Novel substituted pyrimidines as HCV replication (replicase) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.11.091

Compound 1 was identified as a HCV replication inhibitor from screening/early SAR triage. Potency improvement was achieved via modulation of substituent on the 5-azo linkage. Due to potential toxicological concern, the 5-azo linkage was replaced with 5-alkenyl or 5-alkynyl moiety. Analogs containing the 5-alkynyl linkage were found to be potent inhibitors of HCV replication. Further evaluation identified compounds 53 and 63 with good overall profile, in terms of replicon potency, selectivity and in vivo characteristics. Initial target engagement studies suggest that these novel carbanucleoside-like derivatives may inhibit the HCV replication complex (replicase).

Full text of DOI:10.1016/j.bmcl.2011.11.091

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1160–1164
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel substituted pyrimidines as HCV replication (replicase) inhibitors
Cecil D. Kwong a, , Jeremy L. Clark a, Anita T. Fowler a, Feng Geng a, Hollis S. Kezar III a,
⇑
Abhijit Roychowdhury a, Robert C. Reynolds a, Joseph A. Maddry a, Subramaniam Ananthan a,
John A. Secrist III a, Neng-Yang Shih b, John J. Piwinski b, Cheng Li b, Boris Feld b, Hsueh-Cheng Huang b,
Xiao Tong b, F. George Njoroge b, Ashok Arasappan b,
⇑
a Southern Research Institute, Birmingham, AL 35205, USA
b Merck Research Laboratories, 2015 Galloping Hill Rd., Kenilworth, NJ 07033, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Compound 1 was identified as a HCV replication inhibitor from screening/early SAR triage. Potency
improvement was achieved via modulation of substituent on the 5-azo linkage. Due to potential toxico-
logical concern, the 5-azo linkage was replaced with 5-alkenyl or 5-alkynyl moiety. Analogs containing
the 5-alkynyl linkage were found to be potent inhibitors of HCV replication. Further evaluation identified
compounds 53 and 63 with good overall profile, in terms of replicon potency, selectivity and in vivo char-
acteristics. Initial target engagement studies suggest that these novel carbanucleoside-like derivatives
may inhibit the HCV replication complex (replicase).
Received 4 November 2011
Accepted 21 November 2011
Available online 1 December 2011
Keywords:
HCV replication inhibitor
HCV replicase inhibitor
Carbanucleoside-like
5-Alkynyl pyrimidine
5-Azo pyrimidine
Ó 2011 Elsevier Ltd. All rights reserved.
Chronic hepatitis C virus (HCV) infection is a global health bur-
den affecting an estimated 3% of the human population.1 It is the
leading cause of liver cirrhosis, hepatocellular carcinoma and liver
failure in humans. HCV is a positive stranded RNA virus belonging
to the Flaviviridae family. Upon entering hepatocytes, the HCV gen-
ome encodes a polyprotein of approximately 3000 amino acid res-
idues. Post-translational cleavage of the polyprotein into
functional individual viral proteins is mediated by host and viral
proteases, ultimately resulting in viral replication.2 Until recently,
types, with numerous subtypes) and high mutation rate will likely
require a combination of agents to ultimately suppress emergence
of viral resistance resulting in clinical cure.
In our continued interest to discover inhibitors that target HCV
via novel pathways, screening of a proprietary nucleoside-like li-
brary was carried out. From screening and early SAR triage, com-
pound 1 (Fig. 1) was identified as a potential HCV replication
inhibitor with encouraging potency in the cell-based genotype 1b
subgenomic replicon assay.6 Compound 1 exhibited replicon EC50
the standard-of-care (SOC) has been pegylated
a-interferon and
of 0.3 lM, with good selectivity window (MTS CC50 = 25 lM). A
ribavirin combination therapy, which does not target the virus spe-
cifically.3 Moreover, this SOC is effective in less than 50% of geno-
type 1 patients, a population more prevalent in North America,
Europe and Japan, and is accompanied with unwanted side effects.
