Hepatitis C replication inhibitors that target the viral NS4B protein

Article abstract of DOI:10.1021/jm400125h

We describe the preclinical development and in vivo efficacy of a novel chemical series that inhibits hepatitis C virus replication via direct interaction with the viral nonstructural protein 4B (NS4B). Significant potency improvements were realized through isosteric modifications to our initial lead 1a. The temptation to improve antiviral activity while compromising physicochemical properties was tempered by the judicial use of ligand efficiency indices during lead optimization. In this manner, compound 1a was transformed into (+)-28a which possessed an improved antiviral profile with no increase in molecular weight and only a modest elevation in lipophilicity. Additionally, we employed a chimeric "humanized" mouse model of HCV infection to demonstrate for the first time that a small molecule with high in vitro affinity for NS4B can inhibit viral replication in vivo. This successful proof-of-concept study suggests that drugs targeting NS4B may represent a viable treatment option for curing HCV infection.

Full text of DOI:10.1021/jm400125h

Article
pubs.acs.org/jmc
Hepatitis C Replication Inhibitors That Target the Viral NS4B Protein
John F. Miller, Pek Y. Chong, J. Brad Shotwell, John G. Catalano, Vincent W.-F. Tai, Jing Fang,
Anna L. Banka, Christopher D. Roberts, Michael Youngman,† Huichang Zhang, Zhiping Xiong,
Amanda Mathis,‡ Jeffery J. Pouliot, Robert K. Hamatake, Daniel J. Price, John W. Seal, III, Lisa L. Stroup,
Katrina L. Creech, Luz H. Carballo, Dan Todd, Andrew Spaltenstein, Sylvia Furst, Zhi Hong,
and Andrew J. Peat*
GlaxoSmithKline Research and Development, 5 Moore Drive, Research Triangle Park, North Carolina 27709, United States
S
* Supporting Information
ABSTRACT: We describe the preclinical development and in vivo efficacy of a novel chemical
series that inhibits hepatitis C virus replication via direct interaction with the viral nonstructural
protein 4B (NS4B). Significant potency improvements were realized through isosteric
modifications to our initial lead 1a. The temptation to improve antiviral activity while
compromising physicochemical properties was tempered by the judicial use of ligand efficiency
indices during lead optimization. In this manner, compound 1a was transformed into (+)-28a
which possessed an improved antiviral profile with no increase in molecular weight and only a
modest elevation in lipophilicity. Additionally, we employed a chimeric “humanized” mouse model
of HCV infection to demonstrate for the first time that a small molecule with high in vitro affinity
for NS4B can inhibit viral replication in vivo. This successful proof-of-concept study suggests that drugs targeting NS4B may
represent a viable treatment option for curing HCV infection.
However, the error-prone nature of the HCV polymerase
enzyme and the high rate of viral replication will likely
necessitate the combination of at least two drugs with differing
MoAs to provide effective treatment.30
INTRODUCTION
■
Infection with hepatitis C virus (HCV) is well recognized as a
global health issue that affects an estimated 170 million
individuals.1,2 HCV is the leading cause of chronic hepatitis,
liver cirrhosis, and hepatocellular carcinoma and accounts for
approximately 350 000 deaths each year.3,4 The costs associated
with HCV infection place tremendous burden on both the
patients and the healthcare system as a whole.5 Currently, the
standard of care (SoC) involves a combination of pegylated
interferon (IFN), ribavirin, and one of two protease inhibitors
recently approved by the FDA (telaprevir and boceprevir).6−8
Although the cure rates have improved dramatically, many
patients cannot tolerate IFN and remain untreated.9,10 In
addition, IFN is prohibitively expensive in many parts of the
world; thus, there exists a high unmet need to develop IFN-free
treatment regimens.
Tremendous progress has been made in the past decade in
the development of direct acting antivirals (DAAs) for the
treatment of HCV infection.11−15 The HCV genome encodes
for three structural and seven nonstructural (NS) proteins,16−19
several of which have been targeted for pharmacological
intervention. Compounds that directly inhibit the viral NS3/
4A20−22 and NS5B22−24 enzymes, as well as compounds with
an apparent NS5A25 mechanism of action (MoA), have proven
to be clinically effective in reducing viral replication and curing
HCV infection. The discovery of drugs with varying MoAs
enables combination therapy to combat viral resistance,
analogous to the highly active antiretroviral therapy
(HAART) used for the treatment of HIV.26,27 For the first
time, drug cocktails containing oral DAAs show the potential to
cure HCV infection and eliminate the need for IFN.28,29
We recently reported31−33 on a novel series of substituted
imidazo[1,2-a]pyridines that bind to the viral nonstructural
protein 4B (NS4B).34−37 Importantly, a correlation between
binding of NS4B and inhibition of HCV replication in vitro was
observed for this chemical series.31 Our initial lead optimization
effort yielded 1a (Figure 1), a compound with high affinity for
purified NS4B protein and low nanomolar activity against HCV
genotype 1a and 1b replicons (Table 1).31 The antiviral
potency and low dose pharmacokinetic (PK) profile in rat and
dog supported further progression; however, escalating oral
doses of 1a in rats failed to achieve the higher plasma drug
exposures required for preclinical safety studies. Furthermore,
in vitro resistance passaging experiments employing wild-type
HCV replicons revealed that single-point mutations within the
NS4B protein render the virus partially resistant to 1a.31 The
ultimate impact of the resistance mutations on antiviral efficacy
was difficult to predict given the lack of data pertaining to drugs
that bind to NS4B. To date, no clinical or in vivo efficacy data
have emerged to either validate NS4B as a viable target for
treating HCV infection or assess the impact of resistance within
a treatment paradigm.
Special Issue: HCV Therapies
Received: January 25, 2013
© XXXX American Chemical Society
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In Vivo PK and Efficacy of Prodrug 2 in PXB Mice. The
species specificity of the hepatitis C virus has hindered the
development of preclinical animal models of infection.38
Chimpanzees are susceptible to HCV infection;39 however,
we chose to pursue a model involving chimeric “humanized”
mice. The PXB mice are uPA/SCID mice with livers that have
been repopulated with 70−90% human hepatocytes and, as a
result, can be infected with human liver pathogens such as
HCV and HBV.40 We decided to use the PXB model to test
whether an HCV antiviral agent that interacts with NS4B can
inhibit viral replication in vivo.
Prodrug 2 was selected for use in the efficacy study, in part
because of the high plasma drug exposures obtained in the rat
PK studies. To ensure that the prodrug would also perform well
in the efficacy model, the pharmacokinetic profile of 2 was
determined in PXB mice (n = 3/group) prior to infection with
HCV (Figure 2). Oral administration of 2 at doses of 10 and 30
Figure 1. Plasma drug exposures of 1a in rat following po
administration of 300 mg/kg 1a or prodrugs 2−6.
Herein, we describe efforts to (1) improve the PK attributes
of the lead molecule 1a to support preclinical development, (2)
demonstrate that small molecules interacting with NS4B can
inhibit HCV replication in vivo, and (3) identify a new
compound with an improved viral resistance profile relative to
1a.
Figure 2. Plasma concentrations of 1a over 12 h in PXB mice achieved
upon po administration of prodrug 2 at 10 and 30 mg/kg. The dotted
line represents the serum shifted EC90 (14 ng/mL) for 1a versus the
HCV genotype 1a viral strain used to infect the mice.
IN VIVO PROFILING OF THE LEAD 1A
■
Evaluation of Prodrugs of 1a. The preclinical develop-
ment of 1a was contingent upon overcoming its inherently
poor aqueous solubility (11 μg/mL in FaSSIF) and obtaining
higher plasma drug exposures. For example, a 300 mg/kg
suspension dose of crystalline 1a, wet-bead milled to maximize
surface area for faster dissolution, resulted in relatively low
exposure in rat (AUC = 11.3 μg·h/mL, Figure 1). Alternative
dosing formulations failed to achieve the desired outcome, and
therefore, a prodrug strategy was devised. We selected two
chemotypes (phosphate 2 and esters 3−6, Figure 1) that are
cleaved by different enzymes in vivo (phosphatases and
esterases, respectively). For the esters, we also chose varying
degrees of polarity and ionic character, from nonpolar (3) to
polar (4) to acidic (5) to basic (6). The prodrugs 2−6 were
dosed at 300 mg/kg (po) in rats, and the plasma concentrations
of 1a were determined. A side-by-side comparison found that
the phosphate prodrug 2 delivered the highest plasma drug
exposure of 1a (Figure 1) which is likely due to its high FaSSIF
solubility (>1000 μg/mL). Importantly, none of the phosphate
prodrug itself (i.e., 2) was detected in the blood of the rats,
indicating rapid and efficient cleavage of the phosphate moiety
by phosphatases present in the gut wall. In contrast, the esters
gave lower plasma drug levels of 1a and, in some cases,
significant plasma concentrations of uncleaved prodrug. As an
added benefit, the phosphate prodrug 2 formed a stable,
crystalline solid that was easy to synthesize and therefore was
selected for all forthcoming in vivo studies.
mg/kg resulted in a linear increase in plasma drug exposures of
1a (AUC0−12h = 15 and 66 μg·h/mL, respectively). Both dose
groups achieved 12 h plasma concentrations (C12h) of 1a well
above the serum shifted EC90 of 29 nM (14 ng/mL) that was
determined against the HCV genotype 1a viral strain used to
infect the PXB mice. We opted for b.i.d. dosing to ensure that
the plasma drug levels of 1a remained above the serum shifted
EC90 throughout the 7-day study, thereby reducing the
possibility for viral resistance that can occur with monotherapy
treatment regimens. A repeat dose study with compound 2 was
also conducted in PXB mice prior to infection to assess
tolerability. The compound was well tolerated with no evidence
of accumulation or induction of metabolism over 7 days of
treatment (10 and 30 mg/kg b.i.d.). Furthermore, analysis of
liver tissue 12 h after dosing on day 7 revealed modest (5- to 7-
fold) partitioning of 1a into the target organ (see Supporting
Information).
