Synthesis and SAR of geminal substitutions at the C5′ carbosugar position of pyrimidine-derived HCV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.08.111

The installation of geminal substitution at the C5′ position of the carbosugar in our pyrimidine-derived hepatitis C inhibitor series is reported. SAR studies around the C5′ position led to the installation of the dimethyl group as the optimal functionali

Full text of DOI:10.1016/j.bmcl.2012.08.111

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6967–6973
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and SAR of geminal substitutions at the C50 carbosugar position
of pyrimidine-derived HCV inhibitors
Vishal A. Verma a, , Randall Rossman a, Frank Bennett a, Lei Chen a, Stephen Gavalas a,
⇑
Vinay Girijavallabhan a, Yuhua Huang a, Seong-Heon Kim a, Aneta Kosinski a, Patrick Pinto a, Razia Rizvi a,
Bandarpalle Shankar a, Ling Tong a, Francisco Velazquez a, Srikanth Venkatraman a, Joseph Kozlowski a,
Malcolm MacCoss a, Cecil D. Kwong b, Namita Bansal b, Hollis S. Kezar III 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
The installation of geminal substitution at the C50 position of the carbosugar in our pyrimidine-derived
hepatitis C inhibitor series is reported. SAR studies around the C50 position led to the installation of
the dimethyl group as the optimal functionality. An improved route was subsequently designed to access
these substitutions. Expanded SAR at the C2 amino position led to the utilization of C2 ethers. These com-
pounds exhibited good potency, high selectivity, and excellent plasma exposure and bioavailability in
rodent as well as in higher species.
Article history:
Received 2 July 2012
Revised 25 August 2012
Accepted 28 August 2012
Available online 7 September 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Hepatitis C virus
Geminal substitution
HCV inhibitor
Carbosugar
Hepatitis C virus (HCV) is a worldwide health concern, being a
leading cause of liver cirrhosis, hepatocellular carcinoma, and
chronic liver failure.1 Globally, 130–170 million people are infected
with HCV1 and established treatment paradigms suffer from low
response rates (<50% for genotype 1), genotype variability, viral
resistance, and side effect profiles that result in low patient com-
pliance.2 Thus, there is a critical unmet medical need for new ther-
apies that can circumvent these issues. Recently, boceprevir and
telaprevir, two drugs from a new therapeutic class, the NS3 prote-
ase inhibitor, have been approved for HCV treatment.3 In addition
to several other protease inhibitors undergoing clinical trials, novel
compounds targeting the NS5b polymerase, the NS5a protein, and
other cellular targets are in clinical studies.4
ing new pathways with unique resistance profiles have the poten-
tial to become a part of an effective combination regimen.
Our previous studies towards the identification of small mole-
cule inhibitors of HCV replication resulted in the discovery of the
pyrimidine-derived compound 1 (Fig. 1), which inhibited HCV rep-
lication in the subgenomic replicon assay5 (genotype 1b) with an
EC50 = 8 nM, while displaying a low selectivity profile (MTS/
GAPDH = 5/3
lM) and poor rat pharmacokinetics (PK) (AUC0–6h =
0.4
l
M h).6 Moreover, compound 1, as well as other members of
this class, did not exhibit adequate plasma exposure in monkey,
an important higher animal species utilized in dosage predictions,
safety studies, etc. Our current efforts focused on the synthesis of
functionally active compounds that maintained the potency of
the lead while improving selectivity and PK exposure across multi-
ple animal species. Target engagement studies with previous leads
suggest a mechanism of action which inhibits the HCV replication
complex,6b and will be reported in due course.
While recent studies have shown that replacing a benzothiazole
ring with an 5-azabenzothiazole (as in 1) increased the potency of
the series in general, the resulting compounds often displayed low
plasma levels in rats and higher species.6a Substitutions on the aza-
benzothiazole ring were subsequently carried out in order to ad-
dress these PK issues. In an orthogonal effort to circumvent this
A major obstacle for advancing HCV therapies beyond the clinic
is the rapid emergence of resistant mutations. As such, a variety of
therapies targeting mechanistically distinct pathways are required
in order to circumvent resistance to a specific pathway. In addition
to known pathways (e.g., NS3, NS5b, and NS5a), molecules target-
⇑
Corresponding author. Tel.: +1 650 225 5056.
