N-Acetamideindolecarboxylic acid allosteric 'finger-loop' inhibitors of the hepatitis C virus NS5B polymerase: discovery and initial optimization studies

Article abstract of DOI:10.1016/j.bmcl.2009.12.101

SAR studies at the N¹-position of allosteric indole-based HCV NS5B inhibitors has led to the discovery of acetamide derivatives with good cellular potency in subgenomic replicons (EC₅₀ <200 nM). This class of inhibitors displayed improved physicochemical properties and favorable ADME-PK profiles over previously described analogs in this class.

Full text of DOI:10.1016/j.bmcl.2009.12.101

Bioorganic & Medicinal Chemistry Letters 20 (2010) 857–861
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-Acetamideindolecarboxylic acid allosteric ‘finger-loop’ inhibitors of the
hepatitis C virus NS5B polymerase: discovery and initial optimization studies
a
a
a
a
Pierre L. Beaulieu a, , Eric Jolicoeur , James Gillard , Christian Brochu , René Coulombe ,
Nathalie Dansereau b, Jianmin Duan b, Michel Garneau b, Araz Jakalian a, Peter Kühn a, Lisette Lagacé b,
Steven LaPlante a, Ginette McKercher b, Stéphane Perrault a, , Martin Poirier a, Marc-André Poupart a,
Timothy Stammers a, Louise Thauvette b, Bounkham Thavonekham a, George Kukolj a
*
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
SAR studies at the N1-position of allosteric indole-based HCV NS5B inhibitors has led to the discovery of
acetamide derivatives with good cellular potency in subgenomic replicons (EC50 <200 nM). This class of
inhibitors displayed improved physicochemical properties and favorable ADME-PK profiles over previ-
ously described analogs in this class.
Article history:
Received 3 November 2009
Revised 18 December 2009
Accepted 22 December 2009
Available online 4 January 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
HCV NS5B polymerase
HCV replicon inhibitors
Allosteric inhibitors
Antivirals
Hepatitis C virus
The Hepatitis C Virus (HCV) infects millions of people world-
wide leading in some cases to severe and often fatal liver damage
(e.g., cirrhosis or hepatocellular carcinoma). The best current treat-
ment options consist of pegylated interferon and ribavirin combi-
nations and provide limited efficacy against the most prominent
HCV genotype (1a/1b) with significant side effects. In industrial-
ized nations, HCV infection has become the major reason for ortho-
topic liver transplants.1 The NS5B RNA-dependent RNA polymerase
of the hepatitis C virus is a promising target for the development of
novel anti-HCV therapeutics.2 Recently, nucleoside analogs3 and
non-nucleoside allosteric inhibitors2,4 of the enzyme have demon-
strated efficacy in the clinic, particularly in combination with
interferon-based regimens aimed at retarding emergence of resis-
tant strains of the virus. Our own efforts in the field have centered
on benzimidazole-based non-nucleoside allosteric inhibitors of the
enzyme (e.g., compound 1) which were discovered through screen-
ing of our corporate sample collection.5,6 We and others recently
described the progression of this class of inhibitors towards more
potent and cell-permeable indole-based derivatives that inhibited
HCV subgenomic RNA replication in cell culture (replicon assay)
Two strategies were explored to further improve the cell-based
potency of these inhibitors. Incorporation of the indole modifica-
tion into previously described amide analogs8 led to non-ionized
O
N
OH
O
N
IC50 = 1.1 µM
1
EC50 = not active
O
R
N
OH
O
2 (R = H)
3 (R = Me)
IC50 = 0.03 µM
IC50 = 0.016 µM
EC50 = 4.2 µM
EC50 = 6.7 µM
S
H
N
N
O
N
N
H
O
at low l
M concentrations (e.g., compounds 2 and 3, Fig. 1).7
4
OH
IC50 = 0.1 µM
EC50 = 0.05 µM
* Corresponding author. Tel.: +1 450 6824640; fax: +1 450 6828434.
E-mail address: pierre.beaulieu@boehringer-ingelheim.com (P.L. Beaulieu).
Undergraduate chemistry COOP student.
Figure 1. Initial indole-based NS5B inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.101

858
P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 857–861
Table 1
compounds such as 4, a very potent inhibitor of HCV RNA replica-
tion (EC50 = 0.05
M).7a Unfortunately, amide 4 had inadequate
N-Alkylation studies on indole carboxylic acid 2
l
physicochemical properties (high lipophilicity and very poor aque-
ous solubility) that compromised further progression of such
derivatives into development for oral therapy. In contrast, indole
carboxylic acid derivatives 2 and 3 had desirable physicochemical
properties (low MW, good solubility) as well as remarkable intrin-
sic potency against the polymerase enzyme, albeit with modest
replicon potency.
