discovery of ledipasvir (gs-5885): a potent, once-daily oral ns5a inhibitor for the treatment of...

Discovery of Ledipasvir (GS-5885): A Potent, Once-Daily Oral NS5AInhibitor for the Treatment of Hepatitis C Virus InfectionJohn O. Link,*,† James G. Taylor,† Lianhong Xu,† Michael Mitchell,† Hongyan Guo,† Hongtao Liu,†

Darryl Kato,† Thorsten Kirschberg,† Jianyu Sun,† Neil Squires,† Jay Parrish,† Terry Keller,†

Zheng-Yu Yang,† Chris Yang,‡ Mike Matles,‡ Yujin Wang,‡ Kelly Wang,‡ Guofeng Cheng,§ Yang Tian,§

Erik Mogalian,± Elsa Mondou,∥ Melanie Cornpropst,∥ Jason Perry,⊥ and Manoj C. Desai†

†Medicinal Chemistry, ‡Drug Metabolism, §Biology, ±Formulation and Process Development, ∥Clinical Research, and ⊥StructuralChemistry, Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States

*S Supporting Information

ABSTRACT: A new class of highly potent NS5A inhibitors with anunsymmetric benzimidazole-difluorofluorene-imidazole core and distal[2.2.1]azabicyclic ring system was discovered. Optimization of antiviralpotency and pharmacokinetics led to the identification of 39 (ledipasvir,GS-5885). Compound 39 (GT1a replicon EC50 = 31 pM) has anextended plasma half-life of 37−45 h in healthy volunteers and produces arapid >3 log viral load reduction in monotherapy at oral doses of 3 mg orgreater with once-daily dosing in genotype 1a HCV-infected patients. 39has been shown to be safe and efficacious, with SVR12 rates up to 100%when used in combination with direct-acting antivirals having complementary mechanisms.

■ INTRODUCTION

Hepatitis C virus (HCV) infection is a significant public healthconcern, with approximately 170 million infected individualsworldwide, and is the leading cause of liver transplant andhepatocellular carcinoma.1 HCV is the most common chronicblood-borne pathogen in the U.S., and the Center for DiseaseControl and the U.S. Preventative Services Task Force arealigned in recommending that all baby-boomers (individualsborn between 1945 and 1965) undergo testing for HCVinfection.2 Until recently, the standard of care for treatment ofgenotype 1 (GT1) infection (60% of total infections worldwideamong the seven known genotypes, with both GT1a and GT1bas major subtypes)3 consisted of weekly pegylated interferon(PEG) injections and twice-daily oral ribavirin (RBV) for 24 or48 weeks (duration based on response-guided therapy). PEG/RBV treatments achieve up to 54−63% sustained virologicresponse (SVR) in GT1 patients,4 but treatment is accompaniedby considerable toxicity including flu-like symptoms, depression,and anemia.5 Triple therapy containing PEG/RBV combinedwith three-times-daily dosing of the recently approved direct-acting antiviral (DAA) protease inhibitor telaprevir or boceprevirhas improved the GT1HCV SVR rates to 66−79% for treatmentnaıv̈e patients but with increased toxicities including rash(telaprevir) or grade 3 or 4 anemia.6 Prior null responders(patients who attained less than a 1 log viral load reduction onPEG/RBV) are not indicated for retreatment with PEG/RBVbecause they achieve SVR rates <10%, with minimal improve-ment to 29% for patients undergoing triple therapy.7 Finally, agrowing number of patients are being identified as interferon-intolerant or unwilling to take interferon. PEG-free therapy is

necessary to serve a broader patient population, to improveoutcomes, and to provide a safer, simpler regimen.8

We have sought to identify safe oral drugs for combinationtreatment of HCV infection. In addition to programs targetingthe NS3 protease9 and NS5B polymerase (both nucleotides10

and non-nucleotides11), we initiated an NS5A inhibitor programwith the goal of identifying an agent with characteristics thatwould allow its use in combination with DAAs havingcomplementary mechanisms to achieve high SVR rates with ashort treatment duration.Despite significant study, the mechanistic role of NS5A in the

HCV life cycle remains enigmatic.12 NS5A has no knownenzymatic activity and has no homologues in prokaryotes oreukaryotes. Nonetheless, the protein is critical for HCV viability;in clinical monotherapy studies, NS5A inhibitors produce themost rapid viral load declines of any HCV antiviral class. It hasbeen postulated that this rapid decline in HCV RNA is based oninhibition of viral replication (as with NS3 and NS5B inhibitors)and additional inhibition of virion assembly or secretion frominfected cells.13

Early NS5A inhibitors were found empirically throughscreening of the GT1b replicon. Several series of lipophilicproline, proline-mimetic, or alanine-amide inhibitors of theGT1b replicon have been discovered (1−3; Figure 1),14 butthese inhibitors typically have ∼1000-fold weaker activity againstthe GT1a replicon.15 A polyaromatic pyridopyrimidine class (4)

Special Issue: HCV Therapies

Received: September 26, 2013

Article

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affords nanomolar activity against both the GT1a and 1breplicons.16 Dimeric series provide potent GT1b-active stilbenediamide inhibitors (5 discovered from monomer series 1)17 andhighly potent GT1a and 1b-active bis-imidazole biphenylinhibitors including daclatasvir 6 (BMS-790052, GT1a EC50 =50 pM), which achieved clinical proof of concept for the NS5Amechanism.18 NS5A has emerged as an important drug target forthe treatment of HCV infection.19

For our NS5A inhibitor target profile, we sought high potencyand a long plasma half-life to reduce the emergence of viralresistance.20 To achieve these aims, a diverse range of inhibitorstructures was investigated.21 Herein, we describe structuralmodifications and SAR in a symmetric bis-benzimidazole seriesand in an unsymmetric imidazole/benzimidazole series. Wefound that the unsymmetric series posed unique advantages forthe optimization of inhibitor potency and pharmacokinetics. Onthe basis of these studies, we describe the discovery of clinicalcompound 39, a potent and selective HCV NS5A inhibitor withexcellent preclinical and human pharmacokinetics and potentantiviral activity in HCV-infected patients.22

■ CHEMISTRY

Repeatedly used intermediates for the syntheses of these NS5Ainhibitors are shown in Figure 223 and Scheme 1. Coupling ofBoc-proline 13a with diamine 13b using HATU afforded amixture of amides that could be dehydrated at elevatedtemperatures to afford benzimidazole 13 (Scheme 1).24 Miyaura

borylation of 13 generated pinacol boronate 14. Removal of thephenylethyl chiral auxiliary from 15a25 by hydrogenolysisfollowed by Boc protection and hydrolysis of the methyl esterafforded [2.2.1]azabicyclic carboxylic acid 15.C2-symmetric bis-benzimidazoles 16, 17, and 18 were

assembled by palladium-catalyzed cross-couplings via bis(Boc-pyrrolidine) cores (Scheme 2). Suzuki coupling of benzimidazolebromide 13 and boronate ester 14 provided the core of 16.Bromide 13 could also be converted to acetylene 17a bySonogashira coupling. A second Sonogashira coupling between17a and 13 afforded the core of 17, whereas Glaser-type oxidativedimerization of 17a formed the core of 18. For 16−18, as inmany of the following schemes, double Boc-removal followed bydouble peptide-coupling with valine methyl carbamate 11afforded the final compounds.Dibromoarenes were coupled under Suzuki conditions with 2

equiv of 14 to furnish the bis(Boc-pyrrolidine) cores of 19, 20,and 21 (Scheme 3).26

Bromide 13 was coupled to 1,4-benzenediboronic acidbis(pinacol) ester to give boronate 22a (Scheme 4). A secondcoupling between 13 and 22a formed the core of inhibitor 22.Suzuki coupling of 22awith bromoimidazole 9 led to amixture ofhomodimer 23a and cross-coupled product 24a. 23a and 24awere converted to 23 and 24, respectively, by Boc-removal andpeptide-coupling with 11.

Figure 1. Structures of NS5A inhibitors.

Figure 2. Intermediates used in the synthesis of NS5A inhibitors.

