Identification and structure-based optimization of novel dihydropyrones as potent HCV RNA polymerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.06.065

A novel class of non-nucleoside HCV NS5B polymerase inhibitors has been identified from screening. A co-crystal structure revealed an allosteric binding site in the protein that required a unique conformational change to accommodate inhibitor binding. Her

Full text of DOI:10.1016/j.bmcl.2006.06.065

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4834–4838
Identification and structure-based optimization of novel
dihydropyrones as potent HCV RNA polymerase inhibitors
Hui Li,a,* John Tatlock,a Angelica Linton,a Javier Gonzalez,a Allen Borchardt,a
Peter Dragovich,b Tanya Jewell,a Tom Prins,b Ru Zhou,a Julie Blazel,b Hans Parge,a
Robert Love,a Michael Hickey,a Chau Doan,a Stephanie Shi,a Rohit Duggal,a
Cristina Lewisc and Shella Fuhrmana
aPfizer Global Research and Development, La Jolla Laboratories, 10770 Science Center Dr., San Diego, CA 92121, USA
bAnadys Pharmaceuticals, 3115 Merryfield Row, San Diego, CA 92121, USA
cGenentech, Inc., 1 DNA Way, South San Francisco, CA 94098, USA
Received 14 April 2006; revised 17 June 2006; accepted 19 June 2006
Available online 7 July 2006
Dedicated to Professor K. C. Nicolaou on the occasion of his 60th birthday.
Abstract—A novel class of non-nucleoside HCV NS5B polymerase inhibitors has been identified from screening. A co-crystal struc-
ture revealed an allosteric binding site in the protein that required a unique conformational change to accommodate inhibitor bind-
ing. Herein we report the structure–activity relationships (SARs) of this novel class of dihydropyrone-containing compounds that
show potent inhibitory activities against the HCV RNA polymerase in biochemical assays.
Ó 2006 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) infects approximately 3% of
the global population and represents a major public
health problem worldwide. In the US, it has been esti-
mated that there are more than 4 million infections,
mostly with genotype 1a and 1b. Of those individuals
exposed to HCV, 80% become persistently infected,
and about 20–30% of the chronically infected popula-
tion eventually develops progressive liver diseases,
including cirrhosis, hepatocellular carcinoma (HCC),
and liver failure.1 The current standard care of treat-
ment for chronic hepatitis C is based on the combina-
tion of subcutaneous pegylated interferon-a and 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
releases at least 10 structural and non-structural pro-
teins. The HCV NS5B protein has been characterized
as an RNA-dependent RNA polymerase (RdRp)3 and
is essential for viral replication. Because of the consider-
able differences as compared to human DNA and RNA
polymerases, the HCV NS5B is an attractive target for
selective anti-viral therapy.
Development of HCV NS5B polymerase inhibitors,
particularly at allosteric binding sites, has gained
increasing interests in recent years.4 A number of com-
pounds spanning multiple chemotypes have been report-
ed in the literature (Fig. 1). Based on both mutational
sites in the replicon system and protein crystallographic
studies, several different allosteric binding sites of the
HCV NS5B protein have been identified.5 Benzimid-
azole inhibitor 16 has been reported to bind to the
non-catalytic GTP site within the thumb domain. An
X-ray crystal structure with the indole analog 27 also
confirmed this allosteric binding site close to residue
Pro-495. Benzothiadiazine analogs, such as 3,8 inhibit
the initial step of RNA synthesis and induce a resistant
mutation M414T close to the base of the thumb domain.
Another allosteric binding site in the thumb domain was
reported with both substituted phenylalanine 49,10 and
HCV is a positive-sense, single-stranded-RNA virus.
The viral genome encodes a single poly-peptide with
about 3000 amino acid residues, which upon cleavage
Keywords: HCV polymerase; Dihydropyrone; Structure-based drug
design.
*
Corresponding author. Tel.: +1 858 622 6076; e-mail: hui.li@
pfizer.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.065

H. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4834–4838
4835
a conformational rearrangement. During this process
Met-423 and Leu-497 moved away from their original
positions, opening up a new hydrophobic pocket
(pocket A, Fig. 2). Examination of the X-ray co-crystal
structure revealed some key interactions between com-
pound 6 and the HCV polymerase protein. The enol/ke-
tone oxygen of the dihydropyrone forms a direct
hydrogen bond to the backbone amide NH of Ser-476
and a water-mediated hydrogen bond to the amide
NH of Tyr-477 (the donor–donor motif). The lactone
carbonyl of the dihydropyrone is involved in a water-
mediated hydrogen bond to Arg-501. The phenol
functional group forms another hydrogen bond with
Leu-497 through a water molecule, while the phenyl ring
occupies an otherwise hydrophobic pocket (pocket B).
The extension of an additional aromatic ring from the
dihydropyrone, through a sulfur linker, appears to make
limited interactions with the protein (pocket C, Fig. 3).
The preparation of dihydropyrone analogs is outlined in
Scheme 1. Two efficient synthetic approaches toward
key intermediate ketone 11 were utilized. The first start-
ed from substituted phenylpropanoic acids, which upon
reaction with dipyridyl disulfide and triphenylphosphine
at ambient temperature provided pyridyl thioester 8 in
good yields. Further reaction of intermediate 8 with
cyclopentyl magnesium bromide afforded the desired
ketone. The second method utilized a unique palladium
catalyzed Heck reaction protocol between 1-cyclopen-
tyl-2-propen-1-ol 10 and various aryl bromides (or tri-
flates) 9. After carbon–carbon bond formation and the
subsequent enol/ketone isomerization, this route provid-
ed the desired ketone 11 in one single step compatible
with broad R1 variation. Formation of the dihydropy-
rone ring from ketone 11 was straightforward. The
dianion formed from methyl acetoacetate (NaH
followed by n-BuLi) was treated with the ketone to
afford the open chain product 12, which upon reaction
with NaOH in aqueous THF provided the ring-closed
product 13. Chlorination of intermediate 13 at the 3-po-
sition was accomplished by reaction of SO2Cl2 in
CH2Cl2 with high regio-selectivity. The chloro-dihydro-
pyrone product 14 served as a versatile template and
reacted readily with various thiophenols under basic
conditions in DMF to provide sulfur-linked analogs of
general structure 15 in good yields.
Figure 1. Selected HCV NS5B polymerase inhibitors.
thiophene carboxylic acid derivatives 5.11,12 Recently
two new allosteric binding sites,13,14 the anchor grip
and the primer grip sites, were also reported. In this
paper, we report our own effort in the synthesis,
preliminary structure–activity relationship (SAR), and
biological properties of a series of dihydropyrones as
HCV NS5B polymerase inhibitors.
High-throughput screening (HTS) of a proprietary
collection of in-house compounds identified compound
6 (Fig. 2) as an HCV RNA polymerase inhibitor with
modest potency (IC50 = 0.93 lM). Subsequent biochem-
ical experiments demonstrated that compound 6 was a
specific, reversible inhibitor of the HCV NS5B polymer-
ase that bound non-competitively with respect to GTP.
An X-ray co-crystal structure15 of this compound with
NS5B (genotype 1b, BK strain, CD21) triple mutant
(Lys114Asn/Leu47Gln/Phe101Tyr) identified an alloste-
ric binding site close to the junction of thumb and finger
˚
domain, approximately 30 A away from the active site.
Upon inhibitor binding the protein structure underwent
˚
Figure 3. X-ray co-crystal structure (2.2 A resolution) of compound 6
Figure 2. Dihydropyrone lead (6) from high-throughput screening.
bound to the HCV DC21 NS5B protein.

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H. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4834–4838
Table 1. Enzymatic potencies of dihydropyrones: pocket A optimiza-
tion
Compound
R2
R3
IC50 (lM)
16
17
18
19
20
Me
n-Pr
OH
OMe
OH
OH
OH
>100
>50
93
Cyclobutyl
Cyclopentyl
Cyclohexyl
8.2
52
Scheme 1. Synthesis of dihydropyrone derivatives. Reagents and
conditions: (a) PPh3, 2,20-dipyridyl disulfide, CH2Cl2; (b) cyclopentyl
magnesium bromide, THF, ꢀ78 °C; (c) Pd(PPh3)2Cl2, NaHCO3,
NMP, 140 °C; (d) NaH, methyl acetoacetate, followed by n-BuLi,
ꢀ78 °C; (e) NaOH, THF/H2O; (f) SO2Cl2, CH2Cl2; (g) Ar2SH, DMF,
50 °C.
˚
Figure 4. X-ray co-crystal structure of compound 33 (1.58 A resolu-
tion) bound to HCV NS5B DC21 protein.
group of compound 19 at the para position of the phenyl
ring afforded analog 22 with almost equal potency, indi-
cating that a hydrogen bond acceptor is sufficient at this
position to provide the interaction with Leu-497
through a water-mediated hydrogen bond. Extension
from the oxygen with bulkier groups, such as Et or
i-Pr (Table 2, 23 and 24), further increased compound
potency, probably by providing more hydrophobic
interactions. Elimination of the oxygen atom from com-
pound 24 in pocket B resulted in a 3-fold loss of potency
(25, IC50 = 4.3 lM), which can be attributed to the loss
of hydrogen bonding and decrease in hydrophobic
interaction (Fig. 5).
All compounds synthesized were assayed against HCV
genotype 1b (BK strain) NS5B DC21 enzyme to assess
their inhibitory activity (IC50) as previously
described.15
Early exploration was focused on evaluation of binding
features of compound 6 in pockets A and B. Truncation
of the sulfur-linked aniline of compound 6 (pocket C)
resulted in an 8 fold loss in potency in the enzymatic
assay (Table 1, 19). However, this unsubstituted dihyd-
ropyrone simplified chemical synthesis and served as a
good starting point for the further optimization of pock-
ets A and B. Comparison of the co-crystal structure of
compound 6 and the apo structure of the HCV NS5B
protein revealed that the cyclopentyl ring forced a reor-
ganization of residues Met-423 and Leu-497. This result-
ed in the placement of the cyclopentyl group, with a high
degree of complementarity, into the newly formed pock-
et A. As summarized in Table 1, substitutions at this
position with either larger or smaller groups proved to
be detrimental for activity against HCV NS5B enzyme
(compounds 16–18 and 20).
Consistent with the X-ray co-crystal structure, substitu-
tions at the meta position of the phenyl ring in pocket B
were well tolerated and provided a second hydrophobic
interaction (Table 2, 26–29). A simple chlorine substitu-
tion (27, IC50 = 5 lM) afforded 10-fold potency
improvement over unsubstituted derivative 21
(IC50 = 48 lM) and slightly larger groups, such as Et
and i-Pr, further enhanced compound potency to 3.6
and 1.8 lM, respectively. In an attempt to evaluate the
combined substitution effects at both meta and para
positions, compounds 30–33 were prepared. It was
found that a smaller group at the meta position, such
as fluorine, in combination with para-alkyloxy substitu-
tion, afforded the most potent compound 33 with an
IC50 of 0.53 lM.
A close analysis of the pocket B binding site revealed
two smaller hydrophobic pockets, almost perpendicular
to each other (Fig. 4).16 Methylation of the hydroxyl

H. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4834–4838
4837
Table 2. Enzymatic potencies of dihydropyrones: pocket B optimiza-
tion
Table 3. Enzymatic potencies of dihydropyrones: pocket C optimiza-
tion
R7
IC50 (lM)
Compound
R4
R5
IC50 (lM)
Compound
21
22
23
24
25
26
27
28
29
30
31
32
33
H
H
48
35
0.56
H
MeO
EtO
i-PrO
i-Pr
H
9.7
2.9
1.7
4.3
23
H
H
36
37
38
39
0.13
0.28
0.14
0.038
H
Me
Cl
Et
i-Pr
Cl
F
H
5.0
3.6
1.8
1.0
0.89
0.79
0.53
H
H
MeO
MeO
EtO
i-PrO
F
F
improvement (IC50 = 0.13 lM, Table 3, 36). Five-mem-
bered heterocyclic ring systems generally provided bet-
ter inhibitory potency against the HCV NS5B enzyme
and the N-methyl triazole 39 exhibited the best potency
within the series (IC50 = 0.038 lM).
All compounds were tested in replicon assays in the
Huh-7 hepatoma cell line harboring a self-replicating
HCV subgenomic replicon of genotype 1b.17 However,
none of them exhibited any significant antiviral activity
up to 10 lM.
Figure 5. The importance of the enol hydrogen-bond donor.
The dihydropyrone core was essential for the potency of
this series of compounds. Selective O-methylation of
compound 30 afforded compound 34, which was almost
30-fold less potent when compared with the free OH
analog. It is reasonable to assume that a substantial con-
tribution to this loss in potency of the racemate is
accounted for by that enantiomer which places the extra
methyl in the very constrained steric environment to
interfere with the water-mediated hydrogen-bonding
network between the dihydropyrone enol/ketone oxygen
and the donor–donor motif (Ser-476 and Tyr-477) in the
HCV NS5B protein.
In conclusion, we have described the identification of a
novel series of dihydropyrones as potent allosteric inhib-
itors of the HCV NS5B polymerase. Initial optimization
of the series has resulted in more than a 30-fold increase
in compound potency and led to analogs with nanomolar
IC50 against the enzyme. Further efforts to improve com-
pound enzymatic potency and physical/chemical proper-
ties to achieve antiviral activity in the cell-based
replicon system will be reported in future
communications.
Acknowledgments
With a 15-fold potency improvement from the pocket
B optimization, pocket C fragments were re-introduced
to further explore potency enhancement against HCV
NS5B. Examination of the X-ray structure revealed
that this portion of the molecule resided in a shallow
canyon on the protein surface, with a possible p–p
stacking interaction with His-475. A survey of various
arylthio moieties demonstrated that a simple phenyl
ring (Table 3, 35) provided a moderate 2-fold advan-
tage over the corresponding unsubstituted dihydropy-
rone 30. An additional hydroxyl group, at the para
position to the sulfur linker, afforded a 4-fold further
The authors thank Dr. Martin Edwards and Dr. Stacie
Canan for their helpful discussion during manuscript
preparation.
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