Inhibitors of hepatitis C virus polymerase: Synthesis and biological characterization of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines

Article abstract of DOI:10.1021/jm8010965

The hepatitis C virus (HCV) NS5B polymerase is essential for viral replication and has been a prime target for drug discovery research. Our efforts directed toward the discovery of HCV polymerase inhibitors resulted in the identification of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines 2 and 3. The most active compound displayed activity in genotypes 1a and 1b polymerase and replicon cell culture inhibition assays at subnanomolar and low nanomolar concentrations, respectively. It also displayed an excellent pharmacokinetic profile in rats, with a plasma elimination half-life after intravenous dosing of 4.5 h, oral bioavailability of 77%, and a peak liver concentration of 21.8 μg/mL.

Full text of DOI:10.1021/jm8010965

J. Med. Chem. 2009, 52, 1659–1669
1659
Inhibitors of Hepatitis C Virus Polymerase: Synthesis and Biological Characterization of
Unsymmetrical Dialkyl-Hydroxynaphthalenoyl-benzothiadiazines
Rolf Wagner,* Daniel P. Larson,* David W. A. Beno, Todd D. Bosse, John F. Darbyshire,† Yi Gao, Bradley D. Gates,
Wenping He,‡ Rodger F. Henry, Lisa E. Hernandez, Douglas K. Hutchinson, Wen W. Jiang,§ Warren M. Kati, Larry L. Klein,|
Gennadiy Koev, William Kohlbrenner, A. Chris Krueger, Jinrong Liu, Yaya Liu, Michelle A. Long, Clarence J. Maring,
Sherie V. Masse, Tim Middleton, Debra A. Montgomery, John K. Pratt, Patricia Stuart, Akhteruzzaman Molla, and
Dale J. Kempf
Global Pharmaceutical Research and DeVelopment, Abbott, Abbott Park, Illinois 60064
ReceiVed September 2, 2008
The hepatitis C virus (HCV) NS5B polymerase is essential for viral replication and has been a prime target
for drug discovery research. Our efforts directed toward the discovery of HCV polymerase inhibitors resulted
in the identification of unsymmetrical dialkyl-hydroxynaphthalenoyl-benzothiadiazines 2 and 3. The most
active compound displayed activity in genotypes 1a and 1b polymerase and replicon cell culture inhibition
assays at subnanomolar and low nanomolar concentrations, respectively. It also displayed an excellent
pharmacokinetic profile in rats, with a plasma elimination half-life after intravenous dosing of 4.5 h, oral
bioavailability of 77%, and a peak liver concentration of 21.8 µg/mL.
Introduction
The current standard of therapy for chronic HCV infection
consists of combination treatment with pegylated interferon and
ribavirin. While population genetics studies of HCV indicate
an extremely diverse collection of genotypes (1-6) worldwide,
HCV genotype 1 patients comprise ∼75% of HCV infections
in the U.S., Western Europe, and Japan and ∼60% of HCV
infection in Latin America.10 Unfortunately, due to contrain-
dications, only 40% of this population is eligible for therapy
and 75% of this population refuse to undergo therapy, or drop
out of treatment due to severe side effects associated with the
drug regimen. Within those that remain on therapy, the infection
cure rate is estimated to be only 50%.11 For HCV genotype
1-infected individuals with additional risk factors, such as
cirrhosis, HIV coinfection, dialysis treatment, or organ trans-
plants, cure rates are substantially lower.12 Clearly, there is a
compelling need for new agents that will bolster the viral cure
rate for this segment of the patient population.
Hepatitis C virus (HCV) was unambiguously characterized
in 1989 as the organism responsible for non-A and non-B
hepatitis.1 Because of an estimated 170 million people world-
wide chronically infected with HCV, this virus represents a
global public health problem.2 After their initial acquisition of
the virus, HCV-infected individuals pass through an acute phase,
followed in most cases by development of chronic infection.3
The majority of infected individuals remain asymptomatic for
many years after the acute phase, with hepatic fibrosis progress-
ing as the main complication of chronic HCV infection. While
there is tremendous variation in the rate of fibrosis progression
to cirrhosis, with no treatment, the median progression to
cirrhosis is 30 years. Of this population, a substantial fraction
will continue to experience progression of disease into hepa-
tocellular carcinoma.4 Complications due to chronic HCV
infection have now become the predominant driver for liver
transplantation.5 Additionally, the leading cause of death upon
kidney transplantation, which is the most commonly performed
organ allograft, is due to chronic liver disease, most commonly
precipitated by hepatitis C infection.6 In 2004, 2.44 deaths per
100000 people could be attributed to complications due to HCV
infection in the U.S.7 According to one predictive model, this
number is expected to rise to ∼27700 deaths per year by 2010
and peak in 2030 at ∼39800 deaths per year,8 although a more
conservative prediction places the peak mortality level in 2030
at 12900 deaths.9
The nonstructural protein 5B (NS5B) of HCV possesses
RNA-dependent RNA polymerase activity, which is an activity
that is not found in host liver cells.13 The RNA polymerase
plays an essential role in viral replication by directing the
synthesis of both the replicative intermediate minus-strand RNA
as well as the progeny plus-strand RNA molecules.14 In general,
viral polymerases are attractive drug targets, as shown by the
success of viral polymerase inhibitors such as acyclovir for
herpes virus infections, various nucleoside and non-nucleoside
reverse transcriptase inhibitors for HIV, and lamivudine for HBV
infections. More specifically, it has now been shown that
inhibition of NS5B in HCV-infected patients leads to reduction
in viral load. While nucleoside HCV polymerase inhibitors have
garnered the most clinical attention in this mechanistic class,
multiple non-nucleoside inhibitors have already entered into
early trials and are beginning to show significant promise.15
* To whom correspondence should be addressed. (R.W. and D.L.) Phone:
847-937-1138. Fax: 847-938-6603. E-mail: rolf.wagner@abbott.com;
daniel.p.larson@abbott.com. Address: Abbott GPRD, Antiviral Research
Department, R4CQ, Building AP52 200, Abbott Park Road, Abbott Park,
IL 60064-6217.
†
Current Address: Nycomed, Technical Development, Konstanz, Germany.
Current Address: Johnson & Johnson Pharmaceutical Research &
‡
Rationale. Our prior efforts resulted in the discovery of
compound 1.16 Although, this compound demonstrated high in
vitro potency and had a good pharmacokinetic profile, it and
its sodium salt, 1a, were plagued by poor aqueous solubility.
As a response to the solubility limitation of compound 1, a
second-generation core was investigated in which the aza
Development LLC, Preclinical Anti-Infectives, Internal Medicine, 1000 US
Highway 202 South, OMP B206, Raritan, New Jersey 08869.
§
Current Address: Midwestern University, College of Pharmacy, 555
31st Street, Downers Grove, Illinois 60515.
|
Current Address: Institute for Tuberculosis Research, College of
Pharmacy, University of Illinois at Chicago, 833 South Wood Street,
Chicago, Illinois 60612.
10.1021/jm8010965 CCC: $40.75
2009 American Chemical Society
Published on Web 02/18/2009

1660 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Wagner et al.
ate would be the most effective solution to this synthetic
problem. We also determined that resolution of the stereoisomers
at an early stage of the synthesis would be the most efficient
approach.
The chiral synthesis was adapted from the synthesis developed
for nonchiral dialkyl analogues. The synthesis of enantiomeri-
cally pure analogues was accomplished by a 13-step general
procedure.
Figure 1. HCV polymerase inhibitor lead structures.
Shown in Scheme 1, the racemic dialkyl-carboxylic acids 7
and 8 were prepared by reaction of the dianion of carboxylic
acid 4 with either of the appropriate alkyl halide 5 or 6. The
acid chlorides of racemic phenyl acetic acids 7 and 8 were
allowed to react with (S)-phenylglycinol to give diastereomeric
mixtures that were easily separated by flash column chroma-
tography to give amides 9a, 9b, 10a, and 10b. At this point,
the relative stereochemical configuration at the quaternary center
of 10a was determined by a single crystal X-ray experiment.
With the configuration of the phenylglycinol fragment already
established, the absolute stereochemistry could therefore be
assigned. Each of the diastereomeric amides was then hydro-
lyzed to give the enantiomerically pure phenyl acetic acids 11a,
11b, 12a, and 12b.
Scheme 1a
With the necessary enantiomerically pure building blocks in
hand, the remaining syntheses of the optically active thiadiazines
were completed in tandem. Although syntheses of the biologi-
cally active enantiomers are depicted in Scheme 2, the inactive
enantiomers were obtained by the same methodology, and
ultimately the absolute configuration of virologically active 25a
was assigned by analogy to 26a. The corresponding acid
chlorides of carboxylic acids 11a and 12a were allowed to react
with the magnesium enolate of diethyl malonate, and the
resulting ketodiesters 13a and 14a were then cyclized to the
dihydronapthalene esters 15a and 16a by treatment with
methanesulfonic acid. Esters 15a and 16a were decarboxylated
and converted to dithioketene acetals 19a and 20a, which were
treated with (4-amino-3-sulfamoyl-phenyl)-carbamic acid tert-
butyl ester. The syntheses were completed by deprotection of
the aniline group of the tetracyclic compounds 21a and 22a,
followed by reaction with methanesulfonyl chloride provided
25 and 26. Final treatment with aqueous sodium hydroxide gave
the enantiomerically pure target compounds 25a and 26a.
a Key: (a) LiHMDS, THF; (b) (COCl)2, DMF, hexane; (c) (S)-
phenylglycinol, Et3N, CH2Cl2, DMAP; (d) chromatographic resolution; (e)
H2SO4, dioxane.
Results and Discussion
functionality was replaced by a dialkyl motif. We speculated
that the high degree of planarity of the tetracyclic ring system
resulted in the poor solubility characteristics of compound 1.
The intention of the dialkyl replacement was to disturb the
crystal packing forces of the flat tetracyclic system, thereby
improving dissolution rates of the resultant analogues. This
approach resulted in the identification of the racemates 2 and
3.17 The in vitro profiles of these new structures were compa-
rable to compound 1, and anecdotally, we observed these
dialkylated analogues, 2 and 3, to have better solubility
characteristics than 1. The logical path forward in this research
program was to address the chiral aspect of the dialkyl
compounds and develop a more complete biological profile of
the more active enantiomers.
The primary in vitro tools used to screen analogues was a
polymerase enzyme inhibition assay as well as a subgenomic
HCV cell culture system. Although HCV has a very robust
replication rate in vivo, efforts to propagate the virus in cell
culture systems have been largely unsuccessful for genotype 1
despite the success of a genotype 2a infectious system.18 This
difficulty has led to the development of subgenomic HCV
replicons that can be propagated efficiently in the human
hepatoma cell line Huh-7.19 These replicons contain 5′ and 3′
nontranslated regions as well as all of the HCV nonstructural
genes. Similar to conventional tissue culture and in vivo viral
replication, HCV replicon RNA efficiently replicates by under-
going a cycle consisting of translation, polyprotein cleavage (by
the NS3 protease), RNA synthesis (by the NS5B polymerase),
unwinding of the double-stranded RNA (by the NS3 helicase),
and assembly of replication complexes containing the HCV
nonstructural proteins within Huh-7 cells. This system produces
1000-10000 copies of RNA per cell, which is substantially
greater than that estimated for in vivo infection (50-100
particles/cell). However, unlike conventional tissue culture and
in vivo viral infection, this system is limited by the lack of
Chemistry. The primary requirements for the synthesis of
the pure enantiomers of racemates 2 and 3 were 2-fold.
