Allosteric N-acetamide-indole-6-carboxylic acid thumb pocket 1 inhibitors of hepatitis C virus NS5B polymerase-Acylsulfonamides and acylsulfamides as carboxylic acid replacements

Article abstract of DOI:10.1139/cjc-2012-0319

Acylsulfonamide and acylsulfamide as surrogates for the carboxylic acid function of N-acetamide-indole-6-carboxylic acids were evaluated as allosteric inhibitors of hepatitis C virus (HCV) NS5B polymerase. Several analogs displayed excellent antiviral pot

Full text of DOI:10.1139/cjc-2012-0319

66
Allosteric N-acetamide-indole-6-carboxylic acid
thumb pocket 1 inhibitors of hepatitis C virus
NS5B polymerase — Acylsulfonamides and
acylsulfamides as carboxylic acid replacements
Pierre L. Beaulieu, René Coulombe, James Gillard, Christian Brochu,
Jianmin Duan, Michel Garneau, Eric Jolicoeur, Peter Kuhn, Marc-André Poupart,
Jean Rancourt, Timothy A. Stammers, Bounkham Thavonekham,
and George Kukolj
Abstract: Acylsulfonamide and acylsulfamide as surrogates for the carboxylic acid function of N-acetamide-indole-6-
carboxylic acids were evaluated as allosteric inhibitors of hepatitis C virus (HCV) NS5B polymerase. Several analogs
displayed excellent antiviral potency against both 1a and 1b HCV genotypes in cell-based subgenomic replicon assays.
Structure–activity relationships (SAR) are discussed in the context of the crystal structure of an inhibitor Ϫ NS5B
polymerase complex. Absorption, distribution, metabolism, and excretion pharmacokinetic (ADME-PK) properties of this
class of inhibitors are also described.
Key words: hepatitis C virus (HCV), NS5B polymerase, allosteric inhibitors, antivirals, structure–activity relationship.
Résumé : On a évalué les acylsulfonamides et les acylsulfamides comme substituts de la fonction acide carboxylique des
acides N-acétamide-indole-6-carboxylique comme inhibiteurs allostériques de la polymérase du virus de l’hépatite C (VHC)
NS5B. Plusieurs analogues présentent des activités antivirales excellentes contre les génotypes 1a et 1b du VHC dans les
essais de réplication sous-génomiques a base de cellules. On discute de relations structure–activité (RSA) dans le contexte
`
de la structure cristalline d’un complexe inhibiteur – polymérase NS5B. On décrit aussi les propriétés pharmacocinétiques,
telles les taux d’absorption, de distribution, de métabolisme et d’excrétion (PC-ADME), de cette classe d’inhibiteurs.
Mots-clés : virus de l’hépatite C (VHC), polymérase NS5B, inhibiteurs allostériques, antiviraux, relation structure–activité.
[Traduit par la Rédaction]
20–30 years following infection, HCV is now the major cause
of liver transplants in the world, and more than 15 000 people
die annually in the US alone from developing liver diseases
caused by HCV.4
Introduction
The hepatitis C virus (HCV) is a blood-borne ribonucleic
acid (RNA) virus that infects an estimated 170 million people
worldwide and can cause severe liver damage such as cirrhosis
and hepatocellular carcinomas.1 Whereas the spread of this
pathogen is now mostly limited to intravenous drug use and
unsafe medical practice in developing countries, a large pop-
ulation became chronically infected through contaminated
blood products prior to the identification of the etiological
agent in 1989.2 HCV is classified in the Hepacivirus genus of
the Flaviviridae family and, even though six major genotypes
have been characterized, genotypes 1a and 1b comprise ~70%
of documented infections and predominate in the US, Europe,
and Japan.3 As a consequence of a long incubation period of
The HCV (ϩ)-RNA genome encodes a unique polyprotein
of approximately 3000 amino acids, which comprises both
structural (core, E1, E2, p7) and nonstructural (NS2–NS5)
regions. Following processing by host and viral proteases, NS
proteins emerge and harbor enzymatic and other essential
functions necessary for viral replication.5 Several virally en-
coded enzymatic activities have been targeted for HCV anti-
viral therapy. Recently, NS3-4A protease inhibitors have
received approval for use in combination with the standard of
care comprised of the pegylated (PEG) immunomodulatory
cytokine interferon-␣ (IFN-␣) and the broad-spectrum antivi-
Received 17 August 2012. Accepted 26 September 2012. Published at www.nrcresearchpress.com/cjc on 17 January 2013.
P.L. Beaulieu, R. Coulombe, J. Gillard, C. Brochu, E. Jolicoeur, P. Kuhn, M.-A. Poupart, J. Rancourt, T.A. Stammers, and B.
Thavonekham. Department of Chemistry, Boehringer Ingelheim (Canada) Ltd., Research and Development, 2100 Cunard Street, Laval,
QC H7S 2G5, Canada.
J. Duan, M. Garneau, and G. Kukolj. Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., Research and
Development, 2100 Cunard Street, Laval, QC H7S 2G5, Canada.
Corresponding author: Pierre L. Beaulieu (e-mail: pierre.beaulieu@boehringer-ingelheim.com).
This article is part of a Special Issue dedicated to Professor Derrick Clive.
Can. J. Chem. 91: 66–81 (2013)
doi:10.1139/cjc-2012-0319
Published by NRC Research Press

Beaulieu et al.
67
ral agent ribavirin.6 While these new regimens provide patients
with an improved outcome, severe side effects, contraindica-
tions, and treatment failures in hard to treat genotype 1 pop-
ulations are fostering efforts for the search for improved
treatment options. Most notably, the search for interferon-free
regimens using combinations of direct acting antivirals
(DAAs) with improved tolerability and efficacy has become
the main focus of current efforts. Indeed, several combinations
of DAAs encompassing NS3-4A protease, nucleoside and
non-nucleoside NS5B polymerase inhibitors, and NS5A inhib-
itors have demonstrated the potential for improved outcome in
vitro, and more recently in HCV-infected patients.7
Our own research efforts in the field have led to the discov-
ery of BILN 2061 (celuprivir), the first NS3-4A protease
inhibitor to demonstrate antiviral potency in genotype 1 HCV-
infected patients. Faldaprevir, a follow-up analog, is currently
in phase 3 clinical trials in combination with PEG-IFN-␣2a/
ribavirin.8 A complementary program focusing on the discov-
ery of non-nucleoside inhibitors (NNIs) of the NS5B RNA-
dependent RNA polymerase of HCV, led to the development
of BILB 1941, an inhibitor of this enzyme that binds to the
thumb pocket 1 allosteric site, and was the first “finger loop”
inhibitor to provide proof-of-concept for this mechanism in
HCV-infected patients.9 BI 207127 is a more potent follow-up
analog of BILB 1941 that is currently in phase 3 clinical trials
in an interferon-sparing combination with faldaprevir and riba-
virin.10 As part of our continued efforts toward the develop-
ment of anti-HCV therapies, we have been involved in a
search for structurally and chemically diverse back-up com-
pounds for our lead clinical candidates. Recently, we and
others reported the discovery of N-acetamide-6-indolecar-
boxylic acid thumb pocket 1 NS5B inhibitors, which pro-
vided an attractive starting point for the development of
structure–activity relationship (SAR) distinct from previous
series and the discovery of more chemically diverse inhib-
itors.11 We report herein the evaluation of acylsulfonamide
and acylsulfamide isosteres of these indolecarboxylic acid
inhibitors.
domain of the enzyme and the formation of an enclosed active
site that can initiate RNA synthesis.14 Unlike other NS5B
allosteric sites, thumb pocket 1 is highly selective in terms of
its chemotype preferences and their potential to deliver the
potency levels necessary for the development of powerful
antiviral agents. Indeed, only benzimidazole derivatives and
lipophilic isosteric indole analogs have emerged as attractive
starting points for inhibitor design.15
Figure 1 provides some historical background on Boehr-
inger Ingelheim’s thumb pocket 1 inhibitor program. From
screening of our corporate sample collection and preliminary
SAR, benzimidazole 1 was quickly identified as the minimum
core for polymerase inhibition.16a Replacement of the benzimi-
dazole scaffold by an isosteric indole core (compound 2)
resulted in significantly improved intrinsic potency in a bio-
chemical assay using a C-terminally truncated NS5B⌬21 poly-
merase as previously described (half maximal inhibitory
concentration (IC50) ϭ 16 nmol/L).11a,16b Cellular permeability
was also improved for the lipophilic indoles, resulting in
analogs with low micromolar antiviral activity in a cell-based
genotype 1b subgenomic replicon assay (half maximal effec-
tive concentration (EC50) ϭ 5.9 ␮mol/L).16b 6-Indolecarboxylic
acid (2) in turn led to diamide 3 (BILB 1941) with good
antiviral activity in vitro (EC50 ϭ 84 and 153 nmol/L in
genotype 1b and 1a replicons, respectively) and proof-of-
concept in HCV-infected patients.9 While efforts were con-
tinuing toward further optimization of diamide derivatives
such as BILB 1941, a structurally divergent back-up series of
inhibitors was discovered by us and others, which took advan-
tage of the availability of the ring nitrogen atom in indole
derivatives such as 2 as a new vector for exploring interactions
with the protein.11 N-Acetamide indole-6-carboxylic acid de-
rivatives such as 4 provided a chemically diverse starting point
with promising cellular potency (EC50 ϭ 0.36 ␮mol/L) for the
development of an alternative series. To improve the potency
of indole derivatives such as 4 and to address concerns asso-
ciated with the potential for carboxylic acids to form reactive
acylglucuronide metabolites or S-acyl-coenzyme A thioesters
that could lead to toxicity and idiosyncratic allergic responses
in vivo,11c,17 we explored the potential of acylsulfonamide and
acylsulfamide 5 carboxylic acid isosteres as potential NS5B
inhibitors.18 The outcome of this study is reported herein.
Acylsulfonamide and acylsulfamide indole inhibitors were
prepared from the corresponding 6-indolecarboxylic acid de-
rivatives through one of the sequences described in Scheme 1.
2-Bromoindole 616b was alkylated with tert-butyl bro-
moacetate, the tert-butyl protecting group cleaved under acidic
conditions, and the liberated carboxylic acid coupled to
amines under standard amide bond forming protocols to pro-
vide intermediates 7. Introduction of the C-2 aryl or heteroaryl
substituent was accomplished under standard metal-catalyzed
Suzuki–Miyaura cross-coupling conditions using boronic acid
derivatives to provide indole derivatives 8.19 Alternatively, 8
could be accessed through a sequence of steps involving initial
cross-coupling of 2-bromoindole 6 with boronic acids followed
by introduction of the acetamide side chain to provide 9 followed
by amide formation.16b 3-Cyclohexyl-6-indolecarboxylic acid
methyl ester (10)16b can also serve as starting material to
provide intermediate 9 through a sequence involving N-acetam-
ide formation, followed by C-2 bromination, Suzuki–Miyaura
cross-coupling to introduce the C-2 aryl or heteroaryl substituent,
Results and discussion
HCV NS5B is a RNA-dependent RNA polymerase that is
virally encoded and is essential for viral replication. It has no
mammalian counterpart and thus provides an attractive oppor-
tunity for the discovery of specific antiviral agents. The poly-
merase architecture comprises a thumb, finger, and palm
domain, with the latter harboring the enzyme active site.12
NS5B’s key function is to replicate the HCV genome to
initiate production of progeny virus. As with other poly-
merases, it is susceptible to inhibition by nucleoside/nucleo-
tide inhibitors that are anabolized to triphosphate forms and
mimic substrate to effect chain termination. In addition, at
least four allosteric sites have been described, where NNIs can
bind and interfere with enzyme conformational changes that
are necessary for RNA synthesis. These include thumb pocket
1 and 2 as well as two distinct sites located in the palm domain
of the enzyme, in close proximity to the active site. All classes
of HCV NS5B inhibitors (NIs and NNIs) have been validated
in clinical trials and have been the subject of several literature
reviews.13 Thumb pocket 1 inhibitors (also referred to as
finger loop inhibitors) are believed to function by preventing
the closure of a loop that extends from the finger to the thumb
Published by NRC Research Press

