Benzimidazole Thumb Pocket I finger-loop inhibitors of HCV NS5B polymerase: Improved drug-like properties through C-2 SAR in three sub-series

Article abstract of DOI:10.1016/j.bmcl.2010.02.003

SAR at the C-2 position of benzimidazole-based Thumb Pocket I inhibitors of HCV NS5B polymerase revealed parallel activity for distinct sub-series that harbor 5-hydroxytryptophan amides, neutral thiazole isosteres or recently disclosed cinnamic acid diamides. The consistent SAR among the three sub-series suggest a common binding mode to the Thumb Pocket I allosteric site. New inhibitors with sub-micromolar cell-based replicon potency and improved 'drug-like' features are disclosed along with preliminary characterization of their ADME-PK profile.

Full text of DOI:10.1016/j.bmcl.2010.02.003

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1825–1829
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzimidazole Thumb Pocket I finger-loop inhibitors of HCV NS5B
polymerase: Improved drug-like properties through C-2 SAR in three sub-series
b
b
b
a
Pierre L. Beaulieu a, , Nathalie Dansereau , Jianmin Duan , Michel Garneau , James Gillard ,
*
Ginette McKercher b, Steven LaPlante a, Lisette Lagacée b, Louise Thauvette b, George Kukolj b
a Department of Chemistry, Boehringer Ingelheim (Canada) Ltd, Research and Development, 2100 Cunard Street, Laval (Québec), Canada H7S 2G5
b Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd, Research and Development, 2100 Cunard Street, Laval (Québec), Canada H7S 2G5
a r t i c l e i n f o
a b s t r a c t
Article history:
SAR at the C-2 position of benzimidazole-based Thumb Pocket I inhibitors of HCV NS5B polymerase
revealed parallel activity for distinct sub-series that harbor 5-hydroxytryptophan amides, neutral thia-
zole isosteres or recently disclosed cinnamic acid diamides. The consistent SAR among the three sub-ser-
ies suggest a common binding mode to the Thumb Pocket I allosteric site. New inhibitors with sub-
micromolar cell-based replicon potency and improved ‘drug-like’ features are disclosed along with pre-
liminary characterization of their ADME-PK profile.
Received 6 January 2010
Revised 29 January 2010
Accepted 1 February 2010
Available online 6 February 2010
Keywords:
Hepatitis C virus
HCV
Ó 2010 Elsevier Ltd. All rights reserved.
NS5B polymerase
Allosteric inhibitors
Replicon inhibitors
Benzimidazole
Antivirals
Hepatitis C virus (HCV) infections, which are believed to afflict
1–3% of the world population, may lead to serious and often fatal
liver diseases such as cirrhosis and hepatocellular carcinomas
(HCC).1 HCV is the leading cause of liver transplantation in the
world and represents a major concern to public health in both
industrialized and developing countries. The discovery of the virus
20 years ago and the development of diagnostic tools have greatly
contributed to arresting the spread of new viral infections. Never-
theless, it is estimated that the number of newly diagnosed pa-
tients will continue to rise in the next 10 years before reaching a
plateau and eventually decreasing as more effective and better tol-
erated antiviral therapies become available. The current standard
of care, based on combinations of pegylated-interferon and ribavi-
rin, is poorly tolerated and shows sub-optimal efficacy, particularly
against genotype 1 (most abundant in the western world).2
NS5B, the RNA-dependent RNA polymerase of HCV is the en-
zyme that catalyzes replication of the virus’s genome and has been
the focus of extensive research efforts by academia and the phar-
maceutical industry for the development of novel anti-HCV agents.
NS5B polymerase is sensitive to inhibition by both substrate-based
nucleoside analogs as well as non-nucleoside allosteric inhibitors.
Several small molecule candidates from both classes of drugs have
progressed through early stages of clinical development and pro-
vided proof of concept of antiviral efficacy in man.3,4 Allosteric
inhibitors of NS5B are divided into at least four different classes
according to the location of their respective binding sites (referred
to as Thumb Pocket I and II and Palm Sites I and II).4
Our group reported the discovery and subsequent optimization
of benzimidazole-based allosteric inhibitors of HCV NS5B that bind
to Thumb Pocket I of the enzyme (Fig. 1) and were shown by us and
others to restrict conformational changes in the enzyme that are
thought to be required at the initiation stage of RNA synthesis.
