Discovery of a novel series of potent non-nucleoside inhibitors of hepatitis C virus NS5B

Article abstract of DOI:10.1021/jm401266k

Hepatitis C virus (HCV) is a major global public health problem. While the current standard of care, a direct-acting antiviral (DAA) protease inhibitor taken in combination with pegylated interferon and ribavirin, represents a major advancement in recent years, an unmet medical need still exists for treatment modalities that improve upon both efficacy and tolerability. Toward those ends, much effort has continued to focus on the discovery of new DAAs, with the ultimate goal to provide interferon-free combinations. The RNA-dependent RNA polymerase enzyme NS5B represents one such DAA therapeutic target for inhibition that has attracted much interest over the past decade. Herein, we report the discovery and optimization of a novel series of inhibitors of HCV NS5B, through the use of structure-based design applied to a fragment-derived starting point. Issues of potency, pharmacokinetics, and early safety were addressed in order to provide a clinical candidate in fluoropyridone 19.

Full text of DOI:10.1021/jm401266k

Article
pubs.acs.org/jmc
Discovery of a Novel Series of Potent Non-Nucleoside Inhibitors of
Hepatitis C Virus NS5B
Ryan C. Schoenfeld,*,† David L. Bourdet, Ken A. Brameld, Elbert Chin, Javier de Vicente, Amy Fung,
Seth F. Harris, Eun K. Lee, Sophie Le Pogam, Vincent Leveque, Jim Li, Alfred S.-T. Lui, Isabel Najera,
Sonal Rajyaguru, Michael Sangi, Sandra Steiner, Francisco X. Talamas, Joshua P. Taygerly,
and Junping Zhao
Pharma Research & Early Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, New Jersey 07110, United States
S
* Supporting Information
ABSTRACT: Hepatitis C virus (HCV) is a major global public
health problem. While the current standard of care, a direct-acting
antiviral (DAA) protease inhibitor taken in combination with
pegylated interferon and ribavirin, represents a major advancement
in recent years, an unmet medical need still exists for treatment
modalities that improve upon both efficacy and tolerability. Toward
those ends, much effort has continued to focus on the discovery of
new DAAs, with the ultimate goal to provide interferon-free
combinations. The RNA-dependent RNA polymerase enzyme
NS5B represents one such DAA therapeutic target for inhibition
that has attracted much interest over the past decade. Herein, we
report the discovery and optimization of a novel series of inhibitors
of HCV NS5B, through the use of structure-based design applied to a
fragment-derived starting point. Issues of potency, pharmacokinetics, and early safety were addressed in order to provide a
clinical candidate in fluoropyridone 19.
that may ultimately be used in combination with each other as
part of an IFN-free drug cocktail.12,13
INTRODUCTION
■
Hepatitis C virus (HCV) represents a major global public health
problem. Over 150 million individuals suffer from chronic
infection, with over 350 000 deaths each year attributed to
hepatitis C related liver diseases such as cirrhosis and liver
cancer.1 While vaccines exist for some other hepatitis viruses,
there are none available for HCV. Up until 2011, standard of care
(SOC) treatment consisted of pegylated interferon (Peg-IFN) in
combination with the antiviral ribavirin. Numerous short-
comings existed for this treatment regimen, including success
rates of only ∼50% sustained virologic response (SVR) in
patients with HCV genotype 1 (GT-1, by far the most common
genotype worldwide, accounting for ∼75% of all cases) and
generally poor tolerability.2−4 Much effort has recently focused
on the discovery of direct-acting antivirals (DAAs), which can
intervene and interfere at specific points within the viral life
cycle.5−9 The first two DAA therapeutics to reach the market,
NS3/4A protease inhibitors boceprevir and telaprevir, did so in
2011 and are currently being used in combination with the
previous SOC, boosting SVR to 70−80% for GT-1.10,11 Unmet
needs remain, however, to further increase the SVR in both
treatment naive and treatment experienced patients, as well as in
patients comorbid with other infections, such as HIV. Also,
elimination of side effects associated with IFN is highly desirable
in terms of tolerability and patient compliance, and toward that
end much research continues into the discovery of new DAAs
HCV is a virus of the family Flaviviridae and contains a positive
sense, single-stranded RNA genome. The viral life cycle consists
of eight major stages, including entry (endocytosis), uncoating
(fusion), polyprotein synthesis from (+)-RNA, cleavage of the
polyprotein into individual proteins (structural and non-
structural), RNA replication, viral packaging and maturation,
release, and reinfection (Figure 1).14 Any of those stages
represent opportunities for direct-acting antiviral therapeutic
intervention. As already mentioned, inhibitors of the NS3/4A
protease enzyme have now reached the market and are currently
in use in combination with the previous SOC. The RNA-
dependent RNA polymerase enzyme NS5B (enlarged section of
Figure 1) represents another DAA therapeutic target for
inhibition and has been the focus of intense research in recent
years.15−18 Early efforts focused on identification of nucleoside
inhibitors that engage at the catalytic site, ultimately leading to
the discovery of some promising therapeutic agents now in late-
stage clinical trials.19 More recently, multiple allosteric sites have
been identified on NS5B, including the so-called thumb I, thumb
II, palm I, and palm II sites.16 Non-nucleoside inhibitors have
Received: August 15, 2013
© XXXX American Chemical Society
A
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Figure 1. HCV life cycle.14 Reprinted from Clinics in Liver Disease, Vol. 15, Ilyas, J. A., Vierling, J. M., An overview of emerging therapies for the treatment
of chronic hepatitis C, pp 515−536, Copyright 2011, with permission from Elsevier.9
been reported for all of these sites, including several drug
candidates that are currently under clinical evaluation.20−24
Previously, we reported the discovery of a novel series of
benzothiazine-substituted quinolinediones as inhibitors of
NS5B, acting at the palm I allosteric site.25 One example from
that series is compound 1 (Figure 2). Although we identified
RESULTS AND DISCUSSION
■
From a structure-based design point of view, we carefully
considered the X-ray cocrystal structures of 1 and 2 with NS5B
(Figure 3). Both compounds satisfy the large hydrophobic
pocket of the palm I allosteric site, with a p-fluorobenzyl moiety
for 1 and with tert-butylphenyl for 2. Interestingly for 2, the core
benzene ring engages in a classic edge-to-face π-stack with the
side chain of Tyr448, while the tert-butyl group completely fills
the bottom of the hydrophobic pocket, making close hydro-
phobic contact with the side chain of Pro197, among other
residues. Both ligands engage in H-bonding with the adjacent
backbone area, via the quinolinedione oxy anion of 1 and
analogously via the pyridone moiety of 2. As we previously
reported, one of the remarkable features of the highly ligand-
efficient 2 is the donor−acceptor engagement by the 2(1H)-
pyridone moiety with two backbone residues (NH of Tyr448 and
CO of Gln446). In a further comparison of the cocrystal
structures of 1 and 2, superimposing the two structures on each
other (Figure 3C), the major obvious difference is the filling of
the palm I pocket by 1 in the area adjacent to and partially
overlapping with the catalytic pocket. In fact, 1 directly interacts
with the side chain of one of the key catalytic aspartates, Asp318,
in a hydrogen bond via the sulfonamide NH. Our design concept
for the evolution of 2 toward a lead series was to “grow” from the
small template toward the catalytic site of NS5B, adding some of
the key interactions found between 1 and the enzyme that are
absent from 2, while retaining what we felt was a superior, highly
efficient starting point regarding the interactions 2 was already
making with the palm I site. Toward such an end, linkers were
designed to connect the core benzene ring of 2 together with the
highly optimized N-arylmethanesulfonamide fragment present in
1. In the X-ray cocrystal structure of 1, it is apparent that the
carbocyclic ring of the benzothiazine moiety makes an edge-to-
Figure 2. Previously reported Roche inhibitors of NS5B.
highly potent inhibitors, oral exposures in preclinical in vivo
pharmacokinetics studies were not sufficient to reach targeted
levels, and ultimately we looked for new chemotypes from which
to optimize toward a clinical candidate. More recently, we
reported the pioneering use of de novo fragment design that
enabled us to generate a novel, low molecular weight (MW) lead
structure (compound 2, Figure 2).26 Pyridone 2 exhibits potent
inhibition of NS5B enzymatic activity (Table 1), especially
considering its small size. Given the low MW and high ligand
efficiency (LE = 0.46), we felt 2 was a reasonable starting place
from which to develop potent inhibitors of NS5B that would also
possess desirable physicochemical and ADMET properties
required for a safe, orally delivered drug candidate.
B
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Table 1. Core-to-Sulfonamide Linkages
a
b
IC50 measured using GT 1b, Con1 strain (n ≥ 2). EC50 measured using GT 1a or GT 1b stable HCV subgenomic replicon (n ≥ 2).
face π-stack interaction with Phe193 side chain, and all polar
atoms of the sulfonamide moiety make productive hydrogen
bonds to either polar side chains or a bound water molecule.
Molecular modeling using cocrystal structures of 1 and 2 enabled
the in silico evaluation of a range of possible linker compositions
and positions. The goal was to retain the highly optimized
interactions between the N-arylmethanesulfonamide moiety and
residues of the palm I pocket, with particular care taken to ensure
low strain energy of the final bound conformation. A simple two-
atom connection to the position adjacent to the methoxy
substituent of the core benzene ring of 2 satisfied these
requirements.
Extension from the core structure of 2 to the catalytic pocket
resulted in potent inhibitors of NS5B (Table 1). By comparison
of 3 and 4, it can be seen that the optimal connection point of a
two-carbon linker from the core structure 2 to an N-
phenylmethanesulfonamide group is at the position para to the
substituent nitrogen on the pendent benzene ring. Ethylene-
linked 4 not only exhibited potent inhibition of NS5B in the
enzymatic assay but was also efficacious in the cell-based replicon
assay, with EC50 < 50 nM for both genotypes 1a and 1b. We
further evaluated 4 for its physicochemical and ADME properties
(Table 2). Compound 4 demonstrated low solubility (as
measured in a high throughput thermal solubility assay) and
medium to high permeability (as measured in the Caco2 assay).
It was metabolically unstable both in vitro (human and rat liver
microsomes) and in vivo (rat) and gave only modest oral
bioavailability in rat. The reason for low % F may be attributed to
absorption limited by poor solubility, as well as to high first pass
metabolism (iv clearance > liver blood flow). We also evaluated 4
in some of our cytochrome P450-based early safety in vitro assays
(Table 3). It exhibited potent reversible (IC50 = 0.34 μM) as well
as time-dependent (kobs 6-fold greater than ethynylestradiol)
inhibition of CYP3A4. Inhibition of four other CYP isoforms
evaluated (1A4, 2C9, 2C19, 2D6) was not observed (all IC50 > 5
μM, data not shown). Time dependent inhibition (TDI) of
CYP3A4 was especially concerning, as it could indicate the
formation of reactive metabolites, further compounding the risk
for drug−drug interactions (DDIs) from reversible inhibition of
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Figure 3. (A) X-ray cocrystal structure of compound 1 complexed with NS5B at 2.2 Å, showing the palm I allosteric site.25 (B) Compound 2 with NS5B
at 2.8 Å. (C) Overlay 1 and 2 in binding pocket from (A).
Table 2. Physicochemical and ADME Comparison for Linkers
a
b
c
d
e
f
f
g
h
compd
PSA
clogP
Caco2 AB
Caco2 ER
sol.
HLM
RLM
iv Cl
% F
4
75.3
98.1
75.8
93.0
96.8
4.60
2.47
4.85
3.70
3.50
1.5
0.3
3.4
48
<1
<1
<1
<1
185
50
1240
26
82
41
11
0
5
10
12
13
78
103
302
388
0.9
0.3
19
15
40
29
0.4
638
a
b
c
d
e
Polar surface area, calculated in Å2. Calculated log P. Permeability coefficient, in 10−6 cm/s. Ratio of BA/AB permeability coefficients. Thermal
f
aqueous solubility, pH 6.4 (high throughput assay), in μg/mL. Human or rat liver microsome intrinsic clearance, in (μL/min)/mg protein.
g
h
Clearance in rat after 0.5 mg/kg iv injection, in (mL/min)/kg. Bioavailability in rat after 2 mg/kg po administration.
CYP3A4 and even potentially leading to autoimmune reactions
and severe liver toxicities.27
In vitro metabolite identification studies conducted in
microsomal preparations revealed three main regions of
oxidative metabolism/hydroxylation: the ethylene linker, the
pyridone ring, and the tert-butyl group (Figure 4; see also
Supporting Information for metabolite identification study of 4).
