Discovery of novel dialkyl substituted thiophene inhibitors of HCV by in silico screening of the NS5B RdRp

Article abstract of DOI:10.1016/j.bmcl.2007.04.103

A novel 5,4-dialkyl substituted thiophene was discovered by in silico screening of the 3D polymerase crystal structure (1GX6) that demonstrated single digit micromolar HCV inhibition activity in the replicon assay and dose-dependent inhibition in the replicase complex assay. Subsequently, SAR was explored with a small set of dialkyl and tetrahydro-benzo thiophenes. Since these thiophenes inhibit synthesis of both, single- and double-stranded RNAs, their mechanism of action is distinct from other known HCV inhibitors.

Full text of DOI:10.1016/j.bmcl.2007.04.103

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3905–3909
Discovery of novel dialkyl substituted thiophene inhibitors
of HCV by in silico screening of the NS5B RdRp
Shirley Louise-May, Wengang Yang, Xingtie Nie, Dongmei Liu, Milind S. Deshpande,
Avinash S. Phadke, Mingjun Huang* and Atul Agarwal*
Achillion Pharmaceuticals, 300 George Street, New Haven, CT 06511, USA
Received 19 March 2007; revised 26 April 2007; accepted 30 April 2007
Available online 3 May 2007
Abstract—A novel 5,4-dialkyl substituted thiophene was discovered by in silico screening of the 3D polymerase crystal structure
(1GX6) that demonstrated single digit micromolar HCV inhibition activity in the replicon assay and dose-dependent inhibition
in the replicase complex assay. Subsequently, SAR was explored with a small set of dialkyl and tetrahydro-benzo thiophenes. Since
these thiophenes inhibit synthesis of both, single- and double-stranded RNAs, their mechanism of action is distinct from other
known HCV inhibitors.
ꢀ 2007 Elsevier Ltd. All rights reserved.
In the USA alone there are an estimated four million
carriers of the RNA virus Hepatitis C, 80% of which will
develop chronic symptoms and 20–30% will progress on
to the end-stage liver diseases. Current treatment results
in a sustained response rate in about 50% patients.1 The
need for a drug that is well tolerated and targets HCV
directly motivates continued drug discovery efforts such
as the one presented here.
to HCV NS5B protrudes into the active site binding
pocket which likely aids in positioning the 30 end of
the RNA template.11 The hydrophobic C-terminal tail
is believed to act as a membrane anchor and help in
modulating the RNA synthesis as well.12 Two divalent
metal ions coordinated in the palm region drive the
polymerization reaction.13–15 One ion stabilizes the tri-
phosphate group of the incoming nucleotide, while the
other is positioned to activate the 30 hydroxyl group of
the growing RNA chain for nucleophilic attack. This
polymerization process can proceed in the absence of a
primer and from a single nucleotide in vitro.16,17
Hepatitis C is a member of the Flaviviridae family and
exists in six known genotypes and over 12 subtypes.2
The HCV RNA dependent RNA polymerase (RdRp,
NS5B) is a key enzyme in the viral life cycle and cata-
lyzes RNA polymerization.3 Recent clinical studies of
HCV NS5B inhibition by non-nucleoside and active site
nucleoside analogs have validated the HCV RdRp as a
therapeutic target.4
X-ray crystallography and mutational mapping studies
provide evidence for multiple non-nucleoside inhibitor
binding sites in and around the thumb domain.18–23
A
shallow binding pocket that binds at least three distinct
chemotypes is found at the outer base of the thumb do-
˚
main, 30 A away from the active site. Binding of di-
Multiple crystallographic structures of HCV NS5B have
been solved to date5–10 and reveal a common polymer-
ase 3D structure that resembles a right hand made up
of fingers, thumb, and palm domains (Fig. 1). It includes
an active site that is enclosed by the finger and thumb
domains. In addition, a b-hairpin loop that is unique
substituted phenyl-alanines, thiophene-2-carboxylic
acids, and cyclopentyl dihydropyran-ones is found to
be mutationally sensitive to Met423. Two distinct benz-
imidazole analogs interact with a highly conserved
Pro495 and share another allosteric, low affinity GTP
binding site, at the top of the thumb domain. Benzothi-
adiazines and proline sulfonamides are found to bind in
a pocket at the inner surface of the thumb and muta-
tions to Met414 confer resistance to the benzothiadi-
azine series. Substituted benzofurans are found to bind
on the interior of the RdRp palm region interacting with
Keywords: HCV; NS5B inhibitor; Polymerase inhibitor; Virtual scre-
ening; In silico screening; Single-stranded RNA synthesis; Double-
stranded RNA synthesis; Replicase complex assay; Replicon assay.
*
Corresponding authors. Tel.: +1 203 624 7000; fax: +1 203 752 5454;
e-mail addresses: mhuang@achillion.com; aagarwal@achillion.com
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.103