Scheme 1a

a(a) HATU, i-Pr2NEt, DMF; (b) EtOH; (c) bis(pinacolato)diboron,Pd(dppf)Cl2, KOAc, dioxane; (d) Pd(OH)2/C, H2, EtOH; (e) Boc2O,i-Pr2NEt, CH2Cl2; (f) LiOH, THF/MeOH/H2O.

Journal of Medicinal Chemistry Article

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The synthesis of the naphthyl imidazole inhibitor 25 beganwith carboxylic acid 25a, which was converted to bromoketone25b by a three-step sequence (Scheme 5).27 Boc-proline wasalkylated by 25b to give a ketoester that cyclized to imidazole 25cupon heating in the presence of ammonium acetate. The Bocgroup was replaced with valinyl methyl carbamate 11, and thenaphthalene was borylated, providing 25d. 25d and 25e werethen coupled to afford 25.Bromoimidazole 10 was converted to the corresponding

formylimidazole by lithium-halogen exchange and trapping ofthe organometallic species with DMF (Scheme 6). The Ohira−Bestmann reagent28 was employed in the homologation of theresultant aldehyde to acetylene 26a. Boc-removal and peptide-coupling with 11 gave 26b. Suzuki coupling of boronate ester 14and 1,4-dibromobezene afforded a Boc-protected aryl bromide,which was converted to amide 26c. Sonogashira couplingbetween 26c and acetylene 26b provided 26.Sonogashira coupling between 17a and 1,4-diiodobenzene was

followed byMiyaura borylation of the intermediate aryl iodide togive the intermediate boronic ester, which was cleaved to the

boronic acid upon purification by reversed-phase HPLC(Scheme 7). Suzuki coupling of 27a and bromoimidazole 10yielded bis(Boc-pyrrolidine) 27b, which was converted to 27after treatment with TFA and double peptide-coupling with 11.Boronate ester 7 was Suzuki-coupled to 2,5-dibromothio-

phene, and the product was borylated to give 28a (Scheme 8). Asecond Suzuki coupling between 28a and bromobenzimidazole13 provided 28b, which was converted to 28 by the standard two-step procedure.Suzuki coupling of 13 and 4,4′-biphenyldiboronic acid

bis(pinacol) ester yielded biphenyl boronate 29a, which wasthen coupled to bromoimidazole 10 to furnish the core of 29(Scheme 9).

Scheme 2a

a(a) 14, Pd(PPh3)4, K2CO3, H2O/DME; (b) TFA; (c) 11, HATU, i-Pr2NEt, DMF; (d) trimethylsilylacetylene, Pd(PPh3)4, CuI, Et3N,DMF; (e) K2CO3, MeOH; (f) 13, Pd(PPh3)4, CuI, Et3N, DMF; (g)Pd(PPh3)4, CuI, Et3N, DMF.

Scheme 3a

a(a) 2,5-Dibromothiophene, PdCl2(dppf)2, K2CO3, H2O/DME; (b)HCl/dioxane/DCM; (c) 11, HATU, i-Pr2NEt, DMF; (d) 2,6-dibromonaphthalene, Pd(PPh3)4, K2CO3, H2O/DME; (e) 2,6-diiodomobenzo[1,2-b:4,5-b′]dithiophene, Pd(PPh3)4, K2CO3, H2O/PhMe.

Scheme 4a

a(a) 1,4-Benzenediboronic acid bis(pinacol) ester, Pd(PPh3)4, K2CO3,H2O/DME; (b) 13, Pd(PPh3)4, K2CO3, H2O/DME; (c) HCl/dioxane/DCM; (d) 11, HATU, i-Pr2NEt, DMF; (e) 9, Pd(PPh3)4,K2CO3, H2O/DME.

Scheme 5a

a(a) SOCl2; (b) (trimethylsilyl)diazomethane, DCM; (c) HBr/AcOH/EtOAc; (d) Boc-Pro-OH, Et3N, MeCN; (e) NH4OAc, xylenes;(f) TFA/DCM; (g) 11, HATU, i-Pr2NEt, DMF; (h) bis(pinacolato)-diboron, Pd(PPh3)4, KOAc, 1,4-dioxane; (i) Pd(PPh3)4, NaHCO3,H2O/DME.


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Bis-imidazoles with fused tricyclic linkers were prepared fromdibromides 30a, 31a, and 32a (Scheme 10). These dibromideswere converted to corresponding diboronate esters 30b, 31b,and 32b by Miyaura borylation. The imidazoles were attached tothe tricyclic ring systems by Suzuki cross-coupling of thediboronate esters with bromoimidazole 9, en route to inhibitors30, 31, and 32.Difluorofluorene 33a could be formed by treatment of

fluorenone 31a with bis(2-methoxyethyl)aminosulfur trifluoride(Scheme 11). Stille coupling between 33a and tributyl(1-ethoxyvinyl)stannane provided an enol ether that was convertedto bromoketone 33b upon treatment with NBS.29 Alkylation of

Boc-proline by 33b gave an intermediate ketoester that wascyclized to imidazole 33c as described above for the synthesis of25c. Suzuki coupling of 33c with 14 produced 33d, which led to33 after Boc-removal and peptide-coupling with 11.Synthesis of 33 presented several challenges. Fluorination of

31a required forcing conditions employing excess neat bis(2-methoxyethyl)aminosulfur trifluoride at elevated temperatures(bis(2-methoxyethyl)aminosulfur trifluoride is known to bethermally unstable,30 so exercise caution when using thisreagent). The inclusion of an organotin reagent also countedamong the unattractive aspects of this synthesis. Finally, a lack ofselectivity between mono- and dicoupled enol-ether products inthe Stille reaction contributed to the low efficiency of this route.See Scheme 16 for a synthesis of the elaborated difluorofluorenering system that avoids these problems.A HATU-promoted coupling between amine 34a and

piperidine-carboxylic acid 34b afforded a ketoamide that couldbe dehydrated in the presence of ammonium acetate to yieldimidazole 34c (Scheme 12). Boc-removal was followed by valinemethyl carbamate 11 attachment and Miyaura borylation to givecoupling partner 34d. Suzuki coupling between 34d and 8bprovided inhibitor 34. 35 was prepared by a similar sequence tothat for 34, with the exception that [2.2.1]azabicyclic carboxylicacid 15 was employed in place of 34b.[2.2.1]Azabicyclic carboxylic acid 15 was converted to

benzimidazole 36a by a two-step sequence (Scheme 13). Suzukicoupling between 36a and 1,4-benzenediboronic acid bis-(pinacol) ester provided 36b, which produced the core of 36by a second cross-coupling with 8a.

Scheme 6a

a(a) n-BuLi, DMF, THF; (b) dimethyl-1-diazo-2-oxopropylphospho-nate, K2CO3, MeOH/THF; (c) HCl/dioxane; (d) 11, HATU, i-Pr2NEt, DMF; (e) TFA/DCM; (f) 1,4-dibromobenzene, Pd(PPh3)4,K2CO3, H2O/DME; (g) HCl/H2O/MeOH; (h) Pd(PPh3)4, CuI,Et3N, DMF.

Scheme 7a

a(a) 1,4-Diiodobenzene, Pd(PPh3)4, CuI, Et3N, DMF; (b) bis-(pinacolato)diboron, PdCl2(dppf)2, KOAc, 1,4-dioxane; (c) 10,Pd(PPh3)4, PdCl2(dppf)2, K2CO3, DME/H2O; (d) TFA; (e) 11,HATU, i-Pr2NEt, DMF.

Scheme 8a

a(a) 2,5-Dibromothiophene, Pd(PPh3)4, K2CO3, DME/H2O; (b)bis(pinacolato)diboron, Pd(PPh3)4, KOAc, 1,4-dioxane; (c) 13,Pd(PPh3)4, K2CO3, DME/H2O; (d) HCl/dioxane/DCM; (e) 11,HATU, i-Pr2NEt, DMF.

Scheme 9a

a(a) 4,4′-Biphenyldiboronic acid bis(pinacol) ester, Pd(PPh3)4,K2CO3, H2O/DME; (b) 10, Pd(PPh3)4, PdCl2(dppf)2, K2CO3,H2O/DME; (c) TFA; (d) 11, HATU, i-Pr2NEt, DMF.