We refer to this class of compounds as ‘finger-loop inhibitors’ since
they prevent a key intra-molecular interaction of the k1 loop that
extends from the finger domain with the thumb domain of the en-
zyme. This interference with a protein conformational change pre-
vents the formation of a productive enzyme complex for RNA
synthesis.5
In a recent Letter, we reported optimization of the right-hand
side of benzimidazole-based inhibitors and the discovery of novel
diamide derivatives (e.g., compound C1) that displayed excellent
inhibition in biochemical assays with micromolar potency in a
cell-based 1b HCV sub-genomic replicon assay (Fig. 1).6 We now
describe C-2 SAR in this class of inhibitors and some comparison
to previous series of compounds (5-hydroxytryptophans A1 and
thiazoles B1) that, despite significant structural changes on the
right-hand side, provides evidence for parallel SAR and a common
* Corresponding author. Tel.: +1 450 6824640; fax: +1 450 6828434.
E-mail address: pierre.beaulieu@boehringer-ingelheim.com (P.L. Beaulieu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.003

1826
P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1825–1829
S
H
N
N
OH
O
O
O
C2
N
N
N
N
N
H
N
H
O
O
OH
OH
N
H
5-Hydroxytryptophan A1
IC50 = 0.03 µM
Thiazole B1
IC50 = 0.22 µM
O
H
N
N
N
H
O
O
N
O
OH
Diamide C1
IC50 = 0.050 µM
Figure 1. Representative members from three benzimidazole sub-series of finger-loop (Thumb Pocket I) inhibitors.
binding mode for the three sub-series. In addition, the furyl struc-
tural alert in lead inhibitors A1, B1, and C1 was replaced and cellu-
lar potency improved to provide inhibitors with promising in vitro
ADME and rat PK characteristics.
served at this position. Nonetheless, 2- and 3-furyl, 2-N-Me-pyr-
role, 2- and 3-thiophenyl, and 2-pyridyl were identified as
providing the best intrinsic potencies in a full-length NS5B bio-
chemical assay.14
The three benzimidazole sub-series of NS5B inhibitors previ-
ously disclosed contain a 5-carboxybenzimidazole scaffold that
features an optimal N1-cyclohexyl moiety and a small heterocyclic
C-2 substituent. While the N1-position is very sensitive to small
structural modifications, the C-2 position was shown in early work
to tolerate a variety of substituents ranging from small heterocy-
cles7 to large poly-aromatic systems.8 Recent disclosures from
our group revealed how the electron-rich 5-hydroxytryptophan
system may be replaced by a cinnamic acid moiety linked to the
As the series progressed from the short benzimidazole carbox-
ylic acid derivatives to more potent tryptophan-based amides
(sub-series A and B), we had assumed similar inhibitor binding
modes15 and SAR at this position. In order to validate this hypoth-
esis, a series of analogs that contained these privileged C-2 substit-
uents was prepared and tested in both the full-length NS5B and the
C-terminally deleted
D21-NS5B format of the biochemical as-
says.14 Both assays gave similar IC50 values and only data for the
latter are reported in Table 1. As reported previously for the 3-furyl
derivatives A1 and B1, tryptophan analogs that contained a thia-