Foremost, the materials obtained from the syntheses needed to
have a high degree of enantiomeric purity. Additionally, we
required a practical synthesis of multigram quantities of material.
Given the unique challenges presented in an enantioselective
synthesis of the quaternary stereocenter, we determined that an
approach that allowed for the resolution of a racemic intermedi-

Inhibitors of Hepatitis C Virus Polymerase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1661
Scheme 2a
a Key: (a) (COCl)2, DMF, hexane; (b) diethyl malonate, MgCl2, Et3N, CH3CN; (c) CH3SO3H; (d) 1 N HCl, dioxane; (e) (bis-methylsulfanyl-methylene)-
methyl-sulfonium tetrafluoroborate, pyridine, dioxane; (f) tert-butyl 4-amino-3-sulfamoylphenylcarbamate, toluene; (g) HCl/dioxane; (h) CH3SO2Cl, pyridine,
acetone; (i) NaOH, water.
Table 1. Biochemical and Antiviral Activitya
HCV polymerase inhibition IC50 (nM)
HCV replicon EC50 (nM)
cytotoxicity TD50(nM) HCV replicon + 40% human
genotype 1a
genotype 1b
genotype 1a
genotype 1b
0.7
Huh-7 MTT
serum EC50 (nM) 1b
serum effect
1
2.0
1.6
3
>10000
46
66×
n ) 2 (1.5 to 2.7)
n ) 1
n ) 1
n ) 2(0.63-0.86) n ) 2(>10000, >30000)
n ) 2(41.4-50.0)
25a
25b
26a
26b
1.3
0.9
11
3
35300
n ) 1
42
14×
ND
n ) 4 (0.7 to 2.3)
n ) 4 (0.5 to 1.4) n ) 10 (8.5-13.6) n ) 10 (0.8-4.2)
n ) 7 (19.5-58.7)
624
2300
ND
ND
ND
ND
n ) 3 (370-1000) n ) 3 (830-6400)
0.7
0.6
4
2
21700
n ) 5 (14000-33000)
28
17×
ND
n ) 3 (0.5 to 0.9)
n ) 3 (0.5 to 0.6) n ) 10 (2.9-5.8) n ) 5 (1.1-3.0)
n ) 5 (22.0-33.2)
444
518
ND
ND
ND
ND
n ) 3 (405-468)
n ) 3 (395-632)
a ND: not determined.
assembly and release of virus particles. Therefore, the replicon
system is incapable of producing infectious virus, and viral
infection of new cells (entry) does not occur. Nevertheless, the
use of HCV replicons to evaluate HCV inhibitors has been
validated with the NS3 protease inhibitor BILN-2061. This
compound has an EC50 of 3-4 nM against 1a and 1b
subgenomic replicons in cell culture. When this compound was
dosed for 48 h in HCV genotype 1-infected patients, it produced
a 2-3 log10 drop in viral titer.20 The potency of these current
compounds are reported as EC50 values against the subgenomic
genotype 1a and 1b replicons measuring the reduction of HCV
RNA by real time PCR. To establish the effect of nonspecific
protein binding on the antiviral activity, we tested these
compounds in the presence of 5% fetal calf serum (FCS) with
or without 40% human serum.
Preparation and in vitro profiling of the corresponding
optically active isomers of compounds 2 and 3 revealed the (R)-
configured isomers 25a and 26a to be the more active stereoi-
somers. Both compounds 25a and 26a inhibited genotypes 1a
and 1b polymerase activity and HCV replicon replication at low
nanomolar concentrations. These data compare similarly to the
corresponding data obtained for lead compound 1. (Table 1.)

1662 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Wagner et al.
Table 2. Physical Property Comparison of 1a and 26a
solubility at 25 °C (mg/mL)
melting point (°C)
compd
water (pH 4.0)a
water (pH 7.4)b
PEG400
logD (pH 7.4)
non-salt form
sodium salt
1a
26a
not available
<0.0002
<0.00007
0.11 ( 0.004
>191.8
150
1.45 ( 0.02
2.26 ( 0.0045
295-297 decomp.
264-272
>375
129c
a 50 mM citrate buffer. b 50 mM phosphate buffer. c amorphous glass transition temperature.
Figure 2. (a) Rat IV and Oral Pharmacokinetics upon 5 mg/kg dosing of 25a and 26a, 2b. (b) Major metabolite of 25a isolated from rat bile.
Table 3. Rat Pharmacokinetic Parameters of 25a and 26a, iv/Oral Dosing (5 mg/kg)
liver concentrations after
iv dose (5 mg/kg)
AUC
(µg·h/mL) (L/h·kg)
oral dose (5 mg/kg)
oral dosing (5 mg/kg)
CLp
Cmax
AUC
(µg·h/mL)
6 h
12 h
t1/2 (h) Vss (L/kg) Vꢀ (L/kg)
Tmax (h)
(µg/mL)
F (%)
t1/2 (h)
(µg/g)
(µg/g)
25a
26a
1.2
4.5
1.72
2.22
1.97
2.39
5.2
14.9
0.96
0.34
7.6
0.31
0.89
4.2
9
3.3
11.5
62.7
77.3
3.8
21.8
1.43
4.19
unable to calculate
Table 4. Comparison of Dog and Monkey PK Parameters for Compounds 25a and 26a
iv dose
oral dose
species
t1/2 (hr) Vss (L/kg) Vꢀ (L/kg) AUC (µg·h/mL) CLp (L/h ·kg) t1/2 (hr) Cmax (µg/mL) Tmax (hr) AUC (µg·h/mL) F (%)
25a dog (5 mg/kg)
2.7
0.7
1.10
0.19
1.74
0.50
11.8
10.2
0.44
0.51
10.7
1.9
1.94
0.52
0.4
2.7
10.5
1.5
89.3
15.2
monkey (5 mg/kg)
26a dog (2.5 mg/kg)
12.3
2.1
0.38
0.77
0.46
1.77
115.6
10.2
0.02
0.56
14.8
2
5.7
0.48
2.2
2.7
121
1.9
∼100
19
monkey (5 mg/kg)
Table 5. Multispecies in Vitro Microsomal and Hepatocyte Metabolism
of 25a and 26a
ingly, Table 2 shows significantly different phase transition
points of nonsalts 1 and 26. We interpret this observation, in
part, as indicating tighter self-association of 1 vs 26, perhaps
due to the flatter shape of the molecule. In comparison, the
sodium salt form of amorphous 1a shows little change in melting
point, however, the sodium salt of 26a is much higher due to a
combination of its crystallinity and greater ionic character of
the deprotonated diketone sodium salt than the deprotonated
3-ketoamide of 1.
intrinsic clearance
(µL/min/mg)
compd
assay system
rat
dog
monkey
human
25a
microsomes
hepatocytes
microsomes
hepatocytes
36.3
9.0
8.7
1.6
3.5
2.2
2.4
0.5
62.0
29.2
14.4
3.5
29.6
6.8
23.6
2.7
26a
Compound 25a, our initial in vivo study compound, dem-
onstrated an encouraging pharmacokinetic profile in rats (Figures
1 and 2a, Table 3). Following intravenous dosing, the plasma
elimination half-life was 1.2 h and the plasma clearance value
was 0.96 L/h ·kg. Oral dosing resulted in bioavailability of 63%.
A study performed on bile duct cannulated rats indicated that
biliary secretion was the major excretory route in rats. Microso-
mal metabolism studies indicated that the major metabolite
showed an increase in molecular weight of 16 amu. Mass
spectral fragment analysis of this metabolite indicated that it
was formed by oxidation of tertiary carbon of the terminal alkyl
chain, yielding carbinol 27 (Figure 2b). This structure was
confirmed by independent synthesis.21
However, the potencies of compounds 25a and 26a were
attenuated by the presence of 40% human serum in the replicon
assay by 14- and 17-fold, respectively, which compared
favorably to the 66-fold attenuation observed with com-
pound 1.
The subjective observation that the dialkyl-series compounds
had physical characteristics that were superior to the aza-series
compounds was borne out by solubility studies of compounds
1a and 26a (Table 2). In aqueous 50 mM phosphate buffer
adjusted to pH 7.4 at 25 °C, the solubility of compound 26a
exceeded that of compound 1a by at least 3 orders of magnitude.
In acidic aqueous buffer, however, 26 suffered from significant
insolubility and was not detected in the sample indicating a
solubility less than 0.2 µg/mL. In the more organic medium of
PEG400, the solubility differences, between 1a and 26a were
less evident, with some advantage being yielded to 1a. HPLC
was used to determine the LogD at pH 7.4 and provided a
partition coefficient for compound 26a higher than 1a. Interest-
Given the site of metabolism of compound 25a, we rational-
ized that compound 26a might have a superior pharmacokinetic
profile due to the blocking effect of an extra methyl group on
its alkyl side chain.

Inhibitors of Hepatitis C Virus Polymerase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1663
Table 6. Concentrations of 25 and 26 in Rat Liver after Oral dosing
rat plasma concentrations after
oral dosing (5 mg/kg)
rat liver concentrations after
oral dosing (5 mg/kg)
fold over 1b EC50 in presence
of 40% human serum
compd
6 h (nM)
12 h (nM)
6 h (nM)
12 h (nM)
6 h
12 h
25
26
445
2240
135
320
7340
39300
2860
7550
175
1404
68
270
A pharmacokinetic study of compound 26a in rat confirmed
a superior profile to compound 25a. The plasma elimination
half-life after intravenous dosing improved by nearly 4-fold to
4.5 h, and the plasma clearance dropped by 3-fold to 0.34
L/h· kg. While oral bioavailability improved only slightly, it
should be noted that the oral exposure more than tripled to 11.5
µg·h/mL when compared to analogue 25a. One peculiar aspect
to the oral pharmacokinetic behavior of compound 26a relates
to its prolonged oral half-life, precluding our ability to determine
its value in this experiment. Although the solubility character-
istics were improved in comparison to compound 1, we believe
that the relatively flat dose/plasma concentration response curve
is due to dissolution rate limited absorption resultant from
protonation of 26a in the acidic stomach environment. Indeed,
Table 2 reports the significant insolubility of 26a in acidic
aqueous media.
Further characterization of the pharmacokinetics in dog and
monkey underscored the differences of 26a in comparison to
25a. In both species, the iv half-life of 26a was significantly
improved relative to 25a. The iv data for 26a are consistent
with metabolic stability and profiling of 26a in rat, dog, and
monkey where the dog liver microsomes did not produce a key
metabolite that was found in the other species. While this
metabolite was isolated from rat bile and characterized by mass
spectral fragmentation, tentatively assigning the structure as that
shown in Figure 3, lack of production of 28 in the dog was
also reflected in reduced hepatocyte clearance (Table 4 and 5).
Unfortunately, attempts to synthesize metabolite 28 were met
with failure and therefore thwarted our efforts to provide for
an independent verification of this structure.
While the IV PK parameters show a significant improvement
for 26a relative to 25a across all species examined, the
advantage of 26a upon oral administration is less obvious.
Nevertheless, the improved pan-species microsomal/hepatocel-
lular stability of 26a in comparison to 25a (Table 5) cause it to
be the preferred compound to test in human subjects. Demon-
stration of antiviral activity in patients is a key goal of this
program, however, it is impossible to predict with certainty the
pharmacokinetic behavior of 26a in HCV infected human
subjects.
In an attempt to understand liver uptake, hepatic distribution
studies of 25a and 26a were conducted in rats. While reasonable
plasma exposures were achieved upon dosing compounds 25a
and 26a, we were interested in determining the concentrations
of the free acids, 25 and 26, in the target organ. Four groups of
rats were dosed orally at 5 mg/kg with each of 25a and 26a
and sacrificed at the 6 and 12 h time points.
