of each rat by collection into EDTA-containing tubes (Becton Dickinson, Franklin Lakes, NJ) and centrifuged to separate plasma. Plasma was frozen until analysis. For IV studies, compound 32 or 37 was dosed (2 mg/kg) in a vehicle of 90:10 PEG-400/ethanol (v/v) as a 10 min constant rate infusion into the jugular vein with serial blood samples collected before dosing and at 10, 15, 30, 45, and 60 min and 2, 4, 6, 8, and 24 h after dosing (n = 3 rats/dose group). For PO dosing, the compound was administered (6 mg/kg) by gastric gavage as an aqueous solution (90:10 PEG-400/ethanol (v/v) or by a suspension formulation using a spray-dried dispersion (10% w/w drug loading of compound 37 in 0.02:0.5:99.48 docusate/methocel/water). Serial blood samples were collected before dosing and at 15, 30, 45, and 60 min and 2, 4, 6, 8, and 24 h after dosing (n = 3 rats/dose group). Prior to all PO dosing, the rats were fasted overnight with free access to water. For assessing liver exposure, livers were removed from rats at 24 h after IV dosing and 6 (using satellite animals) and 24 h after PO dosing. The tissues were rinsed, blotted dry, weighed, and stored frozen until analysis. Processing included addition of 2 volumes of 80% acetonitrile in Hanks balanced salt solution (HBSS) buffer per gram of tissue, homogenization with a T25 basic S1 generator (IKA Works, Wilminton, NC) using an S25N-8G dispersing tool, and centrifugation (IEC Centra-8R at 3,000 rpm for 10 min). Aliquots of supernatant were removed and stored frozen until analysis by LC/MS/MS. Screening studies of compound 29 were conducted as described above with truncated time points (0 (predose), 0.17 (IV only), 0.5, 1, 2, 4, and 6 h with livers taken at the terminal time point) and with n = 2 rats/dose group. The pharmacokinetics parameters were obtained by noncompartmental analysis of plasma concentration versus time data (KINETICA software, version 2.4, InnaPhase Corporation, Philadelphia, PA). The

peak concentration (Cmax) and time for Cmax (Tmax) were recorded directly from experimental observations. The area under the curve from time zero to the last sampling time (AUC0−T) and the area under the curve from time zero to infinity (AUCINF) were calculated using a combination of linear and log trapezoidal summations. The whole body plasma clearance (CL), steady-state volume of distribution (Vss), apparent terminal t1/2, and mean residence time (MRT) were estimated following intravenous administration. The absolute oral bioavailability (F) was estimated as the ratio of dose-normalized AUC values following PO and IV doses. Computational Methodology. Quantum Chemical Calculations. Geometry optimizations for the different conformations of the model systems included in Figure 7 were performed using the Gaussian 09 program at the MP2/6-31+G** level of theory with default convergence criteria.32,54 Optimized structures were confirmed as minima via vibrational frequency calculations. The electrostatic potential surfaces displayed in Figure 11 were calculated at the MP2/631+G** level, and geometry optimization of the models included PCM solvation (H2O).46 4-Fluorobenzofuran Analogue Model. The A-chain of the X-ray structure of the HCV NS5B 1bC316N protein in complex with the des-fluoro analogue 46 (Figure S1) of the compound shown in Figure 8 and an additional compound occupying the P495 site (PDB ID: 3Q0Z) was imported into Maestro and processed using the Protein Preparation Wizard. The model was then subjected to three stages of energy minimization with MacroModel, all of which utilized the OPLS3 force-field and an implicit water solvation model.55,56 In the first stage, all nonhydrogen atoms were constrained, and 500 steps of PRCG energy minimization were performed. In the second stage, protein backbone atoms were constrained, and the model was subjected to 100 steps of steepest descent energy minimization. Finally, all constraints were removed, and 100 steps of steepest descent energy minimization were performed. Default convergence criteria were included at each stage. The resulting model was modified by replacing the benzofuran 4-position hydrogen atom with a fluorine atom followed by 100 steps of steepest descent energy minimization during which all atoms except the ligand were constrained.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00328. Production of the recombinant protein, crystallization conditions, crystallographic methods, and crystallographic statistics for complexes of HCV gt-1b NS5B protein (PDF) Molecular formula strings (CSV) Accession Codes

