Pyridofuran substituted pyrimidine derivatives as HCV replication (replicase) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.06.021

Introduction of nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzofuran inhibitor 2, resulted in the discovery of the more potent pyridofuran analogue 5. Subsequent introduction of small alkyl and alkoxy ligands into the pyridine ring resulted in further improvements in replicon potency. Replacement of the 4-chloro moiety on the pyrimidine core with a methyl group, and concomitant monoalkylation of the C-2 amino moiety resulted in the identification of several inhibitors with desirable characteristics. Inhibitor 41, from the monosubstituted pyridofuran and inhibitor 50 from the disubstituted series displayed excellent potency, selectivity (GAPDH/MTS CC₅₀) and PK parameters in all species studied, while the selectivity in the thymidine incorporation assay (DNA·CC₅₀) was low.

Full text of DOI:10.1016/j.bmcl.2012.06.021

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5144–5149
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyridofuran substituted pyrimidine derivatives as HCV replication
(replicase) inhibitors
Frank Bennett a, , Hollis S. Kezar III b, , Vinay Girijavallabhan a, Yuhua Huang a, Regina Huelgas a,
Randall Rossman a, Neng-Yang Shih a, John J. Piwinski a, Malcolm MacCoss a, Cecil D. Kwong b,
Jeremy L. Clark b, Anita T. Fowler b, Feng Geng b, Abhijit Roychowdhury b, Robert C. Reynolds b,
Joseph A. Maddry b, Subramaniam Ananthan b, John A. Secrist III b, Cheng Li a, Robert Chase a,
Stephanie Curry a, Hsueh-Cheng Huang a, Xiao Tong a, F. George Njoroge a, Ashok Arasappan a
⇑
⇑
a Merck Research Laboratories, 2015 Galloping Hill Rd, Kenilworth, NJ 07033, USA
b Southern Research Institute, Birmingham, AL 35205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Introduction of nitrogen atom into the benzene ring of a previously identified HCV replication (replicase)
benzofuran inhibitor 2, resulted in the discovery of the more potent pyridofuran analogue 5. Subsequent
introduction of small alkyl and alkoxy ligands into the pyridine ring resulted in further improvements in
replicon potency. Replacement of the 4-chloro moiety on the pyrimidine core with a methyl group, and
concomitant monoalkylation of the C-2 amino moiety resulted in the identification of several inhibitors
with desirable characteristics. Inhibitor 41, from the monosubstituted pyridofuran and inhibitor 50 from
the disubstituted series displayed excellent potency, selectivity (GAPDH/MTS CC50) and PK parameters in
all species studied, while the selectivity in the thymidine incorporation assay (DNAꢀCC50) was low.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 9 May 2012
Revised 4 June 2012
Accepted 6 June 2012
Available online 19 June 2012
Keywords:
HCV replication inhibitor
HCV replicase inhibitor
Carbanucleoside-like
5-Aryl pyrimidine
5-Pyridofuranyl pyrimidine
Hepatitis
An estimated 180 million people worldwide are infected with
hepatitis C virus (HCV), a positive stranded RNA virus belonging
to the Flaviviridae family.1 HCV is the leading cause of liver fibrosis,
cirrhosis and hepatocellular carcinoma leading to liver failure
in humans. Until recently, the standard-of-care (SOC), pegylated
O
H
N
NH2
N
H
N
H
N
O
O
O
a-interferon and ribavirin combination therapy, provided less than
O
50% response rate among patients infected with the most prevalent
strain in North America, Europe and Japan.2 However, response
rates of approximately 70% are achieved in patients infected with
genotypes 2 and 3.3 In addition to the relative ineffectiveness
against genotype 1, this SOC is associated with serious side effects4
underscoring the need for improved therapies, stimulating inten-
sive research worldwide for orally available small molecule drug
candidates that directly target various viral proteins.5 Recently,
the introduction of the NS3 protease inhibitor VictrelisÒ,6
(1, Fig. 1) from our own laboratories and IncivekÒ,7 from Vertex
has provided renewed hope to HCV infected patients. The search
for even more effective treatment continues. In addition to the
NS3 protease inhibitors, NS5B nucleoside and non-nucleoside
EC50 = 0.1 µM
GAPDHCC50>50µM
1
Figure 1. Victrelis (boceprevir) 1.