PXB mice were infected with HCV genotype 1a wild-type
virus and after 5−8 weeks were treated with prodrug 2 (n = 4/
group). Both the 10 and 30 mg/kg (b.i.d.) doses of prodrug 2
produced a rapid and robust drop (∼1 log) in HCV viral
mRNA titers (viral load) within 12 h of treatment (Figure 3).
The maximum viral load reductions (∼4 log units) occurred by
day 4 for both dose groups; however, a dose-dependent
decrease in viral load was apparent by day 7 (relative to baseline
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Figure 4. Structural modification of the initial NS4B compound 1a led
to analogues based on alternative scaffolds (7a−9a).
the 7-CF3-pyrazolo[1,5-a]pyridine derivatives, and therefore,
we explored several synthetic routes toward this scaffold.
Syntheses of the Pyrazolo[1,5-a]pyridine Analogue
(7a). The initial route to 7a required the synthesis of carboxylic
acid 14 and relied on the de novo construction of the
pyrazolopyridine ring system (Scheme 1). Alkylation of
Figure 3. HCV viral RNA titers in PXB mice infected with HCV
genotype 1a virus and treated for 7 days with prodrug 2 (10 or 30 mg/
kg b.i.d.) or the positive control HCV-796 (100 mg/kg b.i.d.).
on day 0). The 30 mg/kg dose maintained viral suppression
from day 4 through day 7, while the lower dose group (10 mg/
kg) showed evidence of viral breakthrough at the end of the
study. As a positive control, we included the non-nucleoside
NS5B polymerase inhibitor HCV-796 which has demonstrated
efficacy in human HCV clinical trials and in a HCV-infected
chimeric mouse model similar to the one described here.41 In
our study, all doses of prodrug 2 reduced HCV viral titers to a
greater extent than did HCV-796, which was given at 100 mg/
kg b.i.d. and produced a maximal viral load drop of ∼2.2 log
units (in good agreement with published results). A more
detailed analysis of the data from this study, including the
results from a 100 mg/kg b.i.d. dose group and the
characterization of resistant viruses, is forthcoming.42 As far
as we are aware, this represents the first in vivo proof of
concept study associated with HCV inhibitors that bind to
NS4B.
The high plasma exposure achieved with prodrug 2 also
enabled the progression of 1a into rat 7-day safety studies
during which an adverse cardiovascular finding was identified
that led to its termination. Encouraged by the robust antiviral
response in the HCV mouse model, a backup effort was
initiated to identify a replacement that would remove the
cardiovascular risk and other limitations associated with 1a.
More specifically, we wanted to avoid the need for a prodrug
and to improve upon the viral resistance profile, especially
against the H94N and V105M mutated viruses found to be
resistant to 1a.31 A substantial number of compounds (>1500)
in the imidazopyridine series had already been profiled as a
result of our initial lead optimization effort, and therefore, we
chose to pursue alternative chemical series.43
Scheme 1. Initial Route to the Pyrazolopyridine Acid 14 and
Final Target Compounds 7a−e
a
a
Reagents and conditions: (a) MeI, K2CO3, DMF (88%); (b) MSH,
CH2Cl2 (62%); (c) DMAD, K2CO3, DMF (49%); (d) H2SO4, 90 °C;
(e) AlBr3, EtSH; (f) H2SO4, MeOH (73% over three steps); (g)
PhN(Tf)2 , DIPEA, CH2 Cl2 (92%); (h) cPrB(OH)2 ,
PdCl2(dppf).CH2Cl2, K3PO4, 1,4-dioxane, 90 °C (84%); (i) NCS,
DMF, 60 °C (94%); (j) 0.5 N NaOH, MeOH, 50 °C (quant); (k) for
7a, 1-(trans-4-hydroxycyclohexyl)-2-piperazinone hydrochloride, T3P,
DIPEA, DMF (99%).
commercially available 2-(trifluoromethyl)pyridine-4-ol 10
with MeI afforded the corresponding methyl ether in high
yield. N-Amination of the pyridine was problematic because of
the low reactivity of the pyridyl nitrogen, a consequence of the
electron-withdrawing o-CF3 substituent. Ultimately, the use of a
highly reactive N-aminating reagent, O-mesitylensufonylhy-
droxylamine (MSH),44,45 was required to effect the desired
transformation.46 Intermediate 11 underwent a 1,3-dipolar
cycloaddition reaction with dimethylacetylene dicarboxylate
(DMAD) to give the pyrazolopyridine 12 in low-to-moderate
yields.47,48 Heating 12 in the presence of H2SO4 led to ester
hydrolysis followed by selective decarboxylation at C(3) of the
pyrazolopyridine.49 Cleavage of the methyl ether followed by
Fischer esterification of the acid gave 13 in 73% yield for the
three-step sequence.
The remaining functional groups, cyclopropyl at C(5) and Cl
at C(3), were installed in a straightforward manner (Scheme 1).
Treatment of 13 with N-phenyltrifluoromethanesulfonamide
gave the corresponding triflate, which underwent Pd-catalyzed
cross-coupling with cyclopropylboronic acid in high yield. The
addition of N-chlorosuccinimide followed by saponification of
the ester gave the required pyrazolopyridine acid 14, which was
IMPROVING POTENCY AGAINST RESISTANT
VIRUSES
■
Identifying Alternative Core Scaffolds. Initially, we
examined isosteric replacements of the imidazopyridine core
and focused on new chemical series based on pyrazolopyridine
7, benzimidazole 8, and benzofuran 9 scaffolds (Figure 4). The
syntheses of analogues 7a−9a were undertaken to allow for a
direct comparison to 1a. The corresponding benzimidazole and
benzofuran analogues (8a and 9a, respectively) were
synthesized in a straightforward manner (see Supporting
Information). However, few reports existed for the preparation
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coupled to the piperazinone amine fragment (a, Figure 4) to
afford the final target compound 7a.
well suited for substrate 17a,b, as the C−H bond at C(5) is the
least sterically hindered. In addition, we anticipated that the
electron-deficient nature of the pyrazolopyridine ring system of
17a,b would facilitate C−H bond activation. By employment of
an Ir catalyst with bispinacolborane, the regioselective
borylation of 17a was accomplished in quantitative yield
(Scheme 3). The boronate ester 20 could be isolated and
The first-generation route (Scheme 1) provided gram
quantities of the pyrazolopyridine acid 14 in 10 steps (14%
overall yield) and was sufficient for establishing structure−
activity relationships (SARs) within the new series. However,
several synthetic issues needed to be addressed prior to the
preparation of multigram batches required for in vivo studies.
First, maintaining a reliable commercial supply of 10 proved to
be capricious. In addition, the required protection/deprotection
of the 4-hydroxyl group was inefficient. As our interest in the
pyrazolopyridine scaffold increased during the course of lead
optimization, an alternative route was sought to overcome these
limitations.
Scheme 3. Alternative Synthesis of 14 Based on Ir-Catalyzed
a
C−H Activation
A search for inexpensive starting materials that were reliably
available in bulk from commercial vendors led us to 2-
(trifluoromethyl)pyridine 15 as a key precursor. Treatment
with MSH gave the N-aminated pyridine 16 that underwent
1,3-dipolar cycloaddition with DMAD to form 17a in low yield
(30%) for the two-step sequence (Scheme 2). Although
a
Reagents and conditions: (a) [Ir(OMe)COD]2, 4,4′-di-tBu-2,2′-
Scheme 2. Alternative Routes to 7-CF3-pyrazolopyridine
17a,b Amenable to Large Scale Production
a
bipyridine; (Bpin)2, hexanes, 80 °C (quant); (b) H2O2; (c) Na2SO3;
(d) aq NaOH (89%, four steps); (e) HCl, HOAc, 90 °C (76%); (f)
MeOH, H2SO4 (quant); (g) Tf2O, DIEA; (h) cPrB(OH)2, Pd(OAc)2,
CataCXium A, K3PO4, toluene/water, 80 °C; (i) 1 M NaOH, then 1
M HCl (86%, three steps); (j) NCS, DCE, 50 °C (69%).
served as a versatile intermediate in Pd-catalyzed cross-coupling
reactions. Alternatively, oxidation of the B−C bond afforded
the phenol 21 that was converted to intermediate 14 in six
steps.
Examination of Alternative Core Series 7a−9a.
Analogues 7a−9a were compared to 1a in the NS4B binding
and HCV replicon assays (Table 1). Substitution of the
imidazopyridine core with other related bicyclic ring systems
resulted in a modest improvement in binding affinity for NS4B
protein. The pyrazolopyridine (7a) and benzofuran (9a) cores
yielded the most significant gains in binding affinity, being
about 4- to 13-fold more potent than the imidazopyridine (1a)
core. The N-methylbenzimidazole (8a) scaffold was also well
tolerated. The increase in binding affinity for NS4B (genotype
1b) protein directly translated to an increase in HCV (genotype
1b) replicon activity. For example, the pyrazolopyridine 7a and
benzofuran 9a analogues were about 4-fold and 9-fold,
respectively, more potent than 1a in the genotype 1b replicon
assay. Improvements of similar magnitude were also observed
against the genotype 1a variant. No cellular toxicity was
observed with these compounds following 2 days of treatment
at 50 μM in Huh7 cells (see Supporting Information).
