Discovery of ledipasvir (GS-5885): A potent, once-daily oral NS5A inhibitor for the treatment of hepatitis C virus infection

Article abstract of DOI:10.1021/jm401499g

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

Full text of DOI:10.1021/jm401499g

Article
pubs.acs.org/jmc
Discovery of Ledipasvir (GS-5885): A Potent, Once-Daily Oral NS5A
Inhibitor for the Treatment of Hepatitis C Virus Infection
John 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 Structural
Chemistry, Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States
S
* Supporting Information
ABSTRACT: A new class of highly potent NS5A inhibitors with an
unsymmetric benzimidazole-difluorofluorene-imidazole core and distal
[2.2.1]azabicyclic ring system was discovered. Optimization of antiviral
potency and pharmacokinetics led to the identification of 39 (ledipasvir,
GS-5885). Compound 39 (GT1a replicon EC50 = 31 pM) has an
extended plasma half-life of 37−45 h in healthy volunteers and produces a
rapid >3 log viral load reduction in monotherapy at oral doses of 3 mg or
greater with once-daily dosing in genotype 1a HCV-infected patients. 39
has been shown to be safe and efficacious, with SVR12 rates up to 100%
when used in combination with direct-acting antivirals having complementary mechanisms.
necessary to serve a broader patient population, to improve
outcomes, and to provide a safer, simpler regimen.8
INTRODUCTION
■
Hepatitis C virus (HCV) infection is a significant public health
concern, with approximately 170 million infected individuals
worldwide, and is the leading cause of liver transplant and
hepatocellular carcinoma.1 HCV is the most common chronic
blood-borne pathogen in the U.S., and the Center for Disease
Control and the U.S. Preventative Services Task Force are
aligned in recommending that all baby-boomers (individuals
born between 1945 and 1965) undergo testing for HCV
infection.2 Until recently, the standard of care for treatment of
genotype 1 (GT1) infection (60% of total infections worldwide
among the seven known genotypes, with both GT1a and GT1b
as major subtypes)3 consisted of weekly pegylated interferon
(PEG) injections and twice-daily oral ribavirin (RBV) for 24 or
48 weeks (duration based on response-guided therapy). PEG/
RBV treatments achieve up to 54−63% sustained virologic
response (SVR) in GT1 patients,4 but treatment is accompanied
by considerable toxicity including flu-like symptoms, depression,
and anemia.5 Triple therapy containing PEG/RBV combined
with three-times-daily dosing of the recently approved direct-
acting antiviral (DAA) protease inhibitor telaprevir or boceprevir
has improved the GT1 HCV SVR rates to 66−79% for treatment
We have sought to identify safe oral drugs for combination
treatment of HCV infection. In addition to programs targeting
the NS3 protease9 and NS5B polymerase (both nucleotides10
and non-nucleotides11), we initiated an NS5A inhibitor program
with the goal of identifying an agent with characteristics that
would allow its use in combination with DAAs having
complementary mechanisms to achieve high SVR rates with a
short treatment duration.
Despite significant study, the mechanistic role of NS5A in the
HCV life cycle remains enigmatic.12 NS5A has no known
enzymatic activity and has no homologues in prokaryotes or
eukaryotes. Nonetheless, the protein is critical for HCV viability;
in clinical monotherapy studies, NS5A inhibitors produce the
most rapid viral load declines of any HCV antiviral class. It has
been postulated that this rapid decline in HCV RNA is based on
inhibition of viral replication (as with NS3 and NS5B inhibitors)
and additional inhibition of virion assembly or secretion from
infected cells.13
Early NS5A inhibitors were found empirically through
screening of the GT1b replicon. Several series of lipophilic
proline, proline-mimetic, or alanine-amide inhibitors of the
GT1b replicon have been discovered (1−3; Figure 1),14 but
these inhibitors typically have ∼1000-fold weaker activity against
the GT1a replicon.15 A polyaromatic pyridopyrimidine class (4)
̈
naıve 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 on
PEG/RBV) are not indicated for retreatment with PEG/RBV
because they achieve SVR rates <10%, with minimal improve-
ment to 29% for patients undergoing triple therapy.7 Finally, a
growing number of patients are being identified as interferon-
intolerant or unwilling to take interferon. PEG-free therapy is
Special Issue: HCV Therapies
Received: September 26, 2013
© XXXX American Chemical Society
A
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Figure 1. Structures of NS5A inhibitors.
a
affords nanomolar activity against both the GT1a and 1b
replicons.16 Dimeric series provide potent GT1b-active stilbene
diamide inhibitors (5 discovered from monomer series 1)17 and
highly potent GT1a and 1b-active bis-imidazole biphenyl
Scheme 1
inhibitors including daclatasvir 6 (BMS-790052, GT1a EC50
=
50 pM), which achieved clinical proof of concept for the NS5A
mechanism.18 NS5A has emerged as an important drug target for
the treatment of HCV infection.19
For our NS5A inhibitor target profile, we sought high potency
and a long plasma half-life to reduce the emergence of viral
resistance.20 To achieve these aims, a diverse range of inhibitor
structures was investigated.21 Herein, we describe structural
modifications and SAR in a symmetric bis-benzimidazole series
and in an unsymmetric imidazole/benzimidazole series. We
found that the unsymmetric series posed unique advantages for
the optimization of inhibitor potency and pharmacokinetics. On
the basis of these studies, we describe the discovery of clinical
compound 39, a potent and selective HCV NS5A inhibitor with
excellent preclinical and human pharmacokinetics and potent
antiviral activity in HCV-infected patients.22
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.
borylation of 13 generated pinacol boronate 14. Removal of the
phenylethyl chiral auxiliary from 15a25 by hydrogenolysis
followed by Boc protection and hydrolysis of the methyl ester
afforded [2.2.1]azabicyclic carboxylic acid 15.
