Allosteric inhibitors of hepatitis C polymerase: Discovery of potent and orally bioavailable carbon-linked dihydropyrones

Article abstract of DOI:10.1021/jm0704447

The discovery and optimization of a novel class of carbon-linked dihydropyrones as allosteric HCV NS5B polymerase inhibitors are presented. Replacement of the sulfur linker atom with carbon reduced compound acidity and greatly increased cell permeation. F

Full text of DOI:10.1021/jm0704447

© Copyright 2007 by the American Chemical Society
Volume 50, Number 17
August 23, 2007
Letters
merase,6 along with HCV NS3/4 protease,7 has attracted the
most attention as a potential therapeutic target. Both nucleoside
and non-nucleoside HCV NS5B polymerase inhibitors, alone
or in combination with Peg-IFN, have demonstrated efficacy
in human clinical trials (Figure 1). A 15-day monotherapy trial
in genotype 1 patients with the Idenix nucleoside inhibitor
valopicitabine 1 (NM-283) resulted in a mean viral load
reduction of 1.2 logs with no apparent resistance development.
When combined with Peg-IFN, valopicitabine achieved a 4.5
log reduction in viral load at 24 weeks.8 Recently, interim results
from an early clinical trial of Roche’s nucleoside inhibitor 2
(R1626) also indicated human efficacy.9 The efforts in develop-
ing non-nucleoside polymerase inhibitors generated equally
encouraging results. Compound 3 (HCV-796), an allosteric-site
inhibitor from Viropharma/Wyeth, was shown to induce a mean
viral load reduction of 1.4 logs in monotherapy and 2-3 logs
in combination therapy with Peg-IFN after 14 days in patients
infected with various genotypes.10 These results constitute proof
of concept for HCV polymerase inhibitors and support the
possibility of replacing ribavirin from the current standard
therapy with a more tolerable alternative.
In the previous communication,11 we have reported the
identification and preliminary optimization of a novel class of
dihydropyrones derived from a high-throughput screening (HTS)
hit (Figure 2, 4) as potent HCV NS5B polymerase inhibitors.
These compounds bind at an allosteric binding site close to the
junction of thumb and finger domain, approximately 30 Å away
from the active site.12 In particular, a more optimized sulfur-
linked analogue, 6-[2-(3-chloro-4-methoxyphenyl)ethyl]-6-cy-
clopentyl-3-[(5,7-dimethyl[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)-
thio]-4-hydroxy-5,6-dihydro-2H-pyran-2-one (Figure 2, 5), exhibits
low nanomolar potency in the biochemical assay against the
isolated HCV NS5B polymerase and is also effective at blocking
the replication of subgenomic HCV RNA in the replicon system.
However, significant hurdles still remain for the development
of such compounds as drug candidates. While single-digit
nanomolar potency was achieved in the enzymatic assay against
the truncated genotype 1b HCV NS5B ∆21 polymerase, the
S-linked dihydropyrones only demonstrated moderate antiviral
activity in the cell-based replicon assay at micromolar concen-
trations.13 Additionally, in the subsequent rat pharmacokinetic
evaluation, 5 showed low bioavailability (2%, Table 1) and poor
Allosteric Inhibitors of Hepatitis C Polymerase:
Discovery of Potent and Orally Bioavailable
Carbon-Linked Dihydropyrones
Hui Li,* Angelica Linton, John Tatlock, Javier Gonzalez,
Allen Borchardt, Mel Abreo, Tanya Jewell, Leena Patel,
Matthew Drowns, Sarah Ludlum, Mike Goble,
Michele Yang, Julie Blazel, Ravi Rahavendran,
Heather Skor, Stephanie Shi, Cristina Lewis, and
Shella Fuhrman
Pfizer Global Research and DeVelopment, La Jolla Laboratories,
10770 Science Center DriVe, San Diego, California 92121
ReceiVed April 14, 2007
Abstract: The discovery and optimization of a novel class of carbon-
linked dihydropyrones as allosteric HCV NS5B polymerase inhibitors
are presented. Replacement of the sulfur linker atom with carbon
reduced compound acidity and greatly increased cell permeation. Further
structure-activity relationship (SAR) studies led to the identification
of compounds, exemplified by 23 and 24, with significantly improved
antiviral activities in the cell-based replicon assay and favorable
pharmacokinetic profiles.
Hepatitis C virus (HCV) is a major public health problem
that infects an estimated 170 million people worldwide.1 It is
the leading cause of liver transplantation in the developed world
and results in over 10 000 deaths annually in the U.S. alone.
Currently there is no vaccine to prevent the disease nor specific
antiviral agent directed against HCV infection on the market.
The standard treatment of chronic hepatitis C is based on the
combination of subcutaneous pegylated interferon (Peg-IFN)
along with the oral nucleoside drug ribavirin. However, serious
side effects and poor response rates, particularly among patients
with genotype 1, render the development of novel anti-HCV
therapy an urgent need.2
In the past decade, particularly after a reliable tissue culture
replication system based on subgenomic replicons was intro-
duced,3 there have been tremendous efforts dedicated to the
development of novel, effective anti-HCV agents.4,5 Among
essential enzymes for HCV replication, the HCV NS5B poly-
* To whom correspondence should be addressed. Phone: 858-622-6076.
E-mail: hui.li@pfizer.com.
10.1021/jm0704447 CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/21/2007

