Aza follow-ups to BI 207524, a thumb pocket 1 HCV NS5B polymerase inhibitor. Part 1: Mitigating the genotoxic liability of an aniline metabolite

Article abstract of DOI:10.1016/j.bmcl.2014.12.028

A series of heterocyclic aza-analogs of BI 207524 (2), a potent HCV NS5B polymerase thumb pocket 1 inhibitor, was investigated with the goal to reduce the liability associated with the release of a genotoxic aniline metabolite in vivo. Analog 4, containing a 2-aminopyridine aniline isostere that is negative in the Ames test was identified, and was found to provide comparable GT1a/1b potency to 2. Although the cross-species PK profile, poor predicted human liver distribution of analog 4 and allometry principles projected high doses to achieve a strong antiviral response in patients, this work has provided a path forward toward the design of novel thumb pocket 1 NS5B polymerase inhibitors with improved safety profiles.

Full text of DOI:10.1016/j.bmcl.2014.12.028

Bioorganic & Medicinal Chemistry Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Aza follow-ups to BI 207524, a thumb pocket 1 HCV NS5B polymerase
inhibitor. Part 1: Mitigating the genotoxic liability of an aniline
metabolite
b
a
a
b
Pierre L. Beaulieu a, , Gordon Bolger , Martin Duplessis , Alexandre Gagnon , Michel Garneau ,
⇑
Timothy Stammers a, George Kukolj b, Jianmin Duan b
a Medicinal Chemistry Department, Boehringer Ingelheim (Canada) Ltd, Research and Development, 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
b Biology Department, Boehringer Ingelheim (Canada) Ltd, Research and Development, 2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of heterocyclic aza-analogs of BI 207524 (2), a potent HCV NS5B polymerase thumb pocket 1
inhibitor, was investigated with the goal to reduce the liability associated with the release of a genotoxic
aniline metabolite in vivo. Analog 4, containing a 2-aminopyridine aniline isostere that is negative in the
Ames test was identified, and was found to provide comparable GT1a/1b potency to 2. Although the
cross-species PK profile, poor predicted human liver distribution of analog 4 and allometry principles pro-
jected high doses to achieve a strong antiviral response in patients, this work has provided a path forward
toward the design of novel thumb pocket 1 NS5B polymerase inhibitors with improved safety profiles.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 26 October 2014
Revised 6 December 2014
Accepted 9 December 2014
Available online xxxx
Keywords:
Hepatitis C virus
NS5B polymerase
Antiviral
Genotoxicity
Ames
In the last years, significant breakthroughs have occurred in the
treatment and cure of disease conditions associated with chronic
hepatitis C virus infection (HCV). The first Direct Acting Antivirals
(DAAs) that enhance efficacy and tolerability of pegylated-inter-
feron/ribavirin based therapies have reached the market and are
providing patients with sustained viral responses (SVR) and cure
in up to 80–90% of genotype 1 (GT1) treatment naïve patients.1
More recently, IFN-free regimens combining two or three DAAs
with complementary modes of actions have shown potential to
further improve cure rates (SVR >90%) and tolerability and are
showing improved efficacy in the harder-to-treat GT1a and cir-
rhotic populations.2,3 To this end, we have been pursuing allosteric
inhibitors of the HCV NS5B polymerase as partners in IFN-free
combination therapy (e.g., with protease inhibitor faldaprevir).4
We have described the discovery and progression to the clinic of
indole-based inhibitors that bind to the thumb pocket 1 allosteric
site of NS5B and prevent inter-domain interactions between the
thumb and finger regions of the protein, resulting in a replica-
tion-deficient enzyme. Proof of concept for this class of inhibitors
was achieved with BILB 1941 (1 Fig. 1) that produced up to
2.5log10 reductions in viremia in HCV GT1 infected patients but
was discontinued because of gastrointestinal intolerance at doses
required to maintain a strong antiviral response (>450 mg TID).5
BI 207524 (2) is a follow-up compound from this class with
improved potency and a cross-species PK profile consistent with
achieving a robust antiviral response at a reduced dose (400 mg
BID), based on our recently published liver-corrected inhibitory