4'
6'
Cl
O
3
N
1'
5
NH2
HN
N
1
HO
OH OH
2
EC50 = 0.3 uM
⇑
Corresponding authors. Tel.: +1 732 594 2602; fax: +1 732 594 2210.
E-mail address: frank.bennett@merck.com (F. Bennett).
Figure 2. Benzofuran inhibitor 2.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.021

F. Bennett et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5144–5149
5145
Table 1
Evaluation of 5-pyridofuran substitution
Cl
Ar
N
N
NHR
HN
OH
HO
HO
Compound #
Ar
R
EC50
(lM)
MTS/GAPDH CC50
>25/—
(
lM)
AUCa
—
(lM.h)
2
H
0.3
O
N
3
H
>25
21/—
—
O
O
N
N
4
5
6
H
H
H
1.7
25/—
25/25
19/—
—
0
0.07
0.4
O
—
N
O
Me
7
H
1.4
>25/25
—
N
O
Me
8
H
H
H
H
0.03
0.01
0.12
0.02
25/9
—
N
O
Et
9
22/12
>25/>25
24/19
0.07
—
N
O
N
10
11
O
O
Me
N
0.05
Et
Me
N
12
13
14
15
16
17
H
0.01
0.01
0.03
0.02
0.01
0.02
22/15
16/13
20/9
0.01
0.01
0
O
Me
Et
N
H
O
Me
Me
N
H
O
Et
Et
N
H
16/25
25/—
0.17
0.1
O
Et
MeO
N
H
O
Me
MeO
N
Me
25/>25
6.5
O
Me
a
AUC0–6 h or AUC0–24 h, po (10 mpk), vehicle ꢁ0.4% mc.

5146
F. Bennett et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5144–5149
Cl
N
inhibitors, and NS5A inhibitors are in various stages of clinical
studies.5
NH2 HCl
OH
20
I
HO
Cl
N
Cl
N
N
Our previous efforts8 towards identification of novel small mol-
ecule inhibitors of hepatitis C virus replication resulted in carbanu-
cleoside-like compounds (e.g., 2; Fig. 2) with modest potency in
the HCV genotype 1b subgenomic replicon assay.9
I
HO
a
N
N
NH2
HN
HO
Cl
NH2
Cl
NH2
b
18
19
HO
OH
21
In this manuscript we will present the syntheses and activities
of a series of analogues derived from logical modifications on the
benzofuran 2, directed towards obtaining more potent anti-HCV
agents with good selectivity relative to cell toxicity, demonstrated
by the MTS and GAPDH assay, and acceptable PK parameters.
Early in this study we decided to introduce nitrogen into the
benzene ring of the benzofuran 2 and synthesized the pyridofuran
analogues (3–6; Table 1). As can be seen from Table 1, while sub-
stitution at the 40-position 3 resulted in complete loss of activity,
the 50-position isomer 4 resulted in a 5- to 6-fold decrease in
potency relative to 2. Potency was retained with the 70-aza deriva-
tive 6. We were encouraged to find significant improvement in the
replicon activity after introduction of nitrogen at the 60-position 5,
and as a result the majority of subsequent modifications were con-
ducted with this bicyclic ring system.
We next introduced small alkyl groups into the pyridine ring
(7–15). Methylation at the 40-position 7 resulted in loss of potency.
We were pleased to find that we could increase the potency with
substitution at the 50-position 8, although this compound did exhi-
bit slightly lower GAPDH CC50. The ethyl analogue 9 was more
potent but displayed similar CC50 profile. Retention of potency
was observed through methylation at the 70-position 10. Interest-
ingly, simply substituting the 70-methyl group in 10 with an ethyl
substituent 11, a 5-fold increase in potency was realized. Extend-
ing to 50,70-disubstituted analogues (12–17) further improvements
in potency could be achieved. As can be seen in Table 1, the major-
ity of our most potent inhibitors were accompanied with differing
levels of cell toxicity demonstrated in the MTS and GAPDH assays;
a notable exception being the 7-methyl analogue 10. This unde-
sired effect could be somewhat alleviated with the incorporation
of 50-alkoxy ligand (16–17) rather than alkyl groups. Initial rat
pharmacokinetic studies10 proved to be disappointing, particularly
for inhibitors 11, 13 and 14. Measurable, albeit low exposure, was
observed in the case of 50-ethyl, 50,70-diethyl and 50-methoxy-70-
methyl derivatives (9, 15 and 16, respectively). Tremendous
improvements in rat PK parameters were observed with the intro-
duction of a methyl group on the 2-amino functionality on the
pyrimidine ring (16 vs 17). This modification resulted in a potent
inhibitor, 17 with good selectivity profiles (MTS EC50/CC50 >1000)
and excellent plasma exposure in rats. Unfortunately, the liver con-
centration11 of 17, another important parameter for HCV inhibi-
tors, was determined to be low, only 274 ng/g at 6 h post-dose.