As shown in Table 6, the results from this experiment indicate
that both drugs are found at high concentrations in rat liver
tissue. The liver to plasma ratios at the 6 h time point were ca.
17-fold for both compounds and increased to slightly better than
20-fold for both compounds at the 12 h time point. After 6 and
12 h, concentrations representing large multiples of the EC50
values are achievable after a single oral dose, with compound
26 achieving a concentration 270-fold of the serum adjusted
replicon EC50 12 h after oral dosing. The improved rat liver
levels of 26 over 25 at all time points is again consistent with
the in vitro metabolism data.
Conclusions
While the in vitro potency of our aza-series provided NS5B
inhibitors of exceptional antiviral potency, the strategy of
exploring the unsymmetrical dialkyl class of inhibitors has
proven to extend the promising properties of the antecedent
series by successfully optimizing their physical characteristics.
We have identified the (R)-configured isoamyl and neo-hexyl
substituted compounds 25a and 26a to be promising drug
candidates for the treatment of HCV infection. These compounds
are potent inhibitors of HCV genotype 1 replicons in the
presence of human serum. By achieving high liver concentra-
tions, a high liver/plasma ratio, good bioavailability, and a long
half-life after oral dosing, compounds 25a and 26a also
demonstrate a desirable pharmacological profile. An important
milestone for either compound would be the demonstration of
antiviral activity in human patients. Unfortunately, human PK
parameters bear on this critical question and will remain
unanswered without further clinical investigation. As an interim
milestone, we were pleased to find that compound 25a provided
the first in vivo chimpanzee validation of the antiviral efficacy
of our dialkyl-hydroxynaphthalenoyl-benzothiadiazine series.22
Further in vivo characterization of compound 26a, including
chimpanzee efficacy studies, will be reported in due course.
Experimental Section
Synthetic Materials and Methods. Unless otherwise specified,
reactions were performed under an inert atmosphere of nitrogen
and monitored by thin-layer chromatography (TLC). All reagents
were purchased from commercial suppliers and used as provided.
Flash column chromatography was carried out on silica gel. All
1H NMR and 13C NMR spectra were recorded on 300 or 500 MHz
instruments, are given ppm (δ), and are referenced to an internal
standard of tetramethylsilane (δ 0.00). 1H-1H couplings are
assumed to be first-order, and peak multiplicities are reported in
the usual manner. Elemental analyses were performed by Robertson
Microlit Laboratories (Madison, NJ) or Qualitative Technologies
Inc. (Whitehouse, NJ).
Solvents used for solubility determinations included PEG 400
(The Dow Chemical Company, Midland MI), water, 50 mM citrate
buffer adjusted to pH 4, and 50 mM phosphate buffer adjusted to
pH 7.4. Buffers were adjusted to an ionic strength of 0.155 M with
NaCl. Solvents used for HPLC analysis and sample preparation
were HPLC grade. Acetonitrile and TFA were used for mobile
phase preparation. Distilled deionized water was obtained from a
Millipore Milli-Q water purification system equipped with a 0.22
µm filter.
Figure 3. Primary rat metabolite of 26a.

1664 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Wagner et al.
2,5-Dimethyl-2-phenylhexanoic acid (7). A solution of lithium
bis(trimethylsilyl)amide (13.94 g, 83.28 mmol) in 40 mL of THF
was cooled in an ice bath. A solution of 2-phenylpropanoic acid
(4.56 mL, 33.3 mmol) in 5 mL of THF was added, followed by
addition of 1-bromo-3-methylbutane (6.66 mL, 53.3 mL.) The
cooling bath was removed, and the reaction solution was stirred
for 30 min. The reaction solution was then heated in a 50 °C oil
bath and stirred for 16 h. After cooling, the reaction solution was
concentrated and the resulting residue was dissolved in 250 mL of
1 N NaOH. The aqueous solution was washed with 2:1 hexane/
i-PrOAc, acidified to pH 1 with 6 N HCl, and extracted 3× with
EtOAc. The combined organic layers were dried over Na2SO4 and
concentrated to give 7.29 g (99%) of a clear, colorless oil. 1H NMR
(CDCl3) δ 7.31 (m, 5H), 1.99 (m, 2H), 1.56 (s, 3H), 1.50 (m, 1H),
1.09 (m, 2H), 0.88 (d, 3H, J ) 6.62), 0.85 (d, 3H, J ) 6.25). 13C
NMR (CDCl3) δ 182.6, 143.0, 128.4, 126.8, 126.2, 49.9, 36.8, 33.5,
28.5, 22.5, 22.5, 22.3. MS (DCI/NH3) m/z 238 (M + NH4)+.
2,5,5-Trimethyl-2-phenylhexanoic acid (8). Following the
procedure for compound 7, except substituting 1-iodo-3,3-dimeth-
ylbutane for 1-bromo-3-methylbutane, the title compound 8 (7.66
mL of dioxane was heated at reflux for 2 d. The reaction solution
was cooled and concentrated. The resulting residue was suspended
in 100 mL of H2O and extracted 3× with Et2O. The combined
organic layers were dried over MgSO4, concentrated, and purified
by chromatography on silica gel with 20% EtOAc in hexane as
1
eluent to give 1.91 g (88%) of a clear, colorless oil. H NMR
(CDCl3) δ 7.31 (m, 5H), 1.99 (m, 2H), 1.56 (s, 3H), 1.50 m, 1H),
1.08 (m, 2H), 0.88 (d, 3H, J ) 6.6), 0.85 (d, 3H, J ) 6.3). 13C
NMR (CDCl3) δ 182.4, 143.1, 128.4, 126.8, 126.2, 49.9, 36.8, 33.5,
28.5, 22.6, 22.5, 22.3. MS (DCI/NH3) m/z 238 (M + NH4)+. Anal.
(C14H20O2) C, H.
(S)-2,5-Dimethyl-2-phenylhexanoic Acid (11b). Following the
procedure for compound 11a, except substituting compound 9b for
compound 9a, the title compound 11b (2.01 g, 93%) was obtained
as a clear, colorless oil. H NMR (CDCl3) δ 7.31 (m, 5H), 1.99 (m,
2H), 1.56 (s, 3H), 1.50 (m, 1H), 1.09 (m, 2H), 0.88 (d, 3H, J )
6.6), 0.85 (d, 3H, J ) 6.3). 13C NMR (CDCl3) δ 182.6, 143.0, 128.4,
126.8, 126.2, 49.9, 36.8, 33.5, 28.5, 22.5, 22.5, 22.3. MS (DCI/
NH3) m/z 238 (M + NH4)+. Anal. (C14H20O2) C, H.
(R)-2,5,5-Trimethyl-2-phenylhexanoic acid (12a). Following the
procedure for compound 11a, except substituting compound 10a
for compound 9a, the title compound 12a (2.55 g, 93%) was
obtained as a clear, colorless oil. 1H NMR (CDCl3) δ 7.31 (m, 5H),
1.97 (m, 2H), 1.55 (s, 3H), 1.08 (m, 2H), 0.85 (s, 9H). 13C NMR
(CDCl3) δ 182.9, 143.0, 128.4, 126.8, 126.2, 49.8, 38.1, 33.8, 30.2,
29.3, 22.1. MS (DCI/NH3) m/z 252 (M + NH4)+. Anal. (C15H22O2)
C, H.
(S)-2,5,5-Trimethyl-2-phenylhexanoic Acid (12b). Following the
procedure for compound 11a, except substituting compound 10b
for compound 9a, the title compound 12b (2.70 g, 96%) was
obtained as a clear, colorless oil. 1H NMR (CDCl3) δ 7.31 (m, 5H),
1.97 (m, 2H), 1.56 (s, 3H), 1.08 (m, 2H), 0.86 (s, 9H). 13C NMR
(CDCl3) δ 182.8, 143.0, 128.4, 126.8, 126.2, 49.8, 38.1, 33.8, 30.2,
29.3, 22.1. MS (DCI/NH3) m/z 252 (M + NH4)+. Anal. (C15H22O2)
C, H.
1
g, 98%) was obtained as a clear, yellow oil. H NMR (CDCl3) δ
7.31 (m, 5H), 1.97 (m, 2H), 1.56 (s, 3H), 1.08 (m, 2H), 0.86 (s,
9H). 13C NMR (CDCl3) δ 182.8, 143.0, 128.4, 126.8, 126.2, 49.8,
38.1, 33.8, 30.2, 29.3, 22.1. MS (DCI/NH3) m/z 252 (M + NH4)+.
(R)-N-((S)-2-Hydroxy-1-phenylethyl)-2,5-dimethyl-2-phenylhex-
anamide (9a) and (S)-N-((S)-2-Hydroxy-1-phenylethyl)-2,5-dimeth-
yl-2-phenylhexanamide (9b). To a solution of compound 7 (7.92
g, 35.9 mmol) in 800 mL of hexane was added DMF (2.78 mL,
35.9 mmol) and (COCl)2 (9.40 mL, 107 mmol). After stirring for
2 h, the reaction mixture was filtered through a plug of celite and
concentrated to an oily residue.
A solution of the residue, (S)-phenylglycinol (7.39 g, 53.9 mmol),
and Et3N (10.5 mL, 75.5 mmol) in 500 mL of CH2Cl2 was stirred
overnight. The reaction mixture was washed with 1 N HCl and
saturated NaHCO3. The organic layer was dried over Na2SO4,
concentrated, and purified by chromatography on silica gel with
10-30% EtOAc in toluene as eluent to give 4.52 g (37%) of a
more polar compound 9a and 3.90 g (32%) of a less polar
(R)-Diethyl 2-(2,5-Dimethyl-2-phenylhexanoyl)malonate (13a).
A solution of diethyl malonate (1.33 g, 8.21 mmol) in 15 mL of
CH3CN was cooled in an ice bath. Magnesium chloride (0.798 g,
8.21 mmol) and Et3N (2.40 mL, 17.2 mmol) were added to the
reaction solution. After stirring for 15 min, the reaction mixture
was removed from the cooling bath and stirred for an additional
2 h.
1
compound 9b. More polar compound 9a: white solid. H NMR
(DMSO-d6) δ 7.41 (d, 1H, J ) 7.7), 7.23 (m, 10H), 4.86 (m, 1H),
4.72 (m, 1H), 3.51 (m, 2H), 1.98 (m, 1H), 1.81 (m, 1H), 1.42 (m,
4H), 0.92 (m, 2H), 0.79 (d, 6H, J ) 6.6). 13C NMR (DMSO-d6) δ
174.7, 145.7, 141.5, 128.0, 127.7, 126.9, 126.5, 126.2, 126.1, 64.2,
55.3, 49.6, 36.5, 33.1, 28.1, 23.6, 22.5, 22.4. MS (ESI) m/z 338
(M - H)-. Anal. (C22H29NO2) C, H, N. Less polar compound 9b:
In a separate flask, to a solution of compound 11a (1.81 g, 8.21
mmol) and DMF (0.70 mL, 9.03 mmol) in 275 mL of hexane was
added (COCl)2 (2.15 mL, 24.6 mmol). After stirring for 2 h, the
reaction mixture was filtered through a plug of celite and concen-
trated to an oily residue. The residue was dissolved in 10 mL of
CH3CN and added to the magnesium malonate solution. The
resulting reaction mixture was removed from the cooling bath and
heated in a 50 °C oil bath for 18 h. The reaction mixture was cooled
and concentrated. The oily residue was suspended in 100 mL of
EtOAc and 30 mL of 1 N HCl and stirred vigorously until all solids
dissolved. The layers were separated, and the aqueous layer was
extracted 3× with EtOAc. The combined organic layers were dried
over Na2SO4, concentrated, and purified by chromatography on
silica gel with 20% EtOAc in hexane as eluent to give 2.41 g (81%)
of a clear, colorless oil. 1H NMR (CDCl3) δ 7.31 (m, 5H), 4.51 (s,
1H), 4.11 (m, 2H), 4.05 (m, 2H), 1.96 (m, 2H), 1.53 (s, 3H), 1.46
(m, 1H), 1.21 (t, 3H, J ) 7.0), 1.16 (t, 3H, J ) 7.4), 1.07 (m, 1H),
0.84 (m, 7H). 13C NMR (CDCl3) δ 200.1, 164.3, 164.3, 139.8,
128.7, 127.5, 127.4, 62.1, 61.9, 60.5, 56.9, 35.0, 32.9, 28.5, 22.5,
22.4, 19.9, 13.8, 13.8. MS (ESI) m/z 361 (M - H)-. Anal.