quotient model (LCIQ).6 In this model, inhibitor liver Ctrough con-
centrations corresponding to P500-fold the EC50 are predicted to
provide a strong antiviral response in infected patients.7
During preclinical development of 2, a genotoxic aniline metab-
olite (3, X = Y = Z = CH) was detected in both human and rat liver
microsome (ꢀ1 ppm/min in HLM in an NADPH-independent man-
ner) and simulated gastric fluid (SGF) incubation studies and
in vivo in rats, thus compromising further progression of this can-
didate.6 A well established medicinal chemistry strategy to address
such a liability consists of replacing the aniline with isosteres (e.g.,
heterocycles) that can modulate the binding affinity of the aro-
matic amine to the CYP1A2 enzyme cavity and stabilize anionic
forms implicated in the formation and stability of the reactive
nitrenium ion that is formed upon metabolic activation.8 The suit-
ability of the replacements can be assessed using computational
methods and verified by testing in the Ames test. One of the strat-
egies implemented in our program to resolve the genotoxic liability
of 2, involved replacement of the right-hand-side 4-amino-2-
ethoxycinnamic acid moiety in 2 by nitrogen-containing isosteres
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2014.12.028
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Beaulieu, P. L.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.028

2
P.L. Beaulieu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
tert-butylacrylate under the usual conditions provided cinnamate
O
H
N
19 that was used to prepare inhibitor 6. Commercially available
2-aminopyrimidine 20 was acetylated and chlorinated to provide
4-chloropyrimidine 21. Treatment with sodium ethoxide resulted
in simultaneous deprotection of the amine function and displace-
ment of the chloro group with ethoxy. The ester functionality
was subsequently reduced to the corresponding benzylic alcohol
that was then oxidized to provide aldehyde 22. Aldehyde 22 was
converted to cinnamate 23 using a Horner–Wadsworth–Emmons
condensation and elaborated to inhibitor 7. Chloropyridazine 24
was prepared in four steps following a literature procedure.11
The ester function was reduced to the corresponding aldehyde
using DIBAL which was homologated to cinnamate ester 25
through a Horner–Wadsworth–Emmons condensation. The chlo-
rine atom was converted to an amine by sequential azide displace-
ment and a two step reduction using triphenylphosphine followed
by hydrolysis to give 26. Aminopyridazine 26 was converted to
analog 8 in the usual manner. Finally, 2-amino-6-chloropyrazine
27 was ethoxylated and iodinated under previously described
conditions to provide iodopyrazine 28 that was converted to
cinnamate 29 and subsequently to inhibitor 9.
N
N
H
O
N
BILB 1941 (1)
1a/1b replicon EC50 = 153 / 84 nM
COOH
OEt
O
H
N
N
N
N
N
H
Cl
O
BI 207524 (2)
1a/1b replicon EC50 = 29 / 11 nM
COOH
OEt
H2N
X
X, Y, Z = CH, N
Y
Z
3
COOH
Figure 1. HCV NS5B polymerase thumb pocket 1 leads.
3 that unlike the parent aniline, were determined in silico to be
devoid of genotoxic potential.9
BI 207524 (compound 2), was initially considered as an attrac-
tive follow-up candidate to BILB 1941 since it combined improved
potency (7 to 8-fold; achieved through decreasing the measured
acidity of the carboxyl function by introduction of the conjugated
ethoxy group) with a predicted human PK profile consistent with
The isosteric aza-BI 207524 analogs investigated in this study
are listed in Table 1 and the synthesis of required right-hand-side
heterocyclic aniline replacements is described in Scheme 1. For
inhibitor 4, 2-amino-6-bromopyridine 10 was treated with
KOtBu/EtOH and regioselectively iodinated to provide 5-iodopyri-
dine 11 which was converted to 4-aminocinnamate 12 using a
standard Heck Pd-catalyzed cross-coupling protocol with ethyl
acrylate. Fragment 12 was then condensed with 1-aminocyclobu-
tane carboxylic acid chloride hydrochloride and converted to
inhibitor 4 as previously described.6 4-Ethoxypyridine N-oxide 13
was converted to 2-amino-4-ethoxypyridine 14 which was then
iodinated and cross-coupled to ethyl acrylate as described for 12,
to provide building block 15 that was converted to inhibitor 5.