We felt that any of these areas could be responsible, at least in
part, for the observed high first pass clearance as well as for the
potential production of reactive metabolites (e.g., quinone
methide type structures from oxidation of the benzylic positions
of the ethylene linker, pyridinedione from the pyridone,
D
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dimethylacetaldehyde from tert-butyl). Our strategy was there-
fore set to address some of the shortcomings of 4 (poor
metabolic stability, DDI/TDI risk) by taking a stepwise approach
to modify each of those three key regions with substitutions and
changes that we expected to confer resistance to oxidation,
through direct steric blocking or through reduction of relative
electron richness or through a combination. Furthermore, we felt
that while compound 4 was quite potent in both biochemical and
cell-based assays, improvement in potency, especially in the cell-
based replicon system, would ultimately lead to lower targeted
exposures in vivo and thus mitigate some of the liabilities a final
candidate may have in terms of limited bioavailability or safety
margins. Optimization of potency was therefore to be addressed
concomitantly with stability and safety.
Table 3. Inhibition of CYP3A4
a
b
c
d
compd
IC50
% TDI
kobs
kobs ratio
4
0.34
>50
>50
1.1
66
4
0.0921
0.0023
0.0003
0.0028
6.01
0.11
0.01
0.15
1.78
5
8
3
10
11
<1
36
0.04
0.0386
a
b
Inhibition of midazolam turnover, in μM. Normalized percent
activity remaining (12 min preincubation with compound at 10 μM).
c
d
Inactivation rate, in min−1. Ratio of inactivation rates between
compound and ethynylestradiol (<1 considered low TDI potential).
Beginning our optimization of 4 by further investigation into
various linker groups (Table 1), we identified carboxamide 5 as
an inhibitor of comparable potency to 4 as measured in the
enzymatic assay and with increased efficacy in the genotype 1b
replicon cellular assay.28 Compound 5 is significantly stabilized
compared to 4 when incubated with liver microsome
preparations (Table 2) and exhibits much reduced DDI and
TDI liabilities (Table 3). Presumably oxidative metabolism has
both been directly blocked at the ethylene linker of 4, as well as
indirectly decreased by potential reduction of partitioning into
Figure 4. Regions of microsomal oxidation of compound 4, as indicated
by the observation of hydroxylated species during metabolite
identification studies.
Figure 5. (A) X-ray cocrystal structure of compound 12 bound in the palm I allosteric site of NS5B at 2.05 Å. (B) Compound 13 with NS5B at 2.05 Å.
E
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Table 4. Pyridone Replacements and Substitutions
a
b
c
Calculated log P. EC50 measured using GT 1a or GT 1b stable HCV subgenomic replicon (n ≥ 2). EC50 and EC90 measured using GT 1b stable
HCV subgenomic replicon in the presence of 40% human serum (n ≥ 2).
the microsomal fractions because of increased overall polarity of
5 (increased PSA, decreased clogP, Table 2). The increased
polarity, however, especially by nature of increased hydrogen
bonding donors and acceptors, leads to reduced permeability and
increased efflux. Reduced permeability, together with poor
solubility, led to essentially no observed bioavailability from po
dosing in rat. From iv dosing, however, it was noted that in vivo
clearance did improve relative to 4 (Table 2).
Next, a variety of two-atom linker analogues of 4 and 5 were
designed and synthesized. Compounds 6−11 (Table 1) are
representative analogues that were predicted via modeling to
maintain the critical interactions made by the N-phenyl-
methanesulfonamide moiety while binding in a low strain energy
conformation. In addition, some of these linkers were designed
to improve solubility by decreasing planarity (6−8) as well as by
introducing an ionizeable moiety (7 and 8). While solubility was
improved (data not shown), significant loss of potency in both
biochemical and cell-based assays was seen as an unacceptable
trade-off. We anticipated a requirement for exposures at
multiples above efficacious concentrations, especially to enable
preclinical toxicological studies. Thus, we set aggressive targets
for potency, especially potency in a serum-shifted cell-based
assay (vida infra), in addition to looking for ideal ADMET
properties to allow for safe, oral delivery. Note that piperidine 8
was also devoid of TDI activity (Table 3), demonstrating that the
1,2-diarylethylene moiety was most likely responsible for the
observed effect in 4. This hypothesis is also supported by the
observed low TDI signal for stilbene analogue 10, as well as by
the moderate TDI signal observed for the 1,2-trans-cyclo-
propane-linked analogue 11, which has stereoelectronic proper-
ties between those of the saturated and unsaturated two-carbon
linkers of 4 and 10, respectively. Compound 10 was also
remarkable to us for its robust potency in the replicon assay, for
both genotypes 1a and 1b. Its improved in vitro (microsomal)
stability compared to 4 is not likely due to a partitioning/polarity
difference (similar PSA, clogP, Table 2), and TDI activity is less
(Table 3). While we were unable to measure in a Caco-2 assay
because of experimental difficulties relating most likely to poor
F
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Figure 6. (A) X-ray cocrystal structure of compound 14 bound in the palm I allosteric site of NS5B at 1.90 Å. (B) Compound 4 with NS5B at 2.09 Å.
compound solubility, we were hopeful that an expected
reasonable permeability of 10 relative to 5 (with fewer H-
bonding groups and lower PSA for 10) would make for a better
starting point for optimization. Thus, considering its advantages
in potency and overall ADMET properties, 10 became our lead
template upon which we could further refine toward a clinical
candidate.
Before we fully committed to the stilbene template of 10,
however, we did design and synthesize a number of analogues of
4 and 5 in which the linker was cyclized onto the aryl ring of the
N-arylmethanesulfonamide terminus. Design ideas were eval-
uated and filtered via modeling, where we looked to further
restrict the rotational freedom of molecules such as 4−11 into
what we believed was the most likely active conformation,
positioning all of the key interactions as seen in the overlay of
crystal structures for 1 and 2 (Figure 3C). Hydrogen bond
accepting moieties were included in the fused ring designs to
engage with the water molecule network observed in the palm I
pocket. Example successful embodiments of this design concept
are oxazole 12 and isocoumarin 13.29 We obtained X-ray
cocrystal structures of 12 and 13 bound with NS5B (Figure 5)
and were pleased to observe them in their predicted binding
modes. Of interest to note is that the water molecule hydrogen-
bonding with the oxazole nitrogen of 12 is effectively replaced by
the isocoumarin carbonyl oxygen of 13. Bicyclic compounds 12
and 13 were both quite potent (Table 1); however, both also
exhibited deficiencies that ultimately led to their deprioritization.
In the case of oxazole 12, poor oral bioavailability was at issue
(Table 2), with near-zero exposure from oral dosing most likely
owing to a combination of solubility-limiting absorption and
efflux-mediated low permeability. In the case of isocoumarin 13,
low stability when incubated with human and rat liver microsome
preparations, as well as observed efflux that could limit oral
exposure (PK experiment not performed), led to its deprioritiza-
tion.
Returning to the stilbene template 10, we next explored
modification of the pyridone ring (Table 4). Although change of
the saturated to unsaturated linker from 4 to 10 resulted in
improved stability to microsomes, as well as reduced TDI
potential, we sought to improve stability further so that our
aggressive exposure targets could be better met. Recalling that
metabolite identification studies had identified the pyridone ring
as one of three sites of oxidation (in addition to the linker and
tert-butyl group), we sought to stabilize the pyridone either by
replacement with more electron deficient heterocycles (14−18)
or by introduction of small substituents to potentially block
oxidation directly (19−22). In either case, we viewed retention
of the 1,3-donor−acceptor motif of the 2(1H)-pyridone as a
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requirement so that the unique engagement with the backbone
amides of residues Gln446 and Tyr448 would be maintained.
Also, at this mature stage of our lead optimization effort we began
to focus more on efficacy in the HCV replicon assay performed in
the presence of 40% human serum (HuS). We targeted potency
in this in vitro system that would allow for exposures in vivo at 5-
fold coverage above the measured EC90. Note that stilbene 10
exhibits a 20-fold shift reduction in efficacy in this assay.
However, we still considered it a very potent compound with
EC50 and EC90 of 21 and 150 nM, respectively, in the presence of
40% HuS. Note also that enzymatic data are no longer included
in Table 4 (and later also not in Table 6), as all of the compounds
14−27 exhibited IC50 between 1 and 3 nM (data not shown),
even for the least active examples, such as 15. Under our
biochemical assay conditions (enzyme and substrate concen-
trations at 3 nM each), all IC50 < 3 nM are indisinguishable, as the
enzyme is effectively titrated with inhibitor at those potencies.
For heteroaromatic replacements of pyridone, examples 14−
18 are representative of the effort. We were able to obtain our
first X-ray cocrystal structure of a stilbene series compound with
14 (Figure 6). A cocrystal structure of the prototype ethylene-
linker 4 is also shown for comparison. It can clearly be seen that
the unsaturated CC linker adopts a more coplanar disposition
between the two aryl rings than does the saturated ethylene
linker, and this bound conformation is very close to the low-
energy ground state conformation typical for stilbenes, which
would help account for the intrinsic potency increase over
diarylethylenes. Note that in the structure for 4 (Figure 6B), it is
curious that the −CH3 group of the methoxy is rotated in a
different direction than that observed in all of our other cocrystal
structures, pushing side chain of Cys366 away from the ligand.
The reason for this observed difference is unclear.
The significant (4- to 7-fold) potency increase for
pyridazinone 14 relative to pyridone 10 in the serum-shifted
replicon assay caught our attention. While decreased lipophilicity
(clogP, Table 4) may be limiting nonspecific binding to serum
proteins for 14, it is also possible that there is an intrinsic potency
increase due to specific interactions with the binding pocket. We
observe what could be an n-to-π* interaction between the ring
nitrogen lone pair and the backbone carbonyl carbon of Gly410
(Figure 7), providing an enthalpic improvement to the binding
energy, although the vector angle for this interaction does not
appear to be ideal. Pyrimidinone 15 is significantly less potent
than the other aza-modified pyridines in Table 4. This
observation could be explained by increased acidity, whereby
an anion form is dominant and thus cannot provide an H-bond
donation to the backbone carbonyl of Tyr448 (we did not
measure the pKa of 15 to test this hypothesis). An alternative
explanation would be that the lone pair of the additional nitrogen
in 15 is not able to make any productive contact with protein
residues, and thus, the cost of desolvation is significantly
detracting from the binding energy.30 Note that uracil analogue
16 recovers potency lost in 15, which could be due to reduction
in relative acidity, the ability to make more productive contacts
with residues in the binding pocket (putatively Gly410 backbone
and Asn411 side chain, in addition to Gln446 and Tyr448
backbone), or both. Pyrazinone 17 exhibits an efficacy profile
similar to that of the parent pyridone 10, whereas triazinone 18 is
slightly less active.
Figure 7. Enlargement from cocrystal structure of 14 with NS5B (Figure
6), showing potential n-to-π* interaction between pyridazinone ring
nitrogen and Gly410 backbone carbonyl.
successful (vida infra). In terms of potency, it was interesting
to see that no loss was observed with the 5-fluoro substitution in
19. The slightly larger substituents chloro and methyl in 20 and
21, respectively, affected modest declines in potency relative to
the unsubstituted pyridone 10, with a trend toward a greater
decline in the genotype 1a replicon assay. We were unable to
obtain cocrystal structures of our molecules complexed with
genotype 1a NS5B, and so structure-based explanations of this
trend can only be speculative. Several residues in the palm I
region near the pyridone ring binding site are changed in
genotype 1a relative to 1b, including Y415F, D444N, and Q446E,
which could play a role in the observed potency differences. Note
that when the substituent is moved from the 5- to the 6-position
(methyl group on 22), potency is restored to the levels observed
for 10 and 19, for both subtypes 1a and 1b.
Because a primary motivation behind the design of pyridone
analogues 14−22 was to improve metabolic stability, we
evaluated them under both in vitro and in vivo conditions
(Table 5). In general, pyridone substitutions (19−22) had a
a
Table 5. Pyridone Replacement in Vitro and in Vivo Stability
b
c
d
e
f
compd
HLM
HH
RLM
RH
iv Cl
14
16
17
18
19
20
21
22
24 (M)
30 (M)
21 (L)
4 (M)
20 (L)
7 (M)
10 (M)
9 (M)
10 (M)
6 (M)
202 (L)
36 (M)
29 (M)
20 (M)
65 (L)
42 (L)
16 (M)
9 (M)
53 (L)
56 (L)
75 (L)
31 (M)
17 (H)
23 (M)
44 (L)
10 (H)
20 (M)
17 (M)
5 (H)
78 (M)
48 (M)
10 (H)
92 (L)
25 (H)
29 (M)
15 (M)
18 (M)
5 (H)
a
b
Stability category H/M/L = high/medium/low. Human liver
microsome intrinsic clearance, in (μL/min)/mg protein (high stability,
c
<6.5; low stability, >35). Human hepatocyte intrinsic clearance, in
d
(μL/min)/106 cells (high stability, <2.8; low stability, >15). Rat liver
microsome intrinsic clearance, in (μL/min)/mg protein (high stability,
e
For single substituents, a sampling of our effort is summarized
by pyridones 19−22. Placing the substituents at the 5-position
(19−21) was intended to directly block the putative site of
metabolic oxidation, a strategy that was at least partially
<15; low stability, >90). Rat hepatocyte intrinsic clearance, in (μL/
f
min)/106 cells (high stability, <5.4; low stability, >29). Clearance in
rat after 0.5 mg/kg iv injection, in (mL/min)/kg (high stability, <20;
low stability, >40).