3970 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Letters
Scheme 1. Synthesis of Carbon-Linked Dihydropyronea
Figure 1. Selected HCV NS5B polymerase inhibitors that demonstrated
efficacy in human clinical trials.
a Reagents and conditions: (a) LDA, -78 °C; (b) CsF, MeOH, 25 °C,
61% for two steps; (c) Pd(PPh3)2Cl2, CuI, diisopropylamine, DMF, 90 °C;
(d) Pd(OH)2, H2, MeOH, 25 °C; (e) K2CO3, MeOH, 45 °C; (f) ArCHO,
MeOH, BH3‚SMe2, 25 °C.
of EC50 to IC50) and the fact that compounds showed low
absorption in rat PK studies turned our attention to the
permeability of this series of compounds. In experimental pKa
determinations, the S-linked dihydropyrones (pKa ≈ 4-5) are
typically 10- to 100- fold more acidic than the corresponding
C-linked analogs (pKa ≈ 5-7). To evaluate the impact of the
single linker atom replacement on the overall profile of these
molecules, the carbon-linked dihydropyrone 6 was prepared
through a convergent synthetic sequence as outlined in Scheme
1.
Figure 2. Dihydropyrones as HCV polymerase inhibitors.
Table 1. Comparison between S-Linked (5) and C-Linked (6)
Dihydropyrones
X ) S (5)
X ) C (6)
IC50 (µM)a
0.036
3.25
90
>100
25
0.020
0.33
17
>100
10
2,2,6-Trimethyl-4H-1,3-dioxin-4-one (7) was deprotonated
with LDA at -78 °C in THF, which upon treatment with TMS-
protected ketone 8 provided acetylene 9. After removal of the
TMS group under CsF conditions in MeOH, the resulting alkyne
10 served as a common intermediate for all further analogue
synthesis. Installation of the aromatic group at the C-terminal
of alkyne 10 was accomplished by a Pd-catalyzed Sonogashira
coupling reaction with a variety of different bromides 11.
Hydrogenation of the triple bond in 12 with Pd(OH)2 in MeOH
generated the saturated 13, which subsequently reacted with
K2CO3 at 45 °C to afford the desired dihydropyrone scaffold
through a ring closure reaction in good overall yield. To generate
the carbon-linked dihydropyrones, we developed a novel,
efficient coupling reaction between the unsubstituted dihydro-
pyrone 14 and aromatic aldehydes in the presence of borane-
dimethyl sulfide complex in MeOH at ambient temperature,
presumably through a condensation/reduction sequence. This
one-step protocol provided the desired carbon-linked analogues
15 in consistently high yields with operational simplicity and
offered a significant advantage over the previously reported
procedure.14
EC50 (µM)a
EC50/IC50
CC50 (µM)
po dose (mg kg-1
)
)
b
iv dose (mg kg-1
5
2
b
Cl (mL min-1 kg-1
)
14.8 ( 2.6
2.0 ( 0.5
1.5 ( 0.2
2.5
8.3 ( 1.1
0.3 ( 0.05
0.5 ( 0.27
48
42
10/6.8
c
d
V
dss (L kg-1
)
T
1/2 effective (h)
Fa (%)e
F (%)f
2
Caco-2 AB/BA
3/2.3
Papp × 10-6 (cm s-1
)
a Genotype 1b. b 70% polyethylene glycol with 30% saline as dosing
vehicle. c Total plasma clearance. d Volume of distribution. e Fraction ab-
sorbed. f Bioavailability.
absorption after oral dosing (2.5% fraction absorbed). In this
report, we describe the discovery and structure-activity rela-
tionships (SAR) of a series of carbon-linked dihydropyrones
that achieved good potency in the biochemical assay and cell-
based replicon system and also demonstrated favorable profiles
in rat pharmacokinetic studies.
The observation that S-linked dihydropyrones generally
exhibit large gaps between their potency in the cell-based
replicon system and enzymatic assays (as measured by the ratio
The compounds synthesized were assayed against the HCV
genotype 1b NS5B polymerase to assess their inhibitory activity
(IC50) as previously described.11 The in vitro antiviral activity