O
R
N
OH
O
The strategy to improve the cell-based potency of compounds 2
and 3 was based on optimization of the N1-indolic substituent
while preserving the ionizable carboxylic acid function in order
to maintain adequate physicochemical properties. We now report
the results of studies directed toward exploring SAR at this newly
accessible position extending from the nitrogen atom of the indole
scaffold of these derivatives (R group in Fig. 2).
a
a
Compds
R
HT-NS5B-
M)
D
21 IC50
1b Replicon EC50
(lM)
(
l
5
6
7
8
9
Ethyl
Allyl
Isopropenyl
Benzyl
2-Picoly
0.07
0.08
0.36
0.25
0.13
0.29
>138
>10
>10
>10
N.T.
N.T.
N.T.
N.T.
10
11
2-Me–C6H4CH2
2,6-Me2–
C6H3CH2
2-(NH2)–
C6H4CH2
3-(NH2)–
C6H4CH2
3-(COOH)–
C6H4CH2
4-(COOH)–
C6H4CH2
CH2COOH
CH2CH2OH
CH2COOEt
CH2CH2COOH
( )-
Initial data from compounds 2 and 37a suggested that substitu-
tion of the indole nitrogen was tolerated. The indole NH did not
contribute to the improved potency that accompanied the replace-
ment of the benzimidazole scaffold by indoles. The possibility of
exploiting new interactions with the enzyme was thus explored
by incorporating highly diverse NH substituents. Table 1 summa-
rizes the highlights from an extensive alkylation study performed
with the 2-(3-furyl) analog 2.9
N-Alkylation of 2 with alkyl groups larger than Me (compound
3), including aliphatics, allylics, benzylic aryls and benzylic hetero-
cyclics (compounds 5–15, Table 1) did not improve enzymatic and
cell-based potencies. Some notable steric effects were observed in
the case of branched alkyls (results not shown) and ortho-disubsti-
tuted benzylic groups (cf. compound 11). On the other hand, SAR
appeared to be somewhat insensitive to electronic/polarity effects
as both basic and acidic substituents were well tolerated (e.g., 12–
15). Initial results exposed a rather unproductive SAR at this posi-
tion and little improvement was achieved relative to compound 3
that had a simple methyl.
12
13
14
15
0.09
0.13
0.07
0.04
2.9
>8
N.T.
N.T.
16
17
18
19
20
0.06
0.10
0.09
0.21
2.8
N.T.
>10
8.5
N.T.
N.T.
CH(CH3)COOH
CH2CONH2
21
0.04
2.4
N.T. (not tested).
a
Values are means of duplicate experiments on two separate weightings.
Alkylation of the indole scaffold with an acetic acid side chain
(compound 16) was also well tolerated and was seen as an oppor-
tunity to introduce further diversity in high-throughput fashion
through the synthesis of amide libraries. The carboxylic acid func-
tion did not contribute specifically to potency as reduction to the
alcohol (17) or esterification (18) maintained activity. The 1C
methylene linker between the indole scaffold and the carboxyl
function appeared to be optimal as extension to the propionic ana-
log decreased potency threefold (cf. 16 vs 19). Consistent with pre-
HO
O
O
O
H
N
N
O
O
O
O
a, b
23
22
1R
2R
N
2R
N
1R
viously observed steric restrictions, substitution at the
a-position
O
of the indole scaffold (20) was not tolerated. Encouragingly how-
ever, primary amide analog 21 seemed to provide a slight improve-
ment in cellular potency compared to 3 despite a small decrease in
NS5B enzymatic inhibition. Compound 21 was selected as starting
point for further studies.10
As mentioned previously, the presence of the carboxylic acid
function in 16 provided an opportunity for rapidly expanding diver-
sity through the formation of amide derivatives as depicted in
Scheme 1.
O
O
O
N
O
N
d
c
OH
O
O
24
25 - 56
Scheme 1. Synthesis of N-acetamidoindole NS5B inhibitors. Reagents and condi-
tions: (a) NaH (1.25 equiv), DMF, 0 °C, 1 h then tert-butylbromoacetate (1.24 equiv),
rt, 18 h (86% yield); (b) TFA, 4 h, rt (99% yield); (c) TBTU or HATU (1.2 equiv), EtiPr2N
(5 equiv), DMF or DMSO, rt, 10 min then 1R2RNH, rt, 1–18 h; (d) 10 N NaOH (5–
10 equiv), 5:1 DMSO-water, 1–2 h, then purification by reversed-phase HPLC (>95%
homogeneity).