Intense efforts were focused in the past decade to discover novel
small molecule direct-acting antivirals (DAA) that inhibit viral rep-
lication. These efforts resulted in the recent regulatory approval of
NS3 protease inhibitors, boceprevir and telaprevir that improved
the cure rates when added to SOC.4 Several other DAAs (NS3 pro-
tease inhibitors, NS5B nucleoside and non-nucleoside inhibitors,
and NS5A inhibitors) are in various stages of clinical trials.5 Despite
these advances, the genetic variability of HCV (at least six geno-
noteworthy feature of the nucleoside-like inhibitor 1 is the pres-
ence of carbocyclic ring instead of the ribose moiety required for
natural nucleosides. Carbocyclic nucleosides are known to have en-
hanced stability toward pyrophosphorylases that hydrolytically
cleave glycosidic bonds.7 The difference in conformation of the
cyclopentane versus ribose ring could also result in a different
Cl
Cl
N
N
N
5
Replicon EC = 0.3 µM
50
µ
MTS CC = 25 M
50
N
1
NH
2
HN
HO
⇑
Corresponding authors. Tel.: +1 205 581 2746; fax: +1 205 581 2726 (C.D.K.);
1
tel.: +1 908 740 2607; fax: +1 908 740 7152 (A.A.).
E-mail addresses: kwong@southernresearch.org (C.D. Kwong), ashok.arasappan@
merck.com (A. Arasappan).
HO
OH
Figure 1. Profile of initial lead 1.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.091

C. D. Kwong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1160–1164
1161
Cl
Ar
Cl
SAR from that of natural nucleosides. Hence we initiated medicinal
chemistry efforts around the core structure 1 with a goal to iden-
tify suitable candidate for further lead optimization.
I
N
N
a
N
NH
N
NH
2
HN
HN
OH
2
Schemes 1–4 describes the chemistry for preparation of target
compounds. SAR studies were initiated with substitution on the
phenyl ring connected to the 5-azo linkage of 1. Synthesis of inhibi-
tors from the 5-azo series is shown in Scheme 1. The key intermedi-
ate 4 was prepared by refluxing a mixture of commercially available
2-amino-4,6-dichloropyrimidine (2), (1R,2S,3R,4R)-2,3-dihydroxy-
4-(hydroxymethyl)-1-amino cyclopentane hydrochloride (3), and
triethylamine in ethanol. The target compounds were subsequently
HO
HO
13
16
HO
OH
HO
b
d
TMS
Cl
Cl
N
N
c
N
NH
N
NH
2
HN
OH
HN
OH
2
HO
HO
Cl
17
18
HO
HO
Cl
NH HCl
N
2.
HO
a
N
N
NH
2
HN
OH
Scheme 4. Reagents and conditions: (a) Arylacetylene, Pd(Ph3P)4, CuI, Et3N, DMF,
18 h (ꢀ78%); (b) TMSacetylene, Pd(Ph3P)4, CuI, Et3N, DMF, 55 °C, 18 h (56%); (c)
tetraethylammonium fluoride dihydrate, MeCN, 2 h (94%); (d) aryl halide,
Pd(Ph3P)4, CuI, Et3N, DMF, microwave, 90 °C, 10 min (50–56%).
+
HO
HO
OH
Cl
N
NH
2
4
2
3
HO
obtained by coupling intermediate 4 with various substituted ben-
zenediazonium halides (commercial or prepared by diazotization
of appropriately substituted anilines with sodium nitrite and HCl)
using established protocol.
Scheme 2 depicts the synthesis of targets from the 5-alkenyl
series, the first group of analogs designed to replace the 5-azo link-
age. Wittig reaction of substituted benzyl triphenylphosphonium
halide and 2-amino-4,6-dichloro-5-formylpyrimidine 7, resulted
in intermediate 8 as mixture of cis/trans isomers (mostly trans).
Treatment of 8 with carbasugar derivative 3 employing previously
described procedures afforded the required compounds as a mix-
ture of cis/trans isomers in a modest yield.
R
R
Cl
N
+
-
N
N
X
2
N
5
N
NH
2
HN
OH
b
HO
6
HO
Scheme 1. Reagents and conditions: (a) EtOH, Et3N, reflux, 16 h (72%); (b)
(commercial or generated in situ from commercially available anilines/NaNO2/HCl),
NaOAc, gl. AcOH, water (ꢀ60%).
5
Preparation of targets with the 5-alkenyl moiety saturated or
cyclopropanated is shown in Scheme 3. Reduction of 10 with cata-
lytic palladium on carbon and cyclohexene at elevated temperatures
gave 11. The cyclopropyl derived target 15 was prepared via Suzuki
conditions.8 Thus, 2-amino-4,6-dichloropyrimidine 2 was iodinated
with iodine monochloride to afford 12, which was subsequently re-
acted with carbasugar 3 in refluxing ethanol to provide 5-iodo inter-
mediate 13. Suzuki reaction of 5-iodo intermediate 13 with
phenylcyclopropyl boronic acid 149 resulted in target 15.