68
Can. J. Chem. Vol. 91, 2013
Fig. 1. Evolution of Boehringer Ingelheim’s thumb pocket 1 allosteric NS5B inhibitors.
and selective cleavage of the tert-butyl ester protecting group.
Acylsulfonamides and acylsulfamides 5 were prepared by sapon-
ification of ester 8 and coupling of the resulting carboxylic acid
with various sulfonamides or sulfamides under standard amide
bond forming conditions. An alternative assembly sequence de-
signed for rapid exploration of amide substituents while main-
taining the acylsulfonamide moiety constant is described in the
Experimental section. It involves the preparation of an indole
6-acylsulfonamide intermediate bearing a free acetic acid side
chain on the indole nitrogen and condensation with amines as
the last step in the sequence (see the Experimental section for
details).
Initial results from the conversion of carboxylic acid 4 to
methyl or phenylacylsulfonamide analogs 11 and 12 (Table 1)
proved encouraging. Comparable intrinsic potencies in the
biochemical enzyme inhibition assay11a,20 (IC50 ϭ 0.015–
0.018 ␮mol/L) were measured for carboxylic acid (4) and
acylsulfonamide/sulfamide derivatives, suggesting that both
functionalities engage in similarly productive interactions with
NS5B. In the cell-based replicon assay, however, despite sim-
ilarities in the calculated pKa and A log P21 values for carbox-
ylic acids and acylsulfonamides, we observed a three- to
Highlights from the evaluation of approximately 40
acylsulfonamide/acylsufamide derivatives are summarized in
Table 1. Phenyl acylsulfonamide (12) served as a reference
point for the investigation of the impact of electronic effects
through substitution of the phenyl ring with electron-
withdrawing and -donating groups at the ortho-, meta-, and
para-positions (results not shown). Despite spanning a calcu-
lated pKa range of 3.7–4.7, there was no significant impact on
overall potency. A similar result was obtained with benzylic
13 or lipophilic aromatic systems such as 2-naphthyl 14. Steric
factors on the other hand appeared to be more important with
respect to cell-based potency, presumably as a result of shield-
ing the polar and ionized acylsulfonamide moiety. The intro-
duction of a lipophilic ortho-methyl group on the phenyl ring
(compound 15) and small sterically encumbering aliphatic
groups such as cyclopropyl 16 and tert-butyl 17 provided
analogs with similar intrinsic potency against the enzyme but sub-
micromolar cell culture potency, possibly because of improved per-
meation of these analogs through cell membranes. tert-Butyl analog
17 had a comparable profile to carboxylic acid 4. Acylsulfamides
(18–21), which have calculated pKa values similar to carboxylic
acids, were also well tolerated, particularly N,N-disubstituted
analogs such as 20 and 21 (EC50 Ͻ 0.5 ␮mol/L). Interestingly,
N-methylation of the acylsulfonamide or replacement of the sul-
fone moiety by a second carbonyl (imide analog) was not toler-
four-fold reduction in a cell-based antiviral assay (EC50
ϭ
0.9–1.5 ␮mol/L).22 A comprehensive SAR study was then
initiated to identify analogs with improved antiviral potency.
Published by NRC Research Press

Beaulieu et al.
69
Scheme 1. Synthesis of inhibitors. Reagents and conditions: (a) NaH (1.25 equiv), DMF, 0 °C for 1 h then tert-butyl bromoacetate (1.24 equiv),
RT, 18 h; (b) TFA, 4 h, RT or 4 N HCl in dioxane, RT, 4 h; (c) TBTU or HATU (1.2 equiv), EtiPr2N (5 equiv), DMF or DMSO, RT, 10 min
then 1R2RNH (1.1 equiv), RT, 1Ϫ18 h; (d) heteroaryl or aryl boronic acid (1.3 equiv), LiCl (2 equiv), Na2CO3 (2.5 equiv), Pd(PPh3)4
(0.04 equiv), degassed toluene/ethanol/water 1:1:1, reflux overnight under argon; (e) 10 N NaOH or LiOH (5Ϫ10 equiv), 5:1 THF–water, 1–2 h;
(f) Oxalyl chloride via acid chloride or, EDAC–HCl, water soluble carbodiimide or carbonyldiimidazole (1.2–2 equiv), DMAP (1.5–2.0 equiv),
sulfonamide or sulfamide (1–1.2 equiv), CH2Cl2, DMF or DMSO, RT (18–48 h); (g) Pyridinium perbromide, THF–CHCl3 (1:1), 0 °C (4 h) then RT
overnight. DMF: dimethylformamide, RT: room temperature, TFA: trifluoroacetic acid, TBTU: O-(benzotriazol-1-yl)-N,N,N=,N=-tetramethyluronium
tetrafluoroborate, HATU: (O-(7-azabenzoriazol-1-yl)-N,N,N=,N=-tetramethyluronium hexafluorophosphate, DMSO: dimethyl sulfoxide,
THF: tetrahydrofuran, EDAC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, DMAP: 4-dimethylaminopyridine.
ated. This could be due to the absence of an ionizable group at
physiological pH in the latter or an unfavorable conformational
change (results not shown).
finger loop of molecule A was blocked by a crystal contact and
cannot open to reveal the allosteric binding site, and thus
remains in an apo conformation (without inhibitor).
The lack of distinctive electronic or steric SAR trends that
emerged from modification of the sulfonamide/sulfamide moi-
ety suggested that this part of the bound inhibitor was likely
solvent-exposed. This was confirmed when compound 12 was
soaked into NS5B⌬21 crystals and an X-ray crystal structure
of the complex was solved. The crystal packing of this com-
plex includes two protein molecules per asymmetric unit (re-
ferred to as A and B). The inhibitor was visible on molecule B
and not observed on molecule A. Inhibitor 12 bound the thumb
pocket 1 allosteric site as previously reported for analogous
indole-6-carboxylic acids.14a,23 Upon binding of compound 12
to molecule B, electron density corresponding to NS5B resi-
dues 18–35 (the ⌳1 finger loop) was apparently displaced and
not seen (Fig. 2A). In the apo structure, this pocket is not
solvent accessible and is sequestered by the residues from the
⌳1 finger loop. Figure 2B depicts the overlap between the ⌳1
finger loop and inhibitor 12 binding sites. In contrast, the
As depicted in Fig. 3A, the hydrophobic protein interactions
made by this class of inhibitor are similar in nature to those
mediated by finger loop residues Ser29, Leu30, and Leu31.
The cyclohexyl ring of 12 occupies the majority of a well-
defined hydrophobic pocket (Leu30 subpocket), the indole
scaffold stacks against proline residues (P495), and the furan
ring lies in a somewhat less-defined hydrophobic area (Leu31
subpocket). A key anchoring point that is critical for inhibitor
potency is provided by a strong hydrogen bond between the
carbonyl moiety of the acylsulfonamide functionality and the
guanidine side chain of Arg503 (Fig. 3A). Similarly, a salt
bridge was observed between the carboxylic acid moiety of
previously reported indole-6-carboxylic acid analogs and the
guanidine group of Arg503.14a,23 Analogs containing a “re-
verse” acylsulfonamide in which the SO2 moiety is attached to
the indole ring were previously explored in a less active
benzimidazole-based series of inhibitors. In this disposition,
Published by NRC Research Press