E-mail addresses: verma.vishal@gene.com, vishal.verma@merck.com (V.A. Ver-
ma).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.111

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V. A. Verma et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6967–6973
previous modifications
Y
N
N
N
N
5
4
5
4
S
N
S
N
Z
Z
2
2
HN
HN
N
N
H
N
NHR
5′
HO
1
′
5
HO
this work
′
1′
HO
OH
HO
OH
1
areas of focus
Replicon (1b) EC50 = 8 nM
MTS/GAPDH CC50 = 5/3 µM
a
Rat AUC0-6h = 0.4 µM.h
Figure 1. Profile of compound 1 and the focus of this work. aPO, 10 mpk, HCl salt.
issue as well as further explore the SAR of these leads, variations in
the carbosugar moiety were targeted. It was hypothesized that the
primary alcohol of the carbosugar could be metabolized in vivo,
leading to lower animal plasma levels.7 Thus, the PK profile could
potentially be improved by incorporating substitution at the 50 po-
sition of the carbosugar, thereby providing steric bulk adjacent to
the primary hydroxyl group and decreasing its metabolic liability.
Starting with phthalimide derivative 2,8 oxidation of the pri-
mary alcohol and methyl Grignard addition gave compound 3
(Scheme 1). Reoxidation of the secondary alcohol and methyl addi-
tion to the resulting ketone provided intermediate 4. Deprotection
of the amine was followed by addition to known chloropyrimidine
59 to give 6, which underwent a Pd/Cu-catalyzed direct coupling
reaction with aryl moieties of the type 710 to provide 8. Oxidation
of the sulfide to the sulfoxide was followed by displacement with a
variety of structurally and electronically diverse amines to give the
penultimate intermediate. Diol deprotection then gave final targets
of the type 9. In order to obtain mono-methyl targets of the type 10
(Fig. 2), steps c and d could be bypassed with the remainder of the
route remaining constant.
Installation of one methyl group onto the C50 position of com-
pound 1 led to analog 10 (as a mixture), which showed a slight
drop in potency but an increase in rat plasma levels (Fig. 2). This
result was encouraging, since it demonstrated that substitution
at this position was tolerated and had a beneficial effect on PK.
The key breakthrough, however, came via further substitution at
C50, resulting in the gem-dimethyl compound 11 (Fig. 2). Com-
pound 11, compared to 1, achieved significantly higher rat plasma
O
O
O
HO
a,b
c,d
N
N
N
O
HO
O
HO
O
O
O
O
O
O
O
2
3
4
I
N
e,f
g
HN
N
SMe
HO
Y
I
N
O
O
N
N
Cl
N
SMe
S
6
5
7
Y
Y
N
N
N
N
S
N
S
N
h-j
R
HN
HN
N
SMe
N
N
H
HO
HO
O
O
HO
OH
8
9
Scheme 1. Initial synthesis of analogs of type 9. Reagents and conditions: (a) TEMPO, PhI(OAc)2, CH2Cl2, rt, 16 h (84%) (b) MeMgBr, THF, ꢀ78 °C, 1 h (55%) (c) TEMPO,
PhI(OAc)2, CH2Cl2, rt, 16 h (59%) (d) MeMgBr, THF ꢀ78 °C, 2 h (28%) (e) N2H4ꢁH2O, EtOH, 70 °C, 2 h (f) 5, iPr2NEt, EtOH, 120 °C, 1.5 h (78%) (g) 7, Pd(PPh3)4, CuI, Cs2CO3, DMF,
110 °C, 2 h (70–80%) (h) mCPBA, CH2Cl2, 0 °C, 30 min (quant) (i) H2NR, dioxane, 120 °C, 16 h (j) HCl, H2O, THF, 0 °C, 3–4 h. (Y = H or Me).

V. A. Verma et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6967–6973
6969
N
N
levels (AUC0–6h = 8.5
l
M h) while maintaining acceptable potency.