Scheme 10a

a(a) Bis(pinacolato)diboron, Pd(PPh3)4, KOAc, 1,4-dioxane; (b) 9,Pd(PPh3)4, NaHCO3, DME/H2O; (c) HCl/dioxane; (d) 11, HATU,i-Pr2NEt, DMF.


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35a was coupled to 22a to provide the Boc-protectedprecursor to 37 (Scheme 14).

Boronate 38a resulted from borylation of bromide 36a(Scheme 15). A Suzuki coupling between 38a and 33c formedthe core of inhibitor 38.

In a novel process, fluorene 39a31 could be difluorinated bytreatment with KHMDS in the presence of N-fluorobenzene-sulfonimide32 (Scheme 16) to give 39b. The iodine-bearingcarbon of 39b was selectively metalated by reaction with i-PrMgCl and converted to single chloroketone 39c by quenchingthe Grignard species with the Weinreb amide 2-chloro-N-methoxy-N-methylacetamide.33 Carboxylic acid 12 was alkylated

Scheme 11a

a(a) Bis(2-methoxyethyl)aminosulfur trifluoride, catalytic EtOH (Caution: Bis(2-methoxyethyl)aminosulf ur trif luoride has the potential to decomposeexothermically under these conditions. Exercise caution. For a safer and more ef f icient synthesis of the dif luorof luorene motif, see Scheme 16); (b) tributyl(1-ethoxyvinyl)stannane, Pd(PPh3)4, PdCl2(PPh3)2, dioxane; (c) NBS, H2O; (d) Boc-Pro-OH, i-Pr2NEt, DMF; (e) NH4OAc, xylenes; (f) 14,Pd(PPh3)4, PdCl2(dppf)2, K2CO3, DME/H2O; (g) TFA; (h) 11, HATU, i-Pr2NEt, DMF.

Scheme 12a

a(a) HATU, i-Pr2NEt, DMF; (b) NH4OAc, xylenes; (c) HCl, H2O/MeOH; (d) 11, HATU, i-Pr2NEt, DMF; (e) bis(pinacolato)diboron,Pd(PPh3)4, KOAc, 1,4-dioxane; (f) 8b, Pd(PPh3)4, K2CO3, H2O/DME.

Scheme 13a

a(a) 4-Bromo-1,2-diaminobenzene, HATU, 4-methylmorpholine,DMF; (b) EtOH; (c) 1,4-benzenediboronic acid bis(pinacol) ester,Pd(PPh3)4, K2CO3, H2O/DME; (d) 8a, Pd(PPh3)4, K2CO3, H2O/DME; (e) HCl/dioxane/DCM; (f) 11, HATU, i-Pr2NEt, DMF.

Scheme 14a

a(a) 22a, Pd(PPh3)4, K2CO3, H2O/DME; (b) HCl/dioxane/DCM;(c) 11, HATU, i-Pr2NEt, DMF.

Scheme 15a

a(a) Bis(pinacolato)diboron, PdCl2(dppf)2, KOAc, 1,4-dioxane; (b)33c, Pd(PPh3)4, K2CO3, H2O/DME; (c) HCl/dioxane; (d) 11,HATU, i-Pr2NEt, DMF.


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with 39c, and the resulting ketoester was condensed to imidazole39d by heating with ammonium acetate. 39dwas coupled to 38a,providing 39e. Boc-removal was followed by peptide couplingwith 11 to afford 39.This is the first reported base-promoted difluorination of the 9

position of a fluorene ring system utilizing an electrophilic sourceof fluorine.34 Crucial to the success of the process are thepremixing of the substrate and the N-fluorobenzenesulfonimideas well as the slow addition of base to this mixture. It isnoteworthy that the base and the fluorinating reagent are inerttoward each other on the time scale of the reaction and that thedifluorinated product is formed in high yield without isolation ofthe monofluorofluorene.

■ RESULTS AND DISCUSSIONThroughout this work, our optimization of potency focused onGT1a activity; accordingly, the discussion here is restricted toreplicon GT1a EC50 values, although the GT1b values areprovided for comparison.35 We pursued a number of symmetricbis-benzimidazole cores (Table 1).21 Directly linked bis-benzimidazole 16 and monoalkyne inhibitor 17 showed noinhibitory activity under the assay conditions (up to 44 nM),whereas diyne 18 had an EC50 of 11 nM. Replacing the alkyneswith a thiophene ring (19) improved potency 6-fold, and thebenzimidazole-phenyl-benzimidazole core in 22 provided furtherimprovements in potency (EC50 = 500 pM). Replacement ofphenyl with biphenyl decreased activity 7-fold. Finally, insertionof fused ring systems provided highly potent naphthyl inhibitor20 (EC50 = 109 pM), with benzdithiophene 21 only 2-fold lessactive. From these studies, inhibitors with fused central ringsystems, as in 20 and 21, were the most potent, whereas lesslipophilic connectors, such as in alkynes 17 or 18, affordedweaker activity.We next sought to determine if an unsymmetric core,

including one benzimidazole and one imidazole, could providepotent inhibitors (Table 2). The directly linked imidazole/benzimidazole was not synthesized because directly linked bis-benzimidazole 16 was not active. We found it striking thatreplacement of the phenyl in 24 by naphthyl (25) resulted in a>600-fold potency enhancement, whereas the differencebetween phenyl and naphthyl linkers in the bis-benzimidazoleseries is <5-fold (Table 1). Furthermore, in the unsymmetricseries, the fused central naphthyl ring affords higher potency thanit does in the symmetric series (e.g., compound 20). Theimportance of the position of the lipophilicity in the linker isdemonstrated by phenyl-alkyne inhibitors 26 and 27, where the

more centrally positioned alkyne in 27 is improved 6-fold inpotency. As in Table 1, introduction of alkynes in the linkeraffords lower potency relative to inhibitors with aromaticelements.Notably, the biphenyl in inhibitor 29 provides the highest level

of potency among the nonfused central connectors in Tables 1 or2. The high potency of biphenyl 29, along with the themethroughout Tables 1 and 2 that fused central ring systemsconsistently provide high potency (compounds 20, 21, and 25),prompted us to study constraint of the biphenyl to form tricyclicfused-ring systems.Initial SAR of central fused tricycles was studied in a symmetric

bis-imidazole series to obviate tricycle desymmetrization andthereby simplify compound synthesis. Fluorene ring-linkedinhibitor 30 (Table 3) suffered a mild loss in potency relativeto biaryl 6 and posed stability concerns because the fluorene ringsystem readily underwent autoxidation upon standing. The

Scheme 16a

a(a) N-Fluorobenzenesulfonimide, KHMDS, THF; (b) i-PrMgCl, 2-chloro-N-methoxy-N-methylacetamide, THF; (c) 12, K2CO3, KI, acetone; (d)NH4OAc, PhMe; (e) 38a, Pd(OAc)2, PPh3, NaHCO3, DME/H2O; (f) HCl/dioxane/DCM; (g) 11, HATU i-Pr2NEt, DMF.

Table 1. In Vitro Activitya

aSee the Experimental Section for detailed assay protocols. bForstandard deviations, see the Experimental Section. cA value of >44 nMmeans that no inhibition was observed at this top well concentration.


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oxidation product, fluorenone 31, lost significant potency (EC50= 300 pM). Blocking oxidation with gem-dimethyl (32) lost evenmore potency, giving some credence to the concept thatsignificant out-of-plane steric bulk as part of the fused linker is

not well-tolerated. We postulated that a smaller, lipophilicblocking group such as a difluoromethylene group might providean optimal connector. We decided to introduce this importantmodification directly in the imidazole/benzimidazole series ofinterest because of the synthetic challenge posed by introductionof the difluoromethylene group. Use of the difluoromethyleneconnector produced the most potent inhibitor in the optimizedunsymmetric series, difluorofluorene 33 (EC50 = 40 pM).We measured the pharmacokinetics of 29 and 33 in rats and

dogs (Table 4) and found that both inhibitors showed similargood half-lives in plasma, low systemic clearance (CL), andmoderate volumes of distribution (Vss) that are greater than totalbody water volume. Unexpectedly, the modest 11% oralbioavailability of biphenyl inhibitor 29 was improved to over35% in difluoroflourene 33. Incorporation of difluorofluorene incompound 33 therefore provided combined improvements inpotency and bioavailability.Next, we assessed terminal heterocycle modifications in the

symmetric bis-imidazole system. Piperidine 34 (Table 5) was lesspotent than the [2.2.1]azabicyclic inhibitor 35. Importantly, wefound that 35 possessed a long half-life of 5.3 h in dog (Table 4).

