Synthesis and SAR of pyridothiazole substituted pyrimidine derived HCV replication inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.06.099

Introduction of a nitrogen atom into the benzene ring of a previously identified HCV replication (replicase) benzothiazole inhibitor 1, resulted in the discovery of the more potent pyridothiazole analogues 3. The potency and PK properties of the compounds were attenuated by the introductions of various functionalities at the R¹, R² or R³ positions of the molecule (compound 3). Inhibitors 38 and 44 displayed excellent potency, selectivity (GAPDH/MTS CC₅₀), PK parameters in all species studied, and cross genotype activity.

Full text of DOI:10.1016/j.bmcl.2012.06.099

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5652–5657
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of pyridothiazole substituted pyrimidine derived HCV
replication inhibitors
Vinay M. Girijavallabhan a, , Carmen Alvarez a, Frank Bennett a, Lei Chen a, Stephen Gavalas a,
⇑
Yuhua Huang a, Seong-Heon Kim a, Aneta Kosinski a, Patrick Pinto a, Razia Rizvi a, Randall Rossman a,
Bandarpalle Shankar a, Ling Tong a, Francisco Velazquez a, Srikanth Venkatraman a, Vishal A. Verma a,
Joseph Kozlowski a, Neng-Yang Shih a, John J. Piwinski a, Malcolm MacCoss a, Cecil D. Kwong b,
Namita Bansal b, Jeremy L. Clark b, Anita T. Fowler b, Hollis S. Kezar III b, Jacob Valiyaveettil 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 a nitrogen atom into the benzene ring of a previously identified HCV replication (repli-
case) benzothiazole inhibitor 1, resulted in the discovery of the more potent pyridothiazole analogues
3. The potency and PK properties of the compounds were attenuated by the introductions of various func-
tionalities at the R1, R2 or R3 positions of the molecule (compound 3). Inhibitors 38 and 44 displayed
excellent potency, selectivity (GAPDH/MTS CC50), PK parameters in all species studied, and cross geno-
type activity.
Received 13 June 2012
Revised 26 June 2012
Accepted 27 June 2012
Available online 15 July 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
HCV replication inhibitor
HCV replicase inhibitor
Carbanucleoside-like
Pyridothiazole
C–H arylation
5-Aryl pyrimidine
Pyridothiazole pyrimidine
Hepatitis C (HCV) is the leading cause of liver cirrhosis, hepato-
cellular carcinoma, and chronic liver failure.1 An estimated 180 mil-
lion people worldwide are infected with HCV. The standard of care
for HCV infection until recently provided low response rates (less
than 50% response rates among patients infected with the most pre-
valent genotype 1 virus), genotype variability, viral resistance, and
side effect profiles that resulted in low patient compliance.2 The re-
cent success of two NS3 protease inhibitors, boceprevir and telapre-
vir, has inspired further efforts to discover novel methods of
treating the disease.3 In addition to several other protease inhibi-
tors undergoing clinical trials, novel compounds targeting the
NS5b polymerase, the NS5a protein, and other cellular targets are
in clinical studies.4 Since the emergence of resistance is a major
obstacle in HCV treatment, it is important to discover therapies
with novel modes of action and unique resistance profiles.
Our early research efforts on small molecule HCV replication
inhibitors led to a series of pyrimidine compounds.5 The lead com-
pound (Fig. 1, 1) was a benzothiazole substituted pyrimidine deriv-
ative that had reasonable potency (replicon assay) and selectivity
(GAPDH/MTS CC50).6 Our earlier SAR5a,b concluded that the 4-car-
basugar and 6-Me group were optimal with regard to replicon
activity; therefore, our goal was to retain these groups and focus
attention on modifications to the benzothiazole ring and the
2-position of pyrimidine. One of the modifications described in
the earlier publication5b was the addition of the methylamino
functionality to the 50-position of the benzothiazole (2). This com-
pound retained potency but displayed suboptimal pharmacoki-
netic properties. Due to the tolerance of polar functionality at the
50-position, we hypothesized that pyridothiazole compound 3
could retain the potency and PK advantages of benzothiazole 1.
⇑
Corresponding author.