(C21H30O5) C, H.
1
white solid. H NMR (DMSO-d6) δ 7.28 (m, 11H), 4.85 (m, 1H),
4.72 (m, 1H), 3.51 (m, 2H), 1.98 (m, 1H), 1.83 (m, 1H), 1.47 (m,
1H), 1.40 (s, 3H), 1.01 (m, 2H), 0.83 (d, 6H, J ) 6.6). 13C NMR
(DMSO-d6) δ 174.7, 145.8, 141.6, 128.0, 127.8, 126.8, 126.5, 126.3,
126.1, 64.3, 55.3, 49.7, 36.3, 33.3, 28.1, 23.9, 22.6. MS (ESI) m/z
338 (M - H)-. Anal. (C22H29NO2) C, H, N.
(R)-N-((S)-2-Hydroxy-1-phenylethyl)-2,5,5-trimethyl-2-phenyl-
hexanamide (10a) and (S)-N-((S)-2-Hydroxy-1-phenylethyl)-2,5,5-
trimethyl-2-phenylhexanamide (10b). Following the procedure for
9a and 9b, the title compounds were prepared from compound 8
and were obtained as 4.95 g (39%) of a more polar compound 10a
and 5.08 g (40%) of a less polar compound 10b. More polar
compound 10a: white solid. 1H NMR (DMSO-d6) δ 7.27 (m, 11H),
4.86 (m, 1H), 4.72 (t, 1H), 3.51 (m, 2H), 1.96 (m, 1H), 1.77 (m,
1H), 1.41 (s, 3H), 0.92 (m, 2H), 0.78 (s, 9H). 13C NMR (DMSO-
d6) δ 174.7, 145.7, 141.4, 127.9, 127.7, 126.9, 126.5, 126.3, 126.1,
64.2, 55.2, 49.4, 37.6, 33.3, 29.8, 29.1, 23.5. MS (ESI) m/z 352
(M-H)-. Anal. (C23H31NO2) C, H, N. Less polar compound 10b:
1
white solid. H NMR (DMSO-d6) δ 7.27 (m, 11H), 4.85 (m, 1H),
(S)-Diethyl 2-(2,5-Dimethyl-2-phenylhexanoyl)malonate (13b).
Following the procedure for compound 13a, except substituting
compound 11b for compound 11a, the title compound 13b (2.83
g, 95%) was obtained as a clear, colorless oil. 1H NMR (CDCl3) δ
7.31 (m, 5H), 4.51 (s, 1H), 4.11 (m, 2H), 4.05 (m, 2H), 1.96 (m,
2H), 1.53 (s, 3H), 1.46 (m, 1H), 1.21 (t, 3H, J ) 7.0), 1.16 (t, 3H,
J ) 7.4), 1.07 (m, 1H), 0.84 (m, 7H). 13C NMR (CDCl3) δ 200.1,
164.3, 164.3, 139.8, 128.7, 127.5, 127.4, 62.1, 61.9, 60.5, 56.9,
4.72 (m, 1H), 3.52 (m, 2H), 1.97 (m, 1H), 1.81 (m, 1H), 1.39 (s,
3H), 1.02 (m, 2H), 0.82 (s, 9H). 13C NMR (DMSO-d6) δ 174.6,
145.8, 141.5, 127.9, 127.7, 126.8, 126.4, 126.3, 126.1, 64.2, 55.2,
49.5, 37.8, 33.1, 29.8, 29.2, 23.8. MS (ESI) m/z 352 (M - H)-.
Anal. (C23H31NO2) C, H, N.
(R)-2,5-Dimethyl-2-phenylhexanoic Acid (11a). A solution of
compound 9a (3.55 g, 9.87 mmol) and 75 mL of 6 N H2SO4 in 75

Inhibitors of Hepatitis C Virus Polymerase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1665
35.0, 32.9, 28.5, 22.5, 22.4, 19.9, 13.8, 13.8. MS (ESI) m/z 361
(M - H)-. Anal. (C21H30O5) C, H.
51.6, 45.2, 38.4, 28.1, 36.9, 36.6, 30.0, 29.8, 29.5, 29.1, 29.0, 28.8,
14.2, 14.2. MS (ESI) m/z 329 (M - H)-.
(R)-Diethyl 2-(2,5,5-Trimethyl-2-phenylhexanoyl)malonate (14a).
Following the procedure for compound 13a, except substituting
compound 12a for compound 11a, the title compound 14a (2.78
g, 90%) was obtained as a clear, colorless oil. 1H NMR (CDCl3) δ
7.31 (m, 5H), 4.52 (s, 1H), 4.12 (m, 2H), 4.04 (m, 2H), 1.95 (m,
2H), 1.53 (s, 3H), 1.21 (m, 3H), 1.15 (m, 3H), 1.08 (m, 1H), 0.82
(m, 10H). 13C NMR (CDCl3) δ 200.2, 164.3, 164.3, 139.7, 128.7,
127.5, 127.4, 62.1, 61.9, 60.6, 56.8, 37.5, 32.1, 30.1, 29.2, 19.8,
13.9, 13.8. MS (ESI) m/z 375 (M - H)-.
(S)-Diethyl 2-(2,5,5-Trimethyl-2-phenylhexanoyl)malonate (14b).
Following the procedure for compound 13a, except substituting
compound 12b for compound 11a, the title compound 14b (2.75
g, 89%) was obtained as a clear, colorless oil. 1H NMR (CDCl3) δ
7.31 (m, 5H), 4.52 (s, 1H), 4.12 (m, 2H), 4.04 (m, 2H), 1.95 (m,
2H), 1.53 (s, 3H), 1.21 (m, 3H), 1.15 (m, 3H), 1.08 (m, 1H), 0.82
(m, 10H). 13C NMR (CDCl3) δ 200.2, 164.3, 164.3, 139.7, 128.7,
127.5, 127.4, 62.1, 61.9, 60.6, 56.8, 37.5, 32.1, 30.1, 29.2, 19.8,
13.9, 13.8. MS (ESI) m/z 375 (M - H)-.
(R)-4-Hydroxy-1-isopentyl-1-methylnaphthalen-2(1H)-one (17a).
A solution of compound 15a (1.50 g, 4.75 mmol) and 15 mL of 1
N HCl in 15 mL of dioxane was heated at reflux for 2 h. The
reaction mixture was cooled and partitioned between 100 mL of
H2O and 150 mL of EtOAc. The layers were separated and the
organic layer was washed with H2O and brine, dried over Na2SO4,
and concentrated. The resulting solid was triturated with warm
hexane and filtered to give 801 mg (69%) of a white solid. 1H NMR
(CDCl3) δ 8.10 (m, 0.5H), 7.66 (m, 0.5H), 7.46 (m, 3H), 6.01 (s,
0.5H), 3.74 (m, 1H), 2.24 (m, 0.5H), 2.03 (m, 0.5H), 1.84 (m, 1H),
1.56 (s, 1.5H), 1.51 (s, 1.5H), 1.42 (m, 0.5H), 1.31 (m, 0.5H), 1.05
(m, 0.5H), 0.80 (m, 4H), 0.72 (d, 1.5H, J ) 6.6), 0.68 (d, 1.5H, J
) 6.6), 0.44 (m, 0.5H). 13C NMR (CDCl3) δ 192.1, 181.5, 147.5,
135.1, 132.0, 131.4, 129.5, 127.3, 127.2, 126.5, 126.3, 125.5, 125.4,
103.8, 53.2, 53.0, 46.9, 40.5, 39.4, 34.1, 33.3, 28.8, 28.2, 28.0, 24.0,
22.4, 22.2. MS (ESI) m/z 243 (M - H)-. Anal. (C16H20O2) C, H.
(S)-4-Hydroxy-1-isopentyl-1-methylnaphthalen-2(1H)-one (17b).
Following the procedure for compound 17a, except substituting
compound 15b for compound 15a, the title compound 17b (1.06
(R)-Ethyl 1-Hydroxy-4-isopentyl-4-methyl-3-oxo-3,4-dihydronaph-
thalene-2-carboxylate (15a). A solution of compound 13a (2.63 g,
7.25 mmol) in 15 mL of methanesulfonic acid was stirred for 18 h.
The reaction solution was poured into 100 mL of H2O, and the
resulting aqueous mixture was extracted 3× with EtOAc. The
combined organic layers were dried over Na2SO4, concentrated,
and purified by chromatography on silica gel with 20% EtOAc in
1
g, 91%) was obtained as a white solid. H NMR (CDCl3) δ 8.10
(m, 0.5H), 7.66 (m, 0.5H), 7.46 (m, 3H), 6.01 (s, 0.5H), 3.74 (m,
1H), 2.24 (m, 0.5H), 2.03 (m, 0.5H), 1.84 (m, 1H), 1.56 (s, 1.5H),
1.51 (s, 1.5H), 1.42 (m, 0.5H), 1.31 (m, 0.5H), 1.05 (m, 0.5H),
0.80 (m, 4H), 0.72 (d, 1.5H, J ) 6.6), 0.68 (d, 1.5H, J ) 6.6), 0.44
(m, 0.5H). 13C NMR (CDCl3) δ 192.1, 181.5, 147.5, 135.1, 132.0,
131.4, 129.5, 127.3, 127.2, 126.5, 126.3, 125.5, 125.4, 103.8, 53.2,
53.0, 46.9, 40.5, 39.4, 34.1, 33.3, 28.8, 28.2, 28.0, 24.0, 22.4, 22.2.
MS (ESI) m/z 243 (M - H)-. Anal. (C16H20O2) C, H.
1
hexane as eluent to give 1.47 g (64%) of a clear, colorless oil. H
NMR (CDCl3) δ 15.15 (s, 0.5H), 15.06 (s, 0.5H), 8.23 (m, 0.5H),
8.16 (m, 0.5H), 7.59 (m, 1H), 7.42 (m, 2H), 4.47 (m, 2H), 2.28
(m, 1H), 1.94 (m, 0.5H), 1.78 (m, 0.5H), 1.64 (s, 1.5H), 1.57 (s,
1.5H), 1.47 (m, 3H), 1.36 (m, 1H), 0.87 (m, 1H), 0.73 (m, 6H),
0.52 (m, 1H). 13C NMR (CDCl3) δ 172.9, 133.0, 126.7, 126.5,
125.4, 62.1, 61.4, 41.7, 39.9, 33.6, 28.8, 28.1, 22.4, 22.1, 14.2, 14.0.
MS (ESI) m/z 315 (M - H)-. Anal. (C19H24O4) C, H.