CHEMISTRY
■
Repeatedly used intermediates for the syntheses of these NS5A
inhibitors are shown in Figure 223 and Scheme 1. Coupling of
Boc-proline 13a with diamine 13b using HATU afforded a
mixture of amides that could be dehydrated at elevated
temperatures to afford benzimidazole 13 (Scheme 1).24 Miyaura
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 benzimidazole
bromide 13 and boronate ester 14 provided the core of 16.
Bromide 13 could also be converted to acetylene 17a by
Sonogashira coupling. A second Sonogashira coupling between
17a and 13 afforded the core of 17, whereas Glaser-type oxidative
dimerization of 17a formed the core of 18. For 16−18, as in
many of the following schemes, double Boc-removal followed by
double peptide-coupling with valine methyl carbamate 11
afforded 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 acid
bis(pinacol) ester to give boronate 22a (Scheme 4). A second
coupling between 13 and 22a formed the core of inhibitor 22.
Suzuki coupling of 22a with bromoimidazole 9 led to a mixture of
homodimer 23a and cross-coupled product 24a. 23a and 24a
were converted to 23 and 24, respectively, by Boc-removal and
peptide-coupling with 11.
Figure 2. Intermediates used in the synthesis of NS5A inhibitors.
B
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a
a
Scheme 2
Scheme 4
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.
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.
a
Scheme 3
a
Scheme 5
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.
a
The synthesis of the naphthyl imidazole inhibitor 25 began
with carboxylic acid 25a, which was converted to bromoketone
25b by a three-step sequence (Scheme 5).27 Boc-proline was
alkylated by 25b to give a ketoester that cyclized to imidazole 25c
upon heating in the presence of ammonium acetate. The Boc
group was replaced with valinyl methyl carbamate 11, and the
naphthalene was borylated, providing 25d. 25d and 25e were
then coupled to afford 25.
Bromoimidazole 10 was converted to the corresponding
formylimidazole by lithium-halogen exchange and trapping of
the organometallic species with DMF (Scheme 6). The Ohira−
Bestmann reagent28 was employed in the homologation of the
resultant aldehyde to acetylene 26a. Boc-removal and peptide-
coupling with 11 gave 26b. Suzuki coupling of boronate ester 14
and 1,4-dibromobezene afforded a Boc-protected aryl bromide,
which was converted to amide 26c. Sonogashira coupling
between 26c and acetylene 26b provided 26.
(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.
boronic acid upon purification by reversed-phase HPLC
(Scheme 7). Suzuki coupling of 27a and bromoimidazole 10
yielded bis(Boc-pyrrolidine) 27b, which was converted to 27
after 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). A
second Suzuki coupling between 28a and bromobenzimidazole
13 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 was
then coupled to bromoimidazole 10 to furnish the core of 29
(Scheme 9).
Sonogashira coupling between 17a and 1,4-diiodobenzene was
followed by Miyaura borylation of the intermediate aryl iodide to
give the intermediate boronic ester, which was cleaved to the
C
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a
a
Scheme 6
Scheme 9
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.
a
a
Scheme 10
(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.
a
Scheme 7
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.
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.
Boc-proline by 33b gave an intermediate ketoester that was
cyclized to imidazole 33c as described above for the synthesis of
25c. Suzuki coupling of 33c with 14 produced 33d, which led to
33 after Boc-removal and peptide-coupling with 11.
a
Scheme 8
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 be
thermally unstable,30 so exercise caution when using this
reagent). The inclusion of an organotin reagent also counted
among the unattractive aspects of this synthesis. Finally, a lack of
selectivity between mono- and dicoupled enol-ether products in
the Stille reaction contributed to the low efficiency of this route.
See Scheme 16 for a synthesis of the elaborated difluorofluorene
ring system that avoids these problems.
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.
A HATU-promoted coupling between amine 34a and
piperidine-carboxylic acid 34b afforded a ketoamide that could
be dehydrated in the presence of ammonium acetate to yield
imidazole 34c (Scheme 12). Boc-removal was followed by valine
methyl carbamate 11 attachment and Miyaura borylation to give
coupling partner 34d. Suzuki coupling between 34d and 8b
provided inhibitor 34. 35 was prepared by a similar sequence to
that for 34, with the exception that [2.2.1]azabicyclic carboxylic
acid 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). Suzuki
coupling between 36a and 1,4-benzenediboronic acid bis-
(pinacol) ester provided 36b, which produced the core of 36
by a second cross-coupling with 8a.