Cl
R
Cl
Cl
N
R
OHC
Cl
a
b
N
N
N
NH
2
HN
OH
HO
N
NH
Cl
N
NH
2
2
9
7
8
HO
Scheme 2. Reagents and conditions: (a) Ph3P+CH2RXÀ, nBuLi, THF, À78 °C (ꢀ40%);
(b) 3, EtOH, Et3N, reflux, 72 h (20–30%).
Synthesis of the 5-alkynyl series targets is shown in Scheme 4.
Sonogashira reaction8 of previously described 5-iodo intermediate
13 with commercially available arylacetylenes resulted in some of
the target compounds of type 16. Alternatively, to expand our
selection of arylacetylenic targets of type 16 the following protocol
was employed. Coupling of the 5-iodinated derivative 13 with
TMS-acetylene under essentially the above described conditions
gave intermediate 17. Subsequent desilylation of 17 to 18 with tet-
raethylammonium fluoride dihydrate in acetonitrile, followed by a
second Sonogashira reaction with appropriately substituted aryl
halides then gave the desired target compounds 16.
Cl
Cl
Ph
Ph
N
N
a
N
NH
2
HN
OH
N
NH
2
HN
OH
HO
HO
10
11
HO
HO
Cl
N
I
Cl
N
I
b
As mentioned above, while screening a nucleoside-like library,
compound 1 was identified with interesting HCV replication
inhibitory activity. In the subgenomic replicon assay compound 1
c
N
NH
HN
2
2
HO
Cl
N
NH
2
13
12
HO
OH
exhibited encouraging potency with an EC50 = 0.3
lM, and
acceptable selectivity (MTS CC50 = 25 M). SAR efforts were
l
Cl
initiated with modifications to the phenyl moiety appended to the
5-azo linkage. Compounds listed in Table 1 were prepared as
described in Scheme 1. Thus, moving the chloro moiety from para
(compd 1) to meta position resulted in an equipotent analog, 19.
However, ortho chloro substituted phenyl analog, 20, and the parent
phenyl derivative, 21 resulted in few fold improvement in replicon
EC50. Substituting the phenyl moiety with fluoro functionality
proved to be advantageous, with analogs 22–24 showing improved
potency while retaining the good selectivity window. Introduction
Ph
Ph
N
B(OH)
2
14
N
NH
2
HN
OH
d
HO
15
HO
Scheme 3. Reagents and conditions: (a) 10% Pd/C, cyclohexene, MeOH, THF, 80 °C,
2 h (ꢀ75%); (b) ICl, gl. AcOH, 5 h (79%); (c) 3, EtOH, Et3N, reflux, 72 h (83%); (d)
phenylcyclopropylboronic acid 14, Pd(Ph3P)4, K2CO3, DMF, 90 °C, 16 h (ꢀ20%).

1162
C. D. Kwong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1160–1164
Table 1
Table 2
Evaluation of 5-azo series
5-Alkenyl series SAR data
R
Cl
N
R
Cl
N
N
N
N
N
NH
2
HN
HO
NH
2
HN
HO
HO
OH
HO
OH
Compd
R
EC50
(lM)
CC50 (lM)
Compd
R
EC50
(
lM)
CC50 (lM)
10
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
Ph
0.9
1.9
1.9
0.6
2.5
0.12
3.3
0.3
0.2
0.5
4.2
5.5
0.6
3.4
1
>25
25
25
25
25
>25
ꢀ30
>25
>25
25
4-MePh
4-MeOPh
3-MeOPh
2-MeOPh
3,4-DiOMePh
4-ClPh
2-ClPh
3-FPh
2-FPh
4-CF3Ph
4-CNPh
3-CNPh
4-Pyridyl
trans-3-Pyridyl
cis-3-Pyridyl
2-Pyridyl
1
4-Cl
3-Cl
2-Cl
H
4-F
3-F
0.3
25
21
13
17
25
24
25
25
25
25
20
7
19
20
21
22
23
24
25
26
27
28
29
30
0.32
0.11
0.1
0.2
0.07
0.1
1.2
0.6
3.8
0.9
2-F
4-CN
3-CN
4-NO2
3-NO2
4-Me
4-OMe
25
>25
>25
25
25
25
0.06
0.02
25
4
2.3
25
of strongly electron withdrawing cyano (25, 26) or nitro (27, 28)
moiety to the phenyl ring resulted in diminished replicon potency.