The PDB code for 5 is 5PZK, 6 is 5PZL, 8 is 5PZM, 18 is 5PZN, 37 is 5PZP, and 46 is 5PZO. CCDC 1541557 contains the supplementary crystallographic data for the unsolvated neat form of 37. The data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac. uk/structures.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Phone: +1 (203) 677-6897. ORCID

Kap-Sun Yeung: 0000-0003-3995-6555 Notes

The authors declare no competing financial interest. 4382



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RNA-dependent RNA polymerase of hepatitis C virus. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 13034−13039. (13) Sofia, M. J.; Chang, W.; Furman, P. A.; Mosley, R. T.; Ross, B. S. Nucleoside, nucleotide, and non-nucleoside inhibitors of hepatitis C virus NS5B RNA-dependent RNA-polymerase. J. Med. Chem. 2012, 55, 2481−2531. (14) Watkins, W. J.; Ray, A. S.; Chong, L. S. HCV Polymerase inhibitors. Curr. Opin. Drug Discovery Dev. 2010, 13, 441−465. (15) 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. Investig. Drugs 2007, 8, 614−634. (16) 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. (17) Maynard, A.; Crosby, R. M.; Ellis, B.; Hamatake, R.; Hong, Z.; Johns, B. A.; Kahler, K. M.; Koble, C.; Leivers, A.; Leivers, M. R.; Mathis, A.; Peat, A. J.; Pouliot, J. J.; Roberts, C. D.; Samano, V.; Schmidt, R. M.; Smith, G. K.; Spaltenstein, A.; Stewart, E. L.; Thommes, P.; Turner, E. M.; Voitenleitner, C.; Walker, J. T.; Waitt, G.; Weatherhead, J.; Weaver, K.; Williams, S.; Wright, L.; Xiong, Z. Z.; Haigh, D.; Shotwell, J. B. Discovery of a potent boronic acid derived inhibitor of the HCV RNA-dependent RNA polymerase. J. Med. Chem. 2014, 57, 1902−1913. (18) (a) McComas, C. C.; Liverton, N. J.; Habermann, J.; Koch, U.; Narjes, F.; Li, P.; Peng, X.; Soll, R.; Wu, H.; Palani, A.; He, S.; Dai, X.; Liu, H.; Lai, Z.; London, C.; Xiao, D.; Zorn, N.; Nargund, R. Preparation of pyridooxazinoindole derivatives and analogs for use as antiviral agents. World Patent Application WO 2013/033971 A1, March 14, 2013. (b) McComas, C. C.; Liverton, N. J.; Habermann, J.; Koch, U.; Narjes, F.; Li, P.; Peng, X.; Soll, R.; Wu, H. Preparation of tetracyclic heterocycle compounds for the treatment of HCV infection. World Patent Application WO 2013/033900 A1, March 14, 2013. (19) A class of analogues of benzofuran 1 with extension from the 2(4-fluorophenyl) substituent can be found in Labadie, S. S.; Lin, C. J. J.; Talamas, F. X.; Weikert, R. J. Heterocyclic antiviral compounds. World Patent Application WO 2009/101022 Al, August 20, 2009. (20) Parcella, K.; Nickel, A.; Beno, B. R.; Sheriff, S.; Wan, C.; Wang, Y.-K.; Roberts, S. B.; Meanwell, N. A.; Kadow, J. F. Discovery and initial optimization of alkoxyanthranilic acid derivatives as inhibitors of HCV NS5B polymerase. Bioorg. Med. Chem. Lett. 2017, 27, 295−298. (21) Burns, C. J.; Del Vecchio, A. M.; Bailey, T. R.; Kulkarni, B. A.; Faitg, T. H.; Sherk, S. R.; BlackLedge, C. W.; Rys, D. J.; Lessen, T. A.; Swestock, J.; Deng, Y.; Nitz, T. J.; Reinhardt, J. A.; Feng, H.; Saha, A. K. Benzofuran compounds, compositions and methods for treatment and prophylaxis of hepatitis C viral infections and associated diseases. World Patent Application WO 2004/041201 A2, May 21, 2004. (22) Shaw, A. N.; Tedesco, R.; Bambal, R.; Chai, D.; Concha, N. O.; Darcy, M. G.; Dhanak, D.; Duffy, K. J.; Fitch, D. M.; Gates, A.; Johnston, V. K.; Keenan, R. M.; Lin-Goerke, J.; Liu, N.; Sarisky, R. T.; Wiggall, K. J.; Zimmerman, M. N. Substituted benzothiadizine inhibitors of hepatitis C virus polymerase. Bioorg. Med. Chem. Lett. 2009, 19, 4350−4353. (23) Howe, A. Y. M.; Cheng, H.; Johann, S.; Mullen, S.; Chunduru, S. K.; Young, D. C.; Bard, J.; Chopra, R.; Krishnamurthy, G.; Mansour, T.; O’Connell, J. Molecular mechanism of hepatitis C virus replicon variants with reduced susceptibility to a benzofuran inhibitor, HCV796. Antimicrob. Agents Chemother. 2008, 52, 3327−3338. (24) Congreve, M.; Chessari, G.; Tisi, D.; Woodhead, A. J. Recent developments in fragment-based drug discovery. J. Med. Chem. 2008, 51, 3661−3680. (25) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand efficiency: a useful metric for lead selection. Drug Discovery Today 2004, 9, 430− 431.