70
Can. J. Chem. Vol. 91, 2013
Table 1. Hepatitis C virus (HCV) NS5B⌬21 enzyme inhibition and
antiviral potency data for acylsulfonamide and acylsulfamide derivatives.
Fig. 2. Panel A shows inhibitor 12 bound to NS5B⌬21 thumb
pocket 1 with the displaced ⌳1 finger loop. The thumb domain is
depicted in green and the palm and finger domains are red and
blue, respectively. Panel B depicts an overlay of apo NS5B with
the ⌳1 finger loop in the closed conformation and inhibitor 12
bound in thumb pocket 1. The inhibitor 12 (in white) and ⌳1
finger loop residues Ser29, Leu30, and Leu31 (ball and stick)
occupy the same region in the thumb domain.
IC50
(␮mol/L)a
EC50
(␮mol/L)a
Compound
X
A log Pb
11
12
13
14
15
CH3
Ph
Bn
2-Naphthyl
0.015
0.018
0.059
0.038
0.028
1.5
3.27
4.85
4.86
5.76
5.37
0.94
0.94
1.05
0.67
16
17
0.017
0.047
0.69
0.37
3.76
4.21
18
19
20
21
NHPh
NHMe
NMe2
0.058
0.045
0.017
0.049
2.1
4.24
2.66
2.87
3.33
1.53
0.34
0.46
aValues are an average of at least two measurements.
bSee ref. 21.
hydrogen bonding to Arg503 may be less favorable and the
compounds were found to be inactive. Therefore, these were
not investigated in the current indole chemotype (unpublished
data). The acylsulfonamide/sulfamide moiety is a good re-
placement of the carboxylate group as a strong hydrogen bond
with Arg503 is maintained. Furthermore, one of the sulfone
oxygen atoms makes an additional weak interaction (2.7 Å)
with the same guanidine side chain of Arg503. In this bound
conformation, the phenyl group is pointing toward solvent,
orthogonal to the protein surface. This is in agreement with the
SAR described in Table 1 and the fact that methyl analog 11
or bulkier substituents (e.g., 2-napthyl group of 14) could be
accommodated with potencies comparable to 12.
Figure 3B depicts the overlay of acylsulfonamide 12 and an
analogous indole-6-carboxylic inhibitor.23 The two molecules
bound similarly to NS5B, and minimal protein movement is
required to accommodate the two classes of inhibitors. The
acylsulfonamide moiety induced a small torsion (~8°) of the
carbonyl out of plane with the indole core bringing it toward
Arg503. A good correspondence was observed for the indole
scaffolds, however, the lipophilic cyclohexyl ring of 12 ap-
pears to insert slightly deeper into its lipophilic pocket (aver-
age shift of ~0.5 Å). Both acetamide substituents project into
the solvent, explaining the lack of improvement in intrinsic
potency through modification of these two susbstituents.
Having identified analogs with overall potency profiles
comparable to the starting 6-indole carboxylic acid 4 and
being less likely to generate reactive metabolites (e.g., acyl-
glucuronides), we next investigated the impact of modifying
the indole acetamide substituent in tert-butyl acylsulfonamide
derivatives. Consistent with this portion of the inhibitor being
exposed to solvent (vide supra), results depicted in Table 2
revealed a rather flat SAR, with most analogs maintaining
intrinsic as well as antiviral potency in a similar range. Inter-
estingly, analogs with a basic amide side chain and ex-
pected to exist as zwitterionic species (e.g., piperazine 25
and 4-aminopiperidine 28) provided the best cellular activities,
despite an increase in polarity (A log P ϳ 3) associated with
the strongly ionized state of these molecules. Similar obser-
vations were made in the carboxylic acid series where zwit-
terionic derivatives displayed improved cell culture activity.11
Although the best analogs at this stage of our investigations
addressed the potential metabolic liability associated with the
carboxylic acid derivatives, they still required a significant im-
Published by NRC Research Press

Beaulieu et al.
71
Fig. 3. Panel A shows inhibitor 12 bound to thumb pocket 1
of HCV NS5B⌬21 with key hydrogen bonds between the
acylsulfonamide moiety and the guanidine side chain of Arg503.
Panel B provides an overlay of an indole-6-carboxylic acid
inhibitor and compound 12.23
Table 2. Hepatitis C virus (HCV) NS5B⌬21 enzyme inhibition
and antiviral potency data for a series of acyl tert-butylsulfonamide
acetamide analogues.
IC50
(␮mol/L)a
EC50
(␮mol/L)a
Compound
A log Pb
17
0.047
0.37
4.21
22
23
0.058
0.10
0.78
0.48
4.52
5.43
24
25
0.073
0.12
0.96
0.31
4.98
2.91
26
27
0.028
0.21
0.67
0.93
4.39
5.40
28
0.047
0.37
3.07
aValues are an average of at least two measurements.
bSee ref. 21.
improvement over clinical candidate BILB 1941. Genotype 1a
HCV is a predominant subtype in the USA and constitutes a hard
to treat patient population for which IFN-based treatments are
often less effective. Representative compounds (29, 31, and 33)
were tested in a genotype 1a subgenomic replicon25 and a greater
than twofold upward shift in antiviral potency was observed
(EC50 ϭ 95, 81, and 42 nmol/L, respectively).
Having identified analogs with improved potency over clin-
ical candidate BILB 1941 and with lower potential to form
reactive metabolites compared with indole-6-carboxylic acid
derivatives such a 4, we evaluated the in vitro absorption,
distribution, metabolism, and excretion (ADME) properties of
representatives of this class of compounds prior to assessing
oral bioavailability in rats. As seen in Table 4, potent zwitter-
ionic inhibitors displayed low aqueous solubility at pH 6.8.
Although these compounds generally showed good metabolic
stability following incubation with human or rat liver micro-
somes (T1/2 Ͼ 100 min), they had poor apical to basolateral
permeability in the Caco-2 model of oral absorption. Conse-
quently, none of the compounds tested in vivo showed plasma
exposure following oral administration to rats. Compounds 40
and 41 were the only two analogs with measurable quantities
of inhibitor detected in the target liver organ, corresponding to
approximately 20-fold in the EC50.
provement in antiviral potency (Ͼ10-fold) to compare favorably
with our first clinical candidate BILB 1941. In addition, the furyl
substituent at the C-2 position of the indole scaffold represented
a potential toxicophore that required replacement.24 Exploiting
ongoing SAR studies that were conducted in parallel in the
carboxylic acid series by us (data to be published separately)
and others,11 we prepared a matrix of compounds featuring
more optimal C-2 substituents and basic acetamide side chains
in combination with our best acylsulfonamide (cyclopropyl)
and acylsulfamide (N,N-dimethylsulfamide). Biological re-
sults are presented in Table 3.
As anticipated from studies performed in the carboxylic
acid series (unpublished data), the building blocks used for this
matrix led to inhibitors that were active in biological assays.
All compounds inhibited NS5B enzymatic activity with
IC50 Ͻ 100 nmol/L and several achieved antiviral po-
tency Ͻ20 nmol/L in the cell-based subgenomic 1b replicon
(32, 41, 43, 44, 45, and 47) representing a four- to eight-fold
Conclusion
In summary, we have evaluated acylsulfonamides and
acylsulfamides as isosteric replacements for the carboxyl function
Published by NRC Research Press

Table 3. Hepatitis C virus (HCV) NS5B⌬21 enzyme inhibition and antiviral potency data for a matrix of acylsulfonamide and acylsulfamide analogues.
IC50
(␮mol/L)a
EC50
(␮mol/L)a
IC50
(␮mol/L)a
EC50
(␮mol/L)a
IC50
(␮mol/L)a
EC50
(␮mol/L)a
Compound
X
Compound
X
Compound
X
29
32
33
t-Bu
Cyclo-Pr
NMe2
0.042
0.052
0.062
0.078
0.017
0.027
30
31
Cyclo-Pr
NMe2
0.069
0.081
0.083
0.055
34
35
Cyclo-Pr
NMe2
0.050
0.039
0.075
0.026
36
37
Cyclo-Pr
NMe2
0.095
0.092
0.088
0.28
38
39
Cyclo-Pr
NMe2
0.080
0.090
0.034
0.023
40
41
Cyclo-Pr
NMe2
0.043
0.044
0.023
0.016
42
43
Cyclo-Pr
NMe2
0.065
0.071
0.050
0.013
44
45
Cyclo-Pr
NMe2
0.066
0.051
0.016
0.013
46
47
Cyclo-Pr
NMe2
0.052
0.047
0.031
0.011
aValues are an average of at least two measurements.