benzimidazole scaffold through an a,a-disubstituted cyclic a-ami-
no acid linker.6 We wished to re-investigate SAR at the C-2 position
of the benzimidazole left-hand side to verify that SAR remained
parallel in the three sub-series and identify a more drug-like
replacement for the furyl heterocycle at C-2.9
zole replacement for the a-carboxylic acid group were all less po-
tent (3–12-fold), independent of the C-2 substituent. This
observation may be rationalized by a key role for the carboxylic
acid. It is believed to be solvent exposed and serves as a conforma-
tion orienting group,12,15 and is favorably hydrated in sub-series A
compared to the more hydrophobic thiazole group present in sub-
series B. However, the charge and high polarity of carboxylic acids
A1–8 were incompatible with cellular permeability and all com-
pounds in this sub-series were inactive in an HCV genotype 1b cel-
lular replicon assay.16 The more lipophilic and neutral thiazole
analogs B1–8, on the other hand provided low-micromolar inhibi-
The synthesis of inhibitors was performed in solution as de-
scribed in Scheme 1. Benzimidazole carboxylic acids with varia-
tions at C-2 or N1 were prepared according to previously
published procedures7,10 and coupled to amines A,11 B,12 or C6
using standard reagents for amide bond formation (TBTU or HATU)
followed by removal of protecting groups by saponification if
necessary.13
C-2 SAR was established early in truncated benzimidazole 5-
tion (EC50 = 2–4 lM) in the cell-based assay, regardless of the C-2
substituent present on the benzimidazole scaffold. As previously
carboxylic acid derivatives7 where a relatively ‘flat’ SAR was ob-
O
O
A, B or C
TBTU or HATU
A, B or C
N
N
N
N
N
H
OH
R
R
DIEA / DMF
then deprotection
(LiOH or NaOH)
OMe
O
S
H
H
N
N
N
H2N
H2N
O
O
H2N
OH
N
H
Et
OH
A
B
C
Scheme 1. Synthesis of inhibitors.

P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1825–1829
1827
Table 1
C-2 and N1 SAR in three benzimidazole sub-series
S
OH
O
O
N
O
O
H
N
N
N
N
H
N
N
H
N
R
N
R
R
H
O
N
OH
COOH
N
N
OH
H
N
H
B
C
A
R
IC50
,
l
Ma
IC50
,
l
Ma
EC50
,
l
Ma,b
IC50
,
l
Ma
EC50
,
l
Ma,b
Calcd Log Pc
0.03 ( 0.01)
0.08 ( 0.02)
0.16 ( 0.1)
0.08 ( 0.02)
0.22 ( 0.04)
0.27 ( 0.1)
0.52 ( 0.17)
0.45 ( 0.06)
3.6 ( 1.5)
3.4 ( 0.7)
2.9 ( 0.5)
2.8 ( 0.6)
0.05 ( 0.02)
0.11 ( 0.01)
0.12 ( 0.05)
0.11 ( 0.05)
1.1 ( 0.4)
1.2 ( 0.3)
1.5 ( 0.5)
1.2 ( 0.4)
4.4
4.7
4.9
4.9
1
O
S
2
3
4
5
O
S
0.06 ( 0.01)
0.73 ( 0.36)
2.7 ( 0.5)
0.09 ( 0.02)
0.7 ( 0.3)
4.3
N
Me
6
7
0.14 ( 0.04)
0.23 ( 0.09)
0.69 ( 0.4)
1.9 ( 0.8)
2.4 ( 0.9)
0.10 ( 0.04)
0.20 ( 0.08)
0.7 ( 0.2)
1.2 ( 0.2)
4.7
5.1
N
1.16 ( 0.28)
O
N
N
O
0.08 ( 0.02)
0.38 ( 0.06)
4.0 ( 1.0)
0.10 ( 0.02)
6.8 ( 2)
4.0
8
a
Values are means of at least three experiments, standard deviation is given in parentheses.
EC50s are corrected for actual compound concentration following the first dilution with assay buffer from 20 mM DMSO stock solutions. The concentration of compound
b
was determined by HPLC after centrifugation of the sample.
c
Log P predictions were calculated using the JChem 5.0.0 software from Chemaxon (see Ref. 17).