While the 4-methyl substituted analog, 29 was essentially equipo-
tent to parent phenyl derivative 21, methoxy substitution at the 4-
position of the phenyl ring (compd 30) resulted in significant
improvement in potency (EC50 = 20 nM) with an excellent selectiv-
ity index (CC50/EC50 > 1000). Our attempts to modify the core struc-
ture by replacing the carbasugar moiety with numerous cyclic and
acyclic alkylamino residues, with and without appended hydroxyl
functionality resulted in significant erosion in replicon activity, thus
highlighting the importance of the carbasugar motif as a potential
recognition element.
While the initial SAR activities were carried out with the screen-
ing lead containing an azo linkage, it was highly desirable to find a
suitable alternative for the azo functionality due to potential toxi-
cological concern. We decided first to replace the 5-azo linkage
with alkenyl moiety. Inhibitors with the 5-alkenyl functionality
were prepared as described in Scheme 2, and the results are shown
in Table 2. Thus, the parent phenyl substituted 5-alkenyl derivative
10 was almost a log less potent than the corresponding 5-azo com-
pound, 21. Introduction of a 4-methyl (31) or 4-methoxy (32) group
on the phenyl ring, substituents that showed the best potency in
the 5-azo series, were less potent as well. Position of the methoxy
group on the phenyl ring had minimal impact on replicon potency,
with 3-methoxy derivative 33 being essentially equipotent and
2-methoxy analog 34, less potent than the parent, 10. However,
introduction of an additional methoxy functionality, the dimethoxy
derivative 35, resulted in restoring most of the replicon potency
were not tolerated. Both compounds displayed significantly dimin-
ished potency when subjected to replicon assay (EC50 > 25 M). The
above described results indicated that sp2-type (or pi-character) at
l
the 5-position was necessary for potency.
Based on the above observations, replacement of the 5-alkenyl
moiety with an alkynyl linkage was deemed appropriate. A series
of 5-alkynyl containing inhibitors were synthesized as described
in Scheme 4, and evaluated in the replicon assay. Results are
shown in Table 3. The parent phenyl alkynyl substituted derivative,
47 displayed good replicon potency, with EC50 = 0.2 lM. Introduc-
tion of a 4-methyl (48) or 4-methoxy (49) functionality on the
phenyl ring provided equipotent analogs. A fluoro group at the 4
or 2-position, 50 and 51, respectively, had minimal impact on the
replicon potency compared to parent 47. Incorporation of a series
of ester (52–54), amide (55–57), sulfone (58, 59) and sulfonamide
(60) groups were studied next. While substituents at the 2-position
of the phenyl ring were essentially equipotent, significant loss in
replicon potency was observed for 4-substitutions, 56 and 59.
Additionally, analogs 55, 57, 58 and 60 displayed very good selec-
tivity (CC50 > 25 lM). Replacement of the phenyl group with small
heterocyclic rings (61–63) was well tolerated, except the imidazo-
lyl analog 64 that resulted in some loss in potency. Installation of
2- or 4-pyridyl group (65 and 67, respectively) on the alkyne moi-
ety resulted in loss in potency, while the 3-pyridyl replacement
(66) restored most of the potency. Hence further modifications
were carried out on the 3-pyridyl analog 66. Introduction of an
ester functionality on the pyridyl motif, compounds 68 and 69,
provided significant improvement in replicon potency (EC50 of
(EC50 = 0.12 lM) with a very good selectivity window. Next, halo
groups were installed on the phenyl ring. Among the halo (chloro
and fluoro) groups studied, 2-chloro (37) and 3-fluoro (38) substi-
tuted inhibitors displayed good EC50, 0.3 and 0.2 lM, respectively.
As observed for the 5-azo series, strongly electron withdrawing
substitutions on the phenyl moiety (40–42) resulted in loss in
potency. Finally, our attempts to replace the phenyl ring with a
pyridyl moiety (43) resulted in no improvement in replicon
potency. The trans and cis-3-pyridyl isomers, 44 and 45, respec-
tively, were essentially equipotent, thus displaying no potency bias
for aromatic group disposition on the alkenyl linkage.