Bis-imidazoles with fused tricyclic linkers were prepared from
dibromides 30a, 31a, and 32a (Scheme 10). These dibromides
were converted to corresponding diboronate esters 30b, 31b,
and 32b by Miyaura borylation. The imidazoles were attached to
the tricyclic ring systems by Suzuki cross-coupling of the
diboronate esters with bromoimidazole 9, en route to inhibitors
30, 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 converted
to bromoketone 33b upon treatment with NBS.29 Alkylation of
D
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a
Scheme 11
a
(a) Bis(2-methoxyethyl)aminosulfur trifluoride, catalytic EtOH (Caution: Bis(2-methoxyethyl)aminosulfur trifluoride has the potential to decompose
exothermically under these conditions. Exercise caution. For a safer and more efficient synthesis of the difluorofluorene 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.
a
Scheme 12
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.
a
Boronate 38a resulted from borylation of bromide 36a
(Scheme 15). A Suzuki coupling between 38a and 33c formed
the core of inhibitor 38.
Scheme 13
a
Scheme 15
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.
35a was coupled to 22a to provide the Boc-protected
precursor to 37 (Scheme 14).
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.
a
Scheme 14
In a novel process, fluorene 39a31 could be difluorinated by
treatment with KHMDS in the presence of N-fluorobenzene-
sulfonimide32 (Scheme 16) to give 39b. The iodine-bearing
carbon of 39b was selectively metalated by reaction with i-
PrMgCl and converted to single chloroketone 39c by quenching
the Grignard species with the Weinreb amide 2-chloro-N-
methoxy-N-methylacetamide.33 Carboxylic acid 12 was alkylated
a
(a) 22a, Pd(PPh3)4, K2CO3, H2O/DME; (b) HCl/dioxane/DCM;
(c) 11, HATU, i-Pr2NEt, DMF.
E
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a
Scheme 16
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.
a
with 39c, and the resulting ketoester was condensed to imidazole
39d by heating with ammonium acetate. 39d was coupled to 38a,
providing 39e. Boc-removal was followed by peptide coupling
with 11 to afford 39.
Table 1. In Vitro Activity
This is the first reported base-promoted difluorination of the 9
position of a fluorene ring system utilizing an electrophilic source
of fluorine.34 Crucial to the success of the process are the
premixing of the substrate and the N-fluorobenzenesulfonimide
as well as the slow addition of base to this mixture. It is
noteworthy that the base and the fluorinating reagent are inert
toward each other on the time scale of the reaction and that the
difluorinated product is formed in high yield without isolation of
the monofluorofluorene.
RESULTS AND DISCUSSION
■
Throughout this work, our optimization of potency focused on
GT1a activity; accordingly, the discussion here is restricted to
replicon GT1a EC50 values, although the GT1b values are
provided for comparison.35 We pursued a number of symmetric
bis-benzimidazole cores (Table 1).21 Directly linked bis-
benzimidazole 16 and monoalkyne inhibitor 17 showed no
inhibitory activity under the assay conditions (up to 44 nM),
whereas diyne 18 had an EC50 of 11 nM. Replacing the alkynes
with a thiophene ring (19) improved potency 6-fold, and the
benzimidazole-phenyl-benzimidazole core in 22 provided further
improvements in potency (EC50 = 500 pM). Replacement of
phenyl with biphenyl decreased activity 7-fold. Finally, insertion
of fused ring systems provided highly potent naphthyl inhibitor
20 (EC50 = 109 pM), with benzdithiophene 21 only 2-fold less
active. From these studies, inhibitors with fused central ring
systems, as in 20 and 21, were the most potent, whereas less
lipophilic connectors, such as in alkynes 17 or 18, afforded
weaker activity.
We next sought to determine if an unsymmetric core,
including one benzimidazole and one imidazole, could provide
potent inhibitors (Table 2). The directly linked imidazole/
benzimidazole was not synthesized because directly linked bis-
benzimidazole 16 was not active. We found it striking that
replacement of the phenyl in 24 by naphthyl (25) resulted in a
>600-fold potency enhancement, whereas the difference
between phenyl and naphthyl linkers in the bis-benzimidazole
series is <5-fold (Table 1). Furthermore, in the unsymmetric
series, the fused central naphthyl ring affords higher potency than
it does in the symmetric series (e.g., compound 20). The
importance of the position of the lipophilicity in the linker is
demonstrated by phenyl-alkyne inhibitors 26 and 27, where the
a
b
See the Experimental Section for detailed assay protocols. For
c
standard deviations, see the Experimental Section. A value of >44 nM
means that no inhibition was observed at this top well concentration.
more centrally positioned alkyne in 27 is improved 6-fold in
potency. As in Table 1, introduction of alkynes in the linker
affords lower potency relative to inhibitors with aromatic
elements.
Notably, the biphenyl in inhibitor 29 provides the highest level
of potency among the nonfused central connectors in Tables 1 or
2. The high potency of biphenyl 29, along with the theme
throughout Tables 1 and 2 that fused central ring systems
consistently provide high potency (compounds 20, 21, and 25),
prompted us to study constraint of the biphenyl to form tricyclic
fused-ring systems.