Attracted by the favorable pharmacokinetic properties that the[2.2.1]azabicyclic ring system imparted in 35, we sought tounderstand the SAR of the azabicyclic ring system in the contextof an unsymmetric core. The more synthetically accessibleimidazole-biphenyl-benzimidazole core was chosen for thisstudy. We discovered matched and mismatched sets where thepotency is markedly better when the [2.2.1]azabicyclic ringsystem is paired with the benzimidazole in 36 rather than withthe imidazole in 37 (Table 6). We considered this differential


aSee the Experimental Section for detailed assay protocols. bForstandard deviations, see the Experimental Section. cA value of >44 nMmeans that no inhibition was observed at this top well concentration.


compd A EC50 (1a, nM)b EC50 (1b, nM)

30 CH2 0.094 0.01331 CO 0.30 0.01832 CMe2 1.2 0.01433 CF2 0.040 0.003

aSee the Experimental Section for detailed assay protocols. bForstandard deviations, see the Experimental Section.

Table 4. Rat and Dog Pharmacokineticsa,b

compd species CL (L/h/kg) Vss (L/kg) t1/2 (hr) MRT (hr)d %F

29 rat 1.04 ± 0.17 1.76 ± 0.17 1.57 ± 0.19 1.71 ± 0.16 11.5 ± 8.7dog 0.78 ± 0.29 2.31 ± 0.37 2.30 ± 0.28 3.00 ± 0.27 n.d.c

33 rat 0.42 ± 0.04 0.93 ± 0.04 1.83 ± 0.22 2.21 ± 0.21 36.7 ± 3.2dog 0.53 ± 0.04 1.96 ± 0.03 2.63 ± 0.18 3.69 ± 0.29 n.d.c

35 rat 0.70 ± 0.02 0.98 ± 0.08 1.49 ± 0.02 1.40 ± 0.07 35.2 ± 10dog 0.05 ± 0.007 0.31 ± 0.007 5.29 ± 0.60 6.60 ± 1.10 n.d.c

36 rat 0.75 ± 0.04 1.81 ± 0.26 2.07 ± 0.19 2.42 ± 0.22 26.3 ± 9.0dog 0.40 ± 0.26 2.03 ± 0.92 4.01 ± 0.82 5.47 ± 1.01 n.d.c

aAll parameters except for %F are from intravenous dosing. bSee the Experimental Section for detailed assay protocols. cn.d., not determined. dMeanresidence time.


compd EC50 (1a, nM)b EC50 (1b, nM)

34 0.45 0.00535 0.21 0.009



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SAR to be an important result; it further demonstrated to us thatthe unsymmetric core series opened opportunities for inhibitoroptimization that would not be available if structures werelimited to a symmetric cores. Unlike in the symmetric coresystem, the [2.2.1]azabicyclic ring system in the matchedunsymmetric case did not demonstrate a loss in potency relativeto the pyrrolidine (36 vs 29). Importantly, the extendedpharmacokinetic half-life that the [2.2.1]azabicyclic ring systemprovided in symmetric core inhibitor 35 translated inunsymmetric core inhibitor 36; both the rat and dog plasmahalf-lives are improved in 36 over those of pyrrolidine analogue29 having the same core (Table 4). To improve upon thepotency of 36, biphenyl was replaced by difluorofluorene,affording inhibitor 38 (EC50 = 56 pM). 38 has a protein-adjustedEC50 of 784 pM, corresponding to a protein-binding shift of 14-fold.36

During final optimization, replacement of the pyrrolidine in 38with a spirocyclopropylpyrrolidine afforded 39 (ledipasvir, GS-5885), the most potent inhibitor in the series (Figure 3).21 39 hasGT1a and 1b EC50 values of 31 and 4 pM, respectively, andprotein-adjusted EC50 values of 210 pM (GT1a) and 27 pM(GT1b). The protein-binding shift is 6.7-fold and is improvedrelative to pyrrolidine 38. Compound 39 is highly protein-boundboth in human serum and in the cell-culture medium (containing10% BSA) of the replicon assay (1.1% unbound fraction in cell-culture medium). Accounting for the low unbound drugconcentration in the replicon assay, the intrinsic EC50 of 39 is310 fM for GT1a and 40 fM for GT1b. 39 is remarkable not onlyon the basis of its high replicon potency but also on the basis of itslow clearance, good bioavailability, and long half-lives (4.7−10.3h) in rat, dog, and monkey and low predicted clearance in human(0.012 L/h/kg) (Figure 3 and Table 7). In 14 day toxicologystudies in rats and dogs with compound 39, there were nosignificant adverse findings. We selected 39 for clinicaldevelopment, projecting that it would be highly efficacious inthe treatment of HCV infection with a long half-life suitable foronce-daily dosing.The pharmacokinetics of 39 were studied in fasted healthy

volunteers in a placebo-matched double-blind phase 1 clinicaltrial at oral doses of 3, 10, 30, 60, and 100 mg (Figure 4 and Table8) and were dose-proportional over the range tested. Long meanplasma half-lives of 37−45 h were observed. Twenty-four hourpostdose drug concentrations were 12−470-fold over the GT1protein-adjusted EC50 and remained over the GT1a protein-adjusted EC50 well past 24 h. These results were consistent withthe potential for both once-daily dosing and low risk for

concentrations of 39 to fall below the protein-adjusted EC50 atthe end of the dosing interval.

■ CONCLUSIONSOptimization of antiviral potency and pharmacokinetic param-eters produced clinical candidate 39, an NS5A inhibitor (EC50 =31 pM GT1a replicon) with a prolonged half-life of up to 45 h inhealthy volunteers and 24 h trough concentrations 12−470 foldover the plasma-protein-adjusted EC50 for doses from 3 to 100mg.39 In further studies, this profile has translated to potentclinical efficacy: in a 3 day monotherapy study in GT1a HCV-infected patients, 39 produced a >3 log median viral loadreduction at doses of 3 mg or greater.22 Even at a 1 mg total dailydose, a 2.4 log median viral load reduction was achieved. Sixphase 2 trials demonstrated 39 to be safe and well-tolerated inover 1000 patients.40 Phase 2 efficacy of 39 in combination withsofosbuvir (GS-7977, an NS5B nucleotide inhibitor)41 and RBVin a 12 week treatment regimen achieved 100% SVR12 in priornull responders.42 39 is now in multiple phase 3 studiescoformulated with sofosbuvir in a single-tablet43 administeredonce-daily with or without RBV.44 NS5A inhibitor 39 has thepotential to be of utility in combination with DAAs ofcomplementary mechanism in all-oral therapy for the treatmentof HCV infection.


compd Y′ A Y EC50 (1a, nM)b EC50 (1b, nM)

36 absent absent CH2 0.16 0.00637 CH2 absent absent 0.66 0.02138 absent CF2 CH2 0.056 0.004


Figure 3. Structure of 39 (A), rat, dog, and cyno pharmacokinetic curves(B), potency and microsomal stability. EC50 values: GT1a = 31 pM,GT1b = 4 pM, intrinsic GT1a = 0.31 pM, intrinsic GT1b = 0.04 pM,protein-adjusted GT1a = 210 pM, protein-adjusted GT1b = 27 pM.Metabolic stability rat, dog, cyno and human microsomes: <9.2, <9.5,<12.7, and <12.7% hepatic extraction, respectively.37 Predicted hepaticclearance on the basis of human hepatocyte stability is 0.012 L/h/kg.38