0.04 and 0.02 lM, respectively), albeit with some loss in selectiv-
ity. Small alkyl introduction on the pyridyl ring was studied next,
with 2-methyl derivative (70) and 2,4-dimethyl analog (72) show-
ing improved profile. Substitution of alkoxy group onto the pyridyl
ring proved advantageous as well. Thus, 4-methoxy substituent,
73, and 4-ethoxyl substituent, 74, were highly potent with EC50
values of 0.03 and 0.02 lM, respectively. Finally, a disubstituted
Modification of the 5-alkenyl linkage, via saturation or cyclo-
propanation of the double bond was studied next. Preparation of
the two derivatives is shown in Scheme 3. Unfortunately, saturation
of the 5-alkenyl group (compd 11) or cyclopropanation (compd 15)
pyridyl moiety containing both alkyl and alkoxy groups, resulted
in inhibitors with very good potency profile; analog 75 dis-
played replicon EC50 = 0.01
(CC50/EC50 = 1000).
lM and excellent selectivity index

C. D. Kwong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1160–1164
1163
Table 3
SAR of 5-alkynyl series
showed acceptable oral AUC levels (1
l
M/h), and minimal target
organ exposure. 4-Methyl and 4-methoxy substituted analogs, 48
and 49, respectively, showed similar in vivo profile. The fluoro
substituted compounds 50 and 51 displayed poor oral exposure.
Interestingly, inhibitor 53 demonstrated good exposure with an
R
Cl
N
N
NH
2
HN
AUC = 4.8
conc6h = 850 ng/g). The isopropyl ester substituted compound, 54
displayed excellent oral exposure (AUC = 20 M/h) and target or-
lM/h, and acceptable target organ distribution (liver
HO
l
HO
OH
gan concentration (liver conc6 h = 4725 ng/g). Inhibitor 58 contain-
ing the sulfone moiety that exhibited good potency and excellent
selectivity did not show any appreciable plasma levels. 2-Thiazolyl
and 3-thiophenyl substituted alkynyl derivatives, 61 and 63,
respectively, displayed similar profile when subjected to rat PK
studies with acceptable oral bioavailability of 18–19%. While the
substituted pyridyl group attached to the alkynyl moiety demon-
strated some of the best replicon potency in this series, most of
those analogs displayed poor oral exposure. Thus, compounds 68,
69, 72, and 75 showed no detectable levels in plasma or liver, while
inhibitors 73 and 76 exhibited minor amounts of plasma exposure
and liver distribution on oral dosing. Based on the good potency,
selectivity and rat oral bioavailability, inhibitor 63 was dosed in
dogs at 2 mpk. Analog 63 demonstrated very good oral exposure
Compd
R
EC50 (
lM)
CC50 (lM)
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
Ph
0.2
15
15
30
23
25
13
20
13
>25
>25
>25
>25
>25
>25
23
25
25
>25
25
25
25
3
4-MePh
4-MeOPh
4-FPh
2-FPh
(2-CO2Me)Ph
(2-CO2Et)Ph
(2-CO2iPr)Ph
(2-CONH2)Ph
(4-CONH2)Ph
(2-CONHEt)Ph
(2-SO2Me)Ph
(4-SO2Me)Ph
(2-SO2NH2)Ph
2-Thiazolyl
2-Thiophenyl
3-Thiophenyl
2-Imidazolyl
2-Pyridyl
0.14
0.18
0.34
0.23
0.13
0.1
0.3
0.2
5
0.2
0.1
1.9
0.25
0.2
0.15
0.1
0.5
0.8
0.4
0.9
0.04
0.02
0.07
0.15
0.05
0.03
0.02
0.01
0.03
in dogs, with an AUC0–24 h = 2.2 lM/h.
Target engagement of the aforementioned HCV replication
inhibitors are not fully understood as yet. Based on some structural
resemblance to nucleosides, it would seem reasonable to hypothe-
size that these compounds are behaving as traditional nucleoside
polymerase inhibitors. However, phosphorylated metabolites were
not detected for compounds from similar structural class when
dosed in replicon cells. This result suggests that the above de-
scribed compounds do not impart their replicon activity via classi-
cal nucleoside-type mechanisms. Furthermore, representative
compounds from the above series displayed only weak inhibitory
3-Pyridyl
4-Pyridyl
(2-CO2Me)-3-pyridyl
(2-CO2Et)-3-pyridyl
(2-Me)-3-pyridyl
3
20
4
15
20
8
10
11
(4-Me)-3-pyridyl
(2,4-diMe)-3-pyridyl
(4-MeO)-3-pyridyl
(4-EtO)-3-pyridyl
(4-MeO-2-Me)-3-pyridyl
(4-EtO-2-Me)-3-pyridyl
activity (IC50 = 200–1000 lM, for 63 and 73) in the polymerase
enzymatic assay, and no activity in the protease or helicase assay
(up to 1 mM, for 63) (manuscript in preparation). Thus, despite
the lack of in vitro enzymatic activities, potent replicon inhibition
can be achieved with these compounds, suggesting a mechanism of
action which inhibits the HCV replication complex (replicase).