Initial SAR of central fused tricycles was studied in a symmetric
bis-imidazole series to obviate tricycle desymmetrization and
thereby simplify compound synthesis. Fluorene ring-linked
inhibitor 30 (Table 3) suffered a mild loss in potency relative
to biaryl 6 and posed stability concerns because the fluorene ring
system readily underwent autoxidation upon standing. The
F
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a
not well-tolerated. We postulated that a smaller, lipophilic
blocking group such as a difluoromethylene group might provide
an optimal connector. We decided to introduce this important
modification directly in the imidazole/benzimidazole series of
interest because of the synthetic challenge posed by introduction
of the difluoromethylene group. Use of the difluoromethylene
connector produced the most potent inhibitor in the optimized
unsymmetric 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 similar
good half-lives in plasma, low systemic clearance (CL), and
moderate volumes of distribution (Vss) that are greater than total
body water volume. Unexpectedly, the modest 11% oral
bioavailability of biphenyl inhibitor 29 was improved to over
35% in difluoroflourene 33. Incorporation of difluorofluorene in
compound 33 therefore provided combined improvements in
potency and bioavailability.
Table 2. In Vitro Activity
Next, we assessed terminal heterocycle modifications in the
symmetric bis-imidazole system. Piperidine 34 (Table 5) was less
potent than the [2.2.1]azabicyclic inhibitor 35. Importantly, we
found that 35 possessed a long half-life of 5.3 h in dog (Table 4).
a
a
b
See the Experimental Section for detailed assay protocols. For
Table 5. In Vitro Activity
c
standard deviations, see the Experimental Section. A value of >44 nM
means that no inhibition was observed at this top well concentration.
a
Table 3. In Vitro Activity
b
compd
EC50 (1a, nM)
EC50 (1b, nM)
34
35
0.45
0.21
0.005
0.009
b
compd
A
EC50 (1a, nM)
EC50 (1b, nM)
a
b
See the Experimental Section for detailed assay protocols. For
standard deviations, see the Experimental Section.
30
31
32
33
CH2
CO
0.094
0.30
1.2
0.013
0.018
0.014
CMe2
CF2
Attracted by the favorable pharmacokinetic properties that the
[2.2.1]azabicyclic ring system imparted in 35, we sought to
understand the SAR of the azabicyclic ring system in the context
of an unsymmetric core. The more synthetically accessible
imidazole-biphenyl-benzimidazole core was chosen for this
study. We discovered matched and mismatched sets where the
potency is markedly better when the [2.2.1]azabicyclic ring
system is paired with the benzimidazole in 36 rather than with
the imidazole in 37 (Table 6). We considered this differential
0.040
0.003
a
b
See the Experimental Section for detailed assay protocols. For
standard deviations, see the Experimental Section.
oxidation product, fluorenone 31, lost significant potency (EC50
= 300 pM). Blocking oxidation with gem-dimethyl (32) lost even
more potency, giving some credence to the concept that
significant out-of-plane steric bulk as part of the fused linker is
a b
,
Table 4. Rat and Dog Pharmacokinetics
d
compd
species
CL (L/h/kg)
Vss (L/kg)
t1/2 (hr)
MRT (hr)
%F
29
rat
1.04 0.17
0.78 0.29
0.42 0.04
0.53 0.04
0.70 0.02
0.05 0.007
0.75 0.04
0.40 0.26
1.76 0.17
2.31 0.37
0.93 0.04
1.96 0.03
0.98 0.08
0.31 0.007
1.81 0.26
2.03 0.92
1.57 0.19
2.30 0.28
1.83 0.22
2.63 0.18
1.49 0.02
5.29 0.60
2.07 0.19
4.01 0.82
1.71 0.16
3.00 0.27
2.21 0.21
3.69 0.29
1.40 0.07
6.60 1.10
2.42 0.22
5.47 1.01
11.5 8.7
c
dog
rat
n.d.
33
35
36
36.7 3.2
c
dog
rat
n.d.
35.2 10
c
dog
rat
n.d.
26.3 9.0
c
dog
n.d.
a
b
c
d
All parameters except for %F are from intravenous dosing. See the Experimental Section for detailed assay protocols. n.d., not determined. Mean
residence time.
G
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a
Table 6. In Vitro Activity
b
compd
Y′
A
Y
EC50 (1a, nM)
EC50 (1b, nM)
36
37
38
absent
CH2
absent
absent
CF2
CH2
0.16
0.66
0.056
0.006
0.021
absent
CH2
absent
0.004
a
b
See the Experimental Section for detailed assay protocols. For
standard deviations, see the Experimental Section.