R
O
Toward this end, alkylation of indole ester 227 with NaH/tert-
butylbromoacetate followed by cleavage of the tert-butyl group
under acidic conditions afforded key intermediate 23 that was cou-
pled to various amines under standard peptide coupling conditions
to provide N-acetamidoindole esters 24 in good yields. Saponifica-
tion under mild basic conditions gave inhibitors 25–56 that were
purified to >97% homogeneity by reversed-phase HPLC. Approxi-
N
OH
O
2 (R = H)
3 (R = Me)
IC50 = 0.03 µM
IC50 = 0.016 µM
EC50 = 6.7 µM
EC50 = 4.2 µM
Figure 2. General structure of indole-based NS5B inhibitors.

P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 857–861
859
Table 2
Table 3
N-Acetamidoindole carboxylic acid inhibitors
Cyclic N-acetamidoindole carboxylic acid inhibitors
2R
2R
1R
N
1R
N
O
O
O
O
N
N
OH
OH
O
O
1R2R
HT-NS5B-
M)
D
21 IC50
1b Replicon EC50
M)
Compds
N
1R2R
HT-NS5B-
M)
D
21 IC50
1b Replicon EC50
M)
a
a
a
a
Compds
N
(
l
(
l
(
l
(l
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
NHMe
NHiPr
0.05
0.05
0.12
0.08
0.06
0.05
0.02
0.05
0.03
0.11
0.07
0.02
0.05
0.02
0.03
2.0
N
N
N
N
N
N
44
45
0.04
0.04
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
0.8
1.7
0.8
N.T.
2.3
1.5
NHCH2(c-C6H11
NHPh
NHCH2Ph
NH(CH2)2OMe
NMe2
NMe(nPr)
NMe(iPr)
NMe(c C6H11
N (iPr)2
NMe(CH2CH2OH)
NMe(CH2CH2OMe)
NMe(CH2CH2NMe2)
NMe(CH2CH2NEt2)
NMe(CH2CH2CH2NMe2) 0.02
NMe[CH2CH2-(4-
pyridyl)]
)
0.82
46
47
48
O
0.03
0.02
0.03
0.60
2.0
OH
COOH
)
>94
1.3
49
0.07
1.3
0.44
0.46
0.58
0.63
OH
50
51
52
N
N
N
N
N
N
N
N
0.04
0.03
0.04
0.45
0.41
0.83
0.03
42
43
NEtCH2-(4-pyridyl)
0.04
N.T.
N
N
0.02
0.13
Me
N.T. (not tested).
53
0.02
1.3
a
Values are means of duplicate experiments on two separate weightings.
OH
54
55
N
N
N
N
0.02
0.03
0.38
0.78
mately 125 amide analogs were prepared in this fashion and the
results for a representative set are shown in Tables 2 and 3.11
Some general conclusions that can be drawn from the data are
presented in Tables 2 and 3. Secondary amides (25–30) exhibited
comparable enzymatic and cellular potencies as unsubstituted
analog 21. IC50s were not significantly affected by the size/nature
of the amide substituent, with <2-fold variation observed between
small or large aliphatic, aromatic or benzylic groups. The first
improvement was noted with the tertiary amide analogs (31–43).
Dimethylation of the acetamide nitrogen (compound 31) provided
an inhibitor with comparable enzymatic potency to N-methylin-
dole 3 but with a fivefold improvement in cell culture and repre-
56
0.02
0.33
N
N
N.T. (not tested).
a
Values are means of duplicate experiments on two separate weightings.
Investigations on 3° acetamide derivatives were extended to
derivatives in which the nitrogen atom was incorporated within
a cyclic system (compounds 44–56, Table 3). Derivatives showed
excellent inhibition of NS5B (all IC50s <40 nM except for 49) but
no improvement was achieved over unsymmetrical 3° amides
(e.g., compound 43, Table 2). Yet again, diverse functionality was
tolerated on the ring systems including neutral, basic and acidic
groups. As described in Table 2, basic derivatives generally im-
proved potency in the cellular replicon assay, with several com-
pounds featuring EC50 values <500 nM (e.g., 50, 51, 54 and 56).