(S)-Ethyl 1-Hydroxy-4-isopentyl-4-methyl-3-oxo-3,4-dihydronaph-
thalene-2-carboxylate (15b). Following the procedure for compound
15a, except substituting compound 13b for compound 13a, the title
compound 15b (1.70 g, 74%) was obtained as a clear, colorless
oil. 1H NMR (CDCl3) δ 15.15 (s, 0.5H), 15.06 (s, 0.5H), 8.23 (m,
0.5H), 8.16 (m, 0.5H), 7.59 (m, 1H), 7.42 (m, 2H), 4.47 (m, 2H),
2.28 (m, 1H), 1.94 (m, 0.5H), 1.78 (m, 0.5H), 1.64 (s, 1.5H), 1.57
(s, 1.5H), 1.47 (m, 3H), 1.36 (m, 1H), 0.87 (m, 1H), 0.73 (m, 6H),
0.52 (m, 1H). 13C NMR (CDCl3) δ 172.9, 133.0, 126.7, 126.5,
125.4, 62.1, 61.4, 41.7, 39.9, 33.6, 28.8, 28.1, 22.4, 22.1, 14.2, 14.0.
MS (ESI) m/z 315 (M - H)-. Anal. (C19H24O4) C, H.
(R)-Ethyl 4-(3,3-Dimethylbutyl)-1-hydroxy-4-methyl-3-oxo-3,4-
dihydronaphthalene-2-carboxylate (16a). Following the procedure
for compound 15a, except substituting compound 14a for compound
13a, the title compound 16a (2.06 g, 86%) was obtained as a clear,
colorless oil. 1H NMR (CDCl3) δ 15.15 (s, 0.5H), 15.03 (s, 0.5H),
8.24 (m, 0.5H), 8.16 (m, 0.5H), 7.59 (m, 1H), 7.41 (m, 2H), 4.47
(m, 2H), 2.28 (m, 1H), 1.94 (m, 0.5H), 1.76 (m, 0.5H), 1.65 (s,
1.5H), 1.57 (s, 1.5H), 1.47 (m, 3H), 0.86 (m, 1H), 0.75 (m, 4.5H),
0.73 (m, 4.5H), 0.51 (m, 1H). 13C NMR (CDCl3) δ 196.5, 192.7,
180.4, 177.8, 173.1, 172.8, 147.8, 143.6, 133.3, 132.6, 131.6, 126.9,
126.6, 126.2, 126.0, 125.9, 124.8, 104.6, 101.4, 62.2, 62.1, 51.6,
45.2, 38.4, 28.1, 36.9, 36.6, 30.0, 29.8, 29.5, 29.1, 29.0, 28.8, 14.2,
14.2. MS (ESI) m/z 329 (M - H)-.
(R)-1-(3,3-Dimethylbutyl)-4-hydroxy-1-methylnaphthalen-2(1H)-
one (18a). Following the procedure for compound 17a, except
substituting compound 16a for compound 15a, the title compound
18a (1.07 g, 87%) was obtained as a white solid. 1H NMR (CDCl3)
δ 8.09 (m, 1H), 7.50 (m, 3H), 6.06 (s, 0.67H), 3.73 (s, 0.67H),
2.25 (m, 0.67H), 2.01 (m, 0.33H), 1.83 (m, 1H), 1.56 (s, 1H), 1.48
(s, 2H), 1.06 (m, 0.33H), 0.82 (m, 4H), 0.70 (s, 6H), 0.41 (m,
0.67H). 13C NMR (CDCl3) δ 192.1, 181.5, 147.5, 132.0, 129.5,
126.3, 125.5, 125.4, 103.9, 46.9, 37.9, 36.3, 29.8, 29.1. MS (ESI)
m/z 257 (M - H)-.
(S)-1-(3,3-Dimethylbutyl)-4-hydroxy-1-methylnaphthalen-2(1H)-
one (18b). Following the procedure for compound 17a, except
substituting compound 16b for compound 15a, the title compound
18b (1.09 g, 89%) was obtained as a white solid. 1H NMR (CDCl3)
δ 8.09 (m, 1H), 7.50 (m, 3H), 6.06 (s, 0.67H), 3.73 (s, 0.67H),
2.25 (m, 0.67H), 2.01 (m, 0.33H), 1.83 (m, 1H), 1.56 (s, 1H), 1.48
(s, 2H), 1.06 (m, 0.33H), 0.82 (m, 4H), 0.70 (s, 6H), 0.41 (m,
0.67H). 13C NMR (CDCl3) δ 192.1, 181.5, 147.5, 132.0, 129.5,
126.3, 125.5, 125.4, 103.9, 46.9, 37.9, 36.3, 29.8, 29.1. MS (ESI)
m/z 257 (M - H)-. Anal. (C17H22O2) C, H.
(R)-2-(Bis(methylthio)methylene)-4-isopentyl-4-methylnaphtha-
lene-1,3(2H,4H)-dione (19a). A solution of compound 17a (953 mg,
3.90 mmol) and pyridine (2.52 mL, 31.2 mmol) in 20 mL of dioxane
was treated with (bis-methylsulfanyl-methylene)-methyl-sulfonium
tetrafluoroborate (4.14 g, 15.6 mmol).23 The reaction solution was
heated in a 100 °C oil bath for 1 h. The reaction mixture was cooled
and partitioned between 100 mL of H2O and 150 mL of EtOAc.
The layers were separated and the organic layer was washed with
H2O and brine, dried over Na2SO4, and concentrated. The resulting
solid was purified by chromatography on silica gel with 15% EtOAc
in hexane as eluent to give 1.20 g of a yellow solid. The product
was immediately used in the following step without further
purification.
(S)-Ethyl 4-(3,3-Dimethylbutyl)-1-hydroxy-4-methyl-3-oxo-3,4-
dihydronaphthalene-2-carboxylate (16b). Following the procedure
for compound 15a, except substituting compound 14b for com-
pound 13a, the title compound 16b (2.04 g, 85%) was obtained as
1
a clear, colorless oil. H NMR (CDCl3) δ 15.15 (s, 0.5H), 15.03
(S)-2-(Bis(methylthio)methylene)-4-isopentyl-4-methylnaphtha-
lene-1,3(2H,4H)-dione (19b). Following the procedure for compound
19a, except substituting compound 17b for compound 17a, the title
compound 19b (1.33 g) was obtained as a yellow solid. The product
was immediately used in the following step without further
purification.
(s, 0.5H), 8.24 (m, 0.5H), 8.16 (m, 0.5H), 7.59 (m, 1H), 7.41 (m,
2H), 4.47 (m, 2H), 2.28 (m, 1H), 1.94 (m, 0.5H), 1.76 (m, 0.5H),
1.65 (s, 1.5H), 1.57 (s, 1.5H), 1.47 (m, 3H), 0.86 (m, 1H), 0.75
(m, 4.5H), 0.73 (m, 4.5H), 0.51 (m, 1H). 13C NMR (CDCl3) δ 196.5,
192.7, 180.4, 177.8, 173.1, 172.8, 147.8, 143.6, 133.3, 132.6, 131.6,
126.9, 126.6, 126.2, 126.0, 125.9, 124.8, 104.6, 101.4, 62.2, 62.1,

1666 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Wagner et al.
(R)-2-(Bis(methylthio)methylene)-4-(3,3-dimethylbutyl)-4-meth-
ylnaphthalene-1,3(2H,4H)-dione (20a). Following the procedure for
compound 19a, except substituting compound 18a for compound
17a, the title compound 20a (1.40 g) was obtained as a yellow
solid. The product was immediately used in the following step
without further purification.
(S)-2-(Bis(methylthio)methylene)-4-(3,3-dimethylbutyl)-4-meth-
ylnaphthalene-1,3(2H,4H)-dione (20b). Following the procedure for
compound 19a, except substituting compound 18b for compound
17a, the title compound 20b (1.40 g) was obtained as a yellow
solid. The product was immediately used in the following step
without further purification.
{3-[(R)-1-Hydroxy-4-methyl-4-(3-methyl-butyl)-3-oxo-3,4-dihy-
dro-naphthalen-2-yl]-1,1-dioxo-1,4-dihydro-1λ6-benzo[1,2,4]thia-
diazin-7-yl}-carbamic Acid tert-Butyl Ester (21a). A solution of
compound 19a (1.32 g, 3.78 mmol) and tert-butyl 4-amino-3-
sulfamoylphenylcarbamate (1.27 g, 3.97 mmol) in 25 mL of toluene
was heated at reflux for 6 h.24 The reaction solution was cooled,
concentrated, and purified by chromatography on silica gel with
30% EtOAc in hexane as eluent to give 1.24 g (61%) of a white
solid. 1H NMR (DMSO-d6) δ 13.78 (s, 1H), 9.89 (s, 1H), 8.15 (m,
2H), 7.76 (m, 2H), 7.66 (m, 2H), 7.54 (m, 1H), 2.23 (m, 1H), 2.06
(m, 1H), 1.57 (s, 3H), 1.50 (s, 9H), 1.31 (m, 1H), 0.80 (m, 1H),
0.72 (d, 3H, J ) 6.6), 0.69 (d, 3H, J ) 6.6), 0.42 (m, 1H). 13C
NMR (DMSO-d6) δ 155.2, 152.7, 138.3, 133.9, 128.3, 127.2, 126.2,
125.9, 123.4, 123.2, 120.3, 109.9, 80.0, 33.2, 29.2, 28.0, 27.6, 22.3,
22.1. MS (ESI) m/z 538 (M - H)-.
7.07 (m, 2H), 2.23 (m, 1H), 2.08 (m, 1H), 1.57 (s, 3H), 1.31 (m,
1H), 0.79 (m, 1H), 0.72 (d, 3H, J ) 6.6), 0.68 (d, 3H, J ) 6.6),
0.41 (m, 1H). 13C NMR (DMSO-d6) δ 154.5, 145.7, 141.1, 134.2,
127.3, 127.1, 126.3, 125.9, 123.7, 123.6, 121.0, 109.5, 100.6, 48.2,
33.2, 29.1, 27.6, 22.3, 22.1. MS (ESI) m/z 438 (M - H)-. Anal.
(C23H26ClN3O4S) C, H, N.
(S)-3-(7-Amino-1,1-dioxo-1,4-dihydro-1λ6-benzo[1,2,4]thiadiazin-
3-yl)-4-hydroxy-1-methyl-1-(3-methyl-butyl)-1H-naphthalen-2-
one Hydrochloride (23b). Following the procedure for compound
23a, except substituting compound 21b for compound 21a, the title
1
compound 23b (1.27 g, 89%) was obtained as a white solid. H
NMR (DMSO-d6) δ 13.58 (s, 1H), 8.16 (m, 1H), 7.77 (m, 2H),
7.55 (m, 1H), 7.49 (m, 2H), 7.07 (m, 2H), 2.23 (m, 1H), 2.08 (m,
1H), 1.57 (s, 3H), 1.31 (m, 1H), 0.79 (m, 1H), 0.72 (d, 3H, J )
6.6), 0.68 (d, 3H, J ) 6.6), 0.41 (m, 1H). 13C NMR (DMSO-d6) δ
154.6, 145.7, 140.4, 134.2, 127.5, 127.3, 126.3, 125.9, 123.9, 123.6,
121.0, 110.0, 100.7, 48.2, 33.2, 29.1, 27.6, 22.3, 22.1. MS (ESI)
m/z 438 (M - H)-. Anal. (C23H26ClN3O4S) C, H, N.