The antiviral activities of the modified core analogues 7a−9a
were also determined against stable NS4B-mutated replicons
that were found to be resistant to 1a (Table 1). In general, the
increased activity in the binding assay was reflected in the
resistant replicon assays. For example, the analogue with the
highest affinity for NS4B (9a) was also the most active in the
H94N and V105M replicon assays. Pyrazolopyridine 7a also
showed a significant improvement over 1a against the H94N
mutated replicon, while 8a showed little difference.
a
Reagents and conditions: (a) MSH, CH2Cl2; (b) DMAD, K2CO3,
D M F ( 3 0 % o v e r t w o s t e p s ) ; ( c ) 0 . 7 e q u i v o f
(TMP)2Zn·2MgCl2·2LiCl, THF, −10 °C, then I2; (d) CuI,
FSO2CF2CO2Me, DMF, 80 °C (86% over two steps).
hundreds of grams of pyrazolopyridine 17a were prepared by
this method, the overall efficiency of the process was low. In
addition, the large scale preparation of MSH was a safety
concern due to the known thermal instability of the
reagent.50,51 Seeking a safe and efficient route to kilogram
quantities of 14, we decided to avoid the N-amination/MSH
reaction and begin with the pyrazolopyridine ring system
already intact. No 7-(trifluoromethyl)pyrazolopyridine ana-
logues were available from commercial vendors; however, the
des-CF3 analogue 18 could be purchased and represented an
attractive starting point. After some optimization, we found that
18 could be efficiently converted to 17b by a two-step
procedure involving selective zincation/iodination at C(7)52,53
followed by Cu-mediated trifluoromethylation of the iodo
intermediate 19 (Scheme 2).54,55 The zincation/iodination/
trifluoromethylation procedure has been carried out success-
fully on 300 g of starting material 18, which provided 17b in
86% isolated yield.56,57
With the 7-CF3 group installed on the pyrazolopyridine core
(17a,b), the introduction of functionality at C(5) was now
required. Iridium-catalyzed activation of aromatic C−H bonds
has proven to be a useful method for the functionalization of
related aromatic ring systems.58−60 The selectivity of the
reaction is dominated by steric effects and therefore appeared
Our effort to optimize antiviral activity was balanced with a
desire to maintain the favorable physicochemical properties
associated with 1a. The increasing MW and lipophilicity of drug
candidates over the past two decades are cited as underlying
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Table 1. NS4B Binding Affinity and HCV Replicon Activity
of Alternate Core Series (7a−9a)
Table 2. Physicochemical Properties and Drug Efficiency
Indices for Analogues 1a and 7a−9a
a
cLogP
b
compd MW
(cLogD)
ChromLogD
BEI LLEcalc LLEmeas
1a
7a
8a
9a
485
485
464
485
3.1
3.3
2.7
4.1
3.5
4.1
4.3
5.1
15.1
16.4
16.4
17.3
4.2
4.6
4.9
4.3
3.8
3.8
3.3
3.3
a
Calculated using the ACD/logD software described in ref 66.
Number of replicates is ≥6. For description of the assay see
b
Supporting Information and ref 65.
conclusions about the development risk associated with
benzimidazole 8a and benzofuran 9a when using cLogP or
MW as a surrogate for measured lipophilicity caution against
the exclusive use of calculated values in guiding lead
optimization decisions.
Comparison of Imidazo- versus Pyrazolopyridine
Series. The low nanomolar activity of 7a against the H94N
mutant replicon and its favorable physicochemical properties
prompted further examination of the pyrazolopyridine core.
Therefore, we combined acid 14 with several previously
optimized amine tail fragments (b−e, Table 3) for which
comparable imidazopyridine analogues had been made.31−33
Analysis of the matched molecular pairs revealed that in all
cases, the pyrazolopyridine analogue was more potent than the
Table 3. NS4B Binding Affinity and HCV Replicon Activity
for Matched Molecular Pairs 1/7b−e
a
For description of the assays and confidence intervals see Supporting
Information and ref 31.
causes for late-stage attrition.61 To help reverse this trend,
various drug efficiency indices have been developed,62 which
allow chemists to relate structural modifications to changes in
both the potency and the development risks associated with a
molecule’s physicochemical properties.63 We utilized the
lipophilic ligand efficiency (LLE)64 index as a guide during
lead optimization, as it emphasizes the development challenges
associated with increasing lipophilicity even in the context of
isosteric replacements. Although the use of calculated proper-
ties allows for prospective analysis and prioritization of target
molecules, we prefer to assess the development risk of an
existing asset based on experimentally determined measure-
ments, in this case, a chromatographically measured log D
designated below as ChromLogD (see Supporting Informa-
tion).65
The relative binding affinities and replicon potencies of core
modifications 7a−9a were therefore considered in light of their
impact on lipophilicity (ChromLogD, Table 2). While all three
core replacements afforded improvements in binding affinity
relative to 1a, the respective LLEmeas (=pIC50(NS4B Binding)
−
ChromLogD) values of 7a−9a indicate that the benzimidazole
8a and benzofuran 9a represent less desirable structural
changes relative to the pyrazolopyridine 7a in that their
increased binding affinity was coupled with excessive increases
in lipophilicity. For comparison, several other commonly
considered drug efficiency indices are reported in Table 2,
including binding efficiency index (BEI = (pIC50/MW) ×
1000) and lipophilic ligand efficiency determined using
cLogP 66 (LLEcalc = pIC50 − cLogP). The discrepant
a
For description of the assays and confidence intervals see Supporting
Information and ref 31.
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corresponding imidazopyridine derivative with respect to NS4B
binding (Table 3). On average, the pyrazolopyridine core
enhanced binding affinity by 0.5 log units over the
imidazopyridine scaffold. All of the tail fragments combined
with the pyrazolopyridine core showed high affinity for NS4B
protein (IC50 < 40 nM), including piperazinone 7b, piperidinol
7c, piperidine 7d, and piperazine 7e. A significant degree of
structural diversity was tolerated at the peripheral end of the tail
(compare 7c with 7e); however, the need for a hydrogen bond
acceptor in the central portion of the tail became apparent.
Optimal binding could be maintained with the H-bond
acceptor positioned in either the central ring (examples 7b
and 7c) or the terminal substituent (examples 7d and 7e).
As seen in the initial survey of core replacements (Table 1),
the increased binding affinity of the pyrazolopyridine series
translated to improved replicon activity (Table 3). For three of
the four matched pairs (1c−7c, 1d−7d, and 1e−7e), the
genotype 1b replicon activity of the pyrazolopyridine analogue
improved 2- to 3-fold over the imidazopyridine counterpart, in
line with the difference observed in the NS4B binding assay. A
notable exception was the 1b−7b pair, for which a 20-fold
difference in replicon genotype 1b activity was observed
between the two compounds with a 15-fold difference in
binding affinity. The same trend was apparent in the H94N and
V105M replicon assays. However, the low nanomolar binding
activity (IC50 = 4 nM) for 7b is at the upper limit of the assay
(i.e., highest resolvable potency) and therefore may not be
accurately determined, which could account for the apparent
discrepancy between the binding and replicon activities. The
exceptional replicon potency appeared unique to the [3.1.0]-
bicyclohexane tail as analogue 7b remains one of the most
potent antiviral compounds we have seen within either
chemical class.
structural similarity with the 4-cyclohexanol derivatives known
to bind NS4B (e.g., 1a and 7a). Unfortunately, the desired
amine fragment appeared unstable under acidic conditions and
further efforts were abandoned.67 There were stability concerns
and a lack of literature precedent related to amine fragment 24,
and therefore, attention shifted to the 2-hydroxybicyclohexane-
amines 25.
Syntheses of [3.1.0]Bicyclohexan-2-ol Tail Analogues.
Allylic oxidation of the pendent cyclopentene ring of 26
occurred opposite the bulky piperazinone substituent, thereby
affording 27 as a racemic mixture (Scheme 4).68 The resulting
Scheme 4. Syntheses of [3.1.0]Bicyclohexan-2-ol Derivatives
a
28a/b, 30a/b, 33a/b
Improving PK of Pyrazolopyridine Series. The antiviral
activity of 7b against the resistant replicons met our desired
criteria, but the increased lipophilicity (ChromLogD = 6.4) was
not optimal as reflected in the significantly eroded LLEmeas
(2.0). In addition, compound 7b exhibited high in vivo
clearance in rats (Cl = 70 mL min−1 kg−1). An analysis of the
metabolites of 7b generated in vitro from rat and human
hepatocytes showed rapid oxidation of the terminal bicyclohex-
ane substituent. We had found previously that the metabolism
of an unsubstituted cyclohexane ring could be reduced by
introducing a hydroxyl group, which ultimately led to 1a.31 To
determine if a similar tactic would improve the metabolic
clearance of 7b, a series of [3.1.0]bicyclohexanol analogues 22
were envisioned that required the preparation of amine
fragments 23−25 (Figure 5). Initially, analogue 23 was deemed
most attractive given the lack of stereogenic centers and
a
Reagents and conditions: (a) SeO2, 450:10:1 dioxane/water/
pyridine, 105 °C (44%); (b) Et2Zn, CH2I2, DCM, 0 °C to rt (86%
for 28, 37% for 33a); (c) chiral SFC chromatography; (d) 1:1 TFA/
DCM, then water (90%); (e) 1:1 TFA/DCM, then 10% aq NaHCO3
(quant).
hydroxyl group directed the cyclopropanaton to the syn-face of
the olefin to give ( )-28,69,70 which upon resolution by chiral
supercritical fluid chromatography (SFC) yielded enantiomers
(+)-28a and (−)-28b. The relative stereochemistry was
1
assigned by two-dimensional H NMR spectroscopy, and the
absolute stereochemistry was confirmed as (S,S,S,S)-(+)-28a by
single crystal X-ray crystallography (see Supporting Informa-
tion).