2,3-Dihydroxypyridine 16 was converted to nitropyridine 17 and
then to 2-amino-5-chloropyridine 18 as described in the literature
for the corresponding methoxy analog.10 Heck cross-coupling with
lower dosing requirements (400 mg BID to achieve
a liver-
corrected IQ = 500, predictive of a strong antiviral response).6 The
success of our genotoxicity mitigating strategy relied on the iden-
tification of compounds that would maintain or improve potency
relative to 2 (particularly against the less sensitive GT1a) as well
as retain its favorable PK profile. Results from biological testing
of BI 207524 and six aza-analogs are shown in Table 1.12 In a
polymerase inhibition assay using
a C-terminally truncated
NS5BD
21 construct,13 all compounds had comparable potency to
2 (IC50 = 60–110 nM), suggesting that introduction of nitrogen
atoms in the aniline moiety was neither detrimental nor beneficial
to interactions with the enzyme binding site. In a cell-based
Table 1
BI 207524 and aza-analogs containing non-genotoxic aniline replacements
O
H
OEt
X
Z
N
N
N
N
N
H
Cl
Y
O
COOH
HLM/RLMc t1/2
(min)
Caco-2d Â10À6
Rat PK C1h/2h or
Liver/plasma
ratioh
LogD
(pH 7.4)
a
b
b
e
Entry
X
Y
Z
IC50
GT1b EC50
(nM)
GT1a EC50
(nM)
TC50
(MTT,
f
(nM)
l
M)
(cm/s)
Cmax
(lM)
2
4
5
6
7
8
9
CH
N
CH
CH
N
CH
CH
N
CH
N
CH
CH
CH
N
CH
N
84
85
85
110
63
80
13/11
15/14
33/—
20/—
40/—
360/—
20/—
29
27
—
—
89
—
38
56
102/104
66/111
13
15
15
7.5
4.3
1.6
13
3.2g
5.9f
1.8f
5.5 (6 h)
2
4.1
>5.9
3.0
3.9
—
>10
>10
>10
>10
>10
145/104
275/143
239/>300
136/227
146/>300
—
5
—
—
5
0.4/0.1e
0.1/0.03e
—
CH
N
N
CH
3.4
3.5
N
100
34
2.1/0.9e
a
b
c
GT1b NS5B
D21 (n P 2).
Luciferase reporter and RT-PCR replicon assay values are reported; see Refs. 6 and 13 (n P 2).
Human and rat microsomal stability at 2
Apical to basolateral permeability.
lM initial concentration.
d
e
Following oral administration to rats as mixtures of four compounds at a dose of 4 mg/kg each in 0.5% Methylcellulose and 0.3% Tween-80 + 1% N-methylpyrrolidone
(NMP).14
f
Administered orally as a single compound at a dose of 5 mg/kg in 0.5% Methylcellulose and 0.3% Tween-80 + 1% N-methylpyrrolidone (NMP).
g
Normalized to 5 mg/kg from a 10 mg/kg dose.
h
Determined at 2 h for cassettes or 8 h for single compound PK.14
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P.L. Beaulieu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
3
H2N
N
OEt
I
H2N
N
OEt
a, b
H2N
N
Br
c
ref 6
4
98%, 73%
COOEt
60%
12
11
10
COOEt
H2N
OEt
OEt
H2N
N
OEt
H2N
OEt
ref 6
c
d
b
N
5
N
N+
O
13
73%
97%
I
COOEt
57%
COOEt
15
14
H2N
OEt
OH
OH
O2N
OEt
OH
H2N
OEt
Cl
e, f
ref 6
g, h
i, c, j
6
N
N
N
N
COOtBu
H2N
COOtBu
91%, 43%, 48%
19
73%, 85%
22% (2 steps)
18
16
17
N
OEt
H2N
N
OH
H2N
N
OEt
AcHN
Cl
Cl
COOEt
N
k, l
p
m
ref 6
N
7
N
N
N
N
n, o
57%
COOEt
95%, 51%
CHO
23
22
COOEt
21
91%, 79%, 90%
r, s, t
15%, 59% (2 steps)
COOtBu
20
H2N
OEt
Cl
OEt
OEt
q, p
ref 6
8
N
N
N
N
COOEt
N
25
26
52% (2 steps)
24
COOtBu
H2N
N
OEt
I
H2N
N
OEt
H2N
N
Cl
b, m
c
ref 6
9
N
N
63%, 50%
N
27
COOEt
68%
28
29
COOEt
Scheme 1. Reagents and conditions: (a) KOtBu (2.4 equiv), EtOH, 130 °C (sealed bomb for 3 days or microwave for 30 min) (98%); (b) N-iodosuccinimide (1 equiv), DMF, 0 °C
then rt 4 h (73%); (c) acrylate (5 equiv), P(o-Tol)3 (0.2 equiv), Et3N (1.5 equiv), Pd(OAc)2 (0.1 equiv), dry DMF, 100 °C, 18 h (60%); (d) tBuNH2 (9 equiv), DCM/a,a,a-
trifluorotoluene (1:1), TsCl (4 equiv in DCM added dropwise over 3 h), 0 °C then rt 4 h (73%); (e) Et2SO4, NaOH (1 equiv), 5 °C, 20 h; (f) HNO3/H2SO4, CHCl3, 10–15 °C; (g) PCl5/
POCl3, 100 °C, 3 h (73%); (h) SnCl2 (4 equiv), concd HCl 80 °C, 1 h (85%); (i) AcCl (1.5 equiv), Et3N (2 equiv), CH2Cl2, rt (91%); (j) 2 N NaOH, tBuOH, reflux, 36 h (48%); (k) Ac2O