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S. Louise-May et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3905–3909
utilizes NS5B within the replicon complex that is iso-
lated from the replicon containing cells. This assay has
been used to determine NS5B inhibition potency of sev-
eral compounds.26–30 Subsequently, one of the active
compounds identified was followed-up by applying sub-
structure and similarity searches and in-house synthesis
of analogs.
A database of lead-like compounds used for virtual
screening was derived from an initial pool of about
two million compounds from the Specs & BioSpecs,
Bionet, Microsource, Available Chemicals Directory
(ACD), Nanoscale, ChemDiv, Orion, Asinex, Interbio-
screen, Timtec, and Chembridge databases. Tripos
SLN Filters32 were used to remove duplicates and reac-
tive functionalities and to select compounds according
to the following criteria; compounds containing at least
one ring, 61 chiral center, 68 rotatable bonds, 275–400
molecular weight, and C logP 63.5. This resulted in a
library of 90,000 molecules. In preparation of the mole-
cules for docking, Schrodinger utilities were used to strip
salts, neutralize, and ionize compounds at the physiolog-
ical pH before energy minimization to a low energy
conformer.
Figure 1. HCV NS5B polymerase: thumb, palm, and fingers domains
are color coded with red, blue, and green colors, respectively. The
active site residues Asp 318 and 319 are displayed with yellow sticks
and catalytic metal ions are in magenta CPK. The predicted binding
pose of compound 1 is also shown in CPK.
Arg200 and loop residues 364–368, a hinge region that is
closed in the apo form of the enzyme but open upon
binding of the inhibitor. Resistance to the benzofurans
is conferred by mutations to Val201 and Cys316. Final-
ly, pyrophosphate mimics bind in the palm region and
presumably interact with bound metal ions to disrupt
binding of incoming RNA nucleotides or activation of
the elongating RNA chain. It is noteworthy that, unlike
nucleosides that are chain elongation inhibitors, all the
known non-nucleoside HCV polymerase ligands are
chain initiation inhibitors.
The 3D structure of HCV NS5B (1GX6)6 was prepared
for virtual screening by deleting the UTP ligand and
mutating seleno-methionine residues to methionines.
This was followed by energy minimization of the model
with the impref utility of Schrodinger suite of soft-
ware.33 The PRCG method was used with a maximum
number of iterations set to 10,000 and gradient conver-
gence criteria set at 0.05 kJ/A mol. The force field used
was OPLS-AA with the GB/SA implicit solvent treat-
ment at a constant dielectric of 1.0. Next, Glide34,35
˚
was used to calculate grids with a 20 A bounding box
centered on residues 421, 422, and 426 and an enclosing
˚
box to fit ligands with up to 30 A length that enclosed
the entire thumb region.
We used virtual screening to dock a library of available
lead-like compounds in the thumb domain of HCV
NS5B for selection and purchase of novel putative li-
gands. In order to identify HCV/NS5B inhibitors, the
purchased compounds were evaluated in an HCV repli-
case complex (RC) assay24,25 (cell-free) and HCV repli-
con assay26 (cell-based). As opposed to the use of
recombinant NS5B in the biochemical assay, RC assay
Compounds were docked flexibly using Glide, allowing
generation of up to 5000 initial conformations and
refinement of 400 poses per ligand with the best scoring
pose retained for each ligand. The virtual screening
S
NH
2
i
N
O
+
+
S
O
O
O
I
O
H
N
H
N
S
S
ii
iii
O
O
O
COOH
O
4
3
Figure 2. Literature procedure for synthesis of amino thiophenes.