While exploring the SAR within the [3.1.0]bicyclohex-2-ol
series, we noted that treatment of (−)-28b with POCl3 resulted
in the efficient epimerization of the 2-hydroxyl group (Scheme
4). The inversion appears to occur via the cyclic intermediate
29, which upon aqueous workup ring-opens to give the
diastereomeric alcohol 30. Other strong acids, such as TFA or
Figure 5. Envisioned [3.1.0]bicyclohexanol derivative 22 (and the
requisite amine fragments 23−25) designed to improve PK.
F
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HCl, also promoted epimerization. By exploitation of this
serendipitous finding, the enantiomerically pure alcohols
(S,R,S,S)-30a and (R,S,R,R)-30b were prepared in high yield
from analogues 28a and 28b, respectively. Taking further
advantage of this pathway, we were able to access another
diastereomer (33) by epimerizing the hydroxyl group before
installing the cyclopropyl moiety. Treating the silyl-protected
ether 31a or 31b with TFA, followed by addition of water,
afforded the epimerized alcohol 32a or 32b, respectively. A
hydroxyl-directed cyclopropanation using Et2Zn and CH2I2
gave the desired enantiomers (R,R,S,R)-33a and (S,S,R,S)-
33b, respectively. Ultimately, we were successful in preparing
six of the eight possible stereoisomers (28a/b, 30a/b, 33a/b).
The low yield of the SeO2 allylic oxidation and the need for
chiral resolution of the resulting racemic mixture necessitated
the development of a more efficient synthesis. By use of the
Evans oxazolidinone, an asymmetric anti-aldol reaction between
34 and cinnamaldehyde installed two of the four desired
stereocenters with a high diastereomeric ratio (19:1 dr) to give
35 (Scheme 5).71 Ring-closing metathesis of the diene 35 with
ing Information). The 11-step route (from 34) provided access
to >100 g of (+)-28a in 35% overall yield.
Comparison of the [3.1.0]Bicyclohexanol Tail Ana-
logues. All six of the [3.1.0]bicyclohexanol isomers displayed
low nanomolar affinity for binding NS4B, which was predictive
of the potent antiviral activity observed in the genotype 1b
replicon assay (Table 4). Interestingly, much larger differences
in activity were observed with the NS4B resistant replicons. For
Table 4. NS4B Binding Affinity and HCV Replicon Activity
of [3.1.0]Bicyclohexan-2-ol Derivatives 28, 30, and 33
a
Scheme 5. Asymmetric Synthesis of (+)-28a
a
Reagents and conditions: (a) trans-cinnamaldehyde, MgCl2, NaSbF6,
TMSCl, TEA (88%); (b) third generation Grubbs catalyst, PhMe, rt
(94%); (c) Et2Zn, CH2I2, DCM (89%); (d) TBSOTf, 2,6-lutidine,
DCM (96%); (e) LiOH, H2O2, THF/water (88%); (f) DPPA, TEA,
PhMe, 80 °C; (g) KOTMS, THF (95% over two steps); (h) Ns-
glycine, EDC, HOBt, DIPEA, DMF (quant); (i) 1,2-dibromoethane,
Cs2CO3, DMF, rt (82%); (j) PhSH, K2CO3, MeCN (84%); (k)
compound 14, 50% T3P/EtOAc, DIPEA, THF, 0 °C to rt (quant); (l)
TBAF, THF, 0 °C to rt (85%).
the third generation Grubb’s catalyst afforded the cyclopentenol
36 in 94% yield. The hydroxyl-directed cyclopropanation of the
olefin followed by protection of the alcohol and cleavage of the
chiral auxiliary gave intermediate 37 as a single diastereomer in
>95% enantiomeric excess. Curtius rearrangement of the
carboxylic acid with DPPA and hydrolysis of the isocyanate
with KOTMS afforded the amine 38 in 95% yield.72 The
piperazinone was formed in two steps from 38 via amide
formation with Nosyl-protected glycine followed by alkylation/
ring closure with dibromoethane. Removal of the Nosyl group
with thiophenol73 gave 39, which was subsequently coupled to
the pyrazolopyridine acid 14. Lastly, the silyl protecting group
was removed with TBAF, thereby affording (+)-28a as a single
isomer in >95% ee, the absolute stereochemistry of which was
confirmed by single-crystal X-ray crystallography (see Support-
a
For description of the assays and confidence intervals see Supporting
Information and ref 31.
G
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example, isomers 30a, 33a, and 33b were essentially inactive
against the H94N and V105M resistant replicons while the
(S,S,S,S) isomer [(+)-28a] exhibited low nanomolar activity.
The (R,R,R,R)-(−)-28b also displayed good antiviral activity,
although it was 5- to 7-fold less potent than its (+)-enantiomer.
With respect to replicon activity, an anti-orientation of the 2-
hydroxyl relative to the piperazinone ring appeared optimal
(e.g., 28a/b), whereas a syn-configuration was detrimental
irrespective of the cyclopropane orientation (e.g., 30a/b and
33a/b).
clearance relative to the unsubstituted bicyclohexane (7b)
derivative across all species (Table 7). The effect was most
a
Table 7. In Vitro Predicted Clearance of 7b and (+)-28a
compd
human
monkey
dog
rat
mouse
7b
18
<5
34
11
30
<5
41
24
80
<5
(+)-28a
a
Clearance units (mL min−1 kg−1). For description of the assay see
Supporting Information.
The impact of these modifications on physiochemical
properties was also examined and compared to 1a (Table 5).
pronounced in human, dog, and mouse hepatocytes, in which
(+)-28a appeared most stable. A more modest improvement
(2- to 3-fold) was seen in rat and monkey hepatocytes. Separate
in vitro studies showed that (+)-28a did not inhibit cytochrome
P450 isozymes (IC50 > 20 μM versus 3A4, 1A2, 2C9, 2C19,
2D6) or generate reactive metabolites in a glutathione-trapping
assay (rat and human microsomes).
Table 5. Physicochemical Properties and Drug Efficiency
Indices for Analogues 1a, 7b, and (+)-28a
a
cLogP
Chrom
b
compd
MW
(cLogD)
LogD
BEI LLEcalc LLEmeas
1a
485
467
483
3.1
4.0
2.8
3.5
6.4
4.6
15.1
18.0
16.8
4.2
4.4
5.3
3.8
2.0
3.5
The improved in vitro metabolic stability of (+)-28a versus
7b translated to reduced clearance in vivo (Table 8). For
7b
(+)-28a
a
f
Calculated using the ACD/logD software described in ref 66.
Number of replicates is ≥6. For description of the assay see
Table 8. In Vivo Pharmacokinetic Profiles of (+)-28a
b
a
b
iv
po
Supporting Information and ref 65.
Cl
Vdss
t1/2
AUC0−α
Introduction of the 2-hydroxyl in (+)-28a dramatically
improved the measured lipophilicity relative to 7b (Chrom-
LogD = 4.6 versus 6.4, respectively). When compared to 1a,
(+)-28a shows a significant increase in the measured log D;
however, the increase in lipophilicity appears roughly
commensurate with the increase in binding affinity, as the
LLEmeas values are comparable (3.8 and 3.5 for 1a and 28a,
respectively). The impact of the increased lipophilicity of
(+)-28a relative to 1a is not negligible, however, and was
reflected in an overall greater binding to plasma proteins across
species (Table 6). The degree of plasma protein binding
species (mL min−1 kg−1
)
(L/kg)
(h)
(μg·h/mL)
F (%)
c
mouse
19
0.6
2.0
0.7
0.7
0.4
2.0
2.0
4.0
1.0
3.1
25
12
d
rat
25
92
e
dog
6.0
3.0
>100
97
e
cyno
25
a
b
Dosed iv at 1 mg/kg in DMSO/20% HP-β-CD (10:90). Dosed
orally at 5 mg/kg in DMSO/20% HP-β-CD (10:90). iv (n = 4); po (n
= 4). iv (n = 3); po (n = 2). iv (n = 3); po (n = 3). Further details
available in Supporting Information.
c
d
e
f
example, the rat clearance of (+)-28a was 3-fold lower than that
of 7b (25 versus 70 mL min−1 kg−1, respectively), which
correlated with the 2-fold improvement in predicted clearance
observed in rat hepatocyte studies. Overall, (+)-28a displayed
low-to-moderate clearance across four species and >90%
bioavailability in all species except mouse. High plasma drug
exposures were also achieved with low doses (5 mg/kg) in both
dog and monkey. The improved pharmacokinetic profile
coupled with the favorable FaSSIF solubility resulted in
increased plasma exposure upon escalation of dose that enabled
progression to 7-day safety studies without the need for a
prodrug.
Table 6. Plasma Protein Binding (%) Across Species for 1a
and (+)-28a
a
compd
human
monkey
dog
rat
mouse
1a
83
94
77
91
66
93
80
96
ND
97
(+)-28a
a
For description of the assay see Supporting Information.
observed for (+)-28a is still considered moderate when
compared to many drug candidates. Ironically, there was a
30-fold improvement in the FaSSIF solubility of (+)-28a
relative to 1a (0.31 versus 0.011 mg/mL) despite the increased
lipophilicity.