E-mail address: vinay.girijavallabhan@spcorp.com (V.M. Girijavallabhan).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.099

V. M. Girijavallabhan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5652–5657
5653
4' R1
NH
6' N
R2
5'
N
Me
N
N
Me
Me
S
6 N
N
5
4
S
S
N
R3
2N
H
HN
N
N
H
HN
N
HO
N
H
HN
N
HO
HO
CF3
CF3
OH OH
OH OH
OH OH
3
2
1
Replicon EC50 = 30 nM
MTS/GAPDH CC50 = 17/25 µM
Replicon EC50 = 30 nM
MTS/GAPDH CC50 = 14/18 µM
Rat PK (HCl, po, 10 mpk):
AUC = 0.3 µM.h
Rat PK (HCl, po, 10 mpk):
AUC = 7.0 µM.h
Figure 1. Optimization of the benzothiazole series.
The sterics of the methylamino functionality suggested that further
substitutions at the 40- and 60-positions might also be tolerated.
Previously it had been determined that the 2-position of the
pyrimidine could be varied with functionalized amines in order
to modulate the potency, selectivity and PK properties of the com-
pounds. Therefore, we developed a synthetic procedure that al-
lowed for the facile incorporation of amines to the 2-position of
the pyrimidine at the end of the synthesis.
(Scheme 1). The open position of the ring was iodinated using io-
dine and sodium hydroxide and further treatment with phospho-
rus oxychloride gave compound 7. The chloro group was then
displaced with commercially available carbasugar 8, followed by
protection of the free secondary alcohols as an acetonide to afford
compound 9. This intermediate was crucial to the convergent syn-
thesis of analogs since it allowed for the incorporation of groups at
both the 2 and 5-positions late in the synthesis.
Our synthesis began with methylation of commercially
available 2-mercapto-6-methylpyrimidin-4(3H)-one (4) using
iodomethane and potassium carbonate to provide compound 5
The next step in the synthesis was the crucial arylation step that
would allow for optimization of the 5-position. Previously, our
group was the first to report the direct arylation of pyridothiazoles
CH3
CH3
CH3
I
N
N
b
N
a
c
N
H
SH
O
N
H
SMe
HO
O
O
N
H
SMe
6
5
4
H3C
N
5
CH3
I
SMe
2
I
N
d
e
N
N
NH
NH2 HCl
N
SMe
N
Cl
HO
S
10
O
O
HO
8 OH
9
7
N
N
N
CH3
N
N
CH3
N
S
S
f
g
N
SMe
HN
N
SOMe
HN
HO
HO
O
O
O
O
11
12
N
N
N
CH3
N
N
CH3
N
S
S
h
N
N
H
HN
N
N
H
HN
CF3
CF3
HO
HO
HCl
HO
OH
O
O
13
14
Scheme 1. Reagents and conditions: (a) Iodomethane, K2CO3, DMSO 70%; (b) iodine, NaOH 83%; (c) POCl3, 120 °C, 88%; (d) (1) triethylamine, EtOH, reflux; (2) 2,2-
dimethoxypropane, methanesulfonic acid, acetone (74% over two steps); (e) Pd(PPh3)4, Cs2CO3, CuI, DMF 62%; (f) m-CPBA, methylene chloride 95%; (g) 2,2,2-
trifluoroethylamine, acetonitrile 85%; (h) 4 M HCl dioxane, MeOH 95%.

5654
V. M. Girijavallabhan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5652–5657
Table 1
Modifications to the 2-position of pyrimidine
N
N
Me
S
N
HN
N
R
HO
OH OH
Liver C6hb (ng/g)
a
Compd
R
EC50 (nM)
MTS/GAPDH CC50
(
lM)
Rat AUC(0–6 h) (lM h)
N
H
CF
3
14
15
16
16
124
52
>25/25
18/25
20/14
0
0
0
0
NH
0
2
NH
0.3
N
17
18
19
557
8
>25/25
5/3
7.5
0.4
0.2
1395
192
N
H
OEt
N
H
9
16/25
368
20
21
125
19/25
1.4
276
N
H
N
H
c
c
1683
>25/25
—
—
N
22
23
168
3
>25/25
14/25
0.2
0
29
0
N
H
N
H
F
N
H
24
25
1
3
10/4
0
0
0
0
F
N
H
25/25
OCF
3
OMe
26
12
19/25
0.1
100
N
H
OMe
N
27
28
301
7
>25/25
>25/25
0.2
0.2
100
104
N
H
N
H
29
30
31
222
6204
3165
>25/25
>25/25
>25/25
0.1
0
77
0
N
H
c
c
N
H
—
—

V. M. Girijavallabhan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5652–5657
5655
Table 1 (continued)
a
Compd
R
EC50 (nM)
114
MTS/GAPDH CC50
>25/25
(
lM)
Rat AUC(0–6 h)
(lM h)
Liver C6hb (ng/g)
N
H
32
33
0.1
116
0
N
H
222
17
25/25
25/25
0
0
F
N
H
34
0
N
N
OMe
N
H
35
36
37
25/25
0.1
17
OMe
OCF
3
17
16
23/24
0.5
1.6
506
N
H
N
H
37
>25/25
1800
OCF
3
a
AUC0–6 h, po (10 mpk), vehicle–0.4% mc.