(2 equiv), pyridine, rt, 1 h (95%); (l) POCl3 (4 equiv), 60 °C, 4 h (51%); (m) NaOEt (21% solution in EtOH, 4 equiv), EtOH, 40 °C, 14 h (91%); (n) LiAlH4 (2.4 M in THF, 1.8 equiv),
THF, 0 °C to rt overnight (79%); (o) MnO2 (10 equiv), CH2Cl2, rt, overnight (90%); (p) triethylphosphonoacetate (1.6 equiv)/NaH (1.5 equiv) in THF, 0 °C, 30 min, then add
aldehyde, 0 °C, 30 min (57%); (q) DIBAL, CH2Cl2, À78 °C, 2 h; (r) NaN3, DMF (15%); (s) PPh3, benzene, reflux, 6 days; (t) 10% AcOH, 70 °C, 5 h.
luciferase reporter GT1b replicon assay,13 potency of pyridine
(4–6), pyrimidine 7 and pyrazine 9 analogs remained within 2 to
3-fold of 2 but pyridazine analog 8 suffered a 25-fold loss in
potency (EC50 = 360 nM). This result is not consistent with the
measured lipophilicity of this analog whose measured LogD of
3.4 at pH 7.4 was comparable to others in this series. The lower
Caco-2 permeability value for 8 (1.6 Â 10À6 cm/s) could be an indi-
cation of the reduced ability of this derivative to cross cell mem-
branes. Comparable values were obtained for compounds 2 and 4
when tested in a replicon assay using RT-PCR for RNA quantifica-
tion. Thumb pocket 1 NS5B inhibitors are typically 2- to 3-fold less
potent in GT1a replicon assays, as previously reported for com-
pound 2.6 This was also observed in the case of aza-analogs. Both
pyridine 4 and pyrazine 9 retained potency comparable to 2 in
the GT1a assay (EC50 = 27, 34 nM, respectively, vs 29 nM), while
pyrimidine 7 was significantly less potent (EC50 = 89 nM) and
was eliminated from further consideration. None of the analogs
tested in the cell-based assays displayed appreciable cytotoxicity
Bioavailability of analogs 4–7 and 9 in rats was evaluated fol-
lowing oral administration either as single compounds (5 mg/kg)
or within a mixture of 4 compounds dosed at 4 mg/kg each.14
Plasma levels are reported in Table 1 (either as Cmax values for sin-
gle compound administration or at 1 and 2 h time points for com-
pound cassettes). Pyridine analog 4 displayed the best profile
overall, with potency, ADME and preliminary rat PK comparable
to 2, albeit with lower partitioning to the liver in this species
(liver/plasma ratio = 2 vs 5). Compound 4 was advanced into
cross-species PK profiling to provide human pharmacokinetic pre-
dictions and estimate the dose required to achieve a strong antivi-
ral response, based on its potency as well as in vitro hepatocyte
and in vivo liver partitioning as previously described.7 Overall,
compound 4 exhibited a similar PK profile to 2 in preclinical animal
species with low clearance and improved exposure and bioavail-
ability in rats and dogs (Table 2). However, the lower in vitro par-
titioning of 4 in human compared to rat hepatocytes (Kp = 3.5 vs
1.5) coupled with
a low in vivo liver/plasma ratio in rats
in the Huh-7 cell line (TC50 >10
opyridazine 29 (as the free carboxylic acid derivatives) were tested
in the Ames test (multi-strain and TA100 strain of Salmonella
typhimurium) at concentrations ranging from 315–5000 lg per
plate with and without S9 metabolic activation. Both fragments
l
M). Aminopyridine 12 and amin-
(Kp = 2.6 vs 5.5) resulted in lower predicted human plasma/liver
partitioning compared to 2 (human Kp = 0.9 vs 3.5).7 It is interest-
ing to note that 4 is significantly more lipophilic than other com-
pounds in this series (LogD >5.9), yet it retains a low Vss across
all species tested, in the range expected for highly protein bound
carboxylic acid derivatives. While compound 4 was also predicted
were found to be non-mutagenic, confirming in silico predictions.