SAR to be an important result; it further demonstrated to us that
the unsymmetric core series opened opportunities for inhibitor
optimization that would not be available if structures were
limited to a symmetric cores. Unlike in the symmetric core
system, the [2.2.1]azabicyclic ring system in the matched
unsymmetric case did not demonstrate a loss in potency relative
to the pyrrolidine (36 vs 29). Importantly, the extended
pharmacokinetic half-life that the [2.2.1]azabicyclic ring system
provided in symmetric core inhibitor 35 translated in
unsymmetric core inhibitor 36; both the rat and dog plasma
half-lives are improved in 36 over those of pyrrolidine analogue
29 having the same core (Table 4). To improve upon the
potency of 36, biphenyl was replaced by difluorofluorene,
affording inhibitor 38 (EC50 = 56 pM). 38 has a protein-adjusted
EC50 of 784 pM, corresponding to a protein-binding shift of 14-
fold.36
During final optimization, replacement of the pyrrolidine in 38
with a spirocyclopropylpyrrolidine afforded 39 (ledipasvir, GS-
5885), the most potent inhibitor in the series (Figure 3).21 39 has
GT1a and 1b EC50 values of 31 and 4 pM, respectively, and
protein-adjusted EC50 values of 210 pM (GT1a) and 27 pM
(GT1b). The protein-binding shift is 6.7-fold and is improved
relative to pyrrolidine 38. Compound 39 is highly protein-bound
both in human serum and in the cell-culture medium (containing
10% BSA) of the replicon assay (1.1% unbound fraction in cell-
culture medium). Accounting for the low unbound drug
concentration in the replicon assay, the intrinsic EC50 of 39 is
310 fM for GT1a and 40 fM for GT1b. 39 is remarkable not only
on the basis of its high replicon potency but also on the basis of its
low clearance, good bioavailability, and long half-lives (4.7−10.3
h) in rat, dog, and monkey and low predicted clearance in human
(0.012 L/h/kg) (Figure 3 and Table 7). In 14 day toxicology
studies in rats and dogs with compound 39, there were no
significant adverse findings. We selected 39 for clinical
development, projecting that it would be highly efficacious in
the treatment of HCV infection with a long half-life suitable for
once-daily dosing.
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 hepatic
clearance 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 top
graph shows intravenous pharmacokinetics, and the bottom graph
shows oral pharmacokinetics.
concentrations of 39 to fall below the protein-adjusted EC50 at
the end of the dosing interval.
CONCLUSIONS
■
Optimization 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 in
healthy volunteers and 24 h trough concentrations 12−470 fold
over the plasma-protein-adjusted EC50 for doses from 3 to 100
mg.39 In further studies, this profile has translated to potent
clinical efficacy: in a 3 day monotherapy study in GT1a HCV-
infected patients, 39 produced a >3 log median viral load
reduction at doses of 3 mg or greater.22 Even at a 1 mg total daily
dose, a 2.4 log median viral load reduction was achieved. Six
phase 2 trials demonstrated 39 to be safe and well-tolerated in
over 1000 patients.40 Phase 2 efficacy of 39 in combination with
sofosbuvir (GS-7977, an NS5B nucleotide inhibitor)41 and RBV
in a 12 week treatment regimen achieved 100% SVR12 in prior
null responders.42 39 is now in multiple phase 3 studies
coformulated with sofosbuvir in a single-tablet43 administered
once-daily with or without RBV.44 NS5A inhibitor 39 has the
potential to be of utility in combination with DAAs of
complementary mechanism in all-oral therapy for the treatment
of HCV infection.
The pharmacokinetics of 39 were studied in fasted healthy
volunteers in a placebo-matched double-blind phase 1 clinical
trial at oral doses of 3, 10, 30, 60, and 100 mg (Figure 4 and Table
8) and were dose-proportional over the range tested. Long mean
plasma half-lives of 37−45 h were observed. Twenty-four hour
postdose drug concentrations were 12−470-fold over the GT1
protein-adjusted EC50 and remained over the GT1a protein-
adjusted EC50 well past 24 h. These results were consistent with
the potential for both once-daily dosing and low risk for
H
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a
Table 7. Compound 39 Pharmacokinetic Parameters in Rat, Dog, and Cyno
b
species
CL (L/h/kg)
Vss (L/kg)
t1/2 (hr)
MRT (hr)
%F
rat
0.43 0.04
0.13 0.02
0.17 0.00
2.66 0.13
1.19 0.13
2.15 0.42
4.67 0.56
7.41 0.80
10.3 1.2
6.19 0.28
9.20 1.35
12.9 2.1
32.5 6.7
53.0 12.4
41.1 3.6
dog
cyno
a
All parameters except for %F are from IV dosing. See the Experimental Section for detailed assay protocols. Intravenous doses were 1.0, 0.2, and 0.5
mg/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.
b
Mean residence time.
(solvent B). High-resolution mass spectra were recorded on a Xevo G2
Q-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 dropwise
over 9 min. On completion of the addition, the reaction mixture was
warmed to 0 °C. After an additional 80 min at 0 °C, TLC indicated
completion of the reaction, and excess base was quenched by addition of
MeOH (30 drops) followed by hexane (20 mL). The suspension was
filtered over Celite and concentrated. The resulting residue was
dissolved 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. The
liquid was concentrated to provide 2-bromo-9,9-difluoro-7-iodo-9H-
fluorene 39b (626 mg, 81% yield) as a pale-yellow-orange solid. The
Figure 4. Single-oral-dose pharmacokinetics of 39 in healthy volunteers.