Liver concn at 6 h.
Not measured.
b
c
using both aryl and heteroaryl bromides and iodides.7 Using this
chemistry, the pyridothiazole 10 was efficiently coupled to the
pyrimidine 9 using Pd(PPh3)4, copper iodide and cesium carbonate
to supply the desired product 11. The use of both palladium and
copper sources were found to be instrumental in the reaction as
well as the choice of base. This intermediate made it possible to
study several substituted amines at the 2-position in order to opti-
mize for potency, selectivity and PK properties. The thiomethyl
group was oxidized to the corresponding sulfoxide using m-CPBA
followed by displacement with a variety of amines to provide com-
pounds of type 13. Deprotection of the acetonide under acidic con-
ditions gave access to the desired inhibitors in good yield.
With an efficient synthesis in hand, our first goal was to opti-
mize the 2-position functionality and then to study modifications
to the pyridothiazole. Table 1 displays the optimization of the
2-amino group of the pyrimidine ring. Compound 14 was synthe-
sized for direct comparison with compounds 1 and 2 and demon-
strated an improved potency (2-fold) compared to compound 1,
but improvements in PK8 were necessary. As can be seen from
compounds 15 and 16, there is a significant loss in potency when
the amino group at the 2- position is unsubstituted or has a smaller
methyl substitution.
to PK properties. Compound 21 is a representative example that
demonstrated that basic substitution was not tolerated. Similarly
N-aryl substitution, such as compound 22, was not tolerated. Ben-
zyl substituted compounds 23–25 displayed a significant improve-
ment in potency, but unfortunately did not provide desirable PK
properties. Phenethylamines such as compound 26 maintained
reasonable activity, but provided no improvements to selectivity
or PK. It was evident from compound 20 that alkyl substitution
adjacent to the nitrogen improved PK properties but lowered po-
tency. Since the benzyl substitutions provided excellent potency,
but lacked adequate PK properties, we decided to investigate fur-
ther substitutions in this series. Incorporation of the N-methyl
group (27) in the more potent benzyl series caused a significant
loss in potency and confirmed the necessity of the N–H for activity.
Compounds 28 and 29 incorporated an alpha-methyl to the ben-
zylic position. The potency discrepancy between the two isomers
clearly indicated that appropriate stereochemistry at the
a-posi-
tion is important. These compounds also provided improvement
in the selectivity window. The more active compound 28 also
exhibited a modest improvement to PK when compared to ben-
zylic compounds 23–25. Di-methyl substitution at the benzylic po-
sition (30) as well as the spirocyclic cyclopropyl 31 were
attempted in order to further improve PK properties, but these
modifications caused a loss in potency. We decided to modulate
the PK of the molecule via substitution of the aromatic ring.
Para-substituted analogs 32–34 retained potency and selectivity
but did not improve PK properties. Meta-substituted analogs such
as 35 lost potency. Addition of the trifluoromethoxy group at the
ortho position (36) provided comparable potency to compound
Compound 17 demonstrated that disubstitution of the nitrogen
was not tolerated with respect to potency, but there was a marked
improvement in PK. Compounds 18–19 showed that the introduc-
tion of substituted alkyl groups gave improved potency and
provided a modest improvement to PK when compared to 14.