(R)-3-(7-Amino-1,1-dioxo-1,4-dihydro-1λ6-benzo[1,2,4]thiadiazin-
3-yl)-1-(3,3-dimethyl-butyl)-4-hydroxy-1-methyl-1H-naphthalen-2-
one Hydrochloride (24a). Following the procedure for compound
23a, except substituting compound 22a for compound 21a, the title
1
compound 24a (1.38 g, 95%) was obtained as a white solid. H
NMR (DMSO-d6) δ 13.58 (s, 1H), 8.16 (m, 1H), 7.77 (m, 2H),
7.55 (m, 1H), 7.49 (m, 2H), 7.07 (m, 2H), 2.23 (m, 1H), 2.08 (m,
1H), 1.57 (s, 3H), 1.31 (m, 1H), 0.79 (m, 1H), 0.70 (m, 9H), 0.41
(m, 1H). 13C NMR (DMSO-d6) δ 154.7, 145.7, 140.3, 134.1, 127.7,
127.4, 126.3, 125.9, 124.0, 123.6, 121.1, 110.2, 37.8, 36.3, 29.7,
29.4, 28.9. MS (ESI) m/z 454 (M - H)-.
{3-[(S)-1-Hydroxy-4-methyl-4-(3-methyl-butyl)-3-oxo-3,4-dihy-
dro-naphthalen-2-yl]-1,1-dioxo-1,4-dihydro-1λ6-benzo[1,2,4]thia-
diazin-7-yl}-carbamic Acid tert-Butyl Ester (21b). Following the
procedure for compound 21a, except substituting compound 19b
for compound 19a, the title compound 21b (1.71 g, 84%) was
(S)-3-(7-Amino-1,1-dioxo-1,4-dihydro-1λ6-benzo[1,2,4]thiadiazin-
3-yl)-1-(3,3-dimethyl-butyl)-4-hydroxy-1-methyl-1H-naphthalen-2-
one Hydrochloride (24b). Following the procedure for compound
23a, except substituting compound 22b for compound 21a, the title
1
obtained as a white solid. H NMR (DMSO-d6) δ 13.78 (s, 1H),
1
9.89 (s, 1H), 8.15 (m, 2H), 7.76 (m, 2H), 7.66 (m, 2H), 7.54 (m,
1H), 2.23 (m, 1H), 2.06 (m, 1H), 1.57 (s, 3H), 1.50 (s, 9H), 1.31
(m, 1H), 0.80 (m, 1H), 0.72 (d, 3H, J ) 6.6), 0.69 (d, 3H, J )
6.6), 0.42 (m, 1H). 13C NMR (DMSO-d6) δ 155.2, 152.7, 138.3,
133.9, 128.3, 127.2, 126.2, 125.9, 123.4, 123.2, 120.3, 109.9, 80.0,
33.2, 29.2, 28.0, 27.6, 22.3, 22.1. MS (ESI) m/z 538 (M - H)-.
{3-[(R)-4-(3,3-Dimethyl-butyl)-1-hydroxy-4-methyl-3-oxo-3,4-di-
hydro-naphthalen-2-yl]-1,1-dioxo-1,4-dihydro-1λ6-benzo[1,2,4]thia-
diazin-7-yl}-carbamic Acid tert-Butyl Ester (22a). Following the
procedure for compound 21a, except substituting compound 20a
for compound 19a, the title compound 22a (1.80 g, 86%) was
compound 24b (1.37 g, 94%) was obtained as a white solid. H
NMR (DMSO-d6) δ 13.58 (s, 1H), 8.17 (m, 1H), 7.77 (m, 2H),
7.55 (m, 1H), 7.48 (m, 1H), 7.04 (m, 2H), 2.23 (m, 1H), 2.08 (m,
1H), 1.59 (s, 3H), 0.79 (m, 1H), 0.71 (s, 9H), 0.39 (m, 1H). 13C
NMR (DMSO-d6) δ 154.7, 145.7, 140.3, 134.1, 127.7, 127.4, 126.3,
125.9, 124.0, 123.6, 121.1, 110.2, 37.8, 36.3, 29.7, 29.4, 28.9. MS
(ESI) m/z 454 (M - H)-.
Sodium (R)-2-(7-Methanesulfonylamino-1,1-dioxo-1,4-dihydro-
1λ6-benzo[1,2,4]thiadiazin-3-yl)-4-methyl-4-(3-methyl-butyl)-3-oxo-
3,4-dihydro-naphthalen-1-olate (25a). A solution of compound 23a
(1.14 g, 2.40 mmol), MsCl (0.744 mL, 9.61 mmol), and pyridine
(1.56 mL, 19.2 mmol) in 30 mL of acetone was stirred for 18 h.
The reaction mixture was diluted with 150 mL of EtOAc and
washed with 0.1 M citric acid, H2O, and brine. The organic layer
was dried over Na2SO4, concentrated, and purified by chromatog-
raphy on silica gel with 2.5% MeOH in CH2Cl2 as eluent. The
resulting solid was suspended in 7 mL of EtOAc, and the mixture
was heated at reflux until the solid completely dissolved. Heptane
(7 mL) was added to the solution at a rate that maintained reflux.
The heat source was removed and the solution was stirred
vigorously overnight. The solid was filtered, washed with a 1 mL
of heptane, and dried under vacuum to give 1.02 g of a pale-yellow
crystalline solid.
1
obtained as a white solid. H NMR (DMSO-d6) δ 13.76 (s, 1H),
9.90 (s, 1H), 8.15 (m, 2H), 7.76 (m, 2H), 7.66 (m, 2H), 7.54 (m,
1H), 2.23 (m, 1H), 2.06 (m, 1H), 1.58 (s, 3H), 1.50 (s, 9H), 0.80
(m, 1H), 0.70 (s, 9H), 0.39 (m, 1H). 13C NMR (DMSO-d6) δ 155.1,
152.6, 138.4, 134.1, 128.1, 127.3, 126.2, 125.9, 123.5, 123.3, 120.4,
109.8, 80.0, 37.8, 36.4, 29.7, 29.5, 28.9, 28.0. MS (ESI) m/z 552
(M - H)-.
{3-[(S)-4-(3,3-Dimethyl-butyl)-1-hydroxy-4-methyl-3-oxo-3,4-di-
hydro-naphthalen-2-yl]-1,1-dioxo-1,4-dihydro-1λ6-benzo[1,2,4]thia-
diazin-7-yl}-carbamic Acid tert-Butyl Ester (22b). Following the
procedure for compound 21a, except substituting compound 20b
for compound 19a, the title compound 22b (1.80 g, 86%) was
1
obtained as a white solid. H NMR (DMSO-d6) δ 13.76 (s, 1H),
To a suspension of the solid in 15 mL of H2O was added 1 N
NaOH (1.97 mL, 1.97 mmol). The suspension was stirred for 2 h
9.90 (s, 1H), 8.15 (m, 2H), 7.76 (m, 2H), 7.66 (m, 2H), 7.54 (m,
1H), 2.23 (m, 1H), 2.06 (m, 1H), 1.58 (s, 3H), 1.50 (s, 9H), 0.80
(m, 1H), 0.70 (s, 9H), 0.39 (m, 1H). 13C NMR (DMSO-d6) δ 155.1,
152.6, 138.4, 134.1, 128.1, 127.3, 126.2, 125.9, 123.5, 123.3, 120.4,
109.8, 80.1, 37.8, 36.4, 29.7, 29.5, 28.9, 28.0. MS (ESI) m/z 552
(M - H)-.
1
and then lyophilized to give 1.06 g (82%) of a yellow solid. H
NMR (DMSO-d6) δ 15.50 (s, 1H), 9.88 (s, 1H), 8.05 (m, 1H), 7.47
(m, 3H), 7.33 (m, 3H), 2.96 (s, 3H), 2.14 (m, 1H), 1.73 (m, 1H),
1.38 (s, 3H), 1.28 (m, 1H), 0.79 (m, 1H), 0.69 (m, 6H), 0.45 (m,
1H). 13C NMR (DMSO-d6) δ 195.5, 180.4, 156.7, 145.0, 135.2,
133.0, 132.4, 130.7, 125.6, 125.6, 125.2, 124.8, 122.9, 118.9, 113.3,
101.6, 48.3, 39.7, 39.1, 33.7, 29.5, 27.7, 22.5, 22.2. MS (ESI) m/z
516 (M - H)-. HRMS (ESI) m/z calcd (C24H27N3O6S2) 518.1414,
found 518.1411 (M + H)+, -0.6 ppm error.
Sodium (S)-2-(7-Methanesulfonylamino-1,1-dioxo-1,4-dihydro-
1λ6-benzo[1,2,4]thiadiazin-3-yl)-4-methyl-4-(3-methyl-butyl)-3-oxo-
3,4-dihydro-naphthalen-1-olate (25b). Following the procedure for
compound 25a, except substituting compound 23b for compound
(R)-3-(7-Amino-1,1-dioxo-1,4-dihydro-1λ6-benzo[1,2,4]thiadiazin-
3-yl)-4-hydroxy-1-methyl-1-(3-methyl-butyl)-1H-naphthalen-2-
one Hydrochloride (23a). A solution of compound 21a (1.60 g,
2.97 mmol) in 2 mL of CH2Cl2 was treated with 15 mL of 4.0 M
HCl in dioxane. The reaction solution was stirred for 1 h and then
concentrated. The resulting white solid was triturated with ether to
1
give 1.29 g (91%) of a white solid. H NMR (DMSO-d6) δ 13.58
(s, 1H), 8.16 (m, 1H), 7.77 (m, 2H), 7.55 (m, 1H), 7.49 (m, 2H),

Inhibitors of Hepatitis C Virus Polymerase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1667
23a, the title compound 25b (1.11 g, 86%) was obtained as a yellow
solid. 1H NMR (DMSO-d6) δ 15.47 (s, 1H), 9.88 (s, 1H), 8.05 (m,
1H), 7.45 (m, 3H), 7.31 (m, 3H), 2.94 (s, 3H), 2.15 (m, 1H), 1.73
(m, 1H), 1.38 (s, 3H), 1.28 (m, 1H), 0.80 (m, 1H), 0.69 (m, 6H),
0.45 (m, 1H). 13C NMR (DMSO-d6) δ 195.5, 180.4, 156.7, 145.0,
135.2, 133.0, 132.4, 130.7, 125.6, 125.6, 125.2, 124.8, 122.9, 118.9,
113.3, 101.6, 48.3, 39.7, 39.1, 33.7, 29.5, 27.7, 22.5, 22.2. MS (ESI)
m/z 516 (M - H)-. HRMS (ESI) m/z calcd (C24H27N3O6S2)
518.1414, found 518.1412 (M + H)+, -0.3 ppm error.
1b-con1 strain. The inhibitory potencies of A-848837 against these
HCV subgenomic replicons were measured based on the reduction
of HCV RNA copy number in the presence of inhibitor over a four
day period as described previously.25 Cytotoxicity was determined
by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) colorimetric assay.26 The protein binding effect on inhibitor
potency was assessed by including 40% human serum in culture
media containing 5% FBS during the four day incubation period.
Solubility Determinations. Approximately 0.5-10 mg of the
lyophilized amorphous powder samples of 1a, 26, and crystalline
26a were weighed using a microbalance (model M5P, Sartorius
Corp., Bohemia, NY) and transferred into 2 mL glass HPLC vials.
Approximately 0.5-1 mL of medium was added into the vials.
The vials were wrapped with aluminum foil and equilibrated with
rotation in a water bath maintained at 25 °C (Vankel Industries)
for 4 days. The final pH of the aqueous solutions was measured
before HPLC assay and crystalline form of residual solid for 26a
was confirmed by powder X-ray diffraction (Rigaku Ultima).