Efficiency indices based on calculated values are also
presented for comparison (BEI and LLEcalc). As seen
previously, the efficiency values derived from MW and
calculated log P/D values may underestimate the risks
associated with developing a compound such as 7b and should
be interpreted with caution.
Pharmacokinetic Profile of (+)-28a. The initial justifica-
tion for pursuing the complex chiral tail of (+)-28a was the
hypothesis that introducing a hydroxyl group would improve
metabolic stability. Therefore, the in vitro metabolism of
(+)-28a was examined in hepatocytes from five species.74,75
Consistent with the findings in the cyclohexane series, adding
the 2-hydroxyl substituent (28a) resulted in a lower predicted
CONCLUSION
■
For the first time, a small molecule that inhibits HCV
replication via an apparent NS4B mechanism of action has
demonstrated in vivo efficacy in a chimeric “humanized” mouse
model of HCV infection. Over a 7-day course of treatment, a
prodrug of compound 1a produced rapid and dose-dependent
decreases in HCV serum RNA that surpassed that of a clinically
validated positive control, HCV-796. The successful proof-of-
concept study indicates that drugs targeting NS4B may
represent viable treatment options for curing HCV infection
that could further expand combination regimens.
Our efforts to optimize antiviral potency while maintaining
favorable physicochemical properties prompted us to examine
isosteric analogues of our lead 1a. Replacement of the
imidazopyridine core with an analogous pyrazolopyridine
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to afford 17a (6.2 g, 30% over two steps) as a tan solid. 1H NMR (400
MHz, DMSO-d6): δ 8.40 (d, J = 8.8 Hz, 1 H) 7.91 (d, J = 6.6 Hz, 1 H)
7.77−7.84 (m, 1 H) 3.95 (s, 3 H), 3.87 (s, 3H). ES-LCMS m/z: 303
(M + 1).
scaffold yielded a modest, but consistent, increase in both
NS4B binding affinity and antiviral activity against HCV
replicons. More significant improvements were realized with
isosteric modifications in the amide (tail) portion of the series,
whereby bridging the terminal cyclohexyl substituent to form a
[3.1.0]bicyclohexane ring afforded an analogue with IC50 < 1
nM in the replicon assays. The temptation to improve antiviral
activity at the cost of increased MW or lipophilicity was
tempered by the judicial use of efficiency indices such as BEI
and LLE during lead optimization. However, a direct
consequence of this approach was the need to construct
densely functionalized fragments as exemplified by a unique
chiral 2-bicyclohexanolamine. In this manner, the original lead
1a was transformed into (+)-28a which possessed an improved
antiviral profile against the resistant NS4B-mutated replicons
and low nanomolar activity against HCV genotypes 3a, 4a, and
5a.43 Compound (+)-28a had no increase in molecular weight
and only a modest elevation in lipophilicity relative to 1a, which
manifested in improved FaSSIF solubility and PK. The need for
a prodrug was thereby eliminated, and (+)-28a progressed into
7-day preclinical safety studies, the results of which will be
published in due course.
5-Hydroxy-7-(trifluoromethyl)pyrazolo[1,5-a]pyridine-2,3-
dicarboxylic Acid (21). A solution of 17a (40.0 g, 0.134 mol),
[Ir(OMe)COD]2 (2.63 g, 3.97 mmol), 4,4′-di-tert-butyl-2,2′-bipyr-
idine (2.06 g, 7.70 mmol), and bis(pinacolato)diboron (33.6 g, 132
mmol) in degassed cyclopentyl methyl ether (320 mL) was heated to
80 °C for 1.3 h. The mixture was allowed to cool to room temperature
and immersed in an ice bath. To the mixture was slowly added 30%
aqueous hydrogen peroxide (27 mL, 625 mmol) while keeping the
temperature at or below 25 °C. After 2 h, 15% aqueous sodium
bisulfite (222 mL) was added, keeping the temperature under 10 °C.
The reaction mixture was left overnight, cooled to 0 °C, and filtered.
The filtrate phases were separated, and the organic phase was isolated.
A solution of 50% aqueous NaOH (37 g) diluted to 232 mL with
water was added. The solution was heated to 80 °C for 2 h. The
solution was cooled to room temperature, and the phases were
separated. The aqueous phase was cooled to 5 °C. HCl (5 equiv) was
added and the mixture held at room temperature overnight. The
suspension was cooled to 5 °C and the solid collected by filtration.
The solid was washed with water and dried at reduced pressure at 60
1
°C overnight to afford 21 (34 g, 89%). H NMR (400 MHz, DMSO-
d6): δ 11.28 (s, 1H), 7.83 (s,1H), 7.30 (s, 1H).
3-Chloro-5-cyclopropyl-7-(trifluoromethyl)pyrazolo[1,5-a]-
pyridine-2-carboxylic Acid (14). (A) 5-Hydroxy-7-
(trifluoromethyl)pyrazolo[1,5-a]pyridine-2-carboxylic Acid. Com-
pound 21 (388 g, 1.34 mol) was added to a reactor along with
AcOH (1552 mL, 27.10 mol) and 12 M aqueous HCl (217 mL, 2.60
mol). The mixture was heated to 90 °C and stirred until the reaction
was determined to be complete by HPLC (4 h). The mixture was
cooled to 20 °C, and water (3880 mL) was added to the reactor. The
suspension was then cooled to 10 °C and stirred overnight. The
resulting solid was collected by filtration, washed with water, and dried
under vacuum at 50 °C for 3 days to afford the title compound (248 g,
76%). 1H NMR (400 MHz, DMSO-d6): δ 13.20 (br s, 1 H), 10.91 (s,
1 H), 7.24 (d, J = 2.3 Hz, 1 H), 7.18 (d, J = 2.3 Hz, 1 H), 6.95 (s, 1 H).
ES-LCMS m/z: 245 (M − 1).
(B) Methyl 5-Hydroxy-7-(trifluoromethyl)pyrazolo[1,5-a]pyridine-
2-carboxylate (13). 5-Hydroxy-7-(trifluoromethyl)pyrazolo[1,5-a]-
pyridine-2-carboxylic acid (0.260 g, 1.06 mmol) was dissolved in
MeOH (5 mL), and concentrated sulfuric acid (3 drops) was added.
The solution was heated under reflux for 1.5 h. Water was added, and
the mixture was extracted with EtOAc. The organic phase was dried
over sodium sulfate and the solvent evaporated to afford 13 as a light
yellow solid (0.28 g, quant). 1H NMR (400 MHz, methanol-d4) δ ppm
7.16 (d, J = 2.4 Hz, 1 H), 7.09 (d, J = 2.3 Hz, 1 H), 6.95 (s, 1 H), 3.95
(s, 3 H). ES-LCMS m/z: 261 (M + 1).
EXPERIMENTAL SECTION
■
All commercially obtained solvents and reagents were used as received.
The purity of the final compounds was determined to be ≥95% by 1H
NMR and LCMS; structural asignments were consistent with the
spectroscopic data. 1H NMR spectra were taken on a Varian (Agilent)
Inova 400 NMR spectrometer. Chemical shifts are reported in parts
per million (ppm, δ) using the residual solvent line as a reference.
Splitting patterns are designated using the following abbreviations: s,
singlet; d, doublet; t, triplet; dd, doublet of doublet; m, multiplet; br,
broad. Coupling constants (J) are reported in hertz (Hz). Mass
spectrometric analyses and compound purity determinations were
conducted on a Waters Acquity UPLC system (Phenomenex Kinetex
column at 40 °C, mobile phase of water with 0.2% v/v formic acid and
acetonitrile with 0.15% v/v formic acid) and Waters Acquity SQD with
alternating positive/negative electrospray ionization scanning from
125 to 1000 amu, with a scan time of 105 ms and an interscan delay of
20 ms. High resolution mass spectrometric analysis was performed on
a Waters qTOF Premiere mass spectrometer using flow injection
operating in W mode. Chiral analytical HPLC was performed on an
Agilent 1100 analytical system. Optical rotations were measured using
a Rudolph Research Analytical Autopol V polarimeter.
1-Amino-2-(trifluoromethyl)pyridinium 2,4,6-Trimethylben-
zenesulfonate (16). A solution of 2-(trifluoromethyl)pyridine (10.0
g, 68.0 mmol) and O-mesitylenesulfonylhydroxylamine (19.0 g, 88.0
mmol; see Mendiola et al. Org. Process Res. Dev. 2009, 13, 263) in
DCM (170 mL) was stirred at room temperature for 4 days, at which
point the reaction was still incomplete. An additional portion (7.3 g,
34 mmol) of O-mesitylenesulfonylhydroxylamine was added, and the
reaction mixture was stirred for another day. No further progress was
observed by NMR. The reaction mixture was evaporated to dryness to
afford crude 16 which was used without further purification. 1H NMR
(400 MHz, DMSO-d6): δ 9.19 (d, J = 6.1 Hz, 1 H) 8.64−8.73 (m, 1
H) 8.58−8.64 (m, 1 H) 8.34−8.42 (m, 1 H) 8.26 (s, 2 H) 2.17 (s, 9
H).