ACKNOWLEDGMENTS We would like to thank colleagues in Lead Profiling for collecting in vitro profiling data, Discovery Analytical Science for performing the key compound characterizations on 37, the Discovery Chemistry Synthesis group for scale-up synthesis, and Process R&D for support. Use of the IMCA-CAT beamline 17-ID and 17-BM at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. Use of the LSCAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology TriCorridor (Grant 085P1000817). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357.

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ABBREVIATIONS ADME, absorption, distribution, metabolism, excretion; ASD, amorphous solid dispersion; BCS, biopharmaceutics classification system; gt, genotype; HAC, heavy atom count; IND, investigational new drug; LE, ligand efficiency; RMSD, rootmean-square deviation; NOAEL, no observed adverse effect level; PAMPA, parallel artificial membrane permeability assay; WT, wild-type

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REFERENCES

(1) WHO Hepatitis C Fact Sheet No. 164, http://www.who.int/ mediacentre/factsheets/fs164/en/ (updated July 2016; accessed February 5, 2017). (2) Centers for Diseases Control and Prevention: Viral Hepatitis Hepatitis C Information, http://www.cdc.gov/hepatitis/hcv/hcvfaq. htm (updated January 27, 2017; accessed February 5, 2017). (3) Choo, Q. L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley, D. W.; Houghton, M. Isolation of a cDNA clone derived from a blood borne non-A, non-B viral hepatitis genome. Science 1989, 244, 359− 362. (4) Smith, D. B.; Bukh, J.; Kuiken, C.; Muerhoff, A. S.; Rice, C. M.; Stapleton, J. T.; Simmonds, P. Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: updated criteria and genotype assignment web resource. Hepatology 2014, 59, 318−327. (5) Scheel, T. K. H.; Rice, C. M. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nat. Med. 2013, 19, 837−849. (6) Bartenschlager, R.; Lohmann, V.; Penin, F. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat. Rev. Microbiol. 2013, 11, 482−496. (7) 2015 Hepatitis C Online, University of Washington; http://www. hepatitisc.uw.edu/page/treatment/drugs (accessed October 7, 2015). (8) Götte, M.; Feld, J. J. Direct-acting antiviral agents for hepatitis C: structural and mechanistic insights. Nat. Rev. Gastroenterol. Hepatol. 2016, 13, 338−351. (9) Meanwell, N. A. 2015 Philip S. Portoghese Medicinal Chemistry Lectureship. Curing hepatitis C virus infection with direct-acting antiviral agents: The arc of a medicinal chemistry triumph. J. Med. Chem. 2016, 59, 7311−7351. (10) Lesburg, C. A.; Cable, M. B.; Ferrari, E.; Hong, Z.; Mannarino, A. F.; Weber, P. C. Crystal structure of the RNA-dependent RNA polymerase from hepatitis C virus reveals a fully encircled active site. Nat. Struct. Biol. 1999, 6, 937−943. (11) Ago, H.; Adachi, T.; Yoshida, A.; Yamamoto, M.; Habuka, N.; Yatsunami, K.; Miyano, M. Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus. Structure 1999, 7, 1417−1426. (12) Bressanelli, S.; Tomei, L.; Roussel, A.; Incitti, I.; Vitale, R. L.; Mathieu, M.; De Francesco, R.; Rey, F. A. Crystal structure of the 4383