a-Branched alkyl groups such as cyclopropyl amino compound
20 demonstrated a loss in potency, but a significant improvement

5656
V. M. Girijavallabhan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5652–5657
Table 2
Optimization of Pyridothiazole
1
R
4'
N
6'
2
R
N
Me
N
S
N
N
H
HN
HO
OCF
3
OH
OH
Compd
R1
R2
EC50 (nM)
MTS/GAPDH CC50
(
lM)
rAUCa
(l
M h)
Liver C6hb (ng/g)
37
38
39
40
41
H
Me
Et
Pr
i-Pr
H
H
H
H
H
16
16
20
20
26
>25/25
>25/25
7/7
13/13
10/11
1.6
1.1c
5.8
0.8
3.2
1800
2816
6000
1259
8500
42
H
26
13/10
3.2
4400
43
44
Cyclobutyl
Cyclopropyl
H
H
16
60
5/5
>25/25
3.3
4900
11,000
5.2c
OMe
d
d
45
H
59
14/23
—
—
O
46
H
34
12/12
0.4
0
—
16
0
—
CN
47
48
49
50
51
52
NH2
H
Me
Et
H
37
38
43
264
703
69
>25/25
>25/25
12/20
24/23
24/21
13/12
d
d
Me
Me
Me
Me
Et
5.6
9800
257
25.0
d
d
Cyclopropyl
Et
—
—
—
—
d
d
a
b
c
AUC0–6 h po (10 mpk), vehicle–0.4% mc.
Liver conc at 6 h.
AUC0–24 h, po (10 mpk).
Not measured.
d
14 as well as measurable improvements to PK. When the trifluoro-
methoxy group was moved to the para position we were encour-
aged to find that compound 37 maintained potency and
displayed improved PK properties. At this point, we decided to con-
tinue our optimization by maintaining the 2-position (R)-4-triflu-
data is summarized in Table 2. Incorporation of a methyl group at
the 40-position of pyridothiazole (compound 38) retained the po-
tency and PK properties of compound 37 including a measurable
improvement to the concentration in the liver at 6 h.10 The corre-
sponding ethyl analog 39 maintained the potency and improved
PK, but displayed lower selectivity. This was also true with other al-
kyl functionalities such as 40–43. The exceptionto this trend was the
cyclopropyl compound 44 which maintained excellent selectivity
while showing significant improvements in PK. Incorporation of es-
ters or nitriles at the 40-position was not optimal as demonstrated by
compounds 45 and 46. Incorporation of a polar functionality, exem-
plified by amino compound 47, provided reasonable potency and
selectivity, but caused a loss in PK. Substitution at the 60-position
of the pyridothiazole, such as compound 48, demonstrated a slight
loss of potency. Compounds 49–52 all contain di-substituted pyri-
dothiazole moeities and displayed a loss in potency and selectivity.
Tables 1 and 2 include several compounds that demonstrate
good potency, PK, and selectivity in the MTS/GAPDH assay. In order
to further prioritize the compounds for advancement, the more
stringent thymidine incorporation assay (DNA CC50) was utilized.
This assay measured the amount that these compounds inhibited
DNA synthesis.11 Table 3 shows the DNA CC50 values for selected
compounds (from Tables 1 and 2) with the best potency, PK or
MTS/GAPDH values. Although, compounds 37, 38, and 44 all had
promising replicon potency and MTS/GAPDH values, the DNA
CC50 selectivity window was much lower for compound 37 and,
therefore, compounds 38 and 44 displayed the best overall profiles.
Compound 38 had encouraging PK in rat and dog, and had a good
selectivity profile (Fig. 2). Although compound 44 was slightly less
potent in the replicon assay, the compound exhibited a higher liver
oromethoxy-a-methylbenzylamino substitution and turning our
attention to the optimization of the pyridothiazole functionality.