(C) 5-Cyclopropyl-7-(trifluoromethyl)pyrazolo[1,5-a]pyridine-2-
carboxylic Acid. DIPEA (176 mL, 1.01 mol) was added to a solution
of 13 (105 g, 0.400 mol) in DCM (1.05 L). The solution was cooled
to 0 °C, and triflic anhydride (75 mL, 0.44 mol) was added at a rate to
maintain the temperature below 10 °C. Once the reaction was
determined to be complete by HPLC, 13% aqueous sodium chloride
was added and the solution was stirred for 20 min. Stirring was
stopped, and the layers were separated. The upper aqueous layer was
1
removed. The organic layer was distilled to /3 volume, and toluene
(1.1 L) was added. Cyclopropylboronic acid (69.2 g, 0.810 mol),
CataCXium A (3.61 g, 10.0 mmol), potassium phosphate tribasic (258
g, 1.21 mol), and water (474 mL) were then added. Lastly, palladium
acetate (2.26 g, 10.0 mmol) was added and the mixture was heated to
80 °C. The reaction was monitored by HPLC until complete, and then
the mixture was cooled to 25 °C. With stirring, additional water (316
mL) was added, and after 10 min the layers were allowed to separate.
The lower aqueous layer was removed and discarded. The organic
layer was diluted with THF (316 mL) and 1 N aqueous NaOH (1106
mL) and heated to 50 °C. When the saponification was determined to
be complete by HPLC, the mixture was cooled to 25 °C and the layers
were allowed to separate. The bottom aqueous layer was isolated and
Dimethyl 7-(Trifluoromethyl)pyrazolo[1,5-a]pyridine-2,3-di-
carboxylate (17a). Dimethyl acetylenedicarboxylate (16.8 mL, 136
mmol) was added by dropwise addition to a 0 °C solution of 16
(crude mixture from previous step, 68 mmol theoretical) and
potassium carbonate (18.8 g, 136 mmol) in DMF (170 mL). Air
was bubbled through the reaction mixture for a few minutes, and
stirring was continued at room temperature overnight. Water (400
mL) was added, and the solution was stirred for 30 min. The resulting
solid was collected by vacuum filtration, washed with water, dried, and
purified by flash chromatography (silica gel, 25−33% EtOAc/hexane)
I
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acidified using 1 N aqueous HCl (1185 mL) and cooled to 10 °C. The
resulting solid was collected by filtration, washed with water, and dried
under vacuum at 50 °C to give the title compound (86 g, 86%).
(D) 3-Chloro-5-cyclopropyl-7-(trifluoromethyl)pyrazolo[1,5-a]-
pyridine-2-carboxylic Acid (14). A mixture of 5-cyclopropyl-7-
(trifluoromethyl)pyrazolo[1,5-a]pyridine-2-carboxylic acid (100 g,
0.370 mol) in 1 L of DCE was heated at reflux and 200 mL of
DCE removed by distillation to ensure dryness of the substrate. The
solution was cooled to 25 °C. N-Chlorosuccinimide (73 g, 0.46 mol)
was added to the reactor, and the solution was heated at 50 °C. When
the reaction was complete by HPLC, the mixture was cooled to 25 °C,
and 1 M aqueous NaOH (1 L) was added. The mixture was stirred for
2 h, and the layers were separated. While stirring, the aqueous layer
was acidified to pH 1 with 6 M aqueous HCl. The suspension was
stirred and cooled to 10 °C. The solid was collected by filtration,
washed with water (500 mL), and dried under vacuum at 50 °C to
standing. 1H NMR (400 MHz, CDCl3): δ 7.25−7.40 (m, 3 H), 7.18−
7.24 (m, 2 H), 6.00−6.08 (m, 1 H), 5.76 −5.85 (m, 1 H), 5.00−5.08
(m, 1 H), 4.65−4.75 (m, 1 H), 4.20−4.35 (m, 2 H), 3.90−4.00 (m, 1
H), 3.29 (dd, J = 13.4 Hz, 3.2 Hz, 1 H), 2.90−3.15 (br s, 1 H), 2.75−
2.90 (m, 3 H). ES-LCMS m/z: 270 (M + 1-H2O).
(D) (4R)-3-{[(1S,2S,3S,5S)-2-Hydroxybicyclo[3.1.0]hex-3-yl]-
carbonyl}-4-(phenylmethyl)-1,3-oxazolidin-2-one. A solution of 36
(330 mg, 1.15 mmol) in DCM (15 mL) was cooled in an ice bath and
treated with 1 M diethylzinc in hexane (5.74 mL, 5.74 mmol) by
dropwise addition. After the mixture was stirred at 0 °C for 20 min,
diiodomethane (0.936 mL, 11.5 mmol) was added dropwise. The
resulting cloudy solution was stirred at 0 °C for 20 min and then
allowed to warm to room temperature. After 6 h, the reaction mixture
was quenched with saturated aqueous ammonium chloride and
extracted with EtOAc. The combined organic extracts were washed
with brine, dried over sodium sulfate, and concentrated to dryness at
reduced pressure. The crude material was purified by flash
chromatography (silica gel, 0−40% EtOAc/hexane) to give the title
compound (308 mg, 89%) as a light brown viscous oil. 1H NMR (400
MHz, CDCl3): δ 7.25−7.40 (m, 3 H), 7.20−7.24 (m, 2 H), 4.84−4.94
(m, 1 H), 4.64−4.74 (m, 1 H), 4.15−4.25 (m, 2 H), 3.57−3.66 (m, 1
H), 3.28 (dd, J = 13.5 Hz, 3.3 Hz, 1 H), 2.84 (dd, J = 13.4 Hz, 9.4 Hz,
1 H), 2.00−2.25 (m, 2 H), 1.60−1.70 (m, 1 H), 1.40−1.48 (m, 1 H),
0.65−0.73 (m, 1 H), 0.45−0.55 (m, 1 H). ES-LCMS m/z: 284 (M + 1
− H2O).
1
constant weight to afford 14 (78 g, 69%) as a white solid. H NMR
(400 MHz, DMSO-d6): δ 13.70 (br s, 1 H), 7.71 (s, 1 H), 7.47 (br s, 1
H), 2.17−2.29 (m, 1 H), 1.05−1.14 (m, 2 H), 0.94−1.03 (m, 2 H).
ES-LCMS m/z: 305 (M + 1).
Asymmetric Synthesis of 4-{[3-Chloro-5-cyclopropyl-7-
(trifluoromethyl)pyrazolo[1,5-a]pyridin-2-yl]carbonyl}-1-
[(1S,2S,3S,5S)-2-hydroxybicyclo[3.1.0]hex-3-yl]-2-piperazinone
(28a). (A) (4R)-3-(4-Pentenoyl)-4-(phenylmethyl)-1,3-oxazolidin-2-
one (34). To a solution of (4R)-4-(phenylmethyl)-1,3-oxazolidin-2-
one (2.50 g, 14.0 mmol) in THF (40 mL) under nitrogen at −78 °C
was added dropwise 1.6 M n-BuLi in hexane (9.17 mL, 14.7 mmol),
and the resulting mixture was stirred at −78 °C for 1 h. Then a
solution of 4-pentenoyl chloride (1.60 mL, 14.1 mmol) in THF (10
mL) was added dropwise. After being stirred at −78 °C for 1 h, the
reaction mixture was allowed to warm to room temperature and stirred
overnight. After being diluted with water, the mixture was extracted
with EtOAc (2 × 60 mL). The combined EtOAc extracts were washed
with brine, dried over sodium sulfate, filtered, and concentrated to
dryness at reduced pressure. The crude residue was purified by flash
chromatography (silica gel, 0−20% EtOAc/hexane) to give 34 (3.29 g,
91%) as a light yellow oil. 1H NMR (400 MHz, CDCl3): δ 7.25−7.39
(m, 3 H), 7.20−7.23 (m, 2 H), 5.82−5.95 (m, 1 H), 5.12 (dd, 1 H, J =
17.1 Hz, 1.7 Hz, 1 H), 5.05 (dd, J = 10.1 Hz, 1.2 Hz, 1 H), 4.65−4.74
(m, 1 H), 4.15−4.25 (m, 2 H), 3.31 (dd, J = 13.3 Hz, 3.3 Hz, 1 H),
2.96−3.18 (m, 2 H), 2.76 (dd, J = 13.3 Hz, 9.6 Hz, 1 H), 2.42−2.54
(m, 2 H).
(B) (4R)-3-[(2S,3S,4E)-3-Hydroxy-5-phenyl-2-(2-propen-1-yl)-4-
pentenoyl]-4-(phenylmethyl)-1,3-oxazolidin-2-one (35). A mixture
of 34 (3.29 g, 12.7 mmol), magnesium chloride (0.121 g, 1.27 mmol),
NaSbF6 (0.985 g, 3.81 mmol), triethylamine (3.54 mL, 25.4 mmol),
trans-cinnamaldehyde (1.94 mL, 15.2 mmol), and TMSCl (2.43 mL,
19.0 mmol) in EtOAc (58 mL) was stirred at room temperature for 17
h. The mixture was diluted with EtOAc and filtered to remove solids.
The filtrate was concentrated to small volume and then diluted with
MeOH (50 mL) and a small amount of EtOAc. Following treatment
with TFA (0.10 mL) the solution was stirred at room temperature for
1 h and then concentrated to dryness at reduced pressure. The residue
was purified by flash chromatography (silica gel, 0−20% EtOAc/
hexane) to give 35 (4.38 g, 88%) as a yellow semisolid. 1H NMR (400
MHz, CDCl3): δ 7.25−7.44 (m, 8 H), 7.13−7.20 (m, 2 H), 6.69 (d, J
= 15.8 Hz, 1 H), 6.33 (dd, J = 15.9 Hz, 6.0 Hz, 1 H), 5.75−5.88 (m, 1
H), 5.12 (d, J = 17.2 Hz, 1 H), 5.05 (d, J = 10.2 Hz, 1 H), 4.63−4.76
(m, 1 H), 4.50−4.60 (m, 1 H), 4.25−4.35 (m, 1 H), 4.13−4.20 (m, 2
H), 3.25 (dd, J = 13.5 Hz, 3.3 Hz, 1 H), 2.82−3.00 (m, 1 H), 2.40−
2.64 (m, 3 H). ES-LCMS m/z: 374 (M + 1 − H2O).