observed for the truncated benzimidazole carboxylic acids, the 3-
furyl analogs A1 and B1 provided the strongest intrinsic inhibition
of the enzyme. Cyclopentyl N1-analogs A8 and B8 were both 2–3-
fold less potent than the corresponding cyclohexyl analogs A1 and
B1 and suggest that addition of the amide substituent on the benz-
imidazole scaffold likewise does not affect binding at this sterically
sensitive position compared to truncated analogs.7
A similar study was conducted in the diamide-cinnamic acid
sub-series C, where we suspected that introduction of a carboxylic
acid moiety at the far right-hand side of the molecules could con-
ceivably affect overall inhibitor binding interactions. As seen in Ta-
ble 1 however, binding was consistent throughout the series of C-2
substituents investigated, with IC50 values ranging from 50 to
200 nM and the trends reflected those of tryptophan sub-series
A. Again, a twofold decrease in potency was observed between
the optimized cyclohexyl analog C1 and its cyclopentyl counter-
part C8. The C-2 SAR detailed in Table 1 are represented graphically
in Figure 2 for the three sub-series. Despite the differences in abso-
lute potency, the trends at C-2 and N1 of the benzimidazole scaf-
fold were consistent as the inhibitor series progressed from
small, less potent truncated benzimidazole 5-carboxylic acids7 to
more elaborated amide derivatives (sub-series A–C).
10
1
A
B
C
0.1
0.01
1
2
3
4
5
6
7
8
C-2 Substituent
Figure 2. Comparison of C-2 SAR in three benzimidazole sub-series. Refer to Table 1
for structure of C-2 substituents (compounds 1–8) and series A–C.
polio virus and a mammalian DNA-dependent RNA polymerase II
isolated from calf-thymus.7
In all cases, no significant inhibition was observed at concentra-
tions up to 250
sub-series C also provided improved potency in the cell-based gt1b
replicon assay with EC50 = 0.7–6.8 M. In particular, the 2-pyridyl
analog C6, with an EC50 = 700 nM, provided improved potency
and a more ‘drug-like’ alternative to 3-furyl analogs such as C1.
Expansion of the cyclobutyl linker ring of C6 to a cyclopentyl ring
lM (IC50 >250 lM). Cinnamic acid derivatives from
l
Representative compounds from sub-series C were tested for
specificity against another RNA-dependent RNA polymerase from

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P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1825–1829
compounds exhibited a significant unbound fraction in the pres-
ence of human plasma protein.
Plasma exposure of compounds C9 and C10 were evaluated in
rat following oral administration of a 5 mg/kg dose.18 While no
C9 plasma exposure was observed, compound C10 was readily de-
O
H
N
N
N
N
H
C9
O
O
O
OH
EC50 = 880 nM
calc. LogP = 4.8
tected in plasma with
a Cmax = 0.58 lM (Tmax = 0.5 h) and
AUC = 0.51 M h. Consistent with the modest AUC, an oral MRT
l
(mean residence time) of 0.74 h was measured, indicative of rapid
clearance from the plasma compartment.
O
H
In conclusion, we established that SAR at the C-2 and N1-posi-
tion on the scaffold of benzimidazole-based Thumb Pocket I inhib-
itors of HCV NS5B polymerase is not affected by the nature of the
right-hand side of the molecule (truncated carboxylic acids or
three distinct amide sub-series) which suggests a common binding
mode of these molecules to the enzyme allosteric site. We also
identified a more ‘drug-like’ replacement for the initial 3-furyl
group that provides slightly improved potency and ADME profile.
A prototype from this series provided modest plasma exposure
and a short MRT after oral administration to rats. Further optimiza-
tion of this class of inhibitors will be reported in the near future.
N
N
N
N
C10
H
O
O
N
OH
EC50 = 550 nM
calc. LogP = 5.1
Figure 3. Cyclopentyl diamide derivatives.
provided compounds C9 and C10 with slight increases in lipophil-
icity17 and sub-micromolar cell culture activity (Fig. 3).
Following the discovery of benzimidazole-cinnamic acid inhib-
itors with sub-micromolar cell-based potency in the replicon assay
and drug-like structural features, the in vitro ADME profiles of ana-
logs such as C9 and C10 were investigated and compared to thia-
zole-based inhibitor B1. Human and rat liver microsome
stabilities, Caco-2 permeability, protein binding and CYP inhibition
data are presented in Tables 2 and 3.