H
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Table 6. tert-Butyl Modifications and Replacements
a
b
c
Calculated log P. EC50 measured using GT 1a or GT 1b stable HCV subgenomic replicon, in nM (n ≥ 2). EC50 and EC90 measured using GT 1b
d
e
(except where noted) stable HCV subgenomic replicon in the presence of 40% human serum (HuS), in nM (n ≥ 2). GT 1a used. Permeability
f
coefficient, in 10−6 cm/s. Ratio of BA/AB permeability coefficients.
larger impact on reducing clearance than did replacement with
heterocyclic analogues containing additional nitrogens (14−18).
Considering stability category as defined in Table 5 (high,
medium, low), data from human liver microsomes and
hepatocytes were in higher concordance with each other than
were the corresponding data from rat. For all of the compounds
reported in Table 5, human in vitro stability fell either within or
very close to the medium range, which represents a significant
improvement relative to the in vitro stability measured for 10
(Table 2). In vitro stability data from rat were not highly
predictive of in vivo results, with no clear advantage to using
either microsomal or hepatocytic systems (both predicting the in
vivo stability category with only ∼50% accuracy). For the
substituted pyridones evaluated, two exhibited high stability in
vivo (19 and 22), with one more in the medium-to-high range
(20).
While several candidates had been identified from the
exploration of pyridone group modifications which exhibited
medium to high stability (18, 19, 20, and 22) in vitro and in vivo,
we decided to extend the effort to block or retard metabolic
oxidation to modifications on the tert-butyl group. Representa-
tive examples of this effort are shown in Table 631 and will be
discussed first in terms of efficacy and cell permeability, before
presenting the effects of these changes on clearance. Hydroxy-
tert-butyl analogue 23 is very potent, especially in the serum-
shifted assay. Oxetane 24 is less potent than 23 but similar to 10
and other potent stilbene series compounds. By comparison of
the polarities (clogP) of 10, 23, and 24, it is not clear that
increased polarity is driving the increased replicon activities of
alcohol 23, as clogP values for 23 and 24 are very close.
Unfortunately, it is more clear for both compounds that
increasing polarity leads to reduced intrinsic permeability, as
well as increased efflux, with 24 fairing slightly better in this
regard than 23. We generally targeted permeability as measured
by the Caco2 assay to be >10−6 cm/s, with efflux ratio of <10, to
advance molecules with the best chance for efficient intestinal
absorption, so data for both 23 and 24 fell outside those targets.
Deuterated tert-butyl analogue 25 was designed to test the
impact of a primary kinetic isotope effect on metabolic oxidation
of tert-butyl CH. Replacement of hydrogens with deuteriums
does not decrease potency (data only available for genotype 1a,
which typically gives lowered measured potencies for this series).
We also explored the 1-chloro-1-cyclopropyl group as a
replacement for tert-butyl that would be a good size mimetic,
without presenting any terminal methyl groups for easy
oxidation. 1-Chloro-1-cyclopropyl appeared to give an advantage
in permeability, with 26 looking much improved relative to oxo-
substituted tert-butyl analogues 23 and 24. Potency drops only 2-
fold (compare 26 to 10, Table 4), showing that the 1-chloro-1-
cyclopropyl moiety is well tolerated in the place of tert-butyl. The
loss of potency is slightly greater when combined with a pyridone
substitution in 27 (3- to 4-fold, compared to data for 19, Table
4), which was designed to see if blocking metabolism at both sites
simultaneously would produce additive or synergistic effects on
stability.
We evaluated promising tert-butyl replacements for stability
(Table 7). Oxetane 24 did show improvement relative to 10 in
Table 7. tert-Butyl Substitution Effect on in Vitro and in Vivo
Stability
a
b
c
d
e
compd
HLM
HH
RLM
RH
iv Cl
50 (L)
24
25
26
27
22 (M)
19 (M)
17 (M)
2 (H)
2 (H)
74 (M)
28 (M)
77 (M)
21 (M)
13 (M)
9 (M)
27 (M)
a
Human liver microsome intrinsic clearance, in (μL/min)/mg protein
b
(high stability, <6.5; low stability, >35). Human hepatocyte intrinsic
clearance, in (μL/min)/106 cells (high stability, <2.8; low stability,
c
>15). Rat liver microsome intrinsic clearance, in (μL/min)/mg
d
protein (high stability, <15; low stability, >90). Rat hepatocyte
intrinsic clearance, in (μL/min)/106 cells (high stability, <5.4; low
e
stability, >29). Clearance in rat after 0.5 mg/kg iv injection, in (mL/
min)/kg (high stability, <20; low stability, >40).
I
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vitro, with all measurements at either medium or high stability. In
vivo stability observed in rat, however, fell in the low range. 1-
Chloro-1-cyclopropyl analogue 26 showed a very similar in vitro
stabilization when incubated with liver microsome preparations
from both human and rat. Combination of the tert-butyl
stabilizing group 1-chloro-1-cyclopropyl with a pyridone
stabilizing substitution (5-fluoro), however, did not result in an
additive or synergistic effect (compare hepatocyte stability for 27
vs 19 from Table 5). Interestingly, one of the most effective
substitution we found for stabilizing the template in vitro through
tert-butyl modification was deuterium, as in the perdeutero
analogue 25, demonstrating that an isotope effect can
significantly impact the rate of metabolic clearance on this
template. Although retarding oxidative metabolism by replacing
or substituting at the tert-butyl group had some effect on overall
rate of clearance (at least in vitro), it appeared that the effect
achieved by stabilizing through pyridone modification alone was
sufficient to reach the desired target of medium to high stability.
We decided to focus on fluoropyridone 19 for further
preclinical development based on its balance of high potency
with medium to high metabolic stability. For a summary of in
vitro data for 19 not already presented, see Table 8. Intrinsic
Table 9. In Vivo Pharmacokinetics for 19 In Rat
b
c
d
e
f
dose/route
0.5 mg/kg iv
Cl
17.4
Vdss
5.12
t1/2
5.3
Cmax
AUC
% F
281
470
138
407
1370
2 mg/kg po, suspension
20 mg/kg po, suspension
12.7
32.4
104
7.2
2.2
2.9
50 mg/kg po,
nanosuspension
a
14 mg/kg po, solution
398
817
4460
33
21
a
50 mg/kg po, solution
10100
a
b
c
1:1 PEG 400/Labrasol. Clearance, in (mL/min)/kg. Volume of
d
e
distribution at steady state, in L/kg. Half-life, in h. Peak
concentration, in ng/mL. Area under curve, in ng·h/mL.
f
Table 8. Additional in Vitro Data for 19
Caco2
CYP3A4
a
b
c
d
e
f
AB
ER
IC50
% TDI
kobs
kobs ratio
0.87
1.8
>50
12
0.0109
0.60
a
b
Permeability coefficient, in 10−6 cm/s. Ratio of BA/AB permeability
c
d
coefficients. Inhibition of midazolam turnover, in μM. Normalized
Figure 8. Exposure curves for compound 19 following oral dosing in rat,
with 5× EC90 line marking 303.5 ng/mL.
percent activity remaining (12 min of preincubation with compound at
e
f
10 μM). Inactivation rate, in min−1. Ratio of inactivation rates
between compound and ethynylestradiol (<1 considered low TDI
potential).
concentrations were again not reached. Considering the low
solubility for this series (for 19, aqueous solubility in pH 6.5
phosphate buffer was 0.1 μg/mL), we viewed it as a strong
possibility that oral absorption was being limited by slow
dissolution. We therefore investigated solution dosing using a
polyethylene glycol/caprylocaproyl macrogol-8 glyceride (Lab-
rasol) vehicle,33 in which compound 19 was completely dissolved
and not merely in suspension. Indeed, increases in bioavailability
and exposures above targeted levels in plasma were observed at
the two doses tested. The increased exposures observed during
these studies from solution formulation support the hypothesis
that absorption was limited because of solubility/dissolution
factors in the previous studies using suspensions. At the high
solution dose (50 mg/kg), plasma concentrations remained
above 303.5 ng/mL for over 8 h (with extrapolation suggesting
up to 12 h, Figure 8), sufficient coverage to enable toxicology
studies in rat. Similar experiments in dog using PEG 400/
Labrasol solution dosing also demonstrated good kinetics and
oral bioavailability (Table 10).
permeability was measured at slightly below the desired target of
10−6 cm/s; however, significant efflux was not observed and the
molecule was still advanced. CYP3A4-mediated DDI and TDI
liabilities were low. Inhibition of four other CYP isoforms (1A4,
2C9, 2C19, 2D6) was not observed (all IC50 > 50 μM, data not
shown).
Further evaluation of 19 included in vivo pharmacokinetic
(PK) studies, as well as in vitro potency studies against a panel of
genotype 1 HCV NS5B clinical isolates. With regard to in vivo
PK, we felt that in order to enable preclinical toxicology studies,
sustained exposures in preclinical species should exceed by at
least several multiples the exposures expected to be useful in
human efficacy studies (i.e., where plasma trough levels, Cmin
,
exceed the 40% human serum-shifted replicon EC90). Thus, a
target Cmin of 303.5 ng/mL, equating to 5-fold above the serum-
shifted EC90 for GT-1b, was considered a minimum requirement
following oral dosing. Furthermore, those levels were targeted to
be maintained for >12 h following a single dose so that b.i.d. or
q.d. dosing regimens could be implemented. Studies in rat are
summarized in Table 9 and in Figure 8. Upon dosing compound
19 as a suspension in our typical vehicle (hypromellose 2910,
polysorbate 80, benzyl alcohol, and water), modest oral
bioavailability (7.2%) was observed at 2 mg/kg, with a nondose
proportional drop-off to 2.1% observed at the higher 20 mg/kg
dose. Importantly, exposures did not cover the targeted plasma
level of 5-fold over serum-shifted EC90, even at Cmax. Moving into
a “nanosuspension”32 and dosing up to 50 mg/kg showed slightly
increased bioavailability (now 2.9%); however, targeted plasma
In addition to profiling 19 for its PK properties, we studied its
ability to inhibit various HCV strains by measuring its activity
Table 10. In Vivo Pharmacokinetics for Compound 19 in Dog
b
c
d
e
f
dose/route
0.5 mg/kg iv
2 mg/kg po, solution
Cl
Vdss
1.11
t1/2
4.9
Cmax
AUC
% F
3.28
666
301
2410
4260
a
45
a
b
c
1:1 PEG 400/Labrasol. Clearance, in (mL/min)/kg. Volume of
d
e
distribution at steady state, in L/kg. Half-life, in h. Peak
concentration, in ng/mL. Area under curve, in ng·h/mL.
f
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against replicons containing NS5B sequences derived from
clinical isolates from treatment naive genotype 1 HCV patients.
Preexisting amino acid polymorphisms may confer reduced
sensitivity to direct-acting antivirals, and drug pressure-induced
selection may increase the prevalence of those strains within a
patient’s quasispecies, leading to increased potential for
resistance.34 Using a transient replicon assay, we measured
EC50 against a number patient-derived NS5B isolates from both
genotypes 1a and 1b (Table 11 and Figure 9).35 The H77 and
Table 11. Antiviral Activity of 19 and PF-00868554 against
GT 1a and GT 1b NS5B Clinical Isolates
a
EC50 (nM)
GT
isolate
19
PF-00868554
1a
H77
1.0 0.3
1.3 0.1
4.1 1.0
3.1 0.1
1.8 0.1
2.3 0.1
1.1 0.1
2.2 0.1
0.4 0.1
6.8 0.5
1.5 0.2
1.8
95.8 9.7
47.0 6.2
42.8 7.6
37.0 1.2
38.8 2.3
26.0 1.2
34.4 3.4
25.0 0.8
22.0 4.9
56.0 6.0
48.0 6.8
39.5
RO-18
RO-22
RO-24
RO-28
RO-32
RO-33
RO-34
RO-35
RO-38
RO-41
b
mean
1b
Con1
RO-1
RO-2
RO-3
RO-4
RO-7
RO-8
RO-9
RO-10
1.4 0.3
0.5 0.1
0.7 0.1
1.7 0.4
0.4 0.1
1.8 0.7
2.0 0.2
3.0 0.1
2.8 0.1
1.3
51.1 11.2
39.3 3.0
21.3 4.9
28.5 7.2
25.5 7.4
20.8 5.4
18.7 4.8
23.5 6.9
39.7 7.2
28.2
Figure 9. Antiviral activity of 19 and PF-00868554 against replicons
from genotype 1 NS5B clinical isolates. Reference strains are shown as
solid shapes, and geometric mean values are shown as dotted lines.
the design of various linkers connecting the hydrophobic core to
the arylsulfonamide that engages the catalytic residues adjacent
to the palm I allosteric site. We identified conformationally
restricted linkers such as in carboxamide 5 and trans-stilbene 10,
predisposed to their active, target-bound conformations in the
ground state, giving them robust intrinsic potency. Evaluation of
linkers based on early safety criteria such as time-dependent
inhibition of CYP3A4, pharmacokinetic criteria such as in vitro
and in vivo stability toward clearance, and physicochemical
criteria such as membrane permeability led to the selection of the
trans-stilbene connector as the preferred template upon which to
further optimize. The results of metabolite identification studies
focused our attention on the pyridone and tert-butyl moieties as
potential regions for tuning properties, especially toward further
improving stability. Substituted pyridones provided the largest
effect in this direction, and fluoropyridone 19 was ultimately
selected as the clinical candidate. While rat in vivo
pharmacokinetics from iv dosing for 19 fell into a desirable
range, low solubility limited oral absorption. In order to achieve
exposures needed to support preclinical toxicology evaluation at
multiples above the human serum-adjusted replicon EC90, we
solved the problem of poor oral absorption by moving to a lipid-
based solution formulation. Finally, we showed that 19 maintains
its robust, low nanomolar potency across 17 different human
HCV NS5B clinical isolates from both genotypes 1a and 1b.