HPLC Analysis. Sample analysis was conducted using an
isocratic reverse phase HPLC method with a PhenomenexLuna 5
µ CN, 250 mm × 4.60 mm or PhenomenexLuna 5 µm C18 (2),
150 mm × 4.60 mm column with detection wavelength was 262
nm. The aqueous mobile phase component was either 0.1% TFA
or 25 mM potassium phosphate, pH 2.5 with acetonitrile used as
the organic component. Beckman Peak Pro software CALS version
8.4a was used to acquire and process the chromatograms. Sample
concentrations were determined against a linear standard curve.
LogD Determinations. Distribution coefficient was measured
between n-octanol and 50 mM phosphate buffer, made isotonic with
NaCl and adjusted to pH 7.4. The compound was first dissolved in
the n-octanol phase and then mixed with the buffer phase. The
mixture was tumbled in a 25 °C water bath at 25 rpm for 2-3
days. The sample was then removed from the water bath and
centrifuged at 3000 rpm for 10 min (Jouan GT 422 centrifuge) to
separate the two phases. The separated phases were transferred into
separate vials by discarding the interface. The buffer phase was
injected into HPLC column without dilution, while the octanol phase
was diluted with mobile phase to achieve a final concentration
within the range of the standard curve and assayed by HPLC. The
distribution coefficient D is expressed as the ratio of concentration
in the octonal phase over that in the buffer phase.
Pharmacokinetic Profiles. 25a and 26a were formulated in
DMSO:PEG400 (10:90; v/v) for single oral dosing of 5 mg/kg
in rat, dog (2.5 mg/kg only for 26a), and monkey. Heparinized
plasma samples were withdrawn at 0.1 (IV only), 0.25, 0.5, 1,
2, 3 (dog only), 4, 6, 9, 12, 15 (dog only), and 24 h postdosing.
Plasma or rat liver homogenate drug concentrations were
determined by a liquid chromatography-mass spectrometry (LC-
MS) assay as follows. Compounds 25a and 26a were separated
from plasma or liver homogenate samples using protein pre-
cipitation with acetonitrile. A 250 µL aliquot of each sample or
spiked standard was combined with 25 µL of internal standard
and 500 µL of acetonitrile. The samples were vortexed vigor-
ously for 1 min, followed by centrifugation for 10 min at 4 °C.
Each supernatant was transferred to a well of a 96-well plate
and evaporated to dryness with a gentle stream of nitrogen. The
samples were reconstituted with sequential aliquots of acetonitrile
and 20 mM ammonium acetate. Compounds 25a or 26a and the
internal standard were separated from each other and coprecipi-
tated contaminants on a 50 mm × 3 mm Clipeus 5 µm column
(Higgins Analytical, Inc.) with a 1:1 acetonitrile:20 mM am-
monium acetate mobile phase at a flow rate of 0.3 mL/min.
Analysis was performed on a Sciex API2000 biomolecular mass
analyzer with a turbo-ionspray interface, in the negative
ion mode. Detection was in the multiple reaction monitoring
(MRM) mode at m/z 516.2f437.0. Compounds 25a or 26a and
internal standard peak areas were determined using Sciex
TurboQuan software. The drug concentration of each sample
was calculated by least-squares linear regression analysis (non-
weighted) of the peak area ratio (parent/internal standard) of
the spiked standards versus concentration. The method, generally
Sodium (R)-4-(3,3-Dimethyl-butyl)-2-(7-methanesulfonylamino-
1,1-dioxo-1,4-dihydro-1λ6-benzo[1,2,4]thiadiazin-3-yl)-4-methyl-3-
oxo-3,4-dihydro-naphthalen-1-olate (26a). A suspension of 26 (223
mg, 0.42 mmol) in ethanol (1.7 mL) was heated to 75 °C and mixed
to dissolve the solids. A solution of sodium ethoxide (31 mg, 0.46
mmol) in ethanol (0.55 mL) was added to the solution via syringe
over 50 min while maintaining the temperature at 70-72 °C. The
solution was cooled to room temperature and mixed for 15 h. The
product was isolated by filtration and dried to give the sodium salt
26a as a white solid (223 mg, 96%). 1H NMR (DMSO-d6) δ 15.50
(s, 1H), 8.05 (m, 1H), 7.46 (m, 3H), 7.31 (m, 3H), 2.94 (s, 3H),
2.16 (m, 1H), 1.71 (m, 1H), 1.39 (s, 3H), 0.80 (m, 1H), 0.70 (s,
9H), 0.44 (m, 1H). 13C NMR (DMSO-d6) δ 196.3, 181.2, 157.4,
145.7, 136.7, 133.8, 132.7, 131.3, 126.4, 126.3, 125.9, 125.6, 123.6,
119.5, 113.8, 102.3, 49.0, 39.7, 39.0, 37.2, 30.6, 30.3, 29.8. MS
(ESI) m/z 530 (M - H)-. HRMS (ESI) m/z calcd (C25H29N3NaO6S2)
554.13900, found 554.13946 (M + Na)+, -0.8 ppm error.
Sodium (S)-4-(3,3-Dimethyl-butyl)-2-(7-methanesulfonylamino-
1,1-dioxo-1,4-dihydro-1λ6-benzo[1,2,4]thiadiazin-3-yl)-4-methyl-3-
oxo-3,4-dihydro-naphthalen-1-olate (26b). Following the procedure
for compound 25a, except substituting compound 24b for com-
pound 23a, the title compound 26b (1.24 g, 93%) was obtained as
a yellow solid. 1H NMR (DMSO-d6) δ 15.48 (s, 1H), 8.05 (m, 1H),
7.46 (m, 3H), 7.30 (m, 3H), 2.93 (s, 3H), 2.16 (m, 1H), 1.71 (m,
1H), 1.39 (s, 3H), 0.81 (m, 1H), 0.70 (s, 9H), 0.44 (m, 1H). 13C
NMR (DMSO-d6) δ 195.5, 180.4, 156.6, 145.1, 136.6, 133.1, 131.7,
130.7, 125.7, 125.7, 125.2, 124.9, 122.9, 118.8, 112.9, 101.7, 48.3,
39.0, 38.3, 36.5, 30.0, 29.7, 29.1. MS (ESI) m/z 530 (M - H)-.
HRMS (ESI) m/z calcd (C25H29N3O6S2) 532.1571, found 532.1570
(M + H)+, <0.1 ppm error.
HCV Polymerase Inhibition Assays. Dilutions of the inhibitors
were incubated with 20 mM Tris-Cl pH 7.4, 2 mM MnCl2, 80 mM
potassium glutamate, 1 mM dithiothreitol, 1 mM ethylene diamine
tetraacetic acid (EDTA), 0.1 mg/ml BSA, 125 uM GTP and 20
nM NS5B, HCV strain 1a (H77, Genbank accession number
AF011751) or 62.5 uM GTP and 50 nM NS5B, HCV strain 1b
(BK) for 15 min at room temperature. The reaction was initiated
by the addition of 20 µM CTP, 20 µM ATP, 0.284 µM (0.5 µCi)
of 5,6-[3H] UTP, 5 nM template RNA, and 0.1 U/µL RNase
inhibitor (RNasin, Promega), and allowed to proceed for 3 h at 30
°C. Reaction volume was 50 µL. The reaction was terminated by
the addition of 1 volume of 4 mM spermine in 10 mM Tris-Cl pH
8.0, 1 mM EDTA. After incubation for at least 15 min at room
temperature, the precipitated RNA was captured by filtering through
a GF/B filter (Millipore) in a 96-well format. The filter plate was
washed five times with 200 µL each of 2 mM spermine, 10 mM
Tris-Cl pH 8.0, and 1 mM EDTA and 2 times with ethanol. After
air drying, 30 µL of Microscint 20 scintillation cocktail (Packard)
was added to each well and the retained cpm were determined by
scintillation counting. The initial rates of 24 inhibitor concentrations
were fit to the tight binding equation 1 to obtain IC50.
2
[ ]
[ ]
V ) kcat S ⁄ 2 K + S
IC + I - E + 4IC E -
(
(
)
(
√
)
(
)
50
m
50
IC + I - E
(1)
)
)
(
50
Replicon Assays with Laboratory Strains. A genotype 1b-con1
strain subgenomic replicon18 and a chimeric genotype 1a-H77/1b-
con1 subgenomic replicon were used in these studies.25 For the
chimeric replicon, the nonstructural genes NS3 (except for the
N-terminal 73 amino acids), NS4A, NS5B, and the 3′ nontranslated
region were derived from the 1a strain H77 and the first 73 amino
acids of NS3 along with all of NS4B and NS5A were from the

1668 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6
Wagner et al.
(11) Malnick, S. D. H.; Beergabel, M.; Lurie, Y. Treatment of chronic hepatitis
C virus infection. Ann. Pharmacother. 2000, 34 (10), 1156–1164, see
also the reference of footnote 4 authored by Pearlman, B. L.
(12) Hoeroldt, B.; Haydon, G.; O’Donnell, K.; Dudley, T.; Nightingale,
P.; Mutimer, D. Results of combination treatment with pegylated
interferon and ribavirin in cirrhotic patients with hepatitis C infection.
LiVer Int. 2006, 26, 650–659. (b) Manns, M. P.; Wedemeyer, H.;
Cornberg, M. Treating viral hepatitis C: efficacy, side effects, and
complications. Gut 2006, 55, 1350–1359. (c) Mallolas, J.; Laguno,
M. Pegylated IFN-R2b plus ribavirin for treatment-na¨ıve patients
coinfected with HCV and HIV. Expert ReV. Anti-Infect. Ther. 2008,
6 (3), 281–289.
evaluated over the concentration range 0-6.4 µg/mL, was linear
(correlation coefficient >0.999), with mean accuracy values from
96-107% of theoretical for the analysis of triplicate standards
at seven separate concentrations. The limit of quantitation was
estimated to be ∼20 ng/mL from a 0.25 mL plasma sample.
Supporting Information Available: ORTEP plot of single
crystal X-ray structure determination of intermediate 10a. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(13) Bartenschlager, R. Candidate targets for hepatitis C virus-specific
antiviral therapy. InterVirology 1997, 40 (5-6), 378–393.
References
(14) Lohmann, V.; Korner, F.; Herian, U.; Bartenschlager, R. Biochemical
properties of hepatitis C virus NS5B RNA-dependent RNA polymerase
and identification of amino acid sequence motifs essential for enzymic
activity. J. Virol. 1997, 71 (11), 8416–8428. (b) Lohmann, V.; Roos,
A.; Korner, F.; Koch, J. O.; Bartenschlager, R. Biochemical and kinetic
analyses of NS5B RNA-dependent RNA polymerase of the hepatitis
C virus. Virology 1998, 249 (1), 108–118. (c) Lohmann, V.; Roos,
A.; Korner, F.; Koch, J. O.; Bartenschlager, R. Biochemical and
structural analysis of the NS5B RNA-dependent RNA polymerase of
the hepatitis C virus. J. Vir. Hepatitis 2000, 7 (3), 167–174. (d)
Ivashkina, N.; Wolk, B.; Lohmann, V.; Bartenschlager, R.; Blum,
H. E.; Penin, F.; Moradpour, D. The hepatitis C virus RNA-dependent
RNA polymerase membrane insertion sequence is a transmembrane
segment. J. Virol. 2002, 76 (24), 13088–13093. (e) Moradpour, D.;
Brass, V.; Bieck, E.; Friebe, P.; Gosert, R.; Blum, H. E.; Barten-
schlager, R.; Penin, F.; Lohmann, V. Membrane association of the
RNA-dependent RNA polymerase is essential for hepatitis C virus
RNA replication. J. Virol. 2004, 78 (23), 13278–13284.