In a previous paper we described the synthesis of a variety of 40
and 60-substituted pyridothiazoles.7,9 These substituted pyrido-
thiazoles were introduced using chemistry described in Scheme 1
in order to optimize the series for potency, selectivity and PK. The
Table 3
Profiles of lead compounds
1
R
N
2
R
N
Me
S
N
N
N
H
HN
HO
OCF
3
OH
OH
Compd R1
R2 EC50 (nM) MTS/GAPDH CC50
(lM) DNA CC50 (lM)
37
38
44
H
Me
Cyp
H
H
H
16
16
60
>25/25
>25/25
>25/25
6
>25
>25

V. M. Girijavallabhan et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5652–5657
5657
N
N
N
N
Me
Me
S
S
N
N
N
H
N
H
HN
N
HN
N
HO
HO
OCF3
OCF3
OH OH
OH OH
38
44
Replicon (1b) EC50 = 16 nM
Replicon (1a) EC50 = 70 nM
Replicon (2a) EC50 = 80 nM
MTS/GAPDH CC50 >25/25 µM
DNA CC50 = >25 uM
Replicon (1b) EC50 = 60 nM
Replicon (1a) EC50 = 400 nM
Replicon (2a) EC50 = 180 nM
MTS/GAPDH CC50 >25/25 µM
DNA CC50 = >25 uM
Full PK Data:
Full PK Data:
Rat (HCl, po, 10 mpk):
AUC = 1.1 µM.h; F = 15%
t1/2 = 2.5 hr; Cl = 40 mL/min/kg
Liver C(6h) = 2816 ng/g
Rat (HCl, po, 10 mpk):
AUC = 5.2 µM.h; F = 18%
t1/2 = 5 hr; Cl = 11 mL/min/kg
Liver C(6h) = 11000 ng/g
Monkey (HCl, po, 3 mpk):
AUC = 0.3 µM.h; F = 7%
t1/2 = 3.3 hr; Cl = 20 mL/min/kg
Monkey (HCl, po, 3 mpk):
AUC = 1.3 µM.h; F = 12%
t1/2 = 4.4 hr; Cl = 7.2 mL/min/kg
Dog (HCl, po, 3 mpk):
AUC = 2.9 µM.h; F = 31%
t1/2 = 4.2 hr; Cl = 9.8 mL/min/kg
Dog (HCl, po, 3 mpk):
AUC = 5.1 µM.h; F = 32%
t1/2 = 5.2 hr; Cl = 4.2 mL/min/kg
Figure 2. Overall profile of compounds 38 and 44.
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.
concentration at 6 h post dose as well as better overall PK in all
species tested compared to compound 38. Both compounds also
demonstrated activity against other genotypes, but with a modest
shift. Inhibitors 38 and 44 had no issues with CYP inhibition (3A4,
4. (a) Flisiak, R.; Parfieniuk, A. Expert Opin. Investig. Drugs 2010, 19, 63; (b)
Birerdinc, A.; Younossi, Z. M. Expert Opin. Emerg. Drugs 2010, 15, 535; (c)
Meanwell, N. A.; Kadow, J. F.; Scola, P. M. Annu. Rep. Med. Chem. 2009, 44, 397.
5. (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.
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.
6. 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.
2D6, 2C9 >20
lM), and were clean in an in-house kinase panel
counterscreen (22 kinases, IC50 >30
lM).
In summary, we have developed a novel class of HCV replication
inhibitors with good potency, selectivity and PK. Improvements to
potency were achieved by adding the pyridothiazole group as a
replacement for the benzothiazole moeity. These modifications
led to the discovery of compounds 38 and 44 which displayed good
potency and selectivity profiles while demonstrating good PK in
multiple animal species. Further optimization of the profile of
these inhibitors by modifications to the carbasugar ring system
will be reported in future publications.
Acknowledgment
We would like to thank Albany Molecular Research Institute
(AMRI) for their contribution to this work.
References and notes
1. Who Health Organization (WHO). Hepatitis C. Fact Sheet No. 164. Revised June
2011. WHO web site [online], <http://www.who.int/mediacentre/factsheets/
fs164/en/> (2011).
2. Manns, M. P.; Foster, G. R.; Rockstroh, J. K.; Zeuzem, S.; Zoulim, F.; Houghton, M.
Nat. Rev. Drug Disc. 2007, 6, 991.
7. Girijavallabhan, V.; Arasappan, A.; Bennett, F.; Huang, Y.; Njoroge, F. G.;
MacCoss, M. Tetrahedron Lett. 2010, 51, 2797.
8. 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.
9. Huang, H.; Bennett, F.; Girijavallabhan, V.; Alvarez, C.; Chan, T. M.; Ostermann,
R.; Senior, M.; Kwong, C.; Bansal, N.; Njoroge, F. G.; MacCoss, M. Tetrahedron
Lett. 2010, 51, 2800.
10. The animals were sacrificed at 6 h and the liver was harvested and processed to
measure the compound concentration (liver C6 h).
11. 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
3. (a) FDA approves Victrelis for Hepatitis C. http://www.fda.gov/NewsEvents/
Newsroom/PressAnnouncements/ucm255390.htm.; (b) Venkatraman, S.;
Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, E.; Liu, Y.-T.; Lovey, R.;
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Microplates (PerkinElmer, Cat
treatment. Various concentrations of an inhibitor in triplicate are added
with [methyl-14C]-thymidine (PerkinElmer, Cat NEC568050UC, final
# RPNQ0163) one day prior to inhibitor
#
concentration 0.5 uCi/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.