The low liver concentration clearly demonstrated that further
derivatization was necessary to realize a molecule with an overall
acceptable profile.
Cl
N
Cl
N
N
N
e
c,d
Boc
N
HN
O
NH2
HN
Boc
O
HO
Boc
Boc
Boc
O
HO
OH
22
23
N
Cl
N
N
Cl
N
OH
I
N
O
N
O
N
Boc
24
N
HN
g
NH2
HN
Boc
O
Boc
HO
f
Boc
Boc
O
O
25
HO
OH
12
Scheme 1. Reagents and conditions: (a) ICl, AcOH (79%); (b) Et3N, EtOH, reflux
(80%); (c) Trimethylsilylacetylene, Pd(PPh3)2Cl2, CuI, Et3N, dioxane, 50 °C (56%); (d)
Et4NFꢀH2O (94%); (e) Boc2O, DMAP; (f) Pd(PPh3)2Cl2, CuI, Et3N, dioxane, 50 °C; g)
4 NꢀHCl in dioxane.
pyrimidine ring 25. Finally, exposure to hydrogen chloride in diox-
ane gave the target compound 12, as the hydrochloride salt.
The hydroxypyridine 24 was prepared in 3 steps, as shown in
Scheme 2. Commercially available 2,6-dimethyl-4-hydroxypyri-
dine 26 was converted to the intermediate methoxymethyl ether.
Ortho-lithiation and treatment of the resulting anion with molecu-
lar iodine gave the desired iodide 27. The MOM protecting group
was removed with trifluoroacetic acid followed by liberation of
the resulting salt with triethylamine to give the desired 3-iodo-
4-hydroxypyridine 24.
In line with our previous studies,8 we replaced the 4-chloro
moiety on the pyrimidine ring with a methyl group. Furthermore,
as shown in Table 1, to realize acceptable exposure in rats, it was
necessary to incorporate at least a small alkyl group on the amino
functionality at the 2-position on the pyrimidine ring. To accom-
modate these requirements a new synthetic route had to be
devised. To this end we envisioned that the methyl sulfide func-
tionality 28 (Scheme 3) once coupled to 20, (as previously shown
for the preparation of 21 in Scheme 1) would provide 29, and the
sulfide could be oxidized to the corresponding sulfone or sulfoxide
at a late stage to be conveniently displaced by a variety of amines.
Indeed, when 28 was exposed to 20 in refluxing ethanol in the
presence of excess triethylamine the desired adduct 29 was ob-
tained. Protection of the 1,2-diol was achieved via the isopropyli-
dene group to provide 30. The next step in the sequence involved
conversion of the iodide to an acetylene under Sonogashira type
conditions with trimethylsilylacetylene to give 31. The methylsul-
fide was oxidized to the intermediate sulfoxide prior to deprotec-
tion of the alkyne with fluoride, providing 32. As previously
shown in Scheme 1, the 5-pyridofuran moiety was constructed
The syntheses of the vast majority of the compounds (3–15),
exemplified by inhibitor 12, is shown in Scheme 1. Iodination of
commercially available dichloropyrimidine 18 with iodine mono-
chloride gave the 5-iodo product 19. Displacement of one of the
identical chloro-substituents in 19 with commercially available
carbasugar 20 was achieved in refluxing ethanol in the presence
of triethylamine, producing adduct 21. Subsequent Sonogashira
type coupling with trimethylsilylacetylene and treatment of the
resulting intermediate with fluoride provided the terminal acety-
lene 22. We found it advantageous to ease the handling of these
polar intermediates by protecting the triol 22 with excess di-tert-
butylpyrocarbonate to give the workable fully protected 23. A sec-
ond Sonogashira reaction, this time in the presence of an appropri-
ately substituted 3-hydroxy-4-iodopyridine 24 installed the
required pyridofuran bicyclic moiety on the 5-position of the
CH3
CH3
CH3
I
I
c
a, b
N
N
N
H3C
OMOM
H3C
OH
H3C
OH
26
27
24
Scheme 2. Reagents and conditions: (a) MOMCl, Et3N, CH2Cl2; (b) nBuLi, followed
by I2, THF; (c) CF3CO2H, CH2Cl2 followed by Et3N.