For panel B: green, SD rat; red, cyno monkey; blue, beagle dog. The topgraph shows intravenous pharmacokinetics, and the bottom graphshows oral pharmacokinetics.


dx.doi.org/10.1021/jm401499g | J. Med. Chem. XXXX, XXX, XXX−XXXH

■ EXPERIMENTAL SECTIONNuclear magnetic resonance (NMR) spectra were recorded on VarianMercury Plus 400MHz and a Varian Unity Plus 500MHz spectrometersat room temperature, with tetramethylsilane as an internal standard.Chemical shifts (δ) are reported in parts per million (ppm), and signalsare reported as s (singlet), d (doublet), t (triplet), q (quartet), m(multiplet), or br s (broad singlet). Purities of the final compounds weredetermined by HPLC and were greater than 95%. HPLC conditions toassess purity were as follows: Agilent HPLC, Phenominex Luna C18, 4.6mm × 250 mm (5 μm); gradient of 0.1% TFA water (A) and 0.1% TFAacetonitrile (B); flow rate, 1.0 mL/min; acquire time, 35 min;wavelength, UV 214 and 254 nm; oven. The preparative HPLC systemincludes two sets of Gilson 332 pumps, a Gilson 156 UV/vis detector,and a Gilson 215 injector and fraction collector, with Trilution LCsoftware. A Phenomenex 250 mm × 21 mm 54u Gemini-NX columnwas used. The mobile phase was a gradient of 0.1% TFA water (A) and0.1% TFA acetonitrile (B). LC/MS was conducted on a ThermoFinnigan MSQ Std using electrospray in both positive and negativemodes, [M + H]+ and [M−H]+, and a Dionex Summit HPLC System(model P680A HPG) equipped with a Gemini 5 μm C18 110A column(30 mm × 4.60 mm), eluting with 0.05% formic acid in 1% acetonitrile/water (solvent A) and 0.05% formic acid in 99% acetonitrile/water

(solvent B). High-resolution mass spectra were recorded on a Xevo G2Q-Tof mass spectrometer with an ESI source.

Compound 39. 2-Bromo-9,9-difluoro-7-iodo-9H-fluorene (39b).2-Bromo-7-iodo-9H-fluorene (39a, 705 mg, 1.90 mmol) and NFSI(1.80 g, 5.70 mmol) were dissolved in THF (9.5 mL) and cooled to−20°C. KHMDS (1.0 M in THF, 5.7 mL, 5.7 mmol) was added dropwiseover 9 min. On completion of the addition, the reaction mixture waswarmed to 0 °C. After an additional 80 min at 0 °C, TLC indicatedcompletion of the reaction, and excess base was quenched by addition ofMeOH (30 drops) followed by hexane (20 mL). The suspension wasfiltered over Celite and concentrated. The resulting residue wasdissolved in 20% DCM/hexane (20 mL) (some solid does not dissolve)and passed over a short silica plug. The plug was rinsed with hexane(∼170 mL) until TLC indicated all desired product was eluted. Theliquid was concentrated to provide 2-bromo-9,9-difluoro-7-iodo-9H-fluorene 39b (626 mg, 81% yield) as a pale-yellow-orange solid. Thematerial is essentially pure by NMR and LCMS. 1H NMR (CDCl3) δ7.94 (1H, d, J = 1.2 Hz), 7.81 (1H, d, J = 7.8 Hz), 7.74 (1H, d, J = 1.4Hz), 7.60 (1H, d, J = 8.3 Hz), 7.41 (1H, d, J = 8.1 Hz), 7.29 (1H, d, J =8.0 Hz). 19F NMR (CDCl3) δ −111.034 (2F, s).

1-(7-Bromo-9,9-difluoro-9H-fluoren-2-yl)-2-chloroethanone(39c). An oven-dried three-necked round-bottomed flask equipped witha 10 mL addition funnel was charged with iodide 2-bromo-9,9-difluoro-7-iodo-9H-fluorene 39b (4.98 g, 12.2 mmol). Anhydrous THF (50 mL)was added, and the mixture was stirred to homogeneity before cooling to−20 °C in an ice/NaCl bath. i-PrMgCl (2 M in THF; 6.8 mL, 13.6mmol) was added dropwise over 4 min. After stirring for 23 min, asolution of theWeinreb amide in anhydrous toluene (15 mL) was addedover 2min. The reactionmixture was warmed to 0 °C, stirred for 10min,and then warmed to room temperature. After stirring for 90 min at roomtemperature, 10% aqueous HCl (50 mL) was added to quench thereaction. The crude reaction mixture was extracted with MTBE (100mL). The organic layer was washed with brine (50 mL), dried overMgSO4, filtered, and concentrated by rotovap. The residue was dried onthe hi-vac for 5 min, DCM (90 mL) was added, and the mixture wasspun on the rotovap (no vacuum) with the bath temp set to 35 °C. Onceall material had dissolved, MeOH (45 mL) was added, and the solutionwas concentrated slowly on the rotovap without allowing contact withthe water bath. Slowly, white material began to precipitate. Once theslurry was thick (volume reduced by approximately 1/3), it was cooled to0 °C in a refrigerator. The crystals were collected by filtration to give 39c(2.84 g, 65% yield) with 97% purity (HPLC). The flask containing themother liquor and the filter funnel were rinsed with 20% DCM/hexane(40 mL) back into the original crystallization flask, and the precipitationon the rotovap was repeated. Filtration provided an additional batch of

Table 7. Compound 39 Pharmacokinetic Parameters in Rat, Dog, and Cynoa

species CL (L/h/kg) Vss (L/kg) t1/2 (hr) MRT (hr)b %F

rat 0.43 ± 0.04 2.66 ± 0.13 4.67 ± 0.56 6.19 ± 0.28 32.5 ± 6.7dog 0.13 ± 0.02 1.19 ± 0.13 7.41 ± 0.80 9.20 ± 1.35 53.0 ± 12.4cyno 0.17 ± 0.00 2.15 ± 0.42 10.3 ± 1.2 12.9 ± 2.1 41.1 ± 3.6

aAll parameters except for %F are from IV dosing. See the Experimental Section for detailed assay protocols. Intravenous doses were 1.0, 0.2, and 0.5mg/kg of body weight in the rat, dog and monkey, respectively. Oral doses were 2, 0.5, and 1.0 mg/kg in the rat, dog and monkey, respectively.bMean residence time.

Figure 4. Single-oral-dose pharmacokinetics of 39 in healthy volunteers.The dotted red line corresponds to the plasma-protein-adjusted GT1aEC50 of 0.21 nM. Curves with increasing plasma exposure correspond tothe following oral doses: 3, 10, 30, 60, and 100 mg. The 3 and 100 mgdose concentrations are 12- and 470-fold over the plasma-protein-adjusted GT1a EC50 at the 24 h time point, respectively.

Table 8. Compound 39 Plasma PK Parameters Following Single-Oral-Dose Administration to Healthy Volunteers

mean (% CV)

cohort 1 cohort 2 cohort 3 cohort 4 cohort 5

compound 39 dose 3 mg (n = 8) 10 mg (n = 8) 30 mg (n = 8) 60 mg (n = 8) 100 mg (n = 8)Cmax (nM) 6.75 (37) 21.3 (36) 82.2 (51) 133 (50) 242 (35)tmax (hr) 5.25 (20) 5 (21) 5.75 (22) 5.5 (17) 5.5 (17)AUClast (nM hr) 140 (62) 558 (34) 2236 (58) 4007 (55) 7048 (33)AUCinf (nM hr) 245 (60) 695 (32) 2717 (60) 5299 (58) 8658 (34)t1/2 (hr) 45.2 (51) 42.4 (29) 37.2 (32) 44.2 (22) 39.5 (23)C24hr (nM) 2.45 (37) 7.94 (34) 31.4 (60) 56.4 (57) 98.8 (35)