Earlier SAR studies at the N1-position of indole 2 led to the iden-
tification of novel acetamideindole NS5B inhibitors10 and the com-
pounds from this study suggest that substituents extending into
this space are likely solvent-exposed and contribute little to inhib-
itor potency relative to a simple methyl group since most com-
pounds had similar IC50 values relative to compound 3. However,
the inclusion of basic, lipophilic functionality in this position pro-
vided zwitterionic species with up to 30-fold increase in cellular
activity. These results justified further expansion on this class
and combinations of representative acetamide side chains with
various C-2 aromatic/heterocyclic substituents were evaluated as
sented the first compound with EC50 <1 lM in this series. The
enhanced replicon potency of 31 relative to compound 3 may, in
part, be attributable to improved cellular permeability (Caco-
2 = 12.8 Â 10À6 cm/s) rather than increased lipophilicity (Log D at
pH 7.4 = 1.45 and 3.26 for 31 and 3, respectively). Based on this
finding, the 3° amide chemotype was expanded but subsequent
evaluation revealed a relatively flat SAR with IC50 values ranging
from 20 to 110 nM. Trends were consistent with the hypothesis
that this part of the molecule is solvent-exposed since polar side
chains were generally preferred to large lipophilic groups. Of great-
er significance however, were the patterns that developed in the
replicon assay. Several analogs, in particular those carrying basic
side chains (e.g., 38, 39 and 43), had EC50 <500 nM. N-Methylpiper-
idine 43 was the most potent inhibitor (EC50 = 130 nM) generated
in this class, and approached the biological potency of compound
4 but with improved physicochemical properties (solubility at pH
7.2 = 290 l
g/mL).12

860
P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 857–861
Table 4
Acetamide–C2 combinations
zwitterions bearing the lipophilic-basic side chain C were more po-
tent than neutral amides A and B. In cell culture assays, 2-furyl
derivatives (57A–C) were significantly less cell-permeable and
had reduced replicon potency relative to the 3-furyl isomers. On
the other hand, more lipophilic thiophene analogs (58A–C, 59A–
C) had EC50 values similar to the corresponding 3-furyl containing
molecules, providing optional C-2 alternatives. In the case of 2-
phenylindole derivatives, only the combination with a lipophilic
and basic acetamide side chain had desirable cell culture potency
(60C). Surprisingly, incorporation of a basic nitrogen atom in the
phenyl ring at C-2 (61A) resulted in a sixfold drop in enzymatic po-
tency. It is possible that intramolecular interactions between the
basic pyridyl nitrogen and the acetamide carbonyl group may
modify the side chain orientation of the latter, resulting in a less
favorable binding conformation. Indeed, the presence of less basic
pyrazine nitrogens at C-2 (e.g., 62) was better tolerated (compare
60C and 62C) but the modification was highly detrimental to cell
culture activity. Finally, the presence of more bulky C-2 groups
(e.g., benzothiophene 63) also negatively impacted on potency.
Finally, representative N-acetamideindoles were tested for
specificity against another RNA-dependent RNA polymerase from
polio virus and a mammalian DNA-dependent RNA polymerase II
isolated from calf-thymus.5 In all cases, no significant inhibition
2R
1R
N
O
O
N
OH
O
3R
N
H3C
CH3
N1R2R
N
N
N
A
B
C
3R
IC50
EC50
(
IC50
M)
EC50
M)
IC50
M)
EC50
a
a
a
a
a
a
(
lM)
l
M)
(l
(l
(
l
(
l
M)
57
58
59
60
61
62
0.04
0.04
0.02
0.04
0.24
0.16
1.5
0.64
0.60
1.4
0.04
0.04
0.03
0.07
1.0
0.03
0.03
0.02
0.04
1.2
O
S
0.33
0.60
0.70
—
1.0
S
0.46
0.38
was observed at concentrations up to 250 lM (IC50 >250 lM).
The identification of a series of compounds with promising anti-
viral efficacy in the replicon assay system merited ADME-PK profil-
ing of a representative set of compounds, in order to evaluate their
potential for further development. The data are summarized in Ta-
ble 5.13
In general, most of the compounds had good metabolic stability
in the presence of human liver microsomes with T1/2 >200 min. In
addition, there was low potential for inhibition of major CYP450
3.4
N
N
N.T.
0.10
0.16
N.T.