F. Bennett et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5144–5149
Me
5147
using the appropriately substituted 3-hydroxy-4-iodopyridine,
again under Sonogashira type conditions. The amino functionality
was conveniently installed by treating the sulfoxide 33 with a vari-
ety of amines in refluxing acetonitrile. For less nucleophilic sub-
strates (e.g., 1,1,1-trifluoroethylamine) it was found necessary to
heat the sulfoxide with neat amine in a sealed tube. Regardless
of the conditions used, the desired products were usually obtained
in good yield. Deprotection of the isopropylidene group to the final
products 35 was conducted with HCl in aqueous dioxane.
I
NH2 HCl
OH
20
N
Me
HO
I
HO
N
SMe
HN
b
N
HO
a
N
Cl
SMe
29
HO
OH
28
TMS
Me
Me
I
N
N
c
Using compounds 9, from mono-substituted pyridofuran series,
and 16, from di-substituted pyridofuran series, as the basis (po-
tency, selectivity window and PK parameters) for further studies,
a selection of the inhibitors prepared and evaluated are shown in
Tables 2 and 3 respectively.
N
SMe
N
SMe
HN
HN
O
HO
HO
30
31
O
O
O
R1
Compounds 36–43 (Table 2) are 4-methyl analogues of 9, differ-
entiated by the substituent on the amino functionality at the
pyrimidine 2-position. Immediately, we saw that the methyl group
was an effective replacement for the 4-chloro moiety. The N-
methyl analogue 36 retained most of the potency and showed an
excellent profile in the cytotoxicity assays. There was a significant
improvement in the rat plasma exposure when compared with 9.
The n-pentyl derivative 37 had similar potency but less desirable
effects in the toxicity assays, and less exposure in rats. The iso-bu-
tyl derivative 38, while displaying good PK properties (AUC), had a
lower potency and selectivity window. Introduction of the neopen-
tyl moiety 39 restored the potency; however the rest of the profile
was similar to that of 38. The N-ethoxyethyl derivative 40 was
highly potent with moderate PK parameters, and displayed lower
GAPDH CC50 levels. The 1,1,1-trifluoroethyl inhibitor 41 retained
good potency, exhibited excellent rat PK properties (both, plasma
exposure and liver concentration) and an acceptable selectivity
window. A similar profile, albeit with a lower AUC, was seen for
the 1,1,1-trifluorobutyl inhibitor, 42. The most potent inhibitor in
this series was the 3,5-dimethoxy analogue 43, with an excellent
selectivity window. Unfortunately, this promising derivative dis-
played extremely poor rat in vivo parameters.
R2
N
Me
Me
OH
I
N
R2
O
R1
N
N
d, e
25
N
SMe
O
N
SMe
O
HN
O
HN
f
HO
HO
32
33
O
O
O
R1
R1
N
Me
N
Me
R2
O
N
R2
g
h
O
N
N
NHR
HN
HO
N
NHR
HN
HO
34
35
O
O
HO
OH
Scheme 3. Reagents and conditions: (a) Et3N, EtOH, reflux; (b) 2,2-dimethoxypro-
pane, MsOH (79% for steps); (c) trimethylsilylacetylene, Pd(PPh3)3Cl2, CuI,
2
dioxane, 50–100 °C; (d) MCPBA (1 equiv), CH2Cl2; (e) Et4NFꢀH2O, MeCN; (f)
Pd(PPh3)2Cl2, CuI, Et3N, dioxane, 50–100 °C; (g) RNH2, heat; (h) HCl, dioxane-H2O.