S. Louise-May et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3905–3909
3907
4,5-dialkyl analogs
Me
H
S
N
1
5
2
4
3
O
B
Et, Me
A
CONH2
CN
COOEt
COOH
4,5,6,7-tetrahydro-benzo analogs
H
N
S
1
2
7
7a
3a
B
6
H, Me, Et
3
5
4
O
A
CONH2
CN
Figure 3. Dialkyl substituted thiophene inhibitors of HCV.
calculation was performed with impact-v20012 on a Li-
nux cluster of four dual processor computers.
Docked ligands were found to cluster in the three known
binding sites of the thumb domain. The top 1318 ligands
that scored ꢀ7.17 (glide score) or better were visually in-
spected to select and purchase a structurally diverse set
of 50 putative ligands, encompassing the known binding
sites. Subsequently, purchased compounds were tested
in the RC and replicon assays.
Figure 4. Inhibition of HCV single-stranded RNA synthesis in a RC
assay. (a) Five compounds tested at a concentration of 100 lM
displayed different inhibitory effects on HCV RNA synthesis. The
positive control was a benzothiadiazine derivative, a known HCV non-
nucleoside inhibitor (testing concentration was 5 lM). DS, double-
stranded; SS, single-stranded. (b) Dose–response of inhibition of the
compound 2 on single-stranded RNA synthesis. The compound was
twofold diluted in DMSO serially before mixing with the reaction
mixture. The controls were the same as (a).
The previously described RC assay24,25 was modified as
following: various concentrations of compounds were
incubated with membrane fractions containing HCV
replicase complexes isolated from HCV replicon cells
in the reaction buffer at 30 ꢁC for 30 min. Viral RNA
synthesis was then initiated by adding ATP, GTP,
UTP, and [a-32P]CTP. After incorporation of nucleo-
tides at 30 ꢁC for 60 min, the reaction was stopped
and RNA was isolated with TRIzol LS Reagent (Invit-
rogen). The isolated RNA was resolved by electrophore-
sis on a 1% agarose gel, dried, and visualized by
autoradiography (Fig. 4).
were refluxed together in ethanol in the presence of pyr-
rolidine for 6 h (Fig. 2). All the volatiles were evapo-
rated and the residue was purified by chromatography
on silica gel to give the aminothiophene I. The amino
group was then converted to the amide using 3-phenyl-
propionylchloride to give compound 3 followed by
hydrolysis of the ester group to give the corresponding
carboxylic acid, compound 4.
Previously described replicon assay31 was modified to
use firefly luciferase as the reporter. Serially diluted com-
pounds were added to the replicon cells seeded in 96-well
plates. The 72 h long treatment was followed by measur-
ing anti-HCV activity of the tested compounds with
Blight-Glo luciferase assay kit (Promega) and the cellu-
lar toxicity was measured with CellTiter 96 Aqueous
One Solution (Promega) Table 1.
The resulting follow-up set led to limited SAR data cap-
tured in eight additional compounds (Fig. 3). Replace-
ment of the amide function in compound 1 by a cyano
(compound 2) results in >2· improvement in potency
in the RC assay. Because of their structural similarity
to known thiophene-2-carboxylic acid inhibitors of
HCV NS5B,37 in addition to the amide and cyano
compounds available for purchase, the ethyl ester (com-
pound 3) and carboxylic acid (compound 4) at the 3-po-
sition were synthesized in-house. Surprisingly, the acid
and ethyl ester were found to be inactive in the RC as-
say. These results imply that the dialkyl substituted
The most active hit identified (compound 1) caused a
dose-dependent inhibition in the RC assay (IC50 50–
100 lM). Following up the initial hit, available analogs
of compound 1 were purchased to explore electronic
properties of the substituents at thiophene 3-position
and steric properties of substituents at thiophene 4-
and 5-positions. In addition, two aminothiophenes, 3
and 4, were synthesized in-house following the literature
procedure.36 3-Pentanone, sulfur, and ethylcyanoacetate

3908
S. Louise-May et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3905–3909
Table 1. Substituents, HCV replicon, and cell-free assay EC50 results for compounds 1–5 and 6–9
Compound
A
B
HCV replicon EC50 (lM) mean (SD)a
HCV replicase complex IC50 (lM)
4,5-Dialkyl analogs
1
2
3
4
5
CONH2
CN
Et
Et
Et
Et
Me
0.92 (0.67)
1.66 (0.79)
ND
50–100b
25c
COOEt
COOH
CONH2
>100
>100
>100
ND
ND
4,5,6,7-Tetrahydro-benzo analogs
6
7
8
9
CONH2
CONH2
CONH2
CN
H
ND
ND
>100
>100
30–50b
>100
Me
Et
Et
1.95 (0.90)
ND
HCV replicon assay. ND, not determined.
a Values are means of three experiments. HCV RC assay: single point (100 lM) experiment, IC50 estimated from single-strand band intensity
reduction on gel; 50% ! ꢁ100 lM, 75% ! ꢁ50 lM, 92.5% ! ꢁ25 lM.
b Ranges reflect N = 2.
c Five point 2· dilution experiment; 100–6.25 lM.
thiophenes have a distinct binding mode or location
from that of the previously reported thiophene-2-car-
boxylic acid HCV NS5B inhibitors.
and the limited SAR explored further supports interac-
tion of compounds with a well-defined binding site. In
addition, inhibition of both single- and double-stranded
RNA synthesis represents a unique mechanism for the
potential treatment of HCV infection. These novel
inhibitors are amenable to parallel synthesis exploration
and offer a reasonable starting point for further drug
discovery efforts.
The inhibition activity of the thiophenes displays a
remarkable sensitivity to the aliphatic substituent
changes. Loss of activity was found for simple substitu-
tions of a methyl group for an ethyl at the 4 (acyclic) or
6 (cyclic) position (compounds 1/5 and 7/8), while activ-
ities of the 4-Et/5-Me (acyclic) and 6-Et (cyclic) substi-
tuted analogs were comparable (compounds 1 and 8)
in the RC assay. Perhaps, this could be explained by a
shape complementarity between aliphatic substituents
and a hydrophobic region of the binding pocket.
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inhibited double-stranded (DS) RNA synthesis. We have
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