The dotted red line corresponds to the plasma-protein-adjusted GT1a
EC50 of 0.21 nM. Curves with increasing plasma exposure correspond to
the following oral doses: 3, 10, 30, 60, and 100 mg. The 3 and 100 mg
dose concentrations are 12- and 470-fold over the plasma-protein-
adjusted GT1a EC50 at the 24 h time point, respectively.
1
material is essentially pure by NMR and LCMS. H NMR (CDCl3) δ
7.94 (1H, d, J = 1.2 Hz), 7.81 (1H, d, J = 7.8 Hz), 7.74 (1H, d, J = 1.4
Hz), 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 with
a 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, 13.6
mmol) was added dropwise over 4 min. After stirring for 23 min, a
solution of the Weinreb amide in anhydrous toluene (15 mL) was added
over 2 min. The reaction mixture was warmed to 0 °C, stirred for 10 min,
and then warmed to room temperature. After stirring for 90 min at room
temperature, 10% aqueous HCl (50 mL) was added to quench the
reaction. The crude reaction mixture was extracted with MTBE (100
mL). The organic layer was washed with brine (50 mL), dried over
MgSO4, filtered, and concentrated by rotovap. The residue was dried on
the hi-vac for 5 min, DCM (90 mL) was added, and the mixture was
spun on the rotovap (no vacuum) with the bath temp set to 35 °C. Once
all material had dissolved, MeOH (45 mL) was added, and the solution
was concentrated slowly on the rotovap without allowing contact with
the water bath. Slowly, white material began to precipitate. Once the
slurry was thick (volume reduced by approximately 1/3), it was cooled to
0 °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 the
mother liquor and the filter funnel were rinsed with 20% DCM/hexane
(40 mL) back into the original crystallization flask, and the precipitation
on the rotovap was repeated. Filtration provided an additional batch of
EXPERIMENTAL SECTION
■
Nuclear magnetic resonance (NMR) spectra were recorded on Varian
Mercury Plus 400 MHz and a Varian Unity Plus 500 MHz spectrometers
at room temperature, with tetramethylsilane as an internal standard.
Chemical shifts (δ) are reported in parts per million (ppm), and signals
are reported as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), or br s (broad singlet). Purities of the final compounds were
determined by HPLC and were greater than 95%. HPLC conditions to
assess purity were as follows: Agilent HPLC, Phenominex Luna C18, 4.6
mm × 250 mm (5 μm); gradient of 0.1% TFA water (A) and 0.1% TFA
acetonitrile (B); flow rate, 1.0 mL/min; acquire time, 35 min;
wavelength, UV 214 and 254 nm; oven. The preparative HPLC system
includes two sets of Gilson 332 pumps, a Gilson 156 UV/vis detector,
and a Gilson 215 injector and fraction collector, with Trilution LC
software. A Phenomenex 250 mm × 21 mm 54u Gemini-NX column
was used. The mobile phase was a gradient of 0.1% TFA water (A) and
0.1% TFA acetonitrile (B). LC/MS was conducted on a Thermo
Finnigan MSQ Std using electrospray in both positive and negative
modes, [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
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
max (nM)
max (hr)
3 mg (n = 8)
6.75 (37)
5.25 (20)
140 (62)
245 (60)
45.2 (51)
2.45 (37)
10 mg (n = 8)
21.3 (36)
5 (21)
30 mg (n = 8)
82.2 (51)
5.75 (22)
2236 (58)
2717 (60)
37.2 (32)
31.4 (60)
60 mg (n = 8)
133 (50)
100 mg (n = 8)
242 (35)
C
t
5.5 (17)
5.5 (17)
AUClast (nM hr)
AUCinf (nM hr)
558 (34)
695 (32)
42.4 (29)
7.94 (34)
4007 (55)
5299 (58)
44.2 (22)
56.4 (57)
7048 (33)
8658 (34)
39.5 (23)
98.8 (35)
t1/2 (hr)
C
24hr (nM)
I
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39c (0.67 g, 15% yield) with 94% purity (HPLC). 1H NMR (CDCl3) δ
8.20 (s, 1H), 8.13 (d, J = 8 Hz, 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 mixture
was heated for 3 h at 60 °C and concentrated. The crude residue was
diluted with EtOAc (100 mL) and washed with water (50 mL) and brine
(50 mL). The solution was dried over Na2SO4, filtered, and
concentrated. The crude material was dissolved in DCM (10 mL),
and hexane (100 mL) was added. The mixture was allowed to crystallize
overnight 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). 13C
NMR (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 (100
mL) was heated to reflux for 7 h. The crude material was cooled to room
temperature and quenched with a mixture of saturated NaHCO3 (100
mL) and EtOAc (150 mL). The organic phase was separated and
washed with water and brine. The light reddish organic phase was stirred
with Na2SO4 in the presence of charcoal. Filtration gave a light yellow
solution, 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 recrystallization
from 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 for
C27H27BrF2N3O2, 542.1, 544.1; found, 542.1, 544.1.