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39c (0.67 g, 15% yield) with 94% purity (HPLC). 1H NMR (CDCl3) δ8.20 (s, 1H), 8.13 (d, J = 8Hz, 1H), 7.84 (s, 1H), 7.67 (m, 2H), 7.53 (d, J= 8 Hz, 1H), 4.72 (s, 2H). 19F NMR (CDCl3) δ −111.44 (s, 2F).(S)-tert-Butyl 6-(5-(7-bromo-9,9-difluoro-9H-fluoren-2-yl)-1H-imi-

dazol-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate (39d). 1-(7-Bromo-9,9-difluoro-9H-fluoren-2-yl)-2-chloroethanone (39c, 1.7 g,4.7 mmol), 12 (1.2 g, 5.1 mmol), K2CO3 (1.3 g, 9.3 mmol), and KI(80 mg, 0.47 mmol) were dissolved in acetone (50 mL). The mixturewas heated for 3 h at 60 °C and concentrated. The crude residue wasdiluted with EtOAc (100mL) and washed with water (50mL) and brine(50 mL). The solution was dried over Na2SO4, filtered, andconcentrated. The crude material was dissolved in DCM (10 mL),and hexane (100 mL) was added. The mixture was allowed to crystallizeovernight to afford (S)-6-(2-(7-bromo-9,9-difluoro-9H-fluoren-2-yl)-2-oxoethyl) 5-tert-butyl 5-azaspiro[2.4]heptane-5,6-dicarboxylate (2.3 g,88% yield) as a mixture of rotamers. 1H NMR (CDCl3) δ 8.13 (s, 1H),8.07−7.97 (m, 1H), 7.79 (s, 1H), 7.67−7.56 (m, 2H), 7.53−7.44 (m,1H), 5.61 (d, J = 16.3 Hz, 0.5H), 5.47 (d, J = 16.2 Hz, 0.5H), 5.29 (d, J =16.2 Hz, 0.5H), 5.15 (d, J = 16.3 Hz, 0.5H), 4.62 (dd, J = 8.7, 3.5 Hz,0.5H), 4.55 (dd, J = 8.7, 4.0 Hz, 0.5H), 3.48−3.28 (m, 2H), 2.43−2.35(m, 1H), 2.17−2.07 (m, 1H), 1.48 (s, 9H) 0.77−0.55 (m, 4H). 13CNMR (CDCl3) δ 190.8, 190.3, 172.2, 172.0, 154.4, 153.7, 143.7−143.4(m), 140.3 (t, J = 25.9 Hz), 138.2 (t, J = 25.4 Hz), 136.9−136.5 (m),135.5, 135.4, 134.7, 134.6, 132.4, 127.7, 124.2, 124.1, 123.2, 123.2, 122.7,121.6 (t, J = 244 Hz), 120.8, 120.8, 80.1, 80.0, 66.0, 65.9, 59.4, 59.0, 54.3,53.7, 38.9, 38.0, 28.4, 28.3, 20.7, 20.0, 12.9, 12.3, 8.8, 8.3. 19F NMR(CDCl3) δ −111.41 (s), −111.43 (s).A mixture of (S)-6-(2-(7-bromo-9,9-difluoro-9H-fluoren-2-yl)-2-

oxoethyl) 5-tert-butyl 5-azaspiro[2.4]heptane-5,6-dicarboxylate (4.6 g,8.2 mmol) and ammonium acetate (6.7 g, 87 mmol) in toluene (100mL) was heated to reflux for 7 h. The crude material was cooled to roomtemperature and quenched with a mixture of saturated NaHCO3 (100mL) and EtOAc (150 mL). The organic phase was separated andwashed with water and brine. The light reddish organic phase was stirredwith Na2SO4 in the presence of charcoal. Filtration gave a light yellowsolution, and concentration yielded a light yellow solid (4 g).Recrystallization from benzene (40 mL) gave 39d (3.1 g, 70% yield)as a white solid. Concentration of the mother liquid and recrystallizationfrom benzene (10 mL) yielded additional (S)-tert-butyl 6-(5-(7-bromo-9,9-difluoro-9H-fluoren-2-yl)-1H-imidazol-2-yl)-5-azaspiro[2.4]-heptane-5-carboxylate (39d, 370 mg, 8.3% yield). 1H NMR (400 MHz,DMSO, mixture of rotomers) δ 12.31−11.78 (m, 1H), 8.15−8.03 (m,1H), 8.02−7.84 (m, 2H), 7.84−7.43 (m, 4H), 5.04−4.84 (m, 1H),3.62−3.21 (m, 2H), 2.42−2.09 (m, 1H), 2.08−1.78 (m, 1H), 1.40 (s,4H), 1.17 (s, 5H), 0.75−0.31 (m, 4H). 19F NMR (376 MHz, CDCl3) δ−103.85 (s), −104.03 (s). MS-ESI+: [M + H]+ calcd forC27H27BrF2N3O2, 542.1, 544.1; found, 542.1, 544.1.(1R,3S,4S)-tert-Butyl 3-(6-(7-(2-((S)-5-(tert-butoxycarbonyl)-5-

azaspiro[2.4]heptan-6-yl)-1H-imidazol-5-yl)-9,9-difluoro-9H-fluo-ren-2-yl)-1H-benzo[d]imidazol-2-yl)-2-azabicyclo[2.2.1]heptane-2-carboxylate (39e). A 250 mL round-bottomed flask was charged with(S)-tert-butyl 6-(5-(7-bromo-9,9-difluoro-9H-fluoren-2-yl)-1H-imida-zol-2-yl)-5-azaspiro[2.4]heptane-5-carboxylate (39d, 2.83 g, 5.21mmol), (1R,3S,4S)-tert-butyl 3-(6-(4,4,5,5-tetramethyl-1,3,2-dioxabor-olan-2-yl)-1H-benzo[d]imidazol-2-yl)-2-azabicyclo[2.2.1]heptane-2-carboxylate (38a, 2.75 g, 6.25 mmol), Pd(OAc)2 (78 mg, 0.348 mmol),and PPh3 (155 mg, 0.589 mmol). DME (56 mL) was added followed bya NaHCO3 aqueous solution (1M, 20 mL, 20 mmol). The reactionmixture was purged with N2 and heated at 93 °C for 4 h under N2. Thereaction was cooled to room temperature and quenched with saturatedNaHCO3 aqueous solution (100 mL). The mixture was extracted withEtOAc (2 × 150 mL). The combined organic solution was washed withbrine (100 mL) and dried over Na2SO4 in the presence of charcoal.Filtration, concentration, and purification by silica gel chromatography(20−100% EtOAc/Hexane) yielded the product (1R,3S,4S)-tert-butyl3-(6-(7-(2-((S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptan-6-yl)-1H-imidazol-5-yl)-9,9-difluoro-9H-fluoren-2-yl)-1H-benzo[d]-imidazol-2-yl)-2-azabicyclo[2.2.1]heptane-2-carboxylate (39e, 3.63 g,90% yield) as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ12.50 (s, 2H), 8.11 (d, J = 1.9 Hz, 1H), 8.07−7.95 (m, 2H), 7.95−7.80

(m, 4H), 7.76 (s, 1H), 7.67−7.48 (m, 3H), 4.98 (dd, J = 16.8, 10.0 Hz,1H), 4.50 (d, J = 9.9 Hz, 1H), 4.28 (s, 1H), 4.19 (s, 1H), 3.51 (d, J = 10.5Hz, 1H), 3.43 (d, J = 9.9 Hz, 2H), 2.74−2.58 (m, 1H), 2.35 (d, J = 10.1Hz, 1H), 2.31−2.19 (m, 1H), 2.04 (t, J = 8.0 Hz, 1H), 2.01−1.52 (m,5H), 1.51−1.24 (m, 10H), 1.16 (d, J = 20.3 Hz, 9H), 0.75−0.50 (m,3H), 0.49−0.33 (m, 1H). HRMS-ESI+: [M + H]+ calcd forC45H49O4N6F2, 775.3778; found, 775.3773.