N
N
Based on the significant PK advantage conferred by the disubstitu-
tion, further exploration of SAR was undertaken in various gemi-
nally substituted series.
S
N
S
N
HN
HN
N
N
H
N
N
H
While the initial set of azabenzothiazole targets was prepared
through the route depicted in Scheme 1, an optimized route was
subsequently designed to intermediate 6 (Scheme 2). Starting with
commercially available intermediate 12, direct double methyl
addition to the acid resulted in only a 15% yield of the desired
product. However, conversion of the acid first to the methyl ester
13, smoothly accomplished with TMSCHN2, was followed by
methyl lithium addition to install the geminal dimethyl group in
63% yield over two steps. Chemoselective deprotection of the
amine could be performed in the presence of the acetonide, which
subsequently underwent addition to chloropyrimidine 5 to give
intermediate 6. This optimized sequence thus shortened the route
by four steps and allowed for greater efficiency. Intermediate 6 was
then converted to final targets using the route shown in Scheme 1.
Utilizing the routes shown above, a variety of azabenzothiazole
targets were prepared (Table 1). Previous data indicated that an
unsubstituted (Y = H) and methyl substituted (Y = Me) azabenzo-
thiazole were optimal and so these were incorporated as the 5-aryl
substituents.6a Exploration of C2 amine substituents in the unsub-
stituted azabenzothiazole case (Y = H) confirmed that improved
plasma levels could be reached through the incorporation of small
alkyl groups (11 and 15), but not with ether containing sidechains
(16). Moreover, there was a slight loss in potency for the ether con-
taining compound, 16. In contrast, methyl substitution of the aza-
benzothiazole (Y = Me: 17–19) led to compounds that exhibited a
similar potency profile, including the C-2 ether containing com-
pound, 19. Additionally, inhibitor 19 displayed significantly higher
rat plasma exposure compared to 16.
HO
HO
HO
OH
HO
OH
10
11
Replicon (1b) EC50 = 20 nM
MTS/GAPDH CC50 = 13/18 µM
Rat AUC0-6ha = 1.3 µM.h
Replicon (1b) EC50 = 20 nM
MTS/GAPDH CC50 = >12/>12 µM
Rat AUC0-6ha = 8.5 µM.h
Figure 2. Structures and biological profiles of compounds 10 and 11. aPO, 10 mpk,
HCl salt.
O
O
NHBoc
NHBoc
a
b
HO
MeO
O
O
O
O
O
12
NHBoc
13
I
N
c,d
HN
HO
N
SMe
HO
O
I
O
O
N
Cl
N
SMe
14
6
5
The installation of the gem-dimethyl group resulted in a series
that demonstrated a clean selectivity profile (MTS/GAPDH > 25 lM
for most compounds, Table 1). Importantly, the geminal methyl
substitution added a significant increase in rat exposure levels,
Scheme 2. Optimized route to gem-dimethyl targets. Reagents and conditions: (a)
TMSCHN2, MeOH, PhMe, rt, 5 min (b) MeLi, THF, ꢀ30 °C, 1 h (63%, two steps) (c)
TFA, CH2Cl2, rt, 2 h (d) 5, Et3N, EtOH, 90 °C, 16 h (82%, two steps).
Table 1
Selected profiles of unsubstituted and substituted azabenzothiazole containing compounds incorporating the gem-dimethyl group
Y
N
N
5
S
N
R
4
2
HN
1
′
N
N
H
5′
HO
HO
OH
9
#
Y
R
Replicon EC50 (nM)
20
MTS/GAPDH CC50
>12/>12
(lM)
AUCa
8.5
(lM h)
11
H
15
16
17
H
30
50
20
25/>25
11
OMe
OMe
H
>25/>25
>25/>25
1.3
10b
Me
18
19
Me
Me
20
20
>25/>25
>25/>25
19b
16b
a
0–6 h, po, 10 mpk
0–24 h, po, 10 mpk.