(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 = 10.5
Hz, 1H), 3.43 (d, J = 9.9 Hz, 2H), 2.74−2.58 (m, 1H), 2.35 (d, J = 10.1
Hz, 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 for
C45H49O4N6F2, 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 4
N HCl in dioxane (2 mL) was added. The reaction mixture was stirred at
room temperature for 20 min. All volatiles were removed in vacuo to
afford 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 used
1
in the next step without further purification. H 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=3.6
Hz, 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 in
DMF (1.5 mL) and DIEA (53.4 mg, 0.414 mmol) was added. A solution
of (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 at
room temperature for 20 min, diluted with EtOAc, and washed with
aqueous 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) to
yield 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). 1H NMR (300 MHz, DMSO-d6)
δ 8.20−7.99 (m, 8H), 7.73 (s, 2H), 7.37−7.27 (m, 2H), 5.25 (dd, J = 7.2
Hz, 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 for
C49H55F2N8O6, 889.4207; found, 889.4214.
Replicon Standard Deviation and Replicates. Standard
deviation and number of replicates for the GT1a EC50 values are in
the following format: compound number (std deviation, no. of
replicates): 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 to
determine compound GT1a antiviral activity and was established as
described previously.45 The compound GT1b antiviral activity was
determined 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-
(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, 5.21
mmol), (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 by
a NaHCO3 aqueous solution (1M, 20 mL, 20 mmol). The reaction
mixture was purged with N2 and heated at 93 °C for 4 h under N2. The
reaction was cooled to room temperature and quenched with saturated
NaHCO3 aqueous solution (100 mL). The mixture was extracted with
EtOAc (2 × 150 mL). The combined organic solution was washed with
brine (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-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-carboxylate (39e, 3.63 g,
1
90% yield) as a light yellow solid. H NMR (400 MHz, DMSO-d6) δ
12.50 (s, 2H), 8.11 (d, J = 1.9 Hz, 1H), 8.07−7.95 (m, 2H), 7.95−7.80
J
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3′/ET, which encodes a subgenomic replicon of the Con-1 strain and
was obtained from ReBLikon.46 The hRluc-Neo gene was PCR
amplified from pF9 CMV hRluc-Neo Flexi (Promega, Madison, WI)
by PCR using Accuprime Super Mix I (Invitrogen, Carlsbad, CA) and
the primers AscI hRLuc Fwd and NotI hRluc Rev. These two primers
have the following sequence and carry restriction sites for subsequent
cloning: AscI hRLuc Fwd: 5′-ACT GAC GGC GCG CCA TGG CTT
CCA 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 was
subcloned into pCR2.1-TOPO (Invitrogen). The resulting plasmid was
digested with AscI and NotI, and the excised fragment (hRluc-Neo) was
ligated using T4 DNA ligase into I389luc-ubi-neo/NS3-3′/ET digested
with the same enzymes. The resulting vector, pCon1/SG-hRlucNeo (G
+I+T), was sequenced to confirm the correct orientation and sequence
of 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 were
determined by measurement of absorbance at 260 nm, and integrity was
verified by 0.8% agarose gel electrophoresis and ethidium bromide
staining. Ten micrograms of in vitro transcribed pCon1/SG-hRlucNeo
(G+I+T) RNA was electroporated into 4 × 106 Huh7-Lunet cells as
described previously.45 Briefly, electroporated cells were plated onto 100
mm cell culture dishes. Twenty-four hours after plating, the media was
replaced with propagation media supplemented with 1.0 mg/mL of
G418 (selection lasted for approximately 3 weeks). G418-resistant
clones were isolated and expanded. HCV replication was quantified
using a commercial Renilla luciferase assay (Promega) per the
manufacturer’s instructions. Clones with the highest luciferase signal-
to-background ratios were selected for validation in high-throughput
antiviral susceptibility assays. The final clonal cell line selected for GT1b
antiviral studies was designated 1b-Rluc-2.
The EC50 values were determined as the testing compound
concentration that caused a 50% decrease in HCV replicon luciferase
signal. EC50 values were obtained using Pipeline Pilot 5.0 software
package (Accelrys, San Diego, CA) by nonlinear regression fitting of
experimental data.
Competitive Protein Binding Assay. Human plasma and cell-
culture medium containing 10% fetal bovine serum (CCM) were spiked
with the test compound at a final concentration of 2 μM. Spiked plasma
(1 mL) and CCM (1 mL) were placed into opposite sides of the
assembled dialysis cells, which are separated by a semipermeable
membrane. The dialysis cells were rotated slowly in a 37 °C water bath
for the time necessary to reach equilibrium. Postdialysis plasma and
CCM weights were measured, and the test compound concentrations in
plasma and CCM were determined with LC/MS/MS.36
Metabolic Stability. Metabolic stability in vitro was determined
using pooled hepatic microsomal fractions (final protein concentration
of 0.5 mg/mL) at a final test compound concentration of 3 μM. The
reaction was initiated by the addition of an NADPH-regenerating
system. Aliquot of 25 μL of the reaction mixture were transferred at
various time points to plates containing a quenching solution. The test
compound concentration in the reaction mixture was determined with
LC/MS/MS. Hepatic intrinsic clearance was calculated as described
previously by Obach,47 and the predicted clearance was calculated using
the well-stirred liver model without protein restriction.