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4
P.L. Beaulieu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
Table 2
Cross-species PK parameters for compounds 2 and 4 at an oral dose of 5 mg/kga and IV dose of 2 mg/kgb
g
Species
LM t1/2 (min)
Cmax
(lM)
AUC (
lM h)
t1/2 (h)
Vss (L/kg)
CL (mL/min/kg)
%F
in vivo liver Kph
5.5
Estimated dose for Ctrough LCIQ500
26
Ratc
104
96
175
102
3.2
4.8
4.1
9.6
26
38
2.4
3.0
4.5
8.6
0.38
0.60
0.38
0.5
2.6
3.6
1.0
0.7
19
72
29
37
Monkeyd,c
Dogc
Humane
3.5i
2.6
400 mg BID
4
Rat
111
32
219
66
5.8
2.6
10
17
8.9
56
1.5
2.0
6.1
2.3
0.28
0.06
0.28
0.1
3.1
1.5
0.9
0.5
42
11
40
16
Monkeyf
Dog
Humane
0.9i
>4000 mg TID
a
b
c
d
e
f
Dosed as an oral suspension in 0.5% Methylcellulose and 0.3% Tween-80 + 1% N-methylpyrrolidone (NMP).
Bolus injection prepared in 70% PEG-400:30% water.
Normalized from a 10 mg/kg oral dose.
Cynomolgous monkeys.
Predicted values derived from allometry principles as described in Ref. 15.
Rhesus monkeys.
g
h
i
Liver microsome half-life at 2 lM starting concentration.
Ratio of compound concentration in liver/plasma at 6–8 h.
Estimated value.6
M.; Arora, S.; Subramanian, G. M.; Zhu, Y.; Dvory-Sobol, H.; Yang, J. C.; Pang, P.
S.; Symonds, W. T.; McHutchison, J. G.; Muir, A. J.; Sulkowski, M.; Kwo, P. N.
Engl. J. Med. 2014, 370, 1483; (b) Afdhal, N.; Zeuzem, S.; Kwo, P.; Chojkier, M.;
Gitlin, N.; Puoti, M.; Romero-Gomez, M.; Zarski, J.-P.; Agarwal, K.; Buggisch, P.;
Foster, G. R.; Bräu, N.; Buti, M.; Jacobson, I. M.; Subramanian, M.; Ding, X.; Mo,
H.; Yang, J. C.; Pang, P. S.; Symonds, W. T.; McHutchison, J. G.; Muir, A. J.;
Mangia, A.; Marcellin, P. N. Engl. J. Med. 1889, 2014, 370; (c) Gane, E. J.;
Stedman, C. A.; Hyland, R. H.; Ding, X.; Svarovskaia, E.; Subramanian, G. M.;
Symonds, W. T.; McHutchison, J. G.; Pang, P. S. Gastroenterology 2014, 146, 736;
(d) Lawitz, E.; Hezode, C.; Gane, E.; Tam, E.; Lagging, M.; Balart, L.; Rossaro, L.;
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to be a low clearance compound in human (IV CL = 0.5 vs 0.7 mL/
min/kg for 2), the significantly lower volume of distribution of 4
in man led to a short predicted human t1/2 = 2.3 h. As a result of
the moderate GT1a potency (EC50 = 27 nM) and short predicted
human t1/2, a strong antiviral response in human would necessitate
administration of >4000 mg three times a day to maintain the
required liver Ctrough concentrations = 500-fold GT1a EC50 (i.e.,
Ctrough ꢀ13.5
l
M).15 Due to these unreasonable exposure and dos-
ing requirements, compound 4 was not considered for further
progression.