b

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V. A. Verma et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6967–6973
new methodology developed for this purpose.12 Tin reagent
R1
=
methoxymethyl(tributyl)stannane was first synthesized from
MOMBr and Bu3SnH.13 Subsequent lithium-tin exchange was fol-
lowed by double addition to the ester to give 22. Finally, cycliza-
tion of the gem-dimethyls to give the C50 cyclopropyl series 23
was accomplished via use of the Kulinkovich reaction.14
NHBoc
a
b
c
HO
R1
The newly synthesized C50 disubstituted compounds in general
maintained good selectivity (Table 2). The gem-diethyl series (25
and 26) had slightly increased potencies compared to the gem-di-
methyl series but the series suffered from lower rat plasma expo-
sure (compound 26) as well as significant solubility
complications.15 The C50 cyclopropyl series (27 and 28) also dis-
played potency increases, yet also exhibited a loss of plasma expo-
sure levels. While the gem-di(CH2OCH3) series (29 and 30)
improved the solubility of the compounds in addition to achieving
a potency gain, this series resulted in a lower selectivity profile as
measured by MTS/GAPDH and the more discerning DNA CC50
incorporation assay16 (vide infra).
O
S
O
21
MeO
O
MeO
or
R1
R1
MeO
R1
HO
N
20
N
22
23
N
N
HO
HN
SMe
O
O
Due to the improved overall profile with respect to potency,
selectivity and PK of the 4-methyl substituted azabenzothiazole
C50-gem-dimethyl series (i.e., 17–19), attention was focused on fur-
ther diverse modifications of the C2 substituent.
As can be seen from the data in Table 3, in general, the high le-
vel of selectivity could be maintained in the gem-dimethyl series
even through the expansion of the C2 substituent to a wide variety
of structures. Potency and PK profiles varied amongst the com-
pounds. The primary amine at the C2 position (32) lost potency,
as well as selectivity. The cyclopropyl amine substituent (33) also
had lower potency but exhibited an excellent rat PK profile
Scheme 3. Synthesis of additional disubstituted C50 compounds. Reagents and
conditions: (a) EtLi, THF, ꢀ78 °C ? ꢀ30 °C, 1 h (34%) (b) MeOCH2SnBu3, nBuLi, THF,
ꢀ78 °C, 2 h (44%) (c) EtMgBr, Ti(OiPr)4, THF, 0 °C ? rt, 16 h (36%).
indicating the success of this overall strategy. Based on the
improvements achieved with the gem-dimethyl group, further
modifications at C50 were thus warranted. The 4-Me azabenzo-
thiazole (Y = Me) was chosen as the C5 aryl substituent in these
studies because the rat in vivo profile was generally better than
the 4-H (Y = H) series and was more tolerant of changes to the C-
2 sidechain (R) with respect to retaining potency.
(AUC0–6h = 58 lM h), exemplifying the potential for this series to
solve the PK hurdle. Methylation of the C2 amine, as in analog
34, led to loss of activity. Introduction of ether moieties at the C2
position, following up on the favorable profile of ether 19, proved
fruitful. Ethers of various types (35–37) displayed good potencies,
selectivities and excellent rat PK profiles. Attempting to capitalize
on the beneficial ether SAR, thioethers such as 38 were synthe-
sized. While retaining good potency, 38 exhibited lower rat expo-
sure levels. The combination of an alkyl and ether substituent, as in
The additional C50 geminally substituted targets were synthe-
sized in a similar fashion as that in Scheme 2, through addition
to methyl esters of type 2011 (Scheme 3). To explore the steric
requirement of the geminal substitution, the gem-diethyl series de-
rived from 21 was synthesized with the use of ethyl lithium. To im-
prove the solubility of the series, compounds of the type 22,
incorporating more polar ether groups, were synthesized through
Table 2
Selected profiles of C50 gem-disubstituted compounds
N
N
5
S
N
Z
Z
R
4
2
HN
1′
N
N
H
5′
HO
HO
OH
24
#
Z
R
Replicon EC50 (nM)
MTS/GAPDH CC50
(l
M)
AUCa
(lM h)
25
26
Et
Et
5
>6/>6
10
OMe
OMe
OEt
10
>25/>25
1.4
Z
Z
27
28
4
7
>25/>25
13/>25
1.6
3.9
HO
HO
29
30
CH2OMe
CH2OMe
2
3
>6/>6b
6/>12
—
—
a
0–6 h, po, 10 mpk.