Beaulieu et al.
73
Table 4. In vitro and in vivo absorption, distribution, metabolism, and excretion (ADME)-PK
(pharmacokinetic) profiles of representative acylsulfonamide and acylsulfamide NS5B inhibitors.
Solubility pH 6.8/2
(␮g/mL)a
HLM/RLM
T1/2 (min)a
Caco-2
(ϫ10Ϫ6 cm/s)b
C1h/2h
(␮mol/L)c
[Liver]2h
(␮mol/L)d
Compound
29
33
40
41
43
Ͼ300/Ͼ300
Ͼ300/Ͼ300
190/101
141/161
288/Ͼ300
Ͻ0.1
Ͻ0.1
0.22
1.4
BLD
BLD
BLD
BLD
BLD
BLD
BLD
0.41
0.49
BLD
0.3/4.0
10/544
4.9/12
4.6/15
Ͻ0.1
HLM, human liver microsomes; RLM, rat liver microsomes; BLD, below limit of detection.
aMeasured on lyophilized amorphous solids using the 24 h shaking flask method and pH 7.2 phosphate buffer.
bThe Caco-2 permeability assays were run without bovine serum albumin (BSA) with both chambers at pH 7.4.
cPlasma concentration at 1 and 2 h following oral administration to rats as mixtures of four compounds at a
dose of 4 mg/kg each.
dLiver concentrations were measured as described in the Experimental section.
of previously described N-acetamide-indole-6-carboxylic acid in-
hibitors of HCV NS5B polymerase that bind in the thumb pocket
1 allosteric site. The new analogues showed improved antiviral
potency in genotype 1a and 1b replicons compared with the
previous clinical candidate, BILB 1941. The binding-site and
protein-inhibitor interactions of an acylsulfonamide analog
were characterized by X-ray crystallography and are consis-
tent with the reported SAR. However, significant improve-
ments in permeability and oral absorption are required prior to
further advancement of this chemical series.
tained on a Micromass Platform LCZ model ZMD 4000 in
electrospray mode. High-resolution mass spectra (HR-MS)
were obtained on a Bruker micrOTOF-Q II in electrospray
positive (ESϩ) ionization mode. Purification of crude material
was performed either by flash column chromatography or by
using a CombiFlash Companion using RediSep Silica or Sili-
caSep columns according to preprogrammed gradient and flow
rate separation conditions in hexane/EtOAc or dichlorometh-
ane (DCM)/MeOH. The final compounds were purified by
preparative HPLC on a Waters 2767 Sample Manager with
pumps 2525, column fluidics organizer (CFO), photodiode
array (PDA) detector 2996, and MassLynx 4.1 using either a
Whatman Partisil 10-ODS-3 column, 2.2 cm ϫ 50 cm or a
YMC Combi-Prep ODS-AQ column, 50 mm ϫ 20 mm inside
diameter (ID), S-5 ␮m, 120 Å, and a linear gradient program
from 2% to 100% AcCN/water (0.06% trifluoroacetic acid
(TFA)). Fractions were analyzed by analytical HPLC, and the
pure fractions were combined, concentrated, frozen, and ly-
ophilized to yield the desired compound as a neutral entity or
the trifluoroacetate salt for basic analogues. Inhibitor HPLC
purity was measured by using a Waters Alliance 2695 sepa-
ration module with a Waters TUV 2487 UV detector. The
column was a Combiscreen ODS-AQ, 5 ␮m, 4.6 mm ϫ
50 mm, linear gradient from 5% to 100% ACN/H2O ϩ 0.06%
TFA in 10.5 min with detection at 220 nm.
Experimental
Instrumentation, analysis, and starting materials
All commercially obtained solvents and reagents were used
as received without further purification. 6-Indolecarboxylic acid
esters 6 and 10 were prepared as previously described.16b
Phenylsulfamide was prepared as previously described.26 All
reactions were carried out under an atmosphere of argon.
Temperatures are given in degrees Celsius. Solution percent-
ages express a weight-to-volume relationship and solution
ratios express a volume-to-volume relationship, unless stated
otherwise. NMR spectra were recorded on a Bruker AVANCE
1
II (400 MHz for H NMR) spectrometer and were referenced
to either DMSO-d6; (2.50 ppm) or CDCl3 (7.27 ppm). Data is
reported as follows: chemical shift (ppm), multiplicity (s ϭ
singlet, d ϭ doublet, t ϭ triplet, br ϭ broad, and m ϭ
multiplet), integration, and coupling constant (J; reported to
the nearest 0.5 Hz). Low-resolution mass spectra were ob-
General procedure for preparing compounds 11–21
Published by NRC Research Press

74
Can. J. Chem. Vol. 91, 2013
Compounds 13–15 were prepared in a similar fashion.
Acid chloride method
To a solution of compound 411a (50.3 mg, 0.16 mmol) in
DCM (1.5 mL) at room temperature (RT) was added a 2.0
mol/L solution of oxalyl chloride in DCM (144 ␮L,
0.29 mmol) and dimethylformamide (DMF; 10 ␮L). After
20 min, volatiles were removed under reduced pressure. The
crude acid chloride intermediate was redissolved in DCM
(1.5 mL) and tert-butyl sulfonamide (23.6 mg, 0.17 mmol),
4-dimethylaminopyridine (DMAP; 14.0 mg, 0.12 mmol), and
triethylamine (TEA; 48 ␮L, 0.35 mmol) were added succes-
sively and the resultant mixture stirred for 30 min. After
evaporation of volatiles, the residue was dissolved in DMSO
(1.5 mL) and the mixture acidified with TFA (44 ␮L). The
desired material was isolated and purified using reversed-
phase preparative (RP-prep) HPLC using an acetonitrile
(ACN)/water gradient incorporating 0.1% TFA as buffer. Fol-
lowing collection, pooling, and lyophilization of relevant frac-
tions, compound 17 was obtained (33 mg, 51% yield) as a
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC)–
HCl method
To a solution of carboxylic acid 411a (20.0 mg, 0.046 mmol) in
DMF (1.0 mL) was added DMAP (11 mg, 0.09 mmol),
N,N-dimethylsulfamide (6.9 mg, 0.056 mmol), and EDAC–
HCl (17.6 mg, 0.092 mmol), and the mixture stirred for 2 d at
RT. The desired material was isolated and purified using
RP-prep HPLC using an ACN/water gradient incorporating
0.1% TFA as buffer. Following collection, pooling, and ly-
ophilization of relevant fractions, compound 20 was isolated
(13 mg, 52% yield) as a white amorphous solid. 1H NMR (400
MHz, DMSO-d6) ␦: 11.58 (s, 1H), 8.07 (d, 1H J ϭ 1.2 Hz),
7.92 (t, 1H, J ϭ 1.6 Hz), 7.85 (s, 1H), 7.83 (m, 1H), 7.64 (dd,
1H, J ϭ 1.6, 8.2 Hz), 6.53 (dd, 1H, J ϭ 0.8, 1.6 Hz),
5.30–5.11 (m, 1H), 5.03 (s, 2H), 3.6–3.4 (m, 8H), 2.92 (s,
6H), 2.75–2.65 (m, 1H), 2.04–1.59 (m, 7H), 1.32 (m, 3H). 13
C
1
white amorphous solid. H NMR (400 MHz, DMSO-d6) ␦:
NMR (100 MHz, DMSO-d6) ␦: 166.3, 166.1, 144.1, 142.5,
136.7, 131.9, 129.3, 123.7, 119.5, 119.4, 118.6, 115.1, 111.9,
111.2, 66.2, 44.8, 42.1, 38.0, 36.3, 32.8, 26.6, 25.6. HR-MS
(APPIϩ) calcd for C27H34N4O6SH [M ϩ H]ϩ: 543.2272;
found: 543.2275.
11.30 (s, 1H), 8.00 (d, 1H, J ϭ 1.1 Hz), 7.90 (t, 1H, J ϭ
1.7 Hz), 7.86–7.80 (m, 2H), 7.59 (dd, 1H, J ϭ 1.4, 8.5 Hz),
6.53 (d, 1H, J ϭ 1.1 Hz), 5.02 (s, 2H), 3.61–3.51 (m, 8H),
2.76–2.65 (m, 1H), 2.00–1.64 (m, 7H), 1.43 (s, 9H), 1.37–
1.22 (m, 3H). HR-MS (positive atmospheric pressure photo-
ionization (APPIϩ)) calcd for C29H37N3O6SH [M ϩ H]ϩ:
556.2476; found: 556.2477.
Compounds 11, 12, 16, 18, 19, and 21 were prepared in a
similar fashion.
Published by NRC Research Press