Outcomes from further development studies of 19 will be
reported in due course.
b
mean
a
EC50 measured using transient HCV subgenomic replicon assay (n ≥
b
2), nM SEM (standard error of the mean). Calculated geometric
mean.
Con1 strains were included as references for genotypes 1a and
1b, respectively. Also for comparison, we evaluated another non-
nucleoside inhibitor of NS5B, PF-00868554, which binds to a
different allosteric site, the “thumb II” site. Pyridone 19 is very
potent against all isolates tested from both genotypes, with EC50
ranging from 0.4 to 6.8 nM. Activity for the thumb II inhibitor
PF-00868554 is also consistent across all isolates tested, with
EC50 measured in the range 18.7−95.8 nM and in agreement
with previously published data.36 Interestingly, the largest shift
observed for 19 relative to a reference strain was for the GT-1a
isolate RO-38 (∼7-fold), which contains the mutation S556G, a
known resistance mutation to palm I non-nucleoside inhib-
itors.35 Serine 556 is present in the palm I binding site (see
Figures 3, 5, and 6). Even considering this shift, 19 is still an
extremely potent inhibitor with an EC50 of 6.8 nM.
CONCLUSION
■
In summary, a novel, highly potent series of non-nucleoside
inhibitors of HCV NS5B has been identified from a fragment-
derived starting point and optimized to a clinical candidate in
fluoropyridone 19. A structure-based approach was applied to
K
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a
Scheme 1
a
Reagents and conditions: (a) Br2, CH2Cl2, 100%; (b) MeI, K2CO3, DMF, 99%; (c) (2-methoxy-3-pyridyl)boronic acid, Pd(PPh3)4, Na2CO3,
MeOH, CH2Cl2, 115 °C (microwave), 30 min, 96%; (d) 4-nitrobenzyltriphenylphosphonium bromide, NaH, 15-crown-5, THF, 78%; (e) H2,
Pd(OH)2, EtOAc, MeOH, 96%; (f) MsCl, pyridine, 82%; (g) 48% HBr, AcOH, 65 °C, 18 h, 51%; (h) Fe, NH4Cl, MeOH, H2O, quant; (i) MsCl,
pyridine, 96%; (j) 48% HBr, AcOH, 60 °C, 18 h, 67%.
a
Scheme 2
a
Reagents and conditions: (a) (2-methoxy-3-pyridyl)boronic acid, Pd(PPh3)4, Na2CO3, MeOH, CH2Cl2, 115 °C (microwave), 35 min, 82%; (b) H2,
Pd/C, EtOAc, MeOH; (c) 4-nitrobenzoyl chloride, Et3N, CH2Cl2, 97%; (d) H2, Pd(OH)2, EtOAc, MeOH; (e) MsCl, pyridine, 99%; (f) 48% HBr,
AcOH, 60 °C, 18 h, 60%.
began with 3-tert-butyl-2-hydroxybenzaldehyde 28, which was
brominated and O-methylated in high yield to produce 5-bromo-
3-tert-butyl-2-hydroxybenzaldehyde 29. Suzuki cross-coupling
with (2-methoxy-3-pyridyl)boronic acid provided biaryl inter-
mediate 30, which was then reacted with a 4-nitrobenzyl Wittig
reagent to give stilbene 31 as the trans isomer. Tandem
hydrogenation of both the nitro and stilbene groups provided
aniline 32 with a saturated ethylene linker. Compound 32 was N-
CHEMISTRY
■
Syntheses of selected representatives from compounds 3−27 are
depicted in Schemes 1−5. Full synthetic details for all
compounds 3−27 that are not outlined here may be found in
the Supporting Information. In Schemes 1 and 2 are depicted the
syntheses of a few different linkers from Table 1: 1,2-
diarylethylene 4 and trans-stilbene 10 (Scheme 1) and
carboxamide 5 (Scheme 2). The sequence toward 4 and 10
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mesylated and selectively O-dealkylated to afford the final
pyridone 4. Alternatively, intermediate 31 could have its nitro
group selectively reduced using elemental iron, resulting in
aniline 33. Mesylation and deprotection as before yielded
stilbene 10. In Scheme 2 is depicted the synthesis of carboxamide
5. First, 5-bromo-1-tert-butyl-2-methoxy-3-nitrobenzene 34 was
cross-coupled under Suzuki conditions to give biaryl 35.
Hydrogenation afforded aniline 36, which was then treated
with 4-nitrobenzoyl chloride in the presence of triethylamine to
provide carboxamide 37 in high yield. Nitro group reduction was
achieved under hydrogenation conditions with catalytic
palladium hydroxide to give 38. Finally, N-mesylation followed
by O-demethylation afforded pyridone 5.
hindering access to the bromine). In Scheme 4, we began with 5-
bromo-3-tert-butyl-2-hydroxybenzaldehyde 29 (from Scheme 1)
and stereoselectively installed the trans-stilbene via Horner−
Wadsworth−Emmons reaction with the benzaldehyde, followed
by reduction of the terminal nitro group with elemental iron to
afford aniline 42 in high yield. N-Mesylation provided 43, which
was then converted to boronate 44, providing a versatile
intermediate that could be used for cross-coupling reactions with
heteroaryl bromides to make pyridones and pyridone replace-
ments. In this example, boronate 44 was reacted with 4-
benzyloxy-5-bromopyrimidine to give a corresponding biaryl
intermediate in 34% yield. Removal of the O-benzyl group via
treatment with HBr in acetic acid at room temperature afforded
the final product pyrimidinone 15 in 71% yield.
In Scheme 5 is depicted the synthesis of compound 24,
containing one of the tert-butyl replacement groups (3-
methyloxetan-3-yl) from Table 6. In the first step, 4,6-
dibromo-2-iodoanisole 45 is coupled to diethyl malonate in
the presence of cesium carbonate and catalytic amounts of
copper(I) iodide and 2-phenylphenol following Buchwald’s
procedure,38 generating α-arylmalonate 46 via selective reaction
at the aryl iodide position. Methylation of 46 at the α-malonate
position followed by reduction of the esters to alcohols with
lithium aluminum hydride at low temperature afforded diol 47.
Ring closure of the 1,3-diol to the oxetane was effected by
subjection to Mitsunobu-type conditions (DIAD, triphenylphos-
phine, toluene) in a sealed tube at 140 °C (microwave), yielding
intermediate 48. Taking advantage of the steric hindrance
differential between the two bromines of 48, a Suzuki cross-
coupling with (2-oxo-1H-pyridin-3-yl)boronic acid occurred
with high regioselectivity at the less hindered aryl bromide to give
the biaryl product 49. Also, we found by this point that the
pyridone group did not need protection to carry through many of
the chemistries that we used to prepare compounds in this series,
thus sparing deprotection steps at the ends of syntheses. The final
step here was cross-coupling with N-[4-[(E)-2-(4,4,6-trimethyl-
1,3,2-dioxaborinan-2-yl)vinyl]phenyl]methanesulfonamide
under Suzuki conditions with microwave heating to 120 °C in a
sealed tube, affording trans-stilbene 24 in 64% yield.
a
Scheme 3
a
Reagents and conditions: (a) Mg, THF, reflux, 45 min followed by
4,5-dichloro-1H-pyridazin-6-one, reflux, 18 h, 77%; (b) H2, Pd/C,
KOH, H2O, DMF, 83%; (c) NBS, DMF, 50 °C, 18 h, 52% (d) N-[4-
[(E)-2-(4,4,6-trimethyl-1,3,2-dioxaborinan-2-yl)vinyl]phenyl]methane-
sulfonamide Pd(PPh3)4, Na2CO3, MeOH, CH2Cl2, 125 °C (micro-
wave), 40 min, 18%.
EXPERIMENTAL SECTION
■
General Information. All reactions were performed in round-
bottom flasks unless otherwise noted. The flasks were fitted with rubber
septa, and reactions were conducted under inert atmosphere. Stainless
steel syringes were used to transfer air- and moisture-sensitive liquids.
Most commercial reagents were purchased from Sigma Aldrich, Alfa
Aesar, Strem, Lancaster, AstaTech, or TCI and used as received.
Solvents were purchased from Mallinckrodt and anhydrous dimethyl-
formamide (DMF) and tetrahydrofuran (THF) from Sigma-Aldrich in a
Sure/Seal system. Flash chromatography was performed using
AnaLogix IntelliFlash 280 or Teledyne Isco CombiFlash Companion
purification systems using silica gel columns RediSep Rf (particle size
40−63 μm powder). Deuterated solvents were purchased from Sigma-
Aldrich. Proton nuclear magnetic resonance (1H NMR) spectra were
recorded on a Bruker Avance 300, 400, or 500 MHz spectrometer at 25
°C. Chemical shifts for protons are reported in parts per million
downfield from tetramethylsilane and are referenced to residual protium
in the NMR solvent (CHCl3 δ 7.27, DMSO δ 2.50). Data are
represented as follows: chemical shift, multiplicity (br = broad, s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling
constants in hertz (Hz), and integration. Liquid chromatography−mass
spectrometry (LC/MS) analyses were carried out using an Agilent 6140
single quadrupole in ES/APCI dual mode and Agilent LC with a Zorbax
SB-C18 2.1 mm × 30 mm, 3.5 μm column using a 0.02% TFA/water−
acetonitrile solvent system. Microwave reactions were carried out in a
In Schemes 3 and 4 are shown two different routes used to
prepare pyridone replacement analogues pyridazinone 14 and
pyrimidinone 15, respectively. Beginning with Scheme 3, 4-
bromo-2-tert-butylanisole is converted to an organomagnesium
reagent under Grignard conditions and then reacted in excess
(2.5 equiv) with 4,5-dichloro-1H-pyridazin-6-one in refluxing
THF. Chlorine substitution occurred at the more reactive 4-
position, with good selectivity and high yield. The remaining
chloro substituent was removed by hydrogenolysis in water/
DMF in the presence of palladium(0) on carbon and potassium
hydroxide to afford pyridazinone 40.37 Selective bromination at
the benzene ring position ortho to the methoxy group was
achieved by treatment with N-bromosuccinimide in DMF at 50
°C for 18 h to give 41. The concise, four-step synthesis of 14 was
completed by a final step cross-coupling with N-[4-[(E)-2-(4,4,6-
trimethyl-1,3,2-dioxaborinan-2-yl)vinyl]phenyl]-
methanesulfonamide under Suzuki conditions and microwave
heating to 125 °C in a sealed tube. Forcing conditions were
required to achieve the modest 18% yield of the final product, as
the bromo group of 41 is quite sterically hindered (bulky tert-
butyl group forces methoxymethyl to rotate away from it, thus
M
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a
Scheme 4
a
Reagents and conditions: (a) 1-(diethoxyphosphorylmethyl)-4-nitrobenzene, 15-crown-5, NaH, THF, 0 °C for 2 h, rt for 18 h, 98%; (b) Fe,
NH4Cl, MeOH, H2O, reflux, 4 h, 95%; (c) MsCl, pyridine, 0 °C, 45 min, 99%; (d) 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1,3,2-dioxaborolane, PdCl2(PPh3)2, KOAc, DMSO, 90 °C, 18 h, 53%; (e) 4-benzyloxy-5-bromopyrimidine, Pd(PPh3)4, Na2CO3, MeOH,
CH2Cl2, 120 °C (microwave), 40 min, 34%; (f) 48% HBr, AcOH, rt, 18 h, 71%.
a
Scheme 5
a
Reagents and conditions: (a) diethyl malonate, CuI, 2-phenylphenol, Cs2CO3, THF, 70 °C, 18 h, 55%; (b) MeI, NaH, THF, 0 °C to rt, 2 days,
84%; (c) LiAlH4, THF, 0 °C, 1 h, 52%; (d) diisopropyl azodicarboxylate, PPh3, toluene, 140 °C, 30 min, 43%; (e) (2-oxo-1H-pyridin-3-yl)boronic
acid, Pd(PPh3)4, Na2CO3, MeOH, CH2Cl2, 140 °C (microwave), 30 min, 66%; (f) N-[4-[(E)-2-(4,4,6-trimethyl-1,3,2-dioxaborinan-2-
yl)vinyl]phenyl]methanesulfonamide Pd(PPh3)4, Na2CO3, MeOH, toluene, 120 °C (microwave), 1 h, 64%.