DNA CC50 = 4 lM.
b

V. A. Verma et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6967–6973
6971
Table 3
Selected profiles of C2 varied, C50 gem-dimethyl compounds
N
N
5
4
S
N
R
2
HN
N
N
H
5′
HO
1′
HO
OH
31
#
R
Replicon EC50 (nM)
MTS/GAPDH CC50
(l
M)
AUCa
(lM h)
17
32
33
20
40
50
>25/>25
6/4
10b
—
H
>6/>6
58
R
=
N
N
H
34
35
36
90
20
20
23/>25
>3/>3
—
18b
6.7b
OMe
OMe
>25/>25
O
37
4
>25/>25
9.9
SMe
38
39
8
>25/>25
>25/>25
1.6
0.7
OMe
20
F
40
41
4
>25/>25
>25/>25
0.4
1.2
F
F
180
OCF3
42
43
10
>12/20
12/7
1.8
—
CF3
NMe2
130
O
44
20
>25/>25
0.2
N
a
0–6 h, po, 10 mpk.
0–24 h, po, 10 mpk.
b
compound 39, also led to a poor PK profile. The trifluorophenyl
substituent (40) improved potency but did not have an acceptable
PK profile. The aryl C2 moiety in 41, an important feature in previ-
ous studies,6a led to loss of activity. Target 42, containing a trif-
luoroalkyl group found in a previous lead,6c led to lower rat
exposure levels. In order to attempt to build upon the favorable
profiles of the polar ether targets, further modifications containing
other polar moieties were probed at the C2 position. However, the
incorporation of an amine in the form of a dimethyl amino group
(43) led to a loss of activity, while introduction of the morpholino
group (44) led to loss of rat plasma exposure.
Compounds exhibiting good HCV replicon potency, selectivity,
and rat plasma exposure were selected for further studies (Table 4).
DNA incorporation studies were performed as an additional mea-
sure of selectivity. Plasma exposure was monitored in multiple
species, including rat, dog and monkey for 24 hours and compound
concentration in the rat liver was measured, an important metric
for anti-HCV compounds. Targets bearing alkyl substitution at
the C2 position (17 and 18) retained a good selectivity profile
and good rat PK, including very high rat liver concentrations. While
levels in dog were acceptable, exposure was curtailed in monkey
(AUC = 0.3–0.5 M h). Linear ether moieties at C2, as in compound
l
19, while displaying good MTS/GAPDH selectivity, unfortunately
suffered from low DNA CC50, as did the more potent pyran deriva-
tive 37. Interestingly, the monkey plasma level for compound 19
was good, indicating the potential for ether substituents to exhibit
good monkey PK. Further profiling the ether series, it was found
that compounds 35 and 36, possessing branching on the C2 ethers,
maintained good selectivity by both the MTS/GAPDH and the DNA
incorporation assays. In rats, these compounds were found to have
acceptable liver concentrations in addition to good plasma levels
and bioavailability. Critically, these compounds also exhibited
plasma coverage and bioavailability across dog and monkey spe-
cies, validating the potential of the ether series to exhibit higher
order animal PK exposure. Analogs 35 and 36 thus represent com-
pounds with the strongest overall profile of potency, selectivity
and total animal PK parameters.
Beginning with HCV replication inhibitors exemplified by 1, it
was found that mono-substitution at the C50 position of the carbo-
sugar was tolerated and led to a moderate improvement in PK.

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V. A. Verma et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6967–6973
Table 4
Full profile, including higher order animal PK parameters, of selected compounds.
#
Ra
Replicon EC50
(nM)
MTS/GAPDH CC50
M)
DNA CC50
M)
Rat AUC /Fb
M h/%)
Rat Liverc
(ng/g)
Dog AUC/Fd
M h/%)
Monkey (Cyno) AUC/Fe
M h/%)
(
l
(
l
(
l
(
l
(l
17
20
>25/>25
25
10/39
9500
4.9/66
0.3/6
18
19
37
20
20
4
>25/>25
>25/>25
>25/>25
>25
7
19/54
16/49
9.9g/—
13700
1900
1800
2.1/32
0.9/18
—
0.5/4
3.7/46
—
OMe
O
3f
35
36
20
20
>3/>3h
25
25
18/81
6.7/58
900
4.2/50
3.2/56
10/50
13/45
OMe
OMe
>25/>25
1300
a
b
c
See compound 31, Table 3 for the full structure.