Metabolic stability was also determined in cryopreserved hepatocytes
using tritiated test compounds. The incubation mixture contained 1 ×
106 hepatocytes/mL and 1 μM tritiated test compound (2.5 μCi). The
incubation was carried out with gentle shaking at 37 °C under a humid
atmosphere of 95% air/5% CO2 (v/v). Aliquots of 50 μL were removed
after 0, 1, 3, and 6 h and added to 100 μL of quenching solution. The
samples were analyzed on a flow scintillation radio detector coupled to
an HPLC system. The metabolites were quantified on the basis of the
peak areas from the radio detector, with the cell-free control samples
used as a reference. Metabolic stabilities in hepatocytes were determined
by measuring the rate of disappearance of the test compound as the
percent of total peak areas of the formed radiolabeled metabolites and
the test compound.
Replicon Antiviral Assays. To determine compound GT1 antiviral
activities, either 1a-57C-RlucP or 1b-Rluc-2 replicon cells were plated at
2000 cells per well in 384-well plates (Greiner Bio-One, Monroe, NC;
cell-culture treated). Compounds were 3-fold serially diluted in DMSO
and added to the cells using an automated instrument (Biotek μFlow
Workstation; 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 a
negative (solvent; no inhibition) control, and a combination of three
HCV inhibitors, including a protease inhibitor, an NS5A inhibitor, and a
nucleoside inhibitor, was used at concentrations >100× EC50 as a
positive control (100% inhibition). Plates were incubated for 3 days at
37 °C in an atmosphere of 5% CO2 and 85% humidity. Culture medium
was aspirated with a Biotek ELX405 plate washer. Twenty microliters of
Dual-Glo luciferase buffer (Promega) was added to each well of the plate
with a Biotek μFlow Workstation. The plate was incubated for 10 min at
room temperature. Twenty microliters of a solution containing a 1:100
mixture of Dual-Glo Stop & Glo substrate (Promega) and Dual-Glo
Stop & Glo buffer (Promega) was added to each well with a Biotek
μFlow Workstation. The plate was incubated at room temperature for
10 min before the luminescence signal was measured with an Envision
plate reader (PerkinElmer, Waltham, MA).
Pharmacokinetics. Pharmacokinetic studies were performed in
̈
̈
male naıve Sprague−Dawley(SD) rats, non-naıve beagle dogs, and
cynomolgus monkeys (three animals per dosing route) following federal
and Institutional Animal Care and Use Committee (IACUC) guidelines.
Intravenous (IV) administration was dosed via infusion over 30 min in a
vehicle containing 5% ethanol, 20% PEG400, and 75% water (pH
adjusted to 3.0 with HCl). Oral dosing was administered by gavage in a
vehicle containing 5% ethanol, 45% PEG 400, and 50% of 50 mM citrate
buffer, pH 3. Blood samples were collected over a 24 h period postdose
into Vacutainer tubes containing EDTA-K2. Plasma was isolated, and
the concentration of the test compound in plasma was determined with
LC/MS/MS after protein precipitation with acetonitrile.
Noncompartmental pharmacokinetic analysis was performed on
plasma concentration data to calculate pharmacokinetic parameters
using the software program WinNonLin (version 5.0.1).
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic procedures and spectroscopic data. This material is
available free of charge via the Internet at http://pubs.acs.org.
The anti-HCV replication activity was determined by the
luminescence signal generated from the reporter Renilla luciferase of
the HCV replicon. The percentage inhibition of the HCV replicon was
calculated using eq 1.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 650-522-1727. E-mail: john.link@gilead.com.
⎛
⎞
⎟
⎠
XC − MB
MD − MB
percent inhibition = 100 1 −
⎜
Notes
⎝
(1)
The authors declare the following competing financial
interest(s): The authors are employees of Gilead Sciences
except for J.S., T.K., E.M., and M.C., who were employed at
Gilead Sciences during this work. All authors are shareholders in
Gilead Sciences.
.where XC is the luminescence signal from compound-treated well, MB is
the average luminescence signal from the positive control wells (100%
inhibition), and MD is the average luminescence signal from DMSO-
treated wells.
K
dx.doi.org/10.1021/jm401499g | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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ACKNOWLEDGMENTS
■
We thank Kathy Brendza and Lorendana Serafini for obtaining
HRMS data.
ABBREVIATIONS USED
■
AUCinf, area under the curve from time zero to infinity; AUClast,
area under the curve from time zero to the last measurable
concentration; %F, bioavailability; C24hr, plasma concentration at
24 h; Cmax, maximum plasma concentration; CL, systemic
clearance; CV, coefficient of variation; DAA, direct-acting
antiviral; dppf, 1,1′-bis(diphenylphosphino)ferrocene; GT1,
hepatitis C virus genotype 1; GT1a, hepatitis C virus genotype
1a, a subtype of hepatitis C virus genotype 1; GT1b, hepatitis C
virus 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 residence
time; n.d., not determined; NS3, hepatitis C virus nonstructural
protein 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, sustained
viral response to treatment regimen 12 weeks after therapy; tmax
time after administration of a drug when the maximum plasma
concentration is reached; Vss, volume of distribution
,
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