Article

(26) Hopkins, A. L.; Keserü, G. M.; Leeson, P. D.; Rees, D. C.; Reynolds, C. H. The role of ligand efficiency metrics in drug discovery. Nat. Rev. Drug Discovery 2014, 13, 105−121. (27) Bissantz, C.; Kuhn, B.; Stahl, M. A medicinal chemist’s guide to molecular interactions. J. Med. Chem. 2010, 53, 5061−5084. (28) Meanwell, N. A. A synopsis of the properties and applications of heteroaromatic rings in medicinal chemistry. Adv. Heterocycl. Chem. 2017, 123, 245−361. (29) Laurence, C.; Brameld, K. A.; Graton, J.; Le Questel, J.-Y.; Renault, E. The pKBHX Database: Toward a better understanding of hydrogen-bond basicity for medicinal chemists. J. Med. Chem. 2009, 52, 4073−4086. (30) cLogP and pKa were calculated by using the molecular property calculator JChem-15.9.7.0 from ChemAxon. (31) Hang, J. Q.; Yang, Y.; Harris, S. F.; Leveque, V.; Whittington, H. J.; Rajyaguru, S.; Ao-Ieong, 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, 15517−15529. (32) Hehre, W. J.; Radom, L.; Schleyer, P. v. R.; Pople, J. A. Ab Initio Molecular Orbital Theory; John Wiley & Sons: New York, NY, 1986, and references cited therein. (33) Qiao, J. X.; Cheney, D. L.; Alexander, R. S.; Smallwood, A. M.; King, S. R.; He, K.; Rendina, A. R.; Luettgen, J. M.; Knabb, R. M.; Wexler, R. R.; Lam, P. Y. S. Achieving structural diversity using the perpendicular conformation of alpha-substituted phenylcyclopropanes to mimic the bioactive conformation of ortho-substituted biphenyl P4 moieties: Discovery of novel, highly potent inhibitors of Factor Xa. Bioorg. Med. Chem. Lett. 2008, 18, 4118−4123 and references cited therein. (34) (a) Ritchie, T. J.; Macdonald, S. J. F. Heterocyclic replacements for benzene: Maximising ADME benefits by considering individual ring isomers. Eur. J. Med. Chem. 2016, 124, 1057−1068. (b) Gleeson, P.; Bravi, G.; Modi, S.; Lowe, D. ADMET rules of thumb II: A comparison of the effects of common substituents on a range of ADMET parameters. Bioorg. Med. Chem. 2009, 17, 5906−5919. (35) Yeung, K.-S.; Parcella, K. E.; Bender, J. A.; Beno, B. R.; GrantYoung, K. A.; Han, Y.; Hewawasam, P.; Kadow, J. F.; Nickel, A. Compounds for the treatment of hepatitis C. World Patent Application WO 2010/030592 A1, March 18, 2010. (36) Olsen, J. A.; Banner, D. W.; Seiler, P.; Sander, U. O.; D’Arcy, A.; Stihle, M.; Müller, K.; Diederich, F. A fluorine scan of thrombin inhibitors to map the fluorophilicity/fluorophobicity of an enzyme active site: evidence for C-F···CO interactions. Angew. Chem., Int. Ed. 2003, 42, 2507−2511. (37) Xing, L.; Keefer, C.; Brown, M. F. Fluorine multipolar interaction: Toward elucidating its energetics in binding recognition. J. Fluorine Chem. 2017, DOI: 10.1016/j.jfluchem.2016.12.013. (38) Belema, M.; Meanwell, N. A. Discovery of daclatasvir, a pangenotypic hepatitis C virus NS5A replication complex inhibitor with potent clinical effect. J. Med. Chem. 2014, 57, 5057−5071. (39) Scola, P. M.; Sun, L.-Q.; Wang, A. X.; Chen, J.; Sin, N.; Venables, B. L.; Sit, S.-Y.; Chen, Y.; Cocuzza, A.; Bilder, D. M.; D’Andrea, S. V.; Zheng, B.; Hewawasam, P.; Tu, Y.; Friborg, J.; Falk, P.; Hernandez, D.; Levine, S.; Chen, C.; Yu, F.; Sheaffer, A. K.; Zhai, G.; Barry, D.; Knipe, J. O.; Han, Y.-H.; Schartman, R.; Donoso, M.; Mosure, K.; Sinz, M. W.; Zvyaga, T.; Good, A. C.; Rajamani, R.; Kish, K.; Tredup, J.; Klei, H. E.; Gao, Q.; Mueller, L.; Colonno, R. J.; Grasela, D. M.; Adams, S. P.; Loy, J.; Levesque, P. C.; Sun, H.; Shi, H.; Sun, L.; Warner, W.; Li, D.; Zhu, J.; Meanwell, N. A.; McPhee, F. The Discovery of asunaprevir (BMS-650032), an orally efficacious NS3 protease inhibitor for the treatment of hepatitis C virus infection. J. Med. Chem. 2014, 57, 1730−1752. (40) Liu, M.; Tuttle, M.; Gao, M.; Lemm, J. A. Potency and resistance analysis of hepatitis C virus NS5B polymerase inhibitor BMS-791325 on all major genotypes. Antimicrob. Agents Chemother. 2014, 58, 7416−7423.