0.06
0.16
10
N
63
0.21
2.5
1.1
1.0
S
isozymes (IC50 P 8 lM). Permeability across Caco-2 cell monolay-
ers was assessed in the apical to basal direction and the values ran-
ged from non-permeable (zwitterions) to highly permeable
(neutral side chains) depending on structural features. The equilib-
rium solubilities in pH 7.2 buffer12 of these inhibitors also spanned
a large range.
Several compounds were screened for oral absorption in rat
using cassette dosing (four compounds per cassettes, 4 mg/kg
each). The most potent compound (43) had no plasma exposure
(consistent with its low Caco-2 permeability). The inhibitor that
exhibited the best overall ADME-PK profile in rat was compound
46 and contained a neutral morpholine acetamide. The compound
was moderately soluble and had reasonable permeability, excel-
N.T. (not tested).
a
Values are means of duplicate experiments on two separate weightings.
described in Table 4. Analogs of compound 22 that bear alternate
C-2 heterocyclic substituents were elaborated to compounds 57–
59 in a similar manner to those described in Scheme 1.7
Though removal of the C-2 substituent (i.e., 3R = H) or the intro-
duction of small C-2 alkyl groups at that position (e.g., Et) led to
substantially decreased intrinsic potency (IC50 >1 lM; results not
shown), several combinations with enzymatic potency comparable
to 3-furyl analogs (IC50 <40 nM) were identified. In most cases,
Table 5
Summary of ADME-PK properties of selected acetamidoindole inhibitors of NS5B polymerase
Solubilitya
(
lg/mL)
Caco-2b (Â10À6 cm/s)
CYP450c IC50
(l
M)
HLM T1/2 (min)
Rat PKe C1h
(lM)
d
31
36
43
46
50
51
52
54
240
1300
290
105
7
12
18
79
60
12.8
BLD
0.2
5.2
1.7
6.5
7.0
1.4
0.2
P26 (3A4)
n.d.
n.d.
P24 (2C9)
>30
P16 (3A4)
P8 (3A4)
n.d.
250
275
n.d.
n.d.
0.2
1.0
0.6
0.3
0.4
0.0
n.d.
0.0f
>300
>300
70
>300
>300
>300
>300
111
59C
60C
P11 (2C19)
n.d.
47
<0.1
n.d.: not determined.
a
Determined with amorphous solids.12
Apical to Basal permeability at pH 7.4.
b
c
IC50 values were determined for 1A2, 2C9, 2C19, 2D6 and 3A4 isozymes. The values represent the lowest IC50 against the most sensitive enzyme (in brackets).
Human liver microsome stability data at 2
Compounds were administered po in suspension (0.3% tween-80/0.5% methocel) as mixtures of 4 compounds (each compound at 4 mg/kg). Plasma concentrations are
d
e
lM compound concentration.
reported for the 1 h time point.
f
Experiment performed at 2 mg/kg po BLD: below limit of detection.

P. L. Beaulieu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 857–861
861
6. (a) 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; (b) 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; Similar lead structures were also discovered by a Japan Tobacco
Ltd research group: (c) Hirashima, S.; Suzuki, T.; Ishida, T.; Noji, S.; Yata, S.;
Ando, I.; Komatsu, M.; Ikeda, S.; Hashimoto, H. J. Med. Chem. 2006, 49, 4721.
7. (a) Beaulieu, P. L.; Gillard, J.; Bykowski, D.; Brochu, C.; Dansereau, N.; Duceppe,
J.-S.; Haché, B.; Jakalian, A.; Lagacé, L.; LaPlante, S.; McKercher, G.; Moreau, E.;
Perreault, S.; Stammers, T.; Thauvette, L.; Warrington, J.; Kukolj, G. Bioorg. Med.
Chem. Lett. 2006, 16, 4987; (b) Harper, S.; Pacini, B.; Avolio, S.; Di Filippo, M.;
Migliaccio, G.; Laufer, R.; De Francesco, R.; Rowley, M.; Narjes, F. J. Med. Chem.
2005, 48, 1314.
lent metabolic stability (RLM T1/2 = 288 min) and low potential for
CYP-mediated drug–drug interactions. At an oral dose of 4 mg/kg,
plasma concentration at the 1 h time point reached 1
lM. Several
piperazine analogs (50–52, 54) were also identified that provided
promising plasma exposures in the 0.3–0.6
dosing.
lM range, 1 h post-
In conclusion, we described how alkylation of the nitrogen atom
of indole-based allosteric inhibitors of HCV NS5B polymerase with a
variety of acetamide side chains led to the discovery of compounds
with generally good cell culture potency in subgenomic HCV RNA
replicon assays. The SAR patterns suggested that the acetamide sub-
stituent is solvent-exposed and allows for the incorporation of
chemical diversity and the modulation of the molecule’s physico-
chemical properties. Many analogs showed improved aqueous solu-
bility and promising ADME-PK profiles in rats and in human in vitro
assays over previously reported series. Further optimization of this
class of compounds will be the focus of future studies.