(15) (a) Parfieniuk, A.; Jaroszewicz, J.; Flisiak, R. Specifically targeted
antiviral therapy for hepatitis C virus. World J. Gastroenterol. 2007,
13 (43), 5673–5681. (b) Liu-Young, G.; Kozal, M. J. Hepatitis C
protease and polymerase inhibitors in development. AIDS Patient Care
STDs 2008, 22 (6), 449–457. (c) Beaulieu, P. L. Non-nucleoside
inhibitors of the HCV NS5B polymerase: progress in the discovery
and development of novel agents for the treatment of HCV infections.
Curr. Opin. InVest. Drugs 2007, 8 (8), 614–634.
(16) See example 432c in Pratt, J. K.; Betebenner, D. A.; Donner, P. L.;
Green, B. E.; Kempf, D. J.; McDaniel, K. F.; Maring, C. J.; Stoll,
V. S.; Zhang, R. Preparation of 1,1-dioxido-4H-1,2,4-benzothiadiazines
as hepatitis C polymerase inhibitors and anti-infective agents U.S.
Patent 2004087577, 2004.
(17) Bosse, T. D.; Larson, D. P.; Wagner, R.; Hutchinson, D. K.; Rockway,
T. W.; Kati, W. M.; Liu, Y.; Masse, S.; Middleton, T.; Mo, H.;
Montgomery, D.; Jiang, W.; Koev, G.; Kempf, D. J.; Molla, A.
Synthesis and SAR of novel 1,1-dialkyl-2(1H)-naphthalenones as
potent HCV polymerase inhibitors. Bioorg. Med. Chem. Lett. 2008,
18 (2), 568–570.
(1) Choo, Q.; Kuo, G.; Weiner, A.; Overby, L.; Bradley, D.; Houghton,
M. Isolation of a cDNA clone derived from a blood-borne non-A,
non-B viral hepatitis genome, Science 1989, 244, (4902), 359-362.
(b) Kuo, G.; Choo, Q.; Alter, H.; Gitnick, G.; Redeker, A.; Purcell,
R.; Miyamura, T.; Dienstag, J.; Alter, M.; Stevens, C. An assay for
circulating antibodies to a major etiologic virus of human non-A, non-B
hepatitis. Science 1989, 244 (4902), 362–364.
(2) Cohen, J. Public health effort unwittingly spread HCV. Science 1999,
285 (5424), 27.
(3) Prince, A. M.; Grady, G. F.; Hazzi, C.; Brotman, B.; Kuhns, W. J.;
Levine, R. W.; Millian, S. J. Long-incubation post-transfusion hepatitis
without serological evidence of exposure to hepatitis B virus. Lancet
1974, 2 (7875), 241–246. (b) Koretz, R. L.; Suffin, S. C.; Gitnick,
G. L. Post-transfusion chronic liver disease. Gastroenterology 1976,
71, 797–803. (c) Mathiesen, L. R.; Skinhoj, P.; Hardt, F.; Nielsen,
J. O.; Sloth, K.; Zoffmann, H. Epidemiology and clinical characteristics
of acute hepatitis types A, B, and non-A non-B. Scand J. Gastroen-
terol. 1979, 14 (7), 849–856. (d) Koretz, R. L.; Stone, O.; Gitnick,
G. L. The long-term course of non-A, non-B post-transfusion hepatitis.
Gastroenterology 1980, 79, 893–898. (e) Kryger, P.; Aldershvile, J.;
Christoffersen, P.; Hardt, F.; Juhl, E.; Mathiesen, L. R.; Nielsen, J. O.;
Poulsen, H. Acute non-A, non-B hepatitiss clinical, epidemiological
and histological characteristics. Scand. J. Infect. Dis. 1980, 12, 165–
169. (f) Mathiesen, L. R.; Hardt, F.; Dietrichson, O.; Purcell, R. H.;
Wong, D.; Skinhoj, P.; Nielsen, J. O.; Zoffmann, H.; Iversen, K. The
role of acute hepatitis type A, B, and non-A non-B in the development
of chronic active liver disease. Scand. J. Gastroenterol. 1980, 15 (1),
49–54.
(4) Poynard, T.; Bedossa, P.; Opolon, P. Natural history of liver fibrosis
progression in patients with chronic hepatitis C. Lancet 1997, 349,
825–832. (b) Yatsuhashi, H.; Yano, M. Towards control of hepatitis
C in the Asia-Pacific regionsNatural history of chronic hepatitis
C. J. Gastroenterol. Hepatol. 2000, 15 (Suppl.), E111-E116. (c)
Pearlman, B. L. Hepatitis C Infection: A Clinical Review. South.
Med. J. 2004, 97 (4), 364–373. (d) Ascione, A.; Tartaglione, T.;
Giuseppe Di Costanzo, G. Natural history of chronic hepatitis C
virus infection. Dig. LiVer Dis. 2007, 39 (Suppl 1), S4-S7.
(18) Wakita, T.; Pietschmann, T.; Kato, T.; Date, T.; Miyamoto, M.; Zhao,
Z.; Murthy, K.; Habermann, A.; Kraeusslich, H.-G.; Mizokami, M.;
Bartenschlager, R.; Liang, T. J. Production of infectious hepatitis C
virus in tissue culture from a cloned viral genome. Nat. Med. 2005,
11 (7), 791–796. (b) Zhong, J.; Gastaminza, P.; Cheng, G.; Kapadia,
S.; Kato, T.; Burton, D. R.; Wieland, S. F.; Uprichard, S. L.; Wakita,
T.; Chisari, F. V. Robust hepatitis C virus infection in vitro. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102 (26), 9294–9299.
(5) Kilpe, V. E.; Krakauer, H.; Wren, R. E. An analysis of liver transplant
experience from 37 transplant centers as reported to Medicare.
Transplantation 1993, 56, 554–561. (b) Alter, M. J. The epidemiology
of acute and chronic hepatitis C. Clin. LiVer. Dis. 1997, 1, 559–568.
(c) Primary Liver Disease of Liver Transplant Recipients 1991 and
1992 (From the UNOS Scientific Registry). UNOS Update 1993, 9,
27.
(19) Lohman, V.; Korner, F.; Koch, J.; et al. Replication of subgenomic
hepatitis C virus RNAs in a hepatoma cell line. Science 1999, 285
(5424), 110–113. (b) Blight, K. J.; Kolykhalov, A. A.; Rice, C. M.
Efficient initiation of HCV RNA replication in cell culture. Science
2000, 290 (5498), 1972–1974.
(20) Lamarre, D.; Anderson, P. C.; Bailey, M.; Beaulieu, P.; Bolger, G.;
Bonneau, P.; Boes, M.; Cameron, D. R.; Cartier, M.; Cordingley,
M. G.; Faucher, A.; Goudreau, N.; Kawai, S. H.; Kukolj, G.; Lagace,
L.; LaPlante, S. R.; Narjes, H.; Poupart, M.; Rancourt, J.; Sentjens,
R. E.; George, R.; Simoneau, B.; Steinmann, G.; Thibeault, D.;
Tsantrizos, Y. S.; Weldon, S. M.; Yong, C.; Llinas-Brunet, M. An
NS3 protease inhibitor with antiviral effects in humans infected with
hepatitis C virus. Nature 2003, 426 (6963), 186–189.
(6) Rao, K. V.; Anderson, R. C. Long-term results and complications in
renal transplant recipients. Observations in the second decade. Trans-
plantation 1988, 45, 45–52.
(7) Wise, M.; Bialek, S.; Finelli, L.; Bell, B. P.; Sorvillo, F. Changing
trends in hepatitis C-related mortality in the United States 1995-2004.
Hepatology 2008, 47 (4), 1128–1135.
(8) Davis, G. L.; Albright, J. E.; Cook, S. F.; Rosenberg, D. M.
Projecting future complications of chronic hepatitis C in the United
States. LiVer Transplant. 2003, 9 (4), 331–338. (b) National Institutes
of Health Consensus Development Conference Panel Statement:
management of hepatitis C. Hepatology 1997, 26 (Suppl. 1), 2S-
10S.
(9) Deuffic-Burban, S.; Poynard, T.; Sulkowski, M. S.; Wong, J. B.
Estimating the future health burden of chronic hepatitis C and human
immunodeficiency virus infections in the United States. J. Vir.
Hepatitis 2007, 14, 107–115.
(10) Kato, N. Genome of human hepatitis c virus (HCV): Gene
organization, sequence diversity, and variation. Microbiol. Comp.
Genomics 2000, 5 (3), 129–151. (b) Zein, N. N. Clinical significance
of hepatitis C virus genotypes. Clin. Microbiol. ReV. 2000, 223–
235. (c) Ramadori, G.; Meier, V. Hepatitis C virus infection: 10
years after the discovery of the virus. Eur. J. Gastroenterol. Hepatol.
2001, 13, 465–471.
(21) See compound 13 in:(a) Krueger, A. C.; Madigan, D. L.; Green,
B. G.; Hutchinson, D. K.; Jiang, W. W.; Kati, W. M.; Liu, Y.;
Maring, C. J.; Masse, S. V.; McDaniel, K. F.; Middleton, T. R.;
Mo, H.; Molla, A.; Montgomery, D.; Ng, T. I.; Kempf, D. J.
Inhibitors of HCV NS5B polymerase: synthesis and structure
activity relationships of unsymmetrical 1-hydroxy-4,4-dialkyl-3-
oxo-3,4-dihydronapthalene benzothiadiazine derivatives. Bioorg.
Med. Chem. Lett. 2007, 17, 2289–2292.
(22) Chen, C-M.; He, Y.; Lu, L.; Lim, H. B.; Tripathi, R. L.; Middleton,
T.; Hernandez, L. E.; Beno, D.W. A.; Long, M. A.; Kati, W. M.;
Bosse, T. D.; Larson, D. P.; Wagner, R.; Lanford, R. E.;

Inhibitors of Hepatitis C Virus Polymerase
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 6 1669
Kohlbrenner, W. E.; Kempf, D. J.; Pilot-Matias, T. J.; Molla, A.
Activity of a potent hepatitis C virus polymerase inhibitor in the
chimpanzee model. Antimicrob. Agents Chemother. 2007, 51, 4290–
4296.
as hepatitis C polymerase inhibitors and anti-infective agents. Patent
WO 2004 0411818, 2004.
(25) Yi, M.; Lemon, S. M. Adaptive mutations producing efficient
replication of genotype 1a hepatitis C virus RNA in normal Huh7
cells. J. Virol. 2004, 78, 7904–7915.
(26) Pauwels, R. J.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.;
Herdewijn, P.; Desmyter, J.; DeClerq, E. Rapid and automated
tetrazolium-based colorimetric assay for the detection of anti-HIV
compounds. J. Virol. Methods 1988, 20, 309–321.
(23) See example 3 Part A in: Wagner, R.; Donner, P. L.; Kempf, D. J.;
Maring, C. J.; Stoll, V. S.; Pu, Y. Preparation of fused thiadiazines as
hepatitis C virus (HCV) polymerase inhibitors. Patent WO 2008
011337, 2008.
(24) See example 414a in Pratt, J. K.; Betebenner, D. A.; Donner, P. L.;
Green, B. E.; Kempf, D. J.; McDaniel, K. F.; Maring, C. J.; Stoll,
V. S.; Zhang, R. Preparation of 1,1-dioxido-4H-1,2,4-benzothiadiazines
JM8010965