(E) (4R)-3-[((1S,2S,3S,5S)-2-{[(1,1-Dimethylethyl)(dimethyl)silyl]-
oxy}bicyclo[3.1.0]hex-3-yl)carbonyl]-4-(phenylmethyl)-1,3-oxazoli-
din-2-one. To a stirred solution of (4R)-3-{[(1S,2S,3S,5S)-2-
hydroxybicyclo[3.1.0]hex-3-yl]carbonyl}-4-(phenylmethyl)-1,3-oxazo-
lidin-2-one (308 mg, 1.02 mmol) and 2,6-lutidine (0.475 mL, 4.09
mmol) in DCM (5 mL) at 0 °C was added TBSOTf (0.587 mL, 2.56
mmol) under nitrogen. The resulting mixture was stirred at 0 °C for
30 min, then room temperature for 1 h. Following addition of MeOH
(0.30 mL), the mixture was poured into water and extracted with ether
(2×). The combined ether extracts were washed with brine, dried over
sodium sulfate, filtered, concentrated, and purified by flash
chromatography (silica gel, 0−20% EtOAc/hexane) to give the title
1
compound (406 mg, 96%) as a colorless oil. H NMR (400 MHz,
CDCl3): δ 7.25−7.38 (m, 3 H), 7.17−7.22 (m, 2 H), 5.02−5.10 (m, 1
H), 4.63−4.73 (m, 1 H), 4.08−4.22 (m, 2 H), 3.57−3.66 (m, 1 H),
3.24 (dd, J = 13.4 Hz, 3.3 Hz, 1 H), 2.77 (dd, J = 13.4 Hz, 9.1 Hz, 1
H), 2.26 (dd, J = 11.9 Hz, 7.8 Hz, 1 H), 1.70−1.82 (m, 1 H), 1.42−
1.50 (m, 1 H), 1.30−1.38 (m, 1 H), 0.90 (s, 9 H), 0.70−0.75 (m, 1 H),
0.40−0.47 (m, 1 H), 0.14 (s, 3 H), 0.08 (s, 3 H). ES-LCMS m/z: 416
(M + 1).
(F) (1S,2S,3S,5S)-2-{[(1,1-Dimethylethyl)(dimethyl)silyl]oxy}-
bicyclo[3.1.0]hexane-3-carboxylic Acid (37). To a solution of (4R)-
3-[((1S,2S,3S,5S)-2-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}bicyclo-
[3.1.0]hex-3-yl)carbonyl]-4-(phenylmethyl)-1,3-oxazolidin-2-one (405
mg, 0.974 mmol) in THF (6 mL) and water (2 mL) at 0 °C was added
30% aqueous hydrogen peroxide (0.796 mL, 7.80 mmol) dropwise,
followed by addition of a solution of lithium hydroxide monohydrate
(167 mg, 3.90 mmol) in water (1 mL). After being stirred for 1 h at 0
°C, the reaction mixture was stirred at room temperature overnight.
The excess hydrogen peroxide was quenched by the addition of
saturated aqueous sodium bisulfite (4 mL). The mixture was treated
with 0.1 N NaOH (0.2 mL) and washed with ether (40 mL). The
aqueous layer was acidified to pH 3 with 1 M aqueous potassium
hydrogen sulfate and extracted with EtOAc (3 × 40 mL). The
combined organic extracts were washed with brine, dried over sodium
sulfate, filtered, and concentrated to give 37 (220 mg, 88%) as a
1
(C) (4R)-3-{[(1S,2S)-2-Hydroxy-3-cyclopenten-1-yl]carbonyl}-4-
(phenylmethyl)-1,3-oxazolidin-2-one (36). A solution of 35 (495
mg, 1.26 mmol) in toluene (60 mL) was degassed three times with
nitrogen and treated with Grubbs third generation catalyst (65 mg,
0.089 mmol). The solution was degassed two more times with
nitrogen and stirred at room temperature overnight. The solution was
then concentrated to dryness at reduced pressure and the residue
subjected to flash chromatography (silica gel, 0−40% EtOAc/hexane)
to give 36 (340 mg, 94%) as a dark brown oil, which solidified upon
colorless oil. H NMR (400 MHz, CDCl3): δ 4.65−4.75 (m, 1 H),
2.30−2.40 (m, 1 H), 1.96−2.10 (m, 2 H), 1.41−1.50 (m, 1 H), 1.32−
1.40 (m, 1 H), 0.91 (s, 9 H), 0.56−0.62 (m, 1 H), 0.41−0.49 (m, 1 H),
0.13 (s, 3 H), 0.11 (s, 3 H).
(G) ((1S,2S,3S,5S)-2-{[(1,1-Dimethylethyl)(dimethyl)silyl]oxy}-
bicyclo[3.1.0]hex-3-yl)amine (38). To a solution of 37 (10.0 g, 39.0
mmol) in toluene (180 mL) were added TEA (6.52 mL, 46.8 mmol)
and diphenylphosphorylazide (8.43 mL, 39.0 mmol). The solution was
heated to 80 °C for 5 h and then cooled to 0 °C in an ice bath. To this
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dx.doi.org/10.1021/jm400125h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
solution was added potassium trimethylsilanolate (10.0 g, 78.0 mmol)
in THF (90 mL) and the mixture stirred at room temperature for 1 h.
The mixture was quenched by addition of 15% aqueous citric acid and
then basified by addition of aqueous sodium hydroxide. The resulting
mixture was extracted with ether. The ether layer was separated, and
the aqueous layer was evaporated to a small volume and extracted
again with ether. The combined ether solutions were washed with a
small amount of saturated brine, dried over sodium sulfate, filtered,
and evaporated to afford 38 (8.86 g, 95%) as a pale yellow oil, which
was used without further purification. 1H NMR (400 MHz, CDCl3): δ
3.92−4.00 (m, 1 H), 2.60−2.70 (m, 1 H), 2.00−2.10 (m, 1 H), 1.50−
1.60 (m, 1 H), 1.32−1.38 (m, 1 H), 1.20−1.29 (m, 1 H), 0.92 (s, 9 H),
0.56−0.60 (m, 1 H), 0.30−0.38 (m, 1 H), 0.12 (s, 3 H), 0.11 (s, 3 H).
(H) N1-((1S,2S,3S,5S)-2-{[(1,1-Dimethylethyl)(dimethyl) silyl]oxy}-
bicyclo [3.1.0]hex-3-yl)-N2-[(4-nitrophenyl)sulfonyl]glycinamide. N-
[(4-Nitrophenyl)sulfonyl]glycine (10.1 g, 39.0 mmol), 38 (8.86 g, 39.0
mmol), HOBT (6.56 g, 42.9 mmol), and DIPEA (8.17 mL, 46.8
mmol) were combined in DMF (187 mL). To this solution was added
EDC (8.22 g, 42.9 mmol), and the reaction mixture was stirred at
room temperature for 5 h. The mixture was diluted with EtOAc and
poured into water. The organic layer was separated and washed with
saturated aqueous sodium bicarbonate followed by brine. After drying
over sodium sulfate, the solution was concentrated to dryness at
reduced pressure. The resulting solid was triturated with ether, filtered,
and dried to afford the title compound in quantitative yield, which was
used without further purification. ES-LCMS m/z: 470 (M + 1).
(I) 1-((1S,2S,3S,5S)-2-{[(1,1-Dimethylethyl)(dimethyl)silyl]oxy}-
bicyclo[3.1.0]hex-3-yl)-4-[(4-nitrophenyl)sulfonyl]-2-piperazinone.
N1-((1S,2S,3S,5S)-2-{[(1,1-Dimethylethyl)(dimethyl)silyl]oxy}-
bicyclo[3.1.0]hex-3-yl)-N2-[(4-nitrophenyl)sulfonyl]glycinamide
(18.3 g, 38.9 mmol), 1,2-dibromoethane (26.8 mL, 311 mmol), and
cesium carbonate (50.7 g, 156 mmol) were combined in DMF (200
mL) and stirred at room temperature for 6 h. The mixture was diluted
with EtOAc, washed with water, brine, dried over sodium sulfate, and
concentrated to dryness at reduced pressure. The resulting solid was
triturated with hexane/ether, filtered, and dried. The filtrate was
concentrated at reduced pressure and the residue purified by flash
chromatography (silica gel, EtOAc/hexane) to afford a solid, which
when combined with the triturated solid, gave the title compound
(23.6 mL, 39.6 mmol). The mixture was allowed to warm to room
temperature and stirred for 2 h. Saturated aqueous sodium bicarbonate
was then added. The mixture was extracted with EtOAc (3×). The
combined organic extracts were washed with brine, dried over sodium
sulfate, and concentrated at reduced pressure to afford the title
compound as a yellow oil in quantitative yield. This material was used
in the next step without further purification. ES-LCMS m/z: 597 (M +
1).