Beaulieu et al.
75
ture was stirred for 2 h and then diluted with ethyl acetate.
The solution was washed successively with saturated
1 mol/L KHSO4 and brine, dried over MgSO4 filtered, and
volatiles evaporated under reduced pressure. Solid residues
were suspended in a mixture of diethyl ether and hexane
and stirred briefly. Solids were filtered, washed with more
diethyl ether/hexane mixture, then air-dried. Compound 52
(520 mg, 89% yield; 96% homogeneity) was obtained as a
pale yellow powder. 1H NMR (400 MHz, DMSO-d6) ␦:
11.27 (s, 1H), 8.16 (s, 1H), 7.95 (s, 1H), 7.90 (s, 1H), 7.85
(d, 1H, J ϭ 8.6 Hz), 7.62 (d, 1H, J ϭ 8.4 Hz), 6.57 (s, 1H),
4.96 (s, 2H), 4.10 (q, 2H, J ϭ 7.0 Hz), 2.68 (m, 1H),
1.94–1.63 (m, 7H), 1.42 (s, 9H), 1.36–1.21 (m, 3H), 1.14
(t, 3H, J ϭ 7.0 Hz). FIA-MS (APPIϩ) m/z: 515.0 [M ϩ H]ϩ.
General procedure for compounds 22–28
Intermediate 49
To a solution of compound 4811a (1.08 g, 3.5 mmol) in tetrahy-
drofuran (THF; 20 mL) was added 2-(trimethylsilyl)ethanol
(751 ␮L, 5.2 mmol) and diethyl azodicarboxylate (DEAD;
825 ␮L, 5.2 mmol). Triphenylphosphine (1.37 g, 5.2 mmol)
was added and the mixture stirred overnight. Volatiles were
removed under reduced pressure and the desired material
purified on silica gel using a 5%–20% ethyl acetate/hexane
gradient to obtain compound 49 as an off-white solid (520 mg,
1
38% yield; 97% homogeneity). H NMR (400 MHz, DMSO-
d6) ␦: 11.38 (s, 1H), 8.04 (s, 1H), 7.96 (s, 1H), 7.87 (s, 1H),
7.80 (d, 1H, J ϭ 8.6 Hz), 7.56 (dd, 1H, J ϭ 1.4, 8.6 Hz), 6.85
(d, 1H, J ϭ 1.4 Hz), 4.38 (t, 2H, J ϭ 8.0 Hz), 2.92 (m, 1H),
2.02–1.70 (m, 7H), 1.47–1.34 (m, 3H), 1.10 (t, 2H, J ϭ
8.0 Hz), 0.10 (s, 9H).
Intermediate 53
To a solution of compound 52 (500 mg, 0.97 mmol) in
DMSO (7 mL) at RT was added 10 mol/L aqueous NaOH
(390 ␮L, 3.88 mmol) and this was stirred for 30 min. The
mixture was diluted with ethyl acetate and washed succes-
sively with saturated 1 mol/L KHSO4 and brine, dried over
MgSO4 filtered, and volatiles evaporated under reduced pres-
sure. The solid residues were suspended in diethyl ether and
stirred briefly. Solids were filtered, washed with more diethyl
ether, then air-dried to give compound 53 (404 mg, 86% yield;
99% homogeneity) as an off-white powder. 1H NMR
(400 MHz, DMSO-d6) ␦: 13.09 (br s, 1H), 11.29 (br s, 1H),
8.16 (s, 1H), 7.92–7.86 (m, 2H), 7.84 (d, 1H, J ϭ 8.6 Hz), 7.62
(d, 1H, J ϭ 8.6 Hz), 6.57 (s, 1H), 4.84 (s, 2H), 2.68 (m, 1H),
1.96–1.64 (m, 7H), 1.43 (s, 9H), 1.37–1.23 (m, 3H). FIA-MS
(APPIϩ) m/z: 487.2 [M ϩ H]ϩ.
Intermediate 50
To a solution of intermediate 49 (939 mg, 2.29 mmol) in
DMF (10 mL) under nitrogen was added sodium hydride
(60% in mineral oil, 96 mg, 2.4 mmol) in one portion and
the solution was stirred until hydrogen evolution had ceased
(about 15 min). To the dark yellow solution was added ethyl
bromoacetate (381 ␮L, 3.44 mmol) and the mixture stirred
for a further 60 min. The reaction was diluted with ethyl
acetate and washed successively with saturated NH4Cl,
water, and brine, dried over MgSO4, filtered through a plug
of silica, and evaporated. The desired material, purified on
silica gel using a 5%–20% ethyl acetate/hexane gradient,
was obtained as a light yellow gum (877 mg, 77% yield;
98% homogeneity). 1H NMR (400 MHz, DMSO-d6) ␦: 8.04
(s, 1H), 7.90 (d, 1H, J ϭ 1.4 Hz), 7.88–7.83 (m, 2H), 7.66
(dd, 1H, J ϭ 1.1, 8.5 Hz), 6.55 (d, 1H, J ϭ 0.8 Hz), 4.96 (s,
2H), 4.39 (t, 2H, J ϭ 8.0 Hz), 4.09 (q, 2H, J ϭ 7.0 Hz),
2.73–2.64 (m, 1H), 1.94–1.66 (m, 7H), 1.45–1.20 (m, 3H),
1.20–1.03 (m, 6H), 0.07 (s, 9H).
Compound 26
To a solution of intermediate 53 (15 mg, 0.031 mmol) in
DMSO (1.0 mL) were added O-(benzotriazol-1-yl)-N,N,N=,N=-
tetramethyluronium tetrafluoroborate (TBTU; 12 mg,
0.037 mmol), TEA (13 ␮L, 0.093 mmol), and N-
(2-methoxyethyl)methyl amine (5 ␮L, 0.047 mmol). This mix-
ture was stirred overnight at RT, following which TFA
(12 ␮L) was added to acidify. The desired material was
isolated and purified using RP-prep HPLC using an ACN/
water gradient incorporating 0.1% TFA as buffer. Following
collection, pooling, and lyophilization of relevant fractions,
compound 26 was obtained (14 mg, 82% yield) as a white
Intermediate 51
Trifluoroacetic acid (2.0 mL) was added to a solution of
compound 50 (850 mg, 1.72 mmol) in DCM (10.0 mL) and
this was stirred at RT overnight. Volatiles were evaporated
under reduced pressure. Solid residues were suspended in
diethyl ether and stirred briefly. Solids were filtered, washed
with more diethyl ether, then air-dried. Intermediate 51
(464 mg, 68% yield; 99% homogeneity) was obtained as an
off-white powder. 1H NMR (400 MHz, DMSO-d6) ␦: 12.60 (s,
1H), 8.04 (s, 1H), 7.89 (d, 1H, J ϭ 1.6 Hz), 7.86 (s, 1H), 7.82
(d, 1H, J ϭ 8.4 Hz), 7.65 (dd, 1H, J ϭ 1.1, 8.3 Hz), 6.55 (d,
1H, J ϭ 1.0 Hz), 4.96 (s, 2H), 4.09 (q, 2H, J ϭ 7.2 Hz), 2.68
(m, 1H), 1.94–1.64 (m, 7H), 1.40–1.22 (m, 3H), 1.14 (t, 3H,
J ϭ 7.2 Hz). Flow-injection analysis (FIA)-MS (APPIϩ) m/z:
396.2 [M ϩ H]ϩ.
1
amorphous solid. H NMR (mixture of rotamers; 400 MHz,
DMSO-d6) ␦: 11.40–11.25 (two s, 1H), 8.01–7.73 (m, 4H),
7.58 (dd, 1H, J ϭ 1.4, 8.4 Hz), 6.54–6.48 (m, 1H), 5.07–4.94
(two s, 2H), 3.60–3.35 (m, 4H), 3.33–2.81 (four s, 6H),
2.78–2.66 (m, 1H), 1.99–1.66 (m, 7H), 1.44 (s, 9H), 1.33 (m,
3H). 13C NMR (100 MHz, DMSO-d6) ␦: 167.7, 167.3, 166.2,
166.0, 144.0, 143.9, 142.2, 136.6, 132.2, 131.9, 129.3, 129.2,
124.7, 124.6, 119.5, 119.4, 119.3, 119.2, 119.1, 118.6, 115.2,
115.1, 112.0, 111.8, 111.2, 69.3, 69.2, 61.3, 58.5, 58.0, 48.0,
46.9, 45.2, 36.3, 34.9, 33.2, 32.7, 26.6, 25.6, 24.2. HR-MS
(APPIϩ) calcd for C29H39N3O6SH [M ϩ H]ϩ: 558.2632;
found: 558.2633.
Intermediate 52
To a solution of compound 51 (448 mg, 1.13 mmol) in
DCM (10 mL) at RT was added a 2.0 mol/L solution of
oxalyl chloride in DCM (1.13 mL, 2.27 mmol) and DMF
(10 ␮L). After 1 h, volatiles were removed under reduced
pressure. The crude acid chloride intermediate was redis-
solved in DCM (10 mL) and tert-butyl sulfonamide
(233 mg, 1.70 mmol), DMAP (138 mg, 1.13 mmol), and
TEA (474 ␮L, 3.40 mmol) were added. The resultant mix-
Compounds 22–25 and 27–28 were prepared in a similar
fashion.
General procedure for the preparation of compounds 34,
35, 40, 41, 46, and 47
Published by NRC Research Press