(41) (a) Reynolds, C. H.; Bembenek, S. D.; Tounge, B. A. The role of molecular size in ligand efficiency. Bioorg. Med. Chem. Lett. 2007, 17, 4258−4261. (b) Reynolds, C. H.; Tounge, B. A.; Bembenek, S. D. Ligand binding efficiency: trends, physical basis, and implications. J. Med. Chem. 2008, 51, 2432−2438. (42) (a) Perola, E. An analysis of the binding efficiencies of drugs and their leads in successful drug discovery programs. J. Med. Chem. 2010, 53, 2986−2997. (b) Hajduk, P. J. Fragment-based drug design: how big is too big? J. Med. Chem. 2006, 49, 6972−6976. (43) Waring, M. J.; Johnstone, C. A quantitive assessment of hERG liability as a function of lipophilicity. Bioorg. Med. Chem. Lett. 2007, 17, 1759−1764. (44) The analysis was based on ten matched molecular pairs. When the hERG inhibition data are determined in the same assay in the same laboratory, a minimum of four matched pairs can be sufficient to identify an activity difference, see: Kramer, C.; Fuchs, J. E.; Whitebread, S.; Gedeck, P.; Liedl, K. R. Matched molecular pair analysis: significance and the impact of experimental uncertainty. J. Med. Chem. 2014, 57, 3786−3802. (45) Johnson, S. R.; Yue, H.; Conder, M. L.; Shi, H.; Doweyko, A. M.; Lloyd, J.; Levesque, P. Estimation of hERG inhibition of drug candidates using multivariate property and pharmacophore SAR. Bioorg. Med. Chem. 2007, 15, 6182−6192. (46) Tomasi, J.; Mennucci; Cammi, R. Quantum mechanical continuum solvation models. Chem. Rev. 2005, 105, 2999−3093 and references cited therein. (47) Gillis, E. P.; Eastman, K. J.; Hill, M. D.; Donnelly, D. J.; Meanwell, N. A. Applications of fluorine in medicinal chemistry. J. Med. Chem. 2015, 58, 8315−8359. (48) The details of pharmacokinetic studies will be the subject of a separate publication. (49) Rumondor, A. C. F.; Dhareshwar, S. S.; Kesisoglou, F. Amorphous solid dispersions or prodrugs: complementary strategies to increase drug absorption. J. Pharm. Sci. 2016, 105, 2498−2508. (50) Meanwell, N. A. Improving drug candidates by design: a focus on physicochemical properties as a means of improving compound disposition and safety. Chem. Res. Toxicol. 2011, 24, 1420−1456. (51) (a) Manuscript in preparation. (b) Eastman, K. J.; Parcella, K.; Yeung, K.