Biotage Initiator microwave synthesizer. Purity of tested compounds
was ≥95% as confirmed by HPLC/MS.
concentrated to afford 29 (3.99 g, 99%) as a yellow solid. 1H NMR (300
MHz, CDCl3) δ 10.30 (s, 1 H), 7.86 (d, J = 2.3 Hz, 1 H), 7.67 (d, J = 2.6
Hz, 1 H), 3.97 (s, 3 H), 1.43 (s, 9 H) ppm.
5-Bromo-3-tert-butyl-2-methoxybenzaldehyde (29). To a
solution of 3-tert-butyl-2-hydroxybenzaldehyde (28) (5.0 g, 28.0
mmol) in CH2Cl2 (20 mL) at 0 °C was added dropwise a solution of
Br2 (1.45 mL, 28.0 mmol) in CH2Cl2 (15 mL) over a period of 30 min.
After the addition was complete, the mixture was stirred for 1 h before
the organic volatiles were removed under reduced pressure to afford 5-
bromo-3-tert-butyl-2-hydroxybenzaldehyde (7.23 g, 100%) as a light
yellowish solid. 1H NMR (300 MHz, CDCl3) δ 11.76 (s, 1 H), 9.84 (s, 1
H), 7.67−7.48 (m, 2 H), 1.44 (s, 9 H) ppm.
3-tert-Butyl-2-methoxy-5-(2-methoxy-3-pyridyl)-
benzaldehyde (30). A sealed tube containing 29 (2.0 g, 7.38 mmol),
(2-methoxy-3-pyridyl)boronic acid (1.68 g, 10.98 mmol), Pd(PPh3)4
(0.66 g, 0.57 mmol), Na2CO3 (1.96 g, 18.49 mmol) in a mixture of
MeOH (11 mL) and CH2Cl2 (3 mL) was heated with a microwave
reactor at 115 °C for 30 min. The reaction mixture was concentrated,
diluted with EtOAc, washed with brine, and dried over Na2SO4.
Chromatography on SiO2 (EtOAc/hexane, 0−10%) gave 30 (2.123 g,
96%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 10.60−10.15
(m, 1 H), 8.25−8.14 (m, 1 H), 7.97−7.88 (m, 1 H), 7.87−7.75 (m, 1 H),
7.70−7.54 (m, 1 H), 7.12−6.91 (m, 1 H), 4.08−3.98(m, 6 H), 1.48 (s, 9
H) ppm.
A mixture of 5-bromo-3-tert-butyl-2-hydroxybenzaldehyde (3.83 g,
15.0 mmol), MeI (2.32 mL, 37.0 mmol), and K2CO3 (6.18 g, 45.0
mmol) in DMF (50 mL) was heated at 50 °C for 1 h and then cooled to
room temperature and diluted with Et2O and water. The organic layer
was thrice washed with water and then brine, dried (MgSO4), and
N
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3-[3-tert-Butyl-4-methoxy-5-[(E)-2-(4-nitrophenyl)vinyl]-
phenyl]-2-methoxypyridine (31). To a slurry of NaH (0.17 g, 4.159
mmol, 60% in mineral oil) in THF (5 mL) containing 0.1 equiv of 15-
crown-5 cooled to 0 °C was added a solution of diethyl (4-
nitrophenyl)phosphonate (1.14 g, 4.16 mmol) in THF (5 mL). When
gas evolution ceased, a slurry of 30 (1.13 g, 3.78 mmol) was added and
stirred at room temperature for 18 h. An additional equivalent of NaH
was added to the partially reacted mixture, and stirring continued for 1 h.
The reaction mixture was partitioned between water and Et2O, and the
organic extract was sequentially washed with water and brine, dried
(Na2SO4), filtered, and evaporated. The crude product was purified over
SiO2 by chromatography (EtOAc/hexane, 50%) to afford 31 as a sole
geometric isomer. 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 8.6 Hz, 2
H), 8.17 (d, J = 4.5 Hz, 1 H), 7.73−7.61 (m, 4 H), 7.57 (d, J = 16.2 Hz, 1
H), 7.53−7.47 (m, 1 H), 7.13 (d, J = 16.7 Hz, 1 H), 7.03−6.95 (m, 1 H),
4.01 (s, 3 H), 3.84 (s, 3 H), 1.45 (s, 9 H) ppm.
4-[2-[3-tert-Butyl-2-methoxy-5-(2-methoxy-3-pyridyl)-
phenyl]ethyl]aniline (32). To a solution of 31 (351 mg, 0.840 mmol)
in EtOAc (15 mL) and MeOH (15 mL) was added Pd(OH)2 (20% on
carbon, 166 mg, 0.237 mmol). H2 gas was bubbled into the solution for
15 min. The mixture was stirred under an atmosphere of H2 for 18 h.
The mixture was filtered, concentrated, and purified over SiO2 by
chromatography (EtOAc/hexane, 5−20%) to give 32 (106 mg, 32%) as
a colorless oil. LC/MS (ES/APCI): 391 (M + H)+.
concentrated. The crude residue was purified over SiO2 by
chromatography (EtOAc/hexane, 40%) to afford N-[4-[(E)-2-[3-tert-
butyl-2-methoxy-5-(2-methoxy-3-pyridyl)phenyl]vinyl]phenyl]-
1
methanesulfonamide (0.178 g, 96%) as a yellow foam. H NMR (400
MHz, CDCl3) δ 8.16 (dd, J = 4.5, 1.5 Hz, 1 H), 7.64 (d, J = 2.5 Hz, 2 H),
7.54 (d, J = 8.6 Hz, 2 H), 7.46 (d, J = 2.0 Hz, 1 H), 7.36 (d, J = 16.7 Hz, 1
H), 7.23 (d, J = 8.1 Hz, 2 H), 7.05 (s, 1 H), 7.02−6.95 (m, 1 H), 6.42 (s, 1
H), 4.00 (s, 3 H), 3.83 (s, 3 H), 3.04 (s, 3 H), 1.45 (s, 9 H) ppm.
A solution of N-[4-[(E)-2-[3-tert-butyl-2-methoxy-5-(2-methoxy-3-
pyridyl)phenyl]vinyl]phenyl]methanesulfonamide (0.14 g, 0.3 mmol),
AcOH (3 mL) and 48% HBr (0.105 mL) was heated at 60 °C for 18 h.
The reaction mixture was cooled to room temperature and diluted with
EtOAc and water to give a precipitate, which was filtered and washed
with water and ether to give 10 (115 mg, 67%) as an off-white solid. 1H
NMR (400 MHz, DMSO-d6) δ 7.84 (d, J = 1.5 Hz, 1 H), 7.67 (dd, J =
6.9, 2.0 Hz, 1 H), 7.58−7.51 (m, 3 H), 7.37 (dd, J = 6.3, 1.8 Hz, 1 H),
7.17 (d, J = 16.4 Hz, 1 H) 7.17−7.07 (m, 3 H), 6.29 (t, J = 6.6 Hz, 1 H),
3.75 (s, 3 H), 2.91 (s, 1 H), 1.39 (s, 9 H) ppm. LC/MS (ES/APCI): 453
(M + H)+.
3-(3-tert-Butyl-4-methoxy-5-nitrophenyl)-2-methoxypyri-
dine (35). A mixture of 5-bromo-1-tert-butyl-2-methoxy-3-nitro-
benzene (34) (0.48 g, 1.7 mmol), (2-methoxy-3-pyridyl)boronic acid
(0.28 g, 1.8 mmol), Pd(PPh3)4 (0.19 g, 0.17 mmol), and Na2CO3 (0.53
g, 5.0 mmol) in CH2Cl2/MeOH (3:1, 8 mL) was subjected to
microwave heating to 115 °C for 35 min. The reaction mixture was
filtered and the filtrate concentrated. The crude residue was purified
over SiO2 by chromatography (EtOAc/hexane, 0−20%) to afford 35
(0.44 g, 82%) as a yellow solid. 1H NMR (300 MHz, CDCl3) δ 8.19 (dd,
J = 4.9, 1.5 Hz, 1 H), 7.87 (d, J = 2.3 Hz, 1 H), 7.72 (d, J = 1.9 Hz, 1 H),
7.61 (dd, J = 7.5, 1.9 Hz, 1 H), 7.00 (dd, J = 7.3, 5.1 Hz, 1 H), 3.99 (s, 3
H), 3.85 (s, 3 H), 1.44 (s, 9 H) ppm.
N-[4-[2-[3-tert-Butyl-2-methoxy-5-(2-oxo-1H-pyridin-3yl)-
phenyl]ethyl]phenyl]methanesulfonamide (4). To a solution of
32 (106 mg, 0.272 mmol) in pyridine (5 mL) at 0 °C was added
methanesulfonyl chloride (0.035 mL, 0.450 mmol). The mixture was
gradually warmed to room temperature and stirred for 1.5 h. The
solution was diluted with EtOAc, washed with saturated CuSO4 solution
and then 1 N HCl solution, and dried over Na2SO4. Purification over
SiO2 by chromatography (EtOAc/hexane, 10−25%) gave N-[4-[2-[3-
tert-butyl-2-methoxy-5-(2-methoxy-3-pyridyl)phenyl]ethyl]phenyl]-
3-tert-Butyl-2-methoxy-5-(2-methoxy-3-pyridyl)aniline (36).
To a solution of 35 (0.98 g, 3.1 mmol) in EtOAc/MeOH (2:1, 30 mL)
was added 10% Pd/C (0.10 g). A stream of H2 was bubbled through the
solution for 20 min, and then the solution was stirred at room
temperature for 16 h. The reaction mixture was filtered and
concentrated to afford 36. 1H NMR (300 MHz, CDCl3) δ 8.12 (dd, J
= 4.9, 1.9 Hz, 1 H), 7.59 (dd, J = 7.36, 1.7 Hz, 1 H), 7.02−6.84 (m, 3 H),
3.97 (s, 3 H), 3.84 (s, 3 H), 3.70 (br s, 2 H), 1.41 (s, 9 H) ppm.
N-[3-tert-Butyl-2-methoxy-5-(2-methoxy-3-pyridyl)phenyl]-
4-nitrobenzamide (37). To a solution of 36 (0.24 g, 0.85 mmol) and
Et3N (0.18 mL, 1.3 mmol) in CH2Cl2 (10 mL) at 0 °C was added 4-
nitrobenzoyl chloride (0.19 g, 1.0 mmol). The resulting mixture was
stirred at 0 °C for 3 h and then warmed to room temperature. The
mixture was then diluted with additional CH2Cl2, washed sequentially
with water and brine, dried (Na2SO4), filtered, and concentrated. The
crude residue was purified over SiO2 by chromatography to afford 37
1
methanesulfonamide (104 mg, 82%) as a colorless oil. H NMR (300
MHz, CDCl3) δ 8.13 (dd, J = 4.9, 1.5 Hz, 1 H), 7.57 (dd, J = 7.3, 1.7 Hz, 1
H), 7.43−7.27 (m, 2 H), 7.25−7.10 (m, 4 H), 6.96 (dd, J = 7.2, 5.3 Hz, 1
H), 6.39 (s, 1 H), 3.98 (s, 3 H), 3.83 (s, 3 H) 2.99 (s, 4 H), 2.97 (s, 3 H),
1.43 (s, 9 H) ppm.
A solution of N-[4-[2-[3-tert-butyl-2-methoxy-5-(2-methoxy-3-
pyridyl)phenyl]ethyl]phenyl]methanesulfonamide (104 mg, 0.222
mmol) and 48% HBr (0.1 mL, 0.87 mmol) in AcOH (3 mL) was
heated at 65 °C for 18 h in a sealed tube. The reaction mixture was
carefully poured into saturated aqueous NaHCO3 ice solution, extracted
with EtOAc, and dried over Na2SO4. Purification by SiO2 preparative
TLC (EtOAc/hexane, 1:1, followed by EtOAc/hexane, 2:1) gave 4 (51
mg, 51%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 11.68 (br s,
1 H), 9.59 (s, 1 H) 7.54 (dd, J = 6.9, 2.1 Hz, 1 H), 7.51 (d, J = 2.2 Hz, 1
H), 7.46 (d, J = 2.2 Hz, 1 H), 7.34 (dd, J = 6.4, 2.0 Hz, 1 H), 7.25 (d, J =
8.5 Hz, 2 H), 7.13 (d, J = 8.5 Hz, 2 H), 6.26 (t, J = 6.8 Hz, 1 H), 3.76 (s, 3
H), 2.93 (s, 3 H), 2.88 (s, 4 H), 1.36 (s, 9 H) ppm. LC/MS (ES/APCI):
455 (M + H)+.