76
Can. J. Chem. Vol. 91, 2013
Intermediate 54
Intermediate 56
To a solution of compound 616b (50.9 g, 151 mmol) in
DMF (300 mL) under nitrogen and cooled to 0 °C was
added sodium hydride (60% in mineral oil, 9.09 g,
227 mmol) in one portion. This was stirred for 30 min then
tert-butyl bromoacetate (33.5 mL, 227 mmol) was added
and the reaction mixture was allowed to warm to room
temperature and stirred for a further 3 h. After quenching
with acetic acid, the product was partitioned between
EtOAc and water. The organic portion was dried over
Na2SO4, filtered, and evaporated under reduced pressure.
Compound 54 was purified by repeated trituration with
dichloromethane/hexane mixtures to afford a yellow solid
(55.5 g, 82% yield). 1H NMR (400 MHz, DMSO-d6) ␦: 8.15
(d, 1H, J ϭ 1.2 Hz), 7.84 (d, 1H, J ϭ 8.6 Hz), 7.68 (dd, 1H,
J ϭ 1.2, 8.6 Hz), 5.11 (s, 2H), 3.88 (s, 3H), 2.94–2.79 (m,
1H), 2.02–1.81 (m, 4H), 1.80–1.64 (m, 3H), 1.48–1.33 (m,
12H). 13C NMR (100 MHz, DMSO-d6) ␦: 167.2, 166.9,
136.3, 129.0, 122.6, 120.0, 119.9, 118.9, 116.0, 111.9, 81.8,
51.9, 46.9, 37.1, 31.7, 27.6, 26.5, 25.5. HR-MS (APPIϩ)
calcd for C22H28BrNO4Na [M ϩ Na]ϩ: 472.1094; found:
472.1084.
Compound 55 from the previous step (15.7 g, 58.7 mmol)
was dissolved in THF (150 mL) and cooled to –78 °C. n-Butyl
lithium (2.3 mol/L in hexanes, 26.8 mL, 61.6 mmol) was
added dropwise. Following this addition, the reaction was
stirred for 40 min and then triisopropylborate (27.0 mL,
117 mmol) was added dropwise. This mixture was stirred for
30 min at –78 °C, then warmed to room temperature over a
period of 3 h. Compound 54 was added as a solid to the
reaction mixture (13.2 g, 29.3 mmol) followed by Pd2(dba)3
(537 mg, 0.587 mmol) and P(2-furyl)3 (817 mg, 3.52 mmol).
Solid potassium phosphate (31.1 g, 146 mmol) was dissolved
in predegassed water (150 mL) and was added, followed by
dimethoxyethane (DME; 300 mL). The mixture was warmed
to reflux and stirred for 10–12 h. The reaction was cooled to
room temperature and concentrated under reduced pressure.
The product was extracted with EtOAc. The combined organic
portions were washed with brine and dried over Na2SO4,
filtered, and evaporated under reduced pressure. Purification
on silica gel, eluting with 5:1 hexane/EtOAc, afforded com-
pound 56 as a light-yellow solid (7.64 g) that was used for the
next step.
Intermediate 55
Intermediate 57
4-Bromo-3-fluorophenol (25.0 g, 131 mmol) was dis-
solved in DMSO (330 mL). 2-Fluoropyridine (14.3 mL,
196 mmol) was added, followed by potassium carbonate
(45.3 g, 328 mmol). The reaction mixture was heated to
120 °C and stirred at this temperature for 30 h. After
cooling to room temperature, water was added and the
compound extracted with EtOAc. The organic portion was
dried over Na2SO4, filtered, and evaporated under reduced
pressure. Purification on silica gel and elution with 10:1
hexane/EtOAc provided compound 55 as a light-yellow oil
(24.6 g, 70% yield; 95% homogeneity) that was used di-
rectly in the next step.
Ester 56 (7.64 g, 13.7 mmol) from the previous step was
added to 4 mol/L HCl/1,4-dioxane (120 mL) and the mixture
was stirred for 3 h at room temperature. The resulting heter-
ogeneous mixture was filtered and the solids washed with
dichloromethane and dried under high vacuum. This material
was resuspended in 10:1 dichloromethane/methanol, sonicated
for 30 min, and then filtered, washed with dichloromethane,
and dried. This procedure was repeated once more to provide
compound 57 as an off-white solid (3.87 g, 26% overall yield
1
from bromoindole 54). H NMR (400 MHz, DMSO-d6) ␦:
8.32–8.27 (m, 1H), 8.10 (d, 1H, J ϭ 1.2 Hz), 8.00–7.93 (m,
1H), 7.90 (d, 1H, J ϭ 8.6 Hz), 7.71 (dd, 1H, J ϭ 1.4, 8.4 Hz),
Published by NRC Research Press