Methyl [(2S)-1-{(6S)-6-[5-(9,9-Difluoro-7-{2-[(1R,3S,4S)-2-{(2S)-2-[(methoxycarbonyl)amino]-3-methylbutanoyl}-2-azabicyclo[2.2.1]-hept-3-yl]-1H-benzimidazol-6-yl}-9H-fluoren-2-yl)-1H-imidazol-2-yl]-5-azaspiro[2.4]hept-5-yl}-3-methyl-1-oxobutan-2-yl]carbamate(39). (1R,3S,4S)-tert-Butyl 3-(6-(7-(2-((S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptan-6-yl)-1H-imidazol-5-yl)-9,9-difluoro-9H-fluoren-2-yl)-1H-benzo[d]imidazol-2-yl)-2-azabicyclo[2.2.1]heptane-2-carbox-ylate (39e, 115 mg, 0.138 mmol) was dissolved in DCM (2 mL), and 4NHCl in dioxane (2mL) was added. The reaction mixture was stirred atroom temperature for 20 min. All volatiles were removed in vacuo toafford the crude HCl salt of 6-(7-(2-((S)-5-azaspiro[2.4]heptan-6-yl)-1H-imidazol-5-yl)-9,9-difluoro-9H-fluoren-2-yl)-2-((1R,3S,4S)-2-azabicyclo[2.2.1]heptan-3-yl)-1H-benzo[d]imidazole, which was usedin the next step without further purification. 1H NMR (400 MHz,DMSO-d6) δ 10.83 (br s, 2H), 10.44 (br s, 2H), 10.33 (br s, 1H), 9.33(br s, 1H), 8.37 (s, 1H), 8.36 (s, 1H), 8.26 (d, J=8.0 Hz, 1H), 8.08 (d,J=0.8 Hz, 1H), 8.06 (d, J=8.0 Hz, 1H), 8.03 (d, J=0.8 Hz, 1H), 8.01 (d,J=8.4 Hz, 1H), 7.98 (dd, J=8.0, 1.2 Hz, 1H), 7.79 (dd, J=8.4, 0.4 Hz, 1H),7.75 (dd, J=8.4, 1.2 Hz, 1H), 5.29 (dd, J=8.0, 7.6 Hz, 1H), 4.82 (d, J=3.6Hz, 1H), 4.19 (s, 1H), 3.65 (d, J=10.8 Hz, 1H), 3.14 (s, 1H), 3.12 (d,J=10.8 Hz, 1H), 2.85 (dd, J=13.2, 9.6 Hz, 1H), 2.23 (dd, J=12.8, 7.6 Hz,1H), 2.11 (m, 1H), 1.99 (d, J=11.2 Hz, 1H), 1.83 (m, 1H), 1.76 (m, 1H),1.71 (d, J=10.8 Hz, 1H), 1.67 (m, 1H), 0.84 (m, 2H), 0.70 (m, 2H).HRMS-ESI+: [M + H]+ calcd for C35H33N6F2, 575.2729; found,575.2729.

The crude HCl salt of 6-(7-(2-((S)-5-azaspiro[2.4]heptan-6-yl)-1H-imidazol-5-yl)-9,9-difluoro-9H-fluoren-2-yl)-2-((1R ,3S,4S)-2-azabicyclo[2.2.1]heptan-3-yl)-1H-benzo[d]imidazole was dissolved inDMF (1.5 mL) and DIEA (53.4 mg, 0.414 mmol) was added. A solutionof (S)-2-(methoxycarbonylamino)-3-methylbutanoic acid (11, 24.2 mg,0.138 mmol), HATU (52.4 mg, 0.138 mmol), and DIEA (17.8 mg,0.138 mmol) in DMF (1 mL) was added. The reaction was stirred atroom temperature for 20 min, diluted with EtOAc, and washed withaqueous bicarbonate solution, aqueous LiCl solution (5%), and brine.The organic phase was dried over sodium sulfate, filtered, concentrated,and purified by reverse-phase HPLC (ACN/H2O with 0.1% TFA) toyield the product methyl [(2S)-1-{(6S)-6-[5-(9,9-difluoro-7-{2-[(1R,3S,4S)-2-{(2S)-2-[(methoxycarbonyl)amino]-3-methylbutano-yl}-2-azabicyclo[2.2.1]hept-3-yl]-1H-benzimidazol-6-yl}-9H-fluoren-2-yl)-1H-imidazol-2-yl]-5-azaspiro[2.4]hept-5-yl}-3-methyl-1-oxobutan-2-yl]carbamate (39, 76 mg, 49% yield). 1HNMR (300MHz, DMSO-d6)δ 8.20−7.99 (m, 8H), 7.73 (s, 2H), 7.37−7.27 (m, 2H), 5.25 (dd, J = 7.2Hz, 1H), 4.78 (s, 1H) 4.54 (s, 1H), 4.16 (m, 1H), 4.02 (m, 1H), 3.87(m,1H), 3.74 (m, 1H), 3.55 (s, 3H), 3.53 (s, 3H), 2.75 (m, 1H), 2.25 (m,2H), 2.09−2.04 (m, 2H), 1.88−1.79 (m, 2H), 1.54 (m, 1H), 0.94 - 0.77(m, 15H) 0.63 (m, 4H). 19F NMR (282 MHz, DMSO-d6) δ −109.1[−74.8 TFA]. HRMS (ESI-TOF) m/z: [M + H]+ calcd forC49H55F2N8O6, 889.4207; found, 889.4214.

Replicon Standard Deviation and Replicates. Standarddeviation and number of replicates for the GT1a EC50 values are inthe following format: compound number (std deviation, no. ofreplicates): 19 (0.26, 4), 22 (0.15, 6), 23 (0.27, 4), 20 (0.036, 28), 21(0.037, 8), 25 (0.010, 8), 26 (0.77, 6), 27 (0.041, 2), 28 (0.069, 8), 29(0.051, 103), 30 (0.016, 4), 31 (0.058, 4), 32 (0.53, 12), 33 (0.008, 27),34 (0.061, 4), 35 (0.095, 78), 36 (0.049, 26), 37 (0.14, 18), 38 (0.017,35), and 39 (0.011, 295).

GT1a and GT1b Replicons. The stable genotype 1a (GT1a)subgenomic replicon cell line 1a-57C-RlucP (H77 strain) was used todetermine compound GT1a antiviral activity and was established asdescribed previously.45 The compound GT1b antiviral activity wasdetermined in the stable GT1b subgenomic replicon cell line 1b-Rluc-2(Con-1 strain). To establish 1b-Rluc-2, replicon plasmid pCon1/SG-hRlucNeo (G+I+T) was generated from plasmid I389luc-ubineo/NS3-


dx.doi.org/10.1021/jm401499g | J. Med. Chem. XXXX, XXX, XXX−XXXJ

3′/ET, which encodes a subgenomic replicon of the Con-1 strain andwas obtained from ReBLikon.46 The hRluc-Neo gene was PCRamplified from pF9 CMV hRluc-Neo Flexi (Promega, Madison, WI)by PCR using Accuprime Super Mix I (Invitrogen, Carlsbad, CA) andthe primers AscI hRLuc Fwd and NotI hRluc Rev. These two primershave the following sequence and carry restriction sites for subsequentcloning: AscI hRLuc Fwd: 5′-ACT GAC GGC GCG CCA TGG CTTCCA AGG TGT ACG-3′ (AscI site underlined) and NotI hRluc Rev:5′-GTC AGT GCG GCC GCT CAG AAG AAC TCG TCA AGA-3′(NotI site underlined). The hRluc-Neo amplification product wassubcloned into pCR2.1-TOPO (Invitrogen). The resulting plasmid wasdigested with AscI and NotI, and the excised fragment (hRluc-Neo) wasligated using T4 DNA ligase into I389luc-ubi-neo/NS3-3′/ET digestedwith the same enzymes. The resulting vector, pCon1/SG-hRlucNeo (G+I+T), was sequenced to confirm the correct orientation and sequenceof the hRluc-Neo fusion gene.Plasmid pCon1/SG-hRlucNeo (G+I+T) was linearized with SpeI