4-[(E)-2-[3-tert-Butyl-2-methoxy-5-(2-methoxy-3-pyridyl)-
phenyl]vinyl]aniline (33). To a suspension of 31 (0.29 g, 0.693
mmol) in MeOH (5 mL) and water (5 mL) was added NH4Cl (0.37 g,
6.93 mmol) followed by Fe powder (0.19 g, 3.464 mmol). The solution
was heated at reflux for 1 h, cooled, and filtered. The filtrate was
concentrated in vacuo. The filtrate was taken up in EtOAc, washed with
brine, dried (Na2SO4), filtered, and evaporated. The crude product was
purified over SiO2 by chromatography (EtOAc/hexane, 30%) to afford
33 (0.26 g, 100%) as a yellow foam. 1H NMR (300 MHz, CDCl3) δ 8.15
(dd, J = 4.9, 1.9 Hz, 1 H), 7.63 (d, J = 2.3 Hz, 2 H), 7.44−7.33 (m, 3 H),
7.17 (d, J = 15.8 Hz, 1 H), 7.02−6.93 (m, 2 H), 6.69 (d, J = 8.3 Hz, 2 H),
4.00 (s, 3 H), 3.83 (s, 3 H), 3.81−3.69 (m, 2 H), 1.44 (s, 9 H) ppm.
N-[4-[(E)-2-[3-tert-Butyl-2-methoxy-5-(2-oxo-1H-pyridin-3-
yl)phenyl]vinyl]phenyl]methanesulfonamide (10). To a solution
of 33 (0.153 g, 0.394 mmol) in pyridine (4 mL) at 0 °C was added
methanesulfonyl chloride (0.037 mL, 0.055 g, 0.473 mmol). Stirring was
continued at 0 °C for 2 h and then at room temperature for an additional
2 h. The reaction mixture was diluted with EtOAc, washed sequentially
with aqueous CuSO4 and 2 N HCl, dried (Na2SO4), filtered, and
1
(0.36 g, 97%) as a light yellow solid. H NMR (400 MHz, CDCl3) δ
8.42−8.35 (m, 3 H), 8.27 (br s, 1 H), 8.16 (dd, J = 5.1, 1.5 Hz, 1 H), 8.10
(d, J = 8.56 Hz, 2 H), 7.66 (dd, J = 7.6, 1.5 Hz, 1 H), 7.40 (d, J = 2.0 Hz, 1
H), 6.98 (dd, J = 7.1, 5.0 Hz, 1 H), 4.00 (s, 3 H), 3.84 (s, 3 H), 1.45 (s, 9
H) ppm.
4-Amino-N-[3-tert-butyl-2-methoxy-5-(2-methoxy-3-
pyridyl)phenyl]benzamide (38). To a solution of 37 (0.36 g, 0.82
mmol) in EtOAc/MeOH (2:1, 15 mL) was added 10% Pd/C (0.035 g).
A stream of H2 gas was bubbled through the solution for 20 min, and the
white solid precipitate that formed was recovered by filtration to afford
38 (quantitative) which was carried onto the subsequent step without
further purification. 1H NMR (300 MHz, CDCl3) δ 8.38 (d, J = 2.3 Hz, 1
H), 8.13 (d, J = 5.0 1.9 Hz, 1 H), 8.08 (br s, 1 H), 7.77 (d, J = 8.7 Hz, 2
H), 7.66 (dd, J = 7.3, 1.7 Hz, 1 H), 7.34 (d, J = 1.9 Hz, 1 H), 6.95 (dd, J =
7.2, 4.9 Hz, 1 H), 6.73 (d, J = 8.3 Hz, 2 H), 4.04 (br s, 2 H), 3.99 (s, 3 H),
3.83 (s, 3 H), 1.44 (s, 9 H) ppm.
N-[3-tert-Butyl-2-methoxy-5-(2-oxo-1H-pyridin-3-yl)phenyl]-
4-(methanesulfonamido)benzamide (5). To a solution of 38 (0.10
g, 0.25 mmol) in pyridine (2 mL) at 0 °C was added methanesulfonyl
chloride (0.034 g, 0.30 mmol). Stirring was continued at 0 °C for 2 h and
then at room temperature for an additional 2 h. The reaction mixture
was diluted with EtOAc, washed sequentially with aqueous CuSO4 and 2
N HCl, dried (Na2SO4), filtered, and concentrated. The crude residue
O
dx.doi.org/10.1021/jm401266k | J. Med. Chem. XXXX, XXX, XXX−XXX

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was purified over SiO2 by chromatography (EtOAc/hexane, 50−100%)
to afford N-[3-tert-butyl-2-methoxy-5-(2-methoxy-3-pyridyl)phenyl]-4-
(methanesulfonamido)benzamide (0.12 g, 99%) as a colorless oil. H
NMR (300 MHz, CDCl3) δ 8.38 (d, J = 1.9 Hz, 1 H), 8.23−8.10 (m, 2
H), 7.94 (d, J = 8.7 Hz, 2 H), 7.65 (dd, J = 7.2, 1.9 Hz, 1 H), 7.40−7.30
(m, 3 H), 6.95 (dd, J = 7.3, 5.0 Hz, 1 H), 6.90 (br s, 1 H), 3.99 (s, 3 H),
3.84 (s, 3 H), 3.10 (s, 3 H), 1.45 (s, 9 H) ppm.
CH2Cl2 (0.6 mL). The tube was flushed with Ar, sealed, and irradiated in
a microwave synthesizer at 125 °C for 40 min. The reaction mixture was
cooled and concentrated in vacuo. The residue was partitioned between
CH2Cl2 (25 mL) and water (5 mL). The organic layer was washed with
brine (5 mL). The aqueous phase was twice extracted with CH2Cl2 (25
mL). The organic layers were combined, dried (Na2SO4), filtered, and
evaporated. The crude product was purified over SiO2 by chromatog-
raphy (EtOAc/hexane, 0−60%) to give 14 (0.175 g, 18%) as a yellow
powder, mp 240.0−242.0 °C. 1H NMR (400 MHz, CDCl3) δ 11.19 (s, 1
H), 8.50 (s, 1 H), 8.25 (d, J = 2.2 Hz, 1 H), 7.93 (d, J = 4.2 Hz, 1 H),
7.56−7.50 (m, 3 H), 7.41 (d, J = 4.2 Hz, 1 H), 7.38−7.30 (m, 3 H), 7.07
(d, J = 16.4 Hz, 1 H), 3.84 (s, 3 H), 3.03 (s, 3 H), 1.45 (s, 9 H) ppm. LC/
MS (ES/APCI): 454 (M + H)+.
4-[(E)-2-(5-Bromo-3-tert-butyl-2-methoxyphenyl)vinyl]-
aniline (42). To a suspension of NaH (0.35 g, 8.73 mmol, 60% mineral
oil dispersion) and dry THF (9 mL) was added 15-crown-5 (0.27 mL,
1.36 mmol), and the resulting mixture was cooled to 0 °C. A solution of
diethyl (4-nitrobenzyl)phosphonate (2.33 g, 8.53 mmol) in dry THF
(10.5 mL) was added slowly while maintaining the temperature at 0 °C.
The mixture was stirred for 15 min, and then a solution of 5-bromo-3-
tert-butyl-2-methoxybenzaldehyde (29) (1.93 g, 7.12 mmol) and dry
THF (21 mL) was added. The reaction mixture was stirred at 0 °C for 2
h and then allowed to warm to room temperature and stirred for 18 h.
The reaction mixture was quenched with water (50 mL) and the
resulting solution thrice extracted with Et2O (3 × 60 mL). The
combined extracts were washed with brine, dried (Na2SO4), filtered, and
concentrated in vacuo. The crude product was purified over SiO2 by
chromatography (EtOAc/hexane, 5%) to afford 5-bromo-1-tert-butyl-2-
methoxy-3-[(E)-2-(4-nitrophenyl)vinyl]benzene (2.70 g, 98%). 1H
NMR (300 MHz, CDCl3) δ 8.24 (d, J = 8.7 Hz, 2 H), 7.66 (d, J = 8.7
Hz, 2 H), 7.60 (d, J = 2.3 Hz, 1 H), 7.51−7.34 (m, 2 H), 7.09 (d, J = 16.6
Hz, 1 H), 3.78 (s, 3 H), 1.40 (s, 9 H) ppm.
A mixture of 5-bromo-1-tert-butyl-2-methoxy-3-[(E)-2-(4-
nitrophenyl)vinyl]benzene (0.788 g, 2.02 mmol), iron (0.471 g, 8.44
mmol), NH4Cl (0.867 g, 16.2 mmol), MeOH (25 mL), and water (25
mL) was heated at reflux for 4 h. The reaction mixture was cooled to
room temperature and filtered. The filtrate was thrice extracted with
EtOAc and the combined extracts were washed with brine, dried
(Na2SO4), filtered, and concentrated in vacuo to afford 42 (0.71 g, 95%)
as a yellow solid which was used without further purification.
N-[4-[(E)-2-(5-Bromo-3-tert-butyl-2-methoxyphenyl)vinyl]-
phenyl]methanesulfonamide (43). To a solution of 42 (0.161 g,
0.45 mmol) and pyridine (5 mL) cooled to 0 °C was added
methanesulfonyl chloride (38 μL, 0.493 mmol). After being stirred for
45 min at 0 °C, the reaction mixture was washed with 1 N HCl and
extracted three times with EtOAc. The organic extracts were washed
with saturated CuSO4 solution three times and once with brine. The
extracts were dried over Na2SO4, filtered, and concentrated to give 43
(193 mg, 99%) as a brown oil. 1H NMR (300 MHz, CDCl3) δ 7.57 (d, J
= 2.3 Hz, 1 H), 7.53 (d, J = 8.3 Hz, 2 H), 7.34 (d, J = 2.3 Hz, 1 H), 7.29−
7.17 (m, 3 H), 6.99 (d, J = 16.4 Hz, 1 H), 6.50 (s, 1 H), 3.77 (s, 3 H), 3.05
(s, 3 H), 1.39 (s, 9 H) ppm.
1
A solution of N-[3-tert-butyl-2-methoxy-5-(2-methoxy-3-pyridyl)-
phenyl]-4-(methanesulfonamido)benzamide (0.12 g, 0.25 mmol), 48%
HBr (40 μL), and AcOH (3 mL) was heated in a sealed tube at 60 °C for
15 h. The reaction mixture was cooled to room temperature, diluted
with EtOAc, and washed with water. Upon addition of saturated
aqueous NaHCO3, a solid precipitated, which was collected and
triturated with Et2O to afford 5 (70 mg, 60%) as a white solid, mp
274.0−276.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 11.74 (br s, 1 H),
10.20 (s, 1 H), 9.87 (s, 1 H), 8.04 (d, J = 8.6 Hz, 2 H), 7.71 (d, J = 2.0 Hz,
1 H), 7.61 (dd, J = 6.8, 1.8 Hz, 1 H), 7.53 (d, J = 2.0 Hz, 1 H), 7.36 (dd, J
= 6.1, 1.5 Hz, 1 H), 7.30 (d, J = 8.6 Hz, 2 H), 6.29 (t, J = 6.6 Hz, 1 H),
3.72 (s, 3 H), 3.10 (s, 3 H), 1.39 (s, 9 H) ppm. LC/MS (ES/APCI): 470
(M + H)+.
5-(3-tert-Butyl-4-methoxyphenyl)-1H-pyridazin-6-one (40). A
dry round-bottom flask was charged with 4-bromo-2-tert-butylanisole
(39) (2.933 g, 12 mmol) and THF (15 mL), and magnesium turnings
(0.29 g, 12 mmol) were added. The reaction mixture was heated to
reflux and stirred for 45 min and then cooled to room temperature. The
resulting solution was added dropwise at room temperature to a stirred
solution of 4,5-dichloro-1H-pyridazin-6-one (0.796 g, 4.8 mmol), THF
(10 mL), and Et2O (20 mL). The reaction mixture was then heated at
reflux for 18 h. The mixture was cooled to 0 °C and quenched with
saturated aqueous NH4Cl and extracted with EtOAc (150 mL). The
aqueous phase was withdrawn and re-extracted with EtOAc (150 mL).