Beaulieu et al.
77
7.38 (t, 1H, J ϭ 8.6 Hz), 7.32 (dd, 1H, J ϭ 2.3, 10.6 Hz),
7.29–7.24 (m, 1H), 7.22–7.13 (m, 2H), 4.93 (d, 1H, J ϭ
18.0 Hz), 4.68 (d, 1H, J ϭ 18.0 Hz), 3.87 (s, 3H), 2.50 (m,
1H), 1.92–1.61 (m, 7H), 1.39–1.17 (m, 3H). 13C NMR (100
MHz, DMSO-d6) ␦: 169.8, 167.1, 161.9, 161.5, 159.0, 156.2,
156.1, 147.6, 140.7, 136.5, 133.4, 133.3, 132.8, 129.4, 122.6,
120.4, 120.1, 119.9, 119.6, 116.9, 114.0, 113.8, 112.4, 112.2,
108.9, 108.6, 51.9, 45.2, 36.5, 32.6, 32.5, 26.6, 26.5, 25.5.
HR-MS (APPIϩ) calcd for C29H27FN2O5H [M ϩ H]ϩ:
503.1977; found: 503.1981.
temperature, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 62 mg,
0.41 mmol) in THF (0.5 mL) and cyclopropylsulfonamide
(5.8 mg, 0.048 mmol) in THF (0.5 mL) were added. This mixture
was heated to 125 °C for 60 min in a sealed tube. The volatiles
were then evaporated under reduced pressure and the residue
dissolved in DMSO (2 mL). The desired material was isolated
and purified using RP-prep HPLC using an ACN/water gradient
incorporating 0.1% TFA as buffer. Following collection, pooling,
and lyophilization of relevant fractions, compound 34 was ob-
Intermediate 58a
To a solution of compound 57 (500 mg, 0.995 mmol) in
DMSO (5.0 mL) was added 4-(diethylamino)piperidine a
(187 mg, 1.19 mmol) and TEA (432 ␮L, 3.10 mmol). Solid
TBTU (415 mg, 1.29 mmol) was added in one portion and
the mixture stirred at room temperature overnight. The
mixture was diluted with tert-butyl methyl ether (t-BME;
50 mL) and 1 mol/L NaOH (45 mL) was added. The phases
were separated. The aqueous layer was further extracted
with t-BME (25 mL). The organic portions were combined,
washed with water and brine, dried over Na2SO4, filtered,
and evaporated to give a white foam (600 mg). This mate-
rial was subsequently dissolved in DMSO (5 mL) and 10
mol/L NaOH (1.0 mL, 10 mmol) was added dropwise. The
mixture was warmed to 50 °C and stirred for 30–60 min,
then cooled to room temperature. Water (15 mL) was
added, followed by the dropwise addition of 1.0 mol/L HCl
until pH 4–5. During this process, a very fine white sus-
pension was formed. The suspension was transferred to a
centrifuge tube of appropriate volume and spun in a bench-
top centrifuge at 3200 rpm for 5 min. The supernatant was
removed and discarded. Water (15 mL) was added to the
sediment, which was resuspended, and the mixture spun
again. The supernatant was discarded and the procedure
repeated once more. The wet solids were collected, dis-
solved in EtOH (30 mL), evaporated under reduced pres-
sure, then dried at 50 °C under a high vacuum to afford
compound 58a (500 mg, 81% overall yield) that was used
directly for inhibitor synthesis.
1
tained (43 mg) as a white amorphous bis-TFA salt. H NMR
(400 MHz, DMSO-d6) ␦: 11.92–11.86 (m, 1H), 9.12–9.00 (br s,
1H), 8.27 (dd, 1H, J ϭ 1.5, 4.8 Hz), 8.07 (d, 1H, J ϭ 14.7 Hz),
7.96 (ddd, 1H, J ϭ 2.0, 7.3, 8.4 Hz), 7.89 (d, 1H, J ϭ 8.6 Hz),
7.66 (dd, 1H, J ϭ 1.4, 8.5 Hz), 7.45–7.23 (m, 3H), 7.23–7.10 (m,
2H), 5.26–5.09 (m, 1H), 4.86–4.64 (m, 1H), 4.38 (m, 1H),
4.0–3.7 (m, 2H), 3.61 (br s, 1H), 3.25–2.85 (m, 6H), 2.64 (m,
1H), 2.08–1.61 (m, 9H), 1.42–1.06 (m, 15H). 13C NMR (100 MHz,
DMSO-d6) ␦: 166.7, 165.5, 165.3, 162.1, 158.3, 157.9, 147.6,
140.7, 136.9, 133.4, 133.0, 129.4, 124.1, 120.4, 120.1,
120.0, 119.8, 118.7, 115.0, 112.3, 112.1, 111.7, 58.2, 44.4,
44.3, 42.7, 36.5, 32.6, 32.4, 31.0, 26.6, 25.7, 25.5, 9.9, 9.8,
9.7, 5.60. HR-MS (APPIϩ) calcd for C40H48FN5O5SH [M ϩ
H]ϩ: 730.3433; found: 730.3425.
Inhibitor 41
In a similar manner to that for intermediate 34, 41 was
made using compound 58b (53 mg, 0.085 mmol) and
N,N-dimethylsulfamide (5.9 mg, 0.048 mmol), heating the
mixture to 125 °C for 120 min in a sealed tube. Inhibitor 41
(50 mg) was obtained as a white amorphous bis-TFA salt.
1H NMR (400 MHz, DMSO-d6) ␦: 11.62 (s, 1H), 9.75 (br s,
1H), 8.32–8.24 (m, 1H), 8.06 (s, 1H), 7.96 (ddd, 1H, J ϭ
2.1, 7.2, 8.3 Hz), 7.87 (d, 1H, J ϭ 8.4 Hz), 7.67 (dd, 1H,
J ϭ 1.4, 8.5 Hz), 7.41–7.28 (m, 2H), 7.26 (ddd, 1H, J ϭ 0.9,
4.9, 7.2 Hz), 7.17 (d, 2H, J ϭ 8.1 Hz), 5.33–5.07 (m, 1H),
4.95–4.72 (m, 1H), 4.46–4.33 (m, 1H), 4.19 (d, 1H, J ϭ
13.6 Hz), 3.59–3.24 (m, 4H), 3.05–2.76 (m, 8H), 1.91–1.54
(m, 7H), 1.43–1.09 (m, 9H). 13C NMR (100 MHz, DMSO-
d6) ␦: 166.4, 162.0, 161.4, 159.0, 158.7, 158.4, 158.0,
Intermediate 58b
In a manner similar to that for intermediate 58a, com-
pound 57 (500 mg, 0.995 mmol) and n-isopropyl piperazine
b (153 mg, 1.19 mmol) provided compound 58b (490 mg,
82% overall yield) that was used directly for inhibitor
synthesis.
Inhibitor 34
To a solution of compound 58a (53 mg, 0.085 mmol) in THF
(2.5 mL) was added carbonyldiimidazole (55 mg, 0.34 mmol)
and this was heated at 50 °C for 1 h. After cooling to room
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78
Can. J. Chem. Vol. 91, 2013
156.1, 156.0, 147.6, 140.7, 136.9, 133.4, 129.2, 124.3,
120.3, 120.1, 119.7, 118.7, 117.9, 116.7, 114.9, 111.6, 57.4,
47.5, 38.0, 36.6, 32.6, 32.4, 26.7, 26.6, 25.5, 16.2. HR-MS
(APPIϩ) calcd for C37H45FN6O5SH [M ϩ H]ϩ: 705.3229;
found: 705.3246.
In a similar fashion, inhibitors 35, 40, 46, and 47 were made
from the appropriated starting materials.
General procedure for the preparation of 30, 31, 36, 37,
42, and 43
methyl morpholine and the methyl ester saponified to pro-
vide the desired indole carboxylic acid intermediate as a
white solid in an 80% yield. Coupling, under conditions
described for 34, with cyclopropylsulfonamide and
N,N-dimethylsulfamide provided inhibitors 42 and 43.
Intermediate 59
Bromoindole 54 (8.00 g, 17.8 mmol) and 4-chloro-3-
fluoro-phenylboronic acid (4.65 g, 26.6 mmol) were dis-
solved in dioxane (200 mL) and 2 mol/L Na2CO3 (32 mL,
64 mmol) was added. Argon gas was bubbled through the
stirred mixture for 1 h. PdCl2(PPh3)2 (630 mg, 0.89 mmol)
was added and bubbling continued for a further 10 min. The
mixture was heated to 100 °C and stirred for 12 h under an
argon atmosphere. The reaction mixture was then cooled to
room temperature and volatiles evaporated under reduced
pressure. Ethyl acetate (200 mL) was added and the layers
separated. The organic portion was washed with brine,
dried over Na2SO4, filtered, and evaporated under reduced
pressure. The compound was partially purified on silica gel,
eluting with 10:1 hexane/EtOAc. The collected material
was triturated with EtOAc/hexane mixtures, filtered, and
dried under high vacuum to afford compound 59 has an
off-white solid (6.65 g) that was used directly in the next
step.
Inhibitor 42
1H NMR (400 MHz, DMSO-d6) ␦: 12.02–11.76 (m, 1H),
9.21 (br s, 1H), 8.04 (s, 1H), 7.89 (d, 1H, J ϭ 8.6 Hz), 7.78
(s, 1H), 7.67 (d, 1H, J ϭ 8.6 Hz), 7.39 (d, 1H, J ϭ 9.5 Hz),
7.21 (d, 1H, J ϭ 7.0 Hz), 5.15–4.90 (m, 2H), 4.31–4.10 (m,
1H), 4.00–3.80 (m, 2H), 3.72–3.25 (m, 3H), 3.23–3.06 (m,
7H), 3.02–2.77 (m, 1H), 2.63–2.52 (m, 1H), 1.97–1.60 (m,
7H), 1.45–0.97 (m, 13H). 13C NMR (100 MHz, DMSO-d6)
␦: 166.7, 166.4, 166.3, 158.2, 158.1, 157.9, 155.6, 136.6,
130.9, 129.4, 127.9, 124.4, 120.0, 120.2, 120.0, 119.4,
118.9, 118.0, 111.7, 70.2, 65.8, 52.4, 48.0, 46.5, 45.0, 36.1,
32.7, 31.0, 26.5, 25.5, 8.7, 8.5, 8.2, 8.1, 5.6. HR-MS
(APPIϩ) calcd for C35H44ClFN4O5SH [M
687.2778; 687.2788.
ϩ
H]ϩ:
Intermediate 60
Ester 59 (6.65 g, 13.3 mmol) from the previous step was
deprotected to give compound 60 in a manner similar to that
described for compound 57. Compound 60 was obtained as
a white solid (5.34 g, 67% overall yield from bromoindole
Inhibitor 43
1H NMR (400 MHz, DMSO-d6) ␦: 11.73–11.50 (m, 1H),
9.36–9.16 (m, 1H), 8.05 (br s, 1H), 7.88 (d, 1H, J ϭ
8.6 Hz), 7.84–7.74 (m, 1H), 7.68 (d, 1H, J ϭ 8.4 Hz), 7.38
(d, 1H, J ϭ 9.2 Hz), 7.20 (d, 1H, J ϭ 7.3 Hz), 5.20–4.86
(m, 2H), 4.31–4.08 (m, 1H), 4.04–3.81 (m, 2H), 3.75–3.25
(m, 3H), 3.23–3.03 (m, 6H), 3.02–2.74 (m, 7H), 2.63–2.53
(m, 1H), 1.94–1.60 (m, 7H), 1.43–1.06 (m, 9H). 13C NMR
(100 MHz, DMSO-d6) ␦: 166.5, 166.4, 166.3, 158.2, 158.1,
157.8, 155.6, 137.7, 136.7, 132.2, 132.1, 129.2, 127.9,
124.5, 124.4, 120.2, 120.0, 119.3, 118.9, 111.5, 70.2, 65.8,
52.4, 51.7, 48.3, 48.0, 46.5, 45.0, 38.0, 36.1, 32.7, 26.5,
25.5, 8.7, 8.5, 8.2, 8.1. HR-MS (APPIϩ) calcd for
C34H45ClFN5O5SH [M ϩ H]ϩ: 690.2887; found: 690.2901.
Inhibitors 30, 31, 36, and 37 were prepared in a similar
fashion.
1
54). H NMR (400 MHz, DMSO-d6) ␦: 13.06 (br s, 1H),
8.09 (d, 1H, J ϭ 1.2 Hz), 7.91 (d, 1H, J ϭ 8.2 Hz), 7.80 (t,
1H, J ϭ 8.2 Hz), 7.71 (dd, 1H, J ϭ 1.6, 8.6 Hz), 7.42 (dd,
1H, J ϭ 2.0, 9.8 Hz), 7.23 (dd, 1H, J ϭ 1.6, 8.2 Hz), 4.83
(s, 2H), 3.89 (s, 3H), 2.62–2.54 (m, 1H), 1.96–1.62 (m,
7H), 1.40–1.13 (m, 3H). 13C NMR (100 MHz, DMSO-d6)
␦: 170.3, 167.0, 158.1, 155.7, 137.4, 136.3, 131.9, 131.8,
131.0, 129.4, 127.9, 127.8, 122.7, 120.5, 120.3, 120.1,
119.8, 119.5, 118.8, 118.6, 112.0, 51.9, 45.3, 36.1, 32.7,
26.5, 25.5. HR-MS (APPIϩ) calcd for C24H23ClFNO4H [M
ϩ H]ϩ: 444.1372; found: 444.1374.
Inhibitors 42 and 43
In a manner similar to that for compounds 58a and 58b,
carboxylic acid 60 was coupled to racemic 2-(diethylamino)
General procedure for the preparation of 29, 32, 33, 38,
39, 44, and 45
Published by NRC Research Press