3. For recent reports on all-oral triple DAA combinations see: (a) Feld, J. J.;
Kowdley, K. V.; Coakley, E.; Sigal, S.; Nelson, D. R.; Crawford, D.; Weiland, O.;
Aguilar, H.; Xiong, J.; Pilot-Matias, T.; DaSilva-Tillmann, B.; Larsen, L.;
Podsadecki, T.; Bernstein, B. N. Engl. J. Med. 2014, 370, 1594; (b) Everson, G.
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Thuluvath, P. J.; Eley, T.; Wang, X.; Huang, S.-P.; McPhee, F.; Wind-Rotolo, M.;
Chung, E.; Pasquinelli, C.; Grasela, D. M.; Gardiner, D. F. Gastroenterology 2014,
146, 420.
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Lett. 2011, 19, 3173; (b) Benigni, R.; Bossa, C. Chem. Rev. 2011, 111, 2507; (c)
Shamovsky, I.; Ripa, L.; Börjesson, L.; Mee, C.; Nordén, B.; Hansen, P.;
Hasselgren, C.; O’Donovan, M.; Sjö, P. J. Am. Chem. Soc. 2011, 133, 16168; (d)
Birch, A. M.; Groombridge, S.; Law, R.; Leach, A. G.; Mee, C. D.; Schramm, C. J.
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9. The genotoxic potential of aniline replacements was assessed computationally
using (i) DEREK by Lhasa Ltd (a) Sanderson, D. M.; Earnshaw, C. G. Hum. Exp.
Toxicol. 1991, 10, 261; (b) Greene, N.; Judson, P. N.; Langowski, J. J.; Marchant, C.
A. SAR QSAR Environ. Res. 1999, 10, 299 (ii) MultiCASE (http://
www.multicase.com/) and (iii) nitrenium ion stability as described in Ref. 8a..
10. Mavunkel, B. J.; Perumattam, J. J.; Lu, Q.; Dugar, S.; Goyal, B.; Wang, D. X.;
Chakravarty, S.; Luedtke, G. R.; Nashashibi, I.; Tester, R.; Tan, X. U.S. Patent
Application 2005/0288299, 2005.
In summary, we have identified analogs of BI 207524 in which
the liability associated with the release of a genotoxic aniline
metabolite has been reduced by replacing the aromatic amine by
6-membered ring nitrogen heterocycles. Although the potency of
pyridine analog 4 was comparable to that of BI 207524, the overall
preclinical ADME-PK profile and lower human plasma to liver
partitioning predicted for this compound, predicted unacceptably
high dosing requirements to achieve a strong antiviral response.
Subsequent optimization efforts focusing on improving liver
partitioning and the cross-species ADME-PK profile of this class of
NS5B thumb pocket 1 inhibitors is reported in the accompanying
Letter (Part 2).16
Acknowledgments
We thank the following colleagues from Boehringer Ingelheim
(Canada) Ltd that participated in generating the data presented
in this Letter: Nathalie Dansereau, Lisette Lagacée, Charles Pellerin
and Louise Thauvette for IC50 and EC50 determinations. Josie De
Marte, Francine Liard, Hélène Montpetit and Christine Zouki for
ADME-PK support. Norman Aubry, Sylvain Bordeleau, Colette Bou-
cher and Serge Valois for analytical support. We also thank Dr. Ray
Kemper (Boehringer Ingelheim Pharmaceuticals Inc, USA) for pro-
viding in silico genotoxicity assessments.
References and notes
1. (a) Jacobson, I. M.; Dore, G. J.; Foster, G. R.; Fried, M. W.; Radu, M.; Rafalsky, V.