Additional studies will be required to further elucidate inhibitor
mechanism.
Selected compounds with good replicon potency from the 5-
alkynyl series were then subjected to rat in vivo studies, and the
results are depicted in Table 4. Animals were dosed at 10 mpk
(PO) or 2 mpk (IV) in a suitable vehicle. Plasma samples were col-
lected at various time points post-dose, and AUC is reported as a
measure of exposure. The animals were then sacrificed at 6 h
post-dose for measuring concentration of the dosed inhibitor in li-
ver, target organ for the viral disease. The parent compound 47
Inhibitor 1 was identified as a screening lead based on the inter-
esting potency in the subgenomic replicon assay, with good selec-
tivity index. While modifications to the core structure, carbasugar
replacement, were not tolerated, selected substitutions on the phe-
nyl ring appended to the 5-azo linkage improved replicon potency.
Potential toxicological issues associated with the azo linkage
prompted the investigation of 5-alkenyl moiety as a suitable alter-
native, albeit with no improvement in potency. Saturation or cyclo-
propanation of the alkenyl group proved detrimental in terms of
potency. Based on the requirement of sp2-like substitution at 5-po-
sition of the pyrimidine ring, alkynyl functionality was explored.
The parent phenyl substituted analog 47 restored replicon potency,
and more importantly demonstrated acceptable oral exposure
when subjected to rat PK study. Furthermore, compounds from
the 5-alkynyl series, 53 and 54, displayed good potency, selectivity
and very good rat plasma and target organ exposure on oral dosing.
Disubstituted pyridyl functionality appended to the 5-alkynyl link-
age resulted in best in vitro profile, with inhibitor 75 exhibiting
Table 4
Rat in vivo data of selected compounds
a
Compd
AUC0–6 h
(l
M/h)
F (%)
Liver conc6 h (ng/g)
47
48
49
50
51
53
54
58
61
63
68
69
72
73
75
76
1
0.73
0.5
0.06
NDb
4.8
—
—
—
—
—
—
—
—
18
19
—
—
—
—
—
—
70
20
<10
<100
<100
850
4725
—
—
—
—
—
20
ND
0.6c
1.6c
ND
ND
ND
0.11
ND
0.03
replicon EC50 = 0.01 lM and excellent selectivity index (CC50/
—
40
—
20
EC50 = 1000). Unfortunately, the potent disubstituted pyridyl ana-
logs evaluated were plagued with poor rat PK profile. Finally,
inhibitors 53 and 63 exhibited good overall profile, in terms of po-
tency, selectivity index, rodent plasma (for 63) and liver exposure
(for 53), and dog plasma exposure (for 63). Thus we have identified
carbanucleoside-like core structure with 5-arylacetylenic linkage
a
b
c
Only PO (10 mpk), vehicle—0.4% mc.
ND—not detected after PO dosing.
IV (2 mpk)/PO (10 mpk), AUC0–24 h
.

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C. D. Kwong et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1160–1164
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optimization efforts to discover HCV replicase inhibitors from the
novel carbanucleoside-like pharmacophore, with improved
in vitro and in vivo profiles will be the subject of future
communication.
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6. (a) Lohmann, V.; Körner, F.; Koch, J.-O.; Herian, U.; Theilmann, L.;
Bartenschlager, R. Science 1999, 285, 110; (b) To measure cell-based anti-HCV
activity, replicon cells (1b-Con1) were seeded at 5000 cells/well in 96-well
plates one day prior to inhibitor treatment. Various concentrations of an
inhibitor in DMSO were added to the replicon cells, with the final concentration
of DMSO at 0.5% and fetal bovine serum at 5% in the assay media. Cells were
harvested 3 days post dosing. The replicon RNA level was measured using real-
time RT-PCR (Taqman assay) with GAPDH RNA as endogenous control. EC50
values were calculated from experiments with 10 serial twofold dilutions of the
inhibitor in triplicate. To measure cytotoxicity (CC50) of an inhibitor, an MTS
assay was performed according to the manufacturer’s protocol (Promega, Cat
#G3580) 3 days post dosing on replicon cells treated identically as in replicon
activity assays.
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