Beaulieu et al.
79
157.1, 156.1, 139.9, 136.4, 132.1, 130.2, 129.6, 126.0, 124.0,
123.7, 119.7, 119.0, 118.7, 118.6, 118.4, 111.5, 58.3, 45.0, 44.4,
44.3, 40.3, 36.1, 32.8, 31.0, 26.6, 26.4, 25.7, 25.5, 9.9, 9.8, 5.6.
HR-MS (APPIϩ) calcd for C41H50N4O5SH [M ϩ H]ϩ:
711.3575; 711.3585.
Intermediate 61
In a similar manner to that described for intermediate
59, bromoindole 54 (10.0 g, 22.2 mmol) and 4-
phenoxyphenylboronic acid (5.20 g, 24.4 mmol) were dis-
solved in DME (220 mL) and the mixture degassed for 30 min
with bubbling argon gas. Pd2(dba)3 (330 mg, 0.355 mmol) and
P(2-furyl)3 (620 mg, 2.66 mmol) were added and degassing
continued for another 15 min. Solid potassium phosphate
(18.8 g, 88.8 mmol) was dissolved in predegassed water
(110 mL) and this solution was added. The reaction was heated
to 85 °C and stirred at this temperature for 16–20 h. After
cooling to room temperature and dilution with EtOAc, the
organic layers were separated, washed with brine, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
product was purified on silica using 80:20 hexane/EtOAc as
eluent. Compound 61 was obtained as a yellow oil (10.8 g)
that was used directly in the next step.
Inhibitor 39
1H NMR (400 MHz, DMSO-d6) ␦: 11.60 (br s, 1H), 9.90 (br
s, 1H), 8.02 (d, 1H, J ϭ 1.2 Hz), 7.86 (d, 1H, J ϭ 8.6 Hz), 7.68
(dd, 1H, J ϭ 1.6, 8.6 Hz), 7.51–7.43 (m, 2H), 7.34 (m, 2H),
7.27–7.20 (m, 1H), 7.20–7.10 (m, 4H), 5.01 (d, 2H, J ϭ
8.2 Hz), 4.45 (d, 1H, J ϭ 11.7 Hz), 4.19 (d, 1H, J ϭ 11.7 Hz),
3.65–3.31 (m, 4H), 3.08–2.84 (m, 8H), 2.75 (br s, 1H), 2.69–
2.56 (m, 1H), 1.97–1.54 (m, 7H), 1.42–1.12 (m, 9H). 13C
NMR (100 MHz, DMSO-d6) ␦: 166.4, 166.3, 158.3, 158.0,
157.2, 156.0, 139.6, 136.5, 132.1, 130.2, 129.5, 125.8, 124.0,
123.9, 119.6, 119.1, 118.7, 118.6, 118.2, 111.5, 57.4, 47.5,
47.3, 44.8, 41.5, 38.0, 36.1, 32.8, 26.6, 25.5, 16.2. HR-MS
(APPIϩ) calcd for C38H47N5O5SH [M ϩ H]ϩ: 686.3371;
found: 686.3388.
Intermediate 62
Compound 61 (10.8 g) from the previous step was con-
verted to compound 62 in a manner similar to that for com-
pound 60. Compound 62 was obtained as a white solid (5.30 g,
49% overall yield from bromoindole 54). 1H NMR (400 MHz,
DMSO-d6) ␦: 13.03 (br s, 1H), 8.04 (s, 1H), 7.88 (d, 1H, J ϭ
8.2 Hz), 7.70 (dd, 1H, J ϭ 1.4, 8.4 Hz), 7.52–7.44 (m, 2H),
7.39–7.33 (m, 2H), 7.27–7.21 (m, 1H), 7.20–7.12 (m, 4H),
4.79 (s, 2H), 3.88 (s, 3H), 2.60 (m, 1H), 1.93–1.65 (m, 7H),
1.39–1.15 (m, 3H). 13C NMR (100 MHz, DMSO-d6) ␦: 170.2,
167.1, 157.4, 155.8, 139.5, 136.1, 132.0, 130.2, 129.7, 125.4,
124.2, 122.2, 119.8, 119.6, 119.4, 118.9, 118.0, 111.9, 51.8,
45.3, 36.1, 32.8, 26.6, 25.5. HR-MS (APPIϩ) calcd for
C30H29NO5H [M ϩ H]ϩ: 484.2119; found: 484.2120.
In a similar fashion, compounds 29, 33, 38, 44, and 45 were
prepared from the appropriate starting materials.
X-ray crystallography
Protein crystallization
HCV strain 1b/J4 NS5B polymerase was purified as a
protein construct that has the C-terminal 21 residues truncated
and the C-terminal addition of a hexahistidine tag (NS5B⌬21).
The protein was concentrated to 7.7 mg/mL and crystallized
by the hanging drop vapor diffusion procedure using
monomethyl ether polyethylene glycol as a precipitant
(PEG5Kmme). In particular, 1 ␮L of NS5B⌬21 (in purifi-
cation buffer (20 mmol/L tris pH 7.3, 300 mmol/L NaCl,
and 10% glycerol)) was add to 1 ␮L of a solution made of
21% PEG5Kmme, 0.1 mol/L 2-(N-morpholino)ethanesul-
fonic acid (MES) pH 5.4, 10% glycerol, and 0.4 mol/L
ammonium sulfate. The resulting 2 ␮L drop was suspended
above a 1 mL reservoir solution made of 21% PEG5Kmme,
0.1 mol/L MES pH 5.4, 10% glycerol, and 0.4 mol/L
ammonium sulfate.
Inhibitors 32 and 39
In the same manner as 42 and 43, compound 62 was
elaborated using appropriate reagents to provide the desired
inhibitors 32 and 39 were made using cyclopropylsulfonamide
and N,N-dimethylsulfamide, respectively, and isolated as their
TFA salts.
Inhibitor 32
1H NMR (400 MHz, DMSO-d6) ␦: 11.85 (s, 1H), 9.10 (br s,
1H), 8.01 (d, 1H, J ϭ 1.3 Hz), 7.87 (d, 1H, J ϭ 8.6 Hz), 7.66
(dd, 1H, J ϭ 1.4, 8.5 Hz), 7.50–7.43 (m, 2H), 7.35 (m, 2H),
7.26–7.16 (m, 3H), 7.15–7.08 (m, 2H), 4.94 (s, 2H), 4.43 (d,
1H, J ϭ 12.5 Hz), 4.03 (d, 1H, J ϭ 12.5 Hz), 3.71–3.55 (m,
1H), 3.28–3.05 (m, 4H), 3.04–2.89 (m, 1H), 2.74–2.57 (m,
2H), 2.07–1.64 (m, 8H), 1.55–1.03 (m, 14H). 13C NMR
(100 MHz, DMSO-d6) ␦: 166.7, 165.8, 158.3, 158.0, 157.6,
Inhibitor soaking
Large single apo NS5B⌬21 crystals were soaked in a
solution containing 1 mmol/L of the inhibitor for 5–6 h and
then flash frozen in liquid nitrogen prior to diffraction data
collection.
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80
Can. J. Chem. Vol. 91, 2013
time–concentration curve with r2 values Ͼ0.99 and a limit of
detection (LD) Յ10 ng/mL. The temporal profiles of drug
concentrations in plasma were analyzed by noncompartmental
methods using WinNonlin (version 3.1; Scientific Consulting,
Inc., Cary, North Carolina).
For liver exposure studies, liver samples were collected 2 h
post the oral dose as follows: The rat liver was perfused with
25 mL ice-cold saline for 1 min prior to collection of the right
lateral lobe. Both plasma and liver samples were extracted and
analyzed by HPLC or LC/MS/MS as previously described.
Data collection and processing
Diffractions were collected on a MicroMax-007 rotating
anode X-ray generator equipped with a Raxis-IVϩϩ image
plate detector (Rigaku/MSC, USA). Data, to a resolution of
2.8 Å, was collected on a single crystal cryogenically cooled
at –180 °C and processed with HKL-2000 software (HKL
Research, USA).
Structure modeling and refinement
Phasing of the experimental data was done by molecular
replacement using a published structure of apo NS5B
(1C2J.pdb). Rotation and translation search as well as further
structure refinement were done using the program CNX (Ac-
celrys, USA). Model building was carried out with software O
(Alwyn Jones, Upsala University, Sweden). The final model
includes two molecules of NS5B (residues A1–A148, A153–
A563, B1–B17, B36–B148, and B153–B563) and one ligand
molecule associated with NS5B molecule B. The final crys-
tallographic R factor is 21.9% and the Rfree factor is 25.7% to
a resolution of 2.7 Å. Data processing and model refinement
statistics are included in the Supplementary data section. The
coordinates and structure factors for the NS5B polymerase in
complex with compound 12 have been deposited in the Protein
Data Bank as entry 4GMC.
Supplementary data
Suppmentary data (proton and mass spectral data for
all inhibitors and data processing and model refinement sta-
tistics for the NS5B – inhibitor 12 complex) are available
with the article through the journal Web site at http://
nrcresearchpress.com/doi/suppl/10.1139/cjc-2012-0319.
Acknowledgments
We thank the following colleagues from Boehringer Ingel-
heim (Canada) who participated in generating the data pre-
sented in this paper: N. Dansereau, L. Lagacée, M. Marquis,
and C. Pellerin for IC50 and EC50 determinations; J. De Marte,
F. Liard, H. Montpetit, and C. Zouki for ADME-PK profiling;
N. Aubry for NMR support; and S. Bordeleau, C. Boucher,
and S. Valois for analytical support.
Biological testing
Inhibition of HCV NS5B⌬21 enzymatic activity was per-
formed as previously described.11a,20 The bicistronic lu-
ciferase reporter replicon, encoding the Con1 genotype 1b
NS2-NS5B coding region, and the experimental procedures
for measuring EC50 values in the experiments reported within
this article have been described elsewhere.11a Compounds
were incubated with cells for 72 h and the relative levels of
luciferase present were determined using the Bright-Glo lu-
ciferase substrate (Promega) on a Packard Topcount instru-
ment. EC50 values were determined by the nonlinear
regression routine NLIN procedure of SAS (EC50). All re-
ported values are the average of at least Ն2 measurements.
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