-S.; Grant-Young, K. A.; Zhu, J.; Wang, T.; Zhang, Z.; Yin, Z.; Beno, B. R.; Sheriff, S.; Kish, K.; Tredup, J.; Jardel, A. G.; Halan, V.; Ghosh, K.; Parker, D.; Mosure, K.; Fang, H.; Wang, Y.-K.; Lemm, J.; Zhuo, X.; Hanumegowda, U.; Rigat, K.; Donoso, M.; Tuttle, M.; Zvyaga, T.; Haarhoff, Z.; Meanwell, N. A.; Soars, M. G.; Roberts, S. B.; Kadow, J. F. The discovery of a pan-genotypic, primer grip inhibitor of HCV NS5B polymerase. MedChemComm 2017, 8, 796−806. (52) Corsi, M.; Faiferman, I.; Merlo-Pich, E.; Ratti, E.; Wren, P. B. 2Amino-7,8- dihydropyrido[2, 3- d] pyrimidin-7-one derivatives as CSBP/RK/p38 kinase inhibitors and their preparation, pharmaceutical compositions and use in the treatment of diseases. World Patent Application WO 2007/147103 A2, December 21, 2007. (53) Lemieux, R. M.; Barbosa, A. J. d. M.; Bentzien, J. M.; Brunette, S.; Richard; Chen, Z.; Cogan, D.; Gao, D. A.; Heim-Riether, A.; Horan, J. C.; Kowalski, J. A.; Lawlor, M. D.; Liu, W.; McKibben, B.; Miller, C. A.; Moss, N.; Tschantz, M. A.; Xiong, Z.; Yu, Hui; Yu, Y. Derivatives of 6,7-dihydro-5H-imidazo[1,2-α]imidazole-carboxylic acid amides as inhibitors of the interaction of CAMs and leukointegrins, and their preparation and use in the treatment of inflammatory diseases. World Patent Application WO 2009/070485 A1, June 4, 2009. (54) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; 4384



Article

Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, revision D.01; Gaussian, Inc.: Wallingford, CT, 2013. (55) (a) Schrödinger Release 2016-2: Maestro; Schrödinger, LLC: New York, NY, 2016. (b) MacroModel; Schrödinger, LLC: New York, NY, 2016. (c) Protein Preparation Wizard; Epik; Schrödinger, LLC: New York, NY, 2016. (d) Impact; Schrödinger, LLC: New York, NY, 2016. (e) Prime; Schrödinger, LLC: New York, NY, 2016. (56) Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, J. Y.; Wang, L.; Lupyan, D.; Dahlgren, M. K.; Knight, J. L.; Kaus, J. W.; Cerutti, D. S.; Krilov, G.; Jorgensen, W. L.; Abel, R.; Friesner, R. A. OPLS3: A force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 2016, 12, 281−296.

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