8. 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.
9. (a) The HT-NS5B
D21 polymerase scintillation proximity assay was performed
as described in Ref. 5 with the following modifications: 12 nM of the soluble
truncated enzyme was used but the reaction had 0.01% w/v BSA and lacked the
previously supplemented 0.33% dodecyl-b-
determinations were performed in duplicates (or more) using Huh-7 cells with
stable subgenomic HCV 1b replicon that encodes modified luciferase
D-maltoside, 0.01% IGEPAL. (b) EC50
a
a
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
05/080388, 2005. Compounds were devoid of cytotoxicity (TC50 >10 lM) at
Acknowledgments
inhibitory concentrations. A sub-set of inhibitors were also tested in a 72 h
subgenomic 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
subtle toxic effects. For an overview and basic protocols on the use of HCV
replicons, see Lohmann, V. In Methods in Mol. Biol., 2nd ed.; Tang, H., Ed.;
‘Hepatitis C: Methods and Protocols’; Humana Press; 2009, Vol. 510, p 145.
10. (a) Beaulieu, P. L.; Brochu, C.; Chabot, C.; Jolicoeur, E.; Kawai, S.; Poupart, M.-A.;
Tsantrizos, Y. WO 04/065367, 2004.; Similar findings were recently reported by
another group: (b) Harper, S.; Avolio, S.; Pacini, B.; Di Filippo, M.; Altamura, S.;
Tomei, L.; Paonessa, G.; Di Marco, S.; Carfi, A.; Giuliano, C.; Padron, J.; Bonelli,
F.; Migliaccio, G.; De Francesco, R.; Laufer, R.; Rowley, M.; Narjes, F. J. Med.
Chem. 2005, 48, 4547.
11. All final inhibitors were purified by C-18 reversed-phase HPLC to >97%
homogeneity and gave 1H NMR, 13C NMR and MS data consistent with their
assigned structure.
12. Solubility data was acquired with amorphous solids after incubation for 24 h in
pH 7.2 buffer using the shaking flask method.
13. A similar derivative was profiled by the Merck group: Giuliano, C.; Fiore, F.; Di
Marco, A.; Padron Velazquez, J.; Bishop, A.; Bonelli, F.; Gonzalez-Paz, O.;
Marcucci, I.; Harper, S.; Narjes, F.; Pacini, B.; Monteagudo, E.; Migliaccio, G.;
Rowley, M.; Laufer, R. Xenobiotica 2005, 35, 1035.
We thank Serge Valois, Norman Aubry, Colette Boucher and Mi-
chael Little for analytical support and Dr. Ma’an Amad, Josie De
Marte, Martin Jutras Hélène Montpetit, François Otis and Manon
Rhéaume for DMPK profiling.
References and notes
1. Shepard, C. W.; Finelli, L.; Alter, M. J. Lancet Infect. Dis. 2005, 5, 558.
2. Beaulieu, P. L. Expert. Opin. Ther. Patents 2009, 19, 145. and references cited
therein.
3. NM283 (Idenix) is the first NI to enter clinical trial: (a) O’Brien, C.; Godofsky, E.;
Rodriguez-Torres, M.; Afdhal, N.; Pappas, S. C.; Pockros, P.; Lawitz, E.; Bzowej,
N.; Rustgi, V.; Sulkowski, M.; Sherman, K.; Jacobson, I.; Chao, G.; Knox, S.;
Pietropaolo, K.; Brown, N. A. Hepatology 2005, 42, 234A; (b) Brown, N. Expert.
Opin. Invest. Drugs 2009, 18, 709.
4. Available clinical data for HCV-796 (ViroPharma/Wyeth, first disclosure on
clinical efficacy for a NNI) has been reviewed in: Huang, Z.; Murray, M. G.;
Secrist, J. A., III Antiviral Res. 2006, 71, 351.
5. McKercher, G.; Beaulieu, P. L.; Lamarre, D.; LaPlante, S.; Lefebvre, S.; Pellerin, C.;
Thauvette, L.; Kukolj, G. Nucleic Acids Res. 2004, 32, 422.