Table 2
Evaluation of C-2 amino substituent on pyrimidine C-4 Me derivatives of inhibitor 9
Et
N
Me
4
O
N
R
2
N
N
H
HN
HO
HO
OH
Compound
R
EC50
(l
M)
MTS/GAPDH CC50
(lM)
AUCa
(l
M.h)
Liverb C6 h (ng.g)
Me
36
37
0.03
0.03
>25/>25
3/3
0.9
936
31
Me
0.48
38
39
0.08
0.03
12/7
4.5
4.0
251
315
C(CH3)3
OEt
11/10
40
41
42
0.004
0.02
0.02
16/4
15/13
1.2
5.3
0.9
492
1732
1000
CF3
CF3
>25/17
OMe
43
0.003
20/>25
0.1
20
OMe
a
AUC0–6 h, po (10 mpk), vehicle ꢁ0.4% mc.
b
Liver conc at 6 h.

5148
F. Bennett et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5144–5149
Table 3
Evaluation of C-2 amino substituent on inhibitor-type 16
1
R
5'
N
Me
4
Me
HO
O
N
R
2
N
N
H
HN
HO
OH
Compound
R
R1
EC50
(lM)
MTS/GAPDH CC50
(lM)
AUCa
(
lM.h)
Liverb C6 h (ng.g)
Me
Me
44
45
OMe
OMe
0.06
0.04
25/>25
21/17
2.7
4.4
310
C(CH3)3
1185
OMe
46
OMe
0.01
25/>25
0.5
178
OMe
47
48
OEt
OEt
0.05
0.05
>25
1.4
6.8
238
C(CH3)3
23/12
2334
49
50
OEt
OEt
0.15
0.02
18/24
25/25
18.5
45.5
8981
10226
OMe
CF3
51
52
OEt
OEt
0.009
0.02
>25/>25
>25/>25
1.1
8.9
285
3015
OMe
53
OEt
0.002
>25/>25
0.35
238
OMe
a
AUC0–6 h, po (10 mpk), vehicle ꢁ0.4% mc.
b
Liver conc at 6 h.
Table 3 shows the C-2 SAR studies carried out on the 4-methyl
Et
pyrimidine derivatives of type 16. As seen before in Table 2, the 4-
methyl group was well tolerated on 50-alkoxy pyridofuran targets
of type 16. The C-2 N-methyl analogues 44 and 47 exhibited good
replicon potency, selectivity indices and moderate plasma expo-
sure (AUC). However, the liver concentrations at the 6 h time point
were poor for inhibitors 44 and 47. The neopentyl inhibitors 45 and
48 showed similar potency, improved plasma exposure and liver
concentrations, and moderate selectivity windows. Again the most
potent derivatives proved to be the 3,5-dimethoxybenzyl ana-
logues 46 and particularly 53, in the 50-methoxy and 50-ethoxy
series respectively. Similar to prior results with the 50-ethyl benzo-
furan 43, the PK parameters were poor for both 50-alkoxy deriva-
tives, 46 and 53.
The N-phenyl analogue 49 proved to be comparatively less ac-
tive. The cyclopropylmethyl derivative 50, methoxyethyl target
51 and 1,1,1-trifluoroethyl compound 52 all displayed excellent
potencies and selectivity windows. Of particular interest was the
cyclopropylmethyl derivative 50, distinguished by its exceptional
rat plasma exposure and liver concentration.