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 3971
Table 3. Profiles of Two Enantiomers of Compound 22
Table 2. Further Optimization of C-Linked Dihydropyrones
enantiomer 1 (23)
enantiomer 2 (24)
IC50 (µM)a
0.003
0.070
0.015
320
10
2
3.7 ( 0.8
0.3 ( 0.8
0.88 ( 0.1
31
0.001
NA
0.032
320
10
2
4.0 ( 1.1
0.17 ( 0.1
0.47 ( 0.2
37
IC50 (µM)b
EC50 (µM)a
CC50 (µM)a
po dose (mg kg-1
c
)
)
iv dose (mg kg-1
c
Cl (mL min-1 kg-1
)
d
e
V
dss (L kg-1
)
T
1/2 effective (h)
F (%)f
a Genotype 1b. b Genotype 1a. c 70% polyethylene glycol with 30% saline
as dosing vehicle. d Total plasma clearance. e Volume of distribution.
f Bioavailability.
advantage in the cell-based replicon assay. Replacement of the
methoxy group in 6 with a methylsulfone provided 19, which
suffered a 3-fold loss in activity in the biochemical and replicon
assays. Incorporation of a gem-dimethylmethanesulfonamide
moiety at the para position of the phenyl ring resulted in 20,
which boosted the replicon activity by 4-fold over 16. Further
exploration in this region led to the discovery of a gem-dimethyl
cyano group that significantly improved compound potency by
making a direct hydrogen bond interaction with nearby Leu 497.
Both 21 and 22 achieved single-digit nanomolar potency against
the HCV polymerase enzyme, while the fluorine analogue (22)
was about 2-fold more potent than the chlorine analogue (21)
in the cell-based replicon assay.
a Genotype 1b.
(EC50) and cytotoxicity (CC50) were determined in the Huh-7
hepatoma cell line harboring a self-replicating HCV subgenomic
replicon of genotype 1b.3
Racemic 22 was separated into its individual enantiomers
using chiral HPLC with a Chiralpak AS-RH column. The two
enantiomers showed comparable potencies in the enzymatic and
replicon assays. The pharmacokinetic properties of enantiomers
23 and 24 were evaluated in rats, and their results are
summarized in Table 3. Both compounds demonstrated low in
vivo clearance and favorable oral bioavailability and half-lives.
The low volumes of distribution exhibited by both enantiomers
were anticipated as a result of the acidic nature of the
dihydropyrone template. On the basis of its potency and
pharmacokinetic profile, 23 was selected to be scaled up to
kilogram-scale for further evaluations.15
In conclusion, we have described the discovery and optimiza-
tion of a novel series of carbon-linked dihydropyrones as potent
allosteric inhibitors of the HCV NS5B polymerase. Replacement
of the sulfur linker with a carbon atom reduced the acidity of
the dihydropyrone scaffold and significantly improved cellular
permeation. This has led to an over 100-fold improvement in
antiviral activities in the cell-based replicon assay and favorable
rat pharmacokinetic profiles.
As summarized in Table 1, replacement of the sulfur linker
atom with carbon had pronounced effects on the overall
compound profiles. While in the enzymatic assay, the C-linked
6 showed only a 2-fold advantage over the S-linked analogue
5 and the improvement in the cell-based replicon system was
10-fold, thus dramatically narrowing the ratio of the EC50 to
IC50 (17 for 6 vs 90 for 5). These findings were well in line
with the permeability of these compounds measured in Caco-2
cell lines: switching from S-linker to C-linker significantly
increased the cellular permeation from low/moderate to high
(Caco-2 AB, 3 × 10-6 cm/s for 5 and 10 × 10-6 cm/s for 6),
and both compounds did not exhibit efflux issues. Encouraged
by these results, 5 and 6 were subjected to rat in vivo
pharmacokinetic evaluation. The C-linked 6 demonstrated a
much better oral absorption, as measured by the fraction
absorbed (Fa ) 48%) and good overall bioavailability at 42%.
In view of its improved potency in the replicon system and
superior pharmacokinetic profile, the C-linked dihydropyrone
6 was judged to be a more appealing lead for further SAR
studies. Optimization of the aromatic region at the left-hand
side of the molecule is summarized in Table 2. While substitu-
tion of the chlorine atom at the meta position in 6 with fluorine
(Table 2, 16) was well tolerated, the corresponding methyl
replacement resulted in a significant reduction in potency,
particularly in the cell-based replicon assay (17, EC50 ) 1.2
Acknowledgment. The authors thank Dr. William Farrell
and Ms. Christy Aurigemma for their help in chiral separation.
Supporting Information Available: Experimental procedures
and characterization data for all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
µM). A larger ethoxy group at the para position (18, IC50
)
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