and purified using a PCR purification kit (Qiagen, Valencia, CA).Replicon RNA was in vitro synthesized with T7MEGAScript reagents(Ambion, Austin, TX) following the manufacturer’s suggested protocol.RNA was purified by column purification using an RNeasy Kit (Qiagen)according to the manufacturer’s instructions. RNA concentrations weredetermined by measurement of absorbance at 260 nm, and integrity wasverified by 0.8% agarose gel electrophoresis and ethidium bromidestaining. Ten micrograms of in vitro transcribed pCon1/SG-hRlucNeo(G+I+T) RNA was electroporated into 4 × 106 Huh7-Lunet cells asdescribed previously.45 Briefly, electroporated cells were plated onto 100mm cell culture dishes. Twenty-four hours after plating, the media wasreplaced with propagation media supplemented with 1.0 mg/mL ofG418 (selection lasted for approximately 3 weeks). G418-resistantclones were isolated and expanded. HCV replication was quantifiedusing a commercial Renilla luciferase assay (Promega) per themanufacturer’s instructions. Clones with the highest luciferase signal-to-background ratios were selected for validation in high-throughputantiviral susceptibility assays. The final clonal cell line selected for GT1bantiviral studies was designated 1b-Rluc-2.Replicon Antiviral Assays. To determine compound GT1 antiviral

activities, either 1a-57C-RlucP or 1b-Rluc-2 replicon cells were plated at2000 cells per well in 384-well plates (Greiner Bio-One, Monroe, NC;cell-culture treated). Compounds were 3-fold serially diluted in DMSOand added to the cells using an automated instrument (Biotek μFlowWorkstation; Biotek, Winooski, VT) at a final concentration of 0.44%DMSO in a total volume of 90 μL. For each drug concentration,quadruple wells were set up in the 384-well plate. DMSO was used as anegative (solvent; no inhibition) control, and a combination of threeHCV inhibitors, including a protease inhibitor, an NS5A inhibitor, and anucleoside inhibitor, was used at concentrations >100× EC50 as apositive control (100% inhibition). Plates were incubated for 3 days at37 °C in an atmosphere of 5% CO2 and 85% humidity. Culture mediumwas aspirated with a Biotek ELX405 plate washer. Twenty microliters ofDual-Glo luciferase buffer (Promega) was added to each well of the platewith a Biotek μFlowWorkstation. The plate was incubated for 10 min atroom temperature. Twenty microliters of a solution containing a 1:100mixture of Dual-Glo Stop & Glo substrate (Promega) and Dual-GloStop & Glo buffer (Promega) was added to each well with a BiotekμFlow Workstation. The plate was incubated at room temperature for10 min before the luminescence signal was measured with an Envisionplate reader (PerkinElmer, Waltham, MA).The anti-HCV replication activity was determined by the

luminescence signal generated from the reporter Renilla luciferase ofthe HCV replicon. The percentage inhibition of the HCV replicon wascalculated using eq 1.

= −−−

⎛⎝⎜

⎞⎠⎟

X MM M

percent inhibition 100 1 C B

D B (1)

.where XC is the luminescence signal from compound-treated well,MB isthe average luminescence signal from the positive control wells (100%inhibition), and MD is the average luminescence signal from DMSO-treated wells.

The EC50 values were determined as the testing compoundconcentration that caused a 50% decrease in HCV replicon luciferasesignal. EC50 values were obtained using Pipeline Pilot 5.0 softwarepackage (Accelrys, San Diego, CA) by nonlinear regression fitting ofexperimental data.

Competitive Protein Binding Assay. Human plasma and cell-culture medium containing 10% fetal bovine serum (CCM) were spikedwith the test compound at a final concentration of 2 μM. Spiked plasma(1 mL) and CCM (1 mL) were placed into opposite sides of theassembled dialysis cells, which are separated by a semipermeablemembrane. The dialysis cells were rotated slowly in a 37 °C water bathfor the time necessary to reach equilibrium. Postdialysis plasma andCCMweights were measured, and the test compound concentrations inplasma and CCM were determined with LC/MS/MS.36

Metabolic Stability. Metabolic stability in vitro was determinedusing pooled hepatic microsomal fractions (final protein concentrationof 0.5 mg/mL) at a final test compound concentration of 3 μM. Thereaction was initiated by the addition of an NADPH-regeneratingsystem. Aliquot of 25 μL of the reaction mixture were transferred atvarious time points to plates containing a quenching solution. The testcompound concentration in the reaction mixture was determined withLC/MS/MS. Hepatic intrinsic clearance was calculated as describedpreviously by Obach,47 and the predicted clearance was calculated usingthe well-stirred liver model without protein restriction.

Metabolic stability was also determined in cryopreserved hepatocytesusing tritiated test compounds. The incubation mixture contained 1 ×106 hepatocytes/mL and 1 μM tritiated test compound (2.5 μCi). Theincubation was carried out with gentle shaking at 37 °C under a humidatmosphere of 95% air/5% CO2 (v/v). Aliquots of 50 μL were removedafter 0, 1, 3, and 6 h and added to 100 μL of quenching solution. Thesamples were analyzed on a flow scintillation radio detector coupled toan HPLC system. The metabolites were quantified on the basis of thepeak areas from the radio detector, with the cell-free control samplesused as a reference. Metabolic stabilities in hepatocytes were determinedby measuring the rate of disappearance of the test compound as thepercent of total peak areas of the formed radiolabeled metabolites andthe test compound.

Pharmacokinetics. Pharmacokinetic studies were performed inmale naıv̈e Sprague−Dawley(SD) rats, non-naıv̈e beagle dogs, andcynomolgus monkeys (three animals per dosing route) following federaland Institutional Animal Care andUse Committee (IACUC) guidelines.Intravenous (IV) administration was dosed via infusion over 30 min in avehicle containing 5% ethanol, 20% PEG400, and 75% water (pHadjusted to 3.0 with HCl). Oral dosing was administered by gavage in avehicle containing 5% ethanol, 45% PEG 400, and 50% of 50 mM citratebuffer, pH 3. Blood samples were collected over a 24 h period postdoseinto Vacutainer tubes containing EDTA-K2. Plasma was isolated, andthe concentration of the test compound in plasma was determined withLC/MS/MS after protein precipitation with acetonitrile.

Noncompartmental pharmacokinetic analysis was performed onplasma concentration data to calculate pharmacokinetic parametersusing the software program WinNonLin (version 5.0.1).

■ ASSOCIATED CONTENT

*S Supporting InformationSynthetic procedures and spectroscopic data. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Phone: 650-522-1727. E-mail: [email protected].

NotesThe authors declare the following competing financialinterest(s): The authors are employees of Gilead Sciencesexcept for J.S., T.K., E.M., and M.C., who were employed atGilead Sciences during this work. All authors are shareholders inGilead Sciences.


dx.doi.org/10.1021/jm401499g | J. Med. Chem. XXXX, XXX, XXX−XXXK

http://pubs.acs.org

mailto:[email protected]

■ ACKNOWLEDGMENTS

We thank Kathy Brendza and Lorendana Serafini for obtainingHRMS data.

■ ABBREVIATIONS USED

AUCinf, area under the curve from time zero to infinity; AUClast,area under the curve from time zero to the last measurableconcentration; %F, bioavailability; C24hr, plasma concentration at24 h; Cmax, maximum plasma concentration; CL, systemicclearance; CV, coefficient of variation; DAA, direct-actingantiviral; dppf, 1,1′-bis(diphenylphosphino)ferrocene; GT1,hepatitis C virus genotype 1; GT1a, hepatitis C virus genotype1a, a subtype of hepatitis C virus genotype 1; GT1b, hepatitis Cvirus genotype 1b, a subtype of hepatitis C virus genotype 1;HATU, (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium 3-oxid hexafluorophosphate); KHMDS,potassium bis(trimethylsilyl)amide; MRT, mean residencetime; n.d., not determined; NS3, hepatitis C virus nonstructuralprotein 3; NS5A, hepatitis C virus nonstructural protein 5A;NS5B, hepatitis C virus nonstructural protein 5B; nM,nanomolar; PEG, pegylated interferon; pM, picomolar; RBV,ribavirin; SEM, [2-(trimethylsilyl)ethoxy]methyl; SVR, sus-tained viral response to treatment regimen; SVR12, sustainedviral response to treatment regimen 12 weeks after therapy; tmax,time after administration of a drug when the maximum plasmaconcentration is reached; Vss, volume of distribution

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