EtO
N
N
Me
Me
N
O
HN
N
Me
O
N
NHCH2CF3
N
NH
HN
HO
HO
HO
OH
41
HO
OH
50
Replicon (1b) EC50 = 0.02 µM
MTS/GAPDH CC50 =15/13 µM
DNA CC50 = 4 µM
Replicon (1b) EC50 = 0.02 µM
MTS/GAPDH CC50 =25/25 µM
DNA CC50 = 5 µM
PK Data: (HCl salt)
Rat (po,10 mpk):
AUC = 5.3µM.h; F = 39%
t1/2 = 4.2 hr; Cl = 27 ml/min/kg
Liver C(6h) = 1732 ng/g
PK Data: (HCl salt)
Rat (po, 10 mpk):
AUC = 45.5µM.h; F = 97%
t1/2 = 3.3 hr; Cl = 8 ml/min/kg
Liver C(6h) = 10226 ng/g
Monkey (po, 3 mpk):
AUC = 5.2 µM.h; F = 69%
t1/2 = 7 hr; Cl = 15 ml/min/kg
Monkey (po, 3 mpk):
AUC =10.2 µM.h; F =100 %
t1/2 = 2.8 hr; Cl = 16 ml/min/kg
Dog (po, 3 mpk):
AUC = 6.4 µM.h; F = 83%
t1/2 = 2.1 hr; Cl = 13 ml/min/kg
Dog (po, 3 mpk):
AUC = 1.6 µM.h; F =15 %
t1/2 = 2.1 hr; Cl = 9.5 ml/min/kg
Based on the potency, selectivity, and rat exposure, both plasma
and liver concentration, inhibitors 41 and 50 from the respective
50-pyridofuran series were selected for further studies in higher
species. The full profile of the two inhibitors, 41 and 50 are shown
in Figure 3. Both molecules exhibited excellent plasma concentra-
tion and bioavailability in monkeys. While 41 displayed high expo-
sure in dogs, the corresponding data for inhibitor 50 proved to be
slightly lower. It should be noted that both compounds showed a
similar, albeit lower, selectivity profile in the more stringent thy-
midine incorporation assay (DNAꢀCC50).
Figure 3. Profile of inhibitors 41 and 50.
case inhibitor 2 resulted in measurable improvements in replicon
potency. Modifications were carried out at the 5-pyridofuran moi-
ety and the pyrimidine core. These studies identified inhibitors 41
and 50 with excellent potency, selectivity with respect to the GAP-
DH/MTS CC50 and desirable PK parameters in all species studied.
However, inhibitors 41 and 50 displayed lower selectivity in the
thymidine incorporation assay. Studies directed towards overcom-
In summary, introduction of a nitrogen atom at the 60-position
of the 5-benzofuran moiety of a carbanucleoside-like HCV repli-

F. Bennett et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5144–5149
5149
G.; Arasappan, A. Bioorg. Med. Chem. Lett. 2012, 22, 1160; (b) Arasappan, A.;
Bennett, F.; Girijavallabhan, V.; Huang, Y.; Huelgas, R.; Alvarez, C.; Chen, L.;
Gavalas, S.; Kim, S.-H.; Kosinski, A.; Pinto, P.; Rizvi, R.; Rossman, R.; Shankar, B.;
Tong, L.; Velazquez, F.; Venkatraman, S.; Verma, V. A.; Kozlowski, J.; Shih, N.-Y.;
Piwinski, J. J.; MacCoss, M.; Kwong, C. D.; Clark, J. L.; Fowler, A. F.; Geng, F.;
Kezar, H. S., III; Roychowdhury, A.; Reynolds, R. C.; Maddry, J. A.; Ananthan, S.;
Secrist, J. A., III; Li, C.; Chase, R.; Curry, S.; Huang, H.-C.; Tong, X.; Njoroge, F. G.
Biorg. Med. Chem. Lett. 2012, 22, 3229.
ing this obstacle while retaining the desirable characteristics of the
aforementioned inhibitors will be the subject of future
communications.
References and notes
1. Lavanchy, D. Liver Int. 2009, 29, 74.
2. Feld, J. J.; Hoofnagle, J. H. Nature 2005, 436, 967.
9. (a) Lohmann, V.; Körner, F.; Koch, J.-O.; Herian, U.; Theilmann, L.;
Bartenschlager, R. Science 1999, 285, 110
3. Zeuzem, S.; Hulcrantz, R.; Bourliere, M.; Goeser, T.; Marcellin, P.; Samchez-
Tapias, J.; Sarazin, C.; Harvey, J.; Brass, C. A.; Albrecht, J. A. J. Hepatol. 2004, 40,
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4. Birerdinc, A.; Younossi, Z. M. Expert Opin. Emerg. Drugs 2010, 15, 535.
5. (a) Flisiak, R.; Parfieniuk, A. Expert Opin. Investig. Drugs 2010, 19, 63; (b)
Meanwell, N. A.; Kadow, J. F.; Scola, P. M. Annu. Rep. Med. Chem. 2009, 44, 397.
6. Boceprevir: (a) FDA press release dated May 13, 2011, http://www.fda.gov/
(b) To measure cell-based anti-HCV activity, replicon cells (1b-Con1) are
seeded at 5000 cells/well in 96-well plates one day prior to inhibitor treatment.