Each extract was washed sequentially with water and brine. The
combined organic extracts were dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was triturated with EtOAc/hexane
(1:1) to afford 5-(3-tert-butyl-4-methoxyphenyl)-4-chloro-1H-pyrida-
zin-6-one (1.087 g, 77%) as a white solid. 1H NMR (300 MHz, CDCl3)
δ 11.25 (br s, 1 H), 7.85 (s, 1 H) 7.53−7.35 (m, 2 H), 6.96 (d, J = 8.3 Hz,
1 H), 3.89 (s, 3 H), 1.39 (s, 9 H) ppm.
A Parr shaker bottle was charged with 5-(3-tert-butyl-4-methox-
yphenyl)-4-chloro-1H-pyridazin-6-one (1.08 g), a solution of KOH
(0.52 g in 11 mL of H2O), and DMF (1.3 mL). To this mixture was
added 10% Pd/C, and the bottle was connected to a Parr shaker and
flushed three times with H2 and then shaken for 18 h at room
temperature under an atmosphere of ∼50 psi of H2. To the resulting
solution was added 5 M KOH to dissolve the precipitate. Then the
solution was filtered through a glass microfiber filter and rinsed with 5 M
KOH and H2O. The filtrate was acidified with concentrated HCl and the
resulting mixture extracted with CH2Cl2 (100 mL). The aqueous layer
was withdrawn and re-extracted with EtOAc. The combined extracts
were dried (Na2SO4), filtered, and evaporated to afford 40 (0.791 g,
83%) as a white powder. 1H NMR (400 MHz, CDCl3) δ 10.78 (br s, 1
H), 7.83 (d, J = 4.0 Hz, 2 H), 7.72 (d, J = 2.0 Hz, 1 H), 7.30 (d, J = 4.0 Hz,
1 H), 6.95 (d, J = 8.6 Hz, 1 H), 3.89 (s, 3 H), 1.40 (s, 9 H) ppm.
5-(3-Bromo-5-tert-butyl-4-methoxyphenyl)-1H-pyridazin-6-
one (41). To a solution of 40 (0.1 g, 0.39 mmol) and DMF (2 mL) was
added NBS (0.069 g, 0.39 mmol), and the resulting solution was stirred
at 50 °C for 18 h. The mixture was concentrated in vacuo and the residue
partitioned between Et2O and water. The aqueous layer was withdrawn
and re-extracted with Et2O. The organic layers were twice washed with
water (5 mL) and once with brine (5 mL). The organic layers were
combined, dried (Na2SO4), filtered, and evaporated. The crude product
was purified over SiO2 by chromatography (EtOAc/hexane, 0 to 30%)
to afford 41 (0.068 g, 52%) as an off-white solid. 1H NMR (300 MHz,
CDCl3) δ 7.97 (d, J = 2.3 Hz, 1 H), 7.91−7.86 (m, 1 H), 7.77−7.73 (m, 1
H), 7.32 (d, J = 4.1 Hz, 1 H), 3.97 (s, 3 H), 1.42 (s, 9 H) ppm.
N-[4-[(E)-2-[3-tert-Butyl-2-methoxy-5-(6-oxo-1H-pyridazin-5-
yl)phenyl]vinyl]phenyl]methanesulfonamide (14). A microwave
tube was charged with 41 (0.068 g), N-[4-[(E)-2-(4,4,6-trimethyl-1,3,2-
dioxaborinan-2-yl)vinyl]phenyl]methanesulfonamide (0.078 g),
Na2CO3 (0.064 g), Pd(PPh3)4 (0.023 g), MeOH (1.8 mL), and
N-[4-[(E)-2-[3-tert-Butyl-2-methoxy-5-(4,4,5,5-tetramethyl-
1 , 3 , 2 - d i o x a b o r o l a n - 2 - y l ) p h e n y l ] v i n y l ] p h e n y l ] -
methanesulfonamide (44). A mixture of 43 (0.476 g, 1.086 mmol),
4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1,3,2-dioxaborolane (0.303 g, 1.19 mmol), PdCl2(PPh3)2 (0.177 g, 0.217
mmol), and KOAc (0.32 g, 3.26 mmol) under an Ar atmosphere was
dissolved in DMSO (25 mL). The reaction mixture was then heated to
90 °C for 18 h. The mixture was cooled to room temperature and
partitioned between EtOAc and water. The aqueous phase was extracted
with EtOAc, and the combined extracts were washed five times with
water and once with brine. The organic layers were dried, filtered, and
evaporated. The crude product was purified over SiO2 by chromatog-
raphy (EtOAc/hexane, 0−30%) to give 44 (0.311g, 53%) as a yellow oil.
1H NMR (300 MHz, CDCl3) δ 7.92 (s, 1 H), 7.78−7.67 (m, 1 H), 7.54
(d, J = 8.3 Hz, 2 H), 7.38−7.06 (m, 4 H), 6.37 (s, 1 H), 3.80 (s, 3 H), 3.04
(s, 3 H), 1.47−1.41 (m, 9 H), 1.38−1.33 (m, 12 H) ppm.
N-[4-[(E)-2-[3-tert-Butyl-2-methoxy-5-(6-oxo-1H-pyrimidin-
5-yl)phenyl]vinyl]phenyl]methanesulfonamide (15). A tube was
P
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charged with 44 (0.130 g, 0.144 mmol), 4-benzyloxy-5-bromopyr-
imidine (0.070 g, 0.264 mmol), Pd(PPh3)4 (0.028 g, 0.024 mmol),
Na2CO3 (0.077 g, 0.726 mmol), MeOH (3 mL), and CH2Cl2 (1 mL).
The tube and solution were sparged with Ar, sealed, and heated at 120
°C for 40 min. The solution was cooled and filtered through CELITE,
and the filtrate was concentrated in vacuo. The crude product was
purified over SiO2 by chromatography (EtOAc/hexane, 0−50%) to
afford N-[4-[(E)-2-[5-(4-benzyloxypyrimidin-5-yl)-3-tert-butyl-2-
methoxyphenyl]vinyl]phenyl]methanesulfonamide (45 mg, 34%) as
an off-white solid. 1H NMR (400 MHz, CDCl3) δ 8.78 (s, 1 H), 8.58 (s,
1 H), 7.67 (d, J = 2.0 Hz, 1 H), 7.56−7.43 (m, 5 H), 7.34 (d, J = 7.6 Hz, 4
H), 7.22 (d, J = 8.6 Hz, 2 H), 6.94 (d, J = 16.7 Hz, 1 H), 6.34 (s, 1 H),
5.53 (s, 2 H), 3.83 (s, 3 H), 3.05 (s, 3 H), 1.39 (s, 9 H) ppm.
To a solution of N-[4-[(E)-2-[5-(4-benzyloxypyrimidin-5-yl)-3-tert-
butyl-2-methoxyphenyl]vinyl]phenyl]methanesulfonamide (0.044 g,
0.081 mmol) and AcOH (1.5 mL) at room temperature was added
48% HBr (30 μL). The mixture was stirred at room temperature for 18
h, diluted with EtOAc, and poured into saturated NaHCO3. The
aqueous layer was extracted with EtOAc, and the combined extracts
were dried, filtered, and evaporated. The crude product was triturated
with EtOAc/Et2O to give 15 (26 mg, 71%) as an off-white solid, mp
253.0−255.0 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.73 (br s, 1 H),
9.83 (s, 1 H), 8.19 (d, J = 11.6 Hz, 2 H), 7.84 (s, 1 H), 7.63 (d, J = 8.1 Hz,
2 H), 7.57 (s, 1 H), 7.34−7.15 (m, 4 H), 3.77 (s, 3 H), 3.01 (s, 3 H), 1.39
(s, 9 H) ppm. LC/MS (ES/APCI): 454 (M + H)+.
solution of 46 (4.1 g, 7.7 mmol) in THF (10 mL). The resulting mixture
was stirred at 0 °C for 20 min. Then iodomethane (3.6 g, 26 mmol) was
added dropwise and the mixture warmed to room temperature and
stirred for 48 h. The reaction mixture was partitioned between Et2O and
saturated aqueous NH4Cl. The organic extract was washed sequentially
with water and brine, dried (Na2SO4), filtered, and concentrated in
vacuo. The crude product was purified over SiO2 by chromatography
(EtOAc/hexane, 0−10%) to afford diethyl 2-(3,5-dibromo-2-methox-
1
yphenyl)-2-methylpropanedioate (2.9 g, 84%) as a pale yellow oil. H
NMR (400 MHz, CDCl3) δ 7.64 (s, 1 H), 7.28 (s, 1 H), 4.30−4.15 (m, 4
H), 3.85 (s, 1 H), 1.81 (s, 3 H), 1.27 (t, J = 7.1 Hz, 6 H) ppm.
To a solution of diethyl 2-(3,5-dibromo-2-methoxyphenyl)-2-
methylpropanedioate (0.919 g, 2.1 mmol) in THF (4 mL) cooled to
0 °C was added dropwise LiAlH4 (2.40 mL, 2.4 mmol, 1.0 M solution in
THF). The reaction mixture was stirred at 0 °C for 1 h, and then
saturated aqueous potassium sodium tartrate was added and the
resulting mixture was stirred at room temperature for 2 h. The mixture
was extracted with EtOAc and the organic extract washed sequentially
with water and brine, dried (Na2SO4), filtered, and concentrated in
vacuo. The crude product was purified over SiO2 by chromatography
(EtOAc/hexane, 0−50%) to afford 47 (0.369 g, 52%) as a pale yellow
oil. 1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 2.5 Hz, 1 H), 7.56 (d, J =
2.0 Hz, 1 H), 4.15−4.06 (m, 2 H), 3.94 (s, 3 H), 3.90 (dd, J = 11.1, 5.6
Hz, 2 H), 2.46 (t, J = 5.6 Hz, 2 H), 2.05 (s, 3 H).
3-(3,5-Dibromo-2-methoxyphenyl)-3-methyloxetane (48). A
tube was charged with 47 (0.34 g, 0.97 mmol) and PPh3 (0.51 g, 1.93
mmol), flushed with Ar, and sealed. A solution of diisopropyl
azodicarboxylate (0.39 g, 1.93 mmol) and toluene (6 mL) was added
via syringe, and the resulting mixture was irradiated in a microwave
reactor at 140 °C for 30 min. The reaction mixture was concentrated in
vacuo and the crude product purified over SiO2 by chromatography
(EtOAc/hexane, 0−10%) to afford 48 (0.138 g, 43%) as a colorless oil.
1H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 2.5 Hz, 1 H), 6.91 (d, J = 2.5
N-[4-[(E)-2-[3-tert-Butyl-5-(5-fluoro-2-oxo-1H-pyridin-3-yl)-2-
methoxyphenyl]vinyl]phenyl]methanesulfonamide (19). A mi-
crowave vial was charged with 43 (0.193 g, 0.442 mmol), (5-fluoro-2-
methoxy-3-pyridyl)boronic acid (0.0771 g, 0.451 mmol), Na2CO3
(0.123 g, 1.16 mmol), Pd(PPh3)4 (0.027 g, 0.023 mmol), MeOH (1.2
mL), and CH2Cl2 (0.3 mL), sealed and irradiated in a microwave
synthesizer at 115 °C for 35 min. The vial was cooled to room
temperature and filtered, and the solid was washed with EtOAc. The
filtrate was washed sequentially with water and brine, dried (Na2SO4),
filtered, and concentrated in vacuo. The crude product was purified over
SiO2 by chromatography (EtOAc/hexane, 20−50%) to afford N-[4-
[(E)-2-[3-tert-butyl-5-(5-fluoro-2-methoxy-3-pyridyl)-2-
methoxyphenyl]vinyl]phenyl]methanesulfonamide (0.127 g, 59%) as a
Hz, 1 H), 4.98 (d, J = 6.1 Hz, 2 H), 4.50 (d, J = 5.6 Hz, 2 H), 3.87 (s, 3
H), 1.76 (s, 3 H) ppm.
3-[3-Bromo-4-methoxy-5-(3-methyloxetan-3-yl)phenyl]-1H-
pyridin-2-one (49). A microwave vial was charged with 48 (0.138 g,
0.411 mmol), (2-oxo-1H-pyridin-3-yl)boronic acid (0.063 g, 0.453
mmol), Na2CO3 (0.131 g, 1.24 mmol), Pd(PPh3)4 (0.047 g, 0.041
mmol), MeOH (3 mL), and CH2Cl2 (1 mL), sealed, and irradiated in a
microwave synthesizer at 115 °C for 30 min. The vial was cooled to
room temperature, filtered, and concentrated in vacuo. The crude
residue was partitioned between CH2Cl2 and water. The organic extract
was washed with brine. The aqueous layers were extracted with CH2Cl2.