0–24 h, po, 10 mpk.
6 h, po, 10 mpk.
0–24 h, po, 3 mpk.
0–24 h, po, 3 mpk.
d
e
f
CC25
.
g
h
0–6 h, po, 10 mpk.
The compound precipitated at >3 lM.
Tung, R. D.; Wei, Y.; Kwong, A. D.; Lin, C. Antimicrob. Agents Chemother. 2006, 50,
899.
Geminal substitution subsequently led to a compound with signif-
icantly higher plasma exposure. Installation of the geminal di-
methyl group on the methyl substituted azabenzothiazoles then
improved the replicon potency and allowed for incorporation of
C2 ethers. Based on the improved initial profile of these gem-disub-
stituted compounds, an optimized route to access additional gem-
inal substitutions was devised and led to a gem-diethyl, a gem-
di(methoxymethyl), and a cyclopropyl series. The introduction of
these geminal substitutions at the C50 position has allowed for
the synthesis of various series that generally exhibit good poten-
cies and selectivity. Further SAR exploration of the C2 position in
the gem-dimethyl series resulted in the discovery of two lead com-
pounds 35 and 36. With good potencies against HCV in the replicon
assay, high selectivity profiles, and broad plasma exposure and bio-
availability in rat, dog, and monkey, these compounds emerged as
lead compounds in the program.
4. (a) Flisiak, R.; Parfieniuk, A. Expert Opin. Investig. Drugs 2010, 19, 63; (b)
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Piwinski, J. J.; MacCoss, M.; Kwong, C. D.; Bansal, N.; Clark, J. L.; Fowler, A. T.;
Kezar, H. S., III; Valiyaveettil, J.; Reynolds, R. C.; Maddry, J. A.; Ananthan, S.;
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R. C.; Maddry, J. A.; Ananthan, S.; Secrist, J. A., III; Shih, N.-Y.; Piwinski, J. J.; Li,
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Pinto, P.; Rizvi, R.; Rossman, R.; Shankar, B.; Tong, L.; Velazquez, F.;
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Kwong, C. D.; Clark, J. L.; Fowler, A. T.; Geng, F.; 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.; Arasappan, A. Bioorg. Med. Chem. Lett. 2012, 22,
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Acknowledgment
We thank the Structural Chemistry group for NMR and MS anal-
ysis. We also thank Albany Molecular Research Institute for their
contributions.
7. (a) Jurowich, S.; Sticht, G.; Käferstein, H. Alcohol 2004, 32, 187; (b) Mayer, S. C.;
Kreft, A. F.; Harrison, B.; Abou-Gharbia, M.; Antane, M.; Aschmies, S.; Atchison,
K.; Chlenov, M.; Cole, D. C.; Comery, T.; Diamantidis, G.; Ellingboe, J.; Fan, K.;
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M.; Kremer, K.; Kubrak, D.; Lin, M.; Lu, P.; Magolda, R.; Martone, R.; Moore, W.;
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References and notes
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V. A. Verma et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6967–6973
6973
15. Compounds were completely soluble at the tested concentrations for all
in vitro assays.
Proliferation Assay (Promega, Cat #G3580) three days post dosing on cells
treated identically as in replicon activity assays. The CC50 is the concentration
of inhibitor that yields 50% inhibition compared to vehicle-treated cells. The
effect of an inhibitor on cellular DNA synthesis is determined by a scintillation
proximity assay. Replicon cells are seeded in 96-well Cytostar-T Scintillating
Microplates (PerkinElmer, Cat #RPNQ0163) one day prior to inhibitor
treatment. Various concentrations of an inhibitor in triplicate are added with
[methyl-14C]-thymidine (PerkinElmer, Cat #NEC568050UC, final concentration
16. 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 of 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
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.