(L) 4-{[3-Chloro-5-cyclopropyl-7-(trifluoromethyl)pyrazolo[1,5-a]-
pyridin-2-yl]carbonyl}-1-[(1S,2S,3S,5S)-2-hydroxybicyclo[3.1.0]hex-
3-yl]-2-piperazinone (28a). TBAF (1 M in THF, 55.3 mL, 55.3
mmol) was added to a solution of 4-{[3-chloro-5-cyclopropyl-7-
(trifluoromethyl)pyrazolo[1,5-a]pyridin-2-yl]carbonyl}-1-
((1S,2S,3S,5S)-2-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}bicyclo-
[3.1.0]hex-3-yl)-2-piperazinone (22.0 g, 36.8 mmol) in THF (350
mL) at 0 °C under nitrogen. The reaction mixture was allowed to
warm to room temperature and stirred for 4 h. Most of the THF was
removed at reduced pressure, and the residue was partitioned between
EtOAc and saturated aqueous sodium bicarbonate. The phases were
separated, and the aqueous solution was extracted with additional
portions of EtOAc (3 × 100 mL). The combined EtOAc solutions
were dried over sodium sulfate and concentrated to dryness at reduced
pressure. The residue was purified by flash chromatography [silica gel,
20−90% EtOAc (containing 5% MeOH)/hexane] to give 28a as a
1
white foam (15.1 g, 85%). Mp 108−111 °C. H NMR (400 MHz,
CDCl3): δ 7.38−7.47 (m, 1 H), 7.06 (br s, 1 H), 4.30−4.63 (m, 4 H),
4.15−4.22 (m, 1 H), 3.85−3.98 (m, 1 H), 3.40−3.54 (m, 2 H), 1.91−
2.05 (m, 3 H), 1.53−1.64 (m, 1 H), 1.42 (br s, 1 H), 1.13−1.22 (m, 2
H), 0.84−0.90 (m, 2 H), 0.75−0.84 (m, 1 H), 0.46−0.58 (m, 1 H).
HRMS m/z calcd for C22H22ClF3N4O3 (M + 1): 483.1411. Found:
483.1414. LCMS purity: >98%. The enantiopurity was determined as
>99% by chiral analytical HPLC [ChiralPak AD-H column (4.6 mm ×
250 mm, 5 μm), isocratic at 70% EtOH/hexane]. The retention time
was in agreement with that for 28a obtained by preparative chiral SFC
of the racemate (see Supporting Information) from the synthetic route
shown in Scheme 4 (tR of 5.9 and 3.5 min for 28a and 28b,
respectively). Specific rotation [α] was +40°, measured at 589 nm and
25 °C. A 50 mg portion of the product was crystallized as follows. The
material was slurried in ethyl ether and the mixture sonicated until the
solid had dissolved. The solution was seeded with a trace of crystalline
28a and the mixture stirred while the ether was allowed to slowly
evaporate to dryness. The resulting solid was dried under vacuum. Mp
134−137 °C. The material was determined to be crystalline by both
polarized light microscopy and PXRD analysis.
1
(15.8 g, 82%) as a yellow solid. H NMR (400 MHz, CDCl3): δ 8.42
(d, J = 8.8 Hz, 2 H), 7.99 (d, J = 8.5 Hz, 2 H), 4.62−4.70 (m, 1 H),
3.84−3.96 (m, 1 H), 3.79 (d, J = 16.3 Hz, 1 H), 3.69 (d, J = 16.4 Hz, 1
H), 3.37−3.44 (m, 3 H), 3.20−3.34 (m, 1 H), 1.90−2.00 (m, 1 H),
1.75−1.83 (m, 1 H), 1.32−1.40 (m, 1 H), 1.24−1.31 (m, 1 H), 0.83
(s, 9 H), 0.64−0.73 (m, 1 H), 0.38−0.48 (m, 1 H), 0.06 (s, 3 H), 0.00
(s, 3 H). ES-LCMS m/z: 496 (M + 1).
ASSOCIATED CONTENT
■
(J) 1-((1S,2S,3S,5S)-2-{[(1,1-Dimethylethyl)(dimethyl)silyl]oxy}-
bicyclo[3.1.0]hex-3-yl)-2-piperazinone (39). A solution of 1-
((1S,2S,3S,5S)-2-{[(1,1-dimethylethyl)(dimethyl)silyl]oxy}bicyclo-
[3.1.0]hex-3-yl)-4-[(4-nitrophenyl)sulfonyl]-2-piperazinone (15.6 g,
31.5 mmol) in MeCN (300 mL) was treated with thiophenol (9.72
mL, 94.0 mmol), followed by potassium carbonate (17.4 g, 126 mmol)
at room temperature under nitrogen. The reaction mixture was stirred
for 11 h, diluted with DCM (200 mL), filtered to remove solids, and
concentrated to dryness at reduced pressure. The residue was purified
by flash chromatography [silica gel, 0−5% MeOH (2 M in ammonia)/
S
* Supporting Information
Experimental details for the syntheses and spectroscopic
characterization of the compounds in this paper. This material
is available free of charge via the Internet at http://pubs.acs.org.
The X-ray crystal structure of compound (+)-28a has been
deposited at the Cambridge Crystallographic Data Centre
(disposition number CCDC 900754).
AUTHOR INFORMATION
1
■
DCM] to give 39 (8.2 g, 84%) as a light-yellow oil. H NMR (400
Corresponding Author
MHz, CDCl3): δ 4.75 (dd, J = 8.0 Hz, 5.1 Hz, 1 H), 3.98 (dt, J = 10.7
Hz, 8.0 Hz, 1 H), 3.52 (s, 2 H), 3.25−3.31 (m, 2 H), 3.01−3.14 (m, 2
H), 2.07 (td, J = 11.6 Hz, 4.9 Hz, 2 H), 1.86 (dd, J = 12.3 Hz, 7.8 Hz, 1
H), 1.35−1.43 (m, 1 H), 1.26−1.33 (m, 1 H), 0.88 (s, 9 H), 0.71−0.77
(m, 1 H), 0.42 (td, J = 7.9 Hz, 5.8 Hz, 1 H), 0.09 (s, 3 H), 0.06 (s, 3
H).
(K) 4-{[3-Chloro-5-cyclopropyl-7-(trifluoromethyl)pyrazolo[1,5-
a]pyridin-2-yl]carbonyl}-1-((1S,2S,3S,5S)-2-{[(1,1-dimethylethyl)-
(dimethyl)silyl]oxy}bicyclo[3.1.0]hex-3-yl)-2-piperazinone. DIPEA
(13.8 mL, 79.0 mmol) was added to a mixture of 14 (8.45 g, 27.7
mmol) and 39 (8.20 g, 26.4 mmol) in THF (250 mL) at room
temperature under nitrogen. The mixture was stirred for 30 min and
then cooled to 0 °C. To the solution was added 50% T3P/EtOAc
*E-mail: andy.j.peat@gsk.com. Phone: 919-483-6137.
Present Addresses
†For M.Y.: Lieber Institute for Brain Development, 855 N.
Wolfe Street, Baltimore, MD 21205, U.S.
‡For A.M.: Salix Pharmaceuticals, Inc., 8510 Colonnade Drive,
Raleigh, NC 27615, U.S.
Notes
The authors declare the following competing financial
interest(s): All of the authors are/were employees of
GlaxoSmithKline where this work was completed.
K
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(11) Asselah, T.; Marcellin, P. Direct Acting Antivirals for the
Treatment of Chronic Hepatitis C: One Pill a Day for Tomorrow.
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ACKNOWLEDGMENTS
■
The authors acknowledge the contributions of the following
GSK scientists to the manuscript: Todd Baughman (reactive
metabolite data), George Dorsey (NMR structure elucidation),
Douglas Minnick (vibrational circular dichroism spectroscopy),
Iris Paulus (chromatographic log D measurements and high
resolution mass spectrometry), Eric Wentz and Yang Zhao
(chromatographic purification), Greg Robertson and Tucker
Sheppard (solubility measurements).
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Acting Antiviral Agents for the Treatment of Hepatitis C Virus
Infection and Perspectives. Gut 2012, 61, i36−i46.
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ABBREVIATIONS USED
■
AcOH, acetic acid; (Bpin)2, bis(pinacolato)diboron; BEI,
binding efficiency index (=(pIC50/MW) × 1000); Chrom-
LogD, chromatographically measured log D; Cl, clearance;
DAA, direct acting antiviral; DIPEA, N,N-diisopropylethyl-
amine; DMAD, dimethylacetylene dicarboxylate; DPPA,
diphenylphosphorylazide; EDC, N-ethyl-N′-(3-
dimethylaminopropyl)carbodiimide hydrochloride; EtOAc,
ethyl acetate; HAART, highly active antiretroviral therapy;
HOBt, N-hydroxybenzotriazole; HP-b-CD, 2-hydroxypropyl-b-
cyclodextrin; IFN, pegylated interferon; KOTMS, potassium
trimethylsilanoate; LLE, lipophilic ligand efficiency; LLEcalc
,
=pIC50 − cLogP; LLEmeas, =pIC50(NS4B Binding) − ChromLogD;
MeCN, acetonitrile; MeOH, methanol; MSH, O-mesitylensu-
fonylhydroxylamine; NS, nonstructural; Ns, 4-nitrophenylsul-
fonyl; NS4B, nonstructural protein 4B; PXRD, powder X-ray
diffraction; SFC, supercritical fluid chromatography; SoC,
standard of care; T3P, 1-propanephosphonic anhydride;
TBSOTf, trimethylsilyl trifluoromethanesulfonate; TEA, trie-
thylamine; TFA, trifluoroacetic acid; TMSCl, trimethylsilyl
chloride
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Infection: Targeting the Non-Structural Proteins of HCV. Curr. Med.
Chem.: Anti-Infect. Agents 2002, 1, 163−176.
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Princ. Med. Chem. 2011, 50, 307−328.
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Recent Clin. Trials 2010, 5, 158−173.
(22) Kwo, P. Y.; Vinayek, R. The Therapeutic Approaches for
Hepatitis C Virus: Protease Inhibitors and Polymerase Inhibitors. Gut
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Journal of Medicinal Chemistry
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