The organic layers were combined, dried over Na2SO4, filtered, and
concentrated. The crude product was purified over SiO2 by
chromatography (EtOAc/hexanes, 0−100%) to afford 49 (0.095 g,
66%) as an off-white powder. 1H NMR (400 MHz, CDCl3) δ 11.79 (br
s, 1 H), 7.80 (d, J = 2.0 Hz, 1 H), 7.54 (dd, J = 7.1, 2.0 Hz, 1 H), 7.35 (dd,
J = 6.6, 2.0 Hz, 1 H), 7.15 (d, J = 2.0 Hz, 1 H), 6.37 (t, J = 6.8 Hz, 1 H),
5.07 (d, J = 5.6 Hz, 2 H), 4.55 (d, J = 5.6 Hz, 2 H), 3.92 (s, 3 H), 1.80 (s, 3
H) ppm.
N-[4-[(E)-2-[2-Methoxy-3-(3-methyloxetan-3-yl)-5-(2-oxo-
1H-pyridin-3-yl)phenyl]vinyl]phenyl]methanesulfonamide
(24). A microwave vial was charged with 49 (0.095 g, 0.271 mmol), N-
[4-[(E)-2-(4,4,6-trimethyl-1,3,2-dioxaborinan-2-yl)vinyl]phenyl]-
methanesulfonamide (0.132 g, 0.408 mmol), Na2CO3 (0.086 g, 0.811
mmol), Pd(PPh3)4 (0.031 g, 0.027 mmol), MeOH (3 mL), and CH2Cl2
(1.5 mL), sealed, and irradiated in a microwave synthesizer at 120 °C for
1 h. The vial was cooled to room temperature, filtered, and concentrated
in vacuo. The crude residue was partitioned between CH2Cl2 and water.
The organic extract was washed with brine. The aqueous layers were
extracted with CH2Cl2. The organic layers were combined, dried over
Na2SO4, filtered, and concentrated. The crude product was purified over
SiO2 by chromatography (MeOH/CH2Cl2, 0−10%) to afford 24 (0.081
g, 64%) as a white powder, mp 279.0−281.0 °C. 1H NMR (300 MHz,
DMSO-d6) δ 11.76 (br s, 1 H), 9.85 (s, 1 H), 7.89 (d, J = 2.1 Hz, 1 H),
7.70 (dd, J = 7.0, 2.1 Hz, 1 H), 7.62 (d, J = 8.7 Hz, 2 H), 7.39 (d, J = 5.3
Hz, 1 H), 7.30−7.12 (m, 5 H), 6.30 (t, J = 6.8 Hz, 1 H), 4.91 (d, J = 5.7
1
yellow oil. H NMR (300 MHz, CDCl3) δ 7.99 (d, J = 3.0 Hz, 1 H),
7.74−7.28 (m, 9 H), 7.03 (d, J = 16.2 Hz, 1 H), 3.98 (s, 3 H), 3.83 (s, 3
H), 3.04 (s, 3 H), 1.45 (s, 9 H) ppm.
A vial was charged with N-[4-[(E)-2-[3-tert-butyl-5-(5-fluoro-2-
methoxy-3-pyridyl)-2-methoxyphenyl]vinyl]phenyl]-
methanesulfonamide (0.087 g, 0.18 mmol), AcOH (2 mL), and 48%
HBr (38 μL), sealed, and heated at 60 °C for 18 h. The mixture was
cooled to room temperature and added to a mixture of ice and saturated
aqueous NaHCO3. The resulting mixture was thrice extracted with
EtOAc and the combined extracts were washed sequentially with
saturated aqueous NaHCO3 and brine, dried (Na2SO4), filtered, and
concentrated in vacuo to afford 19 (0.071 g, 84%) as a solid. 1H NMR
(400 MHz, DMSO-d6) δ 9.82 (s, 1 H), 7.88 (d, J = 2.0 Hz, 1 H), 7.83
(dd, J = 8.8, 3.3 Hz, 1 H), 7.68−7.52 (m, 4 H), 7.35−7.13 (m, 4 H), 3.77
(s, 3 H), 3.01 (s, 3 H), 1.39 (s, 9 H) ppm. LC/MS (ES/APCI): 471 (M +
H)+.
Diethyl 2-(3,5-Dibromo-2-methoxyphenyl)propanedioate
(46). A mixture of 1,5-dibromo-3-iodo-2-methoxybenzene (45) (5.4
g, 14 mmol), diethyl malonate (4.0 g, 25 mmol), CuI (0.13 g, 0.69
mmol), 2-phenylphenol (0.24 g, 1.4 mmol), and Cs2CO3 (6.8 g, 21
mmol) in THF (14 mL) was stirred at 70 °C for 15 h. The resulting
mixture was cooled to room temperature and partitioned between
EtOAc and saturated aqueous NH4Cl. The organic extract was then
washed sequentially with water and brine, dried (Na2SO4), filtered, and
concentrated in vacuo. The crude product was purified over SiO2 by
chromatography (EtOAc/hexane, 0−10%) to afford 46 (4.1 g, 55%) as a
pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J = 2.0 Hz, 1 H),
7.28 (d, J = 2.0 Hz, 1 H), 4.24 (dd, J = 7.1, 4.5 Hz, 4 H), 4.12 (q, J = 7.1
Hz, 1 H), 3.85 (s, 3 H), 1.33−1.17 (m, 6 H) ppm.
2-(3,5-Dibromo-2-methoxyphenyl)-2-methylpropane-1,3-
diol (47). To a suspension of NaH (0.86 g, 22 mmol, 60% dispersion in
mineral oil) in THF (50 mL) cooled to 0 °C was added dropwise a
Q
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Hz, 2 H), 4.45 (d, J = 5.7 Hz, 2 H), 3.74 (s, 3 H), 3.01 (s, 3 H), 1.72 (s, 3
H) ppm. LC/MS (ES/APCI): 467 (M + H)+.
Computational Chemistry. Molecular modeling was performed
using the Molecular Operating Environment (MOE) software.39
Figures were generated using PyMOL, version 1.5.0.4.40
HCV NS5B Polymerase Biochemical Assay Protocol. The
enzymatic activity of HCV polymerase (NS5B570n-Con1) was
measured as described previously26 with only two changes as follows:
concentration of RNA template was 3 nM, and concentration of
NS5B570n-Con1 enzyme was 3 nM.
Stable HCV Replicon Assay. The stable GT 1b Con1 and GT 1a
H77 subgenomic replicon cell lines expressing the renilla luciferase as a
reporter gene (cell lines 2209-23 and pRLucH771b75S/I, respectively)
were used as described by Le Pogam et al.41
Pharmacokinetics. Male Wistar/Han (CRL:WI) rats (Charles
River Laboratories, Hollister, CA or Raleigh, NC) used in these studies
weighed between 180 and 300 g and were surgically implanted with
cannulas in the jugular and/or femoral veins. All studies were conducted
under IACUC approved protocols, and animals were allowed free access
to food and water. Single intravenous bolus doses were administered
into the tail vein or into the jugular cannula of dual-cannulated animals
using dosing formulations consisting of DMSO (<10%)/PEG 400/
water; the remaining cannulas were used for blood collection. Single oral
doses were administered by gavage using a dosing formulation
consisting of hypromellose 2910 (0.5%), polysorbate 80 (0.4%), and
benzyl alcohol (0.9%) in water, unless otherwise specified. Blood was
collected into tubes containing EDTA at typically 0.08 (iv only), 0.25,
0.5, 1, 2, 4, 6, 8, and 24 h after dosing. Plasma concentrations were
determined by a liquid chromatography/tandem mass spectrometry
method (LC/MS/MS) following protein precipitation with acetonitrile.
Transient HCV Replicon Assay Using a Panel of GT 1 NS5B
Clinical Isolates. The GT 1b Con1 and GT 1a H77 transient replicons
in which the wild type NS5B gene has been replaced by NS5B isolates
obtained from untreated patients were used as described by Le Pogam et
al.35
ASSOCIATED CONTENT
■
S
* Supporting Information
Time Dependent CYP3A4 Inhibition. Stock solutions of test
compound, midazolam, and 17α-ethynylestradiol were prepared in 5%
DMSO in acetonitrile. Midazolam, 17α-ethylnylestradiol, and α-
nicotinamide adenine dinucleotide phosphate, reduced form (0-
NADPH), were obtained from Sigma Chemical Co. Human liver
microsomes (HLM, lot 0510222) were purchased from XenoTech, LLC
(Lenexa, KS), prepared from a mixed-gender pool of 50 human livers.
The assay was performed with a two-stage robotic procedure in a 96-well
format using Biomek 2000 (Beckman) for liquid handling. Inactivation
mixtures (180 μL/well) containing 1 mg/mL HLM (Xenotech), 3 mM
MgC12, 1 mM EDTA, and 10 μM test compound in 100 mM potassium
phosphate buffer (pH 7.4) were incubated at 37 °C in a 96-well plate.
After 3 min of preincubation, the reactions were initiated by adding 20
μL of 10 mM NADPH and allowed to proceed up to 20 min. Verapamil
and ethynylestradiol at 10 μM were included as positive controls for
time-dependent CYP3A4 inactivation.42,43 At 0, 2, 6, 12, and 20 min
after the start of the preincubation, an amount of 10 μL of the
preincubation mixtures was transferred to activity assay mixtures (90
μL/well, prewarmed at 37 °C) containing 3 mM MgC12, 1 mM EDTA,
1 mM NADPH, and 10 μM of midazolam in 100 mM potassium
phosphate buffer (pH 7.4). The final mixtures were incubated for 5 min
and quenched with 100 μL of 0.1% formic acid in acetonitrile containing
2 μg/mL 7-hydroxycoumarin as the internal standard. The quenched
mixtures were transferred and further diluted in a deep-well plate. After
centrifugation, the supernatant was analyzed by HPLC−MS/MS.
Metabolite Identification. Human liver microsomal proteins were
purchased from BD Gentest, Woburn, MA. The incubation mixture
contained 50 mM potassium phosphate, pH 7.4, 5 mM magnesium
chloride, 2 mM NADPH, test compound (20 μM), and microsomal
protein (0.5 mg/mL). The total volume of each incubation mixture was
0.5 mL. All reagents except NADPH were combined in a 13 mL round-
bottom glass test tube on ice. The samples were preincubated for
approximately 2 min at 37 °C in a thermostatically controlled shaking
water bath. The reaction was started by the addition of NADPH. The
reaction was stopped at 30 min by quenching with 0.5 mL of methanol.
For each microsomal incubation, one control sample (30 min) without
NADPH was incubated together with the samples. The quenched
samples were kept on ice (10−15 min) and then at room temperature,
vortexed briefly, and centrifuged. The supernatant was transferred to test
tubes, evaporated to partial dryness (∼0.2 mL), then transferred to an
autosampler vial. Samples were analyzed by RP-HPLC/UV/MS. Low
resolution mass spectrometric analysis was carried out on a
ThermoFinnigan iontrap mass spectrometer (model LTQ) equipped
with an electrospray ionization source. The heated capillary temperature
in the source was held at 250 °C. In the standard screening mode, the
trap collected positive m/z 150 to m/z 800 scans. Collision-induced
dissociation tandem MS was conducted with 40% relative collision
energy and an isolation width of 3 amu. Product ion analyses were
performed following collision induced dissociation of selected precursor
ions to obtain substructural fragment ion information. MS3 experiments
were also performed.
Synthetic procedures and spectroscopic data, metabolite
identification study of compound 4 (MS/MS/MS), HCV
NS5B polymerase protein crystallography, and references. This
material is available free of charge via the Internet at http://pubs.
acs.org.
Accession Codes
Atom coordinates and structure factors for HCV NS5B/ligand
complexes have been deposited at the Protein Data Bank under
the codes 4MK7 (2), 4MK8 (4), 4MK9 (12), 4MKA (13),
4MKB (14).
AUTHOR INFORMATION
Corresponding Author
*Phone: +1-908-202-7795. E-mail: ryan.schoenfeld@
chdifoundation.org.
■
Present Address
†R.C.S.: CHDI Management/CHDI Foundation, 155 Village
Boulevard, Suite 200, Princeton, NJ 08540.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge all members of the Analytical
Department in Roche Palo Alto and the Physical Chemistry
Department at Hoffmann-La Roche, Nutley, NJ, for character-
ization of the compounds. The Advanced Light Source is
supported by the Director, Office of Science, Office of Basic
Energy Sciences, of the U.S. Department of Energy under
Contract DE-AC02-05CH11231. Portions of this research were
carried out at the Stanford Synchrotron Radiation Lightsource, a
Directorate of SLAC National Accelerator Laboratory and an
Office of Science User Facility operated for the U.S. Department
of Energy Office of Science by Stanford University. The SSRL
Structural Molecular Biology Program is supported by the DOE
Office of Biological and Environmental Research and by the
National Institutes of Health, National Institute of General
Medical Sciences (including Grant P41GM103393) and the
National Center for Research Resources (Grant P41RR001209).
The contents of this publication are solely the responsibility of
the authors and do not necessarily represent the official views of
NIGMS, NCRR or NIH.
ABBREVIATIONS USED
HCV, hepatitis C virus; NS5B, nonstructural 5B
■
R
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E. J. Sofosbuvir for previously untreated chronic hepatitis C infection. N.
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