Acknowledgements
We thank Colette Boucher, Michael Little and Serge Valois for
analytical support and Dr. Gordon Bolger, Christine Zouki and Josie
De Marte for in vitro ADME data. We are also grateful to Francine
Liard, Manon Rheaume and Hélène Montpetit for rat in vivo data.
Neutral thiazole analog B1 was insoluble at pH 7.2 and exhib-
ited no detectable apical to basolateral permeability in the Caco-
2 cell model. Furthermore, the compound was rapidly metabolized
in the presence of human liver microsomes and displayed strong
inhibition of CYP3A4 in the sub-micromolar range (see Table 3).
In contrast, cinnamic acid analogs C9 and C10 had moderate solu-
bility at pH 7.2 due to the presence of the weakly ionized carbox-
ylic acid function. Both inhibitors showed good permeability of
Caco-2 monolayers, and suggest that the weakly basic benzimid-
azole scaffold and the deactivated carboxylic acid function do not
promote formation of zwitterionic species to a significant extent.
Phase 1 metabolic stability and CYP450 inhibition profiles were
significantly improved with neither compound inhibiting CYP3A4
References and notes
1. (a) Choo, Q.-L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley, D. W.; Houghton, M.
Science 1989, 244, 359; (b) Lavanchy, D. Liver Int. 2009, 29, 74.
2. Foster, G. L.; Mathurin, P. Antiviral Ther. 2008, 13, 3.
3. Brown, N. A. Expert Opin. Investig. Drugs 2009, 18, 709.
4. (a) Beaulieu, P. L. Expert Opin. Ther. Patents 2009, 19, 145; (b) Lin, K.; Hazuda, D.
J.; Otto, M. J.
5. Beaulieu, P. L. Curr. Opin. Drug Discov. Devel. 2006, 9, 618.
6. (a) Goulet, S.; Poupart, M.-A.; Gillard, J.; Poirier, M.; Kukolj, G.; Beaulieu, P. L.
Bioorg. Med. Chem. Lett. 2010, 20, 196; (b) LaPlante, S. et al. submitted.
7. Beaulieu, P. L.; Bös, M.; Bousquet, Y.; Fazal, G.; Gauthier, J.; Gillard, J.; Goulet, S.;
LaPlante, S.; Poupart, M.-A.; Lefebvre, S.; McKercher, G.; Pellerin, C.; Austel, V.;
Kukolj, G. Bioorg. Med. Chem. Lett. 2004, 14, 119.
8. (a) Hirashima, S.; Suzuki, T.; Ishida, T.; Noji, S.; Yata, S.; Ando, I.; Komatsu, M.;
Ikeda, S.; Hashimoto, H. J. Med. Chem. 2006, 49, 4721; (b) Hirashima, S.; Oka, T.;
Ikegashira, K.; Noji, S.; Yamanaka, H.; Hara, Y.; Goto, H.; Mizojiri, R.; Niwa, Y.;
Noguchi, T.; Ando, I.; Ikeda, S.; Hashimoto, H. Bioorg. Med. Chem. Lett. 2007, 17,
3181.
at 30 lM. Despite the relatively high lipophilicity (calcd Log P
4.8–5.4)17 and presence of a free carboxylic acid function, both
9. Kalgutkar, A. S.; Gardner, I.; Obach, R. S.; Shaffer, C. L.; Callegari, E.; Henne, K. R.;
Mutlib, A. E.; Dalvie, D. K.; Lee, J. S.; Nakai, Y.; O’Donnell, J. P.; Boer, J.; Harriman,
S. P. Cur. Drug Metabol. 2005, 6, 651.
Table 2
In vitro ADME profile for compounds B1, C9 and C10
10. Beaulieu, P. L.; Haché, B.; von Moos, E. Synthesis 2003, 1683.
11. Beaulieu, P. L.; Bös, M.; Bousquet, Y.; DeRoy, P.; Fazal, G.; Gauthier, J.; Gillard, J.;
Goulet, S.; McKercher, G.; Poupart, M.-A.; Valois, S.; Kukolj, G. Bioorg. Med.