Various concentrations of an inhibitor in DMSO are added to the replicon cells,
with the final concentration of DMSO at 0.5% and fetal bovine serum at 10% in
the assay media. Cells are harvested three days post dosing. The replicon RNA
level is determined using real-time RT-PCR (Taqman assay) with GAPDH RNA
as endogenous control. EC50 values are calculated from experiments with 10
serial twofold dilutions of the inhibitor in triplicate. To measure cytotoxicity in
replicon cells of an inhibitor, an MTS assay is performed according to the
manufacturer’s protocol for CellTiter 96 Aqueous One Solution Cell
Proliferation Assay (Promega, Cat # G3580) three days post dosing on cells
treated identically as in replicon activity assays. CC50 is the concentration of
inhibitor that yields 50% inhibition compared to vehicle-treated cells. Effect of
NewsEvents/Newsroom/PressAnnouncements/ucm255390.htm.;
(b)
Venkatraman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, E.; Liu,
Y.-T.; Lovey, R.; Hendrata, S.; Huang, Y.; Pan, W.; Parekh, T.; Pinto, P.; Popov, V.;
Pike, R.; Ruan, S.; Santhanam, B.; Vibulbhan, B.; Wu, W.; Yang, W.; Kong, J.;
Liang, X.; Wong, J.; Liu, R.; Butkiewicz, N.; Chase, R.; Hart, A.; Agrawal, S.;
Ingravallo, P.; Pichardo, J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.; Prongay,
A.; Madison, V.; Broske, L.; Cui, X.; Cheng, K.-C.; Hsieh, T. Y.; Brisson, J.-M.;
Prelusky, D.; Korfmacher, W.; White, R.; Bogdanowich-Knipp, S.; Pavlovsky, A.;
Bradley, P.; Saksena, A. K.; Ganguly, A.; Piwinski, J.; Girijavallabhan, V.; Njoroge,
F. G. J. Med. Chem. 2006, 49, 6074.
an inhibitor on cellular DNA synthesis is determined by
proximity assay. Replicon cells are seeded in 96-well Cytostar-T Scintillating
Microplates (PerkinElmer, Cat RPNQ0163) one day prior to inhibitor
a scintillation
#
treatment. Various concentrations of an inhibitor in triplicate are added with
[methyl-14C]-thymidine (PerkinElmer, Cat # NEC568050UC, final concentration
7. Telaprevir: (a) FDA press release dated May 23, 2011, http://www.fda.gov/
NewsEvents/Newsroom/PressAnnouncements/ucm256299.htm.; (b) Perni, R.
B.; Almquist, S. J.; Byrn, R. A.; Chandorkar, G.; Chaturvedi, P. R.; Courtney, L. F.;
Decker, C. J.; Dinehart, K.; Gates, C. A.; Harbeson, S. L.; Heiser, A.; Kalkeri, G.;
Kolaczkowski, E.; Lin, K.; Luong, Y.-P.; Rao, B. G.; Taylor, W. P.; Thomson, J. A.;
Tung, R. D.; Wei, Y.; Kwong, A. D.; Lin, C. Antimicrob. Agents Chemother. 2006, 50,
899.
8. (a) Kwong, C. D.; Clark, J. L.; Fowler, A. F.; Geng, F.; Kezar, H. S., III;
Roychowdhury, A.; Reynolds, R. C.; Maddry, J. A.; Ananthan, S.; Secrist, J. A.,
III; Shih, N.-Y.; Piwinski, J. J.; Li, C.; Feld, B.; Huang, H.-C.; Tong, X.; Njoroge, F.
0.5 lCi/mL media) and incubated for three days. DNA CC50 is the inhibitor
concentration that yields 50% inhibition of labeled thymidine incorporation as
measured by Packard TopCount compared to vehicle-treated cells.
10. Inhibitors were dosed orally at 10 mpk (n = 2 for each compound). The
formulation vehicle was 0.4% MC and plasma samples were taken at
determined time points up to 6 h. Rat plasma exposure is reported as AUC0–6 h
.
11. The animals were sacrificed at 6 h and the liver was harvested and processed to
measure the compound concentration (liver C6h).