V.; Moroz, L.; Craxi, A.; Peeters, M.; Lenz, O.; Ouwerkerk-Mahadevan, S.; De La
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E.; Buti, M.; Horsmans, Y.; Janczewska, E.; Villamil, F.; Scott, J.; Peeters, M.;
Lenz, O.; Ouwerkerk-Mahadevan, S.; De La Rosa, G.; Kalmeijer, R.; Sinha, R.;
Beumont-Mauviel, M. Lancet 2014, 384, 414; (c) Keating, G. M.; Vaidya, A. Drugs
2014, 74, 273; (d) Pawlotsky, J. M. Gastroenterology 2014, 146, 1176.
2. For recent reports on all-oral double DAA combinations see: (a) Afdhal, N.;
Reddy, K. R.; Nelson, D. R.; Lawitz, E.; Gordon, S. C.; Schiff, E.; Nahass, R.; Ghalib,
R.; Gitlin, N.; Herring, R.; Lalezari, J.; Younes, Z. H.; Pockros, P. J.; Di Bisceglie, A.
Please cite this article in press as: Beaulieu, P. L.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.028

P.L. Beaulieu et al. / Bioorg. Med. Chem. Lett. xxx (2015) xxx–xxx
5
11. Xie, L.; Han, B.; Xu Y.; Maynard, G. WO Patent WO 2004/031174, 2004.
12. Inhibitorshad >95% homogeneity asdetermined by reversed-phaseHPLC analysis
and had 1H NMR and mass-spectral data consistent with their structures.
(N = 3 per dose and per route). In the cassette screen experiments, each
‘cassette’ containing 4 compounds at 4 mg/kg for each compound was dosed to
2 rats. Blood samples collected from all time points were placed on ice, and
then centrifuged at 4 °C. The plasma was separated and stored frozen at
approximately À20 °C until analysis. Plasma samples were extracted by solid
phase extraction. Samples were analyzed by HPLC using either a UV diode
array detector between 200 and 400 nm with quantitative determination made
by peak height at the wavelength representing the best signal to noise ratio or
a LC/MS systems using the appropriate retention time and m/z+. Calibration
standards were prepared in blank plasma. The calibration curve was linear to
cover the time-concentration curve with a r2 values >0.99, and a limit of
quantification (LOQ) at 7 ng/mL. The temporal profiles of drug concentrations
in plasma were analyzed by non-compartmental methods using WinNonlin
(version 3.1; Scientific Consulting Inc, Cary, NC).
13. Inhibition of HCV polymerase activity in a biochemical assay was performed as
previously described using
a C-terminal truncated NS5BD21 construct.
Reported values are the average of duplicate measurements. EC50
determinations using the cell-based 1b luciferase reporter assay or replicon
assays using RT-PCR for RNA quantification were performed in duplicates as
described elsewhere.5a,6
14. All rat PK studies were performed at Boehringer Ingelheim (Canada) Ltd PK
studies in dogs and monkeys were performed at LAB Pre-Clinical Research
International Inc., Laval, QC. All protocols involving animal experimentation
were reviewed and approved by the respective Animal Care and Use
Committee of each test facility. In-life procedures were in compliance with
the Guide for the Care and Use of Laboratory Animals from the Canadian
Council of Animal Care. All chemicals used were reagent grade or better.
Animals were fasted overnight prior to dosing. IV dose administration (2 mg/
kg) was performed using a 70% PEG400:30% water dosing solution. Oral dose
administration (10 mg/kg) was performed using a suspension containing 0.3%
Tween-80 and 0.5% methylcellulose, with 1% NMP, as additional solubilizer
15. Duan, J.; Yong, C.-L.; Garneau, M.; Amad, M.; Bolger, G.; De Marte, J.; Montpetit,
H.; Otis, F.; Jutras, M.; Rhéaume, M.; White, P. W.; Linàs-Brunet, M.; Bethell, R.
C.; Cordingley, M. G. Xenobiotica 2012, 42, 164.
16. Beaulieu, P. L.; Bolger, G.; Deon, D.; Duplessis, M.; Fazal, G.; Gagnon, A.;
Garneau, M.; LaPlante, S.; Stammers, T.; Kukolj. G. Duan, J. Bioorg. Med. Chem.
Lett., in press, http://dx.doi.org/10.1016/j.bmcl.2014.12.078.
Please cite this article in press as: Beaulieu, P. L.; et al. Bioorg. Med. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.bmcl.2014.12.028