Chem. Lett. 2004, 14, 967.
12. Beaulieu, P. L.; Bousquet, Y.; Gauthier, J.; Gillard, J.; Marquis, M.; McKercher, G.;
Pellerin, C.; Valois, S.; Kukolj, G. J. Med. Chem. 2004, 47, 6884.
13. All inhibitors in this study were purified to >95% homogeneity by reversed-
phase HPLC and isolated as TFA salts. All compounds were characterized by
mass spectrometry and gave 1H NMR spectra consistent with expected
structures.
Compd
Calcd Log Pa
Solubilityb
g/mL)
Caco-2
(cm/s)
HLM/RLMc
T1/2 (min)
%PBd
(l
B1
C9
C10
5.4
4.8
5.1
<0.1
125
100
N.A.
26/N.A.
77/54
67/17
N.A.
96.2
87.6
3.9 Â 10À6
9.5 Â 10À6
N.A.: not available.
a
Log P predictions were calculated using the JChem 5.0.0 software from
Chemaxon (see Ref. 17).
14. IC50 values are the average of at least three independent determinations unless
indicated otherwise: McKercher, G.; Beaulieu, P. L.; Lamarre, D.; LaPlante, S.;
Lefebvre, S.; Pellerin, C.; Thauvette, L.; Kukolj, G. Nucleic Acids Res. 2004, 32,
422.
b
24 h shaking flask method (pH 7.2 phosphate buffer, amorphous TFA salt).
T1/2 at 10
Protein binding was determined by the equilibrium dialysis method.
c
l
M in human or rat liver microsomes.
d
15. LaPlante, S.; Jakalian, A.; Aubry, N.; Bousquet, Y.; Ferland, J.-M.; Gillard, J.;
Lefebvre, S.; Poirier, M.; Tsantrizos, Y.; Kukolj, G.; Beaulieu, P. L. Angew. Chem.
2004, 43, 4406.
16. EC50 determinations were performed in duplicates (or more) using Huh-7 cells
with a stable sub-genomic HCV 1b replicon that encodes a modified luciferase
reporter gene as previously described in: Tsantrizos, Y. S.; Chabot, C.; Beaulieu,
P. L.; Brochu, C.; Poirier, M.; Stammers, T.; Thavonekham, B.; Rancourt, J. WO
Table 3
CYP inhibition profile for compounds B1, C9 and C10
Compd
IC50 CYP450 inhibition (
l
M)
2D6
05/080388, 2005. Compounds were devoid of cytotoxicity (TC50 >10 lM) at
1A2
2C9
2C19
3A4
inhibitory concentrations. A sub-set of inhibitors were also tested in a 72 h
sub-genomic replicon assay where HCV RNA levels were normalized to total
cellular RNA. Similar EC50 values were determined in both assays and the
quantification of total RNA recovery allowed for an alternative assessment of
cellular homeostasis to eliminate the possibility of antiviral activity due to
B1
C9
C10
>30
>30
>30
2.3
11.5
4.0
2.4
7.7
7.4
4.0
>30
>30
0.44
>30
>30

P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1825–1829
1829
subtle toxic effects. For an overview and basic protocols on the use of HCV
replicons, see: Lohmann, V. Methods Mol. Biol. 2009, 510, 145; Hepatitis C:
Methods and Protocols, 2nd ed.; Tang, H. (Ed.); Humana Press.
17. JChem 5.0.0 (http://www.chemaxon.com) was used for Log P predictions. The
prediction method is based on the algorithm described in Viswanadhan, V. N.;
Ghose, A. K.; Revankar, G. R.; Robins, R. K. J. Chem. Inf. Comput. Sci. 1989, 29,
163.
18. Male Sprague-Dawley rats were starved overnight and dosed by oral gavage at
5 mg/kg using 0.5% methocel and 0.3% Tween-80 as vehicle. Plasma samples
from three animals were pooled at each time points (0–8 h) for analysis.
Compound detection in plasma samples was performed following liquid–liquid
extraction and HPLC analysis using UV detection.